Novel Treatment Strategies for Unconjugated Hyperbilirubinemia

The work described in this thesis was performed at the Department of Pediatrics, Center for Liver, Digestive, and Metabolic Diseases, Beatrix Children’s Hospital, University Medical Center Groningen, The Netherlands and was financially supported by a grant from Hutchison Whampoa, Ltd. Printing of this thesis was financially supported by: Groningen University School for Drug Exploration (GUIDE) University of Groningen (RuG) Nederlandse Vereniging voor Hepatologie Stichting Sanquin Bloedvoorziening Dr Falk Pharma Benelux B.V. Abdiets animal nutrition Woerden Printed by: Gildeprint, Enschede Copyright © F.J.C. Cuperus, Groningen, 2011 ISBN: 978-90-367-5219-0 ISBN: 978-90-367-5220-6 (Ebook)

Novel Treatment Strategies for Unconjugated Hyperbilirubinemia

Proefschrift

 ter verkrijging van het doctoraat in de

Medische Wetenschappen aan de Rijksuniversiteit Groningen

op gezag van de Rector Magnificus, dr. E. Sterken, in het openbaar te verdedigen op

woensdag 7 december 2011 om 11:00 uur

door

Frans Jan Christiaan Cuperus geboren op 23 december 1978

te Groningen

Promotor: Prof. Dr. H.J. Verkade Beoordelingscommissie: Prof. Dr. L. Vitek Prof. Dr. J.P.H. Drenth Prof. Dr. A.F. Bos

Voor mijn ouders

If you are going to try, go all the way. Otherwise don’t even start.

Charles Bukowski

Paranimfen: Martijn Beudel Hester van Meer

Contents

1. Introduction 1 1.1 Bilirubin Metabolism 2 1.2 Bilirubin Properties 6 1.3 Unconjugated Hyperbilirubinemia 11 1.4 Treatment of Unconjugated Hyperbilirubinemia 18 1.5 Scope of this Thesis 23 2. Treatments for Unconjugated Hyperbilirubinemia 41 2.1 Abstract 42 2.2 Introduction 43 2.3 Treatment Strategies 44 2.4 Future Perspectives 59 3. Bile Salt Treatment for Unconjugated 71 Hyperbilirubinemia 3.1 Abstract 72 3.2 Introduction 73 3.3 Animals, Materials, and Methods 74 3.4 Results 78 3.5 Discussion 88 4. PEG Treatment for Unconjugated 95 Hyperbilirubinemia 4.1 Abstract 96 4.2 Introduction 97 4.3 Animals, Materials, and Methods 99 4.4 Results 102 4.5 Discussion 113

5. Combined Treatments for Unconjugated 121 Hyperbilirubinemia 5.1 Abstract 122 5.2 Introduction 123 5.3 Animals, Materials, and Methods 124 5.4 Results 126 5.5 Discussion 131 6. Albumin Treatment for Unconjugated 137 Hyperbilirubinemia 6.1 Abstract 138 6.2 Introduction 139 6.3 Animals, Materials, and Methods 140 6.4 Results 142 6.5 Discussion 148 7. General Discussion 153 7.1 Introduction 154 7.2 Oral Treatment Strategies 154 7.3 Phototherapy and Bilirubin Deposition in the Brain 159 7.5 Conclusions and Future Directions 160 Appendices 165 Summary 166 Samenvatting 169 Dankwoord 176 List of Abbreviations 180 Biografie 183 List of Publications 185

Chapter 1 Introduction

2 Chapter 1

1.1 Bilirubin metabolism

1.1.1 Introduction

ilirubin, the Latinized term for “red bile”, is a yellow-orange colored bile pigment. Accumulation of bilirubin in the body has many causes but, eventually, results from an imbalance between bilirubin production and elimination. Bilirubin accumulation occurs not only in the blood, so-called hyperbilirubinemia, but also in the tissues. The most visible sign of this accumulation is a yellow discoloration of the skin and the sclera, which is referred to as jaundice. Bilirubin can also accumulate in the brain, which is highly dangerous and may lead to permanent neurological damage. In this thesis we will focus on several new pharmacological strategies for excessive (unconjugated) hyperbilirubinemia.

1.1.2 Bilirubin production Bilirubin is produced by the degradation of heme (Figure 1A). The vast majority (~80%) of this heme is derived from hemoglobin, released after the breakdown of red blood cells. The remaining 20% is derived from the catabolism of heme-containing proteins, such as cytochromes, myoglobin, peroxidase, and catalase.[1,2] Bilirubin production occurs in the macrophages of the reticuloendothelial system (RES), which are mainly located in the liver, spleen, and bone marrow. The macrophages of the RES phagocytize heme and degrade it to bilirubin. To do so, these macrophages are equipped with two essential enzymes for heme degradation, namely heme oxygenase and biliverdin reductase.[3] Heme is degraded by microsomal heme oxygenase, which results in the formation of equimolar quantities of iron, carbon monoxide (CO) and blue-green biliverdin IXα.[4] Measurement of CO production, which is excreted via the lungs, can thus be used as a surrogate parameter to quantitate in vivo bilirubin production. The cytosolic biliverdin reductase subsequently converts biliverdin to the yellow-orange bilirubin IXα, also known as unconjugated bilirubin (UCB; Figure 1A).[3] In most non-placental species biliverdin IXα is the end product of heme degradation.[5] It remains unclear why biliverdin IXα, which is non-toxic and water soluble, is converted to the potentially toxic and hydrophobic UCB. One possible explanation could be that, in the fetus, conversion of biliverdin to lipophilic UCB facilitates its excretion, since UCB can readily cross the placenta.[6,7] More important, however, is the role of UCB as a potent anti-oxidant, which may have beneficial effects in neonates.[8] In the fetus, UCB is released to the blood after its production in the RES.

B

Introduction 3

O

!

"

#

O

N N

NN

COOH COOH

!

"

#

N N

NNN N

COOH COOH

Fe

$O

!

"

#

O

N N

N N

COOH COOH

hemeoxygenase

biliverdinreductase

UCB

CB

UCB

FECES

a

HepatocyteSpace of

Disse

ligandin

UGT1A1

MRP2

Heme

UCB

a

b c

b

Liver

Intestine

Blood

Figure 1. Bilirubin metabolism: (a) bilirubin production in the macrophages of the reticuloendothelial system, (b) hepatic clearance of bilirubin, and (c) enterohepatic circulation of bilirubin. and refer to previous panels. CB, conjugated bilirubin; EHC, enterohepatic circulation; ALB, albumin

a b

4 Chapter 1

Once in the blood, the vast majority of UCB (99.9%) is reversibly bound to plasma proteins and transported to the liver. Most bilirubin (>90%) is bound to plasma albumin, and the remainder mostly to apolipoprotein D in high density lipoprotein (HDL) and to alfa-1-fetoprotein in the neonatal period.[9] Only ∼0.1% remains unbound, and is consequently known as free bilirubin (UCBfree). The role of UCBfree in bilirubin-induced neurotoxicity is highly important, since only UCBfree, and not albumin-bound bilirubin, is able to enter the brain (see §1.2.2 for more detail).[10,11] Each human albumin molecule has one high-affinity binding site for UCB.[12,13] UCB can also bind to additional sites on the albumin molecule with a lower affinity. This mainly occurs beyond an albumin:bilirubin molar ratio of 1:1, which, in the presence of physiological albumin levels, roughly corresponds to an UCB concentration of 600 µmol/l.[14,15] UCB is practically insoluble in plasma (<0.1µmol/l) in the absence of albumin or apolipoprotein D.[16]

1.1.3 Hepatic clearance of bilirubin Albumin transports UCB to the liver where it reaches the microcirculation of the sinusoids (Figure 1B). The sinusoidal endothelial layer possesses an uniquely fenestrated architecture, that allows the albumin-UCB complex to enter the subendothelial space of Disse.[17] Once the complex is in direct contact with the hepatocyte membrane, UCB dissociates from albumin and is taken up as UCBfree by the hepatocyte. This uptake is most likely carrier-mediated, involving transporters that have yet to be identified, although diffusional uptake of UCBfree has also been reported.[18-22] In the cytosol, UCB is bound to ligandin (gluthathione-S-transferase; Y-protein) and possibly also to fatty acid binding protein 1 (FABP1; Z-protein), which prevents it from re-entering the circulation (Figure 1B).[23-26]

The next, essential, step in bilirubin catabolism involves conjugation of UCB. Conjugation is of vital importance since only conjugated bilirubin, as opposed to UCB, can be efficiently excreted via the bile. Bilirubin conjugation occurs in the endoplasmatic reticulum, which is the site of the conjugating enzyme bilirubine-uridine diphosphoglucuronosyltransferase (UGT1A1).[27] This enzyme catalyzes the transfer of one molecule of glucuronic acid from UDP-glucuronate to one of the carboxyl side chains of bilirubin, which produces bilirubin-monoglucuronoside. Addition of another glucuronic acid to the second carboxyl side chain of bilirubin produces bilirubin-diglucuronoside, the fully conjugated form of bilirubin.[28] Conjugation converts UCB into a more water-soluble compound by adding one or two polar glucuronic acid groups, and by exposing hydrophilic groups that are normally internalized within the molecule.[22,29]

Introduction 5

Accordingly, conjugation dramatically changes the three-dimensional structure of the bilirubin molecule. Conjugated bilirubin is excreted across the bile canalicular membrane via the multidrug resistance-related protein protein 2 (MRP2, cMOAT, ABCC2).[30-32] This excretion is ATP-dependant and overcomes a steep concentration gradient (i.e. the bilirubin concentration in the bile is approximately 100-fold higher than in the hepatocyte).[32] In human bile ∼80% of the bilirubin species appear as bilirubin diglucuronoside, ∼20% as bilirubin monoglucuronoside, and less then 2% as UCB.[33] Again, this illustrates the importance of bilirubin conjugation, prior to its excretion via the bile.

1.1.4 Enterohepatic circulation of bilirubin Bilirubin transits the biliary tree and enters the intestinal lumen, where most of the conjugates are hydrolyzed to UCB by β-glucuronidase (Figure 1C).[22] This enzyme occurs in the enteric brush border, as well as in the liver and gallbladder,[34] although most of its activity in adults is of bacterial origin.[35,36] UCB, but not conjugated bilirubin, is available for reabsorption from the intestinal lumen. Reabsorbed UCB is transported to the liver via the vena porta, where it is conjugated and excreted once again via the bile. This pathway constitutes the enterohepatic circulation (EHC) of bilirubin (Figure 1C).[37,38] Conditions that increase the intestinal UCB concentration, such as fasting, seem to enhance the reabsorption and EHC of UCB. This leads to a proportional increase in systemic plasma UCB concentrations, due to a relatively low (∼30%) first pass hepatic extraction of UCB from the vena porta. Conditions that increase the plasma UCB concentration are able to reverse the direction of UCBfree transport across the intestinal mucosa (i.e. from the blood into the intestinal lumen). During severe unconjugated hyperbilirubinemia, the direct excretion of UCBfree from the blood into the intestinal lumen indeed becomes a major route to dispose of bilirubin.[39,40] Taken together, the available data suggest that UCBfree transport across the intestinal mucosa occurs passively, and is driven by the concentration gradient between UCBfree in the plasma and UCBfree in the intestinal lumen. Although it has been shown that UCBfree is indeed capable of spontaneous diffusion across membranes,[41] the occurrence of carrier-mediated UCB transport can not be excluded.

The major fraction of intestinal UCB, however, is not reabsorbed, but rather metabolized by the intestinal microflora. Urobilinoids constitute the most important family of these bacterial UCB breakdown products. Several Clostridiae species (C. ramosum, C. difficile, and C. perfrigens) and Bacteroides fragilis are able to convert UCB, but not conjugated bilirubin, to urobilinoids in the colon.[42-47] Bacterial breakdown of UCB starts with the reduction of its tetrapyrrole ring,

6 Chapter 1

which converts UCB into urobilinogen. Urobilinogen can then be further reduced to stercobilinogen. Urobilinogen and stercobilinogen, along with their respective oxidation products urobilin and stercobilin, constitute the largest fraction of the urobilinoids in the intestine.[47] The efficacy of bacterial UCB degradation is illustrated by the observation that urobilinoids are the predominant bile pigment in adult stools.[22,48] A small fraction of the intestinal urobilinoids is reabsorbed from the intestinal lumen and subsequently excreted via the liver or the kidneys.[22,47] In general, almost all bilirubin is excreted via the feces. In human studies using 14C-bilirubin, it was demonstrated that 92% of the daily bilirubin disposal occurs via the feces, which leaves only 8% for renal excretion.[40,48] These studies also showed that only 10% of the fecally excreted label could be recovered as UCB or conjugated bilirubin, which further illustrates the efficacy of bacterial UCB degradation.

1.2 Bilirubin properties

1.2.1 The structure and properties of bilirubin Bilirubin IXα, or UCB, is a tetrapyrrole consisting of two dypyrroles that are joined by a methylene bridge at C10 (Figure 1A).[49,50] The preferential 3D structure of the bilirubin molecule can be visualized as a “partially open book” (Figure 2A). This configuration is stabilized by 6 internal hydrogen bonds between the two dipyrroles, which can be looked upon as the covers of this book.[16,29] Because the hydrophilic -COOH and -NH groups are placed in between the two dipyrroles, and are consequently shielded from interaction with water within the molecule, the resulting rigid configuration renders bilirubin practically insoluble in an aqueous environment.[51] Intact UCB is consequently practically unexcretable via the bile without prior conjugation.[16,22] Conjugation, but also oxidation or interaction with light (photo-isomerization) breaks the internal hydrogen bonds, which increases the hydrophilicity of UCB and allows it to be readily excreted across the bile canalicular membrane.[16,22,29]

Depending on the pH of its environment, UCB can exist as either of three species with different degrees of ionization; diacid, monoacid, and dianion (Figure 2B).[16] With each ionization a hydrogen bond is broken, which exposes the polar –COOH groups that can interact with water. In the plasma, at pH 7.4, >80% is present as diacid (H2B), >18% is present as monoacid (HB-), and <2% is present as the dianion (B2-).[16] Each of the three species may exist either in a bound or in an unbound, or “free”, state (Figure 2B).[16] It is generally accepted that UCB bound to albumin, or other plasma proteins, is not toxic.

Introduction 7

N

N

CO

OO

O

O

O

H

H

HC

N

N

HH

H

N

N

CO

OO

O

O

O

H

H

HC

N

N

HH

H

N

N

CO

OO

O

O

O

H

H

HC

N

N

HH

H

N

N

CO

OO

O

O

O

H

H

HC

N

N

HH

H

B2-

H2B

HB-

B2-

H2B

HB-

Bound(e.g. Albumin)

Free

pH

B2-

H2B

HB-

Free

B2-

H2B

HB-

Bound(e.g. ligandin)

Vascular Extravascular

B2-

H2B

HB-

Bound(e.g. Albumin)

Free Free Bound(e.g. ligandin)

Vascular Extravascular

B2-

H2B

HB-

B2-

H2B

HB-

B2-

H2B

HB-pH

Free

H2B

MRP1H2B

Vascular Extravascular

Export

OxidationConjugationCYP1A1CYP1A2oxidases

UGT1A1

Bindingligandin

a b

c d

high

low

Low

High

Figure 2. Bilirubin properties: (a) The three-dimensional structure of bilirubin. (b) The three species of UCB (H2B, HB-, and B2-) can occur either bound or unbound throughout the body. The pH determines UCB ionisation and thus the contribution of these three species to the total UCB pool. Only the free diacid (H2B) diffuses from the plasma pool into the extravascular (tissue) pool and is consequently considered the toxic species of bilirubin. (c) UCB can redistribute from the plasma to the tissue pool (e.g. the brain) if the amount of free H2B increases in the plasma. This may occur during hypoalbuminemia or in the presence of bilirubin displacing drugs. Additionaly, factors that damage the blood-brain barrier and possibly factors that decrease the pH may further facilitate the entry and precipitation of free H2B into the brain. (d) Cells can decrease their free H2B concentration via export, conjugation, oxidation, and binding.

8 Chapter 1

The reason for this is the inability of the UCB-albumin complex to cross the endothelial layer in the absence of fenestrae.[11,52,53] Of the unbound species, free HB- or B2 are relatively non-toxic, since their polarity prevents them from diffusing across cell membranes.[15,54,55] The free diacid (H2B) is considered to be the toxic form of UCB. The reason for this is that, due to its hydrophobicity (<70 nmol/L in water),[51] free H2B is able to diffuse across cell membranes and enter the central nervous system (CNS) in the presence of a concentration gradient (Figure 2B).[11,41,55-60]

1.2.2 Molecular aspects of bilirubin-induced neurotoxicity Bilirubin can enter the brain if it is not bound to albumin in the blood, or if the blood-brain barrier has been damaged (Figure 2C). The concentration of UCBfree in the blood may increase during hypoalbuminemia, and in the presence of substances that displace H2B from its albumin binding sites (e.g. sulfonamides or free fatty acids).[61,62] Conditions that alter the blood-brain barrier include hyperosmolality, hypercapnia, asphyxia, prematurity, infection, and sepsis.[63] Bilirubin accumulation in the CNS is prevented by the ATP-binding Cassette (ABC) protein MRP1.[60,64-66] This transporter expressed in the capillary endothelium of the brain and in the epithelium of the choroid plexus, actively pumps UCBfree out of the CNS. MRP1 is also expressed in neurons and in astrocytes, which enables them to regulate their intracellular UCBfree concentration (Figure 2D).[60,64-66] MRP1 is not the only defense mechanism of neuronal cells against bilirubin-induced toxicity. Intracellular bilirubin can also be reduced by oxidation, conjugation, and binding to cytosolic ligandin and cellular phospholipids (Figure 2D).[55] In vitro studies have shown that these neuroprotective mechanisms prevent damage if the interstitial UCBfree concentration remains below aqueous saturation (70 nmol/l).[67] A further increase in its concentration, however, may induce neurotoxicity at UCBfree levels as low as 71-85 nmol/l.[67,68] This is especially relevant at a low pH, which may increase precipitation of insoluble H2B aggregates and further increase neurotoxicity. Exposure of neurons to UCBfree can lead to a decrease in their excitability and synaptic transmission by disturbances in calcium homeostasis and/or glutamate efflux,[69,70] and by a direct disruption of plasma and mitochondrial membrane integrity.[71-74] The subsequent release of cytochrome c then triggers apoptosis via the mitochondrial pathway.[75,76] UCBfree exposure may also lead to the release of cytokines from microglial cells and astrocytes, which may further contribute to neurological damage.[77] Taken together these observations suggest that UCBfree disrupts several vital cellular functions, rather than triggering a single cell death pathway. Importantly, less differentiated

Introduction 9

(younger) nerve cells were shown to be more susceptible to UCB induced neurotoxicity in vitro. This may contribute to the increased vulnerability of premature neonates to bilirubin-induced neurotoxicity.[77]

1.2.3 Clinical aspects of bilirubin-induced neurotoxicity Bilirubin predominantly accumulates in the deep nuclei of the brain during unconjugated hyperbilirubinemia. The mechanism underlying this anatomical specificity has not yet been resolved. The precipitation of bilirubin causes a yellow staining of the basal ganglia, the hippocampus, various brain stem nuclei (e.g. oculomotor, cochlear, vestibular, and olivary nuclei), and the cerebellum.[78] This yellow staining is called kernicterus, and was originally described as an autopsy finding in neonates that had died during severe unconjugated hyperbilirubinemia.[79] Although kernicterus used to be a pathological term, it is currently also used to describe the clinical findings that are associated with bilirubin accumulation in the CNS. In its acute form, kernicterus usually consists of three phases.[63,78] The first phase is characterized by poor sucking, stupor, hypotonia, and seizures in the first days of life. The infant subsequently develops a hypertonia of the extensor muscles, which can be accompanied by opisthotonus, retrocollis, fever, and a high-pitched cry. During the third phase, which usually starts after the first week, the hypertonia is gradually replaced by hypotonia. Nowadays, the broader term “acute bilirubin encephalopathy” is often used instead of “acute kernicterus”.[11] The chronic form of kernicterus is mainly characterized by hypotonia in the first year, and movement disorders (e.g. choreoathetosis, ballismus, tremor), vertical gaze paralysis, dental dysplasia, and sensineuronal hearing loss thereafter. A more subtle clinical picture, which involves hearing loss and a mildly impaired neurologic and cognitive performance, is referred to as bilirubin-induced neurological dysfunction (BIND).[78] Currently, the total bilirubin concentration is used to assess the risk of developing kernicterus in neonates and Crigler-Najjar patients. It has become increasingly clear however, that plasma UCBfree concentrations are more closely related to the development of bilirubin-induced neurotoxicity.[11,58,62,80,81] Consequently, it seems reasonable to incorporate the UCBfree concentrations into the clinical evaluation of unconjugated hyperbilirubinemia. The lack of an accurate, reliable, and automated method to determine UCBfree, however, has so far prevented large-scale clinical trials in neonates and Crigler-Najjar patients.

10 Chapter 1

1.2.4 Bilirubin-induced toxicity in other organs The liver is relatively resistant to bilirubin-induced toxicity, which might be due to an avid binding of UCB by cytosolic proteins, and the hepatic ability to rapidly conjugate bilirubin.[82] However, bilirubin toxicity has been described in other organs. Gunn rats develop kidney damage due to the deposition of bilirubin crystals.[83,84] This has also been observed in hyperbilirubinemic infants.[85] As mentioned in §1.2.3, bilirubin deposition in the teeth may cause dental enamel dysplasia.[86] Although in vitro experiments have shown that UCB may influence immune responses and cartilage growth,[87,88] it has remained unclear whether this is physiologically relevant.

1.2.5 Bilirubin as an anti-oxidant Previously we described that both heme oxygenase and biliverdin reductase catalyze the conversion of heme into UCB. The enzyme that catalyzes the first step, heme oxygenase, has an important anti-oxidative role in both humans and animals. This might be due to its ability to convert pro-oxidative heme into biliverdin, a known anti-oxidant. The carbon monoxide that is released during this conversion has also several beneficial effects (i.e. anti-proliferative, anti-inflammatory, and vasodilatory properties). UCB itself, as mentioned, also appears to be a potent anti-oxidant. In the 1950s UCB was already reported to protect linoleic acid and vitamin A from oxidation.[89] Subsequent studies showed that UCB scavenges singlet oxygen and peroxyl radicals,[8,90] and might prevent membrane lipid oxidation.[90] Ames et al. showed in the 1980s that the anti-oxidant capacity of UCB exceeds that of vitamin E in preventing lipid peroxidation.[8,91] These observations were subsequently supported by more clinically oriented studies in both neonates and adults.[92] Neonates with illnesses that increase free radical production (e.g. sepsis, respiratory distress, circulatory failure, and asphyxia), showed a significantly lower increase in plasma bilirubin compared with a control group.[93,94] This might suggest that UCB was consumed to prevent oxidative damage. In adults, many studies have established an negative relationship between plasma bilirubin levels and cardiovascular heart disease (review:[92]). For instance, the prevalence of ischemic heart disease in 50 mildly hyperbilirubinemic patients with Gilbert’s syndrome was 2%, compared with 12% in a comparable middle aged population (n= 2296).[95] Interestingly, UCB inhibits the oxidation of low density lipoprotein, which might contribute to its anti-atherogenic effect.[96] Elevated bilirubin levels were associated with a decreased risk of cancer mortality.[97] Bilirubin might also protect from ischemia-reperfusion injury. In animal studies, exogenous bilirubin was associated with less ischemia-reperfusion injury and graft injury of the liver.[98,99]

Introduction 11

1.3 Unconjugated hyperbilirubinemia

1.3.1 Introduction Hyperbilirubinemia has many causes, but ultimately results from an imbalance between bilirubin production and bilirubin excretion.[100,101] Hyperbilirubinemia can be characterized as predominantly conjugated or predominantly unconjugated. Conjugated hyperbilirubinemia involves the accumulation of bilirubin conjugates, and is consequently caused by a defect downstream of hepatic conjugation. This defect can be localized within the hepatocyte (e.g. inactive MRP2 in Dubin-Johnson syndrome, viral hepatitis) or within the biliary tree (e.g. obstructive jaundice due to malignancies or bile stones). This introductory chapter will be limited, however, to unconjugated hyperbilirubinemia.

Neonates develop a transient elevation in plasma UCB levels within the first postnatal week. This is due to a combination of an elevated erythrocyte turnover (increased production), immature hepatic bilirubin conjugation (decreased hepatic clearance), and a delayed intestinal transit (enhanced EHC of UCB; Figure 1).[63] For the majority of neonates this results in so-called physiological jaundice, which is considered as a transitional phenomenon that may be beneficial, due to the anti-oxidant properties of UCB.[92,102] Jaundice is considered pathological if the levels of UCB rise too fast (e.g. jaundice within the first postnatal day, or an increase in UCB that exceeds 3.4 µmol/L/h), rise too high (e.g. exceeding the 95th percentile for age in hours), or remain elevated for more than 2 weeks (icterus prolongatus). Pathologic unconjugated hyperbilirubinemia is mostly due to an exaggeration of the mechanisms that caused physiological jaundice, e.g. increased production, decreased hepatic clearance, and/or an enhanced EHC of bilirubin.[63] The various pathophysiological causes for unconjugated hyperbilirubinemia, as well as the resulting clinical conditions, are now discussed in more detail.

1.3.2 Increased bilirubin production Neonates produce 7-11 mg UCB per kg per day, whereas adults produce approximately one-third of this amount.[48,103] This relatively high UCB load is due to the combination of a high erythrocyte count, a short erythrocyte life-span (70-90 days), and an enhanced catabolism of fetal hemoglobin (HbF) in neonates compared with adults (Figure 1A). Increased UCB production is a major contributor to physiologic jaundice.[101] Excessive UCB production can further

12 Chapter 1

exaggerate unconjugated hyperbilirubinemia. This causes pathological jaundice and is mostly observed during neonatal hemolytic disease.

Hemolytic disease

Iso-immune mediated hemolysis (e.g. ABO or Rh (D) incompatibility), inherited cell membrane defects (e.g. hereditary spherocytosis), erythrocyte enzymatic effects (e.g. glucose-6-phosphate dehydrogenase deficiency), and sepsis, increase bilirubin production by inducing an acute, or chronic, reduction in the lifespan of erythrocytes.[63] As a result, these conditions may result in a severe, transient unconjugated hyperbilirubinemia in neonates.[101] In adults, hemolysis does not induce such an excessive unconjugated hyperbilirubinemia because of the efficient clearance of bilirubin by the mature liver. Extravasated blood (e.g. from bruises and cephalhematomas), may also transiently increase UCB production in neonates.[63]

1.3.3 Decreased hepatic clearance of bilirubin The levels of ligandin are comparatively low in neonatal hepatocytes (Figure 1B),[104,105] but the resulting decrease in hepatic UCBfree uptake does not have a major impact on the development of physiological jaundice.[106] The immature activity of the hepatic enzyme UGT1A1, however, is considered to be the rate-limiting step in neonatal bilirubin catabolism. The UGT1A1 activity is approximately 1% of adult values directly after birth.[107] This immaturity, combined with the increased UCB load in the first postnatal week, greatly contributes to the pathogenesis of physiological jaundice.[101]

The two most important inherited defects in bilirubin conjugation, Gilbert’s syndrome and Crigler-Najjar’s disease, will now be discussed in more detail.

Gilbert’s syndrome

Gilbert’s syndrome is an inherited defect in bilirubin conjugation that occurs in 7-12% of the population.[108,109] Its mode of inheritance is considered autosomal recessive, although heterozygote carriers do have slightly elevated levels of plasma UCB.[110,111] The disease is characterized by a 70-80%-decrease in UGT1A1 activity, which is mostly due to a polymorphism within the promoter region of the gene (an extra TA in the TATA box in Caucasians).[110,112] Since this decrease does not exceed the reverse capacity of UGT1A1, it will normally not result in jaundice. Concomitant hemolysis, however, may result in a build-up in plasma

Introduction 13

UCB that exceeds 85 µmol/l, which is the threshold for visible jaundice. This occurs for example in newborns with both Gilbert’s syndrome and a glucose-6-phosphate dehydrogenase deficiency.[113] In most patients, however, the disease does not manifest itself until after the second decade in life. Adults with the syndrome suffer from a mild, recurrent, unconjugated hyperbilirubinemia that is exacerbated by conditions that increase the UCB load, such as fasting (increased EHC of UCB) and intercurrent illness.[114] Therapy is not required for Gilbert’s syndrome patients, and the most important aspect of care involves recognition of the disorder and its inconsequential nature. Interestingly, there are indications that patients with Gilbert’s syndrome are protected from developing ischemic heart disease, which would concur with the beneficial anti-oxidant capacity of bilirubin.[95]

Crigler-Najjar’s disease

Crigler-Najjar’s disease is a rare autosomal recessive disorder in bilirubin conjugation that occurs in ∼0.001% of the population (Figure 3).[115] The disease has been classified into two distinct forms. Type I disease is caused by mutations (including deletions, insertions, and premature stop codons) within the five exons of the UGT1A1 gene that prevent the production of (active) UGT1A1.[112,116] In type II disease, the genetic lesions are also located within the 5 exons of the gene, but consist of point mutations. Consequently, the UGT1A1 activity is not completely abrogated, as in type I disease, but rather reduced by approximately 95%.[116]

Patients with Crigler-Najjar disease type I suffer from a permanent and severe unconjugated hyperbilirubinemia.[117] These patients rely on daily phototherapy to prevent kernicterus. The social impact of this regimen, which may take up to 16 hours per 24 hour, tends to decrease compliance over time.[118] The efficiency of phototherapy also decreases with age, due to skin thickening, increased pigmentation, and a decrease in the surface area to body mass ratio.[115,118-120] Exacerbations of jaundice due to fasting or concurrent illness may be treated with additional albumin infusion, exchange transfusion, and oral amorphous calcium phosphate in order to prevent brain damage.[115,118,120] In spite of this intensive treatment, approximately one-fourth to one-half of the patients with Crigler-Najjar disease type I develop mild to severe brain damage. In addition, 9-38% die resulting from complications related to the disease.[118,120-122] Eventually, liver transplantation is the only definitive treatment for Crigler-Najjar type I. In spite of its risks in terms of morbidity and mortality, it has been advocated that liver transplantation should be performed at

14 Chapter 1

a young age to prevent irreversible bilirubin-induced brain damage in Crigler-Najjar type I patients.[118]

Patients with Crigler-Najjar disease type II (or Arias syndrome) still have residual UGT1A1 activity, which results in a milder phenotype.[123] Importantly, these patients are still able to conjugate and excrete bilirubin via the bile to some extent.[124] Bile of type I patients contains essentially no conjugated bilirubin, whereas type II bile contains mono-conjugates and some bi-conjugates.[124] Daily phototherapy is usually not required in type II patients, since the plasma UCB concentration usually remains below 350 µmol/l. If necessary, type II patients can be treated with phenobarbital, which enhances the residual UGT1A1 activity and decreases plasma UCB concentrations by approximately 30%.[123,124]

FECESa

b

UCBBlood

UCB

Intestine

Conjugationdefect

Figure 3. Unconjugated hyperbilirubinemia: (a) Patients with Crigler-Najjar disease type I, and their animal model the Gunn rat, are unable to conjugate bilirubin in the liver due to absent UGT1A1 activity. The resulting severe unconjugated hyperbilirubinemia results in diffusion of free UCB from the blood, across the intestinal mucosa, into the intestinal lumen. This transmucosal UCB diffusion is considered a major excretory pathway for UCB during severe unconjugated hyperbilirubinemia. (b) The Gunn rat.

Introduction 15

1.3.4 Increased enterohepatic circulation of bilirubin In neonates, the high mucosal β-glucuronidase activity results in a relatively complete hydrolysis of conjugated bilirubin in the intestinal lumen. The resulting UCB is either reabsorbed into the EHC, or metabolized by the intestinal microflora (Figure 1C).[37,38] The microflora responsible for UCB metabolism, however, is not fully developed during the first months of life.[125] Almost all intestinal UCB thus becomes available for reabsorption into the EHC.[126] Since the liver extracts only 30% of the UCB load, this increase in EHC will directly contribute to the development of physiological jaundice.[22] Inadequate feeding, breast milk jaundice, and an anatomic or functional intestinal obstruction may further increase the EHC. These conditions can elevate plasma UCB levels to pathological levels, and will be discussed in more detail.

Inadequate feeding

Inadequate feeding may have several causes, but often results from a decreased intake caused by ineffective breastfeeding. This may be due to a variety of maternal and neonatal factors, such as improper technique, engorgement and/or ineffective sucking of the neonate. The decreased intake leads to weight loss and exaggeration of unconjugated hyperbilirubinemia in the first 3-5 postnatal days.[127-129] In animal studies, starvation resulted into decreased fecal UCB excretion, accumulation of UCB in the intestinal lumen, and a subsequent increase in reabsorption of this UCB into the EHC.[130,131] It is therefore highly probable that inadequate feeding exaggerates unconjugated hyperbilirubinemia in neonates via a similar mechanism, i.e. through an increased EHC of the pigment. Indeed, frequent and early feedings in the first postnatal days have been shown to decrease plasma UCB concentrations in neonates, possibly by preventing the accumulation of UCB in the intestinal lumen.[132,133]

Breast milk jaundice

Breast milk jaundice, which must be distinguished from jaundice due to inadequate feeding, typically begins after the first 3-5 postnatal days and peaks within two weeks after birth. It has been postulated that β-glucuronidase, present in human milk but not in infant formula, may be responsible for the exaggeration of unconjugated hyperbilirubinemia, because it would enhance the hydrolysis of intestinal glucuronic acid-bilirubin.[134] This, however, remains highly questionable since the amount of β-glucuronidase in human milk is relatively small compared with the abundance of the enzyme in the neonatal intestinal

16 Chapter 1

mucosa. It is, however, well established that human milk itself enhances intestinal UCB reabsorption, although the responsible constituent remains to be identified.[131]

Intestinal obstruction

Intestinal obstruction (either functional or anatomic) delays the intestinal transit of UCB. This leads to accumulation of UCB in the intestinal lumen, and a subsequent increase in reabsorption of this UCB into the EHC. Obstruction due to a delayed passage of meconium,[135] pyloric stenosis,[136] or Hirschprung’s disease[131] are all associated with increased plasma UCB concentrations. Acceleration of the gastrointestinal transit, by rectal stimulation,[137] frequent feedings,[132,133] or feeding infant formula that contain oligosaccharides, reduces unconjugated hyperbilirubinemia in neonates.[138,139]

1.3.5 The Gunn rat model The Gunn rat, which is used in the animal studies of this thesis, is the well-established animal model for Crigler-Najjar disease type I. In 1938, Gunn described a spontaneously jaundiced strain of Wistar rats, suffering from life-long, severe unconjugated hyperbilirubinemia.[140] Later it was demonstrated that the unconjugated hyperbilirubinemia in these animals was caused by an inherited inability to form bilirubin conjugates (Figure 3).[141,142] The Gunn rat also appeared to be a good model for bilirubin-encephalopathy in humans, especially if the amount of free bilirubin was further increased by concomitant hemolysis or by administration of albumin displacing drugs.[143-146] The neurologic lesions in Gunn rats, which resemble those in humans, include cell loss and gliosis in the auditory nuclei of the brainstem, the oculomotor nuclei, cerebellum, hippocampus, and basal ganglia.[141,147-149] Cerebellar hypoplasia is a prominent feature of UCB-induced neurological damage in Gunn rats, albeit not in humans.[150] Changes in learning behavior, neurological function, and electrophysiology of the auditory nervous system in Gunn rats also resemble the human condition.[147,151] Renal damage has also been described, as it has been in humans.[83-85] The phenotype of Gunn rats is caused by a single frame shift mutation in the Ugt1A1 gene, which leads to the formation of a truncated and inactive enzyme.[141,142] The Gunn rat is thus a phenotypic and genotypic animal model for Crigler-Najjar disease type I and is the most established model to study severe unconjugated hyperbilirubinemia and bilirubin-induced neurological damage.

Introduction 17

1.3.6 Alternative metabolic routes of bilirubin disposal If the physiological disposal of UCB is impaired, as in patients with Crigler-Najjar disease and Gunn rats, UCB may be catabolized via alternative pathways. These include transmucosal diffusion and oxidation of bilirubin.

Transmucosal diffusion

UCB can, in its unbound form, freely diffuse across the intestinal mucosa.[39-41] The direction of this flux is determined by the concentration gradient between UCBfree in the plasma and UCBfree in the intestinal lumen. Accumulation of UCB in the intestine will consequently induce a net flux of UCBfree from the gut lumen into the blood.[130] This situation occurs in conditions that delay the intestinal transit, such as inadequate feeding or intestinal obstruction (see above). The direction of this flux is inversed (i.e. from the blood into the intestinal lumen) if plasma UCB concentrations become excessively elevated. This situation occurs for example in patients with Crigler-Najjar disease and in Gunn rats (Figure 3).[39,115,152,153] In Gunn rats, it has been demonstrated that transmucosal diffusion is the most important alternative route for bilirubin excretion.[39,152] Most experimental treatments used in this thesis were aimed to optimize the efficiency of this excretory pathway during severe unconjugated hyperbilirubinemia. Existing experimental treatments that optimize this pathway will be reviewed in chapter 2.

Bilirubin oxidation

Oxidation converts the hydrophobic UCB into more polar derivatives that can be readily excreted via the bile.[154,155] Interestingly, the majority of bilirubin derivatives in the bile of Gunn rats and in the feces of Crigler-Najjar patients are polar, diazo-negative compounds.[40,48] These compounds could be derived from bacterial UCB reduction, from enhanced UCB oxidation, or from a combination of these two processes. The fecal urobilinogen excretion, however, was similar in Crigler-Najjar patients and age-matched controls, which pleads against an increased bacterial reduction of UCB.[48] CYP1A1 and CYP1A2, the microsomal mixed-function oxidases that catalyze UCB oxidation, however, were markedly up-regulated in the liver of young Gunn rats.[156] The constitutively expressed non-inducible mitochondrial bilirubin oxidase, which can also oxidize bilirubin, has been demonstrated in the mitochondria of the liver, intestine, and kidney of guinea pigs and rats.[157-159] Enzymatic oxidation of UCB has, in addition, been reported in brain, lung, heart, and skeletal muscle.[157] Naturally,

18 Chapter 1

UCB will also be oxidized in its role as anti-oxidant (e.g. to biliverdin and water-soluble compounds).[92] The latter mechanism may evidently also contribute to an increase in bilirubin disposal via oxidation (please refer to paragraph 2.5 for more detail). Although it is thus clear that UCB degradation into diazo-negative compounds contributes greatly to UCB excretion, it has remained unclear whether these compounds are mostly derived from enzymatic UCB oxidation or from other metabolic processes. However, several experimental treatments that increase UCB oxidation have been successfully applied to treat unconjugated hyperbilirubinemia in Gunn rats. These treatments will be reviewed in chapter 2.

1.4 Treatment of unconjugated hyperbilirubinemia

1.4.1 Phototherapy Phototherapy uses light energy to change the shape and structure of bilirubin, which allows it to be excreted without conjugation via the bile.[160-164] Phototherapy was first described by the pediatric resident Cremer in 1958, after a nurse noticed that sunlight apparently decreased jaundice in neonates.[165] Bilirubin absorbs light most strongly in the blue region of the spectrum, near 460 nm.[166-168] Because penetration of the skin increases with wavelength in this region, narrow spectrum blue lights (460-490 nm) are considered to be most effective in the treatment of unconjugated hyperbilirubinemia.[160,166] Absorption of light by the skin changes the configuration of (sub)dermal UCB via three distinct photochemical reactions: configurational photo-isomerization, structural photo-isomerization, and photo-oxidation (Figure 4). Configurational photo-isomerization induces the UCB molecule to twist by opening a double bond at either C4-C5 or C15-C16. This changes its configuration from ZZ, to EZ, ZE, or EE (Z for zusammen, and E for entgegen).[169] Structural photo-isomerization produces lumirubin by intramolecular cyclization.[170] Both reactions increase the water-solubility of UCB by exposing its polar -COOH and -NH groups. Comparable to bilirubin conjugates, the water-soluble photo-isomers are also a substrate for MRP2 in the bile canalicular membrane, and can thus readily be excreted into the bile.[167,168] Finally, photo-oxidation, which seems to play a minor role in UCB catabolism, hydrolyzes UCB into mono- and dipyrroles that can be excreted via the urine.[171] The efficacy of phototherapy depends on the spectrum, irradiance (energy output), and distance of the light source, as well as on the skin area that is exposed to light. All these factors should be routinely evaluated.[160] If necessary, double-sided phototherapy can be used to increase the therapeutic effect. This involves a conventional overhead lamp and a light-emitting blanket, or “bili-blanket”.[172]

Introduction 19

N

N

CO

OO

O

O

O

H

H

HC

N

N

HH

H

NO

H

H

N

N

N

O

O

O

O

O

H

H

H

H

C

C

N

N

N

N HH

H

HH

HO O

O

O

O

O

C

C

LIGHT

Lumibilirubin

4Z,15E-Bilirubin

Oxidation products

1

2

3

 Figure 4. Treatment for unconjugated hyperbilirubinemia. Phototherapy changes the configuration of (sub)dermal UCB via three distinct photochemical reactions: (1) configurational photo-isomerization, (2) structural photo-isomerization, and (3) photo-oxidation. The changes in configuration allow the hydrophilic -COOH and -NH groups within the molecule to interact with the enviroment, which increases the water-solubility of bilirubin.

20 Chapter 1

Short-term phototherapy is considered safe,[166] but some side-effects have been reported. Phototherapy during concomitant cholestasis may produce the “bronze baby syndrome”, due to accumulation of photoproducts that would normally have been excreted via the bile. As a result, the skin, serum, and urine develop a grayish-brown discoloration. This discoloration disappears when the cholestasis resolves or if the phototherapy is discontinued.[173,174] In neonates with cholestatic jaundice rare purpuric and bullous eruptions have been observed during phototherapy.[175,176] In neonates treated with tin mesoporphyrin (an experimental drug that decreases UCB production; please refer to chapter 2), phototherapy induced a mild erythema.[177] Phototherapy may also induce insensible water loss.[178] Finally, porphyria and concomitant use of photosensitizing drugs are considered absolute contraindications to phototherapy.[160,179] Long-term phototherapy has many disadvantages, which have been discussed in § 1.3.3.

1.4.2 Exchange transfusion Currently, the only effective alternative to phototherapy in severely jaundiced neonates is exchange transfusion. This technique involves the removal of relatively small aliquots of blood, and replacing these aliquots with a mixture of red cells and plasma from a donor. Exchange transfusion removes approximately 50% of the plasma UCB, although this amount varies according to the severity of the ongoing hemolysis and the amount of bilirubin that re-enters the circulation from the tissues.[63] This re-entry occurs due to the diffusion of UCBfree from the tissue pool into the plasma pool and decreases the risk of bilirubin-induced neurotoxicity. The term hypobilirubinemic treatment is thus misleading in this respect, since all therapy should be eventually aimed at decreasing the amount of UCB in the tissue, rather than in the plasma pool. Exchange therapy is especially effective during hemolytic disease, since it also decreases the amount of antibodies that target the erythrocytes from the circulation.[63] Exchange transfusion is indicated if, in spite of phototherapy, plasma UCB levels rise too fast, rise above specific concentrations, or if symptoms of acute bilirubin-encephalopathy emerge.[180,181] Exchange transfusions are considerably more dangerous than phototherapy. Significant morbidity occurs in approximately 5-12% of exchange transfusions, whereas the mortality rate is approximately 0.3-2.0%.[181-183] Complications include cardiac arrest, thrombosis, graft vs. host disease, necrotizing enterocolitis, transmission of infectious diseases, coagulopathies, hypoglycemia, and hypocalcaemia.[181-185] Fortunately, there has been a dramatic decrease in the need for exchange transfusions since the introduction of phototherapy.[186,187]

Introduction 21

1.4.3 Albumin infusion Mounting evidence suggests that UCBfree, rather than total bilirubin, is the major determinant of bilirubin deposition and its neurotoxicity. Albumin infusion, prior to exchange transfusion, could thus be a useful strategy to decrease the amount of UCBfree by increasing its binding to albumin.[188] However, the efficacy of albumin administration prior to exchange transfusion has not been firmly established. Some authors have reported that albumin administered one hour prior to an exchange transfusion increased the amount of bilirubin that could be removed from the blood.[188] Other authors, however, have failed to reproduce this effect.[189]

1.4.4 Liver transplantation Liver transplantation is currently the only definitive treatment for Crigler-Najjar disease type I. In 1985 the first successful orthotopic liver transplantation (OLT) in a Crigler-Najjar patient was reported, and several other patients have undergone transplantation since.[118,190] Two types of transplantation have been used: OLT, in which the patients’ own liver is replaced by a donor liver, and auxiliary liver transplantation in which part of the own liver is left in situ and is supported by a donor graft.[191] If successful, a liver transplant completely corrects the hepatic defect underlying Crigler-Najjar disease, which dramatically improves the quality of life.[118] Liver transplantation, however, remains a high-risk procedure, with a one-year survival between 85% and 90%.[192,193] The most common complications include rejection, sepsis, infection, bleeding, and biliary complications.[192,193] Also, to prevent rejection, patients receive life-long immunosuppressive therapy.

1.4.5 Hepatocyte transplantation Hepatocyte transplantation may seem an attractive alternative to liver transplantation, since the architecture of the liver itself is not compromised in Crigler-Najjar disease type I. Also, a complete liver transplant is unnecessary to correct Crigler-Najjar type I, due to the 100-fold overcapacity of UGT1A1.[194] Many techniques of hepatocellular transplantation have been developed in the Gunn rat model.[195] Generally, the best results were obtained when hepatic injury was caused prior to infusion of congeneic hepatocytes via the liver, spleen, portal vein, or via intraperitoneal injection. Hepatic damage provided a regenerative stimulus for the engrafting cells, which resulted in a significant lowering in plasma UCB for up to 12 months.[195] In patients, Fox et al. was the

22 Chapter 1

first to show the feasibility of a hepatocyte transplant in a 10-year old patient, in which the infusion of 7.5 x 109 liver cells via the portal vein decreased plasma bilirubin levels for up to 11 months.[196] However, the restoration in enzyme activity (∼5% of normal) was not sufficient to eliminate the eventual need for liver transplantation. Since this initial report, 6 additional patients were treated with hepatocyte or hepatocyte progenitor cell transplantation, often with multiple infusions.[195,197-200] In all these studies the long-term follow up (if reported) showed a rebound of plasma bilirubin concentrations to pre-treatment levels after 5-6 months, which was probably due to unfavorable immune reactions. In addition, the need for OLT was not eliminated in these patients. Currently, hepatocyte transplantation is thus still hampered by its transient therapeutic effect. Also, immunosuppressive therapy is still required and donor livers, or fetal hepatic progenitor cells, are still required to isolate cells.[195,197-200] Nevertheless, the technique appears to be relatively safe, and could be used to bridge the gap to OLT in Crigler-Najjar patients.

1.4.6 Gene therapy Crigler-Najjar is caused by a deletion in a single gene, and even a partial replacement of UGT1A1 enzyme activity would suffice to successfully treat the disease. This makes the disease an ideal candidate for gene therapy. Numerous strategies of gene transfer, using viral and non-viral vectors, have been developed in the Gunn rat model. Although several of these strategies resulted in a long-term correction in plasma UCB levels, none of them have been translated into a clinical trial. This has been mainly due to concerns regarding long-term safety and vector toxicity. The first gene transfer strategy evaluated in Gunn rats used retroviral vectors.[201,202] Although these vectors efficiently integrate Ugt1A1 into the host’s genome, the technique was not very effective in larger animals. This could be due to the inability of retroviruses to infect non-dividing cells.[203] Another approach involved the use of adenoviral vectors that localize to the liver and do have the ability to infect non-dividing cells. First-generation adenoviral vectors could only correct Ugt1A1 for a limited amount of time, because they are not incorporated into the host’s genome.[204,205] Second-generation adenoviral vectors corrected plasma UCB levels for over 2 years in Gunn rats.[206] These vectors have a lower immunogenicity since all their viral genes have been deleted.[203] Immunogenicity has also been decreased by inducing tolerance for adenoviruses, and by using adenoviral vectors that express immune-modulating molecules.[207-209] Lentiviruses and SV40 viruses incorporate Ugt1A1 into the genome of non-dividing cells. Although results in Gunn rats seem encouraging, these vectors do not specifically localize to the liver and must be administrated via the portal vein.[210-212] Non-viral vectors use receptor-mediated endocytosis to

Introduction 23

transfer Ugt1A1 into the hepatocyte.[213,214] New strategies are currently being developed that decrease lysosomal breakdown of the Ugt1A1, which is a major problem of this technique.[214-216] Another strategy, namely site-directed gene conversion, uses chimeric RNA-DNA molecules to repair Ugt1A1 within the liver. The results in Gunn rats seem encouraging.[217,218] Finally, ex-vivo gene transduction incorporates Ugt1A1 into harvested hepatocytes and subsequently administers these cells into the liver.[203,219] Although the cells are autologous, which prevents rejection, the technique is hampered by the necessity to perform a partial hepatectomy.[202]

The results of gene therapy thus seem promising in Gunn rats. Currently, however, the results of clinical trials must be awaited before any of these strategies can be applied in Crigler-Najjar patients.

1.5 Scope of this thesis Neonates develop a transient elevation in plasma unconjugated bilirubin (UCB) levels during the first postnatal week. This so-called physiological jaundice is due to an increased erythrocyte turnover (increased UCB production), an immature hepatic conjugation (decreased hepatic clearance of UCB), and an increase in intestinal reabsorption of the pigment (increased enterohepatic circulation of UCB). Physiological jaundice is considered a benign and transient phenomenon, that may actually be beneficial due to the anti-oxidant properties of UCB.[8] Underlying pathology, however, may lead to a potentially toxic increase in plasma UCB concentrations. This occurs in conditions such as neonatal hemolytic jaundice (due to excessive UCB production from heme), and Crigler-Najjar disease. Crigler-Najjar disease is characterized by a genetically absent (type I) or impaired (type II) activity of hepatic bilirubine-uridine diphosphoglucuronosyltransferase (UGT1A1).[116,117] This enzyme catalyzes bilirubin conjugation in the hepatocyte, which greatly facilitates the subsequent excretion of the pigment into the bile.[27] Severe unconjugated hyperbilirubinemia may lead to permanent brain damage and death.[78] This is due to the ability of UCBfree, the small fraction of UCB that is not bound to plasma albumin, to passively cross the blood-brain barrier.[11,41,55,57-60] Severe unconjugated hyperbilirubinemia is conventionally treated with phototherapy and/or exchange transfusion. Phototherapy, however, does not always reduce plasma UCB to non-toxic levels. Crigler-Najjar patients are treated with life-long phototherapy that may last up to 16 hours per day. In spite of this intensive regimen, ∼25% of the patients develop permanent brain damage.[118,119] Exchange transfusion is associated with significant morbidity, and even mortality has been reported.[181-183] These considerations show the

24 Chapter 1

need for additional or alternative therapeutic options. In this thesis we consequently focus on new treatment strategies for unconjugated hyperbilirubinemia.

In chapter 2 we review experimental pharmacological treatment strategies for severe unconjugated hyperbilirubinemia. We discuss the state of the art and future perspectives of pharmacological treatment of unconjugated hyperbilirubinemia, thus setting the stage for the subsequent chapters.

In chapter 3 we investigate dietary administration of bile salts to Gunn rats, the well-established animal model for Crigler-Najjar disease type I. During severe unconjugated hyperbilirubinemia UCB is not exclusively excreted via the bile, but may also enter the intestinal lumen via direct diffusion from the blood.[39-41] The efficiency of this excretory pathway is decreased, however, by reabsorption of UCB from the intestinal lumen.[37,38] This reabsorption can be prevented by intestinal capture; the binding of intestinal UCB to orally administered agents. Bile salts can increase the biliary excretion of UCB, but can also bind UCB in the intestinal lumen.[220-223] We investigated whether dietary administration of the bile salts ursodeoxycholic acid, or cholic acid treats unconjugated hyperbilirubinemia in Gunn rats. To gain more mechanistic insight, we used 3H-UCB kinetics to determine the effect of these bile acids on the (fractional) turnover, the pool, and the biliary and transmucosal fluxes of UCB.

In chapter 4 we evaluate whether acceleration of the gastrointestinal transit by the laxative polyethylene glycol treats unconjugated hyperbilirubinemia in Gunn rats. Clinical conditions that delay the gastrointestinal transit, such as fasting, seem to increase plasma UCB levels.[130,223] Conditions that accelerate the gastrointestinal transit, such as frequent feedings in the neonatal period, are associated with a decrease in plasma UCB.[132,133] In theory, acceleration of the gastrointestinal transit could lower the intestinal UCB concentration, thus creating a diffusional shift of the pigment from the blood into the intestinal lumen. A direct relationship between the gastrointestinal transit time and plasma UCB levels, however, has never been established. We investigate whether the gastrointestinal transit time is directly related to plasma UCB concentrations, and whether this relationship can be used to develop a new therapeutic strategy for severe unconjugated hyperbilirubinemia in Gunn rats.

In chapter 5 we investigate, in Gunn rats, whether known hypobilirubinemic treatments (e.g. phototherapy and/or oral capture agents) exert their therapeutic effect by acceleration of the gastrointestinal transit. We also evaluate whether additional treatment with polyethylene glycol can increase the efficacy of these treatments.

Introduction 25

Eventually, all therapy should prevent neurotoxicity by decreasing tissue rather than plasma bilirubin concentrations. UCBfree, the small fraction of UCB that is not bound to plasma albumin, is able to diffuse from the plasma into the tissue pool.[11,58] Decreasing this fraction, for instance by increasing its binding to albumin, would thus be expected to decrease tissue bilirubin levels. In chapter 6 we investigate the effect of phototherapy and phototherapy + albumin infusion on UCB, UCBfree, and tissue bilirubin levels during permanent and acute hemolytic jaundice in Gunn rats.

In chapter 7 we discuss our overall results, place these in a clinical and experimental framework, and discuss future perspectives.

26 Chapter 1

References 1. Bissell DM. Heme catabolism and

bilirubin formation. In: Ostrow JD, editor. Bile pigments and jaundice - Molecular, metabolic and medical aspects. New York: Marcel Dekker Inc.; 1986. p. 133–156.

2. Crawford JM, Hauser SC, Gollan JL. Formation, hepatic metabolism, and transport of bile pigments: a status report. Semin Liver Dis. 1988;8:105–118.

3. Tenhunen R, Ross ME, Marver HS, Schmid R. Reduced nicotinamide-adenine dinucleotide phosphate dependent biliverdin reductase: partial purification and characterization. Biochemistry. 1970;9:298–303.

4. Tenhunen R, Marver HS, Schmid R. Microsomal heme oxygenase. Characterization of the enzyme. J. Biol. Chem. 1969;244:6388–6394.

5. McDonagh AF. Bile pigments: bilatrienes and 5,15-biladienes. In: Dolphin D, editor. The Porphyrins. New York: Academic Press; 1979. p. 293–491.

6. Lester R, Behrman RE, Lucey JF. Transfer of Bilirubin-C14 across Monkey Placenta. Pediatrics. 1963;32:416–419.

7. Schenker S, Dawber NH, Schmid R. Bilirubin Metabolism in the Fetus. J. Clin. Invest. 1964;43:32–39.

8. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science. 1987;235:1043–1046.

9. Goessling W, Zucker SD. Role of apolipoprotein D in the transport of bilirubin in plasma. Am J Physiol Gastrointest Liver Physiol. 2000;279:G356–65.

10. Odell GB. Studies in kernicterus. I. The protein binding of bilirubin. J. Clin. Invest. 1959;38:823–833.

11. Ahlfors CE, Wennberg RP, Ostrow JD, Tiribelli C. Unbound (free) bilirubin: improving the paradigm for evaluating neonatal jaundice. Clin. Chem. 2009;55:1288–1299.

12. Jacobsen J. Binding of bilirubin to human serum albumin - determination of the dissociation constants. FEBS Lett. 1969;5:112–114.

13. Petersen CE, Ha CE, Harohalli K, Feix JB, Bhagavan NV. A dynamic model for bilirubin binding to human serum albumin. J. Biol. Chem. 2000;275:20985–20995.

14. Brodersen R. Bilirubin. Solubility and interaction with albumin and phospholipid. J. Biol. Chem. 1979;254:2364–2369.

15. Brodersen R. Binding of bilirubin to albumin. CRC Crit Rev Clin Lab Sci. 1980;11:305–399.

16. Ostrow JD, Mukerjee P, Tiribelli C. Structure and binding of unconjugated bilirubin: relevance for physiological and pathophysiological function. J. Lipid Res. 1994;35:1715–1737.

17. Sorrentino D, Berk PD. Mechanistic aspects of hepatic bilirubin uptake. Semin Liver Dis. 1988;8:119–136.

18. Zucker SD, Goessling W, Hoppin AG. Unconjugated bilirubin exhibits spontaneous diffusion through model lipid bilayers and native hepatocyte membranes. J. Biol. Chem. 1999;274:10852–10862.

19. Mediavilla MG, Pascolo L, Rodriguez JV, Guibert EE, Ostrow JD, Tiribelli C. Uptake of [(3)H]bilirubin in freshly isolated rat

Introduction 27

hepatocytes: role of free bilirubin concentration. FEBS Lett. 1999;463:143–145.

20. Scharschmidt BF, Waggoner JG, Berk PD. Hepatic organic anion uptake in the rat. J. Clin. Invest. 1975;56:1280–1292.

21. Zucker SD, Storch J, Zeidel ML, Gollan JL. Mechanism of the spontaneous transfer of unconjugated bilirubin between small unilamellar phosphatidylcholine vesicles. Biochemistry. 1992;31:3184–3192.

22. Vitek L, Ostrow JD. Bilirubin chemistry and metabolism; harmful and protective aspects. Curr. Pharm. Des. 2009;15:2869–2883.

23. Habig WH, Pabst MJ, Fleischner G, Gatmaitan Z, Arias IM, Jakoby WB. The identity of glutathione S-transferase B with ligandin, a major binding protein of liver. Proc. Natl. Acad. Sci. U.S.A. 1974;71:3879–3882.

24. Levi AJ, Gatmaitan Z, Arias IM. Two hepatic cytoplasmic protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin, sulfobromophthalein, and other anions. J. Clin. Invest. 1969;48:2156–2167.

25. Wolkoff AW, Goresky CA, Sellin J, Gatmaitan Z, Arias IM. Role of ligandin in transfer of bilirubin from plasma into liver. Am J Physiol. 1979;236:E638–48.

26. Theilmann L, Stollman YR, Arias IM, Wolkoff AW. Does Z-protein have a role in transport of bilirubin and bromosulfophthalein by isolated perfused rat liver? Hepatology. 1984;4:923–926.

27. Schmid R, Hammaker L, Axelrod J. The enzymatic formation of bilirubin glucuronide. Arch. Biochem. Biophys. 1957;70:285–288.

28. Burchell B, Coughtrie MW. UDP-glucuronosyltransferases. Pharmacol Ther. 1989;43:261–289.

29. Lightner A. Structure and Metabolism of Natural and Synthetic Bilirubins. Journal of perinatology. 2001;21:S13.

30. Paulusma CC, Bosma PJ, Zaman GJ, Bakker CT, Otter M, Scheffer GL, et al. Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene. Science. 1996;271:1126–1128.

31. Paulusma CC, Kool M, Bosma PJ, Scheffer GL, Borg ter F, Scheper RJ, et al. A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome. Hepatology. 1997;25:1539–1542.

32. Kamisako T, Leier I, Cui Y, Konig J, Buchholz U, Hummel-Eisenbeiss J, et al. Transport of monoglucuronosyl and bisglucuronosyl bilirubin by recombinant human and rat multidrug resistance protein 2. Hepatology. 1999;30:485–490.

33. Fevery J, Blanckaert N, Leroy P, Michiels R, Heirwegh KP. Analysis of bilirubins in biological fluids by extraction and thin-layer chromatography of the intact tetrapyrroles: application to bile of patients with Gilbert's syndrome, hemolysis, or cholelithiasis. Hepatology. 1983;3:177–183.

34. Saxerholt H, Midtvedt T, Gustafsson BE. Deconjugation of bilirubin conjugates and urobilin formation by conventionalized germ-free rats. Scand J Clin Lab Invest. 1984;44:573–577.

35. Saxerholt H, Midtvedt T. Intestinal deconjugation of bilirubin in germfree and conventional rats. Scand J Clin Lab Invest. 1986;46:341–344.

28 Chapter 1

36. Kent TH, Fischer LJ, Marr R. Glucuronidase activity in intestinal contents of rat and man and relationship to bacterial flora. Proc Soc Exp Biol Med. 1972;140:590–594.

37. Lester R, Schmid R. Intestinal absorption of bile pigments. I. The enterohepatic circulation of bilirubin in the rat. J. Clin. Invest. 1963;42:736–746.

38. Lester R, Schmid R. Intestinal absorption of bile pigments. II. Bilirubin absorption in man. N. Engl. J. Med. 1963;269:178–182.

39. Kotal P, Van der Veere CN, Sinaasappel M, Elferink RO, Vitek L, Brodanova M, et al. Intestinal excretion of unconjugated bilirubin in man and rats with inherited unconjugated hyperbilirubinemia. Pediatr. Res. 1997;42:195–200.

40. Schmid R, Hammaker L. Metabolism and disposition of C14-bilirubin in congenital nonhemolytic jaundice. J. Clin. Invest. 1963;42:1720–1734.

41. Zucker SD, Goessling W, Hoppin AG. Unconjugated bilirubin exhibits spontaneous diffusion through model lipid bilayers and native hepatocyte membranes. J. Biol. Chem. 1999;274:10852–10862.

42. Midtvedt T, Gustafsson BE. Microbial conversion of bilirubin to urobilins in vitro and in vivo. Acta Pathol Microbiol Scand B. 1981;89:57–60.

43. Fahmy K, Gray CH, Nicholson DC. The reduction of bile pigments by faecal and intestinal bacteria. Biochim Biophys Acta. 1972;264:85–97.

44. Moscowitz A, Weimer M, Lightner DA, Petryka ZJ, Davis E, Watson CJ. The in vitro conversion of bile pigments to the urobilinoids by a rat

clostridia species as compared with the human fecal flora. 3. Natural d-urobilin, synthetic i-urobilin, and synthetic i-urobilinogen. Biochem Med. 1971;4:149–164.

45. Vitek L, Kotal P, Jirsa M, Malina J, Cerna M, Chmelar D, et al. Intestinal colonization leading to fecal urobilinoid excretion may play a role in the pathogenesis of neonatal jaundice. J Pediatr Gastroenterol Nutr. 2000;30:294–298.

46. Gustafsson BE, Lanke LS. Bilirubin and urobilins in germfree, ex-germfree, and conventional rats. J Exp Med. 1960;112:975–981.

47. Vitek L, Majer F, Muchova L, Zelenka J, Jiraskova A, Branny P, et al. Identification of bilirubin reduction products formed by Clostridium perfringens isolated from human neonatal fecal flora. J Chromatogr B Analyt Technol Biomed Life Sci. 2006;833:149–157.

48. Bloomer JR, Berk PD, Howe RB, Waggoner JG, Berlin NI. Comparison of fecal urobilinogen excretion with bilirubin production in normal volunteers and patients with increased bilirubin production. Clin. Chim. Acta. 1970;29:463–471.

49. Fischer H, Plieninger H, Weissbarth O. Uber die konstitution des bilirubins und uber bilirubinoide farbstoffe. Hoppe-Seyler´s Zeitschrift für physiologische Chemie. 1941;268:197–226.

50. Bonnett R, Davies JE, Hursthouse MB. Structure of bilirubin. Nature. 1976;262:327–328.

51. Ostrow JD, Mukerjee P. Revalidation and rationale for high pKa values of unconjugated bilirubin. BMC Biochem. 2007;8:7.

52. Bloomer JR, Berk PD, Vergalla J, Berlin NI. Influence of albumin on the

Introduction 29

hepatic uptake of unconjugated bilirubin. Clin Sci Mol Med. 1973;45:505–516.

53. Bloomer JR, Berk PD, Vergalla J, Berlin NI. Influence of albumin on the extravascular distribution of unconjugated bilirubin. Clin Sci Mol Med. 1973;45:517–526.

54. Brodersen R, Stern L. Deposition of bilirubin acid in the central nervous system--a hypothesis for the development of kernicterus. Acta Paediatr Scand. 1990;79:12–19.

55. Ostrow JD, Pascolo L, Shapiro SM, Tiribelli C. New concepts in bilirubin encephalopathy. Eur J Clin Invest. 2003;33:988–997.

56. Diamond I, Schmid R. Experimental bilirubin encephalopathy. The mode of entry of bilirubin-14C into the central nervous system. J. Clin. Invest. 1966;45:678–689.

57. Calligaris SD, Bellarosa C, Giraudi P, Wennberg RP, Ostrow JD, Tiribelli C. Cytotoxicity is predicted by unbound and not total bilirubin concentration. Pediatr. Res. 2007;62:576–580.

58. Ahlfors CE, Amin SB, Parker AE. Unbound bilirubin predicts abnormal automated auditory brainstem response in a diverse newborn population. J Perinatol. 2009;29:305–309.

59. Bratlid D. How bilirubin gets into the brain. Clin Perinatol. 1990;17:449–465.

60. Ostrow JD, Pascolo L, Brites D, Tiribelli C. Molecular basis of bilirubin-induced neurotoxicity. Trends Mol Med. 2004;10:65–70.

61. Harris RC, Lucey JF, Maclean JR. Kernicterus in premature infants associated with low concentrations of bilirubin in the plasma. Pediatrics.

1958;21:875–884.

62. Ahlfors CE, Wennberg RP. Bilirubin-albumin binding and neonatal jaundice. Semin Perinatol. 2004;28:334–339.

63. Dennery PA, Seidman DS, Stevenson DK. Neonatal hyperbilirubinemia. N. Engl. J. Med. 2001;344:581–590.

64. Rigato I, Pascolo L, Fernetti C, Ostrow JD, Tiribelli C. The human multidrug-resistance-associated protein MRP1 mediates ATP-dependent transport of unconjugated bilirubin. Biochem. J. 2004;383:335–341.

65. Falcao AS, Bellarosa C, Fernandes A, Brito MA, Silva RF, Tiribelli C, et al. Role of multidrug resistance-associated protein 1 expression in the in vitro susceptibility of rat nerve cell to unconjugated bilirubin. Neuroscience. 2007;144:878–888.

66. Gennuso F, Fernetti C, Tirolo C, Testa N, L'Episcopo F, Caniglia S, et al. Bilirubin protects astrocytes from its own toxicity by inducing up-regulation and translocation of multidrug resistance-associated protein 1 (Mrp1). Proc. Natl. Acad. Sci. U.S.A. 2004;101:2470–2475.

67. Dore S, Snyder SH. Neuroprotective action of bilirubin against oxidative stress in primary hippocampal cultures. Ann N Y Acad Sci. 1999;890:167–172.

68. Ostrow JD, Pascolo L, Tiribelli C. Reassessment of the unbound concentrations of unconjugated bilirubin in relation to neurotoxicity in vitro. Pediatr. Res. 2003;54:98–104.

69. Silva R, Mata LR, Gulbenkian S, Brito MA, Tiribelli C, Brites D. Inhibition of glutamate uptake by unconjugated bilirubin in cultured cortical rat astrocytes: role of

30 Chapter 1

concentration and pH. Biochem Biophys Res Commun. 1999;265:67–72.

70. Grojean S, Lievre V, Koziel V, Vert P, Daval JL. Bilirubin exerts additional toxic effects in hypoxic cultured neurons from the developing rat brain by the recruitment of glutamate neurotoxicity. Pediatr. Res. 2001;49:507–513.

71. Zhang L, Liu W, Tanswell AK, Luo X. The effects of bilirubin on evoked potentials and long-term potentiation in rat hippocampus in vivo. Pediatr. Res. 2003;53:939–944.

72. Grojean S, Koziel V, Vert P, Daval JL. Bilirubin induces apoptosis via activation of NMDA receptors in developing rat brain neurons. Exp Neurol. 2000;166:334–341.

73. Rodrigues CM, Sola S, Castro RE, Laires PA, Brites D, Moura JJ. Perturbation of membrane dynamics in nerve cells as an early event during bilirubin-induced apoptosis. J. Lipid Res. 2002;43:885–894.

74. Rodrigues CM, Sola S, Brito MA, Brites D, Moura JJ. Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria. J. Hepatol. 2002;36:335–341.

75. Rodrigues CM, Sola S, Silva R, Brites D. Bilirubin and amyloid-beta peptide induce cytochrome c release through mitochondrial membrane permeabilization. Mol. Med. 2000;6:936–946.

76. Rodrigues CM, Sola S, Brites D. Bilirubin induces apoptosis via the mitochondrial pathway in developing rat brain neurons. Hepatology. 2002;35:1186–1195.

77. Brites D, Fernandes A, Falcao AS, Gordo AC, Silva RF, Brito MA. Biological risks for neurological

abnormalities associated with hyperbilirubinemia. J Perinatol. 2009;29 Suppl 1:S8–13.

78. Shapiro SM. Definition of the clinical spectrum of kernicterus and bilirubin-induced neurologic dysfunction (BIND). J Perinatol. 2005;25:54–59.

79. Schmorl G. Zur kenntniss des ikterus neonatorum, insbesondere der dabei auftretenden gehirnveranderungen. Verhandlung Deutsche Pathalogische Gesellschaft. 1903;6:109–115.

80. Wennberg RP, Ahlfors CE, Bhutani VK, Johnson LH, Shapiro SM. Toward understanding kernicterus: a challenge to improve the management of jaundiced newborns. Pediatrics. 2006;117:474–485.

81. Ahlfors CE. Bilirubin-albumin binding and free bilirubin. J Perinatol. 2001;21 Suppl 1:S40–2; discussion S59–62.

82. Schenker S, Hoyumpa AM, W MD. Bilirubin toxicity to the brain (kernicterus) and other tissues. In: Ostrow JD, editor. Bile pigments and jaundice - Molecular, metabolic and medical aspects. New York: Marcel Dekker Inc.; 1986. p. 395–419.

83. Call NB, Tisher CC. The urinary concentrating defect in the Gunn strain of rat. Role of bilirubin. J. Clin. Invest. 1975;55:319–329.

84. Axelsen RA, Burry AF. Bilirubin-associated renal papillary necrosis in the homozygous Gunn rat: light-and electron-microscopic observations. J Pathol. 1976;120:165–175.

85. Broberger U, Aperia A. Renal function in infants with hyperbilirubinemia. Acta Paediatr Scand. 1979;68:75–79.

86. Massler M, Perlstein MA. Prenatal dental enamel dysplasia, with special

Introduction 31

reference to its occurrence in kernicterus. Am J Phys Med. 1956;35:324–325.

87. Rola-Plezczynski M, Hensen SA, Vincent MM, Bellanti JA. Inhibitory effects of bilirubin on cellular immune responses in man. J. Pediatr. 1975;86:690–696.

88. Vassilopoulou-Sellin R, Oyedeji CO, Samaan NA. Bilirubin inhibits cartilage metabolism and growth in vitro. Metab. Clin. Exp. 1989;38:759–762.

89. Bernhard K. Uber eine biologische Bedeutung der Gallenfarbstoffe: Bilirubin und Biliverdin als Antioxydantien fur das Vitamin A und die essentiellen Fettsauren. Helv Chim Acta. 1954;37:306-313

90. McDonagh AF. Is bilirubin good for you? Clin Perinatol. 1990;17:359–369.

91. Stocker R, Glazer AN, Ames BN. Antioxidant activity of albumin-bound bilirubin. Proc. Natl. Acad. Sci. U.S.A. 1987;84:5918–5922.

92. Sedlak TW, Snyder SH. Bilirubin benefits: cellular protection by a biliverdin reductase antioxidant cycle. Pediatrics. 2004;113:1776–1782.

93. Hegyi T, Goldie E, Hiatt M. The protective role of bilirubin in oxygen-radical diseases of the preterm infant. J Perinatol. 1994;14:296–300.

94. Benaron DA, Bowen FW. Variation of initial serum bilirubin rise in newborn infants with type of illness. Lancet. 1991;338:78–81.

95. Vitek L, Jirsa M, Brodanova M, Kalab M, Marecek Z, Danzig V, et al. Gilbert syndrome and ischemic heart disease: a protective effect of elevated bilirubin levels. Atherosclerosis. 2002;160:449–456.

96. Wu TW, Fung KP, Wu J, Yang CC, Weisel RD. Antioxidation of human

low density lipoprotein by unconjugated and conjugated bilirubins. Biochem. Pharmacol. 1996;51:859–862.

97. Temme EH, Zhang J, Schouten EG, Kesteloot H. Serum bilirubin and 10-year mortality risk in a Belgian population. Cancer Causes Control. 2001;12:887–894.

98. Adin CA, Croker BP, Agarwal A. Protective effects of exogenous bilirubin on ischemia-reperfusion injury in the isolated, perfused rat kidney. Am J Physiol Renal Physiol. 2005;288:F778–84.

99. Kato Y, Shimazu M, Kondo M, Uchida K, Kumamoto Y, Wakabayashi G, et al. Bilirubin rinse: A simple protectant against the rat liver graft injury mimicking heme oxygenase-1 preconditioning. Hepatology. 2003;38:364–373.

100. Kaplan M, Muraca M, Hammerman C, Rubaltelli FF, Vilei MT, Vreman HJ, et al. Imbalance between production and conjugation of bilirubin: a fundamental concept in the mechanism of neonatal jaundice. Pediatrics. 2002;110:47-51

101. Maisels MJ, Kring E. The contribution of hemolysis to early jaundice in normal newborns. Pediatrics. 2006;118:276–279.

102. Watchko JF. Neonatal hyperbilirubinemia--what are the risks? N. Engl. J. Med. 2006;354:1947–1949.

103. Maisels MJ, Pathak A, Nelson NM, Nathan DG, Smith CA. Endogenous production of carbon monoxide in normal and erythroblastotic newborn infants. J. Clin. Invest. 1971;50:1–8.

104. Levi AJ, Gatmaitan Z, Arias IM. Deficiency of hepatic organic anion-binding protein, impaired organic amnion uptake by liver and

32 Chapter 1

“physiologic” jaundice in newborn monkeys. N. Engl. J. Med. 1970;283:1136–1139.

105. Mera N, Ohmori S, Itahashi K, Kiuchi M, Igarashi T, Rikihisa T, et al. Immunochemical evidence for the occurrence of Mu class glutathione S-transferase in human fetal livers. J Biochem. 1994;116:315–320.

106. Halamek, LP, Stevenson DK. Neonatal jaundice and liver disease. In: De Young L, editor. Neonatal jaundice and liver disease. St. Louis: Patterson, A.S.; 1997. p. 1345–1389.

107. Kawade N, Onishi S. The prenatal and postnatal development of UDP-glucuronyltransferase activity towards bilirubin and the effect of premature birth on this activity in the human liver. Biochem. J. 1981;196:257–260.

108. Morsche Te RH, Zusterzeel PL, Raijmakers MT, Roes EM, Steegers EA, Peters WH. Polymorphism in the promoter region of the bilirubin UDP-glucuronosyltransferase (Gilbert's syndrome) in healthy Dutch subjects. Hepatology. 2001;33:765.

109. Nowicki MJ, Poley JR. The hereditary hyperbilirubinaemias. Baillieres Clin Gastroenterol. 1998;12:355–367.

110. Bosma PJ, Chowdhury JR, Bakker C, Gantla S, de Boer A, Oostra BA, et al. The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert's syndrome. N. Engl. J. Med. 1995;333:1171–1175.

111. Powell LW, Hemingway E, Billing BH, Sherlock S. Idiopathic unconjugated hyperbilirubinemia (Gilbert's syndrome). A study of 42 families. N. Engl. J. Med. 1967;277:1108–1112.

112. Kaplan M, Hammerman C, Maisels MJ. Bilirubin genetics for the

nongeneticist: hereditary defects of neonatal bilirubin conjugation. Pediatrics. 2003;111:886–893.

113. Kaplan M, Renbaum P, Levy-Lahad E, Hammerman C, Lahad A, Beutler E. Gilbert syndrome and glucose-6-phosphate dehydrogenase deficiency: a dose-dependent genetic interaction crucial to neonatal hyperbilirubinemia. Proc. Natl. Acad. Sci. U.S.A. 1997;94:12128–12132.

114. Felsher BF, Rickard D, Redeker AG. The reciprocal relation between caloric intake and the degree of hyperbilirubinemia in Gilbert's syndrome. N. Engl. J. Med. 1970;283:170–172.

115. Van der Veere CN, Jansen PL, Sinaasappel M, Van Der Meer R, Van der Sijs H, Rammeloo JA, et al. Oral calcium phosphate: a new therapy for Crigler-Najjar disease? Gastroenterology. 1997;112:455–462.

116. Kadakol A, Ghosh SS, Sappal BS, Sharma G, Chowdhury JR, Chowdhury NR. Genetic lesions of bilirubin uridine-diphosphoglucuronate glucuronosyltransferase (UGT1A1) causing Crigler-Najjar and Gilbert syndromes: correlation of genotype to phenotype. Hum. Mutat. 2000;16:297–306.

117. Crigler JF, Najjar VA. Congenital familial nonhemolytic jaundice with kernicterus. Pediatrics. 1952;10:169–180.

118. Van der Veere CN, Sinaasappel M, McDonagh AF, Rosenthal P, Labrune P, Odievre M, et al. Current therapy for Crigler-Najjar syndrome type 1: report of a world registry. Hepatology. 1996;24:311–315.

119. Yohannan MD, Terry HJ, Littlewood JM. Long term phototherapy in Crigler-Najjar syndrome. Arch Dis Child.

Introduction 33

1983;58:460–462.

120. Strauss KA, Robinson DL, Vreman HJ, Puffenberger EG, Hart G, Morton DH. Management of hyperbilirubinemia and prevention of kernicterus in 20 patients with Crigler-Najjar disease. Eur J Pediatr. 2006;165:306–319.

121. Shevell MI, Majnemer A, Schiff D. Neurologic perspectives of Crigler-Najjar syndrome type I. J Child Neurol. 1998;13:265–269.

122. Nazer H, Al-Mehaidib A, Shabib S, Ali MA. Crigler-Najjar syndrome in Saudi Arabia. Am. J. Med. Genet. 1998;79:12–15.

123. Arias IM, Gartner LM, Cohen M, Ezzer JB, Levi AJ. Chronic nonhemolytic unconjugated hyperbilirubinemia with glucuronyl transferase deficiency. Clinical, biochemical, pharmacologic and genetic evidence for heterogeneity. Am J Med. 1969;47:395–409.

124. Sinaasappel M, Jansen PL. The differential diagnosis of Crigler-Najjar disease, types 1 and 2, by bile pigment analysis. Gastroenterology. 1991;100:783–789.

125. Vitek L. Intestinal metabolism of bilirubin in the pathogenesis of neonatal jaundice. J. Pediatr. 2003;143:810; author reply 811–822.

126. Vitek L, Zelenka J, Zadinova M, Malina J. The impact of intestinal microflora on serum bilirubin levels. J. Hepatol. 2005;42:238–243.

127. De Carvalho M, Klaus MH, Merkatz RB. Frequency of breast-feeding and serum bilirubin concentration. Am J Dis Child. 1982;136:737–738.

128. Maisels MJ, Gifford K. Normal serum bilirubin levels in the newborn and the effect of breast-feeding.

Pediatrics. 1986;78:837–843.

129. Bertini G, Dani C, Tronchin M, Rubaltelli FF. Is breastfeeding really favoring early neonatal jaundice? Pediatrics. 2001;107:41-45.

130. Kotal P, Vitek L, Fevery J. Fasting-related hyperbilirubinemia in rats: the effect of decreased intestinal motility. Gastroenterology. 1996;111:217–223.

131. Gartner LM. Breastfeeding and jaundice. J Perinatol. 2001;21 Suppl 1:S25–9; discussion S35–9.

132. Wu PY, Teilmann P, Gabler M, Vaughan M, Metcoff J. "Early" versus “late” feeding of low birth weight neonates: effect on serum bilirubin, blood sugar, and responses to glucagon and epinephrine tolerance tests. Pediatrics. 1967;39:733–739.

133. Wennberg RP, Schwartz R, Sweet AY. Early versus delayed feeding of low birth weight infants: effects on physiologic jaundice. J. Pediatr. 1966;68:860–866.

134. Gourley GR, Arend RA. beta-Glucuronidase and hyperbilirubinaemia in breast-fed and formula-fed babies. Lancet. 1986;327:644–646.

135. Rosta J, Makoi Z, Kertesz A. Delayed meconium passage and hyperbilirubinaemia. Lancet. 1968;292:1138.

136. Etzioni A, Shoshani G, Diamond E, Zinder O, Bar-Maor JA. Unconjugated hyperbilirubinaemia in hypertrophic pyloric stenosis, an enigma. Z Kinderchir. 1986;41:272–274.

137. Cottrell BH, Anderson GC. Rectal or axillary temperature measurement: effect on plasma bilirubin and intestinal transit of meconium. J Pediatr Gastroenterol Nutr. 1984;3:734–739.

34 Chapter 1

138. Mihatsch WA, Hoegel J, Pohlandt F. Prebiotic oligosaccharides reduce stool viscosity and accelerate gastrointestinal transport in preterm infants. Acta Paediatr. 2006;95:843–848.

139. Bisceglia M, Indrio F, Riezzo G, Poerio V, Corapi U, Raimondi F. The effect of prebiotics in the management of neonatal hyperbilirubinaemia. Acta Paediatr. 2009;98:1579–1581.

140. Gunn CH. Hereditary Acholuric Jaundice in a New Mutant Strain of Rats. J.Hered. 1938;29:137–139.

141. Johnson L, Sarmiento F, Blanc WA, Day R. Kernicterus in rats with an inherited deficiency of glucuronyl transferase. AMA J Dis Child. 1959;97:591–608.

142. Labrune P, Myara A, Trivin F, Odievre M. Gunn rats: a reproducible experimental model to compare the different methods of measurements of bilirubin serum concentration and to evaluate the risk of bilirubin encephalopathy. Clin. Chim. Acta. 1990;192:29–33.

143. Shapiro SM. Acute brainstem auditory evoked potential abnormalities in jaundiced Gunn rats given sulfonamide. Pediatr. Res. 1988;23:306–310.

144. Shapiro SM. Reversible brainstem auditory evoked potential abnormalities in jaundiced Gunn rats given sulfonamide. Pediatr. Res. 1993;34:629–633.

145. Ahlfors CE, Shapiro SM. Auditory brainstem response and unbound bilirubin in jaundiced (jj) Gunn rat pups. Biol Neonate. 2001;80:158–162.

146. Rice AC, Shapiro SM. A new animal model of hemolytic hyperbilirubinemia-induced bilirubin encephalopathy (kernicterus). Pediatr. Res. 2008;64:265–269.

147. Swenson RM, Jew JY. Learning deficits and brain monoamines in rats with congenital hyperbilirubinemia. Exp Neurol. 1982;76:447–456.

148. Ahdab-Barmada M, Moossy J. The neuropathology of kernicterus in the premature neonate: diagnostic problems. J Neuropathol Exp Neurol. 1984;43:45–56.

149. Schutta HS, Johnson L. Bilirubin encephalopathy in the Gunn rat: a fine structure study of the cerebellar cortex. J Neuropathol Exp Neurol. 1967;26:377–396.

150. Conlee JW, Shapiro SM. Development of cerebellar hypoplasia in jaundiced Gunn rats: a quantitative light microscopic analysis. Acta Neuropathol. 1997;93:450–460.

151. Schutta HS, Johnson L. Clinical signs and morphologic abnormalities in Gunn rats treated with sulfadimethoxine. J. Pediatr. 1969;75:1070–1079.

152. Hafkamp AM, Havinga R, Ostrow JD, Tiribelli C, Pascolo L, Sinaasappel M, et al. Novel kinetic insights into treatment of unconjugated hyperbilirubinemia: phototherapy and orlistat treatment in Gunn rats. Pediatr. Res. 2006;59:506–512.

153. Van der Veere CN, Schoemaker B, Bakker C, Van Der Meer R, Jansen PL, Elferink RP. Influence of dietary calcium phosphate on the disposition of bilirubin in rats with unconjugated hyperbilirubinemia. Hepatology. 1996;24:620–626.

154. Blanckaert N, Fevery J, Heirwegh KP, Compernolle F. Characterization of the major diazo-positive pigments in bile of homozygous Gunn rats. Biochem. J. 1977;164:237–249.

155. Berry CS, Zarembo JE, Ostrow JD. Evidence for conversion of bilirubin to dihydroxyl derivatives in the gunn rat.

Introduction 35

Biochem Biophys Res Commun. 1972;49:1366–1375.

156. Kapitulnik J, Gonzalez FJ. Marked endogenous activation of the CYP1A1 and CYP1A2 genes in the congenitally jaundiced Gunn rat. Mol.Pharmacol. 1993;43:722–725.

157. Brodersen R, Bartels P. Enzymatic oxidation of bilirubin. Eur J Biochem. 1969;10:468–473.

158. Yokosuka O, Billing B. Enzymatic oxidation of bilirubin by intestinal mucosa. Biochim Biophys Acta. 1987;923:268–274.

159. Cardenas-Vazquez R, Yokosuka O, Billing BH. Enzymic oxidation of unconjugated bilirubin by rat liver. Biochem. J. 1986;236:625–633.

160. Maisels MJ, McDonagh AF. Phototherapy for neonatal jaundice. N. Engl. J. Med. 2008;358:920–928.

161. Ostrow JD. Photocatabolism of labeled bilirubin in the congenitally jaundiced (Gunn) rat. J. Clin. Invest. 1971;50:707–718.

162. Ostrow JD. Photochemical and biochemical basis of the treatment of neonatal jaundice. Prog Liver Dis. 1972;4:447–462.

163. Stoll MS, Zenone EA, Ostrow JD. Excretion of administered and endogenous photobilirubins in the bile of the jaundice gunn rat. J. Clin. Invest. 1981;68:134–141.

164. Stoll MS, Zenone EA, Ostrow JD, Zarembo JE. Preparation and properties of bilirubin photoisomers. Biochem. J. 1979;183:139–146.

165. Cremer RJ, Perryman PW, Richards DH. Influence of light on the hyperbilirubinaemia of infants. Lancet. 1958;271:1094–1097.

166. Ennever JF, McDonagh AF, Speck WT. Phototherapy for neonatal

jaundice: optimal wavelengths of light. J. Pediatr. 1983;103:295–299.

167. Ennever JF. Blue light, green light, white light, more light: treatment of neonatal jaundice. Clin Perinatol. 1990;17:467–481.

168. McDonagh AF, Lightner DA. “Like a shrivelled blood orange--”bilirubin, jaundice, and phototherapy. Pediatrics. 1985;75:443–455.

169. McDonagh AF. Light effects on transport and excretion of bilirubin in newborns. Ann N Y Acad Sci. 1985;453:65–72.

170. Costarino AT, Ennever JF, Baumgart S, Speck WT, Paul M, Polin RA. Bilirubin photoisomerization in premature neonates under low- and high-dose phototherapy. Pediatrics. 1985;75:519–522.

171. Lightner DA, Linnane WP3, Ahlfors CE. Bilirubin photooxidation products in the urine of jaundiced neonates receiving phototherapy. Pediatr. Res. 1984;18:696–700.

172. Mills JF, Tudehope D. Fibreoptic phototherapy for neonatal jaundice. Cochrane.Database.Syst.Rev. 2001;:CD002060.

173. Rubaltelli FF, Jori G, Reddi E. Bronze baby syndrome: a new porphyrin-related disorder. Pediatr. Res. 1983;17:327–330.

174. Kopelman AE, Brown RS, Odell GB. The “bronze” baby syndrome: a complication of phototherapy. J. Pediatr. 1972;81:466–472.

175. Paller AS, Eramo LR, Farrell EE, Millard DD, Honig PJ, Cunningham BB. Purpuric phototherapy-induced eruption in transfused neonates: relation to transient porphyrinemia. Pediatrics. 1997;100:360–364.

176. Mallon E, Wojnarowska F, Hope P,

36 Chapter 1

Elder G. Neonatal bullous eruption as a result of transient porphyrinemia in a premature infant with hemolytic disease of the newborn. J Am Acad Dermatol. 1995;33:333–336.

177. Valaes T, Petmezaki S, Henschke C, Drummond GS, Kappas A. Control of jaundice in preterm newborns by an inhibitor of bilirubin production: studies with tin-mesoporphyrin. Pediatrics. 1994;93:1–11.

178. Maayan-Metzger A, Yosipovitch G, Hadad E, Sirota L. Transepidermal water loss and skin hydration in preterm infants during phototherapy. Am J Perinatol. 2001;18:393–396.

179. Tonz O, Vogt J, Filippini L, Simmler F, Wachsmuth ED, Winterhalter KH. [Severe light dermatosis following photo therapy in a newborn infant with congenital erythropoietic urophyria]. Helv Paediatr Acta. 1975;30:47–56.

180. Practice parameter: management of hyperbilirubinemia in the healthy term newborn. American Academy of Pediatrics. Provisional Committee for Quality Improvement and Subcommittee on Hyperbilirubinemia. Pediatrics. 1994;94:558–565.

181. Jackson JC. Adverse events associated with exchange transfusion in healthy and ill newborns. Pediatrics. 1997;99:E7-13.

182. Patra K, Storfer-Isser A, Siner B, Moore J, Hack M. Adverse events associated with neonatal exchange transfusion in the 1990s. J. Pediatr. 2004;144:626–631.

183. Keenan WJ, Novak KK, Sutherland JM, Bryla DA, Fetterly KL. Morbidity and mortality associated with exchange transfusion. Pediatrics. 1985;75:417–421.

184. Lauer BA, Githens JH, Hayward AR, Conrad PD, Yanagihara RT,

Tubergen DG. Probable graft-vs-graft reaction in an infant after exchange transfusion and marrow transplantation. Pediatrics. 1982;70:43–47.

185. Livaditis A, Wallgren G, Faxelius G. Necrotizing enterocolitis after catheterization of the umbilical vessels. Acta Paediatr Scand. 1974;63:277–282.

186. Steiner LA, Bizzarro MJ, Ehrenkranz RA, Gallagher PG. A decline in the frequency of neonatal exchange transfusions and its effect on exchange-related morbidity and mortality. Pediatrics. 2007;120:27–32.

187. Maisels MJ. Phototherapy--traditional and nontraditional. J Perinatol. 2001;21 Suppl 1:S93–7; discussion S104–7.

188. Odell GB, Cohen SN, Gordes EH. Administration of albumin in the management of hyperbilirubinemia by exchange transfusions. Pediatrics. 1962;30:613–621.

189. Chan G, Schiff D. Variance in albumin loading in exchange transfusions. J. Pediatr. 1976;88:609–613.

190. Kaufman SS, Wood RP, Shaw BWJ, Markin RS, Rosenthal P, Gridelli B, et al. Orthotopic liver transplantation for type I Crigler-Najjar syndrome. Hepatology. 1986;6:1259–1262.

191. Terpstra OT. Auxiliary liver grafting: a new concept in liver transplantation. Lancet. 1993;342:758.

192. Kelly DA. Organ transplantation for inherited metabolic disease. Arch Dis Child. 1994;71:181–183.

193. Muiesan P, Vergani D, Mieli-Vergani G. Liver transplantation in children. J. Hepatol. 2007;46:340–348.

Introduction 37

194. Gupta S, Chowdhary JR. Hepatocyte transplantation: back to the future. Hepatology. 1992;15:156–162.

195. Lysy PA, Najimi M, Stephenne X, Bourgois A, Smets F, Sokal EM. Liver cell transplantation for Crigler-Najjar syndrome type I: update and perspectives. World J Gastroenterol. 2008;14:3464–3470.

196. Fox IJ, Chowdhury JR. Hepatocyte transplantation. Am J Transplant. 2004;4 Suppl 6:7–13.

197. Khan AA, Parveen N, Mahaboob VS, Rajendraprasad A, Ravindraprakash HR, Venkateswarlu J, et al. Treatment of Crigler-Najjar Syndrome type 1 by hepatic progenitor cell transplantation: a simple procedure for management of hyperbilirubinemia. Transplant Proc. 2008;40:1148–1150.

198. Ambrosino G, Varotto S, Strom SC, Guariso G, Franchin E, Miotto D, et al. Isolated hepatocyte transplantation for Crigler-Najjar syndrome type 1. Cell Transplant. 2005;14:151–157.

199. Quaglia A, Lehec SC, Hughes RD, Mitry RR, Knisely AS, Devereaux S, et al. Liver after hepatocyte transplantation for liver-based metabolic disorders in children. Cell Transplant. 2008;17:1403–1414.

200. Ito M, Nagata H, Miyakawa S, Fox IJ. Review of hepatocyte transplantation. J Hepatobiliary Pancreat Surg. 2009;16:97–100.

201. Bellodi-Privato M, Aubert D, Pichard V, Myara A, Trivin F, Ferry N. Successful gene therapy of the Gunn rat by in vivo neonatal hepatic gene transfer using murine oncoretroviral vectors. Hepatology. 2005;42:431–438.

202. Tada K, Chowdhury NR, Neufeld D, Bosma PJ, Heard M, Prasad VR, et al. Long-term reduction of serum bilirubin levels in Gunn rats by

retroviral gene transfer in vivo. Liver Transpl Surg. 1998;4:78–88.

203. Roy-Chowdhury N, Kadakol A, Sappal BS, Thummala NR, Ghosh SS, Lee SW, et al. Gene therapy for inherited hyperbilirubinemias. J Perinatol. 2001;21 Suppl 1:S114–8; discussion S125–7.

204. Askari FK, Hitomi Y, Mao M, Wilson JM. Complete correction of hyperbilirubinemia in the Gunn rat model of Crigler-Najjar syndrome type I following transient in vivo adenovirus-mediated expression of human bilirubin UDP-glucuronosyltransferase. Gene Ther. 1996;3:381–388.

205. Li Q, Murphree SS, Willer SS, Bolli R, French BA. Gene therapy with bilirubin-UDP-glucuronosyltransferase in the Gunn rat model of Crigler-Najjar syndrome type 1. Hum Gene Ther. 1998;9:497–505.

206. Toietta G, Mane VP, Norona WS, Finegold MJ, Ng P, McDonagh AF, et al. Lifelong elimination of hyperbilirubinemia in the Gunn rat with a single injection of helper-dependent adenoviral vector. Proc. Natl. Acad. Sci. U.S.A. 2005;102:3930–3935.

207. Thummala NR, Ghosh SS, Lee SW, Reddy B, Davidson A, Horwitz MS, et al. A non-immunogenic adenoviral vector, coexpressing CTLA4Ig and bilirubin-uridine-diphosphoglucuronateglucuronosyltransferase permits long-term, repeatable transgene expression in the Gunn rat model of Crigler-Najjar syndrome. Gene Ther. 2002;9:981–990.

208. Ilan Y, Attavar P, Takahashi M, Davidson A, Horwitz MS, Guida J, et al. Induction of central tolerance by intrathymic inoculation of adenoviral antigens into the host thymus permits long-term gene therapy in Gunn rats. J. Clin. Invest. 1996;98:2640–2647.

38 Chapter 1

209. Jia Z, Danko I. Long-term correction of hyperbilirubinemia in the Gunn rat by repeated intravenous delivery of naked plasmid DNA into muscle. Mol Ther. 2005;12:860–866.

210. Nguyen TH, Bellodi-Privato M, Aubert D, Pichard V, Myara A, Trono D, et al. Therapeutic lentivirus-mediated neonatal in vivo gene therapy in hyperbilirubinemic Gunn rats. Mol Ther. 2005;12:852–859.

211. Seppen J, van der Rijt R, Looije N, van Til NP, Lamers WH, Oude Elferink RP. Long-term correction of bilirubin UDPglucuronyltransferase deficiency in rats by in utero lentiviral gene transfer. Mol Ther. 2003;8:593–599.

212. Sauter BV, Parashar B, Chowdhury NR, Kadakol A, Ilan Y, Singh H, et al. A replication-deficient rSV40 mediates liver-directed gene transfer and a long-term amelioration of jaundice in gunn rats. Gastroenterology. 2000;119:1348–1357.

213. Wilson JM, Grossman M, Wu CH, Chowdhury NR, Wu GY, Chowdhury JR. Hepatocyte-directed gene transfer in vivo leads to transient improvement of hypercholesterolemia in low density lipoprotein receptor-deficient rabbits. J. Biol. Chem. 1992;267:963–967.

214. Chowdhury NR, Wu CH, Wu GY, Yerneni, P. C., Bommineni VR, Chowdhury JR. Fate of DNA targeted to the liver by asialoglycoprotein receptor-mediated endocytosis in vivo. Prolonged persistence in cytoplasmic vesicles after partial hepatectomy. J. Biol. Chem. 1993;268:11265–11271.

215. Bommineni VR, Chowdhury NR, Wu GY, Wu CH, Franki N, Hays RM, et al. Depolymerization of hepatocellular microtubules after partial hepatectomy. J. Biol. Chem. 1994;269:25200–25205.

216. Wang X, Sarkar DP, Mani P, Steer CJ, Chen Y, Guha C, et al. Long-term reduction of jaundice in Gunn rats by nonviral liver-targeted delivery of Sleeping Beauty transposon. Hepatology. 2009;50:815–824.

217. Kren BT, Bandyopadhyay P, Steer CJ. In vivo site-directed mutagenesis of the factor IX gene by chimeric RNA/DNA oligonucleotides. Nat Med. 1998;4:285–290.

218. Kren BT, Parashar B, Bandyopadhyay P, Chowdhury NR, Chowdhury JR, Steer CJ. Correction of the UDP-glucuronosyltransferase gene defect in the gunn rat model of crigler-najjar syndrome type I with a chimeric oligonucleotide. Proc. Natl. Acad. Sci. U.S.A. 1999;96:10349–10354.

219. Seppen J, Tada K, Ottenhoff R, Sengupta K, Chowdhury NR, Chowdhury JR, et al. Transplantation of Gunn rats with autologous fibroblasts expressing bilirubin UDP-glucuronosyltransferase: correction of genetic deficiency and tumor formation. Hum Gene Ther. 1997;8:27–36.

220. Ostrow JD. Regulation by bile salts of the excretion of conjugated and unconjugated bilirubin in bile. In: Fromm H, Leuschner U, eds. Proceedings of the falk symposium No. 84, held in Berlin, Germany, June 9-10, 1995. Kluwer Academic Publishers, 1996.

221. Rege RV, Webster CC, Ostrow JD. Interactions of unconjugated bilirubin with bile salts. J. Lipid Res. 1988;29:1289–1296.

222. Rege RV, Webster CC, Ostrow JD. Enzymatic oxidation of unconjugated bilirubin to assess its interactions with taurocholate. J. Lipid Res. 1987;28:673–683.

Introduction 39

223. Whitmer DI, Gollan JL. Mechanisms and significance of fasting and dietary hyperbilirubinemia. Semin Liver Dis. 1983;3:42–51.

40 Chapter 1

Chapter 2 Pharmacological Therapies for Unconjugated Hyperbilirubinemia

F.J.C. Cuperus 1 A.M. Hafkamp 1 C.V. Hulzebos 2

H.J. Verkade 1

1Pediatric Gastroenterology and Hepatology, Department of Pediatrics, Center for Liver, Digestive, and Metabolic Diseases, Beatrix Children’s Hospital - University Medical Center Groningen, University of Groningen, The Netherlands. 2Neonatology, Department of Pediatrics, Beatrix Children’s Hospital- University Medical Center Groningen, The Netherlands.

Adapted from: Current Pharmaceutical Design 2009;15:2927-38

42 Chapter 2

2.1 Abstract evere unconjugated hyperbilirubinemia, seen mainly in neonates, may cause kernicterus and death. Conventional treatment for severe unconjugated hyperbilirubinemia consists of phototherapy and exchange transfusion. Phototherapy, however, has several known disadvantages while exchange transfusion is associated with a significant morbidity, and even mortality. These harmful effects indicate the need to develop alternative pharmacological treatment strategies for unconjugated hyperbilirubinemia. Generally, these strategies aim to decrease the plasma concentration of unconjugated bilirubin (UCB) by inhibiting production, stimulating hepatic clearance, or interrupting the enterohepatic circulation of the pigment.

To be considered for routine clinical use, an alternative treatment strategy should be less invasive and at least as effective and safe as phototherapy. Several pharmacological therapies such as metalloporhyrins, clofibrate, bile salts, laxatives and bilirubin oxidase may meet these criteria in the future, but none of them has yet been evaluated sufficiently to allow routine application. This chapter aims to discuss the state of the art and future perspectives in pharmacological treatment of neonatal jaundice.

S

Treatments for unconjugated hyperbilirubinemia 43

2.2 Introduction nconjugated bilirubin (UCB) is produced by the degradation of heme, derived mainly from the catabolism of erythrocyte hemoglobin. After its production, the hydrophobic UCB is bound to albumin in the plasma and transported to the liver. In the liver, the enzyme bilirubin-UDP-glucuronosyltransferase (UGT1A1) conjugates UCB with glucuronic acid. The relatively hydrophilic bilirubin conjugates are readily excreted into the bile and transit the biliary tree to the intestine. In the intestinal lumen, the bilirubin conjugates are partly hydrolyzed to UCB, which can be reabsorbed from the intestine, reduced to urobilinoids, or excreted with the feces (Fig. 1). Reabsorbed UCB is transported to the liver via the vena porta, where it again can be conjugated and excreted via the bile, thus constituting an enterohepatic circulation.

Unconjugated hyperbilirubinemia, the accumulation of UCB in the body, can result from increased production, decreased hepatic clearance, or enhanced enterohepatic circulation of the pigment (Fig. 1). This is illustrated by the transient unconjugated hyperbilirubinemia during the first postnatal week. This so-called physiological jaundice is due to a combination of high erythrocyte turnover (increased UCB production),[1] immature hepatic conjugation of the pigment (decreased hepatic clearance),[2] and a delayed intestinal transit (enhanced enterohepatic circulation of UCB).[3,4] For the majority of neonates, unconjugated hyperbilirubinemia is a benign transitional phenomenon that may be beneficial, due to the potent antioxidant properties of the pigment.[5] Under specific conditions, however, plasma UCB concentrations can rise to hazardous levels. This occurs for example in patients with neonatal hemolytic disease, due to excessive UCB production, or in patients with inherited deficiencies of UCB conjugation,[6,7] due to a severely decreased biliary excretion of the pigment. Crigler-Najjar disease, the most serious of these inherited deficiencies, is characterized by a genetically absent (type I) or decreased (type II) activity of UGT1A1.[6] With severely deficient conjugation, the excretion of bilirubin into the bile is diminished, resulting in a lifelong unconjugated hyperbilirubinemia in Crigler-Najjar patients and in Gunn rats, the animal model for this disease.

The harm from severe unconjugated hyperbilirubinemia is due to the potential deposition of UCB in the central nervous system, causing bilirubin-induced neurological dysfunction (BIND), kernicterus, and death.[8] The conventional treatment for unconjugated hyperbilirubinemia is phototherapy. Phototherapy induces photo-isomerization of the hydrophobic UCB to polar isomers that can readily be excreted into the bile without conjugation.[9] Although generally effective, phototherapy has several disadvantages. Most notably, short-term phototherapy does not always decrease plasma UCB to non-toxic levels in

U

44 Chapter 2

neonates, whereas long-term phototherapy, such as needed for patients with Crigler-Najjar disease type I, becomes less effective with age and has a profound impact on social life.[10,11] Under conditions of very severe unconjugated hyperbilirubinemia or of hyperbilirubinemia with an insufficient response to phototherapy a “rescue” treatment consists of exchange transfusion, in which the hyperbilirubinemic blood is removed and replaced with non-jaundiced blood. Exchange transfusion, however, has a considerable morbidity, especially in sick preterm newborns, and even mortality has been reported.[12]

The potential neurotoxicity of unconjugated hyperbilirubinemia and the disadvantages of present treatments have prompted investigation of alternative treatment strategies. These strategies have concentrated on pharmacological therapies that decreased the production, increased the biliary excretion, and/or interrupted the enterohepatic circulation of UCB. Recently, several excellent reviews have appeared that discuss the clinically available therapies for unconjugated hyperbilirubinemia. Interestingly, and in spite of a large body of experimental and clinical studies, only one systematic review of proven and experimental pharmaceutical treatment strategies has been published recently.[13] In the present review, we critically discuss the background, effectiveness, applicability, and future perspectives of pharmacological treatment options for severe unconjugated hyperbilirubinemia.

2.3 Treatment strategies

2.3.1 Treatments that decrease the production of UCB At the end of their life span, red blood cells are degraded in the reticuloendothelial system (RES), mainly located in the liver, spleen, and bone marrow. In the RES, the released heme is phagocytized by macrophages. These macrophages contain two essential enzymes for heme degradation: heme oxygenase (HO) and biliverdin reductase. The microsomal HO catalyzes the conversion of heme into the blue-green biliverdin, the first step in heme degradation. The cytosolic biliverdin reductase then converts biliverdin to the yellow-orange UCB (Fig. 2A). Heme and biliverdin are water-soluble, non-toxic compounds. It is believed they are converted into the water-insoluble, potentially toxic UCB because of its anti-oxidative properties and ability to cross the placenta.[5,14]

Decreasing UCB production is a rational approach to treat unconjugated hyperbilirubinemia. This strategy has the benefit of actually preventing UCB

Treatments for unconjugated hyperbilirubinemia 45

Figure 1. Unconjugated hyperbilirubinemia can result from increased production ,(decreased hepatic clearance , or enhanced enterohepatic circulation of UCB . After its production from heme, the hydrophobic UCB is transported to the liver. In the liver, the enzyme bilirubin-UDP-glucuronosyltransferase conjugates UCB with glucuronic acid. The relatively hydrophilic conjugated bilirubin (CB) is readily excreted via the bile into the intestine. In the intestinal lumen, CB is partly hydrolyzed to UCB, which can be reabsorbed from the intestine, reduced to urobilinoids (not shown), or excreted with the feces. Reabsorbed UCB is transported back to the liver via the vena porta (the enterohepatic circulation; EHC).

1 23

46 Chapter 2

accumulation, rather than removing already accumulated UCB from the body. UCB production can be decreased via inhibition of HO activity with metalloporphyrins or D-penicillamine (Fig. 2B). Although HO inhibition effectively blocks heme catabolism, the efficient biliary excretion of heme prevents its accumulation in the body.[15] In theory, inhibition of biliverdin reductase could also decrease UCB production. However, inhibitors of biliverdin have not been explored, most likely because their use in neonates would produce green babies.

Metalloporphyrins

Metalloporhyrins are synthetic heme analogues, in which other metals such as zinc (Zn), tin (Sn), and chromium (Cr) replace the central iron atom of heme.[16] Metalloporphyrins competitively inhibit HO, and thus decrease heme degradation and UCB production (Fig. 2B).[17] Of the metalloporphyrins selected from the early in vitro studies, only tin, zinc, manganese, and chromium competitively inhibited HO in vivo,[18] but zinc and chromium were excluded from human use due to detrimental tissue effects.[19-21] Also, zinc-, chromium-, and manganese- protoporphyrins inhibited heme oxygenase, but induced biliverdin reductase during chronic administration in newborn rats.[22]

Clinical research eventually focused on Sn-mesoporphyrin (SnMP) due to its efficacy and its safety profile.[23-27] Kappas et al. reported that preventive SnMP treatment mitigated the development of unconjugated hyperbilirubinemia in 53 infants with a positive Coombs reaction caused by ABO incompatibility, compared with 69 Coombs positive, ABO incompatible infants that did not receive SnMP prophylaxis.[23] Valaes et al. published the results of 5 clinical trials in a total of 517 pre-term infants.[24] This paper showed that a single prophylactic dose of 1-6 µmol/kg SnMP, administered within 24 hours after birth, ameliorated unconjugated hyperbilirubinemia dose-dependently. The maximal dose of 6 µmol/kg reduced the peak plasma bilirubin concentration by 41% and the requirement for phototherapy by 76%. In the most recent clinical trial, the therapeutic use of SnMP (6 µmol/kg) in 40 term hyperbilirubinemic infants shortened the length of hyperbilirubinemia and eliminated the need for phototherapy.[27] In a trial with newborns with glucose-6-phosphate dehydrogenase deficiency, SnMP was administered either in the first day of life (preventive use), or therapeutically after a threshold of plasma bilirubin was reached.[26] Although the bilirubin levels were not particularly elevated in any group, SnMP supplanted the need for phototherapy in both the preventive and the therapeutic group. SnMP has also been used in the management of Crigler-Najjar disease. Regular SnMP treatment in Crigler-Najjar type I patients initially

Treatments for unconjugated hyperbilirubinemia 47

Figure 2. Decreasing UCB production is a potential strategy to treat unconjugated hyperbilirubinemia. At the end of their lifespan, red blood cells are degraded in the reticulo-endothelial system (RES). In the RES, the released heme is phagocytized by macrophages. These macrophages contain two essential enzymes for heme degradation: heme oxygenase and biliverdin reductase. The microsomal heme oxygenase catalyzes the conversion of heme into biliverdin, the first step in heme degradation. The cytosolic biliverdin reductase then converts biliverdin to UCB (A). Metalloporphyrins and D-penicillamine block the conversion of heme into biliverdin, thus decreasing UCB production (B).

48 Chapter 2

decreased plasma bilirubin levels, but the effect was temporary for unknown reasons.[28,29]

Although promising, metalloporphyrins are currently not recommended for routine treatment in newborns due to insufficient evidence and unknown long-term safety.[30] The only known side effect of SnMP treatment that has been observed so far consists of a mild transient erythema due to photosensitivity.[23,24] However, some concerns have risen regarding potential adverse-effects due to the suppression of the cytoprotective effects of HO, especially in critically ill infants,[31,32] and none of the metalloporphyrins are so far available for oral administration. Therefore, the results of further clinical trials regarding safety and efficacy need to be awaited before routine clinical use can be recommended.

D-penicillamine

D-penicillamine is a chelating agent, routinely used for the treatment of Wilson’s disease based on its stimulation of renal copper excretion. However, D-penicillamine also inhibits HO-activity (Fig. 2B) and has been applied for the treatment of unconjugated hyperbilirubinemia.[33] Lakatos et al. published the only clinical trial with D-penicillamine in 1976, which involved 120 full-term infants with ABO-hemolytic disease.[34] In this study, patients were randomized to receive D-penicillamine either within the first 24 hours, or after the third day of life. D-penicillamine decreased plasma bilirubin concentrations by ~16% and decreased the number of exchange transfusions by 91%, but only in the group treated within 24 hours.

Because D-penicillamine is a nitric oxide (NO) donor, any adverse effect related to NO, such as vasodilatation and decreased platelet aggregation, could potentially occur during treatment. Furthermore, chronic D-penicillamine use in adults is associated with serious adverse effects, such as aplastic anemia, Goodpasture’s disease, and myasthenia gravis.[35] Therefore, additional clinical trials will be necessary to evaluate whether short-term administration of D-penicillamine would be an effective and safe treatment for unconjugated hyperbilirubinemia in neonates.

Treatments for unconjugated hyperbilirubinemia 49

2.3.2 Treatments that increase the hepatic clearance of UCB After production in the RES, the hydrophobic UCB is transported to the liver, predominantly bound to albumin (Fig. 3). In the liver, the UCB-albumin complex enters the space of Disse via the endothelial fenestrae. There, UCB is dissociated from albumin and crosses the basolateral membrane via facilitated diffusion, involving transporters that have yet to be identified. Once in the hepatocyte, UCB is bound to ligandin (gluthathione S-transferase; Y-protein) in the cytosol.[36,37] This protein binds UCB with an affinity similar to plasma albumin and thus prevents it reflux into the space of Disse.[38] After diffusion into the cisterna of the endoplasmic reticulum, the hydrophobic UCB is conjugated with one or two glucuronic acids by the microsomal enzyme UGT1A1. Absence of UGT1A1 activity in Crigler-Najjar disease results in a permanent unconjugated hyperbilirubinemia.[6] Finally, bilirubin conjugates are efficiently excreted into the bile via the canalicular ATP-dependent transporter MRP2 (Fig. 3).[39] The nuclear receptor CAR (constitutive androstane receptor) is a key regulator of UCB clearance, since it enhances the activity of ligandin, UGT1A1, and MRP2.[40]

Figure 3. Increasing the hepatic UCB clearance is a potential strategy to lower plasma UCB levels. Phenobarbital, Chinese Herbal Medicine, and Clofibrate achieve this by enhancing the enzymatic steps in hepatic UCB clearance: hepatic uptake and storage via ligandin, hepatic conjugation via UGT1A1, and secretion of the bilirubin conjugates into the bile via MRP2. Alb-UCB, albumin-bound bilirubin.

50 Chapter 2

Increasing the hepatic clearance of bilirubin is a potential strategy to lower plasma UCB levels. This can be achieved by enhancing the three steps in hepatic UCB clearance that are all underactive in human neonates: (1) hepatic uptake and storage via ligandin, (2) hepatic conjugation via UGT1A1, and (3) biliary excretion of conjugated bilirubin via MRP2 (Fig. 3). Below we will discuss the several pharmacological treatments that enhance the biliary excretion of bilirubin by one or more of these mechanisms.

Phenobarbital

Phenobarbital, an anti-epileptic drug, is a CAR agonist that enhances the three steps in hepatic UCB clearance; uptake and storage in the liver, hepatic conjugation, and hepatic excretion of bilirubin. Net uptake and storage is enhanced via an increased concentration of ligandin, conjugation is enhanced via induction of UGT1A1, and biliary secretion is enhanced via induction of Mrp2 (Fig. 3).[40-42]

Phenobarbital has been used to treat neonatal jaundice since the 1960s. Numerous clinical trials have shown that both administration of phenobarbital to pregnant mothers before delivery and phenobarbital administration to neonates after delivery limits the severity of unconjugated hyperbilirubinemia and the need for exchange transfusion (for a complete review [43]). In one of the largest clinical trials, daily administration of 1 g phenobarbital to 310 mothers in the last week of pregnancy decreased the incidence of severe hyperbilirubinemia (bilirubin > 274 µmol/l) and lowered the need for exchange transfusion by 85%.[44]

Nonetheless, phenobarbital is not routinely used to treat neonatal unconjugated hyperbilirubinemia due to several reasons.[45] First, phototherapy is more effective compared with either phenobarbital alone or with phenobarbitone combined with phototherapy.[46] In addition, the bilirubin lowering effect of phenobarbital is not evident until a few days after administration, in contrast to some of the adverse effects, i.e. sedation of the newborn infant.[43] Furthermore, phenobarbital diminishes the oxidative metabolism of UCB in the rat brain, which could lead to an increased neurotoxicity of the pigment.[47] Phenobarbital is useful to distinguish between type I and type II Crigler-Najjar disease. Phenobarbital is not effective in patients with Crigler-Najjar type I because there is no UGT1A1 to induce, and conjugation is essential for the biliary excretion of UCB. In patients with Crigler-Najjar type II disease, who have about 5% of normal baseline UGT1A1 activity, phenobarbital treatment reduces plasma UCB concentrations by 30% or more.[48]

Treatments for unconjugated hyperbilirubinemia 51

Clofibrate

Clofibrate, an activator of peroxisome proliferator-activated receptors (PPARS), is a lipid-lowering drug used in patients with hypercholesterolemia or hyperlipoproteinemia.[49] Clofibrate treatment also increases the hepatic conjugation of UCB via induction of UGT1A1 (Fig. 3).[50,51] Clofibrate treatment in Sprague-Dawley rats increased Ugt1A1 activity and resulted in an 84% increase in the hepatic clearance of IV-administered UCB.[50,52]

In 1981 Lindenbaum et al. published the first randomized placebo-controlled trial with clofibrate, involving 93 full-term neonates with physiological jaundice. Of these 93 neonates, 47 received one preventive oral dose of clofibrate, which significantly lowered plasma UCB levels from the 16th hour after administration and curtailed the duration of jaundice.[53] A similar study in 89 preterm neonates showed comparable results, including indications for a dose-response relationship: the hypobilirubinemic effect appeared to correlate with the plasma concentration of clofibric acid.[54] Three randomized controlled trials, each involving 60 full-term neonates, compared the use of clofibrate and phototherapy with the use of phototherapy alone.[55-57] Clofibrate, as add-on treatment, accelerated the decrease in serum total bilirubin concentrations and decreased the duration of phototherapy in all of these trials. The clinical utility of these results, however, can be disputed based on the methodology: enrollment in these studies was generally 5-8 days postpartum, and therefore after the period in which the peak of neonatal jaundice would normally occur.

Long-term clofibrate treatment has been associated with serious adverse effects. In animals, clofibrate treatment is carcinogenic.[58] Although this effect has not been confirmed in adult humans, it is not known whether long-term carcinogenesis could occur after neonatal treatment.[59] Human clofibrate treatment for hypercholesterolemia or hyperlipoproteinemia has also been associated with an overall increase in non-cardiovascular mortality.[59] In theory, the clofibric acid metabolite p-chlorophenoxyisobutyric acid could displace UCB from albumin, but this has not been confirmed in vitro.[60] Other known side effects of clofibrate include vomiting, diarrhea, increased incidence of gallstones, and muscular myopathy.[61,62] None of these side effects have been reported in the short-term studies in infants described above. Nevertheless, long-term safety issues need to be clarified before its clinical use can be considered, despite the indication that clofibrate is an effective (add-on) therapy for unconjugated hyperbilirubinemia.

52 Chapter 2

Traditional herbal medicine

Various herbs are used in the treatment of neonatal jaundice in traditional medicine throughout Asia. One of the most commonly used concoctions is Yin Zhi Huang, a mixture of four different plant extracts.[63] By now, several studies have elucidated the mechanism underlying its hypobilirubinemic effect (Fig. 3). Administration of Yin Zhi Huang to rats increased the hepatic clearance of IV-administered UCB and induced the enzyme activity of ligandin and Ugt1A1.[64] Yin Zhi Huang seems to act as a CAR agonist, since treatment induced the expression of ligandin, Ugt1A1, and Mrp2 in normal, but not in CAR-knockout mice.[65] Coumarin 6,7-dimethylesculetin (Scoparone), a component of Artesemia capillaris (Yin Chin), which is one of the four plant extracts in Yin Zhi Huang, appeared to be the largest contributor to the effect of Yin Zhi Huang.[65]

The clinical efficacy of Yin Zhi Huang for unconjugated hyperbilirubinemia has not been tested in randomized, controlled clinical trials. Also, there is a legitimate tendency to develop treatments based on individual molecules, rather than mixtures, to guarantee reproducibility and minimize the risk of side effects. Indeed, some herbal concoctions have been able to displace bilirubin from albumin, thereby increasing the concentration of unbound unconjugated bilirubin.[66] Contamination of herbal medicines with heavy metals, i.e. lead and mercury, has been reported.[67] Finally, Yin Zhi Huang treatment is associated with hemolysis in glucose-6-phosphate dehydrogenase-deficient patients.[63] Therefore, clinical use of traditional herbal medicine during neonatal jaundice is not recommended.

2.3.3 Treatments that interrupt UCB’s enterohepatic circulation In the intestine, conjugated bilirubin is extensively hydrolyzed by β-glucuronidases to UCB, which can be converted to urobilinoids or can be reabsorbed into the enterohepatic circulation (Fig. 4A).[68,69] This reabsorption is especially important in human newborns, which do not develop the anaerobic colonic microflora that converts UCB to urobilinoids until 2-6 weeks postnatal.[70] Reabsorption of UCB from the intestinal lumen seems to occur, at least in part, via a passive mode of transport.[71] Intestinal accumulation of UCB, for instance during a delayed passage of meconium or during starvation,[4,72] increases its intestinal reabsorption into the portal venous blood.[3] Because the liver extracts only 30% of the UCB load, this will directly contribute to the pathogenesis of unconjugated hyperbilirubinemia.[73] However, if the plasma UCB concentrations are very high, the concentration gradient will favor UCB diffusion from the blood into the intestinal lumen.[74,75] In Gunn rats, the well-

Treatments for unconjugated hyperbilirubinemia 53

established animal model for Crigler-Najjar disease type 1, the majority of the total bilirubin disposal occurred via this excretory pathway.[74,75]

The transport of UCB across the intestinal membrane thus seems to be predominantly passive, bi-directional, and driven by the concentration gradient between (unbound) plasma UCB and UCB in the intestinal lumen. Theoretically, capturing UCB in the intestinal lumen could enhance the diffusion of UCB from the blood and limit its reabsorption into the enterohepatic circulation. Several capturing strategies that influence the gradient towards net excretion have been tested. (Fig. 4B). Some involve oral administration of insoluble, poorly absorbable solids, or of detergents, that bind UCB. Others work by decreasing the intestinal absorption of endogenous compounds that bind bilirubin. Alternatively, enhancing the transit and fecal elimination of intestinal contents, for example by frequent feeding or laxatives, can also be applied to decrease plasma UCB concentrations (Fig. 4C). Below, we discuss the effectiveness, applicability and future perspectives of the several classes of agents that diminish the enterohepatic circulation of UCB.

BLOOD

EHC INTESTINALCAPTURE

ENHANCINGINTESTINALTRANSIT

Figure 4. Interruption of the enterohepatic circulation of UCB decreases plasma UCB levels. In the intestinal lumen, conjugated bilirubin (CB) is partly hydrolyzed to UCB, which can be reabsorbed from the intestine. Reabsorbed UCB is transported back to the liver via the vena porta. In the liver, this UCB is re-conjugated and excreted via the bile, thus constituting an enterohepatic circulation (EHC) (A). Reabsorption occurs via a passive mode of transport and is, consequently, bi-directional and driven by the UCB concentration gradient between the plasma and the intestinal lumen. Capturing UCB in the lumen with cholestyramine, charcoal, agar, calcium, zinc, and fat limits intestinal reabsorption of the pigment. This induces a shift of UCB from the blood into the intestinal lumen, from where UCB can then be excreted with the feces (B). Accelerating intestinal transit by frequent meals may result in a lower intestinal UCB concentration, which also enhances UCB diffusion from the blood into the intestinal lumen (C).

54 Chapter 2

Cholestyramine

Cholestyramine is an insoluble, quaternary ammonium compound and anion exchanger, known to bind bile salts in the small intestine. In 1962 Lester et al. hypothesized that cholestyramine could lower plasma UCB levels by binding the UCB in the intestinal lumen, thus preventing its reabsorption (Fig. 4B). To prove this hypothesis, four adult Gunn rats were supplemented with dietary cholestyramine, which resulted in a 30-45% drop in plasma UCB levels.[76] Enteral administration of cholestyramine to jaundiced preterm infants, however, did not significantly decrease plasma bilirubin levels compared with controls.[77] Schmid et al. speculated that the discrepancy between animal and human data could be due to a higher affinity between UCB and albumin in the human blood, compared with Gunn rats. This could limit the diffusion of UCB from the blood into the intestinal lumen.[77] Subsequent trials have reported either for,[78-80] or against [81] the effectiveness of cholestyramine as add-on treatment to phototherapy. Side effects of cholestyramine therapy include: hyperchloremic metabolic acidosis, constipation, and diarrhea.[77,79-81] Because its safety and effectiveness remain questionable, treatment of unconjugated hyperbilirubinemia with cholestyramine is not recommended.

Charcoal

In 1964, Ulstrom et al. observed that activated charcoal removed bilirubin from human bile more avidly than did cholestyramine.[82] In that same year, Künzer et al. independently showed that activated charcoal removed nearly all the bilirubin content from human duodenal fluids.[83] Both groups hypothesized that activated charcoal could trap bilirubin in the intestinal tract, prevent its enterohepatic circulation and decrease plasma bilirubin levels in neonates (Fig. 4B). To investigate this hypothesis, Ulstrom et al. administered 11-15 doses of activated charcoal in 30 full-term neonates. Treatment started either 4 hours (n=15) or 12 hours (n=15) after birth. Interestingly, UCB levels decreased, but only in the group that received the first charcoal administration at 4 hours postpartum.[82] Künzer et al. daily administered activated charcoal from the second day after birth in 25 premature neonates, but found no effect on plasma bilirubin levels.[83] Ulstrom et al. concluded that charcoal should be administered soon after birth, possibly because it thus could prevent the intestinal reabsorption of meconial UCB.[82] After these initial reports, the effect of oral charcoal on plasma bilirubin has been investigated in three Gunn rat studies and in one prospective study involving jaundiced newborns.[84-86] In Gunn rats, activated charcoal decreased plasma unconjugated bilirubin levels as effectively as phototherapy.[84-86] In jaundiced neonates, charcoal treatment, started within the first day of life, combined with phototherapy decreased plasma bilirubin levels more effectively compared with phototherapy alone.[87]

Treatments for unconjugated hyperbilirubinemia 55

Side effects of activated charcoal are relatively rare, but include vomiting and dehydration.[84,88] Although activated charcoal seems to have some merit in the treatment of neonatal unconjugated hyperbilirubinemia, more research regarding its safety and effectiveness (e.g. timing of postnatal administration) is necessary before clinical use can be considered. Also, it is unclear whether binding by charcoal limits the intestinal absorption of essential nutrients and other potentially beneficial compounds.

Agar

Agar is a gelatinous substance that is derived from seaweed. In the 1970s, Poland et al. showed that UCB binds to dried agar and that agar thus could act as a trapping agent for the UCB in the intestinal lumen (Fig. 4B).[89,90] In Gunn rats, agar exhibited a protective effect against bilirubin-induced nephropathy.[91] The results of agar in hyperbilirubinemic newborns, however, have been conflicting. Poland et al. initially reported favorable results in term infants.[89] Yet, subsequent papers showed either beneficial effects,[92,93] or no effects[94-99] of agar treatment on decreasing UCB levels or shortening of the duration of phototherapy. However, most of these studies failed to assess the binding affinity for UCB of the ingested agar. Also, many of the studied populations were small and/or heterogeneous.[100] Consequently, although oral agar has appeared to be free from serious side effects in these studies, its effectiveness remains unproven. Thus, treatment of unconjugated hyperbilirubinemia with agar is not recommended currently.

Calcium phosphate

In pigment gallstones there is a strong interaction between calcium and UCB, which is present in the stones as the insoluble salt Ca(HB-)2.[101,102] Van der Veere et al. showed that amorphous calcium phosphate rapidly precipitates UCB in vitro in a dose-dependent fashion.[103] The UCB almost exclusively associated with insoluble amorphous calcium phosphate and not with free calcium ions.[103] Van der Veere et al. subsequently showed that dietary treatment of Gunn rats with calcium phosphate decreased plasma UCB levels by ~35% and transiently increased fecal UCB excretion, in accordance with intestinal capture (Fig. 4B).[104]

Following these promising results, a placebo-controlled double blind cross-over trial was performed in 11 patients with Crigler-Najjar disease.[105] Patients received calcium phosphate supplementation during 3 weeks as adjunct to their usual phototherapy regimen. Calcium phosphate supplementation decreased plasma UCB concentrations by 18% in patients with type I Crigler-Najjar disease, but had no effect in patients with type II disease.[105] Although its

56 Chapter 2

hypobilirubinemic effect is only moderate, calcium phosphate is currently used by a number of Dutch Crigler-Najjar type 1 patients as an adjunct to phototherapy when plasma UCB concentrations reach dangerously high levels (personal communication Dr. M. Sinaasappel, Rotterdam).

The adverse effects of calcium phosphate treatment seem limited. No side effects have been observed in the animal or patient studies, or during routine treatment of patients. However, there are some concerns that prolonged treatment with high doses of calcium phosphate might cause calcium depositions in the kidneys.

Zinc salts

In 2001, Mendez-Sanchez et al. demonstrated that UCB rapidly, and dose-dependently, precipitates with zinc salts in vitro.[106] UCB was hypothesized to associate both with the insoluble zinc salts and with the free zinc cations, but the study did not discriminate between these two possible mechanisms.[107] In the same paper, they showed that dietary administration of zinc sulfate decreased the biliary excretion of UCB in hamsters.[107] These observations suggested that zinc salts might trap UCB in the intestinal lumen (Fig. 4B). To further explore this hypothesis, Mendez-Sanchez et al. administered a single oral dose of zinc sulfate to 10 patients with Gilbert’s syndrome. This inherited condition is characterized by a chronic, mild, unconjugated hyperbilirubinemia related to diminished hepatic UGT1A1 expression. Zinc sulfate enhanced fecal UCB excretion and, moderately (12%-18%), decreased plasma UCB concentrations in these patients.[106] Vitek et al. administered zinc sulfate and zinc methacrylate to Gunn rats, after demonstrating that both zinc salts could precipitate with UCB in vitro. Both zinc sulfate (-36%) and zinc methacrylate (-26%) treatment decreased plasma UCB concentrations.[108] In all three studies zinc sulfate, but not zinc methacrylate, administration increased plasma zinc levels.[106-108] Because elevated plasma zinc levels are associated with diarrhea, vomiting and, eventually, anemia, zinc methacrylate would be the agent of choice for future clinical studies involving zinc salts.[109]

Orlistat / Fat

Gollan et al. had shown in the 1970s that lipid withdrawal from the diet aggravated unconjugated hyperbilirubinemia in Gunn rats and in patients with Gilbert’s disease.[110,111] The underlying mechanism, however, remained unknown. In 2002, Verkade hypothesized that fat, due to its ability to form a hydrophobic association with the pigment, could act as an intestinal trapping agent for UCB (Fig. 4B).[112] In that same year, McDonagh demonstrated in vitro that UCB admixed in buffer completely partitions into an oil phase upon vigorous shaking.[113] In accordance with Verkade’s hypothesis, Nishioka et al. showed

Treatments for unconjugated hyperbilirubinemia 57

that treatment with the lipase inhibitor orlistat increased the fecal excretion of both fat and UCB in Gunn rats, while decreasing their plasma UCB levels by ~40%.[114] Orlistat was used because it decreases the intestinal hydrolysis, and thereby the subsequent absorption, of dietary triglycerides.[114] The resulting increase in fecal fat excretion was strongly, negatively, correlated with plasma UCB levels.[115,116] Orlistat treatment was as effective as phototherapy for reducing plasma UCB levels in Gunn rats, and a combination of orlistat and phototherapy was superior to either treatment alone.[116] In the same study, Hafkamp et al. showed that switching Gunn rats from a low-fat to a high-fat diet, increased fecal fat excretion, and decreased plasma UCB levels by 46%. Using steady-state 3H-UCB kinetics, Hafkamp et al. demonstrated that orlistat treatment induced net transmucosal excretion of UCB into the intestinal lumen, compatible with the hypothesis that fat could act as an intestinal trapping agent for UCB.[117] Still, the presently available data do not clarify whether UCB actually associates with unabsorbed fat and if so, with which class of unabsorbed fat (e.g. partially hydrolyzed triglycerides, fatty acids, phospholipids). In vitro experiments will be needed to characterize the exact mechanism.[117]

The effects of orlistat treatment of Crigler-Najjar patients were less pronounced than observed in Gunn rats. Hafkamp et al. conducted a randomized placebo-controlled double blind cross-over trial in 16 Crigler-Najjar patients.[117] Orlistat was tested during 4-6 weeks as an adjunct treatment to their regular phototherapy and/or phenobarbital regimen. Orlistat treatment decreased plasma UCB concentrations in the whole group by 9%, but the magnitude of the effect was not considered clinically relevant. Interestingly, orlistat treatment decreased plasma UCB by 21% in a subgroup of patients. This clinically relevant response to orlistat treatment appeared to correlate with a relatively low dietary fat intake.[118] It was suggested that apart from dietary fat intake, the responsiveness of Crigler-Najjar patients to orlistat treatment was probably determined by several other factors, which may include gastrointestinal lipase activity levels and the bacterial flora of the patients. Until these factors have been identified, orlistat cannot be recommended as a routine adjunct to conventional treatment for Crigler-Najjar disease. Although some patients reported mild gastrointestinal discomfort, no serious adverse effects were observed in the animal and patient studies. However, orlistat treatment decreased vitamin E levels in Crigler-Najjar patients, indicating that vitamin E supplementation would be required with long-term treatment.

Bile salts

Bile salts are the major organic constituents of the bile. Bile salt administration could lower plasma UCB concentrations for several reasons. First, bile salt administration increased the biliary disposal of organic anions, including UCB, in

58 Chapter 2

rats.[119,120] Secondly, Einarsson et al. showed that treatment in healthy volunteers with the bile salt ursodeoxycholic acid (UDCA) decreased the expiration of 14CO2 from triolein and suggested that UDCA therapy mildly decreased the absorption of fat.[121] This mild fat malabsorption may well decrease plasma bilirubin levels in a similar fashion as orlistat treatment. Finally, Rege and Ostrow showed in vitro that bile salts associate with UCB. Bile salt administration could therefore capture UCB in the intestinal lumen and enhance the fecal excretion of UCB-bile salt complexes (Fig. 4B)[122,123]. The treatment of unconjugated hyperbilirubinemia with bile salts is discussed in chapter 3.

Accelerating intestinal transit

Caloric deprivation, which often occurs in neonates during the initiation of breast-feeding,[4] is associated with unconjugated hyperbilirubinemia.[7] Kotal et al. demonstrated in fasted Gunn rats that this unconjugated hyperbilirubinemia was due to a delayed intestinal transit of stools.[3] This delay resulted in intestinal UCB accumulation and enhanced enterohepatic circulation of UCB. Kotal et al. also showed that feeding non-absorbable bulk to the fasted Gunn rats normalized intestinal transit time and prevented the increase in plasma UCB concentrations.[3] Kotal’s observations are supported by the fact that clinical causes of a delayed intestinal transit, such as a delayed passage of meconium,[72] pyloric stenosis,[124] and Hirschprung’s disease,[125] are also associated with unconjugated hyperbilirubinemia.

Based on these observations, it can be hypothesized that accelerating the intestinal transit time decreases plasma UCB concentrations, presumably by decreasing UCB reabsorption (Fig. 4C). This hypothesis is supported by several publications. In 1966 Wennberg et al. and in 1967 Wu et al. showed that neonates fed within 2-6 hours after birth passed meconium earlier, better maintained their body weights, and had lower plasma bilirubin levels compared with infants initially fed within 24-36 hours after birth.[126,127] In 1982, De Carvalho et al. showed that frequent breast feedings were associated with lower plasma UCB levels.[128] In 1984, Cottrell et al. showed that accelerating meconium passage with rectal stimulation also decreased plasma UCB concentrations.[129] Frequent and early feedings of neonates thus seem to be effective in lowering plasma UCB concentrations by enhancing its fecal elimination. In this thesis we attempted to lower plasma bilirubin in Gunn rats by accelerating the gastrointestinal transit time. The results of these experiments are discussed in chapter 4.

Treatments for unconjugated hyperbilirubinemia 59

2.3.4 Other pharmacological interventions Increasing UCB oxidation

Oxidation converts the hydrophobic UCB into more polar derivatives that can be excreted into the bile without conjugation. When conjugation of UCB is deficient, as in Crigler-Najjar disease or in the Gunn rat model, oxidation can thus serve as an alternate route for UCB excretion.[130,131] The microsomal mixed-function oxidases CYP1A1 and CYP1A2, which catalyze UCB oxidation, were indeed found to be markedly upregulated in the liver of young Gunn rats.[132] Bilirubin can also be oxidized by the constitutive, non-inducible enzyme bilirubin oxidase, found in the mitochondria of the liver, intestine, and kidney of guinea pigs and rats.[133-135]

In 1978, Kapitulnik et al. treated Gunn rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent inducer of microsomal P450-1A1. Although this treatment reduced plasma UCB levels by 60% in Gunn rats, clinical treatment with TCDD was not possible due to its toxicity.[136] Jorritsma et al. showed that induction of CYP1A1 with the natural compound indole-3-carbinol, which is abundant in cruciferous vegetables, decreased plasma UCB levels up to 21% in Gunn rats.[137] The safety of long-term administration of this inexpensive compound to humans has been demonstrated in trials of its prevention of breast cancer in women, but its effects in human neonates are not known. Exogenous bilirubin oxidase from plants has been studied in several publications. In one study, the passage of human or rat blood through an extracorporeal filter containing immobilized bilirubin oxidase degraded more than 90% of the bilirubin in a single pass.[138] In another study, a single dose of bilirubin oxidase, coupled to polyethylene glycol in order to increase its half-life in the blood, was administered intravenously to Gunn rats. This resulted in normalization of plasma UCB levels for a period of 12-48 hours.[139] Although bilirubin oxidase treatment was not associated with adverse effects in animals, it has not yet been applied in clinical trials, human neonates, or Crigler-Najjar patients.

2.4 Future perspectives Conventional treatment for unconjugated hyperbilirubinemia involves phototherapy and exchange transfusion. These treatment options have several disadvantages and are not always available in developing countries. Various pharmacological alternatives have been evaluated. Ideally, an alternative pharmacological intervention should be less invasive, simpler, and at least as effective and safe as phototherapy. However, many of the experimental therapies presently available do not (yet) meet these criteria. Phototherapy currently remains the preferred treatment strategy for unconjugated hyperbilirubinemia.

60 Chapter 2

Nevertheless, several treatment options, such as metalloporhyrins, clofibrate, bile salts, laxatives, indole-3-carbinol, and bilirubin oxidase deserve further investigation to determine their clinical applicability in the near future. Because the harm from unconjugated hyperbilirubinemia is due to its deposition in the central nervous system, future pharmacological treatment strategies should aim more directly at preventing neurological damage. One possible strategy could involve the induction of the ATP-Binding Cassette (ABC) proteins MRP1 and MDR1, which protect the central nervous system by exporting UCB across the blood-brain barrier.[39,140-144] Another strategy could involve the prevention of UCB-induced apoptosis by enhancement of the neurocellular (anti-caspase) defense mechanism against unconjugated hyperbilirubinemia.[145,146] A focus on neurological damage, rather than plasma bilirubin levels, could induce a paradigm shift in the development of new treatment strategies for unconjugated hyperbilirubinemia.

Treatments for unconjugated hyperbilirubinemia 61

References 1. Maisels MJ, Kring E. The

contribution of hemolysis to early jaundice in normal newborns. Pediatrics. 2006;118:276–279.

2. Kawade N, Onishi S. The prenatal and postnatal development of UDP-glucuronyltransferase activity towards bilirubin and the effect of premature birth on this activity in the human liver. Biochem. J. 1981;196:257–260.

3. Kotal P, Vitek L, Fevery J. Fasting-related hyperbilirubinemia in rats: the effect of decreased intestinal motility. Gastroenterology. 1996;111:217–223.

4. Bertini G, Dani C, Tronchin M, Rubaltelli FF. Is breastfeeding really favoring early neonatal jaundice? Pediatrics. 2001;107:41–45.

5. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science. 1987;235:1043–1046.

6. Crigler JF, Najjar VA. Congenital familial nonhemolytic jaundice with kernicterus. Pediatrics. 1952;10:169–180.

7. Gilbert A, Herscher M. Sur les variations de la cholemie physiologique. Presse Med. 1906;14:209–211.

8. Shapiro SM. Definition of the clinical spectrum of kernicterus and bilirubin-induced neurologic dysfunction (BIND). J Perinatol. 2005;25:54–59.

9. Ostrow JD. Photocatabolism of labeled bilirubin in the congenitally jaundiced (Gunn) rat. J. Clin. Invest. 1971;50:707–718.

10. Van der Veere CN, Sinaasappel M, McDonagh AF, Rosenthal P, Labrune P, Odievre M, et al. Current therapy

for Crigler-Najjar syndrome type 1: report of a world registry. Hepatology. 1996;24:311–315.

11. Yohannan MD, Terry HJ, Littlewood JM. Long term phototherapy in Crigler-Najjar syndrome. Arch Dis Child. 1983;58:460–462.

12. Keenan WJ, Novak KK, Sutherland JM, Bryla DA, Fetterly KL. Morbidity and mortality associated with exchange transfusion. Pediatrics. 1985;75:417–421.

13. Dennery PA. Metalloporphyrins for the treatment of neonatal jaundice. Curr. Opin. Pediatr. 2005;17:167–169.

14. McDonagh AF, Palma LA, Schmid R. Reduction of biliverdin and placental transfer of bilirubin and biliverdin in the pregnant guinea pig. Biochem. J. 1981;194:273–282.

15. Kappas A, Simionatto CS, Drummond GS, Sassa S, Anderson KE. The liver excretes large amounts of heme into bile when heme oxygenase is inhibited competitively by Sn-protoporphyrin. Proc. Natl. Acad. Sci. U.S.A. 1985;82:896–900.

16. Stevenson DK, Rodgers PA, Vreman HJ. The use of metalloporphyrins for the chemoprevention of neonatal jaundice. Am J Dis Child. 1989;143:353–356.

17. Maines MD, Kappas A. Enzymatic oxidation of cobalt protoporphyrin IX: observations on the mechanism of heme oxygenase action. Biochemistry. 1977;16:419–423.

18. Drummond GS, Kappas A. Chemoprevention of severe neonatal hyperbilirubinemia. Semin Perinatol. 2004;28:365–368.

62 Chapter 2

19. Fang J, Sawa T, Akaike T, Akuta T, Sahoo SK, Khaled G, et al. In vivo antitumor activity of pegylated zinc protoporphyrin: targeted inhibition of heme oxygenase in solid tumor. Cancer Res. 2003;63:3567–3574.

20. Akins RJ, McLaughlin T, Boyce R, Gilmour L, Gratton K. Exogenous metalloporphyrins alter the organization and function of cultured neonatal rat heart cells via modulation of heme oxygenase activity. J Cell Physiol. 2004;201:26–34.

21. Park RM, Bena JF, Stayner LT, Smith RJ, Gibb HJ, Lees PS. Hexavalent chromium and lung cancer in the chromate industry: a quantitative risk assessment. Risk Anal. 2004;24:1099–1108.

22. Beri R, Chandra R. Chemistry and biology of heme. Effect of metal salts, organometals, and metalloporphyrins on heme synthesis and catabolism, with special reference to clinical implications and interactions with cytochrome P-450. Drug Metab Rev. 1993;25:49–152.

23. Kappas A, Drummond GS, Manola T, Petmezaki S, Valaes T. Sn-protoporphyrin use in the management of hyperbilirubinemia in term newborns with direct Coombs-positive ABO incompatibility. Pediatrics. 1988;81:485–497.

24. Valaes T, Petmezaki S, Henschke C, Drummond GS, Kappas A. Control of jaundice in preterm newborns by an inhibitor of bilirubin production: studies with tin-mesoporphyrin. Pediatrics. 1994;93:1–11.

25. Kappas A, Drummond GS, Henschke C, Valaes T. Direct comparison of Sn-mesoporphyrin, an inhibitor of bilirubin production, and phototherapy in controlling hyperbilirubinemia in term and near-term newborns. Pediatrics. 1995;95:468–474.

26. Valaes T, Drummond GS, Kappas A. Control of hyperbilirubinemia in glucose-6-phosphate dehydrogenase-deficient newborns using an inhibitor of bilirubin production, Sn-mesoporphyrin. Pediatrics. 1998;101:E1.

27. Martinez JC, Garcia HO, Otheguy LE, Drummond GS, Kappas A. Control of severe hyperbilirubinemia in full-term newborns with the inhibitor of bilirubin production Sn-mesoporphyrin. Pediatrics. 1999;103:1–5.

28. Rubaltelli FF, Guerrini P, Reddi E, Jori G. Tin-protoporphyrin in the management of children with Crigler-Najjar disease. Pediatrics. 1989;84:728–731.

29. Galbraith RA, Drummond GS, Kappas A. Suppression of bilirubin production in the Crigler-Najjar type I syndrome: studies with the heme oxygenase inhibitor tin-mesoporphyrin. Pediatrics. 1992;89:175–182.

30. Suresh GK, Martin CL, Soll RF. Metalloporphyrins for treatment of unconjugated hyperbilirubinemia in neonates. Cochrane.Database.Syst.Rev. 2003;:CD004207.

31. Dani C, Masini E, Bertini G, di Felice AM, Pezzati M, Ciofini S, et al. Role of heme oxygenase and bilirubin in oxidative stress in preterm infants. Pediatr. Res. 2004;56:873–877.

32. Willis D, Moore AR, Frederick R, Willoughby DA. Heme oxygenase: a novel target for the modulation of the inflammatory response. Nat Med. 1996;2:87–90.

33. Oroszlan G, Lakatos L, Szabo L, Matkovics B, Karmazsin L. Heme oxygenase activity is decreased by D-penicillamine in neonates. Experientia. 1983;39:888–889.

Treatments for unconjugated hyperbilirubinemia 63

34. Lakatos L, Kover B, Oroszlan G, Vekerdy Z. D-penicillamine therapy in ABO hemolytic disease of the newborn infant. Eur J Pediatr. 1976;123:133–137.

35. Munro R, Capell HA. Penicillamine. Br J Rheumatol. 1997;36:104–109.

36. Levi AJ, Gatmaitan Z, Arias IM. Two hepatic cytoplasmic protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin, sulfobromophthalein, and other anions. J. Clin. Invest. 1969;48:2156–2167.

37. Habig WH, Pabst MJ, Fleischner G, Gatmaitan Z, Arias IM, Jakoby WB. The identity of glutathione S-transferase B with ligandin, a major binding protein of liver. Proc. Natl. Acad. Sci. U.S.A. 1974;71:3879–3882.

38. Wolkoff AW, Goresky CA, Sellin J, Gatmaitan Z, Arias IM. Role of ligandin in transfer of bilirubin from plasma into liver. Am J Physiol. 1979;236:E638–48.

39. Paulusma CC, Bosma PJ, Zaman GJ, Bakker CT, Otter M, Scheffer GL, et al. Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene. Science. 1996;271:1126–1128.

40. Wagner M, Halilbasic E, Marschall HU, Zollner G, Fickert P, Langner C, et al. CAR and PXR agonists stimulate hepatic bile acid and bilirubin detoxification and elimination pathways in mice. Hepatology. 2005;42:420–430.

41. Yaffe SJ, Levy G, Matsuzawa T, Baliah T. Enhancement of glucuronide-conjugating capacity in a hyperbilirubinemic infant due to apparent enzyme induction by phenobarbital. N. Engl. J. Med. 1966;275:1461.

42. Catz C, Yaffe SJ. Barbiturate enhancement of bilirubin conjugation and excretion in young and adult animals. Pediatr. Res. 1968;2:361.

43. Valaes TN, Harvey-Wilkes K. Pharmacologic approaches to the prevention and treatment of neonatal hyperbilirubinemia. Clin Perinatol. 1990;17:245–273.

44. Valaes T, Kipouros K, Petmezaki S, Solman M, Doxiadis SA. Effectiveness and safety of prenatal phenobarbital for the prevention of neonatal jaundice. Pediatr. Res. 1980;14:947–952.

45. Thomas JT, Muller P, Wilkinson C. Antenatal phenobarbital for reducing neonatal jaundice after red cell isoimmunization. Cochrane.Database.Syst.Rev. 2007;:CD005541.

46. Valdes OS, Maurer HM, Shumway CN, Draper DA, Hossaini AA. Controlled clinical trial of phenobarbital and-or light in reducing neonatal hyperbilirubinemia in a predominantly Negro population. J. Pediatr. 1971;79:1015–1017.

47. Hansen TW, Tommarello S. Effect of phenobarbital on bilirubin metabolism in rat brain. Biol Neonate. 1998;73:106–111.

48. Sinaasappel M, Jansen PL. The differential diagnosis of Crigler-Najjar disease, types 1 and 2, by bile pigment analysis. Gastroenterology. 1991;100:783–789.

49. Despres JP, Lemieux I, Robins SJ. Role of fibric acid derivatives in the management of risk factors for coronary heart disease. Drugs. 2004;64:2177–2198.

50. Foliot A, Drocourt JL, Etienne JP, Housset E, Fiessinger JN, Christoforov B. Increase in the hepatic glucuronidation and clearance of

64 Chapter 2

bilirubin in clofibrate-treated rats. Biochem. Pharmacol. 1977;26:547–549.

51. Jemnitz K, Lengyel G, Vereczkey L. In vitro induction of bilirubin conjugation in primary rat hepatocyte culture. Biochem Biophys Res Commun. 2002;291:29–33.

52. Jean F, Foliot A, Celier C, Housset E, Etienne JP. Influence of clofibrate on hepatic transport of bilirubin and bromosulfophthalein in rats. Biochem Biophys Res Commun. 1979;86:1154–1160.

53. Lindenbaum A, Hernandorena X, Vial M, Benattar C, Janaud JC, Dehan M, et al. [Clofibrate for the treatment of hyperbilirubinemia in neonates born at term: a double blind controlled study (author's transl)]. Arch. Fr. Pediatr. 1981;38 Suppl 1:867–873.

54. Lindenbaum A, Delaporte B, Benattar C, Dehan M, Magny JF, Gerbet D, et al. [Preventive treatment of jaundice in premature newborn infants with clofibrate. Double-blind controlled therapeutic trial]. Arch. Fr. Pediatr. 1985;42:759–763.

55. Mohammadzadeh A, Farhat A, Iranpour R. Effect of clofibrate in jaundiced term newborns. Indian J Pediatr. 2005;72:123–126.

56. Eghbalian F, Pourhossein A, Zandevakili H. Effect of clofibrate in non-hemolytic indirect hyperbiliru-binemia in full term neonates. Indian J Pediatr. 2007;74:1003–1006.

57. Zahedpasha Y, Ahmadpour-Kacho M, Hajiahmadi M, Naderi S. Effect of clofibrate in jaundiced full-term infants:a randomized clinical trial. Arch Iran Med. 2007;10:349–353.

58. Reddy JK, Qureshi SA. Tumorigenicity of the hypolipidaemic peroxisome proliferator ethyl-alpha-p-

chlorophenoxyisobutyrate (clofibrate) in rats. Br J Cancer. 1979;40:476–482.

59. WHO cooperative trial on primary prevention of ischaemic heart disease with clofibrate to lower serum cholesterol: final mortality follow-up. Report of the Committee of Principal Investigators. Lancet. 1984;324:600–604.

60. Erkul I, Yavuz H, Ozel A. Clofibrate treatment of neonatal jaundice. Pediatrics. 1991;88:1292–1294.

61. Steiner A, Weisser B, Vetter W. A comparative review of the adverse effects of treatments for hyperlipidaemia. Drug Saf. 1991;6:118–130.

62. Rush P, Baron M, Kapusta M. Clofibrate myopathy: a case report and a review of the literature. Semin Arthritis Rheum. 1986;15:226–229.

63. Fok TF. Neonatal jaundice--traditional Chinese medicine approach. J Perinatol. 2001;21 Suppl 1:S98–S100; discussion S104–7.

64. Yin J, Miller M, Wennberg RP. Induction of hepatic bilirubin-metabolizing enzymes by the traditional Chinese medicine yin zhi huang. Dev Pharmacol Ther. 1991;16:176–184.

65. Huang W, Zhang J, Moore DD. A traditional herbal medicine enhances bilirubin clearance by activating the nuclear receptor CAR. J. Clin. Invest. 2004;113:137–143.

66. Yeung CY, Leung CS, Chen YZ. An old traditional herbal remedy for neonatal jaundice with a newly identified risk. J Paediatr Child Health. 1993;29:292–294.

67. Chan TY. The prevalence use and harmful potential of some Chinese herbal medicines in babies and children. Vet Hum Toxicol.

Treatments for unconjugated hyperbilirubinemia 65

1994;36:238–240.

68. Lester R, Schmid R. Intestinal absorption of bile pigments. I. The enterohepatic circulation of bilirubin in the rat. J. Clin. Invest. 1963;42:736–746.

69. Lester R, Schmid R. Intestinal absorption of bile pigments. II. Bilirubin absorption in man. N. Engl. J. Med. 1963;269:178–182.

70. Vitek L, Kotal P, Jirsa M, Malina J, Cerna M, Chmelar D, et al. Intestinal colonization leading to fecal urobilinoid excretion may play a role in the pathogenesis of neonatal jaundice. J Pediatr Gastroenterol Nutr. 2000;30:294–298.

71. Zucker SD, Goessling W, Hoppin AG. Unconjugated bilirubin exhibits spontaneous diffusion through model lipid bilayers and native hepatocyte membranes. J. Biol. Chem. 1999;274:10852–10862.

72. Rosta J, Makoi Z, Kertesz A. Delayed meconium passage and hyperbilirubinaemia. Lancet. 1968;292:1138.

73. Tiribelli C, Ostrow JD. Intestinal flora and bilirubin. J. Hepatol. 2005;42:170.

74. Kotal P, Van der Veere CN, Sinaasappel M, Elferink RO, Vitek L, Brodanova M, et al. Intestinal excretion of unconjugated bilirubin in man and rats with inherited unconjugated hyperbilirubinemia. Pediatr. Res. 1997;42:195–200.

75. Schmid R, Hammaker L. Metabolism and disposition of C14-bilirubin in congenital nonhemolytic jaundice. J. Clin. Invest. 1963;42:1720–1734.

76. Lester R, Hammaker L, Schmid R. A new therapeutic approach to unconjugated hyperbilirubinaemia.

Lancet. 1962;280:1257.

77. Schmid R, Forbes A, Rosenthal IM, Lester R. Lack of Effect of Cholestyramine Resin on Hyperbilirubinaemia of Premature Infants. Lancet. 1963;282:938–939.

78. Arrowsmith WA, Payne RB, Littlewood JM. Comparison of treatments for congenital nonobstructive nonhaemolytic hyperbilirubinaemia. Arch Dis Child. 1975;50:197–201.

79. Nicolopoulos D, Hadjigeorgiou E, Malamitsi A, Kalpoyannis N, Karli I, Papadakis D. Combined treatment of neonatal jaundice with cholestyramine and phototherapy. J. Pediatr. 1978;93:684–688.

80. Malamitsi-Puchner A, Hadjigeorgiou E, Papadakis D, Kalpoyannis N, Nicolopoulos D. Combined treatment of neonatal jaundice with phototherapy, cholestyramine, and bicarbonate. J. Pediatr. 1981;99:324–325.

81. Tan KL, Jacob E, Liew DS, Karim SM. Cholestyramine and phototherapy for neonatal jaundice. J. Pediatr. 1984;104:284–286.

82. Ulstrom RA, Eisenklam E. The Enterohepatic Shunting of Bilirubin in the Newborn Infant. I. Use of Oral Activated Charcoal to Reduce Normal Serum Bilirubin Values. J. Pediatr. 1964;65:27–37.

83. Kuenzer W, Vahlenkamp H, Jarre W, Fuss W. [on the Treatment of Jaundice in the Newborn with Charcoal.]. Ann Paediatr. 1964;203:247–255.

84. Davis DR, Yeary RA. Activated charcoal as an adjunct to phototherapy for neonatal jaundice. Dev Pharmacol Ther. 1987;10:12–20.

85. Davis DR, Yeary RA, Lee K.

66 Chapter 2

Improved embryonic survival in the jaundiced female rat fed activated charcoal. Pediatr Pharmacol (New York). 1983;3:79–85.

86. Davis DR, Yeary RA, Lee K. Activated charcoal decreases plasma bilirubin levels in the hyperbilirubinemic rat. Pediatr. Res. 1983;17:208–209.

87. Amitai Y, Regev M, Arad I, Peleg O, Boehnert M. Treatment of neonatal hyperbilirubinemia with repetitive oral activated charcoal as an adjunct to phototherapy. J Perinat Med. 1993;21:189–194.

88. James LP, Nichols MH, King WD. A comparison of cathartics in pediatric ingestions. Pediatrics. 1995;96:235–238.

89. Poland RL, Odell GB. Physiologic jaundice: the enterohepatic circulation of bilirubin. N. Engl. J. Med. 1971;284:1–6.

90. Poland RL, Odell GB. The binding of bilirubin to agar. Proc Soc Exp Biol Med. 1974;146:1114–1118.

91. Odell GB, Bolen JL, Poland RL, Seungdambong S, Cukier JO. Protection from bilirubin nephropathy in jaundiced Gunn rats. Gastroenterology. 1974;66:1218–1224.

92. Odell GB, Gutcher GR, Whitington PF, Yang G. Enteral administration of agar as an effective adjunct to phototherapy of neonatal hyperbilirubinemia. Pediatr. Res. 1983;17:810–814.

93. Caglayan S, Candemir H, Aksit S, Kansoy S, Asik S, Yaprak I. Superiority of oral agar and phototherapy combination in the treatment of neonatal hyperbilirubinemia. Pediatrics. 1993;92:86–89.

94. Maurer HM, Shumway CN, Draper DA, Hossaini AA. Controlled trial comparing agar, intermittent phototherapy, and continuous phototherapy for reducing neonatal hyperbilirubinemia. J. Pediatr. 1973;82:73–76.

95. Romagnoli C, Polidori G, Foschini M, Cataldi L, De Turris P, Tortorolo G, et al. Agar in the management of hyperbilirubinaemia in the premature baby. Arch Dis Child. 1975;50:202–204.

96. Windorfer AJ, Kunzer W, Bolze H, Ascher K, Wilcken F, Hoehne K. Studies on the effect of orally administered agar on the serum bilirubin level of premature infants and mature newborns. Acta Paediatr Scand. 1975;64:699–702.

97. Ebbesen F, Moller J. Agar ingestion combined with phototherapy in jaundiced newborn infants. Biol Neonate. 1977;31:7–9.

98. Blum D, Etienne J. Agar in control of hyperbilirubinemia. J. Pediatr. 1973;83:345.

99. Moller J. Agar ingestion and serum bilirubin values in newborn infants. Acta Obstet Gynecol Scand Suppl. 1974;29:61–63.

100. Kemper K, Horwitz RI, McCarthy P. Decreased neonatal serum bilirubin with plain agar: a meta-analysis. Pediatrics. 1988;82:631–638.

101. Moore EW. Biliary calcium and gallstone formation. Hepatology. 1990;12:206S–214S; discussion 214S–218S.

102. Ostrow JD. Unconjugated bilirubin and cholesterol gallstone formation. Hepatology. 1990;12:219S–224S; discussion 224S–226S.

103. Van der Veere CN, Schoemaker B, Van Der Meer R, Groen AK, Jansen

Treatments for unconjugated hyperbilirubinemia 67

PL, Oude Elferink RP. Rapid association of unconjugated bilirubin with amorphous calcium phosphate. J. Lipid Res. 1995;36:1697–1707.

104. Van der Veere CN, Schoemaker B, Bakker C, Van Der Meer R, Jansen PL, Elferink RP. Influence of dietary calcium phosphate on the disposition of bilirubin in rats with unconjugated hyperbilirubinemia. Hepatology. 1996;24:620–626.

105. Van der Veere CN, Jansen PL, Sinaasappel M, Van Der Meer R, Van der Sijs H, Rammeloo JA, et al. Oral calcium phosphate: a new therapy for Crigler-Najjar disease? Gastroenterology. 1997;112:455–462.

106. Mendez-Sanchez N, Martinez M, Gonzalez V, Roldan-Valadez E, Flores MA, Uribe M. Zinc sulfate inhibits the enterohepatic cycling of unconjugated bilirubin in subjects with Gilbert's syndrome. Ann Hepatol. 2002;1:40–43.

107. Mendez-Sanchez N, Roldan-Valadez E, Flores MA, Cardenas-Vazquez R, Uribe M. Zinc salts precipitate unconjugated bilirubin in vitro and inhibit enterohepatic cycling of bilirubin in hamsters. Eur J Clin Invest. 2001;31:773–780.

108. Vitek L, Muchova L, Zelenka J, Zadinova M, Malina J. The effect of zinc salts on serum bilirubin levels in hyperbilirubinemic rats. J Pediatr Gastroenterol Nutr. 2005;40:135–140.

109. Roney N, Osier M, Paikoff SJ, Smith CV, Williams M, De Rosa CT. ATSDR evaluation of the health effects of zinc and relevance to public health. Toxicol Ind Health. 2006;22:423–493.

110. Gollan JL, Bateman C, Billing BH. Effect of dietary composition on the unconjugated hyperbilirubinaemia of Gilbert's syndrome. Gut. 1976;17:335–340.

111. Gollan JL, Hatt KJ, Billing BH. The influence of diet on unconjugated hyperbilirubinaemia in the Gunn rat. Clin Sci Mol Med. 1975;49:229–235.

112. Verkade HJ. A novel hypothesis on the pathophysiology of neonatal jaundice. J. Pediatr. 2002;141:594–595.

113. McDonagh AF. Lyophilic properties of protoporphyrin and bilirubin. Hepatology. 2002;36:1028–1029.

114. Guerciolini R. Mode of action of orlistat. Int J Obes Relat Metab Disord. 1997;21 Suppl 3:S12–23.

115. Nishioka T, Hafkamp AM, Havinga R, vn Lierop PP, Velvis H, Verkade HJ. Orlistat treatment increases fecal bilirubin excretion and decreases plasma bilirubin concentrations in hyperbilirubinemic Gunn rats. J. Pediatr. 2003;143:327–334.

116. Hafkamp AM, Havinga R, Sinaasappel M, Verkade HJ. Effective oral treatment of unconjugated hyperbilirubinemia in Gunn rats. Hepatology. 2005;41:526–534.

117. Hafkamp AM, Havinga R, Ostrow JD, Tiribelli C, Pascolo L, Sinaasappel M, et al. Novel kinetic insights into treatment of unconjugated hyperbilirubinemia: phototherapy and orlistat treatment in Gunn rats. Pediatr. Res. 2006;59:506–512.

118. Hafkamp AM, Nelisse-Haak R, Sinaasappel M, Oude Elferink RPJ, Verkade HJ. Orlistat treatment of unconjugated hyperbilirubinemia in Crigler-Najjar disease: a randomized controlled trial. Pediatr. Res. 2007;62:725–730.

119. Vonk RJ, Jekel P, Meijer DK. Choleresis and hepatic transport mechanisms. II. Influence of bile salt choleresis and biliary micelle binding on biliary excretion of various organic anions. Naunyn Schmiedebergs Arch

68 Chapter 2

Pharmacol. 1975;290:375–387.

120. Ostrow JD. Regulation by bile salts of the excretion of conjugated and unconjugated bilirubin in bile. In: Fromm H, Leuschner U, eds. Proceedings of the falk symposium No. 84, held in Berlin, Germany, June 9-10, 1995. Kluwer Academic Publishers, 1996.

121. Einarsson K, Bjorkhem I, Eklof R, Ewerth S, Nilsell K, Blomstrand R. Effect of ursodeoxycholic acid treatment on intestinal absorption of triglycerides in man. Scand. J. Gastroenterol. 1984;19:283–288.

122. Ostrow JD, Celic L, Mukerjee P. Molecular and micellar associations in the pH-dependent stable and metastable dissolution of unconjugated bilirubin by bile salts. J. Lipid Res. 1988;29:335–348.

123. Rege RV, Webster CC, Ostrow JD. Interactions of unconjugated bilirubin with bile salts. J. Lipid Res. 1988;29:1289–1296.

124. Etzioni A, Shoshani G, Diamond E, Zinder O, Bar-Maor JA. Unconjugated hyperbilirubinaemia in hypertrophic pyloric stenosis, an enigma. Z Kinderchir. 1986;41:272–274.

125. Gartner LM. Breastfeeding and jaundice. J Perinatol. 2001;21 Suppl 1:S25–9; discussion S35–9.

126. Wennberg RP, Schwartz R, Sweet AY. Early versus delayed feeding of low birth weight infants: effects on physiologic jaundice. J. Pediatr. 1966;68:860–866.

127. Wu PY, Teilmann P, Gabler M, Vaughan M, Metcoff J. "Early" versus “late” feeding of low birth weight neonates: effect on serum bilirubin, blood sugar, and responses to glucagon and epinephrine tolerance tests. Pediatrics. 1967;39:733–739.

128. De Carvalho M, Klaus MH, Merkatz RB. Frequency of breast-feeding and serum bilirubin concentration. Am J Dis Child. 1982;136:737–738.

129. Cottrell BH, Anderson GC. Rectal or axillary temperature measurement: effect on plasma bilirubin and intestinal transit of meconium. J Pediatr Gastroenterol Nutr. 1984;3:734–739.

130. Berry CS, Zarembo JE, Ostrow JD. Evidence for conversion of bilirubin to dihydroxyl derivatives in the gunn rat. Biochem Biophys Res Commun. 1972;49:1366–1375.

131. Blanckaert N, Fevery J, Heirwegh KP, Compernolle F. Characterization of the major diazo-positive pigments in bile of homozygous Gunn rats. Biochem. J. 1977;164:237–249.

132. Kapitulnik J, Gonzalez FJ. Marked endogenous activation of the CYP1A1 and CYP1A2 genes in the congenitally jaundiced Gunn rat. Mol.Pharmacol. 1993;43:722–725.

133. Cardenas-Vazquez R, Yokosuka O, Billing BH. Enzymic oxidation of unconjugated bilirubin by rat liver. Biochem. J. 1986;236:625–633.

134. Brodersen R, Bartels P. Enzymatic oxidation of bilirubin. Eur J Biochem. 1969;10:468–473.

135. Yokosuka O, Billing B. Enzymatic oxidation of bilirubin by intestinal mucosa. Biochim Biophys Acta. 1987;923:268–274.

136. Kapitulnik J, Ostrow JD. Stimulation of bilirubin catabolism in jaundiced Gunn rats by an induced of microsomal mixed-function monooxygenases. Proc. Natl. Acad. Sci. U.S.A. 1978;75:682–685.

137. Jorritsma U, Schrader E, Klaunick G, Kapitulnik J, Hirsch-Ernst KI,

Treatments for unconjugated hyperbilirubinemia 69

Kahl GF, et al. Monitoring of cytochrome P-450 1A activity by determination of the urinary pattern of caffeine metabolites in Wistar and hyperbilirubinemic Gunn rats. Toxicology. 2000;144:229–236.

138. Lavin A, Sung C, Klibanov AM, Langer R. Enzymatic removal of bilirubin from blood: a potential treatment for neonatal jaundice. Science. 1985;230:543–545.

139. Sugi K, Inoue M, Morino Y. Degradation of plasma bilirubin by a bilirubin oxidase derivative which has a relatively long half-life in the circulation. Biochim Biophys Acta. 1989;991:405–409.

140. Watchko JF, Daood MJ, Hansen TW. Brain bilirubin content is increased in P-glycoprotein-deficient transgenic null mutant mice. Pediatr. Res. 1998;44:763–766.

141. Hankø E, Tommarello S, Watchko JF, Hansen TWR. Administration of drugs known to inhibit P-glycoprotein increases brain bilirubin and alters the regional distribution of bilirubin in rat brain. Pediatr. Res. 2003;54:441–445.

142. Rigato I, Pascolo L, Fernetti C, Ostrow JD, Tiribelli C. The human multidrug-resistance-associated

protein MRP1 mediates ATP-dependent transport of unconjugated bilirubin. Biochem. J. 2004;383:335–341.

143. Gennuso F, Fernetti C, Tirolo C, Testa N, L'Episcopo F, Caniglia S, et al. Bilirubin protects astrocytes from its own toxicity by inducing up-regulation and translocation of multidrug resistance-associated protein 1 (Mrp1). Proc. Natl. Acad. Sci. U.S.A. 2004;101:2470–2475.

144. Falcao AS, Bellarosa C, Fernandes A, Brito MA, Silva RF, Tiribelli C, et al. Role of multidrug resistance-associated protein 1 expression in the in vitro susceptibility of rat nerve cell to unconjugated bilirubin. Neuroscience. 2007;144:878–888.

145. Lin S, Wei X, Bales KR, Paul AB, Ma Z, Yan G, et al. Minocycline blocks bilirubin neurotoxicity and prevents hyperbilirubinemia-induced cerebellar hypoplasia in the Gunn rat. Eur J Neurosci. 2005;22:21–27.

146. Cesaratto L, Calligaris SD, Vascotto C, Deganuto M, Bellarosa C, Quadrifoglio F, et al. Bilirubin-induced cell toxicity involves PTEN activation through an APE1/Ref-1-dependent pathway. J Mol Med. 2007;85:1099–1112

70 Chapter 2

Chapter 3

Effective Treatment of Unconjugated Hyperbilirubinemia with Oral Bile Salts in Gunn Rats

F.J.C. Cuperus 1 A.M. Hafkamp 1

R.Havinga 1 L.Vitek 2,3

J. Zelenka 3 C. Tiribelli 4

J.D. Ostrow 5 H.J. Verkade 1

1Pediatric Gastroenterology and Hepatology, Department of Pediatrics, Center for Liver, Digestive, and Metabolic Diseases, Beatrix Children’s Hospital - University Medical Center Groningen, University of Groningen, The Netherlands. 24th Department of Internal Medicine, 1st Faculty of Medicine, Charles University, Prague, Czech Republic. 3Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University, Prague, Czech Republic. 4Centro Studi Fegato, AREA Science Park and Department of Life Sciences, University of Trieste, Trieste, Italy. 5Gastroenterology/Hepatology Division, University of Washington School of Medicine, Seattle, WA, USA.

Gastroenterology 2009;13:673-82

72 Chapter 3

3.1 Abstract e tested the hypothesis that oral administration of bile salts, which are known to increase the biliary excretion of unconjugated bilirubin (UCB), decreases unconjugated hyperbilirubinemia in the Gunn rat model. Adult Gunn rats were fed a standard diet or the same diet supplemented with 0.5 wt% ursodeoxycholic acid (UDCA) or cholic acid (CA) for 1 or 6 weeks. UCB and urobilinoids, a family of intestinal UCB breakdown products, were determined in plasma and/or feces. After 6 weeks of treatment, tracer 3H-UCB was administered i.v. to determine steady-state UCB kinetics over the next 60h. One-week treatment with UDCA or CA decreased plasma UCB concentrations by 21% and 30%, respectively (each p<0.01). During the first four days of treatment, both UDCA and CA increased the combined fecal excretion of UCB and urobilinoids (+52% and +32%, respectively; each p<0.01). Prolongation of treatment to 6 weeks caused a persistent decrease in plasma UCB concentrations to ∼40% below baseline (each bile salt p<0.001). 3H-UCB kinetic studies showed that UDCA and CA administration decreased UCB pool size (-33% and -32%, respectively; each p<0.05) and increased UCB fractional turnover (+33% and +25%, respectively; each p<0.05). This chapter demonstrates that dietary bile salt administration induces a large, persistent decrease in plasma UCB concentrations in Gunn rats. Both UDCA and CA enhance UCB turnover by increasing its fecal disposal. These results support the application of oral bile salt treatment in patients with unconjugated hyperbilirubinemia.

W

Bile salt treatment for unconjugated hyperbilirubinemia 73

3.2 Introduction nconjugated hyperbilirubinemia occurs in conditions such as neonatal hemolytic jaundice and Crigler-Najjar disease. Crigler-Najjar disease is characterized by a genetically absent (type I) or decreased (type II) capacity to conjugate bilirubin in the liver,[1] which is essential for efficient biliary excretion of the pigment. Impaired conjugation results in unconjugated hyperbilirubinemia, due to retention of unconjugated bilirubin (UCB) in the body. Severe unconjugated hyperbilirubinemia can lead to deposition of UCB in the central nervous system, causing bilirubin-induced neurological dysfunction (BIND), kernicterus, and death.[2] Unconjugated hyperbilirubinemia is conventionally treated by phototherapy, which induces photo-isomerisation of the hydrophobic UCB to polar isomers that can readily be excreted into the bile.[3] Although generally effective, phototherapy does not always decrease plasma UCB to non-toxic levels. Most importantly, long-term phototherapy, such as needed for patients with Crigler-Najjar disease type I, becomes less effective with age and has a profound impact on social life.[4,5] These considerations favor the development of effective alternative treatments for unconjugated hyperbilirubinemia.

During severe unconjugated hyperbilirubinemia, most UCB does not enter the intestinal lumen via biliary excretion, but rather via direct diffusion across the intestinal mucosa.[6,7] The efficiency of this pathway is decreased, however, by the ability of the intestine to reabsorb UCB from its lumen.[8,9] Several experimental therapies have aimed to prevent reabsorption by oral administration of agents that trap UCB in the intestinal lumen. However, trapping agents tested so far, including agar,[10] cholestyramine,[11] charcoal,[12] amorphous calcium phosphate,[13] zinc salts,[14] and orlistat,[15] have been clinically unsatisfactory, due to side-effects and inconsistent results.

Since bile salts can stimulate biliary excretion of organic anions,[16] including bilirubin in rats,[17] we reasoned that bile salt administration could be relevant for treatment of unconjugated hyperbilirubinemia. Ursodeoxycholic acid (UDCA) treatment in healthy volunteers decreased the expiration of 14CO2 from triolein, suggesting that UDCA also mildly decreases the absorption of fat.[18] Mild fat malabsorption induced by orlistat decreased plasma bilirubin levels in a subset of Crigler-Najjar patients and in homozygous Gunn rats, their well-established animal model.[15,19] Finally, bile salts associate with UCB in vitro, and bile salt administration could therefore also lower plasma UCB concentrations via enhancement of fecal excretion of UCB-bile salt complexes.[20,21]

In the present study we show that dietary administration of UDCA indeed reduces unconjugated hyperbilirubinemia in homozygous Gunn rats. We studied

U

74 Chapter 3

several dosages and administration periods to evaluate the clinical applicability of this potential treatment. We compared the UDCA effects in Gunn rats with those obtained after administration of cholic acid (CA) to evaluate whether effects were bile salt specific. Finally, we studied steady-state kinetics of i.v. administered 3H-UCB, to gain insight in the underlying mechanisms of both UDCA and CA administration.

3.3 Animals, materials, and methods

3.3.1 Animals Homozygous adult male Gunn Rats (RHA/jj, 240-360g), obtained from our breeding colony (University Medical Center Groningen, the Netherlands), were housed in an environmentally controlled facility and fed ad libitum. The Ethics Committee for Animal Experiments of the University of Groningen approved all experimental protocols.

3.3.2 Materials Chemicals

UDCA was a generous gift from Dr. Falk Pharma GmbH (Freiburg, Germany). CA and heptadecanoic acid (C17:0) were obtained from Sigma-Aldrich Co. (St. Louis, MO). Xanthobilirubin-methylester was a generous gift from Dr. J. Fevery (Leuven, Belgium). Urobilin was obtained from Frontier Scientific Inc. (Logan, UT). 3H-labeled UCB (specific activity 6.02 µCi/µmol) was prepared by biosynthetic labeling of 2,3-3H-labeled 5-aminolevulinic acid (specific activity 13 mCi/mmol; Amersham Biosciences, Piscataway, NJ).[22-24] 3H-labeled UCB solution was prepared immediately before injection into Gunn rats as described before.[23]

Diets

The semi-synthetic, purified control diet (code 4063.02) was produced by Hope Farms BV (Woerden, the Netherlands) and contained 13 energy% fat and 5.2 wt% long-chain fatty acids. Diets containing bile salts were identical except for supplementation with UDCA or CA (0.05% - 1.5% by chow weight).

Bile salt treatment for unconjugated hyperbilirubinemia 75

3.3.3 Methods Preliminary dose-response experiment

After a 6-week run-in period on the control diet, Gunn rats were randomly assigned to receive the control diet supplemented with either UDCA or CA (n=6 per group). All animals were housed and fed by dietary group for a period of 10 weeks, during which the dosage of UDCA and CA was increased every 2 weeks. Used dosages: 0.05%; 0.1%; 0.5%; 1%; and 1.5 wt% (by chow weight). Heparinized samples of tail vein blood were obtained under isoflurane anesthesia before and 2, 4, 6, 8, and 10 weeks after dietary randomization for determination of plasma UCB concentrations and, in the last plasma sample, aspartate aminotransferase (AST), alanine aminotransferase (ALT).

Short-term experiment

After a 6-week period on the control diet, individually housed Gunn rats were randomly assigned to receive the control diet or the same diet supplemented with UDCA or CA (each 0.5 wt%; n=6 per group). Food intake and animal weights were determined daily. Heparinized samples of tail vein blood were obtained under isoflurane anesthesia at day 0, 1, 3, 5, and 8 for determination of plasma UCB concentrations. Feces were collected every 24h for 4 days before and for 4 days after dietary randomization to determine fecal excretion of UCB, urobilinoids, and bile acids. Eight days after dietary randomization, the common bile duct was cannulated under pentobarbital anesthesia and bile was collected for 30 minutes under light-protected conditions. Bile flow was determined gravimetrically, assuming a density of 1 g/ml. A 1 ml blood sample was then obtained by puncture of the inferior vena cava to determine AST and ALT.

Long-term experiment

After 6 weeks on the control diet, individually housed Gunn rats were randomly assigned to receive either the control diet or the same diet supplemented with UDCA or CA (each 0.5 wt %; n=6 per group). Food intake and animal weights were determined weekly. Heparinized samples of tail vein blood were obtained under isoflurane anesthesia before and at 2, 4, and 6 weeks after dietary randomization to determine plasma UCB concentrations. At 5 weeks the rats were gavaged with 1 ml (20 mg/ml) carmine red and stools were examined for red staining to assess intestinal transit time. At 6 weeks, the 3H-labeled UCB solution (~0.29 µCi/100g BW) was administered via the penile vein.

76 Chapter 3

Subsequently, heparinized samples of tail vein blood were collected every 12h for 60h for determination of plasma UCB concentrations and feces were collected to determine fecal excretion of urobilinoids, 3H-label, bile salts, calcium, phosphate, and fat. At ~60h after the 3H-UCB-injection, bile was collected for 30 min, followed by vena cava inferior puncture as described above. The intestine was then removed and divided into 5 segments (three equal parts of small intestine, the cecum, and the remaining colon) that were flushed with phosphate buffered saline (pH 7.4) for analysis of UCB and urobilinoids.

Plasma analysis

Blood samples were protected from light and processed immediately. Bilirubin, AST, and ALT levels were determined by routine clinico-chemical spectrophotometry on a P800 unit of a modular analytics serum work area from Roche Diagnostics Ltd. (Basel, Switzerland). Hemoglobin, hematocrit, and reticulocyte counts were determined on a Sysmex-XE-2100 hematology analyzer (Goffin Meyvis, Etten-Leur, The Netherlands). UCB levels were confirmed by reversed-phase HPLC after chloroform extraction as described before.[19] 3H content was determined by liquid scintillation as described previously.[19]

Bile analysis

Bile samples were immediately frozen under argon and processed within 24h. UCB levels were determined by HPLC after chloroform extraction as described above. Urobilinoid levels were determined as zinc complexes of total urobilinoids on a UV-2401PC spectrophotometer (Shimadzu, Duisburg, Germany).[25] Bile salt concentration was determined using the 3α-hydroxysteroid dehydrogenase method[26] and bile salt composition was measured by capillary gas chromatography after conversion of bile salts to methyl ester-trimethylsilyl derivatives.[27] 3H content was determined by liquid scintillation as described previously.[23]

Analysis of feces and intestinal content

Feces and intestinal content were immediately frozen under argon, freeze-dried for 24h, mechanically homogenized, and promptly analyzed for UCB and urobilinoid levels as described previously.[19,25] Bile acid concentration and composition were determined as described before.[26,27] Fatty acid levels in feces

Bile salt treatment for unconjugated hyperbilirubinemia 77

were determined by gas chromatography on a HP-Ultra-1 column from Hewlett-Packard (Palo-Alto, CA) after extraction, hydrolysis, and methylation of aliquots of feces.[19,28] Fecal calcium levels were determined in duplicate 2.5 g aliquots of dried feces. HCl (2.2 M; 25 ml) was added and the mixture was refluxed for 10 minutes at 200°C. After cooling, ammonia (10% w/v) was added to adjust the pH to 4 and the mixture was filtered through grade-1 filter paper from Whatman Ltd. (Kent, England). The filtrate was analyzed spectrophotometrically on a P800 unit of a modular analytics serum work area from Roche Diagnostics Ltd. (Basel, Switzerland). Fecal phosphate levels were determined in duplicate 1.0 g aliquots of dried feces. H2SO4 (96% w/v; 7.5 ml) was added and the mixture was heated at 200°C for 10 minutes and subsequently at 330°C for 60 minutes. After cooling, 5 ml of hydrogen peroxide (33% w/w) was added and the mixture was heated at 330°C for 15 minutes and filtered through grade-1 filter paper (Whatman). The filtrate was analyzed as described above for calcium. 3H content was determined by liquid scintillation as described before.[23]

Calculation of fluxes based on steady-state 3H-UCB kinetics

The natural logarithm of plasma 3H-UCB specific activity (dpm/µmol) was plotted against time and the best-fit linear regression curves were calculated. Fractional turnover of 3H-UCB (%/h) was obtained from the slope of the regression line and bilirubin pool size was calculated by dividing the specific activity at T0h (Y-axis intercept) by the administered dose (dpm) of 3H-UCB. Total turnover was calculated as the product of 3H-UCB fractional turnover and pool size.[29] Fractional biliary and fractional net transmucosal fluxes of UCB and of UCB derivatives were calculated as described previously.[23]

Statistical analysis

Normally distributed data that displayed homogeneity of variance (by calculation of Levene’s statistic), were expressed as mean ± SD, and parametric statistical analysis was used. Analysis of variance (ANOVA) with post-hoc Bonferroni correction was performed for comparisons between groups, and Student t test for comparison of paired data within groups. If the data were not normally distributed, non-parametric Kruskal-Wallis and Mann Whitney U tests (with corrected p-values) were performed for comparison between groups and the data were expressed as median and range. The level of significance was set at p<0.05. Analyses were performed using SPPS 14.0 for Windows (SPSS Inc., Chicago, IL).

78 Chapter 3

3.4 Results

3.4.1 Effects of UDCA and CA treatment: dose dependency Figure 1A shows the effect of increasing dosages of UDCA or CA on plasma bilirubin levels in Gunn rats. The lowest used UDCA or CA dosage of 0.05 wt% (by chow weight) resulted in a mean daily bile salt intake of 32±2 mg/kg bodyweight (BW). This dosage already effectively decreased plasma bilirubin concentrations (-17% and -25%, respectively; each p<0.05). At the highest dose used (1.5 wt%), UDCA and CA decreased plasma bilirubin concentrations by 42% and 50%, respectively (each p<0.001). The rats in both groups did not differ significantly in body weight before the experiments and none of the doses of UDCA or CA affected growth rate (data not shown). We selected a dose of 0.5 wt%, corresponding to a daily bile salt intake of 317±23 mg/kg BW, for further studies to have a substantial effect yet minimize possible bile salt toxicity.

3.4.2 Effects of short-term administration of UDCA and CA Rapid decrease in plasma bilirubin concentrations

Figure 1B shows that 8 days of dietary UDCA or CA administration (each 0.5 wt%) decreased plasma UCB concentrations in Gunn rats by 21% and by 30%, respectively, compared with controls (each p<0.01). Administration of UDCA or CA induced a statistically significant hypobilirubinemic effect within 3 and 5 days, respectively. Mean body weight and growth rate did not differ significantly among rats in the control, UDCA, or CA group either before or during the 8-day experimental period (data not shown).

Rapid increase in fecal and biliary bile salt excretion

Figure 2A shows that dietary UDCA or CA increased fecal bile salt excretion in the first four days of administration (+663% and +466%, respectively; each p<0.001) as compared with the 4-day pre-treatment period. UDCA administration increased fecal excretion of UDCA, lithocholate, and muricholates, while CA administration increased fecal excretion of CA and deoxycholate (Fig. 2B). Figure 2C shows that the increase in fecal bile salt excretion was accompanied by an increase in biliary bile salt secretion (UDCA

Bile salt treatment for unconjugated hyperbilirubinemia 79

Figure 1. UDCA or CA administration decreases plasma UCB concentration in Gunn rats. Gunn rats (n=6 per group) were fed the control diet for 6 weeks, followed by: dietary UDCA or CA supplementation in doses that were increased every 2 weeks for 10 weeks (panel A); dietary UDCA or CA supplementation (0.5 wt%, each) or no supplementation for 8 days (panel B); or dietary UDCA or CA supplementation (0.5 wt%, each) or no supplementation for 6 weeks (panel C). Plasma UCB values at T0 (µmol/l) in panel B: controls, 241 ± 22; UDCA, 260 ± 25; CA, 251 ± 26 (NS). Data represent mean ± SD. *p<0.05; **p<0.01; ***p<0.001, compared with plasma bilirubin at T0 (panel A), or compared with controls (panels B-C). The used dosages of 0.05 wt%; 0.1 wt%; 0.5 wt%; 1.0 wt%; and 1.5 wt%, corresponded with a dietary bile salt intake (mg/24h/kg BW) of respectively 32 ± 2; 62 ± 5; 317 ± 23; and 633 ± 50.

80 Chapter 3

+127%, CA +128%; each p<0.01), measured after 8 days of treatment. Changes in fecal bile salt composition reflected changes in biliary bile salt composition (Fig. 2D).

Rapid increases in fecal, but not biliary, urobilinoid, and UCB excretion

If bile salt administration enhances bilirubin disposal, the fecal excretion of UCB and urobilinoids, a family of intestinally formed bacterial breakdown products of UCB, would be expected to increase upon starting treatment. Figure 3A & B show that UDCA and CA indeed increased the fecal excretion of both urobilinoids (+42% and +48%, respectively; each p<0.01) and UCB (+56%; p<0.05; and +25%; p=0.06; respectively) in the first four days of treatment. The combined fecal excretion of urobilinoids and UCB was increased by 52% with UDCA and 32% with CA treatment (Fig. 3C; each p<0.01). In contrast, UDCA and CA did not influence the biliary excretion of urobilinoids or UCB (data not shown), or the combined biliary excretion of urobilinoids+UCB, after 8 days of treatment (Fig. 3D).

3.4.3 Effects of long-term administration of UDCA and CA Sustained decrease in plasma bilirubin concentrations

Figure 1C shows that 6 weeks of dietary UDCA or CA (each 0.5 wt%) decreased plasma bilirubin concentrations by ~40% in Gunn rats from week 2 onwards, compared with stable values in controls (each p<0.001). Mean body weight and growth rate did not differ significantly among rats in the control, UDCA, or CA group either before or during the 6-week period (data not shown).

Changes in biliary and fecal excretion of bile salts, urobilinoids, and UCB

Table 1 shows that, mimicking the short-term experiment, 6-weeks of UDCA or CA administration increased fecal bile salt (+566% and +652%, respectively; each p<0.001) and fecal urobilinoid excretion (+98% and +103%, respectively; p<0.01, each) compared with controls. UDCA, but not CA, increased the fecal excretion of UCB (+256%; p<0.001; Table 1). Interestingly, only administration of CA, but not UDCA, increased biliary bile salt secretion after 6 weeks of treatment (+306%; p<0.001; Table 1). Changes in fecal urobilinoid and UCB

Bile salt treatment for unconjugated hyperbilirubinemia 81

Figure 2. Short-term UDCA or CA administration to Gunn rats: increases fecal bile salt excretion (panel A); changes the composition of bile salts excreted via the feces (panel B); increases biliary bile salt excretion (panel C); and changes the composition of bile salts excreted via the bile (panel D). Gunn rats (n=6 per group) were fed the control diet for 6 weeks, followed by dietary UDCA or CA supplementation (0.5 wt%, each), or no supplementation for 8 days. Feces were collected during a 4-day period before (pre-treatment period) and after (treatment period) dietary randomization. At 8 days, bile was collected during 30min. Data represent mean ± SD. *p<0.01; **p<0.001; †p<0.05–p<0.001. [Statistical analysis in feces: 4-day pre-treatment period (area under the curve) vs. 4-day treatment period (area under the curve). Statistical analysis in bile: UDCA or CA vs. controls.] LC, lithocholic acid; M, muricholic acid; DC, deoxycholic acid; C, cholic acid; CDC, chenodeoxycholic acid; HDC, hyo- deoxycholic acid; UDC, ursodeoxycholic acid; HC, hyocholic acid.

82 Chapter 3

Figure 3. Short-term UDCA or CA administration to Gunn rats: increases fecal urobilinoid excretion (panel A); increases fecal UCB excretion (panel B); increases fecal urobilinoid + UCB excretion (panel C); and does not affect biliary urobilinoid + UCB excretion (panel D). For experimental setup, please refer to Figure 2. Data represent mean ± SD. †p=0.06; *p<0.05; **p<0.01. [Statistical analysis in feces: 4-day pre-treatment period (area under the curve) vs. 4-day treatment period (area under the curve). Statistical analysis in bile: UDCA or CA vs. controls.]

Bile salt treatment for unconjugated hyperbilirubinemia 83

controls UDCA 0.5% CA 0.5%

Feces

Bile salts (µmol/h/100g BW) 0.3 ± 0.0 2.1 ± 0.1** 2.4 ± 0.4**

UCB (nmol/h/100g BW) 1.6 ± 0.5 5.8 ± 3.8** 2.0 ± 0.6

Urobilinoids (nmol/h/100g BW) 1.8 ± 0.2 3.6 ± 0.7* 3.7 ± 0.5*

UCB + urobilinoids (nmol/h/100g BW) 3.4 ± 0.5 9.4 ± 3.5** 5.7 ± 0.9*

Fat (µmol/h/100g BW) 1.2 ± 0.5 1.1 ± 0.4 0.3 ± 0.0**

Calcium (mmol/h/100g BW) 33 ± 5 34 ± 9 33 ± 4

Phosphate (mmol/h/100g BW) 26 ± 5 24 ± 7 20 ± 4

Transit time (h) 11 ± 1 11 ± 1 9.0 ± 1**

Bile

Bile salts (µmol/h/100g BW) 7.1 ± 1 10 ± 4 29 ± 5**

UCB (nmol/h/100g BW) 11 ± 3 8.2 ± 3 11 ± 2

Urobilinoids (nmol/h/100g BW) 5.9 ± 4 8.7 ± 8 5.1 ± 3

UCB + urobilinoids (nmol/h/100g BW) 17 ± 2 17 ± 10 17 ± 4

excretion were not reflected in the bile, since their biliary excretion was similar among all three groups (Table 1).

No increases in fecal excretion of fatty acids, calcium, or phosphate

An increased fecal excretion of fatty acids, calcium, or phosphate has been associated with decreased plasma UCB concentrations in Gunn rats.[13,19] Table 1 shows that UDCA administration for 6 weeks did not affect fecal fat excretion, whereas CA administration even decreased fecal fat excretion (-75%; p<0.001). Table 1 also shows that UDCA and CA administration did not influence fecal calcium or phosphate excretion.

Table 1. Steady-state excretion of several fecal and biliary components and intestinal transit time 6 weeks after dietary randomisation. For experimental setup, please refer to Figure 4. After 5 weeks of treatment intestinal transit time was determined. Data represent mean ± SD. *p<0.01; **p<0.001, compared with controls.

84 Chapter 3

CA administration decreases intestinal transit time

A decrease in intestinal transit time may decrease plasma UCB concentrations in Gunn rats.[30] CA administration for 6 weeks moderately decreased the intestinal transit time (-18%; p<0.001; Table 1), whereas UDCA administration at the same dosage showed no effect.

3.4.4 3H-Bilirubin turnover studies Decreased pool size and increased fractional turnover of bilirubin

To obtain more detailed mechanistic insights, we determined steady-state 3H-UCB-kinetics in the Gunn rats over 60h, after 6 weeks of treatment with control, UDCA, or CA diet. During the kinetic experiment hematocrit, hemoglobin, reticulocytes, AST, and ALT were unaltered (Table 2). Also, plasma bilirubin levels remained stable and the plasma 3H-UCB specific activity declined in a semi-logarithmic manner in all groups (data not shown). These findings are in accordance with the absence of significant hemolysis and with the presence of first-order steady-state conditions. Analysis of the semi-logarithmic specific activity curves showed that UDCA and CA treatment decreased bilirubin pool sizes by 33% (p<0.01) and 32% (p<0.05), respectively, compared with controls (Table 3). As shown in Figure 4, the pool sizes were strongly, positively correlated with plasma UCB concentrations (y=50*x+40; r=0.91; p<0.001). The fractional turnover of 3H-UCB increased by 33% and 25% in UDCA and CA treated animals respectively, when compared with controls (each p<0.05; Table 3). Fractional turnover was negatively correlated with both plasma UCB

controls UDCA 0.5% CA 0.5%

Hemoglobin (mmol/l) 7.2 ± 0.7 7.3 ± 0.6 7.7 ± 0.4

Hematocrit (v/v) 0.36 ± 0.03 0.37 ± 0.03 0.38 ± 0.03

Reticulocytes (‰) 58 ± 17 49 ± 15 42 ± 8

AST (U/l) 90 ± 23 81 ± 20 75 ± 14

ALT (U/l) 70 ± 22 49 ± 6 50 ± 7

Table 2. Hematological and liver function parameters 6 weeks after dietary randomisation. For experimental setup, please refer to Figure 4. Data represent mean ± SD.

Bile salt treatment for unconjugated hyperbilirubinemia 85

controls UDCA 0.5% CA 0.5%

Plasma bilirubin at T0h (µmol/l) 287 ± 40 187 ± 25 *** 181 ± 14 ***

3H-UCB fractional turnover (%/h) 1.4 ± 0.2 1.9 ± 0.2 ** 1.8± 0.1 *

Bilirubin pool size (µmol/100 g BW) 4.2 ± 0.9 2.8 ± 0.7 * 2.9 ± 0.0 *

Total bilirubin turnover (nmol/h/100 g BW) 59 ± 4 52 ± 8 51 ± 2

Biliary 3H excretion T60h (10^3 dpm/h) 10 ± 1 12 ± 4 11 ± 3

Fecal 3H excretion T0-60h (10^3 dpm/h) 5.0 ± 0.5 5.4 ± 0.5 5.8 ± 0.4

concentrations (y=-177*x+506; r=-0.89; p<0.001), and the calculated bilirubin pool sizes (y=-3.2*x+8.8; r=-0.92; p<0.001). Neither UDCA nor CA administration significantly affected total bilirubin turnover, in accordance with similar UCB production rates in the 3 groups (Table 3).

Increased efficiency of biliary and intestinal transmucosal UCB excretion

In each experimental group, the excretion of UCB into the bile, measured by HPLC, comprised ∼20% of its total turnover. Because the quantitative excretion of bilirubin occurs almost exclusively via the feces, this implies that the remaining

Figure 4. Bilirubin pool sizes and plasma UCB concentrations in Gunn rats were positively correlated. Gunn rats (n=6 per group) were fed the control diet for 6 weeks, followed by dietary UDCA or CA supplementation (0.5 wt%, each), or no supplementation for 6 weeks. After 6 weeks of treatment, tracer 3H-UCB was i.v.-administered to determine UCB kinetics. Plasma was collected every 12h over 60h. Specific activity of plasma bilirubin declined semi-logarithmically with time in each group.

Table 3. Steady-state 3H-bilirubin kinetics 6 weeks after dietary randomisation. For experimental setup, please refer to Figure 4. Data represent mean ± SD. *p<0.05; **p<0.01; ***p<0.001, compared with controls.

86 Chapter 3

∼80% of the UCB disposal occurs via net transmucosal excretion. The treated groups thus excreted similar amounts of UCB via either excretory pathway compared with controls. However, due to smaller bilirubin pool sizes, all fractional fluxes (flux per hour as fraction of the UCB pool size) are increased in the treated groups, compared with controls. Figure 5 shows that UDCA mainly increased the fractional transmucosal UCB flux and the fractional biliary flux of derivatives, whereas CA predominantly increased the fractional biliary flux of UCB.

UDCA, but not CA, administration increases intestinal UCB and urobilinoid content

Table 4 shows that, throughout the bowel, UCB and urobilinoid content tended to be higher in the UDCA-treated animals (+143% and +106%, respectively; p<0.01), compared with controls. By contrast, CA administration did not significantly alter total intestinal content of UCB and urobilinoids.

Figure 5. Fractional biliary and transmucosal fluxes of UCB and UCB-derivatives in Gunn rats, 6 weeks after randomization. Fractional UCB fluxes are expressed as % and fractional UCB-derivative fluxes are expressed as equivalent% of the bilirubin pool size that is excreted per hour. [a] fractional turnover of UCB; [b] fractional biliary UCB excretion; [c] fractional biliary UCB-derivative excretion; [d] fractional fecal excretion of UCB + UCB-derivatives; [e] estimated net transmucosal flux of UCB from the blood into the intestinal lumen, calculated as [a]-[b]. The magnitude of flux [d] equals that of [a] in a steady-state, assuming that UCB turnover equals the fecal excretion of UCB + UCB-derivatives. EHC = enterohepatic circulation. Ovals in upper left of each panel show bilirubin pool size (µmol/100g BW; mean ± SD). For calculation of fractional fluxes, please refer to Methods.

Bile salt treatment for unconjugated hyperbilirubinemia 87

controls UDCA 0.5% CA 0.5%

small intestine (proximal) UCB (nmol) 4 (1 - 11) 2 (1 - 8) 3 (1 - 5)

Urobilinoids (nmol) 2 (1 - 3) 2 (1 - 5) 3 (2 - 5)

small intestine (medial)

UCB (nmol) 18 (13 – 22) 16 ± (4 – 19) 14 ± (1 – 19)

Urobilinoids (nmol) 8 (5 – 9) 9 (3 – 14) 9 (3 – 12)

small intestine (distal)

UCB (nmol) 19 (2 - 50) 90 (23 - 230)* 29 (4 - 127)

Urobilinoids (nmol) 11 (5 - 17) 28 (12 - 43)* 17 (4 - 21)

cecum

UCB (nmol) 10 (7 - 13) 28 (11 - 421)* 19 (13 - 61)*

Urobilinoids (nmol) 47 (25 - 100) 117 (65 - 134)* 106 (64 - 144)

large intesine

UCB (nmol) 14 (7 - 23) 116 (50 - 163)** 8 (4 - 13)†

Urobilinoids (nmol) 34 (23 - 46) 132 (50 - 169)** 38 (16 - 101)†

total intestine

UCB (nmol) 70 (32 - 92) 227 (108 - 790)* 72 (41 - 166)†

Urobilinoids (nmol) 95 (84 – 158) 294 (138 - 340)* 154 (106 - 254)

Table 4. Intestinal UCB and urobilinoid content 6 weeks after dietary randomization. For the experimental setup, please refer to Figure 4. After the 60h-period of the 3H-UCB kinetic study animals were terminated. The intestine was then removed and divided into 5 segments (three equal parts of small intestine, the cecum, and the remaining colon) that were flushed with phosphate buffered saline (pH 7.4) for analysis of UCB and urobilinoids. Data represent median and range. *p<0.05; **p<0.01, compared with controls. †p<0.05, compared with UDCA-treated animals. Non-parametric tests were used.

88 Chapter 3

3.5 Discussion In this study we demonstrate that dietary administration of either UDCA or CA significantly decreases plasma UCB concentrations in Gunn rats. The decrease occurs within 3 days after starting administration, is maximal within 2 weeks and is sustained thereafter.

The conclusion that the administration of UDCA or CA enhances fecal bilirubin disposal is based on two independent analytic moieties, namely biochemical and 3H-kinetic measurements. First, the fecal excretion of UCB and urobilinoids increased promptly upon starting bile salt treatment. Previous studies have shown that biochemical measurements of UCB and urobilinoids in the feces only account for 25-50% of the expected fecal bilirubin disposal.[31] To address this and to investigate possible alterations in UCB metabolism by the treatments, we performed a 3H-UCB kinetic experiment in a steady-state condition.[23] This experiment demonstrated that either bile salt decreased the bilirubin pool size by one-third, mirroring similar decreases in plasma UCB concentrations. Combined, these methodologies indicate that UDCA or CA enhance UCB disposal from the body and that the hypobilirubinemic effects are not due to redistribution among different body compartments.[32]

During steady-state conditions, bilirubin production equals its excretion and both are synonymous with the total turnover of bilirubin (expressed as nmol/h/100g BW; Table 3). If bile salt treatment has no effect on bilirubin production, the total excretory flux of bilirubin is thus similar in the treated groups and the control group during steady-state. To describe differences in the efficiency of bilirubin disposal, the fractional turnover of bilirubin, i.e. the total turnover as percent of the bilirubin pool size (expressed as %/h; Table 3), is preferentially used. The kinetic study showed that the hypobilirubinemic effect of bile salt treatment was not due to a decrease in UCB production, as reflected by the similar total bilirubin turnover, but rather to an increased efficiency of UCB disposal from the body, as reflected by the increased fractional turnover compared with controls.

Fractional turnover, pool size, and total turnover of 3H-UCB of the control animals in this study were reassuringly similar to the values obtained in previous studies of radiolabeled UCB kinetics in Gunn rats.[3,23,32] We calculated steady-state fluxes of UCB and its derivatives using a previously used mathematical model, based on the assumption that the quantitative disposal of bilirubin and its derivatives occurs exclusively via the feces.[23] This assumption seems reasonable since only ∼6% of total bilirubin turnover in Gunn rats is disposed of via the urine.[7] Our calculations showed that only ∼20% of the total bilirubin turnover in each group was disposed of via the bile and ∼80% of the total bilirubin turnover was disposed of via net transmucosal excretion. This result underlines the

Bile salt treatment for unconjugated hyperbilirubinemia 89

previously described importance of the transmucosal excretory route during unconjugated hyperbilirubinemia.[6,7,23]

Analysis of the fractional excretory fluxes (Fig. 5), or flux as a proportion of the bilirubin pool size, suggests that the principal mechanisms differ by which UDCA and CA lower plasma UCB levels. The hypobilirubinemic effect of UDCA is mainly due to an increased fractional transmucosal excretion of UCB, whereas CA increases the fractional biliary excretion of UCB. Unlike the control and CA group, UDCA-fed animals showed a marked increase in UCB content in the colon, i.e. rather distal from the biliary excretion of UCB into the duodenum. Based on this finding we hypothesize that UDCA mainly exerts its hypobilirubinemic effect via intestinal trapping of UCB. This hypothesis is supported by the increased colonic content and the increased fractional biliary flux of urobilinoids in the UDCA group, reflecting enhanced formation and enterohepatic circulation of these bacterial metabolites, probably due to the increased supply of UCB in the intestinal lumen. The mechanism by which CA treatment lowers plasma bilirubin concentrations in our Gunn rats is less clear. Intravenous taurocholic acid administration enhanced biliary UCB excretion in Gunn rats.[17] We observed no increase in biliary excretion of UCB and urobilinoids after 8 days or 6 weeks of CA treatment, although we did not measure directly after starting bile salt treatment. The steady-state 3H-UCB kinetic study and the analysis of intestinal bile pigment content favor greater enhancement by CA of the fractional biliary excretion of UCB, but less effective trapping of UCB in the intestine compared with UDCA. In a binary system, UCB binds less avidly to UDCA than to CA, but in the presence of lecithin, UDCA favors the formation of vesicles over micelles, which enhances solubilization of cholesterol and might conceivably do so for UCB.[33]

We acknowledge that other mechanisms may also contribute to the increase in UCB disposal during bile salt administration. The intestinal transit time was decreased by 18% in the 0.5 wt% CA-treated group, which would decrease contact time for mucosal reabsorption.[30] Increased amounts of fat and amorphous calcium phosphate could lower plasma bilirubin via intestinal trapping.[13,19] However, their fecal excretion was not increased during bile salt treatment. Also, UDCA treatment could affect the expression of relevant transporters.[34]

Among all experimental groups, we observed a strong, positive, linear correlation between bilirubin pool size and plasma UCB concentrations, similar to our findings when treating Gunn rats with orlistat and phototherapy.[23] During treatment of Gunn rats with phototherapy, orlistat, or bile salts, changes in UCB pool sizes are thus well predicted by changes in plasma UCB concentrations. However, 3H-UCB kinetic studies, using our mathematical model (Fig. 5), remain

90 Chapter 3

necessary to estimate the biliary and transmucosal UCB fluxes, and their relationship to UCB pool size.

Mendez-Sanchez et al.[35] showed that in non-jaundiced mice and rats dietary UDCA supplementation increased the enterohepatic UCB circulation. This opposite finding to our study, in which UDCA decreased net UCB intestinal reabsorption, might result from strain-induced differences in bilirubin metabolism. Homozygous Gunn rats cannot conjugate bilirubin and the accumulated UCB diffuses from the plasma into the intestinal lumen.[6,8] This diffusion is inversely directed in non-jaundiced rodents, as used in the study of Mendez-Sanchez, due to lower plasma UCB levels.[8,9] UDCA-induced changes in microfloral hydrolysis of bilirubin conjugates could further enhance this diffusion. The observed differences between rat strains illustrate that therapeutic application of UDCA in patients with an etiology that is incompatible to our animal model, such as chronic liver disease, should be approached with caution.

The rapid and persistent hypobilirubinemic effect of bile salt administration, however, does support the potential clinical applicability of oral UDCA therapy for unconjugated hyperbilirubinemia in humans. The major therapeutic effect was already present in a low dosage (0.05 wt%), corresponding to a clinically applicable dose of approximately 30 mg/kg/day. Bile salt therapy decreased plasma UCB concentrations in Gunn rats as effectively as did phototherapy in our previous experiments.[15,23] All treated Gunn rats ingested comparable amounts of either UDCA or CA, and treatment with neither bile salt induced diarrhea or impaired growth rates. This corresponds with the fact that UDCA treatment is well-established and well-tolerated in pediatric patients.[36]

In conclusion, dietary administration with UDCA or CA induces a rapid and sustained decrease in plasma UCB concentrations in Gunn rats. The mechanism involves stimulation of UCB turnover and its fecal disposal. Present results support the feasibility of oral bile salt treatment in patients with unconjugated hyperbilirubinemia.

Acknowledgement: The authors would like to acknowledge Milan Jirsa (Laboratory of Experimental Hepatology, Institute for Clinical and Experimental Medicine, Prague, Czech Republic) for his help with the preparation of 3H-UCB.

Bile salt treatment for unconjugated hyperbilirubinemia 91

References 1. Crigler JF, Najjar VA. Congenital

familial nonhemolytic jaundice with kernicterus. Pediatrics. 1952;10:169–180.

2. Shapiro SM. Definition of the clinical spectrum of kernicterus and bilirubin-induced neurologic dysfunction (BIND). J Perinatol. 2005;25:54–59.

3. Ostrow JD. Photocatabolism of labeled bilirubin in the congenitally jaundiced (Gunn) rat. J. Clin. Invest. 1971;50:707–718.

4. Van der Veere CN, Sinaasappel M, McDonagh AF, Rosenthal P, Labrune P, Odievre M, et al. Current therapy for Crigler-Najjar syndrome type 1: report of a world registry. Hepatology. 1996;24:311–315.

5. Yohannan MD, Terry HJ, Littlewood JM. Long term phototherapy in Crigler-Najjar syndrome. Arch Dis Child. 1983;58:460–462.

6. Kotal P, Van der Veere CN, Sinaasappel M, Elferink RO, Vitek L, Brodanova M, et al. Intestinal excretion of unconjugated bilirubin in man and rats with inherited unconjugated hyperbilirubinemia. Pediatr. Res. 1997;42:195–200.

7. Schmid R, Hammaker L. Metabolism and disposition of C14-bilirubin in congenital nonhemolytic jaundice. J. Clin. Invest. 1963;42:1720–1734.

8. Lester R, Schmid R. Intestinal absorption of bile pigments. I. The enterohepatic circulation of bilirubin in the rat. J. Clin. Invest. 1963;42:736–746.

9. Lester R, Schmid R. Intestinal absorption of bile pigments. II. Bilirubin absorption in man. N. Engl.

J. Med. 1963;269:178–182.

10. Odell GB, Gutcher GR, Whitington PF, Yang G. Enteral administration of agar as an effective adjunct to phototherapy of neonatal hyperbilirubinemia. Pediatr. Res. 1983;17:810–814.

11. Tan KL, Jacob E, Liew DS, Karim SM. Cholestyramine and phototherapy for neonatal jaundice. J. Pediatr. 1984;104:284–286.

12. Ulstrom RA, Eisenklam E. The Enterohepatic Shunting of Bilirubin in the Newborn Infant. I. Use of Oral Activated Charcoal to Reduce Normal Serum Bilirubin Values. J. Pediatr. 1964;65:27–37.

13. Van der Veere CN, Jansen PL, Sinaasappel M, Van Der Meer R, Van der Sijs H, Rammeloo JA, et al. Oral calcium phosphate: a new therapy for Crigler-Najjar disease? Gastroenterology. 1997;112:455–462.

14. Mendez-Sanchez N, Martinez M, Gonzalez V, Roldan-Valadez E, Flores MA, Uribe M. Zinc sulfate inhibits the enterohepatic cycling of unconjugated bilirubin in subjects with Gilbert's syndrome. Ann Hepatol. 2002;1:40–43.

15. Hafkamp AM, Nelisse-Haak R, Sinaasappel M, Oude Elferink RPJ, Verkade HJ. Orlistat treatment of unconjugated hyperbilirubinemia in Crigler-Najjar disease: a randomized controlled trial. Pediatr. Res. 2007;62:725–730.

16. Vonk RJ, Jekel P, Meijer DK. Choleresis and hepatic transport mechanisms. II. Influence of bile salt choleresis and biliary micelle binding on biliary excretion of various organic anions. Naunyn Schmiedebergs Arch Pharmacol. 1975;290:375–387.

92 Chapter 3

17. Ostrow JD. Regulation by bile salts of the excretion of conjugated and unconjugated bilirubin in bile. In: In: Fromm H, Leuschner U, eds. Proceedings of the falk symposium No. 84, held in Berlin, Germany, June 9-10, 1995. Kluwer Academic Publishers, 1996. In: Fromm H, Leuschner U, eds. Proceedings of the falk symposium No. 84, held in Berlin, Germany, June 9-10, 1995. Kluwer Academic Publishers, 1996; 1996.

18. Einarsson K, Bjorkhem I, Eklof R, Ewerth S, Nilsell K, Blomstrand R. Effect of ursodeoxycholic acid treatment on intestinal absorption of triglycerides in man. Scand. J. Gastroenterol. 1984;19:283–288.

19. Hafkamp AM, Havinga R, Sinaasappel M, Verkade HJ. Effective oral treatment of unconjugated hyperbilirubinemia in Gunn rats. Hepatology. 2005;41:526–534.

20. Ostrow JD, Celic L, Mukerjee P. Molecular and micellar associations in the pH-dependent stable and metastable dissolution of unconjugated bilirubin by bile salts. J. Lipid Res. 1988;29:335–348.

21. Rege RV, Webster CC, Ostrow JD. Interactions of unconjugated bilirubin with bile salts. J. Lipid Res. 1988;29:1289–1296.

22. Bayon JE, Pascolo L, Gonzalo-Orden JM, Altonaga JR, Gonzalez-Gallego J, Webster C, et al. Pitfalls in preparation of (3)H-unconjugated bilirubin by biosynthetic labeling from precursor (3)H-5-aminolevulinic acid in the dog. J Lab Clin Med. 2001;138:313–321.

23. Hafkamp AM, Havinga R, Ostrow JD, Tiribelli C, Pascolo L, Sinaasappel M, et al. Novel kinetic insights into treatment of unconjugated hyperbilirubinemia: phototherapy and orlistat treatment in Gunn rats. Pediatr. Res. 2006;59:506–512.

24. Webster C, Tiribelli C, Ostrow JD. An improved method for isolation of unconjugated bilirubin from rat and dog bile. J Lab Clin Med. 2001;137:370–373.

25. Kotal P, Fevery J. Quantitation of urobilinogen in feces, urine, bile and serum by direct spectrophotometry of zinc complex. Clin. Chim. Acta. 1991;202:1-10

26. Murphy GM, Billing BH, Baron DN. A fluorimetric and enzymatic method for the estimation of serum total bile acids. J Clin Pathol. 1970;23:594–598.

27. Hulzebos CV, Renfurm L, Bandsma RH, Verkade HJ, Boer T, Boverhof R, et al. Measurement of parameters of cholic acid kinetics in plasma using a microscale stable isotope dilution technique: application to rodents and humans. J. Lipid Res. 2001;42:1923–1929.

28. Lepage G, Roy CC. Direct transesterification of all classes of lipids in a one-step reaction. J. Lipid Res. 1986;27:114–120.

29. Zilversmit DB. The design and analysis of isotope experiments. Am J Med. 1960;29:832.

30. Kotal P, Vitek L, Fevery J. Fasting-related hyperbilirubinemia in rats: the effect of decreased intestinal motility. Gastroenterology. 1996;111:217–223.

31. Tiribelli C, Ostrow JD. Intestinal flora and bilirubin. J. Hepatol. 2005;42:170.

32. Cohen AN, Kapitulnik J, Ostrow JD, Zenone EA, Cochrane C, Celic L, et al. Effects of phenobarbital on bilirubin metabolism and its response to phototherapy in the jaundiced Gunn rat. Hepatology. 1985;5:310–316.

33. Salvioli G, Igimi H, Carey MC. Cholesterol gallstone dissolution in

Bile salt treatment for unconjugated hyperbilirubinemia 93

bile. Dissolution kinetics of crystalline cholesterol monohydrate by conjugated chenodeoxycholate-lecithin and conjugated ursodeoxycholate-lecithin mixtures: dissimilar phase equilibria and dissolution mechanisms. J. Lipid Res. 1983;24:701–720.

34. He YJ, Zhang W, Tu JH, Kirchheiner J, Chen Y, Guo D, et al. Hepatic nuclear factor 1alpha inhibitor ursodeoxycholic acid influences pharmacokinetics of the organic anion transporting polypeptide 1B1 substrate

rosuvastatin and bilirubin. Drug Metab Dispos. 2008;36:1453–1456.

35. Mendez-Sanchez N, Brink MA, Paigen B, Carey MC. Ursodeoxycholic acid and cholesterol induce enterohepatic cycling of bilirubin in rodents. Gastroenterology. 1998;115:722–732.

36. Balistreri WF. Bile acid therapy in pediatric hepatobiliary disease: the role of ursodeoxycholic acid. J Pediatr Gastroenterol Nutr. 1997;24:573–589.

94 Chapter 3

Chapter 4 Acceleration of the Gastrointestinal Transit by Polyethylene Glycol Effectively Treats Unconjugated Hyperberbilirubinemia in Gunn Rats

F.J.C. Cuperus 1

A.A. Iemhoff 1

M.Y. van der Wulp 1 R. Havinga 1

H.J. Verkade 1

1 Pediatric Gastroenterology and Hepatology, Department of Pediatrics, Center for Liver, Digestive, and Metabolic Diseases, Beatrix Children’s Hospital - University Medical Center Groningen, University of Groningen, The Netherlands.

Gut 2010;59:373–380

96 Chapter 4

S

4.1 Abstract everal conditions that delay the gastrointestinal transit are associated with unconjugated hyperbilirubinemia. We hypothesized that the gastrointestinal transit time is directly related to plasma unconjugated bilirubin (UCB) concentrations, and that this relationship can be used to develop a new therapeutic strategy for severe unconjugated hyperbilirubinemia in the Gunn rat model. Gunn rats received, for various time periods, oral polyethylene glycol (PEG) with or without conventional phototherapy treatment to accelerate, or oral loperamide to delay the gastrointestinal transit. Gastrointestinal transit time and UCB concentrations in plasma, feces, intestinal content, and bile were determined. Within 36 hours, PEG administration accelerated the gastrointestinal transit by 45% and simultaneously decreased plasma UCB concentrations by 23% (each p<0.001). The decrease in plasma UCB coincided with an increased small intestinal UCB content (+340%, p<0.05) and an increased fecal UCB excretion (+153%, p<0.05). After two weeks, PEG decreased plasma UCB by 41% as single treatment, and by 62% if combined with phototherapy (each p<0.001). Loperamide delayed gastrointestinal transit by 57% and increased plasma UCB by 30% (each p<0.001). Dose-response experiments showed a strong, linear relation between the gastrointestinal transit time and plasma UCB concentrations (r=0.87, p<0.001). This chapter demonstrates that gastrointestinal transit time and plasma UCB concentrations are linearly related in Gunn rats. This relationship can be exploited by pharmacologically accelerating the gastrointestinal transit, which increases transmucosal UCB diffusion and thereby effectively treats unconjugated hyperbilirubinemia. Present results support the feasibility of PEG treatment, either solitary or combined with phototherapy, in patients with severe unconjugated hyperbilirubinemia.

PEG treatment for unconjugated hyperbilirubinemia 97

P

4.2 Introduction atients with Crigler-Najjar disease suffer from a genetically absent (type I) or decreased (type II) capacity to conjugate bilirubin in the liver, resulting in life-long, unconjugated hyperbilirubinemia.[1] Severe unconjugated hyperbilirubinemia can lead to disposition of unconjugated bilirubin (UCB) in the central nervous system, inducing bilirubin-induced neurological damage (BIND), kernicterus, and death.[2] Phototherapy, the conventional treatment for unconjugated hyperbilirubinemia, converts the hydrophobic UCB into polar photo-isomers that can be excreted without conjugation into the bile.[3] Phototherapy, however, does not always lower UCB below toxic levels. Also, long-term phototherapy, as required by Crigler-Najjar patients, has a profound effect on social life and becomes less effective with age.[4,5] In spite of an intensive phototherapy regimen, which may take up more than 12h per day, many Crigler-Najjar patients will eventually develop irreversible brain damage.[4]

The disadvantages of phototherapy have prompted investigation into alternative treatment strategies for unconjugated hyperbilirubinemia. One of these strategies involves stimulation of transmucosal UCB diffusion from the blood into the intestinal lumen.[6-10] In hyperbilirubinemic Gunn rats, the well-established animal model for Crigler-Najjar disease type I, the majority of UCB enters the intestinal lumen via this pathway, rather than via the bile.[10] Transmucosal UCB diffusion thus seems to be a major excretory route for UCB in hyperbilirubinemic conditions. However, the efficiency of transmucosal diffusion with respect to UCB excretion is decreased by reabsorption of UCB from the intestinal lumen.[7,8] Trapping UCB in the lumen can prevent this reabsorption and lowers plasma UCB concentrations in Gunn rats and hyperbilirubinemic patients.[11-16] However, the trapping agents tested so far, including agar,[11] cholestyramine,[12] charcoal,[13] amorphous calcium phosphate,[14] zinc salts,[16] and orlistat[17] have been clinically unsatisfactory due to side-effects and inconsistent results.

We aimed to develop an alternative oral treatment for unconjugated hyperbilirubinemia that enhances transmucosal diffusion, and the subsequent fecal excretion of UCB. Theoretically, this could be achieved by accelerating the gastrointestinal transit, which is expected to decrease the intraluminal UCB concentration. Indeed, several observations in Gunn rats and in human neonates suggest that the gastrointestinal transit is an important regulator for plasma UCB concentrations (Table 1). In Gunn rats, fasting delayed the gastrointestinal transit and simultaneously increased plasma UCB concentrations.[18] In neonates, conditions that delay the gastrointestinal transit, such as fasting,[19,20] pyloric stenosis,[21] and Hirschprung’s disease,[22] were associated with increased

98 Chapter 4

plasma bilirubin concentrations. Only recently Bisceglia et al. showed that feeding infant formula supplemented with prebiotic galactosaccharides and oligosaccharides, a mixture that increases the daily stool frequency and accelerates the gastrointestinal transit,[23] decreased plasma UCB concentrations in neonates.[24] Because these clinical conditions, as well as prebiotic treatment, affect many physiological processes other than the gastrointestinal transit, it has remained unclear whether the observed changes in plasma UCB concentrations were directly related to the gastrointestinal transit time. If the gastrointestinal transit time would indeed directly influence plasma UCB concentrations, we could exploit this relationship to develop new therapeutic strategies for severe unconjugated hyperbilirubinemia.

We presently show that acceleration of the gastrointestinal transit by the laxative polyethylene glycol (PEG) decreases plasma UCB concentrations in hyperbilirubinemic Gunn rats. We demonstrate a strong, positive correlation between the gastrointestinal transit time and plasma UCB concentrations. Additionally, we compared the therapeutic effect of PEG with that of orlistat treatment, and assessed the use of PEG as adjunct treatment to phototherapy, which is the standard treatment for unconjugated hyperbilirubinemia in patients. Current results indicate that pharmacological acceleration of the gastrointestinal transit time could be a feasible strategy to treat patients with severe unconjugated hyperbilirubinemia.

Delayed gastrointestinal transit and increased plasma UCB

Accelerated gastrointestinal transit and decreased plasma UCB

Fasting unconjugated hyperbilirubinemia [18-20, 30, 31] Feeding prebiotic oligosaccharides [23, 24]

Pyloric stenosis [21] Early feeding [31-33]

Hirschprungs’ disease [22] Rectal stimulation [34]

Table 1. Previous studies have indirectly supported a role of the gastrointestinal transit in the regulation of neonatal plasma UCB concentrations. Conditions that are associated with a delayed gastrointestinal transit in neonates, such as fasting, pyloric stenosis, and Hirschprung’s disease, have been associated with increased plasma UCB levels. Bisceglia et al. showed that feeding infant formula supplemented with prebiotic galactosaccharides and oligosaccharides, a mixture that increases the daily stool frequency and accelerates the gastrointestinal transit, decreased plasma UCB concentrations in neonates. Early feeding and rectal stimulation, both associated with an accelerated meconium passage, are also associated with a decrease in plasma UCB. References are detailed at the end of the chapter (reference list).

PEG treatment for unconjugated hyperbilirubinemia 99

4.3 Animals, materials, and methods

4.3.1 Animals Homozygous adult male Gunn rats (RHA/jj; 268-362 g) from our breeding colony were individually housed, fed ad libitum and had free access to water. Food intake, fluid intake and body weight were determined daily during experiments. The Animal Ethics Committee approved all protocols.

4.3.2 Materials Hope Farms BV (Woerden, The Netherlands) produced the semi-synthetic diet (code 4063.02).[17] Gunn rats were fed this diet during a 6-week run-in period and subsequently during the experimental period.[10,17] PEG 4000 (Colofort) was obtained from Ipsen Farmaceutica BV (Hoofddorp, The Netherlands). Orlistat (Xenical®) was obtained from Roche Nederland BV (Woerden, The Netherlands). The PEG solution we used (drinking water solution and gavage solution) was obtained by dissolving one sachet (74 g) of PEG 4000 in 900 ml water. Loperamide, bilirubin, and heptadecanoic acid were obtained from Sigma Chemical Co. (St. Louis, MO). Xanthobilirubin-methyl ester was a gift from dr. J. Fevery (Leuven, Belgium). Urobilin was obtained from Frontier Scientific Inc. (Logan, UT). Carmine red dye was obtained from Macro-imPulse Saveur Ltd. (Stadtoldendorf, Germany). Phototherapy (17 µW/cm2/nm), was administered as described previously.[17]

4.3.3 Methods Short-term treatment

Gunn rats were randomly assigned to receive no treatment (n=6) or PEG (n=6) via drinking water and intragastrical gavage (5.0 ml, every 12h). Heparinized samples of tail vein blood were obtained under isoflurane anesthesia at -12, 0, 12, 24, and 36h for determination of plasma UCB concentrations in the intervention group. Gastrointestinal transit time was determined three days before, and immediately upon starting PEG administration, by measuring the interval between oral gavage and fecal appearance of carmine red marker. Feces were collected before (baseline period) and after the start of PEG treatment for 36h to determine fecal UCB excretion. After 36h bile and intestinal content were collected for analysis of UCB and urobilinoids, as described before.[10] In a separate experiment, Gunn rats (n=7) received orlistat treatment (200 mg/kg

100 Chapter 4

chow) for a period of 36h. Blood samples were obtained at 0 and 36h for determination of plasma UCB.

Long-term treatment

Gunn rats were randomly assigned to receive no treatment (n=6) or PEG (n=6) via drinking water and via intragastrical gavage (2.5ml, every 24h). Heparinized samples of tail vein blood were obtained under isoflurane anesthesia at day 0, 2, 7, and 14 for determination of plasma UCB, sodium (Na), potassium (K), urea (Ur), and creatinine (Creat) concentrations. The gastrointestinal transit time was determined after two weeks of treatment, as described above. Feces were collected during a 3-day period prior to bile canulation to determine fecal UCB, urobilinoids, bile acids, calcium, and fat excretion. After 14 days, bile and intestinal content were collected for analysis of UCB and urobilinoids. In a separate experiment, small intestinal transit was determined by measuring the intestinal progression (as a percentage of the total small intestinal length) of carmine red 15 minutes after its administration in animals that received either no treatment (n=6) or PEG treatment (n=5) during a 2-week period. In two separate experiments, Gunn rats received PEG (as above) with phototherapy (treatment and control group, each n=6) or received orlistat treatment (200 mg/kg chow; treatment group n=7; control group n=11) for 2 weeks. Blood samples and gastrointestinal transit time were determined at identical time points and with identical methods as described above.

Loperamide treatment

Gunn rats were randomly assigned to receive no treatment (n=6) or 7 mg/ml loperamide (n=6) via intragastrical gavage (1 ml, every 24h). Heparinized samples of tail vein blood were obtained under isoflurane anesthesia at day 0, 2, and 7 for determination of plasma UCB concentrations. Gastrointestinal transit time was determined one week after the start of treatment as described above.

Dose-response experiment

Gunn rats were administered PEG via drinking water for 9 days, followed by additional daily PEG administration via intragastrical gavage (2.5 ml and 5 ml, each for 9 days). Heparinized samples of tail vein blood were obtained under isoflurane anesthesia at day 0 and at the end of each 9-day period. The

PEG treatment for unconjugated hyperbilirubinemia 101

gastrointestinal transit time was determined simultaneously, as described above. Feces were collected during a 3-day period before starting PEG administration (baseline period), and a 3-day period before termination at 27 days to determine fecal excretion of UCB, urobilinoids, bile acids, calcium, and fat.

Plasma analysis

Blood samples were protected from light and processed immediately. Total UCB, Na, K, Ur, and Creat were determined by routine spectrophotometry on a P800 unit of a modular analytics serum work area from Roche Diagnostics Ltd. (Basel, Switzerland). UCB concentrations were confirmed by reverse-phased high-performance liquid chromatography (HPLC) after chloroform extraction as described before.[17,25,26]

Bile analysis

Bile samples were protected from light, stored at -80°C under argon directly after collection and processed within 24h. UCB concentrations were determined by reverse-phased HPLC after chloroform extraction.[17,25,26] The biliary bile salt concentration was determined with the 3α-hydroxysteroid dehydrogenase method,[27] and bile acid composition was measured by capillary gas chromatography after conversion of bile acids to methyl ester-trimethylsilyl derivatives.[10] Urobilinoid concentrations were determined as zinc complexes of total urobilinoids on a UV-2401PC spectrophotometer (Shimadzu, Duisburg, Germany).[28]

Feces and intestinal content analysis

Feces and intestinal content were immediately frozen (-20 °C) under argon, freeze-dried for 24h, mechanically homogenized, and thereafter promptly analyzed for UCB and urobilinoid concentrations as described above. Fecal bile acid concentration and bile acid composition were determined as described before.[10,27] Fatty acid concentrations in feces were determined by gas chromatography on a HP-Ultra-1 column from Hewlett-Packard (Palo-Alto, CA) after extraction, hydrolysis and methylation.[29] Fecal calcium concentrations were determined as described previously.[10]

102 Chapter 4

Statistical analysis

Normally distributed data (displaying homogeneity of variance) were analyzed with Student t tests, and expressed as mean ± SD, or as individual data points with the mean. Non-normally distributed data were analyzed with Mann Whitney U tests, and expressed as median and range, or as individual data points with the median. The level of significance was set at p<0.05. Analyses were performed using SPPS 16.0 for Mac (SPSS Inc., Chicago, IL).

4.4. Results

4.4.1 Short-term treatment Rapid decrease in plasma UCB concentrations

Figure 1A shows that oral PEG administration in Gunn rats decreased plasma UCB concentrations by 23% after 36h of treatment, compared with baseline values (p<0.001). PEG administration simultaneously accelerated the gastrointestinal transit (i.e. it decreased gastrointestinal transit time) by 45% within the first day of treatment (p<0.001; Figure 1B). The hypobilirubinemic effect of PEG was apparent within 12h of treatment (Figure 1C). We compared the hypobilirubinemic effect of PEG with that of orlistat, a well-known experimental oral treatment for severe unconjugated hyperbilirubinemia. Orlistat decreased plasma bilirubin by only 10% after 36h (p<0.05; Fig. 2A), which was significantly less compared with PEG (p<0.01).

Rapid increase in fecal UCB excretion

Figure 1D shows that the PEG-induced decrease of plasma UCB concentrations was accompanied by a 153% increase in fecal UCB excretion during the 36h treatment period (p<0.05). Fecal urobilinoids, comprising a family of intestinally formed bacterial breakdown products of UCB, were not quantified in this experiment due to spectrophotometric interference by carmine red dye.

PEG treatment for unconjugated hyperbilirubinemia 103

Figure 1. Short-term PEG administration to Gunn rats: decreases plasma UCB concentrations by 23% after 36 hours of treatment (panel A); accelerates gastrointestinal transit within the first day of treatment (panel B); decreases plasma UCB concentrations within 12 hours after the start of treatment (panel C); and increases fecal UCB excretion during the 36 hours of treatment (Panel D). Gunn rats (n=6) were fed the control diet for 6 weeks, followed by PEG administration (via drinking water and via intragastrical gavage) for a total period of 36h. Gastrointestinal transit time was determined three days before, and directly upon starting PEG administration. Feces were collected before (baseline period) and after starting PEG administration (treatment period) for 36h. Data represent: individual data points with mean (panel A and B), mean ± SD (panel C), or individual data points with median (panel D); *p<0.05; **p<0.01; ***p<0.001, compared with baseline values.

104 Chapter 4

Figure 2. Orlistat administration to Gunn rats decreases plasma UCB concentrations by 10% after 36 hours of treatment (panel A), and by 33% after 2 weeks of treatment (panel B, separate experiments). In the short-term experiment Gunn rats were fed the control diet for 6 weeks followed by the same diet supplemented with orlistat (200 mg/kg chow; n=7) for a period of 36 hours. In the long-term experiment Gunn rats were fed the control diet for 6 weeks followed by the control diet (n=11) or by the control diet supplemented with orlistat (200 mg/kg chow; n=7) for a period of 2 weeks. *p<0.05, compared with baseline; **p<0.001. Data represent mean ± SD

Figure 3. Short-term PEG administration to Gunn rats does not affect: biliary UCB excretion (panel A); biliary urobilinoid excretion (panel B); and bile flow (panel C). Gunn rats (n= 6 per group) were fed control diet for 6 weeks, followed by: control diet (controls) or control diet with PEG administration via drinking water and via intragastrical gavage (5.0 ml, every 12h) for a total period of 36h. At 36h, bile was collected for 30 minutes. Data represent mean ± SD.

PEG treatment for unconjugated hyperbilirubinemia 105

Increase in the small intestinal UCB content, but no effect on biliary UCB excretion

Table 2 shows that 36h of PEG administration increased the UCB content in the medial and distal small intestine, compared with controls (+708% and +205%, respectively; each p<0.05). In the remainder of the bowel, the PEG-induced increase in intestinal UCB content did not reach statistical significance. PEG administration did not significantly influence the urobilinoid content in the intestinal segments (Table 2).

UCB enters the intestinal lumen via transmucosal diffusion or via biliary excretion. Figure 3 shows that the biliary excretion of UCB was not increased after 36h of PEG treatment. Also, PEG administration did not influence biliary urobilinoid excretion or bile flow.

Table 2. Intestinal content composition after 36 hours of PEG administration. Gunn rats (n= 6 per group) were fed the control diet for 6 weeks, followed by: control diet (controls), or control diet with PEG administration (via drinking water and via intragastrical gavage) for a total period of 36h. At 36h, the intestine was removed and divided into 5 segments (three equal parts of small intestine, the cecum, and the remaining colon) that were flushed with phosphate buffered saline (pH 7.4) for analysis of UCB and urobilinoids. Data represent median and range. *p<0.05; **p<0.01.

controls PEG

Small intestine (proximal)

UCB (nmol) 1 (0-3) 1 (1-12)

Urobilinoids (nmol) 0 (0-0) 0 (0-1)

Small intestine (medial)

UCB (nmol) 6 (4-9) 46 (8-113)**

Urobilinoids (nmol) 10 (4-12) 3 (2-4)*

Small intestine (distal)

UCB (nmol) 19 (10-37) 59 (27-250)*

Urobilinoids (nmol) 24 (19-34) 31 (11-77)

Cecum

UCB (nmol) 15 (9-71) 41 (5-76)

Urobilinoids (nmol) 126 (40-463) 305 (106-551)

Large intestine

UCB (nmol) 24 (23-73) 76 (15-278)

Urobilinoids (nmol) 159 (38-1018) 99 (84-194)

Total intestine

UCB (nmol) 69 (49-184) 276 (63-719)

Urobilinoids (nmol) 352 (175-1102) 451 (227-703)

106 Chapter 4

4.4.2 Long-term treatment Sustained decrease in plasma UCB concentrations

Next, we investigated the long-term efficacy of PEG treatment with or without phototherapy. Figure 4A/C shows that 2 weeks of PEG treatment decreased plasma UCB concentrations by 41% and accelerated the gastrointestinal transit by 36% (each p<0.001), compared with controls. Additionally, PEG accelerated the small intestinal transit by 17% (p<0.05; data not shown). Figure 4B/D shows that 2 weeks of PEG treatment combined with continuous phototherapy decreased plasma UCB concentrations by 62%, and accelerated the gastrointestinal transit by 31% (each p<0.001), compared with controls. Combined treatment resulted in an additive therapeutic effect of at least 17% from day 2 onward (p<0.01-0.05, for the different time points), compared with single PEG treatment. Orlistat treatment decreased plasma UCB by 33% after 2 weeks (p<0.001; Fig. 2B), which was not significantly different compared with single PEG treatment. PEG administration did not affect growth rate or food intake. Renal parameters indicated the absence of dehydration (Table 3). PEG administration increased water intake by 120%, compared with controls (p<0.001; Table 3).

controls PEG

Diet and growth

Food intake (g/day) 14 ± 2 14 ± 2

Fluid intake (ml/day) 21 ± 2 47 ± 6**

Body weight (g) 330 ± 34 314 ± 23

Relative growth rate (%) 0% 1%

Renal parameters

Creat (mmol/l) 14 ± 4 12 ± 3

Urea (mmol/l) 8.1 ± 1.7 6.1 ± 0.6*

Na (mmol/l) 143 ± 1 143 ± 1

K (mmol/l) 6.0± 0.6 6.2 ± 0.6

Table 3. Diet, growth, and renal parameters after 2 weeks of PEG administration. For experimental details, please see the Materials and Methods section and Figure 4. Body weight, food intake, and water intake were determined daily. Plasma sodium, potassium, urea and creatinine concentrations were determined after 2 weeks of PEG treatment. Data represent mean ± SD. *p<0.05; **p<0.001.

PEG treatment for unconjugated hyperbilirubinemia 107

Figure 4. Long-term PEG administration to Gunn rats: decreases plasma UCB concentrations by 41% (panel A); and accelerates gastrointestinal transit (panel C) after two weeks of treatment. Long-term PEG administration combined with continuous phototherapy (PT) decreases plasma UCB concentrations by 62% (panel B) and decreases gastrointestinal transit time (panel D) after two weeks of treatment. Gunn rats (n=6 per group) were fed the control diet for 6 weeks, followed by: control diet (controls), control diet with PEG administration (via drinking water and via intragastrical gavage), or control diet with PEG administration combined with continuous phototherapy (17 µW/cm2/nm) for a total period of 2 weeks. Data represent mean ± SD. *p<0.05; **p<0.001, compared with controls.

108 Chapter 4

No effect on fecal UCB and urobilinoid excretion

Long-term hypobilirubinemic treatment, for example with phototherapy or intestinal capture agents, will eventually result in a new steady-state situation in which the transiently increased fecal UCB disposal has returned to baseline values. The UCB turnover, however, will be increased in these treated animals because the UCB pool has been diminished (Figure 5).[10,15] Table 4 shows that after two weeks of treatment the fecal UCB and urobilinoid excretion was indeed similar in controls and PEG-treated animals.

Figure 5. Proposed mechanism: According to previous 3H-UCB kinetic studies, ~95% of the total UCB disposal occurs via the feces in untreated Gunn rats [10, 37]. Approximately 20% of this amount enters the intestinal lumen via the bile (1), whereas ~80% enters the lumen via transmucosal diffusion from the plasma (2) (panel A) [10, 37]. Upon starting PEG administration, the intestinal UCB concentration decreases. This results in a translocation of UCB from the plasma into the intestinal lumen (via transmucosal diffusion), from where it is excreted with the feces. Consequently the intestinal UCB content and the fecal UCB excretion will transiently increase upon starting PEG administration (panel B). However, the plasma UCB concentration decreases during treatment, which results in a simultaneous decrease in transmucosal UCB diffusion and, consequently, in fecal UCB excretion. As soon as the transmucosal diffusion and the fecal excretion of UCB have reached pre-treatment values, the plasma UCB concentrations will not decrease any further (i.e. remain stable) and a new steady-state situation is reached. The new steady-state is characterized by a stable decrease in plasma UCB concentrations and a relative increase in UCB turnover, since the PEG-treated animals have a similar fecal excretion (indicating a similar transmucosal diffusion rate) in the presence of a lower plasma UCB concentration (panel C), compared with untreated animals

PEG treatment for unconjugated hyperbilirubinemia 109

controls PEG

Feces

UCB (nmol/h/100g BW) 4.5 ± 1.6 3.1 ± 1.0

Urobilinoids (nmol/h/100g BW) 3.0 ± 1.0 2.5 ± 1.2

Calcium (mmol/h/100g BW) 19 ± 5 15 ± 5

Fat (µmol/h/100g BW) 0.5 ± 0.2 1.0 ± 0.5

Bile acids (nmol/h/100g BW) 224 ± 89 199 ± 70

Bile

UCB (nmol/h/100g BW) 20 ± 8 15 ± 4

Urobilinoids (nmol/h/100g BW) 16 ± 15 10 ± 4

Bile acids (nmol/h/100g BW) 5066 ± 1331

7290 ± 2441

Bile flow (µl/h/100g BW) 211 ± 29 232 ± 52

Table 4. Excretion of several fecal and biliary components after 2 weeks of PEG administration. Gunn rats (n= 6 per group) were fed the control diet for 6 weeks, followed by: control diet (controls), or control diet with PEG administration (via drinking water and via intragastrical gavage) for a total period of 2 weeks. Feces were collected during a 3 day-period before bile canulation. At 2 weeks bile was collected for 30 minutes. Data represent mean ± SD.

Figure 6. Long-term PEG administration to Gunn rats results in: increased α-M as well as decreased HDC in the bile (panel A), and increased α-M, β-M, ω-M as well as decreased HDC and DC in the feces (panel B). C, cholic acid; M, muricholic acid; DC, deoxycholic acid; CDC, chenodeoxycholic acid; HDC, hyodeoxycholic acid; UDC, ursodeoxycholic acid. For experimental setup, kindly refer to Figure 4 and Table 4 and 5. *p<0.05; **p<0.01, ***p<0.001. Data represent mean ± SD.

70%

60%

50%

40%

30%

20%

10%

0%allo-C !-M DC C CDC HDC UDC "-M #22"-M

control

PEG

***

B

70%

60%

50%

40%

30%

20%

10%

0%allo-C !-M DC C CDC HDC UDC "-M #22"-M

control

PEG

$-M

***

***

***

*

*

***

110 Chapter 4

Table 5. Intestinal content composition after 2 weeks of PEG administration. Gunn rats (n= 6 per group) were fed the control diet for 6 weeks, followed by: control diet (controls), or control diet with PEG administration (via drinking water and via intragastrical gavage) for a total period of 2 weeks. At 2 weeks, the intestine was removed and divided into 5 segments (three equal parts of small intestine, the cecum, and the remaining colon) that were flushed with phosphate buffered saline (pH 7.4) for analysis of UCB and urobilinoids. Data represent median and range. *p<0.05.

Figure 7. Loperamide administration to Gunn rats: increases plasma UCB concentrations (panel A); and increases the gastrointestinal transit time (panel B). Gunn rats (n=6 per group) were fed the control diet for 6 weeks, followed by: control diet (controls) or control diet with loperamide administration (7 mg/ml) via intragastrical gavage (1 ml, every 24h) for a total period of 1 week. Gastrointestinal transit time was determined three days before and 1 week after the start of loperamide administration in all animals. Data represent mean ± SD. *p<0.01; **p<0.001.

controls PEG

Small intestine (proximal)

UCB (nmol) 5 (2 - 9) 3 (1 - 8 )

Urobilinoids (nmol) 1 (0 - 4) 1 (0 - 5)

Small intestine (medial)

UCB (nmol) 14 (6 - 36) 17 (8 - 33)

Urobilinoids (nmol) 8 (0 - 23) 2 (0 - 5)

Small intestine (distal)

UCB (nmol) 37 (15 - 43) 31 (25 - 56)

Urobilinoids (nmol) 42 (21 - 54) 37 (13 - 50)

Cecum

UCB (nmol) 53 (26 - 247) 20 (11 - 58)*

Urobilinoids (nmol) 187 (87 - 349) 146 (18 - 416)

Large intestine

UCB (nmol) 54 (35 - 132) 24 (6 - 43)*

Urobilinoid (nmol) 134 (46 - 344) 186 (114 - 422)

Total intestine

UCB (nmol) 183 (130 - 330) 118 (67 - 160)*

Urobilinoids (nmol) 411 (193 - 640) 455 (180 - 596)

PEG treatment for unconjugated hyperbilirubinemia 111

No effect on fecal calcium, fat, and bile acid excretion

An increased fecal excretion of calcium, fatty acids, or bile acids has been associated with decreased plasma UCB concentrations in Gunn rats.[10,15,17] Table 4 shows that two weeks of PEG administration did not influence the fecal excretion of these compounds, nor did it influence biliary bile acid excretion. Figure 6 shows that PEG treatment did decrease the amount of secondary bile salts in the feces and, to a lesser extent, in the bile.

Decrease in the intestinal UCB content, but no effect on biliary UCB excretion

Table 5 shows that PEG administration for two weeks decreased the total intestinal UCB content by 36%, compared with controls (p<0.05). PEG administration decreased the UCB content in the cecum and in the large intestine (-63% and -56%, respectively; each p<0.05), but did not affect the UCB content in the small intestinal segments. As in the short-term experiment, PEG administration did not affect the amount of urobilinoids in the intestinal segments and did not influence the biliary excretion of UCB, urobilinoids, or the bile flow (Table 4).

4.4.3 Loperamide treatment Sustained increase in plasma UCB concentrations

To study the effect of a delayed gastrointestinal transit on plasma UCB concentrations, Gunn rats were treated daily for one week with loperamide. Figure 7A shows that loperamide increased plasma UCB concentrations by 30% after one week, compared with controls (p<0.001). Loperamide treatment showed a statistically significant effect within 2 days. Loperamide increased the gastrointestinal transit time by 57% after one week (p<0.001, Figure 7B), compared with stable values in controls. Mean body weight and water intake did not differ significantly between the loperamide-treated animals and controls. Loperamide administration decreased the food intake by approximately 40%, compared with the controls (p<0.001; data not shown).

112 Chapter 4

Figure 8. Gastrointestinal transit time and plasma UCB concentrations in Gunn rats were strongly, positively correlated. Gunn rats (n=6) were fed the control diet for 6 weeks, followed by: 9 days of PEG administration via drinking water; 9 days of PEG administration via drinking water and via intragastrical gavage (2.5 ml, every 24h); and 9 days of PEG administration via drinking water and via intragastrical gavage (5.0 ml, every 24h), for a total period of 27 days (three consecutive 9 day-periods). The gastrointestinal transit time was determined at the end of every 9-day period. For experimental setup in the loperamide experiment, please refer to Figure 7. PEG dose-response data points: y=15*x+80; r=0.75; p<0.001 (line not shown). PEG dose-response data points combined with loperamide data points: y=13*x+91; r=0.87, p<0.001.

4.4.4 Dose-response experiment Strong, positive correlation between plasma UCB concentration and the gastrointestinal transit time

To determine the relationship between the plasma UCB concentrations and the gastrointestinal transit time in more detail, we performed a dose-response experiment. We administered PEG in increasing dosages to Gunn rats in three consecutive 9-day periods. Already at the lowest dose (PEG in drinking water) the plasma UCB concentration was decreased by 22% (p<0.01), compared with baseline values. At the highest used dose (PEG in drinking water + daily 5.0 ml gavage), the plasma UCB concentration was decreased by 32% (p<0.001), compared with baseline values. Figure 8 shows the strong, positive correlation between gastrointestinal transit time and plasma UCB concentrations in Gunn rats during the dose-response experiment (y=15*x+80; r=0.75; p<0.001). Upon inclusion of the data obtained in the loperamide experiment, this relationship remained essentially unaffected (y=13*x+91; r=0.87, p<0.001). After 27 days of PEG administration, the fecal excretion of UCB, urobilinoids, calcium, or fat, did not increase whereas the fecal excretion of bile acids decreased (-30%; p<0.01) compared with baseline values (Table 6). PEG treatment did not affect body weight or growth rate (data not shown).

PEG treatment for unconjugated hyperbilirubinemia 113

baseline treatment

Feces

UCB (nmol/h/100g BW) 7.2 ± 6.4 5.4 ± 5.8

Urobilinoids (nmol/h/100g BW) 3.2 ± 0.8 3.3 ± 0.9

Calcium (mmol/h/100g BW) 30 ± 5 26 ± 2

Fat (µmol/h/100g BW) 1.4 ± 0.6 0.9 ± 0.3

Bile salts (nmol/h/100g BW) 233 ± 43 164 ± 20*

Table 6. Excretion of several fecal components after 27 days of administration of increasing amounts of PEG. For experimental details, please see the Materials and Methods section and Figure 8. Feces were collected during a 3 day-period before starting PEG administration (baseline period), and a 3 day-period before termination at 27 days (treatment period). Data represent mean ± SD. *p<0.01.

4.5 Discussion In this study we demonstrate that acceleration of the gastrointestinal transit by PEG administration effectively decreases unconjugated hyperbilirubinemia in Gunn rats. The effect occurred within 12h, was maximal within one week, and was sustained during long-term treatment. Delaying the gastrointesintal transit with loperamide increased plasma UCB levels in Gunn rats, confirming the important role of transit time in bilirubin metabolism.

Our results show that the gastrointestinal transit time and plasma UCB concentrations are linearly related in Gunn rats. This relationship has two important implications. Firstly, it suggests that pharmacological manipulation of the gastrointestinal transit time is a feasible strategy to treat severe unconjugated hyperbilirubinemia. Secondly, it indicates that the gastrointestinal transit time is an important regulator for plasma UCB concentrations under (patho)physiological conditions. Interestingly, our findings corroborate previous observations in animals and humans. Kotal et al. showed in Gunn and Wistar rats that fasting-induced hyperbilirubinemia, a well-known condition in humans,[20,30] was associated with a delayed gastrointestinal transit.[18] Feeding fasted Wistar rats non-absorbable bulk (kaolin dissolved in water with magnesium sulphate) normalized the gastrointestinal transit and prevented the fasting-induced increase in plasma UCB.[18] Human conditions that delay the gastrointestinal passage of meconium, such as fasting,[19,20] pyloric stenosis,[21] and Hirschprung’s disease,[22] are associated with exaggeration of unconjugated

114 Chapter 4

hyperbilirubinemia. Conditions that accelerate the passage of meconium, such as frequent and early feedings,[31-33] or rectal stimulation,[34] seem to lower plasma bilirubin concentrations. Importantly, a recent study showed that supplementing infant formula with prebiotic oligosaccherides, which has been reported to accelerate the gastrointestinal transit,[23] decreased plasma UCB concentrations in neonates.[24] Any specific prebiotic effect on bilirubin metabolism cannot be excluded a priori in this study, but is not very likely since oligosaccharides have no clear effect on bilirubin converting bacteria.[24,35] These observations (Table 1) clearly suggest that maneuvers that modify the gastrointestinal transit influence plasma UCB levels in humans. However, these studies do not show a direct influence of the gastrointestinal transit time on plasma UCB concentrations. In the animal study by Kotal, changes in gastrointestinal transit were secondary to fasting, which increases the possibility of unknown confounders.[18] In the human studies, the gastrointestinal transit time was not quantified. Our data are the first to demonstrate a causal relationship between the gastrointestinal transit time and plasma UCB concentrations, using both pharmacological acceleration and pharmacological inhibition.

Theoretically, the hypobilirubinemic effect of PEG could be mediated by mechanisms other than the acceleration of transit time. It has been demonstrated that an increased fecal excretion of calcium,[15] fat,[17] or bile acids,[10] coincides with a decrease in plasma UCB concentrations, presumably because these agents trap UCB in the intestinal lumen. PEG treatment, however, did not increase the fecal excretion of any of these compounds. The decreased amount of secondary biliary and fecal bile acids could indicate an altered activity of microflora in the colon, which might affect the bacterial degradation of UCB to urobilinoids.[36] PEG treatment, however, did not influence the amount of urobilinoids in the intestinal lumen, the feces, or the bile in any of the experiments, which does not support the mechanistic relevance of this possibility. The altered bile salt composition could, theoretically, also affect the transmucosal UCB diffusion from the blood into the intestinal lumen. However, we previously showed in Gunn rats that the increase in transmucosal UCB diffusion during bile acid feeding does not depend on an altered fecal or biliary bile acid profile, but rather on the increased total fecal bile acid excretion, which remained unaffected in this experiment.[10] Finally, and most importantly, we treated Gunn rats with both PEG and loperamide. By doing so, we could demonstrate a strong, linear relationship between transit time and plasma UCB concentrations. This relationship remained stable during pharmacological manipulation of the gastrointestinal transit that was not only bi-directional, but also occurred via two pharmacologically distinct mechanisms. The contribution of fasting appeared limited in loperamide treatment, since the delay in gastrointestinal transit and increased unconjugated hyperbilirubinemia became apparent (at day 2) prior to any decrease in food intake. Taken together, the data suggest that the

PEG treatment for unconjugated hyperbilirubinemia 115

gastrointestinal transit time in the individual rats may also underlie their inter-individual variation in basal plasma UCB concentrations. It is tempting to speculate that this could be extrapolated to the human conditions, for example in Gilbert’s syndrome or neonatal jaundice.

PEG seemed to decrease plasma UCB levels by enhancing its disposal with the feces, based on the increased fecal UCB excretion in the first 36h of treatment. The enhanced fecal disposal of UCB can only originate from an increase in biliary UCB excretion and/or from an increase in transmucosal UCB diffusion, since UCB exclusively enters the intestinal lumen via these two pathways.[6-10] The increase in intestinal UCB content after 36h of PEG administration, however, was not accompanied by an increase in biliary UCB excretion. This finding strongly suggests that the hypobilirubinemic effect of PEG treatment was due to a selective increase in transmucosal UCB diffusion (Figure 5). Transmucosal UCB diffusion occurs bi-directionally (e.g. from blood to gut lumen and vice-versa) in the small and large intestine of Gunn rats.[6] Normally, the net UCB flux is directed from the blood into the intestinal lumen, thus constituting for ~80% of the total fecal UCB disposal in untreated Gunn rats, as demonstrated by steady-state 3H-UCB kinetic experiments.[10] Its direction is reversed (i.e. from the lumen into the blood) in fasting conditions, which delay the intestinal transit and thereby increase the intestinal UCB concentration. This results in a net reabsorption of UCB into the enterohepatic circulation, as reflected by the marked hyperbilirubinemia and increased biliary UCB excretion in fasted Gunn and Wistar rats.[18] We hypothesize that PEG treatment decreases the intraluminal UCB concentration, by flushing the intestine, which results in enhanced net UCB diffusion into the intestinal lumen. We could not directly validate that PEG decreased the intraluminal UCB concentration, since we needed to rinse the intestine with phosphate buffered saline in order to collect its content. However, the impressive additive therapeutic effect of phototherapy on PEG treatment is in concordance with our hypothesis. The reason for this is that combining two treatments that enhance the same route of disposal (e.g. biliary UCB excretion) will reach the maximal disposal rate sooner than combining treatments that maximize two distinct routes of UCB disposal (e.g. biliary and transmucosal UCB disposal). Previous studies using 3H-labelled UCB have indeed shown that the therapeutic effect of phototherapy, which exclusively increases biliary excretion, is greatly enhanced by treatments that exclusively increase transmucosal UCB excretion.[37,38] The proposed mechanism by which PEG decreases plasma UCB concentrations has been outlined in Figure 5.

Our results support the clinical applicability of oral PEG treatment in patients with severe unconjugated hyperbilirubinemia. PEG treatment decreased plasma UCB more rapidly than did orlistat treatment, which is a well-known experimental oral treatment strategy for unconjugated hyperbilirubinemia. This

116 Chapter 4

rapid decrease supports the clinical feasibility of PEG treatment in hyperbilirubinemic neonates, in which it could prevent hyperbilirubinemia due to delayed meconium excretion. The sustainability of the PEG-induced decrease in plasma UCB concentrations clearly supports its clinical use in Crigler-Najjar patients. Importantly, the combination of phototherapy with PEG resulted in a therapeutic efficacy that was not only superior to single PEG treatment, but also to treatment combinations that were explored in comparable Gunn rat experiments.[10,12,13,15-17,38] PEG treatment was well-tolerated by all animals, and no diarrhea or dehydration was observed. PEG is presently widely applied for the treatment of constipation and is well-tolerated by both adults and children.[39,40] Numerous clinical trials with PEG have shown an absence of serious side effects, and a milder side effect profile compared with other laxatives.[41]

In conclusion, acceleration of the gastrointestinal transit time by PEG effectively treats unconjugated hyperbilirubinemia in Gunn rats. The underlying mechanism involves stimulation of the transmucosal excretion and the subsequent fecal disposal of UCB. Present results support the feasibility of PEG treatment in hyperbilirubinemic patients. The long-term efficacy (e.g. prevention of UCB-induced neurological damage) and safety (e.g. absorption of water and nutrients) of PEG treatment will need to be evaluated in future clinical trials.[42]

Acknowledgement: The authors wish to acknowledge dr. C.V. Hulzebos for valuable discussions and critical reading of the manuscript, and Maarten Hogeweg for his assistance with HPLC determinations.

PEG treatment for unconjugated hyperbilirubinemia 117

References1. Crigler JF, Najjar VA. Congenital

familial nonhemolytic jaundice with kernicterus. Pediatrics. 1952;10:169–180.

2. Shapiro SM. Definition of the clinical spectrum of kernicterus and bilirubin-induced neurologic dysfunction (BIND). J Perinatol. 2005;25:54–59.

3. Ostrow JD. Photocatabolism of labeled bilirubin in the congenitally jaundiced (Gunn) rat. J. Clin. Invest. 1971;50:707–718.

4. Van der Veere CN, Sinaasappel M, McDonagh AF, Rosenthal P, Labrune P, Odievre M, et al. Current therapy for Crigler-Najjar syndrome type 1: report of a world registry. Hepatology. 1996;24:311–315.

5. Yohannan MD, Terry HJ, Littlewood JM. Long term phototherapy in Crigler-Najjar syndrome. Arch Dis Child. 1983;58:460–462.

6. Kotal P, Van der Veere CN, Sinaasappel M, Elferink RO, Vitek L, Brodanova M, et al. Intestinal excretion of unconjugated bilirubin in man and rats with inherited unconjugated hyperbilirubinemia. Pediatr. Res. 1997;42:195–200.

7. Lester R, Schmid R. Intestinal absorption of bile pigments. I. The enterohepatic circulation of bilirubin in the rat. J. Clin. Invest. 1963;42:736–746.

8. Lester R, Schmid R. Intestinal absorption of bile pigments. II. Bilirubin absorption in man. N. Engl. J. Med. 1963;269:178–182.

9. Schmid R, Hammaker L. Metabolism and disposition of C14-bilirubin in congenital nonhemolytic jaundice. J. Clin. Invest.

1963;42:1720–1734.

10. Cuperus FJC, Hafkamp AM, Havinga R, Vitek L, Zelenka J, Tiribelli C, et al. Effective treatment of unconjugated hyperbilirubinemia with oral bile salts in gunn rats. Gastroenterology. 2009;136:673–82 e1.

11. Odell GB, Gutcher GR, Whitington PF, Yang G. Enteral administration of agar as an effective adjunct to phototherapy of neonatal hyperbilirubinemia. Pediatr. Res. 1983;17:810–814.

12. Lester R, Hammaker L, Schmid R. A new therapeutic approach to unconjugated hyperbilirubinaemia. Lancet. 1962;280:1257.

13. Davis DR, Yeary RA, Lee K. Activated charcoal decreases plasma bilirubin levels in the hyperbilirubinemic rat. Pediatr. Res. 1983;17:208–209.

14. Van der Veere CN, Jansen PL, Sinaasappel M, Van Der Meer R, Van der Sijs H, Rammeloo JA, et al. Oral calcium phosphate: a new therapy for Crigler-Najjar disease? Gastroenterology. 1997;112:455–462.

15. Van der Veere CN, Schoemaker B, Bakker C, Van Der Meer R, Jansen PL, Elferink RP. Influence of dietary calcium phosphate on the disposition of bilirubin in rats with unconjugated hyperbilirubinemia. Hepatology. 1996;24:620–626.

16. Vitek L, Muchova L, Zelenka J, Zadinova M, Malina J. The effect of zinc salts on serum bilirubin levels in hyperbilirubinemic rats. J Pediatr Gastroenterol Nutr. 2005;40:135–140.

17. Hafkamp AM, Havinga R, Sinaasappel M, Verkade HJ. Effective oral treatment of unconjugated

118 Chapter 4

hyperbilirubinemia in Gunn rats. Hepatology. 2005;41:526–534.

18. Kotal P, Vitek L, Fevery J. Fasting-related hyperbilirubinemia in rats: the effect of decreased intestinal motility. Gastroenterology. 1996;111:217–223.

19. Bertini G, Dani C, Tronchin M, Rubaltelli FF. Is breastfeeding really favoring early neonatal jaundice? Pediatrics. 2001;107:41–45.

20. Gilbert A, Herscher M. Sur les variations de la cholemie physiologique. Presse Med. 1906;14:209–211.

21. Etzioni A, Shoshani G, Diamond E, Zinder O, Bar-Maor JA. Unconjugated hyperbilirubinaemia in hypertrophic pyloric stenosis, an enigma. Z Kinderchir. 1986;41:272–274.

22. Gartner LM. Breastfeeding and jaundice. J Perinatol. 2001;21 Suppl 1:S25–9; discussion S35–9.

23. Mihatsch WA, Hoegel J, Pohlandt F. Prebiotic oligosaccharides reduce stool viscosity and accelerate gastrointestinal transport in preterm infants. Acta Paediatr. 2006;95:843–848.

24. Bisceglia M, Indrio F, Riezzo G, Poerio V, Corapi U, Raimondi F. The effect of prebiotics in the management of neonatal hyperbilirubinaemia. Acta Paediatr. 2009;98:1579–1581.

25. Lin M, Wu N, Aiken JH. Micellar high-performance liquid separation of serum bilirubin species with direct sample injection. Journal of liquid chromatography. 1995;18:1219–1229.

26. Singh J, Bowers LD. Quantitative fractionation of serum bilirubin species by reversed-phase high-performance liquid chromatography. J.Chromatogr.B 1986;380:321.

27. Murphy GM, Billing BH, Baron DN.

A fluorimetric and enzymatic method for the estimation of serum total bile acids. J Clin Pathol. 1970;23:594–598.

28. Kotal P, Fevery J. Quantitation of urobilinogen in feces, urine, bile and serum by direct spectrophotometry of zinc complex. Clin. Chim. Acta. 1991;202:1–10.

29. Lepage G, Roy CC. Direct transesterification of all classes of lipids in a one-step reaction. J. Lipid Res. 1986;27:114–120.

30. Whitmer DI, Gollan JL. Mechanisms and significance of fasting and dietary hyperbilirubinemia. Semin Liver Dis. 1983;3:42–51.

31. De Carvalho M, Klaus MH, Merkatz RB. Frequency of breast-feeding and serum bilirubin concentration. Am J Dis Child. 1982;136:737–738.

32. Wennberg RP, Schwartz R, Sweet AY. Early versus delayed feeding of low birth weight infants: effects on physiologic jaundice. J. Pediatr. 1966;68:860–866.

33. Wu PY, Teilmann P, Gabler M, Vaughan M, Metcoff J. "Early" versus “late” feeding of low birth weight neonates: effect on serum bilirubin, blood sugar, and responses to glucagon and epinephrine tolerance tests. Pediatrics. 1967;39:733–739.

34. Cottrell BH, Anderson GC. Rectal or axillary temperature measurement: effect on plasma bilirubin and intestinal transit of meconium. J Pediatr Gastroenterol Nutr. 1984;3:734–739.

35. Costalos C, Kapiki A, Apostolou M, Papathoma E. The effect of a prebiotic supplemented formula on growth and stool microbiology of term infants. Early Hum. Dev. 2008;84:45–49.

36. Vitek L, Zelenka J, Zadinova M,

PEG treatment for unconjugated hyperbilirubinemia 119

Malina J. The impact of intestinal microflora on serum bilirubin levels. J. Hepatol. 2005;42:238–243.

37. Ostrow JD. Photochemical and biochemical basis of the treatment of neonatal jaundice. Prog Liver Dis. 1972;4:447–462.

38. Hafkamp AM, Havinga R, Ostrow JD, Tiribelli C, Pascolo L, Sinaasappel M, et al. Novel kinetic insights into treatment of unconjugated hyperbilirubinemia: phototherapy and orlistat treatment in Gunn rats. Pediatr. Res. 2006;59:506–512.

39. Di Palma JA, Cleveland MV, McGowan J, Herrera JL. An open-label study of chronic polyethylene glycol laxative use in chronic constipation. Aliment. Pharmacol. Ther. 2007;25:703–708.

40. Nurko S, Youssef NN, Sabri M, Langseder A, McGowan J, Cleveland M, et al. PEG3350 in the treatment of childhood constipation: a multicenter, double-blinded, placebo-controlled trial. J. Pediatr. 2008;153:254–61, 261 e1.

41. Klaschik E, Nauck F, Ostgathe C. Constipation--modern laxative therapy. Support Care Cancer. 2003;11:679–685.

42. Ponz de Leon M, Iori R, Barbolini G, Pompei G, Zaniol P, Carulli N. Influence of small-bowel transit time on dietary cholesterol absorption in human beings. N. Engl. J. Med. 1982;307:102–10

120 Chapter 4

Chapter 5 Combined Treatment Strategies for Unconjugated Hyperbilribunemia in Gunn Rats

F.J.C. Cuperus 1

A.A. Iemhoff 1

H.J. Verkade 1

1 Pediatric Gastroenterology and Hepatology, Department of Pediatrics, Center for Liver, Digestive, and Metabolic Diseases, Beatrix Children’s Hospital - University Medical Center Groningen, University of Groningen, The Netherlands.

Accepted for publication in Pediatric Research

122 Chapter 5

5.1 Abstract e recently demonstrated that acceleration of the gastrointestinal transit by polyethylene glycol (PEG) treats unconjugated hyperbilirubinemia in jaundiced Gunn rats. It is unclear whether acceleration of gastrointestinal transit also (partly) underlies the therapeutic effects of established hypobilirubinemic treatments, or whether PEG co-treatment might enhance these effects. We treated Gunn rats with phototherapy (17µW/cm2/nm), orlistat (200mg/kg chow), ursodeoxycholate (5g/kg chow), or calcium phosphate (20g/kg chow) either as single treatment, or in combination with PEG. Three weeks of phototherapy, orlistat, ursodeoxycholic acid or calcium phosphate treatment decreased plasma unconjugated bilirubin (UCB) levels by 47%, 27%, 28%, and 45%, respectively (each p<0.001), without a significant impact on gastrointestinal transit time. PEG co-treatment accelerated the gastrointestinal transit in all treatment groups, which resulted in an additive hypobilirubinemic effect of -20% and -26% (final plasma UCB -67% and -53%, respectively) in phototherapy and orlistat-treated animals. PEG co-treatment did not enhance the hypobilirubinemic effect of ursodeoxycholic acid or calcium phosphate. This chapter demonstrates that phototherapy, orlistat, ursodoxycholic acid and calcium phosphate do not exert their hypobilirubinemic effect via acceleration of the gastrointestinal transit. PEG co-treatment enhanced the hypobilirubinemic effects of phototherapy, and of orlistat treatment. Current results support the feasibility of PEG co-treatment during phototherapy in hyperbilirubinemic patients.

W

Combined treatment for unconjugated hyperbilirubinemia 123

5.2 Introduction eonatal hemolytic jaundice and Crigler-Najjar disease are characterized by severe unconjugated hyperbilirubinemia. In Crigler-Najjar patients, a genetically absent (type I) or decreased (type II) ability to conjugate bilirubin within the liver, results in lifelong jaundice.[1,2] Unconjugated bilirubin (UCB) is neurotoxic, and severe unconjugated hyperbilirubinemia is associated with brain damage.[3] This damage occurs because non-protein bound plasma bilirubin (UCBfree) can diffuse across the blood-brain barrier.[4-10] To decrease plasma UCB levels, Crigler-Najjar disease patients rely on lifelong phototherapy, the routine treatment for unconjugated hyperbilirubinemia. Phototherapy, however, becomes less effective with age and eventually fails to prevent bilirubin-induced brain damage in up to 25% of Crigler-Najjar patients.[11,12] This prompted us to develop alternative, and adjunct, treatment strategies for severe unconjugated hyperbilirubinemia.

Severe unconjugated hyperbilirubinemia allows direct transmucosal diffusion of UCBfree from the blood into the intestine.[13-15] The efficacy of this pathway is decreased, however, by the intestines’ ability to reabsorb UCBfree from its lumen.[13,16] We recently hypothesized that acceleration of the gastrointestinal transit might interfere with this ability, and thus enhance the efficacy of transmucosal bilirubin disposal. Indeed, the laxative polyethylene glycol (PEG) proved an effective treatment for unconjugated hyperbilirubinemia in Gunn rats, the animal model for Crigler-Najjars disease.[17] Importantly, our experiments also revealed a strong linear relation between the gastrointestinal transit time and plasma UCB levels in these animals.[14]

Our results raised the question whether other treatments also (partly) rely on acceleration of the gastrointestinal transit for their hypobilirubinemic effects. Interestingly, previous findings have suggested that phototherapy, as well as oral treatments that enhance transmucosal UCB diffusion (e.g. orlistat,[18,19] bile salts,[14] or amorphous calcium phosphate (CaP)[20]), might indeed influence transit time.[21-25] We presently show that neither phototherapy, nor oral treatment with orlistat, ursodeoxycholic acid (UDCA), or CaP affects the gastrointestinal transit time in Gunn rats. We also show that addition of PEG to either phototherapy or orlistat treatment enhances their hypobilirubinemic effect.

N

124 Chapter 5

5.3 Animals, materials, and methods

5.3.1 Animals Homozygous adult male Gunn rats (RHA/jj; 244-375 g; n=60) were obtained from our breeding colony at the University Medical Center Groningen (UMCG, The Netherlands). Animals, housed individually in an environmentally-controlled facility with a diurnal (12/12 hour) light cycle, were fed ad libitum and had free access to water. The Ethics Committee for Animal Experiments of the UMCG approved the experimental protocols for this study.

5.3.2 Materials Diets

Hope Farms BV (Woerden, The Netherlands) produced all diets. The semi-synthetic control diet (code 4063.02), contained 13 energy % fat.[18] Supplemented diets were identical to the control diet, except for the supplementation of orlistat (200 mg/kg chow), UDCA (5 g/kg chow), or CaP (20 g/kg chow). Gunn rats were fed the semi-synthetic purified control diet (4063.02) during a 6-week run-in period prior to the experiments.[14]

Chemicals

PEG 4000 (Colofort) was obtained from Ipsen Farmaceutica BV (Hoofddorp, The Netherlands). Colofort contained per sachet (74 g): 64 g PEG, 5.7 g sodium sulphate (anhydric), 1.68 g sodium bicarbonate, 1.46 g sodium chloride and 0.75 g potassium chloride. We dissolved one sachet of PEG 4000 (Colofort) in 900 mL water to obtain the PEG solution we used in our experiments (drinking water solution and gavage solution). Orlistat (Xenical®) was obtained from Roche Nederland BV (Woerden, The Netherlands). UDCA was a generous gift from Dr. Falk Pharma GmbH (Freiburg, Germany). Carmine red dye was obtained from Macro-imPulse Saveur Ltd. (Stadtoldendorf, Germany).

Phototherapy lamps

Phototherapy devices were designed according the prototype by Ostrow.[26] Two phototherapy lamps (Philips TL-20W/03T) were suspended in a reflective

Combined treatment for unconjugated hyperbilirubinemia 125

framework 20 cm above the bottom of the cage. The light intensity of phototherapy was measured by a Dale 40 phototherapy radiometer (Dale Technologies, Carson City, NV) at 20 cm distance from the light source, and was set to 17 µW/cm2/nm. Gunn rats, shaven on their backs and flanks every 7 days, received continuous phototherapy.[27]

5.3.3 Methods Long-term experiment

Male Gunn rats were randomized (n=5-6 per group) to receive no treatment (controls) or treatment with phototherapy (17 µW/cm2/nm), orlistat (200 mg/kg chow), UDCA (5 g/kg chow), or CaP (20 g/kg chow). Body weight was determined weekly during the experiments. After a 3-week treatment period, which ensured the presence of steady-state conditions, [14,17-19] we determined the gastrointestinal transit time in untreated (controls) and treated Gunn rats by measuring the interval between intragastrical administration of carmine red and its appearance in the feces.[14] Heparinized samples of tail vein blood were obtained under isoflurane anesthesia before (baseline), and 3 weeks after randomization to determine steady-state plasma bilirubin concentrations. After 3 weeks, these treatments were combined with PEG, both via drinking water and via intragastrical gavage (2.5 mL, every 12h). The gastrointestinal transit time was determined again 6 weeks after randomization (3 weeks after the addition of PEG). A final blood sample was then obtained, as described above, for the determination of bilirubin, urea (Ur), creatinine (Creat), aspartate aminotransferase (AST), and alanine aminotransferase (ALT).

Short-term experiment

Male Gunn rats were randomized (n=6-7 per group) to receive no treatment (controls), phototherapy (17 µW/cm2/nm), phototherapy (17 µW/cm2/nm) combined with PEG (via drinking water and via 2.5 mL gavage), orlistat (200 mg/kg chow), or orlistat (200 mg/kg chow) combined with PEG (via drinking water and via 2.5 mL gavage). Body weight was determined daily during the experiments. Carmine red was administered directly after the start of PEG administration in order to determine the gastrointestinal transit as described above. Heparinized samples of tail vein blood were obtained under isoflurane anesthesia every 12-hour for the first 48 hours for the determination of bilirubin, Ur, Creat, AST, and ALT. A final blood sample was obtained 14 days after randomization.

126 Chapter 5

Plasma analysis

Blood samples were protected from light and processed immediately. Bilirubin, Ur, Creat, AST and ALT were determined by routine spectrophotometry on a P800 unit of a modular analytics serum work area from Roche Diagnostics Ltd. (Basel, Switzerland). We previously found that the total bilirubin concentration in Gunn rat plasma, measured by spectrophotometry, equaled the total UCB concentration, measured by high-liquid performance chromatography after chloroform extraction.[18,27]

Statistical analysis

All data were normally distributed, displayed homogeneity of variance by calculation of Levene’s statistic, and were expressed as mean ± SD. Analysis of variance (ANOVA) with post-hoc Bonferroni correction was performed for comparison between groups. Students t test was used for comparison of paired data within groups. The level of significance was set at p<0.05. Analyses were performed using SPPS 16.0 for Mac (SPSS Inc., Chicago, IL).

5.4 Results

5.4.1 Long-term experiment Neither phototherapy nor oral treatments affect gastrointestinal transit time

Since acceleration of gastrointestinal transit decreases plasma bilirubin concentrations in Gunn rats, we assessed whether phototherapy and known oral treatments exert their hypobilirubinemic effects, either partly or exclusively, by acceleration of the gastrointestinal transit. Figure 1 shows that 3 weeks of phototherapy, orlistat, UDCA, or CaP decreased plasma bilirubin concentrations by 47%, 27%, 28%, and 45%, respectively, compared with baseline values (p<0.001). Figure 2 shows that, after 3 weeks of treatment, neither phototherapy, nor oral treatments with orlistat, UDCA, or CaP significantly affected the gastrointestinal transit time, compared with controls.

Combined treatment for unconjugated hyperbilirubinemia 127

PT

Figure 2. Three weeks of continuous phototherapy (PT), or oral treatment with orlistat (Orl), UDCA, or CaP did not significantly affect the gastrointestinal transit time. Gunn rats (n= 5-6 per group) received either no treatment or treatment with phototherapy, orlistat, UDCA, or CaP. After three weeks we determined the gastrointestinal transit time by measuring the interval between intragastrical administration of carmine red and its appearance in the feces.

†

†PT

PT

Time (wks)

Figure 1. Treatment with continuous phototherapy (PT), or oral treatment with orlistat (Orl), UDCA, or CaP significantly decreased plasma bilirubin concentrations after 3 wks and PEG induced an additive therapeutic effect in phototherapy and orlistat treated animals. Gunn rats (n= 5-6 per group received either no treatment or treatment with phototherapy, orlistat, UDCA, or CaP. After three weeks, these treatments were combined with PEG administration for an additional 3 weeks. *p<0.001, compared with baseline values. †p<0.01, compared with single treatment.

128 Chapter 5

Additive therapeutic effect of PEG administration on phototherapy and orlistat treatment

Since neither phototherapy nor any of the oral treatments accelerated the gastrointestinal transit, we assessed whether adjunct PEG treatment could increase their hypobilirubinemic effect. Figure 3 shows that combining phototherapy, orlistat, UDCA, and CaP, with PEG administration for 3 weeks accelerated the gastrointestinal transit by 40%, 33%, 33%, and 28%, respectively, compared with controls (p<0.001; each). Figure 1 shows that 3 weeks of combined treatment of PEG with phototherapy, orlistat, UDCA, or CaP decreased plasma bilirubin concentrations by 67%, 53%, 30%, and 45%, respectively, (p<0.001). Combining phototherapy or orlistat treatment with PEG thus induced an additive therapeutic effect of 20% and 26%, respectively, compared with single treatment (p<0.01). The hypobilirubinemic effect of PEG + phototherapy is in line with our previous results, which showed a 62%-decrease in plasma bilirubin levels after 2 weeks of phototherapy + PEG treatment.(16) Table 1 shows effect of PEG co-treatment on renal function, liver function, and growth rate in the various treatment groups. Addition of PEG to phototherapy, orlistat, UDCA, or CaP did not affect plasma levels of urea, compared with controls, which pleaded against clinically relevant dehydration during PEG co-treatment. PEG + orlistat treatment resulted in a mild increase in plasma creatinine levels compared with the control group, although still within the reference range for Gunn rats.[28] UDCA + PEG treated animals showed a 5%-decrease in growth rate during the entire experiment, compared with a 2.5%-increase in control animals (p<0.01; Table 1).

controls PT + PEG Orl + PEG UDCA + PEG Ca + PEG

Renal parameters

Creat (mmol/L) 17 ± 9 23 ± 12 31 ± 11* 11 ± 3 11 ± 4

Urea (mmol/L) 7.7 ± 2.0 6.0 ± 1.3 7.1 ± 0.9 6.1 ± 1.2 6.9 ± 0.9

Liver parameters

AST (Unit/L) 96 ± 37 74 ± 11 86 ± 17 120 ± 37 98 ± 29

ALT Unit/L) 35 ± 25 21 ± 7 21 ± 10 29 ± 9 28 ± 6

Growth parameters

Growth rate (%, compared with T=0 wks)

2.5 ± 4.7 -1.9 ± 4.5 -2.0 ± 2.7 -5.3 ± 1.4** -1.3 ± 2.2

Table 1. Renal and liver parameters at after 6 weeks of treatment. For experimental setup, please refer to Figure 1. Plasma urea, creatinine, AST, ALT concentrations and growth rate were determined after 6 weeks of treatment. Data represent mean ± SD. *p<0.05, **p<0.01, compared with controls. PT, phototherapy; Orl, Orlistat.

Combined treatment for unconjugated hyperbilirubinemia 129

5.4.2 Short-term experiment Immediate PEG co-treatment with orlistat is effective within days

To further explore its clinical potential, we subsequently determined whether immediate PEG co-treatment could accelerate the hypobilirubinemic effect of phototherapy or orlistat during a short-term period of treatment. Figure 4A shows that phototherapy alone decreased plasma bilirubin concentrations by 35% within 48h (p<0.001), compared with control animals. Immediate co-treatment with PEG accelerated the gastrointestinal transit by 28% (p<0.001; Fig 4B), and decreased plasma bilirubin concentrations by 37% (p<0.001; Fig 4A), compared with controls. Figure 4C shows that orlistat alone decreased plasma bilirubin concentrations by 10% (NS), and that co-treatment with PEG decreased plasma bilirubin by 24% (p<0.001), compared with control animals. PEG thus did not enhance the rapid hypobilirubinemic effect of phototherapy, but did increase that of orlistat treatment by 14% (p<0.05). Continuation of treatment in the PEG + phototherapy-treated animals for 2 weeks, however, did result in a final decrease of 67%, which was virtually identical to the decrease in the long-term experiment. Table 2 shows that there were no differences renal function, liver function or growth rates between the various treatment groups.

PT

Figure 3. Combining phototherapy (PT), orlistat (Orl), UDCA, and CaP, with PEG administration significantly accelerated the gastrointestinal transit. Gunn rats (n=5-6 per group) received either no treatment or treatment with phototherapy, orlistat, UDCA, or CaP. After three weeks, these treatments were combined with PEG administration for an additional 3 weeks. After six weeks we determined the gastrointestinal transit time as described in Fig 2. *p<0.001, compared with controls.

130 Chapter 5

14

12

10

8

6

4

2

0

control

FT

FT+P

EG

Ga

str

oin

testin

al tr

an

sit t

ime

(h

)

**

¶

B

14

12

10

8

6

4

2

0

control

O

rl

O

rl+P

EG

Ga

str

oin

testin

al tr

an

sit t

ime

(h

)

**

D

50%

60%

70%

80%

90%

100%

110%

120%

0 12 24 36 48

* **/§

A

C

50%

60%

70%

80%

90%

100%

110%

120%

0 12 24 36 48

Time (h)

*

*

** **

/†/†

**/§

**/‡ ** **

Time (h)

p= 0.057

*

Figure 4. PEG co-treatment induces an additive hypobilirubinemic effect during orlistat (Orl) treatment (panel A/C), and accelerates the gastrointestinal transit in all treatment groups (panel B/D). Gunn rats (n=6-7 per group) received either no treatment or treatment with phototherapy, phototherapy+PEG, orlistat, or orlistat+PEG. The gastrointestinal transit was determined by administration of carmine red directly after the start of treatment as in figure 2. *p<0.05; **p<0.001, compared with controls. ¶p<0.001, compared with single phototherapy treatment. §p<0.05; ‡p<0.01; †p<0.001, compared with single orlistat treatment. Controls; phototherapy (PT); phototherapy+PEG; orlistat (Orl); orlistat+PEG.

Combined treatment for unconjugated hyperbilirubinemia 131

controls PT PT + PEG Orl Orl + PEG

Renal parameters

Creat (mmol/l) 16 ± 3 13 ± 2 13 ± 2 19 ± 4 16 ± 4

Urea (mmol/l) 8.6 ± 1.8 5.5 ± 0.7** 6.9 ± 1.5 10 ± 2 5.8 ± 0.9*

Liver parameters

AST (Unit/L) 95 ± 26 95 ± 17 88 ± 25 125 ± 45 95 ± 13

ALT Unit/L) 55 ± 21 38 ± 7 43 ± 7 73 ± 12 60 ± 12

Growth parameters

Growth rate (%, compared with T=0 wks)

0.4 ± 2.4 -0.1 ± 0.0 3.7 ± 2.0 4.4 ± 1.4 0.3 ± 2.5

Table 2. Renal and liver parameters at after 2 weeks of treatment. For experimental setup, please refer to Figure 3. Plasma urea, creatinine, AST, and ALT concentrations and growth rate were determined after 2 weeks of treatment. Data represent mean ± SD. *p<0.05; **p<0.01, compared with controls. PT, phototherapy; Orl, Orlistat.

5.5 Discussion In this study we demonstrate that neither routine phototherapy nor experimental oral treatments (e.g. orlistat, UDCA, or CaP), exert their hypobilirubinemic effect via acceleration of the gastrointestinal transit in Gunn rats. We also show that acceleration of the gastrointestinal transit by PEG enhances the therapeutic effects of both phototherapy and orlistat in these animals.

Several studies have suggested a relationship between gastrointestinal transit and plasma bilirubin levels. Conditions that delay the gastrointestinal transit, such as Hirschprungs disease [29] or fasting,[30,31] have been associated with an exaggeration of neonatal jaundice. Conditions that accelerate transit, such as prebiotic supplementation of infant formula, seem to mitigate jaundice in neonates.[32,33] We recently demonstrated that acceleration of the gastrointestinal transit by PEG effectively treated unconjugated hyperbilirubinemia in jaundiced Gunn rats.[17] The strong relationship between the gastrointestinal transit and plasma UCB concentrations in that study prompted us to evaluate the mechanistic contribution of the transit time in phototherapy and in experimental oral treatments. The hypobilirubinemic effects of these treatments have traditionally been linked to different underlying mechanisms. Phototherapy, the routine treatment for neonatal jaundice, has been shown to exert its hypobilirubinemic effect by conversion of UCB into photo-isomers.[26] Experimental oral treatments, such as orlistat, UDCA, or CaP, are

132 Chapter 5

thought to bind UCB within the gut lumen, thereby preventing its reabsorption and interrupting its enterohepatic circulation.[14,27,34] Interestingly, both phototherapy and oral treatments have also been suggested to accelerate the gastrointestinal transit.[35,36] Phototherapy, especially intensive phototherapy, can induce diarrhea. Diarrhea is also a well-known potential side effect of orlistat and UDCA treatment. Orlistat has been shown to accelerate gastric emptying,[22] whereas UDCA treatment accelerated the gastrointestinal transit in dogs, and can improve gastrointestinal motility defects in bile stone patients.[21,37] Theoretically, phototherapy or oral treatments could thus well exert (part of) their hypobilirubinemic effect by acceleration of the gastrointestinal transit. Our Gunn rat model, however, allowed us to demonstrate that neither routine phototherapy nor experimental oral treatments exert their hypobilirubinemic effect via acceleration of the gastrointestinal transit. These findings prompted us to investigate whether PEG co-treatment might induce an additional decrease in plasma bilirubin levels during these treatments. We started PEG co-treatment only after 3 weeks to ensure steady-state conditions, which prevented any interference of non-steady-state plasma bilirubin fluctuations.[14,17-19]

PEG accelerated the gastrointestinal transit in all treatment groups, and induced an additive therapeutic effect in phototherapy and orlistat treated animals. The most impressive effect was observed in the phototherapy + PEG treatment group, which concurred with the hypobilirubinemic result of adding phototherapy to PEG treatment alone in a previous study by us.[17] The additive therapeutic effect (~20%) thus occurred irrespective of whether phototherapy was added to PEG-treatment (previous study), or vice-versa (current study), indicating that both therapies truly complement each other.[17] The efficacy of phototherapy + PEG treatment, which resulted in a final 67% decrease in plasma bilirubin levels, might well result from the distinct mechanisms by which phototherapy and PEG decrease plasma bilirubin levels. Phototherapy predominantly decreases plasma bilirubin levels by enhancing its disposal via the bile,[26] whereas oral treatments, including PEG, selectively enhance net bilirubin diffusion from the blood into the intestinal lumen.[14,27] We and others have demonstrated in 3H-labelled bilirubin studies that a maximal therapeutic effect often results from combining therapies that enhance distinct pathways for bilirubin disposal.[14,27,38] The complementary effect of PEG and phototherapy might thus well result from the combination of their distinct hypobilirubinemic mechanisms. The additive effect of PEG was not observed during the first days of phototherapy treatment. We speculate that this occurred because short-term phototherapy extracted bilirubin at a maximal rate from the body. This rate could thus not be further increased by PEG co-treatment. Short-term orlistat treatment, on the other hand, was notably less effective compared with phototherapy. In agreement with our postulate above, PEG was a useful adjunct strategy in this treatment group.

Combined treatment for unconjugated hyperbilirubinemia 133

PEG co-treatment did not induce an additive therapeutic effect in UDCA or CaP treated animals. This lack of additive effect cannot be attributed to an insufficient acceleration of the gastrointestinal transit, which was comparable between all PEG-treated groups. PEG administration, however, has several effects on the intestinal milieu that might counteract the UDCA or CaP-induced decrease in plasma UCB levels. Firstly, PEG decreases the amount of bile salts in the feces.[14] This, evidently, would counteract the effect of simultaneous UDCA feeding. Secondly, PEG decreases the formation of secondary bile salts, possibly by affecting the microflora that promotes bile salt metabolism. This microflora, however, is also involved in the irreversible degradation of intestinal bilirubin into urobilinoids, which is promoted by UDCA feeding.[14,17] PEG might thus well interfere with the apparent ability of UDCA to promote intestinal bilirubin degradation. Finally, PEG increases the amount of intestinal water and accelerates the gastrointestinal transit. This might well prevent the precipitation of amorphous calcium phosphate with intestinal bilirubin during CaP treatment.[34]

Animals that were treated with UDCA + PEG showed a small (-5%), but significant, decrease in growth rate compared with control animals. Although this was not observed in other groups, it should be emphasized that diarrhea and malabsorption of vitamins (A, D, E, K) have been reported during orlistat, UDCA and CaP treatment.[19,35,36,39] Regular assessments of the nutritional (vitamin) status and/or vitamin supplementation would be indicated if one would combine these treatments with a laxative such as PEG in patient studies. These precautions may not apply for the combination of PEG with phototherapy, which seems safe and deserves further exploration in hyperbilirubinemic patient studies.

In conclusion, our data show that that neither phototherapy, nor orlistat, UDCA, or CaP exerts its hypobilirubinemic effect via acceleration of the gastrointestinal transit. Combining PEG-induced acceleration of the gastrointestinal transit with phototherapy decreased plasma bilirubin levels by 67% in Gunn rats. Our results support the evaluation of PEG co-treatment during phototherapy in clinical trials, for example in Crigler-Najjar patients.

134 Chapter 5

References 1. Crigler JF, Najjar VA. Congenital

familial nonhemolytic jaundice with kernicterus. Pediatrics. 1952;10:169–180.

2. Arias IM, Gartner LM, Cohen M, Ezzer JB, Levi AJ. Chronic nonhemolytic unconjugated hyperbilirubinemia with glucuronyl transferase deficiency. Clinical, biochemical, pharmacologic and genetic evidence for heterogeneity. Am J Med. 1969;47:395–409.

3. Shapiro SM. Definition of the clinical spectrum of kernicterus and bilirubin-induced neurologic dysfunction (BIND). J Perinatol. 2005;25:54–59.

4. Ahlfors CE, Amin SB, Parker AE. Unbound bilirubin predicts abnormal automated auditory brainstem response in a diverse newborn population. J Perinatol. 2009;29:305–309.

5. Ahlfors CE, Wennberg RP, Ostrow JD, Tiribelli C. Unbound (free) bilirubin: improving the paradigm for evaluating neonatal jaundice. Clin. Chem. 2009;55:1288–1299.

6. Ostrow JD, Mukerjee P, Tiribelli C. Structure and binding of unconjugated bilirubin: relevance for physiological and pathophysiological function. J. Lipid Res. 1994;35:1715–1737.

7. Ostrow JD, Pascolo L, Brites D, Tiribelli C. Molecular basis of bilirubin-induced neurotoxicity. Trends Mol Med. 2004;10:65–70.

8. Zucker SD, Goessling W, Hoppin AG. Unconjugated bilirubin exhibits spontaneous diffusion through model lipid bilayers and native hepatocyte membranes. J. Biol. Chem. 1999;274:10852–10862.

9. Ostrow JD, Pascolo L, Shapiro SM,

Tiribelli C. New concepts in bilirubin encephalopathy. Eur J Clin Invest. 2003;33:988–997.

10. Diamond I, Schmid R. Experimental bilirubin encephalopathy. The mode of entry of bilirubin-14C into the central nervous system. J. Clin. Invest. 1966;45:678–689.

11. Van der Veere CN, Sinaasappel M, McDonagh AF, Rosenthal P, Labrune P, Odievre M, et al. Current therapy for Crigler-Najjar syndrome type 1: report of a world registry. Hepatology. 1996;24:311–315.

12. Yohannan MD, Terry HJ, Littlewood JM. Long term phototherapy in Crigler-Najjar syndrome. Arch Dis Child. 1983;58:460–462.

13. Lester R, Schmid R. Intestinal absorption of bile pigments. I. The enterohepatic circulation of bilirubin in the rat. J. Clin. Invest. 1963;42:736–746.

14. Cuperus FJC, Hafkamp AM, Havinga R, Vitek L, Zelenka J, Tiribelli C, et al. Effective treatment of unconjugated hyperbilirubinemia with oral bile salts in gunn rats. Gastroenterology. 2009;136:673–82 e1.

15. Kotal P, Van der Veere CN, Sinaasappel M, Elferink RO, Vitek L, Brodanova M, et al. Intestinal excretion of unconjugated bilirubin in man and rats with inherited unconjugated hyperbilirubinemia. Pediatr. Res. 1997;42:195–200.

16. Lester R, Schmid R. Intestinal absorption of bile pigments. II. Bilirubin absorption in man. N. Engl. J. Med. 1963;269:178–182.

17. Cuperus FJC, Iemhoff AA, van der Wulp M, Havinga R, Verkade HJ.

Combined treatment for unconjugated hyperbilirubinemia 135

Acceleration of the gastrointestinal transit by polyethylene glycol effectively treats unconjugated hyperbilirubinaemia in Gunn rats. Gut. 2010;59:373–380.

18. Hafkamp AM, Havinga R, Sinaasappel M, Verkade HJ. Effective oral treatment of unconjugated hyperbilirubinemia in Gunn rats. Hepatology. 2005;41:526–534.

19. Hafkamp AM, Nelisse-Haak R, Sinaasappel M, Oude Elferink RPJ, Verkade HJ. Orlistat treatment of unconjugated hyperbilirubinemia in Crigler-Najjar disease: a randomized controlled trial. Pediatr. Res. 2007;62:725–730.

20. Van der Veere CN, Jansen PL, Sinaasappel M, Van Der Meer R, Van der Sijs H, Rammeloo JA, et al. Oral calcium phosphate: a new therapy for Crigler-Najjar disease? Gastroenterology. 1997;112:455–462.

21. Colecchia A, Mazzella G, Sandri L, Azzaroli F, Magliuolo M, Simoni P, et al. Ursodeoxycholic acid improves gastrointestinal motility defects in gallstone patients. World J Gastroenterol. 2006;12:5336–5343.

22. Borovicka J, Schwizer W, Guttmann G, Hartmann D, Kosinski M, Wastiel C, et al. Role of lipase in the regulation of postprandial gastric acid secretion and emptying of fat in humans: a study with orlistat, a highly specific lipase inhibitor. Gut. 2000;46:774–781.

23. Ebbesen F, Edelsten D, Hertel J. Gut transit time and lactose malabsorption during phototherapy. I. A study using lactose-free human mature milk. Acta Paediatr Scand. 1980;69:65–68.

24. Ebbesen F, Edelsten D, Hertel J. Gut transit time and lactose malabsorption during phototherapy. II. A study using raw milk from the mothers of the infants. Acta Paediatr Scand.

1980;69:69–71.

25. De Curtis M, Saitta F, Matteoli M, Paludetto R, Ciccimarra F, Guandalini S. Evidence for secretory type diarrhoea in infants treated by phototherapy. Lancet. 1982;319:909.

26. Ostrow JD. Photocatabolism of labeled bilirubin in the congenitally jaundiced (Gunn) rat. J. Clin. Invest. 1971;50:707–718.

27. Hafkamp AM, Havinga R, Ostrow JD, Tiribelli C, Pascolo L, Sinaasappel M, et al. Novel kinetic insights into treatment of unconjugated hyperbilirubinemia: phototherapy and orlistat treatment in Gunn rats. Pediatr. Res. 2006;59:506–512.

28. Call NB, Tisher CC. The urinary concentrating defect in the Gunn strain of rat. Role of bilirubin. J. Clin. Invest. 1975;55:319–329.

29. Gartner LM. Breastfeeding and jaundice. J Perinatol. 2001;21 Suppl 1:S25–9; discussion S35–9.

30. Kotal P, Vitek L, Fevery J. Fasting-related hyperbilirubinemia in rats: the effect of decreased intestinal motility. Gastroenterology. 1996;111:217–223.

31. Bertini G, Dani C, Tronchin M, Rubaltelli FF. Is breastfeeding really favoring early neonatal jaundice? Pediatrics. 2001;107:E41.

32. Mihatsch WA, Hoegel J, Pohlandt F. Prebiotic oligosaccharides reduce stool viscosity and accelerate gastrointestinal transport in preterm infants. Acta Paediatr. 2006;95:843–848.

33. Bisceglia M, Indrio F, Riezzo G, Poerio V, Corapi U, Raimondi F. The effect of prebiotics in the management of neonatal hyperbilirubinaemia. Acta Paediatr. 2009;98:1579–1581.

34. Van der Veere CN, Schoemaker B, Van Der Meer R, Groen AK, Jansen

136 Chapter 5

PL, Oude Elferink RP. Rapid association of unconjugated bilirubin with amorphous calcium phosphate. J. Lipid Res. 1995;36:1697–1707.

35. Filippatos TD, Derdemezis CS, Gazi IF, Nakou ES, Mikhailidis DP, Elisaf MS. Orlistat-associated adverse effects and drug interactions: a critical review. Drug Saf. 2008;31:53–65.

36. Hempfling W, Dilger K, Beuers U. Systematic review: ursodeoxycholic acid--adverse effects and drug interactions. Aliment. Pharmacol. Ther. 2003;18:963–972.

37. Kruis W, Haddad A, Phillips SF.

Chenodeoxycholic and ursodeoxycholic acids alter motility and fluid transit in the canine ileum. Digestion. 1986;34:185–195.

38. Ostrow JD, Wennberg RP, Tiribelli C. Phototherapy for neonatal jaundice. N. Engl. J. Med. 2008;358:2524; author reply 2524–5.

39. Haynes RC. Agents affecting calcification: calcium, parathyroid hormone, calcitonin, vitamin D, and other compounds. In: Goodman Gilman A, Rall TW, Nies AS, Taylor P, editors. The pharmacological basis of therapeutics. New York: Pergamon; 1988. p. 1496–1504.

Chapter 6 Beyond Plasma Bilirubin: the Effects of Phototherapy and Albumin on Brain Bilirubin Concentrations in Gunn Rats

F.J.C. Cuperus 1*

A.B. Schreuder 1*

D.E. van Imhoff 2 L.Vitek 3,4

J. Vanicova 3

R. Konickova 3 C.E. Ahlfors 5

C.V. Hulzebos 2

H.J. Verkade 1

*Both authors contributed equally

1Pediatric Gastroenterology and Hepatology, Department of Pediatrics, Center for Liver, Digestive, and Metabolic Diseases, Beatrix Children’s Hospital- University Medical Center Groningen, University of Groningen, The Netherlands. 2Neonatology, Department of Pediatrics, Beatrix Children’s Hospital - University Medical Center Groningen, The Netherlands. 3Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University, Prague, Czech Republic. 44th Department of Internal Medicine, 1st Faculty of Medicine, Charles University, Prague, Czech Republic. 5Stanford University, School of Medicine, Stanford, California, USA

Submitted

138 Chapter 6

6.1 Abstract evere unconjugated hyperbilirubinemia, as occurs in Crigler-Najjar disease and neonatal jaundice, carries the risk of neurotoxicity. Neurotoxicity is caused by the ability of free bilirubin (UCBfree), the non-protein bound fraction of unconjugated bilirubin (UCB), to translocate into the brain. We hypothesized that higher plasma protein levels would decrease this free bilirubin fraction and thereby prevent its deposition in the brain. Accordingly, we assessed the effect of albumin treatment on plasma UCBfree and brain bilirubin levels during phototherapy in Gunn rat models of chronic and acute jaundice. As a chronic model, resembling patients with Crigler-Najjar disease, we treated adult hyperbilirubinemic Gunn rats for 16 days with phototherapy or with phototherapy + human serum albumin (HSA; 2.5 g/kg i.p. at t=14 days). As an acute model, resembling neonatal jaundice, we induced hemolysis in adult hyperbilirubinemic Gunn rats by acetylphenylhydrazine, and then treated these animals with phototherapy or with phototherapy + HSA (2.5 g/kg i.p. at t=0h) for 48h. In the chronic model, phototherapy decreased plasma UCBfree by 55% and decreased brain bilirubin levels by 45% (each p<0.001). Adjunct HSA treatment enhanced the efficacy of phototherapy; it decreased plasma UCBfree and brain bilirubin levels by 88% and 67%, respectively (each p<0.001). In the acute model, phototherapy decreased plasma UCBfree by 31% (p<0.001), but failed to decrease brain bilirubin. Adjunct HSA decreased plasma UCBfree by 76% (p<0.001) and completely prevented the deposition of bilirubin in the brain during hemolytic jaundice. This chapter demonstrates that adjunct treatment with albumin decreases brain bilirubin levels in phototherapy-treated Gunn rats, during both chronic and acute jaundice. We hypothesize that albumin decreases these levels by lowering UCBfree in the plasma. Present results underline the need to evaluate the use of albumin as an adjunct treatment to phototherapy in Crigler-Najjar patients and in hyperbilirubinemic neonates.

S

Albumin treatment for unconjugated hyperbilirubinemia 139

6.2 Introduction rigler-Najjar patients and hemolytic neonates suffer from unconjugated hyperbilirubinemia.[1] Severe unconjugated hyperbilirubinemia can lead to brain damage. This damage is mediated by the ability of “free” bilirubin (UCBfree), the small (<1%) fraction of unconjugated bilirubin (UCB) not bound to plasma proteins, to cross the blood-brain barrier (BBB).[2-6] Within the brain, bilirubin disrupts several vital cell functions and induces apoptosis and necrosis. Bilirubin-induced neurotoxicity may eventually lead to kernicterus and even death.[3,7,8]

Severe unconjugated hyperbilirubinemia is conventionally treated by phototherapy, which converts UCB into photo-isomers that can readily be excreted in the bile.[9] Phototherapy, however, has some disadvantages. Crigler-Najjar patients, who suffer from permanent inherited unconjugated hyperbilirubinemia, may need up to 16h of treatment per day. In spite of this intensive regimen, up to 25% of these patients will eventually develop brain damage.[10,11] Phototherapy is more effective during neonatal hemolytic jaundice, but may still require additional, potentially dangerous, exchange transfusions.[12] The efficacy of phototherapy is often estimated by its hypobilirubinemic effect. Plasma bilirubin levels, however, correlate poorly with the occurrence of brain damage in individual patients.[6] The reason for this poor correlation lies in the inability of protein-bound bilirubin (>99% of total plasma bilirubin) to leave the circulation.[2,3,5,6] Only UCBfree is able to translocate across the BBB, and thus plays a key role in the pathogenesis of bilirubin-induced brain damage. UCBfree concentrations, however, are not routinely evaluated in phototherapy-treated patients. The main reason for this lies in the inaccuracy of the commercial test, most notably caused by a 42-fold sample dilution that alters bilirubin-albumin binding.

We reasoned that decreasing UCBfree in the plasma could prevent bilirubin deposition in the brain. Human serum albumin (HSA) infusion could, theoretically, achieve this goal by providing additional binding sites for UCBfree in the plasma. Interestingly, HSA treatment has been used in severely jaundiced neonates.[13-17] Its efficacy, however, has been difficult to establish. This difficulty is partly due to the technical shortcomings of the available UCBfree measurement, but mainly results from the obvious inability to assess brain bilirubin levels in humans. Recently, Ahlfors et al. developed an automated UCBfree test with minimal sample dilution, while Zelenka et al. developed a highly sensitive method for tissue bilirubin determinations.[18,19] We now use these techniques to evaluate the effect of HSA treatment on plasma UCBfree and brain bilirubin levels in two well-established animal models. As a chronic model, resembling patients with Crigler-Najjar disease, we treated adult Gunn rats with

C

140 Chapter 6

long-term phototherapy or with photoherapy + HSA.[20] As an acute model, resembling severe hemolytic jaundice, we induced hemolysis by 1-acetyl-2-phenyl-hydrazine (APHZ) in adult Gunn rats, and then treated these animals for 48h with phototherapy or phototherapy + HSA.[21] We now demonstrate that HSA treatment decreases plasma UCBfree and brain bilirubin levels in phototherapy-treated Gunn rats, both during chronic and acute jaundice. We speculate that HSA and phototherapy work in tandem: HSA binds UCBfree within the plasma, and phototherapy then promotes its excretion via the bile. Our results underline the need to evaluate the use of HSA as adjunct to phototherapy in randomized clinical trials.

6.3 Animals, materials, and methods

6.3.1 Animals Homozygous male Gunn rats (RHA/jj; 225-340g, aged 10-12 weeks) from our breeding colony were kept in an environmentally controlled facility and were fed ad libitum with free access to water. Food intake, fluid intake, and body weight were determined regularly. The Animal Ethics Committee of the University of Groningen (Groningen, The Netherlands) approved all experimental protocols.

6.3.2 Materials Hope Farms BV (Woerden, The Netherlands) produced the semi-synthetic control diet (code 4063.02), containing 13 energy% fat and 5.2 wt% long-chain fatty acids.[22] Gunn rats were fed this diet during a 5-week run-in period, and during the experiments.[22] HSA (Albuman®; 200 g/L) was purchased from Sanquin (Amsterdam, The Netherlands). APHZ, horseradish peroxidase type 1, D-glucose, glucose oxidase, hydrogen peroxide, and UCB were purchased from Sigma Chemical Co. (St. Louis, MO). Commercial UCB was further purified according to the method of Ostrow et al.[23] Phototherapy was administered continuously to Gunn rats (shaven on flank and back) via two blue phototherapy lamps (Philips, TL-20W/03T) suspended in a reflective canopy 20 cm above the cage. The phototherapy dose (17 µW/cm2/nm; 380-480 nm) was measured by an Elvos-LM-1010 Lux meter at 20 cm distance.[24]

Albumin treatment for unconjugated hyperbilirubinemia 141

6.3.3 Methods Permanent unconjugated hyperbilirubinemia

Adult Gunn rats were randomized to receive either no treatment (n=7) or phototherapy (17 µW/cm2/nm; n=14) for 16 days. At 14 days, we randomized the phototherapy-treated rats to receive either NaCl 0.9% (w/v; n=7) or HSA (2.5 g/kg, n=7) by means of a single i.p. injection. We determined plasma bilirubin levels from tail vein blood at T=0, 14, and 16 days, and plasma UCBfree at T=16 days under isoflurane anesthesia. After 16 days, all animals were exsanguinated via the descending aorta and flushed via the same port with 100-150 ml NaCl 0.9% under isoflurane anesthesia. The brain, liver, and aliquots of visceral fat were subsequently harvested for the determination of tissue bilirubin levels. These samples were rinsed 2 times in phosphate buffered saline, snap frozen in liquid nitrogen, and immediately stored (wrapped in aluminum foil) at -80 °C until analysis.[19]

Acute unconjugated hyperbilirubinemia

Adult Gunn rats received a single APHZ injection i.p. (15 mg/kg BW; T=-24h) to induce hemolysis. We then randomized these animals after 24h (T=0h) to receive either no treatment (n=6), phototherapy + NaCl 0.9% (17 µW/cm2/nm; n=6), or phototherapy + HSA (2.5 g/kg; n=6) for another 48h. NaCl 0.9% (sham) and HSA were administered as a single i.p. injection at T=0h. We determined plasma bilirubin, UCBfree, and albumin levels from tail vein blood at T=-24, -12, 0, 12, 24, 36, and 48h under isoflurane anesthesia. Hemoglobin (Hb), reticulocyte count, and hematocrit (Ht), were determined at T=-24h and T=48h. After 48h, all animals were exsanguinated and brain, liver, and visceral fat samples were subsequently harvested for the determination of tissue bilirubin levels, as described above.[19]

Plasma analysis

Blood samples were protected from light, stored at -20 °C under argon directly after collection, and processed within 2 weeks. UCB concentrations were determined by routine spectrophotometry on a P800 unit of a modular analytics serum work area from Roche Diagnostics Ltd. (Basel, Switzerland). Hb, Ht, and reticulocytes were determined on a Sysmex XE-2100 hematology analyzer (Goffin Meyvis, Etten-Leur, The Netherlands). We previously found in Gunn rats that the total bilirubin concentration, measured by spectrophotometry, equals the

142 Chapter 6

total UCB concentration, measured by high-liquid performance chromatography after chloroform extraction (coefficient of variation: ~5%).[22,25] UCBfree was determined using a Zone Fluidics system (Global Flopro, Global Fia Inc, WA), as previously described by Ahlfors et al.[18]

Tissue bilirubin analysis

Tissue bilirubin content was determined using HPLC with diode array detector (Agilent, Santa Clara, CA, USA) as described earlier. [19] Briefly, 300 pmol of mesobilirubin in dimethyl sulfoxide (used as an internal standard) was added and samples were homogenized on ice. Bile pigments were then extracted into chloroform/hexane (5:1) solution at pH 6.0, and subsequently extracted in a minimum volume of methanol/carbonate buffer (pH=10) to remove contaminants. The resulting polar droplet (extract) was loaded onto C-8 reverse phase column (Phenomenex, Torrance, CA, USA) and separated pigments were detected at 440 nm. The concentration of bilirubin was calculated as nmol/g of wet tissue weight. All steps were performed under dim light in aluminum-wrapped tubes. We did not specifically measure bilirubin deposition in the brain nuclei, but relied on total tissue bilirubin measurements.

Statistical analysis

Normally distributed data that displayed homogeneity of variance (by calculation of Levene’s statistic), were expressed as means ± SD, and analyzed with parametric statistical tests. Analysis of variance (ANOVA) with post-hoc Tukey correction was performed for comparisons between groups, and the Student t test for comparison of paired data within groups. The level of significance was set at p<0.05. Analyses were performed using PASW Statistics 17.0 for Mac (SPSS Inc., Chicago, IL).

6.4 Results As a model for Crigler-Najjar disease, we first treated permanently jaundiced Gunn rats with routine phototherapy and phototherapy + HSA for 16 days. Figure 1A shows that both treatments decreased plasma UCB levels to a similar extent (46% and 54% at t= 16 days, respectively), compared with untreated controls (p<0.001). Figure 1B shows that phototherapy and phototherapy + HSA decreased plasma UCBfree levels by 55% and 88%, respectively (p<0.001). Finally,

Albumin treatment for unconjugated hyperbilirubinemia 143

figure 1C shows that phototherapy and phototherapy + HSA decreased brain bilirubin concentrations by 45% and 67%, respectively (p<0.001), compared with untreated controls. Adjunct HSA thus lowered plasma UCBfree and brain bilirubin by an additional 33% and 22%, respectively (each p<0.01), compared with phototherapy alone. HSA also decreased hepatic bilirubin levels by an additional 33% (p<0.01), compared with phototherapy alone (figure 2A), but failed to induce a significant additive decrease in visceral fat bilirubin levels (figure 2B). Figure 3A shows that plasma UCB levels correlated well with brain bilirubin levels (y=0.29x-0.60; r2=0.78; p<0.001). Plasma UCB levels above 200 µmol/L however, appeared relatively poor predictors for individual brain bilirubin concentrations. Figure 3B shows the correlation between UCBfree levels and brain bilirubin levels (y=0.24x-0.88; r2=0.86; p<0.001).

As a model for severe acute jaundice we then treated hemolytic Gunn rats with routine phototherapy and phototherapy + HSA for 48 hours. Figure 4A shows that both treatments decreased the severity of hemolytic unconjugated hyperbilirubinemia, compared with untreated hemolytic controls. Phototherapy, however, only decreased plasma UCB levels by 14% at t=36h (p<0.01), while phototherapy + HSA decreased these levels by at least 29% from 36h onward (p<0.001). Figure 4B shows that phototherapy decreased plasma UCBfree levels by

Control PT PT + HSA

0

50

100

150

200

250

300

Control PT PT + HSA

0

1

2

3

4

5

UCB plasma

free(nmol/l)

UCB Brain

(nmol/g)

B C

*

* §/

*

*/#

Control PT PT + HSA

0

50

100

150

200

250

300

UCB plasma

A

* *

( mol/l)

Figure 1. Effects of no treatment (controls), routine phototherapy (PT) or phototherapy + HSA on plasma UCB (panel A), plasma UCBfree (panel B), and brain bilirubin levels (panel C) in Gunn rats at t=16 days. Adult Gunn rats were randomized to receive either no treatment or phototherapy (17 µW/cm2/nm) for 16 days. At 14 days, we randomly injected the phototherapy-treated animals with saline (sham) or with HSA (2.5 g/kg). Plasma bilirubin levels were similar in the phototherapy and phototherapy + HSA group at the time of this injection (data not shown). *p<0.001 compared with controls. #p<0.05, §p<0.01 compared with phototherapy alone.

144 Chapter 6

31% at t=48h (p<0.05), compared with controls, while phototherapy + HSA decreased these levels by at least 41% from 12h onward (p<0.001). Adjunct HSA thereby lowered plasma UCB and plasma UCBfree levels by an additional 14-16%, and 25-47%, respectively (each p<0.01), compared with routine phototherapy. Figure 4C shows that phototherapy + HSA decreased brain bilirubin concentrations by 50% (p<0.001). The resulting brain bilirubin levels were comparable to those of untreated non-hemolytic Gunn rats (figure 1C), which demonstrated that phototherapy + HSA completely prevented the increase in brain bilirubin due to hemolytic jaundice. HSA also decreased hepatic bilirubin

Control PT PT + HSA

0

200

UCB Liver

(nmol/g)

150

100

50

Control PT PT + HSA

0

70

UCB Fat

(nmol/g)

20

60

50

40

30

10

A B

**

*** ***§/

***

Control PT PT + HSA

0

70

UCB Fat

(nmol/g)

20

60

50

40

30

10

D

*

Control PT PT + HSA

0

200

UCB Liver

(nmol/g)

150

100

50

C

*

*** §/

Acut

e ex

perim

ent

Chro

nic e

xper

imen

t

Figure 2. Effects of no treatment (controls), routine phototherapy (PT) or phototherapy + HSA on liver (panel A/C) and visceral fat (panel B/D) bilirubin levels in non-hemolytic (chronic experiment) and hemolytic (acute experiment) Gunn rats. For experimental setup, kindly refer to the Methods section. *p<0.05; **p<0.01; ***p<0.001, compared with controls. §p<0.01, compared with single phototherapy.

Albumin treatment for unconjugated hyperbilirubinemia 145

Figure 4. Effects of no treatment (controls), routine phototherapy (PT) or phototherapy + HSA on plasma UCB (panel A), plasma UCBfree (panel B), and brain bilirubin levels (panel C) in hemolytic Gunn rats at t= 48h. Adult Gunn rats received APHZ i.p. to induce hemolysis, and were randomized after 24h to receive no treatment, phototherapy (17 µW/cm2/nm), or phototherapy + HSA (2.5 g/kg) for 48h. Plasma bilirubin levels were similar in all groups during the 24h-run in after APHZ injection (data not shown). *p<0.05; **p<0.01; ***p<0.001, compared with controls. #p<0.05; §p<0.01; †p<0.001, compared with single phototherapy.

100 150 200 250 300 0

1

2

3

5

4

UCB plasma ( mol/l)

UC

B br

ain

(nm

ol/g

)

50

UCB plasma vs. UCB brain

100 150 200 250 3000 0

1

2

3

5

4

UCBfree plasma (nmol/l)U

CB

bra

in (n

mol

/g)

50

UCB plasma

free vs. UCB brain

A B

r = 0.86; p<0.0012r = 0.78; p<0.0012

Figure 3. The correlation between plasma UCB and brain bilirubin levels (panel A), and the correlation between plasma UCBfree and brain bilirubin levels (panel B) in Gunn rats at t=16 days. Adult Gunn rats were randomized to receive either no treatment or phototherapy (17 µW/cm2/nm) for 16 days. At 14 days, we randomly injected the phototherapy-treated animals with saline (sham) or with HSA (2.5 g/kg).

control, phototherapy, phototherapy + HSA.

A

control

PT

PT + HSA

100

200

400

500

-12 0 12 24 36 48-24

Time (hours)

UCB plasma

( mol/l)

-12 0 12 24 36 48-24

100

200

300

400

500

control

PT

PT + HSA

Time (hours)

UCB plasma

free

(nmol/l)

Control PT PT + HSA

0

2

4

6

8

UCB Brain

(nmol/g)

B C

*

*** #/

***

*****

*

§//†

***/†

** #/

300

*

146 Chapter 6

100 200 300 400 5000

0

1

2

3

4

5

6

8

9

7

UCBfree plasma (nmol/l)U

CB

bra

in (n

mol/g

)

100 200 300 400 5000 0

1

2

3

4

5

6

8

9

7

UCB plasma ( mol/l)

UC

B b

rain

(nm

ol/g

)

UCB plasma vs. UCB brain UCB plasma

free vs. UCB brain

A B

r = 0.50; p<0.0012 r = 0.64; p<0.0012

levels by an additional 36% (p<0.01), compared with routine phototherapy (figure 2C). Phototherapy + HSA, but not phototherapy alone, decreased bilirubin levels in visceral fat by 41%, compared with controls (p<0.05; figure 2D). Figure 4C shows that phototherapy alone failed to decrease brain bilirubin during hemolytic disease. Again, plasma bilirubin levels above 200 µmol/L appeared relatively poor predictors for individual brain bilirubin concentrations (figure 5A). Figure 5B shows that plasma UCBfree levels correlated well with brain bilirubin levels (y=0.0091x+2.32; r2=0.64; p<0.001).

APHZ administration induced a comparable hemolysis in all groups, as indicated by the similar changes in Hb, Ht, and reticulocyte levels (figure 6A-C). Figure 6D shows that a single i.p. HSA injection rapidly increased plasma albumin levels (42% after 12h; p<0.001), compared with untreated controls. Mean growth rates did not differ significantly between experimental and control groups in each experiment (data not shown).

Figure 5. The correlation between plasma UCB and brain bilirubin levels (panel A), and the correlation between plasma UCBfree and brain bilirubin levels (panel B) in Gunn rats at t= 48h. Adult Gunn rats received APHZ i.p. to induce hemolysis, and were randomized after 24h to receive no treatment, phototherapy (17 µW/cm2/nm), or phototherapy + HSA (2.5 g/kg) for 48h control, phototherapy,

phototherapy + HSA.

Albumin treatment for unconjugated hyperbilirubinemia 147

Figure 7. Human serum albumin (HSA) treatment during unconjugated hyperbilirubinemia: (a) Unconjugated hyperbilirubinemia may result in the accumulation of unconjugated bilirubin (UCB) within the brain. Only non-protein bound UCB (UCBfree) is able to move between the blood (e.g. vascular compartment) and the brain (e.g. extravascular compartment). (b) Treatment with HSA decreases UCBfree levels within the blood. This promotes an UCBfree-induced shift of bilirubin from the brain into the circulation. Additional phototherapy subsequently converts this bilirubin into photo-isomers that can readily be excreted with the bile (dashed arrow).

Figure 6. Effects of APHZ injection on Hb (Panel A), Ht (Panel B), and reticulocyte (Panel C) levels, and the effect of human serum albumin injection on plasma albumin levels (Panel D) in Gunn rats. Adult Gunn rats received APHZ i.p. to induce hemolysis, and were randomized after 24h to receive no treatment, phototherapy (17 µW/cm2/nm), or phototherapy + HSA (2.5 g/kg) for 48h. *p<0.001, compared with controls. #p<0.01; §p<0.001, compared with pre-treatment values at T=0h. control, phototherapy, phototherapy + HSA.

APHZ

Contro

l

PT

PT +

HSA

+0

2

4

6

8

10

- +- +-

Time (hours)-24 -12 0 12 24 36 48

APHZ

Contro

l

PT

PT +

HSA

+0

0.1

0.2

0.3

0.4

0.5

- +- +-

APHZ

Contro

l

PT

PT +

HSA

+0

20

40

60

80

- +- +-

35

40

45

50

55

A B

C D

§ § §

#

* **

Hb

Ht

reticulocytes

albumin

(mmol/l) (l/ l)

(‰) (g/l)

§ § §

§§

UCB free

Bound(e.g. Albumin)

Bound(protein)

Blood Brain

UCB freeUCB UCB

HSA

infusion

Bound(e.g. Albumin)

Blood Brain

freeUCB UCB

Bound(protein)

freeUCBUCB

a b

148 Chapter 6

6.5 Discussion In this study we demonstrate that HSA effectively decreases brain bilirubin levels in phototherapy-treated Gunn rats. The decrease was apparent during both chronic and acute hemolytic jaundice. Our results support the feasibility of adjunct HSA treatment during phototherapy in Crigler-Najjar disease and neonatal jaundice.

The rationale of HSA treatment is based on the premises that UCBfree translocates into the brain and, secondly, that i.v. albumin prevents this translocation by binding UCBfree within the plasma. The role of UCBfree translocation became apparent in the 1950s, when sulfisoxazole-treated neonates developed kernicterus in the presence of unusually low plasma bilirubin levels.[26,27] It was soon discovered that sulfisoxazole displaced UCB from albumin, which first suggested the importance of the non-albumin bound UCB fraction.[28] Since then, many studies have supported the critical role of UCBfree in the pathogenesis of bilirubin-induced brain damage.[2,3,5,6] Only recently, Ahlfors et al showed that auditory brainstem response screening, a quantifiable method to evaluate bilirubin-induced neurotoxicity in neonates, correlated with UCBfree rather than with total bilirubin levels.[4] The protective role of HSA administration has also been investigated in neonates. Its efficacy, however, has never been established in randomized controlled trials. Two retrospective studies have shown reduced UCBfree levels in jaundiced neonates after HSA administration.[15,17] One additional small cohort study has shown some protective effect of HSA administration on the development of brain damage, as measured by auditory brainstem response screening.[16] Other studies, however, failed to demonstrate beneficial effects of HSA treatment.[14] Importantly, most human studies did not assess plasma UCBfree, or used methods that seriously underestimate UCBfree levels due to a 42-sample dilution.[14-17,29] The absence of reliable data on UCBfree levels obviously impeded the interpretability of these studies. In addition, human studies are intrinsically limited by the impossibility of measuring brain bilirubin levels. Taken together, these issues demonstrated the need for a more thorough evaluation of HSA administration. Recently, Ahlfors et al. automated and improved the available UCBfree test, while Zelenka et al. developed a sensitive method for tissue bilirubin determinations.[18,19] These newly developed methods allowed us to reliably measure UCBfree and brain bilirubin levels in two well-established animal models. [20,21]

We first investigated the efficacy of HSA treatment in permanently hyperbilirubinemic Gunn rats, as a model for Crigler-Najjar disease. HSA treatment decreased both UCBfree and brain bilirubin levels in these animals. HSA also decreased hepatic, and to a lesser extent visceral fat, bilirubin levels in these animals (figure 2). These results support a model in which only UCBfree is

Albumin treatment for unconjugated hyperbilirubinemia 149

able to move between the vascular and extravascular (tissue) compartment of the bilirubin pool (figure 7). This translocation occurs across both the vascular endothelial cells and the BBB. In this model, HSA administration could act in tandem with phototherapy. HSA first decreases UCBfree within the vascular compartment, and promotes a bilirubin shift from the extravascular pool. The newly recruited intravascular bilirubin is then, after its exposure to photo-isomerization, rapidly transported to the liver and excreted via the bile (figure 7).[30] Our results in acutely jaundiced Gunn rats show that HSA completely prevented the deposition of bilirubin in the brain during hemolysis. Routine phototherapy, however, failed to prevent this deposition. Theoretically, this lack of effect may be time-related: phototherapy only decreased UCBfree after 48h in our acute experiment, but decreased plasma UCB levels within 36h. We cannot exclude the possibility that non-protein bound bilirubin is less readily converted into photo-isomers than the protein bound fraction. Indeed, long-term phototherapy apparently circumvented this delay and decreased both UCBfree and brain bilirubin in permanently jaundiced Gunn rats. Taken together, our results not only demonstrate the benefits of adjunct HSA, but also question the efficacy of phototherapy during acute hemolytic jaundice.

Our present study shows a relatively poor correlation between the higher plasma bilirubin levels (>200 µmol/L) and brain bilirubin levels. This is consistent with clinical evidence that shows a poor predictive value of plasma bilirubin, especially above 300 µmol/L, for neurotoxicity.[6] These results illustrate the limitations of using plasma UCB to assess the individual risk for bilirubin toxicity. UCBfree levels correlated well with individual brain bilirubin levels in our experiment. Yet, the r2-value indicated that even in highly controlled animal studies, a considerable fraction of the variation in brain bilirubin could not be solely related to plasma UCBfree levels. Our data therefore confirm that, apart from UCBfree, other factors (e.g. blood pH, BBB integrity etc.) are also highly important in the pathogenesis of bilirubin-induced neurological damage.[31] It would be interesting to investigate these factors, as well as the accumulation of bilirubin in specific brain regions (since bilirubin predominantly accumulates in the deep nuclei of the brain) during HSA treatment in future animal experiments.

In our study we have used a commercially available HSA solution and found clear proof that it enhanced the therapeutic efficacy of routine phototherapy. This albumin solution is currently widely applied in neonates, without serious side effects.[14-17,29] This greatly increases its therapeutic potential and will facilitate the set-up of future clinical trials. These trials should ideally incorporate UCBfree measurements and auditory brainstem response screening to monitor the efficacy of treatment. UCBfree measurements should be performed according to the recently developed method of Ahlfors et al. that enables an automated and reliable measurement of UCBfree in a clinical setting.[18]

150 Chapter 6

Taken together, our data show that HSA enhances the efficacy of routine phototherapy in phototherapy-treated Gunn rats, both during permanent and acute jaundice. Our study underlines the need to critically evaluate the use of HSA as adjunct to phototherapy in randomized controlled clinical trials. We expect that a focus on tissue, rather than on plasma bilirubin levels, could induce a paradigm shift that will allow the development of increasingly efficient treatment strategies. These strategies will, hopefully, further decrease the burden of bilirubin-induced brain damage in the near future.

Albumin treatment for unconjugated hyperbilirubinemia 151

References 1. Crigler JF, Najjar VA. Congenital

familial nonhemolytic jaundice with kernicterus. Pediatrics. 1952;10:169–180.

2. Diamond I, Schmid R. Experimental bilirubin encephalopathy. The mode of entry of bilirubin-14C into the central nervous system. J. Clin. Invest. 1966;45:678–689.

3. Calligaris SD, Bellarosa C, Giraudi P, Wennberg RP, Ostrow JD, Tiribelli C. Cytotoxicity is predicted by unbound and not total bilirubin concentration. Pediatr. Res. 2007;62:576–580.

4. Ahlfors CE, Amin SB, Parker AE. Unbound bilirubin predicts abnormal automated auditory brainstem response in a diverse newborn population. J Perinatol. 2009;29:305–309.

5. Zucker SD, Goessling W, Hoppin AG. Unconjugated bilirubin exhibits spontaneous diffusion through model lipid bilayers and native hepatocyte membranes. J. Biol. Chem. 1999;274:10852–10862.

6. Wennberg RP, Ahlfors CE, Bhutani VK, Johnson LH, Shapiro SM. Toward understanding kernicterus: a challenge to improve the management of jaundiced newborns. Pediatrics. 2006;117:474–485.

7. Ostrow JD, Pascolo L, Brites D, Tiribelli C. Molecular basis of bilirubin-induced neurotoxicity. Trends Mol Med. 2004;10:65–70.

8. Ahlfors CE, Wennberg RP, Ostrow JD, Tiribelli C. Unbound (free) bilirubin: improving the paradigm for evaluating neonatal jaundice. Clin. Chem. 2009;55:1288–1299.

9. Ostrow JD. Photocatabolism of labeled bilirubin in the congenitally

jaundiced (Gunn) rat. J. Clin. Invest. 1971;50:707–718.

10. Van der Veere CN, Sinaasappel M, McDonagh AF, Rosenthal P, Labrune P, Odievre M, et al. Current therapy for Crigler-Najjar syndrome type 1: report of a world registry. Hepatology. 1996;24:311–315.

11. Yohannan MD, Terry HJ, Littlewood JM. Long term phototherapy in Crigler-Najjar syndrome. Arch Dis Child. 1983;58:460–462.

12. Keenan WJ, Novak KK, Sutherland JM, Bryla DA, Fetterly KL. Morbidity and mortality associated with exchange transfusion. Pediatrics. 1985;75:417–421.

13. Odell GB, Cohen SN, Gordes EH. Administration of albumin in the management of hyperbilirubinemia by exchange transfusions. Pediatrics. 1962;30:613–621.

14. Chan G, Schiff D. Variance in albumin loading in exchange transfusions. J. Pediatr. 1976;88:609–613.

15. Hosono S, Ohno T, Kimoto H, Nagoshi R, Shimizu M, Nozawa M. Effects of albumin infusion therapy on total and unbound bilirubin values in term infants with intensive phototherapy. Pediatr Int. 2001;43:8–11.

16. Hosono S, Ohno T, Kimoto H, Nagoshi R, Shimizu M, Nozawa M, et al. Follow-up study of auditory brainstem responses in infants with high unbound bilirubin levels treated with albumin infusion therapy. Pediatr Int. 2002;44:488–492.

17. Caldera R, Maynier M, Sender A, Brossard Y, Tortrat D, Galiay JC, et al. [The effect of human albumin in

152 Chapter 6

association with intensive phototherapy in the management of neonatal jaundice]. Arch. Fr. Pediatr. 1993;50:399–402.

18. Ahlfors CE, Marshall GD, Wolcott DK, Olson DC, Van Overmeire B. Measurement of unbound bilirubin by the peroxidase test using Zone Fluidics. Clin. Chim. Acta. 2006;365:78–85.

19. Zelenka J, Lenicek M, Muchova L, Jirsa M, Kudla M, Balaz P, et al. Highly sensitive method for quantitative determination of bilirubin in biological fluids and tissues. J Chromatogr B Analyt Technol Biomed Life Sci. 2008;867:37–42.

20. Labrune P, Myara A, Trivin F, Odievre M. Gunn rats: a reproducible experimental model to compare the different methods of measurements of bilirubin serum concentration and to evaluate the risk of bilirubin encephalopathy. Clin. Chim. Acta. 1990;192:29–33.

21. Rice AC, Shapiro SM. A new animal model of hemolytic hyperbilirubinemia-induced bilirubin encephalopathy (kernicterus). Pediatr. Res. 2008;64:265–269.

22. Cuperus FJC, Hafkamp AM, Havinga R, Vitek L, Zelenka J, Tiribelli C, et al. Effective treatment of unconjugated hyperbilirubinemia with oral bile salts in gunn rats. Gastroenterology. 2009;136:673–82 e1.

23. Ostrow JD, Mukerjee P. Solvent partition of 14C-unconjugated bilirubin to remove labeled polar contaminants. Transl Res. 2007;149:37–45.

24. Hafkamp AM, Havinga R, Ostrow JD, Tiribelli C, Pascolo L, Sinaasappel M, et al. Novel kinetic insights into treatment of unconjugated hyperbilirubinemia: phototherapy and orlistat treatment in Gunn rats. Pediatr. Res. 2006;59:506–512.

25. Cuperus FJC, Iemhoff AA, van der Wulp M, Havinga R, Verkade HJ. Acceleration of the gastrointestinal transit by polyethylene glycol effectively treats unconjugated hyperbilirubinaemia in Gunn rats. Gut. 2010;59:373–380.

26. Harris RC, Lucey JF, Maclean JR. Kernicterus in premature infants associated with low concentrations of bilirubin in the plasma. Pediatrics. 1958;21:875–884.

27. Andersen DH, Blanc WA, Crozier DN, Silverman WA. A difference in mortality rate and incidence of kernicterus among premature infants allotted to two prophylactic antibacterial regimens. Pediatrics. 1956;18:614–625.

28. Odell GB. Studies in kernicterus. I. The protein binding of bilirubin. J. Clin. Invest. 1959;38:823–833.

29. Ebbesen F, Brodersen R. Comparison between two preparations of human serum albumin in treatment of neonatal hyperbilirubinaemia. Acta Paediatr Scand. 1982;71:85–90.

30. Ahlfors CE, Wennberg RP. Bilirubin-albumin binding and neonatal jaundice. Semin Perinatol. 2004;28:334–339.

31. Ahlfors EC. Bilirubin-albumin binding and free bilirubin. Journal of perinatology. 2001;21:S40.

Chapter 7 General Discussion, Conclusions, and Future Directions

154 Chapter 7

7.1 Introduction n this thesis we used the Gunn rat model to develop and refine treatment strategies for severe unconjugated hyperbilirubinemia, as occurs in Crigler-Najjar disease and neonatal hemolytic jaundice. Forty years ago, severe unconjugated hyperbilirubinemia often led to irreversible brain damage in these patients. The only available therapy at that time, exchange transfusion, was associated with significant morbidity and mortality.[1-3] Phototherapy, clinically introduced in the late 1960s, proved a safer treatment for unconjugated hyperbilirubinemia.[4,5] Its widespread use decreased the need for exchange transfusions and enabled long-term treatment for Crigler-Najjar patients.[5,6] Although generally effective, phototherapy had (and still has) some disadvantages. The lifelong phototherapy needed in Crigler-Najjar disease may last >16 hours per day, and nevertheless fails to prevent brain damage in ~25% of the patients.[6,7] Neonatal phototherapy is more effective, but may still require additional, and potentially dangerous, exchange transfusions.[1,2] These considerations prompted us to explore alternative treatment strategies for unconjugated hyperbilirubinemia. To provide a basis for this undertaking, we first reviewed the existing treatment strategies, both experimental and conventional (Chapters 1 and 2). Subsequently, we performed several experiments using Gunn rats, the established model for severe unconjugated hyperbilirubinemia.[8-10] This animal model enabled us to develop two novel oral treatment strategies, as described in Chapters 3, 4, and 5. In the final chapter, we investigated the impact of phototherapy and albumin infusion on brain bilirubin levels during acute and permanent jaundice in Gunn rats.

7.2 Oral treatment strategies for unconjugated hyperbilirubinemia The first part of this thesis focused on oral treatment strategies for unconjugated hyperbilirubinemia. We aimed for these strategies to be non-invasive, safe, and at least as effective as phototherapy.

Existing hypobilirubinemic treatments (chapters 1 and 2) are usually based on inhibition of bilirubin production, or on the stimulation of bilirubin disposal via the bile. In chapter 3, however, we decided to focus on an alternative route for bilirubin disposal, namely transmucosal excretion. Transmucosal bilirubin excretion involves the translocation of non-protein bound UCB (UCBfree) from the blood into the intestinal lumen.[11-14] This translocation is caused by the ability

I

General discussion, conclusions, and future directions 155

of UCBfree to passively (and possibly also actively) cross lipid bilayers.[15-17] In hyperbilirubinemic Gunn rats most bilirubin enters the intestinal lumen via this pathway, rather than via the bile.[12] Transmucosal bilirubin excretion, in short, is an important excretory pathway during severe unconjugated hyperbilirubinemia. Its efficacy in actually disposing bilirubin from the body, however, is limited by the intestines’ ability to reabsorb UCBfree from its lumen.[13,14] We, consequently, aimed to decrease this reabsorption. This had been attempted before by binding UCBfree to intestinal compounds, thereby trapping bilirubin within the gut lumen. Most trapping agents, however, including agar,[18] cholestyramine,[19] charcoal,[20] amorphous calcium phosphate,[21] zinc salts,[22] and fat,[23,24] have been clinically unsatisfactory, due to side-effects and inconsistent results. Since bile salts do not only bind UCBfree in vitro, but also increase biliary UCB excretion in vivo, we reasoned that they might be used to treat unconjugated hyperbilirubinemia.[25-27] In chapter 3 we demonstrated that the bile salt ursodeoxycholate (UDCA) did indeed treat severe unconjugated hyperbilirubinemia in Gunn rats. UDCA is a well-established and well-tolerated treatment for various hepatobiliary diseases in neonatal, pediatric and adult patients.[28,29] Our results, therefore, can readily be verified in randomized clinical trials. The observation that even low (and clinically relevant) UDCA dosages decreased plasma UCB concentrations further underlined its clinical applicability. Treatment with cholic acid (CA), a hydrophobic bile salt, yielded similar results as UDCA treatment. Although clinical CA treatment is not feasible due to its toxicity, this did demonstrate that the main therapeutic effects were bile salt type independent.

One of the more important principles of this thesis is that “hypobilirubinemic treatment” is not a goal in itself. Far more important than its hypobilirubinemic effect is a treatments’ ability to prevent neurotoxicity. Neurotoxicity, however, may occur in the presence of relatively low plasma UCB levels. In the 1950s, several sulfisoxazole-treated neonates developed kernicterus in the presence of unusually low plasma bilirubin levels.[30,31] Odell et al. soon discovered that sulfasoxizole displaced UCB form albumin, a discovery that first highlighted the importance of non-protein bound UCB (UCBfree).[32] Since then, many studies have underscored the poor correlation between plasma bilirubin and the individual risk for bilirubin-induced brain damage.[33-36] The reason for this poor correlation lies in the inability of protein-bound bilirubin (>99% of total plasma bilirubin) to leave the circulation. Only its small (<1%) unbound fraction, i.e. UCBfree, is able to translocate across the blood-brain barrier (figure 1A).[37,38] The inability of bilirubin to leave the circulation does, naturally, undermine its potential to predict neurotoxicity.[33,36] This prompted us to extend our evaluation of bile salt treatment beyond plasma bilirubin levels. A radiolabelled 3H-UCB kinetic study demonstrated that bile salt treatment decreased not only plasma, but also tissue bilirubin levels. This decrease was accompanied by an

156 Chapter 7

increase in fecal bilirubin excretion, mostly (∼80%) derived from the transmucosal excretory pathway. Taken together, our data demonstrated that bile salts effectively treat unconjugated hyperbilirubinemia in Gunn rats. The underlying mechanism involved a stimulation of transmucosal and fecal bilirubin excretion, which induced a decrease in the total (plasma + tissue) bilirubin pool size.

The results of chapter 3 confirmed the important contribution of transmucosal bilirubin excretion to its overall turnover. Accordingly, we continued our pursuit of lowering the bilirubin pool via this excretory pathway. This led to an investigation into the role of the gastrointestinal transit in Chapter 4. Several studies had suggested that an acceleration of the gastrointestinal transit, such as prebiotic supplementation of infant formula, seemed to mitigate neonatal jaundice.[39,40] Other studies had linked conditions that delay transit, such as fasting, to an increase in plasma bilirubin levels.[41-43] None of these studies, however, had directly investigated the relationship between transit time and plasma bilirubin levels. We explored this relationship by treating Gunn rats with the laxative polyethylene glycol (PEG), or with loperamide, which delays transit. This approach revealed a linear relationship between gastrointestinal transit and

UCB free

Bound(e.g. Albumin)

Bound(e.g. ligandin)

Blood Brain

UCB freeUCB UCB

HSA

infusion

Bound(e.g. Albumin)

Blood Brain

freeUCB UCB

Bound(e.g. ligandin)

freeUCBUCB

a b

Figure 1. Human serum albumin (HSA) treatment during unconjugated hyperbilirubinemia: (a) Unconjugated hyperbilirubinemia may result in the accumulation of unconjugated bilirubin (UCB) within the brain. Only non-protein bound UCB (UCBfree) is able to move between the blood (e.g. vascular compartment) and the brain (e.g. extravascular compartment). (b) Treatment with HSA decreases UCBfree levels within the blood. This promotes an UCBfree-induced shift of bilirubin from the brain into the circulation. Additional phototherapy subsequently converts this bilirubin into photo-isomers that can readily be excreted with the bile (dashed arrow).

General discussion, conclusions, and future directions 157

plasma bilirubin levels. The strength of this relationship had two major implications. First, it identified the gastrointestinal transit as an important regulator for plasma bilirubin levels in hyperbilirubinemic Gunn rats. Secondly, it suggested that pharmacological manipulation of the transit time might well be used to treat unconjugated hyperbilirubinemia. Acceleration of the gastrointestinal transit by PEG did effectively decrease plasma bilirubin levels in Gunn rats. Simultaneously with the decrease in plasma bilirubin, which occurred within hours, we observed a marked increase in its fecal disposal. This disposal did not originate from an enhanced biliary UCB excretion. Since bilirubin can only enter the intestinal lumen via the bile or via transmucosal excretion, this showed, by inference, that the PEG-induced acceleration in gastrointestinal transit enhanced transmucosal UCBfree excretion. We hypothesized that this enhanced excretion occurred because the PEG-induced acceleration had decreased the intestinal UCBfree concentration. This claim is indirectly supported by the impressive additive effect of phototherapy in PEG-treated animals. The final plasma bilirubin decrease in phototherapy + PEG treated animals was 65%, which was higher than either treatment alone. This decrease might well be due to the distinct hypobilirubinemic mechanisms of phototherapy (enhanced biliary bilirubin excretion) and PEG (enhanced transmucosal bilirubin excretion). These two mechanisms had been previously described to complement each other in Gunn rats.[9,44]

Oral treatment of unconjugated hyperbilirubinemia, as stated previously, is not a novel concept. In 1962 Lester et al. already treated unconjugated hyperbilirubinemia by feeding Gunn rats cholestyramine, which binds UCBfree within the intestinal lumen.[19] Since then many oral treatments have been evaluated. These treatments, however, tended to lower plasma bilirubin levels within days.[18,19,21-23] This, naturally, had limited their therapeutic potential in acutely jaundiced patients. For example, the recently developed oral treatment with orlistat only decreased plasma UCB levels by a modest 10% after 36h of treatment in our study.[24] PEG treatment, however, lowered plasma bilirubin within hours, which sets it apart from other oral treatment strategies. Another major benefit of PEG treatment, namely its additive effect to standard phototherapy, has already been discussed. A (novel) treatment that complements routine phototherapy has an evident clinical advantage. Combining PEG administration with phototherapy resulted in a hypobilirubinemic effect that was not only superior to single PEG treatment, but also to any conventional or experimental treatment combination previously explored in Gunn rat studies.[18,19,21-23] Taken together, our data thus clearly supported the clinical feasibility of PEG treatment, with or without phototherapy, in hyperbilirubinemic patients. PEG is currently widely used as a laxative, without any serious side effects.[45,46] A randomized clinical trial, perhaps initially in phototherapy-

158 Chapter 7

treated Crigler-Najjar patients, thus seems an appropriate continuation of this line of research.

As chapter 4 had identified the gastrointestinal transit time as a major player in bilirubin metabolism, we next sought to evaluate its role in other treatments than PEG or loperamide. In chapter 5 we determined whether routine phototherapy or experimental oral treatments (e.g. orlistat, UDCA, or amorphous calcium phosphate) also partly rely on the transit time for their hypobilirubinemic effects. This notion may seem somewhat far-fetched, since each of these treatments had already been associated with a well-defined mechanism of action. Upon careful examination, however, we noticed that each of these treatments had been troubled with side effects that do affect the gastrointestinal transit.[47-49] Diarrhea, for example, often occurs during orlistat or UDCA administration.[50] This could imply that these side effects are actually beneficial, because they could lower plasma bilirubin by accelerating the gastrointestinal transit. We clearly demonstrated, however, that each of the aforementioned treatments decreased plasma bilirubin levels without affecting the gastrointestinal transit time. These results prompted us to investigate the value of PEG co-treatment, which might complement the underlying hypobilirubinemic mechanisms of phototherapy and the experimental oral treatments. PEG co-treatment accelerated the gastrointestinal transit time in all treatment groups. A complementary therapeutic effect of this acceleration, however, was only observed in orlistat and phototherapy treated Gunn rats. The complementary effect of adding PEG to phototherapy was reassuringly similar to that of adding phototherapy to PEG. This fits with our previous argument (see: discussion on Chapter 4, above), namely that these treatments truly complement each other. The lack of an additive effect during PEG and UDCA or orlistat treatment, although somewhat unexpected, might have several explanations. First, PEG, orlistat, and UDCA all increased the transmucosal excretion of bilirubin. Combining treatments that use a similar route for bilirubin disposal will usually tend to oversaturate their common pathway, whereas treatments that use distinct routes (e.g. biliary and transmucosal bilirubin disposal) will tend to complement each other (see: discussion on Chapter 4, above). An alternative explanation for the lack of effect is that PEG decreased the intraluminal bile salt concentration, which naturally counteracts additional bile salt therapy. Finally, PEG might have also decreased the precipitation of amorphous calcium phosphate (and thus the capture of intestinal bilirubin) by accelerating the transit. Whatever the explanation for these observations might be, the most striking result of Chapters 4 and 5 remains the profound hypobilirubinemic effect of combined PEG and phototherapy treatment. This truly complementary combination deserves an extensive clinical evaluation in the near future.

General discussion, conclusions, and future directions 159

7.3 Phototherapy and bilirubin disposition in the brain Plasma bilirubin levels are, at best, poor predictors for neurological damage. At worst, however, they can provide a false sense of security that may lead to inertia when intervention is needed.[33,36,51] Only UCBfree, and not protein-bound UCB, can cross the blood brain barrier (§ 7.2). We must thus conclude that the small UCBfree fraction (<1% of total plasma bilirubin) plays a major role in the pathogenesis of bilirubin-induced neurotoxicity. We consequently aimed to lower this fraction with human serum albumin (HSA), in order to prevent bilirubin-induced neurotoxicity (Figure 1). The experiment in chapter 6 was primarily designed to evaluate the effect of HSA treatment on brain bilirubin levels of phototherapy-treated Gunn rats. Our experiments were performed in either permanently or acutely (e.g. hemolytic) jaundiced Gunn rats, which served as model for Crigler-Najjar’s disease or neonatal hemolytic jaundice.[52] In non-hemolytic Gunn rats, long-term phototherapy decreased plasma UCB, plasma UCBfree, and brain bilirubin concentrations. Adjunct HSA treatment, however, increased phototherapy’s efficacy by 32-35%. In hemolytic Gunn rats, phototherapy + HSA decreased plasma UCBfree levels, and completely prevented bilirubin accumulation within the brain. A striking finding was the inability of phototherapy alone to protect these animals from bilirubin accumulation within their brains. Taken together, our data showed that HSA provides a synergistic effect to phototherapy in Gunn rats. We speculate that HSA and phototherapy act in tandem. First, HSA decreases UCBfree within the plasma, which promotes a bilirubin shift from the brain into the blood. Phototherapy then converts this bilirubin into photo-isomers that can readily be excreted with the bile (Figure 1). Interestingly, our results also question the efficacy of single phototherapy and, indirectly, the use of the total plasma bilirubin concentration as a marker for bilirubin-induced brain damage. Although HSA treatment has (infrequently) been applied in neonatal jaundice, its effects have remained controversial. This controversy might be partly due to HSA itself, which usually contains preservatives that were described to interfere with the HSA-bilirubin binding.[53] We, however, found no such interference in our FDA-approved HSA solution. We consequently feel that large-scale clinical trials are the next step towards the routine application of HSA in a clinical setting. These trials should incorporate UCBfree measurements and, ideally, a functional marker of brain function, such as auditory brain stem response measurements. These measurements would allow non-invasive monitoring of bilirubin-induced brain damage, and have been well described in neonates.[51,52,54]

160 Chapter 7

7.4 Conclusions and future directions In this thesis we demonstrated that administration of UDCA, PEG, and PEG combined with phototherapy effectively treats unconjugated hyperbilirubinemia in Gunn rats. In addition we showed that HSA infusion synergizes the therapeutic effect of phototherapy in these animals.

The effects of UDCA, PEG, and HSA administration were studied in Gunn rats, the well-established model for unconjugated hyperbilirubinemia. The use of Gunn rats enabled us to study the therapeutic potential and the underlying mechanism of these new treatments. We exclusively investigated FDA-approved compounds, since this should facilitate their future use in a clinical setting. UDCA, PEG, and HSA have all been routinely applied in patients and their use appeared to be safe, well tolerated, and devoid of significant side effects. Consequently, a logical next step would be to determine the effects of UDCA, PEG, and HSA in clinical trials. To our opinion, trials with UDCA and with PEG should first be performed in patients with Crigler-Najjar disease. These patients require life-long phototherapy, and would thus benefit directly if UDCA or PEG proved to be effective. Clinical trials in Crigler-Najjar patients can be difficult, however, due to the low prevalence of this disease.[6] This could be addressed by using a crossover design, and by including both type I and type II patients.[21,23] A third clinical trial should evaluate adjunct HSA infusion in phototherapy-treated neonates. It will be interesting to see whether adjunct HSA will, as we observed in Gunn rats, synergize the therapeutic effect of routine phototherapy in hyperbilirubinemic neonates. In order to study this efficacy, however, it is necessary to evaluate reliable predictors for bilirubin-induced neurological damage, such as UCBfree and auditory brain stem response measurements. These markers are essential, since it is evidently impossible to measure UCB in human tissue.

Continuing our line of research in a more clinical setting could shed more light on the potential of UDCA, PEG, and HSA as alternative treatment strategies in severely hyperbilirubinemic patients. Simultaneously, we should continue to use animal and in vitro studies to increase our knowledge with regard to the underlying mechanisms of these treatments. Our original experiments were not only designed to evaluate the efficacy of UDCA, PEG, and HSA, but also to tried to elucidate their mechanisms. This “mechanistic” approach increased our understanding of bilirubin metabolism during UDCA, PEG, and HSA treatment. Such understanding is not merely a goal on its own, but will also provide a template for the development of future treatments.

General discussion, conclusions, and future directions 161

References 1. Jackson JC. Adverse events

associated with exchange transfusion in healthy and ill newborns. Pediatrics. 1997;99:E7.

2. Patra K, Storfer-Isser A, Siner B, Moore J, Hack M. Adverse events associated with neonatal exchange transfusion in the 1990s. J. Pediatr. 2004;144:626–631.

3. Keenan WJ, Novak KK, Sutherland JM, Bryla DA, Fetterly KL. Morbidity and mortality associated with exchange transfusion. Pediatrics. 1985;75:417–421.

4. Cremer RJ, Perryman PW, Richards DH. Influence of light on the hyperbilirubinaemia of infants. Lancet. 1958;271:1094–1097.

5. Lucey J, Ferriero M, Hewitt J. Prevention of hyperbilirubinemia of prematurity by phototherapy. Pediatrics. 1968;41:1047–1054.

6. Van der Veere CN, Sinaasappel M, McDonagh AF, Rosenthal P, Labrune P, Odievre M, et al. Current therapy for Crigler-Najjar syndrome type 1: report of a world registry. Hepatology. 1996;24:311–315.

7. Yohannan MD, Terry HJ, Littlewood JM. Long term phototherapy in Crigler-Najjar syndrome. Arch Dis Child. 1983;58:460–462.

8. Labrune P, Myara A, Trivin F, Odievre M. Gunn rats: a reproducible experimental model to compare the different methods of measurements of bilirubin serum concentration and to evaluate the risk of bilirubin encephalopathy. Clin. Chim. Acta. 1990;192:29–33.

9. Ostrow JD. Photocatabolism of labeled bilirubin in the congenitally jaundiced (Gunn) rat. J. Clin. Invest.

1971;50:707–718.

10. Gunn CH. Hereditary Acholuric Jaundice in a New Mutant Strain of Rats. J.Hered. 1938;29:137–139.

11. Schmid R, Hammaker L. Metabolism and disposition of C-14 bilirubin in congenital nonhemolytic jaundice. J. Clin. Invest. 1963;42:1720–1734.

12. Kotal P, Van der Veere CN, Sinaasappel M, Elferink RO, Vitek L, Brodanova M, et al. Intestinal excretion of unconjugated bilirubin in man and rats with inherited unconjugated hyperbilirubinemia. Pediatr. Res. 1997;42:195–200.

13. Lester R, Schmid R. Intestinal absorption of bile pigments. I. The enterohepatic circulation of bilirubin in the rat. J. Clin. Invest. 1963;42:736–746.

14. Lester R, Schmid R. Intestinal absorption of bile pigments. II. Bilirubin absorption in man. N. Engl. J. Med. 1963;269:178–182.

15. Zucker SD, Storch J, Zeidel ML, Gollan JL. Mechanism of the spontaneous transfer of unconjugated bilirubin between small unilamellar phosphatidylcholine vesicles. Biochemistry. 1992;31:3184–3192.

16. Zucker SD, Goessling W, Hoppin AG. Unconjugated bilirubin exhibits spontaneous diffusion through model lipid bilayers and native hepatocyte membranes. J. Biol. Chem. 1999;274:10852–10862.

17. McDonagh AF. Controversies in bilirubin biochemistry and their clinical relevance. Seminars in Fetal and Neonatal Medicine. 2010;15:141–147.

18. Odell GB, Gutcher GR, Whitington

162 Chapter 7

PF, Yang G. Enteral administration of agar as an effective adjunct to phototherapy of neonatal hyperbilirubinemia. Pediatr. Res. 1983;17:810–814.

19. Lester R, Hammaker L, Schmid R. A new therapeutic approach to unconjugated hyperbilirubinaemia. Lancet. 1962;280:1257.

20. Davis DR, Yeary RA. Activated charcoal as an adjunct to phototherapy for neonatal jaundice. Dev Pharmacol Ther. 1987;10:12–20.

21. Van der Veere CN, Jansen PL, Sinaasappel M, Van Der Meer R, Van der Sijs H, Rammeloo JA, et al. Oral calcium phosphate: a new therapy for Crigler-Najjar disease? Gastroenterology. 1997;112:455–462.

22. Vitek L, Muchova L, Zelenka J, Zadinova M, Malina J. The effect of zinc salts on serum bilirubin levels in hyperbilirubinemic rats. J Pediatr Gastroenterol Nutr. 2005;40:135–140.

23. Hafkamp AM, Nelisse-Haak R, Sinaasappel M, Oude Elferink RPJ, Verkade HJ. Orlistat treatment of unconjugated hyperbilirubinemia in Crigler-Najjar disease: a randomized controlled trial. Pediatr. Res. 2007;62:725–730.

24. Hafkamp AM, Havinga R, Sinaasappel M, Verkade HJ. Effective oral treatment of unconjugated hyperbilirubinemia in Gunn rats. Hepatology. 2005;41:526–534.

25. Ostrow JD, Celic L, Mukerjee P. Molecular and micellar associations in the pH-dependent stable and metastable dissolution of unconjugated bilirubin by bile salts. J. Lipid Res. 1988;29:335–348.

26. Rege RV, Webster CC, Ostrow JD. Interactions of unconjugated bilirubin with bile salts. J. Lipid Res. 1988;29:1289–1296.

27. Ostrow JD. Regulation by bile salts of the excretion of conjugated and unconjugated bilirubin in bile. In: In: Fromm H, Leuschner U, eds. Proceedings of the falk symposium No. 84, held in Berlin, Germany, June 9-10, 1995. Kluwer Academic Publishers, 1996. In: Fromm H, Leuschner U, eds. Proceedings of the falk symposium No. 84, held in Berlin, Germany, June 9-10, 1995. Kluwer Academic Publishers, 1996; 1996.

28. Balistreri WF. Bile acid therapy in pediatric hepatobiliary disease: the role of ursodeoxycholic acid. J Pediatr Gastroenterol Nutr. 1997;24:573–589.

29. Arslanoglu S, Moro GE, Tauschel HD, Boehm G. Ursodeoxycholic acid treatment in preterm infants: a pilot study for the prevention of cholestasis associated with total parenteral nutrition. J Pediatr Gastroenterol Nutr. 2008;46:228–231.

30. Harris RC, Lucey JF, Maclean JR. Kernicterus in premature infants associated with low concentrations of bilirubin in the plasma. Pediatrics. 1958;21:875–884.

31. Andersen DH, Blanc WA, Crozier DN, Silverman WA. A difference in mortality rate and incidence of kernicterus among premature infants allotted to two prophylactic antibacterial regimens. Pediatrics. 1956;18:614–625.

32. Odell GB. Studies in kernicterus. I. The protein binding of bilirubin. J. Clin. Invest. 1959;38:823–833.

33. Wennberg RP, Ahlfors CE, Bhutani VK, Johnson LH, Shapiro SM. Toward understanding kernicterus: a challenge to improve the management of jaundiced newborns. Pediatrics. 2006;117:474–485.

34. Calligaris SD, Bellarosa C, Giraudi P, Wennberg RP, Ostrow JD, Tiribelli C. Cytotoxicity is predicted by

General discussion, conclusions, and future directions 163

unbound and not total bilirubin concentration. Pediatr. Res. 2007;62:576–580.

35. Ahlfors CE, Wennberg RP. Bilirubin-albumin binding and neonatal jaundice. Semin Perinatol. 2004;28:334–339.

36. Ahlfors CE, Wennberg RP, Ostrow JD, Tiribelli C. Unbound (free) bilirubin: improving the paradigm for evaluating neonatal jaundice. Clin. Chem. 2009;55:1288–1299.

37. Ostrow JD, Pascolo L, Shapiro SM, Tiribelli C. New concepts in bilirubin encephalopathy. Eur J Clin Invest. 2003;33:988–997.

38. Ostrow JD, Pascolo L, Brites D, Tiribelli C. Molecular basis of bilirubin-induced neurotoxicity. Trends Mol Med. 2004;10:65–70.

39. Bisceglia M, Indrio F, Riezzo G, Poerio V, Corapi U, Raimondi F. The effect of prebiotics in the management of neonatal hyperbilirubinaemia. Acta Paediatr. 2009;98:1579–1581.

40. Cottrell BH, Anderson GC. Rectal or axillary temperature measurement: effect on plasma bilirubin and intestinal transit of meconium. J Pediatr Gastroenterol Nutr. 1984;3:734–739.

41. Kotal P, Vitek L, Fevery J. Fasting-related hyperbilirubinemia in rats: the effect of decreased intestinal motility. Gastroenterology. 1996;111:217–223.

42. Rosta J, Makoi Z, Kertesz A. Delayed meconium passage and hyperbilirubinaemia. Lancet. 1968;292:1138.

43. Whitmer DI, Gollan JL. Mechanisms and significance of fasting and dietary hyperbilirubinemia. Semin Liver Dis. 1983;3:42–51.

44. Hafkamp AM, Havinga R, Ostrow JD, Tiribelli C, Pascolo L, Sinaasappel

M, et al. Novel kinetic insights into treatment of unconjugated hyperbilirubinemia: phototherapy and orlistat treatment in Gunn rats. Pediatr. Res. 2006;59:506–512.

45. Corazziari E, Badiali D, Bazzocchi G, Bassotti G, Roselli P, Mastropaolo G, et al. Long term efficacy, safety, and tolerabilitity of low daily doses of isosmotic polyethylene glycol electrolyte balanced solution (PMF-100) in the treatment of functional chronic constipation. Gut. 2000;46:522–526.

46. Nurko S, Youssef NN, Sabri M, Langseder A, McGowan J, Cleveland M, et al. PEG3350 in the treatment of childhood constipation: a multicenter, double-blinded, placebo-controlled trial. J. Pediatr. 2008;153:254–61, 261 e1.

47. Colecchia A, Mazzella G, Sandri L, Azzaroli F, Magliuolo M, Simoni P, et al. Ursodeoxycholic acid improves gastrointestinal motility defects in gallstone patients. World J Gastroenterol. 2006;12:5336–5343.

48. Filippatos TD, Derdemezis CS, Gazi IF, Nakou ES, Mikhailidis DP, Elisaf MS. Orlistat-associated adverse effects and drug interactions: a critical review. Drug Saf. 2008;31:53–65.

49. Kruis W, Haddad A, Phillips SF. Chenodeoxycholic and ursodeoxycholic acids alter motility and fluid transit in the canine ileum. Digestion. 1986;34:185–195.

50. Hempfling W, Dilger K, Beuers U. Systematic review: ursodeoxycholic acid--adverse effects and drug interactions. Aliment. Pharmacol. Ther. 2003;18:963–972.

51. Ahlfors CE, Amin SB, Parker AE. Unbound bilirubin predicts abnormal automated auditory brainstem response in a diverse newborn population. J Perinatol. 2009;29:305–

164 Chapter 7

309.

52. Rice AC, Shapiro SM. A new animal model of hemolytic hyperbilirubinemia-induced bilirubin encephalopathy (kernicterus). Pediatr. Res. 2008;64:265–269.

53. Weisiger RA, Ostrow JD, Koehler RK, Webster CC, Mukerjee P, Pascolo L, et al. Affinity of human serum albumin for bilirubin varies

with albumin concentration and buffer composition: results of a novel ultrafiltration method. J. Biol. Chem. 2001;276:29953–29960.

54. Shapiro SM. Definition of the clinical spectrum of kernicterus and bilirubin-induced neurologic dysfunction (BIND). J Perinatol. 2005;25:54–59.

Appendices Summary, samenvatting, dankwoord, list of abbreviations, biografie, list of publications

166 Appendices

Summary n this thesis we developed and refined treatment strategies for severe unconjugated hyperbilirubinemia in the Gunn rat model. Severe unconjugated hyperbilirubinemia, as occurs in Crigler-Najjar disease and neonatal hemolytic jaundice, is associated with brain damage and death. Routine treatment mainly consists of phototherapy, which converts bilirubin into more readily excretable photo-isomers. Although generally effective, phototherapy has some disadvantages. The lifelong phototherapy in Crigler-Najjar patients, who suffer from inherited jaundice, may last up to 16h per day but fails to prevent brain damage in ~25% of these patients. Neonatal phototherapy, although mostly efficient and safe, sometimes fails to achieve a sufficient decrease in plasma bilirubin levels. This may, ultimately, also lead to permanent brain damage in these patients.

These considerations demonstrate the need for alternative treatment strategies. Most hypobilirubinemic treatments, as described in chapters 1 and 2, stimulate the fecal excretion of bilirubin via the bile. Biliary bilirubin excretion, however, is highly inefficient in patients with Crigler-Najjar disease and, to a lesser extent, in patients with neonatal jaundice. This is due to an inactivated (Crigler-Najjar disease) or immature (neonatal jaundice) isoform of the enzyme UDP-glucuronosyltransferase. This hepatic enzyme catalyzes the transfer of glucuronic acid to bilirubin, thus forming bilirubin monoglucuronoside or diglucuronoside. This so-called conjugated bilirubin is more water soluble, and can readily be excreted into the bile. Bilirubin, however, is not exclusively excreted via the bile, but may also enter the intestinal lumen via direct transmucosal excretion from the blood. The efficiency of this excretory pathway is decreased, however, by reabsorption of unconjugated bilirubin from the intestinal lumen. This reabsorption can be prevented by intestinal capture; the binding of intestinal unconjugated bilirubin to orally administered agents.

In chapter 3 we decided to assess whether oral treatment with the bile salt ursodeoxycholate (UDCA) would enhance the transmucosal excretion of bilirubin. We chose UDCA because it binds bilirubin in vitro, and because it is safely used in the treatment of several biliary diseases. To study bilirubin fluxes, we decided to use radioactively labeled bilirubin, which necessitated the use of an animal model. We consequently assessed the effects of UDCA feeding in Gunn rats, the well-established animal model for unconjugated hyperbilirubinemia. This approach allowed us to demonstrate that UDCA decreases plasma bilirubin levels and enhances its transmucosal excretory pathway. UDCA treatment did not only decrease bilirubin in the circulation, but also lowered the amount of bilirubin in

I

[Geef  de  naam  van  het  bedrijf  op] 167

the tissues. Taken together, these data support the feasibility of UDCA treatment in hyperbilirubinemic patients.

Since chapter 3 demonstrated the importance of transmucosal excretion, we continued our pursuit to enhance this pathway in chapter 4. This chapter evaluated whether acceleration of the gastrointestinal transit by the laxative polyethylene glycol could treat unconjugated hyperbilirubinemia in Gunn rats. Clinical conditions that delay the gastrointestinal transit, such as fasting, seem to increase plasma bilirubin levels. Conditions that accelerate the gastrointestinal transit, such as frequent feedings in the neonatal period, are associated with a decrease in plasma bilirubin. Acceleration of the gastrointestinal transit could, theoretically, lower the intestinal bilirubin concentration. A lower intestinal bilirubin concentration, in turn, could create a diffusional bilirubin shift from the blood into the intestinal lumen. None of the above-described conditions, however, had directly linked the transit time to plasma bilirubin levels. We investigated this link by treating Gunn rats with the laxative polyethylene glycol, or with loperamide (which delays transit). We demonstrated that polyethylene glycol treatment accelerated the gastrointestinal transit and, simultaneously, decreased plasma bilirubin levels. This decrease did not originate from an increased biliary disposal of bilirubin. We thus concluded, by inference, that acceleration of the gastrointestinal transit must enhance the transmucosal excretion of bilirubin, since this is the only alternative excretory pathway that allows bilirubin to enter the intestinal lumen. Loperamide treatment, in contrast, delayed the gastrointestinal transit and thereby increased plasma bilirubin levels.

Our approach demonstrated, for the first time, a linear relationship between the gastrointestinal transit time and bilirubin levels in Gunn rats. The strength of this relationship implicated that pharmacological manipulation of the gastrointestinal transit may well be used to treat unconjugated hyperbilirubinemia. Because polyethylene glycol is a safe and well-tolerated treatment for constipation, we feel that the results in this chapter merit a randomized clinical trial in hyperbilirubinemic Crigler-Najjar patients.

In chapter 5 we investigated, in Gunn rats, whether known hypobilirubinemic treatments (e.g. phototherapy and/or oral capture agents) exert their therapeutic effect by acceleration of the gastrointestinal transit. A review of the literature had shown us that each of these treatments was troubled with side effects, mostly diarrhea, that did affect the gastrointestinal transit. In chapter 5, however, we clearly demonstrated that each of the aforementioned treatments decreased plasma bilirubin without affecting the gastrointestinal transit. We consequently evaluated whether additional treatment with polyethylene glycol could increase the efficacy of these treatments. This experiment demonstrated that phototherapy combined with polyethylene glycol decreased the plasma bilirubin levels in Gunn

168 Appendices

rats by almost 70%. These results identified polyethylene and phototherapy as the most potent hypobilirubinemic treatment combination that has been tested so far in the Gunn rat model.

Eventually, all therapy should prevent neurotoxicity by decreasing tissue rather than plasma bilirubin concentrations. UCBfree, the small fraction of unconjugated bilirubin that is not bound to plasma albumin, is able to diffuse from the plasma into the tissue pool. Decreasing this fraction, for instance by increasing its binding to albumin, would thus be expected to decrease tissue bilirubin levels. In chapter 6 we investigated the effect of phototherapy and phototherapy + albumin infusion on UCBfree and brain bilirubin levels during permanent and acute hemolytic jaundice in Gunn rats. Our results demonstrated a synergistic effect of albumin treatment to phototherapy in Gunn rats. In non-hemolytic Gunn rats, used as a model for Crigler-Najjar disease, we showed that albumin treatment enhanced the phototherapy-induced decrease in plasma UCBfree and brain bilirubin levels by 32-25%. Phototherapy + albumin completely prevented bilirubin accumulation in the brains of hemolytic Gunn rats, used as a model for acute neonatal jaundice. Interestingly, phototherapy alone failed to prevent bilirubin accumulation in the brains of these animals. Our findings, therefore, do not only demonstrate the efficacy of adjunct albumin treatment during phototherapy in Gunn rats, but also question the therapeutic effects of routine phototherapy. UCBfree correlated well with the concentrations of bilirubin in the brain, underlining its potential use as predictor for bilirubin damage in clinical practice. Taken together, our results underline the need for a large-scale clinical trial to evaluate the use of adjunct albumin in phototherapy-treated Crigler-Najjar and neonatal patients.

In the final chapter of this thesis, chapter 7, we place our results in a experimental and clinical framework, and discuss future perspectives.

In this thesis we have developed and refined treatment strategies for unconjugated hyperbilirubinemia in the Gunn rat. These strategies may serve as an alternative for routine treatment, and may well prevent bilirubin-induced brain damage in hyperbilirubinemic patients. We consequently feel that our results can be used as a framework for the development of clinical controlled trials in Crigler-Najjar patients and in severely jaundiced neonates.

[Geef  de  naam  van  het  bedrijf  op] 169

Samenvatting ilirubine is een molecuul met paradoxale eigenschappen. In relatief lage concentraties is het een anti-oxidant, maar in hoge concentraties is het zeer toxisch voor onze lichaamscellen. Vooral hersencellen zijn gevoelig voor bilirubine, en bilirubineophoping in ons lichaam kan leiden tot permanente schade in ons brein (zie ook tekstvak 1). In dit proefschrift onderzochten wij nieuwe behandelstrategieën om deze schade te voorkomen.

Bilirubine wordt gevormd door de afbraak van rode bloedcellen. Deze afbraak, en dus de productie van bilirubine, is een continu proces. De uitscheiding van bilirubine vindt plaats via de lever, welke bilirubine uit de bloedbaan opneemt en transporteert naar de galwegen. Vanuit de galwegen wordt het molecuul dan uitgescheiden in de darm waarna het ons lichaam verlaat met de ontlasting. De uitscheiding van bilirubine via de galwegen en de ontlasting moet uiteraard net zo groot zijn als de aanmaak van bilirubine uit de rode bloedcellen om te voorkomen dat de bilirubinespiegel stijgt of daalt. Als de balans tussen bilirubine aanmaak en uitscheiding echter wordt verstoord, kan het geelgekleurde molecuul zich ophopen in het bloed (hyperbilirubinemie) en in de weefsels. Dit laatste uit zich in geelzucht: een kenmerkende gele verkleuring van de huid en het oogwit. Ernstige ongeconjugeerde hyperbilirubinemie kan leiden tot hersenschade, omdat het toxische bilirubine zich kan ophopen in ons brein. Deze hersenschade komt vooral voor bij patiënten met de ziekte van Crigler-Najjar en bij sommige pasgeborenen. Patiënten met de ziekte van Crigler-Najjar hebben, door een erfelijk defect, een niet (type I) of nauwelijks (type II) werkzame variant van het leverenzym UDP-glucuronosyltransferase. Dit enzym is essentieel voor de uitscheiding van bilirubine, omdat het bilirubine in de lever koppelt aan twee suikergroepen. Pas na deze koppeling, ook wel conjugatie genoemd, kan bilirubine op een efficiënte manier vanuit de lever in de gal worden uitgescheiden. Omdat de ziekte van Crigler-Najjar berust op een genetisch defect, lijdt deze patiëntengroep aan een levenslange ophoping van ongeconjugeerd bilirubine in het bloed (ongeconjugeerde hyperbilirubinemie) en in de weefsels. Ook pasgeborenen ontwikkelen vaak een ongeconjugeerde hyperbilirubinemie, maar deze is slechts in de eerste dagen na de geboorte aanwezig. De oorzaak van deze ‘neonatale geelzucht’ ligt in een tijdelijk verhoogde afbraak van rode bloedcellen, in combinatie met een nog lage activiteit van het eerder genoemde UDP-glucuronosyltransferase. Meestal is neonatale geelzucht niet ernstig, maar in sommige gevallen is er toch behandeling nodig om hersenschade te voorkomen. Dit kan bijvoorbeeld het geval zijn bij bloedgroep-incompatibiliteit tussen moeder en kind. In dat geval zorgen antistoffen van de moeder voor een massale afbraak

B

170 Appendices

LICHT

N

N

CO

O

O

OOO

HH

HC

N

NH

H

H

OOO

Nov

el Tre

atm

ent S

trateg

ies fo

r

Unco

njuga

ted H

yper

bilirubin

emia

Frans C

uperus

Novel Treatment Strategies for

Unconjugated Hyperbilirubinemia

N

N

CO

O

O

O

O

O

H

H

H

C

NNH H

HOOO

Frans Cuperus

3

H O2

H O2

H O2

H O2

H O2

Bilirubine is een molecuul met twee gezichten. Dit is zowel zichtbaar in haar uitwerking op

menselijke cellen (waarover straks meer), als in haar ruimtelijke structuur. Deze structuur

lijkt erg op een boek dat half is geopend. Als u dit proefschrift openslaat, in een hoek van

98º, heeft u dan ook een model van het bilirubinemolecuul in uw handen (Figuur 1A). De ka-

ften van het proefschrift vormen de “buitenkant” van het molecuul en bestaan uit de daarop

afgebeelde pyrroolringen. Waterstofbruggen tussen de pyrroolringen geven het molecuul haar

stabiliteit. Pyrroolringen lossen slecht op in water. Door deze hydrofobe (niet-wateroplosbare)

buitenkant moet bilirubine gekoppeld worden aan 2 suikergroepen (glucuronzuren) voordat

het uitgescheiden kan worden in de gal. Deze koppeling vindt plaats in de lever onder invloed

van het enzym UDP-glucuronosyltransferase. Bij patiënten met de ziekte van Crigler-Najjar

werkt dit enzym niet goed, waardoor bilirubine zich in het lichaam ophoopt. Bilirubineopho-

ping kan gevaarlijk zijn omdat bilirubine, nadat het de bloed-hersenbarrière is gepasseerd,

soms hersenschade veroorzaakt. Deze schade proberen we te voorkomen met fototherapie.

Fototherapie gebruikt lichtenergie om de waterstofbruggen in het molecuul te verbreken.

Hierdoor klapt het molecuul (e.g. dit proefschrift) open, en komen de wateroplosbare (“hydro-

fiele”) groepen aan de binnenkant van het molecuul in contact met de omgeving (Figuur 1B).

Het nu hydrofiele bilirubine kan dan eenvoudig zonder conjugatie worden uitgescheiden. Het

normaal hydrofobe bilirubine kan dus onder invloed van fototherapie omklappen en plots haar

hydrofiele “gezicht” tonen. Een ander voorbeeld van de twee gezichten van bilirubine vinden

we in haar uitwerking op menselijke cellen. In lage concentraties beschermt bilirubine cellen

door haar anti-oxidatieve eigenschappen. Er is bijvoorbeeld beschreven dat mensen met een

milde verhoging van de bilirubinespiegel in het bloed minder hart- en vaatziekten hebben.

In hoge concentraties, echter, zorgt bilirubine voor celschade en celdood, bijvoorbeeld in de

hersenen. Het is deze schade die we in dit proefschrift willen voorkomen.

Tekstvak 1.

Figuur 1A Figuur 1B

[Geef  de  naam  van  het  bedrijf  op] 171

van rode bloedcellen, en dus voor een sterk vergrote bilirubine aanmaak, bij de pasgeborene. Ernstige ongeconjugeerde hyperbilirubinemie is gevaarlijk omdat het gepaard kan gaan met hersenschade. Om deze reden moeten Crigler-Najjar patiënten en neonaten met ernstige geelzucht worden behandeld. Deze behandeling bestaat meestal uit fototherapie. Fototherapie gebruikt blauw licht om het bilirubinemolecuul meer wateroplosbaar te maken. Dit wateroplosbare bilirubine kan dan eenvoudig, en zonder conjugatie, door de lever worden uitgescheiden. Fototherapie werd ontdekt door de opmerkzaamheid van een verpleegkundige, die haar afdelingsarts erop wees dat pasgeborenen aan de raamkant van de zaal minder geelzucht leken te hebben. Deze observatie zorgde er uiteindelijk voor dat fototherapie de standaard behandeling werd van ongeconjugeerde hyperbilirubinemie. Sinds de invoering van fototherapie, eind jaren zestig, is het risico op neurologische schade door ongeconjugeerde hyperbilirubinemie enorm afgenomen. Toch kleven er nog altijd nadelen aan deze vorm van behandeling. Crigler-Najjar patiënten moeten, aangezien zij een permanent defect van het UDP-glucuronosyltransferase enzym hebben, levenslang worden behandeld. Dit houdt in dat deze patiënten dagelijks tot 16 uur onder een soort zonnebank moeten liggen, die met (zichtbaar) blauw licht de huid bestraalt. Deze therapie is niet alleen zeer belastend, maar lijkt ook minder effectief te worden naarmate de patiënt ouder wordt. Door dit verlies van effectiviteit ontwikkelt ongeveer 25% van de Crigler-Najjar patiënten uiteindelijk alsnog bilirubine-geïnduceerde hersenschade. Fototherapie lijkt bij neonatale geelzucht minder belastend te zijn; er is immers sprake van een korte behandelperiode. Soms blijven de bilirubinespiegels in het bloed echter fors stijgen ondanks intensieve fototherapie. Om hersenschade te voorkomen wordt er dan vaak voor gekozen om een wisseltransfusie uit te voeren. Bij een wisseltransfusie wordt het bloed van een pasgeborene vervangen door donorbloed met een normaal bilirubinegehalte. Deze vorm van behandeling is echter niet ongevaarlijk, en wordt dan ook het liefst vermeden.

Om bovenstaande redenen besloten wij een alternatieve behandeling te ontwikkelen voor ongeconjugeerde hyperbilirubinemie. In hoofdstuk 1 en 2 is allereerst een overzicht gemaakt van de reeds bestaande behandelmethoden. Deze behandelingen bleken voornamelijk de bilirubine-uitscheiding via de gal te verhogen. Bilirubine kan het lichaam echter op meer dan één manier verlaten. Als er sprake is van een ernstige ongeconjugeerde hyperbilirubinemie bijvoorbeeld, wordt een groot deel van het in het bloed opgehoopte bilirubine direct getransporteerd naar de darmen. Dit proces heet transmucosale (i.e. over het darmslijmvlies) bilirubine excretie en het speelt een zeer grote rol in de bilirubine-uitscheiding van Crigler-Najjar patiënten. Een gedeelte van het transmucosaal uitgescheiden bilirubine wordt echter direct weer opgenomen in het bloed; er is dus sprake van tweerichtingsverkeer over de darmwand. Deze heropname van bilirubine vermindert, uiteraard, de effectiviteit van de

172 Appendices

transmucosale excretie. Door nu de heropname van bilirubine te remmen ontstaat er eenrichtingsverkeer waardoor de bilirubinespiegel in het bloed daalt, terwijl de hoeveelheid bilirubine in de darm stijgt. Een logische aanpak om deze heropname te remmen zou een orale behandeling zijn met stoffen die bilirubine binden in de darm. Een “pil voor geelzucht” dus, die door bilirubine te binden in de darm de plasma (ongeconjugeerde) bilirubinespiegels op een veilige en effectieve manier verlaagd.

In hoofdstuk 3 besloten we te onderzoeken of het galzout ursodeoxycholaat (UDCA) de transmucosale bilirubine-excretie zou kunnen verhogen. Wij kozen voor UDCA omdat dit lichaamseigen galzout makkelijk bindt aan bilirubine, en bovendien al jaren veilig wordt gebruikt voor de behandeling van verschillende leverziekten. Om de invloed van UDCA op de transmucosale bilirubine-excretie te meten was het nodig om radioactief gelabeld bilirubine te gebruiken (3H-bilirubine). Deze stof kan niet zomaar in mensen worden toegepast en om deze reden besloten we een diermodel te gebruiken, namelijk de Gunn rat. Gunn ratten hebben hetzelfde erfelijke defect als Crigler-Najjar patiënten, en worden gezien als een betrouwbaar model voor ernstige ongeconjugeerde hyperbilirubinemie. Dankzij deze opzet konden wij aantonen dat UDCA de bilirubinespiegel in het bloed verlaagt door haar transmucosale uitscheiding te verhogen. Bovendien verlaagde UDCA niet alleen de bilirubinespiegel van het bloed, maar ook die in de weefsels. Dit laatste is niet onbelangrijk, aangezien juist deze weefselspiegels veel zeggen over het risico op hersenschade.

Nadat we door de resultaten van hoofdstuk 3 overtuigd waren geraakt van het belang van transmucosale bilirubine-excretie, onderzochten we in hoofdstuk 4 of een versnelling van de darmpassage ook zou kunnen leiden tot lagere bilirubinespiegels in het bloed van Gunn ratten. Dit onderzoek was gebaseerd op de aanname dat een versnelde darmpassage ook de bilirubine uitscheiding met de ontlasting zou bevorderen. Ons onderzoek liet inderdaad zien dat behandeling van Gunn ratten met polyethyleen glycol (een laxerend middel) leidde tot een versnelde darmpassage en een verhoogde uitscheiding van bilirubine met de ontlasting. Deze verhoogde uitscheiding ging bovendien gepaard met een forse daling van de hoeveelheid bilirubine in het bloed. De toename van bilirubine uitscheiding met de feces hing niet samen met een toename van bilirubine uitscheiding via de gal. Dit maakt het zeer waarschijnlijk dat een versnelde darmpassage de transmucosale excretie van bilirubine bevorderd, aangezien dit de enige alternatieve manier is waarop bilirubine in de ontlasting kan komen. Mogelijk stimuleert polyethyleen glycol deze excretie door de bilirubineconcentratie in de darm te verlagen. Dit zou zowel de heropname van bilirubine naar de bloedsomloop kunnen verminderen, als het bilirubinetransport van het bloed naar de darm kunnen faciliteren. Door nu andere Gunn ratten met loperamide (een middel tegen diarree) te behandelen, konden we aantonen dat

[Geef  de  naam  van  het  bedrijf  op] 173

een vertraagde darmpassage leidde tot een verlaagde uitscheiding van bilirubine met de feces en een fors hogere bilirubineconcentratie in het bloed. Met behulp van bovenstaande experimenten konden we bovendien bewijzen dat er een lineair verband bestaat tussen de darmpassage tijd en de bilirubinespiegels in het bloed van Gunn ratten. Dit lineaire verband toonde aan dat de uitscheiding van bilirubine direct afhankelijk is van de darmpassagetijd. Deze afhankelijkheid lijkt ook bij patiënten met geelzucht een belangrijke rol te spelen. Eerder onderzoek heeft namelijk aangetoond dat omstandigheden die de darmpassage vertragen, zoals vasten, gepaard gaan met een verhoging van de bilirubinespiegel in het bloed. Om deze reden denken wij dat onze resultaten de mogelijkheid van polyethyleen glycol behandeling bij hyperbilirubinemische patiënten ondersteunen.

In hoofdstuk 5 onderzochten wij of de snelheid van de darmpassage mogelijk ook een rol speelde in andere behandelingen voor ongeconjugeerde hyperbilirubinemie. Om deze reden bepaalden wij de darmpassagetijd in Gunn ratten die behandeld werden met experimentele orale behandelingen of met fototherapie. De aanleiding voor deze proef lag in eerdere observaties waarin al deze behandelingen geassocieerd leken te zijn met een versnelde darmpassage. Wij toonden echter aan dat geen van de door ons onderzochte behandelingen enig effect had op de snelheid van de darmpassage in Gunn ratten. Hierop besloten wij de onderzochte behandelingen te combineren met polyethyleen glycol. Wij redeneerden dat hun werkingsmechanisme kennelijk niets van doen had met de snelheid van de darmpassage, en dat daarom een versnelling van de passagetijd hun bilirubine-verlagende effect zou kunnen complementeren. Deze proef liet inderdaad een complementair effect zien in ratten die behandeld werden met zowel fototherapie als polyethyleen glycol. Deze combinatie zorgde voor een zeer forse (bijna 70%) daling van de plasma bilirubinespiegels in Gunn ratten. Dit maakt de combinatie van fototherapie en polyethyleen glycol de krachtigste behandeling voor ongeconjugeerde hypebilirubinemie die ooit in de Gunn rat is beschreven. Onze bevindingen onderstrepen, naast de resultaten van hoofdstuk 4, eens te meer dat polyethyleen glycol een veelbelovend middel lijkt te zijn voor de behandeling van geelzucht.

Het uitgangspunt van ons onderzoek in hoofdstuk 6 was dat de behandeling van ongeconjugeerde hyperbilirubinemie geen doel op zich hoort te zijn. Een behandeling moet immers in de eerst plaats hersenschade voorkomen, en deze kan ook ontstaan bij relatief lage bilirubinespiegels in het bloed (e.g. een milde ongeconjugeerde hyperbilirubinemie). Dit laatste werd duidelijk in de jaren ’50, toen een cohort pasgeborenen met slechts een milde ongeconjugeerde hyperbilirubinemie ernstige hersenschade ontwikkelde. Al deze patiëntjes bleken te zijn behandeld met het antibioticum sulfisoxazol. Enige tijd later werd ontdekt dat dit middel de binding tussen bilirubine en albumine in het bloed verstoorde.

174 Appendices

Sinds deze observatie hebben vele studies laten zien dat de bilirubinespiegel in het bloed slecht correleert met hersenschade. De reden voor deze slechte correlatie ligt in het feit dat meer dan 99% van het bilirubine in ons bloed gebonden is aan plasma eiwitten (voornamelijk albumine). Deze fractie kan de bloedsomloop hierdoor niet verlaten en zal daarom niet in de hersenen neerslaan. Een zeer klein gedeelte (<1%) van het bilirubine in ons bloed is niet gebonden aan plasma eiwitten en kan de bloed-hersenbarrière wel passeren. Deze vrije, niet-eiwitgebonden fractie (bilirubinevrij) speelt dus een centrale rol in de pathogenese van bilirubine-geïnduceerde hersenschade. In hoofdstuk 6 onderzochten wij daarom of het verlagen van deze fractie, door een injectie met het eiwit albumine, ook daadwekelijk zou leiden tot minder neerslag van bilirubine in de hersenen van Gunn ratten. Dit bleek inderdaad het geval te zijn; Gunn ratten die zowel standaard fototherapie als een albumine-injectie hadden gehad bleken lagere bilirubinevrij spiegels in het bloed en lagere bilirubine spiegels in de hersenen te hebben dan ratten die alleen met fototherapie waren behandeld. Aangezien Gunn ratten een goed model vormen voor de ziekte van Crigler-Najjar, lijkt incidentele behandeling met albumine voor deze patiënten dus aangewezen. Dit zou vooral nuttig kunnen zijn om het gevaar van een plotselinge verergering van de geelzucht, die vaak ontstaat als deze patiënten ziek zijn, te verminderen. Na deze studie besloten we een andere groep Gunn ratten te behandelen met een middel dat rode bloedcellen afbreekt. Deze dieren ontwikkelden een acute hyperbilirubinemie die, net als die bij pasgeborenen, werd veroorzaakt door een toename van de bilirubineproductie uit rode bloedcellen en een verminderde bilirubine uitscheiding door inactief UDP-glucuronosyltransferase. Deze studie liet zien dat de standaardbehandeling voor acute hyperbilirubinemie, namelijk fototherapie, geen significante bescherming bood tegen de neerslag van bilirubine in de hersenen van Gunn ratten. De combinatie van albumine met fototherapie, daarentegen, voorkwam deze neerslag volledig. Zowel in het eerste en in het tweede experiment correleerden de bilirubinevrij concentraties in het bloed uitstekend met de hoeveelheid bilirubine in de hersenen. Bilirubinevrij lijkt dus een goede voorspeller te zijn voor bilirubineaccumulatie in de hersenen. Onze experimenten laten zien dat het loont om niet de ongeconjugeerde hyperbilirubinemie (e.g. de hoeveelheid bilirubine in het bloed), maar de neerslag van bilirubine in de hersenen als uitgangspunt voor behandeling te gebruiken. Aangezien albumine-injecties al veilig worden toegepast in pasgeborenen, lijkt een prospectief gerandomiseerd onderzoek naar de waarde van deze injecties tijdens fototherapie een logische vervolgstap van ons onderzoek.

In hoofdstuk 7, tenslotte, houden we de bevindingen van dit proefschrift tegen het licht van de bestaande literatuur. Bovendien bespreken we potentiele klinische en experimentele toepassingen van deze bevindingen.

[Geef  de  naam  van  het  bedrijf  op] 175

In dit proefschrift hebben wij verschillende nieuwe behandelstrategieën ontwikkeld voor ongeconjugeerde hyperbilirubinemie. Deze strategieën kunnen wellicht de belasting van de huidige behandelmethoden (e.g. fototherapie en wisseltransfusies) voor patiënten verminderen. Bovendien zouden deze nieuwe behandelingen, alleen of in combinatie met fototherapie, de incidentie van bilirubine-geïnduceerde hersenschade verder kunnen terugdringen. De verkregen resultaten kunnen uiteraard dienen als raamwerk voor toekomstig fundamenteel onderzoek, maar rechtvaardigen ook zeker klinische vervolgstudies in Crigler-Najjar patiënten en neonaten.

176 Appendices

I

Dankwoord n Afrika schijnt men te zeggen dat het een dorp kost om een kind op te voeden. Ik zou willen stellen: dat geldt ook voor een onderzoeker. Waarschijnlijk meer dan een dorp. Ik gok meer in de richting van een flinke stad... Al deze mensen hebben direct, of indirect, hun steentje bijgedragen aan mijn interesse voor onderzoek en dus aan dit boekje. Graag wil ik van de gelegenheid gebruik maken om ze hiervoor te bedanken.

Om bij het begin te beginnen wil ik mijn ouders bedanken. Jullie hebben mij, en ik weet niet of jullie je dit nog herinneren, ooit het boek “De Wonderen van het Heelal” gegeven. Volgens mij resulteerde dit in een spreekbeurt met de, enigszins ambitieuze, titel “Het Heelal” in groep 4. Ik geef toe dat ik vanaf toen wat ben gaan downscalen in mijn wetenschappelijke interesses, maar volgens mij is het daar toch allemaal mee begonnen.

Ten tweede wil ik Henkjan Verkade, mijn promotor, bedanken. Vanaf onze eerste ontmoeting, waarin je alle onderzoekslijnen van het kindergeneeskunde lab in 2 minuten en 10 seconden vertelde (“of ga ik nu te langzaam”) was het duidelijk dat je: (a) zeer snel nadacht, (b) vaak direct de crux te pakken hebt. Ik weet in ieder geval dat ik 5 minuten later weer naar huis fietste met een onderzoeksproject over bilirubine. Jouw enthousiasme en analytische blik werkten altijd aanstekelijk. Ook na urenlang geestdodend werk kwam ik altijd met een volledige “mentale reset” en nieuwe ideeën bij je vandaan. Nogmaals hartelijk dank voor je meer dan goede begeleiding!

Dear Don Ostrow, Libor Vitek, and Claudio Tirebelli (a.k.a. the “fellows of yellow”). Thank you for sharing your incredible knowledge on bilirubin metabolism. The best part of research, by far, is the chance to collaborate. Your expertise, encouragment, and enthousiasm have convinced me of this undeniable fact.

Ook wil ik Christian Hulzebos en Deirdre van Imhoff bedanken voor hun betrokkenheid bij het laatste hoofdstuk van mijn proefschrift. Het was een gok om albumine los te laten en ons, met het vrije bilirubine, mee te laten voeren naar de hersenen. We kunnen nu zeggen, na de nodige dagen overwerken, dat deze gok goed heeft uitgepakt. Ik ben meer dan een beetje benieuwd naar wat het verdere onderzoek in neonaten gaat opleveren.

Folkert Kuipers, Edmond Rings, en Frans Stellaard wil ik graag bedanken voor het meedenken met de verschillende proeven die ik, al dan niet terecht, wilde uitvoeren in ons lab. Ik heb veel van jullie mogen leren, hartelijk dank hiervoor.

[Geef  de  naam  van  het  bedrijf  op] 177

Natuurlijk wil ik mijn collega’s van het eerste uur bedanken. In de eerste plaats Anja Hafkamp, mijn voorgangster. Als bergbeklimmer kan ik een goede, en precieze, gids zeker waarderen. Je hebt me kundig behoedt voor een paar valpartijen, mijn dak hiervoor is groot.

Arjan! Het was mooi om slechte grappen te maken, biertjes te drinken, en samen te werken aan het bilirubine front. Het uitzoeken van rattenkeutels voor onze analyses (ja dat is ook onderzoek) werd zo bijna dragelijk. Fantastisch dat “The Dude” straks als getrouwde huisarts abides. Het leven is wat gebeurt terwijl je andere plannen maakt. Mooi dat het nu zo mag lopen..

Ik heb menig verloren uurtje doorgebracht in “the lab nextdoor” van Henk B (nog bedankt Henk!). Ten eerste wil ik Herman, het Orakel uit Haren, bedanken voor zijn onnavolgbare levenslessen, humor, en zijn fantastische persoonlijkheid. Herman, tot in Oslo! Ook Herman’s collega’s: Enge, Jasper, Ingrid, Marchien en alle anderen...bedankt voor jullie hulp en humor.

Bas en Maarten. Mensen begeleiden op de “yellow brick road” van het bilirubineonderzoek was een leuke ervaring. Ik wil jullie hartelijk bedanken voor de pipeteerkunsten en waardevolle inbreng. Helemaal mooi dat jullie (na enige indoctrinatie van mijn kant?) beide gekozen hebben om de M in MLO te gaan vervangen door een H.

Rick, bedankt voor de gouden handjes. Jouw kennis van cannulaties, boeken, vrouwen en voetbal zijn ongeëvenaard op het Westelijk halfrond. Juul (sportklimmer, histologie goeroe, en allround aardig persoon), hartelijk dank voor jouw bijdrage aan mijn Gunn rat experimenten! Iedereen in het kindergeneeskunde lab (ik schrijf de namen niet uit, straks vergeet ik iemand!): enorm bedankt voor jullie raad en daad!

Een promotie betekent eigenlijk opnieuw op kamers gaan, maar dan zonder de pizzadozen en de late nachten. Nu ik terugkijk: die waren er eigenlijk ook gewoon. Goede kamergenoten zijn in ieder geval essentieel. Wat dat betreft niets dan lof. Jannes, Axel, Willemien, Martijn, Karin, Marjan, Anoek, Atta, Sandra, Hester, Robert: jullie waren relaxed in the face of research. Het was mooi om met jullie een klein aantal vierkante meters te mogen delen.

Alle andere lotgenoten in AIO-land; (adem in) Sabina, Esther, Hilde, Anke, Thomas, Kryztof, Margot, Mariëtte, Maxi, Jelske, Gemma, Jaap, Marije, Jurre, Janine, Leonie, Jaap, Niels, Jaana, Jan Fraerk, Thierry, Frank (bedankt voor de mooie grappen tijdens journalclubs), Niels, Marijke, Jelena, Aldo, Antonella, Laura, Lisethe.....(adem uit) en alle anderen die even door de mazen van dit dankwoord heen zijn geglipt: hartelijk dank!

178 Appendices

Ook wil ik mijn opvolgster Andrea hartelijk danken. Het is mooi dat de bilirubine lijn op een goede manier wordt voortgezet. Met jouw praktische blik en energie moet dat zeker gaan lukken: keep me posted!

De leden van de leescommissie, professores Vitek, Bos en Drenth, wil ik hartelijk bedanken voor het beoordelen van dit proefschrift.

Mijn nieuwe collega’s in de kliniek wil ik bedanken voor de collegialiteit en enorme gezelligheid. Het Rijnstate Ziekenhuis is een fantastische rehab voor mensen met een acuut onderzoeks-onttrekkingssyndroom. Ik had me geen betere start in de kliniek kunnen wensen!

Inge, hartelijk dank voor al je energie, positieve vibes, en unieke kijk op de wereld. Ze hebben zeker een goede invloed gehad op dit boekje.

Ewout, dank voor je vriendschap en kennis van het goede leven. Succes als kersverse vader. Hopelijk volgen er nog talloze Lowlandsedities, de kleine mag natuurlijk mee. Ook de rest van de jaarclub: hartelijk bedankt jongens! Sebastiaan, dank voor de slechte films en goede gesprekken.

Robert, ik hoorde voor het eerst van jouw bestaan via onze man in Curaçao: Theo. Toen dacht ik al, die jongen moeten we in de gaten houden. Het is mooi dat we inmiddels met drie man het leven haarfijn menen te analyseren onder het genot van een biertje.

Theo, ik schrijf dit dankwoord in het vliegtuig naar Curaçao. Dit zou door mensen met enig gevoel voor proza prachtig omschreven kunnen worden als iets van een ronde cirkel.... Ik beperk me dan ook even tot een bedankje voor de goede gesprekken en jouw briljante (nee echt...) grappen. Curaçao gaat ongetwijfeld weer mooi worden (als het proefschrift naar de drukker kan).

Hester. Beste paranimf, een betere collega is moeilijk te vinden. Dank voor de vriendschap, humor, en avondjes in de stad. Het onderzoek werd zo een stuk aangenamer. Ik heb straks zowaar tijd om naar Groningen te komen: whisky in de Wolthoorn?

Martijn. Dank voor jouw vriendschap, klimkunsten, paranimfschap, en kennis van het goede leven, plagieer ik even uit het dankwoord van jouw proefschrift. Ik kan het niet beter zeggen, dus: same here! In mijn eigen, wat bescheidener, woorden zou ik zeggen dat simultaan aan dezelfde Simpsons-aflevering denken bij een gegeven situatie toch goede vriendschap moet betekenen. Tot zo op Curaçao... Kan je daar trouwens klimmen?

[Geef  de  naam  van  het  bedrijf  op] 179

Lieve Marit. Ik houd van klimmen, hooggebergte, mooie muziek, wetenschap, oude huizen, Sinterklaas, Curaçao, Volvo’s, Italiaanse ijsjes, Italiaans eten, koken, Casablanca en Lowlands. Zin om wat af te spreken?

Gelukkig vinden we dezelfde dingen mooi in het leven. Bedankt voor de fantastische tijd samen. Ik hoop dat we, nu dit boekje af is, eindelijk nog lang en gelukkig kunnen uitslapen in het weekend.

Lieve zus, dank voor je relativerende humor, en jouw goede kijk op zaken waar ik zelf soms niet direct uitkom. Je gaat, of je nu wil of niet, toch een groot compliment krijgen: een betere zus bestaat gewoon niet!

Lieve ouders. Zonder jullie steun was dit allemaal niet gelukt. Het boekje is niet voor niks aan jullie opgedragen...

180 Appendices

List of abbreviations ABC ATP-binding cassette

ALT alanine-aminotransferase activity

APHZ 1-acetyl-2-phenylhydrazine

AST aspartate-aminotrasferase activity

BBB blood-brain barrier

BIND bilirubin-induced neurological dysfunction

BW body weight

CA cholic acid

CaP calcium phosphate

CAR constitutive androstane receptor

CNS central nervous system

CO carbon monoxide

CYP1A1/2 microsomal mixed-function oxidase 1/2

e.g. for example

EHC enterohepatic circulation

et al. and others

FABP1 fatty acid binding protein 1

Hb hemoglobin

HbF fetal hemoglobin

HDL high density lipoprotein

HO heme oxygenase

HPLC high-performance liquid chromatography

[Geef  de  naam  van  het  bedrijf  op] 181

Ht hematocrit

HSA human serum albumin

i.e. in other words

MRP multidrug resistance-related protein

NS not significant

OATP organic anion transporter

PEG polyethylene glycol

PT phototherapy

r correlation coefficient

SD standard deviation

Sn tin

UCB unconjugated bilirubin

UCBfree free unconjugated bilirubin

UDCA ursodeoxycholic acid

UDPGT uridine-diphosphoglucuronosyltransferase

Zn zinc

182 Appendices

[Geef  de  naam  van  het  bedrijf  op] 183

Biografie Frans J.C. Cuperus werd op 23 december 1978 geboren in Groningen. Na het behalen van zijn gymnasiumdiploma werd hij aanvankelijk uitgeloot voor geneeskunde. Na een jaar te hebben doorgebracht in Frankrijk (Antibes) en Engeland (Londen) kon hij alsnog aan zijn studie geneeskunde in Groningen beginnen. Tijdens deze studie deed Frans Cuperus onderzoek bij dr. Van Laar (neurologie) en begon hij zijn wetenschappelijke stage bij prof. dr. H.J. Verkade. Deze stage zou de basis vormen voor dit proefschrift. Momenteel is Frans Cuperus in opleiding tot maag-, darm-, leverarts en werkzaam in Rijnstate ziekenhuis te Arnhem.

184 Appendices

[Geef  de  naam  van  het  bedrijf  op] 185

List of publications 1. Cuperus FJC, Iemhoff AA, van der Wulp M, Havinga R, Verkade HJ. Acceleration of the gastrointestinal transit by polyethylene glycol effectively treats unconjugated hyperbilirubinaemia in Gunn rats. Gut. 2010;59:373–380.

2. Cuperus FJC, Hafkamp AM, Havinga R, Vitek L, Zelenka J, Tiribelli C, et al. Effective treatment of unconjugated hyperbilirubinemia with oral bile salts in Gunn rats. Gastroenterology. 2009;136:673–82.

3. Cuperus FJC, Hafkamp AM, Hulzebos CV, Verkade HJ. Pharmacological therapies for unconjugated hyperbilirubinemia. Curr. Pharm. Des. 2009;15:2927–2938.

4. Cuperus FJC, Iemhoff AA, Verkade HJ. Combined treatment strategies for unconjugated hyperbilirubinemia in Gunn rats. Pediatr. Res. 2011; PMID: 21857383. Pubmed ahead of print.

186 Appendices