Chapter - 2 Introduction, Bilirubin -...

Chapter - 2 Introduction, Bilirubin

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Section 2.1

Bilirubin

2.1.1 Chemical properties of bilirubin

Figure 2.1.1 Structure of bilirubin

Molecular formula C33H36N4O6

Molar mass 584.66

Solubility Chloroform, DMSO + Na2CO3, DMSO + NaOH

Physical status Orange Crystalline Solid

IUPAC Name: (5Z,5'Z)-5,5'-(5,5'-methylenebis(1H-pyrrole-5,2-diyl)bis(methan-1-yl-

1-ylidene)) bis(1H-pyrrol-2(5H)-one)

http://en.wikipedia.org/wiki/Chemical_formula

http://en.wikipedia.org/wiki/Molar_mass


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2.1.2 Introduction

Bilirubin is the breakdown product of heme moiety of hemeoglobin; other

hemeoproteins include cytochromes, catalase, peroxidase, tryptophan pyrrolase and a

small pool of free heme. In humans, 250–400 mg of bilirubin are produced daily, of

which approximately 20% is produced from non-hemoglobin sources [1].

Increase in concentration of direct reacting bilirubin in blood causes

hyperbilirubinaemia [2], which is toxic under certain conditions inducing jaundice,

hyperbilirubinemia-induced auditory dysfunction and neurotoxicity resulting in brain

damage [3]. On the other hand, mild unconjugated hyperbilirubinaemia behaves as

mild antioxidant [4] and might offer protection against cardiovascular diseases [5] and

tumour development. Recent research survey has reported that low concentration of

direct reacting bilirubin induces stroke in body [6] and sometimes causes cardiac

problems too. Serum bilirubin levels are often enhanced under a variety of clinical

conditions. Bilirubin has attracted the attention of physicians since antiquity. Its

chemistry, metabolism and disposal have been studied systematically during the last

two centuries as a model for hepatic disposal of biologically important organic anions

of limited aqueous solubility [7].

In the circulation of blood, bilirubin is bound to serum albumin, which

prevents its potential toxicity thought to be caused by free bilirubin [8]. Despite its

high-affinity of binding to albumin, bilirubin is rapidly and selectively taken up by the

liver [9], biotransformed upon conjugation with glucuronate [10], and secreted into

bile. Thus bilirubin is converted into bilirubin glucuronic acid in the liver and

excreted along with bile. The metabolism of bilirubin starting from heme proteins to


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its final excretion is represented schematically in Figure 2.1.1. Obstruction to bile

flow results in the appearance of direct reacting bilirubin in blood.

2.1.3 Bilirubin Metabolism

From the formation of bilirubin until its elimination from the body as a waste

product of heme catabolism it undergoes a series of metabolic alterations and

transport processes. Partial or complete failure at any point in this sequence can give

rise to jaundice [11].

Initially enzyme heme oxygenase converts heme to biliverdin. The biliverdin

reductase converts biliverdin to bilirubin. This bilirubin is unconjugated, indirect, and

insoluble in water, therefore it can be detected in the urine. Unconjugated bilirubin

bound to albumin is transported to the liver. In the liver, bilirubin gets conjugated to

glucuronic acid by bilirubin glucuronosyltransferase. This conjugated bilirubin is

direct, and soluble in water. It is then actively transported into bile channels.

Conjugated bilirubin drains into the duodenum and passes through the proximal small

bowel. It is not absorbed in the intestine. In the distal ileum and colon, it is converted

back to unconjugated bilirubin by normal gut bacteria; 80-90% of this product gets

excreted along with the feces, known as stercobilinogen, which imparts feces its

brown color. Ten to twenty percent is passively absorbed, enters the portal venous

blood, and is re-excreted by the liver. A small amount escapes hepatic uptake, filters

across the glomerulus and is excreted in the urine. A small fraction is reabsorbed into

the portal circulation and excreted in the urine as urobilinogen. The schematic

representation of bilirubin metabolism is shown in Figure 2.1.2.


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Figure 2.1.2: Schematic representation of the formation and excretion of

bilirubin


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2.1.4 Formation of bilirubin

Bilirubin is formed from heme by opening the heme ring at α carbon bridge

[12]. This cleavage is catalysed by the enzyme heme-oxygenase (HO) (Figure

2.1.3).Three molecules of O2 are consumed in this reaction in presence of

nicotinamide adenine dinucleotide phosphate hydrogenase (NADPH), a reducing

agent. The α-methene bridge carbon is eliminated as CO and the iron molecule is

released producing biliverdin IXα [13]. This is reduced to bilirubin by the action of

cytosolic biliverdin reductases [14], which require NADH or NADPH for activity.

Cleavage at non-α sites is possible; it is probably non-enzymic and occurs only to a

minor extent. This results in the formation of other isomers; some can be detected in

body fluids, but in small amounts or under special conditions. Because of the

asymmetry in the arrangement of the side chains around the heme molecule, the

methene-bridge carbon atoms, α, β, γ and δ [15] are not equivalent thus four isomeric

bilirubins IX are therefore possible depending on which attacks methene bridge. The

bilirubin found in mammalian bile consists almost exclusively of α - isomer,

designated as bilirubin IXα.


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Figure 2.1.3. Chemistry of the formation of bilirubin from heme protein

2.1.5 Bilirubin fractions

Bilirubin in plasma is not a single homogeneous substance but represents a

variable mixture of isomers and fractions. The various ‘‘bilirubins’’ differ, sometimes

considerably, in their physiological behaviour and their chemical reactions in vitro.

2.1.5.1 Free bilirubin (Bf)

Free bilirubin is the breakdown product of hemoglobin (Hb) of aged

erythrocytes in the reticuloendothelial cells of the spleen [16]. This free bilirubin is

not bound to albumin and its toxic effect is believed to occur even at a concentration


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of 0.005 mg/dL. So far, there has been no really reliable method has been developed

for measuring free bilirubin content in plasma (or alternatively for measuring the free

binding capacity of albumin for free bilirubin).

2.1.5.2 Unconjugated bilirubin (Bu)

The free bilirubin bound to albumin is called unconjugated bilirubin [17]. The

splitting of heme ring at different positions (α, β, γ or δ) leads to the formation of its

various isomers which cannot form hydrogen bonds, and are therefore more readily

water-soluble and get excreted through the urine.

2.1.5.3 Conjugated (glucuronated) bilirubin (Bc)

The free or unconjugated bilirubin bound by albumin [18] is carried to liver,

where it is conjugated with glucuronic acid by the enzyme glucuronyltransferase. The

enzyme, glucuronyltransferase transforms the albumin-bound bilirubin to

monoglucuronide or diglucuronide conjugated bilirubin.

2.1.5.4 Delta-bilirubin (Bd)

Delta-bilirubin [19] arises through a non-enzymatic covalent coupling reaction

between glucuronated bilirubin and albumin, which is nontoxic and excreted neither

in urine nor in bile but is slowly metabolized with a half-life of 20 days.

2.1.6 Reference ranges of bilirubin in serum [20, 21]

Only conjugated form and/or direct bilirubin is filtered by the kidneys and is

also excreted into the urine (called urine bilirubin) [22]. Normal values may vary

from lab to lab. The reference range of bilirubin in blood is mentioned in Table 2.1.1.


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Table 2.1.1. Reference range of bilirubin in serum

Type of bilirubin Serum

mg / dL mmol / L

Conjugated or Direct bilirubin 0.1 to 0.3 1.7 to 5.1

Unconjugated or Indirect bilirubin 0.2 to 0.8 3.4 to 12.0

Total bilirubin 0.3 to 1.0 5.1 to 17.0

2.1.7 Clinical Significance

Any increase in formation or retention of bilirubin (hyperbilirubinemia) [23] by

the body may result in deseased condition called jaundice, which is characterized by

an increase in the bilirubin level in the serum and the presence of a yellowish

pigmentation in the skin. Jaundice may be classified as.


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2.1.8 Analytical significance of bilirubin measurement

Bilirubin is the potentially toxic catabolic product of heme metabolism. There

are elaborate physiologic mechanisms for its detoxification and disposition. Bilirubin

is poorly soluble in water at physiologic pH [28], because of internal hydrogen

bonding that engages all polar groups and gives the molecule an involuted structure.

The intramolecular hydrogen bonding shields the hydrophilic sites of the bilirubin

molecule resulting in a hydrophobic structure.

Water-insoluble, unconjugated bilirubin is associated with all known toxic

effects of bilirubin [29, 30]. Thus, the internal hydrogen bonding is critical in

producing bilirubin toxicity and also in preventing its elimination. Conversion of

unconjugated bilirubin to a water-soluble form by disruption of the hydrogen bonds is

essential for its excretion by the liver and kidney. This is achieved by glucuronic acid

conjugation of the propionic acid side chains of bilirubin. Bilirubin glucuronides are

water-soluble and are readily excreted in bile. Bilirubin is primarily excreted in

normal human bile as diglucuronide.

A low concentration of bilirubin is found in normal plasma, almost all of

which is indirect. The sum of the direct and indirect forms (or conjugated and

unconjugated) is termed as total bilirubin. Routine analytical procedures exist for the

determination of total bilirubin and for the measurement of direct bilirubin. The

indirect fraction is obtained by subtracting the direct value from the total value.

Measurement of total bilirubin and determination of direct and indirect fractions are

important in routine screening and also for the differential diagnosis of jaundice.


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2.1.9 Reference method (CLSI)

Doumas et al. (1985) described a ‘‘Candidate Reference Method’’ for the

measurement of total bilirubin in serum which has been recognized as the reference

method by the Clinical and Laboratory Standards Institute (CLSI), formerly the

National Committee for Clinical Laboratory Standards (NCCLS) [31, 32].

The basis of this method was the Jendrassik-Grof procedure [33, 34, 35],

which was optimized with respect to the concentration of the reagents, the reaction

time and a range of marginal conditions. For testing sample and blank, 0.5 mL of

serum is mixed with 4.0 mL of caffeine reagent. After 10 min, 1.0 mL diazoreagent

(sample) or 1.0 mL sulfanilic acid (blank) is added, and after further 10 min 3.0 mL of

tartrate solution are added. After 10 min, photometry is performed at 598 nm. The

method is calibrated with the Standard Reference Material SRM 916a of the National

Institute of Standards and Technology (NIST), which contains unconjugated bilirubin

at a purity of 99%. The material must be dissolved (by means of dimethyl sulfoxide

and Na2CO3) in human serum or in 40 g/L solution of albumin. The method is linear

up to 25 mg/dL.

The interference through hemoglobin [36] that is typical for the diazo methods

is only apparent in the sample at Hb concentration of 2 g/L. The addition of ascorbic

acid [37, 38] (0.1 mL per 40 g/L solution) before addition of tartrate reagent is

recommended to overcome the interfernce. Addition of ascorbic acid to every sample

is, however, not recommended, since ascorbic acid destroys the diazo reagent.


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2.1.10 Methods other than diazo methods

2.1.10.1 Enzymatic measurement

Bilirubin oxidase (BOX) catalyses the oxidation of bilirubin to biliverdin in

the presence of molecular oxygen [52, 53, 54]. At pH values of 5.0–8.5, the biliverdin

is further oxidized to purple products that finally become colourless. Total bilirubin is

measured with the enzymatic method at pH 8.2 after the addition of sodium dodecyl

sulfate and sodium cholate; the two additives required to release the bilirubin that is

bound to albumin, which then, just like the unbound bilirubin, can be rapidly

converted by BOX. All bilirubin fractions of the serum (including the conjugated

fractions) get oxidized to biliverdin. The total bilirubin can be quantitatively

determined either on the basis of the decrease in the absorption of bilirubin at 425 nm

or through the increase in the absorption of the purple pigment at 450 nm [55]. An

advantage of the enzymatic method is the low amount of sample required. However,

the values obtained for total bilirubin are somewhat lower than those obtained by the

diazo-procedure.

2.1.10.2 Bilirubinometer

Bilirubinometer is a simple filter photometer, which measures the absorption

of plasma at 454 nm near the absorption maximum of bilirubin. Since neonate plasma

contains no lipochromes it likewise absorbs around this wavelength (e.g., carotene),

bilirubin can be determined quantitatively if the spectral interference by hemeoglobin

is compensated. This can be achieved through an additional measurement at 540 nm.

Since the molar extinction coefficient of the hemeoglobin is identical at 455 nm and

540 nm [56, 57], the bilirubin concentration can be calculated from the difference

ΔE = 455 nm – 575 nm.


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2.1.10.3 Transcutaneous bilirubinometry

Transcutaneous bilirubinometry measures the intensity of yellow color in the skin [58,

59, 60] and subcutaneous tissue and correlates it with the serum bilirubin

concentration in newborn Infants.


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Chapter- 2 Introduction, Creatinine

52

Section 2.2

Creatinine

2.2.1 Chemical properties of creatinine

Creatinine exists in two forms as shown in Figure 2.2.1

Figure 2.2.1: Structure of creatinine

IUPAC name : 2-imino-1-methylimidazolidin-4-one

Solubility : Water soluble

Molecular mass : 113.118

Colour : White amorphous solid

Molecular formula : C4H7N3O

Density : 1.09 g cm-3

Melting point : 300 °C


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2.2.2 Introduction

Creatinine is a break-down product of creatine phosphate present in muscle,

and is usually produced at a fairly constant rate by the body (depending on muscle

mass). Chemically, creatinine is a spontaneously formed cyclic derivative of creatine.

In the body, creatinine is formed by a spontaneous and irreversible conversion

of creatine and creatine phosphate [1], which is the source of high-energy phosphate

bonds for the immediate reformation of ATP during muscular contraction.

Creatinine is normally synthesized from arginine, S-adenosyl-methionine and glycine,

primarily by the kidney and liver [2], but recent evidences suggest that the brain and

testis may also be involved in the synthesis of Creatinine [3]. Creatinine is consumed

along with the diet predominantly in meat, fish and other animal products [4]. A

proportion of creatine and phosphocreatine entering the cell is phosphorylated to form

Creatine phosphate, which subsequently provides an immediate source of ATP for

energy requiring processes. Finally, creatine is converted to creatinine and is excreted

completely through urine without any secretion or adsorption.

Creatinine is assumed to be metabolically inert [5]. Conversion of creatinine into

creatine is not possible under the normal physiological conditions; neither it is

reabsorbed nor actively secreted in renal tubules [6]. Blood collects the creatinine

from various parts of the body and enters the kidney, where the creatinine is filtered

and excreted completely through kidneys.

Creatinine is present not only in serum and erythrocytes but is also found in all

bodily secretions, such as sweat, bile, and gastrointestinal fluids. Being ultrafiltrable,

it is also present in the cerebrospinal fluid [7].


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2.2.3 Creatinine metabolism

Synthesis of creatinine in the body starts with the reaction of two amino acids,

Arginine and Glycine [8] in the kidney and in liver [9]. This reaction is catalyzed by

the enzyme L-Arginine-glycine amidinotransferase (AGAT) producing

guanidinoacetic acid and ornithine. Ornithine as a by product is utilized to increase

muscle growth by stimulating the release of anabolic hormones such as growth

hormone and insulin [10]. The guanidinoacetic acid is catalyzed by the enzyme S-

adenosyl-L-methionine:N-guanidinoacetate methyltransferase (GAMT) leading to the

formation of creatine. This creatine is then transported to the skeletal muscles through

the bloodstream. The phosphate of adinosine tri phosphate (ATP) gets transferred to

creatine, generating creatine phosphate, through the action of creatine phosphokinase.

Creatine phosphate is stored in the muscle as high energy phosphate. The reaction is

reversible and when the energy demand is high creatine phosphate donates its

phosphate to adinosine di phosphate (ADP) to yield adinosine tri phosphate [11].

Creatinine is formed in muscle tissues from creatine phosphate by a nonenzymatic

dehydration process and by the loss of phosphate.

Creatinine is transported through the bloodstream to the kidneys. About 1000-

1200 ml of blood passes through kidneys per min. From this volume 120 ml of

plasma/min are filtrated by glomerules of normal kidneys. The kidneys filter out most

of the creatinine and dispose it off along with the urine. The schematic representation

of creatinine metabolism is presented in Figure 2.2.2.


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Figure 2.2.2. Schematic representation of creatinine metabolism in the body


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2.2.4 Formation of creatinine

Creatine (Cr) and phosphocreatine (PCr) have been known to play important

roles as components of an energy buffer mechanism in animal cells. In this system,

ADP and ATP get interconverted (Figure 2.2.3) with the participation of creatine and

phosphocreatine [12]. Catalyzed by creatine kinase, this reversible reaction helps the

cells to maintain adequate ATP levels during large fluctuations in energy demand.

Figure 2.2.3: Interconversion of creatine to phosphocreatine

Creatine is a non-essential dietary molecule; its degradation rate within human

body system is about 1.6% (2 g) per day [13]. The degradation of creatine pool

involves the non-enzymatic conversion (Figure 2.2.4) of creatine to creatinine [14],

most of which is subsequently excreted from the body via the kidneys. A small

proportion of creatinine may get converted into other compounds such as arginine and

guanidinobutyrate [15].


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Figure 2.2.4: Non-enzymatic conversion of creatine to creatinine

2.2.5 Reference range of creatinine in serum and urine sample

Creatinine level in urine is affected by many factors such as age, sex, height,

weight, muscle mass, intake of food, medicines used and many other factors. Normal

urine creatinine concentration ranges from 40 to 300 mg/dL in males and 37-250

mg/dL in females [16]. Lower creatinine values of 17 mg/dL have also been reported

in rare instances. Since creatinine concentration is directly related to creatine in

muscle, excretion through urine varies invariably. The reference ranges of creatinine

[17, 18] in urine, blood and creatinine clearance are presented in Table 2.2.1.


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Table 2.2.1 : Reference ranges of creatinine in blood and urine samples

Male Female

mg/dL mM/L mg/dL mM/L

Urine creatinine 40-300 3.5-26.5 37-250 3.3-22

Blood creatinine 0.6–1.2 0.05-1.06 0.5–1.1 44–97

Creatinine clearance 90–140 ml min-1

87–107 ml min-1

2.2.6 Clinical significance of creatinine

Formation of creatinine is fairly constant [19], about 2% of the whole body

creatine being transformed per day. Creatinine thus formed cannot be reutilised and is

considered as a waste product. Creatinine from various parts of the body is collected

and carried by blood to kidney. The glomeruli in kidney play important role in

filtering and excretion of creatinine from blood.

Blood carrying creatinine and various other metabolites enters the

semipermeable walls of the glomerular capillaries which are almost impermeable to

proteins and large molecules. The filtrate is thus virtually free of proteins and other

cellular elements. The glomerular filtrate is formed by squeezing fluid through the

glomerular capillary. About 20% of renal plasma flow is filtered every minute,

(125 ml.min-1

). This is the glomerular filtration rate (GFR). GFR helps in regulating

the creatinine ratio in blood and urine.

If the filtering rate of the glomerular capillaries of kidney is deficient, then

creatinine level rises in blood. Therefore, creatinine levels in blood and urine are used

to assess the creatinine clearance, which reflects the GFR. The failure of filtration by

glomerular capillaries of kidney indicates the chronic kidney disease [20].


59

Any disease that affects the filtration process of the kidneys will tend to raise

serum creatinine levels in blood over time. Doubling of serum creatinine levels in

blood is an indicative of 50 percent reduction in the filtration rate of the kidneys.

Diseases associated with increased levels of creatinine include

glomerulonephritis, pyelonephritis, diabetic nephropathy [21] and conditions such as

shock and congestive heart failure that reduce blood flow through the kidneys.

2.2.7 Analytical significance of creatinine measurement in serum and urine

Since creatinine is the non toxic waste metabolite, it is neither absorbed nor

adsorbed or secreted in body and is completely filtered out through kidneys.

Measurement of creatinine concentration in blood and urine plays an important role in

identifying many diseases. This test is adopted as routine blood and urine test.

Serum or plasma can be used to measure creatinine concentration in blood.

Increase or decrease in blood creatinine is usually a sign of problems associated with

kidney function which directly reflects on GFR [22]. Other than GFR, increase in the

creatinine concentration in blood relates to many other diseases which directly or

indirectly affects the kidney functioning. Analytical measurement of creatinine

concentration in blood helps in preliminary identification of certain diseases like,

cancer of testis or uterus [23], cholangitis, congestive heart failure, diabetes mellitus,

hypertension, malaria and such other diseases. A recent study suggests that lower-

serum creatinine level is associated with an increased risk for the development of type

2 diabetes [24].

Creatinine excretion in urine is constant and is proportional to total muscle

mass. Urinary excretion of creatinine varies due to many factors especially intake of

creatinine rich foods (e.g., roasted meat). Analytical measurement of creatinine


60

concentration in urine helps in preliminary identification of certain diseases like

hypothyroidism, Alzheimer-type Dementis, hyperthyroidism, muscular dystrophy and

protein malnutrition

Measurement of the ratio of creatinine concentration in blood to that of urine

gives an idea of creatinine clearance. The creatinine clearance provides an estimate of

the amount of plasma that must have flowed through the kidney glomeruli per minute

with complete removal of its content of creatinine to account for the creatinine per

minute actually appearing in the urine. This test needs both blood sample and 24-hour

urine collection from the patient.

Higher or low creatinine clearance by the kidney indicates the malfunctioning

of kidney. Thus creatinine clearance is an indirect way to analyse the chronic kidney

disorder via GFR. Increase or decrease in creatinine clearance by the kidney indicates

the prevalence of certain major diseases like acromegaly, burn, acute/ or chronic

pyelonephritis, heart failure, extrahepatic biliary obstruction, glomerulonephritis,

gout, growth hormone deficiency, liver cirrhosis and many others.

2.2.8 Reference method (CLSI)

Most of the routine serum creatinine assays that are in current use have

evolved based on the reaction initially described by Jaffe in 1886. Over the years,

Jaffe used to report that a red color is produced when creatinine reacts with alkaline

picrat. Folin in 1904 used this “Jaffe’s reaction” to determine creatinine content in

urine. Since then it has been recognized that in addition to creatinine, a number of

other compounds [25] also give the so called Jaffe’s reaction. Jaffe’s assay has since

passed through many phases. Early methods used deproteinised blood. To improve

specificity, creatinine was isolated from common interfering substances by adsorption


61

on to aluminium silicates such as Lloyd’s reagent [26], followed by elution into

alkaline picrate after centrifugation and decanting. Cation exchange resins were also

in use for this purpose [27].

Boyne and his group of innovative biochemists in Australia developed an

interesting solution to detect the false high results produced by the Jaffe assay method

[28]. Their procedure involved incubating approximately 100 μL of serum for 1 hour

in a sample cup containing a mixture of a dried solution of creatininase and

creatinase. By assaying the sample before and after enzymatic treatment, they found

that they could obtain similar accuracy as that of HPLC. The enzymes used in the

assay have now formed the basis for most of the specific routine assays currently in

use which underlies the reaction sequence as shown below:

The hydrogen peroxide generated in the above reaction sequence can be

measured spectrophotometrically using a Trinder’s reaction acceptor, producing a

quinoneimine with high molar absorptivity (eg Roche Creatinine Plus assay), using a

leuco dye (Vitros dry chemistry system), or with blood gas analyser electrodes (eg

new Radiometer dual electrode system which corrects for creatine). Other enzymatic

systems using creatinine deaminase, which convert creatinine to N-methylhydantoin

and ammonia (with various options for measuring ammonia), and creatininase, (with

NADH measured at 340 nm after a creatine kinase reaction sequence), have found

little acceptance and application in routine analysis.


62


63


64


65


66


67

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Chapter- 2 Introduction, Hemoglobin

70

Section 2.3

Hemoglobin

2.3.1 Introduction

Hemoglobin or haemoglobin symbolized as Hb or Hgb is the iron-containing

oxygen-transporting metalloprotein found in red blood cells (RBC) or erythrocytes of

all vertebrates [1] (except Channichthyidae fish family) [2] and also in the tissues of

some invertebrates. Hemoglobin is present in the RBC of the body. Each RBC

contains approximately 280 million hemoglobin molecules. Chemically hemoglobin

is a tetrameric globular protein consisting of two each of alpha and beta chains [3].

The alpha and beta chains are fixed by different loci and are differentially regulated

during human development. Of the four chains of hemoglobin each encloses an iron-

containing heme co-factor, which binds the oxygen. It is the main function of

hemoglobin to capture oxygen from the lungs and deliver the same to all tissues for

respiration, and in turn transport carbon dioxide back from tissues to lungs. One

hemoglobin molecule can transport up to 4 oxygen molecules. In addition,

hemoglobin also plays vital role in regulating blood flow and blood pressure.

Hemoglobin is a conjugated protein having a molecular weight of nearly

64,500 D. Hemoglobin exhibits characteristic features of both tertiary and quaternary

structures of proteins [4]. Most of the amino acids in hemoglobin form alpha helices,

connected by short non-helical segments. Hydrogen bonds stabilize the helical

sections inside this protein causing attractions within the molecule. Hemoglobin's

http://en.wikipedia.org/wiki/Iron

http://en.wikipedia.org/wiki/Vertebrate


71

quaternary structure is derived from its four subunits in roughly a tetrahedral

arrangement [5].

2.3.2 Structure of hemoglobin

Hemoglobin was discovered by Hünefeld in 1840 [6] and its molecular

structure was depicted by X-ray crystallography in 1959 by Max Perutz [7]. Ever

since the discovery of the relation between its structure and function by Max Perutz in

1978 after almost 20 years of research, hemoglobin is presently the most thoroughly

understood protein thus far.

The common hemoglobin molecule is the assembly of four globular protein

consisting of two subunits, two alpha chains, each with 141 amino acids [8] and two

beta chains, each with 146 amino acids [9]. Each subunit is composed of a protein

chain tightly associated with a non-protein heme group. Each protein chain arranges

into a set of alpha-helix structural segment connected together in a globin fold

arrangement; this folding pattern contains a pocket that strongly binds the heme

group. In this protein molecule, the globin portion is approximately 94% while the

heme comprises of 6%.

http://en.wikipedia.org/wiki/X-ray_crystallography

http://en.wikipedia.org/wiki/Molecule

http://en.wikipedia.org/wiki/Heme


72

Figure 2.3.1: Structure of hemoglobin molecule present in red blood cell

A heme group consists of an iron (Fe) ion (charged atom) held in a

heterocyclic ring, known as porphyrin. This porphyrin ring consists of four pyrrole

molecules cyclically linked together by methene bridges with the iron ion being

bound at the center. The iron ion, which is the site of oxygen binding, coordinates

with four nitrogens atoms in the center of the ring, all of which lie in one plane. The

iron is bound strongly to the globular protein via the imidazole ring of the F8 histidine

residue [10] (also known as proximal histidine) below the porphyrin ring.


73

Figure 2.3.2 : Proximal histidine F8 which holds the heme group with globin,

Phorphyrin structure of heme.

The heme molecule contains the iron atom in ferrous (Fe2+

) state to allow the

binding of oxygen. But if the iron is in ferric (Fe3+

) state then there is no binding of

oxygen and it is known as oxyhemoglobin [11.]. In this case the molecule is referred

to as “methemoglobin or ferrihemoglobin”. When O2 binds to the ferrous ion, all the

forces get balanced equally and the iron is pulled back to the center.

Hemoglobin exists either in relaxed state (R-state) or intense state (T-state)

and in these forms they differ both in their structure and affinity for oxygen [12].

They also differ in the number and energy of the interactions between hemoglobin

subunits. In the T-state, constraints between subunits oppose the structural changes

resulting in ligand binding, whereas in the R-state, these constraints are released,

thereby enhancing ligand-binding affinity. Both T and R states of hemoglobin are

shown in Figure 2.2.3.


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Figure 2.3.3: T and R states of hemoglobin

2.3.3 Synthesis of hemoglobin

Synthesis of hemoglobin involves a complex series of steps. The heme part

and globin part are synthesized separately [13.]. The heme part is synthesized in a

series of steps in the mitochondria and the cytosol of immature red blood cells, while

the globin protein parts are synthesized by ribosomes in the cytosol.

2.3.3.1 Heme synthesis

Heme is synthesized in a complex series of steps involving multi enzymes in

the mitochondrion and in the cytosol of the cell. Synthesis of heme starts in

mitochondrion with the condensation of succinyl CoA and glycine by aminolevulic

acid (ALA) synthase [14.] to form 5-aminolevulic acid. This molecule is transported

to the cytosol where a series of reactions produce a ring structure called

coproporphyrinogen III. This molecule returns to the mitochondrion where by an

addition reaction protoporhyrin IX is produced. The enzyme ferrochelatase inserts

iron into the ring structure of protoporphyrin IX to produce heme. This heme is

strongly bound to the globin groups by histidine as shown in Figure 2.3.4.

http://en.wikipedia.org/wiki/Mitochondria


75

Figure 2.3.4: Proximal histidine F8 group attached to iron of heme group

Two distinct globin chains (each with its individual heme molecule) combine

together to form hemoglobin [15]. One of the chains is designated as alpha. The

second chain is called "non-alpha". The human globin genes undergo an orderly

ontogenetic program of expression during their development from embryo to adult

stage. Specific genes for the globin chains are switched on and off to produce

different hemoglobin types [16].

2.3.4 Hemoglobin variants

Normal hemoglobin contains four globin groups 2α and 2β which are

associated with a heme group. Other than α and β globins there are many other

globins are also present. Based on the type of globins several variants of hemoglobin

are present as listed in Table 2.3.1.


76

In humans, there are 5 types of normal hemoglobins are present; others are of

abnormal types. Two types of normal hemoglobins are present in embryos: Hb

Gower-1 and Hb Gower-2. Normal hemoglobin in the fetus is Hb F. The 2 normal

types of hemoglobin in adults are Hb A and HbA2.

Table 2.3.1: Hemoglobin variants

Form Chain

Fraction of total

Adult Hb

Clinical state Reference

HbA α2β2 90% Normal

HbA2 α2δ2 2-5% Normal [17]

HbA1C α2β2-glucose 3-9% Normal [18]

HbF α2γ2 <2% Normal [19]

Gower 1 ζ2ε2 Embryonic Normal [20]

Gower 2 α2ε2 Embryonic Normal [21]

Hb portland ζ2γ2 Embryonic Normal [22]

HbH β4 0 % α Thalassaemia [23]

Hb Barts

γ4 0 % α Thalassaemia

[24] α chain aggregates

insoluble

0 % β Thalassaemia

HbS α2β2 0 %

Sickle cell

disease [25]


77

2.3.5 Clinical significance of hemoglobin

The primary function of hemoglobin is to bind the oxygen from lungs and

transport it to various parts of the body and in turn bring back carbon dioxide to lungs.

Hemoglobin in its tetrameric state has got 4 iron molecules attached to globin group

via a histidine molecule. Each iron molecule can bind with either one oxygen

molecule or one carbon dioxide molecule. Thus each hemoglobin molecule at a time

can transport four oxygen or carbon dioxide molecules. Hemoglobin combines with

oxygen and carbon monoxide cooperatively.

Very high hemoglobin concentration causes high blood viscosity, which

results in compromised oxygen delivery to tissues and also creates cerebrovascular

complications. Studies have also shown that a high maternal hemoglobin

concentration leads to increased risk of poor pregnancy [26].

2.3.6 Reference ranges of hemoglobin

The hemoglobin level is measured in grams per deciliter. The normal ranges

of hemoglobin levels are dependent on the age and gender [27].


78

Table 2.3.2: Reference range of hemoglobin in human blood

Age Male (g / dL) Female (g / dL)

New born 14.7–18.6 12.7–18.3

6 months – 2years 10.3–12.4 10.4–12.4

2 - 12 years 11.0–13.0 10.7–13.3

12 -18 years 11.0–13.3 10.9–13.3

> 18 years 10.9–15.7 10.7–13.5

2.3.7 Analytical significance

Total hemoglobin count is a routine blood test conducted which helps in

diagnosing many abnormalities in the body system. The transport of oxygen in blood

is undertaken by hemoglobin, the largest component of red blood cells. This protein

collects oxygen from respiratory organs, mainly the lungs, and releases it to tissues in

order to generate the energy necessary for cell survival. Hemoglobin is one of the

most refined proteins because its evolution and small mutations in its structure can

produce anemia [28] and other severe pathological conditions. Hemoglobin test is

considered as an important blood test as it is a tool for determining many types of

disorders including certain kinds of blood related diseases.

If the hemoglobin level in blood is lower than the required normal range, then

it is usually indicative of anemic condition [29], which may have many implications,

mainly the anemic person will have a tendency to have low levels of oxygen in the

body. Others include nutritional deficiency, chemotherapy drugs, bone marrow

disorders, loss of blood, abnormal hemoglobin, and kidney failure.


79

On the other hand, abnormally high levels of hemoglobin have varying causes

and implications. Smokers usually will have high hemoglobin levels. This condition

may also true for people living in high altitude areas. Dehydration of body may also

result into a temporary rise in hemoglobin levels. Other possible causes for an

abnormally high hemoglobin level are the abuse of the drug erythropoietin, some

tumors, and advanced lung disorders such as emphysema.

A notable disease that can be indicated by an abnormal hemoglobin level is

sickle cell anemia [30]. This is a genetic disorder in which the type of the hemoglobin

is faulty that also imparts a different shape to the red blood cells. As the name implies,

the red blood cells assume the shape of sickles. The difficulty with these sickle-

shaped red blood cells is in passing through the small blood vessels, which results in

medical problems including anemia. Another blood disorder associated with

hemoglobin is thalassemia in which the globulin molecules themselves are defective.

2.3.8 Analytical methods for the determination of hemoglobin in human blood.

The method of choice for hemoglobin determination is the

cyanmethemoglobin method also called as Drabkin’s method and that is the standard

method approved by clinical and laboratory standard institute (CLSI) [31]. This

method has been adapted widely to date as an international standard method. The

principle behind this method is that when blood is mixed with a solution containing

potassium ferricyanide and potassium cyanide, the potassium ferricyanide oxidizes

iron to form methemoglobin. The potassium cyanide then combines with

methemoglobin to form cyanmethemoglobin, which is a stable color pigment read

photometrically at a wave length of 540 nm. The color is related to the concentration


80

of hemoglobin in the blood. All forms of hemoglobin except sulfhemoglobin can be

measured by this method. The reagent has high stability.

Another method accepted for the measurement of hemoglobin concentration

by CLSI is hemoglobinometry [32]. Multiwavelength laboratory oximeters by the use

of spectrophotometric absorption of a blood specimen determine the percentage of

hemoglobin saturated with oxygen and the percentage of dyshemoglobins. Pulse

oximetry is a noninvasive method of estimating the arterial oxygen saturation and

pulse rate from pulsatile absorption signals derived from a sensor placed on the skin.

The principle is based on the fact that oxy- and deoxyhemoglobin have different

absorption spectra (see Figure 2.3.1), at the commonly used wavelengths of 660 nm

(red light) and 905 to 940 nm (infrared light).


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