The Liver & Tests of Hepatic Function

Study GuideStudy Guide

Pg 90Pg 90

Module Focus It is the primary site for the It is the primary site for the

metabolism of carbohydrates, metabolism of carbohydrates, proteins, lipids, and bile acids.proteins, lipids, and bile acids.

It is the major site for the storage of It is the major site for the storage of iron, glycogen and lipids.iron, glycogen and lipids.

The liver also plays an important The liver also plays an important role in the detoxification of role in the detoxification of molecules foreign to the body & molecules foreign to the body & the excretion of waste products the excretion of waste products such as such as bilirubin, ammonia & urea.bilirubin, ammonia & urea.

Lab tests are generally used as a Lab tests are generally used as a nonspecificnonspecific index of liver function. index of liver function.

Summary of Lab Tests of Liver Function

Total protein, albumin, bilirubin, ALT, Total protein, albumin, bilirubin, ALT, AST, GGT, ALPAST, GGT, ALP

AFP, ammonia, ironAFP, ammonia, iron Urine bilirubin & urobilinogenUrine bilirubin & urobilinogen Hepatitis markersHepatitis markers CBC, retic counts, PT, hemoglobin CBC, retic counts, PT, hemoglobin

electrophoresiselectrophoresis

Hepatic Function

Metabolic/Synthesis:Metabolic/Synthesis: Proteins, carbos, lipids, coag factorsProteins, carbos, lipids, coag factors

DetoxificationDetoxification (protective): (protective): Convert ammonia to urea, bilirubin, Convert ammonia to urea, bilirubin,

drugsdrugs Storage:Storage:

Lipids, glycogen, vitamins, hormonesLipids, glycogen, vitamins, hormones Excretory:Excretory: BilirubinBilirubin

HemoglobinHemoglobin to to bilirubinbilirubin to to urobilinogenurobilinogen

Diseases of the Liver

Tumors, cirrhosis, drugs, alcoholTumors, cirrhosis, drugs, alcohol Hepatitis A, B, CHepatitis A, B, C Cholestatic or obstructive jaundiceCholestatic or obstructive jaundice Reye’s syndromeReye’s syndrome HemochromatosisHemochromatosis Wilson’s diseaseWilson’s disease

Indicators of Metabolic Function (for severity, not diagnosis)

Albumin:Albumin: values due to values due to synthesis by the synthesis by the

liver,liver, causes ascites (the causes ascites (the accumulation of fluid in the accumulation of fluid in the abdominal cavity)abdominal cavity)

Prothrombin time:Prothrombin time: = = clotting time clotting time

Indicators of Hepatic Detoxification & Excretory

Ammonia:Ammonia: Ammonia is Ammonia is very toxic to the brainvery toxic to the brain Normally, it is converted to Normally, it is converted to ureaurea by by

the liverthe liver levels may indicate impending levels may indicate impending

hepatic comahepatic coma

Bilirubin:Bilirubin: derived from breakdown of derived from breakdown of hemoglobin & excreted in feces as hemoglobin & excreted in feces as urobilinogen/urobilinurobilinogen/urobilin

Enzymes Determinations

The majority of enzymes are The majority of enzymes are intracellularintracellular When tissue damage occurs, When tissue damage occurs, enzymes are enzymes are

released into the plasma. released into the plasma. Enzyme tests are the Enzyme tests are the most sensitive testsmost sensitive tests..

Liver enzymes = ALP, GGT, AST, ALTLiver enzymes = ALP, GGT, AST, ALT We will study these tests in the We will study these tests in the next unit.next unit.

Normal Bilirubin Metabolism pg 94

1.1. After 120 days, After 120 days, RBCs RBCs are typically are typically brokenbroken downdown by the RE System. by the RE System.

2.2. The The hemoglobinhemoglobin in the RBCs forms in the RBCs forms heme heme + iron + protein+ iron + protein

3.3. The heme is converted to The heme is converted to unconjugated (indirect) bilirubinunconjugated (indirect) bilirubin

4. Since the 4. Since the unconjugated (indirect) unconjugated (indirect) bilirubin is insolublebilirubin is insoluble, it will attach to , it will attach to albuminalbumin to be transported to the to be transported to the liver. liver.

unconjugated cannot be excreted unconjugated cannot be excreted in the urine in the urine in this form.in this form.

Metabolism in the LIVER

5. Bilirubin is transported into the liver cell.5. Bilirubin is transported into the liver cell. In the liver cell, it combines with In the liver cell, it combines with

glucuronic acidglucuronic acid by the enzyme by the enzyme glucuronyl glucuronyl transferasetransferase to form to form conjugated bilirubinconjugated bilirubin..

The conjugated bilirubin is nowThe conjugated bilirubin is now water water soluble. soluble.

glucuronyl transferaseglucuronyl transferase

Unconjugated + Glucuronic Unconjugated + Glucuronic ConjugatedConjugated

BilirubinBilirubin acid acid BilirubinBilirubin

Bile Ducts

The majority of the conjugated The majority of the conjugated bilirubin that is produced in the liver, bilirubin that is produced in the liver, is is transported into the bile ductstransported into the bile ducts & & the the intestinal tract.intestinal tract.

In the GI tract, bacterial enzymes In the GI tract, bacterial enzymes convertconvert the the conjugated bilirubin to conjugated bilirubin to Urobilinogen.Urobilinogen.

The The urobilinogenurobilinogen then forms then forms UROBILIN UROBILIN which gives the feces its which gives the feces its brown color.brown color.

Expected Normal Valuespg 93

AnalyteAnalyte Reference Reference RangeRange

Total BilirubinTotal Bilirubin < 1.0 mg/dl< 1.0 mg/dl

Direct (conjugated) BilirubinDirect (conjugated) Bilirubin 0 – 0.2 mg/dl0 – 0.2 mg/dl

Indirect (unconjugated) Indirect (unconjugated) BilirubinBilirubin

<0.8 mg/dl<0.8 mg/dl

Urine BilirubinUrine Bilirubin NegativeNegative

Urine UrobilinogenUrine Urobilinogen 1 – 4 mg/day1 – 4 mg/day

Fecal UrobilinogenFecal Urobilinogen 50 – 250 mg/dl50 – 250 mg/dl

Clinical ApplicationsJaundice

pg 92 Signs & SymptomsSigns & Symptoms

Bilirubin = > 3mg/dlBilirubin = > 3mg/dl Causes yellowish discoloration of Causes yellowish discoloration of

skin and scleraskin and sclera Serum appearance = Serum appearance = “icteric”“icteric”

Causes of Altered Bilirubin Levels

Prehepatic jaundicePrehepatic jaundice = = “hemolytic jaundice”“hemolytic jaundice” Excessive hemolysis of RBCs = Excessive hemolysis of RBCs = bilirubin bilirubin

Hepatic Hepatic jaundice jaundice conversion of unconjugated bilirubin to conversion of unconjugated bilirubin to

conjugated bilirubin by the conjugated bilirubin by the liver.liver. PosthepaticPosthepatic jaundice = jaundice = “obstructive jaundice”“obstructive jaundice”

Impairment of bile excretionImpairment of bile excretion Gall stones, pancreatic tumorGall stones, pancreatic tumor

Note:Note: in both pre & post, the function of the in both pre & post, the function of the liver is not impaired.liver is not impaired.

Prehepatic Jaundicepgs 95 & 96

Problem: Problem: RBC destruction exceeds RBC destruction exceeds the ability of the liver to conjugate & the ability of the liver to conjugate & excrete bilirubin.excrete bilirubin.

Causes: Causes: Hemolytic anemias, Hemolytic anemias, transfusion reactions, HDNtransfusion reactions, HDN

Lab Findings:Lab Findings: Increased total bilirubinIncreased total bilirubin Increased (indirect) bilirubinIncreased (indirect) bilirubin Urine bilirubin negativeUrine bilirubin negative fecal urobilinigen & fecal urobilinigen & retic count retic count

Hemolytic Disease of the Newborn (HDN) pg 95

Cause Cause = Usually due to Rh = Usually due to Rh incompatibility of mother & child incompatibility of mother & child (mother is Rh – and baby is Rh (mother is Rh – and baby is Rh ++))

levels of indirect bilirubin due to levels of indirect bilirubin due to excessive breakdown of baby’s RBCs.excessive breakdown of baby’s RBCs.

When the baby’s unconjugated When the baby’s unconjugated bilirubin levels reach >15 – 20 mg/dl, bilirubin levels reach >15 – 20 mg/dl, the bilirubin is either unbound or the bilirubin is either unbound or loosely bound to albumin.loosely bound to albumin.

HDN Continued

Loosely bound or unbound Loosely bound or unbound unconjugated bilirubin has a high unconjugated bilirubin has a high affinity for affinity for brain & CNS tissue.brain & CNS tissue.

Bilirubin that Bilirubin that deposits in brain tissuedeposits in brain tissue may cause irreversible brain damage may cause irreversible brain damage “kernicterus“kernicterus”.”.

May need to perform an exchange May need to perform an exchange transfusion before this occurs.transfusion before this occurs.

Monitor baby’s Monitor baby’s albuminalbumin as well as as well as bilirubinbilirubin levels very closely. levels very closely.

Causes of Hepatic Jaundice

Failure to conjugateFailure to conjugate Defective transport system in or Defective transport system in or

out of hepatocyteout of hepatocyte Hepatitis, cirrhosisHepatitis, cirrhosis Lab values vary depending on Lab values vary depending on

disease process.disease process.

Hepatic Retention Jaundice

Defective transport of bilirubin into Defective transport of bilirubin into hepatocyte or hepatocyte or impaired conjugation.impaired conjugation.

Causes:Causes: Physiologic jaundice of the newborn.Physiologic jaundice of the newborn. The The

baby’s glucuronyl transferase system is baby’s glucuronyl transferase system is not fully developed, especially in premies.not fully developed, especially in premies.

Chronic liver diseases since lever Chronic liver diseases since lever eventually loses its ability to conjugate.eventually loses its ability to conjugate.

Lab FindingsLab Findings: : indirect bilirubin, urine indirect bilirubin, urine bilirubin negativebilirubin negative

Hepatic Regurgitation Jaundice

Defective transport of conjugated Defective transport of conjugated bilirubin out of bile ducts.bilirubin out of bile ducts.

Causes:Causes: Acute viral hepatitisAcute viral hepatitis Neoplasms, cirrhosisNeoplasms, cirrhosis

Lab Findings:Lab Findings: in direct bilirubinin direct bilirubin Urine bilirubin +Urine bilirubin +

Posthepatic Jaundice Problem:Problem: Blockage of bile duct which Blockage of bile duct which

causes blockage of bile (conjugated causes blockage of bile (conjugated bilirubin) flow into intestinal tract bilirubin) flow into intestinal tract “cholestasis” & conjugated backs-up into “cholestasis” & conjugated backs-up into plasma.plasma.

Causes: Causes: Gall stones, neoplasms of Gall stones, neoplasms of pancreas.pancreas.

Lab Findings: Lab Findings: Increases in direct Increases in direct bilirubin, bilirubin, urine bilirubin = +, Clay urine bilirubin = +, Clay colored stoolscolored stools..

Causes of Post hepatic jaundice

“Cholestasis” Gall stones, tumorsGall stones, tumors

Increases in Increases in conjugated bilirubinconjugated bilirubin as it backs up into plasmaas it backs up into plasma

Urine bilirubin is + since Urine bilirubin is + since conjugatedconjugated is water soluble. is water soluble.

Decrease in fecal urobilinogenDecrease in fecal urobilinogen = = clay colored stools.clay colored stools.

Typical Lab Findings in Jaundice

Review summary on pg 93Review summary on pg 93

Prehepatic Jaundice“Hemolytic Anemia”

TotalTotal

BilirubiBilirubinn

Indirect Indirect

BilirubinBilirubinDirect Direct

BilirubinBilirubinUrine Urine

BilirubiBilirubinn

Fecal Fecal UUrobilinogenrobilinogen

Initially Initially NormalNormal

NegativNegativee

In hemolytic anemia, the In hemolytic anemia, the indirect or unconjugated indirect or unconjugated portion is increased due to the portion is increased due to the excessive breakdown of RBCsexcessive breakdown of RBCs..

Indirect bilirubin is Indirect bilirubin is insoluble & can’t be insoluble & can’t be excreted in the urine.excreted in the urine.

Hepatic Jaundice

Total Total BilirubinBilirubin

Direct Direct BilirubinBilirubin

Indirect Indirect BilirubinBilirubin

Urine Urine BilirubiBilirubi

nn

Fecal Fecal UrobilinogUrobilinog

enen

0 or 0 or In hepatic jaundice, In hepatic jaundice, results will be results will be

variablevariable Results are dependent on the Results are dependent on the type of type of

diseasedisease & whether it is an & whether it is an acute or acute or chronic condition.chronic condition.

1.1. Typically, the liver will first lose its ability to Typically, the liver will first lose its ability to transport bilirubin into the bile ducts after it is transport bilirubin into the bile ducts after it is conjugated..conjugated..

2.2. As the disease progresses, the liver will As the disease progresses, the liver will lose lose it’s ability to conjugate.it’s ability to conjugate.

Post Hepatic Jaundice

Total Total BilirubinBilirubin

Direct Direct BilirubiBilirubi

nn

Indirect Indirect BilirubinBilirubin

Urine Urine BilirubiBilirubi

nn

Fecal Fecal UrobilinogenUrobilinogen

NormaNormall

In obstructive jaundice, bilirubin is In obstructive jaundice, bilirubin is conjugated by the liver, but if the conjugated by the liver, but if the bile ducts are blocked, bile ducts are blocked, bilirubin will bilirubin will back up into the serum.back up into the serum.

Because it isBecause it is soluble, it will be soluble, it will be excreted in the urine.excreted in the urine.

Bile ducts are Bile ducts are blocked. blocked. Therfore, Therfore, urobiliogen is urobiliogen is not being not being formed & formed & stools will stools will appear appear clay clay colored.colored.

REMEMBER pg 96

Prehepatic:Prehepatic: indirectindirect bilirubin bilirubin Urine bilirubin negativeUrine bilirubin negative Caused by excessive breakdown of RBCsCaused by excessive breakdown of RBCs

Posthepatic:Posthepatic: directdirect bilirubinbilirubin Urine bilirubin positiveUrine bilirubin positive Fecal urobilinogen will be decreased due Fecal urobilinogen will be decreased due

to blockage of bile ducts.to blockage of bile ducts.

Bilirubin Methods

Jendrassik-GrofJendrassik-Grof Evelyn-MalloyEvelyn-Malloy Direct Spectrophotometric – only Direct Spectrophotometric – only

performed on performed on newborns.newborns.

Jendrassik-Grof

Principle:Principle: Bilirubin couples with Bilirubin couples with diazodiazo to to form a colored complex (azobilirubin)form a colored complex (azobilirubin)

Conjugated bilirubinConjugated bilirubin is is water solublewater soluble and and reacts directlyreacts directly withwith the color- the color-producing agent producing agent DIAZO.DIAZO.

Unconjugated Bilirubin is Unconjugated Bilirubin is attached to attached to albuminalbumin and is and is NOT water solubleNOT water soluble and and cannot react with DIAZO directly.cannot react with DIAZO directly.

Diazo reagent = Sulfanilic acid + Sodium = Sulfanilic acid + Sodium

Nitrate + HClNitrate + HCl

Direct Bilirubin Procedure

Conjugated + Conjugated + DIAZODIAZO

AzobilirubinAzobilirubin (DIRECT) Bilirubin (DIRECT) Bilirubin PinkPink

Conjugated bilirubinConjugated bilirubin is is water solublewater soluble and and reacts directlyreacts directly withwith the color-producing the color-producing agent agent DIAZO DIAZO to produce a to produce a pinkpink compound compound (azobilirubin).(azobilirubin).

Jendrassik-Grof Total Bilirubin Procedure

Caffeine BenzoateCaffeine Benzoate

Conjugated + UnconjugatedConjugated + Unconjugated + + DiazoDiazo AzobilirubinAzobilirubin Bilirubin BilirubinBilirubin Bilirubin AcceleratorAccelerator

A dissociating agent A dissociating agent (accelerator),(accelerator), caffeinecaffeine benzoatebenzoate, is added to the reaction to , is added to the reaction to releaserelease unconjugated bilirubinunconjugated bilirubin from albuminfrom albumin and and make it make it soluble so that it can react with soluble so that it can react with diazo.diazo.

Indirect Bilirubin1.1. Measure the total & direct bilirubin.Measure the total & direct bilirubin.

2.2. Subtract the direct bilirubin from the Subtract the direct bilirubin from the total to obtain the direct total to obtain the direct measurement.measurement.

Example: Example: Total bilirubin = 7.0 mg/dlTotal bilirubin = 7.0 mg/dl

Direct bilirubin = 2.5 Direct bilirubin = 2.5 mg/dlmg/dl

TotalTotal Direct Direct IndirectIndirect

7.0 mg/dl – 2.5 mg/dl = 7.0 mg/dl – 2.5 mg/dl = 4.5 mg/dl4.5 mg/dl

Reference Ranges

Total serum bilirubinTotal serum bilirubin = 0.2 – 1.0 = 0.2 – 1.0 mg/dlmg/dl

Direct bilirubinDirect bilirubin = 0.2 mg/dl = 0.2 mg/dl

Approximately 80% of bilirubin is Approximately 80% of bilirubin is unconjugated in a normal unconjugated in a normal individual.individual.

Specimen Precautions

Bilirubin is photosensitiveBilirubin is photosensitive Serum and urine specimens should be Serum and urine specimens should be

stored in the dark.stored in the dark. Approximately 10% of bilirubin loss per Approximately 10% of bilirubin loss per

30 minutes. 30 minutes.

Remember:Remember: Protect Protect specimens from specimens from

light!light!

DIRECT SPECTROPHOTOMETRIC

Method

NEWBORNS ONLY:NEWBORNS ONLY: ((Cannot be Cannot be performed on adults due to performed on adults due to carotenes carotenes & & other pigments in serum)   other pigments in serum)  

1.1.Dilute serum & measure absorbance at 445 nm Dilute serum & measure absorbance at 445 nm 3.3.Measure absorbance at 575 nm to correct for Measure absorbance at 575 nm to correct for

hemoglobin interference hemoglobin interference

4.4.Subtract Abs. at 575 nm from Abs at 455 nmSubtract Abs. at 575 nm from Abs at 455 nm   

Compare Compare ALBUMINALBUMIN levels to levels to bilirubin levelsbilirubin levels to assess the to assess the ability to transport unconjugated ability to transport unconjugated bilirubin to the liver. If albumin bilirubin to the liver. If albumin levels are insufficient, unconjugated levels are insufficient, unconjugated bilirubin will deposit in CNS tissue bilirubin will deposit in CNS tissue and may cause brain damage and may cause brain damage (KERNICTERUS)(KERNICTERUS) in newborns. in newborns.

PLASMA AMMONIA

Source of ammonia:Source of ammonia: Breakdown of amino-acids obtained Breakdown of amino-acids obtained

from dietary proteins in intestinefrom dietary proteins in intestine Metabolized by liver (ammonia + Metabolized by liver (ammonia +

CO2 UREA)CO2 UREA) Urea excreted by kidneysUrea excreted by kidneys

Purpose

Ammonia levels provide indicators of Ammonia levels provide indicators of hepatic detoxification and excretory hepatic detoxification and excretory ability of the liver.ability of the liver.

Ammonia levels reflect the ability of the Ammonia levels reflect the ability of the liver to convert toxic ammonia liver to convert toxic ammonia byproducts into urea so that they can be byproducts into urea so that they can be excreted by the kidneys.excreted by the kidneys.

SPECIMEN COLLECTION

EDTA plasma,EDTA plasma, place on place on iceice & & analyze analyze ASAPASAP to prevent ammonia contamination to prevent ammonia contamination

SOE = poor venipuncture technique, SOE = poor venipuncture technique, probing, ammonia contamination from probing, ammonia contamination from handlinghandling

  Method:Method: alphaalpha--ketoglutarate + NH4 + ketoglutarate + NH4 + NADPHNADPH ---------------->glutamate + ---------------->glutamate + NADPNADP

(measure decrease in absorbance at 340 (measure decrease in absorbance at 340 nm)nm)

  

Clinical Interpretation

Reference rangeReference range = 20 – 80 = 20 – 80 ug/dl ug/dl or 11 – 35 or 11 – 35 umol/Lumol/L

   levels of ammonia are toxic to levels of ammonia are toxic to CNS CNS - - Reye’s syndromeReye’s syndrome ( if < 5x ( if < 5x normal = 100 % survival), normal = 100 % survival), hepatic hepatic encephalopathy,encephalopathy, hereditary hereditary disorder of urea cycledisorder of urea cycle