2. chapter objective
Upon completion of this chapter the student will be able to:
• Describe the anatomy and physiological role of the liver, including formation of
bilirubin
• Describe bilirubin metabolism, including formation, conjugation and excretion.
• Explain the clinical significance of bilirubin
• Describe methods of analysis of serum bilirubin (Direct & total), sources of errors
and
3. Chapter outline
• Introduction
• Anatomy of the liver
• Physiological role of the liver
• Liver function tests
• Formation & excretion of bilirubin
• Clinical significance of bilirubin
• Determination of serum Bilirubin (Direct & total)
• Interpretation of bilirubin results
4. Introduction…
Anatomy of the Liver
• Liver is a large bi-lobed complex organ receiving a large amount of blood and
nutrients from the GIT system
• K = Kupffer cells
• CV = a central vein
• B = a bile duct –connected
to the biliary tree
• V = branch to portal vein
• A = branch to hepatic artery
• P = parenchymal cells (hepatocytes)
5. Introduction…
Physiological Functions of the Liver
• Metabolism
• Synthesis function
• Protective function
• Conjugation, detoxification and excretion
• Storage function
• Digestion and formation of bile
6. Liver Function Tests
Test of function
• Total protein
• Albumin
• prothrombin time
• Cholesterol
• Triglycerides
• Urea
• Total and direct bilirubin
Tests of injury
• AST, ALT
Tests of obstruction
• Total and direct bilirubin
• ALP, GGT
7. Transaminases
• Transaminases: is a name for a category of enzymes involved in exchange of an
oxygen from α-keto acid and an amine group from an amino acid
• Transaminases are present in almost all tissues both in the cytoplasm and in the
mitochondria
• ALT and AST included here
8. ALT and AST
Are:-
• Intracellular enzymes released from injured hepatocytes
• Signifies hepatic inflammation or hepatocellular necrosis
• Degree of elevation correlates with extent of hepatic injury
ALT More sensitive and specific than AST for liver injury
9. ALT and AST
Alanine Transaminase (ALT) Aspartate Transaminase (AST)
• Produced in hepatocytes
• Very specific marker of hepatocellular
injury
• Relatively low concentrations in other
tissues so more specific than AST
• Levels fluctuate during the day
• Rise may occur with the use of certain
drugs or during periods of strenuous
exercise.
• Occurs in two isoenzymes,
indistinguishable on standard AST
assays.
• The mitochondrial isoenzyme is
produced in hepatocytes
• The cytosolic isoenzyme is present in
skeletal muscle, heart muscle and
kidney tissue.
• Caution must be exercised in its use to
evaluate hepatocellular damage.
• Usually rises in conjunction with ALT
to indicate hepatocellular injury
11. GGT and ALP
Gamma-glutamyl transferase (GGT) Alkaline Phosphatase (ALP)
• Synthesized in ER of hepatocytes and
cholangiocyts(bile duct epithelium)
• It is found in the microsomes of
hepatocytes and biliary epithelial cells.
• Also in kidney, pancreas and intestine.
• Elevation of GGT in association with a
rise in ALP is highly suggestive of a
biliary tract obstruction and is known as
cholestatic
• Subject to rise with hepatic enzyme
induction due to chronic alcohol use or
drugs such as rifampicin and phenytoin.
• Produced in the membranes of cells lining
bile ducts and canaliculi.
• Act to dephosphorylate a variety of
molecules throughout the body.
• Released in response to the accumulation
of bile salts or cholestasis.
• Non-hepatic production in the kidney,
intestine, leukocytes, placenta and bone.
• Physiological rise in pregnancy or in
growing children.
• Pathological rise in Paget’s disease, renal
disease and with bone metastases.
12. GGT and ALP…
• GGT is reasonably specific to the liver and a more sensitive marker for cholestatic
damage than ALP
• GGT may be elevated with even minor, sub-clinical levels of liver dysfunction.
• It can also be helpful in identifying the cause of an isolated elevation in ALP
• GGT is raised in alcohol toxicity(acute and chronic).
• GGT mostly used to tell if elevated ALP is from liver or bone
• If ALP is high but GGT is normal it suggests the source of the ALP is bone since
GGT not produced in bone
14. Measuring principles of GGT and ALP
• The γ-glutamyl transferase catalyzes the transfer of a gamma-glutamyl group from the
colorless substrate, γ-glutamyl-p-nitroaniline, to the acceptor, glycylglycine with
production of the colored product, p-nitroaniline.
• ALP activity is determined by measuring the rate of conversion of p-nitro-
phenylphosphate (pNPP) in the presence of 2-amino-2-methyl-1-propanol (AMP) at pH
10.4.
pNPP + AMP ALP pNP + AMP-PO4
Mg2+
15. Total protein and Albumin
Total serum protein levels are affected by not only changes in one or
more of the individual protein levels, but also by changes in plasma
water
A variety of conditions cause hyperproteinemia, or increased serum
protein.
• Dehydration(hemoconcentration)
• Diarrhea, vomiting
• Inflammation
• Diet
Albumin is the main protein in the blood.
It is synthesized exclusively by the liver
16. Total protein and Albumin
Hypoalbuminemia Hyperalbuminemia
• Malnutrition
• Malabsorption
• Malignancy
• Inflammation ( acute,
chronic)
• Nephrotic syndrome
• Burns
• Exudative skin disease
• Intravenous fluids
• Overhydration
• Cirrhosis
• Pregnancy.
• Higher than normal levels of albumin
may indicate dehydration or
severe diarrhea.
• If the albumin levels are not in the
normal range, it doesn't necessarily
mean a medical condition needing
treatment.
• Certain drugs, including steroids,
insulin, and hormones, can raise
albumin levels
17. Measuring principles of total protein and albumin
• Cupric ions in an alkaline solution react with proteins and polypeptides containing
at least two peptide bonds to produce a violet colored complex read at 540/660 nm
Protein+Cu2 OH- Blue violet complex
• The assay is based on the selective interaction between Bromocresol Green (BCG)
and albumin forming a chromophore that can be detected at 600/800 nm.
Albumin + Bromocresol pH(4.2) Green complex
18. Bilirubin metabolism
• In adults, 250 to 350 mg of bilirubin is produced each day
• Approximately 80% to 85% of this bilirubin is derived from the destruction of
senescent red blood cells by the reticuloendothelial system
• The remaining 15% to 20% comes from the breakdown of nonhemoglobin
proteins, such as myoglobin and the cytochromes
• In reticuloendothelial cells, the microsomal enzyme heme oxygenase cleaves
heme into biliverdin
• Biliverdin is reduced to bilirubin by the cytosolic enzyme
biliverdin reductase before being released into the circulation
• In this unconjugated form, bilirubin is water insoluble and is transported to the
liver tightly bound to albumin.
• When the bilirubin-albumin complex enters the sinusoidal circulation of the liver,
three distinct metabolic phases are recognized: (1) hepatocyte uptake, (2)
conjugation, and (3) excretion into bile
19. Bilirubin metabolism…
• Bilirubin is a yellow bile pigment produced through the breakdown of red blood
cells, which is known as hemolysis
• Unconjugated bilirubin is transported across the sinusoidal membrane of the
hepatocyte into the cytoplasm
• Inside the hepatocyte, unconjugated bilirubin is bound by a cytoplasmic protein, in
this case glutathione S-transferase
• The microsomal enzyme uridine diphosphate–glucuronyl transferase then
conjugates the insoluble unconjugated bilirubin with glucuronic acid to form the
water-soluble conjugated forms, bilirubin monoglucuronide(15%) and bilirubin
diglucuronide (85%)
• Conjugated bilirubin is excreted from the hepatocyte into the bile canaliculus by
an active transport mechanism
• Excretion into bile is the rate-limiting step in bilirubin metabolism
20. Bilirubin metabolism…
• After excretion, bile flows through the biliary ductal collecting system, may or
may not be stored in the gallbladder, and enters in to the intestine
• In the intestine, bilirubin is converted by bacterial enzymes into urobilinogen
• 10% to 20% of the urobilinogen is reabsorbed from the intestine into the portal
circulation, creating an enterohepatic circulation
• This recycled urobilinogen may be re-excreted into the bile by the liver or into
urine by the kidney
• Most (85%) of UBG is oxidized into urobilin, the brown pigment of feces
22. Clinical relevance of bilirubin
Jaundice
• Jaundice describes a yellow discoloration of the sclera and/or skin in response
to elevated bilirubin levels
• Causes of jaundice can be categorized as pre-hepatic, hepatic, or post-
hepatic
Pre-hepatic jaundice is caused by increased hemolysis
• This results in the increased presence of unconjugated bilirubin in the blood as
the liver is unable to conjugate.
• This is caused by:
Tropical disease, e.g. malaria, yellow fever
Genetic disorders, e.g. sickle-cell anemia
Hemolytic anemias
23. Clinical relevance of bilirubin…
Hepatic jaundice is caused by liver impairment
• This causes the decreased ability of the liver to conjugate bilirubin, resulting in
the presence of conjugated and unconjugated bilirubin in the blood
• It can be transport failure(Dubin-Johnson syndrome) and conjugation
failure(Crigler-Najjar syndrome, Gilbert’s syndrome)
Or
• Liver damage can result from:
Viral hepatitis
Hepatotoxic drugs, e.g. paracetamol overdose
Alcohol abuse
24. Dubin–Johnson syndrome
• Is due to a defect in the multiple drug resistance protein 2 gene (ABCC2), located
on chromosome 10
• It is an autosomal recessive disease and is likely due to a loss of binding domain
due to mutation
• Unaffected subjects have a coproporphyrin III to coproporphyrin I ratio around 3–
4:1
• In patients with Dubin–Johnson syndrome, this ratio is inverted
• Analysis of urine porphyrins shows a normal level of coproporphyrin, but the I
isomer accounts for 80% of the total (normally 25%)
Clinical relevance of bilirubin…
25. Gilbert’s Syndrome:
• Gilbert’s syndrome is an inherited disorder where there is hyperbilirubinemia
due to a fault in the UGT1A1 gene leading to a deficiency in UDP-
gluconoryltransferase
• Two bases are inserted into the promoter of the gene
• This faulty gene results in slower conjugation of bilirubin in the liver and so it
builds up in the bloodstream instead of being excreted through the biliary ducts
• Patients are usually asymptomatic and have normal bilirubin levels
• However, under physiological stressors such as illness, alcohol abuse and extreme
exercise, patients can become markedly jaundiced
Clinical relevance of bilirubin
26. Crigler-Najjar syndrome
• Is a rare genetic disorder characterized by an inability to properly convert
unconjugated bilirubin due to mutation
• Caused by a deficiency or complete absence of hepatic microsomal bilirubin-
uridine diphosphate glucuronosyltransferase (bilirubin-UGT) activity
• Mutations lead to the exchange of amino acids, changes of the reading frame or to
stop codons
• Is a severe condition characterized by high levels of bilirubin in the blood
• Crigler-Najjar syndrome is divided into two types
• Type 1 (CN1) is very severe, and affected individuals can die in childhood due to
kernicterus
• Type 2 (CN2) is less severe
Clinical relevance of bilirubin…
27. Post-hepatic jaundice is caused by the blockage of bile ducts
• This results in backflow of conjugated bilirubin into the blood as it cannot
move past the obstruction
• Bile duct obstruction can be caused by:
Gallstones
Hepatic tumors
Clinical relevance of bilirubin…
28. Measurement of bilirubin
• The accurate determination of the types and amounts of bilirubin in serum is
important for diagnostic purposes as well as for therapeutic monitoring
• Only conjugated bilirubin and total bilirubin are measured in the lab
• Measurement techniques can be semiquantitative and quantitative
• Bilirubin is measured by (1) direct spectrophotometry(Icterus index), (2) the direct
diazo reaction, (3) high-performance liquid chromatography (HPLC), and (4)
enzymatic methods
29. Measurement of bilirubin…
Icterus Index Test
• Measures the degree of icterus in plasma or serum and correlates with a
rough estimation for bilirubin concentration
• Take absorbance at 420nm, result is expressed in icterus index units
obtained in comparison with standard potassium dichromate solution of
assigned icterus index value
• Low specificity because of interference due to presence of hemoglobin,
carotene, and different yellow pigments found in sample
30. Direct Diazo(Malloy-Evelyn and Jendrassik-Grof)
• Bilirubin in serum or plasma is commonly measured by photometric methods
based upon the diazo reaction
• Conjugated bilirubin + diazotized sulfanilic acid → azobilirubin + alkaline tartrate
(green to blue-green color)
• Measured with photometer at 555 - 600 nm depending on specific reagent used
• Unsoluble uncojugated-bilirubin requires an accelerating agent to react with the
diazo reagent
• Malloy and Evelyn uses methanol as an accelerator
• Jendrassik-Grof uses a caffeine benzoate accelerator
• Ascorbic acid is used as a stopping agent
Measurement of bilirubin…
31. Enzymatic method
• The development of enzymatic methods for measuring bilirubin was made
possible by the availability of bilirubin oxidase
• contains one atom of copper (Cu”) per enzyme molecule, and is stable between pH
9.2 to 9.7 for 5 d at 4°C
• Bilirubin oxidase (BOX) is completely inhibited by Fez+ (1 mmol/L) and KCN
(0.1 mmol/L
• BOX catalyzes the oxidation of bilirubin to biliverdin by molecular oxygen without
• formation of hydrogen peroxid), and partially inhibited by sodium azide, thiourea,
or NaCl
Measurement of bilirubin…
32. Enzymatic method…
• At pH between 5 to 8.5, biliverdin is further converted to a violet-purple
compound that eventually becomes colorless
• The decrease in absorbance owing to the disappearance of bilirubin is linearly
related to its concentration.
• Conjugated bilirubins are rapidly oxidized over a wide range of pH
Measurement of bilirubin…
34. Quality control
• A normal & abnormal quality control sample should be analyzed along with
patient samples, using Westgard or other quality control rules for acceptance or
rejection of the analytical run
• Assayed known samples
• Commercially manufactured
• Validate patient results
• Detects analytical errors