This document included liver anatomy and its physiological functions. It also explains the measuring principles of liver function tests.

1. Liver Function
Getnet Fetene(MSc in Clinical Chemistry

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

10. ALT and AST measuring principles
• Oxoglutarate + L-Alanine ALT L-Glutamate + Pyruvate
• Pyruvate + NADH + H+ LDH L-Lactate + NAD+
• L-Aspartate + α-Oxoglutarate AST L-Glutamate + Oxalacetate
• Oxalacetate + NADH + H+ MDH L-Malate + NAD+

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

13. GGT and ALP…
Causes of elevation:-
Intrahepatic:
• Medication induced
• Primary biliary cirrhosis
• Alcoholic hepatitis
• Viral hepatitis
Extrahepatic
• Stones
• Biliary stricture
• Malignancy ( pancreatic, duodenal, cholangiocarcinoma)
• Pancreatitis Primary sclerosing cholangitis

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

21. Bilirubin Metabolism
• Production
• Uptake by the hepatocyte
• Conjugation
• Excretion into bile ducts
• Delivery to the intestine.

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…

33. Interferences of LFT
• Hemolysis
• Lipemia
• Anticoagulants(EDTA and ALP)

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

35. Interpretation
Reference Range
AST Range: <39 U/L
ALT range: <45 U/L
ALP range: 34 – 104 U/L
GGT range: 7-64 U/L
BilT range: 0.3-1.0 mg/dL
BilD range: 0.03 – 0.18 mg/dL
TP range: >6.4 g/dL
ALB range: >3.5 g/dL

36. Read
• Measurement of bilirubin by HPLC
• Measuremnet of ALT and AST by colorimetric methods

37. Any questions so far?