This document provides information on liver function tests. It begins with an overview of liver anatomy and functions including synthesis, metabolism, and excretion. It then discusses specific tests that evaluate these hepatic functions and their clinical implications. Details are provided on liver zonation and regeneration. Key proteins and enzymes synthesized by the liver are outlined. Metabolic functions like ammonia, carbohydrate, and xenobiotic metabolism are reviewed. Inherited disorders of bilirubin metabolism that can cause jaundice are also summarized.

1. LIVER FUNCTION TEST
Apeksha Niraula
Junior Resident-III
Department of Biochemistry
BPKIHS

2. Objectives
 Liver: Basic Anatomy
 Functions: Synthesis, Metabolism, Excretion
 Tests assessing synthetic, metabolic and excretory
functions of Liver
 Clinical Implications

3.  Liver Function Test [Misnomer]- describes the measurement of
distinct physiological and biochemical organ functions that have
meaning in the absence of any organ pathology
 Enzymes like Aminotransferases have major and well defined roles
within the cell, but have no functional significance at all in plasma
 They are simply the markers of hepatocyte disruption

4. ANATOMY
 Liver: Wedge-shaped organ; 1.5 kg
 Four lobes: Right and Left lobe anteriorly; Caudate and Quadrate
lobes posteriorly
 Dual blood supply: Arterial Blood from Aorta
Second Source: Portal vein ; collects blood from
gut
 Portal Venous System delivers about 80% of the blood and 20%
Oxygen to the Liver

5. Microscopically, Liver has been divided into 3 Zones
 Zone 1 Nearest the afferent arteriole in the
portal tract
High Oxygen tension, Rich blood supply
Site for Oxidative metabolism (ETC, TCA
Cycle, FA Oxidation), Bile Acid excretion
 Zone 2 Between Zone 1 and 3
Intermediate Blood supply
 Zone 3 Zone surrounding the terminal hepatic
vein in the centrilobular or perivenular
area
Site for Glycolysis, Glutamine Synthesis,
Xenobiotic Metabolism

6. Hepatic Regeneration
 Liver has a great capacity for regeneration is
the rationale for much of the practice of clinical
hepatology
 Initiation by growth factors are now being identified; Epidermal growth factor,
Transforming growth factor α, Hepatocyte growth Factor

7. Functions
 Synthetic Function
 Metabolic Function
 Excretory Function
 Storage Function

8. Synthetic Function
I. Protein Synthesis
 Liver : Primary Site for Plasma Protein Synthesis
 Synthesis occurs in Rough endoplasmic reticulum followed by
release into the hepatic Sinusoids

9. Proteins Synthesised Features Clinical Implications
Albumin Oncotic Pressure, Antioxidant,
Antithrombotic
Decreased in Liver Disease, Ascites,
Cirrhosis
Transthyretin Current Synthetic Ability Decreased in Cirrhosis
Ceruloplasmin Prinicipal Copper containing plasma
protein
Decreased in Wilson’s Disease
Increased in Inflammation,
Cholestasis
α1-Antitrypsin Major Serine Protease Inhibitor
(Serpin), α1- globulin
Decreased in Cirrhosis ; Increased in
Acute Inflammation
α – Fetoprotein Normal Concentration of Fetal Blood,
falls to adult concentration by 1 year
of age
Mild increase is seen in Acute and
Chronic Hepatitis ; Hepatocellular
carcinoma (High)
Immunoglobulins Not synthesized in Liver Increased in Cirrhosis, Autoimmune
Hepatitis

10. Coagulation Proteins
 Coagulation proteins synthesized in Liver
 Fibrinogen (I)
 Prothrombin (II)
 Proaccelerin (IV)
 Proconvertin (VII)
 Christmas Factor (IX)
 Plasma Thromboplastin Antecedent (X)
 Hageman Factor (XI)
 Fibrin-Stabilising Factor (XIII)
 Prekallikrein (PK)
 High-Molecular Weight Kininogen (HMWK)

11.  Prothrombin time Measures activity of Fibrinogen,
Prothrombin, Factors V, VII and X
 All these factors made in the liver; some are Vit K dependent; thus a
prolonged PT indicates presence of significant Liver Disease
 Cholestasis Vitamin K deficiency may cause an increase in PT
 Injection of 10 mg Vit K corrects the coagulation abnormality in few days
 If PT is prolonged due to hepatocellular disease, factor synthesis is
decreased and administration of Vit K doesnot correct the problem

12. Lipid and Lipoprotein Synthesis
 Approximately 33% of the fatty acids originating from adipose tissue enter
the liver, where they undergo esterification into triglycerides or are oxidized
 Excessive esterification results in “Fatty Liver,”
 The relative rates of secretion of bile acids, cholesterol, and lecithin are
important factors in the pathogenesis of cholesterol gallstones

13. Urea Synthesis
 Patients with end-stage liver disease may have low concentrations of urea in
plasma
 These findings suggest that patients with liver disease have an impaired ability
to metabolize protein nitrogen and to synthesize urea.
 Rate of hepatic urea synthesis also depends on exogenous intake of nitrogen and
on endogenous protein catabolism.

14. Metabolic Function
Ammonia Metabolism
 The major source of circulating ammonia is the GI tract
 Aromatic Amino Acids [AAA] are metabolized in the liver while branched chain
amino acid [BCAA] are taken up largely by muscle
 Ratio of BCAA/AAA is decreased in Acute Liver Failure and this alteration forms
the basis of one theory of Hepatic Encephalopathy
 Ammonia enters tissue of the central nervous system by Passive diffusion
 The rate of entry increases in proportion to the plasma concentration and is
dependent on pH

15. Clinical Significance
 Hyperammonemia Toxic Effects in brain
 Inherited deficiencies of urea cycle enzymes are the major cause of
hyperammonemia in infants
 Acquired causes: Advanced Liver Disease and Renal Failure

16. Carbohydrate Metabolism
 Liver: Site for Gluconeogenesis and Glycogen Storage
 Hypoglycemia Common Complication in certain liver
diseases; Reye’s Syndrome, Acute Liver
Failure
 Hyperglycemia due to failure of Liver to store Glycogen and
failure of peripheral tissues to take up glucose
adequately

17. Xenobiotic Metabolism and Excretion
 Xenobiotics: Chemical Substance that are foreign to biological system
 They are cleared and/or metabolized by liver
 Rates of metabolism of these compounds are called as QUANTITATIVE LIVER
FUNCTION TESTS
 As liver disease progresses, quantitative liver function test results gradually
worsen
 Their measurement adds slightly to that obtained by widely used tests such
as bilirubin, albumin, and INR measurement

18. Excretory Function
 Organic compounds of both endogenous and exogenous origin are extracted
from the sinusoidal blood, biotransformed, and excreted into the bile or urine.
 Endogenously produced compounds : Bilirubin and bile acids,
 Determination of the rate of clearance of exogenous compounds, such as
Aminopyrine , Lidocaine and Caffeine

19. Bilirubin
 Bilirubin is the orange-yellow pigment derived from heme
(Ferroprotoporphyrin IX), mainly as a product of red blood cell turnover
 Bilirubin formed in the reticuloendothelium is lipid soluble and virtually
insoluble in water.
 It is extracted and bio-transformed in the liver and excreted in bile and urine

20.  Bilirubin assumes a ridge-tiled configuration stabilized by six
intramolecular hydrogen bonds
 Two additional important structural features have been noted:
I. Z-Z (Trans) confirmation for the double bonds between Carbon 4 and 5
and 15 and 16
II. An Involuted Hydrogen bonded structure in which the proprionic acid-
carboxylic acid groups are hydrogen bonded to the nitrogen atoms of
Pyrrole rings
 On exposure to light, the Z-Z configuration is converted to the E-E(Cis-
confirmation) and other confirmation like 4E-15Z and 4Z-15E
 E-E configuration are more water soluble and easily excreted
Rationale for irradiating jaundiced newborns with 450 nm Light

21.  Total bilirubin: 1.0-1.5 mg/dl
 Conjugated bilirubin (Direct ; glucuronide): 0.2-0.9 mg/dl
 Unconjugated bilirubin ( Indirect; bilirubin - albumin complex): 0.8-1.2 mg/dl
 Delta Bilirubin- Bilirubin bound to Albumin; Bili-albumin
 May account for 90% of Total bilirubin in Hepatocellular and
Cholestatic Jaundice
 Persists for a longer time
 If the plasma bilirubin level exceeds 1mg/dl, the condition is called
hyperbilirubinemia
 Levels between 1 & 2 mg/dl are indicative of latent jaundice

22. Heme
(250 to 400 mg/day)
Heme oxygenase
Biliverdin reductase
Hemoglobin
(70 to 80%) Erythroid cells
Heme proteins
myoglobin, cytochromes
(20 to 25%)
Biliverdin
Bilirubin
NADPH + H+
NADP+
3 [O]
Fe3+ + CO
apoferritinferritin
Indirect
Unconjugated Bilirubin
Albumin
Bilirubin Production

23. Bilirubin Processing
Bile (Gall bladder)

24. Enterohepatic
Circulation of Bilirubin
and Urobilinogen
Bilirubin Excretion

25. Clinical Significance
 Jaundice: Characterised by Hyperbilirubinemia and deposition of bile pigment in
the skin, mucous membranes and sclera with a resulting yellow appearance of
the patient [ICTERUS]
 Defects in bilirubin metabolism resulting in Jaundice can occur at each step of
the metabolic pathway
 Inherited Disorders of Bilirubin Metabolism
 Jaundice of Newborn

26. Inherited Disorders of Bilirubin Metabolism
Unconjugated Hyperbilirubinemia
 Gilbert Syndrome
 Crigler-Najjar (Type I and II) Syndrome
 Lucey-Driscoll Syndrome
Conjugated Hyperbilirubinemia
 Dubin-Johnson Syndrome
 Rotor syndrome

27. Gilbert Syndrome
 Benign condition manifested by mild unconjugated hyperbilirubinemia
 The serum concentration of bilirubin fluctuates between 1.5 and 3 mg/d and
tends to increase with fasting
 Hepatic glucuronyltransferase activity is low as a consequence of a mutation in
the bilirubin-UDP-glucuronosyltransferase (UGT1A1) gene
 Normal Liver Function tests

28. Crigler-Najjar Syndrome (Type I)
 Rare disorder caused by complete absence of UDP-glucuronyltransferase
 Very high concentrations of unconjugated bilirubin often exceeding 20 mg/dL
 Inherited as an autosomal recessive trait
 Most patients die of severe brain damage caused by kernicterus within the first
year of life.
 Phlebotomy and plasmapheresis can reduce the serum bilirubin, but
encephalopathy usually develops.
 Early liver transplantation is the only effective therapy

29. Crigler-Najjar Syndrome (Type II)
 Rare autosomal dominant disorder is characterized by a partial deficiency of
UDP-glucuronyltransferase
 Unconjugated bilirubin is usually 5 to 20 mg/dL
 Unlike the Crigler-Najjar syndrome typeI, type II responds dramatically to
phenobarbital and a normal life is expected
Lucey-Driscoll Syndrome
 Lucey-Driscoll syndrome is a familial form of unconjugated hyperbilirubinemia
caused by a circulating inhibitor of bilirubin conjugation
 The hyperbilirubinemia is mild and lasts for the first 2 to 3 weeks of life

30. Dubin-Johnson Syndrome
 Rare autosomal recessive disorder
 Jaundice with predominantly elevated conjugated bilirubin and a minor elevation
of unconjugated bilirubin
 Excretion of various conjugated organic anions and bilirubin, but not bile vsalts,
into bile is impaired, reflecting the underlying defect in canalicularvexcretion
(mutations in MRP-2 gene encoding Multidrug Resistance Protein2 (MRP2)
 Liver has a characteristic greenish black appearance
 Serum alanine aminotransferase and alkaline phosphatase are usually normal,
and pruritus is absent

31. Rotor Syndrome
 Rotor syndrome is another form of conjugated hyperbilirubinemia
similar to Dubin-Johnson syndrome but without pigment in the liver
 Total urinary coproporphyrins are elevated, with about two thirds
being coproporphyrin I
 Prognosis is excellent

32. Unconjugated Hyperbilirubinemia
 Kernicterus (acute bilirubin encephalopathy) especially in
low birth weight infants
 Kernicterus refers to a neurologic syndrome that results in
brain damage owing to deposition of bilirubin in the basal
ganglia and brain stem nuclei
 Causes of unconjugated hyperbilirubinemia in the neonate
are physiologic jaundice of the newborn, hemolytic disease
and breast milk hyperbilirubinemia.
Jaundice of Newborn

33. Physiologic Jaundice of Newborn
 Bilirubin is usually less than 5 mg/dL, with 90% unconjugated
 Factors causing Hyperbilirubinemia
I. An increased bilirubin load in the newborn
II. Appearance of “shunt” bilirubin, which is bilirubin derived from
ineffective erythropoiesis
III. Decreased conjugation of bilirubin owing to a relative lack of
glucuronyl transferase (conjugating enzyme)
IV. β- glucuronidase in meconium, which hydrolyzes bilirubin
conjugates to unconjugated bilirubin that can be passively
reabsorbed
V. Exposure of breast-feeding infants to pregnanediol,
nonesterified fatty acids

34. Hemolytic disease of the newborn
 Results from maternal-fetal incompatibility of Rhesus blood factors
 Unconjugated Hyperbilirubinemia; Kernicterus
 Glucose-6-phosphate Dehydrogenase (G6PD) deficiency may also lead to
unconjugated hyperbilirubinemia
Breast Milk Hyperbilirubinemia
 Due to α-glucuronidase in breast milk, which hydrolyzes conjugated bilirubin
in the intestine
 The unconjugated bilirubin being more lipophilic is passively absorbed

35. Conjugated Hyperbilirubinemias
 Characterized by hyperbilirubinemia; conjugated
bilirubin exceeds 1.5 mg/dL
 Idiopathic neonatal hepatitis and biliary atresia
 Family history may be helpful in diagnosing α1-antitrypsin
deficiency, cystic fibrosis, galactosemia
 Serologic tests for hepatitis A, B, C and for adenovirus,
Coxsackie virus, Cytomegalovirus, herpes simplex, rubella
and Toxoplasma

36. Isolated Bilirubin 

37. Dye Excretion Tests
 Dye excretion tests [such as bromsulphthalein (BSP) and Indocyanine
green (ICG) clearance] were formerly used as Indicators of liver disease
 With the development of more sensitive and specific indicators of liver
disease, dye excretion tests have become obsolete

38. Drug Clearance Tests
 A variety of drugs that are metabolized by the liver have been used to study
the action of various P450 enzymes
 Aminopyrine is demethylated to form carbondioxide and aminoantipyrine
 With the use of 13C- or 14C-labeled aminopyrine, the resulting isotopically
labeled CO2 is measured in breath as a reflection of functioning liver mass.
 Decrease in metabolism are common in cirrhosis
 Other Drugs used: Lidocaine, Caffeine

39. Transaminases
 Hepatocytes contain high levels of enzymes that can leak into the plasma
when there is liver injury
 Enzymes found in hepatocytes are:
 Cytoplasmic = AST, ALT
 Mitochondrial = ASTm

40. Alanine Aminotransferase (ALT)
 Serum glutamic-pyruvic transaminase (SGPT)
 Cellular Location- Cytosolic
 Primarily used to diagnose liver disease, to monitor the course of treatment for
hepatitis, active postnecrotic cirrhosis, and the effect of drug therapy
 Plasma half-life- 47 hours

41. Increased ALT levels are found in the following conditions:
 Hepatocellular disease
 Active cirrhosis (mild increase)
 Metastatic liver tumor
 Obstructive jaundice or billiary obstruction (mild to moderate increase)
 Viral, infectious or toxic hepatitis (30-50x normal)

42. Aspartate Aminotransferase (AST)
 Serum Glutamic Oxaloacetic Transaminase (SGOT)
 Also reflects damage to the hepatic cell, though less specific
 It may be elevated in other conditions such as a myocardial infarction and muscle
disease
 AST is not a specific enzyme for liver as the ALT, but the ratios between ALT and
AST are useful to physicians in assessing the aetiology of liver enzyme
abnormalities
 Plasma half-life- 17 hours

43.  AST/ALT less than or equal to 2 Viral hepatitis
Mononucleosis
Acute hepatotoxicity
 AST/ALT greater than 2 Alcoholic liver disease
Cirrhosis
Passive Congestion
Bile Duct Obstruction

44. Alkaline Phosphatase
 Source: Liver, bone, placenta and intestine
 Not specific for Liver Disease
 Specificity of ALP can be enhanced by measuring specific Isoenzymes
 Electrophoresis was used to identify the Isoenzymes
 Hepatic> Bone> Intestine> Placenta
 Another method is done by heating the serum at 56oc for 15 mins
Bone and Hepatic Isoenzymes- Heat Sensitive
Placental- Heat Labile

45.  Primary value of an elevated serum level of alkaline phosphatase of liver -
recognition of cholestatic disorders
 4 fold elevation of serum ALP -approximately 75% of patients with chronic
cholestasis, both intrahepatic or extrahepatic
 Whenever in confusion with raised ALP, check for Gamma GT levels; if raised
Confirms Hepatic Disorder

46.  ↑ ALP activity in liver disease are the result of increased synthesis of
the enzymes by cells lining the bile canaliculi, usually in response to
Cholestasis (intra or extra-hepatic)
 ALP from the intestine is increased in a person with inflammatory bowel
disease such as ulcerative colitis
 ALP: Membrane bound enzyme ; in Cholestasis/obstructive Jaundice
Accumulation of bile acid Regurgitation of bile
Fragmentation of Membrane
Increased Plasma ALP

47. Gamma Glutamyl Transferase (GGT) Enzyme
 GGT is used by the body to synthesize glutathione Tri peptide
 GGT is present in liver, kidney, pancreas, intestinal cells and prostrate glands
 Elevated levels (> 10 - 30 IU/l) are observed in :
Chronic alcoholism
Pancreatic disease,
 In liver diseases, GGT elevation parallels that of ALP
 In alcoholic liver disease GGT levels may be parallel to alcohol intake
 Inducible Enzyme: Phenytoin,Barbiturates

48. 5’ Nucleotidase
 Catalyses hydrolysis of AMP Releasing Inorganic Phosphate
 Site: Biliary canalicular membrane
 Increased 5’ NT Cholestasis
 Truly Hepatic Origin
Glutathione S Transferase
 Raised in Hepatocellular Carcinoma
 Half-Life- 90 Minutes

49. Hepatic Storage Function
 Storage of Energy-rich carbohydrate substrates; Glycogen
 Hepatic storage of Glycogen allows the release of glucose to other tissue when
the need exists [ When plasma concentration of glucose decrease]
 Iron storage, Vitamin A,D,E and B12 storage.

50. Tests To Detect Hepatic Fibrosis
 Liver biopsy is the standard for the assessment of hepatic fibrosis
 Noninvasive measures of hepatic fibrosis have been developed
 Hyaluronan is the best to date

51. Hyaluronan
 Hyaluronan is a glucosaminoglycan produced in mesenchymal cells
 Typically degraded by hepatic sinusoidal cells
 Serum levels of hyaluronan are elevated in patients with cirrhosis as a
result of sinusoidal capillarization
 Useful for identifying advanced fibrosis in patients with chronic hepatitis C,
chronic hepatitis B, ALD and NASH

52. Parameter Hemolytic Jaundice
(Pre-Hepatic Jaundice)
Obstructive Jaundice
(Posthepatic Jaundice)
Hepatic Jaundice
(Intrahepatic
Jaundice)
Serum Bilirubin Unconjugated
Bilirubin
Conjugated Bilirubin Both
Van Den Bergh
Reaction
Indirect Positive Direct Positive Biphasic
Serum Enzymes Normal ALT, AST and
ALP
ALP , ALT and AST
marginal
ALT and AST , ALP
marginal
Bilirubin in Urine Not Excreted Excreted Excreted
Urobilinogen in Urine Excretion Normal or Normal or

54. Parameters Analytical Methods
Total Protein Colorimetric Assay( Biuret method)
Albumin Colorimetric Assay using BCG
Bilirubin Diazo Method
ALT Enzymatic Method (IFCC)
AST Enzymatic Method (IFCC)

55. ALP Colorimetric assay using p-Nitrophenol
phosphate
GGT Enzymatic Colorimetric assay
Ammonia Enzymatic Method, Chemical method(Berthelot’s
Reaction)

56. Liver Function Test
Liver chemistry test Clinical implication of abnormality
ALT Hepatocellular damage
AST Hepatocellular damage
Bilirubin Cholestasis, impair conjugation, or biliary obstruction
ALP Cholestasis, infiltrative disease, or biliary obstruction
PT Synthetic function
Albumin Synthetic function
GGT Cholestasis or biliary obstruction
Bile acids Cholestasis or biliary obstruction
5`-nucleotidase Cholestasis or biliary obstruction
LDH Hepatocellular damage, not specific

57. References
 Teitz textbook of Clinical Chemistry and Molecular
Diagnostics; 5th Edition
 Clinical Biochemistry Metabolic and Clinical Aspects; William
J Marshall, 3rd Edition
 Harrison’s Principle of Internal Medicine; 17th Edition

58. To my Moderator Dr. Seraj Ahmed Khan for his
valuable suggestion and guidance during the
preparation of the presentation
Acknowledgement

59. Thank You
The truth is that in clinical medicine, you really don't need a true lab
test of "what percent of liver function remains". You need to know
whether disease is present, and if so, which one