This document discusses liver function tests. It describes the various functions of the liver including metabolic, synthetic, secretory, excretory, detoxifying, storage, protective and miscellaneous functions. Liver function tests are indicated to detect and evaluate liver diseases. The tests are classified into groups based on abnormalities in bile pigment metabolism, synthetic function, serum enzyme activities, carbohydrate and lipid metabolism, detoxicating function, excretory function, amino acid catabolism, drug metabolism and markers of hepatic fibrosis. Specific tests are described including those measuring bilirubin, proteins, clotting factors, enzymes and metabolic products. Interpretations of different test results are provided for various liver conditions.

1. Liver Function Tests
Dr. Roshan Kumar Mahat
(PhD Medical Biochemistry)
Assistant Professor
Department of Biochemistry

2. Functions of Liver
1. Metabolic functions:
 Carbohydrate metabolism
 Protein metabolism
 Lipid metabolism
 Vitamins and minerals metabolism
 Nucleic acid metabolism
2. Synthetic functions:
 Albumin, alpha and beta globulin, prothrombin,
coagulation factors, cholesterol, TG,
apolipoprotein, etc

3. 3. Secretory function
 Secretion of bile in intestine
 Secretion of bilirubin in bile
4. Excretory function
 Certain exogenous dyes like BSP and Rose Bengal
dye are excreted exclusively through live cells.
5. Detoxicating function
 Exogenous substances- drugs, toxic substances
 Endogenous substances-ammonia

4. 6. Storage function
 Vitamin B12, A, D, K, glucose in the form of
glycogen
7. Protective function
 Kupffer cells in the liver remove foreign bodies.
8. Miscellaneous function
 Blood formation in embryo
 Forms blood in adults (abnormal states)

5. INDICATION:
• To detect the presence of liver diseases
• To distinguishes different types of liver
diseases
• To evaluate the extent of live damages or
residual liver function
• To monitor the response to treatment or the
course of the disease.

6. CLASSIFICATION OF LIVER FUNCTION
TESTS
1. Tests based on abnormalities of bile pigment
metabolism
• Serum bilirubin and VD Bergh reaction
• Urine bilirubin
• Urine and faecal urobilinogen
2. Tests based on synthetic function of liver
• Estimation of total plasma proteins, albumin and globulin
and determination of A:G ratio.
• Determination of plasma fibrinogen
• Prothrombin time
• Flocculation test

7. 3.Determination of serum enzyme activities
• Aspartate transaminase
• Alanine transaminase
• Alkaline Phosphatase
• Gamma GT
• Serum 5’-Nucleotidase
• Serum Lactate Dehydrogenase (LDH)
• Serum Isocitrate Dehydrogenase (ICD)
• Serum Cholinesterases
• Serum ornithine carbamoyl transferase (OCT)
• Serum Leucine Amino Peptidase (LAP)
• Serum SHBD (Hydroxy Butyrate Dehydrogenase)
• Serum Aldolase and Phosphohexose Isomerase
• Serum Amylase
• Serum Sorbitol Dehydrogenase (SDH

8. 4. Tests based on liver’s part in
carbohydrate metabolism
• Galactose tolerance test
• Fructose tolerance test
5. Tests based on abnormalities of
lipids:
• Determination of serum cholesterol and ester
cholesterol and their ratio.
• Determination of faecal fats

9. 6. Tests based on detoxicating function
of liver
• Hippuric acid synthesis test.
7. Tests based on excretory function
• Bromsulphthalein test (BSP retention test)
• I131-Rose Bengal test

10. 8. Tests based on amino acid
catabolism
• Determination of blood NH3
• Determination of glutamine in CS fluid
9. Tests based on drug metabolism
• MEGX test
• Antipyrine breath test

11. 10. Tests for metabolic liver disease
(Special tests)
• Ceruloplasmin
• Ferritin and Iron
• Alpha-1 antitrypsin
• Beta-2 microglobulin
• Alpha fetoprotein (AFP)

12. 11. Markers of hepatic fibrosis
• Serum hyaluronic acid
• Procollagen type 1carboxy terminal peptide
(PICP)
• Procollagen type III amino terminal peptide
(PIIINP)
• Matrix metalloproteinases (MMPs)
• Tissue inhibitors of MMPs (TIMPs)
• Transforming growth factor beta-1 (TGF Beta-1)

13. TESTS BASED ON ABNORMALITIES OF
BILE PIGMENT METABOLISM
VD Bergh Reaction and
Serum Bilirubin
Bile Pigments in
Urine/Faeces
Urinary and Faecal
Urobilinogen

14. VD Bergh Reaction and Serum
Bilirubin
• VD Berg reagent contains
Sulfanilic acid
Sodium nitrite
• Was introduced by Dutch physician, Abraham
van den Bergh
• When diazotized sulfanilic acid reacts with
bilirubin, a purple coloured azobilirubin is
produced.

15. • There are three possible responses:
1. A direct positive reaction-given by conjugated
bilirubin
2. An indirect positive reaction- given by
unconjugated bilirubin
3. A biphasic reaction- elevation of both
conjugated and unconjugated bilirubin
 Normal serum gives a negative VD Berg
reaction

16. Interpretation
 Indirect positive
 Hemolytic jaundice
 Direct positive
 Obstructive jaundice
 Biphasic
 Hepatocellular jaundice

18. Bile Pigments in Urine/Faeces
Bile pigments in urine:
 Bilirubin is found in the urine in obstructive
jaundice.
 Bilirubin is not present in urine in most cases of
haemolytic jaundice, as unconjugated bilirubin is
carried in plasma attached to albumin, hence it
cannot pass through the glomerular filter.
 Bilirubinuria is always accompanied with direct
VD Bergh reaction.

19. Bile pigments in faeces:
 Bilirubin is not normally present in faeces.
 Some may be found if there is very rapid passage
of materials along the intestine
 Sometimes it is found in faeces of very young
infants, if bacterial flora in the gut is not
developed.
 It is regularly found in faeces of patients who are
being treated with gut sterilising antibiotics such
as neomycin.
 Biliverdin is found in meconium.

20. Urinary and Faecal Urobilinogen
Faecal urobilinogen:
 Normal=50-250mg/day
 Increased in hemolytic jaundice-dark coloured
stool is passed.
 Decreased or absent if there is obstruction to the
flow of bile in obstructive jaundice-clay-coloured
faeces is passed.
 A complete absence of faecal urobilinogen is
strongly suggestive of malignant obstruction.

21. • Urinary urobilinogen:
 Normal=0.64-4 mg/24hr
 In obstructive jaundice-no urobilinogen is found
in the urine.
The presence of bilirubin in the urine, without
urobilinogen is strongly suggestive of
obstructive jaundice either intrahepatic or
posthepatic.
 In haemolytic jaundice-urobilinogen which
appears in urine in large amounts.
Increased urobilinogen in urine and absence of
bilirubin in urine are strongly suggestive of
haemolytic jaundice.

23. TESTS BASED ON CHANGES IN
PLASMA PROTEINS (TESTS FOR SYNTHETIC FUNCTION
OF LIVER)
Determination of Total Plasma Proteins and
Albumin and Globulin and A:G Ratio
Estimation of Plasma
Fibrinogen
Prothrombin Time
Flocculation Tests

24. Determination of Total Plasma
Proteins and Albumin and Globulin
and A:G Ratio
Normal Ranges:
Total protein : 5-8gm/dl
Albumin : 3.5-5gm/dl
Globulin : 2.5-3.5gm/dl
A/G ratio : 1.5:1 -2.5:1

25. Interpretations:
• quantitative estimations of albumin and globulin
may give normal results in the early stages.
• Qualitative changes may be present, in early
stage rise in β-globulins and in later stages γ-
globulins show rise.
In infectious hepatitis
• normal values are the rule, as long as the
obstructive jaundice is not associated with
accompanying liver cell damage.
In obstructive
jaundice
• the albumin is grossly decreased and the
globulins are often increased, so that A:G ratio is
reversed.
In advanced
parenchymal liver
diseases and in
cirrhosis liver

26. Estimation of Plasma Fibrinogen:
Normal value is 200-400 mg%.
Values below 100 mg% have been
reported in severe parenchymal liver
damage.
• Acute hepatic necrosis
• Poisoning from carbon tetrachloride
• In advanced stages of liver cirrhosis.

27. Determination Prothrombin Time:
• 10-16 Sec
Normal value
• Depending on the degree of liver cells
damage plasma prothrombin time may be
increased from 22 to as much as 150 secs
In
parenchymatous
liver diseases
• Due to absence of bile salts, there may be
defective absorption of vitamin K, hence PT
is increased, as prothrombin formation
suffers
In obstructive
jaundice

28. Other clinical uses:
• Used mostly in controlling anticoagulant
therapy.
• Used to decide whether there is danger of
bleeding at operation in biliary tract diseases.

29. Prothrombin Index:
• Normally index is 70 to 100%.

30. DETERMINATION OF SERUM ENZYME ACTIVITIES
1. Aspartate transaminase
2. Alanine transaminase
3. Alkaline Phosphatase
4. Gamma GT
1. Serum 5’-Nucleotidase
2. Serum Lactate Dehydrogenase (LDH)
3. Serum Isocitrate Dehydrogenase (ICD)
4. Serum Cholinesterases
5. Serum ornithine carbamoyl transferase
(OCT)
6. Serum Leucine Amino Peptidase (LAP)
7. Serum SHBD (Hydroxy Butyrate
Dehydrogenase)
8. Serum Aldolase and Phosphohexose
Isomerase
9. Serum Amylase
10. Serum Sorbitol Dehydrogenase (SDH)

31. AST AND ALT
• Normal: 15-35IU/L
• Increased in hepatocellular injury.
Very high
Infective
hepatitis
Drug toxicity
Ischemic liver
injury
Malignant
infiltration of
liver
Moderate
elevation
Alcoholic
hepatitis
Autoimmune
hepatitis
Chronic active
hepatitis
Minor
elevation
Chronic
hepatitis C
Chronic
hepatitis B
NASH

32. AST/ALT (Normal=0.8), Ratio >2 is seen in
• Alcoholic hepatitis
• Hepatitis with cirrhosis
• Non alcoholic steatohepatitis
• Myocardial infarction
• Erythromycin treatment
ALT>AST is seen in
• Acute hepatocellular injury
• Toxic exposure
• Extrahepatic obstruction (cholestasis)

33. ALP and GGT:
• ALP=23-92 IU/L, GGT=10-47 IU/L
• Markers of cholestasis.
• Increased levels are seen in
– Infiltrating diseases of liver
– Bile duct obstruction due to gall stones or
carcinoma head of pancreas.
• It is better to estimate ALP+GGT+ALT to
confirm liver disease and/or obstruction.

35. 5’-Nucletidase:
• Normal range: 2-17IU/L
• Definite marker of cholestatic liver disease.
• Not affected in bone diseases.
Lactate Dehydrogenase:
 Normal range: 70-240 IU/L
 Increased in infectious hepatitis.

36. Serum isocitrate dehydrogenase
• Normal range: 0.9-4 IU/L
• Increased in liver diseases but not in
obstructive jaundice.
Serum cholinesterases:
• Normal range: 2-5 IU/ml
• This enzyme is formed in liver and serum
activity is reduced in liver cell damage

37. Serum ornithin carbamoyl
transferase:
• Normal =8 to 20 m-IU.
• The enzyme level is markedly elevated 10 to 200-
fold in patients with acute viral hepatitis
depending on the severity and also those with
other forms of hepatic necrosis.
• Relatively slight elevations occur in obstructive
jaundice, cirrhosis of the liver, metastatic
carcinoma, etc.
Serum OCT appears to be a specific and sensitive
measure for hepatocellular injury

38. Serum Leucine Amino Peptidase (LAP)
• Normal range is between 15 to 56 m-IU.
• In viral hepatitis shows mild-to-moderate
increase and ranges from 30.0 to 130.0 m-IU.
• In obstructive jaundice: Marked increase is
seen like alkaline phosphatase. Increase is
more in malignant obstruction than that of
benign obstruction.
• Marked rise has been seen in Liver cell
carcinoma (Hepatoma).

39. Serum SHBD (Hydroxy Butyrate
Dehydrogenase)
• Normal serum HBD between 56 to 125 IU/L
• In liver diseases elevated levels of this
enzyme is observed in acute viral hepatitis.
• Also elevated level is seen in myocardial
infarction

40. Ratio of LDH/SHBD
• To differentiate the liver diseases and acute
myocardial infarction, this ratio has been more
useful.
• Normal= 1.18 to 1.60
• Less than 1.18 is observed in most cases of
myocardial infarction.
• Greater than 1.60 is observed in liver
diseases.

41. Serum Aldolase and Phosphohexose
Isomerase
• These are both markedly increased in serum
of patients with acute hepatitis.
• No increase is found in cirrhosis, latent
hepatitis or biliary obstruction
Serum Amylase
• Studies have shown low serum amylase levels
in liver diseases like acute infectious hepatitis

42. Serum Sorbitol Dehydrogenase (SDH)
• Normal value= less than 0.2 m-IU
• Striking elevation seen in acute viral hepatitis
and carbon tetrachloride poisoning up to 17
m-IU.
• In chronic hepatitis and in obstructive
jaundice: Serum levels of SDH are normal or
only slightly elevated

43. TESTS BASED ON LIVER’S PART IN
CARBOHYDRATE METABOLISM
Galactose Tolerance Test
Fructose Tolerance Test
Epinephrine Tolerance Test

44. • Oral Galactose Tolerance
Test
• IV Galactose Tolerance
Test
Galactose
Tolerance
Test

45. Oral Galactose Tolerance Test
The test is performed in the morning after a
night’s fast.
A fasting blood sample is collected which
serves as “control”. 40 gm of galactose
dissolved in a cup-full of water is given orally.
Further blood samples are collected at ½
hourly intervals for two hours (similar to GTT)

46. Interpretations:
• 3 gm or less of galactose are
excreted in the urine within 3
to 5 hours and the blood
galactose returns to normal
within one hour.
Normal/
obstructive
Jaundice
• The excretion amounts to 4 to 5 gm
or more during the first five hours.
Intrahepatic
(Parenchymatous)
jaundice

47. IV Galactose Tolerance Test
The test is performed in the morning after a night’s
fast.
A fasting blood sample is collected which serves as
“control”. An IV injection of galactose, equivalent
to 0.5 gm/kg body weight is given as a sterile 50
per cent solution.
Blood samples are collected after five minutes, ½
hour, 1 hour, 1½ hours, 2 hours and 2½ hours after
IV injection and blood galactose level is estimated.

48. Interpretations:
• should have a curve
beginning on the average at
about 200 mg galactose/100
dl, falling steeply during the
one hour and reaching a
figure between 0 to 10 mg%
by end of 2 hours
A normal
response/Obstructive
Jaundice
• value is greater than 20 mg/dl
In parenchymatous
diseases

49. Fructose Tolerance Test
50 gm of fructose given to the fasting
patient as for GTT.
Fasting blood sugar is estimated
and blood sugar is estimated in
samples is taken at ½ hourly
intervals for 2½ hours after
taking the oral fructose.

50. Interpretations:
• shows little or no rise in the blood
sugar level.
• The highest blood sugar value reached
during the test should not exceed the
fasting level by more than 30 mg%.
Normal/
obstructive
Jaundice
• Rise in blood sugar is greater than
above, but the increases obtained
are never very great
In infectious
hepatitis and
parenchymatous
liver cells
damage:

51. Epinephrine Tolerance Test
The patient is kept on a high carbohydrate diet
for three days before the test.
After an overnight fast, the fasting blood sugar is
determined. 0.01 ml of a 1 in 1000 solution of
epinephrine per kg body weight in injected.
The blood sugar is then determined in samples
collected at 15 minutes intervals up to one hour.

52. Interpretations:
• in the course of an hour, the rise in
blood sugar over the fasting level
exceeds by 40 mg% or more.
Normal
• the rise is less.
In
parenchymal
hepatic
diseases

53. TESTS BASED ON THE
DETOXICATING FUNCTION OF
THE LIVER
HIPPURIC ACID TEST

54. Hippuric acid test:
• Depends on two factors:
1. Ability of the liver cells to produce and
provide glycine.
2. The capacity of liver cells to conjugate it with
benzoic acid.
For reliable result, renal function must be
normal.

55. •Oral Hippuric acid
test
•IV Hippuric acid test
Methods

56. Oral Hippuric Acid Test: [Procedure]
The patient empties the bladder, the urine
being discarded
The patient is allowed to drink sodium
benzoate (6gm in 200 ml of water) and time is
noted
The bladder is emptied 4 hours later. Any urine
passed during the 4 hours is kept and added to
that passed at the end of 4 hours
The amount of hippuric acid excretes in this 4
hours period is estimated

57. Interpretations:
• at least 3.0 gm of hippuric
acid, expressed as Benzoic
acid or 3.5 gm of sodium
benzoate should be excreted
in health
Normal
• Smaller amounts are found when
there is either acute or chronic liver
damage. Amounts lower than 1.0
gm may be excreted by patients
with infectious hepatitis
Liver
diseases

58. IV Hippuric Acid Test:
Indication:
1. When there is impairment of absorption.
2. If there is accompanying nausea/vomiting.

59. Procedure:
1.77 gm of sodium benozate dissolved in
20 ml of DW as a sterile solution given IV
Shortly before the injection, the patient
empties the bladder, which is discarded.
The bladder is emptied after one hour
and two hours after the injection.

60. Interpretations:
• hippuric acid equivalent to at least
0.85 gm of sodium benzoate, or to
0.7 gm of benzoic acid should be
excreted in the one hour, or
equivalent to 1.15 gm of benzoic
acid in the first two hours
Normal
• Excretion of smaller amounts than
above indicate the presence of liver
damage
Liver
damage

61. TESTS BASED ON EXCRETORY
FUNCTION OF LIVER
BSP Retention Test
(Bromsulphthalein Test)
Rose-Bengal Dye Test
Bilirubin tolerance test

62. BSP Retention Test
• The ability of the liver to excrete certain dyes,
e.g. BSP is utilised in this test.
• In normal healthy individual, a constant
proportion (10 to 15% of the dye) is removed
per minute.
• Removal of BSP by the liver involves
conjugation of the dye as a mercaptide with
the cysteine component of glutathione----rate
limiting.

63. Procedure:
With the patient fasting, inject IV slowly,
an amount of 5 per cent BSP solution,
which contains 5 mg of BSP/Kg, body
weight.
Withdraw 5 to 10 ml of blood, 25 and 45
minutes after the injection and allow the
specimens to clot.
Separate the sera and estimate the
amount of the dye in each sample.
The test is of no value if obstruction of biliary tree exists (obstructive jaundice).

64. Interpretations:
• In normal healthy individual not more than 5
per cent of the dye should remain in the
blood at the end of 45 minutes.
• The bulk of the dye is removed in 25 minutes
and less than 15 per cent is left at the end of
25 minutes.
Normal
• In Parenchymatous liver diseases removal
proceeds more slowly.
• In advanced cirrhosis removal is very slow
and 40 to 50 per cent of the dye is retained in
45 minutes sample
Liver
disease

65. Clinical Significance:
• Useful index of liver damage
• Most useful in liver cell damage without
jaundice
• Most useful in cirrhosis of the liver
• Most useful in chronic hepatitis

66. Rose-Bengal Dye Test
• Rose-Bengal is another dye which can be used
to assess excretory function.
• 10 ml of a 1 per cent solution of the dye is
injected IV slowly.
• Interpretation: Normally 50 per cent or more
of the dye disappears within 8 minutes

67. I131-labelled Rose-Bengal
• I131-Rose-Bengal has been used where isotope
laboratory is present.
I131-labelled Rose-Bengal is administered
IV.
Then count is taken over the neck and
abdomen. Initially, count is more in neck
and practically nil over abdomen.
As the dye is excreted through liver, neck
count goes down and count over
abdomen increases

68. Interpretation:
• In parenchymal liver diseases high count in
the neck persists and there is hardly a rise in
count over abdomen, as the dye is retained.

69. Bilirubin tolerance test:
• 1 mg/kg body weight of bilirubin is injected IV.
• If more than 5 per cent of the injected bilirubin
is retained after 4 hours, the excretory and
conjugating function of the liver is considered
abnormal.
• But the test is not used routinely and
extensively due to its high cost

70. TESTS BASED ON AMINO ACID
CATABOLISM
Determination of blood NH3
Determination of glutamine in
CS Fluid
(An Indirect Liver Function Test)

71. Determination of blood NH3:
• Produced by transamination and deamination.
Other sources
• NH3 is formed from nitrogenous material by
bacterial action in the gut.
• In kidneys, by hydrolysis of glutamine by
glutaminase.
• From pyrimidines catabolism.

72. Interpretations:
• 40 to 75 μg ammonia nitrogen per 100 ml of
blood
Normal
Range
• Increases in NH3 can be found in more
advanced cases of cirrhosis of the liver,
particularly when there are associated
neurological complications. In such cases
blood levels may be over 200 μg/100 ml.
• Very high values may be obtained in hepatic
coma.
In
parenchymal
liver diseases

73. Determination of Glutamine in CS
Fluid:
• Glutamine in CS fluid can be estimated by the
method of Whittaker (1955).
• The glutamine is hydrolysed to glutamic acid
and NH3 by the action of dilute acid at 100
degree centigrade.
• A correction is made for a small amount of
NH3 produced from urea.

74. Interpretation:
• 6-14mg%
Normal range
• found is range from 16 to 28 mg%, but is
usually less than 30 mg%.
In infectious
hepatitis
• the increase is more; depending on the
severity. It varies from 22 to 36 mg% or more
In cirrhosis of
the liver
In hepatic
coma
increase is very high, ranging from 30 to 60 mg% or
more.

75. TESTS BASED ON DRUG
METABOLISM
MEGX Test
Antipyrine breath
test

76. Monoethyl Glycine Xylidine (MEGX)
Test:
• Lidocaine metabolite formation has been
used as an index of hepatic function.

77. Procedure:
An IV bolus injection of a small
lidocaine test dose, 1 mg/ kg, is
given
Blood sample is taken before the
injection. Another blood sample is taken
15 or 30 minutes after the injection.
MEGX is determined in the serum by use
of an automated fluorescence
polarisation immuno-assay within about
20 minutes in both the samples

78. Interpretation:
• The highest MEGX test results are observed in liver donors
with unimpaired organ function and in normal healthy
subjects.
• Liver recipients with uncomplicated postoperative course
show somewhat lower test results.
• In patients with cirrhosis of the liver, the increase of MEGX
concentration in serum is much less marked and decrease
value is dependant on disease severity

79. Antipyrine breath test:
• Antipyrine like lidocaine is also metabolised
by cytochrome P450 system.
• When given orally it is absorbed from
intestine completely, not bound to plasma
proteins and metabolised by liver only.
Procedure:
• C14-labelled aminopyrine (dimethyl amino antipyrine)
is given orally in dosage of 1 to 2 microcurie.
• Breath samples are collected for 2 and 24 hours and
analysed.

80. Interpretation:
• excrete 5 to 8 per cent of
the administered dose in
2 hours.
Normal
subjects
• excretes only 2 to 3 per
cent.
Patients with
hepatitis and
cirrhosis

81. TESTS BASED ON
ABNORMALITIES OF LIPIDS
Cholesterol-Cholesteryl
Ester Ratio
Determination of faecal fats

82. Cholesterol-Cholesteryl Ester Ratio
• 150-250 mg/dl
Normal range
• an increase in total blood cholesterol is common,
but the ester fraction is also raised, so that %
esterified does not change.
Obstructive
Jaundice
• there is either no rise or even decrease in total
cholesterol and the ester fraction is always
definitely reduced.
In parenchymatous
liver diseases
In severe acute
hepatic necrosis
•the total serum cholesterol is usually low and may fall
below 100 mg/dl, whilst there is marked reduction in
the % present as esters.

83. TESTS FOR METABOLIC LIVER
DISEASE (SPECIAL TESTS)
• Ceruloplasmin
• Ferritin and Iron
• Alpha-1 antitrypsin
• Beta-2 microglobulin
• Alpha fetoprotein (AFP)
MARKERS OF HEPATIC FIBROSIS
• Serum hyaluronic acid
• Procollagen type 1carboxy
terminal peptide (PICP)
• Procollagen type III amino
terminal peptide (PIIINP)
• Matrix metalloproteinases
(MMPs)
• Tissue inhibitors of MMPs
(TIMPs)
• Transforming growth factor
beta-1 (TGF Beta-1)