LIVER FUNCTION

TESTS Dr Mir Altaf Ahmad

MBBS, MD(PGI)
Associate Professor
Introduction
• Numerous laboratory investigations
have been proposed in assessment of
liver diseases
• Among these host of tests, the
battery of blood tests: Total bilirubin
and VD Bergh test, total and
differential proteins and A: G ratio and
certain enzyme assays as
aminotransferases, alkaline phosphatase and γ-GGT have become
widely known as Standard Liver Function Tests (LFTs)
• Urine tests for bilirubin and its metabolites and the
prothrombin time (PT) and index (PI) are also often included
under these headings
 Contd…
Second generation LFTs:
• Attempt to improve on this battery of tests
• To gain a genuine measurement of liver function, i.e.,
quantitative assessment of functional hepatic mass
• Include the capacity of the liver to eliminate
 Exogenous compounds such as aminopyrine or caffeine
 Endogenous compounds such as bile acids
 Have gained much importance recently.
 Contd…
• However, such investigations are useful for research purpose
only

• With the advent of sophisticated techniques like

 Ultrasound and CT scanning

 Together with percutaneous and endoscopic
cholangiography and liver biopsy

Routine use of standard LFTs is being questioned now

Functions of the Liver
• Liver is a versatile organ
• Involved as a part in metabolism
• Involved independently in many other biochemical functions
• Regenerating power of liver cells is tremendous

1. Metabolic Functions:
 Principal site where the metabolism of carbohydrates, lipids,
and proteins take place
 (Contd…)
 NH3 is converted to urea
 Principal organ where cholesterol is synthesised, and catabolised
to form bile acids and bile salts

 Esterification of cholesterol takes place solely in liver

 Absorbed monosaccharides other than glucose are converted to
glucose
 Brings about catabolism and anabolism of nucleic acids
 Involved in metabolism of vitamins and minerals to certain
extent
 (Contd…)
2. Secretory Function:
Formation and secretion of bile in the intestine
Bile pigments-bilirubin formed from haem catabolism is
conjugated in liver cells and secreted in the bile
3. Excretory Function:
 Exogenous dyes like BSP (bromsulphthalein)and Rose Bengal dye
are exclusively excreted through liver cells
4. Synthesis of Certain Blood Coagulation Factors:
 Conversion of preprothrombin (inactive) to active prothrombin
in presence of vit. K.
 (Contd…)
 Produces other clotting factors like factor V, VII and X, fibrinogen

5. Synthesis of Other Proteins:

 Albumin is solely synthesised in liver and also to some extent α-
and β-globulins

6. Detoxication Function and Protective Function:

 Kupffer cells of liver remove foreign bodies from blood by
phagocytosis
 (Contd…)
 Liver cells can detoxicate drugs, hormones and convert them into
less toxic substances for excretion

7. Storage Function:
Liver stores glucose in the form of glycogen
It also stores vit. B12, vit A, etc.

8. Miscellaneous Functions:
 Involved in blood formation in embryo
 In some abnormal states, it also forms blood in adult.
Classification of LFTs
 Tests are used in liver and biliary tract diseases
 Classified according to the specific functions of the liver involved:
I. Tests based on abnormalities of bile pigment metabolism:
• Serum bilirubin and VD Bergh reaction
• Urine bilirubin
• Urine and faecal urobilinogen
II. Tests based on liver’s part in carbohydrate metabolism:
• Galactose tolerance test
• Fructose tolerance test
 (Contd…)
III. Tests based on changes in IV. Tests based on abnormalities of
plasma proteins: lipids:
• Estimation of total plasma • Determination of serum
proteins, albumin and globulin cholesterol and ester
and determination of A:G cholesterol and their ratio
ratio. • Determination of faecal
• Determination of plasma fats
fibrinogen
V. Tests based on detoxicating
• Various flocculation tests function of liver:
• Amino acids in urine • Hippuric acid synthesis test.
 (Contd…)
VI. Excretion of injected catabolism:
substances by the liver • Determination of blood NH3
(excretory function):
• Determination of glutamine in
• Bromsulphthalein test (BSP CS fluid (Indirect liver
retention test) function test)
•I131-Rose Bengal test IX. Tests based on drug
VII. Formation of prothrombin metabolism:
by liver: • MEGX test
• Determination of prothrombin • Antipyrine breath test
time and index
X. Determination of serum
VIII. Tests based on amino acid enzyme activities
1. Tests Based on Abnormalities Of Bile
Pigment Metabolism
1. Van Den Bergh Reaction and Serum Bilirubin:
• Detecting and estimating bilirubin in serum
Principle:
 Bilirubin is allowed to react with a freshly prepared solution of
VD Bergh’s diazo-reagent
 Purple compound azo-bilirubin
Diazo-reagent:
• Consists of two solutions:
Solution A: Contains sulphanilic acid in conc. HCl.
 (Contd…)
Solution B: Sodium nitrite in water
Basis of the reaction:
Coupling of diazotised sulphanilic acid and bilirubin if present
produces a “reddish-purple” azo-compound.
VD Bergh Reaction:
 Bilirubin reacts differently with the diazo-reagent
 Depends on whether it has been conjugated or not
Unconjugated Bilirubin
 Water insoluble and does not react in aqueous solution
 (Contd…)
 Requires addition of methyl alcohol to react with diazo reagent
 So called indirect bilirubin

Conjugated bilirubin
 Water soluble
 Reacts directly with aqueous solution of diazo reagent
 So called direct bilirubin

Both conjugated and unconjugated bilirubin is present in increased

amounts
 Purple color is produced immediately
 Color is intensified by addition of alcohol
 Then, the reaction is called biphasic
 (Contd…)
Responses:
Three different responses may be observed:
i. Immediate direct reaction:
Immediate development of colour proceeding rapidly to a maximum.
ii. Delayed direct reaction:
Colour only begins to appear after 5 to 30 minutes and develops
slowly to a maximum
iii. Indirect reaction:
No direct reaction is obtained.
Colour develops after addition of methanol
 (Contd…)
A. Serum Bilirubin
 Measure of intensity of jaundice
 Higher values are found in obstructive jaundice than in
haemolytic jaundice
Usefulness of quantitative estimation of Bilirubin:
In subclinical jaundice:
 Small increases in serum bilirubin 1.0 to 3.0 mg/dl is of
diagnostic value
In clinical jaundice:
 Useful to follow the development and course of the jaundice.
 (Contd…)
Normal levels of Serum Bilirubin:
Total Bilirubin: 0.1 to 1 mg/dL
Direct Bilirubin: 0.1 to 0.4mg/dL
Indirect Bilirubin: 0.2 to 0.7 mg/dL
Interpretations:
A. Pre-hepatic/haemolytic jaundice:
o Increase in unconjugated bilirubin
o Indirect reaction is obtained
o Occasionally it may be a delayed direct reaction
 (Contd…)
B. Hepatic/ hepatocellular jaundice:
o Either or both may be present
o Biphasic
o Viral hepatitis, direct reaction is the rule: intrahepatic obstruction
o Cholestatic jaundice: immediate direct reaction
o Cirrhosis of the liver, results are variable, but an indirect reaction
is usually seen
C. Post-hepatic/obstructive jaundice:
o Conjugated bilirubin is increased
o Immediate direct reaction is obtained
 (Contd…)
B. Bile Pigments in Urine/Faeces (Bilirubinuria)
 Most tests detect oxidized forms of bilirubin like biliverdin (green) and
bilicyanin (blue)
 Bilirubin is detected by Fouchet's test
 Bilirubinuria is seen in “obstructive jaundice” and in “cholestasis”
 Bilirubinuria always accompanied with direct VD Bergh reaction
 In faeces bilirubin is not normally present as bacteria reduce it to
urobilinogen
 Sometimes it is found in faeces of very young infants
 Biliverdin is found in meconium, the material excreted during the first
day or two of life
 (Contd…)
C. Urinary and Faecal Urobilinogen
Faecal Urobilinogen:
 Normal quantity of urobilinogen excreted in the faeces per day is
from 50-250 mg
 Increased in haemolytic jaundice, in which dark-coloured faeces
is passed
 Decreased or absent in obstruction to flow of bile:
Obstructive jaundice: clay-coloured faeces is passed
 Decrease may also occur in extreme cases of diseases affecting
hepatic parenchyma
 (Contd…)
Urine Urobilinogen:
 Urobilinogen by Ehrlich's test
 Normally there are mere traces of urobilinogen in urine
 Average is 0.64 mg, maximum normal 4 mg/24 hours
 Presence of bilirubin in the urine, without urobilinogen is strongly
suggestive of obstructive jaundice either intrahepatic or post
hepatic
 Increased urobilinogen in urine and absence of bilirubin in urine
are strongly suggestive of haemolytic jaundice
 Increased urinary urobilinogen may be seen in damage to the
hepatic parenchyma when enterohepatic circulation suffers
2. Tests Based on Changes in
Plasma Proteins
Normal concentrations of serum proteins are given below:
• Total serum protein = 6 to 8 gm/dl
• Serum albumin = 3.5 to 5.5 gm/dl
• Serum globulin = 2 to 3.5 gm/dl
• Albumin/globulin ratio = 1.2:1 to 2.5:1
1. Determination of Total Plasma Proteins and Albumin and Globulin
and A:G Ratio:
 Site of albumin synthesis
 Some of α-and β-globulins
 Most useful information in chronic liver diseases
 (Contd…)
Interpretations:
 Infectious hepatitis early stage rise in β-globulins and in later
stages γ-globulins show rise
 Advanced parenchymal liver diseases and in cirrhosis liver the
albumin is grossly decreased and globulins are often increased
Increase is usually in γ-globulin fraction
 Serve as a criterion of the degree of damage
2. Estimation of Plasma Fibrinogen
 Normal value is 200-400 mg%
 (Contd…)
Values below 100 mg% have been reported in severe parenchymal
liver damage
o Acute hepatic necrosis
o Poisoning from carbon tetrachloride
o Advanced stages of liver of the cirrhosis
3. Amino acids in urine (Aminoaciduria):
 Daily excretion of amino acid nitrogen in normal health varies
from 80-300 mg
 Aminoaciduria found in severe liver diseases is of overflow type
 Accompanied by increase in plasma amino acids level
3. Tests Based on Changes in Formation of
Prothrombin
Prothrombin is formed in the liver from inactive “preprothrombin”
in presence of vitamin K
 Half life of prothrombin is 6 hours only
 Prothrombin activity is measured as prothrombin time (PT)
 PT indicates the present function of the liver
 Time required for clotting to take place in citrated plasma to
which optimum amounts of “thromboplastin” and Ca++ have been
added
 (Contd…)
 Related inversely to concentration of prothrombin, factors V, VII
and X
 PT is prolonged only when liver loses more than 80% of its
reserve capacity
Normal value:
• Normal levels of prothrombin in control give prothrombin time of
approx 14 seconds (Range: 10-16 Sec)
• Patient results are always expressed as prothrombin time in
seconds to normal control value.
 (Contd…)
o PT increases in advanced parenchymatous liver diseases and
obstructive jaundice from 22 to as much as 150 secs
Prothrombin index: Other of expressing Prothrombin activity

o Normally index is 70 to 100 per cent

4. Value of Serum Enzymes in Liver Diseases

• Quite a large number of enzyme estimations are available
 (Contd..)
Can be divided into 2 groups
I. Most commonly and routinely done in the laboratory:
A. Serum transaminases (amino transferases), and
B. Serum alkaline phosphatase.
II. Not routinely done in the laboratory
A. Serum 5’-Nucleotidase
B. Serum Lactate Dehydrogenase (LDH)
C. Serum Isocitrate Dehydrogenase (ICD
D. Serum Cholinesterases etc.
 (Contd..)
A. Serum Transaminases (Amino transferases)
Interpretations
Normal ranges for these enzymes are as follows:
1. SGOT/Serum glutamate oxaloacetate transaminase
AST/Aspartate Transaminase: 4 to 17 IU/L (7 to 35 units/ml)
2. SGPT/Serum Glutamate Pyruvate Transaminase
ALT/Alanine transaminase: 3 to 15 IU/ L (6 to 32 units/ml)
 Increases in both transaminases are found in liver diseases
 SGPT much higher than SGOT
 Skeletal Muscle/Heart muscles are richer in SGOT
 Liver contains both but more of SGPT
 (Contd..)
 AST may be more than ALT in alcoholic liver disease
 Normal AST: ALT ratio is 0.8.
 A ratio >2 is seen in Alcoholic hepatitis
 Have limited value in differential diagnosis of jaundice because of
considerable overlapping
 Assess severity and prognosis of parenchymal liver diseases especially
acute infectious hepatitis
 Useful as a screening test in outbreak of infectious hepatitis (viral
hepatitis)-in prodromal stage
 Such cases can be isolated and segregated from others
 (Contd..)

 Very high values are also obtained in toxic hepatitis

 Obstructive jaundice (extrahepatic) also increases occur, but
usually do not exceed 200 to 300 IU/L
 Hemolytic Jaundice normal

B. Serum Alkaline Phosphatase

o Found in several organs, mostly in bones and liver
o Then in small intestine, kidney and placenta
o Placental isoenzyme of alkaline phosphatase is heat-stable
 (Contd..)
Interpretations
 Normal range for serum ALP as per King-Armstrong method is 3 to
13 KA Units/100 ml (23 to 92 IU/L)

 Used for many years in differential diagnosis of jaundice

 Rise is usually much greater in cases of obstructive jaundice

 Bile duct obstruction induces the synthesis of enzyme by biliary

tract epithelial cells
 Dividing Line which has been suggested is 35 KA units/100 ml
 (Contd..)
 Higher values are also obtained in space-occupying lesions of liver,
e.g. Abscess
 Serum ALP is found to be normal in haemolytic jaundice
 Increase is due increased synthesis of hepatic ALP
 High ALP with low amino transferase activity is usual in cholestasis
and the converse occurs in non-cholestatic jaundice
II. Other Enzymes (not done routinely)
 Are useful but not routinely done in the laboratory
1. Serum 5’-Nucleotidase:
 Hydrolyses nucleotides with a phosphate group on carbon atom 5’ of
the ribose
 (Contd..)
 Produces adenosine and inorganic PO4

Interpretations
• Normal range is 2 to 17 IU/L
• Serum 5’ nucleotidase and serum ALP is raised in roughly parallel
manner
• Added advantage over serum ALP in that enzyme is not affected in bone
diseases
2. Serum Lactate Dehydrogenase (LDH):
• LDH enzyme is widely distributed
• Plentiful in cardiac and skeletal muscle, liver, kidney and the red blood cells
 (Contd..)
Interpretations
• Normal range is 70-240 IU/L
• Enzyme is less specific-widespread increase in seen in leukemias, pernicious
anaemia, megaloblastic and hemolytic anaemia
• In liver diseases increased activity is seen in infectious hepatitis but not so great
as that of the transaminases
3. Serum Isocitrate Dehydrogenase (ICD)
• Specific enzyme found in liver only
Interpretations
• Normal range is 0.9 to 4.0 IU/L
• Marked increase in ICD activity seen whether it is inflammatory like infectious
hepatitis, malignancy or from taking drugs
 (Contd..)
o Obstructive jaundice normal values are the rule

4. Serum Cholinesterases:
o Enzymes hydrolyse esters of choline to give choline and acid
o Two types have been distinguished:

(a) “True”: Neuromuscular junction

(b) “Pseudo”: Found in various tissues such as liver, heart muscle and intestine
o Present in plasma

Interpretations
• Normal range is 2.17 to 5.17 IU/ml
• Serum activity is reduced in liver cells damage
• Normal serum activity seen in obstructive jaundice cases
 (Contd..)
5. Serum γ-Glutamyl Transferase (γ-GT):
Normal range: 10 to 47 IU/L
• Importance of this enzyme in alcohol abuse has been stressed
• Microsomal enzyme
• Has been found to increase in most of hepatobiliary diseases
• Enzyme induction by drugs such as, phenobarbitone, phenytoin, warfarin and
alcohol
Only two, practical uses
(a) An elevated γ-GT implies that an elevated ALP is of hepatic origin, and
(b) Secondly, it may be useful in screening for alcohol abuse
• Sudden increase in γ-GT in chronic alcoholics suggests recent bout of drinking of
alcohols
 (Contd..)
6. Serum ornithine carbamoyl transferase (OCT):

o Exclusively found in liver

Interpretations:
 Normal healthy individuals are usually very low
 Ranges from 8 to 20 m-IU
 Markedly elevated 10 to 200-fold in patients with acute viral hepatitis
depending on the severity
 Slight elevations occur in obstructive jaundice
 Appears to be a specific and sensitive measure for hepatocellular injury
 (Contd..)
7. Serum Leucine Amino Peptidase (LAP)
 Proteolytic enzyme which splits off N-terminal residues
 N-terminal residue is leucine or related amino acid

Interpretations
• Normal range is between 15 to 56 m-IU
• In viral hepatitis shows mild-to-moderate increase
• Ranges from 30.0 to 130.0 m-IU
• Increases is also seen in cirrhosis of the liver
• In obstructive jaundice marked increase is seen like alkaline phosphatase
• Advantage is that LAP does not rise in osseous involvement.
 (Contd..)
8. SHBD (Serum Hydroxy Butyrate Dehydrogenase):
• Enzyme acting on α-OH butyric acid

Interpretations
• Normal serum HBD between 56 to 125 IU/L
• Elevated levels of this enzyme is observed in
Acute viral hepatitis
Myocardial infarction

Ratio of LDH/SHBD:
 (Contd..)
• Less than 1.18 is observed in most cases of myocardial infarction
• Greater than 1.60 is observed in liver diseases

9. Serum Sorbitol Dehydrogenase (SDH): hepatospecific enzyme

Interpretations
• Normal values for serum found to be less than 0.2 m-IU
• Striking elevation seen up to 17 m-IU in
Acute viral hepatitis
Carbon tetrachloride poisoning
• Chronic hepatitis and in obstructive jaundice levels are normal or only slightly
elevated
 (Contd..)

Enzyme
assays as
per
priorities
useful
in detecting
alterations
in liver
diseases
 5. Tests Based on Carbohydrate
Metabolism
1. Galactose Tolerance Test:
• Detect liver cell injury
• May be used to distinguish obstructive and non-obstructive jaundice
Normally or in obstructive jaundice:
• 3 gm or less of galactose are excreted in the urine within 3 to 5
hours after 40 g of oral test
Intrahepatic (Parenchymatous) jaundice:
• Excretion amounts to 4 to 5 gm or more during the first five hours
Infective, toxic hepatitis and cirrhosis
• Values up to about 30 gm are seen
 (Contd…)
2. Fructose Tolerance Test:
o 50 gm of fructose given to the fasting patient as for GTT

Normal response and obstructive jaundice cases:

o Highest blood sugar value reached during the test should not exceed the
fasting level by more than 30 mg%
Infectious hepatitis and parenchymatous liver cells damage:
o Rise in blood sugar is greater than above

3. Epinephrine Tolerance Test (Storage Function):

 Normally in an hour, rise in blood sugar over the fasting level exceeds by
40 mg% or more
 Parenchymal hepatic diseases the rise is less
6. Tests Based on Abnormalities of Lipids
Cholesterol-Cholesteryl Ester Ratio:
• Plays an active and important role in cholesterol synthesis, esterification, oxidation
and excretion
• Normal total blood cholesterol ranges from 150-250 mg/dl
• Approx. 60 to 70 per cent of this is in esterified form
Parenchymatous liver diseases:
o There is either no rise or even decrease in total cholesterol
o Ester fraction is always definitely reduced

obstructive jaundice:
o Increase in total blood cholesterol is common
o Ester fraction is also raised
o % esterified does not change
 (Contd…)
Severe Acute Hepatic Necrosis:
o Total serum cholesterol is usually low and may fall below 100 mg/dl
o Marked reduction in the % age present as esters

7. Tests Based on the Detoxicating Function of

the Liver:
Hippuric Acid Test of Quick:
Principle:
 Liver removes benzoic acid, administered as sodium benzoate, either orally or IV
 Combines with amino acid glycine to form hippuric acid
 Amount of hippuric acid excreted in urine in a fixed time is determined
 (Contd…)
Test thus depends on two factors:
 Ability of liver cells to produce and provide sufficient glycine
 Capacity of liver cells to conjugate it with benzoic acid
 For reliable result, renal function must be normal
Interpretations:
 Oral hippuric acid test 6.0 gm of sodium benzoate
 Normally, at least 3.0 gm of hippuric acid, expressed as Benzoic
acid or 3.5 gm of sodium benzoate should be excreted
 Smaller amounts are found in acute or chronic liver damage
 Lower than 1.0 gm may be excreted in infectious hepatitis.
 8. Tests Based on Excretory
Function of Liver
BSP Retention Test (Bromsulphthalein Test)
o Based on ability of the liver to excrete certain dyes
o Involves conjugation of dye as a mercaptide with the cysteine
component of glutathione
o Rate-limiting
o Normal healthy individual not more than 5 per cent of the dye
should remain in the blood at the end of 45 minutes
o Parenchymatous liver diseases removal proceeds more slowly
o Advanced cirrhosis removal is very slow: 40 to 50 per cent of the
dye is retained in 45 minutes sample.
 (Contd…)
Contraindication:
o Test is of no value if obstruction of biliary tree exists: (obstructive
jaundice)
Others are
I131-labelled Rose-Bengal
Bilirubin tolerance test
9. Tests Based on Amino Acid
Catabolism
1. Determination of blood NH3:
• Nitrogen part of amino acid is converted to NH3 in the liver mainly by
transamination and deamination
 (Contd…)
Interpretations:
Normal range:
• Blood ammonia varies from 40 to 75 μg ammonia nitrogen per 100
ml of blood
Parenchymal liver diseases:
• Blood levels may be over 200 μg/100 ml
2.Ammonia tolerance test:
Normal healthy persons:
 Little increase is found
 Blood NH3 levels remaining within normal range.
 (Contd…)
Advanced cirrhosis of liver:
 Marked rise to twice initial level or more, exceeding 200 to 300
μg% are seen
3. Determination of glutamine in CS Fluid (An Indirect
Liver Function Test)
Glutamine, the amide of glutamic acid, is formed by glutamine
synthetase by glutamic acid and NH3
Interpretations
Normal range found to be 6.0 to 14.0 mg%
 (Contd…)

Infectious hepatitis found is range from 16 to 28 mg

Cirrhosis of liver:
Increase is more
Varies from 22 to 36 mg% or more.
Hepatic coma:
Increase is very high
Ranging from 30 to 60 mg% or more.
 10. Tests Based on Drug Metabolism
Tests based on drug clearance or metabolite formation
 Kinetics reflect the actual functional state of the liver
Are called dynamic liver function tests

1. MEGX Test
Principle:

 Lidocaine is rapidly converted to its primary metabolite monoethyl glycine

xylidine (MEGX)
 Hepatic microsomal Cytochrome P450 system
 (Contd…)
 Loss of hepatic cytochrome P450 activity
 Major changes in hepatic blood flow (due to portosystemic
shunting)
…..result in decreased MEGX formation
Interpretations
• Highest MEGX test results are observed in normal healthy subjects
• Liver recipients with uncomplicated postoperative course show
somewhat lower test results
• Cirrhosis of liver, increase of MEGX concentration in serum is
much less marked
 (Contd…)
2. Antipyrine breath test:
Antipyrine like lidocaine is also metabolised by Cytochrome P450 system
Absorbed from intestine completely
C14-labelled aminopyrine
Breath samples are collected

Interpretations
• Normal subjects excrete 5 to 8 per cent of the administered dose in 2
hours.
• Patients with hepatitis and cirrhosis excretes only 2 to 3 per cent
Differentiation
of three types
of jaundice
Thank You