LIVER FUNCTION TESTS

Dr. Prabha S.P

Dept. of Biochemistry
PIMS & RC
 THE LIVER

Largest organ in Human Body weighing 1.2 to 1.5 Kg in adult.

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 FUNCTIONS OF LIVER

Metabolic Excretory Synthetic

function function function

Detoxification Storage
function function
1.Synthetic function
a. Synthesis of plasma proteins (albumin, coagulation
factors, many globulins)
b. Synthesis of cholesterol
c. Synthesis of triacylglycerol
d. Lipoprotein synthesis
2. Metabolic function
a. Carbohydrates: Glycolysis; glycogen synthesis; glycogen
metabolism
b. Ketogenesis; fatty acid synthesis and breakdown
c. Protein catabolism
d. Citric acid cycle, production of ATP
Major Functions of Liver
3. Detoxification and excretion
a. Ammonia to urea
b. Bilirubin (bile pigment)
c. Drug metabolites
4. Homeostasis: Blood glucose regulation
5. Storage function: Vitamin A, D, K, B12
6. Production of bile salts; help in digestion
Indications for Liver Function Tests
1. Jaundice
2. Suspected liver metastasis
3. Alcoholic liver disease
4. Any undiagnosed chronic illness
5. Annual check up of diabetic patients
6. Coagulation disorders
7. Therapy with statins to check hepatotoxicity
Classification of Liver Function Tests

• Group I (Tests of hepatic excretory function)

• i. Serum: Bilirubin; total, conjugated, and
unconjugated
• ii. Urine: Bile pigments, bile salts and urobilinogen

• Group II: Liver enzyme panel

• i. Alanine aminotransferase (ALT) (Marker of liver
injury)
• ii. Aspartate aminotransferase (AST) (Marker of
liver injury)
• iii. Alkaline phosphatase (ALP) (Marker of
cholestasis)
• iv. Gamma glutamyl transferase (GGT) (Marker of
cholestasis)
Classification of Liver Function Tests

Group III: Plasma proteins (Tests for synthetic function of

liver)
(Marker, chronic liver diseases)
i. Total proteins
ii. Serum albumin, globulins, A/G ratio
iii. Prothrombin time
iv. Blood ammonia
Classification of Liver Function Tests CONTINUED

Group IV: Special tests (Tests for metabolic liver disease)

i. Ceruloplasmin
ii. Ferritin and iron
iii. Alpha-1 antitrypsin (AAT)
iv. Beta-2 microglobulin (b2M)
v. Alpha fetoprotein (AFP)

Group V: Direct and indirect markers of hepatic fibrosis

i. Serum hyaluronic acid (SHA)
ii. Procollagen type I carboxy terminal peptide (PICP)
iii. Procollagen type III amino terminal peptide (PIIINP)
iv. Matrix metalloproteinases (MMPs)
v. Tissue inhibitors of MMPs (TIMPs)
vi. Transforming growth factor beta-1 (TGF Beta-1)
B. Classification based on Clinical aspects
Group I: Markers of liver dysfunction
i. Serum bilirubin, total, conjugated
ii. Urine: Bile pigments, bile salts and UBG
iii. Total protein, serum albumin and A/G ratio
iv. Prothrombin time
v. Blood ammonia, when indicated

Group II: Markers of hepatocellular injury

vi. Alanine amino transferase (ALT)
vii. Aspartate amino transferase (AST)

Group III: Markers of cholestasis

viii. Alkaline phosphatase
ix. Gamma glutamyl transferase
Laboratory Findings in Serum in Jaundice Cases

Hemolytic Obstructive Hepatocellular

jaundice jaundice jaundice
Total bilirubin Elevated Elevated Elevated
Conjugated Normal Elevated Elevated
bilirubin
Unconjugated Elevated Normal Elevated
Van den Bergh Indirect +ve Direct +ve Biphasic
reaction
Laboratory Findings in Urine in Jaundice Cases

Hemolytic Obstructive Hepatocellular

jaundice jaundice jaundice
Bile pigments Absent +++ ++
Bile salts Absent ++ +
Urobilinogen Elevated Absent Normal or decreased
Positive test Ehrlich’s Fouchet’s +ve Hey’s +ve
+ve
Measurement of Bilirubin
• Bilirubin is estimated by van den Bergh reaction, where
diazotised sulfanilic acid (sulfanilic acid in HCl and
sodium nitrite) reacts with bilirubin to form a purple-
colored complex, azobilirubin.
• Normal serum bilirubin level varies from 0.2 to 0.8
mg/dl.
• The unconjugated bilirubin (bilirubin-albumin complex)
(free bilirubin) (indirect bilirubin) varies from 0.2–0.7
mg/dl and conjugated bilirubin (direct bilirubin) 0.1–0.4
mg/dl.
• When bilirubin is conjugated, the purple color is
produced immediately on mixing with the reagent, the
response is said to be van den Bergh direct positive.
• When the bilirubin is unconjugated, the color is obtained
only when alcohol is added, and this response is known
as indirect positive.
• If both conjugated and unconjugated bilirubins are
present in increased amounts, a purple color is produced
immediately and the color is intensified on adding
alcohol.
• Then the reaction is called biphasic.
• In hemolytic jaundice, unconjugated bilirubin is increased.
• van den Bergh test - Indirect positive.
• In obstructive jaundice, conjugated bilirubin is elevated, and
van den Bergh test is direct positive.
• In hepatocellular jaundice, a biphasic reaction is observed,
because both conjugated and unconjugated bilirubins are
increased.
Urinary Bilirubin
• In all cases of jaundice, urine should be examined for the
presence of bile pigments (bilirubin), bile salts and
urobilinogen.
• Only conjugated bilirubin is soluble in water and is excreted in
urine.
• Hence in prehepatic jaundice, when the unconjugated bilirubin is
increased in blood, it does not appear in urine; hence called
acholuric jaundice.
• But in obstructive jaundice, conjugation of bilirubin is taking
place, which cannot be excreted through the normal passage, and
so it is regurgitated back into bloodstream; this is then excreted
through urine.
• So in obstructive jaundice, urine contains bilirubin; it is called
choluric jaundice.
Urinary Urobilinogen
• In cases of obstruction, bile is not reaching the intestine and so
urobilinogen may be decreased or absent in urine.
• In hepatocellular jaundice, urobilinogen is initially elevated,
then decreases or disappears when the obstructive stage sets in
and reappears when obstruction is cleared.
• Urobilinogen is absent in urine, when there is obstruction to bile
flow.
• The first indication of the recovery is the reappearance of
urobilinogen in urine.
• In hemolytic anemias, urobilinogen is increased.
• Bilirubin is detected by Fouchet's test and urobilinogen by
Ehrlich's test.
Urine Bile Salts
• Normally bile salts (sodium salts of taurocholic acid
and glycocholic acid) are present in the bile; but are
not seen in urine.
• Bile salts in urine are detected by Hay’s test.
• Positive Hay’s test indicates the obstruction in the
biliary passages causing regurgitation of bile salts
into the systemic circulation leading to its excretion in
urine.
Classification Of Jaundice
Type of Bilirubin Class of Jaundice Causes

Abnormal RBC,
Unconjugated Pre Hepatic or Drugs,Toxins
Hemolytic

Unconjugated and Hepatic or Viral or Toxic Hepatitis

Conjugated Hepatocellular Intrahepatic cholestasis

Conjugated Posthepatic or Obstructive Gall Stones, Tumors of Bile

Duct or Pancareas,

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 Tests based on excretory function
Dye excretion test

• Bromosulphthalein [BSP] test

• Nontoxic, almost exclusively excreted by the liver
• 5mg/kg body weight I.V
• Serum level at 45min and 2hours
• Less than 5% dye retained at the end of 45min
Urinary bilirubin

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Tests b/o detoxification function

Hippuric acid
• Collect Benzoic acid +
testfor next 4 hour
urine
• 6gm of sodium benzoate Glycine = HA
dissolved in 250ml water • 6g Sod. Bezoate → 7.5g
• Collect urine for next 4 hour HA
• In healthy person →
4.5g HA excrete in urine
(≈ 60%)
• <3g HA excretion →
hepatic damage
Tests useful to Distinguish Different Types of Jaundice

Speci-men Test Prehepatic or Hepatocellular jaundice Posthepatic or

hemolytic or obstructive or
retention regurgitation
jaundice jaundice

Blood Unconjugated ++ + Normal

bilirubin (van
den Bergh
indirect test)

Blood Conjugated Normal Excretion is rate-limiting. ++

bilirubin (van It is the first impaired
den activity. In early phase, it
Bergh direct is increased
test)

Blood Alkaline Normal 2–3 times increased 10–12 times

phosphatase increased
(40–125 U/L)

Continued
Tests useful to Distinguish Different Types of Jaundice

Speci- Test Prehepatic / Hepatocellular jaundice Posthepatic/

men hemolytic or obstructive /
retention regurgitation
jaundice jaundice
Urine Bile salt Absent Absent Present
(Hay’s test)
Urine Conjugated Absent Present Present
bilirubin
(Fouchet’s)
Urine Urobilinoge +++ Increased in early Absent
ns (Ehrlich phases; later decreased
test) as production is low.

Feces Urobilins ++ Normal or decreased Clay-colored

Classification of Jaundice

Type of Class of Causes

bilirubin jaundice

Unconjugated Prehepatic or Abnormal red cells; antibodies; drugs

hemolytic and toxins; thalassemia;
hemoglobinopathies Gilbert’s
syndrome; Crigler-Najjar syndrome
Unconjugated Hepatic or Viral hepatitis; toxic hepatitis;
and hepatocellular intrahepatic cholestasis
conjugated

Conjugated or Posthepatic Extrahepatic cholestasis; gallstones;

obstructive tumors of bile duct; carcinoma of
pancreas; lymph node enlargement in
porta hepatis
Bilirubin Metabolism
Comparison of Different Types of Jaundices
Serum Albumin Level
• All plasma proteins except immunoglobulins are
synthesized by the liver.
• Serum albumin is quantitatively the most important
protein synthesized by the liver, and reflects the extent
of functioning liver cell mass.
• Since albumin has a fairly long half-life of 20 days, in
all chronic diseases of the liver, the albumin level is
decreased.
• A reversal in A/G ratio is often the rule in
cirrhosis, due to hypoalbuminemia and associated
hypergammaglobulinemia.
• Normal albumin level in blood is 3.5 to 5 g/dl;
and globulin level is 2.5 to 3.5 g/dl.
• The turn-over rates of haptoglobin and transferrin
are lesser than albumin; hence they are useful to
identify the recent changes in liver functions.
Serum Globulins
• Immunoglobulins are produced by B lymphocytes,
• Alpha and beta globulins synthesized mainly by
hepatocytes.
• Gamma globulins increased in chronic liver diseases
(chronic active hepatitis, cirrhosis)
Other Tests Based on Synthetic Function
• Prothrombin time (PT)
• Alpha-fetoprotein (AFP)
• Ceruloplasmin (Cp)
• Transthyretin (Prealbumin)
• Alpha-1 antitrypsin (AAT)
• Haptoglobin
Tests based on Serum Enzymes (Liver Enzyme Panel)

• The enzymes used in the assessment of hepatobiliary

disease may be divided into two groups:
(a) Those indicating hepatocellular damage and
(b) Those indicating cholestasis (obstruction).
Enzymes Indicating Hepatocellular Damage
• Normal serum ALT (alanine amino transferase) is
10-35 IU/L.
• The levels of amino transferases (ALT and AST) in serum are
elevated in all liver diseases.
• Very high levels (more than 1000 units) are seen in acute
hepatitis (viral and toxic).
• The degree of elevation may reflect the extent of hepatocellular
necrosis.
• Lowering of the level of transaminases indicates recovery, but a
sudden fall from a very high level may indicate poor prognosis.
• Elevation of ALT is more in cases of hepatic disease compared to
AST.
• AST > ALT in alcoholic liver disease.
• In alcoholic liver disease, the actual values show only mild elevation;
but a ratio of AST/ALT more than 2 is quite suggestive.
• Moderate elevation of amino transferases often between 100-300
U/L is seen in alcoholic hepatitis, autoimmune hepatitis, Wilson’s
disease and nonalcoholic chronic hepatitis.
• Minor elevation less than 100U/L is seen in chronic viral hepatitis
(hepatitis C), fatty liver and in nonalcoholic steatohepatitis (NASH).
Clinical Significance of AST/ALT Ratio
Normal AST: ALT ratio is 0.8. A ratio >2 is seen in:
o Alcoholic hepatitis
o Hepatitis with cirrhosis
o Nonalcoholic steatohepatitis (NASH)
o Liver metastases
o Myocardial infarction
o Erythromycin treatment

ALT higher than AST is seen in:

o Acute hepatocellular injury
o Toxic exposure
o Extrahepatic obstruction (cholestasis)
Algorithm for Diagnosis of Liver Diseases
Markers of Obstructive Liver Disease
1. Alkaline Phosphatase (ALP)
• Very high levels of alkaline phosphatase (ALP) are noticed in
patients with cholestasis or hepatic carcinoma.
• The bile duct obstruction induces the synthesis of the enzyme by
biliary tract epithelial cells.
• In parenchymal diseases of the liver, mild elevation of ALP is
noticed.
• In hepatitis, inflammatory edema produces an obstructive
phase, during which ALP level is elevated.
• Very high levels of ALP (10-12 times of upper limit) may be
noticed in extrahepatic obstruction (obstructive jaundice) caused
by gallstones or by pressure on bile duct by carcinoma of head of
pancreas.
• Intrahepatic cholestasis may be due to virus (infective hepatitis)
or by drugs (chlorpromazine).
• ALP is produced by epithelial cells of biliary canaliculi and
obstruction of bile with consequent irritation of epithelial cells
leads to secretion of ALP into serum.
• Drastically high levels of ALP (10-25 times of upper limit)
are seen in bone diseases where osteoblastic activity is enhanced.
o Paget's disease (osteitis deformans )
o Rickets
o Osteomalacia
o Osteoblastoma
o Metastatic carcinoma of bone and
o Hyperparathyroidism.
• There are 6 iso-enzymes for ALP. The one, which is inhibited by
phenylalanine is of placental origin.
• It is found in blood in normal pregnancy.
• An iso-enzyme closely resembling the placental form is
characteristically seen in circulation in about 15% cases of
carcinoma of lung, liver and gut and named as Regan iso-enzyme
or carcinoplacental iso-enzyme
Gamma Glutamyl Transferase (GGT)
• Detects alcohol abuse.
• GGT level in alcoholic liver disease roughly parallels the
alcohol intake.
• Elevated levels of GGT are seen in chronic alcoholism, pancreatic
disease, myocardial infarction, renal failure, chronic obstructive
pulmonary disease and in diabetes mellitus.
• In liver diseases, GGT elevation parallels that of ALP and is very
sensitive of biliary tract disease.
• 10-30 IU/L
Other Tests
• 5’ Nucleotidase (NTP)
• Glutathione S transferase (GST)
• Alpha feto protein (AFP) - Tumor marker
• Ammonia
• Immunoglobulins
• Auto antibodies
Clinical Manifestations of Liver Dysfunction
• Jaundice
• Portal Hypertension
a. Presinusoidal – e.g. portal vein thrombosis
b. Sinusoidal – cirrhosis
c. Postsinusoidal – hepatic vein thrombosis
• Ascites
Clinical features of liver failure
Liver
o Loss of metabolic function
o Decreased gluconeogenesis leading to hypoglycemia
o Decreased lactate clearance leading to lactic acidosis
o Decreased ammonia clearance leading to hyperammonemia
o Decreased synthetic capacity leading to coagulopathy
o Portal hypertension

Lungs: Adult respiratory distress syndrome

Adrenal gland: Inadequate glucocorticoid production contributing to hypotension

Brain
o Hepatic encephalopathy
o Cerebral edema
o Intracranial hypertension
Causes of Cholestatic Liver Causes of Cholestatic Liver
Disease Disease Continued

Extrahepatic cholestasis Intrahepatic cholestasis

o Cholelithiasis (stone in o Alcoholic cirrhosis
gallbladder) o Primary biliary cirrhosis
o Carcinoma head of o Nonalcoholic steatohepatitis (NASH)
pancreas o Viral hepatitis (cholestatic phase)
o Portal lymphadenopathy o Protoporphyria
o Chronic pancreatitis o Dubin-Johnson syndrome
o Biliary stricture
o Parasites (liver flukes) Drugs
(rare in India) o Androgens, chlorpromazine
o Chlorpropamide, nitrofurantoin
o Erythromycin, phenytoin
o Cyclosporin, captopril
Causes of Hepatocellular Damage
1. Viruses: HAV, HBV, HCV, herpes, adenovirus
2. Alcohol
3. Toxins: Carbon tetrachloride, chloroform, mushroom, aflatoxin,
arsenic
4. Immunological: Autoimmune hepatitis, NASH
5. General diseases: Wilson’s disease, hemochromatosis, AAT
deficiency, porphyrias, sarcoidosis, amyloidosis
6. Neoplasm: Hepatocellular carcinoma, metastatic liver disease,
lymphoma
7. Bacterial infections: TB, leptospirosis, brucella, abscesses
8. Parasites: Helminths, amoebiasis, plasmodia, leishmania
9. Drugs: Salicylate, tetracyclines, methotrexate, isoniazid,
rifampicin, halothane, methyldopa, valproate
Immunological Tests for Liver Diseases
IgG level is increased in chronic hepatitis, alcoholic and
autoimmune hepatitis. It shows a slow and sustained increase in
viral hepatitis. Ig M shows a marked increase in primary biliary
cirrhosis and a moderate increase in viral hepatitis and cirrhosis. Ig
A is increased in alcoholic cirrhosis and primary biliary cirrhosis.
Autoantibodies: Autoimmune chronic hepatitis is due to
defective suppressor T cells (a type pf immune cells that blocks
the actions of some other types of lymphocytes, to keep the
immune system from becoming over active) leading to the
production of autoantibodies against hepatocyte surface antigens.
Commonly encountered autoantibodies in hepatic autoimmune
disorders are: a) antinuclear antibodies b) antibody against
double-stranded DNA, c) anti-smooth muscle (actin) antibody,
d) asialoglycoprotein receptor autoantibody and e) anti-
mitochondrial antibody.
Liver Regeneration
• About 90% of liver can be removed and still the
remaining liver cells divide replacing the lost cells
within weeks.
• Lipocytes in liver store vitamin A and other retinoids.
• Hepatocytes, when damaged, release a protein which causes
lipocytes to be transformed into myofibroblast-like cells.
• Once activated, these are the sites for the synthesis of connective
tissue and extracellular matrix proteins.
• After severe liver injury, liver cells regenerate.
• But this is chaotic and may result in hepatic fibrosis.
• Repetitive injury leads to fibrosis, otherwise complete
regeneration occurs.