Briefing of Liver Function Test

1. Liver Function Tests
Dr. Nagaraj A Hosamani BNYS, MSc(Med.Biochem)

2. Liver Function Tests
• liver - chief metabolic organ of body
• Group of biochemical tests give different
information liver functions are known are
LIVER FUNCTION TESTS
• Liver function tests are useful in
– detecting the hepatocellular damage & presence
of liver disease
– monitoring therapy & prognosis
• tests are to be selected according to clinical
signs & symptoms

3. Major functions of Liver
• Synthetic function
– Synthesis of plasma proteins (albumin, coagulation factors, many globulins)
– Synthesis of cholesterol, triacyl glycerol, lipoprotein
• Metabolic function
– Carbohydrates : glycolysis, glycogenesis, glycogenolysis, gluconeogenesis
– Ketogenesis; fatty acid synthesis and breakdown
– Protein catabolism
• Detoxification and excretion
– Ammonia to urea
– Bilirubin (bile pigment)
– Cholesterol, drug metabolites
• Storage function : Vitamin A, D, K, B12
• Production of bile salts; help in digestion
• Formation of blood in embryo

4. Classification of Tests based on function
• Tests of hepatic excretory function
– Serum – Bilirubin; total, conjugated, and unconjugated
– Urine – Bile pigments, bile salts and urobilinogen
– Bromosulphthalein test
• Liver enzyme panel (Markers of liver injury/ Cholestasis)
– Alanine amino transferase (ALT)
– Aspartate amino transferase (AST)
– Alkaline phosphatase (ALP)
– Gamma glutamyl transferase (GGT)
• Tests for synthetic function of liver (Plasma proteins)
– Serum total proteins, albumin, globulins, A/G ratio
– Prothrombin time
• Tests for metabolic function: Galactose tolerance test
• Special tests
– Ceruloplasmin, Ferritin, Alpha-1-antitrypsin, Alpha-fetoprotein (AFP),
– Hippuric Acid Test

5. Important Tests based on Clinical aspects
• Serum bilirubin levels - total, conjugated,
unconjugated
• Total protein, serum albumin and A/G ratio
• Prothrombin time
• Serum Enzymes :
– Alanine amino transferase (ALT)
– Aspartate amino transferase (AST)
– Alkaline phosphatase (ALP)
• Blood Ammonia

6. Tests of hepatic excretory function
Serum bilirubin levels:
• Excretory end product of heme catabolism;Excreted through
bile
• Indicator of ability of liver to conjugate & excrete bilirubin
• Helps in differential diagnosis of jaundice
• Types in Blood:
– Conjugated – directed bilirubin
– Unconjugated – indirected bilirubin
• Normal serum bilirubin level: 0.2 – 1.0 mg/dl
– Conjugated bilirubin : 0.2–0.4 mg/dl
– Unconjugated bilirubin : 0.2–0.6 mg/dl
• Test for Bilirubin : van den Bergh reaction
– for measurement of Bilirubin in serum

7. Jaundice
• Yellowish discoloration of sclera, skin
& mucus membrane
– Due to elevation of serum bilirubin > 2 mg/dL
– & deposition in tissues
• Normal serum bilirubin level : < 1.0 mg/dl
• Types based on cause:
– Hemolytic/ Prehepatic jaundice
– Hepatic jaundice
– Obstructive/ Post hepatic jaundice
• Type can be determined
– based on the type of bilirubin elevated
– measured using van den Bergh reaction

8. Types of jaundice & Causes
• Pre- hepatic/ Hemolytic jaundice
– Excessive hemolysis overproduction of bilirubin beyond the ability
of liver to conjugate & excrete
– Unconjugated bilirubin is increased
– Causes: Abnormal red cells, antibodies, drugs & toxins, thalassemia,
hemoglobinopathies
• Hepatic/ Hepatocellular jaundice
– Damage to the liver cells leads to dysfunction
– Both conjugated & unconjugated bilirubin increases
– Causes: Viral hepatitis, cirrhosis, toxic hepatitis ( Chloroform, CCl4)
• Post hepatic/ Obstructive jaundice
– Obstruction in the bile duct
– Conjugated bilirubin is increased
– Causes: Gallstones, tumors of bile duct, carcinoma of pancreas

9. van den Bergh reaction
• Specific reaction to identify the increase in serum
bilirubin
– Normal serum gives a negative van den Bergh reaction
• Principle:
Diazotized sulfanilic acid + Bilirubin  Azobilirubin
( purple-colored complex)
• van den Bergh reagent/ Diazotized sulfanilic acid :
– sulfanilic acid in HCl and sodium nitrite
• Positive van den Bergh reaction can be
– Direct positive
– Indirect positive
– Biphasic

10. van den Bergh reaction
• van den Bergh direct positive reaction:
vdB reagent + Conj. bilirubin Azobilirubin
(purple color within 30 sec)
– High conjugated bilirubin- Soluble in water
– purple color produced immediately on mixing with the reagent
• van den Bergh indirect positive reaction:
vdB reagent + Unconj. bilirubin + Methanol Azobilirubin
(purple color within 30 min)
– High unconjugated bilirubin- insoluble in water, soluble in alcohol
– purple color produced after adding alcohol to the reaction mixture
• van den Bergh biphasic reaction:
vdB reagent + Conj. bilirubin Azobilirubin
(purple color within 30 sec)
vdB reagent + Unconj. bilirubin + Methanol  Azobilirubin
(purple color intensifies)
– Both conjugated & unconjugated bilirubin is increased
– Purple colour produced immediately & color intensifies after addition of
methanol (Direct +Indirect reaction)

11. van den Bergh reaction & Jaundice
• van den Bergh reaction
– Used to differentiate between different types of jaundice
• Indirect positive van den Bergh reaction
– Increased serum unconjugated bilirubin level
– Seen in Hemolytic jaundice
• Direct positive van den Bergh reaction
– Increased serum conjugated bilirubin level
– Seen in Obstructive jaundice
• Biphasic van den Bergh reaction
– Both serum conjugated & unconjugated bilirubin levels are
increased
– Seen in Hepatic/ hepatocellular jaundice

12. Urinary bilirubin
• Only conjugated bilirubin is soluble in water
• Detected by Fouchet’s test
• In Prehepatic jaundice
– Unconjugated bilirubin is increased & not excreted in urine
• In obstructive jaundice
– Conjugated bilirubin is increased in serum
– cannot be excreted through the normal passage
– it is regurgitated back into bloodstream
– Hence excreted in urine
• In hepatic jaundice
– Both Conjugated & unconjugated bilirubin is more& excreted in urine

13. Urinary Urobilinogen
• Urobilinogen detected by Ehrlich's test
• In obstructive jaundice
– bile is not reaching the intestine
– so urobilinogen may be decreased or absent in urine
– The first indication of the recovery is the reappearance of
urobilinogen in urine
• Hepatocellular jaundice
– urobilinogen is initially elevated
– then decreases when the obstructive stage sets in &
– reappears when obstruction is cleared
• Haemolytic jaundice
– urobilinogen is increased

14. Urinary Bile salts
• Bile salts:
– Sodium salts of taurocholic acid and glycocholic acid
• Normally bile salts are present in bile & absent in urine
• Bile salts in urine are detected by Hay’s test
• Positive Hay’s test indicates
– obstruction in the biliary passages
– causing regurgitation of bile salts into systemic circulation
– leading to its excretion in urine
• Obstruction can occur in
– obstructive jaundice
– hepatic jaundice due to obstruction of micro biliary channels
caused by inflammation

15. Features of different types of jaundice
Pre hepatic Hepatic Post hepatic
SERUM
Conjugated bilirubin Normal Increased Increased
Unconjugated bil. Increased Increased Normal
Van den Bergh
reaction
Indirect positive Biphasic Direct positive
URINE
Bilirubin (Conj.) Absent Present Present
Urobilinogen Increased Increased in early phase
Absent in late phase
Absent
Bile salts Absent Absent Present
FECES
Urobilins Increased Decreased Absent
(Clay colored)

16. Liver enzyme panel
• Liver cells contain several enzymes
• In case of cellular damage excess of enzymes enter
circulation
• Measurement in serum provides a reliable estimate of liver
function
• Liver enzyme panel includes,
– Aspartate transaminase (AST)
– Alanine transaminase (ALT)
– Alkaline phosphatase (ALP)
– Gamma glutamyl transferase (GGT)
– 5’- nucleotidase
• No enzyme is absolutely specific for liver function

17. Aminotransferases/ Transaminases
• These indicate hepatocellular damage
• Alanine transaminase (ALT)/ Serum glutamate pyruvate
transaminase (SGPT)
– Cytoplasmic enzyme
– Normal activity in serum: 5-40 IU/L
– More sensitive & reliable for LFT assessment
– Elevation of ALT is more than AST in most cases of hepatic disease
– Normal persons may have elevated ALT levels ( Obese persons)
• Aspartate transaminase (AST)/ Serum glutamate oxaloacetate
transaminase (SGOT)
– Cytoplasmic and mitochondrial enzyme
– Normal activity in serum: 5-45 IU/L
– AST may be more than ALT in alcoholic liver disease
• Estimation cannot identify causes for liver damage

18. Aminotransferases/ Transaminases
• Degree of elevation may reflect the extent of liver damage
• Very high levels (>1000 units)
– seen in acute hepatitis (viral and toxic)
• Moderate elevation of amino transferases (100-300 U/L)
– seen in alcoholic hepatitis, autoimmune hepatitis, Wilson’s disease
and nonalcoholic chronic Hepatitis
• Minor elevation (< 100U/L )
– seen in chronic viral hepatitis (hepatitis C), fatty liver and nonalcoholic
steatohepatitis (NASH)
• A normal value need not rule out minor liver diseases
• Usually, lowering of the level of transaminases indicates recovery

19. Alkaline phosphatase (ALP)
• Mainly derived from bone & cells lining the bile canaliculi
• Normal serum activity level: 3 -13 KA units/dL)
• Mild elevation of ALP is noticed
– In parenchymal diseases of the liver
• Very high levels are noticed in patients with cholestasis
– 10-12 times of upper limit
– caused by gallstones or by pressure on bile duct by carcinoma of
head of pancreas
– Specific Marker of obstructive liver disease
• Drastically high levels of ALP
– 10-25 times of upper limit
– seen in bone diseases where osteoblastic activity is enhanced
– Paget's disease (osteitis deformans ), rickets, osteomalacia

20. Gamma Glutamyl Transferase (GGT)
• Normal serum activity level: 10- 15 U/L
• GGT has high sensitivity to detect alcohol abuse
• GGT level in alcoholic liver disease roughly parallels the
alcohol intake
• Elevated levels of GGT are observed in
– chronic alcoholism, pancreatic disease, myocardial infarction, renal
failure, chronic obstructive pulmonary disease and diabetes mellitus
• In liver diseases, GGT elevation parallels that of ALP and is
very sensitive of biliary tract disease

21. Tests for synthetic function of liver
• Serum total proteins, albumin, globulins, A/G ratio, PT
• Most of the plasma proteins are synthesized by the liver
• Serum albumin
– Quantitatively, most important protein synthesized by the liver
– reflects the extent of functioning liver cell mass
– In all chronic diseases of the liver, the albumin level is
decreased
– A reversal in A/G ratio is often the rule in cirrhosis
• Normal serum albumin level: 3.5 to 5 g/dl
• Normal serum Globulin level: 2.5 to 3.5 g/dl
• Normal A:G ratio: 1.2 to 1.5: 1

22. Tests for synthetic function of liver
• Prothrombin time
– Prothrombin is synthesised by the liver
– The half-life of prothrombin is 6 hours only
– therefore, PT indicates the present function of the liver
– PT is prolonged only when liver loses more than 80% of its
reserve capacity
– Vitamin K deficiency is also a cause for prolonged PT
– In case of liver disease, the PT remains prolonged even after
parental administration of vitamin K

23. Other tests
• Tests for metabolic function:
- Galactose tolerance test
• Special tests:
- Ceruloplasmin
- Ferritin
- Alpha-1-antitrypsin
- Alpha-fetoprotein (AFP)
- Hippuric Acid Test