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The liver performs different kinds of biochemical, synthetic and excretory functions, so no single biochemical test can detect the global functions of the liver.

Liver function tests are a battery of tests for the initial detection and management of liver diseases.
Clinical history and physical examination play an important role to interpret the functions.

Uses of liver function tests

  1. Screening: They are a non-invasive yet sensitive screening modality for liver dysfunction.
  2. They are helpful to recognize the pattern of liver disease.
  3. Assess severity and predict the outcome of certain diseases like primary biliary cirrhosis.
  4. Follow up of certain liver diseases and also helpful in evaluating response to therapy like autoimmune hepatitis.

Limitation of liver function tests

  1. Lack of sensitivity:
    Liver Function Tests may be normal in certain liver diseases.
  2. Lack of specificity and are not specific for any particular disease.
    Except for serum bile acids, the Liver Function Tests are not specific for liver diseases and all the parameters may be elevated for pathological processes outside them.

Classification of liver function tests

A. Tests of the liver’s capacity to transport organic anions and to metabolize drugs
B. Tests that detect injury to hepatocytes. (serum enzyme tests)
C. Tests of the Liver’s biosynthetic capacity

A. Tests of the liver's capacity to transport organic anions and to metabolize drugs.

1. Serum bilirubin

Bilirubin is an endogenous anion derived from hemoglobin degradation from the RBC.
The classification of bilirubin into direct and indirect bilirubin is based on the original van der Bergh method of measuring bilirubin.
Bilirubin is altered by exposure to light so serum and plasma samples must be kept in dark before measurements are made.
When the liver function tests are abnormal and the serum bilirubin levels more than 17µmol/L suggest underlying liver disease.

Types of bilirubin

I. Total Bilirubin: This is measured as the amount, which reacts in 30 minutes after the addition of alcohol. Normal range is 0.2-0.9 mg/dl (2-15µmol/L). It is slightly higher by 3-4 µmol/L in males as compared to females.

II. Direct Bilirubin
This is the water-soluble fraction. Measured by the reaction with diazotized sulfanilic acid in 1 minute and this gives an estimation of conjugated bilirubin. Normal range 0.3mg/dl( 5.1µmol/ L).

III.Indirect Bilirubin
This fraction is calculated by the difference of the total and direct bilirubin and is a measure of the unconjugated fraction of bilirubin.

Diagnostic value of bilirubin levels.

Hyperbilirubinemia results from overproduction/impaired uptake, conjugation Increased conjugated bilirubin: Impaired intrahepatic excretion/regurgitation of unconjugated or conjugated bilirubin from hepatocytes of bile ducts.

Serum bilirubin could be LOWERED by drugs like;

  • Salicylates,
  • Sulphonamides,
  • Free fatty acids

Elevated if the serum albumin increases and the bilirubin shifts from tissue sites to circulation.

Prognostic value of bilirubin levels.
In fulminant hepatic failure, deep jaundice is associated with increased mortality.

2. Urine Bilirubin
The presence of urine bilirubin indicates hepatobiliary disease.
Unconjugated bilirubin is tightly bound to albumin and not filtered by the glomerulus and thus not present in urine.
conjugated bilirubin may be found in urine when the serum bilirubin levels are normal because the renal threshold for conjugated bilirubin is low

3. Urobilinogen
An increase in the urobilinogen in urine is a sensitive indicator of hepatocellular dysfunction.
A good indicator of alcoholic liver damage, well-compensated cirrhosis or malignant disease of the liver. It appears early in urine in viral hepatitis and markedly increased in hemolysis.

Urobilinogen gives a purple reaction to Ehrlich’s aldehyde reagent.
A dipstick containing this reagent allows rough and ready quantification.
Freshly voided urine should be used.

B. Tests that detect injury to hepatocytes( serum enzyme tests)

A. Enzymes detecting hepatocellular necrosis

Aminotransferases.

The aminotransferases or transaminases are specific indicators of hepatocellular necrosis.
These enzymes-

  • Aspartate aminotransferase(AST) /serum glutamate oxaloacetic transaminase-SGOT
  • Alanine aminotransferase( ALT)/serum glutamic pyruvate transaminase-SGPT)

They catalyze the transfer of the á amino acids of aspartate and alanine respectively to the á keto group of ketoglutaric acid.
ALT is primarily localized to the liver but the AST is present in a wide variety of tissues like the heart, skeletal muscle, kidney, brain, and liver.

AST : alanine + α ketoglutarate = oxaloacetate + glutamate
ALT: alanine + α ketoglutarate = pyruvate + glutamate

AST is present in both the mitochondria and cytosol of hepatocytes, ALT is localized to the cytosol.

The cytosolic and mitochondrial forms of AST are true isoenzymes and immunologically distinct.
Large increases in mitochondrial AST occur in serum after extensive tissue necrosis.

Mitochondrial AST is also increased in chronic liver disease.
Virtually no aminotransferases are present in the urine or bile.
Hepatic sinusoids are the primary site for their clearance.

Elevation of aminotransferases
1. Severe elevation ( > 20 Times, 1000 U/L) :
The AST and ALT levels are increased in almost all liver diseases. The highest elevations occur in severe viral hepatitis, drug or toxin-induced hepatic necrosis and circulatory shock.

2. Moderate elevation (3-20 Times): 
They are moderately elevated in acute hepatitis, neonatal hepatitis, chronic hepatitis, autoimmune hepatitis, drug-induced hepatitis, alcoholic hepatitis, and acute biliary tract obstructions.

The ALT is usually more frequently increased as compared to AST except in chronic liver disease.
In uncomplicated acute viral hepatitis, the very high initial levels approach normal levels within 5 weeks of the onset of illness and normal levels are obtained in 8 weeks.
Disproportionately low in patients with Wilson disease.

3. Mild elevation (1-3 Times) :
This is seen in sepsis-induced neonatal hepatitis, extrahepatic biliary atresia (EHBA), fatty liver, cirrhosis, non-alcoholic steatohepatitis(NASH), drug toxicity, myositis, Duchenne muscular dystrophy and even after vigorous exercise

AST: ALT Ratio

The ratio of AST to ALT is of use in Wilson disease, CLD, and alcoholic liver disease and a ratio of more than 2 is usually observed.

The lack of ALT rise is probably due to pyridoxine deficiency.

In NASH the ratio is less than one in the absence of fibrosis on liver biopsy.

In viral hepatitis, the ratio is usually less than one.

The ratio invariably rises to more than one as cirrhosis.

ALT exceeds AST in toxic hepatitis, viral hepatitis, chronic active hepatitis, and cholestatic hepatitis
Falsely low aminotransferase levels seen in patients on long term hemodialysis secondary to either dialysate or pyridoxine deficiency.

Low levels have also been seen in uremia

Other enzymes used to assess hepatocellular damage.

These include glutamate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase, and sorbitol dehydrogenase

B. Enzymes that detect cholestasis.

1. Alkaline Phosphatase

Alkaline phosphatases are a family of zinc metalloenzymes, with a serine at the active center; they release inorganic phosphate from various organic orthophosphates and are present in nearly all tissues.

In the liver, alkaline phosphatase is found histochemically in the microvilli of bile canaliculi and on the sinusoidal surface of hepatocytes.

Alkaline phosphatase from the liver, bone, and kidney are thought to be from the same gene but that from the intestine and placenta are derived from different genes.

In healthy people, most circulating alkaline phosphatase originates from liver or bone.
The reference method uses p- nitrophenol phosphate as a substrate, in an alkaline buffer.
Fresh unhemolysed serum is the specimen of choice for the estimation. Heparinized plasma may also be used.

The test should not be done on plasma if citrate, oxalate or EDTA were used as anticoagulants, they form a complex with zinc and the alkaline phosphatase, causing irreversible enzyme inactivation.

Interpretation

Average values with age and are relatively high in childhood and puberty and lower in middle age and higher again in old age.
Males usually have higher values as compared to females.
The highest levels of alkaline phosphatase occur in cholestatic disorders.

Elevations occur as a result of both intrahepatic and extrahepatic obstruction to bile flow and the degree of elevation does not help to distinguish between the two.

Alkaline phosphatase levels are likely to be very high in EHBA.

In acute viral hepatitis, alkaline phosphatase is usually either normal or moderately increased.
Hepatitis A may present a cholestatic picture with marked and prolonged itching and elevation of alkaline phosphatase.

Tumors may secrete alkaline phosphatase into plasma.

Elevated serum levels of intestinal alkaline phosphatase have been found in patients with cirrhosis, particularly those with blood group type O, and may be associated specifically with an intrahepatic disease as opposed to extrahepatic obstruction.

Hepatic and bony metastasis can also cause elevated levels of alkaline phosphatase.

Other diseases like infiltrative liver diseases, abscesses, granulomatous liver disease, and amyloidosis may also cause a rise in alkaline phosphatase.

Mildly elevated levels of alkaline phosphatase may be seen in cirrhosis and hepatitis of congestive cardiac failure.

Low levels of alkaline phosphatase occur in hypothyroidism, pernicious anemia, zinc deficiency, and congenital hypophosphatasia.

Wilson’s disease complicated by hemolysis and FHF may also have very low levels of alkaline phosphatase.

The ratio of alkaline phosphatase and bilirubin is low in fulminant Wilson disease.
This might be the result of the replacement of cofactor zinc by copper and subsequent inactivation of alkaline phosphatase regardless of the cause of acute hepatic failure a low ratio of alkaline phosphatase to bilirubin is associated with a poor prognosis

Drugs like cimetidine, frusemide, phenobarbitone, and phenytoin may increase levels of alkaline phosphatase.

2. γ Glutamyl transpeptidase
It is a membrane-bound glycoprotein that catalyzes the transfer of γ glutamyl group to other peptides, amino acids, and water.

Large amounts are found in the kidneys, pancreas, liver, intestine, and prostate.
The gene for γ glutamyl transpeptidase is on chromosome 22.
The levels of ã glutamyl transpeptidase are high in neonates and infants up to 1 yr and also increase after 60 yr of life.

Men have higher values. Children more than 4 yr old have serum values of normal adults.
The normal range is 0-30IU/L.

In acute viral hepatitis the levels of γ glutamyl transpeptidase may reach its peak in the second or third wk of illness and in some patients they remain elevated for 6 weeks.
In EHBA GGT is markedly elevated.

Gamma glutaryl transferase is raised only in cholestatic disorders and not bone diseases.
In liver disease, γ glutamyl transpeptidase activity correlates well with alkaline phosphatase levels but rarely the γ glutamyl transpeptidase levels may be normal in intrahepatic cholestasis like in some familial intrahepatic cholestasis.

Other conditions causing elevated levels of γ glutamyl transpeptidase include uncomplicated diabetes mellitus, acute pancreatitis, and myocardial infarction.
Drugs like phenobarbitone, phenytoin, paracetamol, tricyclic antidepressants may increase the levels of γ glutamyl transpeptidase.

Non-hepatic causes of increased levels of the enzyme include anorexia nervosa, Guillain barre syndrome, hyperthyroidism, obesity

As a diagnostic test, the primary usefulness of γ glutamyl transpeptidase is limited to the exclusion of bone disease, as γ glutamyl transpeptidase is not found in bone

These are the other enzymes that are not routinely estimated to detect cholestasis.

1. Serum proteins

The liver is the major source of most serum proteins. The parenchymal cells are responsible for the synthesis of albumin, fibrinogen and other coagulation factors and most of the a and b globulins.

Albumin is the most important protein in plasma synthesized by the liver and is a useful indicator of hepatic function.
Because the half-life of albumin in serum is as long as 20 days, the serum albumin level is not a reliable indicator of hepatic protein synthesis in acute liver disease.

The liver is the only site of synthesis of albumin. The serum levels are typically depressed in patients with cirrhosis and ascites.

In patients with or without ascites, the serum albumin level correlates with prognosis.
Normal serum values range from 3.5g/dl to 4.5 g/dl. The average adult has approximately 300 to 500 g of albumin.

The serum levels at any time reflect its rate of synthesis, degradation, and volume of distribution. Corticosteroids and thyroid hormone stimulate albumin synthesis by increasing the concentration of albumin mRNA and tRNA in hepatocytes.

The serum albumin levels tend to be normal in diseases like acute viral hepatitis, drug-related hepatotoxicity and obstructive jaundice.

Albumin levels below 3g/dl in hepatitis should raise the suspicion of chronic liver disease which usually reflects decreased albumin synthesis.

In ascites, there may be normal synthesis but the levels may appear reduced because of an increased volume of distribution.

Hypoalbuminemia is not specific for liver disease and may occur in protein malnutrition, nephrotic syndrome and chronic protein-losing enteropathies.

2. Prealbumin

The serum prealbumin level is 0.2- 0.3 g/L.
levels fall in liver disease presumably due to reduced synthesis. Because of its short half-life, changes may precede alteration in serum albumin.

The determination of prealbumin has been considered particularly useful in drug-induced hepatotoxicity.

3. Serum ceruloplasmin

It is synthesized in the liver and is an acute-phase protein. The plasma concentration rise in infections, rheumatoid arthritis, pregnancy, non-Wilson liver disease, and obstructive jaundice.

Normal plasma levels are 0.2-0.4g/L.
This is an important diagnostic marker in Wilson disease, in which the plasma level is usually low. Low levels may also be seen in neonates, Menke’s disease, kwashiorkor, marasmus, protein-losing enteropathy, copper deficiency, and aceruloplasminemia.

4. Procollagen III Peptide

The serum concentration of this peptide appears to increase not only with hepatic fibrosis but also with inflammation and necrosis.
Serial measurement of procollagen III may be helpful in the follow up of chronic liver disease.

5. α 1 Antitrypsin
α 1 antitrypsin is a glycoprotein synthesized by the liver and is an inhibitor of serine proteinases, especially elastase.

Its normal concentration is 1- 1.6g/L. it is an acute-phase protein, serum levels increase with inflammatory disorders, pregnancy and after oral contraceptive pills.
Liver disease is usually seen with a deficiency of α 1 antitrypsin, an inherited disorder.
Deficiency should be confirmed by quantitative measurement.

6. α Fetoprotein
This protein, the principal one in fetal plasma in early gestation is subsequently present at very low levels.( <25µg/L)
It is increased in hepatocellular carcinoma (HCC)and more than 90% of such patients have raised levels.
Raised values are also found in other liver diseases like chronic hepatitis, in the regeneration phase of acute hepatitis and in hepatic metastasis.

This is also raised in adenomas associated with tyrosinemia. α fetoprotein elevation is less frequent when HCC arises in no cirrhotic liver.

7. Prothrombin Time
Clotting is the end result of a complex series of enzymatic reactions that involve at least 13 factors.
The liver is the major site of synthesis of 11 blood coagulation proteins: fibrinogen, prothrombin, labile factor, stable factor, Christmas factor, Stuart prower factor, prekallikrein, and high molecular wt kininogen.

Most of these are present in excess and abnormalities of coagulation only result when there is substantial impairment in the ability of the liver to synthesize these factors.

The standard method to assess is the one-stage prothrombin time of quick, which evaluates the extrinsic coagulation pathway.
The results of this test may be expressed in seconds or as a ratio of the plasma prothrombin time to control plasma time.

Normal control usually is in the range of 9-11 seconds.

A prolongation of more than 2 seconds is considered abnormal. The prolonged PT is not specific for liver diseases and is seen in various deficiencies of coagulation factors, DIC, and ingestion of certain drugs.

In acute and chronic hepatocellular disease the PT may serve as a prognostic indicator.
In acute hepatocellular disease worsening of PT suggests an increased likelihood of acute hepatic failure.

The PT is a predictor of outcome in cases of acetaminophen overdosage and acute alcoholic hepatitis.
The prolongation of PT is also suggestive of poor long-term outcomes in chronic liver disease.

If the PT returns to normal or improves by at least 30% within 24 hr of a single parenteral injection of vitamin K1 (5-10 mg), it may be surmised that parenchymal function is good and that hypovitaminosis K was responsible for the original prolongation of PT.

Patients with parenchymal disease, by contrast, will show only minimal improvement. Most patients with an extrahepatic obstruction like EHBA would respond promptly to a single injection of vitamin K.