Laboratory diagnosis of Liver diseases in children

Laboratory tests play a central role in the diagnosis and management of liver disease in children. In this article the usefulness Liver function tests in the diagnosis of liver disease has been dealt with. Though imaging, histology and endoscopy are also important in the diagnosis of liver diseases covering these tests are beyond the scope of this article

Sometimes abnormal laboratory tests may be the only indication of liver dysfunction.  Although the term liver function tests is in common usage, most of the lab test used routinely do not measure the function of the liver except serum albumin and prothrombin time and the rest are indirect tests and not specific to liver diseases alone.

Tests to evaluate liver disease: 

Tests of biochemical activity

       Alanine aminoTransferase (ALT)

·         Aspartate aminoTransferase (AST)

       Lactic Dehydrogenase (LDH)

Tests of cholestasis

·         Gamma Glutamyltranspeptidase (GGT)

·         Alkaline phoshatase (AP)

·         5’nucleotidase

·         Bilirubin (total and fractionation)

·         Urinary urobilinogen

·         Serum and urine bile acids

Tests for synthetic function

·         Albumin and other serum proteins

·         Prothrombin time

·         Ammonia

·         Serum lipids and lipoproteins, cholesterol and triglycerides

 Miscellaneous specific serum tests (a1-antitrypsin, ceruloplasmin, a-fetoprotein, auto antibodies)

 Tests for quantitative function – difficult to perform and not practically useful

LIVER ENZYMES

A liver enzyme is a protein that helps to speed up a chemical reaction in the liver. Liver enzymes have been useful as a screening test for liver diseases, to determine the pattern of liver disease,assess severity and also in the follow up of the patients with liver disease

Enzymes that detect hepatocellular necrosis Aminotransferases Aspartate aminotransferase (AST, formerly SGOT) and Alanine aminotransferase (ALT, formerly SGPT) are the most frequently utilized and specific indicators of hepatocellular necrosis and usually catalyze the transfer of a-amino group to a-keto group.

Aspartate aminotransferase (AST)is a mitochondrial enzyme present in liver, cardiac muscle, skeletal muscle, kidneys, brain, pancreas, lungs leukocytes and erythrocytes whereas Alanine aminotransferase (ALT) is a cytosolic enzyme which is more specific for liver damage and present in higher concentration in liver than in heart/skeletal muscle and normal values of these enzymes range from 5-35 IU/l.

The rise in liver enzymes may be severe ( > 20 times, 1000 U/L) in viral hepatitis, drug or toxin induced hepatic necrosis and circulatory shock, moderate (3-20 times) in acute & chronic hepatitis, neonatal hepatitis, and acute biliary tract obstructions or mild (1-3 times) in  sepsis, Extra Hepatic Biliary Atresia (EHBA), Myositis, Duchenne muscular dystrophy and even after vigorous exercise.

Level of enzyme elevation shows poor correlation regards the extent of necrosis and is of little value in predicting the outcome. Similarly decreasing enzyme level though may indicate recovery can also be due to massive destruction when it indicates poor prognosis.

The ratio of AST and ALT (AST/ALT) may be useful to differentiate certain conditions. The normal ratio is less than or equal to 1 and a ratio of more than 2 is seen commonly in alcoholic hepatitis probably due to pyridoxine deficiency and in acute Wilsons the ratio is usually more than 4 and one should remember that a higher ratio may also be seen incirrhosis due to any cause because of reduced plasma clearance of AST.

Low levels of amino transferases have been seen in patients on long term haemodialysis and also in uremia and the cause probably secondary to either dialysate or pyridoxine deficiency.

Other enzymes like glutamate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase and sorbitol dehydrogenase have also been found to be raised in hepatocellular necrosis but have not found to be clinically significant.

Enzymes that detect cholestasis

Alkaline phosphatase is found histochemically in the microvilli of bile canaliculi and on the sinusoidal surface of hepatocytes and normally serum values range between 45-115 U/L and the increase in serum levels is usually due to overproduction and leakage in blood secondary to bile ductular damage the increase in serum levels may be due to disorders of bone, kidney, intestine, placenta and tumours in addition to cholestatic liver disease and most circulating alkaline phosphatase usually originates from either liver or bone. Physiologically high values are seen in childhood and puberty due to bone growth. Though highest levels of alkaline phosphatase occur in cholestatic disorders, other causes for increase may be infiltrative liver diseases, abscesses, granulomatose liver disease and amyloidosis. Low levels of alkaline phosphatase can occur in hypothyroidism, pernicious anaemia, zinc deficiency and congenital hypophosphatasia.

GGlutamyltranspeptidase (GGT) is a membrane bound glycoprotein and normal value ranges between 0-30 U/L and increasing serum level is due to overproduction and leakage in blood. In addition to liver, the enzyme is also expressed in the kidney, spleen, pancreas, heart, lung and brain except bones and therefore useful in the exclusion of bone diseases. The new borns may have high levels of GGT 5 to 8 times the upper limit of normal. One should also remember that this enzyme is inducible by certain drugs like anticonvulsants and depressed by female sex hormones

Serum GGT is high in all cholestatic disorders and cannot differentiate between intahepatic and extra hepatic causes. Low levels of GGT is found in certain cholestatic disorders like Proressive familial intrahepatic cholestasis (PFIC) types I and II and Inborn errors of bile acid metabolism where GGT has been useful to differentiate these conditions from other causes of cholestasis.

Other enzymes to detect cholestasis are 5’Nucleotidase and Leucineaminopeptidase but not put used commonly.

Bilirubuin

Bilirubin is a yellow tetrapyrrole pigment formed from the degradation of the heme found in hemoglobin in the red cells of the blood which is also found in myoglobin and certain enzymes including catalase, peroxidase and the cytochromes. About 75% of daily production of bilirubin is from the breakdown of red cells and 22% is produced from heme containing enzymes and a small amount 3% from ineffective erythropoeisis and normally less than 1mg/dl beyond neonatal period. Hyperbilirubinemia or increased serum bilirubin may result from overproduction, impaired uptake, conjugation or excretion or regurgitation.

Increased unconjugated bilirubin: This results from overproduction/impaired uptake, conjugation

Increased conjugated bilirubin: Impaired intrahepatic excretion / regurgitation of unconjugated or conjugated bilirubin from hepatocytes into bile ducts.

Serum bilirubin could be lowered by drugs like salicylates, sulphonamides, free fatty acids which displace bilirubin from its attachment to plasma albumin. On the contrary it could be elevated if the serum albumin increases and the bilirubin may shift from tissue sites to circulation.

Level of bilirubin rise may be mild in liver diseases, physiological jaundice and inherited hyperbilirubinemias. Moderate elevations of serum bilirubin is seen with biliary atresia, drugs, viral hepatitis, inherited hyperbilirubinemias

Other causes of hyperbilirubinemia may be due to extra hepatic sources either hemolysis, ineffective erythropoiesis, hematoma or myoglobinemia

But you should remember that bilirubin fractionation is very important to recognize conjugated bilirubinemia/cholestasis which is always pathological.

Urine Bilirubin & UBG

Conjugated bilirubin is water soluble and excreted in the urine and therefore the presence of urine bilirubin indicates hepatobiliary disease. Laboratory methods can detect low levels of bilirubin in urine. Tests strips impregnated with diazo reagent are easy to use to detect bilirubin in urine

Urobilinogen is formed from the degradation of conjugated bilirubin by bacteria in the intestinal lumen and upto 20% undergoes enterohepatic circulation. A small amount is excreted in urine. Increase in the urobilinogen in urine is a sensitive indicator of hepatocellular dysfunction. In cholestatic jaundice with complete biliary obstruction urobilinogen disappears from urine.

Tests of liver synthetic function:

Albumin and other serum proteins

Albumin is the principal serum protein and increases from a mean of 3.41g/dl in the first month of life to a mean of 4.25g/dl by adolescence. It is synthesized only in the rough endoplasmic reticulum of hepatocytes at a rate of 150mg/kg/day and has a half life of approximately 20 days. The major functions of albumin are to maintain intravascular colloid osmotic pressure and to bind and serve as a carrier protein. Decreases in serum levels may result from decreased production by the liver due to significant parenchymal liver disease. Because of the long half-life low serum albumin is taken as a sign of chronic liver disease. Hypoalbuminemia is not specific for liver disease and can occur in other non hepatic causes like poor nutrition, nephritic syndrome, and protein-losing enteropathies.

Serum globulins are often elevated in chronic liver disease including cirrhosis due to any cause and particularly in autoimmune hepatitis and this is due to increase in gamma globulins usually in the setting of low albumin

Abnormalities of coagulation – Prothrombin time (PT)

The liver plays three roles in the control of coagulation (a) the production partly or exclusively of all coagulation factors with the exception of von Willebrand factor, (b) the production and breakdown of factors integral to fibrinolysis such as plasminogen and plasminogen activator, and (c) the clearance of activated clotting factors from the circulation. Synthesis of factors II, VII, IX and X is dependent on adequate supply of vitamin K.

Prothrombin time (PT) is a measure of the time it takes for prothrombin to be converted into thrombin in the presence of other factors. The result is expressed in seconds or as a ratio of the plasma PT to a control PT. Normal range - 9-11 seconds. A prolongation of more than 2 seconds is considered abnormal.The prolonged PT is not specific for liver diseases. In acute and chronic hepatocellular disease the PT may serve as a prognostic indicator. Prothrombin time corrected by inj. Vitamin K indicates cholestasis causing vitamin k deficiency

Other tests for biosynthetic function of the liver are Serum prealbumin, Serum Ceruloplasmin, Procollagen III peptide, A 1 antitrypsin and Alpha feto protein

Prealbumin: The serum prealbumin level is 0.2- 0.3 g/L. these levels fall in liver disease presumably due to reduced synthesis. Determination of prealbumin has been considered particularly useful indrug-induced hepatotoxicity.

Serum Ceruloplasmin: Normal plasma levels are 0.2-0.4g/L. 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. 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.

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.

a1 Antitrypsin: a1 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 (OCP). Liver disease is usually seen with deficiency of a 1 antitrypsin, an inherited disorder. Deficiency should be confirmed by quantitative measurement.

Afeto Protein: This protein, the principal one in fetal plasma in early gestation is subsequently present at very low levels (<25mg/L) It is increased in Hepato Cellular 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 regeneration phase of acute hepatitis and in hepatic metastasis. This is also raised in adenomas associated with tyrosinemia.

Dr. D. Nirmala

Professor and Head

Department of Paediatric Gastroenterology

Institute of Child Health and Hospital for children