Part 1 Liver enzyme(ALT-AST-ALP-GGT) Liver function test Part 1 Liver enzyme(ALT-AST-ALP-GGT)

Liver is the largest Organ of the body weighing about 1.5 Kg. Liver is called kitchen of our body.

Major Metabolic Functions of the Liver Synthetic Function Plasma proteins (albumin, globulins), cholesterol, triglycerides and lipoproteins Detoxification and excretion Ammonia to urea (urea cycle), bilirubin, cholesterol, drug metabolites Storage Function Vitamins A, D, E, K and B12 Production of bile salts Helps in digestion

Liver Function Tests (LFTs) Noninvasive methods for screening of liver dysfunction Help in identifying general types of disorder Assess severity and allow prediction of outcome Disease and treatment follow up

Liver Function Tests (LFTs) Broadly classified as: Tests to detect hepatic injury: Mild or severe; acute or chronic Nature of liver injury (hepatocellular or cholestasis) Tests to assess hepatic function

Classification of LFTs Group I: Markers of liver dysfunction Serum bilirubin: total and conjugated Urine: bile salts and urobilinogen Total protein, serum albumin and albumin/globulin ratio Prothrombin Time

Classification of LFTs Group II: Markers of hepatocellular injury Alanine aminotransferase (ALT) Aspartate aminotransferase (AST)

Classification of LFTs Group III: Markers of cholestasis Alkaline phosphatase (ALP) g-glutamyltransferase (GGT)

Liver Enzymes

Aminotranferases Aminotransferases or transaminases are a group of enzymes that catalyze the interconversion of amino acids and ketoacids (oxoacids) by transfer of amino group The two aminotransferases of greatest clinical significance are: Aspartate aminotransferase (AST), formerly termed glutamate oxaloacetate transaminase (GOT). Alanine aminotransferase (ALT), formerly termed glutamate pyruvate transaminase (GPT). Pyridoxal phosphate (P-5-P) is coenzyme

Aspartate aminotransferase (AST) AST involved in the transfer of an amino group between aspartate and -ketoacids. Mohammed Laqqan

Clinical Significance Aspartate aminotransferase (AST) is an enzyme found primarily in the heart, liver, and muscle. Additional AST is released into the circulation after injury or death of cells. This test is one of several that are performed when there has been damage to the heart muscle, as in myocardial infarction, and in assessing liver damage. Infants levels approximately twice the adult level, these decline to adult levels by approximately 6 months of age. Mohammed Laqqan

Specimen Collection Specimen: Serum, heparin plasma or EDTA plasma Hemolysis should be avoided because it can dramatically increase serum AST concentrations (RBCs contain 15 X the AST activity in serum) Mohammed Laqqan

Assay for Enzyme activity Measurement by Karmen method A coupled reaction involving: pyridoxal-5-phosphate (P-5-P) and malate dehydrogenase (MDH) at 37oC: Decrease in absorbance at 340 nm is determined by continuous monitoring. AST Aspartate + -Ketoglutarate Oxaloacetate + Glutamate Oxaloacetate + NADH + H Malate + NAD MD Mohammed Laqqan

Normal range: 8 – 20 U/L Post AMI Rises 6 – 8 hours Peaks at 24 hours Returns to normal by day 5 AST levels are highest in acute hepatocellular disorders "viral hepatitis, cirrhosis.

Alanine Aminotransferase (ALT) A transferase with enzymatic activity similar to AST Converts alanine + α-ketoglutarate to pyruvate and glutamate Mohammed Laqqan

Clinical Significance It is found in the kidneys, heart, and skeletal muscle tissue but primarily in liver tissue. The test is used mainly in the diagnosis of liver disease and to monitor the effects of hepatotoxic drugs. Often higher than AST with liver damage and tend to remain elevated longer(More liver-specific than AST) Remains normal in AMI Mohammed Laqqan

Specimen Collection Specimen: Serum, heparin plasma or EDTA plasma Hemolysis should be avoided because it can increase serum ALT concentrations (ALT in RBCs is roughly 5 X that of serum) Mohammed Laqqan

Assay for enzyme activity The most common method in use today for measurement of ALT activity utilizes a coupled enzymatic procedure for monitoring disappearance of NADH. In this approach lactate dehydrogenase (LDH) and its required cofactors are added and catalyze the conversion of pyruvate to lactate This causes simultaneous oxidation of reduced nicotinamide adenine dinucleotide (NADH). The disappearance of NADH is followed spectrophotometrically (at 340 nm). Alanine + -Ketoglutarate Pyruvate + Glutamate Pyruvate + NADH + H Lactate + NAD LD ALT

Male: 13-35 Female: 10-30 Normal range (U/L): High serum levels in acute hepatitis (300-1000U/L) Moderate elevation in alcoholic hepatitis (100-300U/L) Minor elevation in cirrhosis, hepatitis C and non-alcoholic steatohepatitis (NASH) (50-100U/L)

Levels of AST & ALT AST is assessed along ALT in monitoring liver damage. These two values normally exist in an approximately 1:1 ratio. As a rough guide: AST>ALT in: alcoholic hepatitis and cirrhosis, metastatic cancer of the liver non-biliary cirrhosis, while ALT>AST in: viral and drug hepatitis, chronic hepatitis C and hepatic obstruction. Mohammed Laqqan

Alkaline Phosphatase (ALP) Phosphatases transfer a phosphate moiety from one group to a second, forming an alcohol and a second phosphate compound. The optimal reaction pH for ALP is between 9 and 10 and varies with the buffer and substrate. ALP requires Mg2+ as an activator Mohammed Laqqan

Isoenzymes Liver isoenzyme (fastest) Bone isoenzyme ALP exists as a number of isoenzymes Major are those found in Liver, bone, placenta, and then intestinal fraction Electrophoresis for isoenzyme analysis Liver isoenzyme (fastest) Bone isoenzyme Placental isoenzyme Intestinal isoenzyme (slowest) Immunochemical methods now available Mohammed Laqqan

Clinical Significance Alkaline phosphatase (ALP) is an enzyme found in the liver, bone, placenta, intestine, and kidneys Primarily in the cells lining the biliary tract and in the osteoblasts involved in the formation of new bone. ALP is normally excreted from the liver in the bile. Increased ALP levels are found most commonly during: periods of bone growth (as in children), in various types of liver disease, and in biliary obstruction. Serum ALP activity primarily reflects changes in bone and liver function, even though higher ALP activities can be found in other organs. Mohammed Laqqan

Specimen Collection Blood should be drawn after a fast of at least 8 hours. Serum or heparinized plasma. Slight hemolysis is tolerable, but gross hemolysis should be avoided. These increases may be caused by: the release of ALP from complexes with lipoproteins, It is best to analyze ALP specimens the same day they are drawn. ALP is inhibited by metal-complexing anticoagulants; EDTA, oxalate, and citrate inhibit the enzyme by complexing Mg2+ and should not be used. Mohammed Laqqan

Assay for Enzyme activity almost all assays for ALP employ p-nitrophenyl phosphate as the substrate. Bowers and Combs method based on absorption of p-nitrophenol at 405 nm At an alkaline pH, p-nitrophenyl phosphate is colorless; the product p-nitrophenol is intensely yellow

Normal range: 40 – 125 U/L

γ-Glutamyltransferase (GGT) Transfers γ-glutamyl residue from γ-glutamyl peptides (usually glutathione) to amino acids, H2O and other small peptides glutathione serves as the γ-glutamyl donor Glutathione + Amino Acid glutamyl-peptide + L-cysteinylglycine High concentrations in liver tissue Also in pancreas and kidney GGT

Diagnostic Significance Increased plasma GGT is associated with Hepatobiliary disease Highest seen in biliary obstruction Alcoholic cirrhosis Used with ALP to differentiate between liver and bone diseases

Assay for enzyme activity γ- glutamyl residue of γ glutamyl-p-nitroanilide is transferred to glycylglycine, releasing p-nitroaniline L-γ-glutamyl-p-nitroanilide + glycylglycine p-nitroaniline + y-glutamyl glycylglycine The rate of liberation of p-nitroaniline is directly related to the GGT activity in the sample and is quantitated by measuring the increase in absorbance at 405 nm. GGT Reference Range: male, 6-45 U/L (37°C); female, 5-30 U/L (37°C)