Liver function test Part 1 Liver enzymeALTASTALPGGT Liver

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 o Plasma proteins (albumin, globulins), cholesterol, triglycerides and lipoproteins • Detoxification and excretion o Ammonia to urea (urea cycle), bilirubin, cholesterol, drug metabolites • Storage Function o Vitamins A, D, E, K and B 12 • Production of bile salts o 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: 1. Tests to detect hepatic injury: • • Mild or severe; acute or chronic Nature of liver injury (hepatocellular or cholestasis) 2. 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)

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: o Aspartate aminotransferase (AST), formerly termed glutamate oxaloacetate transaminase (GOT). o 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: o Serum, heparin plasma or EDTA plasma o Hemolysis should be avoided because it can dramatically increase serum AST concentrations o (RBCs contain 15 X the AST activity in serum) Mohammed Laqqan

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

• Normal range: 8 – 20 U/L • Post AMI o Rises 6 – 8 hours o Peaks at 24 hours o 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: o Serum, heparin plasma or EDTA plasma o Hemolysis should be avoided because it can increase serum ALT concentrations o (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 + NADH + H ALT LD Pyruvate + Glutamate Lactate + NAD

• Normal range (U/L): ▫ Male: 13 -35 ▫ Female: 10 -30 • High serum levels in acute hepatitis (300 -1000 U/L) • Moderate elevation in alcoholic hepatitis (100 -300 U/L) • Minor elevation in cirrhosis, hepatitis C and non-alcoholic steatohepatitis (NASH) (50 -100 U/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: o AST>ALT in: • alcoholic hepatitis and cirrhosis, • metastatic cancer of the liver • non-biliary cirrhosis, o 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 p. H for ALP is between 9 and 10 and varies with the buffer and substrate. • ALP requires Mg 2+ as an activator Mohammed Laqqan

Isoenzymes • • • 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: o 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 Mg 2+ 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 pnitrophenol at 405 nm • At an alkaline p. H, o p-nitrophenyl phosphate is colorless; o 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, H 2 O and other small peptides o glutathione serves as the γ-glutamyl donor GGT Glutathione + Amino Acid glutamyl-peptide + L-cysteinylglycine • High concentrations in liver tissue • Also in pancreas and kidney

Diagnostic Significance • Increased plasma GGT is associated with o Hepatobiliary disease • Highest seen in biliary obstruction o 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 GGT • 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. Reference Range: male, 6 -45 U/L (37°C); female, 5 -30 U/L (37°C)