Enzymes Part 2 M. Zaharna Clin. Chem. 2009

Alanine Aminotransferase (ALT) A transferase with enzymatic activity similar to AST Converts alanine + α-ketoglutarate to pyruvate and glutamate GPT or SGPT= serum glutamic pyruvic transaminase The older terminology was serum glutamic-pyruvic transaminase (SGPT, or GPT) M. Zaharna Clin. Chem. 2009

Alanine Aminotransferase Pyridoxal phosphate is the coenzyme Source is heart, liver, and skeletal muscle Primarily associated with liver disease It is considered the more liver-specific enzyme of the transferases. Pyridoxal phosphate : It is the active form of vitamin B6 M. Zaharna Clin. Chem. 2009

Diagnostic Significance Higher elevations are found in hepatocellular disorders than in extrahepatic or intrahepatic obstructive disorders. In acute inflammatory conditions of the liver, ALT elevations are frequently higher than those of AST and tend to remain elevated longer as a result of the longer half-life of ALT in serum Cardiac tissue contains a small amount of ALT activity, but the serum level usually remains normal in AMI M. Zaharna Clin. Chem. 2009

Assay for enzyme activity The typical assay procedure consists of a coupled enzymatic reaction using LDH in the second reaction NADH is oxidized in the second reaction The change in absorbance is at 340 nm is measured ALT Alanine + -Ketoglutarate Pyruvate + Glutamate Pyruvate + NADH + H Lactate + NAD LDH Reference Range: 6-37 U/L (37°C) M. Zaharna Clin. Chem. 2009

Alkaline Phosphatase (ALP) Belongs to a group of enzymes that catalyze hydrolysis of phosphate monoesters at alkaline pH (9-10) Removes inorganic phosphate from organic phosphate ester & generates alcohol Requires Mg2+ as an activator Phosphomonoesters (or phosphoric esters) are chemical compounds containing one ester bond and a phosphate group. M. Zaharna Clin. Chem. 2009

Alkaline Phosphatase (ALP) Present on all cell surfaces Highest levels are found in: intestine, liver, bone (osteoblasts), spleen, placenta, and kidney In Liver enzyme is located on both sinusoidal and bile canalicular membranes M. Zaharna Clin. Chem. 2009

M. Zaharna Clin. Chem. 2009

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 M. Zaharna Clin. Chem. 2009

Diagnostic Significance Elevations of ALP are of most diagnostic significance in the evaluation of hepatobiliary and bone disorders. Liver Obstructive 3-10 x ULN elevation is due to increased synthesis of the enzyme induced by cholestasis Hepatocellular < 3 x ULN Bone Highest in Paget’s Disease Osteomalacia, rickets, hyperparathyroidism, Healing fractures 1.5 x normal during late pregnancy Low in hypophosphatasia Inherited inadequate bone calcification Osteomalacia is the general term for the softening of the bones due to defective Bone demineralization is a chronic disorder that typically results in enlarged and deformed bones. cholestasis is a condition where bile cannot flow from the liver to the duodenum. M. Zaharna Clin. Chem. 2009

Assay for enzyme activity Different methods due to non-specificity of the enzyme group Bowers and McComb method based on absorption of p-nitrophenol at 405 nm colorless yellow M. Zaharna Clin. Chem. 2009

Acid Phosphatase (ACP) Belong to the same group of phosphatase enzymes as ALP Catalyze hydrolysis of phosphate monoesters at acid (5.0) pH High concentrations in the prostate gland, Found also in bone, liver spleen, kidney, RBC and platelets M. Zaharna Clin. Chem. 2009

Diagnostic Significance ACP used as an aid in the detection of prostatic carcinoma particularly metastatic carcinoma of the prostate Prostatic specific antigen (PSA) now better Total ACP determination relatively insensitive technique Chemical inhibition methods to differentiate the prostatic portion Serum incubated with and without addition of tartarate (inhibits P. ACP) Tartarate inhibits P. ACP M. Zaharna Clin. Chem. 2009

Diagnostic Significance Total ACP – ACP after tartrate inhibition = prostatic ACP Other methods Immunological test Other diagnostics Vaginal washings for rape cases (persist for up to 4 days) Serum levels in bone disease (osteoclasts) Idiopathic thrombocytopenic purpura – platelet destruction ITP= Idiopathic thrombocytopenic purpura M. Zaharna Clin. Chem. 2009

Assay for enzyme activity Same as ALP but at acidic pH P-Nitrophenolphosphate p-nitrophenol + phosphate ion Products are colorless at acidic pH Addition of alkali stops the reaction and transforms the products into chromogens, can be measured spectrophotometery ACP pH 5 M. Zaharna Clin. Chem. 2009

γ-Glutamyltransferase (GGT) Transfers γ-glutamyl residue from γ-glutamyl peptides (usually glutathione) to amino acids, H2O and other small peptides Glutathione + Amino Acid glutamyl-peptide + L-cysteinylglycine High concentrations in liver tissue Also in pancreas and kidney Glutathione, an antioxidant, protects cells from toxins such as free radicals , GGT is involved in the transfer of amino acids across the cellular membrane GGT M. Zaharna Clin. Chem. 2009

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 M. Zaharna Clin. Chem. 2009

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) M. Zaharna Clin. Chem. 2009

Amylase (AMS) Hydrolyzes starch and glycogen Starch = amylose & amylopectin Amylose = long, unbranched α,1-4 linkages Amylopectin = branched chain α,1-6 linkages Glycogen similar to amylopectin but more branches α-Amylase attacks only α-1-4 glycosidic bonds Generates glucose, maltose, and dextrins (branch points) Requires Ca+ and Cl- for activation Cellulose is not attacked by α-Amylase due to α-1-6 bonds Amylose is a linear polymer of glucose Amylopectin is a polysaccharide and highly branched polymer of glucose found in plants M. Zaharna Clin. Chem. 2009

Acinar cells of pancreas and salivary glands major tissue sources AMS is smallest enzyme – 50000 - 55,000 daltons Readily filtered into urine Digestion of starches begins in the mouth Inactivated by stomach acid Pancreatic AMS takes over in intestine Acinar cell can refer to:exocrine cells of the pancreas, which release hydrolytic enzymes into the small intestine (duodenum M. Zaharna Clin. Chem. 2009

Isoenzymes Two isoenzymes are found in normal serum; P isoamylase … derived from pancreas S isoamylase … derived from salivary glands They are identified using electrophoresis chromatography or isoelectric focusing. M. Zaharna Clin. Chem. 2009

Diagnostic Significance In Acute pancreatitis Amylase begin to rise 2-12 hours after onset of attack Peak 24 hours, return to normal 3-5 days 2.5 x ULN Also elevated in salivary gland disorders and Intraabdominal disease (e.g. Perforated peptic ulcer) M. Zaharna Clin. Chem. 2009

Assay for enzyme activity Measurement – 4 different methods Amyloclastic – disappearance of starch substrate Starch substrate with Iodine attached As hydrolyzed Iodine is released = decrease in dark blue color Saccharogenic – appearance of product Classic Somogyi method Starch substrate is hydrolyzed to CHO molecules that have reducing ability Amount of reducing sugars present = amount of AMS present M. Zaharna Clin. Chem. 2009

Assay for enzyme activity Somogyi units A measure of the level of activity of amylase in blood serum. One Somogyi Unit is defined as the number of milligrams of glucose released in 30 minutes at 37°C under specific assay conditions. M. Zaharna Clin. Chem. 2009

Assay for enzyme activity Chromogenic Chromogenic methods use a starch substrate to which a chromogenic dye has been attached, forming an insoluble dye-substrate complex. As AMS hydrolyzes the starch substrate, smaller dye-substrate fragments are produced, and these are water soluble. The increase in color intensity of the soluble dye-substrate solution is proportional to AMS activity. Continuous – coupling enzymes to monitor AMS activity (NADH produced) M. Zaharna Clin. Chem. 2009

Lipase (LPS) Lipase is an enzyme that hydrolyzes the ester linkage of fats to produce alcohols and fatty acids LPS catalyses the partial hydrolysis of dietary triglycerides in the intestine Pancreatic LPS specific for fatty acid residues at position 1 and 3 of triglyceride Accelerated by presence of bile salts & colipase M. Zaharna Clin. Chem. 2009

Lipase (LPS) Primarily found in pancreas Some from stomach and small intestine M. Zaharna Clin. Chem. 2009

Diagnostic Significance Used in diagnosis of acute pancreatitis Similar pattern to AMS but lasts longer May be elevated in other abdominal conditions Normal in salivary gland conditions (AMS↑) M. Zaharna Clin. Chem. 2009

Assay for enzyme activity Cherry-Crandall method – classic Triolein as substrate Titrate amount of fatty acids generated Triglyceride + 2 H2O 2-monoglyceride + 2 fatty acids LPS pH 8.6 - 9 M. Zaharna Clin. Chem. 2009

Assay for enzyme activity Turbidimetric methods Fats in solution = cloudy LPS hydrolysis causes clearing of solution Colorimetric Coupled reactions with peroxidase or glycerol kinase M. Zaharna Clin. Chem. 2009

Glucose-6-Phosphate Dehydrogenase (G6PD) Oxidoreductase catalyzes oxidation of G6P to 6-phosphogluconate or 6-phosphogluconolactone First step in pentose-phosphate shunt of glucose metabolism M. Zaharna Clin. Chem. 2009

Little activity is found in normal serum Primarily focus of RBC Keeps Adrenal cortex, spleen, thymus, lymph nodes, lactating mammary gland, and RBC Little activity is found in normal serum Primarily focus of RBC Keeps NADPH in reduced form Helps to generate glutathione Which in turn protect Hb from oxidation Cells hemolyze without it M. Zaharna Clin. Chem. 2009

Diagnostic Significance Deficiency of G6PD inherited sex-linked trait Drug-induced hemolytic anemia (primaquine) Increased levels reported in AMI and megaloblastic anemia primaquine is a medication used in the treatment of malaria M. Zaharna Clin. Chem. 2009

Assay for enzyme activity G6PD G-6-P + NADP+ 6-phosphogluconate + NADPH + H+ A red cell hemolysate is used to assay for deficiency of the enzyme Serum is used for evaluation of enzyme elevations M. Zaharna Clin. Chem. 2009