Enzymes

1. Enzymes Part 2 M. Zaharna Clin. Chem. 2009

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

3. 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. M. Zaharna Clin. Chem. 2009

4. 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

5. 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

6. 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 M. Zaharna Clin. Chem. 2009

7. 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

8. M. Zaharna Clin. Chem. 2009

9. 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

10. 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 M. Zaharna Clin. Chem. 2009

11. 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 yellow colorless M. Zaharna Clin. Chem. 2009

12. 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

13. 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

14. 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 M. Zaharna Clin. Chem. 2009

15. 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

16. 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 GGT M. Zaharna Clin. Chem. 2009

17. 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

18. 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

19. 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 M. Zaharna Clin. Chem. 2009

20. 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 M. Zaharna Clin. Chem. 2009

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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

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

28. 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

29. 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

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

31. Glucose-6-PhosphateDehydrogenase (G6PD) • Oxidoreductase catalyzes oxidation of G6P to6-phosphogluconate or 6-phosphogluconolactone • First step in pentose-phosphate shunt ofglucose metabolism M. Zaharna Clin. Chem. 2009

32. 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

33. Diagnostic Significance • Deficiency of G6PD inherited sex-linked trait • Drug-induced hemolytic anemia (primaquine) • Increased levels reported in AMI and megaloblastic anemia M. Zaharna Clin. Chem. 2009

34. Assay for enzyme activity G-6-P + NADP+ 6-phosphogluconate + NADPH + H+ G6PD • 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