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109 Total CK (Creatine Kinase) 1. ,t. in muscle, cardiac or brain damage...

109 Total CK (Creatine Kinase) 1. ,t. in muscle, cardiac or brain damage 2. Higher reference ranges in males due to greater muscle mass and physical activity CK Jsoenzymes 1. Different molecular forms of CK Co,nditions Associated with enzyme AFP increase or decrease a. 2 subunits - M and B ❖ CKl = CK-BB - 2 B chains ❖ CK2 = CK-MB - 1 M & 1 B chain Protein.ARsociatea with ❖ CK3 = CK-MM - 2 M chains Copper Transport b. Cardiac muscle= CK-MM and CK- MB c. Skeletal muscle = CK-MM d. Brain, GI, colon, prostate, uterus = Enzymes CK-BB GENERAL e. Trauma to skeletal muscle causes ,t. 1. Organic catalysts responsible for most in total CK and MB isoenzyme, but reactions in the body % activity MB is < 3% (> 6% in MO 2. Enzyme reactions in laboratory measurements affected by: a. Concentrations of reactants b. pH (optimum pH varies with each enzyme) c. Temperature ❖ Optimum 37°C ❖ Rate doubles every ,i. 10 degrees d. Ionic strength e. Cofactors and coenzymes LD (Lactate Dehydrogenase) 3. Enzyme inhibition by substances that can reduce the rate of the enzyme 1. in: reaction a. Myocardial infarction (MI) a. Competitive inhibitor- binds free h. Liver disease enzyme at the active site c. Muscle trauma b. Noncompetitive inhibitor- hinds at a d. R enal infarct site other than the active site e. Hemolytic diseases c. Uncompetitive inhillitor- binds only f. Pernicious anemia to the enzyme substrate complex 2. Sources of error 4. Measurement a. H emolyzed specimens a. Zero-order-kinetics - Large excess b. Prolonged contact of serum to cells of substrate so that the amount of 3. Spectrophotometric method enzyme activity is only rate-limiting a. LD converts pyruvate to lactate factor while oxidizing NADH to NAD b. Catalytic activity rate (not mass or b. Rate of decrease in absorbance of concentration) is directly measured NADH at 340 nm is proportional to c. One international unit ( U or JU) = LD activity amount of enzyme that will cause utilization of substrate or 4. LD isoenzymes production of product at the rate of a. 2 chains (Mand H) 1 µM/ minute b. 4 subunits c. 5 forms (tissue-specific) 110 AST (Aspartate transaminase) c. Disorder s of hepatic biliary tree 1. Found in cardiac muscle, liver, RBCs (obstructive Jaundice due to and other tissues gallstones or malignancy) d. Third trimester of pregnancy 2. ,t.. in MI, liver disease (hepatocellula1· (placenta) damage, cirrhosis, carcinoma), muscle trauma, renal infarct, hemolysis 5. Methods a. Spectrophotometric ALT (Alanine transaminase) ❖ ALP converts p-nitrophenyl 1. ,t.. in liver disease (hepatocellular phosphate to phosphate and P- damage, cirrhosis, carcinoma) nitrophenylate which is measured at 404-410 nm 2. More hver-specific than AST b. Immunoassay for bone isoenzyme GGT (Gamma-glutamyl transferase) 5'NT (5'-Nucleotidase) 1. ,t.. in liver disease (liighest in biliary 1. ,t.. in liver but NOT hone disease obstruction and cirrhosis) 2. ,t.. ALP+ Normal S'NT = bone disease 2. Often ♦ after alcohol intake 3. ,t- ALP +.f- S'NT = liver disease 3. Spectrophotometric method - measure nitroaniline released when Amylase GGT acts on substrate gamma- 1. Produced in salivary and pancreatic glutamyl-p-nitroanilide glands ALP (Alkaline Phosphatase) 2. Requires Ca++ and c1- (dilute elevated ]. Optimum pH= 10; Mg++ activation samples with saline not water) 2. Found in hone, intestinal mucosa , renal 3. Only common enzyme normally tubule cells, biliary tree (liver) , excreted in urine leukocytes, placenta, some tumors 4. Highest elevations seen in pancreatitis 3. Isoenzyme separation: acrylamide gel, and obstruction to pancreatic ducts electrophoresis, chemical or heat (malignancy) (56°C; 10 minutes): Heat Stability: a. Regan (cancer)= rare, most heat 5. Lower elevations seen in obstruction of stable salivary glands (mumps) b. Placental = most heat-stable of 4 6. Methods most common a. Amyloclastic - measure c. Intestinal = inhibited by disappearance of starch substrate L-phenylalanine b. Saccharogenic - measure reducing d. Liver= highest concentrations sugars (glucose and maltose) e. Bone= most heat-labile produced by enzymatic action 4. ,t- in: c. Chromolytic (dye) - measure a. Bone disorders with osteoblastic ahsorbance of soluble dye split from activity insoluble amylase-dye substrate ❖ Paget's (highest ALP values) 7. Urinary amylase remains elevated ❖ Osteoblastic tumors longer than serum in pancreatitis ❖ Rickets ❖ Hyperparathyroidism 8. Opiates (Ex. morphine} cause elevation b. Growing children - rapid skeletal growth (bone) Higbly Elevated ALP & G Various Diseases Biliary Obstruction  11 l Lipase ❖Add tartrate buffer: 1. + in pancreatitis ~ Prostatic ACP inhibited by tartrate 2. Remains elevated longer than amylase I@' RBC ACP not inhibited by tartrate 3. More specific for acute pancreatitis c. Immunoassay for prostatic ACP 4. Methods: 4. Specimen Collection and Handling a. Turbidimetric b. Older method: olive oil substrate; a. Hemolysis results in falsely + results measure fatty acids product b. Storage at room temperature results in loss of enzyme activity; must TE$ remove serum from cells ASAP and stabilize (add disodium citrate t monohydrate or pH to 5.4 with acetic acid) Clinical Significance ofan bicre1JSed Amylase Test Most SpeciJic for Acute Pancreatitis Specimen Handling for Acid Phosphatase Determination ACP (Acid Phosphatase) 1. Sources: primarily prostate; other Cholinesterase tissues: erythrocytes, bone, liver, 1. Erythrocyte acetylcholinesterase and spleen, kidney, platelets plasma pseudocholinesterase 2. Clinical significance 2. Destroys acetylcholine after nerve a. Highest elevations seen in impulse transmission metastasizing carcinoma of prostate; now use PSA instead 3. Severe t results in serious b. + in bone disease or cancers that neuromuscular effects; one of few enzymes in which t is clinically metastasize to hone and in metastasizing breast cancer significant c. Tartrate-resistant portion elevated in hairy cell. leukemia 4. t cholinesterase: organophosphate poisoning and genetic susceptibility to d. Presence in seminal fluid useful in certain anesthetic agents forensic medicine for rape cases; now use PSA instead 3. Methods: a. Spectrophotometric for Total ACP ❖ Phosphate substrate (Ex. p-.nitrophenyl phosphate) Test Helpful in Determining cleaved by ACP to give colored Pesticide Poisoning product (Ex. p-nitrophenol after OH- added; yellow, read at 410nm) Cardiac Markers to Evaluate Possible Acute b. Spectrophotometric for Prostatic Myocardial lnfardion (AMI or Ml) ACP: 1. Myoglobin ❖ Use substrates more specific for a. Produced by muscles including prostatic ACP (Ex. h eart thymolphthalein monophosphate b..+ in muscle damage including AMI and alpha naphthyl phosphate) c. + in renal damage