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2. Enzymes (Table) - Notes taken from the lecture of Mr. Mikhail Valdescona, RMT, MPH
Clinical Chemistry 2 (MDT 3122L)
Our Lady of Fatima University
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OXIDOREDUCTASE LACTATE DEHYDROGENASE (LD) 1.1.1 - Tissue Sources: High: Heart, Liver, Kidney, Skeletal muscles, RBC Less: Brain, Lungs, Smooth muscle - Increase during AMI: rise @12-24hrs, peak @48-72hrs, normal @10 days - during AMI, it will show LD FLIPPED PATTERN (LD-1 > LD-2) ISOENZYME LD-1 (HHHH) LD-2 (HHHM) TISSUE Heart RBC Heart RBC LD-3 (HHMM) Lungs, Pancreas, Spleen, Lymphocytes LD-4 (HMMM) Liver LD-5 (MMMM) Skeletal Muscle LD-6 (ALCOHOL DEHYDROGENASE ) CONDITION Myocardial infarction Hemolytic Anemia Megaloblastic Anemia Acute Renal infarction, Hemolyzed specimen Pulmonary embolism, Pneumonia, Acute Pancreatitis, Lymphocytosis, Carcinoma Hepatic injury/Inflammation Skeletal muscle injury ELEVATION Normal Range: 125-220 U/L CONCENTRATION 14-26% 29-39% Pulmonary Involvement & Carcinoma 20-26% 8-16% Hepatic disorder Grave prognosis, Impending death 6-16% ASSAY METHODS: 1. WACKER METHOD (Forward/Direct Reaction) - reaction @ 8 pH - most commonly used method because it produces a positive rate & not affected by product inhibition 2. WROBLEUSKI LA DUE (Reverse/Indirect Reaction) - reaction @ 7 pH - reaction is 2x faster as the forward reaction - preferred method for Dry Slide Technology 3. WROBLEUSKI CABAUD 4. BERGER BROIDA SOURCES OF ERRORS: Hemolysis results to falsely ↑ LD levels RBC contain 100-150 more LD Stored @25°c within 48hrs TRANSFERASE CREATININE KINASE (CK) Normal Range: Males: 46-171 U/L Females: 34-145 U/L CK-MB: <5% Total CK 2.7.3 - Tissue Sources: High: Skeletal muscles, Heart muscle, Brain tissue Less: Lungs, Thyroid, GIT, Kidney, Liver, Spleen, Pancreas, Uterus, Bladder, Prostate, Placenta - Molecular weight: 82,000 Daltons - occurs in dimer consisting of 2 sub-units that can be separated into 3 Isoenzymes - 3 Isoforms for CK-MM, 2 Isoforms for CK-MB; but the clinical sig. is not well established - associated with ATP generation in Contractile or Transport Systems - used as early diagnostic tool to identify patients with Vibrio vulnificus infections - damage to BBB must occur to allow enzyme release to the peripheral circulation - Increase during AMI: rise @4-8hrs, peak @12-24hrs, normal @48-72hrs. * MACRO-CK - appears to migrate to a position midway between CK-MM & CK-MB - largely comprises CK-BB complexed with IgG & IgA * CK-Mi - bound to the exterior surface of linear mitochondrial membrane of the Muscle, Brain, Liver - it migrates to a point cathodal with CK-MM exists as dimeric molecule of 2 identical subunits - detected in Malignant tumor & Cardiac abnormalities ISOENZYME TISSUE CONDITION Brain, Lungs, Thyroid, CNS shock, Seizure, Carcinoma, CO Poisoning, CK-1 (CK-BB) Stomach, Colon, Reye’s Syndrome, Renal failure “Brain type” Bladder, Prostate CK-2 (CK-MB) “Hybrid type” CK-3 (CK-MM) “Muscle type” Heart Skeletal Muscle Heart Skeletal Muscle Myocardial infarction, Ischemia, Angina, Cardiac Surgery, Duchenne-type muscular dystrophy Reye’s Syndrome, CO poisoning Myocardial infarction Skeletal muscle disorder, Muscular dystrophy, Polymyositis ASSAY METHODS: 1. TANZER-GILBRAG ASSAY (Forward/Direct Method) - reaction @ 9 pH @ 340 nm 2. OLIVER-ROSALKI (Reverse/Indirect Method) - reaction @ 6 pH @ 340 nm - most commonly used method - has faster reaction 3. CK RELATIVE INDEX (CKI) - expression of the % of the Total CK that is attributed to CK-MB - computed to know possible release of CK-MB from non-cardiac tissues when Total CK is very high SOURCES OF ERROR: Hemolysis results to falsely ↑ CK levels >320 mg/dL Hb results to interference Serum must be stored in a dark place because CK is inactivated by light Stored @4°c for 7 days & -20°c for 1 month HYDROLASE ALKALINE PHOSPHATASE (ALP) 3.1.3 - Tissue Sources: Present in all cell surfaces High: Bone, Intestine, Liver, Kidney, Spleen, Placenta - most diagnostic for Hepatobiliary & Bone Disorders - ↑ ALP activity: Paget’s Disease (Osteitis Deformans) - Other Bone disorders: Osteomalacia, Rickets, Hyperparathyroidism, Osteogenic Sarcoma - Complications: Hypertension, Pre-eclampsia, Eclampsia, or threatened Abortion - Pregnancy: ↑ ALP level is detected 16-20 weeks & persist until labor then returns to normal 3-6 days after delivery - ↓ ALP during Inherited Hypophosphatasia - ALP Isoenzymes: derived in Bone, Intestine, Liver, Placenta ASSAY METHODS: 1. ELECTROPHORESIS - most useful single technique for ALP Isoenzyme analysis - Origin > Intestinal > Bone/Placenta > Liver a. LIVER ISOENZYME a1. MAJOR LIVER BAND: Major fraction which is ↑ a2. FAST LIVER / α1-LIVER: Metastatic Carcinoma & Hepatobiliary diseases b. BONE ISOENZYME - ↑ during Osteoblastic Activity - normally ↑ among Children & Adult (>50 y/o) c. INTESTINAL ISOENZYME - common to those with ABO Blood type “B” or “O” (Secretors) - further ↑ after consumption of Fatty meal - ↑ also during Diseases of Digestive tract, Cirrhosis, & Hemodialysis patients 2. HEAT STABILITY - Origin > Placenta > Intestinal > Liver > Bone - ALP activity is measure before & after heating Serum @56°c for 10mins. ↑ due to Bone Phosphatase: residual activity after heating is 20% ↓ than before heating ↑ due to Liver Phosphatase: residual activity after heating is 20% ↑ than before heating 3. SELECTIVE CHEMICAL INHIBITION - used of Phenylalanine to inhibit Intestinal & Placental ALP - however you can’t differentiate Bone from Liver Phosphatase, & Intestinal from Placental Phosphatase ABNORMAL FRACTIONS OF ALP: Carcinoplacental ALPs 1. REGAN ISOENZYME - detected in Carcinoma of the Lung, Breast, Ovary, & Colon - ↑ in Ovarian & Gynecologic cancers - migrates to the same position of the Bone fraction, Heat stable, & inhibited by Phenylalanine 2. NAGAO ISOENZYME - variant of Regan Isoenzyme - same properties with Regan Isoenzyme - detected in Metastatic Carcinoma of Pleural surfaces & in Adenocarcinoma of the Pancreas & Bile duct SOURCES OF ERRORS: Hemolysis results to falsely ↑ LD levels (6x in conc) Diet from Blood type “B” or “O” secretors 25% ↑ following a Fatty meal REFERENCE RANGE: Male & Female (4-15 y/o): 54-369 U/L Males (20-50 y/o): 53-128 U/L (>60 y/o): 56-119 U/L Females (20-50 y/o): 42-98 U/L (>60 y/o): 53-141 U/L HYDROLASE ACID PHOSPHATASE (ALP) Normal Range: 3.1.3 - Tissue Sources: Highest: Prostate Less: Bone, Liver, Kidney, Spleen, RBC, PLT - originally, it aids in the detection of Prostatic Carcinoma - though newer markers such as Prostate-Specific Antigen (PSA) are more useful for screening * THYMOLPHTHALEIN MONOPHOSPHATE - most specific substrate for Prostatic ACP (endpoint determination) * A-NAPTHYL PHOSPHATE: can also be used as Substrate * TARTRATE: as inhibitor (may also inhibit Lysosomal ACP) - may also be ↑ in cases of Paget’s Disease, Gaucher’s Disease, Breast Cancer, Thrombocytopenia - for investigation of Rape (proven useful for Forensic Chemistry) - Vaginal washings are examined for Seminal fluid ACP activity which can persist up to 4 days ASSAY METHODS: METHODS GUTMAN & GUTMAN SHINOWARA BABSON, READ, & PHLIPS RAY & HILLMAN SUBSTRATE Phenyl PO4 END PRODUCTS Inorganic PO4 PNPP Alpha-napthyl PO4 P-Nitrophenol Alpha-Naphtol Thymolphthalein MonoPO4 Free Thymolpthalein SOURCES OF ERRORS: Serum must be separated immediately from the clotted blood to prevent leakage of RBC & Platelets Prolonged standing @ room temp. ↓ activity as a result of CO2 loss thereby ↑ the pH Hemolysis must be prevented If not assayed immediately, serum should be frozen or acidified ↓ 6 pH HYDROLASE AMYLASE Normal Range: Serum: 28-100 U/L Urine: 1-515 U/h 3.2.1 - Tissue Sources: Major: Acinar cells of the Pancreas & Salivary glands Less: Skeletal muscles, Small intestine, & Fallopian tube * SALIVARY AMYLASE: aids in digestion of Starches, but inactivated by the Gastric juices * PANCREATIC AMYLASE: performs major digestive action of Starches once the Polysaccharide reaches the intestine - Other diseases: Salivary Gland lesions (Mumps & Parotitis), Intra-abdominal disease such as Perforated Peptic Ulcer, Intestinal Obstruction, Cholecystitis, ruptured Ectopic Pregnancy, Mesenteric Infarction, Acute Appendicitis - also ↑ in Diabetic Ketoacidosis & Renal Insufficiency - Employed for diagnosis of Acute Pancreatitis - Increase during AP: rise @5-8hrs, peak @24hrs, normal @3-5days * HYPERAMYLESIMIA: occurs among individual with Neoplasmic disease (AMY is 50x ↑ than ULN) * HYPOAMYLESIMIA: combines with Immunoglobulins that is too large to be filtered by the Glomerulus SOURCES OF ERROR: Presence of Plasma Triglycerides can suppress of inhibit Serum Amylase (Amylase can be Normal during Acute Pancreatitis & Hyperlipidemia) Administration of Morphine & other Opiates will lead to false ↑ in Serum Amylase. (Primarily due to constriction of Sphincter of Oddi of the Pancreatic ducts causing regurgitation of Amylase in the serum) SERUM AMYLASE ISOENZYME: PARAMETER P-TYPE ORIGIN Pancreatic tissue FRACTIONS ASSOCIATED CONDITIONS S-TYPE Salivary gland, Fallopian tubes, Lungs S1, S2, S3 P1, P2, P3 Acute Pancreatitis (P3) & Renal failure Salivary Isoenzyme migrate more quickly than the Pancreatic Isoenzyme In normal Serum, IsoAmylases migrate to the A & B Globulin regions of Protein in Electrophoresis where the most commonly observed fractions are P2, S1, S2 ASSAY METHODS: METHODOLOGY AMYLOCLASTIC SACCHAROGENIC CHROMOGENIC CONTINUOUS MONITORING DESCRIPTION Measures the disappearance of Starch Substrate Measures the appearance of the Product Measures the ↑ color from production of Product coupled with a Chromogenic dye Coupling of several enzyme systems to monitor Amylase activity HYDROLASE LIPASE 3.1.1 - Tissue Sources: Pancreas May also be present in the Stomach & Small Intestine - almost exclusively used for diagnosing Acute Pancreatitis - during Acute Pancreatitis, Lipase persists longer than Amylase (2-3 days) - but Lipase remains normal during conditions with salivary involvement - 2 Isoenzymes, in which L2 is the most clinically specific & sensitive - Increase during AP: rise @4-8hrs, peak @24hrs, normal @8-14days Normal Range: ASSAY METHODS: 1. CLASSIC CHERRY-CRANDALL - Olive oil: as Substrate & measured liberated Fatty Acids by Titration after a 24hr incubation 2. TURBIDIMETRIC METHODS - cloudy emulsion of fats are hydrolyzed by Lipase & the rate of clearing is measured 3. COLORIMETRIC METHODS - based on coupled reactions of enzymes such as Peroxidase or Glycerol Kinase SOURCES OF ERRORS: Hemolysis must be avoided because Hemoglobin inhibits Lipase Serum Activity
2. Enzymes (Table) - Notes taken from the lecture of Mr. Mikhail Valdescona, RMT, MPH
Course: Clinical Chemistry 2 (MDT 3122L)
University: Our Lady of Fatima University
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