2. Enzymes (Table) - Notes taken from the lecture of Mr. Mikhail Valdescona, RMT, MPH PDF

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Summary

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) ...

Description

OXIDOREDUCTASE Normal Range: 125-220 U/L

LACTATE DEHYDROGENASE (LD) 1.1.1.27 - 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

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 @ 8.8 pH - most commonly used method because it produces a positive rate & not affected by product inhibition 2. WROBLEUSKI LA DUE (Reverse/Indirect Reaction) - reaction @ 7.2 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: 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

TRANSFERASE

ASPARTATE AMINOTRANSFERASE

Normal Range:

2.6.1.1 - a.k.a “Serum Glutamic Oxaloacetic Transaminase” (SGOT) - Tissue Sources:  High: Skeletal muscle, Cardiac tissue, Liver  Less: Kidney, Pancreas, RBC - limited only to evaluation of Hepatocellular Disorders & Skeletal Muscle Involvement - Increase during AMI: rise @6-8hrs, peak @24hrs, normal @5 days - Peak Elevation during HCD: 24hrs. - not useful in the diagnosis of AMI

ASSAY METHODS: 1. KARMEN METHOD - reaction @ 7.5 pH @ 340nm - uses Malate Dehydrogenase & monitors the change in absorbance @340nm 2. REITMAN & FRANKEL SOURCES OF ERROR:  Hemolysis results to falsely ↑ AST levels AST & ALT:  Rapidly ↑ in almost all Liver Diseases & still ↑ for 2-6 weeks  ↑ level during Viral Hepatitis, Drug-induced Cirrhosis, Hepatic Ischemia  ↑ during AMI, IM, Renal infarction, Progressive Muscular Dystrophy, Diabetic Ketoacidosis, Hyperthyroidism

2 ISOENZYME FRACTION: 1. CYTOPLASM: predominant in serum 2. MITOCHONDRIA: significantly ↑ during Cellular Necrosis

TRANSFERASE

ALANINE AMINOTRANSFERASE

Normal Range: 7-45 U/L

2.6.1.2 - a.k.a “Serum Glutamic Pyruvic Transaminase” (SGPT) - Tissue Sources:  High: Liver  Less: many tissues in the body - ↑ in Hepatocellular Disorders than Extrahepatic or Intrahepatic Obstructive Disorders - ALT levels are frequently ↑ than AST & tend to remain ↑ longer because of longer half-life of ALT in the serum - Cardiac tissue contains small amount ALT activity, but serum level remains normal in AMI unless there’s subsequent Liver damage - Peak Elevation during HCD: 16hrs.

ASSAY METHODS: 1. ELECTROPHORESIS 2. HEAT FRACTIONATION/STABILITY TEST 3. CHEMICAL INHIBITION TEST 4. BOWERS & MC COMB (Szasz Modification) - most specific method - a continuous monitoring technique which required 10.15 pH @ 405nm SOURCES OF ERRORS:  Not affected by Hemolysis  Stable @4°c for 3-4days

HYDROLASE

ALKALINE PHOSPHATASE (ALP) 3.1.3.1 - 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) 3.1.3.2 - 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 ↓ 6.2 pH

TRANSFERASE

Y-GLUTAMYLTRANSFERASE

Normal Range: Males: 6-55 U/L Females: 5-38 U/L

2.3.2.25 - Tissue Sources:  Brain, Kidney, Liver, Pancreas, Prostate - for evaluation of Liver & Biliary disorders - one of the most sensitive enzyme for all Hepatobiliary disorders - ↑ GGT levels: Chronic Alcoholism (2-3 ↑ ULN) - useful for monitoring abstention from alcohol - level return to normal within 2-3 weeks after cessation - may also be ↑ during Acute Pancreatitis, DM, MI - useful in differentiating source of ↑ ALP because GGT remains normal during Pregnancy & Skeletal disorders

SOURCES OF ERRORS:  Not affected by Hemolysis

OXIDOREDUCTASE

GLUCOSE-6-PHOSPHATE-DEHYDROGENASE

Normal Range: 7.9-16.3 U/L

1.1.1.49 - Tissue Sources:  Adrenal cortex, Thymus, Spleen, Lymph Nodes, Lactating Mammary Gland, RBC - research focus is on its role for RBC - it maintains NADPH in its reduced form - adequate conc. of NADPH is required to regenerate Glutathione from the oxidized to reduced - protects Hemoglobin from Oxidation & cell membrane damage - deficiency in G-6-PD results to inadequate supply of NADPH; inability to maintain reduced Glutathione levels; Drug-induced Hemolytic Anemia - when exposed to an oxidant drug, affected individuals experience Hemolytic episodes - ↑ levels of G-6PD in the serum have been reported in MI & Megaloblastic Anemia

ASSAY METHODS:  Red Cell Hemolysate is used to assay for deficiency of the enzyme  Serum is used for evaluation if enzyme ↑

HYDROLASE

AMYLASE

Normal Range: Serum: 28-100 U/L

3.2.1.1 - 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 3.1.1.3 - 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

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...