IBSSD Diagnostic Use of Enzymes 2024 PDF

Diagnostic Use of Enzymes IBSSD 1512 &1522, 2024 Module 1, Lecture 1 Dr. Susan Viselli  Most diagnostically important enzymes have intracellular functions.  Most enzymes are NOT NORMALLY present in the blood!  When membranes are damaged, then...

Diagnostic Use of Enzymes IBSSD 1512 &1522, 2024 Module 1, Lecture 1 Dr. Susan Viselli  Most diagnostically important enzymes have intracellular functions.  Most enzymes are NOT NORMALLY present in the blood!  When membranes are damaged, then enzymes normally contained within their boundaries are released into fluids that normally surround the cells.  Damage to cell structure can result in the intracellular contents spilling into surrounding fluids.  Enzymes and other analytes may be measured in samples prepared from whole blood drawn from a patient. Laboratory assays of enzyme activity Plasma – the fluid, most often use noncellular fraction serum, prepared of blood from blood from a patient. Benefit to the use of Serum – fluid serum – the blood obtained by has already clotted centrifugation of and does not coagulated whole require or contain blood any additives to prevent clotting Plasma Versus Serum Add anticoagulant centrifuge PLASMA Blood cells Blood cells Patient’s centrifuge Add nothing; Blood Sample allow clot to form SERUM M Blood cells clot clot  1. In health – basal or background levels of enzymes are usually due to cell turnover.  2. In pathology (disease) Increased blood plasma enzyme levels (greater than basal levels) may result from: Cell membrane injury (such as in hepatitis, myocardial infarction) Enzyme induction (such as in response to metabolism of certain drugs) Tumor Cell proliferation (such as in bone healing) enzyme abbreviation(s) tissue/organ source alkaline phosphatase ALP or ALK liver, bone osteoblasts gamma glutamyl transferase GGT or GGTP liver, pancreas transaminases: alanine aminotransferase ALT or SGPT liver (serum glutamate-pyruvate transaminase) aspartate aminotransferase AST or SGOT liver, muscle (serum glutamate-oxaloacetate transaminase) lactate dehydrogenase LDH or LD liver, muscle, RBC’s creatine kinase CK or CPK muscle TEST 0630 0645 UNITS REF RANGE GLUCOSE 86 82 MG/DL 70-110 BUN 29 H 13 MG/DL 9-22 CREAT 0.6 L 0.7 MG/DL 0.7-1.5 B/C 48 H 19 MG/DL 10-20 URIC ACID 3.1 L 3.0 L MG/DL 3.9-9.0 PO4 2.7 1.1 L MG/DL 2.5-4.5 CALCIUM 8.1 L 8.9 MG/DL 8.5-10.5 NA 143 140 MEQ/L 138-148 K 4.0 3.8 MEQ/L 3.5-5.0 Cl 110 102 MEQ/L 100-112 CO2 20 L 18 L MEQ/L 23-39 EBAL 17 24 H MEQ/L 8-20 TRIG 261 H 263 H MG/DL 1-160 CHOL 150 152 MG/DL 140-200 BILLI – T 30.4 H 32.6 H MG/DL 0.2–1.0 BILLI – D 16.3 H 17.7 H MG/DL 0.0-0.4 BILLI – I 14.1 H 14.9 H MG/DL 0.0-1.2 ALK 99 100 U/L 30-101 GGT 223 H 187 H U/L 9-38 SGPT 108 H 136 H U/L 0-45 SGOT 309 H 339 H U/L 0-40 LDH 345 H 339 H U/L 60-230 CPK 688 H 614 H U/L 0-255 IRON 211 231 H MCG/DL 60-230 PROTEIN 7.1 6.9 G/DL 6.0-8.0 ALBUMIN 3.5 3.2 L G/DL 3.5-5.5 GLOBULIN 3.6 3.9 G/DL 2.0-4.0 A/G 1.0 0.9 G/DL 0.6-2.4  A group of 8 tests standardized in the USA.  Glucose  Calcium  Sodium  Potassium  CO2  Chloride  BUN  Creatinine  In hospital and ER to give information about status of kidneys, electrolyte and acid/base balance, blood sugar and calcium levels  Can indicate acute problems such as kidney failure, insulin shock, diabetic coma, respiratory distress or heart rhythm changes  Also used in routine health exams  A group of 14 tests standardized in the USA.  Glucose  Calcium  Albumin  Total Protein  Sodium  Potassium  CO2  Chloride  BUN  Creatinine  ALP (alkaline phosphatase)  ALT (alanine aminotransferase)  AST (aspartate aminotransferase)  Bilirubin  As a broad screening tool  To evaluate organ function and to check for diabetes, liver and kidney diseases  Ordered to monitor patients with hypertension and those taking medications that may cause kidney or liver-related adverse reactions  Also in routine health exams  Renal Profile  Liver Profile  Muscle Profile  TEST 0630 0645 UNITS REF RANGE GLUCOSE 86 82 MG/DL 70-110 R BUN 29 H 13 MG/DL 9-22 e CREAT 0.6 L 0.7 MG/DL 0.7-1.5 n B/C 48 H 19 MG/DL 10-20 a URIC ACID 3.1 L 3.0 L MG/DL 3.9-9.0 PO4 2.7 1.1 L MG/DL 2.5-4.5 l CALCIUM 8.1 L 8.9 MG/DL 8.5-10.5 NA 143 140 MEQ/L 138-148 K 4.0 3.8 MEQ/L 3.5-5.0 Cl 110 102 MEQ/L 100-112 CO2 20 L 18 L MEQ/L 23-39 EBAL 17 24 H MEQ/L 8-20 TRIG 261 H 263 H MG/DL 1-160 CHOL 150 152 MG/DL 140-200 BILLI – T 30.4 H 32.6 H MG/DL 0.2–1.0 BILLI – D 16.3 H 17.7 H MG/DL 0.0-0.4 BILLI – I 14.1 H 14.9 H MG/DL 0.0-1.2 ALK 99 100 U/L 30-101 GGT 223 H 187 H U/L 9-38 SGPT 108 H 136 H U/L 0-45 SGOT 309 H 339 H U/L 0-40 LDH 345 H 339 H U/L 60-230 CPK 688 H 614 H U/L 0-255 IRON 211 231 H MCG/DL 60-230 PROTEIN 7.1 6.9 G/DL 6.0-8.0 ALBUMIN 3.5 3.2 L G/DL 3.5-5.5 GLOBULIN 3.6 3.9 G/DL 2.0-4.0 A/G 1.0 0.9 G/DL 0.6-2.4  Renal Profile BUN, CREAT, B/C, uric acid  Liver Profile  Muscle Profile enzyme abbreviation(s) tissue/organ source alkaline phosphatase ALP or ALK liver, bone osteoblasts gamma glutamyl transferase GGT or GGTP liver, pancreas transaminases: alanine aminotransferase ALT or SGPT liver (serum glutamate-pyruvate transaminase) aspartate aminotransferase AST or SGOT liver, muscle (serum glutamate-oxaloacetate transaminase) lactate dehydrogenase LDH or LD liver, muscle, RBC’s creatine kinase CK or CPK muscle  enzyme abbreviation(s) tissue/organ source alkaline phosphatase ALP or ALK liver, bone osteoblasts gamma glutamyl transferase GGT or GGTP liver, pancreas transaminases: alanine aminotransferase ALT or SGPT liver (serum glutamate-pyruvate transaminase) aspartate aminotransferase AST or SGOT liver, muscle (serum glutamate-oxaloacetate transaminase) lactate dehydrogenase LDH or LD liver, muscle, RBC’s creatine kinase CPK or CK muscle  TEST 0630 0645 UNITS REF RANGE GLUCOSE 86 82 MG/DL 70-110 BUN 29 H 13 MG/DL 9-22 CREAT 0.6 L 0.7 MG/DL 0.7-1.5 B/C 48 H 19 MG/DL 10-20 URIC ACID 3.1 L 3.0 L MG/DL 3.9-9.0 PO4 2.7 1.1 L MG/DL 2.5-4.5 CALCIUM 8.1 L 8.9 MG/DL 8.5-10.5 NA 143 140 MEQ/L 138-148 K 4.0 3.8 MEQ/L 3.5-5.0 Cl 110 102 MEQ/L 100-112 CO2 20 L 18 L MEQ/L 23-39 EBAL 17 24 H MEQ/L 8-20 TRIG 261 H 263 H MG/DL 1-160 CHOL 150 152 MG/DL 140-200 BILLI – T 30.4 H 32.6 H MG/DL 0.2–1.0 BILLI – D 16.3 H 17.7 H MG/DL 0.0-0.4 BILLI – I 14.1 H 14.9 H MG/DL 0.0-1.2 ALK 99 100 U/L 30-101 m GGT 223 H 187 H U/L 9-38 u SGPT 108 H 136 H U/L 0-45 s SGOT 309 H 339 H U/L 0-40 LDH 345 H 339 H U/L 60-230 c CPK 688 H 614 H U/L 0-255 l IRON 211 231 H MCG/DL 60-230 e PROTEIN 7.1 6.9 G/DL 6.0-8.0 ALBUMIN 3.5 3.2 L G/DL 3.5-5.5 GLOBULIN 3.6 3.9 G/DL 2.0-4.0 A/G 1.0 0.9 G/DL 0.6-2.4  Renal Profile  Liver Profile  Muscle Profile AST / SGOT, LDH, CPK enzyme abbreviation(s) tissue/organ source alkaline phosphatase ALP or ALK liver, bone osteoblasts gamma glutamyl transferase GGT or GGTP liver, pancreas transaminases: alanine aminotransferase ALT or SGPT liver (serum glutamate-pyruvate transaminase) aspartate aminotransferase AST or SGOT liver, muscle (serum glutamate-oxaloacetate transaminase) lactate dehydrogenase LDH or LD liver, muscle, RBC’s creatine kinase CK or CPK muscle enzyme abbreviation(s) tissue/organ source alkaline phosphatase ALP or ALK liver, bone osteoblasts gamma glutamyl transferase GGT or GGTP liver, pancreas transaminases: alanine aminotransferase ALT or SGPT liver (serum glutamate-pyruvate transaminase) aspartate aminotransferase AST or SGOT liver, muscle (serum glutamate-oxaloacetate transaminase) lactate dehydrogenase LDH or LD liver, muscle, RBC’s creatine kinase CK or CPK muscle  Renal Profile ALP, GGT, ALT, AST, LDH  Liver Profile  Muscle Profile  TEST 0630 0645 UNITS REF RANGE GLUCOSE 86 82 MG/DL 70-110 BUN 29 H 13 MG/DL 9-22 CREAT 0.6 L 0.7 MG/DL 0.7-1.5 B/C 48 H 19 MG/DL 10-20 URIC ACID 3.1 L 3.0 L MG/DL 3.9-9.0 PO4 2.7 1.1 L MG/DL 2.5-4.5 CALCIUM 8.1 L 8.9 MG/DL 8.5-10.5 NA 143 140 MEQ/L 138-148 K 4.0 3.8 MEQ/L 3.5-5.0 Cl 110 102 MEQ/L 100-112 CO2 20 L 18 L MEQ/L 23-39 EBAL 17 24 H MEQ/L 8-20 TRIG 261 H 263 H MG/DL 1-160 CHOL 150 152 MG/DL 140-200 BILLI – T 30.4 H 32.6 H MG/DL 0.2–1.0 BILLI – D 16.3 H 17.7 H MG/DL 0.0-0.4 BILLI – I 14.1 H 14.9 H MG/DL 0.0-1.2 L ALK 99 100 U/L 30-101 GGT 223 H 187 H U/L 9-38 I SGPT 108 H 136 H U/L 0-45 V SGOT 309 H 339 H U/L 0-40 E LDH 345 H 339 H U/L 60-230 CPK 688 H 614 H U/L 0-255 R IRON 211 231 H MCG/DL 60-230 PROTEIN 7.1 6.9 G/DL 6.0-8.0 ALBUMIN 3.5 3.2 L G/DL 3.5-5.5 GLOBULIN 3.6 3.9 G/DL 2.0-4.0 A/G 1.0 0.9 G/DL 0.6-2.4  TEST 0630 0645 UNITS REF RANGE GLUCOSE 86 82 MG/DL 70-110 BUN 29 H 13 MG/DL 9-22 CREAT 0.6 L 0.7 MG/DL 0.7-1.5 B/C 48 H 19 MG/DL 10-20 URIC ACID 3.1 L 3.0 L MG/DL 3.9-9.0 PO4 2.7 1.1 L MG/DL 2.5-4.5 CALCIUM 8.1 L 8.9 MG/DL 8.5-10.5 NA 143 140 MEQ/L 138-148 K 4.0 3.8 MEQ/L 3.5-5.0 Cl 110 102 MEQ/L 100-112 CO2 20 L 18 L MEQ/L 23-39 EBAL 17 24 H MEQ/L 8-20 TRIG 261 H 263 H MG/DL 1-160 CHOL 150 152 MG/DL 140-200 BILLI – T 30.4 H 32.6 H MG/DL 0.2–1.0 L BILLI – D 16.3 H 17.7 H MG/DL 0.0-0.4 BILLI – I 14.1 H 14.9 H MG/DL 0.0-1.2 I ALK 99 100 U/L 30-101 V GGT 223 H 187 H U/L 9-38 E SGPT 108 H 136 H U/L 0-45 SGOT 309 H 339 H U/L 0-40 R LDH 345 H 339 H U/L 60-230 CPK 688 H 614 H U/L 0-255 IRON 211 231 H MCG/DL 60-230 PROTEIN 7.1 6.9 G/DL 6.0-8.0 ALBUMIN 3.5 3.2 L G/DL 3.5-5.5 GLOBULIN 3.6 3.9 G/DL 2.0-4.0 A/G 1.0 0.9 G/DL 0.6-2.4  Renal Profile ALP, GGT, ALT, AST, LDH  Liver Profile Billi T, Billi D, Billi I  Muscle Profile  Renal Profile BUN, CREAT, B/C, uric acid  Liver Profile ALP, GGT, ALT, AST, LDH Billi T, Billi D, Billi I  Muscle Profile AST, LDH, CPK  1. Gaussian distributions  For most tests in the chemistry profile, results for the normal, healthy population will show a Gaussian distribution (bell-shaped curve).  The Gaussian distribution of results is such that:  67% of normal, healthy individuals will give a result within 1 standard deviation of the mean  95% of normal, healthy individuals will give a result within 2 standard deviations of the  99.7% of normal, healthy individuals will give a result with 3 standard deviations of the mean  Most reference ranges are established using the mean + 2 standard deviations of the mean. 2. Skewed Distributions  If a distribution of test results of a normal, healthy sample group is not Gaussian but is instead skewed ▪ values are ordered numerically ▪ the top and bottom 2.5% are dropped to establish the reference limits for that test.  Values for cholesterol in the American population do show a Gaussian distribution, with a mean around 225 mg/dl.  Cholesterol values ≥ 200 mg/dl represent an increased risk of cardiovascular disease  The reference range for cholesterol has been established using epidemiological data.  Cholesterol values above 200 mg/dl are considered to be high.  The reference range for fasting glucose is now reported from 2 SD below to 1 SD above the mean  70 – 100 mg/dl (previously 70-110 mg/dl)  Fasting blood glucose between 100 and 125 mg/dl impaired fasting glucose or pre-diabetes (Diabetes = fasting glucose 126mg/dl or above) B. Variation in Laboratory Tests  Intraindividual and interindividual variation  See information in the lecture notes. Critical Difference  Critical difference is the difference between serial test results that must occur before significance can be claimed.  Isoenzymes are distinct, variant forms of a given enzyme.  All the isoenzymes of the same enzyme catalyze the same reaction but differ in their physical properties.  Different isozymes of an enzyme may be present in different organisms, in different tissues of the same organism, and even in different cells in the same tissue.  Lactate dehydrogenase consists of 4 subunits of 2 different types, H and M.  Only tetramers have catalytic activity.  Different combinations of subunits form isozymes that differ at the level of quaternary structure.  Synthesis of H and M subunits is different in different tissues and therefore different tissues have different proportions of each of the isozymes.  Each of the 5 different isoenzymes of lactate dehydrogenase can catalyze the same chemical reaction:  isozyme  HHHH l1  HHHM l2  HHMM l3  HMMM l4  MMMM l5  Creatine kinase is active as a dimer and has two types of subunits that combine in different ways:  BB (CK1)  MB (CK2)  MM (CK3) 92 96 92 96 LD1 LD5 CK-MB  LD-1 peaks in plasma ~36- CK-MB peaks in plasma 40 hours after a ~18 hours after a myocardial infarction myocardial infarction 24 48 Infarction Hours Protein Initial Elevation Peak Return to normal I MW (Da) Protein Initial Peak Return MW Myoglobin 2-4 hours 6-9 hours 24-36 hours 16,000 CK-MB 6-10 hours 18-24 hours 1 to 3 days 82,000 *Troponin I 3-12 hours 18-24 hours 5 to 9 days 21,000 to 23,000 *Troponin T 3- 12 hours 18-24 hours By 14 days 30,000 to 35,000 LD-1 12-24 hours 36-40 hours 5 to 14 days 135,000  In an enzyme activity assay the amount of enzyme present in a sample is quantified by measuring the formation of reaction product over time.  Samples containing different and unknown enzyme concentration will be compared to standard enzyme concentrations in order to determine the amount of enzyme in each sample.  100 X enzyme R 100 e a 50 50 X enzyme c t i 3 X enzyme o 3 n 2 X enzyme 2 V e 1 X enzyme l 1 o c i t y [Substrate] Therefore…  The more enzyme present, the greater the reaction velocity.  Following the same trend, a lesser amount of enzyme will result in a reduced rate of product formation.  Unwanted effects on enzyme activity may be produced by: pH, temperature, cofactors /coenzymes, activators and inhibitors.  Measurement of enzymes by activity assay ▪ When studying, please note similarities to Michaelis-Menten plots that we covered in Lecture 2 ▪ Now we are varying enzyme concentration to determine how much enzyme is present in a patient’s sample  The amount of enzyme present is measured using an antibody that recognizes and binds to the enzyme.  The antibody is labeled and measurement of the amount of bound, labeled antibody will correlate to the amount of enzyme present.  The enzyme does not have to be functional to be detected immunologically so the unwanted effects on activity are eliminated.  Isoenzymes that originate in different tissues may be distinguished from each other using immunological assays.  In electrophoretic separation charged particles are separated from each other in an electric field.  This technique is useful in separating isoenzymes of a given enzyme which will each have slightly different charges.  In this type of test, the protein (enzyme) sample is placed on a solid support (a gel) which is saturated with a buffer at a specific pH, usually alkaline (above 7).  Most proteins are negatively charged at alkaline pH and migrate toward the positive pole (anode).  For creatine kinase all 3 isozymes have a different charge at pH 8.6 and will migrate to 3 distinct regions. CK LDH