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MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

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1 MLAB 2401: Clinical Chemistry Keri Brophy-Martinez
Measurement of Enzymes & Their Clinical Significance

2 Measurement of Enzyme Activity
Often measured by catalytic activity then related to concentration Enzyme concentration is best measured by its activity or its rate of activity by observing: Substrate depletion Product production Increase/decrease in cofactor/coenzyme Usually performed in zero-order kinetics

3 Measurement of Enzyme Activity
Fixed time Continuous Monitoring/Kinetic Measurement of the amount of substrate utilized over a fixed amount of time or by a fixed amount of serum Problems Long incubation times Possible enzyme denaturation Often a lag phase Possible substrate depletion Multiple measurements of absorbance change are made Advantages Depletion of substrate is observable Improved accuracy

4 Reporting Enzyme Activity
Originally reported as activity units IUB standardized these as international units (IU) IU: the amount of enzyme that will convert one micromole of substrate per minute in an assay system Expressed as units per liter or U/L Conditions: pH, temperature, substrate,activators Katal units(SI): express as moles/second

5 Other Methods to Measure Enzymes
Using Enzyme Mass Measure protein mass NOT catalytic activity Electrophoresis Used to differentiate isoenzymes Time-consuming

6 Enzymes of Clinical Significance

7 Creatine Kinase (CK) Action
Associated with the regeneration and storage of ATP Catalyses the following reaction:

8 Creatine Kinase (CK) Purpose Source
Allows the body to store phosphate energy as creatine phosphate Energy can be released/ provided to muscles by reversing the reaction Source Skeletal muscle Heart Brain Other

9 Creatine Kinase (CK): Structure
Dimer consisting of two subunits Two subunits are further divided into 3 molecular forms or isoenzymes CK-BB: (CK-1)brain type Migrates fast on electrophoresis Small amount found in tissue (brian, lung, bladder, bowel) CK-MB: (CK-2)hybrid type Heart, Skeletal CK-MM: (CK-3)Muscle type Mostly found in healthy people Striated muscle and normal serum

10 Creatine Kinase (CK) Diagnostic Use
Appearance of CK (MB) very sensitive indicator of MI Skeletal muscle disorders such as muscular dystrophy CNS Disorders Cerebrovascular accident(CVA) Seizures Nerve degeneration

11 CK Isoenzymes

12 What’s in a Number?

13 Creatine Kinase: Specimen Collection
Sources of Error Hemolysis Interference of adenylate kinase on CK assays Results in false elevations Exposure to light CK is inactivated by light

14 Creatine Kinase: Reference Range
Affected by: Age Physical activity Race Bed rest (even overnight can decrease CK) Total CK Men: U/L Female: U/L

15 Creatine Kinase Isoenzyme Testing Fractionation of CK Immunoinhibition
Mass Assay Electrophoresis

16 Lactate Dehydrogenase (LDH/LD)
Action Catalyzes a reversible reaction between pyruvate and lactate with NAD as a coenzyme Reaction:

17 Lactate Dehydrogenase (LDH/LD)
Source Skeletal muscle Cardiac muscle Kidney RBCs Widely distributed in the body

18 Lactate Dehydrogenase (LDH/LD): Structure
Tetramer Four polypeptide chains, two subunits (heart & muscle) Five combinations of Isoenzymes

19 Lactate Dehydrogenase (LDH/LD)
Diagnostic Significance Nonspecific Increased Hematologic and neoplastic disorders Liver disease Heart problems

20 Lactate Dehydrogenase (LDH/ LD): Specimen Collection
Sources of Error Hemolysis RBCs contain times that found in serum Analyte stability Run assay asap or store at room temperature Prolonged contact of serum and cells Reference Range U/L

21 Liver Enzymes Transaminases AST ALT Phosphatases ALP

22 Transaminases Retain amino groups during the degradation of amino acids Types Aspartate transaminase (AST) Aka: Glutamic Oxalocetic transaminase (SGOT) Alanine transaminase (ALT) AKA: Glutamic pyruvic transaminase (SGPT)

23 Aspartate Aminotransferase( AST)
Sources Major Heart Liver Muscle Minor RBCs Kidney Pancreas Lung

24 Aspartate Aminotransferase( AST)
Reaction: AST

25 AST: Specimen Collection
Sources of Error Hemolysis Analyte stability Stable at room temp for 48 hours or 3-4 days refrigerated Reference Range 5-30 U/L

26 Alanine Transaminase (ALT)
Transfers an amino group from alanine to alpha-ketoglutarate to form glutamate and pyruvate ALT

27 Alanine Transaminase (ALT)
Sources Liver (Majority) Kidney Heart Skeletal muscle

28 ALT: Specimen Collection
Sources of Error Hemolysis Analyte stability 3-4 days refrigerated Reference Range 6-37 U/L

29 Diagnostic Significance: AST & ALT
Many diseases can cause increases since widely distributed in tissues Liver Hepatitis Cirrhosis Liver cancer Myocardial Infarction AST increases most ALT normal to slightly increased, unless liver damage accompanies Other Pulmonary emboli Muscle injuries Gangrene Hemolytic diseases Progressive Muscular dystrophy

30 Phosphatases Removes phosphates from organic compounds
Functions to facilitate transfer of metabolites across cell membranes Alkaline Phosphatase (ALP) Acid Phosphatase (ACP)

31 Phosphatases: Sources
Alkaline Phosphatase (ALP) Acid Phosphatase (ACP) Bone Liver Kidney Placenta Intestines Prostate gland Seminal fluid Liver Spleen RBCs Platelets

32 Alkaline Phosphatase (ALP)
ALP frees inorganic phosphate from an organic phosphate monoester, resulting in the production of an alcohol at an alkaline pH Maximum activity at pH of

33 Alkaline Phosphatase (ALP)
Diagnostic Significance Elevations seen in During bone activity Paget’s disease Liver disease, especially in obstructive disorders Pregnancy between weeks gestation Decreased levels occur, but not diagnostic

34 Alkaline Phosphatase (ALP): Specimen Collection
Sources of Error Hemolysis Delay in processing, false increases can occur Reference Range (Adult) 30-90 U/L NOTE: Normal increases seen in pregnancy, childhood, adolescence

35 Acid Phosphatase (ACP)
Diagnostic Significance Aids in detection of prostatic carcinoma Other conditions associated with prostate Forensic chemistry: Rape cases Elevated in bone disease

36 Acid Phosphatase (ACP): Specimen Collection
Sources of Error Separate serum from cells asap Decrease in activity seen at room temp Hemolysis Reference Range (prostatic) 0-4.5 ng/mL

37 Gamma-Glutamyltransferase (GGT)
Possibly involved in peptide and protein synthesis, transport of amino acids and regulation of tissue glutathione levels Sources Kidney Brain Prostate Pancreas Liver

38 Gamma-Glutamyltransferase (GGT)
Diagnostic Significance Sensitive indicator of liver damage Increased in patients taking enzyme-inducing drugs such as warfarin, phenobarbital and phenytoin Indicator of alcoholism Elevated in acute pancreatitis, diabetes mellitus and MI

39 GGT: Specimen Collection
Sources of Error Very stable Hemolysis not an issue Reference Range Male: U/L Female: 9-22 U/L

40 Digestive & Pancreatic Enzymes
Amylase Lipase

41 Amylase (AMS) Digestive enzyme that hydrolzes/catalyzes the breakdown of starch and glycogen to carbohydrates Smallest enzyme Sources Acinar cells of pancreas and salivary glands

42 Amylase (AMS) Diagnostic Significance Acute pancreatitis
AMS levels rise 2-12 hours after onset of attack, peak at 24 hrs and return to normal within 3-5 days Salivary gland lesions Mumps

43 Amylase Sources of Error Reference Range
Presence of opiates increases levels Stabile Reference Range Serum: U/L Urine: 1-17 U/h

44 Lipase (LPS) Hydrolyzes triglycerides to produce alcohols and fatty acids Source Pancreas

45 Lipase (LPS) Diagnostic Significance Acute pancreatitis
More specific than amylase LPS persists longer than AMS

46 Lipase: Specimen Collection
Sources of Error Stabile Hemolysis results in false decreases Reference Range 13-60 U/L

47 References Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins. Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .

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