MLAB 2401: Clinical ChemistryKeri Brophy-Martinez

Measurement of Enzymes & Their Clinical Significance

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

Measurement of Enzyme Activity

Fixed time• 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

Continuous Monitoring/Kinetic• Multiple measurements of

absorbance change are made

• Advantages– Depletion of substrate is

observable– Improved accuracy

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

Other Methods to Measure Enzymes

• Using Enzyme Mass– Measure protein mass NOT catalytic activity

• Electrophoresis– Used to differentiate isoenzymes– Time-consuming

ENZYMES OF CLINICAL SIGNIFICANCE

Creatine Kinase (CK)

• Action– Associated with the regeneration and storage of

ATP– Catalyses the following reaction:

Creatine Kinase (CK)

• Purpose– 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

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

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

CK Isoenzymes

What’s in a Number?

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

Creatine Kinase: Reference Range

• Affected by:– Age – Physical activity – Race – Bed rest (even overnight can decrease CK)

• Total CK– Men: 46-180 U/L– Female: 15-171 U/L

Creatine Kinase

• Isoenzyme Testing– Fractionation of CK

• Immunoinhibition• Mass Assay• Electrophoresis

Lactate Dehydrogenase (LDH/LD)

• Action– Catalyzes a reversible reaction between pyruvate

and lactate with NAD as a coenzyme

– Reaction:

Lactate Dehydrogenase (LDH/LD)

• Source– Skeletal muscle– Cardiac muscle– Kidney– RBCs– Widely distributed in the body

Lactate Dehydrogenase (LDH/LD):Structure

• Tetramer– Four polypeptide chains, two subunits (heart &

muscle)– Five combinations of Isoenzymes

Lactate Dehydrogenase (LDH/LD)

• Diagnostic Significance– Nonspecific– Increased• Hematologic and neoplastic disorders • Liver disease • Heart problems

Lactate Dehydrogenase (LDH/ LD):Specimen Collection

• Sources of Error– Hemolysis• RBCs contain 100-150 times that found in serum

– Analyte stability• Run assay asap or store at room temperature

– Prolonged contact of serum and cells• Reference Range

• 140- 280 U/L

Liver Enzymes

• Transaminases– AST– ALT

• Phosphatases– ALP

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)

Aspartate Aminotransferase( AST)

• Sources– Major• Heart• Liver• Muscle

– Minor• RBCs• Kidney• Pancreas • Lung

Aspartate Aminotransferase( AST)

• Reaction:AST

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

Alanine Transaminase (ALT)

• Transfers an amino group from alanine to alpha-ketoglutarate to form glutamate and pyruvate ALT

Alanine Transaminase (ALT)

• Sources– Liver (Majority)– Kidney– Heart– Skeletal muscle

ALT:Specimen Collection

• Sources of Error– Hemolysis – Analyte stability• 3-4 days refrigerated

• Reference Range– 6-37 U/L

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

Phosphatases

• Removes phosphates from organic compounds

• Functions to facilitate transfer of metabolites across cell membranes

• Alkaline Phosphatase (ALP)• Acid Phosphatase (ACP)

Phosphatases: Sources

Alkaline Phosphatase (ALP)

• Bone • Liver• Kidney• Placenta• Intestines

Acid Phosphatase (ACP)

• Prostate gland• Seminal fluid• Liver• Spleen• RBCs• Platelets

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 9.0- 10.0

Alkaline Phosphatase (ALP)

• Diagnostic Significance– Elevations seen in• During bone activity

– Paget’s disease• Liver disease, especially in obstructive disorders• Pregnancy between 16-20 weeks gestation

– Decreased levels occur, but not diagnostic

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

Acid Phosphatase (ACP)

• Diagnostic Significance– Aids in detection of prostatic carcinoma– Other conditions associated with prostate– Forensic chemistry: Rape cases– Elevated in bone disease

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

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

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

GGT:Specimen Collection

• Sources of Error– Very stable– Hemolysis not an issue

• Reference Range– Male: 10-34 U/L– Female: 9-22 U/L

Digestive & Pancreatic Enzymes

• Amylase• Lipase

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

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

Amylase

• Sources of Error– Presence of opiates increases levels– Stabile

• Reference Range– Serum: 30-100 U/L– Urine: 1-17 U/h

Lipase (LPS)

• Hydrolyzes triglycerides to produce alcohols and fatty acids

• Source– Pancreas

Lipase (LPS)

• Diagnostic Significance–Acute pancreatitis•More specific than amylase• LPS persists longer than AMS

Lipase:Specimen Collection

• Sources of Error– Stabile– Hemolysis results in false decreases

• Reference Range– 13-60 U/L

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 .