Measurement of Enzymes and Their Clinical Significance

Report
MLAB 2401: Clinical Chemistry
Keri 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
Continuous Monitoring/Kinetic
• Measurement of the
• Multiple measurements of
amount of substrate utilized
absorbance change are
over a fixed amount of time
made
or by a fixed amount of
• Advantages
serum
– Depletion of substrate is
observable
• Problems
–
–
–
–
Long incubation times
Possible enzyme denaturation
Often a lag phase
Possible substrate depletion
– 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
ALT
pyruvate
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)
Acid Phosphatase (ACP)
•
•
•
•
•
•
•
•
•
•
•
Bone
Liver
Kidney
Placenta
Intestines
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 .

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