Mlab 2401 clinical chemistry keri brophy martinez
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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

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Mlab 2401 clinical chemistry keri brophy martinez

MLAB 2401: Clinical ChemistryKeri Brophy-Martinez

Measurement of Enzymes & Their Clinical Significance


Measurement of enzyme activity

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 activity1

Measurement of Enzyme Activity

Fixed time

Continuous Monitoring/Kinetic

Multiple measurements of absorbance change are made

Advantages

Depletion of substrate is observable

Improved accuracy

  • 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


Reporting enzyme activity

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

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

Enzymes of Clinical Significance


Creatine kinase ck

Creatine Kinase (CK)

  • Action

    • Associated with the regeneration and storage of ATP

    • Catalyses the following reaction:


Creatine kinase ck1

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

CreatineKinase (CK):Structure

  • Dimerconsisting 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 ck2

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

CK Isoenzymes


What s in a number

What’s in a Number?


Creatine kinase specimen collection

CreatineKinase: Specimen Collection

  • Sources of Error

    • Hemolysis

      • Interference of adenylatekinase on CK assays

      • Results in false elevations

    • Exposure to light

      • CK is inactivated by light


Creatine kinase reference range

CreatineKinase: 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

Creatine Kinase

  • Isoenzyme Testing

    • Fractionation of CK

      • Immunoinhibition

      • Mass Assay

      • Electrophoresis


Lactate dehydrogenase ldh ld

Lactate Dehydrogenase (LDH/LD)

  • Action

    • Catalyzes a reversible reaction between pyruvate and lactate with NAD as a coenzyme

    • Reaction:


Lactate dehydrogenase ldh ld1

Lactate Dehydrogenase (LDH/LD)

  • Source

    • Skeletal muscle

    • Cardiac muscle

    • Kidney

    • RBCs

    • Widely distributed in the body


Lactate dehydrogenase ldh ld structure

Lactate Dehydrogenase (LDH/LD):Structure

  • Tetramer

    • Four polypeptide chains, two subunits (heart & muscle)

    • Five combinations of Isoenzymes


Lactate dehydrogenase ldh ld2

Lactate Dehydrogenase (LDH/LD)

  • Diagnostic Significance

    • Nonspecific

    • Increased

      • Hematologic and neoplastic disorders

      • Liver disease

      • Heart problems


Lactate dehydrogenase ldh ld specimen collection

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

Liver Enzymes

  • Transaminases

    • AST

    • ALT

  • Phosphatases

    • ALP


Transaminases

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

Aspartate Aminotransferase( AST)

  • Sources

    • Major

      • Heart

      • Liver

      • Muscle

    • Minor

      • RBCs

      • Kidney

      • Pancreas

      • Lung


Aspartate aminotransferase ast1

Aspartate Aminotransferase( AST)

  • Reaction:

AST


Ast specimen collection

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

Alanine Transaminase (ALT)

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

ALT


Alanine transaminase alt1

Alanine Transaminase (ALT)

  • Sources

    • Liver (Majority)

    • Kidney

    • Heart

    • Skeletal muscle


Alt specimen collection

ALT:Specimen Collection

  • Sources of Error

    • Hemolysis

    • Analyte stability

      • 3-4 days refrigerated

  • Reference Range

    • 6-37 U/L


Diagnostic significance ast alt

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

Phosphatases

  • Removes phosphates from organic compounds

  • Functions to facilitate transfer of metabolites across cell membranes

  • Alkaline Phosphatase (ALP)

  • Acid Phosphatase (ACP)


Phosphatases sources

Phosphatases: Sources

Alkaline Phosphatase (ALP)

Acid Phosphatase (ACP)

Prostate gland

Seminal fluid

Liver

Spleen

RBCs

Platelets

  • Bone

  • Liver

  • Kidney

  • Placenta

  • Intestines


Alkaline phosphatase alp

Alkaline Phosphatase (ALP)

  • ALP frees inorganic phosphate from an organic phosphate monoester, resulting in the production of an alcohol at an alkalinepH

  • Maximum activity at pH of 9.0- 10.0


Alkaline phosphatase alp1

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

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

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

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

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 ggt1

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

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

Digestive & Pancreatic Enzymes

  • Amylase

  • Lipase


Amylase ams

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 ams1

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

Amylase

  • Sources of Error

    • Presence of opiates increases levels

    • Stabile

  • Reference Range

    • Serum: 30-100 U/L

    • Urine: 1-17 U/h


Lipase lps

Lipase (LPS)

  • Hydrolyzes triglycerides to produce alcohols and fatty acids

  • Source

    • Pancreas


Lipase lps1

Lipase (LPS)

  • Diagnostic Significance

    • Acute pancreatitis

      • More specific than amylase

      • LPS persists longer than AMS


Lipase specimen collection

Lipase:Specimen Collection

  • Sources of Error

    • Stabile

    • Hemolysis results in false decreases

  • Reference Range

    • 13-60 U/L


References

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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