Clinical Chemistry Ii - Enzymology.docx

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY CLINICAL ENZYMOLOGY MUSCLE ENZYMES Enzyme

Biochemistry    

Creatine Kinase

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  Aldolase

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 Glycogen Phosphorylase

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Dimeric (2 subunits) each subunit weighs 40000 Da Subunits B & M product on Chromosomes 14 and 19; both have a C-Terminal Lysine Catalyze reversible phosphorylation of Creatine by Adenosine Triphosphate When muscle contracts, catalyzes rephosphorylation of ADP to ATP using Creatine Phosphate as phosphorylation reservoir Isoenzymes  BB (CK-1) – Brain  MB (CK-2) - Heart  MM (CK-3) – Skeletal Muscles  CK-MM1 – No Lysine Residues  CK-MM2 – One Lysine Residue  CK-Mt – Chrom15; Heart;  Macro-CK – complexed with Ig  Type 1 – CK-BB+IgG | CK-MM+IgA  Type 2 – Oligomeric CK-Mt Tetrameric Catalyze splitting of D-fructose-1,6diphosphate to D-glyceraldehyde-3phosphate and dihydroxyacetone-phosphate Determined by three separate gene loci but only two appear to be active simultaneously in most tissues Dimer with two identiocal subunits each weighs 97 000 Da Catalyzes first step in glycogenolysis (Glycogen  Glucose-1-phosphate)

Clinical Significance  

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Activity greatest in Striated Muscle and Heart SERUM CK ↑ in injury, inflammation, necrosis of muscle tissue, intravascualr injections, surgical interventions leading to muscle trauma, normal childbirth, statins, fibrates, antoiretroviral drugs, angiotensinogen II receptor antagonists Greatly ↑ in muscular dystrophies 5x ↑ = Myopathy, Hypothyroidism 200x ↑ = Acute Rhabdomyolysis (crash injury) Not ↑ in Neurogenic Muscle Disorders  Myasthenia Gravis  Multiple Sclerosis  Poliomyelitis & Parkinsonism

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Serum Activity is due almost exclusively to CK-MM activity CK-BB elevated in neonates with brain damage or in very-low birth weight newborns In Endocrine Myopathy, the usual cause is Hypothyroidism, and the major isozyme present is CK-MM. Type 2 Macro-CK is found predominantly in adults with severe illness with malignancy or liver disease and in children with notable tissue distress CK-MB ↑ in Myocardial Infarction and Myocardial Necrosis

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Primary Diseases of Skeletal Muscle Distinguish Neuromusculat atrophies from myopathies Not routinely available

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GP-BB more sensitive than CK-MB and myoglobin for diagnosing myocardial infarction within the first 4 hourss after onset of chest pain. GP-BB is still not heart-specific and its use is limited

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Notes

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Not found in the liver and RBC Neutral pH, formaton of ATP pH 9  CrP Mg2+ is an obligate activating ion Inhibited by  Mn2+, Ca2+, Zn2+, Cu2+  Excess ADP, Citrate, Nitrate, Acetate  F-, I-, Br-, Cl-, Sulfate  Malonate, L-Thyroxine  Urate & Cystine (potent) Ck-Mt – between inner&outer mem of mitochondria

Method of Measurement is Manual ELISA with URL: 10 µg/L

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UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY  

Provide fuel for the energy supply required for muscle contraction Isoenzymes  GP-LL – Brain  GP-BB – Myocardium, Heart  GP-MM – Heart

MUSCLE EZYMES | ANALYTICAL METHODS CREATINE KINASE NAME Tanzer-Gilvarg Assay

Oliver-Rosalki

SUBSTRATE

Reactions

Reagent

NOTES

Creatine and ATP

Creatine + ATP  Creatine-PO4 + ADP ADP + PEP  Pyruvate + ATP Pyruvate + NADH  Lactate + NAD

Creatine Phosphokinase Pyruvate Kinase Lactate Dehydrogenase

Direct Method pH 9.0 340 nm

Creatine Phosphate and ADP

Creatine PO4 + ADP  Creatine + ATP ATP + Glucose  ADP + G6P G6P + NADP  6-Phosphogluconate + NADPH

Creatine Phosphokinase Hexokinase G6PD

Indirect Method pH 6.8 340 nm Most commonly used faster rxn

Szasz Modification of Oliver-Rosalki N-Acetylcysteine – Enzyme Activator EDTA – Chelate Calcium and Increase Sability of reaction Mixture Adenosine Pentaphosphate – Inhibit Adenylate Kinase; in addition to AMP SPECIMEN AND OTHER CONSIDERATIONS 1. 2. 3. 4. 5.

Hemolysis must be avoided as Adenylate Kinase released from RBC lysis will interfere with the assay affecting lag phase and side reactions. Use Serum and Plasma Heparin Stability: < 8 hrs at RT ; 48 hrs at 4C ; 1 month -20C ; > 1 month -80C Imidazole serves as buffer. Urate and Cysteine are potent inhibitors of CK

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UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY 6. CK is light and pH sensitive and is lost with excessive storage. 7. Cleland’s Reagent and Glutathione partially restores lost CK activity. 8. Electrophoresis is the reference method although CK mass unit assays are more sensitive METHODS OF SEPARATING THE ISOENZYMES ELECTROPHORESIS Isozyme Bands Visualized by Incubating the support with a concentrated CK assay mixture using the reverse reaction NADPH formed detected by observing Bluish-White Fluorescence after excitation by longwave UV light at 360 nm Quantified using Fluorescent Densitometry (detect bands of 2 to 5 U/L) Migration at pH 8.6 towards the Anode (+) | MM  MB  BB Abnormal Bands| Macro-CK  CK3 Isoforms  Macro-CK Type 2  CK2 Isoform  Fluorescent Albumin Monoclonal Antibody Sandwich Immunoassay : For CK-MB only ALDOLASE NAME Beisenherz Method

SUBSTRATE

Reactions

Reagent

D-Fructose-1,6Diphosphate (F16DP)

F16DP  D-Glyceraldehyde-3-Phosphate (GLAP) + Dihydroxyacetone Phosphate (DAP) GLAP  DAP DAP + NADH + H+  Glycerol-3-Phosphate + NAD+

Aldolase Triosephosphate Isomerase Glycerol-3-Phosphate Dehydrogenase

NOTES Plasma Sample Avoid Hemolysis Platelet Interference Ambient (48 hrs) Several days (4C)

Reference Interval: 2.5 to 10 U / L

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UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY LIVER ENZYMES Enzyme

Biochemistry 

  Aspartate Aminotransferase

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(SERUM GLUTAMIC OXALOACETIC TRANSAMINASE)

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AST | SGOT

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5-30 U/L

AST: Transfers amino group from Aspartate to an α-ketoacid Pyridoxal-5’-Phosphate and its amino analog pyridoxamine-5’phosphate serves as coenzymes (Prosthetic Group) AST/ALT Ratio – reflect grade of fibrosis in cirrhotic patients

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Alanine Aminotransferase

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ALT: Transfers amino group from Alanine to an α-ketoglutarate

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Clinical Significance Acute Myocardial Infarction  Begins to Rise 6-8 hrs  Peak at 24 hrs  Normalize within 5 days 3x Increase : Pulmonary Emboli Slight to Moderate AST ↑  Acute Pancreatitis  Crushed Muscle Injury  Hemolytic Disease 8x AST: Progressive Muscular Dystrophy and Dermatomyositis AST ↑ > ALT ↑  Alcoholic Hepatitis  Cirrhosis (4-5x both) o ratio > (reduced ALT production and reduced AST clearance)  Liver Neoplasia  Primary/Metastatic Carcinoma of Liver (2-5x Both) Mitochondrial-AST in extensive liver cell degeneration and necrosis Macro-AST no significance Reduced in UREMIA

7x ↑ ALT : most efficient for Liver Injury Reduced in Cirrhosis

Analysis  2 Oxaloglutarate + L-aspartate (in the presence of AST)  L-glutamate + oxaloacetate Oxaloacetate + NADH + H+(in the presence of Malate dehydrogenase)  L-Malate + NAD.  Karmen Method- coupled enzyme assay; kinetic assay; rate reaction principle; measures the decrease in absorbance; Optimum pH of 7.3-7.8; MDH serves as an indicator  Reitman –Frankel- colorimetric method (observation of a brown solution| @ 505 nm); reaction with DNPH (ketoacids is reacted to 2,4dinitrophenyl-hydrazine to form ketoacid hydrazines in the presence of NaOH)  Coupling with Diazonium Salts Sources of Error: Elevated Hemolysis, bilirubin, aceto-acetate and n-acetyl compounds, p-aminophenol, sulfthiazole, isoniazid, ascorbic acid, and lipemia Decreased mercury, cyanide and fluoride   Karmen Method- coupled enzyme assay; kinetic assay; rate reaction principle; measures the decrease in absorbance; Optimum pH of 7.3-7.8; MDH serves as an indicator  Reitman –Frankel- colorimetric method (observation of a brown solution| @ 505 nm); reaction with DNPH (ketoacids is reacted to 2,4dinitrophenyl-hydrazine to form ketoacid hydrazines in the presence of NaOH)

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UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

Glutamate Dehydrogenase

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  Alkaline Phosphatase

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Zinc-Containing Six polypeptide chains Catalyzes removal of Hydrogen from L-Glutamate to form the corresponding ketimino-acid which undergoes spontaneous hydrolysis to 2-oxoglutarate

Catalyzes alkaline hydrolysis of a large variery of naturally occurring and synthetic substances Liberates inorganic phosphate from an organic phosphate ester Isoenzymes (Chrom 1)  Liver  Bone  Placental  Intestinal  Renal Carcinoplacental ALP (Chrom 2)  Regan (Placental)  Nagao (Germ Cell)  Kasahara (Fetal Intestinal)

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↑ due to hepatocellular damage 2x ↑ : Cirrhosis 4-5x ↑ : Chronic Hepatitis Large ↑: Halothane Toxicity (cause ischemia  centrilobular necrosis) Together with m-AST, it is used in estimating severity og liver cell damage.  10-12x Elev  Extrahepatic Biliary Obstruction  Complete Obstrictions  Advnced Primary ALP Activity in the sera originates Liver Cancer mainly from the Liver ALP with most of  Widespread the rest from skeleton. Secondary Normal to Moderate Elevation Hepatic  Infectious hepatitis Metastases  Liver diseases affecting  Transient, parenchymal cells Benign Elev in  Reaction to drug therapy Children and 2-3X Elev infants  Third Trimester of Pregnancy  ↑ Intestinal ALP seen in Liver Cirrhosis  Bone ALP elev due to osteoblastic activity and bone diseases

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NAD+ - preferred coenzyme NADP+ - Hydrogen Acceptor Inhibited by Metal Ions (Ag+ and Hg+) Chelating Agents, and L-Thyroxine More concentrated on lobules

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Measured in fasting state Methodology described on BONE ENZYMES section

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UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY 

5’-Nucleotidase

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Acts on Nucleoside-5’-Phosphates such as AMP and Adenylic Acid, releasing inorganic phosphates Glycoprotein widely distributed principally localized in cytoplasmic membrane of cells Optimum pH is between 6.6 and 7.0

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 γ – Glutamyl transferase

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Catalyze transfer of gammaglutamyl group from peptides and compounds to an acceptor.

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Serum NTP activities reflect hepatobiliary disease with considerable specificity. 3-6x Elev : Biliary Obstruction Moderate : Parenchymal Cell Damage as in infectious Hepatitis Elevation is very often associated with liver disease even if ALT is normal. Normal Level of NTP with High ALT indicates Bone Disease  Small Increase: Non-Alcoholic Fatty Liver Disease  Transient Increase: Drug Sensitive indicator of hepatobiliary Intoxication disease altough not specific of the  Elevated in cause. Alcoholic 5-30x Elevation : Hepatobiliary Hepatitis and Obstruction those receiving 5-15x: Acute and Chronic Pancreatitis anticonvulsants and malignancies especially if such as associated with hepatobiliary phenytoin and obstruction phenobarbital High Elevations:  Elevated in Primary/Secondary(Metastatic) Liver Heavy/Chronic Neoplasm Drinkers Moderate (2-5x) : Infectious Hepatitis  Acute Myocardial Infarction Elev on 4th Day, max after another 4 days

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Glycyglycine 5x more effective as an acceptor than glycine or the tripeptide gly-gly-gly Critical for maintainance of intracellular levels of reduced glutathione Serum Activity primarily from the liver Abundance (Decreasing) 1. Proximal Renal Tubule 2. Liver 3. Pancreas 4. Intestine

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UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY  

Glutathione STransferase 

Dimeric Catalyzes nucleophilic addition of glutathione to the electrophilic centers of a wide variety of chemical structures, accomplishing detoxification reactions Four Main Classes  α – high conc in liver  µ  π  ϴ

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α – GST is released quickly and in large quantities from damaged hepatocytes into bloodstream. Evenly distributed across the liver and is released in all types of hepatocellular damage Valueable than AST in detecting early rejectiion episodes postoperatively in liver transplants Less susceptible to the confounding effects of infections

PANCREATIC ENZYMES Enzyme

Biochemistry 

   α-Amylase

Catalyzes hydrolysis of 1,4-αglycosidic linkages in polysaccharides Calcium Metalloenzymes Optimum pH is at 6.9 to 7.0 Isoenzymes  S-Amylase - Ptyalin - Action terminated by acid in stomach - Inititate hydrolysis of starch  P-Amylase - Released by acinar cells - Nonglycosylated

Clinical Significance  

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Acute Pancreatitis rise within 5-8 hrs of symptom onset, returns normal by 3rd or 4th day. Max conc in 12 to 72 hrs  Greater increaase in Urine AMY and will persist longer than in serum Increases in abdominal disorders and extrapancreatical conditions 4x P-AMY: Biliary Tract Diseases 4x or more: Intra-abdominal events Renal Insufficiency: Increase is prop to extent of renal impairment uusally no more than 5x (both S and P)

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Pancreatic and Intra-Abdominal Diseases – Elev P-AMY Genitourinary Diseases – Elev S-AMY  Ectopic Pregnancy  Salpingitis  Ovarian Malignancy Miscellaneous (S-AMY)  Salivary Gland Lesions  Acute alcohol Abuse  Diabetic Ketoacidosis  Septic Shock  Cardiac Surgery  Tumors  Drugs Macroamylasia – maybe S or P

Notes

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Full activity is displayed only in presence of Chloride, Bromide, Nitrate, Cholate, or Monohydrogen Phosphate Bromide and Chloride as the most effective activators The only Plasma enzyme found in the urine because it is able to pass thorugh the glomeruli (MW 54 000 to 62 000 Da)

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UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Lipase 

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Trypsin  

Single chain glycoprotein with MW of 48 000 Da and IpH of 5.8 Chromosome 10

Hydrolyzes peptide bonds formed by carboxyl groups of lysine or arginine with other amino acids although esters and amides involving these amino acids split more rapidly than peptide bonds Zymogens: Trypsinogen 1 (cationic) and 2 (anionic) Converted to Trypsin by Enterokinase in the Intestinal Tract

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Acute Pancreatitis : Inc within 4-8 hrs after attack, peak at about 24 hrs return to normal within 7 to 14 days (2-50 x elevation) > 3 x , absence of renal failure = Pancreatitis

Normal: TRY 1 = 2x-4x TRY-2 Pancreatic Disease: Ratio is reversed TRY-1 elevated in Chronic Renal Failure TRY-2 10xmore Elev in Acute Pancreatitis and larger amounts excreted in urine. Urine TRY-2 high specificity and Negative Predictive Value but Low PPV

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More pancreas specific Cocnentration in pancreas >5000x than in other tissues

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TRY-1 : 25,800 Da pI = 4.6 to 6.5 TRY-2: 22,900 Da pI > 6.5 TRY-1 Major Form found in serum TRY-2 Rapidly goes autolysis at neutral pH and not stabilized by calcium ions

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BONE ENZYMES Enzyme

Biochemistry

Clinical Significance

Notes

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UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

ACID PHOSPHATASE

- =

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BONE ENZYMES | ANALYTICAL METHODS SUBSTRATE

METHOD

REACTION

NOTES

Acid Phosphatase A-NAPTHYLPHOSPHATE

BABSON-READ & PHILLIPS

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