1. Dr. Manal Basyouni Cardiac Markers 1Dr. Manal Basyouni

2. Objectives List the main cardiac markers (SGOT, LDH, CPK) and their isoenzyme. Explain the importance of cardiac marker in diagnosis and monitoring of diseases. Describe the principles of the tests estimating SGOT, LDH, CPK and perform them. 2 Dr. Manal Basyouni

3. Cardiac markers * Definition : Cardiac marker tests; identify blood chemicals (IC proteins) that are elevated in cardiac damage e.g. myocardial infarction (MI). These markers are not normally present in blood (non function plasma enzymes). Diagnostic Importance: In diagnosis of MI : In most patients; ECG is non diagnostic. Their levels are correlated with infarction size & prognosis. Monitoring of treatment. Types: 1.Myoglobin. 2.Creatine phosphokinase (CPK total, CK-MB). 3.Aspartate Transferase (SGOT or AST). 4.Cardiac Troponin (TnT, TnI). 5.Lactate dehydrogenase (LDH). 3Dr. Manal Basyouni

4. 1- myoglobin: It is IC O2 binding protein in striated & cardiac muscles. It has low MW, release into circulation after MI. Time sequence: It released ; in 1-2 hrs of MI (early diagnosis). peak: 6-8 hrs. persists for: up to 1day. Time sequence: It released ; in 1-2 hrs of MI (early diagnosis). peak: 6-8 hrs. persists for: up to 1day. Significance:  Sensitive marker.  Non specific: increase in skeletal injury & renal failure.  Rapid clearance, to rule out MI from re-infarction. It detected by immunoassays (ELISA) using specific monoclonal antibodies against cardiac isoforms. 4Dr. Manal Basyouni

5. 2- Creatine Kinase (CK-MB) test: Creatine kinase is an IC enzyme responsible for transferring a phosphate group from ATP to creatine. Isoenzymes: It is composed of M and/or B subunits that form CK-MM (skeletal muscle), CK-MB (heart muscle), and CK-BB isoenzyme (brain). Total CK is not myocardial-specific (increase in muscle trauma, dystrophy, convulsion, …..).  MB isoenzyme (CK-2) comprises about 40% of the CK activity in cardiac muscle and 2% or less of the activity in most muscle groups and other tissues.  These isoenzymes can be separated by electrophoresis or chromatography or immuno-inhibition. Time sequence: It released ; in 3–4 hrs of MI. peak: 12 hrs. persists for: up to 3-4days. Time sequence: It released ; in 3–4 hrs of MI. peak: 12 hrs. persists for: up to 3-4days. 5Dr. Manal Basyouni

6. Quantitative determination of total Creatine kinase (CK) activity in serum Creatine phosphate+ ADP CK Creatine + ATP ATP + D-Glucose Glucose 6-P + ADP Hexo Kinase Glucose 6-P + NADP6- phosphogluconate + NADPH +H G-6-phospho-hehydrogenase Specimen: serum (avoid hemolysis). Method: Kinetic method. Principles: deterring CK was based on the rate of ATP formation / unit time. Calculation: CK activity (IU/L)= ∆A/min x factor. *The rate of NADPH formation is indicated by absorption at 340 nm (UV) which is directly proportional to CPK activity (concentration). Normal Levels (measured at 37Cº): 24- 190 U/L. 6Dr. Manal Basyouni

7. Quantitative determination of Creatine kinase –MB activity in serum Background:  CK-MB activity significantly in MI without in total CK.  It is specific & sensitive marker for CM injury. Mycardial damage is high when:  Total CK: (male› 195U/L, female › 170U/L).  CK-MB › 25U/L.  CK-Mb activity: 6% - 25% of total CK activity. 7Dr. Manal Basyouni

8. Detection of CK–MB activity in serum by Immuno- inhibition method  Specimen: serum (avoid hemolysis).  Principle:  Ck activity is measured in the presence of antibody (Ab) against CK-M monomer.  This Ab completely inhibits the activity of CK-MM and half of CK-MB; while not affecting the B subunit activity in CK-BB or CK-MB.  We use the ordinal CK method to quantitatively determine CK- B activity.  The CK-MB activity is obtained by multiplying the CK-B activity by two. % CK- MB activity = CK-MB activity 100 Total Ck activity 8Dr. Manal Basyouni

9. 3- Aspartate Transaminase (AST): This was the first used. It is enzyme catalyzes the exchange of amino and keto groups between alpha AA & alpha keto acid. AST is widely distributed in tissues e.g. heart, liver, RBCs & muscles. It is not specific for heart damage, and it is also one of the liver function tests. liver function tests Time sequence: It released ; in 6–8 hrs of MI. peak: 18- 24 hrs. persists for: up to 4-5 days. Time sequence: It released ; in 6–8 hrs of MI. peak: 18- 24 hrs. persists for: up to 4-5 days. 9Dr. Manal Basyouni

10. Quantitative determination of SGOT (AST) activity in serum Specimen: serum (avoid hemolysis) Method: Kinetic method (change rate of absorbance/ unit time). Principles: L-Aspartate + 2 Oxoglutarate AST Oxaloacetate +Glutamate Oxaloacetate + NADH + H+L- malate + NAD + H2O 1.AST catalyse the transfer of amino group between aspartate and 2 oxoglutarate. 2.The formesd oxaloacetate is then react with NADH+ H by malate dehydrogenase (MDH) to form NAD. 3.AST activity is determined by rate of oxidation of NADH at 340nm. MDH 10Dr. Manal Basyouni

11. Calculation: AST activity (IU/L)= ∆A/min factor. *The rate of NADH reduction is indicated by absorption at 340 nm (UV) which is directly proportional to AST activity (concentration). Normal Levels (measured at 37Cº): 31-37 U/L. 11Dr. Manal Basyouni

12. 4- Troponin test: Troponin C, I, and T are proteins that form the thin filaments of skeletal & cardiac muscle. Skeletal and cardiac forms are structurally distinct: antibodies can be produced to react only with the cardiac forms of TnI & TnT (TnC are identical in both). Time sequence: It released ; in 2–4 hrs of MI. peak: 12 hrs. persists for: up to 3-5 days (TnI), 5-14 days (TnT). Significant:  most sensitive & specific test for myocardial damage.myocardial  Early detectable.  Its level correlated with MI  It used mainly to aid in the diagnosis of chest-pain patients with non- diagnostic electrocardiograms, they are also used as prognostic indicators of a MI. It detected by immunoassays (ELISA) using specific monoclonal antibodies against cardiac isoforms. 12Dr. Manal Basyouni

13. 5- Lactate Dehydrogenase(LDH): Lactate dehydrogenase catalyses the conversion of pyruvate to lactate.pyruvatelactate Isoenzymes: LDH is a tetramer composed of two different subunits, M & H. Five isoenzymes : M4, M3H, M2H2, MH3, and H4, and all five are present in different tissue in different relative amounts. They are differ in chemical structure, physical properties (electrophonic mobility, heat stability) & immunological character. The can be separated by electrophoresis, ion exchange chromatography, heat stability & immunological methods). LDH-1 isozyme is normally found in the heart muscle & LDH- 2 is found predominately in blood serum. LDH-1 / LDH-2 level ( flipped ratio) suggest MI. 13Dr. Manal Basyouni

14. Disadvantages of LDH isoenzyme: Non specific: LDH levels are also high in tissue breakdown or hemolysis. It can mean cancer, meningitis, encephalitis, or HIV.cancermeningitisencephalitisHIV Late marker. Time sequence: It released ; in 10–12 hrs of MI. peak: 3-6 days. persists for: up to 10-14 days. Time sequence: It released ; in 10–12 hrs of MI. peak: 3-6 days. persists for: up to 10-14 days. 14Dr. Manal Basyouni

15. Quantitative determination of Lactate Dehydrogenase activity in serum Specimen: serum (avoid hemolysis) Method: Pyruvate Kinetic method. Principles: Pyruvate + NADH + H+L- lactate + NAD The rate of NADH+ formation is indicated by increasing absorbance at 340nm and is directly proportional to serum LDH activity. LDH Calculation: LD activity (U/L)= ∆A/min Factor. Normal Levels (measured at 37Cº): 24- 190 U/L. 15Dr. Manal Basyouni

16. Serum Cardiac Marker Clinical significance SpecificityDurationPeaksRisesMarkers Very early D NS1 day6-8 hrs1-2 hrs Myoglobin Early DNS4-5days12-24hrs4-6hs CPK Early DS4-5days12-24 hrs4-6hs CK-MB Early DNS5days48-60 hrs6hs AST, SGOT Early DS3-5 days24-48 hrs3-6 hs cTnI Early D, FUS10-14 days24-48 hrs3-6 hs cTnT FU NS14days72-144 hs8-12hs LDH 16Dr. Manal Basyouni

17. Serum Cardiac Markers O- 4 hrs MI: Myoglobin is released. 4- 48 hrs: CPK, CK-MB, cTnI, cTnT, AST. > 48 hrs: LDH, cTnT. O- 4 hrs MI: Myoglobin is released. 4- 48 hrs: CPK, CK-MB, cTnI, cTnT, AST. > 48 hrs: LDH, cTnT. 17Dr. Manal Basyouni

18. Thank you Dr. Manal Basyouni Thank you Dr. Manal Basyouni 18Dr. Manal Basyouni