Demetrio L. Valle Jr., MD, MSc., FPSP, FASCP, IFCAP Anatomic and Clinical Pathologist

Facts About Cardiovascular Diseasesy 2005- 17.5 Million people died of CVD

20 Million (WHO) y Number ONE caused of death in the world, 20% of all deaths y Unhealthy diet, physical inactivity and tobacco smokingy 2015

CARDIAC MARKERSy Lactate Dehydrogenase (LD) y Creatine Kinase (CK) Total and Isoenzymes y CK-MB Activity y CK- MB Isoenzyme, Mass & Relative Index (RI) y CK-MB Isoenzyme Isoforms & Isoforms Ratio y Troponin T y Troponin I y Myoglobin y Others cardiac markers

LACTATE DEHYDROGENASEy Photometric method

Lactate + NAD = Pyruvate + NADH + H+y 30oC is the preferred temperature for enzyme assay at 340 nm y Lactate dehydrogenase (LD) activity is present in all cells of the body with highest concentrations in heart, liver, muscle, kidney, lung, and erythrocytes.

LACTATE DEHYDROGENASEy Moderate to slight increases in LD levels are seen in myocardial infarction (MI), pulmonary infarction, pulmonary embolism, leukemia etc. y Marked elevations in LD activity can be observed in megaloblastic anemia, untreated pernicious anemia, Hodgkin's disease, abdominal and lung cancers, severe shock, and hypoxia.

LACTATE DEHYDROGENASEy Myocardiacl infection assessment y Abnormal after 24-48 hours y Peaks in 3-6 days y Return to normal in 8-14 days

LACTATE DEHYDROGENASE ISOENZYMESy 5 FRACTIONS (ISOENZYMES) y LD1 (H4) - Heart y LD2 (H3M)- Heart y LD3 (H2M2)- Pancreas y LD4 (HM3) y LD5 (M4) - Liver y Methods y - hydroxybutyrate dehydrogenase (HBDH) activity y Electrophoresis y Ion-exchange chromatography

LACTATE DEHYDROGENASE ISOENZYMESy Interpretation y NV (LD1 16-28%, LD2 29-37%, LD3 17-23%, LD4 9-15% and LD5 8-20%) y Myocardial infarctiony

y y

FLIPPED pattern (LD1/LD2 ratio is greater than 1) about 12-24 hours after infarction and remains greater than 1 for as long as 7 days. Samples should be drawn every 24 hours. Two or three samples are needed since the flip pattern will occur within 48 hours of a myocardial infarction.

Creatine Kinase (CK)y Enzyme found in various types of tissues (skeletal, cardiac and brain) y Its concentrations are comparatively high due to its function in energy metabolism. y Conc of Ck in skeletal muscle is 5-10 times higher than that of cardiac muscle y Elevated in AMI, cerebrovascular accident, myositis, skeletal muscle diseases like progressive Duckenne muscular dystrophy

Creatine Kinase Isoenzymesy Electrophoresis on cellulose acetate of agarose gel, differential inhibition, column chromatographym batch absorption and radioimmunoassay y Three isoenzymes y CK-BB (CK1) - Brain y CK-MB (CK2)- Cardiac y CK-MM (CK3)- Skeletal Muscle

CK-MB, Activityy Method Immunoinhibition method y Anti-CK-M inactivates the M sub-unit of CK-MM and CK-MB. Residual B subunit enzyme is measured by the production of NADPH at 340 nm y Appears 4 hours after infarction y Peaks: 12-24 hours y Decline > 48 hours y Normal: y < 16 IU/L

CK-MB Isoenzyme Mass Assayy Gold standard biochemical marker for AMI y Methods: ELISA, IRMA, Chemiluminescent y Rise: 4-6 hr y Peak: 12-24 hr y Normal: > 48 hr y Normal Value (ELISA) y < 4 ng/mL (< 10 ug/L)

CK-MB Isoenzyme with Relative Index (RI)y Relative index (%) relates the CK-MB isoenzyme mass concentration to the total CK activity. y It is used to evaluate increased total CK activity y Formula RI (%) = CK-MB (ug/L) / Total CK (U/L) x 100 y RI > 6% = indicative of cardiac damage y RI < 6% = indicative of skeletal damage

CK-MB Isoforms & Ratioy Develop to improve the sensitivity of the biochemical diagnosis of AMI y Two isoforms of CK-MB isoenzyme y CK-MB1 and CK-MB2 y Have equal levels y Myocardial damage: y CK-MB2 rises above CK-MB1y

CK MB Isoforms ratio produces to be highly sensitive and specific indicator of EARLY AMI

CK-MB Isoforms & Ratioy Rise: 2-6 hr y Peak: 6-12 hr y Normal: 24-36 hr y Normal Value CK-MB1 : 0.5- 1.0 U/L

CK-MB2: 0.5 1.0 U/L Ratio: < 1.5 Isoform ratio

Troponiny Located on the thin filament of striated muscle y Three subunit proteins y TnT tropomyosin binding subunit that binds the troponin complex to tropomyosin along actin. y TnI is the myosin ATPase inhibiting subunit blocking myosin (thick filament) movement in the absence of calcium. y TnC calcium binding subunit

Troponiny Early increase after cardiac injury y Broad diagnostic window y Excellent cardiospecificity y Diagnostic potential in identifying patients with unstable angina pectoris y High risk patients antagonists

therapy from platelet receptor

Troponin Iy Complete cardiospecific y Not detected in adult skeletal muscle y Absent in diseased human skeletal muscle y Indicated for: y AMI y Risk stratification of UAP y Therapy decision making y Minor myocardial damage y Reperfusion

Troponin Iy Also elevated in viral myocarditis, scleroderna or cardiac trauma y Rarely elevated in musculo-skeletal diseases and renal insufficiency y Rise: 4-8 hr y Peak: 14-18 hr y Normal: 5-9 days

Troponin Iy Methods: y ELISA, Chemiluminscent Assay y Normal Values: y 0.0 0.04 ng/mL

Troponin Ty Complete cardiospecific y Present in fetal skeletal muscle y Absent in healthy skeletal muscle y Indicated for: y AMI y Risk stratification of UAP y Therapy decision making y Minor myocardial damage y Reperfusion

Troponin Ty Found in chronic renal disease y Reexpressed in skeletal muscle diseases such as chronic tissue damage. y Rise: 4-8 hr y Peak: 14-18 hr y Normal: 14 days

Troponin Ty Methods: y ELISA, Chemiluminscent Assay y Normal Values: y 0.0 0.04 ng/mL

Myoglobiny Major protein responsible for oxygen supply of striated muscles. y Due to its abundancy in muscle tissue and low molecular weight it is released into blood rapidly as early as 1 hour after cell damage of heart or skeletal muscle y More sensitive than troponins during the first hours after AMI. y Primarily utility

to assist in ruling out an infarct.

Myoglobiny E.g. myoglobin remains within the reference range about 10 hours after chest pain onset AMI can be ruled out with high probability (High negative predictive value) y Lacks cardiospecificity, y Rise: 1-3 hr y Peak: 6-9 hr y Normal: 24-36 hr y Methods: ELISA, Turbidimetry/Nephelometry y Normal Value: o-0.09 ug/mL

CARBONIC ANHYDRASE IIIy Cytoplasmic protein mainly present in skeletal muscle,

only trace amount found in cardiac muscle y Similar rise and fall pattern as myoglobin y Myoglobin: Carbonic anhydrase III ratio useful in determining if the rise of myoglobin is due to skeletal or cardiac muscle.

GLYCOGEN PHOSPHORYLASE (GP)-BBy GP-BB isoenzyme - present in the brain and myocardium y GP-LL and GP MM y Early and specific marker for myocardial necrosis and ischemia y Early diagnosis of acute coronary syndrome and reversible myocardial ischemia y Methods: ELISA and Immunochromatographic

HEART FATTY ACID BINDING PROTEIN (HFABP)y a novel small cytosolic protein that is abundant in the heart. y highly cardiac-specific (i.e. expressed primarily in cardiac tissue), but is also expressed at low concentrations in tissues outside the heart. y can be detected in the blood as early as 1-3 h after onset of chest pain y peak values reached at 6-8 h and plasma levels returning to normal within 24-30 hr

HEART FATTY ACID BINDING PROTEIN (HFABP)y clinical diagnostic value is very limited in the presence of renal failure and skeletal muscle diseases as it is completely renally eliminated the diagnosis of acute myocardial infarction (AMI) may be overestimated

ISCHEMIA MODIFIED ALBUMINy When exposed to ischemic tissue, human serum albumin

loses its ability to bind cobalt, and the structurally altered albumin DUBBED IMA y IMA - can be measured by the albumin cobalt-binding test. y sensitivity of the IMA assay for detecting ischemic chest pain was 82%, compared with sensitivities of 45% for ECG and 20% for cTnT y IMA values were significantly higher in patients with ACS or UA than in those with nonischemic chest pain, and higher in patients with UA than in those with AMI.

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