Cardiac Biomarkers Mahmoud

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Slide 1: 

Cardiac Biomarkers:central points for redefinition Dr. Mahmoud A Abdelwahab, M.D. Consultant Clinical Chemistry; YIACO medical diagnostic center

Cardiac Biomarkers : 

Cardiac Biomarkers Necrotic myocytes loose the membrane integrity and IC macromolecules diffuse into cardiac interstitium and ultimately into cardiac microvasculature and lymphatics and lastly drain into circulating blood. Cardiac Markers (Mahmoud Abdelwahab)

Slide 3: 

Cardiac Markers (Mahmoud Abdelwahab)

The history of biochemical markers : 

The history of biochemical markers 1954 SGOT (AST) 1955 LDH 1960 CPK 1972 CPK isoforms by Electrophoresis 1975 CK - MB by immunoinhibition 1975 Myoglobin 1985 CK - MB Mass immunoassay 1989 Troponin T 1992 Troponin I Cardiac Markers (Mahmoud Abdelwahab)

Ideal Cardiac Marker : 

Ideal Cardiac Marker Diagnostically has; High sensitivity (detection of MI positive cases) High specificity (absent in non-myocardial injury) Rapidly release at a detectible concentration Correlate efficiently with the extent of MI Persists in blood for valuable time (long 1/2 life) Analytically has; High sensitivity (low detectable limit) High specificity (less interferences) Easy, inexpensive and rapidly tested ( short TAT) Cardiac Markers (Mahmoud Abdelwahab)

Cardiac markers types : 

Cardiac markers types Cardiac Markers (Mahmoud Abdelwahab)

Creatine kinase : 

Creatine kinase Muscle Creatine kinase (CK) catalyzes the transfer of phosphate between creatine and ATP/ADP The optimal pH to regenerate ATP is 6.7 while pH for the reverse reaction to regenerate creatine phosphate and using ATP is 9.0 Mg ions activates CK but its excess has an inhibitory effect on CK activity. Also other ions Ca++, Zn+ and Cu+ are inhibitory to its activity. CK in serum is unstable as a result of oxidation of its sulfhydryl group 6.7 9.0

Determining CK (activity) & CK-MB (mass) : 

Total CK activity is determined by a simple enzyme assay (phosphocreatine + ADP  ATP) CK-MB mass is determined by a two-antibody “sandwich” assay. Determining CK (activity) & CK-MB (mass)

Slide 10: 

Spectrophotometric method for CK total activity Reaction intermediates Cardiac Markers (Mahmoud Abdelwahab) + EDTA + Mg Creatine Phosphate + ADP Adenosine Monophosphate Glucose + Hexokinase G6PD (Bacterial)+ NADP Dithioerythritol Creatine + ATP G6P ADP NADPH+H+ Serum CK AK from RBCs GSSG Calcium Reagents

Slide 11: 

Limitation of use of CK: Unstable enzyme affected by storage  (increase pH by CO2 loss), bright day light, temperature and oxidation attacking –SH group (can be minimized by adding thiol agents ) analytical interferences; hemolysis  ATP, AK & G6P that will do side reactions Pre-analytical variables; blacks, children, drugs and exercise increase CK Cardiac Markers (Mahmoud Abdelwahab)

Slide 12: 

Bioluminescence Method (More sensitive method) Creatine phosphate +ADP Creatine + ATP ATP + Luciferin AMP+ Ppi+ CO2+ Oxiluceferin + Light CK pH 6.7 Luciferase Interferences in spectrophotometric method: 1. RBCs – Adenylate Kinase (AK) will result in falsely increase in CK activity. That can be minimized by adding di-adenosine pentaphosphate. 2. GSSG (GR) catalyzes the consumption of NADPH+H+ and result in decrease in CK activity. That can minimized by adding dithioerythritol 3. The inhibitory effect of calcium can be avoided by adding EDTA 4. pH changes affect CK activity but can be optimized through well adjusted buffers e.g. ethane sulfonic acid (MES) 5. Temperature affecting CK activity; at 25 ͦ C activity decreased to be 70 % of the total activity. At 37ͦ C CK increases to 170 % of the total activity.

CK Isoenzymes : 

CK Isoenzymes Isoenzymes (dimers of 2 subunits each is of MW 40 kD. These are B [ Brain from Chr. 14] & M [ Muscle from Chr. 19] CK-3 (MM) in Skeletal muscle CK-2 (MB) 45% in myocardium <2% in skeletal muscle CK-1 (BB) in Brain, Bladder, placenta, prostate, pulmonary and Thyroid gland Macro CKs Type 1 Complex formed between CK-BB and IgG (present in adenoma, bowel diseases, cancers and CV diseases) Type 2 Mitochondrial CK or CK-MM complexes with IgA Cardiac Markers (Mahmoud Abdelwahab)

Slide 14: 

Cardiac Markers (Mahmoud Abdelwahab) Limitation of use of CK isoenzymes in diagnosis of MI Tedious techniques needed; high voltage electrophoresis or HPLC TAT of the accurate result by electrophoresis Difficulty in QC check

Slide 15: 

Cardiac Markers (Mahmoud Abdelwahab) Mass MB assay: Immunological Sandwich technique using two Abs for different epitopes of CK –MB molecule The first Ab is rendered immobile on a matrix (e.g. CrO2 particles) The second Ab conjugate to an enzyme (β- galacto- sidase) Separated bound sandwiches are reacted with their substrate (e.g. Chlorophenol β- Red Galactopyranoside) Liberated end product chlorophenol is measured spectrophotometrically and is proportionate to CK-MB amount (not the activity)

CK- MB isoforms : 

CK- MB isoforms Serum carboxypeptidases cleaves CK-MB carboxy terminal lysine residue yielding MB2 (serum) isoform while MB1 is the original MB isoform. Tissue MB1 isoform excess gives attention towards increase extent of myocardial injury Normally MB2/MB1 ratio is less than 1 On progress of MI the ratio will be flipped Cardiac Markers (Mahmoud Abdelwahab)

CKMB Isoforms : 

CKMB Isoforms Cardiac Markers (Mahmoud Abdelwahab)

Slide 18: 

Cardiac Markers (Mahmoud Abdelwahab) Re-defined CK as marker for myocyte injury; Mass MB is superior to CK electrophoresis separation Current recommendation of WHO for MI diagnosis on using MMB by following a sequential sampling protocol either 2 hrs or at time of admission, 6 – 12 hrs and 24 hrs after admission in cases show no ECG changes and pain is ameliorated For the reperfused patient, MMB is peaked earlier and the time schedule recommended is at time of admission, the time of thrombolytic therapy and then 3, 6 and 12 hrs after therapy. A cut off of 5 μg/L is suggestive as being consistent with AMI. Relative Index (RI) is used at a cut off of 4 % as calculated and equal to ratio between MMB to total CK. CK-MB mass (μg/L) X 100 Total CK activity (U/L)

Slide 19: 

Cardiac Markers (Mahmoud Abdelwahab) The CK-MB isoforms may be analyzed using high-voltage electrophoresis. Automated analyzers with rapid turnaround times are available. The ratio of MB2/MB1 is calculated. Normally, the tissue MB1 isoform predominates; thus, the ratio characteristically is less than 1. A result is positive if MB2 is elevated and the ratio is more than 1.5 The release kinetics of the CK-MB isoforms are rapid. MB2 is detected in serum within 2-4 hours after onset and peaks at 6-9 hours. It is an early marker for AMI.

Lactate Dehydrogenase : 

Lactate Dehydrogenase LDH is a H2 transfer enzyme catalyzes reduction of L-lactate to pyruvate using a carrier nicotinamide adenine dinucleotide (NAD) in an alkaline pH (8.5 – 9.8) L-Lactate + NAD Pyruvate + NADH+H+ Chemistry of LDH; A tetramer of 4 peptide chains of 2 types; M & H forming five isoenzymes; LD1 (H4) LD2 (H3M) LD3 (H2M2) LD4 (HM3) LD5 (M4) Cardiac Markers (Mahmoud Abdelwahab) LDH pH 8.55

LD isoenzymes : 

LD isoenzymes Cardiac Markers (Mahmoud Abdelwahab) Tissue LD activity is ~ 500 times its activity in serum LD1/LD2 ratio normally between 0.47 – 0.74 In Myocardial Injury LD1/LD2 ratio is flipped being > 1

LD inhibiting and interfering factors : 

LD inhibiting and interfering factors Plasma samples as it may contain platelets that is rich in LD Hemolysed samples as RBCs are rich in LD (~ 150 X of serum) EDTA is an inhibitory factor by precipitating Zn+ Competitive inhibitors e.g. Oxalates & Borates Cardiac Markers (Mahmoud Abdelwahab)

Slide 23: 

Cardiac Markers (Mahmoud Abdelwahab) Troponins These are myofibril contractile regulatory apparatus consists of 3 troponins namely; TpC, TpT & TpI. They are sacromeric bound proteins and only 5% are free in the cytoplasm.

Troponin C : 

Troponin C Same isoform for both skeletal and cardiac muscles. No immunological difference make no method to differentiate to be applied for Myocardial injury diagnosis.

Troponin T & I : 

Troponin T & I Different isoforms for cardiac and skeletal tissues Require myocardial necrosis for release from sarcomere. Early rise (4-12 hours after chest pain). Peak 12-24 hours. Continuous release up to 10-14 days 2nd to constant release/necrotic sarcomeres. Unclear excretion pathway.

Troponin I : 

Troponin I The cardiac isoform of troponin I is only found in cardiac muscles (cTnI). Highly bound to the tropomyosin complex in the sarcomere. <5% in cytosol.

Troponin I : 

Troponin I N ,C terminus and central portion. Myocardial necrosis: cleavage of the terminus (more unstable). Different assays with antibodies measuring different terminus (6 assays). Strong binding with troponin C (calcium dependent) may affect measurement. Assays also affected by protein kinases and fibrinogen levels.

Troponin T : 

Troponin T Cardiac troponin T: 4 isoforms. Fetal skeletal muscle: + cardiac troponin isoform. Muscle injury, myopathy, renal failure: re-expression of cardiac troponin T in muscles.

How do troponin compare with ECG in ACS? : 

How do troponin compare with ECG in ACS? Negative troponin and normal ECG, mortality 1%. Negative troponin and ischemic ECG: mortality 4% at 1 month. Troponin and ECG changes complementary.

Role of Tpns in Redefinition of MI : 

Role of Tpns in Redefinition of MI cardiac troponin (I or T) values Defines ischemic presentations as acute, evolving, or recent MI in acute coronary syndrome (ACS) Managing ACS patient with ischemic discomfort Tpn = Dx of non-ST-segment elevation MI (NSTEMI) Nml Tpn = Dx of unstable angina (Consensus document from ESC, ACC, AHA in Circulation, 2000)

Other predictive markers : 

Other predictive markers Am J Emerg Med - 2000 Jan Neg trop but Pos CRP = 5.8% death Neg trop and Neg CRP = 0.36% death Pos trop and Pos CRP = 9.1% death

Slide 33: 

Cardiac Markers (Mahmoud Abdelwahab)

Slide 34: 

Cardiac Markers (Mahmoud Abdelwahab)

Slide 35: 

Cardiac Markers (Mahmoud Abdelwahab)

Properties of individual markers : 

Properties of individual markers Marker Initial Rise Peak Persistence Heart Specificity CK 4 - 6 h 18 - 24 h 24 - 36 h + CK MB 4 - 6 h 16 - 20 h 18 - 30 h ++ Myoglobin 1 - 2 h 4 - 6 h 8 - 12 h + Troponin I 4 - 6 h 18 - 24 h 5 - 7 d ++++ Troponin T 3 - 5 h 18 - 24 h 5 - 7 d ++++

Slide 37: 

New Tools: Fatty Acids Binding Protein (FABP) Small cytoplasmic protein, abundant in heart muscle cells. Normal levels below 1ng/ml. Most sensitive early cardiac marker. <3 hrs to achieve diagnostic performance. 12-24 hrs to return to normal. Current studies ongoing to further evaluate its utility

B Natriuretic Polypeptide : 

B Natriuretic Polypeptide Secreted from the ventricles and atria in proportion to expansion / preload Increased in dyspnoea associated with CHF Degree or class of CHF correlates with BNP level Low levels strong negative predictive value for re-admission Increased BNP at 48 hours post MI is an independent variable for increased mortality Blood assay available now - can be run in 20 - 30 minutes

Natriuretic Peptides : 

Natriuretic Peptides

Natriuretic Peptides: Origin and Stimulus of Release : 

Natriuretic Peptides: Origin and Stimulus of Release ANP = Atrial Natriuretic Peptide BNP = B-type Natriuretic Peptide CNP = C-type Natriuretic Peptide

Slide 41: 

Myoglobin Ubiquitous small-size heme protein released from all damaged tissues. Increases often occur more rapidly than TI and CK. Not utilized often for AMI/cardiac damage assessment because of its very rapid metabolism (short plasma half-life) causing short burst increases that are difficult to assess clinically, as well as its lack of specificity for cardiac tissue.

Conditions for Myoglobin : 

Conditions for Myoglobin Acute myocardial infarction Open heart surgery Skeletal muscle damage, muscular dystrophy, inflammatory myopathies Renal failure, severe uremia Shock and trauma

Clinical Usefulness of Myoglobin : 

Clinical Usefulness of Myoglobin Rapid monitor of success of thrombolytic therapy Negative predictor of MI Due to poor specificity, myoglobin levels do not always predict myocardial injury.

Prognostic Markers and Markers of Risk Stratification : 

Prognostic Markers and Markers of Risk Stratification C-reactive protein Myeloperoxidase Homocysteine Glomerular filtration rate

C-Reactive Protein : 

C-Reactive Protein Pentameric structure consisting of five 23-kD identical subunits Produced primarily in hepatocytes Plasma levels can increase rapidly to 1000x baseline levels in response to acute inflammation “Positive acute phase reactant”

C-Reactive Protein : 

C-Reactive Protein Binds to multiple ligands, including many found in bacterial cell walls Once ligand-bound, CRP can: Activate the classical compliment pathway Stimulate phagocytosis Bind to immunoglobulin receptors

C-Reactive Protein:Risk Factor or Risk Marker? : 

C-Reactive Protein:Risk Factor or Risk Marker? CRP previously known to be a marker of high risk in cardiovascular disease More recent data may implicate CRP as an actual mediator of atherogenesis Multiple hypotheses for the mechanism of CRP-mediated atherogenesis: Endothelial dysfunction via ↑ NO synthesis ↑LDL deposition in plaque by CRP-stimulated macrophages

C-Reactive Protein : 

C-Reactive Protein Multiple roles in cardiovascular disease have been examined Screening for cardiovascular risk in otherwise “healthy” men and women Predictive value of CRP levels for disease severity in pre-existing CAD Prognostic value in ACS

CRP and CV Risk : 

CRP and CV Risk Elevated levels predictive of: Long-term risk of first MI Ischemic stroke All-cause mortality This relationship persists even when data is adjusted for classic CV risk factors (age, tobacco, lipid levels, DM, and HTN)

Limitations to CRP in Screening : 

Limitations to CRP in Screening Low specificity What do you do with an elevated CRP? No evidence that lowering CRP levels decreases CV risk Some medications (e.g.statins) do lower CRP levels; unclear what the implication is

CRP and Existing CAD : 

CRP and Existing CAD Unclear of the role of CRP in stable CAD; acute phase response not present in stable angina Predictive of worse short- and long-term prognosis in non-ST elevation ACS Possibly some correlation with poor outcomes post-CABG and post-PCI

CRP: ACC/AHA Recommendations : 

CRP: ACC/AHA Recommendations Not useful as a public health measure; screening the entire adult population should be avoided It is considered the inflammatory marker most useful in clinical practice Useful in guiding further evaluation in “intermediate risk” patients (10-20% risk of significant CAD in 10 years)

CRP: ACC/AHA Recommendations : 

CRP: ACC/AHA Recommendations Should not determine the application of secondary prevention measures Serial measurements should not be used to gauge efficacy of treatment Measure hs-CRP serum levels in an outpatient setting twice (at least two weeks apart) and average the two levels

Slide 54: 

Cardiac Markers (Mahmoud Abdelwahab) A novel marker of ischemia, ischemia modified albumin (IMA), is produced when circulating serum albumin contacts ischemic heart tissues. IMA can be measured by the albumin cobalt binding (ACB) assay that is based on IMA's inability to bind to cobalt. IMA levels rise within minutes of transient ischemia, peak within 6 hours, and can remain elevated as long as 12 hours. Studies on the use of IMA in patients with chest pain have found sensitivities that ranged from 71-98%, and specificities of 45-65%. Ischemia modified albumin (IMA)

Slide 55: 

Cardiac Markers (Mahmoud Abdelwahab) It is reported that a multimarker approach using the combination of ECG, the TnT, and the IMA levels achieved a sensitivity of 95% for ACS. On calculation of the combination of IMA, myoglobin, CK-MB, and TnI increased the sensitivity to 97% for detecting myocardial ischemia. However, IMA level is also elevated in patients with cirrhosis, certain infections, and advanced cancer, which reduces the specificity of the assay. Further validation and outcome studies are required to evaluate its use in the ED diagnosis of ACS when the ECG and cardiac troponins levels are non-diagnostic.

Myeloperoxidase : 

Myeloperoxidase Released by activated leukocytes at elevated levels in vulnerable plaques Predicts cardiac risk independently of other markers of inflammation May be useful in triage of ACS (levels elevate in the 1st two hours) Also identifies patients at increased risk of CV event in the 6 months following a negative troponin

Homocysteine : 

Homocysteine Intermediary amino acid formed by the conversion of methionine to cysteine Moderate hyperhomocysteinemia occurs in 5-7% of the population Recognized as an independent risk factor for the development of atherosclerotic vascular disease and venous thrombosis Can result from genetic defects, drugs, vitamin deficiencies, or smoking

Homocysteine : 

Homocysteine Homocysteine implicated directly in vascular injury including: Intimal thickening Disruption of elastic lamina Smooth muscle hypertrophy Platelet aggregation Vascular injury induced by leukocyte recruitment, foam cell formation, and inhibition of NO synthesis

Homocysteine : 

Homocysteine Elevated levels appear to be an independent risk factor, though less important than the classic CV risk factors Screening recommended in patients with premature CV disease (or unexplained DVT) and absence of other risk factors Treatment includes supplementation with folate, B6 and B12

Glomerular Filtration Rate : 

Glomerular Filtration Rate The relationship between chronic kidney disease and cardiovascular risk is longstanding Is this the result of multiple comorbid conditions (such as diabetes and hypertension), or is there an independent relationship?

Glomerular Filtration Rate : 

Glomerular Filtration Rate Recent studies have thought to identify whether creatinine clearance itself is inversely related to increased cardiovascular risk, independent of comorbid conditions

Glomerular Filtration Rate : 

Glomerular Filtration Rate Reduced GFR has been associated with: Increased inflammatory factors Abnormal lipoprotein levels Elevated plasma homocysteine Anemia Arterial stiffness Endothelial dysfunction

Slide 64: 

Cardiac Markers (Mahmoud Abdelwahab) We will meet next year for new marker(s) or Will meet just to review what is known today I hope to change my presentation

Slide 65: 

Cardiac Markers (Mahmoud Abdelwahab) Thank You