•Avoid hemolysis - RBCs contain adenylate kinase (AK) which produces ATP - This ATP can then take part in the reaction and cause falsely elevated CK results

The heart has 4 chambers: Left and right atria (upper chambers) Left and right ventricles (lower chambers) The heart pumps blood by contracting and relaxing striated muscle fibers. Proteins in the muscle fibers regulate contractions: •Actin •Myosin •Troponins

enzymes 4 Flashcards by Victoria Penny

Creatine Kinase (CK)

Reaction catalyzed:
CK
𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑒+𝐴𝑇𝑃 ↔ 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑒 𝑃ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒+𝐴𝐷𝑃

Nomenclature:
Transferase (class)
Creatine Kinase (practical name)
CK (abbreviation)

Activators: Mg2+ ( same as ALP & ACP)

Inhibitors: Mg2+( if in high concentrations)

Main function is in muscle where it is involved in formation of high energy storage compound (creatine phosphate ) and generation of ATP.

Slightly higher CK values in males due to an increased muscle mass.

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Creatine Kinase (CK) Analysis

Most common method:
reverse rxn proceeds 2-3x faster than the forward rxn & theres less interferences from side rxns

                                     CK 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑒 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒+𝐴𝐷𝑃 ↔ 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑒+𝐴𝑇𝑃

                  ℎ𝑒𝑥𝑜𝑘𝑖𝑛𝑎𝑠𝑒 𝐴𝑇𝑃+𝐺𝑙𝑢𝑐𝑜𝑠𝑒  ↔ Glucose−6−phosphate+ADP

                                              𝐺− 6−𝑃𝐷  𝐺𝑙𝑢𝑐𝑜𝑠𝑒− 6− 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 +𝑁𝐴𝐷𝑃+ ↔ 6−𝑃ℎ𝑜𝑠𝑝ℎ𝑜𝑔𝑙𝑢𝑐𝑜𝑛𝑎𝑡𝑒+𝑁𝐴𝐷𝑃𝐻

G-6-PD = Glucose-6-Phosphate dehydrogenase

measuring an Increase in absorbance @340 nm, which is directly proportional to CK activity.

optimal pH 6.8

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Creatine Kinase (CK) Isoenzymes

Three isoenzymes each with dimers (subunits)
Dimers consist of the 2 subunits M and/or B

Memory Aid for the highest concentration:
CK-MM = Much More

CK1 = CK-BB
◦ Fastest (most anodal) in electrophoresis
◦ Found in the brain
◦ Usually undetectable

CK2 = CK-MB
◦ Found in heart and diaphragm
◦ Undetectable to trace amounts; <6% of total CK
◦ Increase in CK-MB is the most specific indicator of
myocardial damage

CK3 = CK-MM
◦ Slowest of the three in electrophoresis
◦ Found in skeletal muscle
◦ Major isoenzyme; 94 - 100% of total CK

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Creatine Kinase Isoenzymes

Macro-CK
◦Unusual immunoglobulin-enzyme complex (usually IgG + CK-BB)
◦No clinical significance

CK-MI
◦Mitochondrial CK released during extensive damage to cell walls
◦Indicator of severe illness
◦Not present in normal serum

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Isoenzyme Separation of Creatine Kinase (CK)

Electrophoresis - reference method
Ion exchange chromatography

• Immunoassays:
◦Immuno-inhibition assay can be used to measure CK2 (MB)
◦Total CK measured then anti-M applied which inhibits all M activity in CK3 (MM) and ½ of CK2 (MB) – assumes no CK1 (BB)
◦Difference of two = amount of B chain
◦Multiply by 2 = CK-MB
* just know there is anti-M that inhibits M subunit activity

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Creatine Kinase (CK) - Clinical Significance

Evaluation of cardiac and skeletal muscle disorders

CK1 (CK-BB) increased in:
◦ CNS disorders - damage to the blood/brain barrier allows enzyme to be released
◦ Tumors
◦ Childbirth - placenta & uterine tissue have CK-BB

CK2 (CK-MB) increased in:
◦ Cardiac injury (e.g. AMI)

CK3 (CK-MM) increased in:
◦ Heart and skeletal muscle damage
◦ After physical activity (up to 48 hrs afterwards)
◦ After intramuscular injections (up to a week afterwards)

Total CK increases in most muscle diseases
◦ Marked increase in muscular dystrophy
Esp. Duchenne muscular dystrophy (50 - 100X normal

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CK Sources of Error

•Avoid hemolysis

RBCs contain adenylate kinase (AK) which produces ATP
This ATP can then take part in the reaction and cause falsely elevated CK results

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Creatine Kinase (CK) in Acute Myocardial Infarction (AMI)

CK-MB is the first enzyme to rise after an AMI
Rise 4-8 hours after onset of pain
Peaks 12-24 hours
Normal 2-3 days
CK-MB > 6% of total CK (normally < 6%)Enzyme Elevation Peak Return to Normal
CK 4 - 8 hours 24 hours 3 days
AST 6 8 hours 24 hours 5 days
LD 12 24 hours 72 hours 10 days

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Cardiovascular Disease and Cardiac Markers

Anatomy of the Heart

• The heart is enclosed in a sac called the pericardium.

•Cardiac wall is composed of 3 layers:
1. Epicardium (outer layer)
•Coronary arteries are on epicardium

Middle layer
Endocardium (inner layer)
•Most susceptible to myocardial ischemia

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Anatomy of the Heart

The heart has 4 chambers:
Left and right atria (upper chambers)
Left and right ventricles (lower chambers)
The heart pumps blood by contracting and relaxing striated muscle fibers.
Proteins in the muscle fibers regulate

contractions:
•Actin
•Myosin
•Troponins

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Cardiac Disease - Acute Coronary Syndrome (ACS)

• Acute coronary syndrome (ACS) is a sudden cardiac disorder that varies in severity.
• Includes:
- Angina (chest pain on exertion with reversible tissue injury)
- Unstable angina (with minor myocardial injury)
- Myocardial infarction (with extensive tissue necrosis -irreversible)

•Most conditions are caused an acute event in the coronary artery that obstructs circulation to a region in the heart.

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What is an Acute Myocardial Infarct (AMI)

Occurs when there is a sudden reduction in blood circulation to myocardial tissue.

This results in:

  - Ischemia - lack of blood supply/O2
  - Necrosis - death of cells
   - Release of cellular contents
          - such as cardiac enzymes & proteins into the bloodstream

• Symptoms of decreased blood flow

Pain
Clammy skin
Shortness of breath
Nausea

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Electrocardiogram (ECG)

•Electrocardiograms (ECGs) can record variations in electrical potential caused by the
excitation of the heart muscle.
•In a healthy individual, each cardiac cycle’s electrical potential changes are similar to
every other cycle and include three major components:
-Atrial depolarization (P wave)
-Ventricular depolarization (QRS complex)
-Repolarization (ST segment and T wave)

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ST Segment Elevation Myocardial Infarction (STEMI)

•A patient with any type of myocardial infarction in which the ST segment is elevated in
one or several leads of the ECG

•ECG pattern seen in this case is as follows:
Normal

Hours after infarction,
the ST segment
becomes elevated

Hours to days later,
the T wave inverts
and the Q wave
becomes larger

Days to weeks later,
the ST segment
returns to near
normal

Weeks to months 
later the T wave 
becomes upright 
again, but the large 
Q wave may remain

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Non-ST Segment Elevation Myocardial Infarction (NSTEMI)

A myocardial infarction in which the ST segment is not elevated in any leads of the ECG.
Magnitude of cell death is less than STEMI.
No elevation of the ST segment with increased troponin levels is considered NSTEMI

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Angina

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Angina is a condition marked by severe pain in the chest, often also spreading to the shoulders, arms, and neck, caused by an inadequate supply of blood to the heart.
Unstable ischemia with no evidence of cardiac necrosis (no increase in troponins) is classified as unstable angina.
Unstable angina occurs unpredictably or suddenly increases in severity or frequency

Atherosclerosis

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•Atherosclerosis is the major cause of acute coronary
syndrome.

•Deposits of fatty material (plaques) form on the
inner lining of the coronary arteries that feed the
surface of the heart.

•Plaques are composed of lipid, cell debris, smooth
muscle cells, collagen, and sometimes calcium.

These plaques narrow the artery’s lumen
Atherosclerotic plaque (atheroma) can break open and form blood clots within the vessel which will further block or completely stop blood flow.
Leads to myocardial infarction and ischemic stroke
Irreversible cardiac injury occurs when the occlusion is complete for 15-20 minutes.
Restoring blood flow in first 60-90 minutes allows maximal salvage of tissue.
Patients with STEMI are given clot-dissolving agents (thrombolysis)

Congestive Heart Failure (CHF)

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Syndrome characterized by ineffective pumping of the heart which can lead to accumulation of fluid in the lungs.
Characterized by breathlessness and abnormal sodium and water retention, often leading to edema.
Because heart is not able to pump sufficient blood, organs and other tissues do not receive enough oxygen and nutrients to function properly.

•Natriuretic peptides are biomarkers that will progressively increase with the severity of
disease.

Role of Cardiac Biomarkers in Acute Coronary Syndrome

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Cardiac biomarkers are biological compounds whose measurement is useful in the diagnosis or detection of cardiac disease
Useful when patients have nondiagnostic ECGs.

Characteristics of a clinically useful cardiac marker for detecting AMI:

- Must be rapidly released from heart into circulation.   - Analytical assays must be rapid and able to measure low concentrations in blood samples.    - Should persist in the circulation for several days to provide a late diagnostic time window.

Cardiac Biomarkers

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•Cardiac biomarkers are used to:

Detect cardiac disorders
- Detect risk of developing cardiac disorders
- Monitor the disorder
- Predict the response of a disorder to treatment

AACC Guidelines for Proposed

Biomarkers

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American Association of Clinical Chemistry (AACC) proposed elements for POC guidelines:

Members of emergency departments, primary care physicians, cardiologists, hospital admin, and clinical lab staff should work collectively to develop accelerated protocol for use of biomarkers in evaluation of patients with possible ACS.
Quality assurance measures should be used with monitoring to reduce medical errors and improve patient treatment.
Laboratory should perform biomarker testing with maximum TAT of 1 hour; optimally 30 minutes.
If 1-hour TAT is not possible, should consider implementing POC assays
Performance specifications and characteristics for central lab and POC assays should not differ.
POC assays should provide quantitative results

Laboratory Investigation of AMI

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CK, AST & LD used
Amount of increase roughly equal to size of infarct

• CK and LD isoenzymes are most specific and sensitive for interpretation
◦↑ CK-MB
◦LD-1 > LD-2 (flipped pattern)

CK / LD Isoenzyme Patterns (AMI)

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CK-MB ≥ 6% of total CK, LD-1>LD-2 (flipped pattern)• Confirms AMI
• Found in 80-85% AMI patients
CK-MB ≥ 6% of total, LD-2> LD-1
• Some form of myocardial damage
• Not confirmatory
CK-MB < 6% of total
• No myocardial infarct
CK-MB < 6% of total; LD-1>LD-2
• No myocardial damage
• Seen in non-cardiac disorders (intravascular hemolysis, renal cortex infarct, megaloblastic anemia, hemolyzed sample)

Cardiac Markers

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• Other proteins (not enzymes)

• Include:
◦Myoglobin
◦Troponins
◦Natriuretic peptides

• These are used in the evaluation of AMI and CHF

Myoglobin

* Is a heme protein in striated skeletal and cardiac muscle * Released when muscle is damaged • After AMI;◦ Increases 2-3 hours ◦ Peaks 8-12 hours ◦ Normal 18-30 hours (Kidney filters myoglobin) * Measured serially; if level doubles 1-2 hours after initial level- highly diagnostic of AMI. Early AMI indicator; helps to determine who might benefit from thrombolytic therapy * Disadvantage – Not cardiac specific; elevated in muscular dystrophy, crushing injuries and renal failure

Troponins

• A complex of 3 proteins in striated and skeletal muscle: - Troponin T (tropomyosin-binding component) - Troponin I (inhibitory component) - Troponin C (calcium-binding component) •The complex regulates the interaction of action and myosin and therefore regulates cardiac contraction. •Cardiac TnT and TnI are not in healthy blood - Their presence in blood = cardiac damage •Very specific and sensitive markers of myocardial damage**

Troponins - Analysis

• Cardiac troponins can be measured on serum or plasma by: -ELISA (enzyme-linked immunosorbent assays -Immunoenzymatic assays -POCT •Serial measurements should be taken to diagnose AMI •3-8 hour intervals for 48 hours

Measurement of Cardiac Isoforms of Troponin

• Useful to diagnose AMI:◦ cTnT rises 3-12 hours, peaks 12-24 hours, normal 8-21 days (longest elevated) ◦ cTnI rises 3-12 hours, peaks 12-24 hours, normal 7-14 days * Monitors effectiveness of thrombolytic therapy * Risk assessment of patients with ischemic symptoms◦ e.g. patient with ischemic symptoms → the higher the level of troponin, the greater the chance of an adverse outcome. * Disadvantage of TnT - False positive in renal failure patients & patients with chronic obstructive pulmonary disease (COPD)

Summary of Cardiac Markers and Enzymes

* Most labs use both markers and enzymes to diagnose AMI * Myoglobin is the earliest to rise (2-3 hours) * cTnT stays elevated the longest (8 - 21 days) * CK (CK-MB) is the first enzyme to rise (4-8 hours) * The longest elevated enzyme is LD (10 days) * LD-1 > LD-2 (Flipped pattern) * CK-MB ≥ 6% of total CK

Summary of Cardiac Markers/Enzymes for AMI

Enzyme Elevation Peak Return to Normal CK 4 - 8 hours 24 hours 3 days AST 6 - 8 hours 24 hours 5 days LD 12 - 24 hours 72 hours 10 days First enzyme increased is CK. Longest to stay elevated is LD Marker Elevation Peak Return to Normal Myoglobin 2 - 3 hours 8 - 12 hours 18 - 30 hours Troponin T 3 - 12 hours 12 - 24 hours 8 - 21 days Troponin I 3 - 12 hours 12 - 24 hours 7 - 14 days Myoglobin is 1st to be elevated (before any enzyme). Troponin T remains elevated longer than any enzyme

Natriuretic Peptides

• Several natriuretic peptides have been identified: - Atrial natriuretic peptide (ANP) - B-Type natriuretic peptide (BNP) - C-Type natriuretic peptide (CNP) * BNP is a hormone mainly released from myocardial ventricles (primarily left ventricle) * Pro-BNP is a precursor protein that is continuously produced by the heart in small amounts. - The enzyme corin splits pro-BNP into BNP and an inactive fragment, NT-proBNP - These fragments are released into the blood - BNP and NT-proBNP increase when the ventricles have difficulty pumping sufficient blood to the body.

Natriuretic Peptides Analysis

* Immunoassays have been developed to measure BNP and NT-proBNP * BNP - EDTA whole blood or plasma - Plastic tubes •NT-proBNP - Serum, heparin plasma, EDTA plasma - Glass or plastic tubes acceptable

Markers of Coronary Heart Disease (CHD)

* C-reactive Protein (CRP) | * Homocysteine

C-Reactive Protein (CRP)

* Produced by the liver * Increased in response to inflammation * Nonspecific marker that can indicate conditions such as infection, chronic inflammatory disease, cancer or risk of heart disease •High-sensitivity C-reactive protein (hsCRP) - Inflammatory marker of choice in the evaluation of cardiac heart disease risk - Most useful for those at intermediate risk of having a heart attack • < 1 mg/L Low risk of CVD • 1-3 mg/L Moderate risk • > 3 mg/L High risk

Homocysteine

Homocysteine is a common amino acid (from methionine) found in blood and acquired mostly from eating meat. •High levels are related to early development of heart and blood vessel disease -Elevated levels are considered an independent risk factor for heart disease * B12, B6 and folic acid are needed to metabolize methionine * If vitamin levels are low and protein intake is high, an increase in homocysteine levels will be seen * Increased levels of homocysteine are associated with atherosclerosis

Framingham Risk Score

* An algorithm used to estimate the 10 year cardiovascular risk of a person. * Based on data obtained from the Framingham Heart Study •Can indicate who is most likely to benefit from prevention. ◦ Offered preventative drugs (eg. to lower blood pressure, lower cholesterol)

Summary of Elevated Enzymes/Markers in Common Conditions

``` Acute Hepatitis AST (100X) ALT ALP (3X) GGT LD-4 LD-5 ``` ``` Extra-hepatic Obstructions AST (5X) ALT ALP (10X) GGT (30X) LD-4 LD-5 ``` ``` Metastatic Cancer AST (5X) ALT GGT (30X) LD-4 LD-5 ``` ``` Cirrhosis ALT GGT LD-4 LD-5 ``` ``` AMI: AST LD-1> LD-2 CK-MB Troponin Myoglobin ``` Pancreatic Disease Amylase Lipase GGT Prostatic Disease ACP PSA GGT Bone Disease ALP Muscle Disease AST CK-MM LD-5 LD increased in: megaloblastic anemia, untreated pernicious anemia, Hodgkin disease, abdominal and lung cancers, severe shock, and hypoxia

enzymes 4 Flashcards

(37 cards)