BY3BC1 - Medical Bseiochemistry Lecture 2 - Cardiac Function test LO- - Principles of using serum enzymes, other proteins and metabolites as diagnostic tools - Methods involved in measuring the above - Apply such principles to the diagnosis of the condition's acute myocardial infarction and heart failure RECAP Biomarkers - Tissue specific (i.e. only present within one tissue) - Rapid release in response to damage - Very low in serum of normal individuals - Easily measured with clear universal reference values - If these criteria are difficult to achieve use a battery of test to make a profile (not just one test) Structure for an essay - Diagnosis - Management - Screening Assay of serum biomarkers - Enzyme activity (is good when finding biomarkers for heart conditions) - Colorimetric assay is a better option (a colour change assay conc of protein in direct relationship to the intensity of the colour) - Rate of change in absorbance e.g. aspartate aminotransferase - Aspartate tt 2-oxoglutarate Oxaloacetate tt NADH tt Htt (this example has colourless products) glutamate tt oxaloacetate malate tt NADtt Change in absorbance per m aspartate aminotransferase activitymol/min/ml)= 6.22 x 1 16 NAD+ - Extinction coefficient for NADH = 6.22 mM-1 cm-1 12 --- - 8 Molar absorbance x 10-3 NADH 4 240 280 320 360 Wavelength (nm)
- Quantifying on structure as both products are colourless. The structures being NADH and NADtt (finding protein in the serum is good for biomarkers especially colorimetric proteins - not all proteins produce a colour in this case can look at concentration of the products like NADtt) - Amount of protein/ enzyme: Substrate - Immunological methods e.g. ELISA Substrate - Enzyme-linked immunosorbent assay Substrate Secondary Antibody Conjugate - 4 different types Inhibitor Antigen - Direct ELISA, generate an antibody Primary Antibody Ag Conjugate which binds to the antigen(of interest) attach protein to the DIRECT ELISA antibody so when it meets the substrate a colour is generated. Capture Antibody INDIRECT ELISA SANDWICH ELISA - Indirect ELISA, users a secondary antibody - Sandwich ELISA, more accurate coat plate with capture antibody (left overnight) this way the antigen is already captured to the plate and wash off any proteins or antibody that aren't captured are washed away.Then its similar to indirect ELISA but Substrate COMPETITIVE ELISA a lot more sensitive - Competitive ELISA, aren't used as often- release two antibodies which compete for the same antigen both could have different colours and can check which one gives off more colour - Metabolites. - Enzymatic or chemical conversion to a coloured product - Usually used to determine the concentration of the unknown - Spectrophotometric measurements - Beer-lambert law Linear Range Absorbance - Acute myocardial infarction Concentration - Imbalance between myocardial oxygen supply and demand - Results in muscle injury/ death - Cardiac ischemia (not enough oxygen is getting to the heart its starved) if lasts too long causing a heart attack, risk factors - smoking, obesity, high bp, high cholesterol, poor diet and lack of exercise - Myocardial