Cholesterol and Vitamin C in Biological Samples

It is often possible to quantify the presence of natural molecules in biological samples without actually isolating the molecules. Two such analyses will be completed in this experiment (1) serum cholesterol will be measured by coupling its enzyme-catalyzed oxidation to the peroxidase-catalyzed formation of a chromogen, and (2) the presence of vitamin C in dietary materials will be detected and quantified by redox titration with 2,6-dichlorophenol-indophenol. 

The maximum rate of a reaction (V ) is attained when all the enzyme active sites are saturated with substrate molecules. For the rate to approach VmiX the substrate concentration must be high in fact, it must be much greater than KM. When [S] Ku, the Michaelis-Menten equation becomes 

In words, under substrate-saturating conditions, the initial rate of the enzyme-catalyzed reaction (vQ) is independent of substrate concentration, [S] (Equation El 1.2). However, when the substrate is present in much less than saturating amounts ([S] KM), the Michaelis-Menten equation becomes 

The initial rate of the enzyme-catalyzed reaction is directly proportional to [S] (Equation El 1.3). Most clinical assays using enzymes are performed under the conditions of Equation El 1.3. From further study of this equation, you will note that also depends on enzyme concentration, since there is an enzyme concentration term hidden in Vmax. (If you have forgotten this, review the derivation of the Michaelis-Menten equation in your biochemistry textbook.) This can be used to advantage, because if a reaction used for a clinical analysis is very slow (it probably will be, since [S] is low), extra enzyme can be used so that the reaction will proceed to completion in a reasonable period of time. 

The measurement of serum cholesterol is one of the most common tests performed in the clinical laboratory. Hypercholesterolemia (high blood cholesterol levels) can be the result of a variety of medical conditions. Among the conditions implicated are diabetes mellitus, atherosclerosis, and diseases of the endocrine system, liver, or kidney. High blood cholesterol levels do not point to a specific disease determination of cholesterol is used in conjunction with other clinical measurements mainly for confirmation of a particular diseased condition, rather than for diagnosis of a specific ailment. 

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EXPERIMENT 11 Measurement of Cholesterol and Vitamin C in Biological Samples 371... 

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