Enzymatic methods, automatic measurement

Serum samples were analyzed for concentrations of triacyl-glycerol, total cholesterol, free fatty acids (FFA), and glucose using an enzymatic method in an automatic analyzer. Quantitative analyses of GOT and GPT were also carried out in the same automatic analyzer using an ultraviolet method. The levels of leptin and insulin were measured using an enzyme-linked immunosorbent assay. 

Methods in which some property related to substrate concentration (such as absorbance, fluorescence, chemiluminescence, etc.) is measured at two fixed times during the course of the reaction are known as two-point kinetic methods. They are theoreticahy the most accurate for the enzymatic determination of substrates. However, these methods are technically more demanding than equifibrium methods and all the factors that affect reaction rate, such as pH, temperature, and amount of enzyme, must be kept constant from one assay to the next, as must the timing of the two measurements. These conditions can readily be achieved in automatic analyzers. A reference solution of the analyte (substrate) must be used for calibration. To ensure first-order reaction conditions, the substrate concentration must be low compared to the K, (i.e., in the order of less than 0.2 X K, . Enzymes with high K , values are therefore preferred for kinetic analysis to give a wider usable range of substrate concentration. 

Stat methods Stat methods, which are suitable for slow reactions, involve addition of a reactant to the reaction vessel at a given rate such that a characteristic property of the monitored reaction is maintained constant. In practice, a small amount of one of the reaction ingredients is added until a given value of the monitored parameter (pH, absorbance, luminescence, etc.) is reached. Any deviation from this state as a result of the reaction developing further is immediately compensated for by automatically adding an extra amount of the ingredient in question. The rate of addition is used to measure the reaction rate. This type of method has so far been chiefly used quantitatively with catalyzed (including enzymatic) reactions. 

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