Abstract
A new enzymatic method is described for determination of free fatty acid (FFA) by using acyl CoA synthetase (6.2.1.3).
The principle of this method is as follows: FFA is activated by acyl CoA synthetase in the presence of ATP and CoA, producing acyl CoA, PPi and AMP. AMP thus produced was determined by coupling the reaction in which NADH was oxidized to NAD in the presence of myokinase (2.7.4.3), pyruvate kinase (2.7.1.40) and lactate dehydrogenase (1.1.1.27). The amount of NADH was measured by optical density at 340 nm.
Under these conditions 2 mmoles of NADH are oxidized to NAD when 1 mmol of FFA is activated.
In view of the results so far investigated, 30 mg/l ml of ATP, 6 mg/l ml of CoA and 50μl of the acyl CoA synthetase preparation obtained from rat liver according to the method of Bar-Tana, appeared to be an optimal condition for the reaction.
The standard curve was linear within the concentration of FFA rang from 200μEg/l to 1, 000μEg/l and recovery of FFA from serum specimen was satisfactory when the proposed method was employed.
Since the enzymatic method is specific and simple in comparison with the chemical method, an application of this method for measurment of FFA in a clinical laboratory would be promising.
