Acyl-CoA synthetase, having a high activity to C.sub.16 -C.sub.18 long chain fatty acids, is obtained by culturing strains belonging to various genera. As is known, acyl-CoA synthetase, having a strong activity to C.sub.16 -C.sub.18 long chain fatty acids, is generally obtained from liver of rat; however, it has now been discovered that acyl-CoA synthetase can be obtained from microorganisms and this enzyme is called acyl-CoA synthetase LCF-18. By use of acyl-CoA synthetase LCF-18 of the present invention which has a high activity to C.sub.16 -C.sub.18 long chain fatty acids, serum non-esterified fatty acid of human beings can be accurately determined, and it is very useful for diagnosis of diabetes and so forth.

There is disclosed a method for assaying fatty acids in a system containing albumin together with the fatty acids using acyl-CoA synthetase, characterized by performing the assay in the presence of a water-soluble salt of a dibasic fatty acid having 10 to 18 carbon atoms or a benzenesulfonate optionally having one or more C.sub.1 -C.sub.5 side chains.

There is provided a method for the quantitative analysis of a free fatty acid which comprises (1) the first step of treating a sample containing the free fatty acid with acyl-coenzyme A synthetase in the presence of adenosine triphosphate and coenzyme A to form acyl-coenzyme A, (2) the second step of oxidizing said acyl-coenzyme A, in the presence of oxygen, with acyl-coenzyme A oxidase produced by a microorganism of the genus Candida to form enoyl-coenzyme A and hydrogen peroxide, and (3) the third step of (a) measuring the amount of the formed enoyl-coenzyme A or hydrogen peroxide or (b) measuring the amount of oxygen consumed in said oxidation reaction, to thereby determine the amount of free fatty acid in said sample.

This invention relates to a process for the determination of free fatty acids comprising a reaction system 1 in which acyl coenzyme A synthetase is allowed to act on the free fatty acids in the presence of adenosine triphosphate and coenzyme A to yield acyl coenzyme A and a reaction system 2 in which acyl coenzyme A oxidase is allowed to act on the acyl coenzyme A to form hydrogen peroxide, followed by the determination thereof, said process being characterized in that myokinase is added in the reaction system 1 to make it go rapidly to completion.

A process for measuring the sodium concentration of a biological fluid, such as blood serum or urine, is carried out by fixing the amounts of adenosine-5'-triphosphate (ATP), adenosine triphosphatase (ATPase), magnesium, and potassium, in the presence of a buffer, in enzymatic Reaction I, ##STR1## where ADP is adenosine diphosphate, and measuring the rate of appearance of the products so that the rate of reaction of adenosine triphosphatase can be determined. The reaction rate of this enzyme is directly proportional to the level of sodium present. The sodium concentration of the biological fluid being tested is quantitatively determined by comparing the reaction rate of adenosine triphosphatase to a standard known sodium concentration. Preferably, the reaction rate of adenosine triphosphatase is measured by reacting the ADP product with phosphoenolpyruvate under the catalysis of pyruvate kinase and measuring the pyruvate product of this second reaction. Quantitation of the pyruvate produced determines the activity of adenosine triphosphatase in Reaction I, which is directly proportional to the sodium concentration in the biological fluid being tested. Colorimetric standards of known sodium concentration are used as points of reference. A paper phase procedure for determining sodium levels in biological fluids is also provided.