JPS6317440B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention utilizes free fatty acids (hereinafter referred to as FFA) using enzymes.
(abbreviated as )). In the present invention, FFA refers to a fatty acid whose carboxyl group is not covalently bonded to other compounds. Conventionally, methods for measuring FFA include the Dole method, in which FFA is extracted with an organic solvent and titrated with dilute alkali;
There is the Itaya method, which extracts the metal salt of FFA with a solvent and compares the colors of the metal salt. However, it has been difficult to standardize these methods because the extraction operation using an organic solvent is complicated. As a method for measuring FFA using an enzyme, Takahashi et al. reported a method using acyl coenzyme A synthetase (hereinafter abbreviated as ACS) in Clinical Chemistry Vol. 4, No. 2, pp. 179-185 (1975). . Methods using enzymes have recently become particularly widely used because they have various advantages such as specific reactions and mild conditions. However, in systems where FFA coexists with proteins, especially proteins with strong affinity for FFA such as albumin,
For example, measurement methods using enzymes have been difficult to apply to the measurement of FFA in serum. This is because FFA is tightly bound to albumin, and it is difficult for FFA to be liberated under mild conditions such as when an enzymatic reaction is performed. The nature of this bond between FFA and albumin is not clear, and it is said to be a hydrophobic bond or an ionic bond. The present inventors discovered a protein with strong affinity for FFA,
In particular, as a result of examining substances that dissociate the bond between albumin and FFA to accelerate the reaction rate, it was discovered that benzenesulfonic acid or its water-soluble salt, which may be substituted with a methyl group, is effective. completed. That is, the gist of the present invention is a method for enzymatically measuring fatty acids in a system in which albumin and fatty acids coexist, which is characterized by coexisting benzenesulfonic acid or a water-soluble salt thereof which may be substituted with a methyl group. It consists in the method of measuring fatty acids. Next, the present invention will be explained in detail. In the method of the present invention, FFA is measured by a conventional method except that the above-mentioned benzenesulfonic acids are present in the measurement system. Examples of compounds used in the method of the present invention include:
Benzene sulfonic acid, para-toluene sulfonic acid,
and its sodium, potassium, and ammonium salts. These arylsulfonic acids may be used alone or in combination of two or more. The amount added to the measurement system varies depending on the strength of its effect, influence on the enzyme, solubility, etc., but generally it is 5 to 1000 times (mol) the amount of FFA to be measured.
The degree and concentration in the measurement system are preferably about 0.01 to 5%. These benzenesulfonic acids are
It is introduced into the measurement system by adding it to either a sample containing FFA or a reagent such as an enzyme used for measurement, but it may be added to the system at the time of measurement if desired. The enzyme used for measurement and the measurement method may be any method as long as it is not interfered with by benzenesulfonic acids added to the system. Usually, FFA is reacted with coenzyme A using the above-mentioned ACS, and the coenzyme A or coenzyme A consumed by the reaction is A method is used to measure the amount of FFA-Coenzyme A conjugate produced by the reaction. According to the method of the present invention, FFA can be measured easily and accurately without interference from albumin. The present invention will be explained in more detail with reference to Reference Examples and Examples below. Reference example 1 [Preparation of ACS] European Journal of Biochemistry
Biochemistry) Vol. 93, pp. 197-203 (1979), Triton , hereinafter abbreviated as activator T)
The purified product was purified by phosphocellulose column chromatography and Blue Sepharose (Pharmacia Fine Chemicals Co., Ltd. trademark) column chromatography using 50% glycerin, 10mM potassium phosphate buffer (PH7.4), and activator T0. 063%, 2
-Mercaptoethanol was used as a 2.5 nM solution. Reference example 2 [Acyl coenzyme A oxidase (hereinafter referred to as
(referred to as ACO)] The method described on page 144 of the Abstracts of the Lipid Biochemistry Research Group (Toyama) in 1974 was used with some modifications. Candida utilis IFO 0396
The strain was purified by ammonium sulfate fractionation and DEAE Sephadex column chromatography and used as a suspension in 50mM potassium phosphate buffer (PH7.4) and 50% saturated ammonium sulfate. Reagent (a) Buffer (1); Contains 3.2nM of activator T, 2mM of disodium ethylenediaminetetraacetate, and a dissociating agent such as sodium (paratoluene)sulfonate.
100mM Tris-HCl buffer (PH8.0) (b) Buffer (2); 100mM Tris-HCl buffer containing 60mM magnesium chloride (PH8.0) (c) Enzyme solution (1); Adenosine triphosphate disodium salt
80mg, myokinase 20U in 40ml of the above buffer (1)
(d) Enzyme solution (2): Dissolve 25 mg of coenzyme A in 5 ml of the above buffer (2), and further add 5 ml of the 50% glycerin solution of ACS prepared above at about 4 U/ml. Preparation (e) Enzyme solution (3): Approximately 50 U/ml of ACO prepared above
50% saturated ammonium sulfate suspension (f) coloring liquid; Analytical Clinical Biochemistry
Prepared by modifying the method described in Biochemistry) Volume 6, Page 24 (1969), the composition is 0.05 mg/ml of 4-aminoantipyrine, 0.05% m-acetamino-N,N-diethylaniline, and peroxidase. 100mM phosphate buffer (PH
7.4) Example 1 100μ sample containing 280μM potassium oleate and 2.5% human serum albumin, 400μ enzyme solution (1)
and 100μ of enzyme solution (2) were mixed and held at 37°C for 15 minutes. Next, add 2 ml of coloring solution and enzyme solution to this.
(3) 10μ was added and the mixture was further held at 37°C for 5 minutes. The reaction solution was placed in a cell with an optical path length of 10 mm, and the absorbance (A) at 535 nm was measured. As a control, the absorbance (B) was measured in the same manner by replacing the sample with water. Separately, an albumin-free potassium oleate aqueous solution was prepared as a standard solution, and its absorbance (C) was measured in the same manner as above. The amount of FFA in the sample is calculated using the following formula: FFA in the sample = A-B/C-B x (FFA in the standard solution)

[Table] Example 2 Measurements were carried out in exactly the same manner as in Example 1, except that a standard serum specimen was used as the specimen and a potassium palmitate aqueous solution was used as the standard solution.

[Table] The displayed value of this standard serum was 800±100, and the measured value was slightly lower, but this is thought to be due to the slightly lower concentration of sodium paratoluenesulfonate. Example 3 Sodium p-toluenesulfonate as a dissociating agent,
Sodium n-dodecylbenzenesulfonate and n-
Using sodium dodecylsulfonate, first prepare the enzyme solution (1).
and (2) were mixed and the mixture was allowed to stand at 37° C. for 1 hour before adding the sample, but measurements were carried out in exactly the same manner as in Example 1, except that the sample was added. In addition, as a control experiment, values measured using the above three types of dissociating agents in exactly the same manner as in Experimental Example 1 are also shown.

[Table] When sodium p-toluenesulfonate is used as a dissociating agent, there is almost no change in the absorbance value even if it is mixed with an enzyme and left for about an hour, but n-
It can be seen that the use of sodium dodecylbenzenesulfonate and sodium n-dodecylsulfonate significantly reduces the absorbance. This is thought to be because sodium n-dodecylbenzenesulfonate and sodium n-dodecylsulfonate deactivate the enzyme, and sodium p-toluenesulfonate is extremely stable and cannot be used in the coexistence of albumin.
It is considered to be the optimal dissociating agent when measuring FFA.

Claims (1)

[Claims] 1. A method for enzymatically measuring fatty acids in a system in which albumin and fatty acids coexist, which comprises coexisting benzenesulfonic acid or a water-soluble salt thereof which may be substituted with a methyl group.