1. Field of the Invention
This invention relates to a method, reagent, and reagent kit utilizing a surfactant-dye complex for the demonstration and determination of free fatty acids, compounds which hydrolyze to yield fatty acids, and hydrolase enzymes which act on substrates to produce fatty acids.
2. Description of the Prior Art
Prior methods for determining fatty acids in liquid samples include titration with alkali, determination of the copper salt of such acids after extraction with a suitable solvent, and chromatographic procedures. Such methods are time-consuming and, in general, are not adaptable to rapid, automated analysis techniques.
Further, various methods exist for demonstrating and determining compounds which are hydrolyzable to yield free fatty acids, including triglycerides, other cholesterol esters, and phospholipids. Prior methods for such determinations include enzymic hydrolysis methods, saponification methods, extraction methods, and chromatographic procedures.
Such techniques generally involve a number of processing steps which render the determinations time-consuming and costly, and/or require more than one reagent.
Hall U.S. Pat. No. 4,195,126 (Mar. 25, 1980), the disclosure of which is hereby incorporated by reference, discloses a method for determining free fatty acids and compounds which hydrolyze to yield free fatty acids.
The Hall patent describes an albumin-dye complex which is substantially free of endogenous free fatty acids and which has been pre-treated with a fatty acid. According to the method of the Hall patent, free fatty acids react with the albumin-dye complex to displace dye from the complex, yielding an albumin-fatty acid complex and free dye, the concentration of which is measurable by changes in absorbance or fluorescence. The change in absorbance is thus proportional to the concentration of fatty acid in the sample.
The Hall patent's complex may be used in determining a component in a sample which is capable of being hydrolyzed to yield fatty acid. The component is hydrolyzed by a hydrolase enzyme to yield a fatty acid, which is then reacted with the albumin-dye complex to liberate dye, the concentration of which can be directly measured by the change in absorbance or fluorescence.
The Hall patent also discloses that the complex can be used to determine the activity of a hydrolase enzyme in a liquid sample by adding to the sample a known amount of a compound which is capable of being hydrolyzed to yield a fatty acid, along with a reagent including the albumin-dye complex. The fatty acid thus liberated reacts with the complex to yield free dye, the concentration of which can be measured by observation of the absorbance change.
The albumin-dye complex of the Hall patent is limited in its practical utility, however, since one or more commercially useful hydrolase enzymes, such as Rhizopus arrhizus lipase, is conventionally provided in ammonium sulphate suspension. It has been found that ammonium sulfate interferes with the color development reaction of the Hall complex, thus necessitating desalting and lyophilizing of the lipase preparation obtained from the manufacturer. The lyophilized lipase is stable for only about 48 hours at 4.degree. C.
Further, it has been found that serum albumin depresses absorbance readings of the Hall reagent so that the absorbance of reagent-containing serum, without lipase, is lower than that of the reagent itself.