Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1997-08-21
1999-07-06
Gitomer, Ralph
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435 25, 435810, C12Q 132
Patent
active
059196440
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a method of quantitatively determining sulfate-conjugated bile acid and a kit for determining sulfate-conjugated bile acid. The kit of the invention is particularly useful in the diagnosis of hepatobiliary diseases.
BACKGROUND OF THE INVENTION
It is well known that the bile acid in blood markedly increases due to hepatobiliary diseases. It is therefore an important item to determine the bile acid in blood for evaluating the hepatic function in clinical examinations. It has been clarified that the kinetics of bile acid in urine is similar to that of bile acid in blood. In urine, however, a large proportion of bile acid occurs as a sulfate-conjugated form (sulfate ester, at the 3-position hydroxyl group, of bile acid), which is highly soluble in water, hence the assay of bile acid is not easy.
The present inventors previously developed an enzyme capable of efficiently hydrolyzing the 3.alpha.-position sulfate ester moiety of sulfate-conjugated bile acid, namely bile acid sulfate sulfatase (BSS), and established a method capable of determining the sulfate-conjugated bile acid in biological samples in a simple and easy manner by the enzymatic method using said enzyme as the principal reagent (Japanese Unexamined Patent Publication (Kokai) No.145183/1990).
Subsequently, it was revealed that assaying of urinary sulfate-conjugated bile acid by the above method, as a method for diagnosing of hepatobiliary diseases, is comparable in diagnostic efficiency to blood chemistry test for GOT, GPT, .UPSILON.-GTP, TBA and so on and that said method is useful (Liver, Gallbladder, Pancreas), volume 31 (1995), No. 2, pp. 315-326!.
The assay principle of this prior art method consists in that sulfate-conjugated bile acid is first converted, under the action of BSS, to 3.beta.-hydroxybile acid, the latter is treated with .beta.-hydroxysteroid dehydrogenase (.beta.-HSD) in the presence of nicotinamide adenine dinucleotide (NAD), which is a coenzyme of said .beta.-HSD, and the NADH produced together with 3-oxobile acid is colorimetrically assayed using Nitrotetrazolium Blue (NTB), which is a reductive system indicator generally used as a high sensitive indicator for NADH. When viewed as a clinical test method, however, this method is still unsatisfactory from the viewpoint that a great number of clinical samples should be assayed rapidly without great labor.
Thus, this method, which is directed to urine samples, results in a great irregularity among urine samples as compared with serum samples, with a continuous increase in blank value, which of the extend is also various among urine samples, due to endogenous color developing substances in urine samples. To eliminate the blank value from each assay value, it is therefore inevitable to additionally perform assaying using a blank reagent without BSS. Thus, it is necessary to perform two assays (i.e. sample and blank assays) in parallel for each sample; this makes the procedure complicated. Furthermore, the reductive system indicator Nitrotetrazolium Blue (NTB) and the formazan thereof are scarcely soluble in water, leading to contamination of cells for colorimetry after one use. For repeated use, the cuvettes have to be washed very carefully, and this makes it difficult to apply the method to automated analyzers. Under the present circumstances, therefore, said method has to be performed manually, which places restrictions on the number of samples that can be treated and from the viewpoint of time, among others.
Accordingly, it is an object of the present invention to provide a method for assaying sulfate-conjugated bile acid and a kit therefor, which are suited for application to automated analyzers in general use.
DISCLOSURE OF THE INVENTION
The present inventors made intensive investigations to find out an analytical reagent for automated analyzers which satisfies the conditions (1) that it enables elimination of the blank value of sample in a single assay reaction system, (2) that the overall reaction can be comple
REFERENCES:
patent: 5091305 (1992-02-01), Sugimori et al.
patent: 5100795 (1992-03-01), Sugimori et al.
Ishiyama M., A New Sulfonated Tetrazolium Salt That Produces a HighlyWater Soluble Formazan Dye, Chem Pharm Bull 41(6) 1118-1122, 1993.
Chem. Pharm. Bull., vol. 41, No. 6 (1993) pp. 1118-1122, M. Ishiyama et al.
Adachi Kenichi
Tazuke Yasuhiko
Tsukada Yoji
Gitomer Ralph
Marukin Shoyu Co., Ltd.
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