Quantitative analysis of 3-oxo-5Beta-Steroid and reagent therefor

Abstract: A quantitative analysis of 3-oxo-5β-steroid which comprises acting 3-oxo-5β-steroidΔ⁴-dehydrogenase on a sample in the presence of a reducing chromophoric agent, and measuring the optical density of the chromophoric substance thereby produced. A typical reducing chromophoric agent is a tetrazolium compound. The invention also provides a reagent for the quantitative analysis of 3-oxo-5β-steroid comprising 3-oxo-5β-steroidΔ⁴-dehydrogenase and a tetrazolium compound. The analysis and reagent provide a simple and reliable method for a liver function test.

Description: BACKGROUND OF THE INVENTION Field of the Invention : This invention relates to a quantitative analysis of 3-oxo-5β-steroid by means of an enzymatic method and a reagent therefor. Description of the Background: Among liver function tests those using bile acids have attained an important position in a clinical diagnosis of liver and/or biliary tract diseases in the past several years owing to a wide acceptance of the quantitation of the total bile acids in serum employing 3α-hydroxysteroiddehydrogenase. There has been known for long time, as a liver function test, a loading test comprising the measurement of the metabolic rate after administration of dehydrocholic acid which is a typical of 3-oxo-5β-steroid to the body of a subject [J. Japan Internal Medicine, 21 , 567 (1933)]. The clinical efficiency of this test can be explained as follows. Namely, dehydrocholic acid administered either orally or otherwise at normal or healthy conditions of a subject is partly transferred to the kidney without being captured at the liver and excreted in urine as it is, but the most part of the dehydrocholic acid is captured at the liver, reduced there into reduct dehydrocholic acid, and excreted in bile. The reduct dehydrocholic acid thus excreted in bile is absorbed at intestine and taken into blood, transferred to kidney, and then excreted into urine. If there are any disorders in the liver function of the subject, the partial reduction of dehydrocholic acid may be effected only deficiently, thus giving rise to an increased excretion of dehydrocholic acid and decreased excretion of reduct dehydrocholic acid in urine as compared to a person with a normal liver function. Likewise, the dehydrocholic acid concentration in blood decreases rapidly if the liver functions with a normal hepatic reduction. The rate of decrease in the blood dehydrocholic acid level lowers as the liver function degrades. The measurement of this function is important in order to determine the reductiondetoxication of the liver. The quantitative analysis of dehydrocholic acid has been studied since 1930's. The methods of analysis include those employing paper chromatography, thin-layer chromatography, high performance liquid chromatography and the like [J. Yonago Physic, 3 , 64 (1951); J. Biochem. 29 , 271 (1934)]. The method of tracing labeled dehydrocholic acid has also been proposed [(J. Clin. Invest. 52 , 715 (1973)]. These methods are carried out according to the following procedures. When serum is employed as a sample, 1 - 2 ml of a serum sample is diluted with 9 times by volume of 0.1 N - NaOH physiological saline and passed through a column of Amberlite XAD-2 or the like filler. The column is washed with distilled water and eluted with ethanol to obtain an ethanol solution of bile acids, which is then concentrated by means of an evaporator. The concentrate thus obtained is developed on a thin layer chromatography or a paper chromatography for separation, using a developing solvent, for example, of butanol/acetic acid/water (10:1:1). The detection limit of this method is approximately 10 µg. When an isotope is employed, a 24-hour urine sample, for instance, is passed through the aforementioned column, extracted with ethanol, concentrated, developed by paper chromatography for separation, and then the quantity of the radio isotope is measured. The detection limit is in the range from 1 µg to 100 ng. These methods, however, are not employed in a routine assay because of requirements of complicated pretreatment procedures for extraction, concentration, etc. as well as expensive equipment and materials. In particular, the poor measurement sensitivity in these methods poses following problems. That is, urine does not contain the indicative substance of the amount sufficient for the reliable assay. In case of using serum as a sample, a large amount of the sample is usually needed because the dehydrocholic acid level in blood is not always sufficiently high after administration of this substance. In addition, the assay may be sometimes hindered by the other components in the serum sample. Determination using isotope is not necessarily a preferable method because of requirement of special equipment and devices. A strong need, therefore, has existed for the development of a convenient and precise quantitative analysis of 3-oxo-5β-steroid. The inventors have conducted extensive studies for dissolving the aforementioned problems in the prior art. As a result, it was found that when 3-oxo-5β-steroidΔ⁴-dehydrogenase is acted on 3-oxo-5β-steroid in the presence of a reducing chromophoric agent, 3-oxo-5β-steroid is oxidized into 3-oxo-5β-Δ⁴-steroid, and at the same time, the reducing chromophoric agent is colored and may be conveniently submitted to a sensitive quantitation; (see non-prepublished prior application EP-A-0245528). The finding has led to the completion of this invention. SUMMARY OF THE INVENTION This invention provides the use of a quantitative analysis of 3-oxo-5β-steroid which comprises acting 3-oxo-5β-steroidΔ⁴-dehydrogenase on a sample in the presence of a reducing chromophoric agent and measuring the optical density of the chromophoric substance thereby produced in a loading test, in which a metabolism of dehydrocholic acid, administered in a body, is measured to determine liver functions. The reaction upon which the quantitative analysis of this invention is based is shown in the following scheme: That is, the quantitative analysis of this invention can be carried out by first oxidizing 3-oxo-5β-steroid into 3-oxo-5B-Δ⁴-steroid by the use of 3-oxo-5β-steroidΔ⁴-dehydrogenase (hereinafter abbreviated to "Δ⁴DH") in the presence of a reducing chromophoric agent, and at the same time, reducing said reducing chromophoric agent into a colored substance, and then measuring the optical density of the colored substance. A more complete appreciation of the invention and many of the advantages thereof will be readily obtained as the same becomes better understood by reference to the following description. BRIEF DESCRIPTION OF THE DRAWING Figure 1 is a diagram showing the changes in dehydrocholic acid level in serum on lapse of time, in which the concentration of dehydrocholic acid in serum (µmole/ℓ) is plotted along the ordinate and the time after administration of dehydrocholic acid (min.) is plotted along the abscissa. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Δ⁴-DH (EC.1. 3. 99. 6) useful in the practice of this invention is a dehydrogenase possessing a specificity to 3-oxo-5β-steroid and is widely found in microorganisms such as those belonging to the genus Pseudomonas [J. Chem. Soc.; Chem. Comm. 3 , 115 (1974); J. Biol. Chem. 218 , 675 (1956), ibid. 234 , 2014 (1959); Biochem. Biophy. Acta 56 , 584 (1962)], the genus of Arthrobactor [Eur. J. Biochem. 47 , 555 (1974)]; the genus of Nocardia [Chemical and Pharmaceutical Bulletin 21 , 2794 (1973), ibid. 23 , 2164 (1975); Dissertation Abstracts 35 , 3839 (1975)]; the genus of Corynebacterium (U.S. Patent No. 3,639,212), etc. There is no limitation to the source of the enzyme. The amount of Δ⁴-DH to be used may be, in terms of the reaction concentration, 50 - 10,000 units/ℓ, preferably 300 - 3,000 units/ℓ. Any reducing chromophoric agents may be used for the purpose of this invention so long as their intramolecular potential may change by accepting electrons thereby producing a chromophore which is capable of absorbing only a ray with a specific wave length. Specifically, they may be tetrazolium compounds, including but not limited to, nitroblue tetrazolium (hereinafter abbreviated as "NTB"), 3-(p-indophenyl)-2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (hereinafter abbreviated as "INT"), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (hereinafter abbreviated as "MTT") and 1,1′-(3,3′-dimethoxy4,4′-biphenylene)-bis{5-(4-nitrophenyl)-3-[4-(2-hydroxy-3-(2-hydroxyethyldiethylamino)-propoxy)phenyl]}-2H-tetrazolium chloride (hereinafter abbreviated as "W.S.NTB"). The concentration of the tetrazolium compound may be in the range of 50 - 2,000 µmole/ℓ, preferably, 100 - 1,000 µmole/ℓ. As 3-oxo-5β-steroid to which the quantitative analysis of this invention is applicable may be, beside dehydrocholic acid, 5β-androstan-3,17-dione, Δ¹-5B-androstene-3,17-dione, 5β-pregnane-3,20-dione, 21-hydroxy-5β-pregnane-3,20-dione, etc. Samples usable for the analysis may, therefore, be serum, plasma, urine and the like. In practicing the quantitative analysis of this invention, the sample, reducing chromophoric agent and Δ⁴DH may be added in an arbitrary order to a buffer, and after reaction the optical density of the reaction solution is measured. Any conventional buffers may be used for the quantitative analysis of this invention, including, for example, phosphate buffers, Tris buffers and Good's buffers with a pH range of from 6 to 10. There is no special limitation on the temperature at which the reaction is conducted to the extent that Δ⁴DH may not be deactivated. Usually, a temperature of 20 - 40 °C, preferably of close to 37 °C, may be used. The quantitation of 3-oxo-5β-steroid may be made, upon termination of the reaction by the addition of a terminating solution to the reaction mixture, by measuring the optical density of the colored substance in the mixture, or alternatively, by measuring the increase in the optical density of the colored substance in a prescribed period of time. Inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid or organic acids such as citric acid and acetic acid may be used for a terminating solution. The reagent for the quantitative analysis of this invention may be that consisting of a buffer solution added with 60 - 2,400 µmole/ℓ of the reducing chromophoric agent and 60 - 12,000 units/ℓ of Δ⁴DH. It is possible to prepare a buffer solution added with either one of the reducing chromophoric agent or Δ⁴DH in advance and to add the other when the reagent is used. This invention makes use of the enzymes which are capable of specifically recognizing the structure of 3-oxo-5β-steroid and the colorimetry of the colored substance. The invention thus eliminates the need for the procedures on the samples such as a heat treatment, deproteinization and extraction, thus requiring only a small quantity of the samples, as much as 50 - 200 µℓ in one quantitation, and yet giving an excellent sensitivity. The quantitative analysis of this invention can be applied to a loading test, in which a metabolism of dehydrocholic acid administered in a body is measured, thus providing a very simple and precise liver function test. Other features of the invention will become apparent in the course of the following description of the exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof. EXAMPLES Example 1 0.5 mℓ of 50 mmole/ℓ phosphate buffer solution (pH8) containing 500 µmole/ℓ NTB and 1,500 units/ℓ Δ⁴DH (hereinafter referred to as "reagent") was added with 100 µℓ of samples, and reacted precisely for 10 minutes at 37°C, upon which 0.5 mℓ of the terminating solution (0.1N - HCℓ) was added. After the resultant liquid was allowed to stand for 5 minutes, its optical density at a wave length of 540 nm was measured. The same samples were added to the reagent not containing Δ⁴DH and the above procedures were exactly repeated as a blank test. The samples used were serum added with dehydrochoric acid and diluted to various concentrations with the same serum but not containing dehydrocholic acid. Δ⁴DH used was that separated from Pseudomonas testosteroni cultured in accordance with the method proposed by Levy et al [J. Biol. Chem. 234 , 2014 (1959)], and purified. The result obtained are shown in Table 1 below. Dehydrocholic Acid Concentration (µmole/ℓ) Optical Density at 540 nm 20 0.022 40 0.046 60 0.077 80 0.101 100 0.122 140 0.172 180 0.226 Example 2 The same procedures were repeated on samples of serum added with in various concentrations the reagent of Example 1, but containing 5β-androstan-3,17-dione (a methanol solution) instead of dehydrocholic acid. The result is shown in Table 2. 5β-androstan-3,17-dione Concentration (µmole/ℓ) Optical Density at 540 nm 20 0.065 40 0.132 60 0.201 80 0.270 100 0.332 120 0.403 Example 3 A liver cirrhosis patient and a healthy subject were each intravenously given 1g of dehydrocholic acid. The changes of the dehydrocholic acid level in serum after the administration were determined according to the same procedure as in Example 1 and using the reagent of Example 1. The results are shown in the appended Figure 1. The elimination constant (keℓ), the half-life period (T 1/2) and the area under concentration curve of the compound in blood (AUC) are given in Table 3. Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims: Claims for the following Contracting States : AT, BE, CH, DE, FR, GB, IT, LI, LU, NL, SE Use of a quantitative analysis of 3-oxo-5β-steroid which comprises acting 3-oxo-5β-stereoidΔ⁴-dehydrogenase on a sample in the presence of a reducing chromophoric agent and measuring the optical density of the chromophoric substance thereby produced in a loading test , in which a metabolism of dehydrocholic acid, administered in a body, is measured to determine liver functions. A use as claimed in claim 1 wherein said reducing chromophoric agent is a tetrazolium compound. Claims for the following Contracting States : ES, GR Method for quantitatively determining the 3-oxo-5β-steroid on a sample of human organic fluid, characterized by comprising to treat the sample containing the 3-oxo-5β-steroid to be determined, with a 3-oxo-5β-steroidΔ⁴-dehydrogenase, in the presence of a reducing chromophoric agent, thus producing an oxidizing-reducing reaction in which the 3-oxo-5β-steroid is transformed into a 3-oxo-5β-Δ⁴-steroid, and the reducing chromophoric agent into a coloured substance; and then determining the optical density of the thus obtained reaction solution. Method according to claim 1, characterized by the fact that the 3-oxo-5β-steroid is selected from the group formed by dehydrocholic acid, 5β-androstan-3,17-dione, Δ¹-5β-androstene-3,17-dione, 5β-pregnane-3,20-dione, 21 -hydroxy-5β-pregnane-3,20-dione. Method according to claim 1, characterized by the fact that the reducing chromophoric agent is a tetrazolium compound. Method according to claim 1, characterized by the fact that the reaction is carried out in a buffer providing a pH in the range of from 6 to 10 and with a temperature of 20 - 40 ºC.

Inventors: SHIGIHARA TAKAE, USHIZAWA KOJI, SHIGIHARA, TAKAE, USHIZAWA, KOJI
Applicants: DAIICHI PURE CHEMICALS CO LTD, DAIICHI PURE CHEMICALS CO. LTD.
Application year: 1987
Main classification: C12Q1
IPC codes: C12Q1