JPH0644878B2 - Bile acid measurement reagent - Google Patents

Description

TECHNICAL FIELD The present invention relates to a reagent for measuring bile acid. More specifically, 3α-hydroxysteroid dehydrogenase (hereinafter, abbreviated as 3α-HSDH) having an optimum pH of 8.0 to 9.0 and having substrate specificity for 3α-hydroxysteroid.
And a reagent for measuring bile acid, which comprises a system containing

The reagent for measuring bile acid can be used for measuring the concentration of bile acid in blood as a test method for hepatobiliary diseases in the field of clinical testing.

Conventional technology Conventionally, there have been immunoassay methods, gas chromatographic methods, thin-layer chromatographic methods, high-performance liquid chromatographic methods, etc. for measuring bile acids, but depending on the method, only specific bile acids can be measured. However, there are problems that the pretreatment is complicated and the separation is insufficient, and that the sensitivity is low for measuring the blood concentration. In order to solve these problems, a simple, highly specific and sensitive method using 3α-HSDH has been adopted for the measurement of total bile acid. The enzymatic method for bile acid determination is excellent as a routine routine test method and mass screening method.

3α-HSDH has been described in many reports in the past.

As animal origin, its existence has been proved in rat liver, kidney, testis, prostate, rabbit liver, human prostate, but it is not suitable for industrial use.

On the other hand, examples of microbial origin include Pseudomonas testosterone (Pseudo
monas testosteroni) -derived 3α-HSDH (Nature, 1
Volume 73, p. 1189, 1954, Journal Biological Chemistr
y, 218, 675, 1956, Methods in Enzymology, 5
Volume, 512 pages, 1962, Biochemistry, 4 pages, 1825 pages, 1965
, Pseudo reported by Uejima et al.
domonas putida) and Bacillus suhuerikas (Bacill
us sphaericus) -derived 3α-HSDH (Agricultural
Biological Chemistry, 42, 1577, 1978, 43,
1521, 1979, Japanese Examined Patent Publication No. Sho 60-22914), 3α-HSDH also derived from Bacillus sphaericus (JP-A-54-1789).
4) etc.

However, 3α-HSDH reported by Talalay, P et al.
Has an optimum pH of 9.1 and its properties are different from those of this enzyme because its activity is markedly inhibited by metal ions and p-chloromercuribenzoate. Also, the optimum pH of 3α-HSDH derived from Pseudomonas putida of Ueshima et al. Is 11.
0, metal ions, p-chloromercuribenzoate inhibit the activity. Furthermore, 3α-HSDH derived from Bacillus sphaericus has an optimum pH of 10.0 to 10.5 and requires metal ions for stabilization.

As described above, the previously reported 3α-HSDH is practical,
There were many problems in terms of optimum pH, inhibitors, stability, etc. In particular, the conventional enzyme has a high optimum pH, which is disadvantageous for the measurement of bile acids in biological fluids.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention As described above, an enzyme conventionally used for bile acid measurement, for example, 3α-HSDH derived from Pseudomonas testosterone has an optimum pH of 9.0 or higher. Therefore, it has many disadvantages as a clinical test reagent. That is, in a bile acid measurement kit using this enzyme, the enzyme reaction is usually performed at about pH 7.0 to 8.0, so
It means that an enzyme having an optimum pH farther than H8.0 should be used in a sufficient amount for measurement. Therefore, the increase in the amount of enzyme used is not only in terms of cost but also the adverse effect of the enzyme and the additive, which are often contaminated, due to the large amount used. Moreover, the reagent composition contains β-NAD, and this substance is
The more H is inclined toward the alkali side, the more unstable and decomposes, which hinders the measurement. These problems are also that inspection data is not reflected correctly.

Therefore, an object of the present invention is to provide a reagent for measuring bile acid, which is advantageous for quantifying bile acid in biological fluid.

Means for Solving Problems Due to the above circumstances, the optimum pH is 3α lower than that of the conventional one.
Based on the development for which development of -HSDH is strongly desired, the present inventors have made earnest studies for the purpose of developing 3α-HSDH suitable for measuring bile acids in biological fluids in order to compensate for such drawbacks. As a result, in the course of research, it was unexpectedly found that a microorganism belonging to the genus Nocardia (Nocardia sp. No. Ch2-1, FERM-P No. 6217) produces an enzyme that meets the above-mentioned purpose, and the present invention was completed. Came to do.

The novel 3α-HSDH discovered here has an optimum pH of 8.
It is 0 to 9.0, which not only solves the above-mentioned pH problem associated with the measurement of bile acids, but is not affected by SH reagents such as p-chloromercuribenzoate and metal ions, and does not require a stabilizer. It had excellent properties.

An excellent strain used in producing the enzyme of the present invention is
Nocardia SP No. Ch2-1, FERM No.
6217 and its mycological properties are as follows.

(A) Morphological properties 1) Cell shape and size: It grows in the shape of hyphae in the early stage of culture and diverges. After that, irregular division occurs and the cells become rod-shaped. The size is about 0.8 to 1.0 μ × 1.5 to 4.0 μ.
It does not form aerial hyphae, nor sporangiospores.

2) Gram stainability: Positive 3) Anti-acidity: Positive 4) Motility: None (B) Chemical composition analysis meso-diaminopimelic acid, arabinose, in the cell wall
It contains galactose and does not contain L, L-diaminopimelic acid and glycine.

(C) Growth state in each medium 1) Meat broth agar plate medium: after culturing at 30 ° C. for 4 days, diameter 0.5 to
Form 1.0 mm round colonies. The surrounding area is full or wavy. The surface is smooth and hemispherical, and the central part may be convexly bulged. The color is pale cream and opaque. No dye appears in the medium.

2) Sucrose nitrate agar medium: Medium growth and the color of the colony is white to light cream. No water-soluble dye is emitted.

3) Glucose, asparagine agar medium: Medium growth, the color of the colony is cream. No water-soluble dye is emitted.

4) Glycerin and asparagine agar medium: Medium growth, and the color of the colony is white to light cream. No water-soluble dye is emitted.

5) Starch inorganic salt agar medium: Medium growth, and the color of the colony is white to light cream. No water-soluble dye is emitted.

6) Tyrosine agar medium: Medium growth and colony color is white to light cream. No water-soluble dye is emitted.

7) Nutrient agar medium: Growth is good and the color of the colony is cream. No water-soluble dye is emitted.

8) Yeast malt agar medium: Growth is good and the color of the colony is cream. No water-soluble dye is emitted.

9) Oatmeal agar medium: Medium growth, and the color of the colony is white to light cream. No water-soluble dye is emitted.

(D) Physiological properties 1) Growth temperature: Grow at 15 to 43 ° C. Does not grow at 10 ℃ or 45 ℃. The optimum temperature is 30-35 ° C.

2) Nitrate reducibility: positive 3) Catalase: positive 4) Oxidase: negative 5) Urease: positive 6) Starch hydrolysis: negative 7) Gelatin liquefaction: negative 8) Tyrosine hydrolysis: negative 9) Casein hydrolysis: negative 10 ) Xanthine hydrolysis: Negative 11) DNA degradation: Negative 12) Litmus milk: Alkaline, no peptone, no coagulation.

13) Formation of melanin-like pigment: None 14) Esculin hydrolysis: Positive 15) Tween 20,40,60,80 Hydrolysis: All positive 16) Penicillin resistance test: Tolerance 17) Attitude toward oxygen: Aerobic 18) Inorganic nitrogen source Use of: Both ammonium salt and nitrate are used.

19) NaCl growth range: Grows at 0 to 6%. Does not grow at 7%.

20) Assimilation of various carbon sources (Pridham, Godlieve agar): Assimilates D-glucose, D-fructose, mannose, glycerin and trehalose. L-arabinose, D-xylose, sucrose, inositol,
L-rhamnose, raffinose, D-galactose, D
-Do not assimilate mannite, maltose or sorbit.

21) Production of acid from various sugars: Production of acid from D-glucose, mannose, D-fructose, trehalose and glycerin. L-arabinose, D-xylose, D
No acid is produced from galactose, maltose, saccharose, lactose, D-sorbit, D-mannitol, inosit, starch.

The above-mentioned mycological properties are described in the Bergey's Manual of Determinativ
As a result of examination with reference to the 8th edition of e Bacteriology, the cell wall contains meso-diaminopimelic acid, arabinose, galactose, does not contain L, L-diaminopimelic acid, and glycine, and grows aerobically and well in a mycelial form. This bacterium is Nocardia due to its subsequent fragmentation into a bacillus-like form, acid resistance, and the absence of spore-spores and aerial hyphae.
Is a bacterium belonging to. This bacterium is
Named SP No.Ch 2-1 (Nocardia sp.No.Ch 2-1), and has been deposited in the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Bacteria No. 6217 (FERM -P No.6217). .

In order to produce and accumulate the present enzyme in the culture medium, sugars such as glucose and trehalose can be used as the carbon source in the nutrient medium, and organic substances such as meat extract, peptone and yeast extract can be used as the nitrogen source. The production of 3α-HSDH requires the addition of steroids, cholesterol,
It is preferable to add β-sitosterol and the like. Further, examples of the inorganic substance include phosphoric acid, magnesium, potassium and the like, but the medium components are not limited to the above components, and other components can be arbitrarily used. Culturing is preferably carried out in a medium containing the above components under aerobic conditions at 20 to 40 ° C.

Thus, the accumulated amount of the target substance 3α-HSDH becomes maximum in about 15 to 48 hours. Since this enzyme accumulates in the cells, it is then extracted and purified. This can be achieved by various known methods. For example, the obtained culture broth is filtered with a filter aid, or the cells are collected by centrifugation, and then the cells are crushed with a mortar, dynomill, French press, ultrasonic wave, etc. Then, 3α-HSDH in the cells is extracted. Then, the extract is made into a crude enzyme solution by concentrating by ultrafiltration or by making use of ammonium sulfate salting-out method, organic solvent precipitation method, dialysis method and the like.

The crude enzyme obtained as described above is generally used for adsorption and elution by ion exchange chromatography, gel filtration method based on difference in molecular weight, hydrophobic binding chromatography method, affinity chromatography method, electrophoresis method, etc. Purification is performed by appropriately selecting and combining different enzyme purification methods.

The physicochemical properties of the 3α-HSDH of the present invention thus obtained are as follows.

Action In the presence of coenzymes (NAD, NADP), 3 of steroid
The α-position hydroxyl group is dehydrogenated to produce a 3-ketosteroid.

Substrate specificity Acts well on deoxycholic acid and lithocholic acid. 5α
-Reactivity to steroids and 5β-steroids is equal. The results are shown in Table 1.

Optimum pH The optimum pH is pH 8.0 to 9.0.

The results are shown in Fig. 1.

Stable pH Stable pH range: pH 6.0 to 10.0 after treatment at 40 ° C for 15 minutes. Results are shown in FIG.

Optimum temperature and thermal stability Optimum temperature is 55-60 ℃, pH8.5, 40 ℃ in 20 minutes treatment
It has 100% residual activity up to 60% even when stored at 50 ° C. The results are shown in FIGS. 3 and 4.

Inhibition, activity and stabilization Almost no inhibition by SH reagents such as p-chloromercuribenzoate and monoiodoacetic acid. Also, EDTA, 2
Valent metal ions also do not affect activity. The results are shown in Table 2.

Km value Cholic acid 1.4 × 10 ^-4 M, Deoxycholic acid 1.7 × 10
^-5 M, taurocholic acid 7.1 x 10 ^-5 M, β-NAD 7.4 x
It is 10 ^-5 M.

Molecular weight Sephadex G-100 (registered trademark, manufactured by Pharmacia) was measured by gel filtration.
It is 58,000.

Isoelectric point The isoelectric point is pI 4.4 as measured by sucrose concentration gradient electrophoresis using an ampholine.

Purification method The cell disruption solution of the culture solution is salted out with 40% ammonium sulfate. The precipitate is dissolved in a phosphate buffer solution and dialyzed, then, passed through a DEAE Sepharose (registered trademark, manufactured by Pharmacia) column for adsorption, and eluted with sodium chloride. The active fraction is subjected to ultrafiltration, passed through an aminohexyl sepharose (registered trademark, Pharmacia) column equilibrated with a phosphate buffer, adsorbed, and eluted with sodium chloride. Further, the active fraction concentrated by ultrafiltration is subjected to gel filtration with Sephadex G-100 (registered trademark, manufactured by Pharmacia) and then desalted and concentrated to obtain the present enzyme.

Activity measurement method Sodium cholate (manufactured by Hanai Chemical) solution (10mg / ml)
0.5 ml, β-NAD (Oriental Yeast) solution (30
mg / ml) 0.1 ml, 0.1 M Tris-HCl buffer (pH 8.5)
Transfer 2.8 ml to a quartz cell and add 0.02 ml of enzyme solution to it.
React at 37 ℃ and measure the increase in absorbance at 340nm.

One unit indicates the amount of enzyme that produces 1 micromol of NADH in 1 minute.

As described above, the 3α-HSDH of the present invention differs from the 3α-HSDH derived from Pseudomonas testosterone, Pseudomonas putida, and Bacillus sphaericus in many respects. Table 3 shows these comparisons.

As can be seen from this, since the optimum pH of this enzyme is lower than that of other enzymes, and it is very stable without being affected by SH reagents and metal ions, the 3α-HSD of the present invention.
H has different properties from any of the conventional enzymes and can be determined to be new.

In addition, this enzyme is capable of removing contaminating enzymes, such as 3β, 7α, 12α, 17α-steroid dehydrogenase, which are problematic in the measurement of bile acids, by the above-mentioned purification method, and the amount of enzyme used is small. Combined with the above, only 3α-steroid can be specifically quantified.

The bile acid assay reagent using 3α-HSDH of the present invention determines the conversion amount of NAD, which is a coenzyme of the enzyme, into NADH.
According to the kind of the measuring method such as the UV measuring method based on the ultraviolet absorption of DH, the enzyme fluorescence measuring method, the enzyme colorimetric measuring method, the enzyme amplification measuring method, the bioluminescence reaction system, etc.
By prescribing HSDH, an excellent reagent for measuring bile acid can be obtained.

However, the measurement method selected here depends on the type of biological sample containing bile acid. For example, in blood, cerebrospinal fluid, and ascites, an enzyme fluorometric method or bioluminescence reaction system
For intestinal fluid, any method can be adopted. However, it is necessary to measure gastric juice, urine, feces, etc. after pretreatment.

The usage modes of the preferred reagent composition used in the present invention are described below.

(1) 60 units of 3α-HSDH / β-NAD 0.3mmol / Tris-HCl 0.10mol / Surfactant Add 2.5ml of the above composition to 0.2ml of a sample containing an appropriate amount of 0.156mmol / bile acid at room temperature. After reacting for 15 minutes, measure the absorbance at 340 nm.

(2) Tris 0.13mmol / 3 α-HSDH 100 unit / diaphorase 500 unit / β-NAD 1.0mmol / resazurin 10μmol / bovine serum albumin 12.2g / sample (bile acid up to 2.7μmol /) and NaOH solution 0.1
Add mol / to the above solution and rotate with a rotor 5
Fluorescence intensity of 600 and 300 seconds (fluorescence wavelength 600nm, excitation wavelength 550nm
) Is measured.

(3) 0.02 ml of the sample (up to 12 μmol / bile acid) and 0.02 ml of TCA solution (3 g /) are thoroughly stirred and left at 25 ° C. Next, luciferase 22.3mg /, FMN reductase 2
Unit /, FMN 4μmol /, bovine serum albumin 8g
Mix with 0.39 ml of a liquid containing /, 0.11 mol / of phosphoric acid and 0.2 mmol of decanal, and react for 15 to 20 minutes. Furthermore, β-NAD (0.8mmol /) 0.02ml and 3α-HS
Add 0.05 ml DH (150 units /) and observe the increase in luminescence intensity for the first 20 seconds.

Next, examples of the present invention will be shown, but the present invention is not limited thereto.

Example 1 Nocardia sp. No. Ch 2-1 (FERN-P No. 6217) was added to glucose 5 g /, meat extract 5 g /, yeast extract 0.2 g /.
And 200 ml of a medium (pH 7.2) consisting of a small amount of an antifoaming agent were inoculated into a 500 ml Sakaguchi flask, and cultured at 30 ° C. for 24 hours with shaking. This seed culture solution was mixed with 5 g of cholesterose.
, Glucose 2 g /, meat extract 5 g /, potassium hydrogen phosphate 5 g /, magnesium sulfate 0.2 g /, and defoaming agent 0.5 g //, and planted in a 30 volume jar fermenter containing a medium (pH 7.2) 20. The cells were inoculated and cultured at 30 ° C. for 40 hours with aeration and stirring. The culture solution was centrifuged, and the obtained bacterial cells were suspended in 0.1 M phosphate buffer (pH 7.0) and disrupted with glass beads. This at 1000r.pm
After centrifugation for 10 minutes, a clear cell extract was obtained. Next, ammonium sulfate was added to 40% saturation to precipitate the enzyme, and the precipitate was collected by centrifugation and 20 mM phosphate buffer (pH
6.0) It was dissolved in 100 ml. Further, it was dialyzed with a cellophane tube using the same buffer solution as above for 15 hours to obtain a crude enzyme solution.

This crude enzyme solution was equilibrated with 20 mM phosphate buffer (pH 6.0) to DEAE Sepharose (registered trademark, manufactured by Pharmacia) 10
The solution was passed through a column filled with 0 ml to adsorb the enzyme.
After washing, chromatography was performed with elution bubbles (sodium salt concentration 0 to 0.5 M) that continuously increase the salt concentration in the above buffer solution to obtain an active fraction. The active fraction was desalted and concentrated with an ultrafiltration membrane. This concentrated solution was added to 20 mM phosphate buffer (pH
After passing through a column packed with 50 ml of aminohexyl sepharose (registered trademark, manufactured by Pharmacia) equilibrated with 7.0), the adsorbed enzyme was washed with the same buffer containing 0.1 M sodium chloride, and the salt concentration was continuously adjusted to 0.1 to 0.4 M. Purification was carried out by an elution method in which the concentration was increased. Further, this active fraction was concentrated by an ultrafiltration method and then equilibrated with 10 mM phosphate buffer (pH 8.0) containing 0.2 M sodium chloride, Sephadex G-100 (registered trademark, manufactured by Pharmacia) 2.2 x Gel filtration was performed at 90 cm. After desalting and concentration again by ultrafiltration, slab electrophoresis with 7.5% acrylamide (pH 8.9) was performed, and 3α-HSDH obtained by this purification showed a single band.

Example 2 An appropriate amount of DEAE cellulose (manufactured by Selva Co.) was added to the culture filtrate obtained in Example 1, and the mixture was stirred for 1 hour and filtered through the back paper. 3α-HSDH adsorbed on DEAE cellulose is 0.3 M
After elution with a saline solution, this solution was passed through an octyl agarose column (50 ml volume) for adsorption. After washing, 1
Elution was performed with 1M saline containing sodium cholate, and the eluate was desalted and concentrated by ultrafiltration. Further, it was desalted with Sephadex G-25 (registered trademark, manufactured by Pharmacia). 3α-HSDH obtained by this purification method
Showed a single band on electrophoresis.

Example 3 Bile acid was quantified using the 3α-HSDH obtained in Example 1.

β-NAD (manufactured by Oriental Yeast) 4 μ mole, Triton X-100 (manufactured by Katayama Kagaku) 1.5 mg, 3α-HSDH0.2
3 ml of 0.1 M Tris-HCl buffer (pH 8.5) containing the unit,
Sodium cholate (manufactured by Hanai Chemical) solutions of various concentrations
After adding 0.1 ml and reacting at 37 ° C for 10 minutes, the absorbance at 340 nm was measured.

As shown in Table 4, good results were obtained.

Example 4 β-NAD (Oriental Yeast) 4 μmole, Triton X-100 (Katayama Chemical Co., Ltd.) 1.5 mg, 3α-HSDH 0.2 unit, Nitrotetrazolium Blue (Dojindo Co., Ltd.) 0.05 m
g, 0.1 ml of various concentrations of sodium cholate (manufactured by Hanai Chemical) to 3 ml of 0.1 M Tris-HCl buffer (pH 8.5) containing 3 units of diaphorase (manufactured by Amano Pharmaceutical Co., Ltd.)
After reacting for minutes, the absorbance at 560 nm was measured.

As shown in Table 5, good results were obtained.

Example 5 3α-HSDH [liquid product and freeze-dried powder (3 units / mg activity)] was subjected to stability over time at 4 ° C.

Table 6 shows the stability of the liquid product at each pH and the stability of the freeze-dried powder in terms of relative activity (%).

As is clear from the table, it can be seen that 3α-HSDH retains its activity for a long period of time.

EFFECTS OF THE INVENTION The 3α-HSDH used in the present invention is the conventional 3α-HSD.
Since the optimum pH is lower than that of SDH, the cost can be greatly reduced due to the miniaturization of the enzyme used, and bile acid can be obtained under the stable conditions of coenzymes (NAD, NADP) present in the kit. Can be measured.

Furthermore, since this enzyme is not affected by SH reagents and metal ions at all and has high stability, it has advantages such as excellent reagent storability. Therefore, it is advantageously applied to the determination of bile acids in biological fluids. It is possible to provide accurate inspection data.

[Brief description of drawings]

FIG. 1 is a diagram showing the relationship between pH and activity of 3α-HSDH used in the present invention. FIG. 2 is a diagram showing the relationship between pH and stability of 3α-HSDH used in the present invention. FIG. 3 is a diagram showing the relationship between reaction temperature and activity of 3α-HSDH used in the present invention. FIG. 4 is a diagram showing a relationship between temperature and stability of 3α-HSDH used in the present invention.

Claims (2)

[Claims] 1. Obtained by culturing a Nocardia microorganism,
A reagent for measuring bile acid, which has an optimum pH of 8.0 to 9.0 and comprises a system containing 3α-hydroxysteroid dehydrogenase having substrate specificity for 3α-hydroxysteroid. 2. A strain belonging to the genus Nocardia is Nocardia.
SP No.Ch2-1 (Nocardia sp.No.Ch2-1)
The reagent for measuring bile acid according to claim 1, which is FERM-P No. 6217.