DE2818327A1 - PROCEDURE FOR THE QUANTITATIVE DETERMINATION OF THE TOTAL BALIC ACID CONTENT OF ANALYSIS SAMPLE - Google Patents

Description

uZ: M 698
Case: 214-4

KTOWA HAKKO KOGYO GO., LTD. Tokyo, Japan

"Method for the quantitative determination of the total bile acid content of an analysis sample ¹¹

The quantitative determination of bile acids in serum or

the like is an effective method for the discovery of liver diseases. The determination of bile acid in one Analysis samples have so far been carried out by gas chromatography or with the aid of enzymes. In a known enzymatic In the procedure, a serum solution is passed through a column filled with Amberlit XAD-2 to remove the bile acid to be released into an eluate. The eluate obtained is evaporated and then extracted with methanol. The extract is then used to form cholestenone with 3-oL-hydroxysteroid dehydrogenase offset. After reacting the cholestenone with a fluorescent compound such as hydrazine hydrate, the amount of cholestenone is determined by fluorometric analysis, (a compilation of the main points of the

22. Lecture of the Japanese Society of Clinical Pathology, 1975 »p. 91) · - However, this method has in particular, in the determination of bile acid in a blood test aur " as this requires at least 1 ml of serum. In addition, the necessary evaporation and extraction is difficult u.nd causes errors.

809846/0687

According to an improved method using fluorometric analysis (Clinical Chemistry, Vol. 4, Nos. 3 and 4, (1976), pp. 312-318), 0.2 ml of serum is heated, for example 20 minutes to 67 ⁰ C, and then with a 0.1 M tris-HCl buffer solution (pH 9.0), which contains 3-λ -hydroxysteroid dehydrogenase, diaphorase, nicotinamide adenine dinucleotide and resazurin. The mixture obtained is then incubated at room temperature, for example at 20 ^{° C. for 40 minutes.} Thereupon the fluorescent connection fertilization in the mixture at an emission wavelength of 580 nm (an excitation wavelength of 560 nm) measured. However, this method also has certain disadvantages. In particular, the rate of discovery of bile acid is in this process at most 90 #, impurities in the The analysis sample and the fluorescent substance generally interfere with the measurement, and a complicated apparatus, such as a fluorophotometer, is required.

The invention is based on the object of a method for the quantitative determination of the total bile acid content an analysis sample, such as blood serum, that is more accurate works than the known method and can be carried out quickly.

This object is achieved by the surprising finding that the total bile acid content in an analytical sample can be quantitatively determined by acidifying the sample with an acid, adding 3 ° < -hydroxysteroid dehydrogenase (EC 1.1.1.50) to the acidified mixture (EC 1.1.1.50) (hereinafter referred to as "3ol-HSD"), diaphorase, nicotinamide adenine dinucleotide (hereinafter referred to as "NAD"), a tetrazolium salt and a base or a buffer and then spectrophotometric measurement of the color intensity of the resulting colored formazane can be determined.

The invention thus relates to the subject matter characterized in the patent claims.

t ~ ■■■■■■: ■■ "" "■ '■■. ■

According to the method according to the invention, all steroids with a hydroxyl group in the 30L position can be determined, since 3c *, - HSD is used. Most bile acids have a hydroxyl group in 3a. -Position up. In the human body, the bile acids are either in free or in conjugated form. Glycocholic acid is an example of a conjugated compound of bile acid and glycine. Another example of a conjugated compound is taurocholic acid, in which taurine and a bile acid are conjugated . In all of the conjugated compounds, these can be determined by the method according to the invention if the 3cL position is free, ie carries a hydroxyl group. As a result, the term "total bile acid content" refers to all bile acids which have a hydroxyl group in the 3ol position and their conjugated compounds.

The method according to the invention is suitable for the determination the bile acids in samples such as serum, urine, or bile.

The method according to the invention proceeds according to the following principle zip: The bile acids react with NAD in the presence of 3OCw-HSD to form 3-keto steroids and NAD in a reduced form (hereinafter referred to as "NADHp"). The resulting NADHp reacts with a letrazolium salt in the presence from diaphorase to NAD and a formazan, which is a dye

is. By measuring the absorption of the dye,. that in an amount of 1 or 1 1/2 moles per mole of bile acid arises, at a wavelength of 400 to 600 nm, the amount of bile acid in the sample can be quantified to be determined.

Because enzymes present in the blood such as lactate dehydrogenase, malate dehydrogenase and glutamate dehydrogenase, the implementation inhibit bile acid and NAD, such enzymes must first be inactivated. To inactivate the

_Λ , ORIGINAL IRSPECTED '"

809046/0687

Enzymes will adjust the sample to a pH of 0.1 to 6.0. acidified and then heated ^{to 20 to 4-5 0} C for 1 to 30 minutes. In this stage, enzymes that are active under acidic conditions, such as an acidic protease or peptidase, are preferably added in order to degrade the inactivated enzymes.

After the heat treatment, the solution is treated with a coloring reagent consisting of diaphorase, NAD, a tetrazolium salt, a wetting agent and a buffer, and the resulting mixture is again ^{subjected to a heat treatment at a temperature of 20 to 50 °} C. for 1 to 30 minutes . In this case, the coloring reagent can be added in either a solid or a liquid state.

Then the mixture is rated 3 **. -HSD added and the pH set to 7.5 to 12.5, preferably 9 to 10. The 3tt_-HSD can of course be used as an alkaline solution be used. The absorption of the mixture is then measured at a wavelength of 4-00 to 600 mn. The gallic acid content the sample can then be calculated using a molecular extinction coefficient or using a calibration curve will.

In the method according to the invention, the 3 <* - HSD can also be used together can be used with the coloring solution.

According to the method according to the invention, 0.01 to 1 ml of serum or urine or 0.001 to 0.1 ml of bile are sufficient for the determination of bile acids.

Mineral acids, such as hydrochloric acid and sulfuric acid, and organic acids, such as Citric acid and malic acid. Specific examples of tetrazolium salts, which are suitable as coloring substances are 3- (p-iodophenyl) -2- (p-nitrophenyl) -5-phenyl-2H-tetrazolium chloride (hereinafter referred to as "INT") and 3,3 '- (3,3'-

809846/0687

Dimethoxy-4-, 4- ¹ -biphenylylene ^ bis-Z ^ -Cp-nitrophenyl) -5-phenyl-2H-tetrazolium chloride.7 (hereinafter referred to as "NTB""). They are preferably used at a concentration of 0.1 used up to 50 mM.

To increase the accuracy of the determination, the use of a wetting agent is favorable, since it contains the tetrazolium salt and dissolves denatured proteins. A specific example of a suitable wetting agent is an octylphenoxypolyethoxyethanol. The wetting agent is preferably used in a concentration of 0.001 to 5 percent by weight per volume.

A suitable buffer in the process according to the invention is Borate or tris buffer.

The concentrations of the other constituents of the reaction mixture are preferably 0.1 ΓΕ / 1 to 100,000 ΣΕ / 1 Diaphorase and 0.1 mH to 50 mM NAD. 20th

The concentration of the 30C-HSD is determined by the duration of the conversion. A concentration of 0.0001 ΊΈ / 1 to 100 IU / l is preferred.

Preferred bases for making the reaction mixture alkaline are sodium and potassium hydroxide.

The examples illustrate the invention.

Example i

0.2 ml of serum samples from a patient suffering from cirrhosis of the liver are used. 0.2 ml 0.1N HCl and 0.3 ml distilled water are added to each sample. The resulting mixture is incubated for 10 minutes at 37 ⁰ G.

After the heat treatment, the samples with 2.0 ml of a

809846/0687

coloring solution added. This is a 0.1 M borate puff solution from pH 9 »O and contains (in percent by weight) ent / volume):

Diaphorase (30 ΙΕ / mg). 0.0055 NAD 0.32

UJT 0.026

Octylphenoxypolyethoxyethanol 0.1.

One of the samples is then mixed with 0.3 ml of a 0.1 M borate buffer solution with a pH of 9 »0, which contains 0.1 per cent by weight / volume of 306-HSD (0.984 / mg) another sample with 0.3 ml of distilled water was added. the two mixtures are incubated for 15 minutes at 37 ⁰ G. Thereafter, the absorbance at 5OO is measured using the comparative sample as a blank nm. There is a value of 0,352.erhalten.

The bile acid concentration in the sample is calculated as 352 / uM from the absorption and the molar extinction

4th
coefficient (1.5 χ 10) of the coloring substance, INT, at 500 nm.

Example 2

Example 1 is repeated with the change that instead of 0.2 ml of serum, 0.2 ml of an analytical sample is used which contains a mixture of 10 ml of serum and 0.493 mg (100 / uM) of glycolic acid (O ₀ JBL-NO,. 1.5H_0, Mw = 493). The absorbance value obtained is 0.447.

The increase in the extinction due to the addition of glycolic acid is 0.095, calculated from the difference between the extinctions obtained in Example 2 and Example 1. As a result, the glycolic acid concentration found is 95 / UM. The rate of detection of glycolic acid is calculated as follows:
35

809846/0683

Glycocholic acid detection rate:

_. - · 'found glycocholic acid _{concentration χ} ^ qq used glycocholic acid concentration

■ τ * - * ¹⁰ ° " ^{95 w}

Example 3

0.2 ml of serum samples from a patient suffering from cirrhosis of the liver are used. Each sample is mixed with 0.8 ml 0.01η HOl, which contains 0.1 percent by weight / volume of pepsin (2500 ΙΕ / mg), -. and incubated at 37 ^° C. for 10 minutes. The samples are then each mixed with 2 ml of the following coloring solution and ^{incubated at 37 °} C. for a further 15 minutes.

Me coloring solution is a 0.1 M borate buffer solution with a pH value of 9 »0 and contains (in percent by weight / volume):

Maphorase (3O IS / mg) 0.0055 NTB 0.026 NAD 0.32 3 ^ -HSD (0.984 IU / mg) 0.1 Octylphenoxypolyethoxyethanol 0.1

As a comparison sample, 0.2 ml of distilled water such as treated above. The absorbance of the sample to be examined is measured at 550 nm using the reference sample measured as a blank sample. A value of 0.230 is obtained. The bile acid concentration in the serum is determined from the Absorbance and the molar extinction coefficient (3.64 χ 10) of the coloring reagent, KTB, at 55O nm I9O / 1M calculated.

Example 4

Example 3 is repeated with the change that 0.2 ml of an analysis sample is used instead of 0.2 ml of serum, which is a mixture of 10 ml of serum and 0.493 mg (100 / iM)

805346/0687

Γ »

Glycocholic acid (C ₂ ^H 6 43 ^# 6 ^# 2 ^{1i5H 0i = Hw} ^ ⁹⁵ ^ ^represents · ^It wira obtain an absorbance of 0.348.

The increase in the extinction due to the addition of glycolic acid is calculated from the difference between the extinctions obtained in Example 4 and Example 3 to be 0.118. The retrieved Glykocholsäurekonzentration "is thus 97 / uM. Pursuant / Example 2 calculated retrieval rate of

Glycocholic acid is 97% 10

Example ^.

2 ml of the coloring solution used in Example 1, 0.5 ml of a 0.1 M borate buffer solution of pH 9 »0 and 0.3 ml of the 0.1 M borate buffer solution of pH 9.0, the 0 , 1 percent by weight / volume 3 <* - HSD (0.984 ΙΕ / mg) are mixed and incubated for 10 minutes at 37 ° C. The samples are then each with 0.2 ml of a calibration substance such as sodium deoxycholate (233.4; µM) (hereinafter referred to as "SDC"), chenodeoxycholate (211.4 / µM) (hereinafter referred to as "CDC"), glycolic acid ( 209.1 μΆ) (hereinafter referred to as "GCA") and sodium taurocholate (196.3 / μM) (hereinafter referred to as "STC"). The mixtures obtained are incubated ^{at 37 ° C. for 5 minutes.} The extinctions are measured at 500 nm. The extinctions found and calculated and the rate of detection of the calibration compounds are summarized in Table I.

Table I.

3Q calibration connections

SDC CDC GCA STC

absorbance found 0.230 0201 calculated absorbance 0.233 0.211

Detection rate 99% 95% 35

0.210 0.192 0.209 0.196 100% 98%

L -J

809846/0687

Example6

7 ml urine of a healthy adult man is adjusted to a pH value of 5 and mixed with 0.25 IU ß-glucuronidase (3 IU / mg). The mixture obtained is then incubated for 15 hours at a temperature of 50 ^° C. and then diluted to 10 ml with water.

The procedure described in Example 3 is followed with the change repeats that 0.2 ml of the diluted solution prepared above is used as an analytical sample. as The result is the amount of bile acid in the sample due to the absorbance value of 0.045 measured at 550 nm calculated.

Example7

To determine the reproducibility of the bile acid determination Example 3 is repeated with the change that instead of the serum samples used there, 0.2 ml of serum samples another patient with cirrhosis will. The results are summarized in Table II.

Table II Sample no. Measured GaIl 1 76 2 74 3 75 4th 75 5 76 6th 74 7th 77 8th 75 9 10 76

809646/0687

The table above gives the following mean, standard deviation and coefficient of deviation:

Mean (x): 75.2 / µM

Standard deviation (SD): 1.032

Deviation coefficient (Cv): 1.3%

Examples

It becomes the relationship between that according to the method according to the invention and that in the fluorophötometric .Analyse obtained bile acid value examined. For this purpose, example 3 is carried out using 10 serum samples of various origins repeated.

photo
For fluorometric analysis, the Osuga et al.

in Clinical Chemistry Vol. 4, No. 3 and 4 (1976), pp. 312-318, described procedure carried out. The results are in Table III summarized.

Table III fluorophotometric Trial Fri he bond s- Analysis (x) moderate.procedure
(y) 15 OuM) 1 12 OuM) 7th 2 S. 24 3 22nd 38 4th 40 36 5 35 129 6th 125 16 7th 13th 64 8th 76 11 9 10 7th 10 6th

The correlation coefficient results from the table above and the regression between method (y) according to the invention and that of fluorophotometric analysis (x): Correlation coefficient: 0.99 Regression: y = 1.01x - 0.35.

L. -1

809846/0687

Claims (15)

VOSSIUS · VOSSIUS · HILTL · TAUCHNER · HEUNEMANN SIEBERTSTRASSE A 8OOO MÖNCHEN 86 PHONE: (O89) 4-7 4-O7O CABLE: BENZOUPAT ENT MÖNCHEN -TELEX 5-29 453 VOPAT D u.z .: M 698 (Vo / Ra / H) April 25, 1978 Case: 214-4- KTOWA HAKKO KOGXO 00., LTD.
Tokyo, Japan "Method for the quantitative determination of the total bile acid content an analysis sample " Priority: May 4, 1977, Japan, No. 51575/77 Claims (y. Method for the quantitative determination of the total bile acid content of an analytical sample, characterized in that the sample is acidified, then with 3-tX-hydroxysteroid dehydrogenase, diaphorase, nicotinamide adenine dinucleotide, a tetrazolium salt, a wetting agent and a Base or a buffer are added and the color intensity of the resulting colored formazane is measured spectrophotometrically. 2. The method according to claim. 1, characterized in that the analysis sample from 0.01 to 1 ml of serum or urine or consists of 0.001 to 0.1 ml of bile. 30th 3 ·. Method according to claim 1, characterized in that the sample is brought to a pH of 0.1 with an acid to 6.0 and 1 to 30 minutes at 20 to -5 ° 0 warmed up. 4-. Method according to claim 3, characterized in that to acidify hydrochloric acid, sulfuric acid, citric acid or malic acid is used, 5. The method according to claim 3i, characterized in that To decompose inactivated enzymes, the acidified sample is additionally mixed with one that is active under acidic conditions Enzyme added. 6. The method according to claim 5? characterized in that an acidic protease or peptidase is used as the enzyme. 7. The method according to claim 1, characterized in that the tetrazolium salt is 3- (pJ ° cLph.enyl) -2- (p-nitrophenyl) -5-phenyl-2H-tetrazolium chloride or 3,3 '- (3.3 ^f -Dimethoxy-4,4'-biphenylylene) -bis- ^ 2- (p-nitrophenyl) -5-phenyl-2H-tetrazolium chloride. 7 used. 8. The method according to claim 7 »characterized in that the concentration of the tetrazolium salt in the mixture is 0.1 to 50 mM. 9. The method according to claim 1, characterized in that a borate buffer or iris buffer is used as the buffer. 10. The method according to claim 1, characterized in that the diaphorase concentration in the mixture is 0.1 ΣΕ / 1 to 100,000 IU / 1. 11. The method according to claim 1, characterized in that the concentration of nicotinamide adenine dinucleotide
in the mixture is 0.1 to 50 mM. <^ Ρϊ Λ Λ I f * / ftpB 301848/0 © sucht 1 12. The method according to claim 1, characterized in that the concentration of J> -c * - -hydroxysteroid dehydrogenase in the mixture is 0.0001 IU / l to 100 IU / l. 5 13. The method according to claim 1, characterized in that the curing agent concentration in the mixture from 0.001 to 5 percent by weight per volume. 14. The method according to claim 1, characterized in that the base used is sodium or potassium hydroxide. 15. The method according to claim 1, characterized in that the color intensity is measured at a wavelength of up to 600 nm. 809846/0687