JPH02200199A - Determining composition of bile acid in blood - Google Patents

Abstract

PURPOSE:To readily improve accuracy of determination by making carrier made of polymer material to carry buffer keeping fixed pH, 3alpha-hydroxy steroid dehydrogenase and nicotinamide adenine dinucleotide, etc. CONSTITUTION:3alpha-hydroxy steroid dehydrogenase is dissolved in pH buffer keeping pH 7-9 together with nicotinamide adenine dinucleotide, electron carrier such as diaphorase, tetrazolium salt, ascorbate oxidase and oxalic acid or salt of the acid as lactate dehydrogenase inhibitor, and humectant, as necessary, to obtain a determining composition of bile acid in blood. the composition is impregnated in a carrier made of polymer material such as filter paper and dried to afford the determining composition. Then, desired blood serum is dropped or applied to said composition and reacted, thus generated formazan coloring matter is measured to determine bile acid in blood.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a composition for quantifying bile acids in blood, which is used for clinical diagnostic tests related to hepatobiliary diseases.

(Prior art) Bile acids contained in bile are present in trace amounts in body fluids such as blood and urine, and it is known that the amount changes when a disease occurs in the hepatobiliary system. The amount of bile in the blood has become an effective guideline for diagnosis of hepatobiliary diseases.

Therefore, the determination of bile acids is carried out using blood, especially serum, as a sample, and is used, for example, to determine the presence or absence of hepatobiliary tract disease, to detect non-jaundice disease, to monitor the progress of chronic hepatitis, to determine the prognosis of acute hepatitis, to diagnose liver cirrhosis, It is performed for the purpose of diagnosing cholestasis, congenital biliary atresia, etc.

As a method for quantifying total bile acids in a sample, there is, for example, the method disclosed in Japanese Patent Publication No. 59-DI97.

According to this method, by heat-treating a bile acid-containing sample under acidic conditions, enzymes that interfere with measurement, such as lactate dehydrogenase (hereinafter referred to as rLD), which may be present, are inactivated, and this solution contains A reaction solution containing 3α-hydroxysteroid dehydrogenase (hereinafter referred to as “3α-H5DJ”), nicotinamide adenine dinucleotide (hereinafter referred to as rNAD), diaphorase, and a tetrazolium salt was added, and the solution was adjusted to pH 8-9. The reaction is carried out under alkaline conditions.The reaction in this case is as follows.
It reacts with AD to form a carbonyl group, and thus the bile acid becomes a keto type, and at this time NAD is converted to reduced nicotinamide adenine dinucleotide (hereinafter referred to as rNADHJ).

Bile acid Bile acid (keto type) NADHJ produced by the above reaction reacts with a tetrazolium salt in the presence of diaphorase to convert the tetrazolium salt into formazan, and at this time, NAD
I (itself is oxidized and becomes NAD again.

NADHNAD Tetrazolium salt Formazan The number of moles of formazan produced by this reaction is NAD)
Since the number of moles of l corresponds to the number of moles of bile acids, the total bile acids are determined by measuring the absorbance of this formazan.

Other methods for measuring total bile acids include the reaction formula (1
8) Method for measuring the fluorescence of N A D H generated in 1sct+varz et at, “Cl1n,
Chim, Acta Jiang, 197.19701,
By the NADH produced in the above reaction formula (A),
A method of measuring the fluorescence emitted by reducing resazurin in the presence of diaphorase has been proposed by Mashige et al., Clinical Chemistry J, Vol. 4, p. 312, 1976, etc.

(Problems to be Solved by the Invention and Objectives of the Invention) All of the above conventional methods can arbitrarily and satisfactorily measure bile acids in a sample, but they utilize a reaction in a solution system. Therefore, the operation is complicated and the measurement time is long, so it is not suitable for mass screening or emergency testing where a large number of samples must be tested. Expensive equipment is required to perform quantitative determination by measurement, and these have been regarded as issues to be solved in the future as they make on-site testing impossible.

In order to solve these problems, the present applicant has proposed a test strip and its manufacturing method that make it possible to measure the amount of bile acids in body fluids in a simple manner and in a short time (Japanese Patent Laid-Open No. 6
1-2611199, 62-272994), and the test strips disclosed in these publications contain tetrazolium salt, NAD, and 3α-H2O on a carrier made of a polymeric material.
However, when attempting to quantify bile acids in a sample, such as serum, using this test strip, it may be necessary to use this test strip if the concentrations of LDH, pyruvic acid, lactic acid, or ascorbic acid are relatively high. , LD), pyruvic acid and lactic acid are affected by the following lactate dehydrogenase reaction between NAD impregnated in the test paper and NAD H produced in the main reaction, and ascorbic acid is easily affected by its reducing action, and therefore does not have sufficient measurement accuracy or reliability, so although the above test strips can be used for mass screening, they are not suitable for clinical testing for diagnosis. The reality is that it was practically impossible to do so.

L [lFI lactic acid + N person D + pyruvate ten NADH + )I
“Also, in these patent publications, it is described that when serum is used as a sample, pretreatment is performed, that is, oxamic acid or pyruvic acid is added, but after further testing, the effect of the addition is as follows. As shown in Table 1, in the case of pyruvic acid, the difference between pretreatment and no pretreatment is difficult to recognize, and in the case of oxamic acid, the LD)I inhibitory effect is not sufficient and is not acceptable in practice. It was found that there were no positive errors.

Note: The unit of value in the table is μmol/I, and this test result was obtained when 30 mg/dl of lactic acid coexisted in the sample.
Pyruvate and oxamic acid were added to different types of specimens, and the inhibitory effects of each added substance on LDI were compared with those without pretreatment and without addition. The above-mentioned JP-A-61-268199 and JP-A-62-2
When attempting to quantify bile acids in serum using the test strip disclosed in Publication No. 72994, one of the following two methods must be adopted in order to eliminate the influence of interfering substances. , all of which require a great deal of effort and complicated operations to actually implement.

a) Measure the sample blank value and subtract it in the same way as the measurement method using a reaction in a solution system. That is, a test strip that does not contain 3α-)ISD is prepared separately, and the influence of coexisting substances other than bile acids is determined using this test strip and subtracted.

h) The sample is subjected to pretreatment such as heat treatment under acidic conditions, organic solvent extraction, and ion exchange resin treatment to remove interfering or measurement-interfering substances.

Among the above image processing methods, method a) can provide reasonable results, but it is important to avoid deterioration in measurement accuracy due to the use of two types of test paper (for specimens and for specimen blanks). In particular, bile acids in serum are extremely low, less than 10 ALmol/l in healthy people in the fasting state, and even in clinical diagnosis, several μ+1101 around this IOμ+1101/1. Since identification of the /l concentration is required, there are cases where the purity is insufficient and it is practically unusable.

Therefore, the object of the present invention is to
199 and No. 62-272994, namely, that the measurement operation is simple and the time required for measurement is short, the problem of interference in the sample can be solved. It is an object of the present invention to provide a composition for quantitative determination of bile acids, which can eliminate the superfluous point, which is easily influenced by substances and may have low reliability.

(Means and effects for solving the problem and achieving the object) Therefore, the present inventors have conducted intensive studies to find out what are the interfering substances in the specimen, especially in the serum, that affect the quantitative reaction of bile acids. However, if we discover that ascorbic acid is a component involved in the lactate dehydrogenase reaction, and if we coexist a lactate dehydrogenase inhibitor, especially oxalic acid or its salt, and ascorbic acid oxidase in the reaction system, the above interference can be eliminated. The present invention was completed by discovering that the effects of substances can be avoided.

Therefore, according to the present invention, the above problem is solved by dehydrogenating bile acids in serum by 3α-hydroxysteroid dehydrogenase in the presence of nicotinamide adenine dinucleotide, and in this case, an electron carrier and a tetrazolium salt are added to the reaction system. A composition for quantifying blood bile acids by measuring the degree of coloration of formazan produced in the coexistence of p.

pHM buffer, 3α-hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide, electron carrier, torazolium salt, ascorbate oxidase, and oxalate as lactate dehydrogenase inhibitor. The above object is solved and achieved by a composition for quantifying blood bile acids, which is characterized in that bile acids or a salt thereof are supported on a carrier made of a polymeric material.

In the composition for quantitative determination according to the present invention, the carrier made of a polymeric material is, for example, an absorbent carrier such as filter paper, and this carrier is used for carrying reagents required for quantitative reactions and reagents for removing the influence of interfering substances. It supports everything and is in a dry state. Therefore, when quantifying blood bile acids, if a certain amount of serum is dropped or applied onto this composition, a colored formazan dye will be generated within a few minutes. Bile acids in serum can be determined with high precision by measuring the amount of change in reflected light intensity using a simple reflectance meter. Of course, if a bleach colorimetric table is prepared in advance using a standard serum containing a predetermined amount of bile acids, the color tone developed when using the sample serum is compared with the standard colorimetric table.

So-called visual quantification is also possible.

In the reagent constituting the composition for quantitative determination according to the present invention, the coenzyme NAD is extracted from yeast or animal tissue such as liver, W viscera, etc., and its hydrate or salt 1, such as sodium salt, is 3α-H2O is derived from microorganisms, such as Pseudomonas testostero m: (P
diaphorase derived from microorganisms is used as the electron carrier, such as Bacillus stearothermophilis.
thtrmophilis) and Clostridium kluyveri (ClostridiumklBveri)
) etc., especially Bacillus stearothermophilis from the viewpoint of stability over time.
3-(p-iodophenyl)-2-(p-nitrophenyl)-5-phenyltetrazolium chloride [abbreviation rINTjl, 3(4,
5-dimethyl-2-thiazolyl)-2,5-diphenyl-2totitrazolium [abbreviation [AiTTJ], 3,
3°-33'-dimethoxy-4,4-biphenylylene)-bis(2(p-nitrophenyl)-5-phenyl-
2H-tetrazolium [abbreviated as "nitro-T[l"] 1, etc. can be used, but from the viewpoint of sensitivity, it is preferable to use nitro-TB. Any oxalic acid or its salt can be used as it inhibits the lactate dehydrogenase reaction, which is one of the causes of abnormal color development, but sodium oxalate is preferable in terms of solubility and preparation. Ascorbic acid oxidase, which eliminates the effects of other factors by oxidizing ascorbic acid, is derived from plants, and there are no restrictions on the type of plant from which it is derived.
)II buffer may be any one that provides optimal pH conditions (pH 7-9) for enzymatic reactions and color reactions, such as phosphorus tlJla buffer, boric acid buffer, Tris salt. A buffering agent, a solid buffering agent, etc. can be used. On the other hand, the material of the polymer carrier may be paper made of natural or synthetic fibers, non-woven fabrics, membrane filters, etc., and water-permeable natural or synthetic film-forming polymers such as gelatin, polyvinyl alcohol, etc. You can also use

In addition, in addition to the above-mentioned reagents, the composition for quantitative determination according to the present invention may contain a wetting agent and a viscosity agent as necessary, since the degree of absorption and diffusion of the specimen differs depending on the constituent material of the carrier employed. It may also contain auxiliary agents such as.

When employing a film-forming polymer as a polymeric carrier when preparing a quantitative composition according to the present invention,
When the polymer is dissolved, the above-mentioned reagents are simultaneously dissolved, and this liquid material is further applied uniformly onto another carrier such as a synthetic resin film, and then dried.

In addition, when preparing the composition for quantitative determination according to the present invention, when using filter paper as a carrier made of a polymeric material, all reagents may be
Dissolve the tetrazolium salt in a buffer solution, including a wetting agent if necessary, or dissolve the tetrazolium salt alone in a non-aqueous solvent such as methanol or ethanol, impregnate a filter paper with this solution (in the latter case, two solutions), and then dry. I am forced to do it.

The reagent carrier obtained as described above can be cut and made into appropriate dimensions for use, but in reality, for convenience of use, for example, cut pieces of the above-mentioned quantitative composition are synthesized using double-sided tape. A test piece is made by attaching it to one end of a resin or paper strip. Of course, this test piece is kept in a package and protected from contamination and deterioration of the quantitative composition until it is used.

(Examples, etc.) Next, the present invention will be described specifically and in further detail in relation to production examples, reference examples, and test examples.

11 animals 3α-) ISD 1010, diaphorase 180 II
I, β-NAD ] Omg, nitro blue tetrazolium (nitro-TB) 2 mg, ascorbate oxidase 50011, sodium chloride 30 mg and polyethylene glycol IOB in 50 mM phosphate buffer (pH 8
,0>dissolved in 5 ml. This solution was poured under light shielding.
A 10 x 10 cm filter paper (Toyo Roshi No. 2) was impregnated and immediately lyophilized under reduced pressure. The resulting test paper was cut to obtain a composition for bile acid determination as small pieces of 5 x 5 mm each.

This quantitative composition (small pieces) each has a size of 5 x 10 mm +
The specimen was attached to one end of a polystyrene film strip using double-sided tape.

This test piece was placed in a brown bottle with a stopper, placed in a refrigerator, and stored for 6 months. When the test piece was taken out and the quantitative composition was checked, no discoloration was observed. No decrease was observed.

1L[L (standard calibration curve) The samples are various standard serums that are free from hemolysis, jaundice, and turbidity and have been tested for bile acid concentration, and each sample is compared to the test piece obtained in the manufacturing example. Approximately lθ on the quantitative composition
After removing excess serum that was not impregnated, the change in reflected light intensity was measured using a reflectometer at a wavelength of 555 nm for 2 minutes, and the relationship with the bile acid concentration in the sample was investigated. As shown in the graph of the figure, a good cubic regression relationship was obtained between the two in the range of bile acid concentration from 0 to 150 μmol/I.

Therefore, the graph shown in FIG. 1 can be used as a standard calibration curve.

11 Takumi Yu (Standard Colorimetric Table) The samples are various standard serums that are free of hemolysis, jaundice, and turbidity and have been tested for bile acid concentration, and each sample is used as a test piece obtained in the manufacturing example. on the metering composition of about 10
A standard colorimetric table was prepared based on the bile acid concentration in the sample and the developed color tone.

A normal serum with no hemolysis, yellow thinning, or turbidity observed on the left, with an unknown bile acid concentration, was used as the sample, and each sample was placed on the quantitative composition of the test piece obtained in the manufacturing example. Drop about 10μm,
After removing excess serum that was not impregnated, the amount of change in reflected light intensity was measured as described in Reference Example 1, and the amount of change in reflected light intensity was measured as described in Reference Example 2.
The bile acid concentration in the sample was determined by visually observing the discolored color tone Wt as described in Reference Examples 1 and 2 and comparing it with the standard calibration curve and Rin semi-colorimetric table. The measured values obtained by both methods were consistent, indicating that the bile acid concentration in the sample serum can be measured either by an instrumental method or by a visual method.

(Influence of substances interfering with measurement) A quantitative composition (control composition) was prepared in the same manner as in the production example, but without ascorbate oxidase and sodium oxalate, and also with 3α-H5O, ascorbate oxidase and Quantitative composition without using sodium oxalate (
Control compositions B) were prepared and control test pieces A were prepared by applying these control compositions to one end of a 5 x 60 mm polystyrene film strip with double-sided tape as in the Preparation Example.
and B.

Using both of these control test pieces and the test pieces obtained in the production example, and using standard serum with known bile acid concentrations, samples containing various concentrations of ascorbic acid, and samples in which lactic acid and pyruvic acid coexisted and various types were tested. The results of investigating the influence of ascorbic acid and lactate dehydrogenase (LDH) on samples containing LOH at an active concentration of was as shown in the graphs of FIGS. 2 and 3.

From these figures, it can be seen that the ascorbic acid concentration and lactate dehydrogenase ( The higher the content of LDII), the larger the positive error becomes. It was found that the same phenomenon occurred in the control test specimen B to which the quantitative composition of the present invention was applied, whereas it was found that the test specimen to which the composition for quantitative determination according to the present invention was applied was not affected by these interfering substances.

Therefore, from the above, it is clear that the causes of errors in measurement values are the presence of ascorbic acid and the lactate dehydrogenase reaction.

U Ren Yu (simultaneous reproducibility) Improved method of the method described in JP-A-61-268I99 and JP-A-62-272994 (using both the control test pieces used in Test Example 2 and B, The simultaneous reproducibility of the method of measuring the amount of change in light intensity and subtracting the value from control test piece B as the specimen blank value) and the method of using the test piece according to the present invention obtained in the production example,
The results obtained by measuring the amount of change in reflected light intensity 10 times using the same serum (bile Pi concentration: 12.7 μmol/I) as a sample are as shown in Table 2 below, and the composition according to the present invention When using this method, the CV value was 2.3%, which is good, whereas the improved conventional method showed a CV value about 5 times higher, which is problematic as a clinical quantitative test method for bile acids using serum as a sample. It turned out that there is.

Bile acids were quantified in 48 serum samples by the quantitative method using the quantitative test piece according to the present invention described in the production example described in Shishi-1, and the conventional solution-based quantitative method, and the correlation between the two methods was determined. The results of the investigation are shown in Figure 4, and the correlation coefficient between the two is r・0.9.
966, regression equation y.], 0411x + 0.24, which is an extremely good correlation, and it was therefore found that the composition for quantitative determination according to the present invention is highly practical.

(Effects of the Invention) The composition for quantifying bile acids according to the present invention can be prepared by dropping or applying serum onto it, measuring the amount of change in reflected light intensity using a reflectance meter, and comparing it with a standard calibration curve, or The bile acid concentration can be measured by leaving it for a minute to develop a color and comparing the color tone with a bleach colorimetric table, resulting in a method for quantifying bile acids in blood that is extremely simple to operate and takes an extremely short time.

Furthermore, when using the composition for quantifying bile acids according to the present invention, there is no interference from lactate dehydrogenase or ascorbic acid, and the measurement results show a good correlation with those obtained by conventional solution-based methods, and the measurement accuracy is also improved. Therefore, the composition for quantifying bile acids according to the present invention can be used not only for screening purposes, but also for diagnostic purposes, making it possible to use it in on-site testing and diagnosis.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the results of investigating the relationship between the bile acid concentration in standard serum and the amount of change in reflected light intensity using the quantitative composition according to the present invention, which should be used as a standard calibration curve. Figure 2 shows the results of a test conducted on ascorbic acid-containing serum samples using the quantitative composition according to the present invention and a conventional quantitative composition to investigate the effect of ascorbic acid on the measurement results. FIG. 3 is a graph similar to FIG. 2, except that it shows the results of investigating the influence of lactate membrane 71 coenzyme on measurement results in serum samples in which VL acid and pyruvate coexist. FIG. 4 is a graph showing the correlation between the results of measuring the bile acid concentration in a serum sample using the quantitative composition of the present invention and the results of measuring by a conventional method in which the reaction is carried out in a solution system.

Claims (2)

[Claims] (1) Dehydrogenation of bile acids in a serum sample by 3α-hydroxysteroid dehydrogenase in the presence of nicotinamide adenine dinucleotide, and in this case, the formation of formazan produced by coexisting an electron carrier and a tetrazolium salt in the reaction system. A composition for quantifying blood bile acids by measuring chromaticity, the pH of which is maintained at 7-9.
The buffer, 3α-hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide, electron carrier, tetrazolium salt, ascorbate oxidase, and oxalic acid or its salt as a lactate dehydrogenase inhibitor are made of polymeric materials. A composition for determining blood bile acids, characterized in that the composition is supported on a carrier. (2) The composition for quantifying blood bile acids according to claim (1), wherein the electron carrier is diaphorase.