Method for measuring bilirubin

Abstract: Direct bilirubin can be measured by using as an reaction inhibitor a water-soluble high polymer, nonionic surfactant having a HLB value of 15 or more, etc.

Description: BACKGROUND OF THE INVENTION This invention relates to a method for measuring bilirubin contained in a living body fluid such as plasma, serum or urine. Bilirubin is a compound derived from hemoglobin liberated by lysis of aged erythrocyte. Blood of a living body contains direct bilirubin (mainly glucuronide conjugate form) which reacts directly with a diazo compound to develop a color, and indirect bilirubin (free form) which reacts with a diazo compound only in the presence of an accelerator such as an alcohol. Diagnoses of various liver diseases and diacrisis of jaundice can be made by measuring total bilirubin concentration and the concentration of each of the above two forms of bilirubin in blood, and hence the measurement of bilirubin is an important examination among clinical examinations. At present, a leading method for measuring bilirubin is the so-called diazo method which comprises measuring the purplish red color of azobilirubin formed by the reaction of bilirubin with a diazo compound. The diagnoses of various liver diseases and the diacrisis of jaundice are made on the basis of the concentrations of bilirubins measured mainly by this method. This method, however, is disadvantageous, for example, in that reagent solutions for the method can be used for only about 5 days after their preparation, and that measured values are affected by L-ascorbic acid and hemoglobin which are present in a sample. As other methods for measuring bilirubin, there can be exemplified BOD method utilizing bilirubin oxidase (BOD) as an oxidizing agent, high-performance liquid chromatography method utilizing high-performance liquid chromatography, film method comprising measuring α, β, γ and δ forms of bilirubin by use of a film coated with a mordant, and chemical oxidation method using ferricyanide ions or copper ions as an oxidizing agent (e.g. a method for measuring total or direct bilirubin by use of potassium ferricyanide, a method for measuring direct bilirubin by use of copper ions and thiourea, etc.). All of these methods involve some problem and cannot be said to be preferable to the diazo method. In detail, the BOD method involves no particular problem when used for measuring total bilirubins, but is disadvantageous in that when it is used for measuring direct bilirubin, the measurement should be carried out at a pH outside the optimum pH range for BOD, so that a large amount of the enzyme is unavoidably needed, resulting in a high cost of the measurement. The high-performance liquid chromatography method and the film method are rated high in the precision of measurement but are disadvantageous, for example, in that they require an apparatus for exclusive use and cannot be utilized for measuring many items at the same time for a large number of samples. In the case of the chemical oxidation method, total bilirubins and direct bilirubin cannot be measured at the same pH, so that the measurement is influenced by a change of the absorption curve of bilirubin which accompanies a pH change. Therefore when direct bilirubin is measured utilizing a absorbance value measured by use of standard solutions at the time of measuring total bilirubins, the reliability of measured values is lowered. Furthermore, the BOD method and the chemical oxidation method are disadvantageous also in that a measured value of direct bilirubin obtained by these methods is in poor correlation with that obtained by the diazo method, and hence is difficult to utilize for diagnoses of various liver diseases and diacrisis of jaundice which have been established on the basis of measured values obtained by the diazo method. Under such circumstances, there is desired a method for measuring bilirubin which can be applied to an automatic analyzer, is in good correlation with the diazo method, and is excellent in the stability of reagent solutions. SUMMARY OF THE INVENTION This invention was made in consideration of such conditions and is intended to provide a method for measuring bilirubin which can be applied to an automatic analyzer, is in good correlation with the diazo method, is excellent in the stability of reagent solutions, and can give measured values little affected by substances present in a sample together with bilirubin. This invention provides a method for measuring direct bilirubin which comprises adding a reagent solution comprising a chemical oxidizing agent other than vanadic acid ions and manganese (III) ions and at least one compound selected from the group consisting of hydrazine derivatives, hydroxylamine derivatives, oximes, aliphatic polyvalent amines, phenols, water-soluble high polymers, and nonionic ether type surfactants having a HLB value of 15 or more as a reaction inhibitor to a sample of living body fluid, and measuring an optical change of the sample fluid. This invention further provides a composition for measuring direct bilirubin which comprises a chemical oxidizing agent other than vanadic acid ions and manganese (III) ions, at least one compound selected from the group consisting of hydrazine derivatives, hydroxylamine derivatives, oximes, aliphatic polyvalent amines, phenols, water-soluble high polymers and nonionic ether type surfactants having a HLB value of 15 or more as a reaction inhibitor, and a chelating agent. EP-A-0387784 refers to the use of nonionic surfactants in an oxidative measurement of bilirubin but only describes the use of a nonionic surfactant of HLB value of less than 15 or an ester type surfactant as distinct from an ether type surfactant. JP-A-59160764 discloses determination of bilirubin but not direct bilirubin and also on the users nonionic type surfactant with HLB value less than 15 does not disclose the use of ether type nonionic surfactants. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a correlation graph obtained on the basis of measured values obtained by Example 1 and Referential Example 1: Fig. 1 shows a correlation graph for direct bilirubins. In the method for measuring direct bilirubin of this invention, as the reaction inhibitor which has an ability to prevent indirect bilirubin from oxidization by the oxidizing agent, there are used one or more compounds selected from the group consisting of hydrazine derivatives, hydroxylamine derivatives, oximes, aliphatic polyvalent amines, phenols, water-soluble high polymers, and nonionic ether type surfactants having a HLB value of 15 or more. As these compounds, any compounds can be used without particular limitation so long as they do not affect the measurement of direct bilirubin. Preferable specific examples of the hydrazine derivatives are hydrazine, phenylhydrazine and mineral acid salts (hydrochlorides, sulfates, etc.) thereof. The hydroxylamine derivatives include, for example, hydroxylamine, phenylhydroxylamine, and mineral acid salts (hydrochlorides, sulfates, etc.) thereof. The oximes include, for example, acetoxime, diacetylmonooxime and salicylaldoxime. The aliphatic polyvalent amines include, for example, tetraethylenepentamine, hexamethylenetetramine and triethylenetetramine. Preferable specific examples of the phenols are phenol, p-chlorophenol, p-acetamidophenol, 4-chloro-1-naphthol and β-naphthol. Preferable specific examples of the water-soluble high polymers are polyvinyl alcohols and polyvinyl pyrrolidones. The nonionic ether type surfactants having a HLB value of 15 or more include, for example, Emalgen-123P (a polyoxyethylene lauryl ether, a trade name, Kao Corp.), Emalgen-950 (a polyoxyethylene nonylphenyl ether, a trade name, Kao Corp.), Emalgen-985 (a polyoxyethylene nonylphenyl ether, a trade name, Kao. Corp.) and Triton X-405 (a polyoxyethylene isooctylphenyl ether, a trade name, Rohm and Haas Co.). Although these reaction inhibitors have an inhibitory effect on the oxidation of indirect bilirubin even when used singly, a combination of two or more of them has a higher inhibitory effect on the oxidation of indirect bilirubin in some cases. Therefore, they may be properly chosen depending on purposes. Although the concentration of the reaction inhibitor used for measuring direct bilirubin is not critical so long as it is such that the oxidation of indirect bilirubin can be inhibited, and the concentration depends on the kind of the compound(s) used as reaction inhibitor, the concentration of the reaction inhibitor in a total reagents mixture is usually 0.01 to 5 w/v %. In any method for measuring direct bilirubin by a chemical oxidation method which comprises adding a chemical oxidizing agent to a sample of living body fluid and measuring an optical change such as the degree of change of the color, of the sample fluid, the above-exemplified reaction inhibitors according to this invention can be effectively used regardless of the kind of the chemical oxidizing agent. Of these reaction inhibitors, the water-soluble high polymers and the nonionic surfactants having a HLB value of 15 or more can be used as reaction inhibitors also for measuring direct bilirubin by use of bilirubin oxidase. The presence of a chelating agent in a reagent solution used in this invention is preferable, because it lowers a reagent blank value to improve the precision of analysis, stabilizes the reagent solution, and accelerates the oxidation reaction of bilirubin. Preferable specific examples of the chelating agent used for such purposes are ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), cyclohexanediaminetetraacetic acid (CyDTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylethylenediaminetetraacetic acid (EDTA-OH), triethylenetetraminehexaacetic acid (TTHA), hydroxyethylimino-diacetic acid (HIDA), 1-hydroxyethane-1,1-diphosphonic acid, and alkali metal salts (e.g. lithium salts, sodium salts and potassium salts) thereof and ammonium salts thereof. Although the concentration of the chelating agent used for measuring bilirubin is not critical so long as it does not hinder the measurement of bilirubin, the concentration of the chelating agent in a total reagents mixture is usually 0.02 to 50 mM, preferably 1 to 30 mM, and more preferably 1 to 20 mM. Other reagents (e.g. buffers and antiseptics) used in the measuring method of this invention may, if necessary, be properly selected, for example, from buffers such as phosphoric acid, citric acid, succinic acid, etc., and antiseptics which are used, for instance, in per se well-known chemical oxidation method and BOD method. It is sufficient that the concentration of these reagents, the pH's of reagent solutions, etc. are properly determined in accordance with those employed for reagent solutions used in the per se well-known methods. This invention is conducted, for example, as follows. When direct bilirubin concentration is measured, a buffer solution containing the reaction inhibitor and optionally a chelating agent is first prepared as a first reagent solution, and then mixed with a sample containing bilirubin, for example, a living body fluid such as plasma, serum or urine, and absorbance at a specific wavelength (about 430 to about 460 nm) of the resulting solution is measured (absorbance 1). Subsequently, a second reagent solution containing the specified oxidising agent ions and optionally a chelating agent is added, and the oxidation reaction of bilirubin is carried out at 25° to 40°C for 3 to 15 minutes, after which absorbance at the specific wavelength is again measured (absorbance 2). The thus obtained values of absorbance 1 and absorbance 2 are, for example, multiplied by a correction factor for solution volume, etc., and then the difference of absorbance at the specific wavelength before and after the oxidation reaction is calculated. The total bilirubin concentration in the sample can be determined by using this calculated value and a calibration curve showing the relationship between bilirubin concentration and the change of absorbance at the specific wavelength which has previously been prepared by the same procedure as above by using standard solutions of known bilirubin concentrations. Direct bilirubin concentration in a sample can be measured also by the same procedure as the above procedure of measuring direct bilirubin, except for using as absorbance 1, absorbance measured for a solution obtained by mixing a portion of the first solution with a portion of the sample, and as absorbance 2, absorbance measured for a solution obtained by reacting another portion of the sample with a previously prepared mixture of another portion of the first solution and the second solution. As mentioned above, the reaction inhibitor according to this invention can be effectively used in per se well-known chemical oxidation method, for example, such as chemical oxidation method using ferricyanide ions or copper ions as a chemical oxidizing agent. Therefore, when a buffer solution containing the reaction inhibitor according to this invention and optionally a chelating agent is used as a first reagent solution and a buffer solution containing these chemical oxidizing agent such as ferricyanide ions, copper ions, etc. and optionally a chelating agent is used as a second reagent solution, direct bilirubin concentration can also be measured in good correlation with the diazo method by the same procedure described above. Although a mechanism by which the above compounds inhibit the oxidation of indirect bilirubin is not clear, the following mechanisms, for example, are thought of. Compounds having reducing ability, such as the hydrazine derivative, hydroxylamine derivative, oximes, aliphatic polyvalent amine and phenol absorb the surplus oxidizing agent and hence prevent the gradual oxidation of indirect bilirubin. Compounds such as the water-soluble high polymer and the nonionic surfactant having a HLB value of 15 or more combine with indirect bilirubin in some way or other to prevent the oxidation of indirect bilirubin, so that they improve the specificity for direct bilirubin of the oxidizing agent. In the method for measuring direct bilirubin mentioned above, nonionic surfactants having a HLB value of less than 15 cannot be used. This is because the nonionic surfactant having such a property cannot prevent the oxidation of indirect bilirubin sufficiently, resulting in providing a measured value of direct bilirubin poor in correlation with that obtained by the diazo method. This arises a problem in practical use. This invention is more concretely explained below with reference to Experimental Examples and Examples, which are not by way of limitation but by way of illustration. Experimental Example 1 Selection of an oxidizing agent for bilirubin The following experiment was carried out for finding metal ions useful as an oxidizing agent for bilirubin. [Reagent solutions] First reagent solution 0.1 M acetic acid-sodium acetate buffer (pH 4.0) was used as a first reagent solution (R1, hereinafter the same applied). Second reagent solution 0.1 M acetic acid-sodium acetate buffer (pH 4.0) containing 1 mM predetermined metal ions was used as a second reagent solution (R2, hereinafter the same applied). [Samples] An aqueous solution containing ditaurobilirubin in an amount corresponding to 10 mg/dl of bilirubin was used as a direct-bilirubin-containing sample, and commercially available bilirubin standard serum (containing 10 mg/dl of indirect bilirubin, available from Wako Pure Chemical Industries, Ltd.) was used as an indirect-bilirubin-containing sample. [Procedure] Measurement was carried out by use of a Hitachi automatic analyzer Model 7050 (mfd. by Hitachi, Ltd.) under the conditions shown in Table 1. CHEMISTRY PARAMETERS ASSAY CODE [2POINT]:[15]-[32] SAMPLE VOLUME [ 15] R1 VOLUME [400] [ 50][NO] R2 VOLUME [100][ 50][NO] WAVE LENGTH [505] [450] CALIB. METHOD [ LINEAR ] [0] STD.(1)CONC.-POS. [0.0 ]-[ 1 ] STD.(2)CONC.-POS [10.0] - [ 2 ] STD.(3-6)CONC.-POS [ 0 ]-[ 0 ] UNIT [MG/DL] SD LIMIT [ 0.1) DUPLICATE LIMIT [10000] SENSITIVITY LIMIT [ 0] ABS.LIMIT(INC/DEC) [ 0 ] [DECREASE] PROZONE LIMIT [ 0 ] [LOWER] EXPECTED VALUE [ 0 ]-[10000] INSTRUMENT FACTOR [1.00] [Results] The results obtained are shown in Table 2. Metal ions Blank value Direct-bilirubin-containing sample Indirect-bilirubin-containing sample OD (505 nm/450 nm) [Fe(CN) 6 ] -3 0.030 0.265 0.088 Co 2+ (CoCl 2 ) 0.000 0.034 0.068 Cu 2+ (CuSO 4 ) 0.000 0.079 0.070 Fe 3+ (iron alum) 0.040 0.167 0.059 Ni 2+ (NiSO 4 ) 0.000 0.091 0.088 As is clear from the results shown in Table 2, ferricyanide ions, are excellent as oxidizing agents. For the same samples as used in Example 1, direct bilirubin were measured by use of a commercially available reagent for measuring bilirubin [Bilirubin II-HA Test Wako (mfd. by Wako Pure Chemical Industries, Ltd., diazo method)]. The measurement was carried out according to the standard procedure described in a manual attached to the above commercial reagent. [Standard] An aqueous solution containing ditaurobilirubin in an amount corresponding to 10 mg/dl of bilirubin was used as a direct-bilirubin-containing standard. [Samples] Thirteen human sera frozen and stored after collection were used as samples. [Procedure] The same as in Example 1. [Results] The measurement results are also shown in Table 3. Comparative Example 1 Measurement was carried out by the same procedure as in Example 1 by using the same reagents, reagent solutions and samples as in Example 1, except for using Triton X-100 (a polyoxyethylene isooctylphenyl ether, a trade name, Rohm and Haas Co.; HLB value 13.5) in place of Triton X-405 (a trade name, Rohm and Haas Co.; HLB value 17.9) in the first reagent solution. [Results] The measurement results are also shown in Table 3. Referential Example 2 For the same samples as used in Example 1, direct bilirubin was measured by use of a commercially available reagent for measuring bilirubin [Bilirubin II-HA Test Wako (mfd. by Wako Pure Chemical Industries, Ltd., diazo method)]. The measurement was carried out according to the standard procedure described in a manual attached to the above commercial reagent. [Results] The measurement results are also shown in Table 3. Sample No. Direct bilirubin concentration (mg/dl) Example 1 Example 1 Comparative Example 1 Referential Example 2 1 0.6 0.7 0.5 2 0.9 0.8 0.7 3 0.3 0.3 0.2 4 1.0 2.4 1.1 5 4.7 4.8 4.6 6 1.7 1.9 1.3 7 2.7 4.0 2.6 8 1.7 2.7 2.1 9 2.0 3.2 2.2 10 2.1 3.2 2.3 11 2.1 3.3 2.2 12 2.4 3.0 2.5 13 3.1 3.9 2.7 Table 4 shows correlation coefficients and regression line formulas which were obtained by statistical processing of the direct bilirubin concentrations of the individual samples which were obtained by Referential Example 2 and those obtained by each of Example 1 and Comparative Example 1. Origin of Y Correlation coefficient Regression line formula Example 1 0.980 Y = 0.978X + 0.066 Comparative Example 1 0.943 Y = 1.104X + 0.508 * In all the cases, X is the measured value obtained by Referential Example 2. As is clear from the results shown in Table 3 and Table 4, the measured values of direct bilirubin obtained by using Triton X-405 (a trade name, Rohm and Haas Co.) having a HLB value of 15 or more as the reaction inhibitor were in good correlation with those obtained by the diazo method, but the measured values of direct bilirubin obtained by using Triton X-100 (a trade name, Rohm and Haas Co.) having a HLB value of less than 15 in place of Triton X-405 (a trade name, Rohm and Haas Co.) were poor correlation with those obtained by the diazo method. As is clear from the facts described above, this invention is characterized in that for measuring direct bilirubin, at least one compound selected from the group consisting of hydrazine derivatives, hydroxylamine derivatives, oximes, aliphatic polyvalent amines, phenols, water-soluble high polymers, and nonionic ether type surfactants having a HLB value of 15 or more is used as the reaction inhibitor. This invention provides a method for measuring bilirubin which is free from all of the problems in conventional methods, for example, their low applicability to an automatic analyzer, their poor correlation with the diazo method, and the low stability of reagent solutions.

Claims: A method for measuring direct bilirubin which comprises adding a reagent solution comprising a chemical oxidizing agent other than vanadic acid ions and manganese (III) ions and at least one compound selected from the group consisting of hydrazine derivatives, hydroxylamine derivatives, oximes, aliphatic polyvalent amines, phenols, water-soluble high polymers, and nonionic ether type surfactants having a HLB value of 15 or more as a reaction inhibitor to a sample of living body fluid, and measuring an optical change of the sample fluid. A method according to claim 1 wherein the chemical oxidizing agent is ferricyanide ions and/or copper ions. A composition for measuring direct bilirubin which comprises a chemical oxidizing agent other than vanadic acid ions and manganese (III) ions, at least one compound selected from the group consisting of hydrazine derivatives, hydroxylamine derivatives, oximes, aliphatic polyvalent amines, phenols, water-soluble high polymers and nonionic ether type surfactants having a HLB value of 15 or more as a reaction inhibitor, and a chelating agent. A composition according to claim 3, wherein the chemical oxidizing agent is ferricyanide ions and/or copper ions.

Inventors: TANIMOTO KAZUHITO, TOKUDA KUNIAKI, TANIMOTO, KAZUHITO, TOKUDA, KUNIAKI
Applicants: WAKO PURE CHEM IND LTD, WAKO PURE CHEMICAL INDUSTRIES, LTD
Application year: 1991
Main classification: G01N33
IPC codes: C12Q1, G01N33