JPS60228963A - Measurement of living body component performed by avoiding interference of bilirubin - Google Patents

Abstract

PURPOSE:To obtain good bilirubin interference avoiding action, by using a ferrocyanide compound in combination with albumin and or using both of them in combination with aminopyrine. CONSTITUTION:This measuring method is constituted by using a single-fluid reagent which is prepared by containing one or more kind of other enzyme, which is necessary for inducing enzyme, coenzyme or substrate and oxidase forming hydrogen peroxide to be acted on serum components, and peroxidase and a substance to be oxidized for constituting a detection system and further adding albumin or albumin and aminopyrine and provided with arbitrary buffering action. As the substance to be oxidized, a combination of 4-aminopyrine and a color forming coupler is especially pref. When technique for performing reaction in two stages by using a double-fluid reagent, alubmin can be present in both of first and second stage reactions but a ferrocyanide compound or a ferrocyanide compound and aminopyrine is present only at the time of second stage reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention] The present invention relates to a method for measuring biological components that avoids the interference of bilirubin.

[Prior art]

Bilirubin exists in various forms in serum, including alone, conjugated with glucuronic acid, and sulfuric acid. In normal people, total bilirubin is normally less than about 0.5 m97 dl, but in pathological conditions, it increases markedly and 40 Irvdi It can even reach more than that. This bilirubin interferes in various ways with the measurement of serum components, causing errors in the measured values. That is, directly, the yellow pigment bilirubin is about 450
It has an absorption maximum in the nm range and interferes with the direct method of quantifying serum components using yellow or red color reactions, usually giving abnormally high measured values. For example,
This occurs when quantifying free fatty acids (FFA) in serum, and when quantifying by colorimetry after making a copper complex of FFA and extracting it with dichloroform, the measurement wavelength is 440 nm.
Therefore, the influence of bilirubin cannot be ignored.

In particular, recently, enzyme-based measurement of biological components has been widely used, and in particular, many detection systems using hydrogen peroxide and oxidized systems such as oxidase are in use. In this system, bilirubin competes with the oxidizable substance and has a negative effect on the measured values (Clinical Chemistry, Vol. 8, No. 1, 63-72 (197
9)). For example, an enzymatic reaction system is applied to measure glucose in serum, in which hydrogen peroxide is produced by glucose oxidase, and a chromogenic substance is converted from the chromogenic oxidizable substance by the action of this hydrogen peroxide and 4-fluorooxidase. However, it is known that even in this case, bilirubin competes with the color-forming oxidizable substance and has a negative effect.

Therefore, it has been desired to develop a method that is free from bilirubin interference in measurements using these detection systems.

As prior art related to these problems, methods using heptadrocyan compounds and aminovirine have already been reported. For example, Japanese Patent Application Laid-Open No. 49-50991, B. Fossati (1" FosI! 1ati), etc., Clinical Chemistry 2672.227-231 (1980) describes the detection of ferrocyanine compounds using 4-ruoxidase-hydrogen peroxide-chromogenic oxidizable system. A method is described in which bilirubin is present in the system to avoid interference with bilirubin.

As a function of ferrocyanide, it intervenes between peroxidase and the oxidized substance. Hydrogen peroxide produced by enzymatic oxidation first captures ferrocyanide through the action of peroxidase, oxidizes it to ferricyanide, which oxidizes the oxidized substance and returns to ferroform, forming a pinox reaction system. It is thought that this suppresses the competitive involvement of bilirubin between peroxidase and the oxidized substance.

Furthermore, aminovirine is structurally very similar to 4-aminophenazine and does not have a moiety that undergoes oxidation. Therefore, although it has an affinity for peroxidase with a structure similar to 4-aminophenazine, it has the property of not being oxidized by hydrogen peroxide, so it exists in a form that masks the active site of peroxidase, and selectively performs only the color reaction. It is thought that it is effective in promoting weight loss. There are a variety of components to be detected in biological fluids that are routinely measured, and some are in large amounts, small amounts, or trace amounts relative to the amount of bilirubin present. For example, glucose is a component that is often measured in daily clinical tests.
Total cholesterol, total phospholipids, triglycerides, etc. are components that exist in relatively large amounts, such as uric acid, creatinine, creatine, and phospholipid fractions, zolecithin, sphingomyelin, and putrescine, which is a polyamine fraction. Spermidine is a component that is considered to be in a relatively small amount or trace amount. These enzymatic measurement methods using oxidase are already well known, but the interference of bilirubin appears in relation to the amount of the target component present in bilirubin, and is relatively small compared to bilirubin. Components that are present in large amounts naturally have less interference; conversely, the smaller the amount of the component, the more likely the interference will be. This results in an inviting result.

Therefore, depending on the content of each component to be detected, the means to reduce bilirubin interference will depend on the degree of error in each clinical test value, and in that sense, ferrocyanide, aminobilin It seems that the prior art using . . . alone is effective.

However, with the conventional techniques using these ferrocyanides and aminobilins, it is difficult to detect bilirubin, especially when bilirubin exists not only in a free form but also in various forms, or when the component to be detected is in a small or trace amount. However, the interference effect of bilirubin could not be sufficiently avoided.

[Summary of the Invention] The present inventors improved the insufficient interference avoidance effect of bilirubin using conventional ferrocyanide or aminobilin, and particularly when bilirubin exists in various forms, As a result of intensive research to establish a method for measuring biological components that can sufficiently avoid interference from bilirubin even when the target biological component is in trace amounts, we have found that we can use ferrocyanide in combination with albumin, or By using aminovirine in combination with these two,
It was discovered that a surprisingly good bilirubin interference avoidance effect could be obtained, and the present invention was completed.

[Specific structure of the invention]

According to the present invention, it is possible to achieve sufficiently high accuracy when measuring biological components, particularly serum components, which are constantly coexisted with bilirubin. Such an effect is neither achievable nor expected from prior art techniques using ferrocyanide or aminobilin alone, and it is possible that bilirubin coexists not only in free form but also in various bound forms. This is the first time that the method of the present invention has been able to highly efficiently avoid or reduce the interference of bilirubin in human serum. The effect of removing bilirubin interference in the present invention is particularly remarkable in abnormal bilirubin serum with a high concentration of bilirubin.

The method of the present invention is effective for measuring a wide range of biological components that are affected by the interference effect of bilirubin, but in particular, it uses hydrogen peroxide produced by enzymatic oxidation in a detection system that contains peroxidase to oxidize the oxidized substance. It is effective in a measurement system that detects a target component based on this amount. As the substance to be oxidized, a combination of 4-amino-7enazone (toshiwake 4-aminoantipyrine) and a color-forming cazoler is particularly preferable.

The following systems can be exemplified as measurement systems that can be suitably used in the present invention.

Triglyceride measurement Lipoprotein lipase ■Triglyceride Ricerol + Fatty acid 3-phosphate oxidase Sibide Tetraxyacetone phosphate decaH2O2 2H202 + 4-aminoantipyrine + N + Ethyl-N
-sulfozolovir-m-toluidine 1Δ oxidase
*1) Purple quinone type dye +4H20 Glucose measurement Glucose oxidase ■Glucose + HO 10.

H2O2 + gluconic acid 2H202 + 4-aminoantipyrine + N, N-diethylaniline + H+ Gornimani upper - 1 garlic → purple quinone type pigment *2) +
Measurement of 4HO phospholipids ■Phospholipids (lecithin, sphingomyelin, lecithin)
Acylsphingosyl phosphate, lysophosphatidic acid) Choline +202+H20 已1)7. ni±niinoninigo → 2H202 + betaine 2H20□ + 4-aminoantipyrine + phenol-
−1−−−−−−→Red quinone type dye *3) + peroxidase H20 Measurement of total cholesterol ■ Ester type cholesterol + H2o3ly, X 7
- o-nylesterase free cholesterol + fatty acid free form - Leste-le + 0″v″″te°-″′ Sidase H20 □ Δ4-Cholestenone 2H202 + 4-aminoantipyrine + N,N-jebeloxidase tylaniline + H+--1 1111-→Purple quinone type pigment")+4H20 Measurement of free fatty acids ■Free fatty acids + ATP'l-C: oA 7' COA
v y + p Ni Mg2+ Acyl CoA + Hai + Birophosphate acyl CoA + O”-29 Yoshizushi soil! Ni 2.3
-) Lanceenol CoA 10H2O22H202+
4-aminoantiviral 7-f-N-ethyl-N-sulfoprovir-m-) luidine + H + peroxidase ---> quinone type dye *1) + 4H2゜Measurement of uric acid ■ Uric acid + 02 + 2H20-Ku ■! L: Niza → Allantoin + C02 + H202 2H202 + 4-aminoantipyrine + N-ethyl-N
-Sulfopropyl-m-toluidine+H+--11--
--→Quinone type dye *1) +4H20 Peroxidase Cholinesterase measurement Cholinesterase benzoylcholine + H20 Choline + Benzoic acid choline oxidase Choline + 202 + H2O 2H2O□ + Betaine 2H202 + 4-aminoantipyrine + Phenol --> Quinone type dye * 0-peroxidase *1) (λmax = 550 nm) *2) 2H5 (λm8L! = 560 nm) *3) (λm,, = 500 nm) The measurement method particularly commonly used in the present invention is the measurement method that acts on serum components. Enzymes, coenzymes/or substrates, and, if necessary, necessary to induce the oxidizing enzyme to produce hydrogen peroxide to the active stage. A one-liquid reagent containing one or more other enzymes, oxidase and the oxidizable substance constituting the detection system, and further adding albumin or albumin and aminobilin to provide an arbitrary buffering effect. It can be composed of Further, enzymes, activators, preservatives, surfactants, etc. may be included as necessary.

Alternatively, as a coexisting substance elimination method, the first reagent for eliminating coexisting substances (or interfering substances) is composed of at least an enzyme that directly acts on serum components and a part or all of the chromogenic oxidizable substance. and a second reagent containing at least an enzyme that acts directly on serum components and an oxidizable substance necessary for complete color development. The first reagent may contain a non-chromogenic oxidizable substance for eliminating coexisting substances in serum. Recently, a method of conducting a reaction in two stages using two liquids has been frequently used as a method for clinical chemistry testing, but in this case, albumin is reacted in the first stage and in the second stage.
Although it can be present in any stage reaction, it is preferably present during the first stage reaction. On the other hand, ferrocyanide or ferrocyanide and aminovirine need to be present only during the second stage reaction.

A specific example of a measurement system using such a two-stage reaction is:
A method for measuring creatinine in serum using the following reaction system and reagent composition may be mentioned.

Main Reagent Compositions First reaction solution Creatinamide hydrolase Dercosine oxidase Oxidase Ascorbic acid oxidase Bovine albumin N-Ethyl-N-sulfozolobyl-m-toluidine Second reaction solution Creatinine amidohydrase 4-Aminophonazone Aminovirine Potassium ferrocyanide The present invention also measures not only the above-mentioned sugars, lipids, and nitrogen compounds as target components in biological fluids, but also enzymes when the activity is measured by connecting an oxidizing enzyme to a detection system for components produced by enzymes. It can be used for.

The ferrocyanide ion used in the present invention is commonly used as a thorium or potassium salt. Aminovirine is an N,N-dimethyl substituted product of 4-aminophenazine and has the following structure, but these four methyl groups are ordinary lower alkyl groups or (and) the introduction of an alkyl group, halogeno, etc. to the phenyl group at the 2-position. A similar structure may be used.

Albumin has a variety of origins, including human and bovine, but albumin derived from bovine is commonly used because it is easily available. For each used amount ferrocyanide is 1 μM to 500 μM, preferably 10 μM to 20
0μM0 aminopyrine is 1mM~15mM, preferably 2mM~10mM1albumin is 59/l~
10,! 9/l, preferably 1 g/l to 5 ji
It is preferable to use it within a concentration range of /l.

The present invention will be explained below with reference to Examples, but the embodiments of the present invention are not limited thereto.

Example 1 Effect of addition of albumin, aminobilin, and potassium ferrocyanide on uric acid measurement using high bilirubin serum.

■, Reagent Reagent (A) Urikar-1! #4.2X10'uμ Peroxidase 0.75X10 u/L Lipoprotein Lipase 2.5X10'uμ 4-Aminophenazine
46mM phosphate buffer containing 0.315mM (pH
6,5) Reagent (B) Reagent (A) with 160 μM of potassium ferrocyanide added.

Reagent (C) A reagent containing 2.25 i/l of bovine albumin and 2.25 mM of aminovirine.

Reagent) Reagent (4) contains bovine albumin 2.25.9μ, aminovirine 2.25mM, potassium ferrocyanide 16
0 μMi added.

2. Sample sample (1) Human serum: total bilirubin value 0.9■/dl
(Normal) Sample (2) Human serum: Total bilirubin value 45 m
97 dt (abnormal) 3, add 40 μt each of samples (1) and (2) to 3.27 d each of method reagents (4), (B), (0 and ■), and after heating at 37 ° C. for 10 minutes, The absorbance of each (mAbs) was measured against a reagent blank control at a wavelength of 550 nm.

4.Results As shown in the results, bilirubin normal sample (1)
As for sample (2), there was almost no change in the measured values with reagents (4), (B), and (Cri
), (B), (C) and 0) showed an increase rate of 236 inches, 240% and 296 inches, respectively. This value directly leads to misunderstanding of the clinical test value of the specimen. It is shown that the combination of potassium ferrocyanide, aminovirine, and albumin has a remarkable effect.

Example 2. In order to prove that the measurement values obtained by this measurement method (referred to as this method) using potassium ferrocyanide, aminopyrine, and albumin-added reagents in a correlation method with the uricase-catalase method, the uricase-catalase-hanch method was used for each sample. The correlation with the measured values was determined by the method.

This method is a complicated measurement method in which a serum blank value is obtained for each sample, and then the interference of sibilirubin is subtracted to obtain the actual value for each sample, but in principle it is considered to be able to avoid the interference of bilirubin. It is.

This method was carried out as a two-liquid method in line with actual daily testing, and the uricase-catalase method was carried out manually using Toyobo Co., Ltd., product name Uricolor 400, according to its manual.

1. Reagents (this method) Reaction solution 1 Phosphate IK 46mM K oxidase 1, 5X106uμ lipoglotin linoyase 5X10' u/L Ascorbate oxidase f5X10 u/LN-ethyl-N-sulfopropyl 0.6mM-m -Toluidine bovine albumin 4.5g/1cpH5,8) Reaction solution 2
Phosphate IK 46mM Uricase 8.4X10 u/1 4-aminophenazine 0.63mM Potassium ferrocyanide 0.3mM Aminovirine
4.5mM (pH 8,3)2, sample group (1) normal bilirubin; 19 sample group (2) abnormally high bilirubin; 10 samples 3, method 20 μt of each sample was added to 1.0.8rrLl of reaction solution,
Add 2.0.8 d of the reaction solution after heating at 37°C for 3 minutes,
After further reaction at 37°C for 6 minutes, the wavelength was 546-660 nm.
The uric acid value of each sample was measured using three-wavelength side light using a standard solution as a control.

(Implemented using Toshiba automatic analyzer TBA-380) 4.
Results are shown in the drawing.

As shown here, the correlation with the uricase-catalase method was very good, confirming the accuracy of this method. It has been shown that there is no correlational dissociation between normal bilirubin samples and abnormal samples, and that bilirubin abnormal samples are completely free from interference by bilirubin and can be measured with the same accuracy as normal bilirubin samples.

[Brief explanation of drawings]

The figure is a correlation diagram between the uric acid measurement value by the method of the present invention and the uric acid measurement value by the uricase-catalase method. Fist: Normal serum with bilirubin, ○: Serum with high bilirubin. Engraving of drawings (no change in content) Procedural amendment form) % formula % 1, Indication Patent Application No. 1985-85399 2, Name of the invention Method for measuring biological components by avoiding bilirubin interference 3, Relationship with the case of the person making the amendment Patent applicant name: Yatron 4 Co., Ltd., agent address: Mori Building, 40 Toranomon, 5-13-1 Toranomon, Minato-ku, Tokyo July 31, 1982

Claims (3)

[Claims] (1) In a method for measuring biological components in which hydrogen peroxide generated by an oxidase in a detection system in which peroxidase is present is guided to oxidize the oxidized substance, and the target component is detected using this amount, the detection system is A method for measuring biological components while avoiding interference from bilirubin, characterized by the presence of ferrocyanide ions and albumin. (2) In a detection system in which interfering substances are eliminated in the first-stage reaction, and then the target component is detected in the second-stage reaction, the reaction solution in the first or second stage contains alhumin,
The measuring method according to claim (1), wherein ferrocyanide ions are present in the reaction solution in the step. (3) The measuring method according to claim (1) or (2), wherein the oxidizable substance is a combination of 4-aminophenazine and a color-forming coupler, and aminopyrine is present in the detection system.