JPS6040957A - Stabilization of conjugation-type bilirubin - Google Patents

Abstract

PURPOSE:To obtain a standard liquid for measurement of total bilirubin, direct- type bilirubin in human bodily fluid of special stability, by allowing co-existence of conjugation-type bulirubin and non-ion-type surface-active agent and long- period longevity. CONSTITUTION:To a conjugation-type bilirubin solution where in propionic acid residual radical is combined with glucuronic acid, taurine, etc., non-ion surface active agents of HLB 10-20, such as, polyethylene glycol setylether, polyethylene glycol nonylphenyl ether, etc. are added at a rate of 0.2-10W/V%. Further, when a reducing agent of glutathione, cystine, ascorbic acid is added at 0.0005- 0.02W/V%, stability is improved. The keeping quality lasts innthis state of solution, however, freeze drying of the solution result to elongated stability. The frozen product is melted again in distilled water when using. Thus, a standard liquid of excellent stability suited to diagnosis of hemolitic jaundice, obstructive jaundice.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for stabilizing conjugated bilirubin.

There are two types of bilirubin in body fluids, conjugated and conjugated. Among them, it is mainly those bound with glucuronic acid). Quantitative understanding of the dynamics of bilirubin in the blood has been considered important for the differential diagnosis of jaundice. That is, for example, in hemolytic yellow scars, the blood concentration of unconjugated bilirubin increases, and in obstructive conditions, the blood concentration of conjugated bilirubin increases. In the conventional capacity method, what is called direct bilirubin reflects the concentration of 1-conjugated bilirubin, and what is called 1-indirect bilirubin corresponds to unconjugated bilirubin.

The most widely used method for measuring bilirubin and its fractions (direct and indirect) in body fluids is to measure the light absorption of a pigment (azobilirubin pigment) produced by the reaction of an aromatic diazonium salt with bilirubin. This is what was used ~ This is HIJMANS V in the 1000s
Designed by AN DFiN EIGH et al. 51930
MAItLOY and ILVBLYH and Tl0
This is a quantitative method established by NDRA8 SIK and G'R'OF et al. In recent years, various improved methods have been implemented, all of which utilize differences in solubility in fractionation methods. (i.e. methano) L/-% urea 1 dimethyl sulfoxide 1 caffeine and benzoic acid 1 dyphyllin 1 promoters such as nonionic surfactants - these solubilize unconjugated substances that are difficult to dissolve in aqueous systems. −
total bilirubin (IJ) measured by addition of conjugate and chromogenic quantification of the sum of conjugated and unconjugated substances; (2) total bilirubin, which corresponds to the Levin value and the conjugate measured in the absence of the above-mentioned promoter; Ru.

The standard solutions used in these 1 measurement methods are: 1 Since conjugated bilirubin was difficult to obtain in the past, 1 a calibration curve for total bilirubin was drawn using follicular conjugated bilirubin; 1 this was used to directly measure bilirubin. method has been implemented. In such a method, the bilirubin concentration is calculated directly under the assumption that the presence of the promoter does not affect the extinction coefficient of the azobilirubin pigment,
There is a lack of theoretical clarity. Also, from a practical standpoint, many automatic analyzers are systems that require standard solutions for each of the 11 measurement items, and conventionally there are no suitable standard solutions such as direct bilirubin. In many cases, substitute standards have been used.

An object of the present invention is to provide a stable standard solution using conjugated bilirubin that can be used for the measurement of direct bilirubin and total bilirubin.

In general, bilirubin is chemically unstable.・Conjugated bilirubin is unstable to 1% oxidation (changes to biliverdin)
) Relatively stable to light, 1-follicular synaptic bilirubin is relatively stable to 1-oxidation, 1-unstable to light [Clinical Testing Techniques Book 6
1 Clinical Chemistry Test, 332 pages, published by Igaku Shoin in 1980]
. As a standard solution used for quantitative analysis, it is desired that it has particularly excellent stability.

Recently, a synthetic ditaurin-bilirubin derivative (trade name: bilirubin conjugate) has been released by Porphyrin Products. This compound was shown to exhibit similar reactivity in various diazo methods to bilirubin diglucuronide prepared from human bile. However, when the stability of this bilirubin conjugate water bath solution was investigated, it became clear that it was extremely poor. This instability of bilirubin conjugates is not limited to derivatives based on mono-taurine, but can be said generally to those that have been conjugated to promote water solubility, and mono-glucuronic acid conjugates are similarly unstable. bad. Since it is a well-known fact that bilirubin skeleton is oxidized and changed to biliverdin skeleton, it is easily inferred that it is stabilized by adding a reducing agent. In fact, the aqueous solution of the bilirubin conjugate is stabilized by adding a reducing agent such as ascorbic acid, dithiothreitol, cysteine, and glutathione. However, since these reducing agents significantly inhibit the 1-diazo reaction, it is not appropriate to add a sufficient amount of the reducing agent to ensure sufficient stabilization of the bilirubin standard solution.

The inventors of the present invention have made extensive studies to address the above-mentioned drawbacks and have found that conjugated bilirubin can be significantly stabilized by adding a nonionic surfactant, and have arrived at the present invention. That is, the present invention is a method for stabilizing conjugated bilirubin, which is characterized by allowing a nonionic surfactant to coexist with conjugated bilirupin.

In the present invention, the stability of conjugated bilirubin can be significantly improved by coexisting a nonionic surfactant.

Conjugated bilirubin used in the present invention is bilirubin in which one water-soluble compound is bound to one or two of the two propionic acid residues, and taurine (one glucuronic acid), etc. is bound to one or two of the two propionic acid residues in the body. It has been known. In the present invention, any group may be used as long as it can be made water-soluble for direct diazotization reaction.

The conjugated bilirubin used in the present invention may be a solid or a solution. Conjugated bilirubin solution is pi-1s
, o to 10.5.

As the nonionic surfactant used in the present invention, HLB10
-20 is preferred. A specific example is polyethylene glycol (abbreviated as PIG) cetyl ether k (
HL B 13.0 ) % P I G −/Nylphenyl ether, pH! G-alkyl ether 1PF
IG-lauryl ether, PEG-PPG (polypropylene glycol) copolymer, PEG-X thearyl ether, trade name Pionin D-1420 (manufactured by Takemoto Yushi)
and so on.

The amount of nonionic surfactant used in the present invention is 0.2 to 10 w/v%, preferably 1 to 3 w/v%, to the platform bilirubin solution. Reducing substances to nonionic surfactants 1 For example, glutathione 1 cysteine 1 ascorbic acid 1 isoascorbate The color tone (maximum absorption wavelength) of diazo coloring of conjugated and unconjugated bilirubin is 'I
In the acid azo method, which started with the fflliRYN-MALIiOY method, the presence of protein fragments influences (lower wavelength shift). Therefore, in the case of analysis using serum as a specimen, it is preferable that proteins coexist in the standard solution. Particularly preferred is the addition of normal animal or human serum.

The conjugated bilirubin solution of the present invention is preferably stored and distributed in a lyophilized state in order to maintain it in a usable state for a long period of time.

The present invention will be specifically explained below using examples.

In the examples, the word "regret" simply indicates ψI.

Example L A ditauro2-bilirubin solution having the following composition was prepared.

Pritton-Robinson buffer (pIi9.>) FiD
TA-2Na O, lM ditaurobilirubin 14, zypq/lu additive (as shown in Table 1) 1 Total bilirubin measurement reagent (manufactured by Toyobo ■) at the beginning of the above solution preparation and after storage at 250°C for 3 days The total bilirubin value was measured using a Diamond Color TB""), and the retention rate of the total bilirubin value before and after storage was determined.The results are shown in Table 1, indicating that the method of the present invention has good stability. It has been shown.

Table 1 Examples & Various surfactants shown in Table 2 were selected as one additive in the same composition as in Example 1 (addition amount: 2%). The prepared solution was stored at 40°C and the retention rate of bilirubin value was examined.

The following margins are provided. However, samples 416 and 17.18 are cysteine 1.
Added glutathione 1 dithiothreitol 0.01%.

As shown in Table 2, significant stabilization of 1-conjugated bilirubin was achieved by the addition of various nonionic surfactants and by the combined use of 1-reducing substances.

Example & The composition was the same as Example 1, but the additives were 1 cysteine 0°02% 1 and 1 Pionine D-1420 (nonionic surfactant manufactured by Takemoto Yushi) with the amount added (as shown in Table 3) and stabilized. I saw sex.

Table 3 Example Spring A solution with the following composition was prepared and freeze-dried.

Britton-Robinson buffer pH 9.5 Mannitol 2.0 Tilipid serum (Eiken Chemical controlled serum) 2. Asconosybic acid 0.01% Nonionic surfactant D'-14202.0% ditaurobilirubin 14 , 2 W/dl lyophilized vials were stored refrigerated. Color was developed using a distilled water constant reagent (Diacolor TB and DB manufactured by Toyobo Co., Ltd.). The stability of the degree of color development was good (XIJ<% as shown in FIG. 1), which was sufficient for stable measurements for total and direct bilirubin measurements.

The absorption maximum after color development is 1 total bilirubin 560 nm 1
Direct bilirubin was 54021m, which matched that of the patient's serum.

Example 5 The standard solution prepared in Example 4 was colored using a commercially available reagent kit. As shown in Table 4, both total bilirubin and direct bilirubin showed good color development.

Table 4

[Brief explanation of drawings]

FIG. 1 shows the stability of a gunmetal bilirubin solution prepared according to the present invention. In the figure, 1 is for total bilirubin.
■ indicates the case of direct bilirubin. Patent applicant: Toyobo Co., Ltd.

Claims (1)

[Claims] ■ A method for stabilizing conjugated bilirubin, which comprises coexisting a nonionic surfactant with conjugated bilirubin.