JPS5928664A - Reagent composition for determining triglyceride - Google Patents

Abstract

PURPOSE:To enable determination of triglyceride without being negatively affected by bilirubin contained in body fluid, by adding a specified amt. of ferrocyanide ions. CONSTITUTION:In the reagent compsn. for determining triglyceride contg. ( I ) riboprotein lipase, (II) glycerol kinase and glycerophosphoric acid oxidase or (III) glycerol oxidase, (IV) peroxidase, (V) coupler phenazone, and (VI) an aniline deriv., ferrocyanide ions are added by 0.1-1.0mumol/l. Said ions are selected from sodium, potassium, and the like ferrocyanides, and the composition is preferably within 6.0-8.0 in pH. To determine triglyceride, a compsn. consisting of one, 2, or more solutions can be applied. In the case of 2 or more soln. type, said ferrocyanide ions may be added to either of the reagent soln. to be used before color development and that to be used during it.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a reagent composition for measuring triglycerides in body fluids.

Conventional methods for measuring triglycerides in body fluids include methods using lipoprotein lipase, glycerokinase, glycerophosphate oxidase, peroxidase, 4-aminoantipyrine and aniline derivatives, lipoprotein lipase 1 glycerol oxidase, peroxidase, 4-aminoantipyrine. Also known are methods using aniline derivatives.

In the method of colorimetric measurement using aniline derivatives, the influence of various coexisting substances in body fluids becomes a problem.

Among them, bilirubin exists in high concentration in serum.

The effect on measured values is considered a problem. The correct error due to bilirubin's own absorption in the visible region can be determined by colorimetrically measuring the reddish-purple product (540 to 560 nm) resulting from the oxidative coupling of 4-aminoantipyrine and an aniline derivative.
Negligible [Oxidative coupling red product of 4-aminoantipyrine and phenol derivative (500 nm)]
The colorimetric measurement of will have a positive error. ].

However, the negative error due to the reducing power of bilirubin has not yet been sufficiently avoided. When the pH of the triglycephyte measurement composition is 6.5 or less, the reducing power of bilirubin that affects the pigment system is small.

Ignore it. However, the optimum level of each constituent enzyme 1)
II, stable pH exists in the neutral to alkaline range. Taking these factors into consideration, we have developed a composition for measuring Tribulicella 4)' at pH 6.5 to pH 8.0, in which the oxidatively coupled product of 4-aminoantipyrine and an aniline derivative exhibits sufficient sensitivity and color stability. When quantification is performed using bilirubin, the negative error due to the reducing power of bilirubin becomes large and becomes negligible.

The present inventors have intensively investigated these problems and have determined that triglycephyte in tokoro body fluids was determined by colorimetry.
By using a triglyceride measuring composition containing ferrocyanide ions of 0.1/jmol/J to 1.0 μmol/l, the present invention succeeded in measuring triglyceride without being affected by the negative effects of bilirubin in body fluids. reached.

That is, the present invention provides (1) riboprotein in
(Punishment) In the triglycephite 9 regular reagent composition containing glycerol kinase and briceroliphenazine and (vl) aniline derivative, the ferrocyanide ion is present at a concentration of 0.1 pmo I /l to 1. Opmo I/E
This is a reagent composition for measuring triglycephyte 9, which is characterized in that it contains:

In the present invention, the concentration of ferrocyanide ions is 0.1
If it is less than μm o 1/1, the negative effects of bilirubin cannot be completely avoided. Also, 1.0μjt
If the concentration exceeds mol/l, the change in the rise of the reagent blank over time will become large, causing errors and impairing the reagent performance. 0.1μmot/J~1.0μm o l
The ferrocyanide ion used in the present invention using a ferrocyanide ion concentration of /J is sodium ferrocyanide, potassium ferrocyanide, and F
Other salts containing the e(CN)a= ion may be mentioned.

Lipoprotein lipase used in the present invention is an enzyme that hydrolyzes triglyceride, diglycewide, and monoglycephyte in riboprotein and decomposes it into glycerol and fatty acids.
Pseudomona7. There are some derived from the genus etc.

Glycerokinase used in the present invention acts on glycerol and ATP in the presence of magnesium ions.

It is an enzyme that produces glyceptalophosphate and ADP, and example t
There is one derived from the genus Haselulomananu and Cansida.

One example of glycerophosphate mexidase used in the present invention is derived from the genus Vegiococanu and Aerococus.

Glycerol oxidase used in the present invention is an enzyme that acts on glycerol in the presence of oxygen to generate hydrogen peroxide and glyceraldehyde.
There's something coming to Neurosport on Ju13.

The peroxidase used in the present invention is an enzyme that acts on H2O2, color W, and the body, causes oxidative coupling, and produces a color product and water, and for example, there is one derived from 7+ sous water.

Couplers used in the present invention include 4-aminoantipyrine, 3-methyl-2-benzothiazolinehydrazine, diaminoantipyrine, 1-(p-diethylamino-phenyl)-2,3-diethyl-4-amino-pyrazone-5
,1-(p-amino-phenyl)-2,3-dimethyl-
Examples include 4-aminohydrolone-5,1-(p-acetaminophenyl)-2,3-diethyl-4-amino-pyrazolone-5.

The aniline derivative used in the present invention is aniline, N.

N-dimethylaniline, N,N-dimethylaniline.

N,N-diethyl-m-toluidine, N,N-dimethyl-m-anisidine, N-ethyl-N-(3-methylphenyl)-N-acetylethylenediamine, N-ethyl-N-(β-hydroxyethyl) -m-toluidine,
N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-t/reizin, N-ethyl-N-sulfopropyl-m-))reizin, N-edyl-N-sulfopropyl-xyaniline, N-ethyl -N-(2-hydroxy-3
-sulfopropyl)-3,5-dimethoxyaniline, N
-ethyl-N-sulfopropyl-m-anisidine and the like.

The composition of the present invention preferably has a pH of 6.0 to 8.0.

When measuring triglyceride, the composition of the present invention includes:
It can be applied to a composition of one liquid or (l'i) two or more liquids.In the case of two or more liquids.

Ferrocyan ions may be added to either the pre-coloring solution or the @coloring solution.

The composition of the present invention, compared to the method using phenol,
It is characterized by a low concentration of ferrocyanine ions in consideration of the increase in reagent volume.

When fully determining triglycerides using the composition of the present invention, it is possible to accurately measure triglycerides without being influenced by bilirubin in body fluids.

The present invention will be explained below with reference to examples.

Example 1 1. Sample: Serum A: Water = 8:2 <volume ratio) ■Serum A
: Bilirubin aqueous solution (100W/dJ) = 8:2 (volume ratio) ■ Serum B: Water - 8:2 (volume ratio) ■ Serum B: Bilirubin aqueous solution (100"F/dl) = 8;
2 (volume ratio) ■ 2 reagents: Tris buffer (pH 7,5) riboprotein lipase 200 units/Ml glycerokinase 1.0 units/shoulder t glycerophosphate oxidase 265 peroxidase
7.5〃4-aminoantipyrine 3~/dl
N,N-Diethyl-m-toluidine 20'·'f/dl A reagent was prepared containing the above reagent at a potassium ferrocyanide concentration shown in Table 1 below.

Chapter 1 Table 3.Measurement method Add reagents (A, B) to 20 μl of each sample from ■ to ■.

After adding 3 ml of a, b) and reacting at 37° C. for 10 minutes, absorbance was measured at a wavelength of 545 nm.

The results are shown in Table 2.

Table 2 Although the reagents of the present invention are not affected by bilirubin.

In the case of no addition of potassium ferrocyanide (a) and the case of low concentration of potassium ferrocyanide (b), the results were affected by bilirubin.

Example 2゜1, Sample 2 Serum A: Water = 8:2C volume ratio) ■ Serum A
: Bilirubin aqueous solution (100~/dl) = 8 = 2 (volume ratio) ■ Serum B: Water - 8:2 (volume ratio) ■ Serum B: Bilirubin aqueous solution (100"V/dJ) = 8:
2 (volume ratio) 2. Reagents: Tris buffer (pH 7; 5) riboprotein lipase 200 units/glycerokinase 1.0 units/solution glycerophosphate oxidase 2.5 peroxidase 7.5 4-amino Antipyrine 3/dlN-ethyl-N-(3
-sulfopropyl)-m-anisidine 20
~/dl A reagent containing the potassium ferrocyanide concentration shown in Table 3 below was prepared in the above reagent.

Table 3, each sample of measurement method ω~■ 20 μIK reagent (A, B, a, b
) 3 shoulder t was added and the mixture was reacted at 37° C. for 10 minutes, and then the absorbance was measured at a wavelength of 540 nm.

The results are shown in Table 4.

Table 4 The reagents of the present invention are not affected by bilirubin, but when potassium ferrocyanide is not added (a) or when potassium ferrocyanide is at a low concentration (b), they are affected by bilirubin.

Example 3 Reagents: Tris buffer (pH 7,5) Lipoprotein lipase 200 units/glycerokinase 1.0 Glycerophosphate oxidase 2.5 Peroxidase 7,5 4-aminoantipyrine 3 Flv/d lN, N- Diethyl-m-)luidine 20~/di A reagent containing the above reagent at a potassium ferrocyanide concentration shown in Table 5 below was prepared, and the change over time of the reagent blank was measured.The results are shown in FIG.

Table 5 In the present invention, the reagent blank has a small change in eaves day, and the difference is small compared to the case without additives. When the amount of potassium ferrocyanide added is large, the oral change in the reagent blank is large, and the difference is large compared to when no addition is made.

Example 4゜ Reagent A: Same reagent composition as in Example 3.

Reagent B: N,N-diethyl-m of the reagent composition of Example 3
-) Phenol 12m9/dl instead of luidine.

Sample: Same as Example 2.

Measurement method: 20 μl of each sample from ■ to ■ K reagent (A, B) 3
After adding tttl and reacting at 37°C for 10 minutes,
Absorbance was measured at a wavelength of 540 nm. The results are shown in Table 6.

Table 6 Moreover, the oral changes of the reagent blank are shown in FIG.

In Figure 2, a is potassium ferrocyanide o, 2 μmol
/l containing l reagent B, b is potassium ferrocyanide 0.2
µmol/J Contains/A% C indicates the case of 1 reagent B containing 20.0 µmol/l of potassium ferrocyanide, and trl R drug containing 20.0 µmol/l of potassium ferrocyanide.

/'l) is a ferro whose reagent blank changes little over time.
Although the influence of bilirubin can be avoided by adjusting the concentration of cyanide ion, reagent B requires a high concentration of ferrocyanide ion and the oral change of the reagent blank is large.

[Brief explanation of drawings]

Figures 1 and 2 show the changes over time in the absorbance of the reagent of the present invention and the comparative reagent. Patent applicant: Toyobo Co., Ltd.

Claims (1)

[Claims] (1) Lipoprotein lipase, (I) glycerol kinase and glycerophosphate oxidase or (I)
In a reagent composition for measuring triglycephyte containing l+) glycerol oxidase, (lv) peroxidase, (V) coupler and (vl) aniline derivative, ferrocyanide ion is contained in an amount of 0.1 μmol/1 to 1.
A reagent composition for measuring triglyceride wide, characterized in that it contains 0 tlmo I/l.