FR2535461A1 - REAGENT FOR THE DETERMINATION OF B-LIPOPROTEINS - Google Patents

Abstract

B-LIPOPROTEINS ARE DETERMINED QUALITATIVELY OR QUANTITATIVELY BY USING A COLORIMETRIC REAGENT CONSTITUTED MAINLY BY ONE OF THE DERIVATIVES OF TRIPHENYL-METHANE IN WHICH THE PRESENT CONSTANT OF HYDROGEN SUBSTITUENTS INDICATED IN THE FORMULA (CFINOPROTE I) (CFINOPROTEINS) INDICATED IN THE FORMULA (CFINOPROTEIN I) (CFINOPROTEINS). IN WHICH R AND R, WHICH MAY BE THE SAME OR DIFFERENT, ARE HAL ATOMS, AND X MAY BE H OR A MONOVALENT CATION AND, IN ADDITION, H ARYL ATOMS, WHICH ARE NOT SHOWN, MAY ALSO BE SUBSTITUTED. OF THESE DERIVATIVES OF TRIPHENYLMETHANE, BLUE MORDANT 1 (CI 43830) AND BLUE MORDANT 29 (CI 43825) ARE PARTICULARLY APPROPRIATE. THESE DERIVATIVES ARE DESIGNATED IN THIS APPLICATION BY "B-LIPOPROTEIC COLORANTS". USE OF THIS COLORIMETRIC REAGENT TO IDENTIFY B-LIPOPROTEINS AND ASSAY THEM.

Description

Reagent for the determination of β-lipoproteins.

  The present invention relates to the use of a lipoprotein dye in the identification of i-lipoproteins. More particularly, it relates to the use of a dye for -11 poproteal in a solution.

  buffer and in the presence of cationic or non-cationic surfactants

  appropriate ionic and anionic groups derived from

  metallic or mineral acids, in the

  quantitative or quantitative of lipoproteins.

  Lipoproteins are known to act as

liquid carriers in the blood.

  They can be divided into four main fractions which are differentiated by the composition of the supported liquids, the carrier proteins, their size,

  their density, their electrophoretic mobility and their

antigenic characteristics.

  Three lipoprotein fractions can be

  in the serum taken on an empty stomach, namely lipoprotein

  very low density (VLDL), low density lipoprotein (LDL) and high density lipopritein (HDL) In the post-prandial serum, a fourth fraction is encountered, namely chylomicra, whose presence in subjects with an empty stomach

is pathological.

  The determination of the various fractions of lipoprotein

  These are of considerable clinical interest because they are, inter alia, directly related to the pathology of arteriosclerosis. The conventional methods for determining the various lipoprotein fractions are: 1) Electrophoresis 2) Ultracentrifugation 3) Immunological and immunoelectrophoretic processes

  4) Immunonephelometric processes.

  Colorimetric methods are known in which the concentration of proteins is obtained more or less approximately by determination of cholesterol and

lipids bound to proteins.

  In particular, with regard to LDL, that is to say the proteins that are commonly considered

  to be directly related to the pathology of the arteriosus

  clerose, there are no known processes

metrics for their determination.

  The present inventors have found that lipoproteins react specifically with a dye for lipoproteins and this

  integral part of the present invention.

  Therefore, a first object of the present invention is the use (in the identification of 4-lipoproteins) of a dye for r-lipoproteins which will be called lipoprotein dye selected from triphenylmethane derivatives in which the presence constant of the hydrogen substituents indicated in the general formula (1) is essential:

H O

XOOC C OOX

(I)

H C H

"

R 1 R 2

  wherein R 1 and R 2, which may be the same or different, represent halogen atoms, and X may be hydrogen or a monovalent cation and furthermore, the hydrogens of the aryl ring 1 which are not shown are

also be substituted.

  A second subject of the present invention is a suitable composition comprising: i) a buffer -antoprotein * in buffer solution; ii) a chelating agent capable of sequestering the interfering metal ions present in the sample; in order to obtain a reaction more specific, iii) inorganic and anionic groups, iv) and optionally cationic or non-ionic surfactants Thus, the final composition may include:

  a) A buffer system that can maintain the pH between 3 and 7.

  (b) A chelating agent capable of sequestering metal ions

and in particular iron.

  Especially suitable chelating agents are

  citric acid and sesels / and ethylenediamino acid

  tetracetic acid and its salts which may also act as

buffer pairs.

  c) a cationic or nonionic surfactant and> in particular, cationic surfactants such as

  quaternary ammonium bases in which non-organic groups

  hydrophobic are alkyl groups among them, the

  cetyltrimethylammonium bromide was particularly

appropriate.

  d) Anionic groups such as halides, nitrates, sulphates, phosphates, etc., which can be added to the mixture in the form of salts or

form of acid.

  The efficiency of the process according to the present invention is determined by isolating by chromatography the proteins

  of a set of human serums which under appropriate conditions

  required give a positive reaction with a dye-lipo

  protein and identify them immunologically with the aid of

anti-serum specific Es.

  At the same time, the authors of the present invention verified that other fractionated serum proteins did not give a positive reaction under conditions

  analogous with the p-lipoprotein dye.

  The present invention is illustrated by the examples

  descriptive and non-limiting below.

EXAMPLE 1

  Reactive compositions for identifying lipoproteins A) Reagent: Potassium acetate buffer 0.5 Cetyltrimethylammonium bromide 270 mg / liter Blue 1 for Mordant 70 mg / l, Mg C 12 6 H 20 60 g / l Citric acid 0.026 M citrate tribasic sodium 0.038 M. B) Reagent identical to the previous one in which the Blue 1 for Kordant is replaced by Blue for Mordant 29 with a

concentration of 80 mg / l.

EXAMPLE 2

  Identification of p-lipoproteins in a human serum

  A) 0.1 ml of serum is added to 2 ml of the reagent A above.

  The absorption spectrum of the reaction mixture thus obtained is read with a spectrophotometer compared with a blank test consisting of 2 ml of reagent and 0.1 ml of physiological solution. A peak situated between 640 and 590 nm with an absorption maximum between 600 and 610 nm indicates

  the presence of β-lipoproteins in the serum.

  B) 0.1 ml of serum is added to 2 ml of the aforementioned reagent B in the same way as for example 2 (A) A peak situated between 640 and 770 nm with an absorption maximum between 580 and 590 nm indicates the presence of lipoproteins in the serum.

EXAMPLE 3

  Reagent composition for determining lipoproteins The reagent consists of:

  Chromogenic solution analogous to the reagents of Example 1.

  Solution for blank test identical to the solution

  chromogenic but without lipoprotein dye.

EXAMPLE 4

  Determination of L-lipoproteins in human sera The following quantities are introduced with a pipette into test tubes according to the scheme indicated: Sample Blank 1 Sample White 2 blank

Serum ml 0.05 0.05 -

  Physiological solution 0.05 0.05 Chromogenic solution ml 1.5 1.5 Solution for test at 1.5 1.5 1.5 white After leaving 20 minutes, the optical density of the sample is read at 615-620 nm by 1 (A 1), and the optical density of the blank sample

  is read at 615-620 nm from blank 2 (A 2).

  A, that is, A 1 A 2 'is proportional to the concentration

lipoproteins in the serum.

EXAMPLE 5

  Comparison human sera are tested by the method of

example 4.

  The β-lipoproteins are assayed in the same sera by the radial immunodiffusion method (plates

  provided by BEHRINGWERKE AG).

  The correlation coefficient of the results obtained

by both methods, 0.86 remains.

Claims (4)

  A colorimetric reagent for the determination of the lipoprotein $ comprising as a base reagent a triphenylmethane derivative in which the constant presence of the hydrogen substituents indicated in the general formula (I) is essential: HO O coox (I) H C H R 1 / X R 2   11, wherein R and R, which may be the same or different, are halogen atoms, and X may be hydrogen or a monovalent cation and, in addition, the hydrogens of the aryl ring, which are not shown also be substituted.   Colorimetric reagent according to Claim 1, characterized in that the triphenylmethane derivative is preferably Blue 1 for Mordant (C 1 43830) or Blue 29 for Mordant (C I 43825).   3 Colorimetric reagent according to the claims   prior art, characterized in that it comprises: a) at least one compound of general formula (I), as specified in claim 1, in buffer solution;   (b) a chelating agent capable of sequestering metal ions   liaues that interfere; (c) inorganic anionic groups;   d) and optionally cationic or non-cationic surfactants   Ionic. 4 Use of colorimetric reagent according to   any of the preceding claims to identify P-lipoproteins.