Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-07-20
2004-11-02
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S028000
Reexamination Certificate
active
06811994
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for quantitating triglycerides (TG) in lipids, which are significant in a field of clinical laboratory test, particularly, as a risk factor of arteriosclerosis.
BACKGROUND ART
At present, in a field of clinical laboratory test, cholesterol in high density lipoprotein (HDL) is frequently determined as a risk factor, i.e., negative factor, of arteriosclerosis, while cholesterol in low density lipoprotein (LDL) is also determined as a positive factor. On the other hand, it has been elucidated by many epidemiological searches that hyperlipemia is a primary factor in development of an arteriosclerotic disease which is accompanied by an ischemic heart disease as a major symptom. In addition, among apoproteins E of very low density lipoproteins (VLDL), E1 is incorporated in the receptor, but E2 relating to III type hyperlipemia is not [Arteriosclerosis, 25 (11/12), 415-420 (1998); Journal of Clinical and Experimental Medicine, 172 (5), 276-280 (1995)]. Thus, the lipoproteins have so far been mentioned as a risk factor of arteriosclerosis in various aspects.
In recent years, it has been said that the difference in the kind and quantity of lipids contained in lipoproteins is involved in various diseases. Among them, it has also been reported that LDL, particularly, small dense LDL is a positive factor for closely related arteriosclerosis in a familial hyperlipemia. Small dense LDL is produced from VLDL by action of a TG catabolic enzyme. The ratio of the TG content to the cholesterol content has been altered in the small dense LDL in comparison with normal LDL, and the TG content is increased [Journal of Clinical and Experimental Medicine, 164 (12), 833-836 (1993)]. It has been considered, accordingly, that high TG concentration in LDL (an index of small dense LDL) enhances the risk of arteriosclerosis.
As for a method for specifically quantitating TG in each lipoprotein, for example, a colorimetry or other such method can be considered. In the colorimetry, each lipoprotein is fractionated by ultracentrifugation, and then TG contained in the fraction is treated, for example, with an enzyme system comprising lipoprotein lipase (LPL), glycerol kinase (GK) and glycerol-3-phosphate oxidase to generate hydrogen peroxide, with which a chromogen is developed together with a peroxidase (POD). This method, however, is very troublesome because it requires a great deal of time and effort for ultracentrifugation.
DISCLOSURE OF THE INVENTION
The purpose of the present invention is to provide a method for conveniently quantitating triglycerides (TGs) contained in various lipoproteins.
In serum or plasma samples, there are free glycerol and TG in various lipoproteins. In the invention, TG contained in a particular lipoprotein among these diverse lipoproteins is intended to be specifically quantitated without isolation of the aimed lipoprotein. For this purpose, free glycerol having an influence on the quantitation have to be converted into some inert form somehow, as well as TGs contained in lipoproteins other than the particular one have to be altered in advance or inhibited so that they cannot be involved in the reaction.
The present invention relates to a method for quantitating TGs in a particular lipoprotein which comprises eliminating free glycerol from a sample containing free glycerol and TG in the particular lipoprotein, then allowing the resulting sample to react with lipoprotein lipase (LPL) and an enzyme system which generates hydrogen peroxide from glycerol produced in said reaction, and quantitating the generated hydrogen peroxide.
As for samples containing TGs in lipoproteins, test specimens such as serum, plasma, and the like are exemplified. LPL means an enzyme (EC.3.1.1.34) which has a function decomposing TGs in lipoproteins into glycerol and fatty acids.
The enzyme system which generates hydrogen peroxide from glycerol includes a system containing glycerol kinase (GK) (EC.2.7.1.30) which has a function to convert glycerol into glycerol-3-phosphate in the presence of ATP and glycerol-3-phosphate oxidase (GPO)(EC.1.1.3) which generate hydrogen peroxide from glycerol-3-phosphate, as well as a system containing glycerol oxidase (GO)(EC. 1.1.3.21) which generates hydrogen peroxide from glycerol as a substrate.
In one embodiment of the present invention, the method for eliminating free glycerol comprises decomposing glycerol with an enzyme system, which generates hydrogen peroxide from free glycerol, to yield hydrogen peroxide, and then eliminating said hydrogen peroxide generated.
After removal of glycerol by such a method, TG of a particular lipoprotein can be quantitated as follows. The resulting sample is allowed to react, in the presence of a reagent that inhibits the reaction of lipoproteins other than the particular lipoprotein, with LPL and an enzyme system which generates hydrogen peroxide from glycerol generated in this reaction, and quantitating the generated hydrogen peroxide.
The reagent that inhibits the reaction of lipoproteins other than the particular one includes surfactants which inhibit the reaction of lipoproteins other than the particular one and/or aggregating agent or the like for lipoproteins other than the particular one.
In order to facilitate generation of glycerol from TG of a particular lipoprotein by LPL, it is also possible to use a surfactant and/or an enzyme which allows the reaction of a particular lipoprotein, in the course of quantitation after elimination of free glycerol.
In another embodiment of the present invention, in eliminating free glycerol, it is also possible to choose a method for eliminating free glycerol, which method comprises converting TGs in lipoproteins other than particular one into free glycerol at the same time.
For example, hydrogen peroxide is generated in the presence of a reagent which allows the reaction of lipoproteins other than the particular one using LPL and an enzyme system which generates hydrogen peroxide from free glycerol, and then the resulting hydrogen peroxide is eliminated. Thus, all of TGs other than TG of a particular lipoprotein can be eliminated.
After the elimination reaction, the sample is allowed to react with LPL and an enzyme system which generates hydrogen peroxide from glycerol, and the generated hydrogen peroxide is quantitated. Thus, TG contained in a particular lipoprotein can be quantitated.
The reagent allowing the reaction of lipoproteins other than the particular one includes surfactants which inhibit the reaction of lipoproteins other than the particular one and/or aggregating agents for the particular lipoprotein.
In order to facilitate generation of glycerol from TG of a particular lipoprotein by LPL, it is also possible to add a surfactant and/or an enzyme which allows the reaction of a particular lipoprotein after elimination of free glycerol and TG of lipoproteins other than the particular one.
According to the present invention, the following reagents are provided: reagents for quantitating TG in a particular lipoprotein containing a reagent for inhibiting the reaction of lipoproteins other than the particular one or a reagent for allowing the reaction of lipoproteins other than the particular one, LPL, GK, GPO and peroxidase; or reagents for quantitating TG in a particular lipoprotein containing a reagent for inhibiting the reaction of lipoproteins other than the particular one or a reagent for allowing the reaction of lipoproteins other than the particular one, LPL, GO and peroxidase. In addition to a reagent for allowing there action of lipoproteins other than the particular one, it is possible to add a surfactant and/or an enzyme which allows the reaction of the particular lipoprotein.
In addition, according to the present invention, the following reagents are provided: reagents for quantitating TG in a particular lipoprotein containing a reagent for inhibiting the reaction of lipoproteins other than the particular one or reagent for allowing the reaction of lipoproteins other than the particular one in
Miike Akira
Miyauchi Kazuhito
Murakami Tomomi
Takada Shizuyo
Fitzpatrick ,Cella, Harper & Scinto
Kyowa Medex Co., Ltd.
Saucier Sandra E.
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