Organic compounds -- part of the class 532-570 series – Organic compounds – Carboxylic acids and salts thereof
Reexamination Certificate
1999-03-15
2004-12-21
Gitomer, Ralph (Department: 1651)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carboxylic acids and salts thereof
Reexamination Certificate
active
06833473
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for measuring oxidation parameters of low density lipoproteins (LDL), which methods are rapid, simple to perform, and valid for the determination of LDL oxidation products and LDL antioxidant potential. These methods provide specific means for assessing the oxidative stress in the body of an individual in general and, in particular, for assessing or screening the risk for, and for the diagnosis, management and research of atherosclerosis and coronary heart disease.
BACKGROUND OF THE INVENTION
Oxidation of low-density lipoprotein plays a key role in processes leading to the development of atherosclerosis. LDL oxidation is accompanied by alterations in its biological properties resulting in, for example, accelerated uptake through scavenger receptors in macrophages, altered chemotactic behavior of monocytes, and monocyte-derived macrophages, endothelial cell damage, and increased amounts of mediators of cell proliferation and platelet aggregation (refs. 1-4). All these effects may contribute to the development of atherosclerotic lesions. Therefore, determination of the LDL oxidation related parameters, namely LDL oxidation products, and antioxidant potential, gives more specific information on atherosclerosis-related biochemical phenomena than the commonly used measurements, of which the most common are the measurement of serum cholesterol, LDL and other lipoproteins and the apolipoproteins.
Most of the data on LDL oxidation come from studies where oxidation of LDL fractions, isolated by conventional ultracentrifugation methods, has been monitored by the appearance of conjugated dienes or thiobarbituric acid reactants arising during oxidation of isolated LDL in vitro (5). Thus far, when LDL oxidation has been investigated in humans in vivo, analyses of LDL oxidation products have been based on antibodies raised against in vitro oxidatively damaged LDL (5). The existing methodology is complex and time-consuming and, in addition, the specificity of the immunological analyses can be questioned (3). Therefore, there is still need for single rapid and specific measurement of LDL oxidation that could become part of the laboratory repertoire in the diagnosis and management of atherosclerosis (5).
The immunological methods developed for direct measurement of oxidized LDL may not be specific, as, in addition to oxidized LDL, antibodies seem to recognize also other epitopes (6) and have given contradictory results as well (3). The poor applicability of immunological methods may be a reflection of the chemistry of LDL oxidation: LDL oxidation can be initiated in various different polyunsar fatty acids, and each of these can give rise to a number of different kinds of oxidation products. Due to the multiplicity of oxidation products, development and use of immunological methods is likely to remain problematic also in the future.
The existing methods for measuring the antioxidant potential of LDL are complex and time consuming, and for example only a limited number of analyses can be performed within one working week: LDL is first isolated by ultracentrifugation, whereafter the samples still have to be dialyzed. Another disadvantage is the unprecise recording of results, where changes of the various reaction phases are not always easily detected.
We have developed, for the analysis of LDL oxidation parameters, namely LDL oxidation products and LDL antioxidant potential, methods which are rapid and simple to perform, and can therefore be used for large-scale clinical studies. The validity and clinical applicability of these analytical procedures is clearly indicated by several studies.
SUMMARY OF THE INVENTION
The objects of the present invention are fulfilled by providing a kit for use in the screening of the risk for, the diagnosis, management and research of atherosclerosis and coronary heart disease comprising means for isolating LDL from a serum or plasma sample for the preparation of a LDL fraction, and means for separating the lipids from the LDL fraction to obtain a lipid fraction.
In a preferred embodiment of this invention, the means for isolating the LDL from the serum or plasma sample is a buffered heparin solution and the means for separating the lipids is a chloroform-methanol solution.
According to a further embodiment, the kit comprises a means for use in the determination of the baseline level of conjugated dienes (LDL-BDC) in the lipid fraction. Said means is preferably an organic solvent, and more preferable cyclohexane.
It is a further object of this invention to provide a kit for use in the screening of the risk for, the diagnosis, management and research of atherosclerosis and coronary heart disease comprising means for isolating LDL from a serum or plasma sample for the preparation of a LDL fraction, and means for use in the determination of the antioxidant potential of LDL (LDL-TRAP) in the sample.
According to a preferred embodiment, the means for isolating the LDL from the sample is a buffered heparin solution, the means for use in the determination of the antioxidant potential of LDL in a serum or plasma sample is 2,2′-azobis(2-amidinopropane)HCl (ABAP). The LDL-TRAP is preferably determined by using chemiluminescence.
It is still a further object of this invention to provide a kit for use in the screening of the risk for, the diagnosis, management and research of atherosclerosis and coronary heart disease comprising means for isolating LDL from a serum or plasma sample for the preparation of a LDL fraction, means for separating the lipids from the LDL fraction to obtain a lipid fraction, means for use in the determination of LDL-BDC in the lipid fraction, and means for use in the determination of the antioxidant potential of LDL (LDL-TRAP) in the sample.
Further areas of applicability of the present invention will be apparent from the detailed description given hereinafter.
The kits of the present invention can comprise a combination of the individual components needed to screen the risk for, diagnose, manage and research atherosclerosis and coronary heart disease presented together in a common pack. For this purpose, the kit can comprise separate vials or containers for the necessary reagents and substrates.
According to another aspect of the invention, improved kits and assay methods are provided. The kits are useful for quantifying oxidation parameters in a LDL fraction, optionally in a pre-isolated LDL fraction. The kits include in separate containers reagents for determining baseline levels of conjugated dienes, as described herein, in improved ratios and amounts. Other kits are usefull for quantifying antioxidant potential in a LDL fraction of blood serum or plasma, optionally in a pre-isolated LDL fraction. Such kits contain in separate containers unexpectedly improved and novel formulations, ratios and quantities of reagents for determining total peroxyl radical trapping antioxidant potential. The reagents include precipitants for precipitating an LDL fraction from blood or serum, solvents and solvent mixtures for extracting lipids from the LDL fraction, resuspension solvents and solvent mixtures for resuspending the LDL fractions and/or lipids isolated therefrom, and detection reagents for enabling or improving the detection of measurable assay end products. The improvements in methodology and equivalent instructions with the kits of the invention include sample handling, mixing and centrifugation parameters as are described herein.
Thus in some embodiments, kits for use in quantifying oxidation parameters of lipids in a LDL fraction of blood serum or plasma are provided. The kits include a first container for extracting the lipids from the LDL fraction, the first container containing a solvent which extracts lipids from a LDL fraction and a second container containing an amount of resuspension solvent sufficient to resuspend the extracted lipids. In certain embodiments, the solvent which extracts lipids is chloroform:methanol having a ratio greater than about 2:1, preferably greater th
Gitomer Ralph
Oy Aboatech AB
Wolf Greenfield & Sacks P.C.
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