Chemistry: analytical and immunological testing – Lipids – triglycerides – cholesterol – or lipoproteins
Reexamination Certificate
2000-03-31
2003-07-22
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Lipids, triglycerides, cholesterol, or lipoproteins
C436S161000, C436S164000, C436S172000, C436S173000, C422S067000, C422S070000, C422S082050, C422S082080, C435S011000
Reexamination Certificate
active
06596544
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is related to an application naming Fogelman et al. as inventors entitled “CONTROL OF A GENE INDUCED BY OXIDIZED LIPIDS IN HUMAN ARTERY WALL CELLS”, filed on Mar. 31, 2000 which is incorporated herein by reference in its entirety for all purposes.
FIELD OF THE INVENTION
This invention relates to the diagnosis of atherosclerosis. In particular this invention provides improved assays
BACKGROUND OF THE INVENTION
Cardiovascular disease is a leading cause of morbidity and mortality, particularly in the United States and in Western European countries. Several causative factors are implicated in the development of cardiovascular disease including hereditary predisposition to the disease, gender, lifestyle factors such as smoking and diet, age, hypertension, and hyperlipidemia, including hypercholesterolemia. Several of these factors, particularly hyperlipidemia and hypercholesteremia (high blood cholesterol concentrations) provide a significant risk factor associated with atherosclerosis.
Cholesterol is present in the blood as free and esterified cholesterol within lipoprotein particles, commonly known as chylomicrons, very low density lipoproteins (VLDLs), low density lipoproteins (LDLs), and high density lipoproteins (HDLs). Concentration of total cholesterol in the blood is influenced by (1) absorption of cholesterol from the digestive tract, (2) synthesis of cholesterol from dietary constituents such as carbohydrates, proteins, fats and ethanol, and (3) removal of cholesterol from blood by tissues, especially the liver, and subsequent conversion of the cholesterol to bile acids, steroid hormones, and biliary cholesterol.
Maintenance of blood cholesterol concentrations is influenced by both genetic and environmental factors. Genetic factors include concentration of rate-limiting enzymes in cholesterol biosynthesis, concentration of receptors for low density lipoproteins in the liver, concentration of rate-limiting enzymes for conversion of cholesterols bile acids, rates of synthesis and secretion of lipoproteins and gender of person. Environmental factors influencing the hemostasis of blood cholesterol concentration in humans include dietary composition, incidence of smoking, physical activity, and use of a variety of pharmaceutical agents. Dietary variables include amount and type of fat (saturated and polyunsaturated fatty acids), amount of cholesterol, amount and type of fiber, and perhaps amounts of vitamins such as vitamin C and D and minerals such as calcium.
As indicated above, high blood cholesterol concentration is one of the major risk factors for vascular disease and coronary heart disease in humans. Elevated low density lipoprotein cholesterol (“LDL-cholesterol”) and total cholesterol are directly related to an increased risk of coronary heart disease. Cholesterol and Mortality: 30 Years of Follow-Up from the Framingham Study, Anderson, Castelli, & Levy, JAMA, Vol. 257, pp. 2176-80 (1987).
Although high levels of total cholesterol and LDL-cholesterol are risk factors in developing atherosclerosis and vascular diseases, a deficiency of high density lipoprotein cholesterol (hereafter “HDL-cholesterol”) has recently been recognized as a risk factor for developing these conditions. Several clinical trials support a protective role of HDL-cholesterol against atherosclerosis. A study has shown that for every 1-mg/dl increase in HDL-cholesterol in the blood, the risk for coronary vascular disease is decreased by 3% in women. High-density Lipoprotein Cholesterol and Cardiovascular Disease: Four Prospective American Studies, Gordon, Probstfield, and Garrison et al., Circulation, Vol. 79, pp. 8-15 (1989).
It is widely believed that HDL is a “protective” lipoprotein (Vega and Grundy (1996) Curr. Opin. Lipidology, 7, 209-216) and that increasing plasma levels of HDL may offer a direct protection against the development of atherosclerosis. Numerous studies have demonstrated that both the risk of coronary heart disease (CHD) in humans and the severity of experimental atherosclerosis in animals are inversely correlated with serum HDL cholesterol (HDL-C) concentrations (Russ et al. (1951)
Am. J. Med
., 11: 480-493; Gofman et al. (1966)
Circulation
, 34: 679-697; Miller and Miller (1975)
Lancet
, 1: 16-19; Gordon et al. (1989)
Circulation
, 79: 8-15; Stampfer et al. (1991)
N. Engl. J. Med
., 325: 373-381; Badimon et al. (1989)
Lab. Invest
., 60: 455-461).
While HDL/LDL ratios have appear to provide a good marker for risk of atherosclerosis and heart disease on a population level, HDL and/or LDL measurements have proven to be poor prognostic indicators at an individual level. In particular individuals with high HDL:LDL ratios have been observed with severe atherosclerosis, while conversely, individuals with very low HDL:LDL ratios have been identified with no evidence of atherosclerosis.
SUMMARY OF THE INVENTION
This invention provides novel assays that are prognostic and/or diagnostic for atherosclerosis or risk of atherosclerosis. The assays are based, in part, on elucidation of a mechanism by which HDL affords protection against plaque formation. In particular, it was a discovery of this invention that HDL or components can prevent the oxidation of lipids (e.g. lipids present in LDLs) and can also repair (reduce) already oxidized lipids and thereby reduce the inflammatory response associated with and characteristic of atherosclerotic plaque formation. Moreover it was a discovery of this invention that individuals vary in the ability of their HDL to afford such protection. Thus an assay of HDL protective and/or repair activity provides a highly effective assay for risk of atherosclerosis and its associated pathologies.
Thus, in one embodiment, this invention provides methods of evaluating the risk for atherosclerosis in a mammal by evaluating the ability of the animal's HDL to repair (reduce) oxidized phospholipids. The methods preferably involve providing a biological sample from the mammal where the sample comprises comprising a high-density lipoprotein (HDL) or a component thereof (e.g. apo A-I, paraoxonase, platelet activating factor acetylhydrolase, etc.), contacting the high-density lipoprotein with an oxidized phospholipid; and measuring a change in the amount of oxidized or non-oxidized phospholipid where the absence of change in the amount of oxidized phospholipid indicates the mammal is at risk for atherosclerosis.
The oxidized phospholipid is preferably an oxidized phospholipid that causes a monocytic reaction. Particularly preferred phospholipids include, but are not limited to the oxidized form of lipids selected from the group consisting of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC), 1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (SOVPC), 1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (SGPC), 1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (SEIPC), 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine (Ox-SAPE), 1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylethanolamine (SOVPE), 1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylethanolamine (SGPE), and 1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylethanolamine (SEI PE). In one particularly preferred embodiment, the oxidized phospholipid is a component of (present in) a low density lipoprotein.
The oxidized phospholipid (or reduced phospholipid) can be determined by any convenient method. Such methods include, but are not limited to mass spectrometry, liquid chromatography, thin layer chromatography, fluorimetry, radioisotope detection, antibody detection, and detecting a signal from a label that indicates an oxidized phospholipid. Fluorescent labels (e.g. 2′,7′-dichlorodihydrofluorescine diacetate, rhodamine, cis-par
Fogelman Alan M.
Hama Susan
Navab Mohamad
Hunter Tom
Quine Intellectual Property Law Group P.C.
The Regents of the University of California
Wallenhorst Maureen M.
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