Chemistry: analytical and immunological testing – Lipids – triglycerides – cholesterol – or lipoproteins
Reexamination Certificate
2002-10-01
2004-06-22
Gakh, Yelena G. (Department: 1743)
Chemistry: analytical and immunological testing
Lipids, triglycerides, cholesterol, or lipoproteins
C436S512000, C436S536000, C436S538000, C436S539000
Reexamination Certificate
active
06753185
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods for the analysis and quantification of lipoproteins and, more specifically, to the analysis and quantification of lipoproteins present in biological and clinical samples. In particular, a protocol is disclosed for the multi-dimensional analysis using an absolute equilibrium particle density scale.
BACKGROUND OF THE INVENTION
Lipoproteins have been identified as potential markers for cardiovascular disease risks. For example, the development of artherosclerosis is linked to a dysfunction in lipid metabolism. Cholesterol content and the distribution of cholesterol between high density lipoproteins and low density lipoproteins comprise parameters to determine a “cardiac risk profile” for coronary heart disease. Several classes of lipoproteins have been identified: very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL).
Separation of lipoprotein a, lipoprotein a
−
(reduced species), and apolipoprotein a (apo(a)) using capillary zone electrophoresis has been reported (Hu, A. Z. et al.,
J. Chromatog. A,
717: 33-39, 1995). Sodium borate buffers containing SDS and acetonitrile were used. The method was reported to be advantageous over ELISA and SDS-PAGE assays due to its speed and sensitivity.
A method for profiling plasma lipoproteins based on differences in surface chemical properties was reported using HPCE (Cruzado, I. D. et al.,
J. Cap. Elec.
3(1): 25-29, 1996). Sodium dodecyl sulfate and acetonitrile were used to separate HDL and LDL particles.
High performance capillary electrophoresis was shown to separate LDL, HDL, lipoprotein a (Lp(a)) and Lp(a−) reduction products (Hu, A. Z. et al.,
Am. Lab.
28: 18N-18R, 1996). Benzyl alcohol was used to increase the UV signal intensity of highly hydrophobic lipoproteins.
A combination of capillary electrophoresis and electrospray ionization mass spectrometry was used to prepare a lipoprotein profile and cardiac risk profile analysis (Macfarlane, R. D. et al.,
Electrophoresis
18: 1796-1806, 1997). Ultracentrifugation and image analysis was used to separate the VLDL, LDL, and HDL fractions. Each of the individual fractions was further analyzed using capillary electrophoresis. Mass spectrometry was used to determine the isoform distribution for apoproteins. Lipid profile analyses were performed on nearly 100 clinical samples. Abnormalities in the lipid profiles were observed with samples from every cardiac patient. The analyses were also used to monitor the effects of a statin drug on a hypertriglyceridemic patient.
Capillary electrophoresis was reported as being useful for the characterization and quantitation of apolipoprotein B-100 (Cruzado, I. D. et al.,
J. Lipid Res.,
39: 205-217, 1998). Sucrose gradient ultracentrifugation and capillary electrophoresis were used to analyze serum. The described method was suggested to have the potential for high accuracy due to the elimination of various systematic errors associated with previously used methods.
The use of capillary electrophoresis was suggested as having potential for higher resolution, greater specificity, speed, and automation for detection and quantitation of lipoproteins than other existing methods (Watkins, L. K. et al.,
Methods in Molecular Medicine,
vol. 27:
Clinical Applications of Capillary Electrophoresis,
Ed. Stephen M. Palfrey, pages 99-108, 1999). Samples were separated into lipoprotein classes by ultracentrifugation, and the protein fractions measured by capillary electrophoresis.
Macfarlane and McNeal described the concept of a lipoprotein fingerprint in “What is your Lipoprotein Fingerprint?” (AACC Lipids and Lipoproteins Division Newsletter, vol. 14, no. 4, Fall 2000). The profile involves measurement of the lipoprotein particle density profile of VLDL, LDL, and HDL. The lipoprotein fingerprint measured was a composite of five profiles. Sections of the separated material were obtained and analyzed by capillary zone electrophoresis to identify sub-fractions. MALDI analysis was used to identify small proteins sequestered within the lipoprotein particles.
U.S. Pat. No. 5,783,400 describes a method for the isolation of lipoprotein and subsequent quantification of its mass and cholesterol content. Biological fluids are fractionated using an ultra-centrifuge. The fraction is reacted with immobilized ligand to remove non-Lp(a) substances. Protein concentration, protein isoform determination, and cholesterol content can be obtained from the fractionated material.
U.S. Pat. No. 6,090,921 offers a process for purifying apolipoprotein A or apolipoprotein E from human plasma. Plasma is mixed with polyethylene glycol to precipitate impurities, and the Apo A or Apo E purified by anion-exchange chromatography and gel filtration.
Despite advances made to date, there exists a need for rapid, accurate, and reproducible assays for the separation and identification, and quantitation of lipoproteins.
SUMMARY OF THE INVENTION
Lipoprotein profiles are prepared via an absolute thermodynamic equilibrium density scale using an ultracentrifuge. The use of standards is not required. The lipoprotein subclasses can be identified by their location in the equilibrium density gradient. Individual subclasses can readily be isolated by freezing, cutting, and thawing the centrifuge sample. The presence or absence of a metabolic condition or disease can be correlated by the concentration of one or more lipoprotein subclasses.
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Cornwell at al. “Lipoprotein Pre-Staining and Ultracentrifugal Analysis in a Density Gradient”, Proc. Soc. Exptl. Biol. Med., 1961, v. 107, pp 296-9.
Westhuyzen et al., “Simplified sizing of low-density lipoprotein using polyacrilamide gradient gel electrophoresis of plasma”, Eur. J. Clin. Chem. Clin. Biochem., 1997, v. 35, No. 1, pp. 17-19.
PCT Search Report dated Jan. 6, 2003.
Hu, A.Z. et al.,Journal of Chromatography A, 717: 33-39, 1995, “Characterization of lipoprotein a by capillary zone electrophoresis”.
Cruzado, I.D., et al.,J. Cap.Elec. 3(1):25-20, 1996, “Influence of dodecyl sulfate ions on the electrophoretic mobilities of lipoprotein particles measured by HPCE”.
Hu, A.Z. et al.,Am. Lab. 28: 18N-18R, 1996, “Developing high-performance capillary electrophoresis methods for lipoprotein profiling”.
Macfarlane, R.D. et al.,Electrophoresis18: 1796-1806, 1997, “Development of a lipoprotein profile using capillary electrophoresis and mass spectrometry”.
Cruzado, I.D. et al.,Journal of Lipid Research, 39: 205-217, 1998, “Characterization and quantitation of apolipoprotein B-100 by capillary electrophoresis”.
Watkins, L.K. et al.,Methods in Molecular Medicine, vol. 27:Clinical Applications of Capillary Electrophoresis, Ed. Stephen M. Palfrey, pp. 99-108, 1999.
AACC Lipids and Lipoproteins Division Newsletter, vol. 14, No. 4, Fall 2000, “Treatment of hyperlipidaemic post-menopausal women”.
Cockrill Steven L.
Espinosa Irma L.
Farwig Zachlyn N.
Hosken Brian D.
Macfarlane Ronald D.
Gakh Yelena G.
Howrey Simon Arnold & White , LLP
The Texas A&M University System
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