INTRAMURAL RESEARCH PROJECT Z01 CL 10303-01 CP

October 1, 1998 to September 30, 1999

Title of Project: Development of New Assays for Lipoprotein Testing

Principal Investigator: A.T. Remaley, M.D., Ph.D. (Senior Investigator) CCS, CPD, CC, NIH Bethesda, MD 20892

Other Personnel: M. Sampson, CPD

Collaborating Units: None

Staff-Years: 0.25

Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither (a1) Minors (a2) Interviews

Summary of Work: Lipoprotein subfraction analysis is valuable in estimating the risk for coronary artery disease. The current procedure, however, requires multiple tests and has several manual steps. To reduce the complexity of lipoprotein subfraction analysis and to reduce its cost, we are developing a single tube homogenous assay for measuring serum high-density lipoprotein (HDL)-cholesterol, total cholesterol, and triglyceride. Low-density lipoprotein (LDL)-cholesterol can then be calculated from these parameters by Friedewald equation. We have now developed a working enzymatic assay that utilizes an anti-apoB antibody to block the reactivity of the reporter enzymes to LDL-cholesterol. The assay is performed in a sequential manner so that after the HDL-cholesterol is determined, a detergent is added to disrupt the antibody complex, which allows the subsequent measurement of total cholesterol. Next, the reporter enzymes for measuring total triglyceride are added. In the upcoming year, we plan to work on fully automating the assay and comparing it to standard assays. We also plan to further simplify the assay so it could be performed on a small, dedicated point-of-care device. We also plan to investigate a possible alternative to LDL phenotyping by nondenaturing gel electrophoresis, which is also a costly and complex test. We will explore whether a ratio of surface lipids ( free cholesterol and phospholipid) to neutral lipids (cholesteryl ester and triglyceride) or to the protein content of LDL can be used to estimate LDL size. We plan to first immunoisolate LDL from serum, then conduct a chemical analysis of the various lipids and proteins of LDL. Various ratios of the measured parameters will then be examined to see if they correlate with the LDL phenotype obtained by electrophoresis.