Characterization and immunoassay of apolipoprotein D

• Published in: Atherosclerosis Vol. 39; no. 3; pp. 395 - 409
• Main Authors: Albers, John J., Cheung, Marian C., Ewens, Susan L., Tollefson, John H.
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier Ireland Ltd 01.06.1981
• Subjects: Aging; Apolipoproteins - analysis; Apolipoproteins - immunology; Apolipoproteins D; Cholesteryl ester transfer protein; High density lipoproteins; Hormones - metabolism; Humans; Immunoassay; Immunodiffusion; Quality Control; Radial immunodiffusion; Sex Factors; High density lipoproteins; Cholesteryl ester transfer protein; Radial immunodiffusion
• ISSN: 0021-9150; 1879-1484
• DOI: 10.1016/0021-9150(81)90025-3

Apolipoprotein D (apoD) is a protein component of human plasma high density lipoprotein. It recently has been suggested to be a cholesteryl ester transfer protein, responsible for the transfer of cholesteryl ester from high density lipoproteins (HDL) to low density lipoproteins (LDL) and very low density lipoproteins (VLDL). We characterized apoD and anti-apoD sera and report a simple, specific and precise (CV 6–7%) radial immunodiffusion assay for apoD with application to whole plasma. Purified apoD had an apparent molecular weight of 32,500 ± 700 (n = 11) by sodium dodecyl sulfate electrophoresis and exhibited 3 major isoforms by isoelectric focusing with isoelectric points (pI) and percentage of total apoD as follows: (1) pI 5.20 ± 0.07 (24 ± 19%); (2) pI 5.08 ± 0.05 (55 ± 11%); and (3) pI 5.00 ± 0.05 (21 ± 9%). Immunoadsorption affinity chromatography with antibodies specific for apoD removed essentially all the apoD in plasma and approximately 64% of the lecithin-cholesterol acyltransferase (LCAT) and 11% of the plasma apolipoprotein A-I. However, the plasma did not lose any cholesteryl ester transfer activity assayed as the transfer of [ 14C]cholesteryl ester from labeled HDL to LDL or VLDL; or from labeled LDL to HDL. This suggests that apoD does not contribute significantly to the transfer of cholesteryl ester in whole plasma and that another factor can transfer cholesteryl ester between lipoproteins. Analysis of apoD in fresh plasma from normolipidemic (n = 74) and hyperlipoproteinemic (n = 44) adults indicates that males tend to have slightly higher apoD levels (6.2 ± 1.0 mg/dl, n = 77) than females (5.6 ± 1.4 mg/dl, n = 41) P < 0.01. Normolipidemic subjects had apoD levels (of 6.0 ± 1.1 mg/dl, n = 74) similar to that of hypercholesterolemics (6.3 ± 1.1 mg/dl, n = 15) and dysbetalipoproteinemics (6.1 ± 1.3 mg/dl, n = 10) whereas hypertriglyceridemics had slightly lower levels (5.3 ± 1.2 mg/dl, n = 19, P < 0.02). Subjects with familial LCAT deficiency had apoD levels approximately one-half that of normal levels (2.5 ± 0.6 mg/dl, n = 7). Analysis of apoD levels in frozen plasma from an employee population of the greater Seattle area indicated that among females, women on estrogen had the highest apoD levels (5.5 ± 0.4 mg/dl, n = 9), followed by women not taking medication (5.0 ± 0.9 mg/dl, n = 48), but women on combination oral contraceptives containing estrogen and progesterone had the lowest level (4.5 ± 0.9 mg/dl, n = 35, P < 0.025). These results suggest that estrogen increases apoD levels whereas progesterone decreases apoD levels. Also, apoD levels were significantly correlated with HDL cholesterol levels in males (r = 0.484, P < 0.001, n = 100) and females (r = 0.367, P < 0.001, n = 100).