Quantitation of human apolipoprotein C-Ill and its subspecies by radioimmunoassay and analytical isoelectric focusing: abnormal plasma triglyceride- rich lipoprotein apolipoprotein C-Ill subspecie concentrations in hypertriglyceridemia

A specific, accurate, and sensitive double antibody radioimmunoassay for measuring human apolipoprotein (apo) C-111 has been developed. Anti-apoC-111, developed in rabbits cross-reacted completely with apoC-111 subspecies. Analytical isoelectric focusing of delipidated triglyceride-rich lipoproteins (TRL) was done to assess the percentage of total apoC-111 mass comprised by apoC-HI,, C-IIIl, and C-II12, and the data were used to compute the absolute plasma TRL apoC-111 subspecie concentration. Total plasma apoC-I11 was 11.1 ? 0.9 mg/dl (mean t SEM) in 29 normolipidemic healthy subjects; 21.3 5 4.9, 27.5 t 2.2, and 53.6 t 7 mg/dl in 3, 16, and 13 patients with primary types 111, IV, and V hyperlipoproteinemia, respectively, and significantly ( P < 0.01) higher than normal. Total plasma triglycerides (TG) correlated positively with total plasma apoC-111 ( r = 0.88; P = 0.0001) and TRL apoC-111 ( r = 0.88; P = 0.0001). Progressive hypertriglyceridemia was associated with a rise in the percent of total apoC-I11 in TRL isolated at d < 1.006 g/ml ( r = 0.78; P < 0.0001; n = 43) and a reciprocal decline in the TRLfree plasma fraction (d > 1.006 g/ml). ApoC-111 comprised significantly more of HDLz than HDL, protein (7.3 ? 0.2 versus 1.6 ? 0.276, respectively,P < 0.01). HDL, and HDL, isolated from patients with type IV hyperlipoproteinemia had subnormal apoC-111 as percent of total protein (2.4 0.5 and 0.6 5 0.1, respectively). Total plasma TG correlated negatively with i ) apoC-111 as percent of total HDL protein ( r = -0.67; P = 0.002, n = 20); i i ) apoC111 as percent of total HDLz protein ( r = -0.52;P = 0.019); and Ci) apoC-111 as percent of total HDL, protein ( r = -0.72; P = 0.0004). Plasma TRL apoC-111 subspecie concentrations were significantly higher in the three hypertriglyceridemic groups (primary types 111, IV, and V) compared to normals. TRL apoC-111, levels in patients with type IV and V were comparable (2.4 t 0.3 and 2.2 ? 0.6 mg/dl, respectively). However, TRL apoC-111, and C-IIIz in patients with type V hyperlipoproteinemia were significantly higher ( P < 0.01) than in patients with types IV or 111 hyperlipoproteinemia. Total plasma TG correlated positively with TRL apoC-111, ( r = 0.56; P = 0.0004), TRL apoC-111, ( r = 0.82; P = 0.0001) and TRL apoC-IIIz ( r = 0.76; P = 0.0001). The slope of regression line relating total plasma TG with TRL apoC-111, was significantly 800 Journal of Lipid Research Volume 22, 1981 steeper (P < 0.0001) than that for apoC-111,. Thus, for a given interval of plasma TG, the change in concentration of TRL apoC-111, was much greater than that in TRL apoC-III,.m The development of the RIA and its combined use with analytical isoelectric focusing thus allows quantitation of this important glycopeptide and its subspecies in human plasma and its subfractions. Because apoC-111 inhibits not only tissue lipoprotein lipase but also the hepatic uptake of triglyceride-rich lipoproteins and remnants, the data support the possibility that an abnormal metabolism of apoC-111 subspecies may be linked pathogenetically to elevated plasma TG levels.Kashyap, M. L., L. S. Srivastava, B. A. Hynd, P. S. Gartside, and G. Perisutti. Quantitation of human apolipoprotein C-111 and its subspecies by radioimmunoassay and analytical isoelectric focusing: abnormal plasma triglyceride-rich lipoprotein apolipoprotein C-111 subspecie concentrations in hypertriglyceridemia. J . Lipid Res. 1981. 22: 800-810. Supplementary key words lipoproteins . triglycerides ' high density lipoproteins The protein moiety of human lipoproteins is a heterogeneous mixture of numerous peptides grouped as apolipoproteins A, B, C, and so on. The C group of apolipoproteins is mainly found in the triglyceriderich lipoproteins (i.e., very low density lipoproteins and chylomicrons) and high density lipoproteins (HDL). ApoC consists of three peptides (apoC-I, C-11, and C-111) with distinct differences in structure and physico-chemical properties (1). ApoC-I11 is a peptide (mol wt 9300) that exists in at least three forms. T h e species contain 0, 1, or 2 moles of sialic Abbreviations: TRL, triglyceride-rich lipoprotein; TG, triglyceride; RIA, radioimmunoassay; IEF, isoelectric focusing; HDL, high density lipoprotein; VLDL, very low density lipoprotein; LDL, low density lipoprotein; VHDL, very high density lipoprotein. by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom acid (2) and are known as apoC-III,, C-III,, and C-II12, respectively. Hypertriglyceridemia arising from normal fat digestion and absorption (resulting in chylomicronemia) or from endogenous triglyceride synthesis (resulting in increased levels of very low density lipoproteins) is associated with a transfer of the C apolipoproteins from HDL to TRL (3). The catabolic fate of TRL is mediated initially by lipoprotein lipase. In vitro studies have shown that apoC-I1 increases the activity of this enzyme (4-6) whereas apoC-111 inhibits it (7). Recent evidence with rat, canine, and human lipoproteins or with artificial lipid-protein complexes indicates that the C apolipoproteins may have a very important function in the uptake of TRL particles and remnants by the rat perfused liver. These studies suggest that whereas apoE or its isomers (E3 and E4) promote the recognition of T R L particles or remnants by a specific hepatic receptor, the C apolipoproteins have an opposite effect (8-12). Human apoC-111, has specifically been shown to inhibit this process (8). However, the exact in vivo role of apoCI11 in normal human physiology has not been established. The purpose of this report is to detail a double antibody radioimmunoassay we have developed for the quantitation of human apoC-I11 in plasma or its lipoprotein fractions. Analytical isoelectric focusing has been used to measure the proportions of the desialylated (C-IIIo) and each sialylated (C-111, and C-IIIz) apolipoprotein in delipidated TRL. By measuring total apoC-111 by RIA and its subspecies by analytical IEF, the absolute concentrations of T R L apoC-111,, C-111, and C-II12 have been measured. Their relationship to total plasma triglycerides has been assessed. Additional work related to the amount of radioimmunoassayable apoC-I11 in HDL and its subfractions HDL, and HDL, and its relationship to plasma triglycerides is also presented.