A specific, accurate, and sensitive double antibody radioimmunoassay for measuring human apolipoprotein (apo) C-III has been developed. Anti-apoC-III(1) developed in rabbits cross-reacted completely with apoC-III subspecies. Analytical isoelectric focusing of delipidated triglyceride-rich lipoproteins (TRL) was done to assess the percentage of total apoC-III mass comprised by apoC-III(0), C-III(1), and C-III(2), and the data were used to compute the absolute plasma TRL apoC-III subspecie concentration. Total plasma apoC-III was 11.1 +/- 0.9 mg/dl (mean +/- SEM) in 29 normolipidemic healthy subjects; 21.3 +/- 4.9, 27.5 +/- 2.2, and 53.6 +/- 7 mg/dl in 3, 16, and 13 patients with primary types III, IV, and V hyperlipoproteinemia, respectively, and significantly (P < 0.01) higher than normal. Total plasma triglycerides (TG) correlated positively with total plasma apoC-III (r = 0.88; P = 0.0001) and TRL apoc-III (r = 0.88; P = 0.0001). Progressive hypertriglyceridemia was associated with a rise in the percent of total apoC-III in TRL isolated at d < 1.006 g/ml (r = 0.78; P < 0.0001; n = 43) and a reciprocal decline in the TRL-free plasma fraction (d > 1.006 g/ml). ApoC-III comprised significantly more of HDL(2) than HDL(3) protein (7.3 +/- 0.2 versus 1.6 +/- 0.2%, respectively, P < 0.01). HDL(2) and HDL(3) isolated from patients with type IV hyperlipoproteinemia had subnormal apoC-III as percent of total protein (2.4 +/- 0.5 and 0.6 +/- 0.1, respectively). Total plasma TG correlated negatively with i) apoC-III as percent of total HDL protein (r = -0.67; P = 0.002, n = 20); ii) apoC-III as percent of total HDL(2) protein (r = -0.52; P = 0.019); and iii) apoC-III as percent of total HDL(3) protein (r = -0.72; P = 0.0004). Plasma TRL apoC-III subspecie concentrations were significantly higher in the three hypertriglyceridemic groups (primary types III, IV, and V) compared to normals. TRL apoC-III(0) levels in patients with type IV and V were comparable (2.4 +/- 0.3 and 2.2 +/- 0.6 mg/dl, respectively). However, TRL apoC-III(1) and C-III(2) in patients with type V hyperlipoproteinemia were significantly higher (P < 0.01) than in patients with types IV or III hyperlipoproteinemia. Total plasma TG correlated positively with TRL apoC-III(0) (r = 0.56; P = 0.0004), TRL apoC-III(1) (r = 0.82; P = 0.0001) and TRL apoC-III(2) (r = 0.76; P = 0.0001). The slope of regression line relating total plasma TG with TRL apoC-III(1) was significantly steeper (P < 0.0001) than that for apoC-III(0). Thus, for a given interval of plasma TG, the change in concentration of TRL apoC-III(1) was much greater than that in TRL apoC-III(0). 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-III 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-III 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-III and its subspecies by radioimmunoassay and analytical isoelectric focusing: abnormal plasma triglyceride-rich lipoprotein apolipoprotein C-III subspecie concentrations in hypertriglyceridemia.