X/01/$03.00/0 Vol. 86, No. 7 The Journal of Clinical Endocrinology & Metabolism Copyright 2001 by The Endocrine Society

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1 X/01/$03.00/0 Vol. 86, No. 7 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 2001 by The Endocrine Society Postprandial Remnant-like Lipoproteins in Hypertriglyceridemia* T. C. OOI, M. COUSINS, D. S. OOI, G. STEINER, K. D. UFFELMAN, K. NAKAJIMA, AND I. E. SIMO From the Metabolism and Hormone Laboratory (T.C.O., M.C., D.S.O., I.E.S.), Division of Endocrinology and Metabolism, and Division of Biochemistry (T.C.O., D.S.O.), Department of Laboratory Medicine, Ottawa Hospital Civic Campus, University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9; Division of Endocrinology and Metabolism (G.S., K.D.U.), Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada M5G 1L7; and Otsuka America Pharmaceutical, Inc. (K.N.), Rockville, Maryland ABSTRACT It has been proposed that remnants of chylomicrons and very-lowdensity lipoproteins (VLDL) are atherogenic. We have used an immunochemical method to isolate remnant-like particles (RLP) and measured them in terms of their cholesterol and triglycerides (TG). RLP consist of apob-48-containing triglyceride-rich lipoproteins and remnant-like VLDL containing apob-100. The study aim was to look for information from postprandial RLP data that could not be known from other markers of triglyceride-rich lipoproteins and fasting TG and RLP data alone. A total of 41 subjects were studied. Eight subjects had hypertriglyceridemia (HTG) and low high-density lipoprotein (HDL), 14 had combined hyperlipidemia (CH), 5 had the apo E2/2 genotype receiving gemfibrozil, 10 were normolipidemic (NL) controls, and 4 had hypercholesterolemia. As a whole group, there was correlation among 1) fasting TG, RLP cholesterol (RLP-C), and RLP-TG but not VLDL apo B100, VLDL apo B48 and their respective postprandial responses measured as incremental area under the curve (IAUC), 2) fasting TG and postprandial IAUC of RLP-C and RLP-TG, 3) RLP-C IAUC, RLP-TG IAUC, and TG IAUC, retinyl palmitate (RP) IAUC, and VLDL apo B48 IAUC but not VLDL apo B100 IAUC. The HTG/low HDL-C and CH groups had higher IAUC HYPERTRIGLYCERIDEMIA (HTG) INDICATES the presence of increased triglyceride-rich lipoproteins (TRLs) in plasma. TRLs, however, are heterogeneous, consisting of chylomicrons (CMs) and very low-density lipoproteins (VLDLs) and their remnants. Many studies indicate that remnants are atherogenic while CMs and VLDLs may not be (1 3). Because plasma triglyceride (TG) level is a nondiscriminatory marker of all TRLs, a more accurate assessment of the atherogenic potential of various HTG states may be obtained by measuring subpopulations of TRLs, especially remnants. TRLs are spread over a broad range of size and density, making it impossible to isolate TRL subpopulations by physical means alone. Many studies have simply measured intermediate density lipoprotein (IDL) concentrations Received May 2, Revision received February 5, Accepted March 23, Address all correspondence and requests for reprints to: Dr. T. C. Ooi, Division of Endocrinology and Metabolism, Ottawa Hospital Civic Campus 1053, Carling Avenue, Ottawa, Ontario, Canada K1Y 4E9. E- mail: tcooi@ottawahospital.on.ca. * Supported by a grant from Otsuka America Pharmaceutical Inc. (to T.C.O. and D.S.O.) and a grant from the Heart and Stroke Foundation of Ontario (to G.S.). for RLP-C, RLP-TG, TG, and RP than the NL group. Fasting and postprandial RLP were triglyceride enriched in the HTG/low HDL-C group and to a lesser extent in the CH group. The HTG/low HDL-C and CH groups had a delay in their RLP-C but not RLP-TG peaks suggesting a delay in hepatic clearance of RLP and/or a protracted period of lipolysis and/or processing of RLP. The fasting and postprandial RLP-C/RLP-TG and RLP-C/TG ratios were elevated in the apo E2/2 group in spite of gemfibrozil therapy. The increment in postprandial RLP was, however, not exaggerated. Our data indicate that 1) postprandial RLP lipemia is enhanced in HTG subjects when compared with NL subjects, 2) postprandial RLP lipemia is proportional to fasting RLP and TG levels and mirrors, to a large extent, increases in postprandial TG, RP, and VLDL apo B48 but not VLDL apo B100, 3) there are compositional differences in fasting and postprandial RLP in the three forms of HTG studied, RLP being triglyceride enriched in the HTG/low HDL-C group and to a lesser extent in the CH group, and cholesterol-enriched in the apo E2/2 group, and 4) apo E2/2 subjects had high fasting and postprandial RLP-C concentrations in spite of being on treatment with gemfibrozil and having normal fasting and postprandial TG concentrations. (J Clin Endocrinol Metab 86: , 2001) as a reflection of remnant lipoproteins, but it is clear that remnant lipoproteins are found within a much broader density range. The amount and origin of remnants in fasting and postprandial states have also been examined by measurement of plasma TG (4), chylomicron labeling with retinyl palmitate (5, 6), and quantitation of apoprotein (apo) B-48 and apo B-100 in remnant-containing ultracentrifugal lipoprotein fractions (7 9). Each approach has limitations. In this study, we have employed an immunochemical method for isolation of lipoproteins that have been characterized as remnant-like particles (RLPs) (10). The method uses a monoclonal antibody to apo B (JI-H), which recognizes all apob-100-containing lipoproteins except for those that are apoe enriched. The method also uses an anti-apo A-I antibody, which recognizes high-density lipoprotein (HDL) and CMs containing apo A-I. Thus the unbound RLP contains a subpopulation of apo B-48-containing TRL and remnant-like VLDLs containing apo B-100. The physical, chemical, and receptor-binding properties of RLP have been shown to resemble those of VLDL and CM remnants (11). RLPs are measured in terms of their cholesterol and triglyceride content. 3134

2 POSTPRANDIAL RLP IN HYPERTRIGLYCERIDEMIA 3135 The plasma concentrations of RLP cholesterol (RLP-C) and RLP triglycerides (RLP-TG) have been measured in fasting plasma in several studies as summarized by Cohn et al. (12). Few studies have examined RLP-C and RLP-TG in the postprandial state and mostly in normolipidemic subjects (13 17). Only one study has been published recently with postprandial RLP levels in subjects with HTG (18). The study of RLP concentrations in the postprandial period is important because this is when remnants of intestinal and hepatic origin are formed and metabolized in greater amounts (8, 9). We have studied three groups of HTG subjects, those with HTG and low HDL cholesterol, those with combined hyperlipidemia, and those with the apo E 2/2 genotype and phenotype to address two questions: 1) What additional information can we get about HTG states when postprandial RLP data are obtained along with data of other markers of TRL, namely TG, retinyl palmitate (RP), apo B-100, and apo B-48; and 2) what new information about the HTG states can we get from postprandial RLP data that cannot be known from fasting RLP data alone? Subjects Subjects and Methods This study was approved by the institution s human research committee. We have studied a total of 41 subjects. Twenty-seven of them had one of three HTG states, four had hypercholesterolemia (HC) alone, and the rest were normolipidemic controls (NL, n 10). In view of the small number of HC subjects, their data were included only in analyses of postprandial data involving all 41 subjects as a whole. Subjects were grouped according to fasting plasma lipid levels with the following levels being considered normal: low-density lipoprotein cholesterol (LDL-C) 3.4 mmol/l, TG 2.0 mmol/l, and HDL cholesterol (HDL- C) 0.9 mmol/l. Of the 27 HTG subjects, 14 had elevation of LDL-C and TG [combined hyperlipidemia (CH)], 8 had HTG and low HDL-C but normal LDL-C, and 5 had the apo E2/2 genotype and phenotype. To avoid severe hyperlipidemia after an oral fat load, the apo E2/2 subjects were studied while on gemfibrozil therapy, 600 mg twice daily. On therapy, they had normal fasting plasma LDL-C, TG, and HDL-C, but before gemfibrozil therapy they all had marked HC and HTG. There was no significant difference in apo E allele distribution among all the non-apo E 2/2 groups (P 0.064). Waterworth et al. (19) have recently shown that mean fasting RLP-C and RLP-TG levels in subjects who carried at least one 3 allele were similar to those of the E3/3 group, unlike the E4/4 and E4/2 groups that had higher levels. Of our 32 non-apo E2/2 subjects (and excluding the four HC subjects), only one in the CH group had an apo E4/2 genotype (Table 1), indicating that differences in apo E genotype were unlikely to have contributed to differences in our RLP data. None had diabetes mellitus or hepatobiliary, renal, or pancreatic disease, and apart from the apo E2/2 subjects, none were taking lipidlowering medication for at least 6 weeks before the study. Oral fat tolerance test The vitamin A fat load was given at 0800 h after a 12-h fast and a 36-h abstention from alcohol. We used a fat load formula that was previously reported by us (20). The test drink consisted of 350 ml whipping cream (35% fat), two tablespoons of chocolate-flavored syrup, one tablespoon of granulated sugar, and one tablespoon of instant nonfat dry milk. This volume contained 1,298 kcal, of which 5% were from protein, 26% from carbohydrate, and 69% from fat. It had 453 mg cholesterol and a polyunsaturated/saturated ratio of Each study subject received 350 ml of the mixture per 2 m 2 of body surface area. Vitamin A (Aquasol A, Rorer Canada Inc., St. Laurent, QC), 60,000 U/m 2 of body surface area, was added. The fat load mixture was consumed within 10 min. Blood was drawn before and 2, 4, 6, 8, 10, and 12 h after the fat load. The study subjects consumed only plain water during the study period. Sample handling Blood was collected in EDTA-containing tubes. Plasma was obtained by centrifugation at 2,500 rpm for 12 min at 4 C. All samples for RP were covered with aluminum foil and handled in subdued light. Plasma was ultracentrifuged in a 50Ti fixed-angle rotor (Beckman Coulter, Inc., Mississauga, Canada) at 20,000 rpm for 30 min at 22 C. CM particles in the top fraction were procured by tube slicing. The bottom fraction underwent further ultracentrifugation at d g/ml in a 50Ti fixed-angle rotor (Beckman Coulter, Inc.) at 40,000 rpm for 18 h at 10 C. TABLE 1. Clinical data and fasting plasma levels of study subjects Variable Normolipidemic (n 10) Combined hyperlipidemic (n 14) Hypertriglyceridemic low HDL-C (n 8) Apo E 2/2 (on gemfibrozil therapy) (n 5) Age (yr) Sex (M/F) 5/5 9/5 8/0 4/1 Body mass index (kg/m 2 ) 24 1 b,c 30 1 a 29 1 a 26 1 Cholesterol (mmol/l) b,c a,c,d a,b,d b,c CM Cholesterol (mmol/l) b,c a,c,d a,b,d b,c VLDL Cholesterol (mmol/l) b,c a,c a,b,d c IDI LDI HDL Cholesterol (mmol/l) b,c,d a,c,d a,b a,b TG (mmol/l) b,c a,c,d a,b,d b,c CM TG (mmol/l) b,c a,c,d a,b,d b,c VLDL TG (mmol/l) b,c a,c,d a,b,d b,c IDI LDL HDL TG (mmol/l) b,c,d a,d a a,b HDL-C (mmol/l) b,c a,c a,b,d c LDL-C (mmol/l) b a,c,d b b Apo E alleles ( 2/ 3/ 4[%]) e 25/70/5 4/71/25 0/88/12 100/0/0 Apo E genotype: E3/E3, E4/E3, E3/E2, 3/3 (n 4), 4/3 (n 1) 3/3 (n 7), 4/3 (n 6) 3/3 (n 6), 4/3 (n 2) 2/2 (n 5) E4/E2, E2/E2 3/2 (n 5) 4/2 (n 1) Data are expressed as mean SEM. Variables not normally distributed were normalized by natural logarithm transformation before between-group comparisons by unpaired t test. a Significantly different from normolipidemic group (P 0.05). b Significantly different from CH group (P 0.05). c Significantly different from HTG and low HDL-C group (P 0.05). d Significantly different from apo E 2/2 (gemfibrozil treated) group (P 0.05). e Significance by chi-square analysis of contingency table, excluding apo E 2/2 group.

3 3136 OOI ET AL. JCE&M 2001 Vol. 86 No. 7 The lipoprotein fractions VLDL (d g/ml) and IDL LDL HDL (d g/ml) fractions were isolated by tube slicing. Assay procedures RLP-C and RLP-TG were measured in plasma and lipoprotein fractions by the method of Nakajima et al. (10). RLPs were separated by mixing 5 L plasma with 300 L immunoseparation gel consisting of monoclonal antibodies to apo B-100 and apo A-I. After 2 h of incubation at room temperature, cholesterol and TGs in the unbound fraction were measured by sensitive cholesterol and TG assays on a Cobas Mira (Roche, Laval, Quebec). All RLP assays were done with samples stored at 4 C within 5 days of procurement, without previous freezing. RP in CM and non-cm fractions were measured by high-performance liquid chromatography as previously described (20). Using retinyl acetate as an internal standard, retinyl esters were extracted by a mixture of ethanol, hexane, and water in a 2:10:3 ratio. The hexane layer was removed, evaporated under nitrogen, and the residue dissolved in ethanol. The retinyl esters were separated by high-performance liquid chromatography using a reverse phase column (Varian Canada Inc., St. Laurent, QC) with a step gradient elution by the mobile phase of 100% methanol and 93% methanol at a flow rate of 1.5 ml/min. The effluent was monitored at 325 nm and the RP quantitated by the ratio of retinyl ester peak areas. Cholesterol and TG in plasma and lipoprotein fractions were measured enzymatically (Roche Molecular Biochemicals, Mannheim, Germany, kit no and , respectively) on a Cobas Mira analyzer (RocheDiagnostics, Canada, Laval, Quebec). Fasting HDL-C was measured in the supernate following precipitation of non-hdl with phosphotungstate on a BM/Hitachi 917 analyzer (Roche Diagnostics). Fasting LDL-C was calculated as the difference between the d fraction cholesterol and HDL-C. Apo E genotype was determined by PCR-restriction fragment length polymorphism using the published method of Tsukamoto et al. (21) with minor modifications. Briefly, DNA was extracted from EDTA-blood or buffy coat using the Wizard Genomic Pre DNA isolation kit (Promega Corp., Madison, WI), amplified between bases 2849 and 3071 using TSUK1 and TSUK2 primers (PE Applied Biosystems, Foster City, CA) and digested by restriction enzyme Hhal (Promega Corp.). The apo E phenotype of the five subjects with the apo E2/2 genotype was confirmed as 2/2 by isoelectric focusing. VLDL apo B-100 and apo B-48 concentrations were determined as previously described (22, 23). Briefly, the isolated VLDL was delipidated and run on 4 20% SDS-PAGE along with an LDL standard curve. This LDL standard (d kg/l) contained more than 97.5% of its protein in apo B-100. The electrophoresis was performed using a vertical Xcell II electrophoresis apparatus (Novex, San Diego, CA) connected to a power supply and run for h at 130 V. The gels were stained with Coomassie blue, destained, and scanned using a desktop densitometer supported by gel analysis software (Imagemaster Systems, Pharmacia Biotech). The integrated areas of the VLDL apo B-100 and apo B-48 were then compared with the standard curve and the concentrations determined. Statistical analysis All results are expressed as mean sem. Univariate analysis was used to test parameters for normality of distribution. Data that were not normally distributed were log transformed before the between-group comparisons were made using a two-tailed unpaired t test. The incremental area under the curve (IAUC) was calculated as the increased response above baseline minus any drop below baseline, based on the trapezoid rule. The between-group comparisons of the IAUC and the ratio data were analyzed by the Mann-Whitney U test. The apo E allele and gender distribution among the non-apo E2/2 subjects was analyzed by 2 test. Spearman correlation coefficients (r) were determined to assess the relationship between different parameters. Data were analyzed by using the Statistical Analysis System (version 6.12, SAS Institute, Inc., Cary, NC). Statistical significance was established at P Results Characteristics and basic fasting lipid concentrations There were 29 males and 12 females among the 41 subjects, ranging in age from 30 to 72 yr. Their fasting plasma lipid ranges were as follows: total cholesterol mmol/l, TG mmol/l, HDL-C mmol/l, LDL-C mmol/l, RLP-C mmol/l, and RLP-TG mmol/l. Table 1 shows the characteristics and fasting plasma lipid data of the HTG subgroups and NL controls, excluding the four HC subjects. There was no difference in age among the groups and body mass index was higher in the CH and HTG/low HDL-C groups than the NL control group. There was no significant difference in the distribution of males and females among all the non-apo E2/2 groups (P 0.121). Differences in plasma total cholesterol, TG, HDL-C, and LDL-C were as determined by preset criteria. The relative distribution of cholesterol and TG among lipoprotein classes is strikingly different for the HTG/low HDL-C group. Compared with all other groups, this group had more cholesterol and TG distributed in CM and VLDL (relative to the IDL LDL HDL fraction). This suggests defective lipolysis and/or processing of TRL resulting in accumulation of larger lipoprotein particles. The CH group showed a lipid distribution pattern that was intermediate between the NL controls and the HTG/low HDL-C group, suggesting that the CH phenotype is associated with a less severe lipolytic/processing defect than the HTG/low TABLE 2. Fasting RLP-C and RLP-TG levels and RLP-C/RLP-TG ratio of study subjects Variable Normolipidemic (n 10) Combined hyperlipidemic (n 14) Hypertriglyceridemic low HDL-C (n 8) Apo E 2/2 (on gemfibrozil therapy) (n 5) Plasma RLP-C (mmol/l) b,c,d a,c a,b a CM RLP-C (mmol/l) c c a,b,d c VLDL RLP-C (mmol/l) b,c,d a,c a,b,d a,c IDL LDL HDL RLP-C (mmol/l) d d d a,b,c Plasma RLP-TG (mmol/l) b,c a,c,d a,b,d b,c CM RLP-TG (mmol/l) b,c a,c,d a,b,d b,c VLDL RLP-TG (mmol/l) b,c a,c a,b,d c IDL LDL HDL RLP-TG (mmol/l) Plasma RLP-C/Plasma RLP-TG e b,c,d a,d a,d a,b,c Data are expressed as mean SEM. Symbols denote significance as described in Table 1. Variables were normalized by natural logarithm transformation before between-group comparisons by unpaired t test. CM and IDL LDL HDL are calculated results. RLP lipid analysis in the VLDL subfraction was not done in two normolipidemics, two CH, three HTG with low HDL-C, and two apo E2/2 subjects. e Ratio represents the mean of the ratios of individual subjects, between-group comparisons by Mann-Whitney U test.

4 POSTPRANDIAL RLP IN HYPERTRIGLYCERIDEMIA 3137 HDL-C phenotype. The lipid distribution pattern for the treated apo E2/2 group was similar to that of NL controls. Fasting RLP cholesterol and RLP triglyceride concentrations (Table 2) The HTG/low HDL-C group showed marked elevation of fasting plasma RLP-C and RLP-TG concentrations. Because the RLPs were mostly in the lower density fractions (CM and VLDL) and because none in this group carried the 2 allele, the accumulation of RLP was more likely due to defective lipolysis/processing of TRL than defective hepatic clearance, paralleling the whole plasma data in Table 1. RLP-C and RLP-TG concentrations in the CH group were higher than in NL group but lower than in the HTG/low HDL-C group. In spite of gemfibrozil therapy and a normal fasting plasma TG concentration, the apo E2/2 group had a higher plasma RLP-C but not RLP-TG than the NL group. The fasting plasma RLP-C/RLP-TG mean ratio was highest in the apo E2/2 group (2.42 vs in NL) indicating that the RLPs that accumulate in apo E2/2 subjects were cholesterol enriched, despite gemfibrozil therapy and normal fasting plasma cholesterol and TG levels. This ratio in subjects with CH and HTG/low HDL-C were 0.50 and 0.44, respectively, indicating that RLPs in these groups were TG enriched, consistent with defective hydrolysis/processing of TRL. Fasting VLDL apo B100 and apo B48 (Table 3) Apo B100 and apo B48 were measured in VLDL and not whole plasma because VLDL is more representative of the TRL population, and LDL apo B, which forms the major component of total apo B, is then not measured. The HTG/low HDL-C group showed much higher fasting VLDL apo B100 and apo B48 levels than the NL controls. Because VLDL, CM, and their remnants contain one molecule of either apo B100 (24) or apo B48 (25) per particle, the VLDL apo B100 and apo B48 concentrations reflect the number of VLDL/VLDL remnant particles and CM remnant particles, respectively. Thus, the HTG/low HDL-C group had increased numbers of VLDL and VLDL/CM remnants. The lower apo B100/apo B48 ratio indicates a lower proportion of hepatic to intestinal particles in this group. The CH group showed similar trends as the HTG/low HDL-C group but to a lesser extent. Apo B100 and apo B48 levels were significantly higher than in the NL controls, indicating an increase in the number of VLDL and CM/ VLDL remnants. However, the apo B100/apo B48 ratio is not different from that in NL controls, indicating that hepatic and intestinal particles are proportionally increased. The treated apo E2/2 group showed no difference from NL controls in apo B100 and apo B48 but a lower apo B100/apo B48 ratio, indicating a lower proportion of hepatic to intestinal particles. Postprandial plasma TG, RLP-C, RLP-TG, RP, apo B100, and apo B48 We have examined postprandial responses by calculating the IAUC. We first examined all 41 subjects as a group. A positive correlation was found between fasting plasma TG and postprandial TG IAUC (r 0.67, P ), fasting plasma RLP-C and postprandial RLP-C IAUC (r 0.35, P ), and fasting plasma RLP-TG and postprandial RLP-TG IAUC (r 0.63, P ). There was no significant correlation between fasting VLDL apo B100 and postprandial VLDL apo B100 IAUC, and between fasting VLDL apo B48 and postprandial VLDL apo B48 IAUC. An important finding was a strong relationship between fasting TG concentrations and postprandial RLP-C IAUC (r 0.59, P ) and RLP-TG IAUC (r 0.77, P ) (Fig. 1). There was no correlation between fasting LDL-C and postprandial RLP-C IAUC and RLP-TG IAUC. On the other hand, there was an inverse relationship between fasting HDL-C and postprandial RLP-C IAUC (r 0.50, P ) and RLP-TG IAUC (r 0.66, P ). Table 4 shows the positive correlation among postprandial IAUC of various parameters, including TG, RLP-C, RLP-TG, RP, VLDL apo B100, and VLDL apo B48. Only the IAUC of apo B100 did not show positive correlation with the IAUC of RLP-C, RLP-TG, and RP. The postprandial responses and calculated IAUC for the HTG subgroups and NL controls are shown in Figure 2. The NL controls had their TG, RLP-C, and RLP-TG peaks at or before 6 h after the fat load, and RP seemed to peak after the 6-h mark. The HTG/low HDL-C and CH groups, however, had their RLP-C peaks at 8 h after the fat load and their RLP-TG peaks were at 6 h, coinciding with the TG peaks. The RP peaks for these two groups were also delayed to 8 h after the fat load, but it should be noted that the RP peak for the NL and apo E2/2 groups were also delayed, coinciding with the RP peaks of the HTG/low HDL and CH groups. The postprandial response curves for VLDL apo B100 and VLDL apo B48 were not as pronounced, but they seemed to peak at or before 6 h after the fat load in all groups. The HTG/low HDL-C and CH groups had significantly higher IAUC for TG, RLP-C, RLP-TG, and RP than the NL TABLE 3. Fasting VLDL apo B100 and apo B48 levels and apo B100/apo B48 ratio of study subjects Variable Normolipidemic (n 10) Combined hyperlipidemic (n 14) Hypertriglyceridemic low HDL-C (n 8) Apo E 2/2 (on gemfibrozil therapy) (n 5) Apo B100 (mg/l) b,c a,c,d a,b,d b,c Apo B48 (mg/l) b,c a,c a,b,d c Apo B100/apo B48 e c c a,b Data are expressed as mean SEM. Symbols denote significance as described in Table 1. Variables were normalized by natural logarithm transformation before between-group comparisons by unpaired t test. e Ratio represents the mean of the ratios of individual subjects, between-group comparisons by Mann-Whitney U test.

5 3138 OOI ET AL. JCE&M 2001 Vol. 86 No. 7 The postprandial changes in RLP-C/RLP-TG ratios are shown in panel A of Fig. 3. The treated apo E2/2 group showed the highest fasting and postprandial ratios. These results are consistent with cholesterol enrichment of RLP in the treated apo E2/2 group. The fasting and postprandial ratios in the HTG/low HDL-C and CH groups were slightly lower than in NL controls. The postprandial change in RLP- C/plasma TG ratio is shown in panel B of Fig. 3. The fasting and postprandial ratios were also markedly elevated in the apo E2/2 group but not in the other two HTG groups when compared with NL controls. FIG. 1. Relationship between the fasting plasma TG levels and the IAUC of RLP-C and RLP-TG. The symbol ( ) represents the 10 normolipidemic, 14 CH, 8 HTG with HDL-C, and 4 HC subjects. The symbol ( ) represents the five apo E2/2 genotype (gemfibrozil treated) subjects. The regression line drawn represents the data of the subjects (n 41). group (Fig. 2). The mean IAUC for these parameters were higher in the HTG/low HDL-C group than CH group although the difference did not reach statistical significance. VLDL apo B48 IAUC but not VLDL apo B100 IAUC in the CH group was significantly greater than in the NL control group, but in the HTG/low HDL-C group, both were not significantly higher than in the NL control group. In the treated apo E2/2 group, only the RP IAUC was higher than in the NL group, and only the TG IAUC and RLP-TG IAUC were significantly lower than in the HTG/low HDL-C and CH groups. To determine the relationship between postprandial responses and fasting concentrations, we have calculated the ratio between postprandial IAUC and fasting concentrations for each of the following parameters: TG, RLP-C, RLP-TG, VLDL apo B100, and VLDL apo B48. Uniformly, all the postprandial IAUC/fasting ratios of each parameter in the three HTG groups were not significantly different among all three HTG groups and the NL control group. The only exception was the ratio of RLP-C IAUC to fasting RLP-C in the CH group, which was significantly greater than in the NL group and the other two groups. There was no significant difference in the postprandial RLP-C IAUC/fasting TG and RLP-TG IAUC/fasting TG ratios among all three HTG groups and the NL control group. Discussion The study of TRL remnants has been challenging partly because of difficulties associated with their measurement. Methods used are either nonspecific or technically tedious (12). The immunochemical method used in this study to isolate TRL remnants is an additional tool that has the potential of routine use in a clinical laboratory. It has been well characterized over the past few years (10, 11). This study provides one of the first sets of postprandial RLP data in subjects with HTG. The only other report was published very recently (18). In the few studies that have been reported on postprandial RLP in normolipidemic subjects, the major finding was of increased concentrations of postprandial RLP-C in subjects with coronary artery disease (13 17). The overall objective of our study was to examine postprandial RLP data to look for insights into the pathophysiology of three different forms of HTG. In our whole group analysis (n 41), postprandial RLP-C and RLP-TG responses expressed as IAUC were related to their own fasting levels, consistent with the findings of Marcoux et al. (18), who demonstrated a significant correlation between postprandial RLP levels and fasting RLP levels. In our study, postprandial TG IAUC also correlated with fasting TG but VLDL apo B100 IAUC and VLDL apo B48 IAUC did not correlate with their own fasting levels. These results indicate that fasting lipid levels are better predictors of postprandial lipid responses than fasting VLDL apo B levels are of postprandial responses of lipoprotein particle number. We have also shown that postprandial RLP responses were significantly correlated with fasting TG levels. Because fasting TG is known to be an important determinant of postprandial TG response, it is not surprising that a significant correlation was found between postprandial RLP response and postprandial response of TG as well as of two other markers of TRL, namely, RP and VLDL apo B48. As in the study of Marcoux et al. (18), a large portion of the postprandial increase in total TG was accounted for by an increase in RLP-TG (Fig. 2). These results together indicate that the postprandial RLP response contributes significantly to the overall TRL postprandial response, especially TRL of intestinal origin. VLDL apo B100 IAUC, unlike VLDL apo B48 IAUC, did not correlate with the IAUC of RLP-C and RLP-TG, indicating that postprandial hepatic VLDL production did not parallel postprandial remnant lipid responses, whereas intestinal lipoprotein production did. We speculate that this may be due to the variable delivery of remnant lipids to the liver (with the apo E2/2 and HTG/low HDL-C groups having

6 POSTPRANDIAL RLP IN HYPERTRIGLYCERIDEMIA 3139 TABLE 4. Relationship between postprandial plasma lipids, plasma RP and VLDL apo B IAUC TG IAUC RLP-C IAUC RLP-TG IAUC RP IAUC Apo B100 IAUC RLP-C 0.88 a IAUC RLP-TG 0.97 b 0.85 b IAUC RP 0.75 b 0.75 b 0.78 b IAUC apo B c IAUC apo B a 0.53 b 0.41 a 0.36 c 0.61 b Values represent Spearman correlation coefficients (r). Data represent all groups combined (n 41) except for VLDL apo B100, and VLDL apo B48 (n 39). Statistical significance indicated by a P 0.01, b P 0.001, c P impaired delivery, compared with the NL control and CH groups) resulting in variable assembly of VLDL. On the other hand, the ability of the intestine to produce apo B48 and to load it with lipids to form CM does not seem to be impaired or variable in the group of 41 subjects as a whole. In the HTG subgroup analysis, a striking feature in the postprandial RLP responses in the HTG/low HDL-C and CH groups was the timing of the peaks (Fig. 2). We speculate that the delay in their RLP-C but not RLP-TG peaks may be due to an impairment of receptor-mediated hepatic uptake of RLP allowing more time for further compositional changes to occur. An alternative speculation is that there is protracted lipolysis/processing of RLP (TG depletion and cholesterol enrichment) before their clearance by the liver. In other words, a longer time was required for RLP to reach a compositional state that was suitable for hepatic clearance. In fact, this second speculation is more in keeping with other data in this study showing predominance of larger and less dense RLP especially in the HTG/low HDL-C group but also, to a lesser extent, in the CH group when compared with the NL group. These data include higher proportions of RLP-C and RLP-TG in the CM and VLDL fractions than IDL LDL HDL fraction in the fasting samples (Table 2), more RP in the CM fraction than non-cm fraction (data not shown), and lower fasting and some postprandial RLP-C/RLP-TG ratios than in the NL group (Fig. 3). Our data do not provide a clear conclusion on which mechanism is the dominant one. RP peaks were also delayed when compared with plasma TG peaks, but unlike RLP-C peaks, the delay involved all four groups, including the NL group. The mechanisms responsible for the delay in RP peaks are not clear, but our data are concordant with those of several studies (6, 26, 27). Lemieux et al. (26) have suggested that a possible explanation for the delay in RP peaks relative to TG peaks is that apo B48-containing TRLs are metabolically heterogeneous and that the older TRLs already present in circulation before the fat load might be preferentially removed over the newer postload TRLs, which contain the RP. It is not clear whether the mechanisms causing the delay in RP peaks (relative to TG peaks) in the HTG/low HDL-C and CH groups are the same as those causing delay in the RLP-C peaks. Because the delay in RP peaks involved the NL and apoe2/2 groups whereas the delay in RLP-C peaks did not involve these two groups, it is likely that the mechanisms for the delay in RP are different from those involved in the delay of the RLP-C peaks. The postprandial RLP-C and RLP-TG IAUC shown in Fig. 2 were in themselves unremarkable in that they reflected the responses of postprandial TG and RP. The mean IAUC for these parameters were higher in the HTG/low HDL-C group than CH group, but the differences did not reach statistical significance. To study the extent to which postprandial IAUC of any parameter was determined by the fasting level of that parameter, we calculated the ratio between the postprandial IAUC and fasting levels of TG, RLP-C, RLP-TG, VLDL apo B100, and VLDL apo B48. Interestingly, this revealed that the ratios in the three HTG groups were uniformly not different from those in the NL control group and among themselves indicating that postprandial responses were proportional to their different fasting levels. The exception to this rule was a higher ratio of RLP-C IAUC to fasting RLP-C in the CH group, compared with the NL control group and the CH and apo E2/2 groups. Even so, the RLP-C IAUC to fasting TG ratio was not different from that in all other groups. This finding suggests that postprandial CM and VLDL in the CH group were more readily converted to cholesterol-enriched RLP. The reason for this is unclear. We speculate that in the postprandial period, there is a high output of hepatic and intestinal TRL in the CH group resulting in a disproportionately greater production of cholesterol-enriched RLP. A similar phenomenon was not seen in the HTG/low HDL-C group because of an added problem with lipolysis/processing of TRL. Apart from this exception, the general trend was that postprandial response was proportional to the fasting level. We studied the apo E2/2 group while they were on gemfibrozil therapy because we were concerned with the possibility of excessive postprandial lipemia. Thus, our findings in this group reflect gemfibrozil therapy. Although several studies have already demonstrated elevated RLP-C levels in these patients in the fasting state (18, 28 31), none have provided fasting and postprandial RLP data during therapy with any lipid-altering drug. Our on-treatment data contain a very important observation that has not been reported before. It is that in spite of achievement of normal fasting and postprandial plasma TG levels on fibrate therapy, fasting and postprandial plasma RLP-C levels in these subjects were elevated. In addition, postprandial RP levels were also elevated. Thus, although plasma fasting and postprandial TG levels do not reveal any abnormality, it is evident that there is significant accumulation of RLP, primarily of intestinal origin, even when they are gemfibrozil treated and normotriglyceridemic. In fact, their mean fasting and postprandial (except at 4 h) RLP-C/TG ratio was above 0.23, a cutoff ratio that has been shown to be useful for discriminating patients with familial dysbetalipoproteinemia (31). Additionally, it is clear that the RLPs that accumulate are enriched in cholesterol as indicated by the high fasting and postpran-

7 3140 OOI ET AL. JCE&M 2001 Vol. 86 No. 7 FIG. 2. Line plots of mean SEM postprandial response and bar graphs of mean SEM IAUC of plasma triglycerides, plasma RLP-C, plasma RLP-TG, plasma RP, VLDL apo B100, and VLDL apo B48 following ingestion of a fat load in 10 normolipidemic, 14 CH, 8 HTG with low HDL-C, and 5 apo E/2/ genotype (gemfibrozil treated) subjects. Different letters indicate a significant difference between groups (P 0.05) as described in Table 1.

8 POSTPRANDIAL RLP IN HYPERTRIGLYCERIDEMIA 3141 dial RLP-C to RLP-TG ratios (Table 2 and Fig. 3), and the RLPs are found mostly in the denser lipoprotein fractions (Table 2). These findings are consistent with the known defect in this condition of impaired uptake of TRL remnants (32). This condition is clearly associated with an increased risk of atherosclerosis, and our study highlights the value of measuring fasting and postprandial RLP-C in such patients even in the fibrate-treated state. Although the absolute postprandial RLP-C levels in the apo E2/2 group were high, the postprandial increase was not exaggerated, relative to the fasting level. This again could be due to gemfibrozil therapy, but it may be an inherent feature of the apo E2/2 state because a similar observation has been made in such subjects who are not on any therapy (18). Because the apo E2/2 state is associated with impaired hepatic remnant clearance, the lack of a greater degree of postprandial accumulation of RLP (and TG) was unexpected. One possible explanation for this may be a relatively low postprandial formation of RLP from hepatic VLDL, whose production is diminished by a decrease in delivery of postprandial remnants to the liver because of the apo E2/2 state. On the other hand, there was enhanced formation of remnants from CM as reflected by the exaggerated postprandial RP response shown in Fig. 2. A decrease in hepatic and an increase in intestinal TRL production in the postprandial period are reflected in the low fasting (shown in Table 3) and postprandial (data not shown) VLDL apo B100/apo B48 ratios. Because the liver contributes a larger number of postprandial RLPs than the intestine (as reflected in the absolute fasting and postprandial VLDL apo B100 and apo B48 levels, respectively, shown in Table 3 and Fig. 2), a decrease in the liver s contribution has a significant impact on postprandial RLP response. To summarize, these are one of the first sets of postprandial RLP data in subjects with HTG. Our data indicate that 1) postprandial RLP lipemia is enhanced in HTG subjects when compared with NL subjects, 2) postprandial RLP lipemia is largely proportional to fasting RLP and TG levels and mirrors, to a large extent, increases in postprandial TG, RP, and VLDL apo B48 but not VLDL apo B100, 3) there are compositional differences in fasting and postprandial RLPs in the three forms of HTG studied, RLP being triglycerideenriched in the HTG/low HDL-C group and to a lesser extent in the CH group and cholesterol enriched in the apo E2/2 group, and 4) apo E2/2 subjects had high fasting and postprandial RLP-C concentrations in spite of being on treatment with gemfibrozil and having normal fasting and postprandial TG concentrations. Acknowledgments We thank Drs. R. Havel and Tao Wang for helpful comments. We are grateful to Ms. Ann Port and Ms. Colette Favreau for expert nursing assistance and careful coordination of the study. FIG. 3. Line plots of mean SEM plasma RLP-C/plasma RLP-TG ratio and plasma RLP-C/plasma TG ratio following ingestion of a fat load in 10 normolipidemic, 14 CH, 8 HTG with low HDL-C, and 5 apo E/2/ genotype (gemfibrozil treated) subjects. Different letters indicate a significant difference between groups (P 0.05) as described in Table 1. References 1. Schaefer EJ, Gregg RE, Ghiselli G, Forte TM, Ordovas JM Familial apolipoprotein E deficiency. J Clin Invest. 78: Havel R Triglyceride-rich lipoproteins and atherosclerosis new perspectives. Am J Clin Nutr. 59: Gianturco SH, Bradley WA Triglyceride-rich lipoproteins and their role in atherogenesis. Curr Opin Lipid. 2: Patsch JR, Karlin JB, Scott LW, Smith LC, Gotto AM Inverse relationship between blood levels of high density lipoprotein subfraction 2 and magnitude of postprandial lipemia. Proc Natl Acad Sci USA. 80: Weintraub MS, Eisenberg S, Breslow JL Different patterns of postprandial lipoprotein metabolism in normal, type IIa, type III, and type IV hyperlipoproteinemic individuals. J Clin Invest. 79: Krasinski SD, Cohn JS, Russell RM, Schaefer EJ Postprandial plasma vitamin A metabolism in humans: a reassessment of the use of plasma retinyl esters as markers for intestinally derived chylomicrons and their remnants. Metabolism. 39: Karpe F, Steiner G, Olivecrona T, Carlson LA, Hamsten A Metabolism of triglyceride-rich lipoproteins during alimentary lipemia. J Clin Invest. 91: Cohn JS, Johnson EJ, Millar JS, et al Contribution of apo B-48 and apo B-100 triglyceride-rich lipoproteins (TRL) to postprandial increases in the plasma concentration of TRL triglycerides and retinyl esters. J Lipid Res. 34: Schneeman BO, Kotite L, Todd KM, Havel RJ Relationships between the responses of triglyceride-rich lipoproteins in blood plasma containing apolipoproteins B-48 and B-100 to a fat-containing meal in normolipidemic Humans. Proc Natl Acad Sci USA. 90: Nakajima K, Okazaki M, Tanaka A, et al Separation and determination

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