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Original Articles |
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 20850
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{at}ottawahospital.on.ca
Abstract
It has been proposed that remnants of chylomicrons and very-low-density 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 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.
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, 2, 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 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, 8, 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.
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, 14, 15, 16, 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 and Methods
Subjects
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.
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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 0.06. Each study subject received 350 mL of the mixture per 2 m2 of body surface area. Vitamin A (Aquasol A, Rorer Canada Inc., St. Laurent, QC), 60,000 U/m2 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 = 1.006 g/mL in a 50Ti fixed-angle rotor (Beckman Coulter, Inc.) at 40,000 rpm for 18 h at 10 C. The lipoprotein fractions VLDL (d < 1.006 g/mL) and IDL + LDL + HDL (d > 1.006 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 µL plasma with 300 µL 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. 236691 and 701904, 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 > 1.006 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 1.035–1.050 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 1.5–2.0 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 < 0.05.
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 3.09–6.95 mmol/L, TG 0.62–6.78 mmol/L, HDL-C 0.65–1.74 mmol/L, LDL-C 1.19–5.02 mmol/L, RLP-C 0.12–1.01 mmol/L, and RLP-TG 0.06–2.18 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 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.
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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.
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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 < 0.0001), fasting plasma RLP-C and
postprandial RLP-C IAUC (r = 0.35, P < 0.0257),
and fasting plasma RLP-TG and postprandial RLP-TG IAUC (r = 0.63,
P < 0.0001). 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 < 0.0001) and RLP-TG IAUC (r = 0.77,
P < 0.0001) (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 < 0.0008) and RLP-TG IAUC (r =
-0.66, P < 0.0001).
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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.
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.
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.
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). 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.
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.
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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, 14, 15, 16, 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 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, 29, 30, 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 postprandial 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 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.
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.
Footnotes
1 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.). 
Received May 2, 2000.
Revised February 5, 2001.
Accepted March 23, 2001.
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) 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).
