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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1765-1773

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1765-1773.)
© 1997 American Heart Association, Inc.

Articles

Dietary Fat Clearance in Normal Subjects Is Modulated by Genetic Variation at the Apolipoprotein B Gene Locus

J. Lopez-Miranda; J.M. Ordovas; M.A. Ostos; C. Marin; S. Jansen; J. Salas; A. Blanco-Molina; J.A. Jimenez-Pereperez; F. Lopez-Segura; ; F. Perez-Jimenez

From the Unidad de Lipidos y Arteriosclerosis, Hospital Universitario Reina Sofia, Cordoba, Spain (J.L.-M., M.A.O., C.M., S.J., J.S., A.B.-M., J.A.J.-P., F.L.-S., F.P.-J.), and the Lipid Metabolism Laboratory, USDA Human Nutrition Research Center on Aging, Tufts University, Boston (J.M.O.).

Correspondence and reprint request to Prof. Francisco Perez Jimenez, Unidad de Lipidos y Arteriosclerosis, Hospital Universitario Reina Sofia, Avda. Menendez Pidal, s/n. 14004 Cordoba, Spain.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract Apolipoprotein B (apo B) plays a dominant role in cholesterol homeostasis. Several polymorphic sites within or adjacent to the gene locus for apo B have been detected. The X+ allele (XbaI restriction site present) of the XbaI restriction fragment polymorphism on the apo B gene has been found in some studies to be associated with higher serum cholesterol and/or triglyceride levels and with greater dietary response. The present study was designed to evaluate whether the apo B XbaI polymorphism was associated with the interindividual variability observed during postprandial lipemia. Fifty-one healthy young male volunteers [20 X-/X- (X-), and 31 X+/X- or X+/X+ (X+)], homozygotes for the apo E3 allele, were subjected to a vitamin A-fat load test. Subjects with the X- genotype had significantly greater retinyl palmitate (RP) and apo B-48 postprandial responses on both the large and the small TRL lipoprotein fractions compared with X+ subjects. In summary, subjects with the X-/X- genotype at the apo B locus have a greater postprandial response than X+ subjects. These differences observed in postprandial lipoprotein metabolism could explain some of the reported associations of this polymorphism to coronary heart disease risk.


Key Words: postprandial lipemia • apolipoprotein B • XbaI polymorphism • triglycerides • retinyl palmitate


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Elevated fasting levels of LDL cholesterol and reduced levels of HDL cholesterol (HDL-C) are risk factors for coronary artery disease (CAD), the major cause of death and disability in most industrialized countries.1 2 Subjects in these societies, by eating regular fat-rich meals, are predominantly in a postprandial state throughout the day. In these subjects, the fed state and its effects on lipoprotein metabolism, may be more representative of their physiological status than the fasting state. Since 1979 when Zilversmit proposed the important role of triglyceride-rich lipoproteins (TRL) in the development of atherosclerosis,3 a considerable amount of knowledge on postprandial lipemia has been accumulated and some fasting dyslipidemic conditions, as well as myocardial infarction, have been associated with abnormal postprandial lipoprotein patterns.4 5 6 7 8 9 10 11 12 13 The basic mechanisms involved during alimentary lipemia are relatively well known and the effects of different nutrients on the variability of the postprandial response are under active investigation.14 15 16 17 18 19 20 21 22 23 24 25 26 Less is known, however, regarding the dramatic interindividual variability observed during the postprandial lipemia. Some evidence suggests that genetic variability at the apo E gene locus might affect cholesterol absorption and the postprandial lipemic response.27 28 29 30 31 32 Recent studies indicate that several other gene loci may also be involved in determining this variability.

Apolipoprotein (apo) B plays a dominant role in cholesterol homeostasis. It is required for the assembly and secretion of chylomicrons in intestine and VLDL in liver, and it also acts as the ligand for the recognition of LDL by the LDL receptor. The apo B gene extends over 43 Kb and resides in chromosome 2.33 34 35 36 37 Several polymorphic sites within or adjacent to the gene loci for apo B have been detected, and numerous reports have associated some of these polymorphisms with abnormal lipid levels and/or increased CAD risk.38 39 40 41 42 43 44 Specifically, the X+ allele of the XbaI restriction fragment polymorphism of the apo B gene has been found associated with elevated serum cholesterol and/or triglyceride (TG) levels in several adult populations.39 40 41 45 46 This polymorphism involves the third base of the threonine codon 2488 (ACC->ACT) and, although no amino acid change results from the DNA change creating the XbaI restriction site,47 this polymorphism is strongly associated with the presence of certain Ag epitopes on the LDL particle.48 49 Turnover studies have demonstrated that the presence of the X+ allele is associated with a slower clearance of LDL compared with the presence of the X- allele, suggesting that an alteration in apo B could reduce its binding to the LDL receptor in these subjects50 51 ; however, in a recent study this effect was not found significant.52 The previous hypothesis receives support from an in vitro study showing that LDL from X-/X- subjects was degraded more rapidly by cultured human fibroblasts than LDL from X+/X+ subjects.52 Furthermore, Tikkanen et al53 showed that subjects bearing the X+ allele had a significantly greater LDL-C increase following a high-fat diet than those subjects homozygous for the X- allele. The present study was therefore designed to evaluate whether the apo B XbaI polymorphism could explain some of the interindividual variability observed in postprandial lipoprotein metabolism in subjects homozygous for the apo E3 allele.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Human Subjects
Sixty subjects volunteered to participate in this study. Fifty-one healthy male subjects, 20 X-/X- (X-) and 31 X-X+ or X+X+ (X+), were selected after excluding those who were not homozygotes for the apo E3 allele. They ranged in age from 18 to 49 years. None had diabetes or liver, renal, or thyroid disease. They were not taking medication or vitamins known to affect plasma lipids. Their fasting plasma lipids, lipoproteins, apolipoproteins, age, and body mass index (BMI) are shown in Table 1Down. All studies were carried out in the Research Unit of the Reina Sofia University Hospital. The experimental protocol was approved by the Human Investigation Review Committee of the Reina Sofia University Hospital.


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  		Table 1. Baseline Characteristics of Study Subjects according to Genotype

Vitamin A Fat-Loading Test
After a 12-hour fast, subjects were given a fatty meal enriched with 60 000 U/m2 of body surface area of vitamin A. The fatty meal consisted of 2 cups of whole milk, eggs, bread, bacon, cream, walnuts, and butter. The amounts given were 1 g of fat and 7 mg of cholesterol per kilogram of body weight. The meal contained 65% of calories as fat, 15% as protein, and 25% as carbohydrates and was eaten in 20 minutes. After the meal the subjects ate no calories for 11 hours but were allowed to drink water. Blood samples were drawn before the meal, every hour until the sixth hour, and every 2 hours and 30 minutes until the 11th hour.

Lipoprotein Separations
Blood was collected in tubes containing EDTA to give a final concentration of 0.1% EDTA. Plasma was separated from red cells by centrifugation at 1500g for 15 minutes at 4°C. The chylomicron fraction of TRL (large TRL) was isolated from 4 mL of plasma overlayered with 0.15 mol/L NaCl, 1 mmol/L EDTA (pH 7.4, d 1.006 g/mL) by a single ultracentrifugal spin (20 000 rpm, 30 minutes, 4°C) in a 50-type rotor (Beckman Instruments). Chylomicrons contained in the top layer were removed by aspiration after cutting the tubes and the infranatant was centrifuged at a density of 1.019 g/mL for 24 hours at 45 000 rpm in the same rotor. The nonchylomicron fraction of TRL (also referred as small TRL) was removed from the top of the tube. All operations were done in subdued light. Large and small TRL fractions were stored at -70°C until assayed for retinyl palmitate (RP).

Lipid Analysis
Cholesterol and triglycerides in plasma and lipoprotein fractions were assayed by enzymatic procedures.54 55 Apo A-I and apo B were determined by turbidimetry.56 HDL cholesterol (HDL-C) was measured by analyzing the supernatant obtained following precipitation of a plasma aliquot with dextran sulphate-Mg2+, as described by Warnick et al.57 LDL cholesterol (LDL-C) was obtained as the difference between the HDL-C and the cholesterol from the bottom part of the tube after ultracentrifugation at 1.019 g/mL.

Retinyl Palmitate Assay
The retinyl palmitate (RP) content of large and small TRL fractions was assayed using a method previously described.58 Briefly, different volumes of the various fractions (100 µL for chylomicrons and 100-500 µL for remnant) were placed in 13x100-mm glass tubes. The total volume in each tube was adjusted, as necessary, to 500 µL using normal saline. Retinyl acetate (40 ng in 200 µL of mobile phase buffer) was added to each tube as internal standard. Methanol (500 µL) was added, followed by the addition of 500 µL of the mobile phase buffer for a total volume of 1.7 mL. The mobile phase buffer was prepared fresh on a daily basis by combining 90 mL of hexane, 15 mL of n-butyl chloride, 5 mL of acetonitrile, and 0.01 mL of acetic acid (82:13:5 by volume with 0.01 mL of acetic acid). The tubes were thoroughly mixed after each step. The final mixture was centrifuged at 350g for 15 minutes (at room temperature) and the upper layer was carefully removed by aspiration and placed into individual autosampler vials. The autoinjector was programmed to deliver 100 µL per injection and a new sample every 10 minutes in a custom prepacked silica column SupelcoSil LC-SI (5 µm, 25 cmxmm intradermal) provided by Supelco, Inc. The flow was maintained at a constant rate of 2 mL/min and the peaks were detected at 330 nm. The peak of RP and retinyl acetate was identified by comparing its retention time with a purified standard (Sigma) and the RP concentration in each sample was expressed as the ratio of the area under the RP peak to the area under the RA peak.59 Here, too, all operations were performed in subdued light.

Determination Of Apo B-48 and Apo B-100
Apo B-48 and apo B-100 were determined by SDS-Polyacrylamide gel electrophoresis as described by Karpe and Hamsten.60 In summary, samples containing isolated lipoprotein fractions were delipidated in a methanol/diethyl ether solvent system and the protein pellet was dissolved in 100 to 500 µL of 0.15 mol/L of sodium phosphate, 12.5% glycerol, 2% sodium dodecyl sulfate (SDS), 5% mercaptoethanol, and 0.001% bromophenol blue, pH 6.8, at room temperature for 30 minutes followed by denaturation at 80°C for 10 minutes. Electrophoresis was performed with a vertical Hoefer Mighty Small II electrophoresis apparatus connected to an EPS 400/500 (Pharmacia) power supply on 3 to 20% gradient polyacrylamide gels. The upper and lower electrophoresis buffers contained 25 mmol/L Tris, 192 mmol/L glycine, and 0.2% SDS adjusted to pH 8.5. Apo B-100 derived from LDL was used as a reference protein and for standard curve dilutions. A dilution curve ranging from 0.10 to 2 µg of apo B-100 was applied to four of the gel lanes. Electrophoresis was run at 60 V for the first 20 minutes and then at 100 V for 2 hours. Gels were fixed in 12% trichloroacetic acid for at least 30 minutes and stained in 02% Coomassie G-250-40% methanol-10% acetic acid for at least 4 hours. Destaining was done in 12% methanol-7% acetic acid with four changes of destaining solution for 24 hours. Gels were scanned with a videodensitometer scanner (TDI, Madrid, Spain) connected to a personal computer for integration of the signals. Background intensity was calculated after scanning an empty lane. The coefficient of variation for the SDS-PAGE was 7.3% for apo B-48 and 5.1% for apo B-100.

DNA Amplification and Genotyping
DNA was extracted from 10 mL of EDTA-containing blood. Amplification of a region of exon 26 of the apo B was done by polymerase chain reaction (PCR) with 250 ng of genomic DNA and 0.2 µmol of each oligonucleotide primer (P1, 5'-GGAGACTATTCAGAAGCTAA-3', and P2, 5'-GAAGAGCCTGAAGACTGACT-3') in 50 µL. DNA was denatured at 95°C for 5 minutes followed by 30 cycles of denaturation at 95°C for 1 minute, annealing at 56°C for 1.5 minutes and extension at 70°C for 2 minutes. Twenty microliters of the PCR product were digested with 10 U of restriction enzyme XbaI (BRL) in a total volume of 35 µL. Digested DNA was separated by electrophoresis on an 8% nondenaturing polyacrylamide gel at 150 V for 2 hours. Bands were visualized by silver staining. Samples containing the X- allele characterized by the absence of the polymorphic XbaI cutting site were amplified a second time to verify the genotype.

Amplification of a region of 266-bp of the apo E gene was done by PCR with 250 ng of genomic DNA and 0.2 µmol of each oligonucleotide primer (E1, 5'-GAACAACTGACCCCGGTGGCGGAG-3', and E2, 5'-TCGCGGGCCCCGGCCTGGTACACTGCCA-3') and 10% dimethyl sulfoxide in 50 µL. DNA was denatured at 95°C for 5 minutes followed by 30 cycles of denaturation at 95°C for 1 minute, annealing at 63°C for 1.5 minutes and extension at 72°C for 2 minutes. Twenty microliters of the PCR product were digested with 10 U of restriction enzyme CfoI (BRL) in a total volume of 35 µL. Digested DNA was separated by electrophoresis on an 8% nondenaturing polyacrylamide gel at 150 V for 2 hours. Bands were visualized by silver staining.

Statistical Analysis
The following variables were calculated to characterize the postprandial responses of plasma triglycerides, large TRL, and small TRL to the test meal: Area under the curve (AUC) was the area between the plasma concentration versus time curve and a baseline drawn parallel to the horizontal axis through the 0 hour concentration. We calculated this area with a computer program using the trapezoidal rule. Other variables were the normalized peak concentration, which was the average of the peak and the second highest concentration above the baseline; and the peak time, which was the average of the time to peak concentration and the time to the second highest concentration. Data were tested for statistical significance between genotypes by ANOVA and the Kruskal-Wallis test, and between genotypes and time by ANOVA for repeated measures. BMI and age were introduced as covariates in all analyses. When statistical significance was found, the Tukeys post hoc comparison test was used to identify group differences. A probability value less than .05 was considered significant. Stepwise multiple regression analyses were carried out using small and large TRL-triglycerides, and small and large TRL-RP, and apo B-48 AUC as dependent variables, and age, BMI, apo B genotypes, basal cholesterol, and triglyceride values as independent variables. Discrete variables were divided into classes for analysis. All data presented in text and tables are mean±SD.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
The baseline characteristics of the study subjects are shown in Table 1Up. No significant differences for any of the variables analyzed were detected between subjects with the X+ allele (n=31) and those homozygous for the X- allele (n=20).

Plasma cholesterol and triglyceride responses following the fat load test are presented in Fig 1Down. Significant time effects were seen for both total cholesterol (P=.022) and triglycerides (P<.0001), showing that the cholesterol levels decreased during the postprandial period, whereas the opposite effect was seen for triglycerides. Plasma triglyceride levels remained significantly elevated over the baseline for all time points except the last determination (11 hours). There were no differences in total cholesterol and triglyceride response between X- and X+ subjects (P=.760 and P=.831, respectively). The distribution of plasma triglyceride within the different lipoprotein fractions examined is presented in Fig 2Down. Triglyceride levels in large TRL particles (Fig 2ADown) remained significantly elevated over the baseline in X- subjects during the entire period; however, these values were not significantly different at the end of the experimental period in those subjects with the X+ allele. Triglycerides in the small TRL (Fig 2BDown) fraction increased over baseline during the first 6 hours of the postprandial period for both X- and X+ subjects. No significant genotype effects were observed by ANOVA for repeated measures (interaction between genotype and time) for triglyceride levels in either of the TRL fractions. Subjects with the X- genotype appear to have elevated levels of large TRL and small TRL triglycerides (Fig 2Down) and AUC (Table 2Down); however, these effects did not reach statistical significance.



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  		Figure 1. Postprandial plasma cholesterol and triglyceride response in X- (solid line, {blacksquare}) and X+ subjects (dotted line, {diamondsuit}) (X+ indicates X+/X- or X+/X+ genotypes). For each group, the levels at each time point were averaged. P1, genotype effect; P2, time effect; P3, genotype by time interaction. MANOVA for repeated measures. Tukeys test for normally distributed variables or Kruskal-Wallis test for nonparametric variables.



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  		Figure 2. Postprandial plasma triglyceride response in large TRL (A) and small TRL (B) in X- (solid line, {blacksquare}) and X+ subjects (dotted line, {diamondsuit}) (X+ indicates X+/X- or X+/X+ genotypes). For each group, the levels at each time point were averaged. P1, genotype effect; P2, time effect; P3, genotype by time interaction. MANOVA for repeated measures. Tukeys test for normally distributed variables or Kruskal-Wallis test for nonparametric variables.


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  		Table 2. Area under the Postprandial Curve according to Genotype

The distribution of plasma cholesterol within the different TRL fractions examined is presented in Fig 3Down. Cholesterol in large TRL (Fig 3ADown) was significantly elevated over baseline in X- and X+ subjects during the first 9 hours of the fat load. No significant genotype effect was observed by ANOVA. Cholesterol in the small TRL fraction (Fig 3BDown) increased over the baseline during the first 6 hours of the postprandial period for both X- and X+ subjects and decreased below the baseline at the 11 hours time point. No significant differences were noted between genotypes with regard to postprandial response for this parameter.



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  		Figure 3. Postprandial plasma cholesterol response in large TRL (A) and small TRL (B) in X- (solid line, {blacksquare}) and X+ subjects (dotted line, {diamondsuit}) (X+ indicates X+/X- or X+/X+ genotypes). For each group, the levels at each time point were averaged. P1, genotype effect; P2, time effect; P3, genotype by time interaction. MANOVA for repeated measures. Tukeys test for normally distributed variables or Kruskal-Wallis test for nonparametric variables.

In X- subjects, the plasma LDL cholesterol (Fig 4ADown) levels showed a significant decrease for time points 1-6 hours compared with baseline levels. On the other hand, in X+ subjects, plasma LDL-C levels showed a significant decrease only at 4 hours. There were no significant differences in postprandial LDL-Cholesterol response as demonstrated by ANOVA for repeated measures. In X- subjects, the plasma apo B levels (Fig 4BDown) showed a significant decrease for the 3 and 4 hour time points compared with baseline levels, whereas no significant changes in postprandial apo B was observed in X+ subjects. There were no significant differences in postprandial apo B response.



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  		Figure 4. Postprandial plasma LDL-C (A), apo B (B), HDL-C (C), and apolipoprotein A-I (D), in X- (solid line, {blacksquare}) and X+ subjects (dotted line, {diamondsuit}) (X+ indicates X+/X- or X+/X+ genotypes). For each group, the levels at each time point were averaged. P1, genotype effect; P2, time effect; P3, genotype by time interaction. MANOVA for repeated measures. * indicates statistically significant differences between genotypes at that specific time point. P<.05. Tukeys test for normally distributed variables or Kruskal-Wallis test for nonparametric variables.

HDL-Cholesterol levels (Fig 4CUp) decreased during the first half of the postprandial period (1-5 hours) in X+ subjects and returned to the baseline afterward; however, the changes did not reach statistical significance versus the baseline. In X- subjects, HDL levels remained unchanged except at the 11 hour time point that was significantly elevated over the baseline (P=.0007). No genotype effect could be demonstrated by ANOVA for repeated measures. The postprandial apo AI changes (Fig 4DUp) were similar to those observed for HDL-Cholesterol; however, a significant genotype effect was seen (P=.047), with X- subjects having greater apo A-I levels compared with X+ subjects.

RP and apo B-48 were used as markers for intestinal lipoprotein production. Large and small TRL-RP responses in X- and X+ subjects are shown in Fig 5Down. RP levels in large TRL and small TRL were significantly elevated over the baseline in X- and X+ subjects during the entire period. A significant genotype effect was also observed by ANOVA for repeated measures, with X- subjects showing a significantly greater postprandial response (P=.04) in the large TRL RP (Fig 5ADown) and a greater (P=.023) AUC than X+ subjects (Table 2Up). Furthermore, a significant genotype by time interaction was also observed in this fraction (P=.005). The small TRL RP response (Fig 5BDown) was also significantly greater in X- than X+ subjects, as suggested by a significant genotype effect observed by ANOVA (P=.010) and a greater AUC (P=.007) (Table 2Up).



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  		Figure 5. Postprandial plasma retinyl palmitate response in large TRL (A) and small TRL (B) in X- (solid line, {blacksquare}) and X+ subjects (dotted line, {diamondsuit}) (X+ indicates X+/X- or X+/X+ genotypes). For each group, the levels at each time point were averaged. P1, genotype effect; P2, time effect; P3, genotype by time interaction. MANOVA for repeated measures. * indicates statistically significant differences between genotypes at that specific time point. P<.05. Tukeys test for normally distributed variables or Kruskal-Wallis test for nonparametric variables.

Large and small apo B-48 responses in X- and X+ subjects are shown in Fig 6Down. Apo B-48 levels (as determined by densitometric scanning) in large TRL and small TRL were significantly elevated over the baseline in X- and X+ subjects during the entire period. A significant genotype effect was also observed by ANOVA for repeated measures, with X- subjects showing a significantly greater postprandial response (P=.016) in the large apo B-48 (Fig 6ADown) and a greater (P=.030) AUC than X+ subjects (Table 2Up). The small TRL apo B-48 response (Fig 6BDown) was also significantly greater in X- than X+ subjects, as suggested by a significant genotype effect observed by ANOVA (P=.040) and a greater AUC (P=.040) (Table 2Up). No significant genotype effects were observed for apo B-100 in large or small TRL particles (Table 2Up).



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  		Figure 6. Postprandial apo B-48 response in large TRL (A) and small TRL (B) in X- (solid line, {blacksquare}) and X+ subjects (dotted line, {diamondsuit}) (X+ indicates X+/X- or X+/X+ genotypes). For each group, the levels at each time point were averaged. P1, genotype effect; P2, time effect; P3, genotype by time interaction. MANOVA for repeated measures. * indicates statistically significant differences between genotypes at that specific time point. P<.05 using the Tukeys test.

Multiple regression analyses (Table 3Down) revealed that the XbaI polymorphism at the apo B gene was the only significant (P=.03) predictor of the variability on large TRL-RP postprandial response (AUC) in our study population, accounting for 10.1% of the variance, whereas the apo B genotype (P=.01) and baseline plasma cholesterol (P=.02) were both significant predictors of small TRL-RP postprandial response, accounting for 14.1 and 10% of the variance, respectively. When large TRL apo B-48 was used as a dependent variable using the same variables in the model as for TRL-RP, only the apo B genotype entered the model as a predictor (P=.008) accounting for 20.1% of the variance. When small TRL apo B-48 was introduced in the model as a dependent variable, the XbaI genotype, age, and BMI were significant predictors, accounting for 36.4% of the variance (Table 3Down). The XbaI polymorphism did not enter the model when large or small TRL-TG were used as dependent variables.


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  		Table 3. Multiple Stepwise Regression Analyses


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Interindividual variability in postprandial lipid transport after a standard meal exceeds that observed in the fasting state61 62 and is influenced by numerous environmental and genetic factors affecting the synthesis and catabolism of TRL originating from the liver and intestine. Thus increased postprandial lipemia has been shown in apo A-I Milano carriers,63 and the common variants within the apo E gene locus have also been shown to affect the absorption or clearance of dietary fats,27 28 29 64 with E2 individuals having delayed clearance and E4 individuals having faster clearance as shown by retinyl palmitate concentrations in plasma and the nonchylomicron fraction.32 In order to remove the confounder effect of the variability associated with the apo E gene locus, this study was carried out in subjects homozygous for the apo E3 allele. Using RP and apo B-48 levels to follow clearance of intestinally derived lipoprotein particles, our results show that subjects with the X- genotype at the apo B gene locus have a greater postprandial response to a fat meal than do X+ subjects.

The X+ allele of the XbaI polymorphism has been found associated with increased serum levels of cholesterol and triglycerides by others.39 40 41 45 46 These results could not be confirmed in ours or in other studies,65 possibly due to our reduced sample size or the population studied, young healthy normolipemic males. In agreement with our results, previous studies have shown that in young normolipemic subjects no significant difference was observed between the degradation rate of LDL derived from X- versus X+ subjects.52

Subjects with the X- genotype showed a significantly greater postprandial response in the intestinally derived TRL particles, suggested by the increase in small and large TRL-RP response. Retinyl esters (predominantly RP) are incorporated into the core of chylomicrons in the intestine following the absorption and esterification of vitamin A.66 In the circulation they remain associated with chylomicrons during triglyceride lipolysis and are taken up by the liver within chylomicron remnants.67 Retinyl esters are either stored in the liver or resecreted as unesterified retinol bound to retinol-binding protein.68 The normal liver does not resecrete esterified retinol.69 Since their exchange with other lipoproteins in the circulation is small during the first 8 hours after consumption of a fat load,70 71 retinyl esters have been used as markers for intestinal lipoproteins, thus providing a means for measuring parameters of chylomicrons and chylomicron-remnant metabolism.71 72 73 74 75 In this study we verified the results obtained using retinyl palmitate by determining apo B-48 levels60 in both large and small triglyceride particles. Both markers provided similar results with regard to the XbaI associations with differential postprandial response.

Many factors control the concentration of triglyceride in the circulation. Chylomicron secretion by the intestine, VLDL secretion by the liver, conversion of TRL to triglyceride-depleted lipoproteins, and tissue uptake of triglyceride-depleted lipoproteins are all processes that could be responsible for fluctuations in postprandial triglyceride and lipoprotein concentrations. The greater increases in X- than in X+ subjects in plasma RP concentration in large and small TRL suggest intestinal secretion of TRL as the most plausible mechanism. Since chylomicron catabolism is a relatively fast and efficient process,76 it is unlikely that these phenomena could be due to fluctuations in chylomicron clearance. Several mechanisms can be proposed to explain why TRL secretion by the intestine might not be constant after fat ingestion. Variation in gastric emptying, gastrointestinal motility, or the rate of fat digestion in the intestine,77 potentially could cause postprandial peaks in plasma triglyceride. Postprandial triglyceride peaks could also result from differences in the abilities of the proximal and distal intestine to transport absorbed fat or fluctuations in the availability of phospholipids78 and/or newly synthesized chylomicron apolipoproteins in the enterocyte.79 Alternatively, it has been reported that X+ polymorphism is associated with less efficient binding of LDL to its receptor,50 51 which may result in less efficient down-regulation of the LDL receptor. Consequently, X+ subjects could express more LDL receptor, leading to greater elimination of triglyceride-rich lipoproteins via this receptor and thus to the differences observed with respect to X- carriers.

The DNA change that results in the absence of the XbaI restriction site in the apo B gene occurs at the third base of triplet coding for threonine at amino acid 2488. No amino acid changes result from this base change in apo B-100.47 This polymorphism is in strong linkage disequilibrium with the apo B Val591->Ala polymorphism (Ag a1/d), however, which may be the functional sequence change.80 Another tentative hypothesis relates to the fact that this mutation is close to the editing site at position 2153. In humans, Apo B-48 is synthesized only in the intestine81 and is the product of an intestinal mRNA identical in structure to apo B-100 mRNA except for a single C to U base substitution, the first base of the codon CAA for Gln-2153, changing it to UAA, a stop codon.82 83 The mechanism behind the C to U conversion or editing could involve a form of cytidine deaminase,84 although other possibilities have not been ruled out. The mooring sequence model for the editosome suggests that the recognition and binding sequences of nuclear factors that both identify the specific site for editing and "moor" the editing activity are distal to and different from the sequences in the immediate vicinity of the editing site.84 85 86 The mooring sequences might be a specific primary sequence or a unique secondary structure of apo B mRNA. It is possible that the XbaI mutation, close to the editing site, could affect these "mooring sequences" and modify the editing activity of apo B mRNA that results in changes of apo B-48 and intestinal TRL secretion observed in this study. Alternatively, the XbaI polymorphism may be functionally silent and may be in linkage disequilibrium with a functionally important sequence change in the coding or the regulating regions of the apo B gene.80 Both kinetic and in vitro studies favor the latter hypothesis.

Previous studies have shown that the X- allele was found significantly more frequently among patients with CAD and with peripheral arterial disease than among healthy control subjects.87 88 89 Nevertheless, in some studies, the X- subjects have lower LDL-C and apo B levels than the X+ subjects, although this effect may be dependent on age and sex.90 It is possible that the higher postprandial response observed in this study in subjects with the X- genotype compared with subjects with the X+ genotype could be involved in the higher risk of CAD previously reported in X- subjects. At least five lines of evidence implicate postprandial lipoprotein metabolism in the genesis of CAD. First, receptor-mediated uptake of TRL remnant particles by monocyte-derived macrophages promotes the formation of lipid-laden foam cells in the vessel wall.91 Second, chylomicron and VLDL remnants make a substantial contribution to the lipid content of the vessel wall in cholesterol-fed rabbits.92 Third, the level of HDL-C, an established risk factor for CAD, is dependent on both the metabolism of TRL particles and the extent of postprandial lipemia.93 Fourth, patients with familial dysbetalipoproteinemia are at high risk of CAD and show a build up of TRL remnants of both intestinal and hepatic origin.94 Fifth, cross-sectional case-control studies have found that increased postprandial triglycerides and RP concentrations are associated with angiographically verified CAD and carotid artery wall thickness.5 6

In conclusion, subjects with the X- genotype of the XbaI polymorphism of apo B have a higher postprandial response than do X+ subjects. These differences observed in postprandial lipoprotein metabolism could be involved in the higher risk of coronary heart disease observed in these subjects.


*    Selected Abbreviations and Acronyms
 
Apo = apolipoprotein
AUC = area under the curve
CAD = coronary artery disease
HDL-C = HDL-cholesterol
LDL-C = LDL-cholesterol polymerase chain reaction
RP = retinyl palmitate
TG = triglycerides
TRL = triglyceride-rich lipoproteins


*    Acknowledgments
 
This work was supported by grants from the Consejeria de Agricultura y Pesca, Consejeria de Educacion y Ciencia, Junta de Andalucia, and the Spanish Ministry of Health (FIS 95/1144) and HL54776 (to J.M.O.) from the National Institutes of Health.

Received January 9, 1996; accepted January 20, 1997.


*    References
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 *References
 

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