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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1347-1355.)
© 1996 American Heart Association, Inc.

Articles

Effect of Dietary Fat Saturation on LDL Oxidation and Monocyte Adhesion to Human Endothelial Cells In Vitro

P. Mata; R. Alonso; A. Lopez-Farre; J.M. Ordovas; C. Lahoz; C. Garces; C. Caramelo; R. Codoceo; E. Blazquez; M. de Oya

the Unidad de Investigacion de Lipidos and Instituto de Investigaciones Medicas, Fundacion Jimenez Diaz, Universidad Autonoma, Madrid, Spain, and the Lipid Metabolism Laboratory (J.M.O.), Jean Mayer–United States Department of Agriculture–Human Nutrition Research Center on Aging at Tufts University, Boston, Mass.

Correspondence to Pedro Mata, MD, PhD, Unidad de Lipidos, Fundacion Jimenez Diaz, Avda Reyes Catolicos, 2, 28040 Madrid, Spain.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Forty-two healthy men and women were subjected to four consecutive dietary periods differing in the fat content of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (n-6) [PUFA(n-6)] and (n-3) [PUFA(n-3)]. Plasma lipids, vitamin E, and in vitro LDL oxidation were examined during each period. Adhesion of human monocytes to cultured human endothelial cells was used as a functional test to identify differences in the biological properties of LDL from each dietary period. Consumption of an SFA-rich diet resulted in higher LDL cholesterol (4.06±0.85 mmol/L, P<.05) than did consumption of MUFA- (3.59±0.75 mmol/L), PUFA(n-6)– (3.44±0.77 mmol/L), or PUFA(n-3)– (3.31±0.8 mmol/L) rich diets. HDL cholesterol was lower during both PUFA-rich diets (1.24±0.28 and 1.27±0.28 mmol/L for n-6 and n-3, respectively) than during the SFA- (1.32±0.36 mmol/L) and MUFA- (1.32±0.34 mmol/L) rich diets. LDL resistance to copper-induced oxidation, expressed as lag time, was highest during the MUFA-rich diet (55.1±7.3 minutes) and lowest during the PUFA(n-3)– (45.3±7 minutes) and SFA- (45.3±6.4 minutes) rich diets. LDL induction of monocyte adhesion to endothelial cells was lower during the MUFA-rich diet than the other periods. The highest monocyte adhesion was obtained during the PUFA(n-3) and SFA dietary periods. In conclusion, an MUFA-rich diet benefits plasma lipid levels compared with an SFA-rich diet. Furthermore, this diet results in an increased resistance of LDL to oxidation and a lower rate of monocyte adhesion to endothelial cells than the other dietary fats examined.

Key Words: dietary fat • lipid oxidation • monocyte • low density lipoprotein • monounsaturated fatty acid

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Elevated plasma LDL-C levels represent a recognized risk factor for atherosclerotic cardiovascular disease¹ ; however, the biochemical mechanisms involved in its pathogenesis are not entirely understood. Substantial evidence exists to suggest that LDL may undergo oxidative modification in vivo^{2 3} and that this process may be critical in the initiation and evolution of atherosclerosis.⁴ Several biological effects have been proposed as being involved in the deleterious effect of oxidized LDL, namely, changes in LDL uptake by macrophages, monocyte chemotaxis and adhesion, and nitric oxide–dependent vasodilation.^{4 5}

Dietary fat is one of the most important factors determining plasma LDL-C concentrations,⁶ and diet can modulate the susceptibility of plasma LDL to oxidative modification.⁷ This effect has been attributed to diet-induced changes in the concentration of PUFAs and antioxidants in the LDL particle.^{7 8 9 10 11} LDL shows an increased resistance to the oxidative modification associated with the consumption of diets rich in MUFAs in normolipidemic^{8 9 10} and mildly hypercholesterolemic¹¹ subjects. To our knowledge, no study has compared LDL oxidation–related variables in the same group of subjects consuming diets rich in SFAs, MUFAs, PUFA(n-6), and PUFA(n-3). Moreover, little is known regarding the protective effect of oleic acid (C18:1) in normolipidemic women consuming regular diets. The purpose of this study was to evaluate the effects of four diets, differing only in their fat saturation, on plasma lipid levels, LDL oxidation, and LDL-induced monocyte adhesion to ECs in culture in a group of healthy men and women living in two religious communities.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Study Design
The study consisted of four consecutive 5-week dietary periods. The diets were consumed in the same order for all subjects: SFA, MUFA, PUFA(n-6), and PUFA(n-3). All dietary phases were completed between January and June, and menus were prepared by using similar food items to eliminate variations due to seasonal changes in food supply. The purpose of the study was fully explained to each of the participants, who gave their written consent. This protocol was approved by the Human Studies Committee of the Universidad Autonoma de Madrid.

Population
Fifty-four volunteers from two religious communities were enrolled in the study. Their regular lifestyle contributed to decrease the effect of behavioral changes as possible confounders in this study. Exclusion criteria were the use of drugs, including vitamin supplements, which might potentially affect any of the outcomes of the study. All subjects gave a medical history and underwent a physical examination. None of them had any known metabolic disorder. Nine subjects (7 women and 2 men) were excluded due to baseline cholesterol levels >6.5 mmol/L. Forty-five subjects (20 women and 25 men) started the dietary trial. There were three dropouts during the study due to unscheduled prolonged absence from the community. Data from the 42 subjects (18 women and 24 men) who successfully completed the four dietary phases were used for the final analysis. The women were aged 45.8±9.5 years (range, 23 to 59 years); 10 were postmenopausal. The men were 45.1±16.2 years old (range, 17 to 71 years). Five men smoked and 18 consumed small amounts of alcoholic beverages (<40 g ethanol/d). None of the women smoked or drank alcohol. BMI was 26.5±4.7 in men and 22.8±2.9 in women; these values were maintained during the experimental period. The participants were required to maintain their usual life habits and physical activity and to report in a diary any incident with a potential effect on the outcome of the study, ie, intercurrent illnesses, use of medications, or circumstantial deviations from the diet.

Diets
All menus were prepared by using common food items. Special emphasis was placed on using foodstuffs that were not different from those habitually consumed by the study subjects. Four different diets were designed with seven daily menus rotating weekly. The composition of the diets, which was evaluated by direct chemical analysis (see below) was 15% protein, 50% carbohydrate, and 35% fat (as percent of total energy). This composition, which was maintained constant among the four experimental diets, reflects the diet usually consumed by the participants in the study as well as that consumed in most Mediterranean countries. Dietary fiber (21 to 23 g) and cholesterol (<300 mg/d) were also maintained constant among the dietary phases. Palm oil and butter constituted the major fats used during the SFA period (55% of the total fat intake). Olive oil, rich in oleic acid in its natural cis configuration, accounted for 55% of the fat consumed during the MUFA diet, whereas sunflower oil represented 50% of the PUFA(n-6) and 37.5% of the PUFA(n-3) diets. All diets, except for the PUFA(n-3) period, included the following foods weekly: veal twice, chicken twice, ham or cheese twice, legumes twice, rice twice, pasta once, vegetables seven times, white fish four times, and three eggs. During the PUFA(n-3) period, blue fish (mackerel, salmon, and sardines) were substituted for veal, chicken, and white fish; one fish meal per day was eaten every day of the week. This regimen provided about 3.2 and 4.5 g/d of n-3 fatty acids in women and men, respectively. In addition, the subjects consumed daily a fixed amount of bread, fruit, cookies, jam, milk, and green salad.

Body weight was measured twice a week, and the individual carbohydrate intake was adjusted as needed to prevent weight changes of more than 2%. All meals were prepared at the communities' kitchens and consumed in the dining halls. Individualized and weighed portions were provided for each participant.

Duplicate samples of all meals from randomly selected individuals (one man and one woman) were collected every day during 1 week of each dietary phase and stored at -20°C under nitrogen for chemical analysis of the nutrients (Table 1). The fatty acid composition of the oils used in this study was analyzed by using capillary gas-liquid chromatography at the Food Analysis Laboratory of the Spanish Ministry of Agriculture. By weight, palm oil consisted mainly of palmitic acid (37%), oleic acid (43.6%), and linoleic acid (11.7%). The major fatty acids in olive oil were palmitic acid (9.2%), oleic acid (80%), and linoleic acid (4.7%). The sunflower oil contained palmitic acid (6.8%), oleic acid (29%), and linoleic acid (56%).

View this table: [in this window] [in a new window]   Table 1. Mean Daily Energy and Nutrient Intake During the Four Dietary Periods

Blood Sampling and Analysis
Fasting (12-hour) blood samples were obtained at baseline and twice during the last week of each dietary period. Blood was collected in EDTA-containing (4.0 mmol/L) evacuated tubes. Blood samples were immediately protected from exposure to light and chilled on ice. Plasma was separated by low-speed centrifugation (3000 rpm), and gentamicin and chloramphenicol (0.22 and 0.15 mmol/L, respectively) were added to the samples.

Lipoprotein separation and analysis were performed as recommended by the Lipid Research Clinics.¹² TC and TGs were measured by using enzymatic methods (Boehringer Mannheim) on a Technikon RA-XT autoanalyzer. HDL was measured after precipitation of apoB-containing lipoproteins with phosphotungstic acid–MgCl₂ (Boehringer Mannheim).

LDL Isolation and Analysis of Oxidation-Related Variables
LDL was isolated by sequential ultracentrifugation immediately after separation of plasma.¹² The LDL fraction was dialyzed for 24 hours at 4°C against phosphate-buffered saline (10 mmol phosphate and 0.16 mol saline, pH 7.4) in the dark. The phosphate-buffered saline was maintained oxygen-free by purging with pure nitrogen. All the analyses were completed within 4 days of LDL isolation. LDL protein content was determined by using the method of Bradford.¹³

LDL oxidation was initiated by incubating 100 µg LDL protein with 5 µmol/L Cu in 1 mL phosphate-buffered saline. The kinetics of LDL oxidation were examined by monitoring the change of 234-nm diene absorbance at 37°C for 4 hours at 2-minute intervals on a UV spectrophotometer (Beckman DU-8B UV). Lag time, rate of oxidation, and total amount of conjugated dienes formed per milligram of LDL protein were calculated.¹⁴

The content of TBARS was assessed in freshly isolated LDL¹⁵ ; results were expressed as MDA equivalents (nanomoles MDA per milligram LDL protein).

-Tocopherol levels in plasma and the LDL fraction were measured by using high-performance liquid chromatography¹⁶ with a reverse-phase column (Resolve C18, 5 µm, Waters). Retinyl acetate was used as an internal standard and -tocopherol (Sigma Chemical Co Ltd) as an external standard. Results were expressed in micromoles per liter of -tocopherol for plasma values and in molecules per particle for LDL values.

LDL Fatty Acid Composition
CEs and PL fatty acids were transmethylated and analyzed in a Perkin Elmer Autosystem chromatograph equipped with a capillary column (Supelco SP-2380; 60 mx0.25 mm).¹⁷ Values were expressed as percent of total fatty acid.

Cell Culture and Adhesion Assays
ECs were isolated from human umbilical vein by filling the lumen with 0.5 mg/mL type II collagenase (Sigma) and cultured.¹⁸ The cells were characterized as endothelial by their typical phenotype and cobblestone appearance and by indirect immunofluorescence staining for factor VIII–related antigen. Confluent cells from the first or second passages were used.

Blood was obtained from normolipidemic healthy donors, and monocytes were isolated^{18 19} and used immediately. ECs were preincubated with freshly isolated LDL (50 µg/mL) for 24 hours. LDL-treated human ECs were washed twice with Krebs-Henseleit buffer containing 0.2% bovine serum albumin, and a ⁵¹Cr-labeled monocyte suspension was added (500 µL [3x10⁶ cells]). Adhesion experiments were done in triplicate. Monocytes and ECs were coincubated for 30 minutes at 37°C under static conditions. Loosely adherent or unattached monocytes were removed by washing three times with 1 mL Krebs-Henseleit buffer, and the ECs plus adherent monocytes were lysed in 0.5 mL of 0.1 mol/L NaOH and 0.5 mL of 0.1% sodium dodecyl sulfate. Results were expressed as the percentage of monocytes adhered with respect to the radioactivity contained in a control sample of 3x10⁶ monocytes. Experimental and control samples were measured in triplicate. Adhesion assays were performed immediately after the LDL was isolated from fresh plasma at the end of each dietary period. To examine possible temporal bias in the assay, all samples were also analyzed simultaneously at the end of the study by using LDL frozen at -70°C. No significant differences were found.

Statistical Analysis
Statistical analyses were performed by using the CSS statistical software package (CSS, Statsoft, Inc). MANOVA for repeated measures was used at the 5% significance level to test for effects of each dietary period on the dependent variables (plasma TC, LDL-C, HDL-C, and TG concentrations). Post hoc testing procedures (Tukey post hoc comparison test) were used when significance was found. Except for TG concentrations, all continuous variables were normally distributed as assessed by the Kolmogorov-Smirnov test. TGs were logarithmically transformed to achieve approximately normal distributions, and statistical tests were performed on the transformed values. Using lag period as a dependent variable, we performed stepwise multiple regression analyses to identify independent predictors. Variables included age, BMI, tobacco use, alcohol use, MUFAs/PUFAs in LDL-PL or LDL-CE, and LDL vitamin E. Discrete variables were dichotomized into two classes for the analyses. Monocyte adhesion to ECs was examined by using LDL isolated from 18 randomly selected subjects (9 men and 9 women). All data presented in text and tables are mean±SD.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Diet Composition and Compliance
The composition, fatty acid profile, and cholesterol content of the diets are shown in Table 1. The protein, carbohydrate, and fat contents of the four diets were practically identical.

LDL-CE and LDL-PL fatty acids were assessed to confirm diet compliance and to relate these values to changes in LDL oxidation in vitro (Table 2). Consumption of the SFA diet resulted in slight but significant increases in C16:0 compared with the other three diets. Despite the low proportion of dietary C18:2 during the SFA period, this fatty acid accounted for 52.5% of the total fatty acids in LDL-CE. This proportion was greater than that observed during the MUFA diet (45.4%, P<.05). As expected, C18:1 was significantly increased during the MUFA diet compared with the other diets. C18:2 was significantly elevated during both PUFA diets compared with the SFA and MUFA diets. The CE and PL fractions of LDL isolated from subjects consuming the PUFA(n-3)–rich diet were significantly enriched in the long-chain PUFAs (C20:5 and C22:6) compared with the other three diets. Body weight was maintained constant during the study.

View this table: [in this window] [in a new window]   Table 2. Effects of Diets Enriched in Each of the Experimental Oils on CE and PL Fatty Acid Composition of LDL

Plasma Lipid Levels
The consumption of an SFA-rich diet resulted in elevated plasma TC levels (5.79±0.88 mmol/L, P<.02) compared with the MUFA- (5.22±0.80), PUFA(n-6)– (4.97±0.80), and PUFA(n-3)– (4.83±0.80 mmol/L) rich diets (Table 3). Men displayed higher cholesterol levels than women during the SFA period (5.53±0.83 mmol/L, P<.02 versus the other three diets), whereas similar cholesterol levels were observed during the MUFA (5.04±0.80 mmol/L) and PUFA(n-6) (4.89±0.88) periods. Lower cholesterol levels were found during the PUFA(n-3) period (4.73±0.88 mmol/L, P<.02 versus the other three diets). In women, as for the entire group, the highest cholesterol level was associated with the SFA diet (6.10±0.88 mmol/L). A decrease was observed during the MUFA diet (5.48±0.72 mmol/L, P<.05), and further decreases were noted during both PUFA diets [5.07±0.70 and 4.99±0.72 mmol/L for PUFA(n-6) and PUFA(n-3), respectively, P<.05]. The difference between the latter two diets was not significant. Dietary effects on LDL-C levels paralleled those described for TC. HDL-C levels were lower in men than women. No difference in HDL-C was observed between the SFA (1.32±0.36 mmol/L) and MUFA (1.32±0.34 mmol/L) periods for any subject. During the PUFA(n-6) period, HDL-C levels (1.24±0.28 mmol/L, P<.05) were lower than during the SFA and MUFA periods. HDL-C during the PUFA(n-3) period (1.27±0.28 mmol/L) was significantly lower than during the MUFA period. No other comparisons were significant. When men were analyzed separately, dietary fat saturation did not affect HDL-C levels, whereas in women, both PUFA diets resulted in lower HDL-C levels than the SFA or MUFA diets. The diet-induced changes in plasma TGs were similar to those reported for HDL-C, although the differences between both PUFA diets and the MUFA and SFA diets were significant in both genders.

View this table: [in this window] [in a new window]   Table 3. Effects of Diets Enriched in Each of the Experimental Oils on Fasting Plasma Lipoproteins in 18 Men and 24 Women

LDL Oxidation
Plasma concentrations of vitamin E were similar for all dietary phases: 33.5±8.2, 33.8±8.9, 36.4±9.9, and 38.9±10.0 µmol/L for SFA, MUFA, PUFA(n-6), and PUFA(n-3), respectively. The content of vitamin E on the LDL particle (ie, the number of molecules of vitamin E per LDL particle) at the end of each dietary period is shown in Table 4. No significant difference was noted between the values for the SFA (10.6±2.6) and MUFA (12.4±3.7) periods; however, these values were lower (P<.05) than those obtained during either the PUFA(n-6) (14.2±4.0) or PUFA(n-3) (14.0±3.7) periods.

View this table: [in this window] [in a new window]   Table 4. Effects of Diets Enriched in Each of the Experimental Oils on LDL Vitamin E Levels and Oxidation-Related Variables

TBARS values (expressed as nanomoles MDA per milligram LDL protein) were determined in freshly isolated LDL. Identical values were observed during the SFA (1.15±0.57) and MUFA (1.15±0.35) periods. These values were significantly lower than those obtained during the PUFA(n-6) (1.51±0.50) or PUFA(n-3) (1.69±0.48) periods. Similar results were obtained when we performed the analyses in men and women separately, although the women tended to have lower TBARS values than men for each of the dietary periods. Conjugated dienes (expressed as nanomoles per milligram LDL protein per 4 hours) were lower in LDL isolated from subjects consuming the MUFA (256±47) diet relative to LDL isolated from subjects consuming the PUFA(n-6) (301±61) or PUFA(n-3) (331±50) diets. The difference between the two PUFA-rich diets was not significant. Conjugated dienes measured in LDL isolated during the SFA diet were not significantly different from those found during the MUFA and PUFA(n-6) periods, but they were lower than those determined during the PUFA(n-3) diet. In men, conjugated dienes were significantly elevated only during the PUFA(n-3) period, whereas in women, both PUFA-rich diets resulted in higher values compared with those obtained during the SFA or MUFA diets.

Consumption of the MUFA diet was associated with an increased lag time in LDL oxidation (55.1±7.3 minutes) relative to all other dietary periods (P<.05). No significant differences were observed between the SFA (45.3±6.4 minutes), PUFA(n-6) (47.1±8.4 minutes), or PUFA(n-3) (45.3±7.0 minutes) diets. Similar effects were observed in men and women (Table 4).

When the data obtained from all dietary phases were pooled, women had significantly lower TBARS values than men (1.25±0.39 versus 1.47±0.59 nmol MDA/mg LDL protein, respectively, P=.006). The same results were observed for conjugated dienes (273±61 for women and 297±63 nmol/mg LDL protein for men, P=.017). No significant differences were noted for lag time (47.4±8.3 for women and 48.9±8.36 minutes for men, P=.27). The LDL-CE MUFA-to-PUFA ratio (CE-MUFA/PUFA) was similar in men (0.16±0.07) and women (0.14±0.064, P=.06). Similar findings were observed for the LDL-PL fraction; however, the vitamin E content of the LDL particle was greater in women than men (13.7±4.3 versus 12.1±3.2 vitamin E molecules/LDL particle, P=.027).

Stepwise multiple regression analysis was performed by using LDL lag time, conjugated dienes, and TBARS as dependent variables and LDL-PL MUFA/PUFA, LDL vitamin E, age, menopausal status in women, alcohol, smoking, and BMI as predictors (Table 5). These analyses demonstrated that the most significant predictor of lag time in men was LDL-PL MUFA/PUFA, accounting for 14.9% of the variability. The only other significant predictor in this model was LDL vitamin E, which explained an additional 14.5% of the variability. Both variables were directly associated with the dependent variable. In women, LDL-PL MUFA/PUFA and LDL vitamin E accounted for 21.5% and 17.5%, respectively, of the variability in lag time. None of the other variables had a significant contribution to the model. By using conjugated dienes as the dependent variable, LDL-PL MUFA/PUFA and LDL vitamin E levels accounted for 16.4% and 4.4% of the variability in women, whereas in men, LDL-PL MUFA/PUFA and smoking accounted for 10.1% and 5.3% of the variability. LDL vitamin E levels did not enter the model in men. Only LDL-PL MUFA/PUFA and LDL vitamin E levels were significant predictors of TBARS variability in this study. Similar analyses were performed by using LDL-CE MUFA/PUFA in the model instead of LDL-PL MUFA/PUFA. The results were parallel to those presented in Table 5; however, the percent of the variability accounted for by LDL-CE MUFA/PUFA was slightly lower than that for LDL-PL MUFA/PUFA (data not shown).

View this table: [in this window] [in a new window]   Table 5. Multiregression Models for LDL Oxidation-Related Variables

Monocyte Adhesion to ECs in Culture
To determine the effects of the different diets on monocyte adhesion to ECs in culture, LDL was isolated at the end of each dietary phase from plasma from 18 randomly selected subjects and incubated with ECs in culture before being exposed to monocytes isolated from healthy donors. Adhesion was 9.1±2.0%, 6.6±0.7%, 7.9±1.2%, and 13.2±2.9% for the SFA, MUFA, PUFA(n-6), and PUFA(n-3) diets, respectively (Fig 1). No statistical difference was obtained between the SFA and PUFA(n-6) dietary periods. Although the MUFA diet was associated with lower adhesion than that obtained during the SFA and PUFA(n-3) periods, the difference between the MUFA and PUFA(n-6) periods did not reach significance. The PUFA(n-3) dietary period was associated with a significantly greater adhesion than the other three phases. Similar effects were noted when men and women were analyzed separately.

View larger version (33K): [in this window] [in a new window]   Figure 1. Bar graph shows percent monocyte adhesion to ECs in culture after incubation of the ECs with LDL isolated from subjects at the end of each dietary phase. Results are mean±SD. *P<.05 vs MUFA; P<.05 vs PUFA(n-3).

The content of 18:1 in the PL fraction of the isolated LDL was inversely correlated with the percent monocyte adhesion (r=-.5138, P=.0005), whereas the content of 18:2 in this same lipid fraction was positively associated with the percent monocyte adhesion (r=.3179, P=.04). A significant inverse correlation was seen between the LDL MUFA/PUFA ratio and percent adhesion for both CE (P=.023; Fig 2A) and PL (P=.0001; Fig 2B) fatty acids.

View larger version (18K): [in this window] [in a new window]   Figure 2. Plots show relations between LDL-CE MUFA/PUFA (A) or LDL-PL MUFA/PUFA (B) and percent monocyte adhesion to ECs in culture.

Stepwise multiple regression analyses were performed by using adhesion-related measurements as dependent variables and LDL-PL MUFA/PUFA, LDL vitamin E, age, menopausal status in women, alcohol, smoking, and BMI as predictors. These analyses demonstrated that the most significant predictor of adhesion variability was LDL-PL MUFA/PUFA, accounting for 31.8% of the variability in monocyte adhesion to ECs in culture.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This controlled dietary study showed that dietary fatty acids produce significant changes in LDL fatty acid composition and plasma lipoprotein levels in healthy men and women consuming a regular diet. Moreover, these dietary changes modified the susceptibility of LDL to undergo in vitro oxidative modification and monocyte adhesion to human ECs in vitro.

The isocaloric substitution of SFA by MUFA, PUFA(n-6), or PUFA(n-3) resulted in significant decreases in plasma TC and LDL-C levels. The fact that the most significant reduction in TC was achieved with the PUFA(n-3) diet suggests that this phenomenon is related to the degree of unsaturation.^{20 21 22} HDL-C levels were also lower during the PUFA diets than either the SFA or MUFA diets. We have reported^{17 23} that PUFA-enriched regular diets decreased HDL-C levels compared with an MUFA-enriched diet and that this effect was maintained during the entire 4- to 6-month experimental period. Similar results using liquid diets have also been reported.²⁴ In the present study, the HDL-C lowering associated with the PUFA-enriched diets was more pronounced in women than men, suggesting a gender effect on HDL-C response to diet modification as reported by Clifton and Nestel.²⁵

Our data suggest that the increase in the MUFA content of the diet and the associated relative increase in oleic acid and decrease in linoleic acid content of LDL appear to have the strongest effect on determining the resistance of LDL to oxidative modification as measured by several parameters. In our study design, the variability in LDL vitamin E levels depended exclusively on its natural presence in the foods used to prepare the menus; consequently, our purpose was not directed to assess the effects of vitamin E supplementation on LDL oxidation–related parameters. However, under these conditions, the vitamin E content of LDL still had a significant contribution (14.0% in men and 17.5% in women) to the variability in lag time values.

TBARS and conjugated dienes in LDL were significantly lower in women than men; no differences were noted regarding menopausal status. This finding was associated with a greater concentration of vitamin E in the LDL isolated from women. No gender differences were demonstrated in LDL-CE or LDL-PL MUFA/PUFA, the major determinants of lag time variability. These data suggest that the higher levels of HDL-C found in women may have a vitamin E "sparing" effect, as other investigators have suggested.²⁶

Studies in normolipidemic men have found that MUFA-rich diets induce an increased resistance of LDL to oxidative modification.^{8 9 10} In general, these studies used supplemented liquid diets or higher total fat content. Our results show that LDL enriched in MUFAs derived from common foods was less prone to oxidation in both men and women; in contrast, the PUFA(n-3) diet showed the highest conjugated dienes formation. Suzukawa et al²⁷ also found that a fish oil–supplemented diet increased LDL susceptibility to oxidation compared with a corn oil–supplemented diet.

To date, the relative effects of SFAs on LDL oxidation compared with all other major classes of fatty acids [MUFA, PUFA(n-6), and PUFA(n-3)] have not been examined in humans. The results generated by the inclusion of an SFA-enriched diet in our experimental design provided additional support for the critical role of LDL-CE MUFA/PUFA and LDL-PL MUFA/PUFA in conditioning the susceptibility of LDL to oxidation. Lag time was similar for the SFA, PUFA(n-6), and PUFA(n-3) periods and significantly shorter than during the MUFA period. Simultaneously, LDL-PL MUFA/PUFA and LDL-CE MUFA/PUFA were significantly decreased during those three dietary periods compared with the MUFA period. However, the rate of conjugated dienes formation was similar during the SFA and MUFA diets, suggesting that LDL differences in C18:1 or C18:2 may influence the lag time before conjugated dienes formation begins.

A central finding in this study was the relation observed between diet-induced changes in LDL composition and those observed in the adhesion of monocytes to ECs, an early event in the development of atherosclerosis.^{28 29} A direct relationship has been found between diet-induced hypercholesterolemia and monocyte adhesion and infiltration of the arterial endothelium.^{30 31} However, it remains to be shown whether these findings in experimental models are applicable to the physiological events that occur in humans. More precisely, the effects of human LDL with different fatty acid compositions on the adhesion of monocytes to ECs is still unknown. In this regard, this study provided an appropriate experimental setting, including the strict control of diet composition and the characterization of the fatty acid composition of LDL. We used native, freshly isolated LDL, which closely resembles the in vivo situation. Furthermore, the experiments were done using LDL from the same subjects undergoing the different dietary phases, which allowed a more meaningful interpretation of the results.

This study demonstrated that ECs incubated with LDL isolated during the MUFA phase had the lowest level of monocyte adhesion. Conversely, LDL obtained during the PUFA(n-3) phase induced the highest monocyte adhesion. There were no significant differences between the MUFA and PUFA(n-6) diets. No individual correlation existed in our study between the degree of LDL oxidation susceptibility, as assessed by either TBARS, conjugated dienes, or lag time, and the percentage of monocyte adhesion. However, Watson et al³² have shown that mildly oxidized LDL and more specifically, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoryl-choline, are indeed responsible for inducing monocyte-endothelial interactions. Consequently, some caution should be taken in interpreting the results obtained using traditional oxidation measurements such as TBARS and conjugated dienes.

Our results can be interpreted in light of the changes in the fatty acid composition of the LDL particle induced by the diets. Our data show a significant negative association between percent monocyte adhesion to ECs and the 18:1 content in the PL fraction of LDL. Conversely, a positive association was found regarding the 18:2 content of the same lipid fractions. Similar findings have been shown in vitro.³³ An inverse relationship was observed between the CE and PL MUFA-to-PUFA ratio and the degree of monocyte adhesion. Although both relations were statistically significant, the magnitude of the effect was greater for the relative content of MUFAs and PUFAs on PLs (P=.0001) than for this parameter on CEs (P=.023), suggesting that the fatty acid content of the lipoprotein surface may be important for its interaction with cellular membranes.

Our data support the hypothesis that an important determinant of LDL interaction with ECs is their fatty acid composition. Changes in the LDL fatty acid composition may produce alterations in membrane properties of ECs³³ that may in turn influence the surface expression or function of adhesion proteins.^{34 35 36}

In summary, the consumption of both SFA- and PUFA-rich diets increased the susceptibility of LDL to oxidative modification, and n-3 fatty acids produced the greatest monocyte adhesion to ECs in vitro, thus suggesting an increased atherogenic risk. This latter observation contradicts epidemiological findings that link the consumption of diets high in fish to a lower incidence of cardiovascular disease,^{37 38} although this association has not been consistent in all studies.³⁹ Whitman et al⁴⁰ have demonstrated that LDL from fish oil–fed animals was more susceptible to in vitro peroxidation, but it was not necessarily more atherogenic in vivo. Therefore, further clarification is required, and long-term human studies need to be undertaken using noninvasive assessment of atherogenesis to properly address the role of dietary fatty acids in the development of atherosclerosis.

Reduction of dietary intake of SFAs appears to be the key to reducing the risk of atherosclerosis in humans.⁴¹ In the past, dietary recommendations have suggested the replacement of excess SFAs with PUFAs. But replacement of SFAs with MUFAs may also lower LDL-C levels. Our study provides further support to the epidemiological evidence⁴² that suggests that MUFA-enriched diets confer a beneficial effect by decreasing the risk of degenerative diseases, primarily coronary heart disease. In this regard, besides their beneficial effect on the plasma lipoprotein profile, these diets may positively affect the properties of LDL particles that are related to their oxidizability and promotion of monocyte adhesion. Nevertheless, it remains to be demonstrated whether this form of dietary modification can slow or inhibit the development of atherosclerosis in humans.

	Selected Abbreviations and Acronyms

 

BMI = body mass index HDL-C = HDL cholesterol LDL-C = LDL cholesterol MDA = malondialdehyde MUFA = monounsaturated fatty acid PL = phospholipid PUFA = polyunsaturated fatty acid SFA = saturated fatty acid TBARS = thiobarbituric acid–reactive substances TC = total cholesterol TG = triglycerides

	Acknowledgments

 
This work was supported in part by grants FIS 93/0428, FIS 95/0838, and CICYT SAL 93/0516. We are indebted to the participants in the study for their great cooperation and enthusiasm. We thank Jose R. Garcia-Hierro for the analysis of the diets. Olive and sunflower oils were kindly supplied by Aceites Toledo, SA, Spain, and palm oil by Agra, SA, Spain. R. Alonso is a visiting professor from the University of Valparaiso, Chile, and a Fellow of the Conchita Rabago Foundation.

	Footnotes

 
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994.

Received January 3, 1996; revision received April 2, 1996;

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