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The Journal of Nutrition Vol. 128 No. 10 October 1998,
pp. 1703-1709
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Lipid Metabolism Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111; * Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06268; and Department of Family Medicine and Community Health, Tufts University School of Medicine, Boston, MA 02111
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The effect of the fatty acid composition of reduced-fat diets on the in vitro oxidation of LDL was examined in 14 moderately hypercholesterolemic [low density lipoprotein (LDL) > 3.36 mmol/L] postmenopausal female and male subjects (age 44-78 y). Each subject consumed each of five reduced-fat diets [30 energy percent (E%) fat, 17 E% protein and 53 E% carbohydrate] enriched in beef tallow, canola oil, corn oil, olive oil or rice bran oil (20 E%) for 32-d periods. In vitro oxidation of LDL was assessed by incubating LDL with hemin and hydrogen peroxide, and measuring the time required for the reaction to reach maximum velocity (lag time). LDL lag times were 93.2 ± 25.8, 95.9 ± 26.4, 104.2 ± 32.7, 108.0 ± 26.6 and 113.1 ± 24.0 min for corn oil, beef tallow, rice bran oil, canola oil and olive oil periods, respectively. When the data from all dietary phases were pooled, LDL 
-tocopherol level (r = 0.30, P = 0.01) and plasma 18:1/18:2 ratio (r = 0.22, P = 0.08) were positively related to resistance of LDL to oxidation. Differences induced by the dietary perturbations in LDL content of 
-cryptoxanthin, lutein/zeaxanthin, lycopene, -carotene or -carotene, and LDL particle size were not related to resistance of LDL to oxidation. In conclusion, in middle-aged and elderly moderately hypercholesterolemic subjects, the consumption of reduced-fat diets enriched in animal fat or vegetable oils with a relatively wide range of fatty acid profiles did not alter the in vitro susceptibility of LDL to oxidation. The advantages of reducing the saturated fat content of the diet were reflected in lower total and LDL cholesterol levels.
Oxidative modification of low density lipoprotein (LDL)6 has been reported to increase the in vitro atherogenicity of the particle (Esterbauer et al. 1990 The fatty acid composition of the diet has been reported to affect the in vitro susceptibility of LDL to oxidation. This may be due to a number of factors including the fatty acid composition of the particle, the particle size, the antioxidant concentration in the particle itself or as yet unidentified factors. LDL particles formed during the consumption of a diet high in polyunsaturated fatty acids have been reported to be more susceptible to oxidation than after consumption of a diet high in saturated or monounsaturated fatty acids (Abbey et al. 1993 The aim of this study was to examine the effect of reduced-fat diets varying in fatty acid composition resulting from the substitution of one predominant fat source for another on the in vitro susceptibility of LDL to oxidation in middle-aged and elderly subjects with moderately elevated plasma LDL cholesterol levels. These data were related to plasma fatty acid patterns, LDL particle size and the antioxidant content of the LDL particle.
Subjects.
Fourteen subjects (8 postmenopausal females, 6 males) with plasma LDL cholesterol levels >3.36 mmol/L on at least two occasions before the start of the study were eligible for participation in this study. Before enrollment, subjects underwent a medical history, physical examination and had clinical chemistry analyses performed. The subjects had no evidence of chronic illness, did not smoke and were not taking medications known to affect plasma lipid levels, vitamin and mineral preparations, or, for the females, hormone replacement therapy. If subjects reported using dietary supplements at the time of screening and wanted to be considered for participation in the study, they were required to terminate use and were re-evaluated after a 2-mo period. Subjects gave their informed consent and the protocol was approved by the Human Investigation Review Committee of New England Medical Center and Tufts University.
Experimental design and diets.
This study included five 32-d phases, each separated by periods during which the subjects consumed their habitual diet. The vegetable oil-enriched diets, canola, corn, olive and rice bran, were designed to meet Step 2 guidelines (The Expert Panel 1993) and were provided to the study subjects in randomized order. The beef tallow-enriched diet, due to its relatively high level of saturated fatty acids, did not conform to Step 2 guidelines. Each of the experimental diets was designed to provide 17% of energy (E%) as protein, 53% E% carbohydrate and 30 E% fat, of which two thirds (20 E%) was contributed by each of five experimental fats (canola, corn, rice bran, olive and beef tallow). With the exception of the experimental fats, all other foods in the diet were the same. The experimental fats were incorporated into various food mixtures. All food and drink were provided by the Metabolic Research Unit of the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University for consumption on site or packaged for take out in amounts that resulted in a stable body weight. Deviations of ±1 kg resulted in an adjustment of energy level of the diet. Triplicate preparations of each complete meal cycle (3 d) for each diet phase were analyzed by Hazleton Laboratories America, Madison, WI. Subjects were required to report to the Metabolic Research Unit at least three times per week, have blood pressure and weight measured, and consume at least one meal on site at each visit. The subjects were encouraged to maintain their habitual level of physical activity. Previous work had demonstrated that within the context of the study design, the length of the study period resulted in stable plasma lipid levels (Schaefer et al. 1996 Biochemical analysis.
Blood was collected in tubes containing EDTA (10 mg/L) after a 14-h fast; plasma was separated by centrifugation at 1100 × g at 4°C. Very LDL (VLDL; d < 1.006 kg/L) were isolated from plasma by ultracentrifugation at 109,000 × g for 18 h at 4°C. Plasma and the 1.006 kg/L infranatant fraction were assayed for total cholesterol and triacylglycerols with an Abbott Spectrum CCX analyzer using enzymatic reagents from Abbott Diagnostics (Dallas, TX) (McNamara and Schaefer 1987 Preparation of LDL.
A modified single discontinuous density gradient ultracentrifugation in a near-vertical rotor (NVT90, Beckman Instruments, Palo Alto, CA) (Chung et al. 1980 Measurement of the in vitro oxidation of LDL.
LDL was oxidized with hemin and H2O2 within 2 h of isolation as previously described (Balla et al. 1991 Fatty acid analysis.
Fatty acid methyl esters were prepared from lipid extracts of plasma as previously described (LePage and Roy 1986 LDL particle size.
LDL particle size was determined as previously described (Campos et al. 1992 Statistical analysis.
The data were analyzed using the Statistical Analysis System (SAS Institute, Cary, NC) version 6.03 for a VAX mainframe 11/780 and version 6.08 for personal computer. ANOVA (PROC GLM) for repeated measures was followed by Tukey's t test at the P < 0.05 level. Results are presented as means ± SD. Pearson's correlation coefficients were calculated to explore the relationship between variables. In addition, a covariance analysis using PROC GLM was used to predict changes in lag time as a function of within-subject changes in continuous variables such as plasma fatty acids, LDL, vitamin E and carotenoid concentrations. Results are presented in the form of a multiple regression equation (Hegsted et al. 1993 The mean age of the study subjects was 63 ± 12 y (range 44-78 y) ; mean body mass index was 27.2 ± 4.3 kg/m2. The mean plasma LDL cholesterol level at the time the subjects were screened for entry into the study was 4.25 ± 0.60 mmol/L (164 ± 23 mg/dL). Other plasma lipid variables were unremarkable at entry into the study and have been previously reported (Lichtenstein et al. 1994a
The aim of this study was to examine the effect of diets consistent with Step 2 guidelines (<30% fat, <7% saturated fatty acids) (The Expert Panel 1993) and one diet of comparable fat level but high in saturated fat on the susceptibility of LDL to oxidation in middle-aged and elderly subjects with moderately elevated plasma LDL cholesterol levels. The only difference among the reduced fat experimental diets was the predominant fat source. The data on susceptibility of LDL to oxidation were evaluated relative to plasma fatty acid patterns, LDL particle size and antioxidant content of the LDL particle.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Steinberg et al. 1989). There is strong evidence that this process occurs in vivo (Palinski et al. 1989, Ylä-Herttuala et al. 1989). Oxidized LDL are taken up by specific receptors on macrophages that do not recognize unmodified LDL (Rohrer et al. 1990, Sparrow et al. 1989). Uptake via these receptors is not down-regulated by the internal cholesterol content of the cell and can result in cholesterol accumulation and foam cell formation (Brown and Goldstein 1983, Steinberg et al. 1989). Additionally, oxidized LDL are chemotactic for monocytes and cytotoxic; they inhibit macrophage mobility and may alter vascular tone (Esterbauer et al. 1997, Steinberg 1997).
, Bonanome et al. 1992, Reaven et al. 1991, 1993b and 1994).
SUBJECTS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). A portion of this work that addressed different experimental questions was published previously (Lichtenstein et al. 1994a and 1994b).
). HDL cholesterol was measured in the supernatant fraction after precipitation of apolipoprotein (apo) B-containing lipoproteins by a dextran sulfate magnesium procedure as previously described (Warnick et al. 1982). LDL cholesterol level was calculated by subtracting HDL cholesterol from the 1.006 kg/L infranatant fraction cholesterol. Lipid assays were standardized through the Lipid Standardization Program of the Centers for Disease Control (CDC), Atlanta, GA. Within-assay and between-assay CV were <2% for cholesterol and <2.5% for triacylglycerols.
) was used to isolate LDL from previously frozen plasma stored at 
80°C. (Cuchel et al. 1996). Briefly, plasma density was adjusted to 1.35 kg/L with solid Kbr, and the sample was layered under 9 g/L NaCl (pH 7.2) with phenylmethylsulfonyl fluoride (0.5 mmol/L) in Beckman Optiseal polyallomer tubes (13 × 48 mm, capacity 4.9 mL). The samples were immediately centrifuged in a Beckman L8-80 ultracentrifuge at 339,000 × g at 4°C for 90 min. The LDL fraction was isolated using a gradient fractionator (Hoefer Scientific Instruments, San Francisco, CA). Immediately after isolation, the cholesterol concentration was determined by the modified enzymatic method previously described (Shireman and Durieux 1993). The cholesterol concentration of the samples was then adjusted to 1.68 mmol/L with 9 g/L NaCl. A pool of plasma frozen under the same conditions as the experimental samples was included in all of the LDL isolation and oxidation assays and remained stable during the analytical period. Within 2 h of isolation of the LDL fraction, the susceptibility to oxidation was assessed.
, Belcher et al. 1993). The final assay concentrations were 0.56 mmol/L of LDL cholesterol, 5 µmol/L hemin and 50 µmol/L H2O2 in 9 g/L NaCl, in a final assay volume of 0.15 mL. Each sample was analyzed in quadruplicate. The oxidation of LDL was monitored by measuring the decreasing absorbance of hemin (405 nm) with a MR600 Microplate Reader (Dynatech Laboratories, Chantilly, VA) controlled by the Immunosoft software (Dynatech) for 150 min. LDL from all experimental periods were oxidized within that time period. Samples from each experimental period for an individual subject were analyzed within the same assay. The resistance of LDL to oxidation is expressed as lag time to oxidation, which was assessed by calculating the time required for the reaction to reach maximum velocity or Vmax (Belcher et al. 1993). The longer the lag time, the more resistant the sample was to oxidation. The interassay CV was 8%.
). The fatty acid methyl esters were analyzed on a Hewlett-Packard (San Fernando, CA) 5890 gas-liquid chromatograph fitted with a 105-m fused-silica capillary column and liquid-phase RTX 2330 (Restek, Port Matilda, PA) and were detected with a flame-ionization detector as previously described (Lichtenstein and Chobanian 1990). Peak identification was validated by chromatography of mixtures of authenticated fatty acid methyl esters.
). Whole plasma was subjected to electrophoresis using 2-16% polyacrylamide agarose gradient gels (PAA 2-16 %, Pharmacia, Piscataway, NJ). Scanning was performed using an LKB Ultrascan XL laser densitometer (LKB Instruments, Paramus, NJ) and LKB GSXL software was used for peak integration. The percentage of relative area of each LDL peak was calculated. The LDL score is an estimate of LDL size and represents the relative areas under all LDL peaks. A lower particle score corresponds to larger LDL particle size.
-Tocopherol and carotenoid analysis.
-Tocopherol and carotenoid concentrations (lutein + zeaxanthin, -cryptoxanthin, lycopene, - and -carotene) in LDL were analyzed using the reverse-phase high performance liquid chromatography (HPLC) method as previously described (Barua et al. 1993). The HPLC system included a Waters pump (Milford, MA) operating at a flow rate of 1.5 mL/min, a 5 µm C18 stainless-steel resolve column (Waters), an autosampler (Millipore, Milford, MA), two absorbance detectors (Thermo Separation Products, Fremont, CA; Varian 2550, Varian, Palo Alto, CA), which were connected in series for simultaneous detection of all analytes, and a dual channel integrator (Perkin Elmer, Norwalk, CT). The mobile phase consisted of acetonitrile/methylenechloride/methanol/n-butanol (90:15:10:0.1, v/v/v/v) with 0.013 mol/L ammonium acetate. Retinyl hexanoate and -apo-8'-carotenyl-myristate in methanol were used as internal standards for tocopherols and carotenoids, respectively. The analyte peaks were identified by retention times and quantified using standard curves of external standards (Sigma Chemical, St. Louis, MO) for each analyte.
).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
and 1994b), as have the plasma fatty acids and lipid response data. The new data are those of in vitro susceptibility of LDL to oxidation and the antioxidant levels.
View this table:
Table 1.
Composition of the study diets as determined by chemical analysis1
-linolenic acid than the other experimental diets. Conversely, the beef tallow- and olive oil-enriched diets had a markedly lower proportion of polyunsaturated fatty acids than the canola, corn and rice bran oil-enriched diets. These data demonstrate that there was a broad range of fatty acid intakes among the diet phases.
View this table:
Table 2.
Plasma lipid concentrations in middle-aged and elderly female and male subjects fed each of the experimental
diets at the end of each 32 d diet phase1
-linolenic acid (18:3) after consumption of the diet enriched in canola oil and a somewhat intermediate fatty acid pattern after consumption of the diet enriched in rice bran oil.
View this table:
Table 3.
Plasma fatty acid patterns in middle-aged and elderly female and male subjects fed each of the experimental
diets at the end of each 32 d diet phase1
View this table:
Table 4.
LDL lag time, plasma 18:1/18:2 ratio, LDL particle score and selected antioxidant constituents in LDL particles isolated from middle-aged and elderly female and male subjects fed each of the experimental diets for 32 d1
View larger version (30K):
[in a new window]
Fig 1.
The susceptibility of LDL isolated from middle-aged and elderly female and male subjects fed each of the experimental diets for 32 d to oxidation after challenging the lipoprotein fraction with hemin and hydrogen peroxide. Each point represents the lag time of LDL observed for an individual. Each line represents a complete data set from one individual subject.
-Tocopherol is the major antioxidant in LDL (Esterbauer et al. 1991). The data from this study suggest that there were no significant differences on the basis of dietary fat consumed on the -tocopherol content of LDL. Differences in the levels of the other antioxidants assessed, -cryptoxanthin, lutein + eaxanthin, lycopene, -carotene and -carotene, were identified on the basis of dietary fat; however, none of these differences were related to LDL lag time.
-tocopherol concentration (r = 0.30, P = 0.01), and the correlation with plasma 18:1/18:2 ratio approached significance (r = 0.22, P = 0.08).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
, Belcher et al. 1993). Hemin has been demonstrated to readily intercalate into LDL particles and cause a rapid oxidation of LDL in vitro (Balla et al. 1991). The resistance of LDL to oxidation was quantitated by measuring the time required for the reaction to reach maximum velocity, which was near the midpoint of the propagation phase. A decrease in hemin absorbance has been reported to parallel the increase in the concentration of conjugated dienes and thiobarbituric acid-reactive substances (Belcher et al. 1993). Because hemin readily intercalates into the LDL particles, the assay assesses the effect of the total LDL particle composition on susceptibility to oxidation. The Cu2+-mediated assay assesses the formation of conjugated dienes and is thought to assess the effect of the surface components of the LDL particle on susceptibility to oxidation (Tribble et al. 1995).
), and there is no indication that the measure of LDL oxidation used would have influenced the outcome of the work. The lack of an effect of dietary fat type on the susceptibility of LDL to oxidation may have been a reflection of the more modest changes in the total fat and fatty acid profile of the diet, consistent with (or slightly greater than) the magnitude of shifts that might be seen after a patient is counseled to reduce the total and saturated fat content of his/her diet.
and Abbey et al. (1993) reported increased oxidative degradation of LDL after subjects consumed high fat diets (45 and 34 E%, respectively) relatively high in polyunsaturated fatty acids (30 and 12 E%, respectively) compared with monounsaturated fatty acids (30 and 17 E%, respectively). Louheranta et al. (1996) reported a positive association (r = 0.294) of dietary linoleic acid and susceptibility of LDL to oxidation as assessed using hemin and hydrogen peroxide in participants of the Kuopio Atherosclerosis Prevention Study. In contrast, similar findings to those reported in this study have been reported by Sarkkinen et al. (1993) comparing the susceptibility of LDL to oxidation after subjects consumed a diet low in total and polyunsaturated fat relative to a Step 1 diet (8 vs. 3 E% polyunsaturated fat, respectively) and Turpeinen et al. (1995) after subjects consumed high fat diets (38 E%) enriched in either polyunsaturated fatty acids (sunflower oil, 13 E%) or monounsaturated fatty acids (rapeseed oil, 15 E%).
, Dejager et al. 1993, Reaven et al. 1994 and 1996, Tribble et al. 1992). In this study, there were no significant differences in LDL particle score among the diet periods and no correlation between resistance of LDL to oxidation and LDL particle size. In the absence of diet-induced hypertriglyceridemia, perhaps the minor shifts in the particle score observed in this study were not of a sufficient magnitude to alter the physical properties of the particles. However, from a practical perspective, within the context of diets recommended for moderately hypercholesterolemic subjects, the results of this study suggest that LDL particle size is not sufficiently changed to have an effect on the in vitro oxidation of LDL.
-carotene content of the LDL particle were observed. However, these differences were not associated with variation in the susceptibility of the LDL particle to oxidation. The relationship between -carotene and susceptibility of LDL to oxidation has previously been investigated by either supplementing diets with -carotene or assessing habitual -carotene intake. Princen et al. (1992), Reaven et al. (1993a) and Shaish et al. (1995) have reported that supplementation with -carotene dramatically increases the -carotene content in LDL; however, there was no evidence that increased -carotene content altered the resistance of the particle to oxidation. Additionally, these investigators (Princen et al. 1992, Reaven et al. 1993a), and others (Croft et al. 1995, Frei and Gaziano 1993, Tribble et al. 1992), found no relationship between unsupplemented LDL -carotene concentrations and susceptibility of LDL to oxidation. Less information is available on the effect of carotenoids other than -carotene on the susceptibility of LDL to oxidation. Although we found small but significant differences in antioxidant content among LDL isolated after the subjects consumed the different diets, these differences appeared to result either from an insufficient magnitude to alter the susceptibility of LDL to oxidation or a lack of major protective effect in the antioxidants identified in the LDL particle.
-tocopherol has been shown to be the most powerful antioxidant in the LDL particle (Esterbauer et al. 1991). -Tocopherol supplementation has been reported to result in a dramatic increase in the LDL -tocopherol concentration and significantly increased resistance to oxidation (Jialal et al. 1995, Princen et al. 1992 and 1995, Reaven et al. 1993a and 1993c). Differences in LDL -tocopherol concentrations, presumably due to variations in dietary intake, in subjects who have not been taking -tocopherol supplements, have not been related to altered susceptibility nor do they correlate with in vitro LDL oxidation (Babiy et al. 1990, Esterbauer et al. 1992, Raal et al. 1995). The exception to this finding is in patients with vitamin E-deficient LDL or active variant angina (Miwa et al. 1995). On the basis of differences in dietary fat, the results of this study confirm the observation of lack of effect on LDL -tocopherol concentration in unsupplemented individuals and susceptibility of LDL to oxidation. However, there was a positive relationship of LDL -tocopherol concentration and resistance of LDL to oxidation (P = 0.01) when the data from all of the diet phases were combined. This result suggests an effect of LDL -tocopherol concentration and susceptibility to oxidation and the possibility that these differences may be related to individual variability among subjects, attributable to as yet unidentified causes.
reported that oleic acid was negatively correlated with the rate of LDL oxidation, whereas linoleic acid was positively correlated with the extent of LDL oxidation. Similar relationships after dietary modification have been reported by others (Bonanome et al. 1992, Reaven et al. 1993b and 1994). When all of our data from the five different diet phases were pooled, there was a trend towards a positive correlation between the plasma 18:1/18:2 ratio and susceptibility of LDL to oxidation (P = 0.08). As noted for LDL -tocopherol concentration, although the diet-induced changes in plasma 18:1/18:2 ratio were not of sufficient magnitude to alter LDL oxidation, variations in the plasma 18:1/18:2 ratio among individuals due to factors in addition to dietary fat were of sufficient magnitude to alter susceptibility of LDL to oxidation. In hindsight, LDL fatty acid profiles, rather than plasma fatty acid profiles, should have been determined. However, there does not appear to be any indication that differences between the two measures would have had a significant effect on the outcome of the study.
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FOOTNOTES |
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Manuscript received 2 February 1998. Initial reviews completed 25 March 1998. Revision accepted 28 May 1998.
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ACKNOWLEDGMENTS |
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The authors thank the staff of the Metabolic Research Unit for the expert care provided to the study subjects, and they gratefully acknowledge the cooperation of the study subjects without whom this investigation would not have been possible.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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-tocopherol supplementation on LDL oxidation. A dose-response study.
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15:190-198-carotene in vivo protects low density lipoprotein from lipid peroxidation in vitro. Effect of cigarette smoking.
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12:554-562-tocopherol on the susceptibility of LDL and LDL subfractions to oxidative modification in humans.
Arterioscler. Thromb.
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14:557-566-carotene.
Arterioscler. Thromb.
1993a;
13:590-600-tocopherol and racemic -tocopherol in humans. Effects on lipid levels and lipoprotein susceptibility to oxidation. Arterioscler. Thromb. 13: 601- 608.This article has been cited by other articles:
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  S. Yu-Poth, T. D. Etherton, C. C. Reddy, T. A. Pearson, R. Reed, G. Zhao, S. Jonnalagadda, Y. Wan, and P. M. Kris-Etherton Lowering Dietary Saturated Fat and Total Fat Reduces the Oxidative Susceptibility of LDL in Healthy Men and Women J. Nutr., September 1, 2000; 130(9): 2228 - 2237. [Abstract] [Full Text]
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S.-C. Lu, W.-H. Wu, C.-A. Lee, H.-F. Chou, H.-R. Lee, and P.-C. Huang LDL of Taiwanese Vegetarians Are Less Oxidizable than Those of Omnivores J. Nutr., June 1, 2000; 130(6): 1591 - 1596. [Abstract] [Full Text]
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