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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:201-207.)
© 1996 American Heart Association, Inc.

Articles

Effects of Diet and Exercise on Qualitative and Quantitative Measures of LDL and Its Susceptibility to Oxidation

Christopher M. Beard; R. James Barnard; David C. Robbins; Jose M. Ordovas; Ernst J. Schaefer

From the Department of Physiological Science, University of California, Los Angeles (C.M.B., R.J.B.); The Medlantic Research Institute, Washington, DC (D.C.R.); and the Lipid Metabolism Laboratory, US Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, Mass (J.M.O., E.J.S.).

Correspondence to R. James Barnard, PhD, Dept. of Physiological Science, University of California, Los Angeles, PO Box 951527, Los Angeles, CA 90095. E-mail jbarnard@physci.ucla.edu.

	Abstract

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Abstract The purpose of this study was to investigate the effects of an intensive diet and exercise program on the quantity and quality of LDL as well as its susceptibility to in vitro oxidation. The diet was low in fat (<10% kcal) and cholesterol (<100 mg/d), while high in complex, unrefined carbohydrates (>70% kcal) and fiber (35 g/1000 kcal). The study was composed of 80 participants in a 3-week residential program where food was provided ad libitum and there was daily aerobic exercise, primarily walking. In each subject, preparticipation and postparticipation fasting blood samples were drawn and LDL was isolated via density gradient ultracentrifugation. LDL particle diameter was determined by gradient gel electrophoresis of serum (n=23). Isolated LDL was either separated into 6 subfractions by saline gradient equilibrium ultracentrifugation (n=26) or subjected to in vitro copper oxidation (n=32). Significant reductions (P<.01) in serum levels of cholesterol (20%), LDL-cholesterol (20%), HDL-cholesterol (17%), triglycerides (26%), and glucose (16%) as well as in body weight (4%) were noted for the total population. The mean particle diameter of the LDL increased (24.2±0.2 to 25.1±0.14 nm, P<.01) and was correlated with the reduction in serum triglycerides (r=.58, P<.01). Six of 22 subjects changed in LDL phenotype from B (25.5 nm) to A (>25.5 nm). The percentage of LDL-cholesterol carried in the more dense subfractions fell significantly, while that carried by the less dense fractions increased. Initial oxidation levels fell (21%), while the lag time before copper-induced oxidation increased (13%). Reductions were observed in both the rate of oxidation (16%) and peak oxidation (20%). All of these changes should result in a dramatic reduction in the risk for atherosclerosis and its clinical sequelae.

Key Words: LDL phenotype • antioxidants • LDL density • LDL subfractions

	Introduction

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Epidemiological studies have firmly established a relationship between the incidence of atherosclerosis and dietary saturated fat, total dietary fat, and plasma lipid levels.¹ The current model for the development of atherosclerosis points to an interaction between plasma LDL, endothelial cells of the arterial lumen, and monocytes.^{2 3} LDL particles isolated from lesions and normal artery walls are different from plasma LDL, having undergone lipid peroxidation.^{3 4 5} Cholesterol oxides and oxidized lipids in lipoproteins may be derived from exogenous and endogenous sources.^{6 7} Factors influencing the oxidizability of lipoproteins include apo B structure, particle glycosylation, particle lipid and fatty acid composition, particle size, and modification of dietary fatty acid and antioxidant intake.^{6 8}

LDL mean particle diameter has been classified into two phenotypes: A and B.⁹ Phenotype A particles have peak diameters >25.5 nm with a skewing toward the large particles, while phenotype B particles have peak diameters <25.5 nm with a skewing toward the smaller particles. Mean particle diameter has been inversely correlated with plasma triglycerides in both men and women.¹⁰ Smaller, more dense subfractions of LDL have been associated with an increased risk of atherosclerosis.^{11 12} Several studies have supported the hypothesis that the smaller, more dense particles are more susceptible to oxidation, and thus more atherogenic.^{13 14 15 16} This may be due to intrinsic changes in particle structure or the ability of small, dense LDL to interact with glycocalyx-bound molecules.^{15 16}

In previous studies, we have documented that a low-fat, high-complex-carbohydrate, high-fiber diet in concert with daily aerobic exercise reduces serum total and LDL cholesterol as well as triglycerides.^{17 18} It was the intent of this study to investigate the effects of such a protocol on the physical characteristics of LDL and its susceptibility to oxidation. These manipulations were hypothesized to result in a reduction in mean LDL particle density, increased average LDL diameter, and increased resistance of LDL to oxidation.

	Methods

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Subjects and Design
The total sample population (N=80) was drawn from participants in the Pritikin Longevity Center 3-week residential program. There were 38 women and 42 men with an average age of 58±2 years. Diseases under drug therapy included 3 subjects with hypercholesterolemia, 3 with combined hyperlipidemia, 6 with diabetes, 1 with diabetes and hypertension, and 1 with hypertension.

Due to the availability of subjects, the amount of blood provided, and the multiple analyses involved, the study was conducted in three phases. Each phase was designed to involve approximately 25 subjects and included all participants enrolled in any given 3-week session. Lipid changes were measured in all three phases. Phase 1 also included LDL particle size, phase 2 included LDL isolation and density determinations, and phase 3 included LDL isolation, oxidation, and antioxidant determinations.

Program
Once enrolled, the subjects underwent education and orientation to the program. The subjects had ad libitum access to high-complex-carbohydrate/low-fat foods and were encouraged to exercise for up to 1.5 hours daily. The program and nutrient values have been described in detail previously.^{17 18} In brief, fat provided <10% of calories, protein 10% to 20%, and carbohydrate 70% to 80%. Daily cholesterol intake was <100 mg, fiber at least 35 g, and sodium no more than 1600 mg.

Formal exercise classes were held 5 days per week and included 15 to 20 minutes of stretching, flexibility, and muscle conditioning followed by 45 minutes of aerobic exercise on a treadmill or exercycle at the age-adjusted or disease-dictated training heart rate. The participants also were encouraged to walk on their own, especially on the weekends.

Immediately before the start and immediately at the conclusion of the 3-week program, 12-hour fasting blood samples were taken in two 10-mL Vacutainers (Becton-Dickinson Vacutainer Systems) containing SST clot activating gel between 6:30 AM and 8 AM. The samples were allowed to clot and the serum separated by high-speed centrifugation for 15 minutes. One tube was analyzed for serum lipids while the second tube constituted the experimental sample from which LDL was isolated. Prior to serum separation in tube No. 2, a protease inhibitor cocktail was added to prevent degradation of the apolipoproteins. The components of the cocktail and their final concentrations were as follows: hexadimethrine bromide (24 µg/mL), benzamidine (2 mmol/L), e-aminocaproic acid (5 mmol/L), and soybean trypsin inhibitor (20 µg/mL).

Determination of Serum Lipids and Glucose
Triglyceride, HDL-cholesterol, total cholesterol, and glucose levels were measured using standard enzymatic procedures on an Olympus Autoanalyzer (Smith-Kline Beecham Laboratories). The LDL-cholesterol was calculated as follows: (LDL-cholesterol)=(total cholesterol)-[HDL-cholesterol+(triglycerides/5)], as described by Friedewald et al,¹⁹ except when triglyceride values were >400 mg/dL. Tests were all run on the day of blood collection along with standards from the Centers for Disease Control and Prevention and control samples.

LDL Isolation and Analysis
The LDL was isolated from serum by a saline gradient ultracentrifugation sequential flotation procedure first published by Schumaker and Puppione²⁰ and modified by Chatterton et al.²¹ The blood was drawn and allowed to clot as previously described. EDTA, sodium azide, and gentamicin sulfate were added to final concentrations of 0.04% (wt/vol), 0.05% (wt/vol), and 0.005% (wt/vol), respectively. These concentrations were maintained throughout the isolation procedure.

The density of the solution was adjusted to 1.063 g/mL (20°C) by addition of a saturated NaCl solution. The assumption was made that serum is approximately 6% protein (by volume) and has the same density as aqueous 0.195 mol/L NaCl. LDL, IDL, and VLDL were isolated by centrifugation at 150 335g for 22 hours at 20°C in a Ti-70 rotor and L8-70M ultracentrifuge (Beckman). The top 5 mL of each tube (containing VLDL, IDL, and LDL) was collected, the density of the background salt solution adjusted to 1.019 g/mL (20°C), and the run repeated. VLDL and IDL were then removed in the top 6 to 10 mL of each tube. The remaining fluid was discarded by withdrawing it in a Pasteur pipette until no more than 1 mL of fluid remained in the bottom of the tube. The LDL pellet was then gently resuspended using a 200 µL Pipetman (Rainin Instrument Co, Inc) and stored under N₂ gas at 4°C until use within the next 24 hours. If the sample was to be used at a more distant time, the sample was stored at -70°C with added EDTA and a 50% sucrose solution to prevent damage to or aggregation of LDL particles.^{22 23}

All saline solutions were degassed under house vacuum and saturated with nitrogen prior to addition to the lipoprotein suspensions. Polycarbonate bottles were used in the Ti-70 rotor and each was layered with nitrogen before capping. During the removal of lipoproteins from the tubes, care was taken to avoid air bubbling through the solutions. The protein content of the isolated LDL was determined by the micro-Lowry method²⁴ using an assay kit from Biorad. The cholesterol and triglyceride analyses were performed using enzymatic, colorimetric procedures adapted from those described by Warnick.²⁵ All enzymes were obtained from International Bioproducts Inc.

LDL Size Determination
Frozen serum from 23 subjects (7 women, 16 men) was used to determine the effect of the program on mean LDL particle diameter. The size of the LDL particles was determined via electrophoresis as adapted from the procedure described by Krauss and Burke.²⁶ The serum was adjusted to 20% (wt/vol) sucrose and 10 µL placed in each lane of a polyacrylamide gradient gel (2% to 16%, Isolab, Inc). The sample was run at 12°C to 14°C and 125 V for 24 hours in Tris-boric acid-Na₂ EDTA buffer, pH 8.3 along with two standards supplied by Dr R. Krauss, Berkeley, Calif, and two control samples. Pre and post samples were run at the same time. The gels were fixed in a solution containing methanol, acetic acid, and Coomassie brilliant blue R-250. They were then destained with 20% methanol followed by 9% acetic acid. The gels were then analyzed by densitometry. The diameter of the absorbance maxima was calculated from a calibration curve using the Krauss standards of known diameters. The error for this procedure has been shown to be 2.09%.

LDL Density Determination
The serum of 26 subjects (11 women, 15 men) was used to isolate LDL samples, which were then separated into 6 subfractions using the procedure reported by Shen et al²⁷ as modified by Chatterton et al.²¹ The LDL sample was raised to a density of 1.040 g/mL in a volume of 3.42 mL by using a saturated NaCl solution. The LDL sample was then gently layered between 4.29 mL of 1.063 g/mL saline and 4.29 mL of 1.019 g/mL saline in Beckman Ultraclear centrifuge tubes for an SW-41 rotor. All solutions contained EDTA, sodium azide, and gentamicin sulfate as described previously. After centrifugation (197 000g) for 42 hours at 20°C, the tubes were fractionated by piercing the bottom of the tube and injecting Flourinert (3M Co.), an inert, dense fluorocarbon. This permitted the removal of fractions from the top of the tube using the ISCO density gradient fractionator (model 185, ISCO). The density of each fraction was determined with an Abbe-3L refractometer (Bausch & Lomb) on a saline standard run with each spin. The lipoprotein fractions were stored under nitrogen at -70°C for later cholesterol and protein analyses.

In Vitro Oxidation
The resistance of the LDL particles to oxidation was assayed using the LDL from 32 subjects (20 women, 11 men) by use of a continuous monitoring procedure of the oxidation process adapted from that described by Esterbauer et al.²⁸ Prior to oxidation, the isolated LDL samples were dialyzed overnight against a 1x Dulbecco's phosphate-buffered saline solution devoid of calcium and magnesium salts (Irvine Scientific). The buffer was changed twice and the oxidation experiment was performed within 24 hours of dialysis. The EDTA-free LDL sample was diluted to 0.25 mg LDL protein/mL with the 1x phosphate-buffered saline solution described above. To initiate oxidation, an aqueous 200 µmol/L CuSO₄ solution was added to a final concentration of 2 µmol/L CuSO₄. The reaction was conducted at room temperature and the optical density obtained every 10 minutes until a maximal absorbance was recorded for three recordings or until the absorbance began to decay. The spectrophotometer was set at 234 nm, the maximal absorbance for conjugated dienes.²⁸ The initial reading at time zero for each sample was used as a self-balancing value and subtracted from subsequent readings. The spectrophotographic analyses were analyzed for a lag phase, a propagation phase, and a decomposition phase. The end of the lag phase was defined as the intercept of straight lines drawn along the slopes of the lag and propagation phases.²⁸ The concentration of conjugated dienes present in the samples was a calculated value as described previously.^{6 28}

LDL Antioxidant Analysis
The content of -tocopherol was determined from frozen LDL samples using the high-performance liquid chromatography methodology described by Bieri et al.²⁹ The content of ß-carotene was determined by the colorimetric method described by Roels et al.³⁰

Data Analysis
All data are expressed as mean±SEM. The data were analyzed using ANOVA followed by Tukey's post hoc test or by a repeated-measures paired t test when appropriate. Pearson simple correlation procedures were used to assess the relationship between changes in variables. All statistics were run using the SAS statistical package (SAS Institute, Inc). Treatment effects are considered significant at a value of P<.05.

	Results

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Serum and Weight Data
The serum data analyzed from 79 of the 80 subjects showed significant reductions (P<.01) in all areas as shown in Table 1. The serum results for men and women also were analyzed. As expected, women had significantly higher HDL (1.6±0.05 versus 1.2±0.05 mmol/L) and lower body weight (85±4 versus 112±5 kg) upon entry into the program than men. The percent change in body weight (4% in women versus 5% in men) was significantly different between the sexes. However, the percent changes in the lipid and glucose values were not significantly different.

View this table: [in this window] [in a new window]   Table 1. Serum Values and Body Weight of Total Sample

The serum lipid data were also analyzed for each of the three phases. There were no significant differences for any of the cholesterol reductions among the three groups but there was a significant difference in triglyceride reductions. Phase 2 achieved only a 19% reduction compared with 32% and 28% for phases 1 and 3, respectively.

LDL Particle Diameter
The mean diameter of the LDL particles increased from 24.2±0.2 to 25.1±0.14 nm (P=.01). The mean increase was 0.9±0.19 nm. The mean diameter in men increased from 24.2±0.28 to 25.0±0.16 nm (P<.05), while the mean diameter in women increased from 24.4±0.23 to 25.4±0.26 nm (P<.01). The difference between the change or increase in men and women was not statistically significant. As can be seen in Fig 1, the change in LDL size was correlated with the change in triglycerides (r=.58, P=.01). There was also a significant correlation (r=.54) between the triglyceride level and LDL particle size for the pre data.

View larger version (14K): [in this window] [in a new window]   Figure 1. Correlation between change in triglycerides and change in LDL particle size.

The increase in LDL size resulted in an altered phenotype in some subjects. Entering the program, one of the 23 subjects was classified as phenotype A, while after the program, 7 subjects were classified as phenotype A. The subjects who converted from phenotype B to A had triglyceride decreases ranging from 0.25 to 2.2 mmol/L.

LDL Subfraction Composition
The fractions collected were as follows with density in milligrams per deciliter in parentheses: 1 (1.021), 2 (1.027), 3 (1.034), 4 (1.044), 5 (1.053), and 6 (1.063). The distribution of cholesterol in the subfractions changed significantly. The percent of cholesterol in subfractions 4 and 6 fell while that of 2 and 3 rose (Fig 2). Changes in the ratio of cholesterol to protein did not change significantly in any fraction. Similar results were observed for both men and women.

View larger version (14K): [in this window] [in a new window]   Figure 2. Effect of lifestyle modification on the percent of cholesterol in the various LDL subfractions.

Oxidation of LDL Particles
The results of subjecting the isolated LDL particles to the oxidizing agent, copper, may be seen in Table 2. The value for initial diene concentration was not calculated because the initial absorbance was used as the standard in the calculation of the various diene values. No standard for diene concentration was used because the LDL particle itself absorbs in the 234-nm UV wavelength, and the most accurate calculations are obtained from the initial absorbance of the LDL. The initial spectrophotometer reading did, however, fall significantly (21%), suggesting reduced basal levels of oxidation. There was a significant increase in the lag phase (13%) and the time to peak oxidation increased by 6%, approaching statistical significance (P=.06). The peak oxidation of the particles fell (20%), as did the rate of oxidation (17%). No significant change was noted in -tocopherol levels, while ß-carotene was significantly elevated (46%) in LDL isolated after the program. The increase in ß-carotene, however, did not correlate with the change in lag time. Similar results were obtained for men and women for all oxidation data.

View this table: [in this window] [in a new window]   Table 2. Summary of Oxidation Data

	Discussion

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The reductions in body weight, serum lipids, and serum glucose reported in this study are consistent with prior results of Barnard et al^{17 18 31 32 33} using the same diet and exercise protocol on a variety of subjects and by others^{34 35} using similar dietary guidelines. There is a possibility of transitory effect given the 3-week study period; however, if such lifestyle changes are maintained over a 4- to 5-year period, the changes in serum values remain to the degree that subjects maintain adherence to the dietary guidelines.^{31 32 34}

Considerable controversy has been caused by the tendency for very-low-fat diets to reduce serum HDL levels. In the United States and other western countries there is an inverse relationship between the risk for atherosclerosis and HDL-cholesterol levels.¹ In countries traditionally consuming low-fat, high-complex-carbohydrate diets, this correlation is not found.^{36 37} In such countries, HDL-cholesterol levels tend to be below the minimal safety standards set by the National Cholesterol Education Program, yet coronary artery disease (CAD) is rare. Research by Brinton et al^{38 39} suggests that reduction in HDL-cholesterol due to a reduction of fat in the diet is due primarily to a modification in apo A-1 production by the liver and intestines, while differences in HDL between individuals on diets high in fat content appear to be due to an elevated clearance of apo A-1 and HDL particles associated with a reduced ratio of lipoprotein lipase to hepatic lipase.³⁹

The effects of the exercise component of the program probably played a major role in the reductions in serum triglycerides and glucose. Aerobic exercise has been shown to reduce serum triglycerides independent of other factors.⁴⁰ It also accelerates the clearance of glucose and increases insulin sensitivity of skeletal muscle.³³ Some recent studies^{41 42 43 44} have reported that serum triglyceride and glucose/insulin levels rise in response to low-fat, high-carbohydrate diets. These studies used glucose, fructose, and other simple or refined carbohydrates as a significant portion of the diet, fed the subjects isocalorically, and did not include exercise. Dietary studies⁴⁵ comparable with the present one that did not include exercise also showed reductions in triglyceride levels but less than the 26% found in this study.

The reduction in serum LDL should significantly reduce the risk for CAD. Further reduction in CAD risk is also indicated from the qualitative changes in the remaining LDL. Austin et al¹¹ reported that individuals with LDL phenotype B had a threefold increase in myocardial infarction risk and that this phenotype was associated with reduced HDL-cholesterol and increased plasma triglyceride, VLDL, and IDL levels. Subsequent studies by others^{46 47} have confirmed that subjects with CAD generally exhibit the smaller, more dense type B LDL more often than do non-CAD control subjects. The size of LDL appears to be influenced by both genetics (33% to 50% of variation)^{10 48} and lifestyle factors such as body weight, smoking, and plasma triglyceride, apo B, and VLDL levels.^{46 48 49 50 51} The increase in mean particle diameter and the fact that 26% of the population showed a shift from the B phenotype to the A phenotype in this study are very significant from a clinical point of view. Based on a 6-fraction separation of LDL, this represents an increase in mean particle diameter across 2 to 3 discrete LDL size subfractions.⁵⁰ These changes are in opposition to those reported by Dreon et al,⁴² who used primarily refined carbohydrates and fed isocalorically.

Several studies^{11 46 47} have reported that plasma triglyceride level is the single most significant predictor of LDL phenotype. The significant correlation between serum triglycerides and LDL particle size found for the pre data supports these earlier studies. In the Framingham study, change in LDL phenotype over a 3- to 4-year period was highly correlated with change in triglycerides.⁵⁰ Thus, it was no surprise to find that the change in particle size in the present study was significantly correlated with the change in triglycerides. However, merely reducing triglycerides may not necessarily alter phenotype. Several drug intervention trials aimed at reducing triglycerides reported no shift from phenotype B to phenotype A in the presence of highly significant triglyceride reductions.^{52 53} Others⁵⁴ have reported changes with drug therapy. Beltz et al⁴¹ and Stacpoole et al⁴⁴ have indicated that there is an altered production of VLDL on a diet low in fat such that larger, less dense, triglyceride-rich particles are produced and metabolized into larger, less dense LDLs. Thus, diet may be more effective than drugs for determining LDL size and density.

Groups that are at an increased risk of presenting with CAD often exhibit a greater percentage of total LDL-cholesterol in the smaller, more dense subfractions 4 to 6.^{55 56} The shift in distribution of cholesterol from the dense fractions to the less dense in this study suggests a reduction in atherogenic risk from a molecular standpoint. Unfortunately, the studies of LDL particle size and subfraction shift were performed on separate groups, so correlational analysis could not be performed. The decrease in small, dense particles agrees with an earlier study by Williams et al,⁵⁷ which also emphasized the importance of exercise. When subjects were placed on a calorie-restriction diet, only a slight reduction in small, dense particles was found. When aerobic exercise was added to the calorie restriction, a much more significant decrease in dense LDL particles was noted. The present study combined exercise with a low-fat, high-complex-carbohydrate diet and did not stress caloric restriction, which would be more compatible with long-term eating habits.

The data obtained from in vitro oxidation show that the resistance of LDL to oxidation did increase as shown by an increase in the lag phase, reduced rate of oxidation, and lower peak oxidation values. Earlier studies have reported a significant effect of size and subfraction upon the resistance of LDL to in vitro oxidation.^{14 16} Regnström et al⁵⁸ reported that the severity of CAD was independently associated with the susceptibility of isolated LDL to copper-induced oxidation in men. The lack of any correlation between the increase in ß-carotene and the changes in in vitro LDL oxidation was not a complete surprise. Some have found a significant increase in resistance of LDL to in vitro oxidation after dietary supplementation with high levels of vitamins.^{59 60} However, others have found no relationship between the antioxidant content and the oxidation profile of LDL in those with and without CAD.⁶¹ It has been suggested that the normal levels of unsupplemented antioxidants may be unable to counteract the impact of the many other pro-oxidant factors, and this may account for some of the poor correlation between both -tocopherol and ß-carotene and in vitro oxidation.^{62 63}

Thus, it would appear that changes in particle characteristics play a larger role in the reduction in oxidation. Tribble et al,¹⁴ de Graaf et al,¹⁵ and Chait et al¹⁶ have all reported that small, dense LDL particles are oxidized more easily and to a greater degree than are large, less dense particles. The reason has not been elucidated but may be a combination of residence time in the plasma, protein content, glycosylation, lipid content, membrane fluidity, and numerous other factors. The reduced amount of oxidized LDL measured initially may have been due to the increase in ß-carotene, a reduction in residence time, or a reduced dietary intake of oxidized lipid on the very-low-fat diet. It is known that diet can be a source of oxidized lipid,^{7 64 65} which may initiate or sensitize lipoproteins to oxidative stress in vitro. The diet was free of common sources of oxidized lipid such as reheated meats and fried food. This protocol also reduced serum glucose, a molecule that has been linked to an increased oxidizability of LDL in diabetics.^{66 67 68} The actual mechanism for such an effect is unclear but may be an altered interaction of the LDL with endothelial cells or affinity for the LDL receptor.

In summary, the diet and exercise protocol used in this study significantly altered both the quantity and quality of LDL. On average, LDL was reduced by 20%. The mean LDL particle diameter increased, there was a shift from LDL phenotype B to LDL phenotype A, there was a significant increase in cholesterol carried by large LDL at the expense of small, and the resistance of LDL to oxidation was increased. Most of the changes can be attributed to dietary factors, though exercise likely played a role in reducing serum triglyceride and glucose concentrations. All of these changes should result in a dramatic reduction in risk for atherosclerosis and its clinical sequelae and may also help to explain our recent observation of increased coronary vasodilatory reserve.⁶⁹

	Acknowledgments

 
We thank Robert Pritikin, director of the Pritikin Longevity Center, for recommending that his participants volunteer for this study. We also thank the volunteers for donating their blood.

Received August 22, 1995; accepted November 27, 1995.

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