Original Research

Effects of Dietary Fat and Endurance Exercise on Plasma Cortisol, Prostaglandin E2, Interferon- and Lipid Peroxides in Runners
Jaya T. Venkatraman, PhD, CNS, FACN, Xiaohong Feng, MS, and David Pendergast, EdD Department of Physical Therapy, Exercise and Nutrition Sciences (J.T.V., X.F.), Department of Physiology (D.P.), State University of New York at Buffalo, Buffalo, New York Key words: cortisol, diet fat, exercise, interferon- , prostaglandins, peroxides, runners
Objective: Exercise and the neuroendocrine and oxidative stress it elicits on immune function is modulated by dietary fat intake. The effects of increasing dietary fat on endurance exercise-induced alterations (80% of VO2max for 2 hours) in the plasma levels of cortisol and prostaglandin E2 (PGE2), interferon- (IFN- ) and lipid peroxides were investigated. As higher levels of cortisol, PGE2 and lipid peroxides could be immunosuppressive, the effects of different levels of dietary fat on these measures in runners were determined. Methods: Healthy trained runners (males and females) consumed serially 15% fat diet (of daily energy), 30% fat diet and 40% fat diets for four weeks each. In the last week of each diet period the subjects ran to exhaustion at 80% of their VO2max and blood was drawn pre- and post-run. Cortisol, IFN- , PGE2 and lipid peroxides were determined using standard techniques. Results: Pre-exercise levels of plasma cortisol were elevated, IFN- was unchanged and PGE2 and lipid peroxides decreased on the 40%F diet compared to 30%F and 15%F. Post-exercise levels of plasma cortisol (p 0.004), PGE2 (p 0.0057) and lipid peroxide levels increased (p 0.0001) after endurance exercise on all diets. The rates of increase of plasma cortisol levels during exercise were similar on all three diets. Although absolute cortisol levels were higher in the high fat group, the rate of increase of plasma cortisol level during exercise was similar on each diet. The dietary fat levels did not affect IFN- , however, PGE2 and lipid peroxides decreased with increasing fat at baseline at 40%F level (p 0.01; 30%F vs. 40%F: p 0.002; 15%F vs. 40%F: p 0.007). Conclusions: Data from the present study suggest that higher levels of fat in the diet, up to 40%, increase endurance running time without adverse effects on plasma cortisol, IFN- , and lipid peroxide levels.

INTRODUCTION
Intense exercise has been associated with both immunosuppression and oxidative stress. It has also been suggested that high fat diets may lead to increased neuroendocrine and oxidative stress and further immunosuppression [1]. The elevated oxygen consumption (VO2) and associated stress during aerobic exercise is reported to result in increased neuroendocrine stress (increased cortisol, prostaglandins (PGs), interferon(IFN- ) and the generation of free radicals. Both the intensity and duration of the exercise determine the level of these stresses. Restriction of dietary fat intake to 16% of total energy intake has been shown to be detrimental to endurance performance [2,3] as it compromises intramuscular fat stores [4] in

trained runners. Low fat diets also compromise the intake of micronutrients and fats instrumental in immune function. Intramuscular fat stores [4], endurance performance [2,3] and micro- and macronutrients can be restored by higher fat (40%) intake [5]. Although high fat diets may improve performance, it has been suggested that they may compromise immune function [1]. Fat intake may affect cortisol, PGs, IFN- and lipid peroxides [1,6]. Our observations in a previous study [7] on the suppression of proliferation response in peripheral blood mononuclear (PBMN) cells to T cell mitogens after exercise, elicited our interest in examining whether the decrease in proliferation response could be related to increases in the level of PGE2 after intense exercise. We have previously reported increases in

Address reprint requests to: Jaya T. Venkatraman, Ph.D., Associate Professor, Nutrition Program, Department of Physical Therapy, Exercise and Nutrition Sciences, State University of New York at Buffalo, 15 Farber Hall, Buffalo, NY 14214. E-mail: jtv@acsu.buffalo.edu

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natural killer (NK) cell number with exercise and dietary fat [7]. We propose that the increase in NK cells would be associated with an increase in plasma IFN- levels as suggested by studies on animal models [1]. Neuroendocrine factors released in situations of stress, such as intense exercise, are suggested to be responsible for the exercise-induced changes in the immune system [8 12]. At the present time, there are no reports on the effects of dietary fat on exercise-induced alterations in neuroendocrine hormones. Recent evidence suggests that elevated oxygen consumption may increase free radical activity [13]. Little information is available on immunological conditions modulating the susceptibility to peroxidation [14]. Evidence indicates that free radicals play an important role as mediators of skeletal muscle damage and inflammation after strenuous exercise. Oxidative stress inflicted by monocytes/macrophages is recognized as an important immunosuppressive mechanism in several human diseases. IL-2 biosynthesis is suppressed by oxidative stress [15]. NF- B is activated by oxidative stress causing activation of genes responsible for synthesis of inflammatory cytokines. Oxidative stress may up-regulate inflammatory cytokines thereby causing immunosuppression [16]. Both IFN- and cortisol may attenuate oxidative stress [17,18] while PGs of the E series may increase oxidative stress [19]. The overall aim of this study was to determine if increasing dietary fat intake from 15% to 30% and then from 30% to 40% of total energy and the resultant increase in exercise time would affect the neuroendocrine and oxidative stress responses in training runners. We hypothesized that a low-fat diet (15%) would result in higher cortisol, PGE2 and lipid peroxides, and decrease IFN- prior to and after an endurance run of short duration compared to a diet higher in fat (30%) and with longer running time (40%). In addition, we propose that increasing dietary fat intake further (40% of total energy) would not significantly affect cortisol, PGE2, IFN- or lipid peroxides. and men, respectively. The mean age of the runners was 35 3 years; height was 1.67 .02 m and 1.81 .03 m, and the body masses were 58.0 2.2 kg and 72.6 3.0 kg for women and men, respectively. The subjects completed a questionnaire to document training distance, medical history, and dietary pattern.

Experimental Diets
Diets were designed such that total energy intake equaled estimated energy expenditure. Three levels of dietary fat were studied: 15% energy from fat, 30% energy from fat, and 40% energy from fat. Based on the fact that a high fat diet is postulated to suppress the immune system and increase the risk of cardiovascular disease in the current experiment we were required by the Human Subjects Committee to increase the percent fat stepwise (15% to 30% to 40%). The level of protein intake was set at 15% of total energy intake for all diets. The diets were based on an initial consultation including three-day food-intake records, activity records, and a list of food preferences. Based on this information, three different seven-day menus were generated for each subject using Auto-Nutritionist IV (First Databank, San Bruno, CA) [5,7,20]. The diets were designed to meet all the United States Recommended Dietary Allowances (USRDA), including energy requirements, and had a fatty acid ratio of 1:1:1 (saturated:monounsaturated:polyunsaturated). The subjects were given the seven-day menus as examples, but they selected their own food. Each diet was continued for 28 to 31 days. The subjects recorded their food intake after each meal and returned the records weekly for review and to check compliance with the diets. The diets were analyzed for nutritional composition with Auto-Nutritionist IV for macronutrients, micronutrients, and fatty acids composition. Increasing fat intake progressively protected the subjects from potential negative affects of 40%-fat on immune or blood lipoprotein responses, although none of the subjects had to be eliminated from the study. The non-random order introduced a potential order effect error. The number of subjects in each diet group was as follows: 15%F7 women and 7 men; 30%F9 women and 6 men; 45%F 8 women and 6 men. The differences in subject numbers for the three diet interventions reflect the unavailability of the data for selected subjects at various time intervals. The missing data was due to failure to follow the diet, missed blood samples, subject injury or scheduling conflicts at one point, but not at other points, in the protocol.

METHODS AND MATERIALS

Subjects
The data presented in this paper were collected as part of a large project that examined the effects of increasing the level of dietary fat from 15% to 40% of total energy intake on the performance, biochemical and metabolic aspects, cardiovascular risk factors, and immune status of well-trained runners. All procedures were performed according to the guidelines of American College of Sports Medicine (ACSM) and were approved by the Human Subjects Committee of the School of Medicine at the State University of New York at Buffalo, Buffalo, NY. The subjects were informed about the procedures and risks associated with the study and gave written consent. The subjects were highly fit but not elite distance runners with VO2max values of 50 2 and 58 3 (mL/min/kg) for women

Protocol for Endurance Performance

Data on VO2max were collected at the beginning of the study. Data on endurance performance were collected at the beginning and end of each diet period. Subjects walked on a treadmill for five minutes at about 30% of their VO2max, then speed was increased to 9.7 or 11.3 km/hour (women and men

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respectively) at zero grade (40% VO2max) for five minutes, and then speed was increased to 60% of their VO2max for 10 minutes. After that, the subjects were stopped for blood mea surement. The subjects then began running at 80% VO2max, and ran until voluntary exhaustion. The testing was carried out at the same time of day, day of the week and month to avoid circadian variations and menstrual-cycle variations in women on each diet.

RESULTS
Increasing the dietary fat level from 15% to 40% did not affect body weight (57 1.8 and 72 2.2 kg for women and men respectively) body fat (18 1.2 and 16 0.8% for women and men respectively), resting heart rate or systolic (118 7 mm Hg) and diastolic (73 5 mm Hg) blood pressure.

Collection of and Analysis of Blood

Blood was collected from the antecubital vein of the runners in heparinized tubes at base line (resting level) before and immediately after each test. The blood was centrifuged at 10,000 g for 10 minutes at 4 C. Clear plasma was stored at 70 C for analyses. Cortisol ELISA kit was purchased from Neogen Corporation (Lexington, KY). The kits were supplied with 96 well microtiter plates pre-coated with anti-cortisol rabbit antibody. Cortisol was extracted from plasma by adding 1 mL of ethyl ether to 100 L of plasma. The manufacturers procedures were followed explicitly. The plates were read at 450 nm in a microplate reader (Biotek Instruments Inc., Winooski, VT), and a standard curve was constructed to quantitate the concentration of cortisol in the samples. Plasma PGE2 was determined by the ELISA technique utilizing kits purchased from Neogen Corporation and following their protocol. The kits were supplied with 96 well microtiter plates pre-coated with anti-PGE2 rabbit antibody. The plates were read at 450 nm in a microplate reader, and a standard curve was constructed to quantify the concentration of PGE2 in the samples. IFN- level in plasma was analyzed by ELISA, utilizing kits purchased from Genzyme Diagnostics (Cambridge, MA) and following their protocol. The plates were read at 450 nm in a microtiter plate reader, and a standard curve was constructed to quantify IFN- concentrations in the samples. The lipid-compatible formulation of the Peroxoquant peroxide assay kit was purchased from Pierce Chemical Co. (Rockford, IL) and used following their protocol. The microtiter plates were read at 600 nm. A standard curve was constructed to quantify the concentration of H2O2.

Composition of the Diets

Total energy consumption (Table 1) was not different between men and women when adjusted for body mass (36 7 and 37 6 Kcal/d/kg for women and men, respectively). In spite of the fact that the subjects were requested to consume the same number of calories of food on each diet, there was an increase of 435 27 Kcal, on average, with increasing level of fat in the diets (Table 1). The percent of protein in the diets of the men was significantly higher compared to that of the diets of the women. Protein consumption was significantly higher with 40%F, compared to 15%F and 30%F diets. The total amount of fat consumption was significantly greater for men than women on all three diets (p 0.0001). The level of polyunsaturated fatty acids (PUFA) was lower in all the diets when compared with saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA). The PUFA/SFA (P/S) ratio was in the range of 0.46 0.75 in the diets and decreased as the level of fat increased. The levels of oleic acid (18:1), and linoleic acid (18:2 -6) were higher in the diets of men compared with women (Table 1). The levels of oleic acid and linoleic acid increased as the level of dietary fat increased. The level of vitamin E, as predicted from the nutrition analysis, increased with the increase in the level of dietary fat, with significantly greater intakes occurring in men compared to women (Table 2). The amount of vitamin A intake was not affected by an increasing level of dietary fat, while the amount of -carotene intake was significantly higher (p 0.02) in the 15%F compared with the 30%F and 40%F diets in both women and men. Calcium and zinc intake increased with the increase in dietary fat intake. The intake of zinc by the runners was lower than the RDA values (15 mg/day). In male subjects, the level of iron was higher on all diets than the RDA values (10 mg/day). Vitamin C intake was much higher than RDA values (60 mg/day) in both men and women and significantly greater in men than in women (p 0.037). Selenium intake, although significantly higher in men compared with women (p 0.0001), was around RDA values (0.07 mg/day for men and 0.055 mg/day for women).

Statistical Analysis
The values are presented as mean SEM. Statistical analyses of the data were carried out using Statview 4.0/Super ANOVA package software (Abacus Concepts, Berkeley, CA). Data were analyzed by analysis of variance using 3 (diets) 2 (gender) 2 (exercise) factorial design; where a significant F ratio was found, Fishers protected least significant difference test was used to describe differences in the means among groups (p 0.05). Wherever there was no significant effect of gender, data collected on women and men were pooled.

Exercise Performance
On the 15%F diet, the women ran 39.2 11.2 minutes and the men ran 44.3 9.6 minutes. There was no significant difference between the times of the women and men, as they both ran at the same % of VO2max. Increasing dietary fat to

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Table 1. Macronutrients and Lipid Components of Diets with 15%, 30% and 40% of Total Calories from Fat for Male and Female Endurance Runners
Women 15%* (n7) Kcal/day Protein (Kcal/d) Protein (g/d) Fat (g/d) Fat (%) SFA (g/d) MUFA (g/d) PUFA (g/d) SFA (%) MUFA (%) PUFA (%) PUFA/saturates Oleic acid (g/d) LA (g/d) LNA (g/d) Cholesterol (mg/d) 1708 150 282 29 70.5 7.3 31.6 4.0 15.6 0.9 9.2 1.4 9.1 1.4 6.6 0.7 36.0 1.7 36.9 1.4 27.0 2.1 0.75 6.0 0.9 4.3 0.6 0.5 0.07 126 9 30%* (n9) 2011 145 282 23 70.5 5.8 68.5 6.6 30.6 0.9 23.5 2.7 22.0 2.0 12.5 0.9 40.1 1.1 38.1 0.5 21.9 0.9 0.55 18.2 1.7 8.9 0.8 0.5 0.07 170 25 40%* (n8) 2553 153 332 16 83 4 106 10.8 41.0 2.3 36.0 4.1 33.4 3.7 18.9 2.1 40.7 1.1 37.8 0.8 21.5 0.5 0.53 26.6 3.4 12.7 1.4 0.9 0.17 260 16 15%* (n7) 2201 211 362 24 90.5 6 37.6 4.7 14.0 1.2 10.4 1.1 11.2 1.6 7.4 0.8 37.4 2.1 36.4 2.0 26.2 0.6 0.70 7.2 0.8 7.8 3.5 2.4 2.10 153 21 Men 30%* (n6) 2674 265 391 28 97.8 7 104 12.8 29.0 2.0 35.9 4.6 33.8 3.9 18.3 2.2 40.2 1.0 38.1 1.0 21.7 1.3 0.54 26.0 3.8 13.0 1.6 0.8 0.17 263 27 40%* (n6) 3097 186 421 21 105.3 5 150 13.4 39.7 2.4 53.0 4.6 50.3 4.6 24.3 2.2 41.6 1.0 39.3 1.0 19.1 0.7 0.46 39.1 4.1 17.5 1.6 1.4 0.45 401 39

Data generated by dietary intake analysis by Nutritionist IV software. Values Mean SEM. * % of the total energy intake from fat. SFA saturated fatty acids, MUFA monounsaturated fatty acids, PUFA day; Kcal/d kilocalories/day.

polyunsaturated fatty acids, LA

linoleic acid, LNA

linolenic acid, g/d

grams per

Table 2. Vitamin and Mineral Components of Diets with 15%, 30% and 40% of Total Calories from Fat for Male and Female Endurance Runners
Women 15%* (n7) Vit. A (RE) -carotene ( g/d) Vit. E (mg/d) Vit. E acetate (mg/d) Vit. C (mg/d) Calcium (mg/d) Iron (mg/d) Zinc (mg/d) Selenium ( g/d) 1730 1149 10.2 5.0 159 657 15.1 6.9 50 407 433 2.3 1.1 16 55 1.6 0.6 5 30%* (n9) 1133 481 11.0 6.7 144 856 12.9 8.7 48 164 104 1.0 0.8 13 107 0.7 0.5 4 40%* (n8) 1234 470 15.9 7.2 136 1004 15.7 10.6 60 209 160 2.0 0.6 11 84 1.0 0.4 9 15%* (n7) 1982 1136 7.8 5.1 214 861 16.9 8.6 71 542 452 1.6 0.9 41 121 1.4 0.7 5 Men 30%* (n6) 1441 554 11.4 7.4 180 965 19.5 11.4 82 214 143 1.2 0.9 24 69 1.9 0.8 11 40%* (n6) 1428 463 17.6 10.3 161 1227 19.3 13.1 84 222 181 2.7 0.7 14 89 2.9 0.7 7

Data generated by analyzing dietary intake records using Nutritionist IV databank program. Values Mean SEM. * % Kcal of total Kcal intake from fat, Vit. vitamin, RE retinol equivalents, mg/d milligrams per day,

g/d

micrograms per day.

30% significantly increased (p 0.05) the running time to exhaustion 19% for women (46.6 13.5 min) and 24% for men (54.9 12.8 min). Increasing the dietary fat to 40% did not significantly increase endurance time, when compared to the time on the 30% fat diet for either women or men. There were no significant differences in VO2s running at 40%, 60% O2max (39.2 1.1 and 46.7 2.1 for women and and 80% of V men, respectively). There were no significant differences in heart rates (161 2 beats/min) or the respiratory gas exchange ratios (0.95 0.01) during the endurance run among the three diet groups or between women and men.

Plasma Cortisol Level

As no significant gender effects were observed, the cortisol data for men and women were pooled. The plasma cortisol level ranged between 2.325.03 ng/mL (Fig. 1). Pre-run baseline plasma cortisol levels increased with the increase in dietary fat. Plasma cortisol levels were significantly higher after the endurance run compared to baseline levels on all diets (p 0.004). The post-exercise plasma cortisol level significantly increased with increasing dietary fat (15% F vs. 40% F p 0.002; 30% F vs. 40% F p 0.012). At post exercise points,

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15%F and 40%F diets. The level of dietary fat did not significantly affect the plasma PGE2 levels, except it was significantly lower at pre-exercise on the 40%F diet.

Plasma IFN- Level

There were no significant differences in IFN- baseline levels in the subjects among the three diets (95.9 5.3 pg/mL). Plasma IFN- level after exercise was not significantly affected by diet (89.0 2.8). No significant effects specific to gender, the endurance run or the level of dietary fat were found at p 0.05.

Plasma Lipid Peroxide Level

Fig. 1. Effects of the level of dietary lipids and the endurance run on plasma cortisol levels in endurance runners. Values are mean SEM. Cortisol level significantly higher after endurance run compared to before run. * Significantly higher compared to 15%F and 30%F.

the percent increase in cortisol levels from baseline was 56%, 39% and 13.3% for 15% fat, 30% fat and 40% fat diets respectively. Although the post-run plasma cortisol levels increased with increase in dietary fat intake, the rate of increase of cortisol during exercise was lower on the higher fat diets (15% F: 0.04 ng/mL/min, and 40% F: 0.02 ng/mL/min).

As no significant effect of gender was observed in plasma lipid peroxides, the data on men and women were pooled. The plasma lipid peroxide levels ranged between 0.19 1.65 nmoles/mL before the endurance run and 5.8 12.4 nmoles/mL after the endurance run (Fig. 3). The pre-exercise baseline level of lipid peroxide was significantly lower on the 40%F diets than on the 30%F and 15%F diets. The levels of lipid peroxides were significantly higher after the endurance run in subjects (p 0.0001) on all three diets. The pre- and post-exercise plasma lipid peroxide concentrations were significantly lower on the 40%F compared to 15%F (p 0.007) and 30%F group (p 0.002).

Plasma PGE2 Level

As no significant gender difference was observed in PGE2 levels, the PGE2 data on men and women were pooled. Plasma PGE2 levels ranged between 0.551.13 ng/mL (Fig. 2). Preexercise baseline PGE2 levels were significantly lower on the 40%F diet than on the 30%F and 15%F diets. Plasma PGE2 levels were significantly higher after the endurance run when compared to levels before the endurance run (p 0.005) on the

DISCUSSION
The present study confirmed the results of previous studies [25,7,20] that trained runners on a low fat diet (15%) run significantly shorter times at 80% of their VO2max than runners on higher fat diets (30% to 40%). Increased running times after

Fig. 2. Effects of dietary fat level and exercise on plasma PGE2 in endurance runners. Values are mean SEM. PGE2 levels significantly higher after endurance run compared to before the run. * Significantly lower compared to 15%F and 30%F.

Fig. 3. Effects of dietary fat level and exercise on plasma lipid peroxides in endurance runners. Values are mean SEM. Lipid peroxides level significantly higher after endurance run compared to before. # Significantly lower compared to 15%F and 30%F before endurance. Significantly lower compared to 15%F and 30%F after endurance.

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high fat diets are due to the increased stores of intramuscular fat [4], which when coupled with the enzymatic adaptations increase lipid oxidation [21], thus sparing glycogen [2]. A diet that increases intramuscular fat stores without compromising glycogen stores improves performance but at the price of higher oxygen consumption. In addition, when the subjects were on the 15% diet, their intake of nutrients, especially calcium, iron, zinc and vitamin E were below recommended levels, that were corrected when the runners ate the higher fat diets (30% and 40%). As these micronutrients are essential to immune function and vitamin E is a major lipid-soluble antioxidant, when athletes consume a low fat diet, they may be more susceptible to oxidative stress and infections after intense exercise. The data presented here show that plasma cortisol, PGE2 and lipid peroxide levels increased significantly after endurance exercise on all diets. Although plasma cortisol levels were higher on the higher energy and fat diets, the rate of increase in cortisol during acute exercise did not differ among the diets; nevertheless, the absolute level of circulating cortisol was highest in the 40% fat group, both pre- and post-exercise. Results from the present study suggest that endurance exercise and increases in levels of energy and dietary fat increased plasma cortisol levels. A study conducted on hockey players suggested that a reduced-fat, high-carbohydrate diet increased concentrations of plasma cortisol and testosterone in the experimental group (55% carbohydrate, less than 30% fat) compared to controls (45% carbohydrate, 40% fat) [22]. When the plasma cortisol increases observed in the present study were adjusted for endurance run time, the rate of increase in plasma cortisol levels were lower with increasing dietary fat intake in women while the rate of cortisol increase was similar for the 15%F and the 40%F diets in men. The plasma concentration of cortisol increases only in relation to exercise of long duration, resulting in suppressive effects on certain cytokines and immune cells [8,12]. Hydrocortisone is reported to inhibit superoxide generation [18]. Isoprostanes produced during peroxidation by membrane lipids may mediate the effects of free radicals and reactive oxygen species [19]. Cyclopentenone PGs are potential inducers of intracellular oxidative stress leading to cell degeneration [23]. Our data showed that plasma PGE2 levels were significantly higher after endurance exercise (15%F and 40%F groups) compared to before exercise. Our data also showed that plasma PGE2 levels were not significantly different among the 15%F, 30%F, and 40%F groups. In the present study, when the level of dietary fat was increased, the amount of calories coming from PUFA was lower than those coming from saturated FA and MUFAs. It is also known that the plasma PGE2 level increases after strenuous exercise. If these two effects were independent, we would expect the plasma PGE2 level of the 40%F group to be significantly higher than that of the 15%F and 30%F group. But our data showed a significant reduction in pre-exercise plasma PGE2 level on the 40% fat diet compared to the 15 and 30% fat diets. The post-exercise PGE2 was significantly lower on the 40% fat diet compared to the 15% fat diet. This finding is in agreement with PGE2 levels reported by other investigator [24]. Current literature suggests that it may be the type of fat and not the amount of fat that influences PGE2 and other immune indices. This indicates that dietary fat may help reduce the stress caused by exercise and, therefore, has less or no adverse effects on well-trained athletes who engage in exercise. The magnitude of the antioxidant defense system enhancement depends on training loads. Because of their training status, triathletes did not suffer from oxidative damage after they finished a long distance triathlon race [25]. The level of energy intake and fat intake had little effect on IFN- . Results from the present study suggest that exercise or dietary fat levels had no significant effects of plasma IFNlevel. Similar observations have been previously reported [26,27]. A possible explanation for this may be that IFN- is produced by helper T cells (CD4 ) and natural killer (CD16 ) cells. CD4 subset decreases and CD16 subset increases after exercise. Interferon- is synthesized several hours after its induction and immediately released into the extra-cellular environment, where it is rapidly utilized by its receptors [24]. Higher energy and fat diets (30%F to 40%F) significantly decreased the levels of PGE2 lipid peroxides. Cytokines such as IL-1 released from sites of inflammation cross the blood-brain barrier and drive the hypothalamus-pituitary-adrenal axis so that cortisol is released into circulation to exert indiscriminate systemic anti-inflammatory effects [28]. The cytokine profiles in Th2 are linked to changes of humoral balance between cortisol and dehydroepiandrosterone. Intense physical activity increases skeletal muscle capability for oxidizing pyruvate and fatty acid in order to increase energy production (i.e. oxygen consumption). Increased VO2 could lead to increases in free-radical production. However, exercise and training appear to augment the bodys antioxidant defense system [29]. Athletes who restrict energy intake or go on low fat diets to achieve a low body mass (e.g. endurance runners) may not have adequate vitamin or mineral status to maintain the anti-oxidant system [30]. PUFA are generally more susceptible to peroxidation compared to SFA and MUFA. In the present study, fat levels were increased, but the PUFA level stayed constant (only 23% of total fat), while saturated fats were the highest (40%) and MUFA were 37%. Perhaps the type of fats in the diet might contribute to preventing exercise-induced lipid peroxides. Antioxidant vitamins may also play a role in reducing exerciseinduced lipid peroxides. Also, dietary analysis showed that vitamin E intake increased with increasing fat intake and vitamin C intake was higher than RDA values. In another study, exercise-induced lipid peroxidation during graded aerobic exercise was measured in seven healthy men and women [31]. In those healthy individuals, physical exercise induced lipid peroxidation transiently, and removal of lipid

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peroxidation byproducts occurred during recovery. Those with genetic deficiency in antioxidant enzymes, those who respond poorly to oxidative stress or those who have overwhelming plasma oxidative stress might require additional antioxidant supplementation. In vivo urate levels before exercise may be a factor influencing lipid peroxidation during exhaustive exercise and urate may serve as an important free radical scavenger in vivo [32]. Exercise increases blood xanthine oxidase activity in rats. Inhibiting xanthine oxidase prevented increases in plasma activity of cytosolic enzymes activities after exhaustive exercise [33]. Xanthine oxidase may be responsible for free radical production and tissue damage during exhaustive exercise. The immune system is closely linked to the neuroendocrine system, and various messengers, including hormones, neurotransmitters and cytokines, regulate cellular and humoral immunity [7,20,34,35]. In general, regular exercise at moderate intensity may increase immune competence, while intense exercise, prolonged training sessions, and competitions may suppress the immune system [36 38]. Epidemiological studies demonstrate that persons involved in activities such as prolonged intense training sessions or competitions experience an increased incidence of infectious diseases and allergy-related disorders [39,40]. Exercise, estrogens, and substitution with PUFAs for SAF are beneficial in preventing atherosclerosis but these factors are also capable of inducing oxidative stress [41]. Under certain conditions an oxidative stress may be beneficial for inducing antioxidant enzymes in arterial walls [41]. Increasing fat from 15% to 40% of energy did not change adiposity, body weight, heart rate, blood pressure, plasma triglycerides, total cholesterol, LDL cholesterol, ApoB or ratio of ApoA1/ApoB [42]. Compared to those eating 40% fat diet, subjects on 15% fat diet had lower HDL cholesterol and ApoA1 and higher total cholesterol/HDL-cholesterol ratio suggesting 40% fat diet maintained favorable CHD risk factors in runners while a 15% fat diet lowered ApoA1 and HDL-cholesterol and raised total cholesterol/HDL-cholesterol ratio. The changes described above may have been influenced by factors other than the dietary fat intake. The order of the diets was not randomized in the present study. To eliminate any health risk to the subjects, immunological and blood lipoprotein levels had to be evaluated at each stage prior to increasing dietary fat intake. Although this introduced a potential order effect, progressing to higher fat diets over a two-month period would have exaggerated any negative effects of higher fat. As no negative effects were seen, even with the potential bias, the findings of this study would seem valid [42]. In spite of the prescription of the diets, day-to-day diet records and weekly follow-up revealed the subjects caloric intake increased as dietary fat increased. We cannot eliminate potential effects of the differences in the three diets on the parameters reported in this paper. The present study suggests that increasing total energy intake and dietary fat increases endurance run time without adverse effects on plasma cortisol, PGE2, IFN- , and lipid peroxide concentrations. Plasma cortisol levels increased, but when expressed as the rate of increase in cortisol concentration, the levels were lower on the 40%F diet compared to 15%F and 30%F diets. These data when combined with previously published data from the same subjects [7,20] demonstrate that low fat diets (15% to 16%) compromised cellular and plasma immune function, as well as aspects of hormonal and oxidative stress responses when compared to 30%F diets. Increasing dietary fat intake to 40% did not increase oxidative stress, IFN- , cortisol, PGE2 levels or lipid peroxidation suggesting 40% fat in the diet of endurance runners may not be immunosuppressive.

ACKNOWLEDGMENTS
The authors wish to thank Dr. P. J. Horvath, Dr. Nadine Fisher, Stacie Ryer-Calvin, and Colleen Eagen for their support.

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