Journal of the American College of Nutrition, Vol. 22, No. 6, 519-523 (2003)
Published by the American College of Nutrition
Orange Juice Ingestion and Supplemental Vitamin C Are Equally Effective at Reducing Plasma Lipid Peroxidation in Healthy Adult Women
Carol S. Johnston, PhD, FACN,
Candice L. Dancho, MS and
Gail M. Strong, MS
Department of Nutrition, Arizona State University East, Mesa, Arizona
Carol Johnston, Ph.D., Department of Nutrition, Arizona State University East, 7001 E. Williams Field Rd, Mesa, AZ 85212. E-mail: carol.johnston{at}asu.edu
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ABSTRACT
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Objective: To directly examine the contribution of vitamin C to the antioxidant potential of fruits and vegetables, the antioxidant effect of orange juice consumption (8 and 16 fl. oz.) was compared to the antioxidant effect of supplemental vitamin C (dosage equivalent to that supplied by 8 fl. oz. of orange juice).
Methods: Subjects (n = 11; 28.6 ± 2.1 years) received each treatment in a 3 x 3 randomized crossover design, and each two-week treatment was preceded by a two-week washout. During the entire trial, subjects restricted fruit and vegetable consumption to 
3 servings per day except the vitamin C-rich foods (items containing >20 mg/serving), which were restricted to 3 servings per week. A fasting blood sample was collected at the end of each washout and each treatment period.
Results: Following washouts, plasma vitamin C and lipid peroxidation (plasma TBARS) were similar by treatment group and averaged 25.4 ± 3.6 µmol/L and 3.82 ± 0.10 nmol/mL respectively. Plasma vitamin C concentrations were similar following each treatment period, 37.9 ± 8.1, 45.8 ± 9.4, and 38.3 ± 12.4 µmol/L for the 8 and 16 fl. oz. orange juice treatments and the supplement treatment, respectively. All intervention treatments reduced plasma TBARS as compared to pretreatment values: -47% (p = 0.013), -40% (p = 0.083), and -46% (p = 0.015) for the 8 and 16 fl. oz. orange juice treatments and supplement treatment respectively.
Conclusions:These data indicate that the regular consumption of 8 fl. oz. orange juice or supplemental vitamin C (
70 mg/day) effectively reduced a marker of lipid peroxidation in plasma.
Key words: vitamin C, orange juice, oxidative stress, lipid peroxidation
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INTRODUCTION
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Diets rich in fruits and vegetables are consistently associated with a decreased risk of cardiovascular disease and cancer [1–5]. The health effects afforded by fruits and vegetables are attributed to the many biologically active phytochemicals present in these foods, including both nutrient and non-nutrient compounds which possess detoxification, immunostimulating, antiviral, anticancer, and antioxidant properties [2]. Much of the research has focused on the antioxidant capacity of fruits and vegetables in particular since oxidative damage is believed to play a key role in cardiovascular disease, cancer initiation, cataract formation, inflammatory diseases, neurologic disorders, and the aging process in general.
Block et al. [6] recently examined the correlation between fruit and vegetable intake and plasma levels of antioxidants in 116 males who were non-smokers and non-supplement users. Of the antioxidants tested (ascorbic acid, beta-carotene, beta-cryptoxanthin, and alpha- and gamma-tocopherol), the strongest association with fruit and vegetable intake was noted for ascorbic acid, and the investigators concluded that ascorbic acid is an important component of the protective effect seen for fruits and vegetables in numerous epidemiologic studies. Moreover, Szeto et al. [7] measured the total antioxidant capacity and the total ascorbic acid content of vitamin C-rich fresh fruits and vegetables (eg., oranges, grapefruits, kiwi, mango, cabbage, turnip greens, and cauliflower) and determined that vitamin C accounted for 35% to 75% of the antioxidant power of the food. We recently correlated the antioxidant power and vitamin C content of the eight most commonly consumed fruits and vegetables in the American diet and demonstrated that vitamin C accounted for 61% of the variability of the antioxidant power of these foods [8].
To directly examine the contribution of vitamin C to the antioxidant potential of fruits and vegetables, we compared the antioxidant effect of orange juice consumption to that of supplemental vitamin C (each contributing approximately the same amount of ascorbic acid, 63–69 mg). Subjects consumed a vitamin C-restricted diet throughout the randomized, crossover trial, and lipid peroxidation in plasma was estimated by measuring the thiobarbituric acid reactive substances (TBARS) in plasma, a summary measure of total circulating oxidation.
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METHODS
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Eleven healthy, non-smoking women (21 to 39 years of age) were recruited from a college campus. During the entire 12-week trial, subjects were instructed not to use dietary supplements and to limit fruit and vegetable consumption to 3 servings per day except the vitamin C-rich fruits and vegetables (items containing >20 mg vitamin C per serving) which were restricted to 3 servings per week. A list of vitamin C-rich fruits and vegetables was provided to the subjects, and all subjects maintained daily fruit and vegetable records which a dietitian reviewed weekly. Subjects were instructed to read all food labels and to avoid vitamin C fortified foods. A list of vitamin E-rich foods was also given to subjects, and subjects were instructed to keep their intake of vitamin E foods constant throughout the study. All participants provided written informed consent, and the study was approved by the Institutional Review Board at Arizona State University.
Subjects completed three daily dietary treatments in a randomized, 3 x 3 crossover design: 1 cup (8 fl. oz.) orange juice, 2 cups (16 fl. oz.) orange juice, and vitamin C supplement. Each treatment period lasted two weeks and was preceded by a two-week washout period. A two-week period of vitamin C restriction (washout) as imposed by this protocol has been shown to lower plasma vitamin C concentrations in free-living, healthy subjects by 50% [9,10]. Restricting dietary vitamin C prior to the experimental treatment served to reduce vitamin C concentrations in plasma and, thus, highlight the physiological response to the experimental treatment. Since the physiological response to vitamin C repletion is rapid (taking fewer than 10 days) [10], we felt that a two-week experimental treatment period was appropriate for this investigation.
Orange juice, chilled, ready-to-drink, was purchased from local retailers weekly and distributed to subjects weekly. Hence, orange juice was unsealed by the subjects and used for one week and discarded, and a new container was unsealed and used the second week. Orange juice contained 72 mg vitamin C per cup as indicated by label claim. Subjects were instructed to use measuring cups to accurately determine portions. Vitamin C capsules were prepared in our laboratories to contain 72 mg ascorbic acid (Trader Joe’s, Pasadena CA) each, a level comparable to 1 c orange juice. Using two levels of orange juice ingestion permitted evaluation of a potential dose-effect of fruit consumption on health indicators.
A fasting, morning blood sample was collected from each subject at the initiation of the study and after each two-week washout and two-week treatment period. Blood, collected in EDTA-treated Vacutainers, was immediately processed, and an aliquot of plasma was frozen (-45°C) for analysis of lipid peroxidation using the TBARS assay as described by Yagi [11]. To increase assay specificity, interfering substances such as glucose and water-soluble aldehydes were eliminated by extracting plasma lipids with a phosphotungstic acid-sulfuric acid system prior to their reaction with the thiobarbituric acid reagent [11]. A separate aliquot of plasma was immediately mixed with equal volumes 10% TCA, and the supernatant frozen (-45°C) for vitamin C analysis using the colorimetric 2,4-dinitrophenylhydrazine method [12]. Same-lot containers of orange juice as provided to the subjects were analyzed colorimetrically for both total and oxidized vitamin C as described by Schaus et al. [13]. Orange juice was unsealed and analyzed at the start of each week, stored in the original container at 4°C, and analyzed again on day 7. A random sample (n = 10) of the vitamin C capsules were analyzed for both total and oxidized vitamin C [13].
Data are reported as the mean ± SD. Following a significant multivariate analysis of variance repeated measures test, the Bonferroni post-hoc test for multiple comparisons was used to determine differences in mean plasma vitamin C and in mean plasma TBARS by two-week intervention treatment and by group. p values <0.05 were considered significant. The vitamin C content of the orange juices and the supplement are reported as reduced vitamin C (ascorbic acid) as calculated by subtracting oxidized vitamin C from total vitamin C. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS for Windows, Version 10.0, SPSS Inc., Chicago IL).
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RESULTS
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The mean age of subjects (n = 11) was 28.6 ± 7.0 years; ten subjects had normal body mass indexes (BMI range, 19.0 to 23.6 kg/m2), and one subject was overweight (BMI = 29.1 kg/m2). Based on daily food logs, subjects averaged 1.1 servings of fruits and vegetables per day during the 12-week trial (range: 0.3–1.5 servings/day). Self-reported compliance to the intervention treatments, the daily ingestion of orange juice or supplement as prescribed, was 97%.
The ascorbic acid content of the orange juice was determined at the start and at the end of each issue period (one week) and values were averaged to give the approximate vitamin C content per cup. The average vitamin C content of the orange juice was 63.3 ± 17.7 mg ascorbic acid per 8 fl.oz. Chemical analysis of the vitamin C supplements indicated an average of 68.6 ± 10.5 mg ascorbic acid per capsule.
Plasma vitamin C concentrations did not differ by group at pre-intervention (following the two-week washout), and values ranged from 21.3 ± 7.4 to 27.4 ± 6.7 and 27.5 ± 10.1 µmol/L for the supplement treatment and the 8 and 16 fl. oz. orange juice treatments, respectively (Fig. 1). At post-intervention, plasma vitamin C concentrations rose for each treatment: +28% (p = 0.143), +66% (p = 0.000), and +80% (p = 0.001) for the 8 and 16 fl. oz. orange juice treatments and the supplement treatment, respectively (Fig. 1). Plasma TBARS were similar among groups at pre-intervention (3.86 ± 1.18, 3.70 ± 1.22, and 3.89 ± 1.64 nmol/mL for the 8 and 16 fl. oz. orange juice treatments and the supplement treatment, respectively), and all intervention treatments reduced plasma TBARS as compared to pretreatment values: -47% (p = 0.013), -40% (p = 0.083), and -46% (p = 0.015) for the 8 and 16 fl. oz. orange juice treatments and supplement treatment respectively (Fig. 2).
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Fig. 1. Plasma vitamin C concentrations in free-living subjects (n = 11) immediately prior to and after a two-week diet intervention: 8 fl.oz. orange juice daily (66 mg ascorbic acid), 16 fl.oz. orange juice daily (122 mg ascorbic acid); and 1 vitamin C supplement daily (69 mg ascorbic acid/capsule). All subjects received each two-week treatment proceeded by a two-week washout period in a randomized, crossover fashion. There were no significant differences among groups at pre-intervention or at post-intervention. An asterisk denotes a significant change from pre-intervention (p < 0.05).
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Fig. 2. Lipid peroxidation (plasma TBARS) in free-living subjects (n = 11) immediately prior to and after a two-week diet intervention (see Fig. 1 legend for intervention protocol). There were no significant differences among groups at pre-intervention or at post-intervention. An asterisk denotes a significant change from pre-intervention (p < 0.05).
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DISCUSSION
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This study demonstrated that orange juice ingestion (8 fl.oz./day) and supplemental vitamin C (70 mg/day) were equally effective at reducing lipid peroxidation in plasma. Thus dietary vitamin C exerted a strong antioxidant effect in vivo regardless of whether the source was a supplement or fruit. In this trial, plasma vitamin C concentrations explained 21% of the variation in antioxidant protection of plasma (r = -0.46, p < 0.01). Since dietary vitamin C increases glutathione levels in vivo [14,15] and promotes vitamin E recycling [16], vitamin C may alter the level of other antioxidants in vivo which in turn help to reduce levels of lipid peroxides in plasma [17].
Surprisingly, 1 cup orange juice was slightly more effective than 2 cups orange juice in reducing lipid peroxidation in plasma. To our knowledge no other study has investigated the possibility of a dose response for fruit and vegetable ingestion on oxidative stress in vivo. Kurowska et al. [18] examined the HDL-cholesterol-raising effect of 1, 2, or 3 cups orange juice incorporated sequentially into the daily diets of middle-aged women and men with moderately elevated plasma total and LDL-cholesterol. Only the consumption of 3 cups orange juice daily significantly impacted on HDL-cholesterol (+21%), but this level of juice consumption also significantly raised serum triglycerides (+30%), a potential health concern. Markers of in vivo oxidative stress were not determined in this study; however, plasma vitamin C levels were remarkably low throughout the entire trial: 9, 19, 27, and 33 µmol/L at baseline and after the 1, 2, and 3 cup juice intervention respectively [18]. One cup orange juice containing 75 mg vitamin C (as reported by the authors) would be expected to raise vitamin C concentrations to 35 µmol/L [19,20]. Pasteurization, storage, and handling significantly affect both the vitamin C and the flavanone content of orange juice [21–23]; hence, potential health benefits afforded by orange juice consumption may depend on the freshness and quality of the juice, factors that should be addressed by direct analyses of juices used in clinical trials.
In the present report, we provided subjects with orange juice on a weekly basis, and all juice was analyzed for ascorbic acid over the course of its use by subjects. We believe that since ascorbic acid is highly labile and sensitive to heat, oxygen, and light, ascorbic acid is a good marker of juice quality. Daily orange juice consumption (1 cup) by subjects adhering to a low vitamin C diet maintained plasma vitamin C concentrations at about 38 µmol/L. Consumption of 2 cups orange juice daily raised plasma vitamin C concentrations to 46 µmol/L. The lack of additional plasma antioxidant protection for subjects ingesting 2 cups orange juice versus 1 cup orange juice may relate to a leveling effect. All intervention treatments (1 or 2 cups orange juice, or vitamin C supplement) reduced plasma TBARS from 3.8 nmol/mL to 2.1 nmol/mL. The reference value for young women established by Yagi [11] is 2.98 ± 0.05 nmol/mL. Thus further reductions in plasma lipid peroxidation in healthy adults may not be easily achieved. In conditions characterized by elevated oxidative stress, however, such as positive smoking status, hypertension, cardiovascular disease, diabetes, or inflammation, mean TBARS concentrations are much higher than in healthy subjects (from 4.3 to 6.1 nmol/mL) [24–26]. Under these circumstances, greater intakes of vitamin C or fruits and vegetables than used in the present study may show added benefits. These possibilities should be addressed in future investigations.
Oxidative stress in vivo has been linked to a wide variety of pathological conditions. Plasma lipid peroxidation, but not total blood cholesterol, was associated with in increased risk of cardiovascular morbidity in old people [27] and with the severity of cardiovascular disease in dialyzed patients [28]. Lipid peroxidation is increased in Alzheimer disease, and individuals with mild cognitive impairment displayed increased lipid peroxidation before the onset of symptomatic dementia [29]. In diabetics, hyperglycemia promotes lipid peroxidation increasing risk for coronary heart disease [30]. Thus, regular vitamin C consumption, in the form of food or supplement, may reduce risk for disease pathology by reducing oxidative stress in vivo.
Yet, some fruits and vegetables low in vitamin C possess high antioxidant power in vitro and in vivo. Cao et al. [31] reported that strawberries (240 g), raw spinach (294 g), and red wine (300 mL) raised serum total antioxidant capacity in elderly women 12%, 25%, and 11%, respectively during the four hours post-dose, and that a single oral dose of vitamin C (1250 mg) increased serum total antioxidant capacity 21% during the four hours post-dose. Each of these treatments was formulated to provide equal antioxidant capacity in vitro, but the vitamin C content of the treatments varied from 0 mg (wine) to approximately 40 mg and 120 mg (spinach and strawberries). The particular antioxidant profile of specific fruits and vegetables is variable just as the vitamin C content of specific fruits and vegetables varies; thus, regular ingestion of a variety of fruits and vegetables would provide ample vitamin C and the phytochemical "cocktail" that, in combination, are likely to be the most effective protection against oxidative stress.
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ACKNOWLEDGMENTS
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This work was funded by the Lloyd S. Hubbard Nutrition Research Fund of the Arizona State University Foundation.
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FOOTNOTES
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This work was presented in part at Experimental Biology ’02, April 2002, New Orleans, Louisiana.
Received November 20, 2002.
Accepted June 22, 2003.
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ABSTRACT
INTRODUCTION
