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Nutrient Interactions and Toxicity

Lutein Interacts with Ascorbic Acid More Frequently than with -Tocopherol to Alter Biomarkers of Oxidative Stress in Female Zucker Obese Rats^1,2

Shirley Blakely³, Arnetra Herbert^*, Michelle Collins, Mamie Jenkins, Geraldine Mitchell, Erich Grundel, Karen R. O’Neill and Frederick Khachik^**

Food and Drug Administration, College Park, MD 20740; ^* Centers for Disease Control and Prevention, Atlanta, GA 30341; Department of Molecular and Biochemical Nutrition, University of California, Berkeley, CA 94704; and ^** Department of Chemistry, University of Maryland, College Park, MD

³To whom correspondence should be addressed. E-mail: sblakely{at}cfsan.fda.gov.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The influence of dietary lutein, with and without moderate amounts of vitamin C (VC) or vitamin E (VE), on biomarkers of oxidative stress was examined in rats. Nine groups of immature Zucker obese (fa/fa) and lean female rats (8/group) consumed ad libitum for 8 wk the AIN-93G diet (Control) to which was added either dl--tocopherol acetate (VE) at 0.60 mg/kg or ascorbic acid (VC) at 0.75 mg/kg diet. Each of these diets contained lutein oil (FloraGlo) at 0.5 (Lut0.5) or 1.0 (Lut1.0) mg/kg diet. Weight gain, food efficiency and relative liver weight were higher in obese than in lean rats. Although liver malondialdehyde (MDA) concentrations were significantly higher in obese than in lean rats, levels were significantly lower in obese rats fed VE, VE-Lut and VC-Lut0.5 compared with other obese groups. The accumulation of -tocopherol in liver was 6- and 3-times greater in the VE and VE-Lut1.0 groups, respectively, compared with the obese and lean control groups. Lutein reduced the activity of superoxide dismutase (SOD) in obese rats, independent of VC or VE, and raised the activity of glutathione peroxidase to higher levels in lean rats when combined with VC. Plasma insulin levels were dramatically higher in obese compared with lean rats, but significantly lower in obese rats fed VC-Lut0.5, VE-Lut1.0 and Lut1.0 compared with the Control group. These results suggest that lutein independently reduces the activity of SOD and alters more biomarkers of oxidative stress when combined with vitamin C than with vitamin E, and that vitamin E reduces liver lipid peroxidation in obese rats when the accumulation of liver -tocopherol is very high.

KEY WORDS: • obesity • antioxidants • carotenoids • lutein • ascorbic acid • -tocopherol

There is ample evidence that oxidative stress influences the onset and progression of a number of chronic diseases including cardiovascular disease, diabetes and cancer (1,2). Oxidative stress occurs when unstable reactive oxygen species (free radicals such as lipid hydroperoxides, superoxide anions and singlet oxygen) produced during cellular respiration exceed the antioxidant capacity of the cell. This results in an imbalance between the production of reactive oxygen species and their removal (3). Antioxidants from dietary and endogenous sources reduce and remove reactive oxygen species and thereby may lower the risk of chronic disease. Increased antioxidant status is achieved through frequent consumption of fruits and vegetables, which has been shown to be associated with lower risks of chronic disease in humans (4). Lifestyle factors have been shown to be inversely associated with antioxidant status (5,6).

Lutein, one of the most common carotenoids in green leafy vegetables, has shown promise in inhibiting the formation of aberrant crypt foci in rats (7) and blocking the progression of atherosclerotic lesions in apolipoprotein E-null mice (8). Several studies suggest a role for lutein and its stereoisomer, zeaxanthin, in the prevention of age-related macular degeneration (9–11). The ability of these carotenoids to absorb the harmful short-wavelength blue light and protect the macular region of the human retina from light-induced oxidative damage has been postulated as a possible mechanism for this beneficial effect (11). Hammond et al. (12) recently reported an inverse correlation between macular pigment density and concentrations of lutein and zeaxanthin in which significantly lower levels were found in obese compared with nonobese subjects. The allylic hydroxyl group on the epsilon end-group of lutein contributes greatly to its potent antioxidant properties. Compared with other carotenoids, all trans-lutein is rapidly absorbed in rodents (13) and humans (14).

Results of several reports suggest that the ingestion of mixtures of antioxidants such as carotenoids and vitamin E or C may be more effective than the ingestion of single carotenoids. Taylor et al. (15) reported dramatic benefits of supplemental intakes of vitamin C, carotenoids and folate in cortical and posterior subcapsular lens opacities in human subjects. Tauler et al. (16) reported that the basal neutrophil antioxidant defense enzymes, catalase and superoxide dismutase (SOD),³ were enhanced by supplementation with a mixture of ß-carotene and vitamins C and E. A high intake antioxidant vitamins was reported by Gale et al. (17) to prevent the initiation and progression of early atherosclerotic lesions in men.

In the present study, we investigated the effects of dietary lutein, fed separately or in combination with ascorbic acid (vitamin C) or -tocopherol (vitamin E), on biomarkers of oxidative stress in Zucker (fa/fa) obese and lean rats. Our purpose was to determine whether lutein is more effective when combined with moderate amounts of the nonpolar free-radical quencher, -tocopherol, or the water-soluble electron-donor, ascorbic acid. Although adequate levels of ascorbic acid are obtained in rodents through endogenous synthesis and adequate -tocopherol is obtained through the rodent diet, supplemental, subpharmacologic levels of these nutrients were studied. The Zucker (fa/fa) obese rat model mimics human obesity and prediabetes mellitus in that it is hyperinsulinemic, insulin resistant and hypertriglyceridemic (18,19). Because hyperinsulinemia and insulin resistance are associated with increased oxidative stress, it was also of interest to examine the influence of these antioxidants on insulin and the adrenal stress hormone, corticosterone (20,21).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animals.

The 144 (72 lean and 72 obese) 4- to 6-wk-old female viral antibody–free Zucker obese (fa/fa) and lean rats (Harlan Sprague Dawley, Indianapolis, IN) used in the study were housed individually in suspended wire mesh–bottomed cages. Lighting in the animal room was on a 12-h light:dark cycle with lights on 0700–1900 h. The temperature was maintained at 18–26°C and relative humidity at 40–70%. After a 1-wk acclimation period, obese and lean rats were assigned to one of the nine experimental groups. Animal care conformed to the principles and guidelines set forth by the National Academy of Sciences (22).

Diets.

All diets were formulated from the AIN-93G (23) diet, which was modified to contain tocopherol-stripped soybean and corn oil at 70 and 5.0 g/kg of diet, respectively. This modified AIN-93G diet served as the Control. Lutein (Lut) was added to the diet at two levels: 0.5 mg/kg (Lut0.5) and 1.0 mg/kg of diet (Lut1.0). The lutein preparation, FloraGlo (Kemin Foods, Des Moines, IA), consisted of a liquid suspension in corn oil of 20% all-trans lutein and 0.86% all-trans zeaxanthin. According to the manufacturer, lutein FloraGlo also contains vitamin E, rosemary and citric acid as preservatives. The lutein FloraGlo displaced a portion of the tocopherol-stripped corn oil equal to its weight when added to the diet. dl--Tocopherol acetate (Dyets, Bethlehem, PA), vitamin E (VE), was added separately to the diet at 0.6 mg/kg (VE) or in combination with each of the lutein concentrations, hereafter referred to as VE-Lut0.5 or VE-Lut1.0. This level of vitamin E, in combination with the 0.015 mg/kg (75 IU/kg) present in the AIN-93-VX vitamin mix (23), resulted in a total vitamin E concentration of 0.615 mg/kg of diet, which was 40 times the requirement. Ascorbic acid (Dyets), vitamin C (VC), was added separately to the diet at 0.75 mg/kg (VC) or in combination with each of the lutein concentrations, hereafter referred to as VC-Lut0.5 or VC-Lut1.0. Ascorbic acid and dl--tocopherol acetate displaced a portion of the cornstarch equal to their weight when added to the diet. The levels of antioxidants used in the test diets in this study represented moderate, subtoxic and subpharmacologic levels in the diet and were based on levels used in previous studies for lutein (24,25), ascorbic acid (26) and dl--tocopherol acetate (16). The diet mixing procedure was standardized to optimize the integrity of the antioxidants. Premixes of treatment compounds were prepared before mixing in the bulk batches. Fresh diet was prepared every 14 d and used within that period. Purity and stability of the treatment compounds (lutein, ascorbic acid and -tocopherol) and homogeneity of mixtures were verified for each batch through chemical analysis. All diets were stored at -20°C until fed to the rats. Rats consumed their respective diets and reverse osmosis deionized water ad libitum.

At the end of the 8-wk feeding period, rats were weighed and killed by asphyxiation with carbon dioxide. Blood was collected from the inferior vena cava and placed into centrifuge tubes coated with sodium EDTA to prevent clotting. Plasma was separated by centrifugation at 600 x g for 20 min and stored at -70°C until analysis. The livers and other organs were removed, weighed, frozen using liquid N₂ and stored at -70°C until assayed.

Assays.

The specific activity of liver SOD [EC 1.15.1.1] was determined using the method described by Prohaska et al. (27), which employs deactivation of MnSOD with ethanol/chloroform (25:15, v/v) and measures CuZn-SOD activity continuously by following the kinetics of pyrogallol oxidation on a Beckman DU-620 spectrophotometer (Columbia, MD) at 320 nm. SOD activity is the amount of enzyme needed to inhibit the autooxidation rate of pyrogallol by 50%. Glutathione peroxidase (GSH-px) [EC 1.11.1.9] activity was measured in liver using the method of Asayama et al. (28), which continuously measures the kinetics of the inhibition of NADPH oxidation to NADP by t-butyl hydroperoxide at 340 nm on a Beckman DU-620 spectrophotometer. GSH-Px activity is the amount of enzyme needed to convert H₂O₂ to H₂O per minute. Protein concentration was determined for each of the SOD and GSH-Px fractions using the Bradford protein assay (29) (Bio-Rad Laboratories, Richmond, CA).

Liver lipid peroxidation was assessed as MDA production by a modification of the method of Ohkawa et al. (30). Lipid peroxides in liver, induced in the presence of ferrous sulfate, react with thiobarbituric acid to form MDA. The concentration of lipid peroxides is expressed as MDA.

The total concentration of glutathione (GSH) in liver was determined by a modification of the method of Tietze (31), which measures both forms of GSH, oxidized and reduced, on a spectrophotometer (DU-70, Beckman) at 412 nm.

Plasma lutein, retinol and -tocopherol were extracted from the Control, Lut and VE diets by the method of Khachik et al. (32). Due to limiting amounts of plasma from the lean rats, vitamin C groups were not analyzed. Liver lutein and -tocopherol were extracted by the method of Olson (33). Plasma and tissue -tocopherol was analyzed using a Shimadzu RF-551 (Columbia, MD) spectrofluorometric detector (_ex290 nm, _em330 nm). Plasma and tissue retinol and lutein were analyzed using a SPD-M10A Shimadzu diode array detector. Lutein was detected at 450 nm using a YMC (Wilmington, NC) C₃₀ carotenoid column (25 x 0.46 cm; 3 µm) protected by a guard column. All components were eluted with 100% methanol at a flow rate of 1.5 mL/min. Ascorbic acid was determined spectrophotometrically in the presence of 2,4-dinitrophenylhydrazine and thiourea after extraction of blood in 4 volumes of 0.31mol/L trichloroacetic acid and of liver in 9 volumes of 0.38 mol/L metaphosphoric acid (34). All analyses of lutein, ascorbic acid, -tocopherol and retinol were conducted under yellow lights.

Plasma insulin and corticosterone levels were determined by a RIA (Insulin or Corticosterone RIA Kits, ICN Biomedicals, Horsham, PA), which uses the double antibody technique. The concentrations of the hormones were quantified using a precalibrated standard curve (35).

Statistical analyses.

Data were analyzed using a four-way factorial ANOVA to examine the effects of treatments with lutein, vitamin E, vitamin C, lutein plus vitamin E or lutein plus vitamin C in lean vs. obese rats. Normality and homogeneity tests of all data indicated that data were normally distributed and variances were equal. The Ryan-Einot-Gabriel-Welsch Multiple Range test was used to compare three or more means in a step-down multiple range test (36). All statistical analyses were conducted using the SAS Computerized Program (SAS Institute, Cary, NC). Values of P 0.05 are considered significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Characteristics of the lean and obese Zucker rats.

At the beginning of the study, Zucker obese (fa/fa) rats were already significantly heavier than their lean counterparts (data not shown). By the end of the 8-wk feeding period, body weight gain in obese rats was >100% greater than in lean rats (Table 1). In obese rats, there was a trend (P = 0.03) for increasing weight gain as lutein concentrations in the diet increased from 0.5 to 1.0 mg/kg, but in those fed VE-Lut, there was a decrease in weight gain with increasing lutein concentrations (P = 0.03). Food efficiency (g gain/g feed) was significantly higher in obese than in lean rats, but was not affected by the dietary treatments. Relative liver weight (g/100 g body) was higher in obese than in lean rats, due to the fatty liver feature of this model (Table 1), but was not significantly affected by the dietary treatments.

View this table: [in this window] [in a new window]   TABLE 1 Weight gain, food efficiency and relative liver weights in lean and obese rats fed diets containing various levels of lutein (Lut), -tocopherol (VE) and ascorbic acid (VC) for 8 wk1

Liver antioxidant status, as measured by GSH concentrations, was lower and lipid peroxidation (MDA concentration) was higher in obese than in lean rats (Table 2). Liver GSH levels did not differ due to dietary treatments. In obese rats, MDA was significantly lower in groups fed the VE diets than in other diet groups. A significant VC x Lut interaction was observed for liver MDA concentrations in obese rats such that VC-Lut0.5 fed rats had significantly lower MDA concentrations than either the Lut0.5 or the Control group.

View this table: [in this window] [in a new window]   TABLE 2 Hepatic glutathione and malondialdehyde (MDA) concentrations in lean and obese rats fed diets containing various levels of lutein (Lut), -tocopherol (VE), and ascorbic acid (VC) for 8 wk1

View this table: [in this window] [in a new window]   TABLE 3 Hepatic superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) specific activity in lean and obese rats fed diets containing various levels of lutein (Lut), -tocopherol (VE), and ascorbic acid (VC) for 8 wk1

Hepatic lutein, -tocopherol and ascorbic acid.

Concentrations of liver lutein and -tocopherol, but not ascorbic acid, were lower in some groups of obese rats compared with their lean counterparts (Table 4). Liver lutein concentrations were significantly enhanced in lean rats fed VE-Lut1.0. A weight xVC-Lut1.0 interaction (P= 0.02, not shown in Table 4) revealed that VC enhanced lutein levels in lean but not obese rats. Levels were higher in lean than in obese rats (Table 4). In obese rats, liver lutein concentrations did not differ due to dietary treatments. Lutein was not detected in the livers of rats not fed lutein in the diet.

View this table: [in this window] [in a new window]   TABLE 4 Liver lutein, -tocopherol, and ascorbic acid concentrations in Zucker obese and lean rats fed diets containing various levels of lutein (Lut), -tocopherol (VE) and ascorbic acid (VC) for 8 wk1

Liver ascorbic acid concentrations increased significantly with VC supplementation above that produced during endogenous biosynthesis in both lean and obese rats. Levels were higher in the Lut1.0 group than in all other non-VC supplemented groups.

Plasma lutein and vitamin concentrations.

Obese rats had significantly higher plasma concentrations of -tocopherol and retinol than did lean rats (Table 5). Plasma lutein levels were significantly higher (P 0.014) in obese than in lean rats, but did not differ due to dietary supplementation. -Tocopherol levels in plasma increased with VE feeding in both lean and obese rats; however, levels increased in a dose-dependent fashion in lean rats fed VE-Lut diets and decreased similarly in obese rats fed VE-Lut diets. Plasma retinal concentrations were significantly higher in obese than in lean rats, but significantly lower in Lut1.0, VE-Lut0.5 and VE-Lut1.0 compared with all other obese diet groups. Blood ascorbic acid concentrations ranged from 29 ± 3 to 38 ± 3 µmol/L in lean and 32 ± 6 to 44 ± 6 µmol/L in obese rats and did not differ due to dietary treatments or weight class (data not shown).

View this table: [in this window] [in a new window]   TABLE 5 Plasma lutein, -tocopherol, and retinol concentrations in Zucker obese and lean rats fed diets containing various levels of lutein (Lut) and -tocopherol (VE) for 8 wk1

Plasma insulin, but not corticosterone, concentrations were significantly influenced by obesity, in that levels were two- to eightfold higher in obese than in lean rats (Table 6). The rats had continuous access to their diets until they were killed. Plasma insulin levels were significantly lower in obese rats fed the VC-Lut0.5, VE-Lut1.0, Lut0.5 and Lut1.0 diets compared with rats fed the Control diet. There were no dietary effects on plasma insulin levels in lean rats. Corticosterone concentrations, which are indicative of increased carbohydrate metabolism during adrenal stress, did not differ due to weight class. Corticosterone levels were significantly higher in the Control lean and obese groups than in any of the treated groups. In lean and obese rats, lutein feeding reduced corticosterone levels more in obese VE-Lut groups than in Lut groups. In lean rats, the Lut x VE interaction resulted in a dose-dependent increase in corticosterone levels that was more exaggerated in VE-Lut than in Lut groups. The lowest plasma corticosterone levels were in obese rats fed VC-Lut1.0 and in lean rats fed Lut0.5, VE-Lut0.5, VC-Lut0.5 and VC-Lut1.0.

View this table: [in this window] [in a new window]   TABLE 6 Plasma corticosterone and insulin concentrations in Zucker lean and obese rats fed diets containing various levels of lutein (Lut), -tocopherol (VE), and ascorbic acid (VC) for 8 wk1, 2

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

For a number of the variables in this study, the effects were more dramatic when lutein was fed in combination with ascorbic acid rather than with -tocopherol. Lutein is readily absorbed in humans (40) and animals (13) and has been reported to decompose more slowly in biological systems than other carotenoids (41). These may be distinctive features of the potency of lutein as an antioxidant. Lutein may also be less likely to become a prooxidant (43) compared with arotene. Endogenous synthesis of ascorbic acid by rodents is established (26), but supplementation with ascorbic acid appeared to have a synergistic effect with lutein on some of the variables measured in this study.

The effects of lutein on biomarkers of oxidative stress were nonlinear with increasing lutein concentration in the diet where the most effective level was frequently at the lower concentration in the diet. This was the case with MDA concentrations and insulin in obese rats and GSH-px in lean rats. The highest GSH-px activity occurred when vitamin C was fed in combination with lutein at the higher concentration. Although lutein at the levels fed in the current study has been shown to be readily absorbed in rodents (13), the most effective dose of lutein on biomarkers of oxidative stress is currently unknown. Some biomarkers were altered when lutein was combined with antioxidants, especially vitamin C, suggesting that the most effective lutein dose is different when administered in combination with vitamin C or vitamin E.

Although the level of vitamin C in the diet was sufficient to raise liver ascorbic acid levels above those in the lean and obese nonsupplemented groups, it is unclear whether the low and nonpharmacologic levels in the diets of rodents were sufficient. Ascorbic acid has been demonstrated to be effective as an antioxidant against aqueous peroxyl radicals when administered at 1 g/kg diet (44) and to have a sparing effect on GSH and -tocopherol (26). Evidence of the sparing effect of ascorbic acid on liver GSH concentrations was not apparent in the present study because liver GSH was significantly lower in obese than in lean rats, but was not enhanced by ascorbic acid. Had the level of ascorbic acid been higher, findings related to alterations in biomarkers of oxidative stress might have been more numerous.

The effects of lutein in raising GSH-px activity in lean rats was enhanced when fed in combination with vitamin C. The effects of lutein on liver SOD activity in obese rats were independent of vitamin E or C. There is a paucity of information related to the effects of lutein on endogenous enzymatic antioxidants in the rat liver. The increase in SOD activity paralleled MDA concentrations in the present study and differed from the findings of Soltys et al. (37) who reported that liver SOD did not differ between obese and lean rats. These authors also reported higher catalase activity in fatty livers from obese Zucker rats compared with livers from lean rats. The lower liver GSH concentrations and hyperinsulinemia in obese rats in the present study compared with lean are consistent with these findings (37).

Evidence from this study suggests that lutein interacts with both vitamins C and E in altering biomarkers of oxidative stress. The combined effects of lutein and vitamin C altered hepatic MDA concentrations and hyperinsulinemia in obese rats and plasma corticosterone concentrations and hepatic GSH-px activity in lean rats, whereas those of lutein and vitamin E affected hyperinsulinemia in obese rats and plasma corticosterone in lean and obese rats. Lutein in combination with ascorbic acid affected biomarkers of oxidative stress more frequently than lutein in combination with vitamin E. Vitamin E greatly reduced hepatic MDA levels in obese rats, independent of lutein. Interactions between vitamin E and lutein increased liver lutein concentrations in lean rats and reduced liver -tocopherol concentrations in lean and obese rats. The findings from this study add to the body of knowledge that obese Zucker rats have increased biomarkers of oxidative stress, compared with their lean counterparts, and that the ingestion of moderate amounts of lutein, vitamin C or vitamin E effectively reduces the obesity-induced biomarkers. These results suggest that the effectiveness of lutein as an antioxidant in body systems other than the eye may be enhanced when combined with vitamin C or vitamin E. Future studies might examine further the relationship between lutein and ascorbic acid in altering biomarkers of oxidative stress.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 02, April 2002, New Orleans, LA [Blakely, S. R., Herbert, A., Collins, M., Mitchell, G., Jenkins, M. & Grundel, E. (2002) Lutein, ascorbic acid and -tocopherol reduce obesity-induced oxidative stress in Zucker obese rats. FASEB J. 16: LB57 (abs.)].

² Sponsored by the Office of Women’s Health, Food and Drug Administration, Rockville, MD

⁴ GSH, glutathione; GSH-px, glutathione peroxidase; Lut, lutein; MDA, malondialdehyde; SOD, superoxide dismutase; VC, vitamin C; VE, vitamin E.

Manuscript received 21 March 2003. Initial review completed 18 April 2003. Revision accepted 26 June 2003.

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