The Journal of Nutrition Vol. 128 No. 11 November 1998, pp. 1989-1994

Supplementation with Canthaxanthin Affects Plasma and Tissue Distribution of - and -Tocopherols in Mice^1,2

Paola Palozza, Gabriella Calviello, Simona Serini^*, Piera Moscato, and Gianna Maria Bartoli^{*, 3}

Institute of General Pathology, Catholic University, 00168 Rome, Italy and ^* Department of Biology, Tor Vergata University, 00133 Rome, Italy

	ABSTRACT

Abstract Introduction Methods Results Discussion References

The effects of oral doses of canthaxanthin on tissue distribution of - and -tocopherols were investigated in three experiments in male and female Balb/c mice. Mice were assigned to receive canthaxanthin [7 or 14 µg/(g body weight·d)] or placebo (olive oil) by gavage for different periods of time (0, 1, 2, 4 and 6 wk). A 2 wk-treatment with canthaxanthin resulted in incorporation of the carotenoid in all tissues analyzed, including liver, spleen, kidney, lung and heart. In liver, the maximum accumulation of the carotenoid was reached after 2 wk of dosing in female mice and after 6 wk in male mice. Canthaxanthin incorporation was accompanied by changes in - and -tocopherol concentrations in plasma and tissues. These included the following: 1) a significant increase (P < 0.001) in -tocopherol concentration in spleen (21 and 27% in male and female mice, respectively) after 2 wk and in liver (~50% in both male and female mice) after 6 wk; 2) a significant decrease in -tocopherol concentration in plasma (P < 0.05) and tissues (P < 0.001) after 2 wk of treatment. In female mice, this decrease was 55% in plasma, 43% in liver, 44% in kidney, 71% in lung and 70% in heart. In male mice, the decrease was observed only in plasma (30%), kidney (54%) and heart (46%). In liver, the decrease in -tocopherol concentration was both dose- and time-dependent and significantly (P < 0.001) greater in female than in male mice. We conclude that dietary administration of canthaxanthin modifies tocopherol status in murine tissues.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Canthaxanthin, a 4,4' diketo--carotene commonly used as a feed additive, has been reported to act as an anticarcinogenic agent by a mechanism not involving pro-vitamin A activity (Grubbs et al. 1991, Katsumura et al. 1996, Tanaka et al. 1995). Several alternative hypotheses have been proposed to explain the antitumor effects of this carotenoid, including its ability to act as an antioxidant (for reviews, see Burton and Ingold 1984, Krinsky 1993, Palozza and Krinsky 1992a and 1992b), to potentiate immune responses (Bendich and Shapiro 1986), to enhance gap junctional communication directly (Zhang et al. 1992) or through the formation of 4-oxo-retinoic acid (Hanusch et al. 1995). It has also been suggested that canthaxanthin supplementation may interfere with the metabolism of lipophilic compounds, including other carotenoids (Kostic et al. 1995, White et al. 1993 and1994) and -tocopherol (Bendich and Shapiro 1986, Blakely et al. 1991, Tang et al. 1995, Woodall et al. 1996). In particular, it has been demonstrated that rats (Bendich and Shapiro 1986, Blakely et al. 1991) and ferrets (Tang et al. 1995), supplemented with canthaxanthin, exhibited low levels of -tocopherol in plasma and/or tissues. Similarly, animal (Lambert et al. 1994, Xu et al. 1992, Woodall et al. 1996) and human (Mobarhan et al. 1994, Xu et al. 1992) studies have documented that dietary -carotene can reduce plasma -tocopherol levels (Bendich and Shapiro 1986) and conversely, that dietary -tocopherol can cause significant decreases in plasma and hepatic concentrations of -carotene (Alam et al. 1990). Moreover, it has been reported that feeding chicks canthaxanthin increased resistance to lipid peroxidation primarily by enhancing membrane -tocopherol levels in liver (Mayne and Parker 1989). Such interference between carotenoids and tocopherols could alter the protective ability of cells against oxidative stress and, consequently, modify the biological effects of carotenoids in vivo and their preventive role in chronic diseases (Mayne 1996). Therefore, in light of the possible role of carotenoids as chemopreventive agents, it is helpful to determine how dose and time of carotenoid administration may affect tissue distribution of tocopherols.

Laboratory animal species, such as rats and mice, do not accumulate dietary carotenoids as efficiently as humans (Mathews-Roth 1977, van Vliet 1996). However, several studies have shown that mice given carotenoid-fortified supplements do accumulate them in serum and tissues (Kornhauser et al. 1994, Wamer et al. 1985). We found that dietary administration of canthaxanthin to Balb/c mice at a dose of 14 µg/ (g body weight·d) for 2 wk resulted in an accumulation of the carotenoid in plasma and tissues and in a prolonged survival of Balb/c mice bearing thymoma cells, derived from a highly malignant tumor of lymphatic origin of the BALB/c mouse (Palozza et al. 1997). More xanthophyll was accumulated than -carotene and other carotenoids (unpublished data).

The purpose of this study was to determine whether canthaxanthin supplementation affects the homeostasis of fat-soluble compounds such as -and -tocopherol. The effects of canthaxanthin on tocopherols were evaluated by supplementing male and female Balb/c mice with different doses of the carotenoid for different periods of time and by measuring plasma and tissue levels of canthaxanthin, - and -tocopherols.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Materials.  Canthaxanthin (kindly provided by Hoffmann-La Roche, Basel, Switzerland) was administrated to mice as stock solutions in olive oil (1.5 and 3.0 g/L). To improve the solubility of the carotenoid in the oil, canthaxanthin was first dissolved in tetrahydrofuran (THF) and olive oil was then added. Finally, THF was evaporated under a stream of nitrogen. Canthaxanthin solutions were prepared fresh daily. The percentage of all-trans-canthaxanthin in the total material used was 97%. This concentration was verified spectrophotometrically at 470 nm (E^1% _1cm = 2200) and the HPLC analysis revealed the presence of a unique peak, corresponding to all-trans-canthaxanthin. Ammonium acetate and butylated hydroxytoluene (BHT) were obtained from Sigma Chemical (St Louis, MO); tocol was kindly provided by Hoffmann-La Roche, Basel, Switzerland; THF (99%) was purchased from Aldrich Chemical (Milwaukee, WI) and always used under a nitrogen atmosphere; hexane, methanol, acetonitrile and ethanol were obtained from Fluka Chemika-Biochemika (Buchs, Switzerland). All of the solvents used were HPLC grade.

Animals.  Female or male inbred Balb/c mice, aged 6 wk, were allocated separately to cages housing five mice per cage in a room with a 12-h light:dark cycle, at a temperature of 25°C, and at a constant humidity. The average body weight of mice was 22.5 ± 1.0 g (females) and 23.3 ± 2.0 g (males). Throughout the experiments, the animals were fed a nonpurified commercial diet (Altromin-Rieper, Rieper, Bz, Italy) with the following composition (g/100 g): crude protein, 23; fat, 5.5; fiber, 5; mineral, 8; carbohydrates, 58.5; water, 12. The diet provided (g/kg): (n-6) polyunsaturated fatty acids (18:2), 7.5 and (n-3) polyunsaturated fatty acids (18:3, 18:4, 20:5 and 22:6), 1.02. The vitamin mixture added to the diet (g/kg) was 2.5: all-rac--tocopheryl acetate, 0.1; retinyl palmitate, 0.06; cholecalciferol, 0.00005; vitamin C, 0.1; choline chloride, 1; biotin, 0.2; folic acid, 0.01; DL-methionine, 3.5; vitamin B-12, 0.00003; calcium pantothenate, 0.00005; and thiamin·HCl, 0.00015. The mineral mixture (g/kg) was 0.52 and contained: Fe, 0.12; Mg, 0.06, Zn 0.02; Cu, 0.005, Co, 0.0004; and Se, 0.0005. The animals were given free access to water. Mice were examined and weighed at the start of the experiment and twice a week during the entire period of study. All animal procedures were reviewed and approved by the Ministry of Health, Veterinary Service, Rome, Italy.

Experimental studies.  The mice were randomly assigned to one of the following experimental groups:

Experiment 1.  Distribution in tissues. Separate groups of male and female mice, consisting of 15 mice each, received canthaxanthin 14 µg/(g body weight·d) or olive oil as a vehicle (control group) for 2 wk. Canthaxanthin and olive oil were administrated daily by gavage. This dose (14 µg/g) represented the maximum dose of canthaxanthin that can be dissolved in an amount of olive oil tolerated by the animals. At the end of the treatment, all mice were killed by cervical dislocation. Blood was collected. Liver, spleen, kidney, lung and heart were excised, rinsed briefly with ice-cold saline, weighed, frozen in liquid nitrogen and stored at 80°C.

Experiment 2.  Time response. Separate groups of male and female mice, consisting of 15 mice each, received canthaxanthin or olive oil for 1, 2, 4 or 6 wk. The carotenoid was administrated at the dose of 14 µg/(g body weight·d) as indicated above. At the end of the treatment, the animals were killed and plasma and liver were collected.

Experiment 3.  Dose response. Three separate groups of 15 female mice received canthaxanthin at the concentrations 0 (olive oil, control), 7 or 14 µg/(g body weight·d) for 2 wk. At the end of the experiment the animals were killed and liver was isolated.

Plasma separation.  Blood was obtained by intracardiac puncture and placed in heparin tubes on ice. Plasma was separated from blood by centrifugation at 800 × g for 30 min and stored at 80°C.

Extraction of canthaxanthin and tocopherols.  Duplicate aliquots of plasma (100 µL) were denatured by addition of an equal volume of absolute ethanol containing BHT (1 g/L). As an internal standard for the evaluation of the tocopherols, 250 µL of tocol in hexane (5 mg/L) was added to each sample. The samples were extracted three times with 2 mL of hexane and the combined hexane layers were evaporated to dryness under nitrogen.

Weighed tissue samples (0.2 g) were homogenized in absolute ethanol (1:5, wt/v) containing BHT (1 g/L) by an Ultraturrax homogenizer (6 times, 15 s each). After the addition of deionized water (1:2, wt/v), the samples were extracted two times with hexane (1:5, wt/v) (White et al. 1993). The combined hexane layers were evaporated to dryness under nitrogen. Residues of plasma and tissue extracts were reconstituted with methanol (60 µL).

Analysis of canthaxanthin and tocopherols.  For canthaxanthin analysis, 20-µL aliquots were injected into an HPLC system. Chromatography was carried out with a LC-18-DB Supelcosil column, 15 cm × 0.46 cm, 3-µm particle size (Supelco, Bellefonte, PA). A C18-DB Supelcosil precolumn, 2 cm × 0.46 cm, 5-µm packing, was used. Mobile phase was 85% acetonitrile/15% methanol, containing 0.1 g/L ammonium acetate. Flow rate was 1 mL/min and the detection was at 460 nm. The retention time of the compound was 7.2 min. Canthaxanthin concentration in samples was calculated as all-trans-canthaxanthin from a calibration curve generated from a peak height of canthaxanthin in calibration samples (Palozza et al. 1997).

For tocopherol analysis, 20-µL aliquots were analyzed by reverse-phase HPLC with fluorescence detection on a Perkin-Elmer 650-LC fluorescence detector (Perkin-Elmer, Norwalk, CT) with excitation at 295 nm and emission at 340 nm. Tocopherols, as well as the internal standard, tocol, were eluted with 100% methanol on a C18, 15 cm × 0.46 cm, 3-µm particle size column (Alltech Associates, Deerfield, IL). The retention times of - and -tocopherols were 9.1 and 10.4 min, respectively. Tocopherol concentration in samples was calculated from a calibration curve generated from a peak height of tocopherols in calibration samples (Palozza et al. 1992a).

Canthaxanthin did not interfere with the tocopherol assay because fluorescence peaks obtained by methanol solutions of - and -tocopherols were identical to those obtained by the same solutions containing different amounts of canthaxanthin.

Statistical analysis.  Multifactorial ANOVA was used to determine the effects of treatment, gender, time or tissues and their interactions on the measured parameters in Experiments 1 and 2. One-way ANOVA was used to determine differences in tissue concentration of canthaxanthin and -tocopherol in Experiment 3. When significant values were found, post-hoc comparisons of means were made using the Tukey's Honestly Significant Difference test. Differences, analyzed using Minitab Software (Minitab, State College, PA), were considered significant at P < 0.05.

	RESULTS

Abstract Introduction Methods Results Discussion References

The mice appeared to be healthy and showed no signs of discomfort during the feeding trials. Feed efficiencies did not differ among the groups. No significant differences in body weights of canthaxanthin- and control (olive oil)-treated groups were found.

Distribution in tissues.  The treatment with canthaxanthin for 2 wk resulted in incorporation of the carotenoid in all tissues analyzed (Fig. 1). No traces of canthaxanthin were found in tissues after treatment with olive oil for the same period of time (data not shown). The highest concentrations of canthaxanthin were found in liver and in spleen. Lower amounts of the carotenoid were found in kidney, lung and heart. The hepatic and splenic incorporations of canthaxanthin were significantly higher (P < 0.001) in female than in male mice. In plasma, canthaxanthin concentrations were 8.5 ± 0.5 and 11.0 ± 0.6 nmol/L in male and female mice, respectively (P < 0.05).

View larger version (33K): [in this window] [in a new window]   Fig 1. Canthaxanthin distribution in tissues of male and female mice treated with the carotenoid at a dose of 14 µg/(g body weight·d) for 2 wk. Values are the means ± SEM, n = 15. The gender/organ interaction was significant (P < 0.02). Values not sharing a letter are significantly different, P < 0.001.

In both male and female mice, 2-wk canthaxanthin supplementation increased -tocopherol levels only in spleen (Table 1). In female mice, canthaxanthin supplementation significantly decreased -tocopherol concentration in all tissues, except spleen. In male mice, a decrease in -tocopherol level was significant (P < 0.001) only in plasma, kidney and heart.

View this table: [in this window] [in a new window]   Table 1. Effect of 2-wk oral administration of canthaxanthin [Cx, 14 µg/(g body weight·d)] on the concentrations of -tocopherol and -tocopherol in plasma and tissues of male and female Balb/c mice1,2

Time-response.  Canthaxanthin concentration increased significantly in liver of both male and female mice after 1 wk of treatment (Fig. 2). After this period, female mice had higher (P < 0.005) levels of canthaxanthin than male mice. The maximum accumulation of the carotenoid was reached after 2 wk of dosing in female mice and after 6 wk in male mice. During the experiment, no traces of canthaxanthin were found in mice treated with olive oil.

View larger version (18K): [in this window] [in a new window]   Fig 2. Canthaxanthin concentration in liver of male and female mice treated with the carotenoid at a dose of 14 µg/(g body weight·d) for various periods. Values are the means ± SEM n = 15. There was a significant gender/time interaction (P < 0.002). Values not sharing a letter are significantly different, P < 0.005.

Hepatic -tocopherol concentration was modified by canthaxanthin treatment, and its maximum accumulation was reached at 6 wk in both male and female mice (Fig. 3). Concomitantly, a progressive decrease in the hepatic concentration of -tocopherol was observed. It started after 4 wk in males and after 1 wk in females. Female mice exhibited a total depletion of the antioxidant at 4 wk.

View larger version (17K): [in this window] [in a new window]   Fig 3. Concentration of -tocopherol (A) and -tocopherol (B) in liver of male and female mice treated with the carotenoid at a dose of 14 µg/(g body weight·d) for various periods. Values are the means ± SEM, n = 15. There was a significant treatment/time interaction (P < 0.0001) for -tocopherol and significant gender/treatment and treatment/time interactions (P < 0.005) for -tocopherol. Values not sharing a letter are significantly different, P < 0.001.

The levels of canthaxanthin and - and -tocopherols in plasma were not further modified by prolonging the treatment until 6 wk (data not shown).

Dose-response.  A 2-wk treatment of female mice with canthaxanthin at a dose of 14 µg/(g body weight·d) resulted in significantly (P < 0.05) greater carotenoid concentration in liver (Fig. 4A) and a lower (P < 0.05) concentration of -tocopherol (Fig. 4B) than in mice administered a dose of 7 µg/(g body weight·d). No differences in the hepatic concentration of -tocopherol were observed at the two doses of the carotenoid (data not shown).

View larger version (34K): [in this window] [in a new window]   View larger version (28K): [in this window] [in a new window]   Fig 4. Concentration of canthaxanthin (A) and -tocopherol (B) in liver from female mice treated with the carotenoid at 0, 7 or 14 µg/(g body weight·d) for 2 wk. Values are the means ± SEM, n = 15. Values not sharing a letter are significantly different, P < 0.05.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Oral supplementation with canthaxanthin modified the endogenous concentration of both -tocopherol and -tocopherol in murine tissues. The mice accumulated the carotenoid in all tissues analyzed. After a 2 wk-treatment, the highest amount of canthaxanthin was found in liver, in which carotenoid incorporation was dose-dependent in female mice. When the period of administration was prolonged for >2 wk, no further accumulation of canthaxanthin was observed in this organ in female mice. In contrast, an increase in hepatic accumulation of the carotenoid was observed at 6 wk of treatment in male mice. Shapiro et al. (1984) found a similar trend of -carotene accumulation in rat liver. The observation that canthaxanthin was not further stored after a 2-wk treatment suggests that mice could adjust the intestinal absorption from excessive oral intake or increase hepatic metabolism of canthaxanthin, as suggested by Tang et al. (1995). Spleen, as well as liver, is an important organ of canthaxanthin accumulation, confirming the results obtained by other investigators (Bendich and Shapiro 1986, Krinsky et al. 1990, Mathews-Roth et al. 1990). Canthaxanthin was more efficiently accumulated by female than by male mice, as shown by the levels of the carotenoid in plasma and in liver. These data suggest a possible difference between male and female mice in intestinal absorption and/or metabolism of canthaxanthin. Sex differences in the content of canthaxanthin were recently demonstrated by us in the same animal model (Palozza et al. 1997). These data also confirm previous observations in humans, showing that antioxidant nutrients, including -carotene and other carotenoids, are present in a higher amount in females than in males (Kaplan et al. 1987).

Canthaxanthin treatment interfered with the metabolism of both - and -tocopherols. This carotenoid increased the concentrations of -tocopherol in spleen and liver, the two major organs of canthaxanthin storage. This increase was apparent in liver after 6 wk of treatment and was similar in male and female mice. These results agree with those reported by Mayne and Parker (1989), who showed that dietary canthaxanthin increased fourfold the hepatic concentration of -tocopherol in vitamin E-deficient chicks. Conversely, ferrets (Tang et al. 1995) and rats (Blakely et al. 1991) supplemented with canthaxanthin had decreased hepatic concentrations of -tocopherol. The discrepancies of these findings may be due to the different animal models as well as the dose and time of canthaxanthin administration. In this study, we supplemented canthaxanthin for 6 wk as did Mayne and Parker, whereas Tang et al. prolonged the treatment for 2 y. Moreover, Blakely et al. (1991) supplemented canthaxanthin to the diet of rats for 8 wk, but at a much higher canthaxanthin concentration [2 g/kg diet, which corresponds to an oral dose of ~100 µg/(g body weight·d), estimated from food intakes and body weights] than that administered by gavage to mice in this study [14 µg/(g body wt·d)].

High levels of -tocopherol were also found in spleen after 2 wk of canthaxanthin supplementation, whereas no changes in the concentrations of this antioxidant were found in kidney, heart and lung. This observation suggests that the modifications in -tocopherol levels by canthaxanthin are tissue specific.

Although canthaxanthin treatment increased -tocopherol concentrations in spleen and liver in our study, it did not modify significantly -tocopherol levels in plasma. This could reflect a dynamic redistribution of the antioxidant among different organs consequent to canthaxanthin accumulation. In agreement with our data, canthaxanthin supplementation did not affect plasma levels of -tocopherol in ferrets (Tang et al. 1995) or chicks (Woodall et al. 1996). Conversely, in other experimental models, a decrease in plasma levels of -tocopherol due to dietary supplementation with canthaxanthin (Bendich and Shapiro 1986, Blakely et al 1991) and/or -carotene (Bendich and Shapiro 1986, Lambert et al. 1994, Xu et al. 1992) has been reported.

The major finding of this study is the observation that oral supplementation with canthaxanthin decreased -tocopherol concentration in plasma and tissues. In liver, this reduction was time-dependent and significantly greater in female than in male mice.

The mechanisms by which canthaxanthin reduces the concentration of -tocopherol and increases that of -tocopherol in tissues are not fully understood. Canthaxanthin and tocopherols might compete for intestinal absorption or for binding with lipoproteins. Evidence for interactions between -carotene and other lipid-soluble nutrients, such as other carotenoids, has been reported (Kostic et al. 1995, White et al. 1993 and 1994). The possibility that tocopherols and carotenoids may influence one another is suggested by recent research on ferrets, indicating that -tocopherol promotes the intestinal absorption of intact -carotene (Wang et al. 1995).

On the other hand, the finding that, in our study, canthaxanthin supplementation induces different effects on -tocopherol and -tocopherol could be due to the different storage of the two tocopherols in tissues as well as to their different susceptibilities to oxidation. -Tocopherol is stored less efficiently in tissues than -tocopherol, and -tocopherol disappears more quickly than -tocopherol (Chow et al. 1971, Griffiths 1959, Peake and Bieri 1971). In agreement with the hypothesis that carotenoids may affect tocopherols in tissues through a modulation of cell oxidative status, we previously demonstrated that carotenoids altered the loss of tocopherols induced by different sources of free radicals in lipid homogeneous solution (Palozza and Krinsky 1991), in isolated membranes (Palozza and Krinsky 1992c) and in intact cells (Palozza et al. 1996).

Canthaxanthin intake may also interfere with other antioxidant mechanisms. Schwartz et al. (1993) demonstrated that -carotene supplementation reduced the concentration of glutathione and the activity of superoxide dismutase and S-glutathione transferase in tumor cells. Such a reduction did not occur if the cells were incubated together with both -carotene and vitamin E.

We conclude that canthaxanthin supplement, at the dose given, modifies the concentrations of - and -tocopherols in murine tissues. The effects of carotenoid administration on tocopherols were gender-, dose- and time-dependent and tissue-specific. This finding could be important in light of the possible role of carotenoids as potential chemopreventive agents. The changes in tocopherol concentrations induced by carotenoids could alter the protective ability of cells against oxidative stress and, consequently, modify the biological effects of carotenoids in vivo and their preventive role in chronic diseases (Mayne 1996). However, further studies are required to clarify the mechanisms responsible for this interference and to establish the importance of this finding in humans.

	ACNOWLEDGMENT

The authors thank Hoffmann-La Roche for the generous donation of canthaxanthin.

	FOOTNOTES

Manuscript received 23 December 1997. Initial reviews completed 9 February 1998. Revision accepted 8 June 1998.

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