Comparison of bioavailability between β-cryptoxanthin and β-carotene and tissue distribution in its intact form in rats

Abstract

The bioavailability of two intact carotenoids in several tissues of β-cryptoxanthin- and β-carotene-fed rats (20 mg/kg of diet) was investigated. Although metabolites of provitamin A are not included in our study, β-cryptoxanthin was found at higher concentrations in majority of the tissues. The results show that the bioavailability of intact β-cryptoxanthin seemed to be higher than that of β-carotene.

Key words:

• β-cryptoxanthin
• β-carotene
• absorption
• tissue distribution
• bioavailability

β-cryptoxanthin is a carotenoid pigment particularly found in Satsuma mandarin (Citrus unshiu Marc.).¹⁾ We have previously reported that serum β-cryptoxanthin reflected the frequency of Satsuma mandarin consumption.^2,3) β-cryptoxanthin might be easily absorbed, and it can survive for relatively long periods in the human body in its intact form. We also found from a nutritional epidemiological survey that individuals with a high serum β-cryptoxanthin or β-carotene level had lower risk for lifestyle-related diseases.⁴⁻⁹⁾ We assume from these results that absorbed β-cryptoxanthin and β-carotene were accumulated in several organs in their intact form and may have provided a protective effect against oxidative stress in several tissues. However, little is known about the differences between β-cryptoxanthin and β-carotene regarding absorption and tissue distribution in their intact form. We compared in this study the bioavailability of intact β-cryptoxanthin and β-carotene in rats after supplementing β-cryptoxanthin or β-carotene to a standard commercial diet.

Twenty milligrams of β-cryptoxanthin (Shikoku Yashima Pure Chemicals Co., Tokushima, Japan) or β-carotene (Sigma-Aldrich Co., Tokyo, Japan) was dissolved in 10 g of corn oil (Wako Pure Chemical Industries, Osaka, Japan) and mixed with one kilogram of a standard CE-2 commercial diet (Clea Japan, Tokyo, Japan). Thirty male and female Wistar rats (four-weeks old and with a 75–85 g body weight) were purchased from Clea Japan (Tokyo, Japan). All animals were maintained in an environmentally controlled room under a 12:12-h light–dark cycle and fed a standard CE-2 diet for one week before the tests. Five males and five age-matched female rats were fed with β-cryptoxanthin diet (20 mg/kg of diet), and 10 others were fed with β-carotene diet (20 mg/kg of diet) for four weeks (n = 5 for each group). The 10 other age-matched male and female rats were used for a control group to determine the basal carotenoid levels in the serum and tissues. Food and water were available ad libitum. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals adopted by the National Institute of Fruit Tree Science. After four weeks of supplementation of the β-cryptoxanthin or β-carotene diet, male blood samples were obtained from the abdominal vena cava under diethyl ether anesthesia. Serum was separated from the blood cells by centrifugation. After blood collection, the male rats were euthanized by decapitation; the liver, spleen, kidney, pancreas, lung, heart, testis, and brain were then removed and stored at −80 °C until needed for analyses. The uterus and ovary were collected from age-matched female rats. All the rats were dissected after 3 h of fasting, because, in rodents, carotenoids might be rapidly eliminated and deposited in the liver.^10,11) The concentrations of intact β-cryptoxanthin and β-carotene in the serum and tissues were evaluated by HPLC.¹²⁾

The carotenoid contents in the standard diet supplemented with β-cryptoxanthin or β-carotene are summarized in Table 1. The major carotenoids in the standard diet were lutein, zeaxanthin, and β-carotene. A small amount of β-cryptoxanthin was also detected in the standard diet (0.07 mg/kg). Table 2 shows the serum and tissue carotenoid concentrations in the control diet-fed rats after four weeks. In the group of rats receiving the control diet, none of the five carotenoids could be detected in the serum and most tissues, with the exception of the liver and ovary. Although lutein and zeaxanthin were not detected in the liver of the group receiving the control diet, α- and β-carotene and β-cryptoxanthin were detected in small amounts. In contrast, the α- and β-carotene contents in the ovary were similar to those in the liver. However, β-cryptoxanthin was not detected in the ovary.

Table 1. Carotenoid contents of the standard and β-cryptoxanthin- and β-carotene-supplemented diets.¹

	Carotenoid content
	Lutein	Zeaxanthin	α-carotene	β-carotene	β-cryptoxanthin
Control diet (mg/kg)	1.15 (0.01)	0.27 (0.00)	0.02 (0.00)	0.26 (0.01)	0.07 (0.00)
β-cryptoxanthin-supplemented diet (mg/kg)	1.14 (0.01)	0.27 (0.00)	0.02 (0.00)	0.26 (0.01)	20.11 (0.01)
β-carotene-supplemented diet (mg/kg)	1.15 (0.01)	0.28 (0.00)	0.02 (0.00)	20.21 (0.01)	0.08 (0.00)

¹ Data are presented as the means ± SEM of three observations.

Table 2. Serum and tissue carotenoid contents of the control diet-fed rats.¹

	Lutein	Zeaxanthin	α-carotene	β-carotene	β-cryptoxanthin
Serum (ng/mL)	ND	ND	ND	ND	ND
Liver (ng/g of issue)	ND	ND	13.7 (3.8)	67.8 (14.5)	26.9 (9.6)
Kidney (ng/g of tissue)	ND	ND	ND	ND	ND
Spleen (ng/g tissue)	ND	ND	ND	ND	ND
Heart (ng/g of tissue)	ND	ND	ND	ND	ND
Pancreas (ng/g of tissue)	ND	ND	ND	ND	ND
Lung (ng/g of tissue)	ND	ND	ND	ND	ND
Brain (ng/g of tissue)	ND	ND	ND	ND	ND
Testis (ng/g of tissue)	ND	ND	ND	ND	ND
Uterus (ng/g of tissue)	ND	ND	ND	ND	ND
Ovary (ng/g of tissue)	ND	ND	8.5 (2.3)	59.0 (8.2)	ND

Note: ND, not detectable.

¹ Data are presented as the means ± S.E.M. of five observations.

In the groups of β-cryptoxanthin or β-carotene diet-fed rats, there was no evidence of any influence of the chronic β-cryptoxanthin or β-carotene treatment on the body weight when compared with the control diet-fed rats during the experimental period (data not shown). We therefore concluded that the amounts of diet ingested were not different among the three groups. Fig. 1 summarizes the serum and tissue intact carotenoid concentrations of the β-cryptoxanthin or β-carotene diet-fed rats after four weeks of supplementation. In the group of rats receiving the β-cryptoxanthin diet, there was a wide variation in the tissue levels of intact β-cryptoxanthin, liver having the highest level. The ovary was second, followed by the spleen, uterus, kidney, testis, pancreas, brain, and heart. The lung had the lowest level. Intact β-cryptoxanthin was also detected in the serum. In the group of β-carotene diet-fed rats, intact β-carotene was also detected in the serum, liver, ovary, pancreas, uterus, testis, kidney, and brain. The heart had the lowest level. The intact β-carotene content in the ovary of the β-carotene diet-fed rats was about two and a half times higher than that of the β-cryptoxanthin diet-fed rats. Furthermore, the intact β-carotene content in the pancreas also tended to be higher than that in the rats receiving the β-cryptoxanthin diet. However, no intact β-carotene was detected in the spleen and lung of the β-carotene diet-fed rats. Our results show that absorbed β-cryptoxanthin was accumulated in various organs in its intact form and varied widely within the tissues when compared with that of β-carotene. This indicates that the various physiological functions of β-cryptoxanthin seemed to be related to the higher bioavailability of intact β-cryptoxanthin.

Fig. 1. Serum and tissue carotenoid concentrations in rats fed the β-cryptoxanthin- or β-carotenoid-supplemented diet.

Note: All data are presented as the mean ± SEM of five observations. The unfilled column indicates the results for the group fed the β-carotene diet after four weeks of supplementation. The filled column indicates the results for the group fed the β-cryptoxanthin diet after four weeks of supplementation. The statistical significance for the two groups was tested by using an unpaired t-test. Abbreviations: β-CX, β-cryptoxanthin; β-CA, β-carotene; ND, not detectable; NS, not significant.

The conversion of retinol from provitamin carotenoids by the human body is actively regulated by the amount of retinol available to the body.¹³⁾ It therefore seems that a substantial amount of intact β-carotene and β-cryptoxanthin, except for a requisite amount as provitamin A, was also absorbed and released into the blood circulation and then accumulated in several tissues. Some previous studies have reported that β-carotene was distributed in its intact form in such tissues as the liver, adrenal, ovary, heart, kidney, lung, skin, and brain of rats after chronic ingestion of β-carotene.^14,15) In this present study, the contents of β-cryptoxanthin and β-carotene in the supplemented diets were about 20 mg/kg, this dose being extremely high compared with the standard basal diet (0.26 mg/kg for β-carotene and 0.07 mg/kg for β-cryptoxanthin). We therefore concluded that a substantial amount of supplemented β-cryptoxanthin or β-carotene was absorbed and released into the blood circulation and then accumulated in several tissues in its intact form. However, the breakdown products of provitamin A such as retinoid are not included in our study, and further future work will be required.

The liver had the highest levels of β-cryptoxanthin and β-carotene in this study. Carotenoids are mainly accumulated in the liver and combined into lipoprotein for release into the blood circulation. Ingested carotenoids could participate in an antioxidant defense system when present in high concentrations of free radical species in the liver, and these physiological functions of carotenoids could inhibit the development of a liver dysfunction. The liver might therefore be a valuable target organ for carotenoids. We have previously found from a cross-sectional Mikkabi study that a high level of serum β-cryptoxanthin or β-carotene was associated with a lower risk for such liver diseases as alcoholic- and non-alcoholic-related liver dysfunction.^4,5) These epidemiological surveys enabled us to find that an inverse association of the serum carotenoid concentration with serum liver enzymes was more evident with β-cryptoxanthin than with β-carotene. We also found that the dietary intake of β-carotene was higher than that of β-cryptoxanthin, although the serum concentration of β-carotene was significantly lower than that of β-cryptoxanthin.⁹⁾ In addition, lutein was the highest consumed carotenoid in our nutritional epidemiological surveys, although the serum lutein concentration was significantly lower than that of β-cryptoxanthin and was not associated with the risk of a liver dysfunction.^4,5) The bioavailability of lutein seems to have been extremely low when compared with that of β-cryptoxanthin or β-carotene. Our previous findings from human studies further support the hypothesis that β-cryptoxanthin would be more easily absorbed than other carotenoids and accumulated in several organs, and that the various physiological functions of β-cryptoxanthin might be related to the higher bioavailability of intact β-cryptoxanthin.

In contrast, β-carotene in our study was markedly accumulated with 393.28 (ng/g of tissue) in the ovary of rats fed the diet supplemented with β-carotene, this content being about two and a half times higher than that of β-cryptoxanthin. Furthermore, a small amount of α-carotene was also detected in the ovary of the β-cryptoxanthin- or β-carotene-supplemented diet-fed rats (data not shown). The bioavailability of α- and β-carotene in the ovary seems to have been higher than that of other carotenoids. We have no clear explanation for this, but we conclude that the ovary might have a selective α- and β-carotene-uptake mechanism. We also found that the spleen accumulated a high level of β-cryptoxanthin (100.65 ng/g of tissue). In contrast, β-carotene was not detected in the spleen of the group fed the β-carotene-supplemented diet. We have no clear explanation for this, but we conclude that β-cryptoxanthin stored in erythrocytes might have been removed from old erythrocytes and accumulated in the spleen. Further studies are necessary to provide more detailed information about the characteristics of β-cryptoxanthin.

Funding

This work was supported by research and development projects for application in promoting a new policy for agriculture, forestry, and fisheries from Ministry of Agriculture, Forestry and Fisheries of Japan [grant number 22027] and by a grant from the Council for the Advancement of Fruit Tree Science.