In vivo tissue uptake of intravenously injected water soluble all-trans β-carotene used as a food colorant

Water soluble β-carotene (WS-BC) is a carotenoid form that has been developed as a food colorant. WS-BC is known to contain 10% of all-trans β-carotene (AT-BC). The aim of the present study was to investigate in vivo tissue uptake of AT-BC after the administration of WS-BC into rats. Seven-week-old male rats were administered 20 mg of WS-BC dissolved in saline by intravenous injection into the tail vein. At 0, 6, 24, 72, 120 and 168 hours (n = 7/time), blood was drawn and liver, lungs, adrenal glands, kidneys and testes were dissected. The levels of AT-BC in the plasma and dissected tissues were quantified with HPLC. After intravenous administration, AT-BC level in plasma first increased up to 6 h and returned to normal at 72 h. In the testes, the AT-BC level first increased up to 24 h and then did not decrease but was retained up to 168 h. In the other tissues, the level first increased up to 6 h and then decreased from 6 to 120 or 168 h but did not return to normal. The accumulation of WS-BC in testes but not in the other 5 tissues examined may suggest that AT-BC was excreted or metabolized in these tissues but not in testes. Although WS-BC is commonly used as a food colorant, its effects on body tissues are still not clarified. Results of the present study suggest that further investigations are required to elucidate effects of WS-BC on various body tissues.


Findings
Carotenoids are one of the main groups of coloring substances in nature [1][2][3]. The advantages to add carotenoids as food colorants are: high tinctorial potency, safety, stability, compatibility and availability. As color conveys a concept of freshness and wholesomeness by an ingrained color-taste expectancy relationship, the technical challenge of the food industry has been to create suitable application forms of carotenoids for food coloring needs [1][2][3][4]. Several application forms of the carotenoids have been developed for coloring both fat-based (margarine, cheese, butter, etc.) and water-based (juice, beverages, etc.) foods [4]. Because carotenoids are fat soluble, three approaches were used to overcome this disadvantage: 1) reduction in crystal size; 2) preparation of emulsions in liquid and beadlet forms; and 3) development of colloidal preparations [4]. Since various application forms of βcarotene have been developed and are most widely used as food colorants, people intake β-carotene easily in their daily life. β-Carotene is one of the provitamin A carotenoids, which is cleaved to retinal, followed by its conversion to retinyl ester within the small intestine [5][6][7][8]. Vitamin A and its analogs (retinoids) are needed to maintain normal growth and development, immunity, reproduction and other essential physiologic processes [8][9][10]. Besides the provitamin A activity, β-carotene has other important biological functions such as quenching of singlet oxygen, interrupting peroxidation, reducing the free radicals and so on [3,6]. Many epidemiological studies over a long period have reported a negative relationship between β-carotene intake and chronic disease [reviewed in [6,11]]. However, two large recent trials found that pharmacological levels of β-carotene increased lung cancer incidence and deaths in smokers [12,13] and asbestos workers [13]. A larger trial with healthy American men, however, found no effect of β-carotene on several types of malignant neoplasms except an increased risk for thyroid and bladder cancer [14]. These contradictory reports [6,[11][12][13][14] suggest a possible dual response of β-carotene, whereby it promotes health when taken at dietary levels, but may have adverse effects when taken at higher doses [15]. The tissue distribution of β-carotene is still not clearly defined [15]. In the present study, we aimed to examine rat in vivo tissue uptake of all-trans β-carotene (AT-BC). The studies of β-carotene distribution are difficult because several factors, such as the solubility conditions and in vivo digestion, influence its absorption. Therefore, the choice of solvent and the method of β-carotene administration have been controversial [16]. In the present study, we used water soluble β-carotene (WS-BC) which has been commonly used as one of the application forms of food colorants. The WS-BC is considered to overcome the β-carotene insolubility and its absorption difficulties.
Dry β-carotene beadlets (Trade name: Dry β-Carotene 10% Cold Water-Soluble) were kindly donated by Hoffmann La Roche Japan, Co., Ltd. (Tokyo, Japan). The beadlets contained 10% of AT-BC. In addition, the beadlets consisted of vehicle (starch, gelatin, sucrose and plant oil) and antioxidants (vitamin E and vitamin C). In this paper, the dry β-carotene beadlets refer to as water soluble β-carotene (WS-BC). The reagents used were purchased from Wako Pure Chemical Industries Inc. (Osaka, Japan). The solvents for high performance liquid chromatography (HPLC) were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Sprague-Dawley male rats (7 weeks old, body weight 210-230 g) were purchased from CLEA Japan, Inc. (Tokyo, Japan) and housed under constant temperature on a 12-hour light/dark cycle. Before the intravenous injection of WS-BC, rats were fed standard laboratory diet (CE-2, CLEA Japan, Inc., Tokyo, Japan) and given access to tap water ad libitum for 5 days. To make WS-BC solution, 20 mg of WS-BC was dissolved in saline, and 1 ml of this solution was injected into the tail vein of the rat. Thus, the actual amount of AT-BC administered into the rat was 2 mg. The intravenous injection of WS-BC was performed as described previously [16]. At 0, 6, 24, 72, 120 and 168 hours after the intravenous administration of WS-BC, blood was drawn from the abdominal vein and centrifuged to obtain plasma. Rats were sacri-ficed, and liver, lungs, adrenal glands, kidneys and testes were removed and frozen in liquid nitrogen. For each time point, 7 rats were used. The removed organs and plasma were kept at -80°C until further analysis. All experimental protocols were conducted in accordance with Japanese Act on Welfare and Management of Animals (Act No. 105 of October 1, 1973). The quantification of AT-BC in the tissues was performed using HPLC as described before [16]. Data were presented as means ± SD. Statistical analyses were carried out using Origin (Microcal Software Inc., USA) or Excel (Microsoft, USA) software. Differences were considered significant at the level of p < 0.05.
The mean (± SD) AT-BC levels in the tissues were examined at 6 time points after the intravenous administration ( Figure 1). The AT-BC levels in all tissues increased up to 6 h ( Figure 1). From this, it may be assumed that the AT-BC circulated in the blood stream and was distributed to these tissues within 6 h. In plasma, the AT-BC level decreased rapidly after 6 h and the level at 72 h was not significantly different from that at 0 h ( Figure 1A). This may suggest that all the AT-BC administered in plasma was distributed to other body tissues or excreted from blood by 72 h. The AT-BC level in the liver decreased over a period of 6 h to 120 h ( Figure 1B). In the lung, adrenal gland and kidney, the AT-BC levels decreased gradually from 6 h to 120 or 168 h ( Figure 1C, D and 1E). This may indicate that the AT-BC was excreted from these tissues or was metabolized to the possible metabolites of AT-BC, retinoids and carotenoid isomers [5][6][7][8]15]. The AT-BC levels in lung, adrenal gland and kidney at 120 and/or 168 h were still significantly different from those at 0 h ( Figure  1C, D and 1E). This observation could suggest that some amount of AT-BC was still retained at 120 or 168 h in these tissues. In the testes, AT-BC level first increased up to 24 h and then did not decrease but was retained up to 168 h ( Figure 1F).
In the present study, in vivo tissue uptake of AT-BC was examined after the intravenous administration of WS-BC into rats. The in vivo emulsifying conditions have been shown to affect β-carotene absorption [15,17]. In our pilot experiments, when the rats were fed a refined diet containing WS-BC, the level of AT-BC in serum, liver and lung showed a wide variation between animals (data not shown). This may suggest that in vivo tissue uptake of AT-BC is affected by individual absorption conditions of WS-BC. Therefore, oral administration of WS-BC is considered difficult to examine in vivo tissue uptake. Furthermore, in our previous work involving intravenous administration of the emulsified AT-BC crystals in solvent, high levels of AT-BC accumulated in the lung [16]. This was suggested to be due to the trapping of the solvent used for dissolving AT-BC [16]. To overcome these problems of absorption and insolubility of AT-BC, in the present study, WS-BC Time dependent changes in AT-BC levels in 6 tissues after WS-BC intravenous administration in rat was dissolved in saline and administered intravenously. However, like our previous study [16], in some tissues including lung, accumulation of a large amount of AT-BC was not found in the present study. This would suggest that the WS-BC was not trapped by the solvent in the present study whereas it was in the previous study [16].
The antioxidant properties of β-carotene are strictly dependent on oxygen partial pressure (OPP) [18,19]. In vitro experiments at different OPP have demonstrated ambiguous behavior of β-carotene [18]. At OPP less than the oxygen pressure in normal air, β-carotene behaved as an antioxidant whereas at higher values, it was found to lose its antioxidant activity and actually showed a pro-oxidant effect [18]. A number of reports have now confirmed this phenomenon in purified systems [20], microsomes [21], cell lines [22] and bacteria [23]. In addition, after βcarotene administration to rats, cytochrome P450 isoforms were induced and reactive oxygen species (ROS) were increased in kidney, lung, intestine and liver, with liver being the most affected tissue [19]. From these findings, it was suggested that β-carotene may have two contradicting behaviors, antioxidant and pro-oxidant [19]. Oxidative stress plays a major contributory role in pathogenesis of many generative and chronic diseases [19]. Many epidemiological studies have been reported which show a negative relationship between dietary β-carotene intake and chronic disease [6,11]. On the other hand, recent intervention trials suggest that β-carotene supplementation may promote health when taken at dietary levels but have adverse effects when taken in higher amounts [11,15]. The conflicting behavior of β-carotene may explain why the contradictory results were obtained in different studies.
In the present study, the observation of accumulation of AT-BC over a period of 168 h after the intravenous administration of WS-BC ( Figure 1F) suggests that under certain conditions, testes may have the ability to store AT-BC for several days. In an immunohistochemical study [7], human β-carotene 15,15'-mono-oxygenease (BCO1), which is involved in the symmetrical cleavage of β-carotene into two retinal molecules, was detected in steroidogenic cells in testis, ovary, and adrenal gland [24]. The level of mRNA of carotene cleavage enzyme (CCE), which cleaves provitamin A carotenoid to retinol, was found to be highest in testis among 4 tissues examined including the small intestine [8]. From these observations, it was suggested that in testis, β-carotene could act as a local source of retinoids, which have been shown to be important during proliferation, differentiation, and maturation of germinal cells [7,8,24]. In male rats treated with fenvalerate [25] or cadmium [26], administration of β-carotene was reported to ameliorate the induced toxicity in the testis. Moreover, β-carotene administration increased semen quality [25]. Overall, from these findings, it appears that β-carotene may be essential for the function of testes.
Because β-carotene is commonly used commercially to color food, people intake it easily in their daily life. Since this provitamin has an ambiguous behavior as becoming antioxidant or pro-oxidant depending on its partial oxygen pressure, results of the present study suggest that further investigations are required to elucidate its effect on body tissues under various physiological conditions.