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Supplement: Proceedings of Symposium to Honor the Memory of James Allen Olson

Oxidative Conversion of Carotenoids to Retinoids and Other Products^1,2

National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan

³To whom correspondence should be addressed. E-mail: nagao{at}nfri.affrc.go.jp.

	ABSTRACT

TOP ABSTRACT LITERATURE CITED

 
In vertebrates, provitamin A carotenoids are converted to retinal by ß-carotene-15,15'-dioxygenase. The enzyme activity is expressed specifically in intestinal epithelium and in liver. The intestinal enzyme not only plays an important role in providing animals with vitamin A, but also determines whether provitamin A carotenoids are converted to vitamin A or circulated in the body as intact carotenoids. We have found that a high fat diet enhanced the ß-carotene dioxygenase activity together with the cellular retinol binding protein type II level in rat intestines. Flavonols with a catechol structure in the B-ring and 2,6-di-tert-butyl-4-methylphenol inhibited the dioxygenase activity of pig intestinal homogenates and the conversion of ß-carotene to retinol in Caco-2 human intestinal cells. Thus, the bioavailability of dietary provitamin A carotenoids might be modulated by the other food components ingested. Regulation of the dioxygenase activity and its relation to the retinoid metabolism as well as to lipid metabolism deserve further study. In contrast to enzymatic cleavage, it is known that both retinal and ß-apocarotenals are formed in vitro from ß-carotene by chemical transformation, which cleaves conjugated double bonds at random positions under various oxidative conditions. Moreover, recent studies have indicated that the oxidation products formed by chemical transformation might have specific actions on the proliferation of certain cancer cells. We have found that lycopene, a typical nonprovitamin A carotenoid, was cleaved in vitro to acycloretinal, acycloretinoic acid and apolycopenals in a nonenzymatic manner, and that the mixture of oxidation products of lycopene induced apoptosis of HL-60 human promyelocytic leukemia cells. Thus, it is worth evaluating the formation of oxidation products and their biological actions, in order to elucidate the underlying mechanisms of the beneficial effects of carotenoids on human health.

KEY WORDS: • ß-carotene • dioxygenase • lycopene • oxidation • vitamin A

The oxidative metabolites of carotenoids work as essential molecules in a wide variety of living organisms. Carotenoids are converted to biologically active products such as abscisic acid, trisporic acid, and retinoic acid in plants, fungi, and animals, respectively. Their formation is mediated by enzymes that catalyze cleavage reactions against specific double bonds of carotenoids. Moreover, carotenoids vulnerable to oxidation have the potential to be converted to biologically active compounds by chemical transformation. In mammals, ß-carotene-15,15'-dioxygenase catalyzes conversion of ß-carotene to retinal (1,2). Although the cleavage enzyme plays a crucial role in vitamin A formation, its properties have not yet been fully revealed. The regulatory mechanism of the dioxygenase in particular remains to be clarified in terms of the nutrition of pro-vitamin A carotenoids. Dietary ß-carotene solubilized in mixed micelles with bile components and hydrolyzates of dietary lipids is absorbed in intestinal cells. Retinal formed from ß-carotene is further converted to retinyl ester by retinal reductase and lecithin-retinol acyltransferase with the aid of cellular retinol binding protein type-II (CRBP-II), and thereafter incorporated into chylomicron (3). Thus, the cleavage of ß-carotene in intestinal cells is closely linked to lipid and retinoid metabolism, and the regulation of the cleavage enzyme activity should be considered in this context. Moreover, the cleavage enzyme is located in the intestinal cells, which are directly exposed to various food components. Actions of dietary phytochemicals on the dioxygenase activity might affect the bioavailability of provitamin A carotenoids derived from fruits and vegetables.

In contrast to the specific cleavage of certain carotenoids by the enzymes, carotenoids can be oxidized to a number of compounds by chemical transformation, because of the high reactivity of conjugated double bonds to active oxygen species. Oxidation products formed in vitro have been extensively identified, especially as products when carotenoids work as antioxidants in oxidative conditions (4–6). These products would be formed in biological tissues by their reaction with active oxygens, which are generated even in normal physiological conditions, and could be further metabolized and finally eliminated from the body. Recent reports showing the specific actions of the oxidation products of carotenoids on the growth of several cancer cells (7,8) have enforced the need for elucidating oxidation products in biological tissues. Some of the products would also be interesting as markers which could indicate the antioxidant nature of carotenoids in vivo. Thus, oxidation products of carotenoids and their metabolites in biological tissues are important to understand the metabolism and actions of carotenoids in terms of human health. In this paper, the enzymatic conversion of ß-carotene to vitamin A, the nonenzymatic cleavage of lycopene, and the biological actions of the oxidation products of lycopene are described.

Enzymatic conversion of ß-carotene to vitamin A

ß-Carotene can be converted to two molecules of retinal by cleavage, specifically at its central double bond, catalyzed by ß-carotene-15,15'-dioxygenase. The retinal formed is further metabolized to retinoic acid or retinol. In addition to this central cleavage pathway, an eccentric cleavage pathway was proposed for vitamin A formation from ß-carotene. In this eccentric pathway, ß-carotene is cleaved to retinal and ß-apocarotenals with different chain lengths by cleavage at a random position of its conjugated double bonds. The aldehydes were further cleaved to the short-chain carbonyl compounds or oxidized to retinoic acid by the ß-oxidation pathway (9). In addition to these pathways, it was suggested that ß-carotene was converted to retinol and retinoic acid by rat liver cytosol, not through retinal as an intermediate (10). However, several studies have confirmed the central cleavage of ß-carotene for vitamin A formation (11–13). We have examined the extent to which central and eccentric cleavage pathways contribute to vitamin A formation with pig intestinal homogenate (14). The enzyme reaction and extraction of carotenoids and retinoids have been carefully conducted to avoid the nonenzymatic oxidation of ß-carotene and retinal, since they are extremely vulnerable to oxidative degradation. Retinal was accurately quantified by separating each geometric isomer on a normal phase column by HPLC, because all-trans retinal formed from all-trans ß-carotene was readily isomerized to cis isomers. Moreover, the low recovery of retinal due to formation of a Schiff base with amino compounds present in tissue homogenate was overcome by formaldehyde treatment prior to extraction. Thus, the improvements enabled an accurate estimation of the stoichiometry, and >94% of the ß-carotene consumed was converted to retinal in our conditions. No formation of ß-apocarotenals was found. These results clearly indicated that the enzyme preparation of pig intestinal mucosa converted ß-carotene to retinal exclusively by central cleavage.

Although ß-carotene-15,15'-dioxygenase plays essential roles in providing vertebrates with vitamin A, regulation of the enzyme activity has not been well clarified. We have developed a facile assay method for this enzyme activity to investigate the detailed properties of the enzyme and its regulatory mechanism. The method was simplified by reducing extraction procedures and eliminating solvent evaporation, and enabled us to detect even small activities in brain, kidney and lung of rats (15). In the intestinal cells, the enzyme determines whether provitamin A carotenoids are converted to vitamin A or delivered through lymph to tissues as intact carotenoid. The intestinal cells are exposed to various dietary phytochemicals, some of which might directly modify the physiological function of intestine. To evaluate possible effects of dietary components on metabolism of ß-carotene in intestine, we have evaluated the effects of dietary antioxidants and phytochemicals on the dioxygenase activity in vitro by the assay method developed. A synthetic antioxidant, 2,6-di-tert-butyl-4-methylphenol (BHT), strongly inhibited the activity at the level of 10^-6 mol/L (a mixed-type inhibition). The flavonoids such as luteolin, quercetin and rhamnetin with a catechol structure in B-ring remarkably inhibited the enzyme activity noncompetitively. The enzyme inhibition was also indicated in the cultured Caco-2 human intestinal cells by the significantly reduced conversion of ß-carotene to retinol in the presence of BHT and rhamnetin. The results suggested that the dietary antioxidants present in foods together with carotenoids might affect the bioavailability of provitamin A carotenoids (16).

Dietary lipids might be another factor regulating the dioxygenase activity. Absorption of ß-carotene to intestinal cells requires the solubilization in mixed-micelles composed of bile salts, phosphatidylcholine, cholesterol and hydrolyzates of dietary fats (17–19). Thus, the ß-carotene level in intestinal cells available for the dioxygenase reaction would be enhanced by dietary fats, which could facilitate the solubilization processes of carotenoids in digestive tracts. Moreover, both the mRNA and protein levels of CRBP-II, which is an important carrier-protein binding retinol or retinal for subsequent enzyme reactions up to retinyl ester, were reported to increase in the jejunum of rats fed a diet with a high content of unsaturated fatty acids (20). Therefore, the enhancement of the dioxygenase by dietary fat could be of biological relevance in terms of efficient conversion of ß-carotene to vitamin A. In order to confirm this hypothesis, we have examined the effects of dietary fats on the dioxygenase activity in rat intestines and livers. The enrichment of olive oil and soybean oil in the diet significantly enhanced the dioxygenase activity, as well as the CRBP-II level, in rat intestines (21). The results suggest the modulation of the dioxygenase expression by dietary fatty acids, and the close linkage of the dioxygenase reaction to the subsequent retinoid metabolism in intestinal cells. The effects of fatty acid on the transcriptional regulation of CRBP-II have been intensively studied in relation to the peroxisome proliferator-activated receptors (22). The regulatory mechanism of the dioxygenase on the molecular level deserves future studies, because the cloning of the dioxygenase has recently been achieved by Lintig and Wyss (23,24).

Oxidative cleavage of carotenoids

There are few reports about metabolic fate of carotenoids other than provitamin A carotenoids in mammals. The detailed analysis of human plasma showed eight kinds of metabolites or oxidation products of carotenoids, such as anhydrolutein and 2,6-cyclolycopene-1,5-diol (25). These metabolites still have a carbon skeleton of their parental carotenoids. However, little is known about the in vivo formation of metabolites of lower molecular weight, except for retinoids. The biological tissues would produce active oxygen species, especially through the mitochondria, even in normal tissues. It is likely that the hydrophobic lipid peroxy radicals among the active oxygen species would react with carotenoids to produce oxidation products, and thereafter the oxidation products might be further metabolized to be eliminated from the body. Formations of oxidation products with lower molecular weight from ß-carotene have been extensively studied in vitro. Eccentric cleavage products such as retinal and ß-apocarotenals, as well as epoxy carotenoids, were produced by autoxidation of ß-carotene dissolved in solvent (4). Similarly, oxidation of ß-carotene with a radical generating reagent and singlet oxygen gave ß-apocarotenals (5,6). The cleavage reaction at the random position of conjugated double bonds to carbonyl compounds may occur through a dioxetane intermediate (4,6). Moreover, 5,8-endoperoxy-2,3-dihydro-ß-apocarotene-13-one was isolated from ß-carotene oxidized with 3-chloroperoxybenzoic acid (8). Canthaxanthin was oxidized in cell culture medium to 4-oxo-retinoic acid (7). Thus, these chemical cleavages would occur in biological tissues when carotenoids were present in the tissues exposed to oxidative stress. Indeed, the cleavage of double bond at C9–C10 was suggested to occur by identification of 3-hydro-4-oxo-7,8-dihydro-ß-ionone as a urinary metabolite of canthaxanthin in rats (26), and 3-hydroxy-4-oxo-ß-ionone as one of the astaxanthin metabolites in the primary culture of rat hepatocyte (27).

Although the in vitro oxidation products of ß-carotene were well characterized as mentioned above, little knowledge was available about the oxidation products of nonprovitamin A carotenoids. We have evaluated the oxidation products of lycopene, a typical dietary nonprovitamin A carotenoid, especially with respect to cleavage products (28). Lycopene solubilized in toluene, aqueous Tween 40 or phosphatidylcholine liposomal suspension was autoxidized at 37°C. In the three conditions tested, a number of oxidation products formed from lycopene. The following eight products were identified in the carbonyl compound fraction: 3,7,11-trimethyl-2,4,6,10-dodecatetraenal, 6,10,14-trimethyl-3,5,7,9,13-pentadecapentaen-2-one, acycloretinal (3,7,11,15-tetramethyl-2,4,6,8,10,14-hexadecahexaenal), apo-14'-lycopenal, apo-12'-lycopenal, apo-10'-lycopenal, apo-8'-lycopenal and apo-6'-lycopenal. These were a series of products formed by cleavage in the respective eleven conjugated double bonds of lycopene (Fig. 1). Acycloretinal, a cleavage product at the central double bond of lycopene, was an analogous compound to retinal. In addition to these carbonyl compounds, an acidic compound was isolated from the oxidation mixture of lycopene, and was identified as acyloretinoic acid. The amount of acycloretinoic acid formed in toluene was comparable to those of lycopenals in toluene, whereas formation of retinoic acid was suppressed in aqueous media such as liposomal suspension and aqueous Tween 40. However, liver homogenate showed the oxidizing activity of acycloretinal to acycloretinoic acid, comparable to conversion of retinal to retinoic acid. Therefore, acycloretinoic acid might form enzymatically from acycloretinal produced from lycopene in biological tissue exposed to oxidative stress. Thus, taken together, the results in our study and the previous reports suggest that the cleavage reaction at the conjugated double bonds by autoxidation, radical mediated oxidation, and singlet oxygen occurs in any carotenoid with a long-chain of conjugated double bonds. These cleavage products may be produced in vivo, and thereafter metabolized to be eliminated from the body if the tissues are exposed to an oxidative stress.

View larger version (15K): [in this window] [in a new window]   FIGURE 1 Cleavage products formed from lycopene by autoxidation.

The eccentric cleavage and other oxidation products formed under oxidative conditions may have specific actions on certain cancer cells. The autoxidation of canthaxanthin in cell culture medium produced 4-oxo-retinoic acid, which could activate the promoter of the retinoic acid receptor (RARß) gene and enhance gap junctional communication (7,29). 5,8-Endoperoxy-2,3-dihydro-ß-apocarotene-13-one, an oxidation product of ß-carotene, inhibited the growth of breast cancer cells (8). 5,6-Epoxy-ß-carotene was reported to have a significantly greater differentiation-inducing activity than ß-carotene toward NB4 human leukemia cells (30). These results strongly suggest that oxidation products as well as intact carotenoids have potentially biological effects on human health. However, adverse effects of carotenoids may be mediated by their oxidation products. ß-Apo-8'-carotenal induced P450 in rodents, which might activate carcinogens and enhance catabolism of retinoic acids (31,32). ß-Carotene oxidation products enhanced the binding of benzo[a]pyrene metabolites to DNA (33). ß-Apo-10'-carotenal and retinal inhibited Na⁺-K⁺-ATPase (34). Carotenoid UV-photo degradation products of ß-carotene and lycopene suppressed the immune function of human peripheral blood mononuclear cells (35). Thus, oxidation products might have biological actions related to human health, whether beneficial or harmful.

Some retinoid-like substances with the same biological activity as retinoic acid might form in the oxidation mixture of carotenoids by oxidative cleavage of conjugated double bonds, as described above. In order to examine this possibility, we evaluated the effects of oxidation products of carotenoids on the growth and differentiation of HL-60 human promyelocytic leukemia cells (36), which are well known to be differentiated to granulocyte by all-trans retinoic acid. ß-Carotene, lycopene and their oxidation mixtures were supplemented to the medium for HL-60 cells. Acyclic carotenoids homologous to lycopene, such as -carotene, phytofluene and phytoene, present in tomato products and human plasma, were also evaluated. The oxidation mixture of the carotenoids was prepared by autoxidation in toluene at 37°C for 24 h. No carotenoid preparation induced differentiation in HL-60 cells. However, -carotene and phytofluene at 10 µmol/L inhibited the cell growth during 120 h to 3.7 and 22.6% of the growth in control culture, respectively, although they were extremely unstable in the culture medium. The oxidation mixture of -carotene and phytofluene more strongly inhibited the cell growth than did the intact carotenoids. Surprisingly, the oxidation mixture of lycopene remarkably inhibited the cell growth, but the intact lycopene did not. The strong growth inhibition observed in the presence of -carotene, phytofluene and the oxidation mixture of lycopene was associated with the induction of apoptosis in HL-60 cells. The apoptosis induction was confirmed by detection of chromatin condensation and nuclear fragmentation, the DNA ladder. The addition of -tocopherol to the medium did not eliminate growth inhibition by the oxidation mixture of lycopene. The ß-carotene oxidation mixture with the same oxidation degree as lycopene oxidation mixture did not inhibit cell growth. These results suggested that oxidation products specifically formed from lycopene might induce apoptosis rather than the oxidative stress through the reactive oxygen species formed in the oxidation mixture. A similar observation was obtained in LNCaP human prostate cancer cells. Lycopene, -carotene and phytofluene inhibited cell growth of LNCaP cells, while their oxidation mixtures showed more remarkable growth inhibition than the corresponding intact carotenoids. Although isolation and identification of active compounds to induce apoptosis or inhibit cell proliferation are still needed to clearly demonstrate the involvement of oxidation products, these results strongly suggest that oxidation products as well as carotenoid per se exert biological actions in the human body.

	FOOTNOTES

 
¹ Presented as part of the James Allen Olson Memorial Symposium, "Functions and Actions of Retinoids and Carotenoids" held at Iowa State University, June 21–24, 2001 to honor the memory of James Allen Olson. This conference was supported by the U.S. Department of Agriculture; National Institutes of Health; Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University (ISU); Department of Food Science and Human Nutrition, ISU; College of Liberal Arts and Sciences, ISU; F. Hoffmann-La Roche; Kemin Foods, L.C., Procter & Gamble Company; Lipton; Best Foods; BASF; SmithKline Beecham; Cognis Corporation; Allergen and INEXA. Guest editor for this symposium was Norman I. Krinsky, Department of Biochemistry, School of Medicine, and the Jean Mayer Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111-1837.

² This work was supported by the Bio-Renaissance Program of the Ministry of Agriculture, Forestry and Fisheries, Japan and by the PROBRAIN project of Bio-oriented Technology Research Advancement Institution.

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