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Is β-carotene an antioxidant?
Medical Hypotheses (1997) 48, 183-187
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Is 13-carotene an antioxidant? D. V. CRABTREE, A. J. ADLER Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA (Correspondence to Donald V. Crabtree, Schepens Eye Research Institute, 20 Staniford Street, Boston MA 02114, USA. Tel: (001) 617-742-3140, x405; Fax: (001) 617-720-1069; E-mail: [email protected])
Abstract - - An hypothesis is presented that is opposed to the conventional viewpoint that [3-carotene is an in vivo free-radical scavenger. It is suggested that there are biochemical reasons w h y [3-carotene, other carotenoids, and especially their metabolites may be harmful to mammalian systems. Finally, the hypothesis that the macular pigment carotenoids, lutein and zeaxanthin, are free-radical scavengers is challenged.
Introduction
Properties of in vivo antioxidants
Efficacy of the putative antioxidant, [3-carotene, has become controversial. Since the influential paper by Burton and Ingold (1), this provitamin-A carotenoid has been considered a free-radical scavenger. One study has shown it to be a more effective free-radical scavenger than a-tocopherol (2). Furthermore, the suggestion has been made that [3-carotene reduces the risk of cancer and may increase longevity in individuals consuming more-than-typical quantities of it (3). Two recent epidemiological studies on smokers (4,5) have provoked reappraisal of that litany. In the Finnish study (4), the investigators increased (with capsules) the average [3-carotene plasma level to 17 times above baseline. They were surprised to find that the incidence of cancer increased among subjects who received the [3-carotene. More recently, a similar study (5) in the USA was ended early because the results were parallel to the Finnish study. What went wrong?
A recent article on the biology of free-radical scavengers (6), which discusses what the properties of an ideal in vivo free-radical scavenger should be, is particularly germane (for an elementary review on free radicals see Ref. 7). In that article (6), seven significant properties are described. Although the focus of that article was to ascertain whether vitamin C has those properties, the suggested criteria also apply to other in vivo antioxidants. Briefly, the properties are: adequate amount in the body, versatility, suitability for compartmentalization, availability, regenerability, conservation by kidneys, and tolerable toxicity. Pertinent to our discussion are two important properties: regenerability and toxicity. Just because a compound is capable of acting as an antioxidant in chemical, in vitro, or ex vivo systems does not necessarily imply that it functions as an in vivo antioxidant in humans. After an antioxidant has completed its task, it
Date received 1 March 1996 Date accepted9 April 1996 183
184 must either remain unchanged (as when I]-carotene quenches singlet oxygen (8)), be recycled (as vitamin E probably is by vitamin C (9)), or be excreted by an orderly and precise metabolic pathway. (Please note that ground-state oxygen has two unpaired electrons and hence is a radical; however, singlet oxygen, which is an excited state of oxygen, does not have any unpaired electrons and hence is not a radical (10).) Furthermore, the oxidation product(s) formed when an antioxidant is oxidized should be less toxic to the organism than the oxidized substrate that is reduced by the antioxidant. These important points have been virtually ignored in the field of antioxidants. Recommending that people supplement with a putative antioxidant without considering how it will be metabolized is analogous to recommending the building of a chemical-manufacturing facility without considering how the waste products will be dealt with.
MEDICAL HYPOTHESES
implies that the number of potential products formed from [3-carotene acting as an in vivo free-radical scavenger (at low partial pressures of oxygen) is close to infinite. It also implies that these products will have diverse and complicated chemical structures. Furthermore, although it is stated that the carbon-centered radical is resonance-stabilized, no proof of this is provided (e.g. with electron spin resonance spectroscopy). In addition, the paper (1) states that, at high partial pressures of oxygen (e.g. the partial pressure of oxygen in the human lung, 160mmHg), ~-carotene itself undergoes a fairly facile autoxidation (in chlorobenzene). Or, to be more explicit, at the partial pressure of oxygen in the human lung, the much-cited paper (1) indicates that 13-carotene is a pro-oxidant! Although Burton and Ingold (1) do not present any data regarding the products formed during their experiments, Handelman et al (16) showed that numerous products are formed when [3-carotene (in a simple chemical system) is subjected to oxidative stress. Implications for [3-carotene Although the oxidation products formed from l~-carotene or other carotenoids reacting with free Since the cardinal paper by Burton and Ingold (1), radicals are of little importance in chemical or ex ~i-carotene has been considered to be a free-radical vivo systems (unless they continue the free-radical scavenger, particularly at low partial pressures of chain reaction), they are of utmost importance in vivo. oxygen (_< 15 mmHg). Analysis of the meager data (1) Metabolic pathways for the regeneration or excretion indicates that the effect of low oxygen tension in a of the potentially infinite number of (toxic) oxidation chemical system (which was neither in vitro nor ex products of [3-carotene (or other carotenoids) are not vivo) is small and mostly occurs at 5 millimolar 13- known (17). Because of the large number of possible carotene. A typical physiological value for 13-carotene products formed from the oxidation of 13-carotene in concentration in human plasma is 0.43 _+0.31 micro- vivo (only a few of which have been characterized molar (11). Also, in the human adrenal gland, the tissue (18)), regeneration of 13-carotene would require an in which ~-carotene probably has the highest concen- extensive and diverse array of enzymes. Excretion tration, the range is 0.94-160 ~tg/g wet weight (12). of the oxidized metabolites of I]-carotene (rather (With the assumption of homogeneity, 160 ~tg/g wet than regeneration) most probably requires extensive weight is equivalent to 0.3 millimolar.) Others have oxidative processing (to enhance their solubility in shown that the antioxidant capacity of 13-carotene is water), if damage of the organs associated with this the same at 160 and 15 mmHg oxygen (13). But the task is to be avoided. As a consequence, not only is concentration of 13-carotene and the partial pressure it doubtful that 13-carotene is regenerated from its of oxygen are both irrelevant. Although 13-carotene oxidation products, but many of the products formed is an effective singlet-oxygen quencher in chemical as the result of its reaction with free radicals are systems (8) and may perform that function in green undoubtedly more toxic than the starting materials. plants (14), it would make a disastrous in vivo freeUnfortunately, except for their transformation to radical scavenger in either plants or animals. vitamin A (19), the metabolic pathways associated Oxidation of 13-carotene yields a large variety with the catabolism of ~-carotene, other carotenoids, of potentially toxic products (1,15,16). According to and their oxidation products has been mostly ignored. Burton and Ingold (1), 'The inhibiting, resonance- Consistent with the lack of study of this important stabilized, carbon-centered radical is probably formed topic, in the Finnish smokers study (4), analyses of by the addition of an ROO ° (and, perhaps also of an urine, feces or blood for the catabolites of [3-carotene R °) radical to the conjugated system of ~-carotene, were not reported. Furthermore, a smoking-animal rather than by an initial H-atom abstraction.' This model that could have been used to study the metawas stated in the context of 13-carotene serving as an bolism of ~-carotene under conditions analogous to antioxidant at low partial pressures of oxygen (and that of the human smokers, and hence could have hence the mention of R'). This statement strongly predicted the probable outcome of the human studies
185
IS [~-CAROTENEAN ANTIOXIDANT?
was (astonishingly) not established (20). It seems appropriate that, prior to use of an antioxidant in large-scale intervention studies in humans, a prudent course of action is to first understand its absorption, metabolism, and how it and its metabolites are excreted. The results of a recent ex vivo study comparing human plasma before and after supplementation with [3-carotene are consistent with the possibility that [3-carotene may be a pro-oxidant rather than an antioxidant (in human plasma) (21). This possibility is particularly relevant for smokers, since cigarette tar contains high concentrations of radicals (22) that may combine with [3-carotene in the respiratory system (where the partial pressure of oxygen is high) to form compounds that may initiate free-radical chain reactions. Although many carotenoids effectively quench singlet oxygen in chemical systems (23), it has not been proved that humans accumulate carotenoids for this purpose. Since there is only about one carotenoid molecule for every two low-density lipoprotein (LDL) particles (24), it is doubtful that LDL particles carry carotenoids to quench singlet oxygen. (For comparison, there are approximately six molecules of the free-radical scavenger, c~-tocopherol, per LDL particle (24).) Whether other organs, such as the adrenal glands, which contain relatively high concentrations of carotenoids (12), are particularly vulnerable to inadvertent production of singlet oxygen is unknown (25).
Accumulators versus non-accumulators of carotenoids: relevance for intake
Some mammals accumulate carotenoids in their tissues but many do not (26). Somewhat surprisingly, mammals that are primarily vegetarians such as elephants, hares, sheep and white-tailed deer do not accumulate carotenoids in their tissues (26). Even among humans, a sizable percentage of people do not accumulate carotenoids well (27). Furthermore, other than a simple transformation from one carotenoid to another that may occur in the human retina (28), evidence to show that mammals synthesize carotenoids does not exist (26,28). If these compounds could play a major role in enhancing the fitness of mammals, why are they not found in the tissues of so many vegetarians or synthesized by obligate carnivores? It would be of interest to compare the longevity of humans that easily accumulate carotenoids to those that do not. Consistent with carotenoids not being found in the tissues of so many vegetarians (that have survived millions of years of natural selection), one
might anticipate that humans who are poor accumulators of carotenoids live longer and are healthier. Although we do not intend to imply that a diet rich in fruit and vegetables should be avoided - because of the potential toxicity of carotenoids and their metabolites - we strongly recommend that supplementation with carotenoids should only be done under the supervision of a physician (e.g. for erythropoietic protoporphyria (29)). How are the ideas presented above consistent with the large number of epidemiological studies that indicate that low intake of fruits and vegetables, and hence low intake of carotenoids, is consistently associated with increased risk for various cancers (30)? The majority of these studies have focused only on diet (30). The epidemiological studies that have measured components of the plasma have usually measured total carotenoids or [3-carotene (30). Although, on average, plasma levels of [3-carotene are an indicator of the amount of food consumed that contains it (19,27), when one considers the extremely large number of compounds that food contains (most of which have not been characterized), it is presumptuous to conclude that [3-carotene or other carotenoids are responsible for the reduction in the risk of cancer in humans who consume a diet rich in fruit and vegetables. Many other factors may contribute to the reduction in risk (31). Recently, sulforaphane, that was isolated from broccoli, was shown to have anticarcinogenic activity (32). In murine hepatoma cells in culture, it was a potent inducer of phase-2 detoxication enzymes and it reduced the incidence of mammary tumors in rats (32). Whether it or similar compounds are responsible for the reduction in risk of cancer associated with a diet high in fruit and vegetables remains to be verified. In regard to recommending a diet high in broccoli, one might first ask: Why does broccoli contain a compound that induces phase-2 detoxication enzymes? One reasonable hypothesis is that it also contains toxic compounds that require these enzymes. We would not rule out the possibility that some of those toxic compounds are carotenoids or subsequent catabolites of carotenoids. It may be that the reason many animals do not accumulate carotenoids in their tissues is because they lack a sufficient array of enzymes that can detoxify their metabolites.
Lutein and zeaxanthin in the retina
Lutein and zeaxanthin are two carotenoids that are found in the retina of primates (33). They are concentrated in the central retina in a region referred to as the 'macula lutea' (yellow spot) (34). While it has
186 been known since the late 1940s that carotenoids were responsible for this yellow spot (35), their function is still not well delineated. Several functions, including that of being antioxidants, have been suggested (36, 37). Because the oxygen tension is particularly low in the region of the photoreceptor axons where the carotenoids are concentrated (36), and as it has been suggested that ~-carotene (and hence other similar carotenoids, such as lutein and zeaxanthin) may be effective free-radical scavengers at low partial pressures of oxygen (1), the free-radical scavenger hypothesis has been particularly tantalizing (36). For the same reasons as for [~-carotene (the metabolites lack regenerability, are difficult to excrete, and are potentially toxic), it is our contention that, in the macula lutea, the intended function of the macular pigment carotenoids is not that of free-radical scavengers. Whether or not their intended purpose is to quench singlet oxygen is uncertain. If that was the intent, then the more appropriate carotenoid would be lycopene, which is 3 - 4 times more effective at that task (23). Other carotenoids (e.g. u-carotene, [3carotene and canthaxanthin, which are absent from the retina) are also more effective at quenching singlet oxygen than either lutein or zeaxanthin (23). Furthermore, although the macula may be vulnerable to the inadvertent production of singlet oxygen, there is no evidence for its presence (25). Besides light and oxygen, an appropriate sensitizer is needed to produce singlet oxygen (25). Again, there is not any evidence for the presence of such a sensitizer in the region of the retina where the carotenoids are localized.
Conclusion In summary, we suggest that [3-carotene's primary in vivo function is to serve as a reservoir for retinol, retinaldehyde and retinoic acid. Although ~-carotene may have other functions, regardless of its concentration, or the partial pressure of oxygen, its intended function is not that of a free-radical scavenger. It does not meet the requirements: regenerability and tolerable toxicity. Since it is capable of effectively quenching singlet oxygen in chemical systems (8,23), it may inadvertently do so in vivo. Nonetheless, there is little evidence in support of its intended function being that of an in vivo singlet-oxygen quencher in humans. Although the function of lutein and zeaxanthin in the macula lutea is still open to speculation, for the same reasons as for 13-carotene, it is doubtful that their intended function is that of free-radical scavengers. Because of the unfortunate negative results associated with recent clinical trials on putative anti-
MEDICAL HYPOTHESES
oxidants, we suggest that approval of future placebocontrolled clinical trials on putative antioxidants should include the following constraints: (A) A n appropriate animal model should first be established. For example, animals could be trained to smoke prior to receiving dosages of antioxidants that will increase their plasma or tissue concentrations of an antioxidant (or its metabolites) significantly above normal; (B) Knowledge of the metabolic pathways associated with the putative antioxidant should be reasonably well understood in both an animal model and in humans; (C) Periodic analysis of the plasma, urinary and possibly fecal catabolites of the antioxidant should be included in the human studies.
Acknowledgement This research was supported by National Institutes of Health grant EY04368 (AJA).
References 1. Burton G W, Ingold K U. [~-Carotene:an unusual type of lipid antioxidant. Science 1984; 224: 569-573. 2. Vile G F, Winterbourn C C. Inhibition of adriamycinpromoted microsomal lipid peroxidation by beta-carotene, alpha-tocopherol and retinol at high and low oxygen partial pressures. FEBS Lett 1988; 238: 353-356. 3. Ames B N, Shigenaga M K, Hagen T M. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA 1993; 90: 7915-7922. 4. The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N Engl J Med 1994; 330: 1029-1035. 5. Peterson K. 'Natural' cancer prevention trial halted. Science 1996; 271: 441. 6. Rose R C, Bode A M. Biology of free radical scavengers: an evaluation of ascorbate. FASEB J 1993; 7:1135-1142. 7. Halliwell B. Free radicals and antioxidants: a personal view. Nutr Rev 1994; 52: 253-256. 8. Foote C S, Denny R W. Chemistry of singlet oxygen. VII. Quenching by ~-carotene.J Am Chem Soc 1968; 90: 6233-6235. 9. Packer J E, Slater T F, Wilson R L. Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 1979; 278: 737-738. 10. Chan H W-S. The mechanism of autoxidation. In: Chan H W-S, ed. Autoxidation of Unsaturated Lipids. London: Academic Press, 1987: 1-16. 11. Nierenberg D W, Nann S L. A method for determiningconcentrations of retinol, tocopherol, and five carotenoids in human plasma and tissue samples. Am J Clin Nutr 1992; 56: 417-426. 12. Kaplan L A, Lau J M, Stein E A. Carotenoid composition, concentrations, and relationships in various human organs. Clin Physiol Biochem 1990; 8: 1-10. 13. KennedyT A, Liebler D C. Peroxyl radical scavenging by [~carotene in lipid bilayers: effect of oxygen partial pressure. J Biol Chem 1992; 267: 4658-4663. 14. Young A J. The photoprotective role of carotenoids in higher plants. Physiol Plant 1991; 83: 702-708.
IS ~-CAROTENEAN ANTIOXIDANT? 15. Britton G. Structure and properties of carotenoids in relation to function. FASEB J 1995; 9: 1551-1558. 16. Handelman G J, van Kuijk F J G M, Chatterjee A, Krinsky N I. Characterization of products formed during the autoxidation of p-carotene. Free Radic Biol Med 1991; 10: 427-437. 17. Parker R S, Swanson J E, Marmor B e t al. Study of 15-carotene metabolism in humans using C-13 ~-carotene and high precision isotope ratio mass spectrometry. Ann NY Acad Sci 1993; 691: 86-95. 18. Khachik F, Englert G, Daitch C E, Beecher G R, Tonucci L H, Lusby W R. Isolation and structural elucidation of the geometrical isomers of lutein and zeaxanthin in extracts from human plasma. J Chromatogr 1992; 582: 153-166. 19. Goodman D S. Overview of current knowledge of metabolism of vitamin A and carotenoids. JNCI 1984; 73: 1375-1379. 20. Malone W F. Studies evaluating antioxidants and B-carotene as chemopreventives. Am J Clin Nutr 1991; 53:305S-313S. 21. Gaziano J M, Hatta A, Flynn M e t al. Supplementation with B-carotene in vivo and in vitro does not inhibit low density lipoprotein oxidation. Atherosclerosis 1995; 112: 187-195. 22. Pryor W A, Stone K. Oxidants in cigarette smoke: radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite. Ann NY Acad Sci 1993; 686: 12-28. 23. Di Mascio P, Murphy M E, Sies H. Antioxidant defense systems: the role of carotenoids, tocopherols, and thiols. Am J Clin Nutr 1991; 53: 194S-200S. 24. Ingold K U, Bowry V W, Stocker R, Walling C. Autoxidation of lipids and antioxidation by tx-tocopherol and ubiquinol in homogeneous solution and in aqueous dispersions of lipids: unrecognized consequences of lipid particle size as exemplified by oxidation of human low density lipoprotein. Proc Natl Acad Sci USA 1993; 90: 45-49. 25. Gal D. Hunt for singlet oxygen under in vivo conditions. Biochem Biophys Res Commun 1994; 202: 10-16. 26. Goodwin T W. The biochemistry of the carotenoids, Vol II.
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Animals, 2nd edn. New York: Chapman and Hall, 1984: 173-187. Bowen P E, Garg V, Stacewicz-Sapuntzakis M, Yelton L, Schreiner R S. Variability of serum carotenoids in response to controlled diets containing six servings of fruits and vegetables per day. Ann NY Acad Sci 1993; 691: 241-243. Bone R A, Landrum J T, Hime G W, Cains A, Zamor J. Stereochemistry of the human macular carotenoids. Invest Ophthalmol Vis Sci 1993; 34: 2033-2040. Mathews-Roth M M. Beta-carotene therapy for erythropoietic protoporphyria and other photosensitivity diseases. Biochimie 1986; 68: 875-884. Ziegler R G. Vegetables, fruits, and carotenoids and the risk of cancer. Am J Clin Nutr 1991; 53: 251S-259S. Ziegler R G. Carotenoids, cancer, and clinical trials. Ann NY Acad Sci 1993; 691: 110-119. Zhang Y, Kensler T W, Cho C-G, Posner G H, Talalay P. Anti-carcinogenic activities of sulforaphane and structurally related synthetic norbornyl isothiocyanates. Proc Natl Acad Sci USA 1994; 91: 3147-3150. Bone R A, Landrum J T, Tarsis S L. Preliminary identification of the human macular pigment. Vision Res 1985; 25: 1531-1535. Snodderly D M, Handelman G J, Adler A J. Distribution of individual macular pigment carotenoids in central retina of macaque and squirrel monkeys. Invest Ophthalmol Vis Sci 1991; 32: 268-279. Waid G. The photochemistry ofvision. Doc Ophthalmol 1949; 3: 94-127. Snodderly D M. Evidence for protection against age-related macular degeneration by carotenoids and antioxidant vitamins. Am J Clin Nutr 1995; 62 (suppl): 1448S-1461S. Schalch W. Carotenoids in the retina - a review of their possible role in preventing or limiting damage caused by light and oxygen. In: Emerit I, Chance B, eds. Free Radicals and Aging. Basel: Birkhauser Vedag, 1992: 280-298.
© PearsonProfessionalLtd 1997
Is 13-carotene an antioxidant? D. V. CRABTREE, A. J. ADLER Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA (Correspondence to Donald V. Crabtree, Schepens Eye Research Institute, 20 Staniford Street, Boston MA 02114, USA. Tel: (001) 617-742-3140, x405; Fax: (001) 617-720-1069; E-mail: [email protected])
Abstract - - An hypothesis is presented that is opposed to the conventional viewpoint that [3-carotene is an in vivo free-radical scavenger. It is suggested that there are biochemical reasons w h y [3-carotene, other carotenoids, and especially their metabolites may be harmful to mammalian systems. Finally, the hypothesis that the macular pigment carotenoids, lutein and zeaxanthin, are free-radical scavengers is challenged.
Introduction
Properties of in vivo antioxidants
Efficacy of the putative antioxidant, [3-carotene, has become controversial. Since the influential paper by Burton and Ingold (1), this provitamin-A carotenoid has been considered a free-radical scavenger. One study has shown it to be a more effective free-radical scavenger than a-tocopherol (2). Furthermore, the suggestion has been made that [3-carotene reduces the risk of cancer and may increase longevity in individuals consuming more-than-typical quantities of it (3). Two recent epidemiological studies on smokers (4,5) have provoked reappraisal of that litany. In the Finnish study (4), the investigators increased (with capsules) the average [3-carotene plasma level to 17 times above baseline. They were surprised to find that the incidence of cancer increased among subjects who received the [3-carotene. More recently, a similar study (5) in the USA was ended early because the results were parallel to the Finnish study. What went wrong?
A recent article on the biology of free-radical scavengers (6), which discusses what the properties of an ideal in vivo free-radical scavenger should be, is particularly germane (for an elementary review on free radicals see Ref. 7). In that article (6), seven significant properties are described. Although the focus of that article was to ascertain whether vitamin C has those properties, the suggested criteria also apply to other in vivo antioxidants. Briefly, the properties are: adequate amount in the body, versatility, suitability for compartmentalization, availability, regenerability, conservation by kidneys, and tolerable toxicity. Pertinent to our discussion are two important properties: regenerability and toxicity. Just because a compound is capable of acting as an antioxidant in chemical, in vitro, or ex vivo systems does not necessarily imply that it functions as an in vivo antioxidant in humans. After an antioxidant has completed its task, it
Date received 1 March 1996 Date accepted9 April 1996 183
184 must either remain unchanged (as when I]-carotene quenches singlet oxygen (8)), be recycled (as vitamin E probably is by vitamin C (9)), or be excreted by an orderly and precise metabolic pathway. (Please note that ground-state oxygen has two unpaired electrons and hence is a radical; however, singlet oxygen, which is an excited state of oxygen, does not have any unpaired electrons and hence is not a radical (10).) Furthermore, the oxidation product(s) formed when an antioxidant is oxidized should be less toxic to the organism than the oxidized substrate that is reduced by the antioxidant. These important points have been virtually ignored in the field of antioxidants. Recommending that people supplement with a putative antioxidant without considering how it will be metabolized is analogous to recommending the building of a chemical-manufacturing facility without considering how the waste products will be dealt with.
MEDICAL HYPOTHESES
implies that the number of potential products formed from [3-carotene acting as an in vivo free-radical scavenger (at low partial pressures of oxygen) is close to infinite. It also implies that these products will have diverse and complicated chemical structures. Furthermore, although it is stated that the carbon-centered radical is resonance-stabilized, no proof of this is provided (e.g. with electron spin resonance spectroscopy). In addition, the paper (1) states that, at high partial pressures of oxygen (e.g. the partial pressure of oxygen in the human lung, 160mmHg), ~-carotene itself undergoes a fairly facile autoxidation (in chlorobenzene). Or, to be more explicit, at the partial pressure of oxygen in the human lung, the much-cited paper (1) indicates that 13-carotene is a pro-oxidant! Although Burton and Ingold (1) do not present any data regarding the products formed during their experiments, Handelman et al (16) showed that numerous products are formed when [3-carotene (in a simple chemical system) is subjected to oxidative stress. Implications for [3-carotene Although the oxidation products formed from l~-carotene or other carotenoids reacting with free Since the cardinal paper by Burton and Ingold (1), radicals are of little importance in chemical or ex ~i-carotene has been considered to be a free-radical vivo systems (unless they continue the free-radical scavenger, particularly at low partial pressures of chain reaction), they are of utmost importance in vivo. oxygen (_< 15 mmHg). Analysis of the meager data (1) Metabolic pathways for the regeneration or excretion indicates that the effect of low oxygen tension in a of the potentially infinite number of (toxic) oxidation chemical system (which was neither in vitro nor ex products of [3-carotene (or other carotenoids) are not vivo) is small and mostly occurs at 5 millimolar 13- known (17). Because of the large number of possible carotene. A typical physiological value for 13-carotene products formed from the oxidation of 13-carotene in concentration in human plasma is 0.43 _+0.31 micro- vivo (only a few of which have been characterized molar (11). Also, in the human adrenal gland, the tissue (18)), regeneration of 13-carotene would require an in which ~-carotene probably has the highest concen- extensive and diverse array of enzymes. Excretion tration, the range is 0.94-160 ~tg/g wet weight (12). of the oxidized metabolites of I]-carotene (rather (With the assumption of homogeneity, 160 ~tg/g wet than regeneration) most probably requires extensive weight is equivalent to 0.3 millimolar.) Others have oxidative processing (to enhance their solubility in shown that the antioxidant capacity of 13-carotene is water), if damage of the organs associated with this the same at 160 and 15 mmHg oxygen (13). But the task is to be avoided. As a consequence, not only is concentration of 13-carotene and the partial pressure it doubtful that 13-carotene is regenerated from its of oxygen are both irrelevant. Although 13-carotene oxidation products, but many of the products formed is an effective singlet-oxygen quencher in chemical as the result of its reaction with free radicals are systems (8) and may perform that function in green undoubtedly more toxic than the starting materials. plants (14), it would make a disastrous in vivo freeUnfortunately, except for their transformation to radical scavenger in either plants or animals. vitamin A (19), the metabolic pathways associated Oxidation of 13-carotene yields a large variety with the catabolism of ~-carotene, other carotenoids, of potentially toxic products (1,15,16). According to and their oxidation products has been mostly ignored. Burton and Ingold (1), 'The inhibiting, resonance- Consistent with the lack of study of this important stabilized, carbon-centered radical is probably formed topic, in the Finnish smokers study (4), analyses of by the addition of an ROO ° (and, perhaps also of an urine, feces or blood for the catabolites of [3-carotene R °) radical to the conjugated system of ~-carotene, were not reported. Furthermore, a smoking-animal rather than by an initial H-atom abstraction.' This model that could have been used to study the metawas stated in the context of 13-carotene serving as an bolism of ~-carotene under conditions analogous to antioxidant at low partial pressures of oxygen (and that of the human smokers, and hence could have hence the mention of R'). This statement strongly predicted the probable outcome of the human studies
185
IS [~-CAROTENEAN ANTIOXIDANT?
was (astonishingly) not established (20). It seems appropriate that, prior to use of an antioxidant in large-scale intervention studies in humans, a prudent course of action is to first understand its absorption, metabolism, and how it and its metabolites are excreted. The results of a recent ex vivo study comparing human plasma before and after supplementation with [3-carotene are consistent with the possibility that [3-carotene may be a pro-oxidant rather than an antioxidant (in human plasma) (21). This possibility is particularly relevant for smokers, since cigarette tar contains high concentrations of radicals (22) that may combine with [3-carotene in the respiratory system (where the partial pressure of oxygen is high) to form compounds that may initiate free-radical chain reactions. Although many carotenoids effectively quench singlet oxygen in chemical systems (23), it has not been proved that humans accumulate carotenoids for this purpose. Since there is only about one carotenoid molecule for every two low-density lipoprotein (LDL) particles (24), it is doubtful that LDL particles carry carotenoids to quench singlet oxygen. (For comparison, there are approximately six molecules of the free-radical scavenger, c~-tocopherol, per LDL particle (24).) Whether other organs, such as the adrenal glands, which contain relatively high concentrations of carotenoids (12), are particularly vulnerable to inadvertent production of singlet oxygen is unknown (25).
Accumulators versus non-accumulators of carotenoids: relevance for intake
Some mammals accumulate carotenoids in their tissues but many do not (26). Somewhat surprisingly, mammals that are primarily vegetarians such as elephants, hares, sheep and white-tailed deer do not accumulate carotenoids in their tissues (26). Even among humans, a sizable percentage of people do not accumulate carotenoids well (27). Furthermore, other than a simple transformation from one carotenoid to another that may occur in the human retina (28), evidence to show that mammals synthesize carotenoids does not exist (26,28). If these compounds could play a major role in enhancing the fitness of mammals, why are they not found in the tissues of so many vegetarians or synthesized by obligate carnivores? It would be of interest to compare the longevity of humans that easily accumulate carotenoids to those that do not. Consistent with carotenoids not being found in the tissues of so many vegetarians (that have survived millions of years of natural selection), one
might anticipate that humans who are poor accumulators of carotenoids live longer and are healthier. Although we do not intend to imply that a diet rich in fruit and vegetables should be avoided - because of the potential toxicity of carotenoids and their metabolites - we strongly recommend that supplementation with carotenoids should only be done under the supervision of a physician (e.g. for erythropoietic protoporphyria (29)). How are the ideas presented above consistent with the large number of epidemiological studies that indicate that low intake of fruits and vegetables, and hence low intake of carotenoids, is consistently associated with increased risk for various cancers (30)? The majority of these studies have focused only on diet (30). The epidemiological studies that have measured components of the plasma have usually measured total carotenoids or [3-carotene (30). Although, on average, plasma levels of [3-carotene are an indicator of the amount of food consumed that contains it (19,27), when one considers the extremely large number of compounds that food contains (most of which have not been characterized), it is presumptuous to conclude that [3-carotene or other carotenoids are responsible for the reduction in the risk of cancer in humans who consume a diet rich in fruit and vegetables. Many other factors may contribute to the reduction in risk (31). Recently, sulforaphane, that was isolated from broccoli, was shown to have anticarcinogenic activity (32). In murine hepatoma cells in culture, it was a potent inducer of phase-2 detoxication enzymes and it reduced the incidence of mammary tumors in rats (32). Whether it or similar compounds are responsible for the reduction in risk of cancer associated with a diet high in fruit and vegetables remains to be verified. In regard to recommending a diet high in broccoli, one might first ask: Why does broccoli contain a compound that induces phase-2 detoxication enzymes? One reasonable hypothesis is that it also contains toxic compounds that require these enzymes. We would not rule out the possibility that some of those toxic compounds are carotenoids or subsequent catabolites of carotenoids. It may be that the reason many animals do not accumulate carotenoids in their tissues is because they lack a sufficient array of enzymes that can detoxify their metabolites.
Lutein and zeaxanthin in the retina
Lutein and zeaxanthin are two carotenoids that are found in the retina of primates (33). They are concentrated in the central retina in a region referred to as the 'macula lutea' (yellow spot) (34). While it has
186 been known since the late 1940s that carotenoids were responsible for this yellow spot (35), their function is still not well delineated. Several functions, including that of being antioxidants, have been suggested (36, 37). Because the oxygen tension is particularly low in the region of the photoreceptor axons where the carotenoids are concentrated (36), and as it has been suggested that ~-carotene (and hence other similar carotenoids, such as lutein and zeaxanthin) may be effective free-radical scavengers at low partial pressures of oxygen (1), the free-radical scavenger hypothesis has been particularly tantalizing (36). For the same reasons as for [~-carotene (the metabolites lack regenerability, are difficult to excrete, and are potentially toxic), it is our contention that, in the macula lutea, the intended function of the macular pigment carotenoids is not that of free-radical scavengers. Whether or not their intended purpose is to quench singlet oxygen is uncertain. If that was the intent, then the more appropriate carotenoid would be lycopene, which is 3 - 4 times more effective at that task (23). Other carotenoids (e.g. u-carotene, [3carotene and canthaxanthin, which are absent from the retina) are also more effective at quenching singlet oxygen than either lutein or zeaxanthin (23). Furthermore, although the macula may be vulnerable to the inadvertent production of singlet oxygen, there is no evidence for its presence (25). Besides light and oxygen, an appropriate sensitizer is needed to produce singlet oxygen (25). Again, there is not any evidence for the presence of such a sensitizer in the region of the retina where the carotenoids are localized.
Conclusion In summary, we suggest that [3-carotene's primary in vivo function is to serve as a reservoir for retinol, retinaldehyde and retinoic acid. Although ~-carotene may have other functions, regardless of its concentration, or the partial pressure of oxygen, its intended function is not that of a free-radical scavenger. It does not meet the requirements: regenerability and tolerable toxicity. Since it is capable of effectively quenching singlet oxygen in chemical systems (8,23), it may inadvertently do so in vivo. Nonetheless, there is little evidence in support of its intended function being that of an in vivo singlet-oxygen quencher in humans. Although the function of lutein and zeaxanthin in the macula lutea is still open to speculation, for the same reasons as for 13-carotene, it is doubtful that their intended function is that of free-radical scavengers. Because of the unfortunate negative results associated with recent clinical trials on putative anti-
MEDICAL HYPOTHESES
oxidants, we suggest that approval of future placebocontrolled clinical trials on putative antioxidants should include the following constraints: (A) A n appropriate animal model should first be established. For example, animals could be trained to smoke prior to receiving dosages of antioxidants that will increase their plasma or tissue concentrations of an antioxidant (or its metabolites) significantly above normal; (B) Knowledge of the metabolic pathways associated with the putative antioxidant should be reasonably well understood in both an animal model and in humans; (C) Periodic analysis of the plasma, urinary and possibly fecal catabolites of the antioxidant should be included in the human studies.
Acknowledgement This research was supported by National Institutes of Health grant EY04368 (AJA).
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