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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:241S-245S, January 2004

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

Alpha and Omega of Carotenoid Cleavage1

M. R. Lakshman2

Lipid Research Laboratory, VA Medical Center and the George Washington University, Washington, D.C.

2To whom correspondence should be addressed. E-mail: Raj.lakshman{at}med.va.gov.


   ABSTRACT
TOP
 ABSTRACT
SUMMARY
 LITERATURE CITED
 
In early 1900s, based on indirect evidence, Steenbock and Morton independently predicted that ß-carotene could be the biological precursor of vitamin A, although this notion was contested by others. In the 1930s, Thomas Moore showed the in vivo formation of vitamin A from ß-carotene. But it was not until Jim Olson and DeWitt Goodman independently showed in 1965 the formation of retinal, the aldehyde form of vitamin A from ß-carotene in cell-free extracts of liver and intestine, that this vital pathway of ß-carotene was recognized. Despite compelling evidence in several experimental systems for the central cleavage of ß-carotene to retinal by many investigators, there were some careful independent studies by Glover et al., Ganguly et al., Hansen and Meret and Krinsky et al. showing the eccentric cleavage of ß-carotene resulting in the formation of apocarotenoids both in vivo and in vitro. In an attempt to resolve this controversial issue, we revisited this problem in 1989 and showed beyond doubt the formation of retinal as the sole enzymatic product of a cytosolic enzyme from rabbit and rat intestinal mucosa by mass spectrometry and tracer analysis of the crystallized product. This was confirmed in 1996 by Nagao using the pig intestinal extract. Yeum et al. confirmed in 2000 that retinal is the sole product of ß-carotene cleavage in the presence of {alpha}-tocopherol, and that the observed formation of apocarotenoids occurs only in the absence of an antioxidant like {alpha}-tocopherol. In the same year, Barua and Olson also concluded from their in vivo studies in rats that central cleavage is by far the major pathway for the formation of vitamin A from ß-carotene. ß, ß-Carotene 15,15'-dioxygenase (EC 1.13.11.21) is the key enzyme that cleaves ß-carotene into two molecules of retinal. It is a cytosolic enzyme primarily localized in the duodenal mucosa although it has been found in liver. It is a 66 kDa sulfhydryl protein, requires molecular oxygen and is activated by ferrous ions. It is highly specific for 15:15' ethylenic bond of carotenoids although it has fairly broad specificity towards a number of carotenoids with at least one intact ß-ionone ring. The dioxygenase was recently cloned from Drosophila melanogaster and from the chicken intestine. The recombinant protein was found to form retinal as the sole cleavage product of ß-carotene. No apo-carotenoids were formed. Therefore, it is unequivocally proven that the major, if not the sole, pathway of ß-carotene cleavage to vitamin A is by oxidative cleavage of the central ethylenic bond of ß-carotene to yield two molecules of retinal. Most recently, human dioxygenase has also been cloned. Thus, the wisdom, vision and epoch-making mission of Jim Olson in the science of ß-carotene metabolism have been accomplished. I have no doubt that the impact of his original discovery of the dioxygenase and its importance in vitamin A nutriture should be forthcoming in the near future.

KEY WORDS: • ß-carotene • dioxygenase • central cleavage • eccentric cleavage • vitamin A

In the early 1900s, based on indirect evidence, Steenbock et al. (1) and Morton et al. (2) independently predicted that ß-carotene could be the biological precursor of vitamin A, although this notion was contested by others. In the 1930s, Thomas Moore (3) showed the in vivo formation of vitamin A from ß-carotene. But it was not until Olson et al. (4) and Goodman et al. (5a, 5b) independently showed in 1965 the formation of retinal, the aldehyde form of vitamin A, from ß-carotene in cell-free extracts of liver and intestine, that this vital pathway of ß-carotene was recognized. Despite compelling evidence in several experimental systems for the central cleavage of ß-carotene to retinal by many investigators (6–10), there were some careful independent studies by Glover et al. (11), Sharma et al. (12), Hansen and Maret (13) and Krinsky and his associates (14,15) showing the eccentric cleavage of ß-carotene resulting in the formation of apocarotenoids both in vivo and in vitro.

In an attempt to resolve this controversial issue, we revisited this problem (16) and showed beyond doubt the formation of retinal as the sole product of a cytosolic enzyme preparation from rabbit and rat intestinal mucosa by mass spectrometry and tracer analysis of the crystallized product.

Central cleavage

It has been previously observed (10) that there may be accompanying activities in the cytosol fraction of the intestine which could catalyze the further metabolism of retinal to retinol or retinoic acid. Furthermore, the crude cytosol fraction from the rat intestine, kidney, testes and liver has also been shown to convert {alpha}-carotene to retinoic acid (17). In view of these observations, we decided to fractionate the cytosol fraction of the rabbit intestinal mucosa. As shown in Table 1 (16) we accomplished an overall 238-fold purification of the dioxygenase following some simple purification steps such as ammonium sulfate fractionation and heat inactivation of other proteins followed by cold acetone fractionation. The â-carotene dioxygenase activity was localized in the 45–60% acetone pellet fraction. More importantly, this fraction was devoid of either retinal reductase or retinal oxidase activity (data not shown). Since our aim was to test whether or not retinal was the product of ß-carotene cleavage, we used the 45–60% acetone pellet fraction of the intestinal mucosa throughout this study.


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  		TABLE 1 Summary of rabbit intestinal dioxygenase purification1

 
The details of the enzyme assay have been described (16). As shown in Figure 1, the HPLC analysis of the lipid extract from the incubation of ß-carotene with the rabbit intestinal enzyme preparation (Fig. 1A) showed a peak with a retention time of 7.99 min corresponding to authentic retinal (O-ethyl) oxime with a retention time of 8.1 min (Fig. 1B). In contrast, as shown in Figure 1C, the extract from the boiled enzyme incubation mixture failed to show any HPLC peaks corresponding to retinal (O-ethyl) oxime or retinal or retinol. The absorption spectrum in ethanol of the O-ethyl oxime derivative of the enzymatic product corresponded very well with that of authentic retinal with an absorption maximum at 363 nm. The enzyme preparation from rat intestinal mucosa also showed similar results (data not shown).



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  		FIGURE 1 HPLC profiles of the lipid extracts from the standard assay mixtures from: (A) Rabbit BCC enzyme, (B) Authentic retinal (O-ethyl) oxime, (C) Boiled rabbit BCC enzyme. The experimental details and the HPLC conditions are given in the text. Detection wavelength, 360 nm.

 
Furthermore, the mass spectrum of the o-ethyl oxime derivative of the enzymatic product (Fig. 2A) was identical to that (Fig 2B) of authentic retinal (O-ethyl) oxime (m/z 327: 45%, m+. and m/z 282: 100%, m- ethoxy). These results conclusively prove the identity of the enzymatic product as retinal.



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  		FIGURE 2 Mass spectra of crystallized retinal (O-ethyl) oxime: (A) Authentic; (B) Enzymatic Product.

 
Table 2 summarizes the recovery of radioactivity in crystalline retinal (O-ethyl) oxime formed after the lipid extract from the incubation of [15,15'-14C] ß-carotene with the dioxygenase from rabbit intestine. Nearly 87% of the original radioactivity in the final ethanol solution containing the concentrate of retinal (O-ethyl) oxime was recovered in its crystalline form with a concomitant recovery of the product (1st crystallization). It is clear that even after three crystallizations the specific activity of the isolated retinal (O-ethyl) oxime remained constant (1343 dpm/mg). This is further evidence that the identity of the enzymatic product is retinal.


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  		TABLE 2 Co-crystallization of enzymatically formed [14C]retinal as its O-ethyloxime with authentic nonradioactive retinal O-ethyloxime1, 2

 
As shown in Table 3 the growth-promoting activity of a given carotenoid and its enzymatic cleavage rate to retinal are roughly comparable.


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  		TABLE 3 Cleavage of various carotenoids by carotenoid 15,15'-dioxygenase of rabbit intestine1

 
Taking ß-carotene activity as 100%, it can be seen that {alpha}-carotene had about 50% activity. Interestingly, Bis-3–3'-dehydro-{alpha}-carotene showed activity equal to {alpha}-carotene giving rise to 2 molecules of dehydro-retinal. To our knowledge, this is the first demonstration of the enzymatic formation of this prominent pigment found in freshwater fish. It was somewhat surprising that monoepoxy-{alpha}-carotene was not cleaved by dioxygenase since it shows some growth-promoting activity. On the other hand, it was understandable that the diepoxy-{alpha}-carorene was not cleaved by the enzyme since it has no growth-promoting activity. The hydroxy-carotenoid zeaxanthin yielded a product that was not identified.

Species specificity of the dioxygenase (Table 4) revealed that, compared to the rabbit enzyme, the guinea-pig (both are herbivores) enzyme was five times more active, whereas the enzyme activity from most other species (omnivores) varied between 50 and 100% of the rabbit enzyme activity. The only exception was the cat (a carnivore) enzyme, which showed no activity at all. This is consistent with the observation that cats became vitamin A-deficient when maintained on a vitamin A-free diet supplemented with ß-carotene (18).


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  		TABLE 4 Activity of intestinal dioxygenase in several species1

 
Subsequent studies (19,20) have confirmed that retinal is the sole product of ß-carotene cleavage by intestinal extracts from guinea pigs and pigs. Significantly, Nagao et al. (20) clearly demonstrated the stoichiometry of this enzymatic reaction to yield 2 moles of retinal per mole of ß-carotene cleaved (Table 5).


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  		TABLE 5 The stoichiometry of conversion of ß-carotene to retinal by pig intestinal extract1

 
Krinsky’s group (21) re-evaluated this cleavage reaction using the postmitochondrial fraction of rat intestinal homogenates both in the absence and presence of {alpha}-tocopherol. They also confirmed that in the presence of {alpha}-tocopherol, retinal is the sole product of ß-carotene cleavage and that the previously observed formation of apocarotenoids (14,15) occurs only in the absence of an antioxidant like {alpha}-tocopherol (Table 6).


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  		TABLE 6 Stoichiometry of the conversion of ß-carotene to retinal and ß-apocarotenals by the postmitochondrial fraction of rat intestine1–4

 
Barua and Olson (22) also conducted some in vivo studies on the conversion of ß-carotene to vitamin A in both vitamin A-sufficient and deficient rats. They also concluded that central cleavage is by far the major pathway for the formation of vitamin A from ß-carotene since the total amount of apocarotenoids formed was <5% of total vitamin A formed.


   SUMMARY
TOP
 ABSTRACT
 SUMMARY
LITERATURE CITED
 
Based on all these studies, some of the properties of the dioxygenase can be summarized as follows: ß,ß-carotene 15,15'-dioxygenase (EC 1.13.11.21) is the key enzyme that cleaves ß-carotene into two molecules of retinal. It is a cytosolic enzyme of 66 kDa primarily localized in the duodenal mucosa although it has been found in the liver. It is a sulfhydryl enzyme that is protected by GSH or DTT and inhibited by sulfhydryl binding agents such as PCMB, heavy metals, etc.

Ferrous ions activate it and iron chelators such as bipyridyl, and o-phenanthroline inhibit. It is highly specific for the central double bond but has fairly broad specificity towards a number of carotenoids and apocarotenoids with at least one intact ß-ionone ring. The Km for ß-carotene is 2–10 µM and Vmax is 0.5–2.0 nmoles of ß-carotene cleaved per mg per h.

The dioxygenase has been recently cloned from Drosophila melanogaster (23) and from the chicken intestine (24). The recombinant protein was found to form retinal as the sole cleavage product of ß-carotene. No apo-carotenoids were formed. Therefore, it is unequivocally proven that the major, if not the sole, pathway of ß-carotene cleavage to vitamin A is by oxidative cleavage of the central ethylenic bond of ß-carotene to yield two molecules of retinal. Most recently, human dioxygenase has also been cloned (25). Thus, Jim Olson’s epoch-making discovery of ß-carotene dioxygenase as the key enzyme responsible for the biotransformation of ß-carotene to vitamin A has withstood the test of time over the years and is indeed a landmark in vitamin A research.


   ACKNOWLEDGMENTS
 
This article is dedicated to the memory of my mentor, Professor James A. Olson. I also wish to thank the authors and publishers for the permission to reproduce the original tables of their publications presented in this article.


   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. Back


   LITERATURE CITED
TOP
 ABSTRACT
 SUMMARY
 LITERATURE CITED
 

1. Steenbock, H. (1919) White corn vs yellow corn and a probable relationship between the fat soluble viamins and yellow plant pigments. Science 50:352.[Free Full Text]

2. Drummond, J. C., Ahmad, B. & Morton, R. A. (1930) Further observations on the relation of carotenes to vitamin A. J. Soc. Chem. Ind. 49:291T.

3. Moore, T. (1930) Vitamin A and carotene VI. The conversion of carotene to vitamin A in vivo. Biochem. J. 24:692.

4. Olson, J. A. & Hayaishi, O. (1965) The enzymatic cleavage of ß-carotene into vitamin A by soluble enzymes of rat liver and intestine. Proc. Natl. Acad. Sci. U.S.A. 54:1364-1369.[Free Full Text]

5. Goodman, D. S. & Huang, H. S. (1965) Biosynthesis of vitamin A with rat intestinal enzymes. Science 149:879-880.[Abstract/Free Full Text]

5. Goodman, D. S., Huang, H. S. & Shiratori, T. (1966) Mechanism of biosynthesis of vitamin A from ß-carotene. J. Biol. Chem. 242:1929-1932.

6. Goodman, D. S., Huang, H. S., Kanai, M. & Shiratori, T. (1967) The enzymatic conversion of all-trans ß-carotene into retinal. J. Biol. Chem. 242:3543-3554.[Abstract/Free Full Text]

7. Lakshman, M. R., Pope, J. L. & Olson, J. A. (1968) The specificity of a partially purified carotenoid cleavage enzyme of rabbit intestine. Biochem. Biophys. Res. Commun. 33:347-352.[Medline]

8. Lakshman, M. R., Chansang, H. & Olson, J. A. (1972) Purification and properties of carotenoid 15,15'-dioxygenase of rabbit intestine. J. Lipid. Res. 13:477-482.[Abstract]

9. Gawienowski, A. M., Stacewicz, M. & Longley, R. (1974) Biosynthesis of retinal in bovine corpus luteum. J. Lipid. Res. 15:375-379.[Abstract]

10. Singh, H. & Cama, H. R. (1974) Enzymatic cleavage of carotenoids. Biochim. Biophys. Acta. 370:49-61.[Medline]

11. Glover, J. & Redfearn, E. R. (1954) The mechanism of transformation of ß-carotene into vitamin A in vivo. Biochem. J. 58:15-16.[Medline]

12. Sharma, R. V., Mathur, S. N., Dmitrovskii, A. A., Das, R. C. & Ganguly, J. (1977) Studies on the metabolism of ß-carotene and apo-ß-carotenoids in rats. Biochim. Biophys. Acta. 485:183-194.

13. Hansen, S. & Maret, W. (1988) Retinal is not formed in vitro. Biochemistry 27:200-206.[Medline]

14. Wang, X. D., Tang, G. W., Fox, J. G., Krinsky, N. I. & Russell, R. M. (1991) Enzymatic conversion of ß-carotene into ß-apocarotenals and retinoids by human, monkey, ferret and rat tissues. Arch. Biochem. Biophys. 285:8-16.[Medline]

15. Tang, G., Wang, X.-D., Russell, R. M. & Krinsky, N. I. (1991) Characterization of ß-apo-13-carotenone and ß-apo-14'-carotenal as enzymatic products of the excentric cleavage of ß-carotene. Biochemistry 30:9829-9834.[Medline]

16. Lakshman, M. R., Mychovsky, I. & Attlesey, M. (1989) Enzymatic conversion of all-trans- ß-carotene to retinal by a cytosolic enzyme from rabbit and rat intestinal mucosa. Proc. Natl. Acad. Sci. U.S.A. 86:9124-9128.[Abstract/Free Full Text]

17. Napoli, J. L. & Race, K. R. (1988) Biogenesis of retinoic acid from ß-carotene. J. Biol. Chem. 263:17372-17377.[Abstract/Free Full Text]

18. Ahmad, B. (1931) The fate of carotene after absorption in animal organism. Biochem. J. 25:1195-1204.

19. Devery, J. & Milborrow, B. V. (1994) ß-carotene-15,15'-dioxygenase (1.13.11.21) isolation reaction mechanism and improved assay procedure. Brit. J. Nutr. 72:397-414.[Medline]

20. Nagao, A., During, A., Hoshino, C., Terao, J. & Olson, J. A. (1996) Stoichiometric conversion of all-trans-ß-carotene to retinal by pig intestinal extract. Arc. Biochem. Biophys. 328:57-63.[Medline]

21. Yeum, K. J., Ferreira, A. L., Smith, D., Krinsky, N. I. & Russell, R. M. (2000) The effect of {alpha}-tocopherol on the oxidative cleavage of ß-carotene. Free Radic. Biol. Med. 29:105-114.[Medline]

22. Barua, A. B. & Olson, J. A. (2000) ß-carotene is converted primarily to retinoids in vivo. J. Nutr. 130:1996-2001.[Abstract/Free Full Text]

23. von Lintig, J. & Vogt, K. (2000) Filling the gap in vitamin A research. J. Biol. Chem. 275:11915-11920.[Abstract/Free Full Text]

24. Wyss, A., Wirtz, G., Woggon, W. D., Brugger, R., Wyss, M., Friedlein, A., Bachmann, H. & Hunziker, W. (2000) Cloning and expression of ß-carotene-15, 15'-dioxygenase. Biochim. Biophys. Res. Commun. 271:334-336.[Medline]

25. Yan, W., Jang, G. F., Haeseleer, F., Esumi, N., Chang, J., Kerrigan, M., Campochiaro, M., Campochiaro, P., Palczewski, K. & Zack, D. J. (2001) Cloning and characterization of a human ß, ß-carotene-15,15'-dioxygenase that is highly expressed in the retinal pigment epithelium. Genomics 72:193-202.[Medline]




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