Nutrient Metabolism

␤-Carotene Is Converted Primarily to Retinoids in Rats In Vivo1,2 Arun B. Barua3 and James A. Olson Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011

ABSTRACT ␤-Carotene might be converted oxidatively to vitamin A– active products in animals by the following three possible routes: 1) central cleavage, 2) sequential excentric cleavage or 3) random cleavage. Central cleavage

is strongly favored by stoichiometric studies with tissue homogenates in vitro. To examine the relative importance Downloaded from https://academic.oup.com/jn/article/130/8/1996/4686395 by guest on 29 September 2021 of these pathways in rats in vivo, an oral dose (5.6 ␮mol) of all-trans ␤-carotene in oil was given to vitamin A–deficient (ϪA) and to vitamin A–sufficient (ϩA) adult female Sprague-Dawley rats. Serum and several tissues were analyzed before and 3 h after dosing. The primary products of ␤-carotene found in the intestine, serum and liver were retinol, retinyl esters and retinoic acid. Two minor oxidation products of ␤-carotene, namely, 5,6-epoxy- ␤-carotene and a partially characterized hydroxy-␤-carotene, were present in the stomach and its contents as well as in intestinal preparations. In the intestine, including its contents, of ϪA rats, very minor amounts of 5,6- epoxyretinyl palmitate and of ␤-apocarotenals (8Ј,10Ј,12Ј,14Ј) were identified. The total amount of the ␤-apocarotenoids, however, was Ͻ5% of the retinoids formed in the intestine from ␤-carotene during the same period. Another ␤-carotene derivative, with a spectrum similar to that of semi-␤-carotenone, citranaxanthin and ␤- apo-6Ј-carotenal, was also found in the intestinal extract of a ϪA rat. ␤-Apocarotenals, ␤-apocarotenols, ␤-apocarotenyl esters and ␤-apocarotenoic acids were not detected in tissues of ϩA rats nor in other tissues of ϪA rats. These findings agree with the view that central cleavage is by far the major pathway for the formation of vitamin A from ␤-carotene in healthy rats in vivo. J. Nutr. 130: 1996–2001, 2000.

KEY WORDS: ● ␤-carotene ● retinoids ● ␤-apocarotenals ● rats ● central cleavage

Although vitamin A has been known as an essential mi- are depicted in Figure 1. The polyene chain of the carotenoid cronutrient for almost 90 years, vitamin A deficiency is still a might also be cleaved randomly, probably by nonspecific li- major health problem in much of the world (Underwood poxygenases and chemical oxidants. In oxidative stress, central 1994). The major dietary source of vitamin A in humans cleavage tends to be depressed and other oxidative transfor- consuming primarily vegetarian diets is provitamin A carot- mations of carotenoids tend to be enhanced (Gomboeva et al. enoids, of which ␤-carotene is the major component. 1998, Yeum et al. 1995). In mammalian tissues in vitro, provitamin A carotenoids Most past studies on ␤-carotene cleavage have been con- are converted mainly into vitamin A by central oxidative ducted with tissue homogenates in vitro. Although the inter- cleavage, which is catalyzed by the enzyme carotenoid 15,15Ј- pretation of in vivo studies is challenging (Glover et al. 1948, dioxygenase (EC 1.13.11.21) (Devery and Milborrow 1994, Mattson et al. 1947, West and Castenmiller 1998), we decided Duszka et al. 1996, Goodman and Huang 1965, Lakshman et to determine the extent to which retinal, ␤-apocarotenals and al. 1989, Lakshman and Okoh 1993, Lakshmanan et al. 1972 their metabolites, as well as other products, arise as a result of Nagao et al. 1996, Olson and Hayaishi 1965, Olson 1999, van administering ␤-carotene to both vitamin A-sufficient (ϩA)4 Vliet et al. 1996). A minor pathway in healthy mammals in and vitamin A–deficient (ϪA) rats in vivo. Our findings are vivo and in vitro is stepwise oxidative cleavage from one end summarized in this paper. of the polyene chain, presumably via a sequence of ␤-apocarotenals, to yield retinal (Ganguly and Sastry 1985, Gessler MATERIALS AND METHODS et al. 1998, Glover 1960, Sharma et al. 1977, Tang et al. 1991, Chemicals. Methanol, dichloromethane, 2-propanol, acetoni- Wang et al. 1991, Wang and Krinsky 1998) These pathways trile and ethyl acetate were supplied by Fisher Scientific (Fair Lawn, NJ). HPLC-grade solvents were used whenever available. All-trans ␤-carotene in the form of water-soluble beadlets, ␤-apo- 1 Presented in part at the 12th International Carotenoid Symposium, 18–23 8Ј-carotenal, ␤-apo-10Ј-carotenal and ␤-apo-12Ј-carotenal were gifts July 1999, Cairns, Australia [Barua, A. B. & Olson, J. A. (1999) Conversion of from Hoffmann-La Roche, (Nutley, NJ). The oral dose of ␤-carotene all-trans ␤-carotene into vitamin A and other products by the rat in vivo,p.62 was prepared as follows: ␤-carotene beadlets (1 g, 10% ␤-carotene, (abs.)]. wt/wt) were ground with water (3 mL). When a clear solution was 2 Supported in part by National Institutes of Health-DK39733, U.S. Depart- obtained, peanut oil (3 mL) was added and ground well to obtain a ment of Agriculture-NRICGP 97–37200-4290 and U.S. Department of Agriculture/ CDFIN 96–34115-2835. Journal Paper No. J-18838 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 3335, and supported by Hatch Act and State of Iowa Funds. 4 Abbreviations used: ϩA, vitamin A-sufficient; ϪA, vitamin A-deficient; RA, 3 To whom correspondence should be addressed. retinoic acid.

1996 ␤-CAROTENE METABOLISM IN RATS IN VIVO 1997

plemented diet contained retinyl palmitate (14,000 IU/kg, or 14.7 ␮mol/kg diet). The vitamin A–supplemented ICN diet contained only 3.5% of the retinyl palmitate (400,000 IU or 420 ␮mol/kg diet) present in the AIN-76A and AIN-93 diets (Reeves 1997). Although it supports growth, this ICN diet does not induce vitamin A storage in intestinal tissue and minimizes that in the liver. The weights of rats were recorded at regular intervals. The rats fed the ϪA diet showed signs of vitamin A deﬁciency, e.g., a weight plateau, after 5 wk. Blood (0.5 mL) was collected from the tail vein of each rat before administering the ␤-carotene. Rats from each group (n ϭ 3) were killed at zero time to obtain baseline values. Then, each rat (ϩA and ϪA) was given a single oral dose of ␤-carotene (5.6 ␮mol or 3 mg) in 0.18 mL peanut oil. The ϪA and ϩA rats (n ϭ 3/group) were killed under ether anesthesia 3 h after

the dose. To maximize the formation of ␤-apocarotenals, one ϪA rat Downloaded from https://academic.oup.com/jn/article/130/8/1996/4686395 by guest on 29 September 2021 was killed 1 h after dosing. Blood, collected from the heart, was allowed to clot, and serum was obtained by centrifugation at 1200 ϫ g for 15 min. The stomach, small intestine and liver were removed and weighed. Serum and tissues were kept frozen at Ϫ20°C. Serum. Retinoids and carotenoids in serum were extracted under yellow light at 4°C by a slight modification of a published procedure (Barua et al. 1998). In brief, serum (500 ␮L) was mixed with ethanol (1 mL), dilute acetic acid (3.3 mol/L, 0.1 mL), retinyl acetate in ethanol (internal standard, 7 ␮mol/L, 20–100 ␮L) that contained BHT (46 ␮mol/L), ethyl acetate (1 mL) and hexane (1 mL). The mixture was vortexed (30 s) and then centrifuged (1200 ϫ g) for 1 min. The supernatant solution was removed, and the pellet was extracted with hexane (1 mL). The pooled extracts were vortexed with water (0.5 mL) and then centrifuged, as indicated above. The organic extract was evaporated to dryness under a slow stream of argon. The residue was dissolved in a mixture of 2-propanol/dichloromethane (2:1, v/v; 100 ␮L). The recovery of the internal standard, retinyl acetate, was 88–95%. All reported serum values were correspondingly corrected. The efficiency of extraction of ␤-apo-8Ј-carotenal was similar to retinyl acetate under the same conditions. Liver, small intestine with its contents and stomach with its contents. The extraction procedure was a slight modification of a published procedure (Barua et al. 1998). The tissues were first chopped and minced. Liver (0.2–0.5 g) or 1 g small intestine or stomach (including contents) was placed in a mortar. The tissue was ground to a powder with anhydrous sodium sulfate. After the addition FIGURE 1 Central and stepwise cleavage pathways for the oxi- of 2 volumes (v/wt) of 2-propanol/dichloromethane (1:1), the mix- dative conversion of all-trans ␤-carotene to retinal. Semi-␤-carotenone, ture was ground further with a pestle and then allowed to stand for citranaxanthin, and ␤-apo-6Ј-carotenal, which may possibly be inter- 2–3 min. The extract was filtered, and the residue was extracted with mediates, are in brackets. dichloromethane 3–4 times, as described. The pooled extract, after being filtered, was evaporated to dryness in a rotary evaporator, and the residue was dissolved in 2-propanol/dichloromethane (1:1, 0.5 uniform solution. The purity and concentration of the ␤-carotene mL). An aliquot (50–100 ␮L) was analyzed by HPLC. The recovery solution were checked by spectrophotometry and by HPLC. of the internal standard, retinyl acetate, was 88–95%. All reported Derivatives. The ␤-apo-carotenals were reduced to the corre- tissue values were correspondingly corrected. The recovery of ␤-apo- sponding ␤-apo-carotenols by treatment with NaBH4 (Barua and 8Ј-carotenal under the same conditions was similar to retinyl acetate. Ghosh 1972). ␤-Apo-carotenoic acids were prepared by oxidation Reverse-phase gradient HPLC. For reverse-phase gradient with Tollen’s reagent, as described in the conversion of retinal to HPLC (Barua et al. 1998), Waters Associates (Milford, MA) pumps retinoic acid (RA) (Barua and Barua 1964). 5,6-Epoxyretinyl palmi- (model 510), an autosampler (WISP model 717 Plus), a pump control tate was prepared by treating retinyl palmitate with 3-chloroperoxy- module, a photodiode array detector (model 996) and the Millenium benzoic acid (Barua 1999). The ␤-apo-carotenols and ␤-apo-carote- 2010 chromatography manager were used. A Rainin (Woburn, MA) noic acids were purified by TLC on silica gel plates, and then tested Microsorb-MV 3 ␮mC (3.6 ϫ 100 mm) column was used. A for purity by HPLC. The oximes of ␤-apocarotenals were prepared by 18 15-min linear gradient of methanol/water (7:3, v/v containing 10 reaction with hydroxylamine, as described for the preparation of retinal oximes (Landers 1989), and tested by HPLC for purity. mmol/L ammonium acetate) to methanol/dichloromethane (4:1, v/v) Animals. Weanling female Sprague-Dawley rats were obtained or to acetonitrile/dichloromethane/methanol (95:10:5, v/v/v) at a through University Laboratory Animal Resources. All experiments flow rate of 0.6 mL/min was followed by isocratic elution with the with animals were in accord with NIH Guidelines for the Use of latter solvent mixture for another 30 min. The gradient was then Animals and were approved by the University Committee on the Use reversed to initial conditions in 5 min. Thereafter, the column was of Animals in Research. The rats were kept in individual cages, and equilibrated with the initial solvent for 10 min before the next fed either a vitamin A–deficient diet (Diet No. 904646) (ϪA) or a injection was made. vitamin A–sufficient diet (ϩA) of the same composition, both sup- The limit of detection for retinol and retinyl esters by HPLC was plied by ICN, Cleveland, OH. Both diets (g/kg) contained sucrose 1.8 pmol and for RA and ␤-carotene was 3.3 pmol when an injected (325), cornstarch (325), vitamin-free casein (180), brewer’s yeast aliquot of 100 ␮L was used. (80), cottonseed oil (50), and salt mixture #2 (40). Both diets also Statistical analysis. Means were compared using Student’s t test contained viosterol (4400 IU/kg), whereas only the vitamin A–sup- (Snedecor and Cochran 1989). 1998 BARUA AND OLSON

TABLE 1

An oral dose (5.6 ␮mol) of ␤-carotene in oil increases the concentrations of retinoids and ␤-carotene at 3 h in the serum of vitamin A–deﬁcient and vitamin A–sufﬁcient rats1

Vitamin A status Time Retinol Retinyl esters Retinoic acid ␤-Carotene

h ␮mol/L

Deﬁcient 0 0.061 Ϯ 0.005 ND2 ND ND 3 2.02 Ϯ 0.78 0.27 Ϯ 0.03 0.091 Ϯ 0.03 0.31 Ϯ 0.03 Sufﬁcient 0 1.05 Ϯ 0.15 ND ND ND 3 1.26 Ϯ 0.16 0.06 Ϯ 0.04 Յ0.01 0.34 Ϯ 0.08

1 Mean values Ϯ SD, n ϭ 3. 2 ND, not detected. Downloaded from https://academic.oup.com/jn/article/130/8/1996/4686395 by guest on 29 September 2021

RESULTS found in the stomach contents. The other carotenoid resem- bled a monohydroxy-␤-carotene. Its spectrum was identical to Carotenoids and retinoids in serum after a dose of - ␤ that of ␤-carotene, and its retention time was the same as that carotene of ␤-cryptoxanthin. Retinoids were not detected in the stom- Before dosing, the serum of ϪA rats had only a trace of ach and its contents. retinol, whereas the serum of ϩA rats had normal retinol Small intestine. In the small intestine, including its con- levels (Table 1). After the dose of ␤-carotene, the retinol level tents, ␤-carotene rose from nondetectable amounts at zero in ϪA rats increased significantly (P Ͻ 0.01) at 3 h, whereas time to relatively high amounts at 3 h (Table 2). Retinoids that in the serum of ϩA rats rose only slightly (P Ͻ 0.10) also increased markedly. Although the overall amounts of (Table 1). Retinyl esters reached much higher concentrations retinol plus its esters that were present 3 h after dosing were (P Ͻ 0.001) at3hintheserum of ϪA rats than of ϩA rats. not different in ϪA and ϩA rats, the amount of RA present RA also increased more (P Ͻ 0.01) at3hintheserums of ϪA in ϪA rats at 3 h was much higher than that found in ϩA rats rats than in that of ϩA rats (Table 1). Serum ␤-carotene (P Ͻ 0.001). A typical chromatogram, obtained at 330 nm for concentrations rose similarly in ϪA and ϩA rats. No other retinoids and 445 nm for carotenoids, of the small intestinal metabolites of ␤-carotene, such as the ␤-apocarotenals, were extract of ϪA rats 3 h after the dose of ␤-carotene is shown in detected in any serum sample of either ϪAorϩA rats. Figure 2A and B. The spectra of RA, retinol, retinyl palmitate and ␤-carotene, which were the major peaks in these chro- Carotenoids and retinoids in tissues matograms, are shown in Figure 2 C. The unmarked peak Stomach. In the stomach, including its contents, in both eluting at about 24 min in Figure 2A was tentatively identified ϪA and ϩA rats, ␤-carotene rose from undetectable amounts as 5,6-epoxyretinyl palmitate by its chromatographic behavior at zero time to 13.5 Ϯ 0.53 nmol/g in ϪA rats and to 7.2 and ␭max (325, 313, 295 nm). The peaks eluting near 30 min Ϯ 0.65 nmol/g in ϩA rats at 3 h. were retinyl esters (␭max ϭ 326 nm) of unidentified fatty acid Two other carotenoids were present in stomach extract at composition. Because of the large amount of ␤-carotene in the 3hatϳ10% of the concentration of ␤-carotene. One of these analyzed aliquot (Fig. 2B), however, considerable absorption compounds was identified as 5,6-epoxy-␤-carotene on the ba- in this elution region was also noted at 330 nm (Fig. 2A). A sis of its coelution with and spectral properties (␭max 475, 445 small amount of retinyl ester, probably retinyl stearate, was nm) similar to authentic 5,6-epoxy-␤-carotene (Barua 1999). also present in this peak at 330 nm (Fig. 2A). Only trace The 5,6-epoxy group of the carotenoid was further character- amounts of other products were present. ized by a hypsochromic shift in spectra on treatment with 0.1 Liver. The carotenoid and retinoid compositions of the mol/L HCl, indicative of the isomerization of the 5,6-epoxy livers of ϪA and ϩA rats before and 3 h after the dose of group to a 5,8-furanoid group (␭max 455, 425 nm) (Barua ␤-carotene are presented in Table 3.InϪA and ϩA rats, at 1999). The 5,8-furanoid form of epoxy-␤-carotene was not 3 h, retinol and retinyl esters increased as expected from

TABLE 2

An oral dose (5.6 ␮mol) of ␤-carotene increases the amounts of retinoids and ␤-carotene at3hinthesmall intestine and its contents of vitamin A–deﬁcient and vitamin A–sufﬁcient rats1

Vitamin A status Time Retinol Retinyl esters Retinoic acid ␤-Carotene

h nmol/g wet intestine

Deﬁcient 0 ND2 ND ND ND 3 3.69 Ϯ 1.13 6.55 Ϯ 4.39 2.46 Ϯ 0.38 10.9 Ϯ 0.82 Sufﬁcient 0 Ͻ0.10 Ͻ0.10 Ͻ0.10 ND 3 4.42 Ϯ 1.19 4.63 Ϯ 2.23 0.10 Ϯ 0.02 5.8 Ϯ 1.5

1 Mean values Ϯ SD, n ϭ 3. 2 ND, not detected. ␤-CAROTENE METABOLISM IN RATS IN VIVO 1999 Downloaded from https://academic.oup.com/jn/article/130/8/1996/4686395 by guest on 29 September 2021

FIGURE 3 Separation of products of ␤-carotene in a concentrated extract of the intestine and its contents from a vitamin A–deﬁcient (ϪA) rat 1 h after oral dosing with ␤-carotene. (A) HPLC separation with detection at 425 nm. (B) Spectra of retinal and ␤-apocarotenoid frac- tions. Abbreviations: EC, epoxy-␤-carotene; OH, monohydroxy-␤-carotene; RAL, retinal; 14Ј,12Ј,10Ј,8Ј, ␤-apo-carotenals. X, the uniden- tiﬁed carotenoid with the spectrum of semi-␤-carotenone.

Minor products of ␤-carotene cleavage in the small intestine, including its contents. In the chromatogram (Fig. 2B) of intestinal extracts, which included the lumen contents, FIGURE 2 Separation by HPLC of ␤-carotene and its products several tiny peaks that absorbed at 445 nm eluted between from the intestine and its contents of vitamin A–deficient (ϪA) rats 3 h retinol (20 min) and epoxy-␤-carotene (30 min). In prelimi- after oral dosing with ␤-carotene. (A) Peaks detected at 330 nm. (B) Peaks detected at 445 nm. (C) Spectra of major detected peaks. nary studies, these peaks were slightly more prominent at 1 h Abbreviations: BC, ␤-carotene; EC, epoxy-␤-carotene; RA, retinoic than at 3 h. To examine these compounds more fully, a acid; ROL, retinol; RP, retinyl palmitate fivefold (5 g) greater amount of the intestine and its contents from a ϪA rat was extracted at 1 h after dosing and a fourfold larger aliquot of the final extract was chromatographed. As baseline values; the increase was significant (P Ͻ 0.001) for shown in Figure 3A, thirteen peaks appeared in this region ϪA rats but not for ϩA rats because of their endogenous (20–28 min); seven of these have been tentatively identified, reserves. Furthermore, the liver concentrations of ␤-carotene, namely, retinal, four ␤-apocarotenals (8Ј,10Ј,12Ј and 14Ј), RA and retinal (0.88 Ϯ 0.50 vs. Ͻ0.1 Ϯ 0.1 nmol/g) were 5,6 epoxy-␤-carotene, and a hydroxycarotenoid. The absorp- 1% higher (P Ͻ 0.05) at3hinϩA than in ϪA rats. tion maxima and E1cm values of the ␤-apocarotenoids are

TABLE 3

An oral dose (5.6 ␮mol) of ␤-carotene increases the amounts of retinoids and carotenoids at3hintheliver of vitamin A–deﬁcient and vitamin A–sufﬁcient rats1

Vitamin A status Time Retinol Retinyl esters Retinoic acid ␤-Carotene

h nmol/g wet liver

Deﬁcient 0 0.01 Ϯ 0.002 7.86 Ϯ 2.3 ND2 ND 3 6.1 Ϯ 1.3 30.2 Ϯ 0.50 0.18 Ϯ 0.03 2.92 Ϯ 0.56 Sufﬁcient 0 11.3 Ϯ 2.5 73.8 Ϯ 11.5 Ͻ0.01 ND 3 21.8 Ϯ 9.8 96.4 Ϯ 30.4 2.42 Ϯ 0.76 24.9 Ϯ 3.9

1 Mean values Ϯ SD, n ϭ 3 or 2 (sufﬁcient, 0 h). 2 ND, not detected. 2000 BARUA AND OLSON

TABLE 4

Absorption maxima and extinction coefﬁcients of ␤-apocarotenoids1

Aldehydes ␤-Apocarotenoid Citation2 or ketones Alcohols Acids Oximes Reference

1% nm (Ecm)

14Ј Ref 405 (1708) 392 Tang et al. 1991 Obs 409 390 12Ј Ref 414 (2160) 377 (2120) 399 (2620) Ruegg et al. 1959 393 (2095) 418 (2530) Obs 414 375, 395 409 404, 418 10Ј Ref 435 (2190) 403 (1835) 430 (2235) 421 (2700) Ruegg et al. 1959

424 (1905) 443 (2500) Downloaded from https://academic.oup.com/jn/article/130/8/1996/4686395 by guest on 29 September 2021 Obs 433 404, 425 436 424, 445 8Ј Ref 457 (2640) 426 (2690) 448 (2515) 444 (2780) Ruegg et al. 1959 453 (2440) ϳ472 471 (3030) Obs 462 428, 458 443, ϳ468 443, 470 6Ј Ref 473 443 (2250) 458 (2495) Isler et al. 1959 471 (1855) 495 (1990) Citranaxanthin Ref 463 (2145) 442, 469 Yokoyama and White, 1965 Semi-␤-carotenone Ref 467 (1849) 442, 471 Yokoyama and White, 1968 Obs 467–472 443, 471 457, 476

1 Absorption maxima of reference compounds were determined in hexane, whereas those of ␤-carotene products were determined in the HPLC eluant, e.g., mixtures of methanol/water/dichloromethane. 2 Reference values from the literature (Ref) are cited first, followed by the values observed (Obs) in the current studies. summarized in Table 4, and the spectra of six of the isolated this tissue and its contents. Retinol and retinyl esters were ␤-carotene metabolites are shown in Figure 3B. Each of the major products at 3 h, together with smaller amounts of RA ␤-apocarotenals isolated from the intestinal preparation was (Table 2). The net increase of retinoids (12.7 Ϯ 4.8 nmol/g) reduced to its alcohol and converted to its oxime. As shown in in the intestine of ϪA rats at 3 h was somewhat higher than Table 4, the observed absorption maxima of these derivatives in ϩA rats (9.15 Ϯ 2.9 nmol/g), in keeping with the obser- agreed well with literature values, particularly in view of the vation that the activity of the intestinal central cleavage fact that different solvents were used in these determinations. enzyme is increased in vitamin A deficiency (van Vliet et al. The observed absorption maxima for the ␤-apo-carotenoic 1996). acids (8Ј,10Ј,12Ј) in Table 4, however, were derived from Retinal and several ␤-apo-carotenals were also detected at chemically oxidized reference compounds, not from ␤-caro- 3 h as very minor metabolites in the extracts of small intestines tene metabolites. and their contents of A rats, but not of A rats. By use of 1% Ϫ ϩ By use of the E1cmvalues for the reference compounds, the a sensitive photodiode array detector during HPLC and a amounts of various ␤-apocarotenals present per gram of intes- concentrated extract of the intestine of a ϪA rat at 1 h, these tinal tissue at 1 h were as follows (nmol/g): 14Ј (0.09), 12Ј products were characterized by their spectra, by their retention (0.04), 10Ј (0.12), and 8Ј (0.05), or a total of 0.30 nmol/g. In time on HPLC, and by the formation and spectral analysis of the same extract, the concentration of retinal was 0.28 nmol/g. two different chemical derivatives. The total amount of the At3hinϩA rats, however, these compounds were not ␤-apocarotenals present at 1 h was ϳ0.30 nmol/g, or Ͻ5% of detected. Furthermore, no ␤-apocarotenyl esters, which are the total retinoids present at the same time. Furthermore, no found in significant amounts in human sera after the oral ␤-apocarotenoic acids or ␤-apocarotenyl esters, which are administration of 8Ј-␤-apocarotenal (Zeng et al. 1992), or major metabolites of the ␤-apocarotenals (Zeng et al. 1992), ␤-apocarotenoic acids were detected in the intestinal extract were detected. In this regard, ␤-apo-8Ј-carotenal is converted of ϩAorϪA rats at 3 h. very slowly, if at all, to retinal in intestinal homogenates in vitro (Nagao et al. 1996). DISCUSSION In addition to the more common ␤-apocarotenals (8Ј,10Ј, Orally administered ␤-carotene is converted to several 12Ј,14Ј), we identified a HPLC peak (ϳ0.05 nmol/g) with a products in rats in vivo. After the single oral dose of ␤-caro- spectrum, chromatographic behavior and derivatives similar to tene, small quantities of 5,6-epoxy-␤-carotene and of a polar those of carotenoids containing a 6Ј carbonyl group, e.g., carotenoid resembling monohydroxy-␤-carotene were identi- ␤-apo-6Ј-carotenal (Isler et al. 1959), citranaxanthin fied in the stomach and its contents. These products were most (Yokoyama and White 1965) and semi-␤-carotenone likely formed by chemical oxidation in the presence of oxygen, (Yokoyama and White 1968). We favor semi-␤-carotenone as although 5,6-epoxy-␤-carotene has also been identified as a the primary product because its formation by a dioxygenase is product of ␤-carotene incubation with intestinal homogenates analogous to that of all other carotenoid cleavage products. (Handelman et al. 1991, Tang et al. 1991). No retinoids were Semi-␤-carotenone, although formed biologically in citrus detected in the stomach and its contents. (Yokayama and White 1968), has not been suggested previ- Because the small intestine is a major site of the conversion ously as a possible product of ␤-carotene metabolism in mam- of ␤-carotene to vitamin A in vivo (Glover et al. 1948, mals in vivo. Further study is clearly necessary, however, to Mattson et al. 1947, Parker et al. 1994), this study focused on elucidate the structure of this compound. the isolation and characterization of products of ␤-carotene in The extent to which these ␤-apocarotenals arise by chem- ␤-CAROTENE METABOLISM IN RATS IN VIVO 2001 ical oxidation in the lumen of the intestine or by enzymatic Glover, J., Goodwin, T. W. & Morton, R. A. (1948) Studies in vitamin A. VIII. Conversion of ␤-carotene into vitamin A in the intestine of the rat. Biochem. cleavage in the mucosa is unclear. Because the ␤-apo-carote- J. 43: 512–518. nals were not identified in ϩA rats nor in tissues of ϪA rats Gomboeva, S. B., Gessler, N. N., Shumaev, K. B., Khomich, T. I., Moiseenok, other than the intestine, ␤-apo-carotenals clearly were not A. G. & Bykhovskii, V. Y. (1998) Some natural and synthetic antioxidants as formed as artifacts of the isolation and extraction procedures. stabilizers of ␤-carotene conversion into vitamin A. Biochemistry (Moscow) 63: 185–190. The serum of ϪA rats expectedly showed an increase in all Goodman, D. S. & Huang, H. S. (1965) Biosynthesis of vitamin A with rat retinoids and ␤-carotene 3 h after dosing, whereas that of ϩA intestinal enzymes. Science (Washington, DC) 149: 879–880. rats showed lesser effects. Of particular note is that serum Handelman, G. J., Van Kujik, F.J.G.M., Chatterjea, A. & Krinsky, N. I. (1991) Characterization of products formed during the oxidation of ␤-carotene. Free retinol in ϪA rats peaked at3hataconcentration almost Radic. Biol. Med. 10: 427–437. twice that in ϩA rats. Furthermore, the serum RA concen- Isler, O., Guex, W., Ruegg, R., Ryser, G., Saucy, G., Schwieter, U., Walter, M. & tration at 3 h was much higher in ϪA than in ϩA rats, in Winterstein, A. (1959) 95. Synthesis in der carotinoid-reihe: carotinoide von typus destorularhodins. Helv. Chim. Acta 42: 864–871. keeping with our observation that retinoyl ␤-glucuronide is Kaul, S. & Olson, J. A. (1998) Effect of vitamin A deficiency on the hydrolysis also converted more rapidly to RA in ϪA than in ϩA rats of retinoyl ␤-glucuronide to retinoic acid by rat tissue organelles in vitro. Int. J. Vitam. Nutr. Res. 68: 232–236.

(Barua et al. 1998, Kaul and Olson 1998). Downloaded from https://academic.oup.com/jn/article/130/8/1996/4686395 by guest on 29 September 2021 Lakshman, M. R., Mychkovsky, I. & Attlesey, M. (1989) Enzymatic conversion All retinoids in the livers of ϪA rats increased markedly at of all-trans ␤-carotene to retinal by a cytosolic enzyme from rabbit and rat 3 h after dosing with ␤-carotene. Although the liver concen- intestinal mucosa. Proc. Natl. Acad. Sci. U.S.A. 86: 9124–9128. trations of retinoids were much higher initially in ϩA rats, the Lakshman, M. R. & Okoh, C. (1993) Enzymatic conversion of all-trans ␤-car- total mean increment of stored retinoids at3hinthelivers of otene to retinal. Methods Enzymol. 214: 256–269. Lakshmanan, M. R., Chansang, H. & Olson, J. A. (1972) Purification and ϩA rats (35.5 nmol/g) was higher than that of ϪA rats (28.6 properties of carotene 15,15Ј-dioxygenase of rabbit intestine. J. Lipid Res. 13: nmol/g). 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