In vitro measurement of beta-carotene cleavage activity: methodological considerations and the effect of other carotenoids on beta-carotene cleavage.
In view of controversies about assessment of the beta-carotene cleavage activity, methodological aspects and problems of the dioxygenase assay are described. Using rat and hamster intestinal preparations the method was optimized on retinal formation, the only cleavage product we could demonstrate. It appeared that the cell fraction with the highest cleavage activity was the 9,000 g supernatant (S-9). Maximal retinal formation was obtained with SDS, taurocholate and egg lecithin in the buffer and 3 micrograms beta-carotene dissolved in acetone. Ethanol, THF/DMSO (1:1) or propylene glycol as solvent for beta-carotene reduced retinal formation to 55, 24, and 19%, respectively. Retinal formation increased proportionally with the amount of protein S-9 used and was linear up to 40-60 minutes of incubation. Incubation with alpha-carotene or beta-cryptoxanthin resulted in a retinal formation of 29 and 55% of the amount formed from beta-carotene. Addition of 9 micrograms of lutein to an incubation with 3 micrograms beta-carotene reduced retinal formation, while lycopene had no effect. In conclusion, the beta-carotene cleavage assay with S-9 as enzyme source described in this report, seems a useful tool to study (dietary) determinants of beta-carotene cleavage activity, but for other purposes adaptation of the method is required.
van Vliet
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T
Department of Physiology and Kinetics, TNO Nutrition and Food Research Institute, AJ Zeist, The Netherlands.
van Schaik
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F
Schreurs
W H
WH
van den Berg
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H
D000818:Animals; D002338:Carotenoids / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology*; D006224:Cricetinae; D065366:Cryptoxanthins; D003600:Cytosol / Q000201:enzymology; D005260:Female; D007413:Intestinal Mucosa / Q000187:drug effects / Q000201:enzymology*; D007421:Intestine, Small / Q000201:enzymology; D000077276:Lycopene; D008297:Male; D008647:Mesocricetus; D010105:Oxygenases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012172:Retinaldehyde / Q000378:metabolism; D014316:Tritium; D024341:Xanthophylls; D019207:beta Carotene; D050559:beta-Carotene 15,15'-Monooxygenase
C493357:Bco1 protein, rat; D065366:Cryptoxanthins; D024341:Xanthophylls; D019207:beta Carotene; D014316:Tritium; D002338:Carotenoids; C041635:alpha-carotene; D010105:Oxygenases; D050559:beta-Carotene 15,15'-Monooxygenase; D012172:Retinaldehyde; D000077276:Lycopene