[47]
CAROTENOID
CLEAVAGE: ALTERNATIVE
PATHWAYS
433
[47] C a r o t e n o i d C l e a v a g e : A l t e r n a t i v e P a t h w a y s
By L. E. GERBER and K. L. SIMPSON Introduction
Although it is well-established that carotene and other carotenoids require cleavage to shorter products for nutritional value as vitamin A, the nature of the cleavage remains in dispute. Some argue strongly for central cleavage by a putative 15,15'-dioxygenase (EC 1.13.11.21) with production of retinol directly. ~ Others argue strongly for excentric cleavage to 8'-, 10'-, and 12'-apo-fl-carotenals followed by serial oxidation to retinal by mechanisms similar to/3-oxidation. 2 The best evidence for excentric cleavage of carotenoids is the demonstration of the presence of 8'-, 10'-, and 12'-fl-apocarotenals in small intestinal tissue taken from animals previously consuming carotenoids. 3-5 Other evidence is provided by observations of the oxidative fission of carotenoids in model systems devoid of cells or cellular extracts. 6-9 In addition, recent evidence suggests that excentric cleavage of carotenoids is enhanced by some factors present in cell-free small intestinal extracts.l° Oxidative fission of/3-carotene using alkaline permanganate as a catalyst was demonstrated by Karrer and Solmssen 6 and revealed that the major products were apo-/3-carotenals. Glover and Redfearn 7 and Hasani and Parrish 8 have also demonstrated the chemical oxidation of/3-carotene to 8'-, 10'-, and 12'-apo-/3-carotenals. A detailed study of the site of initial attack of/3-carotene by molecular oxygen in a model system devoid of biological material indicates that an enzyme system would be needed in order to produce central fission. 9
l j. A. Olson, J. Nutr. 119, 105 (1989). 2 j. Ganguly and P. S. Sastry, World Rev. Nutr. Diet. 45, 198 (1985). 3 G. N. Festenstein, Ph.D. Thesis, University of Liverpool, England, 1951. 4 A. Winterstein and B. Hegedus, Chimia 14, 18 (1960). 5 R. V. Sharma, S. N. Mathur, A. A. Dmitrovskii, R. C. Das, and J. Ganguly, Biochim. Biophys. Acta 485, 183 (1977). 6 p. Karrer and L. Solmssen, Heir. Chim. Acta. 20, 682 (1937). 7 j. Glover and E. R. Redfearn, Biochem. J. 58, 15 (1954). 8 S. M. A. Hasani and P. B. Parrish, J. Agric. Food Chem. 15, 943 (1967). 9 A. H. E1-Tinay and C. O. Chichester, J. Org. Chem. 35, 2290 (1970). l0 S. Hansen and W. Maret, Biochemistry 27, 200 (1988).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
434
ENZYMOLOGY AND METABOLISM
[47]
Isolation of 8'-, 10'-, and 12'-apo-[J-carotenal from intestinal tissue of horses, chickens, and rats has been accomplished. Festenstein 3 isolated both 10'- and 12'-apo-[J-carotenals from horse intestine. Both Winterstein and Hegedus 4 and Sharma et al. 5 have isolated various apo-/3-carotenals from rat small intestines, whereas only Sharma et al. 5 have isolated apo[J-carotenals from chicken small intestine. A recent report by Hansen and Maret ~° demonstrated the production of 8'-, I0'-, and 12'-apo-[J-carotenals from [J-carotene when the crude enzyme extract of rat small intestine was incubated with [J-carotene. It is of interest that the production of both 8'- and 12'-apo-[J-carotenals was approximately twice as high for small intestinal tissue taken from vitamin A-deficient rats compared to vitamin A sufficient rats. Their protein-free control incubations also showed production of 8'-, 10'-, and 12'-apo-/3carotenals, as found in investigations of model systems of carotenoid cleavage devoid of biological materials. Assay Method: General
Purified/3-carotene is cleaved in the presence of a cell-free homogenate fraction from the small intestines to form 8'-, I0'-, and/or 12'-apo-/3carotenals. These cleavage products are extracted from lyophilized samples with acetone, separated by HPLC, and detected by spectrophotometry at a wavelength of 450 nm. Preparation o f Cell-Free Homogenate Fraction
As identified previously in this series, H intestinal mucosa scrapings are homogenized in 0.1 M potassium phosphate buffer, pH 7.7, and centrifuged at 2000 g for 20 min at 4 °. The resulting supernatant is then centrifuged at 104,000 g for 60 min at 4° to obtain a soluble fraction containing cleavage activity. Cleavage activity can be further purified by ammonium sulfate precipitation isolating the 20-45% ammonium sulfate precipitation fraction. Purity of[J-Carotene
The purity of commercially available [J-carotene is assessed by highperformance liquid chromatography (HPLC),~2 and the material is recrystallized, if necessary, using a modification of the procedure of Britton and 11 D. S. Goodman and J. A. Olson, this series, Vol. 15, p. 462. 12 G. Arroyave, C. O. Chichester, H. Flores, J. Glover, L. A. Mejia, J. A. Olson, K. L. Simpson, and B. A. Underwood, "Biochemical Methodology for the Assessment of Vitamin A Status." The Nutrition Foundation, Washington, D.C., 1982.
[47]
CAROTENOID CLEAVAGE: ALTERNATIVE PATHWAYS
435
Goodwin. 13The purity of the recrystallized/3-carotene is then assessed by HPLC before use. Procedures for the proper handling and analysis of carotenoids have been published) 2
Incubation of /3-Carotene with Cell-Free Ammonium Sulfate Fraction Assay procedures are those outlined by Goodman and Olson for/3carotene conversion. H The cell-free homogenate fraction previously isolated is dissolved in 10 mM potassium phosphate buffer, pH 7.7. The resulting solution is dialyzed for several hours against a large solution of phosphate buffer to remove ammonium sulfate. A 1-ml aliquot of this solution is used for each incubation. An additional amount of 200/zmol potassium phosphate buffer, pH 7.7, is added as well as several chemicals which enhance/3-carotene cleavage. These enhancing factors include 30 /zmol nicotinamide, 10/~mol glutathione, 12/zmol sodium glycocholate, and 400/zg egg lecithin. In addition, a nonpolar antioxidant such as atocopherol should be added in small amounts. To the resulting incubation mixture is added a total of 1/xg/3-carotene in 50/.d of acetone. Incubations are carried out either in amber Erlenmeyer flasks or in clear Edenmeyer flasks under red light. After 1 hr of shaking at 37° in a water bath, the reaction is stopped by rapid freezing and the sample lyophilized.
Extraction and Detection of Carotenoids, Retinoids, and Apocarotenals Lyophilized samples are extracted 3 times with acetone using pyrogal1ol as antioxidant. The pooled extracts are evaporated with a rotary evaporator at 37 ° and resolubilized in the appropriate carrier system prior to chromatography.~2 For retinoid analysis, extracts are redissolved in hexane/ethyl acetate/methanol (95 : 4 : 1). Retinoids are then detected after HPLC separation using a Spherisorb-S-5-CN column (5/.~m, 0.46 × 25 cm) with a carrier solvent of hexane/ethyl acetate/methanol (95:4 : 1) at a flow rate of 1 ml/min. Detection of retinol, retinal, and retinoic acid is accomplished at 350 nm. For analysis of/3-carotene and 8'-, 10'-, and 12'apo-/3-carotenal, extracts are redissolved in acetonitrile/dichloromethane (7 : 3). A Zorbax ODS column (10/zm, 0.46 × 25 cm) is used for separation with a carrier solvent system of acetonitrile/dichloromethane (7 : 3) at 1 ml/min. Detection is accomplished at 450 nm. Biological Significance It appears that evidence of excentric cleavage of/3-carotene in mammalian systems is undeniable. Further experiments are required using the 13 G. Britton and T. W. Goodwin, this series, Vol. 18C, p. 654.
436
ENZYMOLOGY AND METABOLISM
[48]
described techniques to determine if the reactions are enhanced by enzymatic activity and what enzyme is responsible for the cleavage. Clearly, apo-fl-carotenals are present in the small intestine, but enhanced production in small intestine tissue owing to enzymatic activity may not be a normal physiological event. Acknowledgment The above manuscript is contribution #2502 of the Rhode Island Agricultural Experiment Station, Kingston, RI.
[48] I s o e n z y m e s o f A l c o h o l D e h y d r o g e n a s e in Retinoid Metabolism B y X A V I E R PARI~S a n d PERE JULIA
Introduction I all-trans-Retinol + NAD + ~ all-trans-retinal + NADH + H ÷
Alcohol dehydrogenase (alcohol : NAD + oxidoreductase, EC 1.1.1.1) (ADH) is an enzyme widely distributed in animals, plants, and microorganisms. 2 It catalyzes the reversible oxidation of a great variety of alcohols to the corresponding aldehydes and ketones. 3 ADH exhibits several isoenzymes in most species studied. In mammals the isoenzymes have been grouped in three classes.4 Class I shows a wide substrate specificity, a low Km for ethanol, and low Ki for pyrazole. It is abundant in liver. Class III is a glutathione-dependent formaldehyde dehydrogenase. 5 Its activity with alcohols is restricted to long-chain primary alcohols, is not inhibited by pyrazole, and is detected in most tissues. Class II shows intermediate properties. In general, ADH isoenzymes are very active toward longchain hydrophobic alcohols) Retinol and retinal are, therefore, subSupported by Grant 86/0156 of Direcci6n General de Investigaci6n Cientffica y Trcnica (Spain). We acknowledge Dr. B. L. Vallee and Dr. W. P. Dafeldecker for the gift of the CapGapp-Sepharose. 2 H. Jfrnvall, B. Persson, and J. Jeffery, Eur. J. Biochem. 167, 195 (1987). 3 R. Pietruszko, in "Biochemistry and Pharmacology of Ethanol" (E. Majchrowicz and E. P. Noble, eds.), Vol. 1, p. 87. Plenum, New York, 1979. 4 B. L. Vallee and T. J. Bazzone, lsozymes: Curr. Top. Biol. Med. Res. 8, 219 (1983). 5 M. Koivusalo, M. Baumann, and L. Uotila, FEBS Lett. 257, 105 (1989).
METHODS IN ENZYMOLOGY,VOL. 189
Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin any formreserved.
CAROTENOID
CLEAVAGE: ALTERNATIVE
PATHWAYS
433
[47] C a r o t e n o i d C l e a v a g e : A l t e r n a t i v e P a t h w a y s
By L. E. GERBER and K. L. SIMPSON Introduction
Although it is well-established that carotene and other carotenoids require cleavage to shorter products for nutritional value as vitamin A, the nature of the cleavage remains in dispute. Some argue strongly for central cleavage by a putative 15,15'-dioxygenase (EC 1.13.11.21) with production of retinol directly. ~ Others argue strongly for excentric cleavage to 8'-, 10'-, and 12'-apo-fl-carotenals followed by serial oxidation to retinal by mechanisms similar to/3-oxidation. 2 The best evidence for excentric cleavage of carotenoids is the demonstration of the presence of 8'-, 10'-, and 12'-fl-apocarotenals in small intestinal tissue taken from animals previously consuming carotenoids. 3-5 Other evidence is provided by observations of the oxidative fission of carotenoids in model systems devoid of cells or cellular extracts. 6-9 In addition, recent evidence suggests that excentric cleavage of carotenoids is enhanced by some factors present in cell-free small intestinal extracts.l° Oxidative fission of/3-carotene using alkaline permanganate as a catalyst was demonstrated by Karrer and Solmssen 6 and revealed that the major products were apo-/3-carotenals. Glover and Redfearn 7 and Hasani and Parrish 8 have also demonstrated the chemical oxidation of/3-carotene to 8'-, 10'-, and 12'-apo-/3-carotenals. A detailed study of the site of initial attack of/3-carotene by molecular oxygen in a model system devoid of biological material indicates that an enzyme system would be needed in order to produce central fission. 9
l j. A. Olson, J. Nutr. 119, 105 (1989). 2 j. Ganguly and P. S. Sastry, World Rev. Nutr. Diet. 45, 198 (1985). 3 G. N. Festenstein, Ph.D. Thesis, University of Liverpool, England, 1951. 4 A. Winterstein and B. Hegedus, Chimia 14, 18 (1960). 5 R. V. Sharma, S. N. Mathur, A. A. Dmitrovskii, R. C. Das, and J. Ganguly, Biochim. Biophys. Acta 485, 183 (1977). 6 p. Karrer and L. Solmssen, Heir. Chim. Acta. 20, 682 (1937). 7 j. Glover and E. R. Redfearn, Biochem. J. 58, 15 (1954). 8 S. M. A. Hasani and P. B. Parrish, J. Agric. Food Chem. 15, 943 (1967). 9 A. H. E1-Tinay and C. O. Chichester, J. Org. Chem. 35, 2290 (1970). l0 S. Hansen and W. Maret, Biochemistry 27, 200 (1988).
METHODS IN ENZYMOLOGY, VOL. 189
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
434
ENZYMOLOGY AND METABOLISM
[47]
Isolation of 8'-, 10'-, and 12'-apo-[J-carotenal from intestinal tissue of horses, chickens, and rats has been accomplished. Festenstein 3 isolated both 10'- and 12'-apo-[J-carotenals from horse intestine. Both Winterstein and Hegedus 4 and Sharma et al. 5 have isolated various apo-/3-carotenals from rat small intestines, whereas only Sharma et al. 5 have isolated apo[J-carotenals from chicken small intestine. A recent report by Hansen and Maret ~° demonstrated the production of 8'-, I0'-, and 12'-apo-[J-carotenals from [J-carotene when the crude enzyme extract of rat small intestine was incubated with [J-carotene. It is of interest that the production of both 8'- and 12'-apo-[J-carotenals was approximately twice as high for small intestinal tissue taken from vitamin A-deficient rats compared to vitamin A sufficient rats. Their protein-free control incubations also showed production of 8'-, 10'-, and 12'-apo-/3carotenals, as found in investigations of model systems of carotenoid cleavage devoid of biological materials. Assay Method: General
Purified/3-carotene is cleaved in the presence of a cell-free homogenate fraction from the small intestines to form 8'-, I0'-, and/or 12'-apo-/3carotenals. These cleavage products are extracted from lyophilized samples with acetone, separated by HPLC, and detected by spectrophotometry at a wavelength of 450 nm. Preparation o f Cell-Free Homogenate Fraction
As identified previously in this series, H intestinal mucosa scrapings are homogenized in 0.1 M potassium phosphate buffer, pH 7.7, and centrifuged at 2000 g for 20 min at 4 °. The resulting supernatant is then centrifuged at 104,000 g for 60 min at 4° to obtain a soluble fraction containing cleavage activity. Cleavage activity can be further purified by ammonium sulfate precipitation isolating the 20-45% ammonium sulfate precipitation fraction. Purity of[J-Carotene
The purity of commercially available [J-carotene is assessed by highperformance liquid chromatography (HPLC),~2 and the material is recrystallized, if necessary, using a modification of the procedure of Britton and 11 D. S. Goodman and J. A. Olson, this series, Vol. 15, p. 462. 12 G. Arroyave, C. O. Chichester, H. Flores, J. Glover, L. A. Mejia, J. A. Olson, K. L. Simpson, and B. A. Underwood, "Biochemical Methodology for the Assessment of Vitamin A Status." The Nutrition Foundation, Washington, D.C., 1982.
[47]
CAROTENOID CLEAVAGE: ALTERNATIVE PATHWAYS
435
Goodwin. 13The purity of the recrystallized/3-carotene is then assessed by HPLC before use. Procedures for the proper handling and analysis of carotenoids have been published) 2
Incubation of /3-Carotene with Cell-Free Ammonium Sulfate Fraction Assay procedures are those outlined by Goodman and Olson for/3carotene conversion. H The cell-free homogenate fraction previously isolated is dissolved in 10 mM potassium phosphate buffer, pH 7.7. The resulting solution is dialyzed for several hours against a large solution of phosphate buffer to remove ammonium sulfate. A 1-ml aliquot of this solution is used for each incubation. An additional amount of 200/zmol potassium phosphate buffer, pH 7.7, is added as well as several chemicals which enhance/3-carotene cleavage. These enhancing factors include 30 /zmol nicotinamide, 10/~mol glutathione, 12/zmol sodium glycocholate, and 400/zg egg lecithin. In addition, a nonpolar antioxidant such as atocopherol should be added in small amounts. To the resulting incubation mixture is added a total of 1/xg/3-carotene in 50/.d of acetone. Incubations are carried out either in amber Erlenmeyer flasks or in clear Edenmeyer flasks under red light. After 1 hr of shaking at 37° in a water bath, the reaction is stopped by rapid freezing and the sample lyophilized.
Extraction and Detection of Carotenoids, Retinoids, and Apocarotenals Lyophilized samples are extracted 3 times with acetone using pyrogal1ol as antioxidant. The pooled extracts are evaporated with a rotary evaporator at 37 ° and resolubilized in the appropriate carrier system prior to chromatography.~2 For retinoid analysis, extracts are redissolved in hexane/ethyl acetate/methanol (95 : 4 : 1). Retinoids are then detected after HPLC separation using a Spherisorb-S-5-CN column (5/.~m, 0.46 × 25 cm) with a carrier solvent of hexane/ethyl acetate/methanol (95:4 : 1) at a flow rate of 1 ml/min. Detection of retinol, retinal, and retinoic acid is accomplished at 350 nm. For analysis of/3-carotene and 8'-, 10'-, and 12'apo-/3-carotenal, extracts are redissolved in acetonitrile/dichloromethane (7 : 3). A Zorbax ODS column (10/zm, 0.46 × 25 cm) is used for separation with a carrier solvent system of acetonitrile/dichloromethane (7 : 3) at 1 ml/min. Detection is accomplished at 450 nm. Biological Significance It appears that evidence of excentric cleavage of/3-carotene in mammalian systems is undeniable. Further experiments are required using the 13 G. Britton and T. W. Goodwin, this series, Vol. 18C, p. 654.
436
ENZYMOLOGY AND METABOLISM
[48]
described techniques to determine if the reactions are enhanced by enzymatic activity and what enzyme is responsible for the cleavage. Clearly, apo-fl-carotenals are present in the small intestine, but enhanced production in small intestine tissue owing to enzymatic activity may not be a normal physiological event. Acknowledgment The above manuscript is contribution #2502 of the Rhode Island Agricultural Experiment Station, Kingston, RI.
[48] I s o e n z y m e s o f A l c o h o l D e h y d r o g e n a s e in Retinoid Metabolism B y X A V I E R PARI~S a n d PERE JULIA
Introduction I all-trans-Retinol + NAD + ~ all-trans-retinal + NADH + H ÷
Alcohol dehydrogenase (alcohol : NAD + oxidoreductase, EC 1.1.1.1) (ADH) is an enzyme widely distributed in animals, plants, and microorganisms. 2 It catalyzes the reversible oxidation of a great variety of alcohols to the corresponding aldehydes and ketones. 3 ADH exhibits several isoenzymes in most species studied. In mammals the isoenzymes have been grouped in three classes.4 Class I shows a wide substrate specificity, a low Km for ethanol, and low Ki for pyrazole. It is abundant in liver. Class III is a glutathione-dependent formaldehyde dehydrogenase. 5 Its activity with alcohols is restricted to long-chain primary alcohols, is not inhibited by pyrazole, and is detected in most tissues. Class II shows intermediate properties. In general, ADH isoenzymes are very active toward longchain hydrophobic alcohols) Retinol and retinal are, therefore, subSupported by Grant 86/0156 of Direcci6n General de Investigaci6n Cientffica y Trcnica (Spain). We acknowledge Dr. B. L. Vallee and Dr. W. P. Dafeldecker for the gift of the CapGapp-Sepharose. 2 H. Jfrnvall, B. Persson, and J. Jeffery, Eur. J. Biochem. 167, 195 (1987). 3 R. Pietruszko, in "Biochemistry and Pharmacology of Ethanol" (E. Majchrowicz and E. P. Noble, eds.), Vol. 1, p. 87. Plenum, New York, 1979. 4 B. L. Vallee and T. J. Bazzone, lsozymes: Curr. Top. Biol. Med. Res. 8, 219 (1983). 5 M. Koivusalo, M. Baumann, and L. Uotila, FEBS Lett. 257, 105 (1989).
METHODS IN ENZYMOLOGY,VOL. 189
Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin any formreserved.
