Nutrient Requirements
and Interactions
JUDY D. RIBAYA-MERCADO, JAMES G. FOX, * WILLIAM D. ROSENBLAD, * MICHAEL C. BLANCO* AND ROBERT M. RUSSELL U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts Uniuersity, Boston, MA 02111, and *Diuision of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139 amount of intact ß-carotene(-15-25%) is also ab sorbed into the lymph (Blomstrand and Werner 1967, Goodman et al. 1966). In most other species (i.e., rats, pigs, chickens, guinea pigs, rabbits), very little or no ß-caroteneis absorbed intact; it is largely converted to vitamin A before absorption (Bondi and Sklan 1984, Ganguly et al. 1953, Thompson et al. 1950). Horses and certain breeds of cattle are able to absorb intact ßcarotene (Bondi and Sklan 1984). However, they are not convenient laboratory animal models. We previously reported that the ferret is a suitable animal model because ferrets have the capacity to absorb intact dietary ß-carotene(Ribaya-Mercado et al. 1989). We also reported that it can store absorbed ß-carotenein liver, and to a much lesser extent, in adipose tissue (Ribaya-Mercado et al. 1989). Because of their convenient size, domesticated ferrets can be easily handled and maintained in the laboratory setting. Thus, the ferret seems to be a promising model for future studies of ß-caroteneabsorption and metabolism. In this study, we report serum concen trations of carotenoids (ß-carotene, a-carotene, lycopene) and retinoids (retinol, total and individual retinyl esters) achieved by feeding ferrets diets supple mented with ß-carotenefor 3 wk. Concentrations of ß-carotene,retinol and total retinyl esters in other selected tissues are also reported.
ABSTRACT The concentrations of ß-carotene,retinol and retinyl esters in serum and selected tissues of ferrets fed diets supplemented with ß-carotene(80 ng/g wet diet) for 3 wk were determined. The initial concen tration of serum ß-carotenewas 0.011 ±0.006 nmol/L (mean ±SEM); at the end of the experimental period it was 5.75 ±1.60 umol/L. No significant differences in serum retino! and total retinyl esters were observed be tween ß-carotene-fedand control ferrets that had been fed an unsupplemented diet. The predominant retinyl esters in serum were retinyl stéarate(53%) and retinyl palmitate (35%). Of the tissues analyzed after licarotene feeding, the liver contained the highest concen tration of ß-carotene (78.8 ±18.8 nmol/g). Other tissues that contained ß-carotenein amounts ranging from 17 to 20 nmol/g were adrenals, small intestine, stomach and colon; lesser amounts (6.9 nmol/g) were found in kidneys. Amounts ranging from 1.2 to 2.3 nmol/g were found in muscle, bladder, adipose tissue, lungs and skin; only 0.37 and 0.34 nmol/g were present in brain and eyes, respectively. Thus, like humans, ferrets have the capacity to absorb intact ß-caroteneand to store this compound in tissues, especially the liver. However, compared with humans, ferrets have elevated concentrations of retinyl esters in serum, liver and other tissues. J. Nutr. 122: 1898-1903, 1992. INDEXING KEY WORDS:
•ß-caroiene•vitamin A •retinyl esters •retinol •ferrets
Among the dietary carotenoids all-trans-ß-carotene has the highest provitamin A activity (Goodman 1984). This compound is converted to vitamin A in the intestinal mucosa (Goodman and Huang 1965). The efficiency of conversion varies among different species. In humans most dietary ß-caroteneis con verted to vitamin A in the gut; however, a substantial
'Supported in part by U.S. Department of Agriculture contract no. 53-3KO6-5-10. The contents of this publication do not neces sarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of commercial products imply en dorsement by the United States government. 2An abstract describing preliminary data has been published [Ribaya-Mercado, J. D., Fox, J. G., Rosenblad, W. & Russell, R. M. (1990) Dietary ß-carotenesupplementation and tissue levels of ßcarotene, retinol, and retinyl esters in ferrets. J. Am. Coll. Nutr. 9: 526 (abs. 20)].
0022-3166/92 $3.00 ©1992 American Institute of Nutrition. Received 12 March 1992. Accepted 12 May 1992.
1898
Downloaded from https://academic.oup.com/jn/article-abstract/122/9/1898/4769470 by Tulane University Library, Serials Acquisitions Dept. user on 20 January 2019
ß-Carotene, Retinol and Retinyl Ester Concentrations in Serum and Selected Tissues of Ferrets Fed ß-Carotene1'2
ß-CAROTENE-FED FERRETS
MATERIALS AMD METHODS and diets
Male ferrets (Marshall Farms, North Rose, NY), 8 mo old and weighing 1234 ±98 g (mean ±SEM),were used in these experiments. They were housed individ ually in suspended stainless steel cages and were given free access to a standard diet and water. For 3 wk, the experimental ferrets were fed a basal diet consisting of dry cat food (Purina Cat Chow,3 Ralston Purina, St. Louis, MO) moistened with water (1.5 mL/ g dry food), to which was added 80 ug of ß-carotene(as 824 ug of ß-carotenebeadlets, Hoffmann-LaRoche, Nutley, NJ) per gram of wet diet. Control ferrets were fed the moistened cat food only. Diets were prepared weekly and refrigerated. Using extraction and analytical procedures described below for tissues, we analyzed the moistened basal diet and found it to contain 0.33 ug ß-caroteneand 17.3 ug retinyl esters/g wet wt. The diet also contained 0.005 ug a-carotene and 0.016 ug lycopene/g wet wt. Because the average daily food intake during the 3-wk period was 193 ±8 g, the average intakes of ß-caroteneand retinyl esters from the basal diet were 0.064 ±0.003 and 3.3 ±0.14mg/d, respectively; the daily intakes of a-carotene and lycopene were about 0.97 and 3.1 ug, respectively; and the daily intake of supplemental ß-carotenewas 15.44 ±0.64 mg. Thus the total ß-caroteneintake of the experimental ferrets was -15.5 mg/d. Blood was ob tained from jugular veins for baseline serum measure ments. After 3 wk, the animals were killed with carbon dioxide. Blood was obtained by cardiac puncture, and the following tissues were quickly excised: liver (midsection of the left lateral lobe), adrenal glands (entire right and left), small intestine (mid-jejunum), stomach (greater curvature from cardia to pylorus), colon (descending), kidney (right kidney), muscle (section of rectus femorus), bladder (whole), skin (ventral ab domen), adipose tissue (subcutaneous, from ventral abdomen), lung (right medial lobe), brain (right hemi sphere) and eye (entire right eye). The serum and tissues were frozen at -80°Cuntil analyzed for carotenoids and retinoids. All animal procedures were re viewed and approved by the Animal Care and Use Committee at Tufts University and the Committee on Animal Care at the Massachusetts Institute of Technology. Analytical methods For the extraction of serum and tissue carotenoids and retinoids, procedures we have already described (Ribaya-Mercado et al. 1989) were used with some
modifications. The reverse-phase gradient HPLC pro cedures previously described (Ribaya-Mercado et al. 1989) were used for the detection and measurement of these compounds. Serum extraction. A 200-uL aliquot of serum was treated with 2 mL of CHC13:CH3OH (2:1, v/v). In ternal standards (150 uL) consisting of Y-carotene (Hoffmann-LaRoche) and retinyl acetate (Sigma Chemical, St. Louis, MO) were added, followed by 500 uL of 8.5 g/L saline. The tube was vortexed and centrifuged at 684 x g at 4°Cfor 10 min. The CHC13 and aqueous layers were separated, and the aqueous layer was extracted with 3 mL of hexane. The tube was vortexed and centrifuged at 684 x g at 4°Cfor 10 min, and the hexane layer was combined with the CHC13 extract. The combined extracts were dried under nitrogen in a water bath and the residue was redissolved in 150 uL of ethanol. The tube was vor texed and centrifuged at 684 x g at 4°Cfor 2 min. A 50-uL aliquot was injected onto the HPLC column. Tissue extraction. The procedures for tissue ex traction were similar to those we have described for liver and adipose tissue (Ribaya-Mercado et al. 1989) with some modifications. The tissues were weighed (about 0.05 g for liver; 0.1 g for kidney and adrenal; 0.25 g for eye; 0.3 g for lung and whole bladder; 0.5 g for small intestinal mucosa, colon, stomach, skin, subcutaneous adipose tissue, brain and muscle), minced and homogenized in 5 mL of CHC13:CH3OH (2:1, v/v) using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). The Polytron probe was rinsed with 5 mL of CHC13:CH3OH, and the wash was added to the homogenate. Internal standards (0.3 to 0.9 mL) of ycarotene and retinyl acetate were added, followed by 2 mL of 8.5 g/L saline. The mixture was vortexed and centrifuged at 684 x g at 4°Cfor 10 min. The CHC13 layer was separated, and the aqueous layer was extracted with 3-5 mL of hexane. After vortexing and centrifuging the tube at 684 x g at 4°Cfor 10 min, the hexane layer was combined with the CHC13 extract and the combined extracts dried under nitrogen in a water bath. The residue was redissolved in 0.15 to 5 mL of ethanol,
Composition (g/100 g): crude protein, not 12; calcium, not
and Interactions
JUDY D. RIBAYA-MERCADO, JAMES G. FOX, * WILLIAM D. ROSENBLAD, * MICHAEL C. BLANCO* AND ROBERT M. RUSSELL U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts Uniuersity, Boston, MA 02111, and *Diuision of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139 amount of intact ß-carotene(-15-25%) is also ab sorbed into the lymph (Blomstrand and Werner 1967, Goodman et al. 1966). In most other species (i.e., rats, pigs, chickens, guinea pigs, rabbits), very little or no ß-caroteneis absorbed intact; it is largely converted to vitamin A before absorption (Bondi and Sklan 1984, Ganguly et al. 1953, Thompson et al. 1950). Horses and certain breeds of cattle are able to absorb intact ßcarotene (Bondi and Sklan 1984). However, they are not convenient laboratory animal models. We previously reported that the ferret is a suitable animal model because ferrets have the capacity to absorb intact dietary ß-carotene(Ribaya-Mercado et al. 1989). We also reported that it can store absorbed ß-carotenein liver, and to a much lesser extent, in adipose tissue (Ribaya-Mercado et al. 1989). Because of their convenient size, domesticated ferrets can be easily handled and maintained in the laboratory setting. Thus, the ferret seems to be a promising model for future studies of ß-caroteneabsorption and metabolism. In this study, we report serum concen trations of carotenoids (ß-carotene, a-carotene, lycopene) and retinoids (retinol, total and individual retinyl esters) achieved by feeding ferrets diets supple mented with ß-carotenefor 3 wk. Concentrations of ß-carotene,retinol and total retinyl esters in other selected tissues are also reported.
ABSTRACT The concentrations of ß-carotene,retinol and retinyl esters in serum and selected tissues of ferrets fed diets supplemented with ß-carotene(80 ng/g wet diet) for 3 wk were determined. The initial concen tration of serum ß-carotenewas 0.011 ±0.006 nmol/L (mean ±SEM); at the end of the experimental period it was 5.75 ±1.60 umol/L. No significant differences in serum retino! and total retinyl esters were observed be tween ß-carotene-fedand control ferrets that had been fed an unsupplemented diet. The predominant retinyl esters in serum were retinyl stéarate(53%) and retinyl palmitate (35%). Of the tissues analyzed after licarotene feeding, the liver contained the highest concen tration of ß-carotene (78.8 ±18.8 nmol/g). Other tissues that contained ß-carotenein amounts ranging from 17 to 20 nmol/g were adrenals, small intestine, stomach and colon; lesser amounts (6.9 nmol/g) were found in kidneys. Amounts ranging from 1.2 to 2.3 nmol/g were found in muscle, bladder, adipose tissue, lungs and skin; only 0.37 and 0.34 nmol/g were present in brain and eyes, respectively. Thus, like humans, ferrets have the capacity to absorb intact ß-caroteneand to store this compound in tissues, especially the liver. However, compared with humans, ferrets have elevated concentrations of retinyl esters in serum, liver and other tissues. J. Nutr. 122: 1898-1903, 1992. INDEXING KEY WORDS:
•ß-caroiene•vitamin A •retinyl esters •retinol •ferrets
Among the dietary carotenoids all-trans-ß-carotene has the highest provitamin A activity (Goodman 1984). This compound is converted to vitamin A in the intestinal mucosa (Goodman and Huang 1965). The efficiency of conversion varies among different species. In humans most dietary ß-caroteneis con verted to vitamin A in the gut; however, a substantial
'Supported in part by U.S. Department of Agriculture contract no. 53-3KO6-5-10. The contents of this publication do not neces sarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of commercial products imply en dorsement by the United States government. 2An abstract describing preliminary data has been published [Ribaya-Mercado, J. D., Fox, J. G., Rosenblad, W. & Russell, R. M. (1990) Dietary ß-carotenesupplementation and tissue levels of ßcarotene, retinol, and retinyl esters in ferrets. J. Am. Coll. Nutr. 9: 526 (abs. 20)].
0022-3166/92 $3.00 ©1992 American Institute of Nutrition. Received 12 March 1992. Accepted 12 May 1992.
1898
Downloaded from https://academic.oup.com/jn/article-abstract/122/9/1898/4769470 by Tulane University Library, Serials Acquisitions Dept. user on 20 January 2019
ß-Carotene, Retinol and Retinyl Ester Concentrations in Serum and Selected Tissues of Ferrets Fed ß-Carotene1'2
ß-CAROTENE-FED FERRETS
MATERIALS AMD METHODS and diets
Male ferrets (Marshall Farms, North Rose, NY), 8 mo old and weighing 1234 ±98 g (mean ±SEM),were used in these experiments. They were housed individ ually in suspended stainless steel cages and were given free access to a standard diet and water. For 3 wk, the experimental ferrets were fed a basal diet consisting of dry cat food (Purina Cat Chow,3 Ralston Purina, St. Louis, MO) moistened with water (1.5 mL/ g dry food), to which was added 80 ug of ß-carotene(as 824 ug of ß-carotenebeadlets, Hoffmann-LaRoche, Nutley, NJ) per gram of wet diet. Control ferrets were fed the moistened cat food only. Diets were prepared weekly and refrigerated. Using extraction and analytical procedures described below for tissues, we analyzed the moistened basal diet and found it to contain 0.33 ug ß-caroteneand 17.3 ug retinyl esters/g wet wt. The diet also contained 0.005 ug a-carotene and 0.016 ug lycopene/g wet wt. Because the average daily food intake during the 3-wk period was 193 ±8 g, the average intakes of ß-caroteneand retinyl esters from the basal diet were 0.064 ±0.003 and 3.3 ±0.14mg/d, respectively; the daily intakes of a-carotene and lycopene were about 0.97 and 3.1 ug, respectively; and the daily intake of supplemental ß-carotenewas 15.44 ±0.64 mg. Thus the total ß-caroteneintake of the experimental ferrets was -15.5 mg/d. Blood was ob tained from jugular veins for baseline serum measure ments. After 3 wk, the animals were killed with carbon dioxide. Blood was obtained by cardiac puncture, and the following tissues were quickly excised: liver (midsection of the left lateral lobe), adrenal glands (entire right and left), small intestine (mid-jejunum), stomach (greater curvature from cardia to pylorus), colon (descending), kidney (right kidney), muscle (section of rectus femorus), bladder (whole), skin (ventral ab domen), adipose tissue (subcutaneous, from ventral abdomen), lung (right medial lobe), brain (right hemi sphere) and eye (entire right eye). The serum and tissues were frozen at -80°Cuntil analyzed for carotenoids and retinoids. All animal procedures were re viewed and approved by the Animal Care and Use Committee at Tufts University and the Committee on Animal Care at the Massachusetts Institute of Technology. Analytical methods For the extraction of serum and tissue carotenoids and retinoids, procedures we have already described (Ribaya-Mercado et al. 1989) were used with some
modifications. The reverse-phase gradient HPLC pro cedures previously described (Ribaya-Mercado et al. 1989) were used for the detection and measurement of these compounds. Serum extraction. A 200-uL aliquot of serum was treated with 2 mL of CHC13:CH3OH (2:1, v/v). In ternal standards (150 uL) consisting of Y-carotene (Hoffmann-LaRoche) and retinyl acetate (Sigma Chemical, St. Louis, MO) were added, followed by 500 uL of 8.5 g/L saline. The tube was vortexed and centrifuged at 684 x g at 4°Cfor 10 min. The CHC13 and aqueous layers were separated, and the aqueous layer was extracted with 3 mL of hexane. The tube was vortexed and centrifuged at 684 x g at 4°Cfor 10 min, and the hexane layer was combined with the CHC13 extract. The combined extracts were dried under nitrogen in a water bath and the residue was redissolved in 150 uL of ethanol. The tube was vor texed and centrifuged at 684 x g at 4°Cfor 2 min. A 50-uL aliquot was injected onto the HPLC column. Tissue extraction. The procedures for tissue ex traction were similar to those we have described for liver and adipose tissue (Ribaya-Mercado et al. 1989) with some modifications. The tissues were weighed (about 0.05 g for liver; 0.1 g for kidney and adrenal; 0.25 g for eye; 0.3 g for lung and whole bladder; 0.5 g for small intestinal mucosa, colon, stomach, skin, subcutaneous adipose tissue, brain and muscle), minced and homogenized in 5 mL of CHC13:CH3OH (2:1, v/v) using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). The Polytron probe was rinsed with 5 mL of CHC13:CH3OH, and the wash was added to the homogenate. Internal standards (0.3 to 0.9 mL) of ycarotene and retinyl acetate were added, followed by 2 mL of 8.5 g/L saline. The mixture was vortexed and centrifuged at 684 x g at 4°Cfor 10 min. The CHC13 layer was separated, and the aqueous layer was extracted with 3-5 mL of hexane. After vortexing and centrifuging the tube at 684 x g at 4°Cfor 10 min, the hexane layer was combined with the CHC13 extract and the combined extracts dried under nitrogen in a water bath. The residue was redissolved in 0.15 to 5 mL of ethanol,
Composition (g/100 g): crude protein, not 12; calcium, not
