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Effect of nonfat milk on the utilization of carotene and vitamin A
Effect of Nonfat Milk on the Utilization Carotene and Vitamin Al
of
M. G. Vavich, J. W. Stull, N. Raica and A. R. Kexnmerer From the Departments of Agricultural Biochemistry College of Agriculture, University of Arizona,
and Dairy Husbandry, Tucson, Arizona
Received October 22, 1954
Studies on the utilization of vitamin A and carotene have assumed considerable importance in recent years. Vavich and Kemmerer (1) have reported that the carot,ene in cantaloupe is considerably more available to rats than carotene in alfalfa. Bentley and Morgan (2) have found that the nature of the diet accompanying ingestion of vit.amin A or carotene affects t,he efficiency of their utilization. Sobel el al. (3) have observed that vitamin A is more efficiently utilized when dispersed in aqueous media than in oily solutions. The findings of Baumann et al. (4), Deuel et al. (5), Moore et al. (6), and James and El Gindi (7) have indicated that protein influences carotene and vitamin A utilization. In the experiments reported in this paper the effect of nonfat milk upon the utilization of vitamin A and carot.enewas studied. EXPERIMENTAL Cnrotene was purified by recrystallizing a mixture of 900%j3- and 10% a-carotene from benzene with methanol. This purified carotene was first dissolved in a minimum amount of chloroform and then diluted to volume with cottonseed oil* containing 1% cr-tocopherol. Vitamin A acetate-in-oil solutions were prepared similarly except that the vitamin A was not recrystallized since E:Fm at 325 mp was 1495. This indicated a satisfnctory product. The oil solutions of carotene or vitamin A were diluted with an equal weight of polyoxyethylene sorbitan monooleate (Tween 80). Then 40 g. of the oil-Tween * .4rizona Agricultural Experiment Station Technical Paper No. 339. This investigation forms part of the Western Regional Research Project on the nutritional status of selected population groups which was financed in part from funds approprinted under the Research amd Marketing Act of 1946. * Wesson oil. 310
UTILIZATION
OF
CAROTENE
AND
VITAMIN
A
311
solution was partially emulsified by stirring with 2 1. of nonfat milk or distilled water preheated to 100°F. This emulsion was transferred immediately to a small gravity-fed supply tank of a homogenizer3 and recirculated through the machine at single-stage pressure of 3000 lb./sq. in. for 1 min. The outlet line was fitted so as to minimize splash and thus avoid incorporation of air. Reconstituted nonfat milk emulsion was prepared by adding powdered nonfat milk to the water emulsion of carotene and stirring by hand until the powder was dissolved. Albino rats of the Sprague-Dawley strain were used as experimental animals. The basal diet was the U.S.P. XIV vitamin A test diet to which irradiated yeast was added to provide vitamin D. When the rats were 1214 days old, the regular breeding stock diet was replaced with the basal diet. At 28 days of age the rats were caged individually and weighed every 3 days thereafter. When the animals showed no further weight gain they were equalized in groups according to litter, sex, and weight. Supplements of carotene or vitamin A emulsions were given daily to each rat in 5-ml. port,ions. Water was withheld until the supplement was consumed; thereafter free access to water was allowed. An equal amount of U.S.P. test diet was fed to each rat. The amount was determined from the smallest amount consumed by any rat in 24 hr. This varied from 8 to 10 g. per day and was adjusted on a daily basis for the entire feeding period of 13 days. The carotene and vitamin A supplements were fed for 12 consecutive days; the rats were killed on the 14th day, and t.heir livers were removed immediately and blotted with filter paper to remove adhering blood. Each liver was then weighed and stored individually in 50 ml. of 12% alcoholic KOH at 40°F. until analyzed by the method of Vavich and Kemmerer (8). The vitamin A content of the kidneys was not determined since previous work (8) had shown that the amount of vitamin A in the kidneys was so small in comparison with the amount in the liver that it did not affect results of experiments on utilization. Hooth (9) also noted this fact in the statement of “The sex difference in kidney storage [of vitamin A] cannot account for differential liver storage be. cause t,he kidney difference is too small and the liver difference ant,icipates the kidney difference.” DISCUSSION
The dat’a in Table I show that carotene emulsified in either fresh, fluid nonfat milk or reconstituted nonfat milk was utilized more effectively by rats for hepatic storage of vitamin A than carotene emulsified in water. The differences between the milk and water emulsions were not significant statistically when 32 pg. of carotene was fed daily but mere highly significant when the higher levels of 56 or 60 pg. of carotene were fed. The data in Table I also show that the deposition of vitamin A in the liver is not increased by milk when vitamin A acetate is fed, Actually, at the 52qg. level of vitamin A acetate the utilization of vitamin A was significantly decreased by reconstituted nonfat milk. 3 Manton-Gaulin,
Everett,
Mass
312
VAVICH,
STULL,
RAICA
AND
KEMMERER
TABLE I Influence of Nonfat Milk and Vitamin Blz on Vitamin A Storage in Livers of Rate Fed Emu&fled Carotene or Vitamin A
=
wt. of rats at start
Emulsion
-
-
Gain in wt. --
Pg.ldaY
c07otme
1\To. 0 f rats __
Vit.
A in liver
-_
g.
g.
144 146 150
23 16 21
10 10 10
51.5 f 59.1 f 52.9 f
4.4 9.oa
~g.lliPer
Water Nonfat milk Reconstituted
nonfat milk
32 32 32
Water Reconstituted
nonfat milk
60 60
153 156
21 22
10 10
67.3 f 82.8 f
5.0 2.6"
56 56 56 56 56
109 109 113 110 111
35 34 35 41 40
12 12 12 12 12
41.2 37.4 44.4 63.5 65.1
3.4 4.0" 5.1a 4.55 3.86
Water Water Water Nonfat Nonfat
+ 0.05 pg. BIG + 0.60 pg. BIZ milk milk + 0.60 pg. BIZ
--
.Vitamin
Water Nonfat milk Reconstituted Water Nonfat milk Reconstituted
f f * f f
3.3Q
A
nonfat milk
30 30 30
155 143 156
13 22 16
10 10 10
120.1 i 132.0 i 105.8 f
9.9 2.8" 10.0"
nonfat milk
52 52 52
151 156 153
23 23 22
10 10 10
156.6 f 166.3 f 123.2 f
10.8 6.5" 7.4b
-
-
= Difference from water; not significant. b Difference from water; significant.
The findings that milk had a different effect upon high than upon low dosages of carotene and a different effect upon carotene than upon vitamin A can be rationalized from previous reports. Vavich and Kemmerer (10) have reported that xanthophylls inhibited carotene utilization by chicks when 130 pg. of carotene was fed daily but not when 65 pg. was fed daily. The same kind of effect was observed for vitamin A by Ellingson et al. (11) who found that the liver storages of vitamin A by rats were equal for water emulsions and oil solutions at dosages of 10, 20, and 40 U.S.P. units, but at higher dosages water emulsions gave a higher storage. High and Wilson (12) found that vitamin BI, increased carotene utilization by rats but that it did not affect vitamin A
UTILIZATION
OF
CAROTENE
AND
VITAMIN
A
313
utilization. Johnson and Baumann (13) have shown that desiccated thyroid or thiourea produced an effect upon carotene utilization that was the direct opposite of that produced upon vitamin A utilization. In view of the experiments of High and Wilson (12) it appeared that the favorable effect of milk on carotene utilization possibly might be explained on the basis of its vitamin B 12 content which, according to Collins ct al. (14), varies from 3 to 12 pg./l. of milk. To evaluate the effect of vitamin Blz on carotene utilization, enough vitamin Blz was added t’o both the daily feedings of the milk and water emulsions of carotene to give a daily supplement of 0.05 and 0.6 pg. of B~z . The 0.05~pg. supplement was approximately the amount of Blz that is furnished by 5 ml. of milk. Neither level of vitamin Blz increased the utilization of carotene for deposition of vitamin A in the liver (Table I). The experiments showing that reconstituted nonfat milk exerts the same favorable effect upon carotene utilization as does fluid skim milk (Table I) indicate that possible differences in particle size in the milk and water emulsions do not account for the difference in liver vitamin A storage. Dissolving powdered nonfat milk directly into water emulsions of carotene would not appreciably change t’he size of the particles in the emulsion. The favorable effect of milk found in these experiments is not very likely caused by the increased amount of dietary casein supplied by the milk as might be indicated from the work of ,James and El Gindi (7) who rcport,ed t,hat addition of casein to a basal diet gave bet’ter carotene utjilization than addit’ion of other prot’eins. The basal diet in the study reportSed here contained 18% casein, and the amount of casein in the milk in which the carotene was administered is very small in cornparison with that afforded by the basal diet. The factors in milk Ohat cause the increased liver storage of vitamin A from carotene cannot be determined from these experiments. However, these experiments do show that some property of milk exerts a favorahle effect, on carotene utilization. SUMMARY
Rats fed 60 pg. daily of carotene significantly more vitamin A in amount of carotene homogenized fed daily, there was no difference the water and milk homogenates.
homogenized in nonfat milk deposited their livers than rats fed the same in water. When 32 pg. carotene was in liver storage of vitamin A between When vitamin A per se was fed, there
314
VAVICH,
STULL,
RAICA
AND KEMMERER
was no significant difference in vitamin A storage between the milk and water homogenates except for reconstituted nonfat milk which significantly lowered liver storage of vitamin A when 52 pg. of vitamin A was fed. REFERENCES 1. VAVICH, M. G., ASD KEMYERER, A. R., Food Research 16, 494 (1950). 2. BENTLEY, L. S., AND MOROAN, A. F., J. ~Vufrition 31, 333 (1946). 3. SOBEL, A. E., SHERMAN, M., LICHTBLAU, J., SKOW, S., AND KRAMER, B., J. Nutrition 36, 225 (1948). 4. DAUMANN, C. A., FOSTER, E. G., AND MOORE, P. R., J. Biol. Chem. 142. 597 (1942). 5. DEUEL, H. J., JR., HRUBETZ, M. C., JOHNSTON, C. II., ROLLX~N, H. S., AXD GEIGER, E., J. Nutrition 31, 187 (1946). 6. MOORE, T., SHAR~AX, I. M., AND WARD, It. J., Biochem. J. 62, xii (1952). 7. JA~XES, W. H., AND EL GINDI, I. M., J. Nutrilim 61, 97 (1953). 8. VAVICH, M. G., AND KEINERER, A. R., J. Nutrition 40.605 (1950). 9. BOOTH, V. H., J. Nutrition 48, 13 (1952). 10. Vawct1, M. G., AND KEMMERER, A. It., Arch. Biochem. 26,295 (1950). 11. ELLINGSON, R. C., MCDONALD, F. G., hlASSExGALE, 0. K., AND Cox, W. H., Pediafrics 8, 107 (1951). 12. HIGH, E. G., ASD WILSON, S. S., J. Nutrition 60.203 (1953). 13. JOHXSOS, R. M., AND BAUMANX, C. A., -1. Riol. Chem. 171,513 (1947). 14. COLLINS, R. A., HARPER, A. E., SCHREIBER, M., AND E~LVEHJEM, C. A., J. Nutrilion 43, 313 (1951).
of
M. G. Vavich, J. W. Stull, N. Raica and A. R. Kexnmerer From the Departments of Agricultural Biochemistry College of Agriculture, University of Arizona,
and Dairy Husbandry, Tucson, Arizona
Received October 22, 1954
Studies on the utilization of vitamin A and carotene have assumed considerable importance in recent years. Vavich and Kemmerer (1) have reported that the carot,ene in cantaloupe is considerably more available to rats than carotene in alfalfa. Bentley and Morgan (2) have found that the nature of the diet accompanying ingestion of vit.amin A or carotene affects t,he efficiency of their utilization. Sobel el al. (3) have observed that vitamin A is more efficiently utilized when dispersed in aqueous media than in oily solutions. The findings of Baumann et al. (4), Deuel et al. (5), Moore et al. (6), and James and El Gindi (7) have indicated that protein influences carotene and vitamin A utilization. In the experiments reported in this paper the effect of nonfat milk upon the utilization of vitamin A and carot.enewas studied. EXPERIMENTAL Cnrotene was purified by recrystallizing a mixture of 900%j3- and 10% a-carotene from benzene with methanol. This purified carotene was first dissolved in a minimum amount of chloroform and then diluted to volume with cottonseed oil* containing 1% cr-tocopherol. Vitamin A acetate-in-oil solutions were prepared similarly except that the vitamin A was not recrystallized since E:Fm at 325 mp was 1495. This indicated a satisfnctory product. The oil solutions of carotene or vitamin A were diluted with an equal weight of polyoxyethylene sorbitan monooleate (Tween 80). Then 40 g. of the oil-Tween * .4rizona Agricultural Experiment Station Technical Paper No. 339. This investigation forms part of the Western Regional Research Project on the nutritional status of selected population groups which was financed in part from funds approprinted under the Research amd Marketing Act of 1946. * Wesson oil. 310
UTILIZATION
OF
CAROTENE
AND
VITAMIN
A
311
solution was partially emulsified by stirring with 2 1. of nonfat milk or distilled water preheated to 100°F. This emulsion was transferred immediately to a small gravity-fed supply tank of a homogenizer3 and recirculated through the machine at single-stage pressure of 3000 lb./sq. in. for 1 min. The outlet line was fitted so as to minimize splash and thus avoid incorporation of air. Reconstituted nonfat milk emulsion was prepared by adding powdered nonfat milk to the water emulsion of carotene and stirring by hand until the powder was dissolved. Albino rats of the Sprague-Dawley strain were used as experimental animals. The basal diet was the U.S.P. XIV vitamin A test diet to which irradiated yeast was added to provide vitamin D. When the rats were 1214 days old, the regular breeding stock diet was replaced with the basal diet. At 28 days of age the rats were caged individually and weighed every 3 days thereafter. When the animals showed no further weight gain they were equalized in groups according to litter, sex, and weight. Supplements of carotene or vitamin A emulsions were given daily to each rat in 5-ml. port,ions. Water was withheld until the supplement was consumed; thereafter free access to water was allowed. An equal amount of U.S.P. test diet was fed to each rat. The amount was determined from the smallest amount consumed by any rat in 24 hr. This varied from 8 to 10 g. per day and was adjusted on a daily basis for the entire feeding period of 13 days. The carotene and vitamin A supplements were fed for 12 consecutive days; the rats were killed on the 14th day, and t.heir livers were removed immediately and blotted with filter paper to remove adhering blood. Each liver was then weighed and stored individually in 50 ml. of 12% alcoholic KOH at 40°F. until analyzed by the method of Vavich and Kemmerer (8). The vitamin A content of the kidneys was not determined since previous work (8) had shown that the amount of vitamin A in the kidneys was so small in comparison with the amount in the liver that it did not affect results of experiments on utilization. Hooth (9) also noted this fact in the statement of “The sex difference in kidney storage [of vitamin A] cannot account for differential liver storage be. cause t,he kidney difference is too small and the liver difference ant,icipates the kidney difference.” DISCUSSION
The dat’a in Table I show that carotene emulsified in either fresh, fluid nonfat milk or reconstituted nonfat milk was utilized more effectively by rats for hepatic storage of vitamin A than carotene emulsified in water. The differences between the milk and water emulsions were not significant statistically when 32 pg. of carotene was fed daily but mere highly significant when the higher levels of 56 or 60 pg. of carotene were fed. The data in Table I also show that the deposition of vitamin A in the liver is not increased by milk when vitamin A acetate is fed, Actually, at the 52qg. level of vitamin A acetate the utilization of vitamin A was significantly decreased by reconstituted nonfat milk. 3 Manton-Gaulin,
Everett,
Mass
312
VAVICH,
STULL,
RAICA
AND
KEMMERER
TABLE I Influence of Nonfat Milk and Vitamin Blz on Vitamin A Storage in Livers of Rate Fed Emu&fled Carotene or Vitamin A
=
wt. of rats at start
Emulsion
-
-
Gain in wt. --
Pg.ldaY
c07otme
1\To. 0 f rats __
Vit.
A in liver
-_
g.
g.
144 146 150
23 16 21
10 10 10
51.5 f 59.1 f 52.9 f
4.4 9.oa
~g.lliPer
Water Nonfat milk Reconstituted
nonfat milk
32 32 32
Water Reconstituted
nonfat milk
60 60
153 156
21 22
10 10
67.3 f 82.8 f
5.0 2.6"
56 56 56 56 56
109 109 113 110 111
35 34 35 41 40
12 12 12 12 12
41.2 37.4 44.4 63.5 65.1
3.4 4.0" 5.1a 4.55 3.86
Water Water Water Nonfat Nonfat
+ 0.05 pg. BIG + 0.60 pg. BIZ milk milk + 0.60 pg. BIZ
--
.Vitamin
Water Nonfat milk Reconstituted Water Nonfat milk Reconstituted
f f * f f
3.3Q
A
nonfat milk
30 30 30
155 143 156
13 22 16
10 10 10
120.1 i 132.0 i 105.8 f
9.9 2.8" 10.0"
nonfat milk
52 52 52
151 156 153
23 23 22
10 10 10
156.6 f 166.3 f 123.2 f
10.8 6.5" 7.4b
-
-
= Difference from water; not significant. b Difference from water; significant.
The findings that milk had a different effect upon high than upon low dosages of carotene and a different effect upon carotene than upon vitamin A can be rationalized from previous reports. Vavich and Kemmerer (10) have reported that xanthophylls inhibited carotene utilization by chicks when 130 pg. of carotene was fed daily but not when 65 pg. was fed daily. The same kind of effect was observed for vitamin A by Ellingson et al. (11) who found that the liver storages of vitamin A by rats were equal for water emulsions and oil solutions at dosages of 10, 20, and 40 U.S.P. units, but at higher dosages water emulsions gave a higher storage. High and Wilson (12) found that vitamin BI, increased carotene utilization by rats but that it did not affect vitamin A
UTILIZATION
OF
CAROTENE
AND
VITAMIN
A
313
utilization. Johnson and Baumann (13) have shown that desiccated thyroid or thiourea produced an effect upon carotene utilization that was the direct opposite of that produced upon vitamin A utilization. In view of the experiments of High and Wilson (12) it appeared that the favorable effect of milk on carotene utilization possibly might be explained on the basis of its vitamin B 12 content which, according to Collins ct al. (14), varies from 3 to 12 pg./l. of milk. To evaluate the effect of vitamin Blz on carotene utilization, enough vitamin Blz was added t’o both the daily feedings of the milk and water emulsions of carotene to give a daily supplement of 0.05 and 0.6 pg. of B~z . The 0.05~pg. supplement was approximately the amount of Blz that is furnished by 5 ml. of milk. Neither level of vitamin Blz increased the utilization of carotene for deposition of vitamin A in the liver (Table I). The experiments showing that reconstituted nonfat milk exerts the same favorable effect upon carotene utilization as does fluid skim milk (Table I) indicate that possible differences in particle size in the milk and water emulsions do not account for the difference in liver vitamin A storage. Dissolving powdered nonfat milk directly into water emulsions of carotene would not appreciably change t’he size of the particles in the emulsion. The favorable effect of milk found in these experiments is not very likely caused by the increased amount of dietary casein supplied by the milk as might be indicated from the work of ,James and El Gindi (7) who rcport,ed t,hat addition of casein to a basal diet gave bet’ter carotene utjilization than addit’ion of other prot’eins. The basal diet in the study reportSed here contained 18% casein, and the amount of casein in the milk in which the carotene was administered is very small in cornparison with that afforded by the basal diet. The factors in milk Ohat cause the increased liver storage of vitamin A from carotene cannot be determined from these experiments. However, these experiments do show that some property of milk exerts a favorahle effect, on carotene utilization. SUMMARY
Rats fed 60 pg. daily of carotene significantly more vitamin A in amount of carotene homogenized fed daily, there was no difference the water and milk homogenates.
homogenized in nonfat milk deposited their livers than rats fed the same in water. When 32 pg. carotene was in liver storage of vitamin A between When vitamin A per se was fed, there
314
VAVICH,
STULL,
RAICA
AND KEMMERER
was no significant difference in vitamin A storage between the milk and water homogenates except for reconstituted nonfat milk which significantly lowered liver storage of vitamin A when 52 pg. of vitamin A was fed. REFERENCES 1. VAVICH, M. G., ASD KEMYERER, A. R., Food Research 16, 494 (1950). 2. BENTLEY, L. S., AND MOROAN, A. F., J. ~Vufrition 31, 333 (1946). 3. SOBEL, A. E., SHERMAN, M., LICHTBLAU, J., SKOW, S., AND KRAMER, B., J. Nutrition 36, 225 (1948). 4. DAUMANN, C. A., FOSTER, E. G., AND MOORE, P. R., J. Biol. Chem. 142. 597 (1942). 5. DEUEL, H. J., JR., HRUBETZ, M. C., JOHNSTON, C. II., ROLLX~N, H. S., AXD GEIGER, E., J. Nutrition 31, 187 (1946). 6. MOORE, T., SHAR~AX, I. M., AND WARD, It. J., Biochem. J. 62, xii (1952). 7. JA~XES, W. H., AND EL GINDI, I. M., J. Nutrilim 61, 97 (1953). 8. VAVICH, M. G., AND KEINERER, A. R., J. Nutrition 40.605 (1950). 9. BOOTH, V. H., J. Nutrition 48, 13 (1952). 10. Vawct1, M. G., AND KEMMERER, A. It., Arch. Biochem. 26,295 (1950). 11. ELLINGSON, R. C., MCDONALD, F. G., hlASSExGALE, 0. K., AND Cox, W. H., Pediafrics 8, 107 (1951). 12. HIGH, E. G., ASD WILSON, S. S., J. Nutrition 60.203 (1953). 13. JOHXSOS, R. M., AND BAUMANX, C. A., -1. Riol. Chem. 171,513 (1947). 14. COLLINS, R. A., HARPER, A. E., SCHREIBER, M., AND E~LVEHJEM, C. A., J. Nutrilion 43, 313 (1951).