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The effects of several milk components and similar compounds on the utilization of carotene by the rat
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
83,
376-380 (1959)
The Effects of Several Milk Components and Similar Compounds on the Utilization of Carotene by the Rat’,’ N. R&a, From
Jr., M. G. Vavich
and A. R. Kemmerer
the Department of Agricultural Biochemistry, Agriculture, University of Arizona, Tucson, Received
October
College Arizona
of
8, 1958
In a previous communication from this laboratory, Vavich et al. (1) reported that nonfat milk and reconstituted nonfat milk significantly increased carotene utilization by albino rats as measured by the liver vitamin A storage technique. High and Wilson (2) reported that vitamin B~z had a favorable effect on carotene utilization. Vavich et al. (1) and Mayfield and Roehm (3), using different approaches, could not verify High and Wilson’s results. It would seem then that the favorable effect of milk on carotene utilization cannot be explained solely by its vitamin B1z content. Fraps (4) and James and El Gindi (5), among others, have reported increased carotene utilization when casein was included as the principal dietary protein source. Vavich and Kemmerer (6) reported that carotene utilization was not influenced when diet intake was restricted to one-half of the control rat diet intake. In view of the results reported by Vavich et al. (l), which were obtained with a casein diet in which the additional casein contributed by the milk-carotene emulsion was insignificant by comparison, it seemedthat other factors may have been of greater significance. The purpose of this investigation was to determine the influence of the nonfat milk components and similar nonmilk components on carotene utilization. 1 Abstracted from a portion of the dissertation submitted by N. Raica, Jr., in partial fulfillment of the requirements for the degree of Doctor of Philosophy. * Arizona Agricultural Experiment Station Technical Paper No. 494. This investigation forms part of the Western Regional Research Project on the nutritional status of selected population groups which was financed in part by the Western Regional Research Project W-4 from funds appropriated under the Research and Marketing Act of 1946.
376
UTILIZATION
OF CAROTENE
TABLE Znjluence
Supplement
Water Caseina Water Caseina Water Gelatin” Water CaseinGelatin”
of Casein
Sex
M M F F M M M M M
I
or Gelatin on Liver Vitamin pg. of Emulsified Carotene
No. of rats
6 6 6 6 5 5 6 6 6
Initial weight
377
Weight gained
A Storage Daily
Liver weight
9.
8.
9.
84 82 89 89 86 88 87 97 90
59 55 42 36 59 56 52 49 48
7.52 7.54 7.22 6.00 7.71 8.24 8.00 8.25 8.04
Carotene excretedc
in Rats
Fed 60
Total vitamin
Ab
pg./liver
47.9 51.1 44.0
92.1 108.4 85.2 94.9 90.8 56.8 83.3 77.7 40.3
f f i f f f f f f
6.9 8.4 8.2 5.0 8.3 6.0d 5.5 9.3 4.8’
Q 3.57; in emulsified carqtene. b Including standard error. c Per cent of ingested carotene. d Difference from water significant: P < 0.05. e Difference from water highly significant: P < 0.01.
EXPERIMENTAL Emulsified carotene solutions, liver analyses, and experimental rats were prepared and treated as described by Vavich et al. (1). Casein-carotene emulsion was prepared by soaking go-mesh Vitamin-Test casein in distilled water prior to the addition of the carotene and subsequent emulsification. Gelatin-carotene emulsion was prepared by adding cold carotene emulsion to a given amount of gelatin4 and, after about 1 hr., heating until the gelatin just dissolved. The caseinand gelatin-carotene emulsion contained 3.5yo protein. Sugar-carotene emulsions were prepared by adding 5% of the given sugar to cold carotene emulsion. All emulsions were stored in the refrigerator and were analyzed for carotene at the start, the midpoint, and the end of the experiment. The analysis of the carotene emulsions was as follows: 5 ml. or 5 g. was hydrolyzed in 25 ml. of 12y0 alcoholic KOH, extracted with petroleum ether, dried with anhydrous Na2S04 , diluted to volume, and read at 440 rnp. Liquid emulsion supplements were pipetted and gelatin emulsion supplements were weighed daily into small cups. Five ml. or 5 g. contained the desired daily carotene supplement and was withheld until all of the carotene supplement was consumed. In general, the time required for consumption of the supplements was 6-12 hr.
RESULTS
AND DISCUSSION
In Table I it may be seenthat casein had no significant effect on carotene utilization. Gelatin on the other hand significantly decreased carotene utilization in each trial. In Table II data show that when equal amounts 3 Nutritional 4 Swift and
Biochemicals Corporation, Company, Chicago, Illinois.
Cleveland,
Ohio.
378
Injluence
RAICA,
Storage
Water Gas-GeP Cas-Lactb Lactalbuminc Water Cas-Gel” Cas-Lactb Lactalbuminc
Sex
AND
KEMMERER
TABLE II Casein-Lactalbumin and Lactalbumin
of Casein-Gelatin,
Supplement
VAVICH
in Rats
No. of rats
M M M M F F F F
5 5 5 5 5 5 5 5
Fed
Initial weight
60 pg.
of Carotene
Weight gained
on Liver
Vitamin
A
Daily
Liver weight
9.
9.
9.
73 71 66 66 67 70 69 69
49 42 56 43 45 36 42 34
7.10 6.50 6.13 6.00 5.66 6.01 5.81 5.45
Total vitamin Ad pg./liver
90.7 33.7 36.2 45.9 109.0 32.6 44.0 48.8
f f f f f f f f
9.0 4.6” 3.66 5.3e 8.8 4.7. 2.78 6.1”
n 1.75% casein and 1.75yo gelatin in emulsified carotene. b 3.5rr/, casein and 0.5% lactalbumin in emulsified carotene. c 0.5ojc lactalbumin in emulsified carotene. d Including standard error. e Difference from water highly significant: P < 0.01.
of casein and gelatin were added to the carotene supplement a significant decrease in carotene utilization was also obtained. Explanation of these results cannot be made on the basis of the report by Ganguly et al. (7) that the absorption of vitamin A and carotenoids may be dependent upon the protein carriers present in the circulating blood stream. James and El Gindi (5) used this assumption to explain the favorable results obtained on carotene utilization with casein and the poor results obtained with other proteins. Since casein was supplied as the primary source of protein in the diet, it seemsunlikely that the small amount of additional casein in the carotene supplement should contribute greatly toward this end. On the other hand, it also seemsvery unlikely that the small amount of gelatin in the supplements should greatly alter the protein carriers. It may be that the digestion products of gelatin in “intimate” association with the carotene or formed vitamin A prevent the proper adsorption on the systemic carriers. That carotene absorption from the intestine is not impaired by gelatin is shown by the data in Table I. If anything, casein-supplemented carotene caused a higher excretion of carotene in the fecesthan gelatin-supplemented carotene. When 0.5 % lactalbumin5 or 3.5 % casein-lactalbumin was added to the carotene emulsion, it also significantly decreased carotene utilization, as shown in Table II. 5 Nutritional
Biochemicals
Corporation,
Cleveland,
Ohio.
UTILIZATION
OF
379
CAROTENE
In an attempt to determine the influence of the amino acid compositions of casein and of gelatin on carotene utilization, experiments were performed in which a partial equalization of the amino acids was made. When casein was supplemented with glycine and hydroxyproline to make it more like gelatin, there was no significant difference in carotene utilization between plain casein and amino acid-supplemented casein. A further experiment was tried in which commercial Vitamin-Test casein was compared with freshly extracted casein as prepared by Dunn (8). No differences in carotene utilization were found between the two casein preparations. When certain sugars were added to the carotene emulsion at a concentration of 5% (Table III), it was found that lactose, fructose, and galactose had no significant effect on carotene utilization, while glucose and raffinose significantly decreased carotene utilization. The decreased utilization caused by raffinose may be due to the nonabsorption of the sugar by rats (9). TABLE Injluence
of Certain
Sugars
on Liver
III
Vitamin
A Storage
in Rats Fed Emulsified
Carotene
a? $
Supplement”
$
Total
vitamin
Ab
2 .-
9.
9.
9.
rg.ldw
pg./liver
Water Lactose
M M
6 6
76 80
55 52
6.93
7.64
60 60
83.1 82.2
f f
5.3 5.0
Water Lactose
F F
6 6
90 90
48 44
8.17 7.93
62 62
55.4 48.0
f f
4.6 4.5
Water Fructose Galactose Glucose Raffinose
M M M M M
6 6 6 6 6
90 84 89 88 87
59
51 50 47 41
8.35 7.56 7.56 7.52 6.76
55 55 55 55 55
54.5 51.8 57.2 40.0 32.6
f f f f f
5.3 3.0 5.0 2.5c 4.5d
Water Fructose Galactose Glucose Raffinose
F F F F F
6 6 6 6 6
92 89 80 80 81
30 35 29 31 29
6.09 5.90
55 55 55 55 55
76.8 59.2 64.1 56.8 53.0
f f f f f
3.5 7.3 3.8 4.Bc 2.3d
Q Sugars: b Including c Difference d Difference
5.20 5.20 5.40
5% in emulsified carotene. standard error. from water significant: P < 0.05. from water highly significant: P < 0.01
380
RAICA, VAVICH AND KEMMERER
A possible explanation as to why glucose should differ in its effect from fructose, galactose, and lactose may be found in work reported by Cori and Cori (10) and Goldberg et al. (11). They reported that glycogen synthesized from fructose, in viva, was greater than from other monosaccharides and more stable than that synthesized from glucose. Further evidence was indicated in the work of Wolf et al. (12) in which it was reported that in the vitamin A-deficient rat there is impaired utilization of S-carbon fragments for glycogen formation. From this, it appears that the mode of utilization of certain sugarsmay influence carotene utilization or the storage of vitamin A in the liver. It is concluded that the favorable effect of nonfat milk on the utilization of carotene cannot be ascribed to any of the individual components tested, SUMMARY
AND CONCLUSIONS
When 3.5 % casein or 5% lactose, fructose, or galactose was added to carotene-water emulsion, no significant effect on carotene utilization was noted in rats fed 60 pg. carotene daily. When 3.5 % gelatin or 0.5 % lactalbumin or 5% glucose or raffinose was added, carotene utilization was significantly decreased. The effect of gelatin could not be explained by increased excretion of carotene. REFERENCES 1. VAVICH, M. G., STULL, J. W., RAICA, N., AND KEMMERER, A. R., Arch. Biochem. Biophys. 66, 310-14 (1955). 2. HIGH, E. G., AND WILSON, S. S., J. Nutrition 60, 203-12 (1953). 3. MAYFIELD, H. L., AND ROEHM, R. R., J. Nutrition M),483-93 (1956). 4. FRAPS, G. S., Arch. Biochem. 10, 4859 (1946). 5. JAMES, W. H., AND EL GINDI, I. M., J. Nutrition 61, 97-108 (1953). 6. VAVICH, M. G., AND KEMMERER, A. R., J. Nutrition 40, 605-10 (1950). 7. GANGULY, J., KRIMSKY, N. I., MEHL, J. W., AND DEUEL, H. J., JR., Arch. Biothem. Biophys. 38, 275-82 (1952). 8. DUNN, M. S., Biochem. Preparations 1, 224 (1949). 9. CLARKE, F., SOLKOT, R., AND CORLEY, R. C., J. Biol. Chem. 131, 135-8 (1939). 10. CORI, C. F., AND CORI, G. T., J. Biol. Chem. 76, 755 (1926). 11. GOLDBERG, L., KAHAN, E., AND KAHAN, J., Arch. intern. pharmacodynamie 112, 73-83 (1957). 12. WOLF, G., WAGLE, S. R., AND JOHNSON, B. C., Federation Proc. 16, 272 (1957).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
83,
376-380 (1959)
The Effects of Several Milk Components and Similar Compounds on the Utilization of Carotene by the Rat’,’ N. R&a, From
Jr., M. G. Vavich
and A. R. Kemmerer
the Department of Agricultural Biochemistry, Agriculture, University of Arizona, Tucson, Received
October
College Arizona
of
8, 1958
In a previous communication from this laboratory, Vavich et al. (1) reported that nonfat milk and reconstituted nonfat milk significantly increased carotene utilization by albino rats as measured by the liver vitamin A storage technique. High and Wilson (2) reported that vitamin B~z had a favorable effect on carotene utilization. Vavich et al. (1) and Mayfield and Roehm (3), using different approaches, could not verify High and Wilson’s results. It would seem then that the favorable effect of milk on carotene utilization cannot be explained solely by its vitamin B1z content. Fraps (4) and James and El Gindi (5), among others, have reported increased carotene utilization when casein was included as the principal dietary protein source. Vavich and Kemmerer (6) reported that carotene utilization was not influenced when diet intake was restricted to one-half of the control rat diet intake. In view of the results reported by Vavich et al. (l), which were obtained with a casein diet in which the additional casein contributed by the milk-carotene emulsion was insignificant by comparison, it seemedthat other factors may have been of greater significance. The purpose of this investigation was to determine the influence of the nonfat milk components and similar nonmilk components on carotene utilization. 1 Abstracted from a portion of the dissertation submitted by N. Raica, Jr., in partial fulfillment of the requirements for the degree of Doctor of Philosophy. * Arizona Agricultural Experiment Station Technical Paper No. 494. This investigation forms part of the Western Regional Research Project on the nutritional status of selected population groups which was financed in part by the Western Regional Research Project W-4 from funds appropriated under the Research and Marketing Act of 1946.
376
UTILIZATION
OF CAROTENE
TABLE Znjluence
Supplement
Water Caseina Water Caseina Water Gelatin” Water CaseinGelatin”
of Casein
Sex
M M F F M M M M M
I
or Gelatin on Liver Vitamin pg. of Emulsified Carotene
No. of rats
6 6 6 6 5 5 6 6 6
Initial weight
377
Weight gained
A Storage Daily
Liver weight
9.
8.
9.
84 82 89 89 86 88 87 97 90
59 55 42 36 59 56 52 49 48
7.52 7.54 7.22 6.00 7.71 8.24 8.00 8.25 8.04
Carotene excretedc
in Rats
Fed 60
Total vitamin
Ab
pg./liver
47.9 51.1 44.0
92.1 108.4 85.2 94.9 90.8 56.8 83.3 77.7 40.3
f f i f f f f f f
6.9 8.4 8.2 5.0 8.3 6.0d 5.5 9.3 4.8’
Q 3.57; in emulsified carqtene. b Including standard error. c Per cent of ingested carotene. d Difference from water significant: P < 0.05. e Difference from water highly significant: P < 0.01.
EXPERIMENTAL Emulsified carotene solutions, liver analyses, and experimental rats were prepared and treated as described by Vavich et al. (1). Casein-carotene emulsion was prepared by soaking go-mesh Vitamin-Test casein in distilled water prior to the addition of the carotene and subsequent emulsification. Gelatin-carotene emulsion was prepared by adding cold carotene emulsion to a given amount of gelatin4 and, after about 1 hr., heating until the gelatin just dissolved. The caseinand gelatin-carotene emulsion contained 3.5yo protein. Sugar-carotene emulsions were prepared by adding 5% of the given sugar to cold carotene emulsion. All emulsions were stored in the refrigerator and were analyzed for carotene at the start, the midpoint, and the end of the experiment. The analysis of the carotene emulsions was as follows: 5 ml. or 5 g. was hydrolyzed in 25 ml. of 12y0 alcoholic KOH, extracted with petroleum ether, dried with anhydrous Na2S04 , diluted to volume, and read at 440 rnp. Liquid emulsion supplements were pipetted and gelatin emulsion supplements were weighed daily into small cups. Five ml. or 5 g. contained the desired daily carotene supplement and was withheld until all of the carotene supplement was consumed. In general, the time required for consumption of the supplements was 6-12 hr.
RESULTS
AND DISCUSSION
In Table I it may be seenthat casein had no significant effect on carotene utilization. Gelatin on the other hand significantly decreased carotene utilization in each trial. In Table II data show that when equal amounts 3 Nutritional 4 Swift and
Biochemicals Corporation, Company, Chicago, Illinois.
Cleveland,
Ohio.
378
Injluence
RAICA,
Storage
Water Gas-GeP Cas-Lactb Lactalbuminc Water Cas-Gel” Cas-Lactb Lactalbuminc
Sex
AND
KEMMERER
TABLE II Casein-Lactalbumin and Lactalbumin
of Casein-Gelatin,
Supplement
VAVICH
in Rats
No. of rats
M M M M F F F F
5 5 5 5 5 5 5 5
Fed
Initial weight
60 pg.
of Carotene
Weight gained
on Liver
Vitamin
A
Daily
Liver weight
9.
9.
9.
73 71 66 66 67 70 69 69
49 42 56 43 45 36 42 34
7.10 6.50 6.13 6.00 5.66 6.01 5.81 5.45
Total vitamin Ad pg./liver
90.7 33.7 36.2 45.9 109.0 32.6 44.0 48.8
f f f f f f f f
9.0 4.6” 3.66 5.3e 8.8 4.7. 2.78 6.1”
n 1.75% casein and 1.75yo gelatin in emulsified carotene. b 3.5rr/, casein and 0.5% lactalbumin in emulsified carotene. c 0.5ojc lactalbumin in emulsified carotene. d Including standard error. e Difference from water highly significant: P < 0.01.
of casein and gelatin were added to the carotene supplement a significant decrease in carotene utilization was also obtained. Explanation of these results cannot be made on the basis of the report by Ganguly et al. (7) that the absorption of vitamin A and carotenoids may be dependent upon the protein carriers present in the circulating blood stream. James and El Gindi (5) used this assumption to explain the favorable results obtained on carotene utilization with casein and the poor results obtained with other proteins. Since casein was supplied as the primary source of protein in the diet, it seemsunlikely that the small amount of additional casein in the carotene supplement should contribute greatly toward this end. On the other hand, it also seemsvery unlikely that the small amount of gelatin in the supplements should greatly alter the protein carriers. It may be that the digestion products of gelatin in “intimate” association with the carotene or formed vitamin A prevent the proper adsorption on the systemic carriers. That carotene absorption from the intestine is not impaired by gelatin is shown by the data in Table I. If anything, casein-supplemented carotene caused a higher excretion of carotene in the fecesthan gelatin-supplemented carotene. When 0.5 % lactalbumin5 or 3.5 % casein-lactalbumin was added to the carotene emulsion, it also significantly decreased carotene utilization, as shown in Table II. 5 Nutritional
Biochemicals
Corporation,
Cleveland,
Ohio.
UTILIZATION
OF
379
CAROTENE
In an attempt to determine the influence of the amino acid compositions of casein and of gelatin on carotene utilization, experiments were performed in which a partial equalization of the amino acids was made. When casein was supplemented with glycine and hydroxyproline to make it more like gelatin, there was no significant difference in carotene utilization between plain casein and amino acid-supplemented casein. A further experiment was tried in which commercial Vitamin-Test casein was compared with freshly extracted casein as prepared by Dunn (8). No differences in carotene utilization were found between the two casein preparations. When certain sugars were added to the carotene emulsion at a concentration of 5% (Table III), it was found that lactose, fructose, and galactose had no significant effect on carotene utilization, while glucose and raffinose significantly decreased carotene utilization. The decreased utilization caused by raffinose may be due to the nonabsorption of the sugar by rats (9). TABLE Injluence
of Certain
Sugars
on Liver
III
Vitamin
A Storage
in Rats Fed Emulsified
Carotene
a? $
Supplement”
$
Total
vitamin
Ab
2 .-
9.
9.
9.
rg.ldw
pg./liver
Water Lactose
M M
6 6
76 80
55 52
6.93
7.64
60 60
83.1 82.2
f f
5.3 5.0
Water Lactose
F F
6 6
90 90
48 44
8.17 7.93
62 62
55.4 48.0
f f
4.6 4.5
Water Fructose Galactose Glucose Raffinose
M M M M M
6 6 6 6 6
90 84 89 88 87
59
51 50 47 41
8.35 7.56 7.56 7.52 6.76
55 55 55 55 55
54.5 51.8 57.2 40.0 32.6
f f f f f
5.3 3.0 5.0 2.5c 4.5d
Water Fructose Galactose Glucose Raffinose
F F F F F
6 6 6 6 6
92 89 80 80 81
30 35 29 31 29
6.09 5.90
55 55 55 55 55
76.8 59.2 64.1 56.8 53.0
f f f f f
3.5 7.3 3.8 4.Bc 2.3d
Q Sugars: b Including c Difference d Difference
5.20 5.20 5.40
5% in emulsified carotene. standard error. from water significant: P < 0.05. from water highly significant: P < 0.01
380
RAICA, VAVICH AND KEMMERER
A possible explanation as to why glucose should differ in its effect from fructose, galactose, and lactose may be found in work reported by Cori and Cori (10) and Goldberg et al. (11). They reported that glycogen synthesized from fructose, in viva, was greater than from other monosaccharides and more stable than that synthesized from glucose. Further evidence was indicated in the work of Wolf et al. (12) in which it was reported that in the vitamin A-deficient rat there is impaired utilization of S-carbon fragments for glycogen formation. From this, it appears that the mode of utilization of certain sugarsmay influence carotene utilization or the storage of vitamin A in the liver. It is concluded that the favorable effect of nonfat milk on the utilization of carotene cannot be ascribed to any of the individual components tested, SUMMARY
AND CONCLUSIONS
When 3.5 % casein or 5% lactose, fructose, or galactose was added to carotene-water emulsion, no significant effect on carotene utilization was noted in rats fed 60 pg. carotene daily. When 3.5 % gelatin or 0.5 % lactalbumin or 5% glucose or raffinose was added, carotene utilization was significantly decreased. The effect of gelatin could not be explained by increased excretion of carotene. REFERENCES 1. VAVICH, M. G., STULL, J. W., RAICA, N., AND KEMMERER, A. R., Arch. Biochem. Biophys. 66, 310-14 (1955). 2. HIGH, E. G., AND WILSON, S. S., J. Nutrition 60, 203-12 (1953). 3. MAYFIELD, H. L., AND ROEHM, R. R., J. Nutrition M),483-93 (1956). 4. FRAPS, G. S., Arch. Biochem. 10, 4859 (1946). 5. JAMES, W. H., AND EL GINDI, I. M., J. Nutrition 61, 97-108 (1953). 6. VAVICH, M. G., AND KEMMERER, A. R., J. Nutrition 40, 605-10 (1950). 7. GANGULY, J., KRIMSKY, N. I., MEHL, J. W., AND DEUEL, H. J., JR., Arch. Biothem. Biophys. 38, 275-82 (1952). 8. DUNN, M. S., Biochem. Preparations 1, 224 (1949). 9. CLARKE, F., SOLKOT, R., AND CORLEY, R. C., J. Biol. Chem. 131, 135-8 (1939). 10. CORI, C. F., AND CORI, G. T., J. Biol. Chem. 76, 755 (1926). 11. GOLDBERG, L., KAHAN, E., AND KAHAN, J., Arch. intern. pharmacodynamie 112, 73-83 (1957). 12. WOLF, G., WAGLE, S. R., AND JOHNSON, B. C., Federation Proc. 16, 272 (1957).