- Email: [email protected]
The Absorption of Vitamin A Natural Esters and of Carotene by Young Male Holstein Calves1
JOURNAL OF DAIRY SCIENCE VOLUME X X X V
APRIL, 1952
NUMBER 4
T H E A B S O R P T I O N OF V I T A M I N A N A T U R A L E S T E R S A N D O F CAROTENE BY YOUNG MALE H O L S T E I N CALVES 1 MAGNAR RONNINGz AND C. B. KNODT Pennsylvania Agricultural Experiment Station, State College The importance of vitamin A and its precursors in the rations of d a i r y calves has been well established. On the basis of the research published, however, information is needed relative to the site of absorption of these compounds in obtaining an understanding of the mechanisms involved in their utilization. P o p p e r and Volk (10) determined that absorption of vitamin A in rats was most rapid in the u p p e r part of the small intestine. Barrick et al. (3) indicated that the small intestine was most important in the absorption of vitamin A by sheep. Eden and Sellers (5) demonstrated that vitamin A was absorbed primarily in the duodenal area of the small intestine in sheep and in rats. The absorption of carotene and its subsequent conversion to vitamin A is not well understood and there appears to be considerable species variation in the mechanism of utilization of carotene (1, 3, 4, 6, 9). I t was the purpose of this experiment to obtain information concerning the site of absorption of vitamin A n a t u r a l esters and carotene by y o u n g dairy calves. EXPERIMENTAL
Male Holstein calves were left with their dams for 24 hr. and then were placed on a vitamin A- and carotene-deficient diet for a period of 7 days. The purpose of this stabilization period was to deplete the digestive tract and its contents of carotene and vitamin A. The experimental ration consisted of a mixture of 0.2 lb. d r y whole milk replacement, 1.8 lb. water and 3.0 lb. skimmilk fed twice daily. No hay, grain or additional fluids were made available. The calves were penned individually on wire screens to prevent eating of bedding. A f t e r completion of this stabilization period, the calves were administered one of the following preparations dispersed in the experimental diet to insure more homogeneous distribution in the digestive t r a c t : Group A, 5.0 g. commercial corn oil; group B, 2.0 g. concentrated fish liver oil containing 608,000 I. U. vitamin A per g r a m ; group C, 5.0 g. cottonseed oil containing 255,000 I. U. vitamin Received for publication Sept. 9, 1951. 1 Data contained in this publication are from a thesis submitted by the senior author to the Graduate School of The Pennsylvania State College in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Authorized for publication as paper no. 1681 in the Journal Series of The Pennsylvania Agricultural Experiment Station. s Present address: Department of Dairying, Oklahoma A. & M. College, Stillwater, Okla. 283 Copyright 1952, by the A:M~RICANDAIRYSCIENCEASSOCIATIOI~
284
I~[.kGNAR R O N N I N G A N D C. B. K N O D T
A equivalent in the f o r m of fl-carotene per g r a m ; group D, 0.5 g. of the above v i t a m i n A oil as used in g r o u p B ; and t r e a t m e n t E, 1.25 g. of the above carotene oil used in group C. The total amount of oil was adjusted by adding corn oil as needed so t h a t each calf received a total of 5.0 g. oil. The concentrate oil was a d d e d to 1 lb. of skimmilk of the ration, heated to 37 ° C. and dispersed by vigorous shaking. This m i x t u r e then was dispersed in the total test meal by thorough stirring and a sample for analysis was taken while the m a t e r i a l was swirling in the bucket. E i g h t hr. a f t e r feeding this meal the calves were stunned with a blow on the head a n d bled to death by severing the left carotid artery. The body cavity was exposed, the esophagus tied off and the digestive t r a c t ligated as soon as possible in the following places to p r e v e n t as much as possible the movement of material f r o m one portion to a n o t h e r : Between the r u m e n and a b o m a s u m ; the pyloric and ileocecal valves; and at several places throughout the small intestine. The t r a c t then was removed intact a n d t a k e n to the l a b o r a t o r y and p r e p a r e d for analysis. The contents of the rumen, abomasum and the large intestine, including the cecum, were removed a n d identified as R, AB a n d LI, respectively. Because of a lack of sufficient material, the contents of the reticulum were included with those of the r u m e n for analysis. Similarly, the contents of the omasum were included with those of the abomasum. The small intestine was divided into three sections a p p r o x i m a t e l y equal in length and the contents removed and identified as SI-1, 2 and 3, beginning f r o m the duodenal end. Blood samples were taken for v i t a m i n A and carotene analysis prior to administration and at time of slaughter. The livers were removed a n d prep a r e d f o r analysis for v i t a m i n A and carotene. The mesenteric membranes, adhering f a t t y tissue a n d l y m p h nodules were removed f r o m the outer surface of the digestive t r a c t a n d the inner surfaces washed thoroughly with a 0.9 per cent saline solution. The mucous tissue of the a b o m a s u m and of the three sections of the small intestine were removed by c a r e f u l l y scraping the inner surfaces with a knife. The two layers of these sections and the tissues of the r u m e n and of the large intestine were p r e p a r e d for analysis b y grinding a n d macerating. Analyses for carotene and v i t a m i n A were made on the test meal, digestive t r a c t contents, tissues of the digestive t r a c t walls and livers a f t e r saponification and the extraction of the nonsaponifiable material with ethyl ether. The concentrations of v i t a m i n A and carotene of the feed and digestive t r a c t contents are reported in terms of I. U. per g r a m of d r y matter. The concentrations of these compounds in the tissues are presented on the basis of I. U. per g r a m of fresh tissue. The Carr-Price reaction was used in the analyses for v i t a m i n A, while the carotene was measured colorimetrically in petroleum ether, using the E v e l y n colorimeter according to suggestions b y the Association of V i t a m i n Chemists. Chromatographic purification (2) of the carotene extract was not employed since it h a d been p r e d e t e r m i n e d t h a t interfering pigments were not concentrated sufficiently to affect the final results. Blood analyses for v i t a m i n A
285
VITAMIN A ABSORPTION
and carotene were made according to a combination of methods proposed by Moore (8) and Kimble (7). The per cent d r y m a t t e r of the feed and of the digestive t r a c t contents were determined by using a B r a b e n d e r semiautomatic moisture tester. The samples were dried at 100 ° C. with forced d r a f t f o r 4 hr. which h a d been p r e d e t e r m i n e d as the time required f o r constant weight• Three calves were subjected to each of the d i e t a r y t r e a t m e n t s described. RESULTs
The concentration of v i t a m i n A and carotene in the digestive t r a c t contents f r o m the various sections are presented in figure 1. These levels m a y be corn120 115 IIC IO5
Ioc 95 9~ 85 8¢
7~ 70
8 65
i~\
HIOH LEVEL VITAMIN & LOW LEVEL VITAMIN A HIOH LEVEL CAROTENE "4 LOW LEVEL CAROTENE
:E 60
d 5~5~ 50 45 40 35 3O 25 20 15 I0
0L
R
Ab
Sl-I
SI'~
SI-3
L!
SECTIOh
FIG. 1.
Concentration of administered compound in digestive tract contents I.U./g. D.M.
p a r e d with the average levels of the a d m i n i s t r e d compound present in the feed, which were as follows as determined chemically: H i g h level v i t a m i n A, 5330 I. U. per g r a m d r y m a t t e r ; low level v i t a m i n A, 938 I. U. p e r g r a m d r y m a t t e r ; high level carotene, 3079 I. U. v i t a m i n A equivalent per g r a m d r y m a t t e r ; a n d low level carotene, 939 I. U. v i t a m i n A equivalent per g r a m d r y matter. The test meal a n d the digestive t r a c t contents of the control g r o u p showed only traces of v i t a m i n A a n d carotene values and these in all p r o b a b i l i t y were due to the
MAGNAR RONNING AND C. B. KNODT
286
presence of v e r y small amounts of interfering substances. The chemically determ i n e d values of vitamin A a n d carotene of the feed were a p p r e c i a b l y lower than the calculated values, which indicates t h a t considerable loss in potency was suffered during the time of dispersing the oil with the feed. Some oxidative destruction m a y have taken place while shaking the heated m i x t u r e and f u r t h e r loss m a y have resulted f r o m adherence of some oil to the utensils used. V//,~
HIGH Lc-'VEL VITAMIN A HIGH LEVEL CAROTENE LOW LEVEL VITAMIN A
G
LOW LEVEL ¢AROTENE
-4'
0~
Ab
FIG. 2.
$I'l
SEGTION
SI-~.
51-3
LI
Concentration of carotene G.I. tract tissue I . U . / g .
I0
m GONTROI,. ~7-'/"~HIGH LEVEL ~"-'~HSGH LEVEL LOW LEVEL ~ LOW LEVEL
I
VXTAMINA CAROTENE VITAMIN A CAROTENE
V-
I FIG. 3.
Ab
Sl-i
SECTION
SI-2
$1-3
L~
Concentration of vitamin A G.I. tract tissue I . U . / g .
A v e r y high concentration of vitamin A in the abomasum resulted from the administration of this compound at both levels of dosage. The lowest level of v i t a m i n A was observed in the u p p e r one-third of the small intestine in the low-dosage g r o u p a n d in the middle one-third of the small intestine in the highdosage group. There a p p e a r e d to be a concentrating effect upon vitamin A in
287
VITAMIN A ABSORPTION
the lower one-third of the snlall intestine and in the large intestine, which was particularly evident in the high-dosage group. The carotene levels of the digestive tract contents of the two groups administered this compound were quite comparable, although the low-dosage group received only about one-third as much carotene as the high-dosage group. The concentration of carotene was lowest in the middle one-third of the small intestine G
6
~MUSCULAR WALLHIGHLEVEL I///IMUCOUS LAYERHIGHLEVEL I:f,:'-:::lMUSCULARWALLLOWLEVEL {'--'--]MUCOUS LAYERLOW LEVEL
~ 4
Ab
]~](;. 4.
Sl-I
SECTION
t
61-2
$1-3
The distribution of carotene between the mucous layer and muscular wall.
I0
~MUSGULAR WALL HIGH LEVEL 177-71.ucous ~YER NIG. LEVEL ~MUSCUL*R WALL LOW LEVEL F'----]MUCOUS ~YER low reVEL
/7 //
8
/
Cs .P
-"
4
E
Ab
SI-I
SI-2
Sl-3
SECTION
FIG. 5.
The d i s t r i b u t i o n of v i t a m i n A b e t w e e n t h e m u c o u s l a y e r a n d m u s c u l a r w a l l .
and highest in the abomasmn. The concentration of carotene was quite high in the large intestine, approaching that observed in the abomasum. A great deal of individual variation was observed in the levels of carotene in the contents of the rumen, influenced probably by the passage of liquid material into this section. The average d r y matter content of the digestive tract contents was 61.5 per cent of that in the feed. This had the effect of increasing the concentration of
288
~IAGN'AR R O N N I N G AND C. B. K N O D T
v i t a m i n A a n d carotene p e r u n i t g r a m of d r y m a t t e r . The total a m o u n t of these compounds, however, was somewhat less in the digestive t r a c t contents t h a n in the feed. The recovery of v i t a m i n A was 83.7 a n d 65.4 p e r cent in the high- a n d l o w - a d m i n i s t r a t i o n groups, a n d the recovery of carotene was 21.6 a n d 64.3 per cent in the high- a n d low-intake groups, respectively. The concentrations of carotene a n d v i t a m i n A in the tissues of the various p o r t i o n s of the digestive t r a c t are s u m m a r i z e d in figures 2 a n d 3. The total a m o u n t of these compounds in the tissues did not a p p e a r to be r e l a t e d to the c o n c e n t r a t i o n s of v i t a m i n A or carotene in the c o r r e s p o n d i n g digestive t r a c t contents. A t both levels of dosage the carotene concentrations were highest in the r u m e n tissue a n d lowest in the mid-section of the small intestine. The conc e n t r a t i o n of carotene in the tissues of the digestive t r a c t wall was considerably h i g h e r in the high-dosage group t h a n in the lower level intake group. However, at both levels of carotene dosage, the carotene concentrations in the digestive t r a c t contents were n e a r l y the same. The c o n c e n t r a t i o n of v i t a m i n A in the tissues of the walls of the digestive t r a c t was highest in the abomasum a n d became p r o g r e s s i v e l y lower t h r o u g h o u t the r e m a i n d e r of the tract, in the high-dosage group. A t the low-level intake, the digestive t r a c t tissue levels of v i t a m i n A were quite u n i f o r m , with somewhat higher levels in the u p p e r two-thirds of the small intestine t h a n elsewhere. TABLE 1 FiTeights and carotene and vitamb~ A contents of calf livers
Calf
Treatmenta
(no.)
Wt.
Carotene~
Vitamin A
(a.)
(I. ~7.)
(I. V.)
282 289 292 X
A
776 878 735 796
498 2,520 809 1,276
30,410 85,078 2,793 39,427
278 285 291 X
B
825 790 763 793
2,368 869 1,099 1,445
61,215 6,565 10,758 26,179
280 286 290 X
C
924 838 897 886
1,654 2,506 484 1,548
2,495 38,715 4,575 15,262
287 294 296 X
D
1,199 958 1,024 1,060
2,290 1,494 2,202 1,995
54,914 40,332 77,414 57,553
288 295 297 X
E
824 706 179 570
1,673
72,759 1,483 3,365 25,869
777
177 876
a A, Control; B, highdevel vitamin A; C, high-level carotene; D, low-level vitamin A; E, low-level carotene. b Vitamin A equivalent.
VITAMIN
289
A ABSORPTION
The distribution of carotene and v i t a m i n A between the mucous layer a n d the muscular wall is presented in figures 4 a n d 5. The concentration of carotene in these two tissue layers was nearly equal. V i t a m i n A, however, was predomin a n t l y higher in the mucous layer as compared to the muscular tissue. The concentration of v i t a m i n A was highest in the mucous layer in the u p p e r two-thirds of the small intestine in both dosage groups. F r o m the data summarized in table 1, liver storage of v i t a m i n A a p p a r e n t l y was not affected by a n y of these t r e a t m e n t s during the 8-hr. period following administration. The highest concentration of vitamin A was f o u n d in the liver of a calf of the control g r o u p and only one of the four s u p p l e m e n t e d groups showed a higher average liver concentration of the vitamin t h a n did the control group. I t should be noted in table 1 t h a t even a f t e r the period of stabilization there were large variations in the concentrations of liver carotene a n d v i t a m i n A. The changes in blood plasma v i t a m i n A and carotene resulting f r o m the t r e a t m e n t s employed in this experiment are presented in table 2. A great deal TABLE
2
Blood plasma carotene and v~tamin A Carotene
Calf
Vitamin A
(v/loo ~l.)
(v/ioo ~nl.)
Treatment a e -. aPdrm
Slaughter
Change
e -. aPdrm
11.66 15.55 7.77 11.66
10.69 14.57 7.77 11.01
- 0.97 - 0.98 0.00 - 0.65
14.16 23.32 3.91 13.80
13.74 23.32 2.91 13.32
-
Slaughter
Change
(nO.) 282 289 292 X
A
278 285 291 X
B
20.40 11.66 9.72 13.93
19.43 9.72 6.80 11.98
-
0.97 1.94 2.92 1.95
12.91 7.77 3.91 8.20
46.64 13.32 14.16 24.71
+ 33.73 + 5.55 + 10.25 + 16.51
280 286 290 X
C
6.80 13.60 4.86 8.42
7.77 15.55 3.89 9.07
+ + +
0.97 1.95 0.97 0.65
0.00 12.91 8.61 7.17
1.42 16.24 11.24 9.63
+ + + +
L42 3.33 2.63 2.46
287 294 296 X
D
7.77 1.94 6.80 5.50
6.80 4.86 5.83 5.83
+ +
0.97 2.92 0.97 0.33
5.25 11.66 11.33
4.58 17.49 19.57 13.88
+ + +
0.67 5.83 2.50 2.55
288 295 297 X"
E
14.57 1.94 3.89 6.80
17.49 10.69 4.86 11.01
+ 2.92 + 8.75 + 0.97 +4.21
26.90 3.91 5.25 12.02
24.80 5.58 6.25 12.21
+ + +
2.10 1.67 1.00 0.19
a A, Control; B, high-level vitamin E, low-level carotene.
A;
17.07
C, h i g h - l e v e l c a r o t e n e ;
-
D, l o w - l e v e l v i t a m i n
0.42 0.00 1.00 0.48
A~
of individual variation was observed in the changes in blood plasma levels of both v i t a m i n A- and carotene-fed calves. One calf of the v i t a m i n A - a d m i n i s t e r e d groups did not exhibit an increase in blood plasma v i t a m i n A. Only one calf
290
I~IAGNAR RONNING AND C. B. KNODT
of the carotene groups showed an appreciable increase in blood plasma carotene. Changes in blood plasma vitamin A resulting from carotene administration were relatively small and quite inconsistent under the conditions of these experiments. The increases in blood plasma vitamin A resulting from the administration of this compound at both levels of intake were significantly correlated to the concentrations of vitamin A in the mucous layers of the tissues of the walls of the small intestine. The calculated coefficient of correlation was r = 0.97, which was significant with P > 0.01. No other relationships could be shown between blood plasma changes and concentrations of carotene or vitamin A in the digestive t r a c t tissues or contents. SUMMARY AND CONCLUSIONS
The concentrations of vitamin A and carotene in the digestive tract contents a n d tissues after a single administration of either compound at a high or low level to y o u n g calves have been studied. F u r t h e r observations were made with respect to the effect of such administrations upon liver storage and upon blood plasma carotene and vitamin A. The resulting data indicate that the most active absorption of vitamin A takes place in the u p p e r two-thirds of the small intestine. The concentration of vitamin A in the digestive tract contents after the administration of one single dose was lowest in this area. The mucous tissues of the u p p e r two-thirds of the small intestine were more concentrated with vitamin A than those elsewhere. A highly significant correlation coefficient was calculated between the increase in blood plasma vitamin A and the concentration of vitamin A in the mucous tissue of the small intestine after the administration of a single dose of vitamin A. Absorption seemed to be limited in the lower one-third of the small intestine and in the large intestine, since there appeared to be a concentration effect upon the vitamin A that passed into these two sections. The low concentration of carotene in the middle one-third of the small intestine after a single administration of this compound might indicate that absorption takes place most rapidly in this area. The low concentration of carotene in the tissues of the digestive tract wall may be evidence of carotene conversion, but concurrent increases in vitamin A could not be demonstrated u n d e r these conditions. A large single dose of vitamin A or of carotene did not affect liver storage of vitamin A during the 8-hr. period following administration. The administration of vitamin A in a single dose increased blood plasma vitamin A in all instances but one. Carotene administrations resnlted in inconsistent and relatively small changes in blood plasma carotene and vitamin A in these experinlents. REFERENCES (l)
(2)
AH.~IAD, B. T h e F a t e of Carotene a f t e r A b s o r p t i o n in the A n i m a l O r g a n i s m . t / i o e h e m J., 25: 1195-1°04. 1931. ASSOCIATION OF "VITAMIN CHEMISTS. Methods of Vitamin Assay. I n t e r s c i e n c e Publishers, Inc., N . Y . 1947.
VITAMIN A ABSORPTION
29~
(3) BhR~ICK, E. R , ANDREWS, F. N., AND BULLARD, J . F . The Absorption of Carotene and: V i t a m i n A f r o m Various Levels of the Gastro-intestinal Tract of Sheep. J. A n i m a l Sei., 7: 539. 1948. (4) BAUI~iANN, C. A., STEENROCK, H., BEESON, V. M., AND RUPEL, I. W. The II~flue~ce of Breed and Diet of Cows on the Carotene and V i t a m i n A Content of Butter. J. Biol. Chem., 105: 167-176. 1934. (5) EDEN, E., AND SELLERS, K. C. The Absorption of Vitamin A in R u m i n a n t s and Rats. Biochem. J., 44: 264-267. 1949. (6) G0ODWIN, T. W., AND GREGORY, R . A . Carotene Metabolism in Herbivores. Biochem. J., 43: 505-512. 1948. (7) KIMRLE, ~V[. S. The Photoelectric Determination of V i t a m i n A and Carotene in H u m a n Plasma. J. Lab. and Clin. Med., 24: 1055-1065. 1939. (8) Mool~E, L. A. The Determination of Blood P l a s m a Carotene in the Bovine U s i n g a Photoelectric Colorimeter. J. Dairy Sci., 22: 501-511. 1939. (9) MOORE, T. The Conversion of Carotene to V i t a m i n A in vivo. Biochem. J., 24: 696-702. 1930. (]{)) POPPER, I~., AND YOLK, B. W. Absorption of V i t a m i n A in the Rat. Arch. Path., 38: 71-75. 1944.
APRIL, 1952
NUMBER 4
T H E A B S O R P T I O N OF V I T A M I N A N A T U R A L E S T E R S A N D O F CAROTENE BY YOUNG MALE H O L S T E I N CALVES 1 MAGNAR RONNINGz AND C. B. KNODT Pennsylvania Agricultural Experiment Station, State College The importance of vitamin A and its precursors in the rations of d a i r y calves has been well established. On the basis of the research published, however, information is needed relative to the site of absorption of these compounds in obtaining an understanding of the mechanisms involved in their utilization. P o p p e r and Volk (10) determined that absorption of vitamin A in rats was most rapid in the u p p e r part of the small intestine. Barrick et al. (3) indicated that the small intestine was most important in the absorption of vitamin A by sheep. Eden and Sellers (5) demonstrated that vitamin A was absorbed primarily in the duodenal area of the small intestine in sheep and in rats. The absorption of carotene and its subsequent conversion to vitamin A is not well understood and there appears to be considerable species variation in the mechanism of utilization of carotene (1, 3, 4, 6, 9). I t was the purpose of this experiment to obtain information concerning the site of absorption of vitamin A n a t u r a l esters and carotene by y o u n g dairy calves. EXPERIMENTAL
Male Holstein calves were left with their dams for 24 hr. and then were placed on a vitamin A- and carotene-deficient diet for a period of 7 days. The purpose of this stabilization period was to deplete the digestive tract and its contents of carotene and vitamin A. The experimental ration consisted of a mixture of 0.2 lb. d r y whole milk replacement, 1.8 lb. water and 3.0 lb. skimmilk fed twice daily. No hay, grain or additional fluids were made available. The calves were penned individually on wire screens to prevent eating of bedding. A f t e r completion of this stabilization period, the calves were administered one of the following preparations dispersed in the experimental diet to insure more homogeneous distribution in the digestive t r a c t : Group A, 5.0 g. commercial corn oil; group B, 2.0 g. concentrated fish liver oil containing 608,000 I. U. vitamin A per g r a m ; group C, 5.0 g. cottonseed oil containing 255,000 I. U. vitamin Received for publication Sept. 9, 1951. 1 Data contained in this publication are from a thesis submitted by the senior author to the Graduate School of The Pennsylvania State College in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Authorized for publication as paper no. 1681 in the Journal Series of The Pennsylvania Agricultural Experiment Station. s Present address: Department of Dairying, Oklahoma A. & M. College, Stillwater, Okla. 283 Copyright 1952, by the A:M~RICANDAIRYSCIENCEASSOCIATIOI~
284
I~[.kGNAR R O N N I N G A N D C. B. K N O D T
A equivalent in the f o r m of fl-carotene per g r a m ; group D, 0.5 g. of the above v i t a m i n A oil as used in g r o u p B ; and t r e a t m e n t E, 1.25 g. of the above carotene oil used in group C. The total amount of oil was adjusted by adding corn oil as needed so t h a t each calf received a total of 5.0 g. oil. The concentrate oil was a d d e d to 1 lb. of skimmilk of the ration, heated to 37 ° C. and dispersed by vigorous shaking. This m i x t u r e then was dispersed in the total test meal by thorough stirring and a sample for analysis was taken while the m a t e r i a l was swirling in the bucket. E i g h t hr. a f t e r feeding this meal the calves were stunned with a blow on the head a n d bled to death by severing the left carotid artery. The body cavity was exposed, the esophagus tied off and the digestive t r a c t ligated as soon as possible in the following places to p r e v e n t as much as possible the movement of material f r o m one portion to a n o t h e r : Between the r u m e n and a b o m a s u m ; the pyloric and ileocecal valves; and at several places throughout the small intestine. The t r a c t then was removed intact a n d t a k e n to the l a b o r a t o r y and p r e p a r e d for analysis. The contents of the rumen, abomasum and the large intestine, including the cecum, were removed a n d identified as R, AB a n d LI, respectively. Because of a lack of sufficient material, the contents of the reticulum were included with those of the r u m e n for analysis. Similarly, the contents of the omasum were included with those of the abomasum. The small intestine was divided into three sections a p p r o x i m a t e l y equal in length and the contents removed and identified as SI-1, 2 and 3, beginning f r o m the duodenal end. Blood samples were taken for v i t a m i n A and carotene analysis prior to administration and at time of slaughter. The livers were removed a n d prep a r e d f o r analysis for v i t a m i n A and carotene. The mesenteric membranes, adhering f a t t y tissue a n d l y m p h nodules were removed f r o m the outer surface of the digestive t r a c t a n d the inner surfaces washed thoroughly with a 0.9 per cent saline solution. The mucous tissue of the a b o m a s u m and of the three sections of the small intestine were removed by c a r e f u l l y scraping the inner surfaces with a knife. The two layers of these sections and the tissues of the r u m e n and of the large intestine were p r e p a r e d for analysis b y grinding a n d macerating. Analyses for carotene and v i t a m i n A were made on the test meal, digestive t r a c t contents, tissues of the digestive t r a c t walls and livers a f t e r saponification and the extraction of the nonsaponifiable material with ethyl ether. The concentrations of v i t a m i n A and carotene of the feed and digestive t r a c t contents are reported in terms of I. U. per g r a m of d r y matter. The concentrations of these compounds in the tissues are presented on the basis of I. U. per g r a m of fresh tissue. The Carr-Price reaction was used in the analyses for v i t a m i n A, while the carotene was measured colorimetrically in petroleum ether, using the E v e l y n colorimeter according to suggestions b y the Association of V i t a m i n Chemists. Chromatographic purification (2) of the carotene extract was not employed since it h a d been p r e d e t e r m i n e d t h a t interfering pigments were not concentrated sufficiently to affect the final results. Blood analyses for v i t a m i n A
285
VITAMIN A ABSORPTION
and carotene were made according to a combination of methods proposed by Moore (8) and Kimble (7). The per cent d r y m a t t e r of the feed and of the digestive t r a c t contents were determined by using a B r a b e n d e r semiautomatic moisture tester. The samples were dried at 100 ° C. with forced d r a f t f o r 4 hr. which h a d been p r e d e t e r m i n e d as the time required f o r constant weight• Three calves were subjected to each of the d i e t a r y t r e a t m e n t s described. RESULTs
The concentration of v i t a m i n A and carotene in the digestive t r a c t contents f r o m the various sections are presented in figure 1. These levels m a y be corn120 115 IIC IO5
Ioc 95 9~ 85 8¢
7~ 70
8 65
i~\
HIOH LEVEL VITAMIN & LOW LEVEL VITAMIN A HIOH LEVEL CAROTENE "4 LOW LEVEL CAROTENE
:E 60
d 5~5~ 50 45 40 35 3O 25 20 15 I0
0L
R
Ab
Sl-I
SI'~
SI-3
L!
SECTIOh
FIG. 1.
Concentration of administered compound in digestive tract contents I.U./g. D.M.
p a r e d with the average levels of the a d m i n i s t r e d compound present in the feed, which were as follows as determined chemically: H i g h level v i t a m i n A, 5330 I. U. per g r a m d r y m a t t e r ; low level v i t a m i n A, 938 I. U. p e r g r a m d r y m a t t e r ; high level carotene, 3079 I. U. v i t a m i n A equivalent per g r a m d r y m a t t e r ; a n d low level carotene, 939 I. U. v i t a m i n A equivalent per g r a m d r y matter. The test meal a n d the digestive t r a c t contents of the control g r o u p showed only traces of v i t a m i n A a n d carotene values and these in all p r o b a b i l i t y were due to the
MAGNAR RONNING AND C. B. KNODT
286
presence of v e r y small amounts of interfering substances. The chemically determ i n e d values of vitamin A a n d carotene of the feed were a p p r e c i a b l y lower than the calculated values, which indicates t h a t considerable loss in potency was suffered during the time of dispersing the oil with the feed. Some oxidative destruction m a y have taken place while shaking the heated m i x t u r e and f u r t h e r loss m a y have resulted f r o m adherence of some oil to the utensils used. V//,~
HIGH Lc-'VEL VITAMIN A HIGH LEVEL CAROTENE LOW LEVEL VITAMIN A
G
LOW LEVEL ¢AROTENE
-4'
0~
Ab
FIG. 2.
$I'l
SEGTION
SI-~.
51-3
LI
Concentration of carotene G.I. tract tissue I . U . / g .
I0
m GONTROI,. ~7-'/"~HIGH LEVEL ~"-'~HSGH LEVEL LOW LEVEL ~ LOW LEVEL
I
VXTAMINA CAROTENE VITAMIN A CAROTENE
V-
I FIG. 3.
Ab
Sl-i
SECTION
SI-2
$1-3
L~
Concentration of vitamin A G.I. tract tissue I . U . / g .
A v e r y high concentration of vitamin A in the abomasum resulted from the administration of this compound at both levels of dosage. The lowest level of v i t a m i n A was observed in the u p p e r one-third of the small intestine in the low-dosage g r o u p a n d in the middle one-third of the small intestine in the highdosage group. There a p p e a r e d to be a concentrating effect upon vitamin A in
287
VITAMIN A ABSORPTION
the lower one-third of the snlall intestine and in the large intestine, which was particularly evident in the high-dosage group. The carotene levels of the digestive tract contents of the two groups administered this compound were quite comparable, although the low-dosage group received only about one-third as much carotene as the high-dosage group. The concentration of carotene was lowest in the middle one-third of the small intestine G
6
~MUSCULAR WALLHIGHLEVEL I///IMUCOUS LAYERHIGHLEVEL I:f,:'-:::lMUSCULARWALLLOWLEVEL {'--'--]MUCOUS LAYERLOW LEVEL
~ 4
Ab
]~](;. 4.
Sl-I
SECTION
t
61-2
$1-3
The distribution of carotene between the mucous layer and muscular wall.
I0
~MUSGULAR WALL HIGH LEVEL 177-71.ucous ~YER NIG. LEVEL ~MUSCUL*R WALL LOW LEVEL F'----]MUCOUS ~YER low reVEL
/7 //
8
/
Cs .P
-"
4
E
Ab
SI-I
SI-2
Sl-3
SECTION
FIG. 5.
The d i s t r i b u t i o n of v i t a m i n A b e t w e e n t h e m u c o u s l a y e r a n d m u s c u l a r w a l l .
and highest in the abomasmn. The concentration of carotene was quite high in the large intestine, approaching that observed in the abomasum. A great deal of individual variation was observed in the levels of carotene in the contents of the rumen, influenced probably by the passage of liquid material into this section. The average d r y matter content of the digestive tract contents was 61.5 per cent of that in the feed. This had the effect of increasing the concentration of
288
~IAGN'AR R O N N I N G AND C. B. K N O D T
v i t a m i n A a n d carotene p e r u n i t g r a m of d r y m a t t e r . The total a m o u n t of these compounds, however, was somewhat less in the digestive t r a c t contents t h a n in the feed. The recovery of v i t a m i n A was 83.7 a n d 65.4 p e r cent in the high- a n d l o w - a d m i n i s t r a t i o n groups, a n d the recovery of carotene was 21.6 a n d 64.3 per cent in the high- a n d low-intake groups, respectively. The concentrations of carotene a n d v i t a m i n A in the tissues of the various p o r t i o n s of the digestive t r a c t are s u m m a r i z e d in figures 2 a n d 3. The total a m o u n t of these compounds in the tissues did not a p p e a r to be r e l a t e d to the c o n c e n t r a t i o n s of v i t a m i n A or carotene in the c o r r e s p o n d i n g digestive t r a c t contents. A t both levels of dosage the carotene concentrations were highest in the r u m e n tissue a n d lowest in the mid-section of the small intestine. The conc e n t r a t i o n of carotene in the tissues of the digestive t r a c t wall was considerably h i g h e r in the high-dosage group t h a n in the lower level intake group. However, at both levels of carotene dosage, the carotene concentrations in the digestive t r a c t contents were n e a r l y the same. The c o n c e n t r a t i o n of v i t a m i n A in the tissues of the walls of the digestive t r a c t was highest in the abomasum a n d became p r o g r e s s i v e l y lower t h r o u g h o u t the r e m a i n d e r of the tract, in the high-dosage group. A t the low-level intake, the digestive t r a c t tissue levels of v i t a m i n A were quite u n i f o r m , with somewhat higher levels in the u p p e r two-thirds of the small intestine t h a n elsewhere. TABLE 1 FiTeights and carotene and vitamb~ A contents of calf livers
Calf
Treatmenta
(no.)
Wt.
Carotene~
Vitamin A
(a.)
(I. ~7.)
(I. V.)
282 289 292 X
A
776 878 735 796
498 2,520 809 1,276
30,410 85,078 2,793 39,427
278 285 291 X
B
825 790 763 793
2,368 869 1,099 1,445
61,215 6,565 10,758 26,179
280 286 290 X
C
924 838 897 886
1,654 2,506 484 1,548
2,495 38,715 4,575 15,262
287 294 296 X
D
1,199 958 1,024 1,060
2,290 1,494 2,202 1,995
54,914 40,332 77,414 57,553
288 295 297 X
E
824 706 179 570
1,673
72,759 1,483 3,365 25,869
777
177 876
a A, Control; B, highdevel vitamin A; C, high-level carotene; D, low-level vitamin A; E, low-level carotene. b Vitamin A equivalent.
VITAMIN
289
A ABSORPTION
The distribution of carotene and v i t a m i n A between the mucous layer a n d the muscular wall is presented in figures 4 a n d 5. The concentration of carotene in these two tissue layers was nearly equal. V i t a m i n A, however, was predomin a n t l y higher in the mucous layer as compared to the muscular tissue. The concentration of v i t a m i n A was highest in the mucous layer in the u p p e r two-thirds of the small intestine in both dosage groups. F r o m the data summarized in table 1, liver storage of v i t a m i n A a p p a r e n t l y was not affected by a n y of these t r e a t m e n t s during the 8-hr. period following administration. The highest concentration of vitamin A was f o u n d in the liver of a calf of the control g r o u p and only one of the four s u p p l e m e n t e d groups showed a higher average liver concentration of the vitamin t h a n did the control group. I t should be noted in table 1 t h a t even a f t e r the period of stabilization there were large variations in the concentrations of liver carotene a n d v i t a m i n A. The changes in blood plasma v i t a m i n A and carotene resulting f r o m the t r e a t m e n t s employed in this experiment are presented in table 2. A great deal TABLE
2
Blood plasma carotene and v~tamin A Carotene
Calf
Vitamin A
(v/loo ~l.)
(v/ioo ~nl.)
Treatment a e -. aPdrm
Slaughter
Change
e -. aPdrm
11.66 15.55 7.77 11.66
10.69 14.57 7.77 11.01
- 0.97 - 0.98 0.00 - 0.65
14.16 23.32 3.91 13.80
13.74 23.32 2.91 13.32
-
Slaughter
Change
(nO.) 282 289 292 X
A
278 285 291 X
B
20.40 11.66 9.72 13.93
19.43 9.72 6.80 11.98
-
0.97 1.94 2.92 1.95
12.91 7.77 3.91 8.20
46.64 13.32 14.16 24.71
+ 33.73 + 5.55 + 10.25 + 16.51
280 286 290 X
C
6.80 13.60 4.86 8.42
7.77 15.55 3.89 9.07
+ + +
0.97 1.95 0.97 0.65
0.00 12.91 8.61 7.17
1.42 16.24 11.24 9.63
+ + + +
L42 3.33 2.63 2.46
287 294 296 X
D
7.77 1.94 6.80 5.50
6.80 4.86 5.83 5.83
+ +
0.97 2.92 0.97 0.33
5.25 11.66 11.33
4.58 17.49 19.57 13.88
+ + +
0.67 5.83 2.50 2.55
288 295 297 X"
E
14.57 1.94 3.89 6.80
17.49 10.69 4.86 11.01
+ 2.92 + 8.75 + 0.97 +4.21
26.90 3.91 5.25 12.02
24.80 5.58 6.25 12.21
+ + +
2.10 1.67 1.00 0.19
a A, Control; B, high-level vitamin E, low-level carotene.
A;
17.07
C, h i g h - l e v e l c a r o t e n e ;
-
D, l o w - l e v e l v i t a m i n
0.42 0.00 1.00 0.48
A~
of individual variation was observed in the changes in blood plasma levels of both v i t a m i n A- and carotene-fed calves. One calf of the v i t a m i n A - a d m i n i s t e r e d groups did not exhibit an increase in blood plasma v i t a m i n A. Only one calf
290
I~IAGNAR RONNING AND C. B. KNODT
of the carotene groups showed an appreciable increase in blood plasma carotene. Changes in blood plasma vitamin A resulting from carotene administration were relatively small and quite inconsistent under the conditions of these experiments. The increases in blood plasma vitamin A resulting from the administration of this compound at both levels of intake were significantly correlated to the concentrations of vitamin A in the mucous layers of the tissues of the walls of the small intestine. The calculated coefficient of correlation was r = 0.97, which was significant with P > 0.01. No other relationships could be shown between blood plasma changes and concentrations of carotene or vitamin A in the digestive t r a c t tissues or contents. SUMMARY AND CONCLUSIONS
The concentrations of vitamin A and carotene in the digestive tract contents a n d tissues after a single administration of either compound at a high or low level to y o u n g calves have been studied. F u r t h e r observations were made with respect to the effect of such administrations upon liver storage and upon blood plasma carotene and vitamin A. The resulting data indicate that the most active absorption of vitamin A takes place in the u p p e r two-thirds of the small intestine. The concentration of vitamin A in the digestive tract contents after the administration of one single dose was lowest in this area. The mucous tissues of the u p p e r two-thirds of the small intestine were more concentrated with vitamin A than those elsewhere. A highly significant correlation coefficient was calculated between the increase in blood plasma vitamin A and the concentration of vitamin A in the mucous tissue of the small intestine after the administration of a single dose of vitamin A. Absorption seemed to be limited in the lower one-third of the small intestine and in the large intestine, since there appeared to be a concentration effect upon the vitamin A that passed into these two sections. The low concentration of carotene in the middle one-third of the small intestine after a single administration of this compound might indicate that absorption takes place most rapidly in this area. The low concentration of carotene in the tissues of the digestive tract wall may be evidence of carotene conversion, but concurrent increases in vitamin A could not be demonstrated u n d e r these conditions. A large single dose of vitamin A or of carotene did not affect liver storage of vitamin A during the 8-hr. period following administration. The administration of vitamin A in a single dose increased blood plasma vitamin A in all instances but one. Carotene administrations resnlted in inconsistent and relatively small changes in blood plasma carotene and vitamin A in these experinlents. REFERENCES (l)
(2)
AH.~IAD, B. T h e F a t e of Carotene a f t e r A b s o r p t i o n in the A n i m a l O r g a n i s m . t / i o e h e m J., 25: 1195-1°04. 1931. ASSOCIATION OF "VITAMIN CHEMISTS. Methods of Vitamin Assay. I n t e r s c i e n c e Publishers, Inc., N . Y . 1947.
VITAMIN A ABSORPTION
29~
(3) BhR~ICK, E. R , ANDREWS, F. N., AND BULLARD, J . F . The Absorption of Carotene and: V i t a m i n A f r o m Various Levels of the Gastro-intestinal Tract of Sheep. J. A n i m a l Sei., 7: 539. 1948. (4) BAUI~iANN, C. A., STEENROCK, H., BEESON, V. M., AND RUPEL, I. W. The II~flue~ce of Breed and Diet of Cows on the Carotene and V i t a m i n A Content of Butter. J. Biol. Chem., 105: 167-176. 1934. (5) EDEN, E., AND SELLERS, K. C. The Absorption of Vitamin A in R u m i n a n t s and Rats. Biochem. J., 44: 264-267. 1949. (6) G0ODWIN, T. W., AND GREGORY, R . A . Carotene Metabolism in Herbivores. Biochem. J., 43: 505-512. 1948. (7) KIMRLE, ~V[. S. The Photoelectric Determination of V i t a m i n A and Carotene in H u m a n Plasma. J. Lab. and Clin. Med., 24: 1055-1065. 1939. (8) Mool~E, L. A. The Determination of Blood P l a s m a Carotene in the Bovine U s i n g a Photoelectric Colorimeter. J. Dairy Sci., 22: 501-511. 1939. (9) MOORE, T. The Conversion of Carotene to V i t a m i n A in vivo. Biochem. J., 24: 696-702. 1930. (]{)) POPPER, I~., AND YOLK, B. W. Absorption of V i t a m i n A in the Rat. Arch. Path., 38: 71-75. 1944.