FAT-SOLUBLE VITAMIN S XLII. THE ABSORPTION AND STORAGE OF VITAMIN A IN THE RAT* BY C. A. BAUMANN, BLANCHE M. RIISING, AND H. STEENBOCK (From the Department of Agricultural Chemistry, University of Wisconsin, Madison) (Received for publication, May 28, 1934) The apparent importance of vitamin A reserves for the well being of the animal has led us to make a detailed study of the absorption and storage of this vitamin. The rat was used as the experimental animal. The vitamin A determinations were made on the unsaponifiable extracts by means of the Carr-Price tech- nique (1). A solution of SbCL in CHC13 saturated at 0” was used as the reagent. The results were calculated according to the method of Moore (2). Analyses for vitamin A when added as halibut liver oil to rat tissues or extracts of rat tissues gave values ranging from 95 to 104 per cent of the amount added. The method therefore appeared sufficiently accurate for our purpose. Identical results were obtained whether alcohol extracts of the tissues, or the tissues themselves, were saponified directly. Occasionally the calorimetric results were verified by means of spectrophotometric determinations. An inspection of the vitamin A stores of animals maintained on our stock diet (3) showed that approximately 95 per cent of the total vitamin A was present in the liver. Small amounts of vita- min A were detected in kidneys and lungs, whereas brain, blood, muscle, and the organs of digestion gave negative results. These results are in complete harmony with those of others (4-6). * Presented in part before the American Institute of Nutrition, Proceed- ings of the First Annual Meeting, New York, March 28, 1934 (J. Nutrition, 7, suppl., 9 (1934)). Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station. 705 by guest on May 6, 2020 http://www.jbc.org/ Downloaded from
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FAT-SOLUBLE VITAMIN S
XLII. THE ABSORPTION AND STORAGE OF VITAMIN A IN THE RAT*
BY C. A. BAUMANN, BLANCHE M. RIISING, AND H. STEENBOCK
(From the Department of Agricultural Chemistry, University of Wisconsin, Madison)
(Received for publication, May 28, 1934)
The apparent importance of vitamin A reserves for the well being of the animal has led us to make a detailed study of the absorption and storage of this vitamin. The rat was used as the experimental animal. The vitamin A determinations were made on the unsaponifiable extracts by means of the Carr-Price tech- nique (1). A solution of SbCL in CHC13 saturated at 0” was used as the reagent. The results were calculated according to the method of Moore (2). Analyses for vitamin A when added as halibut liver oil to rat tissues or extracts of rat tissues gave values ranging from 95 to 104 per cent of the amount added. The method therefore appeared sufficiently accurate for our purpose. Identical results were obtained whether alcohol extracts of the tissues, or the tissues themselves, were saponified directly. Occasionally the calorimetric results were verified by means of spectrophotometric determinations.
An inspection of the vitamin A stores of animals maintained on our stock diet (3) showed that approximately 95 per cent of the total vitamin A was present in the liver. Small amounts of vita- min A were detected in kidneys and lungs, whereas brain, blood, muscle, and the organs of digestion gave negative results. These results are in complete harmony with those of others (4-6).
* Presented in part before the American Institute of Nutrition, Proceed- ings of the First Annual Meeting, New York, March 28, 1934 (J. Nutrition, 7, suppl., 9 (1934)).
Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station.
Vitamin A was never found in the non-hepatic tissues of an ani- mal unless the liver also contained vitamin A. Tissues of rats showing deficiency symptoms contained no vitamin A, nor could the vitamin be detected in the non-hepatic tissues of rats which had been fed a low vitamin A diet until their livers just ceased to give a blue color with SbC&. Extracts from such tissues showed no selective spectral absorption at 328 m+ A determination of vitamin A in the liver, therefore, was deemed adequate as an index of the vitamin A content of the animal.
Effect of Age and of Maternal Diet upon Liver Stores of Vitamin A-Variations in the vitamin A content of rats with age, reported previously (6, 7), have been confirmed. F,or these demonstrations female rats were fed our stock ration (3) plus greens and 5 per cent butter fat. These rats were allowed to suckle their young; the ration was available to both. Ten young selected at 7 day intervals were used for each determination, except with the new born young and those 7 days old, in which case thirty animals were used instead.
In the new born young the stores of vitamin A were found to be very low, averaging 7 blue units1 per liver; or 21 blue units per gm. of tissue. This decreased during the suckling period, as no vitamin A could be detected in the livers at an age of 1 or 2 weeks (Chart 1). After 3 weeks the increase in storage was rapid and regular so that at the end of the 7th week 206 blue units were contained per liver. This increase was absolute as well as relative. It amounted to 1.5 units per gm. of liver tissue at 3 weeks and 25.8 units per gm. at 7 weeks.
The addition of vitamin A to the diet of the pregnant female was found to increase the vitamin A content of the young. When 6 drops of halibut liver oil (9000 blue units) were fed daily to female rats during the last 5 days of their pregnancy, their stores in the liver increased from 5000 to 27,000 blue units per liver. The livers of the new-born from these females contained 20 blue units (60 units per gm. of tissue), whiIe the young from mothers on the stock ration alone, it will be recalled, averaged only 7 blue units per liver. The females from which these young were taken had
1 With our instrument 3 blue units were approximately equivalent to 1 international unit of vitamin A.
been kept on large meshed screen so that the young dropped through the meshes at birth, with no opportunity to suckle.
I 210
Chart 1 I
I The Vitamin A Content ---- 180 of Rat Livers --P I
150
120
90
60
0 inlreelcs 2 3 4 5 6 7
I Age
CHART 1
The vitamin A content of the young was also found to be in- creased by increasing the vitamin A intake of the mother during lactation. For this demonstration female rats on the stock ration
were given a supplement of 9000 blue units of halibut liver oil daily with a medicine dropper during the 1st week of lactation. At the end of the 1st week the young were found to contain an average of 120 blue units per liver. Without this supplement no vitamin A could be detected. Dann (8) likewise found that milk is a more important channel of vitamin A transmission to the young than the placenta.
The livers of stock animals, approximately 1 year of age, varied in their vitamin A content from 2000 to 8000 blue units per liver, or from 200 to 800 units per gm. of tissue. No variations depend- ent upon sex were observed, nor could the vitamin storage be correlated with the number of litters which had been reared successfully. Infections of the respiratory tract, likewise, appeared without effect on the liver stores. That respiratory infections are not necessarily prevented by high stores of vitamin A was clearly demonstrated to us in the winter of 1932-33 when an epidemic of respiratory trouble appeared in our stock colony. Many adults died of infections of the respiratory tract. Analyses of the livers of such animals, however, revealed a storage reserve of from 2000 to 8000 blue units-the same range as in our normal animals.
Utilization of Vitamin A by the Rat-To ascertain the amount of vitamin A which must be ingested to produce storage, rats 40 to 50 gm. in weight were fed our standard low vitamin A diet (9). After 4 weeks when they showed incipient symptoms of ophthalmia, they were fed a halibut liver concentrate dissolved in cottonseed oil. Upon analysis of the livers an occasional faint trace of color was obtained from a rat which had received 25 blue units daily for 4 weeks. Animals which had received 50 blue units daily revealed an average storage of 50 units per liver. Inasmuch as 3 blue units daily restore growth and cure ophthalmia in vitamin A-depleted animals, it is obvious that a large excess of vitamin must be fed before storage results.
In a second series we determined the rate of depletion of vitamin A stores in our animals after their transference to a low vitamin A diet. By way of preparation, forty rats, 70 to 110 gm. in weight, were fed halibut liver oil equivalent to 1000 blue units daily, for 8 days. The vitamin A content of their livers was found to average 1826 units. The remaining animals were then put on the low
vitamin A ration and were killed for analysis at intervals. The
number of units which were lost daily was found to vary directly with the amount in storage (Table I).
Vitamin A Losses after Ingestion-To determine the ratio of vitamin A stored to that ingested we proceeded as follows: Rats 21 to 28 days old, weighing 40 to 50 gm., were fed our low vitamin A diet until they showed a loss in weight and severe symptoms of ophthalmia. They were then fed single doses of halibut oil in
TABLE I
Rate of Depletion of High Stores of Vitamin A
No. of animals Average blue units
1826 (1600-2100) 1255
480 195 52
Daily loss of blue units per period
18 11
9 7
TABLE II
Relation of Ingested and of Stored Vitamin A to Growth and Survival of Rats on a Low Vitamin A Diet
Single dpses of balibu;ltfeYer 011
units ingested
100 500
1000 5000
Blue units in liver
Range Average
0 0 14 32 23 68-150 105
320-560 425
Biological efficiency
Growth on low Survival on low vit&in A diet vitamin A diet
days days
11 31 35 75 78 122 96 192
amounts ranging from 100 to 5000 blue units. After 3 days the amount of vitamin A in the livers was determined. Another series of depleted animals was fed similar levels of oil, but these were continued on the low vitamin A diet, and the growth response as well as the survival period determined. The results presented in Table II show that only about 10 per cent of the ingested vitamin A was recovered from the livers on the higher levels of administra-
tion. On the lowest level, i.e. on 100 blue units of halibut liver oil, no storage resulted. As had been observed by others (6, 10) the amount of storage in the liver runs parallel within certain limits to the amount of supplement.
A large number of experiments in which halibut liver oil was fed to rats showed that in normal animals the average amount of ingested vitamin recovered in the liver was 20 per cent. Recently Davies and Moore (11) reported that 15 per cent of ingested vitamin A was stored in the liver, whereas the results of McCoord and Lute-Clausen (6) indicate that 25 to 70 per cent of the ingested vitamin may be stored. All workers, therefore, agree that the losses of ingested vitamin A are large.
The ratio of ingested to stored vitamin A was the same whether cottonseed oil, mineral oil, or ethyl laurate was used as a diluent. When the vitamin concentrate was suspended in water, however, storage was relatively small.
The losses of vitamin A incurred by incomplete absorption from the digestive tract were next investigated. Ten rats were fed our low vitamin A diet plus 5000 blue units of halibut liver oil each and the feces were collected for the following 48 hours. Only 160 blue units were found to be excreted by each rat over the 2 day period. Feces collected both before and after this 2 day period were found to be free from vitamin A. Unfortunately the deter- minations could not be considered quantitative since an unknown chromogen in the extract gave a red color with the reagent. This substance was also present in the contents of the cecum. Never- theless, it was obvious that fecal excretion could account for only a small portion of the vitamin.
That much of the vitamin A disappeared by destruction in the tract was indicated by several experiments. The first experiment correlated the rate of absorption from the digestive tract with the rate of storage in the liver. Young rats 50 to 60 gm. in weight were fed our low vitamin A ration for 2 days and were then given 5000 blue units of halibut liver oil by stomach tube. Some animals were killed at once, and others after intervals of 3, 6, 12, 24, or 48 hours. The livers as well as the stomachs, intestines, ceca, and their respective contents were removed and analyzed as before. Most of the absorbed vitamin was found to have been transferred to the liver between the 3rd and 6th hours, after which
the increases in liver stores were relatively small (Table III). Nevertheless, nearly 40 per cent of the ingested vitamin was still present in the tract after 6 hours. 52 per cent remained unac- counted for. After 48 hours the liver stores had increased to 500 blue units, 10 per cent of the amount administered. Only 18 units of vitamin A were located in tissues other than the liver. The rapid absorption of vitamin A by rats parallels a rapid absorp- tion by children, as observed by Clausen (12), who reports that the vitamin A content of the blood reaches a maximum 4 to 5 hours after its administration:
Destruction of vitamin A was also revealed by a series of experi- ments in which there were fed equal amounts of halibut liver oil
TABLE III
Rate of Absorption of Vitamin A
Thgd~
hrs.
0 3 6
12 24 48
Stomach
4852 1600 1178
137 25
0
Blue units
Intestine Cecum Liver
0 0 2 2877 3 14
641 88 440 178 840 450
67 124 456 29 3 496
over a 2 week period, but in which the size of the dose was varied. One group received one dose of 4000 blue units; another two doses of 2000 units each-spaced a week apart; a third received four doses of 1000 units each at 3 day intervals. Other groups received eight and sixteen doses during the 2 week period, the total dosage being 4000 units in each case.
When the vitamin was administered in four or in eight equal doses over the 2 week period, an optimal storage, viz. 480 blue units, resulted. When the vitamin was administered in larger or in smaller doses, 350 units or less were stored. Evidently excessively large doses were mostly lost due to incomplete absorp- tion whereas small doses were mostly destroyed in the digestive tract. Destruction of vitamin A in the tract was also suggested
by the observation that it required more vitamin A to produce storage in the liver than to satisfy the daily requirements of the animal, as measured by the rate of disappearance of liver stores.
E$ect of Previous Intake of Vitamin A on Vitamin A Xtorage- To determine to what extent the storage of vitamin A may be influenced by the specific nutritive state of the animal, two groups of five rats each were fed our low vitamin A diet for 2 days, and then given 4000 blue units of halibut liver oil. The first group had been on a vitamin A-free diet for 6 weeks and showed severe symptoms of ophthalmia; the second group was composed of nor- mal rats, whose litter mates had a fair reserve of vitamin A per liver. When killed 2 days after the vitamin A had been fed, the stores of the normal animals had increased to 517 blue units, whereas the stores of the animals given the low vitamin A ration had increased to only 352 units. Evidently the vitamin A had been poorly absorbed by the depleted animals or else it was more rapidly metabolized.
The effect of vitamin A depletion upon vitamin A storage was tested out in a second experiment. Various levels of a standard cod liver oil solution were fed to depleted rats for 8 weeks. With 3 blue units daily growth was restored and ophthalmia was cured; with 1.5 or 0.75blue units daily ophthalmia was not cured. Vita- min A storage had not occurred in any case. 4000 blue units of halibut liver oil were then fed to six animals of each group. After 2 days the amount of storage was found to parallel in a general way the vitamin intake of the preceding weeks, in spite of the fact that the levels of intake had been in themselves too small to result in storage. For example, animals which had pretiously received 1, 0.5, and 0.25 international unit of vitamin A per day yielded respectively 405, 336, and 200 units. Apparently there had been produced in effect a vitamin deficit which had to be covered before storage could occur. This suggested an increased rate of destruction or metabolism by the tissues as the deficiency was increased.
Relation between Growth and Storage-Although, in general, the growth and survival of rats paralleled their storage of vitamin A, prolonged growth and survival were frequently encountered in the absence of its storage. For example, 100 blue units of halibut liver oil, when fed to a vitamin A-depleted rat, were sufficient to
cure ophthalmia, to restore growth for 11 days, and to prolong life for 30 days (Table II). Nevertheless, this level of vitamin intake was not sufficient to cause storage. The same phenomenon was encountered when animals with low stores of vitamin A were depleted until they showed deficiency symptoms. Rats 50 to 60 gm. in weight on our low vitamin A ration showed 30 blue units per liver. At the end of 2 weeks vitamin A was no longer detect- able. However, growth of the animals did not cease until 4 weeks later. Growth in the absence of stored vitamin A was also demon- strated when small amounts of the vitamin were administered. For example, vitamin A-depleted rats which received 3 blue units of cod liver oil daily for 8 weeks, grew throughout the experimental period, without storage of any vitamin A whatsoever.
The discrepancy between the incidence of deficiency symptoms and the exhaustion of vitamin A reserves could be explained if vitamin A were present in the tissues in a form which escaped detection by the usual methods. Vitamin A, then, would in effect be merely a precursor of the active compound. Another suggestion is that vitamin A may not take part directly in ordinary metabolic processes but function indirectly as, for example, in the detoxication of certain products of metabolism.
It was hoped that the incubation of vitamin A with tissue preparations might throw some light on the relatively rapid dis- appearance of vitamin A when fed to depleted animals. Consist- ent results, however, were not obtained. When the conditions of incubation were mild, as for example when the vitamin was exposed to the tissues for short periods of time, at low temperatures, or in the presence of an antioxidant such as hydroquinone, only slight destruction of the vitamin was noted. When comparatively drastic conditions were employed, such as an exposure of an aqueous suspension of the vitamin to minced tissue for 24 or 48 hours at 37”, or an exposure of the tissue extracts to relatively large volumes of air, marked losses of vitamin A were noted, but the results obtained with tissues from depleted animals did not differ from those obtained with the tissues of normal rats. Oc- casional differences between normal tissues and those low in vita- min A were obtained by adding vitamin A to the minced tissues immersed in a 3- to 4-fold volume of 95 per cent alcohol and incu- bating for 24 or 48 hours at room temperature.
1. As measured by the SbC13 test, 95 per cent of the total vitamin A of the rat was found stored in the liver. The remainder was located in lung and in kidney tissue. On our regular stock ration only traces of vitamin A were found in rats under 3 weeks of age; thereafter the increase in the stores was rapid and regular.
2. The vitamin A content of new born rats was increased slightly by raising the vitamin A intake of the mothers during pregnancy; raising it during lactation increased the vitamin stores of the nursing young markedly.
3. The minimum daily dose of vitamin A necessary to produce storage in the liver was between 25 and 50 blue units. However, when storage had been attained, the reserves were depleted at the rate of 7 to 18 blue units daily, depending upon the amount stored.
4. When vitamin A was fed in the form of halibut liver oil, the amount stored in the liver was found to parallel the amount admin- istered, but only 10 to 20 per cent of the vitamin could be accounted for. When equal amounts of vitamin A were fed to normal and to vitamin A-depleted rats, the liver storage was greatest in the normal animals. When equal amounts of vitamin A were fed to animals in various stages of depletion, the amount stored was inversely proportional to the state of depletion.
5. Whereas the growth and survival of rats on a vitamin A- deficient diet paralleled both their previous vitamin intake and their vitamin A stores, the period of growth was longer than could be predicted on the basis of liver stores alone. Prolonged growth in the absence of stored or dietary vitamin A was observed.
6. Absorption and storage of vitamin A to a large extent took place within 6 hours after the ingestion of the vitamin. Vitamin A losses, due to destruction in the digestive tract, were large. Fecal elimination of vitamin A was small.
BIBLIOGRAPHY
1. Carr, F. H., and Price, E. A., Biochem. J., 20, 497 (1926). 2. Moore, T., Biochem. J., 24, 692 (1930). 3. Steenbock, H., Science, 68, 949 (1923). 4. Moore, T., Biochem. J., 26, 275 (1931). 5. Sherman, H. C., and Boynton, L. C., J. Am. Chem. Sot., 47, 1646
(1925). 6. McCoord, A. B., and Lute-Clausen, E. M., J. Nutrition, 7, 557 (1934).
7. Sherman, H. C., and Storms, L. B., J. Am. Chem. Sac., 47, 1653 (1925). 8. Dann, W. J., Biochem. J., 26, 1072 (1932). 9. Steenbock, H., Nelson, M. T., and Black, A., J. Biol. Chem., 62, 275
(1924-25). 10. Steenbock, H., Sell, M. T., and Nelson, E. M., J. Biol. Chem., 66, 327
(1923). 11. Davies, A. W., and Moore, T., Biochem. J., 28, 288 (1934). 12. Clausen, S. W., J. Am. Med. Assn., 101, 1384 (1933).
