University College, London.)

Transcription

1 : THE FATE OF CAROTENE INJECTED INTO THE CIRCULATION OF THE RAT. BY J. C. DRUMMOND AND R. J. MACWALTER. (From the Department of Physiology, Pharmacology and Biochemistry, University College, London.) (Received October, 93.) THE following results were obtained during an investigation having as its main objective the determination of the amount of vitamin A formed in the liver from the various isomers of carotene, although this was not achieved for reasons which will be apparent later. It has been claimed that the three natural isomers of carotene, a, and y, possess respectively the relative values, 2, as sources of vitamin A in the animal organism; a claim which can be correlated in a very interesting manner with the accepted views regarding the molecular structure of the compounds concerned [Kuhn, Brockmann, Scheunert and Schieblich, 933]. Attractive as this hypothesis may appear it cannot we think be unreservedly accepted in view of the errors associated with feeding tests such as those which formed the basis of the claims, and of our own experience with them for such a purpose. It being our view that many of the irregular results in the feeding tests are due to differences in the extent to which carotene is absorbed from the intestinal tract, an attempt was made to eliminate this source of error. It has been shown [Drummond, Gilding and Macwalter, 93] that carotene introduced directly into the circulation in the form of aqueous colloidal suspensions is rapidly removed from the blood stream by the Kupffer cells of the liver. It was proposed, therefore, to study the fate of the isomeric forms of carotene after administration in this manner; the only reservation being that in so doing one could not necessarily assume that this mode of administration reproduced the conditions obtaining when carotene is absorbed from the alimentary tract in the normal manner. Actually, our knowledge of the physical condition of carotene occurring normally in the blood, tissue fluids and organs of the body, is

2 THE FATE OF INJECTED CAROTENE. 237 very meagre. On the fact that the pigment cannot always be extracted by direct treatment with solvents such as ether, Palmer [95, 922] has based the view that it may be present in the form of a complex with proteins (carotoalbumins). The force of this argument has been weakened by van der Bergh and Muller [920] having demonstrated that similar behaviour may be shown by simple aqueous colloidal suspensions of carotene, an observation we have ourselves repeatedly confirmed. The suggestion that carotene may be absorbed from the intestine in the form of a water-soluble, diffusible complex with bile acids has been advanced by Drummond [932-3], Euler and Klussmann [933], and recently by Greaves and Schmidt [93], whose experiments on rats appear to show that the absorption of fl-carotene from the intestine is, to a large extent, dependent on the presence of bile acids. A complex of this nature can readily be prepared by fusing carotene with an excess of desoxycholic acid [Euler and Klussmann, 933], or, as E. R. Smith has discovered in our laboratory, by heating under reflux an excess of carotene with a solution of desoxycholic acid in dilute aqueous sodium carbonate'. We are also able to confirm the observation that the solutions of the complex contain carotene in a form more protected against oxidation than when it is in simple solution in the usual fat solvents. The general plan of the experiments was as follows. Young adult rats depleted of vitamin A were to have injected into the portal circulation carotene in the form of an aque6us colloidal suspension. After a short interval, during which practically all the pigment would be abstracted from the circulation by the liver, a lobe of the liver would be removed and the incision in the abdomen closed. After a suitable lapse of time the remainder of the liver would be removed for comparative analyses. It was first necessary, however, to ascertain whether under normal conditions vitamin A is uniformly distributed in the liver of the rat and to determine whether a substance abstracted from the circulation by the Kupifer cells of the liver, as carotene is known to be under such conditions, is deposited uniformly in the organ. EXPERIMENTAL. () Method. (a) Analytical. Liver samples were immediately freed as far as possible from blood and desiccated by grinding with anhydrous sodium sulphate. The dried material was then rapidly extracted by Unpublished work. The nature of this complex is being further investigated in this laboratory.

3 238 J. C. DRUMMOND AND R. J. MACWALTER. cyclohexane in a continuous extractor and the solvent removed to the required extent in a current of nitrogen. Total "fat" was estimated by weighing the solvent-free extract; vitamin A by the use of the Hilger " Vitameter A," in a few of the earlier experiments by the SbCl3 reaction; and carotene by colorimetric comparison against potassium dichromate solution in a Hellige colorimeter, employing a blue glass filter to increase the accuracy of the comparisons. (b) Operative. The rats were placed under ether anaesthesia and a medial incision of about in. made in the abdo inal wall. The injection of the colloidal carotene solution was usually made directly into the portal vein, a very fine needle being used to lessen the risk of haemorrhage. The solution usually contained approximately 0 05 p.c. and was prepared by pouring slowly a hot saturated solution of carotene in acetone into the requisite amount of isotonic glucose solution heated in a water bath and agitated by a rapid stream of nitrogen gas. This modification of the procedure of Fodor and Schoenfeld [93] enables one rapidly to prepare relatively strong carotene suspensions which are sufficiently stable to be sterilized by boiling. About 3 min. after injection of the carotene a lobe was tied off and removed. In practically every case recovery was satisfactory. An examination of Table I having given us reasonable assurance that it should be possible to detect the formation of vitamin A from carotene, provided sufficient was administered, it was necessary to ascertain whether after injection the pigment would be evenly deposited in the liver. Table II gives the amounts of carotene found in the three main lobes of rat livers from 3 to 0 min. after the injection of a colloidal suspension of the pigment directly into the circulation. The rats employed for this test had been kept for 9 or 0 weeks on a lipochromefree diet, and control estimations showed that only a negligible amount of yellow fat-soluble pigment was present in the liver before the infusion. As the results showed that the distribution of carotene was reasonably uniform a series of experiments was made in order to follow the formation of vitamin A from injected,-carotene. On the assumption that the carotene retained by the liver was quantitatively converted into vitamin A, and that the initial reserve of vitamin A did not exceed the value represented by E/g. = 0, an initial concentration of 3*2 mg./00 g. would produce an increase of vitamin A concentration of the order of 50 p.c., an increase far larger than the variations that might be encountered between the respective lobes.

4 THE FATE OF INJECTED CAROTENE. 239 TABLE I. Distribution of fat and vitamin A in lobes of rat liver. A. =anterior lobe, P.R. = posterior right, P.L. = posterior left lobe. A. FAT. (i) Healthy rats previously fed for 9 months on adequate diets containing 2-8 p.c. cod-liver oil. A P.R P.L (ii) Rats deprived of vitamin A for varying periods of from 5 to 2 weeks. A P.R P.L X B. VITAMIN A. (i) Healthy rats. (a) Estimations by SbCl3 reaction. Values represent blue units per g. of liver. A P.R P.L (b) Estimations by " Vitameter A." The numbers given represent extinction coefficient (E) at 3280 A. at such a concentration that g. of liver is contained in c.c. of extract. A P.R * 79- P.L (ii) Vitamin A-deficient rats. (a) Estimations by SbCl3 reaction. A P.R P.L (b) Estimations by "Vitameter A." A P.R P.L TABLE II. Carotene in mg. per 00 g. of liver. A P.R X P.L It will be seen from Table III that in every case except Exp. 2 they complied with the above minimal requirements. Even in Exp. 2 a similar increase might be expected, although the initial concentration of vitamin in the liver was rather high. The results were not a little disturbing; for whereas the one column appears to show a disappearance of the injected carotene from the liver, the other not only fails to demonstrate a significant increase in vitamin A but actually in several cases records a substantial loss of this substance.

5 20 Rat No J. C. DRUMMOND AND R. J. MACWALTER. TABLE III. Carotene mg./g. liver Initial Final Change * * X05 0* X002 0*020 Vitamin A E/g. liver Initial Final Change * ' '7 8*0 9.7 F * Repetition of the experiment having provided confirmation of this curious observation it was clearly imperative that the influence of the operation of partial hepatectomy should be investigated. In these cases carotene was not injected, the operation consisting solely of a removal of the greater part of one lobe of the liver. The remaining lobes were analysed at various periods after the operation. Rat No Time between injection and final analyses days Time between operation and final analyses days 2 2 TABLE IV. t- Initial 2* * * Vitamin A E/g. liver Final *0 * * *6 Change * *55-305

6 THE FATE OF INJECTED CAROTENE. In every case (Table IV) the removal of a lobe of the liver apparently brought about a reduction in the concentration of vitamin A in the residual tissue, the loss amounting in some of the rats to 50 p.c. or even more of the vitamin present before the operation. The rapidity with which this effect was produced is perhaps the most remarkable feature, for we have evidence that prior to the operation the rate of disappearance of the reserves of A from the livers of some of the animals, as a result of maintenance on an A-deficient diet, was surprisingly slow. This is illustrated by the history of the group of rats from which Nos., 7 and 37 were drawn. These animals had been reared on an adequate diet for 2 months before being given the A-deficient food mixture. They were, therefore, healthy young adults before the restriction was imposed, and it is not surprising that for a long time there was no apparent sign of impairment of well-being or development; indeed, after 7 months on the defective diet but few of these rats showed any clear signs of A deficiency. That at this age the stores of vitamin A in the liver are utilized much more slowly than in younger animals is generally recognized, but we we're surprised to learn from a series of random estimations made in September that the reserves were not significantly lower than those determined 3 months earlier (Table V). TABLE V. Values for E/g. liver. June September In striking contrast with this apparently slow rate of utilization there stands the remarkably rapid disappearance of as much as 50 p.c. of the vitamin stored in the liver when a lobe is removed. So far as we are aware there is only one recorded observation of a similar character and there can be little doubt that the two are closely related. Best and Huntsman [932] in studying the curious effect of choline in preventing the deposition of fat in the livers of rats fed on certain diets carried out a series of control experiments in which saline was administered in exactly the same manner as the choline solution. The group which received the saline injections had an average liver fatty acid content of 7. p.c., whilst the comparable group receiving the same diet, but no injections of saline, had the average fatty acid content of 5-6 p.c. These authors remarked "...it is apparent that the frequent PH. LXXXIII. 6 2

7 22 J. C. DRUMMOND AND R. J. MACWALTER. handling or subcutaneous injection interfered with deposition of liver fat." It would be premature to discuss these observations until much more information is available, but it is already clear that another important fragment of evidence has been provided in support of the view that the "fat metabolism" of the liver is every bit as active an ebb and flow of change as is that concerning the carbohydrate cycle. SUMMARY.. The distribution of fat and of vitamin A in the three main lobes of the rat liver is reasonably uniform. 2. Carotene injected into the portal circulation of rats in the form of aqueous colloidal suspensions is uniformly taken up by the liver lobes. 3. Carotene taken up in this manner tends to disappear from the liver during the next few days. Contrary to expectation this disappearance was not correlated with the formation of vitamin A, because in many of the animals a reduction in the amount of the vitamin also occurred after the injection and operation of partial heptectomy.. Experiments showed that the removal of one lobe of the liver may of itself bring about a substantial fall in the amount of vitamin A stored in the residual lobes. The expenses of this investigation were defrayed from a grant made by the Medical Research Council, which we desire gratefully to acknowledge. REFERENCES. Bergh, H. van der and MIuller, P. (920). Proc. Acad. Sci. Amst. 22, 78. Best, C. H., Huntsman, M. Elinor (932). J. Physiol. 75, 05. Drummond, J. C. (932-3). The Harvey Lectures, 28, 202. Drummond, J. C., Gilding, H. P. and Macwalter, R. J. (93). J. Physiol. 82, 75. Drummond, J. C. and Macwalter, R. J. (933). Biochem. J. 27, 32. Euler, H. and Klussmann, E. (933). Ark. Kemi. Min. Geol. B, No. 7. Fodor, A. and Schoenfeld, R. (93). Biochemn. Z. 223, 23. Greaves, J. D. and Schmidt, C. L. (93). J. biol. Chem. 05, Proc. xxxi. Kuhn, R., Brockmann, H., Scheunert, A. and Schieblich, M. (933). Z. physiol. Chem. 22, 29. Palmer, L. S. (95). J. biol. Chem. 23, 27. Palmer, L. S. (922). Carotinoids and Related Pigments. A.C.S. Monograph No. 9, New York.