Experimental. Method of estimation. It was impossible to use the digitonin method of estimation owing to. injections of cholesterol.

Transcription

1 THE CHOLESTEROL CONTENT OF NORMAL TISSUES AND THE EFFECT OF INTRAVENOU,S INJECTIONS OF CHOLESTEROL THEREON. BY E. NOBLE CHAMBERLAIN, Beit Memorial Fellow. (Department of Bio-Chemistry, University of Liverpool.) FOR convenience this paper will be divided into two sections, the first dealing with the cholesterol content of normal tissues in the rabbit, and the second with such modifications as were produced by intravenous injections of cholesterol. The first section was undertaken with the object of obtaining such light on the physiology of cholesterol as could be derived from a rough comparison of the cholesterol content of different tissues, and also of providing adequate controls to the experiments in Section II. The second section was expected to give information on problems of storage and metabolism, and the part taken by different organs in these processes. SECTION I. THE CHOLESTEROL CONTENT OF NORMAL TISSUES. Many estimations of blood cholesterol have been made, but the estimations of cholesterol in other tissues available in the literature are still very few, especially in the case of the suprarenals and the spleen. As the writer's work has been principally concerned with these organs, most attention has been paid to them in the present investigation, though estimations have also been made in the liver, brain, muscle, kidneys, lungs, and blood, for comparison with those of other workers. Experimental. Method of estimation. It was impossible to use the digitonin method of estimation owing to the expense of this substance, and the large number of experiments made. The method adopted, therefore, was that described by Grigaut(1). This consists in boiling the weighed tissue in a flask containing 70 p.c. alcohol, and 1 p.c. sodium hydrate until the tissue is dissolved, and is about 15 c.c. in volume. It may be mentioned here that in the case of some tissues, notably the liver, complete solution cannot be obtained, though the semisolid suspension is more or less uniformly distributed through the solution. These 15 c.c. are then put into a separating funnel graduated to 15 PH. LXVI. 17

2 250 E. NOBLE CHAMBERLAIN. and 30 c.c., cooled under running water, and then extracted twice with ether. The ethereal extract is washed each time with distilled water, and then evaporated to dryness. The residue is dissolved in chloroform and compared colorimetrically with a standard solution of cholesterol. The colour reaction used was that of Liebermann and Burchard, which consists in the addition of 2 c.c. of acetic anhydride, and 0-2 c.c. of pure sulphuric acid to the chloroform extract of cholesterol, and the subsequent development of a green colour. It was found essential to the proper development of this colour that all reagents and containers should be quite free from water, and that a definite fixed amount of sulphuric acid should be used in all cases, and not a "few drops" as sometimes advocated. A number of duplicate estimations were made to test the adequacy of the results for the present purposes. When the samples were taken from the same finely minced organ the results never differed by more than 10 p.c. With samples not previously mixed by mincing, it was found that the results might differ as much as 18 p.c., doubtless because samples *taken from different parts of an organ are not identical. This was par- -±icularly the case with the liver. Owing to these discrepancies in certain of the estimations it should be mentioned that in comparing the results in Section II with the controls of Section I, no variation of less than 20 p.c. has been accounted of any significance. This rule allows a wide margin for experimental error, for it will be seen that the differences upon which any arguments were based, were at least 40 p.c., and sometimes as much as 300 p.c. Where duplicate observations have been made the average between the two results has always been used. In the case of the suprarenals and spleen the whole organ was used in the estimations to avoid errors of sampling. All results are based on the wet weight of the tissue. RESULTS. The results,are given in tabular form. The findings are expressed in "milligrams per cent." (i.e. per 100 grm. of fresh tissue), except in the case of the suprarenals and brain where grams per cent. is more convenient. In addition to the percentage, the total amount of cholesterol is given for those tissues where it was possible to use the whole organ for the estimation.

3 CHOLESTEROL IN TISSUES. 251 Rabbit No Weight in grm TABLE I. Cholesterol content of normal ti8sues. Stprarenals. Total cholesterol Weight Grm. of Average per kilo of adrenal percentage Total body adrenals per kilo cholesterol cholesterol weight in grm. body weight (gri.) (mg.) (mg.) *226 0* *127 6* * *213 6* * * * * * *209 6* * * *270 0* * * * * Average TABLE IL Cholesterol content of normal tissues. Rabbit Weight No. in grm Spleen. Total Weight Grm. of cholesterol of spleen Total per kilo spleen per kilo Cholesterol cholesterol body weight in grm, body weight (mg. p.c.) (mg.) (mg.) * * * * * i * * *0 0* *2 0* * *650 0* * *760 0* *9 1* * *2 1*7 Average

4 252 E. NOBLE CHAMBERLAIN. TABLE III. Cholesterol content of normal tissues. Rabbit Weight Liver Muscle Brain Lung Kidney Blood No. in grm. (mg. p.c.) (mg. p.c.) (grm. p.c.) (mg. p.c.) (mg. p.c.) (mg. p.c.) *2 Average DISCUSSION. Varying with their richness in cholesterol the tissues examined fall into three groups. Firstly, the brain and suprarenals, which contain respectively an average percentage of 1.8 and 7-3 grm., and are thus the richest in cholesterol. Secondly, the spleen, liver, kidneys, and lungs, which do not differ materially in their content, containing an average of between 300 and 500 mg. p.c. in the majority of cases. Lastly, blood and muscle may be classed together as being the poorest in cholesterol, the blood containing an average of 78 mg. p.c. and the muscles an average of 64 mg. p.c. Suprarenals. From the tables one striking fact stands out, namely that compared with other tissues the suprarenals contain a very variable amount of cholesterol. The percentage varies roughly from 4 to 11 grm. whilst the total amount in the glands varies from 9 to 60 mg. This probably indicates that the glands take an active part in cholesterol physiology, the amount found in the glands reflecting varied physiological states. The suprarenals contrast forcibly with the brain in this respect, as the brain, though also containing a large amount of cholesterol, shows only small variations. The variations in other tissues are not sufficiently great to give any clue as to whether they take an active part in cholesterol physiology.

5 COWLOLESTEROL IN TISSUES. The cholesterol of whole blood was estimated in most cases by Leib off's method (2), which, like,grigaut's method for the tissues, gives values for the total cholesterol. A few estimations were also made on the blood serum by Grigaut's method. Four such estimations gave an average of 37 mg. p.c., about half the value usually obtained in rabbits for whole blood. This is an interesting contrast with human blood where the amounts in serum and whole blood are approximately equal. Results of other authors. The results given here for brain, liver, kidneys, lungs, and blood, agree moderately well with those recorded by Ellis and Gardner (3) who used the more accurate digitonin method of estimation, and also with those given by Baumann and Holly(4) for kidney. The few estimations made by Landau and Mc Nee (5) for the suprarenals led them to regard 10 p.c. as an abnormally high value. The present larger series of observations show that 10 p.c. is by no means uncommon, and must be regarded as falling within physiological limits. Since completion of this work Dr E. J. Baumann of New York has kindly allowed me to compare some of his unpublished figures for the cholesterol content of the suprarenals with my own. The method of estimation was similar, and the results are in close agreement. SECTION II. THE EFFECTS OF INTRAVENOUS INJECTIONS OF CHOLESTEROL. 253 The effect of feeding cholesterol to animals has been investigated by numerous workers (Landau and McNee(5); Rothschild(6); Sternberg(7); Zinserling(s)), who, however, have paid most attention to the histological changes produced, and have made very few chemical estimations. The injection of cholesterol directly into the blood stream has been attempted only on a few occasions, and whilst the histological changes produced are described in some detail no chemical analyses are given. Dewey(9) records experiments of this nature, and notes an infiltration of the liver with cholesterol, a variable infiltration of the suprarenals, but no apparent increase in the spleen. Since, however, the staining reactions of a substance such as cholesterol can be greatly modified by its association or combination with other substances in the tissues, histological methods are of little quantitative value. The objects of the present work, therefore, were to ascertain by chemical estimations what gross modifications could be produced in the cholesterol content of various organs by intravenous injections of this

6 254 E. NOBLE CHAMBERLAIN. substance. It was hoped that the results would throw light on certain aspects of the physiology of cholesterol. Experimental. It was found difficult to prepare emulsions of cholesterol which were sufficiently perfect to pass through the capillaries without untoward results though numerous methods were tried. Those which were unsatisfactory, as judged by the size of the particles under the microscope, and by the fact that injected animals died, need not be mentioned here. The following method, however, usually gave good results: grm. of oleic acid and 0 75 grm. of cholesterol were weighed into the same test-tube and acetone was added in sufficient quantities (about 10 c.c.) to dissolve the whole when heated. The acetone solution was then poured little by little into a measuring cylinder containing 16-4 c.c. N/10 NaOH, i.e. the amount required to neutralise the oleic acid. The vessel was constantly agitated during the operation. A fine emulsion resulted which was concentrated by heating over a water bath until the volume was about 18 c.c., so that the emulsion contained approximately 4 p.c. of cholesterol.' The emulsion was used in amounts of 2 to 7 c.c. and injected into the veins of the ears very slowly. In the majority of cases there were no ill effects, though occasionally the animal showed signs of collapse for a few minutes, and then recovered. If the emulsions were kept longer than 14 days they seemed to deteriorate, and it is advisable that they should be made up fresh in small quantities as required. RESULTS. The effects of the cholesterol injections will be seen in the various tables. They may be discussed best under the headings of the different tissues. Blood. The blood cholesterol was estimated in numerous cases just before the animal was killed. The blood was taken from the opposite ear to the one used for cholesterol injection. The cholesterol content was raised in all cases except one, but not to such a marked degree as was anticipated. The average increase was about 50 p.c. These findings accord with those of Dewey(9), but are opposed to those of Weltmann and Biach(lo), who stated that a substance such as cholesterol which gains access to rabbit serum cannot be readily eliinated. The rapidity with which cholesterol is removed from the blood, however, is variable, for in one case (Rabbit 109) the content was normal

7 CHOLESTEROL IN TISSUES j hours after the injection, whilst in another (Rabbit 84) the cholesterol was still raised to 151 mg. p.c. (normal 78 mg. p.c.) 48 hours after the injection. TABLE IV. The effects of intravenous injections of cholesterol. Period Milli- between Blood Liver Lung Kidney Muscle Brain grams injec- chole- chole- chole- chole- chole- choleof chole- tion and sterol sterol sterol sterol sterol sterol Rabbit Weight sterol deatb (mg. (mg. (mg. (mg. (mg. (grm. No. in grm. injected (hours) p.c.) p.c.) p.c.) p.c.) p.c.) p.c.) i ij i Average TABLE V. The effects of intravenous injections of cholesterol on the spleen. Grm. of Total cholesterol Weight spleen Total per kilo of per kilo Chole- chole- body Rabbit Weight spleen body sterol sterol weight No. in grm. in grm. weight (mg. p.c.) (mg.) (mg.) Average

8 256 E. NOBLE CHAMBERLAIN. TABLE VI. The effects of intravenous injections of cholesterol on the suprarenals. Total Weight Grm. of cholesterol of adrenal Total per kilo Rabbit Weight suprarenals per kilo Cholesterol cholesterol body weight No. in grm. in grm. body weight (gri. p.c.) (mg.) (ig.) * * * * *5 7* * * * * Average Suprarenals. The German school supposes that the suprarenals are concerned in storing cholesterol, and that the amount present in the glands is proportionate to the amount in the blood. If this were so hyper-cholesterinaemia would be expected to result in an increased cholesterol concentration in the suprarenals. In the present experiments, however, no such increase was found. On the contrary there was a definite decrease. The average percentage decreased from 7-3 in normal animals to 4-6 in cholesterol injected animals. Also the total amount of cholesterol in the glands was correspondingly diminished. This was. not due to any variation in the weight of the suprarenals, since their average weight proportionate to the body weight remained constant. It is possible that part of the decrease was due to the sodium oleate with which the cholesterol was administered since injections of this substance alone gave a similar though much smaller decrease, but it is improbable that the whole could have been due to the sodium oleate. The French authors, contrary to the German, regard the suprarenals as mainly concerned in the synthesis of cholesterol, and as it has been shown (Fraser and Gardner, Nomura, Sano, and others (11, 12, 13)) that the body may also derive cholesterol from food, it seems reasonable to suppose that a balance is normally maintained between the exogenous and endogenous supply, so that an increase in exogenous cholesterol might be expected to produce a diminished endogenous production. If such a theory were correct the decrease in the cholesterol of the

9 CHOLESTEROL IN TISSUES. 257 suprarenals in the present experiments might be interpreted as indicating a diminished production on the part of the glands due to an increase in exogenous supply following the intravenous injection. Spleen. Of all the tissues examined this organ showed the greatest changes. The percentage of cholesterol was do'ubled, and, as at the same time the average weight of the organ was considerably increased, the total amount of cholesterol in the spleen was three or four times its normal value. It is evident, therefore, that, weight for weight, the power of removing excess cholesterol from the blood is greater in the spleen than in any other tissue. The question arises as to whether this property is exercised by the spleen in the normal animal. It may be suggested that by use of this function the spleen may normally regulate the level of the blood cholesterol, continually removing small excesses. In support of this theory it is to be noted that most authors (Eppinger, King, Sotti and Torri(14, 15, 16)) find that removal of the spleen produces hyper-cholesterinaemia, though Bodansky(17) found no such increase in blood cholesterol, nor did Randles and Knudson(18) after splenectomy in rats. In criticism of the theory it may be urged that in the experiments described here, the spleen merely removes cholesterol as it would remove any other foreign particulate substance, for example, silver or Indian ink, from the blood stream. It should be pointed out, however, that even in cases of hyper-cholesterinaemia where there is reason to believe that the cholesterol is in a similar form to that in which it normally circulates, a great excess is found in the spleen. This is illustrated by a case reported recently by Dyke(19) where hyper-cholesterinaemia of 1250 mg. p.c. was present, associated with great enlargement of the spleen which contained 41 times the normal percentage of cholesterol. Liver. The liver also showed an increase in its cholesterol content following intravenous injections, but this was very much smaller than in the case of the spleen. Nevertheless it is evident that the removal of small amounts by such a large organ as the liver would be equivalent to the removal of a very considerable total amount. It is possible that the liver may also take'a part in maintaining the blood cholesterol at a constant level. Brain, kidneys, lungs, and muscle. None of these tissues showed any material change in its average cholesterol' content after intravenous injection.

10 258 E. NOBLE CHAMBERLAIN. The effect of repeated injections of cholesterol. Four experiments were made in which in place of the usual single dose of cholesterol two to four injections were made extending over a period of two to four days. The results were similar to those obtained by single injections, the spleen showing the most marked increase in cholesterol, and the liver a lesser increase. One important difference, however, will be observed. Instead of the customary decrease in the cholesterol content of the suprarenals the average value is alnost identical with that of normal buck rabbits. Evidently the influence exerted by the cholesterol injections on the suprarenals is not sustained if the injections are repeated. TABLE VII. Effects of repeated injections of cholesterol. Supra- Chole- renals Liver Spleen Kidney sterol chole- chole- chole- chole- Weight in- sterol sterol sterol sterol Rabbit in jected Period over Period since (grm. (mg. (mg. (mg. No. grm. (mg.) which given last dose p.c.) p.c.) p.c.) p.c.) hours 24 hours - -' days 24 hours hours 3 days days 23 hours Average Effects of reticulo-endothelial blockade. On histological grounds it has been suggested by Rothschild(2o), Zinserling(s), McMeans2l), and Khalatov(22) that cholesterol can be taken up by the Kupfer cells of the liver, and it seemed probable in the present experiments that the reticulo-endothelial elements of both the spleen and the liver might be responsible for the removal of the excess of cholesterol. If this were the case, a blockade of the R.E. system, followed by intravenous injections of cholesterol, would be expected to interfere with the removal of cholesterol by these organs. Experiments were, therefore, made in which the R.E. system was blocked by intravenous injections of Indian ink for several days, followed by the usual injection of cholesterol. Bedson(23), in his work on blood platelets, found blockade of this description to be effective. It will be seen from the tables, however, that the blockade did not prevent the accumulation of cholesterol in the spleen, though the degree of infiltration was not quite so marked as usual. In the case of the liver the degree of cholesterol infiltration was not materially altered.

11 CHOLESTEROL IN TISSUES. 259 TABLE VIII. Effect of sodium oleate injections on tissue cholesterol content. Total Total chole- Supra- chole- sterol renals sterol per Spleen Liver Kidney Brain Sodium chole- in kilo chole. chole- chole- chole- Weight oleate sterol supra- body sterol sterol sterol sterol Rabbit in solution (grm. renals weight (mg. (mg. (mg. (grm. No. grm. injected p.c.) (mg.) (mg.) p.c.) p.c.) p.c.) p.c.) c.c p.c c c p.c c.c p.c c.c p.c c.c p.c c.c p.c. Average TABLE IX. Effect of Indian ink injections on tissue cholesterol content. Supra. Grm. of Blood Liver Spleen Lung renals Indian chole- chole- chole- chole- chole- Rabbit ink Period of sterol sterol sterol sterol sterol No. injected action (mg. p.c.) (mg. p.c.) (mg. p.c.) (mg. p.c.)(grm.p.c.) days hours days Average Effect of blockade of the R.E. system with Indian ink followed by injection of cholesterol Amount Supraof Liver Spleen renals Kidney Grm. of chole- Period chole- chole- chole- chole- Indian Period sterol of sterol sterol sterol sterol Rabbit ink of injected action (mg. (mg. (grm. (mg. No. injected action (mg.) (hours) p.c.) p.c.) p.c.) p.c.) hours i days days 200 6j Average The experiments cannot be regarded as conclusive and are capable of two interpretations, either that the power of the spleen and liver to remove excess of cholesterol does not depend on their reticulo-endothelial

12 260 E. NOBLE CHAMBERLAIN. elements, or alternatively that the blockade was not complete. The latter explanation seems the more probable, especially in the light of previous histological work, and of the slight decrease in the amount of cholesterol removed by the spleen after blockade. SUMMARY. 1. Tables are given showing the cholesterol content of the suprarenals, spleen, brain, liver, kidneys, lungs, muscle, and blood in normal buck rabbits. 2. The great variations in the amount of cholesterol in the suprarenals as compared with other tissues suggest that these organs take an active part in cholesterol physiology. 3. Intravenous injections of cholesterol resulted in: (a) A definite hyper-cholesterinsamia, though less than would have been expected, showing that the cholesterol has been removed rapidly from the circulation. (b) The suprarenals showed no increase in cholesterol, but on the contrary a definite decrease. This, it is argued, is in opposition to the idea of storage in these organs, and indirectly supports the theory of synthesis. (c) The spleen showed a marked increase in its percentage and total amount of cholesterol, removing proportionate to its weight a greater amount of cholesterol than any other tissue. It is suggested that the spleen may be concerned normally in maintaining the blood cholesterol at a fixed value by continually removing small excesses. (d) The liver takes up a smaller amount of cholesterol as judged by the percentage of cholesterol present. It may also normally have the function of maintaining the blood cholesterol constant. (e) The effects of reticulo-endothelial blockade did not decide the question as to whether the power of the spleen and liver to remove cholesterol depends on their reticulo-endothelial elements. (f) The lungs, kidneys, brain, and muscle, showed no appreciable change after intravenous injection of cholesterol. I wish to express my gratitude to Prof. W. Ramsd e n in whose department this work has been carried out, and who has always given me most valuable help and advice. My thanks are also due to Miss T. Hughes for her kind help in the preparation of this paper.

13 CHOLESTEROL IN TISSUES. 261 REFERENCES. 1. Grigaut, A. Compt. Rend. Soc. de Biol. 71. p Leiboff. Journ. Biol. Chem. 61. p Ellis and Gardner. Proc. Roy. Soc. B, 85. p Baumann and Holly. Amer. Journ. Physiol. 75. p Landau and McNee. Beitriige z. Path. Anat. 58. p Rothschild. Proc. New York Path. Soc. 14. p Sternberg. Beitrige z. Path. Anat. 60. p Zinserling. Beitriige z. Path. Anat. u. Allgem. Path. 71. p Dewey. Arch. Int. Med. 17. p Weltmann and Biach. Zeitschr. f. Exp. Path. u. Ther. 14. p Fraser and Gardner. Proc. Roy. Soc. B, 98. p Nomura. Tohoku Journ. Exp. Med. 5. p Sano. Ibid. 4. p Eppinger. Berl. Klin. Woch King. Arch. Int. Med. 14. p Sotti and Torri. Arch. Ital. de Biol. 65. p Bodansky. Journ. Biol. Chem. 63. p Randles and Knudson. Ibid. 66. p Dyke. Journ. Path. and Bact. 31. p Rothschild. Proc. New York Path. Soc. 14. p McMeans. Journ. Med. Research, 33. No Khalatov. Petrograd Diss. p Bedson. Brit. Journ. Exp. Path. 7. No. 5. p