metabolism, as in Fr6hlich's syndrome; and by physiological facts, such Coope and Mottram (1914).

Transcription

1 THE EFFECT OF PITUITRIN ON THE FATTY ACID OF THE LIVER. By R. COOPE AND E. N. CHAMBERLAIN'. (From the Department of Biochemistry, University of Liverpool.) THE work described in this paper was undertaken to investigate some of the factors affecting the mobilisation of fat from the depots and its transference to the liver, and the influence upon it of certain of the internal secretions. The possibility that extract of the posterior lobe of the pituitary gland might produce a fatty infiltration of the liver was suggested by the clinical association of pituitary disease with striking anomalies of fat metabolism, as in Fr6hlich's syndrome; and by physiological facts, such as the increased activity of the gland in the later stages of pregnancy, at which time a definite fatty infiltration of the liver had been found by Coope and Mottram (1914). Mottram (1909), and Coope and Mottram (1914), having shown that the fat content of the liver was very constant in rabbits, the experiments were confined almost entirely to these animals, usually weighing from 1 to 2 kgms. Pregnant and lactating animals were discarded. The rabbits had been kept singly in cages, usually for not less than a month, and were fed on a constant and liberal diet of oats, bran and green vegetables. The preparation used was mainly Parke Davies and Co.'s "pituitrin," though in a few experiments other preparations were employed. It was noted that injections of even 4 c.c. of pituitrin had no appreciable ill-effect either on the general health, or on appetite. This was important as excluding the possibility that any fatty infiltration of the liver obtained was due to hunger. The pituitrin was injected subcutaneously into the flank. At first the dosage and times of injection were tentative, small repeated doses being given in the earlier experiments, usually within the 24 hours before death. Later, the effect of single, larger doses (3 or 4 c.c.) given at varying intervals before death, was studied, and a "time curve" was plotted. The pituitrin was usually mixed with 5 or 6 c.c. of a warmed saturated solution of gum arabic, which was found to prevent marked local reaction, apt to occur with injection of 3 or 4 c.c. of pituitrin alone. It was 1 Who received a part-time grant from the Medical Research Council.

2 70 R. COOPE AND E. N. CHAMBERLAIN. thought, too, that the gum would spread the absorption of the extract over a longer period. The animal was weighed just before death. It was killed by a blow on the back of the head. The liver was quickly excised, weighed after removal of the gall bladder and obvious connective tissue, and minced by a special mincer into a fine cream, which was then well mixed in order that the two aliquot samples taken might be truly representative of the whole liver. In some of the earlier experiments, neglect of this precaution, with a view to saving time or material, resulted in discrepancies in the final percentages of fatty acid, which although small were outside the limits of experimental error. Differences in the amount of fat in different parts of the liver have previously been pointed out by Do wler and Mottram (1918). Only the pure non-volatile fatty acids were estimated, Mottram's (1910) method being used for the purpose. Small pieces of the liver were taken and examined histologically. A sample of blood was taken from the heart immediately after death -some was used for an estimation of sugar by the F o 1 in - W u method, and the rest was oxalated and centrifuged for naked eye evidence of lipaemia. Urine was withdrawn from the bladder, and tested for sugar and ketone bodies. Control rabbits. The fatty acid content of the liver was estimated in six control rabbits (Table I A). The mean content was 2X86 p.c. of fresh liver. The figure agrees quite closely with those of earlier work, as for example the "normals" in the pregnancy experiments previously mentioned, where the mean in six animals was 2-62 p.c. It is justifiable, therefore, to regard a percentage higher than 3 as an abnormal fat content of the liver under the conditions of feeding and management described. Rabbits injected with pituitrin. The results with pituitrin are given also in Table I. As the size of the liver varies considerably even in animals of the same body weight, both body weight and total weight of the liver were taken, and the ratios of liver fatty acid to body weight, and to (body weight)2/3 estimated. No obvious relation between these ratios and the percentages of fatty acid in the liver was found. The averages of these estimations are given in Table II, and show that even had the factors of body weight or of surface area to be taken into consideration, the above-mentioned ratios would not contradict the conclusions drawn from the percentages. The experiments with rabbits given pituitrin with gum not more than 24 hours before death (Table I B a) form the only consistent series

3 C. Pitulitrin 1 2 Exp. With gum Doses 26 3of 3c.( 27 3of2c.( Without gum 3 4 B of of j oflc.c PITUITRIN AND LIVER FAT. TABLE I. Percentage of fatty acid in liver of rabbit. B. Pituitrin injected in one dose of 4 c.c., except as mentioned a. Injected with gum Hours of Exp. action p.c. A. Controls Ex ;p. p.c k k 7-25 Mean * * 30 2* * 3 c.c. injected b. Injected without gum injected in successive doses * Hours from 21t 6k 4-88 injection * 3 c.c. injected t 5 c.c. injected first last p.c. c. Rabbit fed previously on carrots 48 18k k k 3-69 d. Pituitrin boiled for 6 hours, and c.c injected with gum.c k e. Pituitrin boiled with gum for 11 hours for which the mean results can fairly be compared with the normal mean, shown in Table I A. A. Controls *54 B. Single large dosewithgum (action 24 hours or less) TABLE II. Mean results compared. F.A. in liver Total liver ILi' ver F.A. Liver F.A. P.C. F.A. (g.) Body weight (Body weight)2/3 74 x *6 x It is seen that in the "pituitrinised" animals, the percentage of liver fatty acid is approximately double that of the controls. Even if we lump together all the 24 experiments in which pituitrin was given, with their great differences in time, frequency, amount and conditions of dosage, the effect on the fatty acid content of the liver is still markedly shown by a mean percentage of In a series of experiments on animals in which. it is impossible to 71

4 72 R. COOPE AND E. N. CHAMBERLAIN. control all disturbing factors, it cannot be expected that each individual experiment will show the same degree of response, or even show the characteristic infiltration at all. Exp. 7 is of interest as illustrating this point. We regard the above experiments, therefore, as amply justifying a conclusion that extracts of posterior lobe of the pituitary body produce a well marked fatty infiltration of the liver. The general appearance of the liver in the "pituitrinised" animals was that of a fatty liver. It was softer and paler than the normal dark brown liver; it was frequently mottled, yellowish in tinge, and in some cases almost fluid after mincing. The histological evidence agreed throughout with the chemical analyses. Pieces were frozen in formol-gum, and stained with either Sudan III or Scharlach R. The fat in these infiltrations was remarkable for its equality of distribution. There was only a slight tendency to accentuation at the centre and periphery of the lobule. Specimens of livers showing high percentages of fatty acid were also stained (paraffin sections being prepared for this purpose) with haematoxylin and eosin, to demonstrate the cell structure. In no case was there any degeneration either of nucleus or of protoplasm. The effect of the medium in which the pituitrin was given. In the majority of the experiments, the pituitrin was mixed with 5 or 6 c.c. of gum arabic solution before injection; to the rabbits of Table I B b the pituitrin was given alone. A comparison of these results, together with Exps. 28, 29 and 30 (Table I C) with those of Table I B a (with gum), suggests that the effect was less constant than when gum was added. Two explanations may be offered. It was our experience that the gum prevented any serious local reaction such as was liable to result from a large dose of pituitrin alone and might well interfere with the proper absorption of the drug. It is also possible, though we have no proof of this, that when given with gum, the pituitrin is absorbed more slowly, and its effect is spread over a longer period. It will be shown later that when pituitrin is given with gum, the effect does not pass off until about 24 to 30 hours after the injection. Exps. 21 and 29 strongly suggest that without gum, absorption is more rapid, and that the onset and disappearance of the fatty infiltration of the liver are correspondingly accelerated'. The period of action of the pituitrin, and the degree offatty infiltration produced. In the experiments with single doses, the pituitrin was allowed to act for various periods of time before the animal was killed. Fig. 1 1 Two later experiments, however, have shown, four hours after injection of 4 c.c. pituitrin with gum, 4-25 p.c. and 6-11 p.c. fatty acid in the liver.

5 PITUITRIN AND LIVER FAT. shows the time relations of the liver infiltration when pituitrin was given with gum (3 or 4 c.c. pituitrin in 5 or 6 c.c. of gum arabic solution). Since it is based on comparatively few experiments, it must be regarded as affording at best a rough approximation to the true curve. 9 8 M 73 ;, 0 16 I 0X Gum arabic solution alone does not produce fatty infiltration of the liver. In Exp. 2, 8 c.c. of the gum solution was injected subcutaneously into an animal which was killed 18 hours later. The fatty acid content of the liver was 2*59 p.c., a normal figure. Further controls are afforded by the experiments with "deactivated" pituitrin given with gum (Table III, Exps. 31, 32 and 33). Experiments with "deactivated" pituitrin. A series of experiments was undertaken to show whether pituitrin which had been " deactivated " produced any fatty infiltration. For this purpose the method of Dale and Dudley (1921) was adopted. They found that both pressor and oxytocic principles were destroyed by boiling for 6 hours with 0 5 p.c. HCI. Previously H. S. Adams (1917) had shown that the oxytocic principle could be destroyed by boiling at 1000 C., if the H. ion concentration was of the order N x 10-. Pituitrin was boiled for 6 hours on a water bath (a reflux condenser being used) with an equal volume of 1 p.c. HCl, and then approximately neutralised with N/5 NaOH. From this preparation, the equivalent of 3 or 4 c.c. of pituitrin was taken, mixed with 5 c.c. of the gum solution, injected into three rabbits in the usual way, and allowed to act for 16 to 17 hours. A preparation treated as above, save for the boiling, was injected into a control animal, and showed that this particular batch of pituitrin produced the usual fatty infiltration. Another sample was

6 74 R. COOPE AND E. N. CHAMBERLAIN. boiled for 6 hours with an equal quantity of distilled water and similarly injected. The results are given in Table III. TABLE III. "Deactivation" experiments. Exp. Dose of pituitrin Hours of action Liver F.A. (P.C.) Control. Active pituitrin 12 4 c.c. 17j 5-82 Control. Pituitrin boiled, no acid 24 4c.c. 16a "Deactivation" 31 3 c.c c.c. 16j c.c Mean 3-02 It is thus clear that the substance which causes the fatty infiltration is either the same as the pressor and oxytocic substances, or is destroyed by the same treatment. With injection of pituitrin boiled for I{ hours with gum arabic (Exp. 25, Table I B e) there was for some reason absence of the expected infiltration. Whether this apparent "deactivation" occurs regularly under such treatment must be left undecided. Experiments with other tissue extracts. Further control experiments were made to see if injection of tissue extracts other than pituitrin produced fatty infiltration of the liver. "Antuitrin," a preparation of the anterior lobe of the pituitary gland, prepared by the same firm (Parke Davies and Co.), and by a similar method, was thought specially suitable for the purpose. It was given in single doses of 3 c.c. with gum, in the same way as the pituitrin, and the animal killed 15 to 18 hours later. The results are shown in Table IV. TABLE IV. Experiments with antiitrin Exp. Liver F.A. (P.C.) Mean 2-98 The figures must be compared with those of Table I B a. It is clear that antuitrin produces no such effect as was obtained with pituitrin. It is true that in Exps. 35 and 36, there is a slight increase of the fatty acid percentage above the normal. In Exp. 35, however, the liver was small for the size of the animal (49 g. for a rabbit of 2 kilos.), and in any case the small increase in either experiment may well be due to inclusion of small amounts of posterior lobe material in the " antuitrin."

7 PITUITRIN AND LIVER FAT. Experiments made with other tissue extracts have so far also failed to produce fatty infiltration of the liver. It is hoped to publish an account of these in another paper. The absence of fatty infiltration after injection of "deactivated" pituitrin affords further support to the view that the infiltration is due to some special constituent of the posterior lobe of the pituitary body. Blood and urinary results in rabbits. No attempts have been made to estimate the blood sugar during the experimental period, lest the taking of samples should upset the animal, and so prejudice the issue. In samples taken immediately after death, the blood sugar varied so widely both in normal and in pituitrinised animals as to make it clear that nervous or other disturbances deprived the results of all significance. The range of variation was from 90 to 500 mg. per 100 c.c., yet in no case was there any glycosuria. Throughout the whole of the experiments with pituitrin, the only one in which a lipaemia was observable by the naked eye was rabbit 13. This was also the only case in which there was excess of acetone in the urine (a trace, by Rothera's test). Sugar was always absent in the urine removed from the bladder at death. Experiments with rats. Table V shows, although the experiments are few in number, that under appropriate conditions, pituitrin produces the same effect in rats as in rabbits; the dose necessary, relative to the surface area, appears however to be much larger. TABLE V. Experiments with rats. Total Hours of No. of body weight action of /O Exp. rats (g.) Dose of pituitrin pituitrin F.A. mn liver 37 3 (6) (6) c.c. each (6T) 1 c.c.,, (6) - I c.c. each with gum (V) 722 c.c.,,,, 15j (6) c.c.,,,, 15i 4-46 Three or four rats were used for each experiment, and the livers minced together to give enough material for accurate estimation. With doses of 1 c.c. of pituitrin, whether alone or with gum, no infiltration occurred. When the dose was increased to 2 c.c. per rat, and given with gum, fatty infiltration of the liver was found both histologically and chemically in 151 hours. It should be added that one rat, not included in the above experiments, died 20 minutes after injection of 2 c.c. of pituitrin. The effect of carrot feeding on the pituitrin effect in rabbits. Two ex- 75

8 76 R. COOPE AND E. N. CHAMBERLAIN. periments were made on rabbits (Table I B e, Exps. 22 and 23) which had previously received a high carbohydrate diet. Rabbit 23 was fed for nine days on carrots only, and then injected with 4 c.c. of pituitrin with gum. During the actual experimental period it received the ordinary diet. Eighteen hours after the injection, the liver contained 3-69 p.c. of fatty acid, as against 3-96, 4-11 and 6*49 p.c. in the comparable experiments 8, 9 and 14 respectively. Rabbit 22 was fed for 14 days before death on the usual diet with the addition of as much carrot as it would eat; 151 hours after a similar injection of 4 c.c., the liver contained 3-72 p.c. of fatty acid, as against 7-25 and 8-36 p.c. in the comparable experiments 15 and 16 (Table I B a). In both experiments, therefore, the fat content, although increased, was considerably less than in the rabbits injected with similar doses but without previous feeding with carrots. The glycogen of the liver was estimated only in Exp. 22, and was found by Pfluiger's method to be 2S52 p.c. Discussion. Carraro (1908) found that repeated injections of pituitary extract produced actual necrosis of the liver cells, with "fatty degeneration." In our own experiments there was nothing to suggest damage to the liver cell, even when the fatty acid content was as high as 8 p.c. Moreover, the disappearance of the pituitrin effect within about 30 hours indicates that the accumulation of fat was a physiological process. There is much to suggest that increased secretion of pituitrin in the living animal leads to increased transfer of fat from depots to liver, and that deficient secretion results in accumulation of fat in the depots. Thus it is well known that in the later stages of pregnancy, both in man and other mammalia, there is increased activity of the pituitary gland, often associated with a definite hyperplasia; and D ixon and Marshall (1925) have recently shown in experimental animals that the cerebrospinal fluid contains an increasing amount of the oxytocic constituent of pituitary secretion in the later stages of pregnancy. Coope and Mottram (1914) had also found with rabbits that an infiltration of the liver with depot fat occurred shortly before parturition. Conversely, the massive accumulation of depot fat is one of the most characteristic features of Fr6hlich's syndrome, a condition associated with destructive lesions of the gland, and therefore presumably with deficiency of posterior lobe secretion. Although the two experiments with carrot-fed rabbits appear at first sight to support the view of Rosenfeld (1895, 1903) that accumulation of fat and of glycogen respectively in the liver are mutually antagonistic,

9 PITUITRIN AND LIVER FAT. it should be realised that this view was based entirely on observations of highly pathological livers, and may fail to establish itself as a general proposition'. So far as the absence of glycosuria and hyperglycemia can justify any inference concerning the glycogen content of the liver, there was nothing to suggest that the pituitrin fat-liver had been depleted of glycogen. In our experiments glycosuria was never found, and Burn (1923) has shown that the prevalent view that pituitrin causes hyperglycaemia is incorrect. Further experiments on this aspect of the problem are in progress. SUMMARY. 1. Injection of extract of posterior lobe of the pituitary body into rabbits and rats is followed by a well-marked increase in the amount of fatty acid in the liver. 2. The time relations of this effect are more consistent if the extract is given with gum arabic solution. 3. The fatty acid infiltration after pituitrin with gum reached its maximum between 10 and 15 hours, and disappeared before the 30th hour. 4. The infiltration does not occur if the pituitrin has been previously submitted to such treatment as destroys its oxytocic and pressor constituents. 5. Control experiments with extracts of other tissues have, up to the present, failed to produce fatty infiltration of the liver. We wish to express grateful acknowledgment for much helpful advice given by Prof. R am s d en throughout this investigation. We are indebted also to the Medical Research Council for a grant covering part of the expenses. REFERENCES. Adams. Journ. Biol. Chem Burn. This Journ Carraro. Arch. per la Se. med Coope and Mottram This Journ Dale and Dudley. Journ. Pharmacol. and Exp. Ther Dixon and Marshall. This Journ Dowler and Mottram. Ibid Mottram. Ibid ; Rosenfeld. Ztschr. f. klin. Med ; Ergebn. d. Physiol abt In this connection see Mottram, this Journ , ; and Mottram and Dowler, ibid

10 78 R. COOPE AND E. N. CHAMBERLAIN. APPENDIx. Body weight and liver weight of rabbits. The figures which follow represent-number of experiment (in italics); body weight in grams; liver weight in grams. Table IA. 1, 1420, 61F65; 2, 1427, 40-38; 3, 1980, 57-41; 4, 2020, 5810; 5, 1922, 89-40; 6, 1720, 35*82. Table I Ba. 7, 1470, 72-63; 8, 1830, 73-71; 9, 2020, ; 10, 1420, 48-56; 11, 2020, 61-47; 12, 1836, 42410; 13, 2490, 78-90; 14, 1930, 62-23; 15, 1320, 49-62; 16, 2110, 68-40; 17,?, 81-40; 18, 1560, Table I Bb. 19, 1750, 57*71; 20, 1310, 42-70; 21, 2620, 73'84. Table I Bc. 22, 2030, 75-60; 23, 1480, d, 24, 2541, e, 25, 2160, Table I C. 26, 1310, 42-36; 27, 1820, 63-48; 28, 2750, 79-55; 29, 1182, 39-80; 30, 2002, Table III. 31, 1880, 56-27; 32, 1810, 46-26; 33, 1710, Table IV. 34, 1920, 84-40; 35, 1975, 48-80; 36, 1135,