obtained from oxen'. In one experiment (V) the glands were given

Transcription

1 ON THE INFLUENCE OF PITUITARY GLAND SUB- STANCE ON METABOLISM. BY JOHN MALCOLM. (From the Physiological Laboratory, University of Edinburgh.) Historical. In 1897 Schiff(1) published a report on some metabolism experiments in which he administered pituitary substance (Merck's tablets) to a healthy man, to a man suffering from paralysis agitans, and to a case of acromegaly. He found that there was no influence on the nitrogen in any case, but that the phosphorus outpuit in the two diseased cases was increased. The increase took place in the fieces, and he attributed it to katabolism of bony tissue. Neither calcium nor magnesium was estimated and the conclusion come to assumes that bone was the only phosphorus-rich tissue which was affected, whereas it is possible that the increase may have been due to nuclein. Moraczewski (2) (1901) examined the metabolism of a case of acromegaly under the influence of various therapeutic agents including pituitary gland tablets (Merck). He found that on simiple diet alone there was a marked retention of nitrogen, phosphorus, calcium, magnesium, and chlorine. This he puts down to the increased growth of the tissues, soft as well as bony. On giving pituitary the nitrogen output was increased, also the phosphorus, while the calcium was unaffected or even slightly retained. Oswald (3) (1902) experimented on a dog with some pituitary gland preparations and found no effect on the nitrogen or phosphorus in the urine. The experiments detailed here were begun early in 1902 and are given in chronological order. The pituitary substance used was obtained from oxen'. In one experiment (V) the glands were given fresh and entire (freed from all connective tissue, etc.). A little chloroform was added to the jar in which the glands were sent, and the jar kept in ice; fresh supplies were sent twice weekly. In the other 1 I have to thank the firm of Messrs Burroughs, Wellcome and Co. for their kindness in supplying me with material. J. M.

2 PITUITARY GLAND. experiments the "glandular" and "nervous" portions were separated, dried at 700 C. or thereby, and stored in bottles till required. Analysis of the pituitary glands gave the following results:-the average weight of the fresh gland in the ox is 2 grm. of which onesixth consists of the "nervous" portion when separated at the cleft. The water-percentage of the entire gland is about 77, of the "glandular" part 73S5, of the "nervous" part 85. The dried substance gave the following percentages: Glandular" "Nervous" Nitrogen (as N) 13'3 12'3 Phosphorus (as P) 072 0'8 The percentage of calcium in the entire gland (dry)= 0123 (Ca). 271 Mode of Experiment. A large (21 kilo) bitch was the subject of experiment in each case. She was confined to a kennel with access only to a large rectangular tray which sloped gently to a central opening. The urine was collected in a glass vessel placed beneath this opening. A glass tube of about one inch diameter, tapered at the upper end, was placed in this vessel, the lower end being open and the upper end connected by narrow rubber tubing to a recording tambour which wrote on a vertical drum revolving once in twelve hours. When urine was passed, it rose in the glass tube, driving the air before it, and so gave a record of the time of micturition. The urine of 24 to 60 hours collected in this way was made up to a round number of c.c.'s with distilled water, and sampled. In Exps. II, III, IV and V, the volume of the urine was taken by notitng the number of c.c.'s necessary. An advantage of the use of this "urine-clock" is that the animal is kept under perfectly normal conditions as regards micturition. Fceces. The feeces were collected from the tray whenever observed, kept under a close-fitting bell-jar till two or three days' feeces had accumulated, chloroform or thymol being added; tlhey were then weighed, dried as a whole on a water-bath to constant weight, ground to a fine powder and a sufficient amount kept for analysis. To limit the faeces of one period from another 0'5 grm. pure carbon was given some hours before a meal. Exercise. The dog was exercised by being made to run up and down the laboratory stairs (15 feet) a fixed number of times for each experiment, usually fifteen to twenty. Food. Various food-stuffs were used, but in each case the caloric value of the diet was estimated directly by the bomb calorimeter and the necessary daily amount calculated from this-allowing fifty-five 18-2

3 MIALCOLM. calories per kilo per day in accordance with Pfliiger's experience. In each case, except Exp. I, the experiment was only begun after the animal had been fed on the diet for two to three weeks. The amount of common salt given was carefully measured out daily, and in the experiments on calcium excretion distilled water was used in cooking the food. Analyses. The methods used for analyses were-for nitrogetn, Kjeldahl-for phosphoruis, Neumann's (4) method of incineration with HNO3 and H2S04 followed by his titration method-for chlorides, Volhard (von Mering's modification for urine of dog). In estimating the calcium and magnesium in urine in the later experiments a modification of Neubauer's method was found to be more convenient and quite as exact as the original, viz.:-500 c.c. of the urine were taken, some ammonium chloride solution added (20 c.c. of a 20 O/o solution), then 25 c.c. of strong ammonia (880); the resulting precipitate of calcium and magnesium phosphate was filtered after twelve or more hours' standing, washed several times with 1 in 3 ammonia, and then dissolved off the filter-paper with weak HCI, and the calcium precipitated by the successive additions of ammonia till alkaline, acetic acid till acid, and finally ammonium oxalate. The magnesium was estimated in the usual way in the filtrate from the calcium oxalate by adding ammonia and some sodium phosphate, filtering, washing, and incinerating the resulting precipitate of ammonium-magnesium-phosphate. The advantage of the modified form of the method lies in the diminished bulk of the filtrate from the Ca-oxalate, and the larger amounts of Ca and Mg dealt with. Almost every figure given in the tables of analyses was controlled. Exp. I. In this, the influence of the " glandular " part was tried. Horse-flesh and oatmeal formed the diet. The former was bought in large quantity, mineed, weighed out into daily portions and sterilized in glass jars by heating on three successive days till the centre of the mass reached 700 C., the jars being closed by a paraffined cork provided with a short glass tube plugged with cotton wool. The oatmeal was made into porridge and the meat added while still hot. The whole ration was given in two meals at 9 a.m. and 9 p.m. Table I gives the data. TABLE I. ("Glandular " part.) Dog. About 21 kilo weight. Aet. 2 years. Ittake. 200 grm. oatmeal = grm. N grm. P 250 grm. horse-flesh= 8'213 grm. N grm. P During Periods IV, V 15 grm. dried "glandular" part of pituitary were given (2, 3, 4,.and 6 grm. on four successive days). This entails an additional intake during the time of 0'5 grm. N per day and grm. P= grn. N and grm. P.

4 Output. Urine PITUITARY GLAND. 273 Fices Amount Nitrogen Phospliorus NaCi Total amt. Nitrogen Phosphorus Time per day Total amt. Total amt. Total amt. Tinme (dry) Total amt. Total amt. No. liours c.c. grnis. grms. grms. days gris. grmis. grms. I II X III Average per day 11A IV P * V * Average per day 11P VI P Average per day 11P P '393 * 15 grm. dried "glandular" pituitary. RESULTS. Nitrogen Balance. There is a slight retention of nitrogen during the "glandular" peliod. Thus during the whole normdal, pre-pituitary period (I-III) the average output per day -( ) = as against 12'340 of intake, leaving a + balance of -023 per day; during the pituitary ("glandular") period the otutput per day = (11A ) = as against intake,giving a + balance of *624 per day; during the post-pituitary period, output = (I ) = 11575, intake = 12340, giving a + balance of *765 per day, but as some vomiting occurred during this period, one can scarcely lay any stress on the result. Phosphorus Balance. Output during the pre-pituitary period -(0' ) = 1-476, intake = a - balance of *008; during the pituitary period, output = (1, ) = 1P531, intake = 1P495 = a - balance of *036 per day; during the post-pituitary period, output = ( ) = 1,350, intake = = + *118 per day (vomiting may account for some of this). Chlorine. The amount of chlorine given as NaCl was perhaps unnecessarily large in this experiment. The excretion by the urine shows a slight retention. Water. The amount of water passed in urine and faeces was not specially measured in this experiment, so that no definite conclusion can be drawn from the amount of urine. The experiment as a whole shows that the " glandular " lobe of the pituitary, when administered in the dry form, tends to cause a retention of nitrogen, and probably chlorine, while the oiutput of phosphorus is increased.

5 274 J.! MALCOLM. Exp. II. In this experiment the influence of the " nervous" portion (dried) was investigated in a similar way. The food consisted of dog-biscuit, finely ground, mixed and weighed out in daily amounts of 280 grm. It was prepared by pouring a measured amount of boiling water over and stirring. The amount of urine was carefully noted. Table II gives the data. TABLE If. ("INervous" part.) Intake. 280 grm. dog-biscuit meal daily c.c. water. Nitrogen= grm.; Phosphorus = grm. During the pituitary periods (V-VIII) 10 grm. dried " nervous " portion of pituitary were given over three days. The intake during this interval is therefore = grm. N, and 1P =1P513 grm. P. Output. Urine Fices Total Total Total Total ault. Total Total Time volume Nitrogen Phosphorus Time (dry) Nitrogen Phosphorus No. hours c.c. grms. grms. days grms. grmis grms. I II III IV.' Average per day V VI VII i * VIII Average per day IX X XI XII Average per day 913, * 10 grm. dried "nervous" part of pituitary. RESULTS. Nitrogen Balance. Output in normal, pre-pituitary period = ( ) = 9-148; intake , giving a - balance of -088 per day. In the pituitary period (V-VIII) output = ( P597) = 9-300; intake = , giving a + balance of practically -170 per day. This is more marked in the post-pituitary period where the outpu1t = ( P634) = 8 670; intake = = per day. In the two latter cases the diminution is due to the output in the urine. Phosphorus Balance. Although the total phosphorus given in this experiment exceeded that of Exp. I, it was evidently in a less soluble form as shown by the relative amounts in urine and faeces in the two tables; thus, in Exp. I, 670/0 of the P-otutput takes place in the urine, in Exp. II, 41 0/0. This probably accounts for the negative balance of

6 PITUITARY GLAND. 275 the normal, pre-pituitary period (IJIV) where the output = (0( ) = 1P529; intake = 1P486 = per day. In the pituitary ("nervous ") period, output = ( ) = 1P584; intake = = per day. In the post-pituitary period, output =( ) = 1-372; intake = 1486 = a + balance of *114 per day. Water. The amount of uirine shows a slight diminution during the pituitary period which is still, apparent in the next period. The amount of solids in the faeces shows a distinct increase in the same two periods. Exp. III. In this experiment the points studied were the output of water in urine and feces and the output of calcium. The food consisted of dog-biscuit meal and was prepared in the same way as in Exp. II. The faces were weighed before as well as after drying. The calcium in the food and in the ftaces was estimated by simple incineration and extraction with HCI. In this extract the iron was precipitated by neutralizing with NH., and then acidifying with acetic acid, filtering and washing. The Ca was precipitated in the combined filtrate and washings by ammonium oxalate and the precipitate incinerated and weighed as CaO in the usual way. Table III gives the results in summarized form, each of the periods I, II, etc., being formed of three sub-periods. The whole experiment lasted about six weeks. TABLE III. Intake. 280 grin. dog-biscuit meal c.c. water daily. Total N = 13-4 grm., Ca= grm., Mg=0A472 per day. Output. Normal { Urine Feces Fieces Fines Time Volume Total Ca Time (moist) (dry) Total Ca Total Mg No. hours c.c. grms. days grms. grmi. grms. gris. I II III Average per day " Glandular" IV * Average per day Normal V Average per day " Nervous " VI Average per day Normal VII Average per day * Ca estimation of a sub-period lost; -03 is the amount in 96 hours' urine

7 MALCOLM. RESULTS. Calcium Balance. The intake of calcium in the food was unfortunately larger than necessary, and the animal retained a considerable portion of it during the normal periods. In the pre-pittuitary period (I-III) output = (0*006: ) = ; intake =-825 = a + balance of *181 grm. daily. In the "glandular" period (IV) 0-5 grmi. dried "glandular" pituitary was given daily for six consecutive days; output = ( ) = 2'0385; intake = 1P825 (disregarding the very small amount in 0'5 grm. substance) = a - balance of *213 grm. daily. In the next (normal) period (V) the balance returns to that of (I-III), output = ( P523) = 1P5296; intake = 1P825 = + '295. During the " nervous" pituitary period (VI) output = (0W ) = 1P9614; intake = 1P825 = a - balance of *136. This effect is still apparent in the post-pituitary period where output = (0' P916) _ 1-925; intake-= 1825 = - '100. Magnesium. The magnesium was estimiated in the faeces a conisiderable time after the experiment was finished, and when the urine was no longer available. The significance of the ratio (Mg: Ca) as an index of the state of bone metabolism was pointed out by I. MunkO(), and more recently it was used by Cronheim and Muller (6) in investigatitng growth of bone in infants. The Mg in the faeces is increased as much as the Ca under the influence of the "glandular" portion, while it is not increased to the same extent as the Ca under the influence of the " nervous " portion; thus the (Mg: Ca) ratio in faeces of the normal period II -III is 1 : 4'4, of period IV (" glandular ") 1 :4'5, of period V (normal) 1 : 4-4, of period VI ("nervous") 1 : 5'1, and of period VII (post-" nervous ") 1 : 5 4, showing that under the influence of the " nervous" portion there was probably an increased katabolism of bone. Water. The amount of water in the urine and faeces was examined to see whether the diuretic effect of intravenous injection of pituitary extracts observed by Schiifer and Magnus(7), took place when the gland substance was administered orally, and also to see whether there was any gross effect on carbohydrate metabolism. Since the dog sweats very little, the only other source of water loss is the breath, and as the output by this channel depends on the absolute humidity of the atmosphere, an attetnpt was made to estimate this daily, but from want of time it had to be abandoned. The output of water is shown in Table IV.

8 PITUITARY GLAND. 277 TABLE IV. Period Volume of urine Water in faces Total water No. per day cc. per day c.c. per day c.c. I-III normal IV "glandular" V normal VI " nervous)) VII normal It is evident from these figures that there is no diuretic effe.ct from the oral administration of pituiitary in this case, the differences that do occur being too small to allow one to draw conclusions fromn them. There is certainly ino diuresis sullc as one frequently sees whenl thyroid substance is similarly given, and hence in all probability tio effect on carbohydrate mnetabolism. The only other point of interest in this experiment is the excretioln of solids in the faces. There is a distinct increase in the solids of the fieces in both " glandular " and " nervous" periods which, in the latter case, continues for somne tinme. Exp. IV. In this experiment the influenice of the fresh gland substance in its entirety was similarly studied. The food consisted of oatmeal made into porridge to which dried and powdered horse-flesh was added. This flesh-meal was supplied by a dog-food company (Eldin Chem. Co., Loanhead, Midlothian) and, although the amount of calcium present was above the normal percentage found in pure horse-flesh, the total amount of Ca given was barely sufficient to cover the calcium-need of the dog. All the arrangements in this experiment were similar to those of the previous ones except that the fices were not limited off with carbon but simply collected for so many days at a time. Results are given in Table V. RESULTS. Nitrogen Balance. Output in the pre-pituitary period - ( ) = ; intake = , giving a + balance of -149 grm. daily. In the pituitary period, the balance becomes distinctly niegative, in contrast to the influence of the dried substance; output - ( ) = ; intake = = -815 grm. daily. In the post-pituitary period this negative balance is still evident, output = ( ) = ; intake = =-339 grm. laily. Calcium Balance. During the pre-pituitary period output = (0' ) = 0 633; intake = = a - balance of grm. daily. It is perhaps due to this that there is less effect on the calcium excretion than in Exp. III where the Ca-intake was more than sufficient to cover the need of the dog. In this experiment it was evidently too small.

9 278 TABLE V. J. MA LCOLM. (Fresh gland substance.) Intake. 190 grm. oatmeal=3-282 grm. N, grm. Ca, grm. Mg. 90 grin. dried flesh=12'738 grm. N, 0'2057 grm. Ca, grm. Mg. Total= grm. N, grm. Ca, grm. Mg. During the pituitary period (14 days) there was given about 25 grm. fresh substance daily, in all 336 grm. containing grm. N, grm. Ca (Mg estimation lost). Total intake during this period is, accordingly, grm. N, grn. Ca, and grm. Mg. Output. Urine Time Volunie Total Total Total No. hours c.c. Nitrogen Calcium Magnesium I '030 0' II. 48&5 1S III. 47* '1307 Normal IV V ' VI Average per day VII VIII IX X XI XII Average per day 758 XIII XIV Average per day , ' , ' t About 25 grm. pituitary daily (fresh substance) Normal Feces No. Time days Weight (dry) grms. I II. 3 78,9 III IV Total N grms * Total Ca grms Total Mg grins } Normal Average per day 24-3 V VI VII VIII Average per day 25-8 IX. 3 68,8 Average per day * I 25 grm. fresh pituitary daily Normal

10 PITUITA RY GLAND. 279 In the pituitary period output = ( ) = 06768; intake = 0'4337 = a - balance of grm. daily. In the post-pittuitary period output = ( ) = 0491; intake = = a - balance of 0(152 grm. daily. Magnesium Balance. Since the Mg-intake was sufficient in amouint the balance is more perfect than in the case of the Ca. In the pre-pituitary period output = ( '244) = ; intake = grm. daily. In the pituitary period output = ( ) ; intake = = or rather less to allow for the Mg in pituitary substance. In the post-pituitary period output =-( ) = ; intake = 0(3004 = grm. daily. TABLE VI. SUMMARY. (Summary.) Average Nitrogen Pliospborus Calcium Magnesium Ratio amount of balance balance balance balance Mg Ca dry feces per day per day per day per day in fwces per day Exp. I. Normal " Glandular" +*624 + '036 Normal + *765 + *118 (some vomiting) - - ExP. II. Normal ' " Nervous" + *170 - * Normal '114 28'2 Exp. III. Normal : 44 34'0 "Glandular" *213-1: Normal +'295-1 : "Nervous" : 5B Normal : 5' Exp. IV. Normal '011-24x3 Fresh gland - ' ' Normal Nitrogen. The "glandular portion of the pituitary, when administered orally in the dried form, caused a slight retention of nitrogen; the dried "nervous" portion had a similar effect. The fresh entire gland in large doses has an opposite effect and increases the output of nitrogen. Phosphorus. The " glandular " portion caused a retention of P, while the " nervous " caused a loss followed by a retention. Calcium and magnesium. Both the dried "glandular" and dried "nervous)) portions gave an increased output of calcium (on Ca-rich diet), but while the excretion of Ca in the former case was accompanied

11 280 J. MALCOLM. by an increased output of Mg (in faeces at any rate), the latter was not, or not to the same extent, so accompanied; this points to the " nervous " portion having had a katabolising influence on bony tissue. Fresh gland substance gave no increased Ca-output (diet poor in Ca) but rather a tendency in the opposite direction. The Mg otutput was at first increased -to a greater extent thani can be accounted for by the increased katabolism of proteid tissue which took place simultaneously (if one may so interpret the increased N-output). In the post-pituitary period the balance swings in the opposite direction. This corresponds to the effect of the (dried) " glandular" portion (see Table III) which forms fivesixths of the entire gland. In the case of nitrogen and calcium the fresh gland has an opposite effect to that of the dried, pointing to the probable existence of more than one active substance. The "nervous" portion is probably the miore active as shown by the duration of the effect, e.g. increased amount of fmces and increased Ca-outptut, lasting over the succeedinig normal period, while with the "'glandular " portion effects usually cease with the administratioui. The expenses of this research have been entirely defrayed by gratits from the Moray Research Fund of this University. REFERENCES. 1. Schiff.LWiener klin. Wochen. x. S v. Moraczewski. Zeit. f. klin. Med. XLIII. S Oswald. Virchow's Archiv, CLXIX. S Neumann. Zeit. f. physiol. Chemie, xxxvii. S Mun k. Pfluiger's Archiv, LVIII. S Cronheim u. Muller. Jahrbuch f. Kinderheilkunde, LVII. S Schiifer and Magnus. Journal of Physiol. xxvii. p. iii