phosphorus-containiing bodies-the nucleins-makes it a matter of no phosphates, are used in the synthesis of the nucleins, and the

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1 CONTRIBUTIONS TO THE STUDY OF THE META- BOLISM OF PHOSPHORUS IN THE ANIMAL BODY. BY D. NOEL PATON, M.D., F.R.C.P.E., J. CRAUFURD DUNLOP, M.D., F.R.C.P.E. AND R. S. AITCHISON, M.D., F.R.C.P.E. (From the Laboratory of the Royal College of Physicians of Edinburgh.) CONTENTS. 1. Preliminary. 2. The Absorption and Excretion of Phosphorus. i. General considerations and previous work. ii. Normal intake and output of phosphorus in dogs on mixed diet, and in goat. iii. Subcutaneous injection of phosphate of soda. iv. Influence of milk secretion. 3. The Distribution of Phosphorus in the Milk of the Goat. 4. The Influence of the Administration of Glycerophosphate of Lime in the Excretion of Phosphorus. 5. General Conclusion. 6. Appendix-Methods and Analyses. 1. PRELIMINARY. THE great importance in the life of all protoplasm of the complex phosphorus-containiing bodies-the nucleins-makes it a matter of no little interest to.attempt to follow the change of that element within the body. There is now no doubt that in plants and in the lower animals the simple inorganic compounds of phosphorus, such as the phosphates, are used in the synthesis of the nucleins, and the investigations carried out by one of us1 on the exchanges of phosphorus in the salmon show that even in fishes a similar synthesis is effected. As yet we know little of the progress of such synthesis in the mainmalia. Rohmann2, as the result of investigations carried on in his I Report to the Scottish Fishery Board on Investigations on the Life History of the Salmon in Fresh Water, Also this Journal, xxii. p Berl. klin. Wochensch. 1898, p. 789.

2 METABOLISM OF PHOSPHOR US. 213 laboratory, has come to the conclusion that in dogs at least the inorganic phosphorus compounds are hardly used at all, but that the phosphorus of organic compounds, such as nutrose and vitellin, is alone available in the metabolism. As a preliminary to the study of the metabolism of phosphorus a knowledge of its channels of absorption and excretion is required. 2. ABSORPTION AND EXCRETION OF PHOSPHORUS. (i) General considerations and previous work. Dogs. The observations of Bischoff' are the ground-work of our present knowledge on this subject. By experiments upon dogs he showed that in the starving animal the proportions of N to P,O, in the urine is as 6A4 to 1 while in muscle it is as 7-6 to 1. When the animal was fed on lean beef the proportion in the urine was as 8-1 to 1. On such a diet nearly all the phosphorus of the food was excreted in the uirine, only a very small amount appearing in the faeces. On a diet of bread the proportion of N to P2,O was 3-7 to 1, but a much larger proportion of the phosphorus appeared in the faeces. On a meat diet 900/0 of the phosphorus and 98 /0 of the nitrogen were excreted in the urine while on a diet of bread 850/o of the phosphorus and 920/0 of the nitrogen were excreted in the same way. Such observations would seem to indicate that in dogs the absorption of phosphorus in the quantities in which it exists in these foods is very complete, and that the kidney is the chief channel of excretion. In the human subject on a mixed diet North2 found that about equal quantities of the phosphorus of the food were excreted by the kidney and by the bowel. In the food the proportion of N to P,O, was as 4-5 to 1, while in the urine it was as 7 to 1. Some older observations of Siewert' made on himself on a diet of meat and vegretable and with 1 grm. of CaO added to the food do not give this difference between the nitrogen and phosphorus of the intake and of the urine. In his experiments the proportion was roughly, intake 4: 1; urine 5: 1. These results are the more interesting because lime salts have been supposed to form an insoluble compound with phosphoric acid in the intestine and thus to prevent its absorption. I Ztsch. f. Biol. iii. p Proc. of the Royal Soc. xxxvi. p Ztsch. f. ges. Naturwis. xxxi. p. 466, quoted by Atwater, Digest of Metabolism Experiments

3 214 D. N. PATON, J. C. DUNLOP AND R. S. AITCHISON. Herbivora. It has been for long known that in herbivorous animals, in the food of which the proportion of phosphorus to nitrogen is very high-in cereals as much as 1 to 2, the urine contains only very small quantities of the former element. The following figures taken from Atwater's Digest of Metabolism Experiments show this very clearly. P205 in grms. P205 in grms. Food Urine Faeces Gain Food Urine Faeces Gain Oxen' 143x Sheep Such results indicate either that the absorption of phosphorus in herbivorous animals is very imperfect, or that the waste phosphorus is excreted by some other channel than the urine. The observations on sheep seem to tell strongly against the former hypothesis for it is incredible that these animals should live without any waste of their phosphorus-containing tissues. Ludwig long ago expressed the view that in herbivorous animals the phosphates might be excreted by the bowel. We are not aware of any direct experiments on this point, but several series of observations at least suggest that even in the dog phosphorus may be in part excreted by the intestine. Voit3 found that the faeces of starving dogs contains phosphates. Kolpakcha4 found phosphorus still present in the faeces of dogs fed upon a phosphorus free diet; while Falck5 found, after injecting phosphate of soda into the vein of a dog, that in two of his three experiments less than 700/0 was recovered in the urine. (ii) Normal intake and output of phosphorus in dogs on mixed diet. Exp. I. In the dog used by Noel Paton, Dunlop and Macadam6, in their observations on the influence of diphtheria toxin on the metabolism the intake and output of nitrogen and phosphorus were studied from June 1st to June 5th with the following results:- 1 Emery and Kilgore. North Caroline St. Bul. 118, p Jordan, Maine Station Rep , p Hermann's Handbuch, v. p Quoted by Atwater, op. cit. 5 Virch. Arch. LIV. p This Journal, xxiv. p

4 METABOLISM OF PHOSPHOR US. 215 TABLE I.1 Balance of intake and output of nitrogen and phosphoric acid in dog on diet of Indian meal and milk. NITROGEN Intake Output Days Food Urine Fseces Total ' ' Totals PHOSPHORIC AcID Intake Output Food Urine FaeceS2 Tot ' ' ' '88 There appeared in the urineof the nitrogen excreted 84 per cent. of the phosphoric acid excreted 32 The proportion of N to P205 was about 2: 1 in the food and 5: 1 in the urine. The following table gives the amount of phosphorus in the food in organic and inorganic combination. In its construction the analyses of cows' milk given by Siegfried3 have been employed, while the inorganic phosphorus of the meal is the phosphorus which is dissolved by weak acetic acid. Indian meal. Total P205 Inorganic,, Organic,, Phosphorus as P205 in dog's food. Per cent. Total Milk Total P '742 Inorganic, Organic it P205 in organic combination=1-456 inorganic,, = Per cent Exp. V. p. 219 gives on a somewhat similar diet an excretion of 430/0 of the phosphorus of the food in the urine. Goat. See Exp. IV. p Here the proportion of nitrogen to phosphoric acid was 2 to 1 in the food and 99 to 1 in the urinie. In dogs on a vegetable diet only a comparatively small portion, and in goats practically none of the phosphorus of the food is excreted in the urine. 1 Throughout this paper weights are expressed in grms. and fluid measures in c.cms. 2 The phosphoric acid was determined in a sample of the mixed faces of the period. 3 Ztsch. f. phys. Chem. XXII. p Total

5 216 D. N. PATON, J. C. DUNLOP AND R. S. AITCHISON. In order to determine how far this may be due to the excretion of phosphorus by channels other than the kidneys the following experiments were performed. (iii) Injection of phosphates of soda. The general plan of these experiments was to bring a goat as representing the herbivora, and a dog as representing the carnivora, into nitrogen and phosphorous equilibrium, to estimate day by day the excretion by the urine of phospborus as phosphoric acid, to administer stubcutaneously a solution containing a definite amount of phosphate of soda, and to ascertain to what extent it is excreted by the kidneys. Exp. II. Goat. A goat was selected because it is easily kept and fed and it yields a sufficient quantity of urine and milk for analysis. TABLE II. Influence of subcutaneous injection. of phosphate of soda in the goat. Urine Milk Date N P206 N P vi , grms Na2HPO4=1 5 grms P205 injected subcutaneously 1. VII ' ' grms Na2PHO4=3 5 grms P2Ob injected A subcutaneously ' The evidence is thus conclusive that in the goat P205 thus injected is not excreted in the urine or in the milk-that it must be excreted by the intestine. Exp. III. Dog. Our object being to study the excretion of phosphorus on a mixed diet the animal was fed on Spratt's dog biscuits and milk-two biscuits and 700 c.c. of milk per diem. It was kept in a cage with a sloping zinc floor and the urine was collected in a vessel placed below. The faeces were hard and formed and were removed as- soon as possible after they were passed. There was no admixture of feces with the urine.

6 METABOLISM OF PHOSPHOR US. 217 TABLE III. Excretion of phosphoric acid after subcutaneous injection of phosphate of soda in the dog. Urine Date Amount P205 vi P92 1. vii P grms Na2HPO4=17 grm P P The average dailv excretion of phosphoric acid for the 30th June to 2nd July and for the 5th and 6th was 185 grm. After 1P7 grm. of P205 it rose to 1'96-a rise of only Ol grm. Falck1 after injecting phosphate of soda inito the jugular vein of a bitch recovered the following amounts in the urine. Inj ected Na2HPO4 Excreted expressed as in urine Per cent. No. of P20 P205 excreted hours Exp. I II , III These and the present observations show that in the goat and to a less extent in the dog phosphates must be excreted by channels other than the kidney. (iv) Influence of lactation on the excretion and absorption of phosphorus. That in herbivora there is an excretion of phosphorus by the intestinal mucous membrane as well as an absorption by this channel is further demonstrated by thie proportions of phosphiorus in the various excretions in the goat during lactation and after nilk secretion is stopped. Exp. IV. The weight of the goat througrhout the experiment was 39 to 39.5 kilos. I Loc. cit.

7 218 D. N. PATON, J. C. DUNLOP AND R. S. AITCHISON. TABLE IV. Excretion of nitrogen and phosphorus in the goat durintg and after lactation. A. During lactation. Nitrogen Intake per diem grms. hay ,, oatmeal 2-77 Totals 1103 Output Nitrogen Pi Date Urine Milk Feeces Totals Urine 20.x ' '30 1' ' '10 1' ' ' '518 0' X46 1'821 6' '050 Intake per dien. Output B. Milk secretion stopped grns. hay 100,, oatmeal Totals Nitrogen Date Urine Faeces Totals 23. xi ' 'hosphorus (as P205) * iosphorus (as P205) Milk Faeces Totals ' ' * piliospliorus Nitrogen ((s P20;s) X12 2'77 1X X22 Phosphcorus (as P205) Urine Feeces Totals 0o ' Average of of whole. The results of these observations may be tabulated as follows: With milk secretion. N N. Intake. Output. A rrine Faeces Milk Total Average daily 2f26 7'91 1*84 12* Without milk secretion. N N. Intake. Output. Urine Feces Milk Total Average daily P205 4*22 P2o5 Urine Fseces Milk -N Total P205 Urine Feeces Milk Total 0'

8 METABOLISM OF PHOSPHOR US. 219 Thus a smaller proportion of the phosphorus taken appears in the faeces during lactation. A study of the nitrogen gives no indication of any increase in absorption during lactation, and the evidence thus seems to point to a diminution in the excretion of phosphorus by the bowel due to a retention of a certain proportion for the formation of the phosphorus compounds of the milk. This seems to indicate that it is not imperfect absorption of phosphorus that induces the absence of phosphoric acid from the urine of herbivora, but its excretion by the bowel. The appended table gives the distribution of phosphorus in organic and inorganic combination in the food of the goat. Phosphorus compounds of goat's food. DA_ AA T_ A AZ rer cent. In aiet rer cen t. In diet Hay. Oatmeal. Total 0' Total *11 Inorganic 0' Inorganic Organic Organic 0'98 0'98 The diet contained phosphorus in organic combination 3-10 grm.,,,,,,,, inorganic,, 1-13 grm. The following observation is of interest as showing the difference between the goat which excretes practically all its phosphorus by the bowel, and the dog which excretes only a portion by that channel. Exp. V. A pregnant spaniel bitch got daily Nitrogen Oatmeal 300 grins. = 6-37 Bovril 10 grms. = 0-52 Milk 700 grms. = 3-27 Totals P20t, '07 1'40 4'80 TABLE V. Excretion Date 13. vii Average 5. vni 6 Average in the urine of nitrogen antd phosphorus in the dog, before and during lactation. Urine N. P * ' , Pups born on 8th. Urine Date N. P viii Average

9 220 D. N. PATON, J. C. DUNLOP AND R. S. AITCHISON. Here the excretion of phosphorus in the urine is diminished proportionately to the diminution in the nitrogen excretion, as the result of the withdrawal of the element for the production of milk. 3. THE NATURE OF THE PHOSPHORUS COMPOUNDS OF MILK. It has been for long known that a certain quantity of the phosphorus of milk is in organic combination in caseinogen and in lecithin, and it has been very generally assumed that the remaining portion is in inorganic combination. But the investigations of Siegfried', Wittmaack' and otbers, have shown that a portion of this phosphorus supposed to be in inorganic combination is really part of a somewhat complex organic substance, which, unlike inorganic phosphates, is not precipitated by ammonia and calcic chloride. This substance readily yields carnic acid, which according to Siegfried has the empirical formula C1QH,5N305 and is identical with anti-peptone. If this be so phosphocarnic acid is of the nature of a nuclein or pseudo-nuclein. Wittmaack's investigations show that in 1000 c.c. of cow's milk about 0 09 grms. of the total 1-.5 grm. of P205 is as phosphocarnic acid. In the samne amount of human milk of the 047 grms. P205 present, grms. are in this combination. In cow's milk the caseinogen of 1000 c.c. contains 0-58 grms. P205, and in human milk there is in the same quantity 0-16 grms. of P205, in this form. The most recent investigations on the phosphorus in lecithin in milk is by Stoklasas. He shows that in 1000 c.c. of cow's milk there is grms. P205 in this combination and in human milk grms. The object of our investigations was not to determine the proportion of phosphorus in these various compounds but to ascertain the proportion of organically combined to inorganically combined. Method. (A) The total phosphorus as P205 was determined in 25 c.c. of milk in the usual manner. (B) 50 c.c. of milk were mixed with 100 c.c. methylated spirit and tannic acid and left standing for a month. About 500 c.c. of water acidulated with acetic acid was added, and the precipitate extracted repeatedly with water to about 1000 c.c. This was filtered off. To the filtrate ammonia and calcic chloride were Ztschz. f. phys. Chem. xxii. p lbid. xxii. p Ztsch. f. phys. Chen. xxiii. p

10 METABOLISM OF PHOSPHORUS. added, and after standing for 24 hours the precipitate was filtered off and washed with a weak solution of calcic chloride and ammonia. The phosphorus in the precipitate was determined gravimetrically in the usual way, care being taken to wash the lime salts out of the molybdate precipitate with 100/0 nitrate of ammonia solution. The residue containing the casein and lecithin was burned with caustic potash and nitre, and the phosphorus determined gravimetrically. The P205 in phosphocarnic acid compounds was determined by evaporating the filtrate from the inorganic phosphate and estimating the phosphorus as P205 in the usual way; care being taken to wash out the lime from the molybdate precipitate. TABLE VI. Distribution oj phosphorus in milk of the goat. Milk of 7. vi 8. vll 16.I 17.v x Casein and lecithin Inorganic Phosphocarnic acid Not determined Total Total on 16. vii and 17. x by Dr Aitchison's independent analyses and If these analyses of the milk of the goat are compared with the analyses of Siegfried, Stoklasa, etc., of the milk of the cow and of the human subject, the following results are obtained. Orgic P205 Inorganiic P20r Total P206 0/0 of organic P205 Human Cow Goat OBSERVATIONS ON THE ADMINISTRATION OF GLYCERO- PHOSPHATE OF LIME. 221 Glycerophosphoric acid is one of the simplest organic compounds of phosphorus. It is also a normal product of the decomposition of lecithin, and as lecithin is split in the intestine it is a normal constituent of the contents of the gut. The glycerophosphate of lime which was supplied to us by Roberts and Son is a very soluble salt and it was hoped by its use to prevent the formation of the ordinary insoluble phosphate of lime in the intestine. It has been claimed for this salt that it is very readily absorbed.

11 222 D. N. PATON, J. C. DUNLOP AND R. S. AITCHISON. Two observations were made upon the dog and one on the goat during lactation. Exp. VI. Spaniel bitch. Diet per diem, 2 Spratt's dog biscuits and 700 c.c. milk. (This is a continuation of Exp. III.) TABLE VII. Excretion of nitrogen and phosphorus as P2O0 after glycerophosphate of limte in dog. Urine Date N. P vii 7* , , , grms. glycerophosphate of lime by mouth= 1 6 grm. P , Exp. VII. Setter bitch. Diet per diem, 500 grms. oatmeal and 700 c.c. milk. TABLE VIII. Excretion of nitrogen and phosphorus as PO05 after glycerophosphate of lime in dog. Date N. P20 14.xn grms. glycerophosphate of lime by mouth=4 grms. P Exp. VIII. In the goat during milk secretion the following observation was carried out as a continuation of Exp. IV. On the 27th and 28th the animal got 10 grms. of glycerophosphate of lime-20 grms. in all, equivalent to 4 grms. of P205 TABLE IX. Excretion of nitrogen and phosphorus as P,O5 after administration of glycerophosphate of lime. Output Nitrogen Phosphorus (as P205) Date Urine Milk Fices Totals Urine Milk Feces Totals 28. x X49 11' , , , X86 8X X713 4X54 5X293 1.xi o Average daily excretion. Nitrogen Phosphorus (as P205) Urine Milk Fices Totals Urine Milk Fices Totals Before (Table IV. A p.218) After (Table IX.)

12 METABOLISM OF PHOSPHORUS. This shows practically no increase in the excretion of phosphorus in the milk, nearly all the phosphoruis taken being recovered in the feeces. D. CONCLUSIONS In dogs on a vegetable diet, a large proportion of the phosphorus of the food is not excreted in the urine. 2. In dogs a large proportion of phosphoric acid injected subcutaneously as phosphate of soda is not excreted in the urine. 3. In the goat none of the phosphoric acid injected subcutaneously is excreted in the urine. 4. In the goat practically none of the phosphoric acid formed in the body or contained in the food is excreted in the urine. 5. In the goat during lactation the excretion of phosphorus by the bowel is diminished to meet the requirements of milk formation. 6. From this it may be concluded that in the normal condition, with the animal in phosphorus equilibrium, the absorption and excretion of phosphorus bv the intestine are equal. 7. In the dog during lactation there is a diminished excretion of phosphorus in the urine. 8. The milk of the goat contains a very high proportion of phosphorus and the percentage of organically combined phosphorus is lower than in the milk of the human subject or of the cow. 9. The administration of the soluble glycerophosphate of lime by the mouth causes no increased excretion of phosphorus in the urine in the dog, or in the urine or milk of the goat. 6. APPENDIX. For the general arrangement of the experiments and for most of the phosphorus analyses Dr Noel Paton is responsible. Dr Aitchison undertook the analysis of the phosphorus in the milk and feces of Exps. I., IV. and VII. while Dr Dunlop carried out, or superintended the carrying out, of the nitrogen analyses. We have to thank Prof. Aitken of the Royal Veterinary College for analyses of the phosphorus of hay. METHODS. Goat. Collection of urine. A special stall was constructed with a sloping zinc floor so arranged that any urine passed upon it was collected in a vessel behind it. Throughout the observation the animal never micturated in the stall, but did so thrice daily into a basin when it was allowed to come out of the stall for exercise. All contamination of the urine was thus avoided. Collection offawces. These were collected from-the zinc floor of the cage and at the end of each 24 hours they were weighed, rubbed up in a mortar and a sample of about 30 grms.

13 224 D. N. PATON, J. C. DUNLOP AND R. S. AITCHISON. taken and dried after the addition of a little sulphuric acid. The dry residue was weighed and used for analyses. Collection of milk. The goat was milked twice daily into a clean glass vessel. Food. To secure uniformity of composition and ease of analyses the food consisted of chopped hay and oatmeal with water. On this diet the animal's health remained good for prolonged periods. Methods of analyses. The phosphorus in the urine was determined as P205 by titrating 100 c.c. with nitrate of uranium in the usual way. On several occasions the urine was evaporated and ignited with caustic potash and nitre and the phosphoric acid determined. Concordant results were obtained. The phosphorus of the food, milk and freces was determined gravimetrically as pyrophosphate of magnesium. The nitrogen of food, milk, urine and feces was determined by K j eldahl 's method. Analyses of foods. HAY. 1. Sample used in October, Nitrogen. 2 grms. taken. Against 40c.c. N 10 H2S , 28-2, 28 0, 27-8c.c. utsed=_8260/0. Phosphorus. Prof. Aitken's analysis. Moisture /0. P Sample used in November, Nitrogen. 2 grms. taken. N Against 50c.c SO4 42-8, 42-2='5250/0. Against 40c.c. acid, 32-0, 32-1, 32-7, 32-3=-5410/0. Average /0. Phosphorus. Prof. Aitken's analysis. Moisture P205 0X312. OATMEAL. Phosphorus. A = 5 grms. B = 5 grms. Phosphate of magnesia weighed. A= grm. B = grm. Average P205= /0. Glycerophosphate of lime (Roberts). A. 2 grms. B. 2 grms. Phosphate of magnesia weighed. A B = P205 = grm. = 20 0/0.