Clearly the best method of deciding whether the liver is at fault. mammals excision of the liver is, unfortunately, such a difficult and

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1 CARBOHYDRATE METABOLISM IN DUCKS. BY G. B. FLEMING. (From the Physiology Department, University of Glasgow.) THE following investigation was undertaken in the attempt to throw light on the seat of the metabolic error in diabetes mellitus. Although we know various means by which glycosiiria can be produced in animals, we have little definite knowledge regarding the modus operandi of these measures. Nevertheless from the available facts it seems fairly certain that the error can be ascribed to one of the following processes of metabolism: (1) Defective storing of glycogen in the liver; (2) Too rapid conversion of glycogen or proteins into glucose; (3) Defective oxidation of carbohydrate in the tissues. In short the question resolves itself intowhether the fault lies in the liver or the tissues. Until about the year 1909 the general weight of opinion was in favour of the view that the defective process consisted in the inability of the tissues to burn carbohydrate, but von Noorden (5) and others working in conjunction with him produced evidence that it was the liver that was at fault. More recently Starling(6) has shown that no difference in the utilisation of carbohydrates can be detected in the excised hearts of diabetic and normal dogs. Clearly the best method of deciding whether the liver is at fault would be to excise that organ in animals previously rendered diabetic and then to see whether the tissues could utilise carbohydrate. In mammals excision of the liver is, unfortunately, such a difficult and dangerous operation that it is almost certain that any results obtained are open to the objection that the animals are moribund before the tests as to whether the tissues are utilising carbohydrate can be applied. In ducks, however, the portal veins can be ligatured near the liver and the blood diverted through the vein of Jacobsen into the systemic circulation. It was thought that in these birds, after eliminating the hepatic functions by obstructing the portal circulation through the liver and rendering them diabetic, evidence might be obtained regarding

2 CARBOH YDRATE METABOLISM. 237 the behaviour of the tissues to carbohydrate. But before this could be done it was essential to prove that the carbohydrate metabolism of these birds is similar to that of mammals. It is well known that protein metabolism is carried on differently in birds and mammals. For example, in birds nitrogen is excreted chiefly in the form of uric acid and instead of creatinin being a constant constituent of the urine creatin seems to take its place. Contradictory results have been obtained regarding the regulation of carbohydrate metabolism. Noel Paton(7) found that excision of the pancreas in ducks did not produce glycosuria, while Kausch(4) found that hyperglycaemia was constantly present in ducks in which the pancreas had been completely excised. According to Noel Paton the injection of adrenalin does not always lead to glycosuria though in some cases sugar is detected in the urine. In view of these conflicting results it was evident that if ducks were to be used to investigate the site of the metabolic error in diabetes mellitus, their carbohydrate metabolism must first be proved to be analogous to that of mammals. I. Experiments to show that the carbohydrate metabolism is alike in birds and mammals. The quantity of sugar in the blood is a more reliable guide to disturbances of carbohydrate metabolism than the quantity of sugar in the urine, and this is especially true in birds since they seem able to retain larger quantities of sugar in the blood without the occurrence of glycosuria than is the case in mammals. In my experiments the birds employed were for the most part ducks but on a few occasions geese were used. The pancreas of these birds lies in the loop of intestine formed by the duodenum. It is supplied by the blood vessels which also supply the duodenum and these vessels run through the substance of the gland. Consequently it is a matter of great difficulty to remove the pancreas without impairing the blood supply to the duodenum. Noel Paton dissected the pancreas from the blood vessels with special forceps, while Kausch removed the duodenum and pancreas en masse. I have tried both these methods and, as the resuilts show, the latter is by far the more satisfagtory. In all the experiments the urine was collected uncontaminated with faeces by making an artificial anus in the lower part of the animal's intestine and closing the distal end of gut so that feeces passed through the artificial opening and urine alone through the cloaca. The blood sugar was estimated hours after the operation.

3 238 G. B. FLEMING. From c.c. of blood were drawn off into a vessel containing oxalate solution; the proteins were removed by Sc hen c k's(lo) method and the sugar estimated by the Pfluiger Alhin gravimetric method. (a) The normal sugar content. In normal ducks the average percentage of glucose in the blood was found to be (Table I). (b) The effect of excising the pancreas on the blood sugar. In three birds from which the pancreas had been removed by Noel Paton's method, the average percentage of sugar in the blood was (Table II), that is, only slightly above the normal figure. In six ducks from which the pancreas and duodenum were removed en masse, the average percentage of sugar in the blood was (Table III). In only one case did sugar appear in the urine. TABLE I. Normal ducks Exp. p.c * * * *117 Average 075 or, excluding 12, which is abnormally high, Mean is *07 Percentage of sugar in blood of ducks. TABLE IL TABLE III. Partial excision Excision of of pancreas pancreas and duodenum Exp. p.c. Exp. p.c. Goose * *202 Average *201 Average *272 * Partial pancreatectomy. TABLE IV. Injection of 2 c.c..1 p.c. adrenalin Exp. p.c * *258* *167 * *184 Goose * *221 Average *235 From these results it seems clear that the removal of the pancreas in the duck produces hyperglyctemia quite comparable to that produced in mammals by a similar procedure. In ducks, however, hyperglycaemia does not seem to lead to glycosuria as readily as it does in mammals. The possibility that removal of the duodenum might have some effect on the quantity of sugar in the blood was taken into consideration and in one duck the duodenum alone was removed; in this case the sugar of the blood was found to be , a figure which was reached in some of the normal ducks (Table I). (c) The effect of injecting adrenalin on the quantity of sugar in the

CARBOHYDRATE METABOLISM. 239 blood. It is well known that in mammals injection of adrenalin produces hyperglyca3mia and glycosuria. In five ducks and in one goose this was also found to occur. Two c.

4 CARBOHYDRATE METABOLISM. 239 blood. It is well known that in mammals injection of adrenalin produces hyperglyca3mia and glycosuria. In five ducks and in one goose this was also found to occur. Two c.c. of 1/1000 adrenalin (Parke Davis) were injected subcutaneously. Within an hour of the injection blood was drawn off, and in all the experiments it was found to contain excessive quantities of sugar. In three normal ducks and in one goose after injecting adrenalin the blood sugar averaged /; in two ducks which had had their pancreas incompletely removed it was /0 and /0. The average amount of sugar in the blood in these six experiments was /0 (Table IV). Glycosuria was present in three cases. From these experiments it is clear that both removal of the pancreas and injection of adrenalin affect the carbohydrate metabolism of birds and mammals in the same way. II. The utilisation of carbohydrate as indicated by the respiratory exchange in birds. Having demonstrated that hyperglyeaemia can be produced in birds by the injection of adrenalin and by excision of the pancreas, the next step was to find out in what way these procedures affected the carbohydrate metabolism in the tissues. If carbohydrate be utilised in the tissues the respiratory quotient is high (about 1), while if protein or fat be used it is low (between 0 7 and 0.8). Thus by determining the respiratory exchange it is possible to decide whether carbohydrates or proteins or fats are being utilised. Hyperglyceemia may be produced in three ways: (1) by defective storage in the carbohydrate storehouses (the liver); (2) by excessive production of glucose from protein and possibly from fat; (3) by defective combustion of glucose. In the first case the respiratory quotient will be high because the tissues will be able to utilise the carbohydrate which is being supplied to them in abundance. In the second it will be low because the process of converting proteins or possibly fats into carbohydrate will require oxygen and although it will be glucose that is being burned in the tissues this glucose will have been manufactured from proteins and fats. In the third case it will be low because the metabolism will be carried on at the expense of proteins and fats. In this series of experiments the respiratory exchange was first estimated in normal ducks that had recently been fed, second in ducks that had not been fed for about 20 hours, third in fasting ducks before and after the injection of adrenalin, fourth in fasting ducks after excision

5 240 G. B. FLEMING. of the pancreas, fifth in ducks which had had their pancreas excised and which had subsequently been fed on glucose, sixth in fasting ducks whose portal veins had been tied, seventh in fasting ducks after excision of the pancreas and ligature of the portal veins. All the experiments on the respiratory exchange were performed by Haldane's(3) method. This method consists in estimating the quantity of C02 and H20 given off by the animal supplied with air which has been freed from CO2 and water. The oxygen utilised is estimated by weighing the animal before and after the experiment and subtracting the loss of weight of the animal from the weight of C02 and water exhaled. The air was drawn through the apparatus at a rate of about 600 litres per hour. For a few minutes before the commencement of each experiment, dry air was passed through the box with the animal in situ in order to bring the air in the apparatus to a constant composition. The respiratory exchange in normal ducks. It will be seen from Table VI that in fasting animals the respiratory quotient is low while if the animal has recently been fed the respiratory quotient approaches unity (Table V). The ducks were fed on a mixture of maize and oat-meal. TABLE V. The respiratory exchange in ducks that have been recently fed. CO2 gms. 02 gims. per hour per hour Exp. per kilo per kilo R.Q *87 Fed 3 hours before experiment ,, 1 3 3{ *84,,3 4 4* ,, I, ,, *85 4,. Average These results show that the respiratory exchange in ducks is influenced by the diet in the same way as it is in mammals. The respiratory exchange after hyperglyccemia had been produced by the injection of adrenalin. In each experiment the respiratory quotient was first estimated in the normal fasting duck. This part of the experiment was usually done between 11 a.m. and 1 p.m. In the afternoon between 2 and 4 o'clock, 2 c.c. adrenalin solution (1/1000) were injected and the respiratory exchange was immediately determined. Each experiment lasted.about 45 minutes. From Table VI it will be seen that after adrenalin the respiratory quotient was raised and that there was as a rule a slight increase in C02 output and a slight diminution in

CARBOHYDRATE METABOLISM. 241 the 02 intake as compared with the figures obtained in the fasting animal immediately before the injection of adrenalin.

6 CARBOHYDRATE METABOLISM. 241 the 02 intake as compared with the figures obtained in the fasting animal immediately before the injection of adrenalin. The conclusion to be drawn from these experiments seems to be that within threequarters of an hour after injecting adrenalin the power of the tissues to burn carbohydrate is in no way impaired. The results obtained by various other workers in this field have been somewhat contradictory. Falta and Bernstein(land 2) found that in man the respiratory quotient was at first increased after giving adrenalin but within twenty minutes it fell to normal. The C02 output and the Exp TABLE VI. Exp Average TABLE VII. The respiratory exchange in fasting ducks before and after the injection of adrenalin solution. Fasting Fasting after adrenalin CO2 gms. 02 gims. CO2 gms. 02 gms. per hour per hour per hour per hour per kilo per kilo R.Q. per kilo per kilo R.Q * * * * * * * * *85 * * * * "ll * * * * *59 * X29 *84 The respiratory exchange in fasting ducks after injection of adrenalin solution. Adrenalin Adrenalin Fasting First half hour Second half hi C02 gms. 02 gms. C02gMs. 02 gms. C02gMs. 02 gms. per hour per hour per hour per hour per hour per hour per kilo per kilo R.Q. per kilo per kilo R.Q. per kilo per kilo * * * Averafye * intake were both increased but the former began to fall after about five minutes. In two experiments I estimated the respiratory exchange after injecting adrenalin in two periods of half an hour each; the results are shown in Table VII. It will be seen that there was a distinct fall in the respiratory quotient during the second half hour period. The results obtained by Wilenko(1i) after injecting adrenalin into rabbits are apparently in opposition to those obtained by Falta and Bernstein and myself. He found that the respiratory quotient was low even when glucose was given with the adrenalin. Wilenko, however, did cour R.Q. *82 *76.79

7 242 G. B. FLEMING. not estimate the respiratory exchange till one or two hours after he had injected adrenalin and, as shown by Falta and Bernstein and myself (Table VIII), the respiratory quotient falls rapidly after the initial rise. This accounts for his low readings. TABLE VIIL The respiratory quotient one hour after giving glucose solution. R.Q. before R.Q. after Exp. glucose glucose 25 *80 *91 Glucose given by rectum ,,, duodenum , *80, *80-85 Average *77.90 The explanation of the rise in the respiratory quotient after adrenalin is not altogether clear. We know that hyperglyceemia is produced, but the increase in the quantity of sugar in the blood lasts longer than the rise in the respiratory quotient. If there was no impairment of the glycolytic power of the tissues we should expect the rise in the respiratory quotient and increase in the quantity of sugar in the blood to be synchronous. Possibly adrenalin has a double action. It may mobilise carbohydrate and also inhibit the pancreatic internal secretion. If this were so it is possible that at first the excessive carbohydrate in the blood might be in part oxidised in the presence of the pancreatic internal secretion already in the blood, but later, as the pancreatic internal secretion failed the tissues would be unable to utilise the carbohydrate circulating in the blood and the respiratory quotient would fall in spite of the existence of hyperglyceemia. Cause of the low respiratory quotient in diabetes. The concluding experiments in this investigation were carried out with the object of determining whether the low respiratory quotient always found in diabetes mellitus is due to defective combustion of carbohydrate in the tissues or to an abnormal activity of the liver causing excessive formation of carbohydrate from protein and possibly fat. Until recently it has been taken for granted that the low respiratory quotient in diabetes mellitus indicates defective combustion of carbohydrate in the tissues. Within the last few years, however, opinion has begun to veer round to the other alternative, namely that hyperglyceemia and glycosuria may be due to abnormal processes in the liver causing excessive conversion of protein and fat into sugar. The experiments of Porges and Solo man (8) support this view. These workers estimated the respiratory exchange in animals whose livers had been excluded from the circulation.

8 CARBOHYDRATE METABOLISM. 243 This was done by ligaturing the aorta, the inferior vena cava, the hepatic and the portal veins. They found that the respiratory quotient was high although the animals had been fasting for some time. They next tried similar experiments on dogs which had previously been rendered diabetic by removal of the pancreas; again the respiratory quotient was found to be high. Interesting though these experiments are they are open to the criticism that the animals were more dead than alive when the respiratory exchange was determined and results obtained under such circumstances cannot be accepted as conclusive. Some time before Porges and Soloman carried out their work Scaffidi(s) estimated the respiratory exchange in ducks after ligaturing the portal veins. He found a slight rise in the respiratory quotient: but again in these experiments the results are open to doubt, since the animals lived some weeks after the operation and on post nortem examination, collateral circulation was found to have developed. In my experience, if the portal circulation is completely obstructed, ducks only live twenty to thirty hours. Scaffidi states that he tied the portal vein and two veins running between the stomach and the liver, but there are numerous veins not only between the stomach and the liver but also between the lower part of the cesophagus and the liver, and in addition there are vascular connections in the peritoneal bands between the thoracic walls and the liver. To cut the portal circulation completely off from the liver all these connections must be severed. Moreover in many of Scaffidi' s experiments the animals were not fasting. In their most recent experimenis Starling and his co-workers failed to find any difference in the utilisation of sugar between the excised diabetic and normal heart. These conclusions so far as they go favour the view that the metabolic disturbance in diabetes mellitus is not in the muscles, and support von Noorden and his pupils in the suggestion that the error lies in the hepatic functions. III. The respiratory exchange after ligature of the portal veins. I have endeavoured to determine whether tying the portal vein in ducks which are otherwise normal raises the respiratory quotient. The fortunate circumstance that in these birds the portal blood can be diverted into the general circulation through the vein of Jacobsen allowed the respiratory experiments to be carried on with them in a fairly satisfactory condition, thus obviating the fallacies of Porges' experiments, and by taking the utmost care that all the portal veins were ligatured the fallacy of incomplete exclusion of the portal blood from PH. LIII. 16

9 244 G. B. FLEMING. the liver was as far as possible avoided. The ducks as a rule were operated on about 10 a.m. and the respiratory experiments carried out at about 3 p.m. on the same day. This gave ample time for them to recover from the anawsthetic; it was usually found that, within an hour of the operation they were standing up in the cage, seemingly quite comfortable. The ducks were anasthetised with ether; the abdomen was opened, the portal vein exposed and ligatured between the vein of Jacobsen and, the liver. The bile ducts were not included in the ligature. The vessels between the stomach and oesophagus and the left.lobe of the liver were then tied and the bands of peritoneum between the parietes and the liver divided. The abdomen was then closed. TABLE IX. The respiratory exchange in ducks after removal of the pancreas. Fasting After glucose by the duodenum C02gisn. 02 gms. C02 gis. 02 gms. per hour per houir per hour per hour Exp. per kilo per kilo R.Q. per kilo per kilo R.Q * x75 1*68 *71 2* *76 TABLE X. The respiratory exchange in ducks after ligature of the portal veins. C02 gms. 02 gms. per hour per hour Exp. per kilo per kilo R.Q *11 * *26 2*52 * * * *25 * i90 * *75 1*81 *70 Average 1V *65 TABLE XI. The respiratory exchange in ducks after removal of the pancreas and ligature of the portal veins. C02 gms. 02 gms. per hour per hour Exp. per kilo per kilo R.Q * *94 * *51 * *82 Average *72 In Table X the results of six experiments are recorded. It will be seen that the average output of CO2 and intake of oxygen per kilo per hour is low as compared with the normal fasting duck (Table VI) and also that the respiratory quotient is slightly lower than normal.

10 CARBOHYDRATE METABOLISM. 245 In a few cases when the animals were obviously very ill very low respira- *tory quotients were obtained and the intake of oxygen and output of C02 were also abnormally low. These results have not been recorded as the animals were clearly not in a condition to give a fair idea of the respiratory exchange. IV. The respiratory exchange after combined excision of the pancreas and ligature of the portal veins. Before determining the respiratory exchange after this combined operation, it was thought advisable to find whether excision of the pancreas alone influences the respiratory exchange. It is well known that in mammals the hyperglyceamia resulting from excision of the pancreas does not lead to a rise in the respiratory quotient even if carbohydrate be given in the diet. In two ducks after removal of the pancreas I found the respiratory quotient to be low, and in one of them in which I performed a second respiratory experiment after running 8 gm. of glucose in solution into the peripheral part of the duodenum the respiratory quotient was still low (Table IX). To make sure that glucose administered in this way caused a rise in the respiratory quotient in normal ducks, an artificial opening was made into the duodenum in five ducks and 8 gm. of glucose dissolved in water was run into each. The respiratory quotient was determined before and after the glucose was given. It will be seen (Table VIII) that in each case the respiratory quotient rose after the administration of glucose. Unfortunately the records of the duration of the respiratory experiments were lost so it is impossible to give the oxygen and C02 in grams per kilo per hour. In four cases in which the pancreas was removed and the portal veins ligatured the respiratory quotient was found to be low, average 0*72 (Table XI). This is higher than in the cases where the portal veins alone were ligatured but none the less it indicates a metabolism in which fats are being utilised and not carbohydrates. These results point to the conclusion that in pancreas diabetes the liver is not the organ where the disturbed metabolic processes hold sway but that the fault lies in the power of the tissues to utilise carbohydrate. CONCLUSIONS. 1. In ducks the respiratory exchange is influenced by the diet in the same way as it is in mammals. 2. In ducks the injection of adrenalin or removal of the pancreas produces hyperglycaemia. 16-2

11 246 G. B. FLEMING. 3. Within half an hour of the injection of adrenalin the respiratory quotient is raised and during the next half hour it falls. 4. The rise in the respiratory quotient after the injection of adrenalin in the fasting animal indicates that there is a mobilisation of carbohydrate but the persistence of hyperglycaemia after the respiratory quotient has fallen suggests that adrenalin may cause inhibition of the pancreatic internal secretion. 5. Neither ligature of the portal veins, nor excision of the pancreas, nor the combination of these procedures causes a rise in the respiratory quotient, even when glucose is administered. 6. The evidence indicates that in pancreatic diabetes the tissues are unable to utilise carbohydrate rather than that the liver is the sole seat of the metabolic error. REFERENCES. (1) Bernstein and Falta. Deutsche Kongress f. inn. Med. p (2) Falta. Die Erkrankungen der Blutdrrusen, (Berlin.) (3) Haldane. Journ. of Physiol. 13, p (4) Kausch. Arch. f. Exp. Path. und Pharm. 37, pp (5) v. Noorden. Zuckerkrankheit, (Berlin.) (6) Paterson and Starling. Journ. of Physiol. 47, p (7) Paton. Journ. of Physiol. 32, p (8) Porges and Soloman. Biochem. Ztschr. 27, p (9) Scaffidi. Biochem. Ztschr. 41, p (10) Schenck. Pfliiger's Arcb. 55, p (11) Wilenko. Biochem. Ztschr. 42, p

Edinburgh.) IN a previous paper, I recorded observations on rabbits and dogs which

THE EFFECT OF ADRENALIN ON SUGAR AND NITROGEN EXCRETION IN THE URINE OF BIRDS. BY D. NOEL PATON.. (From the Research Laboratory of the Royal College of Physicians, Edinburgh.) IN a previous paper, I recorded