during the apnceic pause, when the H2CO3 concentration of the blood is investigators, and the results of these researches have been most thoroughly

Transcription

1 CHANGES IN THE HYDROGEN ION CONCENTRATION OF THE BLOOD PRODUCED BY PULMONARY VENTILATION. By T. H. MILROY (Department of Physiology, Queen's University, Belfast). (Received for publication 16th February 1914.) DURING rapid ventilation of the lungs with air, the carbonic acid content of the blood is lowered, and if, after a sufficient interval, the ventilation be stopped, the respiratory movements cease until the alveolar carbonic acid pressure and therefore the carbonic acid concentration of the blood have risen again to the stimulating level for the respiratory centre. That the carbonic acid plays the predominant part in exciting the centre to action has been shown by many experiments. The withdrawal of carbonic acid from the blood during excessive ventilation must lead to a diminution in the hydrogen ion concentration, (H+), from the decrease in the concentration of H2CO3, unless there be during this period an increase in the acid products of incomplete combustion of the material used by the tissues during their activity. So also during the apnceic pause, when the H2CO3 concentration of the blood is rising there must be an increase in the (H+) unless there be an increased alkali transport from tissues to blood during this period. The means which the organism possesses of maintaining a constancy in the reaction of the blood and tissue fluids have been very carefully studied by many investigators, and the results of these researches have been most thoroughly analysed by Henderson (1). This paper will deal in the main with the quantitative changes in the (H+) of the blood produced by excessive pulmonary ventilation with air or other gas inixture, and will also refer to the subsequent changes in ionic concentration during the pause and after the respirations have resumed their normal character. It is necessary to refer in some detail to the method which has been employed to determine the (H+) of the blood, namely, the electrometric method, as the recent workers in this subject have directed attention to certain inaccuracies which are difficult to avoid in the case of blood estimations. One must, however, omit very many important points in connection with the rnethod, as a full description would occupy too much space, and the method as applied to blood has already been explained by many others, especially within recent years by Soirensen (2) and Hasselbalch (3). There are certain difficulties attached to the use of the hydrogen

2 142 Milroy electrode in the case of such a fluid as blood. In the first place, one cannot saturate this fluid with hydrogen in the usual way by passing a stream of the gas through the blood, as by this procedure the carbonic acid would be removed from the blood, with a resultant lowering of the (H+). In the second place, owing to the rich oxygen content of the blood, the platinum hydrogen plate undergoes partial depolarisation, and so the readings cannot be constant until the blood is completely reduced. And in the third place, as hydrogen cannot be passed through the blood, and as the fluid requires to be saturated with the gas at atmnospheric pressure, a certain amount of time must elapse in the case of a hydrogen electrode with a stationary hydrogen atmosphere before such a viscous fluid as blood can be saturated. To prevent an alteration in the (H+) from a withdrawal of carbonic acid, Hober passed through the blood hydrogen with a percentage of carbonic acid approximating to that existing in the blood. This in many respects rather inconvenient method has not been employed to any great extent. Hasselbalch (5) then introduced another method, using an electrode which enables one to remove the first specimen of the blood from the electrode after gaseous equilibrium has been established between the blood and the stationary hydrogen atmosphere, and to replace this with a fresh specimen of the same blood while still retaining the original gas in the electrode. It is true that the partial pressure of the hydrogen is too low, as it is mixed with carbonic acid; but this introduces a very small error, as the measured potential varies only as the logarithm of the hydrogen pressure. Michaelis and Davidoff (6) and many others referred to by Sorensen adopted the method of the hydrogen electrode, with stationary hydrogen atmosphere and minimal contact between platinum plate and blood, without, however, replacing the first specimen of blood by a second or third one, regarding the lowering of (1H+) from the loss of carbonic acid from the blood as being too small to introduce anything beyond an inappreciable fall in (H+). Michaelis first directed attention to the advantage of ininimal contact between plate and blood in order to obtain rapidly constant readings of the potential. Hasselbalch and his co-workers adopted this plan of minimal contact. Now, as regards the preliminary depolarisation of the plate, this is bound to occur when a hydrogen plate is brought into contact with a fluid so rich in' oxygen as arterial blood. In Hasselbalch and Lundsgaard's earlier papers sufficient attention was not directed to the depolarisation as a factor leading to inconstant readings. All who have used the electrode must have been troubled by this error, and Konikoff (7) has recently drawn attention to it. At the outset of my experiments, I was so much troubled by this depolarisation that for the last year I have made use of blood plasma instead of the entire blood. The long periods referred to by Hasselbalch as necessary before constant readinigs can be obtained were undoubtedly due mainly to the very slow reduction of the blood. The

3 Changes in the Hydrogen Ion Concentration of the Blood >ip changes in the readings noticeable on shaking the electrode after a constant reading has been previously arrived at are due to a renewed depolarisation with a consequent fall in potential followed by a rise as the hydrogen film again forms on the plate surface. Constant readings are more quickly attained by minimal contact between the plate and the blood owing to better reduction of the superficial film of blood and the less general depolarisation of the plate surface. Hasselbalch's method of replacing the first by a second or third specimen of blood leads naturally to renewed depolarisation, although the final readings may be and probably are correct. It is necessary, however, to have complete reduction of the blood in order to obtain absolutely constant readings. As regards the difficulties entailed in the saturation of a viscous fluid like blood with hydrogen, these can be removed by frequent shaking of the electrode. The method suggested by Wilke (8), namely, the use of a platinum capillary electrode with a high internal pressure of hydrogen, does not appear satisfactory unless one knows the actual pressure of the hydrogen on the outer side of the capillary. Very briefly reference may now be made to the method which has been adopted in carrying out the electrometric measurements. In the first place, as regards the charging of the electrode (one of the Hasselbalch type) the hydrogen, which was obtained from the action of aluminium amalgam (9) on water, was passed for about an hour through the usual series of liquids to render it oxygen-free and saturated with water vapour, passing it finally through a specimen of blood plasma into the empty electrode. While the hydrogen was still passing through, blood plasma was quickly introduced into the electrode until the plate was approximately half immersed in the fluid; the electrode was now closed and carefully sealed with melted paraffin at any place where there might be a tendency to leakage. It was then shaken gently for ten minutes, allowed to stand for about five minutes, and finally, before connecting up with the calomel electrode through the strong KCI solution (3 5 N) in the contact dish, the tube used for dipping into the latter was opened with the end submlerged in a small quantity of the blood plasma. The hydrogen electrode and the calomel electrode were now connected up through the contact fluid and an estimation of the e.m.f. carried out in the usual way. The calomel electrode was of the form employed by Pauli, and was prepared as described by Loomis and Acree (10). The estimations were all made by the usual zero method, using a calibrated potentiometer, accumulator, standard Weston cell ( volt), and an Ayrton-Mather galvanometer of the d'arsonval type. Accurate details as regards the measurement of the e.m.f. of the electrode system are given in Soirensen's article already referred to. The hydrogen and calomel electrodes were frequently subjected to controls as described by Loomiis and Acree. As regards the method of obtaining the blood, it was taken from the

4 144 Milroy carotid artery with as little agitation as possible, into a centrifuge tube containing two or three milligrammes of hirudin dissolved in a very small quantity of saline. The tube when filled was closed by a paraffined cork and put into the centrifuge. The plasma after separation was taken from the tube, every precaution being taken to prevent loss of carbonic acid, and introduced into the electrode. The after treatment of the electrode has been already described. As soon as a constant reading of the e.m.f. had been obtained, the electrode was removed from the contact dish, the connecting tube closed, and the electrode again shaken and allowed to stand for a short time before the second reading was taken. This was repeated until the readings were absolutely constant. This was the case usually after the third shaking. In some cases Hasselbalch's procedure was adopted introducing a second specimen of plasma while retaining the gas mixture, but this was found to be unnecessary, as the differences between the two final readings were so small and a larger amount of blood was required to be taken than was advisable, three or four specimens being required in each ventilation experiment, as will be afterwards described. Before each specimen of plasmna was introduced the electrode was charged afresh with hydrogen, and when the estimations in a series were completed the plasma was washed well out of the electrode with conductivity water and the latter then saturated with hydrogen. The platinum plates were thus kept ready for further estimations. It was found necessary to replatinise the plates for a short time, more frequently than is necessary when standard solutions are used. The estimations were in most cases made at 15 C. and corrected for 180, in a few cases also at 380, but the former were more satisfactory. An air thermostat of the Hasselbalch type did not prove very satisfactory for estimations at 38, as it was difficult to maintain a constant temperature in the electrode system. In cases where it is advisable to replace the blood plasma used in the first case by a second specimen of the same, the electrode used by Konikoff is very useful, although I have mainly used the Hasselbalch type. The slight fall in (H+) due to a loss of CO2 from blood to hydrogen atmosphere may, in my opinion, be disregarded, and so by the use of one specimen only a continual depolarisation of the plate by the fresh oxygenholding fluid is avoided. My aim was to investigate the (H+) changes produced by a ventilation period and subsequent thereto. Therefore a specimen of blood was taken just before ventilation, a second one just at the close of a ventilation period, a third one at the close of the pause, and in many cases a fourth one after breathing had resumed its normal character. In some cases one or other of the samples was omitted in order to see whether the effects differed with variations in the loss of blood. The same electrode system was used for each sample in the series, the hydrogen electrode being charged afresh before the introduction of the new specimen.

5 Changes in the Hydrogen Ion Concentration of the Blood 145 The effect of variations in the composition of the gas mixture used for ventilation was also studied. It is advisable before giving the experimental results to refer to the calculation of (H+) from the e.m.f. The hydrogen ion concentration of the blood was calculated from the e.m.f. in the usual way, making use of Nernst's well-known formula: 7rp= r log CO, where 7r = e.m.f. of the unknown fluid in the electrode system, 7ro = e.m.f. of a hydrogen electrode when the (H+) of the fluid (CO = 1) is normal, and C and CO= the respective H+ concentrations of the two fluids. 7ro may be taken as *3377 volt at 180 C. Therefore 7p= log!. p C may therefore be calculated when ;r is measured. Or if p be put in place of C (10--P), the results may be stated in terms of Sorensen's negative hydrogen ion exponent written by him ph+. Thus 7rp- '3377 P P0577 As one is dealing with solutions much below normal ionic concentration, PH+ is a negative exponent, so that a rise in ph+ signifies a fall in the hydrogen ionic concentration (H+). The results of the estimations in the various experiments will be given for convenience both as (H+) and as p1+. The animals were anasthetised with ether, and pulmonary ventilation was carried out by connecting the trachea tube with the tubes of the Meyer's pump. In the smaller animals 100 c.c. of air or oxygen, as the case might be, were driven in at a rate of 67 times per minute for varying periods, and the examination of the blood made in the way and at the times above mentioned. The first experiments refer to ventilation with air, and finally with oxygen, as the addition of the latter produced a longer apnceic pause. I. Cat, kgm. 15 min. ventilation with air, followed by 2 min. 15 sec. ventilation with oxygen. The apnoeic pause was 3 min. 10 sec. in duration. Specimens of blood taken. (H+) x 1O-7N. a. Before ventilation b. After ventilation c. At end of aprcea d. 10 min. later.. *3890 7'41

6 146 Milroy II. Cat, kgm. Air Apnoea 2 inin. 50 sec. veintilation 5 min. Oxygen 2 min. a. b. C. d. Specimeens of blood taken. Before ventilation After ventilation End of apncea. 5 niii. later (H+) x 10- N. *4169 * * III. Cat, kgm. 16 Apnoea 2 min. 45 sec. Specimens of blood taken. min. air. 2 min. 10 sec. oxygen. (H+) x 10- N. a. b. C. d. Before velntilation After ventilation Encd of apnca. 6 min. later * ' IV. Cat, kgn. 9 min. air. 2 min.oxygen. Apncea 2 min. Specimens of blood taken. (H+)x10 N. a. Before ventilation b. After ventilation c. End of apncea d. 9 min. later V. Cat, kgm. 10 min. air. 1 lain. 25 sec. oxygen. nearly 4 min. Apnoea a. b. C. d. Specimens of blood taken. Before ventilation After ventilation End of apncea. 7 min. later (H+) x *47 ~~~~ VI. Dog, kgm. 15 min. air (200 c.c. per stroke of pump 67 times per mninute) and oxygen finally for 1 min. Apnoeic pause 3 min. 25 sec. Specimens of blood taken. (H+) x 10- N. PH+ a..... b. c. After ventilation End of apncea '60

7 Changes in the Hydrogen Ion Concentration of the Blood VII. Cat, kgm. 30 min. air. 3 min. oxygen. Apncea 1 min. 3 sec. 147 Specinmens of blood taken. (H+) x 10-7 N. PH+ a.... b. After ventilation c. End of apnoea. *1820 * As the results obtained by ventilation with air were of the same character as those just referred to when the air period was followed by a short one with oxygen, the former will now be given before consideration of the nature of the effects produced by the latter. VIII. Cat, kgm. Air ventilation 15 min. Apnceic pause 1 min. 30 sec. Specimens of blo xod taken. (H+) x 19- N. a. Before ventilation b. After ventilation c. End of apncea. d. 15 min. later *3236 *1445 *2399 * IX. Cat, kgm. Ventilation with air for 21 min., one sample of blood being taken after 6 min. ventilation and a second after 21 min. ventilation. Apnceic pause 3 min., but sample of blood was taken shortly before the close of the pause. Specimens of blood taken. (H+) x 10-7 N. a. Before ventilation b. After 6 min. ventilation bl. After 21 min. ventilation.. * c. Shortly before end of apncea * X. Dog, kgin. 200 c.c. air pumped per minute for 15 min. Apncea 2 min. in at a rate of 67 Specimens of blood taken. (H+) x 10-7 N. a. Before ventilation b. After ventilation c. End of apncea _~~~~

8 148 Milroy XI. Catj kgm. Air 30 min. Apneea 1I min. Specimens of blood taken. (H+) x 10-7 N. P a. Before ventilation. * b. After ventilation c. End of apncea XII. Cat, kgm. Air 10 min. Apnoea 1 min. 30 sec. Specimens of blood taken. (H+) x 10-7 N. PH+ a. Before ventilation 0. * b. After ventilation c. End of apncea d. 6 nmin. later In this case, when sample d was taken the breathing was more forcible than at the beginning before ventilation. In all the experiments referred to, ventilation, whether with air alone or with air and oxygen, has led to a fall in (II+), and in all those cases an apnceic pause has followed this fall in hydrogen ion concentration. In certain comparatively rare cases no pause follows the excessive pulmonary ventilation, and in one case where this occurred I examined the blood and found that the hydrogen ion concentration had not fallen at the close of the ventilation period. This is of interest as perhaps shedding light upon the absence of an apnoeic pause after excessive respiration, noticed by Boothby (11) in his own case. XIII. Cat, kgm. Air ventilation 9 min. No pause followed, but samples of blood were taken at 1 min. 45 sec. after stoppage of pump, and again 10 min. later. Control specimens were taken shortly after the first ones, passing the second specimen in after the reading for the first one had been taken. Specimnens of blood taken. (H+) x 10 -' N. PH+ 1st 2nd 1st 2nd specimen. specimen. specimen. specimen. a. Before ventilation.. * b. After ventilation... *3715 * d. 1 min. 45 sec. later while breathing was going on.... *2884 *3467 7, d1. 10 min. later *

9 Changes in the Hydrogen Ion Concentration of the Blood 149 It is evident that ventilation has not produced the usual effect upon the (H+), and the slight differences in (H+) at different periods after the ventilation, viz. between *35 x 1O-7 and *28 x 1O-7, are such as might occur under normal conditions in the blood from changes in the amplitude of the respirations. This departure from the usual effect produced by ventilation may have been due to some circulatory disturbance such as Boothby refers to. On the other hand, the ventilation may have led to the usual lowering in H2CO3 concentration, and accompanying this there may have been a rise in (H+) due to the appearance of acid products of incomplete oxidation. In the air and oxygen experiments there is produced by the ventilation as a rule a fall in (H+) of from *14 to *17 x 1O-7, followed by a rise during the pause, which usually reaches, before breathing again starts, the level before ventilation, or may reach a distinctly higher one, as in Experiment IX., when the blood was slightly on the acid side of the neutral point. Experiments VIII.-XI. were carried out in order to see whether any difference in the results was observable if one or other of the blood samples in the series was omitted, thus leading to a diminished loss of blood. It is evident that in these cases also the results are of the samie character as those obtained when a complete series was taken. In the air ventilation experiments there is a fall in the (H+) of froin *13 to *18 x 1O-7. Just at the close of the ventilation the (H+) lies between *10 and *19 x 10-7 in the various experiments. At the end of the pause there is a rise in (H+), the final value of which varies according to the original reaction of the blood. The hydrogen ion concentration at the end of the pause may be taken as varying from *20 to *35 x It very rarely exceeds the original (H+), before ventilation. The (H+) at the end of a pause produced by air and oxygen ventilation may rise, on the other hand, distinctly above the original value, and is rarely more than slightly below it. In all probability, therefore, oxygen raises the threshold of excitation of the respiratory centre for the hydrogen ion stimulus. Brief reference will now be made to the effects produced by air ventilation followed by a short period of ventilation with air to which carbonic acid has been added. It is well known that the addition of carbonic acid to the air used for ventilation prevents the apnoeic pause, when the CO2 content of the gas mixture is at or above alveolar CO pressure. It is interesting, therefore, to determine whether the fall in the (H+) produced by ordinary air ventilation is prevented, as one would expect, by giving at the conclusion of a period of air ventilation a gas mixture rich in carbonic acid. [XIV.

10 150 Milroy XIV. Dog, kgm. Air ventilation (200 c.c. at usual rate) for 12 min., then 1 min. with 12 per cent. carbonic acid in air. Very forcible breathing started immediately on stoppage of the pump. Samples of blood were taken before ventilation and at different times after stoppage of the pump. Specimens of blood taken. (H+) x 10- N. PH a. Before ventilation b. Just at close of ventilation * c. 2 min. later d. 31 min. later XV. Cat, kgm. Air ventilation for 12 min., followed by 2 min. 15 sec. ventilation with 12 per cent. carbonic acid in oxygen. On stoppage of the pump there was an inappreciable pause, and the breathing was much less forcible than after the carbonic acid air ventilation. Specimens of blood taken. (H+) x 10 N. Before ventilation.. * At close of ventilation min. later min. later. * In both cases the finial ventilation with a 12 per cent. CO2 gas mixture produced a rise in (H+) instead of the fall observed in all cases of ventilation with air or air and oxygen. The respirations, which started immediately on stoppage of the pump, at least in the case of the air-co2 mixture, were forcible and led to a rapid fall in the (H+) from the removal of the excess of carbonic acid in the blood. In the case of the oxygen-carbonic acid ventilation there was a very short apncea, followed by respirations which were less forcible than in the preceding case, and therefore the excess of CO2 was not so quickly removed, with the result that the fall in (H+) was less rapid. The oxygen had evidently led to a decrease in the excitability of the centre, as in both cases the ventilation led to the same rise in (H+), namely, about 12 x The forcible breathing which usually follows inhalation of gas mixtures rich in CO2 is often interrupted by periods when the respirations are less in amplitude, these being evidently due to the overaction of the respiratory centre leading to an excessive washing out of the carbonic acid and so to a lowering of the (H+). In conclusion, I may refer briefly to certain recent investigations dealing with differences in the (H+) of the blood due to variations in the H2C53 concentration in the same. Michaelis and Davidoff, in the paper

11 Changes in the Hydrogen Ion Concentration of the Blood 151 already referred to, were able to recognise a difference in the hydrogen ion concentration of arterial and venous blood in the rabbit. Carotid artery. Jugular vein. PH at 180 P at 18 a. b. a. b. 7, So that during ordinary respiratory exchange the (H+) of venous blood falls in becomiing arterialised to the extent of from -05 to *08 x They obtained rather more alkaline values for human venous blood than Hasselbalch and Lundsgaard, but Hasselbalch, in his most recent paper, gives values more nearly resembling those of Michaelis and Davidoff. The experiments of Hasselbalch and Lundsgaard, and Lundsgaard (13) alone, on the (H+) of blood in gaseous equilibrium with gas mixtures containing CO2 at different partial pressures are of very great interest and importance. The following results were obtained for ox blood at 38.50, for the corpuscles and for the serum of the same: Defibrinated ox blood. Blood corpuscles. Mm. C0O H+ Mill. C02. PH Serum. Mm. C02. PH , In the first set there was a rise in (H+) from *281 8 x 10-7 to *4898 x 10-7 when the CO2 pressure was raised from 20 to 40 mimi. With a further rise in CO2 pressure the ionic rise became less marked. This rise of approximately *2 x 10-7 produced by increased H2CO3 concentration is similar to VOL. VIII., NOS. 2 AND

12 152 Milroy the fall produced by air or air and oxygen ventilation brought about by the removal of CO2 and so decreased H2CO3 concentration. Again, in Hdber's experiments (loc. cit.), where various hydrogen-co2 mixtures were passed through the blood, the results show the same rise in (H+) with increasing H2CO3 concentration. Although the blood specimens were from different animals and consequently were scarcely uniform, the general effect is quite definite. C02 per cent. (H+) X 10-7 N. - in hydrogen. 0 * ,6 ' '44 * '18 * '81 * '15 *49 7'31 4'26 * '51 *79 7' '89 7o05 Thus a rise in the C02 percentage from 1'6 to 6 per cent. leads to a rise in the (H+) of the blood from *31 to *79 x These experiments of Hasselbalch, Lundsgaard, and Hober deal with the changes which altered H2CO3 concentration produces in the blood in vitro. In the living animal such changes might not be observable owing to rapid replacement of C02 by the tissues or from some other cause. Hasselbalch (14) studied the changes in the reaction of the rabbit's blood during natural pulmonary ventilation, especially as regards their relationship to the alveolar carbonic acid pressure. He found that with increased natural ventilation theph+ rose (i.e. (H+) fell) and the alveolar carbonic acid pressure fell. In his first experiment with an alveolar ventilation of 945 c.c. per minute, the P.+ was 7'42, while with a ventilation of 1139 c.c. per minute it was found to be 7'46. He also found that a rise in the percentage of carbonic acid in the gas mixture inhaled led to a rise in the hydrogen ion concentration of the blood. For the evidence which this author has brought forward that the degree of ventilation seems to be dependent upon the hydrogen ionic concentration rather than the alveolar carbonic acid pressure, his paper should be consulted. The papers which I have referred to in the course of this contribution to the subject give the full literature on the subject of the reaction of the blood, and so I have omitted reference to many papers of great importance. As to the effect produced by an increased H2C003 concentration of the blood on the ratios of the various salts of the alkalies in the blood to one another and the influence of the protein content on the carbonic acid transport, attention may be directed to the extremely interesting article by Henderson already referred to.

13 Changes in the Hydrogen Ion Concentration of the Blood 153 I may now shortly summarise my results:- 1. Pulmonary ventilation with air or air and oxygen produces a rapid fall in hydrogen ion concentration, this apparently reaching its lowest level after very short periods of ventilation. 2. This fall in (H+) is always associated with cessation of breathing when the artificial ventilation ceases. 3. During the apnceic pause the (H+) rapidly rises approximately to its old level prior to ventilation, but may be below or, more rarely, slightly above that point.. 4. The effects produced on the (H+) by the subsequent breathing depend upon the amplitude and frequency of the respirations, hyperpncea leading to a fall and diminished breathing to a rise in (H+). 5. Ventilation with gas mixtures rich in carbonic acid, even when given only for short periods after the main ventilation with air, lead to a rise in (H+) instead of a fall, and no apnceic pause follows the period of artificial ventilation. 6. It is probable that in cases where air ventilation is not followed by an apnceic pause the (H+) has not fallen as a result of the ventilation. 7. The extremely short duration of all those (H+) changes in the blood, and their close relationship to simultaneously occurring and easily recognisable variations in the H2C03 concentration, make it extremely likely that these are causally related to one another, and that the activity of the respiratory centre is very largely dependent upon the hydrogen ion concentration of the blood. 8. The excitability of the centre as gauged by its threshold of excitation by (H+) is evidently lowered by iniereased oxygen absorption. LITERATURE. (1) HENDERSON, Ergebnisse der Physiologie, 1909, p (2) S6RENSEN, Ergebnisse der Physiologie, 1912, p. 393 (giving the literature up to this date). (3) HASS-ELBALCH, Biochem. Zeitschr., 1913, Bd. xlix. p (4) HOBER, Arch. f. d. ges. Physiologie, L903, Ed. xcix. p (5) HASSELBALCH, Biochem. Zeitschr., 1911, Ed. xxx. p (6) MICHAELIs and DAVIDOFF, Biochem. Zeitschr., 1912, Bd. xlvi. p (7) KONIKOFF, Biochem. Zeitschr., 1913, Bd. li. p (8) WILKE, Zeitsch. f. Elektrochemie, 1913, Bd. mix. p (9) WISLICENUS, Journ. f. prakt. Chem., 1896, Bd. liv. p. 54. (10) LooMIs and ACREE, Amer. Chem. Journ., 1911, vol. xlvi. p (11) BOOTHBY, Journ. Physiol., 1912, vol. xlv. p (12) HASSEILBALCH and LUNDSGAARD, Biochem. Zeitschr., 1912, Bd. xxxviii. p. 77. (13) LUNDSGAARD, Biochem. Zeitschr., 1912, Bd. xli. p (14) HASSEILBALCH, Skand. Arch., 1912, Bd. xxvii.-xxviii. p. 13.