(i.e. tyrosine minus C02), also known as tyramine. of arterial pressure; and have further shown that these bases are either

Transcription

1 THE PRESSOR BASES OF NORMAL URINE. By WILLIAM BAIN. (From the Physiological Laboratory, King's College, London.) (With nine figures in the text.) (Received for publication 25th February 1914.) IN previous communications (1) I have adduced evidence that the normal urine of adults contains certain bases which, when injected into the blood-stream of ansesthetised or pithed animals, produce a marked rise of arterial pressure; and have further shown that these bases are either reduced in amount or entirely absent from the urine of patients with high blood-pressure. The non-excretion of these bases leads to their retention in the blood, and is an important factor in the causation of hypertension. The bases doubtless arise in the intestines as a result of the putrefaction of proteins, and their amount can be considerably diminished by a marked decrease in protein intake. Suggestions as to their chemical nature have been advanced; it is probable they are similar to or identical with those obtained by Barger and Walpole (2) from putrefying meat, and by Rosenheim (3) from putrefying placenta. This conclusion was based upon the general reactions, both chemical and physiological, of the material obtained from the urine. In my previous papers I have suggested that the most abundant pressor base in the urine is iso-amylamine (i.e. leucine minus C02); this is soluble in ether. In some cases this appears to be the only pressor base present. But in some urines a second base can be obtained, after ether extraction, by extraction with amylic alcohol, and this I have suggested is p-hydroxyphenylethylamine (i.e. tyrosine minus C02), also known as tyramine. During the last two years I have made attempts to identify these bases with greater exactitude, and I propose to describe my experiments and results under the following heads:- 1. Methods. 2. The identification of the ether-soluble base. 3. The existence of a second pressor base in certain urines. 4. The alleged pressor effect of trimethylamine. 5. Conclusions. 1. METHODS. The method used for the isolation of the bases depends on an observation previously published, namely, that charcoal possesses the power of adsorbing them from the urine. The urine was accordingly shaken up

2 230 Bain in quantities of two and a half litres at a time with blood charcoal (Fleming's first quality); an addition of 5 gms. of this charcoal per 100 c.c. of urine was found to be sufficient to remove them entirely; the shaking was performed in a bottle-shaker worked by an electric motor. After thorough shaking, the mixture was filtered, and a colourless filtrate obtained. The bases were next extracted from the charcoal by boiling it with dilute hydrochloric acid. This was again filtered, and the bases were found in the filtrate. The filtrate was made alkaline with sodium hydrate, and then repeatedly shaken with ether; after the removal of the ethersoluble base, the residue was reacidified with hydrochloric acid, and having been made alkaline with powdered sodium bicarbonate was extracted with amylic alcohol; this extract also contained basic pressor material. One hundred and twenty litres of normal urine were worked up by the charcoal method just described. Taking first the ether extracts, the bases were removed by extraction with acid water; this extract was concentrated on the water bath and evaporated to complete dryness by allowing it to stand in vacuo in a dessicator over sulphuric acid. A crystalline brownish residue (A) was obtained, and in this crude condition its total weight was 0-64 gm.' The amylic alcohol extract was treated in the same way, and the crystalline residue (B) obtained from it weighed gms.l Before proceeding to the chemical investigation of these residues I thought it advisable to test their physiological activity, for owing to unavoidable reasons I was obliged to be absent from the laboratory for eight months, during the whole of which time they had been kept in the dessicator. For this purpose A was dissolved in 60 c.c. of distilled water. One cubic centimetre of this was diluted to 6 c.c. with physiological saline solution, and 2 c.c. of this diluted final solution, containing 3 mgms. of the original crude residue, was injected into the jugular vein of a pithed cat. The accompanying record (fig. 1), taken from the carotid, shows that I was dealing with a powerful presor substance. Residue B was similarly diluted, and 1 c.c., containing 7-5 mgms. of the original residue, was injected in the same way. The next tracing (fig. 2) shows that the base or bases contained in the amylic alcohol extract produced as usual a less powerful effect. Although the amount of material available for chemical examination was very small, an attempt was made to separate out the bases in a pure condition. On the assumption that one had to deal with bases of the nature of those obtained from putrefying protein material, it was to be expected that, at any rate, part of them, namely, those representing aliphatic amines, would be volatile with steam, while the phenolic bases, if present in the extract after treatment with sodium carbonate, would not be volatile with 1 No significance is attached to these figures. The crude material furnishes no index of the amount of bases in it.

3 The Pressor Bases of Normal Urine steam, but would remain behind. The solutions were therefore subjected to steam distillation in an alkaline solution. The distillate was caught in decinormal acid. In the distillaiion of the ether-soluble material an accident occurred; owing to a loose connection in the distilling apparatus, much of the distillate was lost, and although I subsequently worked up what re- FIG. 1.-Pithed cat. Injection of 2 c.c. of solution prepared from ether-soluble base as described in text. The injection was made into the external jugular vein, and the blood-pressure record was taken from the carotid artery. Time tracing marks seconds, and also serves as the abscissa. The depression in the time tracing marks the period of the injection. All the tracings to be read from left to right. miained, the findings were not so conclusive as those I subsequently obtained in a second experiment which was fully successful, and which will be described later. The results with the amylic alcohol extract I will leave to section 3 of this paper, in which the existence of a second pressor base in the urine will be discussed (see p. 238). Before describing the second attempt to obtain the bases from a large VOL. VIII., NOS. 2 AND

4 232 Bain quantity of urine, I may add that we endeavoured in some preliminary experiments to isolate the bases from the urine by ineans of phosphotungstic acid, as it was thought that this might prove to be a simpler method than the charcoal one. It is not necessary to go into the details of the phosphotungstic method, for it was finally abandoned. It was, however, shown that all the pressor substances in the urine are precipitable by this reagent, but, on the separation of the bases from the phosphotungstic precipitate by Jacobs' method (4), it was found that their pressor action, though present, was greatly reduced, and it was therefore evident that somewhere in the FIG. 2. -Similar experiment oin the same animal in which the injection solution was )repared from the crude amylic alcohol extract as described in text. treatment the active principle had been either decomposed or lost. We therefore fell back upon the charcoal method, and our first object was to obtain the ether-soluble base in amounts sufficient for analysis. Four hundred litres of urine were worked up by the charcoal method, and the hydrochloric acid extracts of the ether extractions were kept in a stock bottle until the whole extraction was finished. This stock solution was kept just acid to congo-red until required for chemiical investigation; its total volume was 3 litres. A prelimninary experimient was first made in order to ascertain the result of steam distillation; accordingly, 100 c.c. of this stock solution were placed in a 500 c.c. round-bottomed Jena flask, and made alkaline to phenolphthalein with sodiuimi hydroxide. The contents of tlle flask were then steam distilled for 35 minutes. The apparatus had previously been

5 The Pressor Bases of Normal Urine 233 steamed through for 20 minutes to remove any impurities. The distillate was collected in decinormal hydrochloric acid, a drop of methyl-red being used as indicator. After distillation the contents of the receiver were evaporated to dryness over a water bath, and finally over soda lime in a vacuum dessicator. The residue was finally dissolved in normal saline solution for injection (Solution 1). FIG. 3.-Rise of arterial blood-pressure in pithed cat produced by intravenous injection of the base prepared from the steam distillate. The thick mark on the abscissa shows the period of injection. Time = 5 seconds. The contents of the distillation flask were then repeatedly extracted with ether. This was necessary since simple evaporation of the acidified residue would have given a product containing a comparatively large amount of sodium chloride, which would have rendered the preparation of an injection solution a matter of some difficulty. The combined ether extracts were then extracted with acid water and evaporated to dryness in the usual way. A solution of this in normal saline was prepared for injection purposes as before (Solution 2). These two solutions were used for injection

6 234 Bain and also another prepared for purposes of comparison from an equal volume of the untreated stock solution. The steam distillate extract (Solution 1) gave a considerable rise of blood-pressure (fig. 3), more than equal to the rise given by a corresponding amount of the stock solution (fig. 4). The extract from the distilling flask (Solution 2) had no effect upon the blood- FIG. 4.-Same experiment: here the injection was a corresponding amount of the stock solution to that shown in fig. 3 of the steam distillate prepared from it. In each case the actual volume of injected fluid was 2 c.c., and this was equivalent to about 666 c.c. of the original urine. pressure. Hence the base is entirely volatile with steam and is not destroyed by it. It was also found that distillation at a low temperature (390 C.) and diminished pressure (13 7 mrm. Hg) yielded a distillate which contained the pressor material, but in view of the fact that this substance is not destroyed at a high tenmperature, it was decided to adopt the simpler method of ordinary steam distillation for the whole of the stock solution. The base contained in the steamyn distillate was then isolated by convert-

7 The Pressor Bases of Normal Urine ing it into the oxalate. The steam distillate was first carefully rendered alkaline; the base present was removed by extraction with 3 lots of 50 c.c. of ether, and the combined ether extracts dried over anhydrous sodium sulphate. To the dry ethereal solution was added an ethereal solution of anhydrous oxalic acid. A thin cloudy precipitate slowly formed, which, on shaking the solution, formed into a gelatinous precipitate, which under the microscope had no crystalline appearance. This precipitation is identical in every particular with the preparation of the acid oxalate of iso-amylamine from iso-amylamine itself by a similar method. After standing for a few minutes out of contact with air, the whole was filtered through a small hardened filter and the precipitate was washed dry with ether, and finally dried over sulphuric acid in a vacuum dessicator for 24 hours. Fromi the whole of the steam distillate, which measured over 3 litres and represented 400 litres of urine, the total quantity of the crude oxalate obtained weighed 0-08 gm. 2. THE IDENTIFICATION OF THE ETHER-SOLUBLE BASE. (a) Melting-point.-The melting-point of the acid oxalate of isoamylamine is 1690 C. The melting-point of the crude oxalate of the ethersoluble base from the urine was 159 to 162 ; this was then recrystallised twice from a mixture of alcohol and acetone, and finally gm. of a substance consisting of needle-shaped crystals was obtained, identical in appearance with a synthetic specimen of the acid oxalate of isoamylamine. The melting-point determinations then made gave the following results: Determination 1. Determination 2. Melting-point of product from urine C C. Melting-point of synthetic acid oxalate of iso-amylamine C C. Mixed melt of the above two C o C. 235 The product from urine, even after two crystallisations, did not, however, melt sharply, and prior to melting, darkened at about 1500 C. But recrystallisation was not further continued, as we feared to risk losing any more of the small amount of material at our disposal. (b) Solubilities.-Not only did the product from urine resemble the synthetic oxalate in macroscopic and microscopic appearance, but it exhibits identical solubilities. It is readily soluble in water, also in alcohol, very slightly soluble in acetone, and insoluble in ether. (c) Elementary Analysis.-Two portions of 16-5 mgms. of the twice recrystallised acid oxalate were dried for four days over sulphuric acid in a vacuum dessicator. As this aniount was too small for ordinary

8 236 Bain analysis, a micro-analysis of the substance, by Pregl's method, was made by Dr H. Weil of Munich. The following figures were returned by him: Determination A.-6O000 mgms. of product gave mgms. of carbon-dioxide and mgms. of water. This corresponds to per cent. of carbon and 9.99 per cent. hydrogen. Determination B mgms. gave mgms. of carbon-dioxide and 4080 mgms. of water. This corresponds to per cent. of carbon and 9-12 per cent. hydrogen. Nitrogen Analysis mgms. gave 0279 c.c. of nitrogen at 170 and 711 mm. pressure; this corresponds to 9-65 per cent. nitrogen. Results- Carbon. Hydrogen. Nitrogen. A.. B Calcuilated for C5Hl3N(COOH) The discrepancies here shown between the calculated figures and these actually found, and which are most marked in the cases of hydrogen and nitrogen, are not sufficiently pronounced to militate against the view FiG. 5.-Pithed cat; 2 c.c. of the solution of the synthetic acid oxalate (=0,01 gramme) of iso-amylamine was injected into the external jugular vein between the two vertical marks on the abscissa. The abscissa has been raised 4 centimetres. Time = seconds. that we are really dealing with an acid oxalate of iso-amylamine; they rather indicate that two crystallisations are not sufficient to obtain the substance in a state of absolute purity, as we had already suspected from the melting-point determinations.

9 The Pressor Bases of Normal Urine 237 (d) Physiological Activity.-The following solutions were made: gm. of the substance isolated from urine was dissolved in 3 c.c. normal saline solution gm. of synthetic acid oxalate of iso-amylamine was dissolved in 9-8 c.c. normal saline. Both solutions were therefore of equal strength. The solutions were warmed to, 370 C. prior to injection. The synthetic extract was injected first. This produced a considerable rise of blood-pressure. A second injection of the same extract gave a much smaller rise (fig. 5), and this is confirmatory of the observation made by Dale and Dixon, who noted this type of immunity set up by the injection of iso-amylamine (5). The injection of the substance isolated FIG. t.-same experiment. The injection made in this case was the same quantity of the acid oxalate prepared from the ether-soluble and steamvolatile base of the urine. The rise of arterial pressure is almost exactly similar to that shown in fig. 5. from the urine produced a rise (fig. 6) identical with the second injection of the synthetic product. This experiment was performed, in my absence from the laboratory, by Professor Halliburton. Professor Dixon, who was present, stated that the results were precisely similar to those he had himself previously obtained with iso-amylamine. We therefore conclude, on the grounds of crystalline appearance, solubilities, melting-point, elementary analysis, and physiologicali activity, that the substance we were dealing with was iso-amylamine oxalate, and that the pressor base in urine, which 'I's soluble in ether and is volatile with steam, is iso-amylamine.

10 238 Bain 3. THE EXISTENCE OF A SECOND PRESSOR BASE IN CERTAIN URINES. After the iso-amylamine has been extracted by the use of ether, pressor material is still present in the residue, and can be extracted therefrom by amylic alcohol. The rise of pressure produced by the injection of the material prepared by the use of amylic alcohol is, however, always a small one, and in my previous papers I have noted several specimens of urine in which it is altogether absent. Arguing from the solubility of this second base in amylic alcohol, and from the fact that in protein putrefaction, p-hydroxyphenylethylamine or tyramine is a constant companion of iso-amylamine, I felt in my earlier work justified in concluding that tyramine is the second pressor base of the urine. Seeing, however, that tyramine is, weight for weight, a much more powerful pressor substance than iso-amylamine, it is evident that if tyramine occurs, it must be present in very minute quantities. If, therefore, it is absorbed from the intestine in equivalent amount to iso-amylamine, it must be largely destroyed in the body before it reaches the kidney. Barger and Walpole (6), working with 30 litres of human urine, found that " nearly all, if not all, the presor action is due to a substance which is volatile with steam," and therefore conclude that phenolic bases, such as tyramine, are absent, or nearly absent. It was suggested to me that the pressor action I had found in amylic alcohol extracts was due to a previous imperfect extraction with ether, and that I was really dealing with the remains of iso-amylamine, the ether-soluble base. It therefore became necessary to re-investigate the question, and, in discussing it, it will be convenient now to return to the amylic alcohol extract prepared from 120 litres of mixed human urine, referred to on p. 230 of this paper as residue B. A tracing of the pressor effect it produced is shown in fig. 2. This material was subjected to steam distillation, and the substances prepared from the distillate produced a distinct rise of blood-pressure. It was therefore evident that the amylic alcohol did contain some iso-amylamine which had not been previously removed by ether extraction, and that part of the rise of pressure at any rate, shown in fig. 2, was due to contamination with this substance. But after steam distillation, the residue was again extracted with amylic alcohol from the sodium carbonate solution, and a final solution obtained for injection, as previously described, produced a slight but decided rise of arterial pressure (fig. 7). I therefore felt justified in adhering to my original view that a mlinute quantity of a pressor substance is present in the urine, which is not volatile with steam. The problem was then attacked in a different way. Four litres of urine were treated with charcoal in the usual way, and the pressor material extracted from the charcoal with 0-3 per cent hydrochloric acid. The clear solution so obtained, which contained all the pressor material

11 The Pressor Bases of Normal Urine 239 in*the form of hydrochlorides, was then rendered alkaline with sodium hydrate, and extracted with ether, twenty times. The first fifteen extracts FIG. 7.-Pithed cat. Rise preceded by a slight fall of arterial pressure produced by the injection of material prepared from the amylic alcohol extract, after all bases volatile with steam had been distilled off. The amount injected corresponded to that yielded by 666 c.c. of urine. The lowering of the time tracing (= seconds) indicates the period of injection. The drawing of an abscissa line was omitted in this experiment. Compare this tracing with that shown in fig. 2, in which approxniately the same dose was injected of the material prepared from the crude amylic alcohol extract. were united and kept separate from the last five, which were also united. The residue was then reacidified with dilute hydrochloric acid, rendered alkaline with sodium carbonate, and extracted five times with successive portions of 20 c.c. of pure (Kahlbaum's pyridine-free) amylic alcohol; FIG. R.-Pithed cat. During the period (=3 seconds) between the two vertical lines an injection was made into the external jugular vein of a solution prepared from the amylic alcohol extract after the material had been freed from the ether-soluble base by extracting it twenty times with ether. The resulting rise of arterial pressure is very small. The actual amount injected corresponded to 750 c.c. of urine. The abscissa has been raised 4 centimetres. these five extracts were also united. The three solutions so obtained were then prepared in the usual way for injection. The dose injected in each case corresponded to the material obtained from two-thirds of a litre of urine. The injection solution prepared from the first fifteen ether extrac-

12 240 Bain 240 Bi tions caused as usual a large rise of arterial pressure; the injection of the solution prepared from the last five ether extractions caused no rise of pressure, showing that the preliminary extractions had completely removed the ether-soluble base. The injection of the solution prepared from the amylic alcohol extract caused the very small rise of pressure shown in fig. 8. Fio. 9. -Pithed cat. Pressor preceded by depressor effect of the intravenous injection of a solution prepared from the amylic alcohol extract after removal of the ether-soluble base by twenty-five previous extractions with ether. The amount injected corresponded to 750 c.c. of urine. No time tracing was taken in this experiment, but the rate of the (li-nm was approximately the same as in the previous records. This somewhat questionable proof of the existence of a second pressor base led me to repeat the experiment in an even more drastic way, only this time I used a specimen of urine (my own), which previous work led me to expect would contain a larger amount of the second base; in this case twenty-five extractions with ether were performed before amylic alcohol was employed. The first twenty ether extractions mixed together yielded plenty of pressor material. The last five ether injections gave, as

13 The Pressor Bases of Normal Urine 241 before, a negative result, showing that the first twenty extractions had removed all the ether-soluble base; a blank experiment, in which the same quantity of amylic alcohol was carried through the same process, also caused no alteration in blood-pressure when injected, showing that any effect in an amylic alcohol extract could not be due to the alcohol or to any impurity in it. The crucial experiment of injecting the material obtained from the amylic alcohol extract was then performed, with the result seen in the next figure (fig. 9). It was quite evident that in this urine the evidence in favour of a second pressor principle is quite conclusive, only the rise of pressure is preceded by a depressor effect. A slight indication of a preliminary fall of pressure is also seen in fig. 7. Abelous and his colleagues (7), who were the earliest to work at the pressor material of the urine, gave to it the name uro-hypertensine; they also showed that a depressor principle (uro-hypotensine) is present as well. It appears possible that the fall of pressure so clearly seen in fig. 9 may be due to this latter substance. The conclusion I draw from this part of my work is that, although a second pressor substance may be absent or nearly so from mhany urines, there are certain urines which do contain it in small quantities. In view of the small quantity present, and the enormous volume of urine necemsary to obtain it in amount sufficient for identification, no attempt was made to isolate it. It may, however, be added that Millon's reagent, the formaldehyde reaction, and the sodium benzene azo-sulphonate reaction all gave negative results, so that the substance or substances present cannot be tyramine or,b-imido-azolethylamine, the bases derived by the removal of C02 from tyrosine and histidine respectively. 4. THE ALLEGED PRESSOR EFFECT OF TRIMETHYLAMINE. In a previous paper (9) I mentioned trimethylamine as a substance possessing pressor properties. Abelous also (8) states that this substance raises the blood-pressure. The sample I originally employed was a commercial preparation of trimethylamine hydrochloride (Kahlbaum). But as I found that triethylamine caused no effect on arterial pressure, I decided to reinvestigate the action of trimethylamine and to purify the material which produced the rise in pressure. For this purpose it was separated into several fractions by recrystallisation from absolute alcohol. Four fractions were obtained in this way, and the remainder of the salt was recovered from the mother liquor by precipitation with ether. On comparing the physiological activity of the fractions as well as the final amount recovered, it was found that the power to elevate blood-pressure was equally distributed over all the specimens. If, therefore, the pressor principle was an impurity, it could not be separated from the trimethylamine by fractional crystallisation. I therefore prepared from another quantity of the same original material the platinum salt of trimethylamine, and

14 242 Bain purified it by Eisenberg's method (10). On analysis this salt was found to contain the theoretical platinum percentage. The base was next liberated from the platinum salt in the usual way and its activity tested physiologically. There was no doubt that in this case one was dealing with absolutely pure trimethylamine. On injecting it into the blood-stream it caused no alteration in blood-pressure. Pure trimethylamine, therefore, possesses no pressor action, and any such action which is possessed by commercial specimens, is due to impurities. It may be assumed that the source of commercial trimethylamine is probably the molasses obtained in the manufacture of beet sugar. It is difficult at present to say to which of the large number of bases known to be present in molasses this pressor activity is due. 5. CONCLUSIONS. 1. The best method of separating the pressor bases from the urine is the charcoal method. 2. The most abundant base present is soluble in ether, and is volatile with steam. It was isolated as an oxalate, which, in crystalline form, melting-point, and solubilities, is shown to be identical with the oxalate of iso-amylamine, and this conclusion is confirmed by elementary analysis. 3. After ether extraction, amylic alcohol dissolves out from the residue a small amount of pressor material. Some of this pressor effect, described in my previous papers, is, however, due to an imperfect removal of the ether-soluble base; but when this is removed by steam distillation, the residue in some urines still produces a slight rise of blood-pressure. 4. After very thorough ether extraction, there is evidence of this second pressor principle in the extract of some urines, although it may be absent or nearly absent in many cases. 5. On account of the very minute quantities of this second base, no attempts were made to isolate it. It, however, does not appear to be tyramine (p-hydroxyphenylethylamine). 6. The pressor effect produced by the injection of this second base may be preceded by a fall of blood-pressure, which may possibly be due to admixture with Abelous's uro-hypotensine. 7. Trimethylamine is not a pressor substance; its pressor action, previously described, is due to impurities in commercial specimens. I take this opportunity of expressing my thanks for much suggestive help from the staff at the King's College Laboratory, Professor Halliburton, Dr Rosenheim, and Mr Myers-Ward. The latter performed for me some of the injection experiments described. I am also greatly indebted to my assistant, Mr J. C. Drummond, B.Sc., for the painstaking way in which he performed his tedious duties in working up the large quantities of urine employed; I owe a great deal to his knowledge of chemical technique, without which my results would have been impossible. My

15 The Pressor Bases of Normal Urine 243 thanks are also due to those who supplied me with urine, especially to Dr Mott, who kindly obtained a large quantity for me from Claybury Asylum. REFERENCES. (1) BAIN, W., Lancet, August 27, 1909; April 30, 1910; May 27, (2) BARGER and WALPOLE, Journ. Physiol., 1909, vol. xxxviii. p (3) ROSENHEIM, O., ibid., p (4) JACOBS, Jourti. Biol. Chem., 1912, vol. xii. p (5) DALE and DIXON, Journ. Physiol., 1909, vol. xxxix. p. 28. (6) BARGER and WALPOLE, loc. cit., p (7) ABEILOUs, RIBAUT, SOULTE, and TOUGAN, Compt. rend. soc. biol., 1906, vol. i. pp. 460, 463. (8) ABELOUs and BARDIER, ibid., 1909, p (9) BAIN, Lancet, April 30, (10) EISENBERG, Berichte d. deutsch. chem. Ges., vol. xiii. p