conditions of infusion, an increase of the histamine concentration could

Transcription

1 : EXCRETION OF HISTAMINE IN HUMAN URINE. By H. M. ADAM. From the Departments of Pharmacology and Therapeutics, University of Edinburgh. (Received for publication 4th November 1949.) IT is now known that histamine is a normal constituent of mammalian urine [Anrep, Ayadi, Barsoum, Smith and Talaat, 1944; Gaddum, 1948; Rosenthal and Tabor, 1948; Hunter and Dunlop, 1948; Urbach, 1949]. In human urine it occurs mainly in a conjugated and inactive form: free histamine is found only in traces. Recently, Urbach [1949] has obtained evidence by paper partition chromatography that the conjugate is probably identical with N-acetyl histamine [4(,B-acetylamino-ethyl)imidazole]. In the dog, 3 to 5 per cent. of histamine given by mouth is excreted as the conjugate. When, however, histamine is injected subcutaneously it appears in the urine in the free form [Anrep et al., 1944]. The object of the present investigation was to study in man the excretion of histamine given orally and by intravenous infusion. At the same time an attempt was made to discover whether, under the conditions of infusion, an increase of the histamine concentration could be detected in the blood as well as in the urine. Rose [1940] failed to detect an increase in samples of whole blood after the subcutaneous injection of 1 mg. histamine base in man. Emmelin, Kahlson and Wicksell [1941] observed in the dog that a measurable rise of the plasma histamine occurred only when the rate of intravenous infusion was 2-5,tg./kg./min. According to Weiss, Robb and Ellis [1932], this rate of infusion would be likely to have toxic effects in man. The present study was carried out on four men, designated D.H., McG., McF. and M.W. METHODS. 1. Pharmacological. (a) Extraction of Histamine from Urine. A simplified form of the charcoal method [Anrep et al., 1944] was used. Two stages in the extraction procedure were omitted: the final alcoholic extraction, and the treatment of the aqueous solution of the 281

2 282 Adamn extract with alumina. Further, the elution was carried out at a raised temperature, which obviated the need for repeated washing of the charcoal with each lot of acid alcohol. No relaxing substance was encountered in extracts of human urine prepared by this method. Reagents.-These were similar to those employed in the original method. The activated charcoal was obtained from Messrs. Sutcliffe, Speakman & Co. Ltd., Leigh, Lancs. It contained iron which was removed by boiling in concentrated HCI (A.R.) for 15 minutes in the proportion of 1 g. charcoal to 10 ml. of acid. The charcoal was then washed with distilled water until free from acid, and dried in a desiccator. Absolute ethyl alcohol was purified by refluxing with solid NaOH and redistillation. Procedure.-Take 10 ml. of filtered, neutral urine and shake it with 0-25 g. charcoal in a centrifuge tube for five minutes. Centrifuge the suspension at 2000 r.p.m. and discard the supernatant. Resuspend the charcoal in 10 ml. distilled water and repeat the centrifuging. After decanting the supernatant, add 10 ml. of 95 per cent. acid alcohol (95 ml. absolute alcohol + 3 ml. ION HC1 + 2 ml. distilled water) to the charcoal and stir with a glass rod to obtain thorough mixing. Immerse the tube for 15 minutes in a water-bath at C. and stir the suspension from time to time. Next decant the suspension on to a No. 12 Whatman filter-paper and filter by gravity into a 50 ml. measuring cylinder. Wash the tube clean with three successive lots, each of 10 ml. of warm 75 per cent. acid alcohol (75 ml. absolute alcohol + 3 ml. ION HC1 +22 ml. distilled water), and pour each lot on to the layer of charcoal on the filter-paper. One lot can be conveniently warmed up, while the other is filtering, by placing the tube in the water-bath for 3 minutes. Heat is applied to the filter funnels to speed up filtration. The eluate has a final volume of about 35 ml., and can be stored overnight at 00 C. without loss of activity. Divide the eluate into two equal portions and take each to dryness. The evaporations are carried out under reduced pressure on a water-bath at 600 C. Fig. 1 shows the type of apparatus used. Wash one residue with 10 ml. absolute alcohol and set it aside for the estimation of free histamine. The other residue is hydrolysed in the presence of 10 ml. 1ON HC1 (A.R.). For this purpose, fit an air condenser to the flask and immerse the flask in a boiling water-bath for 11 hours. Then distil off the acid and wash the hydrolysed extract with 10 ml. absolute alcohol. At this stage the dried extracts can, if necessary, be stored in a desiccator for several days without loss of activity. Finally, take up the two extracts, hydrolysed and unhydrolysed, in three lots of 1-5 ml. each of hot distilled water. Scrape the flask clean with a glass rod fitted with a rubber policeman. Transfer each wash by dropping pipette to a 10 ml. graduated centrifuge tube. Add one drop of a 0 1 per cent. solution of neutral red to the suspension and neutralise it with 1-2 drops of 2N

3 Excretion of Histamine in Human Urine 283 NaOH delivered from a fine capillary. Make the volume of the suspension up to 5 ml. with 5-85 per cent. NaCl and centrifuge at 2000 r.p.m. for 5 minutes. Decant the supernatant and, if need be, make a final adjustment of the ph before proceeding to the assay. b C FIG. 1.-Apparatus for evaporation of extracts. (a) Screw-clip on rubber connection; (b) glass capillary; (c) rubber connection; (d) ground-glass cone with bubble trap (e) and side arm (g). This part (d, e, g) is adapted from a distillation tube (M15, Quickfit and Quartz). (f) 150 ml. spherical flask (B24, Quickfit and Quartz). The side arm (g) is inserted into a manifold which is connected to a trap and water pump. This method was tested by recovering histamine acid phosphate added to different samples of urine. A control sample of each urine was extracted at the same time. In the present experiments the recovery of conjugated histamine was assumed to be the same as for histamine added to urine. (b) Extraction of Histamine from Blood. Code's modification [1937] of the method of Barsoum and Gaddum [1935] was used. Citrated or heparinised blood samples of 10 ml. were centrifuged at 3000 r.p.m. for 15 minutes and the plasma and cells extracted separately. The final volume of the solution of each extract used for the assay was 10 ml. VOL. XXXV, NO

4 284 Adam (c) Estimation and Identification of Histamine. The histamine values are all calculated in terms of the base, on the assumption that this represents one-third of the weight of the phosphate. The extracts were tested on a strip of guinea-pig ileum suspended in 2 ml. Tyrode's solution containing atropine (0.1,ug./ml.) and were compared with a standard solution of histamine acid phosphate (British Drug Houses Ltd.). Mepyramine maleate (May & Baker Ltd.) was used as a test for histamine in the extracts [Reuse, 1948]. 2. Clinica7. (a) Administration of Histamine. Oral.-The dose of histamine was dissolved in 0-9 per cent. NaCl to give a concentration of 0*33 mg./ml. The solution was run slowly into the stomach through a Ryle tube. The subject was in a fasting state and had previously emptied the bladder. Intravenous Infusion.-After being at rest for 20 minutes the subject voided the bladder, and was immediately connected by one arm to a saline intravenous drip infusion. A control sample of venous blood was removed from the other arm. The saline infusion was then stopped, and a solution containing 10 ug. of histamine per ml. of 0 9 per cent. NaCl was infused by gravity from a graduated aspirator bottle of 1 litre capacity. To this was connected a drip chamber previously calibrated by relating drop rate to volume flow in ml. per minute. The flow rate was regulated by adjusting a screw-clip placed between the aspirator bottle and the drip chamber. In this way, quantities of 3-3 and 5 mg. of histamine were infused in 2 to 3 hours. The total amount infused after an interval of time could be read approximately on the scale attached to the bottle, and the rate of infusion (in,ug./min.) estimated at any moment by counting the drop rate per minute. (b) Effects of Histamine. The pulse rate and arterial blood-pressure were recorded at intervals of 5 to 10 minutes before and during the intravenous infusion of histamine. The onset and severity of headache, and extent of flushing, were also noted. The infusion rate was increased at varying times, but usually not beyond the limit which produced intolerable headache or a large fall in blood-pressure. The second venous blood sample was removed towards the end of the infusion. (c) Collection of Urine Samples. The urine was collected directly into chemically clean, stoppered 750 ml. conical flasks at intervals of 6 hours. The sample was

5 Excretion of Histamine in Human Urine immediately stored at 0 C. and, if necessary, acidified. Under these conditions free histamine is stable for at least 24 hours, and conjugated histamine for several weeks. In an earlier experiment (D.H.) 24-hour collections were made, and chloroform (approx. 0.5 per cent.) was added as a preservative. A preliminary routine examination of the urine, and an experiment to test for the recovery of added histamine, were carried out in each subject. In none was there any evidence of abnormality of the urine or of failure to recover the expected amount of histamine. RESULTS. 1. Recovery of Histamine added to Urine. Table I contains a summary of the results of experiments on different urines to which histamine was added in concentrations of 100 and 500,ug. per litre. Table II shows one set (500,ug. per litre) of results in detail. The initial value is that of the control sample of urine; the final, of the urine after the addition of histamine; the difference represents the recovery, and is expressed in the last column as a percentage of the amount added. Within each series, the difference between the means of the recoveries for free and total histamine is not statistically significant. Hence the extracts did not differ in their content of substances that might be expected to interfere with the assay of histamine. The recovery of histamine added to the test solutions after these had been assayed confirmed this view: the mean recoveries were 91 per cent. (range ) and 92 per cent. (range 85-95) for the unhydrolysed and hydrolysed tests respectively. These values fell short of 100 per cent. recovery, but were mostly within the limits of the experimental error of the assay. If inhibitory substances were present in the extracts, they did not wholly account for the incomplete recovery of histamine added to urine. Experiments on the extraction of histamine in similar concentrations from Tyrode solution and distilled water showed that a loss of 10 to 15 per cent. might be expected, because of incomplete elution from the charcoal. There is no evidence that conjugated histamine, in the concentrations encountered in the present experiments, interferes with the recovery of free histamine. TABLE L.-RECOVERY OF HISTAMINE ADDED TO URINE. Concentration micrograms per litre Number of estimations Percentage recovery. Total.. Total.. Mean S.D. for single observation S.E. of mean

6 286 Expt. TABLE Urine. 1 Total Total 2 Total 3 Total 4 Total 5 Total 6 Total 7 Total 8 Total 9 Total 10 Total 11 Total Adam I.-RECOVERY OF HISTAMINE ADDED TO URINE. Concentration 500,ug. per litre. Initial. Micrograms histamine per litre urine. Final < Difference. Percentage recovery J duplicate It should be mentioned that the unhydrolysed extract may exhibit actions which disappear after treatment with concentrated HCl. These are seen after a full dose (0-2 ml.) of the extract is added to the bath: there is some frothing, a slight increase in the rhythmic activity of the gut, and an unusually large contraction after washing out. 2. Accuracy of the Method. The method can be used to estimate histamine in human urine in concentrations of not less than 100,ug. per litre. For example, the mean recovery for free histamine at this level is 74*8,ug. per litre, and

7 Excretion of Histamine in Human Urine 287 the limits of error (P = 0 95) for a single observation are ,ug. per litre. When the concentration is 500,ug. per litre the mean recovery is 83-6 per cent. or 418,ug. per litre, and the corresponding limits of error are ,ug. per litre. The amount of free histamine normally present in the urine is less than can be estimated within the limits of error already mentioned. After the administration of large doses of histamine, however, the concentration of free or conjugated histamine in the urine may be expected to reach 100 pg. per litre, and even to exceed it. Excretion of Histamine in Urine. (a) After Oral Administration. This was studied in subjects D.H., McG. and M.W., who were on an ordinary hospital diet. D.H. received two doses of histamine and the 4o0 a a 0 LIS 7"S ISO. so 42.0 {0 FIG. 2.-Subject D.H. Total histamine excretion following: (A) 67 mg. histamine orally; (B) 133 mg. histamine orally; (C) 3-3 mg. histamine by slow intravenous infusion. urine was collected daily. McG. and M.W. were given single doses, and the urine was collected at six-hourly intervals to find out how quickly conjugated histamine was formed and excreted. The results of these experiments are given in Tables 1II, IV and V, and are shown graphically in figs. 2 and 3. The values obtained for McG. and M.W. show that about 50 per cent. of the conjugated histamine which appears in the urine does so in the first 6 hours; that excretion proceeds exponentially and is nearly

8 288 Adam complete in 24 hours. It may also be noted that the excretion of free histamine is slightly increased in the first 6 hours. In man, as in the dog, the amount of conjugated histamine excreted is independent of changes in the urinary output. (b) During and after Intravenous Infusion. This was studied in subjects D.H., McG. and McF., and the results are shown in Tables III, VI and VII, and in figs. 2 and 3. In subject A SO - - zcdmstpa m -fore NHIsSl o B so -B L 1*w FIG. 3. Subject McG#. Total histamine excretion following: (A) 133 mg. histamine orally; (B) 5 mg. histamine by slow intravenous infusion. McF., whose exrcretion was comparatively low, difficulties arose in the control of the infusion rate: for a period it ran at 0fi82,ug./kg./min. and the blood-pressure fell steeply. It mnay be that during this period of low blood-pressure urine formation was suppressed, and that histamine, which would have been excreted in the urine, disappeared in other ways. Anrep et al. [1944] found this to be so in the dog after large doses were injected subcutaneously. D.H. and McG. each excreted about the same proportion of the dose given. McG.'s excretion curve (fig. 3) shows that all the histamine exrcreted was present in the first six-hourly sample. McF. continued to exrcrete small quantities in the succeeding six-hourly samples, but these are of doubtful significance.

9 Excretion of Histamine in Human Urine 289 Day TABLE III.-EXCRETION OF HISTAMREm Urine vol. ml D.H. cet. 40. B.W. 80 kg. Histamine,ug.. Conjugated. < 20 > IN URINE PER 24 HouRs. Percentage of dose excreted per 24 hours. 67 mg. histamine in 200 ml. 0 9 per cent. NaCl orally <13 <18 <16 < > mg. histamine in ml. 0 9 per cent. NaCl orally. < I > * mg. histamine in 330 ml. 0 9 per cent. NaCl infused intravenously in 120 minutes. 85 < 17 < 13 0 > 30 0 > 13 2*60 Hours TABLE IV.-EXCRETIoN OF HISTAMINE IN URINE PER 6 HouRs BEFORE AND AFTER ORAL DosE. McG. aet. 48. B.W. 60 kg. Urine vol. Histamine,ug. ml Conjugated. > 2 >7 > 3 > mg. histamine in ml. 0 9 per cent. NaCl ) < 3 < 4 483) i 37 > 9 > 6 Percentage of dose excreted. 0*67

10 290 Hours Adam TABLE V.-EXCRETION OF HISTAMNE IN URINE PER 6 HOuRS BEFORE AND AFTER ORAL DOSE. M.W. aet. 57. B.W. 78 kg. Urine vol. mi.. Histamine pg. Conjugated. 770 < 8 > mg. histamine in ml. 0-9 per cent. NaCl } < 4 > < 5 > < 4 > < 4 > 12 Percentage of dose excreted. 1.0 TABLE VI.-EXCRETION OF HISTAMINE IN URINE PER 6 HouRs BEFORE AND AFTER INTRAVENOUS INFUSION. McF. aet. 32. B.W. 68 kg. Urine vol. Histamine gg. Percentage Hours. - - of dose ml.. Conjugated. excreted. Blood histamine pg. per ml. Cells. Plasma < < 4 > < > < mg. histamine in 500 ml. 0 9 per cent. NaCl infused intravenously in 180 minutes < TABLE VII.-EXCRETION OF HISTAMINE IN URINE PER 6 BEFORE AND AFTER INTRAVENOUS INFUSION. HouRs McG. aet. 48. B.W. 68 kg. Percentage of dose excreted. Hours. Urine vol. Histamine pg. ml.. Conjugated mg. histamine in Blood histamine pg. per ml. Cells. Plasma. < 4 > < 5 > 2 < 3 > 2 0*05 < ml. 0 9 per cent. NaCl infused intravenously in 170 minutes *2 0*05 < < 3 >

11 Excretion of Histamine in Human Urine 291 The Blood Histamine during Infusion. Venous blood samples were removed before and during the infusion from subjects McG. and McF. No increase in the histamine content of the cells or of the plasma was detected. At the time when the infusion sample was removed the rates were approximately 0-48 and 028 Htg./kg./min. for McG. and McF. respectively, and the drug was producing characteristic effects on the circulation. HEADACHEC sor A L 0B 120= ~ SySTOLIC X so - $0-o DlRSTOLIC i 2 to o 120 Ito MINUTES FIG. 4.-Subject McG. Effects of 5 mg. histamine given by slow intravenous infusion. (A) Remove control blood sample (10 ml.) and start histamine infusion. (B) Remove second blood sample (10 ml.). Other Effects of the Infusion. When histamine is infused intravenously for several hours, its action on the cardiovascular system frequently diminishes [Weiss et al., 1932]. In the present study it was confirmed that the intensity of the skin flush and of the headache decreased during the course of the infusions, which were given at an increasing rate (fig. 4). Weiss et at. ascribe this effect not only to the chemical inactivation of histamine but also to a physiological inactivation through the formation of a substance or substances antagonistic to histamine. In a few incidental observations it has not been possible to demonstrate that either the blood histaminase or the plasma adrenaline is increased at the end of an infusion.

12 292 Adam DIscusSION. The results obtained in man for the excretion of administered histamine confirm in a general manner those recorded by Anrep et al. [1944] for the dog. When histamine was given by mouth, the excretion of the conjugate varied between 0-17 and 1 per cent. of the dose; in the dog, Anrep et al. found 3 to 5 per cent. In man, as in the dog, there was only a slight increase of the free histamine which appeared in the first six-hourly sample. When histamine was given by intravenous infusion in doses of 3*3-5 mg., the excretion in man varied between 0-6 and 2-6 per cent.; in the dog the same authors found 175 per cent. (calculated from their figures) after subcutaneous injection of 10 mg. of the acid phosphate, but they were unable to find a relation between the dose injected and the proportion excreted because of anuria of variable duration. It is confirmed also that free histamine is normally present, but only in traces, in human urine. According to Urbach [1949], conjugated histamine is formed in the intestine by the action of bacteria: some of it is absorbed and appears in the urine. In paper chromatograms of extracts of urine and faeces which were made after the ingestion of histamine diphosphate, it behaved like acetyl histamine [4(,8-acetylamino-ethyl)imidazole]. The chromatogram of urine collected at 1 hours, and of a stool collected six hours, after an oral dose of histamine diphosphate showed a pronounced acetyl histamine spot. These findings agree with the results of the present experiments, which show that about half of the conjugated histamine excreted in 24 hours appears in the urine in the first sixhourly sample. The conjugation of histamine has also been found to occur in the tissues of the rat [Millican, Rosenthal and Tabor, 1949]. These authors injected histamine subcutaneously in rats deprived of the alimentary canal below the level of the stomach, and obtained conjugated histamine in the urine. In man, however, there is no evidence to suggest that conjugation takes place outside the alimentary tract. It appears that free histamine infused intravenously is more easily detected in the urine than in the venous blood; probably because it is concentrated in the kidneys. Failure to detect it in the venous blood by methods at present available confirms the findings of Rose [1940] and Emmelin et al. [1941]. In seeking evidence for the theory that liberated or newly formed histamine contributes to the pathogenesis of certain diseases, it may be useful to examine the urine as well as the blood.

13 Excretion of Histamine in Human Urine 293 SUMMARY. 1. A simplification of the method of Anrep et al. for the estimation of histamine in human urine is described. 2. It is not possible by this method to obtain a reliable estimation of the amount of free histamine normally present in human urine, but the effects of the administration of histamine may be followed. 3. When 133 mg. of histamine was given by mouth to three men, it was mainly conjugated histamine that appeared in the urine. 4. When mg. was infused intravenously in three men, free histamine appeared in the urine. 5. In two of these experiments no changes were detected in the histamine content of the plasma or cells obtained from the antecubital vein. It is concluded that investigations of the excretion of histamine are more likely to give information about the release of histamine in the body than investigations on venous blood. ACKNOWLEDGMENTS. I wish to thank Professor J. H. Gaddum for advice and criticism, Professor D. M. Dunlop for permission to make observations on patients in his wards, Dr. R. B. Hunter for valuable assistance with the intravenous infusions, and Dr. Kapeller-Adler for estimations of the blood histamine and histaminase. REFERENCES. ANREP, G. V., AYADI, M. S., BARsoIm, G. S., SMIm, J. R., and TALAAT, M. M. (1944). J. Physiol. 103, 155. CODE, C. F. (1937). Ibid. 89, 237. EMMELIN, N., KAHLsoN, G., and WICKSELL, F. (1941). Acta physiol. scand. 2, 123. GADDUM, J. H. (1948). Brit. med. J. 1, 867. HUNTER, R. B., and DUNLOP, D. M. (1948). Quart. J. Med. 41, 271. MILLICAN, R. C., ROSENTHAL, S. M., and TABOR, H. (1949). J. Pharmacol. 97, 4. REUSE, J. J. (1948). Brit. J. Pharmacol. 3, 174. ROSE, B. (1940). Science, 92, 454. RosENTHAL, S. M., and TABOR, H. (1948). J. Pharmacol. 92, 425. URBACH, K. F. (1949). Proc. Soc. exp. Biol. N.Y. 70, 146. WEISS, S., ROEB, G. P., and ELLIs, L. B. (1932). Arch. intern. Med. 49, 360.