OBSERVATIONS ON THE URINARY EXCRETION OF HISTA-

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1 OBSERVATIONS ON THE URINARY EXCRETION OF HISTA- MINE IN URTICARIA. By H. M. ADAM, R. B. HUNTER1 and T. W. G. KINNEAR. From the Departments of Pharmacology and Therapeutics, University of Edinburgh. (Received for publication 29th May 1950.) THE pharmacological changes that occur in the skin in urticaria are not clearly known. According to Lewis and Grant [1924] the appearance of wheals in the skin may be the result of the liberation of a histaminelike substance. Evidence in support of this hypothesis has been sought in various ways: by the extraction of histamine from the skin [Harris, 1927], the liberation of histamine in the allergic skin reaction [Katz, 1942], and, more recently, by the action of synthetic antihistamine drugs [Feinberg, 1946; Hunter, 1947; Bovet and Bovet-Nitti, 1948]. Apart from the study of local reactions, it has been shown that systemic effects, comparable with those of histamine, may also occur in urticaria factitia [Lewis and Harmer, 1927; Kalk, 1929] and in allergic reactions characterised by whealing of the skin [Horton and Brown, 1929]. It seemed likely, therefore, that some of the H-substance released at the site of the wheal was absorbed into the general circulation. Rose [1941] estimated the histamine content of the blood in cases of urticaria and angioneurotic aedema, but found that it was diminished, rather than increased, during attacks. Recently one of us [Adam, 1950] reported that histamine given by slow intravenous infusion, at a rate which produced systemic effects, could be detected more easily in the urine than in the venous blood. In the present work an attempt was made to discover (a) whether urticarial subjects differed from healthy subjects in their excretion of histamine administered by mouth and by intravenous infusion, and (b) whether the daily excretion of histamine in the urine of subjects with urticarial wheals was raised in comparison with that of subjects without urticaria. The blood histamine was also estimated in two urticarial subjects who received histamine by intravenous infusion. METHODS. 1. Pharmacological. Free and conjugated histamine in the urine were estimated by a simplified method based on that of Anrep, Ayadi, Barsoum, Smith and I Present address: Department of Pharmacology and Therapeutics, University of St. Andrews. VOL. XXXVI, NO

2 50 Adam, Hunter and Kinnear Talaat [1944], and Adam [1950]. When histamine was added to the urine in a concentration of 100 jug. per litre, this method gave a mean recovery of 74-8 per cent. as free histamine, and the limits of error (P = 0.95) for a single observation were ,ug. per litre. Histamine in the blood-cells and plasma was estimated by Code's modification [1937] of the method of Barsoum and Gaddum [1935]. The extracts were tested on a strip of guinea-pig ileum suspended in 2 ml. Tyrode's solution containing atropine (0.1,ug./ml.) in comparison with a standard solution of histamine acid phosphate (British Drug Houses, Ltd.). At the end of several of the assays Mepyramine maleate (May & Baker, Ltd.) was added to the bath in doses of 1-5 jig. All the histamine values are given as the base which is taken to represent one-third of the weight of the acid phosphate. 2. Clinical. The methods used for the administration of histamine and for collection of the urine have already been described [Adam, 1950]. The excretion of administered histamine was followed in three men with chronic urticaria. They are referred to as W.A., W.R. and D.B., and all received histamine by intravenous infusion; W.A. and W.R. also received it by mouth. The urine was collected at six-hourly intervals before, during and after the administration of histamine. These patients, like all the others, were on an ordinary hospital diet. The daily excretion of histamine was studied in 9 subjects with urticaria, in 10 with other diseases and in 10 normal or convalescent subjects. The urine collections were made on two successive days. Seven of the urticarial cases had chronic urticaria which varied in duration from several weeks to years; 2 had acute urticaria with cedema which came on suddenly and for the first time during treatment of an infection with penicillin. Treatment of the urticarial patients with synthetic antihistamine drugs curtailed the number of histamine estimations that could be usefully made. These drugs were excreted in the urine and the amounts present in the extracts interfered with the assay. All these patients presented a varying number of urticarial wheals during the period of urine collection. RESULTS. 1. Excretion of Administered Histamine. (a) After Oral Administration.-The results for W.A. and W.R. are given in Tables I and II. The value for conjugated histamine was obtained by subtracting the free from the total histamine. When the concentration of free histamine in the test was less than could be assayed (usually < 0-01 ug./ml.), this was recorded as a trace (tr.).

3 Urinary Excretion of Histamine in Urticaria 51 TABLE I.-CHRONIC URTICARIA. EXCRETION OF HISTAMINE IN URINE BEFORE AND AFTER ORAL DOSE. W.A. cet. 38. B.W. 68 kg. Urine vol. Histamine,g. per 6 hours. Hours. ml. Total. Free. Conjugated tr. < tr. < tr. < mg. histamine in 400 ml. 0 9 per cent. NaCl tr mean values mean fvalues Percentage of dose excreted TABLE II.-CHRONIC URTICARIA. EXCRETION OF HISTAMINE IN AND AFTER ORAL DOSE. W.R. cet. 16. B.W. 52 kg. Hours Urine vol. Histamine pg. per 6 hours. ml Total. Free. Conjugated tr. < tr. < tr. < tr. < mg. histamine in 400 ml. 0-9 per cent. NaCl tr tr. 250 URINE BEFORE Percentage of dose excreted. 1'2 (b) After Intravenous Infusion.-The results are set out in Tables III, IV and V, which also contain the blood histamine values for W.A. and D.B. An arterial blood sample was taken from D.B. by puncture of the femoral artery. The blood samples were removed after the infusion had run for over two hours, and the pharmacological action of histamine on the pulse rate, blood pressure and skin colour was well established.

4 52 Adam, Hunter and Kinnear TABLE III.-CHRONIC URTICARIA. EXCRETION OF HISTAMINE IN URINE BEFORE AND AFTER INTRAVENOUS INFUSION. W.A. crt. 38. B.W. 68 kg. Urine vol. Histamine pg. per 6 hours. ml Total. Free. Conjugated. Percentage of dose excreted. Blood histamine pg. per ml. Cells. Plasma mean J values mean J values 0-02 < mg. histamine in 552 ml. 0 9 per cent. NaCl infused intravenously in 155 minutes tr Onset of urticarial reaction. 6 0 tr. 0 tr. < < 0-01 Hours TABLE IV.-CHRONIC URTICARIA. EXCRETION OF HISTAMINE IN URINE BEFORE AND AFTER INTRAVENOUS INFUSION. W.R. cet. 16. B.W. 52 kg. Urine vol. Histamine pg. per 6 hours. ml. Total. Free. Conjugated tr. tr. tr. tr. < 6 11 < 9 < 8 Percentage of dose excreted. 5 mg. histamine in 500 ml. 0 9 per cent. NaCl infused intravenously in 185 minutes Onset of urticarial reaction tr. < tr. < tr. < 6

5 Urinary Excretion of Histamine in Urticaria 53 TABLE V.-CHRONIC URTICARIA. EXCRETION OF HISTAMINE IN URINE BEFORE AND AFTER INTRAVENOUS INFUSION. D.B. cet. 35. B.W. 67 kg. Urinavo.e g per_6_hours p oet Blood histamine p Hours.eUrine vol. e r 6 hours. Pecte g. per ml. m. Total. Free. Conjugated. excreted. Cells. Plasma tr < 0.01 (venous sample) 7-25 mg. histamine in 725 ml. 0 9 per cent. NaCl infused intravenously in 145 minutes < 0-01 (arterial sample) tr tr. 46 mean tr. 46 fvalues mean f values 2. Effects of Administered Histamine. After oral administration there were no obvious effects, and after intravenous infusion the action on the pulse rate, arterial blood pressure and skin vessels was similar to that observed in men without urticaria [Adam, 1950]. However, in W.A. and W.R., urticaria of unusual severity occurred 3 and 18 hours, respectively, after the infusion was ended. The lesions were distributed over the whole skin, excluding the palms and soles, and were most numerous on the trunk. At first there were many flares which contained near the centre a wheal 2-3 mm. wide. The wheals grew to their full size in several hours. The flare then appeared as a narrow well-defined ring surrounding the wheal. This was usually paler, about 1-2 cm. in width and distinctly raised. The attack in W.A. subsided after about 48 hours and in W.R. after about 24 hours. The urine passed during the period of the attack, excepting one sample (W.A., hours, Table III), showed no increase in the excretion of either free or conjugated histamine. 3. Histamine in the Urine of Patients with and without Urticaria. The results are presented in Tables VI, VII and VIII. When the histamine equivalent of the extract was less than 0-01 ug./ml. it was assumed for the purpose of the calculations that no histamine was present in the sample.

6 54 Adam, Hunter and Kinnear TABLE VI.-URINE HISTAMINE OF SUBJECTS WITH URTICARIA (9). Subject. Sex. Age. Histamine utg. per Urine 24 hours. Number of Duration vol. wheals. urticaria. ml. Toa.Fe o-urticaria. H.S. F. H.S. 3 F jugated years I.M. F months M.T. F tr months tr. 76 McC. F year W.R. M tr lj years tr. 34 W.A. M tr years D.B. M year T.B. M Acute urticaria 48 hours on 5th day of penicillin treatment for a pulp infection. F.M. M Acute urticaria 48 hours on 13th day of penicillin treatment for syphilis. Mean of 17 estimations S.E The mean value for the excretion of free histamine in the urticarial group (35-8,ug./24 hours) differs significantly from the mean in the group with various diseases (15-1 ug/24 hours; P < 0-02) and from the mean in the group of normal and convalescent subjects (8-6,ug./24 hours; P < 0-001). The higher mean value in the urticarial group is due mainly to the contribution of the two cases with acute urticaria. If these are omitted, the mean excretion in chronic urticaria does not differ significantly from that in the control groups. The means for the excretion of conjugated histamine in the urticarial and control groups do not differ significantly. It may be added that the differences in the excretion of either free or conjugated histamine in the control groups are not statistically significant.

7 Urinary Excretion of Histamine in Urticaria 55 TABLE VII.-URINE HISTAMINE OF SUBJECTS WITH VARious DISEASES (10). Urine Subject. Sex. Age. vol. ml. HIistamnine,ug. per 24 hours. Total. Free. Conjugated. T.R. M Disease. Lobar pneumonia T.S. M Addison's disease (controlled by DOCA implant) F.M. F tr tr. 9 D.D. F T.S. M J.W. M tr. 14 A.D. M tr. tr tr. tr. 0 M.P. F tr M.S. F S. F Mean of 19 estimations S.E. 15*4 24' Erythema bullosum Anorexia nervosa Ulcerative colitis Sprue syndrome T.B. spondylitis Menieres disease Diabetes mellitus Pernicious aneemia DISCUSSION. It appears that men with chronic urticaria excrete administered histamine in the same way as men without urticaria [Adam, 1950]. When histamine was given by mouth, the amounts excreted in the urine as the conjugate were 0*55 and 1*2 per cent. of the dose. In each case the sample collected from 6-12 hours gave the highest value for the conjugated histamine; but the largest amount of free histamine was present in the sample from 0-6 hours (see figure). The absorption of free histamine from the gut of these subjects was no greater than that previously observed in normal subjects. When histamine was infused intravenously in doses of mg. in 2-3 hours, the amounts excreted in the urine were 10, 1.1 and 1.4

8 56 Adam, Hunter and Kinnear TABLE VIII.-URINE HISTAMINE OF NORMAL AND CONVALESCENT SUBJECTS (10). Subject. Sex. Age. Urine vol. ml. D.S. M D.L. M A.L. M R.H. M D.H. M M.W. McG. McF. H.A. McK. M M M M F Histamine,ug. per 24 hours. Total. Free. Conjugated tr. 18 tr. 20 tr tr tr. 23 tr. 34 tr. 40 tr tr. 15 tr. 30 tr Mean of 20 estimations S.E ' per cent. of the dose. There was no detectable rise in the blood or plasma histamine concentration, which suggests that in these subjects histamine disappeared from the blood as quickly as in normal subjects. Failure to detect an increase in the concentration of histamine in the venous blood, while the drug was being infused, may mean that the histamine was removed from the circulation during a single passage through the capillaries. It is also possible that the rise in concentration was less than could be detected by the method used. On the first view, a detectable rise would be expected in the arterial blood, but the concentration in a single sample of arterial blood was found to be no higher than the values obtained for venous blood samples. The urticarial reactions which developed in W.A. and W.R. several hours after the infusion was completed are of considerable interest.

9 ULrinary Excretion of Histamine in Urticaria They may have occurred by chance, or have been related in somle way to the infusion proceduire. On the other hancd, it is possible that they were a late effect of the infused histamine. If this were so, it might be r- 350 I A FREE t TOTAL CONJUGATED I HISTAMINE 0 I.10 z 0 v ce z D Vz zh I.., z D 0 H J D I I 100 I U- h.1-mommmm HOURS B U BLOC D HISTAMINE LIL F. L 24 C -II 4=I 48 HOURS J Subject WA. Totol hlishtlilin(' excret ion following: (A) 1:33 nig. histamnine orallv; (131.) ni-. if 1hisfillillm)nb 1(sow inti-xvenoitis infilsion; (C) Olnset of lirticainil r((( tioli (1 histaiiuil' /og. JIil. suip)posed that some of the histamine not excreted in the urine wnas held iii the skin in a form that could(t be released durinig the next attack of urticaria, which was thus miiade imiore severe than it otherwise would have been. Histamwine inijected into the skin [Lewis, 1927] or introduced into it by ionitophoresis [Abramson and Gorin, 1940] is not

10 58 Adam, Hunter and Kinnear rapidly destroyed, possibly because there is no histaminase in skin [Best and McHenry, 1930]. Free histamine did not appear in the urine in larger amounts during these urticarial reactions. By analogy with infusion experiments, it may be concluded that if histamine passed from the skin wheals into the circulation, the amount was certainly less than at the rate of 5 mg. of the base in 2-3 hours. The method used for the estimation of histamine would be expected to detect 1 per cent. (50,ug.) of this quantity in the urine. Only when the urticarial reaction occurred in its severest form and was accompanied by cedema of the subcutaneous tissues, was the excretion of free histamine unusually high. Thus, the amount of free histamine that subject T.B. (Table IV) excreted in 24 hours was equivalent to the amount that would be expected in the urine after the slow intravenous infusion of 5 mg. of the base. Among the cases of chronic urticaria only two, H.S. and McC., gave high values for the excretion of free histamine. Others had wheals, but only a trace of free histamine in the urine. Yet, in the group as a whole, the higher values seem to be related in a general way to the severity of the urticaria. The evidence presented supports the conception that histamine is either liberated or newly formed in urticaria, but it is inconclusive. Reliable evidence is unlikely to be obtained until a more sensitive method is devised for the estimation of free histamine in the urine. SUMMARY. 1. The effect on the urinary histamine of histamine given by mouth or by intravenous infusion was studied in three men with chronic urticaria and found not to differ from that recorded in normal men. Two of these men who received histamine intravenously developed some hours later an urticarial reaction of unusual severity. 2. In two of the intravenous infusions no changes were detected in the histamine content of the plasma or cells obtained from the antecubital vein; no changes were detected in a single sample withdrawn from the femoral artery. The possible significance of this result is discussed. 3. The mean daily excretion of free histamine in a group of nine cases of urticaria differed significantly from the mean in a group of ten patients with other diseases and in a group of ten normal and convalescent subjects. The mean daily excretion of conjugated histamine did not differ significantly in the three groups. These results support the conception that histamine is either liberated or newly formed in urticaria.

11 Urinary Excretion of Histamine in Urticaria 59 ACKNOWLEDGMENTS. We wish to thank Professor D. M. Dunlop for permission to make these observations on patients in his wards, and Dr. R. M. Marquis for collection of the arterial blood sample. REFERENCES. ABRAMSON, H. A., and GORIN, M. H. (1940). Symposia on Quantitative Biology, 8, 272. ADAM, H. M. (1950). Quart. J. exp. Physiol. 35, 281. ANREP, G. V., AYADI, M. S., BARsOuM, G. S., SMITH, J. R., and TALAAT, M. M. (1944). J. Physiol. 103, 155. BEST, C. H., and McHENRY, E. W. (1930). Ibid. 70, 349. BOVET, D., and BOVET-NITTI, F. (1948). Medicaments du Systeme Ve'getatif, p S. Karger, Bale. CODE, F. C. (1937). J. Physiol. 89, 257. FEiNBERG, S. M. (1946). J. Amer. med. Ass. 132, 12, 702. HARRIs, K. E. (1927). Heart, 14, 161. HORTON, B. T., and BROWN, G. E. (1929). Amer. J. med. Sc. 178, 191. HUNTER, R. B. (1947). Lancet, 252, 672. KALK, H. (1929). Klin. Wschr. 8, 64. KATZ, G. (1942). Proc. Soc. exp. Biol. N.Y. 49, 272. LEWIS, T., and GRANT, R. T. (1924). Heart, 11, 210. LEWIS, T., and HARMER, I. M. (1927). Ibid. 14, 19. ROSE, B. (1941). J. Allergv. 12, 327.