Subsequent investigations served to differentiate the melanophoric. (Received 26 May 1937)

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1 429 J. Physiol. (I937) 90, I2.796:6I2.46I.269 SOME CHARACTERISTICS OF THE ACTION OF URINE UPON AMPHIBIAN MELANOPHORES BY S. H. RAZA AND W. R. SPURRELL From the Physiological Laboratory, Guy's Hospital, S.E. 1 (Received 26 May 1937) OUR knowledge of the role of the pituitary in controlling the pigmentary effector system of Amphibia arose from the discovery by Hogben & Winton [1922a] that the colour changes in Rana were largely due to fluctuations in the activity of a pituitary secretory mechanism under the influence of variations in temperature, light and humidity. They further showed that suitable extracts of the posterior lobe of the pituitary of mammals and birds as well as of Amphibia possessed a specific local action on the melanophores of the frog, producing maximal expansion both in the intact animal and in the isolated limb or piece of skin. This melanophoric stimulant possessed properties which differentiated it from the "pressor" component of pituitary extract. They showed (1922b] that this reaction was highly specific, nicotine in relatively large doses being the only drug which they found to produce a similar expansion. Subsequent investigations served to differentiate the melanophoric principle from other products of the pituitary and thus the idea of a specific melanophoric hormone, secreted by the pituitary, became established. In recent years the possibility that this substance might be identified in the body fluids of subjects suffering from pituitary dysfunction has attracted attention. Many observers have reported that the tlrine of certain individuals possessed the power of promoting expansion of the frog's melanophores. Colin & Drouet [1933] obtained positive reactions with urines of patients suffering from pituitary tumours, hyperthyroidism, and in three cases with retinal hwemorrhages and chloride retention. Drouet et al. [1933] obtained positive reactions from patients suffering from migraine. Konsuloff [1934] obtained nine positive results from nine pregnancy urines and suggested that the reaction might be employed

2 430 S. H. RAZA AND W. R. SPURRELL as a pregnancy test. In these and similar records, the observers appear to accept the reaction as being due to the appearance of melanophoric hormone in the urine. Jores [1933] made a detailed study of the melanophoric principle. By comparison with the Voegtlin standard extract of pituitary, he made quantitative studies of the melanophoric response with preparations subjected to various chemical and physical manipulations and applied to small isolated fragments of standardized frog's skin. In this way he was able to classify a large number of properties which justified the establishment of the melanophoric hormone as a separate entity exerting a highly specific influence upon the melanophores of the frog. In the light of this knowledge he [Jores, 1936] examined the melanophoric reactions produced by certain urines and came to the conclusion that they did not contain the melanophoric hormone because they differed in certain characteristic properties. Incubation with trypsin, irradiation with ultra-violet light and shaking up with charcoal all remove or inactivate the melanophoric hormone of pituitary extract, but these manipulations did not invariably abolish the urinary reaction. Furthermore, melanophoric hormone, injected into the circulation, could be identified in the blood but not in the urine, whereas the blood of subjects with a positive urine contained no melanophoric hormone. For these reasons he concluded that the urinary reaction was non-specific, i.e. it was due to some condition other than the presence of the melanophoric hormone. Our attention was drawn to this urinary reaction by Konsuloff's suggestion that it might be employed as a test for pregnancy. We have examined the urines of many subjects, pregnant and non-pregnant, male and female, and the results of these examinations are presented in this paper. Furthermore we have examined some of the characteristics of this reaction in the light of Jores's views on non-specificity. METHOD As the reaction of the medium affects the degree of melanophoric response, all urines tested have been adjusted to ph 7X2 unless otherwise stated. As test object the contracted dermal melanophores of Rana have been employed and we have used various methods of assay of the melanophor expanding activity, namely that on hypophysectomized frogs [Konsuloff, 1934], on intact pale frogs [Hogben & Slome, 1931], on Ringer-perfused limbs [Fenn, 1934] and on isolated skin [Jores, 1933]. All methods give comparable results but we have found intact pale frogs

3 MELANOPHORE EXPANSION BY URINE or Ringer-perfused limbs most convenient. To test the activity of urines, we have injected c.c. into the dorsal lymph sacs of pale frogs in groups of four, taking the "melanophoric index" [Hogben & Slome, 1931] of the web as our indicator. We have arbitrarily classified our reactions as " strong " if the alteration in index exceeds 2 and as " weak " if the change lies below that figure. In our studies of the factors affecting the reaction we have used Ringer-perfused limbs. RESULTS Incidence ofthe melanophoric reaction in urine. The urines of46 pregnant and 52 non-pregnant healthy adults have been examined and the results are recorded in Table I. Attempts to correlate the occurrence of positive reactions with age, sex or colour have failed to demonstrate any relationship. TABLE I. Incidence of urinary reactions Proportion of positive No. of No. of positive reactions Source of specimen cases reactions p.c. Male Female (non-pregnant) Average (non-pregnant) Female (pregnant) "weak" 98 Characteristics of the reaction. In order to determine some of the characteristics of the urinary reaction for comparison with the properties of the melanophoric hormone, pooled pregnancy urines were employed. The potency of the urine was first tested by the perfusion method, the urine then subjected to various conditions and the resulting potency tested by a repetition of perfusion. The reaction appears unaffected by acidification to ph 5, but further acidification leads to a progressive diminution in the response elicited. Boiling the urine with N/1O HCI for half an hour does not abolish the reaction. Treatment with alkali seems to enhance the reaction. The reaction is completely abolished if the urine is shaken up with charcoal or a fine precipitate of benzoic acid. Dialysis through collodion or cellophane yields a dialysate which is quite inert and the residue within the dialyser is also inactive. This suggests that adsorption is responsible for the removal of the active component. Incubation with trypsin and alkali for 2 hours usually has no effect upon the reaction; in two cases, however, the reaction was markedly diminished or abolished. Control incubation of the urine with trypsin, 431

4 432 S. H. RAZA AND W. R. SPURRELL alkali and powdered fibrin showed that, with the exception of the abovementioned two cases, protein digestion did not occur and the urines presumably contained some antitryptic factor. Attempts were made to concentrate and separate the active principle concerned. Positive urines were concentrated in vacuo but no very definite increase in potency was demonstrated; apparently this was not due to any gross destruction of the principle, for redilution of the concentrate to its original volume gave reactions similar to that of the original urinary sample. All attempts to separate the principle have failed. Adsorption on charcoal, collodion and benzoic acid effectively removes the active principle, but the recovery of the principle from the adsorption compound has not been achieved despite numerous modifications in method. DIsCUSSION The expansion of the melanophores of the frog can be produced by the urines of a large percentage of normal subjects so that any necessary association between a positive reaction and some disturbance of the pituitary need not be considered. In pregnancy the incidence of positive reactions is markedly increased, but its common occurrence in the nonpregnant invalidates the reaction as a pregnancy test. Konsuloff's small series of pregnancy urines gave 100 p.c. positive reactions but Jores quotes a larger series in which 62 p.c. were positive: the discrepancy between that figure and our 98 p.c. may depend upon the sensitivity of the test employed and his figure may be related to the 60 p.c. of "strong" positive reactions we obtained. All attempts to correlate the distribution of positive reactions in non-pregnant subjects with such variants as age, sex or colour have failed, nor does it appear to be related to urinary reaction or specific gravity. The question as to whether the reaction depends upon the presence of the specific melanophoric hormone or upon some non-specific principle can only be answered by a comparison of the known properties of the hormone with the characteristics of the urinary reaction or of its isolated principle. At the present time the results of such a comparison are inconclusive, but certain points of similarity and apparent difference have been established. The most important property of the melanophoric hormone is its capacity for promoting the expansion of the frog's melanophores and in this capacity the urinary reaction is apparently similar. The melanophoric hormone is stable in moderately acid solutions, slowly destroyed in strong

5 MELANOPHORE EXPANSION BY URINE 433 acid, stable and even of enhanced potency in alkali; with all these characteristics the urinary reaction corresponds. The melanophoric hormone is adsorbed by charcoal and will not pass through a collodion membrane: again the urinary reaction corresponds. Melanophoric hormone is destroyed by incubation with trypsin but usually the urinary reaction withstands such digestion. This difference was one which led Jo res to doubt the specificity of the reaction, but it receives a simple explanation when it is realized that these very urines contain some antitryptic principle. What is really more significant is the fact, observed by Jores and ourselves, that in some urines the positive reaction is completely abolished by trypsin, thus suggesting that there is a component of the reaction which can, under certain circumstances, be destroyed by tryptic digestion. Our experiments suggest that the requisite circumstance is the absence of the antitryptic property in urine, for protein digestion proceeds normally in those urines in which trypsin abolishes the melanophoric reaction. Another fact which led Jores to doubt the specificity of the reaction was his ability to regain the melanophoric principle after adsorption with kaolin and his inability to regain anything from similar adsorbing agents used to inactivate a positive urine. We have failed in like manner to regain any active principle after adsorption from a positive urine, but the wellrecognized difficulties of elution do not make this discrepancy quite so formidable, especially when it is appreciated that recovery of the melanophoric hormone itself has failed after adsorption with agents other than kaolin. The fact that the urinary reaction is abolished by dialysis and by adsorption on charcoal, benzoic acid and collodion suggests that it depends upon the presence of some substance or substances of large molecular size. The abolition of the reaction by trypsin, when the urine is not antitryptic, suggests that this substance is related to protein. As far as these investigations go, there is nothing really incompatible with the idea that the urinary reaction depends upon the presence of melanophoric hormone in the urine. It may be in a form which differs from that found in direct pituitary extracts but it agrees with many of the characteristics of the hormone. The question cannot be settled until the active principle can be separated from the urine.

6 434 S. H. RAZA AND W. R. SPURRELL SUMMARY 1. Melanophoric expansion in the frog can be produced by the urines of 38 p.c. of normal people. 2. In pregnancy the percentage of positive reactions is increased to The characteristics of the reaction are not incompatible with its dependence upon the presence of the melanophoric hormone in the urine. REFERENCES Colin, R. & Drouet, P. L. (1933). Rev. fran9. Endocrin. 2, 161. Drouet, P. L., Mathieu, U. L. & Colleson (1933). Bull. Soc. medi. H6p. Paris, 49, Fenn, W. 0. (1924). J. Physiol. 59, 35P. Hogben, L. T. & Slome, D. (1931). Proc. Roy. Soc. B, 108, 10. Hogben, L. T. & Winton, F. R. (1922a). Ibid. 93, 318. Hogben, L. T. & Winton, F. R. (1922 b). Biochem. J. 16, 619. Jores, A. (1933). Z. ges. exp. Med. 87, 266. Jores, A. (1936). Klin. Wschr. 15 (2), Konsuloff, St (1934). Ibid. 13, 776.