PUBLIC HEALTH REPORTS

VOL. 38

MARCH 16, 1923

No. 11

STUDIES ON THE BIO-ASSAY OF PITUITARY EXTRACTS.1

Concerning the Use of a Desiccated Infundibular Powder as a Standard in the Physiological Evaluation of Pituitary Extracts.

By MAURICE I. SMITH, M. D., Pharmacologist, and WM. T. MCCLOSKY, A. B., Assistant Pharmacologist, Division of Pharmacology, Hygienic Laboratory, United States Public Health Service.

INTRODUCTION.

It is needless to enter here into a discussion concerning the necessity for accurately standardizing extracts prepared from the infundibular lobe of the pituitary gland so as to insure uniformity in potency of the various commercial products. This has been sufficiently emphasized by various workers in this field, and the matter has been considered of so great importance that the Ninth Decennial Revision of the United States Pharmacopoeia adopted a method for the bioassay of pituitary extracts and made it obligatory that the commercial products conform to a certain standard (1). Several years have elapsed since this went into effect; nevertheless, pituitary extracts from various commercial houses of to-day show as much variation in potency as prevailed before the method of assay was adopted. Obviously, either the commercial firms failed to adhere to the prescribed standard or else the standard or method is unreliable. It appears that there is truth in both deductions. Several years of experience with this method of assay of pituitary extracts convinced workers in this field of the unreliability of the standard (Beta-iminazolylethylamine, or, briefly, histamine) originally proposed by Roth in 1914 (2). Expressions of dissatisfaction with the standard are found in the publications of Fenger (3), Pittenger and Vanderkleed (4), Eckler (5), and others. In 1918, Spaeth wrote in reference to this standard that his "experiments confirm the evidence that histamine, on account of its deterioration and for other reasons, is not a practical standard" (6). Consequently we find that one commercial firm standardizes its pituitary extracts by a method which it finds most suitable, without regard for what other manufacturers do, and thus one firm puts out extracts of a potency that bears no relation whatever to that of extracts made by other firms.

1 A detailed report of this work will appear in a Bulletin of the Hygienic Laboratory in the near future. 32152°-23-1

(493)

In 1918 Spaeth (loc. cit.), not being satisfied with histamine as a standard for the bio-assay of pituitary extracts, suggested potassium chloride as a standard. He based this upon his observation that the reaction of the isolated uterus of the guinea pig to potassium chloride is qualitatively of the same order as that to pituitary extracts. Spaeth's experiments led him to believe that there is also a very close quantitative relationship in the reaction of the isolated uterus of the guinea pig to certain concentrations of potassium chloride and of pituitary extracts. This did not find confirmation in the hands of Nelson (7). During the months of March and April, 1922, one of us (M. I. S.) carried out a series of observations on the use of potassium chloride as a standard, and it was found that out of a series of experiments upon the isolated uterus of the guinea pig with a given pituitary extract it was possible to get a number of experiments yielding perfectly concordant results, giving a definite ratio of activity between the extract and potassium chloride. Many experiments, however, were encountered showing variations from the established ratio all the way up to 500 per cent. It has not been possible to define clearly the conditions under which consistent results could be obtained with uniformity, and it was therefore concluded that there is no constant parallelism in the irritability of different uteri or of the same uterus under different conditions toward potassium chloride and pituitary extracts.

Perhaps the clearest criticism against the use of both histamine and potassium chloride as standards in the bio-assay of pituitary extracts was brought out recently by Burn and Dale (8) in a publication which appeared at a time when the work detailed in this paper had progressed so far as to enable us to draw some very definite conclusions.

We believe that any attempt at utilizing an artificial standard for the bio-assay of pituitary extracts would be doomed to failure unless it were known definitely that the mechanism of pharmacologic action of the standard and of pituitary extracts on the uterine muscle, the test object, were identical in every respect. This is admittedly impossible with our present state of knowledge; for even if we had definite knowledge of the action of the proposed standard, we should have no exact information as to the mode of action of pituitary extracts on the uterine muscle. Much less do we know what influences slight deviations in the composition of the medium bathing the isolated uterine segment might have upon its reactivity to pituitary extracts. In our judgment, preparations from the pituitary gland alone, and, since the chemistry of its active principles is virtually unknown, preparations that represent the entire infundibular lobe only, should be used as a standard for the physiological assay

of pituitary extracts by the isolated uterus of the guinea pig, which, as is generally recognized, is the most useful test object.

With this aim in view we have attempted to discover a preparation of the infundibular portion of the pituitary gland which would show a reasonable degree of uniformity in potency, which would keep a reasonable length of time without deterioration, and which could be prepared without an undue amount of chemical manipulation.

THE METHOD AND TECHNIQUE OF THE BIO-ASSAY OF PITUITARY EXTRACTS.

In carrying out the assay of pituitary extracts we have, in general, used the method described by Dale and Laidlaw (9) in 1912.

During the early part of this work we were frequently confronted with the difficulty of the rapidly increasing sensitiveness of the uterine preparation toward pituitary extracts, so that successive reactions to a given dose of the extract rapidly increased to a maximum. This made it impossible to get more than a very few, and often only approximate, comparisons, and at the end of an experiment there would still be some doubt as to the relative potency of the two extracts examined. We suspected that the oxygen which we were using contained some impurity which tended to augment the tone of the uterine muscle and to sensitize it to pituitary extracts. We then attempted to wash the oxygen in an effort to free it of its hypothetic impurity, and we discovered that by washing the oxygen through a solution of sodium bicarbonate we obtained incomparably better results. We then proceeded to determine, by means of indicators such as are used in hydrogen ion concentration work, whether there is a measurable amount of acidity in the oxygen. The results were negative; but it was observed that as the oxygen. bubbled through the solution of sodium bicarbonate a small amount of carbon dioxide was blown over with it, and it was concluded that it is the small amount of carbon dioxide admixed with the oxygen that tends to maintain the tone and irritability of the uterine muscle at a uniform level. Whether the effect is due to the direct action of carbon dioxide on the uterine muscle or to an indirect effect, owing to the maintenance of an optimum balance of ions in the bathing fluid, can not be answered at present.

We did not attempt to work out in detail the optimum concentration of carbon dioxide. This would obviously require a very large number of experiments. In our experience, 500 c. c. of a 2 per cent solution of sodium bicarbonate, renewed every 7 to 10 days, has given us very good results.

We have used throughout this work guinea pigs weighing not over 250 grams, nor under 180 grams, most generally 200 to 210 grams.

We are convinced, however, that the age of the animal is a far better guide than its weight. We have frequently encountered useless preparations obtained from small guinea pigs, not weighing much over 200 grams, especially if brought into the laboratory from outside breeders. We therefore use as a routine, guinea pigs of our own stock, which are weaned and segregated at the age of 10 to 14 days, yielding very useful preparations at the age of 3 to 5 weeks. It is seldom (if ever) that uteri obtained from such animals have to be discarded. Trendelenburg and Borgmann (10) have called attention to the great convenience of working with quiescent uteri obtained from young virgin guinea pigs, and we can confirm them. Such uteri require very little weighting, are easily adjusted, and show a considerable degree of sensitiveness to pituitary extracts.

The animal is killed by a blow on the head, and at once both horns of the uterus are removed and placed in Locke's solution. One entire horn, freed from the broad ligament, ovary, and fallopian tube, is suspended in the bath of Locke's solution at 38° C. We have never seen the necessity for retaining the ovary and fallopian tube, as some workers do, but have felt, on the contrary, that it might introduce an error or at least some difficulty in the interpretation of results, owing to gradual stretching of the fallopian tube during the course of an experiment. Since we know of no special advantage for their retention, we have uniformly discarded them.

After complete relaxation, which takes from 15 to 30 minutes, the preparation is so adjusted as to write a base line with but slight spontaneous movements. With small uteri of young animals, this is readily accomplished by the weight of the lever or a small additional weight if necessary. We have found the Harvard aluminium heart lever, with a magnification of approximately 4, to answer the purpose very well. This gives such a degree of magnification on the tracing as to make it easy to note the effect of small differences in dosage. The extracts to be tested are always prediluted with Locke's solution, so that the dose added to the bath of 100 c. c. is about 0.5 c. c., and never exceeds 1 c. c. We usually make the dilutions of such order as to require nearly equal volumes of standard and unknown to elicit equivalent reactions. Each dose is carefully measured with pipettes accurately graduated to 0.01 c. c. After an equivalent dose of the unknown is found for a given dose of the standard, the respective doses are then increased or decreased, or both, by 10 to 20 per cent. This not only confirms the earlier finding, but also gives assurance of the sensitivity of the uterine segment to small increases or decreases of dosage.