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 (Council 
 
 MEDICAL RESEARCH 
 COUNCIL 
 
 REPORTS ON BIOLOGICAL 
 STANDARDS 
 
 I. On the Physiological Standardization of 
 Extracts of the Posterior Lobe of the 
 Pituitary Body 
 
 BY 
 
 J. H. BURN, M.A., M.B. 
 
 AND 
 
 H. H. DALE, G.B.E., M.D., F.R.S. 
 
 
 LONDON 
 
 PUBLISHED BY HIS MAJESTY'S STATIONERY OFFICE 
 
 1922
 
 MEDICAL RESEARCH COUNCIL 
 
 The VISCOUNT GOSCHEN, C.B.E. (Cliairmari). 
 
 WILLIAM GRAHAM, LL.B. 
 
 The Rt. Hon. F. B. MILDMAY, (Treasurer). 
 
 SIR FREDERICK W. ANDREWES, O.B.E., D.M., F.R.S. 
 
 Professor T. R. ELLIOTT, C.B.E., D.S.O., M.D., F.R.S. 
 
 HENRY HEAD, M.D., F.R.S. 
 
 Professor F. GOWLAND HOPKINS, D.Sc., F.R.S. 
 
 Major-General SIR WILLIAM B. LEISHMAN, K.C.M.G., C.B., F.R.S. 
 
 Professor D. NOEL PATON, M.D., F.R.S. 
 
 SIR CUTHBERT S. WALLACE, K.C.M.G., C.B., F.R.C.S. 
 
 SIR WALTER M. FLETCHER, K.B.E., M.D., Sc.D., F.R.S. 
 
 (Secretary). 
 
 15 YORK BUILDINGS, 
 
 ADELPHI, W.C. 2.
 
 8imedical 
 Library 
 
 WK 
 
 INTRODUCTION 
 
 
 IT has been part of the consistent policy of the Council to promote 
 researches which have as their object the attainment of well-based 
 methods of estimating the quantitative and qualitative values of 
 biological substances used either in medical research work itself or in 
 the preventive and curative work of the medical profession. The 
 investigations of this kind which they have supported from public 
 funds are chiefly centred at their National Institute for Medical 
 Research, at Hampstead, which in that respect is designed to fulfil 
 in the biological sphere of medicine functions analogous to those of 
 the National Physical Laboratory. So far as means and men are 
 available, there are being actively prosecuted here systematic studies 
 of biological methods of measurement and assay necessary for the 
 determination of ' standards ' of value, in the absence of which the 
 scientific progress of medicine has been, and still is being, greatly 
 hampered. It is the design of the Council to publish the results of 
 work of this kind as they become available, and they hope that the 
 Eeport now presented may be only the first of an extended series. 
 
 The importance to the public and to the medical profession of the 
 subject treated in the present Report is very great, and no words 
 need be added to enforce the grave observations made here by 
 Dr. Dale as the outcome of his work with Dr. Bum. Extracts of 
 the pituitary gland are used widely and increasingly in medical and 
 surgical practice throughout the country. They provide, in particular, 
 a remedy which, in the various difficulties or accidents of childbirth, 
 is at once highly valuable and, like many other valuable remedies, 
 often dangerous. Rightly used, it is a weapon in the hands of the 
 doctor by which he may avoid difficulties or save life ; wrongly or 
 inaccurately used, the dangers it brings to mother and child may be 
 extreme. Both the benefits and the dangers which this drug can bring 
 must obviously depend chiefly upon the factor of dose, and the experi- 
 ments described in this Report show that the commercial preparations 
 used by medical practitioners, and naturally, or at least habitually, 
 regarded by them as equivalent to one another, may actually so vary 
 that the most potent may have 80 times the strength of the least 
 potent. A doctor accustomed to one preparation may, when forced 
 to use its apparent equivalent in an emergency, be under danger 
 therefore of giving, 80 times the intended dose, and the most serious 
 or even fatal consequences may follow. 
 
 It is clear that the manufacturers are by no means chiefly to 
 blame for the position which is displayed so clearly here. It is 
 pointed out that no official guidance has been given to them in this 
 country as to the strength of extract most suitable for therapeutic 
 use, and no standard of activity has been available for common 
 reference, though all the extracts examined at the National Institute 
 were described as being physiologically standardized. No dissent is 
 possible from Dr. Dale's view that the present position is ' deplorable '. 
 
 (2126) Ps. 23792. Wt. 11958. 363/626. 1500. 11/22. O.U.P. A 2
 
 It is a position, however, which fortunately is quite unnecessary 
 and easily to be avoided. The United States Pharmacopoeia has 
 officially laid down a theoretical strength for this extract. This Eeport 
 recommends the adoption of the same theoretical strength for this 
 country, and indicates a method by which a preparation of uniform 
 activity can be made and used as a standard of reference. It may be 
 noted that the British Pharmacopoeia has not yet recognized the 
 existence of this potent and common remedy. 
 
 It is earnestly hoped that early action may be taken as the result of 
 representations made in detail by the Medical Kesearch Committee in 
 1916 to Mr. Asquith's Eeconstruction Committee, and later in 1919 
 to the President of the Local Government Board (subsequently the 
 Minister of Health), upon the urgent need for official standards of 
 value and authenticity for drugs of this kind and for the numerous 
 biological preparations, sera, and the like, used in medical practice. 
 The Committee on those occasions expressed their conviction that 
 the absence of the possibilities of control which would be given by 
 the determination and imposition of standards was ' discreditable 
 to our national position in the world of science and a source of grave 
 danger to the community '. The Departmental Committee appointed 
 to advise the Minister of Health upon this subject in April, 1920, has 
 already reported strongly in favour of immediate measures, and 
 has formulated a detailed scheme. (Command paper 1156, 1921, 
 Stationery Office, price 2d.) 
 
 In the meantime, a Committee appointed jointly by the Obstetrical 
 Section of the Royal Society of Medicine and the Medical Research 
 Council is conducting a thorough investigation into the doses of 
 pituitary extract appropriate for various medical and surgical uses, 
 and more particularly for administration to women in labour. The 
 Council agree with Dr. Dale that ' it will be truly lamentable if the 
 results of this important inquiry, when in due course they are 
 published, should be deprived of all accurate meaning by the failure 
 of the manufacturers to bring all the makes of pituitary extract to 
 such a uniform standard of activity as, without serious trouble, 
 could be ensured '. 
 
 MEDICAL RESEARCH COUNCIL, 
 15 YORK BUILDINGS, 
 ADELPHI, 
 LONDON, W.C. 2. October 20, 1922.
 
 ON THE PHYSIOLOGICAL STANDARDIZATION 
 OF EXTRACTS OF THE POSTERIOR LOBE 
 OF THE PITUITARY BODY 
 
 BY 
 
 J. H. BUEN, M.A., M.B. 
 
 AND 
 
 H. H. DALE, C.B.E., M.D., F.R.S., 
 
 (From the National Institute for Medical Research, Hampstead) 
 
 CONTENTS 
 
 PAGE 
 
 I. INTRODUCTION .......... 6 
 
 II. THE CHOICE OF A PHYSIOLOGICAL METHOD ..... 7 
 
 III. DETAILS OF THE METHOD ........ 10 
 
 (1) The Apparatus .10 
 
 (2) The Locke-Ringer Solution . . . .14 
 
 (3) The Choice of a Guinea-Pig ....... 15 
 
 (4) The Preparation of the Uterus ...... 16 
 
 (5) Method of Carrying out the Test ...... 17 
 
 IV. SUGGESTED ARTIFICIAL STANDARDS . . . . . . .17 
 
 (1) Experiments with Histamine . . . . . .19 
 
 (2) Experiments with Potassium Chloride ..... 24 
 
 V. THE USE OF AN EXTRACT MADE FROM PERFECTLY FRESH PITUITARY 
 
 GLAND AS A STANDARD . . . . ... .29 
 
 (1) Method of Making the Extract 29 
 
 (2) Method of Performing the Test ... 33 
 
 VI. THE REGULAR ACTIVITY OF PERFECTLY FRESH GLASTDS . . . 35 
 
 VII. THE ACTIVITY OF COMMERCIAL EXTRACTS ...... 37 
 
 (1) Effect of Drying on Activity ....... 38 
 
 (2) Examination of Commercial Samples ..... 39 
 
 (3) The Proportion of the Original Activity present in the Finished 
 
 Products ......... 
 
 (4) The Relative Activities of the different Commercial Extracts 
 
 (5) The Comparative Pressor Activities of the above Samples 
 
 VIII. CONCLUDING REMARKS ........ 
 
 IX. REFERENCES . .......
 
 I. INTRODUCTION. 
 
 MUCH has been written in the past two decades concerning the 
 necessity for employing a physiological method for measuring, and 
 adjusting to a common standard, the activity of potent remedies, 
 when the active principles are unknown, or of such a nature that 
 they cannot be estimated by chemical methods. In no case is the 
 necessity less open to discussion than in that of the now widely used 
 watery extract of the posterior lobe of the pituitary body. The 
 active principles are unknown and what little is known as to their 
 nature indicates that they are very unstable, so that they are easily 
 destroyed in the process of preparing the extract. The extract 
 is essentially a remedy for emergencies. There are physicians 
 who discount the value of physiological standards for the activity 
 of digitalis, for example, on the ground that the real test is made 
 in each case, by determining the reaction of the individual patient 
 to the tincture which happens to be in use. The obstetrical physician 
 faced with a post-partuin haemorrhage, or the surgeon called to deal 
 with a post-operative paralysis of the bowel, cannot take time to 
 discover by experiment what dose, if any, of a particular sample 
 of the pituitary extract will produce an effect sufficient to save the 
 patient's life. Abundant proof has already been produced to show 
 that the commercial preparations of the extract exhibit an enormous 
 range of variation in activity ; and we shall make, from our own 
 experience, an addition to the evidence on this point. There is 
 a pressing need for the determination of the proper dosage of the 
 extract on a scientific basis. In the first instance it was indicated 
 in a crudely empirical manner, and a dose which was found effective 
 and safe post partum soon came to be used without discrimination, 
 for accentuating or accelerating the pains, for example, in the 
 earlier stages of labour. The growing record of accidents from 
 ruptured uteri and retained placentae, has evoked a response from 
 some manufacturers, who have put up a half-strength extract 
 (usually corresponding to a 5 per cent, extract of fresh gland) and 
 labelled it ' obstetrical '. Apparently the medical practitioner, when 
 using a remedj^ put up in sterile solution in sealed ampoules, cannot 
 be prevented from regarding the size of the ampoule as an indication 
 of the dose. Nothing effective, however, has been done, or even 
 attempted, towards a settlement of the question as to what dose is 
 safe and effective for each particular condition. Nor is investigation 
 of this important matter possible, under conditions in which extracts 
 are put out under various trade names, with ostensible values 
 varying from 1 part of fresh substance in 5, to 1 part in 20 of water, 
 while the actual strength even of theoretically equivalent extracts 
 varies, as we shall show, much more widely. One observer mav 
 recommend ' 1 c.cm. of pituitary extract '* as the safe dose for 
 a certain purpose, on the strength of experience with a ' 5 per cent.' 
 extract of a certain make ; another worker, accepting this advice, 
 may give, in similar circumstances, 1 c.cm. of a ' 20 per cent.' extract 
 of another make a dose which in theory is 4 times, and in fact 
 is quite possibly 10, 50, or even some hundreds of times as great. 
 It is clear that no scale of dosage can be determined until some
 
 reasonable uniformity is obtained among the preparations used. 
 This can only be effected, at present, by physiological comparison 
 with a standard, and there is no certain prospect of a chemical 
 method being ever available. 
 
 II. THE CHOICE OP A PHYSIOLOGICAL METHOD. 
 
 If we could be assured that all the physiological actions of the 
 extract, or that all those which are of therapeutic importance, were 
 due to a single active principle, any method of determining the 
 relative concentration of that principle, present in a sample of the 
 extract, would be admissible. Provided that the chosen physio- 
 logical reaction measured this with accuracy, it need have no more 
 direct relation to the therapeutic effect desired than a chemical test, 
 such as a colour-reaction, would have. The action of the extract 
 on the melanophores of fish-scales, for example, might provide 
 a suitable basis of measurement, as suggested by Spaeth (1918 a), 
 if we knew that only one active principle was concerned in the 
 therapeutic effects, and if this could be shown to be responsible 
 for the whole action on the melanophores. 
 
 The position is, in fact, much less simple. The evidence available 
 points clearly to the participation of at least two active principles, 
 in producing even the two physiological effects of the extract 
 which are most familiar and of the greatest therapeutic importance 
 the action on the blood pressure and the action on the uterus. We 
 do not know whether the principle acting on arterial plain muscle 
 is wholly without action on that of the uterus, or the uterine stimulant 
 wholly free from pressor action. All that we can properly demand 
 of any method is that it shall measure one or other of these effects 
 with reasonable accuracy. In the case of an action such as that on 
 melanophores, we have no proper ground for assuming that it has any 
 relation either to the action on the uterus or to that on the arteries ; 
 it may well be due to a constituent which has no special action on 
 either. Moreover, it is by no means easy to obtain satisfactory 
 evidence of the identity of the constituents responsible for two 
 different effects. In the case of the actions on the blood pressure 
 and the uterus, we are certainly dealing with principles which are 
 very similar as regards the conditions of their solubility and stability. 
 Conditions such as staleness of the material, or slow heating in the 
 making of the extract, which lead to deficiency in one kind of 
 activity, lead to a deficiency in both. The impression is easily 
 formed that the two types of activity always vary in a parallel 
 manner. In an earlier communication Dale and Laidlaw (1912) 
 stated that the correspondence was so invariable, in an experience 
 which had already covered a very large number of samples, that it 
 was safe to assume, for practical purposes, that only one active 
 principle was concerned, and to use the measurement of one type of 
 activity as an index of both. Even now it can be stated that depar- 
 tures from this rule are rare ; but it can also be stated that they do, 
 beyond question occur. Two extracts have been examined by one 
 of us (H.H.D.) which were indistinguishable in their pressor action, 
 though one of them was about four times as active as the other on
 
 8 
 
 the uterus ; .and Dudley's recent work (1919, 1922) has demonstrated 
 the possibility of making at least a partial separation of the two types 
 of activity. A very large body of evidence, showing invariable 
 correspondence over a very wide range of samples, would, therefore, 
 be needed before any action, other than those directly concerned 
 in the practical uses of the extract, could safely be adopted as an 
 index of therapeutic potency. 
 
 This being so, the choice lies between the action on the bloocl 
 pressure and that on the uterus as a measure of therapeutic activity. 
 Both measurements, indeed, ought to be made for absolute assurance 
 of satisfactory activity in both directions. For practical purposes, 
 however, it will probably be sufficient to adopt one or the other test, 
 with an occasional control to ensure that no part of the process of 
 making or sterilizing the extract is destroying one principle at 
 a disproportionate rate. 
 
 Of the two kinds of activity available for measurement, that on 
 the blood pressure was probably the first to be applied, though no early 
 description is available giving details of its application. Eoth (1914) 
 compared several extracts by this method and by the uterus method, 
 and concluded that the latter lent itself to greater accuracy. 
 Hamilton (1916) gave an account of the test, as used by him on 
 dogs anaesthetized with chloretone, in which he claimed to be able 
 to detect a difference of 10 per cent, in activity. Our own experience 
 with the blood-pressure test has been confined to experiments on the 
 spinal cat, in continuation of those described by Dale and Laidlaw 
 (1912). We have not observed such a delicacy of discrimination 
 as that mentioned by Hamilton, but we have no reason to doubt its 
 possibility in an occasional, particularly favourable, preparation. 
 Apart, however, from the question of its accuracy, which in our 
 experience is usually much lower than that of the test on the uterus, 
 there is an objection to the blood-pressure test, on whatever species 
 it is made, and under any form of anaesthetic. Howell (1898) first 
 described the greatly reduced sensitiveness of the blood pressure 
 to a second injection, given within an hour of the first. With small 
 doses the phenomenon is not so pronounced, but, however small 
 the dose chosen, the effects following the first few injections show 
 a rapid decline, until a condition is produced in which further equal, 
 small doses, 'given at regular intervals, cause practically equal 
 results. At this stage the comparison of a preparation with the 
 standard is made. Given a sufficient accuracy of comparison no 
 objection could be raised to this procedure, if we had certain know- 
 ledge that the pressor action was entirely due to one principle. We 
 have, in fact, no such knowledge. It is not inconceivable, or even 
 improbable, that more than one substance may contribute to the 
 total pressor effect. Dudley's recent work (1922) affords strong 
 evidence in favour of this supposition, and suggests that tolerance 
 is rapidly established for the action of the most potent of the pressor 
 principles, leaving a remnant of more constant response to a less 
 active principle, to which the tolerance acquired is relatively trivial. 
 If this is the case, the blood-pressure method, in the only form in 
 which it can afford anything like a quantitative comparison, must 
 give an estimate, not of the total pressor activity, but of the part
 
 of it due to a subsidiary active principle. The objection is of 
 sufficient importance to determine choice in favour of another 
 method, if one can be found which is equally sensitive in discrimina- 
 tion and free from suspicion of any kind. 
 
 The test on the isolated uterus of the guinea-pig is certainly free 
 from this objection. Unless the successive doses are given with 
 altogether unreasonable rapidity no tolerance is produced ; on the 
 contrary, the tendency is for the plain muscle to become gradually 
 more sensitive in reaction as the experiment proceeds. A further 
 and extensive experience of the method has confirmed the conclusion, 
 drawn by Dale and Laidlaw in connexion with their first description 
 of it, that it will detect differences of activity within much narrower 
 limits than those which the blood-pressure method usually reveals. 
 It has the advantage that the particular form of activity which it 
 determines is the one which has given to the extract its widest use 
 in therapeutics. The manipulations involved are of the simplest 
 kind, and, since the use of the living animal is not involved, the test 
 can be carried out in any laboratory possessing the necessary equip- 
 ment, and by anybody who is endowed with the necessary patience. 
 The method is apt to be tedious, and the fact that it calls for no 
 special skill in manipulation makes it more so. The experimenter, 
 during all but a small fraction of the period of some hours needed to 
 complete an accurate comparison, is reduced to the role of a mere 
 spectator ; yet, if he is to get the best results which the method can 
 jdeld, he must not allow his attention to be seriously diverted or his 
 procedure to be hurried. Eesults cannot indeed be obtained more 
 rapidly by the blood-pressure method, if they are to have any 
 quantitative significance ; but the more complicated and varied 
 technique keeps the attention of the experimenter occupied, and 
 makes the experiment less tedious. 
 
 The test on the uterus, since its first description, has been widely 
 used, and apparently with varying success. Some workers, such as 
 Hamilton, have dismissed it as giving merely a qualitative indication, 
 as contrasted with the quantitative results obtained with the blood- 
 pressure method. This is not the experience of the majority, but 
 many have met with difficulties, and much discussion has arisen on 
 details. Much of the more .recent discussion has centred round 
 attempts, mistaken in our opinion, to use stable chemicals to provide 
 an arbitrary standard of activity. 
 
 More than a year of continuous work with the isolated uterus has 
 had the effect of strengthening our conviction that, when the method 
 is properly used, it will give results far more accurate than those 
 obtainable by other methods. Attention to certain details, which, 
 for the most part, are not those to which other workers have attached 
 importance, has rendered difficulty and failure increasingly rare. 
 Fenger (1916) states that ' often three or four guinea-pigs have to 
 be killed before a satisfactory strip is obtained '. In our last 200 
 experiments we have only on some 10 occasions had to reject pre- 
 parations as unsuitable. 
 
 Our conclusion, therefore, is the converse of that reached by 
 Hamilton. We regard the test on the guinea-pig's uterus as by far 
 the most suitable for quantitative comparisons with a standard ; 
 
 2126 A 2
 
 10 
 
 but, for reasons already given, we believe that it should occasionally 
 be supplemented by a test on the blood pressure, which, though less 
 accurately quantitative, suffices for assurance that the pressor 
 activity is not disproportionately defective. The choice of method 
 being made in favour of the test on the isolated uterus of the guinea- 
 pig, we have now to define the conditions of its performance, and the 
 standard with which comparison is to be made. 
 
 FIG. A. Sectional diagram of whole apparatus X 1/12. 
 
 III. DETAILS OF THE METHOD. 
 (1) The Apparatus (Figures A and B) 
 
 The apparatus which we have used is essentially the same as that 
 described by Dale and Laidlaw (1912), but improvements have been 
 made in detail, which have given such increased convenience as to 
 make a full redescription desirable. The main improvements are in 
 the method of fixing the experimental glass chamber in the copper 
 thermostat bath, in the method of regulating the temperature, and 
 in the recording lever. 
 
 The thermostat bath (A) which we have in use is of stout copper,
 
 12 
 
 and is cylindrical in shape, being approximately 18 cm. in both 
 diameter" and depth. The bath is flat-bottomed and stands on the 
 table which also supports the recording drum. A hole in the table 
 allows the passage of the glass tube leading to and from the testing 
 vessel (B). In the present form of the apparatus we use a cylindrical 
 glass vessel of about 3-5 cm. internal diameter, i.e. with a sectional 
 area of about 9-5 sq. cm. When the vessel is filled to a depth of 
 10-11 cm. its content is, therefore, approximately 100 c.cm. At its 
 lower end it is narrowed shacply to join the connecting tube, which 
 has a diameter of about 1 cm. This passes through the bottom of 
 the thermostat bath, being held tightly by a packed gland (G) 
 a method of fixing the testing vessel which we have found much 
 more convenient than the rubber cork indicated in Dale and Laidlaw's 
 figure. Immediately after passing the gland the tube is connected 
 to a wide-bore glass tap (c), and this again to a glass T-piece (D), 
 enabling the testing vessel to be emptied and refilled from the 
 reservoir of warm saline, by opening the tap and spring clip E or P 
 shown in the diagram. The extra T-piece (H), enabling the system, 
 as far as F, to be filled with warm saline prior to a change of solution,, 
 is exactly as in Dale and Laidlaw's apparatus. 
 
 The method of controlling the temperature of the thermostat is 
 one which we have found most convenient and effective. The 
 copper rod and Bunsen burner of the earlier apparatus have been 
 abandoned. The bath has now a wide copper tube (j), closed at the 
 inner end, soldered at its mouth to the wall of the bath, and pro- 
 jecting into the interior of the bath near the bottom, sufficiently far 
 from the centre to avoid contact with the testing vessel. A small 
 blind tunnel is thus formed, passing nearly across the bath. This 
 accommodates a cylindrical, carbon-filament electric glow-lamp (K). 
 The lamp socket is attached to a cylindrical hollow brass holder (L), 
 through which the insulated flex passes to the lamp socket. This 
 brass holder passes through two simple friction-guides (M) which 
 are fixed to the table, and which fix the height and direction of the 
 lamp so that it can be drawn by a touch in and out of the tunnel 
 in the side of the tank, the temperature of the water in which is 
 controlled by the extent to which the lamp is inserted. With the 
 laboratory at its ordinary temperature and the bath filled in the 
 first instance with water at 37 C., the lamp will keep the temperature 
 remarkably steady for hours, when inserted so that about half its 
 length lies within the tunnel. A slight withdrawal if the temperature 
 tends to rise, a slightly deeper insertion if it tends to fall, is all that 
 is necessary ; careful watching of the thermometer, and an occasional 
 small adjustment suffice to keep the temperature within 0-5 e C., and 
 it would be easy to keep it within 0-1, if such accuracy had any 
 advantage. The control is so easy that we have not thought it 
 worth while to devise a mechanism for making it automatic, though 
 this should not be difficult if a thermostat on this principle were to 
 be used for observations needing a more accurate regulation of 
 temperature. The principle is not at all new ; it was used by 
 Professor A. N. Eichards when working with one of us in 1917, 
 and is doubtless familiar to many workers ; but this particular 
 method of applying it has proved so convenient for this special
 
 13 
 
 purpose that it seems worth while to describe it in detail for the 
 benefit of others. 
 
 The testing vessel bears graduation marks corresponding to 
 contents of 100 and 125 c.cm., measured from the closed tap c. 
 Before each experiment the thermostat bath is filled with water 
 at 37 C. up to the mark on the testing vessel corresponding to the 
 volume of 125 c.cm. measured from the tap. At each change of the 
 fluid the level of the water gives a useful guide to approximate 
 adjustment, and final adjustment is made to the 125 c.cm. mark. 
 
 The lever (N) which we have used recently differs from that given 
 in the earlier description. The fulcrum pin, w r ith conical end- 
 bearings, passes through the centre of a small circular slab of ebonite 
 (o). Bather less than the upper half of the circle is cut away, leaving 
 a flat surface to which the lever is attached. The lever is made of 
 a slip of thin magnalium, folded so as to have an L-section, w r hich 
 gives it rigidity in both horizontal and vertical planes. The horizontal 
 portion is attached near its middle to the flat surface of the ebonite 
 sector and is pierced with a series of holes. The vertical portion is 
 cut by a series of corresponding grooves. The holes and grooves 
 enable the hook terminating the thread from the uterus to be 
 attached, or small weights to be hung, at any desired distance from 
 the fulcrum. One end of the lever projects over the mouth of the 
 testing vessel, and the other carries a delicate frontal writing-point 
 (p) of the type described by our colleague Lovatt Evans (1919). 
 The frontal writing eliminates the difficulty of using an ordinary 
 cylindrical recording drum for the long curves drawn by the con- 
 tracting uterus, and the absence of circular distortion enables the 
 comparison of the heights of successive curves to be made with much 
 greater accuracy. With the adoption of the frontal lever the ink- 
 writing point, formerly used, has been abandoned in favour of the 
 glass point writing on a delicately smoked surface. As in the earlier 
 pattern a small brake is used for fixing the lever while the fluid is 
 changed. This consists, as before, of a small Bowden cable (Q), 
 made for the shutter-release of a photographic camera, on pressing 
 which a small steel rod impinges radially on the under, circular 
 surface of the ebonite segment to which the lever is attached, and 
 so fixes the lever. 
 
 The lever which we have actually in use has a total length of 
 19 cm. The distances from the fulcrum to the hinge of the frontal 
 writing point and to the point of attachment of the thread from 
 the uterus are, respectively, 10-5 and 8-5 cm. ; so that the records 
 of contraction are made with a magnification of only about 5 : 4. 
 The whole horn of the uterus, w r hich we always use, gives contractions 
 of such length as to require no magnification ; indeed, any greater 
 magnification would bring the summits of the curves beyond the 
 range of reasonable accuracy of the frontal lever. We adjust the 
 weighting according to the size or strength of the horn being used, 
 a small weight being hung in notches of the lever at different distances 
 from the fulcrum. It is impossible to give a general indication of the 
 effective load required. It is necessary to load each preparation to 
 such an extent that, when unstimulated, it relaxes to the full extent 
 of its length, but not so heavily as to obliterate, or seriously impede
 
 14 
 
 the small, automatic rhythm which the relaxed uterus normally 
 shows. The average effective load (i. e. the appropriate load for 
 application at the same distance from the fulcrum as the attachment 
 of the thread from the uterus) is about 0-5 gm. ; but as little as 
 0-2 gm. will in some cases suffice, and more than 0-5 gm. may be 
 needed. 
 
 (2) The Locke-Ringer Solution. 
 
 For some years past we have used a saline solution of the same 
 composition for this purpose. Its formula was gradually established 
 by deliberate trial, assisted by accident, as the most suitable. It 
 differs on two small points from the Locke solution in most general 
 use for isolated mammalian hearts, &c. (1) The proportion of 
 sodium bicarbonate is 0-05 per cent., in place of the more usual 
 0-015 per cent. This proportion of the bicarbonate was indicated 
 by Dale and Laidlaw in their description of the method in 1913, 
 but special attention was not drawn to it. We know of one case 
 in which other workers have fallen into difficulty, with the method 
 through use of a Einger solution containing only 0-015 per cent, of 
 the bicarbonate. (2) The other difference from an ordinary Ringer's 
 solution which we have found advantageous 4 , and which was adopted 
 after the publication of Dale and Laidlaw's paper, is the addition 
 of a minute proportion of magnesium chloride. Magnesium chloride 
 as a constituent of a Ringer solution was first used by Tyrode, in the 
 now familiar fluid, in which segments of rabbits' intestine preserve 
 such a regular rhythm and remain so long excitable. We tried 
 Tyrode's solution for the uterus many years ago, and found that it 
 depressed the excitability to the pituitary extract to a degree which 
 was inconvenient. W T e found, however, that the introduction of 
 a smaller proportion of magnesium into the solution we had pre- 
 viously used had a favourable effect in weakening the tendency 
 to irregular spontaneous rhythm and preserving the excitability 
 of the plain muscle to pituitary extract. Kochmann (1921) has 
 recently recommended, as facilitating the testing of pituitary 
 extracts on the uterus, reduction of the calcium chloride till the 
 spontaneous rhythm is completely suppressed. Dale and Laidlaw 
 pointed out in 1912 that reduction of the calcium to one-half the 
 normal strength a much smaller reduction than that advocated 
 by Kochmann produced a condition in which the responses to 
 successive equal doses of the pituitary extract showed a rapid 
 decline. We regard any method which involves the suppression of 
 automatic rhythm by reduction of calcium as quite unsuitable for 
 our purpose. 
 
 The formula of the Ringer's solution which we have now used for 
 some j-ears is as follows : 
 
 NaCl 0-9 gm. / 
 
 KC1 0-042 
 
 CaCl 2 0-024 
 
 NaHC0 3 .... 0-05 
 
 MgCl 2 0-0005 
 
 Dextrose .... 05 
 
 Water . 100 c.crn.
 
 15 
 
 It is possible that others may find slight changes in the formula 
 advantageous. The only point on which we wish to insist is the 
 desirability of stating the composition of the Ringer's solution used, 
 when comparative results are presented. This is particularly 
 important when an artificial standard of comparison is used. Changes 
 in the composition of the Ringer's solution modify the sensitiveness 
 of the plain muscle to the pituitary extract ; they also modify its 
 sensitiveness to substances like histamine and potassium chloride, 
 but not necessarily to the same extent, or even in the same direction. 
 We shall return to this point in considering the choice of a standard. 
 
 (8) The Choice of a Guinea-Pig. 
 
 There has been some controversy as to the most suitable size of 
 the guinea-pig furnishing the uterus for this test. Dale and Laidlaw 
 recommended that young virgins of not more than 350 gm. weight 
 should be used. Roth (1914) stated that they should preferably 
 weigh from 275 to 350 gm., while other workers have obtained 
 better results with younger animals weighing from 175 to 250 gm. 
 When the strength of pituitary extract is indicated in terms of 
 an artificial standard such as histamine, it may be of impor- 
 tance to prescribe the age of the guinea-pigs to be used by 
 indication of a limited range of body weight. It is quite possible 
 that the uteri from guinea-pigs of 200 gm. may show a different 
 average ratio of sensitiveness to pituitary extract and histamine 
 from that exhibited by uteri from older guinea-pigs of, say, 350 gm. 
 For the method as we use it, however, any guinea-pig's uterus is 
 suitable, provided only that it shows a delicacy of grading in its 
 responses -to different doses of the same pituitary preparation, 
 constancy in the height of contraction by which it responds to the 
 same sub-maximal dose, and freedom from hampering automatic 
 activity. We most commonly obtain such uteri from guinea-pigs 
 weighing between 200 and 300 gm. In smaller guinea-pigs the 
 uterus is usually too delicate and inert ; in larger ones it is apt to be 
 too sensitive, and prone to automatic contractions ; but we have 
 often obtained useful preparations from animals outside these limits 
 of weight. There is a matter more important than age or weight, 
 to which we have not seen definite reference in the writings of other 
 workers. It is agreed that the uterus from the parous guinea-pig 
 is too excitable and irregular in activity, and virgins are always 
 chosen, but it does no* seem to be generally recognized that the 
 uterus of the virgin on ' heat ' is unsuitable. The condition is 
 easily recognized in life by the swollen and moist condition of the 
 vulva, and, when such an animal is killed, the state of the uterus is 
 also easily detected. Whereas, in the condition of sexual rest, the 
 uterus is found to be slender, pale and almost fully extended, the 
 oestral organ is short, thick and vascular, being so contracted that 
 the fallopian tube is extended and almost straight. The horn of 
 such a uterus will often relax rapidly in Ringer's solution, but it is 
 excitable, and prone to acquire a large, irregular, automatic rhythm. 
 At certain times of the year the majority of young females taken 
 from a mixed pen are found to be in this condition, which renders
 
 16 
 
 the uterus unsuitable for the accurate conduct of the test. This is, 
 no doubt, the cause of the difficulty which some workers have 
 experienced in finding a suitable preparation ; it has at times, in 
 the past, almost driven us to the conclusion that the test could be 
 properly carried out only at certain times of the year. The difficulty 
 was one which obviously had to be eliminated, if the test was to 
 have general utility ; and by a simple expedient we have eliminated 
 it almost completely from our practice. If the young females are 
 separated at an early age, and kept in pens or cages quite separate 
 from the males, it is", in our experience, very unusual to find one in 
 which the uterus is unsuitable, on account of oestral activity, until 
 they have attained a weight well over 300 gm. 
 
 Our recommendations as regards method of choosing guinea-pigs 
 are, therefore, as follows : the young females should be segregated, as 
 soon as they are ready to leave the mother ; they should be used, 
 preferably, when between 200 and 300 gm. in weight ; and the 
 absence of oestral activity should be verified by external inspection, 
 before the animal is killed. 
 
 (4) The Preparation of the Uterus. 
 
 We have never found reason for departing from Dale and Laidlaw's 
 original recommendation to use the whole horn of a uterus, suspended 
 between a cervical tag at one end and the ovary at the other. 
 Workers in the Washington Laboratory have been inclined to insist 
 on the use of a portion of the horn ' measuring from 1 to 2 centi- 
 metres in length ' for the official test, and to attribute the varying 
 results obtained by other workers, partly to the choice of greater 
 portions of the uterus, with or without ovaries. There may be 
 some unexplained need for this rigidity of prescription if the strength 
 of the pituitary extract is to be laid down in terms of equivalence 
 to an artificial standard ; though we must admit some personal 
 doubt as to the exact significance of ' 1 to 2 centimetres ', seeing 
 that the same horn may show a physiological variation in total 
 length from 1 to 5 centimetres or more. A prescription of the middle, 
 upper, or lower half, third or quarter of the horn, whichever is 
 intended, would, we think, be clearer. But, whatever be the exact 
 intention, we can see no objection to the use of any special part of 
 the organ by a worker w r ho finds that he can obtain with it clearer 
 and more regular results than with the whole. For our own part, 
 having tried the preparation with the ovary and fallopian tube 
 removed, we are definitely of opinion that we obtain a finer delicacy 
 of discrimination, between nearly equal doses, when it is retained. 
 With the range of dosage in which we find that the finest differences 
 are detected, i.e. with doses about 60 to 80 per cent, of that pro- 
 ducing a maximal effect, the curve is broken near its summit by 
 a series of small contractions and relaxations, when the ovary forms 
 one of the points of suspension. We find that similarity in the 
 depth of this indentation of the summit is an additional point of 
 some value in enabling us to judge of the exact equivalence of two 
 doses. With a smaller dose, not only is the absolute maximum 
 lower, but the indentation is deeper ; with a larger dose, the indenta- 
 tion becomes nearly extinguished.
 
 17 
 
 The retention of the ovary seems to us to have only one possible 
 disadvantage. If the preparation needs more than the average 
 weighting to secure proper relaxation and a duly small rhythm, 
 there is a tendency for the upper delicate portion to undergo gradual 
 stretching, with a consequent lowering of the level of full relaxation, 
 as the experiment proceeds. A continuously falling base line makes 
 accurate comparison more difficult. In such cases we have found 
 it useful, when once full relaxation has been obtained, to introduce 
 a small stop which limits the fall of the lever to this point. Each 
 curve then starts from exactly the same level, and we have obtained 
 some very accurate and concordant determinations under such 
 conditions. 
 
 (5) Method of Carrying out the Test. 
 
 It will be evident, from the description of our apparatus given 
 above, that we have adhered to the method of making the final 
 dilution of the extract in the actual testing vessel. The material 
 for test is prediluted, usually from 100 to 200 times, with Ringer's 
 solution, so that the dose to be added to the bath shall not be too 
 small for reasonable accuracy of measurement, or so large that its 
 addition, unwarmed, will sensibly affect the temperature of the 
 solution in the testing vessel. We aim at working with a dose of 
 0-3-0-6 c.cm. We are aware that a more perfect accuracy of dilution 
 can be attained by the method adopted by Roth (1914) and others, 
 of making the required dilutions in bulk, and placing them in 
 aspirators in the same large warming-tank as the testing vessel, so 
 that, in changing from pure Ringer's solution to the dilution of 
 extract, no measurement of volume is involved. This method, 
 however, is obviously much more cumbrous. Whenever a small 
 change of dosage is required, a new dilution must be made on a large 
 scale, and time given for it to come to the required temperature. 
 If we correctly interpret the description, it would appear, moreover, 
 that the bath must be emptied of Ringer's solution and refilled with 
 the dilution whenever a dose is given. If this is the case, we should 
 expect the stimulus of exposure to form an undesirable complication 
 to the effect of the dilution of extract. In any case, we are convinced 
 that the method of making the dilution by adding a small measured 
 volume directly to the testing vessel, though less ideally accurate, 
 involves no such error as the method itself is capable of detecting. 
 Successive equal doses of the same preparation produce indis- 
 tinguishable results ; indeed, if they do not, we know that we are 
 dealing with a uterus which is unsuitable for the test, and discard it 
 a necessity which is fortunately rare in our experience. Our method, 
 on the other hand, gives a freedom and elasticity in the choice and 
 change of dose, which we regard as essential to the test as we carry 
 it out. Further details of procedure can more suitably be given 
 when we have discussed the choice of a standard. 
 
 IV. SUGGESTED ARTIFICIAL STANDARDS. 
 
 The determination of the strength of the extract is necessarily 
 comparative. A rough idea of the activity could be obtained, no 
 doubt, by finding the weakest dilution which would suffice to produce 
 
 2126 A 3
 
 18 
 
 contraction to the maximum. It is admitted, however, by all who 
 have used the method, that different preparations of the uterus 
 exhibit wide differences in sensitiveness, so that the only satisfactory 
 basis, for accurate measurement of activity, is a determination of the 
 dilution which produces an effect identical with that produced by 
 a standard preparation. 
 
 There would obviously be great advantages in the use of a stable 
 standard of known chemical constitution, which could be prepared 
 perfectly pure, and weighed out exactly. In the course of their early 
 work with the test Dale and Laidlaw made a few attempts to use 
 substances of this kind as a standard. Among others they tried 
 histamine, on the action of which they had, at the time, been recently 
 working. They observed what appeared to be obvious differences 
 in the relative sensitiveness of different uteri to histamine and to the 
 pituitary extract. One preparation would show a high degree of 
 sensitiveness to histamine and a low degree to the pituitary extract, 
 and in another the reverse relation seemed to hold. They accord- 
 ingly suggested, as the only immediate possibility, the use, as an 
 empirical standard, of a simple extract of the fresh substance of the 
 infundibular pituitary lobes of oxen, sterilized in ampoules, and 
 renewed at frequent intervals. 
 
 The suggestion was admittedly not ideal in every way, and several 
 attempts have been made to replace it by the use of pure chemical 
 standards. Both (1914) worked out with great care a method in 
 which histamine was used as the standard, and this method was 
 subsequently prescribed officially in the U.S.P. IX. Several workers 
 published criticisms of the method, which was found difficult to use. 
 It has further been suggested that the strength of pituitary extract, 
 officially indicated in terms of equivalence to histamine, is much 
 below that of a good preparation. 
 
 Trendelenburg and Borgmann (1920), apparently without knowledge 
 of Eoth's publications, also suggested the use of histamine as a 
 standard. 
 
 A later publication, by Spaeth (1918 b), from the same laboratory 
 as that of Koth (the Hygienic Laboratory of the Public Health 
 Department, Washington), seems to withdraw the suggestion of 
 histamine as a suitable standard, not on account of the difficulties 
 which formed the main ground of external criticism, but because 
 further investigation had produced evidence that histamine was not 
 indefinitely stable, either in sterilized solution, or even as the dry 
 hydrochloride. 
 
 We may say at once that, as the result of some years of continuous 
 experience, we have observed nothing to give any support to this 
 alleged instability of histamine. We have been accustomed to use 
 the diphosphate, which forms large, well-formed crystals, free from 
 water of crystallization and not hygroscopic. We have for years 
 used solutions prepared from the same sample, and have also had 
 experience of numerous samples, without observing anything which 
 could create even a suspicion of deterioration, or of variation in the 
 activity of this pure salt. We know, on the other hand, that solutions, 
 unless sterilized with great care, readily support the growth of 
 bacteria and moulds, with a consequent rapid loss of activity. We 
 can only suppose that the samples of the hydrochloride, available to
 
 19 
 
 American workers, were incompletely dry, so that decomposition 
 by micro-organisms became possible ; or that some unknown im- 
 purity in some way affected the stability of the salt or its solutions. 
 
 Histamine, as a standard of reference for the activity of pituitary 
 extracts cannot therefore, in our opinion, be rejected on the ground 
 of its supposed instability. Spaeth, however, for this reason sug- 
 gested its replacement by potassium chloride, which is beyond 
 question stable, and which, in relatively high concentrations, produces 
 contraction of the uterine muscle which, superficially, are not wholly 
 dissimilar from those produced by the pituitary extract. We con- 
 sidered it desirable, therefore, to give both of these suggested 
 standards a thorough trial. 
 
 In forming a conclusion as to the suitability of a substance, other 
 than the pituitary extract itself, to serve as a standard of activity 
 for this extract, there are tw r o points to be kept clearly in mind. 
 
 1. Preparations of the isolated uterus from different guinea- 
 pigs show, under conditions otherwise identical, a rather wide 
 variation of sensitiveness to the pituitary extract. It is, indeed, 
 the existence of such variation which creates the need for 
 a standard of reference. It is obvious that the use as a standard 
 of some other substance will be completely fallacious, unless 
 the sensitiveness of the uterine muscle to this standard substance 
 shows a strictly parallel variation. 
 
 2. During the course of an experiment the uterine muscle 
 usually exhibits a progressive increase of excitability to the 
 pituitary extract. Since each test involves the trial of a series 
 of doses, until equivalent doses of the standard and of the 
 preparation being tested are discovered, it is evident that an 
 accurate final result can only be obtained if any change in 
 excitability to the pituitary extract, occurring during the course 
 of an experiment, is accompanied by an exactly parallel change 
 in excitability to the standard substance. 
 
 We accordingly carried out a series of experiments to determine 
 whether histamine or potassium chloride was suitable, in these 
 respects, for use as a stable standard of activity. 
 
 (1) Experiments with Histamine. 
 
 The salt used was histamine diphosphate C 5 H 9 N 3 (H 3 P0 4 ) 2 . The 
 molecular weight is 307, while that of histamine base is 111 ; so that 
 10 parts of base are contained in 27-7 of the salt. It is our custom, 
 for convenience of comparison with results obtained with other 
 salts of histamine, to make up our solutions and express dosage in 
 terms of the free base. 28 mgm. of the diphosphate dissolved in 
 1 litre of Einger's solution gives, with sufficient approximation to 
 accuracy, a solution containing 0-01 mgm. per c.cm. Addition of 
 0-5 c.cm. of such a solution to a testing vessel containing 125 c.cm. 
 gives a final dilution of one part of histamine base in 25 millions. 
 (The ' standard ' dilution suggested by Both, and adopted by the 
 U.S.P. IX, was 1 part of histamine hydrochloride in 20 millions, 
 which is equivalent to 1 part of the base in 26-5 millions.) We 
 prepared a fresh quantity of the 1 in 100,000 histamine solution on 
 each day on which the test was carried out. The weighing and
 
 20 
 
 measurement were done accurately, and variation in the strength 
 of the solutions so prepared can be excluded. 
 
 The pituitary extract, which formed the test preparation for 
 standardization, was prepared from desiccated posterior-lobe sub- 
 stance, the method used being similar to that described later. The 
 extract was so made that 100 c.cm. represented 0-05 gin. of the 
 dry substance, and a sufficient quantity for the whole series of tests 
 was prepared in one operation. Immediately after nitration, and 
 while still hot, the extract was distributed into clean test-tubes. 
 These were plugged with cotton-wool and immersed for 5 minutes 
 in a common boiling water-bath. They were then stored in the 
 refrigerator, at a temperature just below 0C, till required. For each 
 
 o$H cJ 3P 
 
 I 
 
 SA. 
 
 o-2*P 0-3 H 
 
 FIG. 1. Comparison of pituitary extract, P (0-05 per cent.), with histamine solution, 
 H, 1 in 100,000. Change of Ringer at arrow. Note equivalence of 0-5 c.cm. H and 
 0-3 c.cm. P in first part of tracing, and the inferiority of 0-8 c.cm. H to 0-25 c.cm. P 
 in later part. 
 
 of the tests a fresh tube of the extract was drawn from the cold 
 store. In the following records ' H ' represents the solution of 
 histamine, 1 in 100,000, and ' P ' represents the pituitary extract, 
 0-05 per cent. The equivalents recorded do not represent all the 
 doses administered, but the approximations obtained at different 
 stages of the experiment. In some cases a rough equivalence was 
 obtained quite early. In these cases further doses were administered 
 to discover whether equality was maintained. In other cases the 
 earlier doses merely indicated certain limits, and, with these as guide 
 further doses were tried, with a view to obtaining an exact match 
 between those limits. 
 
 In all the records, as reduced for reproduction, 1-5 mm. represents 
 approximately 1 minute, when the drum is in motion. 
 
 (a) Guinea-pig weighed 250 gm. 
 the experiment were as follows : 
 
 The results at different stages of 
 
 1. 
 
 2. 
 
 
 
 
 5c 
 5c 
 
 .cm. 
 .cm. 
 
 H 
 H 
 
 < 
 
 0-8 c 
 0-3 c 
 
 .cm. 
 .cm. 
 
 P. 
 P. 
 
 (See Fig. ] 
 
 -) 
 
 3. 
 
 
 
 75 
 
 c.cm 
 
 .H 
 
 < 
 
 0-3 c 
 
 .cm. 
 
 P. 
 
 
 
 
 4. 
 
 
 
 75 
 
 c.cm 
 
 .H 
 
 < 
 
 0-25 
 
 c.cm 
 
 .P. 
 
 (See 
 
 Fig. 
 
 1.) 
 
 5. 
 
 
 
 8c 
 
 .cm. 
 
 H 
 
 < 
 
 0-2 c 
 
 .cm. 
 
 P. 
 
 

 
 21 
 
 (fc) Guinea-pig weighed 250 gm. 
 
 1. 0-5 c.cm. H < 0-4c.cm. P. 
 
 2. 0-5 c.cm. H > 0-3 c.cm. P. 
 
 3. 0-5 c.cm. H = 0-35 c.cm. P. 
 
 (c) Guinea-pig weighed 250 gm. 
 
 1. 0-5 c.cm. H < 0-5 c.cm. P. 
 
 2. 0-5 c.cm. H - 04 c.cm. P. 
 
 3. 0-5 c.cm. H - 0-35 c.cm. P. 
 
 4. 0-8 c.cm. H = 0-2 c.cm. P. 
 
 (See Fig. 2.) 
 
 FIG. 2. Determination on another uterus. 
 
 Comparison of same pituitary extract (P) and histamine solution (H) as in Fig. 1. 
 Note equivalence at early stage of 0-5 c.cm. H and 0-35 c.cm. P, and at a slightly 
 later stage of 0-8 c.cm. H and 0-2 c.cm. P. 
 
 1 
 
 . 0-5 
 
 c.cm. 
 
 H 
 
 = 
 
 
 
 5 c.cm. 
 
 P. 
 
 (See 
 
 Fig. 3.) 
 
 
 2 
 
 . 0-5 
 
 c.cm. 
 
 H 
 
 < 
 
 
 
 4 c.cm. 
 
 P. 
 
 
 
 
 3 
 
 . 0-5 
 
 c.cm. 
 
 H 
 
 < 
 
 
 
 3 c.cm. 
 
 P.) 
 
 /At 
 
 this stage 
 
 0-5 c.cm. H = 
 
 4 
 
 . 0-5 
 
 c.cm. 
 
 H 
 
 > 
 
 
 
 25 
 
 c.cm 
 
 .P.) 
 
 ) (approx.) 
 
 275 c.cm. P. 
 
 5 
 
 . 0-5 
 
 c.cm. 
 
 H 
 
 < 
 
 
 
 25 
 
 c.cm 
 
 .P. 
 
 
 
 
 6 
 
 . 0-7 
 
 c.cm. 
 
 H 
 
 = 
 
 
 
 25 
 
 c.cm 
 
 .P. 
 
 
 
 
 7 
 
 . 0-75 c.cm. H > 
 
 
 
 25 
 
 c.cm 
 
 .P. 
 
 
 
 
 8 
 
 . 0-7 
 
 c.cm. 
 
 H 
 
 > 
 
 
 
 3c 
 
 .cm. 
 
 PJ 
 
 (R 
 
 
 
 9 
 
 . 0-6 
 
 c.cm. 
 
 H 
 
 < 
 
 
 
 3c 
 
 .cm. 
 
 P.) 
 
 ( * 
 
 ig. .) 
 
 
 () 
 
 Guinea-pig 
 
 weighed 210 
 
 gm. 
 
 
 
 
 
 
 1. 
 
 0-5 c 
 
 .cm. 
 
 H 
 
 
 > 
 
 0-3 c 
 
 .cm. 
 
 P. 
 
 (See Fig. 4.) 
 
 
 2. 
 
 0-5 c 
 
 .cm. 
 
 H 
 
 
 > 
 
 04 c 
 
 .cm. 
 
 P -l 
 
 At this stage 0-5 c.crn. H 
 
 
 3. 
 
 0-5 c 
 
 .cm. 
 
 H 
 
 
 < 
 
 0-5 c 
 
 .cm. 
 
 P4 
 
 = (approx 
 
 .) 045 c.cm. P. 
 
 
 4. 
 
 0-5 c 
 
 .cm. 
 
 H 
 
 
 < 
 
 04 c 
 
 ,cm. 
 
 P. 
 
 
 
 
 5. 
 
 0-75 
 
 c.cm 
 
 .H 
 
 
 < 
 
 04 c 
 
 .cm. 
 
 P. 
 
 
 
 
 6. 
 
 0-75 
 
 c.cm 
 
 .H 
 
 
 < 
 
 0-3 c. 
 
 ,cm. 
 
 P. 
 
 (See Fig. 4.) 
 
 A superficial inspection of these results is sufficient to show that 
 histamine does not fulfil either of the conditions, above laid down, 
 for a suitable standard for the pituitary extract.
 
 22 
 
 If we restrict our attention, in the first instance, to the earlier 
 stages of each experiment, and accept as most correct the first 
 equation of activities obtainable, we obtain, in the series of five 
 experiments, the following equivalents of the pituitary extract for 
 0-5 c.cm. of the histamine solution : 
 
 (a) 0-3, (6) 0-35, (c) 04, (d) 0-5, (e) 045 c.cm. 
 
 0-5P 0-5H o-5? 0-7H 0-3 P 0~6H 
 
 FIG. 3. Determination on another uterine horn. 
 
 Same solutions of pituitary extract and histamine as previous figures. There was 
 first an equivalence between 0-5 c.cm. P and 0-6 c.cm. H, while later 0-3 c.cm. was 
 less than 0-7 c.cm. H, but greater than 0-6 c.cm. H. 
 
 FIG. 4. Same solutions of pituitary extract and histamine as in Figs. 1-3. At 
 first 0-5 c.cm. H is greater than 0-4 c.cm. P, while later 0-75 c.cm H is less than 
 0-3 c.cm. P. 
 
 Experiment (d) gives us an equivalence practically the same as that 
 demanded by the U.S.P. IX. This stipulates for a 10 per cent, 
 extract of fresh gland substance, which, in a dilution of 1 in 10,000, 
 shall match a solution of histamine hydrochloride 1 in 20,000,000. 
 We may take the fresh gland substance as containing about 20 per cent, 
 of dry matter, so that a 10 per cent, extract of fresh gland is equivalent 
 to a 2 per cent. (1 in 50) extract of dry gland substance. This diluted 
 10,000 times gives a fluid containing the extract from 1 part of dry 
 gland in 500,000. We start with a dry gland extract diluted to 0-05 per 
 cent.,i.e.to represent 1 part of dry gland in 2,000. We add 0-5 c.cm. of
 
 23 
 
 this to 125 c.cm., thus further diluting 250 times, and making the final 
 dilution 1 in 500,000, which, in this experiment, is initially equivalent 
 to 1 part in 25 millions of histamine base, which, as we have seen, is 
 practically the same as 1 in 20 millions of histamine hydrochloride. 
 
 We may conclude, then, that an equivalence such as that found 
 in the initial stages of (d) would entitle the manufacturer to pass 
 a batch of extract as of standard strength according to U.S. P. IX. 
 If, however, the test at his disposal was similar to (a), in which 
 0-5 c.cm. of the histamine solution was initially matched by 0-3 c.cm. 
 of the pituitary dilution, he would be required to add 2 parts of 
 water to every 3 of the extract before issuing it ; and (&), (c), and (e) 
 would entitle him to make smaller, but still important dilutions. 
 We may add that we have not made an effort to find examples of 
 extreme discrepancy ; on the contrary, we gave the proposed 
 standard every chance to justify itself, by conducting the whole of 
 the series of experiments within a few weeks, on as uniform a batch 
 of guinea-pigs as we could collect. We have no doubt that, by 
 throwing our net wider, we could have produced some far more 
 striking discrepancies. But, with the greatest pains to secure 
 uniformity of material and conditions, and basing our estimates on 
 the earliest match obtainable in each experiment, we still find such 
 wide discrepancies between individual determinations made on the 
 same material, that we are forced to the conclusion that the uteri 
 from different guinea-pigs, even under the best conditions, vary inde- 
 pendently in their sensitiveness to histamine, and to pituitary extract 
 respectively, and that these variations are such that the use of histamine 
 as a standard for pituitary extract is unsatisfactory, on this ground alone. 
 
 When we come to consider the second condition, which we indicated 
 as necessary for the satisfactory use of an artificial standard, viz. that 
 in any one experiment the sensitiveness of the uterus to the standard 
 should show a change parallel to its change (usually an increase) of 
 sensitiveness to the pituitary extract, it is at once evident that 
 histamine fails even more conspicuously to fulfil our demand. In 
 every experiment in which the comparison was continued for a long 
 time, i. e., in all except (b), there is obviously a progressive increase 
 in sensitiveness to the pituitary extract, and a progressive decrease 
 in sensitiveness to histamine. The extent of the change involved 
 can be appreciated from inspection of Figs. (1), (2), (3), and (4). 
 Keference to Experiment (a) (Pig. (1) ) will be sufficient. Here we 
 have, initially, 0-5 c.cm. H = 0-3 c.cm. P. As we have indicated 
 above, on the U.S.P. standard, the manufacturer should, on this 
 finding, dilute 3 parts to 5 before issue. Finally, however, 0-8 c.cm. H. 
 fails to produce as big an effect as 0-2 c.cm. P, so that, if this be 
 accepted as the true equivalence, a more than fourfold dilution is 
 required. There seems, indeed, a tendency to greater regularity in 
 the finding of this final 4 : 1 ratio, than in the initial match. It 
 might be suggested that this later evaluation is the true one ; it is 
 certainly obtained at a stage of the experiment at which, in comparing 
 one pituitary extract with another, we expect to obtain the most 
 accurate discrimination and the most definite equivalence. The 
 suggestion, however, cannot be accepted. The extract with which 
 we were working in this experiment had, in terms of dry gland
 
 24 
 
 substance, what we regard as a very low level of activity. The dry 
 material from which it was made had been kept in the laboratory 
 for about two years ; and, though it had apparently attained a 
 relative stability, and yielded very uniformly active extracts, it 
 had lost approximately three-quarters of the activity of the freshly 
 dried infundibular substance. The fact that the lower initial value 
 we obtained for the activity of the extract, in comparison with 
 histamine, would make it of standard activity, according to the 
 U.S. P. IX, strongly confirms the suggestion, made from several 
 quarters, that the standard was, in any case, much too low. To 
 accept a determination which would make this weak extract four 
 times as strong as the U.S. P. standard would be to reduce the 
 latter to an absurdity. Nor would a new definition enable these 
 late values to be accepted. There is no finality in the whole procedure. 
 With the sensitiveness of the uterus to the two preparations con- 
 stantly changing, and moving, on the whole, in divergent directions 
 in the two cases, it is possible to get almost any value, according to 
 the length of the experiment, and the stage at which an approximate 
 match is accepted as giving the correct result. Clearly then, on the 
 further ground that, in the course of an experiment, the sensitiveness of 
 the uterus to histamine and to pituitary extract changes in opposite 
 directions, histamine is unsuitable for use as a standard. 
 
 (2) Experiments with Potassium Chloride. 
 
 We tested Spaeth's suggestion (1918 b) to use potassium chloride 
 as a standard, in a series of experiments made on similar lines to 
 those on the use of histamine. The extract of pituitary substance 
 was made by an identical procedure from the same sample of dried 
 gland, and again a supply was tiiade sufficient for the whole series, 
 distributed into tubes, sterilized, and stored below C. The 
 potassium chloride solution was made up to a concentration of 
 19-68 per cent. According to Spaeth's indication, 0-5 c.cm. of this 
 solution, added to 100 c.cm. of Einger's solution, raising the con- 
 centration from 0-042 per cent, to 0-14 per cent., should produce 
 a contraction of the uterus suitable for comparative testing. Our 
 testing vessel held 125 c.cm., and in most cases a smaller dose of the 
 KC1 solution than 0-5 c.cm. sufficed to produce a nearly maximal 
 effect. Four complete tests were carried out. In the records K 
 represents the strong KC1 solution, P, as before, pituitary extract 
 diluted to represent 0-05 per cent, of dry gland substance. 
 
 (a) Guinea-pig weighed 275 gr. 
 
 0-2 c.cm. K < 0-4 c.cm. P. 
 
 0-3 c.cm. K > 0-3 c.cm. P. ) 
 
 0-3 c.cm. K < 0-4 c.cm. P. \ (See Fig. 5.) 
 
 0-3 c.cm. K = 0-35 c.cm. P. j 
 
 (Interval of 30 minutes.) 
 
 0-3 c.cm. K < 0-35 c.cm. P. ) 
 
 0-3 c.cm. K < 0-3 c.cm. P. I (See Fig. 5.) 
 
 0-35 c.cm. K= 0-8 c.cm. P. j 
 
 Later. 
 
 0-3 c.cm. K < 0-25 c.cm. P.
 
 (a) 
 
 FIG. 5. Comparison of the same pituitary extract (P) (strength 0-05 per cent.) as 
 used in Figs. 1-4, with KC1 solution (K) of strength 19-68 per cent. 
 
 In section (a) an equivalence between 0-3 c.crn. K and 0-35 c.cm. P is seen. In 
 section (b) 0-35 c.cm. K is equivalent to 0-3 c.cm. P. In section (c) is seen a curious 
 effect apparently following the action of KC1 on the isolated uterus. The first two 
 doses in section (c) are from different tubes of the same pituitary extract. They are 
 equivalent in action. Following a dose of 0-3 c.cm. K, another dose of the pituitary 
 extract is much less in its effect. See text. 
 2126 A *
 
 26 
 
 In this experiment, in its later stages, the opportunity was taken to 
 compare two tubes of the pituitary extract, with the result that an 
 exact equivalence of effect was obtained with successive doses ; 
 0-3 P! =0-3 P 2 , and 0-25 Pj -0-25 P 2 . At this stage a dose of 0-3 K, 
 following two doses of 0-25 P I} gave a definitely smaller effect than 
 these ; it was found, however, that another dose of 0-25 P, following 
 0-3 K, gave a subnormal effect, while with a further dose of P the 
 reaction returned to the former level (see Fig. 5, section c). This 
 apparent depression of the reaction to a submaximal dose of pituitary 
 extract, by an immediately preceding reaction to KC1, was observed 
 again on repeating the sequence ; and it occurred at a stage when the 
 uterus was responding to successive, equal doses of pituitary extract 
 with mechanical regularity. 
 
 (6) Guinea-pig weighed 250 gm. 
 
 Irregular results were obtained at first, but, when response became 
 fairly uniform, the following comparisons were obtained : 
 
 04 c.cm. K > 0-6 c.cm. P. 
 
 0-3 c.cm. K = 0-6 c.cm. P. 
 
 04 c.cm. K < 0-6 c.cm. P. 
 
 04 c.cm. K < 0-5 c.cm. P. 
 
 04 c.cm. K > 0-5 c.cm. P. 
 
 04 c.cm. K > 0-fi c.cm. P. ] , Q -rr r ,. 
 
 04 c.cm. K - 0-7 c.cm. P. I < So0 Fl * ^ 
 
 (c) Guinea-pig weighed 300 gm. 
 
 0-3 c.cm. K < 0-3 c.cm. P. 
 
 0-3 c.cm. K < 0-25 c.cm. P. 
 
 04 c.cm. K < 0-3 c.cm. P. 
 
 04 c.cm. K = 0-25 c.cm. P. 
 
 04 c.cm. K - 0-25 c.cm. P. 
 
 0-3 c.cm. K < 0-25 c.cm. P. 
 
 (d) Guinea-pig weighed 250 gm. ' 
 
 0-5 c.cm. K < 0-5 c.cm. P. 
 
 0-6 c.cm. K = 0-5 c.cm. P. 
 
 0-6 c.cm. K < 04 c.cm. P. 
 
 0-7 c.cm. K > 04 c.cm. P. 
 
 0-6 c.cm. K = 04 c.cm. P. (See Fig. 7.) 
 
 The results show both the defects which were observed in the attempt 
 to use histamine as a standard. Different ratios between the 
 activities of KC1 and of the pituitary extract are obtained in the 
 different experiments, and the same ratio does not hold throughout 
 the course of a single experiment. In the latter respect the results 
 with the potassium standard are less disconcerting than those with 
 histamine. Variation in the course of an experiment is observed ; 
 for example, in experiment (a) we find, initially, a good match between 
 0-3 K and 0-35 P, and later between 0-35 K and 0-3 P. The ratio has 
 become inverted, but the discrepancy is not so glaring as in some of 
 the histamine tests. In experiment (c) the ratio seems to remain 
 unchanged. When change occurs it seems rather to take the form 
 of a capricious variation than that of the progressive divergence seen
 
 27 
 
 with histamine. The fact that a dose of pituitary extract may have 
 its effect modified by an immediately precedent response to KC1 
 possibly accounts for some of the anomalies. When the results of 
 different experiments are compared, on the other hand, the dis- 
 crepancies obtained with KC1 as standard are at least as great as 
 those with histamine. If we take the records of the above series and 
 calculate, from the first match obtained in each experiment, the 
 volume of P apparently equivalent to 0-5 c.cm. K, we obtain the 
 following results: (a) 0-58 c.cm., (&) 1-0 c.cm., (c) 0-31 c.cm., 
 (d) 041 c.cm. Spaeth's suggestion is that the effect of a commercial 
 extract diluted to 1 in 4,000 should be equal to that of his standard 
 potassium dilution, which he fixes at 0-14 per cent. This dilution 
 would be produced by the addition of 0-5 c.cm. K in our experiment. 
 The dilution of an ordinary extract, representing 10 per cent, of 
 
 FIG. 6. Comparison of pituitary extract (P) and 
 KC1 solution (K) of the same strengths as in 
 Fig. 5, pn a different uterus. Equivalence between 
 0-4 c.cm. K and 0-7 c.cm. P. 
 
 FIG. 7. Comparison of same 
 pituitary extract (P) and KOI 
 solution (K) as in Figs. 5 and 6. 
 Equivalence between 0-6 c.crn. 
 K and 0-4 c.cm. P. 
 
 fresh gland substance, to the extent of 1 in 4,000 would be equivalent 
 to 1 part of dry gland substance in 200,000, as shown above. To 
 obtain this dilution with our extract we should have to add 
 1-25 c.cm. P. So that, on the lowest estimate of its activity (&),it is 
 too strong, and needs dilution in the proportion of 100 to 125. On 
 the estimate given by experiment (c) it is many times too strong, 
 and needs dilution in the proportion of 31 to 125 fourfold, while 
 the results of (a) and (d) would indicate approximately twofold 
 and threefold dilutions. These trials of potassium chloride us 
 a standard were carried out on a uniform series of guinea-pigs, 
 without any attempt to discover the extreme range of variation of 
 the results obtainable by the method with one pituitary extract. 
 The results are not selected, but recorded exactly as they wnv 
 obtained. If we had desired to exhibit wider discrepancies we could 
 doubtless have obtained them in a longer series. It seemed to us, 
 however, that these four experiments, carefully conducted under the 
 best conditions, showed that the expression obtained, in any one 
 experiment, for the activity of pituitary extract in terms of potassium 
 chloride, was purely accidental. We accordingly conclude, ihat utt>ri
 
 28 
 
 from different yuinea-piyx rari/ independently in their sensitiveness to 
 potassium chloride and to pituitary extract respectirely. and that these 
 ruriations are of such an order that potas.^iutn <-ltln> idr ran not be used 
 as a standard for the actirity of jiituitafi/ c.Hract. 
 
 Incidentally \\e may note that the lowest estimate for the activity 
 of our extract gives it rather more activity, in relation to the amount 
 Of dry gland suhstance represented, than that suggested by Spaeth 
 for adoption as a standard strength. As mentioned al>o\e. our 
 extract was made from old dried material, having only ahout one 
 quarter of the activity present in the infundibular substance imme- 
 diately after drying. Spaeth's suggested sta.nda.i-d could not. there- 
 fore, in our opinion be, in any case, regarded as representing a proper 
 level of activity for an extract purporting to be a 10 per cent, extract 
 of fresh infundibular lobe, even if the indications obtained by his 
 test were more constant for the same preparation. 
 
 Wide as the discrepancies are between our individual attempts to 
 estimate the activity of the same extract in relation to either of tie 
 standards, we believe that they would have been much wider if we 
 had varied the composition of the Ringer's solution. We regard as 
 fundamentally unsound such a method as that proposed by Koch- 
 mann (1921). who reduces the calcium of his Ringer's solution and 
 adds magnesium chloride, until even the ut eru.-. from an old guinea-pig 
 no longer shows spontaneous contractions. He then proceeds to 
 estimate the activity of a series of pituitary preparations by com- 
 paring the heights of the contractions produced by a standard dose 
 of each, with a scale produced by varying doses of hist a mine. He 
 gives no evidence of any precaution to ascertain that the effects of 
 several successive doses of the same preparation would be identical. 
 Under the conditions of his method we think this very improbable. 
 This is an extreme example of faith in the mechanical accuracy of 
 comparison obtainable between the pituitary extract and an artificial 
 standard. In minor degrees, however, w r e believe that small varia- 
 tions in the composition of Ringer's solution, which would be -very 
 difficult to exclude from the practice of widely scattered laboratories, 
 would introduce systematic differences between the estimates of 
 different workers using the same artificial standard, in addition to 
 the variations inseparable from the method. In our own series the 
 composition of the Ringer's solution has been constant, the guinea- 
 pigs have formed a reasonably uniform series as regards age and 
 weight, all conditions have been kept rigidly the same, and all the 
 experiments have been carried out in the space of a few weeks by the 
 same practised workers. We do not think that conditions more 
 favourable to the performance of the suggested standards could have 
 been devised ; yet we are driven to the conclusion that their use gives 
 no closer estimate of the value of a pituitary extract than could be 
 obtained by some simpler expedient, such as the determination of 
 the highest dilution capable of producing a maximal contraction of 
 the uterus. Such a method would obviously suffer from the large 
 inaccuracy due to variation in the sensitiveness of different prepara- 
 tions ; but if an extremely rough estimate were sufficient, we believe 
 it would be better to choose a method suffering from such an obvious 
 and frankly recognized inaccuracy, than to use standards which are
 
 29 
 
 supposed to eliminate this source of error, but, in actual fact, give no 
 trustworthy information concerning the sensitiveness of the prepara- 
 tion to the pituitary active principle. We do not regard it as 
 impossible that, in due course, a stable chemical standard will be 
 discovered which does give this information ; if it were, we should 
 strongly advocate its adoption. Meanwhile we hope to show, in the 
 next section, that an empirical standard, prepared from the pituitary 
 substance itself, can be made with reasonable uniformity ; and that 
 by its use it is already possible to make a much closer estimate of 
 the activity of a pituitary extract, than with any of the artificial 
 standards yet suggested. 
 
 V. THE USE OF AN EXTRACT MADE FROM THE PERFECTLY 
 FRESH PITUITARY GLAND AS A STANDARD. 
 
 Our conclusion being that, at present, no satisfactory artificial 
 standard can be found, we have to inquire whether any preparation 
 of sufficiently certain uniformity can be made from the infundibular 
 substance itself. The ideal standard, as Dale and Laidlaw pointed 
 out, would be a dry, stable sample of the infundibular substance, or 
 of some active preparation therefrom, from which an extract or 
 solution could be prepared from time to time, according to an 
 accurately prescribed routine. We are not without hope that such 
 a preparation may eventually be available. Some years of observa- 
 tion will be required, however, before it is possible to claim absolute 
 stability for such a standard. We know already that the infundibular 
 substance, ' dried ' in an ordinary commercial sense, and kept in the 
 laboratory in a stoppered bottle, loses an important part of its 
 activity in the space of a year or tw r o. The question has still to be 
 answered whether such material, if treated like a standard antitoxic 
 serum, by drying to absolutely constant weight over phosphorus 
 pentoxide, and sealing in perfectly dry vacuum, will remain stable. 
 It is clear, however, that such a possibility cannot be put to the test 
 unless some other kind of reasonably good standard is available for 
 comparison. We believe that such can be found in the simplest 
 possible kind of preparation, namely, the simple, heat-coagulated 
 watery extract from the perfectly fresh infundibular substance of the 
 ox pituitary body. Seasonal variations in the glands have often been 
 alleged as a probable cause of the lack of uniformity in the strength 
 of the commercial extracts. We can find no experimental basis for 
 the suggestion, which seems to have arisen from an assumed analogy 
 with other ductless glands. Fenger reports that he was unable to 
 detect any such seasonal fluctuations in the activity of the pituitary 
 posterior lobe. Our own experiments, definitely directed to this 
 point, and extending over a period of many months, have convinced 
 us that no variations, appreciable by the methods available, occur 
 in the activity of the perfectly fresh material from the only species 
 which we have investigated, namely the ox. 
 
 (1) Method of Making the Extract. 
 
 (a) Procuring the material. This was obtained direct from the 
 slaughter-house, the glands, as soon as dissected, being handed to
 
 30 
 
 a messenger who brotight them straight to the laboratory. In our 
 earlier series of comparisons we took no special precautions to keep 
 the glands cold during this transit, so that they were, for about two 
 hours, at the variable temperature of the air before being dissected 
 and worked up. No differences were detected between the values 
 of the extracts from glands thus treated, the comparison extending 
 to six samples obtained at intervals between August and December. 
 A seventh sample yielded an extract of distinctly lower activity, but 
 the cause of this, which will be discussed later, was clearly ascertained, 
 and had no connexion with changes in the glands between killing and 
 extraction. It appeared to us unlikely, however, that no change 
 occurred during the interval of exposure to a temperature of about 
 1 8 C. It seemed more probable that some deterioration had occurred 
 but, under the conditions of collection, had been sufficiently constant 
 to escape detection by the test. To ascertain whether this was so we 
 had one small batch of glands divided into two lots immediately on 
 receipt from the slaughterer. One lot was brought to the laboratory 
 as before,' without artificial cooling ; the other lot was dropped at 
 once into a wide test-tube, which was kept immersed in a freezing 
 mixture contained in a Dewar (thermos) flask, so that the glands 
 were quickly frozen and brought thus to the laboratory. Both lots 
 were kept longer than usual in the laboratory, and when 3| hours 
 had elapsed since removal from the carcase, they were dissected 
 simultaneously, extracted in precisely the same manner, and tested 
 against one another. The extract from the cooled glands had about 
 30 per cent, greater activity than that from those transported at 
 air temperature. Since local variations, both of transmission-time 
 and temperature, are bound to occur, we recommend that the standard 
 extract should be made from glands which are cooled to near 
 freezing point as soon as they are obtained from the carcase, and kept 
 thus until they are dissected for extraction, the time between 
 slaughter and extraction being, in any case, made as short as possible. 
 We found that the posterior lobes from 6 to 8 glands gave a convenient 
 volume of ' standard ' extract. Smaller quantities are more difficult 
 to handle ; larger are undesirable for reasons to be indicated later. 
 
 (b) Preparation for extraction. The glands are dissected as rapidly 
 as possible, care being taken that nothing but pure infundibular lobe 
 substance is retained. Occasionally, in the region of the pars inter- 
 media, small masses of colloid material are found. These should not 
 be included in the material to be extracted, and they are easily 
 detached. In one gland we found a piece of the colloid material 
 sufficiently large to be worked up by itself into a separate small 
 volume of extract, an extract being made under exactly parallel 
 conditions from the posterior lobe of the same gland. The extract 
 from the colloid material was entirely inert on uterus and blood 
 pressure ; that from the infundibular lobe was quite normal in 
 activity. The observation is worthy of record, in criticism of the 
 suggestion that the colloid, formed by the pars intermedia, represents 
 the active principle, and that its presence in the posterior lobe, on its 
 way to secretion, confers on the latter its physiological activity. 
 It is quite clear that the colloid does not yield the active principle 
 to the ordinary methods of extraction, and there is no reason to
 
 31 
 
 suppose that it is in any way connected with the kinds of activity 
 with which we are concerned. We found no difficulty in separating 
 the posterior lobe, which shells out cleanly from such inert admixtures. 
 
 Strictly speaking, a standard method of extraction should include 
 a standard of fineness for the subdivision of the material to be 
 extracted. We have none to offer. Fenger tested the effect of 
 careful mincing on the yield of active principle, and found, as might 
 be expected, that fine division increased the yield of activity. If it 
 should prove possible to make a stable, dry standard, this difficulty 
 will not arise, since the dried material can be pulverized finely and 
 perfect extraction secured. Meanwhile, in using the fresh, moist 
 gland substance, we have not thought it desirable to add such aids 
 to subdivision as sand, glass, or Kieselguhr, and therewith to intro- 
 duce unknown possibilities of adsorption of the active principles. 
 We have been content to take the 6 to 8 dissected posterior lobes 
 and mince them together, with thorough mixing of their substance, 
 by snipping as finely as possible with small dissecting scissors. 
 
 The material, thus minced and mixed as rapidly as possible, is 
 weighed out into suitable portions for extraction. The weighing 
 is conveniently done by taking the half of a microscopic glass slide, 
 weighing this accurately, placing about 1-5 gm. of the minced gland 
 substance on it, and weighing again. 
 
 (c) Process of extraction. The slide, with the weighed pituitary 
 substance on it, is introduced into a 200 c.cm. flask, with a short 
 wide neck. Distilled water is added in the proportion of 40 c.cm. 
 to 1 gm. of gland substance. The latter is washed off the slide and 
 distributed in the water by shaking, and the mixture, with occasional 
 agitation, is allowed to stand for one hour at the laboratory tempera- 
 ture (i. e. not above 20 C.). At the end of this period of cold 
 extraction a half normal solution of acetic acid is added, in the 
 proportion of 0-2 c.cm. to 1 gm. of the gland substance. The neck 
 of the flask is then plugged \vith cotton-wool and the belly is plunged 
 into a large bath of briskly boiling water. The object is to bring the 
 contents of the flask to a temperature near 100 C. in the minimum 
 time. The flask is kept in the boiling water for ten minutes, and is 
 then removed and cooled under the tap. The cooled extract is 
 filtered through a folded paper, and the coagulum is not washed. 
 The filtrate represents a ' 2-5 per cent, extract of fresh gland substance '. 
 
 Determinations carried out by our colleague Dr. H. W. Dudley 
 showed, on two samples, ratios of 18-6 and 194 of dry substance to 
 100 of fresh substance. As a convention w r e have adopted 20 per cent, 
 as expressing, with sufficient accuracy, the proportion of dry material 
 present in a known weight of fresh gland. Our ' 2-5 per cent, extract 
 of fresh gland ' is, therefore, termed a ' 0-5 per cent, extract ' in 
 terms of dry gland substance. We have alw r ays diluted it tenfold 
 with water, and distributed the dilution in a series of test-tubes. 
 These, after plugging with cotton-wool, are placed in the boiling 
 water bath for three minutes and then stored in the refrigerator 
 until required. Such brief additional heating does not impair the 
 activity perceptibly, and secures such relative sterility as is 
 adequate for keeping the extract for a week or two in the 
 refrigerator. The standard, as stored for brief periods in this
 
 32 
 
 approximately sterile condition, represents, therefore, a 0-25 per cent. 
 extract of the fresh, moist gland substance, or a 0-05 per cent, extract 
 in terms of dry gland substance. 
 
 It is neoeeaxy to insist that, if uniformity of result is to be obtained, 
 the method of extraction must be kept constant in every detail. 
 Mention has already been made of the difference made by keeping 
 the glands thoroughly chilled between the time of their removal and 
 the preparation of the extract. We believe that a more thorough 
 comminution of the material would probably result in more complete 
 extraction, and might render the preliminary cold extraction un- 
 necessary. It seems highly probable, also, that the deterioration 
 detected in the undissected gland material at the ordinary tempera- 
 ture, likewise proceeds during the preliminary extraction of the fresh 
 minced material with water at the room temperature. We have 
 found, however, by experiment, that it is more than counterbalanced 
 by the more perfect extraction which this preliminary soaking 
 effects when the gland substance is simply minced as finely as 
 possible with scissors. Admittedly this is a compromise ; but it is 
 a convenient one, and we have found it to give extracts of very 
 regular activity when the indicated time limit is strictly observed. 
 
 A much more important source of variation, which does not so 
 readily suggest itself, is the rate at which the suspension of gland 
 substance in acidulated water is heated to the coagulation point. 
 We came upon this source of error accidentally, though it might 
 have been predicted by a more thorough consideration of the factors 
 involved. The fall of activity, occurring slowly at the ordinary 
 temperature, may be attributed with some confidence to the action 
 of an autolytic enzyme. This again may be assumed to act with 
 much greater rapidity at temperatures between 30 and 50 C. The 
 longer the period of heating through this range, the greater the fall 
 of activity which may be expected from this action. We have clear 
 evidence of this deleterious effect of slow heating. In the directions 
 given above, the size and shape of the vessel indicated, in relation 
 to the volume of fluid, and the stipulation that the water bath 
 into which it is plunged must be large and briskly boiling, are all 
 designed to bring the mixture as rapidly as possible to a temperature 
 above that at which autolytic enzymes are put out of action. In 
 this way, as we have found by experience, we obtain the maximum 
 activity which can be extracted from the glands under the given 
 conditions with regard to relative volume of water, mincing of glands, 
 &c. If we prescribed any but the minimum possible period for 
 attaining the ' lethal ' temperature, it would be necessary to prescribe 
 not only a standard total time of heating up, but a uniform rate of 
 rise, so that the time at each intervening temperature would be 
 constant. This we regard as impracticable. We therefore choose, 
 in this instance, the procedure giving the optimum preservation of 
 activity, as being the easiest method of obtaining a constant result. 
 As regards the other conditions the volume of liquid in proportion 
 to weight of gland substance, &c. these are chosen for convenience, 
 and fixed, not with the idea that they give us the whole of the 
 activity of the gland substance, but that they give us a sufficiently 
 constant proportion. Using a larger relative volume we should
 
 33 
 
 probably secure greater completeness of extraction, subject, however, 
 to a less constant loss by slower heating, unless the vessels used were 
 inconveniently large. 
 
 No purpose would be served by an attempt to obtain the whole of 
 the activity in solution, in making our standard. No process of 
 manufacture on a large scale preserves or can be expected to preserve 
 a proportion nearly so large as that obtained on a small scale by the 
 above process. Our sole aim is to obtain a constant standard of 
 reference, and we have reason to believe that this can be achieved 
 by a rigid adhesion to the above directions. 
 
 (2) Method of Performing the Test. 
 
 . 
 
 The horn of a suitable uterus, having been carefully dissected and v t^ 
 
 suspended in the bath of warm Ringer's solution, must be left to ' 
 itself until relaxation is approximately complete. This will usually 
 take from 20 to 30 minutes. Any necessary adjustment of the weight 
 of the lever is then made. A first dose of the standard preparation 
 is then given and the effect recorded. As a first dose we ordinarily *.S'^ * 
 use 0-5 c.cm. of a 50-fold dilution of the standard fresh extract. The 
 undiluted extract represents, it will be remembered. 2-5 per cent, of . O r , 
 the fresh infundibular substance, or, approximately, 0-5 per cent, 
 of the dry weight ; so that the 50-fold dilution is an 0-05 per cent, 
 or 0-01 per cent, extract, according to the notation adopted. For 
 convenience we have usually recorded the strength in terms of dry 
 weight, assuming this to be one-fifth of the moist weight. We start, 
 then, with a dose of 0-5 c.cm. of 0-01 per cent, extract, added to 
 the bath containing 125 c.cm., giving a dilution of 1 part of original 
 dry weight in 2| millions. The effect varies much with different 
 samples of uterine muscle. With an initially sensitive preparation 
 the result will be a contraction to a maximum, maintained for a few 
 minutes. In such a case the contents of the bath are changed for 
 fresh Ringer's solution and time allowed for complete relaxation. 
 This process, after such a first dose, is frequently less rapid than at 
 a later stage. After 15-20 minutes, however, the muscle should be 
 relaxed and ready for a second dos?. which, in such a case, is smaller 
 than the first. After a few trials a dose is found usually 0-3 to 
 04 c.cm. of the indicated dilution to which the muscle responds 
 regularly, with identical contractions which are less, but not widely 
 less, than the maximum of which it is capable. In the case of a less 
 sensitive preparation, especially with the delicate horn from the 
 uterus of a very young guinea-pig, the first effect of 0-5 c.cm. of the 
 dilution may be very small. In such a case it is well, after change 
 to pure Ringer's solution and relaxation, to repeat the same dose. 
 It will usually produce a much larger response. If after 3 or 4 such 
 doses the response is still widely submaximal, the dose must be 
 increased ; but it is desirable to establish the fact, that the regular 
 response of the accustomed muscle to such a dose is widely sub- 
 maximal, before such an increase is made. The aim in any case is 
 to establish, for the particular preparation, the close which is about 
 60-70 per cent, of that needed to produce contraction to the 
 maximum.
 
 34 
 
 When this dose has been fixed, it is essential to make sure, not 
 only that the response to it is regular, but that an increase or decrease 
 of the dose by 20 per cent, produces a clear increase or decrease in 
 the response. By this stage of the test it will have become necessary 
 to fix the time interval between doses. This should be so chosen as 
 to allow complete relaxation, and persistence of the relaxed con- 
 dition for about 2 minutes before the next dose is given. The 
 suitable interval will vary, with different preparations, from 8 to 
 12 minutes, with an average of about 10 minutes. When once chosen 
 it must not be arbitrarily or irregularly varied, though change in 
 the rapidity of reaction and relaxation may make desirable a definite 
 shortening of the interval at a later stage of the test. 
 
 It will be seen that these preliminary tests, to fix the suitable, 
 submaximal dose, and to demonstrate the possibility of accurate 
 
 5 o 4 0-5 Q 5 04 05 <?A 64$ '&4 '043 '0-<- 
 
 FA F, r * A ' 5 F , F J F < $ F, 5 fj F A f, F A f 
 
 FIG. 8. A comparison of two extracts from fresh infundibular material. 
 
 Fj is the extract referred to in Table II as F,, and F 2 is that referred to in the same 
 table as F 5 . In each case for testing, a 50-fold dilution of a 2-5 per cent, extract of 
 the fresh material is used, and the figures under the tracing represent the amount of 
 each extract in c.cm. added to the bath of 125 c.cm. The tracing shows our regular 
 procedure in comparing two preparations. After finding doses producing equivalent 
 contractions of the uterine horn, as in the first four seen above, we then demonstrate 
 that by slightly varying the doses contractions of correspondingly different heights 
 are produced. 
 
 differentiation at this level of dosage, will necessarily occupy an 
 hour or two, at the rate of 5 to 6 doses per hour. Similarly, when 
 the standard dose is thus fixed, the determination of the exactly 
 equivalent dose of the preparation to be tested cannot be made 
 quickly. In fixing this equivalence we alternate doses of the unknown 
 with those of the standard extract, keeping the same constancy of 
 time interval. A few ' sighting shots ' suffice to indicate the range 
 within which the appropriate dose will be found, and the discrepancy 
 is gradually narrowed, by changing the dose either of the standard 
 or of the unknown extract, until doses are found in which the two, 
 given in an alternated series of 3 or 4 doses, produce curves of exactly 
 the same height on the record. It is then desirable to make sure 
 that reduction or increase of the dose of the unknown produces 
 curves definitely lower or higher than that produced by the fixed 
 dose of the standard (see Fig. 8). 
 
 We are aware that the procedure thus laid down, in which as 
 much as five hours may be required for the completion of a com-
 
 35 
 
 parison, makes a heavy demand on the patience of the observer. 
 We are convinced, however, that results of real value can be obtained 
 from this test in no other way. Occasionally the worker will, by 
 good fortune, obtain a preparation which settles down to regular 
 activity almost immediately ; or a lucky shot at the appropriate 
 dose of the unknown extract may enable him rapidly to establish 
 an equivalent for the standard. In such ways the time needed for 
 a test may be much reduced, but any attempt to accelerate the 
 determination, by departure from the essential lines of the procedure 
 above described, will, in the end, result in untrustworthy estimates 
 and wasted time. It is, with us, a rare experience to have to abandon 
 a test because the preparation will not give a response of sufficient 
 constancy or a discrimination sufficiently fine. One determination, 
 carefully and conscientiously carried through, withput stint of time 
 or patience, is of far more value in result, and far less expensive in 
 material, or even in time, than six in which impatience has yielded 
 a series of rough estimates. 
 
 VI. THE REGULAR ACTIVITY OF PERFECTLY FRESH GLANDS. 
 
 The experiments on this point were conducted between August 
 1921 and March 1922. As a standard of reference, having presumably 
 an approximate stability, we used, on several occasions during the 
 test, an extract made from the old specimen of dried infundibular 
 substance, previously mentioned in connexion with the tests with 
 histamine and potassium chloride. In this series, however, the dried 
 material was only used for occasional confirmation of results obtained, 
 mostly, by direct comparison of successive batches of extract from 
 fresh glands with one another. In the case of the first two batches 
 made, the interval August to October was considered too wide 
 for such direct comparison to be trustworthy. The extract of the 
 18th of August had, in fact, been destroyed before that of the llth of 
 October was prepared. Comparison in this case was, therefore, 
 made only with reference to the dry gland extract, which is called 
 ' A ' throughout these tests, the fresh gland extracts being called 
 Fj, F 2 , &c., in the order of the dates of preparation. Table I shows 
 the details of the comparison of Fj and F 2 with A. 
 
 TABLE I. 
 
 Date of Dilutions (in terms of dry Equivalent doses Equivalents 
 
 preparation. material) used in the comparisons, of these dilutions. of activity. 
 
 Fresh gland 
 
 extract. Extract ' A ' 
 
 F! Aug. 18/21 0-025 0-05 % 0-3F, =0-45A F, =3A 
 
 F 2 Oct. 11/21 0-01% 0-02% 0-75F 2 =M25A F 2 =3A 
 
 It will be seen that these two first preparations of the fresh gland 
 extract were equal in activity, on the assumption that the old sample 
 of dried gland lost no activity between these dates. In the case of 
 the later extracts, which were prepared at shorter intervals, each 
 was compared with its predecessor, F 4 being compared with ' A ' as 
 well. Table II shows the results (see also Fig. 8).
 
 36 
 TABLE II. 
 
 Extracts 
 
 Dates of 
 
 Dilutions used 
 
 .Doses found 
 
 Equivalents of 
 
 corn-par fd. 
 
 preparations. 
 
 in the test. 
 
 equivalent. 
 
 activity. 
 
 Fo & F 
 
 Oct. 11 & 18 
 
 0-01 % 
 
 0-8F 2 =O8F 3 
 
 F 2 =F S 
 
 F* & F 
 
 Oct. 18 & 25 
 
 0-01 % 
 
 06F 3 =O6F 4 
 
 F s =F 4 
 
 F 4 3 & A 4 
 
 Oct. 25 
 
 0-01 % F 4 
 
 0-7F 4 =0-4A 
 
 F 4 =3A 
 
 
 
 0-05 % A 
 
 
 
 F 4 F 5 
 
 Oct. 25 & Nov. 29 
 
 0-01 % 
 
 0-5F 4 =0-5F D 
 
 F 4 =F 5 
 
 F 5 &F C 
 
 Nov. 29 & Dec. 6 
 
 0-01 % 
 
 1-OFr, = 1-4F 6 
 
 F 5 =1-4F 6 
 
 F 6 &F 7 
 
 Dec. 6 & Dec. 13 
 
 0-01 % 
 
 0-5F^ =0-35F 7 
 
 F 6 =0-7F 7 
 
 / T? 1 _ A 17 \ 
 
 Since F 2 had previously been found equal in activity to F 1? it is 
 seen that, out of the extracts made from seven samples of fresh 
 glands, obtained between August and December, only F 6 , the sample 
 of the 6th of December, showed any departure from strict uniformity 
 of activity. It formed such a remarkable exception that a careful 
 scrutiny was made of the process of its preparation. It had happened 
 that our colleague, Dr. Dudley, who made all the other extracts, was 
 absent on this date, and that one of us (J. H. B.) had made this 
 extract, with a supposedly strict observance of Dudley's routine. 
 On investigation, the only discrepancy discovered was that Dudley 
 had heated his extract, after the preliminary soaking in the cold, by 
 plunging into a small boiling water bath, while Burn had placed it 
 in the same water bath before this boiled, about 5 minutes being 
 occupied in bringing it to the boil. It seemed probable that the 
 rapid action of an autolytic ferment during the period of heating 
 between 30 and 50 was responsible for this greatly inferior activity 
 of the more slowly heated sample. 
 
 The next sample of fresh glands, F 8 , was accordingly divided into 
 two portions after mincing. Both were made up and treated in 
 exactly the same way, up to the process of heating on the water 
 bath. One extract was then heated as rapidly as possible, by plung- 
 ing the relatively capacious round-bottomed flask containing it into 
 a large bath of briskly boiling water. The other extract was placed 
 in a cold water bath which was then heated, about 20 minutes being 
 taken to reach the boiling point. The two extracts were then 
 compared with one another and with F 7 . 
 
 The test established the following equivalents : 
 
 F^ (rapid heating) =1-5 F 7 . 
 
 F 8b (slow heating) = 0-66 F 7 . 
 
 F^ = 2-25 F 8b (see Fig. 9). 
 
 It will be seen that Fga, for which precautions w r ere taken to secure 
 very rapid heating to 100, had 50 per cent, more activity than F 7 , 
 while the slowly heated F gb fell even further below the activity level 
 of F 7 , than F 6 , which was slowly heated by accident. We decided, 
 therefore, to adopt the most rapid heating easily obtainable in the 
 preparation of the standard extract, as the only means of obtaining 
 a practical uniformity with respect to this important detail. 
 
 In later experiments the effect of chilling during transit from the 
 slaughter-house, as already described, was twice tested, the result 
 showing, in each case, a 30 per cent, superiority for the chilled 
 specimen over that transported at ordinary temperature. The results
 
 37 
 
 obtained in these experiments were not strictly comparable with 
 those of the earlier extracts from glands transported at the ordinary 
 temperature, since the interval between slaughter and extraction was 
 deliberately lengthened, in order to make obvious any superiority due 
 to transport below = C. These last experiments, moreover, were 
 carried out at an interval of 3 months after the latest of the earlier series, 
 and it was not thought desirable to make a direct comparison of the 
 new extract with one which had been kept so long, even at C. There 
 was, however, no obvious departure from the level maintained by the 
 earlier fresh extracts, as judged by comparison with extracts from the 
 old sample of dried material which has been several times mentioned. 
 Our conclusion is, therefore, that extracts of perfectly fresh infundibular 
 material, if prepared by a method kept exactly constant in all its details, 
 furnish a sufficiently constant standard of reference for the biological 
 testing of pituitary extracts. 
 
 l 04 01 
 
 -. 
 
 FIG. 9. The tracing shows the comparison between the fresh gland extract F fa 
 and F 8b (see text). For testing, a .50-fold dilution of a 2-5 per cent, extract of fresh 
 gland was used, and the figures below the tracing represent the amount of each 
 extract in c.cm., added to the bath of 125 c.cm. It is seen that 0-4 c.cm. of the 
 extract prepared by " rapid heating ' is as active as 0-9 c.cm. of the extract of the same 
 glands prepared by ' slow heating '. 
 
 VII. THE ACTIVITY OF COMMERCIAL EXTRACTS. 
 
 From the demonstration, given in the previous section, of the 
 strictly constant activity of extracts from the perfectly fresh gland 
 material, made by a uniform conservative method, it is an obvious 
 corollary that, if all extracts made for therapeutic use could be 
 prepared under such conditions, no standardization would be neces- 
 sary, or, indeed, possible. Each batch of extract would be, in itself, 
 the standard which we propose to apply. 
 
 The fact has to be faced, however, that it would be impossible, on 
 these lines, to meet the world's demand for this preparation. Small- 
 scale working is of the essence of the method which we have recom- 
 mended for the preparation of the ' Standard fresh gland extract '. 
 For good or ill, the use of the pituitary extract, particularly in 
 obstetrics, has grown with such rapidity and to such dimensions, 
 that the supply of raw material is scarcely adequate to meet the 
 demand. As in the case of other pharmaceutical preparations, we 
 have to accept organized mass-production, with its disadvantages as 
 well as its convenience and efficiency.
 
 38 
 
 Several of the precautions, which we found essential for the pro- 
 duction of extracts of uniformly high activity, are hardly applicable 
 to large-scale working. Even where the slaughtering and the work- 
 ing up of the carcases are centralized and organized, the time between 
 the death of the animal and the completion of the dissection process 
 will probably be materially longer than in a laboratory, where only 
 six glands have to be dissected. 
 
 In other cases there is no room for doubt that the time between 
 slaughtering and working up may vary very widely, some makers 
 having to depend for their supplies of glands upon scattered and 
 distant sources. In many cases economy in working will be favoured 
 by making extracts in bulk, at intervals not too frequent. This 
 necessitates the accumulation and storage of gland material in the 
 dried form, to be worked up in accordance with demand. It is 
 possible that the activity of gland substance, if dried absolutely to 
 constant weight, may be indefinitely stable. It is certain that that 
 of the substance, dried in the ordinary, factory sense, is not so. 
 Further, the heating up of a large volume of the \vatery suspension 
 to coagulation point cannot, if carried out by ordinary methods, 
 have the momentary brevity which we found essential to the pre- 
 servation of the activity. Under ordinary conditions, the time taken 
 to reach the temperature of safety for the active principles may be 
 expected to vary with the volume to be heated. 
 
 For all these reasons, it was not to be expected that any of the 
 extracts on the market would exhibit such a high proportion of 
 activity, in relation to the weight of gland substance used, as those 
 which we prepared on the small scale. We have not, in fact, found 
 any which does so. It is obvious, also, that variations in the activity 
 of the finished product are to be expected, unless the ' physiological 
 standardization ' applied by the makers has eliminated them. 
 
 Before we proceed to the evidence concerning the activity of 
 commercial extracts, we may dispose of one additional point. We 
 mentioned above the imperfect stability of incompletely dried 
 material, as a possible source of defective or variable activity in 
 commercial extracts. The question arose, however, whether the 
 drying process, in itself, could be effected without loss. 
 
 (1) Effect of Drying on Activity. 
 
 The drying and the preparation of the equivalent extracts were 
 kindly carried out for us by our colleague, Dr. H. W. Dudley. 
 
 We found that drying the fresh gland material over phosphorus 
 pentoxide in vacuo was not accompanied by any loss of activity, 
 whether the drying was carried out at C. or 40 C. In the latter 
 case the drying took place very rapidly, and this presumably com- 
 pensated for any greater tendency to deterioration of the moist 
 material at the higher temperature, which might have been expected. 
 On the other hand, drying at room temperature in vacuo over 
 sulphuric acid was accompanied by a significant loss of activity. We 
 have no clear evidence as to the cause of the loss under thes : e con- 
 ditions. Our object, however, was to discover whether drying, in 
 itself, was necessarily accompanied by loss, and this question is
 
 89 
 
 answered in the negative by the experiments on drying over phos- 
 phorus pentoxide. 
 
 The following are the details of the comparisons : 
 
 1. Equivalent extracts made directly from fresh glands (F) and 
 from the same material dried at C. over P 2 5 (Dj). Tested in 
 dilutions representing 0-05 per cent, of dried material. 
 
 04 c.cm. F = 04 c.cm. D r 
 
 2. Equivalent extracts made from fresh glands (F) and from 
 material dried over H,jS0 4 at 15 C. (D 2 ). Tested in dilutions repre- 
 senting 0-01 per cent, of dried material. 
 
 0-75 c.cm. F - 1-12 c.cm. D 2 . 
 
 3. Equivalent extracts made from fresh glands (F) and from 
 material dried over P 2 5 at 40 C. (D 3 ). 
 
 1-0 c.cm. F = 0-8 c.cm. D 3 . 
 
 It will be seen that, in the last experiment, the extract from dried 
 material was slightly but distinctly more active than that from the 
 corresponding fresh material. Presumably the drying facilitated 
 a slightly more complete extraction of the active principle. 
 
 (2) Examination of Commercial Samples. 
 
 Samples of five different makes of the extract, which are current 
 on the English market, were obtained directly from the manu- 
 facturers. Three were of British and two of American manufacture. 
 In response to our request, the manufacturers, in all cases, courteously 
 gave us confidential information as to the methods of manufacture 
 and standardization. The following are the details, relevant to our 
 estimates of the activity of these preparations, which were furnished 
 with the samples supplied to us by the manufacturers as repre- 
 sentative of their respective products. 
 
 Preparation P. Made from dry material by a special process of 
 extraction. The strength arrived at is such as to represent a 20 per 
 cent, extract of the fresh infundibular substance. The extract is 
 made, in the first place, stronger than this. It is then physiologically 
 tested in comparison with another well-known commercial pre- 
 paration, the test on the blood pressure, and, more recently, that 
 on the uterus, being employed. The activity of the extract is then 
 adjusted by dilution. The sample supplied to us corresponded, 
 after this adjustment, to a 5-6 per cent, extract of dry substance. 
 
 Preparation Q. This is stated to represent approximately a 20 per- 
 cent, extract of the fresh gland substance. The extract, however, 
 is made from the dried material, and, according to the figures given 
 to us by the manufacturers, in the preparation of the sample supplied, 
 one part of this was taken to represent six parts of the fresh sub- 
 stance, instead of five parts, as we have assumed in most of our 
 calculations. The preparation is standardized by the uterus method. 
 
 Preparation R. Made from dried glands, or from fresh glands 
 frozen and kept in cold storage. Theoretical strength, a 10 per cent, 
 extract of fresh .infundibular substance. Standardized by uterus 
 method, and by the blood-pressure method.
 
 40 
 
 Preparation S. This is stated to be a 10 per cent, extract prepared 
 from fresh infundibular substance. It is standardized by the uterus 
 method. 
 
 Preparation T. This also is stated to be a 10 per cent, extract of 
 the fresh infundibular substance. Standardization is carried out by 
 the test on the blood pressure, though it appears that a test on the 
 uterus is sometimes used as a control. 
 
 It should be noted that, whilst two of the preparations in the above 
 list are of American manufacture, in no instance is reliance placed 
 on the test with histamine as a standard, prescribed by the United 
 States Pharmacopoeia. In two instances mention has been made 
 to us of the use of this test as a supplementary control, but for their 
 own satisfaction the makers, even in using the test on the uterus, 
 appear in all cases to have used some pituitary preparation as 
 a standard. In one case a previous satisfactory batch is used, in 
 one a ' standard ' extract, frequently renewed, and in one a ' specially 
 prepared, highly active, stable powder '. The experience of manu- 
 facturers, therefore, has already brought their practice into accord 
 with the suggestion made by Dale and Laidlaw, and strongly sup- 
 ported by our own recent experience, here recorded, that the only 
 suitable standard yet available for the activity of pituitary extract 
 is a preparation of the pituitary gland itself. That this is the case 
 when methods based on the pressor action are concerned, had never 
 been called in question. 
 
 There are two different questions which can be decided by com- 
 parison of the activity of these different commercial extracts with 
 the standard fresh extract, and with one another. In the first place, 
 we can find, in each case, what proportion of the activity originally 
 present in the gland substance used in the preparation is still present 
 in the extract as marketed. In the second place we can determine 
 the relative activities of the different preparations to one another, 
 irrespective of the amount of gland substance used. The two ques- 
 tions are not identical, because there is, among the manufacturers 
 whose products we have examined, no agreement as to the pro- 
 portion of gland to be used. In the United States the Pharmacopoeia 
 has attempted to fix the proportion, in such a way that the pre- 
 paration should represent a 10 per cent, extract of fresh infundibular 
 substance. In this country some workers aim at a 20 per cent. 
 extract ; but two samples having this ostensible value were made from 
 dry infundibulum in the respective proportions of 5-6 and 3-5 per cent. 
 We may take the results bearing on these two points separately. 
 
 It will be seen that we have not, for all the commercial samples 
 examined, figures of direct comparison with what we have termed 
 the ' Standard Fresh Extract '. The experiments, by which we 
 arrived at the suggested conditions for the preparation of this 
 extract, were proceeding at the same time as the examination of the 
 earlier samples supplied by the manufacturers. In some cases, 
 therefore, we only made a determination of the value of the 
 sample in terms of one of the earlier fresh extracts, in other cases 
 in terms of an extract made, under Standard conditions, from the 
 old dried material, which we have called extract ' A ' above. Since, 
 however, we have accurate records of the value of these in terms of
 
 41 
 
 the Standard now proposed, a very simple calculation gives us the 
 activity, of the manufacturers' samples in question, in relation to 
 this Standard. In some other cases we have only determined the value 
 of a sample in relation to one or more of the other commercial samples. 
 Since, however, the relation of these to the Standard was already 
 known, the value of the unknown sample in terms of the Standard 
 could easily be calculated with an accuracy sufficient for our purpose. 
 
 (3) Tlie Proportion of the Original Activity present in the Finished 
 
 Products. 
 
 A word is needed as to the basis of calculation used in these 
 comparisons. It is desirable, for simplicity, to express the theoretical 
 strengths of all the extracts compared in terms either of the weight 
 of fresh moist infundibular substance, or of the weight of dry sub- 
 stance represented. If all the extracts had been simply labelled in 
 terms of fresh gland substance, this might have furnished the more 
 convenient notation, our own Standard extract being made from 
 the fresh material. We had, however, at our disposal definite 
 information as to the proportionate weights of the dry material used 
 in preparing some of the extracts, and the figures made it clear that 
 estimates of the activity preserved, based on an assumed identity 
 of the proportion of gland substance, \vould in these cases be mis- 
 leading. It seemed unavoidable, in these cases, to express the yield 
 in terms of the known weight of dry substance. This, however, 
 involved the expression of the strengths of the other extracts under 
 test, and of our Standard extract, in terms of dry substance, though 
 some of them were made from the fresh material, and in others the 
 weight of dry material was not accurately known. We have adopted 
 what is. frankly, a conventional notation, in reckoning, in all such 
 cases, that 5 parts of fresh gland substance are equivalent to 1 part 
 of dry. Thus our Standard fresh extract is actually a 2-5 per cent, 
 extract of the fresh gland substance, but we call it, for purposes of 
 comparison, 0-5 per cent, extract of dry substance ; similarly a 
 commercial sample described as a 10 per cent, extract of fresh gland 
 substance is reckoned as a 2 per cent, extract of dry substance. We 
 are aware that this assumption is not strictly accurate, but it seemed 
 to afford the best method of arriving at a common basis, and it will 
 be seen that the differences to w r hich we attach significance, in com- 
 paring the yields of activity, are of such an order that this minor 
 inaccuracy of calculation could not affect them. 
 
 Preparation P. Sample 1. This preparation, being theoretically 
 a 20 per cent, extract of fresh substance, and actually a 5-6 per cent, 
 extract of dried substance, was diluted 200 times, giving a 0-028 per 
 cent, extract of dried substance. It was compared against F 4 , which 
 was diluted 2| times. F 4 being made to represent a 0-05 per cent, 
 extract of dry material, on the assumption that 1 part of dry cor- 
 responds to 5 parts of fresh gland, our dilution is an extract repre- 
 senting 0-02 per cent, of dry gland, as obtained by the method used 
 for F 4 . The equivalent doses, when added to the bath containing 
 125 c.cm., were 
 
 0-25 c.cm. P (0-028 per cent.) =0-3 c.cm. F 4 (0-02 per cent.).
 
 42 
 
 Therefore, at theoretically equal concentrations, 
 
 7P = 6F 4 
 
 or P = 7 F 4 . 
 
 Now F 4 has only 70 per cent, of the activity of the corresponding 
 frsh extract, prepared under the standard conditions now proposed. 
 Therefore, the value of P, expressed in percentage of that of a 
 ' standard extract ' from the same amount of gland, is 
 
 - x - = 60 per cent. 
 
 That is to say, in making this sample of P from dried material, the 
 manufacturers obtained 60 per cent, of the activity which can be 
 extracted from the equivalent of fresh (or carefully dried) material 
 by our standard method. 
 
 This same sample of P was also compared against Extract A, made 
 from the old dried material, and the following equivalent was 
 obtained : 
 
 04 c.cm. P (0-014 per cent.) = 0-55 c.cm. A (0-025 per cent.). 
 
 .; 56 P = 137-5 A. 
 
 P = 2-45 A. 
 
 But A = J Standard. (See under Q.) 
 0.45 
 
 .-. P = - - X 100 = 61 per cent. Standard. 
 4 
 
 The two comparisons give practically identical values for this sample. 
 Sample 2. We had no exact data as to the weight of dry gland 
 used, so that we have taken the 20 per cent, extract of fresh gland to 
 represent a 4 per cent, extract of dry gland. The comparison was 
 made with Standard fresh extract, with the following result : 
 
 0-7 c.cm. P (0-02 per cent.) = 0-7 c.cm. Standard (0-01 per cent.). 
 
 i. e. 2 P = 1 Standard 
 or P = 50 per cent, of Standard. 
 
 Preparation Q. Sample 1. This also theoretically represents a 
 20 per cent, extract of fresh substance, but is actually a 3-5 per cent, 
 extract of dried substance. It was first tested against Extract A, 
 made from the old dried powder. The extract made from this, under 
 standard conditions, has been repeatedly found to have almost 
 exactly one quarter of the activity of the standard fresh extract. 
 The dilutions of Q used for testing were a 200 times and a 400 times 
 dilution, with dry gland equivalents of 0-0175 and 0-00875 per cent, 
 respectively. Extract A was used in dilutions corresponding to 
 0-025 and 0-05 per cent, of dry substance. 
 
 The following were the equivalents found : 
 
 (1) 1-2 c.cm. Q (0-00875 per cent.) =1-0 c.cm. A (0-025 per cent.). 
 
 (2) 0-6 c.cm. Q (0-0175 per cent.) = 0-4 c.cm. A (0-05 per cent.). 
 
 (3) In Experiment (2) a further comparison was made between 
 A and F 4 , with the following result : 
 
 4A (0-05 per cent.) = 0-7 F 4 (0-01 per cent.), 
 i.e. 20 A = 7F 4
 
 or A =F 4 . 
 
 7 
 But F 4 = .r of Standard 
 
 7 7 
 
 .'. A = on x in = practically J Standard. 
 20 1U 
 
 From (1) 105 Q = 250 A. 
 
 or Q = 24 A. 
 Frdm (2) 105 Q = 200 A 
 
 orQ = 1-9 A. 
 
 The correspondence is not very good. If we take as average 
 Q - 2-2 A, 
 
 then Q = - X = 55 per cent, of the Standard. 
 
 Sample 2. The second sample, examined some six months later, 
 was compared directly with standard fresh extract. We had no 
 accurate indication of the proportion of dried gland used for this 
 sample. We have assumed, as in other cases, a ratio of 1 : 5, the 
 dilutions of the sample Q and of the standard being expressed in 
 dry gland substance on this basis. The following was the equivalent 
 obtained : 
 
 1-0 c.cm. Q (0-02 per cent.) = 0-25 c.cm. Standard (0-05 per cent.), 
 i.e. 200 Q = 125 Standard. 
 
 .'. Q = 62-5 per cent, of Standard. 
 
 Preparation R. This theoretically represents a 10 per cent, extract 
 of fresh gland substance, though it is sometimes made from dried 
 gland, in a proportion not exactly known to us. We have again 
 assumed a ratio of 1 : 5 for dry weight to fresh weight. 
 
 Sample 1 . This sample was first compared twice with Extract A, 
 and the equivalents obtained gave us 
 
 (1) E = 1-9 A. 
 
 (2) E = 2-1 A. 
 Average E = 2-0 A. 
 
 2 
 Since A = Standard, E = j X 100 = 50 per cent, of Standard. 
 
 Six months later the same sample was compared with the Standard 
 fresh extract, with the following result : 
 
 0-45 c.cm. E (0-02 per cent.) = 0-5 c.cm. Standard (0-01 per cent.) 
 
 (See Fig. 10.) 
 
 i. e. 9 E =5 Standard. 
 
 R = 55 per cent, of Standard. 
 
 These tests, which show a fair correspondence, indicate that in 
 making this first sample of E the manufacturers obtained practically 
 the same proportionate yield of activity as the makers of Q in pre- 
 paring the first sample of that product.
 
 44 
 
 Sample 2. This was compared only with the Standard fresh 
 extract, with the following result : 
 
 0-5 c.cm. R (0-02 per cent.) = 0-6 c.cm. Standard (0-01 per cent.), 
 i. e. 10 R = 6 Standard 
 
 /. R =60 per cent. Standard. 
 
 Preparation S. This is said to be a 10 per cent, extract prepared 
 from fresh infundibular substance. 1 The ratio 1 : 5 for dry to fresh 
 weight was assumed. A few preliminary trials showed a very definite 
 inferiority to the extracts previously examined, in theoretically 
 equivalent dilutions. The value of S has not been measured directly 
 in relation to the Standard, but its relation to the first samples of 
 P and R was carefully determined, these being diluted according to 
 the indications given above. 
 
 xi. -^ ^ ofc. "^ rv\ c\y -<J- ^^^ ^ oj -<^ ""^ 
 
 //37 il-5o tf-o * * ~ "> % * ^ * Q 
 
 0-U- -.? 'T O-3 b'5 '0-4 05 b t b'45'o-5 V 0-45 '0-6 'O'S '06> OS O-fT b-5" '0-jT ' 
 
 St R sr R Sr * Sr R K sr * * ^ R SA * st ~ * s 
 
 FIG. 10. A comparison of a commercial preparation (R) with our Standard fresh 
 gland extract (St). 
 
 The Standard is a 50-fold dilution of a 2-5 per cent, extract of the fresh glands, and 
 this dilution represents a 0-01 per cent, extract of the dry material present. ' R ' is 
 theoretically a 10 per cent, extract of fresh gland substance ; it was diluted 100 times ; 
 this dilution represents a 0-02 per cent, of the dry material present, so that the 
 dilution is theoretically twice as strong as that made from the Standard extract. 
 The tracing shows that 0-45 c.cm. R =0-5 c.cm. St, so that R contains activity =55 per 
 cent, of St. The figures above the tracing are the times at which the corresponding 
 doses were put into the bath. 
 
 (1) 0-5 c.cm. S (0-04 per cent.) = 0-5 c.cm. P (0-014 per cent.). 
 (S being diluted 50 times, P 400 times.) 
 
 i. e. 20 S = 7 P 
 or S = 0-35 P. 
 
 Since P = 60 per cent. S = 21 per cent. Standard. 
 
 (2) 0-7 c.cm. S (0-02 per cent.) = 0-3 c.cm. R (0-02 per cent.). (See 
 
 Fig. 11.) 
 
 c 3R 
 i. e. S = -y- 
 
 Since R = 50 per cent. S = = = 21 per cent. Standard. 
 
 1 Subsequent information suggests that we may, by mistake, have been supplied 
 with a sample representing a 6 per cent, extract of the fresh gland, instead of 10 per cent, 
 as originally stated. If this were the case the figure for the percentage yield of available 
 activity should be 35 instead of 21 per cent, for preparation S. In the legend to Fig. 11, 
 R should, in that case, be 1 -4 times, instead of ' more than twice ' as strong as S ; in 
 Table III the figure for S would be 35.
 
 45 
 
 Both these comparisons, therefore, give the same value for S in 
 relation to the Standard. 
 
 Preparation T. This is also said to be a 10 per cent, extract of the 
 fresh infundibular substance. It was tested on several occasions 
 against P, Q, and E, the results all showing a very low activity for T. 
 The comparison against E may be quoted. Assuming a 1 : 5 ratio 
 
 FIG. 11. Comparison of two oommerical preparations R and 8. Both represented 
 10 per cent, extracts of fresh gland substance, and both were diluted 100 times for 
 testing. So far from equal doses producing contractions of the same height, the 
 tracing shows that a dose of 0-3 c.cm. R is equivalent to 0-7 c.cm. S, so that R is more 
 than twice as strong as S. 
 
 05" 
 7t 
 
 FIG. 12. Comparison of commercial extracts R and T. Both represented 10 per 
 cent, extracts of fresh glands. When both were diluted 100 times, 0-4 c.cm. R gave 
 a good contraction, but 0-4 c.cm. T produced no effect whatever. A fresh sample of T 
 was taken and diluted only 10 times, and 0-3 c.cm. of this gave the response recorded 
 in the tracing as 5-0 T. Finally 1-0 c.cm. of a 10-fold dilution of T was approximately 
 equal to 0-4 c.cm. of a 100-fold dilution of R. 
 
 for dry to fresh weight in each case, and diluting T 10 fold and E 
 100 fold, we found : 
 
 1 c.cm. T (0-02 per cent.) =04 c.cm. E (0-02 per cent.). (See Fig. 1 2.) 
 So that T - 0-04 E. 
 
 Since E = 50 per cent. Standard, T = 2 per cent. Standard. 
 
 These various results may be tabulated. If we assume that our 
 
 1 Or 1-4 times. See footnote, p. 44.
 
 46 
 
 standard method extracts the whole of the activity of the perfectly 
 fresh gland substance, the following are the percentages, present in 
 the final products, of the activity originally present in the gland 
 substance used in making these different samples : 
 
 TABLE III. 
 
 Proportion of activity present in 
 
 Preparation. final product. (Standard = 100). 
 
 P. 1st sample 60 
 
 2nd 50 
 
 Q. 1st sample 55 
 
 2nd 62-5 
 
 K. 1st sample 50 (or 55) 
 
 2nd 60 
 
 S. 21 l 
 
 T. 2 
 
 It will be seen that P, Q, and K form a group in which the amount 
 of original activity preserved varies between about 50 and 60 per 
 cent. In the manufacture of S a much more serious loss nearly 
 80 per cent 2 has occurred, while in the case of T only a trace of the 
 activity remains. It is, perhaps, of interest to note that S and T 
 were samples of the only two preparations, concerning which we have 
 been informed that they are wholly prepared from the fresh gland 
 substance. The manufacturers of E state that they use fresh as 
 well as dry material, but we have no information as to the form in 
 which the gland was extracted in preparing the samples of E which 
 we have tested. The poor activity of the preparations which we 
 know to have been made from fresh glands may be a coincidence. 
 On the other hand, it is possible that the activity of autolytic 
 enzymes, during the heating up of a large volume, may play a more 
 serious part in lowering activity when fresh material is used. We 
 presume that, when finely powdered dry material is used, this could 
 be added directly to water already at a temperature above the 
 danger zone ; such a procedure would probably be inapplicable to 
 the sticky mass of pulped fresh material. These, however, are purely 
 speculative suggestions, and our only concern is to draw the attention 
 of the makers to this possibility of a serious loss of activity in 
 preparation, even when perfectly fresh material is used. 
 
 (4) The Relative Activities of tlie different Commercial Extracts. 
 
 While the results given above indicate the possibility of wide 
 discrepancies between the amounts of "activity obtained by different- 
 makers from the same quantities of gland material, they do not 
 reveal the full extent of the variation in activity among the different 
 extracts presented for therapeutic use. The labels, indeed, in some 
 cases give indications of the theoretical strengths of the extracts. 
 This, however, is not always done. In any case we have reason to 
 believe that the medical practitioner, finding two different pre- 
 parations of the pituitary gland, both dispensed in sealed ampoules 
 containing 1 c.cm., usually assumes that they are equivalent, or 
 intended to be so. We have, further, mentioned above the fact that 
 the proportions of dry gland substance used in preparing even 
 
 1 Or 35. See footnote, p. 44. z Or 65 per cent. See footnote, p. 44.
 
 47 
 
 theoretically equivalent extracts are by no means necessarily 
 identical with different makers. If we take the various extracts, as 
 the majority of practitioners do, as being theoretically equivalent, 
 and compare them merely with regard to their activities, without 
 any reference to the amount of gland represented, the contrasts are 
 even more glaring than those tabulated above. In addition to the 
 comparison of S and T with other preparations, already quoted, we 
 may record the following direct comparisons of E with P and Q. 
 
 In all cases the comparisons refer to the first samples of these 
 preparations tested : 
 
 (1) 0-25 c.cm. P (dil. 200 fold) = 04 E (dil. 100 fold). 
 
 i. e. at equal dilutions, P = 3-2 E. 
 
 A very similar relation is obtained from the figures given earlier. 
 P is a 5-6 per cent, extract, containing 60 per cent, of the available 
 activity. E, on the assumption that it is a 2 per cent, extract (of 
 dried material), shows 50 per cent, of available activity. This gives 
 the activities P : E : : 5-6 x 60 : 2 x 50 
 
 i. e. P - 3-36 E. 
 
 (2) 0-5-c.cm. E (dil. 100 times) = 0-55 c.cm. Q (dil. 200 times). 
 
 i. e. at equal dilutions Q = 1-8 E. 
 
 Again, from earlier comparisons, E, when taken as a 2 per cent, 
 extract, shows 50 per cent, of available activity, and Q, a 3-5 per 
 cent, extract, shows 55 per cent. 
 
 Whence Q : E : : 3-5 x 55 : 2 x 50 
 i. e. Q = 1-9 E. 
 
 We have already, from direct comparisons, for S and T, which 
 like E theoretically represent 10 per cent, extracts of fresh substance, 
 
 S = 043 E 
 T - 0-04 E. 
 
 We have, therefore, expressions for the relative activities of all 
 these first samples, supplied directly by the manufacturers as 
 representative, in terms of one of them, that of preparation E. If 
 we represent the activity of the weakest, T, as unity, we obtain the 
 following relative values. 
 
 TABLE IV. 
 
 The figures show the relative activities of the representative samples 
 of infundibular extract from five different makers. 
 
 T = 1 
 S = 11 
 R = 25 
 Q = 45 
 P = 80 
 
 (5) The Comparative Pressor Activities of the above Samples. 
 
 As already mentioned, we have not succeeded in obtaining results 
 of the same quantitative accuracy with the test on the blood 
 pressure as with that on the isolated uterus. Hamilton carries out 
 the blood-pressure test on the dog under chloretone. We have used
 
 48 
 
 the cat, pithed under preliminary anaesthesia with ether, and then 
 kept under artificial respiration with pure air. We regard the 
 results as sufficiently accurate to detect only the occurrence of any 
 wide disproportion in the extent to which the pressor and oxytocic 
 principles have been preserved. 
 
 1. Comparison of the fresh extract F 2 with extract A, made from 
 an old sample of dried infundibulum. The test on the isolated 
 uterus has shown that F 2 = 3 A. On the blood pressure equal rises 
 were obtained with 0-5 c.cm. F 2 (0-1 per cent, dry gland) and 0-3 c.cm. 
 A (0-5 per cent.) 
 
 5 F 2 = 15 A. 
 or F 2 = 3 A. 
 
 2. Comparison of samples P and Q. (By uterus test, 
 
 P : Q : : 80 : 45). 
 
 For the blood-pressure test both extracts were diluted 16 times. 
 0-25 c.cm. Q < 0-25 c.cm. P. 
 
 No exact match was obtained, but P was apparently about twice 
 as strong as Q a result which is not discordant with that on the 
 uterus. 
 
 3. Comparison of samples P and E. 
 At equal dilutions (8 times) 
 
 0-25 c.cm. P > 1-0 c.cm. E. 
 or P > 4 E. 
 
 This is not in full accord with the result of the tests of the uterus, 
 which gave P = 3-2 E. 
 
 A second test on the blood pressure gave, with equal dilutions 
 (16 times) 
 
 0-75 c.cm. P = 2-0 c.cm. E. 
 P = 2-66 E. 
 
 So that the two tests on the blood pressure give values for the ratio 
 P : E which lie on either side of that given by the tests on the uterus. 
 
 4. Comparison of samples P and S. 
 At equal dilutions 
 
 0-25 c.cm. P > 0-7 c.cm. S 
 and, later, 
 
 0-6 c.cm. P < 1-6 c.cm. S. 
 
 i. e., 0-25 c.cm. P first produces a somewhat larger effect than 0-7 
 c.crn. S and later a somewhat smaller one than 0-66 c.cm. S. 
 
 If we take 0-7 c.cm. S as the approximate equivalent of 0-25 c.cm.P, 
 
 P = 3-5 S. 
 
 This is a clearly different ratio from that obtained from the test on 
 the uterus, which gave 
 
 P:S::80:11. 
 
 5. Preparation T. This, which had a very low degree of activity 
 on the uterus, had no pressor action at all. Its effect on the blood 
 pressure, in any dose, was purely depressant.
 
 49 
 
 Since preparation T has no pressor action, we cannot, as in the 
 case of the activities on the uterus, take it as unity. We can, how- 
 ever, make a rough comparison of the relative values obtained by 
 the two methods, if we equate the pressor action of the strongest 
 preparation P with its oxytocic activity, calling both 80. (Table V). 
 
 TABLE V. 
 
 Preparation. Relative oxytocic Relative pressor 
 
 (Manufacturers' samples), activities, taking activities, talcing 
 
 T = 1. P = 80. 
 
 P 80 80 
 
 Q 45 circa 40 
 
 R 25 15-30 
 
 S 11 circa 23 
 
 T 1 
 
 VIII. CONCLUDING REMARKS. 
 
 The figures given above speak for themselves. The infundibular 
 extract is a remedy which is at once valuable and dangerous, and 
 the fact that such variations can occur in the activity of prepara- 
 tions, habitually regarded by the medical profession as equivalent, 
 is deplorable. We have purposely abstained from giving the names 
 of the manufacturers concerned, or details which would enable their 
 products to be identified from our descriptions. We have adopted 
 this policy for two reasons. In the first place we do not take the 
 view that the manufacturers are chiefly to blame for the position 
 which we have displayed. No official guidance has been given to 
 them in this country as to the strength of extract most suitable 
 for therapeutic use, and no standard of activity has been available, 
 though all the extracts we have examined were described as ' physio- 
 logically standardized '. We are satisfied that in all cases a con- 
 scientious attempt has been made to control the activity by a physio- 
 logical test ; we gather that in the cases of two members of the 
 above series, the testing was not carried out by the manufacturers' 
 own staff, but by external and presumably independent authorities. 
 We thought it our duty to draw the maker's attention to the quality 
 of the preparation which we have called T, and have since received 
 from the same source preparations corresponding sufficiently well in 
 activity with others, such as B, having the same theoretical strength. 
 
 In the second place, past experience seems to indicate that the 
 publication of the names of makers, in connexion with figures 
 showing the activities of their preparations, may result in adver- 
 tising claims which are not truly justifiable. We are by no means 
 convinced that the sample of P, in the above list, is a better pre- 
 paration than that of E. The proportion of available activity 
 extracted in its manufacture is but little greater ; its much greater 
 activity is due to the fact that nearly three times as much gland is 
 represented in the same volume. To the practitioner accustomed 
 to, E, P would be dangerously strong, while one accustomed to P 
 would find E unduly weak ; but the manufacturer would not, in 
 either case, be entitled to claim superiority for his product. 
 
 The position revealed by Table V is not only deplorable ; we 
 are convinced that it is quite unnecessary. It will be clear, from
 
 50 
 
 what has been already said, that two different factors are involved 
 in the variation. (1) The proportion of gland substance, of which 
 the activity is supposed to be represented, is different in the case of 
 different preparations. (2) The actual yield of activity, obtained 
 from the same weight of gland substance, is widely different in the 
 case of different preparations. We may take the two points separ- 
 ately. 
 
 (1) To secure uniformity of theoretical strength, nothing more is 
 needed than the adoption of an agreed formula. For reasons already 
 given, we do not think that the need is properly met by an indication, 
 such as most manufacturers formerly gave, of the amount of gland 
 substance represented in a given volume of each preparation. The 
 medical man, in the hurry of an emergency, will not stop to read 
 labels and make calculations. Whatever the preparation available, 
 he will give the dose of ' pituitary extract ' which he has previously 
 found effective. The fact that several manufacturers have now 
 abandoned the custom of stating the supposed strength of their 
 extracts, suggests that they have learned that it has no value as 
 a guide to dosage. The omission, however, makes the position 
 perfectly clear. We may have two manufacturers issuing, -with the 
 same indications and for the same purposes, preparations of which 
 one is theoretically twice as strong as the other ; one of them will 
 have the advantage of being able to make twice as much extract 
 as the other from the same quantity of gland, while the other will, 
 perhaps, find grounds for a claim that his preparation has ' unique ' 
 or ' exceptional ' potency a claim which ought properly to be 
 regarded as a severe condemnation of his product. What is wanted 
 is not competition to produce the greatest possible activity in 
 proprietary preparations, but conformity of all makers to a common 
 standard. By whomever it is prepared, the pituitary extract is, 
 essentially, a sterilized watery decoction of the infundibular substance, 
 and we can see no proper obstacle to agreement on a uniform 
 theoretical strength for this decoction. 
 
 The United States Pharmacopoeia has officially settled this 
 theoretical strength at 10 per cent, of the fresh infundibular substance. 
 The British Pharmacopoeia has not yet recognized the existence of 
 this potent remedy. We strongly recommend the general adoption, 
 by British manufacturers, of the theoretical strength laid down by 
 the United States Pharmacopoeia, so that the practitioner may be 
 justified in his usual assumption, that the dose contained in the same 
 volume is, at least, supposed to be the same, whatever preparation 
 he uses. 
 
 (2) If this recommendation is adopted, there is further needed 
 a physiological assay, to ensure that the proportion of activity 
 extracted by different makers from the standard proportion of gland 
 shall be reasonably uniform. The United States Pharmacopoeia 
 has attempted to meet this need also. Unfortunately, as we have 
 shown above, it has prescribed a standard which, so far as it can be 
 applied at all, would result in the production of an extract having 
 only a small fraction of the activity present in a 10 per cent, decoction 
 of fresh gland substance, or of the equivalent of substance carefully 
 dried when fresh. If the extract is defined as a 10 per cent, extract,
 
 51 
 
 the standard of activity demanded should be that obtained in such 
 an extract by the manufacturer who uses sound material and proper 
 skill. In proposing as the standard of reference our ' Standard fresh 
 extract ', prepared on a small scale, we do not suggest that prepara- 
 tions nearly approaching it in activity, in corresponding dilutions, 
 could be prepared on the manufacturing scale with any regularity. 
 We have clear evidence to the contrary ; and if they cannot be 
 produced with regularity, they should not, if occasionally produced, 
 be placed on the market without dilution. Our measurements seem 
 to indicate 60 per cent, of the activity of our Standard extract, as 
 a level which should be without difficulty attainable by any manu- 
 facturer who has the necessary equipment and staff for the careful 
 extraction of perfectly fresh material, or of material which has been 
 dried at low temperature when perfectly fresh, and used without 
 unduly long storage. A 10 per cent, extract w T ould need four-fold 
 dilution to bring it to the same theoretical concentration as our 
 ' Standard ' 2-5 per cent, extract of fresh gland substance. We 
 suggest that, at corresponding dilutions, no commercial extract 
 should have less than 55 per cent., or more than 65 per cent., of the 
 activity of our ' Standard ' extract on the isolated guinea-pig uterus. 
 If the activity is outside these limits, we recommend that it should 
 be adjusted by dilution or blending to the 60 per cent, level of 
 activity. 
 
 A further test on the blood pressure of the cat or dog is desirable, 
 to ensure that the pressor principle has not been preferentially 
 destroyed by some accident of preparation ; but we cannot, from 
 our own experience, recommend this test as a basis for quantitative 
 adjustment. 
 
 With regard to the keeping qualities of the Standard extract, 
 it will be clear, from data given above, that we found its activity 
 not perceptibly diminished daring a fortnight, or even longer, in the 
 refrigerating chamber, after approximate sterilization in test-tubes. 
 Our recommendation, however, is that the Standard should be 
 freshly prepared as often as required. We do not assume that it 
 will be necessary to make a new batch of the Standard for the assay 
 of every batch of the commercial extract. For limited periods we 
 believe that a comparison of each batch with its accurately assayed 
 predecessor should suffice for the maintenance of uniformity without 
 significant error. On the other hand, it is evident that such a process 
 cannot be indefinitely repeated, without ultimate danger of serious 
 departure from the original standard of activity, in one direction or 
 the other. For this reason it is essential that there should be an 
 independent Standard, which can be reproduced at any time, and 
 used as a check against the development of different standards by 
 the different makers, which the use of the batch-to-batch method 
 alone would inevitably entail. 
 
 If the procedure which we have indicated is conscientiously 
 followed, we believe that there should be no real difficulty in the 
 maintenance of a practical uniformity among the pituitary extracts 
 produced by all manufacturers who have the requisite equipment 
 and staff. When once that is secured, it will be possible to deal 
 with the urgent question of therapeutic dosage. From the first
 
 52 
 
 introduction of the extract into practice the information on this 
 important point has been of the most unsatisfactory kind. The 
 tendency of the practitioner, perhaps naturally, is to inject the 
 whole contents of an ampoule, in any case in which the administra- 
 tion of the pituitary extract is indicated. There is an obvious 
 possibility that a dose which is safe and necessary in one condition 
 may be extremely dangerous in another ; but it is useless even to 
 discuss the question of dosage, when a dose, apparently the same as 
 another, may be anything up to 80 times as powerful. 
 
 We understand that a committee appointed by the Obstetrical 
 Section of the Royal Society of Medicine, with the co-operation of 
 the Medical Research Council, is conducting a thorough investigation 
 into this question of the doses appropriate to different conditions. 
 It will be truly lamentable if the results of this important inquiry, 
 when in due course they are published, should be deprived of all 
 accurate meaning by the failure of the manufacturers to bring all 
 the makes of pituitary extract to such a uniform standard of activity 
 as, without serious trouble, could be ensured. 
 
 We wish to thank our colleague, Dr. H. W. Dudley, who has 
 prepared for us the majority of the pituitary extracts used in the 
 above investigation, and also Miss F. M. Durham, who has carried 
 out several of the tests. 
 
 IX. REFERENCES. 
 
 DALE, H. H., and LAIDLAW, P. P. (1912). A method of standardizing pituitary 
 
 (infundibular) extracts. J. Pharmacol. d: Exper. Therap., 4, 75. 
 DUDLEY, H. W. (1919). Some observations on the active principles of the pituitary 
 
 gland. J. Pharmacol. & Exper. Therap., 14, 295. 
 DUDLEY, H. W. (1922). In the press. 
 EVANS, C. L. (1919). A simple frontal writing point. J. Physiol. 53, Proc. Physiol. 
 
 Soc., Ixiii. 
 FENCER, F. (1916). The composition and physiological activity of the pituitary 
 
 body. J. Biol. Chem., 25, 417. 
 
 HAMILTON, H. C. (1916). Pituitary Standardization. J. Lab. <t- Clin. M., 2, 120. 
 Ho WELL, W. H. (1898). The physiological effects of extracts of the hypophysis 
 
 cerebri and infundibular body. J. Exper. M., 3, 245. 
 KOCHMANN, M. (1921). Zur Wertbestimmung der Hypophysenpraparate und anderer 
 
 Wehenmittel. Ztschr. f. physlol. Chem., 115, 305. 
 ROTH, G. B. (1914). Pituitary Standardization. Public Health Service, Hygienic 
 
 Lab. Bull. No. 100. 
 SPAETH, R. A. (1918 a). The standardization of pituitary extracts. J. Pharmacol. 
 
 <L- Exper. Therap., 11, 209. 
 (1918b). The standardization of pituitary extracts. Public Health Service, 
 
 Hygienic Lab. Bull. No. 115. 
 TREKDELENBURG, P., and BORGMANN, E. (1920). Titrierung von Hypophysenextrakten 
 
 am ausgeschnittenen Uterus. Biochem. Ztschr., 106, 239. 
 
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 HUMPHREY MILFORD 
 OXFORD UNIVERSITY PRESS 
 
 LONDON EDINBURGH GLASGOW COPENHAGEN 
 
 NEW YORK TORONTO MELBOURNE CAPE TOWN 
 
 BOMBAY CALCUTTA MADRAS SHANGHAI
 
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