Using this assay and the parathyroidectomized rat plasma calcium assay, a

Transcription

1 79 J. Physiol. (I955) I30, A MOUSE URINE PHOSPHATE ASSAY FOR PARATHYROID HORMONE, WITH CERTAIN APPLICATIONS By BERYL M. A. DAVIES,*t A. H. GORDON AND MARJORIE V. MUSSETT From the National Institute for Medical Research, Mill Hill, London, N. W. 7 (Received 28 February 1955) Since the parathyroid hormone has been shown to act directly on the bones to cause the liberation of bone salts (Barnicot, 1948) and also to cause inhibition of phosphate reabsorption by the kidney tubules (Brull, 1936; Harrison & Harrison, 1941; Cargill & Witham, 1949; Kleeman & Cooke, 1951; Jacobs & Verbanck, 1953) and since it has been claimed that these two activities of the hormone are chemically distinguishable (Stewart & Bowen, 1952; Davies & Gordon, 1953a), it would be of interest to have a satisfactory assay based on the renal phosphate action of the parathyroid hormone to supplement the existing calcium assays (dog serum calcium assay, Collip & Clark, 1925a; parathyroidectomized rat plasma calcium assay, Davies, Gordon & Mussett, 1954). Earlier attempts to devise such assays have been reported by Tepperman, L'Heureux & Wilhelmi (1947), who measured the fall in serum inorganic phosphate in normal rats, and by Davies & Gordon (1953 b), using changes in plasma or urine phosphate in parathyroidectomized rats. None of these assays was very successful. The present paper describes an assay using about 200 saline-loaded normal mice, based on the increase in the rate ofurine phosphate excretion during 31 hr after the injection of parathyroid hormone. A correction for dependence of response on urine volume is introduced. Using this assay and the parathyroidectomized rat plasma calcium assay, a comparison has been made of the ratio of renal phosphate activity/plasma calcium activity in parathyroid extracts made according to Collp & Clark (1925b) and according to L'Heureux, Tepperman & Wilhelmi (1947). The significance of these ratios in relation to that for Parathormone (Eli Lilly Co) is discussed. * This work formed part ofa thesis accepted for the degree ofph.d., ofthe University oflondon. t Present address: Radioisotopes Unit, Postgraduate Medical School, Ducane Road, W. 12.

2 80 BERYL M. A. DAVIES AND OTHERS Mice METHODS g albino mice were used. Mice below 15 g weight proved less sensitive to injected hormone and older mice had too high a control urine phosphate excretion to be useful for this work. The animals were of the 'P' strain and were bred and maintained at the National Institute for Medical Research, Mill Hill, London. They were kept in a thermostatically controlled animal house, temperature F. The diet used was a M.R.C. cube diet, no. 41 (Bruce & Parkes, 1949). The mice received food and water ad lib. Although sex appeared to cause no difference in the phosphate responses obtained, either all male or all female animals were used in a single assay. Mice could be used for more than one assay (see later). Hydration of mice and urine collection. It was necessary to see that the mice received fresh drinking water daily to ensure they were in a state of adequate hydration prior to use. 1 ml. of 0.9% NaCl at 380 C per 5 g body weight was also given intraperitoneally to each mouse at the commencement of the experiment. A 10 ml. syringe, fitted with a tap for suction filling was used. Water, 1.36% NaCl or 1.27% sodium sulphate were not more efficient diuretics than 0.9% NaCl. Urine collection, from groups of sixteen mice, was started 15 min after hormone administration and was continued for 3i hr. This interval before the start of the collection period allowed any leakage of intraperitoneal saline to stop. The amount of preformed urine included in the sample was probably small, as the mice often urinated on or soon after being handled. Aluminium metabolism cages, 9 in. by 8 in. were used. These had i in mesh floors and rested on aluminium funnels with 1 in. wide necks. The urine was separated from the faeces by the use ofa pear-shaped bulb sus. pended over a collecting cylinder. No food was given in the metabolism cages, but water bottles were provided. Glass traps prevented any leakage of water into the urine samples. Hormone Standard hormone was 'Parathormone' supplied by the Eli Lilly Co. Hormone doses are expressed in U.S.P. units (see also p. 86). Assays were carried out on HCl-acetone preparations of ox parathyroid glands, made according to L'Heureux et al. (1947), and on acetic acid extracts made from acetone powder of the glands (Davies & Gordon, 1953a). All hormone injections were given subcutaneously, immediately after the administration of the intraperitoneal saline. Pho8phate estimations Urine inorganic phosphorus was estimated by the method of Allen (1940) and 20,1O. samples delivered from a semi-automatic constriction pipette as described by Levy (1936). EXPERIMENTAL Selection of the increase in rate of urine phosphate excretion during 3j hr as a measurement of the activity of injected parathyroid hormone In preliminary work, consecutive 7 ml. samples were taken from groups of twelve mice per metabolism cage and sample times were recorded. These experiments showed that a hr collection period was necessary to produce a satisfactory urine volume and to obtain an adequate similarity in urine volume from all cages. The routine procedure adopted was to obtain 3-5 hr samples from sixteen mice per metabolism cage, and to measure the rate of urine phosphorus excretion. The number of mice was increased from twelve to sixteen in an attempt to diminish the effect of inaccuracies due to incomplete urine collection from the metabolism cages, and to reduce the effect of animal variation.

3 PHOSPHATE ASSAY FOR PARATHYROID HORMONE 81 Dependence of response on urine volume In preliminary work it became apparent that, at any one dose level, when the volume of the urine sample was large the rate of phosphorus excretion was large. To get more precise information about this positive correlation between the mg P excreted per hour and volume, a series of experiments were carried out in which a total of about 550 mice per dose level were used. As urine was collected from sixteen mice per metabolism cage this yielded an average of thirty-five values for the response to one specific amount of hormone. Doses of 0, 1P0 and 3 0 units of Parathormone were used. The same animals never received the same dose more than once. From this work it became apparent that there was a high positive correlation between mg P/hr (h) and volume at each dose level (Fig. 2, p. 92). Since this correlation existed it seemed possible that variations of volume between different cages, or those due to different doses of hormone, might unduly affect the assay results. Therefore, two other metameters, apart from h, were considered. One of these was the direct measurement of mg P/ml., which obviously takes some account of volume, but is not independent of it. There was found to be a negative correlation between mg P/ml. and volume (i.e. high phosphate content occurred with low urine volume). The third expression H, is a value of mg P/hr corrected for dependence on urine volume and was shown to be uncorrelated with the latter. The derivation of H is given in an appendix, together with an explanation of why this or some similar correction is necessary. Log dose-response relationship Four curves relating response with log. dose have been constructed, using doses of 0.1, 0 33, 1P0 and 3 0 units of parathormone. Three cages of mice were used per dose level per day. Two experiments were carried out on one day only, and in the other two a cross-over test was carried out on 2 days, so that those mice which had received a high dose on the first day received a low dose on the second day and vice versa. The four log. dose-response lines were each subjected to statistical analysis three times, using h, mg P/ml. and H as response. Details of the two crossover experiments are given in Table 1, but the following points relate to all four lines: (i) There were no significant departures from linearity, whichever measure of response was used (column 5). (ii) The high values for the linearity variance ratio indicated a significant slope in each case (column 4). (iii) The slope of the log. dose-response line varied in each experiment (from 1*34 to 2*58 when H was used as response) (column 1). This indicated the 6 PHYSIO. CXXX

4 82 BERYL M. A. DAVIES AND OTHERS importance of adopting an assay design in which animals treated with standard hormone would be incorporated. (iv) The use of H gave the highest slopes associated with the smallest error variance, as shown by the small values of A =4(error variance). slope (column 2). In two cases the use of h led to the largest values of A and in the other two mg P/ml. gave the highest A. We should therefore expect that in the interpretation of an assay H would give the smallest limits of error to the estimate of potency. TABLE 1. Comparison of the results obtained when three different expressions for response are used in the statistical analysis of log. dose-response lines Variance ratios Departures Within dose from high/low Linearity linearity Date of Slope A (4, 4) D.F. (1, 16) D.F. (2, 16) D.F. experiment Response (1) (2) (3) (4) (5) May 1953 h <1 mg/ml * H '17 39* Oct h <1 mg/ml * H * * * Variance ratio is significant at 5% level when greater than 6-4. The effect of four doses of hormone has been measured on each date. Three cages of mice were used per dose level on each of 2 days. (v) The variance between cages at each dose level was calculated, and was found to increase with increasing dose. The ratio of variances within the highest and the lowest dose (3.0 and 0.1 units) has been taken as the criterion of this increase (column 3). This variance ratio was never significant when h was used as response, but was significant once when H and three times when mg P/ml. was used. A typical log. dose response line is illustrated in Fig. 1. The effect. of repeated Parathormone injections on the urine phosphate response A set of 192 mice, divided into twelve groups, were used in a series of experiments to see whether repeated injections of 10 unit of hormone per mouse would lead to a decrease in the animal's sensitivity. Half the number of animals were used throughout the experiments as controls. The other ninety-six animals received a total of eight injections of 1F0 U.S.P. unit of Parathormone over a period of 7 weeks. The interval between injections was from 2 to 7 days. In each experiment, the rate of phosphorus excretion was measured in both the hormone-treated and the control mice and the mean values so obtained expressed as a ratio (i.e. mg P/hr/sixteen hormone-injected mice/mg P/hr/ sixteen control mice). This ratio was calculated once using the values before

5 PHOSPHATE ASSAY FOR PARATHYROID HORMONE 83 correction for dependence of rate of phosphorus excretion on urine volume (h values) and again using values corrected for the effect of urine volume (H values). In neither case did the ratio alter significantly over the whole period. Using the corrected form for the response, the ratio ranged from 14 to 2-0, but there was no tendency for the ratio to alter consistently with time. Thus, it can 5-0 H 0 45 h x _ O 4-0/ 0. v XI ~~~~~~~~~x Log. dose in 0-1 U.S.P. unit Fig. 1. Log. dose-response curve and effect of correction for urine volume. Each point is an average value from six cages of mice. sixteen animals/cage. x, Average mg P/hr (h) at each dose level; 0, average mg P/hr corrected for volume (H) at each dose level. be concluded that repeated dosing with 1-0 unit of hormone does not affect the responsiveness of the mice to injected parathyroid hormone, at least when an interval of a few days is left between each dose. It is, however, noticeable that for both the control and hormone treated groups, volume, observed (h) and corrected (H) rate of phosphorus excretion all decreased slightly but definitely as the experiment proceeded. Hence the routine procedure adopted was that one set of mice was never used for more than one month or for more than four assays or experiments, in order to 6-2

6 84 BERYL M. A. DAVIES AND OTHERS ensure that different assays were not carried out under unduly different conditions. Design of assay A twin cross-over assay (Smith, Marks, Fieller & Broom, 1944) was adopted, three cages each containing sixteen mice were used per dose and two dose levels were employed. Within one assay the ratios between doses were the same for standard and unknown hormone preparations. The value and the ratio of the standard doses were varied in different assays. The assay was commenced between 9.0 a.m. and 10.0 a.m. and was carried out in two parts, an interval of not less than 2 and not more than 7 days being left between the two halves. This interval was used in order that the second injection of hormone would not be given to animals with an already increased urine phosphate excretion. Mice which had received standard hormone for their first injection received the unknown preparation for their second injection and vice versa, and in addition the dose levels were also reversed in the second part of the assay. The responses obtained from each individual cage of mice in a 3.5 hr period were expressed both as mg P/ml. and as H values, i.e. as mg P/hr/ sixteen mice, corrected for dependence on urine volume. The potency was calculated, using each of these expressions as response (Tables 3 and 4). In each individual assay the dose ratio was selected on the basis of the results obtained from a preliminary test on the unknown preparation. If the extract appeared not to be very active, the ratio chosen was small to ensure both that the low dose would still produce a measurable response and that an excessive amount of material would not be required for the high dose. Investigation of the validity of the mouse urine phosphate assay method In order to test the validity of the assay method and to decide which of the three metameters considered gave the most accurate estimate of potency, four 'known-unknown' assays were carried out. The 'unknown preparations' were in fact dilutions of Parathormone, the dilutions being carried out by independent workers, who chose the concentrations and only revealed them after the completion of the experiment. The only condition laid down was that the dilution must be within such a range of Parathormone concentrations that measurable responses would occur. This unknown dilution of Parathormone was regarded as containing x units/0*2 ml. Doses of x and x/3 units of 'unknown preparation' were assayed against doses of 1F5 and 0.5 units of Parathormone (standard hormone). Results were expressed as mg P/ml. and as h and H values and potencies for the 'unknown' solutions calculated. From these potencies, values for x could be obtained. Comparisons of experimentally determined values of x together with the 5 % limits of error are given in Table 2.

7 PHOSPHATE ASSAY FOR PARATHYROID HORMONE 85 It will be seen that the true values of x lay well within the limits of error of the estimated values of x whichever method of calculation was used. These limits were, however, so wide when uncorrected mg P/hr was used as response, that no confidence could be placed on such assay results. The other two methods both gave values of x which corresponded well with the true values and had reasonable limits of error. The use of H generally gave the closest limits as was suggested by the values of A calculated from the log. dose-response lines. TABLE 2. Comparison of the results obtained when three different expressions for response are used to estimate the potencies of solutions of Parathormone which have been diluted to an unrevealed value (x units/02 ml.) (Experimental details are given in the text) mg P/hr (h) mg P/ml. Corrected mg P/hr (H) Actual,_ A _--- A- A value 5% limits of 5% limits of 5% limits of Assay of x x error x error x error * ,129 1* * x 10-1L Invalid-slope not significant * * Specificity of the urine phosphate response In order to test the specificity of the mouse urine phosphate response, a comparison was made between the action of acetic acid extracts of ox parathyroid and salivary (parotid) glands made by the same method (Davies & Gordon, 1953 a). The salivary gland extract, when injected on 2 days in a cross-over experiment at a dose level of 6 mg/mouse into a total of twelve cages of mice, failed to cause any increase in urine-phosphate excretion (H values). On the other hand, three acetic acid extracts of different batches of ox parathyroid glands, assayed at levels of 0-25 and 0 75 mg/mouse, were found to have an average activity of 42 units per 100 g of fresh glands, assuming that 1 ml. of Parathormone (Eli Lilly Co) contains 100 units of phosphate activity. Results of mouse urine phosphate assays The results of a representative assay are shown in Table 3. A comparison of the potencies and limits of error, obtained from seven assays, and using mg P/ml. and H as response is given in Table 4. It is interesting to note that the two measures of response led to potencies which were very similar, and certainly not significantly different. In most cases the limits of error were noticeably narrower when H was used as response. In the statistical analysis of each assay a t test was done between the slopes of the log. dose-response lines for standard and test substances. In assays III and IV this value of t was significant when mg P/ml. was used, and in all except assay V the value of t was reduced by the use of H.

8 86 BERYL M. A. DAVIES AND OTHERS TABLE 3. Mouse urine phosphate assay (no. IV) of an ox parathyroid extract (OPG 4) D mg of preparation OPG 4 were equivalent to 0-24 mg dry wt. Sixteen mice were used per metabolism cage Rate of urine phosphorus excretion corrected for dependence on urine volume (H values) Metabolism (Corrected cage no. mgp/hr/16 mice) Observed rate of urine phosphorus excretion Vol. (h values) I: )ose per mouse (ml.) (mgp/hr/16 mice) 1st half of assay ol )*2 U.S.P. unit;l :Parathormone l 0 U.S.P. unit (28 6*0 olf Parathormone *7 D mg OPG *8 5D mg OPG nd half of assay t; D mg OPG D mg OPG *9 4.5 (25 2 5*5 L*0 U.S.P. unit oif Parathormone )-2 U.S.P. unit ( oif Parathormone * *93 7*15 6* * *50 5* *68 4* Average H values for the complete assay: 0-2 units of Parathormone, H=4-60, D mg OPG 4, H=4 59; 1.0 unit of Parathormone, H=5.93, 5D mg OPG 4, R=6-81. Results of statistical analysis: potency = ; U.S.P. units/mg of OPG 4 = 1-23; limits of error calculated at the 5% level and expressed as a percentage of the potency = %. Comparison of the ratio of renal phosphate activity/plasma calcium activity in various types of parathyroid extracts Using Parathormone as the standard hormone, both a mouse urine phosphate assay and a parathyroidectomized rat plasma calcium assay (Davies et al. 1954) were carried out on a hydrochloric acid preparation of ox parathyroid gland prepared according to Collip & Clark (1925b) and on an acetone- HCl preparation made according to L'Heureux et al. (1947) in order that the ratio of renal phosphate activity/plasma calcium activity might be calculated. Parathormone is standardized before issue and its calcium activity is 100 U.S.P. units/ml. For the purposes of these experiments, in which one batch of Parathormone was used, 1 ml. of Parathormone was defined as giving 100 units of phosphate activity, i.e. phosphate activity was measured in terms of

9 PHOSPHATE ASSAY FOR PARATHYROID HORMONE 87 TABLE 4. Mouse urine phosphate assays of unknown parathyroid preparations Potency of Potency in Limits of error Assay Unknown unknown U.S.P. units/ expressed as % no. preparation Date of test preparation* mg. of potencyt Using mg P/ml. as response I OPG vii II OPG Viii F III OPG x $ 46* IV OPG xii : *1 V OPG ii * (test 1) VI OPG ii. 54 0*89 1*33 56*0-168*4 (test 2) VII OPG 6 8. iii *8-226*7 Using corrected mg P/hr (H) as response I OPG vii II OPG viii * III OPG x IV OPG xii. 53 1' V OPG ii * (test 1) VI OPG ii * (test 2) VII OPG 6 8. iii. 54 0* * Doses of standard and unknown preparation varied in different assays. t Limits were calculated at the 5% level. : These estimates are not valid, since the log. dose-response lines for standard and test showed a significant deviation from parallelism. The two estimates of potency obtained by assaying an extract (OPG 5) twice were not significantly different. U.S.P. units. Hence it was possible to obtain values for the phosphate and calcium activity of the tested extracts expressed in the same units and to compare the experimentally determined ratios with that characteristic for Parathormone which, as defined for this work, was 1.0. In the calculation of these ratios the phosphate response was measured as H. The results of this experiment are shown in Table 5. DISCUSSION As mentioned above, Tepperman et al. (1947) attempted to devise an assay for parathyroid hormone, based on the decreases in serum inorganic phosphate of normal rats caused by injected Parathormone. Difficulties encountered included variation in response between individual animals and between groups of rats used at different times. The sensitivity of the method was not great, 100 U.S.P. units being needed to produce a 21 % fall in serum phosphate in the most reactive set of rats. Further, Davies & Gordon (1953b), in a study of the influence of the hormone on the levels of plasma inorganic and urine phosphate of parathyroidectomized rats, found these animals to be very much more sensitive to sub-

10 88 BERYL M. A. DAVIES AND OTHERS cutaneous injections of Parathormone than normal rats. Thus, maximal urine or plasma responses could be obtained with only 2 or 3 units of Parathormone. However, the extremely wide animal and seasonal variation precluded the development of a satisfactory assay based on either of these effects. More recently, Kenny, Vine & Munson (1954) have described an assay, based on the increase in the six-hourly urine phosphate excretion of individual parathyroidectomized rats, but as yet no details of the design or accuracy of the assay have been published. TABLE 5. Comparison of the ratio of renal phosphate activity/plasma calcium activity in a commercial extract (Parathormone), a hydrochloric acid extract and an acetone-hydrochloric acid extract of ox parathyroid glands Both renal phosphate and plasma calcium activities were expressed as U.S.P. units/mg. Except where specified, mouse urine phosphate assays and parathyroidectomized rat plasma calcium assays were always employed; in them Parathormone was used as the standard hormone. Ratio of: Renal phosphate activity Extract Method of preparation Number of assays performed plasma calcium activity Commercial hormone Not known None: standardized before issue by dog 1.0 (Parathormone) serum calcium assay and contained 100 U.S.P. units/ml. For the purposes of these experiments, it was taken that its renal phosphate activity was 100 units/ml. A hydrochloric acid Collip & Clark (1925b) 2 phosphate assays, 1 calcium assay 3*1 extract (OPG 4) An acetone- L'Heureux, Wilhelmi 2 phosphate assays, 2 calcium assays 2'6 hydrochloric acid & Tepperman (1947) extract (OPG 2) The present work was based on the changes caused by parathyroid hormone in the urine phosphate excretion of normal mice. An investigation of these increases in phosphate excretion was only possible if adequate volumes of urine could be obtained during the short period in which the hormonal response developed. This difficulty was met by ensuring that the mice always received fresh drinking water, keeping them in a thermostatically controlled animal house and injecting them with large amounts of saline just prior to the hormone treatment. It was assumed that though the mouse, like the rat, would be relatively insensitive to parathyroid hormone on a weight basis, yet its small size might more than compensate for this disadvantage. In fact responses adequate as the basis of an assay method were obtained in normal mice by the use of similar small amounts of hormone as had previously been found to give maximal increases in the urine phosphate excretion of parathyroidectomized rats. Thus, the assay method was based on responses to small doses of hormone ( U.S.P. units) which caused the urine phosphate excretion of normal mice to increase by about 5-80 %. The variability of the responses was satisfactorily small when urine was collected from large groups of animals.

11 PHOSPHATE ASSAY FOR PARATHYROID HORMONE 89 In considering the effect of parathyroid hormone on urine phosphate excretion of mice it was necessary, as shown above, to consider the dependence of response on urine volume. The results of assaying unknown dilutions of Parathormone suggest that either the use of mg P/ml. or the expression H lead to valid estimates of potency but that uncorrected mg P/hr is not a useful measure. Theoretically it would seem that H is the better measure of response, since it is uncorrelated with volume. It has been shown that the use of H leads to narrower limits of error of the potency, to response lines which deviate less from parallelism and to a more constant within-dose variance than does the use of mg P/ml. Hence our routine calculations of potency have been based on H. TABLE 6. Comparison of phosphate and calcium assays for parathyroid hormone by various workers Limits of error Times expressed as Total no. of used in % of the Authors Type of assay animals the assay potency* CoUip & Clark (1925a); Dog serum calcium Bliss & Rose (1940) Biering (1950) Normal rat serum 20t calcium Davies, Gordon & Parathyroidectomized 18 or Mussett (1954) rat plasma calcium Davies, Gordon & Mouse urine phosphate 192 (12 cages) Mussett (present work) * Limits were calculated at the 5% level. t All on unknown preparation. The advantages of the mouse urine phosphate assay method are that it involves no elaborate techniques and that it is relatively cheap, since it is carried out with small animals which may be used in more than one assay. As shown in Table 6, the accuracy of the mouse assay method based on H is comparable with, though slightly less good, than that of the dog plasma calcium assay (Collip & Clark, 1925 a; Bliss & Rose, 1940), and rather similar to that of the parathyroidectomized rat plasma calcium assay (Davies et al. 1954). Our figures suggest that, if dose levels of an extract being tested were such as to give a potency of 1.0, the use of about twice as many cages of mice as are specified in the present assay (i.e. 2 x 192 mice) would give an accuracy as great as that of the dog serum calcium method. Since it takes into account the variations in the volumes of the urine samples, the present assay allows the activity of a parathyroid extract to be tested without the measurement of the hormonal action being obscured by the simultaneous action of any antidiuretic substance and probably of any pressor substance which might also be present in the extract unless these substances occur in very high concentration. As only urine samples with a volume range of ml. were obtained in the experiments with Parathormone the results

12 90 BERYL M. A. DAVIES AND OTHERS of which were used to derive the expression H for measuring response, extracts which are strongly antidiuretic and produce volumes less than 15 ml. cannot reasonably be tested using the present form of this assay. In practice, such strongly antidiuretic effects were rarely encountered at the hormone levels necessary to give useful urine phosphate responses. No assays were carried out using any dose level of an unknown preparation which, in preliminary testing had produced urine volumes 50 % lower than those of control urine samples. The mouse urine phosphate assay throws no light on the action ofthe injected hormone on the cells of the renal tubules: for this, clearance measurements would be necessary and these would be extremely difficult to obtain, especially if individual blood and urine samples had to be collected from large numbers of mice. The assay is also probably not sensitive enough to test the level of circulating parathyroid hormone in humans. The fact that parathyroid extracts prepared at this Institute and a commercial preparation have each been shown to have different hormonal activity when assayed by the mouse urine phosphate method than when assayed by the parathyroidectomized rat plasma calcium method confirms statements that parathyroid hormone has two distinct physiological activities, and that different preparations can possess different amounts of each. By means of the above pair of assays it should be possible to test the claim of Stewart & Bowen (1952) mentioned above to have obtained by formaldehyde treatment preparations of the hormone no longer able to cause increases in plasma calcium level while still retaining power to increase renal phosphate excretion. This has not yet been done, and meanwhile a preliminary report has appeared by Talmage, Buchanan & Kraintz (1953) that such material did not lead to either of the two effects. A possible explanation of this discrepancy is that Talmage et al., working with dogs, may have used too short a urine collection period (20 min) to allow for the complete development of the phosphate response after injection of a test substance. Some further work on the properties of diffusates of acetic acid extracts of parathyroid glands similar to those mentioned by Davies & Gordon (1953a) has recently been carried out. As will be described in another paper (Davies & Gordon, 1955) such solutions have been found to be active by the present assay and in the only case where, in addition, activity in respect to plasma calcium levels was tested, a positive result was obtained. This suggests that our earlier failure to obtain a calcium effect with rather similar material (Davie,s & Gordon, 1953 a) may have been due to the presence of unduly high levels of impurities. It was not possible to conclude from the results in Table 5 whether Colliptype preparations always contain the highest ratio of renal phosphate activity, since only a single Collip-type extract and a single L'Heureux-type extract

13 PHOSPHATE ASSAY FOR PARATHYROID HORMONE 91 were tested. However, the very fact that the ratios differed from that pertaining to Parathormone suggests the necessity of all extracts being routinely standardized for both phosphate and calcium activity before use. In this connexion, it is interesting to recall that Ellsworth & Howard in 1934 found that an injection of 20 U.S.P. units caused a larger urine phosphate response in the normal human than that obtained by some present-day workers who have repeated the experiment (Milne, 1951; Dent, 1953). A possible explanation is that Ellsworth and Howard's extract may have contained a higher proportion of renal phosphate factor, as the extracts used by all the workers had been standardized for calcium activity only. This same view has been put forward by Kenny et al. (1954) in a recently published note. Other work (Gordon, unpublished observations) has shown that the calcium activity of parathyroid hormone is unstable under unexpectedly mild conditions. It seems likely that modifications in the manufacturing conditions would all have been directed towards increasing the content of calcium activity. Thus, the present-day product with a higher calcium purity would have been extracted from a smaller quantity of ox glands and therefore might contain a smaller content of phosphate activity than did the commercial preparation used by Ellsworth & Howard (1934). SUMMARY 1. A two-day cross-over assay has been developed using 192 saline-loaded normal mice and based on the increase in urine phosphate excretion during the 31 hr after the injection of parathyroid hormone. 2. A correction is used which frees responses from their dependence on urine volume. This dependence was established by a series of preliminary experiments with Parathormone which showed: (i) At any one dose level, the larger the volume the greater was the rate of phosphate excretion. (ii) The magnitude of the correlation was greatest at the highest levels of urine phosphate. (iii) Mean volume decreased with increasing response. 3. The results of seven assays are reported. Using corrected responses the average limits of error were %. These are slightly wider than those reported by Bliss & Rose (1940) for the dog serum calcium assay (79-126%) and very similar to those obtained in the parathyroidectomized rat plasma calcium assay (64-158%) (Davies et al. 1954). 4. Using this mouse assay and the parathyroidectomized rat plasma calcium assay, the ratio of renal phosphate activity/calcium mobilizing activity has been determined for extracts made by different procedures. In each case the value of the ratio differed from that for Parathormone (commercial hormone). Consequently the necessity for the standardization of hormonal preparations in terms of both calcium and phosphate activities is suggested. The authors wish to express their thanks to Sir Charles Harington, F.R.S., for his interest in this work.

14 92 BERYL M. A. DAVIES AND OTHERS APPENDIX Correction of responme for variation in urine volume It has been stated earlier that a series of experiments were carried out with the object of obtaining more information about the dependence of mg P excreted per hour (h) on volume (v). From this work it became apparent that: (i) At any one dose level, there was a high correlation between response and urine volume; the larger the volume the greater was the rate of phosphorus excretion. (ii) The magnitude of this correlation was influenced by the actual value of the phosphorus excretion, being greater at the higher levels of urine phosphorus which were obtained in response to large doses of hormone. E8 0~ c6 0xl ol 4; --fp=ato@. 2) Of Urine volume (v) (mi.) Fig. 2. Correlation between rate of urine phosphorus exeretion and urine volume. Each line was constucted using values obtained from about 35 urine samples. x, Mean urine volume and mean response at each dose level (v), hi). (iii) Mean volume was found to decrease with increasing response. Thus, the mean volume was smallest at the highest dose level. These conclusions were confirmed by examination ofthe responses obtained in individual assays. These three statements mean that if volume is plotted as abscissa and rate of phosphorus excretion as ordinate, regression lines with positive slopes will be obtained at each dose level of Parathormone (including the control level), that the slope of the lines will vary (being greatest at highest dose level) and that for each line, the mean volume will lie at a different distance from the ordinate, the nearest being that for the highest dose level (Fig. 2).

15 PHOSPHATE ASSAY FOR PARATHYROID HORMONE 93 Since the slopes for the volume-response regression lines varied with different doses it was possible neither to eliminate the effect of varying volume by the use of co-variance nor to use some simple correction as in the parathyroidectomized rat plasma calcium assay (Davies et al. 1954), as this type of adjustment is also based on a common slope. As explained below, the equation adopted for calculating the corrected response was H= 2vh + 7 (25-v), where h is the mg P/hr/sixteen mice and v the urine volume for sixteen mice (one cage) and hi and i the corresponding mean values for one dose level. Derivation of H To overcome points (i) and (ii) it is necessary to develop a corrected response, which is uncorrelated with volume. Graphically, this means that the volume-response line must be rotated to a horizontal position about some point. The obvious point to choose is the mean (7i) as this is easily calculated, and is by definition a point on the regression. The rotation could be done exactly ifthe slope (b) was known, but to calculate b for every volumeresponse line would be tedious and also inaccurate, for in an assay there are only six points at each dose level. It was however noticed that the relationship b=2_7(1) closely approximated to the actual slopes found in the large-scale work at three dose levels. The correction is based on this empirical equation. The general equation for the slope of the volume-response regression is b= h-. (2) Eliminating b between (1) and (2) 2;5h (3) v +v (3 If there were no animal or other experimental variation, every response (h) measured at a particular dose level would be the same. The best estimate of this true value of h is i. Since it is impracticable to remove all types of variation, and only that due to differences in volume is dealt with here, the corrected values of h h hcor.v+v2; 2v3h(4 (4) will be distributed about hi just as the original h values were scattered about the volume response regression line. Fig. 3 shows the fitted regressions of h on v and h,,r. on v calculated from thirtyone responses to 1 unit and thirty-six responses to 3 units of Parathormone. Correlation between hcor. and volume is not significant in either case. The effect of this first part of the correction is to reduce greatly the within-dose variation without altering the level of response. In our experience the average hcor., at a particular dose level, has never differed more than 2% from the corresponding 7i. A second stage in the correction is required if responses to different doses are to be compared on a common volume basis (point (iii)). A volume of 25 ml. was chosen, and all hcor. values adjusted to it. This was about the average volume generally encountered, and by always adopting this value rather than by using the mean volume of all the urine samples in each separate experiment, the responses from different assays are directly comparable. This part of the correction can be explained graphically as moving the h,,r. regression from its position through (vh) through a vertical distance d until the mean corrected h coincides with a volume of 25 ml. It will be seen that d 0= 25 -v-~ to)

16 94 BERYL M. A. DAVIES AND OTHERS Combining (1) and (5) h d =- (25 -fv). The value d is constant for one dose level within one particular assay. Its object is to adjust each h value to a common volume of 25 ml. without affecting the scatter of individual values about their mean. The corrected responses are finally expressed as H = h.r + d, the effect of H can be seen by the thick dotted lines in Fig. 3. A comparison of h and corresponding H values for one assay is made in Table 3. The effect of using H in the interpretation of assays and log. dose response lines is illustrated in Tables 1, 2 and ~~~~~~~~~~~~~~~~~~~ (4 bo llll-l----.s~~~~.cr Urine volume (v) (ml.) Fig. 3. Effect of correction for urine volume on the correlation between response and volume. For definition of h, Co,r. and H see text. 0, Mean urine volume and mean response at each dose level (vi, Ei); x, average corrected response (H) at a common volume (25 ml.). REFERENCES ALLEN, R. J. L. (1940). The estimation of phosphorus. Biochvem. J. 54, BARIueCOT, N. A. (1948). The local action of the parathyroid and other tissues on bone in intracerebral grafts. J. Anawt., L,ond., 82, BIERING, A. (1950). Bio.As8ay of Parathyroid HormJone on Rat,s, p. 55. Aarhus: Universitetsforlaget. BuIss, C. I. & ROSE, C. L. (1940). The assay of parathyroid extract from the serum calcium of dogs. Amer. J. Hyg. 31A, BRUCE, H. M. & PARKES, A. 8. (1949). Feeding and breedinlg of laboratory animals. IX. A complete cubed diet for mice and rats. J. Hyg., Camb., 47, BRULL, L. (1936). M6canisme de l'action de la Parathormone sur l'elimination urinaire du phosphore. C.R. Soc. Biol., Paris, 122, CAGSILL, W. H. &; WITHAM, A. C. (1949). Parathormone and the tubular reabsorption of glucose and phosphate in man. Fed. Proc. 8, COLLI, J. B. & CLARK, E. P. (1925a). Further studies on the physiological action of a parathyroid hormone. J. biol. Cihem. 64,

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