International Standard Preparation Research Articles

Studies on the mechanism of ACTH-secretion with special reference to the bioassay of C.R.F. in vitro

The author has tried to establish a convenient and accurate bioassay method for the determination of C.R.F. (corticotrophin relasing factor) activity of the hypothalamic extract. The principle of the method lies in the combination of two in vitro techniques. In the first step three pituitary halves taken from the freshly sacrified female rats were incubated in vitro for the liberation of ACTH into the incubation medium. In the second step the expected ACTH secretion into the medium was determined by the in vitro assay technique of ACTH-like activity following the description of Tsuji and Yasui, which is characterised through its specificity, high sensitivity and reproductbility (Folia Endo-crinologica Japonica. 41 : 643, 1965).The method and the results obtained are summarized as follows : 1) As the first test, tissue for the liberation of ACTH three pituitary halves taken from three different rats were placed in two flasks and used in the form of pooled samples. One flask served as test and the other flask as the control. Preincubation of pituitary halves for 60 minutes brought about high sensitivity and well standardized reactivity of the pooled pituitary tissues. After preincubation for sixty minutes three halves of the above-described pituitary halves were placed in the fresh incubation medium after tho-rough washing. A standardized portion of the rat hypothalamus encircling the median eminence was cut out with a razor blade and crude extract was placed with cold galcial acetic acid following the description of Vernikos-Danellis. This extract (henceforth abr. M.M.E.) was added to the incubation medium, in which 3 halves of rat pituitary halves are placed. After final incubation for 60 minutes the expected ACTH secretion in the medium was determined after Tsuji-Yasui's bioassay method of ACTH. The characteris-tic of this last step lies in the use of pooled samples of four beef adrenocortical slices weigh-ing about 100 mg in total as test material. The two-step procedures made it possible to determine the C.R.F. activity of M.E.E. qualitatively and even semi-quantitatively. The sensitivity of the method for C.R.F. activity was in the level of M.E.E. prepared from one animal.2) At first the author has tried to verify the specifictiy of this assay technique through the exclusion of possible sources of error originating from this M.E.E.The following points were checked : i) No ACTH-like activity was observed in the M.E.E. ii) No ACTH destroying or potentiating effect was observed in the M.E.E. iii) C.R.F. contained in the M.E.E. was stable for heating up to 100°C for 30 minutes, henceforth all experiments were carried out using M.E.E. heated to 100°C for 30 minutes. This procedure excludes various enzymic actions. iv) No C.R.F. activity was shown in the crude extract of brain cortex. v) It was proved that the M.E.E. acts directly upon the incubated pituitary tissues and induces the release of ACTH. The simultaneous examination of ACTH content of the incubated pituitary tissues revealed the decrease of the ACTH content in the tissue.3) To verify the presence of ACTH itself in the incubation medium, in which pituitary tissues were placed, a 4 point assay was carried out with the incubation medium and ACTH (Third international standard preparation of ACTH). There was always a 'exact parallelism. In the following experiments for the assay of expected ACTH secretion in the incubation medium two control flasks with two concentrations of ACTH standard preparation, i.e, 10-2 and 10-3 mU/flask were incubated simultaneously for the guarantee of the ACTH assay.4) The specificity of this assay system for the determination of C.R.F. activity was clearly demonstrated in the comparative studies, in which crude extract from brain cortex, serotonin, pitressin, histamine, acetylcholin and bradykinin were used as the test material.

Immunoassay of serum human chorionic gonadotropin by quantitative complement fixation and its comparison with the Delfs bioassay and two International Standard preparations

A complement fixation radioimmunoassay is described which has the advantage of giving direct quantitation of international units (IU) of human chorionic gonadotropin (hCG) in a given serum sample. With this method unlike other immunoreactive techniques the complement can be fixed quantitatively with the number of IUs of hCG directly related to the number of complement units fixed. None of 10 hCG negative specimens studied to determine effects of dilutaion and anticoagulated vs. clotted blood gave measurable amounts of hCG. Progressive dilutions and presence of anticoagulant gave the same results as clotted blood in the same dilutions. Sera from hysterectomized and nonpregnant patients fixed the same amount of antibody (O-2 U) as controls containing no antibody. Thus no measurable amounts of hormone within assay limits (.5 IU of hCG/ml) for nonpregnant serum are to be found. Quantitative measure of impurities cross-reactions and artifacts which may produce pro- or anticomplementary activity are inherent in this technique. Compared with other complement fixation methods this does not require back titration of antibody to relate it to the International Standard. The reagents necessary are simple and commercially available and the method seems practical for any general hospital laboratory. This pilot study indicated that values obtained by this immunoassay compare exactly with those from a Delf bioassay method.

A METHOD FOR THE DETERMINATION OF HUMAN CHORIONIC GONADOTROPHIN IN URINE EXTRACTS.

A method is described for the preparation in rabbits of a specific antiserum to human chorionic gonadotrophin. The ability of this antiserum to fix complement in the presence of urinary human chorionic gonadotrophin is demonstrated. The standardization of antiserum against the international standard preparation of human chorionic gonadotrophin, the determination of antiserum specificity using appropriate controls, and the value of antiserum in the diagnosis of pregnancy and conditions associated with high urinary levels of human chorionic gonadotrophin is emphasized.By the complement-fixation test it is possible to detect a minimum of 1.5 international units of human chorionic gonadotrophin contained in 0.1 ml. of a 1 in 2 dilution of urine extract. In the Hogben pregnancy test, using 2.5 ml. of urine extract per animal, a minimum concentration of 70 international units of human chorionic gonadotrophin is required to give a positive response. The ease and economy of the complement-fixation test are described and results are compared with those of the Hogben test.

FATE OF SYNTHETIC OXYTOCIN ANALOGUES IN THE RAT

The disappearance of phe(3)-oxytocin, val(3)-oxytocin and oxytocin from the circulation of male rats was shown to be due to qualitatively similar mechanisms, that is, it depends on uptake in the kidneys and organs of the splanchnic vascular area. However, compared to phe(3)-oxytocin and oxytocin (whose half-lives were essentially similar) val(3)-oxytocin took twice as long to reach half its initial blood concentration. In lactating rats the mammary glands probably participated in the uptake of phe(3)-oxytocin, but the rate of disappearance of val(3)-oxytocin was not different from that in non-lactating animals. In male nephrectomized rats without splanchnic circulation, phe(3)-oxytocin, unlike val(3)-oxytocin, was quickly distributed in a volume equal to two-thirds of the total body water. Using oxytocin as the standard, val(3)-oxytocin and phe(3)-oxytocin were more potent when assayed on a superfused uterus or on a rat uterus in vivo than when assayed by the pharmacopoeial method (1958) on the isolated uterus in an organ bath. The difficulties of assaying oxytocin analogues against oxytocin (or the international standard preparation) are discussed.

THE SUBCUTANEOUS ASSAY OF CORTICOTROPHIN A

The subcutaneous method of assay of corticotrophin (U. S. P. XV, 1955) is used instead of the adrenal ascorbic acid depletion method (Sayers et al., 1948), when corticotrophin is to be given extravascularly, i. e. subcutaneously or intramuscularly in clinical treatment. The reason for this is, that the latter method does not measure the degree of extravascular inactivation of the hormone. which may vary considerably depending upon the chemical treatment of the pituitary glands in the process of purification. Thus it was shown clinically (Wolfson, 1953) and experimentally (Fisher et al., 1953), that crude corticotrophin has an extravascular inactivation rate which is about three times that of corticotrophin A. Quantitative subcutaneous assays have fully confirmed these findings (Rerup, 1957). The present international corticotrophin standard preparation, however, is of the crude corticotrophin type, and assays of corticotrophin A against this standard revealed a lack of parallelism in the dose-response curves from

Further observations on the estimation of urinary gonadotropins in non-pregnant human subjects.

One of the great difficulties in estimating the excretion of gonadotropins in non-pregnant subjects is the fact that no International Standard preparation is available for comparative assay of these substances. Accordingly it has been necessary to express results of clinical assays in terms of arbitrary “animal” units, a unit being defined as that quantity necessary to produce a given effect. Arbitrary units employed in intact and hypophysectomized rats have included the doses necessary to induce vaginal cornification, to cause histologic changes in the ovaries, and and to induce a 50–100 per cent increase in the weight of ovaries, uterus, seminal vesicles and the ventral lobe of the prostate (1–4). In intact immature mice most investigators have adopted as a unit the quantity of extract causing a 100–200 per cent increase in uterine weight (5–8). It is now generally recognized that the error of results expressed without reference to a standard preparation may be threefold or more, and that little quantitative significance can be attached to data calculated in this way.

The preparation and maintenance of standards of radioactivity

A historical survey is made of the preparation and measurement of international radium standards. The need for standards of other radioisotopes is explained. Standardization is subdivided into primary and secondary methods; 4πβ counting, β-γ and γ-γ coincidence counting, gas counting, and microcalorimetry are discussed as primary methods, and the maintenance of standards thus obtained is considered. The present state of international comparison of several radioisotopes is reported and discussed together with a policy for wider international standardization.

SOURCES OF ERROR IN CLINICAL BIOASSAYS OF SERUM CHORIONIC GONADOTROPIN

ESTIMATES of the serum concentration of human chorionic gonadotropin (H.C.G.) in international units throughout normal and abnormal pregnancy have been made by several workers using different bioassay methods (1–8). Implicit in these estimations are the assumptions 1) that the hormone present in the serum of pregnant women and the international standard preparation are qualitatively identical, and 2) that other constituents of serum do not contribute in smy way to the effect of H.C.G. on the target organs. However, it has been recently reported that the effect of H.C.G. on the rat uterus and ovary is greatly increased when the hormone is administered in human plasma instead of in saline (9, 10, 11). Since this observation appeared to cast doubts on the validity of estimates of serum H.C.G. by these two methods, it was felt necessary to ascertain whether results obtained by the prostatic weight and seminal-vesicle weight methods could be criticized on the same grounds.

Pure all-trans vitamin A acetate and the assessment of vitamin A potency by spectrophotometry.

THE international unit of vitamin A activity corresponds with 0.3 µgm. of vitamin A alcohol or 0.345 µgm. of vitamin A acetate1. The International Standard Preparation is a solution of the all-trans acetate in cottonseed oil, and it serves a dual purpose as a biological and a spectrophotometric standard of reference. The properties of the acetate are therefore important.

A Hyaluronidase Unit

DESPITE our deficient knowledge of the physical and chemical properties of both hyaluronic acid and hyaluronidase, it seems desirable to have a hyaluronidase unit, which can be used until international standard preparations are available for comparison, in order to be able to correlate the numerous investigations being carried out with this substance. All investigations so far suggest that the viscosimetric method of estimation at present affords the best possibilities for quantitative analysis of a biological material.

New International Standard for Vitamin B1

IT is announced that the first International Standard for Vitamin Bx, which consisted of an adsorbate of the antineuritic vitamin, made from rice polishings, on fuller's earth, has now been replaced by a preparation of crystalline vitamin Ba hydrochloride. In recent years, progress in the study of the antineuritic vitamin has been rapid, and this change in the form of the international standard has been made possible by the synthetic preparation of the vitamin in pure crystalline form. Through the generosity of four manufacturers, an adequate quantity of the new crystalline material was placed at the disposal of the National Institute for Medical Research, Hampstead, to enable a new standard to be prepared consisting of the pure crystalline substance. Extensive international investigations of the properties of this material, and, in particular, the determination of its potency in terms of the original international standard by a variety of methods, have now been completed, and the members of the International Conference on Vitamin Standardization have unanimously recommended that the sample be adopted as the Second International Standard for Vitamin B15 and that the international unit be defined as the antineuritic activity of 3 micrograms of the international standard preparation. This recommendation has been adopted by the Permanent Commission on Biological Standardisation of the Health Organisation of the League of Nations.