J. R. Daly

A sensitive bioassay for adrenocorticotrophic hormone in human plasma.

A technique for the bioassay of adrenocorticotrophic hormone (ACTH) in human plasma is described, and its experimental validation discussed.

The cytochemical bioassay of hormones.

Publisher Summary The evidence summarized in this chapter shows that it is possible to precisely measure the biochemical changes induced in target cells when the specific hormone acts on these cells with the modern techniques of cellular biology and cytochemistry. By measuring such effects, it is possible to assay the biological activity of the hormone with a degree of sensitivity that exceeds that of most currently available radioimmunoassays by two to three orders of magnitude. The increase in the use of cytochemical bioassays have resulted in the increase in their validity tests and applicability scope. The development of a cytochemical section assay for adrenocoticotropin (ACTH) has resulted in extension of these assays to routine purposes. It is a model for similar assays for other hormones. But the principle that the effect of bioreactive molecules on their target tissues can be measured very sensitively opens further vistas. These techniques can be applied to the detection, and assay, of other biologically active substances, such as pharmacologically active substances or toxic substances. These techniques can be readily used to study the analysis of end-organ sensitivity and the effects of hormones or their products on the other tissues in the body can be readily studied by these new techniques.

THE CYTOCHEMICAL BIOASSAY OF CORTICOTROPHIN

The procedures developed in cytochemistry offer special advantages for hormone assay. Maintenance of the target organ in culture (Trowell, 1959) prior to exposure to the hormone, allows it to be removed from the hormonal environment of the parent animal and to return to an apparently basal state. This has been shown to be particularly useful in the assay of trophic hormones, for it eliminates both the necessity of keeping the test animal in very strictly controlled environmental conditions prior to its use in the assay, and of preliminary hypophysectomy. Furthermore, the exhibition of the hormone to the target organ is carried out within the culture system at the end of the culture period, and this allows very precise regulation of the period of exposure to the hormones action. Thus activity/time relationships can be studied and the period of exposure made optimal for the assay. The measurement of a chemical change in individual cells makes it possible to isolate the specific hormone-sensitive cells within the target organ, thus avoiding any diluting or masking effect produced by other cells in the tissue which are not involved in the response. The achievement of this isolation by optical means, with a microdensitometer, prevents the cells being exposed to the potentially damaging effects of separation by chemical or mechanical methods, and the normal histological relationships of the cells are preserved. These features of the cytochemical approach to hormone assay are probably important in contributing to the very great sensitivity of the assays developed so far. The approach should also prove very versatile, being theoretically applicable to the assay of any hormone for which a target cell can be identified and in which an appropriate hormonally-induced chemical reaction can be measured. The first hormone to which the technique was applied was corticotrophin (ACTH). This hormone is eminently suitable as a model system to test the feasibility of cytochemical bioassay, because it has a clearly identifiable target organ readily accessible to surgery, and a great deal is known about the chemical changes which occur in the adrenal cortex under the influence of ACTH. The guinea-pig was chosen as the assay animal because it is easy to handle, small enough

Comparison of Effects of Long-term Corticotrophin and Corticosteroid Treatment on Responses of Plasma Growth Hormone, ACTH, and Corticosteroid to Hypoglycaemia

The development of the highly sensitive cytochemical bioassay for ACTH has permitted the measurement of plasma ACTH levels during the insulin hypoglycaemia test (I.H.T.) in patients treated with corticosteroids and corticotrophin. The ACTH, corticosteroid, and growth hormone (GH) responses in the I.H.T. were measured in three groups of 12 rheumatoid arthritis patients. One group was receiving long-term corticotrophin treatment, the second was undergoing long-term corticosteroid treatment, and the third had never received systemic hormone therapy. The increments in plasma ACTH, corticosteroids, and GH were diminished in the corticosteroid-treated group, as were increments in plasma GH and ACTH in the corticotrophin-treated group; but in this group the corticosteroid increment was normal. Examination of the area under the curve of the ACTH response showed that the total amount of ACTH secreted was normal though the rate of secretion was reduced. In the corticosteroid-treated group both rate and total secretion were diminished.

PLASMA CORTICOTROPHIN LEVELS DURING INSULIN‐HYPOGLYCAEMIA : COMPARISON OF RADIOIMMUNOASSAY AND CYTOCHEMICAL BIOASSAY

Development of a highly sensitive bioassay for ACTH has enabled bioassayable ACTH to be measured in plasma during the course of the insulin hypoglycaemia test for the first time. Comparison with radioimmunoassay (RIA) on the same plasma samples shows that the latter consistently overstimates the true, or biologically active, ACTH level. This overstimate appears to be most marked when ACTH levels are highest and during the fall in plasma levels which occurs following the ACTH peak. This may limit the value of the RIA of ACTH, in some situations.

CORTICOSTEROID AND GROWTH-HORMONE RESPONSE TO HYPOGLYCÆMIA IN PATIENTS ON LONG-TERM TREATMENT WITH CORTICOTROPHIN

Abstract Ten patients with rheumatoid arthritis who had been receiving corticotrophin (A.C.T.H.) therapy for up to 17 years, and ten control patients with rheumatoid arthritis untreated by either A.C.T.H. or steroids, were given insulin-hypoglycaemia tests. The rises in circulating corticosteroid and growth-hormone (G.H.) levels were compared in the two groups and found to be diminished in the A.C.T.H.-treated patients, the G.H. impairment being the more striking. It is concluded that long-term A.C.T.H. therapy suppresses the pituitary, but that, owing to possible adrenal hypertrophy, plasma-corticosteroid levels are less affected than those of G.H. This may explain the apparent differences between the effect of A.C.T.H. and corticosteroids on the hypothalamo-pituitary-adrenal axis.

RESPONSE OF HUMAN BREAST-CANCER TISSUE TO STEROID HORMONES IN VITRO

Abstract Biopsy tissue from sixteen patients with carcinoma of the breast was maintained in Trowells T8 culture medium to which oestradiol, œstrogen antagonists (testosterone and drostanolone), or both oestradiol and drostanolone had been added. This in-vitro test differentiated two types of malignant tissue: five specimens survived well in the medium without additives, but seven did not. These seven specimens survived well in the presence of œstradiol, but not when œstrogen antagonists (alone or with oestradiol) were added.

POSSIBLE UNNECESSARY PROLONGATION OF CORTICOSTEROID THERAPY IN RHEUMATOID ARTHRITIS

Abstract Thirty-five patients who had been receiving long-term corticosteroids for rheumatoid arthritis were assessed 5 years after an attempt at steroid withdrawal. Of the ten from whom steroids had been withdrawn completely, five had been replaced on steroids and two had died. Of the twenty-five who had remained on steroids, six had died and all had continued corticosteroid therapy in essentially the same dosage throughout the 5 years. On the basis of clinical and laboratory data it is suggested that eight of these should have undergone another attempt at withdrawal. It is possible that corticosteroid therapy is being continued in certain patients with rheumatoid arthritis with insufficient clinical justification, and it is recommended that the dose of steroids should be frequently assessed in all cases with a view to possible reduction.

THE CYTOCHEMICAL BIOASSAY OF CORTICOTROPIN (ACTH)

The conventional criteria of reliability for any assay method are precision, accuracy, sensitivity, and specificity. Of these, the last-specificity-if not always the most difficult to achieve, is usually the most difficult to demonstrate and, particularly perhaps in endocrinology, is conceptually the most complex. This is because a hormone (like an enzyme) is primarily defined in terms of its activity, and its postulated existence may in some cases be only an inference from observed metabolic or behavioral effects. In hormone assay, therefore, bioassays, that is assays based on the quantification of such effects, have a certain logical, and often also historical, priority over other types of assay. But a hormone not only produces effects, it is also, of course, a substance with a particular chemical structure. Thus, another approach to hormone assay is the use of some form of analytic “probe” to seek and quantify this structure. The by now classical (although still, in fact, astonishingly recent) technique of structural assay in endocrinology is radioimmunoassay (RIA). Thus, the “functional” assay-bioassay-and the “structural” assay-radioimmunoassay-each present different specificity problems.? It is possible that widely different molecules may produce similar biologic effects, a well recognized instance of this being the enhanced synthesis of thyroxine produced both by thyroid-stimulating hormone and by the structurally very different thyroidstimulating immunoglobulin that circulates in Grave’s disease. On the other hand, molecules, the structure of which is so similar that they cannot be distinguished in a radioimmunoassay system, may differ widely in biologic activity. Whilst it is true that niceties of technique may reduce these specificity problems, for example by observation of different time courses of the response in a bioassay system or by refinement of the antibody in a radioimmunoassay SYStem, full characterization of certain endocrine states requires investigation by both structural and functional assays. Radioimmunoassay and bioassay are thus complementary.

STUDIES ON THE MECHANISM OF CYTOCHEMICAL BIOASSAYS

In the early studies which led to the cytochemical bioassay for corticotrophin (ACTH) (Chayen et al., 1971) we observed that we could measure the effect of graded concentrations of ACTH, from 0.005 to 5.0 pg/ml, acting for 3 min (later changed to 4 min) on segments of guinea-pig adrenal gland. How was it possible that the maximum effect occurred at such a low concentration as 5 pg/ml and that even 0.05 pg/ml produced a considerable response in this tissue when the normal physiological concentrations of ACTH in rodents, as in man, varied from about 20 pg/ml upwards? Either the response we were measuring was only a concomitant of the effect of the hormone or the phenomenon, if analysed, would tell us something about the mechanism of action of these hormones. In the later studies on gastrin, where the measured effect is the enhancement of carbonic anhydrase activity, this effect is in fact the production of hydrogen-ions which is the end-product of gastrin stimulation; hence it was unlikely to be merely a correlate and consequently the phenomenon of tissue responding to 1/1000 of the physiological level of a hormone was worthy of deeper analysis.