John E.T. Corrie

The preparation of 125I-labelled bile acid ligands for use in the radioimmunoassay of bile acids

A general method for the preparation of 125I-labelled bile acid-histamine or 125I-labelled bile acid-tyramine conjugates is presented. The method is simple, quick and produces ligands in good yield (30%). The characteristics of a radioimmunoassay for conjugated chenodeoxycholic acid, based on an 125I-labelled ligand prepared by the method, are also described. The assay produced values for fasting serum concentrations of conjugated chenodeoxycholic acid that agree well with previous data.

Effect of the benzodiazepines diazepam, des-N-methyldiazepam and midazolam on corticosteroid biosynthesis in bovine adrenocortical cells in vitro; location of site of action

Diazepam and midazolam inhibited cortisol and aldosterone synthesis in bovine adrenal cells in vitro. The biologically active metabolite des-N-methyldiazepam did not. Midazolam was a more potent inhibitor (IC50: 6 micrograms/ml) than diazepam (IC50: 13 micrograms/ml) in ACTH-stimulated cells. Both compounds inhibited steroidogenesis at several points in the biosynthetic chain; the greatest effects were on 17 alpha hydroxylation and 21 hydroxylation. Diazepam had a relatively greater effect on 17 alpha hydroxylation; midazolam on 21 hydroxylation. Both were less potent inhibitors of 11 beta hydroxylation and had little apparent effect on side chain cleavage. Thus microsomal hydroxylation is more vulnerable to benzodiazepines than mitochondrial hydroxylation. It is suggested that the drugs act by competing with steroid mixed function oxidases for cytochrome P-450. The plasma concentrations required for these effects are high in relation to therapeutic levels but may be achieved, for example, during acute infusions or when they are used in combination with imidazole drugs such as cimetidine.

Generally applicable 125iodine-based radioimmunoassays for plasma progesterone

Two methods are described for estimation of plasma progesterone, both employing a heterologous-bridge radioimmunoassay system with anti-sera raised against a progesterone 11 alpha-hemisuccinate conjugate and a radioiodinated progesterone 11 alpha-glucuronide-tyramine conjugate as tracer. Separation techniques based on double antibody methods have been employed to improve assay precision, and the assays described are sensitive, precise, accurate and robust and well suited to the measurement of progesterone levels for routine monitoring of luteal function. Present evidence suggests that the majority of laboratories which already use such antisera could readily adopt this assay system with its advantages of improved performance and gamma-labelled tracer.

Development and validation of an improved radioimmunoassay for serotonin in platelet-rich plasma

A radioimmunoassay (RIA) using a 125I-tracer is described for measurement of serotonin (5-hydroxytryptamine, 5-HT) in human platelet-rich plasma (PRP). Antisera were raised against 5-HT-succinamate conjugated to bovine albumin and, to improve assay sensitivity, the analyte was made chemically similar to the immunogen by conversion to N-acetylserotonin prior to assay, using the specific amino reagent N-acetoxysuccinimide. The assay shows good correlation with a high-pressure liquid chromatography (HPLC) reference method (5-HT RIA = 1.007 X 5-HT HPLC + 29.3, r = 0.936, p less than 0.001, n = 40), indicating that no significant cross-reactions were detected. Samples of PRP are diluted 1/20 to fall within the working range (80-15% B/B0) of the assay, which is 4.75-325 nmol/l, (0.95-65.0 pmol/tube), corresponding to 95-6500 nmol/l in PRP. Intra- and interassay coefficients of variation were 5.0-10.5% and 12.0-21.2% respectively for serotonin concentrations of 250-2,500 nmol/l added to platelet-poor plasma. With this improved assay, it is possible to analyse up to 100 samples/day, compared with 10-20 samples/day by HPLC.

Monoclonal antibodies to testosterone: the effect of immunogen structure on specificity.

A 15 beta-thioalkyl derivative of testosterone conjugated to bovine serum albumin (BSA) was synthesised and used to produce monoclonal antibodies. These antibodies were evaluated using 15 beta-(2-carboxyphenylthio)-testosterone [125I]histamine as radioligand. Out of 1368 hybrids, 5 secreted anti-testosterone antibodies. These were compared with monoclonal antibodies derived from immunisation with testosterone-3-carboxymethyloxime-BSA. The first group of monoclonal antibodies all showed very low cross-reactivity with 5 alpha-dihydrotestosterone (less than 2.8%) indicating that this site of linkage is a good choice for discriminating between differences at the 4-5 position in the A-ring on the testosterone molecule. However they generally showed much higher cross-reactivity with progesterone and androstenedione than monoclonal antibodies raised to the 3-linked immunogen. Nevertheless within each fusion there were monoclonal antibodies with markedly different specificities. None of these antibodies could be considered suitable for use in a testosterone immunoassay, but it does suggest that an antibody with an improved specificity profile could be found using the monoclonal antibody approach.

The production and assessment of monoclonal antibodies to cortisol

In an extensive series of experiments, Balb/C mice and Lou rats were immunised with 3-O-(carboxymethyl)oximinocortisol conjugated to bovine serum albumin. The spleen cells from selected animals were fused with cells from mouse or rat plasmacytoma lines. Out of many hundreds of hybridomas screened, more than seventy produced antibody that bound 125I-labeled cortisol. These cultures were investigated further for stability of antibody production, affinity for cortisol and cross-reactivity with other steroids. An unexpected but consistent finding was that immunised rats produced antibody which cross-reacted with 11-deoxycortisol to a level greater than 100% and this characteristic was reproduced by rat-rat hybridomas. Strategies designed to improve the chances of generating non-cross-reactive anti-cortisol monoclonal antibodies did not appear to be successful. Nevertheless, several monoclonals were identified with properties that suggest they may be useful for the development of sensitive and specific cortisol assays.

Development and application of a direct radioimmunoassay for aldosterone in saliva

A previously described direct radioimmunoassay for plasma aldosterone has been modified to enable direct measurement of the steroid in saliva. The specificity of the method has been demonstrated by assay after high pressure liquid chromatographic purification of saliva extracts. Assay of matched plasma and saliva samples taken from normal subjects during unrestricted and controlled sodium intakes, either under basal conditions or while undergoing ACTH stimulation or dexamethasone suppression, confirms that salivary aldosterone values provide a good reflection of levels in plasma. Mean salivary aldosterone values are approximately one-third of those in plasma. Sampling immediately upon waking appears to provide reliable values for salivary aldosterone, and the potential application of this technique to the screening of hypertensive patients is discussed.

DEXAMETHASONE-SUPPRESSIBLEHYPERALDOSTERONISM: STUDIES ON OVERPRODUCTION OF 18-HYDROXYCORTISOL IN THREE AFFECTED FAMILY MEMBERS

We report a newly diagnosed family in which a father and his two sons were found to be hypertensive and to have the rare familial condition dexametha‐sone‐suppressible hyperaldosteronism (DSH). All three patients became normotensive on dexamethasone treatment alone and have been successfully maintained on low doses of the drug for 6 months since diagnosis. Each of the patients had extremely high plasma and urinary concentrations of the recently discovered steroid 18‐hydroxycortisol, which were more than ten times higher than the upper normal limit. Plasma levels were readily suppressed by dexamethasone treatment. The hypothesis that 18‐hydroxycortisol might derive from 18‐hydroxylation of recirculating Cortisol was tested by measuring plasma 18‐hydroxycortisol levels during low‐dose and high‐dose hydrocortisone infusions, in a normal subject and in one of the patients with DSH. During the high‐dose infusions (with plasma Cortisol levels of 3000‐5000 nmol/1) there was net production of 18‐hydroxycortisol within 8 h, but this was not observed during the low‐dose infusions (plasma Cortisol levels 300‐400 nmol/1). The origin of 18‐hydroxycortisol remains uncertain: these findings do not support the recirculation theory, but lend weight to the alternative hypothesis that 18‐hydroxycortisol is produced in transitional adrenocortical tissue. This steroid is of considerable value in the differential diagnosis of primary hyperaldosteronism and may also be important as a marker of transitional adrenal cell function.

Factors affecting the secretion of 18-hydroxycortisol, a novel steroid of relevance to Conn's syndrome.

A recently developed radioimmunoassay for direct measurement of 18‐hydroxycortisol (18‐OH‐F) in plasma and urine has been used to study the physiology of this newly described steroid in normal subjects. Plasma levels of 18‐OH‐F show a circadian variation similar to that of cortisol and are increased and suppressed by administration of ACTH and dexamethasone respectively. A clear increase was observed in response to sodium restriction but despite this, angiotensin II infusion failed to cause a rise in 18‐OH‐F levels and a possible explanation is discussed. The results are interpreted in terms of a proposed biosynthetic pathway involving 18‐hydroxylation of cortisol during a second passage through the adrenal gland.

The origin and significance of 18-hydroxycortisol: Studies in hyperaldosteronism and in bovine adrenocortical cells in vitro

18-Hydroxycortisol has been suggested as a marker compound for a transitional zone between the adrenocortical zonae glomerulosa and fasciculata. The control of secretion of 18-hydroxycortisol has been compared with those of cortisol and aldosterone in normal subjects and patients with primary hyperaldosteronism. Comparisons were also made in isolated bovine zona glomerulosa and zona fasciculata cell preparations. Although there was considerable cross-contamination between fractions, 18-hydroxycortisol secretion occurred with equal facility in both fractions but depended on the availability of cortisol as substrate. Changes in secretion during stimulation following those of cortisol. It is concluded that, in vivo, 18-hydroxycortisol derives mainly from the zona fasciculata. The relevance of these findings to primary hyperaldosteronism and to the nature of the transition is discussed.