Diana Riad-Fahmy

SALIVARY CORTISOL LEVELS IN TRUE AND APPARENT HYPERCORTISOLISM

Total plasma cortisol measurements may be misleading when there are variations in the plasma cortisol‐binding protein capacity resulting from drugs, pregnancy or congenital alterations in cortisol‐binding globulin (CBG). Salivary cortisol levels, which represent the free component of plasma cortisol, are less affected by alterations in protein binding and have been used in the investigation of hypothalamic‐pituitary‐adrenal disorders. This study compares these two indices of adrenal function in conditions of true hypercortisolism and spurious hypercortisolism (resulting from oral contraceptive medication or pregnancy). The circadian variation of cortisol in plasma and saliva was studied in six patients with unequivocal hypercortisolism and compared with normal volunteers. In the normal group, plasma and salivary cortisol levels taken at 0900 h were significantly higher than those taken at 2400 h. Patients with Cushings syndrome failed to show a significant difference between plasma and salivary cortisol levels collected at 0900 and 2400 h. Five patients with pituitary‐dependent Cushings disease, one patient with an adrenal carcinoma causing Cushings syndrome and seven normal subjects each received a dexamethasone suppression test using a continuous infusion of dexamethasone sodium phosphate at a rate of 1 mg/h. There was no significant difference in the half‐life disappearance rate of endogenous cortisol in either plasma or saliva comparing grouped data from patients with pituitary‐dependent Cushings disease with that of normal subjects. Failure of suppression of both plasma and salivary cortisol levels was observed in the one patient with adrenal carcinoma during dexamethasone infusion. The correlation between plasma and salivary cortisol measurements during the dexamethasone infusion was r=0·989 (P < 0·001) for normals and r= 0·982 (P < 0·001) for Cushings patients. Measurements of plasma and salivary cortisol were compared in pregnancy and during low dose oestrogen therapy; both these conditions are known to be associated with elevated cortisol binding globulin concentrations. A significant difference was demonstrated between the 0900 h plasma cortisol levels of normal subjects and those both in the third trimester of pregnancy of the oestrogen containing contraceptive pill. No difference was observed in the 0900 h salivary cortisol between control and pregnant or oestrogen treated groups.

A Rapid Assay for 17αOH-Progesterone in Plasma, Saliva and Amniotic Fluid Using a Magnetisable Solid-Phase Antiserum:

A radioimmunoassay suitable for measurement of 17αOH-progesterone concentrations in small aliquots of plasma (20 μL), amniotic fluid (20 μL) and saliva (200 μL) is described. The assay features an antiserum raised against a 17αOH-progesterone-3-(O-carboxymethyl)oxime/BSA conjugate coupled to a magnetisable, solid-phase support; the homologous radioligand is a 125I-iodohistamine conjugate. This combination of a γ-emitting ligand and a magnetic-separation procedure has the advantage of reducing assay time and cost; it also allows processing of plasma and saliva samples in the same assay batch. The method has satisfactory sensitivity, precision and accuracy. Data derived from clinical studies of patients with congenital adrenal hyperplasia indicate the usefulness of this assay in routine practice.

A radioimmunoassay for ethinyl oestradiol in plasma incorporating an immunosorbent, pre-assay purification procedure

A radioimmunoassay for plasma ethinyl oestradiol, featuring an immunosorbent extraction procedure, is described. Ethinyl oestradiol (EE2) was extracted using a non-specific, anti-oestrogen serum, raised to an oestradiol-17-hemisuccinate conjugate. The antiserum, coupled to microcrystalline cellulose, selectively extracted EE2 but not norethisterone (NE), thus conferring specificity on a radioimmunoassay which has previously exhibited unacceptably high cross-reactivity with the synthetic progestagen, norethisterone, often used concomitantly with ethinyl oestradiol. This radioimmunoassay was shown to fulfil accepted assay validation criteria. Levels in subjects not receiving EE2 were less than 25 pmol/l. Circulating concentrations of EE2 could therefore be accurately determined in patients receiving low-dose combined preparations (EE2 35 μg; NE 500 μg).

SALIVARY 17α-HYDROXYPROGESTERONE IN CONGENITAL ADRENAL HYPERPLASIA

A specific radioimmunoassay having the sensitivity (4 pg/tube) and precision required for the routine determinations of 17α‐hydroxyprogesterone concentrations in mixed whole saliva, parotid fluid and plasma has been developed. The correlation between 17α‐hydroxyprogesterone concentrations in matched samples of saliva and parotid fluid was excellent (r = 0.98): therefore saliva, being easier to collect, was used exclusively in later studies. The median 17α‐hydroxyprogesterone concentration in mixed whole saliva collected from thirty‐two healthy children was 390 pmol/litre ranging from 90–1520 pmol/litre. In a group (n=14) of treated CAH patients having a C21 hydroxylase deficiency, 17α‐hydroxyprogesterone showed a 20 fold greater variation, ranging from 67 pmol/litre in patients receiving excessive glucocorticoid dosage to 26300 pmol/litre in inadequately treated patients. A close correlation (r = 0.91) in 17α‐hydroxyprogesterone levels was observed in matched samples of saliva and plasma collected between 09.00 and 10.00 h from these patients. Concentrations of 17α‐hydroxyprogesterone in saliva therefore could well replace those in plasma for monitoring treatment of these patients. Matched samples of mixed whole saliva, parotid fluid and plasma were also collected from one inadequately treated female patient at frequent intervals over a 24 h period. Two other patients, one male and one female, collected matched samples at 30 min intervals for 4 h following Synacthen stimulation. The pattern of 17α‐hydroxyprogesterone in these samples suggests that salivary steroid concentrations are of potential value in assessing endocrine function in conditions such as CAH, where frequent sampling over prolonged periods is required.

A Simple Robust Assay for Testosterone in Male Plasma Using an 125I-Radioligand and a Solid-Phase Separation Technique

A radioimmunoassay for testosterone in male plasma utilising a gamma-emitting radioligand and a solid-phase antiserum is described. The radioligand is testosterone-3-(O-carboxymethyl)-oxime coupled to 125I-iodohistamine, and the solid-phase antiserum is prepared by coupling antitestosterone-3-bovine serum albumin to cyanogen bromide activated cellulose. The new procedure retains much of the specificity associated with a published, specific radioimmunoassay using an antiserum raised against testosterone-11α-BSA and a tritium radioligand and incorporating a dextran-coated charcoal separation procedure; values obtained by the two procedures are in excellent agreement (r = 0·98, n = 20). The combination of an 125I-radioligand and a solid-phase separation technique greatly increases sample throughput and has the further advantage of reduced running costs and a greater potential for automation. The method gives satisfactory levels of sensitivity, precision, and accuracy.

A sensitive solid phase enzymeimmunoassay for norethisterone (norethindrone) in saliva and plasma.

A sensitive, solid phase enzymeimmunoassay suitable for determining norethisterone in small aliquots of plasma (10 microliters) and saliva (100 microliters) has been developed. A solid phase antiserum raised against a norethisterone-11 alpha-hemisuccinyl/bovine serum albumin conjugate was prepared by coupling to cyanogen bromide activated cellulose. A norethisterone/horseradish peroxidase conjugate was used as enzyme label, o-phenylenediamine/hydrogen peroxide being the substrate for colour development. The assay had a lower limit of sensitivity of 3 pg/assay tube and satisfied accepted validation criteria. Norethisterone concentrations determined by enzymeimmunoassay and by a well established radioimmunoassay were in excellent agreement in both plasma (r = 0.993, n = 20) and saliva (r = 0.989, n = 15). Plasma and salivary norethisterone concentrations determined in healthy volunteers reached peak values at about 1 hour after administering a norethisterone-containing oral contraceptive preparation. The maximum values achieved in saliva (775--1430 pmol/l) were only approximately 3% of those observed in plama. Since salivary norethisterone concentrations reflected those in plasma, they may be useful in fertility control programmes and pharmacokinetic studies.

Radioimmunoassay of blood-spot 17α-hydroxyprogesterone in the management of congenital adrenal hyperplasia

A robust assay for routine measurement of blood-spot 17α-hydroxyprogesterone (17-OHP) concentrations has been developed using a magnetisable, solid-phase antiserum and an 125I-radioligand. The working range of this assay (13.5–500 nmol/L) is well suited for the initial diagnosis of congenital adrenal hyperplasia (CAH) and for monitoring replacement therapy in CAH patients. Data derived from multiple blood-spot samples, collected on two consecutive days, provide 17-OHP profiles. These profiles have been used to construct a chart allowing a rapid visual assessment of the efficacy of replacement therapy in CAH patients. Measurement of 17-OHP in the blood-spots of over-treated patients and accurate determination of normal range values in healthy infants relied on development of a sensitive assay (range 1.7–34 nmol/L). In the blood-spots of normal male (n=50) and female (n=50) infants collected 5–7 days after birth, 17-OHP concentrations were 7.62±2.55 nmol/L and 7.32±2.87 nmol/L respectively. Retrospective measurement of this steroid in samples from known CAH patients (n=4), which had values ranging from 224 to 2145 nmol/L, support a role for measurement of blood-spot 17-OHP in high-risk screening programmes.

Improvement of antisera for cortisol immunoassay by reduction of endogenous hormone concentrations

Markedly elevated concentrations of endogenous steroids in antisera may reduce the potential sensitivity of immunoassays. Attempts to raise antisera containing reduced amounts of cortisol by adrenalectomy, or concomitant administration of synthetic corticosteroid during immunisation, have met with indifferent success. Removal of cortisol, using dextran-coated charcoal in buffer of optimum molarity, may increase the average affinity constant of the antiserum, and improve the assay sensitivity and specificity.

A simple direct solid-phase enzymeimmunoassay for cortisol in plasma

A simple, direct solid-phase enzyme-labelled immunoassay for plasma cortisol was established using horseradish peroxidase/cortisol 21-hemisuccinate conjugate as ‘enzyme label’. The antiserum, raised against a cortisol 21-hemisuccinate/bovine serum albumin conjugate, was coupled to cellulose to facilitate separation of free and bound steroid. Solvent extraction was avoided by the use of heat denaturation of the cortisol-binding globulin. This assay had a lower limit of sensitivity of 16·6 nmol/l and satisfied the standard criteria of accuracy and precision. Cortisol concentrations determined by enzymeimmunoassay were in excellent agreement with a gas liquid chromatography/mass spectrometry procedure (r = 0·98, n = 19) and also with the radioimmunoassay in current use (r = 0·95, n = 20). Cortisol levels after ACTH stimulation and dexamethasone suppression in various subjects are presented. This enzymeimmunoassay is particularly applicable to the routine determination of plasma cortisol in small clinical laboratories or in those with a fluctuating workload.

A Sensitive, Specific, Solid-Phase Enzymeimmunoassay for Plasma Progesterone

A homologous enzymeimmunoassay (EIA) for plasma progesterone, using a horseradish peroxidase conjugate as enzyme label and an antiserum raised against a progesterone-11α-hemisuccinyl/BSA conjugate, is described. The antiserum was covalently linked to microcrystalline cellulose to facilitate separation of bound and free steroid; this solid-phase antiserum was stable for at least nine months when stored at 4°C. The freeze-dried enzyme label is also stable, having retained both enzymic and immunological activity for about four years. The EIA developed was specific and had the sensitivity (4·8 pg/tube) required for determining progesterone concentrations in plasma samples collected at any time during the menstrual cycle. EIA of plasma samples provided results which were in good agreement with a well validated radioimmunoassay (RIA). The specificity and inter- and intra-assay coefficients of variation in the EIA were strictly comparable with those of the RIA. The method described has been in use for two years and has been assessed in external quality assurance programmes established by the World Health Organization and the United Kingdom Department of Health and Social Security.