Measurement of diethylstilbestrol in plasma from patients with cancer of the prostate.

A specific radioimmunoassay has been developed for diethylstilbestrol (DES), using an antiserum raised against DES monocarboxymethyl ether and a tritium-labeled radioligand. Prior to radioimmunoassay, a fraction enriched in DES is obtained from a dichloroethane extract of plasma using Sephadex LH-20. The specificity of the assay is good, and the sensitivity (130 pg/ml) is adequate for accurate determination of DES in plasma from prostatic cancer patients treated with the drug. The precision is satisfactory, with an interassay coefficient of variation of approximately 10% at concentrations of approximately 1 ng/ml, and the blank values are negligible. Excellent agreement (r = 0.96) is observed between data obtained by radioimmunoassay and those obtained by a procedure using gas chromatography-high-resolution mass spectrometry. DES concentrations in the plasma of six treated (1 mg DES three times daily) patients were in the range 0.15 to 6.0 ng/ml. Increases in plasma concentration were observed within 2 hr of administration, with secondary rises occurring 5 to 6 hr later. Plasma testosterone concentrations were low in four of the patients; in a single subject, relatively high levels of testosterone were further elevated following administration of luteinizing hormone-releasing hormone.

Comparisons of plasma and salivary cortisol determinations for the diagnostic efficacy of the dexamethasone suppression test.

The current status of the saliva dexamethasone suppression test (DST) is discussed and results from the literature reviewed. Evidence is presented that demonstrates that the efficacy of the salivary-based test is equal to that of the plasma DST provided that specifically developed radioimmunoassays are used for determination of salivary cortisol. Such evidence relied on measurement of cortisol in 300 matched samples of plasma and saliva provided by patients admitted to a routine psychiatric ward over a 2-year period. The results according to diagnosis (DSM-III categories) were in line with those generally reported. The influence of anticholinergic medication was examined: this had no significant effects on the performance of the plasma or salivary-based DST.

Anxiety and the dexamethasone suppression test monitored with saliva.

To assess the effect of anxiety on response to the Dexamethasone Suppression Test (DST), cortisol concentrations were determined in patients who had various diagnoses, with anxiety as a secondary characteristic. Saliva was collected before and after venepuncture at 4:05 PM following completion of the Leeds Questionnaire at 3:00 PM. Matrix effects, which caused an initial artefactual decrease in cortisol levels in some saliva samples in patients with high anxiety, could be eliminated by repeated freezing/thawing. There was no significant difference between salivary cortisol concentrations before and after venepuncture, indicating that variability in response to the DST is not a correlate of anxiety and stressful venepuncture. There was no association between anxiety scores and plasma or salivary cortisol values: thus, anxiety is unlikely to be a major contributory cause of nonsuppression of hypercortisolemia in the DST.

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.

Improved production of oestradiol antibodies of high affinity and specificity by hormonal manipulation of the immunised animals and affinity chromatography of the antisera.

The titre, affinity and specificity of oestradiol antisera were increased by hormonal manipulation of immunised rats. Castration was as effective as daily injection of the aromatase inhibitor, 1,4,6-androstene-3,17-dione. Chromatography on the affinity matrix oestrone-3-CME/Sepharose-4B was less successful.

Improved production of high-affinity antisera to progesterone by castration and adrenalectomy of the immunised animals.

The titre and affinity of antisera to progesterone were increased by ablation of the progesterone-secreting organs of immunised rats. The animals were maintained for several months following castration and adrenalectomy by daily fludrocortisone.

Characterization of profiles of salivary progesterone concentrations during the luteal phase of fertile and subfertile women.

Salivary progesterone concentrations were measured in daily samples collected between 08.00 and 09.00 h throughout the menstrual cycle of women with a history of fertility. The luteal-phase salivary progesterone profiles in these normally menstruating, healthy women were characterized using a computer program based on a cumulative sum procedure. This method of statistical analysis led to the development of a 'progesterone boundary diagram', the inner and outer domains of which distinguished between the profiles of salivary progesterone considered compatible with fertility, and those observed in subfertile women attending an infertility clinic.

'Maternity blues' and hormone levels in saliva.

Psychological symptoms were monitored for 5 days post partum, and specimens of saliva were taken for assays of cortisol, progesterone and oestradiol. Of 40 normal primiparous mothers, the 5 who experienced most severe 'maternity blues' were matched with 5 who were symptom-free. On the day of symptoms, concentrations of progesterone and oestradiol were significantly higher in the experimental group (P less than 0.02 and less than 0.05, respectively), but mean cortisol concentrations did not differ between the groups. This pilot study has demonstrated the potential value of using a non-invasive technique (obtaining specimens of saliva) in conjunction with determinations of a predictable mood change.

Seasonal variation and the results of the dexamethasone suppression test.

There is controversy over the presence of a circannual rhythm in cortisol values in samples provided by depressed patients after a standard dexamethasone suppression test (DST). Post-DST cortisol values from patients admitted to an acute psychiatric ward over a 2-year period have therefore been analysed by appropriate statistical tests. No evidence was found for significant seasonal variation.

A sensitive enzymeimmunoassay with a fluorimetric end-point for the determination of testosterone in female plasma and saliva.

A fluorimetric enzymeimmunoassay has been developed having the sensitivity (500 fg/assay tube) required for determining testosterone concentrations in female plasma and saliva samples. The assay featured a solid-phase antiserum raised against an 11 alpha-hydroxytestosterone-11-hemisuccinate bovine serum albumin conjugate, an 11 alpha-hydroxytestosterone-11-hemisuccinate horseradish peroxidase conjugate as the "enzyme label", and p-hydroxyphenylacetic acid as the substrate for the development of fluorescence. Specificity was ensured by "extracting" testosterone from samples with a solid-phase anti testosterone-3-/0-carboxymethyl/-oxime serum. The assay was shown to satisfy accepted validation criteria providing results in good agreement with routine radioimmunoassay procedures in both plasma (r greater than 0.98, n=28) and saliva (r greater than 0.99, n=28). In saliva samples collected at 2 hourly intervals by normal healthy women (n=5) testosterone concentrations showed a well defined circadian rhythm: the mean testosterone concentration in early morning samples (174 pmol/litre) fell by 83% in late evening collections. In healthy female volunteers (n=7), mean daily throughout one complete cycle ranged from 50 to 218 pmol/litre. Following dexamethasone administration testosterone concentrations in plasma fell by approximately 50%, and salivary concentrations were undetectable after one hour. This enzymeimmunoassay may be useful in studies of female infertility.

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.

PIP: This paper reports the development of a solid-phase enzymeimmunoassay for quantitating levels of norethisterone (norethindrone) in small portions of plasma (10 mcliters) and saliva (100 mcliters). The assay system uses an antiserum against norethisterone-11 alpha-hemisuccinyl/bovine serum albumin conjugate, prepared by coupling to cyanogen bromide-activated cellulose. Enzyme label was norethisterone/horseradish peroxidase conjugate. The assay's sensitivity at its lower limit was 3 pg/tube. When these enzyme immunoassay results were compared with a standard radioimmunoassay, good agreement for both plasma (r=.99) and saliva (r=.98) was found. Healthy volunteers were then given a norethisterone-containing contraceptive orally, and their plasma and saliva levels of the agent were measured. The plasma and salivary values both peaked at 1 hour postadministration. Maximum salivary levels were but 3% of plasma levels; however, since they reflected, in ratio, concentrations in plasma, salivary measurements may prove useful in fertility control programs and pharamacokinetic studies.

A sensitive solid phase enzymeimmunoassay for testosterone in plasma and saliva.

A sensitive, solid phase enzymeimmunoassay suitable for determining testosterone concentrations in samll aliquots of plasma (20 microliter) and saliva (200 microliter) has been developed. A solid phase antiserum raised against a testosterone-11 alpha-hemisuccinate/bovine serum albumin conjugate was prepared by coupling to cyanogen bromide activated cellulose. The "enzyme label" was a covalently linked testosterone/horseradish peroxidase conjugate. The assay had a lower limit of sensitivity of 4pg/assay tube and satisfied accepted criteria of specificity and precision. Testosterone concentrations determined by enzyme-immunoassay were in excellent agreement not only with a gas liquid chromatography/mass spectrometry procedure (r=0.96, n=12) but also with the radioimmunoassay in routine use (r=0.95, n=12). The EIA can therefore replace RIA in both the small clinical laboratory and high throughput service centres for determining plasma and salivary testosterone concentrations. In normal males salivary testosterone concentrations reflected circulating steroid levels and indicated the possibility of assaying saliva rather than plasma in clinical studies.

Testosterone levels during systemic and inhaled corticosteroid therapy.

Testosterone has importance both as a sex hormone and as an anabolic steroid promoting bone formation. Osteoporosis is associated with both hypogonadism and corticosteroid therapy. Testosterone levels are reduced by long term prednisolone treatment. Although high dose inhaled corticosteroid therapy may cause a variety of systemic effects including adrenal suppression, dermal thinning and a reduction in total bone calcium, its effect on testosterone levels is not known. Testosterone, luteinizing hormone, follicle stimulating hormone and sex hormone binding globulin were therefore measured in 35 male patients with respiratory disease attending an outpatient clinic (median age 58, range 21-75 years). They were grouped according to steroid therapy and compared with 19 age matched controls. Mean (SD) testosterone levels were 33% lower in 12 men on long term oral prednisolone [14.5 (6.0) nmol 1-1] than in controls [21.7 (6.3) nmol 1-1], but were not significantly reduced in 10 patients on low dose inhaled beclomethasone [200-800 micrograms day-1: 19.7 (3.7)] nor in 13 men taking high dose inhaled beclomethasone [1500-2,250 micrograms day-1: 17.9 (5.6)]. Levels of luteinizing hormone, follicle stimulating hormone and sex hormone binding globulin were similar in all four groups. These cross sectional data confirm that long term systemic corticosteroid therapy reduces testosterone levels. However, testosterone was reduced by only 18% (NS) by long term inhaled corticosteroids. Other mechanisms to explain the disordered bone metabolism should now be explored.

Clinical utility of the dexamethasone suppression test assessed by plasma and salivary cortisol determinations.

Concentrations of cortisol in saliva and plasma were compared in matched samples during a standard 1 mg dexamethasone suppression test. The study involved 185 routine psychiatric admissions, all of whom were classified according to DSM-III criteria. In a group of 122 matched samples of saliva and plasma, there was a Pearson correlation coefficient of 0.867 and a Spearman rank correlation coefficient of 0.869. Medication with anticholinergic side effects had little effect on the correlation and was not associated with major difficulties in salivary sampling due to "dry mouth." In a group of 178 diagnoses where both saliva and plasma were obtained, results were almost identical. Although nonsuppression was found in all psychiatric conditions, there was a very significant association with major depressive episode with melancholia.

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-11 alpha-BSA and a tritium radioligand and incorporating a dextra-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.

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

A robust assay for routine measurement of blood-spot 17 alpha-hydroxyprogesterone (17-OHP) concentrations has been developed using a magnetizable, 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 overtreated 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.

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 micrograms; NE 500 micrograms).

A rapid assay for 17 alpha OH-progesterone in plasma, saliva and amniotic fluid using a magnetisable solid-phase antiserum.

A radioimmunoassay suitable for measurement of 17 alpha OH-progesterone concentrations in small aliquots of plasma (20 microL), amniotic fluid (20 microL) and saliva (200 microL) is described. The assay features an antiserum raised against a 17 alpha OH-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 gamma-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 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-11 kappa-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 degrees 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 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 programme established by the World Health organization and the United Kingdom Department of Health and Social Security.

A robust assay for dexamethasone in plasma using a heterologous 125I radioligand and a magnetizable solid-phase antiserum.

An assay using an antiserum raised against a dexamethasone 21-hemisuccinate conjugate and the heterologous radioligand dexamethasone 21-(carboxymethyl) ether was developed, validated, and used to study the pharmacokinetics of this steroid for 12 h following administration to patients with congenital adrenal hyperplasia. Coupling the antiserum to magnetizable cellulose allowed rapid separation of bound/free steroid. A C-21 rather than a C-3 antiserum was used to minimize interference with a main metabolite, 6 beta-hydroxydexamethasone. Close correspondence of assay (0.35 nmol/L) and curve (0.25 nmol/L) sensitivities suggests that interference by matrix effects is minimal. This was confirmed by good agreement in data from the in-house assay and that of a reference procedure. Good precision was demonstrated by the precision profile and Shewhart chart quality control data. The latter also demonstrated the assay was robust and reliable in routine practice.