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.

A comparison of menstrual cycle profiles of salivary progesterone in British and Thai adolescent girls.

Menstrual-cycle profiles of salivary progesterone concentration, obtained by radioimmunoassay of daily samples collected throughout the cycle, were obtained from Thai (n = 232) and British (n = 130) adolescent girls up to 4 years postmenarche. These profiles were graded from 1 to 5 ranging, respectively from concentrations at the detection limit of the assay to profiles generally observed for the mature premenopausal woman. Contingency table analysis of the grade frequencies for Thai-British pairs of girls matched for chronological age and age at menarche (n = 2 x 90) demonstrated that British girls had more mature cycles (22/90) than Thais (11/90) (P less than 0.05) particularly in the first 2 years postmenarche (P less than 0.01). For these matched pairs of girls there was no evidence to support the view that girls with an early age of menarche develop their profiles more quickly following menarche than those with a late age of menarche, as previously reported and which was thought to be important in the development of breast cancer. The findings of this study also suggest that adolescent girls in Britain develop their menstrual cycle profiles of salivary progesterone more quickly than their Thai counterparts and this may be of value in formulating hypotheses regarding any role that ovarian progesterone secretion may have on subsequent breast cancer risk.

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.

The use of salivary progesterone assays in the assessment of ovarian function in postmenarcheal girls.

Menstrual cycle profiles of salivary progesterone were derived from daily samples obtained from 75 postmenarcheal girls aged between 12 and 17 years. Data were analysed to establish the proportion of girls exhibiting ovulatory cycles in each of several age groups following menarche or in groups based solely on chronological age. 'Luteal'-phase profiles of salivary progesterone, which were similar in shape to those characteristic of mature premenopausal women, were taken to indicate the probable occurrence of ovulation. The percentage of cycles in those subjects judged to be ovulatory, grouped according to gynaecological age, rose from 14% in the first year to 56% in the fourth year. Those classified according to chronological age, and where sufficient data permitted reliable estimates of the percentage of subjects ovulating, rose from 11% at 12 years of age to 64% for girls aged 16. These data are in reasonable concordance with published data using plasma progesterone and urinary methods for establishing ovulatory incidence, and indicate the value of salivary progesterone measurements in studies of ovarian function in adolescence.

Transient increases in progesterone in daily and 2-hourly saliva specimens from adolescent girls.

Daily progesterone profiles obtained from saliva specimens from 101 peri- and postmenarcheal girls showed episodic 'spikes' which reached up to 600 pmol/l from baselines of approximately 40 pmol/l. The authenticity of progesterone in the 'spike' specimens was established using gas chromatography-mass spectrometry. 'Spikes' of progesterone occurred in 5.3% of premenarcheal saliva specimens but in only 1-2% of specimens from girls who were 4-6 years postmenarche. Two-hourly specimens from 53 pre- and postmenarcheal girls had 'spike' frequencies similar to those measured in daily specimens.

Progesterone concentrations in samples of saliva from adolescent girls living in Britain and Thailand, two countries where women are at widely differing risk of breast cancer.

Menstrual-cycle patterns of salivary progesterone concentration were obtained for 131 and 245 adolescent girls up to 4 years postmenarche living in Britain and Thailand respectively. These patterns were graded on a scale of 1 (little or no activity) to 5 (activity similar to that exhibited by the mature premenopausal woman) and the frequency of these grades within groups of girls from each centre was analysed. The major finding was that British girls exhibited a predominance of higher grades of progesterone activity when compared with their Thai counterparts (n = 2 x 58) when matched for chronological and gynaecological ages (P approximately 0.002). This was particularly so for the girls from these two matched groups in the gynaecological age range 2-4 years (P approximately 0.03). The major contribution to this significant difference between the two groups is attributed to the greater effect of chronological age on progesterone secretion in the British girls (P approximately 0.03) compared with the Thai girls (P approximately 0.29). These findings may have implications for facilitating our understanding of the reason for the differing risk of breast cancer in women in both countries.

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.

Non-insulin-dependent diabetic patients (NIDDMs) do not demonstrate the dawn phenomenon at presentation.

A dawn rise of plasma glucose (PG) and/or insulin, the 'dawn phenomenon', has been commonly reported in treated diabetic patients and normal subjects. To evaluate the effect of treatment on this phenomenon in non-insulin-dependent diabetics (NIDDMs), PG, C peptide, immunoreactive insulin (IRI), growth hormone (GH), cortisol, epinephrine, and norepinephrine were measured hourly between 24.00 and 09.00 h in 17 newly diagnosed untreated NIDDMs (group 1). The study was repeated in 11 patients after a year of treatment (group 2). The PG levels did not change significantly at any time from 03.00 to 08.00 h in group 1 but increased continuously from 6.7 +/- 0.5 mmol/l at 04.00 h to 7.8 +/- 0.5 mmol/l at 08.00 h (P less than 0.01) in group 2. IRI and C peptide decreased significantly after 07.00 h in both groups. GH and catecholamine changes were similar in group 1 and group 2. Cortisol levels showed a nadir at 02.00 h and a peak after 07.00 h in both groups. Our results demonstrate no dawn rise of mean PG, IRI and C peptide in newly diagnosed untreated NIDDMs but a significant rise of PG in the early morning period in NIDDMs after a year of treatment with diet alone and diet plus sulphonylureas. Therefore other factors such as treatment and/or duration of the diabetes may play an important role in the pathogenesis of the dawn phenomenon.

Some observations on the determination of cortisol in human plasma by radioimmunoassay using antisera against cortisol-3-BSA.

Antisera were obtained from rabbits immunised against cortisol-3-BSA with a view to examining their application in a radioimmunoassay of the steroid. One such antiserum was studied in detail; cross-reactivity with other C21 steroids normally present in human plasma was negligible and it proved possible to establish a radioimmunoassay which satisfied all criteria of reliability. The specificity of the cortisol determination achieved in human plasma was examined by performing measurements with and without including an initial Sephadex LH-20 column chromatographic purification step; the values obtained were in excellent agreement both for normal plasma and for that obtained following adrenal stimulation with ACTH.

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.

The use of multivariable standard curves in the radioimmunoassay of testosterone and 5alpha-dihydrotestosterone.

Multivariable calibration curves have been used to enable testosterone and 5alpha-dihydrostestosterone to be assayed directly in plasma extracts without further pre-purification of the sample. Two antisera were used, both with relatively high, but different affinities for the substances measured, and with relatively low affinity towards all other substances tested. The antisera were obtained from rabbits immunized against testosterone-3-BSA and 5alpha-dihydrostestosterone-3-BSA. The technique was of adequate precision, accuracy and specificity. The last was examined by comparison of values obtained by the present method and those obtained following prepurification by thin layer chromatography.

The use of multivariable standard curves in the radioimmunoassay of testosterone and 5alpha-dihydrotestosterone.

Multivariable calibration curves have been used to enable testosterone and 5alpha-dihydrotestosterone to be assayed directly in plasma extracts without further pre-purification of the sample. Two antisera were used, both with relatively high, but different affinities for the substances measured, and with relatively low affinity towards all other substances tested. The antisera were obtained from rabbits immunized against testosterone-3-BSA and 5alpha-dihydrotestosterone-3-BSA. The technique was of adequate precision, accuracy and specificity. The last was examined by comparison of values obtained by the present method and those obtained following pre-purification by thin layer chromatography.

A specific enzymeimmunoassay for progesterone in human plasma.

An enzymeimmunoassay for plasma progesterone was established using progesterone covalently linked to the enzyme, horseradish peroxidase, as the 'label'. Separation of free and bound steroid was effected by Sepharose-coupled antiprogesterone-11alpha-hemisuccinyl bovine serum albumin antiserum (Sepharose-antisera). The enzymeimmunoassay satisfied the normal criteria of specificity, precision and accuracy. Comparison of assay results obtained by radioimmunoassay (with and without thin-layer chromatography) and enzyme-immunoassay (with and without thin-layer chromatography) showed excellent agreement of results in all cases (r greater than 0.98). This enzymeimmunoassay is particularly applicable to the routine determination of plasma progesterone in the smaller clinical laboratory.

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.

The detection and measurement of D-norgestrel in human milk using Sephadex LH 20 chromatography and radioimmunoassay.

An accurate sensitive method for the assay of D-norgestrel in human milk is described. The steroid is isolated from an ether extract of milk by Sephadex LH 20 chromatography in the system iso-octane-benzene-methanol (70:20:10 v/v). The radioimmunoassay utilises a specific antibody produced in rabbits against D-'norgestrel 3-(O-carboxymethyl) - oxime coupled to bovine serum albumin with D-norgestrel 3-(0-carboxymethyl) -oxime/ [125I]-iodohistamine conjugate as radioligand. Accuracy, sensitivity and blank value are satisfactory. Milk samples were obtained from three subjects treated with 30 microgram/day D-norgestrel, treatment commencing two weeks following parturition. Significant amounts of D-norgestrel were found, ranging between 92-135 pg/ml milk at the end of the first two-week treatment regimen. In two of three subjects, lower, but significant concentrations (53 pg and 35 pg/ml respectively) of steroid were found at the end of four weeks treatment. In the third subject, D-norgestrel could not be detected at this time. As a check on the specificity of the assay, three samples were submitted to additional chromatographic purification on alumina thin layer in the system benzene-cyclohexane-ethanol (70:27:3 v/v). Although this additional chromatographic step yielded somewhat lower values, agreement between the respective sets of results was good. The significance and implications of these findings are discussed.

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.