Biomedical subjects
Publications and source records attributed to G F Read.
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We report a radioimmunoassay sensitive enough to determine 17 alpha-hydroxyprogesterone concentrations in 200 microliter of parotid fluid or mixed whole saliva. Because the correlation of concentrations in matched samples of parotid fluid and saliva was excellent (r = 0.98), we exclusively used saliva, which is easier to collect, in later studies. The assay is specific; saliva samples assayed with and without thin-layer chromatographic purification showed no significant difference. The assay is also precise, and has a lower limit of sensitivity of 4 pg per assay tube. In 14 patients having congenital adrenal hyperplasia from a C21-hydroxylase enzyme deficiency, all of whom were receiving cortisol replacement therapy, the range in 17 alpha-hydroxyprogesterone concentrations observed in saliva (67-26,300 pmol/L) was about 20-fold that seen in 32 healthy children (90-1520 pmol/L). The close correlation (r = 0.91) between 17 alpha-hydroxyprogesterone concentrations in matched samples of saliva and plasma from these patients indicates that determination of steroids in saliva could well replace determination in plasma. This concept is supported by 17 alpha-hydroxyprogesterone concentrations monitored throughout 24 h from one patient and following stimulation with synthetic corticotropin in another patient.
We report a specific radioimmunoassay that has the required sensitivity (7 pg per assay tube) for determining progesterone concentrations in 400 microL of mixed saliva collected from normal women. The assay is precise: intra and inter-assay variation (CV) never exceeded 11.0 and 8.0%, respectively. The assay was used to determine progesterone in saliva samples collected daily for not less than 28 days by normal women and by patients having abnormal ovarian function. Four normal women provided matched saliva and plasma samples for accurate dating of the menstrual cycle by plasma progesterone, estradiol, lutropin, and follitropin. Nine further subjects collected saliva samples only, and from these data a provisional "normal range" was established. Progesterone concentrations in saliva during the follicular phase of the cycle were low (less than 100 pmol/L) but rose beginning on day 12 to reach peak values of 230-550 pmol/L on day 21. Thereafter, progesterone concentrations in saliva declined to values generally less than 170 pmol/L at the commencement of menses. Saliva samples from three patients attending an infertility clinic were also studied to assess ovarian function.
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We describe an enzyme immunoassay for progesterone in which we use a progesterone-11alpha-hemisuccinyl-horseradish peroxidase conjugate as the "label" and an antiserum raised in rabbits to a progesterone-11alpha-hemisuccinyl-bovine serum albumin conjugate. In this assay, antibody-bound and free steroid are separated by using a second antibody precipitation procedure. The assay has a lower limit of sensitivity of 10 pg/assay tube and satisfies the usual criteria of specificity, precision, and accuracy. Results obtained with a comparison radioimmunoassay and our procedure agreed well (r = 0.98). Eighty samples can be assayed per day. It is not only well suited for surveys where accurate determination of progesterone concentrations in small plasma aliquots is required, but also for monitoring ovulation induction in patients attending infertility clinics.
A radioimmunoassay is reported for the tricyclic antidepressant drug, clomipramine (Anafranil, Geigy). Antisera generated in rabbits to clomipramine conjugated to bovine serum albumin at positions 10 and 11 were specific, cross reacting less than 5% with the pharmacologically active metabolite, desmethylclomipramine. Specificity was confirmed by the good agreement in titres observed when the samples were assayed with and without a pre-assay thin-layer chromatographic purification. Intra- and inter-assay variations were less than 8 and 13%, respectively, with a sensitivity of 175 ng/liter. Results obtained agreed well with those reported by other groups using double radioisotope derivative assays. The method has a high throughput rate, one technician can assay 200 samples in duplicate in a working week; and it is sufficiently precise, sensitive, and specific for use in routine minitoring of the drug in plasma and for checking patient compliance with dosage regimen.
We describe a direct radioimmunoassay for cortisol in 10-microliter volumes of parotid saliva or whole saliva. Binding proteins are absent from these fluids, as demonstrated by the excellent correlation between results for samples assayed directly and by a comparison procedure involving extraction with 1,2-dichloroethane. The direct assay is specific, precise, and had a lower limit of sensitivity of 4 pg per assay tube. Comparison of cortisol concentrations in plasma, parotid saliva, and whole saliva in persons undergoing investigations for assessing adrenal function, including stimulation with cosyntropin (Synacthen) and suppression with dexamethasone, indicated that changes in plasma cortisol concentration were accurately and immediately reflected in saliva from either the parotidgland or whole saliva. A marked circadian rhythm has also been demonstrated for cortisol in parotid-gland saliva and whole saliva. We had to modify the 1,2-dichloroethane extraction procedure for accurate determination of cortisol in parotid saliva and whole saliva of patients undergoing treatment with metyrapone.
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.
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.
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 radioimmunoassay for plasma cortisol featuring the gamma-emitting radioligand 125I-iodohistamine, coupled to cortisol-3-(O-carboxymethyl)-oxime, is described. The new procedure retains much of the specificity associated with the use of anti-cortisol-3-BSA sera with tritium-labelled radioligands, and has the further advantages that running costs are lower and there is a greater potential for automation. Cortisol values obtained by this procedure agree well with those obtained by a published specific radioimmunoassay using the tritiated cortisol radioligand. Specificity of the procedure was checked by comparing values obtained with and without thin-layer chromatography purication: correlation was excellent (r = 0.96). Satisfactory levels of sensitivity, precision, and accuracy were obtained.
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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.
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.
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This study was carried out to investigate the relationship between the level of training and the impairment of ovarian function among female recreational distance runners, and its reversibility. Thirty-six female distance runners self-recorded, for 7 consecutive days each month, the duration and distance of daily running from October 1989 to May 1990 in Great Britain. During the last 3 months of the survey, saliva samples were taken for progesterone assay and a subsample was measured for body composition. No trend in weight loss was observed over the season of training. Amenorrheic (AM) and oligomenorrheic (Oligo) runners had a significantly lower body mass index than eumenorrheic (EU) and irregularly menstruating (IM) runners. The amenorrheic and oligomenorrheic subjects did not show any rise in progesterone, at any time, during the 3 months of sampling. The eumenorrheic subjects showed evidence of a rise in progesterone, though the mean level was always significantly lower than that of sedentary controls. The most severely impaired runners (AM and Oligo) ran more than EU or irregularly menstruating runners in this sample, had lower body weight, a younger age and had a significantly lower body mass index (BMI). They tended to run faster during training sessions than those with apparently normal menstrual cycle or just irregular periods. It is suggested that low BMI, which is an indicator of body energy stores, reflects the intensity of regular training runs among female athletes with a stable body weight. It is possible that repeated elevations of beta-endorphins or other suppressors of gonadotropin release, secreted above a level of training commonly exceeded by long distance runners, when concurrent with energy restriction, could contribute to impairment of menstrual cycle.