Double Isotope Derivative Assay Research Articles

Testosterone analysis in prostate cancer patients.

Testosterone is an essential steroid hormone associated with a wide variety of biological processes in humans. In prostate cancer, androgen signaling is an important driver of tumor cell growth. Depletion of gonadal testosterone, achieved by surgical or chemical castration, prevents androgenic signaling and temporally reduces, stops or reverses tumor growth before inevitable progression to castration-resistant prostate cancer occurs. Additional treatment strategies targeting androgenic signaling have become available, although these are without curative intent. While circulating testosterone is also associated with disease risk and potential clinical utility, the main use in the clinical lab is monitoring adequate castration and subsequent resistance to therapy. Adequate castrate testosterone concentrations are currently based on over 50 year-old double-isotope derivative assays that are disputed in automated immunoassay (IA) analysis. The debate has been further fueled with the introduction of mass spectrometry-based assays for testosterone, offering a substantial increase in sensitivity and specificity. In this review, we discuss testosterone regulation and androgen deprivation therapy in prostate cancer. We provide an overview of the developments in testosterone analysis for monitoring adequate castration and resistance to therapy. Current clinical practice and future clinical utility will be discussed. Finally, clinical and research recommendations will be presented.

A double-isotope derivative assay for glucosamine, galactosamine and mannosamine in nanomole and picomole amounts

A micro-assay for glucosamine, galactosamine, and mannosamine in mixtures containing amino acids is described. The procedure involves the reduction of these hexosamines in the presence of 14C-labelled standards, followed by their reaction with 3H-dansyl chloride. By a combination of thin-layer electrophoresis and thin-layer chromatography on a single plate, the dansylated hexosamine derivatives are both separated from those of the amino acids, and resolved from one another. The 3H/14C ratio of the hexosamine derivatives is linearly related to the amount of hexosamine originally present. The method will assay as little as 400 pmoles of any one of these three hexosamines.

Radioimmunoassay of cortisone in the adult Atlantic salmon, Salmo salar L

A radioimmunoassay (RIA) was developed to measure cortisone in Atlantic salmon plasma. Diatomaceous earth microcolumn chromatography was used to separate the cortisone from other steroids. The RIA method following chromatography was validated by analyzing each of six samples of blood by double isotope derivative assay (DIDA) and RIA. The RIA gave significantly lower plasma cortisone concentrations ( P < 0.05) than the DIDA: the correlation coefficient was 0.84. Accuracy of the RIA was also demonstrated by addition of radioinert cortisone and measurement of increasing volumes of plasma. Specificity of the antisera was very good. Sensitivity of the RIA was 11 pg/RIA tube or 0.025 μg cortisone/dl plasma corrected for recovery and fraction assayed. Plasma cortisone concentrations in adult Atlantic salmon ranged from 2 to 26 μg/dl. The mean plasma cortisone concentration of salmon in fresh water was not significantly different from that in sea water. The mean cortisone/cortisol ratio in both adult and juvenile Atlantic salmon was between 1.40 and 1.92 indicating that cortisone and not cortisol may be the principal corticosteroid in Atlantic salmon.

Isolation and identification of testosterone from the serum and testes of the American lobster ( Homarus americanus)

Testosterone was isolated and quantified from male lobster serum and testes by solvent extraction, sequential thin-layer chromatography, and high-performance liquid chromatography. The identity of the isolated steroid was established by its isopolarity and isomorphicity with authentic radiolabeled testosterone and its acetate derivative. The concentrations of testosterone were determined by high-performance liquid chromatography, by a double-isotope derivative assay, and by radioimmunoassay. The concentrations of testosterone as determined by the three methods were the same and were 0.3 ng/ml and 14.3 ng/g in lobster serum and testes, respectively.

Radioimmunoassay of cortisol in the adult Atlantic salmon, Salmo salar L

A radioimmunoassay (RIA) was developed to measure cortisol in Atlantic salmon plasma. The method was validated by analyzing each of six samples of blood by three different methods: double-isotope derivative assay (DIDA) and RIA with and without chromatographic purification of the plasma extract. The RIA without chromatography gave significantly lower plasma cortisol concentrations ( P < 0.01) than the DIDA: the correlation coefficient was 80.3%. The RIA with chromatography tended to give lower values than the DIDA but higher values than the nonchromatographic RIA. 11-Deoxycortisol which was definitively identified in the plasma of Atlantic salmon by DIDA did not interfere with the RIA.

The presence of steroids in the sera of the Pacific hagfish, Eptatretus stouti, and the sea lamprey, Petromyzon marinus

Cortisol, 11-deoxycortisol, corticosterone, 11-dehydrocorticosterone, and testosterone were identified by double-isotope derivative assays (DIDA) in the sera of Pacific hagfish and of sea lamprey. 17α-Hydroxy-20β-dihydroprogesterone was rigorously identified in the serum of ACTH-treated sea lamprey. No cortisone or 11-deoxycorticosterone was found in either fish. Circulating levels of cortisol, 11-deoxycortisol, and corticosterone in individual hagfish were also measured by a radioimmunoassay method. Multiple injections of ACTH in hagfish consistently elevated serum concentration of corticosterone. ACTH effects on other steroids were variable.

Corticosteroids of the coelacanth Latimeria chalumnae smith: A provisional study on their identity

An extract of frozen tissue cut from an area of the coelacanth kidney, presumed to be the site of adrenocortical cells, was analyzed by double isotope derivative assay for adrenocorticosteroids. Evidence for the presence of 11-deoxycorticosterone, cortisol, and corticosterone was obtained. No 11-deoxycortisol or cortisone was detected. Incubation of the tissue with exogenous radioactive steroid precursors and identification of some transformation products indicated the presence of active enzyme systems including 5-ene-3β-hydroxysteroid dehydrogenase, 5-ene-4-ene-isomerase, 20α-hydroxysteroid dehydrogenase, 11β-hydroxysteroid dehydrogenase, and pregnene-5α- and 5β-hydrogenases.

The determination of cortisol in fish plasma by radioimmunoassay

A radioimmunoassay (RIA) method using antiserum to cortisol-21-succinyl-BSA was devised to determine cortisol in fish plasma using the plasma of cod Gadus morhua. Steroid extracts were purified by thin layer chromatography before RIA. Saturated ammonium sulfate was used to separate protein-bound from free [ 3H]cortisol. The method is precise and accurate and validated by double isotope derivative assay (DIDA).

Reliable routine method for the determination of plasma amitriptyline and nortriptyline by gas chromatography

A gas chromatographic method has been developed for the determination of amitriptyline and nortriptyline in plasma. OV-17 is used in a 1 m long packed column, with a flame ionization detector and an electronic integrator. Five internal standards are added. The base-specific extraction procedure and the method of calibrating the chromatograph are described in detail. The accuracy, precision and reliability of the method are demonstrated by the results of nearly 700 determinations of each drug, at concentrations ranging from 5 to 400 ng/ml in the plasma. An interlaboratory comparison with a double radioactive isotope derivative assay for nortriptyline has also shown satisfactory agreement.

Identification of cortisol in plasma of the embryonic chick

Cortisol has been identified by double isotope derivative assay in plasma of 15 day old embryonic chicks. To our knowledge this represents the first definitive identification of cortisol in avian peripheral plasma; the concentration was 64.5 ng/100 ml plasma.

A comparison of 11-hydroxytestosterone and 11-ketotestosterone in blood of ambisexual and gonochoristic teleosts

Samples of whole blood or plasma from four ambisexual and two gonochoristic teleost species were investigated for the occurrence of 11β-hydroxytestosterone and 11-ketotestosterone. The steroids were identified and quantified by double-isotope derivative assays (DIDA) after initial separation and purification by thin-layer and paper chromatography. In all samples from the ambisexual fish 11β-OH-testosterone was found in respectable quantities, whereas 11-ketotestosterone could not be detected in these animals. In the gonochoristic winter flounder and Atlantic salmon, 11-ketotestosterone was the predominant androgen, and compared to the ambisexual fish the concentration of 11β-hydroxytestosterone was extremely low.

Effects of sublethal cadmium on maturation and testosterone and 11-ketotestosterone production in vivo in the brook trout.

A double-isotope derivative assay using [“C]steroid tracers and [‘H]acetic anhydride as tritiating agent was used to determine testosterone and li-ketotestosterone levels in the blood plasma of male brook trout maintained in freshwater containing 1 ppb cadmium (Cd) and control fish during various stages of sexual maturation. Plasma testosterone and 1 1-ketotestosterone levels were correlated to approaching period of functional maturity in the control fish,reached a peak of 0.25 and 2.68 Mg/i00 ml plasma, respectively, and gradually declined with the onset of testicular regression. 11-Ketotestosterone levels (2.34-3.96 �g/100 ml) in Cd-treated fish were higher than in the controls (p <0.05) and remained elevated during testicularregression. Testosterone levels in the treated fish increased 2 wk later than the control and also remained elevated throughout. There was no apparent effect of the level of Cd used on the onset of secondary sexual characteristics and spermatogenesis but the testes of Cd-treated fish regressed at least 2 wk earlierthan the control. The results suggested an impairment in the clearance and/or utilizationof testosterone and li-ketotestosterone by Cd-treated fish. The impaired clearance may also be related to the abnormal vasculature and hemorrhagic necrosis observed in the testes of some of the Cd-treated fish.

Corticosteroid levels in the mother and fetus of the rabbit during gestation.

The cortisol and corticosterone levels in the fetal and maternal plasma were measured during the last 10 days of gestation by a double isotope derivative assay. The cortisol levels in the fetal plasma increased from 0.89 μg/100 ml to 1.38 μg/100 ml between day 22 and 30 and decreased to 0.69 μg/100 ml in the newborn. Cortisol level in maternal plasma on day 22 (0.61 μg/100 ml) was higher than in the nonpregnant female (0.26 μg/100 ml) and a gradual increase was observed between day 22 and day 30 (1.83 μg/100 ml). Postpartum, the cortisol concentration decreased to nonpregnant levels. Plasma corticosterone levels fluctuated considerably both in the fetal and maternal plasma; the fetal plasma corticosterone decreased from 1.18 μg/100 ml on day 22 to 0.45 μg/100 ml on day 27 with an increase on day 28 (2.52 μg/100 ml) followed by a decrease on day 29 and 30. Blood corticosterone in the pregnant rabbit were similar to the values found in the nonpregnant female (2.42 μg/100 ml) up to day 26, but a decrease was...

Modification of rabbit adrenal steroid biosynthesis by prolonged ACTH administration.

A series of in vitro experiments using 4-14C-pregnenolone and 4-14C-progesterone as precursors were performed to determine the effects of prolonged adrenocorticotrophic hormone (ACTH) administration on adrenal steroid biogenesis in rabbits. The adrenal steroids were isolated by paper chromatography and quantitated by the double isotope derivative assay method. In the control group, there was no distinction between the roles of pregnenolone and progesterone as precursors of adrenal steroids. Following the administration of 25 U of porcine ACTH for 21 days, however, 17α-hydroxylated steroids became quantitatively important. Prolonged exogenous ACTH treatment enhanced the specific activities of the 17α-hydroxycorticosteroids from both precursors, but the specific activities were enhanced more from pregnenolone than from progesterone. Under experimental conditions more than twice as much pregnenolone was metabolized via 17α-hydroxypregnenolone bypassing the progesterone step than under control conditions. Pro...

Plasma progesterone concentration in the hen in relation to the ovulatory cycle.

Synopsis The concentration of peripheral plasma progesterone was measured by a double isotope derivative assay in hens slaughtered at various stages during the ovulatory cycle. A peak value was obtained 2 to 6 h before ovulation and basal levels were reached at ovulation. No secondary peaks were observed during the cycle. Similar results were obtained using a protein‐binding assay on plasma from individual birds taken serially during the cycle. Birds which did not ovulate on a particular day showed no peak of progesterone. In two birds sampled at the beginning of a laying cycle, the progesterone peak was of longer duration.

A sensitive double isotope derivative assay to measure release of amino acids from brain in vitro.

I-DIMETHYLAMINONAPHTHALENE-5 sulphonic acid, used as its chloride derivative dansyl chloride, forms highly fluorescent derivatives with compounds containing amino groups. Dansyl chloride has proved useful in peptide sequence studies1–4 as well as in the qualitative analysis of mixtures of amino acids, amines and related compounds5–7. Recently, it has been used for the quantitative micro analysis of amino acids8–10 and such assays have been made even more sensitive by the use of radioactively labelled dansyl chloride11. Its use for quantitative amino acid analysis, however, presents problems of undesirable side reactions5,11,12, variation in the degree to which different amino acids are dansylated5,11 and variation due to changes in the ratio of reagent to substrate11. We have used high specific activity 14C-amino acids as internal standards to overcome these sources of error when using 3H-dansyl chloride, so that the ratio between 3H and 14C radioactivity depends upon the ratio of 14C-amino acid to non-radioactive amino acid present in the sample. By this technique we can measure minute amounts of amino acids in microgram samples of brain tissue and it can be used to measure release of endogenous amino acids from brain slices in vitro.

Peripheral plasma progesterone and oestradiol-17 levels before and after puberty in gilts.

Summary. A double isotope derivative assay and a simple competitive protein-binding assay were used to measure peripheral plasma progesterone levels during the pig oestrous cycle. In view of the similarity between the results, the more convenient protein-binding method was employed to determine progesterone levels during prepuberty, the oestrous cycle, early and late pregnancy, parturition and lactation. Peripheral plasma oestradiol-17β levels were measured during the oestrous cycle, early pregnancy and around the time of parturition.

Corticosteroids in plasma of elasmobranchs

1. 1. Free corticosteroids were quantified by double isotope derivative assay in thirteen plasma samples from seven species of elasmobranchs. 2. 2. In each instance, four batoids and three selachians, 1α-hydroxycortico-sterone was the principal plasmatic corticosteroid. 3. 3. In both samples from one selachian, the dogfish Squalus acanthias, there were relatively high levels of corticosterone. The corticosterone concentration was very low or below detectable levels in all other plasma samples. 4. 4. 11-Deoxycorticosterone was detected in all seven samples in which it was sought. In all batoids the plasma levels of 11-deoxycorticosterone exceeded those of corticosterone. 5. 5. 11-Dehydrocorticosterone was detected in the plasma of five samples and was below the detectable level in two samples. 6. 6. Cortisol and cortisone were below the detectable levels in all plasma samples. This finding confirms other evidence that 17α-hydroxylation does not occur to a significant extent in elasmobranchs.

Hypophysial corticotropic function and its hypothalamic control in Bufo bufo (L.) evaluated by the plasma concentration of corticosterone.

Corticosterone was measured by a double isotope derivative assay. The quantifying reagent was [ 35 S]4-toluene sulfonic anhydride ([ 35 S]Tosan). Addition of [ 3 H]corticosterone made possible the correction for loss throughout the procedure. Reliability criteria and applicability of the method are discussed. Corticosterone was demonstrated in peripheral plasma of adult female toads at concentrations of 0.5–1.8 μg per 100 ml of plasma (sample volume: 300–1000 μl). The plasma concentration of this hormone was used as a measure of the secretory activity of the adrenocortical tissue. After extirpation of the pars distalis of the pituitary, corticosterone could not be detected (the limit of detection for a single determination was 0.5 ng). One hour after the injection of 0.4 or 2 mU porcine ACTH into “pars distalis-ectomized” toads, corticosterone concentrations were similar to the level in the unoperated control group, and doses of 10 or 50 mU ACTH caused stimulation up to ten times this control level. The results indicate that the pars distalis stimulates the secretory activity of the adrenocortical tissue, probably by a hormone similar to mammalian ACTH. After transplantation of the pars distalis to an eye muscle, corticosterone could not be detected. On the basis of the corticosterone determinations, corticotropic function thus seems to be dependent on the normal structural connections between the central nervous system and the pars distalis. The location of central nervous structures regulating the corticotropic function was investigated by transection of the brain stem at various levels. Plasma corticosterone was below or slightly above the limit of detection after transection between the optic chiasma and the median eminence, whereas concentrations comparable to those of the unoperated control groups were measured after transection rostral to the optic chiasma. These results indicate that a hypothalamic area is involved in the control of the corticotropic function, and that this area is located at the level of the optic chiasma.

Identification and metabolic clearance of cortisol and cortisone in amarine teleost, the sea raven Hemitripterus americanus Gmelin (family Scorpaenidae).

SUMMARY Cortisol and cortisone were identified and their levels determined in sea raven plasma by a double isotope derivative assay involving acetylation with [1-3H]acetic anhydride, purification by thin-layer and paper chromatography, followed by recrystallization to constant 3H: 14C ratios. The mean level of cortisol in four plasma samples was 7·2±1·0 μg/100 ml (range 4·0–9·2) and the mean level for cortisone in three samples was 1·1 ± 0·2 μg/100 ml (range 0·7–1·5). Metabolic clearance rates (MCR) were determined for both corticosteroids by the method of continuous infusion over an 8-h period. The mean MCR for cortisol in five fish was 126 ± 17 ml/kg/h, and for cortisone 449 ± 48 ml/kg/h in six fish. The mean percentage conversion of [1,2-3H]cortisol to cortisone was 9·4 ± 2·9%. There was no evidence of any significant conversion of cortisone to cortisol.