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Serotonin Metabolism in Functional Somatic Illness
Published in Peter Manu, The Psychopathology of Functional Somatic Syndromes, 2020
The pre-tryptophan challenge data indicated that in the premenstrual syndrome group, the allopregnanolone levels increased from 5 ng/mL in the follicular phase to 11 ng/mL at the time of ovulation and 12 ng/mL during the luteal phase. In contrast, the corresponding levels (5,5, and 7 ng/mL) measured in the control group were significantly lower in the second half of the cycle (p < 0.05). The behavior of pregnenolone and tetrahydrocorticosterone followed a similar pattern. After the tryptophan challenge, only the concentrations of allopregnanolone showed a significant increase. This change was most striking during the luteal phase in the premenstrual syndrome group. The authors felt that this increase “strongly suggests that there is a relationship between central serotonergic activity and circulating allopregnanolone concentrations” and reflects “lower baseline serotonergic activity in this group of women” (Rasgon et al., 2001, p. 145).
Steroid Δ4-Reductases: their Physiological Role and Significance
Published in Ronald Hobkirk, Steroid Biochemistry, 1979
There have been a number of interesting studies from the laboratories of Colby and Kitay on corticosterone Δ4-5α-reductase in the rat adrenal cortex. This enzyme is under the control of a number of hormones. There is a relationship between the levels of this enzyme and the secretory products of the adrenal cortex, e.g., when enzyme activity is high corticosterone secretion decreases and the rates of 5α-dihydrocorticosterone and 3β,5α-tetrahydrocorticosterone secretion increase. Hypophysectomy leads to an increased adrenal 5α-reductase activity in 1 week and this response appears to play a significant role in the decreased responsiveness of the adrenal to ACTH when corticosterone secretion is the measured end point.34 Several days of ACTH administration leads to decreased enzyme activity and increased corticosterone secretion in response to ACTH injection.34 Administration of growth hormone and prolactin as well as the steroid hormone testosterone and estradiol all lead to decreases in corticosterone Δ4-5α-reductase activity.35,36 The effects of several of these hormones are probably additive. The pineal gland is also involved in the control of the adrenal Δ4-5α-reductase; removal of the pineal leads to a decrease in enzyme activity and a decrease in the secretion of reduced corticosterone metabolites.37 The effect appears to be a direct one of melatonin rather than one that is mediated by the adenohypophysis as direct addition of this compound to adrenal slices stimulates Δ4-5α-reductase activity.38 The levels of corticosterone in the circulation are, of course, kept constant by feedback control but the total steroid secretion by the adrenal cortex changes dramatically with the changes in Δ4-5α-reductase activity. The enzyme does not appear to be specific for corticosterone39 but its physiological role with other steroids is unknown.
Synthesis, Enzyme Localization, and Regulation of Neurosteroids
Published in Sheryl S. Smith, Neurosteroid Effects in the Central Nervous System, 2003
For example, we determined the concentration (10 iM) of the steroid anesthetic alphaxalone to inhibit [35S]TBPS binding to a total rat brain homogenate by 50%, and included that concentration in a binding assay with a rat brain sagittal slice analyzed by autoradiography:27 the convulsant binding in some regions, such as the cerebellum, colliculus, and dentate gyrus, was inhibited even more than 50%, while other regions, such as the parietal and occipital cortex, were little inhibited and possibly enhanced (Figure 2.1). This was followed up with a major study on heterogeneity of binding characteristics of GABAR, using multiple radioligands and multiple modulators. Both allopreganolone and alphaxalone were shown to have differential maximal enhancement on four polypeptide subunits (a, 51 kDA > 53-kDa band; p, 55 kDa > 58-kDa band) identified on SDS-PAGE following photoaffinity labeling of purified total rat brain GABAR with [3H]muscimol or [3H]flunitrazepam.28 The same study showed that the extent of enhancement by alphaxalone of [3H]flunitrazepam binding varied from zero (e.g., cerebellar granule cell layer) to ca. 2-fold (pyriform cortex, lateral septum, globus pallidus); the enhancement of [3H]muscimol binding likewise varied from 4-fold (deep layers of neocortex) to 2.5-fold (superficial cortex, dentate gyrus) to nil (cerebellum).28 Some differences in this steroid modulation of GABAR were observed with respect to regional variation when comparing rat brain to postmortem human brain, using the same two radioligands and the neuroactive steroids alphaxalone and 3a-5α-tetrahydrocorticosterone (THDOC). The latter steroid (3 to 10 yM) inhibited binding of [3H]muscimol in temporal lobe neocortex and occipital cortex, while enhancing it in the hippocampus and frontal and sensorimotor cortex; alphaxalone enhanced [3H]muscimol binding everywhere examined, but to a varying extent, and did not enhance [3H]flunitrazepam binding; instead, it had either no effect or inhibition, depending on the region.29
Melatonin mends adverse temporal effects of bright light at night partially independent of its effect on stress responses in captive birds
Published in Chronobiology International, 2020
Quantitative determination of FCORT levels was completed by enzyme immunoassay DetectX corticosterone kit (Arbor Assays, Michigan, USA). According to the manufacturer’s instructions, the kit is expected to detect corticosterone in several species with concurrent validation in different samples including bird droppings (Alm et al. 2014). A volume of 50 μl from each sample was incubated with both 25 μl corticosterone-peroxidase and 25 μl polyclonal antibody to corticosterone in microplate wells for one hour with shaking at 250 rpm at room temperature. Then, wells were rinsed four times with wash buffer and 100 μl TMB substrate was added to the plate. After 30 min incubation at room temperature, the color reaction was stopped and optical density was measured at 450 nm (PowerWave HT, Biotec, Winooski; USA). Data were analyzed using 4PLC Gen5™ Data Analysis Software (Version 2, Bioteck, Winooski; USA). The standard curve detection range was 78.125 pg/ml – 10000 pg/ml. The analytical sensitivity of FCORT is 18.6 pg/ml with a detection limit of 16.9 pg/mL. The intra- and inter-assay coefficients were 259–2460.6 pg/ml (CV: 4.8–6.3%) and 267.9–2618.3 pg/ml (CV: 7.5–9.9%), respectively. Mean recovery was 98.9%. The cross-reactivities of the assay reported by Arbor Assays were corticosterone (100%), desoxycorticosterone (12.3%), tetrahydrocorticosterone (0.76%), aldosterone (0.62%), cortisol (0.38%), progesterone (0.24%), cortisone (< 0.08%), and estradiol (< 0.08%). FCORT concentration was expressed as ng/g DM · 4 h.
Insights into thermal stress in Japanese quail (Coturnix coturnix): dynamics of immunoendocrine and biochemical responses during and after chronic exposure
Published in Stress, 2018
Franco Nicolas Nazar, Emiliano Ariel Videla, Maria Emilia Fernandez, Maria Carla Labaque, Raul Hector Marin
Plasma corticosterone concentrations were determined by enzyme immunoassay (EIA) using polyclonal antibodies, standards, and their corresponding horseradish peroxidase conjugates (anti-corticosterone CJM006, Department of Population Health and Reproduction, C. Munro, UC Davis, CA). The polyclonal CJM006 antibody cross-reacts with: corticosterone 100%, desoxycorticosterone 14.25%, progesterone 2.65%, tetrahydrocorticosterone 0.90%, testosterone 0.64%, cortisol 0.23%, prednisolone 0.07%, 11-desoxycortisol 0.03%, prednisone <0.01%, cortisone <0.01%, and oestradiol <0.01%. The assays were performed according to the general technique described by Munro & Lasley (1988). Briefly, plasma samples were assayed in duplicates using flat-bottom microtiter plates. These were first coated with 50 µl of the antibody stock diluted in coating buffer (50 mM bicarbonate buffer, pH 9.6, 1: 15000) and were incubated overnight at 4 °C. Later, plates were washed to remove any unbound antibody and immediately after, 50 µl of samples, standards, and controls were added in duplicates, followed by 50 µl of horseradish peroxidase conjugate diluted in EIA buffer (1:70,000). Plates were then incubated at room temperature (21 °C) for 2 h in an orbital shaker. Following incubation, plates were washed and blotted dry, and 100 µl of substrate solution (50 mM citrate, 1.6 mM hydrogen peroxide, and 0.4 mM 2,20-azino-di-(3-ethylbenzthiazoline sulfonic acid) diammonium salt, pH 4.0) were added to each well. Absorbance was measured at 405 nm using a microplate reader (Thermo Electron Corporation, Waltham, MA). For all hormone determinations, the intra- and inter-assay coefficients of variation were <12 and 5.1%, respectively. Assay sensitivity was 0.08 ng/ml.