Neurosteroids and ms

Neurosteroids were isolated separately in a two steps procedure by using ethyl acetate-n-hexane (90:10) as the first step to extract the unconjugated steroids, then the steroid fractions were further purified by SPE. All steroids were derivatized with 2-nitro-4-trifluoromethylphenylhydrazine (2-NFPH) and analyzed by HPLC-MS ( APCI) using selected-ion monitoring. Methyltestosterone was chosen as the internal standard. Results The linear calibration curve of DHEA was obtained in the concentration range of - microg x L(-1). The linear calibration curves of PREG and AP were obtained in the concentration range of - microg x L(-1). The concentrations of DHEA, PREG and AP in male rat brain regions were ( +/- ), ( +/- ), ( +/- ) ng x g(-1) for frontal cortex, ( +/- ), (6 +/- 3), ( +/- ) ng x g(-1) for hippocampus, ( +/- ), (9 +/- 5), ( +/- ) ng x g(-1) for amygdale, ( +/- ), ( +/- ), ( +/- ) ng x g(-1) for striatum, ( +/- ), (18 +/- 9), ( +/- ) ng x g(-1) for nucleus accumbens, ( +/- ), (27 +/- 12), ( +/- ) ng x g(-1) for pituitary gland, ( +/- ), ( 16 +/- 10), and (0. 8 +/- ) ng x g(-1) for hypothalamus, respectively.

Steroid hormones that are produced de novo, metabolized and accumulated in the CNS are called ‘neurosteroids’. Neurosteroids are endogenous neuroprotectants, reduce inflammation and increase neuronal and glial differentiation and myelination in the CNS [1]. They may therefore influence multiple pathological aspects of MS.  In the last two years Dr. Luchetti investigated how neurosteroid synthetic pathway and hormone receptors change in Multiple sclerosis (MS) pathology [See figure].  In this study, pathway of neurosteroid synthesis and hormone receptors is analyzed by qPCR and immunohistochemistry in different lesions (. chronic active, chronic inactive, remyelinating areas) and normal appearing white matter of MS patients [4].  Sex differences in neurosteroid synthesis in the MS lesions are also investigated in relation to different aspects of MS pathology such as neuroinflammation, neurodegeneration and de/remyelination processes. Alterations of neurosteroid synthetic pathway and hormone receptor are also evaluated in  MS lesion of female and male patients with different course of the disease (. relapsing remitting–secondary progressive versus primary progressive MS). She recently obtained the first evidence that the neurosteroid biosynthetic pathways are indeed changed in MS tissue, and that these changes are partly gender specific [4]. These data support a key role for neurosteroids in MS pathology.

Description:   We provide consultation and services for the following: 1. Multiparameter cell phenotyping up to 11-color antibody combinations. 2. Analysis applications such as cell activation, proliferation, cell cycle/DNA content, apoptosis, surface marker expressions, intracellular expressions, quantification of internal, regulatory and structural proteins and quantification of reporter genes (GFP, DsRed, mCherry, etc). 3. High-speed cell sorting of live and biohazardous samples, single-cell deposition into 96-well plates. Core instruments include: MoFlo XDP sorter, FACSAria II sorter, FACSCanto analyzers.

This article describes the development and validation of a sensitive and robust method for the determination of neurosteroids in sea lamprey, an ancestral animal in vertebrate evolution. Chemical derivatization was used to enhance the detection of neurosteroids containing ketone function by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Iminooxy derivatives of 12 oxosteroids and three internal standards were monitored by positive electrospray tandem mass spectrometry using the neutral loss of sulfate. Limit of quantification, extraction recovery and matrix effect were first evaluated and SPE using C18 sorbent was selected after comparison with liquid/liquid extraction and protein precipitation. Matrix effect ranged from % to % in plasma and from % to % in the brain. Recovery values ranged from % to % in plasma and from % to % in the brain. Chromatographic separation was achieved by reverse phase chromatography (×100mm, µm particle size, C18) with a binary gradient between methanol and % formic acid in water. Limit of quantification ranged from 5 to 10pg/mL and was up to 80 times lower than those for non-derivatized steroids. Accuracy and precision parameters were determined and inter- and intra-day at three concentrations: 50, 500 and 5000pg/mL. This method was applied to analyze real samples. progesterone (P), pregnenolone (P5), 7-hydroxy-pregnenolpne (7P5), 17-hydroxy-pregnenolpne (17P5)dehydroepiandrosterone (DHEA), androstenedienone (A4), testosterone (T), dihydrotestosterone (DHT), allopregnanolone (THP), 11-hydroxy-androstenedienone (11A4) and 11-Deoxycortisol (S) were measured in sea lamprey brain and plasma matrixes.

Neurosteroids and ms

neurosteroids and ms

This article describes the development and validation of a sensitive and robust method for the determination of neurosteroids in sea lamprey, an ancestral animal in vertebrate evolution. Chemical derivatization was used to enhance the detection of neurosteroids containing ketone function by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Iminooxy derivatives of 12 oxosteroids and three internal standards were monitored by positive electrospray tandem mass spectrometry using the neutral loss of sulfate. Limit of quantification, extraction recovery and matrix effect were first evaluated and SPE using C18 sorbent was selected after comparison with liquid/liquid extraction and protein precipitation. Matrix effect ranged from % to % in plasma and from % to % in the brain. Recovery values ranged from % to % in plasma and from % to % in the brain. Chromatographic separation was achieved by reverse phase chromatography (×100mm, µm particle size, C18) with a binary gradient between methanol and % formic acid in water. Limit of quantification ranged from 5 to 10pg/mL and was up to 80 times lower than those for non-derivatized steroids. Accuracy and precision parameters were determined and inter- and intra-day at three concentrations: 50, 500 and 5000pg/mL. This method was applied to analyze real samples. progesterone (P), pregnenolone (P5), 7-hydroxy-pregnenolpne (7P5), 17-hydroxy-pregnenolpne (17P5)dehydroepiandrosterone (DHEA), androstenedienone (A4), testosterone (T), dihydrotestosterone (DHT), allopregnanolone (THP), 11-hydroxy-androstenedienone (11A4) and 11-Deoxycortisol (S) were measured in sea lamprey brain and plasma matrixes.

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