Evaluation of neuroactive steroid levels by liquid chromatography-tandem mass spectrometry in central and peripheral nervous system: effect of diabetes.

The nervous system is a target for physiological and protective effects of neuroactive steroids. Consequently, the assessment of their levels in nervous structures under physiological and pathological conditions is a top priority. To this aim, identification and quantification of pregnenolone (PREG), progesterone (PROG), dihydroprogesterone (DHP), tetrahydroprogesterone (THP), testosterone (T), dihydrotestosterone (DHT), 5alpha-androstan-3alpha, 17beta-diol (3alpha-diol), 17alpha- and 17beta-estradiol (17alpha-E and 17beta-E) by liquid chromatography and tandem mass spectrometry (LC-MS/MS) has been set up. After validation, this method was applied to determine the levels of neuroactive steroids in central (i.e., cerebral cortex, cerebellum and spinal cord) and peripheral (i.e., brachial nerve) nervous system of control and diabetic rats. In controls only the brachial nerve had detectable levels of all these neuroactive steroids. In contrast, 17alpha-E in cerebellum, 17alpha-E, 17beta-E, DHP and THP in cerebral cortex, and 17alpha-E, 17beta-E and DHP in spinal cord were under the detection limit. Diabetes, induced by injection with streptozotocin, strongly affected the levels of some neuroactive steroids. In particular, the levels of PREG, PROG and T in cerebellum, of PROG, T and 3alpha-diol in cerebral cortex, of PROG, DHT and 3alpha-diol in spinal cord and of PREG, DHP, THP, T, DHT and 3alpha-diol in brachial nerve were significantly decreased. In conclusion, the data here reported demonstrate that the LC-MS/MS method allows the assessment of neuroactive steroids in the nervous system with high sensitivity and specificity and that diabetes strongly affects their levels, providing a further basis for new therapeutic tools based on neuroactive steroids aimed at counteracting diabetic neuropathy.

Short acidic peptides isolated from wheat sprout chromatin and involved in the control of cell proliferation. Characterization by infrared spectroscopy and mass spectrometry.

Low molecular weight peptides were isolated from the chromatin of wheat sprouts. Following gel filtration the peptide fraction shows a sharp inhibiting activity on the growth of HeLa cancer cells. Infrared (IR) spectroscopy and mass spectrometry have been utilized to characterize the wheat sprout peptides in an attempt to recognize the peptide sequence involved in the control of cell growth. The quantitative presence of a peptide with MH+=572 appears proportional to the cell growth inhibition activity. This compound has been subjected to extensive mass spectrometry analysis. The automatic computational analysis of the ions of second, third and fourth generations indicate a peptide sequence, AcHis-Asp-Ser-Glu-, that binds at the C-terminal a molecule of ethanolamine. Moreover, the results show that some sequences of the wheat sprout peptide family are present in the peptide fractions isolated from several other tissues, thus supporting the hypothesis of ubiquitous regulatory peptides.

Cigarette smoke negatively and dose-dependently affects the biosynthetic pathway of the n-3 polyunsaturated fatty acid series in human mammary epithelial cells.

Maternal smoking during pregnancy has been associated with a reduced content of n-3 long-chain PUFA (LC-PUFA) in breast milk, thereby reducing the intake of key nutrients by the infants. We postulated that the mammary gland is affected by maternal smoking in the process of n-3 LC-PUFA secretion into milk. This prompted us to investigate the effects of cigarette smoke on the synthesis of n-3 LC-PUFA in vitro by using a line of healthy epithelial cells from the human mammary gland, MCF-10A. Cells were exposed to cigarette smoke under controlled conditions by adding to the medium aliquots of horse serum containing smoke components, as analyzed by GC-MS. The major findings concern the inhibition of both the conversion of the precursor 14C-ALA (alpha-linolenic acid) to n-3 LC-PUFA and of the A5 desaturation step (assessed by HPLC analysis with radiodetection of n-3 FAME) following exposure to minimal doses of smoke-enriched serum, and the dose-dependent relationship of these effects. The data indicate that exposure to cigarette smoke negatively affects the synthesis of n-3 LC-PUFA from the precursor in mammary gland cells.