RESUMO
Quantitation of progesterone (P(4)) in biological fluids is often performed by radioimmunoassay (RIA), whereas liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been used much less often. Due to its autoconfirmatory nature, LC-MS/MS greatly minimizes false positives and interference. Herein we report and compare with RIA an optimized LC-MS/MS method for rapid, efficient, and cost-effective quantitation of P(4) in plasma of cattle with no sample derivatization. The quantitation of plasma P(4) released from three nonbiodegradable, commercial, intravaginal P(4)-releasing devices (IPRD) over 192 h in six ovariectomized cows was compared in a pairwise study as a test case. Both techniques showed similar P(4) kinetics (P > 0.05) whereas results of P(4) quantitation by RIA were consistently higher compared with LC-MS/MS (P < 0.05) due to interference and matrix effects. The LC-MS/MS method was validated according to the recommended analytical standards and displayed P(4) limits of detection (LOD) and quantitation (LOQ) of 0.08 and a 0.25 ng/mL, respectively. The high selective LC-MS/MS method proposed herein for P(4) quantitation eliminates the risks associated with radioactive handling; it also requires no sample derivatization, which is a common requirement for LC-MS/MS quantitation of steroid hormones. Its application to multisteroid assays is also viable, and it is envisaged that it may provide a gold standard technique for hormone quantitation in animal reproductive science studies.
Assuntos
Cromatografia Líquida/veterinária , Progesterona/sangue , Espectrometria de Massas em Tandem/veterinária , Animais , Bovinos , Cromatografia Líquida/métodos , Sincronização do Estro/métodos , Feminino , Espectrometria de Massas em Tandem/métodosRESUMO
Spontaneous Ca(2+) events have been observed in diverse stem cell lines, including carcinoma and mesenchymal stem cells. Interestingly, during cell cycle progression, cells exhibit Ca(2+) transients during the G(1) to S transition, suggesting that these oscillations may play a role in cell cycle progression. We aimed to study the influence of promoting and blocking calcium oscillations in cell proliferation and cell cycle progression, both in neural progenitor and undifferentiated cells. We also identified which calcium stores are required for maintaining these oscillations. Both in neural progenitor and undifferentiated cells calcium oscillations were restricted to the G1/S transition, suggesting a role for these events in progression of the cell cycle. Maintenance of the oscillations required calcium influx only through inositol 1,4,5-triphosphate receptors (IP(3)Rs) and L-type channels in undifferentiated cells, while neural progenitor cells also utilized ryanodine-sensitive stores. Interestingly, promoting calcium oscillations through IP(3)R agonists increased both proliferation and levels of cell cycle regulators such as cyclins A and E. Conversely, blocking calcium events with IP(3)R antagonists had the opposite effect in both undifferentiated and neural progenitor cells. This suggests that calcium events created by IP(3)Rs may be involved in cell cycle progression and proliferation, possibly due to regulation of cyclin levels, both in undifferentiated cells and in neural progenitor cells.