Sensitive isotope dilution liquid chromatography/tandem mass spectrometry method for quantitative analysis of bumetanide in serum and brain tissue
We have developed and validated a simple and sensitive stable isotope dilution liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for the quantification of bumetanide in human serum. Samples were prepared with a simple acetonitrile based protein precipitation. The supernatant was then analyzed directly using LC-MS/MS. Chromatographic separation was achieved on a C18 reversed phase column using a methanol and water gradient. The detection was performed in selected reaction monitoring (SRM) mode via a positive electrospray ionization (ESI) interface. The method had a lower limit of quantification (LLOQ) of 1 ng/mL, linearity up to 1250 ng/mL, intra- and inter-day precision less than 10%, and accuracy within ±10%. This method was also demonstrated to be suitable for the analysis of bumetanide in rat serum and brain tissue. Bumetanide concentrations in rat serum and brain were determined for samples collected at several intervals following intraperitoneal (i.p.) injection of bumetanide, and were used to calculate bumetanide permeability through the blood-brain barrier.
Available from: Margery A Barrand
- "Thus, total concentrations in the brain as small as 2 % of those in plasma might reflect either slow penetration across the blood–brain barrier or a lack of binding in brain tissue or both. Low concentrations with rapid removal from the brain  would imply little binding and rapid penetration. No data for penetration of cariporide into cells or across the blood–brain barrier appear to have been published . "
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ABSTRACT: Ions and water transported across the endothelium lining the blood-brain barrier contribute to the fluid secreted into the brain and are important in maintaining appropriate volume and ionic composition of brain interstitial fluid. Changes in this secretion process may occur after stroke. The present study identifies at transcript and protein level ion transporters involved in the movement of key ions and examines how levels of certain of these alter following oxidative stress. Immunohistochemistry provides evidence for Cl(-)/HCO3 (-) exchanger, AE2, and Na(+), HCO3 (-) cotransporters, NBCe1 and NBCn1, on brain microvessels. mRNA analysis by RT-PCR reveals expression of these transporters in cultured rat brain microvascular endothelial cells (both primary and immortalized GPNT cells) and also Na(+)/H(+) exchangers, NHE1 (primary and immortalized) and NHE2 (primary cells only). Knock-down using siRNA in immortalized GPNT cells identifies AE2 as responsible for much of the Cl(-)/HCO3 (-) exchange following extracellular chloride removal and NHE1 as the transporter that accounts for most of the Na(+)/H(+) exchange following intracellular acidification. Transcript levels of both AE2 and NHE1 are increased following hypoxia/reoxygenation. Further work is now required to determine the localization of the bicarbonate transporters to luminal or abluminal membranes of the endothelial cells as well as to identify and localize additional transport mechanisms that must exist for K(+) and Cl(-).
Pflügers Archiv - European Journal of Physiology 09/2013; 466(5). DOI:10.1007/s00424-013-1342-9 · 4.10 Impact Factor
Available from: Zeng-You Ye
- "In addition to having a diuretic effect in the kidney, inhibiting NKCC1 activity with bumetanide could act in the brain to reduce the sympathetic drive (Li et al., 2011). "
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ABSTRACT: Hypertension is a major risk factor for coronary artery disease, stroke, and kidney failure. However, the etiology of hypertension in most patients is poorly understood. Increased sympathetic drive emanating from the hypothalamic paraventricular nucleus (PVN) plays a major role in the development of hypertension. Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) in the brain is critically involved in maintaining chloride homeostasis and in neuronal responses mediated by GABA(A) receptors. Here we present novel evidence that the GABA reversal potential (E(GABA)) of PVN presympathetic neurons undergoes a depolarizing shift that diminishes GABA inhibition in spontaneously hypertensive rats (SHRs). Inhibition of NKCC1, but not KCC2, normalizes E(GABA) and restores GABA inhibition of PVN neurons in SHRs. The mRNA and protein levels of NKCC1, but not KCC2, in the PVN are significantly increased in SHRs, and the NKCC1 proteins on the plasma membrane are highly glycosylated. Inhibiting NKCC1 N-glycosylation restores E(GABA) and GABAergic inhibition of PVN presympathetic neurons in SHRs. Furthermore, NKCC1 inhibition significantly reduces the sympathetic vasomotor tone and augments the sympathoinhibitory responses to GABA(A) receptor activation in the PVN in SHRs. These findings suggest that increased NKCC1 activity and glycosylation disrupt chloride homeostasis and impair synaptic inhibition in the PVN to augment the sympathetic drive in hypertension. This information greatly improves our understanding of the pathogenesis of hypertension and helps to design better treatment strategies for neurogenic hypertension.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 06/2012; 32(25):8560-8. DOI:10.1523/JNEUROSCI.1346-12.2012 · 6.34 Impact Factor
British dental journal 08/2006; 201(1):61. DOI:10.1038/sj.bdj.4813815 · 1.08 Impact Factor
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