Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4).

The effect of cholesterol was investigated on the OCTN1 transport activity measured as [14C]-tetraethylamonium or [3H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli. Removal of cholesterol from the native transporter by MßCD before reconstitution led to impairment of transport activity. A similar activity impairment was observed after treatment of proteoliposomes harboring the recombinant (cholesterol-free) protein by MßCD, suggesting that the lipid mixture used for reconstitution contained some cholesterol. An enzymatic assay revealed the presence of 10 µg cholesterol/mg total lipids corresponding to 1% cholesterol in the phospholipid mixture used for the proteoliposome preparation. On the other way around, the activity of the recombinant OCTN1 was stimulated by adding the cholesterol analogue, CHS to the proteoliposome preparation. Optimal transport activity was detected in the presence of 83 µg CHS/ mg total lipids for both [14C]-tetraethylamonium or [3H]-acetylcholine uptake. Kinetic analysis of transport demonstrated that the stimulation of transport activity by CHS consisted in an increase of the Vmax of transport with no changes of the Km. Altogether, the data suggests a direct interaction of cholesterol with the protein. A further support to this interpretation was given by a docking analysis indicating the interaction of cholesterol with some protein sites corresponding to CARC-CRAC motifs. The observed direct interaction of cholesterol with OCTN1 points to a possible direct influence of cholesterol on tumor cells or on acetylcholine transport in neuronal and non-neuronal cells via OCTN1.

Human organic cation transporter 2 (hOCT2): Inhibitor studies using S2-hOCT2 cells.

Highly expressed in kidney and located on the basolateral membrane, human organic cation transporter 2 (hOCT2) can transport various compounds (i.e. drugs and toxins) into the proximal tubular cell. Using cultured proximal tubule cells stably expressing hOCT2 (i.e. S2-hOCT2 cells), we sought to probe different compound classes (e.g. analgesics, anti-depressants, anti-psychotics, disinfectant, herbicides, insecticides, local anesthetic, muscarinic acetylcholine receptor antagonist, sedatives, steroid hormone, stimulants and toxins) for their ability to inhibit (14)C-TEA uptake, a prototypical OCT2 substrate. Aconitine, amitriptyline, atropine, chlorpyrifos, diazepam, fenitrothion, haloperidol, lidocaine, malathion, mianserin, nicotine and triazolam significantly inhibited (14)C-TEA uptake; IC50 values were 59.2, 2.4, 2.0, 20.7, 32.3, 13.2, 32.5, 104.6, 71.1, 17.7, 52.8 and 65.5µM, respectively. In addition, aconitine, amitriptyline, atropine, chlorpyrifos, fenitrothion, haloperidol, lidocaine, and nicotine displayed competitive inhibition with Ki values of 145.6, 2.5, 2.4, 24.8, 16.9, 51.6, 86.8 and 57.7µM, respectively. These in vitro data support the notion that compounds pertaining to a wide variety of different drug classes have the potential to decrease renal clearance of drugs transported via hOCT2. Consequently, these data warrant additional studies to probe hOCT2 and its role to influence drug pharmacokinetics.

Chemical standards in ion mobility spectrometry.

In ion mobility spectrometry (IMS), reduced mobility values (K(0)) are used as a qualitative measure of gas phase ions, and are reported in the literature as absolute values. Unfortunately, these values do not always match with those collected in the field. One reason for this discrepancy is that the buffer gas may be contaminated with moisture or other volatile compounds. In this study, the effect of moisture and organic contaminants in the buffer gas on the mobility of IMS standards and analytes was investigated for the first time using IMS directly coupled to mass spectrometry. 2,4-Dimethylpyridine, 2,6-di-tert-butylpyridine (DTBP), and tetrabutylammonium, tetrapropylammonium, tetraethylammonium, and tetramethylammonium chlorides were used as chemical standards. In general, the mobility of IMS standard product ions was not affected by small amounts of contamination while the mobilities of many analytes were affected. In the presence of contaminants in the buffer gas, the mobility of analyte ions is often decreased by forming ion-molecule clusters with the contaminant. To ensure the measurement of accurate reduced mobility values, two IMS standards are required: an instrument and a mobility standard. An instrument standard is not affected by contaminants in the buffer gas, and provides an accurate measurement of the instrumental parameters, such as voltage, drift length, pressure, and temperature. The mobility standard behaves like an analyte ion in that the compound's mobility is affected by low levels of contamination in the buffer gas. Prudent use of both of these standards can lead to improved measurement of accurate reduced mobility values.

Assessment of ion transfer amperometry at liquid-liquid interfaces for detection in CE.

In this research, ion transfer across the interface between two immiscible electrolyte solutions (ITIES) was used as a method of detection in a CE separation system. This method allows for the electrochemical detection of ionic analytes that cannot be easily oxidized or reduced. Method development revealed that the optimal separation conditions for three model ions (tetraethylammonium, tetrabutylammonium, and benzensulfonate) were found to be 5 mM sodium tetraborate buffer pH 9.2 with a separation voltage of 20 kV using a 40 cm, 50 microm id fused silica capillary. Constant potential amperometry and pulsed amperometric detection were applied at the ITIES in which the organic phase was gelled. A miniaturized ITIES within a pipette tip was investigated, which resulted in improved separation efficiency and LOD. To demonstrate the ability of the system to detect substances of bioanalytical interest, the beta-adrenergic receptor blockers timolol and propranolol were detected. The simplicity of the detection platform means that it may be useful for analytical situations not requiring trace or ultratrace detection capabilities.

Subnanomolar ion detection by stripping voltammetry with solid-supported thin polymeric membrane.

Subnanomolar limits of detection (LODs) are obtained for stripping voltammetry based on ion transfer at the interface between the aqueous sample and the thin polymeric membrane supported with a solid electrode. It has been predicted theoretically that a lower LOD can be obtained for a more lipophilic analyte ion, which can be preconcentrated at a higher equilibrium concentration in the solid-supported thin polymeric membrane to enhance a stripping current response. This study is the first to experimentally confirm the general theoretical prediction for both cationic and anionic analytes. Proof-of-concept experiments demonstrate that a subnanomolar LOD of (8 +/- 4) x 10(-11) M tetrapropylammonium is significantly lower than a LOD of less lipophilic tetraethylammonium. Importantly, stripping voltammetry of the cationic analytes is enabled by newly introducing an oxidatively doped poly(3,4-ethylenedioxythiophene) film as the intermediate layer between a plasticized poly(vinyl chloride) membrane and a Au electrode. On the other hand, an undoped poly(3-octylthiophene) film is used as an intermediate layer for voltammetric detection of a lipophilic inorganic anion, hexafluoroarsenate, an arsenical biocide found recently in wastewater. A LOD of (9 +/- 2) x 10(-11) M hexafluoroarsenate thus obtained by ion-transfer stripping voltammetry is comparable to a LOD of 80 pM by inductively coupled plasma mass spectrometry with anion-exchange chromatography. Great sensitivity for a lipophilic ion is potentially useful for environmental analysis because high lipophilicity of an ion is relevant to its bioaccumulation and toxicity.

Hybrid mass spectrometers for tandem mass spectrometry.

Mass spectrometers that use different types of analyzers for the first and second stages of mass analysis in tandem mass spectrometry (MS/MS) experiments are often referred to as "hybrid" mass spectrometers. The general goal in the design of a hybrid instrument is to combine different performance characteristics offered by various types of analyzers into one mass spectrometer. These performance characteristics may include mass resolving power, the ion kinetic energy for collision-induced dissociation, and speed of analysis. This paper provides a review of the development of hybrid instruments over the last 30 years for analytical applications.

[Advances in nonaqueous capillary electrophoresis].

Advances in nonaqueous capillary electrophoresis are reviewed with 71 references in this paper. Capillary electrophoresis is generally performed in aqueous buffer. In fact, it can provide some advantages to use organic solvent as a separation medium instead of water. The choice of organic solvent and electrolyte, the detection mode and solute-additive interactions are summarized. Furthermore, the separation of inorganic ions, neural compounds, organic acids, pharmaceuticals, metabolites and chiral substance is also described.