From a non-targeted metabolomics approach to a targeted biomarkers strategy to highlight testosterone abuse in equine. Illustration of a methodological transfer between platforms and laboratories

In order to overcome the challenge associated with the screening of Anabolic-Androgenic Steroids abuses in animal competitions, a non-targeted liquid chromatography coupled to high resolution mass spectrometry based metabolomics approach was implemented on equine urine samples to highlight potential biomarkers associated with the administration of such compounds, using testosterone esters as model steroids. A statistical model relying on four potential biomarkers intensity could be defined to predict the status of the samples. With a routine application perspective, the monitoring of the highlighted potential biomarkers was first transferred into high-throughput liquid chromatography-selected reaction monitoring (LC-SRM). The model's performances and robustness of the approach were preserved and providing a first demonstration of metabolomics-based biomarkers integration within a targeted workflow using common benchtop MS instrumentation. In addition, with a view to the widespread implementation of such biomarker-based tools, we have transferred the method to a second laboratory with similar instrumentation. This proof of concept allows the development and application of biomarker-based strategies to meet current doping control needs.     

Lipoprotein(a) and Oxidized Phospholipids Promote Valve Calcification in Patients With Aortic Stenosis

Background

Lipoprotein(a) [Lp(a)], a major carrier of oxidized phospholipids (OxPL), is associated with an increased incidence of aortic stenosis (AS). However, it remains unclear whether elevated Lp(a) and OxPL drive disease progression and are therefore targets for therapeutic intervention.

Opening of ATP-sensitive potassium channels causes generation of free radicals in vascular smooth muscle cells

Recent evidence suggests that opening of mitochondrial K_ATP channels in cardiac muscle triggers the preconditioning phenomenon through free radical production. The present study tested the effects of K_ATP channel openers in a vascular smooth muscle cell model using the fluorescent probe MitoTracker (MTR) Red™ for detection of reactive oxygen species (ROS). Rat aortic smooth muscle cells (A7r5) were incubated with 1 μM reduced MTR (non-fluorescent) and the MTR oxidation product (fluorescent) was quantified. Thirty-minute pretreatment with either diazoxide (200 μM) or pinacidil (100 μM), both potent mitochondrial K_ATP channel openers, increased fluorescent intensity (FI) to 149 and 162 % of control (p < 0.05 for both), respectively, and the K_ATP channel inhibitor 5-hydroxydecanoate (5HD) blocked it. Valinomycin, a potassium-selective ionophore, raised FI to 156 % of control (p <: 0.05). However, 5HD did not affect the valinomycin-induced increase in FI. Inhibition of mitochondrial electron transport (myxothiazol) or uncoupling of oxidative phosphorylation (dinitrophenol) also blocked either valinomycin- or diazoxide-induced increase in FI, and free radical scavengers prevented any diazoxide-mediated increase in fluorescence. Finally the diazoxide-induced increase in fluorescence was not blocked by the PKC inhibitor chelerythrine, but was by HMR 1883, a putative surface K_ATP channel blocker. Thus opening of K_ATP channels increases generation of ROS via the mitochondrial electron transport chain in vascular smooth muscle cells. Furthermore, a potassium-selective ionophore can mimic the effect of putative mitochondrial KATP channel openers. We conclude that potassium movement through KATP directly leads to ROS production by the mitochondria. Received: 7 January 2002, Returned for revision: 31 January 2002, Revision received: 21 February 2002, Accepted: 14 March 2002