Capillary electrophoresis (CE) with contactless conductivity detection (C4D) represents a strong tool for determining amino acids in clinical samples. This chapter provides detailed instructions for CE/C4D determination of the branched-chain amino acids (BCAAs) valine, isoleucine, and leucine in human plasma, which can be readily employed in physiological studies. Baseline separation of all the BCAAs is achieved on a short separation length equal to 18 cm in optimized background electrolyte consisting of 3.2 M acetic acid dissolved in 20% v/v methanol with addition of 1.0% v/v INST-coating solution. The analysis time does not exceed 3 min and the limit of detection is 0.4 μM for all BCAAs. The pretreatment of human plasma is very simple and is based on fourfold plasma dilution by acetonitrile and subsequent filtration. Only 50 μL of plasma is used for the analysis. The high sensitivity of the CE/C4D method is achieved by injecting a large volume of sample, combined with application of negative pressure to flush the acetonitrile zone out of the capillary.

• Keywords
• Branched-chain amino acids, Capillary electrophoresis, Contactless conductivity detection, Human plasma, Large-volume sample stacking, Pressure-assisted analysis, Rapid determination,
• MeSH
• Electric Conductivity * MeSH
• Electrophoresis, Capillary methods   MeSH
• Physiology * MeSH
• Calibration MeSH
• Humans MeSH
• Reference Standards MeSH
• Amino Acids, Branched-Chain blood   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amino Acids, Branched-Chain MeSH

Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70-4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes.

• MeSH
• Cell Line MeSH
• Citric Acid Cycle drug effects   MeSH
• Citrate (si)-Synthase drug effects   metabolism   MeSH
• Glutamate Dehydrogenase drug effects   metabolism   MeSH
• Isocitrate Dehydrogenase drug effects   metabolism   MeSH
• Ketoglutarate Dehydrogenase Complex antagonists & inhibitors   metabolism   MeSH
• Muscle, Skeletal enzymology   MeSH
• Rats MeSH
• Malate Dehydrogenase drug effects   metabolism   MeSH
• Membrane Potential, Mitochondrial drug effects   MeSH
• Onium Compounds pharmacology   MeSH
• Rats, Wistar MeSH
• Pyruvate Dehydrogenase Complex drug effects   metabolism   MeSH
• Mitochondria, Muscle drug effects   enzymology   MeSH
• Triphenylmethyl Compounds pharmacology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Citrate (si)-Synthase MeSH
• Glutamate Dehydrogenase MeSH
• Isocitrate Dehydrogenase MeSH
• Ketoglutarate Dehydrogenase Complex MeSH
• Malate Dehydrogenase MeSH
• Onium Compounds MeSH
• Pyruvate Dehydrogenase Complex MeSH
• triphenylmethylphosphonium MeSH Browser
• Triphenylmethyl Compounds MeSH