Bserved pronounced uptake of M1 into endothelial cells and monocytes/macrophages in vitro. The uptake was decreased by phloretin, suggesting a facilitated transport mechanism [13]. Although the partitioning of compounds into red blood cells has received significantly less interest than the plasma protein binding, erythrocytes constitute a important compartment for distribution [14,15]. We recently analyzed the plasma protein binding of different maritime pine bark polyphenols and observed pronounced variations in the binding tendency [16]. Even though catechin and taxifolin displayed protein binding close to 100 , low binding about 30 was observed for M1 and its structurally related metabolite M2 (d(3methoxy4hydroxyphenyl)cvalerolactone). The goal from the present investigation was to analyse thePLOS One particular | www.plosone.orgUptake of a Bioactive Metabolite into Erythrocytesbinding of selected PycnogenolH constituents and also the metabolite M1 to human erythrocytes to gain further insight into the disposition of these compounds.Supplies and Methods Chemical compounds and reagentsFerulic acid, (six)taxifolin, caffeic acid, pcoumaric acid, glutathione, glutathioneStransferase (EC 2.five.1.18), phloretin, and 2,29azobis(2amino propane (AAPH), cytochalasin B from Drechslera dematioidea, D ()glucose, ethylene glycolbis(2aminoethylether)N,N,N9,N9tetraacetic acid (EGTA), have been all obtained from SigmaAldrich (Taufkirchen, Germany). 4(2Hydroxyethyl)piperazine1ethanesulfonic acid (HEPES) was bought from Gerbu (Wieblingen, Germany). The metabolite M1 (d(3,4dihydroxyphenyl)cvalerolactone) was synthesized by M. Rappold as part of his diploma thesis [17]. Methanol (HPLC grade) was obtained from Merck (Darmstadt, Germany), acetonitrile (HPLC grade) was from Fisher Scientific (Schwerte, Germany).Buy958451-91-7 Ultrapure MilliQ water was made use of for all aqueous solutions. All other chemical substances had been bought from SigmaAldrich.933708-92-0 structure obtained right after a single freezethaw cycle (280uC). The haemolysis was calculated from the absorption in the cell supernatant in relation for the absorption from the completely haemolysed sample.PMID:23833812 In all experiments the percentage of haemolysed erythrocytes was below 3 more than the whole experimental period.Uptake of M1 into human erythrocytesPacked red blood cells had been incubated having a threefold volume of PBS buffer with 100 mM Dglucose for 30 min at 37uC and centrifuged for five min at 2,000 g temperated to 4uC (Mikrofuge 22 R, Beckmann CoulterTM, Krefeld, Germany). Thereafter these cell pellets were washed twice with the threefold volume of cold PBS buffer (4uC) containing one hundred mM Dglucose and centrifuged for five min at 2,000 g (4uC). 43 mL of these packed glucosesaturated cells had been mixed with PBS buffer to acquire a hematocrit of 0.043. The cells had been subsequently incubated with different concentrations of M1 (0.30 mM) for 1 min by rocking (Mini Rocker MR1, Hartenstein, Wurzburg, Germany) in closed reaction tubes (Eppendorf, Hamburg, Germany) at room temperature. In parallel handle experiments were carried out accordingly for each and every variable without cells to monitor the stability of M1 during the experimental procedures. Similar towards the process described by Leitch and Carruthers [21] the reaction was interrupted by adding a cold stop solution (4uC) containing 150 mM KCl, five mM MgCl2, five mM EGTA, 5 mM HEPES, 20 mM cytochalasin B and 200 mM phloretin in PBS buffer (pH 7.four), followed by a centrifugation in the cell preparations and matched controls for 5 min at two,000 g (4uC). The supernatants were harvested and i.