Administration of -hydroxybutyrate, an MCT substrate with affinity similar to that of GHB, significantly inhibited lactate uptake and rate of metabolism in hepatocytes and a rat liver perfusion model at a concentration of 10 mM (Metcalfe et al

Administration of -hydroxybutyrate, an MCT substrate with affinity similar to that of GHB, significantly inhibited lactate uptake and rate of metabolism in hepatocytes and a rat liver perfusion model at a concentration of 10 mM (Metcalfe et al., 1986). series high-performance liquid chromatography system with binary pump and autosampler (Agilent Systems, Santa Clara, CA) connected to a PerkinElmer Sciex API 3000 triple quadrupole tandem mass spectrometer having a TurboIonSpray (Applied Biosystems, Foster City, CA). Chromatographic separation was achieved by injecting 7 l of sample on an Xterra MS C18 column (250 2.1 mm i.d., 5-m particle size; Waters, Milford, MA). Mobile phone phase A consisted of 5:95 acetonitrile-water with 0.1% acetic acid and mobile phase B consisted of 95:5 acetonitrile-water with 0.1% acetic acid. The flow rate was 200 l/min with the following gradient elution profile: 100 to 68% A over 7 min; 68 to 10% A over 3 min; and 10 to 100% over 5 min for a total run time of 15 min. The mass spectrometer was managed inside a positive ionization mode with multiple reaction monitoring. Q1/Q3 ratios for the parent/product ions of GHB and GHB-d6 were 105.2/87.2 and 111.1/93.2, respectively. The mass spectrometer guidelines were optimized at a declustering potential of 18 V, focusing potential of 100 V, collision energy of 20 V, entrance potential of 10 V, and collision cell exit potential of 5 V. The ion aerosol voltage was arranged at 5500 V with temp at 350C. Nebulizer and curtain gas circulation were arranged at 10 and 8 ml/min, respectively. The retention time for GHB was 4.15 min. The data were analyzed using Analyst software version 1.4.2 (Applied Biosystems). Regression analysis of peak area ratios of GHB/GHB-d6 to GHB concentrations was used to assess linearity of the curve. The intraday and interday precision and accuracy were identified using quality control (QC) samples at 10 g/ml (low QC), 125 g/ml (medium QC), and 400 g/ml (high QC). For dedication of the intraday precision and accuracy, quality control samples were analyzed in triplicate on each day, whereas for the interday precision and accuracy, quality control samples were analyzed 24, 25-Dihydroxy VD2 on three different days. A calibration curve was run on each analysis day along with the quality settings. The precision was determined by the coefficient of variance, and accuracy was measured by comparing the calculated concentration with the known concentration. Urine samples were prepared and analyzed for GHB using a previously explained LC-MS/MS method (Felmlee et al., 2010b). Plasma lactate concentrations were determined using a YSI 1500 Sport Lactate Analyzer (YSI, Inc., Yellow Springs, OH). Data and Statistical Analysis. Pharmacokinetic guidelines were identified via noncompartmental analysis using WinNonlin 5.2 (Pharsight, Mountain View, CA). The area below the plasma concentration-time curve (AUC) was identified using the trapezoidal method. Total clearance (Cl) was identified as dose/AUC. Renal clearance (ClR) was identified as 0.05. One-way analysis of variance followed by Dunnett’s or Tukey’s post hoc checks was used to determine statistically significant variations in mean pharmacokinetic and pharmacodynamic guidelines between groups. Combined checks were used to determine statistically significant changes in respiratory guidelines compared with baseline. In determining the effects of l-lactate only on respiration, the average of the last hour of respiratory measurements was compared with the individual average baseline ideals. Mean steady-state lactate plasma concentrations were calculated as the average of hourly ideals beginning at 60 min. Results Plasma GHB LC-MS/MS Assay. The lower limit of quantification for GHB in plasma was found to be 5 g/ml with suitable error in precision and accuracy of less than 20%. The endogenous concentrations of GHB in plasma are negligible compared with GHB concentrations acquired after administration of the lowest dose in our studies (Fung et al., 2004); consequently, the endogenous concentrations were not included in the calculation of GHB concentrations in plasma. The standard curve for GHB ranged from 5 to 500 g/ml based on regression 24, 25-Dihydroxy VD2 analysis of peak area ratios of GHB/GHB-d6 to GHB concentrations having a correlation coefficient ( 24, 25-Dihydroxy VD2 0.05). Uncooked plethysmography traces showing the switch in respiratory pattern with GHB administration are demonstrated in Fig. 2. As a result of this experiment, respiratory rate was regarded as the primary parameter of interest for assessment of receptors involved and potential treatment strategies. It was also determined with this experiment that 1500 mg/kg GHB was the maximal dose that may be given without causing death; therefore, this dose was utilized for further investigation. TABLE 2 Nonlinear pharmacokinetics of GHB GHB was given intravenously. Data are offered as mean (S.D.); = 4 to 6 6. One-way analysis of variance followed by Tukey’s post hoc test was used to determine statistically significant variations in pharmacokinetic guidelines. 0.05). b Significantly different from 200 Rabbit Polyclonal to NM23 and 600 mg/kg GHB ( 0.05). Open in a separate windowpane Fig. 24, 25-Dihydroxy VD2 1. Dose-dependent effects of GHB on actions of.