The albumin concentration was determined in all

admission samples and the concentration of albumin and creatinine was determined in all serial samples. These analyses were conducted by Queensland Health Forensic and Scientific Services at Princess Alexandra Hospital, Brisbane, Australia. This service is accredited by the National Association of Testing Authorities, Australia and certified to International Standards (ISO 9001). The MCPA concentration–time profile in patients providing the most serial samples was Bcl-2 inhibitor constructed using the total and free MCPA concentrations. A plot of the free versus total MCPA concentration was then constructed using data from all admission and serial plasma samples to determine whether protein binding Inhibitor Library research buy was saturable and the approximate concentration at which this occurred. The bound MCPA concentration was calculated as the difference between the free and total concentration at each time point. A Scatchard plot was constructed using the bound and free MCPA concentrations to estimate the number of apparent protein binding sites. Here, following visual inspection, a one-phase (linear) relationship suggests one-site binding,

a two-phase relationship suggests two-site binding, and so on (Kermode, 1989, Molinoff et al., 1981 and Motulsky and Christopoulos, 2005). In the case of two-site binding the relationship between free and bound concentrations is quantified by nonlinear regression using a two-site binding hyperbola model as follows: Bound concentration=Bmax1⋅CuKd1+Cu+Bmax2⋅CuKd2+Cu

Here, Cu is the free (unbound) plasma concentration of MCPA and Kdi and Bmaxi are the affinity constant and maximum density (concentration of saturation) of binding at the ith site ( Molinoff et al., 1981 and Motulsky and Christopoulos, 2005). This analysis was initially conducted using the combined population data. To account for possible inter-individual variability in protein binding, the analysis was also conducted by global fitting. Global fitting is a computational regression method which incorporates best-fit data for individuals when determining the best-fit data for the group as a whole. Finally, because chlorophenoxy compounds Non-specific serine/threonine protein kinase largely bind to albumin ( Braunlich et al., 1989 and Rosso et al., 1998), this regression was also conducted relative to the concentration of albumin (g/L) to determine the extent to which this influenced the fit. When determining the protein binding properties of MCPA it was assumed that binding was at equilibrium at the time of measurement. To determine whether saturation of protein binding influences clearance in humans the plasma apparent elimination half-life was determined prior to and following the concentration where saturation was calculated to occur.