13C/12C isotope ratios (expressed as ��13CPDB values in per mil difference vs. the Pee Dee Belemnite standard) in the CO2 of the breath samples Volasertib aml were analysed by using a validated breath 13C-analyser (Heliview, Medichems, Seoul, South Korea) based on isotope ratio mass spectrometry (IRMS) (Stellaard and Geypens, 1998). Blood samples were centrifuged and plasma was stored at ?20oC until analysis. From 0.5 mL, plasma endogenous bicarbonate was removed by acidification with acetic acid and subsequent evaporation under nitrogen (Rembacz et al., 2007). Then, urea was converted to CO2 using the enzyme urease (1% urease in ethylenediaminetetraacetic acid (EDTA) buffer) (Kloppenburg et al., 1997). After incubation, phosphoric acid (1M) was added to convert dissolved 13C-bicarbonate in the solution to 13CO2 in the vapour phase.

Finally, the 13C/12C isotope ratio was determined in CO2 in the headspace using the same IRMS instrumentation. Urea concentrations in plasma were determined using the Roche Modular? analyser (F. Hoffmann-La Roche Ltd., Basel, Switzerland). Calculation of the concentration of 13C-urea in plasma The ��13CPDB values obtained from the plasma samples were converted to atom percentage excess 13C by the equations described by Schellekens et al. (2008). The concentration of 13C-urea in a plasma sample at any time point can then be calculated by Equation (1): (1) The concentration versus time graph is described by Equation (2): (2) where a is the slope of the curve. Subsequently, several kinetic parameters were deducted, based on the assumption that 13C-urea kinetics may be described by a one-compartment model (Kloppenburg et al.

, 1997). The lag time (starting point of release) of coated capsules is defined as the time point at which the area under the curve (AUC) is 5% of the AUC at t= 12 h (t5%). Based on Equation (2), the intercept with the y-axis (t= 0 h) of the curve in the steady state phase represents the fictitious (13C-urea)t= 0 value. According to conventional Batimastat kinetics theory, the apparent urea distribution volume (UDV in L?kg?1 Lean Body Mass (LBM)) is then calculated by Equation (3): (3) Then, the elimination rate constant (kel) is calculated by Equation (4), in which a is the slope of the semi-logarithmic concentration versus time graph in the steady state phase. (4) Subsequently, the half-life (t1/2) is calculated by Equation (5): (5) The pulsatile release properties are reflected by the so-called pulse time, defined as the time period between the lag time (t5%) and tmax. The availability of 13C-urea in urease-poor segments is expressed by the not fermented fraction (Fnot fermented).