Rate-limiting Processes Research Articles

(Invited) Are Nanotube Networks the Key to High-Rate, High Capacity Lithium Ion Batteries?

Increasing the energy storage capability of lithium-ion batteries necessitates maximization of their areal capacity. This requires thick electrodes performing at near-theoretical specific capacity. However, achievable electrode thicknesses are restricted by mechanical instabilities, with high-thickness performance limited by the attainable electrode conductivity. Here we show that forming a segregated network composite of carbon nanotubes with a range of lithium storage materials (for example, silicon, graphite and metal oxide particles) suppresses mechanical instabilities by toughening the composite, allowing the fabrication of high-performance electrodes with thicknesses of up to 800 μm. Such composite electrodes display conductivities up to 1 × 104 S m−1 and low charge-transfer resistances, allowing fast charge-delivery and enabling near-theoretical specific capacities, even for thick electrodes. The combination of high thickness and specific capacity leads to areal capacities of up to 45 and 30 mAh cm−2 for anodes and cathodes, respectively. Combining optimized composite anodes and cathodes yields full cells with state-of-the-art areal capacities (29 mAh cm−2) and specific/volumetric energies (480 Wh kg−1 and 1,600 Wh l−1).However, such thick electrode might be expected to show poor rate performance. Optimisation of the combination of areal capacity and rate performance would be simplified if a simple mechanistic model linked both parameters to electrode properties. Here we demonstrate an equation which can fit capacity versus rate data, outputting three parameters which fully describe rate performance. Most important is the characteristic time associated with charge/discharge which can be linked by a second equation to physical electrode/electrolyte parameters via various rate-limiting processes. We fit these equations to ~200 data sets, deriving parameters such as diffusion coefficients or electrolyte conductivities. It is possible to show which rate-limiting processes are dominant in a given situation, facilitating rational design and cell optimisation. In addition, this model predicts the upper speed limit for lithium/sodium ion batteries, yielding a value that is consistent with the fastest electrodes in the literature. We show that this equation completely describes much experimental rate data.

In situ oxidation studies of Cu thin films: Growth kinetics and oxide phase evolution

A comprehensive understanding of the oxidation of Cu thin films in the low-temperature regime is of fundamental interest and particularly relevant for applications in the fields of micro- and nanoelectronics, sensors, catalysis, and solar cells. The current study reports on the oxidation kinetics of PVD grown Cu thin films (20–150 nm thick) and the oxide phase evolution from Cu2O to CuO upon thermal oxidation in the temperature range of 100–450 °C. XRD investigations in the laboratory and at the synchrotron show that the oxide phase formation critically depends on the oxidation conditions such as temperature and oxygen partial pressure. The real-time synchrotron XRD measurements reveal that the formation of the CuO phase only starts after complete oxidation of the Cu films to Cu2O films. In situ resistance measurements were performed to follow the oxide growth rate of Cu2O on Cu films in the temperature range of 100–300 °C in air and in 10 mbar pO2. It is found that the oxidation kinetics of Cu films to Cu2O films follows the linear rate law, which is attributed to surface reaction controlled oxidation. The oxygen dissociation rate at the gas–solid interface is the rate-limiting process. A dramatic decrease in the linear oxidation rate is observed at low oxygen partial pressures. The fundamental differences between the oxidation rate-limiting processes of Cu as compared to other transition metal films are discussed.

Modeling of a Gas-Phase, Bipolar Membrane CO2 Electrolyzer

Aqueous electrochemical carbon dioxide reduction electrodes encounter prohibitive mass-transport limitations due to the low solubility of CO2 in aqueous electrolyte which limits cells to ~10 mA/cm2 reaction rates (1, 2). While C2+ products are desirable they remain economically challenging with low Faradaic efficiencies and high cell overpotentials (3). Alkaline environments are capable of facilitating preferential C-C bond formation (4); for example, the highest faradaic efficiencies for C2 products (5) and C3 products (6) have been reported under alkaline conditions. Unfortunately, carbon dioxide fouls alkaline reactors through the formation of carbonate species. Bipolar membrane-based CO2 electrolyzers, which combine gas phase acidic reduction and alkaline oxygen evolution, offer a unique opportunity to address the engineering and technological challenges of CO2 reduction. Such a system may preclude large anodic activation overpotentials while avoiding carbonate generation as the cathodic reaction proceeds in acid environment. In the gas-phase bipolar membrane CO2 reduction reactor, fundamental questions remain regarding optimal choice of cell architecture, catalyst, and operating conditions. A model of the cell was developed and used to explore rate-limiting processes and process parameters effects on reaction kinetics which dictate Faradaic efficiencies and, therefore, economic viability of the reactor. Fundamental benefits and challenges of the reactor were assessed and contrasted with competing cell architectures in context of the ultimate parameter to be optimized, cost per kilogram of averted CO2. R. Singh, E. L. Clark, and A. T. Bell, Phys. Chem. Chem. Phys., 17 (2015)Higgins et al., ACS Energy Letters, 4 (1) (2018)S. Bushuyev et al., Joule, 2 (2018)Wang et al., ACS Catal., 8 (8) (2018)Ma et al., J. Power Sources, 301 (2016)Ren et al., J. Phys. Chem. Lett., 7 (2016)

Interpretation of Impedance Spectra of Solid Oxide Fuel Cells: L-Curve Criterion for Determination of Regularization Parameter in Distribution Function of Relaxation Times Technique

The distribution function of the relaxation times (DRT) technique is a powerful tool for the interpretation of impedance spectra of solid oxide fuel cells (SOFCs) because it enables direct identification of rate-limiting processes through mathematical calculations. Selection of an appropriate regularization parameter (λreg) is critical for obtaining meaningful deconvolution solutions in DRT analysis. Herein, we introduce an L-curve criterion as a reliable graphical tool for determination of the λreg value. In a parametric plot of log(solution norm) versus log(misfit norm), the λreg value at the corner of the L curve provides an optimum balance between the regularization error and perturbation error. In a case study on La0.6Sr0.4CoO3−δ-based cathodes, the DRT technique guided by the L-curve criterion enabled identification of rate-limiting processes and clarification of reaction pathways. The analytical methodology presented in this article provides guidelines for the effective use of the DRT technique for the impedance analysis of SOFCs.

Physical and chemical characteristics of hematite nanoparticles prepared using microwave-assisted synthesis and its application as adsorbent for Cu, Ni, Co, Cd and Pb from aqueous solution

Hematite nanoparticles were synthesized using the microwave-assisted hydrothermal technique and from aqueous solution. The structure of the prepared material was characterized using x-ray fluorescence (XRF), X-ray diffraction (XRD), electron spin resonance (ESR), transmission electron microscopy (TEM), and electron diffraction (ED). The average particle size was found to be 18 nm while the XRD and ESR results confirmed the formation of hydrogenated hematite phase. XRD matched the hexagonal phase of Fe1.9H0.06O3 in which two ESR absorptions at Landé g-factor of 2.022 and 2.424 are attributed electron splitting at Fe3＋ and H+ sites, respectively. Radial distribution function (RDF) showed that the lattice parameters equal to a=5.82 Å and c=13.7 Å. Inductively coupled plasma optical emission spectroscopy (ICP-OES) was used to determine the ability of the material to adsorb Cu, Ni, Co, Cd and Pb ions from their aqueous solution. The effect of hydrogen concentration (pH)and the kinetic analysis showed that pseudo-second-order model of adsorption is suitable for rate-limiting processes around the particles surface that may involve chemisorption and physicochemical interactions between ionic species. Sorption isotherm study showed that the Langmuir isotherm model for single mono-layer sorption was more suitable and the maximum sorption capacity attained by the prepared hematite nanoparticle was around 100 mg/g and independent on the ion.

Combined high-precision ∆48 and ∆47 analysis of carbonates

High-precision analysis of the excess abundance (relative to the stochastic distribution) of mass 48 isotopologues in CO2 evolved from acid digestion of carbonates (∆48) has not been possible until recently due to the relatively low natural abundance of 18O. Here we show that the 253 Plus™ gas source mass spectrometer equipped with Faraday cups and 1013 Ω resistors can perform combined ∆47 and ∆48 analyses on carbonates with external reproducibilities (1SD) of 0.010 ‰ and 0.030 ‰, respectively.~10 mg aliquots of five carbonate reference materials (ETH 1, ETH 2, ETH 3, ETH 4, and Carrara) are digested with phosphoric acid at 90 °C using a common acid bath. The evolved CO2 is purified using an automated gas preparation system (including cryotraps and a GC) and analyzed for its ∆47 and ∆48 compositions using the dual inlet system of a 253 Plus™ gas source mass spectrometer. Raw ∆47 and ∆48 values are finally normalized to the Carbon Dioxide Equilibrium Scale (CDES).In ∆47, CDES 90°Cvs. ∆48, CDES 90°C space, calcite reference materials Carrara, ETH 3 and ETH 4 agree with the equilibrium curve for calcite after adding semi-empirically determined 90 °C acid fractionation factors of 0.196 ‰ (for ∆47) and 0.136 ‰ (for ∆48) to theoretical ∆63 and ∆64 data. Agreement between measured and theoretically expected ∆48, CDES 90°C highlights the accuracy of our high-precision clumped isotope analytical setup. Combined analysis of the abundances of mass 47 and mass 48 isotopologues in CO2 evolved from acid digestion of natural carbonates has excellent potential for the determination of accurate and highly precise paleotemperatures as well as for the identification of rate-limiting kinetic processes involved in biomineralization. A formation temperature of 15(±2) °C is obtained on the 95 % confidence level for the Upper Cretaceous chalk sample ETH 3.

Quantifying the factors limiting rate\xa0performance in battery electrodes

One weakness of batteries is the rapid falloff in charge-storage capacity with increasing charge/discharge rate. Rate performance is related to the timescales associated with charge/ionic motion in both electrode and electrolyte. However, no general fittable model exists to link capacity-rate data to electrode/electrolyte properties. Here we demonstrate an equation which can fit capacity versus rate data, outputting three parameters which fully describe rate performance. Most important is the characteristic time associated with charge/discharge which can be linked by a second equation to physical electrode/electrolyte parameters via various rate-limiting processes. We fit these equations to ~200 data sets, deriving parameters such as diffusion coefficients or electrolyte conductivities. It is possible to show which rate-limiting processes are dominant in a given situation, facilitating rational design and cell optimisation. In addition, this model predicts the upper speed limit for lithium/sodium ion batteries, yielding a value that is consistent with the fastest electrodes in the literature.

Solid-phase reactive inter-diffusion of Mg/B multilayers

A better understanding of the reaction of Mg and B in the solid-phase regime is needed for the low-temperature synthesis of MgB2 films. Here, we study the kinetics of reactive inter-diffusion of Mg and B multilayers on glassy carbon substrates in the temperature range of 400−650°C. Results show that, at these temperatures, inter-diffusion is characterized by a single activation energy of ∼0.45eV. The formation of the superconducting MgB2 phase with critical temperatures of 25–31 K occurs at reaction temperatures of 450°C and above, with the rate of inter-diffusion obeying a power law with a kinetic exponent of ∼0.3. This suggests that rate-limiting processes are the nucleation and growth of MgB2 grains rather than diffusion and interfacial reactions. Implications of these results to the low-temperature synthesis of MgB2 films are discussed.

Outer Coordination Sphere Proton Relay Base and Proximity Effects on Hydrogen Oxidation with Iron Electrocatalysts

The intra- and intermolecular deprotonation reactions of [(CpC5F4N)Fe(PEtNR′PEt)H]+ (PEtNR′PEt = (Et2PCH2)2NR′) complexes are rate-limiting processes in the electrocatalytic oxidation of hydrogen and can be mediated by an amine base in the outer coordination sphere (OCS) by facilitating the matching of energy of deprotonation intermediates. A series of complexes with different N-substituents (R′ = Me, Et, CH2OMe, CH2N(Ph)Me, and (CH2)nNMe2; n = 1–4) was examined computationally using density functional theory to determine the influence of the identity and proximity of the OCS base on the intramolecular deprotonation. Complexes with an OCS dimethylamino substituent were the most suitably energy matched. The number of carbon atoms between the secondary and OCS nitrogen atoms dictated the strength of the hydrogen-bonding interaction and ring strain following deprotonation of the iron hydride. The previously reported [(CpC5F4N)Fe(PEtNR′PEt)H]+ (R′ = (CH2)3NMe2) electrocatalyst was predicted to have the most f...

Role of native defects and the effects of metal additives on the kinetics of magnesium borohydride.

Magnesium borohydride (Mg(BH4)2) has been considered as a potential material for hydrogen storage. In this paper, density functional theory studies have been carried out on native point defects and electrically active impurities (Ni and Ti) in Mg(BH4)2. A detailed analysis of the geometrical structures, energetics and migration of the defects reveals that hydrogen related defects are charged and their formation energies are Fermi-level dependent. We propose a specific mechanism for the decomposition of Mg(BH4)2: the self-diffusion of Mgi2+ is the rate-limiting process for decomposing Mg(BH4)2. Moreover, Ni and Ti impurities can tailor the hydrogen desorption kinetics of Mg(BH4)2 by shifting the Fermi level, but the effects of impurities on shifting the Fermi levels are related to how the impurity is incorporated into Mg(BH4)2.

Prediction of bioavailability and toxicity of complex chemical mixtures through machine learning models

Empirical data from a 6-month mesocosms experiment were used to assess the ability and performance of two machine learning (ML) models, including artificial neural network (NN) and random forest (RF), to predict temporal bioavailability changes of complex chemical mixtures in contaminated soils amended with compost or biochar. From the predicted bioavailability data, toxicity response for relevant ecological receptors was then forecasted to establish environmental risk implications and determine acceptable end-point remediation. The dataset corresponds to replicate samples collected over 180 days and analysed for total and bioavailable petroleum hydrocarbons and heavy metals/metalloids content. Further to this, a range of biological indicators including bacteria count, soil respiration, microbial community fingerprint, seeds germination, earthworm's lethality, and bioluminescent bacteria were evaluated to inform the environmental risk assessment. Parameters such as soil type, amendment (biochar and compost), initial concentration of individual compounds, and incubation time were used as inputs of the ML models. The relative importance of the input variables was also analysed to better understand the drivers of temporal changes in bioavailability and toxicity. It showed that toxicity changes can be driven by multiple factors (combined effects), which may not be accounted for in classical linear regression analysis (correlation). The use of ML models could improve our understanding of rate-limiting processes affecting the freely available fraction (bioavailable) of contaminants in soil, therefore contributing to mitigate potential risks and to inform appropriate response and recovery methods.

Growth bottlenecks of microalga Dunaliella tertiolecta in response to an up-shift in light intensity

ABSTRACTMicroalgae are a potential source of valuable products including drugs, chemicals, food supplements, biofuels, animal feed and fertilizers. In outdoor culture, biomass productivity is dependent on how well the microalgae respond to fluctuations in light during the day. Dunaliella tertiolecta is a halotolerant, photoautotrophic microalga. Our objective is to identify rate-limiting steps in growth as D. tertiolecta acclimates to changing light intensity. We studied the physiological, metabolic and transcriptomic changes of D. tertiolecta in a low dilution rate chemostat culture to determine rate-limiting processes in cell growth in response to changes in light intensity. Upon a 10-fold increase in light intensity from 40 to 400 µmol photons m–2 s–1 (HL), the photosynthetic efficiency and maximum photosynthetic rate (PR) per cell, or per chlorophyll, did not change significantly during lag (2 h) and exponential growth (8 h) phases, which suggests that the photosynthetic system was not the growth rate limiting step in response to higher light intensity. The expression of most genes in 2 h and 8 h samples was similar. Some genes involved in synthesis of cellular structures, protein processing and the cell cycle might be bottlenecks in the HL response, because they were expressed only at 8 h. It might not be practical to increase biomass by genetic modification of D. tertiolecta because of the numerous pathways and genes that are potential bottlenecks. Instead, future research can be directed to improving carbon sequestration by channelling it to storage compounds or excretion as extracellular glycerol.

Ignition and self-propagating reactions in Al/Pt multilayers of varied design

The different rate-limiting processes underlying ignition and self-propagating reactions in Al/Pt multilayers are examined through experiments and analytical modeling. Freestanding, ∼1.6 μm-thick Al/Pt multilayers of varied stoichiometries and nanometer-scale layer thicknesses ignite at temperatures below the melting point of both reactants (and eutectics) demonstrating that initiation occurs via solid-state mixing. Equimolar multilayers exhibit the lowest ignition temperatures when comparing structures having a specific bilayer thickness. An activation energy of 76.6 kJ/mol at. associated with solid state mass transport is determined from the model analysis of ignition. High speed videography shows that equimolar Al/Pt multilayers undergo the most rapid self-sustained reactions with wavefront speeds as large as 73 m/s. Al- and Pt-rich multilayers react at reduced rates (as low as 0.3 m/s), consistent with reduced heat of reaction and lower adiabatic temperatures. An analytical model that accounts for key thermodynamic properties, preliminary mixing along interfaces, thermal transport, and mass diffusion is used to predict the wavefront speed dependencies on bilayer thickness. Good fits to experimental data provide estimates for activation energy (51 kJ/mol at.) associated with mass transport subject to high heating rates and thermal diffusion coefficient of premixed interfacial volumes (2.8 × 10−6 m2/s). Pt dissolution into molten Al is identified as a rate-limiting step underlying high temperature propagating reactions in Al/Pt multilayers.

Assessment and optimization of collective variables for protein conformational landscape: GB1 β-hairpin as a case study.

Collective variables (CVs), when chosen judiciously, can play an important role in recognizing rate-limiting processes and rare events in any biomolecular systems. However, high dimensionality and inherent complexities associated with such biochemical systems render the identification of an optimal CV a challenging task, which in turn precludes the elucidation of an underlying conformational landscape in sufficient details. In this context, a relevant model system is presented by a 16-residue β-hairpin of GB1 protein. Despite being the target of numerous theoretical and computational studies for understanding the protein folding, the set of CVs optimally characterizing the conformational landscape of the β-hairpin of GB1 protein has remained elusive, resulting in a lack of consensus on its folding mechanism. Here we address this by proposing a pair of optimal CVs which can resolve the underlying free energy landscape of the GB1 hairpin quite efficiently. Expressed as a linear combination of a number of traditional CVs, the optimal CV for this system is derived by employing the recently introduced time-structured independent component analysis approach on a large number of independent unbiased simulations. By projecting the replica-exchange simulated trajectories along these pair of optimized CVs, the resulting free energy landscape of this system is able to resolve four distinct well-separated metastable states encompassing the extensive ensembles of folded, unfolded, and molten globule states. Importantly, the optimized CVs were found to be capable of automatically recovering a novel partial helical state of this protein, without needing to explicitly invoke helicity as a constituent CV. Furthermore, a quantitative sensitivity analysis of each constituent in the optimized CV provided key insights on the relative contributions of the constituent CVs in the overall free energy landscapes. Finally, the kinetic pathways connecting these metastable states, constructed using a Markov state model, provide an optimum description of the underlying folding mechanism of the peptide. Taken together, this work offers a quantitatively robust approach toward comprehensive mapping of the underlying folding landscape of a quintessential model system along its optimized CV.

Direct numerical simulation of flame stabilization assisted by autoignition in a reheat gas turbine combustor

A three-dimensional direct numerical simulation (DNS) is performed for a turbulent hydrogen-air flame, represented with detailed chemistry, stabilized in a model gas-turbine combustor. The combustor geometry consists of a mixing duct followed by a sudden expansion and a combustion chamber, which represents a geometrically simplified version of Ansaldo Energia’s GT26/GT36 sequential combustor design. In this configuration, a very lean blend of hydrogen and vitiated air is prepared in the mixing duct and convected into the combustion chamber, where the residence time from the inlet of the mixing duct to the combustion chamber is designed to coincide with the ignition delay time of the mixture. The results show that when the flame is stabilized at its design position, combustion occurs due to both autoignition and flame propagation (deflagration) modes at different locations within the combustion chamber. A chemical explosive mode analysis (CEMA) reveals that most of the fuel is consumed due to autoignition in the bulk-flow along the centerline of the combustor, and lower amounts of fuel are consumed by flame propagation near the corners of the sudden expansion, where the unburnt temperature is reduced by the thermal wall boundary layers. An unstable operating condition is also identified, wherein periodic auto-ignition events occur within the mixing duct. These events appear upstream of the intended stabilization position, due to positive temperature fluctuations induced by pressure waves originating from within the combustion chamber. The present DNS investigation represents the initial step of a comprehensive research effort aimed at gaining detailed physical insight into the rate-limiting processes that govern the sequential combustor behavior and avoid the insurgence of the off-design auto-ignition events.

The impact of kinetic parameters on cellulose hydrolysis rates

Abstract Mechanistic kinetic models are vital tools to elucidate rate-limiting interactions and processes hampering complete hydrolysis of cellulose by cellulases. Properly formulated, the models should ultimately simulate experimental cellulose hydrolysis time courses given that model parameters are known a priori. Here, we review reported values of critical parameters in cellulose hydrolysis (catalytic, adsorption/desorption, complexation/decomplexation rate constants and apparent processivity) and examine how estimation methods may impact the magnitudes and interpretation of the values. The sensitivity of the extents and rates of cellulose hydrolysis to the range of reported values were examined by conducting simulations with a general mechanistic model of TrCel7A hydrolysis of cellulose. When the simulation was conducted at enzyme-limiting conditions, initial bursts in cellulose hydrolysis rates were evident and highly sensitive to specific hydrolysis rates of productively bound enzymes and dissociation rates of non-productively bound enzymes. Under substrate limiting conditions, hydrolysis rates were significantly reduced and no initial burst was observed, with more pronounced sensitivity of hydrolysis rates to the range of kinetic values. Enzyme-substrate interaction rate constants reviewed here only capture short-time hydrolysis rate outcomes and future work to elucidate mechanisms of cellulose hydrolysis must also incorporate how substrate-limitations evolve over the course of hydrolysis.

Membrane electroporation: chemical thermodynamics and flux kinetics revisited and refined.

The chemical thermodynamic concept for membrane electroporation is critically revisited. The hysteresis in the electric field dependence of the rapid in-field electroporation events (on the in-field hysteresis branch) and the slower post-field pore resealing process (zero-field hysteresis branch) is a typical ensemble property involving rapid single-pore opening-closing events that are temporally and spatially distributed. In the case of spherical membrane shells in homogeneous external fields, the acting local field is dependent on the polar-angular position. Hence, the experimental state distribution constant and the ensemble rate coefficients are statistical position averages; they are cosine square averages of the polar angle. Advanced flux analysis uses the concept of time-dependent flux coefficients reflecting the kinetics of the rate-limiting structural processes of electroporation and membrane resealing. The explicit integral flux equations rationalize the sigmoid onset of the in-field kinetics and quantify the post-field-stretched exponentials as exponentials of exponentials. Finally, the new analytical proposal for the evaluation of the electric field strength dependence of global cell electroporation data starts with the low-field range and continues with iterative parameter optimisation over the entire field strength range.

Reaction mechanisms at 4H-SiC/SiO2 interface during wet SiC oxidation

The reaction processes at the interface between SiC with 4H structure (4H-SiC) and SiO2 during wet oxidation are investigated by electronic structure calculations within the density functional theory. Our calculations for 4H-SiC/SiO2 interfaces with various orientations demonstrate characteristic features of the reaction depending on the crystal orientation of SiC: On the Si-face, the H2O molecule is stable in SiO2 and hardly reacts with the SiC substrate, while the O atom of H2O can form Si–O bonds at the C-face interface. Two OH groups are found to be at least necessary for forming new Si–O bonds at the Si-face interface, indicating that the oxidation rate on the Si-face is very low compared with that on the C-face. On the other hand, both the H2O molecule and the OH group are incorporated into the C-face interface, and the energy barrier for OH is similar to that for H2O. By comparing the calculated energy barriers for these reactants with the activation energies of oxide growth rate, we suggest the orientation-dependent rate-limiting processes during wet SiC oxidation.

Evaluating the kinetic basis of plant growth from organs to ecosystems.

Contents Summary 37 I. Introduction 37 II. Predictions for metabolic kinetics 38 III. Kinetics of net photosynthesis 38 IV. Kinetics of plant growth 40 V. Hypotheses for higher-level kinetic decoupling 41 VI. Conclusions 42 Acknowledgements 42 References 42 SUMMARY: Understanding how temperature influences the scaling of physiological rates through levels of biological organization is critical for predicting plant responses to climate. Metabolic theory predicts that many rates increase exponentially with temperature following an activation energy (E) of 0.32eV for photosynthesis. Here, I evaluate this prediction for net photosynthesis and organ, individual, and ecosystem growth. Observed E for photosynthesis varied widely but was not statistically different from predictions, while E for organs was greater than predicted, and E for individuals and ecosystems only weakly characterized temperature responses. I review several hypotheses that may underlie these results. Understanding how multiple rate-limiting processes coalesce into a single E that characterizes metabolic responses to temperature, and how to best estimate E from unimodal data, remain important challenges.

Importance of the Unstirred Water Layer and Hepatocyte Membrane Integrity In Vitro for Quantification of Intrinsic Metabolic Clearance.

Prediction of clearance-a vital component of drug discovery-remains in need of improvement and, in particular, requires more incisive assessment of mechanistic methodology in vitro, according to a number of recent reports. Although isolated hepatocytes have become an irreplaceable standard system for the measurement of intrinsic hepatic clearance mediated by active uptake transport and metabolism, the lack of prediction reliability appears to reflect a lack of methodological validation, especially for highly cleared drugs, as we have previously shown. Here, novel approaches were employed to explore fundamental experimental processes and associated potential limitations of in vitro predictions of clearance. Rat hepatocytes deemed nonviable by trypan blue staining showed undiminished metabolic activity for probe cytochrome P450 (P450) substrates midazolam and propranolol; supplementation with NADPH enhanced these activities. Extensive permeabilization of the plasma membrane using saponin showed either full or minimal P450 activity, depending on the presence or absence of 1 mM NADPH, respectively. The shaking of incubations facilitated P450 metabolic rates up to 5-fold greater than static incubation, depending on intrinsic clearance, indicating the critical influence of the unstirred water layer (UWL). Permeabilization allowed static incubation metabolic rates to approach those of shaking for intact cells, indicating an artificially induced breakdown of the UWL. Permeabilization combined with shaking allowed an increased metabolic rate for saquinavir, resolving the membrane permeability limitation for this drug. These findings advance the interpretation of the rate-limiting processes involved in intrinsic clearance measurements and could be critical for successful in vitro prediction.