Effects of p67phox on the mitochondrial oxidative state in the kidney of Dahl salt-sensitive rats: optical fluorescence 3-D cryoimaging.

The goal of the present study was to quantify and correlate the contribution of the cytosolic p67(phox) subunit of NADPH oxidase 2 to mitochondrial oxidative stress in the kidneys of the Dahl salt-sensitive (SS) hypertensive rat. Whole kidney redox states were uniquely assessed using a custom-designed optical fluorescence three-dimensional cryoimager to acquire multichannel signals of the intrinsic fluorophores NADH and FAD. SS rats were compared with SS rats in which the cytosolic subunit p67(phox) was rendered functionally inactive by zinc finger nuclease mutation of the gene (SS(p67phox)-null rats). Kidneys of SS rats fed a 0.4% NaCl diet exhibited significantly (P = 0.023) lower tissue redox ratio (NADH/FAD; 1.42 ± 0.06, n = 5) than SS(p67phox)-null rats (1.64 ± 0.07, n = 5), indicating reduced levels of mitochondrial electron transport chain metabolic activity and enhanced oxidative stress in SS rats. When fed a 4.0% salt diet for 21 days, both strains exhibited significantly lower tissue redox ratios (P < 0.001; SS rats: 1.03 ± 0.05, n = 9, vs. SS(p67phox)-null rats: 1.46 ± 0.04, n = 7) than when fed a 0.4% salt, but the ratio was still significantly higher in SS(p67phox) rats at the same salt level as SS rats. These results are consistent with results from previous studies that found elevated medullary interstitial fluid concentrations of superoxide and H2O2 in the medulla of SS rats. We conclude that the p67(phox) subunit of NADPH oxidase 2 plays an important role in the excess production of ROS from mitochondria in the renal medulla of the SS rat.

A mechanistic mathematical model for the catalytic action of glutathione peroxidase.

Glutathione peroxidase (GPx) is a well-known seleno-enzyme that protects cells from oxidative stress (e.g., lipid peroxidation and oxidation of other cellular proteins and macromolecules), by catalyzing the reduction of harmful peroxides (e.g., hydrogen peroxide: H2O2) with reduced glutathione (GSH). However, the catalytic mechanism of GPx kinetics is not well characterized in terms of a mathematical model. We developed here a mechanistic mathematical model of GPx kinetics by considering a unified catalytic scheme and estimated the unknown model parameters based on different experimental data from the literature on the kinetics of the enzyme. The model predictions are consistent with the consensus that GPx operates via a ping-pong mechanism. The unified catalytic scheme proposed here for GPx kinetics clarifies various anomalies, such as what are the individual steps in the catalytic scheme by estimating their associated rate constant values and a plausible rationale for the contradicting experimental results. The developed model presents a unique opportunity to understand the effects of pH and product GSSG on the GPx activity under both physiological and pathophysiological conditions. Although model parameters related to the product GSSG were not identifiable due to lack of product-inhibition data, the preliminary model simulations with the assumed range of parameters show that the inhibition by the product GSSG is negligible, consistent with what is known in the literature. In addition, the model is able to simulate the bi-modal behavior of the GPx activity with respect to pH with the pH-range for maximal GPx activity decreasing significantly as the GSH levels decrease and H2O2 levels increase (characteristics of oxidative stress). The model provides a key component for an integrated model of H2O2 balance under normal and oxidative stress conditions.

A database of thermodynamic quantities for the reactions of glycolysis and the tricarboxylic acid cycle.

Analysis of biochemical systems requires reliable and self-consistent databases of thermodynamic properties for biochemical reactions. Here a database of thermodynamic properties for the reactions of glycolysis and the tricarboxylic acid cycle is developed from measured equilibrium data. Species-level free energies of formation are estimated on the basis of comparing thermodynamic model predictions for reaction-level equilibrium constants to previously reported data obtained under different experimental conditions. Matching model predictions to the data involves applying state corrections for ionic strength, pH, and metal ion binding for each input experimental biochemical measurement. By archiving all of the raw data, documenting all model assumptions and calculations, and making the computer package and data available, this work provides a framework for extension and refinement by adding to the underlying raw experimental data in the database and/or refining the underlying model assumptions. Thus the resulting database is a refinement of preexisting databases of thermodynamics in terms of reliability, self-consistency, transparency, and extensibility.

BISEN: Biochemical Simulation Environment.

SUMMARY: The Biochemical Simulation Environment (BISEN) is a suite of tools for generating equations and associated computer programs for simulating biochemical systems in the MATLAB computing environment. This is the first package that can generate appropriate systems of differential equations for user-specified multi-compartment systems of enzymes and transporters accounting for detailed biochemical thermodynamics, rapid equilibria of multiple biochemical species and dynamic proton and metal ion buffering. AVAILABILITY: The software and a user manual (including several tutorial examples) are available at bbc.mcw.edu/BISEN.

Relating pulmonary oxygen uptake to muscle oxygen consumption at exercise onset: in vivo and in silico studies.

Assessment of the rate of muscle oxygen consumption, UO(2m), in vivo during exercise involving a large muscle mass is critical for investigating mechanisms regulating energy metabolism at exercise onset. While UO(2m) is technically difficult to obtain under these circumstances, pulmonary oxygen uptake, VO(2p), can be readily measured and used as a proxy to UO(2m). However, the quantitative relationship between VO(2p) and UO(2m) during the nonsteady phase of exercise in humans, needs to be established. A computational model of oxygen transport and utilization--based on dynamic mass balances in blood and tissue cells--was applied to quantify the dynamic relationship between model-simulated UO(2m) and measured VO(2p) during moderate (M), heavy (H), and very heavy (V) intensity exercise. In seven human subjects, VO(2p) and muscle oxygen saturation, StO(2m), were measured with indirect calorimetry and near infrared spectroscopy (NIRS), respectively. The dynamic responses of VO(2p) and StO(2m) at each intensity were in agreement with previously published data. The response time of muscle oxygen consumption, tauUO(2m) estimated by direct comparison between model results and measurements of StO(2m) was significantly faster (P < 0.001) than that of pulmonary oxygen uptake, tauVO(2p) (M: 13 +/- 4 vs. 65 +/- 7 s; H: 13 +/- 4 vs. 100 +/- 24 s; V: 15 +/- 5 vs. 82 +/- 31 s). Thus, by taking into account the dynamics of oxygen stores in blood and tissue and determining muscle oxygen consumption from muscle oxygenation measurements, this study demonstrates a significant temporal dissociation between UO(2m) and VO(2p) at exercise onset.

Shear augmented dispersion of a solute in a casson fluid flowing in a conduit.

The unsteady dispersion of a solute in a Casson fluid flowing in a conduit (pipe/channel) is studied using the generalized dispersion model of Gill and Sankarasubramanian. With this approach, the entire dispersion process is described appropriately in terms of a simple diffusion process with the effective diffusion coefficient as a function of time, in addition to its dependence on the yield stress of the fluid. The results are accurate up to a first approximation for small times, but verified with Sharp to be exact for large times. The model brings out mainly the effect of yield stress, or equivalently, the plug flow region on the overall dispersion process. It is found that the rate of dispersion is reduced (i.e., the effective diffusivity decreases) due to the yield stress of the fluid, or equivalently, the plug flow region in the conduit. Also, the effective diffusivity increases with time, but eventually attains its steady state value below a critical time [0.48(a2/Dm) for dispersion in a pipe and 0.55(a2/Dm) for dispersion in a channel-the critical transient time for a Newtonian fluid-where "a" is the radius of the pipe and Dm is the molecular diffusivity]. At steady state, for dispersion in a pipe with the plug flow radius one tenth of the radius of the pipe, the effective diffusivity is reduced to about 0.78 times of the corresponding value for a Newtonian fluid at equivalent flow rates; for dispersion in a channel, the reduction factor is about 0.73 confirming the earlier result of Sharp. Further, the location of the center of mass of a passive species over a cross section is found to remain unperturbed during the course of dispersion and for different values of the plug flow parameter (i.e., the yield stress of the fluid). The study can be used as a starting first approximate solution for studying the dispersion in the cardiovascular system or blood oxygenators.

Flow in a catheterized curved artery with stenosis.

The fluid mechanics of blood flow in a catheterized curved artery with stenosis is studied through a mathematical analysis. Blood is modelled as an incompressible Newtonian fluid and the flow is assumed to be steady and laminar. An approximate analytic solution to the problem is obtained through a double series perturbation analysis for the case of small curvature and mild stenosis. The effect of catheterization on various physiologically important flow characteristics (i.e. the pressure drop, impedance and the wall shear stress) is studied for different values of the catheter size and Reynolds number of the flow. It is found that all these flow characteristics vary markedly across a stenotic lesion. Also, increase in the catheter size leads to a considerable increase in their magnitudes. These results are used to obtain the estimates of increased pressure drop across an arterial stenosis when a catheter is inserted into it. Our calculations, based on the geometry and flow conditions existing in coronary arteries, suggest that, in the presence of curvature and stenosis, and depending on the value of k (ratio of catheter size to vessel size) ranging from 0.1 to 0.4, the pressure drop increases by a factor ranging from 1.60 to 5.16. But, in the absence of curvature and stenosis, with the same range of catheter size, this increased factor is about 1.74-4.89. These estimates for the increased pressure drop can be used to correct the error involved in the measured pressure gradients using catheters. The combined effects of stenosis and curvature on flow characteristics are also studied in detail. It is found that the effect of stenosis is more dominant than that of the curvature. Due to the combined effect of stenosis, curvature and catheterization, the secondary streamlines are modified in a cross-sectional plane. The insertion of a catheter into the artery leads to the formation of increased number of secondary vortices.

Estimation of increased flow resistance in a narrow catheterized artery--a theoretical model.

The changed flow pattern in a narrow catheterized artery is studied and an estimate of the increased flow resistance is made. The anomalous behaviour of blood in small blood vessels has been taken into account by modelling blood as a Casson fluid possessing some finite yield stress. Both the cases of steady and pulsatile flow situations are studied. The pulsatile flow is analysed by considering the pressure gradient as a periodic function of time with small inertial effects. The resulting quasi-steady non-linear coupled implicit system of differential equations governing the flow are solved using a perturbation analysis, where it is assumed that the Womersley frequently parameter is small (alpha < 1) which is reasonable for physiological situations in small blood vessels as well as in coronary arteries. The effect of pulsatility, catheter radius and yield stress of the fluid on the yield plane locations, velocity distribution, flow rate, shear stress and frictional resistance are investigated. Because of the yield stress theta, two yield surfaces are found to be located in the flow field. Depending on the ration kappa (catheter size/vessel size) ranging from 0.3 to 0.7 (which is widely used in coronary angioplasty procedures), the frictional resistance to flow in large blood vessels, where the effect of yield stress can be neglected (i.e. theta = 0), increases by a factory ranging from 3 to 33. In small blood vessels with the same range of catheter size and an unit pressure gradient, frictional resistance increase was by a factor of 7-21 when theta = 0.05 and 11-294 when theta = 0.1. For small values of kappa and theta, the frictional resistance increased to several hundred times thus implying that the combined effect of increased catheter radius and yield stress is to obstruct the fluid movement considerably.