A compartment model to predict in vitro finite dose absorption of chemicals by human skin.

Dermal uptake is an important and complex exposure route for a wide range of chemicals. Dermal exposure can occur due to occupational settings, pharmaceutical applications, environmental contamination, or consumer product use. The large range of both chemicals and scenarios of interest makes it difficult to perform generalizable experiments, creating a need for a generic model to simulate various scenarios. In this study, a model consisting of a series of four well-mixed compartments, representing the source solution (vehicle), stratum corneum, viable tissue, and receptor fluid, was developed for predicting dermal absorption. The model considers experimental conditions including small applied doses as well as evaporation of the vehicle and chemical. To evaluate the model assumptions, we compare model predictions for a set of 26 chemicals to finite dose in-vitro experiments from a single laboratory using steady-state permeability coefficient and equilibrium partition coefficient data derived from in-vitro experiments of infinite dose exposures to these same chemicals from a different laboratory. We find that the model accurately predicts, to within an order of magnitude, total absorption after 24 h for 19 of these chemicals. In combination with key information on experimental conditions, the model is generalizable and can advance efficient assessment of dermal exposure for chemical risk assessment.

The mismatch of narratives and local ecologies in the everyday governance of water access and mosquito control in an urbanizing community.

Mosquito-borne disease presents a significant threat to urban populations, but risk can be uneven across a city due to underlying environmental patterns. Urban residents rely on social and economic processes to control the environment and mediate disease risk, a phenomenon known as everyday governance. We studied how households employed everyday governance of urban infrastructure relevant to mosquito-borne disease in Bengaluru, India to examine if and how inequalities in everyday governance manifest in differences in mosquito control. We found that governance mechanisms differed for water access and mosquitoes. Economic and social capital served different roles for each, influenced by global narratives of water and vector control.

Physiologically Based Pharmacokinetic (PBPK) Modeling of Metabolic Pathways of Bromochloromethane in Rats.

Bromochloromethane (BCM) is a volatile compound and a by-product of disinfection of water by chlorination. Physiologically based pharmacokinetic (PBPK) models are used in risk assessment applications. An updated PBPK model for BCM is generated and applied to hypotheses testing calibrated using vapor uptake data. The two different metabolic hypotheses examined are (1) a two-pathway model using both CYP2E1 and glutathione transferase enzymes and (2) a two-binding site model where metabolism can occur on one enzyme, CYP2E1. Our computer simulations show that both hypotheses describe the experimental data in a similar manner. The two pathway results were comparable to previously reported values (V(max⁡) = 3.8 mg/hour, K(m) = 0.35 mg/liter, and k(GST) = 4.7 /hour). The two binding site results were V(max⁡(1) ) = 3.7 mg/hour, K(m⁡(1) ) = 0.3 mg/hour, CL(2) = 0.047 liter/hour. In addition, we explore the sensitivity of different parameters for each model using our obtained optimized values.

Evaluation of two different metabolic hypotheses for dichloromethane toxicity using physiologically based pharmacokinetic modeling for in vivo inhalation gas uptake data exposure in female B6C3F1 mice.

Dichloromethane (DCM, methylene chloride) is a lipophilic volatile compound readily absorbed and then metabolized to several metabolites that may lead to chronic toxicity in different target organs. Physiologically based pharmacokinetic (PBPK) models are useful tools for calculation of internal and target organ doses of parent compound and metabolites. PBPK models, coupled with in vivo inhalation gas-uptake data, can be useful to estimate total metabolism. Previously, such an approach was used to make predictions regarding the metabolism and to make subsequent inferences of DCM's mode of action for toxicity. However, current evidence warrants re-examination of this approach. The goal of this work was to examine two different hypotheses for DCM metabolism in mice. One hypothesis describes two metabolic pathways: one involving cytochrome P450 2E1 (CYP2E1) and a second glutathione (GSH). The second metabolic hypothesis describes only one pathway mediated by CYP2E1 that includes multiple binding sites. The results of our analysis show that the in vivo gas-uptake data fit both hypotheses well and the traditional analysis of the chamber concentration data is not sufficient to distinguish between them. Gas-uptake data were re-analyzed by construction of a velocity plot as a function of increasing DCM initial concentration. The velocity (slope) analysis revealed that there are two substantially different phases in velocity, one rate for lower exposures and a different rate for higher exposures. The concept of a "metabolic switch," namely that due to conformational changes in the enzyme after one site is occupied - a different metabolic rate is seen - is also consistent with the experimental data. Our analyses raise questions concerning the importance of GSH metabolism for DCM. Recent research results also question the importance of this pathway in the toxicity of DCM. GSH-related DNA adducts were not formed after in vivo DCM exposure in mice and DCM-induced DNA damage has been detected in human lung cultures without GSH metabolism. In summary, a revised/updated metabolic hypothesis for DCM has been examined using in vivo inhalation data in mice combined with PBPK modeling that is consistent with up-to-date models of the active site for CYP2E1 and suggests that this pathway is the major metabolizing pathway for DCM metabolism.

Development of an updated PBPK model for trichloroethylene and metabolites in mice, and its application to discern the role of oxidative metabolism in TCE-induced hepatomegaly.

Trichloroethylene (TCE) is a lipophilic solvent rapidly absorbed and metabolized via oxidation and conjugation to a variety of metabolites that cause toxicity to several internal targets. Increases in liver weight (hepatomegaly) have been reported to occur quickly in rodents after TCE exposure, with liver tumor induction reported in mice after long-term exposure. An integrated dataset for gavage and inhalation TCE exposure and oral data for exposure to two of its oxidative metabolites (TCA and DCA) was used, in combination with an updated and more accurate physiologically-based pharmacokinetic (PBPK) model, to examine the question as to whether the presence of TCA in the liver is responsible for TCE-induced hepatomegaly in mice. The updated PBPK model was used to help discern the quantitative contribution of metabolites to this effect. The update of the model was based on a detailed evaluation of predictions from previously published models and additional preliminary analyses based on gas uptake inhalation data in mice. The parameters of the updated model were calibrated using Bayesian methods with an expanded pharmacokinetic database consisting of oral, inhalation, and iv studies of TCE administration as well as studies of TCE metabolites in mice. The dose-response relationships for hepatomegaly derived from the multi-study database showed that the proportionality of dose to response for TCE- and DCA-induced hepatomegaly is not observed for administered doses of TCA in the studied range. The updated PBPK model was used to make a quantitative comparison of internal dose of metabolized and administered TCA. While the internal dose of TCA predicted by modeling of TCE exposure (i.e., mg TCA/kg-d) showed a linear relationship with hepatomegaly, the slope of the relationship was much greater than that for directly administered TCA. Thus, the degree of hepatomegaly induced per unit of TCA produced through TCE oxidation is greater than that expected per unit of TCA administered directly, which is inconsistent with the hypothesis that TCA alone accounts for TCE-induced hepatomegaly. In addition, TCE-induced hepatomegaly showed a much more consistent relationship with PBPK model predictions of total oxidative metabolism than with predictions of TCE area-under-the-curve in blood, consistent with toxicity being induced by oxidative metabolites rather than the parent compound. Therefore, these results strongly suggest that oxidative metabolites in addition to TCA are necessary contributors to TCE-induced liver weight changes in mice.

A physiologically based pharmacokinetic model for trichloroethylene in the male long-evans rat.

A physiologically based pharmacokinetic (PBPK) model for trichloroethylene (TCE) in the male Long-Evans (LE) rat was needed to aid in evaluation of neurotoxicity data collected in this rodent stock. The purpose of this study was to develop such a model with the greatest possible specificity for the LE rat. The PBPK model consisted of 5 compartments: brain, fat, slowly perfused tissue, rapidly perfused viscera, and liver. Partition coefficients (blood, fat, muscle, brain, liver) were determined for LE rats. The volumes of the brain, liver, and fat compartments were estimated for each rat, with tissue-specific regression equations developed from measurements made in LE rats. Vapor uptake data from LE rats were used for estimation of Vmaxc. As blood flow values for LE rats were not available, values from Sprague-Dawley (SD) and Fischer-344 (F344) rats were used in separate simulations. The resulting values of Vmaxc were used to simulate tissue (blood, liver, brain, fat) TCE concentrations, which were measured during (5, 20, 60 min) and after (60 min of TCE followed by 60 min of air) flow-through inhalation exposures of LE rats to 200, 2000, or 4000 ppm TCE. Simulation of the experimental data was improved by use of F-344 blood-flow values and the corresponding Vmaxc (8.68 mg/h/kg) compared to use of SD flows and the associated Vmaxc (7.34 mg/h/kg). Sensitivity analysis was used to determine those input parameters with the greatest influence on TCE tissue concentrations. Alveolar ventilation consistently (across exposure concentration, exposure duration, and target tissue) had the greatest impact on TCE tissue concentration. The PBPK model described here is being used to explore the relationship between measures of internal dose of TCE and neurotoxic outcome.

A comparison of Haber's rule at different ages using a physiologically based pharmacokinetic (PBPK) model for chloroform in rats.

Haber's rule as commonly interpreted in inhalation toxicology, can be stated as exposure concentration times duration equals a constant biological effect, or C x t=k. In other words, identical products of concentration and duration lead to the same effect. The goals of this paper are to develop a biological and pharmacokinetic modeling approach for chloroform, and to evaluate Haber's rule for different ages by taking into account the physiological changes due to growth and aging in rats. Three-dimensional dose-response surfaces for liver toxicity were generated for each age group of interest: adolescent, adult, and senescent rats. The three-dimensional surfaces were then characterized with a generalized description of Haber's rule for each age group. The simulations suggest that adolescent rats need higher exposure levels in order to achieve similar levels of liver damage compared to adults or senescent rats, if the comparison is made using the same exposure length. In summary, a pharmacokinetic modeling approach with a biological framework including the chemical's mode of action, was used to relate concentration, exposure duration and effect. Major advantages of this approach include: the potential ability to extrapolate to humans, the inclusion of aging in the simulations, and the ability to summarize the results using a generalized form of Haber's rule.

Physiologically based pharmacokinetic models for the transport of trichloroethylene in adipose tissue.

In this paper we present three physiologically based pharmacokinetic (PBPK) models for the systemic transport of trichloroethylene (TCE), with a focus on the adipose, or fat tissue. TCE is a widespread environmental contaminant, and has been shown to produce toxic effects in both animals and humans. A key characteristic of TCE is its tendency to accumulate in fat tissue, which has a major impact on the overall systemic disposition of TCE. Here we use PBPK models to predict the dynamics of TCE in the various tissues and organs, including the adipose tissue. The first model utilizes the standard 'perfusion-limited' compartmental model for the fat tissue, while the second model uses a 'diffusion-limited' model to describe the transport through the adipose tissue. Both of these ODE models are based on 'well-mixed' and rapid equilibrium assumptions, and do not take into account the specific and largely heterogeneous physiology of adipose tissue. The third model we discuss is a PBPK hybrid model with an axial-dispersion type model for the adipose tissue. This PDE-based model is designed to capture key physiological heterogeneities of fat tissue, including widely varying fat cell sizes, lipid distribution, and blood flow properties. Model simulations demonstrate that this model may be well-suited to predict the experimental behavior of TCE in adipose tissue using parameter estimation techniques.

Application of modelling techniques to the planning of in vitro arsenic kinetic studies.

A kinetic model describing the hepatic methylation of arsenite [As(III)] was developed on the basis of limited data from in vitro mechanistic studies. The model structure is as follows: sequential enzymic methylation of arsenite to its monomethylated (MMA) and dimethylated (DMA) products by first-order and Michaelis-Menten kinetics, respectively; uncompetitive inhibition of the formation of DMA by As(III); and first-order reversible binding of As(III), MMA and DMA to cytosolic proteins. Numerical sensitivity analysis was used to evaluate systematically the impact of changes in input parameters on model responses. Sensitivity analysis was used to investigate the possibility of designing experiments for robust testing of the uncompetitive inhibition hypothesis, and for further refining the model. Based on the sensitivity analysis, the MMA concentration is the most important response on which to focus. The parameters V(max) and k(i) can be reliably estimated by using the same concentration time-course data at intermediate initial arsenite concentrations of 1--5microM at 30 +/- 5 minutes. K(m) must be estimated independently of V(max), since the two parameters are highly correlated at all times, and the optimal experimental conditions would include lower initial concentrations of arsenite (0.1--0.5microM) and earlier time-points (about 8--18 minutes). The use of initial arsenite concentrations much above 5microM would not yield additional useful information, because the sensitivity coefficients for MMA, protein-bound MMA, DMA and protein-bound DMA tend to become extremely small or exhibit erratic trends. Overall trends in the sensitivity analysis indicated the desirability of performing measurements at times shorter than 60 minutes. This work demonstrates that physiological modelling and sensitivity analysis can be efficient tools for experimental planning and hypothesis testing when applied in the earliest phases of kinetic model development, thus allowing more-efficient and more-directed experimentation, and minimising the use of laboratory animals.

Sensitivity analysis of a physiological model for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): assessing the impact of specific model parameters on sequestration in liver and fat in the rat.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has dose-dependent tissue distribution because of induction of CYP1A2, a TCDD-binding protein, in the liver. Induction requires transcriptional activation of the CYP1A2 gene product by TCDD and the Ah receptor. An empirical model for dose-dependent distribution (Carrier et al., 1995, Toxicol. Appl. Pharmacol. 131, 253-266) included two simple descriptors: one for the maximum liver sequestration (Fmax) and the other for body burden leading to half maximum sequestration (Kd). Physiologically based pharmacokinetic (PBPK) models include specific parameters for protein receptors, protein binding, tissue solubility, and protein induction. We have applied a PBPK model to define two macroscopic constants related to these dose-response curves, i.e., the inflection point, and the maximum values of these curves. The dose-response curves generated from the PBPK model were for the proportion sequestered in liver and the liver to fat concentration ratio. Our analysis assessed the specific biological factors in the PBPK model that governed the values of these two macroscopic constants. For the fraction in liver, the Hill coefficient (a shape exponent describing the relationship between the Ah receptor-TCDD complex with the DNA receptor) resulted in the largest shift in inflection when using PBPK model parameters specific for TCDD. For the liver to fat ratio, the inflection point was most affected by the number of available Ah receptors. Conventional normalized sensitivity coefficients for the liver-to-fat ratio at the maximum were highest for the fat-to-blood partition coefficient, CYP1A2 binding affinity, and maximum extent of induction of CYP1A2. A similar pattern was observed for the liver fraction, except that the sensitivity coefficients were much smaller. The behavior of different TCDD congeners was evaluated by altering the value of key parameters. Our results demonstrate that the inflection point is more related to characteristics of DNA binding/induction steps of the Ah receptor-DNA complex than by the CYP1A2 affinity of TCDD or concentrations of CYP1A2. Surprisingly, the maximum is more sensitive to changes in CYP1A2 concentrations and affinity for TCDD. In addition, the analysis showed that the liver-to-fat ratio is a more useful experimental measure than is proportion in liver because the ratio responds with similar sensitivity over a much wider range of input parameters.

Effects of aging on cardiac output, regional blood flow, and body composition in Fischer-344 rats.

The purpose of this study was to determine the effects of maturation and aging on cardiac output, the distribution of cardiac output, tissue blood flow (determined by using the radioactive-microsphere technique), and body composition in conscious juvenile (2-mo-old), adult (6-mo-old), and aged (24-mo-old) male Fischer-344 rats. Cardiac output was lower in juvenile rats (51 +/- 4 ml/min) than in adult (106 +/- 5 ml/min) or aged (119 +/- 10 ml/min) rats, but cardiac index was not different among groups. The proportion of cardiac output going to most tissues did not change with increasing age. However, the fraction of cardiac output to brain and spinal cord tissue and to skeletal muscle was greater in juvenile rats than that in the two adult groups. In addition, aged rats had a greater percent cardiac output to adipose tissue and a lower percent cardiac output to cutaneous and reproductive tissues than that in juvenile and adult rats. Differences in age also had little effect on mass-specific perfusion rates in most tissues. However, juvenile rats had lower flows to the pancreas, gastrointestinal tract, thyroid and parathyroid glands, and kidneys than did adult rats, and aged rats had lower flows to the white portion of rectus femoris muscle, spleen, thyroid and parathyroid glands, and prostate gland than did adult rats. Body mass of juvenile rats was composed of a lower percent adipose mass and a greater fraction of brain and spinal cord, heart, kidney, liver, and skeletal muscle than that of the adult and aged animals. Relative to the young adult rats, the body mass of aged animals had a greater percent adipose tissue mass and a lower percent skeletal muscle and skin mass. These data demonstrate that maturation and aging have a significant effect on the distribution of cardiac output but relatively little influence on mass-specific tissue perfusion rates in conscious rats. The old-age-related alterations in cardiac output distribution to adipose and cutaneous tissues appear to be associated with the increases in percent body fat and the decreases in the fraction of skin mass, respectively, whereas the decrease in the portion of cardiac output directed to reproductive tissue of aged rats appears to be related to a decrease in mass-specific blood flow to the prostate gland.

Toxicity of linoleic acid hydroperoxide to Saccharomyces cerevisiae: involvement of a respiration-related process for maximal sensitivity and adaptive response.

Linoleic acid hydroperoxide (LoaOOH) formed during free radical attack on long-chain unsaturated fatty acids is an important source of biomembrane damage and is implicated in the onset of atherosclerosis, hepatic diseases, and food rancidity. LoaOOH is toxic to wild-type Saccharomyces cerevisiae at a very low concentration (0.2 mM) relative to other peroxides. By using isogenic mutant strains, the possible roles of glutathione (gsh1 and gsh2), glutathione reductase (glr1), respiratory competence ([rho0] petite), and yAP-1p-mediated expression (yap1) in conferring LoaOOH resistance have been examined. Respiration-related processes were essential for maximal toxicity and adaptation, as evidenced by the fact that the [rho0] petite mutant was most resistant to LoaOOH but could not adapt. Furthermore, when respiration was blocked by using inhibitors of respiration and mutants defective in respiratory-chain components, cells became more resistant. An important role for reduced glutathione and yAP-1 in the cellular response to LoaOOH was shown, since the yap1 and glr1 mutants were more sensitive than the wild type. In addition, total glutathione peroxidase activity increased following treatment with LoaOOH, indicating a possible detoxification role for this enzyme. Yeast also showed an adaptive response when pretreated with a nonlethal dose of LoaOOH (0.05 mM) and subsequently treated with a lethal dose (0.2 mM), and de novo protein synthesis was required, since adaptation was abolished upon treatment of cells with cycloheximide (25 microg ml-1). The wild-type adaptive response to LoaOOH was independent of those for the superoxide-generating agents paraquat and menadione and also of those for the organic hydroperoxides cumene hydroperoxide and tert-butyl hydroperoxide. Pretreatment with LoaOOH induced resistance to hydrogen peroxide, while pretreatment of cells with malondialdehyde (a lipid peroxidation product) and heat shock (37 degrees C) gave cross-adaptation to LoaOOH, indicating that yeast has effective overlapping defense systems that can detoxify fatty acid hydroperoxides directly or indirectly.

Determination of parameters responsible for pharmacokinetic behavior of TCDD in female Sprague-Dawley rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic member of a class of planar and halogenated chemicals. Improvements in exposure assessment of TCDD require scientific information on the distribution of TCDD in target tissues and cellular responses induced by TCDD. Since 1980, several physiologically based pharmacokinetic (PBPK) models for TCDD and related compounds have been reported. Some of these models incorporated the induction of a hepatic binding protein in response to interactions of TCDD, the Ah receptor, and DNA binding sites and described the TCDD disposition in a biological system for certain data sets. Due to the limitations of the available experimental data, different values for the same physical parameters of these models were obtained from the different studies. The inconsistencies of the parameter values limit the application of PBPK models to risk assessment. Therefore, further refinement of previous models is necessary. This paper develops an improved PBPK model to describe TCDD disposition in eight target tissues. The interaction of TCDD with the Ah receptor and with hepatic inducible CYP1A2 were also incorporated into the model. This model accurately described the time course distribution of TCDD following a single oral dose of 10 microg/kg, as well as the TCDD concentration on Day 3 after six different doses, 0.01, 0.1, 0.3, 1, 10, and 30 microg TCDD/kg, in target tissues. This study extends previous TCDD models by illustrating the validity and the limitation of the model and providing further confirmation of the potential PBPK model for us in optimal experimental design and extrapolation across doses and routes of exposure. In addition, this study demonstrated some critical issues in PBPK modeling.

Physiologically based pharmacokinetic estimated metabolic constants and hepatotoxicity of carbon tetrachloride after methanol pretreatment in rats.

A single 6-hr exposure to inhaled methanol (MeOH) has been shown to enhance carbon tetrachloride (CCl4) hepatotoxicity. The objective of the present study was to use gas uptake data and the development of a physiologically based pharmacokinetic model (PBPK) to determine in vivo changes in CCl4 metabolism resulting from MeOH pretreatment. Adult male F344 rats (167-197 g) were exposed to 10,000 ppm MeOH (constant concentration) via inhalation for 6 hr. Individual rats were exposed using gas uptake techniques to CCl4 alone or to CCl4 either 24 or 48 hr after initiation of MeOH pretreatment. The following initial concentrations were used for CCl4: 0, 25, 100, 250, and 1000 ppm with exposures lasting 6 hr. Vmax (metabolic rate) was estimated from gas uptake data and Km (Michaelis constant) was assumed constant after methanol pretreatment. For CCl4 alone, Vmax was 0.11 mg/hr (Vmaxc = 0.37 mg/hr/kg) and Km was 1.3 mg/liter. Vmax was 0.48 mg/hr (Vmaxc = 1.6 mg/hr/kg) for the 24-hr MeOH + CCl4 group and Vmax was 0.18 mg/hr (Vmaxc = 0.6 mg/hr/kg) for the 48-hr MeOH + CCl4 group. For CCl4 alone, serum markers of hepatotoxicity alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) were increased significantly only at 1000 ppm CCl4. Both serum markers of hepatotoxicity in the 24-hr MeOH + CCl4 group increased as a function of CCl4 concentration when compared with 0 ppm CCl4 controls. The maximum increase occurred at 1000 ppm CCl4, where ALT and SDH increased by 392- and 286-fold, respectively. At 100, 250, and 1000 ppm CCl4, ALT and SDH values for the 24-hr MeOH + CCl4 groups were significantly increased relative to control (0 ppm CCl4), CCl4 alone, and 48-hr MeOH + CCl4. ALT and SDH levels in the 48-hr MeOH + CCl4 groups were not statistically different from the respective CCl4 alone groups.

Modeling the cytochrome P450-mediated metabolism of chlorinated volatile organic compounds.

Comparative molecular field analysis (CoMFA), a three-dimensional quantitative structure-activity relationship (3D-QSAR) paradigm, has been used to analyze the metabolic rates, as intrinsic clearance, of a series of chlorinated volatile organic compounds (VOCs). A comparison between 3D-QSAR and conventional Hansch-type QSAR is provided. To develop predictive 3D-QSARs for metabolism, the standard CoMFA model based on steric and electrostatic potential fields must be supplemented with hydropathic and molecular orbital information also in the form of three-dimensional fields. A mechanistic interpretation of chlorinated VOC metabolism by cytochrome P450 isozymes is provided as a rationalization for the inclusion of multiple fields in the CoMFA 3D-QSAR model. Models of this type have practical utility in the development of generalized physiologically-based pharmacokinetic models, as well as the rational, structure-based, design and/or selection of compounds for use in the in vivo and in vitro metabolic studies.

Applications of sensitivity analysis to a physiologically based pharmacokinetic model for carbon tetrachloride in rats.

Physiologically based pharmacokinetic (PBPK) models developed from gas uptake experiments have been used to estimate metabolic parameters for volatile organic compounds. Due to the potential application of PBPK models to estimate metabolic bioactivation constants in humans, it is important to understand the complex nature of these models and the resulting estimates. Adult male F344 rats (165-205 g) were individually exposed to carbon tetrachloride (CCl4) in gas uptake systems. Three rats at each concentration were exposed for 6 hr to initial concentrations of 25, 100, 250, and 1000 ppm CCl4. Partition coefficient determinations were performed by the vial equilibration technique and used as model inputs. Computer optimizations with the means of each initial chamber concentration at each time point resulted in an estimate of Vmax of 0.11 mg/hr (Vmaxc = 0.37 mg/hr/kg) and Km of 1.3 mg/liter. To determine the effect of individual animal variation in Vmax, optimizations were also performed with the mean +/- SD, resulting in Vmax estimates of 0.09 and 0.12 mg/hr, respectively. Similar analysis resulted in Km estimates of 0.98 and 1.58 mg/liter. The results of the sensitivity analysis were concentration dependent for CCl4. These results show Vmax and Km to be most accurately detected at lower initial chamber concentrations. Results of the sensitivity analysis at the lowest concentration established the following model input hierarchy: blood to air partition > fat partition and fat volume fraction > slowly perfused partition, ventilation rate, cardiac output, fat blood flow percentage > liver blood flow percentage and slowly perfused blood flow percentage. Further sensitivity analysis determined Vmax and Km to be highly correlated when using gas uptake technology and point to the need to an independent estimate for either constant. In summary, the application of sensitivity analysis to PBPK modeling resulted in an increased understanding of factors governing the estimation of metabolic parameters.

Time shift in ventilation-induced density fluctuation of arterial blood.

In an artificially ventilated dog, the varying tracheal pressure causes a density fluctuation in the blood sampled from the aorta. We cross-correlated the tracheal pressure with the density to determine the time shift or delay of the latter from the former waveform for a ventilation frequency in the range of 6-30 CPM. The delay time was found to be 29% of the mean transit time (MTT) of the pulmonary vasculature and independent of the ventilation frequency. A comparison of this percentage with the reported arterial-to-capillary-to-venous fractional volumes of the lung suggested that the delay time may be the MTT time for blood flowing through the venous network of the lung and the cross-correlation may serve as an in vivo means to partition the MTT of the pulmonary vasculature at its capillaries. These results and an analysis on the deformation of the viscoelastic, pulmonary capillaries indicated that the tracheal pressure, acting primarily through the viscous part of the viscoelasticity, deforms the capillaries to produce the density fluctuation in blood outflowing from the lung.

A density method to quantify pulmonary microvascular hematocrit.

We perfused the left lower lobe of a dog with autologous blood having a hematocrit Ha. When the vascular pressure perfusing the lobe was elevated, we observed a transient increase in the density of venous blood. Converting the density increase to a rise in hematocrit, we could calculate a volume (Vr) of red blood cells (RBC) over their normal outflow that was released by the lobe as a result of the elevation. We measured the weight gain of the lobe to determine the increase in pulmonary vascular volume, V' - V. We found that the ratio, Vr/Ha/(V' - V), is 0.11 +/- 0.02. To determine the implication of this ratio, we divided the lobular vasculature into an arterial, microvascular, and venous compartment. Due to the Fahraeus effect, the tube hematocrit in the microvascular compartment (Hc) is lower than that of two macrovascular compartments, Ha. An analysis on the balance of RBC and plasma flows through the lobe identified the volume Vr as (Vc' - Vc) (Ha - Hc) with Vc' - Vc being the volumetric increase of the lobular microvascular compartment. Based on the reported volumetric fractional change of microvascular compartment, we estimated that the microvascular (tube) hematocrit in pulmonary capillaries is 80% (ranging from 78 to 82%) of the hematocrit perfusing the lobe. Since the additional RBC volume (Vr) being released from the lobe cannot be accounted for by transcapillary filtration or capillary recruitment, we conclude from this analysis that the measurement of the transient density change in pulmonary outflow can be used to quantify the microvascular hematocrit of the lung.