Transport and metabolism of palmitate in the rat liver. Net flux and unidirectional fluxes across the cell membrane.

The unidirectional fluxes of palmitate across the liver cell membrane and metabolic uptake rates were measured employing the multiple-indicator dilution technique. The following results were obtained: (1) Influx and net uptake rates do not vary proportionally to each other when albumin and palmitate concentrations are varied. (2) Efflux is significant for albumin concentrations in the range between 1.5 and 500 microM. (3) At 150 microM albumin net uptake rates are proportional to the total (bound plus free) extracellular palmitate concentration in the range from 10 to 600 microM; the dependence of influx rates on the palmitate concentration is rather concave up. (4) When albumin and palmitate are both varied at an equimolar ratio, pseudo-saturation appears in the net uptake rates; the influx rates also show pseudo-saturation, but with a declining tendency at the higher concentrations. (5) The intracellular palmitate concentration is strongly influenced by albumin. At very low concentrations of the protein (1.5 microM) the intracellular concentration is practically equal to the extracellular one; at physiological albumin concentrations, however, the intracellular palmitate concentration is less than 2% of the extracellular one. (6) Saturation of net uptake with respect to the intracellular palmitate concentration was not observed with concentrations up to 46 microM.

A method of dynamic analysis of iodine-123-metaiodobenzylguanidine scintigrams in cardiac mechanical overload hypertrophy and failure.

Cardiac sympathetic neuronal degeneration accompanies mechanical overload heart failure. We hypothesized that sympathetic nerve and myocyte failure share a common etiology and that 123I-metaiodobenzylguanidine (MIBG) might provide a precise method of detecting failure in chronic mechanical overload. Our aim was to develop a method for the dynamic analysis of 123I-MIBG scintigrams which could yield a quantitative index of myocardial sympathetic neuronal function in this condition. We performed serial 123I-MIBG scintigraphy in 33 volunteers, 10 orthotopic cardiac transplant recipients and 26 patients with chronic mechanical overload of the left ventricle. We constructed a compartmental model in which total heart activity represents the sum of cardiac sympathetic vesicular and cytosolic pools. Patients with antecedent mechanical overload heart failure or myocardial dysfunction had accelerated myocardial egress of tracer that we ascribed to a specific impairment in vesicular storage rather than to a more rapid turnover of an intact vesicular pool.

Inhibition of monosaccharide transport in the intact rat liver by stevioside.

The transport and metabolism of D-glucose and D-fructose in the isolated perfused rat liver and the influence of stevioside and its derivatives were investigated. The transport parameters were measured by the multiple indicator dilution technique. The maximal exchange rate of D-glucose was 700 mumol X min-1 X ml-1 and the Km was 38 mM. Stevioside and its derivatives (isosteviol and steviolbioside) inhibited D-glucose and D-fructose transport across the cell membrane. The half-maximal effect at 1 mM D-glucose occurred at 0.8 mM stevioside. The inhibitory action of stevioside was of mixed type. Isosteviol was more potent than stevioside (half-maximal effect at 0.4 mM), whereas steviolbioside was less active (50% inhibition at 2.5 mM). Stevioside was without effect on D-glucose metabolism, except for transient changes in D-glucose release, reflecting changes in the intracellular concentration. D-Fructose consumption, however, was specifically affected (half-maximal effect at 2.8 mM), as well as all parameters depending on D-fructose transformation (D-glucose production, L-lactate and pyruvate production, and extra oxygen uptake). In livers releasing D-glucose from endogenous glycogen, strong inhibition of transport increased the intracellular to extracellular D-glucose concentration ratio (Ci/Ce). The control values of Ci/Ce, representing an average over the total intracellular water space, were always smaller than unity. The latter observation may indicate that D-glucose does not have access to the whole intracellular water space.

The multiple indicator dilution method and its utility in risk assessment.

The multiple-indicator dilution (MID) technique entails the injection of a mixture of labeled indicators into the blood vessel immediately at the entrance of an organ, e.g., the liver, kidney, heart, or lung, and characterization of outflow dilution profiles from timed venous samples. The mathematical basis of the method encompasses linear systems of partial differential equations that are formulated for flow- or barrier-limited transport combined with intracellular metabolism/excretion. The concept can be generalized to include metabolites. MID experiments are useful for determining tissue partition coefficients as well as kinetic parameters such as membrane permeabilities or metabolic/excretory intrinsic clearances, factors that affect the mean residence times or exposure of solutes to the organ. The main utility of the MID method lies in its role in identifying the basic mechanisms of the interaction of organs with vascular components. The concentration dependence in transport and removal is revealed by the rate coefficients upon varying the input concentrations of unlabeled substances into the organ at steady state. The data obtained with MID experiments can be incorporated into physiologically based pharmacokinetic (PBPK) models such as those used for biological risk assessment. This is especially pertinent in the case where diffusional barriers appear within organs. The insight gained from the MID organ approach may be useful for PBPK models with more realistic representation of organ kinetics.

Effects of flow, perfusion pressure, and oxygen consumption on cardiac capillary exchange.

The roles of blood flow, local oxygen consumption, and perfusion pressure on cardiac transcapillary exchange were characterized in closed-chest anesthetized dogs by use of the multiple-indicator dilution technique. Occlusion of the carotid arteries or injection of dipyridamole increased coronary flow to significantly higher values compared with a group of animals in a basal state obtained in a previous study. Carotid occlusion resulted in a significant increase in perfusion pressure and myocardial oxygen consumption, whereas these two variables were significantly reduced after dipyridamole. For the whole group of animals, the capillary permeability-surface area product for sucrose increased with coronary flow, which appeared to be the important controller for this microcirculatory exchange parameter. Perfusion pressure and myocardial oxygen consumption also regulated permeability-surface area product values, although to a lesser extent than flow. The heterogeneity of transit times in the capillaries was reduced at high coronary flow values, despite large differences in the cardiac utilization of oxygen. The data suggest that cardiac capillary exchange responds mostly to hemodynamic changes originating at the precapillary level.

Kinetics of pulmonary uptake of serotonin during exercise in dogs.

The multiple indicator-dilution technique was employed in the exercising dog to evaluate the effect of increasing activity on the pulmonary extraction and kinetics of removal of tracer 3H-labeled serotonin (5-HT) and on the measured central blood volume and tracer-accessible extravascular lung water. 51Cr-labeled red blood cells, 125I-labeled albumin, and 14C-labeled 1,8-octanediol were injected with labeled 5-HT at rest and at two increasing levels of exercise (lower and higher in 9 dogs). Blood flow approximately tripled at the highest level of exercise, and the central blood volume increased linearly with increasing blood flow. The tracer-accessible extravascular lung water increased in the transition from rest to low-level exercise and stabilized at an average proportion of 0.85 of the gravimetric extravascular lung water at the higher values of blood flow. The average labeled 5-HT extraction at rest was 42 +/- 11%, and this slowly decreased with increase in flow. The calculated permeability-surface area product for 5-HT increased approximately directly with increasing blood flow. We conclude that exercise results in an increase in the central blood volume that is accompanied by an increase in the tracer-accessible extravascular lung water (lung tissue recruitment) over low exercise levels, with no change at higher levels of exercise, and that the pulmonary capillary surface area subserving 5-HT uptake increases almost linearly with flow over the range of flows attained.

Transport, binding, and metabolism of sulfate conjugates in the liver.

Sulfate conjugates are a heterogeneous class of polar, anionic metabolites that result from the conjugation of endogenous and exogenous compounds. Sulfate conjugates exhibit a high degree of binding to albumin, the extent of which usually exceeds those of their parent compounds. Preponderant direct and indirect evidence suggests that sulfation activity is slightly higher in the periportal than in the perivenous (centrilobular) region of the liver, but recent immunohistochemical studies imply that specific isoforms of the sulfotransferases may also be preferentially localized in the perivenous region. Entry of sulfate conjugates into the liver cell is poor unless discrete carriers are present. Although known transport carriers exist for the sulfated bile acids, the specificity of the carriers for drug sulfate conjugates is presently unknown. The removal of sulfates is usually by way of biliary excretion while, on occasion, sulfates can be desulfated and participate in futile cycling with their parent compounds. The binding, transport, and hepatic elimination of various drug sulfate conjugates are examined. Non-recirculating studies carried out in the perfused rat liver with the multiple indicator dilution technique under varying input sulfate conjugate concentrations have provided essential information on the effects of vascular (red blood cells and plasma protein) binding on transport and removal of the conjugates. These studies clearly demonstrate the need to study protein binding, transmembrane transfer characteristics across the liver basolateral (sinusoidal) and canalicular membranes, and enzyme zonation in a distributed-in-space fashion in order to properly define the handling of sulfate conjugates in the liver.

Patterns of contraceptive use and pregnancy among young Hispanic women on the Texas-Mexico border.

Our objective was to identify significant family planning and health access problems of young Hispanic women on the Texas-Mexico border. Samples of 300 young Hispanic women living in each of the twin cities of El Paso and Juarez were interviewed regarding their knowledge, attitudes, and experiences with respect to birth control, pregnancy, maternal and child health, and health-care services. Knowledge and attitudes of the women about birth control technology were assessed along with their beliefs regarding the use of such technology. Results showed that contraceptive knowledge and usage patterns for young Hispanic women in Juarez and El Paso were significantly different. They relied on different types of birth control methods and also differed with respect to confidence in these methods and related medical services. Both groups reflected positive attitudes toward both child bearing and use of birth control although Juarez women were significantly more favorable toward child bearing. Both groups overwhelmingly favored female doctors. The young women studied have accepted the need for birth control, prefer fewer children, and have some degree of confidence in medical services. Their knowledge and use of reliable versus unreliable birth control devices appear to be major areas requiring culturally sensitive intervention.

PIP: Social work students administered a questionnaire to 600 randomly selected adolescent females from El Paso, Texas, and Juarez, Mexico, to examine their knowledge and use of contraceptive methods and their attitudes about pregnancy, contraception, and child care. 11.7% of the El Paso respondents and 16.7% of the Juarez respondents had been pregnant at some time. Oral contraceptives (OCs) were the most known contraceptive method. Women in El Paso were more likely to be familiar with OCs (98% vs. 91.3%), condoms (93.7% vs. 74.7%), vaginal methods (79.7% vs. 67.8%), and withdrawal (65.3% vs. 44.5%) than were those in Juarez (p = .001). On the other hand, more Juarez women than El Paso women knew about injectables (79.8% vs. 34.1%), sterility or tubal ligation (89.9% vs. 63.3%), vasectomy (73.2% vs. 69%), Billing's method (19.2% vs. 5.3%), and the rhythm method (73.6% vs. 36.4%) (= = .001). Many women from both cities used ineffective methods. Women in Juarez were much more likely to use the rhythm method than those in El Paso (26.4% vs. 3.4%; p = .001). Women from both cities, particularly those in Juarez (p = .001), believed more information on contraception was needed. They felt strongly that men should also be responsible for family planning and approve of women using contraceptives. They believed that couples should have fewer children. Juarez women has a more positive view of pregnancy and childbearing than did El Paso women. El Paso women were more likely to visit a physician in the last year for reasons other than check-ups (mean number of visits, 1.88 vs. 1.25; p = .005). Most women preferred to receive maternal and child health/family planning services from a female physician (56% in Juarez and 64.3% in El Paso). These findings indicated that cultural differences in contraceptive knowledge and usage as well as confidence in various methods and related medical services existed. They emphasized the need for culturally relevant education, research, and service programs concerning adolescents in the Texas-Mexico border region.

The influence of albumin on palmitate transport in the isolated perfused rat liver.

Unidirectional fluxes (influx and efflux) and the net flux of palmitate across the hepatocyte membrane were measured in the intact rat liver employing the multiple indicator dilution technique. At albumin concentrations in the range between 0.1 and 0.5 mM the influx rate was 2.3 times greater than that of the net flux. The rate of efflux was somewhat higher than the net flux, indicating that palmitate undergoes an exchange process across the liver cell membranes with efflux to the extracellular albumin site being significant. At lower albumin concentrations, however, the influx/net flux ratio approached unity, implying that transport becomes a rate-limiting factor for metabolism.

An analysis of the population genetics of potential multi-drug resistance in Wuchereria bancrofti due to combination chemotherapy.

Currently, annual mass treatments with albendazole (ABZ) plus ivermectin (IVM) or diethylcarbamazine (DEC) are administered under the Global Programme to Eliminate Lymphatic Filariasis (GPELF). Drug resistance against both ABZ and IVM is prevalent in nematodes of veterinary importance, raising awareness that if anthelmintic resistance were to develop among Wuchereria bancrofti populations, this would jeopardize GPELF's goals. Genetic structure was incorporated into an existing transmission dynamics model for lymphatic filariasis (LF) to investigate the potential development of concurrent resistance to ABZ and IVM. The resultant models explore the impact of different inheritance modes of resistance to ABZ and IVM on the likely risk of treatment failure under our model assumptions. Results indicate that under ABZ+IVM combination, selection for resistance to one drug is enhanced if resistance to the other drug is already present. Excess parasite homozygosity may increase selection for dominant IVM resistance via enhancing the frequency of recessive ABZ resistance. The model predicts that if multiple resistance genes are associated with different efficacy properties of a drug combination, then examining changes at single loci may be misleading. Sampling schemes in genetic epidemiological surveys investigating the frequency of an allele under selection should consider host age, as individuals of different ages may acquire parasites at different rates.

Population genetics of concurrent selection with albendazole and ivermectin or diethylcarbamazine on the possible spread of albendazole resistance in Wuchereria bancrofti.

The Global Program for the Elimination of Lymphatic Filariasis (GPELF) intends to achieve its aims through yearly mass treatments with albendazole (ABZ) combined with ivermectin (IVM) or diethylcarbamazine (DEC). The use of ABZ and IVM separately to combat parasites of veterinary importance has, on many occasions, resulted in widespread drug resistance. In order to help predict the spread of potential ABZ resistance alleles through a population of Wuchereria bancrofti, we have developed a mathematical model that incorporates population genetics into EPIFIL, a model which examines the transmission dynamics of the parasite. Our model considers the effect of the combined treatments on the frequency of a recessive allele, which confers ABZ resistance. The model predicts that after 10 yearly treatments with ALB and DEC, 85% coverage and an initial resistance allele frequency of 5%, the frequency of the resistance genotype will increase from 0.25 to 12.7%. If non-random mating is assumed, the initial genotype frequency will be 2.34% and will increase to 62.7%. ABZ and IVM combination treatment may lead to weaker selection for this genotype. Treatment coverage, initial allele frequencies and number of treatments also affect the rate of selection.

Free fatty acid uptake by polyethylene: what can one learn from this?

Previous experiments have shown that fatty-acid uptake by isolated hepatocytes is inhibited by albumin, but this inhibition was less than expected from the decrease in the equilibrium concentration of fatty acid. The possible explanation of this observation by the effects of codiffusion of protein-bound and unbound fatty acid across the unstirred layer surrounding these isolated cells has recently been challenged on the basis of experiments in which uptake by monolayers of hepatocytes was compared with that by a polyethylene sheet [F.J. Burczynski et al., Am. J. Physiol. 257 (Gastrointest. Liver Physiol. 20): G584-G593, 1989]. In the present report, we reevaluate the theoretical basis for interpretation of these experiments by solving the differential equations describing diffusion into a sheet behind a linear barrier. The diffusion coefficient for palmitate in polyethylene is estimated to be approximately 10(-9) cm2/s. We conclude that when proteins are absent from the aqueous phase, diffusion across the unstirred layer is rate limiting for removal of fatty acids by cellular monolayers, and also rate limiting for net flux across the water-polyethylene interface. In contrast, if the aqueous phase contains either 5 microM albumin or 125 microM beta-lactoglobulin, diffusion within the polyethylene sheet will become rate limiting. The net flux of fatty acids into a polyethylene sheet becomes insensitive to an increase in protein concentration if the latter rises above a certain threshold. The polyethylene data provide no additional insight into the manner in which hepatocytes take up free fatty acids.

Hepatic uptake of protein-bound ligands: effect of an unstirred Disse space.

Uptake of protein-bound substances by the liver was modeled considering concurrent depletion of unbound ligand (i.e., not bound to protein)along the length of a sinusoid as well as within a 0.5-micron unstirred boundary layer at the surface of the hepatic parenchymal cells. The development completes a previous exploration of Weisiger et al. [Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G872-G884, 1991]. Ligand is carried across the unstirred layer by albumin, producing a deviation from binding equilibrium inside and outside the unstirred layer. The resulting differential equations have a closed solution. In the case of tight binding, the unbound ligand in the sinusoid is in a quasi-steady state, and the unbound fraction becomes constant along the flow path, except for a very short section at its beginning. During hepatic oleate uptake, the unbound oleate concentration rises from 39% of the equilibrium value at 0.1 microM albumin and 0.01 microM oleate to 78% at 0.5 microM albumin and 0.05 microM oleate. diffusion through the unstirred layer and across the cell membrane was found, in the model, to contribute to the overall resistance to oleate uptake in a complementary fashion.

The multiple indicator-dilution method for the study of enzyme heterogeneity in liver: theoretical basis.

The theoretical basis of the use of the multiple indicator dilution technique to account for the heterogeneous distribution (or zonation) of enzymes in the liver was explored. The microcirculation was assumed to consist of identical capillaries perfused in parallel, with enzymatic activities for drug metabolism being distributed uniformly over the upstream half (periportal or pp) or the downstream half (perivenous or pv) of the flow path, whereas all other transport/removal processes were assumed to be homogeneously distributed. Outflow dilution profiles for parent drug and metabolite were estimated by inversion of Laplace transforms or by a finite difference method. The areas under the curves for parent and metabolite, the mean transit times of parent (MTT) and metabolite (MTTM, mean time from injection of parent to exit of metabolite from organ), and their relative dispersions (CV2 or CVM2) were estimated from analytical expressions. When the influx-efflux ratio (or cellular-sinusoidal distribution ratio) for metabolite was equal to or smaller than that of the parent, the MTTM ranking was: pp < homogeneous < pv. The ranking was reversed when the influx-efflux ratio for metabolite greatly exceeded that for the parent. The presence of elimination pathways for the metabolite reduced its MTTM and CVM2, more for pp than for homogeneous and pv cases. The theory can be applied to determine enzyme zonation in multiple indicator dilution studies with use of the area under the curve for the metabolite and MTTM during prograde (from portal vein to hepatic vein) and retrograde (from hepatic vein to portal vein) perfusion.

Linkage between phosphoglycerate kinase and Xg in a large German kindred.

In a family with an extremely rare PGK variant, linkage with Xg was investigated. The analysis suggested that linkage might prove measurable. The estimate of the recombination fraction was 0.07 and the 90% probability limits were 0.05--0.28. The height of the antilog curve was 19.

Distributed-in-space product formation in vivo: enzymic kinetics.

Distributed-in-space Michaelis-Menten enzymic conversion of precursor substrate to product within an organ is explored when all formed product is released into the bloodstream. In the nonlinear saturating enzymic case, length-wise precursor concentration profiles are found to vary from a falling exponential to a slowly declining linear profile, with rise in input concentration. The vascular profile for product rises in complimentary fashion; the sum of precursor and product is constant along the length. Symmetric permeability barriers produce stepdowns in precursor concentration from blood to tissue and, for product, converse stepdowns from tissue to blood are produced. Tracer precursor, introduced as an impulse input within this steady state, is converted to product with details of its distribution varying with the number of barriers for precursor. During this conversion, reversible tracer precursor association with enzyme leads to an enzymic space effect perceived as a saturating additional compartment, largest at tracer bulk levels, and decreasing with increase in underlying bulk concentration. While tracer product is not delayed by enzyme association, its outflow profile varies with the amount and location of enzyme, the enzymic rate constants, and the barriers for product between enzyme and the blood.

Distributed-in-space product formation in vivo: linear kinetics.

Distributed-in-space conversion of precursor substrate to product within an organ is explored when all formed product is released into the bloodstream. When precursor removal kinetics are linear (conversion occurring proportionately to concentration) and the process is uniformly distributed along the length, exponentially decreasing concentration profiles for precursor result. The vascular profile for product rises in complementary fashion; the sum of precursor and product is constant along the length. Symmetric permeability barriers are found to produce concentration stepdowns of precursor from blood to tissue and, for product, converse stepdowns from tissue to blood. Tracer precursor, introduced as an impulse input, within this steady state, is converted to product, details varying with the number of barriers. Nevertheless, a particular common feature is found in the solutions. The analytic expression for locally generated tracer product is found, in each case, to contain the impulse response to tracer product introduced at the origin. Therefore, to simplify experimental analysis and to superpose a set of constraints on computational approaches to parameter estimation, one should introduce, simultaneously with tracer precursor, tracer product labeled in an identifiably different manner.

Increased hepatocyte permeability surface area product for 86Rb with increase in blood flow.

Liver cell recruitment (the equivalent of capillary recruitment in other organs) was explored by carrying out multiple indicator dilution experiments with labeled rubidium across the liver of the anesthetized dog under basal conditions and after bleeding with saline replacement infusion, which increases liver blood flow. A mixture of 51Cr-labeled red blood cells (a vascular reference), 22Na (which immediately equilibrates in the extracellular space, the sum of the sinusoidal plasma and Disse or interstitial spaces, the expected distribution space for labeled rubidium in the absence of cellular entry), and 86Rb was injected into the portal vein, and normalized outflow patterns, expressed as outflowing fractions of each injected tracer per milliliter versus time, were obtained. In relation to the labeled red blood cell curve, the labeled sodium curve is displaced by flow-limited distribution into the Disse or interstitial space; it is lower on the upslope, reaches a lower and delayed peak, and decays more slowly. The early part of the labeled rubidium curve lies within the labeled sodium curve; it reaches a much reduced peak, and the later return of tracer entering cells is so slow that it is obscured by recirculation. Modeling of the concentrative cellular uptake of rubidium from the Disse space provided an influx permeability surface area product for labeled rubidium. This increases with flow over the observed flow range, demonstrating that sinusoidal recruitment occurs with increase in hepatic blood flow.