Perioperative risk factors for pulmonary complications after liver transplantation.

Using monofactorial and multivariate logistic regression analyses, the correlation of perioperative risk factors with postoperative pulmonary complications (PPCs) within 1 month after orthotopic liver transplantation (OLT) was investigated. Data on 107 patients (median age 46.8 years, 72% male) with end-stage liver disease who received OLT were retrospectively analysed. The incidence of PPCs was 60.7%. Overall mortality was 13.1% and pulmonary causes accounted for 85.7% of deaths. Mortality was 18.5% and 4.8% for patients with and without pulmonary complications, respectively. Independent risk factors for PPCs were a preoperative model for end-stage liver disease (MELD) score > or =25, intraoperative fluid transfusion volume > 10 1 and intraoperative blood transfusion volume > 4 l. A fluid balance of < or = -300 ml for > or =2 days of the first 3 days after surgery was protective. Other variables studied did not predict PPCs. It was concluded that improving the patient's preoperative medical condition, restricting intraoperative transfusion volumes and maintaining a negative fluid balance in the first 3 days after operation may decrease PPCs.

Is there a Brachyury the Second? Analysis of a transgenic mutation involved in notochord maintenance in mice.

A new phenotype mapping to the t-complex, which is designated Brachyury the Second (T2), is characterized by a slightly shortened tail in heterozygotes and homozygous failure to form an organized notochord with subsequent abnormal development of posterior somites and neural tube. The phenotype of T2 superficially resembles that of Brachyury; however, there are several important differences. Brachyury homozygotes fail to make posterior somites, notochord, floor plate, and a placental connection, resulting in death by 10.5 days of development. In contrast, T2 homozygotes make posterior somites, scattered notochord cells, and floorplate and achieve an allantoic connection. However, despite making a maternal connection, T2 homozygotes cease development at E11.5 and die soon after. We have cloned and analyzed the transgene insertion site, which maps within 100 kb of the Brachyury gene, but does not seem to physically interrupt nor affect transcription from that locus. The existence of a second gene mapping near Brachyury and affecting the same developmental processes was alluded to over 50 years ago and has been debated ever since. An embryological description of T2 is presented, as is a discussion of the implications of a single, larger Brachyury locus versus two closely linked genes coordinately regulating axial development.

Palmitate uptake by cardiac myocytes and endothelial cells.

The mechanisms regulating the cellular uptake of long-chain fatty acids are poorly understood. Although there is evidence that hepatocytes facilitate the uptake of ligands from the protein-bound fraction, it is not known whether cardiac myocytes also facilitate the uptake process. The present studies were designed to address the role of albumin in the uptake of long-chain fatty acids by cardiac myocytes isolated from adult male rats. At low albumin concentrations (1 microM), the myocyte palmitate clearance rate did not exceed that predicted by the diffusion-reaction model. At high albumin concentrations (300 and 600 microM), the clearance ratio test was used to determine whether myocytes facilitate the uptake of palmitate. As with the low albumin concentrations, the diffusion-reaction model accounted for the overall clearance rates. Because endothelial cells might be involved in enhancing fatty acid transport into myocytes, we also determined the effects of endothelial cells on palmitate uptake by cardiac myocytes. Based on cell number, the palmitate clearance rate by endothelial cells in the presence of albumin was only 7% of the cardiac myocyte clearance rate. Combining the endothelial cells with the myocytes did not result in any synergistic effect on the palmitate clearance rate.

Intralobular zonal heterogeneity and hepatic indicator dilution curves.

Conventional interpretation of hepatic indicator dilution curves rests on the assumption, among others, that every hepatocyte operates with the same rate constants. When this assumption is false, owing to intralobular zonal variation in surface-to-volume ratios and/or to zonal differences in permeability, the apparent rate constants recoverable from outflow transients are wrong estimates of average liver performance. We develop the theoretical basis for this conclusion and illustrate by example how it can confuse the interpretation of experimental data. The analysis proceeds from vascular and extracellular reference curves recorded from perfused rat livers and from a simple model of intralobular architecture in which highly arborized periportal sinusoids have a larger surface-to-volume ratio than the less-branched vasculature around the central vein. The experimental data and the model, applied to a wide range of hypothetical solutes, are used to compare the true average rate constants for uptake, efflux, and intracellular removal with the apparent values recoverable from outflow curves. When zonal differences in surface-to-volume ratios are the sole source of heterogeneity, the wrong estimates prove of little practical importance. By contrast, assigning larger regional variations in permeability leads to substantial errors. The confusion arising from such errors may be qualitative as well as quantitative. The presence of heterogeneity and thus the risk of interpretive error appears unrecognizable from outflow curves.

Palmitate uptake by hepatocyte suspensions: effect of albumin.

The clearance ratio test has been used to determine whether hepatocytes isolated from female rat livers facilitate the dissociation rate of albumin-palmitate complexes. This test requires knowledge of the clearance rate and unbound palmitate fraction at two protein concentrations. The unbound fractions have been estimated using the heptane-water partition ratio method. These fractions were determined to deviate from the expected linear relationship because of radiolabeled impurities that were still present after purification of the [3H]palmitic acid. With the use of experimentally determined partition ratio values, the palmitate clearance ratios were statistically greater than those predicted by the diffusion-reaction model at albumin concentration combinations above 99 microM/49 microM. Experimental clearance rates also exceeded those predicted by the diffusion-reaction model, but only at albumin concentrations greater than 198 microM. These data support the proposal that hepatocytes facilitate the dissociation of albumin-palmitate complexes at high albumin concentrations.

Uptake of organic anions by hepatocyte monolayers: codiffusion versus facilitated dissociation.

We used the clearance ratio test to determine whether the presence of albumin enhances the uptake of palmitate by hepatocyte monolayers. If uptake is facilitated, then the ratio of palmitate clearance by hepatocytes in the presence of 600 and 300 microM albumin (i.e., Clt600/Clt300) will exceed the square root of the ratio of the unbound fractions at 600 and 300 microM albumin (i.e., square root of alpha 600/alpha 300). The latter value was determined using the heptane-water partitioning technique. The hepatocyte clearance ratio (0.92 +/- 0.06) exceeded the predicted value (0.80) by 15% (p < 0.05). The observed clearance rate also exceeded that predicted by the diffusion-reaction model.

Facilitated dissociation of albumin-fatty acid complexes by rat hepatocytes.

Experiments were conducted comparing palmitate clearance by hepatocyte monolayers, polyethylene and that predicted by the diffusion-reaction model. The objective was to learn whether the albumin-dependent enhancement of unbound palmitate clearance was wholly attributable to codiffusion of bound and unbound fatty acid across an unstirred fluid layer, or whether the uptake flux was also contributed by facilitated dissociation of albumin-fatty acid complexes at the cell surface. We use a model-independent formulation of the facilitation phenomenon to explore this question at low concentrations of albumin. The central finding that facilitation occurs takes into consideration albumin-dependent changes in solution viscosity, endogenous albumin secretion and the adsorption of albumin to the polyethylene surface.

A parameter identification problem arising from a model of canalicular bile formation.

We develop a simple mathematical model for bile formation and analyze some features of the model that suggest the design for future physiological experiments. The mathematical model results in a boundary value problem for a system of functional differential equations depending on several physical parameters. From the observability of the boundary values we can identify, both qualitatively and quantitatively, some of these physical parameters. This identification then suggests physical experiments from which one could infer some of the bile transport phenomena that are not, at present, directly observable. The mathematical parameter identification problem is solved by converting the boundary value problem to a transition time problem for a quadratic system of ordinary differential equations on the plane where we are able to employ some special properties of quadratic systems in order to obtain a solution.

On the design and interpretation of experiments to elucidate albumin-dependent hepatic uptake.

The liver's apparently anomalous extraction of organic anions tightly bound to albumin continues to provoke controversy and confusion. Decisive experiments have proved difficult to design, and mathematical models have usually been constructed to defend one or another putative mechanism to the exclusion of others. To stimulate more decisive experiments and as an aid to interpreting those already reported, we discuss a general formulation of the problem that predicts the clearance pattern to be expected when facilitated dissociation and codiffusion are joint determinants of the uptake flux. The results provide an approach to modeling the various mechanisms by which the concentration of bound ligand at the cell surface could be a driving force for uptake. Further we present new calculations to clarify the interpretation of net ligand clearance when the removal of free ligand is the result of bidirectional fluxes into and out of an unstirred sink. Applied to a previously published comparison of the uptake performances of hepatocytes and polyethylene, the new calculations support the inference that facilitated dissociation of albumin-palmitate complexes occurs at or near the hepatocyte surface.

Beta-lactoglobulin enhances the uptake of free palmitate by hepatocyte monolayers: the relative importance of diffusion and facilitated dissociation.

We compared the uptake of bound palmitate by rat hepatocytes to its uptake by polyethylene using beta-lactoglobulin (BLG) as the binding protein. The experiments were designed to supply a direct measure of the protein-dependent change in the diffusive conductance of extracellular fluid without determining the diffusion coefficients for free and bound fatty acid or the off-rate constant for protein binding. Rate-limiting dissociation in the stirred phase of extracellular fluid was excluded. The results obtained with BLG are strikingly similar to those previously obtained with albumin and provide additional circumstantial evidence that when the free fraction is small, palmitate uptake is partially driven by the concentration of bound fatty acid. Because this phenomenon is not specific for the binding protein, it may reflect direct exchange of ligand between the binding protein in extracellular fluid and the putative transport protein in the hepatocyte plasma membrane.

Palmitate uptake by cultured hepatocytes: albumin binding and stagnant layer phenomena.

We compared uptake of palmitate by hepatocyte monolayers with uptake by polyethylene membranes under conditions of identical binding and stirring. Hepatocytes and polyethylene display similar clearances when the fatty acid is free, reflecting partial rate limitation by diffusion across the unstirred water layer. When palmitate is 99.8% bound to albumin, however, hepatocytes clear free fatty acid about seven times faster than does polyethylene. We analyzed the uptake of palmitate by polyethylene at two different pHs to isolate the diffusive resistance of the unstirred layer and to show that codiffusion of bound and free palmitate to the hepatocyte surface accounts for only approximately 20% of the albumin-dependent increment in the clearance of free palmitate. The clearance data are supported by independent measurements of the stagnant layer thickness obtained from indicator dilution data and by an electrochemical method. The findings suggest that hepatocytes facilitate the dissociation of albumin-palmitate complexes. Alternatively, albumin may modulate the uptake capacities of hepatocytes and/or polyethylene.