Transfer of fatty acids between intracellular membranes: roles of soluble binding proteins, distance, and time.

Soluble fatty acid binding proteins (FABPs) are thought to facilitate exchange of fatty acids between intracellular membranes. Although many FABP variants have been described, they fall into two general classes. "Membrane-active" FABPs exchange fatty acids with membranes during transient collisions with the membrane surface, whereas "membrane-inactive" FABPs do not. We used modeling of fatty acid transport between two planar membranes to examine the hypothesis that these two classes catalyze different steps in intracellular fatty acid transport. In the absence of FABP, the steady-state flux of fatty acid from the donor to the acceptor membrane depends on membrane separation distance (d) approaching a maximum value (J(max)) as d approaches zero. J(max) is one-half the rate of dissociation of fatty acid from the donor membrane, indicating that newly dissociated fatty acid has a 50% chance of successfully reaching the acceptor membrane before rebinding to the donor membrane. For larger membrane separations, successful transfer becomes less likely as diffusional concentration gradients develop. The mean diffusional excursion of the fatty acid into the water phase (d(m)) defines this transition. For d<>d(m), aqueous diffusion is rate limiting. All forms of FABP increase d(m) by reducing the rate of rebinding to the donor membrane, thus maintaining J(max) over larger membrane separations. Membrane-active FABPs further increase J(max) by catalyzing the rate of dissociation of fatty acids from the donor membrane, although frequent membrane interactions would be expected to reduce their diffusional mobility through a membrane-rich cytoplasm. Individual FABPs may have evolved to match the membrane separations and densities found in specific cell lines.

Affinity of human serum albumin for bilirubin varies with albumin concentration and buffer composition: results of a novel ultrafiltration method.

Albumin binding is a crucial determinant of bilirubin clearance in health and bilirubin toxicity in certain disease states. However, prior attempts to measure the affinity of albumin for bilirubin have yielded highly variable results, reflecting both differing conditions and the confounding influence of impurities. We therefore have devised a method based on serial ultrafiltration that successively removes impurities in [(14)C]bilirubin until a stable binding affinity is achieved, and then we used it to assess the effect of albumin concentration and buffer composition on binding. The apparent binding affinity of human serum albumin for [(14)C]bilirubin was strongly dependent on assay conditions, falling from (5.09 +/- 0.24) x 10(7) liters/mol at lower albumin concentrations (15 microm) to (0.54 +/- 0.05) x 10(7) liters/mol at higher albumin concentrations (300 microm). To determine whether radioactive impurities were responsible for this change, we estimated impurities in the stock bilirubin using a novel modeling approach and found them to be 0.11-0.13%. Formation of new impurities during the study and their affinity for albumin were also estimated. After correction for impurities, the binding affinity remained heavily dependent on the albumin concentration (range (5.37 +/- 0.26) x 10(7) liters/mol to (0.65 +/- 0.03) x 10(7) liters/mol). Affinities decreased by about half in the presence of chloride (50 mm). Thus, the affinity of human albumin for bilirubin is not constant, but varies with both albumin concentration and buffer composition. Binding may be considerably less avid at physiological albumin concentrations than previously believed.

Induction of hepatic cytosolic fatty acid binding protein with clofibrate accelerates both membrane and cytoplasmic transport of palmitate.

The role of liver cytosolic fatty acid binding protein (L-FABP) in fatty acid transport and metabolism is unclear. Female liver contains substantially more L-FABP than male liver. Female liver also has a different fatty acid transport phenotype, including more rapid uptake, efflux and cytoplasmic transport. However, it is not known if the greater levels of L-FABP are responsible for these differences. We therefore determined whether increasing L-FABP using clofibrate causes male liver to acquire a female transport phenotype. The multiple indicator dilution (MID) method was used to estimate the rate constants for influx, efflux and cytoplasmic diffusion of palmitate in isolated perfused rat livers. Clofibrate treatment increased cytosolic concentrations of L-FABP 4.2+/-0.8-fold, the rate of cytoplasmic diffusion of palmitate 4.3+/-1.7-fold, and the steady-state palmitate extraction 1.5+/-0.3-fold (mean+/-S.E.). Influx and efflux constants were both increased (by 44% and 79%, respectively) to levels typical of female livers. These data suggest that clofibrate-induced elevation of cytosolic L-FABP not only stimulates intracellular diffusion but also influx and efflux of fatty acids. Possible mechanisms include reducing fatty acid binding to cytoplasmic membranes, induction of membrane fatty acid carriers, and catalyzing fatty acid exchange between aqueous cytoplasm and the plasma membrane.

Saturable stimulation of fatty acid transport through model cytoplasm by soluble binding protein.

To better define the role of soluble binding proteins in the cytoplasmic transport of amphipathic molecules, we measured the diffusional mobility of a fluorescent long-chain fatty acid, 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate), through model cytoplasm as a function of soluble binding protein concentration. Diffusional mobilities were correlated with the partition of the fatty acid between membrane and protein binding sites. Cytoplasm was modeled as a dense suspension of liposomes, and albumin was used as a model binding protein. Albumin saturably increased NBD-stearate mobility through the membrane suspension approximately eightfold. Fatty acid mobility in the absence of albumin was identical to the mobility of the membrane vesicles (1.99 +/- 0.33 x 10(-8) cm(2)/s), whereas the mobility at saturating concentrations was identical to the mobility of albumin (1.65 +/- 0.12 x 10(-7) cm(2)/s). The protein concentration producing half-maximal stimulation of NBD-stearate diffusion (42.8 +/- 0.3 microM) was unexpectedly greater than that required to solubilize half of the NBD-stearate (17.9 +/- 3.0 microM). These results support a proposed mechanism for cytoplasmic transport of small amphipathic molecules in which aqueous diffusion of the protein-bound form of the molecule largely determines the transport rate. However, slow interchange of fatty acid between the binding protein and membranes also appears to influence the transport rate in this model system.

Sex differences in multiple steps in hepatic transport of palmitate support a balanced uptake mechanism.

Hepatic clearance of long-chain fatty acids is substantially faster in females than in males, a fact that may underlie known gender-related differences in lipoprotein metabolism and associated disease states. To further investigate the transport steps responsible for this difference, we used a novel method combining multiple-indicator dilution and steady-state measurements of palmitate extraction from albumin solutions. We found that cytoplasmic transport of palmitate is sufficiently slow (diffusion constants 9.0 and 5.9 x 10(-9) cm2/s for male and female liver, respectively) that the steady-state concentration of palmitate in the center of the cell should be approximately 0.5 of that found in the cytoplasm just beneath the plasma membrane. Previous studies in cultured liver cells using nonphysiological fatty acids have shown more rapid cytoplasmic transport in females. This sex difference reflects higher concentrations of cytosolic fatty acid-binding protein, which acts as a carrier system to transport fatty acids across cell water layers. The current study confirmed slow cytoplasmic diffusion rates in intact perfused rat liver using a physiological fatty acid and found a similar female-to-male ratio. Female liver also had a greater influx rate constant and a larger vascular volume than male liver but had a similar rate of metabolism. Rapid cytoplasmic diffusion enhances movement of palmitate into deeper layers of the cell cytoplasm, thus reducing efflux. The larger sinusoidal volume in females not only permits more dissociation of palmitate from albumin within the sinusoids but also may generate a greater permeability-surface area product. These multiple sex-related differences combine to produce a nearly twofold greater steady-state uptake rate by female liver.

Nontraditional effects of protein binding and hematocrit on uptake of indocyanine green by perfused rat liver.

We used a novel parameter-free approach to study the role of protein binding in the hepatic clearance of indocyanine green (ICG) from reconstituted pig blood by perfused rat liver. Either perfusate total plasma protein concentration or hematocrit was changed. By analyzing protein concentration ratios or plasma volume ratios relative to ratios of intrinsic hepatic clearance of ICG (K), it was possible to evaluate current models of hepatic uptake of protein-bound ligands without precise knowledge of some of the model parameters. A four-fold increase in the total plasma protein concentration produced only a 36% decrease in K. This was substantially less than predicted by the traditional model, where K is proportional to the free concentration of ligand. Because an unstirred water layer effect could not by itself account for the observations, the effects of binding disequilibrium in the sinusoids or uptake directly from the bound pool had to be considered. To discriminate, hematocrit was increased from 15% to 29%, causing a 20% decrease in the sinusoidal plasma volume. A significant reduction in K strongly suggested a sinusoidal binding disequilibrium effect. The dissociation rate constant predicted by this model was confirmed by in vitro measurement, further supporting this interpretation. The simple experimental design and its parameter-free evaluation provide a new tool for investigating the hepatic uptake of protein-bound ligands.

When is a carrier not a membrane carrier? The cytoplasmic transport of amphipathic molecules.

After entering the cell, small molecules must penetrate the cytoplasm before they are metabolized, excreted, or can convey information to the cell nucleus. Without efficient cytoplasmic transport, most such molecules would efflux back out of the cell before they could reach their targets. Cytoplasmic movement of amphipathic molecules (e.g., long-chain fatty acids, bilirubin, bile acids) is greatly slowed by their tendency to bind intracellular structures. Soluble cytoplasmic binding proteins reduce this binding by increasing the aqueous solubility of their ligands. These soluble carriers catalyze the transport of hydrophobic molecules across hydrophilic water layers, just as membrane carriers catalyze the transport of hydrophilic molecules across the hydrophobic membrane core. They even display the kinetic features of carrier-mediated transport, including saturation, mutual competition between similar molecules, and countertransport. Recent data suggest that amphipathic molecules cross the cytoplasm very slowly, with apparent diffusion constants 10(2) to 10(4) times smaller than in water. By modulating the rate of cytoplasmic transport, cytosolic binding proteins may regulate transport and metabolism of amphipathic molecules. Storage diseases may cause hepatocellular dysfunction by disrupting normal cytoplasmic transport.

Intrahepatic blood flow distribution in the perfused rat liver: effect of hepatic artery perfusion.

Variations in blood flow to different sinusoids within the liver can prevent uniform uptake of solutes from plasma and contribute to cellular ischemia in low-flow states. However, the degree of variability and the role of hepatic artery perfusion in maintaining uniform flow are poorly defined. We used an indicator dilution technique to compare the distribution of sinusoidal transit times in isolated rat livers perfused through the portal vein alone with livers perfused using both portal vein and hepatic artery. Physiological flow rates were used in each case (1.2 +/- 0.3 ml.min-1.g liver-1), but the second group received 32% of flow through the hepatic artery. Intralobular flow heterogeneity was further assessed by gamma counting of small (approximately 100 mg) pieces of the liver after bolus injection of approximately 5 mCi of a highly extracted compound ([125I])triiodothyronine) into the portal vein. Hepatic artery perfusion had no significant effect on mean sinusoidal transit time or intrahepatic distribution volume for 51Cr-labeled red blood cells or 125I-albumin. Analysis of the outflow profiles indicated that hepatic artery perfusion did not affect transit time dispersion. However, heterogeneity of flow to individual portions of the liver, measured as the coefficient of variation, increased from 19 to 30%. These results indicate relatively uniform perfusion of the sinusoids in the portally perfused rat liver and that additional perfusion of the hepatic artery does not further improve hemodynamics. These results have significance for the design and interpretation of transport studies with the use of the perfused rat liver model.

Endothelin induced contractility of stellate cells from normal and cirrhotic rat liver: implications for regulation of portal pressure and resistance.

Hepatic stellate cells are similar to tissue pericytes and have been shown to be contractile. In this study, we examined the effects of known mediators of stellate cell contraction on portal pressure in rat livers after carbon tetrachloride induced injury (including cirrhosis) and investigated the contractility of stellate cells as a function of liver injury. Sarafotoxin S6C, an endothelin B (ETB) receptor agonist, had minor effects on portal pressure when perfused into normal livers at concentrations known to elicit stellate cell contraction (2 nmol/L). In contrast, both endothelin-1 (2 nmol/L) and angiotensin II (8.6 nmol/L) caused a rapid and pronounced rise in portal pressure. The effects of sarafotoxin S6C (a potent stellate cell contractile agonist) on portal pressure were greater in cirrhotic than normal liver, whereas those of angiotensin II were unchanged after liver injury. Endothelin-1 and sarafotoxin S6C induced contractility of stellate cells increased in proportion to the degree of liver injury. Contractility was greatest for stellate cells isolated from cirrhotic livers, a population of cells that displayed the most activated phenotype, as measured by immunoblot of smooth muscle alpha actin. Autoradiography of cirrhotic livers after perfusion with 125I-endothelin-1 revealed binding to cells in both sinusoidal spaces and collagenous fibrotic bands, consistent with known locations of stellate cells. Finally, the mixed endothelin-A (ETA) and ETB receptor antagonist, bosentan, reduced portal pressure in portal hypertensive animals, consistent with its inhibitory effect on stellate cell contraction. We conclude that stellate cell contractility increases with progressive liver injury and is proportional to the degree of stellate cell activation, becoming most prominent in the cirrhotic liver. Endothelin-stimulated contraction of stellate cells in cirrhotic liver may contribute to increased intrahepatic resistance and portal pressure.

Recovery of hepatic drug extraction after hypothermic preservation.

BACKGROUND: To determine whether liver preservation before transplantation impairs hepatic drug metabolism, hepatic extraction of drugs with different metabolic pathways (fentanyl, morphine, and vecuronium) in isolated rat livers was measured either immediately or after 24 h of hypothermia at 4 degrees C using a standard preservation-reperfusion sequence. METHODS: Isolated rat livers were perfused via the portal vein for 30 min to document initial viability. Test livers (n = 5) were perfused with iced Belzer solution, stored for 24 h at 4 degrees C, and flushed with 6% hetastarch. After hypothermic preservation for 24 h, or in control livers (n = 5) immediately after the 30-min perfusion, livers were perfused single-pass at a constant flow rate with solutions containing fentanyl, morphine, and vecuronium at 37 degrees C. Perfusate and bile samples were obtained at regular intervals for 64 min, after which liver tissue was harvested for analysis. Drug concentrations were measured using radioimmunoassay and gas chromatography. Metabolic capacity of the liver was estimated from the extraction fraction of each drug at steady-state. RESULTS: After warming to 37 degrees C, preserved livers consumed oxygen and produced bile at rates similar to that of control livers. Hypothermic preservation did not affect extraction of fentanyl and morphine. Vecuronium extraction was initially less in preserved livers, but this difference disappeared as the preserved livers returned to 37 degrees C (< 16 min). Biliary excretion and tissue concentrations of vecuronium were similar in each group. CONCLUSIONS: Hypothermic preservation does not significantly impair extraction of these drugs in this liver preservation model. If these results apply to human liver transplantation, little danger of drug accumulation exists during the early postoperative period if hepatic function is normal.

Effects of gender and pregnancy on hepatocellular uptake of palmitic acid: facilitation by albumin.

The human serum albumin (HSA)-dependent unbound clearance (Clu) of [3H]palmitic acid (PA) by hepatocyte suspensions isolated from immature and mature male and female and pregnant female rats was studied. The Clu values obtained experimentally were compared with the predictions of a noncompartmental diffusion-reaction (Bass-Pond) theory for the cellular uptake of protein-bound ligands. In all groups, as the concentration of HSA (Ca) was increased, there was a striking increase in Clu. These enhancement factors were predicted by the theory. Adult females had higher Clu values at high Ca values than males or immature females. Furthermore, at high Ca values, Clu in pregnant animals was twice as high as in the nonpregnant animals and four times as high as in the aged-matched males. The absolute values of Clu obtained experimentally in both pregnant and nonpregnant females exceeded the maximal predictions of the theory, using reasonable values of all of the parameters. Thus, according to current data on the physicochemical characteristics of the uptake system, the study demonstrates that some specialized process exists to facilitate hepatocellular uptake of fatty acid from albumin, and that it is potentiated by the female sex hormones.

Sex differences in intracellular fatty acid transport: role of cytoplasmic binding proteins.

Female liver clears long-chain fatty acids from plasma more rapidly than male liver, and yet the basis for this sex difference is poorly understood. We tested the hypothesis that cytosolic fatty acid binding protein (FABP), which is more concentrated in female liver, may enhance fatty acid utilization by increasing the rate of transport through the cytoplasm. We modified the technique of fluorescence recovery after laser photobleaching to measure the cytoplasmic diffusion rate of the fluorescent long-chain fatty acid 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate) in cultured hepatocytes from female and male rats. NBD-stearate was used because its hepatic handling is similar to natural fatty acids. After uptake, NBD-stearate distributed uniformly in the cytoplasm but was excluded from the nucleus. Intracellular transport occurred by diffusion with no detectable convective flux. The cytoplasmic diffusion rate at 37 degrees C was 65% greater in female cells than in male cells (mean +/- SE, 5.03 +/- 0.37 vs. 3.05 +/- 0.21 x 10(-9) cm2/s respectively; P < 0.001) and was two to three orders of magnitude slower than for either unbound NBD-stearate or FABP in water. A correspondingly greater fraction of cellular NBD-stearate was found in the aqueous cytosol in females (35.1 +/- 7.0 vs. 18.2 +/- 2.7%), suggesting that FABP reduces binding of NBD-stearate to immobile cytoplasmic membranes. These data indicate that intracellular transport of NBD-stearate, a typical amphipathic molecule, is slowed by binding to cytoplasmic membranes. The primary function of soluble binding proteins such as FABP may be to enhance the diffusive fluxes of their ligands by reducing membrane binding. If cytoplasmic transport of rapidly metabolized fatty acids such as palmitate is similarly slow, substantial concentration gradients could develop within the cytoplasm of hepatocytes at steady state. By catalyzing these diffusive fluxes, FABP may regulate fatty acid metabolism.

Calibration of albumin-fatty acid binding constants measured by heptane-water partition.

Most measurements of binding affinity of albumin for long-chain fatty acids are based on heptane-water partition. In this method, equilibrium partition of fatty acid between heptane and an albumin-containing buffer is calibrated using the partition ratio between heptane and buffer in the absence of protein. In the current study, we used a variety of techniques to examine potential problems with this approach. Hydrophobic impurities in commercial [3H]palmitate preparations were incompletely removed by standard purification techniques. These impurities contributed from 5% of the total radioactivity in the heptane phase at low albumin concentrations (5 microM) to 62% at higher albumin concentrations (500 microM), thus confounding determination of binding affinity. These were identified by gas chromatography/mass spectroscopy as radio-labeled glycerol monopalmitate and monostearate. When albumin was not present, the partition ratio was similar to values reported by others. However, our results varied by a factor of four (265-1,119) depending on how the solutions were prepared. Although a true equilibrium partition must not depend on starting conditions, the partition ratio after 24-72 h was > 2x as large when tracer [3H]palmitate was added to the heptane phase than when it was added to the aqueous phase. Results also depended on the relative volumes of heptane and buffer used, approaching a maximum of 1,445 +/- 112 for very low heptane/buffer volume ratios. Much of this variability was due to hydrophilic impurities in [3H]palmitate, which ranged from 0.2 to 1.2% in commercial lots down to 0.1-0.5% after alkaline ethanol extraction and < 0.05% after thin-layer chromatography (TLC).(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic taurocholate uptake is electrogenic and influenced by transmembrane potential difference.

Uptake of the bile acid taurocholate by hepatocytes is coupled to Na+ influx. The stoichiometry of uptake, however, is uncertain, as is the influence of the transmembrane electrical potential difference (PD) on this process. In this study, we examined the relationship between taurocholate extraction and PD (measured using intracellular microelectrodes) in perfused liver, and we measured taurocholate-induced transport current in cultured hepatocytes using patch-clamp recording techniques. In the perfused liver under basal conditions, PD averaged -28.4 +/- 0.6 (SE) mV, and extraction of 1, 50, and 300 microM taurocholate was 0.95 +/- 0.02, 0.98 +/- 0.01, and 0.41 +/- 0.03, respectively. When the Na+ chemical gradient was decreased by replacing perfusate Na+ with choline, the membrane depolarized to -17.2 +/- 1.1 mV, and taurocholate extraction markedly decreased at all taurocholate concentrations (P < 0.01). When perfusate Na+ concentration was held constant at 137 mM, membrane depolarization induced by substitution of gluconate for perfusate Cl- (-17.9 +/- 0.6 mV) or Cl- for nitrate (-10.3 +/- 2.1 mV) significantly decreased extraction of 300 microM taurocholate. Abrupt exposure to taurocholate produced a concentration-dependent membrane depolarization in the presence of Na+, but not in its absence (P < 0.001). In cultured hepatocytes, exposure to 100 microM taurocholate produced an inward current of -0.056 +/- 0.016 pA/pF at a holding potential of -40 mV. This current was Na+ dependent, and it increased twofold as holding potential was changed from -20 to -50 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Extending the multiple indicator dilution method to include slow intracellular diffusion.

The traditional multiple indicator dilution (MID) method is extended to incorporate cytoplasmic concentration gradients due to slow intracellular diffusion of the permeable molecule. The new model is governed by a system of partial differential equations that are solved using Laplace transformation. An analysis of the transformed solution shows that the traditional MID method is a special case of the extended model. We then use simulation analysis to show that the traditional MID model and the new diffusion model generate similar outflow curves. However, when the traditional MID equations were used to analyze outflow curves generated using a system in which intracellular diffusion is slow compared to other transport processes, the recovered rate constants for the transmembrane and excretion processes were incorrect. The diffusion model permits estimation of the rate of intracellular transport of amphipathic molecules from suitable indicator dilution data.

A new method for quantitating intracellular transport: application to the thyroid hormone 3,5,3'-triiodothyronine.

After entering cells from plasma, molecules must permeate through the cytoplasm before they can be metabolized or excreted. If sufficiently slow, cytoplasmic transport may determine the overall rate of cellular elimination at steady state. Cytoplasmic transport of amphipathic molecules should be particularly slow because of extensive binding to intracellular membranes and proteins. Traditional transport models assume that molecules become instantly available for metabolism and canalicular excretion after entering the cell and thus cannot be used to assess cytoplasmic transport. We therefore extended the traditional multiple-indicator dilution (MID) method of Goresky to explicitly incorporate cytoplasmic transport and used the resulting model to estimate the rate constant for cytoplasmic transport of the amphipathic thyroid hormone 3,5,3'-triiodothyronine (T3). We chose T3 because control studies indicated that it is neither metabolized nor excreted during the brief period of an MID experiment (40-90 s). The traditional MID model was unable to account for the data unless we postulated rapid metabolism or excretion of T3. In contrast, the new diffusion MID model fit the data closely without this false assumption and gave values for the influx and efflux rate constants that agreed with previously published data. The half-time for equilibration of T3 across the cytoplasm of the hepatocyte averaged 50 s. This corresponds to an effective cytoplasmic diffusion constant of 3.1 x 10(-8) cm2/s, which is > 100 times slower than expected for free T3 in water. Our results indicate that cytoplasmic transport of this model amphipathic compound is much slower than membrane transport.(ABSTRACT TRUNCATED AT 250 WORDS)