The presence of cysteine and methionine groups together with an ability to bind long-chain fatty acid (LCFA) oxidation products makes liver fatty acid binding protein (L-FABP) an attractive candidate against hepatocellular oxidative stress. In this report, we show that pharmacological treatment directed at modulating L-FABP level affected hepatocellular oxidant status. L-FABP expressing 1548-hepatoma cells, treated with dexamethasone or clofibrate, decreased and increased intracellular L-FABP levels, respectively. Oxidative stress was induced by H2O2 incubation or hypoxia-reoxygenation. The fluorescent marker, dichlorofluorescein (DCF), was employed to measure intracellular reactive oxygen species (ROS). Hepatocellular damage was assessed by lactate dehydrogenase (LDH) level. Dexamethasone treatment resulted in a significant increase in DCF fluorescence with higher LDH release compared to control cells. Clofibrate treatment, however, resulted in a significant decrease in both parameters (p<0.05). Drug treatments did not affect cytosolic activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), or catalase suggesting that the differences between treated and control cells may likely be associated with varying L-FABP levels. We conclude that L-FABP may act as an effective endogenous cytoprotectant against hepatocellular oxidative stress.

Determination of the BSA-palmitate high-affinity binding constant ( K a) traditionally relied on the heptane-water partitioning technique. We used this technique to calculate K a for the BSA-[3H]palmitate complex, to determine if K a was independent of protein concentration, and to determine if the unbound [3H]palmitate concentration is constant at different BSA concentrations using constant BSA-to-palmitate molar ratios (range 1:1 to 1:4). After extensive extraction of non-[3H]palmitate radiolabeled substances, the heptane-to-buffer partition ratio, in the absence of BSA, was 702 ± 19 (mean ± SD, n = 6). This value was much lower than the predicted value of 1,376 and was highly dependent on which phase (organic or aqueous) initially contained the [3H]palmitic acid. The data were consistent with the notion of self-association of [3H]palmitate in the aqueous phase. K afor the BSA-[3H]palmitate complex was determined to be similar (2.2 ± 0.1) × 108M−1 (mean ± SD, P > 0.05) at all BSA concentrations studied. At each BSA-to-palmitate molar ratio, the equilibrium unbound ligand concentration was constant only at low BSA concentrations (<10 μM) and at low BSA-to-palmitate molar ratios (i.e., 1:1 and 1:2). At higher BSA concentrations and molar ratios, the unbound ligand concentration increased with an increase in protein concentration. Hepatocyte uptake using the manufacturer-supplied radiolabeled product was significantly higher than with the purified product, suggesting that a non-[3H]palmitate radiolabel is also a substrate for the uptake process.

1 Studies were directed at determining whether hepatocytes, isolated from female Sprague-Dawley rats, facilitate the uptake of protein-bound long-chain fatty acids. We postulated one form of facilitated uptake may occur through an ionic interaction between the protein-ligand complex and the cell surface. These interactions are expected to supply additional ligand to the cell for uptake. 2 The clearance rate of [ 3 H]-palmitate in the presence of a 1 -acid-glycoprotein (pI=2.7), albumin (pI=4.9) and lysozyme (pI=11.0) was investigated. Palmitate uptake was determined in the presence of protein concentrations that resulted in similar unbound ligand fractions (=0.03). The experimental clearance rates were compared to the theoretical predictions based upon the diusion-reaction model. 3 By use of our experimentally determined equilibrium binding and dissociation rate constants for the various protein-palmitate complexes, the diusion-reaction model predicted clearance rates were 4.9 ml s 71 /10 6 cells, 4.8 ml s 71/10 6 cells and 5.5 ml s 71 /10 6 cells for a 1 -acid-glycoprotein, albumin and lysozyme, respectively; whereas the measured hepatocyte palmitate clearance rates were 1.2+0.1 ml s 71 / 10 6 cells, 2.3+0.3 ml s 71/10 6 cells and 7.1+0.7 ml s 71 /10 6 , respectively. 4 Hepatocyte palmitate clearance was signi®cantly faster (P50.01) in the presence of lysozyme than albumin which was signi®cantly faster than a 1 -acid-glycoprotein (P50.01). The marked dierence in clearance rates could not be explained by considering dierences in solution viscosity. 5 Our results are consistent with the notion that ionic interactions between protein-ligand complexes and the cell surface facilitate the ligand uptake by decreasing the diusional distance of the unbound ligand and/or by facilitating the protein-ligand dissociation rate.

These results suggest that, at physiological albumin concentrations, most of the LCFA uptake is mediated from that bound to albumin by a hepatocyte basolateral membrane transport protein, and uptake of unbound LCFA occurring by passive diffusion contributes a minor component.

Understanding the driving forces for the hepatic uptake of endogenous and exogenous substrates in isolated cells and organs is fundamental to describing the underlying hepatic physiology/pharmacology. In this study we investigated whether uptake of plasma protein-bound [3H]-palmitate across the hepatocyte wall is governed by the transmembrane electrical potential difference (PD). Uptake was studied in isolated hepatocytes and isolated perfused rat livers (IPL). Protein-binding and vasoactive properties of the different perfusates were determined using in vitro heptane/buffer partitioning studies and the multiple indicator dilution (MID) technique in the IPL, respectively. Altering hepatocyte PD by perfusate ion substitution resulted in either a substantial depolarization (-14 +/- 1 mV, n = 12, mean +/- S.E., substituting choline for Na+) or hyperpolarization (-46 +/- 3 mV, n = 12, mean +/- S.E., substituting nitrate for Cl-). Perfusate ion substitution also affected the equilibrium binding constant for the palmitate-albumin complex. IPL studies suggested that, other than with gluconate buffer, hepatic [3H]-palmitate extraction was not affected by the buffer used, implying PD was not a determinant of extraction. [3H]-Palmitate extraction was much lower (p < 0.05) when gluconate was substituted for Cl- ion. This work contrasts with that for the extraction of [3H]-alanine where hepatic extraction fraction was significantly reduced during depolarization. Changing the albumin concentration did not affect hepatocyte PD, and [3H]-palmitate clearance into isolated hepatocytes was not affected by the buffers used. MID studies with vascular and extravascular references revealed that, with the gluconate substituted buffer, the extravascular volume possibly increased the diffusional path length thus explaining reduced [3H]-palmitate extraction fraction in the IPL.