Further Observations on the Measurement of Fatty Acid Incorporation by Erythrocyte Ghosts to Quantify Unbound Palmitate Concentration in Albumin–Palmitate Mixtures

Pond, Susan M.; Gordon, R. A.; Simi, A.; Winzor, Donald J.

doi:10.1006/abio.1996.0234

Cited by 3 publications

(2 citation statements)

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“…Competition method involving partitioning of LCFA between erythrocyte and aqueous phases. The main advantage of this method is that it reflects binding of LCFA by specific sites in the erythrocyte membrane, not just general partitioning into the erythrocyte membrane (20). The major problem of the method, like the organic solvent partition method, is that it is difficult to define the extremely low unbound LCFA monomer concentration (rev.…”

Section: Reviewmentioning

confidence: 99%

Cellular uptake and intracellular trafficking of long chain fatty acids

McArthur

Atshaves

Frolov

et al. 1999

Journal of Lipid Research

331

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While aspects of cellular fatty acid uptake have been studied as early as 50 years ago, recent developments in this rapidly evolving field have yielded new functional insights on the individual mechanistic steps in this process. The extremely low aqueous solubility of long chain fatty acids (LCFA) together with the very high affinity of serum albumin and cytoplasmic fatty acid binding proteins for LCFA have challenged the limits of technology in resolving the individual steps of this process. To date no single mechanism alone accounts for regulation of cellular LCFA uptake. Key regulatory points in cellular uptake of LCFA include: the aqueous solubility of the LCFA; the driving force(s) for LCFA entry into the cell membrane; the relative roles of diffusional and protein mediated LCFA translocation across the plasma membrane; cytoplasmic LCFA binding protein-mediated uptake and/or intracellular diffusion; the activity of LCFA-CoA synthetase; and cytoplasmic protein mediated targeting of LCFA or LCFA-CoAs toward specific metabolic pathways. The emerging picture is that the cell has multiple, overlapping mechanisms that assure adequate uptake and directed intracellular movement of LCFA required for maintenance of physiological functions. The upcoming challenge is to take advantage of new advances in this field to elucidate the differential interactions between these pathways in intact cells and in tissues. -McArthur, M.

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Section: Reviewmentioning

confidence: 99%

Cellular uptake and intracellular trafficking of long chain fatty acids

McArthur

Atshaves

Frolov

et al. 1999

Journal of Lipid Research

331

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“…Previously, we have used the method described here to find the number of binding sites on BSA and K a for five different long-chain fatty acids (15) and for the cannabinoid N-arachidonoylethanolamide (anandamide) (16) and the method has been used by others to study the palmitate-albumin interaction (17). …”

Section: Introductionmentioning

confidence: 99%

Direct determination of unbound lipophilic ligands in aqueous solutions

Bojesen

2004

Biol. Proced. Online

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Due to their hydrophobic nature, lipophilic compounds are always bound to proteins when transported in the organism. The transfer of such compounds between their binding proteins and cells as well as intracellular trafficking is mediated by a very low water-phase concentration of monomers. The use of protein filled resealed red cell membranes (erythrocyte ghosts) as semipermeable bags enables us to determine directly such water-phase concentrations in a biological system where the lipophilic compound is in equilibrium with the compound bound to its binding protein. Equilibrium dissociation constants (K d 's) and number of binding sites are determined by regression analyses of data. We describe the method with the hydrophobic anion arachidonate and the neutral Narachidonoylethanolamide as examples.

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Binding of [³H]palmitate to BSA

Elmadhoun

Wang

Templeton

et al. 1998

American Journal of Physiology-Gastrointestinal and Liver Physi

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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.

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Further Observations on the Measurement of Fatty Acid Incorporation by Erythrocyte Ghosts to Quantify Unbound Palmitate Concentration in Albumin–Palmitate Mixtures

Cited by 3 publications

References 1 publication

Cellular uptake and intracellular trafficking of long chain fatty acids

Cellular uptake and intracellular trafficking of long chain fatty acids

Direct determination of unbound lipophilic ligands in aqueous solutions

Binding of [³H]palmitate to BSA

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Further Observations on the Measurement of Fatty Acid Incorporation by Erythrocyte Ghosts to Quantify Unbound Palmitate Concentration in Albumin–Palmitate Mixtures

Cited by 3 publications

References 1 publication

Cellular uptake and intracellular trafficking of long chain fatty acids

Cellular uptake and intracellular trafficking of long chain fatty acids

Direct determination of unbound lipophilic ligands in aqueous solutions

Binding of [3H]palmitate to BSA

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Binding of [³H]palmitate to BSA