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 [H-3]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 muM) to 62% at higher albumin concentrations (500 muM), thus confounding determination of binding affinity. These were identified by gas chromatography/mass spectroscopy as radiolabeled 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 [H-3]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 [H-3]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). However, at least some criteria of equilibrium were not satisfied even for the most highly purified samples, and purification by TLC unexpectedly reduced the partition ratio severalfold. Similar criteria of validity were not documented in prior studies of albumin binding to long-chain fatty acids using the heptane-water method. Our data suggest that binding affinities reported in these studies may need to be recalibrated upward by a factor of 2 or more.