Calibration of albumin-fatty acid binding constants measured by heptane-water partition
| Autor: | Richard A. Weisiger, L. P. Johnson, Frank J. Burczynski, C. K. Davis, S. M. Pond |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Time Factors
Physiology Palmitic Acid Analytical chemistry Serum albumin Palmitic Acids Gas Chromatography-Mass Spectrometry Heptanes chemistry.chemical_compound Physiology (medical) Fatty acid binding Methods Serum Albumin chemistry.chemical_classification Heptane Chromatography Hepatology biology Fatty Acids Gastroenterology Aqueous two-phase system Reproducibility of Results Water Fatty acid Partition coefficient Dissociation constant chemistry Calibration biology.protein Chromatography Thin Layer Gas chromatography |
| Zdroj: | American Journal of Physiology-Gastrointestinal and Liver Physiology. 265:G555-G563 |
| ISSN: | 1522-1547 0193-1857 |
| DOI: | 10.1152/ajpgi.1993.265.3.g555 |
| Popis: | 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) |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|