LONG-CHAIN FATTY-ACID-BINDING TO ALBUMIN - REEVALUATION WITH DIRECTLYMEASURED CONCENTRATIONS

In studies on uptake of fatty acids (FA) into organs, the unbound (or free) fatty acid fraction is commonly calculated from the concentratio n bound to albumin and from published binding constants. However, ther e is some dispute on the methods used for determining those binding co nstants. We developed a method allowing direct measurement of unbound FA by extending the previous studies of Svenson et al. [1] and Reed et al. [2]. Albumin was coupled to a solid phase (Sepharose 4B), loaded with FA and equilibrated with an aqueous solution. Laurate, palmitate and oleate concentrations in the aqueous phase were determined at diff erent molar ratios of FA to albumin (r) and at different temperatures. FA albumin-binding constants (K-1) increase with chain length and dec rease with temperature, in accordance with data obtained by others. Ho wever, the unbound concentrations measured are markedly lower than tho se obtained from binding constants, and the resulting K-1 values marke dly higher. This difference is presumed to result from (1) our direct measurement of unbound FA and (2) utilizing different more physiologic al conditions. Recalculating kinetic parameters from published FA upta ke data, we found considerably different K-m and V-max values compared to the original data. Thus, the FA-binding characteristics measured i n this study may influence the interpretation of FA uptake substantial ly.