Unbound free fatty acid levels in human serum

Total serum free fatty acids (FFAt) levels provide an important measure of the physiologic state. Most of the FFA in serum is bound to albumin; a small portion, however, is unbound. This study presents the first measurements of serum unbound free fatty acid (FFAu) concentrations. These measurements were made possible by the development of the fluorescence probe ADIFAB (acrylodated intestinal fatty acid binding protein). In the present study ADIFAB was shown to provide the correct value of the sum of the [FFAu] of each molecular species of long chain FFA: 1) in aqueous mixtures of FFA and ADIFAB; 2) in mixtures of FFA, human serum albumin, and ADIFAB; and 3) in serum and ADIFAB. Human serum [FFAu] were measured in 283 samples from healthy donors and yielded a mean value of 7.5 nM with a standard deviation of 2.5 nM. This measured value is as much as 1000-fold smaller than [FFAu] values previously estimated. The distribution of [FFAu] values was found to be invariant with donor gender and age. Average [FFAu] values do, as expected, exhibit a small (1.5 nM) but significant (P < 0.001) increase after overnight fasting. FFAu levels were found to be well correlated with total serum FFA or, equivalently, [FFAt]/[HSA]. Given their ease and accuracy, and because [FFAu] values increase exponentially with [FFAt]/[HSA], [FFAu] measurements could provide a sensitive method for assessing the physiologic state.