Identification of saturated and unsaturated fatty acids released during microsomal incubations.

1. In vitro clearance in liver microsomes is routinely measured in drug discovery and development for new chemical entities. Literature reports indicate that long chain fatty acids such as arachidonic, linoleic and oleic acids may be released over a period of time during microsomal incubations. Some fatty acids have been shown to interfere with oxidative and conjugative reactions in microsomes, thus potentially inhibiting microsomal clearance of compounds. 2. The present study was aimed at deciphering the fatty acids present or released from microsomes. Analytical methods were developed to characterize and quantitatively assess the fatty acids without chemical derivatization in rat, monkey and human liver microsomes. Additionally, incubations with uridine-5'-diphosphoglucuronic acid (UDPGA) were utilized to trap the released fatty acids as their glucuronate esters, which were characterized and confirmed by high-resolution LC-MS/MS. 3. Our results indicate for the first time that timnodonic, trans-eicosenoic, gondoic, behenic, and nervonic acid were released during microsomal incubations. Additionally, α- and γ-linolenic, timnodonic, palmitoleic, linoleic, arachidonic, palmitic, oleic, and stearic acid were identified as their corresponding acyl-glucuronides in rat, monkey and human liver microsomes, providing the first direct evidence that the released fatty acids are capable of forming glucuronides under incubation conditions.