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. 2014 Mar;55(3):573–582. doi: 10.1194/jlr.D044230

Fig. 5.

Fig. 5.

Recovery of long-chain aldehydes in presence of proteins. A: Effect of methanol on standards. C13-al (triangles) and C17-al (open squares) (1.5 and 2 nmol, respectively) were derivatized following the optimized protocol in the presence of 33% (v/v) methanol. Upon derivatization, equal volumes of water, water/methanol, or pure methanol were added to the assay to obtain the indicated methanol concentration. After centrifugation, the supernatant was subjected to RP-HPLC, and the fluorescent derivatives were detected by fluorimetry (Ex 390 nm, Em 460 nm). Values represent means of two determinations. B: Effect of methanol on standards in presence of proteins. Brain homogenates (2 mg tissue) were spiked with C13-al (triangles) and C17-al (open squares) before derivatization. Upon derivatization, mixtures were processed as described for (A). Integrated peak areas of C13-al and C17-al derivatives are plotted versus final methanol concentration. C. Recovery of IS in presence of proteins. Increasing amounts of brain and liver homogenates were spiked with C13-al and C17-al before derivatization. Water/methanol or methanol was added to the derivatized sample in order to have a final concentration of either 30% or 60% (v/v). Derivatives were separated by RP-HPLC. Recoveries of C13-al and C17-al derivatives were plotted versus amount of tissue per assay. Open triangles: C13-al, 60% methanol; filled triangles: C13-al, 30% methanol; open squares: C17-al, 60% methanol; filled squares: C17-al, 30% methanol. D. Recovery of IS in presence of proteins. Increasing amounts of brain and liver homogenates were spiked with C13-al (triangles) and C17-al (open squares) before derivatization. Methanol was added to the derivatized sample to a final concentration of 60% (v/v). Derivatives were separated by RP-HPLC. Integrated peak areas of C13-al and C17-al derivatives were plotted versus amount of tissue per assay. E. Analysis of endogenous aldehydes. Increasing amounts of brain and liver homogenates were spiked with C17-al before derivatization and separation of the fluorescent derivatives by RP-HPLC. The ratio of endogenous C16-al (squares) and C18-al (open triangles) over C17-al standard was plotted versus amount of tissue per assay.