Low-temperature improved-throughput method for analysis of brain fatty acids and assessment of their post-mortem stability

Fatty Acid Composition of Frontal, Temporal and Parietal Neocortex in the Normal Human Brain and in Alzheimer’s Disease

“…By use of a previously validated [23] procedure that involves multiple-sample mechanical homogenization of tissue samples, protection of polyunsaturated fatty acids by addition of BHT, and the methylation and saponification of samples in small sealed glass vials overnight at room temperature we have been able to perform the largest and most comprehensive analysis yet reported of the fatty acid composition of the cerebral cortex in the normal elderly and in AD. This has revealed a significant reduction in stearic acid (18:0) and an increase in oleic acid (18:1n-9) in the frontal and temporal cortex in AD, reflecting loss of normal variation in the content of these two fatty acids between frontal, temporal and parietal neocortex in the diseased brains.…”

“…As previously described [23], 50 mg tissue samples were added to 350 ll of methanol at 0°C and homogenized in a Bertin Technologies Precellys 24 mechanical homogenizer (Stretton Scientific, Stretton, UK) at 6,000 rpm for 2 9 30 s with 15-25 2.3 mm Biospec Products zirconiasilica beads (Stratech Scientific, Newmarket, UK) in a screw cap 2 ml microcentrifuge tube (Fisher Scientific, Loughborough, UK) that had been washed by vortexing with 1 ml of chloroform three times. The sample was cooled on ice after homogenization.…”

“…The two upper phases (containing the free fatty acids derived from the lipid extract) were combined and dried under N 2 . Methyl esters of the free fatty acids were then prepared in a sealed tube with a solution of 2.5% concentrated H 2 SO 4 in anhydrous methanol overnight at room temperature, as described previously [23,24].…”

“…Fatty acid methyl esters were analyzed by gas chromatography and identified by co-chromatography with authenticated standards and gas chromatography-mass spectrometry as described previously [23,25]. 200 nmol heptadecanoic acid (C17:0) that had been added during the lipid extraction was used as the internal standard and quantified by flame ionization detection as described [25].…”

“…We recently described a low-temperature relatively high-throughput method for analysis of fatty acids in postmortem brain tissue [23] and demonstrated that there is no significant change in the fatty acid composition of human brain tissue under conditions simulating post-mortem delay for at least 3 days. In the present study we have used this method to determine the fatty acid content of frontal, temporal and parietal neocortex from a large number of AD and control brains.…”