Ion-Mobility Mass Spectrometry Assay for Incorporation of Phytanic Acid into Muscle Phospholipids

Abstract

Human dysferlinopathies (Limb-Girdle Muscular Dystrophy 2b, Myoshi Myopathy) are muscle-wasting syndromes caused by mutations in the dysferlin protein. Dysferlin loss impairs sarcolemmal repair that may contribute to disease progression. One therapeutic approach is to treat subjects with compounds that fortify the natural membrane tendency to reseal after damage, and minimize the inflammation that impedes regeneration and amplifies tissue destruction. Phytanic acid is a saturated branched chain fatty acid comprised of a 16 carbon aliphatic chain with 4 methyl groups (4ME 16:0); it is incorporated into phospholipids and triglycerides. Model phospholipid bilayers containing phytanic acid exhibit greater electrical stability than bilayers composed of straight-chain phospholipids. Phytanic acid-containing phospholipids may improve the resistance of the muscle fiber sarcolemma to stretch-induced damage. To measure phytanic acid incorporation into muscle phospholipids, dysferlin-deficient A/J mice were maintained on a defined diet supplemented with 2% phytol for three weeks; control animals received the defined diet without phytol. Muscle tissue lipids were analyzed by mass spectrometry. The muscle phosphatidylcholine species profiles were similar between phytol and control diet, except that some additional species were detected in the phytol diet muscle. The two most abundant novel species (m/z 790.7 and 862.7) are tentatively identified as PC 20:0-16:0 and PC 20:0-22:6. To confirm the presence of phytanic acid, the ion mobility of these species was compared with diphytanoyl PC standard and endogenous straight chain PC phospholipids. The ion mobility is intermediate between PC species containing either zero or two phytanic acid chains, consistent with having one phytanic acid chain. We estimate that these species represent 5% of PC. Using this methodology, we will identify phytanic acid containing species in the other classes, in order to determine the total amount of phytanic acid-containing phospholipid incorporated in the muscle.