4000 qtrap mass spectrometer

All tissue processing, preparation and extraction occurred in a fume hood, under a red light. Liver tissue (500 mg) was homogenized in 1ml of Dulbecco’s phosphate-buffered saline solution using a Precellys homogenizer (Bertin Technologies, Rockville, MD). Tissue homogenate was added to a glass screw-capped tube covered by aluminum foil. After adding the internal standard (250 µl, 4,4-retinyl acetate) and 0.025 M KOH to the homogenate, retinoids were extracted in hexane, and dried under a nitrogen stream. Retinoid extracts were reconstituted in 200 µl of acetonitrile and separated by HPLC using an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA) using Phenomenex Synergi 4 µ Max-RP 80A column (4 µm, 3×150 mm) as previously described (11 (link)). Retinol and retinoic acid were identified using a 4000 Q TRAP mass spectrometer (Applied Biosystems) coupled with the HPLC. The mass spectrometer was controlled using Analyst version 1.5.1 software and was operated in a MRM mode. Optimum positive APCI (Atmospheric pressure chemical ionization) conditions included: curtain gas, 30.0; collision gas, medium; nebulizer current, 3; temperature, 500°C; and GS1, 60. Peak area measurements were used to quantify retinoid amounts using standard curves based on retinol or retinoic acid and normalized using the internal standard.

Membrane fluidity was quantified by fluorescence depolarization as described (Crockett and Hazel 1995 (link)) (see supplementary material). Change in polarization (excitation=356 nm, emission=430 nm) was measured between 2 and 40°C using a Perkin-Elmer LS-50B spectrophotometer. Temperatures were elevated at 2°C intervals (for myelin) and 5°C intervals (for synaptic membranes and mitochondria) at a rate of ~0.3°C min^-1.
Lipids were extracted from myelin as described (Bligh and Dyer 1959 (link)) (see supplementary material). Extracts were sent to the Kansas Lipidomics Research Center for phospholipid analysis, and a diacyl polar lipid profile dataset was generated by quadrupole mass spectrometry using an Applied Biosystems 4000 QTRAP mass spectrometer as described (Xiao et al. 2010 (link)). Relative abundances of the major phospholipid classes were compared between species. The unsaturation index (UI) was calculated as described (Grim et al. 2010 (link)). Polar lipid compositions were not analyzed in synaptic membranes due to lack of sufficient material.
Cholesterol was quantified in myelin and synaptic membranes using a Cayman assay kit and normalized to total phospholipid content, which was measured as hydrolyzed inorganic phosphate in membranes as described (Rouser et al. 1970 (link)) (see supplementary material).