Supplementary Methods
Metabolite measurements
Metabolite measurements were performed by the NIH West Coast Metabolomics Center using
validated protocols and standard operating procedures. For quality control purposes, 30 standard
metabolites were included in the samples in each randomized batch. FAMEs C08 - C30, a mixture of
fatty acid methyl esters that are used as ‘Retention Index Markers’ served as internal references for
Retention Index (RI) calibration, the LECO GCTOF Pegasus III (LECO Corporation, St. Joseph, MI)
proprietary deconvolution protocol for peak finding, and the BinBase database
(http://fiehnlab.ucdavis.edu/db/) for metabolite annotation. Sample derivatization with
methoxylamine hydrochloride in pyridine and N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA)
was performed as described previously. GC/TOF-MS analysis was performed using an Agilent 6890N
gas chromatograph (Palo Alto, CA, USA) interfaced to a time-of-flight (TOF) Pegasus III mass
spectrometer (Leco, St. Joseph, MI, USA). Automated injections were performed with an MPS2
programmable robotic multipurpose sampler (Gerstel, Mühlheim an der Ruhr, Germany). The GC
system was fitted with both an Agilent injector and a Gerstel temperature-programmed injector,
cooled injection system (model CIS 4), with a Peltier cooling source. An automated liner exchange
(ALEX) designed by Gerstel was used to minimize matrix effects occuring in complex sample analysis.
Multiple baffled liners for the GC inlet were deactivated with 0.5-µl injections of MSTFA. The Agilent
injector temperature was held constant at 250°C while the Gerstel injector was programmed (initial
temperature 50°C, hold 0.1 min, and increased at a rate of 10°C/s to a final temperature of 330°C,
hold time 10 min). Injections of 1 µl were made in split (1:5) mode (purge time 120 s, purge flow 40
ml/min). Chromatography was performed on an Rtx-5Sil MS column (30 m x 0.25 mm i.d., 0.25 µm
film thickness) with an Integra-Guard column (Restek, Bellefonte, PA, USA). Helium carrier gas was
used at a constant flow of 1 ml/min. The GC oven temperature program was 50°C initial temperature
with 1 min hold time and ramping at 208°C/min to a final temperature of 330°C with 5 min hold time.
Both the transfer line and source temperatures were 250°C. The Pegasus III TOF mass spectrometer
ion source operated at –70 kV filament voltage. After a solvent delay of 335 s, mass spectra were
acquired at 17 scans per second with a mass range of 50 to 500 m/z. We quantified 394 metabolites
in plasma and 240 metabolites in CSF, with 36.3% (143) in plasma and 41.7% (100) in CSF of known
identity.