Aa standards

AA standards, OA standards, FFA standards, internal standards (IS; norvaline, ¹³C₁-leucine, ¹³C₁-phenylalanine, ¹³C₂-succinic acid, 3,4-dimethoxybenzoic acid, and lauric-d₂-acid), ethyl chloroformate (ECF), and triethylamine (TEA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-Methyl-N-tert-butyldimethylsilyl trifluoroacetamide (MTBSTFA) was obtained from Pierce (Rockford, IL, USA). Diethyl ether, ethyl acetate, toluene, and dichloromethane were purchased from Kanto Chemical Co. Inc. (Chuoku, Tokyo, Japan), and other reagents, including sulfuric acid, sodium hydroxide, and sodium chloride, were manufactured by Deajung (Gyeongido, Korea). All chemicals were analytical reagent grade.

Plasma nitrogen containing compounds were analyzed at Molecular Structure Facility, Proteomics Core of Genome Center (Davis, CA) with Li-based Hitachi 8900 according to established methods (Cooper et al., 2001 ). Briefly, plasma samples were thawed at 20 to 22 °C, acidified with 2% sulfosalicylic acid (Sigma #247006) final concentration and incubated at 20 to 22 °C for 15 min before overnight freezing the samples at −20 °C. Next day, the acidified samples were diluted with 100 nmol/mL aminoethylcysteine (AE-Cys) Li diluent (Pickering Labs, #Li220 Mountainview, CA) prior to the 50 μL injection. Free AA were separated using ion-exchange chromatography with a secondary post-column reaction with ninhydrin (WAKO, #299-70501). Column and buffers were supplied by Hitachi (Hitachi High-Technologies Corporation, Tokyo, Japan), and ninhydrin was supplied by Wako (FUJIFILM Wako Chemicals U.S.A. Corporation, Richmond, VA). Calibration of the Hitachi AA Analyzer (Model 8900, Japan) was performed using AA standards (Sigma–Aldrich, St. Louis, MO). Absorbance was recorded at both 570 and 440 nm after the reaction with ninhydrin to determine the response factor for each individual AA and to quantify levels relative to the known AA standards. The included internal standard (AE-Cys) was used to correct for any variations in injection volume caused by auto-sampler (integrated part of Hitachi 8900).