Quantification of Amino Acids in Mice Serum and CSF

Male mice (8–11 weeks of age) were anesthetized with isoflurane inhalation. Blood was collected by cardiocentesis, and serum was prepared after coagulation and centrifugation. CSF samples were collected by ventricular puncture with glass capillaries. Serum and CSF amino acid levels were measured using high-performance liquid chromatography (HPLC), amino acid labeling with 4-fluoro-7-nitrobenzofurazn (NBD-F; Dojindo, Kumamoto, Japan), and thiol labeling with 4-fluoro-7-sulfobenzofurazn (SBD-F; Dojindo) as previously described [28 (link),29 (link)]. NBD-F labeling of tryptophan (Trp) resulted in a fluorescence wave change, and Trp was detected by its fluorescence (excitation: 295 nm; emission: 340 nm). Asparagine was not measurable because its peaks were too close to those of NBD-F derivatives. Because the minimum volume required for all our assays (NBD-F labeling, SBD-F labeling, and Trp detection) is 8 µL (the routine serum volume used was 25 µL), equal CSF volumes (3–5 µL) of two–three mice were pooled. For the detection of thiol-containing amino acids, serum and CSF samples were reduced to cleave disulfide bonds before SBD-F labeling, and the total levels of Hcy, Cys, and GSH (tHcy, tCys, and tGSH, respectively) were measured [28 (link),29 (link)].