Abstract: This paper describes a simple and sensitive reversed-phase HPLC method for the determination of total homocysteine, total cysteine, total glutathione (GSH+GSSG), and neuroactive amino acids (Asp, Glu, Tau, GABA) using precolumn derivatization with ortho-phtaldialdehyde and fluorimetric detection at 360 and 470 nm for emission and excitation, respectively. Derivatization was performed with ortho-phthaldialdehyde in the presence of 2-mercaptoethanol after alkylation of the free sulfhydryl groups with iodoacetic acid. For determination of total aminothiols, the disulfide bonds were reduced and protein-bound thiols were released by addition of dithiothreitol to the plasma sample. The advantage of this method is the simultaneous determination of both homocysteine/cysteine/glutathione and neuroactive amino acids in the sample. The plasma levels of studied compounds were determined in 14 healthy volunteers (20-45 years old) and 55 patients with chronic hepatitis C (20-49 years old) and the resulting numbers were in a good agreement the studies published earlier. The calibration curves were linear over a concentration range of 5-100 μM in plasma (r2=0.985-0.996). The intraday and interday coefficients of variation were 3-6% and 4-7%, respectively. The recovery of the standards added to the plasma samples ranged from 94 to 102%. The limits of detection (LOD) were 0.2-0.5 ng per 10 μl injection volume (signal-to-noise ratio of 3).
Reference: Pozdeev V.K., Pozdeyev N.V., Determination of total aminothiols and neuroactive amino acids in plasma by high performance liquid chromatography with fluorescence detection, Biomeditsinskaya khimiya, 2010, vol:
6. Ventura P., Rosa M.C., Abbati G., Marchini S., Grandone E., Vergura P., Tremosini S., Zeneroli M.L. (2005) Liver Int., 25, 49-56. CrossRef Scholar google search
7. Wang W., Rusin O., Xu X., Kim K.K., Escobedo J.O., Fakayode S.O., Fletcher K.A., Lowry M., Schowalter C.M., Lawrence C.M., Fronczek F.R., Warner I.M., Strongin R.M. (2005) J. Am. Chem. Soc., 127, 15949-15958. CrossRef Scholar google search
34. Palmio J., Huuhka M., Saransaari P., Oja S.S., Peltola J., Leinonen E., Suhonen J., Keranen T. (2005) Psychiatry Res., 137, 183-190. CrossRef Scholar google search
35. Dagilas A., Kimiskidis V., Aggelopoulou M., Kapaki E., Fitili C., Libitaki G., Papagiannopoulos S., Kazis D., Kazis A., Aidonis A. (2005) Otol. Neurotol., 26, 476-480. CrossRef Scholar google search