Reversed Phase HPLC Determination of Total Homocysteine, Cysteine, Cysteinyl Glycine, Glutathione in Plasma of Epileptic Patients

Abstract

The fast and accurate reversed-phase HPLC method was predicated on qualitative and quantitative estimation of the total concentrations of the essential thiols in the plasma of adults volunteers compared with epileptic patients, which is l-cysteine (Cys) total homocysteine (tHys), cysteinyl glycine (Cys-Gly), glutathione (GSH) and cysteamine (Cstm), respectively. The plasma samples of (50) epileptic patients were classified into three groups; the non-anti-epileptic drug consisted of 20 patients (12 male, eight female) who were mainly diagnosed with idiopathic generalized epilepsy. The mean age was 34 ± 13 (range 19–48) years. The second group consisted of 30 patients receiving two types of anti-epileptic drugs, 18 patients (12 male and six female) receiving Carbamazepine, and 12 patients (7 males and five female) receiving phenytoin. The mean age of the patients was 32 ± 11 Compared with 31 healthy volunteers (20 male and 11 female). The mean age of the controls was 31 ± 9 (range 26–49 years). The mean duration of treatment of patients receiving the anti-epileptic drug was six months). All measurement was done using pre-column derivatization with bromobimane, and the derivative of each standard mixture were baseline separated on C18-DB (50 x 4.6 mm ID) column, 3μm particle size. The regression coefficients for the separated and standard deviation SD within-run ranged from 0.09 to 8.40 μmol/L, and between-run ranged from 0.15to 9.16 μmol/L; the analytical procedure gave good linearity in the range between 1.25 to 20 µmole/ l, the detection limit was 0.1 µmole /L for all the thiol groups. Analytical recovery was 96.9–107.4 %; the mean concentration of plasma cysteine and total homocysteine was slightly higher in males than females; it was 221± 75 for adult males and 190± 44 μmol/L for adult females, while t-homocysteine was (10.55 ±2.45 vs. 9.79 ± 1.88 μmol/L, the results observed that the value of cysteine and homocysteine were significantly higher in epileptic patients using Carbamazepine and phenytoin than in healthy volunteers. Mean values for glutathione were lower, while cysteinyl glycine showed no significant difference in healthy and epileptic patients and no sex- and age-dependent.