The Study Of Methodology And Clinical Applications Of Simultaneous Determination Of Homocysteine And Other Related Thiols In Plasma By High Performance Liquid Chromatography

Objective: 1. A method for the determination total homocysteine (tHcy), totalcysteine (tCys), total cysteinylglycine (tCysGly), total cysteine (tCys) inplasma by reverse phase high performance liquid chromatography andFluorescence Detection by pre-column derivatization was developed. 2. To study the effects on the plasma tHcy, tCys, tCysGly, tGSHconcentrations of different blood anticoagulant and the various stabilizingagents,together with the time and temperature before centrifugation. 3. To study the prevalence of plasma tHcy, tCys, tCysGly, tGSH inpre-hemodialysis (Pre-HD) and post-hemodialysis (Post-HD) of uremiapatients and theirs relationship each other. Methods: 1. Thiols in plasma and internal standard- N-acetylcysteirea werereduced by tris-(2-carboxylethyl)-phosphine (TCEP), then derivated by7-fluorbenzo -2-oxa-1,3-diazole-4-sulfonate (SBD-F). The derivativeswere separated using a methanol /phosphate buffer solution (25 mmol/L,pH 3.0) gradient on C8 column and detected at λex 380 nm, λem 510 nm. 2. Blood samples were collected into tubes containing EDTA,EDTA/NaF, EDTA/3DA, Sodium citrate/NaF, Oxalate/NaF, SodiumHeparin/NaF and stored at room temperature or on crush ice. Aliquots ofblood were centrifuged at 0, 3,6,24,48 hours,the plasma was removedand measured by HPLC-FD. Blood samples were stored on crush ice withEDTA used as basaline sample. 3. Blood samples from 54 healthy subjects and 26 uremia patientswere collected into tubes containing EDTA,and stored on crush iceimmediately. plasma was removed within one hour. Determined theplasma tHcy, tCys, tCysGly, tGSH concentrations by HPLC-FD. Theconcentration of Cr, UA and other parameters were determined byAutomatic Biochemistry Analyzer. Results: 1. The linearitys of the assay of tHcy, tCys, tCysGly, tGSH were 1.0～160.0 μmoL/L, 30.0～1040.0 μmoL/L, 2.0～300.0 μmoL/L, 1.0～130.0μmoL/L respectively, the regressions ranged from 0.9994 to 0.9999. for thethiols, the detection limit ranged from 0.10 to 2.0 μmoL/L, the within-dayprecision were <3.0%, The between-day precision were <6.0% exceptthe tGSH was 13.21%, the average recoveries were 98.1%～103.2%. 2. ① The concentrations of tHcy, tCys, tCysGly, tGSH rise rapidlywhen the collection of blood into anticoagulant EDTA and placing at roomtemperature. ② plasma tHcy, tCys, tCysGly, tGSH concentrations stayed stable for6h when the blood collected in EDTA and stored on ice. ③ plasma tHcy, tCys, tCysGly concentrations stayed stable for 6 hexcept for tGSH increasing within 3 h in whole blood when the bloodcollected in EDTA/3DA or EDTA/NaF at room temperature. ④ Blood samples could be kept for 6 h prior to separation of bloodcelle without observable change in tHcy , tCys , tCysGly , tGSHconcentrations when collected in Sodium citrate/NaF, in Oxalate/NaFSodium, in Heparin/NaF at room temperature. 3. ① There was a significant increase in plasma tHcy of male thanfemale (p<0.0001), while there was no significant change in tCys,tCysGly, tGSH concentrations. ② There were significant increase in tHcy and tCys repectively inpatients before dialysis compared to healthy subjects (p<0.0001), incontrast , GSH was significantly lower than healthy subjects (p<0.0001). ③ There were significant decrease in tHcy and tCys in uremia patientsbefore dialysis than after dialysis,while there was no significant change intGSH or tCysGly concentrations. ④ there were significant positive correlation between plasma tHcy andtCys (r=0.4582, p<0.01), creatinine (r=0.7536, p<0.01) too. ⑤ There was a significant decrease in plasma Tc, HDL, and LDLrepectively in uremia patients compared to healthy subjects, while therewas a significant increase in TG concentrations. Conclusion: 1. This method was implemented determination simultaneously ofplasma tHcy, tCys, tCysGly, tGSH for the first time in the interior. It isaccurate, sensitive, rapid, specific and suitable for the routine clinicalpractice and the research practice. 2. The study investigated the stability of plasma tHcy, tCys, tCysGly,tGSH in...