The Study Of Methodology And Applications Of Determination Of Methionine Metabolites In Blood Sample By Hplc

Objective:1. To established a method for simultaneous determination S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in red blood cells (RBCs) by HPLC with programmed wavelength ultraviolet (UV) detection2. The developed HPLC-UV method was applied to determine the concentrations of SAM and SAH in RBCs of folate deficiency and healthy mice. Study the metabolism of SAM and SAH in folate deficiency mice.3. To established a method determination of plasma total homocysteine (tHcy) by precolumn derivatization high performance liquid chromatography with fluorescence detection (HPLC-FD).4. The developed HPLC-FD method was applied to determine plasma tHcy in patients with hypertension and healthy controls. Investigate the change of tHcy catabolism in hypertension patients.Methods:1. Simultaneous determination of SAH and SAM in RBCs of mice: After purification treatment of the RBCs samples, An Agilent ZORBA×Eclipse×DB-C8(150mm×4.6mm,5μm) column was used for the sample analysis. The temperature was operated at35℃. Separation was carried out using the mobile phase consisting of3mmol/L1-heptanesulfonic acid in40mmol/L NaH2PO4buffer (pH3.72) and methanol (95:5,v/v) at a flow rate of0.9ml/min. The eluates were monitored by the programmed wavelength detection setting at450nm from0to8.00min and at260nm from8.01to16.00min. The SAM and SAH in red blood cells of ten healthy mice and nineteen folate deficiency mice were tested by the developed method.2. Tris-(2-carboxylethyl)-phosphine (TCEP) and N-(1-pyrenyl) maleimide (NPM) were used as the reduced reagent and derivatization reagent for the plasma tHcy analysis, respectively. Separation was carried out on An Agilent Hypersil C-18column(250mm×4.0mm;5μm) in gradient elution mode. The mobile phase consisted of15mmol/L sodium acetate solution(A), acetonitrile(B) and300ml water containing lml acetic acid and lml phosphoric acid(C). The temperature was set at25℃. The eluates were monitored by the fluorescence detection at an excitation wavelength of330nm and an emission wavelength of380nm. The concentration of plasma tHcy in seven patients with hypertension and seven healthy controls were determined by this developed method.Results: 1. The retention time of SAH and SAM were12.2min and13.6min respectively. The linearities were from0.25to3μg/ml for SAH,0.5to10μg/ml for SAM. The detection limits were0.15fig/ml for SAH,0.07fig/ml for SAM. The mean recoveries range for SAM and SAH were80%to116%. The intra-day coefficient of variation (CV) rang for SAM and SAH were1.0%to6.4%, while inter-day CV rang for SAM and SAH were1.3%to7.3%.2. The metabolism of SAH in the red blood cells from the folate deficiency group was significantly different from that of the controls (P <0.01). No significant difference were found in the concentration of SAM between the two group.3. The retention time of Hcy was5.8min. The mean recovery of tHcy was (102±4.9)%. The linearity for tHcy was from0.500to100μmol/L The detection limit for tHcy was0.0160μmol/L. The intra-day and inter-day coefficient of variation (CV) for tHcy were less than4%.4. The concentration of tHcy in the plasma from the hypertension patient group was significantly different from that of the control group (P <0.05)Conclusion:1. A new method of HPLC with programmed wavelength ultraviolet detection have been developed to simultaneous determination of SAM and SAH in red blood cells of mice. The method is simple, accurate, and provide a reliable method for SAH metabolism research in mice.2. In this paper, we found the metabolism of SAH in folate deficiency mice were abnormal.3. In this research, we established an HPLC method using Tris-(2-carboxylethyl)-phosphine (TCEP) as the reduced reagent N-(1-pyrenyl) maleimide (NPM) as derivatization reagent for plasma tHcy determination. Gradient elution mode was used to shorter the analysis time. The developed method is simple, fast, sensitive, wich can meet the determination requirements of plasma samples. The presented method is well suitable for research and clinical routine measurement.