| Objective：1. To establish a reversed-phase high performance liquidchromatography-fluorescence detection (RP-HPLC-FLD) method forsimultaneously determination of hepatoma-associated amino acidenantiomers with pre-column derivatization. The concentration alterationsof amino acid enantiomers in plasma of hepatocellular carcinoma (HCC)patients and healthy controls have been investigated and the metabolicprofiles of hepatoma-associated amino acid enantiomers between the twogroups have been explored.Methods：Separation was carried out on a Phenomenex Gemini C18column(250mm×4.6mm,5m) with a programmed gradient elution.O-phthaldialdehyde (OPA) and N-acetyl-L-cysteine (NAC) were served asthe pre-column derivatization reagents. The mobile phase was consisted of 20.0mmol/L sodium dihydrogen phosphate (pH6.80) and acetonitrile. Theflow rate was1.0ml/min. The column temperature was30℃. Injectionvolume was10l. The eluted solution was monitored using a fluorescencedetector with excitation wavelength at350nm and emission wavelength at450nm.Concentrations of hepatoma-associated amino acid enantiomers(L-threonine (L-Thr), D-threonine (D-Thr), L-alanine (L-Ala), D-alanine(D-Ala), L-tyrosine (L-Tyr), D-tyrosine (D-Tyr), L-valine (L-Val), D-valine(D-Val), Total-methionine (T-Met), L-phenylalanin (L-Phe) andD-phenylalanine (D-Phe)) in the plasma of23HCC patients and20healthycontrols were determined by reversed-phase high-performance liquidchromatography (RP-HPLC). The metabolic profiles of L-amino acid andD-amino acid in HCC patients and healthy controls plasma had beeninvestigated, which might provide basic evidence for the early diagnosisand treatment of HCC patients.Results：The retention times of D-Thr, L-Thr, D-Ala, L-Ala, L-Tyr, D-Tyr,L-Val, D-Val, T-Met, D-Phe, L-Phe were5.91,6.45,8.70,9.11,13.20,13.92,16.39,18.38,19.10,23.43and24.02min, respectively. Thepresented method exhibited an excellent linearity for all the analytes overtheir respective concentration ranges. The limit of detection (LOD) forD-Thr, L-Thr, D-Ala, L-Ala, L-Tyr, D-Tyr, L-Val, D-Val, T-Met, D-Phe, L-Phe were9.77，11.4，3.50，4.11，6.79，7.81，1.86，1.95，2.39，7.10and7.32pg/ml, respectively. The recovery of the substances determinedwas between90.22and109.44%，intra-day precision was between1.32and4.20%and inter-day precision was between2.12and4.51%.2. The variance between the two groups had been analyzed using thetwo independent sample t test. Compared with healthy control subjects, theHCC patient plasma showed higher levels of L-Tyr and lower levels ofD-Thr, L-Thr, D-Ala, L-Ala, D-Val, L-Val and T-Met, however,there was no obvious change in the level of D-Tyr and L-Phe. Multivariatestatistical analysis showed that the differences between the two groups havestatistical significance.Conclusions:1. We have developed a reversed-phase high performance liquidchromatography-fluorescence detection (RP-HPLC-FLD) method withpre-column derivatization to simultaneously determinehepatoma-associated amino acid enantiomers. This method is simple,accurate and suitable for applicability to routine scientific research andclinical measurement.2. In this research, we have investigated the metabolic profiles ofhepatoma-associated amino acid enantiomers in the plasma of the healthycontrols and HCC paients. The results demonstrated that there was obviousdifference between the two groups. Alterations in L-amino acid and D-amino acid concentrations in plasma of the two groups can contribute tothe discoveries of new diagnostic markers as well as provide references forthe pathogenic mechanism, early diagnosis and novel therapeutic targets ofHCC. |
PDF Full Text Request