| Part ?:An HPLC protocol to determine free neuroactive amino acids in body fluids of depressive patientsBackground:Amino acid(AA)neurotransmitters such as glutamic acid(Glu),and y-aminobutyric acid(GABA)may serve as biomarkers for mood disorders.Ion-exchange high performance liquid chromatography(IE-HPLC)are mostly used to quantitatively determine low levels of free amino acids(AAs)-of interest as putative neuroactive compounds-in human plasma.Its use is,however,severely limited,since it shows slow or insufficient AA separation,or that the AA levels are below the limit of detection(LOD),especially in the cerebrospinal fluid(CSF)of major depressive disorder(MDD)patients.Although a reversed-phase HPLC(RP-HPLC)coupled with extra advanced equipment such as mass spectrum may improve the sensitivity of the measuring system,this is not a practical procedure for clinical studies.We made some modifications for RP-HPLC protocol used for measuring Glu,GABA and histamine in the rat brain(called for 'rotine protocol' in this paper)in order to develop an RP-HPLC with fluorescence detection(FLD)(RP-HPLC-FLD)protocol allowing determination of AA levels in CSF and in plasma of mood disorder patients.Methods:In the present study,an optimal RP-HPLC-FLD by pre-column derivatization have been achieved for separating free amino acids in human postmortem CSF by a number of alterations in a routine protocol,which included i)AAs standards or sample(including deproteinization)preparation,using borate buffer instead of HClO4 as dissolving solution for standard AAs and for CSF-samples,using methanol instead of HClO4 as deproteinization reagent;ii)using O-phthaldialdehyde(OPA)-?-mercaptopropionic acid(MPA)instead of OPA-Thiofluor as derivatizing solution,and iii)using acetonitrile-free acetate buffer with tetrahydrofuran(THF)and triethanolamine(TEA)in the mobile phase to eliminate the tailed and overlaid peaks,and keep to detection for a long time.The efficacy of the protocol was evaluated by determining postmortem CSF-Glu and GABA levels of 17 mood disorder patients which included 8 major depressive disorder(MDD)and 9 bipolar disorder(BD)patients,together matched with 19 controls.In addition,the efficacy of the protocol was tested for plasma and CSF AA levels in 4 melancholic MDD patients.Results:23 AA standards and AAs in the postmortem CSF were well separated in 40 minutes and showed significantly narrower and higher HPLC signal peaks by the improved protocol.The control parameters of this improved HPLC show the results of Glu and GABA in the postmortem CSF,as follows:the limit of the detections of Glu and GABA are 1 pg/?L and 0.5 pg/?L,respectively;the limit of the quantifies of Glu and GABA are 3.68 pg/?L and1.03 pg/?L,respectively;the recoveries for Glu and GABA are 102.71%and 104.41%,respectively;the intra-day and inter-day coefficients of variation are(1.99-3.82)%and(3.26-7.83)%,respectively.Plasma and CSF AAs of melancholic MDD patients were also well separated by the improved protocol.It was found that postmortem CSF-Glu and GABA levels were significantly decreased in MDD(P?0.0209)but not in BD patients(P?0.4030)compared to control group,and that CSF-Glu and GABA levels were significantly decreased in MDD(P=0.0056,both)compared to BD group.In the present results,the samples were relatively small and the depression group of CSF samples had used antidepressants.In the control group,both CSF-Glu and GABA levels showed significant positive correlations with postmortem delay(PMD)(rho=0.826,P=0.000;rho=0.887,P=0.000,respectively),and CSF-GABA levels showed a significant negative correlation with age(rho=-0.567,P=0.011).There were no sex differences in CSF Glu or GABA levels either in controls(12 male and 7 female)(P?0.422)or in depressive patients(MDD+BD)(12 male and 5 female)(P>0.155).The sex difference did not do because of 1 female in BD group.In MDD(4 male and 4 female),CSF-GABA showed higher level in female than that in males,only an increasing trend and no significance(P=0.083).Conclusion:This improved HPLC protocol is applicable for detection of neuroactive AAs in body fluids of patients with mood disorder,and is the strong protocol to search for the AA biomarker in mood disorder.Diminished CSF-Glu and GABA levels may serve as biomarkers for MDD.Part ?:Effect of regulation of membrane estrogen receptors-NO pathway on corticotropin-releasing hormone expressionBackground:Women are more vulnerable to depression,which has been considered as one of the top threaten to human health by the WHO.According to clinical research,the number of female depression patients is twice as male patients.The hypothalamus-pituitary-adrenal(HPA)axis participates in stress response,and is believed to be the final common pathway of depression.We and others have found that estrogen can increase the release of corticotropin-releasing hormone(CRH).Estrogen binding to its receptor may act as a nuclear factor and increase the transcription of CRH,leading to the activation of the HPA axis.On the other hand,there is also evidence suggesting that membrane estrogen receptors(mER)could stimulate CRH through a series of signaling pathways.Nitrogen oxide(NO),which is synthesized by nitric oxide synthase(NOS)locally in cells,is one of these putative pathways.Some studies have indicated that the estrogen-NO-pathway may play a key role in the pathology of depression.Our research aimed to study how the mER-NOS-NO pathway may regulate the transcription and release of CRH.Methods:1)We used SK-N-SH cells which express endogenously CRH,ERa,ER?,GPR30,NOS1,NOS2 and NOS3.We exposed the cells with E2-BSA at different concentrations and determined the concentration of CRH in the cell culture medium,and CRH-,ERs-,NOSs-mRNA and NO expression in the cells.The E2-BSA complex stays outside of cells,but it will not affect the binding of E2 to membrane ER.Subsequently we applied SNP(NO donor)or 7-Ni(specific NOS1 inhibitor)with E2-BSA to the cells,and performed the same tests.In addition,added E2 to the cell medium and measure CRH-mRNA as a comparison of E2-BSA.2)We transfected HEK293T cells with plasmids containing human CRH promoter with luciferase,and added E2-BSA to the cells after 24h of transfection.We determined the activity of CRH promoter with luciferase at 0,0.5h and 24h after the stimulation by E2-BSA.Result:E2-BSA elevated the cellular NO in SK-N-SH cells,increased the release of CRH after 0.5h,and increased CRH-mRNA after 24h.SNP also increased CRH and CRH-mRNA.7Ni diminished the effect of E2-BSA on NO,CRH and CRH-mRNA.E2-BSA increased the activity of the CRH promoter in the absence of NOS1.Conclusions:E2-BSA binds to the mER in SK-N-SH cells and elevates the levels of cellular NO,CRH release and CRH-mRNA expression.However,NOS 1-NO pathway is not the only pathway that conducts signals from mER to CRH activity. |
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