?url_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Adc&rft.relation=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F19758%2F&rft.title=MONITORING+THE+METABOLISM+OF+THIOLS%2FDISULFIDES+IN+THE+EXTRACELLULAR+SPACE+OF+ORGANOTYPIC+HIPPOCAMPAL+SLICE+CULTURES+BY+ONLINE+MICROFLUIDIC+ANALYSIS+COUPLED+WITH+ELECTROOSMOTIC+SAMPLING&rft.creator=Wu%2C+Juanfang&rft.description=Sulfur-containing+compounds%2C+such+as+glutathione+(GSH)%2C+cysteamine+(CSH)%2C+homocysteine+(Hcy)%2C+cysteine+(Cys)+and+their+disulfides%2C+are+critical+for+maintaining+various+cellular+functions.+Concentration+profiles+of+these+compounds+in+biological+samples+are+useful+for+evaluating+cellular+physiological+status+and+diagnosing+diseases.+Traditional+quantitative+methods+involve+tedious+sample+pretreatment+and+are+not+suited+to+dealing+with+submicroliter+samples.+We+have+developed+a+microfluidic+system+for+online+analysis+of+thiol-containing+compounds+in+the+extracellular+space+of+rat+organotypic+hippocampal+slices+cultures+(OHSCs).+This+system+is+also+capable+of+monitoring+ectoenzymatic+activity+involved+in+the+metabolism+of+thiols%2Fdisulfides.+%0D%0AA+microfluidic+chip+capable+of+derivatization%2C+multiple+injection%2C+separation%2C+and+quantitation+was+designed+and+coupled+to+a+homemade+confocal+laser+induced+fluorescence+detector+with+a+multifunctional+control+program.+The+microfluidic+system+was+evaluated+in+an+on-chip+kinetic+study+of+the+reduction+of+oxidized+glutathione+(GSSG)+catalyzed+by+glutathione+reductase+(GR%2C+EC+1.8.1.7)+followed+by+derivatization+of+GSH+with+ThioGlo-1.+The+separation+of+analytes+was+successfully+achieved+within+4.5+s+and+9+mm+separation+length.+The+linear+range+of+the+system+is+up+to+50+%C2%B5M+(GSH).+The+mass+and+concentration+detection+limits+are+10-18+mol+and+4.2+nM%2C+respectively.+Based+on+GSH+growth+rates%2C+the+apparent+Michaelis+constants+of+GR+were+determined+to+be+40+%C2%B1+11+%CE%BCM+(GSSG)+and+4.4+%C2%B1+0.6+%CE%BCM+(%CE%B2-NADPH).+Electroosmotic+sampling+of+the+extracellular+fluid+of+OHSCs+was+further+coupled+to+a+microfluidic+device+for+in+situ+quantitation+of+endogenous+aminothiols+in+OHSCs%2C+by+which+CSH+(10.6+%C2%B1+1.0+nM)%2C+Hcy+(0.18+%C2%B1+0.01+%CE%BCM)+and+Cys+(11.1+%C2%B1+1.2+%CE%BCM)+were+separated+and+evaluated+simultaneously.+Using+this+method%2C+we+also+observed+the+complete+biodegradation+of+coenzyme+A+(CoA)+in+the+extracellular+space+of+OHSCs+and+determined+the+kinetic+parameters+of+this+sequential+multi-enzyme+reaction+in+in+situ.+Metabolism+of+cystamine+(CSSC)+and+pantethine+(PSSP)+in+OHSCs+have+also+been+investigated+and+the+percentage+yields+of+CSH+from+CSSC+and+PSSP+after+~55+s+exposure+time+to+OHSCs+were+91%25+%C2%B1+4%25+and+0.01%25+-+0.03%25%2C+respectively%2C+which+explains+the+differences+of+the+drugs+in+clinical+effectiveness+and+toxicity.%0D%0A&rft.date=2013-10-18&rft.type=University+of+Pittsburgh+ETD&rft.type=PeerReviewed&rft.format=application%2Fpdf&rft.language=en&rft.identifier=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F19758%2F1%2FJuanfang_Wu_dissertation.pdf&rft.identifier=++Wu%2C+Juanfang++(2013)+MONITORING+THE+METABOLISM+OF+THIOLS%2FDISULFIDES+IN+THE+EXTRACELLULAR+SPACE+OF+ORGANOTYPIC+HIPPOCAMPAL+SLICE+CULTURES+BY+ONLINE+MICROFLUIDIC+ANALYSIS+COUPLED+WITH+ELECTROOSMOTIC+SAMPLING.++Doctoral+Dissertation%2C+University+of+Pittsburgh.++++(Unpublished)++