Wolfhardt Freinbichler1, Bashkim Misini1, Maria Alessandra Colivicchi2, Wolfgang Linert1, Keith F Tipton3, Laura Della Corte4. 1. Institute for Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163-AC, A-1060 Vienna, Austria. 2. Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino (NEUROFARBA), Università degli Studi di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy. 3. School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland. 4. Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino (NEUROFARBA), Università degli Studi di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy. Electronic address: laura.dellacorte@unifi.it.
Abstract
BACKGROUND: Free, non-protein bound, Fe(II), which can catalyse the formation of the toxic highly-reactive oxygen species (hROS), has been implicated in several neurodegenerative conditions. The determination of free Fe(II) and Fe(III) in samples obtained from microdialysis experiments has been limited by the small amounts of sample available. NEW METHOD: This work describes the development of a HPLC, with absorbance detection, method, based on the complexation of Fe(II) with bathophenanthroline disulfonate (BS), which allows a complete extracellular iron analysis with the small sample amounts that are available from in vivo microdialysis in rat brain. RESULTS: Microdialysis experiments using 6-hydroxydopamine stimulation, showed that basal-as well as evoked levels of extracellular Fe(II) and total iron could be determined in parallel with measurements of hROS formation. COMPARISON WITH EXISTING METHODS: Although a spectrophotometric BS-based assay has been reported for use in microdialysis samples from large animals, the present procedure is applicable to the small sample sizes available from studies in rat brain. It is simpler than the alternative, involving inductively-coupled plasma mass spectrometry. CONCLUSIONS: The procedure described is simple and sensitive, giving a linear response in the Fe(II) concentration range of 50 -2000 nM. A 20 min microdialysis sample (flow-rate 3 μl/min) yields sufficient material for triplicate determinations of the evoked release of Fe(II) and total iron whilst leaving sufficient sample volume for determining hROS and amine or amino-acid neurotransmitter release.
BACKGROUND: Free, non-protein bound, Fe(II), which can catalyse the formation of the toxic highly-reactive oxygen species (hROS), has been implicated in several neurodegenerative conditions. The determination of free Fe(II) and Fe(III) in samples obtained from microdialysis experiments has been limited by the small amounts of sample available. NEW METHOD: This work describes the development of a HPLC, with absorbance detection, method, based on the complexation of Fe(II) with bathophenanthroline disulfonate (BS), which allows a complete extracellular iron analysis with the small sample amounts that are available from in vivo microdialysis in rat brain. RESULTS: Microdialysis experiments using 6-hydroxydopamine stimulation, showed that basal-as well as evoked levels of extracellular Fe(II) and total iron could be determined in parallel with measurements of hROS formation. COMPARISON WITH EXISTING METHODS: Although a spectrophotometric BS-based assay has been reported for use in microdialysis samples from large animals, the present procedure is applicable to the small sample sizes available from studies in rat brain. It is simpler than the alternative, involving inductively-coupled plasma mass spectrometry. CONCLUSIONS: The procedure described is simple and sensitive, giving a linear response in the Fe(II) concentration range of 50 -2000 nM. A 20 min microdialysis sample (flow-rate 3 μl/min) yields sufficient material for triplicate determinations of the evoked release of Fe(II) and total iron whilst leaving sufficient sample volume for determining hROS and amine or amino-acid neurotransmitter release.