title: A novel method for non-transferrin-bound iron quantification by chelatable fluorescent beads based on flow cytometry.
abstract: The reliable measurement of non-transferrin-bound iron (NTBI) in serum has proved to be difficult and generally time consuming. We have sought a simple and fast method for such a determination. We adopted a fluorescence assay and designed a fluorescent dye with a chelating agent attached to sense iron. To avoid autofluorescence from serum samples, the iron probes were linked to beads and the autofluorescence could be separated and excluded from the measurement by flow cytometry due to the size difference between beads and serum proteins. Fluorescent beads containing both fluorescent and chelating moieties have been synthesized. The nature of the chelating function has been systematically investigated using four different chelators: bidentate hydroxypyranone, bidentate hydroxypyridinone, hexadentate hydroxypyranone and hexadentate hydroxypyridinone, each with different iron affinity constants. Competition studies demonstrate that the hexadentate hydroxypyridinone-based beads are capable of scavenging most of low molecular mass and albumin-bound iron but negligible amounts of iron from transferrin and ferritin. Serum samples from 30 patients with different types of disease and normal volunteers were measured. The concentrations of NTBI fall in the range -0.41 to +6.5 μM. The data have been compared with those obtained from the traditional 'NTA' method.
authors.forename: Yongmin
authors.initials: Y
authors.lastname: Ma
authors.affiliation: ‡London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K.
authors.forename: Maria
authors.initials: M
authors.lastname: Podinovskaia
authors.affiliation: §Department of Haematology, University College London, London WC1E 6BT, U.K.
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Evans
authors.affiliation: ∥Department of Chemistry, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Emma
authors.affiliation: ‡London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K.
authors.forename: Ulrich E
authors.initials: UE
authors.lastname: Schaible
authors.affiliation: §Department of Haematology, University College London, London WC1E 6BT, U.K.
authors.forename: John
authors.initials: J
authors.lastname: Porter
authors.affiliation: †King's College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, U.K.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Hider
mesh_terms: D005293:Ferritins / Q000097:blood / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D007501:Iron / Q000097:blood*; D007502:Iron Chelating Agents / Q000138:chemical synthesis / Q000737:chemistry*; D019190:Iron Overload / Q000097:blood; D011485:Protein Binding; D011714:Pyrans / Q000138:chemical synthesis / Q000737:chemistry; D011728:Pyridones / Q000138:chemical synthesis / Q000737:chemistry; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D007502:Iron Chelating Agents; D011714:Pyrans; D011728:Pyridones; D012709:Serum Albumin; D014168:Transferrin; D005293:Ferritins; D007501:Iron