title: Mitochondrial localization and characterization of 99Tc-SESTAMIBI in heart cells by electron probe X-ray microanalysis and 99Tc-NMR spectroscopy.
abstract: As the development of targeted intracellular magnetic resonance contrast agents proceeds, techniques for the quantitative analysis of the subcellular compartmentation and characterization of metallopharmaceuticals must also advance. To this end, the subcellular distribution and chemical state of hexakis (2-methoxyisobutyl isonitrile) technetium-99 (99Tc-SESTAMIBI), the ground state of the organotechnetium radiopharmaceutical used for the noninvasive evaluation of myocardial perfusion and viability by scintigraphy, has been determined by a novel application of electron probe X-ray microanalysis (EPXMA) and 99Tc-NMR spectroscopy. In cryopreserved cultured chick heart cells equilibrated in 36 microM 99Tc-SESTAMIBI, EPXMA imaging of mitochondria yielded a respiratory uncoupler-sensitive characteristic 99Tc X-ray peak representing 32.0 +/- 2.9 nmoles Tc/mg dry weight, while EPXMA of cytoplasm or nucleus showed no peak significantly greater than the threshold detectability limit of approximately 1 nmole/mg dry weight. Technetium-99 NMR spectroscopy of heart cells equilibrated with 99Tc-SESTAMIBI showed a single peak at -45.5 ppm with no evidence of significant line broadening or chemical shift compared to aqueous chemical standards, indicating that the majority of the complex exists unbound within the mitochondrial matrix. These data quantitatively demonstrate the localization of this lipophilic cationic organometallic complex within mitochondria in situ, consistent with a sequestration mechanism dependent on membrane potentials. Furthermore, this study establishes the general feasibility of combined EPXMA and NMR spectroscopy for the direct subcellular localization and characterization of metallopharmaceuticals, techniques that are readily applicable to MR contrast agents.
authors.affiliation: Department of Radiology, Harvard Medical School, Brigham and Women's Hospital, Boston, MA 02115.
authors.forename: D P
authors.initials: DP
authors.lastname: Piwnica-Worms
authors.forename: J F
authors.initials: JF
authors.lastname: Kronauge
authors.forename: A
authors.initials: A
authors.lastname: LeFurgey
authors.forename: M
authors.initials: M
authors.lastname: Backus
authors.forename: D
authors.initials: D
authors.lastname: Hockett
authors.forename: P
authors.initials: P
authors.lastname: Ingram
authors.forename: M
authors.initials: M
authors.lastname: Lieberman
authors.forename: B L
authors.initials: BL
authors.lastname: Holman
authors.forename: A G
authors.initials: AG
authors.lastname: Jones
authors.forename: A
authors.initials: A
authors.lastname: Davison
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D002642:Chick Embryo; D015925:Cryopreservation; D003593:Cytoplasm / Q000737:chemistry; D004577:Electron Probe Microanalysis; D009682:Magnetic Resonance Spectroscopy; D008854:Microscopy, Electron; D008929:Mitochondria, Heart / Q000737:chemistry* / Q000648:ultrastructure; D009206:Myocardium / Q000648:ultrastructure; D011188:Potassium / Q000032:analysis; D012964:Sodium / Q000032:analysis; D013667:Technetium; D017256:Technetium Tc 99m Sestamibi
chemical_list: D013667:Technetium; D017256:Technetium Tc 99m Sestamibi; D012964:Sodium; D011188:Potassium