title: Imaging of alkaline phosphatase activity in bone tissue.
abstract: The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP) using a small imaging molecule in combination with (19)Flourine magnetic resonance spectroscopic imaging ((19)FMRSI). 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using (19)Fluorine magnetic resonance spectroscopy ((19)FMRS) and (19)FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. (19)FMRS and (19)FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. (19)FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized (19)FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of (19)FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, (19)FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications.
authors.affiliation: Bone Cell Biology and Imaging Laboratory, Hospital for Special Surgery, New York, New York, United States of America.
authors.forename: Terence P
authors.initials: TP
authors.lastname: Gade
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Motley
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Beattie
authors.forename: Roshni
authors.initials: R
authors.lastname: Bhakta
authors.forename: Adele L
authors.initials: AL
authors.lastname: Boskey
authors.forename: Jason A
authors.initials: JA
authors.lastname: Koutcher
authors.forename: Philipp
authors.initials: P
authors.lastname: Mayer-Kuckuk
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D001842:Bone and Bones / Q000166:cytology* / Q000201:enzymology* / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D005110:Extracellular Space / Q000378:metabolism; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D051381:Rats
chemical_list: C119285:6,8-difluoro-4-methylumbelliferyl phosphate; D006923:Hymecromone; D000469:Alkaline Phosphatase