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 19Flourine magnetic resonance spectroscopic imaging (19FMRSI). 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 19Fluorine magnetic resonance spectroscopy (19FMRS) and 19FMRSI. 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. 19FMRS and 19FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. 19FMRS 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 19FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of 19FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, 19FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications.
Highlights
Imaging is one of the most important diagnostic tools in the clinical evaluation of patients with musculoskeletal conditions [1,2]
MRS Detection of Hydrolysis of difluoro-4methylumbelliferyl phosphate (DiFMUP) In Vitro To assess the feasibility of DiFMUP detection by 19Fluorine magnetic resonance spectroscopy (19FMRS), the compound was prepared in alkaline (ALP-buffer) or physiological solution
DiFMUP was activated via hydrolysis in the presence of purified alkaline phosphatase (ALP)
Summary
Imaging is one of the most important diagnostic tools in the clinical evaluation of patients with musculoskeletal conditions [1,2]. The majority of current clinical musculoskeletal imaging strategies are designed to detect abnormalities in bone morphology as indicators of disease. Radiographs and computed tomography measure tissue mineral density to visualize bone anatomy. Current MR imaging techniques measure proton (1H, water, lipid) relaxation and density to visualize musculoskeletal anatomy. Nuclear imaging agents, including Na18F for positron emission tomography and bisphosphonate tracers for bone scanning, principally target matrix/ mineral composition through physio-chemical uptake [3,4,5]. More recent experimental approaches focused on fluorescent bisphosphonates as static probes for optical detection and validated their potential as structural markers of bone turnover [6,7,8]
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