Imaging of Alkaline Phosphatase Activity in Bone Tissue

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

ALP-dependent DiFMUP activation on the surface of ALP-positive osteoblastic bone cells and in the presence of rat tibial bone.

<p>A: Histochemistry detected ALP expression as violet-red staining on 7F2 cells as shown in low and high magnification images 1 and 2, respectively. In contrast, no ALP expression was detectable on the MC3T3-E1#4 cells shown also in low (3) and high magnification (4). B: Activation (hydrolysis) of DiFMUP occurred on 7F2 cells (closed squares), but not on ALP-negative MC3T3-E1#4 cells (open triangles). No-cell background (Bkg) measurements are also presented (open circles). C: Following the separation of cells (CE) and medium (M) and a single wash step (W), the vast majority of the hydrolysis product was found in the medium. Mean values and standard deviations are shown (n = 3). D: DiFMUP in physiological solution was activated in a time-dependent fashion in the presence of highly purified rat tibia bone (closed squares). Addition of the ALP inhibitor levamisole reduced DiFMUP activation (open squares). E: DiFMUP in alkaline solution was activated by a single highly purified tibia bone chip (closed squares). Presence of levamisole significantly suppressed the activation (open squares). Mean values and standard deviations are shown (n = 3).</p

Non-invasive imaging of ALP activity in rat tibia cortical bone.

<p>A: RARE ¹H images of the bone sample anatomy including the rat tibia cortical bone core within the glass vial. B and C: ¹⁹FMRSI-derived parametric maps of regional DiFMUP and hydrolysis product concentrations overlaid onto RARE ¹H images of the bone sample (B and C, respectively). D: ¹⁹FMRSI-derived parametric maps of regional ALP concentration and activity overlaid onto RARE ¹H images of the bone sample.</p

Pharmacokinetics of ALP-dependent DiFMUP activation in the presence of rat tibial bone.

<p>A: Representative serial ¹⁹FMR spectra of DiFMUP and its hydrolysis product in the presence of rat bone. Spectral acquisition was initiated 6 minutes after addition of DiFMUP (20–25 mM range) to highly purified rat bone chips and serial spectra were acquired with 10 min temporal resolution. Peak assignments are as described for previously demonstrated spectra including (1) sodium fluoride, (2,4) DiFMUP and (3,5) DiFMUP hydrolysis product. B: The measured and modeled time course kinetics of unbound DiFMUP (x, —) and its hydrolysis product (x, —) for the ¹⁹FMR spectra seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022608#pone-0022608-g003" target="_blank">Fig. 3A</a>. The modeled ALP bound DiFMUP (—) is also shown for comparison.</p

Basic concept and specific detection of the ALP substrate DiFMUP by magnetic resonance spectroscopy.

<p>A: In this study, imaging is based on the use of DiFMUP. This small imaging molecule prototype contains two MR-detectable fluorine atoms in direct vicinity to a phosphate group. Prior to hydrolyzation by ALP, DiFMUP is expected to yield a spectrum characteristic of two discrete signals from the fluorine atoms since the molecule is not symmetrical, and hence the fluorine atoms exist in distinct chemical environments. If ALP is present and catalyzes the exchange of the phosphate to a hydroxy group, a significant change in the environment next to the fluorine atoms occurs and is expected to result in a chemical shift variation of the fluorine signals that is detectable by ¹⁹FMRSI. An added advantage of DiFMUP is the fluorescent property of its hydrolysis product; this allows for the convenient measurement of DiFMUP activation. This feature is not essential for the presented imaging paradigm, however it is used to monitor ALP activity in several subsequent figures. B: The two-peak MR signal of DiFMUP at alkaline pH 9.8 relative to a sodium fluoride (NaF) standard. C: An identical DiFMUP spectrum characteristic was observed under physiological pH 7.4 conditions. D and E: The hydrolysis product of DiFMUP exhibits fluorescence properties, which allows one to record DiFMUP activation. Substantial activation of 50 µM (D) and 5 mM (E) DiFMUP in the presence of ALP was seen in both alkaline and physiological solutions. F and G represent MR spectra of DiFMUP and its hydrolysis product, respectively. The discrete spectrum of the hydrolysis product is seen including resonances from non-equivalent fluorine atoms at −17 ppm and −41 ppm relative to the sodium fluoride reference resonance.</p