Development of a quantification method for adenosine in tumors by LC-MS/MS with dansyl chloride derivatization.

Adenosine is known to be an important signaling molecule in many physiological processes and has recently been shown to be an important molecule in oncology. A fit for purpose method has been developed for the quantification of adenosine in murine tumor samples using pre-column derivatization and liquid chromatography-mass spectrometry (LC-MS/MS). To overcome adenosine quantification challenges, derivatization with dansyl chloride was employed. This derivatization technique, following protein precipitation and liquid-liquid extraction, improved the sensitivity and selectivity of adenosine in tumor samples through the reduction of endogenous interference and matrix effects. This method utilizes a mouse plasma calibration curve, qualified over a range of 0.019 µM-37 µM. The 15 min derivatization incubation time and 1 min chromatographic run time allow for higher throughput. The following established method overcomes challenges associated with the quantification of low molecular weight, polar, endogenous molecules, such as adenosine, using derivatization and LC-MS/MS. With the additional analysis of murine tumors, this method will contribute to the understanding of the impact adenosine plays in the tumor microenvironment and the bearing it has on targeted cancer therapies.

Material properties of interstitial lamellae reflect local strain environments.

The objective of this study was to determine if the material properties of bone at the lamellar level are related to the predominant mode and magnitude of mechanical strain experienced in situ. The tibia and first metatarsal bones of five unpaired cadaveric lower extremities were instrumented with strain gauge rosettes and subjected to repeated loading trials in an apparatus that replicates the muscle forces and external loads experienced by the foot and shank while walking. The spatial distributions of axial strain within diaphyseal cross-sections taken from each bone were subsequently determined. Nanoindentation measurements were then performed on the same cross-sections to determine the compressive elastic moduli of individual lamellae located within osteonal, interstitial, and outer circumferential microstructures. Twenty percent of the variance in interstitial elastic modulus within cross-sections of diaphyseal bone was explained by local strain magnitude. Lamellae residing in regions of compressive strain displayed significantly higher compressive elastic modulus values than those located in predominantly tensile regions (19.9 +/- 1.6 GPa compared to 17.9 +/- 1.7 GPa, p < 0.05). Elastic moduli of interstitial lamellae were 11% greater than those of osteonal or outer circumferential lamellae, irrespective of strain or anatomical location (p < 0.001). Differences exist in the material properties of individual bone lamellae located within different anatomical regions and different microstructures, and these differences are related to the distribution of axial strain. These findings suggest that mechanical strain, or another closely related variable, may influence the design and ultimate mechanical behavior of the extra-cellular matrix found in lamellar bone. This tissue heterogeneity is of potential importance in bone fragility and adaptation.