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At present, post-flight measurements are used to establish the effectiveness of the spaceflight bone loss and kidney stone prevention program. A preventive approach would be preferable, where ongoing in-flight measurements provide feedback on countermeasure effectiveness, allowing for adjustments in the countermeasure program during flight. Urinary calcium excretion is a reliable indicator of bone loss and kidney stone formation risk and is relatively easy to quantify. Urinary calcium levels increase profoundly in space. Recorded concentrations from the Skylab mission have shown 2-to-4-fold increases relative to preflight baselines. Urinary calcium excretion in clinical labs is often measured by collecting urine over a 24-hour period, but calcium concentration measurements taken from just the first void of the day provide similar information. Additionally, spot measurements of urinary calcium taken when an astronaut is voiding throughout the day provide key operational information with minimal impact on crew time, power, or stowage.
We developed a device that measures urinary calcium concentration fluorimetrically using the marker calcein. Calcium binds with calcein to form a fluorescing complex, and the magnitude of the fluorescence signal is proportional to the calcium concentration of the sample. The assay we developed uses an ultra-compact disposable assay: a small capillary tube (an optrode) with a fixed amount of calcein reagent coated onto the interior wall. 20 uL of urine is drawn into the capillary tube which is then inserted into a compact handheld fluorimeter to measure urinary calcium concentration. The device reports the optical signal as a voltage on its LCD display, which can then be converted to the equivalent calcium concentration based on a calibration fit.
In this study, the prototype fluorometer and optrode system were tested on urine samples to assess accuracy. 42 volunteers were recruited into the study and provided urine samples. Each urine sample was analyzed using a hospital clinical analyzer (Roche Cobas) for urinary calcium and urinary magnesium concentration and with the prototype for urinary calcium concentration. Using samples from the first 22 participants, we determined the correlation between the hospital analyzer and our prototype device to be R2=0.79 when excluding samples with high magnesium outliers (those greater than 120 mg/L magnesium concentration). Without excluding outliers, the correlation drops to 0.55. Magnesium reduces the accuracy of the prototype by interfering with the creation of calcein-calcium fluorescing complex. The intensity of the calcein-calcium fluorescence is the basis of the device reported urinary calcium concentration value. Magnesium interference can be suppressed by alkalizing the binding environment.
With the remaining 20 samples, we carefully alkalized the urine samples to a pH of 13.5 before measuring with the prototype. With this adjusted method the correlation improved from 0.55 to 0.67 when including all samples. With high magnesium outliers removed, correlation was R2=0.78, a level similar to before implementing the careful alkalizing procedure. From our correlation analysis, we have noticed that the prototype device tends to read lower than the clinical analyzer. In a highly alkaline environment, calcium may precipitate out of the solution and can suppress the device reading. In the Biosatellite missions where a similar technology was used, potassium citrate was used to prevent precipitation of calcium. We studied the effect of potassium citrate and did not see a dramatic improvement with the addition of this reagent.
Overall, the study showed a good correlation between the calcium measurements from the optrode and from the clinical analyzer (R2=0.79). The main issue is measurement range. Device accuracy improvements can be gained by extending the linear range of the device to minimize or remove the need to dilute the sample to within the current measurable range of the device. This can be accomplished by loading more reagent into the capillary tubes, or to manufacture different capillary tubes for different concentration ranges (e.g., tubes for the 0-30 mg/L range, tubes for the 30-60 mg/L range).
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Abstracts for Journals and Proceedings
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Lan L, Knaus DA, Devoy C, Phillips SD, Fellows AM, Buckey JC. "Ultra-Compact Device for Monitoring Bone Loss and Kidney Stone Risk." Presented at the 2021 NASA Human Research Program Investigators’ Workshop, Virtual, February 1-4, 2021. Abstracts. 2021 NASA Human Research Program Investigators’ Workshop, Virtual, February 1-4, 2021. , Feb-2021
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Articles in Peer-reviewed Journals
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Thamer S, Buckey JC Jr. "First void urinary calcium for tracking bone loss and kidney stone risk in space." Aerosp Med Hum Perform. 2022 Jul;93(7):546-50. https://doi.org/10.3357/AMHP.5979.2022 ; PMID: 35859310 , Jul-2022
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