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Project Title:  Monitoring of Bone Loss Biomarkers in Human Sweat: A Non-Invasive, Time Efficient Means of Monitoring Bone Resorption Markers under Micro and Partial Gravity Loading Conditions Reduce
Fiscal Year: FY 2012 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 05/20/2008  
End Date: 05/20/2012  
Task Last Updated: 03/27/2013 
Download report in PDF pdf
Principal Investigator/Affiliation:   Clarke, Mark  Ph.D. / University of Houston 
Address:  Health and Human Performance 
3855 Holman St, Garrison Rm 104 
Houston , TX 77204 
Email: mclarke@mail.uh.edu 
Phone: 713-743-9854  
Congressional District: 18 
Web:  
Organization Type: UNIVERSITY 
Organization Name: University of Houston 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
O'Connor, Dan  University of Houston 
Project Information: Grant/Contract No. NNX08AQ37G 
Responsible Center: NASA JSC 
Grant Monitor: Maher, Jacilyn  
Center Contact:  
jacilyn.maher56@nasa.gov 
Solicitation / Funding Source: 2007 Crew Health NNJ07ZSA002N 
Grant/Contract No.: NNX08AQ37G 
Project Type: GROUND 
Flight Program:  
TechPort: Yes 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Human Research Program Elements: (1) HHC:Human Health Countermeasures
Human Research Program Risks: (1) Osteo:Risk Of Early Onset Osteoporosis Due To Spaceflight (No longer used, July 2020)
Human Research Program Gaps: (1) Osteo05:We need an inflight capability to monitor bone turnover and bone mass changes during spaceflight (IRP Rev E)
Flight Assignment/Project Notes: NOTE: End date is now 5/20/2012 (Ed., 11/14/2011)

NOTE: New end date is 11/20/2011 per NSSC information (Ed., 5/31/2011)

Task Description: We propose to validate that the rate and extent of unloading-induced bone loss in humans can be assessed by monitoring the levels of two bone resorption markers in sweat, namely ionized calcium and collagen break-down products. Initial testing will be carried out in a young healthy population (at rest and during activity) and then in a clinical population undergoing active bone loss, namely spinal cord injury patients. All groups will include both male and female participants. Biomarker concentration will be determined in contemporaneous samples of sweat, blood, and urine collected during both short (24 hr) and long-term studies (multiple sessions over a period of months) to define the relationship between biomarker levels in the respective biological samples. Several different sweat collection techniques will be investigated to determine the most appropriate and efficient means of sample collection suitable for deployment during a space flight mission. These experiments will also include investigation of the most appropriate biomarker analysis techniques that allow for future deployment in micro- or partial gravity environments. This near-real-time monitoring approach may also provide the information required to justify modifying an ineffective bone loss countermeasure prescription during a mission. One of the approaches tested will be a novel, micro-fabricated fluid collection capillary array, known as the micro-fabricated sweat patch (MSP) device, specifically developed for use in microgravity. The MSP technology was initially developed because of its potential to become an autonomous, solid-state collection/analysis device worn on the skin of an astronaut requiring little or no crew interaction to perform its monitoring function.

Research Impact/Earth Benefits: Loss of bone mass, density, and structural integrity is a significant health risk in a variety of populations such as the elderly, post-menopausal women, young female athletes, and astronauts. Such changes in overall bone quality lead to a greater risk of bone fracture and potentially a reduced rate of bone healing after injury. The ability to monitor biomarkers of bone remodeling (e.g., ionized calcium, collagen cross links) using sweat as an analytical sample provides an attractive alternative to the more invasive and costly measures presently employed such as a bone density scans by dual-energy X-ray absorptiometry (DXA), 24 hour urine collection protocols, or whole blood analyses. The development of a non-invasive, skin-mounted monitoring device which allows the quantitation of ionized calcium and/or collagen cross links in sweat will allow bone loss to be monitored in a wide variety of terrestrial populations that to date have not easily been monitored outside of a clinical setting. This particular project focuses on validating the concept that sweat analysis can be used as a non-invasive means of monitoring bone loss in crew members during periods of mechanical unloading under altered gravitational conditions. In addition, this project is also investigating the best technical approach to collecting a sweat sample which is specifically applicable to the space flight environment while utilizing well-accepted, clinically validated analytical methods. Development of a technology capable of real-time monitoring of biomarkers of bone loss that satisfies the criteria required for use in the space flight environment (i.e., non-invasive/non-intrusive, passive, small, light-weight, low power) has many direct applications in various populations here on Earth.

Task Progress & Bibliography Information FY2012 
Task Progress: The overall goal of this project was to validate the concept that the rate and extent of unloading-induced bone loss in humans can be assessed by monitoring the levels of two bone resorption markers in sweat, namely ionized calcium (Ca2+) and total collagen cross-links (T-CCL) (i.e., the pyridinium cross-links PYD & DPD) The original funded project plan called for a phased approach consisting of three phases; the first phase focused on selection of the most appropriate and efficient means of collecting a sweat sample from an individual compatible with the microgravity environment of space flight coupled with biochemical validation that these sweat samples contained bone resorption markers at levels capable of being detected using standard laboratory analysis techniques; the second phase focused on validation of the concept that bone resorption marker levels detected in sweat samples accurately and consistently reflected circulating levels and/or urine levels of these biomarkers; and the third phase focused on longitudinal assessment of bone resorption marker level in sweat, blood, and urine in young and old populations undergoing active bone formation or bone loss, respectively.

After successful completion of Phase I, preliminary data generated during Phase II indicated that the NASA criterion measure for bone loss during space flight (i.e., 24 hr urinary ionized calcium excretion), while related to calcium and T-CCL levels in sweat samples actively produced during defined exercise, were not predictive of 24 hr urine calcium excretion rates. After consultation and review of the preliminary Phase II results by representatives of the NASA-Johsnon Space Center-Human Research Program (JSC-HRP), the focus of Phase II was redirected to explore collection of a 24 hr sweat sample, rather than collection of a discrete, exercise-induced “active” sweat sample, to determine if a 24 hr integrated sweat sample was predictive of biomarker concentrations found in 24 hr urine samples. This redirection of effort required the identification and validation of additional commercially available absorbant materials which did not contain endogenous biomarker signal as well as a means of extracting the biomarkers from the absorbant material compatible with fluid handling limitations in the space flight environment. After identifying and developing such a collection method, this approach was then utilized to answer the question of whether or not biomarker levels in an integrated 24 hr sweat sample was predictive of those found in a concurrent 24 hr urine sample in a convenience of healthy individuals.

After successful completion of Phase II, the NASA-JSC-HRP program indicated that they wished to deploy the sweat monitoring technology in a NASA bed-rest campaigns being performed at the GCRC at University of Texas Medical Branch (UTMB) instead of in young and old subject populations as originally planned. Unfortunately however, during the last 9 months of the project NASA had to postpone bed-rest operations resulting in a joint decision by NASA-HRP and the Principal Investigator to utilize spinal-injured patients recruited from the Texas Medical Center (rather than NASA bed rest subjects) in which to test the 24 hr sweat monitoring technology as means of assessing bone loss. The resulting time delay surrounding availability of bed rest subjects and the subsequent decision to utilize spinal chord injury (SCI) patients, coupled with the additional requirement to seek Committee for the Protection of Human Subjects (CPHS) approval for testing in a new subject population resulted in NASA granting a one year no-cost extension to the project. Limited data gathered in the final year of the project provides evidence that biomarker levels in a 24 hr integrated sweat sample are predictive of those levels found in 24 hr urine samples in SCI patients. These data indicate that 24 hr sweat sample collection (using a collection and analysis scheme compatible with space flight operations) in a terrestrial human population undergoing active bone loss in a similar fashion to crew members during space flight is a non-invasive, time-efficient alternative to on-orbit 24 hr urine void collection as means of assessing biomarkers of bone loss. In addition, the ability to perform this type of sample collection using a microgravity compatible approach to liquid sample handling and the use of simple colorimetric based analysis techniques is of notable operational relevancy. Our data and validated methodologies suggest that sweat biomarker analysis (as an operationally compatible means of assessing and/or monitoring bone loss in crew members during space flight) should be considered for further development as a “real-time” analytical method for assessment of space flight-induced bone loss and a valid means of monitoring the efficacy of “in-flight” bone loss countermeasures.

[Editor's note 3/27/2013: No Task Book report received. Progress section and Bibliography compiled from PI's Final Technical Report submitted January 2013]

Bibliography Type: Description: (Last Updated: 03/08/2018)  Show Cumulative Bibliography Listing
 
Abstracts for Journals and Proceedings Clarke MS, Babcock LW, Diak D, O'Connor DP. "Sweat Analysis for Assessment of Bone Loss Biomarkers." 2012 NASA Human Research Program Investigators’ Workshop, Houston, TX, February 14-16, 2012.

2012 NASA Human Research Program Investigators’ Workshop, Houston, TX, February 14-16, 2012. , Feb-2012

Abstracts for Journals and Proceedings Clarke MS, Knoblauch MA, O'Connor DP. "Monitoring Biomarkers of Bone Loss in Human Sweat." 18th IAA Humans in Space Symposium, Houston, TX, April 11-15, 2011.

18th IAA Humans in Space Symposium, Houston, TX, April 11-15, 2011. , Apr-2011

Abstracts for Journals and Proceedings Clarke MS, Knoblauch MA, O'Connor DL. "Sweat and Biomarkers --can sweat be used to monitor biomarkers of bone loss?" 2010 NASA Human Research Program Investigators’ Workshop, Houston, TX, February 3-5, 2010.

2010 NASA Human Research Program Investigators’ Workshop, Houston, TX, February 3-5, 2010. , Feb-2010

Abstracts for Journals and Proceedings Clarke MS, Knoblauch MA, O'Connor DL. "Monitoring of biomarkers of bone loss in human sweat - a non-invasive, time efficient means of monitoring bone resorption markers under micro- and partial gravity loading conditions." 2009 NASA Human Research Program Investigators’ Workshop, League City, TX, February 2-4, 2009.

2009 NASA Human Research Program Investigators’ Workshop, League City, TX, February 2-4, 2009. , Feb-2009

NASA Technical Documents Clarke MS, Feeback DL. "Microgravity-Compatible Sweat Collection and Analysis Device." MSC-23625-1 NASA Tech Briefs. , Jul-2011
Project Title:  Monitoring of Bone Loss Biomarkers in Human Sweat: A Non-Invasive, Time Efficient Means of Monitoring Bone Resorption Markers under Micro and Partial Gravity Loading Conditions Reduce
Fiscal Year: FY 2011 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 05/20/2008  
End Date: 05/20/2012  
Task Last Updated: 10/26/2011 
Download report in PDF pdf
Principal Investigator/Affiliation:   Clarke, Mark  Ph.D. / University of Houston 
Address:  Health and Human Performance 
3855 Holman St, Garrison Rm 104 
Houston , TX 77204 
Email: mclarke@mail.uh.edu 
Phone: 713-743-9854  
Congressional District: 18 
Web:  
Organization Type: UNIVERSITY 
Organization Name: University of Houston 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
O'Connor, Dan  University of Houston 
Project Information: Grant/Contract No. NNX08AQ37G 
Responsible Center: NASA JSC 
Grant Monitor: Baumann, David  
Center Contact:  
david.k.baumann@nasa.gov 
Solicitation / Funding Source: 2007 Crew Health NNJ07ZSA002N 
Grant/Contract No.: NNX08AQ37G 
Project Type: GROUND 
Flight Program:  
TechPort: Yes 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Human Research Program Elements: (1) HHC:Human Health Countermeasures
Human Research Program Risks: (1) Osteo:Risk Of Early Onset Osteoporosis Due To Spaceflight (No longer used, July 2020)
Human Research Program Gaps: (1) Osteo05:We need an inflight capability to monitor bone turnover and bone mass changes during spaceflight (IRP Rev E)
Flight Assignment/Project Notes: NOTE: End date is now 5/20/2012 (Ed., 11/14/2011)

NOTE: New end date of 5/20/2012 is Pending as of 10/27/2011 (Ed.)

NOTE: New end date is 11/20/2011 per NSSC information (Ed., 5/31/2011)

Task Description: We propose to validate that the rate and extent of unloading-induced bone loss in humans can be assessed by monitoring the levels of two bone resorption markers in sweat, namely ionized calcium and collagen break-down products. Initial testing will be carried out in a young healthy population (at rest and during activity) and then in a clinical population undergoing active bone loss, namely spinal cord injury patients. All groups will include both male and female participants. Biomarker concentration will be determined in contemporaneous samples of sweat, blood and urine collected during both short (24 hr) and long-term studies (multiple sessions over a period of months) to define the relationship between biomarkers levels in the respective biological samples. Several different sweat collection techniques will be investigated to determine the most appropriate and efficient means of sample collection suitable for deployment during a space flight mission. These experiments will also include investigation of the most appropriate biomarker analysis techniques that allow for future deployment in micro- or partial gravity environments. This near-real-time monitoring approach may also provide the information required to justify modifying an ineffective bone loss countermeasure prescription during a mission. One of the approaches tested will be a novel, micro-fabricated fluid collection capillary array, known as the micro-fabricated sweat patch (MSP) device, specifically developed for use in microgravity. The MSP technology was initially developed because of its potential to become an autonomous, solid-state collection/analysis device worn on the skin of an astronaut requiring little or no crew interaction to perform its monitoring function.

Research Impact/Earth Benefits: Loss of bone mass, density and structural integrity is a significant health risk in a variety of populations such as the elderly, post-menopausal women, young female athletes and astronauts. Such changes in overall bone quality lead to a greater risk of bone fracture and potentially a reduced rate of bone healing after injury. The ability to monitor biomarkers of bone remodeling (e.g. ionized calcium, collagen cross links) using sweat as an analytical sample provides an attractive alternative to the more invasive and costly measures presently employed such as a bone density scans by DXA, 24 hour urine collection protocols or whole blood analyses. The development of a non-invasive, skin-mounted monitoring device which allows the quantitation of ionized calcium and/or collagen cross links in sweat will allow bone loss to be monitored in a wide variety of terrestrial populations that to date have not easily been monitored outside of a clinical setting. This particular project focuses on validating the concept that sweat analysis can be used as a non-invasive means of monitoring bone loss in crew members during periods of mechanical unloading under altered gravitational conditions. In addition, this project is also investigating the best technical approach to collecting a sweat sample which is specifically applicable to the space-flight environment while utilizing well-accepted, clinically validated analytical methods. Development of a technology capable of real-time monitoring of biomarkers of bone loss that satisfies the criteria required for use in the space-flight environment (i.e. non-invasive/non-intrusive, passive, small, light-weight, low power) has many direct applications in various populations here on Earth.

Task Progress & Bibliography Information FY2011 
Task Progress: Our initial studies in Year 1 of this project indicated that there was significant individual variation in the amount and rate of sweating observed between subjects based on: 1) the anatomical site sampled (i.e. fore-head, shoulder, fore-arm); 2) level of activity (i.e. “active” sweating during exercise; and 3) the concentration of biomarkers relative to the onset of “active” sweating. In addition, the existing sweat collection technology previously employed to collect sweat (i.e. commercial absorptive pad) exhibited significant analytical issues based on sub-optimal analyte retention and analyte extraction efficiency. To overcome these difficulties we developed a standardized sweat collection protocol in which subjects performed a defined exercise protocol at a fixed level of intensity (i.e. 30 min of aerobic exercise at an intensity of 40% of their age-predicted VO2max) under similar environmental conditions and hydration status. Sweat samples were collected at the fore-head using micro-fabricated polymer capillary arrays which collect sweat as an unadulterated liquid sample. This approach allows both determination of the volume of sweat produced and has the advantage of minimal residual analyte retention within the array due to its polymer construction unlike the commercially available absorptive sweat collection technology. Sweat samples were initially collected at discrete time intervals to determine when during exercise-induced sweating did biomarker concentration reach “steady-state” levels. These experiments indicated that biomarker concentrations (i.e. Ca2+ and T-CCL) in “active” sweat reached a “steady state” after 15 min of exercise period regardless of the volume of sweat produced by a particular individual. In addition, analyte concentration (i.e. T-CCL) in actively produced sweat was found to be highly correlated (R = 0.92) with plasma levels of the same analyte during the period sweat was being collected. However, comparison of analyte concentration produced during actively produced sweat and 24 hr urine voids in the same individual indicated, indicated that although related in a linear fashion (R = 0.61), sweat analyte excretion during the 15 min period of exercise was not predictive of total 24hr urine analyte excretion. As 24 hr urine void collection provides an integrated value for whole body biomarker excretion rates, comparison of the amount of biomarker excreted during a 15 min period of “active” sweating from 7.5cm of skin surface at the fore-head appears inappropriate as a means of predicting total calcium and T-CCL excreted in a 24 hr urine void.

As the overall goal of this project is to determine whether or not excretion of bone loss biomarkers in sweat is predictive of bone loss biomarker levels detected in 24 hr urine voids, we revisited the possibility of collecting a sweat sample over a 24 hr period in order to provide a more appropriate comparison. During Years 1 and 2 of this project we investigated the accuracy of the existing commercially available absorptive patch sweat collection technology with regard to analyte concentration relative to unadulterated liquid sweat samples collected in a contemporaneous fashion at the same anatomical site. These data indicated that the cellulose matrix of the commercially available absorptive patch posed significant analytical problems.

These issues were that the cellulose matrix: 1) contained significant amounts of endogenous calcium signal; 2) resulted in significant retention of sweat-derived T-CCL that could not be extracted from the matrix using the standard method (i.e. extraction in ddH2O); and 3) that extraction efficiency from the matrix was variable between identical samples. To address these analytical issues during Year 2 of this project we investigated the use of two new absorptive materials, a glass fiber material and a polypropylene woven material, for the collection of sweat over a 24 hr period. Our initial experiments focused on the extraction efficiency of four bone loss biomarkers of interest, namely calcium, T-CCL, NTx and osteocalcin (OST). We also modified the extraction protocol to utilize acidified ddH2O (pH 5.0) or a Tris buffer (pH 5.0) to enhance the solubility of these biomarkers in the extraction buffer and reduce ionic interactions within the matrix of the absorptive material. Unlike the original commercially available cellulose absorptive pad technology, efficient recovery of known amounts of all analytes was achieved using our modified extraction method from both the glass fiber and woven polypropylene materials.

During Year 2 of this project, utilizing glass fiber absorptive pads (7.5cm2 in area) and our modified extraction protocol, we collected sweat samples from a total of 18 normal healthy subjects over a period of 24 hr at three separate anatomical locations, the shoulder, hip and fore-arm. We also collected 24hr urine voids from these subjects during the same 24 hr period. When the total amount of calcium excreted in sweat over a 24 hr period into a 7.5cm2 glass fiber absorptive pad was compared to the total amount of calcium excreted in a 24hr urine void obtained from the same individual, our results indicate that there is a near linear correlation between the two values in sweat collected from the shoulder (R = 0.90) and fore-arm (R=0.86) and contemporaneous 24 hr urine voids. A similar relationship between 24hr sweat T-CCL and 24hr urine void T-CCL levels was observed at the shoulder (R = 0.99) in a sub-set of these subjects (N=6) for which this analysis has been completed. Our data indicate that calcium and T-CCL levels detected in 24hr sweat samples are highly correlated with levels detected in 24hr urine voids from healthy individuals, suggesting that bone loss biomarker concentration in the criterion sample (i.e. 24 hr urine void) can be accurately predicted from that found in a sweat sample collected over the same 24 hr period using an absorptive pad technology.

Year 3 of this project has focused on validating this approach to monitoring bone loss biomarkers in a terrestrial population undergoing active bone loss. Due to issues with gaining access to NASA bed rest subjects, and after agreement with HRP, we decided to switch our focus to a clinical population undergoing active bone loss, namely spinal cord injured patients. This change in focus and the subsequent time delay in obtaining CPHS approval for this study, added to difficulties in recruiting subjects based on our initial inclusion criteria (i.e. 6 – 18 month post injury, injury site T4 or below) necessitated a no cost extension to the project of 12 months (new project end date May 2012). We have now broadened our study inclusion criteria by including SCI patients who are more than 18 months post injury. To help in recruitment to our study, we have begun collaborating with an ongoing SCI rehabilitation study being carried out at UH which attracts SCI patients of all types. During Year 3 of the project we have also developed a new protocol suitable for deployment on-orbit for the extraction and collection of analytes from the absorptive sweat patch based on the Salivette™ technology previously validated for use on-orbit. We are also testing the ability of the iSTAT analysis technology (previously flown and validated on Shuttle and ISS) to perform Ca2+ analysis in sweat samples.

Bibliography Type: Description: (Last Updated: 03/08/2018)  Show Cumulative Bibliography Listing
 
 None in FY 2011
Project Title:  Monitoring of Bone Loss Biomarkers in Human Sweat: A Non-Invasive, Time Efficient Means of Monitoring Bone Resorption Markers under Micro and Partial Gravity Loading Conditions Reduce
Fiscal Year: FY 2010 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 05/20/2008  
End Date: 11/20/2011  
Task Last Updated: 06/23/2010 
Download report in PDF pdf
Principal Investigator/Affiliation:   Clarke, Mark  Ph.D. / University of Houston 
Address:  Health and Human Performance 
3855 Holman St, Garrison Rm 104 
Houston , TX 77204 
Email: mclarke@mail.uh.edu 
Phone: 713-743-9854  
Congressional District: 18 
Web:  
Organization Type: UNIVERSITY 
Organization Name: University of Houston 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
O'Connor, Dan  University of Houston 
Project Information: Grant/Contract No. NNX08AQ37G 
Responsible Center: NASA JSC 
Grant Monitor: Goodwin, Thomas  
Center Contact:  
thomas.j.goodwin@nasa.gov 
Solicitation / Funding Source: 2007 Crew Health NNJ07ZSA002N 
Grant/Contract No.: NNX08AQ37G 
Project Type: GROUND 
Flight Program:  
TechPort: Yes 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Human Research Program Elements: (1) HHC:Human Health Countermeasures
Human Research Program Risks: (1) Osteo:Risk Of Early Onset Osteoporosis Due To Spaceflight (No longer used, July 2020)
Human Research Program Gaps: (1) Osteo05:We need an inflight capability to monitor bone turnover and bone mass changes during spaceflight (IRP Rev E)
Flight Assignment/Project Notes: NOTE: New end date is 11/20/2011 per NSSC information (Ed., 5/31/2011)

Task Description: We propose to validate that the rate and extent of unloading-induced bone loss in humans can be assessed by monitoring the levels of two bone resorption markers in sweat, namely ionized calcium and collagen break-down products. Initial testing will be carried out in three populations (at rest and during activity) that have different constitutive levels of bone remodeling, namely young normal healthy individuals, air-force cadets undergoing regular heavy resistance training and healthy elderly individuals. All groups will include both male and female participants. Biomarker concentration will be determined in contemporaneous samples of sweat, blood and urine collected during both short (24 hr) and long-term studies (six months)to define the relationship between biomarkers levels in the respective biological samples. Bone mineral density (BMD) measures will be incorporated in the long-term studies to test the predictive value of sweat biomarkers with regard to overall bone remodeling. Future testing will utilize subjects undergoing bed-rest simulations of micro- or partial gravity loading conditions. Several different sweat collection techniques will be investigated to determine the most appropriate and efficient means of sample collection suitable for deployment during a space flight mission. These experiments will also include investigation of the most appropriate biomarker analysis techniques that allow for future deployment in micro- or partial gravity environments. This near-real-time monitoring approach may also provide the information required to justify modifying an ineffective bone loss countermeasure prescription during a mission. One of the approaches tested will be a novel, micro-fabricated fluid collection capillary array, known as the micro-fabricated sweat patch (MSP) device, specifically developed for use in microgravity. The MSP technology was initially developed because of its potential to become an autonomous, solid-state collection/analysis device worn on the skin of an astronaut requiring little or no crew interaction to perform its monitoring function.

Research Impact/Earth Benefits: Loss of bone mass, density and structural integrity is a significant health risk in a variety of populations such as the elderly, post-menopausal women, young female athletes and astronauts. Such changes in overall bone quality lead to a greater risk of bone fracture and potentially a reduced rate of bone healing after injury. The ability to monitor biomarkers of bone remodeling (e.g. ionized calcium, collagen cross links) using sweat as an analytical sample provides an attractive alternative to the more invasive and costly measures presently employed such as a bone density scans by DXA, 24 hour urine collection protocols or whole blood analyses. The development of a non-invasive, skin-mounted monitoring device which allows the quantitation of ionized calcium and/or collagen cross links in sweat will allow bone loss to be monitored in a wide variety of terrestrial populations that to date have not easily been monitored outside of a clinical setting. This particular project focuses on validating the concept that sweat analysis can be used as a non-invasive means of monitoring bone loss in crew members during periods of mechanical unloading under altered gravitational conditions. In addition, this project is also investigating the best technical approach to collecting a sweat sample which is specifically applicable to the space-flight environment while utilizing well-accepted, clinically validated analytical methods. Development of a technology capable of real-time monitoring of biomarkers of bone loss that satisfies the criteria required for use in the space-flight environment (i.e. non-invasive/non-intrusive, passive, small, light-weight, low power) has many direct applications in various populations here on Earth.

Task Progress & Bibliography Information FY2010 
Task Progress: Our initial studies in Year 1 of this project indicated that there was significant individual variation in the amount and rate of sweating observed between subjects based on: 1) the anatomical site sampled (i.e. fore-head, shoulder, fore-arm); 2) level of activity (i.e. “active” sweating during exercise; and 3) the concentration of biomarkers relative to the onset of “active” sweating. In addition, the existing sweat collection technology previously employed to collect sweat (i.e. absorptive pad) exhibited significant analytical issues based on sub-optimal analyte retention and analyte extraction efficiency.

To overcome these difficulties we developed a standardized sweat collection protocol in which subjects performed a defined exercise protocol at a fixed level of intensity (i.e. 30 min of aerobic exercise at an intensity of 40% of their age-predicted VO2max) under similar environmental conditions and hydration status. Sweat samples were collected at the fore-head using micro-fabricated polymer capillary arrays which collect sweat as an unadulterated liquid sample. This approach allows both determination of the volume of sweat produced and has the advantage of minimal residual analyte retention within the array due to its polymer construction unlike the existing absorptive sweat collection technology. Sweat samples were initially collected at discrete time intervals to determine when during exercise-induced sweating did biomarker concentration reach “steady-state” levels. These experiments indicated that biomarker concentrations (i.e. Ca2+ and T-CCL) in “active” sweat reached a “steady state” after 15 min of exercise period regardless of the volume of sweat produced by a particular individual. In addition, analyte concentration (i.e. T-CCL) in actively produced sweat was found to be highly correlated (R = 0.92) with plasma levels of the same analyte during the period sweat was being collected. However, comparison of analyte concentration produced during actively produced sweat and 24 hr urine voids in the same individual indicated, that although related in a linear fashion (R = 0.61), that sweat analyte excretion during the 15 min period of exercise was not predictive of total 24hr urine analyte excretion. As 24 hr urine void collection provides an integrated value for whole body biomarker excretion rates, comparison of the amount of biomarker excreted during a 15 min period of “active” sweating from 7.5cm of skin surface at the fore-head may be inappropriate and thus account for the poor correlations observed between total calcium and T-CCL in sweat versus 24 hr urine voids.

As the overall goal of this project is to determine whether or not excretion of bone loss biomarkers in sweat is predictive of bone loss biomarker levels detected in 24 hr urine voids, we revisited the possibility of collecting a sweat sample over a 24 hr period in order to provide a more appropriate comparison. During Year 1 of this project we investigated the accuracy of the existing absorptive patch sweat collection technology with regard to analyte concentration relative to unadulterated liquid sweat samples collected in a contemporaneous fashion at the same anatomical site. These data indicated that the cellulose matrix of the absorptive patch posed significant analytical problems. These issues were that the cellulose matrix: 1) contained significant amounts of endogenous calcium signal; 2) resulted in significant retention of sweat-derived T-CCL that could not be extracted from the matrix using the standard method (i.e. extraction in ddH2O); and 3) that extraction efficiency from the matrix was variable between identical samples.

To address these analytical issues we investigated the use of two new absorptive materials, a glass fiber material and a polypropylene woven material, for the collection of sweat over a 24 hr period. Our initial experiments focused on the extraction efficiency of four bone loss biomarkers of interest, namely calcium, T-CCL, NTx and osteocalcin (OST). We also modified the extraction protocol to utilize acidified ddH2O (pH 5.0) which theoretically would enhance the solubility of these biomarkers in the extraction buffer and reduce ionic interactions within the matrix of the absorptive material. Unlike the original cellulose absorptive pad technology, efficient recovery of known amounts of all analytes was achieved using our modified extraction method from both the glass fiber and woven polypropylene materials.

Utilizing glass fiber absorptive pads (7.5cm2 in area) and our modified extraction protocol, we have now collected sweat samples from subjects over a period of 24 hr at three separate anatomical locations, the shoulder, hip and fore-arm. We also collected 24hr urine voids from these subjects during the same 24 hr period. To date we have collected and analyzed samples from 8 subjects and have an additional 12 subjects enrolled in the study. When the total amount of calcium excreted in sweat over a 24 hr period into a 7.5cm2 glass fiber absorptive pad was compared to the total amount of calcium excreted in a 24hr urine void obtained from the same individual, our preliminary results indicate that there is a near linear correlation between the two values in sweat collected from the shoulder (R = 0.94) and fore-arm (R=0.91) and contemporaneous 24 hr urine voids. A similar relationship between 24hr sweat T-CCL and 24hr urine void T-CCL levels was observed at the shoulder (R = 0.99) in these subjects.

Our data concerning the relationship between both calcium and T-CCL levels detected in 24hr sweat samples as compared to those detected in 24hr urine voids from the same individual suggests that bone loss biomarker concentration in the criterion sample (i.e. 24 hr urine void) can be accurately predicted from that found in a sweat sample collected over the same 24 hr period using an absorptive pad technology. The use of a glass fiber absorptive pad and modified extraction buffer ensures consistent and efficient analyte extraction from the pad. We are presently expanding the number of subjects in our data set and also including NTx analysis in that data set. From an operational perspective, we are also investigating a number of separate approaches to performing the liquid-based extraction procedure under microgravity conditions and testing the ability of the iSTAT analysis technology (previously flown and validated on Shuttle and ISS) to perform the sample analysis.

Bibliography Type: Description: (Last Updated: 03/08/2018)  Show Cumulative Bibliography Listing
 
 None in FY 2010
Project Title:  Monitoring of Bone Loss Biomarkers in Human Sweat: A Non-Invasive, Time Efficient Means of Monitoring Bone Resorption Markers under Micro and Partial Gravity Loading Conditions Reduce
Fiscal Year: FY 2009 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 05/20/2008  
End Date: 05/19/2011  
Task Last Updated: 04/09/2009 
Download report in PDF pdf
Principal Investigator/Affiliation:   Clarke, Mark  Ph.D. / University of Houston 
Address:  Health and Human Performance 
3855 Holman St, Garrison Rm 104 
Houston , TX 77204 
Email: mclarke@mail.uh.edu 
Phone: 713-743-9854  
Congressional District: 18 
Web:  
Organization Type: UNIVERSITY 
Organization Name: University of Houston 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
O'Connor, Dan  University of Houston 
Project Information: Grant/Contract No. NNX08AQ37G 
Responsible Center: NASA JSC 
Grant Monitor: Meck, J@n  
Center Contact: 281-244-5405 
janice.v.meck@nasa.gov 
Solicitation / Funding Source: 2007 Crew Health NNJ07ZSA002N 
Grant/Contract No.: NNX08AQ37G 
Project Type: GROUND 
Flight Program:  
TechPort: Yes 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Human Research Program Elements: (1) HHC:Human Health Countermeasures
Human Research Program Risks: (1) Osteo:Risk Of Early Onset Osteoporosis Due To Spaceflight (No longer used, July 2020)
Human Research Program Gaps: (1) Osteo05:We need an inflight capability to monitor bone turnover and bone mass changes during spaceflight (IRP Rev E)
Task Description: We propose to validate that the rate and extent of unloading-induced bone loss in humans can be assessed by monitoring the levels of two bone resorption markers in sweat, namely ionized calcium and collagen break-down products. Initial testing will be carried out in three populations (at rest and during activity) that have different constitutive levels of bone remodeling, namely young normal healthy individuals, air-force cadets undergoing regular heavy resistance training and healthy elderly individuals. All groups will include both male and female participants. Biomarker concentration will be determined in contemporaneous samples of sweat, blood and urine collected during both short (24 hr) and long-term studies (six months)to define the relationship between biomarkers levels in the respective biological samples. Bone mineral density (BMD) measures will be incorporated in the long-term studies to test the predictive value of sweat biomarkers with regard to overall bone remodeling. Future testing will utilize subjects undergoing bed-rest simulations of micro- or partial gravity loading conditions. Several different sweat collection techniques will be investigated to determine the most appropriate and efficient means of sample collection suitable for deployment during a space flight mission. These experiments will also include investigation of the most appropriate biomarker analysis techniques that allow for future deployment in micro- or partial gravity environments. This near-real-time monitoring approach may also provide the information required to justify modifying an ineffective bone loss countermeasure prescription during a mission. One of the approaches tested will be a novel, micro-fabricated fluid collection capillary array, known as the micro-fabricated sweat patch (MSP) device, specifically developed for use in microgravity. The MSP technology was initially developed because of its potential to become an autonomous, solid-state collection/analysis device worn on the skin of an astronaut requiring little or no crew interaction to perform its monitoring function.

Research Impact/Earth Benefits: Loss of bone mass, density and structural integrity is a significant health risk in a variety of populations such as the elderly, post-menopausal women, young female athletes and astronauts. Such changes in overall bone quality lead to a greater risk of bone fracture and potentially a reduced rate of bone healing after injury. The ability to monitor biomarkers of bone remodeling (e.g. ionized calcium, collagen cross links) using sweat as an analytical sample provides an attractive alternative to the more invasive and costly measures presently employed such as a bone density scans by DXA, 24 hour urine collection protocols or whole blood analyses. The development of a non-invasive, skin-mounted monitoring device which allows the quantitation of ionized calcium and/or collagen cross links in sweat will allow bone loss to be monitored in a wide variety of terrestrial populations that to date have not easily been monitored outside of a clinical setting. This particular project focuses on validating the concept that sweat analysis can be used as a non-invasive means of monitoring bone loss in crew members during periods of mechanical unloading under altered gravitational conditions. In addition, this project is also investigating the best technical approach to collecting a sweat sample which is specifically applicable to the space-flight environment while utilizing well-accepted, clinically validated analytical methods. Development of a technology capable of real-time monitoring of biomarkers of bone loss that satisfies the criteria required for use in the space-flight environment (i.e. non-invasive/non-intrusive, passive, small, light-weight, low power) has many direct applications in various populations here on Earth.

Task Progress & Bibliography Information FY2009 
Task Progress: During the first nine months of this project we have demonstrated definitively that human sweat contains significant amounts of both ionized calcium and total collagen cross-links that can be detected using standard analytical laboratory techniques. In addition, calcium levels in sweat samples approach that found in urine when sweat is produced under active sweating conditions. We have also discovered significant technical issues with the use of the commercial OsteoPatch/Pharmchek (OPD) technology for collecting sweat samples, namely that (1) the device contains significant amounts of endogenous calcium within the evaporative pad and (2) non-uniform extraction of both calcium and T-CCL occurs from the evaporative pad during “reconstitution” of sweat constitutents. Tasks 1-3 for Year One are essentially complete. Additional data will be gathered using our newest batch of microfabricated sweat patch (MSP) devices with regard to sweating rate to augment the data already gathered for Task 3. Task 4 (i.e. comparison of biomarker concentration in urine and sweat) is ongoing and will now focus on samples collected using the MSP technology due to the technical issues identified with the OPD technology and the unsuitability of the catch pocket device (CPD) technology for deployment in altered gravity conditions.

Bibliography Type: Description: (Last Updated: 03/08/2018)  Show Cumulative Bibliography Listing
 
 None in FY 2009
Project Title:  Monitoring of Bone Loss Biomarkers in Human Sweat: A Non-Invasive, Time Efficient Means of Monitoring Bone Resorption Markers under Micro and Partial Gravity Loading Conditions Reduce
Fiscal Year: FY 2008 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 05/20/2008  
End Date: 05/19/2011  
Task Last Updated: 06/26/2008 
Download report in PDF pdf
Principal Investigator/Affiliation:   Clarke, Mark  Ph.D. / University of Houston 
Address:  Health and Human Performance 
3855 Holman St, Garrison Rm 104 
Houston , TX 77204 
Email: mclarke@mail.uh.edu 
Phone: 713-743-9854  
Congressional District: 18 
Web:  
Organization Type: UNIVERSITY 
Organization Name: University of Houston 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
O'Connor, Dan  University of Houston 
Project Information: Grant/Contract No. NNX08AQ37G 
Responsible Center: NASA JSC 
Grant Monitor:  
Center Contact:   
Solicitation / Funding Source: 2007 Crew Health NNJ07ZSA002N 
Grant/Contract No.: NNX08AQ37G 
Project Type: GROUND 
Flight Program:  
TechPort: Yes 
No. of Post Docs:  
No. of PhD Candidates:  
No. of Master's Candidates:  
No. of Bachelor's Candidates:  
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Human Research Program Elements: (1) HHC:Human Health Countermeasures
Human Research Program Risks: (1) Osteo:Risk Of Early Onset Osteoporosis Due To Spaceflight (No longer used, July 2020)
Human Research Program Gaps: (1) Osteo05:We need an inflight capability to monitor bone turnover and bone mass changes during spaceflight (IRP Rev E)
Task Description: We propose to validate that the rate and extent of unloading-induced bone loss in humans can be assessed by monitoring the levels of two bone resorption markers in sweat, namely ionized calcium and collagen break-down products. Initial testing will be carried out in three populations (at rest and during activity) that have different constitutive levels of bone remodeling, namely young normal healthy individuals, air-force cadets undergoing regular heavy resistance training and healthy elderly individuals. All groups will include both male and female participants. Biomarker concentration will be determined in contemporaneous samples of sweat, blood and urine collected during both short (24 hr) and long-term studies (six months)to define the relationship between biomarkers levels in the respective biological samples. Bone mineral density (BMD) measures will be incorporated in the long-term studies to test the predictive value of sweat biomarkers with regard to overall bone remodeling. Future testing will utilize subjects undergoing bed-rest simulations of micro- or partial gravity loading conditions. Several different sweat collection techniques will be investigated to determine the most appropriate and efficient means of sample collection suitable for deployment during a space flight mission. These experiments will also include investigation of the most appropriate biomarker analysis techniques that allow for future deployment in micro- or partial gravity environments. This near-real-time monitoring approach may also provide the information required to justify modifying an ineffective bone loss countermeasure prescription during a mission. One of the approaches tested will be a novel, micro-fabricated fluid collection capillary array, known as the micro-fabricated sweat patch (MSP) device, specifically developed for use in microgravity. The MSP technology was initially developed because of its potential to become an autonomous, solid-state collection/analysis device worn on the skin of an astronaut requiring little or no crew interaction to perform its monitoring function.

Research Impact/Earth Benefits: 0

Task Progress & Bibliography Information FY2008 
Task Progress: New project for FY2008.

Bibliography Type: Description: (Last Updated: 03/08/2018)  Show Cumulative Bibliography Listing
 
 None in FY 2008