Clinical trial NCT02580604
Conditions: Osteoporosis
Sponsor: University of Colorado, Denver
Start date: 2015-11-01; Completion date: 2025-07-31
Enrollment: 12 participants; Allocation: NON_RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
Declines in serum calcium during exercise may cause increases in markers of bone resorption. This study will determine if preventing the decline serum ionized calcium experienced at the onset of exercise through the use of a "calcium clamp" also prevents increases in blood biomarkers associated with bone resorption.

Detailed description:
Participants will undergo two identical walking exercise sessions on a treadmill. One session will occur until continuous calcium infusion and the other will be under continuous saline infusion. C-telopeptide, parathyroid hormone, total calcium, and serum ionized calcium will be monitored to distinguish differences in bone activity under the two conditions.

Arms: arm 1: All participants will have a continuous calcium infusion during exercise at their first collection visit. The second collection visit will always be a volume-matched half-normal saline infusion.
Interventions: intervention 1: Calcium Chloride/Calcium Gluconate intervention 2: Half-Normal Saline
Intervention descriptions: intervention 1: Calcium (as either calcium chloride or calcium gluconate in half normal saline) will be delivered via IV during exercise to keep serum ionized calcium levels above baseline. The clamp is modeled after the hyperglycemic glucose clamp. intervention 2: This is the placebo comparative infusion. Half-normal saline will be infused at the second collection visit following the identical infusion schedule determined during the calcium infusion visit.

Protocol documents:
Prot_SAP_000.pdf: 
COMIRB Protocol 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX: F-490 TELEPHONE: 303-724-1055 FAX: 303-724-0990 
Project Title: PTH And Calcium Responses to Exercise in Older Adults Experiment 2 (PACE 
Sr. 2)  
Principal Investigators: Sarah Wherry, PhD & Wendy Kohrt, PhD 
Version Date: 05/13/2024 
 
1. Specific aims and hypotheses 
 
Physical activities that load the skeleton through ground- and joint-reaction forces are essential 
to the maintenance of bone health. Although exercise training is typically thought to have 
favorable skeletal effects, there is evidence that it can sometimes lead to a decrease in bone 
mineral density (BMD). For example, we found a decline in hip BMD of -1.5% in male road 
cyclists over a year of training and competition,1 a rate of loss similar to that of postmenopausal 
women. The decrease was not explained by changes in body weight or composition. Although 
cycling may not be an optimal bone-building activity because it is weight-supported, rather than 
weight-bearing, it remains unclear why it would cause an accelerated loss of BMD. An exercise 
associated loss of bone mass has also been observed in response to weight-bearing exercise. 
Male college basketball players were found to have a decrease in bone mass over a year of 
training and competition.2 Interestingly, the loss was reversed the following year when additional 
calcium (Ca) supplementation was provided during practices. The mechanisms by which exercise 
training sometimes results in a loss of BMD, even when 
loading forces are seemingly high (e.g., basketball), are 
not known, but could include such possibilities as low 
energy availability, hypogonadism, metabolic acidosis, 
excess cortisol exposure, or disrupted Ca metabolism. 
The global aim of the proposed research is to 
investigate a novel and plausible mechanism for 
exercise-related bone loss. We postulate that the 
disruption of Ca homeostasis during acute exercise is 
a trigger for the activation of bone resorption. The 
working model (Fig 1) portends that excessive dermal Ca 
loss (i.e., sweating) causes a decline in serum ionized Ca 
(iCa; the unbound fraction) and triggers an acute increase 
in parathyroid hormone (PTH).3 PTH can defend serum 
Ca through multiple pathways, including reduced urinary Ca excretion, increased intestinal Ca 
absorption, and increased bone resorption to mobilize skeletal Ca. If an increase in bone 
resorption occurs repeatedly over multiple exercise sessions (i.e., exercise training), and is not 
accompanied by appropriate loading forces to stimulate bone formation, this could lead to a 
decrease in BMD over time (or diminish the increase in BMD).  
 
 
 
 


1.1 Overarching Hypothesis 
 
The overarching hypothesis for this line of research is that exercise that causes a decline in 
serum Ca as a result of dermal Ca loss stimulates bone resorption via an increase in 
PTH. The working model for this line of research assumes that an exercise bout can activate 
both bone formation and resorption through distinct mechanisms. Whereas the loading 
characteristics of an activity are the primary determinant of how effectively bone formation is 
stimulated,2 the metabolic characteristics of an activity may determine the extent to which bone 
resorption is stimulated. Under this paradigm, the net effect of exercise training on BMD would 
reflect the cumulative effects of how individual exercise bouts disrupt the balance between bone 
resorption and formation. Although many studies have assessed changes in markers of bone 
resorption and formation from the beginning to the end of an exercise training intervention 
lasting weeks to months, there is a paucity of knowledge of the acute effects of exercise on bone 
metabolism in humans. We will target this knowledge gap through experiments to exploit the 
working model by which we postulate exercise activates bone resorption (Fig 1). If the proposed 
experiment supports the global hypothesis, the long-term goal will be to determine whether the 
acute activation of bone resorption in response to exercise compromises the expected increases 
in BMD in response to exercise training. 
 
1.2 Specific Aims (SA) and Hypotheses (H) 
 
SA2: Determine whether preventing the decline in 
serum iCa during exercise via intravenous 
Caadministration (i.e., iCa clamp) prevents an 
increase in serum PTH and c-telopeptide (CTX). 
EXP2 will manipulate serum Ca level during 
exercise to determine whether this is the trigger for 
downstream steps in Figure 1. This will be achieved 
by having participants perform two identical 
exercise bouts under conditions of saline (Fig 2, left) 
vs Ca infusion (Fig 2, right). 
 
H2: When compared with saline infusion, 
intravenous Ca infusion during exercise to prevent a 
decline in serum iCa will attenuate the increases in 
serum PTH (primary) and CTX (secondary). 
 
Interpretation: Our expectation is that preventing a 
decline in serum iCa during exercise will prevent the 
increase in PTH. The goal of the iCa clamp will be 
to increase serum iCa by ~0.10 mg/dL above the pre-exercise level and maintain this during 
exercise; this should generate a decrease in PTH (Fig 2, right). If preventing the decline in serum 
iCa during exercise is not fully effective in preventing the increases in serum PTH and CTX, this 
will suggest that other mechanisms, such as increased metabolic acidosis,4 contribute to the 
stimulation of PTH during exercise. Future experiments would challenge this possibility. 
 


2. Significance of this research 
 
Exercise is recommended for both the prevention and treatment of osteoporosis. Although there 
have been no randomized controlled trials to document the anti-fracture efficacy of exercise, 
animal studies demonstrate that even small increases in bone mass and density (4-6%) in 
response to mechanical loading generate very large improvements in bone strength (60-90%).5 
This is in contrast to pharmacologic therapies with proven anti-fracture efficacy, which generate 
increases in bone strength (10-20%) in animals that are only proportional to the increases in bone 
mass.6,7 Such findings suggest that exercise is an important strategy for building and maintaining 
bone strength. Accordingly, identifying novel factors that either compromise or enhance the 
skeletal adaptations to exercise is of high priority. 
 
It is our hypothesis that the beneficial effects of exercise on BMD in humans may be attenuated 
by metabolic responses to exercise, and specifically the disruption of Ca homeostasis under 
conditions of high dermal Ca loss. Importantly, despite the vast literature on the effects of 
exercise on bone in rodents, it is unlikely the proposed mechanism has ever been considered 
because of the lack of a sweating response in rodents. However, as will be discussed further in 
the Research Strategy section, there is compelling preliminary evidence from our lab and from 
others to support the proposed mechanism as a modifier of bone metabolism in humans. 
 
The new concept being advanced is that the skeletal response to exercise is determined by 
both the mechanical loading characteristics and metabolic characteristics of the exercise. 
Our premise is that exercise can acutely activate both bone formation and bone resorption, and 
that it is the balance of these processes with repeated exercise bouts that is a determinant of the 
BMD adaptation to exercise training. Under this paradigm, activities such as basketball, soccer, 
and cycling could all have similar metabolic characteristics (i.e., could trigger the cascade in Fig 
1), but the more favorable loading characteristics of basketball and soccer could explain the 
higher BMD in such athletes than in cyclists.8 The proposed disruption of Ca homeostasis 
during vigorous exercise training could underlie the increased risk of stress fracture during basic 
training in military recruits.9 This is supported by the finding that Ca (2000 mg/d) and vitamin D 
(800 IU/d) supplements taken with morning and evening meals reduced the incidence of stress 
fractures in Navy recruits.9 The proposed experiments will develop mechanistic evidence for this 
contention and could lead to improved recommendations regarding the timing and dose of Ca 
supplementation to prevent unfavorable bone metabolism responses to exercise. We believe that 
acute increases in PTH and bone resorption during and after exercise could increase risk for 
stress fracture and diminish the potential benefits of exercise on BMD. However, we also 
recognize that transient increases in PTH in response to exercise could have favorable effects on 
bone metabolism, similar to the benefits of transient increases in PTH with pharmacologic 
therapy.10-12 In future intervention trials, it will be important to advance the proposed acute 
exercise studies to more chronic interventions that challenge our hypotheses. 
 
 
 
 
 
 


3. Research Strategy 
 
SA: Determine whether preventing the decline in serum iCa during exercise via 
intravenous Ca administration (i.e., iCa clamp) prevents an increase in serum PTH and 
CTX. The experiment will manipulate serum iCa level during exercise to determine 
whether this is the trigger for downstream steps in Fig 1. 
 
Rationale 
 
The goal is to isolate the decline in 
serum iCa during exercise as the 
trigger for the increase in PTH. In 
healthy adults, Ca-sensing receptors in 
the parathyroid glands are exquisitely 
sensitive to serum iCa. Small increases 
in iCa in response to Ca infusion result 
in a decline in serum PTH within 
minutes (Fig 3).11 
 
 
Importantly, changes in total serum Ca levels during 
exercise, which are more commonly measured than 
iCa, do not reflect the changes in iCa (Fig 4).12 Our 
lab is currently in the process of investigating this 
protocol in younger adults. However, to the best of 
our knowledge, this will be the first study to 
evaluate whether preventing the decline in serum 
iCa during exercise prevents an increase in PTH and 
CTX in healthy older adults.  
 
H: When compared with saline infusion, 
intravenous Ca infusion during exercise to prevent a 
decline in serum iCa will prevent the increases in 
serum PTH and CTX. 
 
In recent discussions with colleagues who study osteocytes, it has been noted that the changes we 
observe with exercise may be due to osteocytic osteolysis (bone resorption by the osteocyte)13,14, 
rather than the assumed bone resorption by the osteoclast. Osteocytes are known to contribute to 
the regulation of bone remodeling in response to various stimuli, such as mechanical loading.15 
However, until the discovery of the osteoclast, osteocytes were also believed to be involved in 
calcium homeostasis in response to acute fluctuations in serum calcium.14,16 Recently, research 
has resurrected the idea that osteocytes may contribute to mineral homeostasis, but the majority 
of research was done in vitro, which may not have direct implications for human physiology.14 
Studying osteocytic osteolysis in vivo in humans is challenging because the majority of osteocyte 
research focuses on osteocyte isolation and culture.17,18 However, osteoporosis drugs that have 
direct action on osteoclasts (i.e. bisphosphonates) have the potential to determine the 
contribution of the osteocyte to calcium homeostasis via studies of exercise in older adults. 


 
Although we are generating the scientific basis to suggest that the decline in serum iCa during 
exercise is the driver behind increases in both PTH and CTX, our research to date does not 
provide information regarding which bone cell is responsible for regulating calcium homeostasis 
and increasing bone resorption during exercise. Given that this research is ongoing in older 
adults, it is possible to recruit participants who are already taking bisphosphonate medications 
and conduct the clamp experiments in this group. Because bisphosphonates slow bone loss by 
increasing osteoclast apoptosis, if those on bisphosphonate medication respond similarly to 
healthy older adults not taking bone altering medications (i.e., the decrease in iCa during 
exercise stimulates increases in PTH and CTX), the osteocyte is likely the cell responsible for 
regulating mineral homeostasis and increasing bone resorption. However, if we do not see 
similar responses (i.e., a failure of CTX to increase in response to a decline in iCa and increase in 
PTH), this will suggest that the osteoclast is the cell primarily responsible for calcium 
homeostasis and bone resorption during exercise. Knowing which cell type is responsible for the 
large and sustained increases in CTX that we observe during long duration, high intensity 
exercise can help create targeted exercise and supplementation strategies to preserve bone health 
with age. Further, if those on bisphosphonates do respond differently, it will provide preliminary 
evidence to design more personalized exercise interventions for those taking those medications.  
 
Accordingly, we will repeat the calcium clamp (methods detailed below) in older women 
currently taking a bisphosphonate medication to generate pilot data as part of the larger research 
project. We will compare the iCa, PTH, and CTX responses between the saline and control 
conditions. We will also compare these results to those from the ongoing studies in healthy older 
adults not taking a bisphosphonate. 
 
Approach 
 
Study Overview. Healthy older adult men and women will undergo two 1-hour exercise bouts 
with concomitant intravenous infusion of saline or CaCl2 (Fig 5). Because of a shortage of 
CaCl2, Ca gluconate may be used in some experiments. The exercise bouts will be held 
approximately 1 week apart. 
 
Participants will be asked to 
record their dietary intake the 
day before the first exercise bout 
and repeat that intake the day 
before the second exercise bout. 
A pre-exercise meal that 
contains <100 mg Ca will be consumed 4 hours before exercise (same meal on both days; Figure 
5). The macronutrient composition of the meal is as follows: CHO/protein/fat of 50%/15%/35%. 
Meals will contain 575 kcal for men and 335 kcal for women (20% of daily caloric intake).   
 


Participants will be instructed to avoid Ca supplements for ~24 hours prior to testing. Blood 
samples will be obtained at 15-min intervals starting 15 min before exercise until 30 min after 
exercise, and then at 1 hour intervals starting 1 hour after exercise and ending 4 hours after 
exercise for the measurement of iCa, PTH and CTX. Samples will also be obtained every 5 min 
during exercise for the measurement of iCa (on the Ca and saline days), which will be used to 
adjust the infusion of CaCl2 or Ca gluconate.  
 
Urine samples will be collected before exercise, immediately after exercise, 2 
hours after exercise and 4 hours after exercise for the measurement of urine Ca concentration. 
 
Following completion of the testing visit, participants will receive a boxed lunch. 
 
Subjects. Participants will be healthy older adult women and men aged 60-80 y who are 
accustomed to vigorous walking (i.e., walking for intervals over 1 hour in duration at an intensity 
that causes sweating). Volunteers will undergo an orientation session and a screening session. 
The screening session will include a medical history, physical exam, blood chemistries (complete 
metabolic panel, complete blood count, TSH, 25-hydroxy-vitamin D (25(OH)D), dual-energy x-
ray absorptiometry (DXA) scan, and graded exercise test (GXT) on a treadmill for measurement 
of VO2peak. Exclusion criteria are described in the section on the Protection of Human Subjects. 
 
Blinding and randomization. Because of the nature of the experiments, neither the research team 
nor the participants will be blinded because of the need to adjust the rate of CaCl2 or Ca 
gluconate infusion during the iCa clamp. However, samples (other than iCa) will be processed in 
a way that ensures the individual analyzing the samples will blinded to the test condition. The 
order of the test conditions will not be randomized. The iCa clamp will be performed first for all 
participants, for reasons described below. 
 
Procedures. 
 
GXT. The walking GXT will be done to 
determine whether the BP/ECG responses to 
exercise are normal and measure VO2peak, 
which will be used to determine the exercise 
intensity for the two exercise bouts. VO2peak 
will be measured by indirect calorimetry using a 
TruMax2400 metabolic cart (ParvoMedics, 
Sandy UT). GXT tests will be supervised by 
either the study physician (Dr. Rebecca Boxer) 
or the CTRC PA.  
 
DXA. Participants will undergo a DXA (Hologic, Bedford, MA) for the total body, left hip 
(unless metal/injury prevents this; in which case, right hip will be used), and lumbar spine. All 
three scans take approximately 30 minutes to complete, and the total radiation exposure is the 
equivalent of one day on Earth. All scans will be conducted by a certified technician who has 
undergone the appropriate training. 


Exercise bout. The exercise bout will be 1 hour of walking at ~80% of walking VO2peak. 3- to 
5-min warm-up at a self-selected intensity will precede the exercise bout and a 3- to 5-min cool-
down at a self-selected intensity will follow the exercise bout. This intensity and duration is 
expected to result in a significant decrease in serum iCa and increase in serum PTH (Fig 6). The 
first exercise bout will be the iCa clamp. The volume of fluid infused intravenously will be 
recorded and the same volume will be delivered in the same manner during the subsequent saline 
infusion. Participants will be allowed to drink water ad libitum.  
Blood sampling and analysis. Two indwelling intravenous catheters will be positioned 
approximately 30 minutes before the start of exercise for serial blood sampling. Baseline samples 
will be obtained 15 minutes before exercise, immediately before the start of exercise and then 
every 15 minutes during exercise and for 1 hour following exercise. Additional blood samples 
will be obtained at 5-min intervals during exercise for iCa.  
 
Intact PTH will be measured using an Immulite two-site EIA (DPC). CTX and will be measured 
by ELISA (Nordic Bioscience). Ionized Ca will be measured using an iSTAT point-of-care 
whole blood analyzer (Abbott Point of Care, Inc; Princeton, NJ). The iSTAT analyzer will also 
measure hemoglobin and hematocrit, to allow adjustments for hemoconcentration), and pH, a 
potential modulator of PTH secretion.4,19 
 
Despite the wealth of information generated from preclinical (i.e., animal) studies of the effects 
of acute exercise or loading on bone metabolism, there have been relatively few studies that 
measured changes in bone metabolism in response to acute exercise in humans, as reviewed in 
201020 and since.20-23 Recent studies evaluated the effects of training status23 and exercise 
intensity20 on the metabolic response of bone to acute exercise, but to the best of our knowledge 
no studies have specifically focused on dermal Ca loss as the determinant of the metabolic 
response. By obtaining blood samples every 15 minutes during and after exercise, we should be 
able to capture the dynamic regulation of PTH by serum iCa because small changes in iCa 
trigger an increase in PTH within minutes.24 Obtaining samples up to 4 hours after exercise 
should be sufficient time to observe a peak in CTX.25 
 
Urine sampling and analysis. Urine samples will be collected immediately before and after 
exercise and 2 and 4 hours after exercise. Urinary Ca concentrations will be measured with an 
ion-sensitive electrode. 
 
Data management. Ms. Anusha Guntupalli, who is the data manager for the PI’s research group, 
will create and maintain the database for all of the experiments in a secure centralized SQL 
server that meets the institutional requirements for security and regulatory compliance. 
Whenever possible, data will be uploaded electronically from the point of collection (e.g., core 
lab chemistries) to the database. When key outcomes data must be entered manually, they will be 
double-entered to provide a means of doing quality assurance checks. The user interface for 
manual entry will be Research Electronic Data Capture (REDCap), which is a secure, web-based 
application developed at Vanderbilt University for building and managing online surveys and 
databases. REDcap is available to investigators at UCD for use at no charge. Data entered via 
REDcap will eventually be merged electronically into the central SQL server to facilitate data 
analysis. Data verification and integrity for the database are imposed at the field, table, and 
relationship levels within the SQL backend. We have minimized data input error by eliminating 


manual input of most clinical data by using electronic upload. All data are automatically backed 
up to off-site servers on a nightly schedule. Voucher copies of the database and supporting 
documents are copied to permanent media and stored in a secure location on a semi-annual basis. 
 
Sample size estimate and power calculation. The primary outcome for SA2 is the change in 
serum PTH from the beginning to the end of exercise. The expectation is that PTH will increase 
under the saline condition by 74±63 pg/mL, as observed in previous studies using a similar 
exercise intervention (Fig 4).11 Based on our hypothesis, we expect that serum PTH will be 
unchanged (or decrease) during the iCa clamp condition. Thus, the expected difference in the 
PTH response under the two experimental conditions is 74±63 pg/mL. Assuming the within-
subject correlation between exercise bouts is 0.5, a paired t test at the 0.05 level with a sample 
size of 10 will provide 91% power to detect this magnitude of difference between conditions. For 
a sample size of 8 (i.e., if some of the iCa clamps are not successful), the power is 81%. In order 
to distinguish any potential sex differences, we will enroll both men and women (12 men and 12 
women to allow for attrition). To generate pilot data, we will enroll 6 women taking a 
bisphosphonate medication for comparison with the women who are enrolled in the study and 
not taking a bisphosphonate. Because the purpose of this is to generate pilot data, sample size is 
based on what is feasible given available funds.  
 
Statistical analysis plan. The primary statistical test will be a paired t test comparing the change 
in serum PTH from the beginning to end of exercise in the saline condition with the change in 
the iCa clamp condition. Secondary analyses will include similar comparisons of the changes in 
serum iCa and CTX. For the primary and secondary outcomes, we will also estimate the changes 
in slope over time and the integrated responses using data obtained before, during, and after 
exercise. Depending on the appearance of changes over time, we will use linear or nonlinear 
mixed models.6 Regression analyses will be used to explore the associations of change in serum 
iCa with the changes in serum PTH and CTX in a manner that informs the working model 
depicted in Figure 1 (i.e.,upstream events as mediators of downstream events).10 Potential 
confounding variables, such as training history or conditioning, will be collected to allow for 
adjustment of the model, if necessary.  
 
For the comparison between the bisphosphonate and non-bisphosphonate users, the primary test 
will be a paired t test comparing the change in serum CTX from the beginning to end of exercise 
in the saline condition with the change in the iCa clamp condition. Secondary analyses will 
include similar comparisons of the changes in serum iCa and PTH. Independent t test analyses 
will be conducted on the calcium and saline conditions between groups (no bisphosphonate vs. 
bisphosphonate) to determine if the responses to the calcium clamp and/or saline infusions are 
different. Given the small sample size, negative findings will be interpreted with caution. We 
will be looking for trends and differences between groups to determine in further investigation of 
osteocytic osteolysis in humans is warranted.  
 
Data interpretation and limitations. The expectation is that preventing a decline in iCa will 
prevent an increase in PTH and the activation of bone resorption. If PTH increases during the 
iCa clamp, this could indicate that: 1) Some factor other than serum iCa mediated the increase in 
PTH. Because metabolic acidosis is known to stimulate PTH,4,19 we will measure pH 


to evaluate this possibility; or 2) The iCa clamp was not successful in preventing a decline in 
iCa. Serum iCa concentrations will be measured in real-time during the clamp, so there will be 
immediate knowledge of whether the clamp was successful. Clamps in which the iCa 
concentration decreases below the baseline level will not be stopped because of the information 
that can potentially be gained regarding fluctuations of iCa during exercise and the PTH 
response. However, secondary statistical analyses will be performed using only data from 
successful clamps. If we do not have 8 subjects with successful clamps (needed to achieve >80% 
power), we will enroll additional volunteers; the likelihood that this will occur is low. 
 
For the comparison of the bisphosphonate and non-bisphosphonate users, it is possible that 
calcium homeostasis during exercise is regulated by both the osteoclast and osteocyte. If this is 
the case, we may see an intermediate CTX response that would indicate diminished osteoclast 
activity and normal osteocyte activity; this data would be difficult to interpret. However, the goal 
of this study is to determine if there is a preliminary signal for osteocytic osteolysis. Even if the 
results are not what we expect, it will provide some indication on if osteocytic osteolysis is a 
contributing factor to the bone resorption observed during exercise. 
 
4. Protection of human subjects 
 
4.1. Eligibility criteria  
 
Volunteers who are eligible and willing to participate will undergo the procedures described in 
the Protocol. They will be healthy adults aged 60-80 y. There will be no discrimination by race 
or ethnicity for enrollment in the trial. Based on the 2010 U.S. Census, the ethnic composition of 
the Greater Denver Metropolitan area is 21.8% of Hispanic or Latino origin and the 
racial composition is 0.1% Native Hawaiian or Pacific Islander, 1.0% American Indian or 
Alaskan Native, 3.8% Asian, 5.2% Black or African American, 78.6% White, 3.5% multi-racial, 
and 7.8% other. Therefore, we plan that ~22% of our participants will be of Hispanic or Latino 
ethnicity and ~21% will be non-white or multi-racial. 
 
Inclusion and exclusion criteria. Healthy adults aged 60-80 y will be enrolled in the experiments. 
To ensure that participants are familiar with the type of exercise to be performed, volunteers will 
be recreationally active (i.e., engage in moderate- or vigorous-intensity physical activities at least 
2 days per week based on self-report) and accustomed to walking bouts of 1 hour duration (based 
on self-report). Volunteers will be excluded from participation for the following: 
 
• use of medications in the past 6 months known to affect bone metabolism (e.g., 
bisphosphonates, thiazide diuretics, oral glucocorticoids) 
• BMD t score < -2.5 at the total hip or lumbar spine 
• known disease or condition associated with intestinal malabsorption 
• moderate or severe renal impairment defined as an estimated glomerular filtration rate of 
<60 mL/min/1.73m2 based on the Modification of Diet in Renal Disease (MDRD) 
equation 
• chronic hepatobiliary disease, defined as liver function tests (AST, ALT) >1.5 times the 
upper limit of normal; if such values are obtained on initial screening and thought to be 
transient in nature, repeated testing will be allowed 


• thyroid dysfunction, defined as an ultrasensitive TSH <0.5 or >5.0 mU/L; volunteers with 
abnormal TSH values will be re-considered for participation in the study after follow-up 
evaluation by the PCP with initiation or adjustment of thyroid hormone replacement 
• serum calcium <8.5 or >10.3 mg/dL 
• serum 25(OH)D <20 ng/mL; volunteers with abnormal serum 25(OH)D values may be 
re-considered for participation in the study if serum 25(OH)D is >20 ng/mL after vitamin 
D supplementation 
• uncontrolled hypertension defined as resting systolic BP >150 mmHg or diastolic BP>90 
mmHg; participants who do not meet these criteria at first screening will be re-evaluated, 
including after follow-up evaluation by the PCP with initiation or adjustment of anti-
hypertensive medications 
• history of type 1 or type 2 diabetes 
• cardiovascular disease; subjective or objective indicators of ischemic heart disease (e.g., 
angina, ST segment depression) or serious arrhythmias at rest or during the graded 
exercise test (GXT) without follow-up evaluation will be cause for exclusion; follow-up 
evaluation must include diagnostic testing (e.g., stress echocardiogram or thallium stress 
test) with interpretation by a cardiologist 
• diagnosis or history of asthma 
 
The addition 6 women enrolled for the bisphosphonate comparison group will meet all the above 
criteria with two exceptions: 
• must be taking a bisphosphonate for a minimum of one year 
must have a t-score ≥ -3.0 at the total hip or lumbar since osteoporosis is often the diagnostic 
criteria necessary to be prescribed a bisphosphonate 
 
4.2. Potential risks  
 
Exercise: The potential risks of exercise include development of ventricular arrhythmia, 
myocardial infarction, cardiac arrest, asthma, and death, as well as the less serious problems of 
injury to tendons, ligaments, joints, and muscles.  
 
Venipuncture: There is a small risk of local hematoma, infection, syncope, and thrombosis 
associated with intravenous blood sampling. 
 
Confidentiality and privacy: The use of questionnaires, interviews, and collection of personal 
medical information poses a risk to confidentiality and privacy and may cause embarrassment. 
 
4.3. Adequacy of protection against risks 
a. Recruitment and informed consent 
 
Volunteer recruitment. Recruitment of volunteers for the study will rely on strategies that have 
proven successful in attracting volunteers to intervention trials the PI and CO-Is have conducted. 
Our intention is that the study cohort will be comprised of men and women with a race/ethnic 
distribution similar to that of the Denver metropolitan area. The recruitment strategies will be 
conducted by the research coordinator and the research assistant and will include: 
 


• radio advertisements – utilization of radio stations that target a demographic (age, race, 
ethnicity) consistent with the study population 
• newspaper advertisements – the major publication in the Denver Metropolitan has a 
Health and Fitness section that runs once per week; advertisements in these sections have 
targeted appropriate individuals for our previous studies 
• suburban newspaper advertisements – there are many suburban journals in the Metro area 
that are issued on a weekly or monthly basis; we specifically advertise in those that target 
neighborhoods in relative proximity to our exercise training facility and those that target 
persons of under-represented minorities 
• university advertisements – advertisements will be placed on a quarterly basis in the 
University of Colorado Denver (UCD) publications that target faculty/staff and alumni 
• e-mail bulletins – system-wide (UCD, University of Colorado Hospital, The Childrens 
Hospital) email advertisements will be distributed quarterlyelectronic networks – 
electronic networks, such as Craigs List, have been a productive source of study 
volunteers 
• study pamphlets – study pamphlets describing the study will be printed for a variety of 
purposes (for study participants to give to family and friends who may be interested in 
participating; to place in the UCD Primary Care Clinics and other relevant clinics; to 
place in community recreation centers; to hand out when the investigators give 
community lectures) 
• RECRUIT- this offers a variety of recruitment (RECRUIT) tools which are intended to 
assist research teams in their recruitment efforts by providing a mechanism for teams to 
connect with UCHealth patients using My Health Connection. We will specifically use 
Epic Recruitment to recruit potential participants. Patients have the opportunity through 
My Health Connection to click the “I’m Interested” button and be on a list in Epic where 
interventionist can further determine eligibility for the study and reach out to them 
through secure messaging. 
 
Participants will receive volunteer remuneration, although this will not be advertised as a study 
benefit. The amount of remuneration in the study will be $100 for the completion of the exercise 
test.  
 
Informed consent process. The first interaction with volunteers is typically by telephone or 
email, initiated by individuals who have seen or heard an advertisement and call to learn more 
about the study. A brief screening interview is conducted, either by phone or by email (form 
containing the questions in the phone script). Callers who meet general study criteria and are 
interested in learning more about the study are scheduled for an orientation session. The 
consenting process is part of the orientation session, which typically takes ~60 min. Volunteers 
are either sent a copy of the consent form in advance or are given one at the start of the 
orientation; time is allowed for review of the consent form. Volunteers then meet with a member 
of the research team to a) have the study and what is expected of them explained in detail; b) 
discuss their reasons for wanting to participate to determine whether they are realistic; c) discuss 
any practical problems (e.g., scheduling conflicts) that could interfere with participation; d) have 
their questions answered; and e) demonstrate their ability to provide informed consent by 
describing their understanding of the major study goals and what is expected of participants.  
 


Volunteers are given a signed copy of the consent form and a signed copy is maintained in the 
study chart for each participant. The HIPAA regulations are also explained at this session. 
The member of the research group who will obtain consent and HIPAA authorization from 
volunteers will have complied with IRB and HIPAA education requirements. The PI will have 
the responsibility of ensuring that research personnel are prepared to convey information to 
volunteers specific to the study protocol. 
 
b. Protections against risk 
 
Plans to minimize risks. 
 
Exercise testing: The risks of exercise testing will be minimized by 
adhering to the following strategies: 
 
Endpoints for maximal exercise tests: In asymptomatic individuals who do not develop 
cardiovascular abnormalities, the endpoint for the maximal exercise tests will be severe fatigue 
that forces cessation of exercise.  
 
The criteria that will be used to stop the exercise test before volitional fatigue include 
thedevelopment of: (a) ST-segment depression of more than 0.2 mV that is either horizontal, 
downsloping, or slowly upsloping (less than 1 mV/sec) and lasts for 0.08 sec, or ST-segment 
elevation greater than 0.1 mV; (b) chest pain or discomfort; (c) serious arrhythmias, including 
multifocal PVCs, ventricular tachycardia, frequent (>10/min) PVCs or couplets, or sustained 
atrial tachyarrhythmias; (d) A-V block or other conduction defects; (e) a fall of systolic blood 
pressure of 10 mmHg or greater from the peak level with increasing exercise intensity; (f) 
diastolic blood pressure above 110 mmHg or systolic above 220 mmHg; (g) dizziness; (h) ataxic 
gait; and (i) pallor or cyanosis. 
 
Training of personnel: Study personnel assisting with the administration of screening cycling 
tests will have BLS certification. A physician will be immediately available in the facility if not 
directly administering these screening tests. A physician will interpret ECG and BP responses for 
all screening cycling tests. 
 
Screening of volunteers: To minimize risks of exercise testing in subjects, testing will be 
conducted only after the volunteer is examined by a clinician and after a resting ECG is obtained 
and evaluated. During exercise testing, the ECG is monitored constantly and BP is measured 
frequently. Exercise tests are terminated if any of the ACSM absolute or relative stopping criteria 
are met. The AHA-recommended emergency equipment and supplies will be available, 
including: automated electronic defibrillator; portable oxygen tank, nasal cannula, ventimask, 
non-rebreathing mask, and appropriate tubing to connect to the oxygen tank; oral airways; bag-
valve-mask hand respirator; syringes and needles; IV tubing, solutions, and stand; and adhesive 
tape. The Exercise Research Laboratory operates as a 911 emergency response facility, 
as do all other UCH outpatient clinics. 
 
 
 


Submaximal exercise tests: The intensity for the submaximal, 1-hour exercise tests for all 
experiments will be individualized, based on performance during the maximal exercise tests, 
such that the relative intensity is similar for all participants. Eligible participants will be 
accustomed to the usual discomforts associated with vigorous exercise. For all experiments, and 
particularly for the experiment in a warm environment, participants will be allowed (and 
encouraged) to drink water ad libitum. 
 
Venipuncture: The risks of hematoma and infection are minimized by having trained personnel 
perform the procedures using sterile techniques. 
 
Confidentiality and privacy: The risks will be minimized by not including personal identifying 
information on the forms, when possible, and by conducting interviews and collection of 
personal information in a private setting. 
 
Potential benefits to the subjects and others 
 
The benefits to an individual participant in the study are not known. There is potential benefit 
from the assessment of health status by medical history and physical examination, and screening 
for cardiovascular disease. The potential benefits of the study to women and men in general 
could be significant. The study is expected to lead to the scientific discovery of the role that 
calcium metabolism during exercise affects changes in bone mineral density (BMD). This may 
lead to the development of strategies to maximize improvements in BMD with exercise. In light 
of these potential benefits, the risks to subjects are reasonable. 
 
Importance of knowledge to be gained 
 
The current exercise recommendations for the prevention and treatment of osteoporosis have 
evolved from preclinical research (i.e., animal models) on the loading characteristics of an 
activity that are important for the activation of bone formation (i.e., dynamic, high magnitude 
bone-loading forces, high strain rate). The current application is focused on whether vigorous 
exercise also activates bone resorption. Importantly, the proposed mechanisms to be investigated 
are thought to be triggered by the dermal loss of calcium during exercise (i.e., sweating), which 
has not been addressed in preclinical studies because most laboratory animals do not lose 
calcium through this route. This line of research could lead to strategies to enhance the benefits 
of exercise on osteoporosis prevention. 
 
4.4 Data and safety monitoring plan 
 
The data and safety monitoring for the proposed experiments will be carried out by the research 
team and an independent Research Monitor. Kerry Hildreth, MD, Assistant Professor of 
Medicine in the Division of Geriatric Medicine, will serve as the Research Monitor. 
Responsibilities and Authorities of the Research Monitor 
 
The Research Monitor will: 1) approve the protocol and any changes to the protocol that have an 
impact on participant; 2) monitor recruitment, consenting, enrollment, and testing of study 
participants; 3) review anticipated and unanticipated problems; 4) review study progress and data 


quality; and 5) submit reports on these activities and recommended action items to the PI, local 
IRB, and sponsor (if applicable). Meetings with the Research Monitor will be held at least 
annually, or more frequently if requested by the IRB, Sponsor, or Monitor. 
 
Because of the nature of the studies (i.e., mechanistically-driven physiological experiments, not 
clinical trials), the Research Monitor will monitor safety only and not efficacy. In the event the 
Research Monitor determines an experiment should be stopped for reasons of safety, this will be 
communicated to the PI, IRB, and Sponsor. 
 
The investigators will promptly report all anticipated (i.e., identified in the consent form) and 
unanticipated problems involving risk to subjects or others to the Research Monitor for review. 
The monitor will confirm whether the investigators have appropriately categorized the problem 
as anticipated or unanticipated. The Research Monitor will have the authority to discuss the 
research protocol with the investigators, interview human subjects, and consult with others 
outside of the study about the research. 
 
 
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