Effect of Blueberries on Bone Turnover

Study Overview
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Brief Summary
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This study uses a bone labeling calcium tracer methodology to compare the dose-response effect of blueberry consumption on calcium retention and bone loss. Post-menopausal women will receive food or beverage products containing freeze-dried blueberries in the amount equivalent to 0.75 (low), 1.5 (medium), and 3 cups (high) of fresh blueberries per day over a 42-day period. The hypothesis is that the polyphenolics found in blueberries will reduce calcium loss from bones.

Detailed Description
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Participants will be dosed with Ca-41, a rare long-lived radioisotope of calcium. After the equilibration of tracer in the body and its deposition in bones (150 days), subjects will be randomized to one of 6 dose sequences, all of which will begin with a 42-day baseline period. During baseline, 24-hour urine will be collected every 14 days. Following baseline, subjects will enter a 42-day intervention period with one of three doses of blueberries equivalent to 0.75 (low), 1.5 (medium), and 3 cups (high) of blueberries per day. Each dose will be provided in the form of freeze-dried blueberry powder incorporated in 2-4 foods or beverages per day. During the intervention, 24-hour urine will be collected weekly for 6 weeks except week 1. After intervention, subjects will enter a 42-day washout period, during which 24-hour urine will be collected every 3 weeks. The entire study duration will be 444 days for subjects who have not been dosed with Ca-41 previously. In a crossover design, all subjects will complete three 42-day intervention periods corresponding to the three doses of blueberries (low, medium, and high), each followed by a 42-day washout period. The dose-response effect of continuous blueberry consumption over a 42-day period on bone resorption in healthy post-menopausal women will be studied by measuring the loss of Ca-41 in urine by Accelerator Mass Spectrometry.

Official Title
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Berries and Bones: The Effect of Polyphenolic Metabolites From Blueberries on Bone Turnover

Conditions
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Osteoporosis, Postmenopausal, Bone Loss, Age-related

Intervention / Treatment
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* Dietary Supplement: Blueberry Baseline
* Dietary Supplement: Blueberry Low
* Dietary Supplement: Blueberry Medium
* Dietary Supplement: Blueberry High


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Female subject is healthy Subject is > 4 years past the onset of natural menopause or total hysterectomy Exclusion Criteria: History of metabolic bone disease or low trauma fractures; Subject taking osteoporosis treatment drugs or glucocorticoids within 6 months of the beginning of the study; Subjects taking bisphosphonates within 2 years of the beginning of the study; History of cancer, thromboembolisms, clotting disorders, uncontrolled hypertension, abnormal thyroid function, malabsorption syndrome, seizure disorders, or heart attack; BMI > 35 kg/m2; Subjects who will not comply with study interventions ; Subjects who will not stop taking natural product supplements of their own selection.

Ages Eligible for Study
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Minimum Age: 45 Years
Maximum Age: 70 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Prevention
Allocation: Randomized
Intervention Model: Crossover Assignment
Interventional Model Description: This partially randomized crossover design model will include participants assigned first to a control baseline period followed by three intervention phases with a low, medium or high daily dose of blueberry products in a randomized order.
Masking: Single


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Placebo Comparator: Blueberry baseline<br>No blueberry products provided as part of the usual dietary intake | Dietary Supplement: Blueberry Baseline<br>* Before the study beginning, eligible participants will receive a radioactive tracer, Ca-41, by iv infusion. A total of 150 days will be required for the equilibration of tracer in the body i.e. elimination from soft tissue and deposition in the bone. After equilibration, a baseline period of 42 days will occur, during which no blueberry products will be provided.<br>|
| Active Comparator: Blueberry Low<br>One blueberry product per day containing an equivalent of 0.75 cups of fresh blueberries provided as part of usual dietary intake for 42 days | Dietary Supplement: Blueberry Low<br>* One food or beverage product containing freeze-dried blueberry powder equivalent to 0.75 cups of fresh blueberries will be provided daily for 42 days.<br>|
| Active Comparator: Blueberry Medium<br>Two blueberry products per day containing an equivalent of 1.5 cups of fresh blueberries provided as part of usual dietary intake for 42 days | Dietary Supplement: Blueberry Medium<br>* Two food or beverage products containing freeze-dried blueberry powder equivalent to 1.5 cups of fresh blueberries will be provided daily for 42 days.<br>|
| Active Comparator: Blueberry High<br>Four blueberry products per day containing an equivalent of 3 cups of fresh blueberries provided as part of usual dietary intake for 42 days | Dietary Supplement: Blueberry High<br>* Four food or beverage products containing freeze-dried blueberry powder equivalent to 3 cups of fresh blueberries will be provided daily for 42 days.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Ca-41 / Ca ratio in 24-hour urinary excretion to estimate calcium loss from bone | Urinary Ca-41 excretion will be expressed as Ca-41/Ca ratio, which is unit-less, and converted to a percent change from the baseline value. 24-hour urine will be collected approximately every 2 weeks during baseline (week 0, 2, 4, and 6), weekly (except for week 1) during the low, medium, and high blueberry dose interventions completed in a randomized order (weeks 8-12, 20-24, 32-36) and every 3 weeks during the washout periods (weeks 15, 18, 27, 30, 39, 42). Ca-41/Ca ratios will be measured by Accelerator Mass Spectrometry. | From the beginning of baseline (week 0) to the end of the 3rd washout period (week 42) | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Fasting blood and urine analysis of blueberry polyphenolic metabolites | Polyphenol concentrations in urine and serum will be expressed in molar units and compared against reference values and across the study periods. Polyphenolic metabolites will be measured by LC-MSMS. Fasting blood and urine will be collected at the start and end of baseline (weeks 0 and 6), at the end of the low, medium, and high dose blueberry interventions (weeks 12, 24, and 36), and at the end of the 3 washout periods (weeks 18, 30, 42). | Weeks 0, 6, 12, 18, 24, 30, 36, 42 | 
| Serum and urine biochemical markers of bone metabolism: calcium concentration | Calcium concentration in urine and serum will be expressed in mg/L and measured by Atomic Absorption Spectrophotometry. Fasting blood and urine will be collected at the start and end of baseline (weeks 0 and 6), at the end of the low, medium, and high dose blueberry interventions (weeks 12, 24, and 36), and at the end of the 3 washout periods (weeks 18, 30, 42). | Weeks 0, 6, 12, 18, 24, 30, 36, 42 | 
| Serum biochemical markers of bone metabolism: Insulin Dependent Growth Factor-1 (IGF-1) | IGF-1 will be expressed in ng/mL and measured by ELISA. Fasting blood will be collected at the start and end of baseline (weeks 0 and 6), at the end of the low, medium, and high dose blueberry interventions (weeks 12, 24, and 36), and at the end of the 3 washout periods (weeks 18, 30, 42). | Weeks 0, 6, 12, 18, 24, 30, 36, 42 | 
| Serum biochemical markers of bone metabolism: Osteoprotegrin | Osteoprotegerin will be expressed in pmol/L and measured by ELISA. Fasting blood will be collected at the start and end of baseline (weeks 0 and 6), at the end of the low, medium, and high dose blueberry interventions (weeks 12, 24, and 36), and at the end of the 3 washout periods (weeks 18, 30, 42). | Weeks 0, 6, 12, 18, 24, 30, 36, 42 | 
| Serum biochemical markers of bone metabolism: RANK ligand | RANK-L will be expressed in pmol/L and measured by ELISA. Fasting blood will be collected at the start and end of baseline (weeks 0 and 6), at the end of the low, medium, and high dose blueberry interventions (weeks 12, 24, and 36), and at the end of the 3 washout periods (weeks 18, 30, 42). | Weeks 0, 6, 12, 18, 24, 30, 36, 42 | 
| Serum biochemical markers of bone metabolism: 25(OH) Vitamin D | 25(OH) Vitamin D will be expressed in ng/mL and measured by LC/MS. Fasting blood will be collected at the start and end of baseline (weeks 0 and 6), at the end of the low, medium, and high dose blueberry interventions (weeks 12, 24, and 36), and at the end of the 3 washout periods (weeks 18, 30, 42). | Weeks 0, 6, 12, 18, 24, 30, 36, 42 | 
| Serum biochemical markers of bone metabolism: Sclerostin | Sclerostin will be expressed in ng/mL and measured by ELISA. Fasting blood will be collected at the start and end of baseline (weeks 0 and 6), at the end of the low, medium, and high dose blueberry interventions (weeks 12, 24, and 36), and at the end of the 3 washout periods (weeks 18, 30, 42). | Weeks 0, 6, 12, 18, 24, 30, 36, 42 | 
| Urine biochemical markers of bone metabolism: N-terminal telopeptide (NTX) | NTX will be expressed in ng/mL and measured by ELISA. Fasting urine will be collected at the start and end of baseline (weeks 0 and 6), at the end of the low, medium, and high dose blueberry interventions (weeks 12, 24, and 36), and at the end of the 3 washout periods (weeks 18, 30, 42). | Weeks 0, 6, 12, 18, 24, 30, 36, 42 | 
| Serum biochemical markers of bone metabolism: procollagen I intact N-terminal (PINP) | PINP will be will be expressed in ug/L and measured by ELISA. Fasting blood will be collected at the start and end of baseline (weeks 0 and 6), at the end of the low, medium, and high dose blueberry interventions (weeks 12, 24, and 36), and at the end of the 3 washout periods (weeks 18, 30, 42). | Weeks 0, 6, 12, 18, 24, 30, 36, 42 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Bone Health, Calcium, Osteoporosis, Polyphenolics