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 Table of Contents  
STUDY PROTOCOL
Year : 2017  |  Volume : 2  |  Issue : 3  |  Page : 53-58

Correlating oxidative stress-related factors with bone metabolic markers in older adult male patients exhibiting degenerative osteoporosis in the high-altitude hypoxic area of China: study protocol for a non-randomized controlled trial


1 Department of Bone Traumatology, Affiliated Hospital of Qinghai University, Xining, Qinghai Province, China
2 Department of Orthopedics, Qinghai Province Reserve Brigade Hospital, Xining, Qinghai Province, China

Date of Web Publication17-Oct-2017

Correspondence Address:
Jian-wen Ma
Department of Bone Traumatology, Affiliated Hospital of Qinghai University, Xining, Qinghai Province
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2542-3975.216582

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  Abstract 

Background and objectives: Hypoxia is an important factor that affects bone formation and regulates bone growth. Therefore, many older adult patients living in high-altitude hypoxic areas exhibit osteoporosis. Oxidative stress-related hypoxia-inducible factors can induce abnormal expression of various factors including vascular endothelial growth factor (VEGF), insulin-like growth factor, and endothelin. However, it remains unclear whether these factors influence changes in bone metabolic markers. This study protocol aimed to investigate the correlation between oxidative stress-related factors and bone metabolic markers in older adult male patients with degenerative osteoporosis who reside in the high-altitude hypoxic area of China.
Design: A prospective, single-center, non-randomized, controlled trial.
Methods: One hundred and twenty older adult male patients with degenerative osteoporosis residing in the high-altitude area of China who receive treatment at the Affiliated Hospital of Qinghai University of China between January 2015 and February 2018 are being included in the osteoporosis group. One hundred and twenty healthy older adult males who concurrently received physical examination are being included in the control group. One day after admission, serum levels of hypoxia-inducible factor 1-alpha (HIF-1α), HIF-2α, VEGF, osteocalcin, and tartrate-resistant acid phosphatase 5b (TRACP 5b) were measured using an enzyme-linked immunosorbent assay. Bone mineral density in L1–4 segments, right femoral neck, and the greater trochanter of the femur was detected using dual-energy X-ray absorptiometry.
Outcome measures: The primary outcome measure of this study is serum HIF-1α levels at 1 day after admission. Secondary outcome measures include serum levels of HIF-1α, HIF-2α, VEGF, osteocalcin, and TRACP 5b at 1 day after admission, as well as the correlation between serum levels of oxidative stress indicators (HIF-1α, HIF-2α, and VEGF) and bone metabolic markers (osteocalcin and TRACP 5b) at 1 day after admission.
Discussion: Findings from this study aim to validate the correlation between oxidative stress-related factors and bone metabolic markers in older adult male patients with degenerative osteoporosis who reside in the high-altitude area of China.
Ethics and dissemination: This study was approved by the Ethics Committee of the Affiliated Hospital of Qinghai University of China (approval No. QHY1402G). The study was performed in accordance with the Declaration of Helsinki. Participants are informed of the study protocol and procedures, and sign an informed consent. Participant recruitment, blood sampling, and data collection began in January 2015 and will be ended in February 2018. Outcome measure analysis and trial completion will be in March 2018. Results will be disseminated through presentations at scientific meetings and/or by publication in peer-reviewed journals.
Trial registration: This trial was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR-ROC-17012848).

Keywords: clinical trial; plateau region; hypoxia; osteoporosis; hypoxia-inducible factor 1-alpha; hypoxia-inducible factor 2-alpha; vascular endothelial growth factor; osteocalcin; tartrate-resistant acid phosphatase 5b


How to cite this article:
Ma Jw, Li Dc, Zhang Zg, Li Y, Wang Yb, Cao Zq. Correlating oxidative stress-related factors with bone metabolic markers in older adult male patients exhibiting degenerative osteoporosis in the high-altitude hypoxic area of China: study protocol for a non-randomized controlled trial. Clin Trials Degener Dis 2017;2:53-8

How to cite this URL:
Ma Jw, Li Dc, Zhang Zg, Li Y, Wang Yb, Cao Zq. Correlating oxidative stress-related factors with bone metabolic markers in older adult male patients exhibiting degenerative osteoporosis in the high-altitude hypoxic area of China: study protocol for a non-randomized controlled trial. Clin Trials Degener Dis [serial online] 2017 [cited 2017 Dec 15];2:53-8. Available from: http://www.clinicaltdd.com/text.asp?2017/2/3/53/216582


  Introduction Top


In patients with osteoporosis, bone loss, bone microstructure destruction, and osteosis directly increase bone fragility, particularly in older adult individuals.[1],[2] Residents from Qinghai Province of China, which is located in a high-altitude area, are exposed to a lifelong hypoxic environment.[3] Hypoxia has been reported to be an important factor that affects bone formation and regulates bone growth.[4],[5],[6],[7],[8],[9],[10] Clinical evidence suggests that hypoxia leads to difficulties in bone healing and decreased bone mineral density, and can result in osteoporosis, which increases the risk of chronic bone injury.[11],[12],[13],[14],[15] Therefore, hypoxia is an important risk factor for osteoporosis and it is very important to effectively prevent and treat osteoporosis in high-altitude hypoxic environments.

Oxidative stress-related hypoxia-inducible factors can induce abnormal expression of various factors including vascular endothelial growth factor (VEGF), insulin-like growth factor, and endothelin.[16] However, it remains unclear whether these factors influence changes in bone metabolic markers.

This study aimed to investigate the correlation between serum levels of oxidative stress-related factors and bone metabolic markers in older adult male patients with degenerative osteoporosis who reside in a high-altitude hypoxic area.


  Methods/Design Top


Study design

A prospective, single-center, non-randomized controlled trial.

Study setting

Affiliated Hospital of Qinghai University of China.

Study participants

One hundred and twenty older adult male patients with degenerative osteoporosis who received treatment in the Affiliated Hospital of Qinghai University of China (osteoporosis group), and 120 healthy elderly males who concurrently received physical examination (control group) are included in this study.

Diagnostic criteria for osteoporosis group

Osteoporosis is defined as bone mineral density less than 30% of peak bone mineral density, as evaluated in males from the Qinghai province of China, and osteoporosis-caused pain lasting for over 3 weeks.[17]

Inclusion criteria for osteoporosis group



Patients with osteoporosis presenting with all of the following conditions will be considered for inclusion in this study:

  • Male
  • Loading pain
  • Aged between 60–70 years
  • Unbearable spontaneous low back pain
  • Able to sit up, turn body over, and walk with bearable pain
  • Unable to sleep because of pain


Exclusion criteria for osteoporosis group

Patients presenting with one or more of the following criteria will be rejected for this study:

  • Presence of vertebral compression fracture on X-ray film
  • Definitive diagnosis of rheumatoid arthritis, osteomalacia, hyperthyroidism, hyperparathyroidism, or other bone metabolic disease
  • External fixation with gypsum for repair of fracture, or within 6 months after removal of gypsum
  • Pain-inducing disease such as lumbar disc herniation, deformed vertebral arthritis, lumbar spinal stenosis, vertebral spondylolisthesis, or ankylosing spondylitis
  • Unable to judge for curative effects, provide disease condition, or unable to cooperate with treatment because of mental illness, senile dementia, or severe neurosis
  • No daily life self-care ability or long-term bed rest
  • Continuous administration of activated vitamin D preparations, calcitonin, corticosteroid hormone, androgen, estrogen or other hormonal preparation within 3 months prior to participation in this trial
  • Administration of selective estrogen receptor modulator within 6 months prior to participation in this trial
  • Severe kidney or liver disease, or history of cancer


Inclusion criteria for control group

Participants presenting with both of these conditions will be considered for inclusion in this study:

  • Healthy older adult males who have resided in a high-altitude hypoxic environment for over 10 years
  • Good compliance


Exclusion criteria for control group

Participants presenting with one or more of the following conditions will be rejected for this study:

  • Hyperthyroidism, hypoparathyroidism, or other bone metabolic diseases
  • Diabetes mellitus
  • Kidney or liver disease


Withdrawal criteria for osteoporosis and control groups

Participants presenting with one or more of the following conditions will be withdrawn from this study:

  • Unable to follow program referral, or cannot comply with any of the terms of the study protocol
  • Potential risks from imaging examinations


Expected total trial duration

Based on the number of patients with osteoporosis previously admitted to our hospital, a time period of 3 years is considered adequate for participant recruitment.

Recruitment

Patients from clinics and wards who received treatment during January 2015 to February 2018 in the Affiliated Hospital of Qinghai University of China are being considered for inclusion in this study. Healthy older adult people who concurrently received physical examination in this hospital are being included as healthy controls. Participants voluntarily participated in the trial after reading a recruitment leaflet. Only eligible participants are being included after signing informed consent.

Assignment

A non-randomized grouping method is used. Osteoporosis and control groups (n = 120 participants/group) are designated to detect serum markers and bone mineral density. This is an open-label study, and blind grouping is not used. Study flow chart is shown in [Figure 1].
Figure 1: Study flow chart.

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Detection of serum markers

At 1 day after admission, 5 mL of peripheral venous blood will be collected in the morning and centrifuged. Supernatant is collected to harvest serum samples. Serum levels of hypoxia-inducible factor 1-alpha (HIF-1α), HIF-2α, osteocalcin, VEGF, and tartrate-resistant acid phosphatase 5b (TRACP 5b) are measured using an enzyme-linked immunosorbent assay (ELISA; USCN, Wuhan, China). ELISA reader is from Bio-Tek (EXL-200, Winooski, VT).

Outcome measures

Primary outcome measure

HIF-1α level: HIF-1α is a core regulator of hypoxia induction and repair of the intracellular oxygen environment.[18],[19],[20],[21] Higher serum levels of HIF-1α indicate more severe hypoxia.

Secondary outcome measures

HIF-2α level: This hypoxia-inducible factor 1 subunit is used to evaluate the severity of body hypoxia.

VEGF level: Reflects the state of hypoxia and antagonizes hypoxia-induced apoptosis of vascular endothelial cells.[22],[23]

Osteocalcin level: Changes with aging and bone metabolism rates; higher levels indicate increased bone metabolism rate, and vice versa.[24],[25]

TRACP 5b level: Good marker of bone resorption and osteoclast activity under various physiological and pathological conditions.[26],[27]

Correlation between serum levels of oxidative stress indicators and bone metabolic markers: A correlation analysis method is used to correlate serum levels of oxidative stress indicators (HIF-1α, HIF-2α, and VEGF) with bone metabolic markers (osteocalcin and TRACP 5b).

Audits

Initial stage

The study protocol should be approved by the Ethics Committee of the Affiliated Hospital of Qinghai University of China, and a protocol agreement should be signed. Researchers should fully understand the study protocol and clinical operation.

During the trial

During participant recruitment, regular audits are necessary to ensure included participants are eligible, the trial is being performed in strict accordance with the study protocol, and related data are complete.

Final stage

At the end of all trial procedures, a final visit is required to ensure relevant records are complete and accurate.

Statistical analysis

Statistical design, method, and analysis principle

All data are statistically analyzed using SPSS13.0 software (SPSS, Chicago, IL) and follow the intention-to-treat principle. Normally distributed measurement data are expressed as the mean ± SD. Non-normally distributed measurement data are expressed as lower quartile (q1), median, and upper quartile (q3). Two-sample t-test is used to evaluate normally distributed serum indicators and bone metabolic markers in the two groups. A Pearson correlation analysis is used. Mann-Whitney U test is used to evaluate non-abnormally distributed serum indicators and bone metabolic markers in the two groups. Spearman rank correlation is used to test the correlation between serum levels of oxidative stress indicators and bone metabolic markers.

Sample size

In accordance with our experience,[27] we hypothesized that the HIF-1α level in osteoporosis and control groups was 12 and 8 μg/L, respectively, with a standard deviation of 2 μg/L. Taking β = 0.2, power = 80%, α = 0.05 (two-sided), the final effective sample size of n = 199 per group was calculated. Assuming a participant loss rate of 20%, we require 239 participants per group. At the end of expected recruitment, at least 120 participants per group are included.

Inspection level

An α = 0.05 (two-sided) will be used for inspection level.

Estimated participant loss rate

The estimated participant loss rate is no more than 20%.

Processing method for missing data

If a participant’s records are lost, the participant is rejected from this study. Corresponding numbers of new cases are supplemented.

Baseline analysis

The baseline data of included participants are shown in [Table 1].
Table 1: Patient$s baseline data

Click here to view


Data management

Researchers complete the case report form for each case. After the completed case report form is reviewed by an inspector, data input and management are performed. After data transfer, contents recorded in the case report form are not modified.

After the database is confirmed to be correct, data are audited by a data manager, the researchers in charge, and statisticians. Finally, the database is password-protected, after which the data are not altered.

Quality control of the clinical trial

Participating researchers must undergo unified training. Unified data recording methods and evaluation criteria are used. During the clinical trial, sponsor inspectors conduct regular periodic visits to the research center to ensure strict adherence to all aspects of the research program. In addition, the original data are checked to ensure the contents of the case report forms are correct and complete.

Ethical considerations and informed consent

This clinical trial follows the relevant laws and regulations of the Declaration of Helsinki . This manuscript is prepared and modified according to SPIRIT guidelines (Additional file 1 [Additional file 1]). The researchers take the responsibility of providing the independent ethics committee with the clinical trial protocol and informed consent forms, and providing patients with related informational materials. The trial cannot be initiated until approval from ethics committee is received. This study was approved by the Ethics Committee of the Affiliated Hospital of Qinghai University of China (approval No. QHY1402G).


  Results Top


Trial status

This trial was registered with the Chinese Clinical Trial Registration (registration number: ChiCTR-ROC-17012848). Participant recruitment and data analysis are expected to be completed by February 2018.

Preliminary experiments

Preliminary experiments have been concluded.[28] Detection of serum indicators and bone metabolic markers were detected in 60 older adult male patients with degenerative osteoporosis and 58 healthy controls. In the osteoporosis group, HIF-1α levels positively correlated with VEGF and osteocalcin levels (P < 0.05), but were negatively correlated with bone mineral density and TRACP 5b level (P < 0.05). In addition, VEGF levels were negatively correlated with bone mineral density (P < 0.05).


  Discussion Top


Significance of this study

Findings from this study intend to confirm that for older adult male patients with degenerative osteoporosis residing in high-altitude areas, monitoring of bone mineral density and detection of hypoxia-inducible factor should be applied in addition to treatment of primary osteoporosis.

Limitations of this study

Patient cohort sample size is small and random grouping is not used. Serum indicators and bone metabolic markers are relatively simple indicators. All of these influence the accuracy of results. In future studies, these factors will be improved.[29],[30],[31]

Evidence for contribution to future studies

This study aimed to investigate the correlation between oxidative stress-related factors and bone metabolic markers in older adult male patients with degenerative osteoporosis who reside in a high-altitude hypoxic area. Findings from this study intend to confirm that for older adult patients with degenerative osteoporosis residing in high-altitude hypoxic areas, providing oxygen to improve hypoxia-induced injury along with calcium supplementation and increased exercise to increase bone mineral density can effectively prevent and treat osteoporosis.

 
  References Top

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Author contributions
Conception and design of study protocol: JWM and DCL; conduction of study protocol: ZGZ, YL, YBW and ZQC; data collection: JWM and DCL; trial evaluation: YL and ZQC.
Conflicts of interest
None declared.
Research ethics
The study protocol was approved by the Ethics Committee of Affiliated Hospital of Qinghai University of China (approval No. QHY1402G). The study followed international and national regulations in accordance with the Declaration of Helsinki and relevant ethical principles.
Declaration of participant consent
The authors certify that they will obtain participant consent forms. In the form, participants will give their consent for their images and other clinical information to be reported in the journal. Participants understand that their names and initials will not be published, and while due efforts will be made to conceal their identity, anonymity cannot be guaranteed.
Data sharing statement
Datasets analyzed during the current study are available from the corresponding author on reasonable request.
Plagiarism check
Checked twice by iThenticate.
Peer review
Externally peer reviewed.
Additional file
Additional file 1: SPIRIT checklist.


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