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CLINICAL INVESTIGATION

CLINICAL INVESTIGATION Circulating serum level of retinoic acid and hip fractures among postmenopausal women Xiao-Bin Li,

Circulating serum level of retinoic acid and hip fractures among postmenopausal women

Xiao-Bin Li, MD,* Tao Liu, MD, Lei Fan, MD, Qiang Gao, MD, Qiang Peng, MD, Teng Cai, MD, and Li-Min Wang, MD*

PURPOSE: The aim of this study was to evaluate the serum levels of retinoic acid (RA), an active form of vitamin A, in postmenopausal women with hip fractures from Zhengzhou, China. METHODS: This was a case-control study from the Af li- ated Hospital of Zhengzhou University. Serum samples were drawn from 375 postmenopausal women who were diagnosed as having hip fracture and 750 matched controls without fracture. Serum RA levels were evaluated as both a continuous variable and a categorical variable in quintiles. RESULTS: The results showed that the serum levels of RA were signicantly (P = .039) higher in patients with hip frac- ture compared with controls. In univariate and multivariate logistic regression analysis, for each 1 ng/mL increase of serum level of RA, the unadjusted and adjusted risk of hip fracture would be increased by 5% (odds ratio [OR] = 1.05; 95% condence interval [CI] = 1.00-1.10; P = .035) and 2% (OR = 1.02; 95% CI = 0.95-1.11; P = .096), respectively. In multivariate models comparing the fth with the third quin- tiles of RA, the RA was associated with hip fracture, and adjusted risk of hip fracture would be increased by 52% (OR = 1.52; 95% CI = 1.13-1.42; P = .011). CONCLUSIONS: The results of our study suggest that sub- clinical higher levels of RA may increase the risk of hip fracture in postmenopausal women, particularly among the top quintile of serum RA. J Am Geriatr Soc 00:1 6, 2018.

Key words: retinoic acid; vitamin A; hip fracture; post- menopausal women; Chinese

From the *Department of Orthopedics, The First Af liated Hospital of Zhengzhou University, Zhengzhou, China; and the Department of Orthopedics, Henan Provincial People s Hospital, Zhengzhou, China.

Address correspondence to Li-Min Wang, No. 1 Jianshe East Rd, Zhengzhou 453100, Henan Province, China E-mail: walmzzun13@163.com.

DOI: 10.1111/jgs.15667

O steoporosis is a major health threat. Men and women lose bone at a rate of 0.3% to 0.5% per year starting in

midlife. However, women experience accelerated bone loss at a rate of 3% to 5% per year for approximately 5 to 7 years after menopause, placing them at a particularly increased risk for fracture. 1 Furthermore, a previous study found that the rate of hip fracture had been increasing rapidly in Beijing, China, and the rates over the age of 50 years increased 58% in women and 49% in men from 2002 to 2006. 2 Previous studies had suggested that effects of toxic amounts of vitamin A stimulated bone resorption, inhibited bone formation, and might contribute to osteoporosis and

hip fractures. 3 In humans, chronic vitamin A toxicity can impair bone remodeling and bone abnormalities 4 and has been linked with decreased bone density and osteoporosis. 5 Some studies had indicated an increased risk of hip fracture 4,6 and low bone density 4,7 in women with a high dietary intake of vitamin A. In growing animals, hypervitaminosis A can lead to bone fractures 8 and other skeletal anomalies. 9 Although studies in animals and epidemiologic studies have indicated that a high vitamin A intake is associated with increased bone fragility, few biological markers of vitamin A status have thus far been used to assess the risk of fractures in humans. Feskanich et al 4 reported that long- term intake of a diet high in retinol may promote the devel- opment of osteoporotic hip fractures in women. However, another study found no evidence to support any skeletal harm associated with increased serum indexes of retinol exposure or modest retinol supplementation in this popula- tion. 10 In addition, serum retinol is a poor measure of vita- min A status because it is subject to homeostatic control. 11 Retinoic acid (RA) is the biologically active form of vitamin A, and its signaling is mediated by the RA and reti- noid X receptors. 12 Hotchkiss et al 13 found that bone min- eral density (BMD), bone mineral content, bone diameter, and cortical thickness of the femur were reduced in rats treated daily with 10 or 15 mg/kg all- trans-RA (ATRA) or 30 mg/kg 13- cis -RA. Retinoic acid induced osteoporosis was one of the most common causes of secondary osteopo- rosis. 14 Therefore, this study was performed to evaluate the serum levels of RA in postmenopausal women with hip fractures from Zhengzhou, China.

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PATIENTS AND METHODS

Included Participants

From January 1, 2017, to April 30, 2018, all postmenopausal women with hip fracture from the Department of Orthopedics of the First Afliated Hospital of Zhengzhou University (Zhengzhou, China) were recruited to participate in the study. All the fractures were conrmed by x-ray examination, and all x-ray examinations were done by the same radiologist. Patients with the following conditions were excluded: (1) those with fractures caused by a trafc accident or secondary to can- cer; (2) patients with calcium or vitamin A/D supplements; (3) patients with other metabolic disorders or internal disease, such as hyperthyroidism, chronic kidney disease, and autoim- mune diseases; (4) those with bilateral hip arthroplasty, hypo- calcemia, leucopenia, or impaired hepatic/renal function; and (5) those who died or were referred during research. For each fracture case, we sampled two healthy post- menopausal women matched for age at screening (+/1 year), race or ethnicity, and body mass index (BMI; +/1). This was a random population sample and came from a commu- nity located around the hospital. All control subjects were physically active (at work or able to perform home duties) within 1 month of the date of fracture diagnosis in the patient. If more than one potential control met the criteria, the control whose date of serum collection was closest to that of the patient was selected. Both groups comprised post- menopausal women who were not using estrogens or other bone-active therapies. This study was approved by the ethics committee of the First Afliated Hospital of Zhengzhou Uni- versity. All participants were informed of the study protocol, and their written informed consents were obtained according to the Declaration of Helsinki.

Clinical Information

At admission, all women answered a standardized question- naire designed to document putative risk factors of fractures. The collected information included age, BMI, race, current tobacco or alcohol use, history of falls during the past year, hip fracture type (cervical or trochanteric), cognitive impair- ment, neurologic impairment, number of concomitant dis- eases (hypertension, diabetes mellitus, neurologic disorders, hyperlipoproteinemia, and cardiovascular disease), surgical procedure type (arthroplasty or internal xation), and family history of fragility fractures. Cognitive impairment was assessed with the Mini-Mental State Examination (MMSE), and a score < 24 indicates the presence of a cognitive impair- ment. 15 Among the included patients, we identied the sub- group who sustained a concomitant fracture of the upper limb. 16 In this subgroup, a single fall resulted in both a hip and an upper limb fracture. In addition, total hip BMD of the proximal femur was measured using dual x-ray absorptiome- try (Hologic QDR2000/QDR4500; Hologic, Inc. Bedford MA, USA) in all the participants.

Laboratory Testing

Blood samples of participants were obtained at 8:00 AM on the day after admission. After centrifugation, aliquots of the serum samples were immediately stored at 80 C before the

assay. The serum levels of calcium, phosphorous, C-reactive protein (CRP), and alkaline phosphatase (ALP) were mea- sured by enzymatic assays (Autoanalyzer Model 7600 II; Hitachi, Tokyo, Japan) on the day of sample collection. Serum levels of RA were batch analyzed using a 96-plate commercial RA enzyme-linked immunosorbent assay (ELISA) kit (catalog number MBS705877; MyBioSource, San Diego, CA). This ELISA is a competitive immunoassay for the quantitative determination of RA and considered to be a sensitive and reliable assay. 17,18 No signicant cross- reactivity or interference between human RA and analogues was observed. Each sample was assayed for all time periods of participation in the same batch to avoid differences due to interassay variance. The relevant standard curve had the range of 0.8 to 10.0 ng/mL. The intra-assay and interassay coefcients of variation were 5.0% to 8.0% and 6.0% to 9.5%, respectively. We further validated the measurement of serum RA level by using the liquid chromatography/tandem mass spectrometry (LC-MS/MS) method, according to the previous report method 19 in a small sample (N = 50). The duplicate measures of between-serum RA by the ELISA and LC-MS/MS methods were highly correlated (r = 0.9685).

Statistical Analysis

The results are expressed as percentages for categorical var- iables and as median (interquartile range [IQR]) for contin- uous variables. Proportions were compared using the χ 2 test or the Mann-Whitney test between groups, as appropriate. Correlations among continuous variables were assessed by the Spearman rank-correlation coef cient. The relationship between serum levels of RA and risk of fractures was evaluated using univariate and multivariate regression analysis. For multivariate analysis, the possible con- founders included age, BMI, race, smoker, alcoholics, history of falls during the past year, number of concomitant diseases, MMSE, neurologic impairment, and serum levels of calcium, phosphate, ALP, and CRP. The results were expressed as adjusted odds ratios (ORs), with the corresponding 95% con- dence intervals (CIs). For a more detailed exploration of RA and risk of fractures, we also used multivariate analysis models to estimate adjusted ORs and 95% CIs of risk of frac- tures for RA quintiles (with third RA quintile as reference). The receiver operating characteristic (ROC) curve was used to evaluate the accuracy of serum RA in predicting fractures. The area under the curve (AUC) was calculated as a measure of the accuracy of the test. Last, the relation- ship between serum levels of RA and risk of hip fracture, combined with upper limb fractures, was also evaluated using univariate and multivariate regression analysis. All statistical analyses were done with SPSS for Windows, Ver- sion 22.0 (SPSS Inc, Chicago, IL); the ROCR package (ver- sion 1.0-2; http://rocr.bioinf.mpi-sb.mpg.de/); and GraphPad Prism 5.0 (GraphPad Software, CA, USA). Statis- tical signi cance was de ned as P < .05.

RESULTS

Patient Characteristics

Of the 405 patients, 30 were excluded (18 because of either major trauma or cancer and 12 because of death or transfer

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to other hospitals) and 375 patients were included in the study. The median age of patients was 66 (IQR, 58-75) years, and 92.8% were Chinese Han. The summary statis- tics of the study subjects are presented in Table 1. As shown in Table 1, we found that patients with hip fracture more frequently had a history of falls during the past year, con- comitant diseases, an MMSE score lower than 24 and neu- rologic impairment, low total hip BMD, and high serum levels of calcium, CRP, and ALP when compared with controls.

Main Results

The results showed that the serum levels of RA were signi - cantly ( P = .039) higher in patients with hip fracture com- pared with controls (3.63 [IQR, 1.52-5.39] ng/mL vs 3.19 [IQR, 1.26-5.01] ng/mL; Z = 2.062; Figure 1). There was a modest positive correlation between levels of RA and cal- cium ( r = 0.115, P = .017) and CRP ( r = 0.105, P = .030). In addition, there was a signi cant negative correlation between RA levels and BMD ( r = 0.139, P = .004). There were no correlations between serum levels of RA and other factors, such as age, race, BMI, cognitive impairment, neu- rologic impairment, infections, hip fracture type, family his- tory of fragility fracture, phosphorous, or ALP ( P > .05).

RA and Risk of Hip Fracture

In logistic regression analysis, we calculated the OR of RA levels compared with other risk factors. As a continuous variable, RA was associated with an increased risk of hip

fracture. In univariate and multivariate logistic regression analysis, for each 1 ng/mL increase of serum level of RA, the unadjusted and adjusted risk of hip fracture would be increased by 5% (OR = 1.05; 95% CI = 1.00-1.10;

P

= .035) and 2% (OR = 1.02; 95% CI = 0.95 1.11;

P

= .096), respectively. This relationship was con rmed in

the dose-response model. Furthermore, data showed that the hip fracture was associated with different concentra- tions of RA quintiles from a group with the third quintiles of serum RA (group 3, 27.4%) to a group with the highest quintiles (group 5, 42.6%) (Figure 2). Similarly, we also used multivariate analysis models to estimate adjusted ORs and 95% CIs of hip fracture for RA quintiles (with the third quintile as reference). In multivariate models compar- ing the fth with the third quintiles of RA (Table 2 and Figure 3), the RA was associated with hip fracture, and adjusted risk of hip fracture would be increased by 52% (OR = 1.52; 95% CI = 1.13-1.42; P = .011). In addition, comparing the rst, second, and fourth quintiles with the third quintile of RA (Table 2 and Figure 3), the RA was associated with hip fracture, and adjusted risk of hip frac- ture would be increased by 11%, 3%, and 28%, respec- tively. However, those results were not statistically signi cant ( P > .05, all). Using ROC curves, RA level at 3.4 ng/mL predicted the hip fracture with the highest sensitivity and speci city (53.6% and 56.1%, respectively; AUC = 0.54; 95% CI =

0.50-0.57; P = .039). RA levels had a higher predictive accu- racy compared with CRP (AUC = 0.50; 95% CI = 0.45-0.55;

P

= .031), calcium (AUC = 0.48; 95% CI = 0.43-0.53;

P

= .025), and ALP (AUC = 0.49; 95% CI = 0.44-0.54;

P = .029). When RA was added to the model containing established signicant risk factors, Area Under the Receiver Operating Characteristic curve (AUROC) (SE) was increased

from 0.60 (0.026) to 0.63 (0.023). A signi cant difference

in the AUC between the established risk factors alone and

the addition of RA was observed (difference, 0.03 [0.003]; P = .04).

Subgroup Analysis

In this study, 29 patients with hip fracture had concomitant

upper limb fractures. The results indicated that the serum RA levels were signi cantly ( P = .009) higher in patients with hip fracture with upper limb fracture compared with those without upper limb fracture (3.94 [IQR, 1.85-6.14] ng/mL vs 3.15 [IQR, 1.274.95] ng/mL). In univariate and multivariate logistic regression analysis, for each 1 ng/mL increase of serum level of RA, the unadjusted and adjusted risk of hip fracture would be increased by 10% (OR =

1.10; 95% CI = 1.02-1.18; P = .016) and 5% (OR = 1.05; 95% CI = 1.00-1.11; P = .039), respectively. Again, using ROC curves, RA level at 4.1 ng/mL predicted the hip frac- ture with upper limb fracture with the highest sensitivity and specicity (70.1% and 72.4%, respectively; AUC = 0.65; 95% CI = 0.59-0.72; P = .006). RA levels had a higher predictive accuracy compared with ALP (AUC = 0.55; 95% CI = 0.50-0.61; P = .003), calcium (AUC = 0.53; 95% CI = 0.48-0.60; P = .001), and CRP (AUC = 0.58; 95% CI = 0.51-0.66; P = .010).

DISCUSSION

Previous studies had showed that high intake of dietary vitamin A and serum retinol levels were associated with increased risk of hip fractures in humans. 4,6,7,20 However, there was substantial lack of consensus, possibly because of differences in method or study population. 21,22 RA, a meta- bolic product of vitamin A (retinol), has been reported to play an important role in osteoporosis and fractures. To the best of our knowledge, this is the rst time serum levels of RA were measured in Chinese postmenopausal women with hip fractures and their association with fracture risk was assessed. Our main ndings were the following: (1) serum levels of RA were higher in patients with hip fractures com- pared with normal controls; (2) high RA levels were associ- ated with low BMD levels; (3) comparing the fth with the third quintiles of RA, the RA was associated with hip frac- ture, and adjusted risk of hip fracture would be increased by 52% (OR = 1.52; 95% CI = 1.13-1.42; P = .011).

Results from observational studies of the association between vitamin A intake or serum concentration and BMD or fracture were mixed. 11 A weak association was found between retinol and femoral neck BMD in osteoporotic women. 23 A biochemical marker of retinol intake in relation to fracture risk had been reported in a cohort study 20 ; this study reported a 64% increase in risk of any osteoporotic fracture for men in the top quintile of serum retinol relative to those in the middle quintile. Similarly, another study found that elevated serum-retinol levels are associated with an increased risk of low bone mass and, thus, with osteopo- rotic fractures. 24 However, no association between vitamin

A or retinol intake and the risk of hip or total fractures was

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Table 1. Baseline Characteristics of Patients With Hip Fracture and Controls

Parameters

Patients

Controls

P Value 1

No.

375

750

Age, median (IQR), y

66 (58-75)

66 (58-75)

BMI, median (IQR), kg/m 2

26.5 (24.1-29.1)

26.9 (24.2-28.7)

.75

Han, No. (%)

348 (92.8)

703 (93.7)

.55

Smoker, No. (%)

67 (17.9)

127 (16.9)

.70

Alcoholics, No. (%)

32 (8.5)

59 (7.9)

.63

History of falls during the past year, median (IQR), No.

2 (1-3)

1 (0-2)

.033

Total hip BMD, median (IQR), g/cm 2

0.69 (0.66-0.71)

0.77 (0.75 0.78)

<.001

Hip fracture type, No. (%)

Femoral head fracture

168 (44.8)

Femoral neck fracture

155 (41.3)

Subtrochanteric

52 (13.9)

Concomitant upper-limb fractures, No. (%)

29 (7.7)

Concomitant diseases, median (IQR), No.

1 (1-2)

1 (0-1)

.018

MMSE score <24, No. (%)

61 (16.3)

71 (9.5)

.001

Neurologic impairment, No. (%)

71 (18.9)

88 (11.7)

.001

Hospital stay, median (IQR), d

12 (7-24)

Laboratory ndings, median (IQR)

Serum calcium, mmol/L

2.35 (2.20-2.43)

2.54 (2.32-2.71)

.048

Serum phosphate, mmol/L

1.42 (1.10-1.69)

1.50 (1.21-1.75)

.108

Serum ALP, IU/L

169 (107-286)

155 (98-243)

.043

Serum CRP, mg/L

4.8 (3.2-9.8)

4.2 (2.8-8.6)

.036

Serum RA, ng/mL

3.63 (1.52-5.39)

3.19 (1.26-5.01)

.039

Abbreviations: ALP, alkaline phosphatase; BMD, bone mineral density; BMI, body mass index; CRP, C-reactive protein; IQR, interquartile range; MMS E, Mini-Mental State Examination; RA, retinoic acid. 1 Compared using the χ 2 test or Mann-Whitney U test.

observed in postmenopausal women in the study by Caire- Juvera et al. 25 Similarly, Holvik et al 26 found no evidence of an adverse effect of high serum retinol on hip fracture, and Vestergaard et al 27 suggested that risk of fracture was not associated with vitamin A analogue treatment. Furthermore, another study suggested that inverse U-shaped association of retinol intake with BMD and bone maintenance observed in this cohort raises the concern that either too little or too much retinol may adversely affect bone health. 7 Consistent with this, we also found that either too little or too much RA may adversely affect bone health (Table 2 and Figure 2). Similarly, Opotowsky and Bilezikian 28 reported that both low and high serum vitamin A concentrations may be

that both low and high serum vitamin A concentrations may be Figure 1. Distribution of serum

Figure 1. Distribution of serum retinoic acid (RA) levels in postmenopausal women with hip fractures and normal con- trols. All data are medians and interquartile ranges. P values refer to Mann-Whitney U tests for differences between groups.

associated with an increased risk of hip fracture. The incon- sistencies of the previously described ndings might be due, in part, to difculties in obtaining an accurate assessment of vitamin A intake or status, 29 heterogeneity in terms of age or sex, 10 or different follow-up between exposure assessment and fracture. 20 Studies in rats had shown that supplementation with vitamin A was associated with an improvement in BMD 30 or had no effect on mineralization. 31 In this study, a nega- tive association between RA status and BMD had been found. However, other studies had reported a positive, rather than a negative, association between retinol status and BMD in postmenopausal women. 32 The mechanism by which RA (vitamin A) affects bones was unclear. Some possible mechanisms can be proposed.

bones was unclear. Some possible mechanisms can be proposed. Figure 2. The incidence rate for hip

Figure 2. The incidence rate for hip fractures and normal women according to the baseline retinoic acid (RA) quintiles (Qs).

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Table 2. Logistic Regression Model for Serum Concentrations of RA Using Hip Fracture as the Dependent Variables

RA Quintiles, ng/mL

Patients/Controls

Crude OR (95%CI); P Value 1

Multivariable-Adjusted OR; P Value 1,2

Quintile 1 (<1.06)

70/158

1.18 (0.78-1.76); .44

1.11 (0.65-1.82); .63

Quintile 2 (1.06-2.46)

65/158

1.09 (0.72-1.64); .69

1.03 (0.61-1.77); .76

Quintile 3 (2.47-4.02)

62/164

Reference

Reference

Quintile 4 (4.03-5.71)

83/142

1.55 (1.04-2.31); .032

1.28 (0.92-2.05); .12

Quintile 5 (>5.71)

95/128

1.96 (1.32-2.91); .001

1.52 (1.13-2.42); .011

Abbreviations: CI, con dence interval; OR, odds ratio; RA, retinoic acid. 1 P value for the trend <.001. 2 Adjusted for age, body mass index, race, smoker, alcoholics, history of falls during the past year, number of concomitant diseases, Mini-Mental Stat e Exami- nation score, neurologic impairment, and serum levels of calcium, phosphate, alkaline phosphatase, and C-reactive protein.

First, RA had been shown to both inhibit osteoblast activity and stimulate osteoclast formation and bone resorption. 33,34 Second, long-term ingestion of large amounts of vitamin A could lead to hypercalcemia. 35 Serum calcium might, thus, be regarded as having a role in the development of osteopo- rosis. 35,36 In this study, a positive association between RA status and calcium had been found. Third, RA increased pro- liferation of human osteoclast progenitors and inhibited receptor activator of nuclear factor κ B ligand (RANKL)- stimulated osteoclast differentiation by suppressing receptor activator of nuclear factor κ B (RANK). 37 Fourth, an interac- tion between vitamin A and vitamin D had been established. Vitamin A had been shown to be a weak antagonist of the actions of vitamin D to maintain normal serum calcium levels. 38,39 In a recent study in humans, a vitamin A dose corresponding to approximately one serving of liver was shown to severely diminish the ability of vitamin D to increase intestinal calcium absorption. 40 However, the ability of ATRA to cause bone resorption was not dependent on vitamin D3, dietary calcium, or dietary phosphorus. 38 Some limitations should be noted. First, the risk factors for fractures, including daily diet, physical activity, and physical performance, were not collected. Second, serum levels of RA in males were not tested. However, a previous study reported that the risk of fracture was highest among men with the highest levels of serum retinol. 20 Third, the relationship between serum RA and the concentration of RA in bone was uncertain. In addition, we did not obtain data on the prefracture levels of serum RA. It could be that an element of the fracture leads to the higher RA levels.

an element of the fracture leads to the higher RA levels. Figure 3. Multivariate logistic regression

Figure 3. Multivariate logistic regression model for serum reti- noic acid (RA) quintiles using hip fracture as the dependent variables. The third RA quintile was used as reference. CI indi- cates con dence interval; OR, odds ratio.

Interestingly, the patients with two fractures had even higher levels of RA than those with one fracture. Further study should be performed to resolve these questions. Finally, it is well known that the intestinal calcium absorption is decreased in postmenopausal osteoporosis and vitamin D may play an important role in postmenopausal osteoporosis. However, we did not measure serum vitamin D and vitamin Dbinding protein in this sample. Vitamin D and vitamin Dbinding protein may play a role in the process of fracture constant. 41,42 Interestingly, a previous study did not nd any interaction between retinol and 25-hydroxyvitamin D. 26 In addition, a previous study showed that the risk of fracture was not associated with the level of serum beta carotene. 20 This study did not evaluate the level of serum beta carotene in hip fractures in postmenopausal women and did not ana- lyze the role of beta carotene in the ndings of this study.

CONCLUSION

The results of our study suggest that subclinical higher levels of RA may increase the risk of hip fracture in post- menopausal women, particularly among the top quintile of serum RA. Monitoring the alterations in the serum levels of RA could be applied clinically as independent risk factors for hip fracture. Further studies are necessary to con rm this association.

ACKNOWLEDGMENTS

We express our gratitude to all the participants who partici- pated in this study and thereby made this work possible. This study was approved by the ethics committee of the First Afliated Hospital of Zhengzhou University. All par- ticipants were informed of the study protocol, and their written informed consents were obtained according to the Declaration of Helsinki. Financial Disclosure: This study was supported by a grant from science and technology project of Henan Prov- ince (2016HN16011). Conict of Interest: The authors have no con icts of interest. Author Contributions: All authors have contributed signi cantly, and all authors agree with the content of the article. L.M. Wang had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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Study concept and design: X.B. Lin, T. Liu, L. Fan,

Q.

Gao, Q. Peng, T. Cai, L.M. Wang. Acquisition of data: X.B. Lin, T. Liu, L. Fan. Analysis and interpretation of data: Q. Gao, Q. Peng,

T.

Cai, L.M. Wang.

Drafting of the manuscript: X.B. Lin, T. Liu, L.M. Wang. Critical revision of the manuscript for important intel- lectual content: L. Fan, Q. Gao, Q. Peng, T. Cai. Administrative, technical, or material support: T. Liu, L. Fan, Q. Gao, Q. Peng, T. Cai. Obtain funding: X.B. Lin. Study supervision: L.M. Wang. Sponsors Role: The funding organizations had no role in the design and concept of the study; the collection, man- agement, analysis, and interpretation of the data; or the preparation, review, or approval of the manuscript.

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