Endocrine Care 697
Low Levels of Free Testosterone Correlated with Bone Mineral Densities of Femoral Necks in Aged Healthy Shanghainese Men
Affiliations
Key words ▶ LC-MS/MS ● ▶ total testosterone ● ▶ free testosterone ● ▶ bone mineral density ● ▶ X-ray absorptiometry ●
T. Bo1, 2, J. M. Zhu2 1 2
Jiangsu University, Zhenjiang, Jiangsu Province, China Shanghai Xuhui Central Hospital & Shanghai Clinical Center, Chinese Academy of Sciences – Orthopaedics, Shanghai, China
Abstract
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The aim of the study was to investigate the influence of serum testosterone concentrations on bone mineral densities (BMDs) in healthy aged men in Shanghai area. Eighty-four participants, registered in the physical examination center of our hospital were included. Liquid chromatography tandem mass spectrometry (LC-MS/ MS) was used to measure concentrations of total testosterone (TT) and free testosterone (FT) in serum. BMDs of the lumbar spine, femoral neck, the trochanter major, and Ward’s triangle were determined with dual energy X-ray absorptiometry (DXA). The correlations of TT and FT with BMDs at the different skeleton sites were analyzed; stratified analyses by age were performed with 10-year range groups and the changing trends of TT and FT with increasing age were
further investigated. In addition, we performed a stratified quartile TT and FT analysis of their correlations with BMDs of different bones in each group. The average age of the participants was 71.8 ± 9.6 years (50–90 years). In a stratified analysis by age, no significant TT changes with increasing age was found, but there was a significant decrease of FT in men older than 80 years (p < 0.05). In a stratified FT quartile analysis, FT in the first quartile group correlated significantly with BMDs of the lumbar spine, femoral neck and Ward’s triangle; however, there was only a significant correlation between FT and BMD of the femoral neck after adjustment for age and body mass index (BMI). FT blood serum concentrations decreased significantly in healthy men aged over 80 and positively correlated with BMDs of femoral necks.
received 17.12.2013 accepted 24.04.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1375687 Published online: May 27, 2014 Horm Metab Res 2014; 46: 697–701 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence J. M. Zhu Shanghai Xuhui Central Hospital & Shanghai Clinical Center Chinese Academy of Sciences – Orthopaedics 966 Huai Hai Middle Road Shanghai 200030 China Tel.: + 86/136/41629 279 Fax: + 86/021/200 031 [email protected]
Introduction
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The importance of estrogen for the maintenance of female bone minerals has been widely accepted, and accelerated bone loss generally occurs in postmenopausal women due to plunge of estrogen. Testosterone is considered to play a similar role in male bones and the discovery of androgen receptors in bone tissue has provided evidence [1]. In addition, men with hypogonadism often had reduced BMD [2], and androgen deprivation therapies for patients with prostate cancer lead to osteoporosis and increased fracture risk [3]. In a testosterone and BMD correlation study, Fink et al. demonstrated that there was a significant association between low level of testosterone and bone mineral density [4], whereas other studies have reported no correlation between testosterone levels and BMD [5]. The reason for this discrepancy might be that BMD is affected by multiple factors besides testosterone, including physical activity, chronic dis-
ease, socioeconomic status, nutritional intake, age, and BMI; the latter 2 have also attracted broad attention. Results from a comparison between 2 studies without control of these factors may be unconvincing. In addition, for the determination of testosterone, immunoassay and enzyme-linked methods were mostly employed [6, 7], but recent studies [8, 9] have revealed that there is obvious errors for immunoassay for the low testosterone concentrations and the results based on these methods could be questionable. Meanwhile LC-MS/MS is the gold standard for the detection of testosterone since researchers reported its higher sensitivity and specificity compared to conventional radioimmunoassays for quantifications of low sex hormone concentrations [8, 10]. Ammonium sulfate precipitation is a reference method for detection of FT [11], but the procedure is cumbersome, which limited its routine use. In another method [12], free testosterone (FT) is calculated by total testosterone (TT) and sex hormone binding globulin, but the
Bo T, Zhu JM. Testosterone with Bone Mineral Density … Horm Metab Res 2014; 46: 697–701
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Authors
698 Endocrine Care
Subjects and Methods
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Subjects From July 2012 to August 2013, healthy aged men (50–90 years old) registered in the physical examination center of the Shanghai Xuhui district central hospital were prospectively involved. Exclusion criteria were endocrine disorders affecting the bone metabolism (diabetes, thyroid or parathyroid dysfunctions, growth hormone deficiency, Cushing syndrome) as well as history of chronic kidney diseases, chronic gastrointestinal diseases, blood system tumors, bone tumors or tumor bone metastasis, hip or lumbar fractures, other osteopathies, and history of taking drugs affecting the bone metabolism. This study was approved by the hospital ethics council and written informed consents were obtained from all participants.
Grouping criteria 1) Ten years was set as an age group range and subjects were divided into 4 groups: 50–59, 60–69, 70–79 and 80–89 years old; and 2) based on quartiles of total and FT, subjects were divided into 4 groups as above.
Measurements of bone mineral densities
with Q1/Q3 ion transitions at m/z 292.1/109.0 amu. The source parameters were set as follows: curtain gas 10, CAD 10, GS1 28, GS2 60, NC 4, and source temperature at 300 °C. The decluster potential, entrance potential, collision energy, and collision cell exit potential were optimized as 74 V, 10 V, 36 V, and 10 V by infusion of standard testosterone and testosterone-d3 solutions (both 1 μg/ml in methanol) to the mass spectrometer. Instrument control as well as data acquisition and processing were performed with Analyst 1.5 software (Applied Biosystems/MDS Sciex).
FT determination by ultrafiltration LC-MS/MS Aliquot of 0.5 ml serum was diluted with 0.5 ml HEpES buffer. After equilibrating at room temperature for 5 min, the mixture was transferred to a Centrifree® UF device and immediately centrifuged (1 800 g) at 25 °C in a fixed angle rotor for 1 h. Testosterone-2,2,4,6,6-d5 (40 fmol) was added to 0.5 ml of the ultrafiltrate, the mixture was vortex-mixed for 5 s, and then incubated for 5 min at room temperature. Testosterone was extracted with 1 ml of methyl tert-butyl ether (MTBE), which was evaporated under a stream of nitrogen at 40 °C and the residue was dissolved in 1 ml of 90 % methanol to which 1 ml of heptane was added. After shaking, the top heptane layer was discarded and the bottom methanol layer was transferred to clean tubes and evaporated to dryness. The residues were dissolved in 70 μl 50 % methanol and 50 μl aliquots were analyzed by LC-MS/MS.
Statistical analysis The SPSS16.O statistical software was used for data processing and analysis. Measurement data with normal distribution are presented as mean ± standard deviation; analysis of variance was performed for comparison of data among groups, and Scheffe test was used for post hoc multiple comparisons. Pearson correlation was employed for bivariate correlations and a partial correlation analysis was performed after age and BMI were adjusted. A p < 0.05 was considered as statistically significant.
Results
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Basic data of the participants
We used a dual energy X-ray absorptiometry apparatus (Hologic Inc. ASY-0049 type. Bedford, USA) to measure bone mineral densities of the lumbar spine (L1–L4), femoral neck, the trochanter major, and Ward’s triangle. The resulting data are presented as g/cm2.
In total, 84 healthy aged subjects registered in our hospital were included and the average age was 71.8 ± 9.6 (50–90 years old) ▶ Table 1). (●
Testosterone measurements by LC-MS/MS TT determination
In the overall analysis, TT significantly correlated with bone mineral density of Ward’s triangle (p = 0.043), but the signifi-
Chromatographic separations were achieved with an analytical Chromolith Speed Rod column (50 × 4.6 mm, 5 μm, Merck) at a flow rate of 1.0 ml/min with 0.2 % formic acid in methanol and 0.2 % formic acid in water (70:30, v/v) as mobile phase in an Agilent 1200 liquid chromatography system (Santa Clara, CA, USA) consisting of a G1312A binary pump, a G1367B Hip ALS autosampler, a G1379B degasser, and a G1316A TCC column. The liquid chromatography system was coupled to an API 4000 triple quadrupole mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada) equipped with an atmospheric pressure chemical ionization (APCI) source. Testosterone was detected in positive multiple reaction monitoring (MRM) mode using primary Q1/Q3 ion transitions at m/z 289.1/109.0 amu and secondary ion transitions at m/z 289.1/97.0 amu. Testosterone-d3 was detected
The overall analysis of correlations between TT, FT, and bone mineral density at different sites
Table 1 Basic characteristics of the study participants. Mean ± standard deviation Number of subjects Age (years) Body weight (kg) Height (m) BMI (kg/m2) Total testosterone (ng/ml) Free testosterone (pg/ml) BMD of the lumbar spine (g/cm2) BMD of the femoral neck (g/cm2) BMD of the trochanter major (g/cm2) BMD of Ward’s triangle (g/cm2)
Bo T, Zhu JM. Testosterone with Bone Mineral Density … Horm Metab Res 2014; 46: 697–701
84 71.8 ± 9.6 67.4 ± 3.7 1.71 ± 0.02 23.2 ± 1.2 4.14 ± 0.93 10.72 ± 1.28 1.024 ± 0.079 0.770 ± 0.034 0.758 ± 0.038 0.648 ± 0.051
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accuracy is also questionable. Recently, ultrafiltration in combination with LC-MS/MS for detection of FT [13] was launched by Canadian scholars and its sensitivity and specificity have been confirmed after comparison with the ammonium sulfate precipitation method. The new approach is simple, reliable and can be used routinely. Here we use an LC-MS/MS method for detection of both of TT and FT, which is routinely applied in the clinical laboratory in our hospital. This method has the lower limit of quantification at 0.02 ng/ml for TT and at 1 pg/ml for FT, which is sensitive enough to determine the low level of testosterone in the aged people. We adopted LC-MS/MS and DXA for investigating the changing trend of testosterone serum concentrations with increasing age and its correlation with bone mineral densities of the lumbar spine, the femoral neck, the trochanter major, and Ward’s triangle in healthy aged men in Shanghai area.
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Stratified analysis by age Based on age, all 84 participants were divided into 50–59, 60–69, 70–79, and 80–89 year old groups. There was no significant difference of TT levels among the groups in variance analyses (p = 0.078), but we detected significant differences of FT, BMD of the lumbar spine, BMD of the trochanter major, BMD of the femoral neck, and BMD of Ward’s triangle among the groups (p < 0.05). Further analysis by post hoc multiple comparison (Scheffe test) suggested that FT, BMD of the lumbar spine, BMD of the trochanter major, and BMD of Ward’s triangle in the 80–90 year-old group were significantly different than in the 50–59, 60–69, and 70–79 year-old groups (p < 0.05). The BMD of the femoral neck in the 80–90 year-old group was significantly different from that in the 50–59 and 70–79 year-old groups ▶ Table 3). (p < 0.05) (●
Stratified analysis by TT quartiles In order to clarify correlations between testosterone and bone mineral density, all subjects were divided into 4 groups ( < 3.53 ng/ ml, 3.53–3.91 ng/ml, 3.91–4.76 ng/ml, and > 4.76 ng/ml) based on quartile values of TT (3.53, 3.91, 4.76 ng/ml) in serum concentrations. As a result of it, we did not find significant BMD differences of the lumbar spine, femoral neck, trochanter major, and Ward’s ▶ Table 4). triangle among the groups (● Analysis of bivariate correlations suggested that there were no significant correlations between TT and BMD of the lumbar spine, the femoral neck or Ward’s triangle in the < 3.53 ng/ml group, but they were all close to statistical significance (p = 0.090, p = 0.097, and p = 0.055). In the other groups, no significant correlations of ▶ Table 5). TT with BMD at different bone sites were detectable (●
Stratified analysis by quartiles of FT Next, all 84 subjects were divided into 4 groups ( < 9.81 pg/ml, 9.81–10.71 pg/ml, 10.71–11.46 pg/ml, and > 11.46 pg/ml) based Table 2 Correlations of total testosterone (TT) and free testosterone (FT) with BMD at different bone sites.
TT FT
Lumbar
The trochanter
Femoral
spine
major
neck
Ward’s triangle
r = 0.116 p = 0.293 r = 0.177 p = 0.108
r = 0.044 p = 0.694 r = 0.208 p = 0.057
r = 0.136 p = 0.219 r = 0.156 p = 0.157
r = 0.221 p = 0.043* r = 0.086 p = 0.435
*After adjustment for age and BMI, p = 0.103
on their FT quartiles (9.81 pg/ml, 10.71 pg/ml, 11.46 pg/ml), and except for the BMD of the lumbar spine (p = 0.042), we did not find significant BMD differences of the femoral neck, the trochanter ▶ Table 6). major, and Ward’s triangle among the groups (● Analysis of bivariate correlation suggested that there were significant correlations between FT and BMDs of the lumbar spine, the femoral neck, and Ward’s triangle in the < 9.81 pg/ml group (p < 0.05), but after adjustment for age and BMI, a significant correlation only remained between FT and BMD of the femoral neck ▶ Table 7). (p < 0.05) (●
Discussion
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To determine the low level of testosterone in samples from the aged men, the sensitivity is considered as the most important for the method. However, most of the immunoassay-based method cannot be competent to such a tough task. Therefore, accurate and sensitive measurement of low level of testosterone is essential for correct diagnosis and appropriate treatment in clinical setting. Here we present an LC-MS/MS method with single step liquid-liquid extraction sample preparation procedure and APCI detection that allow the rapid and extremely sensitive measurement of serum TT and FT. Some recent studies suggested that testosterone concentrations remain stable in men aged less than 70 years and then decrease significantly [14, 15]. Our study results showed that the TT serum concentrations had a decreasing trend with increasing age, but without statistical significance, and FT values were only significantly lower in elderly men over 80 years. Our finding is supported by recent research, in which testosterone levels did not decrease stably with increasing age and mainly became obvious in men over 70 years old [10, 16]. The decreased levels were more apparent in FT, while TT was relatively stable, which might be a result of increased sex hormone binding protein expression and is in line with our results. The average FT level in our group was 10.72 ± 1.28 pg/ml, which was considerably lower than those reported in other research study (about 20 pg/ml) [10]. However, this study was done with a Caucasian population, whereas Orwoll et al. [14] reported that the level of FT in Asians was lower than that in other races and recently, Yoshimura et al. [17] noted that the average FT serum concentrations in healthy Japanese men older than 60 years was about 8.8 pg/ml and similar with our results. Our overall analysis of correlations between testosterone and BMDs showed that neither TT nor FT significantly correlated with BMDs at different bone sites, although there was a significant correlation of TT with BMD of the Ward’s triangle (p = 0.043), which disappeared after adjustment for age
Table 3 By age stratified analysis.
Number Age Body weight Height BMI TT FT BMD of the lumbar spine BMD of the trochanter major BMD of the femoral neck BMD of Ward’s triangle
50–59
60–69
70–79
80–89
10 53.1 ± 2.8 71.6 ± 2.7 1.71 ± 0.03 24.5 ± 0.9 4.42 ± 1.15 10.76 ± 1.36 1.085 ± 0.084 0.781 ± 0.023 0.778 ± 0.041 0.683 ± 0.041
21 64.9 ± 2.7 68.1 ± 3.5 1.70 ± 0.02 23.5 ± 1.1 4.31 ± 1.06 10.71 ± 1.64 1.002 ± 0.086 0.778 ± 0.035 0.754 ± 0.036 0.637 ± 0.044
35 75.6 ± 2.1 66.9 ± 3.6 1.71 ± 0.02 22.9 ± 1.0 4.20 ± 0.75 10.87 ± 1.20 1.023 ± 0.077 0.775 ± 0.025 0.767 ± 0.030 0.668 ± 0.042
18 82.8 ± 1.7 65.9 ± 3.0 1.71 ± 0.03 22.7 ± 1.0 3.65 ± 0.85 10.45 ± 0.89 1.017 ± 0.058 0.742 ± 0.042 0.735 ± 0.041 0.604 ± 0.047
Bo T, Zhu JM. Testosterone with Bone Mineral Density … Horm Metab Res 2014; 46: 697–701
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cance disappeared after adjustment for age and BMI (p = 0.103). No significant correlation was found between FT and bone min▶ Table 2). eral density at all other bone sites (●
700 Endocrine Care
< 3.53 3.53–3.91 3.91–4.76 > 4.76
< 9.81 9.81–10.71 10.71–11.46 > 11.46 F-value p-value
The trochanter major
Femoral neck
Ward’s triangle
1.006 ± 0.074 1.021 ± 0.066 1.050 ± 0.088 1.019 ± 0.084 1.171 0.326
0.762 ± 0.039 0.779 ± 0.046 0.770 ± 0.056 0.768 ± 0.069 0.872 0.459
0.752 ± 0.039 0.753 ± 0.047 0.770 ± 0.049 0.757 ± 0.057 1.159 0.331
0.631 ± 0.053 0.655 ± 0.053 0.647 ± 0.051 0.659 ± 0.050 1.216 0.309
Lumbar spine
The trochanter major
Femoral neck
Ward’s triangle
r = 0.379 p = 0.090 r = 0.449 p = 0.410 r = 0.102 p = 0.660 r = 0.290 p = 0.203
r = 0.317 p = 0.161 r = 0.437 p = 0.348 r = 0.262 p = 0.251 r = 0.001 p = 0.995
r = 0.372 p = 0.097 r = 0.284 p = 0.424 r = 0.129 p = 0.578 r = 0.178 p = 0.439
r = 0.425 p = 0.055 r = 0.106 p = 0.649 r = 0.244 p = 0.287 r = 0.355 p = 0.114
Lumbar spine
The trochanter major
Femoral neck
Ward’s triangle
0.991 ± 0.078 1.060 ± 0.090 1.018 ± 0.065 1.027 ± 0.070 2.868 0.042*
0.757 ± 0.038 0.777 ± 0.051 0.766 ± 0.032 0.777 ± 0.055 1.639 0.187
0.746 ± 0.044 0.772 ± 0.054 0.754 ± 0.056 0.761 ± 0.045 1.830 0.148
0.638 ± 0.060 0.669 ± 0.048 0.639 ± 0.046 0.647 ± 0.052 1.635 0.188
Lumbar spine
The trochanter major
Femoral neck
Ward’s triangle
r = 0.534 p = 0.013* r a = 0.584 p a = 0.099 r = 0.025 p = 0.914 r = 0.249 p = 0.277 r = 0.051 p = 0.826
r = 0.164 p = 0.477
r = 0.515 p = 0.017* r a = 0.777 p a = 0.000** r = 0.176 r = 0.446 r = 0.355 p = 0.114 r = 0.311 p = 0.170
r = 0.479 p = 0.028* r a = 0.691 p a = 0.101 r = 0.391 p = 0.080 r = 0.119 p = 0.608 r = 0.078 p = 0.736
Table 4 Comparison of BMDs at different bone sites among the 4 quartile TT groups.
Table 5 Correlation of TT with BMDs at different bone sites in each quartile TT group.
Table 6 Comparison of BMD at different bone sites among the 4 FT quartile groups.
*p < 0.05
< 9.81
9.81–10.71 10.71–11.46 > 11.46
r = 0.064 p = 0.782 r = 0.057 p = 0.806 r = 0.043 p = 0.852
Table 7 Correlation of FT with BMD at different bone sites in each quartile FT group.
r a refers to the r-value after age and BMI adjustment p a refers to the p-value after age and BMI adjustment *p < 0.05 **p < 0.01
and BMI (p = 0.103). Similarly, a significant correlation only remained between FT and BMD of the femoral neck (p < 0.05) after adjustment was made for age and BMI in the stratified analysis by FT quartile. To our knowledge, FT level was reported to related significantly to the rate of change for BMD at femoral neck during the first 3 years and to incidence of osteoporosis after adjustment for age and BMI in a study by Yoshimura et al. [17]. The authors did not mention whether the adjustment for age and BMI lead to change of relation between FT levels and BMD in lumbar spine, which was observed in our study. However, age did have an effect on BMD in this report, and BMI was also reported to correlate with BMD, the adjustment for them would be meritorious. Meanwhile, there seems to be a trend toward association between TT and BMD of Ward’s triangle, or FT and lumbar spine, Ward’s triangle after age and BMI adjustment, which may be attributed to the fact that testosterone is an important but may not be the single influencing factor on BMD. Effect of transder-
mal testosterone treatment on bone and muscle in elder men with low bioavailable testosterone was investigated by Kenny et al. [18]. They found that when testosterone levels before treatment were < 10.5 nmol/l, the increase of BMD was 3.4 ± 1.2 %; when testosterone levels before treatment were < 7 nmol/l, the increase of BMD was 5.9 ± 2.2 %. These data showed that testosterone at low concentrations have a significant effect on BMDs and a correlation of high testosterone serum concentrations and BMDs is not obvious. When testosterone concentrations maintain stable at a relative high level, the correlation of testosterone with BMD is not easy to detect without avoiding shielding effects of other factors by precise design, because BMD is not only influenced by sex hormones but also by age, BMI, and other factors [19]. Thus, we further analyzed the correlation of testosterone with BMD in quartile groups of TT and FT. The results showed that FT in the first quartile group independently correlated positively with the BMD of the femoral neck. In addition, we found significant BMD differences only in the lumbar spine among the
Bo T, Zhu JM. Testosterone with Bone Mineral Density … Horm Metab Res 2014; 46: 697–701
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< 3.53 3.53–3.91 3.91–4.76 > 4.76 F-value p-value
Lumbar spine
4 quartile FT groups, which suggested that increases of testosterone concentrations caused little BMD changes when they were already high, thereby confirming our hypothesis. Snyder et al. noted, that BMD improvements only occurred when testosterone levels were < 2 ng/ml [20]. In an epidemiological study [16], Cauley et al. found that major hip bone loss over 1.8 years occurred only in men in the lowest FT category, which also proved that only low levels of FT are correlating with BMDs. Kaufman et al. found that TT maintained relatively stable, but FT significantly decreased in elderly men [21]. We suggest that a decrease of FT correlates with BMD, which is supported by previous studies [5, 22]. However, FT correlated different with lumbar spine, femoral neck, the trochanter major and Ward’s triangle and only the BMD of the femoral neck correlated independently with low FT levels after adjustment for age and BMI. Men with complete androgen insensitivity syndrome had different degrees of spine and hip BMD reductions [23] and a random, double-blind, placebo control trial revealed that improvement of BMD mainly occurred in the lumbar spine and femoral neck after testosterone therapy [18]. A similar result was also reported in another previous study [24], but some degenerative changes, such as osteophytosis or sclerotic alterations occur more frequently in the lumbar spine than in the femoral neck and these changes increase BMDs complicating the correlation of testosterone and BMD in these bones. Our study has some limitations. First, the small sample seemed to be the most severe weakness, but subjects like our healthy aged men were hard to collect, because people at this age frequently suffer from diseases and most of them have affected BMDs. Second, as a static study, we could not investigate the dynamic BMD changes with decreasing testosterone concentrations, while following of a particular cohort would give more information about correlations between testosterone and BMDs.
Conclusions
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In conclusion, based on LC-MS/MS method, we analyzed the correlation of testosterone (TT and FT) with BMD. Our conclusions are that after the age of about 80 years old, FT serum concentrations decrease significantly in healthy men in Shanghai area and positively correlated with the BMD of the femoral neck.
Acknowledgements
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The investigations were carried out at Shanghai Xuhui Central Hospital & Shanghai Clinical Center, Chinese Academy of Sciences.
Conflicts of Interest
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The authors declare that they have no conflicts of interest in the authorship or publication of this contribution.
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Endocrine Care 701
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Low Levels of Free Testosterone Correlated with Bone Mineral Densities of Femoral Necks in Aged Healthy Shanghainese Men
Affiliations
Key words ▶ LC-MS/MS ● ▶ total testosterone ● ▶ free testosterone ● ▶ bone mineral density ● ▶ X-ray absorptiometry ●
T. Bo1, 2, J. M. Zhu2 1 2
Jiangsu University, Zhenjiang, Jiangsu Province, China Shanghai Xuhui Central Hospital & Shanghai Clinical Center, Chinese Academy of Sciences – Orthopaedics, Shanghai, China
Abstract
▼
The aim of the study was to investigate the influence of serum testosterone concentrations on bone mineral densities (BMDs) in healthy aged men in Shanghai area. Eighty-four participants, registered in the physical examination center of our hospital were included. Liquid chromatography tandem mass spectrometry (LC-MS/ MS) was used to measure concentrations of total testosterone (TT) and free testosterone (FT) in serum. BMDs of the lumbar spine, femoral neck, the trochanter major, and Ward’s triangle were determined with dual energy X-ray absorptiometry (DXA). The correlations of TT and FT with BMDs at the different skeleton sites were analyzed; stratified analyses by age were performed with 10-year range groups and the changing trends of TT and FT with increasing age were
further investigated. In addition, we performed a stratified quartile TT and FT analysis of their correlations with BMDs of different bones in each group. The average age of the participants was 71.8 ± 9.6 years (50–90 years). In a stratified analysis by age, no significant TT changes with increasing age was found, but there was a significant decrease of FT in men older than 80 years (p < 0.05). In a stratified FT quartile analysis, FT in the first quartile group correlated significantly with BMDs of the lumbar spine, femoral neck and Ward’s triangle; however, there was only a significant correlation between FT and BMD of the femoral neck after adjustment for age and body mass index (BMI). FT blood serum concentrations decreased significantly in healthy men aged over 80 and positively correlated with BMDs of femoral necks.
received 17.12.2013 accepted 24.04.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1375687 Published online: May 27, 2014 Horm Metab Res 2014; 46: 697–701 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence J. M. Zhu Shanghai Xuhui Central Hospital & Shanghai Clinical Center Chinese Academy of Sciences – Orthopaedics 966 Huai Hai Middle Road Shanghai 200030 China Tel.: + 86/136/41629 279 Fax: + 86/021/200 031 [email protected]
Introduction
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The importance of estrogen for the maintenance of female bone minerals has been widely accepted, and accelerated bone loss generally occurs in postmenopausal women due to plunge of estrogen. Testosterone is considered to play a similar role in male bones and the discovery of androgen receptors in bone tissue has provided evidence [1]. In addition, men with hypogonadism often had reduced BMD [2], and androgen deprivation therapies for patients with prostate cancer lead to osteoporosis and increased fracture risk [3]. In a testosterone and BMD correlation study, Fink et al. demonstrated that there was a significant association between low level of testosterone and bone mineral density [4], whereas other studies have reported no correlation between testosterone levels and BMD [5]. The reason for this discrepancy might be that BMD is affected by multiple factors besides testosterone, including physical activity, chronic dis-
ease, socioeconomic status, nutritional intake, age, and BMI; the latter 2 have also attracted broad attention. Results from a comparison between 2 studies without control of these factors may be unconvincing. In addition, for the determination of testosterone, immunoassay and enzyme-linked methods were mostly employed [6, 7], but recent studies [8, 9] have revealed that there is obvious errors for immunoassay for the low testosterone concentrations and the results based on these methods could be questionable. Meanwhile LC-MS/MS is the gold standard for the detection of testosterone since researchers reported its higher sensitivity and specificity compared to conventional radioimmunoassays for quantifications of low sex hormone concentrations [8, 10]. Ammonium sulfate precipitation is a reference method for detection of FT [11], but the procedure is cumbersome, which limited its routine use. In another method [12], free testosterone (FT) is calculated by total testosterone (TT) and sex hormone binding globulin, but the
Bo T, Zhu JM. Testosterone with Bone Mineral Density … Horm Metab Res 2014; 46: 697–701
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Authors
698 Endocrine Care
Subjects and Methods
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Subjects From July 2012 to August 2013, healthy aged men (50–90 years old) registered in the physical examination center of the Shanghai Xuhui district central hospital were prospectively involved. Exclusion criteria were endocrine disorders affecting the bone metabolism (diabetes, thyroid or parathyroid dysfunctions, growth hormone deficiency, Cushing syndrome) as well as history of chronic kidney diseases, chronic gastrointestinal diseases, blood system tumors, bone tumors or tumor bone metastasis, hip or lumbar fractures, other osteopathies, and history of taking drugs affecting the bone metabolism. This study was approved by the hospital ethics council and written informed consents were obtained from all participants.
Grouping criteria 1) Ten years was set as an age group range and subjects were divided into 4 groups: 50–59, 60–69, 70–79 and 80–89 years old; and 2) based on quartiles of total and FT, subjects were divided into 4 groups as above.
Measurements of bone mineral densities
with Q1/Q3 ion transitions at m/z 292.1/109.0 amu. The source parameters were set as follows: curtain gas 10, CAD 10, GS1 28, GS2 60, NC 4, and source temperature at 300 °C. The decluster potential, entrance potential, collision energy, and collision cell exit potential were optimized as 74 V, 10 V, 36 V, and 10 V by infusion of standard testosterone and testosterone-d3 solutions (both 1 μg/ml in methanol) to the mass spectrometer. Instrument control as well as data acquisition and processing were performed with Analyst 1.5 software (Applied Biosystems/MDS Sciex).
FT determination by ultrafiltration LC-MS/MS Aliquot of 0.5 ml serum was diluted with 0.5 ml HEpES buffer. After equilibrating at room temperature for 5 min, the mixture was transferred to a Centrifree® UF device and immediately centrifuged (1 800 g) at 25 °C in a fixed angle rotor for 1 h. Testosterone-2,2,4,6,6-d5 (40 fmol) was added to 0.5 ml of the ultrafiltrate, the mixture was vortex-mixed for 5 s, and then incubated for 5 min at room temperature. Testosterone was extracted with 1 ml of methyl tert-butyl ether (MTBE), which was evaporated under a stream of nitrogen at 40 °C and the residue was dissolved in 1 ml of 90 % methanol to which 1 ml of heptane was added. After shaking, the top heptane layer was discarded and the bottom methanol layer was transferred to clean tubes and evaporated to dryness. The residues were dissolved in 70 μl 50 % methanol and 50 μl aliquots were analyzed by LC-MS/MS.
Statistical analysis The SPSS16.O statistical software was used for data processing and analysis. Measurement data with normal distribution are presented as mean ± standard deviation; analysis of variance was performed for comparison of data among groups, and Scheffe test was used for post hoc multiple comparisons. Pearson correlation was employed for bivariate correlations and a partial correlation analysis was performed after age and BMI were adjusted. A p < 0.05 was considered as statistically significant.
Results
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Basic data of the participants
We used a dual energy X-ray absorptiometry apparatus (Hologic Inc. ASY-0049 type. Bedford, USA) to measure bone mineral densities of the lumbar spine (L1–L4), femoral neck, the trochanter major, and Ward’s triangle. The resulting data are presented as g/cm2.
In total, 84 healthy aged subjects registered in our hospital were included and the average age was 71.8 ± 9.6 (50–90 years old) ▶ Table 1). (●
Testosterone measurements by LC-MS/MS TT determination
In the overall analysis, TT significantly correlated with bone mineral density of Ward’s triangle (p = 0.043), but the signifi-
Chromatographic separations were achieved with an analytical Chromolith Speed Rod column (50 × 4.6 mm, 5 μm, Merck) at a flow rate of 1.0 ml/min with 0.2 % formic acid in methanol and 0.2 % formic acid in water (70:30, v/v) as mobile phase in an Agilent 1200 liquid chromatography system (Santa Clara, CA, USA) consisting of a G1312A binary pump, a G1367B Hip ALS autosampler, a G1379B degasser, and a G1316A TCC column. The liquid chromatography system was coupled to an API 4000 triple quadrupole mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada) equipped with an atmospheric pressure chemical ionization (APCI) source. Testosterone was detected in positive multiple reaction monitoring (MRM) mode using primary Q1/Q3 ion transitions at m/z 289.1/109.0 amu and secondary ion transitions at m/z 289.1/97.0 amu. Testosterone-d3 was detected
The overall analysis of correlations between TT, FT, and bone mineral density at different sites
Table 1 Basic characteristics of the study participants. Mean ± standard deviation Number of subjects Age (years) Body weight (kg) Height (m) BMI (kg/m2) Total testosterone (ng/ml) Free testosterone (pg/ml) BMD of the lumbar spine (g/cm2) BMD of the femoral neck (g/cm2) BMD of the trochanter major (g/cm2) BMD of Ward’s triangle (g/cm2)
Bo T, Zhu JM. Testosterone with Bone Mineral Density … Horm Metab Res 2014; 46: 697–701
84 71.8 ± 9.6 67.4 ± 3.7 1.71 ± 0.02 23.2 ± 1.2 4.14 ± 0.93 10.72 ± 1.28 1.024 ± 0.079 0.770 ± 0.034 0.758 ± 0.038 0.648 ± 0.051
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accuracy is also questionable. Recently, ultrafiltration in combination with LC-MS/MS for detection of FT [13] was launched by Canadian scholars and its sensitivity and specificity have been confirmed after comparison with the ammonium sulfate precipitation method. The new approach is simple, reliable and can be used routinely. Here we use an LC-MS/MS method for detection of both of TT and FT, which is routinely applied in the clinical laboratory in our hospital. This method has the lower limit of quantification at 0.02 ng/ml for TT and at 1 pg/ml for FT, which is sensitive enough to determine the low level of testosterone in the aged people. We adopted LC-MS/MS and DXA for investigating the changing trend of testosterone serum concentrations with increasing age and its correlation with bone mineral densities of the lumbar spine, the femoral neck, the trochanter major, and Ward’s triangle in healthy aged men in Shanghai area.
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Stratified analysis by age Based on age, all 84 participants were divided into 50–59, 60–69, 70–79, and 80–89 year old groups. There was no significant difference of TT levels among the groups in variance analyses (p = 0.078), but we detected significant differences of FT, BMD of the lumbar spine, BMD of the trochanter major, BMD of the femoral neck, and BMD of Ward’s triangle among the groups (p < 0.05). Further analysis by post hoc multiple comparison (Scheffe test) suggested that FT, BMD of the lumbar spine, BMD of the trochanter major, and BMD of Ward’s triangle in the 80–90 year-old group were significantly different than in the 50–59, 60–69, and 70–79 year-old groups (p < 0.05). The BMD of the femoral neck in the 80–90 year-old group was significantly different from that in the 50–59 and 70–79 year-old groups ▶ Table 3). (p < 0.05) (●
Stratified analysis by TT quartiles In order to clarify correlations between testosterone and bone mineral density, all subjects were divided into 4 groups ( < 3.53 ng/ ml, 3.53–3.91 ng/ml, 3.91–4.76 ng/ml, and > 4.76 ng/ml) based on quartile values of TT (3.53, 3.91, 4.76 ng/ml) in serum concentrations. As a result of it, we did not find significant BMD differences of the lumbar spine, femoral neck, trochanter major, and Ward’s ▶ Table 4). triangle among the groups (● Analysis of bivariate correlations suggested that there were no significant correlations between TT and BMD of the lumbar spine, the femoral neck or Ward’s triangle in the < 3.53 ng/ml group, but they were all close to statistical significance (p = 0.090, p = 0.097, and p = 0.055). In the other groups, no significant correlations of ▶ Table 5). TT with BMD at different bone sites were detectable (●
Stratified analysis by quartiles of FT Next, all 84 subjects were divided into 4 groups ( < 9.81 pg/ml, 9.81–10.71 pg/ml, 10.71–11.46 pg/ml, and > 11.46 pg/ml) based Table 2 Correlations of total testosterone (TT) and free testosterone (FT) with BMD at different bone sites.
TT FT
Lumbar
The trochanter
Femoral
spine
major
neck
Ward’s triangle
r = 0.116 p = 0.293 r = 0.177 p = 0.108
r = 0.044 p = 0.694 r = 0.208 p = 0.057
r = 0.136 p = 0.219 r = 0.156 p = 0.157
r = 0.221 p = 0.043* r = 0.086 p = 0.435
*After adjustment for age and BMI, p = 0.103
on their FT quartiles (9.81 pg/ml, 10.71 pg/ml, 11.46 pg/ml), and except for the BMD of the lumbar spine (p = 0.042), we did not find significant BMD differences of the femoral neck, the trochanter ▶ Table 6). major, and Ward’s triangle among the groups (● Analysis of bivariate correlation suggested that there were significant correlations between FT and BMDs of the lumbar spine, the femoral neck, and Ward’s triangle in the < 9.81 pg/ml group (p < 0.05), but after adjustment for age and BMI, a significant correlation only remained between FT and BMD of the femoral neck ▶ Table 7). (p < 0.05) (●
Discussion
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To determine the low level of testosterone in samples from the aged men, the sensitivity is considered as the most important for the method. However, most of the immunoassay-based method cannot be competent to such a tough task. Therefore, accurate and sensitive measurement of low level of testosterone is essential for correct diagnosis and appropriate treatment in clinical setting. Here we present an LC-MS/MS method with single step liquid-liquid extraction sample preparation procedure and APCI detection that allow the rapid and extremely sensitive measurement of serum TT and FT. Some recent studies suggested that testosterone concentrations remain stable in men aged less than 70 years and then decrease significantly [14, 15]. Our study results showed that the TT serum concentrations had a decreasing trend with increasing age, but without statistical significance, and FT values were only significantly lower in elderly men over 80 years. Our finding is supported by recent research, in which testosterone levels did not decrease stably with increasing age and mainly became obvious in men over 70 years old [10, 16]. The decreased levels were more apparent in FT, while TT was relatively stable, which might be a result of increased sex hormone binding protein expression and is in line with our results. The average FT level in our group was 10.72 ± 1.28 pg/ml, which was considerably lower than those reported in other research study (about 20 pg/ml) [10]. However, this study was done with a Caucasian population, whereas Orwoll et al. [14] reported that the level of FT in Asians was lower than that in other races and recently, Yoshimura et al. [17] noted that the average FT serum concentrations in healthy Japanese men older than 60 years was about 8.8 pg/ml and similar with our results. Our overall analysis of correlations between testosterone and BMDs showed that neither TT nor FT significantly correlated with BMDs at different bone sites, although there was a significant correlation of TT with BMD of the Ward’s triangle (p = 0.043), which disappeared after adjustment for age
Table 3 By age stratified analysis.
Number Age Body weight Height BMI TT FT BMD of the lumbar spine BMD of the trochanter major BMD of the femoral neck BMD of Ward’s triangle
50–59
60–69
70–79
80–89
10 53.1 ± 2.8 71.6 ± 2.7 1.71 ± 0.03 24.5 ± 0.9 4.42 ± 1.15 10.76 ± 1.36 1.085 ± 0.084 0.781 ± 0.023 0.778 ± 0.041 0.683 ± 0.041
21 64.9 ± 2.7 68.1 ± 3.5 1.70 ± 0.02 23.5 ± 1.1 4.31 ± 1.06 10.71 ± 1.64 1.002 ± 0.086 0.778 ± 0.035 0.754 ± 0.036 0.637 ± 0.044
35 75.6 ± 2.1 66.9 ± 3.6 1.71 ± 0.02 22.9 ± 1.0 4.20 ± 0.75 10.87 ± 1.20 1.023 ± 0.077 0.775 ± 0.025 0.767 ± 0.030 0.668 ± 0.042
18 82.8 ± 1.7 65.9 ± 3.0 1.71 ± 0.03 22.7 ± 1.0 3.65 ± 0.85 10.45 ± 0.89 1.017 ± 0.058 0.742 ± 0.042 0.735 ± 0.041 0.604 ± 0.047
Bo T, Zhu JM. Testosterone with Bone Mineral Density … Horm Metab Res 2014; 46: 697–701
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cance disappeared after adjustment for age and BMI (p = 0.103). No significant correlation was found between FT and bone min▶ Table 2). eral density at all other bone sites (●
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< 3.53 3.53–3.91 3.91–4.76 > 4.76
< 9.81 9.81–10.71 10.71–11.46 > 11.46 F-value p-value
The trochanter major
Femoral neck
Ward’s triangle
1.006 ± 0.074 1.021 ± 0.066 1.050 ± 0.088 1.019 ± 0.084 1.171 0.326
0.762 ± 0.039 0.779 ± 0.046 0.770 ± 0.056 0.768 ± 0.069 0.872 0.459
0.752 ± 0.039 0.753 ± 0.047 0.770 ± 0.049 0.757 ± 0.057 1.159 0.331
0.631 ± 0.053 0.655 ± 0.053 0.647 ± 0.051 0.659 ± 0.050 1.216 0.309
Lumbar spine
The trochanter major
Femoral neck
Ward’s triangle
r = 0.379 p = 0.090 r = 0.449 p = 0.410 r = 0.102 p = 0.660 r = 0.290 p = 0.203
r = 0.317 p = 0.161 r = 0.437 p = 0.348 r = 0.262 p = 0.251 r = 0.001 p = 0.995
r = 0.372 p = 0.097 r = 0.284 p = 0.424 r = 0.129 p = 0.578 r = 0.178 p = 0.439
r = 0.425 p = 0.055 r = 0.106 p = 0.649 r = 0.244 p = 0.287 r = 0.355 p = 0.114
Lumbar spine
The trochanter major
Femoral neck
Ward’s triangle
0.991 ± 0.078 1.060 ± 0.090 1.018 ± 0.065 1.027 ± 0.070 2.868 0.042*
0.757 ± 0.038 0.777 ± 0.051 0.766 ± 0.032 0.777 ± 0.055 1.639 0.187
0.746 ± 0.044 0.772 ± 0.054 0.754 ± 0.056 0.761 ± 0.045 1.830 0.148
0.638 ± 0.060 0.669 ± 0.048 0.639 ± 0.046 0.647 ± 0.052 1.635 0.188
Lumbar spine
The trochanter major
Femoral neck
Ward’s triangle
r = 0.534 p = 0.013* r a = 0.584 p a = 0.099 r = 0.025 p = 0.914 r = 0.249 p = 0.277 r = 0.051 p = 0.826
r = 0.164 p = 0.477
r = 0.515 p = 0.017* r a = 0.777 p a = 0.000** r = 0.176 r = 0.446 r = 0.355 p = 0.114 r = 0.311 p = 0.170
r = 0.479 p = 0.028* r a = 0.691 p a = 0.101 r = 0.391 p = 0.080 r = 0.119 p = 0.608 r = 0.078 p = 0.736
Table 4 Comparison of BMDs at different bone sites among the 4 quartile TT groups.
Table 5 Correlation of TT with BMDs at different bone sites in each quartile TT group.
Table 6 Comparison of BMD at different bone sites among the 4 FT quartile groups.
*p < 0.05
< 9.81
9.81–10.71 10.71–11.46 > 11.46
r = 0.064 p = 0.782 r = 0.057 p = 0.806 r = 0.043 p = 0.852
Table 7 Correlation of FT with BMD at different bone sites in each quartile FT group.
r a refers to the r-value after age and BMI adjustment p a refers to the p-value after age and BMI adjustment *p < 0.05 **p < 0.01
and BMI (p = 0.103). Similarly, a significant correlation only remained between FT and BMD of the femoral neck (p < 0.05) after adjustment was made for age and BMI in the stratified analysis by FT quartile. To our knowledge, FT level was reported to related significantly to the rate of change for BMD at femoral neck during the first 3 years and to incidence of osteoporosis after adjustment for age and BMI in a study by Yoshimura et al. [17]. The authors did not mention whether the adjustment for age and BMI lead to change of relation between FT levels and BMD in lumbar spine, which was observed in our study. However, age did have an effect on BMD in this report, and BMI was also reported to correlate with BMD, the adjustment for them would be meritorious. Meanwhile, there seems to be a trend toward association between TT and BMD of Ward’s triangle, or FT and lumbar spine, Ward’s triangle after age and BMI adjustment, which may be attributed to the fact that testosterone is an important but may not be the single influencing factor on BMD. Effect of transder-
mal testosterone treatment on bone and muscle in elder men with low bioavailable testosterone was investigated by Kenny et al. [18]. They found that when testosterone levels before treatment were < 10.5 nmol/l, the increase of BMD was 3.4 ± 1.2 %; when testosterone levels before treatment were < 7 nmol/l, the increase of BMD was 5.9 ± 2.2 %. These data showed that testosterone at low concentrations have a significant effect on BMDs and a correlation of high testosterone serum concentrations and BMDs is not obvious. When testosterone concentrations maintain stable at a relative high level, the correlation of testosterone with BMD is not easy to detect without avoiding shielding effects of other factors by precise design, because BMD is not only influenced by sex hormones but also by age, BMI, and other factors [19]. Thus, we further analyzed the correlation of testosterone with BMD in quartile groups of TT and FT. The results showed that FT in the first quartile group independently correlated positively with the BMD of the femoral neck. In addition, we found significant BMD differences only in the lumbar spine among the
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< 3.53 3.53–3.91 3.91–4.76 > 4.76 F-value p-value
Lumbar spine
4 quartile FT groups, which suggested that increases of testosterone concentrations caused little BMD changes when they were already high, thereby confirming our hypothesis. Snyder et al. noted, that BMD improvements only occurred when testosterone levels were < 2 ng/ml [20]. In an epidemiological study [16], Cauley et al. found that major hip bone loss over 1.8 years occurred only in men in the lowest FT category, which also proved that only low levels of FT are correlating with BMDs. Kaufman et al. found that TT maintained relatively stable, but FT significantly decreased in elderly men [21]. We suggest that a decrease of FT correlates with BMD, which is supported by previous studies [5, 22]. However, FT correlated different with lumbar spine, femoral neck, the trochanter major and Ward’s triangle and only the BMD of the femoral neck correlated independently with low FT levels after adjustment for age and BMI. Men with complete androgen insensitivity syndrome had different degrees of spine and hip BMD reductions [23] and a random, double-blind, placebo control trial revealed that improvement of BMD mainly occurred in the lumbar spine and femoral neck after testosterone therapy [18]. A similar result was also reported in another previous study [24], but some degenerative changes, such as osteophytosis or sclerotic alterations occur more frequently in the lumbar spine than in the femoral neck and these changes increase BMDs complicating the correlation of testosterone and BMD in these bones. Our study has some limitations. First, the small sample seemed to be the most severe weakness, but subjects like our healthy aged men were hard to collect, because people at this age frequently suffer from diseases and most of them have affected BMDs. Second, as a static study, we could not investigate the dynamic BMD changes with decreasing testosterone concentrations, while following of a particular cohort would give more information about correlations between testosterone and BMDs.
Conclusions
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In conclusion, based on LC-MS/MS method, we analyzed the correlation of testosterone (TT and FT) with BMD. Our conclusions are that after the age of about 80 years old, FT serum concentrations decrease significantly in healthy men in Shanghai area and positively correlated with the BMD of the femoral neck.
Acknowledgements
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The investigations were carried out at Shanghai Xuhui Central Hospital & Shanghai Clinical Center, Chinese Academy of Sciences.
Conflicts of Interest
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The authors declare that they have no conflicts of interest in the authorship or publication of this contribution.
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