The reference ranges of bone turnover markers (BTMs) were important during the treatment of osteoporosis, and the associations with bone mineral density (BMD) were controversial. The aim of this study was to establish the reference ranges of N-terminal procollagen of type l collagen (P1NP), osteocalcin (OC), and beta C-terminal cross-linked telopeptides of type I collagen (
Bone mineral density (BMD) is used to diagnose osteoporosis and to predict the risk of osteoporotic fracture in postmenopausal women [
BTMs include several enzymes and their decomposition products that come from bone cells and bone matrix components. Among BTMs that reflect the activity of osteoblast cells are serum osteocalcin (OC) in form of N-terminal midmolecule fragment (N-MID) and undercarboxylated osteocalcin (UcOC), N-terminal procollagen of type l collagen (P1NP), and C-terminal procollagen of type l collagen (P1CP). The P1NP that is by the N-terminal enzymatic hydrolysis and is a specific marker that reflected in osteoblasts activity. Its conversion rate is also higher than the soft tissue sources, so the determination of the P1NP can reflect bone formation. BTMs that reflect the activity of osteoclast cells include cross-linked N-telopeptide of type l collagen (NTX), cross-linked C-telopeptide of type l collagen (CTX), tartrate-resistant acid phosphatase (TRAP), and pyridinoline (Pyd). Because it is not being degraded in the blood and excreted by the kidneys, CTXs directly reflect bone matrix collagen degradation. During mature collagen degradation, the C-terminal peptide
Most reference ranges have been provided by commercial laboratory kits using approximately 100–200 observations [
In total, 2,799 individuals aged 20–79 yrs (790 men and 2,009 women) were recruited in March 2009 from the local population in communities of Shanghai. The inclusion criteria were as follows: (1) healthy men and women according to the following excluding criteria; (2) blood tests in this study were accorded with the laboratory normal reference range. The following criteria were used to exclude individuals from the study: (1) serious effects from cerebrovascular disease; (2) diabetes mellitus; (3) chronic renal disease and chronic liver disease; (4) evidence of other metabolic or inherited bone diseases; (5) rheumatoid arthritis or collagen disease; (6) recent major gastrointestinal disease; (7) significant disease of any endocrine organ that would affect bone mass; (8) hyperthyroidism; (9) any neurological or musculoskeletal condition; and (10) any form of calcium and vitamin-D therapy in three months or taking anti-osteoporotic drugs (e.g., bisphosphonates, selective estrogen receptor modulators, and calcitonin). Postmenopausal women who had experienced early menopause (before 45 yrs of age) were excluded. This study was approved by the Ethics Committee of the Shanghai Sixth People’s Hospital, Shanghai Jiao Tong University.
In addition, 520 healthy postmenopausal women with normal liver and kidney function were collected in communities of Shanghai. We carried BMD and the following BTMs in 520 postmenopausal women.
Age, body weight, height, and age of menarche and amenorrhea were recorded. All subjects were medically examined and interviewed using a standardized questionnaire to collect information on life style, smoking habits, the level of physical activity during leisure time, and use of vitamins and medications. All healthy subjects included in the present study had (1) normal blood counts and (2) normal results for liver and kidney function tests.
All biochemical markers were measured at the same time point using a single lot of reagents in one batch, following both the manufacturer’s protocol and specialized assay laboratory quality control procedures. Fasting blood samples were collected for the measurement of the serum levels of calcium, phosphate, albumin, glucose, insulin, cholesterol, triglycerides (TG), blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT), aspartate transaminase (AST), r-glutamyl transpeptidase (r-GT), alkaline phosphatase (ALP), and B ultrasound of the spleen, kidneys, hepatobiliary system, and pancreas.
Serum 25-hydroxyvitamin D [25(OH)D], PTH,
520 postmenopausal women’s BMD (g/cm2) of the left proximal femur including the total hip, the femoral neck, the trochanter, and Ward’s triangle was measured using dual-energy X-ray absorptiometry (DXA) on a Lunar Prodigy GE densitometer (Lunar Corp, Madison, WI, USA). Both of the scanners were calibrated daily, and the coefficient of variability (CV) values for the DXA measurements at L1-4, the total hip, and the femoral neck were 1.39%, 0.7%, and 2.22%, respectively, for the Lunar Prodigy. The long-term reproducibility of the DXA data during the trial, which was based on phantom measurements that were repeated weekly, was 0.45%.
The anthropometric characteristics were presented as the means ± SD, and BTMs were presented as geometric means with 95% confidence intervals (95% CI). Data were analyzed using SPSS 11.0 (SPSS Inc., Chicago, IL, USA). The BTMs ALP, OC, P1NP, PTH, and
A total of 2,799 subjects were screened for entry into the study by measuring indicators of liver and kidney function and blood calcium and phosphorus: 148 failed the initial screening, leaving 2,651 subjects. 25(OH)D concentrations were divided into four subgroups according to the following criteria: severely deficient (<10 ng/mL), deficient (10–20 ng/mL), insufficient (20-30 ng/mL), and sufficient (≥30 ng/mL). The prevalence of vitamin D insufficiency was 84% in males and 89% in females. The prevalence of vitamin D deficiency was 30% in males and 46% in females. Based on 25(OH)D concentrations, we excluded the subject whose 25(OH)D concentrations were <10 ng/mL in 2651 cases, leaving 705 males and 1836 females last.
Recent studies containing data from larger cohorts of healthy premenopausal women have reported reference intervals for which the age range was 30–45 yr [
Anthropometric characteristics and other variables in the men and women aged 35–45 yr used to calculate the reference intervals of the bone turnover markers.
Characteristics | Men ( |
Women ( |
---|---|---|
Age (year) | 39.8 ± 3.2 (35–45) | 39.4 ± 3.2 (35–45) |
Height (cm) | 173.2 ± 5.9 (165.8–180.1) | 162.0 ± 4.9 (153.7–172.5) |
Weight (kg) | 75.0 ± 12.5 (61.6–85.5) | 58.3 ± 7.1 (44.7–68.3) |
BMI (kg/m2) | 25.4 ± 3.1 (20.8–26.4) | 22.2 ± 2.6 (17.9–24.6) |
Medians, geometric means, and 95% reference intervals for the bone turnover markers in 35–45-year-old subjects.
Measurement | Women ( |
Men ( |
||||
---|---|---|---|---|---|---|
Median | Geometric mean | 95% reference interval | Median | Geometric mean | 95% reference interval | |
ALP (U/L) | 53.00 | 54.80 | 29.55–82.04 | 63.00 | 63.87 | 35.52–94.16 |
OC (ng/mL) | 13.90 | 15.05 | 4.91–22.31 | 16.57 | 18.01 | 5.58–28.62 |
P1NP (ng/mL) | 32.90 | 35.22 | 13.72–58.67 | 42.43 | 44.01 | 16.89–65.49 |
|
0.210 | 0.241 | 0.112–0.497 | 0.378 | 0.400 | 0.100–0.612 |
25(OH)D (ng/mL) | 20.58 | 20.98 | 10.97–32.15 | 22.89 | 23.55 | 11.11–34.43 |
PTH (pg/mL) | 35.02 | 37.30 | 15.52–66.78 | 30.39 | 33.92 | 14.61–63.22 |
Values were obtained from 35–45-year-old healthy men and women.
Geometric means and 95% confidence intervals (95% CI) for the age-specific measurements of bone turnover markers in healthy women.
Age | Number |
|
P1NP (ng/mL) | OC (ng/mL) | PTH (pg/mL) |
---|---|---|---|---|---|
20–24 | 145 | 0.412* (0.354–0.470) | 54.97* (47.87–62.06) | 21.23* (18.72–22.47) | 37.20 (35.07–39.33) |
25–29 | 386 | 0.335* (0.304–0.367) | 45.94* (41.38–50.49) | 18.48* (17.19–19.77) | 35.70 (34.29–37.10) |
30–34 | 247 | 0.280* (0.230–0.313) | 40.21* (35.78–44.63) | 16.80* (15.33–18.26) | 36.45 (34.76–38.13) |
35–39 | 210 | 0.250 (0.221–0.284) | 35.93 (31.72–40.13) | 15.37 (13.95–16.78) | 38.10 (35.99–40.21) |
40–44 | 178 | 0.239 (0.201–0.271) | 34.27 (30.50–38.04) | 13.84 (12.48–15.27) | 38.55 (36.17–40.94) |
45–49 | 104 | 0.278* (0.221–0.310) | 36.35* (31.10–41.58) | 14.84* (12.88–16.79) | 38.99 (36.09–41.89) |
50–54 | 138 | 0.470* (0.399–0.541) | 52.72*(45.54–56.90) | 23.46* (20.44–26.46) | 35.83 (33.58–38.08) |
55–59 | 184 | 0.474* (0.407–0.540) | 50.21* (43.85–56.57) | 24.94* (22.19–27.68) | 36.50 (34.76–38.24) |
60–64 | 51 | 0.441* (0.307–0.584) | 47.07* (34.78–59.36) | 26.40* (20.28–32.60) | 37.92 (32.71–43.13) |
65–69 | 70 | 0.471* (0.356–0.585) | 50.61* (38.11–63.11) | 25.57* (22.90–28.22) | 36.13 (32.72–39.55) |
70–74 | 77 | 0.431* (0.297–0.565) | 50.04* (37.94–62.13) | 26.50* (20.82–32.17) | 35.57 (30.94–38.21) |
75–79 | 66 | 0.384* (0.284–0.482) | 45.44* (35.60–54.47) | 24.11* (18.85–29.37) | 36.25 (29.70–38.81) |
Reference valuea | 406 | 0.242 (0.218–0.297) | 35.22 (30.67–40.10) | 15.05 (13.81–16.59) | 37.30 (33.18–40.92) |
All values are presented as the geometric means (95% CI).
*
aReference values were calculated from the 35–45-year-old cohort as the geometric mean (95% CI).
Geometric means and (95% CI) for the age-specific measurements of bone turnover markers in healthy men.
Age | Number |
|
P1NP (ng/mL) | OC (ng/mL) | PTH (pg/mL) |
---|---|---|---|---|---|
20–24 | 43 | 0.745* (0.464–1.024) | 64.44* (41.81–87.05) | 25.21* (17.57–28.86) | 31.77 (25.61–39.94) |
25–29 | 72 | 0.504* (0.415–0.551) | 51.81* (43.42–60.20) | 21.08* (18.12–24.06) | 34.73 (31.14–38.33) |
30–34 | 93 | 0.419* (0.353–0.482) | 44.68* (38.25–51.10) | 18.51* (15.97–21.05) | 34.06 (30.46–37.67) |
35–39 | 102 | 0.402 (0.333–0.470) | 44.22 (37.64–57.01) | 18.34 (15.66–21.02) | 35.51 (32.40–38.62) |
40–44 | 117 | 0.402 (0.318–0.485) | 43.98 (36.78–51.78) | 17.85 (14.89–20.79) | 35.09 (31.89–38.29) |
45–49 | 45 | 0.340* (0.252–0.428) | 36.62* (29.37–43.88) | 16.02* (11.97–20.05) | 30.01 (25.92–34.20) |
50–54 | 53 | 0.342* (0.267–0.414) | 39.59* (32.23–46.93) | 16.69* (13.72–19.65) | 34.60 (30.86–38.35) |
55–59 | 64 | 0.353* (0.278–0.427) | 36.93* (30.62–43.23) | 15.58* (13.07–18.09) | 35.80 (31.57–40.02) |
60–64 | 45 | 0.393* (0.276–0.508) | 41.19* (31.63–50.13) | 20.95* (16.32–25.66) | 36.94* (31.27–42.60) |
65–69 | 26 | 0.352* (0.218–0.485) | 39.39* (27.48–50.87) | 19.43* (12.28–25.51) | 37.44* (30.58–44.31) |
70–74 | 21 | 0.383* (0.167–0.598) | 40.00* (26.65–53.33) | 21.84* (15.95–27.73) | 37.45* (30.52–44.38) |
75–79 | 24 | 0.355* (0.208–0.501) | 40.10* (25.60–54.69) | 20.86* (14.72–27.00) | 36.34* (29.98–42.70) |
Reference valuea | 226 | 0.400 (0.310–0.479) | 44.01 (37.27–52.97) | 18.01 (14.91–20.97) | 33.92 (30.21–38.17) |
All values are presented as the geometric means (95% CI).
*
aReference values were calculated from the 35–45-year-old cohort as the geometric mean (95% CI).
Table
The geometric means and 95% confidence intervals (95% CI) of the age-specific BTMs in healthy men are shown in Table
In 520 postmenopausal women correlation between BTMs and BMD is given in Table
Standardized correlation coefficients (Betastd) between BTMS and BMD in 520 postmenopausal women.
BTMs | L1-4 BMD | Femoral neck BMD | Total hip BMD | |||
---|---|---|---|---|---|---|
Not adjusted | Adjusted | Not adjusted | Adjusted | Not adjusted | Adjusted | |
|
−0.155** | −0.157** | −0.188** | −0.182** | −0.207** | −0.195** |
P1NP | −0.197** | −0.201** | −0.169** | −0.193** | −0.220** | −0.217** |
OC | −0.191** | −0.188** | −0.178** | −0.176** | −0.209** | −0.199** |
Data adjusted age, height, body weight, and YSM are given.
**
It is important to establish reference intervals for BTMs from a sample of the healthy young population to use such markers to correctly assess bone turnover in subjects of various ages. In this study, we chose women aged 35–45 yrs for reference interval calculations because these women have achieved peak bone mass and are not yet perimenopausal. Women under 30 yrs of age were excluded to reduce the chance of including women with elevated bone turnover due to skeletal immaturity. In addition, women over the age of 45 were excluded because several studies have demonstrated that BTMs are increased in perimenopausal women because of the estrogen deficiency that occurs after spontaneous menopause in an increase in bone remodeling [
In the present study, the age-related analysis of all subjects revealed a significant negative correlation between each of the bone turnover markers and age. In women, the bone formation markers OC and P1NP decreased with increasing age until the age of 44 before sharply increasing in the 50- to 59-year-old age group. The bone resorption marker
In the cohort of men we studied, age-related changes in BTMs were discovered. The bone formation markers OC and P1NP decreased with increasing age until the age of 59 and then abruptly increased in the 60- to 69-year-old group. The bone resorption marker
Previous reports have indicated that 25(OH)D levels are negatively correlated with serum PTH, OC, P1NP, and
The study found that OC,
Our study has the following strengths: (1) the sample size was large enough (greater than 600 men and 1,000 women) to avoid sample error; (2) the random selection of women from the local population, thereby covering a wide age-range and being representative of the Chinese population; and (3) the participants were well characterized and the exclusion criteria were detailed and evidence based to ensure a more precisely characterized sample population. Thus, numerous factors to minimize biological variability were considered and controlled.
Certain limitations of this study must also be acknowledged. First, we did not measure BMD in 2,799 subjects (790 males and 2,009 females). Second, the purpose of this study was to establish the reference intervals for BTMs, for which we analyzed a sufficient number of young men and women; however, compared with the number of young subjects, the sample size of elderly subjects was not adequate, particularly when examining elderly men. Finally, the cross-sectional nature of the study design is a limitation.
The present study provides reference interval values for BTMs in healthy men and women. These reference ranges are based on data showing that BTMs are stable between the ages of 35 and 45 yrs. These results will contribute to the appropriate assessment of bone turnover in Shanghai area population and offer a comparison to measurements obtained in other populations. This study also found that BTMs correlated with BMD in Chinese postmenopausal women and suggested that BTMs were the key determining factors of early BMD decreases.
The authors have declared that no conflict of interests exists.
The authors acknowledge the excellent assistances of Yao-Hua Ke and Wen-Zhen Fu with blood extraction and analysis. This study was supported by the National Science Foundation of China (NSFC) (Grants nos. 81170803, 30800387, 81070692, and 81000360), the Program of Shanghai Chief Scientists (Project no.: 08XD1403000), STCSM10DZ1950100, and Academic Leaders in Health Sciences in Shanghai (XBR 2011014). The authors would like to thank all of the patients who participated in this study.