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Reference intervals for serum concentrations of three bone turnover markers for men and women
Bone 57 (2013) 399–404
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Original Full Length Article
Reference intervals for serum concentrations of three bone turnover markers for men and women J. Michelsen a, H. Wallaschofski a, N. Friedrich a, C. Spielhagen a, R. Rettig b, T. Ittermann c, M. Nauck a, A. Hannemann a,⁎ a b c
Institute of Clinical Chemistry and Laboratory Medicine, University Medicine Greifswald, Greifswald, Germany Institute of Physiology, University Medicine Greifswald, Greifswald, Germany Institute for Community Medicine, University Medicine Greifswald, Greifswald, Germany
a r t i c l e
i n f o
Article history: Received 18 March 2013 Revised 10 September 2013 Accepted 17 September 2013 Available online 27 September 2013 Edited by: Felicia Cosman Keywords: Bone turnover marker Reference intervals Germany Osteoporosis Fracture
a b s t r a c t Objective: Bone turnover markers (BTMs) reflect the metabolic activity of bone tissue and can be used to monitor osteoporosis therapy. To adequately interpret BTMs, method-specific reference intervals are needed. We aimed to determine reference intervals for serum concentrations of intact amino-terminal propeptide of type I procollagen (PINP), bone-specific alkaline phosphatase (BAP) and carboxy-terminal telopeptide of type I collagen (CTX). Material and methods: We established a healthy reference population of 1107 men as well as 382 pre- and 450 postmenopausal women, who participated in the first follow-up of the Study of Health in Pomerania. Serum PINP, BAP and CTX concentrations were measured on the IDS-iSYS Automated System (Immunodiagnostic Systems, Frankfurt am Main, Germany). The reference interval was defined as the central 95% range. We determined age-specific reference intervals for PINP, BAP, and CTX for men by quantile regression. Reference intervals for women were age-independent. Results: Reference intervals for men for PINP and CTX decreased with age (25–29 year-old men: PINP 31.1– 95.9 ng/mL, CTX 0.12–0.83 ng/mL; 75–79 year-old men: PINP 15.7–68.1 ng/mL, CTX 0.05–0.58 ng/mL). The reference interval for men for BAP did not significantly change with age (25–29 year-old men: 7.4–27.7 ng/mL; 75–79 year-old men: 7.6–24.4 ng/mL). The reference intervals for 30–54 year-old premenopausal women were: PINP 19.3–76.3 ng/mL, BAP 6.0–22.7 ng/mL, and CTX 0.05–0.67 ng/mL. The reference intervals for 50–79 year-old postmenopausal women were: PINP 18.2–102.3 ng/mL, BAP 8.1–31.6 ng/mL, and CTX 0.09– 1.05 ng/mL. Conclusion: An intensively characterized, large reference population free of bone-related diseases allowed us to determine robust reference intervals for serum concentrations of PINP, BAP and CTX. Our normative data may aid to interpret bone turnover in adult men and pre- and postmenopausal women. © 2013 Elsevier Inc. All rights reserved.
Introduction The population in the industrialized countries is ageing [1]. The rise in mean age is accompanied by an increasing prevalence of osteoporosis and osteoporotic fractures [2]. Osteoporotic fractures, including hip and vertebral fractures, cause substantial pain, have detrimental effects on quality of life, are associated with disability and mortality and produce
Abbreviations: ATC, Anatomical Therapeutic Chemical Classification System; BAP, bone-specific alkaline phosphatase; BMD, bone mineral density; BMI, body mass index; BTM, bone turnover marker; CTX, carboxy-terminal telopeptide of type I collagen; PINP, amino-terminal propeptide of type I procollagen; PTH, parathyroid hormone; SHIP, Study of Health in Pomerania. ⁎ Corresponding author at: Institute of Clinical Chemistry and Laboratory Medicine, University Medicine Greifswald, Ferdinand-Sauerbruch-Straße NK, D-17475 Greifswald, Germany. Fax: +49 3834 865502. E-mail address: [email protected] (A. Hannemann). 8756-3282/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bone.2013.09.010
high costs [3,4]. Thus, the main target of any osteoporosis therapy is to prevent fractures [3,4]. In clinical routine, the efficacy of osteoporosis therapy is usually assessed by measuring BMD1 [3]. However, BMD changes slowly, often over several months or even years [5]. Earlier effects of osteoporosis therapy can be monitored by the determination of BTMs,2 e.g. two or three months after initiation of oral bisphosphonate therapy [6–9]. BTMs reflect the metabolic activity of bone tissue [6], can be measured in serum or urine samples, and allow for the assessment of bone formation and bone resorption processes [7]. Various bone formation and bone resorption markers have been suggested for clinical use [5,10], e.g. intact PINP,3 BAP,4 or CTX.5 PINP 1 2 3 4 5
BMD, bone mineral density. BTM, bone turnover marker. PINP, amino-terminal propeptide of type I procollagen. BAP, bone-specific alkaline phosphatase. CTX, carboxy-terminal telopeptide of type I collagen.
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is cleaved from procollagen and is released into the circulation during bone formation, while CTX is a degradation product of type I collagen and is released into the circulation during bone resorption [5]. BAP is a glycoprotein involved in bone mineralization whose serum concentration is strongly determined by osteoblast activity [5]. PINP and CTX were recommended as the most informative BTMs for monitoring of osteoporosis in the current European guidance for the diagnosis and management of osteoporosis in postmenopausal women [3]. To interpret BTM concentrations, population-, sex-, and methodspecific reference intervals are needed [6,9,11]. Currently, reference intervals for serum PINP, BAP, and CTX concentrations have neither been established for the German population nor for measurement with the IDS-iSYS Multi Discipline Automated Analyser. For males, PINP and CTX reference intervals have been established in Spanish men older than 50 years of age [12]. BAP reference intervals have not yet been established for men at all. For females, previous studies reported large differences between pre- and postmenopausal serum BTM concentrations [11,13]. BTMs increase during the menopausal transition due to increased osteoclastic activity which in turn is due to decreasing estrogen levels [14]. Accordingly, the intake of sex hormones for menopausal hormone therapy or contraception may alter BTM concentrations [11,15,16]. The majority of previous studies that determined reference intervals for serum PINP [15–19], BAP [16,18,19] or CTX [15–19] concentrations, were limited to premenopausal women. There is only one study [20] that reported reference intervals for PINP and CTX for postmenopausal women. BAP reference intervals for postmenopausal women have not yet been established. We aimed to determine sex- and age-specific reference intervals for serum PINP, BAP and CTX concentrations for adult healthy Caucasian men as well as for pre- and postmenopausal women living in northeast Germany. Additionally, we aimed to analyze the impact of sex hormone therapy on serum BTM concentrations. Material and methods The Study of Health in Pomerania (SHIP) The present study is based on data from the first follow-up of the SHIP.6 SHIP is a population-based cohort study in northeast Germany. In the baseline study (SHIP-0), which was conducted between October 1997 and May 2001, 4308 men and women from a representative sample of 7008 subjects were examined. All subjects were invited to participate in the first follow-up (SHIP-1), which took place between March 2003 and July 2005 with 3300 subjects being re-examined. All SHIP participants gave written informed consent. Further details on study design and sampling have been previously reported [21]. The study was reviewed by an external scientific review board and conformed to the principles of the Declaration of Helsinki as reflected by an a priori approval of the Ethics Committee of the Board of Physicians Mecklenburg-West Pomerania at the University of Greifswald. All SHIP participants underwent standardized medical examinations including blood sampling and an extensive computer-aided personal interview. Data on socio-demographic characteristics and medical histories was collected. Intake of medication was recorded and classified using the ATC7 code. All women were divided according to menopausal status in pre- or postmenopausal. Women younger than 40 years of age and women between 40 and 60 years of age who reported menstrual cycling were defined as premenopausal, all other women were defined as postmenopausal. It was not possible to define perimenopause as the respective information was not collected. Blood sampling was performed between 8.00 a.m. and 8.00 p.m. More than half of all blood samples (58.2%) were taken before 12 a.m., another third (31.0%) between 12:00 a.m. and 2:59 p.m., and one6 7
SHIP, Study of Health in Pomerania. ATC, Anatomical Therapeutic Chemical Classification System.
tenth (10.8%) after 3:00 p.m. Blood samples were taken from the cubital vein of mostly non-fasting participants in the supine position. Serum aliquots were stored at −80 °C. Serum PINP, BAP, and CTX concentrations were determined on the IDS-iSYS Multi-Discipline Automated Analyser (Immunodiagnostic Systems Limited, Frankfurt am Main, Germany). For each analyte three concentrations of control material were measured by skilled technical personnel. Serum PINP concentrations were measured with the IDS-iSYS Intact PINP assay. During the course of the study the coefficients of variation were 7.33% at low concentrations, 5.67% at medium concentrations, and 7.88% at high concentrations, respectively, of control material. The assay manufacturer reports good linearity in the range of 5.8–203.8 ng/mL, with variations between −3% and +3% of predicted and measured PINP concentrations. Serum BAP concentrations were measured with the IDS-iSYS Ostase + BAP assay. During the course of the study the coefficients of variation were 12.28% at low concentrations, 13.47% at medium concentrations, and 13.23% at high concentrations, respectively, of control material. The assay manufacturer reports good linearity in the range of 9.7–71.0 ng/mL, with variations between −2% and +7% of predicted and measured BAP concentrations. The assay demonstrates a comparable cross-reactivity to liver isoenzyme as the Tandem R Ostase assay [22], as it was developed using the same monoclonal antibody. The assay provides a result of 6.9 ng/mL BAP for each 100 U/L liver enzyme. Serum CTX concentrations were measured with the IDS-iSYS CTX-I (CrossLaps) assay. During the course of the study, the coefficients of variation were 12.23% at low concentrations, 10.40% at medium concentrations, and 11.40% at high concentrations, respectively, of control material. The assay manufacturer reports good linearity in the range of 0.245–5.293 ng/mL, with variations between −6% and +3% of predicted and measured CTX concentrations. Reference population To generate a healthy reference population we excluded all subjects with missing data on serum PINP, BAP, or CTX concentrations (n = 39), or with any of the following conditions: renal disease defined as estimated glomerular filtration rate (Cockcroft-Gault) b30 mL/min (n = 30), hyperparathyroidism defined as serum parathyroid hormone concentration N120 pg/mL (n = 24), hyperthyroidism defined according to local reference intervals [23] as serum thyroid-stimulating hormone concentration b0.25 mU/L and serum free thyroxine concentration N18.9 pmol/L (n = 70), or a selfreported history of cancer (n = 189), a self-reported physician's diagnosis of osteoporosis (n = 207), or a self-reported history of liver disease (n = 41). We further excluded all pregnant women (n = 12), all subjects with serum 25-hydroxy vitamin D concentration b 10 ng/mL (n = 342), and all subjects (n = 85) who reported intake of any of the following medication: bisphosphonates, strontium ranelate, vitamin D, calcium, parathyroid hormone, calcitonin, steroids, selective estrogen receptor modulators, testosterone, anticonvulsants, heparin, antiandrogens, or aromatase inhibitors. Moreover, we excluded all premenopausal women using oral contraceptives (n = 126) and all postmenopausal women using hormone replacement therapy (n = 55). Furthermore, we excluded subjects with a serum osteocalcin concentration N100 ng/mL (n = 2) and, due to small numbers in the respective age groups, all subjects older than 79 years of age (n = 58), all premenopausal women younger than 30 years of age (n = 36) or older than 54 years of age (n = 3), and all postmenopausal women younger than 50 years of age (n = 44). This resulted in a reference population of 1939 subjects, including 1107 men as well as 382 premenopausal women (aged 30–54 years) and 450 postmenopausal women (aged 50–79 years). Statistical analyses Continuous data are expressed as median (1st-3rd quartile) and nominal data are expressed as percentage. We report median instead
J. Michelsen et al. / Bone 57 (2013) 399–404
of mean values for the three BTM serum concentrations as they are not normally distributed. Group comparisons were performed using Kruskal–Wallis tests. P-values b0.05 were considered statistically significant. Reference intervals were defined as the central 95% range between the 2.5th and 97.5th percentiles of serum PINP, BAP, and CTX concentrations, respectively. Scatterplots with LOESS curves and 95% confidence intervals (CI) were used to assess the relation between the three BTMs and age. The LOESS procedure is a nonparametric method to fit a local regression function. It produces a smooth curve fitted to the data set and allows a graphical evaluation of the relation between the BTMs and age. Agerelated changes in the BTMs were further tested for statistical significance with quantile regression models [24]. Reference limits for men were determined as a function of age by quantile regression, while reference limits for women were determined disregarding age. The agerelated changes of the serum PINP concentration in men were modeled by fitting a quadratic equation (including age and age squared), while the models for the serum BAP, and CTX concentrations were fitted by a linear equation (including age). Reference limits for men for serum PINP, BAP, and CTX concentrations were estimated for each single year of age. Subsequently, mean values for five-year age groups were calculated. Next to age-related changes we tested the influence of sex hormones for oral contraception or hormone therapy, of month or daytime of blood sampling, as well as of 25-hydroxy vitamin D and parathyroid hormone concentrations on the three BTMs within quantile regression models. All statistical analyses were performed with SAS 9.1.3 (SAS Institute Inc., Cary, NC, USA).
Results Characteristics of the study population, including median age, BMI,8 the proportion of diabetes mellitus, as well as median concentrations of 25-hydroxy vitamin D, parathyroid hormone, and BTMs are presented in Table 1. Age-specific (5-year age groups) median serum concentrations of PINP, BAP, and CTX are presented in Table 2. Scatterplots with LOESS curves for men as well as pre- and postmenopausal women are displayed in Fig. 1. The LOESS curves for serum PINP and CTX in men indicated decreasing concentrations with increasing age at an age range of 25 to 55 years and a plateau at older ages. In contrast to serum PINP and CTX concentrations, serum BAP concentrations in men hardly changed with age. The LOESS curve indicated a very modest decrease in serum BAP concentrations in men between 25 and 40 years of age and after 70 years of age. Reference intervals for men were determined with quantile regression models fitted with parameters presented in Supplemental Table 1. Reference intervals for men are presented in Table 3. For men aged 25–54 years, upper and lower reference limits for the serum PINP concentration decreased continuously with increasing age. For older men, the upper and lower reference limits remained relatively stable. Reference intervals for the serum BAP concentration were stable throughout the observed age range, with very small decreases in lower and upper reference limits between the ages 25–79. The lower CTX reference limits decreased strongly with increasing age between 25 and 54 years of age and were relatively stable for older men, while the upper reference limits continued to decrease over the whole age range of 25–79 years. Reference curves for men are visualized in Supplemental Fig. 1. The LOESS curves for all three BTMs for premenopausal women between 30 and 54 years of age indicated relatively stable concentrations over the observed age range (Fig. 1). Also quantile regression models did not indicate significant age-related changes in the three BTMs in premenopausal women. In postmenopausal women serum BTM concentrations were markedly higher than in premenopausal women 8
BMI, body mass index.
401
Table 1 Characteristics of the reference population. Characteristics
Men (N = 1107)
Women, premenopausal (N = 382)
Women, postmenopausal (N = 450)
Age [years] BMI [kg/m2] Diabetes mellitus [%] 25-hydroxy vitamin D [μg/L] Parathyroid hormone [pg/mL] PINP [ng/mL] BAP [ng/mL] CTX [ng/mL]
53.0 (41.0–64.0) 27.9 (25.5–30.7) 10.1
40.0 (36.0–45.0) 25.0 (22.3–28.6) 1.1
62.0 (56.0–68.0) 28.2 (25.1–32.2) 14.22
19.2 (14.6–25.1)
19.0 (14.2–27.5)
18.1 (13.7–23.5)
33.7 (25.7–43.2)
28.9 (21.9–38.6)
36.7 (27.4–46.0)
38.0 (29.2–49.4) 14.0 (11.3–16.8) 0.28 (0.19–0.41)
36.7 (28.2–46.4) 10.8 (8.9–13.6) 0.23 (0.16–0.34)
46.4 (32.7–62.0) 16.3 (13.1–20.4) 0.37 (0.25–0.56)
BAP, bone-specific alkaline phosphatase; BMI, body mass index; CTX, carboxy-terminal telopeptide of type I collagen; PINP, intact amino-terminal propeptide of type I procollagen Data are median (1st–3rd quartile) or proportions.
(Table 2). The LOESS curves in postmenopausal women indicated an increase in the serum BTM concentrations between ages 50 and 55 followed by a decrease until age 60. In Europe, the median age of natural menopause is about 54 years [25]. These age-related fluctuations in the three BTMs may thus be due to the menopausal transition. In postmenopausal women older than 60 years of age the three BTMs were relatively stable. Small decreases in serum PINP and CTX concentrations were observed in women older than 75 years of age. Quantile regression models did not reveal age-related changes in the three BTMs in postmenopausal women. Reference intervals for pre- and postmenopausal women were thus determined disregarding age (Table 4). The upper reference limits for all three BTMs were substantially higher for post- than for premenopausal women. The lower reference limits for serum BAP and CTX concentrations were also higher for post- than for premenopausal women but to a lesser extent. In a sensitivity analysis we included only postmenopausal women between 60 and 75 years of age to assess whether the fluctuation in the BTMs in early menopause affected the reference intervals. The reference intervals for the three BTMs were very similar to those obtained in all postmenopausal women (Table 4). Therefore, we assume that the reference intervals obtained in the main analyses are valid for all postmenopausal women. Month of blood sampling was not associated with serum PINP, BAP, or CTX concentrations in men or pre- or postmenopausal women, regardless whether or not subjects with 25-hydroxy Vitamin D concentrations b10 ng/mL (n = 276) were included. Daytime of blood sampling (in hours) was also not associated with serum PINP or BAP concentrations. However, we detected an association between daytime of blood sampling and the serum CTX concentration in premenopausal women (Supplemental Figs. 2–4). As this association was not statistically significant (p = 0.46) and vanished when women with blood sampling after 5:00 p.m. were excluded from the analyses, daytime of blood sampling was not further considered in the analyses. There were no associations between serum concentrations of PTH9 and PINP or BAP. Yet, there were positive associations between serum concentrations of PTH and CTX. Quantile regression models yielded the following results for each 10 pg/mL increase in PTH: men estimate 0.02 ng/mL (p b0.001), premenopausal women estimate 0.03 ng/mL (p b 0.001), postmenopausal women estimate 0.02 ng/mL (p = 0.004). Moreover, there were a negative association between the serum 25hydroxy Vitamin D and BAP concentration in premenopausal women as well as a positive association between the serum 25-hydroxy Vitamin D and CTX concentration in men. We cannot exclude that the associations between the serum 25-hydroxy Vitamin D and BAP or CTX concentrations were statistical artefacts since they were observed only in subsamples of the population. 9
PTH, parathyroid hormone.
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Table 2 Serum PINP, BAP and CTX concentrations in men (N = 1107) as well as in premenopausal (N = 382) and postmenopausal women (N = 450) according to 5-year age groups. Age group [years]
25–29 30–34 35–39 40–44 45–49 50–54
50–54 55–59 60–64 65–69 70–74 75–79
N
PINP [ng/mL]
BAP [ng/mL]
CTX [ng/mL]
Men
Premenopausal Women
Men
Premenopausal Women
p
Men
Premenopausal Women
p
Men
Premenopausal Women
p
49 79 113 113 123 127
– 75 96 96 72 43
53.4 (47.9–63.0) 48.9 (37.8–57.7) 43.2 (33.7–58.0) 43.4 (36.4–52.0) 33.9 (29.0–44.3) 33.8 (27.2–46.4)
– 38.3 (28.2–47.9) 37.8 (30.3–50.2) 36.2 (27.3–45.5) 35.7 (26.9–44.2) 36.4 (29.5–42.1)
– b0.01 0.01 b0.01 0.41 0.83
14.6 (12.3–18.2) 14.5 (12.1–18.4) 13.5 (11.7–15.9) 14.7 (12.5–16.9) 13.9 (11.4–16.6) 13.2 (10.6–16.4)
– 10.8 (9.1–13.6) 11.3 (9.3–13.6) 10.6 (8.8–12.5) 10.6 (8.5–13.1) 11.9 (9.6–15.4)
– b0.01 b0.01 b0.01 b0.01 0.14
0.50 (0.28–0.61) 0.35 (0.26–0.45) 0.30 (0.21–0.44) 0.32 (0.22–0.42) 0.26 (0.16–0.34) 0.25 (0.16–0.42)
– 0.22 (0.14–0.33) 0.25 (0.17–0.33) 0.23 (0.16–0.31) 0.23 (0.15–0.38) 0.24 (0.17–0.38)
– b0.01 b0.01 b0.01 0.69 0.65
Men
Postmenopausal Women
Men
Postmenopausal Women
p
Men
Postmenopausal Women
p
Men
Postmenopausal Women
p
127 118 132 114 85 54
65 98 114 81 55 37
33.8 (27.2–46.4) 35.3 (27.9–45.3) 34.4 (27.9–42.4) 34.0 (25.9–40.8) 33.2 (26.3–42.6) 33.6 (24.2–44.8)
50.6 (32.9–65.0) 53.3 (35.1–74.2) 47.4 (35.2–59.8) 45.1 (30.7–57.8) 42.4 (30.8–63.3) 38.5 (28.3–52.3)
b0.01 b0.01 b0.01 b0.01 b0.01 0.13
13.2 (10.6–16.4) 14.1 (11.3–17.2) 14.4 (11.9–17.1) 13.4 (10.7–16.8) 12.3 (10.9–14.7) 12.8 (10.5–15.7)
15.8 (12.5–21.0) 17.6 (14.8–20.9) 16.2 (13.3–20.4) 15.8 (12.8–18.8) 15.8 (12.7–21.4) 16.1 (11.8–18.1)
b0.01 b0.01 b0.01 b0.01 b0.01 b0.01
0.25 (0.16–0.42) 0.28 (0.19–0.40) 0.24 (0.17–0.35) 0.25 (0.18–0.36) 0.24 (0.17–0.33) 0.24 (0.18–0.41)
0.40 (0.25–0.59) 0.44 (0.30–0.62) 0.32 (0.23–0.51) 0.38 (0.23–0.55) 0.37 (0.26–0.60) 0.33 (0.22–0.44)
b0.01 b0.01 b0.01 b0.01 b0.01 0.04
BAP, bone-specific alkaline phosphatase; CTX, carboxy-terminal telopeptide of type I collagen; PINP, intact amino-terminal propeptide of type I procollagen. Data are median (1st–3rd quartile). Sex differences were tested with Kruskal-Wallis tests.
Premenopausal women taking oral contraceptives (n = 91) had lower serum PINP, BAP, and CTX concentrations than premenopausal women not taking oral contraceptives. Likewise, postmenopausal women taking sex hormones (n = 48) had lower serum BTM concentrations than postmenopausal women not taking hormone therapy (Supplemental Table 2).
Discussion In the present study sex-specific reference intervals for serum PINP, BAP, and CTX concentrations measured on the IDS-iSYS Multi Discipline Automated Analyser were established. The reference intervals are based on an extensively characterized, large study population of 1939
Caucasian men and women between 25 and 79 years of age living in northeast Germany. Reference intervals for serum PINP and CTX concentrations in men were age-dependent in our study population. Olmos et al. [12] reported age-independent reference intervals for serum PINP (15–78 ng/mL) and CTX concentrations (0.069–0.760 ng/mL), based on data from 660 Spanish men aged 50–92 years. Although the reference intervals were independent of age, the authors report that men older than 80 years had up to 22% higher CTX concentrations than men 50–60 years of age [12]. Our results, obtained in men from a different age range, provided a complementary message. To the best of our knowledge, reference intervals for men for BAP have not yet been published. Szulc et al. [26] determined mean serum BAP concentrations in a cohort of 934 French men aged 19–85 years.
Fig. 1. Scatterplots showing the relation between serum concentrations of intact amino-terminal propeptide of type I procollagen (PINP), bone-specific alkaline phosphatase (BAP), and carboxy-terminal telopeptide of type I collagen (CTX) and age in men as well as in pre- and postmenopausal women. Individual values (grey dots) and trend lines (solid black lines) with 95% confidence intervals (dashed black lines) from LOESS regression models are displayed.
J. Michelsen et al. / Bone 57 (2013) 399–404 Table 3 Reference intervals for serum PINP, BAP, and CTX concentrations in men. Age group
Reference intervals for men
[years]
PINP [ng/mL]
BAP [ng/mL]
CTX [ng/mL]
25-29 30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 70-74 75-79
31.1–95.9 27.3–90.2 24.1–85.1 21.3–80.7 19.1–77.0 17.3–73.9 16.0–71.4 15.2–69.6 14.9–68.5 15.0–68.0 15.7–68.1
7.4–27.7 7.4–27.4 7.4–27.0 7.5–26.7 7.5–26.4 7.5–26.1 7.5–25.7 7.5–25.4 7.6–25.1 7.6–24.8 7.6–24.4
0.12–0.83 0.11–0.81 0.11–0.78 0.10–0.76 0.09–0.73 0.08–0.71 0.08–0.68 0.07–0.66 0.06–0.63 0.05–0.61 0.05–0.58
BAP, bone-specific alkaline phosphatase; CTX, carboxy-terminal telopeptide of type I collagen; PINP, intact amino-terminal propeptide of type I procollagen Reference intervals were determined by quantile regression.
They [26] detected an age-related decline in serum BAP concentrations for men between 19 and 40 years, with highest levels in men younger than 20 years of age. In men older than 40 years of age, serum BAP concentrations were relatively stable [26]. In two further studies [27,28], including 528 25–75 year-old men from Norway [28], and 179 20–79 year-old men from the U.K. [27] age-related changes in BAP concentrations were not detected. In the present study agerelated changes in serum BAP concentrations were marginal and not statistically significant. Since our study population did not include men under the age of 25 years we might have missed an age-related change in serum BAP concentrations in very young men. Moreover, there is cross-reactivity of the BAP assay with the liver isoenzyme. Whether this cross-reactivity may have contributed to mask any agerelated changes in the serum BAP concentration in men, could not be assessed in the present study. Next to the age-specific reference intervals for men, we determined age-independent reference intervals for pre- and postmenopausal women. Several investigators proposed that antiresorptive therapy in postmenopausal women should be targeted to reach serum BTM concentrations within the lower half of the reference interval of 35–45 year old women [15,19], as bone health is at its best at this age. We found that reference intervals for postmenopausal women were considerably higher than reference intervals for premenopausal women. The increases in BTMs after menopause are well known and largely due to decreased estrogen concentrations which in turn lead to increased osteoclastic activity [14,29]. Several studies provided reference intervals for serum PINP [11,15,17,18], BAP [11,17,18], and CTX concentrations [11,15,17,18], respectively, in premenopausal women. Ardawi et al. [11] provided reference intervals for premenopausal Saudi-Arabian women for PINP (22.3–42.9 ng/mL) and CTX (0.16–0.27 ng/mL). These reference intervals [11] were obtained from 765 women aged 35–45 years and are distinctly narrower than our reference intervals. This may be due to the different laboratory methods applied [17], ethnical differences [6,30], or the different age ranges in the studies. Similar factors may have Table 4 Reference intervals for serum PINP, BAP, and CTX concentrations in women. Women
premenopausal postmenopausal Sensitivity analysis — postmenopausal ages 60–75
N
382 450 263
Reference intervals for women PINP [ng/mL]
BAP [ng/mL]
CTX [ng/mL]
19.3–76.3 18.2–102.3 18.2–99.2
6.0–22.7 8.1–31.6 8.2–31.2
0.05–0.67 0.09–1.05 0.08–0.99
BAP, bone-specific alkaline phosphatase; CTX, carboxy-terminal telopeptide of type I collagen; PINP, intact amino-terminal propeptide of type I procollagen. Reference intervals were determined as the 2.5th and 97.5th percentiles.
403
contributed to the small differences between our reference intervals for premenopausal women and those reported by Adami et al. [15], Eastell et al. [17], and Glover et al. [18]. Only one study [20] reported reference intervals for serum PINP and CTX concentrations in postmenopausal women. This study [20] included 1080 postmenopausal Spanish women aged 44–93 years. The authors [20] reported reference intervals of 19–100 ng/mL for serum PINP and of 0.112–1.018 ng/mL for serum CTX concentrations, which are quite similar to our reference intervals. While there are no reference intervals for BAP, a study including 1083 postmenopausal women from Japan [13] reported a mean serum BAP concentration of 15.7 ng/mL. This is in good agreement with our median BAP concentration in postmenopausal women. In the SHIP study region, intake of oral contraceptives and estrogens for postmenopausal hormone therapy is common. Estrogens are known to inhibit osteoclast activity [14] and thus to alter BTM concentrations [11,15,16]. We found that women taking oral contraceptives or hormone therapy had lower serum BTM concentrations than women not using these drugs. Women on oral contraceptives or hormone therapy were therefore excluded from the reference population. In the present study, we also assessed the effects of month and daytime of blood sampling on each of the three BTMs. Seasonal variation in BTMs is a plausible phenomenon as bone turnover is influenced by vitamin D synthesis [31,32], which varies with the seasons [32]. However, previous studies reported no or only small seasonal variations in serum PINP [33], BAP [32–35], and CTX concentrations [31–33]. Moreover, there was no difference in serum BTM concentrations between subjects with or without vitamin D supplementation [19]. Our results support the notion that serum PINP, BAP, and CTX concentrations do not significantly vary in healthy adult men and women throughout the year. Regarding daytime of blood sampling, previous studies reported no or only small circadian variations in serum PINP [5,36] and BAP concentrations [5,32,34]. Also in the present study there was no circadian variation in serum PINP or BAP concentrations. On the other hand, marked circadian variations in serum CTX concentrations were reported [31] with highest concentrations in the early morning and lowest values in the afternoon [31]. The amplitude of daily variations in serum CTX concentrations was reported to range from 20% to 90% [31]. We detected a weak association between serum CTX concentrations and blood sampling time in premenopausal women but not in postmenopausal women in men. The reasons for the lack of a circadian rhythm in serum CTX concentration in postmenopausal women and in men are unclear, but may be related o the fasting status of our subjects. It has been shown that fasting decreases serum CTX concentration [5]. The majority of the SHIP-1 participants were non-fasting before blood sampling. Therefore, the presented CTX reference intervals need to be interpreted with caution, keeping in mind that they were obtained from non-fasting subjects. The present study was performed in a large sample of healthy subjects from the general population of northeast Germany. The large number of men as well as pre- and postmenopausal women allowed us to determine robust reference intervals. Moreover, our study is strengthened by the extensive characterization of our study participants, including a detailed personal interview regarding lifestyle, medical history and drug use as well as a large array of medical examinations. This allowed us to identify a healthy study population with respect to bone metabolism. Three major limitations need to be mentioned. First, we did not obtain BMD measurements. Thus, we had to rely on the participants' selfreported diagnoses and our reference population may have included some participants with unknown osteoporosis. Second, menopausal status was assessed according to age and self-reported menstrual cycling. For this reason, it is possible, that some participants were falsely classified as pre- or postmenopausal. However, we expect the influence of these limitations on our reference intervals to be small, as we could
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rely on a large number of study subjects and as our results are comparable to previous studies. Third, in clinical practice, BTMs should be determined from fasting, morning blood samples. Due to the large number of participants in our epidemiological study, it was logistically not possible to perform blood sampling at the same time of day or to repeat blood sampling with fasting subjects. In summary, we provide age and gender-specific reference intervals for the determination of PINP, BAP, and CTX concentrations in serum performed by IDS-iSYS Multi Discipline Automated Analyser. Our normative data may aid to interpret bone turnover in adult men, pre- and postmenopausal women.
Funding This work was funded by grants from the German Federal Ministry of Education and Research (BMBF, Grants 01ZZ0403, 01ZZ0103, 01GI0883), the Ministry for Education, Research and Cultural Affairs as well as the Ministry of Social Affairs of the Federal State of Mecklenburg-West Pomerania. This work is also part of the research project Greifswald Approach to Individualized Medicine (GANI_MED). The GANI_MED consortium is funded by the Federal Ministry of Education and Research and the Ministry of Cultural Affairs of the Federal State of Mecklenburg–West Pomerania (03IS2061A). Furthermore, we received an independent research grant for determination of serum samples from Immunodiagnostic Systems.
Appendix A. Supplementary material Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bone.2013.09.010.
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Original Full Length Article
Reference intervals for serum concentrations of three bone turnover markers for men and women J. Michelsen a, H. Wallaschofski a, N. Friedrich a, C. Spielhagen a, R. Rettig b, T. Ittermann c, M. Nauck a, A. Hannemann a,⁎ a b c
Institute of Clinical Chemistry and Laboratory Medicine, University Medicine Greifswald, Greifswald, Germany Institute of Physiology, University Medicine Greifswald, Greifswald, Germany Institute for Community Medicine, University Medicine Greifswald, Greifswald, Germany
a r t i c l e
i n f o
Article history: Received 18 March 2013 Revised 10 September 2013 Accepted 17 September 2013 Available online 27 September 2013 Edited by: Felicia Cosman Keywords: Bone turnover marker Reference intervals Germany Osteoporosis Fracture
a b s t r a c t Objective: Bone turnover markers (BTMs) reflect the metabolic activity of bone tissue and can be used to monitor osteoporosis therapy. To adequately interpret BTMs, method-specific reference intervals are needed. We aimed to determine reference intervals for serum concentrations of intact amino-terminal propeptide of type I procollagen (PINP), bone-specific alkaline phosphatase (BAP) and carboxy-terminal telopeptide of type I collagen (CTX). Material and methods: We established a healthy reference population of 1107 men as well as 382 pre- and 450 postmenopausal women, who participated in the first follow-up of the Study of Health in Pomerania. Serum PINP, BAP and CTX concentrations were measured on the IDS-iSYS Automated System (Immunodiagnostic Systems, Frankfurt am Main, Germany). The reference interval was defined as the central 95% range. We determined age-specific reference intervals for PINP, BAP, and CTX for men by quantile regression. Reference intervals for women were age-independent. Results: Reference intervals for men for PINP and CTX decreased with age (25–29 year-old men: PINP 31.1– 95.9 ng/mL, CTX 0.12–0.83 ng/mL; 75–79 year-old men: PINP 15.7–68.1 ng/mL, CTX 0.05–0.58 ng/mL). The reference interval for men for BAP did not significantly change with age (25–29 year-old men: 7.4–27.7 ng/mL; 75–79 year-old men: 7.6–24.4 ng/mL). The reference intervals for 30–54 year-old premenopausal women were: PINP 19.3–76.3 ng/mL, BAP 6.0–22.7 ng/mL, and CTX 0.05–0.67 ng/mL. The reference intervals for 50–79 year-old postmenopausal women were: PINP 18.2–102.3 ng/mL, BAP 8.1–31.6 ng/mL, and CTX 0.09– 1.05 ng/mL. Conclusion: An intensively characterized, large reference population free of bone-related diseases allowed us to determine robust reference intervals for serum concentrations of PINP, BAP and CTX. Our normative data may aid to interpret bone turnover in adult men and pre- and postmenopausal women. © 2013 Elsevier Inc. All rights reserved.
Introduction The population in the industrialized countries is ageing [1]. The rise in mean age is accompanied by an increasing prevalence of osteoporosis and osteoporotic fractures [2]. Osteoporotic fractures, including hip and vertebral fractures, cause substantial pain, have detrimental effects on quality of life, are associated with disability and mortality and produce
Abbreviations: ATC, Anatomical Therapeutic Chemical Classification System; BAP, bone-specific alkaline phosphatase; BMD, bone mineral density; BMI, body mass index; BTM, bone turnover marker; CTX, carboxy-terminal telopeptide of type I collagen; PINP, amino-terminal propeptide of type I procollagen; PTH, parathyroid hormone; SHIP, Study of Health in Pomerania. ⁎ Corresponding author at: Institute of Clinical Chemistry and Laboratory Medicine, University Medicine Greifswald, Ferdinand-Sauerbruch-Straße NK, D-17475 Greifswald, Germany. Fax: +49 3834 865502. E-mail address: [email protected] (A. Hannemann). 8756-3282/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bone.2013.09.010
high costs [3,4]. Thus, the main target of any osteoporosis therapy is to prevent fractures [3,4]. In clinical routine, the efficacy of osteoporosis therapy is usually assessed by measuring BMD1 [3]. However, BMD changes slowly, often over several months or even years [5]. Earlier effects of osteoporosis therapy can be monitored by the determination of BTMs,2 e.g. two or three months after initiation of oral bisphosphonate therapy [6–9]. BTMs reflect the metabolic activity of bone tissue [6], can be measured in serum or urine samples, and allow for the assessment of bone formation and bone resorption processes [7]. Various bone formation and bone resorption markers have been suggested for clinical use [5,10], e.g. intact PINP,3 BAP,4 or CTX.5 PINP 1 2 3 4 5
BMD, bone mineral density. BTM, bone turnover marker. PINP, amino-terminal propeptide of type I procollagen. BAP, bone-specific alkaline phosphatase. CTX, carboxy-terminal telopeptide of type I collagen.
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is cleaved from procollagen and is released into the circulation during bone formation, while CTX is a degradation product of type I collagen and is released into the circulation during bone resorption [5]. BAP is a glycoprotein involved in bone mineralization whose serum concentration is strongly determined by osteoblast activity [5]. PINP and CTX were recommended as the most informative BTMs for monitoring of osteoporosis in the current European guidance for the diagnosis and management of osteoporosis in postmenopausal women [3]. To interpret BTM concentrations, population-, sex-, and methodspecific reference intervals are needed [6,9,11]. Currently, reference intervals for serum PINP, BAP, and CTX concentrations have neither been established for the German population nor for measurement with the IDS-iSYS Multi Discipline Automated Analyser. For males, PINP and CTX reference intervals have been established in Spanish men older than 50 years of age [12]. BAP reference intervals have not yet been established for men at all. For females, previous studies reported large differences between pre- and postmenopausal serum BTM concentrations [11,13]. BTMs increase during the menopausal transition due to increased osteoclastic activity which in turn is due to decreasing estrogen levels [14]. Accordingly, the intake of sex hormones for menopausal hormone therapy or contraception may alter BTM concentrations [11,15,16]. The majority of previous studies that determined reference intervals for serum PINP [15–19], BAP [16,18,19] or CTX [15–19] concentrations, were limited to premenopausal women. There is only one study [20] that reported reference intervals for PINP and CTX for postmenopausal women. BAP reference intervals for postmenopausal women have not yet been established. We aimed to determine sex- and age-specific reference intervals for serum PINP, BAP and CTX concentrations for adult healthy Caucasian men as well as for pre- and postmenopausal women living in northeast Germany. Additionally, we aimed to analyze the impact of sex hormone therapy on serum BTM concentrations. Material and methods The Study of Health in Pomerania (SHIP) The present study is based on data from the first follow-up of the SHIP.6 SHIP is a population-based cohort study in northeast Germany. In the baseline study (SHIP-0), which was conducted between October 1997 and May 2001, 4308 men and women from a representative sample of 7008 subjects were examined. All subjects were invited to participate in the first follow-up (SHIP-1), which took place between March 2003 and July 2005 with 3300 subjects being re-examined. All SHIP participants gave written informed consent. Further details on study design and sampling have been previously reported [21]. The study was reviewed by an external scientific review board and conformed to the principles of the Declaration of Helsinki as reflected by an a priori approval of the Ethics Committee of the Board of Physicians Mecklenburg-West Pomerania at the University of Greifswald. All SHIP participants underwent standardized medical examinations including blood sampling and an extensive computer-aided personal interview. Data on socio-demographic characteristics and medical histories was collected. Intake of medication was recorded and classified using the ATC7 code. All women were divided according to menopausal status in pre- or postmenopausal. Women younger than 40 years of age and women between 40 and 60 years of age who reported menstrual cycling were defined as premenopausal, all other women were defined as postmenopausal. It was not possible to define perimenopause as the respective information was not collected. Blood sampling was performed between 8.00 a.m. and 8.00 p.m. More than half of all blood samples (58.2%) were taken before 12 a.m., another third (31.0%) between 12:00 a.m. and 2:59 p.m., and one6 7
SHIP, Study of Health in Pomerania. ATC, Anatomical Therapeutic Chemical Classification System.
tenth (10.8%) after 3:00 p.m. Blood samples were taken from the cubital vein of mostly non-fasting participants in the supine position. Serum aliquots were stored at −80 °C. Serum PINP, BAP, and CTX concentrations were determined on the IDS-iSYS Multi-Discipline Automated Analyser (Immunodiagnostic Systems Limited, Frankfurt am Main, Germany). For each analyte three concentrations of control material were measured by skilled technical personnel. Serum PINP concentrations were measured with the IDS-iSYS Intact PINP assay. During the course of the study the coefficients of variation were 7.33% at low concentrations, 5.67% at medium concentrations, and 7.88% at high concentrations, respectively, of control material. The assay manufacturer reports good linearity in the range of 5.8–203.8 ng/mL, with variations between −3% and +3% of predicted and measured PINP concentrations. Serum BAP concentrations were measured with the IDS-iSYS Ostase + BAP assay. During the course of the study the coefficients of variation were 12.28% at low concentrations, 13.47% at medium concentrations, and 13.23% at high concentrations, respectively, of control material. The assay manufacturer reports good linearity in the range of 9.7–71.0 ng/mL, with variations between −2% and +7% of predicted and measured BAP concentrations. The assay demonstrates a comparable cross-reactivity to liver isoenzyme as the Tandem R Ostase assay [22], as it was developed using the same monoclonal antibody. The assay provides a result of 6.9 ng/mL BAP for each 100 U/L liver enzyme. Serum CTX concentrations were measured with the IDS-iSYS CTX-I (CrossLaps) assay. During the course of the study, the coefficients of variation were 12.23% at low concentrations, 10.40% at medium concentrations, and 11.40% at high concentrations, respectively, of control material. The assay manufacturer reports good linearity in the range of 0.245–5.293 ng/mL, with variations between −6% and +3% of predicted and measured CTX concentrations. Reference population To generate a healthy reference population we excluded all subjects with missing data on serum PINP, BAP, or CTX concentrations (n = 39), or with any of the following conditions: renal disease defined as estimated glomerular filtration rate (Cockcroft-Gault) b30 mL/min (n = 30), hyperparathyroidism defined as serum parathyroid hormone concentration N120 pg/mL (n = 24), hyperthyroidism defined according to local reference intervals [23] as serum thyroid-stimulating hormone concentration b0.25 mU/L and serum free thyroxine concentration N18.9 pmol/L (n = 70), or a selfreported history of cancer (n = 189), a self-reported physician's diagnosis of osteoporosis (n = 207), or a self-reported history of liver disease (n = 41). We further excluded all pregnant women (n = 12), all subjects with serum 25-hydroxy vitamin D concentration b 10 ng/mL (n = 342), and all subjects (n = 85) who reported intake of any of the following medication: bisphosphonates, strontium ranelate, vitamin D, calcium, parathyroid hormone, calcitonin, steroids, selective estrogen receptor modulators, testosterone, anticonvulsants, heparin, antiandrogens, or aromatase inhibitors. Moreover, we excluded all premenopausal women using oral contraceptives (n = 126) and all postmenopausal women using hormone replacement therapy (n = 55). Furthermore, we excluded subjects with a serum osteocalcin concentration N100 ng/mL (n = 2) and, due to small numbers in the respective age groups, all subjects older than 79 years of age (n = 58), all premenopausal women younger than 30 years of age (n = 36) or older than 54 years of age (n = 3), and all postmenopausal women younger than 50 years of age (n = 44). This resulted in a reference population of 1939 subjects, including 1107 men as well as 382 premenopausal women (aged 30–54 years) and 450 postmenopausal women (aged 50–79 years). Statistical analyses Continuous data are expressed as median (1st-3rd quartile) and nominal data are expressed as percentage. We report median instead
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of mean values for the three BTM serum concentrations as they are not normally distributed. Group comparisons were performed using Kruskal–Wallis tests. P-values b0.05 were considered statistically significant. Reference intervals were defined as the central 95% range between the 2.5th and 97.5th percentiles of serum PINP, BAP, and CTX concentrations, respectively. Scatterplots with LOESS curves and 95% confidence intervals (CI) were used to assess the relation between the three BTMs and age. The LOESS procedure is a nonparametric method to fit a local regression function. It produces a smooth curve fitted to the data set and allows a graphical evaluation of the relation between the BTMs and age. Agerelated changes in the BTMs were further tested for statistical significance with quantile regression models [24]. Reference limits for men were determined as a function of age by quantile regression, while reference limits for women were determined disregarding age. The agerelated changes of the serum PINP concentration in men were modeled by fitting a quadratic equation (including age and age squared), while the models for the serum BAP, and CTX concentrations were fitted by a linear equation (including age). Reference limits for men for serum PINP, BAP, and CTX concentrations were estimated for each single year of age. Subsequently, mean values for five-year age groups were calculated. Next to age-related changes we tested the influence of sex hormones for oral contraception or hormone therapy, of month or daytime of blood sampling, as well as of 25-hydroxy vitamin D and parathyroid hormone concentrations on the three BTMs within quantile regression models. All statistical analyses were performed with SAS 9.1.3 (SAS Institute Inc., Cary, NC, USA).
Results Characteristics of the study population, including median age, BMI,8 the proportion of diabetes mellitus, as well as median concentrations of 25-hydroxy vitamin D, parathyroid hormone, and BTMs are presented in Table 1. Age-specific (5-year age groups) median serum concentrations of PINP, BAP, and CTX are presented in Table 2. Scatterplots with LOESS curves for men as well as pre- and postmenopausal women are displayed in Fig. 1. The LOESS curves for serum PINP and CTX in men indicated decreasing concentrations with increasing age at an age range of 25 to 55 years and a plateau at older ages. In contrast to serum PINP and CTX concentrations, serum BAP concentrations in men hardly changed with age. The LOESS curve indicated a very modest decrease in serum BAP concentrations in men between 25 and 40 years of age and after 70 years of age. Reference intervals for men were determined with quantile regression models fitted with parameters presented in Supplemental Table 1. Reference intervals for men are presented in Table 3. For men aged 25–54 years, upper and lower reference limits for the serum PINP concentration decreased continuously with increasing age. For older men, the upper and lower reference limits remained relatively stable. Reference intervals for the serum BAP concentration were stable throughout the observed age range, with very small decreases in lower and upper reference limits between the ages 25–79. The lower CTX reference limits decreased strongly with increasing age between 25 and 54 years of age and were relatively stable for older men, while the upper reference limits continued to decrease over the whole age range of 25–79 years. Reference curves for men are visualized in Supplemental Fig. 1. The LOESS curves for all three BTMs for premenopausal women between 30 and 54 years of age indicated relatively stable concentrations over the observed age range (Fig. 1). Also quantile regression models did not indicate significant age-related changes in the three BTMs in premenopausal women. In postmenopausal women serum BTM concentrations were markedly higher than in premenopausal women 8
BMI, body mass index.
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Table 1 Characteristics of the reference population. Characteristics
Men (N = 1107)
Women, premenopausal (N = 382)
Women, postmenopausal (N = 450)
Age [years] BMI [kg/m2] Diabetes mellitus [%] 25-hydroxy vitamin D [μg/L] Parathyroid hormone [pg/mL] PINP [ng/mL] BAP [ng/mL] CTX [ng/mL]
53.0 (41.0–64.0) 27.9 (25.5–30.7) 10.1
40.0 (36.0–45.0) 25.0 (22.3–28.6) 1.1
62.0 (56.0–68.0) 28.2 (25.1–32.2) 14.22
19.2 (14.6–25.1)
19.0 (14.2–27.5)
18.1 (13.7–23.5)
33.7 (25.7–43.2)
28.9 (21.9–38.6)
36.7 (27.4–46.0)
38.0 (29.2–49.4) 14.0 (11.3–16.8) 0.28 (0.19–0.41)
36.7 (28.2–46.4) 10.8 (8.9–13.6) 0.23 (0.16–0.34)
46.4 (32.7–62.0) 16.3 (13.1–20.4) 0.37 (0.25–0.56)
BAP, bone-specific alkaline phosphatase; BMI, body mass index; CTX, carboxy-terminal telopeptide of type I collagen; PINP, intact amino-terminal propeptide of type I procollagen Data are median (1st–3rd quartile) or proportions.
(Table 2). The LOESS curves in postmenopausal women indicated an increase in the serum BTM concentrations between ages 50 and 55 followed by a decrease until age 60. In Europe, the median age of natural menopause is about 54 years [25]. These age-related fluctuations in the three BTMs may thus be due to the menopausal transition. In postmenopausal women older than 60 years of age the three BTMs were relatively stable. Small decreases in serum PINP and CTX concentrations were observed in women older than 75 years of age. Quantile regression models did not reveal age-related changes in the three BTMs in postmenopausal women. Reference intervals for pre- and postmenopausal women were thus determined disregarding age (Table 4). The upper reference limits for all three BTMs were substantially higher for post- than for premenopausal women. The lower reference limits for serum BAP and CTX concentrations were also higher for post- than for premenopausal women but to a lesser extent. In a sensitivity analysis we included only postmenopausal women between 60 and 75 years of age to assess whether the fluctuation in the BTMs in early menopause affected the reference intervals. The reference intervals for the three BTMs were very similar to those obtained in all postmenopausal women (Table 4). Therefore, we assume that the reference intervals obtained in the main analyses are valid for all postmenopausal women. Month of blood sampling was not associated with serum PINP, BAP, or CTX concentrations in men or pre- or postmenopausal women, regardless whether or not subjects with 25-hydroxy Vitamin D concentrations b10 ng/mL (n = 276) were included. Daytime of blood sampling (in hours) was also not associated with serum PINP or BAP concentrations. However, we detected an association between daytime of blood sampling and the serum CTX concentration in premenopausal women (Supplemental Figs. 2–4). As this association was not statistically significant (p = 0.46) and vanished when women with blood sampling after 5:00 p.m. were excluded from the analyses, daytime of blood sampling was not further considered in the analyses. There were no associations between serum concentrations of PTH9 and PINP or BAP. Yet, there were positive associations between serum concentrations of PTH and CTX. Quantile regression models yielded the following results for each 10 pg/mL increase in PTH: men estimate 0.02 ng/mL (p b0.001), premenopausal women estimate 0.03 ng/mL (p b 0.001), postmenopausal women estimate 0.02 ng/mL (p = 0.004). Moreover, there were a negative association between the serum 25hydroxy Vitamin D and BAP concentration in premenopausal women as well as a positive association between the serum 25-hydroxy Vitamin D and CTX concentration in men. We cannot exclude that the associations between the serum 25-hydroxy Vitamin D and BAP or CTX concentrations were statistical artefacts since they were observed only in subsamples of the population. 9
PTH, parathyroid hormone.
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Table 2 Serum PINP, BAP and CTX concentrations in men (N = 1107) as well as in premenopausal (N = 382) and postmenopausal women (N = 450) according to 5-year age groups. Age group [years]
25–29 30–34 35–39 40–44 45–49 50–54
50–54 55–59 60–64 65–69 70–74 75–79
N
PINP [ng/mL]
BAP [ng/mL]
CTX [ng/mL]
Men
Premenopausal Women
Men
Premenopausal Women
p
Men
Premenopausal Women
p
Men
Premenopausal Women
p
49 79 113 113 123 127
– 75 96 96 72 43
53.4 (47.9–63.0) 48.9 (37.8–57.7) 43.2 (33.7–58.0) 43.4 (36.4–52.0) 33.9 (29.0–44.3) 33.8 (27.2–46.4)
– 38.3 (28.2–47.9) 37.8 (30.3–50.2) 36.2 (27.3–45.5) 35.7 (26.9–44.2) 36.4 (29.5–42.1)
– b0.01 0.01 b0.01 0.41 0.83
14.6 (12.3–18.2) 14.5 (12.1–18.4) 13.5 (11.7–15.9) 14.7 (12.5–16.9) 13.9 (11.4–16.6) 13.2 (10.6–16.4)
– 10.8 (9.1–13.6) 11.3 (9.3–13.6) 10.6 (8.8–12.5) 10.6 (8.5–13.1) 11.9 (9.6–15.4)
– b0.01 b0.01 b0.01 b0.01 0.14
0.50 (0.28–0.61) 0.35 (0.26–0.45) 0.30 (0.21–0.44) 0.32 (0.22–0.42) 0.26 (0.16–0.34) 0.25 (0.16–0.42)
– 0.22 (0.14–0.33) 0.25 (0.17–0.33) 0.23 (0.16–0.31) 0.23 (0.15–0.38) 0.24 (0.17–0.38)
– b0.01 b0.01 b0.01 0.69 0.65
Men
Postmenopausal Women
Men
Postmenopausal Women
p
Men
Postmenopausal Women
p
Men
Postmenopausal Women
p
127 118 132 114 85 54
65 98 114 81 55 37
33.8 (27.2–46.4) 35.3 (27.9–45.3) 34.4 (27.9–42.4) 34.0 (25.9–40.8) 33.2 (26.3–42.6) 33.6 (24.2–44.8)
50.6 (32.9–65.0) 53.3 (35.1–74.2) 47.4 (35.2–59.8) 45.1 (30.7–57.8) 42.4 (30.8–63.3) 38.5 (28.3–52.3)
b0.01 b0.01 b0.01 b0.01 b0.01 0.13
13.2 (10.6–16.4) 14.1 (11.3–17.2) 14.4 (11.9–17.1) 13.4 (10.7–16.8) 12.3 (10.9–14.7) 12.8 (10.5–15.7)
15.8 (12.5–21.0) 17.6 (14.8–20.9) 16.2 (13.3–20.4) 15.8 (12.8–18.8) 15.8 (12.7–21.4) 16.1 (11.8–18.1)
b0.01 b0.01 b0.01 b0.01 b0.01 b0.01
0.25 (0.16–0.42) 0.28 (0.19–0.40) 0.24 (0.17–0.35) 0.25 (0.18–0.36) 0.24 (0.17–0.33) 0.24 (0.18–0.41)
0.40 (0.25–0.59) 0.44 (0.30–0.62) 0.32 (0.23–0.51) 0.38 (0.23–0.55) 0.37 (0.26–0.60) 0.33 (0.22–0.44)
b0.01 b0.01 b0.01 b0.01 b0.01 0.04
BAP, bone-specific alkaline phosphatase; CTX, carboxy-terminal telopeptide of type I collagen; PINP, intact amino-terminal propeptide of type I procollagen. Data are median (1st–3rd quartile). Sex differences were tested with Kruskal-Wallis tests.
Premenopausal women taking oral contraceptives (n = 91) had lower serum PINP, BAP, and CTX concentrations than premenopausal women not taking oral contraceptives. Likewise, postmenopausal women taking sex hormones (n = 48) had lower serum BTM concentrations than postmenopausal women not taking hormone therapy (Supplemental Table 2).
Discussion In the present study sex-specific reference intervals for serum PINP, BAP, and CTX concentrations measured on the IDS-iSYS Multi Discipline Automated Analyser were established. The reference intervals are based on an extensively characterized, large study population of 1939
Caucasian men and women between 25 and 79 years of age living in northeast Germany. Reference intervals for serum PINP and CTX concentrations in men were age-dependent in our study population. Olmos et al. [12] reported age-independent reference intervals for serum PINP (15–78 ng/mL) and CTX concentrations (0.069–0.760 ng/mL), based on data from 660 Spanish men aged 50–92 years. Although the reference intervals were independent of age, the authors report that men older than 80 years had up to 22% higher CTX concentrations than men 50–60 years of age [12]. Our results, obtained in men from a different age range, provided a complementary message. To the best of our knowledge, reference intervals for men for BAP have not yet been published. Szulc et al. [26] determined mean serum BAP concentrations in a cohort of 934 French men aged 19–85 years.
Fig. 1. Scatterplots showing the relation between serum concentrations of intact amino-terminal propeptide of type I procollagen (PINP), bone-specific alkaline phosphatase (BAP), and carboxy-terminal telopeptide of type I collagen (CTX) and age in men as well as in pre- and postmenopausal women. Individual values (grey dots) and trend lines (solid black lines) with 95% confidence intervals (dashed black lines) from LOESS regression models are displayed.
J. Michelsen et al. / Bone 57 (2013) 399–404 Table 3 Reference intervals for serum PINP, BAP, and CTX concentrations in men. Age group
Reference intervals for men
[years]
PINP [ng/mL]
BAP [ng/mL]
CTX [ng/mL]
25-29 30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 70-74 75-79
31.1–95.9 27.3–90.2 24.1–85.1 21.3–80.7 19.1–77.0 17.3–73.9 16.0–71.4 15.2–69.6 14.9–68.5 15.0–68.0 15.7–68.1
7.4–27.7 7.4–27.4 7.4–27.0 7.5–26.7 7.5–26.4 7.5–26.1 7.5–25.7 7.5–25.4 7.6–25.1 7.6–24.8 7.6–24.4
0.12–0.83 0.11–0.81 0.11–0.78 0.10–0.76 0.09–0.73 0.08–0.71 0.08–0.68 0.07–0.66 0.06–0.63 0.05–0.61 0.05–0.58
BAP, bone-specific alkaline phosphatase; CTX, carboxy-terminal telopeptide of type I collagen; PINP, intact amino-terminal propeptide of type I procollagen Reference intervals were determined by quantile regression.
They [26] detected an age-related decline in serum BAP concentrations for men between 19 and 40 years, with highest levels in men younger than 20 years of age. In men older than 40 years of age, serum BAP concentrations were relatively stable [26]. In two further studies [27,28], including 528 25–75 year-old men from Norway [28], and 179 20–79 year-old men from the U.K. [27] age-related changes in BAP concentrations were not detected. In the present study agerelated changes in serum BAP concentrations were marginal and not statistically significant. Since our study population did not include men under the age of 25 years we might have missed an age-related change in serum BAP concentrations in very young men. Moreover, there is cross-reactivity of the BAP assay with the liver isoenzyme. Whether this cross-reactivity may have contributed to mask any agerelated changes in the serum BAP concentration in men, could not be assessed in the present study. Next to the age-specific reference intervals for men, we determined age-independent reference intervals for pre- and postmenopausal women. Several investigators proposed that antiresorptive therapy in postmenopausal women should be targeted to reach serum BTM concentrations within the lower half of the reference interval of 35–45 year old women [15,19], as bone health is at its best at this age. We found that reference intervals for postmenopausal women were considerably higher than reference intervals for premenopausal women. The increases in BTMs after menopause are well known and largely due to decreased estrogen concentrations which in turn lead to increased osteoclastic activity [14,29]. Several studies provided reference intervals for serum PINP [11,15,17,18], BAP [11,17,18], and CTX concentrations [11,15,17,18], respectively, in premenopausal women. Ardawi et al. [11] provided reference intervals for premenopausal Saudi-Arabian women for PINP (22.3–42.9 ng/mL) and CTX (0.16–0.27 ng/mL). These reference intervals [11] were obtained from 765 women aged 35–45 years and are distinctly narrower than our reference intervals. This may be due to the different laboratory methods applied [17], ethnical differences [6,30], or the different age ranges in the studies. Similar factors may have Table 4 Reference intervals for serum PINP, BAP, and CTX concentrations in women. Women
premenopausal postmenopausal Sensitivity analysis — postmenopausal ages 60–75
N
382 450 263
Reference intervals for women PINP [ng/mL]
BAP [ng/mL]
CTX [ng/mL]
19.3–76.3 18.2–102.3 18.2–99.2
6.0–22.7 8.1–31.6 8.2–31.2
0.05–0.67 0.09–1.05 0.08–0.99
BAP, bone-specific alkaline phosphatase; CTX, carboxy-terminal telopeptide of type I collagen; PINP, intact amino-terminal propeptide of type I procollagen. Reference intervals were determined as the 2.5th and 97.5th percentiles.
403
contributed to the small differences between our reference intervals for premenopausal women and those reported by Adami et al. [15], Eastell et al. [17], and Glover et al. [18]. Only one study [20] reported reference intervals for serum PINP and CTX concentrations in postmenopausal women. This study [20] included 1080 postmenopausal Spanish women aged 44–93 years. The authors [20] reported reference intervals of 19–100 ng/mL for serum PINP and of 0.112–1.018 ng/mL for serum CTX concentrations, which are quite similar to our reference intervals. While there are no reference intervals for BAP, a study including 1083 postmenopausal women from Japan [13] reported a mean serum BAP concentration of 15.7 ng/mL. This is in good agreement with our median BAP concentration in postmenopausal women. In the SHIP study region, intake of oral contraceptives and estrogens for postmenopausal hormone therapy is common. Estrogens are known to inhibit osteoclast activity [14] and thus to alter BTM concentrations [11,15,16]. We found that women taking oral contraceptives or hormone therapy had lower serum BTM concentrations than women not using these drugs. Women on oral contraceptives or hormone therapy were therefore excluded from the reference population. In the present study, we also assessed the effects of month and daytime of blood sampling on each of the three BTMs. Seasonal variation in BTMs is a plausible phenomenon as bone turnover is influenced by vitamin D synthesis [31,32], which varies with the seasons [32]. However, previous studies reported no or only small seasonal variations in serum PINP [33], BAP [32–35], and CTX concentrations [31–33]. Moreover, there was no difference in serum BTM concentrations between subjects with or without vitamin D supplementation [19]. Our results support the notion that serum PINP, BAP, and CTX concentrations do not significantly vary in healthy adult men and women throughout the year. Regarding daytime of blood sampling, previous studies reported no or only small circadian variations in serum PINP [5,36] and BAP concentrations [5,32,34]. Also in the present study there was no circadian variation in serum PINP or BAP concentrations. On the other hand, marked circadian variations in serum CTX concentrations were reported [31] with highest concentrations in the early morning and lowest values in the afternoon [31]. The amplitude of daily variations in serum CTX concentrations was reported to range from 20% to 90% [31]. We detected a weak association between serum CTX concentrations and blood sampling time in premenopausal women but not in postmenopausal women in men. The reasons for the lack of a circadian rhythm in serum CTX concentration in postmenopausal women and in men are unclear, but may be related o the fasting status of our subjects. It has been shown that fasting decreases serum CTX concentration [5]. The majority of the SHIP-1 participants were non-fasting before blood sampling. Therefore, the presented CTX reference intervals need to be interpreted with caution, keeping in mind that they were obtained from non-fasting subjects. The present study was performed in a large sample of healthy subjects from the general population of northeast Germany. The large number of men as well as pre- and postmenopausal women allowed us to determine robust reference intervals. Moreover, our study is strengthened by the extensive characterization of our study participants, including a detailed personal interview regarding lifestyle, medical history and drug use as well as a large array of medical examinations. This allowed us to identify a healthy study population with respect to bone metabolism. Three major limitations need to be mentioned. First, we did not obtain BMD measurements. Thus, we had to rely on the participants' selfreported diagnoses and our reference population may have included some participants with unknown osteoporosis. Second, menopausal status was assessed according to age and self-reported menstrual cycling. For this reason, it is possible, that some participants were falsely classified as pre- or postmenopausal. However, we expect the influence of these limitations on our reference intervals to be small, as we could
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rely on a large number of study subjects and as our results are comparable to previous studies. Third, in clinical practice, BTMs should be determined from fasting, morning blood samples. Due to the large number of participants in our epidemiological study, it was logistically not possible to perform blood sampling at the same time of day or to repeat blood sampling with fasting subjects. In summary, we provide age and gender-specific reference intervals for the determination of PINP, BAP, and CTX concentrations in serum performed by IDS-iSYS Multi Discipline Automated Analyser. Our normative data may aid to interpret bone turnover in adult men, pre- and postmenopausal women.
Funding This work was funded by grants from the German Federal Ministry of Education and Research (BMBF, Grants 01ZZ0403, 01ZZ0103, 01GI0883), the Ministry for Education, Research and Cultural Affairs as well as the Ministry of Social Affairs of the Federal State of Mecklenburg-West Pomerania. This work is also part of the research project Greifswald Approach to Individualized Medicine (GANI_MED). The GANI_MED consortium is funded by the Federal Ministry of Education and Research and the Ministry of Cultural Affairs of the Federal State of Mecklenburg–West Pomerania (03IS2061A). Furthermore, we received an independent research grant for determination of serum samples from Immunodiagnostic Systems.
Appendix A. Supplementary material Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bone.2013.09.010.
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