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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 1 95-101
Copyright © 2000 by The Endocrine Society

Original Studies

Physiologic Fluctuations of Serum Estradiol Levels Influence Biochemical Markers of Bone Resorption in Young Women

A. Zittermann, I. Schwarz, K. Scheld, T. Sudhop, H. K. Berthold, K. von Bergmann, H. van der Ven and P. Stehle

Department of Nutrition Science (A.Z., I.S., K.S., P.S.), Department of Clinical Pharmacology (T.S., H.K.B., K.v.B.), and Department of Gynecological Endocrinology (H.v.d.V.), University of Bonn, 53115 Bonn, Germany

Address correspondence and requests for reprints to: Armin Zittermann, Ph.D., Associate Professor, Department of Nutrition Science, University of Bonn, Endenicher Allee 11–13, 53115 Bonn. E-mail: a.zittermann{at}uni-bonn.de


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We investigated the effect of physiologic variations in sex hormone levels during the menstrual cycle on biomarkers of bone turnover. Blood and 24-h and fasting urine samples were obtained in nine women (age, 25.1 ± 3.0 yr) with regular menstrual cycles during the early follicular period (t1), 3 days before ovulation (t2), 3 days after ovulation (t3), at the midluteal period (t4) and again during the early follicular period of the next cycle (t5). All subjects had a calcium intake covering current dietary recommendations (above 1.000 mg/day, standardized food record).

Serum calcium, phosphorus, calcitriol, 24-h and 2-h fasting urinary calcium, and phosphorus excretion remained constant during the menstrual cycle. Serum 25-hydroxyvitamin D3 levels decreased slightly from the beginning until the end of the study (P < 0.05), indicating low cutaneous vitamin D synthesis during wintertime.

The serum levels of sex hormones showed typical monthly variations, with the lowest estradiol (E2) levels at t1 and t5. Fasting 2-h pyridinoline (Pyd) concentrations (a marker of bone resorption) fell from t1 to t3 and rose again at t5 (P < 0.01). Similar variations were observed for the resorption marker deoxypyridinoline (Dpd; P < 0.05). The amplitude of the two biomarkers was 32% and 33%, respectively. The serum levels of the carboxyterminal propeptide of type I collagen (a marker of bone formation) showed an inverse cyclic pattern, as compared with the pyridinium cross-links. Low concentrations were observed at t1; a rise occurred until t3 and was followed by a decrease until t5 (P < 0.05). A similar cyclic pattern was observed for serum PTH levels, with the highest concentrations at t3 (P < 0.05).

Dpd and Pyd values were significantly correlated with serum E2 levels (r = 0.52; P < 0.0001 and r = 0.50; P < 0.001, respectively). Neither progesterone nor LH nor FSH was correlated with Pyd or Dpd levels.

The data suggest that normal menstrual cycling in young women is associated with monthly fluctuations in bone turnover. This physiological effect of the menstrual cycle is most probably related to variations in serum E2 concentrations.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
SOME STUDIES have been performed during the last decade to investigate the impact of cyclic fluctuations in female sex hormones and in endogenous androgens on biomarkers of bone turnover (1, 2, 3, 4, 5). However, these analyses exhibited inconsistent results: significant variations were found in the bone formation markers osteocalcin and carboxyterminal propeptide of type I procollagen (PICP) in two studies (1, 4), whereas others observed constant serum levels of these biomarkers of osteoblastic activity during the menstrual cycle (2, 5).

In one study (2), small (but significant) variations of the bone resorption marker s-ICTP (serum Pyd cross-linked-terminal telopeptide of type I collagen) were observed, whereas urinary excretion of the resorption markers Dpd and Pyd remained constant during the menstrual cycle. In contrast, two recent studies (3, 4) found significant cyclic changes in renal Dpd and several other bone resorption markers, and an association between biomarkers of bone turnover and sex steroid hormones has been suggested from these results. Nevertheless, no correlation between bone resorption markers and sex hormones was found (3, 4). Thus, it is still unclear whether monthly variations in bone resorption in young healthy women are mediated by sex hormones.

Because these earlier studies did not account for the short-term nutritional status of the volunteers, it is possible that hormonal effects on bone tissue may be masked by nutritional factors, e.g. by differences in calcium (Ca) intake (6, 7).

We therefore performed a study with a group of healthy women, during one menstrual cycle, to analyze several biomarkers of bone metabolism under standardized nutrient intake conditions throughout the study period.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Ten healthy Caucasian women (age, 25.1 ± 3.0 yr; body mass index, 21.1 ± 2.1 kg/m2) were enrolled in the study. Exclusion criteria (questionnaire) were: intake of oral contraceptives; and amenorrhea and eating disorders, like anorexia nervosa and bulimia. All subjects had had regular menstrual cycles for at least 3 months (mean cycle length, 28.0 ± 2.9 days). Pregnancy was excluded by use of standard tests 1 day before actual examinations.

Written informed consent was given by each subject. The study protocol was approved by the ethical committee of the University of Bonn.

Study protocol

Design. The study was performed in the winter season, from January 6 until February 17, at a geographic latitude of 51° N. Serum and urine analyses were performed on day 3 after onset of menstruation (t1), 3 days before ovulation (t2), 3 days after ovulation (t3), during the midluteal phase (t4), and again 3 days after onset of the next menstruation (t5). The time of ovulation was calculated according to the method of Knaus and Ogino by using the mean duration of the last three menstrual cycles and subtracting 14 days. Midluteal phase was calculated by the day of ovulation plus 7 days. A rise in serum progesterone (P4) levels above 5 ng/mL during the luteal period was assumed to indicate ovulatory cycles.

Nutrient intake. Nutrient intake was assessed by a prospective standardized food record, which had to be completed by the subjects on each day before the actual examination. Mineral water and fruit juices consumed had to be specified; intake of Ca supplements had to be listed. All participants were asked to include Ca-rich foods in their daily diet. Nutrient content of the diets was quantified using a computer program based on the Bundeslebensmittelschluessel (BLS II.1). We measured 24-h renal nitrogen excretion to determine the reliability of nutrient intake, as estimated by the food record.

Sampling. Serum was collected (days t1–t5) from the vena cubitalis in serum monovettes before breakfast. One day before blood sampling, 24-h urine (0700 to 0700 h) was collected. On the next morning, a 2-h urine sample (second spontaneous urine after morning urine) was obtained after a 12-h overnight fast, at 0900 h, before breakfast. Aliquots of blood and urine samples were frozen immediately, at -20 C, until analysis.

Analytical procedures

All samples were measured during the same assay sequence. The serum concentrations of LH, FSH, P4, estradiol (E2), and sex hormone binding globulin (SHBG) were measured by micro enzyme-linked immunoassays (ELISAs) using an autoanalyzer (Abbott Laboratories, Wiesbaden, Germany). Serum levels of estrone (E1) were analyzed using an ELISA supplied by IBL (Hamburg, Germany), and intact PTH levels were determined using an ELISA test kit from DRG Diagnostics (Marburg, Germany). Serum 25-hydroxyvitamin D (25-OH-D) metabolites (25-OH-D2 and 25-OH-D3) were separated and quantified after solid-phase extraction with C18 cartridges (8) using high-performance liquid chromatography and UV-detection at 264 nm. The detection limit was 1.5 ng/mL for each metabolite. Serum calcitriol was determined by a radio receptor assay using a calf thymus cytosol binding protein (9). Serum carboxyterminal PICP was measured by means of an ELISA, with a commercial kit supplied by Biermann GmbH (Bad Nauheim, Germany). Coefficients of variation (CVs) for all assays described above were below 10%. Blood and urine Ca, sodium (Na), and phosphorus (P) concentrations were analyzed using atomic absorption spectrometry (Ca and Na) and a colorimetric test kit (P; BioMerieux, Nürtingen, Germany). CVs were below 2.5%. Urinary nitrogen was determined by high sensitive chemiluminescence (10), with an imprecision below 3%. The total concentrations of Dpd and Pyd were measured in the 2-h urine samples after acid hydrolysis of protein-bound cross-links, by high-performance liquid chromatography, as described by Uebelhardt et al. (11). The intraassay CVs were 6.7% and 7.4% and the interassay CVs were 10.1% and 11.6%, respectively. Urinary creatinine was analyzed by the Jaffé reaction. All results for urinary calcium, P, Pyd, and Dpd were expressed per urinary creatinine excretion.

Statistical methods

Statistical analyses were performed with the Statistical Package for the Social Sciences (SPSS, Inc./PC+, Chicago, IL). Data were evaluated using the Friedman test. In the case of significant differences between the sampling time points, Wilcoxon-Wilcox test was used to further specify these differences (12). To assess interrelationships between variables, Pearson’s correlation coefficient and nonlinear regression analyses were used. P values below 0.05 (two-tailed test) were considered significant. Except when explicitly indicated, data are presented as means ± SD.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Total energy consumption and nutrient relations were constant during the study period (Table 1Go). Mean Ca and P intakes were well above 1,000 mg/day. Urinary nitrogen excretion was not different during the five investigational periods. Assuming that 2 g of nitrogen are excreted via extrarenal routes, reported nitrogen intake was slightly higher than nitrogen excretion. Urine volume and urinary water, Na, and Cr excretion remained unchanged during the menstrual cycle (Table 1Go).


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  		Table 1. Nutrient intake and urinary water, nitrogen, and sodium excretion

 
P4 values of one subject did not exceed 1.5 ng/mL during the entire cycle. Consequently, the data of this subject were excluded from further evaluations. P4 values of the remaining nine participants showed typical variations during the menstrual cycle (Table 2Go). Serum LH and serum FSH levels were within the normal range. Serum E1 levels increased during the end of the follicular period, decreased after ovulation, and increased again during the midluteal period (Fig. 1Go). A similar, but more pronounced, cyclic pattern was observed for E2 serum levels. Serum SHBG levels remained constant throughout the menstrual cycle. Sex hormone levels were similar on examinations t1 and t5.


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  		Table 2. Mean levels of P4, LH and FSH during the study period ( ± SD)

 


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  		Figure 1. Mean values (±SE) for serum levels of E1 (A), E2 (B), and SHBG (C) on day 3 of the follicular period (t1), 3 days before ovulation (t2), 3 days after ovulation (t3), at midluteal period (t4), and on day 3 of the next follicular period (t5). *, P < 0.05 vs. t1 and t5; **, P < 0.025 vs. t1 and t5.

 
The mean duration of the menstrual cycle was 28.3 ± 3.0 days and was similar to the cycle length of the participants during the last 3 months before the study.

Serum Ca and P, as well as urinary electrolyte excretions, were comparable among the five examinations (Table 3Go). Mean serum 25-OH-D3 concentrations decreased by 2 ng/mL from the beginning until the end of the study (Table 3Go). Serum 25-OH-D2 levels were below the detection limit of 1.5 ng/L in all subjects. Serum calcitriol levels remained unchanged, whereas serum PTH was significantly increased at t3 (see Fig. 3Go).


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  		Table 3. Biochemical parameters of calcium and phosphorus metabolism

 


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  		Figure 3. Mean values (±SE) for serum PTH levels on day 3 of the follicular period (t1), 3 days before ovulation (t2), 3 days after ovulation (t3), at midluteal period (t4), and on day 3 of the next follicular period (t5). *, P < 0.05 vs. t5.

 
Urinary Pyd and Dpd excretion was influenced by the hormonal status (Fig. 2Go). Pyd and Dpd concentrations were lowest 3 days after ovulation (t3) and highest during the early follicular phase (t1). The mean amplitude for Dpd was 33%. Pyd showed a variation during the menstrual cycle similar to that of Dpd. The ratio between Pyd and Dpd remained constant (between 3.34:1 and 3.53:1) at each time point. Serum PICP levels reached a minimum during the early follicular period and a maximum at t3. The mean amplitude for PICP was 12% (Fig. 2Go).



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  		Figure 2. Mean values (±SE) for serum levels of PICP (A) and for urinary levels of Dpd (B) and Pyd (C) on day 3 of the follicular period (t1), 3 days before ovulation (t2), 3 days after ovulation (t3), at midluteal period (t4), and on day 3 of the next follicular period (t5). *, P < 0.05 vs. t5; **, P < 0.01 vs. t5.

 
Dpd values were correlated with Pyd concentrations (r = 0.82; P < 0.0001) and PICP levels (r = 0.36; P < 0.025). Moreover, data showed a nonlinear inverse correlation of E2 serum concentrations with renal Dpd (see Fig. 4Go) and a similar association with Pyd concentrations (r = 0.50, P < 0.001) but no correlation between the collagen cross-links and the serum levels of E1, LH, FSH, P4, 25-OH-D3, PTH, or calcitriol.



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  		Figure 4. Association between renal Dpd and serum E2 values (r = 0.52, P < 0.0001; regression equation: Dpd/Cr = 42,7·E2-0.231).

 
Results of P4, E2, SHBG, and biomarkers of bone turnover of the subject with the presumed anovulatory cycle are presented in Table 4Go. Highest PICP and lowest Pyd concentrations occurred during high E2 levels at t4, whereas PICP levels were lowest and Pyd and Dpd were highest on low E2 levels at t5.


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  		Table 4. Concentrations of selected sex hormones, SHBG, and biormarkers of bone turnover in the subject with the presumed unovulatory cycle

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Bone metabolism is a complex interplay of hormonal status and nutrient availability. Several micro- and macronutrients, like dietary Ca (13, 14), Na (15), protein (16), and soybean-phytoestrogens (17), are known to influence bone turnover and bone mineral density in a dose-dependent manner. Reliable studies to evaluate the effect of hormonal factors on bone turnover thus require standardization and monitoring of the diet. As shown in Table 1Go, nutrient intake of our volunteers was unchanged during the study period and well within actual recommendations (18). Any changes in biomarkers of bone turnover (Fig. 2Go) were therefore related to hormonal fluctuations.

In contrast to these results, no hormone-related variations in Dpd and Pyd levels were found in a Danish population previously studied (2). Although Gorai et al. (4) recently reported significant monthly variations in fasting urinary Dpd excretion in Japanese women, they observed low Dpd concentrations during the follicular period and high concentrations during the early luteal period. These data are in contrast to previous data published by the same group (3), as well as in controversy with our findings. Possible explanations for these inconsistent results may include different timing of sample collection (6), different determinations of renal Dpd excretion (measurement of total or only free Dpd), no batched samples, inadequate lag time from the last calcium intake on the previous evening (6), and variations in daily consumption of soybean-phytoestrogens, which may cause changes in bone metabolism (17).

During the entire period of our study, the urinary Pyd-to-Dpd ratio was constant, at a level of approximately 3.5:1 (Fig. 2Go). Such a ratio between Pyd and Dpd concentrations has also been found in bone samples, whereas cartilage collagen has a Pyd-to-Dpd ratio of 10:1 (19). Thus, our data indicate that the surplus of urinary cross-link excretion during the follicular phase, as compared with the midcycle period, exclusively occurred from bone collagen breakdown.

PICP, a marker of bone formation (20, 21), showed an inverse cyclic fluctuation, compared with Dpd and Pyd values (Fig. 2Go). Normally, a temporal and spatial coupling exists between resorption and formation in the skeleton, with resorption preceding formation during the remodeling sequence (22). Thus, the increase in bone formation at t3 (reflected by PICP) may be caused by increased bone resorption (reflected by Dpd) at t1. However, there was a time lag of approximately 2 weeks between t1 and t3. Production of type I collagen extracellular matrix occurs primarily within the first 10 days of the osteoblast development sequence (23). The highest PICP values occurred 4 days after the E2 peak of a normal menstrual cycle (2). Because osteoblasts exhibit estrogen receptors and estrogens are able to enhance collagen synthesis (24, 25), the PICP peak at t3 (Fig. 2Go) may be the result of a direct estrogen effect on bone formation. In addition, an indirect estrogen mechanism could explain the increase in PICP at t3. The enhanced PTH serum level at t3 (Fig. 3Go) may be responsible for an increase in PICP serum levels (26). An in vitro study indicated that estrogen might stimulate PTH secretion (27). Thus, it may be that the slight rise in PICP and the pronounced fall in Dpd values at midcycle represent a true uncoupling of bone resorption and formation processes.

Changes in osteoclastic and osteoblastic activity are the basis for building up, maintaining, or loosing bone matrix. Strategies to optimize premenopausal bone mineralization are regarded as important measures to reduce the risk of osteoporosis in elderly subjects (28). However, data on factors that influence bone turnover and bone mass in the general population of premenopausal women, including those associated with hormonal status, are scanty. As illustrated in Fig. 4Go, the excretion of the bone resorption marker Dpd was inversely associated with serum E2 levels, especially in the lower physiologic range of E2 serum values. Clinical trials have indicated that estrogen status has a profound effect on collagen cross-link excretion. A 62% increase in fasting urinary Pyd and an 82% increase in fasting urinary Dpd excretion has been found in postmenopausal women, compared with age-matched premenopausal women. Pyd and Dpd concentrations return to premenopausal values after 6 months of estrogen-containing hormone replacement therapy (29).

The adverse effects on bone mass of estrogen deficiency associated with amenorrhea is well established, even in young women (30, 31). From results of postmenopausal women, an E2 serum level of 60 pg/mL has been suggested to arrest bone loss (32). In our study in premenopausal women, an E2 serum level of 60 pg/mL, as observed at t5, was associated with a significantly enhanced renal excretion of bone resorption markers (Figs. 1Go and 2Go). Our data indicate that, even in eumenorrhoic subjects, bone turnover is influenced by physiologic changes in serum E2 levels. Sowers et al. (33) could demonstrate that, in healthy premenopausal women with a normal menstrual cycle, subtle interindividual variations in E2 serum levels, measured during the luteal phase, are associated with differences in bone mass at the femoral neck. Individual E2 serum levels can be reduced during different phases of the menstrual cycle by a high intake of dietary fiber or by phytoestrogens containing soy products (34, 35). These alterations may probably raise the risk of low premenopausal bone mass (34). Because asymptomatic ovulatory disturbances associated with decreases in spinal bone density are a frequent finding, even in premenopausal women without amenorrhea (36), future investigations should evaluate the impact of transient low physiologic E2 serum levels in this context. Moreover, hypoestrogenemia and bone mineral loss can occur in premenopausal women who are on GnRH agonist therapy, and in premenopausal breast cancer patients after chemotherapy (37, 38). Carefully designed prospective clinical trials, with well-defined objectives and endpoints, are required to learn about harm of estrogen therapy and about E2 serum threshold values necessary for bone preservation.

Despite the variations in markers of bone turnover and PTH serum levels, we could not find monthly changes in serum Ca and calcitriol levels, 24-h urinary Ca, or fasting urinary Ca excretion (Table 3Go), the latter reflecting endogenous, bone-derived Ca (39). Moreover, the fall in serum 25-OH-D3 levels from the beginning until the end of the study did not follow the cyclic variations of the biomarkers. The decrease in 25-OH-D3 levels is more in agreement with the observation that, at a latitude of 51° N, only little cutaneous vitamin D production occurs from mid-October to mid-April (40). It can not be excluded that, in our study, systemic changes in Ca metabolism were too small to be detected. Serum Ca levels are regulated homeostatically; and, even in postmenopausal women, the increase in serum Ca is small, compared with perimenopausal levels (41). Moreover, renal Ca output is not the only excretion route for Ca. A decreased Ca retention during the follicular period, as presumed by the enhanced resorption and reduced bone formation processes (Fig. 2Go), can occur via an enhanced endogenous fecal Ca loss or by a decreased Ca absorption, variables which were not determined in our study.

In summary, we observed monthly variations in bone resorption and bone formation. Our data suggest that this rhythm is determined, at least in part, by the cyclic fluctuations in serum E2 levels. Further studies should be performed to explore factors influencing estrogen status and bone turnover of eumenorrhoic premenopausal women and to elucidate their effect on bone mineral density.

Received December 3, 1998.

Revised May 26, 1999.

Revised August 23, 1999.

Accepted September 1, 1999.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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