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American Journal of Clinical Nutrition, Vol. 86, No. 3, 781-789, September 2007
© 2007 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Changes in biochemical indexes of bone metabolism and bone mineral density after a 12-mo dietary intervention program: the Postmenopausal Health Study1,2,3

Yannis Manios, George Moschonis, George Trovas and George P Lyritis

1 From the Department of Nutrition and Dietetics, Harokopio University, Athens, Greece (YM and GM) and the Laboratory for the Research of the Musculoskeletal System, School of Medicine, University of Athens, Athens, Greece (GT and GPL)

2 Supported by a research grant from Friesland Foods Hellas.

3 Reprints not available. Address correspondence to Y Manios, Department of Nutrition and Dietetics, Harokopio University, 70, Eleftheriou Venizelou Avenue, 176 71 Kallithea, Athens, Greece. E-mail: manios{at}hua.gr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: In southern Europe, calcium supplementation alone is a common practice for osteoporosis prevention.

Objective: We examined whether calcium supplementation could be as effective in achieving favorable bone mass changes in postmenopausal women as is a holistic dietary approach including dairy products fortified with calcium and vitamin D3.

Design: A sample of 101 postmenopausal women were randomly assigned to a dairy intervention group (n = 39) who received daily {approx}1200 mg Ca and 7.5 µg vitamin D3 via fortified dairy products and attended biweekly nutrition education sessions; a calcium-supplemented group (n = 26) who received a total of 1200 mg Ca/d; and a control group (n = 36).

Results: The increases observed in serum concentrations of insulin-like growth factor I were greater in the dairy intervention group than in the 2 other groups, especially during the first 5 mo of intervention (P = 0.034). The decreases and increases observed during 5 and 12 mo, respectively, in serum 25-hydroxyvitamin D3 were significant in all groups (P = 0.050). Serum parathyroid hormone increased only in the control group, and serum type 1 collagen cross-linked C-telopeptide decreased only in the dairy intervention group during both 5 and 12 mo of intervention (P = 0.035 and 0.047, respectively). The dairy intervention group had greater improvements in pelvis (P = 0.040), total spine (P = 0.001), and total-body (P = 0.001) bone mineral density than did the other 2 groups.

Conclusion: The application of a holistic intervention approach combining nutrition education and consumption of fortified dairy products for 12 mo can induce more favorable changes in biochemical indexes of bone metabolism and bone mineral density than can calcium supplementation alone.

Key Words: Bone metabolism • calcium and vitamin D • fortified dairy products • health education • postmenopausal women


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Osteoporosis and the subsequent risk of bone fracture occur most commonly among postmenopausal women who are middle-aged and older. These disorders account for a significant burden of morbidity and mortality worldwide and have become a major public health problem (1). Although no exact data on the prevalence of osteoporosis among Greek women exist, epidemiologic data show that the incidence of hip fracture, the most dramatic complication of osteoporosis, in older persons in Greece has increased significantly: from 1977 to 1992, age-adjusted incidence in Greek persons aged >50 y increased by 80.9% (2).

The adequate intake of certain nutrients that are essential for bone metabolism, such as calcium and vitamin D, plays an important role in maintaining bone mass. With increasing age, however, both dietary calcium intake and intestinal calcium absorption decrease (3). Furthermore, in the elderly, serum concentrations of 25-hydroxyvitamin D3 [25(OH)D3] decline, mostly because of decreased sunlight (ultraviolet B irradiation) exposure, which leads to a limited capacity for cutaneous vitamin D synthesis (4). Combined with low dietary intake of vitamin D from staple foods, especially in countries without mandatory fortification policy (5), these factors contribute to lower concentrations of 25(OH)D3 and consequently to accelerated bone loss and greater risk of bone fracture (6, 7). It has been reported that meeting daily dietary requirements of calcium and vitamin D produces a significant reduction in the incidence of bone fracture (8, 9).

Although low bone mineral density (BMD) has been identified as one of the stronger predictors of future bone fracture, the serum concentrations of several biomarkers of bone remodeling have also been proposed as important predictors of BMD loss (10). According to recent evidence, supplementation with calcium or vitamin D or both has been reported to exert a significant favorable effect on BMD and on several bone-remodeling biomarkers (10-12) and has been associated with a lower risk of fracture (10). An abundance of clinical trials have indicated the beneficial effects of supplementation with calcium or vitamin D or both on the prevention of bone loss and on bone remodeling indexes (10, 11, 13-17), but very few studies have examined the effect of increases in the intakes of these micronutrients through fortified foods and especially through dairy products (7, 18-20). Even scarcer are the data coming from dietary intervention trials with fortified foods in Mediterranean populations (21). The common beliefs in Mediterranean populations are that, because of the sunny climate, there is no need for additional vitamin D intake, and calcium supplementation alone may be adequate to prevent bone loss. However, studies conducted in this region have shown a high prevalence of vitamin D deficiency (22-24). The substantial risk of vitamin D deficiency in southern European countries, particularly during the winter months, and the alleviation of concerns about vitamin D toxicity due to supplementation doses exceeding the current upper level (16), emphasize the need for "safe" directions on adequate sun exposure and the use of fortified foods and supplements.

The aim of the current study was to examine whether supplementation with calcium, as generally believed, could be as effective in achieving favorable changes in biochemical indexes of bone metabolism and bone mineral density in apparently healthy postmenopausal women as is a holistic dietary approach, based on the Mediterranean diet profile, using fortified dairy products with calcium and vitamin D3.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sampling
First screening
The study was initiated in July 2004, when volunteers were invited to participate by informational brochures and posters distributed in public buildings and community centers in 3 municipalities of Greater Athens—namely, Nea Smyrni, Kallithea, and Neo Iraklio. Through this initial screening, conducted in the premises of the aforementioned settings and with the cooperation of the local authorities, a sample of 307 Greek postmenopausal women volunteered to participate. The first screening comprised a short questionnaire that primarily focused on gathering information on the women's medical history, demographic status, and dietary, physical activity, and smoking habits. In addition, the bone status of all volunteers was assessed by using calcaneal quantitative ultrasound (QUS) measurement, carried out with the Clinical Bone Sonomater (SAHARA; Hologic Inc, Waltham, MA). Through this initial screening, those women having an estimated BMD T-score lower than –2.5, those who were taking medications (ie, thiazide diuretics or glucocorticoids) or dietary supplements (eg, calcium, magnesium, phosphate, or vitamin D) that affect bone metabolism, those who had any bone-degenerative chronic disease (ie, diabetes, nephrolithiasis, heart disease, cancer, hyperthyroidism or hypothyroidism, hyperparathyroidism, or impaired renal and liver function), and those who smoked smoking or were <1 y past menopause were excluded from the second screening of the study.

Second screening
After the initial screening, 134 women (aged 55–65 y) who met the inclusion criteria were identified and were invited to participate in the second screening (September–October 2004). At the second screening, all volunteers underwent dual-energy X-ray absorptiometry [(DXA) DPX-MD; GE Medical Systems Lunar, Madison, WI] measurement, as well as hematologic and biochemical examinations including a hematologic profile, erythrocyte sedimentation rate, and serum concentrations of calcium, phosphorus, glutamic-oxaloacetic and glutamic-pyruvic transaminases, alkaline phosphatase, and creatinine. Those women found to be osteoporotic, according to the data provided by the DXA examination, or who had abnormal values on the abovementioned blood indexes, were excluded from the study. This second screening yielded 112 eligible women who proceeded in the intervention study. The intervention component of the study was initiated in October 2004.

Study groups and measurements
These 112 eligible women were randomly assigned to 3 groups, a dairy intervention group (DG), a calcium-supplemented group (CaG), and a control group (CG). The DG consisted of 42 women, who were encouraged to consume on a daily basis 3 portions of low-fat dairy products fortified with calcium and vitamin D3 (one portion equals 250 mL milk and 200 g yogurt), which were provided to them. The dairy products, produced by Friesland Foods Hellas (Athens, Greece) were milk and yogurt enriched with calcium (33%), which provided a total of 400 mg Ca/portion. The extra calcium source was concentrated milk protein, a natural source of milk calcium. The protein content of milk was 12.5 g/portion, and that of yogurt was 15 g/portion. Each portion provided 2.5 µg vitamin D3. To ensure compliance with the intervention scheme, health and nutrition education sessions were held biweekly at Harokopio University. The aims of the sessions held for the DG were to increase awareness of the subjects of health issues, primarily those related to osteoporosis, and to motivate them to change certain lifestyle and dietary habits to improve their health status. The first sessions primarily focused on educating the subjects about the pathophysiology of osteoporosis and about the risk factors (health-related behaviors) related to the development of the disease. Gradually, the sessions became more interactive, and emphasis was placed on guiding and assisting the subjects in changing their dietary habits according to the Mediterranean diet profile (25). To avoid excess caloric intake, subjects in the DG were also advised to replace other dairy products in their diet with those provided, so that the new dietary scheme they were advised to follow was low in fat but efficient in providing the recommended daily intake of calcium and vitamin D (ie, {approx}1200 mg Ca/d and 7.5 µg vitamin D/d).

The CaG consisted of 30 women, who received an extra 600 mg supplementary Ca/d (87% calcium lactate gluconate and 13% calcium carbonate). This extra quantity was added to the estimated dietary calcium intake and was intended to provide a total minimum of 1200 mg/d. The CG consisted of 40 women who underwent no intervention as they continued with their usual diet. All 3 study groups were given the results of medical examination and DXA analysis during scheduled meetings at the university immediately after each screening phase. The procedures followed during these meetings—including explanation of the results, simple dietary and lifestyle advice, and discussion sessions—were standard for all groups.

All subject provided written informed consent. The study was approved by the Ethics Committee of Harokopio University.

Assessment of the effectiveness of the intervention
During the intervention period, the subjects from the 3 study groups were invited to go through certain examinations that primarily focused on the assessment of behavioral and clinical indexes. The data obtained from the second screening of the study were used as baseline data. A midterm examination took place in March 2005, after 5 mo of intervention, and the final screening took place in September–October 2005, after 12 mo of intervention. At the baseline, midterm, and final examinations, numerous measurements were obtained.

Anthropometric measures
Anthropometric measurements were made during the initial screening and at the baseline and follow-up examinations. At all of those time points, body weight and standing height were measured by using a digital scale (Seca, Hamburg, Germany) with an accuracy of ±100 g and a commercial stadiometer (Leicester Height Measure; CMS Instruments, Oxford, United Kingdom) to the nearest 0.5 cm, respectively, while the subjects wore light clothing and no shoes. Body mass index (BMI; in kg/m2) was calculated.

Nutritional assessment
At the baseline, midterm, and final examinations, the 24-h recall technique was used to collect information on dietary intake for a total of 3 d—2 weekdays and 1 weekend day, preferably a Sunday. These data were obtained during scheduled interviews with the study participants at the university. All interviewers were rigorously trained to minimize interviewer effects. Respondents were asked to recall the type and amount of any foods and beverages consumed during the previous day (ie, 24 h) in a chronological order—ie, from the time they woke up in the morning to the same time on the day of the examination. To improve the accuracy of food descriptions and portion sizes, standard household measures (eg, cups and tablespoons) and picture food models (Western Dairy Council, Thornton, CO) were used during interviews to define amounts when appropriate. Food intake data were analyzed by using NUTRITIONIST V diet analysis software (version 2.1; First Databank, San Bruno, CA), which was extensively amended to include food composition tables for Greek foods and recipes (26, 27) and chemically analyzed commercial food items widely consumed in Greece.

To remove the effects of day-to-day within-subject variability in dietary intakes and to estimate the distribution of usual intakes, we used the National Research Council (NRC) method (28, 29). The adjusted (usual) intake was calculated with the following equation:

Formula 1(1)

Physical activity assessment
Physical activity was assessed with a 3-d activity interview questionnaire. Respondents reported the time spent in various physical activities during 2 consecutive weekdays and 1 weekend day. The questionnaire classified all work, sport, and leisure activities into 4 categories on the basis of their average intensity relative to the effect on the cardiovascular system (low to high) and by subgrouping activities according to their effect on bone mass (low to high) (30). The aim of the questionnaire was to determine the frequency and duration (ie, h/session) per week of these physical activities. The total amount of time spent per week in activities categorized as having an intensity of >4 metabolic equivalents was defined as time spent in moderate-to-vigorous physical activity (MVPA). Similarly, the time spent per week in activities categorized as having an intensity of <2 metabolic equivalents was defined as time spent in sedentary activities.

Measurement of sunlight exposure by personal ultraviolet dosimetry
Sunlight exposure measurements were carried out by using biological-spore dosimeters (Viospor blue line, type I, 0.2–12 minimal effective dose; Biosense, Bornheim, Germany) with inactivated repair-deficient spores of Bacillus subtilis. Each woman was provided with one dosimeter, which was worn on the right shoulder outside of the clothing. The shoulder was chosen as the placement site because shoulders are among the body sites with the greatest exposure to sunlight (31). The measurement period was 7 consecutive days (Monday–Sunday) at each time point of examination. The dosimeters were kept in their metal casings overnight, applied in the morning, and unpinned at night. Exposure was measured in J/m2 body surface.

Biochemical analyses
Early morning venous blood samples were obtained from each subject for biochemical screening tests after a 12-h overnight fast. Professional staff performed venipuncture to obtain a maximum of 25 mL blood. The blood was collected in evacuated tubes without added anticoagulant and was kept at room temperature for {approx}2 h, after which it was allowed to clot; this blood was designated for serum separation. Serum was separated by centrifugation at 3000 rpm for 15 min at 41 °C (Universal 32R centrifuge; Hettich Laboratories, Tuttlingen, Germany). A part of the collected serum was used for biochemical analyses, and 1.5-mL aliquots of the remaining serum were pipetted into plastic Eppendorf tubes and stored at –80 °C.

Biochemical analyses included a chemiluminescence immunoassay to assess serum concentrations of osteocalcin, 25(OH)D3, insulin-like growth factor I [(IGF-I) Nichols Advantage; Nichols Institute Diagnostics, San Clemente, CA], and intact parathyroid hormone (PTH; Elecsys Roche Diagnostics, Mannheim, Germany). Serum type I collagen cross-linked C-telopeptide (CTx) was measured by using an electrochemiluminescence immunoassay (Elecsys Roche Diagnostics). The interassay CVs were 8.6% for osteocalcin, 4.7% for CTx, 8% for 25(OH)D3, 6.7% for IGF-I, and 5.0% for PTH.

Bone mineral density and total body-composition measurements
Bone mineral density (BMD) of the lumbar spine (L2–L4) and total body composition (lean and fat mass) were measured at baseline and at the final screening by using DXA and DXA analysis software (version 4.6; GE Medical Systems Lunar). A daily quality assurance check was performed by using an aluminum phantom provided by the manufacturer. The scans were performed in the morning by an experienced technician, who was blinded to the therapy.

Statistical analysis
All data are reported as means ± SEs or as the mean (and 95% CI) percentage change from baseline. The Kolmogorov-Smirnov test was used to determine normality of distribution of the examined variables. Differences in baseline characteristics among the 3 groups of women were evaluated by using the one-way analysis of variance (ANOVA). Repeated-measures ANOVA was used to evaluate the significance of the differences among groups at baseline and at 12-mo follow-up (treatment effect), the significance of the changes observed within each group (time effect), and the effect of the treatment x time interaction. The between-group factor was the study groups (ie, DG compared with CG compared with CaG); the within-group factor was the time point of measurement. Analysis of covariance (ANCOVA) was used to compare the 5- and 12-mo values after adjustment for baseline values. In the case of biochemical indexes of bone metabolism and BMD, adjustments were also made for dietary intake indexes. A paired-sample t test was used to compare the mean values at 5 and 12 mo of intervention with the mean values at baseline within each group. To test for the effect of dietary confounders to the biochemical and BMD indxes, standardized beta (ß) coefficients were estimated. All P values reported were 2-tailed. Statistical analysis was conducted with SPSS statistical software (version 13.0; SPSS Institute, Chicago, IL). The level of statistical significance was set at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Eleven of the 112 women initially assigned to participate in the study could not be reexamined at follow-up. This group of 11 subjects comprised 3 subjects from the DG who dropped out for personal reasons, 4 subjects from the CG who could not be located or who were not available to participate at follow-up examinations, and 4 subjects from the CaG who dropped out because of side effects such as bloating, constipation, and intestinal discomfort apparently related to the supplement used. Consequently, the number of subjects with full baseline and follow-up data was 39 in the DG, 36 in the CG, and 26 in the CaG. The sample sizes in the 3 groups were adequate because we achieved statistical power of >90% for standardized differences between groups in the main outcomes of the present study (ie, bone remodeling and BMD indexes) of > 0.9 at a probability of type I error < 0.05. The mean age of these women was 60.5 ± 5.1 y (range: 55–65 y), and the average time elapsed since menopause was 9.5 ± 5.6 y.

The baseline characteristics of the 101 study participants with full data at baseline and at both follow-up examinations are summarized in Table 1Go. No differences were observed between the 3 groups examined, which indicated homogeneity in baseline demographic, anthropometric, and body-composition characteristics.


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  		TABLE 1. Descriptive characteristics of study groups at baseline

 
The values of certain behavioral indexes related to bone metabolism (ie, dietary indexes, physical activity, and sunlight exposure) at baseline and after 5 and 12 mo of intervention are summarized in Tables 2Go and 3Go. Significant (P ≤ 0.001) treatment x time interactions were observed for protein, phosphorus, magnesium, and vitamin D intakes, which showed a significant increase in these variables only in the DG. Similarly, a significant (P < 0.001) increase was found for calcium intake in both the DG and the CaG. With respect to time spent in MVPA and in solar ultraviolet B irradiation exposure, there were no significant differences among groups at any time point of examination.


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  		TABLE 2. Dietary intakes (energy and macronutrients), physical activity, and sunlight exposure in women in the dairy intervention (n = 39), calcium-supplemented (n = 26), and control (n = 36) groups at baseline and after 5 and 12 mo of intervention1

 

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  		TABLE 3. Micronutrient intakes in women in the dairy intervention (n = 39), calcium-supplemented (n = 26), and control (n = 36) groups at baseline and after 5 and 12 mo of intervention1

 
The changes observed in the examined biochemical indexes of bone metabolism are shown in Table 4Go. Significant treatment x time interaction effects were found for serum IGF-I (P = 0.019), 25-hydroxyvitamin D [25(OH)D] (P = 0.050), PTH (P = 0.035), and CTx (P = 0.047), all of which have more favorable changes in the DG than in the other 2 groups. The ANCOVA (adjusted for baseline values and dietary intake indexes) showed that the DG had significantly (P = 0.032) higher IGF-I concentrations and a tendency toward higher 25(OH)D concentrations (P = 0.066) after 5 mo of intervention than did the CG and the CaG. Furthermore, the DG was found to have significantly (P = 0.026) lower serum PTH concentrations at 5 mo of follow-up than did the other 2 groups. Within groups, serum IGF-I concentrations increased significantly only in the DG after 5 mo of intervention, whereas serum PTH increased significantly only in the CG after both 5 and 12 mo of intervention. Furthermore, serum CTx decreased significantly over 5 and 12 mo of intervention only in the DG. The changes in serum 25(OH)D3 concentrations observed during 5 and 12 mo of intervention were significant within all groups ( P < 0.05 for all changes within groups).


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  		TABLE 4. Changes in biochemical indexes of bone metabolism in women in the dairy intervention (n = 39), calcium-supplemented (n = 26), and control (n = 36) groups1

 
The changes observed in BMD at different skeletal sites are shown in Table 5Go. Significant treatment x time interaction effects were observed for pelvis (P = 0.040), total spine (P < 0.001), and total-body (P < 0.001) BMD. Specifically, the ANCOVA (adjusted for baseline values and dietary intake indexes) showed that the DG had significantly higher pelvis (P = 0.029), total spine (P = 0.001), and total-body (P = 0.001) BMD than did the CG and the CaG. Within groups, pelvis, total spine, and total-body BMD increased significantly over the 12-mo intervention only in the DG. In contrast, significant decreases in total spine and total-body BMD were seen in the CG (P < 0.05 for all changes within groups).


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  		TABLE 5. Changes in bone mineral density (BMD) at various skeletal sites in women in the dairy intervention (n = 39), calcium-supplemented (n = 26), and control (n = 36) groups1

 
The ANCOVA showed that, at the 5-mo examination,vitamin D intake was significantly associated with serum IGF-I (ß = 0.34, P = 0.008), 25(OH)D (ß = 0.22, P = 0.032), and PTH(ß = –0.37, P = 0.042), whereas, at the 12-mo examination, it was significantly associated with serum CTx (ß = –0.64, P = 0.050) and pelvis BMD (ß = 0.37, P = 0.042). Furthermore, at the 5-mo examination, serum PTH was significantly related to calcium (ß = –0.45, P = 0.034) and magnesium (ß = –0.26, P = 0.043) intakes. Finally, at the 12-mo examination, magnesium intake was significantly associated with pelvis BMD (ß = 0.38, P < 0.001) and protein intake was significantly associated with serum IGF-I (ß = 0.16, P = 0.034) and CTx (ß = –0.26, P = 0.022) concentrations.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Changes in behavioral indexes related to bone metabolism
The current study showed certain favorable dietary changes in the DG and to a lesser extent in the CaG. More specifically, both the DG and the CaG increased calcium intake to >1000 mg/d, reaching the recommended adequate intake of 1200 mg/d (29). Furthermore, greater intakes of protein, magnesium, phosphorus and vitamin D were observed for the DG than for the other 2 groups. The changes in the abovementioned micronutrients were mainly delivered by the increased consumption of fortified dairy products provided to the DG. These favorable dietary changes are indicative of the effectiveness of the Health and Nutrition Education component of the program in increasing the ability of the population under study to comply with the given dietary instructions. This increased compliance is also reflected in the total energy intake in the DG, which did not differentiate from that of the other 2 groups over the intervention period. As did the present study, other intervention studies conducted with middle-aged women and including nutrition education as an integral part of their intervention scheme, also reported high adherence to the dietary guidelines provided (32), whereas compliance was considerably lower in intervention programs not followed by regular nutritional education sessions (7). No changes in physical activity were observed in the groups, despite the fact that subjects in the DG were encouraged to increase their daily physical activity levels. Similar studies have also confronted difficulties in motivating middle-aged women whose lifestyle is already sedentary to become more active (19).

Changes in biochemical indexes of bone metabolism
The significant effect of the applied dairy intervention on the examined biochemical indexes of bone metabolism should mainly be attributed to the dietary changes observed in the present study, because physical activity levels remained unchanged in all study groups. This effect was also indicated by the significant associations derived from the ANCOVA between biochemical and dietary indexes that changed significantly over the intervention period (ie, vitamin D, calcium, magnesium, and protein). Specifically, our results regarding IGF-I are concordant with those in other reports showing significant positive associations between increases in protein and vitamin D dietary intakes and serum concentrations of IGF-I (33-36). This anabolic hormone-like peptide has been reported by recent studies to stimulate bone formation activity in postmenopausal women, basically via the differentiation of osteoblasts (37).

In the case of serum 25(OH)D3 concentrations, a significant decline was observed during the winter months, and this decline was followed by a significant increase until the end of the intervention in all study groups. These changes, especially the decrease in serum 25 (OH)D3 concentrations during the winter—ie, the months when exposure to sunlight is limited—were similar to those reported for other current population groups (6, 7, 38). However, changes in serum concentrations of 25 (OH)D3 were more favorable in the DG, who had a milder decrease during the winter period and a higher increase during the summer period than did the other 2 groups. Although the major source of 25(OH)D3 endogenous synthesis is ultraviolet B irradiation, through photoconversion of 7-dehydrocholesterol to previtamin D3 in the skin, measurements of sunlight exposure via personal ultraviolet dosimetry in the present study showed no significant differences among groups. These data indicate that seasonal variations in serum 25(OH)D3 concentrations were greater for women in the CG and CaG than for women in the DG, who received supplementation with an additional amount of vitamin D3. The positive effect of dietary vitamin D intake on serum 25(OH)D3 concentrations was also indicated by the significant association between these 2 variables derived from the ANCOVA. Nonetheless, the amount of vitamin D3 supplemented to the DG was obviously not enough to entirely prevent reduction during the winter months. Contrary to the current study, other recent studies of vitamin D supplementation have reported dose-dependent increases in serum 25(OH)D concentrations during the winter months with limited sun exposure ranging from 38.4 to 88 ng/mL after daily oral supplementation of 100 to 250 µg vitamin D3 (16, 39). Furthermore, Heaney et al (39) showed that the calculated oral intake required to sustain the serum 25(OH)D3 concentration present at baseline (ie, in the autumn) was 12.5 µg/d. Therefore, recent proposals to substantially raise the requirement for vitamin D intake in susceptible population groups appear sensible (9, 40).

Seasonal variations were also observed for serum PTH concentrations but only in the CG, who maintained a low dietary calcium intake (ie, < 700 mg/d) throughout the intervention period. In contrast, the changes observed in the other 2 groups did not differ significantly from baseline. This lack of difference should be attributed to the additional calcium supplementation provided to the DG and the CaG that improved calcium status and suppressed PTH secretion. Another regulating mechanism for serum PTH concentrations in the DG probably was the serum concentrations of 25(OH)D3, which were maintained at >25 ng/mL. Evidence has suggested that a serum concentration of 25(OH)D that is adequate to prevent compensatory hypersecretion of PTH ranges from 25 to 40 ng/mL (41). The findings of the current study are in agreement with similar dietary intervention studies providing either calcium supplements (with or without vitamin D) or fortified dairy products to postmenopausal women, which showed that suppression of serum PTH was positively associated with the dose of supplemented calcium or vitamin D (or both) as well as with basal serum 25(OH)D3 concentrations (7, 13, 18, 42).

The more favorable dietary and biochemical changes observed in the DG than in the other 2 groups may have mediated the significant 35% and 23% reductions observed in serum osteocalcin and CTx concentrations, respectively. These changes, which were consistent with those reported by other dietary intervention studies (7, 18, 21, 43), indicate that the consumption of fortified dairy products by apparently healthy postmenopausal women can lead to a reduction in the rate of bone remodeling. Suppression of this process has been associated with prevention of bone loss from several skeletal sites that are susceptible to fracture as well as from the total body (10, 44, 4).

BMD changes
The decreases in the serum concentrations of bone remodeling indexes in the DG coincided with the more favorable BMD changes also observed in that group. According to the data derived from DXA measurements, the current study showed that subjects in the DG had higher increases in total body, total spine, and pelvis BMD than did the other 2 groups. In agreement with these findings, other similar dairy intervention studies conducted with postmenopausal white or Asian women also showed favorable bone mass changes at the total body, lumbar spine, total hip, and femoral neck skeletal sites (7, 18, 46). Ensuring an intake of {approx}1200 mg Ca/d in the CaG did not induce any bone mass changes in all skeletal sites examined that were more favorable than the changes seen in the CG. Similarly, Prince et al (19) did not report any BMD changes after providing 1000 mg Ca/d for 1 y to postmenopausal women. In contrast, data from other intervention studies using calcium supplements as a primary mode of treatment showed that calcium doses >1600 mg/d were more likely to prevent bone loss from the total body, lumbar spine, femoral neck, and greater trochanter (7, 42).

In conclusion, although the current intervention program did not succeed in increasing the levels of physical activity and sunlight exposure of apparently healthy postmenopausal women, it was proven successful in improving their compliance with the dietary recommendations provided. Furthermore, our findings showed that the application of a holistic approach combining nutrition education and consumption of fortified dairy products for a period of 12 mo induced favorable changes in biochemical indexes of bone remodeling, calciotropic hormones, and pelvis, total spine, and total-body BMD. In contrast, no such favorable changes in either biochemical indexes or BMD were obtained in the CaG, the group that was supplemented only with the recommended amount of calcium. The favorable changes observed in the DG may not be attributed solely to the greater intakes of calcium and vitamin D but also to other, less studied ingredients of dairy products. Recent research has highlighted the important roles of magnesium and other micronutrients (47) and of milk protein (48) in bone metabolism. It has been suggested that the effect of dairy products on bone health may be greater than can be accounted for by any single constituent and that milk ingredients as a whole may be more effective than the sum of their individual parts (47).


   ACKNOWLEDGMENTS
 
We thank Eva Grammatikaki, Ioanna Katsaroli, and Paraskevi Kannelou (dietitians) and Sofia Tanagra (technician) for their valuable contributions to data collection and processing.

The authors' responsibilities were as follows—YM, GM, and GPL: designed the study and collected the data; YM, GM, and GT: conducted the literature review and performed the statistical analyses; YM, GM, GT, and GPL: writing and revising the manuscript. YM works as a part-time scientific consultant for Friesland Foods Hellas. None of the other authors had any personal or financial conflict of interest. The study sponsor had no role in the study design; the collection, analysis, or interpretation of the data; the writing of the manuscript; or the revision and submission of the manuscript.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication November 7, 2006. Accepted for publication April 16, 2007.




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