Abstract
Research in the field of nutrition allows considering the establishment of a real prevention of osteoporosis. The value of fruits is discussed. Indeed, red fruits are particularly interesting for their high content in anthocyans, endowed with antioxidant and anti-inflammatory properties.
Fifty-six postmenopausal women (less than 6 years) aged 50–65 years, without HRT, were included in a controlled, randomized, double-blind placebo, prospective study, after a medical examination and a blood test. Throughout the 3-month study period, they kept their eating habits, limiting however consumption of red fruits. They were randomized into two groups of 28 subjects receiving either 0 or 120 mg of anthocyans daily, from blueberry extract. Those polyphenols were provided at the dose of 0 or 60 mg of active molecule in 100 ml of fermented milk (two bottles of 100 ml/day covering 25 and 20 % RDA for calcium and vitamin D, respectively).
Consumption of the milk enriched in polyphenols significantly improved serum bALP activity (an osteoblastic marker), without significant modification of CTX, a marker for bone resorption. This favorable orientation of bone metabolism could be explained by the contribution of anthocyans, the only noticeable difference between the two test foods. This finding is independent of the initial calcium and vitamin D consumption.
In conclusion, consumption of fermented milk enriched with calcium and vitamin D, containing blueberry, for 3 months, has corrected the insufficiency of vitamin D of postmenopausal women and resulted in improved bone formation, as indicated by the rise of a biomarker of osteoblastic activity. This benefit is probably related to the presence of blueberries (rich in polyphenols and phenolic acids).
Introduction
Among the manifestations of senescence, impairment of the musculoskeletal system is particularly debilitating and greatly speeds up entry into the dependence. Thus, given the aging of populations globally and in the industrialized countries specifically, osteoporosis, the primary skeletal disorder, is considered to be a major public health and socioeconomic problem worldwide [1, 2].
So far, hormone replacement therapy was currently used as prophylaxis. Nevertheless, because of side effects, health professionals strongly advocate new strategies of proven clinical value for prevention, to provide a wide array of treatments. For that purpose, research in nutrition over the past 30 years has led to an exciting progress supporting the hypothesis that, by modulating specific target functions in the body, diet can help to achieve optimal health by reducing the risk of disease. In this context, although calcium and vitamin D are required nutrients for bone development [3], it has been recognized that the human diet contains a complex array of naturally occurring bioactive molecules, the phytochemicals endowed with antioxidant and anti-inflammatory properties. Actually, antioxidant nutrients may enhance bone formation and reduce the production of free radicals which contribute to bone resorption, leading to a bone-sparing effect. This is why they could play a strategic role in the prevention of osteoporosis as well [4–6]. Indeed, recent observations suggest that consumption of dried plum, rich in phenolic compounds, is associated with increased markers for bone formation in postmenopausal women [7]. Besides, a prospective study has shown a positive correlation between tea consumption and bone mineral density in elderly [8].
In this regard, red fruits (red grapes, blueberry, cassis, cherry, cranberry, elderberry) are particularly interesting for their high anthocyanin content (from a few 100 mg to several g/kg of fresh weight; blueberry 25–495 mg/kg) and their widespread consumption (leading to a mean daily of intake of such compounds from 25 to 215 mg) [9]. Moreover, recent studies have demonstrated the benefit of blueberries to prevent various age-related chronic diseases such as cancer, diabetes, hyperlipidemia, hypertension, neurodegeneration, obesity through their apoptosis, antioxidant, anti-inflammation, and antiangiogenesis effects [10].
The objective of this study was thus to evaluate the effect of consumption of a fermented milk (providing 25 and 20 % RDA of calcium and vitamin D) enriched with a blueberry extract rich in anthocyanins, on bone biomarkers in postmenopausal women.
Subjects and Methods
Subjects
For this prospective randomized, double-blind, placebo-controlled trial, 56 postmenopausal women were recruited. They were 50–65 years old (postmenopausal for at least 1–5 years), without hormone replacement therapy. The volunteers were informed of the study procedures and gave informed written consent before enrollment in the clinical trial. Throughout the study period, they kept their eating habits, limiting however red fruits. They were randomized into two groups (a placebo group vs. an intervention group) of 28 subjects each and received either 0 or 120 mg of anthocyanins/day.
Study Design
The duration of the study was 3 months and was conducted at the Clinical Investigation Center (CIC INSERM Clermont-Ferrand France) which has the ministerial approval for biomedical research without direct individual benefit (N° 03046MHC; 2007-A00940-53).
The anthocyanins (amount equivalent to 22.5 g of blueberries) were provided at the dose of 0 or 60 mg of active drug/100 ml of calcium- and vitamin D-enriched fermented milk (allowing covering 25 and 20 % of the RDA for calcium and vitamin D, respectively). The volunteers were asked to consume two bottles per day.
A food record and a questionnaire on lifestyle, to assess tobacco and alcohol consumption and physical activity level as well, were established at baseline. A full clinical examination was also performed at the screening visit.
Throughout the study, biomarkers to evaluate bone metabolism and inflammatory status were assessed. Blood was collected at 0, 45, and 90 days, after an overnight fasting. The record of intercurrent events was carried out at the same time (45 and 90 days), to ensure good tolerance of the food test.
Biochemical Analysis
Blood samples were collected at baseline, 45 and 90 days. The samples were stored at −80 °C and analyzed. Biochemical tests were performed by using electrochemiluminescence-based immunoanalysis (Roche Diagnostics GmbH, Germany), according to standard methods. Concentrations of bone-specific alkaline phosphatase (a marker of bone formation) were measured by enzyme immunoassay on paramagnetic particle DXI; serum concentrations of C-telopeptide cross-links (a marker of bone resorption), serum CRP (a marker of inflammation), and serum 25-hydroxyvitamin D (25(OH)D) with the Roche Modular Analytics E immunoassay system.
Statistical Analysis
The significance of the treatment effects on the main criteria between the supplemented and control group was performed by Student’s t-test (after log transformation of data, if necessary). P values <0.05 were considered significant.
Results are expressed as mean ± SEM. Analyses of repeated measurements for bone markers were performed using SAS version (SAS Institute Inc.). The percent changes in serum bALP and in serum CTX were calculated [(post intervention value − baseline value)/baseline value × 100 %] for each group.
Results
Of the 56 subjects included in the study, 1 dropped out for personal reasons.
Baseline characteristics of the subjects who completed the study are presented for each group in Table 37.1. No difference was observed between the two groups at baseline, except for alcohol consumption which is slightly higher in the supplemented group (p < 0.026) and which could explain the higher plasma levels of total alkaline phosphatase (71.50 ± 3.15 vs. 71.04 ± 2.90 IU/l) in this group. Indeed, anthropometric data, plasma biomarker for bone metabolism, and vitamin D status were similar.
The analysis of food records completed over 5 days and collected before the start of the study showed that the two groups were similar in their protein, carbohydrates, fats, vitamins, and trace element consumption. With regard to calcium intake, the average consumption (mg/day) was 827 ± 228 for women in the treated group and 854 ± 297 mg for the placebo group, which is far below the RDA in postmenopausal women (1,200 mg/day). Nevertheless, whatever the experimental group, a significant dispersion was observed (from 338 to 1,423 mg/ day).
At the end (after 3 months of test foods consumption), vitamin D status was significantly improved. Indeed, serum 25(OH)D levels were increased by 72 % in the placebo group and 101 % in the intervention group. Regarding the primary outcome, i.e., bone remodeling, consumption of the milk enriched in polyphenols significantly increased osteoblast activity (as shown by higher serum bALP concentrations), without significant modification in CTX values, a marker for bone resorption (Fig. 37.1). CRP did not significantly change throughout the study, whatever the test food was.
Discussion
Broad-based preventive strategies designed to lower the risks of osteoporosis need to be established and implemented. This is why the concept of a healthy diet providing adequate amounts of various potent micronutrients deserves mention [11]. We targeted anthocyanins from blueberry as a possible way to achieve this goal. Indeed, a recent interest in food phenolics has increased greatly owing to their anti-inflammatory and free radical scavenging abilities. We thus assessed the effect of consumption of a fermented milk (providing 25 and 20 % RDA of calcium and vitamin D) enriched with a blueberry extract rich in anthocyanins, on bone biomarkers in postmenopausal women.
The volunteers enrolled in the present study are representative of the general population of this age. Indeed, their vitamin D status was relatively low, since 50 % of them had a level <40 nmol/l, which is consistent with the results found in other studies. Actually, in France, prevalence of vitamin D insufficiency (defined as 25(OH)D concentrations below 30 nmol/l) reached 15–30 % in the SUVIMAX study [12] (general population) and more than 59 % in the DHE age study [13] (60–79 years healthy subjects), with the lowest values being seen in winter. As a matter of fact, it is considered that serum level from 100 to 150 nmol/l would be necessary to maintain the integrity of bone in osteoporosis prevention strategies [12] and at least 50 nmol/l to optimize intestinal calcium absorption and to avoid secondary hyperparathyroidism (clinical chemistry laboratories now defining vitamin D insufficiency as a serum 25(OH)D level less than 75 nmol/l) [14–16]. In this study, conducted over 3 months, consumption of fermented milk fortified with calcium and vitamin D (20 % of the RDA) resulted in an improvement of vitamin D status, whatever the group, plasma 25 (OH)D level reaching levels above 75 nmol/l. These results agree with those published by Golombick and Diamond [17] who obtained a normalization of serum 25(OH)D (70 nmol/l) after a supplementation providing both calcium (500 mg/day) and vitamin D (500 UI/day) for 3 months in postmenopausal women. This is also consistent with the study conducted in the elderly by Romagnoli et al. [18]. In the same way, 25(OH)D has been shown to play a role in determining bone density, not just in elderly people, but in peri- and postmenopausal women as well [19]. Indeed, healthy postmenopausal women with vitamin D intakes of 100 IU daily can significantly reduce late wintertime bone loss and improve net bone density of the spine over 1 year by increasing their intake of vitamin D [20]. Again, in women in late menopause [21] or in pre- and postmenopausal women (<5 years), Di Daniele et al. [22] confirmed the preventive role of calcium and vitamin D supplementation on bone loss, especially when baseline level is low. On another hand, these changes in vitamin D status were not associated with any variation of plasma CRP concentrations in both groups. In fact, in the NHANES study, vitamin D supplementation elicited reduced CRP levels only among adults with low serum 25(OH)D (<21 ng/ml) [23].
With regard to the primary outcome, bone remodeling, dynamic changes in bone turnover, estimated by measurement of bone biochemical markers, can account for a major portion of anti-fracture efficacy of antiresorptive agents [24]. These parameters are known to be early and predictive of fracture risk, from 3 to 6 months of treatment [25]. In the present work, compared to what is observed in the placebo group, consumption of the fermented milk enriched with calcium, vitamin D, and a blueberry extract significantly improved serum bALP (a marker for osteoblast activity), without any significant change in CTX levels (a marker of bone resorption), only a trend toward a decrease being observed for this parameter. This positive effect on bone metabolism could be explained by the contribution of anthocyanins in the blueberry extract, i.e., the only difference between the two test foods. Effectively, in a population from the Aberdeen Prospective Osteoporosis Screening Study (APOSS) of Scottish women 54.8 years old, between 1997 and 1999, high dietary anthocyanin intakes (22 μg/day) were associated with increased BMD and decreased markers of bone resorption (PYD and DPD) [26]. Actually, such a bone-sparing activity of blueberry has recently been demonstrated in an ovariectomized rat model for menopausal osteoporosis as well [27]. According to this study, this effect could be due to suppression of the ovariectomy-induced increase in bone turnover, as evidenced by lowered femoral mRNA levels of alkaline phosphatase and type 1 collagen, and increased bone mineral density (compared to what is measured in Sham animals). More precisely, such a protection could result from the phenolic compounds of blueberry such as flavonoids (anthocyanins, catechin, quercetin, epicatechin) or phenolic acids (gallic and caffeic acids) [28, 29]. As a matter of fact, the effect of grape seed proanthocyanidin extract on bone formation has already been documented using mandibular condyles of rats [30–32]. Indeed, a mixture of phenolic acids found in the serum of young rats fed with blueberries was able to significantly stimulate osteoblast differentiation through Wnt signaling pathway, and the p38 MAPK/b-catenin signaling cascade appeared to be a critical molecular determinant of the positive skeletal effects of blueberry [33]. Furthermore, blueberry polyphenol-derived phenolic acids may also contribute to the prevention of osteoblast senescence. Indeed, short-term feeding of a diet containing blueberry, just prior to puberty, can prevent OVX-induced bone loss later on, in adult rats, and early exposure of osteoblastic cells or mesenchymal stromal cells to dietary blueberry can maintain long-term cytoskeletal stability by regulation of myosin and Runx2 genes [34]. The molecular targets (i.e., Sirt 1) have been identified in the senescence pathway whereby blueberry prevents senescence and differentiation to adipocytes [35]. Finally, it has been shown, in healthy adults, that anthocyanin supplementation isolated from bilberries (300 mg/day for 3 weeks) significantly reduced plasma concentration of NF-kB-related proinflammatory cytokines and chemokines, suggesting that anthocyanins possess anti-inflammatory effects as well [36].
Conclusion
In conclusion, this is the first clinical trial demonstrating the bone-sparing potential of blueberry in postmenopausal women. More precisely, consumption of fermented milk enriched with calcium and vitamin D, and such red fruits, for 3 months:
-
Has corrected vitamin D status (initially low)
-
Has resulted in a favorable orientation of bone metabolism, as indicated by the rise of a biomarker of osteoblast activity, whereas CTX tended to decrease
This benefit is most likely related to the presence of blueberries (rich in polyphenols); the only difference with the placebo group in which bALP was lower. Consequently, these original results demonstrate that, in addition to calcium and vitamin D, other nutrients must be considered in the nutritional prevention strategies of osteoporosis. The nutritional prevention of osteoporosis must thus evolve to new concepts which include the potential exerted by certain micronutrients, even though these promising data need to be implemented by longer term trials allowing bone mineral density assessment.
Abbreviations
- bALP:
-
Bone alkaline phosphatase
- CRP:
-
C-reactive protein
- CTX:
-
C-telopeptide cross-links
- DPD:
-
Deoxypyridinoline
- IU:
-
International unit
- 25(OH)D:
-
25-hydroxyvitamin D
- PYD:
-
Pyridinoline
- RDA:
-
Recommended dietary allowance
- SEM:
-
Standard error to the mean
References
Seeman E. Pathogenesis of bone fragility in women and men. Lancet. 2002;359(9320):1841–50.
Maravic M, Le Bihan C, Landais P, Fardellone P. Incidence and cost of osteoporotic fractures in France during 2001. A methodological approach by the national hospital database. Osteoporos Int. 2005;16(12):1475–80.
Rizzoli R, Boonen S, Brandi ML, Burlet N, Delmas P, Reginster JY. The role of calcium and vitamin D in the management of osteoporosis. Bone. 2008;42(2):246–9.
Dew TP, Day AJ, Morgan MR. Bone mineral density, polyphenols and caffeine: a ressaissement. Nutr Res Rev. 2007;20:89–105.
Das AS, Mukherjee M, Mitra C. Evidence for a prospective anti-osteoporosis effect of black tea (Camellia sinensis) extract in a bilaterally ovariectomized rat model. Asia Pac J Clin Nutr. 2004;13(2):210–6.
Horcajada MN, Coxam V. Flavonols and isoflavones prevent bone loss in the ovariectomized rat a model for postmenopausal osteoporosis. In: Burckard F, editor. Nutritional aspects of osteoporosis. San Diego: Academic; 2001. p. 325–40.
Arjmandi BH, Khalil DA, Lucas EA, et al. Dried plums improve indices of bone formation in postmenopausal women. J Womens Health Gend Based Med. 2002;11(1):61–8.
Hegarty VM, May HM, Khaw KT. Tea drinking and bone mineral density in older women. Am J Clin Nutr. 2000;71(4):1003–7.
Wu X, Beecher GR, Holden JM, Haytowitz DB, Gebhardt SE, Prior RL. Concentrations of anthocyanins in common foods in the United States and estimation of normal consumption. J Agric Food Chem. 2006;54(11):4069–75.
Chen CF, Li YD, Xu Z. Chemical principles and bioactivities of blueberry. Yao Xue Xue Bao. 2010;45(4):422–9.
Wauquier F, Leotoing L, Coxam V, Guicheux J, Wittrant Y. Oxidative stress in bone remodelling and disease. Trends Mol Med. 2009;15(10):468–77.
Chapuy MC, Preziosi P, Maamer M, et al. Prevalence of vitamin D insufficiency in an adult normal population. Osteoporos Int. 1997;7(5):439–43.
Souberbielle JC, Cormier C, Kindermans C, et al. Vitamin D status and redefining serum parathyroid hormone reference range in the elderly. J Clin Endocrinol Metab. 2001;86(7):3086–90.
Heaney RP. Vitamin D, and calcium interactions: functional outcomes. Am J Clin Nutr. 2008;88(2):541S–4.
Holick MF. Vitamin D, deficiency. N Engl J Med. 2007;357(3):266–81.
Bischoff-Ferrari HA, Willett WC, Orav EJ, Lips P, Meunier PJ, Lyons RA, Flicker L, Wark J, Jackson RD, Cauley JA, Meyer HE, Pfeifer M, Sanders KM, Stähelin HB, Theiler R, Dawson-Hughes B. A pooled analysis of vitamin D dose requirements for fracture prevention. N Engl J Med. 2012;367(1):40–9.
Golombick T, Diamond T. The effect of a combined oral calcium and vitamin D supplement for treating mild to moderate vitamin D deficiency in postmenopausal women. Clin Interv Aging. 2008;3(1):183–6.
Romagnoli E, Mascia ML, Cipriani C, et al. Short and long-term variations in serum calciotropic hormones after a single very large dose of ergocalciferol (vitamin D2) or cholecalciferol (vitamin D3) in the elderly. J Clin Endocrinol Metab. 2008;93(8):3015–20.
Khaw KT, Sneyd MJ, Compston J. Bone density parathyroid hormone and 25-hydroxyvitamin D concentrations in middle aged women. BMJ. 1992;305(6848):273–7.
Dawson-Hughes B, Dallal GE, Krall EA, Harris S, Sokoll LJ, Falconer G. Effect of vitamin D supplementation on wintertime and overall bone loss in healthy postmenopausal women. Ann Intern Med. 1991;115(7):505–12.
Dawson-Hughes B, Harris SS, Dallal GE, Lancaster DR, Zhou Q. Calcium supplement and bone medication use in a US Medicare health maintenance organization. Osteoporos Int. 2002;13(8):657–62.
Di Daniele N, Carbonelli MG, Candeloro N, Iacopino L, De Lorenzo A, Andreoli A. Effect of supplementation of calcium and vitamin D on bone mineral density and bone mineral content in peri- and post-menopause women; a double-blind, randomized, controlled trial. Pharmacol Res. 2004;50(6):637–41.
Amer M, Qayyum R. Relation between serum 25-hydroxyvitamin D and C-reactive protein in asymptomatic adults (from the continuous National Health and Nutrition Examination Survey 2001 to 2006). Am J Cardiol. 2012;109(2):226–30.
Srivastava AK, Vliet EL, Lewiecki EM, et al. Clinical use of serum and urine bone markers in the management of osteoporosis. Curr Med Res Opin. 2005;21(7):1015–26.
Garnero P. Biomarkers for osteoporosis management: utility in diagnosis, fracture risk prediction and therapy monitoring. Mol Diagn Ther. 2008;12(3):157–70.
MC Donald CJ, Fraser WD, Reid DM, MC Donald HM. Dietary anthocyanidin intakes are associated with increased bone mineral density and decreased markers of bone resorption in a population of Scottish women. In: 8th international symposium on nutritional aspects of osteoporosis (Isnao) proceedings, Lausanne; 2012, p. 35.
Devareddy L, Hooshmand S, Collins JK, Lucas EA, Chai SC, Arjmandi BH. Blueberry prevents bone loss in ovariectomized rat model of postmenopausal osteoporosis. J Nutr Biochem. 2008;19(10):694–9.
Sellappan S, Akoh CC, Krewer G. Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries. J Agric Food Chem. 2002;50(8):2432–8.
Taruscio TG, Barney DL, Exon J. Content and profile of flavanoid and phenolic acid compounds in conjunction with the antioxidant capacity for a variety of northwest Vaccinium berries. J Agric Food Chem. 2004;52(10):3169–76.
Kamitani Y, Maki K, Tofani I, Nishikawa Y, Tsukamoto K, Kimura M. Effects of grape seed proanthocyanidins extract on mandibles in developing rats. Oral Dis. 2004;10(1):27–31.
Kojima K, Maki K, Tofani I, Kamitani Y, Kimura M. Effects of grape seed proanthocyanidins extract on rat mandibular condyle. J Musculoskelet Neuronal Interact. 2004;4(3):301–7.
Ishikawa M, Maki K, Tofani I, Kimura K, Kimura M. Grape seed proanthocyanidins extract promotes bone formation in rat’s mandibular condyle. Eur J Oral Sci. 2005;113(1):47–52.
Chen JR, Lazarenko OP, Wu X, et al. Dietary-induced serum phenolic acids promote bone growth via p38 MAPK/beta-catenin canonical Wnt signaling. J Bone Miner Res. 2010;25(11):2399–411.
Zhang J, Lazarenko OP, Blackburn ML, et al. Feeding blueberry diets in early life prevent senescence of osteoblasts and bone loss in ovariectomized adult female rats. PLoS One. 2011;6(9):e24486.
Zhang J, Lazarenko OP, Blackburn ML, Badger TM, Ronis MJ, Chen JR. Blueberry consumption prevents loss of collagen in bone matrix and inhibits senescence pathways in osteoblastic cells. Age (Dordr). 2012; [Epub ahead of print].
Karlsen A, Retterstol L, Laake P, et al. Anthocyanins inhibit nuclear factor-kappaB activation in monocytes and reduce plasma concentrations of pro-inflammatory mediators in healthy adults. J Nutr. 2007;137(8):1951–4.
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Davicco, MJ., Puel, C., Lebecque, P., Coxam, V. (2013). Blueberry in Calcium- and Vitamin D-Enriched Fermented Milk Is Able to Modulate Bone Metabolism in Postmenopausal Women. In: Burckhardt, P., Dawson-Hughes, B., Weaver, C. (eds) Nutritional Influences on Bone Health. Springer, London. https://doi.org/10.1007/978-1-4471-2769-7_37
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