A Vitamin D, Calcium and Leucine-Enriched Whey Protein Nutritional Supplement Improves Measures of Bone Health in Sarcopenic Non-Malnourished Older Adults: The PROVIDE Study

Loss of muscle mass and function (i.e. sarcopenia), and decline in bone mineral density (BMD) and bone strength (i.e. osteopenia and osteoporosis) lead to alterations in musculoskeletal health with advanced age [1]. Both disorders increase the risk of fractures, loss of independence, and mortality [2] but the risk is highest in those with so-called osteosarcopenia where both disorders coincide [3]. A bilateral cross-talk between muscle and bone has been suggested [4], with underlying causal factors that partly overlap.

Like muscle, bone is a dynamic tissue that responds to the external and internal environments to which it is exposed during an individual’s lifetime. While a considerable variations in bone mass is considered genetically determined (up to 70%), lifestyle factors such as nutrition and exercise are well-established modifiable factors of bone mass. Of the modifiable nutritional factors, dietary protein, vitamin D and calcium are well known [5]. In a study of 82 patients (mean age, 80.7 years) with recent osteoporotic hip fracture, protein deficiency was associated with accelerated bone resorption and impaired bone formation [6]. Adequate protein intake increases insulin-like growth factor 1 (IGF-1) and promotes calcium absorption, potentially slowing bone loss [7]. Garnero et al. reported an association between increased fracture risk and low-IGF-1 levels, independent of BMD, in healthy postmenopausal women [8]. A recent meta-analysis in adults (≥ 18 years) showed positive trends of higher protein intake (defined as either > 90 g protein/day, 25% and 30% of total energy from protein/day or 1.4 g protein kg⁻¹/day) on lumber spine BMD [9]. Apart from the absolute protein intake level, there is some evidence that the intake of specific amino acids is associated with higher spine and forearm BMD, with the strongest association observed for leucine [10]. Moreover, dietary essential amino acid supplementation in rats on a low-protein diet increased bone strength through significant (2–4%) increases in lumbar spine and tibia BMD, trabecular architecture, and cortical thickness, likely mediated via an increase of IGF-1 [11]. This is also reflected by the dietary recommendation for prevention of age-related deterioration of musculoskeletal health in postmenopausal women by The European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO). The ESCEO recommends a daily protein intake of 1.0–1.2 g/kg with at least 20 to 25 g of high-quality protein at each main meal, adequate vitamin D intake at 800 IU/day to maintain serum 25(OH)D above 50 nmol/L as well as calcium intake of 1000 mg/day. These recommendations for protein and vitamin D are in line with those from the PRO-TAGE study group [12] and Institute of Medicine [13] for older adults.

In a previous publication [14], we described the intervention effects of a targeted nutritional supplement containing whey protein enriched with leucine, vitamin D, calcium and a full range of micronutrients on macronutrient intake and muscle mass and lower-extremity function in older sarcopenic adults. A nutritional intervention can improve the functional capacity in older adults with an inadequate nutritional status. To establish an (detectable) increase in muscle mass, in addition to improvement of muscle quality, a sufficient 25(OH)D status and protein intake may be required as we have previously shown [15]. Therefore, we hypothesised that providing the same nutritional intervention in sarcopenic older adults would improve markers of musculoskeletal health. Here, we report the efficacy of this supplement for improving serum 25(OH)D and reducing parathyroid hormone (PTH) as well as altering biochemical markers of bone formation (Osteocalcin; OC, Procollagen type 1 amino-terminal propeptide; P1NP), resorption (carboxy-terminal collagen crosslinks; CTX), serum IGF-1 and total body BMD.

Between June 30, 2010, and May 30, 2013, 1240 older adults were screened for participation, 380 of whom were randomized to the active or control groups. After the 13-week intervention, 302 participants completed all three study visits (79% completion rate). The reason for drop-out of 78 study participants are: adverse events (n = 45), a serious adverse event (n = 2; not related to product intake), withdrawal of informed consent (n = 15), lost to follow up (n = 2), protocol deviation (participant taking calcium supplements), another reason (n = 13) [14]. During the RCT there was no significant difference in the number of adverse events between the test group (45 subjects; 25.0%) and the control group (54 subjects; 27.7%) (p = 0.562). The most commonly reported related adverse events were gastro-intestinal (such as abdominal pain and nausea) or metabolic and nutritional disorders (such as hyperglycaemia). No treatment-related serious adverse events occurred during the RCT. The mean age of the population at enrolment was 77.7 years (65% women) and living independently (87%). Baseline characteristics were similar in both groups (Table 1). At baseline, the  % of participants with 25(OH)D concentrations below 50 nmol/L was 51% and 53% in the control and active group, respectively (p > 0.05). All participants had low muscle mass, a mean SPPB score of 7.5, a mean BMI of 26.1 kg/m², and were non-malnourished based on the Mini Nutritional Assessment Short-Form (99.5%). Ten per cent of the participants reported osteoporosis in their medical history, similar in both groups. Intervention compliance was high (median: 93%) from baseline to follow-up, and did not differ between treatment groups. Body weight increased equally and significantly (by ~ 1 kg) over the 13-week intervention in both active and control treatment groups [data not shown] [14].

Total body BMD increased in the active group only, and the improvement was significantly different from control (p = 0.033) (Table 2). Serum 25(OH)D concentrations increased on average by 26 nmol/L (p < 0.001) in the active group and decreased on average by 6 nmol/L in the control group after 13 weeks (Table 2). The prevalence of serum 25(OH)D concentration below 50 nmol/L decreased from 53 to 8% in the active group, whilst the proportion of those in the control group with serum 25(OH)D below 50 nmol/L did not decrease during the study (51% at baseline and 66% at week 13). Serum PTH concentrations showed significant treatment differences (p < 0.001) with decreased concentrations in the active treatment group and increased concentrations in the control group after 13 weeks (Table 2). Changes in serum CTX concentrations were significantly different (p < 0.001) between the two interventions, with lower values in both treatment groups after 13 weeks, but the decline was more marked in the active treatment group. Serum calcium (corrected for albumin) increased slightly after 13 weeks in the active treatment group only (p < 0.001; Table 2). Serum IGF-1 change during intervention was significantly different between active and control groups (p < 0.001; Table 2) with decreased concentrations in the control group and significantly increased concentrations in the active group. There were no statistical significant treatment differences in change from pre- to post-intervention values in serum OC and P1NP. Analyses corrected for age, sex and study site showed very similar and consistent results (Table 2).

The findings of this 13-week nutritional intervention among non-malnourished older sarcopenic adults showed important improvements in vitamin D status (by 55%), decreased PTH (by 21%) and increased IGF-1 (by 11%) concentrations in those participants who consumed the vitamin D (1600 IU/day), calcium (1000 mg/day), and leucine-enriched whey protein (40 g/day whey protein and 6 g/day total leucine) supplement. In addition, concentrations of the bone resorption marker CTX declined in both treatment groups although the decline was more pronounced in the active group. We postulate that the ***reduction in CTX in the control group [and to some degree in the active treatment group] might be explained by an increase in energy intake (and thus energy availability) over the 13 weeks in the participants.

Mean daily energy intake increased by 166 kcal/day and 165 kcal/day during the 13 weeks intervention period in the active and control groups, respectively [14]. A dose–response inverse relationship between energy availability and bone resorption has been demonstrated in young adult women [20] and dietary energy restriction leads to raised bone resorption in post-menopausal women [21]. Validating our assumption is the fact that participants on control and active treatment gained approximately 1 kg during the intervention suggesting a net positive energy balance in the study participants. Circulating concentrations of bone formation markers OC and P1NP did not change in either group during the intervention. Despite the lack of change in bone formation markers with active treatment, the observed large increase in circulating 25(OH)D (> 30 nmol/l) and concomitant reduction in PTH concentration in those participants receiving active treatment may have potential benefits to bone health. Indeed, consumption of the active product showed small but significant improvements in total body BMD after 13 weeks of treatment in comparison to those on the control product. However, whether the observed changes in total body BMD are transient or sustained after supplementation remains unclear. The observed difference in IGF-1 concentrations between the treatment groups may also offer some explanation as to why those receiving active treatment had significantly higher BMD after the intervention. It is known that IGF-1 regulates key signalling pathways in bone including those involved in cell survival, protein synthesis and energy utilization [22], thus potentially mediating the apparent positive effect of protein supplementation on BMD. Although serum calcium adjusted for albumin was lower in the active treatment group at baseline, a small statistically significant increase was observed in those participants on active treatment after 13 weeks. All serum calcium values remained within the normal range on the 13-weeks supplementation. Moreover, no (serious) adverse events point at hypercalcemia were recorded during the study.

Similar nutritional interventions in older adults support combined protein and vitamin D/calcium supplementation in ameliorating bone turnover but in particular bone resorption. In a study of 89 vitamin D deficient institutionalized women ≥ 60 years old living in 10 French nursing homes, Bonjour et al. [23] observed a 20% decrease in PTH and an 8% decrease in CTX after consumption of a vitamin D (400 IU) and calcium-fortified (800 mg) yoghurt twice daily, over 56 days. These effects are similar to the active intervention in our study that resulted in a 21% and 14% decrease of PTH and CTX, respectively. A significant decline in PTH and bone resorption markers was also observed after 84 days of vitamin D (400 IU) and calcium-fortified (800 mg) yoghurt consumption twice daily in a community-dwelling population of 57 women aged > 60 years, (mean age 73 years, BMI 30 kg/m²) living in a sheltered accommodation housing in Hull (England) [24]. Similar effects of calcium and/or vitamin D fortified milk on bone parameters have been observed in Asian populations. A 16-weeks calcium or calcium/vitamin D fortified milk trial in post-menopausal Asian women demonstrated reductions in PTH and bone turnover of about 10% and ≥ 20% respectively [25, 26]. Two longer term intervention studies with a duration between 18 and 30 months using vitamin D fortified dairy with or without calcium have shown beneficial effects on bone turnover and/or total and site-specific BMD in postmenopausal women [26, 27]. In an 18-month placebo-controlled randomized intervention in post-menopausal Chinese women receiving calcium-fortified milk, providing 900 mg calcium and 256 IU vitamin D daily, BMD at the hip was improved by ~ 2% with treatment, but no change was observed at the lumbar spine [26]. Total body BMD was maintained in 50 post-menopausal women over 30 months when provided calcium/vitamin D fortified dairy foods (~ 1200 mg calcium and 300 IU vitamin D) daily, relative to a decline in BMD in the control group (0.3% vs. − 1.8%) [27]. These alterations in BMD are comparable to those observed in our study, i.e. ~ 2% increase in BMD. One possibility which may explain the improvement in BMD observed in the active treatment group after 13 weeks in our study is the concomitant improvement seen in IGF-1 concentrations after protein supplementation. IGF-1 is a growth-promoting protein that plays essential roles in growth and development including influencing skeletal growth in all stages of life. It is well known that the secretion IGF-1 declines during adulthood to older age until only low levels are detected in adults ≥ 60 years however, the biological significance of this decline is unclear. Whilst the precise mechanism by which circulating IGF-1 functions to maintain bone mass is not fully understood, evidence suggests that circulating IGF-1 is strongly and positively associated with BMD at several sites in older women from the Framingham cohort [28]. Moreover, IGF-1 can also be considered as the cross road of the nutritional, inflammatory and hormonal pathways in the frailty process [29] and one of the main actors of the cross-talk between bone and muscle [4].

The improvement in total body BMD in this study compliments the improvements previously observed in muscle mass and lower-extremity function in the same study [14], and adds further support to the hypothesis that osteopenia and sarcopenia are inextricably linked [30]. Moreover, the observations in this study may offer a potential benefit for older people at risk of fragility fractures. The 13-weeks intervention phase in this study, although sufficient to observe changes in bone resorption markers, may not have been long enough to detect corresponding changes in bone formation. In addition, we cannot conclude whether the beneficial intervention effects on bone are due to high protein, high leucine, vitamin D, calcium and/or other micronutrients in the active treatment such as magnesium and phosphorus which have a well-established role in bone metabolism or indeed whether there is a ‘synergistic effect’ due to the combination of bone related nutrients in the active product. Despite the relatively high phosphorus content of the supplement it is our view that given the likelihood of suboptimal dietary intakes of vitamin D and protein in these participants [and the general geriatric population] the positive effects on vitamin D status, PTH, IGF-1 and BMD observed in the active treatment group is most likely due to a combination of vitamin D and protein supplementation. Analysing the nutritional intake of a subset of the PROVIDE study participants [31], it appeared that the dietary intake of phosphorus was sufficient (mean intakes = 1196 mg/day, which are well above current reference intakes) suggesting that additional phosphorus to already replete phosphorus diets may not be a large contributor to the positive effects on bone health in this study.

In conclusion, consuming a vitamin D, calcium and leucine- enriched whey protein supplement for 13 weeks improved vitamin D status, suppressed serum PTH and had small but significant effects on BMD in sarcopenic non-malnourished older adults. This study shows proof-of-principle that a specific nutritional intervention alone might benefit vitamin D status and bone health in older adults.