The additional intake of 1 g calcium (via CaP) per day was used in other studies due to beneficial effects on intestinal metabolism [10, 13]. The additional daily intake of 10 μg vitamin D3 was chosen, because 5 μg/d was the previously recommended vitamin D intake in Germany . In 2012, the German Nutrition Society set up this value to 20 μg/d for adolescents and adults under 65 years , because the UV index in Germany is lower than 3 for about six months of the year . A rule of thumb says, that indexes lower than 3 are insufficient to produce vitamin D in the skin . In the present study, the total intake of vitamin D (supplement, defined diet and bread) was approximately 15 μg/d in the vitamin D3-supplemented groups, and therefore below the actual recommendation of the German Nutrition Society .
However, after vitamin D3 supplementation, the 25-(OH)D concentration significantly increased after eight weeks (Figure 2). The mean increases in the vitamin D3 and in the CaP + vitamin D3 groups were 22.5 nmol/l and 19.8 nmol/l, respectively (Figure 2). The present results indicate, that the supplementation with 10 μg/d was enough to increase 25-(OH)D significantly. Only 5% of participants treated with vitamin D3 had 25-(OH)D concentrations below 50 nmol/l after eight weeks of intervention. Furthermore, the mean increase is in accordance with other vitamin D3 supplementation studies [17, 18].
Interestingly, in the CaP + vitamin D3 group the increase in 25-(OH)D was significant after four and eight weeks, but in the vitamin D3 group only after eight weeks (Figure 2). It seems that the combination with CaP leads to a faster increase in 25-(OH)D concentration. A low calcium intake could lead to a higher turnover of vitamin D metabolites (higher production of 1,25-(OH)2D, due to PTH increase), while a high calcium intake could be 25-(OH)D sparing . These findings were based on rat experiments, in which the half-life of 25-(OH)D was longer when calcium intake was high. Furthermore, in a rat experiment conducted by Anderson et al., animals fed high calcium combined with vitamin D3 had significantly higher serum 25-(OH)D concentrations compared to animals fed low calcium combined with vitamin D3. In the present study, plasma calcium and PTH were not significantly affected by the interventions. Only the 1,25-(OH)2D concentration decreased compared to placebo after eight weeks of CaP (CaP alone and CaP + vitamin D3) supplementations. From the increase of 25-(OH)D concentration after four weeks of CaP + vitamin D3 supplementation it can be concluded, that the combination of vitamin D3 and calcium is more effective than vitamin D3 alone.
In the present study, we determined different biomarkers of bone metabolism, like urinary CTX, serum CTX and urinary DPD for bone resorption and plasma alkaline phosphatase, serum P1NP and plasma osteocalcin for bone formation. None of the bone metabolism markers changed related to the CaP, vitamin D3 or CaP + vitamin D3 supplementations. Literature indicates that a modulation of bone metabolism through calcium and/or vitamin D supplementations occurred especially in older subjects with a vitamin D deficiency [12, 21]. In studies involving subjects with no or only marginal vitamin D deficiency, no beneficial effects on bone metabolism were observed [22, 23].
Due to CaP supplementation, the faecal excretion of calcium and phosphorus significantly increased. This is based on the formation of amorphous calcium phosphate, a well-known process in the intestine [8, 24]. Thus, only a minor part of the calcium can be absorbed . The unchanged excretion of calcium and phosphorus in urine and the comparable plasma concentration confirm this suggestion. Vitamin D is known to increase the absorption of calcium and phosphate in the human gut, in order to maintain the calcium homeostasis. After supplementation of vitamin D3 alone, only the renal excretion of phosphorus significantly increased after eight weeks compared to placebo. In phosphorus equilibrium, the amounts of intestinally absorbed phosphorus is equal to the excreted phosphorus by the kidney . Thus, the increased excretion indicates higher phosphorus absorption due to vitamin D3 supplementation.
In a rat experiment, it has been shown that magnesium is also able to form an insoluble complex with calcium and phosphate . When this formation occurs in the human gut, the absorption of magnesium would be inhibited. After supplementation with high amounts of phosphorus, Greger et al. showed a significant increase in faecal excretion of magnesium, but simultaneously a decrease of magnesium in urinary excretion . The authors concluded that magnesium metabolism was not negatively influenced by high phosphorus doses . In the present study, CaP supplementation had no harmful effects on magnesium metabolism. After eight weeks of vitamin D3 supplementation, the renal excretion of magnesium increased compared to placebo. Hardwick et al. reviewed the literature concerning magnesium absorption and suggested that a significant amount of magnesium absorption is vitamin D-dependent .
Literature indicates, that calcium supplementation can decrease iron absorption, both from haem and non-haem iron . This decrease in iron absorption has been shown mostly in short-term studies [30–32]. In contrast, Minihane and Fairweather-Tait supplemented calcium carbonate in a short-term (1200 mg calcium carbonate for one day) and in a long-term study (1200 mg calcium carbonate over six months) and showed that the non-haem iron absorption decreased only in the short-term supplementation . By supplementing 1200 mg calcium as calcium carbonate over six months, they found no changes in any of the haematologic indexes (i.e. haemoglobin, plasma ferritin). The mechanism proposed by the authors is an adaptive response in the intestinal mucosa cell: the lower supply of iron to plasma after calcium intake may modify the developing enterocytes in order to stimulate specific proteins . Thus, the dietary iron is used more efficiently, when developing cells are mature . The unchanged results of faecal iron and of iron status on the present study indexes (transferrin, transferrin saturation, ferritin) confirm the results of Minihane and Fairweather-Tait, that long-term CaP supplementation does not negatively influence the parameters of iron metabolism. The significantly higher ferritin concentration in the CaP + vitamin D3 is due to two participants with concentrations in the upper limits of normal. In all, the presented results underline that supplementation with CaP and/or vitamin D3 did not negatively influence the metabolism of magnesium and iron.