Editorial: the role of vitamin D metabolites in the evaluation of bone health: are they physiologically relevant?

There is significant interest in determining whether vitamin D supplementation increases bone density. In the Calgary Vitamin D study, participants with sufficient vitamin D levels were randomized to 400 IU, 4000 IU, or 10 000 IU vitamin D3 daily for 36 months. HR-pQCT analyses demonstrated that supplementation with 4000 IU or 100 000 IU leads to significant decreases in radial total (Tt) BMD, while the 10 000 IU dose resulted in a decline in tibial TtBMD, showing that higher vitamin D doses do not improve bone health relative to the lower vitamin D dose.1 Vitamin D is generated in the skin and circulates to the liver, where it is hydroxylated to form 25-hydroxyvitamin D3 [25 (OH)D3], the predominant circulating form of vitamin D. 25 (OH)D3 is transported to the kidney, where it is hydroxylated by CYP27B1 (1-alpha-hydroxylase) to generate the active form of vitamin D, or 1,25 dihydroxyvitamin D3 [1,25 (OH)2D3]. In the kidney, the CYP24A1 (24-hydroxylase) enzyme hydroxylates 25(OH)D3 or 1,25(OH)2D3 to generate 24,25 dihydroxyvitamin D3 [24,25(OH)2D3] and 1,24,25 trihydroxyvitamin D3 [1,24,25(OH)3D3] respectively. Traditionally, 1,25(OH)2D3 is regarded as the key hormonally active form of vitamin D. Therefore, it is hypothesized that the decrease in bone density observed in the higher vitamin D supplementation groups in the Calgary Vitamin D study could be due to increases in serum 1,25(OH)2D3 levels, which increases osteoclastogenesis. Secondary analyses were performed on plasma from the Calgary Vitamin D Study to quantitate 25(OH)D3, 1,25(OH)2D3, 24,25(OH)2D3, and 1,24,25(OH)3D3 by liquid chromatography-tandem mass spectroscopy (LC–MS/MS), over 36 months. Plasma levels of all vitamin D metabolites except 1,25(OH)2D3 increased in a dose dependent manner; 1,25(OH)2D3 levels remained unchanged regardless of supplement dose. Corresponding with this, linear regression analyses demonstrated a significant negative slope between 1,24,25(OH)3D3 and radial TtBMD. In controlling for sex, all metabolites except for 1,25(OH)2D3 had significant negative slopes with tibial TtBMD. Limitations of their analyses are that this is not a placebo-controlled study and the analyses are not controlled for other factors that may impact bone density, like age, BMI, race, and ethnicity. Linear regression analyses demonstrated that there was a significant effect of sex on radial and tibial TtBMD for all metabolites at 12 months. By 36 months, sex continued to have a significant effect on radial TtBMD for 25(OH)D3 and 24,25(OH)2D3. For both time points, females had a steeper decline in TtBMD with increasing metabolite levels compared with males. In contrast to these results, treatment of men and women with calcitriol to prevent transplant related bone loss and to treat osteopenia/osteoporosis2 did not result in differing effects on bone density. Although the current manuscript shows that high levels of vitamin D metabolites may have sex-dependent effect on bone mass, it is unclear what the effect of sex is on vitamin D metabolite action. In 1973, Holick et al. purified 1,24,25(OH)3D3 and demonstrated that it increases intestinal calcium reabsorption and bone calcium mobilization.3 It is estimated that 1,25(OH)2D3 is 10-fold more potent than 1,24,25(OH)3D3.4 Despite this, 1,24,25(OH)3D3 improved rickets in rats and increased cancellous bone mass in ovariectomized (OVX) rats. Treatment of OVX rats with higher doses of 1,24,25(OH)3D3 did not block skeletal mineralization.5 Since 24,25(OH)2D3 is generated from the catabolism of 1,25(OH)2D3, it is often described as the “inactive” form of vitamin D. Despite this, 24,25(OH)2D3 is essential for regulating multiple biological and skeletal processes independent of 1,25(OH)2D3. 24,25(OH)2D3 increases serum calcium and phosphate levels,3 bone mass, and skeletal mineralization.6 Moreover, 24,25(OH)2D3 plays an essential role in regulating the hatching of chicken eggs7 and is an important modulator of fracture healing.8 Although these animal studies demonstrate the biological activity for vitamin D metabolites, there are scarce data showing the physiologic relevance for these molecules in modulating human bone health and disease. Treating individuals affected with X-linked hypophosphatemia (XLH) with 24,25(OH)2D3 improved rickets in children and mineralization in adults9 and treating patients with renal insufficiency with 24,25(OH)2D3 improved skeletal mineralization.10 Children affected with severe osteogenesis imperfecta had higher 24,25(OH)2D3 levels, but it is unclear as to the significance of this elevation in metabolite levels.6 Moreover, administration of high dose boluses of vitamin D3 to vitamin D sufficient older subjects increases the incidence of fractures and falls.11 However, treating this population with lower daily doses of vitamin D3 had no effect on bone density.11 It would be informative to measure the levels of vitamin D metabolites in patients treated with bolus or daily doses of vitamin D3 to determine if the effects of vitamin D3 supplementation observed in these studies are associated with changes in circulating levels of metabolites. These clinical studies suggest that vitamin D metabolite supplementation in humans can result in meaningful changes in skeletal health. However, it is unclear as to what plasma levels of these metabolites are considered “normal” and necessary to impact biological activity. Also, it is not known whether supraphysiologic levels of these vitamin D metabolites have direct detrimental effects on the skeleton. Despite the current study suggesting that higher levels of vitamin D metabolites are associated with lower TtBMD, administration of high doses of 24,25(OH)2D3 to mice with XLH12 or human subjects with chronic renal insufficiency,10 did not result in increased osteoclast surface area. Further studies are required to determine if high levels of these vitamin D metabolites have any direct impact on enhancing bone resorption and decreasing bone density. This study did not provide analyses on whether the increase in plasma levels of vitamin D metabolites correlate with changes in bone resorption or formation markers. Another limitation is that it is not reported whether the plasma levels of these vitamin D metabolites correlate with serum calcium, phosphate, and parathyroid hormone levels, all of which are regulated by vitamin D and may impact bone mineralization. It is uncertain if measurements of plasma levels of vitamin D metabolites can be used in a practical and meaningful way in clinical practice, or if these analyses will be limited to research study analyses. Future studies will be needed to identify whether vitamin D metabolites have a direct and essential impact on bone health. Eva S. Liu: Conceptualization, Writing – original draft, Writing – review and editing. Meryl S. LeBoff: Conceptualization, Writing – review and editing. Eva Liu, MD served on the clinical advisory board for Ascendis Pharma and serves on the Data Safety Monitoring Board for Inozyme. This is supported by grants (R01 AR076973 to Dr. Liu and R01 AR060574, R01 AR070854, and R01 AR059775, to Dr. LeBoff) from the National Institute of Arthritis and Musculoskeletal and Skin Diseases. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.