Potential insufficiency of vitamin D in healthy individuals: divergent performances of C3-epi-D3 levels and percentages

Accumulating evidence offers valuable insights into the overestimation of 25-hydroxyvitamin D [25(OH)D] levels due to the presence of C3-epimeric 25-hydroxyvitamin D3 (C3-epi-D3) and the relatively reduced bioavailability of 25(OH)D2. This study focuses on healthy populations to assess the relevance of C3-epi-D3 generation and vitamin D potential insufficiency (VDPI). Among 5494 healthy participants with total 25(OH)D sufficiency involved in this study, VDPI was assessed after excluding non-bioavailable C3-epi-D3 levels and converting 25(OH)D2 to equivalent 25(OH)D3 levels based on differential bioavailability. The gender, age, and seasonal characteristics of C3-epi-D3 levels and percentages were investigated, as well as their relationships with 25(OH)D3 levels and vitamin D potential insufficiency. Both C3-epi-D3 levels and percentages presented similar gender, age, and seasonal variations, characterized by higher values in males and peak values during summer (all P < 0.001), while negligible variation from adolescent to longevous periods (all P > 0.05). After adjusting for the aforementioned variations, both forward and reverse restricted cubic spline (RCS) revealed a linear positive correlation between C3-epi-D3 and 25(OH)D3 levels. Conversely, %C3-epi-D3 decreased in an L-shaped trend as 25(OH)D3 levels increased, while 25(OH)D3 levels demonstrated a U-shaped correlation with increasing %C3-epi-D3. Notably, RCS revealed divergent performances of C3-epi-D3 levels and percentages, levels exceeding 1.10 ng/mL protected against VDPI, while percentages surpassing 4.47% increased VDPI risk. The consistent findings were further substantiated by the forest plot across gender, age, and seasonal subgroups. Healthy individuals remain vigilant about vitamin D insufficiency, given the presence of C3-epi-D3 and lower bioavailability of 25(OH)D2. C3-epi-D3 percentage, not its level, accurately reflects this condition, consistent with its negligible bioavailability.