Case report: Three patients with substantial serum levels of 3-epi-25(OH)D including one with 3-epi-25(OH)D2 while on high-dose ergocalciferol

Abstract

Context: We report the presence of substantial concentrations of 3-epi-25(OH)D3 in two patients and a third patient with 3-epi-25(OH)D2.

Patients: The first patient, a 66-year-old female receiving cholecalciferol 4000 IU daily was originally found to have 53 ng/mL of 25(OH)D3 and almost an equal amount of 3-epi-25(OH)D3. Subsequently, the patient had four additional samples, each of which has similar levels of both 25(OH)D3 and 3-epi-25(OH)D3. The second patient, a 7-year-old male receiving cholecalciferol 1000 IU daily, had a 25(OH)D3 concentration of 37 ng/mL and 3-epi-25(OH)D3 of approximately 10 ng/mL. The third and most intriguing patient, a 55-year-old female was receiving ergocalciferol 50,000 IU twice weekly for approximately 3 months, at which time her serum 25(OH)D2 was 64 ng/mL and her 3-epi-25(OH)D2 was approximately 32 ng/mL. This patient's physician changed her vitamin D therapy to cholecalciferol 1000 IU daily, discontinuing ergocalciferol, and a second specimen was collected 5 months later. Analysis of this last specimen found both 25(OH)D3 and 25(OH)D2 at concentrations of 12 and 24 ng/mL respectively, plus corresponding 3-epimer peaks for both 25(OH)D3 and 25(OH)D2 observed chromatographically.

Conclusion: The presence of a substantial concentration of 3-epi-25(OH)D in these three patients documents that one cannot assume 3-epi is a trivial metabolite of 25(OH)D for all patients. In addition, the appearance of 3-epi-25(OH)D3 when the last patient changed her vitamin D supplementation from ergocalciferol to cholecalciferol demonstrates that the 3-epimer is probably an endogenous metabolite of 25(OH)D in humans.

Figure 1.

A, HPLC-UV chromatogram (absorbance 275 nm) showing peaks detected for the internal standard (lauraphenone), 25(OH)D₃, and 3-epi-25(OH)D₃ for patient 1. Note the horizontal bars on the chromatogram, which represent the expected retention time windows for 25(OH)D₃ (red rectangle) and 25(OH)D₂ (blue rectangle), respectively. B, HPLC-MS/MS chromatogram obtained for the same specimen. Three major peaks noted in this chromatogram are the internal standard [25(OH)D₃.d6] in green; 25(OH)D₃, which coelutes with the internal standard; and 3-epi-25(OH)D₃ (second red peak). The MRM ions for 25(OH)D₃ (red peaks) are 383.3/211.2, for hexadeuterated 25(OH)D₃ (green peak) are 389.3/211.1, and for 25(OH)D₂ (small blue peak) are 395.3/209.1.

Figure 2.

A, HPLC-MS/MS chromatogram for patient 3, during period when she was receiving high-dose ergocalciferol. The prominent peaks (blue) are 25(OH)D₂ and the later-eluting 3-epi-25(OH)D₂. Note the very minor peaks (red) for 25(OH)D₃ and 3-epi-25(OH)D₃. B, HPLC-MS/MS chromatogram for patient 3, 5 months after discontinuing ergocalciferol treatment. This chromatogram shows the presence of 25(OH)D₃, 3-epi-25(OH)D₃ (red arrows), 25(OH)D₂, and 3-epi-25(OH)D₂, (blue arrows). The MRM ions for 25(OH)D₃ (red peaks) are 383.3/211.2, for hexadeuterated 25(OH)D₃ (green peak), are 389.3/211.1, and for 25(OH)D₂ (blue peaks) are 395.3/209.1.