Bone development in growing female mice fed calcium and vitamin D at lower levels than is present in the AIN-93G reference diet

Jenalyn L Yumol; C Brent Wakefield; Sandra M Sacco; Philip J Sullivan; Elena M Comelli; Wendy E Ward

doi:10.1016/j.bonr.2018.05.004

Bone development in growing female mice fed calcium and vitamin D at lower levels than is present in the AIN-93G reference diet

Bone Rep. 2018 May 19:8:229-238. doi: 10.1016/j.bonr.2018.05.004. eCollection 2018 Jun.

Authors

Jenalyn L Yumol¹, C Brent Wakefield², Sandra M Sacco², Philip J Sullivan², Elena M Comelli^{2

3

4}, Wendy E Ward^{1

2

3}

Affiliations

¹ Department of Health Sciences, Faculty of Applied Health Sciences, Brock University, St. Catharines, Ontario, Canada.
² Department of Kinesiology, Faculty of Applied Health Sciences, Brock University, St. Catharines, Ontario, Canada.
³ Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Ontario, Canada.
⁴ Joannah and Brian Lawson Centre for Child Nutrition, University of Toronto, Ontario, Canada.

Abstract

Background: The AIN-93G reference (REF) diet is used to allow the comparison within and between studies of different research groups but its levels of vitamin D (vit D) and calcium (Ca) may be higher than required for healthy bone structure and bone mineral density (BMD).

Objective: To determine if lower dietary levels of Ca (3.5, 3 or 2.5 g Ca/kg diet) at 1 of 2 levels of vit D (100 or 400 IU/kg diet) supports similar development of bone structure and BMD compared to AIN-93G reference (REF) diet in female CD-1 mice at 2 and 4 months of age.

Methods: Within a trial, weanling female mice (n = 12-15/group) were randomized to 1 of 4 diets until necropsy at 4 months of age: Trial 1: 100 IU vit D/kg + 3.5, 3 or 2.5 g Ca/kg diet or 1000 IU vit D/kg + 5 g Ca/kg diet (REF); and Trial 2: 400 IU vit D/kg + 3.5, 3 or 2.5 g Ca/kg diet or 1000 IU vit D/kg + 5 g/kg diet (REF). At age 2 and 4 months, in vivo bone structure and BMD were assessed using micro-computed tomography (μCT) at the proximal and midpoint tibia. At age 4 months, lumbar vertebra 4 (L4) and mandible structure were analyzed ex vivo, femur strength at midpoint and neck was assessed and serum 25(OH)D₃ and PTH were quantified.

Results: For Trial 1 (100 IU vit D/kg), there were no differences in tibia structure at age 2 and 4 months nor L4 or mandible structure or femur strength at the midpoint or neck at 4 months of age despite lower serum 25(OH)D₃ among all groups compared to REF. For Trial 2 (400 IU vit D/kg), mice fed 2.5 g Ca/kg diet had lower (p < 0.05) Ct.Ar/Tt.Ar and Ct.Th at the tibia midpoint compared to REF. Furthermore, Ct.Th. was greater in REF and 3.5 g Ca/kg diet compared to 2.5 g Ca/kg diet at age 2 but not 4 months of age. At L4, BV/TV was lower (p < 0.05) in the 3 g Ca/kg diet group compared to REF at age 4 months. There were no differences among groups for serum 25(OH)D₃ or femur strength at the midpoint or neck. Serum PTH was not elevated compared to REF in either Trial.

Conclusion: Lowering both dietary vit D (100 IU/kg) and Ca (2.5 g/kg) in AIN-93G diet did not result in differences in bone development of female CD-1 mice at early adulthood. Translational relevance of bone studies conducted using the AIN-93G diet may be affected by its high vit D and Ca content.

Keywords: AIN93G; ANOVA, analysis of variance; BMC, bone mineral content; BMD, bone mineral density; BV/TV, percent bone volume; Bone mineral density; Bone structure; Ca, calcium; Calcium; Conn.Dn, connectivity density; Ct.Ar/Tt.Ar, cortical area fraction; Ct.Th, cortical thickness; DA, degree anisotropy; Ec.Pm, endocortical perimeter; Ecc, mean eccentricity; ISO, isoflavones; L4, lumbar vertebrae 4; Ma.Ar, medullary area; Micro-computed tomography; P, phosphorus; PBM, peak bone mass; PTH, parathyroid hormone; Ps.Pm, periosteum perimeter; REF, AIN-93G reference diet; ROI, region of interest; SEM, standard error mean; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; Vitamin D; vit D, vitamin D; μCT, micro computed tomography.