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Genetically Modified Medicago truncatula Lacking Calcium Oxalate has Increased Calcium Bioavailability and Partially Rescues Vitamin D Receptor Knockout Mice Phenotypes

Xiangkai Li, Jian Yang, Jay Morris, Ashley Hester, Paul A. Nakata and Kendal D. Hirschi

Background: How the distribution and sequestered form of plant macro/micro-nutrients influence their
bioavailability, and ultimately impact human health, are poorly understood. The legume Medicago truncatula has a portion of its tissue calcium (Ca) sequestered in the form of the Ca oxalate (CaOx) crystal which reduces its nutritional value in terms of Ca bioavailability. The calcium oxalate deficient 5 (cod5) mutant has a total Ca content similar to wild-type (WT) plants, but sequesters less of its tissue Ca in the form of the CaOx crystal. Previous short-term mice feeding studies suggest that this difference is responsible for the improved Ca bioavailability of the cod5
plants compared to WT plants.

Objectives: To perform long term feeding studies with Vitamin D Receptor Knockout (VDR-KO) mice and the
nutritionally improved cod5 line to assess the impact of increased Ca bioavailability on VDR-KO Ca deficiency phenotypes.

Methods: To assess the ability of diets containing cod5 plant material as the sole Ca source to rescue the Ca deficiency phenotypes of the VDR-KO mice we conducted both short term and long term experiments. Specifically, Ca absorption and utilization were measured short term (24-hour) in the hind limb bones and duodenum tissue of VDR-KO mice that were fed either an intrinsically 45Ca labeled cod5 or WT Medicago diet. Long term (20-day) bodyweight gain and change in Bone Mineral Density (BMD) were also measured over a 20 day period in VDR-KO mice fed either a cod5 or WT Medicago diet.

Results: In the 24-hour feeding study, 45Ca incorporation was found to be 46.3% (male) or 53.9% (female)
higher in hind limb bones (P<0.01); and 32.5% (male) or 38.5% (female) higher in duodenums (P<0.01) in VDR-KO mice fed cod5 than those fed WT plants. In the 20-day feeding study, the VDR-KO mice (male) fed cod5 gained 38.1% more bodyweight than those fed WT plants (P=0.06). The increase of BMD after 20 days in the VDR-KO mice (male) fed cod5 diets was 22.5% higher than those fed WT diets (P=0.17).

Conclusions: Our study confirms and extends an earlier study by showing that cod5 Medicago not only had
higher Ca bioavailability but it can also rescue, in part, the VDR-KO Ca deficiency phenotypes. Thus, the removal of CaOx from a plant-based diet appears to be a viable long-term dietary option to boost bioavailable Ca levels and help combat Ca related disorders.