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EXPERIMENTAL MODEL OF SUBCLINICAL VITAMIN K DEFICIENCY: ADAPTATION OF THE STUDY OF OSTEOCALCIN

Radi Lukanov, Silvia Gancheva, Elizabet Miladinova, Dessyslava Nikolaeva, Maria Zhelyazkova-Savova

Abstract

Introduction: Vitamin K (VK), comprising VK1 and VK2, is involved in the post-translational gamma glutamyl-carboxylation of VK dependent proteins (Gla-proteins). The VK dependent factors of coagulation are carboxylated in the liver with the participation of VK1. Other Gla-proteins, among which osteocalcin, are dependent on the extrahepatic pool of VK2. To study the physiological functions of osteocalcin, we utilized warfarin as a VK antagonist to impair the carboxylation of osteocalcin, based on the original method of Price et al. (1994). An added suitable dose of VK1 was supposed to prevent bleeding, yet allowing the effect of warfarin on osteocalcin.

Materials and Methods: To establish the necessary doses of drugs, we conducted two consecutive experiments each including 20 male Wistar, divided into a control and an experimental group. Initially, the experimental rats were treated with high dose subcutaneous warfarin with simultaneous administration of VK1 by gavage. Because of hemorrhages that developed in some rats, the dose of warfarin was reduced after the first week and the dose of VK1 was increased. Six rats out of ten survived at the end of the 4 weeks of treatment. In the second 4 weeks of the experiment it was confirmed that the selected doses were well-tolerated. Subsequently, plasma levels of uncarboxylated (ucOC) and carboxylated (cOC) osteocalcin were determined using ELISA kits.

Results: As expected, the levels of cOC in the experimental group were significantly lower and those of ucOC were about ten times higher compared to the control group. With the doses of warfarin and VK1 thus determined, we were able to selectively inhibit the carboxylation of osteocalcin by VK2, while avoiding the risk of bleeding.

Conclusion: The model of subclinical VK deficiency can be useful as a tool to study the physiological roles of Gla proteins and particularly of osteocalcin.


Keywords

vitamin K, deficiency


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