Aki Igarashi

Effect of vitamin K2 (menaquinone-7) in fermented soybean (natto) on bone loss in ovariectomized rats

Abstract: The effect of dietary vitamin K2 (menaquinone-7) on bone loss in ovariectomized (OVX) rats was investigated. OVX rats were freely given experimental diets containing menaquinone-4 (MK-4; 12 mg/100 g diet) or menaquinone-7 (MK-7; 18.1 mg/100 g diet) for 24 days; MK-4 and MK-7 were equal in molar concentrations. This feeding caused a remarkable increase of MK-4 and MK-7 concentrations in the serum and femur of OVX rats. OVX-induced decrease in the femoral dry weight and femoral calcium content was prevented by the feeding of dietary MK-4 or NK-7. In separate experiments, OVX rats were freely given experimental diets containing the fermented soybean (natto; including 9.4 μg MK-7/100 g diet) without or with added MK-7 (37.6 μg/100 g diet) for 77 days. Feeding produced a significant elevation of MK-4 and MK-7 concentrations in the serum of OVX rats. In this case, a significant increase in the femoral MK-4 content was observed but MK-7 was not detected in the femoral tissues. OVX-induced decreases in the femoral dry weight and femoral calcium content were significantly prevented by the feeding of diets containing natto with MK-7 added (37.6 μg/100 g diets). This study demonstrates that the intake of dietary MK-7 has a preventive effect on bone loss caused by OVX. This effect may be partly caused by MK-4, which is formed by degradation of MK-7.

Stimulatory effect of zinc acexamate administration on fracture healing of the femoral-diaphyseal tissues in rats

The effect of zinc acexamate on fracture healing of the femoral-diaphyseal tissues in rats was investigated in vivo. Zinc acexamate (0.3 and 10.0 mg Zn/100 g body weight per day) was orally administered to rats (4 weeks old) surgically fractured the femoral diaphysis for 14 to 28 days. Calcium content and alkaline phosphatase activity in the femoral-diaphyseal tissues were significantly decreased in rats with fracture healing, while bone acid phosphatase activity and protein content were markedly increased. The administration of zinc acexamate (10.0 mg Zn/100 g) for 28 days caused a significant increase in calcium content, alkaline and acid phosphatases activities, protein and deoxyribonucleic acid (DNA) contents in the femoral-diaphyseal tissues of rats with fracture healing. With the lower dose (3.0 mg Zn/100 g), zinc compound had a partial effect on bone components. Femoral mineral density in rats with fracture healing was significantly increased by the administration of zinc acexamate (10.0 mg Zn/100 g) for 28 days. Femoral-diaphyseal zinc content was significantly decreased in rats with fracture healing. This decrease was completely restored by the administration of zinc acexamate (10.0 mg Zn/100 g) for 28 days. The present study suggests that the supplement of zinc compound stimulates fracture healing of the femoral-diaphyseal tissues in rats.

Enhancement of albumin expression in bone tissues with healing rat fractures

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral‐diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [ 2002 ] Int. J. Mol. Med. 9:503–508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral‐diaphyseal tissues with fracture healing were cultured for 48 h in a serum‐free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N‐terminal sequencing of 66 kDa protein indicated that its N‐terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral‐diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum‐free medium containing either vehicle, parathyroid hormone (1–34) (10−7 M), insulin‐like growth factor‐I (10−8 M) or zinc acexamate (10−4 M), medium albumin was significantly increased in the presence of those bone‐stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral‐diaphyseal and ‐metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral‐diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components. J. Cell. Biochem. 89: 356–363, 2003.