Naoko Tsugawa

Generation of 1,25-dihydroxyvitamin D 3 in Cyp27b1 knockout mice by treatment with 25-hydroxyvitamin D 3 rescued their rachitic phenotypes

We have reported that 25-hydroxyvitamin D3 [25(OH)D3] binds to vitamin D receptor and exhibits several biological functions directly in vitro. To evaluate the direct effect of 25(OH)D3 in vivo, we used Cyp27b1 knockout (KO) mice, which had no detectable plasma 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] when fed a diet containing normal Ca and vitamin D. Daily treatment with 25(OH)D3 at 250 μg kg-1 day-1 rescued rachitic phenotypes in the Cyp27b1 KO mice. Bone mineral density, female sexual cycles, and plasma levels of Ca, P, and PTH were all normalized following 25(OH)D3 administration. An elevated Cyp24a1 mRNA expression was observed in the kidneys, and plasma concentrations of Cyp24a1-dependent metabolites of 25(OH)D3 were increased. To our surprise, 1,25(OH)2D3 was detected at a normal level in the plasma of Cyp27b1 KO mice. The F1 to F4 generations of Cyp27b1 KO mice fed 25(OH)D3 showed normal growth, normal plasma levels of Ca, P, and parathyroid hormone, and normal bone mineral density. The curative effect of 25(OH)D3 was considered to depend on the de novo synthesis of 1,25(OH)2D3 in the Cyp27b1 KO mice. This suggests that another enzyme than Cyp27b1 is present for the 1,25(OH)2D3 synthesis. Interestingly, the liver mitochondrial fraction prepared from Cyp27b1 KO mice converted 25(OH)D3 to 1,25(OH)2D3. The most probable candidate is Cyp27a1. Our findings suggest that 25(OH)D3 may be useful for the treatment and prevention of osteoporosis for patients with chronic kidney disease.

Associations between serum 25-hydroxyvitamin D3 level and skeletal muscle mass and lower limb muscle strength in Japanese middle-aged subjects

Objectives One of the important risk factors of falling is decreased muscle mass and muscle strength. Recently, there has been an increasing concern on the role of vitamin D in muscle strength and physical activity. Aim of our study is to examine the relationships between vitamin D status and muscle mass and muscle strength in middle-aged healthy adults. Methods Subjects were 40 healthy volunteers aged 42.0 ± 10.6 years old. Evaluation was made for serum vitamin D3 metabolites including 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] concentrations, lower limb muscle strength, and dietary intake by food frequency questionnaire. Body composition was measured by dual-energy X-ray absorptiometry (DXA), and appendicular skeletal mass index (ASMI) was calculated as skeletal muscle mass/squared height. Results 70% of the subjects had vitamin D insufficiency/deficiency (serum total 25(OH)D < 20 ng/mL), and female subjects had significantly lower serum total 25(OH)D level compared with males. Vitamin D insufficiency/deficiency group had significantly higher body fat, lower SMI and muscle strength, probably reflecting higher percentage of female subjects. Serum vitamin D3 metabolites levels were significantly correlated with whole and site-specific ASMI, and lower limb muscle strength, except for the correlation between serum 24,25(OH)2D3 concentration and lower limb muscle strength. In addition, serum 25(OH)D3 level was a positive significant predictor for both ASMI and lower limb muscle strength, while serum 24,25(OH)2D3 level was not their significant predictor. Conclusions Serum 25(OH)D3 level was significantly correlated with both skeletal muscle mass and lower limb muscle strength.

Determination of menadione by liquid chromatography-tandem mass spectrometry using pseudo multiple reaction monitoring

This study aimed to develop a menadione (MD) determination method employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a pseudo multiple reaction monitoring (MRM) technique, wherein two quadrupoles are used to monitor the same ion. Detection limits of 40 and 2 pg were obtained for MD and its deuterium-labeled form, respectively, whereas MD intra- and inter-assay coefficient of variation values were determined as 5.4 - 8.2%, with the corresponding recoveries equaling 90.5 - 109.6%. The developed method enables determination of MD in urine, plasma, cell extract, and culture media, demonstrating that pseudo multiple reaction monitoring can achieve quantification of compounds forming no suitable product ions, such as MD.