Explore the vast range of titles available.

Key enzyme in vitamin K metabolism Vitamin K, an important factor in blood clotting and bone metabolism, is present in the diet principally as phylloquinone (PK) from plants. One form of the vitamin, menaquinone-4 or MK-4, has a highly specific tissue distribution in the brain, kidney and pancreas in humans and in rats, suggestive of local synthesis from phylloquinone. An enzyme catalysing that synthesis has now been identified: UbiA prenyltransferase containing 1 (UBIAD1) is a human homologue of an Escherichia coli enzyme. Previously its function was unclear, although it is a candidate gene in Schnyder crystalline corneal dystrophy. The discovery of a human MK-4 enzyme able to biosynthesize the hormonally active form of vitamin K is of relevance to work on human vitamin K requirements and bone health. These authors identify the human enzyme responsible for menaquinone-4 biosynthesis, a naturally occurring form of vitamin K. They find that UbiA prenyltransferase containing 1 , a human homologue of a prenyltransferase gene from Escherichia coli , encodes an enzyme that can convert vitamin K derivatives into menaquinone-4. Vitamin K occurs in the natural world in several forms, including a plant form, phylloquinone (PK), and a bacterial form, menaquinones (MKs). In many species, including humans, PK is a minor constituent of hepatic vitamin K content, with most hepatic vitamin K content comprising long-chain MKs. Menaquinone-4 (MK-4) is ubiquitously present in extrahepatic tissues, with particularly high concentrations in the brain, kidney and pancreas of humans and rats 1 , 2 , 3 . It has consistently been shown that PK is endogenously converted to MK-4 (refs 4–8 ). This occurs either directly within certain tissues or by interconversion to menadione (K 3 ), followed by prenylation to MK-4 (refs 9–12 ). No previous study has sought to identify the human enzyme responsible for MK-4 biosynthesis. Previously we provided evidence for the conversion of PK and K 3 into MK-4 in mouse cerebra 13 . However, the molecular mechanisms for these conversion reactions are unclear. Here we identify a human MK-4 biosynthetic enzyme. We screened the human genome database for prenylation enzymes and found UbiA prenyltransferase containing 1 ( UBIAD1 ), a human homologue of Escherichia coli prenyltransferase menA . We found that short interfering RNA against the UBIAD1 gene inhibited the conversion of deuterium-labelled vitamin K derivatives into deuterium-labelled-MK-4 (MK-4-d 7 ) in human cells. We confirmed that the UBIAD1 gene encodes an MK-4 biosynthetic enzyme through its expression and conversion of deuterium-labelled vitamin K derivatives into MK-4-d 7 in insect cells infected with UBIAD1 baculovirus. Converted MK-4-d 7 was chemically identified by 2 H-NMR analysis. MK-4 biosynthesis by UBIAD1 was not affected by the vitamin K antagonist warfarin. UBIAD1 was localized in endoplasmic reticulum and ubiquitously expressed in several tissues of mice. Our results show that UBIAD1 is a human MK-4 biosynthetic enzyme; this identification will permit more effective decisions to be made about vitamin K intake and bone health.

Recent studies have suggested that vitamin D activities involve vitamin D receptor (VDR)-dependent and VDR-independent effects of 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) and 25-hydroxyvitamin D 3 (25(OH)D 3 ) and ligand-independent effects of the VDR. Here, we describe a novel in vivo system using genetically modified rats deficient in the Cyp27b1 or Vdr genes. Type II rickets model rats with a mutant Vdr (R270L), which recognizes 1,25(OH) 2 D 3 with an affinity equivalent to that for 25(OH)D 3 , were also generated. Although Cyp27b1 -knockout (KO), Vdr- KO, and Vdr (R270L) rats each showed rickets symptoms, including abnormal bone formation, they were significantly different from each other. Administration of 25(OH)D 3 reversed rickets symptoms in Cyp27b1 -KO and Vdr (R270L) rats. Interestingly, 1,25(OH) 2 D 3 was synthesized in Cyp27b1 -KO rats, probably by Cyp27a1. In contrast, the effects of 25(OH)D 3 on Vdr (R270L) rats strongly suggested a direct action of 25(OH)D 3 via VDR-genomic pathways. These results convincingly suggest the usefulness of our in vivo system.

Fibroblast growth factor 23 (FGF23) exerts its effect by binding to its cognate FGF receptor 1 (FGFR1) in the presence of its co-receptor Klotho. Parathyroid glands express both FGFR1 and Klotho, and FGF23 decreases parathyroid hormone gene expression and hormone secretion directly. In uremic patients with secondary hyperparathyroidism (SHPT), however, parathyroid hormone secretion remains elevated despite extremely high FGF23 levels. To determine the mechanism of this resistance, we measured the expression of Klotho, FGFR1, and the proliferative marker Ki67 in 7 normal and 80 hyperplastic parathyroid glands from uremic patients by immunohistochemistry. All uremic patients had severe SHPT along with markedly high FGF23 levels. Quantitative real-time reverse transcription PCR showed that the mRNA levels for Klotho and FGFR1correlated significantly with their semi-quantitative immunohistochemical intensity. Compared with normal tissue, the immunohistochemical expression of Klotho and FGFR1 decreased, but Ki67 expression increased significantly in hyperplastic parathyroid glands, particularly in glands with nodular hyperplasia. These results suggest that the depressed expression of the Klotho–FGFR1 complex in hyperplastic glands underlies the pathogenesis of SHPT and its resistance to extremely high FGF23 levels in uremic patients.

Vitamin D deficiency is observed worldwide and represents a health hazard for mothers, infants and elderly persons. We know that many young Japanese women experience vitamin D insufficiency; however, there is a lack of knowledge regarding the serum 25-hydroxyvitamin D [25(OH)D] profile of pregnant Japanese women and of the association between maternal 25(OH)D level and maternal bone mass during pregnancy and lactation. In this longitudinal study, 160 pregnant Japanese women were enrolled; of them, 68 have been followed-up from the first trimester through at least 1 year of breast-feeding. We estimated serum 25(OH)D levels, intact PTH levels, calcaneus quantitative ultrasound (QUS: T score) scores, bone mineral density at the distal one-third of the radius, dietary intakes according to the Food Frequency Questionnaire, and sunlight exposure times. We found that Vitamin D deficiency is prevalent in Japanese women, irrespective of pregnancy or lactation, and our analysis suggested that 25(OH)D levels and BMI in the first trimester were related to the lactating women’s bone mass from after delivery to 1 year after delivery.

Populations of East Asian countries have been known to have low calcium intakes and low serum 25(OH)D concentrations, suggesting that Ca and vitamin D (VitD)-deficiencies are commonly observed. These nutritional imbalances may lead to low peak bone mass (PBM). The low PBM seen in Ca/VitD-deficient individuals may lead to osteoporosis, as well as an increased risk of fracture. A survey was conducted in young Japanese women (n = 296, 21.2 ± 2.3 years old) on their Ca/VitD intakes and serum 25(OH)D levels, which demonstrated a significant positive correlation between VitD intake and serum 25(OH)D levels (R2 = 0.020, P = 0.016), and the proportion with serum 25(OH)D over 20 ng/mL was significantly increased with VitD intake (P = 0.013). Serum 25(OH)D was negatively correlated to serum intact parathyroid hormone (R2 = 0.053, P < 0.001). On receiver operating characteristic curve analysis, the VitD intake threshold for maintaining 25(OH)D levels at 20 ng/mL or higher was 11.6 μg/day or greater. It was suggested that the recommended VitD intake allowance, defined in the Adequate Intakes as 5.5 μg/day, may not be sufficient to maintain serum 25(OH)D levels for bone health.

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a significant role in vitamin K2 (MK-4) synthesis. We investigated the enzymological properties of UBIAD1 using microsomal fractions from Sf9 cells expressing UBIAD1 by analysing MK-4 biosynthetic activity. With regard to UBIAD1 enzyme reaction conditions, highest MK-4 synthetic activity was demonstrated under basic conditions at a pH between 8.5 and 9.0, with a DTT ≥0.1 mM. In addition, we found that geranyl pyrophosphate and farnesyl pyrophosphate were also recognized as a side-chain source and served as a substrate for prenylation. Furthermore, lipophilic statins were found to directly inhibit the enzymatic activity of UBIAD1. We analysed the aminoacid sequences homologies across the menA and UbiA families to identify conserved structural features of UBIAD1 proteins and focused on four highly conserved domains. We prepared protein mutants deficient in the four conserved domains to evaluate enzyme activity. Because no enzyme activity was detected in the mutants deficient in the UBIAD1 conserved domains, these four domains were considered to play an essential role in enzymatic activity. We also measured enzyme activities using point mutants of the highly conserved aminoacids in these domains to elucidate their respective functions. We found that the conserved domain I is a substrate recognition site that undergoes a structural change after substrate binding. The conserved domain II is a redox domain site containing a CxxC motif. The conserved domain III is a hinge region important as a catalytic site for the UBIAD1 enzyme. The conserved domain IV is a binding site for Mg2+/isoprenyl side-chain. In this study, we provide a molecular mapping of the enzymological properties of UBIAD1.

Calcium (Ca) absorption from the intestinal tract is promoted by active vitamin D (1α,25D3). Vitamin D not only promotes Ca homeostasis, but it also inhibits bone resorption and promotes osteogenesis, thus playing a role in the maintenance of normal bone metabolism. Because 1α,25D3 plays an important role in osteogenesis, vitamin D formulations, such as alfacalcidol (ALF) and eldecalcitol (ELD), are used for treating osteoporosis. While it is known that, in contrast to ALF, ELD is an active ligand that directly acts on bone, the reason for its superior osteogenesis effects is unknown. Cyp27b1-knockout mice (Cyp27b1-/-mice) are congenitally deficient in 1α,25D3 and exhibit marked hypocalcemia and high parathyroid hormone levels, resulting in osteodystrophy involving bone hypocalcification and growth plate cartilage hypertrophy. However, because the vitamin D receptor is expressed normally in Cyp27b1-/-mice, they respond normally to 1α,25D3. Accordingly, in Cyp27b1-/-mice, the pharmacological effects of exogenously administered active vitamin D derivatives can be analyzed without being affected by 1α,25D3. We used Cyp27b1-/-mice to characterize and clarify the superior osteogenic effects of ELD on the bone in comparison with ALF. The results indicated that compared to ALF, ELD strongly induces ECaC2, calbindin-D9k, and CYP24A1 in the duodenum, promoting Ca absorption and decreasing the plasma concentration of 1α,25D3, resulting in improved osteogenesis. Because bone morphological measurements demonstrated that ELD has stronger effects on bone calcification, trabecular formation, and cancellous bone density than ALF, ELD appears to be a more effective therapeutic agent for treating postmenopausal osteoporosis, in which cancellous bone density decreases markedly. By using Cyp27b1-/-mice, this study was the first to succeed in clarifying the osteogenic effect of ELD without any influence of endogenous 1α,25D3. Furthermore, ELD more strongly enhanced bone mineralization, trabecular proliferation, and cancellous bone density than did ALF. Thus, ELD is expected to show an effect on postmenopausal osteoporosis, in which cancellous bone mineral density decreases markedly. In the future, this study may enable the development of next-generation active vitamin D derivatives with higher affinity for bone than ELD.

There is currently insufficient information on serum 25-hydroxyvitamin D (25OHD) and parathyroid hormone (PTH) concentrations, and bone mineral status in healthy adolescents to allow reference values to be set. This study aimed to provide comparable data on vitamin D status in Japanese adolescents and to assess sex differences in susceptibility to vitamin D insufficiency. Serum 25OHD and PTH concentrations were measured in 1,380 healthy adolescents (aged 12–18 years). Subjects completed a questionnaire on exercise history, diet, and lifestyle factors. Calcaneal stiffness was evaluated by quantitative ultrasound. Serum 25OHD concentrations in boys and girls were 60.8 ± 18.3 and 52.8 ± 17.0 nmol/L, respectively. Approximately 30 % of boys and 47 % of girls had suboptimal 25OHD concentrations (<50 nmol/L). Serum PTH concentration was negatively correlated with serum 25OHD concentration in boys, but negatively correlated with calcium intake rather than serum 25OHD in girls. In contrast, the increment in calcaneal stiffness as a result of elevation of serum 25OHD was higher in girls than in boys. As vitamin D deficiency is common in Japanese adolescents, it was estimated that intakes of ≥12 and ≥14 μg/day vitamin D would be required to reach 25OHD concentrations of 50 nmol/L in boys and girls, respectively. Moreover, the results of the present study indicate that vitamin D deficiency has a greater association with calcaneal stiffness in girls than in boys.

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.

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) is a vitamin K2 biosynthetic enzyme. We previously showed the lethality of this enzyme in UBIAD1 knockout mice during the embryonic stage. However, the biological effects of UBIAD1 deficiency after birth remain unclear. In the present study, we used a tamoxifen-inducible systemic UBIAD1 knockout mouse model to determine the role of UBIAD1 in adult mice. UBIAD1 knockout resulted in the death of the mice within about 60 days of administration of tamoxifen. The pancreas presented with the most prominent abnormality in the tamoxifen-induced UBIAD1 knockout mice. The pancreas was reduced remarkably in size; furthermore, the pancreatic acinar cells disappeared and were replaced by vacuoles. Further analysis revealed that the vacuoles were adipocytes. UBIAD1 deficiency in the pancreatic acinar cells caused an increase in oxidative stress and autophagy, leading to apoptotic cell death in the tamoxifen-induced UBIAD 1 knockout mice. These results indicate that UBIAD1 is essential for maintaining the survival of pancreatic acinar cells in the pancreas.