We synthesized new vitamin K2 analogues with ω-terminal modifications of the side chain and evaluated their selective differentiation of neuronal progenitor cells into neurons in vitro. The result of the assay showed that the menaquinone-3 analogue modified with the m-methylphenyl group had the most potent activity, which was twice as great as the control. This finding indicated that it is possible to obtain much more potent compounds with modification of the structure of vitamin K2.

We synthesized new vitamin K analogues with demethylation or reduction of the double bonds of the side chain of menaquinone-4 (MK-4) and evaluated their SXR-mediated transcriptional activity as well as the extent of their conversion to MK-4. The results indicated that the analogue with the methyl group deleted at the 7′ site of the side chain part affected conversion activity to MK-4. In contrast, a decrease in the number of the double bonds in the side chain moiety appeared to decrease the SXR-mediated transcriptional activity.

Vitamin K2 is a ligand for a nuclear receptor, steroid and xenobiotic receptor (SXR), that induces the gene expressions of CYP3A4. We synthesized vitamin K2 analogues with hydroxyl or phenyl groups at the ω-terminal of the side chain. The up-regulation of SXR-mediated transcription of the target gene by the analogues was dependent on the length of the side chain and the hydrophobicity of the ω-terminal residues. Phenyl analogue menaquinone-3 was as active as the known SXR ligand rifampicin.

Vitamin K2 has been demonstrated to induce gene expression related to bone formation through a nuclear steroid and xenobiotic receptor (SXR). We synthesized new vitamin K analogues with the same isoprene side chains symmetrically introduced at the 2 and 3 positions of 1,4-naphthoquinone and evaluated the transcriptional activity of the target gene. The transcriptional activity was related to the length of the side chain which allowed optimal interaction with ligand-binding domain of SXR.

To reveal an essential biological role of menaquinone-4, we have clarified that dietary PK was converted to menaquinone-4 (MK-4) in animal tissues using deuterated vitamin K analogues. However, the kinds of analogue converted into MK-4 have not been elucidated. In this study, we examined structure–activity relationships in the conversion of several vitamin K analogues, with a substituted side chain, into MK-4 using cultured human cell lines. The results differed with the side chain of the analogues, that is, (1) the length of the isoprene unit and (2) the number of double bonds in the side chain. These findings would be useful for clarifying the mechanism of conversion of other vitamin K homologs into MK-4 as well as related enzymes.We examined structure–activity relationship in the conversion of several vitamin K analogues, with a substituted side-chain, into menaquinone-4 using cultured human cell lines.

Vitamin K is an essential nutrient and a cofactor for the carboxylation of specific glutamyl residues of proteins to γ-glutamyl residues, which activates osteocalcin related to bone formation. Among vitamin K homologues, menaquinone-4 (MK-4) is the most active biologically, up-regulating the gene expression of bone markers, and thus has been clinically used in the treatment of osteoporosis in Japan. Recently, we confirmed that MK-4 was converted from dietary phylloquinone (PK), and then accumulated in various tissues at high concentrations. This system should play an important role in biological functions including bone formation, however, the pathway by which MK-4 is converted remains unclear. In this study, we studied the mechanism of MK-4’s conversion with chemical techniques using deuterated analogues.

It has been reported that vitamin K supplementation effectively prevents fractures and sustains bone mineral density in osteoporosis. However, there are only limited reported data concerning the association between vitamin K nutritional status and bone mineral density (BMD) or fractures in Japan. The objectives were to evaluate the association between plasma phylloquinone (K1) or menaquinone (MK-4 and MK-7) concentration and BMD or fracture in Japanese women prospectively. A total of 379 healthy women aged 30–88 years (mean age, 63.0 years) were consecutively enrolled. Plasma K1, MK-4, MK-7, and serum undercarboxylated osteocalcin (ucOC) concentrations, BMD, and incidence of vertebral fractures were evaluated. In stepwise multiple linear regression analyses, L2–4 BMD and a bone turnover marker, log K1, concentrations were independently correlated with vertebral fracture incidence. When subjects were divided into low and high K1 groups by plasma K1 concentration, the incidence of vertebral fracture in the low K1 group (14.4%) was significantly higher than that in the high K1 group (4.2%), and its age-adjusted RR was 3.58 (95% CI, 3.26–3.93). L2–4 BMD was not different between the two groups. These results suggest that subjects with vitamin K1 insufficiency in bone have increased susceptibility for vertebral fracture independently from BMD.

1α,25-Dihydroxyvitamin D3 (1α,25-D3) has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, the major limitation to its clinical use is that it causes hypercalcemia. Therefore, vitamin D analogs with potent cell regulatory effects but with weaker calcemic effects than 1α,25-D3 are required. Among them, 22-oxa-1α,25-D3 and 19-nor-1α,25-D3 have anti-cancer effects with relatively low calcemic effects. Modifications at the C-2α position of the A-ring also produced analogs with a unique biological profile. Not only the side-chain but also the A-ring modification thus generates a unique analog with potent cell regulatory effects and low calcemic activity as well. We report here that the hybrid 1α,25-D3 analog, synthesized via the highly regio- and stereo-selective ring opening 2α-fluorination and catalytic asymmetric carbonyl-ene cyclization, with 2α-fluoro, 19-nor, and 22-oxa modification exhibits unique cell regulatory activities against the development of metastatic lung carcinoma.Hybrid 1α,25-dihydroxyvitamin D3 (1α,25-D3) analog, synthesized via the highly regio- and stereo-selective ring opening 2α-fluorination and catalytic asymmetric carbonyl-ene cyclization, with 2α-fluoro, 19-nor, and 22-oxa modification exhibits unique cell regulatory activities against the development of metastatic lung carcinoma.

Sensitive quantification method for fat-soluble vitamins in human breast milk by liquid chromatography–tandem mass spectrometry was developed. Vitamins A, D and E were extracted from 10.0 mL of breast milk after saponifying by basic condition. Vitamin K derivatives were extracted from 3.0 mL of breast milk after lipase treatment. The corresponding stable isotope-labeled compounds were used as internal standards. For the determination of vitamin D compounds, derivatization with a Cookson-type reagent was performed. All fat-soluble vitamins were determined by liquid chromatography–tandem mass spectrometry in the positive ion mode. The detection limits of all analytes were 1–250 pg per 50 μL. The recoveries of fat-soluble vitamins were 91–105%. Inter-assay CV values of each vitamin were 1.9–11.9%. The mean concentrations of retinol, vitamin D3, 25-hydroxyvitamin D3, α-tocopherol, phylloquinone and menaquinone-4 were 0.455 μg/mL, 0.088 ng/mL, 0.081 ng/mL, 5.087 μg/mL, 3.771 ng/mL, and 1.795 ng/mL, respectively (n = 82). This method makes possible to determine fat-soluble vitamins with a wide range of polarities in human breast milk. The assay may be useful for large-scale studies.

Background & aimsFew studies have investigated the association between vitamin K status and bone health in adolescents. We established a novel method for estimating the vitamin K status in adolescents by curvature analysis using the serum concentrations of undercarboxylated osteocalcin (ucOC)—a sensitive biomarker of vitamin K status in the bone. We also compared the vitamin K concentrations required for good bone health and for normal blood coagulation.MethodWe enrolled 1183 healthy adolescents. For the curvature analysis, we used a logarithmic regression equation obtained from vitamin K intake and serum ucOC or plasma abnormal prothrombin (PIVKA-II) concentrations (marker for blood coagulation). The cut-off point was determined to be the vitamin K intake that showed the highest curvature.ResultsIn adolescents, the serum ucOC concentration was negatively correlated with vitamin K intake. In the curvature analysis, requirement of vitamin K intake for good bone health and normal blood coagulation were 155–188 μg/d and 62–54 μg/d [1 μg/(kg d)], respectively; the latter result was consistent with that of a previous report.ConclusionOur novel method is useful for estimating the vitamin K status; moreover, this method showed that bone metabolism requires more vitamin K than blood coagulation.

•We produced Cyp27b1−/− mice and analyzed their phenotypes.•Vdr−/− mice exhibited alopecia and Cyp27b1−/− mice exhibited cartilage dysplasia.•Maintenance of the hair cycle is associated with 25D3 via Vdr.•1α,25D3 controls chondrocyte proliferation and differentiation independent of Vdr.The active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1α,25D3), plays an important role in the maintenance of calcium (Ca) homeostasis, bone formation, and cell proliferation and differentiation via nuclear vitamin D receptor (VDR). It is formed by the hydroxylation of vitamin D at the 1α position by 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1) in the kidney. However, Cyp27b1−/− mice, deficient in CYP27B1, and VDR-deficient mice (Vdr−/−) have not been extensively examined, particularly in a comparative framework. To clarify the physiological significance of 1α,25D3 and VDR, we produced Cyp27b1−/− mice and compared their phenotypes with those of Vdr−/− mice. Cyp27b1−/− mice exhibited hypocalcemia, growth defects, and skeletogenesis dysfunction, similar to Vdr−/− mice. However, unlike Cyp27b1−/− mice, Vdr−/− mice developed alopecia. Cyp27b1−/− mice exhibited cartilage mass formation and had difficulty walking on hindlimbs. Furthermore, a phenotypic analysis was performed on Cyp27b1−/− mice provided a high Ca diet to correct for the Ca metabolic abnormality. In addition, the effects of 1α,25D3 that are not mediated by Ca metabolic regulatory activity were investigated. Even when the blood Ca concentration was corrected, abnormalities in growth and cartilage tissue formation did not improve in Cyp27b1−/− mice. These results suggested that 1α,25D3 directly controls chondrocyte proliferation and differentiation. Using Cyp27b1−/− mice produced in this study, we can analyze the physiological effects of novel vitamin D derivatives in the absence of endogenous 1α,25D3. Accordingly, this study provides a useful animal model for the development of novel vitamin D formulations that are effective for the treatment and prevention of osteoporosis.Figure optionsDownload full-size imageDownload high-quality image (268 K)Download as PowerPoint slide