Vitamin D Status in Japanese Young Women in 2016-2017 and 2020: Seasonal Variation and the Effect of Lifestyle Including Changes Caused by the COVID-19 Pandemic.

Avoidance of sunlight and self-restraint due to the COVID-19 pandemic may contribute to reduced vitamin D status. This study provides comparable data on vitamin D status in Japanese young women and assesses the effect of lifestyle, including changes caused by the COVID-19 pandemic, on vitamin D status. In study 1, 39 young healthy Japanese women aged 21-25 y were recruited from May 2016-June 2017. Serum 25-hydroxyvitamin D (25OHD) concentration and diet and lifestyle information were obtained from participants each month (n=124). In study 2, using the same parameters as study 1, young women aged 21-23 y (n=10) were recruited in September 2020. In the results of study 1, we found the frequencies of vitamin D deficiency (25OHD<20 ng/mL) in spring, summer, fall, and winter were 90.5%, 62.5%, 81.5%, and 91.3%, respectively. The substantial difference of serum 25OHD concentration was obtained in spring (Δ3.6 ng/mL) and summer (Δ5.1 ng/mL) depending on the frequency of sunscreen use (0-2 d/wk, 3-7 d/wk). In study 2, serum 25OHD concentration in September 2020 was extremely lower than in September 2016 (13.2 ng/mL vs. 21.7 ng/mL). The number of days spent outside in 2020 decreased drastically compared with 2019. In conclusion, vitamin D deficiency was highly common in Japanese women in their early 20s, and frequent sunscreen use contributed to low vitamin D status. Moreover, because the decrease in days outside due to the COVID-19 pandemic obviously resulted in a decline in vitamin D status, both appropriate sunbathing and increased dietary vitamin D intake are recommended to young women.

Comparison of Vitamin D and 25-Hydroxyvitamin D Concentrations in Human Breast Milk between 1989 and 2016-2017.

BACKGROUND: Breast milk is considered the optimal source of nutrition during infancy. Although the vitamin D concentration in human breast milk is generally considered poor for infants, vitamin D in breast milk is an important source for exclusively breastfed infants. Increases in vitamin D insufficiency and deficiency in lactating mothers may reduce vitamin D concentrations in breast milk. This study aimed to compare vitamin D and 25-hydroxyvitamin D (25OHD) concentrations in breast milk collected in 1989 and 2016-2017 and simultaneously analyze them with liquid chromatography-tandem mass spectrometry (LC-MS/MS); the association between the lifestyle of recent lactating mothers (2016-2017) and vitamin D status in human breast milk was also evaluated. METHOD: Lactating mothers were recruited from three regions of Japan in 1989 (n = 72) and 2016-2017 (n = 90), and milk from 3-4 months was collected in summer and winter. The samples were strictly sealed and stored at -80℃ until measurement. Breast milk vitamin D and 25OHD concentrations were analyzed by LC-MS/MS. Vitamin D intake, sun exposure, and sunscreen use of the lactating mothers in 2016-2017 were assessed. RESULTS: Both vitamin D and 25OHD concentrations in breast milk were higher in the summer regardless of the survey year. Significantly lower vitamin D and 25OHD concentrations were observed in 2016-2017 compared with 1989 in summer, but no survey year difference was observed in winter. The stepwise multiple regression analyses identified season, daily outdoor activity, and suntan in the last 12 months as independent factors associated with vitamin D3 concentrations. CONCLUSION: The results suggest that low vitamin D status in recent lactating mothers may have decreased vitamin D and 25OHD concentrations in breast milk compared with the 1980s. These results are helpful for developing public health strategies to improve vitamin D status in lactating mothers and infants.

Study on structure-activity relationship of vitamin K derivatives: Conversion of the naphthoquinone part into another aromatic ring and evaluation of their neuronal differentiation-inducing activity.

We synthesized novel vitamin K derivatives by converting the naphthoquinone group to benzene derivatives and benzoquinone. We evaluated their neuronal differentiation activities to investigate the effect of the quinone moiety on this process. We observed that the 1,4-quinone as well as the side chain part play important roles in neuronal differentiation. We also performed QSAR analysis to predict the compounds which would have higher differentiation activity.

Eldecalcitol is more effective in promoting osteogenesis than alfacalcidol in Cyp27b1-knockout mice.

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.

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.

Nongenomic effects of 1α,25-dihydroxyvitamin D3 on cartilage formation deduced from comparisons between Cyp27b1 and Vdr knockout mice.

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.

Association between vitamin D status and serum parathyroid hormone concentration and calcaneal stiffness in Japanese adolescents: sex differences in susceptibility to vitamin D deficiency.

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.

Functional characterization of the vitamin K2 biosynthetic enzyme UBIAD1.

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.

Vitamin K2 biosynthetic enzyme, UBIAD1 is essential for embryonic development of mice.

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K2 biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1(-/-)) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1(-/-) embryonic stem (ES) cells failed to synthesize vitamin K2 but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1(+/-) mice developed normally, exhibiting normal growth and fertility. Vitamin K2 tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K2 synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1(+/-) E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1(-/-) mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1(+/-) mice. These results suggest that UBIAD1 is responsible for vitamin K2 synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K2, but may have additional functions beyond the biosynthesis of vitamin K2.

Menadione (vitamin K3) is a catabolic product of oral phylloquinone (vitamin K1) in the intestine and a circulating precursor of tissue menaquinone-4 (vitamin K2) in rats.

Mice have the ability to convert dietary phylloquinone (vitamin K1) into menaquinone-4 (vitamin K2) and store the latter in tissues. A prenyltransferase enzyme, UbiA prenyltransferase domain-containing 1 (UBIAD1), is involved in this conversion. There is evidence that UBIAD1 has a weak side chain cleavage activity for phylloquinone but a strong prenylation activity for menadione (vitamin K3), which has long been postulated as an intermediate in this conversion. Further evidence indicates that when intravenously administered in mice phylloquinone can enter into tissues but is not converted further to menaquinone-4. These findings raise the question whether phylloquinone is absorbed and delivered to tissues in its original form and converted to menaquinone-4 or whether it is converted to menadione in the intestine followed by delivery of menadione to tissues and subsequent conversion to menaquinone-4. To answer this question, we conducted cannulation experiments using stable isotope tracer technology in rats. We confirmed that the second pathway is correct on the basis of structural assignments and measurements of phylloquinone-derived menadione using high resolution MS analysis and a bioassay using recombinant UBIAD1 protein. Furthermore, high resolution MS and (1)H NMR analyses of the product generated from the incubation of menadione with recombinant UBIAD1 revealed that the hydroquinone, but not the quinone form of menadione, was an intermediate of the conversion. Taken together, these results provide unequivocal evidence that menadione is a catabolic product of oral phylloquinone and a major source of tissue menaquinone-4.

A novel method based on curvature analysis for estimating the dietary vitamin K requirement in adolescents.

BACKGROUND & AIMS: Few 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. METHOD: We 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. RESULTS: In 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. CONCLUSION: Our novel method is useful for estimating the vitamin K status; moreover, this method showed that bone metabolism requires more vitamin K than blood coagulation.

High prevalence of hypovitaminosis D and K in patients with hip fracture.

Although hip fracture is considered to be associated with hypovitaminosis D and K, few reports have previously studied both of them. We have studied the vitamin D- and K-status as well as the general nutritional status in ninety-nine patients with hip fracture. Mean serum concentration of 25hydroxy-vitamin D (25OH-D) in female fractured patients was only approximately 9 ng/mL, suggesting severe vitamin D deficiency. There was no significant difference between the two groups in serum concentration of intact parathyroid hormone in both genders and serum 25OH-D levels in the male subjects. Plasma concentrations of phylloquinone (vitamin K1; PK) and menaquinone-7 (MK-7) were significantly lower in the fractured group than in the control group in both genders. Logistic regression analysis indicated that circulating concentrations of albumin, PK and 25OH-D were the significant and independent determinants of fracture risk, with their higher concentrations associated with decreased fracture risk. Finally, principal component analysis (PCA) was performed to summarize the clinical parameters into smaller numbers of independent components. Three components were obtained, each representing the overall nutritional status, the vitamin D status, and the vitamin K status. In conclusion, our study has shown that patients with hip fracture have vitamin D and K deficiency independent of general malnutrition.

Hypovitaminosis D and K are highly prevalent and independent of overall malnutrition in the institutionalized elderly.

There have been methodological problems for studying hypovitaminosis D and K in the elderly. First, studies were done either by evaluating food intake or measuring their circulating levels, but rarely by both in Japan. In this paper, vitamin D and K intakes and their circulating levels were simultaneously determined. Second issue is whether hypovitaminosis D and K are independent of general malnutrition, prevalent in the elderly. We tried to statistically discriminate them by principal component analysis (PCA). Fifty institutionalized elderly were evaluated for their circulating 25 hydroxy-vitamin D (25OH-D), intact parathyroid hormone (PTH), phylloquinone (PK), menaquinone-7 (MK-7) levels, and their food intake. Although average vitamin D intake (7.0 microg/day) exceeded the Japanese Adequate Intake (AI) of 5.0 microg/day, average serum 25OH-D concentration was in the hypovitaminosis D range (11.1 ng/mL). Median vitamin K intake was 168 microg/day, approximately 2.5 times as high as AI for vitamin K. Nevertheless, plasma PK and MK-7 concentrations were far lower than those of healthy Japanese elderly over 70 years old. PCA yielded four components; each representing overall nutritional, vitamin K2, vitamin D, and vitamin K1 status, respectively. Since these components are independent of each other, vitamin D- and K-deficiency in these subjects could not be explained by overall malnutrition alone. In summary, institutionalized elderly had a high prevalence of hypovitaminosis D and K, and the simultaneous determination of their circulating level and dietary intake is mandatory in such studies. PCA would yield fruitful results for eliminating the interference by confounders in a cross-sectional study.

[In vivo metabolism of vitamin K: in relation to the conversion of vitamin K1 to MK-4].

Phylloquinone is a major form (>90%) of dietary vitamin K, but the form of vitamin K that exists at the highest concentrations in tissues of animals and humans is menaquinone-4 (MK-4) . Despite this great difference, the origin of tissue MK-4 had not been clarified until recently. We demonstrated that deuterium-labeled phylloquinone was converted into deuterium-labeled MK-4 in mice and this conversion occurred following an oral or enteral administration, but not parenteral administration. By the oral route, the phylloquinone with the deuterium-labeled side chain (phytyl side-chain) was clearly converted into menaquinone-4 with a non-deuterium-labeled side chain (geranylgeranyl side-chain), implying that phylloquinone was converted into menaquinone-4 via integral side-chain removal. Our results suggest that cerebral menaquinone-4 originates from phylloquinone intake and the release of menadione from phylloquinone in the intestine followed by the prenylation of menadione into menaquinone-4 in the intestine or tissues.

Design and synthesis of biologically active analogues of vitamin K2: evaluation of their biological activities with cultured human cell lines.

Novel omega-oxygenated vitamin K(2) analogues were efficiently synthesized and their biological activities were evaluated. Some were biologically active and the side-chain played an important role in gamma-carboxylation and apoptosis-inducing activity. The results provide useful information on the structure-activity relationship of vitamin K(2) analogues for the development of new drugs.

Conversion of phylloquinone (Vitamin K1) into menaquinone-4 (Vitamin K2) in mice: two possible routes for menaquinone-4 accumulation in cerebra of mice.

There are two forms of naturally occurring vitamin K, phylloquinone and the menaquinones. Phylloquinone (vitamin K(1)) is a major type (>90%) of dietary vitamin K, but its concentrations in animal tissues are remarkably low compared with those of the menaquinones, especially menaquinone-4 (vitamin K(2)), the major form (>90%) of vitamin K in tissues. Despite this great difference, the origin of tissue menaquinone-4 has yet to be exclusively defined. It is postulated that phylloquinone is converted into menaquinone-4 and accumulates in extrahepatic tissues. To clarify this, phylloquinone with a deuterium-labeled 2-methyl-1,4-naphthoquinone ring was given orally to mice, and cerebra were collected for D NMR and liquid chromatography-tandem mass spectrometry analyses. We identified the labeled menaquinone-4 that was converted from the given phylloquinone, and this conversion occurred following an oral or enteral administration, but not parenteral or intracerebroventricular administration. By the oral route, the phylloquinone with the deuterium-labeled side chain in addition to the labeled 2-methyl-1,4-naphthoquinone was clearly converted into a labeled menaquinone-4 with a non-deuterium-labeled side chain, implying that phylloquinone was converted into menaquinone-4 via integral side-chain removal. The conversion also occurred in cerebral slice cultures and primary cultures. Deuterium-labeled menadione was consistently converted into the labeled menaquinone-4 with all of the administration routes and the culture conditions tested. Our results suggest that cerebral menaquinone-4 originates from phylloquinone intake and that there are two routes of accumulation, one is the release of menadione from phylloquinone in the intestine followed by the prenylation of menadione into menaquinone-4 in tissues, and another is cleavage and prenylation within the cerebrum.

Low plasma phylloquinone concentration is associated with high incidence of vertebral fracture in Japanese women.

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.

Nutritional effects of gamma-glutamyl carboxylase gene polymorphism on the correlation between the vitamin K status and gamma-carboxylation of osteocalcin in young males.

Vitamin K is a cofactor for gamma-glutamyl carboxylase (GGCX), which is an essential enzyme for the gamma-carboxylation of vitamin K-dependent proteins such as osteocalcin (OC). Associations among dietary vitamin K intake, vitamin K status, and bone metabolism have not been thoroughly investigated. Recently, it has been reported that single nucleotide polymorphisms of GGCX (R325Q, 974G>A) were associated with age-related bone loss and the kinetic affinity for the substrate. In the present study, we investigated the associations among dietary vitamin K intake, the level of serum vitamin K, and the ratio of undercarboxylated OC (ucOC) to intact OC. The subjects were 60 healthy young male volunteers (mean age, 22.6 y; standard deviation, 1.6). Dietary nutrient intake was assessed by consecutive individual 3-d food records taken before the day of blood examinations. Serum concentrations of vitamin K (phylloquinone: PK, menaquinone 4: MK-4, and menaquinone 7: MK-7), ucOC, and intact OC were measured. All subjects were genotyped for polymorphism (R325Q) presence. Dietary vitamin K intake from vegetables was significantly correlated with the level of serum PK, and vitamin K intake from fermented beans, natto, was also significantly correlated with the level of serum MK-7. The ratio of ucOC to intact OC showed a negative association with the total vitamin K intake (r=-0.331, p=0.010) and serum MK-7 (r=-0.394, p=0.002). Interestingly, grouped by the GGCX genotype, a significant interaction between the ratio of ucOC to intact OC with serum MK-7 was observed in 325R homozygotes (r=-0.572, p=0.003), but not in heterozygotes, nor in 325Q homozygotes. This is the first report to suggest the effects of the single nucleotide polymorphism R325Q in the GGCX gene on the correlation between the level of serum MK-7 and gamma-carboxylation of serum OC.

Quantification of fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry.

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 microL. 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, alpha-tocopherol, phylloquinone and menaquinone-4 were 0.455 microg/mL, 0.088 ng/mL, 0.081 ng/mL, 5.087 microg/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.

Efficient synthesis and biological evaluation of omega-oxygenated analogues of vitamin K2: study of modification and structure-activity relationship of vitamin K2 metabolites.

Novel omega-oxygenated vitamin K2 analogues, which are candidates for metabolites of vitamin K2 homologues, were efficiently synthesized and their apoptosis-inducing activity was evaluated. We revealed that some of those analogues were biologically active and the side-chain part played an important role in apoptosis-inducing activity. Our results can provide useful information to develop the structure-activity relationship of vitamin K2 analogues for new drugs based on vitamin K.