Plasma Response to Deuterium-Labeled Vitamin K Intake Varies by TG Response, but Not Age or Vitamin K Status, in Older and Younger Adults.

Background: Phylloquinone is the primary form of vitamin K in the diet and circulation. Large intra- and interindividual variances in circulating phylloquinone have been partially attributed to age. However, little is known about the nondietary factors that influence phylloquinone absorption and metabolism. Similarly, it is not known if phylloquinone absorption is altered by the individual's existing vitamin K status. Objective: The purpose of this secondary substudy was to compare plasma response with deuterium-labeled phylloquinone intake in older and younger adults after dietary phylloquinone depletion and repletion. Methods: Forty-two older [mean ± SD age: 67.2 ± 8.0 y; body mass index (BMI; in kg/m2): 25.4 ± 4.6; n = 12 men, 9 women] and younger (mean ± SEM age: 31.8 ± 6.6 y; BMI: 25.5 ± 3.3; n = 9 men, 12 women) adults were maintained on sequential 28-d phylloquinone depletion (∼10 µg phylloquinone/d) and 28-d phylloquinone repletion (∼500 µg phylloquinone/d) diets. On the 23rd d of each diet phase, participants consumed deuterated phylloquinone-rich collard greens (2H-phylloquinone). Plasma and urinary outcome measures over 72 h were compared by age group, sex, and dietary phase via 2-factor repeated-measures ANOVA. Results: The plasma 2H-phylloquinone area under the curve (AUC) did not differ in response to phylloquinone depletion or repletion, but was 34% higher in older than in younger adults (P = 0.02). However, plasma 2H-phylloquinone AUC was highly correlated with the serum triglyceride (TG) AUC (r2 = 0.45). After adjustment for serum TG response, the age effect on the plasma 2H-phylloquinone AUC was no longer significant. Conclusions: Plasma 2H-phylloquinone response did not differ between phylloquinone depletion and repletion in older and younger adults. The age effect observed was explained by the serum TG response and was completely attenuated after adjustment. Plasma response to phylloquinone intake, therefore, seems to be a predominantly lipid-driven effect and not dependent on existing vitamin K status. More research is required to differentiate the effect of endogenous compared with exogenous lipids on phylloquinone absorption. This trial was registered at clinicaltrials.gov as NCT00336232.

Double strand DNA-based determination of menadione using a Fe3O4 nanoparticle decorated reduced graphene oxide modified carbon paste electrode.

In this work an electrochemical label free DNA biosensor (ds-DNA) for the determination of menadione (MD) was developed. The biosensor was constructed using a modified nanocomposite consisting of Fe3O4 nanoparticles decorated reduced graphene oxide (Gr) on a carbon paste electrode (CPE). Scanning electron microscope (SEM), energy dispersive X-ray (EDAX) and Fourier transform infrared (FT-IR) spectroscopy confirmed the structure of the synthesized nanocomposites (electrode composition). The Gr-Fe3O4 nanocomposites formed a sensitive layer with large surface area. Electrochemical studies revealed that modification of the electrode surface with ds-DNA and Gr- Fe3O4 nanocomposite significantly increases the oxidation peak currents and reduces the peak potentials of MD. Under the optimum conditions, calibration curve was linear in the range of 0.3-100.0 nM with a detection limit of 0.13 nM. The relative standard deviation for 50.0 nM was 3.90% (n = 5). The proposed biosensor was successfully applied to the determination of MD.

Quantification of menadione from plasma and urine by a novel cysteamine-derivatization based UPLC-MS/MS method.

Menadione, as the crucial component of vitamin Ks, possessed significant nutritional and clinical values. However, there was still lack of favourable quantification strategies for it to date. For improvement, a novel cysteamine derivatization based UPLC-MS/MS method was presented in this work. The derivatizating reaction was proved non-toxic, easy-handling and high-efficient, which realized the MS detection of menadione under positive mode. Benefitting from the excellent sensitivity of the derivatizating product as well as the introduction of the stable isotope dilution technique, the quantification could be achieved in the range of 0.05-50.0ng/mL for plasma and urine matrixes with satisfied accuracy and precision. After analysis of the samples from healthy volunteers after oral administration of menadione sodium bisulfite tablets, the urinary free menadione was quantified for the very first time. We believe the progress in this work could largely promote the exploration of the metabolic mechanism of vitamin K in vivo.

Vitamin E decreases extra-hepatic menaquinone-4 concentrations in rats fed menadione or phylloquinone.

SCOPE: The mechanism for increased bleeding and decreased vitamin K status accompanying vitamin E supplementation is unknown. We hypothesized that elevated hepatic α-tocopherol (α-T) concentrations may stimulate vitamin K metabolism and excretion. Furthermore, α-T may interfere with the side chain removal of phylloquinone (PK) to form menadione (MN) as an intermediate for synthesis of tissue-specific menaquinone-4 (MK-4). METHODS AND RESULTS: In order to investigate these hypotheses, rats were fed phylloquinone (PK) or menadione (MN) containing diets (2 µmol/kg) for 2.5 weeks. From day 10, rats were given daily subcutaneous injections of either α-T (100 mg/kg) or vehicle and were sacrificed 24 h after the seventh injection. Irrespective of diet, α-T injections decreased MK-4 concentrations in brain, lung, kidney, and heart; and PK in lung. These decreases were not accompanied by increased excretion of urinary 5C- or 7C-aglycone vitamin K metabolites, however, the urinary α-T metabolite (α-CEHC) increased ≥ 100-fold. Moreover, α-T increases were accompanied by downregulation of hepatic cytochrome P450 expression and modified expression of tissue ATP-binding cassette transporters. CONCLUSION: Thus, in rats, high tissue α-T depleted tissue MK-4 without significantly increasing urinary vitamin K metabolite excretion. Changes in tissue MK-4 and PK levels may be a result of altered regulation of transporters.

Age group and sex do not influence responses of vitamin K biomarkers to changes in dietary vitamin K.

Inadequate vitamin K intake has been associated with abnormal soft tissue calcification. Older adults may have insufficient intakes of vitamin K and respond less to vitamin K supplementation compared with younger adults. However, little is known about the determinants that influence the response to vitamin K supplementation. Our primary objective was to assess dietary and nondietary determinants of vitamin K status in healthy younger and older adults. In a nonrandomized, nonmasked study, 21 younger (18-40 y) and 21 older (55-80 y) men and women consumed a baseline diet (200 µg phylloquinone/d) for 5 d, a phylloquinone-restricted diet (10 µg phylloquinone/d) for 28 d, and a phylloquinone-supplemented diet (500 µg phylloquinone/d) for 28 d. Changes in vitamin K status markers in response to vitamin K depletion and repletion were studied and the influences of BMI, body fat, and circulating TG were assessed by including them as covariates in the model. Despite baseline differences in measures of vitamin K status, plasma phylloquinone tended to increase (P = 0.07) and the percentage of uncarboxylated osteocalcin and uncarboxylated prothrombin both improved with phylloquinone supplementation (P < 0.007), regardless of age group or sex. Only the excretion of urinary menadione, a vitamin K metabolite, was greater among younger adults in response to depletion than in older adults (P = 0.012), regardless of sex. Adiposity measures and circulating TG did not predict response of any measures. In conclusion, poor vitamin K status can be similarly improved with vitamin K supplementation, regardless of age group or sex.

Measurement of menadione in urine by HPLC.

Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C(30) column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H(2)O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R(2)) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine.

Menadione is a metabolite of oral vitamin K.

Phylloquinone is converted into menaquinone-4 and accumulates in extrahepatic tissues. Neither the route nor the function of the conversion is known. One possible metabolic route might be the release of menadione from phylloquinone by catabolic activity. In the present study we explored the presence of menadione in urine and the effect of vitamin K intake on its excretion. Menadione in urine was analysed by HPLC assay with fluorescence detection. Urine from healthy male volunteers was collected before and after administration of a single dose of K vitamins. Basal menadione excretion in non-supplemented subjects (n 6) was 5.4 (sd 3.2) microg/d. Urinary menadione excretion increased greatly after oral intake of the K vitamins, phylloquinone and menaquinone-4 and -7. This effect was apparent within 1-2 h and peaked at about 3 h after intake. Amounts of menadione excreted in 24 h after vitamin K intake ranged, on a molar basis, from 1 to 5 % of the administered dose, indicating that about 5-25 % of the ingested K vitamins had been catabolized to menadione. Menadione excretion was not enhanced by phylloquinone administered subcutaneously or by 2',3'-dihydrophylloquinone administered orally. In archived samples from a depletion/repletion study (Booth et al. (2001) Am J Clin Nutr 74, 783-790), urinary menadione excretion mirrored dietary phylloquinone intake. The present study shows that menadione is a catabolic product of K vitamins formed after oral intake. The rapid appearance in urine after oral but not subcutaneous administration suggests that catabolism occurs during intestinal absorption. The observations make it likely that part of the menaquinone-4 in tissues results from uptake and prenylation of circulating menadione.