Phylloquinone Concentrations Research Articles

Effect of Vitamin K1 on Cell Growth Inhibition and Apoptosis on the U937 Cell Line

This experiment was conducted in order to verify the role of Vitamin K1 as a cell growth inhibitor on the U937 cell line. This experiment was performed in two parts—one with a lesser concentration of Vitamin K1, and the other with a range of concentrations from low-to-high. Through the remaining number of U937 cells, as well as cell areas, it was concluded that the presence of Vitamin K1 reduces the number of cancer cells. It was also concluded that as Vitamin K1 concentration increases, so does the frequency and effects of apoptosis.

C-Met-Akt pathway-mediated enhancement of inhibitory c-Raf phosphorylation is involved in vitamin K1 and sorafenib synergy on HCC growth inhibition

Sorafenib is an FDA-approved agent for treatment of human hepatocellular carcinoma (HCC), but tumor shrinkage is minor. We therefore developed a strategy to combine K vitamins with sorafenib to treat HCC, and found that this combination enhanced sorafenib-induced HCC cell growth inhibition. To explore the mechanisms involved, we examined the role of Raf kinase, since both vitamins K and sorafenib were reported to inhibit tumor cell growth via Raf signaling pathway. We found that whereas lower concentration of vitamin K1 (25 μM) or sorafenib (2.5 μM) alone slightly induced c-Raf phosphorylation at both Ser-43 and Ser-259, combination vitamin K1 plus sorafenib resulted in strong c-Raf phosphorylation at these two serine residues. A Raf kinase activity assay confirmed that combination vitamin K1 plus sorafenib had a synergistic inhibitory effect on it. Since c-Raf phosphorylation at Ser-43 and Ser-259 can be regulated by either PKA or Akt kinase, we examined the effects of both vitamin K1 and sorafenib on their phosphorylation. Although vitamin K1 or sorafenib alone induced PKA phosphorylation, no enhanced phosphorylation effects on PKA were found using this combination. However, vitamin K1 enhanced sorafenib-induced c-Met phosphorylation at Tyr-1349, a DEP-1 protein phosphatase acting site, and consequently induced phosphorylation of PI3K-Akt. Both PI3K inhibitor Ly294002 as well as dominate negative Akt plasmid transfection antagonized vitamin K1 plus sorafenib actions on c-Raf phosphorylation and cell growth inhibition, suggesting that c-Met-PI3K-Akt signaling pathway mediated inhibitory c-Raf phosphorylation may play a central role in vitamin K1 plus sorafenib synergy in inhibiting HCC cell growth.

The Bioavailability of a Mixed Micellar Preparation of Vitamin K1, and its Procoagulant Effect in Anticoagulated Rabbits

We have investigated the pharmacokinetics and procoagulant activity of a new, mixed-micellar preparation of vitamin K1 (MM-K) in male New Zealand White rabbits. Oral administration of MM-K alone caused a significant (P less than 0.01) increase in the plasma concentrations of vitamin K1 as measured by normal-phase high-performance liquid chromatography (HPLC). Maximum plasma concentrations of vitamin K1 (450 ng mL-1, range 133-824 ng mL-1) were recorded at 3.3 h (range 3-5 h), and were significantly (P less than 0.05) greater than those seen after administration of an existing polyethoxylated castor oil preparation (PE-K; Konakion), which were 260 ng mL-1, range 198-390 ng mL-1 (tmax 0.8 h, range 0.4-1.2 h). AUC after MM-K (4.6 micrograms mL-1 h-1, range 2.1-6.3 micrograms ML-1 h-1) was also significantly (P less than 0.05) greater than after PE-K (1.6 micrograms mL-1 h-1, range 1.0-2.1 micrograms ML-1 h-1). However, the bioavailability of vitamin K1 after administration of MM-K was poor (9.4%), and there was considerable intra-individual variability between the concentrations of vitamin K1 recorded in the plasma samples. Both preparations of vitamin K1 stimulated clotting factor synthesis in rabbits anticoagulated with the potent and long-acting coumarin, brodifacoum. Maximum stimulation of clotting factor synthesis by vitamin K1 after MM-K was 87%, range 44-124% (%PCA). The maximum was seen later (tmax 12 h) than after PE-K (PCA 82%, range 47-125%; tmax 5 h). However, there was considerable intra-individual variability in response to both MM-K and PE-K.(ABSTRACT TRUNCATED AT 250 WORDS)

An investigation of sex-linked differences to the toxic and to the pharmacological actions of difenacoum: studies in mice and rats.

We have investigated the actions of the coumarin anticoagulant, difenacoum, in male and female rats and mice. In our first experiment difenacoum (0.5 mg kg-1) killed 50% of male mice within 9 days of its administration, whereas no female mice died during this study. In a second group of experiments, the anticoagulant effect of difenacoum in male and female rats was determined. Under resting conditions, the prothrombin complex activities (PCA) of male and female rats were not significantly different. Over the first 24 h after administration of difenacoum (0.4 mg kg-1 i.p.), there was a monoexponential fall in PCA in both sexes. However, 6, 12 and 24 h after difenacoum, the PCA in male rats was significantly (P less than 0.05) lower than in female rats. PCA began to recover over the subsequent 48 h in both sexes, during which time there was marked variability in recovery in female rats. The difference between the onset of action of difenacoum in male and female rats did not appear to be due to a greater rate of elimination of the drug in female rats, since the plasma concentrations of difenacoum 24 h after its administration were the same in both sexes. The concentration of vitamin K1 in rat liver was also investigated. Vitamin K1 levels were 35.1 +/- 18.6 ng (g liver)-1 (male), and 29.4 +/- 5.4 ng (g liver)-1 (females) in control rats, but 24 h after difenacoum, vitamin K1 levels were either very low, or undetectable in all rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-dependent differences in the effect of phenprocoumon on the vitamin K1-epoxide cycle in rats.

Abstract The anticoagulant activity and the pharmacokinetics of phenprocoumon as well as the effect of phenprocoumon on the vitamin K1-epoxide cycle in younger (12 weeks) and older (36 weeks) male inbred Lewis rats has been examined in a study of the mechanism responsible for the increase in the responsiveness to oral anticoagulant drugs (OAD's) with increasing age. After a single i.v.-dose of phenprocoumon (0†355 mg kg−1 the anticoagulant effect obtained was greater in older than in younger rats. There were no differences between younger and older rats in the rate of elimination, volume of distribution and in the free fraction and free concentration values of phenprocoumon in plasma and liver. After i.v.- injection of 64·3 μg kg−1 [3 H]vitamin K1 and different doses of phenprocoumon (0·02 to 3 mg kg−1) the [3 H]vitamin K1 concentration in the liver decreased and the [3 H] vitamin K1-2, 3-epoxide concentration increased dependent on the dose and the liver concentration of phenprocoumon. These changes were more pronounced in the older than in the younger rats. Concentration-response curves gave similar EC50-values for both age-groups but a 1·6-fold higher maximal response (expressed as vitamin K1-epoxide/vitamin K1 ratios) in the older rats. Since OAD's exert their anticoagulant effect most probably by inhibiting an enzyme (vitamin K1-epoxide reductase) which regenerates vitamin K1 from the epoxide metabolite and since the vitamin K1-epoxide/vitamin K1 ratios in the liver may reflect the degree of inhibition of the epoxide reductase by OAD's, our results may indicate that the inhibitory effect of phenprocoumon on this enzyme is more pronounced in older than in younger rats. This could explain the age-dependent differences in the anticoagulant activity.

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.

Differential Effects of Vitamin K1 on AFP and DCP Levels in Patients with Unresectable HCC and in HCC Cell Lines

DCP is a useful HCC tumor marker, which reflects a defect in vitamin K metabolism. We tested the hypothesis that vitamin K treatment of HCC patients might suppress this marker and possibly AFP also. HCC patients who had both elevated AFP and DCP were included. A phase I cohort was treated with escalating vitamin K1 intravenous weekly doses and a 27-patient phase II cohort was then treated with a fixed oral daily dose. A maximum tolerated dose was not reached up to 100-fold the normal vitamin K1 dose. No toxicities were found up to 1,000 mg/infusion. In the phase II cohort, 93% of patients had tumor marker responses by decreased DCP levels, but only 22% had responses by decreased AFP levels. CT scans showed 11% of patients had PRs, 59% had stable tumors and 29.6% had tumor progression. Mechanism studies showed that vitamin K1 induced phosphorylation of JNK and c-Jun and caspase-mediated apoptosis. Vitamin K1 was non-toxic at high doses, strongly inhibited plasma DCP levels, but weakly suppressed AFP levels. The results provide evidence that the two tumor markers are not directly linked and that DCP levels may not reflect HCC cell growth, as DCP levels were decreased in patients without AFP change, and were suppressed in vitro at 1% of the vitamin K1 concentration needed to inhibit AFP.

Plasma vitamin K concentration in horses supplemented with several vitamin K homologs1

The effect of several vitamin K homologs on plasma vitamin K concentration was determined to assess their potential as a vitamin K supplement for adult horses. Sixteen Thoroughbred horses consisting of 8 mares and 8 geldings, aged 8.4 ± 3.6 yr and weighing 520.8 ± 36.1 kg, were allocated to 4 groups (n = 4). Each group was given phylloquinone, menaquinone-4, or menadione at 58 µmol/d, or no vitamin K supplement for 7 d. Plasma samples were collected before feeding, and 2, 4, and 8 h after feeding on d 7, and plasma concentrations of phylloquinone and menaquinone-4 were determined. Plasma phylloquinone concentration was greater in the phylloquinone group than in the other groups (P < 0.001). The phylloquinone concentration quadratically increased (P < 0.001) after feeding in the phylloquinone group but no changes in the plasma phylloquinone concentration were observed after feeding in the other groups. Plasma menaquinone-4 concentration was greater (P < 0.001) in the menadione group than the other groups, including the menaquinone-4 group. Menaquinone-4 concentration did not change (P = 0.192) after feeding in each group. Menaquinone-4 has been considered the most potent vitamin K homolog for bone metabolism; therefore, the present experiment indicates that menadione is a good source of vitamin K for bone health in horses because it is the only vitamin K homolog that increased the plasma concentrations of menaquinone-4.

Vitamin K status in spaceflight and ground-based models of spaceflight.

Bone loss is a well-documented change during and after long-duration spaceflight. Many types of countermeasures to bone loss have been proposed, including vitamin K supplementation. The objective of this series of studies was to measure change in vitamin K status in response to microgravity under a variety of spaceflight and spaceflight analog (model) conditions, including long-duration spaceflight studies (n = 15), three bed rest studies (n = 15, 49, and 24), and a 14-day saturation dive (n= 6). In crew members who flew 2–6 months on the International Space Station, in-flight and postflight plasma phylloquinone concentrations were unchanged from the preflight mean. Consistent with this finding, urinary γ-carboxyglutamic acid (GLA), a measure of vitamin K-dependent protein turnover, did not change in response to flight. Serum undercarboxylated osteocalcin (%ucOC), a measure of vitamin K function, was generally unchanged in response to flight. Spaceflight findings were corroborated by findings of no changes in phylloquinone, urinary GLA, or %ucOC during or after bed rest in three separate bed rest studies (21–90 days in duration) or after a 14-day saturation dive. The data presented here do not support either a need for vitamin K supplementation during spaceflight or the suggestion of using vitamin K as a bone loss countermeasure in spaceflight. © 2011 American Society for Bone and Mineral Research.

Effect of oseltamivir treatment on anticoagulation: a cross‐over study in warfarinized patients

To investigate whether oseltamivir enhances the anticoagulant effect of warfarin and to evaluate any pharmacokinetic (PK) interaction between the agents. Twenty volunteers (mean age 62 years) receiving daily warfarin and with INR values of 2.0-3.5 during the previous 2 weeks were randomized to concomitant oseltamivir 75 mg twice daily for 4.5 days or warfarin alone in a two-way cross-over design with a 4-8 day wash-out. Anticoagulant effects were assessed by calculating overall [AUEC(0,96 h)] and observed maximum effect (E(max) ) increase from baseline in INR, decrease from baseline in factor VIIa, and change in vitamin K₁ concentrations. Plasma pharmacokinetics of (R)- and (S)-warfarin and oseltamivir were also assessed. For both treatments, changes in INR and factor VIIa during treatment were small; for net AUEC(0,96 h), least square mean values were -9.53 (oseltamivir + warfarin) and -1.69 h (warfarin alone) for INR (difference -7.84 h, 90% CI -18.86, 3.17 h), and 1.56 and 0.54 kIU l⁻¹ h, respectively, for factor VIIa (difference, 1.01 kIU l⁻¹ h; 90% CI -1.18, 3.21). Differences between the treatments in E(max) increase from baseline for INR, decrease from baseline for factor VIIa and change from baseline in vitamin K₁ concentration were not statistically significant. Oseltamivir did not alter warfarin pharmacokinetics. Oseltamivir was well tolerated in this study with no clinically significant adverse safety findings. Concomitant administration of oseltamivir for 4.5 days to volunteers on daily warfarin had little or no effect on warfarin pharmacokinetics and no effect on pharmacodynamics.

Influence of dietary menadione nicotinamide bisulphite (vitamin K3) and phylloquinone (vitamin K1) on Atlantic salmon (Salmo salar L.) tissue levels, determined by high-performance liquid chromatography with fluorescence detection

The aim of the present study was to investigate the retention of menadione nicotinamide bisulphite (MNB; vitamin K3) and phylloquinone (vitamin K1) in Atlantic salmon (Salmo salar L.). Another objective was to find a reliable method for determination of menadione in fish feed, and to include and validate more matrices in the methods for phylloquinone and menaquinones (vitamin K2). Duplicate tanks of Atlantic salmon (∼93 g) were fed four levels (0–1000 mg menadione kg−1 feed) of MNB for 9 weeks. The concentration of menadione and phylloquinone in the feed and the concentration of phylloquinone and menaquinone-4 (MK-4) in the tissues were determined. The analysed concentration of dietary menadione found in feed indicated a substantial loss of MNB during feed production. This assumption was supported by screening 15 commercial fish feed samples which also revealed menadione concentrations far below the recommended level. MNB fed salmon showed only a minor increase in liver MK-4 concentration, compared to salmon fed phylloquinone which had a considerably higher level of liver phylloquinone, indicating a higher retention of phylloquinone compared to menadione in Atlantic salmon. Due to highly varying stability and bioavailability of the different vitamin K derivatives, vitamin K supplementation in fish feed needs a revision.

Vitamin K epoxide reductase (VKORC1) gene mutations in osteoporosis: A pilot study

Susceptibility to osteoporosis seems to be influenced genetically. Previous studies on the effects of genetic polymorphisms on bone mineral density (BMD) showed controversial results. Vitamin K hydrochinon is an important cofactor for gamma carboxylation of osteocalcin. The reduction of vitamin K to vitamin K hydrochinon depends on the vitamin K epoxide reductase complex subunit 1 (VKORC1). We evaluated the impact of polymorphisms in VKORC1 on BMD and fractures. In this single-center study, 184 individuals (141 female subjects and 43 male subjects, mean age: 63.2 +/- 14.3 years) were recruited. In all, 149 of 184 could be genotyped by allele-specific polymerase chain reaction (PCR) for the VKORC1 variants 3673G>A or 9041G>A. The genotypes were correlated with clinical parameters. Vitamin K(1) concentrations were determined by high-performance liquid chromatography (HPLC); carboxylated (GlaOC) and undercarboxylated osteocalcin (GluOC) was determined by enzyme-linked immunosorbent assays (ELISAs). The 9041 GG and GA genotypes were significantly more frequent in patients with low BMD (P = 0.012). Thus, carriers of at least 1 G-allele seem to have a higher risk for low BMD. No statistically significant association was found for the 3673 G>A variant and BMD. GluOC concentrations were higher in patients who carried a 3673 GA and GG genotypes (P = 0.07). For both variants, no association with fractures could be observed. In our cohort, a genetic variation in the 3'-region of the VKORC1 gene (9041 AG and GG) was associated significantly with low BMD. This finding suggests that VKORC1 may play a role in osteoporosis. The results of our pilot study should be confirmed as our findings may be important for treatment decisions.

Adulthood Obesity Is Positively Associated with Adipose Tissue Concentrations of Vitamin K and Inversely Associated with Circulating Indicators of Vitamin K Status in Men and Women1–3

Increased adiposity is associated with increased storage of several fat-soluble nutrients. However, the extent to which vitamin K is stored in fat and the association between vitamin K status and adiposity are unknown. Our objectives in this study were to determine whether vitamin K is stored in human adipose tissue and the association between vitamin K status and body fat in older men and women. In study A, the vitamin K concentration of subcutaneous and visceral adipose tissue was quantified in samples taken from 16 gastric bypass patients [13 women, 3 men, age 40 ± 10 y (mean ± SD)] using HPLC. In study B, cross-sectional associations between percent body fat (%BF) and circulating measures of vitamin K status were examined in 260 women and 183 men [age = 68 ± 5 y]. The phylloquinone (K1) concentrations in subcutaneous and visceral adipose tissue were 148.2 ± 71.8 and 175 ± 112 nmol/kg, respectively, which is higher than the reported concentrations of other organs known to store vitamin K. There was an inverse association between %BF and plasma K1 in women (P-trend < 0.001). Higher %BF was associated with greater circulating concentrations of uncarboxylated prothrombin, indicative of lower hepatic utilization of vitamin K in both men (P-trend = 0.02) and women (P-trend = 0.002) but not with the percentage of undercarboxylated osteocalcin. Adipose tissue contained high concentrations of vitamin K, and increased adiposity was associated with poorer vitamin K status in older adults. Additional studies are needed to further explore the relationships among body fat, storage of vitamin K in adipose tissue, and implications for vitamin K status and function.

Abstract 5407: Inhibitory Raf-1 phosphorylations at Ser-43 and Ser-259 are induced in vitamin K1 enhancement of Sorafenib-mediated HCC cell growth inhibition

Abstract Sorafenib, a multi-kinase inhibitor, is an FDA-approved agent for treatment of human hepatocellular carcinoma (HCC). However, tumor shrinkage is minor. K vitamins can activate protein kinase A (PKA), which in turn can mediate inhibitory Raf phosphorylation. We developed a new strategy to combine naturally occurring K vitamins with Sorafenib to treat HCC, and found that K vitamins were able to enhance the inhibition of HCC cell growth in vitro and in vivo by Sorafenib. To explore the mechanisms involved, we examined the role of Raf-1 kinase, because it has been reported that Sorafenib inhibits tumor cell growth as a direct inhibitor of Raf, whereas vitamin K2 can induce PKA activity, which is a Raf-1 upstream kinase. We found that while lower doses of vitamin K1 (10 μM) or Sorafenib (2.5 μM) alone slightly induced Raf-1 phosphorylation at both Ser-43 and Ser-259 in Hep3B cells, combination vitamin K1 plus Sorafenib treatment resulted in strong Raf-1 phosphorylation at these two Raf-inhibitory serine residues. Raf-1 protein kinase activity assay showed that lower concentrations of vitamin K1 or Sorafenib alone had little, if any effects on Raf-1 activity, combination vitamin K1 plus Sorafenib at the same low concentrations significantly inhibited Raf-1 activity, judged by strong inhibition of MEK, and of ERK phosphorylation, its down stream targets. Since Raf-1 phosphorylation at Ser-43 and Ser-259 can be regulated by either PKA or Akt kinase, we examined the effects of both vitamin K1 and Sorafenib on their phosphorylation. Although both vitamin K1 and Sorafenib alone induced PKA phosphorylation, no synergistic phosphorylation effects on PKA were found using the combination. However, vitamin K1 enhanced Sorafenib-induced Akt phosphorylation, which was associated with enhanced c-Met tyrosine phosphorylation at Tyr-1349, a known phospho-Akt regulator. Our data suggest that enhanced inhibitory Raf-1 phosphorylation at Ser-43 and Ser-259 may play a central role in vitamin K1 plus Sorafenib synergy in inhibiting HCC cell growth. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5407.

Influence of dietary weight‐loss on vitamins D and K in obese post‐menopausal women

Obese persons are at risk for fat‐soluble vitamin (FSV) insufficiency. It is also plausible that weight loss is associated with change in FSV status. Hence, our aim was to examine associations between change in weight status and change in vitamin D and K status in 89 post‐menopausal obese women (mean±SD BMI=33.1±3.6kg/m2, age=58±6y), following a hypocaloric weight‐loss program. All women received 200IU/d vitamin D3 and 80 mcg/d vitamin K1. On average, baseline serum 25‐hydroxyvitamin‐D (25(OH)D) and vitamin K1 concentrations were within normal ranges (51±18nmol/L, 1.3±1.0nmol/L respectively). After 20‐weeks, women lost 11.1±5.3% of initial body weight and 8.0±3.2kg of fat mass. 25(OH)D increased by 10±14nmol/L (p&lt;0.01, season‐adjusted), but serum vitamin K1 did not change (p=0.85, triglyceride‐adjusted). Using linear models, with change in FSV status as outcomes and change in weight‐status as primary exposures in separate analyses, there was no association between change in 25(OH)D and change in weight, BMI, or fat mass [unstd B(p‐value)= −0.51(0.15), −0.98(0.32), −0.60(0.19) respectively] or between change in serum K1 and change in weight, BMI, or fat mass [unstd B(p‐value)= −0.04(0.17), −0.10(0.16), −0.06(0.10) respectively]. Our data do not suggest moderate weight loss is associated with change in serum measures of vitamin D or K. Supported by NIA R01AGDK20583, P30AG21332 &amp; USDA ARS #58‐1950‐7707.

Quantitative determination of plasma vitamin K1 by high-performance liquid chromatography coupled to isotope dilution tandem mass spectrometry

Plasma vitamin K1 (phylloquinone) determination is commonly used for the diagnosis of vitamin K deficiency in patients suffering from lipid malabsorption. Moreover, current evidence that adequate vitamin K intake, and correspondingly adequate plasma vitamin K1 concentration, could also be of importance in relation to bone and brain diseases emphasizes the need to improve the current analytical methods. We developed a liquid chromatography coupled to tandem mass spectrometry method using a stable isotope ring-D4-labeled internal standard of vitamin K1 and operating in the multiple reaction monitoring mode by the selection of a precursor and product ions. The atmospheric pressure chemical ionization (APCI) method was shown to be more sensitive than electrospray ionization. After a single-step extraction with cyclohexane, chromatographic separation was performed on a C18 column with an isocratic mobile phase. The linearity was up to 5400 ng/L, and the limit of detection was 14 ng/L. Intra- and interrun precision were 2.4% and 8.3%, respectively, for the lower limit of the reference range. Recovery was better than 98%. The method is simple and reliable, allowing accurate vitamin K1 measurement in plasma samples from healthy subjects and patients suffering from vitamin K deficiency.

Absorption of vitamin K2 by dogs after oral administration of a soft gelatin capsule formulation containing a new emulsion-type vehicle.

This study has evaluated the performance of a newly developed vehicle for administration of a drug in a soft gelatin capsule. The absorption of vitamin K2 in dogs after oral administration of the vitamin in a soft gelatin capsule containing the newly developed vehicle was compared with absorption after administration of a control formulation prepared by encapsulating the contents of a commercially available vitamin K2 capsule (Glakay capsules 15 mg) in the same type of soft gelatin. Under non-fasted conditions the profile of the plasma concentration of vitamin K2 against time for the test formulation was comparable with that for the control formulation in non-fasted dogs. Under fasted conditions, however, both the maximum concentration (Cmax) and the area under the plot of concentration against time (AUC) were significantly smaller for the test formulation than for the control formulation. The Cmax and AUC for the test formulation were about 10 times larger for non-fasted dogs than for fasted dogs whereas values for the control formulation were about twice as large. These results suggest that both formulations might require the presence of food or digestive fluid components, or both, for better absorption of vitamin K2. It seems that although the performances of the test and control formulations were comparable in the presence of these components, the control formulation works better in their absence. It should be also noted that, in contrast with the results from the absorption tests, the dispersibility of the test vehicle in water was much better than that of the control vehicle. This suggests that dispersibility does not significantly affect vitamin K2 absorption. In conclusion, although the new vehicle did not perform better than the control vehicle in terms of vitamin K2 absorption, the performance of the control formulation was comparable for non-fasted dogs. Because the new vehicle contains considerably less surfactant than the vehicles currently used in soft gelatin capsules, it could be a safer alternative for use under non-fasted conditions.

The effect of different meals on the absorption of stable isotope-labelled phylloquinone.

Few studies have investigated the absorption of phylloquinone (vitamin K1). We recruited twelve healthy, non-obese adults. On each study day, fasted subjects took a capsule containing 20 microg of 13C-labelled phylloquinone with one of three meals, defined as convenience, cosmopolitan and animal-oriented, in a three-way crossover design. The meals were formulated from the characteristics of clusters identified in dietary pattern analysis of data from the National Diet and Nutrition Survey conducted in 2000-1. Plasma phylloquinone concentration and isotopic enrichment were measured over 8 h. Significantly more phylloquinone tracer was absorbed when consumed with the cosmopolitan and animal-oriented meals than with the convenience meal (P = 0.001 and 0.035, respectively). Estimates of the relative availability of phylloquinone from the meals were: convenience meal = 1.00; cosmopolitan meal = 0.31; animal-oriented meal = 0.23. Combining the tracer data with availability estimates for phylloquinone from the meals provides overall relative bioavailability values of convenience = 1.00, cosmopolitan = 0.46 and animal-oriented = 0.29. Stable isotopes provide a useful tool to investigate further the bioavailability of low doses of phylloquinone. Different meals can affect the absorption of free phylloquinone. The meal-based study design used in the present work provides an approach that reflects more closely the way foods are eaten in a free-living population.

Measurement of Deuterium-Labeled Phylloquinone in Plasma by High-Performance Liquid Chromatography/Mass Spectrometry

Phylloquinone (vitamin K(1)) is a lipophilic compound present in plasma at low concentrations, which presents technical challenges for determining its bioavailability or metabolic fate using stable isotopes. We developed a method to simultaneously measure unlabeled and deuterium-labeled phylloquinone concentrations in plasma specimens using high-performance liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization (LC-APCI/MS). Phylloquinone was extracted from plasma using hexane, further purified by solid-phase extraction, and then quantified using high-performance liquid chromatography with an APCI/MS as a detector. Plotting the expected versus the measured amount of serial dilutions of either unlabeled or labeled phylloquinone gave correlation coefficients (R) of 0.999 for both compounds. The minimum detectable concentrations of unlabeled and labeled phylloquinone were 0.05 and 0.08 pmol/injection, respectively. Pooled plasma samples spiked with between 0.5 and 32 nmol phylloquinone/L gave average recoveries of 96.7% with 5.4% relative standard deviation (RSD) for unlabeled phylloquinone and 96.2% with 6.6% RSD for labeled phylloquinone. Plasma phylloquinone concentrations determined by LC-fluorescence and LC-APCI/MS methods from healthy subjects (n = 17) were not statistically different (P = 0.13). The LC-APCI/MS method is a sensitive technique for simultaneous determination of both unlabeled and labeled phylloquinone and can be applied to bioavailability studies.

The external quality assurance of phylloquinone (vitamin K1) analysis in human serum

The vitamin K external quality assurance scheme (KEQAS) aims to assist in the harmonization of phylloquinone (vitamin K(1)) analysis in order to improve the comparability of clinical and nutritional studies. Serum samples were despatched to 17 groups from eight countries during 2000-2006. Using pilot data (1996-1999), an analytical performance target of 20% absolute difference from the all-laboratory trimmed mean (ALTM) was assigned and formed the basis for interlaboratory comparison. Assay specificity, analytical bias and assay performance were evaluated. From 21 batches of samples distributed, 414 results were reported of which 2.7% were outliers. The mean interlaboratory absolute difference from the ALTM was 21.7% with 47% of groups consistently meeting the performance target. The mean interlaboratory coefficient of variation was 29.6%. The false positive rate for phylloquinone depleted samples was high at 35%. Bias was found to be independent of HPLC-detector type (fluorescence vs electrochemical). Assay characteristics for the measurement of phylloquinone in human serum compare favourably with methods for analytes at equivalent concentrations. The high proportion of false positive results suggest that poor assay specificity at low phylloquinone concentrations is a common problem, which in the clinical setting could lead to underreporting of vitamin K deficiency.