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The health-promoting properties of vitamin K stimulate the growing interest in this compound, which translates into the development of new analytical methodologies for its determination. New, more efficient methods of its isolation are sought, paying increasingly more attention to the methods within currently available extraction techniques that, owing to the optimization of the process, not only increase the extraction efficiency but are also economical and environmentally friendly. This article proposes a procedure for the extraction and analysis of one of the vitamin K vitamers, i.e., vitamin K1, using PLE and LC-MS/MS. It has been shown that the PLE technique can be optimized with a mathematical model-accelerating and reducing the costs of the extraction process-which, together with process automation, bodes well for industrial applications. The optimized process was used to extract vitamin K1 from various vegetables, showing very different contents of the test compound ranging from 1.22 to 114.30 µg/g dry weight for avocado and spinach, respectively. In addition, by showing the effect of water within the material subjected to extraction on the variable yield of vitamin K1, attention was drawn to the need to standardize the analytical methods used in assessing the quality of food products.

The possibility of controlling periorbital hyperpigmentation disorders is one of the most important research goals in cosmetic preparations. In the current investigation, 1% vitamin K (Vit K) was incorporated into a Chitosan/alginate hydrogel which aimed to increase the dermal delivery and anti-pigmentation effect. The Vit K-hydrogel was evaluated using several different tests, including volume expansion/contraction analysis, differential scanning calorimetry (DSC), scanning electron microscopy (SEM), ultraviolet (UV) absorbance spectroscopy, and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Vit K hydrogel's drug release profile showed a steady increase over time. Furthermore, the modified Vit K hydrogel formulations showed no harmful effects in an in vitro cytotoxicity study. The Vit K hydrogel was tested for dermal irritation on Wistar rats, and the hydrogel was found to be non-irritating. Furthermore, Vit K-hydrogel inhibited melanin formation (31.76 ± 1.14%) and was remarkably higher than free Vit K. In addition, Vit K-hydrogel inhibited L-dopa auto-oxidation to a greater extent (94.80 ± 2.41%) in comparison with Vit K solution (73.95 ± 1.62%). Vit K-hydrogel enhanced percutaneous transport of Vit K, according to in vitro percutaneous absorption findings, suggesting that this innovative formulation may provide new therapeutic options for periorbital hyperpigmentation.

Determination of the various forms of vitamin K, which are involved in coagulation and other physiological processes in humans, is challenging and no standardized method is yet available. Therefore, a reliable and practical method was developed to quantify vitamin K levels in serum and additionally in lipoprotein fractions to clarify its distribution. The LC-MS/MS method for the determination of vitamin K 1 and the three main isoforms of vitamin K 2 (MK-4, MK-7, MK-9) was combined with a gradient ultracentrifugation technique to allow the separation of lipoprotein fractions. The chromatographic separation was carried out on a Kinetex™ C18 column using a mobile phase consisting mainly of methanol. The target analytes were detected by electrospray ionization mass spectrometry. The separation of all four substances was achieved after a simple sample preparation technique based on miniaturized liquid-liquid extraction. Our method of only 8.5 min revealed the levels of the major forms of vitamin K in 59 human and 12 rat sera and confirmed our hypothesis that vitamin K is primarily (about 50 %) found in the high-density lipoprotein fraction. The median concentrations of vitamin K 1 , MK-4, MK-7, and MK-9 were found to be 1.19, 2.98, 0.43, and < 0.71 nmol/L in human serum and 1.74, 6.75, less than 0.2, and less than 0.5 nmol/L in rat serum, respectively., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

[Q A - - Q A ] Fourier transform infrared difference spectra have previously been obtained using purple bacterial reaction centers from Rhodobacter sphaeroides with unlabeled, 18 O and 13 C isotope labeled phylloquinone (PhQ, also known as vitamin K 1 ) incorporated into the Q A protein binding site (Breton, (1997), Proc. Natl. Acad. Sci. USA94 11318-11323). The nature of the bands in these spectra and the isotope induced band shifts are poorly understood, especially for the phyllosemiquinone anion (PhQ - ) state. To aid in the interpretation of the bands in these experimental spectra, ONIOM type QM/MM vibrational frequency calculations were undertaken. Calculations were also undertaken for PhQ - in solution. Surprisingly, both sets of calculated spectra are similar and agree well with the experimental spectra. This similarity suggests pigment-protein interactions do not perturb the electronic structure of the semiquinone in the Q A binding site. This is not found to be the case for the neutral PhQ species in the same protein binding site. PhQ also occupies the A 1 protein binding site in photosystem I, and the vibrational properties of PhQ - in the Q A and A 1 binding sites are compared and shown to exhibit considerable differences. These differences probably arise because of changes in the degree of asymmetry of hydrogen bonding of PhQ - in the A 1 and Q A binding sites., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gary Hastings reports financial support was provided by US Department of Energy Basic Energy Sciences., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Vitamin K is an essential lipophilic vitamin that acts as a coenzyme in several metabolic pathways. Accurate measurement of apolar metabolites transported by lipoproteins in serum matrices requires high-recovery extractions of vitamin K and its derivatives following standardized protocols. Conventionally developed methods in this field have predominantly employed solid-phase extraction for the measurement of vitamin K and its derivatives. In this study, our objective was to develop an enzyme-assisted extraction method for the precise measurement of vitamin K and its derivatives. Our methodology involved mixing 450 µL of serum samples with 50 µL of an internal standard and 50 µL of a lipase enzyme solution. Following vortexing, the mixture was incubated at 37°C for 15 min to activate the enzymes. The enzyme reaction was subsequently quenched with a mixture of 250 µL of methanol and 1 mL of hexane, followed by centrifugation at 12,000  g for 5 min. The upper phase was collected, concentrated using a concentrator device, and dissolved in a 100 µL solution of methanol/acetone/isopropanol (7:1:1, v/v/v) for analysis. Spectrum analysis was performed using the open-source MZmine 3 software, and a reference interval was established using the Python programming language on the Google Colab platform. The developed method for measuring vitamin K and its derivatives exhibited limit of detection and limit of quantitation values of 0.005 and 0.01 ng/mL, respectively. In conclusion, our study presents a precise and reliable method for the measurement of vitamin K and its derivatives using enzyme-assisted extraction.

Vitamin K is an essential micronutrient required for blood coagulation, regulation of vascular calcification and bone mineralization. Plasma and serum measurements of vitamin K 1 (phylloquinone, K 1 ) made using high-performance liquid chromatography with fluorescence detection, or tandem mass spectrometry are used clinically and in population studies to assess vitamin K status. Standard reference materials provide a validation tool for laboratories, helping assure clinical diagnosis and the comparability of data from different populations. We manufactured two K 1 standard reference materials, in 2009 (KEQAS SRM-001) and in 2019 (KEQAS SRM-002). The target concentrations of K 1 were assigned to each SRM using the All Laboratory Trimmed Mean of results reported by selected laboratories enrolled in the Vitamin K External Quality Assurance Scheme (KEQAS). The assigned concentrations of K 1 for KEQAS SRM-001 and SRM-002 were 0.25 and 0.36 μg/L respectively. In 2019 KEQAS SRM-001 was re-analysed simultaneously with KEQAS SRM-002 to provide traceability between the two standards, therefore aiding comparability of analysis performed using these materials. Both standards were stored as aliquots at -80°C in the dark; annual re-analysis of the materials indicated that K 1 is stable for at least 12 years in these conditions., (© 2022 John Wiley & Sons, Ltd.)

Photosystem I (PS I) has a symmetric structure with two highly similar branches of pigments at the center that are involved in electron transfer, but shows very different efficiency along the two branches. We have determined the structure of cyanobacterial PS I at room temperature (RT) using femtosecond X-ray pulses from an X-ray free electron laser (XFEL) that shows a clear expansion of the entire protein complex in the direction of the membrane plane, when compared to previous cryogenic structures. This trend was observed by complementary datasets taken at multiple XFEL beamlines. In the RT structure of PS I, we also observe conformational differences between the two branches in the reaction center around the secondary electron acceptors A 1A and A 1B . The π-stacked Phe residues are rotated with a more parallel orientation in the A-branch and an almost perpendicular confirmation in the B-branch, and the symmetry breaking PsaB-Trp673 is tilted and further away from A 1A . These changes increase the asymmetry between the branches and may provide insights into the preferential directionality of electron transfer., (© 2021. The Author(s).)

Vitamin K epoxide reductases (VKORs) constitute a major family of integral membrane thiol oxidoreductases. In humans, VKOR sustains blood coagulation and bone mineralization through the vitamin K cycle. Previous chemical models assumed that the catalysis of human VKOR (hVKOR) starts from a fully reduced active site. This state, however, constitutes only a minor cellular fraction (5.6%). Thus, the mechanism whereby hVKOR catalysis is carried out in the cellular environment remains largely unknown. Here we use quantitative mass spectrometry (MS) and electrophoretic mobility analyses to show that KO likely forms a covalent complex with a cysteine mutant mimicking hVKOR in a partially oxidized state. Trapping of this potential reaction intermediate suggests that the partially oxidized state is catalytically active in cells. To investigate this activity, we analyze the correlation between the cellular activity and the cellular cysteine status of hVKOR. We find that the partially oxidized hVKOR has considerably lower activity than hVKOR with a fully reduced active site. Although there are more partially oxidized hVKOR than fully reduced hVKOR in cells, these two reactive states contribute about equally to the overall hVKOR activity, and hVKOR catalysis can initiate from either of these states. Overall, the combination of MS quantification and biochemical analyses reveals the catalytic mechanism of this integral membrane enzyme in a cellular environment. Furthermore, these results implicate how hVKOR is inhibited by warfarin, one of the most commonly prescribed drugs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In our previous study, an antimutagenic compound from spinach (Spinacea oleracea L.), ethoxy-substituted phylloquinone (ESP) was isolated and characterized. The current study deals with elucidation of the possible mechanism of antimutagenicity of ESP against ethyl methanesulfonate (EMS) deploying model systems such as human lymphoblast (TK +/- or TK6) cell line (thymidine kinase gene mutation assay) and Escherichia coli MG1655 (rifampicin resistance assay). Findings of the study ruled out the possibility of direct inactivation of EMS by ESP. DAPI competitive binding assay indicated the DNA minor groove binding activity of ESP. Interestingly, ESP did not display major groove binding or intercalating abilities. Further, proteomics study using 2-D gel electrophoresis in E. coli and subsequent studies involving single gene knockout strains revealed the possible role of tnaA (tryptophanase) and dgcP (diguanylate cyclase) genes in observed antimutagenicity. These genes have been reported to be involved in indole and cyclic-di-GMP biosynthesis, respectively, which eventually lead to cell division inhibition. In case of TK +/- cell line system, ADCY genes (adenylate cyclase), a functional analogue of dgcP gene, were found to be transcriptionally up-regulated. The generation/doubling time were significantly higher in E. coli or TK +/- cells treated with ESP than control cells. The findings indicated inhibition of cell proliferation by ESP through gene regulation as a possible mechanism of antimutagenicity across the biological system. Cell division inhibition actually provides additional time for the repair of damaged DNA leading to antimutagenicity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The vitamin K epoxide reductase (VKORC1) enzyme is of primary importance in many physiological processes, i.e., blood coagulation, energy metabolism, and arterial calcification prevention, due to its role in the vitamin K cycle. Indeed, VKORC1 catalyzes reduction of vitamin K epoxide to quinone and then to hydroquinone. However, the three-dimensional VKORC1 structure remains experimentally undetermined, because of the endoplasmic reticulum membrane location of this enzyme. Here we present a molecular modeling investigation of the VKORC1 enzymatic site structure and function, supported by in vitro enzymatic assays. Four VKORC1 mutants were designed in silico (F55G, F55Y, N80G, and F83G) based on a previous study that identified residues F55, N80, and F83 as being crucial for vitamin K epoxide binding. F55G, N80G, and F83G nonconservative mutants were all predicted to be inactive by molecular modeling analyses. However, the F55Y conservative mutant was expected to be active compared to wild-type VKORC1. In vitro enzymatic assays performed on recombinant proteins assessed our molecular modeling hypotheses and led us to describe the role of accurate VKORC1 active site residues with respect to VKORC1. Residues F55, N80, and F83 appeared to act in a concerted manner to keep vitamin K epoxide close to the C135 catalytic residue. Residues F55 and N80 prevent naphthoquinone head rotation away from the active site, assisted by residue F83 that prevents vitamin K from sliding outside the enzymatic pocket, through hydrophobic tail stabilization. Our results thus highlighted the specific functions of VKORC1 catalytic pocket residues and evidenced the ability of our structural model to predict biological effects of VKORC1 mutations.

Vitamin K 1 is one of the important hydrophobic vitamins in fat-containing foods. Traditionally, lipase is employed in the determination of vitamin K 1 to remove the lipids, which makes the detection complex, time-consuming, and insensitive. In this study, the determination of vitamin K 1 in fat-containing foods was developed based on ultrasound-assisted extraction (UAE), solid-phase extraction (SPE) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The optimal conditions for extraction of vitamin K 1 were material-liquid ratio of 1:70 (g/mL), extraction temperature of 50 °C, extraction power of 700 W, extraction time of 50 min, material-wash fluid ratio of 1:60 (g/mL), and 8 mL of hexane/anhydrous ether (97:3, v / v ) as the elution solvent. Then, vitamin K 1 was analyzed on a ZORBAX SB-C18 column (50 mm × 2.1 mm, 1.8 μm) by gradient elution with water (0.01% formic acid) and methanol (0.01 formic acid + 2.5 mmol/L ammonium formate) as the mobile phase. The limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.16 μg/kg, respectively. Calibration curve was linear over the range of 10-500 ng/mL ( R 2 > 0.9988). The recoveries at three spiked levels were between 80.9% and 119.1%. The validation and application indicated that the proposed method was simple and sensitive in determination of vitamin K 1 in fat-containing foods.

Vitamin K 1 and vitamin K 2 play very important biological roles as members of chains of electron transport as antioxidants in membranes and as cofactors for the posttranslational modification of proteins that participate in a number of physiological functions such as coagulation. The interaction of these vitamins with dimyristoylphosphatidylcholine (DMPC) model membranes has been studied by using a biophysical approach. It was observed by using differential scanning calorimetry that both vitamins have a very limited miscibility with DMPC and they form domains rich in the vitamins at high concentrations. Experiments using X-ray diffraction also showed the formation of different phases as a consequence of the inclusion of either vitamin K at temperatures below the phase transition. However, in the fluid state, a homogeneous phase was detected, and a decrease in the thickness of the membrane was accompanied by an increase in the water layer thickness. 2 H NMR spectroscopy showed that both vitamins K induced a decrease in the onset of the phase transition, which was bigger for vitamin K 1 , and both vitamins decreased the order of the membrane as seen through the first moment ( M 1 ). 1 H NOESY MAS-NMR showed that protons located at the rings or at the beginning of the lateral chain of both vitamins K interacted with a clear preference with protons located in the polar part of DMPC. On the other hand, protons located on the lateral chain have a nearer proximity with the methyl end of the myristoyl chains of DMPC. In agreement with the 2 H NMR, ATR-FTIR (attenuated total reflectance Fourier transform infrared spectroscopy) indicated that both vitamins decreased the order parameters of DMPC. It was additionally deduced that the lateral chains of both vitamins were oriented almost in parallel to the myristoyl chains of the phospholipid.

The primary objective of this review is to propose an approach for the biosynthesis of phylloquinone (vitamin K 1 ) based upon its known sources, its role in photosynthesis and its biosynthetic pathway. The chemistry, health benefits, market, and industrial production of vitamin K are also summarized. Vitamin K compounds (K vitamers) are required for the normal function of at least 15 proteins involved in diverse physiological processes such as coagulation, tissue mineralization, inflammation, and neuroprotection. Vitamin K is essential for the prevention of Vitamin K Deficiency Bleeding (VKDB), especially in neonates. Increased vitamin K intake may also reduce the severity and/or risk of bone fracture, arterial calcification, inflammatory diseases, and cognitive decline. Consumers are increasingly favoring natural food and therapeutic products. However, the bulk of vitamin K products employed for both human and animal use are chemically synthesized. Biosynthesis of the menaquinones (vitamin K 2 ) has been extensively researched. However, published research on the biotechnological production of phylloquinone is restricted to a handful of available articles and patents. We have found that microalgae are more suitable than plant cell cultures for the biosynthesis of phylloquinone. Many algae are richer in vitamin K 1 than terrestrial plants, and algal cells are easier to manipulate. Vitamin K 1 can be efficiently recovered from the biomass using supercritical carbon dioxide extraction.

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.

Vitamin K family molecules-phylloquinone (K1), menaquinone (K2), and menadione (K3)-act as γ-glutamyl carboxylase (GGCX)-exclusive cofactors in their hydroquinone state, activating proteins of main importance for blood coagulation in the liver and for arterial calcification prevention and energy metabolism in extrahepatic tissues. Once GGCX is activated, vitamin K is found in the epoxide state, which is then recycled to quinone and hydroquinone states by vitamin K epoxide reductase (VKORC1). Nevertheless, little information is available concerning vitamin K1, K2, or K3 tissue distribution and preferential interactions towards VKORC1. Here we present a molecular modeling study of vitamin K1, menaquinones 4, 7 (MK4, MK7), and K3 structural interactions with VKORC1. VKORC1 was shown to tightly bind vitamins K1 and MK4 in the epoxide and quinone states, but not in the hydroquinone state; five VKORC1 residues were identified as crucial for vitamin K stabilization, and two other ones were essential for hydrogen bond formation. However, vitamin MK7 revealed shaky binding towards VKORC1, induced by hydrophobic tail interactions with the membrane. Vitamin K3 exhibited the lowest affinity with VKORC1 because of the absence of a hydrophobic tail, preventing structural stabilization by the enzyme. Enzymatic activity towards vitamins K1, MK4, MK7, and K3 was also evaluated by in vitro assays, validating our in silico predictions: VKORC1 presented equivalent activities towards vitamins K1 and MK4, but much lower activity with respect to vitamin MK7, and no activity towards vitamin K3. Our results revealed VKORC1's ability to recycle both phylloquinone and some menaquinones, and also highlighted the importance of vitamin K's hydrophobic tail size and membrane interactions.

One of the fundamental problems in biophysics is whether the protein medium at room temperature can be properly treated as a fluid dielectric or whether its dynamics is determined by a highly ordered molecular structure resembling the properties of crystalline and amorphous solids. Here, we measured the recombination between reduced A 1 and the oxidized chlorophyll special pair P 700 over a wide temperature range using preparations of photosystem I from the cyanobacterium Synechococcus sp. PCC 7002 depleted of the iron-sulfur clusters. We found that the dielectric properties of the protein matrix in early electron transfer reactions of photosystem I resemble the behavior of solids that require an implicit treatment of electron-phonon coupling even at ambient temperatures. The quantum effects of electron-phonon coupling in proteins could account for a variety of phenomena, such as the weak sensitivity of electron transfer in pigment-protein complexes to changing environmental conditions including temperature, driving force, polarity, and chemical composition.

The purpose of this study was to evaluate the stability of vitamin K1 oral liquids in Sterile Water for Injection when stored in amber glass bottles and amber plastic syringes under refrigerated conditions. Four 100-mL batches of vitamin K1 in Sterile Water for Injection were prepared in amber glass bottles to protect from light. One of the batches was divided into 1-mL aliquots, using amber plastic oral syringes, and capped. The prepared bottles and syringes were stored in a laboratory refrigerator. On each day of sampling, 1-mL aliquots were removed from each bottle and mixed with an equal volume of ethanol. Likewise, the contents of sample syringes were mixed with ethanol to achieve an assay concentration of 0.5 mg/mL. Recovery of vitamin K1 in the compounded samples was quantified against a United States Pharmacopeia reference standard. Quantification was achieved using a stability-indicating high-performance liquid chromatography with ultraviolent light detection method. Product stability is defined as 90% to 110% of the initial concentration. The percent recovery in the Sterile Water for Injection preparations in glass bottles remained above 90% for the 105-day duration of the study, but some samples stored in amber plastic syringes fell below 90% on day 21. Furthermore, a statistically significant difference (2-way ANOVA, P < 0.0001) emerged between syringes at day 0 and day 30, and this trend continued through the day 60, 90, and 105 samples. The only statistically significant difference found within the bottle-stored samples occurred on day 105 (versus zero, P = 0.0465), but the recovery on day 105 still exceeded 90%. Vitamin K1 in Sterile Water for Injection, stored in a refrigerated amber glass bottle, is stable for 105 days. This preparation can also be stored in amber plastic syringes, but this decreases the beyond-use date to 14 days., (Copyright© by International Journal of Pharmaceutical Compounding, Inc.)

Vitamin K was originally discovered as a cofactor required to activate clotting factors and has recently been shown to play a key role in the regulation of soft tissue calcification. This property of vitamin K has led to an increased interest in novel methods for accurate vitamin K detection. Molecularly Imprinted Polymers (MIPs) could offer a solution, as they have been used as synthetic receptors in a large variety of biomimetic sensors for the detection of similar molecules over the past few decades, because of their robust nature and remarkable selectivity. In this article, the authors introduce a novel imprinting approach to create a MIP that is able to selectively rebind vitamin K₁. As the native structure of the vitamin does not allow for imprinting, an alternative imprinting strategy was developed, using the synthetic compound menadione (vitamin K₃) as a template. Target rebinding was analyzed by means of UV-visible (UV-VIS) spectroscopy and two custom-made thermal readout techniques. This analysis reveals that the MIP-based sensor reacts to an increasing concentration of both menadione and vitamin K₁. The Limit of Detection (LoD) for both compounds was established at 700 nM for the Heat Transfer Method (HTM), while the optimized readout approach, Thermal Wave Transport Analysis (TWTA), displayed an increased sensitivity with a LoD of 200 nM. The sensor seems to react to a lesser extent to Vitamin E, the analogue under study. To further demonstrate its potential application in biochemical research, the sensor was used to measure the absorption of vitamin K in blood serum after taking vitamin K supplements. By employing a gradual enrichment strategy, the sensor was able to detect the difference between baseline and peak absorption samples and was able to quantify the vitamin K concentration in good agreement with a validation experiment using High-Performance Liquid Chromatography (HPLC). In this way, the authors provide a first proof of principle for a low-cost, straightforward, and label-free vitamin K sensor.

Owing to its limited aqueous solubility, Phytomenadione (vitamin K) undergoes a low bioavailability (50%) with a large inter-individual variability after oral administration. Therefore, the aim of this work was to incorporate vitamin K into nanostructure lipid carrier systems to improve its aqueous solubility and bioavailability. Phytomenadione was used as a liquid lipid; Precirol ATO5, and Compritol ATO were used as solid lipids; Labrasol and Cremophore EL as water soluble surfactants; Capryol 90 and Lauroglycol as lipid soluble surfactants. Eight formulas were prepared and characterized for their particle sizes, zeta potential, entrapment efficiencies, and drug release. Those formulas had particle sizes ranging from 25.4 to 68.3 nm. The best formula, consisting of 15% Phytomenadione, 45% Precirol ATO5, 30% Cremophore EL, and 10% Lauroglycol 90, was selected for stability study and characterized by the techniques mentioned above and scanning electron microscopy. It had the highest drug loading and an acceptable in vitro release profile (94.54% within 30 min). This formula was also chemically and physically stable, and it recorded a relative bioavailability of 645.5% in rabbits compared to the commercial conventional tablet. This formula could be a promising carrier regarding its ease of preparation, dosage form versatility and enhanced bioavailability.

A unique electron-accepting analog of vitamin K 1 found in photosystem I in several species of oxygenic photosynthetic microorganisms was confirmed to be 5'-hydroxyphylloquinone (1) through stereo-uncontrolled synthesis. Furthermore, the stereochemistry of 1 obtained from Synechococcus sp. PCC 7942 was assigned to be 5'S using proline-catalyzed stereocontrolled reactions.

Quinones serve as redox active cofactors in bacterial photosynthetic reaction centers: photosystem I, photosystem II, cytochrome bc 1 , and cytochrome b 6 f. In particular, ubiquinone is ubiquitous in animals and most bacteria and plays a key role in several cellular processes, e.g., mitochondrial electron transport. Their experimentally measured redox potential values for one-electron reduction E m (Q/Q ·- ) were already reported in dimethylformamide (DMF) versus saturated calomel electrode but not in water versus normal hydrogen electrode (NHE). We calculated E m (Q/Q ·- ) of 1,4-quinones using a quantum chemical approach. The calculated energy differences of reduction of Q to Q ·- in DMF and water for 1,4-quinone derivatives correlated highly with the experimentally measured E m (Q/Q ·- ) in DMF and water, respectively. E m (Q/Q ·- ) were calculated to be -163 mV for ubiquinone, -260 mV for menaquinone and phylloquinone, and -154 mV for plastoquinone in water versus NHE.

Quinones can accept two electrons and two protons, and are involved in electron transfer and proton transfer reactions in photosynthetic reaction centers. To date, the pK a of these quinones in aqueous solution have not been reported. We calculated the pK a of the initial protonation (Q ·- to QH · ) and the second protonation (QH - to QH 2 ) of 1,4-quinones using a quantum chemical approach. The calculated energy differences of the protonation reactions Q ·- to QH · and QH - to QH 2 in the aqueous phase for nine 1,4-quinones were highly correlated with the experimentally measured pK a (Q ·- /QH · ) and pK a (QH - /QH 2 ), respectively. In the present study, we report the pK a (Q ·- /QH · ) and pK a (QH - /QH 2 ) of ubiquinone, menaquinone, phylloquinone, plastoquinone, and rhodoquinone in aqueous solution.

Background: Phylloquinone is the most abundant form of vitamin K in US diets. Green vegetables are considered the predominant dietary source of phylloquinone. As our food supply diversifies and expands, the food groups that contribute to phylloquinone intake are also changing, which may change absolute intakes. Thus, it is important to identify the contributors to dietary vitamin K estimates to guide recommendations on intakes and food sources. Objective: The purpose of this study was to estimate 1 ) the amount of phylloquinone consumed in the diet of US adults, 2 ) to estimate the contribution of different food groups to phylloquinone intake in individuals with a high or low vegetable intake (≥2 or <2 cups vegetables/d), and 3 ) to characterize the contribution of different mixed dishes to phylloquinone intake. Methods: Usual phylloquinone intake was determined from NHANES 2011-2012 (≥20 y old; 2092 men and 2214 women) and the National Cancer Institute Method by utilizing a complex, stratified, multistage probability-cluster sampling design. Results: On average, 43.0% of men and 62.5% of women met the adequate intake (120 and 90 μg/d, respectively) for phylloquinone, with the lowest self-reported intakes noted among men, especially in the older age groups (51-70 and ≥71 y). Vegetables were the highest contributor to phylloquinone intake, contributing 60.0% in the high-vegetable-intake group and 36.1% in the low-vegetable-intake group. Mixed dishes were the second-highest contributor to phylloquinone intake, contributing 16.0% in the high-vegetable-intake group and 28.0% in the low-vegetable-intake group. Conclusion: Self-reported phylloquinone intakes from updated food composition data applied to NHANES 2011-2012 reveal that fewer men than women are meeting the current adequate intake. Application of current food composition data confirms that vegetables continue to be the primary dietary source of phylloquinone in the US diet. However, mixed dishes and convenience foods have emerged as previously unrecognized but important contributors to phylloquinone intake in the United States, which challenges the assumption that phylloquinone intake is a marker of a healthy diet. These findings emphasize the need for the expansion of food composition databases that consider how mixed dishes are compiled and defined., Competing Interests: Author disclosures: SGH, EGF, KJB, RLB, DBH, CHG, and SLB, no conflicts of interest., (© 2017 American Society for Nutrition.)

Due to lack of suitable bioanalytical methods in previous literature, for simultaneous estimation of Vitamin K1 isomers, in compliance with the current regulatory expectation, we aimed to develop a sensitive and rapid method with UFLC-APCI-MS/MS (ultrafast liquid chromatography - tandem mass spectrometry) using human plasma. A simple and cost effective procedure was implemented with the combination of protein precipitation and liquid extraction, to isolate the targets from plasma sample, while achieving an insignificant matrix effects and high recovery (≥88.2%). A short 9.0min run time per sample was accomplished by using water in methanol (1.0% v/v) and acetonitrile, which pumped at 0.8mL/min, on to the COSMOSIL ® packed column, for separating the trans and cis isomers of Vitamin K1 along with the corresponding stable labeled D7 internal standards (ISs). The analytes and ISs were quantified, at their parent to product ion mass transitions of 451.3 →187.1m/z and 458.1→194.3m/z respectively, using an APCI (atmospheric pressure chemical ionization) source of the tandem mass, in MRM (multiple reaction monitoring) mode. Performance of the method over the calibration range: 0.1-150.0ng/mL, while using a low sample volume (0.3mL), was successfully evaluated through full method validation in compliance with the latest regulations. Fully validated method with significant results was applied to human pharmacokinetic study, and had a potential to further advance the clinical research programs and generic drug development of Vitamin K1, intended for the regulatory submission., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Vitamin K-dependent carboxylation, an essential posttranslational modification catalyzed by gamma-glutamyl carboxylase, is required for the biological functions of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylation, reduced vitamin K (KH 2 ) is oxidized to vitamin K epoxide (KO). KO must be recycled back to KH 2 by the enzymes vitamin K epoxide reductase and vitamin K reductase in a pathway known as the vitamin K cycle. Our current knowledge about the enzymes of the vitamin K cycle is mainly based on in vitro studies of each individual enzymes under artificial conditions, which are of limited usefulness in understanding how the complex carboxylation process is carried out in the physiological environment. In this chapter, we review the current in vitro activity assays for vitamin K cycle enzymes. We describe the rationale, establishment, and application of cell-based assays for the functional study of these enzymes in the native cellular milieu. In these cell-based assays, different vitamin K-dependent proteins were designed and stably expressed in mammalian cells as reporter proteins to accommodate the readily used enzyme-linked immunosorbent assay for carboxylation efficiency evaluation. Additionally, recently emerged genome-editing techniques TALENs and CRISPR-Cas9 were used to knock out the endogenous enzymes in the reporter cell lines to eliminate the background. These cell-based assays are easy to scale up for high-throughput screening of inhibitors of vitamin K cycle enzymes and have been successfully used to clarify the genotypes and their clinical phenotypes of enzymes of the vitamin K cycle., (© 2017 Elsevier Inc. All rights reserved.)

Vitamin K epoxide reductase (VKOR) catalyzes the reduction of vitamin K quinone and vitamin K 2,3-epoxide, a process essential to sustain γ-carboxylation of vitamin K-dependent proteins. VKOR is also a therapeutic target of warfarin, a treatment for thrombotic disorders. However, the structural and functional basis of vitamin K reduction and the antagonism of warfarin inhibition remain elusive. Here, we identified putative binding sites of both K vitamers and warfarin on human VKOR. The predicted warfarin-binding site was verified by shifted dose-response curves of specified mutated residues. We used CRISPR-Cas9-engineered HEK 293T cells to assess the vitamin K quinone and vitamin K 2,3-epoxide reductase activities of VKOR variants to characterize the vitamin K naphthoquinone head- and isoprenoid side chain-binding regions. Our results challenge the prevailing concept of noncompetitive warfarin inhibition because K vitamers and warfarin share binding sites on VKOR that include Phe55, a key residue binding either the substrate or inhibitor.

Although warfarin is the most widely used anticoagulant worldwide, the mechanism by which warfarin inhibits its target, human vitamin K epoxide reductase (hVKOR), remains unclear. Here we show that warfarin blocks a dynamic electron-transfer process in hVKOR. A major fraction of cellular hVKOR is in an intermediate redox state containing a Cys51-Cys132 disulfide, a characteristic accommodated by a four-transmembrane-helix structure of hVKOR. Warfarin selectively inhibits this major cellular form of hVKOR, whereas disruption of the Cys51-Cys132 disulfide impairs warfarin binding and causes warfarin resistance. Relying on binding interactions identified by cysteine alkylation footprinting and mass spectrometry coupled with mutagenesis analysis, we conducted structure simulations, which revealed a closed warfarin-binding pocket stabilized by the Cys51-Cys132 linkage. Understanding the selective warfarin inhibition of a specific redox state of hVKOR should enable the rational design of drugs that exploit the redox chemistry and associated conformational changes in hVKOR.

During in vitro analysis, spinach (Spinacea oleracea L.) leaf extracts displayed varying antimutagenicity when analyzed in models including human lymphoblast (TK +/- ) cell line (thymidine kinase gene mutation assay) and Escherichia coli MG1655 (rifampicin resistance assay) against chemically (ethyl methanesulfonate and 5-azacytidine) induced mutagenicity. Highest antimutagenicity was displayed by the quinone extract. The principal bioactive compound exhibited fluorescence in TLC at 366 nm (termed C4) resolved at R f 0.32 and t R 15.2 min in TLC and HPLC, respectively. On the TLC plate, three spots (C1-C3), observed at 254 nm, displayed comparatively lesser antimutagenicity. Furthermore, biochemical and spectroscopic analyses using MALDI-TOF MS and NMR indicated the nature of the potent compound (C4) as an ethoxy-substituted phylloquinone derivative [2-ethoxy-3-((E)-3,7,11,15-tetramethylhexadec-2-enyl)naphthalene-1,4-dione]. The C4 compound did not display any cytotoxicity and hence possesses significant nutraceutical-based intervention possibility to combat the onset of mutation-associated disease(s).

Phylloquinone is a redox active naphthoquinone involved in electron transport in plants. The function of this reduced form remains unclear due to its instability, which has precluded detection. Herein, a simple method that permits the stabilization of the reduced form of phylloquinone by di-O-methylation and HPLC detection is described., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Purpose: To develop a stable micellar formulation of vitamin K for oral delivery, because the commercial and clinically used formulation of vitamin K (Konakion® MM) destabilizes at gastric pH resulting in low bioavailability of this vitamin in neonates with cholestasis., Methods: Mixed micelles composed of EPC, DSPE-PEG 2000 and glycocholic acid, with and without vitamin K, were prepared by a film hydration method. The influence of pH on the stability of the micelles was analyzed by dynamic light scattering (DLS). The critical micelle concentration (CMC) was determined by fluorescence spectroscopy using pyrene and the morphology was evaluated by transmission electron microscopy . Caco-2 cells were used to study the cytocompatibilty., Results: Mixed micelles with mean diameters from 7.1 to 11.0 nm and a narrow size distribution (PDI < 0.2) were obtained after 3 membrane extrusion cycles. Konakion® MM formed aggregated particles at gastric pH, which was avoided through steric stabilization by introducing PEG. TEM showed that mixed micelles had a spherical size (diameter of around 10 nm) with a narrow size distribution in agreement with the DLS results. The loading capacities for vitamin K of mixed micelles with varying molar fractions of DSPE-PEG and EPC (from 0/100 to 50/50 (mol/mol)) were 10.8-5.0 w%, respectively. The mixed micelles showed good cytocompatibility at concentrations of glycocholic acid between 0.12 and 1.20 mM., Conclusions: Mixed micelles with superior stability to Konakion® MM at low pH were obtained by introducing DSPE-PEG 2000. These are therefore attractive oral formulations for vitamin K.

2-methyl-6-phytyl-1, 4-benzoquinol methyltransferase (MPBQ-MT) is a vital enzyme catalyzing a key methylation step in both α/γ-tocopherol and plastoquinone biosynthetic pathway. In this study, the gene encoding MPBQ-MT was isolated from lettuce (Lactuca sativa) by rapid amplification of cDNA ends (RACE), named LsMT. Overexpression of LsMT in lettuce brought about a significant increase of α- and γ-tocopherol contents with a reduction of phylloquinone (vitamin K1) content, suggesting a competition for a common substrate phytyl diphosphate (PDP) between the two biosynthetic pathways. Besides, overexpression of LsMT significantly increased plastoquinone (PQ) level. The increase of tocopherol and plastoquinone levels by LsMT overexpression conduced to the improvement of plants' tolerance and photosynthesis under high light stress, by directing excessive light energy toward photosynthetic production rather than toward generation of more photooxidative damage. These findings suggest that the role and function of MPBQ-MT can be further explored for enhancing vitamin E value, strengthening photosynthesis and phototolerance under high light in plants.

The objective of this study was to develop a rapid, sensitive, and specific analytical method to study vitamin K1 in fruits and vegetables. Accelerated solvent extraction and solid phase extraction was used for sample preparation. Quantification was done by liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization in selected reaction monitoring mode with deuterium-labeled vitamin K1 as an internal standard. The precision was estimated as the pooled estimate of three replicates performed on three different days for spinach, peas, apples, banana, and beetroot. The repeatability was 5.2% and the internal reproducibility was 6.2%. Recovery was in the range 90-120%. No significant difference was observed between the results obtained by the present method and by a method using the same principle as the CEN-standard i.e. liquid-liquid extraction and post-column zinc reduction with fluorescence detection. Limit of quantification was estimated to 0.05 μg/100g fresh weight., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Tocopherol (vitamin E) and phylloquinone (vitamin K1) are lipid-soluble antioxidants that can only be synthesized by photosynthetic organisms. These compounds function primarily at the thylakoid membrane but are also present in chloroplast lipid droplets, also known as plastoglobules (PG). Depending on environmental conditions and stage of plant development, changes in the content, number and size of PG occur. PG are directly connected to the thylakoid membrane via the outer lipid leaflet. Apart from storage, PG are active in metabolism and likely trafficking of diverse lipid species. This review presents recent advances on how plastoglobules are implicated in the biosynthesis and metabolism of vitamin E and K., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Two functional electron transfer (ET) chains, related by a pseudo-C2 symmetry, are present in the reaction center of photosystem I (PSI). Due to slight differences in the environment around the cofactors of the two branches, there are differences in both the kinetics of ET and the proportion of ET that occurs on the two branches. The strongest evidence that this is indeed the case relied on the observation that the oxidation rates of the reduced phylloquinone (PhQ) cofactor differ by an order of magnitude. Site-directed mutagenesis of residues involved in the respective PhQ-binding sites resulted in a specific alteration of the rates of semiquinone oxidation. Here, we show that the PsaA-F689N mutation results in an ∼100-fold decrease in the observed rate of PhQA(-) oxidation. This is the largest change of PhQA(-) oxidation kinetics observed so far for a single-point mutation, resulting in a lifetime that exceeds that of the terminal electron donor, P700(+). This situation allows a second photochemical charge separation event to be initiated before PhQA(-) has decayed, thereby mimicking in PSI a situation that occurs in type II reaction centers. The results indicate that the presence of PhQA(-) does not impact the overall quantum yield and leads to an almost complete redistribution of the fractional utilization of the two functional ET chains, in favor of the one that does not bear the charged species. The evolutionary implications of these results are also briefly discussed., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Fourier transform infrared difference spectroscopy (FTIR DS) has been widely used to study the structural details of electron transfer cofactors (and their binding sites) in many types of photosynthetic protein complexes. This review focuses in particular on work that has been done to investigate the A₁cofactor in photosystem I photosynthetic reaction centers. A review of this subject area last appeared in 2006 [1], so only work undertaken since then will be covered here. Following light excitation of intact photosystem I particles the P700⁺A⁻(1) secondary radical pair state is formed within 100ps. This state decays within 300ns at room temperature, or 300μs at 77K. Given the short-lived nature of this state, it is not easily studied using "static" photo-accumulation FTIR difference techniques at either temperature. Time-resolved techniques are required. This article focuses on the use of time-resolved step-scan FTIR DS for the study of the P700⁺A⁻(1) state in intact photosystem I. Up until now, only our group has undertaken studies in this area. So, in this article, recent work undertaken in our lab is described, where we have used low-temperature (77K), microsecond time-resolved step-scan FTIR DS to study the P700⁺A⁻(1) state in photosystem I. In photosystem I a phylloquinone molecule occupies the A₁binding site. However, different quinones can be incorporated into the A1 binding site, and here work is described for photosystem I particles with plastoquinone-9, 2-phytyl naphthoquinone and 2-methyl naphthoquinone incorporated into the A₁binding site. Studies in which ¹⁸O isotope labeled phylloquinone has been incorporated into the A1 binding site are also discussed. To fully characterize PSI particles with different quinones incorporated into the A1 binding site nanosecond to millisecond visible absorption spectroscopy has been shown to be of considerable value, especially so when undertaken using identical samples under identical conditions to that used in time-resolved step-scan FTIR measurements. In this article the latest work that has been undertaken using both visible and infrared time resolved spectroscopies on the same sample will be described. Finally, vibrational spectroscopic data that has been obtained for phylloquinone in the A1 binding site in photosystem I is compared to corresponding data for ubiquinone in the QA binding site in purple bacterial reaction centers. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Many proteins in chloroplast are regulated through the disulfide bond/thiol transformation to realize their activities. A homologue of VKOR (Vitamin K epoxide reductase) in Arabidopsis chloroplast is found to catalyze the disulfide bond formation in vivo and to mediate the specific phylloquinone reduction in vitro. It is also called LTO1 (Lumen Thiol Oxidoreductase 1). Investigations about functions and essential amino acid residues of AtVKOR have important theoretical significance to clarify the chloroplast redox regulation mechanism. In this study, several amino acids in the VKOR domain of AtVKOR were identified to be involved in binding of phylloquinone. Site-directed mutagenesis was used to study the function of these positions. The results suggested that residues Ser77, Leu87, Phe137 and Leu141 were quite important in the binding and catalyzing the reduction of phylloquinone. These residues were also involved in the electron transferring and disulfide bond formation of substrate proteins by motility assays in vivo, suggesting that the binding of phylloquinone not only affected the delivery of electrons to phylloquinone but also affected the whole electron transfer process. The conserved cysteines in the AtVKOR domain also played critical roles in phylloquinone reduction. When each of the four conserved cysteines was mutated to alanine, the mutants lost reduction activity entirely, suggesting that the four conserved cysteines played crucial roles in the electron transfer process.

Molecular dynamics (MD) calculations, a semi-continuum (SC) approach, and quantum chemistry (QC) calculations were employed together to investigate the molecular mechanics of ultrafast charge separation reactions in Photosystem I (PS I) of Thermosynechococcus elongatus. A molecular model of PS I was developed with the aim to relate the atomic structure with electron transfer events in the two branches of cofactors. A structural flexibility map of PS I was constructed based on MD simulations, which demonstrated its rigid hydrophobic core and more flexible peripheral regions. The MD model permitted the study of atomic movements (dielectric polarization) in response to primary and secondary charge separations, while QC calculations were used to estimate the direct chemical effect of the A(0A)/A(0B) ligands (Met or Asn in the 688/668 position) on the redox potential of chlorophylls A(0A)/A(0B) and phylloquinones A(1A)/A(1B). A combination of MD and SC approaches was used to estimate reorganization energies λ of the primary (λ₁) and secondary (λ₂ ) charge separation reactions, which were found to be independent of the active branch of electron transfer; in PS I from the wild type, λ₁ was estimated to be 390 ± 20mV, while λ₂ was estimated to be higher at 445 ± 15mV. MD and QC approaches were used to describe the effect of substituting Met688(PsaA)/Met668(PsaB) by Asn688(PsaA)/Asn668(PsaB) on the energetics of electron transfer. Unlike Met, which has limited degrees of freedom in the site, Asn was found to switch between two relatively stable conformations depending on cofactor charge. The introduction of Asn and its conformation flexibility significantly affected the reorganization energy of charge separation and the redox potentials of chlorophylls A(0A)/A(0B) and phylloquinones A(1A)/A(1B), which may explain the experimentally observed slowdown of secondary electron transfer in the M688N(PsaA) variant. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Unlike the other fat-soluble vitamins, vitamin K circulates in the human bloodstream at very low levels because of a low intake in the diet. Mammals have developed an efficient recycling system, known as vitamin K-epoxide cycle, which involve quinone, hydroquinone and epoxide forms of the vitamin. Phylloquinone (K(1)) is the main homologue, while menaquinone-4 (MK-4) is both a member of the vitamin K(2) family and metabolite of K(1) in extra-hepatic tissues. Notwithstanding the recent advances, many aspects of the complex vitamin K physiology still remain to be investigated. Therefore, there is a critical need to develop more reliable analytical methods for determining the vitamin K and its metabolites in biological fluids and tissues. Nevertheless, relatively low concentrations, unavailability of some authentic standards and occurrence of interfering lipids make this a challenging task. The method proposed in the present paper can directly and accurately estimate K(1), K(1) 2,3-epoxide (K(1)O), and MK-4 in human serum and plasma at concentrations in the ng/L-μg/L range, using labelled internal standards and a quadrupole linear ion trap instrument operated in multiple reaction monitoring (MRM) mode. High sensitivity was achieved by removing signal "endogenous suppressors" and making the composition of the non-aqueous mobile phase suitable to support the positive atmospheric pressure chemical ionization of the analytes. An excellent selectivity resulted from the combination of some factors: the MRM acquisition, the adoption of an identification point system, an extraction optimized to remove most of the lipids and a tandem-C18 column-system necessary to separate isobaric interferences from analytes. The method was validated according to the Food and Drug Administration (FDA) guidelines and its accuracy was assessed by analysing 9 samples from the Vitamin K External Quality Assessment Scheme (KEQAS). Its feasibility in evaluating vitamin K status in human serum was also tested by monitoring a group of six healthy subjects and a group of six patients under oral anticoagulant therapy (OAT). Warfarinised patients did not show deficiency of K1 but levels comparable with those of healthy people and an accumulation of K1O up to 3.760μg/L. MK-4 was not detected in either of the two groups., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Vitamin K1 (VK1) is a very lipophilic and photosensitive molecule contained in some vegetables. Recently, the use of VK1 on the skin has been proposed for different pharmaceutical or cosmeceutical applications. In this study, an innovative strategy for the administration of VK1 on the skin was proposed. In particular, to overcome the drawbacks associated with a VK1-containing fatty ointment available on the market, an aqueous formulation suitable to be administered by nebulization was developed. The use of liposomes encapsulating VK1 enabled issues due to the lipophilicity of VK1 to be overcome. Thus, different liposomal formulations, with different VK1 concentrations, were prepared and characterized in terms of size, zeta potential, VK1 encapsulation into liposomes, and stability of the formulations during storage. After a first phase of screening, the selected formulation was tested by a portable device for nebulization. No alteration of the vesicle characteristics following the liposome supply through the nebulizer was found. Finally, permeation studies were carried out on pig-excised skin in Franz cells and the newly developed formulation was compared to a marketed VK1-containing ointment. In this test, an enhanced VK1 accumulation into the skin was found when using nebulized liposomes. In conclusion, in order to administer VK1 on the skin, the newly developed formulation could be a valid alternative to the products available on the market today. In particular, the use of liposomes could facilitate the multiple administrations per day by aerosol, but also increase, compared to a semi-solid preparation, the accumulation of VK1 into the epidermis and dermis.

In contrast to other fat-soluble vitamins, dietary vitamin K is rapidly lost to the body resulting in comparatively low tissue stores. Deficiency is kept at bay by the ubiquity of vitamin K in the diet, synthesis by gut microflora in some species, and relatively low vitamin K cofactor requirements for γ-glutamyl carboxylation. However, as shown by fatal neonatal bleeding in mice that lack vitamin K epoxide reductase (VKOR), the low requirements are dependent on the ability of animals to regenerate vitamin K from its epoxide metabolite via the vitamin K cycle. The identification of the genes encoding VKOR and its paralog VKOR-like 1 (VKORL1) has accelerated understanding of the enzymology of this salvage pathway. In parallel, a novel human enzyme that participates in the cellular conversion of phylloquinone to menaquinone (MK)-4 was identified as UbiA prenyltransferase-containing domain 1 (UBIAD1). Recent studies suggest that side-chain cleavage of oral phylloquinone occurs in the intestine, and that menadione is a circulating precursor of tissue MK-4. The mechanisms and functions of vitamin K recycling and MK-4 synthesis have dominated advances made in vitamin K biochemistry over the last five years and, after a brief overview of general metabolism, are the main focuses of this review.

Unlabelled: Quinone compounds act as membrane resident carriers of electrons between components of the electron transport chain in the periplasmic space of prokaryotes and in the mitochondria of eukaryotes. Vitamin K is a quinone compound in the human body in a storage form as menaquinone (MK); distribution includes regulated amounts in mitochondrial membranes. The human brain, which has low amounts of typical vitamin K dependent function (e.g., gamma carboxylase) has relatively high levels of MK, and different regions of brain have different amounts. Coenzyme Q (Q), is a quinone synthesized de novo, and the levels of synthesis decline with age. The levels of MK are dependent on dietary intake and generally increase with age. MK has a characterized role in the transfer of electrons to fumarate in prokaryotes. A newly recognized fumarate cycle has been identified in brain astrocytes. The MK precursor menadione has been shown to donate electrons directly to mitochondrial complex III., Hypothesis: Vitamin K compounds function in the electron transport chain of human brain astrocytes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The synthesis of phylloquinone (vitamin K1) in photosynthetic organisms requires a thioesterase that hydrolyzes 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA) to release 1,4-dihydroxy-2-naphthoate (DHNA). Cyanobacteria and plants contain distantly related hotdog-fold thioesterases that catalyze this reaction, although the structural basis of these convergent enzymatic activities is unknown. To investigate this, the crystal structures of hotdog-fold DHNA-CoA thioesterases from the cyanobacterium Synechocystis (Slr0204) and the flowering plant Arabidopsis thaliana (AtDHNAT1) were determined. These enzymes form distinct homotetramers and use different active sites to catalyze hydrolysis of DHNA-CoA, similar to the 4-hydroxybenzoyl-CoA (4-HBA-CoA) thioesterases from Pseudomonas and Arthrobacter. Like the 4-HBA-CoA thioesterases, the DHNA-CoA thioesterases contain either an active-site aspartate (Slr0204) or glutamate (AtDHNAT1) that are predicted to be catalytically important. Computational modeling of the substrate-bound forms of both enzymes indicates the residues that are likely to be involved in substrate binding and catalysis. Both enzymes are selective for DHNA-CoA as a substrate, but this selectivity is achieved using divergent predicted binding strategies. The Slr0204 binding pocket is predominantly hydrophobic and closely conforms to DHNA, while that of AtDHNAT1 is more polar and solvent-exposed. Considered in light of the related 4-HBA-CoA thioesterases, these structures indicate that hotdog-fold thioesterases using either an active-site aspartate or glutamate diverged into distinct clades prior to the evolution of strong substrate specificity in these enzymes.

The distance and relative orientation of functional groups within protein domains and their changes during chemical reactions determine the efficiency of biological processes. In this work on electron transfer proteins, we report the results of orientation resolving dipolar high-field EPR spectroscopy on the charge-separated state P700•+ A1•– (P700, primary electron donor; A1, phylloquinone electron acceptor) in Photosystem I (PS I). Pulsed high-field EPR spectroscopy at W-band (95 GHz, 3.4 T) with extensions to PELDOR (pulsed electron–electron double resonance) and RIDME (relaxation-induced dipolar modulation enhancement) was utilized to obtain the parameters describing the three-dimensional structure of the laser-flash-induced transient radical pair P700•+ A1•– in a frozen solution of deuterated PS I from the cyanobacterium Synechocystis sp. PCC 6803, which is performing oxygenic photosynthesis. The measured distances and relative orientations of the weakly coupled radical ions in the radical pair P700•+ A1•– are compared with previously reported geometries and with those of the precursor cofactors P700 and A1 known from X-ray crystallography. Cyclic electron transfer was found to proceed exclusively via the A-branch of the cofactor chain of PS I at cryogenic temperature. The position and orientation of the reduced phylloquinone coincide with those of the precursor, revealing that no substantial orientational changes of the phylloquinone molecule upon charge separation occur. Several distinct orientations of the P700•+ g-tensor axes with respect to the molecular frame of the primary donor were found experimentally, which we explain by several conformational substates of the P700•+ radical structure having slightly different electron spin density distributions.

When the quinone, vitamin K1 (VK1), is electrochemically reduced in aqueous-acetonitrile solutions (CH3CN with 7.22 M H2O), it undergoes a two-electron reduction to form the dianion that is hydrogen-bonded with water [VK1(H2O)y(2–)]. EPR and voltammetry experiments have shown that the persistent existence of the semiquinone anion radical (also hydrogen-bonded with water) [VK1(H2O)x(–•)] in aqueous or organic–aqueous solutions is a result of VK1(H2O)y(2–) undergoing a net homogeneous electron transfer reaction (comproportionation) with VK1, and not via direct one-electron reduction of VK1. When 1 mM solutions of VK1 were electrochemically reduced by two electrons in aqueous-acetonitrile solutions, quantitative EPR experiments indicated that the amount of VK1(H2O)x(–•) produced was up to approximately 35% of all the reduced species. In situ electrochemical ATR-FTIR experiments on sequentially one- and two-electron bulk reduced solutions of VK1 (showing strong absorbances at 1664, 1598, and 1298 cm(–1)) in CH3CN containing <0.05 M H2O led to the detection of VK1(–•) with strong absorbances at 1710, 1703, 1593, 1559, 1492, and 1466 cm(–1) and VK1(H2O)y(2–) with strong absorbances at 1372 and 1342 cm(–1).

The phylloquinone acceptor PhQ(A) in photosystem I binds to the protein through a single H-bond to the backbone nitrogen of PsaA-L722. Here, we investigate the effect of this H-bond on the electron transfer (ET) kinetics by substituting threonine for PsaA-L722. Room temperature spin-polarized transient EPR measurements show that in the PsaA-L722T mutant, the rate of PhQ(A)(-) to F(X) ET increases and the hyperfine coupling to the 2-methyl group of PhQ(A) is much larger than in the wild type. Molecular dynamics simulations and ONIOM type electronic structure calculations indicate that it is possible for the OH group of the Thr side chain to form an H-bond to the carbonyl oxygen atom, O(4) of the phylloquinone, and that this results in an increase in the 2-methyl hyperfine couplings as observed in the transient EPR data. The Arrhenius plot of the PhQ(A)(-) to F(X) ET in the PsaA-L722T mutant suggests that the increased rate is probably the result of a slight change in the electronic coupling between PhQ(A)(-) and F(X). The strong deviation from Arrhenius behavior observed at ∼200 K can be reproduced using a semiclassical model, which takes the zero-point energy of the mode coupled to the ET into account. However, since the change in slope of the Arrhenius plot occurs at the protein glass transition temperature, it is argued that it could be the result of a change in the protein relaxation dynamics at this temperature rather than quantum mechanical effects.

It is not known how plants cleave the thioester bond of 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA), a necessary step to form the naphthoquinone ring of phylloquinone (vitamin K(1) ). In fact, only recently has the hydrolysis of DHNA-CoA been demonstrated to be enzyme driven in vivo, and the cognate thioesterase characterized in the cyanobacterium Synechocystis. With a few exceptions in certain prokaryotic (Sorangium and Opitutus) and eukaryotic (Cyanidium, Cyanidioschyzon and Paulinella) organisms, orthologs of DHNA-CoA thioesterase are missing outside of the cyanobacterial lineage. In this study, genomic approaches and functional complementation experiments identified two Arabidopsis genes encoding functional DHNA-CoA thioesterases. The deduced plant proteins display low percentages of identity with cyanobacterial DHNA-CoA thioesterases, and do not even share the same catalytic motif. GFP-fusion experiments demonstrated that the Arabidopsis proteins are targeted to peroxisomes, and subcellular fractionations of Arabidopsis leaves confirmed that DHNA-CoA thioesterase activity occurs in this organelle. In vitro assays with various aromatic and aliphatic acyl-CoA thioester substrates showed that the recombinant Arabidopsis enzymes preferentially hydrolyze DHNA-CoA. Cognate T-DNA knock-down lines display reduced DHNA-CoA thioesterase activity and phylloquinone content, establishing in vivo evidence that the Arabidopsis enzymes are involved in phylloquinone biosynthesis. Extraordinarily, structure-based phylogenies coupled to comparative genomics demonstrate that plant DHNA-CoA thioesterases originate from a horizontal gene transfer with a bacterial species of the Lactobacillales order., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Phylloquinone (PK) is converted into menaquinone-4 (MK-4) via side chain removal-addition. Stable isotope use is an effective approach to identify the tissue location of this conversion, which is currently unknown. Following a 14-d PK-deficient diet, male Fischer 344 rats (8 mo; n = 15) were fed 1.6 mg deuterium-labeled PK (L-PK) per kg diet for 0 (control), 1 d (PK-1d), and 7 d (PK-7d). Both L-PK and deuterium-labeled MK-4 (L-MK-4) were detected in tissues in PK-1d and PK-7d, although the results varied. Whereas some tissues had an overall increase in MK-4 in response to L-PK, total brain, testes, and fat MK-4 concentrations did not. In contrast, L-MK-4 concentrations increased in all 3 tissues. The deuterium label was found only on the L-MK-4 naphthoquinone ring, confirming the need for side chain removal for the formation of MK-4. Labeled menadione (MD) was detected in urine and serum in PK-1d and PK-7d, confirming its role as an intermediate. A Caco-2 cell monolayer model was used to study the role of the enterocytes in the conversion process. Neither MK-4 nor MD was detected in Caco-2 cells treated with PK. However, when Caco-2 cells were treated with MD, MK-4 was formed. Similarly, MK-4 was formed in response to MD-treated 293T kidney cells, but not HuH7 liver cells. These data demonstrate that MK-4 is the predominant form of vitamin K in multiple tissues, but there appears to be a tissue-specific regulation for the conversion of PK to MK-4.

Light-induced electron transfer reactions in the chlorophyll a/d-binding Photosystem I reaction centre of Acaryochloris marina were investigated in whole cells by pump-probe optical spectroscopy with a temporal resolution of ~5ns at room temperature. It is shown that phyllosemiquinone, the secondary electron transfer acceptor anion, is oxidised with bi-phasic kinetics characterised by lifetimes of 88±6ns and 345±10ns. These lifetimes, particularly the former, are significantly slower than those reported for chlorophyll a-binding Photosystem I, which typically range in the 5-30ns and 200-300ns intervals. The possible mechanism of electron transfer reactions in the chlorophyll a/d-binding Photosystem I and the slower oxidation kinetics of the secondary acceptors are discussed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Thylakoid membranes contain two types of quinones, benzoquinone (plastoquinone) and naphthoquinone, which are involved in photosynthetic electron transfer. Unlike the benzoquinone, the chemical species of naphthoquinone present (phylloquinone, menaquinone-4 and 5'-monohydroxyphylloquinone) varies depending on the oxygenic photosynthetic organisms. The green alga Chlamydomonas reinhardtii has been used as a model organism to study the function of the naphthoquinone bound to PSI. However, the level of phylloquinone and the presence of other naphthoquinones in this organism remain unknown. In the present study, we found that 5'-monohydroxyphylloquinone is the predominant naphthoquinone in cell and thylakoid extracts based on the retention time during reverse phase HPLC, absorption and mass spectrometry measurements. It was shown that 5'-monohydroxyphylloquinone is enriched 2.5-fold in the PSI complex as compared with thylakoid membranes but that it is absent from PSI-deficient mutant cells. We also found a small amount of phylloquinone in the cells and in the PSI complex and estimated that accumulated 5'-monohydroxyphylloquinone and phylloquinone account for approximately 90 and 10%, respectively, of the total naphthoquinone content. The ratio of these two naphthoquinones remained nearly constant in the cells and in the PSI complexes from logarithmic and stationary cell growth stages. We conclude that both 5'-monohydroxyphylloquinone and phylloquinone stably co-exist as major and minor naphthoquinones in Chlamydomonas PSI.

The phylloquinones of photosystem I (PS I), A(1A) and A(1B), exist in near-equivalent protein environments but possess distinct thermodynamic and kinetic properties. Although the determinants responsible for the different properties of the phylloquinones are not completely understood, the strength and geometry of hydrogen bond interactions are significant factors in tuning and control of function. This study focuses on characterizing the hydrogen-bonding interactions of the phylloquinone acceptor, A(1A), by (1)H and (14)N HYSCORE spectroscopy. Photoaccumulation of PS I complexes at pH 8.0 results in the trapping of the phyllosemiquinone anion, A(1A)(-), on the A-branch of cofactors. The experiments described here indicate that A(1A)(-) forms a single H-bond. Using a simple point dipole approximation, we estimate its length to be 1.6 ± 0.1 Å. The value of the (1)H isotropic hyperfine coupling constant suggests that the H-bond has significant out-of-plane character. The (14)N HYSCORE spectroscopy experiments support the assignment of a H-bond wherein, the (14)N quadrupolar coupling constant is consistent with a backbone amide nitrogen as the hydrogen bond donor.