Vitamin K (phylloquinone and menaquinones) in foods - Optimisation of extraction, clean-up and LC-ESI-MS/MS method for quantification.

Growing evidence of vitamin K's importance in human health beyond blood coagulation and bone health necessitates its further research. A method involving extraction, lipase treatment, clean-up, and detection and quantification by LC-ESI-MS/MS of phylloquinone (PK), menaquinone-4 (MK-4), menaquinone-7 (MK-7) and menaquinone-9 (MK-9) was developed, and single-laboratory validated. The matrices included in the validation were hazelnut, cheese, broccoli, and pork. The LC-method runtime was 9 min. The LOQ for PK, MK-4 and MK-7 was 0.5 µg/100 g food, while for MK-9 it was 2.5 µg/100 g food. The intra- and inter-day precision was <15% for endogenous and spiked levels, except for low content at 4 times the LOQ. Trueness was assessed to be in the range 94-125% for spiking at levels approximately 4 and 10 times LOQ. It is further shown that deuterium labelled MK-7 can be used as an internal standard for MK-9.

A cell-based high-throughput screen identifies drugs that cause bleeding disorders by off-targeting the vitamin K cycle.

Drug-induced bleeding disorders contribute to substantial morbidity and mortality. Antithrombotic agents that cause unintended bleeding of obvious cause are relatively easy to control. However, the mechanisms of most drug-induced bleeding disorders are poorly understood, which makes intervention more difficult. As most bleeding disorders are associated with the dysfunction of coagulation factors, we adapted our recently established cell-based assay to identify drugs that affect the biosynthesis of active vitamin K-dependent (VKD) coagulation factors with possible adverse off-target results. The National Institutes of Health (NIH) Clinical Collection (NCC) library containing 727 drugs was screened, and 9 drugs were identified, including the most commonly prescribed anticoagulant warfarin. Bleeding complications associated with most of these drugs have been clinically reported, but the pathogenic mechanisms remain unclear. Further characterization of the 9 top-hit drugs on the inhibition of VKD carboxylation suggests that warfarin, lansoprazole, and nitazoxanide mainly target vitamin K epoxide reductase (VKOR), whereas idebenone, clofazimine, and AM404 mainly target vitamin K reductase (VKR) in vitamin K redox cycling. The other 3 drugs mainly affect vitamin K availability within the cells. The molecular mechanisms underlying the inactivation of VKOR and VKR by these drugs are clarified. Results from both cell-based and animal model studies suggest that the anticoagulation effect of drugs that target VKOR, but not VKR, can be rescued by the administration of vitamin K. These findings provide insights into the prevention and management of drug-induced bleeding disorders. The established cell-based, high-throughput screening approach provides a powerful tool for identifying new vitamin K antagonists that function as anticoagulants.

Microwave Assisted Cloud Point Extraction for the Determination of Vitamin K Homologues in Vegetables by Liquid Chromatography with Tandem Mass Spectrometry.

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) with a triple quadrupole (QqQ) is proposed for determining the vitamin K homologues, phylloquinone (PK), menaquinone-4 (MK) and menadione (MD), in vegetables. The analytes were isolated from the samples (1-1.5 g) by ultrasound assisted extraction using acetonitrile (2 mL), and the liquids were submitted to microwave assisted cloud point extraction with Triton X-45. The enrichment factors were between 20 and 50, depending on the vitamin homologue in question. The analytes were determined by LC-ESI-QqQ-MS/MS in the multiple reaction monitoring (MRM) mode, providing unequivocal identification and quantification, with limits of detection of 0.8, 1.0, and 16 ng/g for MK, PK, and MD, respectively. Recovery assays for samples spiked at two concentration levels, between 40 and 600 ng/g depending on the compound, provided recoveries in the 90-114% range. Only PK was detected in the samples analyzed, at concentrations in the 90-2350 ng/g range.

[On the history of vitamin K, dicoumarol and warfarin]. / Pionerer bag vitamin K, dikumarol og warfarin.

The history of the discovery and development of vitamin K and its antagonists, the oral anticoagulants dicoumarol and warfarin, are fascinating, triumphant landmarks in the annals of medicine. Vitamin K was found by Carl Peter Henrik Dam and Fritz Schønheyder from the University of Copenhagen. The discovery was initiated by Dam, by a lucky choice of chicks in the dissertation of sterol metabolism, since the vitamin is not formed by intestinal bacteria in these animals. In these experiments the lack of an unknown factor in the synthetic diet caused internal bleeding similar to that found in scurvy, but the bleeding was not reversed by vitamin C and it could not be explained by the lack of classical vitamins. In 1935 the unknown antihaemorrhagic factor was named vitamin K and a few months later the phenomenon was also observed by H.J. Almquist and E.L.R. Stokstad in Berkeley. The activity of the factor was determined by bioassay in different extracts of green vegetables and alfalfa by Dam and Schønheyder. Vitamin K was isolated in 1939 by Dam and Paul Karrer in Zurich and the structure was determined by Edward Adelbert Doisy. Dam and Doisy were awarded the Nobel Prize in 1943. A dramatic story starts the discovery of dicoumarol. In the 1920s cattle in Canada began dying of internal bleeding with no obvious precipitating cause. Frank W. Schofield, a veterinary pathologist in Alberta, found that the mysterious disease was connected to the consumption of spoiled sweet clover hay and noted a prolonged clotting time. Ten years after a farmer traveled in a blizzard with his dead cow and a milk can of the unclotted blood to the University of Wisconsin. Only the door to the biochemical department of Karl Paul Link was open. This event started the isolation of the anticoagulant agent dicou- marol which was formed by microbial induced oxidation of coumarin in the mouldy sweet clover hay. More than hundred dicoumarol-like anticoagulants were synthesized by Link and his co-workers. A potent hemorrhagic agent named warfarin was first used as an effective rat poison. However, warfarin became the drug of choice and the break- through in the treatment of thromboembolic diseases. Today new oral anticoagulants are competing with warfarin.

Regulation of osteoclastogenesis by Simon extracts composed of caffeic acid and related compounds: successful suppression of bone destruction accompanied with adjuvant-induced arthritis in rats.

Simon extracts are vitamin K(1)-rich food materials extracted from the leaves of the Simon sweet potato. Although vitamin K is known to stimulate bone formation, we postulated that Simon extracts also contain unknown biological compounds having the ability to regulate bone resorption. Here we prepared the vitamin K-free fraction from the Simon extracts and investigated the ability of this fraction on the differentiation of osteoclasts. A remarkable inhibitory effect of osteoclastogenesis was observed when osteoclast precursors were treated with this fraction in rat bone marrow culture systems as well as in a pure differentiation system using murine osteoclast precursor cell line. The vitamin K-free Simon extracts markedly suppressed severe bone destruction mediated by abundant osteoclasts associated with adjuvant-induced arthritis in rats. High performance liquid chromatography (HPLC) analysis revealed that the vitamin K-free Simon extracts contained three types of low molecular weight inhibitors for osteoclastogenesis; caffeic acid, chlorogenic acids and isochlorogenic acids. Among these substances, caffeic acid showed the most powerful inhibitory effects on osteoclastogenesis. Caffeic acid significantly suppressed expression of NFATc1, a key transcription factor for the induction of osteoclastogenesis. Our current study enlightened a high utility of the Simon extracts and their chemical components as effective regulators for bone resorption accompanied with inflammation and metabolic bone diseases.

Simultaneous determination of vitamin K analogs in human serum by sensitive and selective high-performance liquid chromatography with electrochemical detection.

OBJECTIVES: We report a sensitive and selective method for the simultaneous determination of vitamin K1 and K2 analogs (VKs) with high-performance liquid chromatography in which a platinum catalyst reduction column and an electrochemical detector operated in the oxidation mode are incorporated into the detection system. We also applied this trace analysis method to the simultaneous determination of VKs in human serum to investigate the physiologic and pathophysiologic roles of VKs in the bone metabolism. METHODS: Our high-performance liquid chromatographic method with postcolumn catalyst reduction and electrochemical detection was applied for the simultaneous determination of VKs in human serum samples. After separation of VKs on a reversed-phase separation column by using a mixture of ethanol and methanol (1:1, v/v), containing 0.025 M of sodium perchlorate as the mobile phase, the VKs were reduced once in a platinum catalyst reduction column connected online and then monitored quantitatively by an electrochemical detector with a glassy carbon working electrode operated in the oxidation mode (+0.6 V versus Ag/AgCl). RESULTS: VKs were clearly separated from each other within 80 min. The detection limits at a signal-to-noise ratio of 3 were 2 to 10 pg for VKs. Quantitative recovery from serum was obtained in the range of 86% to 91% for VKs. The average coefficients of variation of within-day and between-day assays were 1.6% to 2.1% and 2.4% to 3.6%, respectively, for all VKs. CONCLUSIONS: This method was sensitive and selective for detection of VKs and was satisfactory in the simultaneous determination of VKs in small volumes of human serum.

Separation of vitamins by supercritical fluid chromatography with water-modified carbon dioxide as the mobile phase.

Supercritical fluid chromatography (SFC) has become a technique for solving problems that are difficult to be monitored by other chromatographic methods. However, the most widely used fluid, is no more polar than hexane. Polar samples which are difficult to be analyzed with pure supercritical CO(2) because of their high polarity can be separated by adding polar modifiers to supercritical CO(2). In this paper various vitamins were well separated using water-modified supercritical CO(2) fluid. The amount of water dissolved in supercritical CO(2) was measured using an amperometric microsensor made of a thin film of perfluorosulfonate ionomer (PFSI).

Determination of phylloquinone and menaquinones in animal products with fluorescence detection after postcolumn reduction with metallic zinc.

A high-performance liquid chromatographic (HPLC) method for the determination of phylloquinone and menaquinones in foods of animal origin is described. The K vitamers were quantified with a fluorescence detector after postcolumn reduction with metallic zinc using K1(25) as an internal standard. Extraction was done either with 2-propanol-hexane (meat and fish products) or with acid hydrolysis method (dairy products). Prior to quantification, sample extracts were purified by semipreparative HPLC; in addition, the fats of cheese and rainbow trout samples were removed with lipase hydrolysis. By this method the phylloquinone and menaquinones (MK-4 to MK-10) present in a few representative samples of different animal food groups were determined. HPLC-MS was used to confirm the identification of K vitamers. Long-chain menaquinones were found from bovine and pig livers as well as from various cheeses. The total vitamin K contents calculated as the sum of quantified K vitamers were in general low (mean content 10-100 ng/g); the highest amount was analyzed in chicken meat (600 ng/g).

Testosterone 5 alpha-reductase inhibitors, menaquinone 7 produced by a Bacillus and phenazine methosulfate.

Menaquinone 7 (MW: 649, C46H64O2), a natural electron acceptor for steroid ring A dehydrogenations, produced by Bacillus sp. SNU-299, was isolated as a rat prostate testosterone 5 alpha-reductase inhibitor with an IC50 value of 4.0 x 10(-5) M from the cultured broth. Phylloquinone was as active as the purified microbial metabolite with an IC50 value of 6.6 x 10(-4) M. On the basis of this evidence, the inhibitory activities of electron carriers, menadione, phenazine methosulfate, and 2,6-dichlorophenolindophenol, for rat prostate testosterone 5 alpha-reductase were tested, and the IC50 values were 3.1 x 10(-6) M, 4.9 x 10(-8) M, 8.9 x 10(-5) M, respectively. A product of the 5 alpha-reductase enzyme reaction and an electron and proton carrier, NADP+, inhibited the 5 alpha-reduction by rat prostate testosterone 5 alpha-reductase with an IC50 value of 9.2 x 10(-5) M. However, the inhibition effect of a proton carrier, carbonylcyanide-m-chlorophenylhydrazone, for rat prostate testosterone 5 alpha-reductase was substantially inactive.

Dihydrophencomycin methyl ester, a new phenazine derivative from a marine Streptomycete.

The novel 5,10-dihydrophencomycin methyl ester (4) and the known microbial metabolites (2-hydroxyphenyl)-acetamide (1), menaquinone MK9 (II, III, VIII, IX-H8) (2), and phencomycin (3a) were isolated from an unidentified marine Streptomyces sp. and the structures were elucidated by NMR methods. Compound 4 shows weak antibiotic activity against Escherichia coli and Bacillus subtilis.

Isolation and identification of menaquinone-9 from purified nitrate reductase of Escherichia coli.

On the basis of the observation that nitrate reductase from Escherichia coli is sensitive to UV irradiation with an action spectrum indicative of a naphthoquinone (F. Brito and M. Dubourdieu, Biochem. Int. 15:1079-1088, 1987), we extracted and characterized quinone components from two different preparations of purified nitrate reductase. A soluble form of nitrate reductase, composed of alpha and beta subunits, was purified after release from the membrane fraction by heat treatment, and a detergent-solubilized form, containing alpha, beta, and gamma (cytochrome bNR) subunits, was purified in the presence of Triton X-100. Extraction of soluble alpha beta form with chloroform-methanol yielded several UV-absorbing components, which were characterized as menaquinone-9 with an oxidized side chain and further photodestruction products of the menaquinone. The total amount of menaquinone extracted into the organic phase was estimated to be 0.97 mol/mol of alpha beta dimer. Extraction of the detergent-solubilized alpha beta gamma form by a similar procedure yielded two naphthoquinone-like components which were characterized by mass spectrometry as the oxidized forms of menaquinone-9 and demethylmenaquinone-9. In this case, the molar ratio of total naphthoquinone to the alpha beta dimer was estimated to be greater than 6:1. When cytochrome bNR and detergent were eliminated from the detergent-solubilized enzyme by heat treatment and ion-exchange chromatography, only menaquinone-9 could be identified in the organic extract of the active alpha beta product. These results suggest that menaquinone-9 is specifically bound to the alpha beta dimer and may be the UV-sensitive component in the pathway of electron transfer catalyzed by nitrate reductase.

Quantitation of vitamin K in human milk.

A quantitative method was developed for the assay of vitamin K in human colostrum and milk. The procedure combines preparative and analytical chromatography on silica gel in a nitrogen atmosphere followed by reversed phase high performance liquid chromatography (HPLC). Two HPLC steps were used: gradient separation with ultraviolet (UV) detection followed by isocratic separation detected electrochemically. Due to co-migrating impurities, UV detection alone is insufficient for identification of vitamin K. Exogenous vitamin K was shown to equilibrate with endogenous vitamin K in the samples. A statistical method was incorporated to control for experimental variability. Vitamin K1 was analyzed in 16 pooled milk samples from 7 donors and in individual samples from 15 donors at 1 month post-partum. Vitamin K1 was present at 2.94 +/- 1.94 and 3.15 +/- 2.87 ng/mL in pools and in individuals, respectively. Menaquinones, the bacterial form of the vitamin, were not detected. The significance of experimental variation to studies of vitamin K in individuals is discussed.

Structure and function of a menaquinone involved in electron transport in membranes of Clostridium thermoautotrophicum and Clostridium thermoaceticum.

Clostridium thermoaceticum and Clostridium thermoautotrophicum contain the same menaquinone. Its structure, determined by thin-layer chromatography, UV absorption spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy, was found to be MK-7 (2-methyl-3-heptaprenyl-1,4-naphthoquinone). The menaquinone is located in the cytoplasmic membranes and is involved in redox reactions of two b-type cytochromes present in the clostridia. These reactions were studied with right-side-out membranes prepared from C. thermoautotrophicum by using CO as an electron donor. In intact membranes, both cytochromes were reduced, whereas after inactivation of the menaquinone by exposure of the membranes to UV irradiation, reduction of the low-potential cytochrome (Eo', -200 mV) but not of the high-potential cytochrome (Eo', -48 mV) occurred. The reduction of the high-potential cytochrome in UV-irradiated membranes was restored following the addition of oxidized menaquinone and with an excess of CO. The addition of oxidized menaquinone to reduced membranes resulted initially in a preferential oxidation of the low-potential cytochrome. The results obtained indicate that the menaquinone acts between the two b-type cytochromes in an electron transport chain.

[Analysis of the products of drug decomposition. XLII. Determining the levels of vitamin K in pharmaceutical preparations in the presence of decomposition products]. / Analiza produktów rozkladu leków. XLII. Oznaczanie witamin K w preparatach farmaceutycznych w obecnosci produktów rozkladu.

Chromatographic-spectrophotometric methods for the determination of vitamins K1, K3 and K4 in pharmaceuticals (tablets, dragees, injection solutions) in the presence of decomposition products have been elaborated. The TLC method on Kieselgel HF 254 (Merck) was applied to separation of the active substances from decomposition products, using the following solvent systems: benzene--ethyl acetate (9:1) for vitamin K1, cyclohexane--chloroform--metanol--acetic acid (2:15:3:1) for vitamin K3, and benzene--acetone (9:1) for vitamin K4. After elution from the plates, vitamin K1 was determined in ethanol at lambda = 251 nm, vitamin K3 in water at lambda = 234 nm and vitamin K4 in methanol at lambda = 225 nm. Maximum sensitivity of the elaborated methods came up to 7 X 10(-5) g, and standard deviations of single determinations ranged from 1.99% to 3.70%.

Intermediates of tocopherol biosynthesis in the unicellular alga Scenedesmus obliquus. The presence of three isomeric methylphytylbenzoquinones.

Three isomers of methylphytylbenzoquinone have been isolated from lipids of the unicellular alga Scenedesmus obliquus, the most abundant being 2-methyl-6-phytylbenzoquinone (65% of the total). The 2-methyl-3-phytyl and 2-methyl-5-phytyl isomers amounted to 8 and 27% respectively. Previously problems have been encountered in the separation of the 3-phytyl and the 6-phytyl isomers, but in the present study it was found that they separated readily as quinols. Phytyl plastoquinone was also found and the relevance of these compounds to the biosynthesis of alpha-tocopherol is discussed. As well as phylloquinone, a hydroxyphylloquinone was detected, and studies indicated that it is the 5' carbon atom to which the hydroxy group is attached. Such a compound has been found by workers using other unicellular algae.

Quinones from Archaebacteria, I. New types of menaquinones from the thermophilic archaebacterium Thermoproteus tenax.

From the archaebacterium Thermoproteus tenax, strain Kra-1 a mixture of 6 quinones of menaquinone and phylloquinone type with isopentyl side chains, MK-6(12H), MK-6-(10H), MK-5(10H), MK-5(8H), MK-4(8H), MK-4(6H) and two analogous quinones, containing in addition a methyl group in the naphthoquinone system, were isolated and characterized.

Identity of the quinone in Bacillus alcalophilus.

Every Bacillus species so far examined contains menaquinone as the sole quinone. In contrast, the alkalophilic Bacillus alcalophilus has been reported to be unusual in containing ubiquinone rather than menaquinone. In this communication, we demonstrate that B. alcalophilus, like all the other bacilli, contains menaquinone as the only quinone.

Isolation and characterization of a novel vitamin-K from Eubacterium lentum.

A novel fat-soluble vitamin K like molecule was isolated from the prokaryote, Eubacterium lentum, and its structure investigated by mass spectrometry and proton nuclear magnetic resonance spectrometry. On the basis of these studies the novel quinone is shown to be 2,5 and 6- or 2,7 and 8-trimethyl-3-farnesylfarnesyl-1,4-naphthoquinone.