Vitamin K1 Epoxide Research Articles

Substituted vitamin K epoxide analogs. New competitive inhibitors and substrates of vitamin K1 epoxide reductase

2- and 3-substituted vitamin K 2,3-epoxide analogues were synthesized and tested as inactivators, inhibitors, and substrates for beef liver microsomal vitamin K1 epoxide reductase. 2-(X)-3-phytyl-1,4-naphthoquinone 2,3-epoxides, where X is hydroxymethyl, chloromethyl, fluoromethyl, difluoromethyl, and formyl were all competitive inhibitors, but none was an inactivator. Only the 2-hydroxymethyl analogue was reduced to a quinone that was stable enough under the conditions of the experiment to be detected. Vitamin K1 epoxide analogues with modified phytyl chains (1'-hydroxy, 3'-fluoro with isomerized double bond, 1'-hydroxy and 1'-fluoro with saturated double bond, and the corresponding unsubstituted chains) were synthesized. All of the analogues were competitive inhibitors of vitamin K1 epoxide reductase. The nonfluorinated analogues also were shown to be substrates, being reduced to the corresponding quinone without enzyme inactivation. At least one other enzyme besides vitamin K1 epoxide reductase in beef liver microsomes also metabolizes all of these analogues.

Effect ofN-Ethylmaleimide on Beef and Rat Liver Vitamin K1Epoxide Reductase

There is little difference in the extent of inactivation of beef liver microsomal vitamin K1 epoxide reductase by N-ethylmaleimide (NEM) whether or not the microsomes are pre-treated with dithiothreitol (DTT). The rat liver microsomal enzyme, however, is inactivated by NEM to a much greater extent if the microsomes are pre-treated with DTT. The beef liver enzyme activity is protected from NEM inactivation by the substrate, vitamin K1 epoxide. Ping-pong kinetics are exhibited by the beef liver enzyme. These results support a mechanism for vitamin K1 epoxide reductase in which the function of the required dithiol is to reduce an active site disulfide bond; however, the geometry of the active sites of the enzyme from rat and beef may be different.

Vitamin K, 2,3-Epoxide And Quinone Reduction: Mechanism And Inhibition

The chemical and enzymatic pathways of vitamin K1 epoxide and quinone reduction have been investigated. The reduction of the epoxide by thiols is known to involve a thiol-adduct and a hydroxy vitamin K enolate intermediate which eliminates water to yield the quinone. Sodium borohydride treatment resulted in carbonyl reduction generating relatively stable compounds that did not proceed to quinone in the presence of base. NAD(P)H:quinone oxidoreductase (DT-diaphorase, E.C. 1.6.99.2) reduction of vitamin K to the hydroquinone was a significant process in intact microsomes, but 1/5th the rate of the dithiothreitol (DTT)-dependent reduction. No evidence was found for DT-diaphorase catalyzed reduction of vitamin K1 epoxide, nor was it capable of mediating transfer of electrons from NADH to the microsomal epoxide reducing enzyme. Purified diaphorase reduced detergent- solubilized vitamin K1 10(-5) as rapidly as it reduced dichlorophenylindophenol (DCPIP). Reduction of 10 microM vitamin K1 by 200 microM NADH was not inhibited by 10 microM dicoumarol, whereas DCPIP reduction was fully inhibited. In contrast to vitamin K3 (menadione), vitamin K1 (phylloquinone) did not stimulate microsomal NADPH consumption in the presence or absence of dicoumarol. DTT-dependent vitamin K epoxide reduction and vitamin K reduction were shown to be mutually inhibitory reactions, suggesting that both occur at the same enzymatic site. On this basis, a mechanism for reduction of the quinone by thiols is proposed. Both the DTT-dependent reduction of vitamin K1 epoxide and quinone, and the reduction of DCPIP by purified DT-diaphorase were inhibited by dicoumarol, warfarin, lapachol, and sulphaquinoxaline.

Temporal Variation in the Effects of Warfarin on the Vitamin K Cycle

In this in vivo study, the time-dependent effect of oral sodium warfarin was studied in male rats synchronized under a 12-hr light-dark cycle (light 0600-1800). Groups of 5 animals received an oral dose of 500 micrograms/kg of warfarin or saline at 0600 or 1800 and 1 mg/kg of vitamin K 8 hr later and the rats were sacrificed 240 min after vitamin K administration. The activities of the vitamin K reductase and vitamin K epoxide reductase were measured indirectly by determining the content of vitamin K1 and vitamin K epoxide reductase in the plasma and liver. The data obtained in control rats indicated that vitamin K and vitamin K 2,3 epoxide concentrations in plasma and liver were higher (P less than 0.05) at 1800 than at 0600. Warfarin had a greater (P less than 0.05) inhibitory effect on the vitamin K and vitamin K-epoxide reductases at 0600 compared to 1800; plasma levels of S- and R-warfarin did not vary with time of administration. The findings suggest that the activity of both reductases under control conditions, and the warfarin-induced inhibition of these enzymes varied depending on the time of drug administration.

Inhibition of phylloquinone epoxide reductase by BHT quinone methide, salicylic acid and α-tocopherolquinone

Inhibition of phylloquinone epoxide reductase by BHT quinone methide, salicylic acid and α-tocopherolquinone

Effect of N-methyltetrazolethiol on liver microsomal gamma-glutamylcarboxylation: modification of the in vitro action of N-methyltetrazolethiol.

Vitamin K-dependent gamma-glutamylcarboxylation activity in rat liver microsomes was monitored using the incorporation of 14CO2 into exogenous pentapeptide and endogenous protein substrates as indicators. Detergent solubilization of the microsomal enzymes was required for the activity to develop, but higher concentrations of detergent inhibited the enzymatic reaction. Pyridoxal-5'-phosphate (PAL-P) and dithiothreitol (DTT) stimulated the enzyme activity. The enzyme activity was observed when the hydroquinone form of vitamin K or the quinone form plus NADH was employed as the cosubstrate, but little activity was detected in the reaction system containing vitamin K-epoxide plus NADH plus DTT. N-Methyltetrazolethiol (NMTT), the substituent at the 3'-position of several beta-lactam antibiotics, inhibited the enzyme activity in vitro only in the reaction system containing NADH. Addition of DTT diminished the in vitro action of NMTT, while PAL-P and detergent did not affect it. The results indicate that the in vitro inhibitory action of NMTT is observable under some specific and restricted assay conditions. This paper also discusses the differences between the in vitro action and the in vivo effect of NMTT.

Effect of N-Methyltetrazolethiol on Liver Microsomal gamma-Glutamylcarboxylation: Modification of the In Vitro Action of N-Methyltetrazolethiol

Vitamin K-dependent gamma-glutamylcarboxylation activity in rat liver microsomes was monitored using the incorporation of 14CO2 into exogenous pentapeptide and endogenous protein substrates as indicators. Detergent solubilization of the microsomal enzymes was required for the activity to develop, but higher concentrations of detergent inhibited the enzymatic reaction. Pyridoxal-5’-phosphate (PAL-P) and dithiothreitol (DTT) stimulated the enzyme activity. The enzyme activity was observed when the hydroquinone form of vitamin K or the quinone form plus NADH was employed as the cosubstrate, but little activity was detected in the reaction system containing vitamin K-epoxide plus NADH plus DTT. N-Methyltetrazolethiol (NMTT), the substituent at the 3’-position of several ôeia-lactam antibiotics, inhibited the enzyme activity in vitro only in the reaction system containing NADH. Addition of DTT diminished the in vitro action of NMTT, while PAL-P and detergent did not affect it. The results indicate that the in vitro inhibitory action of NMTT is observable under some specific and restricted assay conditions. This paper also discusses the differences between the in vitro action and the in vivo effect of NMTT.

Enantiomers of warfarin and vitamin K1 metabolism.

The effect of the individual enantiomers of warfarin at steady state (1 mg daily) was investigated in five healthy volunteers. Both enantiomers produced a significant increase in prothrombin time, but the increase with S warfarin (1.8 +/- 0.8 s, mean +/- s.d.) was greater than with R warfarin (1.0 +/- 0.3 s), despite lower steady state plasma concentrations of S warfarin, due to its more rapid clearance. Following the administration of vitamin K1, the maximum plasma concentration and area under the plasma concentration time curve values for the metabolite vitamin K1 2,3-epoxide were greater after S warfarin than after R warfarin. The greater anticoagulant potency of S warfarin is reflected by a greater degree of inhibition of vitamin K1 epoxide reductase.

Vitamin K-dependent carboxylation and vitamin K metabolism in liver. Effects of warfarin.

The systems involved in vitamin K-dependent carboxylation and vitamin K metabolism have been extensively studied in rat liver. To determine how clinically applicable this information is, similar in vitro studies were completed using human liver. One major difference exists in the pathways that provide reduced vitamin K1 cofactor for the carboxylation reaction. The coumarin-sensitive DT-diaphorase (EC.1.6.99.2) in human liver appears to play a relatively minor role in the dehydrogenase pathway. However, similar to rat liver, the human liver contains a warfarin-insensitive enzyme in this dehydrogenase pathway. The data suggest that this enzyme is responsible for the antidotic effect of vitamin K1 in cases of coumarin intoxication. Human vitamin K epoxide reductase, which constitutes the other pathway for vitamin K1 reduction, has kinetic and enzymological characteristics that are very similar to the rat enzyme. This enzyme exhibited similar activity in rat and human microsomes. Initial velocities for vitamin K1 epoxide reduction in rat and human microsomes were 20 and 32 pmol/mg X min, respectively. The human enzyme is highly sensitive to warfarin inhibition. The mechanism for this inhibition appears to be similar to what has been proposed for the rat enzyme. Also, a vitamin K-dependent carboxylation system is described that allows both pathways to support the carboxylation reaction with reduced vitamin K1 cofactor. The effect of warfarin on this in vitro system is consistent with the current model for the mechanism of action of coumarin anticoagulant drugs in the rat.

A study of factors which determine the pharmacological response to vitamin K in coumarin anticoagulated rabbits

The pharmacological response to vitamin K has been determined by measuring prothrombin complex activity (P.C.A.) in male New Zealand White rabbits anticoagulated (P.C.A. <20%) with the long acting 4-hydroxycoumarin brodifacoum, at a dose (10 mg/kg) which produces maximum antagonism of vitamin K 1. Thus, according concurrent concepts, this animal model may be used to assess vitamin K requirements in the absence of a functional vitamin K-epoxide reductase. After intravenous administration of vitamin K 1 (1 mg/kg) P.C.A. reached a maximum (64±19%) at 3 hr and then declined at a rate which corresponds to complete inhibition of clotting factor synthesis. Vitamin K 2 (1 mg/kg) stimulated clotting factor synthesis for 2 hr, while cis-vitamin K 1, vitamin K 3, vitamin K 1 2,3-epoxide and oral administration of vitamin K 1 were ineffective. Plasma concentrations of vitamin K 1 fell steeply during the 12 hr following administration of a pharmacological dose, and then declined with a terminal half-life of 18.9 ± 9.0 hr. Comparison of the phamacodynamic and pharmacokinetic data indicated that plasma concentrations in the range 0.4–1.0 μg/ml are required for clotting factor synthesis in the limiting situation of maximum antagonism of vitamin K by coumarin anticoagulants. These findings explain why frequent and repeated administration of vitamin K 1 may be necessary during coumarin poisoning.

Formation of 3-Hydroxy-2,3-dihydrovitamin K1 In Vivo: Relationship to Vitamin K Epoxide Reductase and Warfarin Resistance

Hydroxy vitamin K [3(2)-hydroxy-2,3-dihydrovitamin K1] has been identified as a quantitatively important metabolite of injected vitamin K epoxide in vivo. The metabolite has been isolated and identified by comparison of its UV, mass spectra and high-performance liquid chromatography (HPLC) retention times with those of synthetic standards, and by its characteristic conversion to vitamin K quinone on treatment with the base triethylamine. This metabolite is formed from the vitamin K epoxide, not from the vitamin K quinone and can represent up to 3.5% of dose and 13% of hexane-extractable metabolites present in liver 1 hour after injection of 330 µg vitamin K1 epoxide per kilogram body weight. It is formed in both normal and warfarin-resistant rat strains, but to a significantly greater extent in the latter. Unlike the hydroxy vitamin K formed by warfarin-resistant rat liver microsomes in vitro, the metabolite formed from racemic vitamin K epoxide in vivo was not optically active, nor was its formation inhibited by coumarin anticoagulants under conditions that completely blocked vitamin K epoxide reduction in vivo. On this basis, hydroxy vitamin K formation in vivo differs from its formation in vitro; it is not a product of vitamin K epoxide reductase in vivo, but of some other possibly non-enzymatic reaction.

Stereospecificity of vitamin K-epoxide reductase.

Vitamin K epoxide can occur as a pair of optical isomers due to the asymmetry of the oxirane ring substituents. The stereoselectivity of vitamin K-epoxide reductase for the oxirane ring configuration was determined by recovery of the partially resolved unreacted substrate following incubations of racemic vitamin K epoxide with rat liver microsomes. The substrate ws enriched for the (--)-enantiomer, but selectivity for the biologically relevant (+)-enantiomer was low. This result was confirmed by direct comparison of the rates of reaction for the racemic substrate and (+)-vitamin K epoxide. The selectivity of vitamin K-epoxide reductase for the cis- or trans-phytyl configuration of the vitamin K side chain was also low. These results suggest an enzyme-active site which is open toward the 2,3-positions and is able to bind the substrate in two opposite orientations with respect to the positions of the methyl and phytyl side chain substituents.

Inhibition of phylloquinone epoxide-dependent carboxylation of microsomal proteins from rat liver by 2,6-di- tert-butyl-4-methylene-2,5-cyclohexadienone

In a study of the effects of butylated hydroxytoluene (BHT) on the carboxylation process involved in the conversion of rat-liver microsomal protein to prothrombin, 2,6-di- tert-butyl-4-methylene-2,5-cyclohexadienone, a major hepatic metabolite of BHT, did not inhibit carboxylation reactions dependent on vitamin K 1 hydroquinone or on phylloquinone (vitamin K 1) but did inhibit phylloquinone epoxide-dependent carboxylation. BHT administered to rats in the diet increased the level of microsomal prothrombin precursor and the extent of vitamin K-dependent carboxylation in the presence of dithiothreitol. These results suggest that 2,6-di- tert-butyl-4-methylene-2,5-cyclohexadienone inhibited phylloquinone epoxide reductase and, as a result, the prothrombin precursor accumulated in the microsomes of rats treated with BHT.

Quantitation of Radio-Labelled Vitamin K1 and Vitamin K1 Epoxide in Plasma by High Pressure Liquid Chromatography

A convenient, accurate and reproducible high pressure liquid chromatographic method for the quantitation of radio-labelled vitamin K1 and vitamin K1 epoxide in plasma is described. The method involves the determination of total ether extractable radioactivity, and a chromatographic separation to determin the relative quantities of radio-labelled vitamin K1 and vitamin K1 epoxide. The method is useful over a wide range of ratios of the two compounds, and has a coeffcient of variation of approximately 5%.

Effect of warfarin on the metabolism of phylloquinone (vitamin K1):dose-response relationships in man.

1. The dose—response relationship between the oral anticoagulant, warfarin, and its effect on the metabolism of phylloquinone (vitamin K1) has been examined in normal male volunteer subjects. 2. In each study the subject received a single, oral dose of warfarin and, 2 h later, an intravenous injection of [1′,2′-3H2]phylloquinone. Changes in the metabolism of phylloquinone were assessed by the fractionation and chromatographic separation of labelled phylloquinone and its metabolites in plasma and urine samples. 3. Increasing doses of warfarin did not affect the rate of disappearance of injected phylloquinone from the plasma but caused the accumulation of increasing amounts of the metabolite, phylloquinone epoxide. 4. Increasing doses of warfarin were found to decrease the proportion of labelled conjugates excreted in the urine as glucuronides and to block progressively the excretion of the normal aglycones of phylloquinone. At the same time there was a progressive increase in the excretion of at least three abnormal aglycones of phylloquinone. 5. The doses or plasma concentrations of warfarin were related to the increase in phylloquinone epoxide in the plasma and to the decrease in the proportion of normal aglycones of phylloquinone in urine by typical log dose—response curves, which were linear over the therapeutic range. 6. The nature of the metabolites detected suggested that the dose—response curves reflected the progressive inhibition by warfarin of the enzyme, phylloquinone epoxide reductase. 7. The results are consistent with the hypothesis that the pharmacological action of oral anticoagulants is closely linked to their ability to inhibit the cyclic interconversion of vitamin K and vitamin K epoxide.

Inhibition of prothrombin synthesis and epoxidation of vitamin K 1 by anticoagulants invitro

A number of 4-hydroxycoumarin and indanedione anticoagulatns completly inhibited prothrombin synthesis invitro at concentrations of less than 10 mM. Since the high concentration of vitamin K1 used to stumulate prothrombin synthesis made it unlikely that the anticoagulants were inhiting prothrombin synthesis by prevention the regeneration of vitamin from vitamin K1 epoxide, this suggested a second site of action of anticoagulants. This second site may be the epoxication of vitamin K since all the anticoagulants inhibited the epoxidation at concentrations less than 7 mM. 3-Phenyl-4-hydroxycoumarin, Dicumarol, Marcoumar and phenylindanedione were the most potent in inhibiting prothrombin synthesis and epoxidation while Warfarin, 4-hydrocoumarin and indanedione were the least effective.

Warfarin and metabolism of vitamin K 1

To further examine the hypothesis that warfarin inhibits prothrombin synthesis by interfering with the cyclic interconversion of vitamin K 1 and phylloquinone epoxide, the metabolism of tracer doses of labeled vitamin K 1 was studied in anticoagulant-treated rats. A tracer dose of [ 3H]K 1 initially turned over with a half-life of 2.2 hr in the liver, but after 5 hr the degradation rate decreased considerably. Warfarin caused hepatic [ 3H]K 1 levels to drop to 50 per cent of controls 5 min after administration of the vitamin, and over 10 hr the concentration of [ 3H]K 1 was 26–36 per cent of control levels. This decrease in vitamin K 1 was not large enough to account for the inhibition of prothrombin synthesis by warfarin. Thirty-five min after warfarin administration, there was more [ 3H]phylloquinone epoxide in the liver than labeled vitamin and over 10 hr the [ 3H]epoxide: [ 3H]K 1 ratio varied from 1.6 to 3.0. In addition to increasing the relative amount of [ 3H]epoxide, warfarin and phenylindanedione also increased hepatic metabolites more polar than vitamin K 1 or epoxide. However, warfarin did not increase plasma or urinary radioactivity. The urine was the principal excretory route for metabolites of vitamin K 1. 2-Diethylaminoethyl-2,2, diphenylvalerate hydrochloride (SKF 525-A) inhibited the turnover of [ 3H]K 1 but did not decrease the [ 3H]polar metabolites in the liver in control or warfarin-treated rats. The drug also did not inhibit the epoxidation of the vitamin to phylloquinone epoxide. The metabolism of vitamin K 1 was not altered by vitamin K deficiency or chronic administration of the vitamin. After a small dose of warfarin (35 μg/100 g body wt), plasma prothrombin decreased for 14 hr and then slowly rose but did not reach 90 per cent of normal until 3 days after administration of anticoagulant. The epoxide: K 1 ratio in the liver, measured by injecting a tracer dose of [ 3H]K 1, was 0.90 at 6 hr when prothrombin synthesis was completely blocked. The ratio dropped to 0.52 just before prothrombin started to rise at 14 hr and remained around 0.5 during the slow climb of plasma prothrombin toward the normal level. These low ratios were unexpected in animals in which prothrombin synthesis was partially or completely blocked. Hepatic epoxide: K 1 ratios of 11.4 and 1.3 were observed in warfarin-treated rats in which prothrombin synthesis was stimulated by injections of 0.1 mg [ 3H]K 1 plus 0.4 mg [ 3H]epoxide and 0.1 mg [ 3H]K 1 respectively. Therefore, there appears to be a lack of correlation of epoxide: K 1 ratios and prothrombin synthesis.

Ineffectiveness of phylloquinone epoxide as an inhibitor of prothrombin synthesis in the rat

Phylloquinone epoxide (vitamin K 1-oxide), a metabolite of phylloquinone, does not inhibit prothrombin synthesis when administered in high doses to Sprague-Dawley and warfarin-resistant rats. Further, it does not accumulate to presumed inhibitory levels in the livers of rats given physiological doses of 3H-phylloquinone when they are anticoagulated with warfarin. These data do not support the Bell-Matschiner hypothesis that warfarin exerts its action by inhibiting the vitamin K oxide reductase which results in the accumulation of vitamin K oxide and the inhibition of vitamin K at its active site. Rather, our data support the view that vitamin K and warfarin combine at different sites with a single regulatory protein which serves as a conformational switch for prothrombin synthesis.