Measurement of menadione in urine by HPLC

Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C₃₀ column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H₂O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R²) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3 pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine.     

Nature, intracellular distribution and formation of terpenoid quinones in maize and barley shoots

1. Maize and barley shoots have been shown to contain phylloquinone, plastoquinone, α-tocopherol (and γ-tocopherol in maize), α-tocopherolquinone and ubiquinone-9. 2. No solanesol was detected in any tissue examined. 3. In maize shoots plastoquinone and α-tocopherolquinone were localized in the chloroplast; ubiquinone was in the mitochondria. 4. Etiolated (dark-grown) shoots contained smaller amounts of phylloquinone and plastoquinone; α-tocopherolquinone was entirely absent; ubiquinone and α-tocopherol concentrations were unaffected. 5. On illumination of etiolated shoots the chloroplastidic quinones phylloquinone, plastoquinone and α-tocopherolquinone were synthesized in step with chloroplast development. α-Tocopherolquinone was not formed at the immediate expense of α-tocopherol.     

Asymmetric incorporation of 4-(2′-carboxyphenyl)-4-oxobutyrate into phylloquinone by Zea mays

Radioactivity from 4-(2′-carboxyphenyl)-4-oxobutyrate-[2-¹⁴C] and 4-(2′-carboxyphenyl)- 4-oxobutyrate-[3-¹⁴C] was incorporated into C-3 and C-2 respectively of phylloquinone in maize shoots. These results show that this substrate is incorporated in the same asymmetric manner into phylloquinone as it is into the bacterial menaquinones.     

Sulfated Purine Alkaloid Glycosides from the Pupal Case Built by the Bruchid Beetle Bruchidius dorsalis Inside the Seed of Gleditsia japonica

Three new sulfated isoguanine alkaloid glycosides, designated as saikachinoside A monosulfate ( 1 ), saikachinoside A disulfate ( 2 ), and locustoside B disulfate ( 3 ), have been isolated from the pupal case of the wild bruchid seed beetle Bruchidius dorsalis (Chrysomelidae, Bruchinae) infesting the seed of Gleditsia japonica Miq . (Fabaceae). Their structures were determined by spectroscopic methods and the inhibitory activity of 2 and 3 against acid phosphatase was evaluated.     

UDP-glucuronosyltransferases 1A6 and 1A10 catalyze reduced menadione glucuronidation

Menadione (2-methyl-1,4-naphthoquine), also known as vitamin K3, has been widely used as a model compound in the field of oxidative stress-related research. The metabolism of menadione has been studied, and it is known that menadione undergoes a two-electron reduction by NAD(P)H:Quinone oxidoreductase 1 (NQO1) after which the reduced form of menadione (2-methyl-1,4-naphthalenediol, menadiol) is glucuronidated and excreted in urine. To investigate which human UDP-glucuronosyltransferase (UGT) isoforms participate in the glucuronidation of menadiol reduced by NQO1 from menadione, we first constructed heterologously expressed NQO1 in Sf9 cells and tested the menadiol glucuronidating activity of 16 human recombinant UGT isoforms. Of the 16 UGT isoforms, UGTs 1A6, 1A7, 1A8, 1A9, and 1A10 catalyzed menadiol glucuronidation, and, of these, UGTs 1A6 and 1A10 catalyzed menadiol glucuronidation at much higher rates than the other UGTs. Menadiol was regioselectively glucuronidated in the manner of 4-position > 1-position by UGTs 1A7, 1A8, 1A9, and 1A10. In contrast to these UGTs, only UGT1A6 exhibited 1-menadiol-preferential glucuronidating activity. The results suggest possible detoxification pathways for quinones via NQO1 reduction followed by UGT glucuronidation.     

Bioluminescence of the arm light organs of the luminous squid Watasenia scintillans

The squid Watasenia scintillans emits blue light from numerous photophores. According to Tsuji [F.I. Tsuji, Bioluminescence reaction catalyzed by membrane-bound luciferase in the “firefly squid”, Watasenia scintillans, Biochim. Biophys. Acta 1564 (2002) 189–197.], the luminescence from arm light organs is caused by an ATP-dependent reaction involving Mg²⁺, coelenterazine disulfate (luciferin), and an unstable membrane-bound luciferase. We stabilized and partially purified the luciferase in the presence of high concentrations of sucrose, and obtained it as particulates (average size 0.6–2 µm). The ATP-dependent luminescence reaction of coelenterazine disulfate catalyzed by the particulate luciferase was investigated in detail. Optimum temperature of the luminescence reaction is about 5 °C. Coelenterazine disulfate is a strictly specific substrate in this luminescence system; any modification of its structure resulted in a very heavy loss in its light emission capability. The light emitter is the excited state of the amide anion form of coelenteramide disulfate. The quantum yield of coelenterazine disulfate is calculated at 0.36. ATP could be replaced by ATP-γ-S, but not by any other analogues tested. The amount of AMP produced in the luminescence reaction was much smaller than that of coelenteramide disulfate, suggesting that the reaction mechanism of the Watasenia bioluminescence does not involve the formation of adenyl luciferin as an intermediate.     

Bioluminescence of the arm light organs of the luminous squid Watasenia scintillans

The squid Watasenia scintillans emits blue light from numerous photophores. According to Tsuji [F.I. Tsuji, Bioluminescence reaction catalyzed by membrane-bound luciferase in the "firefly squid", Watasenia scintillans, Biochim. Biophys. Acta 1564 (2002) 189-197.], the luminescence from arm light organs is caused by an ATP-dependent reaction involving Mg2+, coelenterazine disulfate (luciferin), and an unstable membrane-bound luciferase. We stabilized and partially purified the luciferase in the presence of high concentrations of sucrose, and obtained it as particulates (average size 0.6-2 microm). The ATP-dependent luminescence reaction of coelenterazine disulfate catalyzed by the particulate luciferase was investigated in detail. Optimum temperature of the luminescence reaction is about 5 degrees C. Coelenterazine disulfate is a strictly specific substrate in this luminescence system; any modification of its structure resulted in a very heavy loss in its light emission capability. The light emitter is the excited state of the amide anion form of coelenteramide disulfate. The quantum yield of coelenterazine disulfate is calculated at 0.36. ATP could be replaced by ATP-gamma-S, but not by any other analogues tested. The amount of AMP produced in the luminescence reaction was much smaller than that of coelenteramide disulfate, suggesting that the reaction mechanism of the Watasenia bioluminescence does not involve the formation of adenyl luciferin as an intermediate.     

Incorporation of shikimate and 4-(2′-carboxyphenyl)-4-oxobutyrate into phylloquinone

The patterns of incorporation of d-[G-¹⁴C]shikimate and variously labelled ¹⁴C-4-(2′-carboxy-phenyl)-4-oxobutyrate into the naphthoquinone nucleus of phylloquinone by maize shoots have been investigated. The results show that (a) the alicyclic ring and C-7 of shikimate give rise to Ring A and either C-1 or C-4, and (b) the phenyl ring, 2′-carboxy and C-4, and C-2 and -3 of 4-(2′-carboxyphenyl)-4-oxobutyrate give rise to Ring A, C-1 and -4 and C-2 and -3. Radioactivity from α-[1-¹⁴C]naphthol, 1,4-[1,4-¹⁴C]naphthoquinone and [Me-¹⁴C]menadione is not incorporated into phylloquinone to any significant extent.     

Chlorophyll a phytylation is required for the stability of photosystems I and II in the cyanobacterium Synechocystis sp. PCC 6803

In oxygenic phototrophic organisms, the phytyl ‘tail’ of chlorophyll a is formed from a geranylgeranyl residue by the enzyme geranylgeranyl reductase. Additionally, in oxygenic phototrophs, phytyl residues are the tail moieties of tocopherols and phylloquinone. A mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking geranylgeranyl reductase, ΔchlP, was compared to strains with specific deficiencies in either tocopherols or phylloquinone to assess the role of chlorophyll a phytylatation (versus geranylgeranylation). The tocopherol‐less Δhpt strain grows indistinguishably from the wild‐type under ‘standard’ light photoautotrophic conditions, and exhibited only a slightly enhanced rate of photosystem I degradation under strong irradiation. The phylloquinone‐less ΔmenA mutant also grows photoautotrophically, albeit rather slowly and only at low light intensities. Under strong irradiation, ΔmenA retained its chlorophyll content, indicative of stable photosystems. ΔchlP may only be cultured photomixotrophically (due to the instability of both photosystems I and II). The increased accumulation of myxoxanthophyll in ΔchlP cells indicates photo‐oxidative stress even under moderate illumination. Under high‐light conditions, ΔchlP exhibited rapid degradation of photosystems I and II. In conclusion, the results demonstrate that chlorophyll a phytylation is important for the (photo)stability of photosystems I and II, which, in turn, is necessary for photoautotrophic growth and tolerance of high light in an oxygenic environment.     

Menadiol diphosphate,a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry

Summary Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce³⁺ ions and the DAB−Ni−H₂O₂ procedure yielded better results than Ca²⁺ ions in the Co−(NH₄)₂S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce³⁺ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT. Supported by the Alexander von Humboldt-Stiftung and the Deutsche Forschungsgemeinschaft (Sfb 174)     

Menadiol diphosphate, a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry.

Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce3+ ions and the DAB-Ni-H2O2 procedure yielded better results than Ca2+ ions in the Co-(NH4)2S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce3+ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT.     

Biosynthesis of phytoquinones. Incorporation of l-[Me-14C3H]methionine into terpenoid quinones and chromanols in maize shoots

1. Radioactivity from l-[Me-(14)C,(3)H]methionine is incorporated into phylloquinone, plastoquinone, gamma-tocopherol, alpha-tocopherol, alpha-tocopherolquinone and ubiquinone in maize shoots. 2. Comparative studies with other terpenoids (squalene and beta-carotene) and chemical degradation of selected quinones (ubiquinone and plastoquinone) established that all the radioactivity is confined to nuclear methyl substituents. 3. In ubiquinone 76% of the radioactivity is in the methoxyl groups and 24% in the ring C-methyl group. 4. Taking the phytosterols as an internal reference and accepting the atomic ratio of (14)C/(3)H transferred from l-[Me-(14)C,(3)H]methionine to the supernumerary group at C(24) to be 1:2 the ratio of all the quinones and chromanols examined approached 1:3. After allowing for the fact that for plastoquinone, gamma-tocopherol, alpha-tocopherol and alpha-tocopherolquinone one nuclear methyl group is formed from the beta-carbon of tyrosine, these results show that one nuclear C-methyl group for phylloquinone, plastoquinone and gamma-tocopherol, two nuclear methyl groups for alpha-tocopherol and alpha-tocopherolquinone and one nuclear methyl and two methoxyl groups for ubiquinone are formed by the transfer of intact methyl groups from methionine. 5. From a comparison of the incorporation of (14)C radioactivity into these compounds it would appear that the methylation reactions involved in phylloquinone and plastoquinone biosynthesis take place in the chloroplast, whereas those involved with ubiquinone biosynthesis occur else-where within the cell.     

集胞藻 PCC 6803中Sll0875蛋白参与调控光系统I活性的生物功能研究

叶绿醌是由1个萘醌环和1个半不饱和植基侧链组成的一类光系统Ⅰ（photosystem Ⅰ,PSⅠ）特有的辅因子。目前,在蓝藻中对其生物合成途径的研究主要集中在萘醌环的形成方面,而对其植基侧链的合成尚缺乏相关报道。本研究通过与近期在拟南芥中发现的1种催化植基单磷酸形成植基二磷酸的激酶（VTE6）进行同源序列比对,在集胞藻 PCC 6803中发现1个与之高度同源的蛋白质Sll0875。研究发现,在Sll0875缺失突变体中,叶绿醌和生育酚的含量缺失,叶绿素的含量降低（P<0.05）,且该突变体在无葡萄糖培养基中生长迟缓。进一步利用叶绿素荧光、P700氧化还原动力学、77K低温荧光光谱和免疫印迹分析等方法分析了该蛋白质的缺失对PSⅠ功能的影响。研究表明,在突变体Δsll0875中, PSⅠ活性下降,PSⅠ亚基含量与野生型相比显著降低（P<0.01）。这一结果表明,叶绿醌的缺失影响了PSⅠ复合物的累积,导致PSⅠ功能受损,从而影响了蓝藻正常的生长和发育。本研究在蓝藻中证实植醇磷酸化途径对叶绿醌合成的重要性,为进一步研究蓝藻中叶绿醌在PSⅠ复合物的合成、组装和稳定等过程中的作用奠定基础。     

Studies on the metabolites of phylloquinone (vitamin K1) in the urine of man

The nature of the metabolites excreted in the urine was investigated up to 48 h after oral and intravenous administration of 0.3 to 1.3 mg [1′,2′-³H₂]phylloquinone. The metabolites were water-soluble of which the major fraction consisted of glucuronide conjugates. A chromatographic comparison of the aglycone fragments released by β-glucuronidase and by dilute HCl revealed the presence of at least three labelled aglycones. The major aglycones obtained by enzyme hydrolysis consisted of at least two closely related organic acids which were not separated by adsorption thin-layer chromatography but one of which on treatment with dilute acid yielded a neutral metabolite with the chromatographic properties of phylloquinone γ-lactone. The results suggest that phylloquinone γ-lactone, the only previously isolated urinary metabolite of phylloquinone, is an artifact produced by the conditions of acid hydrolysis. Although the acid labile aglycone was the minor component of the two acid metabolites, its proportion in urine extracts as measured by conversion to the lactone, increased with the time after administration of labelled phylloquinone.     

Prenylation and methylation reactions in phylloquinone (vitamin K1) synthesis in Capsicum annuum plastids

The biosynthesis of phylloquinone (vitamin K₁) was examined using Capsicum fruit chloroplasts and chromoplasts (apparently phylloquinone free). In both cases, the synthesis of phylloquinone from -naphthoquinone, dihydro--naphthoquinone, 1,4-dihydroxy-2-naphthoic acid (as precursors of the ring moiety) and (S)-adenosyl-L-methionine was achieved. In the presence of phytylpyrophosphate, the biosynthesis of phylloquinone in both organelles is particularly enhanced when 1,4-dihydroxy-2-naphthoic acid is used.     

Selective inhibition of glutathione S-transferases by 17 beta-estradiol disulfate.

Enzyme activity of homogeneous glutathione S-transferases A, B, and C with reduced glutathione and 1-chloro-2,4-dinitrobenzene was inhibited in varying degrees by 50 μm concentrations of monosulfate and disulfate derivatives of several steroids. In contrast, transferase AA activity was not affected. Of the inhibitors tested, estradiol-3,17-disulfate and estradiol-3-sulfate were the most inhibitory, followed by pregnenolone sulfate, estradiol-17-sulfate, dehydroisoandrosterone sulfate, and cortisol sulfate. Transferases A and C were most affected, especially by estradiol disulfate and estradiol-3-sulfate, which exhibited essentially complete inhibition at a concentration of μm. Double reciprocal plots of estradiol disulfate inhibition with respect to 1-chloro-2,4-dinitrobenzene concentration showed uncompetitive inhibition with transferases A and C and noncompetitive inhibition with transferase B (ligandin). With reduced glutathione as the variable substrate, transferases A and C exhibited noncompetitive inhibition kinetics, while transferase B showed partial noncompetitive kinetics.     

Partial Purification and Characterization of the Quinol Oxidase Activity of Arum maculatum Mitochondria

The menadiol oxidase activity of Arum maculatum mitochondria has been solubilized and fractionated. A preparation has been obtained which has an increased specific activity and a greatly decreased polypeptide composition when compared to the mitochondria. This preparation retains normal inhibitor sensitivities in that the oxidation of menadiol remains insensitive to cyanide and is inhibited by aromatic hydroxamates. Metal analyses of the preparation showed that only iron was closely correlated with the oxidase activity. No unusual lipid components were detected in the preparation. The results are discussed in relation to chemical quinol oxidation mechanisms and to several recent hypotheses concerning the nature of the higher plant alternative oxidase.     

Steroid Compounds from the Far Eastern Starfish Diplopteraster multipes

Sodium salt of (20R)-3,4-dihydroxycholest-5-ene-21-yl sulfate and disodium salts of (20R)-4-hydroxycholest-5-ene-3,21-diyl disulfate, (20R)-24-methylcholest-5,24(28)-diene-3,21-diyl disulfate, (20R)-24-methyl-5-cholest-24(28)-ene-3,21-diyl disulfate, (20R)-cholest-5-ene-3,21-diyl disulfate, (20R)-5-cholestane-3,21-diyl disulfate, and (20R)-3-hydroxycholest-5-ene-2,21-diyl disulfate were isolated from the far eastern starfish Diplopteraster multipes and characterized. These compounds differ structurally from sulfated polyhydroxysteroids in other starfish species. At the same time, they are typical secondary metabolites of Ophiuroidea and have some structural features characteristic of the ophiuroid-isolated steroids, namely the 3-hydroxy (or 3-sulfoxy) and 21-sulfoxy groups. These data support the opinion of some taxonomists that starfishes and ophiuroids are phylogeneteically related classes and are closer to each other than to other classes of the Echinodermata phylum.