Conversion of Phylloquinone (Vitamin K1) into Menaquinone-4 (Vitamin K2) in Mice

There are two forms of naturally occurring vitamin K, phylloquinone and the menaquinones. Phylloquinone (vitamin K1) is a major type (>90%) of dietary vitamin K, but its concentrations in animal tissues are remarkably low compared with those of the menaquinones, especially menaquinone-4 (vitamin K2), the major form (>90%) of vitamin K in tissues. Despite this great difference, the origin of tissue menaquinone-4 has yet to be exclusively defined. It is postulated that phylloquinone is converted into menaquinone-4 and accumulates in extrahepatic tissues. To clarify this, phylloquinone with a deuterium-labeled 2-methyl-1,4-naphthoquinone ring was given orally to mice, and cerebra were collected for D NMR and liquid chromatography-tandem mass spectrometry analyses. We identified the labeled menaquinone-4 that was converted from the given phylloquinone, and this conversion occurred following an oral or enteral administration, but not parenteral or intracerebroventricular administration. By the oral route, the phylloquinone with the deuterium-labeled side chain in addition to the labeled 2-methyl-1,4-naphthoquinone was clearly converted into a labeled menaquinone-4 with a non-deuterium-labeled side chain, implying that phylloquinone was converted into menaquinone-4 via integral side-chain removal. The conversion also occurred in cerebral slice cultures and primary cultures. Deuterium-labeled menadione was consistently converted into the labeled menaquinone-4 with all of the administration routes and the culture conditions tested. Our results suggest that cerebral menaquinone-4 originates from phylloquinone intake and that there are two routes of accumulation, one is the release of menadione from phylloquinone in the intestine followed by the prenylation of menadione into menaquinone-4 in tissues, and another is cleavage and prenylation within the cerebrum. There are two forms of naturally occurring vitamin K, phylloquinone and the menaquinones. Phylloquinone (vitamin K1) is a major type (>90%) of dietary vitamin K, but its concentrations in animal tissues are remarkably low compared with those of the menaquinones, especially menaquinone-4 (vitamin K2), the major form (>90%) of vitamin K in tissues. Despite this great difference, the origin of tissue menaquinone-4 has yet to be exclusively defined. It is postulated that phylloquinone is converted into menaquinone-4 and accumulates in extrahepatic tissues. To clarify this, phylloquinone with a deuterium-labeled 2-methyl-1,4-naphthoquinone ring was given orally to mice, and cerebra were collected for D NMR and liquid chromatography-tandem mass spectrometry analyses. We identified the labeled menaquinone-4 that was converted from the given phylloquinone, and this conversion occurred following an oral or enteral administration, but not parenteral or intracerebroventricular administration. By the oral route, the phylloquinone with the deuterium-labeled side chain in addition to the labeled 2-methyl-1,4-naphthoquinone was clearly converted into a labeled menaquinone-4 with a non-deuterium-labeled side chain, implying that phylloquinone was converted into menaquinone-4 via integral side-chain removal. The conversion also occurred in cerebral slice cultures and primary cultures. Deuterium-labeled menadione was consistently converted into the labeled menaquinone-4 with all of the administration routes and the culture conditions tested. Our results suggest that cerebral menaquinone-4 originates from phylloquinone intake and that there are two routes of accumulation, one is the release of menadione from phylloquinone in the intestine followed by the prenylation of menadione into menaquinone-4 in tissues, and another is cleavage and prenylation within the cerebrum. Vitamin K is a cofactor for γ-glutamyl carboxylase (GGCX), 2The abbreviations used are: GGCX, γ-glutamyl carboxylase; Gla, γ-carboxyglutamic acid; PK, phylloquinone; MK, menaquinone; LC-MS/MS, liquid chromatography-tandem mass spectrometry; K3-d8, D-labeled menadione; HPLC, high-performance liquid chromatography; APCI, atmospheric pressure chemical ionization; MRM, multiple reaction monitoring; MEM, minimal essential medium; E, embryonic day; L-DMEM, low glucose Dulbecco's modified Eagle's medium; FCS, fetal calf serum. an enzyme that converts specific glutamic acid residues in several substrate proteins to γ-carboxyglutamic acid (Gla) residues (1Furie B. Furie B.C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (995) Google Scholar, 2Wallin R. Hutson S.M. Trends Mol. Med. 2004; 10: 299-302Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 3Furie B. Furie B.C. New Eng. J. Med. 1992; 326: 800-806Crossref PubMed Scopus (457) Google Scholar). Gla residues serve to form calcium-binding groups in proteins and are essential for their biologic activity. Gla-containing proteins are involved in blood coagulation (4Suttie J.W. CRC Crit. Rev. Biochem. 1980; 8: 191-223Crossref PubMed Scopus (155) Google Scholar), bone metabolism (5Price P.A. Annu. Rev. Nutr. 1988; 8: 565-583Crossref PubMed Scopus (151) Google Scholar), vascular repair (6Benzakour O. Kanthou C. Blood. 2000; 95: 2008-2014Crossref PubMed Google Scholar), prevention of vascular calcification (7Shanahan C.M. Proudfood D. Farzaneh-Far A. Weissberg P.L. Crit. Rev. Eukaryot. Gene Expr. 1998; 8: 357-375Crossref PubMed Scopus (148) Google Scholar), regulation of cell proliferation, and signal transduction (8Tsaioun K.I. Nutr. Rev. 1999; 57: 231-240Crossref PubMed Scopus (48) Google Scholar). Vitamin K undergoes a cyclic interconversion, the vitamin K cycle, comprising reduction of the vitamin K quinone form into the hydroquinone, oxidation to 2,3-epoxide (vitamin K epoxide), and reduction to the quinone. The formation of Gla from glutamate is coupled with the conversion of the hydroquinone to the vitamin K epoxide. Both of these activities occur in GGCX. The warfarin-sensitive microsomal enzyme, vitamin K epoxide reductase, recycles the vitamin K epoxide back to the hydroquinone, thus completing the vitamin K cycle (9Stafford D.W. J. Thromb. Haemostasis. 2005; 3: 1873-1878Crossref PubMed Scopus (309) Google Scholar, 10Carlisle T.L. Suttie J.W. Biochemistry. 1980; 19: 1161-1167Crossref PubMed Scopus (73) Google Scholar, 11Bristol J.A. Ratcliffe J.V. Roth D.A. Jacobs M.A. Furie B.C. Furie B. Blood. 1996; 88: 2585-2593Crossref PubMed Google Scholar). Natural vitamin K exists in two molecular forms, phylloquinone (PK) or vitamin K1 and the menaquinones (MKs). All forms of vitamin K have 2-methyl-1,4-naphthoqinone as a common ring structure, but individual forms differ in the length and degree of saturation of a variable aliphatic side chain attached to the 3-position. PK is a single compound and contains a mono-unsaturated side chain of four isoprenoid residues and is found primarily in plants in association with chlorophyll. MKs can be classified into 15 types based on the length of their unsaturated side chains. They are denominated as MK-n, where n denotes the number of isoprenyl residues in the side chain (12Suttie J.W. Annu. Rev. Nutr. 1995; 15: 399-417Crossref PubMed Scopus (197) Google Scholar). The MKs most commonly found in foods are menaquinone-4 (MK-4, vitamin K2), which is regarded as a short-chain menaquinone, and the long-chain menaquinones MK-7 through MK-10 exclusively synthesized by bacteria and gut microflora in mammals (13Seegers W.H. Thromb. Diath. Haemorrh. 1965; 14: 213-228PubMed Google Scholar). Menadione or vitamin K3 (K3) is a synthetic compound lacking a side chain but is believed to be biologically active by virtue of its conversion into MK-4 in the body before being active as a cofactor for GGCX (14Taggart W.V. Matschiner J.T. Biochemistry. 1969; 8: 1141-1146Crossref PubMed Scopus (58) Google Scholar). PK is known to be selectively distributed in a number of hepatic and non-hepatic tissues. In rats fed a conventional laboratory chow diet, the heart contains as much PK as the liver, but the brain appears to have low concentrations. Interestingly, MK-4 is found in most tissues. In general, tissue concentrations of MK-4 exceed those of PK except for liver, where relatively low MK-4 levels are found. Exocrine organs, such as the pancreas and the salivary gland, contain large amounts of MK-4. The brain also contains high MK-4 concentrations. Similar patterns of the tissue-specific distribution of vitamin K are observed in animals and humans (15Thijssen H.H.W. Drittij-Reijnders M.J. Br. J. Nutr. 1994; 72: 415-425Crossref PubMed Scopus (120) Google Scholar, 16Thijssen H.H.W. Drittij-Reijnders M.J. Br. J. Nutr. 1996; 75: 121-127Crossref PubMed Scopus (129) Google Scholar). Will et al. (17Will B.H. Usui Y. Suttie J.W. J. Nutr. 1992; 122: 2354-2360Crossref PubMed Scopus (55) Google Scholar) reported that the livers of chicks fed PK as a sole source of vitamin K contained as much MK-4 as PK. Thijssen et al. (18Thijssen H.H.W. Drittij-Reijnders M.J. Fischer M.A.J.G. J. Nutr. 1996; 126: 537-543Crossref PubMed Scopus (94) Google Scholar) reported that extrahepatic tissues contained more MK-4 in rats fed a PK-rich diet than in rats fed an MK-4-rich diet. Because gut flora do not produce as much MK-4 as MKs with a long side chain (n = 6–9) and tissue MK-4 concentrations are not significantly different between normal rats and germ-free rats, and additionally, MK-4 is not abundantly present in normal food products, the MK-4 in rat and human tissues may originate from the conversion of PK in the body (19Davidson R.T. Foley A.L. Engelke J.A. Suttie J.W. J. Nutr. 1998; 128: 220-223Crossref PubMed Scopus (117) Google Scholar, 20Roden J.E. Drittij-Reijnders M.J. Vermeer C. Thijssen H.H.W. Biochim. Biophys. Acta. 1998; 1379: 69-75Crossref PubMed Scopus (65) Google Scholar). However, this speculation has yet to be demonstrated correct since the first report by Billeter and Martius in 1960 (21Billeter M. Martius C. Biochem. Z. 1960; 333: 430-439Google Scholar). If the speculation is correct, two possible routes for the conversion of PK to MK-4 can be taken into account; one is the desaturation of the phytyl side chain of phylloquinone to produce the geranylgeranyl group of MK-4, and removal of the phytyl side chain to release K3, followed by geranylgeranylation to form MK-4. In the latter, two possible pathways are postulated; one is that the side-chain removal occurs during intestinal absorption and then the released K3 is transferred to tissues via the bloodstream and thereafter is prenylated to form MK-4 (22Thijssen H.H.W. Vervoort L.M.T. Schurgers L.J. Shearer M.J. Br. J. Nutr. 2006; 95: 260-266Crossref PubMed Scopus (127) Google Scholar). The alternative is that, after the transfer of PK into tissues, side-chain cleavage and geranylgeranylation occur simultaneously within tissues. In the present study, we examined which routes are responsible for the conversion of PK to MK-4. Stable isotope-labeled compounds are particularly useful for distinguishing the behavior of exogenous compounds from that of the corresponding endogenous compounds on the basis of structural assignments by NMR spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). We synthesized deuterium (D)- or heavy oxygen (18O)-labeled forms of PK and MK-4 in our laboratory. Using these compounds, we were able to obtain unequivocal evidence of the origin of MK-4 in the cerebra of mice. In this report, we present evidence that cerebral MK-4 originates from not only systemic conversion comprising the release of menadione from PK in the intestine and the prenylation of menadione into MK-4 in the cerebra but also the in-cell conversion of PK into MK-4 in cerebra. Our findings suggest that MK-4, a transcriptional regulator of steroid and xenobiotic receptor-mediated signaling (23Ichikawa T. Horie-Inoue K. Ikeda K. Blumberg B. Inoue S. J. Biol. Chem. 2006; 281: 16927-16934Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) as well as a cofactor for GGCX, is not simply a dietary nutrient, but should be regarded as an active form of vitamin K that may contribute to neural functions in mammals. Materials—PK and MK-4 were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. D-labeled menadione (K3-d8) was purchased from C/D/N Isotopes, Inc. (Quebec, Canada). PK epoxide, MK-4 epoxide, 18O-labeled PK (PK-18O), and 18O-labeled MK-4 (MK-4-18O) were respectively synthesized in our laboratory as reported previously (24Suhara Y. Murakami A. Nakagawa K. Mizuguchi Y. Okano T. Bioorg. Med. Chem. 2006; 14: 6601-6607Crossref PubMed Scopus (19) Google Scholar, 25Suhara Y. Kamao M. Tsugawa N. Okano T. Analyt. Chem. 2005; 77: 757-763Crossref PubMed Scopus (96) Google Scholar). D-labeled PK (PK-d7 and PK-d9), D-labeled PK epoxide (PK epoxide-d7), D-labeled MK-4 (MK-4-d7 and MK-4-d9), and D-labeled MK-4 epoxide (MK-4 epoxide-d7) were also synthesized in our laboratory. The synthesis and unambiguous physicochemical assignments of these D-labeled vitamin K compounds will be described elsewhere. Culture media and antibiotics were purchased from Nakalai Tesque, Kyoto Japan. D-labeled chloroform (CDCl3, 99.8%, NMR analytical grade) was obtained from EURISO-TOP (Gif-Sur-Yvette, France). Organic solvents of HPLC grade were purchased from Wako Pure Chemical Industries, Ltd. Animals and Diets—Male and Female C57BL/6 mice at the age of 7 weeks were obtained from Japan SLC, Inc., Hamamatsu, Japan and were fed a commercially available normal laboratory chow diet (F-2 diet, Oriental Yeast Co., Ltd., Tokyo, Japan) for 1 week. They were housed 5 per cage with a 12-h light-dark cycle under controlled environmental conditions (temperature: 20 ± 2 °C, humidity: 50 ± 5%). All animals were allowed free access to diet and deionized water for a period of 1 week. The protocols for the experiments were approved by the Guidelines for the Care and Use of Laboratory Animals of Kobe Pharmaceutical University. Measurements of PK, MK-4, and Their Respective Epoxides in Tissues and Plasma of Mice—At 8 weeks of age, blood was withdrawn by heart puncture under light diethyl ether anesthesia, and plasma was obtained by centrifugation at 3000 rpm for 10 min. Immediately after death, tissues were excised, immersed and rinsed in ice-cold saline, and stored at –80 °C until the assays. Tissue concentrations of PK, MK-4, and their respective epoxides were assayed as previously described (24Suhara Y. Murakami A. Nakagawa K. Mizuguchi Y. Okano T. Bioorg. Med. Chem. 2006; 14: 6601-6607Crossref PubMed Scopus (19) Google Scholar, 25Suhara Y. Kamao M. Tsugawa N. Okano T. Analyt. Chem. 2005; 77: 757-763Crossref PubMed Scopus (96) Google Scholar). Because vitamin K is sensitive to light, all procedures were performed under a dark light. Briefly, tissues (wet weight, 1–2 g) were pulverized thoroughly with anhydrous sodium sulfate (1:10, w/v) and transferred into a brown-colored glass tube with a Teflon-lined screw cap. The homogenates were added to 0.1 ml of ethanol containing PK-18O and MK-4-18O as internal standards, 0.9 ml of ethanol, and 9 ml of acetone, then mixed thoroughly with a Voltex mixer for 3 min, and allowed to stand for 5 min. This procedure was repeated three times. The resulting mixture was centrifuged at 3000 rpm for 5 min at 4 °C, and the upper layer was transferred into a small brown-glass tube and evaporated dry under reduced pressure. The residue was dissolved in 2 ml of water and 6 ml of hexane, mixed thoroughly, and centrifuged at 3000 rpm for 5 min at 4 °C. The upper layer was loaded onto a Sep Pak Vac Silica cartridge column (Waters). The column was eluted with 5 ml of hexane to remove concomitants, and thereafter the vitamin K-containing fraction was eluted with 5 ml of hexane and diethyl ether (97:3) solution. The eluate was evaporated under reduced pressure, and the residue was dissolved in 60 μl of methanol. An aliquot of this solution was subjected to APCI3000 LC-MS/MS (Applied Biosystems, Foster City, CA). The HPLC analyses were conducted with a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) consisting of a binary pump (LC-10AD liquid chromatography), automatic solvent degasser (DGU-14A degasser), and autosampler (SIL-10AD autoinjector). Separations were carried out using a reversed-phase C18 column (Capcell PAK C18 UG120, 5 μm; 4.6 mm inner diameter × 250 mm, Shiseido, Tokyo, Japan) with a solvent system consisting of an isocratic solvent A (25 min) and then a linear gradient from 0 to 50% ethanol (50 min). Solvent A contained methanol/0.1% acetic acid aqueous (95:5, v/v) and was delivered at 1.0 ml/min. This mobile phase was passed through the column at 1.0 ml/min. The column was maintained at 35 °C with a column oven (CTO-10AC column oven). All MS data were collected in the positive ion mode with atmospheric pressure chemical ionization (APCI). The following settings were used: corona discharge needle voltage, 5.5 kV; vaporizer temperature, 400 °C; sheath gas (high purity nitrogen) pressure, 50 p.s.i.; and transfer capillary temperature, 220 °C. The electron multiplier voltage was set at 850 eV. Identification and quantification were based on MS/MS using a multiple reaction monitoring (MRM) mode. The range for the parent scan was 400–500 atomic mass units. MRM transitions (precursor ion and product ion, m/z) and retention time (min) for each analyte were as follows: MK-4: precursor ion, 445.4; product ion, 187.2; retention time, 20.8; MK-4-epoxide: precursor ion, 461.3; product ion, 161.0; retention time, 14.6; MK-4-18O: precursor ion, 449.3; product ion, 191.2; retention time, 20.8; PK: precursor ion, 451.3; product ion, 187.1; retention time, 41.3; PK epoxide: precursor ion, 467.4; product ion, 161.2; retention time, 27.2; and PK-18O: precursor ion, 455.4; product ion, 191.3; retention time, 41.3. Calibration, using internal standardization, was done by linear regression with five different concentrations, 12.5, 50, 200, 800, and 1600 ng/ml. Isolation and Identification of MK-4-d7 in Cerebra of Mice Orally Given PK-d7—At 8 weeks of age, 200 female mice were orally given PK-d7 as a single dose of 10 μmol/kg body weight, and their cerebra were collected at 24 h post-administration and stored at –80 °C for analysis. The cerebra (total wet weight, 60 g) were divided into 40 portions (each ∼1.5 g) for the purification of MK-4-d7. MK-4-d7 in each cerebral sample was extracted and purified with the same procedures as described in the measurement of MK-4-d7 in tissues. Subsequently, the sample solution dissolved in 100 μl of methanol was subjected to purification using a Shimadzu HPLC system consisting of a C-R4A Chromatopac, SPD-6A UV spectrophotometric detector, and LC-6A liquid chromatograph. Separations were carried out using a reversed-phase C18 column (COSMOSIL 5C18 ARII, 10 mm inner diameter × 250 mm, Nakalai Tesque) with a mobile phase containing methanol/ethanol (95/5). This mobile phase was passed through the column at 4.0 ml/min. Samples with a peak corresponding to authentic MK-4 were collected and evaporated dry, then re-dissolved in the mobile phase. After further purification of the combined samples with the same HPLC system, the sample was pure enough for 1H NMR, D NMR, and LC-MS/MS analyses. The 500-MHz H NMR and D NMR spectra of the putative MK-4 and MK-4-d7 were measured on a Varian VNS-500 (H: 500 MHz, D: 77 MHz). The sample was first dissolved in 40 μl of CDCl3 in a nanoprobe for 1H NMR spectrometry, and then after evaporation with a stream of nitrogen gas, the residue was dissolved in CHCl3 for D NMR spectrometry. The LC-MS/MS analysis was carried out with the same method as described above. Comparison of MK-4-d7 Accumulation in Cerebra of Mice Administered PK-d7 or K3-d8 via Four Routes—At 8 weeks of age, 5 female mice in each group were given orally, intravenously, or enterally, either PK-d7 or K3-d8 as a single dose of 10 μmol/kg body weight or intracerebroventricularly either PK-d7 or K3-d7 as a single dose of 0.1 μmol/kg body weight. To examine whether the conversion of PK-d7 or K3-d8 into MK-4-d7 occurs in a dose-dependent manner, 5 female mice in each group were given orally either PK-d7 or K3-d8 as a single dose of 0.1, 1.0, or 10 μmol/kg body weight. After 24 h, the mice were killed, and cerebra were removed and stored at –80 °C for analysis. The measurements of MK-4-d7 and MK-4 epoxide-d7 in cerebra were carried out with the same LC-APCI-MS/MS method as described above. Comparison of the Conversion of PK-d7 or K3-d8 to MK-4-d7 in Mouse Cerebral Slice Culture and Embryonic Primary Culture—At 8 weeks of age, female mice were sacrificed by aortic exsanguination under light diethyl ether anesthesia. Cerebra were excised and cut into 1-mm-thick slices. Two slices were placed on a stainless steel mesh in a culture dish (60-mm inner diameter) and cultured in 6 ml of medium containing minimum essential medium (MEM): Hepes buffered-saline solution (2:1) with 25% horse serum (Invitrogen) in the presence or absence (vehicle; ethanol) of PK-d7 or K3-d8 (10–5 m) at 37°C in 5% CO2 in a humidified atmosphere for 24 h. The slices were washed with cold Ca,Mg-free phosphate-buffered saline 2 times and homogenized using a Dounce-type homogenizer with 1 ml of MilliQ water. The homogenates (20 μl) were used to determine protein concentrations with a BCA protein assay kit (Pierce). Using other homogenates, the measurements of MK-4-d7 and MK-4 epoxide-d7 were carried out with the same LC-APCI-MS/MS method as described above. Primary cultures of mouse cerebral hemispheres on embryonic day (E) 14 were prepared as described previously with minor modifications (26Fabre M. Langley O.K. Bologa L. Delaunoy J.P. Lowenthal A. FerretSena V. Vincendon G. Sarlieve L.L. Dev. Neurosci. 1985; 7: 323-339Crossref PubMed Scopus (15) Google Scholar). Fetuses (E14) were removed from pregnant C57BL/6J mice. Embryonic cerebral hemispheres were collected and incubated in the L-15 dissociation medium (Sigma) containing trypsin (0.25 mg/ml) and DNase I (0.1 mg/ml). After treatment, cells were dispersed by repeated trituration and suspended in low glucose Dulbecco's modified Eagle's medium (L-DMEM) with 10% FCS. The suspended cells were plated at a density of 8 × 106 cells/well on polyethyleneimine (Sigma)-coated 6-well tissue culture plates. The cells were cultured in L-DMEM with 10% FCS at 37 °C in 5% CO2 in a humidified atmosphere for 2 days. Then, the cells were incubated with or without PK-d7 or K3-d8 (10–6 m) in the L-DMEM at 37 °C in 5% CO2 in a humidified atmosphere for 24 h. The amounts of MK-4-d7 and MK-4 epoxide-d7 generated from PK-d7 or K3-d8 by the cells were measured by the LC-APCI-MS/MS method described above. Embryonic cerebral cells were cultured in L-DMEM with 10% FCS for 2 days and thereafter divided into two sub-culture groups, one for neurons and another for astrocytes (27Mains R.E. Patterson P.H. J. Cell Biol. 1973; 59: 329-345Crossref PubMed Scopus (307) Google Scholar, 28Mary E. Hatten M.E. Liem R.K.H. J. Cell Biol. 1981; 90: 622-630Crossref PubMed Scopus (129) Google Scholar). For the neurons, the primary cerebral cells were cultured in L-DMEM containing 10% FCS along with 50 μm 1-β-d-arabinofuranosylcytosine for 2 days. For the astrocytes, the primary cells were dissociated by incubation in 0.1% trypsin-EDTA solution and cultured in L-DMEM medium containing 10% FCS for 2 days. The cells were suspended in L-DMEM containing 10% FCS, plated at a density of 107 cells/plate (60 mm), and cultured for 2 days. The cells that reached confluency were dissociated by incubation in 0.1% trypsin-EDTA solution. The cells were suspended in L-DMEM containing 10% FCS and plated onto 6-well tissue culture plates at a density of 106 cells/well for 2 days to reach confluency. Both cells were treated with the culture medium containing PK-d7 or K3-d (10–6 m) for 24 h. After incubation, the cells were collected and washed with cold Ca,Mg-free phosphate-buffered saline 3 times and then refrigerated at –30 °C. After being warmed to room temperature, cells were lysed in 1 ml of Ca,Mg-free phosphate-buffered saline. With this procedure, 20 μl of cell lysate was analyzed for protein determination. To the cell lysate in a brown screw-capped tube were added PK-18O and MK-4-18O as internal standards. The measurements of MK-4-d7 and MK-4 epoxide-d7 in cerebra were carried out with the same LC-APCI-MS/MS method as described above. Concentrations of PK, MK-4, and Their Epoxides in Plasma and Tissues of Mice—As shown in Table 1, four species of K-vitamins were found in all tissues examined, although their levels varied greatly while only PK and MK-4 were detected, and at low levels, in plasma. Basically, tissue-distribution patterns of K-vitamins did not differ between male and female mice, although the levels of MK-4 and MK-4 epoxide were relatively higher in the females. Concentrations of MK-4 and its epoxide were much higher than those of PK and its epoxide in all tissues examined. Relatively high PK levels were found in thyroid gland, aorta, heart, and adrenal gland, but most other tissues contained <50 pmol/g tissue. In contrast, relatively high MK-4 levels were found in almost all the tissues except for heart, lung, liver, muscle, and bowel content in male mice, and liver and bowel content in female mice, where concentrations were <50 pmol/g tissue. There was no consistent correlation between the tissue levels of PK and MK-4 or PK epoxide and MK-4 epoxide in male or female mice. The laboratory chow given to the mice was assayed for PK and MK-4 by LC-APCI-MS/MS. PK and MK-4 concentrations were 212.2 ± 0.9 and 2.2 ± 0.5 pmol/g diet, respectively. Thus, it is not conceivable that the high concentrations of MK-4 and its epoxide in tissues of mice originated from the intake of laboratory chow.TABLE 1Tissue distribution of vitamin K in mice fed a normal dietPKPK epoxideMK-4MK-4 epoxideMaleFemaleMaleFemaleMaleFemaleMaleFemalepmol/g or mlCerebrum1.4 ± 0.71.1 ± 0.40.1 ± 0.00.1 ± 0.1106.0 ± 8.2252.5 ± 10.313.4 ± 1.028.9 ± 1.7Cerebellum4.8 ± 1.826.2 ± 21.50.1 ± 0.10.4 ± 0.2200.5 ± 17.5487.7 ± 28.236.3 ± 3.081.5 ± 4.1Medulla oblongata7.6 ± 2.822.4 ± 15.50.1 ± 0.10.4 ± 0.1116.2 ± 8.3253.3 ± 5.522.4 ± 1.639.4 ± 1.9Eye10.3 ± 3.815.3 ± 5.20.2 ± 0.21.0 ± 0.558.0 ± 4.4103.3 ± 6.122.0 ± 4.131.7 ± 3.1Thyroid gland134.9 ± 45.5274.8 ± 140.313.9 ± 6.260.8 ± 34.6247.3 ± 30.4370.3 ± 64.0188.2 ± 53.7256.0 ± 26.1Aorta62.2 ± 32.6109.2 ± 45.21.2 ± 0.713.4 ± 10.4124.0 ± 13.7220.0 ± 8.832.5 ± 4.952.3 ± 2.5Heart4.9 ± 2.380.4 ± 76.30.5 ± 0.37.7 ± 4.646.4 ± 4.2107.7 ± 10.05.6 ± 0.811.7 ± 1.9Thymus7.5 ± 1.29.0 ± 3.90.1 ± 0.13.9 ± 3.5131.2 ± 5.5232.5 ± 12.056.2 ± 3.9104.4 ± 11.9Lung2.1 ± 0.45.5 ± 2.90.1 ± 0.00.1 ± 0.038.1 ± 4.267.1 ± 5.413.3 ± 1.934.8 ± 3.4Liver1.2 ± 0.11.5 ± 0.20.1 ± 0.00.1 ± 0.018.2 ± 2.735.0 ± 2.21.8 ± 0.44.4 ± 0.4Pancreas3.0 ± 0.63.2 ± 0.40.1 ± 0.00.2 ± 0.0520.1 ± 47.4829.4 ± 56.7184.5 ± 15.5355.3 ± 35.0Spleen4.6 ± 0.93.9 ± 0.30.1 ± 0.10.3 ± 0.150.9 ± 5.0100.6 ± 9.025.0 ± 3.651.3 ± 4.8Kidney1.1 ± 0.11.7 ± 0.20.3 ± 0.20.2 ± 0.166.1 ± 4.9212.7 ± 20.87.6 ± 1.030.6 ± 3.2Adrenal gland50.7 ± 10.950.7 ± 10.61.2 ± 1.21.0 ± 0.5148.6 ± 14.7417.5 ± 139.615.1 ± 7.277.6 ± 11.1Stomach7.8 ± 1.27.7 ± 1.10.3 ± 0.10.3 ± 0.190.4 ± 6.1171.7 ± 14.426.5 ± 5.470.7 ± 10.6Duodenum13.2 ± 8.85.0 ± 0.51.1 ± 0.90.2 ± 0.182.7 ± 6.0172.8 ± 11.533.6 ± 3.178.1 ± 11.9Small intestine3.1 ± 0.619.7 ± 16.70.3 ± 0.10.2 ± 0.157.8 ± 7.893.6 ± 6.817.1 ± 1.730.4 ± 3.7Large intestine5.5 ± 2.13.1 ± 0.70.1 ± 0.00.1 ± 0.060.1 ± 5.4106.1 ± 13.123.3 ± 4.356.8 ± 4.7Bone3.2 ± 1.42.2 ± 0.50.1 ± 0.00.1 ± 0.069.8 ± 9.2122.7 ± 10.232.7 ± 3.859.9 ± 3.5Skin12.7 ± 6.26.1 ± 1.30.2 ± 0.10.2 ± 0.150.2 ± 7.5128.2 ± 26.216.1 ± 1.421.3 ± 2.6Muscle2.2 ± 0.81.4 ± 0.2NDaND, not detected.ND19.1 ± 1.556.3 ± 7.54.1 ± 0.612.6 ± 1.2Fat17.4 ± 7.510.3 ± 2.1NDND155.2 ± 37.2423.0 ± 60.13.3 ± 2.022.3 ± 2.9Bowel content24.9 ± 2.927.8 ± 2.12.4 ± 0.24.0 ± 1.113.6 ± 3.724.6 ± 4.70.6 ± 0.30.3 ± 0.1Testis5.5 ± 2.50.1 ± 0.0185.3 ± 3.374.3 ± 4.4Seminal vesicle2.2 ± 0.60.1 ± 0.0184.2 ± 9.1109.5 ± 9.1Ovary16.2 ± 3.40.5 ± 0.2363.4 ± 35.670.5 ± 7.2Uterus4.9 ± 1.20.2 ± 0.1219.6 ± 31.891.5 ± 12.6Plasma0.6 ± 0.00.6 ± 0.0NDND0.7 ± 0.11.2 ± 0.1NDNDa ND, not detected. Open table in a new tab Isolation and Identification of MK-4-d7 in Cerebra of Mice Orally Given PK-d7—We next examined whether orally given PK-d7 accumulates in cerebra as a converted form of MK-4-d7 in mice. 200 female mice were orally given PK-d7 at a single dose of 10 μmol/kg body weight, and cerebra were collected at 24 h post-administration. We subjected an aliquot of the lipid extract from the cerebra to LC-APCI-MS/MS and confirmed that the peaks correspond to MK-4 epoxide, MK-4, PK epoxide, and PK on the MRM chromatogram (Fig. 1). If PK-d7 was converted into MK-4-d7 in the body, then the MK-4 peak shown in Fig. 1B should include both MK-4 and MK-4-d7, because they gave the same retention time on the MRM chromatogram, and therefore, we decided to isolate and purify the peak. After purification by HPLC as noted under “Experimental Procedures,” we finally obtained the MK-4 fraction containing MK-4 and MK-4-d7 in amounts of 3.8 and 2.0 μg, respectively. Because tissue MK-4 has not yet been identified on the basis of structural assignments in animals and humans, and no information was available about whether the purified MK-4-d7 in the presence of endogenous MK-4 can be identified by D NMR, we first analyzed the MK-4 fraction by 1H NMR spectroscopy. The 1H NMR spectra of authentic MK-4 and the MK-4 fraction are shown in Fig. 2, A and B, respectively. Consequently, the values of resonance derived from the 2-methyl-1,4-naphthoquinone ring and the geranylgeranyl side chain of the MK-4 fraction entirely coincided with those of authentic MK-4. The D NMR spectra of authentic MK-4-d7 and the