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• W2914501047 abstract "Vitamin K (VK), in both its phylloquinone and menaquinone forms, has been hypothesized to undergo ω- and β-oxidation on its hydrophobic side chain in order to generate the observed urinary metabolites, K acid I and K acid II, which are excreted primarily as glucuronide conjugates. Synthetic standards of K acid I, K acid II, and a putative intermediate metabolite, menaquinone (MK)1 ω-COOH, were used to develop and optimize a new atmospheric pressure negative chemical ionization LC-MS/MS assay for the quantitation of these compounds in urine from untreated individuals and subjects treated with a high dose VK supplement. VK catabolites were extracted from urine, deconjugated, and converted to their methyl ester derivatives using previously reported methodology. The assay showed a high degree of sensitivity, with limits of detection below 10–50 fmol of metabolite per milliliter of urine, as well as an inter-assay precision of 8–12%. Metabolite standards provided unambiguous evidence for MK1 ω-COOH as a new human urinary metabolite of VK. This assay provides a minimally invasive, highly sensitive, and specific alternative for monitoring VK status in humans. Vitamin K (VK), in both its phylloquinone and menaquinone forms, has been hypothesized to undergo ω- and β-oxidation on its hydrophobic side chain in order to generate the observed urinary metabolites, K acid I and K acid II, which are excreted primarily as glucuronide conjugates. Synthetic standards of K acid I, K acid II, and a putative intermediate metabolite, menaquinone (MK)1 ω-COOH, were used to develop and optimize a new atmospheric pressure negative chemical ionization LC-MS/MS assay for the quantitation of these compounds in urine from untreated individuals and subjects treated with a high dose VK supplement. VK catabolites were extracted from urine, deconjugated, and converted to their methyl ester derivatives using previously reported methodology. The assay showed a high degree of sensitivity, with limits of detection below 10–50 fmol of metabolite per milliliter of urine, as well as an inter-assay precision of 8–12%. Metabolite standards provided unambiguous evidence for MK1 ω-COOH as a new human urinary metabolite of VK. This assay provides a minimally invasive, highly sensitive, and specific alternative for monitoring VK status in humans. The designation vitamin K (VK) is given to a number of structurally similar compounds that contain a common 2-methyl-1,4-naphthoquinone head group substituted with a variable alkyl chain at the 3-position of the quinone. Vitamin K1, or phylloquinone (PK), has a phytyl group attached to the 3-carbon, while vitamin K2 encompasses a subset of molecules that contain one or more 5-carbon isoprenyl moieties linked in a linear fashion to this position. These latter compounds are collectively known as menaquinones (MKs), or MKn, where n denotes the number of isoprenyl units in the alkyl side chain. Vitamin K3, or menadione (MN), is unsubstituted at the 3-position. The two most biologically relevant K vitamers are PK and MK4, which both contain a C20 alkyl side chain that varies only in degree of unsaturation (Fig. 1). PK is biosynthesized in plants and bacteria, but not in animals, thus humans must acquire it by consuming green vegetables (1Booth S.L. Suttie J.W. Dietary intake and adequacy of vitamin K.J. Nutr. 1998; 128: 785-788Crossref PubMed Scopus (286) Google Scholar). By contrast, humans and animals are capable of converting PK into MK4. The mechanism by which this is believed to occur involves first, cleavage of the phytyl side chain of PK in gut endothelium to form MN as an intermediate. MN is then coupled to geranyl geraniol, a terminal allylic C20-isoprenyl alcohol, through the action of UBIAD1, producing MK4 (2Nakagawa K. Hirota Y. Sawada N. Yuge N. Watanabe M. Uchino Y. Okuda N. Shimomura Y. Suhara Y. Okano T. Identification of UBIAD1 as a novel human menaquinone-4 biosynthetic enzyme.Nature. 2010; 468: 117-121Crossref PubMed Scopus (230) Google Scholar, 3Okano T. Shimomura Y. Yamane M. Suhara Y. Kamao M. Sugiura M. Nakagawa K. Conversion of phylloquinone (vitamin K1) into menaquinone-4 (vitamin K2) in mice: two possible routes for menaquinone-4 accumulation in cerebra of mice.J. Biol. Chem. 2008; 283: 11270-11279Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 4Thijssen H.H. Vervoort L.M. Schurgers L.J. Shearer M.J. Menadione is a metabolite of oral vitamin K.Br. J. Nutr. 2006; 95: 260-266Crossref PubMed Scopus (126) Google Scholar). Unlike PK, MK4 can be acquired by eating animal products such as meat and liver. Longer chain menaquinones in humans (MK5–MK13) are believed to be the product of either bacterial synthesis by human intestinal flora or dietary intake of foods such as cheese and fermented soybeans (5Elder S.J. Haytowitz D.B. Howe J. Peterson J.W. Booth S.L. Vitamin K contents of meat, dairy, and fast food in the U.S. diet.J. Agric. Food Chem. 2006; 54: 463-467Crossref PubMed Scopus (110) Google Scholar). Physiologically, PK and MK4, in their reduced dihydroquinone forms, are utilized as essential cofactors by the enzyme, γ-glutamyl carboxylase (GGCX), in the carboxylation of poly-Glu residues in a number of vital proteins, including several involved in blood coagulation, bone mineralization, and the inhibition of vascular calcification (6Fusaro M. Mereu M.C. Aghi A. Iervasi G. Gallieni M. Vitamin K and bone.Clin. Cases Miner. Bone Metab. 2017; 14: 200-206Crossref PubMed Google Scholar, 7Vermeer C. Vitamin K: the effect on health beyond coagulation - an overview.Food Nutr. Res. 2012; 56Crossref PubMed Scopus (122) Google Scholar). The resulting γ-carboxy glutamate residues produced in these proteins show an increased affinity for calcium ions, the binding of which induces a conformational change that results in a more biologically active form of the VK-dependent protein (8Wallin R. Stanton C. Hutson S.M. Intracellular maturation of the gamma-carboxyglutamic acid (Gla) region in prothrombin coincides with release of the propeptide.Biochem. J. 1993; 291: 723-727Crossref PubMed Scopus (18) Google Scholar). Correspondingly, low physiological levels of VK have been associated with increased patient risk for both osteoporosis and vascular calcification, the latter of which has been linked to a decrease in the activated form of matrix Gla protein (9Gallieni M. Fusaro M. Vitamin K and cardiovascular calcification in CKD: is patient supplementation on the horizon?.Kidney Int. 2014; 86: 232-234Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 10Palermo A. Tuccinardi D. D'Onofrio L. Watanabe M. Maggi D. Maurizi A.R. Greto V. Buzzetti R. Napoli N. Pozzilli P. et al.Vitamin K and osteoporosis: myth or reality?.Metabolism. 2017; 70: 57-71Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 11Wuyts J. Dhondt A. The role of vitamin K in vascular calcification of patients with chronic kidney disease.Acta Clin. Belg. 2016; 71: 462-467Crossref PubMed Scopus (19) Google Scholar). As a result of the GGCX carboxylation mechanism, the naphthoquinone moiety of VK is epoxidized at its 2- and 3-position and must be cycled back to its dihydroquinone form, which occurs through successive two electron reductions catalyzed by the enzyme, VK oxidoreductase, before the cofactor can again be utilized by GGCX (12Rettie A.E. Tai G. The pharmocogenomics of warfarin: closing in on personalized medicine.Mol. Interv. 2006; 6: 223-227Crossref PubMed Scopus (102) Google Scholar). VK (in both PK and MK forms) is apparently removed from this homeostatic cycle primarily through initial cytochrome P450 (CYP)4F2-mediated metabolism (13Edson K.Z. Prasad B. Unadkat J.D. Suhara Y. Okano T. Guengerich F.P. Rettie A.E. Cytochrome P450-dependent catabolism of vitamin K: omega-hydroxylation catalyzed by human CYP4F2 and CYP4F11.Biochemistry. 2013; 52: 8276-8285Crossref PubMed Scopus (62) Google Scholar, 14McDonald M.G. Rieder M.J. Nakano M. Hsia C.K. Rettie A.E. CYP4F2 is a vitamin K1 oxidase: An explanation for altered warfarin dose in carriers of the V433M variant.Mol. Pharmacol. 2009; 75: 1337-1346Crossref PubMed Scopus (259) Google Scholar). CYP4F2 oxidizes the terminus of the side chain, converting the ω-methyl group first to a primary alcohol, which can then be further oxidized to a carboxylic acid. The resultant VK acid metabolites contain a long hydrocarbon chain that is similar in structure to a fatty acid and is presumed to similarly undergo degradation via the β-oxidation pathway. K acids I and II, which were found to be the major VK metabolites isolated from human and rat urine (subsequent to deconjugation), are the apparent end products of VK catabolism, produced after several rounds of β-oxidative side chain truncation (15Landes N. Birringer M. Brigelius-Flohe R. Homologous metabolic and gene activating routes for vitamins E and K.Mol. Aspects Med. 2003; 24: 337-344Crossref PubMed Scopus (46) Google Scholar, 16Shearer M.J. Barkhan P. Studies on the metabolites of phylloquinone (vitamin K 1) in the urine of man.Biochim. Biophys. Acta. 1973; 297: 300-312Crossref PubMed Scopus (30) Google Scholar, 17Tadano K. Yuzuriha T. Sato T. Fujita T. Shimada K. Hashimoto K. Satoh T. Identification of menaquinone-4 metabolites in the rat.J. Pharmacobiodyn. 1989; 12: 640-645Crossref PubMed Scopus (16) Google Scholar) (Fig. 1). K acids I and II, therefore, have obvious potential for use as biomarkers for osteoporosis as well as chronic kidney disease (CKD), because CKD is strongly associated with increased vascular calcification in patients. In fact, quantitation of the urinary K acids can give a more complete analysis of a patient's overall VK levels than can be obtained from direct measurement of their VK plasma concentrations, due to the physiological compartmentalization that exists between the major K vitamers (18Ferland G. Doucet I. Mainville D. Phylloquinone and menaquinone-4 tissue distribution at different life stages in male and female Sprague-Dawley rats fed different VK levels since weaning or subjected to a 40% calorie restriction since adulthood.Nutrients. 2016; 8: 141Crossref PubMed Scopus (13) Google Scholar, 19Gentili A. Cafolla A. Gasperi T. Bellante S. Caretti F. Curini R. Fernandez V.P. Rapid, high performance method for the determination of vitamin K(1), menaquinone-4 and vitamin K(1) 2,3-epoxide in human serum and plasma using liquid chromatography-hybrid quadrupole linear ion trap mass spectrometry.J. Chromatogr. A. 2014; 1338: 102-110Crossref PubMed Scopus (43) Google Scholar). PK is the more prominent form of VK in plasma, evidenced by the relative importance of PK in blood coagulation, while MK4 is more concentrated in tissues, such as kidneys, brain, and lungs, which likely contributes to its relative importance in other key biological processes like bone mineralization and the inhibition of vascular calcification (10Palermo A. Tuccinardi D. D'Onofrio L. Watanabe M. Maggi D. Maurizi A.R. Greto V. Buzzetti R. Napoli N. Pozzilli P. et al.Vitamin K and osteoporosis: myth or reality?.Metabolism. 2017; 70: 57-71Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 20Groenen-van Dooren M.M. Soute B.A. Jie K.S. Thijssen H.H. Vermeer C. The relative effects of phylloquinone and menaquinone-4 on the blood coagulation factor synthesis in vitamin K-deficient rats.Biochem. Pharmacol. 1993; 46: 433-437Crossref PubMed Scopus (28) Google Scholar, 21Schurgers L.J. Dissel P.E. Spronk H.M. Soute B.A. Dhore C.R. Cleutjens J.P. Vermeer C. Role of vitamin K and vitamin K-dependent proteins in vascular calcification.Z. Kardiol. 2001; 90: 57-63Crossref PubMed Scopus (80) Google Scholar). Thus, direct plasma analysis can only give an accurate read of a patient's PK levels, whereas the quantitation of urinary K acid concentrations would reflect the patient's total systemic exposure to VK. A published method exists for the quantitation of K acids I and II in urine, but the assay relies upon electrochemical detection (ECD) of these metabolites and is therefore of limited utility to most laboratories, which lack the capability to run such an assay (22Harrington D.J. Soper R. Edwards C. Savidge G.F. Hodges S.J. Shearer M.J. Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection.J. Lipid Res. 2005; 46: 1053-1060Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Therefore, in this study, we describe the first LC-MS/MS assay for the accurate and precise quantitation of K acids I and II in human urine and report the identification of a new VK metabolite, MK1 ω-COOH (Fig. 1). The VK supplement, Koncentrated K, was generously gifted to us by Dr. Patrick Theut, President of Red Foot Associates (Manistique, MI). The MK1 internal standard as well as K acid I, K acid II, and MK1 ω-COOH were previously synthesized (23Fujii S. Shimizu A. Takeda N. Oguchi K. Katsurai T. Shirakawa H. Komai M. Kagechika H. Systematic synthesis and anti-inflammatory activity of omega-carboxylated menaquinone derivatives–Investigations on identified and putative vitamin K(2) metabolites.Bioorg. Med. Chem. 2015; 23: 2344-2352Crossref PubMed Scopus (24) Google Scholar, 24Teitelbaum A.M. Scian M. Nelson W.L. Rettie A.E. Efficient Syntheses of Vitamin K Chain-Shortened Acid Metabolites.Synthesis (Stuttg.). 2015; 47: 944-948Crossref PubMed Scopus (6) Google Scholar). Organic solvents were obtained from Fisher Scientific (Pittsburgh, PA), and all other chemicals (including unlabeled MK4 and PK) were purchased from Sigma-Aldrich (St. Louis, MO). Urine creatinine content was measured using a creatinine colorimetric assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's protocol. Six healthy adult male subjects were recruited for the VK supplementation study. Each subject provided a single time-point baseline urine sample immediately prior to the oral ingestion of two capsules of the VK supplement, Koncentrated K (reported to contain 25 mg MK4, 5 mg PK, 0.5 mg MK7, and 2 mg of astaxanthine per capsule). After swallowing the Koncentrated K capsules, the subjects immediately consumed one half pint of 2% milk, to aid in absorption of vitamin, and then collected their urine for the next 24 h postsupplementation. Urine samples were subsequently aliquoted and kept frozen at −80°C until analysis. All subjects provided written informed consent for the study, which was approved by the University of Washington Institutional Review Board. This study abides by the Declaration of Helsinki principles. Urine processing and deconjugation was based closely on the methodology of Harrington, et al. (22Harrington D.J. Soper R. Edwards C. Savidge G.F. Hodges S.J. Shearer M.J. Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection.J. Lipid Res. 2005; 46: 1053-1060Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) with minor modifications. Menaquinone-1 (MK1; internal standard, 100 pmol) was premixed into 2.5 ml of urine, which were then passed through a Bond Elut 3 ml C18 SPE cartridge (Agilent Technologies). The column was washed with 2.5 ml of water and the VK metabolites were eluted with 2 ml of methanol. Concentrated HCl (500 μl) was added to the methanol eluate and the samples were shaken in a water bath at room temperature overnight in the dark. The deconjugated VK metabolites were next diluted with water and extracted into dichloromethane, which was then washed with water to remove traces of inorganic acid. The carboxylic acid moieties of these aglycone catabolites were then converted to their methyl ester derivatives through the addition of excess etheric diazomethane, and solvent was removed under a nitrogen gas stream. Sample residues, taken up in 2 ml hexane, were further purified by loading onto a normal phase Bond Elut LRC-Si 500 mg SPE column (Agilent) and eluting with 10 ml of 15% ether in hexane subsequent to an 8 ml hexane column wash. After solvent evaporation via nitrogen gas stream, sample residues were dissolved in 100 μl of isopropyl alcohol for LC-MS analysis. Quantitative calibration curves (supplemental Figs. S6–S8) were prepared by spiking concentrated standard mixtures, containing variable amounts of K acid I, K acid II, and MK1 ω-COOH, into 2.5 ml aliquots of a baseline human urine sample (0.05 to 500 pmol/ml final concentrations above endogenous urine levels). The standard urine solutions, prepared in duplicate, were worked-up and analyzed in identical fashion to the samples described above. Because the presence of endogenous catabolites in these urine samples blocked our ability to determine limits of detection for the assay using this type of curve, limits of detection were instead estimated from a different standard curve, which was prepared by spiking the metabolites into buffer rather than urine (at variable concentrations between 0.001 and 100 pmol/ml). Methanolic stock solutions of the K acid catabolites were stored at −80°C and, along with the endogenous K acid conjugates present in urine, proved to be stable at this temperature for at least 12 months, as judged by the strong consistency found between numerous standard curves performed on various aliquots of the same baseline human urine sample within this time frame. LC-MS/MS analyses were conducted on a Waters Xevo TQ-S tandem quadrupole mass spectrometer (Waters Co., Milford, MA.) coupled to an ACQUITY Ultra Performance LC™ (UPLC™) system with integral autoinjector (Waters). The Xevo was operated in atmospheric pressure negative chemical ionization (APCI−) MS/MS [selected reaction monitoring (SRM)] mode at a source temperature of 150°C and a probe temperature of 500°C, and the following mass transitions for the methyl ester derivatives of each metabolite were monitored in separate ion channels: m/z 240 > 185 (MK1, internal standard), m/z 312 > 195 (K acid I), m/z 286 > 185 (MK1 ω-COOH), and m/z 284 > 195 (K acid II). The cone voltages were set to 50 V for K acid I, K acid II, and MK1 ω-COOH and to 60 V for MK1. Optimized collision energies were set to 40, 30, 40, and 18 eV, respectively, for the ester derivatives of K acid I, K acid II, MK1 ω-COOH, and MK1. Compounds were separated by injecting 5 μl of sample onto a Shim-pack XR-ODS 2.2 μ, 2.0 × 75 mm UPLC column (Shimadzu Scientific, Columbia, MD), set to a temperature of 50°C, using a binary solvent gradient of 0.1% aqueous formic acid (solvent A) and methanol (solvent B) with a constant flow rate of 0.35 ml/min. The initial solvent B concentration was set to 65% where it was kept for 3 min, then increased linearly to 98% over 4 min, and there maintained for an additional 5 min. The K acid I, K acid II, and MK1 ω-COOH metabolites were quantified by comparing peak area ratios (relative to the internal standard, MK1) to ratios from the appropriate calibration curve, determined with synthesized metabolite standards (23Fujii S. Shimizu A. Takeda N. Oguchi K. Katsurai T. Shirakawa H. Komai M. Kagechika H. Systematic synthesis and anti-inflammatory activity of omega-carboxylated menaquinone derivatives–Investigations on identified and putative vitamin K(2) metabolites.Bioorg. Med. Chem. 2015; 23: 2344-2352Crossref PubMed Scopus (24) Google Scholar, 24Teitelbaum A.M. Scian M. Nelson W.L. Rettie A.E. Efficient Syntheses of Vitamin K Chain-Shortened Acid Metabolites.Synthesis (Stuttg.). 2015; 47: 944-948Crossref PubMed Scopus (6) Google Scholar), using linear regression analysis. The intra- and inter-assay variability studies were performed on two different single time-point urine samples collected from the same healthy adult male subject (unsupplemented with VK) approximately 6 months apart. For the intra-assay variability study, 3 nmol of MK1 standard was spiked into 30 ml of urine, which was mixed thoroughly by vortex, and the urine was then aliquoted into 11 × 2.5 ml samples. These samples were processed and the deconjugated VK catabolites, as their methyl ester derivatives, were quantified by LC-MS/MS as described above. The inter-assay variability study was carried out over a month-long period and involved four identical experiments, each performed roughly 1 week apart. In each experiment, 3.2 nmol of MK1 standard were spiked into 32 ml of urine, which was then aliquoted into 12 × 2.5 ml samples (48 samples total) and the VK catabolites were again processed and quantified as described. Urine, collected from healthy human adult male subjects, was processed for VK metabolite quantitation using the optimized methodology of Harrington et al. (22Harrington D.J. Soper R. Edwards C. Savidge G.F. Hodges S.J. Shearer M.J. Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection.J. Lipid Res. 2005; 46: 1053-1060Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Briefly, urine samples were premixed with internal standard, desalted on a C18 SPE column, and the eluents (containing the VK catabolites primarily as their glucuronide conjugates) (16Shearer M.J. Barkhan P. Studies on the metabolites of phylloquinone (vitamin K 1) in the urine of man.Biochim. Biophys. Acta. 1973; 297: 300-312Crossref PubMed Scopus (30) Google Scholar, 17Tadano K. Yuzuriha T. Sato T. Fujita T. Shimada K. Hashimoto K. Satoh T. Identification of menaquinone-4 metabolites in the rat.J. Pharmacobiodyn. 1989; 12: 640-645Crossref PubMed Scopus (16) Google Scholar, 22Harrington D.J. Soper R. Edwards C. Savidge G.F. Hodges S.J. Shearer M.J. Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection.J. Lipid Res. 2005; 46: 1053-1060Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) were hydrolyzed in methanolic HCl. The aglycone metabolites, already partially converted to their methyl ester derivatives during acidic deconjugation, were fully esterified with etheric diazomethane and the evaporated sample residues were further purified on a silica SPE column prior to LC-MS/MS analysis. We were able to confirm the assertions of Harrington et al. (22Harrington D.J. Soper R. Edwards C. Savidge G.F. Hodges S.J. Shearer M.J. Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection.J. Lipid Res. 2005; 46: 1053-1060Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) that this procedure generates highly predictable and repeatable recoveries of K acids I and II, as their methyl ester derivatives, with no detectable lactonization products (data not shown). Synthetic standards of K acid I, K acid II, and MK1 ω-COOH were utilized in the development of an optimized LC-MS/MS quantitation assay. Multiple types of MS ionization were tested on both the underivatized and methyl ester versions of these metabolite standards, and it was found that APCI− provided consistently higher sensitivity of detection for all compounds than was observed with electrospray ionization (positive or negative). It has been reported previously that VK compounds lacking a carboxylic acid or even alcohol functionality can still ionize well by APCI− MS because the quinone moiety of these molecules is capable of forming a highly-stabilized radical anion (Fig. 2) (13Edson K.Z. Prasad B. Unadkat J.D. Suhara Y. Okano T. Guengerich F.P. Rettie A.E. Cytochrome P450-dependent catabolism of vitamin K: omega-hydroxylation catalyzed by human CYP4F2 and CYP4F11.Biochemistry. 2013; 52: 8276-8285Crossref PubMed Scopus (62) Google Scholar). Thus, conversion of the K acid metabolites to their methyl ester derivatives does not result in significant loss of sensitivity despite the fact that this technique utilizes negative chemical ionization. Both PK and MK4 fragment, by APCI− MS/MS, primarily via homolytic bond cleavage between the first and second carbons of the side chain attached to position 3 of the quinone, resulting in a very prominent 3-methyl-menadione fragment anion, at m/z = 185 (Fig. 2A) (13Edson K.Z. Prasad B. Unadkat J.D. Suhara Y. Okano T. Guengerich F.P. Rettie A.E. Cytochrome P450-dependent catabolism of vitamin K: omega-hydroxylation catalyzed by human CYP4F2 and CYP4F11.Biochemistry. 2013; 52: 8276-8285Crossref PubMed Scopus (62) Google Scholar). We found that the methyl ester derivatives of both K acid I and K acid II generate the same major fragment ion as the parent vitamins (supplemental Figs. S1, S2). Interestingly, MK1 ω-COOH shows a different APCI− MS/MS fragmentation profile, generating a major fragment ion at either m/z = 197 at relatively low collision energy (CE) (CE = 20 V) or m/z = 195 at higher CE (CE = 40 V) with only a negligible ion at mass 185 (supplemental Fig. S3). This is perhaps due to the fact that the side chain double bond is conjugated to the carboxy ester moiety of MK1 ω-COOH, but is isolated (or absent) in the other VK species. We propose that the m/z 197/195 fragment ions may result from a mechanism involving an initial side-chain double bond isomerization followed by radical ionization, hydrogen atom transfer, and then chain truncation/cyclization as shown in Fig. 2B. Conjugation with the carboxy ester moiety of the MK1 ω-COOH derivative should enable base-catalyzed double bond isomerization to occur more readily for this compound compared with VK analogs possessing an isolated double bond at this position, perhaps explaining the difference in the MK1 ω-COOH derivative's fragmentation profile. We used this fragmentation data to develop a highly sensitive APCI− MS/MS (SRM) assay where we analyzed for the three VK catabolite derivatives using transitions from their respective molecular ions to either their m/z 185 or 195 fragments. While we initially monitored the K acid I ester in the m/z 312 > 185 SRM channel, we found that human urine contains two additional compounds (perhaps double bond regio-isomers of K acid I) that show up in this same channel, bracketing the K acid I ester, and which proved difficult to fully separate from the target metabolite by LC-MS/MS (supplemental Fig. S4). Because the K acid I ester also produces a minor m/z 195 fragment ion that is absent in the more prominent of the two bracketing compounds (in terms of the 312 > 185 mass transition), we chose to instead analyze the K acid I ester in the m/z 312 > 195 SRM channel, resulting in some loss of sensitivity, but allowing for better overall precision of metabolite quantitation. As outlined in the Materials and Methods section, VK catabolites within unknown urine samples were identified and quantified, after processing, by fitting their LC-MS/MS measured peak area ratios (relative to the internal standard, MK1) to standard curves using linear regression analysis. The assay showed a high degree of sensitivity for all three VK catabolites, with limits of detection below 10 fmol/ml urine for K acid II and MK1 ω-COOH and a limit of detection below 50 fmol/ml urine for K acid I. The intra- and inter-assay precision of quantitation for the urinary VK catabolites was tested, respectively, on two single-time point urine samples collected from the same human adult male subject roughly six months apart. Comparison of sample urine LC-MS/MS chromatograms to chromatograms obtained from urine spiked with synthetic catabolite standards allowed for the unambiguous identification of MK1 ω-COOH as a new VK metabolite (Fig. 3). The urinary concentrations determined for the three catabolites in both of these experiments, along with their precisions of measurement, are listed in Table 1.TABLE 1Intra- and inter-assay reproducibility for the quantitation of VK catabolite concentrations in human urine by LC-MS/MSIntra-Assay Reproducibility (Sample A, n = 11)Inter-Assay Reproducibility (Sample B, n = 48)K Acid IK Acid IIMK1 ω-COOHK Acid IK Acid IIMK1 ω-COOHMean (pmol/ml)1.404.370.0651.352.590.045SEM (pmol/ml)0.040.080.0030.020.030.001Coefficient of. variation (%)8.46.416.211.07.812.0Samples A and B represent two separate urine samples, collected roughly 6 months apart from the same healthy adult male subject. Concentrations are listed in picomoles of metabolite per milliliter of urine. Inter-assay reproducibility was assessed through four identical experiments conducted over a month-long period. Open table in a new tab Samples A and B represent two separate urine samples, collected roughly 6 months apart from the same healthy adult male subject. Concentrations are listed in picomoles of metabolite per milliliter of urine. Inter-assay reproducibility was assessed through four identical experiments conducted over a month-long period. Six human adult male subjects were each given two capsules of the VK supplement, Koncentrated K, and urine was collected prior and subsequent to supplementation as described in the Materials and Methods section. LC-MS/MS analysis showed a minimum 50-fold increase in concentration, postsupplementation, for all three VK catabolites in each of the six subjects. In order to account for differences in urine dilution levels between subjects, creatinine concentrations were determined for each sample and these were used to normalize the VK catabolite concentrations, as shown in Table 2. The large increase in postsupplementation size for the assigned MK1 ω-COOH peak provides additional confirmation of its status as a newly identified VK metabolite (Fig. 3).TABLE 2Urinary VK catabolite concentrations determined in six healthy adult male subjects before and after taking two capsules of the VK supplement, Koncentrated KSubjectPresupplement0-24 Hours Postsupplement (0–24 h)K Acid IK Acid IIMK1 ω-COOHK Acid IK Acid IIMK1 ω-COOH(nmol/mmol creatinine)(nmol/mmol creatinine)10.242 ± 0.0140.987 ± 0.057BLQ100.1 ± 11.8127.9 ± 17.12.06 ± 0.2920.102 ± 0.0130.465 ± 0.0310.0031 ± 0.000632.5 ± 5" @default.
• W2914501047 created "2019-02-21" @default.
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• W2914501047 date "2019-04-01" @default.
• W2914501047 modified "2023-10-18" @default.
• W2914501047 title "A new LC-MS assay for the quantitative analysis of vitamin K metabolites in human urine" @default.
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