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Methods |
* Centre for Haemostasis and Thrombosis, The Haemophilia Reference Centre, St. Thomas' Hospital, London, UK
Department of Biological Sciences, University of Essex, Colchester, UK
Institute of Hepatology, Liver Failure Group, Department of Medicine, University College, London, UK
Published, JLR Papers in Press, February 1, 2005. DOI 10.1194/jlr.D400033-JLR200
1 To whom correspondence should be addressed. e-mail: domonic.harrington{at}gstt.nhs.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.999) and high sensitivity with an on-column detection limit of <3.5 fmol (<1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.
Abbreviations: 5C-aglycone, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone; 7C-aglycone, 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone; ECD, electrochemical detection; Gla, 
-carboxyglutamate; IS, internal standard; K1, phylloquinone; K3, menadione; MK, menaquinone; SPE, solid-phase extraction
Supplementary key words phylloquinone • menaquinones • urinary vitamin K metabolites • aglycones • high-performance liquid chromatography with electrochemical detection
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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90% and 10% of vitamin K intake, respectively (1). Dietary MK-n mainly comprises MK-4, MK-7, MK-8, and MK-9 (2), although MKs with longer side chains up to MK-13 are present in human liver (3, 4). Menadione (abbreviated K3) is a synthetic vitamin K homolog that lacks the side chain at the 3 position and that, despite toxicity concerns and restricted biological activity, is still available in some countries as a pharmaceutical vitamin K preparation in the form of menadiol sodium phosphate (5) or similar water-soluble salt. The biological activity of K3 in vivo depends entirely on its prenylation to MK-4 (6, 7).
At the cellular level, the cofactor role of vitamin K for the posttranslational conversion of specific peptide-bound glutamate to -carboxyglutamate (Gla) is well established, as is the intimately associated metabolic cycle whereby the vitamin K 2,3-epoxide metabolite generated during -glutamyl carboxylation is salvaged (8, 9).
Other aspects of the intermediary metabolism of vitamin K, including the processes leading to vitamin K catabolism and excretion, are much less understood. During the 1970s, human studies using radiolabeled tracer and unlabeled pharmacological doses showed that K1 was rapidly and extensively catabolized via the urine and bile (10–12). These studies established that the major aglycone metabolites of K1 were two side chain-shortened carboxylic acids with the structures 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone; side chain length of 5 carbon atoms; structure IV in Fig. 1) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone; side chain length of 7 carbon atoms; structure III in Fig. 1) (10–12). Both 5C- and 7C-aglycones were excreted as water-soluble conjugates, mainly with glucuronic acid.
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-carboxylation status of specific Gla proteins such as prothrombin and osteocalcin, representing hepatic and bone -carboxylation capacity, respectively. In addition, measurements of urinary free Gla offer an overall assessment of the -carboxylation status of Gla proteins. Each of these status assessments has a number of methodological and interpretational drawbacks (23–25). Although useful in many situations, the measurement of circulating vitamin K to assess tissue stores has the major disadvantage that only K1 is commonly measured, to the detriment of MKs. MKs from the diet (1, 2) and possibly from intestinal synthesis (3, 4) may make a significant contribution to total daily vitamin K intake (1), and the liver stores of vitamin K are predominately long-chain MKs (3, 4). Here, we describe the development of chromatographic techniques to quantify the two major urinary aglycones of vitamin K with high sensitivity, enabling their measurement at low physiological concentrations. We chose electrochemical detection (ECD) in the redox mode for the final analytical HPLC stage because this method is especially suitable for quinone compounds and has been used successfully for the determination of vitamin K in serum (26, 27). To determine whether the 5C- and 7C-aglycones are common to all K vitamins, we obtained urine samples from adults before and after supplementation with different doses of K1, MK-4, MK-7, and K3. We also analyzed urine samples from newborn infants, who, as a group, are known to have precariously low vitamin K stores and are routinely given vitamin K prophylaxis at birth.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-lactone form of the 7C-aglycone [2-methyl-3-(5'-carboxy-3'-hydroxy-3'-methylpentyl)-1,4-naphthoquinone lactone], also known as vitamin K -lactone, was a gift to M.J.S. from Hoffmann-La Roche and Co. (Basel, Switzerland). Organic solvents were of HPLC grade (Rathburns Chemicals, Walkerburn, Scotland). Water for ECD was purified using a Purite Neptune water purification system (Jencons-PLS, Leighton Buzzard, UK). Potassium hydroxide (Ultra grade), 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), and ethylenediaminetetraacetic acid (disodium salt, dihydrate, Analar grade) were obtained from Sigma-Aldrich (Dorset, UK). Anhydrous sodium acetate, glacial acetic acid, and 3 M HCl, all AristaR grade, were obtained from BDH (Lutterworth, Leicstershire, UK). Nitrogen gas (oxygen free) for removal of solvents was obtained from BOC (Guildford, Surrey, UK).
Apparatus
Solid-phase extraction (SPE) procedures were performed using IsoluteTM SPE C18 (100 mg, 1 ml reservoir) cartridges (Jones Chromatography, Mid Glamorgan, UK) and Sep-PakTM silica plus cartridges (Waters Ltd., Elstree, Hertfordshire, UK) with a SPE vacuum manifold.
For HPLC, we used a Gynkotek model 480 pump with pulse damper, a WatersTM 717 plus Autosampler, and an in-line DeJour X-Act HPLC degassing unit. For on-line detection, we used a DECADE electrochemical detector (Antec, Leyden, The Netherlands) equipped with an in-line cell (model 5011) containing dual porous graphite coulometric electrodes in series (ESA Analytical Ltd., Aylesbury, Buckinghamshire, UK) and an amperometric wall jet electrode situated immediately downstream (Antec model VT-03). Chromatographic data were captured using a data chromatography manager (Waters).
Urine collection and human subject protocols
Twenty four hour and spot urine collections were made into plastic containers. Owing to the photosensitive nature of the metabolites, the urine containers were protected from sources of strong light. Urine collections were stored at room temperature for the duration of the collection period, and aliquots were frozen at –70°C until analysis. No significant metabolite loss occurred under these conditions.
We investigated urinary 5C- and 7C-aglycone excretion in healthy adult volunteers. Measurements were made in spot or 24 h urine collections in both unsupplemented and vitamin K-supplemented subjects. For the supplementation studies, the subjects took different forms of vitamin K orally at doses previously used for the treatment or prevention of several pathologies. The vitamin K compounds used were K1 (2 mg and 50 mg) (29); two homologs representative of the MK series, MK-4 (45 mg) (30) and MK-7 (1 mg) (31); and K3 (20 mg) (5). The amounts of 5C- and 7C-aglycone excreted in the urine were measured before and after supplementation.
A number of spot urine samples were also collected from newborn infants (two males, one female) before and after intramuscular K1 prophylaxis with 1 mg of Konakion Neonatal (Roche, Basel, Switzerland).
All adult volunteers and guardians of newborns gave full written informed consent before participation in these investigations.
Assay procedure
A flow chart showing the extraction and analytical procedures is shown in Fig. 2
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0.5 ml/min, and the eluent was discarded (32). The conjugated vitamin K metabolites were then eluted into a clean tapered polypropylene test tube (12 ml capacity) with 2 ml of a methanolic stock solution containing 0.15 µg of the IS, and the eluent was evaporated to dryness under a stream of N2 at 50°C. Conjugated urinary vitamin K metabolites were hydrolyzed overnight at room temperature (in the dark) with 1.1 ml of methanolic HCl [prepared by combining 1 volume of concentrated (35%) HCl with 4 volumes of methanol] (33). The metabolites, now as their aglycones, were extracted into chloroform by the addition of 1.1 ml of chloroform and 1.0 ml of water to the sample tube (34). The upper methanolic-aqueous layer was removed and discarded. To prevent acid-catalyzed lactonization of the 7C-aglycone during subsequent drying procedures, the lower chloroform layer was washed with 10 ml of deionized water to remove any residual mineral acid. After brief shaking and time to allow the two phases to separate, the upper aqueous layer was discarded and the lower chloroform layer was evaporated to dryness under a stream of N2 at 50°C.
Methylation of aglycones
The carboxylic acid forms of the vitamin K aglycones were converted to their methyl ester derivatives by reaction with freshly prepared ethereal diazomethane generated on a small scale by a modification of a method described elsewhere (35). In brief, a mixture of 5 M KOH (20 ml) and diethyl ether (15 ml) was placed into a small, screw-capped bottle (100 ml capacity; Duran), and solid MNNG was gradually added until sufficient diazomethane was generated to turn the upper ether layer yellow (caution: only perform in a well-ventilated fume hood). A volume of 0.5 ml of this ethereal diazomethane was then added to each urine extract. Complete methylation of the carboxylic acid group of the aglycone metabolites was achieved at room temperature within 5 min, after which the diethyl ether was removed under a stream of N2 at 50°C.
Normal-phase SPE of methylated aglycones
Further fractionation of the urine extract was achieved by normal phase-SPE using Sep-PakTM cartridges. Each cartridge was attached to a 10 ml glass syringe with a Luer end fitting and activated by drawing through 2 ml of n-hexane. Each extract was dissolved in 2 ml of n-hexane and then pipetted into the glass syringe attached to the activated cartridge. The n-hexane was drawn through the cartridge and the eluent was discarded. Another volume of 8 ml of n-hexane was added to the sample tube and the washings were drawn through the cartridge and discarded as before. The vitamin K metabolites and IS were then eluted from the cartridge with 10 ml of n-hexane-diethyl ether (85:15, v/v) and collected into a fresh stoppered, tapered test tube of polypropylene. The solvent was removed under a stream of nitrogen at 50°C.
Reversed-phase HPLC-ECD
The final separation of the methylated aglycones was achieved by reversed-phase HPLC using a Thermo Hypersil-Keystone HyPURITY C18 column (3 µm particle size, dimensions 100 x 4.4 mm; Hichrom, Reading, Berkshire, UK) and a mobile phase consisting of 60:40 (v/v) methanol-0.05 M sodium acetate buffer (pH 3.0; containing 0.1% EDTA) (26). The flow rate was 1.0 ml/min. Effluent from the column passed through the coulometric dual-electrode cell in which the twin, porous graphite electrodes were set at a negative potential (–1.2 V), thereby reducing the urinary vitamin K quinone metabolites and IS to their quinol states. The effluent then passed to the amperometric wall jet electrode set at +0.3 V, which resulted in the reoxidation of the quinol forms of metabolites and IS to their respective quinones. Chromatograms were generated by monitoring the current (nanoamperes) at the wall jet electrode.
Quantification of 5C- and 7C-aglycone metabolites
From methanolic stock solutions of the 5C- and 7C-aglycones and the IS (methyl ester forms), we prepared a series of standards containing all three analytes with spectrophotometrically determined (EM mM = 18.9 at 248 nm) weight ratios of 5C-aglycone/IS and 7C-aglycone/IS ranging from 0.003 to 0.121. Each chromatographic run included the direct injection of 10 µl of each calibration standard. For routine analyses (i.e., for physiological urinary concentrations of metabolites), the SPE fractionated extracts were reconstituted in 40 µl of methanol and a volume of 10 µl was injected onto the reversed-phase HPLC system. For the determination of the much higher levels after vitamin K supplementation, the extract was reconstituted in 100 µl of methanol and 10 µl was injected. Metabolite concentrations were quantified by the method of peak height ratios. For each chromatogram generated, the peak heights of 5C- and 7C-aglycones were expressed as a ratio to the IS peak. A calibration plot of peak height ratios of the standards versus their weight ratios gave excellent linearity (typically, r2 0.999) and was used to calculate the equivalent weight ratios of aglycone/IS for each unknown. Multiplication of this weight ratio by the amount of IS originally added gave the amounts of aglycones in the volume of urine processed.
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RESULTS |
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After the initial SPE stage, conjugated metabolites of vitamin K were hydrolyzed with methanolic HCl. At room temperature, hydrolysis of conjugated metabolites with methanolic HCl was complete within 16 h. After hydrolysis, the lipid-soluble aglycones and IS were efficiently extracted into chloroform after the addition of greater volumes of chloroform and water to form a two-phase system, as described for the final stage of the Bligh and Dyer (34) total lipid extraction technique. A second extraction of the methanolic digest with chloroform did not improve the recovery of aglycones.
Methyl ester derivatization of carboxylic acid aglycones
There were two main reasons for adopting a pre-HPLC methylation procedure to quantitatively convert the aglycone carboxylic acids to their respective methyl esters. First, exposure to methanol during the chromatographic procedures resulted in some inadvertent methylation, which was enhanced by the use of methanolic HCl to deconjugate the aglycones. Second, the reduced polarity of these methylated derivatives improved their retention in our reversed-phase HPLC separation system.
SPE purification of methylated aglycones
An additional SPE purification stage using silica cartridges was introduced to remove interfering compounds, which prevented baseline resolution of the 5C- and 7C-aglycones in the final HPLC-ECD analytical stage. Optimization of the composition of the eluting solvent was carried out using a standard mixture containing known concentrations of the 5C-aglycone, 7C-aglycone, and IS. A diethyl ether concentration of 15% diethyl ether in n-hexane (v/v) was found to give quantitative recoveries of the methylated aglycones and the IS while producing good baseline resolution on HPLC-ECD. Recovery of the vitamin K aglycones and IS after this and previous purification stages was consistently >90% for each analyte, with no interanalyte bias.
Optimization of HPLC-ECD conditions
Hydrodynamic voltammograms of the 5C- and 7C-aglycone methyl esters together with the IS are shown in Fig. 3
. A voltage of –1.2 V was selected as the applied potential in the upstream flow-through coulometric twin electrodes for quantitative reduction of the quinone forms of metabolites and IS to their corresponding quinols (this was the maximum possible voltage to the electrodes of the ESA coulometric cell when controlled by the DECADE instrument). In selecting +0.3 V as the applied potential for the downstream amperometric electrode (for the reoxidation of the respective quinols), a minimal degree of assay sensitivity was sacrificed. However, this reduced interferences from coextracted and cochromatographed substances and resulted in reduced background current.
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8, 12, and 32 min, respectively. Both reductive and oxidative electrochemical cells were found to be robust and foul free after prolonged use.
Calibration, sensitivity, and precision of the HPLC-ECD assay
The injection of varying amounts of the 5C- and 7C-aglycones and the IS gave good peak shapes and a linear detector response within and beyond the mass range used for the quantification of the aglycones. Typical chromatograms of a calibration standard solution and urinary extracts from both unsupplemented and vitamin K-supplemented subjects are shown in Fig. 4
. The on-column lower limit of detection of 5C- and 7C-aglycones (defined as 5x baseline noise) was <3.5 fmol (<1 pg). The intra-assay and interassay coefficients of variation for 5C- and 7C-aglycone determination in urine from a healthy, nonsupplemented volunteer are shown in Table 1.
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position relative to the carboxyl group of the side chain. Organic acids with this ,-unsaturated structure are unstable to mineral acids and undergo cyclization to a five member -lactone ring structure. As has been demonstrated in radioisotopic studies, the 7C-aglycone in its carboxylic acid form is readily converted to vitamin K -lactone (structure V in Fig. 1) under aqueous acidic conditions, such as when mineral acid is used to release the aglycones from their conjugated forms in urine (10–12). To determine whether we could demonstrate -lactonization by HPLC, we subjected 0.5 ml aliquots of urine (urine collected from a volunteer who had ingested 50 mg of K1) to hydrolysis with 1 ml of aqueous 3 M HCl at 70°C for 60 min. After aqueous HCl hydrolysis, the aglycones were extracted with chloroform and subjected to the usual assay procedure. Treatment with mineral acid completely abolished the peak that cochromatographed with the 7C-aglycone standard and resulted instead in an extra peak that cochromatographed with authentic vitamin K -lactone (data not shown). A peak of vitamin K -lactone was not found in urine samples after hydrolysis of the metabolite conjugates with methanolic HCl or enzyme preparations of ß-glucuronidase (also containing sulfatase).
Confirmation of chromatographic peak identification
Identification of the 5C- and 7C-methyl ester aglycone chromatographic peaks isolated from urine was initially achieved by confirming that they had chromatographic and chemical properties identical to those of the authentic synthetic standards. The chemical similarities between analytes and standards included their hydrodynamic voltammograms, their sensitivity to light, and, for the 7C-aglycone, the lability toward mineral acid and conversion to a compound chromatographically indistinguishable from vitamin K -lactone. Definitive confirmation of correct peak assignment was provided by in vivo studies that confirmed that the chromatographic measurements responded to supplementation with different forms of vitamin K.
Typical concentrations (corrected for creatinine) of 5C- and 7C-aglycones in urine samples collected on 2 consecutive days from five healthy, fasting adults (subjects A to E) are shown in Table 2. Table 2 also shows the increase in urinary concentrations of both aglycones in response to supplementation with K1, MK-4, MK-7, and K3 in a dose-dependent manner. These studies provided confirmation of the chromatographic peak identity for the respective aglycones.
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DISCUSSION |
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10% for both 5C- and 7C-aglycones. Although we used the same principle of redox mode electrochemical detection as used for a previous assay of the parent K vitamins (27, 36), the metabolite assay has proved to be much less susceptible to periodic loss of sensitivity. In the serum vitamin K assay, this often rapid loss of sensitivity is thought to be attributable to passivation of the electrodes by adsorbed species of vitamin K and coextracted nonpolar lipids (27, 36). The lack of significant electrode passivation in the present assay may be related to the greater polarity of the metabolites and coextracted urinary components, which makes them less likely to adsorb to the carbon electrodes than are the highly lipophilic vitamin K and other lipids present in serum extracts. The 5C- and 7C-aglycones measured in this assay were derived by the chemical hydrolysis of conjugates by methanolic HCl. This method of hydrolysis was previously used for the isolation of the urinary aglycones of ubiquinone-7 (33) and K1 (37) from rabbit urine. Previous work from our laboratory has suggested that the majority of the 5C- and 7C-aglycones in human urine are excreted as glucuronides (10, 11). We also confirmed in this study (data not shown) that incubation of urine with ß-glucuronidase released the same two 5C- and 7C-aglycones and gave chromatograms identical to those after methanolic HCl hydrolysis. This does not exclude the possibility of other conjugates being present, because K3, which lacks the side chain, has been shown to be excreted as both glucuronide and the sulfate conjugates of the quinol form in rats (38, 39) and rabbits (40). For routine quantitative analyses, we chose methanolic HCl over enzymic hydrolysis for reasons of its greater convenience and efficiency and to ensure the complete hydrolysis of all conjugates present.
The results in Table 2 show that in both unsupplemented and supplemented adults, the excretion of the 5C-aglycone is greater than the excretion of the 7C-aglycone. This is in agreement with early radioisotopic measurements of these two aglycones after the administration of tritium-labeled K1 to adults (10, 11). In the latter studies, the intravenous administration of single doses of 45–1,000 µg of labeled K1 resulted in a gradually increasing proportion of the 7C-aglycone (assayed as vitamin K -lactone after acid hydrolysis) from an initial 13% to some 30–40% in successive urine collections made over the first 24 h (10). In rabbits, the administration of a huge (91 mg) intravenous dose of K1 changed the percentage proportion of 7C-aglycone (in 24 h urine collections) from 13% in control animals to 57% after supplementation (37). These data suggest that whereas physiological amounts of K1 are largely metabolized to the terminal 5C-aglycone, vitamin K supplementation may lead to a greater urinary excretion of the less extensively metabolized 7C-aglycone. Interestingly, one of the infants studied here (Infant A in Table 2) excreted predominantly the 7C-aglycone after supplementation with 1 mg of K1. Further support that large doses may overload the pathway comes from the previous isolation and definitive identification of a 10C side chain length aglycone after a very large K1 dose (12). No evidence for this 10C-aglycone was seen on our chromatograms. Although the preliminary supplementation studies shown in Table 2 using the present assay showed dose-related increases in the urinary concentrations of both 5C- and 7C-aglycones, there was no consistent pattern in their relative concentrations. The supplementation studies with K1 and members of the MK series (MK-4 and MK-7) clearly indicate that the 5C- and 7C-aglycones are common to the major naturally occurring forms of vitamin K (Table 2). Although the excretion of different forms of vitamin K as these two common catabolites has not been formally demonstrated before, it was to be expected in view of previous work that has shown that the isoprenoid side chains of ubiquinone-7 (33), K1 (11, 37), 
-tocopherol (37, 41), and -tocopherol (42) are all shortened in the same way and excreted as equivalent metabolites.
The proposed degradation pathway (Fig. 1) of vitamin K by successive 
- and ß-oxidation is supported by the previous unambiguous identification of 5C- and 7C-aglycones (11), by the finding of the expected 10C side chain intermediate (12), and by analogy to the findings of others that the same degradation pathway operates for similar isoprenoid compounds (33, 41, 42).
The increase in 5C- and 7C-aglycone excretion in response to supplementation with K3 is noteworthy. The generation of MK-4 from K3 through the addition of a geranyl-geranyl side chain is well described (6, 7) and is the probable source of the increased excretion of the 5C- and 7C-aglycones after K3 supplementation. It may be possible to use this metabolite assay as an index of the tissue conversion of K3 to MK-4.
Our ongoing work is directed at evaluating the present assay as a new marker of vitamin K status. An obvious advantage over current serum measurements of vitamin K is that urinary excretion reflects the degradation of all K vitamins, whereas most serum assays are only directed at the assay of a single vitamer, K1. Thus, the measurement of urinary excretion might be suitable as a measure of overall vitamin K status. The relative contribution to total urinary excretion from K1 and MKs remains to be determined. It seems likely that the majority of daily variability stems from excretion of K1, because this is by far the major dietary form, and long-chain MKs are thought to have a slower turnover (43). In addition to the prerequisite for normal renal function, a possible disadvantage of urinary measurements as a marker of vitamin K stores is that metabolites of vitamin K are also excreted via the bile, so that urinary excretion will not reflect the total elimination of vitamin K from the body.
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ACKNOWLEDGMENTS |
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Manuscript received November 15, 2004 and in revised form January 24, 2005.
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REFERENCES |
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-carboxylated osteocalcin concentration in normal individuals. J. Bone Miner. Metab. 18: 216–222.[CrossRef]
-tocopherol. Chem. Pharm. Bull. 22: 176–182.
-tocopherol metabolites in -tocopherol transfer protein-deficient patients. J. Lipid Res. 41: 1543–1551.-tocopherol in rats. J. Lipid Res. 25: 40–48.[Abstract]
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  D. J. Harrington, S. L. Booth, D. J. Card, and M. J. Shearer Excretion of the Urinary 5C- and 7C-Aglycone Metabolites of Vitamin K by Young Adults Responds to Changes in Dietary Phylloquinone and Dihydrophylloquinone Intakes J. Nutr., July 1, 2007; 137(7): 1763 - 1768. [Abstract] [Full Text] [PDF]
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