A new LC-MS assay for the quantitative analysis of vitamin K metabolites in human urine

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.

Keywords: beta-oxidation; liquid chromatography-mass spectrometry; menaquinone; omega-oxidation; phylloquinone.

Fig. 1.

Proposed VK (PK and MK4) catabolic pathway. Both K vitamers are known to undergo successive ω-oxidations, catalyzed primarily by CYP4F2, which result in the production of their respective ω-carboxylic acid metabolites (13, 14). It is believed that these VK acid metabolites then enter the fatty acid β-oxidation pathway, going through several rounds of oxidative side-chain cleavage, which eventually leads to the observed urinary VK metabolites, K acid I and K acid II. MK1 ω-COOH is a putative metabolic intermediate between K acids I and II.

Fig. 2.

Hypothetical APCI⁻ ionization and fragmentation mechanisms for VK analogs. Initial ionization occurs via addition of an electron to the quinone ring, generating a radical anion, as shown. Most VK species then form a major m/z 185 fragment ion via homolytic cleavage between the second and third carbons of the side chain (A). However, the methyl ester of MK1 ω-COOH instead forms major fragment ions at either mass 197 (at relatively low CE) or 195 (high CE) (supplemental Fig. S3). This could occur through a mechanism involving base-catalyzed double bond isomerization followed by cyclization and aromatization as shown in pathway B.

Fig. 3.

APCI⁻ LC-MS/MS (SRM) chromatograms of VK catabolites K acid II (m/z 286 > 185, red lines), K acid I (m/z 312 > 195, blue lines) and MK1 ω-COOH (m/z 284 > 195, black lines), analyzed as their respective methyl ester derivatives. A: Chromatogram obtained from a human urine sample spiked with synthetic standards of the three derivatized VK catabolites. B: Chromatogram obtained from a human urine sample showing basal (i.e., presupplement) levels of VK catabolites. C: Chromatogram of the urinary extract, obtained from the same subject as in chromatogram B, collected 0–24 h after the subject ingested two capsules of Koncentrated K. All data in the figure were normalized to the height of the internal standard peak, MK1 (peak height ratio).

Fig. 4.

Hypothetical alternative mechanisms for the conversion of MK1 ω-COOH into the observed urinary metabolite, K acid II.