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Journal of Lipid Research, Vol. 46, 1053-1060, May 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

Dominic J. Harrington^1,*, Robin Soper, Christine Edwards, Geoffrey F. Savidge^*, Stephen J. Hodges and Martin J. Shearer^*

^* 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

¹ To whom correspondence should be addressed. e-mail: domonic.harrington{at}gstt.nhs.uk

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C₁₈) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C₁₈) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r² 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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This article has been cited by other articles:

				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]