Menadione (vitamin K3) is a catabolic product of oral phylloquinone (vitamin K1) in the intestine and a circulating precursor of tissue menaquinone-4 (vitamin K2) in rats

Abstract

Mice have the ability to convert dietary phylloquinone (vitamin K1) into menaquinone-4 (vitamin K2) and store the latter in tissues. A prenyltransferase enzyme, UbiA prenyltransferase domain-containing 1 (UBIAD1), is involved in this conversion. There is evidence that UBIAD1 has a weak side chain cleavage activity for phylloquinone but a strong prenylation activity for menadione (vitamin K3), which has long been postulated as an intermediate in this conversion. Further evidence indicates that when intravenously administered in mice phylloquinone can enter into tissues but is not converted further to menaquinone-4. These findings raise the question whether phylloquinone is absorbed and delivered to tissues in its original form and converted to menaquinone-4 or whether it is converted to menadione in the intestine followed by delivery of menadione to tissues and subsequent conversion to menaquinone-4. To answer this question, we conducted cannulation experiments using stable isotope tracer technology in rats. We confirmed that the second pathway is correct on the basis of structural assignments and measurements of phylloquinone-derived menadione using high resolution MS analysis and a bioassay using recombinant UBIAD1 protein. Furthermore, high resolution MS and (1)H NMR analyses of the product generated from the incubation of menadione with recombinant UBIAD1 revealed that the hydroquinone, but not the quinone form of menadione, was an intermediate of the conversion. Taken together, these results provide unequivocal evidence that menadione is a catabolic product of oral phylloquinone and a major source of tissue menaquinone-4.

Keywords: Conversion; Enzymes; Intestine; Menadione; Menaquinone-4; Metabolism; Phylloquinone; Rat; UBIAD1; Vitamin K.

FIGURE 1.

Time course changes in the concentrations of PK-d₇, MK-4-d₇, and their respective epoxides in the intestine (A), liver (B), and cerebrum (C) of mice orally administered PK-d₇. Female mice aged 10 weeks were orally administered PK-d₇ as a single dose of 10 μmol/kg of body weight after a 12-h fast. At 0, 3, 6, 12, 24, 48, and 72 h postadministration, the mice were sacrificed, and the intestine, liver, and cerebrum were collected to measure PK-d₇, MK-4-d₇, and their respective epoxides by APCI-LC-MS/MS as described under “Experimental Procedures.” Results represent the means for 6 mice (values shown as broken lines) and standard errors (vertical bars).

FIGURE 2.

Time course changes in the concentrations of PK-d₇ (A), MK-4-d₇ (B), and MD-d₇ (C) in the bile, lymph, and serum collected from cannulated rats orally administered PK-d₇ as a single dose of 10 μmol/kg of body weight are shown. PK-d₇ and MK-4-d₇ in the samples were measured by APCI-LC-MS/MS as described under “Experimental Procedures.” MD-d₇ was measured by the in vitro bioassay using rhUBIAD1 with GGPP-d₅ as described under “Experimental Procedures.” Except for the TLD experiment, which included three rats, the results represent the means for four rats (values shown as broken lines) and the standard errors (vertical bars). The abbreviations BD, TLD, PV, and IVC represent bile from bile duct-, lymph from thoracic lymph duct-, serum from portal vein-, and serum from inferior vena cava-cannulated rats, respectively. HR-MS analysis of authentic MD (D), MD-d₈ (E), and PK-d₇-derived MD (F) in the lymph collected as described above is shown. A lipid extract of lymph was dissolved in 100 μl of methanol, and an aliquot of the solution was used for HR-MS analysis. MS was performed on an Exactive Orbitrap mass spectrometer (Thermo Scientific), which was operated in the positive ion mode as described under “Experimental Procedures.”

FIGURE 3.

Concentrations of PK-d₇ (A), MK-4-d₇ (B), their respective epoxides, and MD-d₇ (C) in serum collected by heart puncture 6 h postadministration from cannulated rats orally administered PK-d₇ as a single dose of 10 μmol/kg body of weight are shown. PK-d₇, MK-4-d₇, and their epoxides in the samples were measured by APCI-LC-MS/MS as described under “Experimental Procedures.” MD-d₇ was measured by in vitro bioassay using rhUBIAD1 with GGPP-d₅ as described under “Experimental Procedures.” Concentrations of PK-d₇, MK-4-d₇, and their respective epoxides in small intestine (D), liver (E), heart (F), and cerebrum (G) collected 6 h postadministration from cannulated rats orally administered PK-d₇ as a single dose of 10 μmol/kg of body weight are shown. PK-d₇, MK-4-d₇, and their respective epoxides in tissues were measured by APCI-LC-MS/MS as described under “Experimental Procedures.” Except for the TLD experiment, which included three rats, the results represent the means for four rats (values are shown as columns) and the standard errors (vertical bars). The abbreviations BD, TLD, PV, and IVC represent bile duct-, thoracic lymph duct-, portal vein-, and inferior vena cava-cannulated rats, respectively.

FIGURE 4.

Time course changes in concentrations of PK (A), MK-4 (B), and MD (C) in serum collected from healthy adult volunteers orally administered PK capsules as a single dose of 40 mg. PK and MK-4 in the samples were measured by APCI-LC-MS/MS as described under “Experimental Procedures.” MD was measured by in vitro bioassay using rhUBIAD1 with GGPP-d₅ as described under “Experimental Procedures.” Error bars represent S.E.

FIGURE 5.

Conversion of PK-d₇ to MK-4-d₇ in the rat small intestine epithelial cell line IEC-6. IEC-6 cells were cultured for 3 days at a density of 5 × 10⁶ cells/well in 6-well tissue culture plates in low glucose DMEM containing 10% FCS at 37 °C in 5% CO₂ in a humidified atmosphere. The cells were then incubated with PK-d₇ (1 μm) at 37 °C for 24 h. The cells were collected, and the lipids were extracted using 3 ml of hexane/diethyl ether (97:3). MK-4-d₇ and its epoxide were measured by APCI-LC-MS/MS as described under “Experimental Procedures.” N.D., not detected. Error bars represent S.E.

FIGURE 6.

¹H NMR analysis and HR-MS analysis of menadione labeled with deuterium at the 2-methyl group (MD-d₃)-derived MK-4 generated by the incubation of MD-d₃ with rhUBIAD1 in the presence of GGPP. MD-d₃ (2 × 10⁻⁴ m) was incubated at 37 °C for 3 h with rhUBIAD1 (10 μg as protein weight of the microsomes prepared from UBIAD1 baculovirus-infected Sf9 cells) in the presence of GGPP (2 × 10⁻⁴ m) and DTT (1 mm) in 100 mm Tris-HCl buffer (pH 7.8). After incubation, the lipids were extracted with 10 ml of acetone/ethanol (9:1) solution. The extract was centrifuged at 2,500 rpm for 5 min at 4 °C, and the supernatant (2.5 ml) was evaporated under reduced pressure. This procedure was repeated 1,200 times. All the residues were then redissolved in 2 ml of hexane, evaporated under reduced pressure, and then dissolved in 60 μl of CDCl₃ to yield the resulting samples. An aliquot of the solution was used for ¹H NMR analysis. A and B, ¹H NMR spectra of authentic MK-4-d₃ and MD-d₃-derived MK-4, respectively. The number and letter H in each spectrum refer to the chemical shift (ppm) and the respective position of the proton in the 2-methyl-1,4-naphthoquinone ring or the side chain of MK-4-d₃. After ¹H NMR analysis of MD-d₃-derived MK-4, the sample solution was evaporated under reduced pressure, dissolved in 100 μl of methanol, and used for HR-MS analysis. C and D, HR-MS spectra of authentic MK-4-d₃ and MD-d₃-derived MK-4, respectively. MS was performed on an Exactive Orbitrap mass spectrometer (Thermo Scientific) operating in the positive ion mode as described under “Experimental Procedures.”

FIGURE 7.

Pathways for the conversion of MD-d₃ into MK-4-d₃ by UBIAD1. The present study demonstrated that UBIAD1 converted MD-d₃ to MK-4-d₃ but not MK-4-d₂ (Route 2), indicating that the hydroquinone, but not the quinone form of MD, is an intermediate in the course of conversion of PK to MK-4.

FIGURE 8.

The side chain of PK is cleaved to release MD during intestinal absorption followed by delivery of MD through a mesenteric lymphatic system and blood circulation to local tissues. After MD is reduced to the hydroquinone form by redox enzyme(s), it is converted to MK-4 by UBIAD1 (working hypothesis).