The Enzymatic Conversion of Phylloquinone to Menaquinone-4.
Al Rajabi, Ala.
forms of vitamin K share the naphthoquinone ring, but differ in the side chain. The
naphthoquinone ring is the active site for the enzyme cofactor activity of vitamin K.
Mammals have the ability to convert dietary phylloquinone into MK-4 and store the latter
in specific tissues. This suggests that MK-4 plays a role beyond that of an enzyme
cofactor. Several functions unique to ... read moreMK-4 have been proposed. Recently, UBIAD1 was
identified as the enzyme catalyzing prenylation of menadione with geranylgeranyl side
chain forming MK-4. However, the exact mechanism by which phylloquinone is converted to
MK-4 and the tissue localization(s) for this conversion are not known. We hypothesized
that the phylloquinone's phytyl side chain is removed and a preformed geranylgeranyl
side chain is added to the resultant menadione to form MK-4. The overall goal of this
thesis was to provide direct evidence that menadione is the intermediate in this
conversion. Menadione detection would provide insight into the location and mechanism by
which phylloquinone is converted to MK-4. A highly reproducible HPLC method was
developed and validated to measure menadione in urine. Archived urine samples of
participants in a human phylloquinone supplementation study were analyzed (n=367).
Phylloquinone supplementation resulted in a significant increase in urinary menadione
secretion (P<0.0001). These results lend support to the hypothesis that menadione is
an intermediate in the conversion of phylloquinone to MK-4. Furthermore, changes in
urinary menadione excretion were significantly correlated with changes in biomarkers of
vitamin K status, including serum phylloquinone and percent serum undercarboxylated
osteocalcin (P=0.0008 and 0.02, respectively). In a separate metabolic study of 42
healthy men and women, we used deuterium-labeled (-d) collard greens to confirm that
urinary menadione-d (MW 173-174) was formed from phylloquinone-d. For an intake of 255
nmol phylloquinone-d, up to 14.9%± 10.0% (mean± SD) was recovered as
menadione-d in urine. The urinary menadione extraction method developed as part of this
thesis, was subsequently modified to extract menadione from different matrices including
rat urine, serum, tissues, cells and cell culture media. New menadione detection methods
were developed using both LC/MS and LC-APCI-MS/MS. Male Fischer 344 rats (8 months;
n=15) were fed 1.6 mg of phylloquinone-d (MW 459-463) per kg diet for 0 (control), 1 and
7 days. Using LC/MS, phylloquinone-d and MK-4-d (MW 446-449) were detected in tissues
after 1 day. MK-4-d carried the d-label on the naphthoquinone ring, but not on the side
chain, confirming the need for side chain removal for its formation. Using LC/MS,
unconjugated menadione-d (MW 173-174) was measured in urine. Using LC-MS/MS,
unconjugated menadione-d was detected in serum of rats (n=4/5 at day 1; n= 3/5 at day
7). A Caco-2 cell culture model was used to study the role of the enterocytes in the
conversion process. Neither MK-4 nor menadione was detected in cells treated with
phylloquinone. However, when treated with menadione, MK-4 was formed. These data suggest
that enterocytes are not the location for the phytyl side chain removal step of this
conversion. In conclusion, menadione can be measured in both urine and serum. This is
the first time unconjugated menadione has been detected in biological materials. We
demonstrated that newly formed MK-4 is synthesized from the dietary phylloquinone by the
means of replacing the phylloquinone's phytyl side chain with the geranylgeranyl side
chain. This conversion does not appear to occur in the enterocytes. Menadione is
produced from dietary phylloquinone. Our findings support that menadione is an
intermediate in phylloquinone to MK-4 conversion. Future studies are required to
determine the exact location and function of this
Thesis (Ph.D.)--Tufts University, 2011.
Submitted to the Dept. of Biochemical and Molecular Nutrition.
Advisor: Sarah Booth.
Committee: John Suttie, and Sang-Woon Choi.
Keywords: Nutrition, Biochemistry, and Molecular biology.read less