Dietary vitamin K is remodeled by gut microbiota and influences community composition

Abstract

Vitamins have well-established roles in bacterial metabolism. Menaquinones (MKn, n = prenyl units in sidechain) are bacterially produced forms of vitamin K produced by the gut microbiota and consumed in the diet. Little is known about the influence of dietary vitamin K quinones on gut microbial composition and MKn production. Here, male and female C57BL6 mice were fed a vitamin K deficient diet or vitamin K sufficient diets containing phylloquinone (PK, plant-based vitamin K form), MK4, and/or MK9. DNA was extracted from cecal contents and 16S sequencing conducted to assess microbial composition. Cecal microbial community composition was significantly different in vitamin K deficient female mice compared to females on vitamin K sufficient diets (all p < .007). Parallel trends were seen in male mice, but were not statistically significant (all p > .05 but <0.1). Next, stable isotope-labeled vitamin K quinones were supplemented to male and female C57BL6 mice (²H₇PK, ¹³C₁₁MK4, ²H₇MK7, ²H₇MK9) and to an in vitro fermentation model inoculated with human stool (²H₇PK, ²H₇MK4, ²H₇MK9, or vitamin K precursor ²H₈-menadione). Vitamin K quinones in feces and culture aliquots were measured using LC-MS. In vivo, supplemented vitamin K quinones were remodeled to other MKn (²H₇- or ¹³C₆-labeled MK4, MK10, MK11, and MK12), but in vitro only the precursor ²H₈-menadione was remodeled to ²H₇MK4, ²H₇MK9, ²H₇MK10, and ²H₇MK11. These results suggest that dietary vitamin K deficiency alters the gut microbial community composition. Further studies are needed to determine if menadione generated by host metabolism may serve as an intermediate in dietary vitamin K remodeling in vivo.

Keywords: Gut microbiota; menaquinone; metabolism; micronutrient; stable isotope; vitamin K.

Figure 1.

Chemical structures of vitamin K (VK) quinones. (a) All VK quinones share a common naphthoquinone ring (menadione (MD), considered a provitamin K) and can vary in length and saturation of the sidechain. Phylloquinone (PK) is produced by plants and has a mostly saturated sidechain, whereas menaquinones (MKn) are largely produced by bacteria and have unsaturated (n = number of prenyl units) sidechains. Stable-isotope label placement on B) deuterium (²H)-labeled and C) carbon-13 (¹³C)-labeled quinones used and/or detected in the stable isotope-labeled animal study (Study 2) and in vitro fermentation study (Study 3)

Figure 2.

Non-metric multidimensional scaling (NMDS) ordination of mouse cecal microbial communities in the unlabeled vitamin K supplementation study (Study 1). (a) Male and female mice had significantly different cecal microbial communities. Microbial communities were also significantly different by diet in both (b) female and (c) male mice. PK = phylloquinone, MK4 = menaquinone-4, MK9 = menaquinone-9, VK def = vitamin K deficient

Figure 3.

Microbial taxa that were significantly enriched across different diet groups in female mice of the unlabeled vitamin K animal study (Study 1, FDR-corrected p ≤ 0.05). Assessed with Kruskal-Wallis tests across diet groups for all ASVs with a mean abundance threshold of 0.001 (the minimum mean value needed in at least one of the factor levels for an ASV to be retained in the analysis). *indicates a pairwise comparison p < .05. No taxa were significantly enriched across diet groups in male mice after FDR adjustment. PK = phylloquinone, MK4 = menaquinone-4, MK9 = menaquinone-9, VK def = vitamin K deficient

Figure 4.

Unlabeled and stable isotope labeled (²H₇- or ¹³C₆-) MK10-MK12, shown as mean % total MK10-MK12 (a and c) and mean concentrations (b and d), in male and female mice in the labeled animal study (Study 2). Error bars on the stacked bar charts represent the standard error of the mean. Unlabeled and stable isotope-labeled MK4 were excluded from the analysis because MK4 deriving from host intestinal conversion could not be differentiated from bacterially produced MK4

Figure 5.

Production of ²H-labeled MKn at each time point in the ²H-MD supplemented vessel in Study 3. ²H-labeled MKn were not recovered in any of the other supplemented vessels. Stacked bars represent the mean (n = 3 experiments) concentrations, with error bars representing standard deviation from the mean. ²H₇MK11 concentrations are corrected for interference