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The Journal of Nutrition Vol. 128 No. 2 February 1998,
pp. 220-223
,, and, 3
* Department of Nutritional Sciences and Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI 53706
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The ability of male rats to accumulate menaquinone-4 (MK-4) in tissues when fed a vitamin K-deficient diet supplemented with intraperitoneal phylloquinone (K) as the sole source of vitamin K for 14 d was assessed. In both conventionally housed controls and gnotobiotic rats, supplementation with the equivalent of 1500 µg vitamin K/kg diet increased (P < 0.001) tissue MK-4 concentrations above those of controls fed a vitamin K-deficient diet. MK-4 concentrations were ~5 ng/g (11 pmol/g) in liver, 14 ng/g in heart, 17 ng/g in kidney, 50 ng/g in brain and 250 ng/g in mandibular salivary glands of gnotobiotic rats. MK-4 concentrations in conventionally housed rats were higher than in gnotobiotic rats in heart (P < 0.01), brain (P < 0.01) and kidney (P < 0.05) but lower in salivary gland (P < 0.05). Cultures of a kidney-derived cell line (293) converted K to the epoxide of MK-4 in a manner that was dependent on both time of incubation and concentration of vitamin K in the media. A liver-derived cell line (H-35) was less active in carrying out this conversion. These data offer conclusive proof that the tissue-specific formation of MK-4 from K is a metabolic transformation that does not require bacterial transformation to menadione as an intermediate in the process.
KEY WORDS: vitamin K · phylloquinone · menaquinone-4 · gnotobiotic rats · cell culture
The metabolic role of vitamin K is as a cofactor for an enzyme that converts specific glutamyl residues in a limited number of proteins to We recently have observed (Will et al. 1992 Further study of the apparent conversion of dietary phylloquinone to MK-4 in the rat has shown that liver and plasma have low MK-4 concentrations, but that in extrahepatic tissues such as brain, pancreas, salivary gland and sternum, the concentrations of MK-4 exceed those of phylloquinone (Sundaram et al. 1996 The available data suggest that the route to MK-4 synthesis may not be through the liberation of menadione from phylloquinone by intestinal bacteria and subsequent realkylation of menadione in target tissues but that the conversion is tissue mediated. Definitive evidence that microbial metabolism is not involved, however, is lacking. The experiments reported here demonstrate that phylloquinone is converted readily to MK-4 in both germ-free rats (Gustafsson 1959 Rats.
Animal studies were approved by the University of Wisconsin-Madison Research Animal Resources Center. Rats housed under gnotobiotic conditions (Sprague-Dawley strain males) were obtained from Taconic Farms (Germantown, NJ). Germ-free status was monitored and certified throughout the study by a microbiologist at the University of Wisconsin-Madison Gnotobiotic Facility. Age-matched Sprague-Dawley males used as controls for this experiment were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and were housed in conventional wire-bottom cages in the Department of Biochemistry Animal Facility. Rats in this experiment were 7-wk old (~200 g) at the beginning of the experiment. A second experiment comparing menadione and phylloquinone as sources of MK-4 used weanling Sprague-Dawley rats.
Diets.
Vitamin K-deficient diet (TD 81053)5 was obtained from Teklad Research Diets (Madison, WI). All diet was prepackaged in double-wrap steripaks before sterilization by SteriGenics (Schaumburg, IL). Diet packs were irradiated using a dose of 47.4-53.6 kGy. A phylloquinone solution was prepared by appropriate dilution of injectable phylloquinone (AquaMephyton, 10 g/L, Merck, West Point, PA) into sterile saline. On alternate days, both the gnotobiotic and positive controls (+K) rats received an intraperitoneal injection of 3.3 µmol (1500 µg) phylloquinone/kg diet consumed. During the 14-d experiment, each rat received seven injections for a total of 630 µg phylloquinone. The negative control group ( Assays.
Biological prothrombin was determined by assay of thrombin catalyzed amidolysis of the chromogenic peptide substrate S2238 (Chromgenix, Molnda, Sweden) as described by Shah et al. (1984) Cell culture conditions.
The H-35 rat hepatoma cell line and human 293 transformed kidney cell line (American Type Culture Collection, Rockville, MD) were cultured in T-75 flasks (Corning Laboratories, Corning, NY) at 37°C in humidified 5% CO2. Cells were cultured in 10 mL Dulbecco's modified Eagle's medium (DMEM; Sigma Chemical, St. Louis, MO) with 10% fetal bovine serum (Hyclone Labs, Logan, UT). AquaMephyton was added at 0, 2.2 or 22 µmol/L of media and warfarin (obtained from the Wisconsin Alumni Research Foundation, Madison, WI) was added to media at 3.3 µmol/L as noted. After incubation, cells were lysed by sonication in a 2-mL volume of deionized water. Fifty microliters of cell lysate were analyzed for protein determination by the method of Lowry et al. (1951) Statistical analysis.
One-way analysis of variance and Tukey-Kramer tests (Fisher and Van Belle 1993 Vitamin K metabolism in gnotobiotic rats.
To assess the influence of an active gut flora on menaquinone production, male rats were fed a vitamin K-deficient diet (
Conversion of phylloquione to MK-4 in cultured cells.
Because the results of the gnotobiotic study indicated that bacterial metabolism is not needed for phylloquinone to serve as a precursor to MK-4 synthesis, a direct demonstration of the cellular conversion of these two forms of vitamin K was attempted. When a transformed kidney cell line (293) was incubated in the presence of 1 mg/L (2.2 µmol/L) phylloquinone, there was an increase in cellular K and KO, and menaquinone-4 epoxide (MKO-4) and a much smaller amount of MK-4 were formed (Table 1). In the presence of warfarin, less K and KO accumulated in cells, and neither MK-4 nor MKO-4 could be detected. The conversion of phylloquinone to menaquinone-4 was not observed in a rat hepatoma cell line (H-35) incubated in the same concentration of phylloquinone.
Menaquinone-4 is not a major constituent of the spectrum of compounds with vitamin K activity produced by intestinal bacteria (Fernandez and Collins 1987
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
-carboxyglutamyl (Gla)4 residues. The well-characterized proteins involved in this posttranslational modification include plasma clotting factors II (prothrombin), VII, IX and X, plasma proteins C and S and two proteins (osteocalcin and matrix Gla protein) first found in bone (Suttie 1996
). The metabolic requirement for vitamin K is satisfied by dietary phylloquinone (2-Me-3-phytyl-1,4-naphthoquinone) present in plants and, to an undetermined extent, by a bacterially produced series of 2-Me-3-polyisoprenyl homologues called menaquinones (MK-n) produced in the lower bowel (Suttie 1995). Menadione, commonly used in animal diets, is converted to 2-Me-3-geranyl-geranyl-1,4-naphthoquinone (MK-4) when administered to animals (Taggart and Matschiner 1969).
) that the livers of chicks fed phylloquinone as a sole source of vitamin K contain as much MK-4 as phylloquinone, and this observation has been confirmed by other investigators (Guillaumont et al. 1992, Sakamoto et al. 1996, Thijssen and Drittij-Reijnders 1994). These observations have reconfirmed some of the conclusions of previous studies carried out in the early 1960s with rather low specific activity radiolabeled vitamin K. At that time evidence for the conversion of dietary phylloquinone to tissue MK-4 was presented (Billeter and Martius 1960). These data were confusing, and in subsequent studies (Billeter et al. 1964), it was concluded that phylloquinone was cleaved to form menadione by bacteria in the gut and that menadione was absorbed and converted to MK-4 in rat and pigeon tissues. The alkylation of menadione to MK-4 by geranyl-geranyl pyrophosphate subsequently has been demonstrated in vitro (Dialameh et al. 1970).
, Thijssen and Drittij-Reijnders 1994). Relatively high concentrations of MK-4 also have been reported in human extrahepatic tissues (Thijssen and Drittij-Reijnders 1996), and it has been demonstrated in the rat that these extrahepatic tissues contain more MK-4 after phylloquinone administration than after MK-4 administration (Thijssen et al. 1996).
) and in aseptic mammalian cell cultures.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
K) received only the vitamin K-deficient diet with no vitamin K supplementation for 14 d. In a second experiment, weanling male rats were fed the same vitamin K-deficient diet supplemented with 2 µmol phylloquinone or menadione/kg diet.
. At the termination of the experiment, rats were killed by CO2 asphyxiation, and the desired tissues removed, frozen in liquid N2 and stored at 80°C for analysis. Determination of phylloquinone, MK-4 and MKO-4 content of tissues was carried out as described by Kindberg and Suttie (1989).
. The remaining sample was denatured with 1 mL ethanol and extracted with 6 mL hexane. The hexane extract was dried under a stream of filtered air. Dried extracts were redissolved in 250 µL methanol and vitamin K concentration measured using reverse-phase high performance liquid chromatography (HPLC) with postcolumn zinc reduction and fluorometric detection by a modification of the method of Haroon et al. (1986). The HPLC system consisted of a gradient controller model 680, two model 510 pumps, a WISP model 712 automatic injector, and a scanning fluorescence detector model 474 (Waters, Milford, MA). The sample, in 100% methanol, was injected onto a Zorbax ODS 4.6 mm I.D. × 25 cm column (Mac-Mod Analytical, Chadds Ford, PA) at a 1 mL/min flow rate. The solvent was increased to 150 mL/L methylene chloride in methanol through a 10-min nonlinear (late-ramping) gradient and remained at 150 mL/L methylene chloride in methanol for an additional 9 min before returning to initial solvent conditions (100% methanol) via a 2-min linear gradient. Peak heights of samples were compared with those of known standards.
) were performed using an InStat statistical software program (Graph Pad, San Diego, CA). All values are reported as means ± SD.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
K control) for 14 d, or the same diet supplemented with phylloquinone, and were housed in either conventional wire-bottom cages or under germ-free conditions. After 14 d the plasma prothrombin concentration of the +K control rats was 101.7 ± 3.9% of a pooled reference standard compared with 89.8 ± 3.5% for the gnotobiotic rats (P < 0.05) and 47.8 ± 10.8% (P < 0.001) for the K control rats.
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Fig 1.
Effect of phylloquinone supplementation on rat tissue phylloquinone (upper panel) and menaquinone-4 (MK-4, lower panel) concentrations. Seven-week-old rats were fed a vitamin K-deficient diet for 14 d ( K control) or were supplemented with the equivalent of 1500 µg phylloquinone/kg diet by intraperitoneal injection (gnotobiotic and +K control group). There were six rats/group and values are means ± SD for phylloquinone and MK-4. Phylloquinone supplementation increased phylloquinone concentrations in all tissues (P < 0.001). Plasma concentrations of MK-4 were below the detection level in all groups (ND). In all other tissues, phylloquinone supplementation increased MK-4 concentration (P < 0.001). Tissue concentrations of MK-4 differed in gnotobiotic and +K control groups at P < 0.05 (*) or P < 0.01 (**).
K control group. Concentrations of MK-4 were ~5 ng/g (11 pmol/g) in liver, 14 ng/g in heart, 17 ng/g in kidney, 50 ng/g in brain and 250 ng/g in mandibular salivary gland. Menaquinone-4 concentration in heart, kidney and brain were higher in the +K control group than in the gnotobiotic group. Concentrations of MK-4 did not differ in liver. In salivary gland, the tissue with the highest MK-4 concentration, the highest concentration was observed in the gnotobiotic group. In addition to the MK-4 present in these tissues, menaquinone-4-epoxide (MKO-4) was detected at a concentration 15-30% that of MK-4. These data establish that active gut flora are not required for the conversion of phylloquinone to MK-4 in rat tissues.
, Taggart and Matschiner 1969, Thierry et al. 1970), the ability of the rat to maintain tissue MK-4 concentration when phylloquinone, menadione or both were available as an MK-4 precursor was assessed. The data in Figure 2 again demonstrate that liver, heart and kidney contain relatively high concentrations of phylloquinone when it is the dietary source of vitamin K. The groups fed phylloquinone had higher concentrations of tissue phylloquinone than the menadione-fed group for all tissues. The presence of menadione in the phylloquinone supplemented diet did not influence phylloquinone tissue concentrations. Phylloquinione and menadione supplementation resulted in similar concentrations of MK-4 in tissues (Fig. 2), and supplementation with both forms of the vitamin increased tissue MK-4 concentrations in all tissues; differences were significant in heart and kidney.
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Fig 2.
Tissue phylloquinone (upper panel) and menaquinone-4 (MK-4, lower panel) concentrations of rats fed phylloquinone or menadione. Weanling male rats were fed a diet containing 2 µmol phylloquinone/kg (group 1), 2 µmol menadione/kg diet (group 3), or both forms of the vitamin (group 2) for 7 d. There were five rats/group, and values are means ± SD. Phylloquinone supplementation increased tissue phylloquinone in all tissues (P < 0.001). MK-4 concentrations in group 1 or group 3 were less than in group 2 at P < 0.01 (*).
View this table:
Table 1.
Formation of menaquinone-4 and menaquinone-4 epoxide in cultured human kidney (293) and rat liver (H-35) cells1,2
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Fig 3.
Accumulation of menaquinone-4 epoxide in human kidney 293 cells incubated in the presence of 2.2 and 22 µmol/L of phylloquinone. Values are means ± SD for three flasks/time point.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
, Ramotar et al. 1984). It, however, has been demonstrated to be a product of the tissue metabolism of menadione by Taggart and Matschiner (1969). The data presented here confirm recent reports (Guillaumont et al. 1992, Sakamoto et al. 1996, Thijssen and Drittij-Reijnders 1994, Thijssen and Drittij-Reijnders 1996, Will et al. 1992), suggesting that phylloquinone can be converted to menaquinone-4 by a number of species. The presence of similar amounts of MK-4 in the tissues of gnotobiotic and conventionally housed rats fed phylloquinone clearly indicate that bacterial action is not required for the conversion of phylloquinone to menaquinone-4.
, Taggart and Matshincer 1969) and the in vivo production of MK-4 from phylloquinone (Thijssen et al. 1996).
) and are consistent with the cell culture data.
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FOOTNOTES |
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-carboxyglutamic acid; K, phylloquinone; KO, phylloquinone 2,3-epoxide; MK-4, menaquinone-4; MKO-4, menaquinone-4 2,3-epoxide.
Manuscript received 25 July 1997. Initial reviews completed 26 September 1997. Revision accepted 27 October 1997.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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