ITO Michiko Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku Unive, 農学部, 教務職員 (60250734)
FURUKAWA Yuji Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku Unive, 農学部, 教授 (60005626)
|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1996 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1995 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Phylloquinone (PK=VK_1) and the menaquinones (MK-n, VK_2) are naturally occurring forms of vitamin K.Most of the menaquinone series are synthesized microbiologically, but we have reported elsewhere that menaquinone-4 (MK-4) is an usual in being synthesized by the conversion of ingested phylloquinone or menadione (VK_3) in the major tissues of germ-free rats or mice (which lack an intestinal microflora). The present research was undertaken to clarify the mechanism of MK-4 formation from naphthoquinone ring and isoprenoid side-chain in major tissues of rats.
In the first experiment, the distribution of phylloquinone and MK-4 in various tissues were assessed after the oral administration of phylloquinone. Wistar rats were fed a vitamin-K-deficient diet for 9 days, fasted for 24h and then given phylloquinoe orally at 4mg/kg body weight. Rats were sacrificed 0,6,12, and 24h after the administration, and an analysis was made of the vitamin K analogues in the plasma, liver, brain, testis, kidn
ey, and spleen. The phylloquinone concentration in plasma and the tissues reached a peak 6h after the oral administration of phylloquinone. By contrast, the concentration of MK-4 peaked in the liver, plasma, kidney, and spleen at 12h, and in brain and testis at 24h. This data suggests that the ingested phylloquinone was probably converted into MK-4 within the tissues themselves, rather than via hepatic metabolism. The evidence for this is that, after phylloquinone administration, (i) in each of the tissues, the MK-4 concentration increased much more slowly than that of phylloquinone, and (ii) the MK-4 concentration in the plasma and liver reached only much lower levels than those seen in other tissues.
In the second and third experiments in which the labeled phylloquinone was used, it was shown that [side-chain-^3H]-label of phylloquinone was not incorporated into the side-chain of MK-4, however, the radioacitivity originated from the [ring-8]-^<14>C-phylloquinone was detected in the MK-4 fraction of various tissues. These phenomena indicate that the isoprenoid side-chain of phylloquinone once separate from its maphthoquinone ring, then it may be replaced by other isoprenoid side chain such as geranyl-geranyl group within each tissue, and suggest that K_3 must be detected in the midst of the reaction. Actually, the radioactivity from [ring-8]-^<14>C-phylloquinone was clearly detected in the K_3 fraction of heart. In the final experiment, the converted MK-4 fraction was collected and purified, then the identification of estimated MK-4 molecule was undertaken by the high-resolution mass spectrometry, and it was finally identified as MK-4.