Clarification of the mechanism of menaquinone-4 formation from naphthoquinone ring and isoprenoid side-chain in major tissues of rats

叶绿醌（PK=VK_1）和甲基萘醌（MK-n, VK_2）是维生素k的天然存在形式。大多数甲基萘醌系列是微生物合成的，但我们在其他地方报道过，甲基萘醌-4 （MK-4）是在无菌大鼠或小鼠（缺乏肠道微生物群）的主要组织中由摄入的叶绿醌或甲基萘醌（VK_3）转化合成的常见形式。本研究旨在阐明萘醌环和类异戊二烯侧链在大鼠主要组织中形成MK-4的机制。在第一个实验中，我们在口服叶绿醌后，评估了叶绿醌和MK-4在各组织中的分布。Wistar大鼠饲喂维生素k缺乏饲料9 d，禁食24h，然后按4mg/kg体重口服叶绿醌。分别于给药后0、6、12、24h处死大鼠，分析血浆、肝脏、脑、睾丸、肾脏、脾脏中维生素K类似物的含量。血浆和组织中叶绿醌浓度在口服叶绿醌6h后达到峰值。MK-4在大鼠肝脏、血浆、肾脏和脾脏中的浓度在12小时达到峰值，在大鼠脑和睾丸中的浓度在24小时达到峰值。这一数据表明，摄入的叶绿醌可能在组织内部转化为MK-4，而不是通过肝脏代谢。这方面的证据是，在给药叶绿醌后，(i)在每个组织中，MK-4浓度的增加都比叶绿醌慢得多，（ii）血浆和肝脏中的MK-4浓度仅比其他组织中的MK-4浓度低得多。在使用标记的叶绿醌的第二次和第三次实验中，我们发现叶绿醌的[侧链-^3H]-标记并未掺入MK-4的侧链中，但在MK-4的各个组织中检测到源自[环-8]-^<14> c -叶绿醌的放射性。这些现象表明，叶绿醌的类异戊二烯侧链一旦与其甲基醌环分离，就可能在每个组织内被香叶基-香叶基等其他类异戊二烯侧链所取代，并提示在反应过程中必须检测到K_3。实际上，[ring-8]-^<14> - c -叶绿醌的放射性在心脏K_3部分被明显检测到。在最后的实验中，收集转化后的MK-4馏分进行纯化，然后用高分辨率质谱法对估计的MK-4分子进行鉴定，最终鉴定为MK-4。少

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 … More 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. Less