Gastaldi G, Ferrari G, Verri A, Casirola D, Orsenigo M N, Laforenza U
Institute of Human Physiology, University of Pavia, 27100 Pavia, Italy.
J Nutr. 2000 Oct;130(10):2556-61. doi: 10.1093/jn/130.10.2556.
In isolated rat enterocytes, both normoenergized (normal) and de-energized with rotenone, riboflavin intracellular metabolic processes, operating in association with a membrane-specific transport mechanism, were investigated. The contents of unlabeled (endogenous) and labeled (exogenous) flavins [riboflavin (RF), flavin mononucleotide (FMN), flavin adenindinucleotide (FAD)] were determined by HPLC before and after incubation with tritiated RF. In normoenergized enterocytes, total labeled RF content (i.e., total uptake, the sum of RF membrane transport and intracellular metabolism) increased steadily to a plateau after 20 min incubation; FMN and FAD contents reached a plateau between 3 and 20 min, whereas free RF content increased constantly. The phosphorylated forms prevailed over the free form ( approximately 60% of total flavins). In de-energized enterocytes, RF total uptake was significantly lower than in normoenergized enterocytes and reached a plateau after only 3 min incubation. FMN and FAD contents were significantly lower than in normoenergized enterocytes, and free RF represented the prevailing form of flavins (70% of total RF ). In both normoenergized and de-energized enterocytes, the contents of unlabeled total RF, FMN and FAD decreased significantly after 20 min incubation, whereas free RF increased significantly only in normoenergized enterocytes. After 20 min incubation, the RF structural analog 8-dimethyl-amino-8-demethyl-RF caused a significant decrease of all flavin contents, whereas 5'-deoxy-RF decreased only the total and free RF contents. Results directly confirmed the leading role of metabolic processes such as phosphorylation in RF transport by isolated small intestinal enterocytes.
在分离的大鼠肠上皮细胞中，研究了正常供能（正常状态）以及用鱼藤酮使其失去能量供应的细胞中，与膜特异性转运机制相关的核黄素细胞内代谢过程。在用氚标记的核黄素孵育前后，通过高效液相色谱法测定未标记（内源性）和标记（外源性）黄素[核黄素（RF）、黄素单核苷酸（FMN）、黄素腺嘌呤二核苷酸（FAD）]的含量。在正常供能的肠上皮细胞中，孵育20分钟后，总标记RF含量（即总摄取量，RF膜转运与细胞内代谢之和）稳步增加至平稳状态；FMN和FAD含量在3至20分钟之间达到平稳状态，而游离RF含量持续增加。磷酸化形式占主导地位，超过游离形式（约占总黄素的60%）。在失去能量供应的肠上皮细胞中，RF总摄取量显著低于正常供能的肠上皮细胞，仅孵育3分钟后就达到平稳状态。FMN和FAD含量显著低于正常供能的肠上皮细胞，游离RF是黄素的主要形式（占总RF的70%）。在正常供能和失去能量供应的肠上皮细胞中，孵育20分钟后，未标记的总RF、FMN和FAD含量均显著下降，而游离RF仅在正常供能的肠上皮细胞中显著增加。孵育20分钟后，RF结构类似物8 - 二甲基氨基 - 8 - 去甲基 - RF导致所有黄素含量显著下降，而5'-脱氧 - RF仅降低总RF和游离RF含量。结果直接证实了诸如磷酸化等代谢过程在分离的小肠肠上皮细胞转运RF中的主导作用。
