以 Caco-2 細胞模式探討 ferric reductase 的電子來源與影響攝鐵能力之成份

Abstract

已知小腸細胞鐵還原酵素 ferric reductase 是影響鐵吸收率的重要因子。本實驗目的在於利用 Caco-2 細胞模式，探討 ferric reductase 反應的電子來源及影響其活性之成份，並評估這些成份對細胞攝鐵能力之影響。 Caco-2 細胞在滿盤後 13-18天進行攝鐵實驗，以 100 M FeCl3 提供鐵源，處理 6 小時之後，測量細胞 ferric reductase 酵素活性及鐵蛋白含量，作為攝鐵能力之指標。培養槽有單槽與雙槽兩種，使用雙槽培養皿時，細胞接種於內槽形成具有 tight junction 之單槽，鐵源均添加於上層，維生素 C 等實驗成份則依實驗設計而添加於上層或下層。 單槽實驗結果顯示，以不添加任何維生素 C 為控制組， 1 mM 還原型維生素 C (AA) 可使 ferric reductase 活性升高 12 倍，使細胞鐵蛋白含量升高 10 倍，其效應與劑量有正相關性。 1 mM 氧化型維生素 C (DeHA) 會使酵素活性升高 3-4 倍，並使鐵蛋白含量增高 5-6 倍。 AA 對酵素活性的影響約為 DeHA 的 4 倍，對鐵蛋白的影響則約為 1-2 倍。 以 DeHA 處理細胞時，若同時利用 BSO 降低 GSH 含量， 可使酵素活性及鐵蛋白含量分別降低 60% 及 50%；若利用 BCNU 降低細胞 GSH 含量，也可使酵素活性及鐵蛋白含量則分別降低 60% 及 40% ；可見 GSH 與鐵之吸收有關，小腸細胞會利用細胞內 GSH 還原氧化型維生素 C 而增加 ferric reductase 活性與促進鐵吸收。以 quercetin 或單寧酸處理細胞時，都會使 ferric reductase 活性上升，但鐵蛋白含量卻低於控制組，表示此兩種物質會藉由化學作用抑制鐵質進入細胞。 雙槽實驗結果顯示， AA 無論添加於上層或下層，均可使 ferric reductase 活性與鐵蛋白含量顯著高於未添加者； DeHA 添加於上層也可使酵素活性與鐵蛋白含量顯著高於未添加者，但添加於下層則無效應。添加於下層之 AA 無法拮抗 quercetin 或單寧酸對鐵吸收的抑制作用。針對 carbonyl Fe、FeCl3、Fe-NTA 三種鐵劑，下層未添加 AA 時，鐵蛋白含量為 Fe-NTA > FeCl3 > carbonyl Fe；添加 AA 時， carbonyl Fe 與 FeCl3 處理組之鐵蛋白含量顯著增加，以 FeCl3 顯著高於 carbonyl Fe ，但 Fe-NTA 與前述兩者並無顯著差異；顯示利用 Caco-2 細胞評估食物鐵可利用率時，細胞維生素 C 含量不足可能成為限制因素而有低估三價鐵之利用率的疑慮。

Studies have implicated duodenal ferric reductase activity as an important factor in regulation of intestinal iron absorption. Caco-2 cell model was used to study the electron source of ferric reductase and the effect of ascorbic acid on accuracy of iron bioavailability estimation. In the iron uptake experiments, Caco-2 cells were conducted after 13-18d post-confluence and treated with 100 M of FeCl3 for six hours. Cellular ferric reductase activity was assayed to be an indicator of reducing ability and ferritin level was assayed to be an indicator of iron status in Caco-2 cells. Compared to the control group, ferric reductase activity and ferritin levels showed respective 3- to 4-fold and 5- to 6-fold increase when dehydroascorbic acid (DeHA) was added. When DeHA + BSO or DeHA + BCNU were added, both ferric reductase activity decrease 60%, and ferritin levels showed respective 50% and 40% decrease compared with the group which only added DeHA. This study demonstrated that cellular vitamin C mediated iron uptake enzymatically in addition to extracellular reduction. Cellular GSH level can also affect the ability of cellular iron uptake. Ferric reductase activity was very high when cells were treated with quercetin or tannic acid, but the ferritin level was lower than control group both. To assess the effect of ascorbic acid on accuracy of iron bioavailability estimation, cells were seeded in insert plate and ascorbic acid were added in basolateral site. The ferric reductase activity and ferritin level were both significantly higher in the group that was added ascorbic acid in basolateral site. This result indicates the importance of electron source on estimation of iron bioavailability.