Miyayama Takamitsu, Ogra Yasumitsu, Osima Yousuke, Suzuki Kazuo T
Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba, 260-8675, Japan.
Anal Bioanal Chem. 2008 Apr;390(7):1799-803. doi: 10.1007/s00216-008-1894-2. Epub 2008 Feb 8.
Minute amounts of tissue supernatants from mouse neonates bearing a mutation in the copper (Cu)-transporter gene, Atp7a, were injected into narrow-bore HPLC coupled with an inductively coupled plasma-mass spectrometer (ICP-MS) to examine Cu metabolism. In the 14-day-old mutant neonates, Cu accumulated in the intestine in the metallothionein (MT)-bound form, and mRNA expression of the two MT isoforms was increased. Meanwhile, Cu in the MT-bound form (Cu-MT) was depleted in the liver and mRNA expression decreased in comparison with wild-type mice. These results suggest that Cu is not secreted by intestinal microvillus cells into bloodstream due to the defect of Atp7a, and systemic depletion of Cu occurred. On the other hand, in the kidneys of mutant mice, Cu accumulated in the MT-bound form despite the fact that mRNA expression of the two MT isoforms was low. Part of Cu-MT in microvillus cells may be released into bloodstream at turnover and be preferably taken up by the kidneys. Consequently, the mRNA expression of MT isoforms was not always coincident with the amounts of MT proteins binding Cu, and narrow bore HPLC-ICP-MS used for MT protein determination is a complementary technique to real-time RT-PCR used for MT mRNA determination in Cu speciation.
将携带铜（Cu）转运蛋白基因Atp7a突变的小鼠新生幼崽的微量组织上清液注入与电感耦合等离子体质谱仪（ICP-MS）联用的窄孔高效液相色谱仪中，以检测铜代谢情况。在14日龄的突变新生幼崽中，铜以金属硫蛋白（MT）结合的形式在肠道中积累，并且两种MT同工型的mRNA表达增加。与此同时，与野生型小鼠相比，肝脏中MT结合形式的铜（Cu-MT）减少，mRNA表达降低。这些结果表明，由于Atp7a缺陷，肠道微绒毛细胞无法将铜分泌到血液中，从而导致全身铜缺乏。另一方面，在突变小鼠的肾脏中，尽管两种MT同工型的mRNA表达较低，但铜仍以MT结合的形式积累。微绒毛细胞中的部分Cu-MT可能在周转时释放到血液中，并优先被肾脏摄取。因此，MT同工型的mRNA表达并不总是与结合铜的MT蛋白量一致，用于MT蛋白测定的窄孔HPLC-ICP-MS是用于铜形态分析中MT mRNA测定的实时RT-PCR的补充技术。
