Narrow-bore HPLC-ICP-MS for speciation of copper in mutant mouse neonates bearing a defect in Cu metabolism.
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.
Miyayama
Takamitsu
T
Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba, 260-8675, Japan.
Ogra
Yasumitsu
Y
Osima
Yousuke
Y
Suzuki
Kazuo T
KT
D000818:Animals; D000831:Animals, Newborn; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003300:Copper / Q000032:analysis / Q000172:deficiency / Q000378:metabolism*; D005260:Female; D020869:Gene Expression Profiling; D006579:Heterozygote; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008659:Metabolic Diseases / Q000235:genetics / Q000378:metabolism*; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013997:Time Factors
D012333:RNA, Messenger; D003300:Copper; D008668:Metallothionein