Microanalysis of non-heme iron in animal tissues.
The method recommended by the Iron Panel of the International Committee for the Standardization in Haematology for measurement of serum iron was adapted for measurement of non-heme iron in animal tissues. The method developed was designed specifically to facilitate measurement of non-heme iron using as little as 10 mg of tissue, in a final reaction volume of 60 microl. In this assay, tissue homogenates are treated with hydrochloric acid and trichloroacetic acid and heated at 95 degrees C. Non-heme iron is released and protein is precipitated. Following centrifugation, iron in the supernatant is reacted with ferrozine in the presence of the reducing agent thioglycolic acid, and the complex is quantified by spectrophotometry. The method was validated by analysis of two Standard Reference Materials (bovine liver), comparing results of this assay against certified values and concentrations determined by flame atomic absorption spectrometry following acid digestion. Results using this method for analysis of non-heme iron in guinea pig tissues (liver, kidney and heart) compared favorably with those obtained using micro-scale adaptations of three published reference methods. The new method was more sensitive, required less time, and was less cumbersome than the three published methods to which it was compared.
Rebouche
Charles J
CJ
Department of Pediatrics, University of Iowa, Iowa City, IA 52242, USA. charles-rebouche@uiowa.edu
Wilcox
Cari L
CL
Widness
John A
JA
D000818:Animals; D005297:Ferrozine / Q000737:chemistry; D006168:Guinea Pigs; D006418:Heme / Q000737:chemistry; D006851:Hydrochloric Acid / Q000737:chemistry; D007501:Iron / Q000032:analysis* / Q000737:chemistry; D007502:Iron Chelating Agents / Q000737:chemistry; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D009206:Myocardium / Q000737:chemistry; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013054:Spectrophotometry, Atomic / Q000379:methods*; D013864:Thioglycolates / Q000737:chemistry; D014238:Trichloroacetic Acid / Q000737:chemistry
D007502:Iron Chelating Agents; D013864:Thioglycolates; D005297:Ferrozine; D006418:Heme; D014238:Trichloroacetic Acid; C017487:2-mercaptoacetate; D007501:Iron; D006851:Hydrochloric Acid