Kinetics of iron import into developing mouse organs determined by a pup-swapping method.

The kinetics of dietary iron import into various organs of mice were evaluated using a novel pup-swapping approach. Newborn pups whose bodies primarily contained (56)Fe or (57)Fe were swapped at birth such that each nursed on milk containing the opposite isotope. A pup from each litter was euthanized weekly over a 7-week period. Blood plasma was obtained, and organs were isolated typically after flushing with Ringer's buffer. (56)Fe and (57)Fe concentrations were determined for organs and plasma; organ volumes were also determined. Mössbauer spectra of equivalent (57)Fe-enriched samples were used to quantify residual blood in organs; this fraction was excluded from later analysis. Rates of import into brain, spleen, heart, and kidneys were highest during the first 2 weeks of life. In contrast, half of iron in the newborn liver exited during that time, and influx peaked later. Two mathematical models were developed to analyze the import kinetics. The only model that simulated the data adequately assumed that an iron-containing species enters the plasma and converts into a second species and that both are independently imported into organs. Consistent with this, liquid chromatography with an on-line ICP-MS detector revealed numerous iron species in plasma besides transferrin. Model fitting required that the first species, assigned to non-transferrin-bound iron, imports faster into organs than the second, assigned to transferrin-bound-iron. Non-transferrin-bound iron rather than transferrin-bound-iron appears to play the dominant role in importing iron into organs during early development of healthy mice.

Speciation of iron in mouse liver during development, iron deficiency, IRP2 deletion and inflammatory hepatitis.

The iron content of livers from (57)Fe-enriched C57BL/6 mice of different ages were investigated using Mössbauer spectroscopy, electron paramagnetic resonance (EPR), electronic absorption spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS). About 80% of the Fe in an adult liver was due to blood; thus removal of blood by flushing with buffer was essential to observe endogenous liver Fe. Even after exhaustive flushing, ca. 20% of the Fe in anaerobically dissected livers was typical of deoxy-hemoglobin. The concentration of Fe in newborn livers was the highest of any developmental stage (∼1.2 mM). Most was stored as ferritin, with little mitochondrial Fe (consisting primarily of Fe-S clusters and haems) evident. Within the first few weeks of life, about half of ferritin Fe was mobilized and exported, illustrating the importance of Fe release as well as Fe storage in liver function. Additional ferritin Fe was used to generate mitochondrial Fe centres. From ca. 4 weeks of age to the end of the mouse's natural lifespan, the concentration of mitochondrial Fe in liver was essentially invariant. A minor contribution from nonhaem high-spin Fe(II) was observed in most liver samples and was also invariant with age. Some portion of these species may constitute the labile iron pool. Livers from mice raised on an Fe-deficient diet were highly Fe depleted; they were devoid of ferritin and contained 1/3 as much mitochondrial Fe as found in Fe-sufficient livers. In contrast, brains of the same Fe-deficient mice retained normal levels of mitochondrial Fe. Livers from mice with inflammatory hepatitis and from IRP2(-/-) mice hyper-accumulated Fe. These livers had high ferritin levels but low levels of mitochondrial Fe.

Low-molecular-mass metal complexes in the mouse brain.

The presence of labile low-molecular-mass (LMM, defined as <10 kDa) metal complexes in cells and super-cellular structures such as the brain has been inferred from chelation studies, but direct evidence is lacking. To evaluate the presence of LMM metal complexes in the brain, supernatant fractions of fresh mouse brain homogenates were passed through a 10 kDa cutoff membrane and subjected to size-exclusion liquid chromatography under anaerobic refrigerated conditions. Fractions were monitored for Mn, Fe, Co, Cu, Zn, Mo, S and P using an on-line ICP-MS. At least 30 different LMM metal complexes were detected along with numerous P- and S- containing species. Reproducibility was assessed by performing the experiment 13 times, using different buffers, and by examining whether complexes changed with time. Eleven Co, 2 Cu, 5 Mn, 4 Mo, 3 Fe and 2 Zn complexes with molecular masses <4 kDa were detected. One LMM Mo complex comigrated with the molybdopterin cofactor. Most Cu and Zn complexes appeared to be protein-bound with masses ranging from 4-20 kDa. Co was the only metal for which the "free" or aqueous complex was reproducibly observed. Aqueous Co may be sufficiently stable in this environment due to its relatively slow water-exchange kinetics. Attempts were made to assign some of these complexes, but further efforts will be required to identify them unambiguously and to determine their functions. This is among the first studies to detect low-molecular-mass transition metal complexes in the mouse brain using LC-ICP-MS.

Changing iron content of the mouse brain during development.

Iron is crucial to many processes in the brain yet the percentages of the major iron-containing species contained therein, and how these percentages change during development, have not been reliably determined. To do this, C57BL/6 mice were enriched in (57)Fe and their brains were examined by Mössbauer, EPR, and electronic absorption spectroscopy; Fe concentrations were evaluated using ICP-MS. Excluding the contribution of residual blood hemoglobin, the three major categories of brain Fe included ferritin (an iron storage protein), mitochondrial iron (consisting primarily of Fe/S clusters and hemes), and mononuclear nonheme high-spin (NHHS) Fe(II) and Fe(III) species. Brains from prenatal and one-week old mice were dominated by ferritin and were deficient in mitochondrial Fe. During the next few weeks of life, the brain grew and experienced a burst of mitochondriogenesis. Overall brain Fe concentration and the concentration of ferritin declined during this burst phase, suggesting that the rate of Fe incorporation was insufficient to accommodate these changes. The slow rate of Fe import and export to/from the brain, relative to other organs, was verified by an isotopic labeling study. Iron levels and ferritin stores replenished in young adult mice. NHHS Fe(II) species were observed in substantial levels in brains of several ages. A stable free-radical species that increased with age was observed by EPR spectroscopy. Brains from mice raised on an Fe-deficient diet showed depleted ferritin iron but normal mitochondrial iron levels.