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

Abstract

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.

Figure 1. 5 K, 0.05 T Mössbauer spectra of 96 wk flushed mouse liver (A) and mouse blood (B)

C is the difference spectrum, A minus B; D – F are simulations using the parameters mentioned in the text: D, Ferritin; E, CD; F, NHHS Fe^II; The red line in B is a simulation with parameters of HS haems. The field was applied parallel to the gamma radiation.

Figure 2. Iron concentration of flushed mouse liver

Circles, with blood contribution included; squares, with blood contribution re moved. Concentrations are listed in Table S1.

Figure 3

5 K, 0.05 T Mössbauer spectrum (upper) and EPR spectrum (lower) of isolated liver mitochondria. The solid line in the upper panel is a simulation with parameters given in the text and in Table S2. EPR simulations were performed using Spin count (http://www.chem.cmu.edu/groups/hendrich/facilities/) and the g values listed in the text.

Figure 4. Mössbauer spectra of flushed livers isolated at different ages

A, 1 dy; B, 1 wk; C, 3 wk; D, 3 wk Fe-deficient; E, 63 wk IRP2(−/−); F, 56 wk diseased. Red lines are simulations using parameters in the text and percentages in Table S2. Spectra were collected at 5 K and 0.05 T field applied parallel to the γ radiation.

Figure 5. Diseased liver (upper) and model of liver development (lower)

The examined liver section was stained with haematoxylin and eosin. Dark small punctate structures are inflammatory response cells. In the model, the newborn liver (0 wk) contains a high concentration of Fe, primarily in the form of ferritin (brown circles); a small amount of mitochondria (yellow and brown ovals) is also present. During the first few weeks of life, there is a massive exodus of Fe occurring while the organ is both growing and generating mitochondria. This causes the concentration of Fe in the organ to decline, achieving a minimum at ~3 wks. The Fe concentration gradually recovers over time. The distribution of that Fe is essentially invariant for the majority of the animal’s adult life.