Timothy J. Peters

Mössbauer spectroscopic studies of human haemosiderin and ferritin

Ferritin and haemosiderin isolated from iron-overloaded human spleens have been investigated by 57Fe Mössbauer spectroscopy at temperatures between 1.3 and 200 K and also in applied magnetic fields. Virtually identical spectra were obtained from both materials at the high and low-temperature ends of this range, and also at 4.2 K in an applied magnetic field of 10 T; this indicates that both must contain iron in a closely similar chemical form. The difference between the two materials lies in the temperature dependence of their Mössbauer spectra in the intermediate temperature range, between 10 and 100 K. The temperature dependence of the Mössbauer spectra is characteristic of superparamagnetic behaviour, which occurs when a magnetically ordered material is present in the form of small particles. The details of this temperature dependence are related to the distribution of particle sizes and the magnetic anisotropy constant of each substance. Electron microscopy shows the haemosiderin cores to be markedly smaller on average than those of ferritin. Combining the Mössbauer spectroscopy and electron microscopy data we have shown that the magnetic anistropy constant of haemosiderin is considerably larger than that of ferritin. This is thought to result from the smaller core size and less symmetrical protein shell of the former. These data are consistent with the proposal that haemosiderin is derived from ferritin.

Studies on the Concentration and Intracellular Localization of Iron Proteins in Liver Biopsy Specimens from Patients with Iron Overload with Special Reference to their Role in Lysosomal Disruption

Summary. Liver biopsies were collected from control subjects and patients with iron overload due to either primary or secondary haemochromatosis. They were analysed for iron proteins by cation exchange chromatography and flameless atomic absorption spectrophotometry.

Mössbauer spectroscopy, electron microscopy and electron diffraction studies of the iron cores in various human and animal haemosiderins.

Mössbauer spectroscopy has indicated significant differences in the iron-containing cores of various haemosiderins. In the present study, haemosiderin was isolated from a number of animal species including man. In addition, haemosiderin was isolated from patients with primary idiopathic haemochromatosis or with secondary (transfusional) iron-overload. The iron cores of the animal and normal human haemosiderin appear to be very similar by Mössbauer spectroscopy, and the electron diffraction data indicate a ferrihydrite structure similar to that of ferritin cores. The haemosiderin isolated from secondary iron-overload shows anomalous behaviour in its temperature-dependent Mössbauer spectra. This can be understood in terms of the microcrystalline goethite structure of the cores as indicated by electron diffraction. The haemosiderin cores obtained in the case of primary haemochromatosis have an amorphous Fe(III) oxide structure and show Mössbauer spectra characteristic of a magnetically disordered material, which only orders at very low temperatures.

Structural specificity of haemosiderin iron cores in iron-overload diseases

Haemosiderin iron cores isolated from patients with secondary haemochromatosis have a goethite‐like (α‐FeOOH) crystal structure whereas those from patients with primary haemochromatosis are amorphous Fe (III) oxide. Haemosiderin cores isolated from normal human spleen are crystalline ferrihydrite (5Fe2O3·9H2O). The disease‐specific structures are significantly different from the ferrihydrite structure of associated ferritin cores. The results are important in understanding the biological processing of iron in pathological states and in the clinical treatment of iron‐overload diseases.

Iron K-edge absorption spectroscopic investigations of the cores of ferritin and haemosiderins

The extended X-ray absorption fine structure (EXAFS) associated with the iron K-edge has been measured and interpreted for ferritin and haemosiderin extracted from horse spleen, and haemosiderin extracted from the livers of humans with treated primary haemochromatosis, and from the spleens of humans with treated secondary haemochromatosis. For ferritin, the data are consistent with, on average, each iron atom being in an environment comprised of approx. six oxygen atoms at 1.93 +/- 0.02 A, approx. 1.5 iron atoms at 2.95 +/- 0.02 A and approx. 1.1 iron atoms at 3.39 +/- 0.02 A, with a further shell of oxygens at approx. 3.6 A. Iron in horse spleen haemosiderin is in an essentially identical local environment to that in horse spleen ferritin. In contrast, the EXAFS data for primary haemochromatosis haemosiderin indicate that the iron-oxide core is amorphous; only a single shell of approx. six oxygen atoms at approx. 1.94 +/- 0.02 A being apparent. Secondary haemochromatosis haemosiderin shows an ordered structure with approx. 1.4 iron atoms at both 2.97 +/- 0.02 and 3.34 +/- 0.02 A. This arrangement of iron atoms is similar to that in horse spleen haemosiderin, but the first oxygen shell is split with approx. 2.9 atoms at 1.90 +/- 0.02 A and approx. 2.7 at 2.03 +/- 0.02 A, indicative of substantial structural differences between secondary haemochromatosis haemosiderin and horse spleen haemosiderin.

A Mössbauer spectroscopic study of the form of iron in iron overload.

There are major differences in the temperature dependence of the Mössbauer spectra of ferritin and haemosiderin extracted from the organs of humans suffering from transfusional iron overload. Iron overload can also occur in animal systems as a result of artificial treatments or dietary factors. None of the animal systems which were investigated in the present study showed evidence in their Mössbauer spectra for the presence of the haemosiderin found in transfusional iron overload in humans. This suggests that the haemosiderin which occurs in the case of human transfusional iron overload may be specific to that situation.

SEPARATION AND ASSAY OF IRON PROTEINS IN NEEDLE-BIOPSY SPECIMENS OF HUMAN-LIVER

A micro method has been developed for the separation of the principal classes of iron proteins in needle biopsy specimens of human liver. The iron content of the fractions was determined by automated flameless atomic absorption spectrophotometry in a one step procedure. The levels of total iron, transferrin-, ferritin-, haemprotein- and haemosiderin-iron are reported for control tissue.

Biochemical studies of the iron cores and polypeptide shells of haemosiderin isolated from patients with primary or secondary haemochromatosis.

Haemosiderin isolated from different iron-loading syndromes, primary haemochromatosis (PHC) and secondary haemochromatosis (SHC) biochemically exhibited differences in both their iron core and peptide composition. The rate of release of iron from PHC haemosiderin to oxalate was 3-fold greater than that from SHC haemosiderin. The major peptides separated by SDS-PAGE showed a major band at Mr 20,000 for PHC haemosiderin and at Mr 15,000 for SHC haemosiderin.

Transamination pathways influencing l-glutamine and l-glutamate oxidation by rat enterocyte mitochondria and the subcellular localization of l-alanine aminotransferase and l-aspartate aminotransferase

Using analytical subcellular fractionation techniques, 12% of the total L-alanine aminotransferase activity and 26% of the total L-aspartate aminotransferase activity was localized in enterocyte mitochondria. Alanine and aspartate were products from the oxidation of glutamine and glutamate by enterocyte mitochondria. At low concentrations, malate stimulated aspartate synthesis but was inhibitory at higher concentrations. The malate inhibition of aspartate synthesis, which increased in the presence of pyruvate, was accompanied by an increase in alanine synthesis. With glutamine as substrate in the presence of pyruvate and malate, alanine synthesis was increased by 127% on addition of purified L-alanine aminotransferase, in spite of large amounts of glutamate generated. It was concluded that when pyruvate is available the important route for glutamine or glutamate oxidation by transamination was via L-alanine:2-oxoglutarate aminotransferase and not via L-aspartate:2-oxoglutarate aminotransferase. Results suggested that mitochondria may account for 50% of alanine production from glutamine in the enterocyte despite the relatively low activity of L-alanine aminotransferase therein.