Iron-Overload Diseases


BioMag

Transmission electron micrograph of iron oxide particles encapsulated in the spherical protein shell of ferritin. Each particle is about 7nm across. The iron oxide particles appear dark because they are electron dense. The protein shells are not seen in this image. Note the regularity of the size and shape of the particles.


About the Project:

Iron-overload diseases are usually genetic and are extremely common in many parts of the world. In areas such as South East Asia they constitute a major public health problem. In Australia, the gene for hemochromatosis is carried by about 1% of the population. Iron accumulates in patients and precipitates in various parts of the body in the form of iron oxyhydroxide particles. Some of our research over the past few years has contributed to the discovery that there are three major mineral structures of iron oxyhydroxide that can be found in iron-overloaded tissues.

Most living systems are able to store iron in the form of nanoscale particles of iron(III) oxyhydroxide encapsulated by the protein ferritin. Each molecule of ferritin consists of 24 subunits which are assembled to form an approximately spherical cage-like structure of external diameter 12 nm [1]. The internal cavity of the structure, within which the iron(III) oxyhydroxide particle is stored, has a diameter of approximately 8 nm. Thus the cavity limits the number of iron atoms in each particle to a maximum of approximately 4500. The protein shell surrounding each particle renders it water-soluble. Under some circumstances, e.g. pathological conditions of iron-overload, insoluble iron(III) oxyhydroxide particles are found to be deposited in biological tissue [2, 3]. This form of iron deposit is often termed hemosiderin. Hemosiderin may be a degradation product of ferritin or may be formed independently. Hemosiderin is not associated with well formed protein cavities and as such the particle size distribution may be less restricted when compared with ferritin.

The structure of the iron(III) oxyhydroxide particle of most ferritins is based on that of the mineral ferrihydrite (5Fe2O3.9H2O) with varying amounts of phosphate incorporated [2]. In some cases, such as the bacterioferritins from Azotobacter vinelandii and Pseudomonas aeruginosa, the phosphate content is large so that the composition is better described as a hydrated iron(III) oxyphosphate [4, 5]. The degree of crystallinity of the particles within ferritins can also vary from the well ordered structure of ferrihydrite through to a non-crystalline state as determined by electron diffraction measurements. The degree of crystallinity and particle size distribution of the inorganic particles in a native ferritin depend on the source of the ferritin and have also been observed to vary with pathological condition [6].

The variations in structure of the mineral particles in hemosiderins are even more marked than those in ferritins [2, 7, 8]. Three different particle structures have been identified, namely (i) a structure based on that of ferrihydrite (ii) a highly defect structure based on that of the mineral goethite (a-FeOOH), and (iii) a non-crystalline iron(III) structure. The particle size distributions in hemosiderins are often difficult to measure because of aggregation. However, transmission electron microscope observations clearly indicate that the distributions are generally broader than those of ferritins. Like ferritins, the structural and compositional characteristics of hemosiderin vary with biological source and pathological condition [2, 7-9].

The methodology used to assign these solid state structures to the hemosiderins was based on the biochemical isolation of the hemosiderins from tissue samples followed by characterisation using Mössbauer spectroscopy and electron diffraction. All three structures have been observed to occur in human tissues under varying pathological conditions. This chemical speciation of hemosiderin iron oxides is of importance because the different structures are expected (i) to have different degrees of toxicity, (ii) to have different reactivities with chelating drugs, (iii) to reflect different mechanisms of iron deposition. Subsequent to the discovery of the three different structures, several workers have used Mössbauer spectroscopy of frozen or freeze dried tissue specimens to elucidate the structures of the iron oxide deposits present in situ. This has proved to be a powerful technique, allowing characterisation of iron oxide structures in many more tissue specimens without the need for biochemically isolating the iron oxide deposits from the tissue. The three different iron oxide structures can be differentiated with the use of Mössbauer spectroscopy by virtue of their different temperature dependent magnetic behaviour. Thus, Mössbauer spectroscopic measurements generally need to be made at several different sample temperatures in order to characterise the magnetic behaviour sufficiently for determination of the structures present. A disadvantage of the Mössbauer spectroscopic technique is that each spectrum requires at least 24 hrs (and usually more) for data collection. Thus one sample may require 1 week or more for full characterisation.

We have made the first ever use of AC-magnetic susceptibility measurements to characterise the temperature dependent magnetic behaviour of hemosiderins [12]. We show that this technique yields greater detail about the temperature dependent magnetic behaviour of hemosiderins and that the data can be collected on a time-scale one order of magnitude smaller than that required for Mössbauer spectroscopy. This opens the way for larger scale studies of the structure of iron oxide deposits in iron loaded tissues. In addition, a detailed knowledge of the magnetic properties of pathological tissue iron oxide deposits is necessary for the interpretation of magnetic resonance images of iron loaded tissues [10, 11].

This project has been carried out in collaboration with Prof K.V.Rao and Dr J.L. Costa-Krämer at the Royal Institute of Technology, Stockholm, Prof J. Webb and D.J. Macey, Murdoch University, Perth, and Prof P. Pootrakul, Mahidol University, Bangkok.

 

References:

[1] P.M. Harrison and P. Arosio. The ferritins: molecular properties, iron storage function and cellular regulation, Biochim. Biophys. Acta. 1275 (1996) 161-203.

[2] T.G. St. Pierre, J. Webb and S. Mann (1989) in Biomineralization: chemical and biochemical perspectives (S. Mann, J. Webb, R.J.P. Williams, eds.), pp. 295-344, VCH, Weinheim.

[3] T. Iancu. Ferritin and hemosiderin in pathological tissues, Electron Microsc. Rev. 5 (1992) 209-229.

[4] S. Mann, J.V. Bannister and R.J.P. Williams. Structure and composition of ferritin cores isolated from human spleen, limpet (patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells, J. Mol. Biol. 188 (1986) 225-232.

[5] G. Watt, R.B. Frankel, G.C. Papaefthymiou, K. Spartalian and E.I. Stiefel. Redox Properties and Mössbauer spectroscopy of Azotobacter vinelandii bacterioferritin, Biochem. 25 (1986) 4330-4336.

[6] T.G. St. Pierre, K.C. Tran, J. Webb, D.J. Macey, B.R. Heywood, N.H. Sparks, V.J. Wade, S. Mann and P. Pootrakul. Organ specific crystalline structures of ferritin cores in b-thalassaemia/haemoglobin E, Biol. Metals 4 (1991) 162-165.

[7] D.P.E. Dickson, N.M.K. Reid, S. Mann, V.J. Wade, R.J. Ward and T.J. Peters. Mössbauer spectroscopy, electron microscopy and electron diffraction studies of the iron cores in various human and animal haemosiderins, Biochim. Biophys. Acta. 957 (1988) 81-90.

[8] S. Mann, V.J. Wade, D.P.E. Dickson, N.M.K. Reid, R.J. Ward, M. O'Connell and T.J. Peters. Structural specificity of haemosiderin iron cores in iron-overload diseases, FEBS Lett. 234 (1988) 69-72.

[9] T.G. St. Pierre, W. Chua-anusorn, J. Webb, D. Macey, P. Pootrakul, The form of iron oxide deposits in thalassemic tissues varies between different groups of patients: a comparison between Thai b-thalassemia/hemoglobin E patients and Australian b-thalassemia patients, Biochim. Biophys. Acta 1407 (1998) 51-60.

[10] P. Gillis and S.H. Koenig. Transverse relaxation of solvent protons induced by magnetized spheres: application to ferritin, erythrocytes, and magnetite, Magn. Reson. Med. 5 (1987) 323-345.

[11] S.H. Koenig (1990) in Iron biominerals (R.B. Frankel, R.P. Blakemore, eds.), pp. 359-372, Plenum Press, New York.

[12] Allen, P.D., St Pierre, T.G., Chua-anusorn, W., Ström, V., and Rao, K.V. (2000) Low-frequency low-field magnetic susceptibility of ferritin and hemosiderin. Biochim. Biophys. Acta, 1500, 186-196.




Contact Information

To contact us directly, send e-mail to Tim St.Pierre (stpierre@physics.uwa.edu.au)


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