^Thalassemia Center, Faculty of Graduate Studies and Division of Hematology, ... Medicine, Slriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
Hyperfine Interactions 71 (1992) 1279-1282
1279
CORE STRUCTURES OF HAEMOSIDERINS DEPOSITED IN VARIOUS ORGANS IN yff-THALASSAEMIA/HAEMOGLOBIN E DISEASE T.G. St. P I E R R E ' , K . C . T R A N ' , J . W E B B ' , D . J . M A C E Y ' , P . P O O T R A K U L ' and D . P . E . D I C K S O N '
'School of Mathematical and Physical Sciences and ^School of Biological and Environmental Sciences, Murdoch University, Perth, W.A. 6150, Australia ^Thalassemia Center, Faculty of Graduate Studies and Division of Hematology, Department of Medicine, Slriraj Hospital, Mahidol University, Bangkok 10700, Thailand ''Department of Physics, University of Liverpool, Liverpool L69 3BX, U.K.
ABSTRACT: Mossbauer spectra were recorded of tissue from pthalassaemia/haemoglobin E spleen, liver, pancreas and heart and of crude haemosiderins (insoluble iron fractions) isolated from the organs. Iron in the crude haemosiderins from the spleen and heart remains paramagnetic below 4.2K indicating that the iron is in a non-crystalline form. Superparamagnetic behaviour of the crude haemosiderins from the pancreas and liver indicate the presence of fenihydrite cores with some cores with a stiiicture based on defect-goethite. INTRODUCTION p-thalassaemia/haemoglobin E (p-thal/HbE) disease is a double heterozygote for the pthalassaemia gene and haemoglobin E gene and is very common in South East Asia /I/. It is characterized by increased erythropoietic activity of up to 10 or more times normal which leads to increased gastrointestinal absorption of iron /2,3/. This results in iron overload being a constant complication in severe cases of the disease /4-7/. Iron is deposited in the organs in the form of haemosiderin and ferritin. Haemosiderin contains iron in the form of insoluble mineral granules associated with some protein while ferritin, which is soluble, consists of a multisubunit protein shell surrounding an iron-mineral core /8,9/. MATERIALS AND METHODS Specimens of spleen, liver, heart and pancreas obtained from p-thal/HbE patients (post-mortem) were supplied by the Thalassemia Center, Siriraj Hospital, Bangkok, Thailand. The tissues were homogenized and about 1 cm^ of the homogenate from each specimen was transferred to a 19 mm .diameter nylon sample holder and was then frozen for M()ssbauer spectroscopy using liquid nitrogen. About 50-60g of tissue from each organ was homogenized on ice in 220ml of 0.15M NaCl saturated with thymol and containing 0.12mM PMSF. After fdtering through gauze, the homogenate was centrifuged at 5000g for 30 minutes. The pellet was then washed by centrifugation in a further 220ml of 0.15M NaCl at 5000g. This pellet was considered to be crude haemosiderin i.e. it contained all of the insoluble iron components of the tissue plus some cell debris. Each sample of crude haemosiderin was then transfened to a 19mm diameter nylon sample holder and frozen ready for Mossbauer spectroscopy. Mossbauer spectra were recorded using a 57Co in rhodium source moving in constant acceleration mode with a symmetric double ramp wave form. Spectra were subsequently folded to eliminate the parabolic background. The velocity scale was calibrated with reference to the spectrum of an a-iron foil at room temperature. The centre of the six line pattern was taken as zero velocity. Sample temperatures down to 12K were achieved using a closed cycle helium gas expansion refrigerator (Air Products Expander Module DE 202). Temperatures of 4.2K and below were obtained using a pumped liquid helium bath ciyostat. © J.C. Baltzer A.G., Scientific Publishing Company
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RESULTS Mossbauer spectra of whole tissue samples from p-thal/HbE spleen and pancreas are shown in Figures 1 and 2. At 78K the spleen tissue has a spectrum that appears to be a broad singlet. The parameters of the singlet are co^isistent with high-spin Fe(III). At 12K a small sextet component is apparent. Spectra of the heart tissue were vei7 similar. Fig . 1 78K
j -IS
-10
-5
a
IZK
^
i0
5
-15
-10
-5
0
5
Velocity (nn/s) V e l o c i t y (nn/s) Mossbauer spectra of tissue samples from P-thalassaemia/haenioglobin E spleen (Fig. 1) and pancreas (Fig. 2). Fig. 4
i0aK
7SK
70K
Q ca
IZK -10
1! 5~ VeIoci ty (nn/s ) -5
12K
Mossbauer spectra of the crude haemosiderin from p - t h a l a s s a e m i a / h a e m o g l o b i n E spleen (Fig. 3) and pancreas (Fig. 4).
1. 3K
-15
Mi
5 IF VeIoc i ty (nn/s ) ^5
i
T.G.St. Pierre et al. / Haemosiderins in P-thalassaemia/haemoglobin
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At 78K the pancreas tissue has a spectrum that consists of a superposition of a central doublet with broad lines and a magnetic hyperfine sextet of broad lines. The spectral parameters are consistent with the presence of high-spin Fe(IlI). Lowering the temperature to 14K increases the intensity of the sextet component while that of the doublet decreases. The p-thal/HbE liver tissue gives similar spectra (Table 1). Mossbauer spectra of the crude haemosiderin from 3-thal/HbE spleen are shown in Figure 3. At 78K the spectrum appears as an asymmetric doublet. As the temperature is lowered to 1.3K the doublet becomes symmetric although the lines remain broad. No magnetic splitting is observed. The spectral parameters are given in Table 1. A Mossbauer spectrum of the crude haemosiderin from p-thal/HbE leart was recorded at 4.2K only due to the small amount of sample available. The spectral parameters are similar to those of the spleen tissue. Mossbauer spectra of the crude haemosiderin from p-thal/HbE pancreas are shown in Figure 4. At 1(X)K the spectrum consists of a central quadnipole-split doublet and a small magnetically-split sextet component. Lowering the temperature to 60K has vei^ little effect on the spectrum. However, over the temperature range from 60K to 1.3K the sextet component increases in intensity at the expense of the central doublet. At 1.3K the central doublet is almost completely extinguished. The spectra of the crude ^haemosiderin from p-thal/HbE liver are very similar except that the sextet appears between lOOK and %K. Spectral parameters for p-thal/HbE pancreas crude haemosiderin are given in Table 1. Table 1. Mossbauer spectral parameters of tissue samples from P-thalassaemia/haemoglobin E organs and the crude haemosiderins isolated from the organs. non-magnetic component Sample
T
5
AEQ
A%
magnetic component 8
2E
Bhf
A%
approx eiTor spleen tissue pancreas tissue spleen hsd.
0.2 0.02 0.03 3 0.02 0.06 0.5 3 . 78 0.31 100 12 0.38 68 0.60 -0.41 46.7 32 78 0.45 0.66 83 0.45 -0.23 46.4 17 14 0.48 0.58 23 0.46 -0.12 48.4 77 78 0.27 100 12 0.28 100 1.3 0.48 0.72 100 pancreas 78 0.42 0.62 82 0.57 -0.26 46.0 18 hsd. 12 0.34 42 0.47 -0.12 48.7 58 1.3 0.26 5 0.46 -0.13 50.1 95 T is the sample temperature (K), 5 is the chemical isomer shift (mm/s), AEQ is the quadrupole splitting (mm/s), A % is the percentage spectral area of each subcomponent, e is the quadrupole perturbation of the magnetic-hyperfine-split component defined experimentally as (A2 -Ai)/4 where Aj and A2 are the differences (in mm/s) between the two lowest and the two highest energy absorption lines respectively, Bhf is the magnetic hyperfine field (T). The errors given for each parameter are estimates and vary somewhat depending on the intensity of the spectral component. DISCUSSION Comparison of the Mossbauer spectra of the p-thal/HbE haemosiderins with those for haemosiderins isolated from the livers and spleens from patients with idiopathic haemochromatosis, patients with transfusional iron-overioad, and normal humans and animals /lO/ indicate that there are at least three different forms of haemosiderin present in p-thal/HbE disease. The crude haemosiderins from the p-thal/HbE spleen and heart give spectra similar to those for the haemosiderin from a clinically treated idiopathic haemochromatosis liver /lO/ although in the present case there is no magnetic splitting observed even at 1.3K. This foim of haemosiderin is thought to comprise non-crystalline hydrated iron-
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oxyhydroxide cores. The Mossbauer spectra of the crude haemosiderins from the p-thal/HbE pancreas and liver have a small but significant magnetically split sextet component at liquid nitrogen temperature (78K). This spectral characteristic has been previously observed for haemosiderins isolated from the spleen / I I / and liver /lO/ of patients with secondary haemochromatosis due to regular blood transfusions. The presence of the sextet component at 78K is indicative of haemosiderin cores based on a defect goethite structure /lO/. The feirihydrite stnicture of mammalian ferritin cores and normal human and animal haemosiderins gives rise to Mossbauer spectra with a mean superparamagnetic blocking temperature of about 30-40K and the sextet is completely extinguished above 60K /10,8/. Thus the spectral data indicate that the crude haemosiderins from p-thal/HbE spleen and heart contain noncrystalline iron-oxyhydroxide cores while those from p-thal/HbE pancreas and liver contain mostly ferrihydrite cores with some defect-goethite-like cores. The Mossbauer spectra of the whole tissue samples appear to be a superposition of the spectra of the cnide haemosiderin and the ferritin /12/ isolated from each organ. This indicates that haemosiderin (as defined by the isolation procedures outlined in this paper) and ferritin are the major forms of iron in each organ studied. However, a quantitative comparison of the whole tissue spectra of the pancreas and liver samples with those of their respective haemosiderins show that the sextet to doublet area ratio at 78K is greater in the whole tissue spectra. This indicates that the relative yield during isolation of the haemosiderin with the defect goethite cores is lower than that for the haemosiderin with the ferrihydritg-^ cores. Electron microscopy of the haemosiderins shows that while some of the cores are well dispers others appear to be aggregated (data not shown). Laige aggregates of haemosiderin would tend to l)c trapped more easily in the gauze during the filtration step in the isolation procedure. Thus the haemosiderin cores based on the defect goethite structure may have a greater tendency to aggregate than those with the fenihydrite stnacture. The observation of three different fonns of haemosiderin in one disease state suggests at least three different mechanisms of haemosiderin fomiation. The different mechanisms may be influenced by factors such as cell type, flux of iron into the cell, and the time span of deposition. These factors need further investigation. ACKNOWLEDGEMENTS We thank P. Fallon and P. Yansukon for their valuable technical assistance with electron microscopy and organ collection respectively. We would also like to thank the Australian Research Council, the Cooley's Anemia Foundation, and the British Council for financial support of this work. REFERENCES III P. Wasi, Clinics in Haematology, 10 (1981) 707-729 121 P. Pootrakul, S. Hungsprenges, S. Fucharoen, D. Baylink, E. Thompson, E. English, M. Lee, J. Burnell and C. Finch, N. Engl. J. Med. 304 (1981) 1470-1473 /3/ P. Pootrakul, K. Kitcharoen, P. Yansukon, P. Wasi, S. Fucharoen, P. Charoenlarp, G. Brittenham, M.J. Pippard and C.A. Finch, Blood 71 (1988) 1124-1129 /4/ N. Bhamarabravati, S. Na-Nakorn, P. Wasi, and S. Tuchinda, Am. J. Clin. Path. 47 (1967) 745-758 75/ D. Sonakul, M. Sookanek and P. Pacharee, J. Med. Ass. Thailand 61 (1978) 72 /6/ P. Pootrakul, R. Rugkiatsakul and P. Wasi, Brit. J. Haematol. 46 (1980) 143-145 fll P. Pootrakul, V. Vongsmasa, P. La-ongpanich and P. Wasi, Acta Haematol. 66 (1981) 244-250 /8/ T.G. St. Pieire, J. Webb and S. Mann, in Biomineralization: chemical and biochemical perspectives, eds S. Mann, J. Webb and R.J.P. Williams (VCH Verlagsgesellschaft, Weinheim 1989) pp 295-344 191 G.C. Ford, P.M. Harrison, D.W. Rice, J.M.A. Smith, A. Treffry, J.L. White and J. Yariv, Phil. Trans. R. Soc. Lond. B 304 (1984) 551-565 /lO/ D.P.E. Dickson, N.M.K. Reid, S.Mann, V.J. Wade, R.J. Ward and T.J. Peters, Biochim. Biophys. Acta 957 (1988) 81-90 /II/ S.H. Bell, M.P. Weir, D.P.E. Dickson, J.F. Gibson, G.A. Sharp and T.J. Peters, Biochim. Biophys. Acta 787 (1984) 227-236 /12/ T.G. St. Piene, K.C. Tran, J. Webb, D.J. Macey and P. Pootrakul, in Mechanisms and phylogeny of mineralization in biological systems, eds S. Suga and H. Nakahara, (SpringerVerlag, Tokyo 1991) 291-295
