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Influence of iron-saturation of plasma transferrin in iron distribution in the brain
Neurochemistry International 41 (2002) 223–228
Influence of iron-saturation of plasma transferrin in iron distribution in the brain Atsushi Takeda∗ , Keiko Takatsuka, Naoki Sotogaku, Naoto Oku Department of Medical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan Received 29 October 2001; accepted 24 January 2002
Abstract Based on the evidence that iron distribution in the peripheral tissues is changed by iron-saturation of plasma transferrin, the influence of iron-saturation of plasma transferrin in iron delivery to the brain was examined. Mouse plasma was pre-incubated with ferric chloride in citrate buffer to saturate transferrin and then incubated with 59 FeCl3 . Peak retention time of 59 Fe was transferred from the retention time of transferrin to that of mercaptalbumin, suggesting that iron may bind to albumin in the plasma in the case of iron-saturation of transferrin. When mice were intravenously injected with ferric chloride in citrate buffer 10 min before intravenous injection of 59 FeCl3 , 59 Fe concentration in the plasma was remarkably low. 59 Fe concentration in the liver of iron-loaded mice was four times higher than in control, while 59 Fe concentration in the brain of iron-loaded mice was approximately 40% of that of control mice. Twenty-four hours after intravenous injection of 59 FeCl3 , brain autoradiograms also showed that 59 Fe concentrations in the brain of iron-loaded mice were approximately 40–50% of those of control mice in all brain regions tested except the choroid plexus, in which 59 Fe concentration was equal. These results suggest that the fraction of non-transferrin-bound iron is engulfed by the liver, resulting in the reduction of iron available for iron delivery to the brain in iron-loaded mice. Transferrin-bound iron may be responsible for the fraction of iron in circulation that enters the brain. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Iron-saturation of transferrin; Hemochromatosis; Hypotransferrinemia; Choroid plexus
1. Introduction Iron is an essential trace metal for brain development (Pollitt and Leibel, 1982; Pollitt and Metallinos-Katsaras, 1990). Transferrin, a serum glycoprotein, has long been considered as an important molecule for the transport of iron (Fishman et al., 1987). Transferrin is synthesized primarily in the liver, but significant amount is also produced in the brain. In the brain, transferrin mRNA exists in oligodendrocytes and also in the choroid plexus in concentrations equal to that found in liver (Bennett and Giarman, 1965; Bloch et al., 1985; Aldred et al., 1987; Espinosa de los Monteros et al., 1990). Iron is found in oligodendrocytes in high density and required for myelin production (Hill and Switzer, 1984; Connor et al., 1990). Iron uptake is the highest during postnatal development at a time period that coincides with peaks in brain growth and myelin production (Taylor and Morgan, 1990), and an insufficient iron supply results in hypomyelination (Larkin and Rao, 1990). A recent paper suggests the existence of a functional difference between transferrin synthesized in the brain and in ∗
Corresponding author. Tel.: +81-54-264-5700; fax: +81-54-264-5705. E-mail address: [email protected] (A. Takeda).
other tissues such as liver and a specific role of transferrin in oligodendrocyte maturation and in the myelinogenesis (de Arriba Zerpa et al., 2000). Hereditary hemochromatosis is characterized by the triad of increased iron absorption by gastrointestinal cells, high or total iron-saturation of plasma transferrin, and abnormal tissue iron deposition, especially in the liver (Dadone et al., 1982; Edwards et al., 1982). The similar pattern of liver iron deposition was also observed in the hypotransferrinemic (HP) mice (Bernstein, 1987; Takeda et al., 2002), which have a point mutation or small deletion in the transferrin gene and produce <1% of the normal circulating level of plasma transferrin. The liver iron deposition in the two disorders of hereditary hemochromatosis and hypotransferrinemia may be due to saturation or lack of transferrin. In the brain of hereditary hemochromatosis, iron deposition has not been described as a pathological phenomenon and this disease is not usually associated with neurological symptom (Nielsen et al., 1995). Dickinson and Connor (1995, 1996) demonstrated that the intensity of iron staining in the brain of adult HP mice is not different from that of control mice and that 59 Fe accumulation in the brain is similar between control and adult HP mice. Moreover, in normal and iron-deficient mice heterozygotic for
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hypotransferrinemia, brain iron concentration does not correlate significantly with either transferrin levels or total iron-binding capacity in the plasma (Malecki et al., 1998). Based on these findings, it is important to clarify the effect of iron-saturation of plasma transferrin on iron distribution in the brain. Craven et al. (1987) reported peripheral tissue distribution of non-transferrin-bound iron in the case of transient saturation of plasma transferrin by intravenous injection of ferric citrate. The present paper deals with iron distribution in the brain in the case of the transient saturation of plasma transferrin.
2. Experimental procedures 2.1. Chemicals 59 FeCl
3 (110–925 MBq, i.e. 3–25 mCi/mg Fe) was purchased from Amersham Pharmacia Biotech Plc., Buckinghamshire, UK.
2.2. Animals Male ddY mice (10-week-old, 35–40 g body weight, usual mouse strain) were purchased from Japan SLC Inc., Hamamatsu, Japan. Mice were housed under standard laboratory conditions (23 ± 1 ◦ C, 55 ± 5% humidity). Mice had access to tap water and were fed a conventional mouse chow diet (Oriental Yeast Co. Ltd., Yokohama, Japan) ad libitum. The lights were automatically turned on at 8:00 h and off at 20:00 h. All experiments were carried out in accordance with the principles of laboratory animal care of the National Institute of Health and the University of Shizuoka. 2.3. HPLC analysis To saturate plasma transferrin with iron in vitro, the plasma (80 l) obtained from mice was incubated for 10 min at 37 ◦ C with 10 l of vehicle or FeCl3 (2 g Fe/10 l citrate buffer (0.1 M, pH 6.6)) and then incubated for 10 min at 37 ◦ C with 10 l of 59 FeCl3 (156.7 kBq, i.e. 4.2 Ci/0.33 g Fe/10 l citrate buffer (0.1 M, pH 6.6)). Aliquot (10 l) of the samples was analyzed by HPLC using an Asahipak GS-520P column (21.5 mm × 500 mm, Asahi Chemical Industry Co., Kawasaki, Japan; eluent, 10 mM Tris–HCl buffer (pH 7.4) + 0.09% NaCl + 0.05% NaN3 ; flow rate, 6.0 ml/min). The experiment was done in duplicate. Iron-saturation of plasma transferrin was also performed in vivo according to the procedure of Craven et al. (1987). Mice were intravenously injected with 0.1 ml of vehicle or FeCl3 (70 g Fe per mouse) in 0.1 M citrate buffer (pH 6.6) and intravenously injected with 59 FeCl3 (370 kBq, i.e. 10 Ci/0.66 g Fe/0.2 ml citrate buffer (0.1 M, pH 6.6) per mouse) 10 min after the injection. The blood was collected from control or iron-loaded mice 10 min after injection of 59 FeCl and centrifuged (2800 rpm, 4 ◦ C) for 10 min to ob3
tain the plasma. Aliquot (10 l) of the plasma was analyzed by HPLC as described earlier. 2.4. Brain autoradiography Mice (n = 4) were intravenously injected with 0.1 ml of vehicle or FeCl3 (70 g Fe per mouse) in 0.1 M citrate buffer (pH 6.6) and then intravenously injected with 59 FeCl (185 kBq, i.e. 5.0 Ci/1.14 g Fe/0.2 ml citrate 3 buffer (0.1 M, pH 6.6) per mouse) 10 min after the injection. The blood was collected from the common carotid arteries of the mice under deep diethyl ether anesthesia 24 h after injection of 59 FeCl3 . The mice were decapitated and the brain, liver, spleen and bone were excised. The brain excised from the calvarium was frozen immediately with dry ice, fixed quickly with ice-cold 4% sodium carboxymethyl cellulose on each specimen stage, and sliced at 300 m thickness at −20 ◦ C with a microtome (Cryostat HM505E, Microm Laborgerate GmbH, Heidelberg, Germany). The serial coronal slices were dried in a Cryostat at −20 ◦ C. The distribution of radioactivity in each area of the slices was determined by autoradiography (Bio-imaging Analyzer BAS 2000, Fuji Photo Film Co. Ltd., Tokyo, Japan) after exposure to the imaging plates (Fuji imaging plate, 20 cm × 40 cm, Fuji Photo Film Co. Ltd.) for approximately 7 days. The exact time of exposure was determined by taking account of the physical decay. Radioactivity (photo-stimulated luminescence (PSL)/mm2 ) in each area from the autoradiograms of the selected slices was measured quantitatively with a Bio-imaging Analyzer, and corrected according to PSL/mm2 of internal standards in each autoradiogram. 2.5. γ -Ray counting The blood, liver, spleen and bone obtained from control and iron-loaded mice were weighed and counted for the radioactivity in a ␥-counter (Packard 5530, Packard Instrument Co., Inc., Meriden, CT).
3. Results 3.1. Binding of 59 Fe to plasma proteins To study iron-binding ligands in the case of iron-saturation of plasma transferrin, 59 FeCl3 was incubated for 10 min at 37 ◦ C with control and iron-loaded plasma. 59 Fe was detected at the retention time of transferrin in control plasma, while 59 Fe was detected at the retention time of mercaptalbumin in iron-loaded plasma (Fig. 1). 59 Fe was scarcely detected at the retention time of transferrin in iron-loaded plasma. A minor peak of 59 Fe was detected at 20 min retention time in both groups. To study the clearance of iron and iron-binding ligands in the plasma in the case of iron-saturation of plasma
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Fig. 1. HPLC profile of iron-loaded plasma incubated with 59 FeCl3 . The plasma was pre-incubated for 10 min at 37 ◦ C with vehicle or FeCl3 in citrate buffer and then incubated for 10 min at 37 ◦ C with 59 FeCl3 . Aliquot of the samples was analyzed by HPLC. The experiment was done in duplicate. Arrows represent eluting position of transferrin (Tf) and mercaptalbumin (Alb(mercapt)).
transferrin in vivo, moreover, 59 FeCl3 was intravenously injected into control and iron-loaded mice that were intravenously pre-injected with ferric chloride in citrate buffer. 59 Fe-binding to the plasma proteins was assayed by HPLC 10 min after injection of 59 FeCl3 (Fig. 2). 59 Fe was detected at the retention time of transferrin in control mice. On the other hand, 59 Fe was not detected other than a little peak at the retention time of transferrin in iron-loaded mice, because the clearance of 59 Fe in the plasma was remarkably higher in iron-loaded mice (0.66 ± 0.09% dose/g plasma) than in control mice (12.9 ± 1.3% dose/g plasma).
3.2.
59 Fe
distribution in the brain
Ten minutes after intravenous injection of 59 FeCl3 , 59 Fe concentration in the liver of iron-loaded mice (13.1 ± 2.6% dose/g liver) was much higher than in control mice (3.0 ± 0.4% dose/g liver), while 59 Fe concentration in the brain of iron-loaded mice (0.057 ± 0.009% dose/g brain) was lower than in control mice (0.13 ± 0.02% dose/g brain). Because the 59 Fe concentration in the brain of control mice was increased 24 h after injection, compared to that 10 min after injection (data not shown), brain autoradiography was
Fig. 2. HPLC profile of plasma of iron-loaded mice injected with 59 FeCl3 . Control and iron-loaded mice were intravenously injected with 59 FeCl3 . Ten minutes after injection, the blood was collected and then centrifuged to obtain the plasma. Aliquot of the plasma was analyzed by HPLC. The experiment was done in duplicate. Arrows represent eluting position of transferrin (Tf).
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Fig. 3. 59 Fe-imaging of mouse brain. Control and iron-loaded mice were intravenously injected with 59 FeCl3 . Twenty-four hour after injection, the radioimaging was performed on selected coronal slices of control and iron-loaded mouse brain. The experiment was done in quadruplicate and the four brain images in each group were almost identical. Red means high emission representing high 59 Fe concentration. The schemes (left-hand side) show the brain maps. LV, lateral ventricle; 3V, third ventricle; 4V, fourth ventricle.
performed 24 h after intravenous injection of 59 FeCl3 . 59 Fe was largely concentrated in the lateral, the third and the fourth ventricles, including the choroid plexus, with only small amounts in the cerebral aqueduct (Fig. 3). In the brain of control mice, 59 Fe was extensively distributed in the brain and was concentrated highly in regions such as areas around the ventricles. When radioactivity distribution in the brain autoradiograms was measured quantitatively with a Bio-imaging Analyzer, the radioactivity in the choroid plexus (the lateral ventricles) of iron-loaded mice was equal to that of control mice (Fig. 4). The radioactivity in other brain regions of iron-loaded mice was significantly lower than in control mice. Blood radioactivity of iron-loaded mice was approximately 1/10 of that of control mice (Fig. 5). The radioactivity
in the spleen and bone of iron-loaded mice was significantly lower than in control mice, while the radioactivity in the liver of iron-loaded mice was significantly higher than in control mice.
4. Discussion Craven et al. (1987) reported that >90% of the injected dose of 59 Fe is not bound to transferrin by transient iron-saturation of this plasma protein. Plasma clearance of non-transferrin-bound 59 Fe is estimated to be very high. In iron-loaded rats, >80% of the injected radioactivity is eliminated from the plasma by 30 s. As a matter of fact, in the present study, 10 min after intravenous injection of 59 FeCl3 , the radioactivity in the plasma of iron-loaded mice was
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Fig. 4. 59 Fe distribution in mouse brain. Each value (mean ± S.D.), which was measured with a Bio-imaging Analyzer, represents the radioactivity (PSL/mm2 ) in brain autoradiograms obtained in Fig. 3 (n = 4). Asterisks represent significant difference (∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001: t-test) from control group.
remarkably low and the radioactivity in transferrin fraction of iron-loaded mice was much less than in control mice. To search ligands for 59 Fe eliminated rapidly from the plasma in iron-loaded mice, 59 FeCl3 was incubated for 10 min at 37 ◦ C with iron-loaded plasma and 59 Fe-binding to the plasma proteins was assayed by HPLC. A major peak of the radioactivity was detected at the retention time of mercaptalbumin in iron-loaded plasma, unlike the case in control plasma. There is the possibility that iron binds to albumin in the plasma in the case of iron-saturation of transferrin. Craven et al. (1987) demonstrated that non-transferrin-bound iron migrates more rapidly toward the cathode on polyacrylamide gel electrophoresis. In the case of iron-saturation of transferrin, iron might loosely bind to albumin and/or low molecular weight ligands, e.g.
Fig. 5. 59 Fe distribution in the peripheral tissues of mice. Control and iron-loaded mice were intravenously injected with 59 FeCl3 . Each value (mean ± S.D.) represents the radioactivity 24 h after injection of 59 FeCl (n = 4). Asterisks represent significant difference (∗∗ P < 0.01; 3 ∗∗∗ P < 0.001: t-test) from control group.
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citrate and ascorbate, in the plasma. In the serum of HP mice, the presence of non-transferrin-bound iron species (>150, 40–80 and 1–5 kDa) is reported by Simpson et al. (1992). The non-transferrin-bound iron species is distinguished from known extracellular iron-binding proteins and haem-proteins. On the other hand, 10 min after intravenous injection of 59 FeCl , 59 Fe concentration in the liver of iron-loaded mice 3 was four times higher than in control, while 59 Fe concentration in the brain of iron-loaded mice was approximately 40% of that of control mice. Twenty-four hours after intravenous injection of 59 FeCl3 , brain autoradiograms also showed that 59 Fe concentrations in the brain of iron-loaded mice were approximately 40–50% of those of control mice in all brain regions tested except the choroid plexus, in which 59 Fe concentration was equal. These results suggest that the fraction of non-transferrin-bound iron is engulfed by the liver, resulting in the reduction of iron available for iron delivery to the brain in iron-loaded mice. Transferrin-bound iron may be responsible for the fraction of iron in circulation that enters the brain. The finding that 59 Fe was concentrated in the choroid plexus in spite of the rapid clearance of 59 Fe in the plasma seems to be significant. There is the possibility that the choroid plexus is involved in iron delivery to the brain parenchyma and/or iron homeostasis in the brain. Increased iron concentrations in the brain have been demonstrated in a range of neurodegenerative diseases, e.g. Alzheimer’s disease and Parkinson’s disease (Double et al., 2000; Pinero et al., 2000). Excess iron is toxic; free iron can be cytotoxic, by catalyzing the production of hydoxyl radical from hydrogen peroxide (Zaleska and Floyd, 1985). Oxidative stress has been proposed as a pathogenic mechanism in Alzheimer’s disease and Parkinson’s disease. Recently, Benveniste et al. (2001) report that inflammation mediated by activated microglia, in which the -amyloid protein is involved, is an important component of Alzheimer’s disease pathophysiology. Iron can promote the aggregation of -amyloid protein (Mantyh et al., 1993). On the other hand, brain transferrin levels decrease with age and the decrease is dramatic when Alzheimer’s and Parkinson’s disease are superimposed on the aging process (Loeffler et al., 1995). The transferrin/iron ratio, a possible index of iron mobilization capacity, is decreased in the globus pallidus and caudate putamen in both Alzheimer’s and Parkinson’s disease. The decrease in transferrin levels in neurodegenerative diseases has been suggested as the cause of increased brain iron concentrations in these diseases (Craelius et al., 1982; Thompson et al., 1988; Yehuda and Youdim, 1988; Dexter et al., 1989; Connor et al., 1992a,b; Good et al., 1992). Brain transferrin is considered to be involved in the management of iron in the brain (Takeda et al., 2001). Hereditary hemochromatosis is not usually associated with neurological symptom (Nielsen et al., 1995; Demarquay et al., 2000). Sotogaku et al. (2000) demonstrated that iron concentration in the brain, unlike liver,
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scarcely increases following persistent iron overloading. Based on this observation and the results of the present paper, it is likely that non-transferrin-bound iron present in circulation of hemochromatosis patients is of little significance for causing pathological iron accumulation in the brain. References Aldred, A.R., Dickson, P.W., Marley, P.D., Schreiber, G., 1987. Distribution of transferrin synthesis in brain and other tissues. J. Biol. Chem. 262, 5293–5297. Bennett, D.S., Giarman, N.J., 1965. Schedule of appearance of 5-hydroxy-tryptamine (serotonin) and associated enzymes in the developing rat brain. J. Neurochem. 12, 911–918. Benveniste, E.N., Nguyen, V.T., O’Keefe, G.M., 2001. Immunological aspects of microglia: relevance to Alzheimer’s disease. Neurochem. Int. 39, 381–391. Bernstein, S.E., 1987. Hereditary hypotransferrinemia with hemosiderosis, a murine disorder resembling human atransferrinemia. J. Lab. Clin. Med. 110, 690–705. Bloch, B., Popovici, T., Levin, M., Tuil, D., Kahn, A., 1985. Transferrin gene expression visualized in oligodendrocytes of the rat brain using in situ hybridization and immunohistochemistry. Proc. Natl. Acad. Sci. U.S.A. 82, 6706–6710. Connor, J.R., Menzies, S.L., St. Marttin, S.M., Mufson, E.J., 1990. Cellular distribution of transferrin, ferritin, and iron in normal and aged human brains. J. Neurosci. Res. 27, 595–611. Connor, J.R., Menzies, S.L., St Marttin, S.M., Mufson, E.J., 1992a. A histochemical study of iron, transferrin, and ferritin in Alzheimer’s diseases brains. J. Neurosci. Res. 31, 75–83. Connor, J.R., Snyder, B.S., Beard, J.L., Fine, R.E., Mufson, E.J., 1992b. Regional distribution of iron and iron-regulatory proteins in the brain in aging and Alzheimer’s disease. J. Neurosci. Res. 31, 327–335. Craelius, W., Migdal, M.W., Luessenhop, C.P., Sugar, A., Mihalakis, I., 1982. Iron deposits surrounding multiple sclerosis plaques. Arch. Pathol. Lab. Med. 106, 397. Craven, C.M., Alexander, J., Eldridge, M., Kushner, J.P., Bernstein, S., Kaplan, J., 1987. Tissue distribution and clearance kinetics of non-transferrin-bound iron in the hypotransferrinemic mouse: a rodent model for hemochromatosis. Proc. Natl. Acad. Sci. U.S.A. 84, 3457– 3461. Dadone, M.H., Kushner, J.P., Edwards, C.Q., Bishop, D.T., Skolnick, M.H., 1982. Hereditary hemochromatosis: analysis of laboratory expression of the disease in 18 pedigrees. Am. J. Clin. Pathol. 78, 196–207. de Arriba Zerpa, G.A., Saleh, M.C., Fernandez, P.M., Guillou, F., Espinosa de los Monteros, A., de Vellis, J., Zakin, M.M., Baron, B., 2000. Alternative splicing prevents transferrin secretion during differentiation of a human oligodendrocyte cell line. J. Neurosci. Res. 61, 388–395. Demarquay, G., Setiey, A., Morel, Y., Trepo, C., Chazot, G., Broussolle, E., 2000. Clinical report of three patients with hereditary hemochromatosis and movement disorders. Mov. Disord. 15, 1204–1209. Dexter, D.T., Wells, F.R., Lees, A.J., Agid, F., Agid, Y., Jenner, P., Marsden, C.D., 1989. Increased nigral iron content and alterations in other metal ions occurring in brain in Parkinson’s disease. J. Neurochem. 52, 1830–1836. Dickinson, T.K., Connor, J.R., 1995. Cellular distribution of iron, transferrin and ferritin in the hypotransferrinemic (HP) mouse brain. J. Comp. Neurol. 355, 67–80. Dickinson, T.K., Connor, J.R., 1996. Distribution of injected iron-59 and manganese-54 in hypotransferrinemic mice. J. Lab. Clin. Med. 128, 270–278. Double, K.L., Gerlach, M., Youdim, M.B., Riederer, P., 2000. Impaired iron homeostasis in Parkinson’s disease. J. Neural. Transm. Suppl. 60, 37–58.
Edwards, C.Q., Dadone, M.H., Skolnick, M.H., Kushner, J.P., 1982. Hemochromatosis. Clin. Haematol. 11, 411–436. Espinosa de los Monteros, A., Kumar, S., Scully, S., Cole, R., de Vellis, J., 1990. Transferrin gene expression and secretion by rat brain cells in vitro. J. Neurosci. Res. 25, 576–580. Fishman, J.B., Rubin, J.B., Handrahan, J.V., Connor, J.R., Fine, R.E., 1987. Receptor-mediated transcytosis of transferrin across the blood-brain barrier. J. Neurosci. Res. 18, 299–304. Good, P.F., Perl, D.P., Bierer, L.M., Schmeidler, J., 1992. Selective accumulation of aluminum and iron in the neurofibrillary tangles of Alzheimer’s disease: a laser microprobe (LAMMA) study. Annu. Neurol. 31, 286–292. Hill, J.M., Switzer, R.C., 1984. The regional distribution and cellular localization of iron in the rat brain. Neuroscience 11, 595–603. Larkin, E.C., Rao, A., 1990. Importance of fetal and neonatal iron: adequacy for normal development of the central nervous system. In: Brain, Behavior and Iron in the Infant Diet. Springer, New York, pp. 43–62. Loeffler, D.A., Connor, J.R., Juneau, P.L., Snyder, B.S., Kanaley, L., DeMaggio, A.J., Nguyen, H., Brickman, C.M., LeWitt, P.M., 1995. Transferrin and iron in normal, Alzheimer’s disease, and Parkinson’s disease brain regions. J. Neurochem. 65, 710–716. Malecki, E.A., Devenyi, A.G., Beard, J.L., Connor, J.R., 1998. Transferrin response in normal and iron-deficient mice heterozygotic for hypotransferrinemia; effects on iron and manganese accumulation. Biometals 11, 265–276. Mantyh, P.W., Ghilardi, J.R., Rogers, S., DeMaster, E., Allen, C.J., Stimson, E.R., Maggio, J.E., 1993. Aluminium, iron, and zinc ions promote aggregation of physiological concentrations of -amyloid peptide. J. Neurochem. 61, 1171–1174. Nielsen, J.E., Jensen, L.N., Krabbe, K., 1995. Hereditary haemochromatosis: a case of iron accumulation in the basal ganglia associated with a Parkinsonian syndrome. J. Neurol. Neurosurg. Psych. 59, 318–321. Pinero, D.J., Hu, J., Connor, J.R., 2000. Alterations in the interaction between iron regulatory proteins and their iron responsive element in normal and Alzheimer’s diseased brains. Cell Mol. Biol. (Noisy-le-grand) 46, 761–776. Pollitt, E., Leibel, R.L. (Eds.), 1982. Iron Deficiency: Brain Biochemistry and Animal Behavior. Raven, New York. Pollitt, E., Metallinos-Katsaras, E., 1990. Iron deficiency and behavior: constructs, methods, and validity of the findings. In: Wurtman, R.J., Wurtman, J.J. (Eds.), Nutrition and the Brain, Vol. 8. Raven, New York, pp. 101–146. Simpson, R.J., Cooper, C.E., Raja, K.B., Halliwell, B., Evans, P.J., Aruoma, O.I., Singh, S., Konijn, A.M., 1992. Non-transferrin-bound iron species in the serum of hypotransferrinemic mice. Biochim. Biophys. Acta 1156, 19–26. Sotogaku, N., Oku, N., Takeda, A., 2000. Manganese concentration in mouse brain after intravenous injection. J. Neurosci. Res. 61, 350–356. Takeda, A., Takatsuka, K., Connor, J.R., Oku, N., 2002. Abnormal iron delivery to the bone marrow in neonatal hypotransferrinemic mice. Biometals 15, 33–36. Takeda, A., Takatsuka, K., Conner, J.R., Naoto, O., 2001. Abnormal iron accumulation in the brain of neonatal hypotransferrinemic mice. Brain Res. 912, 154–161. Taylor, E.M., Morgan, E.H., 1990. Developmental changes in transferrin and iron uptake by the brain in the rat. Dev. Brain Res. 55, 35–42. Thompson, C.M., Marksberry, W.R., Ehmann, W.D., Mao, Y.Y., Vance, D.E., 1988. Regional brain trace-element studies in Alzheimer’s disease. Neurotoxicology 9, 1–8. Yehuda, S., Youdim, M.B.H., 1988. Brain iron deficiency: biochemistry and behavior. In: Youdim, M.B.H. (Ed.), Brain Iron, Neurochemical and Behavioral Aspects. Taylor & Francis, London, pp. 89–114. Zaleska, M.M., Floyd, R., 1985. Regional lipid peroxidation in rat brain in vitro: possible role of endogeneous iron. Neurochem. Res. 10, 397– 410.
Influence of iron-saturation of plasma transferrin in iron distribution in the brain Atsushi Takeda∗ , Keiko Takatsuka, Naoki Sotogaku, Naoto Oku Department of Medical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan Received 29 October 2001; accepted 24 January 2002
Abstract Based on the evidence that iron distribution in the peripheral tissues is changed by iron-saturation of plasma transferrin, the influence of iron-saturation of plasma transferrin in iron delivery to the brain was examined. Mouse plasma was pre-incubated with ferric chloride in citrate buffer to saturate transferrin and then incubated with 59 FeCl3 . Peak retention time of 59 Fe was transferred from the retention time of transferrin to that of mercaptalbumin, suggesting that iron may bind to albumin in the plasma in the case of iron-saturation of transferrin. When mice were intravenously injected with ferric chloride in citrate buffer 10 min before intravenous injection of 59 FeCl3 , 59 Fe concentration in the plasma was remarkably low. 59 Fe concentration in the liver of iron-loaded mice was four times higher than in control, while 59 Fe concentration in the brain of iron-loaded mice was approximately 40% of that of control mice. Twenty-four hours after intravenous injection of 59 FeCl3 , brain autoradiograms also showed that 59 Fe concentrations in the brain of iron-loaded mice were approximately 40–50% of those of control mice in all brain regions tested except the choroid plexus, in which 59 Fe concentration was equal. These results suggest that the fraction of non-transferrin-bound iron is engulfed by the liver, resulting in the reduction of iron available for iron delivery to the brain in iron-loaded mice. Transferrin-bound iron may be responsible for the fraction of iron in circulation that enters the brain. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Iron-saturation of transferrin; Hemochromatosis; Hypotransferrinemia; Choroid plexus
1. Introduction Iron is an essential trace metal for brain development (Pollitt and Leibel, 1982; Pollitt and Metallinos-Katsaras, 1990). Transferrin, a serum glycoprotein, has long been considered as an important molecule for the transport of iron (Fishman et al., 1987). Transferrin is synthesized primarily in the liver, but significant amount is also produced in the brain. In the brain, transferrin mRNA exists in oligodendrocytes and also in the choroid plexus in concentrations equal to that found in liver (Bennett and Giarman, 1965; Bloch et al., 1985; Aldred et al., 1987; Espinosa de los Monteros et al., 1990). Iron is found in oligodendrocytes in high density and required for myelin production (Hill and Switzer, 1984; Connor et al., 1990). Iron uptake is the highest during postnatal development at a time period that coincides with peaks in brain growth and myelin production (Taylor and Morgan, 1990), and an insufficient iron supply results in hypomyelination (Larkin and Rao, 1990). A recent paper suggests the existence of a functional difference between transferrin synthesized in the brain and in ∗
Corresponding author. Tel.: +81-54-264-5700; fax: +81-54-264-5705. E-mail address: [email protected] (A. Takeda).
other tissues such as liver and a specific role of transferrin in oligodendrocyte maturation and in the myelinogenesis (de Arriba Zerpa et al., 2000). Hereditary hemochromatosis is characterized by the triad of increased iron absorption by gastrointestinal cells, high or total iron-saturation of plasma transferrin, and abnormal tissue iron deposition, especially in the liver (Dadone et al., 1982; Edwards et al., 1982). The similar pattern of liver iron deposition was also observed in the hypotransferrinemic (HP) mice (Bernstein, 1987; Takeda et al., 2002), which have a point mutation or small deletion in the transferrin gene and produce <1% of the normal circulating level of plasma transferrin. The liver iron deposition in the two disorders of hereditary hemochromatosis and hypotransferrinemia may be due to saturation or lack of transferrin. In the brain of hereditary hemochromatosis, iron deposition has not been described as a pathological phenomenon and this disease is not usually associated with neurological symptom (Nielsen et al., 1995). Dickinson and Connor (1995, 1996) demonstrated that the intensity of iron staining in the brain of adult HP mice is not different from that of control mice and that 59 Fe accumulation in the brain is similar between control and adult HP mice. Moreover, in normal and iron-deficient mice heterozygotic for
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hypotransferrinemia, brain iron concentration does not correlate significantly with either transferrin levels or total iron-binding capacity in the plasma (Malecki et al., 1998). Based on these findings, it is important to clarify the effect of iron-saturation of plasma transferrin on iron distribution in the brain. Craven et al. (1987) reported peripheral tissue distribution of non-transferrin-bound iron in the case of transient saturation of plasma transferrin by intravenous injection of ferric citrate. The present paper deals with iron distribution in the brain in the case of the transient saturation of plasma transferrin.
2. Experimental procedures 2.1. Chemicals 59 FeCl
3 (110–925 MBq, i.e. 3–25 mCi/mg Fe) was purchased from Amersham Pharmacia Biotech Plc., Buckinghamshire, UK.
2.2. Animals Male ddY mice (10-week-old, 35–40 g body weight, usual mouse strain) were purchased from Japan SLC Inc., Hamamatsu, Japan. Mice were housed under standard laboratory conditions (23 ± 1 ◦ C, 55 ± 5% humidity). Mice had access to tap water and were fed a conventional mouse chow diet (Oriental Yeast Co. Ltd., Yokohama, Japan) ad libitum. The lights were automatically turned on at 8:00 h and off at 20:00 h. All experiments were carried out in accordance with the principles of laboratory animal care of the National Institute of Health and the University of Shizuoka. 2.3. HPLC analysis To saturate plasma transferrin with iron in vitro, the plasma (80 l) obtained from mice was incubated for 10 min at 37 ◦ C with 10 l of vehicle or FeCl3 (2 g Fe/10 l citrate buffer (0.1 M, pH 6.6)) and then incubated for 10 min at 37 ◦ C with 10 l of 59 FeCl3 (156.7 kBq, i.e. 4.2 Ci/0.33 g Fe/10 l citrate buffer (0.1 M, pH 6.6)). Aliquot (10 l) of the samples was analyzed by HPLC using an Asahipak GS-520P column (21.5 mm × 500 mm, Asahi Chemical Industry Co., Kawasaki, Japan; eluent, 10 mM Tris–HCl buffer (pH 7.4) + 0.09% NaCl + 0.05% NaN3 ; flow rate, 6.0 ml/min). The experiment was done in duplicate. Iron-saturation of plasma transferrin was also performed in vivo according to the procedure of Craven et al. (1987). Mice were intravenously injected with 0.1 ml of vehicle or FeCl3 (70 g Fe per mouse) in 0.1 M citrate buffer (pH 6.6) and intravenously injected with 59 FeCl3 (370 kBq, i.e. 10 Ci/0.66 g Fe/0.2 ml citrate buffer (0.1 M, pH 6.6) per mouse) 10 min after the injection. The blood was collected from control or iron-loaded mice 10 min after injection of 59 FeCl and centrifuged (2800 rpm, 4 ◦ C) for 10 min to ob3
tain the plasma. Aliquot (10 l) of the plasma was analyzed by HPLC as described earlier. 2.4. Brain autoradiography Mice (n = 4) were intravenously injected with 0.1 ml of vehicle or FeCl3 (70 g Fe per mouse) in 0.1 M citrate buffer (pH 6.6) and then intravenously injected with 59 FeCl (185 kBq, i.e. 5.0 Ci/1.14 g Fe/0.2 ml citrate 3 buffer (0.1 M, pH 6.6) per mouse) 10 min after the injection. The blood was collected from the common carotid arteries of the mice under deep diethyl ether anesthesia 24 h after injection of 59 FeCl3 . The mice were decapitated and the brain, liver, spleen and bone were excised. The brain excised from the calvarium was frozen immediately with dry ice, fixed quickly with ice-cold 4% sodium carboxymethyl cellulose on each specimen stage, and sliced at 300 m thickness at −20 ◦ C with a microtome (Cryostat HM505E, Microm Laborgerate GmbH, Heidelberg, Germany). The serial coronal slices were dried in a Cryostat at −20 ◦ C. The distribution of radioactivity in each area of the slices was determined by autoradiography (Bio-imaging Analyzer BAS 2000, Fuji Photo Film Co. Ltd., Tokyo, Japan) after exposure to the imaging plates (Fuji imaging plate, 20 cm × 40 cm, Fuji Photo Film Co. Ltd.) for approximately 7 days. The exact time of exposure was determined by taking account of the physical decay. Radioactivity (photo-stimulated luminescence (PSL)/mm2 ) in each area from the autoradiograms of the selected slices was measured quantitatively with a Bio-imaging Analyzer, and corrected according to PSL/mm2 of internal standards in each autoradiogram. 2.5. γ -Ray counting The blood, liver, spleen and bone obtained from control and iron-loaded mice were weighed and counted for the radioactivity in a ␥-counter (Packard 5530, Packard Instrument Co., Inc., Meriden, CT).
3. Results 3.1. Binding of 59 Fe to plasma proteins To study iron-binding ligands in the case of iron-saturation of plasma transferrin, 59 FeCl3 was incubated for 10 min at 37 ◦ C with control and iron-loaded plasma. 59 Fe was detected at the retention time of transferrin in control plasma, while 59 Fe was detected at the retention time of mercaptalbumin in iron-loaded plasma (Fig. 1). 59 Fe was scarcely detected at the retention time of transferrin in iron-loaded plasma. A minor peak of 59 Fe was detected at 20 min retention time in both groups. To study the clearance of iron and iron-binding ligands in the plasma in the case of iron-saturation of plasma
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Fig. 1. HPLC profile of iron-loaded plasma incubated with 59 FeCl3 . The plasma was pre-incubated for 10 min at 37 ◦ C with vehicle or FeCl3 in citrate buffer and then incubated for 10 min at 37 ◦ C with 59 FeCl3 . Aliquot of the samples was analyzed by HPLC. The experiment was done in duplicate. Arrows represent eluting position of transferrin (Tf) and mercaptalbumin (Alb(mercapt)).
transferrin in vivo, moreover, 59 FeCl3 was intravenously injected into control and iron-loaded mice that were intravenously pre-injected with ferric chloride in citrate buffer. 59 Fe-binding to the plasma proteins was assayed by HPLC 10 min after injection of 59 FeCl3 (Fig. 2). 59 Fe was detected at the retention time of transferrin in control mice. On the other hand, 59 Fe was not detected other than a little peak at the retention time of transferrin in iron-loaded mice, because the clearance of 59 Fe in the plasma was remarkably higher in iron-loaded mice (0.66 ± 0.09% dose/g plasma) than in control mice (12.9 ± 1.3% dose/g plasma).
3.2.
59 Fe
distribution in the brain
Ten minutes after intravenous injection of 59 FeCl3 , 59 Fe concentration in the liver of iron-loaded mice (13.1 ± 2.6% dose/g liver) was much higher than in control mice (3.0 ± 0.4% dose/g liver), while 59 Fe concentration in the brain of iron-loaded mice (0.057 ± 0.009% dose/g brain) was lower than in control mice (0.13 ± 0.02% dose/g brain). Because the 59 Fe concentration in the brain of control mice was increased 24 h after injection, compared to that 10 min after injection (data not shown), brain autoradiography was
Fig. 2. HPLC profile of plasma of iron-loaded mice injected with 59 FeCl3 . Control and iron-loaded mice were intravenously injected with 59 FeCl3 . Ten minutes after injection, the blood was collected and then centrifuged to obtain the plasma. Aliquot of the plasma was analyzed by HPLC. The experiment was done in duplicate. Arrows represent eluting position of transferrin (Tf).
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Fig. 3. 59 Fe-imaging of mouse brain. Control and iron-loaded mice were intravenously injected with 59 FeCl3 . Twenty-four hour after injection, the radioimaging was performed on selected coronal slices of control and iron-loaded mouse brain. The experiment was done in quadruplicate and the four brain images in each group were almost identical. Red means high emission representing high 59 Fe concentration. The schemes (left-hand side) show the brain maps. LV, lateral ventricle; 3V, third ventricle; 4V, fourth ventricle.
performed 24 h after intravenous injection of 59 FeCl3 . 59 Fe was largely concentrated in the lateral, the third and the fourth ventricles, including the choroid plexus, with only small amounts in the cerebral aqueduct (Fig. 3). In the brain of control mice, 59 Fe was extensively distributed in the brain and was concentrated highly in regions such as areas around the ventricles. When radioactivity distribution in the brain autoradiograms was measured quantitatively with a Bio-imaging Analyzer, the radioactivity in the choroid plexus (the lateral ventricles) of iron-loaded mice was equal to that of control mice (Fig. 4). The radioactivity in other brain regions of iron-loaded mice was significantly lower than in control mice. Blood radioactivity of iron-loaded mice was approximately 1/10 of that of control mice (Fig. 5). The radioactivity
in the spleen and bone of iron-loaded mice was significantly lower than in control mice, while the radioactivity in the liver of iron-loaded mice was significantly higher than in control mice.
4. Discussion Craven et al. (1987) reported that >90% of the injected dose of 59 Fe is not bound to transferrin by transient iron-saturation of this plasma protein. Plasma clearance of non-transferrin-bound 59 Fe is estimated to be very high. In iron-loaded rats, >80% of the injected radioactivity is eliminated from the plasma by 30 s. As a matter of fact, in the present study, 10 min after intravenous injection of 59 FeCl3 , the radioactivity in the plasma of iron-loaded mice was
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Fig. 4. 59 Fe distribution in mouse brain. Each value (mean ± S.D.), which was measured with a Bio-imaging Analyzer, represents the radioactivity (PSL/mm2 ) in brain autoradiograms obtained in Fig. 3 (n = 4). Asterisks represent significant difference (∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001: t-test) from control group.
remarkably low and the radioactivity in transferrin fraction of iron-loaded mice was much less than in control mice. To search ligands for 59 Fe eliminated rapidly from the plasma in iron-loaded mice, 59 FeCl3 was incubated for 10 min at 37 ◦ C with iron-loaded plasma and 59 Fe-binding to the plasma proteins was assayed by HPLC. A major peak of the radioactivity was detected at the retention time of mercaptalbumin in iron-loaded plasma, unlike the case in control plasma. There is the possibility that iron binds to albumin in the plasma in the case of iron-saturation of transferrin. Craven et al. (1987) demonstrated that non-transferrin-bound iron migrates more rapidly toward the cathode on polyacrylamide gel electrophoresis. In the case of iron-saturation of transferrin, iron might loosely bind to albumin and/or low molecular weight ligands, e.g.
Fig. 5. 59 Fe distribution in the peripheral tissues of mice. Control and iron-loaded mice were intravenously injected with 59 FeCl3 . Each value (mean ± S.D.) represents the radioactivity 24 h after injection of 59 FeCl (n = 4). Asterisks represent significant difference (∗∗ P < 0.01; 3 ∗∗∗ P < 0.001: t-test) from control group.
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citrate and ascorbate, in the plasma. In the serum of HP mice, the presence of non-transferrin-bound iron species (>150, 40–80 and 1–5 kDa) is reported by Simpson et al. (1992). The non-transferrin-bound iron species is distinguished from known extracellular iron-binding proteins and haem-proteins. On the other hand, 10 min after intravenous injection of 59 FeCl , 59 Fe concentration in the liver of iron-loaded mice 3 was four times higher than in control, while 59 Fe concentration in the brain of iron-loaded mice was approximately 40% of that of control mice. Twenty-four hours after intravenous injection of 59 FeCl3 , brain autoradiograms also showed that 59 Fe concentrations in the brain of iron-loaded mice were approximately 40–50% of those of control mice in all brain regions tested except the choroid plexus, in which 59 Fe concentration was equal. These results suggest that the fraction of non-transferrin-bound iron is engulfed by the liver, resulting in the reduction of iron available for iron delivery to the brain in iron-loaded mice. Transferrin-bound iron may be responsible for the fraction of iron in circulation that enters the brain. The finding that 59 Fe was concentrated in the choroid plexus in spite of the rapid clearance of 59 Fe in the plasma seems to be significant. There is the possibility that the choroid plexus is involved in iron delivery to the brain parenchyma and/or iron homeostasis in the brain. Increased iron concentrations in the brain have been demonstrated in a range of neurodegenerative diseases, e.g. Alzheimer’s disease and Parkinson’s disease (Double et al., 2000; Pinero et al., 2000). Excess iron is toxic; free iron can be cytotoxic, by catalyzing the production of hydoxyl radical from hydrogen peroxide (Zaleska and Floyd, 1985). Oxidative stress has been proposed as a pathogenic mechanism in Alzheimer’s disease and Parkinson’s disease. Recently, Benveniste et al. (2001) report that inflammation mediated by activated microglia, in which the -amyloid protein is involved, is an important component of Alzheimer’s disease pathophysiology. Iron can promote the aggregation of -amyloid protein (Mantyh et al., 1993). On the other hand, brain transferrin levels decrease with age and the decrease is dramatic when Alzheimer’s and Parkinson’s disease are superimposed on the aging process (Loeffler et al., 1995). The transferrin/iron ratio, a possible index of iron mobilization capacity, is decreased in the globus pallidus and caudate putamen in both Alzheimer’s and Parkinson’s disease. The decrease in transferrin levels in neurodegenerative diseases has been suggested as the cause of increased brain iron concentrations in these diseases (Craelius et al., 1982; Thompson et al., 1988; Yehuda and Youdim, 1988; Dexter et al., 1989; Connor et al., 1992a,b; Good et al., 1992). Brain transferrin is considered to be involved in the management of iron in the brain (Takeda et al., 2001). Hereditary hemochromatosis is not usually associated with neurological symptom (Nielsen et al., 1995; Demarquay et al., 2000). Sotogaku et al. (2000) demonstrated that iron concentration in the brain, unlike liver,
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