J Neural Transm (1988) 74:199-205 Journal oJm Neural Transmission (cid:14)9 by Springer Verlag 1988 Increased iron (II1) and total iron content in post mortem substantia nigra of parkinsonian brain Short Note E. Sofic ~, P. Riederer 1, H. Heinsen 1, H. Beckmann 2, G. P. Reynolds 2, G. Hebenstreit 3, and M. B. H. Youdim 4 Clinical Neurochemistry, Department of Psychiatry, University of Wiirzburg, Wfirzburg, Federal Republic of Germany 2 Department of Pathology, University of Nottingham, Nottingham, U.K. 3 Nieder6sterreichisches Landeskrankenhaus ftir Psychiatrie und Neurologie, Mauer-Ohlig, Mauer, Austria 4 Department of Pharmacology, Rappaport Family Research Institute, Faculty of Medicine, Technion, Haifa, Israel Accepted August 31, 1988 Summary. Significant differences in the content of iron (III) and total iron were found in post mortem substantia nigra of Parkinson's disease. There was an increase of 176% in the levels of total iron and 255% of iron (III) in the substantia nigra of the parkinsonian patients compared to age matched controls. In the cortex (Brodmann area 21), hippocampus, putamen, and globus pallidus there was no significant difference in the levels of iron (Ill) and total iron. Thus the changes in total iron, iron (III) and the iron (II)/iron (III) ratio in the parkinsonian substantia nigra are likely to be involved in the pathophysiology and treatment of this disorder. Keywords: Total iron, iron (III), iron (II), iron (II)/iron (III) ratio, Parkinson's disease, neurotoxicity. Introduction Nigrostriatal degeneration in Parkinson's disease (PD), neuronal cell death in the substantia nigra (SN) and loss of innervation in the corpus striatum is accompanied by a significant reduction especially of dopamine (DA) (Ehringer and Hornykiewicz, 1960) and catecholamine related enzymes (Birkmayer and Riederer, 1985). The activity of the rate limiting enzyme, tyrosine hydroxylase (TH) is considerably reduced in the striatum and SN of PD (Lloyd et al., 1975; Nagatsu et al., 1977; Riederer et al., 1978). Optimum function of TH requires
`
`Pharmacosmos A/S v. Luitpold Ex. Pharmaceuticals, Inc., IPR2015-01490
`
`Luitpold Pharmaceuticals, Inc., Ex. 2052, P. 1
`
`

`
`200 E. Sofic et al. a sufficient amount of tyrosine, oxygen, tetrahydrobiopterin and activation by iron (II) (Carlsson, t974; Kaufmann, 1977; Ikeda et al., 1965; Nagatsu et al., 1981 ; Rausch et al., 1988). Administration of the co-enzyme tetrahydrobiopterin to patients with PD as a trial for enzyme stimulation did not show any benefit (Birkmayer and Riederer, 1985). In contrast, intravenously applied iron in form of a special ferric-ferrous complex was reported to have considerably thera- peutical effect in most Parkinsonian patients treated so far (Birkmayer and Birkmayer, 1986). However, at present the molecular mode of action of this iron preparation is unclear. It is known, however, that compared with other regions of the brain the basal ganglia, globus pallidus, and SN are especially enriched with iron deposits (Spatz, 1922). Owing to its large neuron density with high DA turnover and metabolic activity, SN is sensitive to toxic influences (Riederer et al., 1985; Halliwell and Gutteridge, 1985). Monoamine oxidase (MAO) has a high activity in this area and generates hydrogen peroxide (H202) directly during deamination (Youdim, 1988). H202 rapidly crosses ceil mem- branes and in many cells it may be toxic. It is excessive H202 when in contact with reduced transitional metals Fe (II) and Cu (I) that liberates free radicals, such as the highly reactive cytotoxic hydroxyl ('OH) or superoxide (02) radicals which in turn enhance lipid peroxidation (Halliwell and Gutteridge, 1985). Increased availability of non-reactive iron (Riederer et al., 1988 a, b; Dexter et al., 1987; Drayer et al., 1986; Youdim, 1988) and a shift (oxidation) of iron (II) to iron (III) may be an additional factor of enhanced vulnerability of SN towards neurotoxic events. Therefore we have examined the concentration of total iron, iron (II), iron (III), and the iron (II)/iron (III) ratio in various brain areas of PD and controls. Postmortem tissue and methods Brain tissue (SN, putamen, hippocampus, globus pallidus, and cortex (Brodmann area 21 ; BA21) from 8 patients with PD [-4 male, 4 female; mean age 75.3 years; range 66 to 86 years, postmortem time, 40.74-26.6 (range 11-78) hours; duration of PD 7.5 (cid:127) 3.4 (range: 2-12) years] and from neurologically normal control subjects [4 male, 4 female; mean age 71.3:k 12.5 years; range 51-91 years, postmortem time, 26.1+23.3 (range 5-79) hours] were obtained at autopsy and dissected according to a standard protocol by a neuroana- tomist. The brain areas were quickly frozen at --80 ~ until analysis. Diagnosis was con- firmed in all cases by pathological and neuropathological examination. In PD drug therapy consisted of combined L-DOPA therapy (I-DOPA plus the peripherally acting decarbo- xylase inhibitor benserazide, amantadine sulfate, and anticholinergics). Controls had died without any evidence of neurological or psychiatric disease. All brains were examined histologically by routine staining methods and were diagnosed by a neuropathologist. Drug treatment consisted of cardiovascular active drugs and antibiotics. In six cases of either controls or PD the examination of DA in the striatum showed a severe depletion of the amine ranging between 90.4% (CN) and 97% (putamen) indicating a near total denervation. The cause of death (PD group) was bronchopneumonia (n = 6), pulmonary embolism after leg vein thrombosis and hypertensive heart disease (n= 1), and cardic arrest after colon carcinoma (n = 1). In controls the cause of death was bronchopneumonia (n = 2), myocardial infarction (n= 1), pulmonary thromboembolism and arteriosclerotic cerebro-vascular dis-
`
`Pharmacosmos A/S v. Luitpold Ex. Pharmaceuticals, Inc., IPR2015-01490
`
`Luitpold Pharmaceuticals, Inc., Ex. 2052, P. 2
`
`

`
`Iron content of human brain regions 201 ease (n = 1), coronary arteriosclerosis and old infarction (n = 1), coronary thrombosis (n = 1), cor pulmonale, chronic bronchitis and emphysema (n = 1), and pleural me sothelioma (n = 1). The determination of total iron, and iron (II) in brain tissue was done using a mod- ification of the spectrophotometric method by Siedel et al. (1984). Iron (II) was then determined by using the iron (II) chelator ferrozine | (commercial kit obtained from Boeh- ringer Mannheim GmbH, Federal Republic of Germany). Tissue samples (50-80 rag) were homogenized in 1.0 ml hydrochloric acid, pH 2.5, containing pepsine and 50 ~tl 80 mmol/1 ferrozine | The homogenates were divided in two portions. In the first portion Fe (II) was determined. In the second portion 10 mg of granulated ascorbic acid was added to reduce Fe (III) to Fe (II). These homogenates were incubated for 20 min at 37 ~ and centrifuged at 10,000 g, 4 ~ for 15 rain. Then absorbances of the supernatants and iron standards were read against sample blank at 578 nm within 30 min. Data were given as gg/g fresh weight. All results were analysed using Student's t-test and Wilcoxon's Rank Sum test. Results We have compared the total content of iron, iron (II), iron 0ID, and the iron (II)/iron (III) ratio in corresponding samples of Parkinson's disease and matched controls (Table 1). In putamen, cortex (BA 21), hippocampus, and globus pal- lidus there was no significant difference in the levels of total iron, iron (II), iron (III), and of the iron (II)/iron (III) ratio. Table 1. Total ferrous and ferric iron in parkinsonian brain Fe + + Fe + + + Total Fe + + iron Fe + + + Substantia nigra Control (8) 324-7.0 164-4.2 484-8.2 2.454-0.54 P.D. (8) 434-8.0 424-4.8** 854-11.1' 1.064-0.17"* Putamen Control (8) 65+14 31+5.6 964-19 2.44+0.56 P.D. (8) 474-10 314-8.5 784-17 1.884-0.45 Gl. Pallidus Control (6) 274-6.3 534-12 814-18 0.54-0.04 P.D. (6) 294-7.7 674- 16 974-24 0.44-0.05 Hippocampus Control (6) 104-0.8 154-2.7 244-3.3 0.84-0.14 P.D. (6) 114-1.9 13+2.4 25+3.9 0.94-0.10 Cortex (BA 21) Control (6) 15+2.6 14+2.2 28+5.4 0.94-0.16 P.D. (6) 16+2.7 134-2.5 284-5.1 1.14-0.24 Data as gg/g fresh weight; means + seem; number of regions in ) Wilcoxon Rank Sum Test; * p < 0.040; ** p < 0.0019 In six cases putamen showed a near total depletion of dopamine (mean loss 97%) indicating a severe grade of degeneration, equivalent to grade 4 + in neuropathological examinations
`
`Pharmacosmos A/S v. Luitpold Ex. Pharmaceuticals, Inc., IPR2015-01490
`
`Luitpold Pharmaceuticals, Inc., Ex. 2052, P. 3
`
`

`
`202 E. Sofic et al. However, a significant increase in the concentration of total iron and iron (III) and a significant decrease of the iron (II)/iron (III) ratio was found in SN of PD. It is, however, interesting to note that in controls the concentrations of iron in hippocampus and cortex (BA 21) is much lower than those found in other brain regions (Table 1). Furthermore, the ratio between iron (II) and iron (III) is shifted towards iron (II) in the nigro-striatal system, while it is around the ratio of 1 in the other three brain areas. Discussion Metals, especially iron (II) have always been suspected to act in a bimodal manner either as important cofactors for enzymes like TH (Kaufman, 1977) or to be involved in processes leading to cell death (Halliwelt and Gutteridge, 1985). For these reasons we examined the distribution of iron, iron (II) and iron (III) in brains from subjects with PD. The results of the present study clearly show that there were profound differences of total iron and the iron (II)/iron (III) ratio in SN of PD. However, this finding is to some extent at variance with recent findings by Dexter et al. (1987a, b) who reported increased iron in Brodmann's area 10 and SN. Furthermore, these data (Table 1 ; Dexter et al., 1987a, b) contrast in part with earlier reports by Earle (1968) stating that iron was above the control values in caudate nucleus and globus pallidus. The reason for this discrepancy is not known, but may be related to the methods used for determination of iron and the severity of PD. The latter aspect seems to be most relevant, as recent findings by Drayer et al. (1986), Rutledge et al. (1987), and Riederer et al. (1988a) do show a dependence of stages of severity as determined by the percentage of nerve cell loss or by clinical observations ("Parkinson plus", i.e. PD combined with dementia or other complicating factors; Fischer et al., 1983) and the increase of iron in SN and other brains areas. Although copper, zinc and calcium and possibly magnesium show rather uniform regional concentrations (Greiner et al., 1975; Ule et al., 1974; Riederer et al., 1988a), that of iron has a marked characteristic distribution. Thus, the highest iron contents are present in globus pallidus > putamen > substantia nigra > hippocampus and BA 21 confirming earlier reports (Spatz, 1922; V61kl and Ule, 1972). While this distribution per se might contribute to increase the vulnerability of the nigrostriatal system to endogenous or exogenous neuro- toxins, it is suggested that excess iron (II) itself in SN and putamen may additionally provoke a process of degeneration expecially when the membrane integrity breaks down. The significant increase in the iron (II)/iron (III) turnover in SN of PD may be an indirect indication of enhanced oxidative processes. The processes by which iron is transported across the blood brain barrier (BBB) and deposited in such high concentrations in a rather circumscribed topography is not known. There are three possible explanations; a) either iron uptake is increased via the breakdown of BBB in some individuals prone to
`
`Pharmacosmos A/S v. Luitpold Ex. Pharmaceuticals, Inc., IPR2015-01490
`
`Luitpold Pharmaceuticals, Inc., Ex. 2052, P. 4
`
`

`
`Iron content of human brain regions 203 PD; b) the brain iron turnover, which in any case is extremely slow as compared with liver (see Ben-Shachar et al., 1986), is further decreased resulting in the accumulation in substantia nigra, c) the transport across to CSF is decreased and d) it could not be excluded, that the increased iron content was due to the medication rather than to the disease. This possibility does not appear to be remote since DOPA, dopamine and to a smaller extent, metabolites like HVA are capable of chelating iron and could conceivably lead to increased tissue retention of iron. However, an increase in nigral iron concentration was also found in formalin-fixed Parkinsonian brains long before the clinical use of L- DOPA (Earle, 1968). Furthermore, and in agreement with the findings by Dexter et al. (1987a, b) the decrease of copper (Riederer et al., 1988a) argues against an influence of L-DOPA, which significantly increases copper content in ex- perimental studies (Donaldson et al., 1974). Whatever the mechanisms are, it is now apparent that increased availability of iron may contribute to the neu- rodegenerative aspect of this disease (Crichton, 1979; Switzer, 1982; Halliwell and Gutteridge, 1985). However, it cannot be decided by now whether this is of primary or secondary importance. Acknowledgement The authors are grateful to Mrs. Rosalinde Schreiner for her excellent technical assistance and to Mrs. Judith Philipp for the preparation of the manuscript. References Ben-Shachar D, Ashkenazi A, Youdim MBH (1986) The long term consequences of early iron deficiency. Int J Dev Neurosci 4:81-88 Birkmayer W, Riederer P (1985) Die Parkinson-Krankheit, 2nd edn. Springer, Wien New York Birkmayer W, Birkmayer JGD (1986) Iron, a new aid in the treatment of Parkinson patients. J Neural Transm 67:287-292 Carlsson A (1974) The in vivo estimation of rates of tryptophan and tyrosine hydroxylation: effects of alterations in enzyme environment and neuronal activity. In: Wolstenholme GEW, Fitzsimons DW (eds) Aromatic amino acids in the brain. Elsevier, Excerpta Medica, Amsterdam London New York, pp 126-134 Crichton RR (1979) Interaction between iron metabolism and oxygen activation. In: Oxygen free radicals and tissue damage. Ciba Foundation Symposium. Excerpta Medica, Am- sterdam, p 57 Dexter DT, Jenner P, Marsden CD (1987a) Alterations in the content of iron and other metal ions in Parkinsonian brain. Br J Pharmacol 91 : P 427 Dexter DT, Wells FR, Agid F, Agid Y, Lees AJ, Jenner P, Marsden CD (1987b) Increased nigral iron content in postmortem parkinsonian brain. Lancet ii: 1219-1220 Donaldson J, Cloutier T, Minnich JL, Barbeau A (1974) Trace metals and biogenic amines in rat brain. In: McDowelt F, Barbeau A (eds) Second Canadian-American Conference on Parkinson's disease. Adv Neurol 5:245-252 Drayer BP, Olanow W, Burger P, Johnson GA, Herfkens R, Riederer S (1986) Parkinson plus syndrome: diagnosis using high field MR imaging of brain iron. Radiology 159: 493-498 Earle KM (1968) Studies on Parkinson's disease including X-ray fluorescent spectroscopy of formalin fixed brain tissue. J Neuropathol Exp Neurol 27:1-14
`
`Pharmacosmos A/S v. Luitpold Ex. Pharmaceuticals, Inc., IPR2015-01490
`
`Luitpold Pharmaceuticals, Inc., Ex. 2052, P. 5
`
`

`
`204 E. Sofic et al. Ehringer H, Hornykiewicz O (1960) Verteilung von Noradrenalin und Dopamin im Gehirn des Menschen und ihr Verhalten bei Erkrankungen des extrapyramidalen Systems. Wien Klin Wochenschr 38:1236-1239 Fischer PA, Schneider E, Jacobi P (1983) Klinische Bilder des Parkinson-Syndroms und ihre Verl/iufe. In: Gfinshirt H, Berlit P, Haack G (eds) Pathophysiologie, Klinik und Therapie des Parkinsonismus. Editiones "Roche", Basle, pp 51-65 Greiner AC, Chan SC, Nicolson GA (1975) Human brain contents of calcium, copper, magnesium, and zinc in some neurological pathologies. Clin Chim Acta 64:211-213 Halliwell B, Gutteridge JMC (1985) Free radicals in biology and medicine. Calendon Press, Oxford Ikeda M, Levitt M, Udenfriend S (1965) Hydroxylation of phenylalanine by purified preparations of adrenal and brain tyrosine hydroxylase. Biochem Biophys Res Commun 18:482-488 Kaufman S (1977) Mixed function oxygenases--general considerations. In: Usdin E, Weiner N, Youdim MBH (eds) Structure and function of monoamine enzymes. Marcel Dekker Inc., New York Basle, pp 3-22 Lloyd KG, Davidson L, Hornykiewicz O (1975) The neurochemistry of Parkinson's disease: effect of L-DOPA therapy. J Pharmacol Exp Ther 195:453-464 Nagatsu T, Kato T, Numata Y, Ihuta K, Sano M, Nagatsu I, Kondo Y, Inagaki S, Ilzuka R, Hori A, Narabayashi H (1977) Phenylethanolamine-N-methyltransferase and other enzymes of catecholamine metabolism in human brain. Clin Chim Acta 75:221 232 Nagatsu T, Namaguchi T, Koto T, Sugimoto T, Matsuura S, Akino M, Nagatsu I, Iizuka R, Narabayashi H (1981) Biopterin in human brain and urine from controls and parkinsonian patients: application of a new radioimmunoassay. Clin Chim Acta 109: 305 Rausch WD, Hirata Y, Nagatsu T, Riederer P, Jellinger K (1988) Tyrosine hydroxylase activity in caudate nucleus from Parkinson's disease. Effects of iron and phosphorylating agents. J Neurochem 50:202-208 Riederer P, Rausch WD, Birkmayer W, Jellinger K, Seemann D (1978) CNS modulation of adrenal tyrosine hydroxylase in Parkinson's disease and metabolic encephalopathies. J Neural Transm [Suppl] 14:121-131 Riederer P, Sofic E, Rausch WD, Kruzik P, Youdim MBH (1985) Dopaminforschung heute und morgen-- L-DOPA in der Zukunft. In: Riederer P, Umek H (eds) L-DOPA- Substitution der Parkinson-Krankheit. Springer, Wien New York, pp 127-144 Riederer P, Sofic E, Rausch WD, Schmidt B, Youdim MBH (1988a) Transition metals, ferritin, glutathione and ascorbic acid in Parkinsonian brains. J Neurochem, in press Riederer P, Rausch WD, Schmidt B, Kruzik P, Konradi C, Sofic E, Danielczyk W, Fischer M, Ogris E (1988b) Biochemical fundamentals of Parkinson's disease. Mount Sinai J Med 55:21-28 Ruttedge JN, Hilal SK, Silver A J, Defendini R, Fahn S (1987) Study of movement disorders and brain iron by MR Am J Neuroradiol 8:397-411 Siedel J, Wahlefeld AW, Ziegenhorn J (1984) Improved, ferrozine-based reagent for the determination of serum iron (transferrin iron) without deproteinisation. Clin Chem 30: 975 Switzer III RC (1982) Iron-rich areas of brain as targets for damage in certain induced and naturally occuring neurological disorders. In: Saltman P, Hegenauer J (eds) The biochemistry and physiology of iron. Elsevier, Amsterdam, pp 569-574 Spatz H (1922) fiber den Eisennachweis im Gehirn, besonders in Zentren des extrapy- ramidal-motorischen Systems. Z Ges Neurol Psychiat 77:261-390 Ule G, V61kl A, Berlet H (1974) Spurenelemente im menschlichen Gehirn. Z Neurol 206: 117-128 VSlkl A, Ule G (1972) Spurenelemente im menschlichen Gehirn. Z Neurol 202:331-338
`
`Pharmacosmos A/S v. Luitpold Ex. Pharmaceuticals, Inc., IPR2015-01490
`
`Luitpold Pharmaceuticals, Inc., Ex. 2052, P. 6
`
`

`
`Iron content of human brain regions 205 Youdim MBH (1985) Brain iron metabolism: biochemicals and behavioural aspects in relation to dopaminergic neurotransmission. In: Lajtha A (ed) Handbook of neuro- chemistry, vo110. Plenum Press, New York, pp 731-755 Youdim MBH (1988) Iron in the brain: implications for Parkinson's and Alzheimer's diseases. Mount Sinai J Med 55:97-102 Authors' address: Dipl.-Ing. Mag. Dr. E. Sofic, Clinical Neurochemistry, Department of Psychiatry, University of Wiirzburg, F/ichsleinstrasse 15, D-8700 Wfirzburg, Federal Republic of Germany. Received June 8, 1988
`
`Pharmacosmos A/S v. Luitpold Ex. Pharmaceuticals, Inc., IPR2015-01490
`
`Luitpold Pharmaceuticals, Inc., Ex. 2052, P. 7