J Neural Transm [P-D Sect] (1992) 4:283-290
__ Journal o f Neural Transmission 9 Springer-Verlag 1992 Printed in Austria
Peripheral blood cell activities of m o n o a m i n e oxidase B and superoxide dismutase in Parkinson's disease H. Checkoway l, L. G. Costa 1, J. S. Woods 1, A. F. Castoldi 1, B. O. Lund1, and P. D. Swanson2 1Department of Environmental Health, School of Public Health and Community Medicine, and 2Department of Medicine, Neurology Division, School of Medicine, University of Washington, Seattle, WA, U.S.A. Accepted March 23, 1992
Summary. Monoamine oxidase type B (MAO-B) and superoxide dismutase (SOD) are two enzyme stystems that are potentially relevant to an oxidative stress model of Parkinson's disease (PD) causation. Activities of MAO-B in platelets (nmol/108 cells/hr) and total SOD in lymphocytes (U/mg protein) were assayed among 28 cases of idiopathic PD and 22 controls. As anticipated, MAO-B was lowest in PD cases on selegiline (L-deprenyl) therapy (mean 1.10). There was a slight deficit of MAO-B among male cases not taking selegiline compared to controls (3.78 vs. 4.15), but the opposite trend was observed for females (6.18 vs. 4.16). SOD was slightly higher in cases (7.40), than controls (6.81). Excess SOD among PD cases was seen irrespective of gender, age, or selegiline treatment, although none of the differences was statistically significant. Future research on SOD should take advantage of the availability of assays specific for the cytosolic and mitochondrial forms of the enzyme. Keywords: Parkinson's disease, monoamine oxidase B, superoxide dismutase, biomarkers. Introduction The identification of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) as a cause of Parkinsonism in intravenous drug abusers (Langston et al., 1983) has greatly stimulated experimental and epidemiologic research on exogenous etiologic agents (Snyder and D'Amato, 1986; Tanner, 1989). Environmental toxicants, including some pesticides (Golbe etal., 1990; Hertzman et al., 1990) and toxic metals (Uitti etal., 1989; Ngim and Devathasan, 1989), have been examined as risk factors for Parkinson's disease (PD). The interaction between
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age-related loss of dopaminergic cells and toxic environmental exposures (Calne and Langston, 1983), especially exogenous agents that induce oxidative stress (Olanow, 1990) is a plausible model of PD induction. There is an increasing emphasis on the characterization of biochemical profiles that confer particular vulnerabilities to insults from environmental toxicants. This approach has generally relied on measurements of bioactivating or detoxifying enzyme activities in accessible tissues (e.g., peripheral blood cells) in PD cases and controls. Monoamine oxidase is a mitochondrial enzyme that exists in neuronal and glial cells in two forms, A and B, with different substrate specificities (Johnson, 1968). MAO-B is an important catabolic enzyme for a number of biogenic amines, including dopamine (Cohen, 1986). Moreover, it has been established that the metabolism of MPTP to the ultimate neurotoxicant, 1-methyl-4-phenylpyridinium ion (MPP +), requires MAO-B (Langston et al., 1984). Thus, MAO-B may be predicted to be a relevant activating enzyme for other pro-neurotoxicants with MPTP-like effects (Maret et al., 1990). The platelet is a convenient source of MAO-B, and platelet MAO-B activity appears to be a reliable indicator of activity in nervous tissue (Lee et al., 1989). There have been several previous studies of platelet MAO-B activity among PD cases, some showing relative excesses compared to controls (Danielwicz et al., 1988; Bonucelli etal., 1990), and others indicating either relative deficits (Zeller etal., 1976) or no discernible differences (Mann etal., 1983; Yong and Perry, 1986). The direction of differences appears to be related to the substrate used to quantify MAO-B activity. For example, excess MAO-B activity in platelets of PD cases was observed with phenylethylamine as substrate (Humfrey et al., 1990); however, deficits of platelet activity were found among PD cases when either 4-hydroxy- or 3, 4-dihydroxyphenylethylamine (Humfrey etal., 1990) or dopmaine (Steventon et al., 1990) was used as substrate. The number of hydroxyl groups on the substrate (dopamine has two) may therefore be a critical determinant of peripheral blood cell MAO-B activity. Free radical formation and detoxification processes have been postulated to be significant contributors to PD induction. The catabolism of dopamine by MAO-B generates hydrogen peroxide (Cohen, 1986) which, on reaction with transition metals (e.g., Fe), is converted to the highly reactive hydroxyl radical (Olanow, 1990). MAO-B activity may be enhanced in the presence of hydrogen peroxide, thus leading to an increased activation of MPTP-like toxicants (Riederer etal., 1990). Support for a free radical mechanism derives from observations of relative deficiencies of mitochondrial NADH-ubiquinone reductase and NADPH cytochrome c reductase in post-mortem substantia nigra of PD cases (Schapira et al., 1990). Moreover, the toxicity of MPP + may be mediated b y free radical formation (Sinha et al., 1986). The potential relevance of free radicals to PD induction provides the rationale for exploring biomarkers of susceptibility that relate to their formation and detoxification. Superoxide dismutase (SOD) is an enzyme system that converts superoxide radicals to hydrogen peroxide, which may promote oxi-
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dative destruction to lipids, proteins and D N A (Lunec, 1990). SOD in brain exists in two forms, Cu, Z n - d e p e n d e n t (cytosolic) and M n - d e p e n d e n t (mitochondrial). Rosetti etal. (1988) reported that b o t h the M A O - B inhibitor, selegiline (L-deprenyl), and SOD inhibited free radical f o r m a t i o n in M P T P - t r e a t e d m o u s e m i t o c h o n d r i a l preparations. In h u m a n P D cases, elevated cytosolic and m i t o c h o n d r i a l SOD activities have been f o u n d in various brain regions, including substantia nigra (Saggu et al., 1989; A d a m s and Odunze, 1991). Preferentially elevated gene expression for cytosolic SOD has been detected in the n e u r o m e l a n i n - p i g m e n t e d cells of the substantia nigra, which m a y be m o s t affected by P D degenerative processes (Ceballos etal., 1990). The widespread distribution of SOD a m o n g m a n y cell types and in serum (Ohno etal., 1990) indicates that activity in peripheral blood cells m a y reflect an adaptative response to oxidative stress in target tissue. Martilla etal. (1988) detected significant excesses of cytosolic SOD in substantia nigra and basal nuclei of P D cases, but no discernible difference in peripheral lymphocytes. We u n d e r t o o k a pilot study c o m p a r i n g M A O - B activity in platelets and SOD activity in lymphocytes a m o n g P D cases and a control group of other neurological service patients. The goal of this study was to evaluate case-control differences of M A O - B and SOD, and to explore for possible indications that SOD m a y be a useful b i o m a r k e r of risk.
Subjects and methods Twenty-eight (28) idiopathic PD cases (19 males and 9 females) aged 39 to 83 yr (mean 66) were identified from the University of Washington Neurology Clinic patient population. The control group consisted of 22 Neurology Clinic patients free of PD. Control patient diagnoses included cerebral infarct (4), headaches (3), seizures (2), non-PD essential tremors or movement disorders (3), Meige's Sndrome (1), epilepsy (1), parasthesias (1), progressive supranuclear palsy (1), spino-cerebellar degeneration (1), peripheral neuropathy (1), and 4 patients for whom no definitive diagnoses could be determined. The control group included 10 males and 12 females aged 35 to 79 yr (mean 61). All cases and controls were Caucasians. Study subjects were volunteers who were informed of the purposes of the study according to approved Institutional Review Board criteria for research on human subjects. Information on MAO inhibitor treatment (selegiline) and other medications was obtained by asking study subjects themselves and by reviewing patient charts. Ten of the PD cases were on selegiline therapy, 14 were taking carbidopa-levodopa, and 4 were not taking any specific PD medication. All except 4 controls were receiving some form of medication, including, anti-depressants, anti-hypertensives, and analgesics. Ten (10)mL of venous blood was drawn into EDTA-treated tubes. Platelets were isolated and prepared for the MAO-B assay by method of Young etal. (1986). Blood samples were centrifuged for 10rain at 200 x g to obtain the platelet rich plasma (PRP). The platelets were counted and the PRP was centrifuged at 16,000 x g for 10min at 4 ~ The supernatant was discarded, and the pellet was homogenized for 15 sec in NA +/K + buffer containing 10raM dithiothreitol 2.5 mM EDTA, and 0.5 mg/mL bo~ne serum albumin. The homogenates were frozen and stored at - 80 ~ until final use. a4C-phenylethylamine (New England Nuclear, Boston, MA) was used to assay MAO-B. Lymphocytes for the SOD activity assay were separated from venous blood according to the method of Boyum (1968). Total SOD (Cu, Zn- plus Mn-dependent) activity in
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lymphocytes was measured by monitoring the rate of ferricytochrome c reduction at 550 nm in reaction mixtures containing a superoxide-generating system (hypoxanthine and xanthine oxidase) as described by McCord and Fridovitch (1969). The technicians who performed the MAO-B and SOD assays were blinded as to the case or control status of study subjects. Statistical comparisons between cases and controls were made using t-test for unpaired samples.
Results
T h e M A O - B activity (nmol/10 s platelets/hour) d a t a are s u m m a r i z e d in Table 1. M e a n activity (1.10) was m a r k e d l y lower a m o n g the 10 P D cases on selegiline therapy; thus, statistical c o m p a r i s o n s were restricted to cases n o t t a k i n g selegiline at the time o f the study a n d controls. Platelet M A O - B activity was n o t m e a s u r e d for 5 subjects (4 cases a n d 1 control) because o f difficulties e n c o u n t e r e d in platelet separation. Male cases d e m o n s t r a t e d a small deficit o f activity relative to controls (3.78 vs. 4.15); however, the reverse t r e n d was seen for females a m o n g w h o m activity was higher in cases t h a n controls (6.18 vs. 4.16). A n anticipated relative excess o f M A O - B in females ( M u r p h y etal., 1976) was f o u n d only for the cases. S O D activity in l y m p h o c y t e s was d e t e r m i n e d for 27 cases a n d 20 controls (problems with l y m p h o c y t e p r e p a r a t i o n p r e v e n t e d assays for o t h e r 3 subjects, 1 case a n d 2 controls). A relative excess o f S O D activity was seen in cases c o m p a r e d to controls (7.40 vs. 6.81), a n d the difference persisted o n stratification by gender a n d age (Table 2). Selegiline has been s h o w n to increase S O D activity in rat brain (Carillo et al., 1989), a n d the selegiline-treated cases h a d a higher m e a n l y m p h o c y t e S O D activity t h a n the o t h e r P D cases (7.62 vs. 7.26). N o n e o f the differences was statistically significant, however.
Table
1. Monoamine oxidase B activity (nmolq0 8 hr) in platelets of PD-cases and controls
Group
Mean
(S.E.M.)t
P-Value**
PD cases on selegiline (N = 10) All subjects - cases* (N = 14) - controls (N = 21) Males -cases* (N = 10) -controls (N = 10) Females -cases* (N = 4) -controls (N = 11)
1.10
(0.64)
4.46 4.15
(0.58) (0.29)
0.64
3.78 4.15
(0.50) (0.32)
0.54
6.18 4.16
(1.37) (0.48)
0.24
* Cases not on selegiline; -~ standard error of mean; ** P-value for t-test, cases vs. controls
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Table 2. Superoxide dismutase activity (U/mg protein) in lymphocytes of PD cases and controls Group All subjects - cases ((N = 27) - controls (N = 20) PD cases - taking selegiline (N -- 10) - n o t taking selegiline (N = 17) Males - cases (N = 19) -controls (N = 8) Females - cases (N = 8) -controls (N = 12) Age < 50 - cases (N = 2) - controls (N = 6) Age ~> 50 -cases (N = 25) - controls (N = 14)
Mean
(S.E.M.)t
P-Value**
7.40 6.81
(0.21) (0.29)
0.11
7.62 7.26
(0.30) (0.28)
0.40
7.42 6.96
(0.28) (0.62)
0.52
7.35 6.71
(0.27) (0.26)
0.11
7.20 6.72
(0.50) (0.31)
- *
7.41 6.85
(0.39) (0.22)
0.23
t Standard error of mean, ** P-value for t-test, cases vs. controls, * too few cases for t-test result
Discussion Inferences d r a w n f r o m the d a t a o b t a i n e d in this study are limited to some extent by the use o f o t h e r neurological service patients as controls a n d by the relatively small n u m b e r s of subjects. (Other neurological service patients were selected as controls primarily for convenience.) It is possible t h a t b l o o d cell M A O - B a n d S O D activities m a y be related p a t h o p h y s i o l o g i c a l l y to other neurological conditions a m o n g the controls. F u r t h e r m o r e , like nearly all p r i o r studies o f this type, we m a d e only a single d e t e r m i n a t i o n o f e n z y m e activities; thus, the stability o f the d a t a remains in d o u b t , a n d we c a n n o t d i s c o u n t the possibility t h a t i n d u c t i o n o f M A O - B or S O D m a y be influenced by e n v i r o n m e n t a l factors. In fact, there is evidence t h a t cigarette s m o k i n g ( Y o n g a n d Perry, 1986) a n d the industrial solvent, styrene, ( C h e c k o w a y etal., 1992) m a y lower platelet M A O B activity. L o n g i t u d i n a l studies with r e p e a t e d samplings o f b i o m a r k e r s , ideally before a n d after disease onset, are n e e d e d to resolve these tissues, a l t h o u g h such studies m a y suffer f r o m severe feasibility constraints. A n o t h e r limitation is the possibility o f c o n f o u n d i n g effects o f m e d i c a t i o n s o n the M A O - B a n d S O D assays a m o n g cases a n d controls. Ideally, only u n m e d i c a t e d subjects s h o u l d be studied. Previous studies o f p l a t e l e t M A O - B activity assayed using p h e n y l e t h y l a m i n e
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as substrate have yielded mixed results; thus, the failure to detect consistent differences in this study was not surprising. The markedly lower activity seen in platelets of PD cases on selegiline therapy supports the utility of the platelet as a valuable indicator cell. However, the interpretation of peripheral MAOB activity as a possible susceptibility biomarker remains an open question. Alternative explanations for relative excesses or deficits in PD case platelets have been suggested. In view of the roles of MAO-B in dopamine catabolism and subsequent hydrogen peroxide generation, and in MPTP activation, it might be predicted that excessive activity might confer increase risk. On the other hand, the active metabolite of MPTP, MPP +, does not cross the blood-brain barrier readily; hence, a relative deficit of MAO-B activity in peripheral tissues may represent the high risk profile for MPTP analogs (Sturman etal., 1991). Other possible explanations for lower MAO-B activity among cases are that the failure to metabolize dopamine efficiently may either result in toxic dopamine concentrations in nervous tissue or may stimulate other catabolic pathways for dopamine that generate toxic metabolities (Williams et al., 1991). The dependence of MAO-B activity on substrate is a further complicating factor, as relative activity in PD case platelets appears to vary inversely with the number of hydroxyl groups on the substrate (Humfrey et al., 1990). An oxidative stress model of PD causation has gained favor in recent years. Increased SOD may suggest an enhanced adaptation to superoxide free radicals with an accelerated rate of hydrogen peroxide formation. We observed only slight, and statistically non-significant, excesses of total SOD activity in lymphocytes of PD cases. Nonetheless, the excess persisted irrespective of gender, age, or selegiline treatment. The availability of specific probes for the mitochondrial and cytosolic forms of SOD (Nakano et al., 1990) should be useful for clarifying the role of SOD as a biomarker of PD risk in future research.
Acknowledgements This research was supported by Grant ES-04696 from NIEHS. Dr. Castoldi received support from the Clinica del Lavoro (Pavia, Italy). The authors are grateful to M. Linde for enrolling study subjects and data collection, to M. Finley for manuscript preparation, and to J. Picciano for data management.
References Adams JD, Odunze IN (1991) Oxygen free radicals: their involvementin disease processes. Ann Clin Biochern 27:161-169 Bonucelli U, Piccini P, Del Dotto P, Pacifici GM, Corsini GU, Muratorio A (1990) Platelet monoamine oxidase B activity in Parkinsonian patients. J Neurol Neurosurg Psychiatry 53:854-855 Boyum A (1968) Isolation ofmononuclear cells and granulocytes from human blood. Scand J Lab Invest 21 l-Supp197]: 77-89 Calne DB, Langston JW (1983) Aetiology of Parkinson's disease. Lancet ii: 1457-1459 Carillo M, Kanai S, Kitani K (1991) ( - ) Deprenyl induces activities of both superoxide dismutase and catalase but not glutathione peroxidase in the striatum of young rats. Life Sci 48:517-521
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Ceballos I, Lafon M, Javoy-Agid F, Hirsch E, Nicole A, Sinet PM, Agid Y (1990) Superoxide dismutase in Parkinson's disease. Lancet 335:1035-1036 Checkoway H, Costa LG, Camp J, Coccini T, Daniell WE, Dills R (1992) Peripheral markers of neurochemical function among styrene-exposed workers. Br J Ind Med (in press) Cohen G (1986) Monoamine oxidase, hydrogen peroxide, and Parkinson's disease. In: Yahr MD, Bergmann KJ (eds) Advances in neurology, vo145. Raven Press, New York, pp 119-125 Danielczyk W, Streifler M, Konradi C, Riederer P, Moll G (1988) Platelet MAO-B activity and psychopathology of Parkinson's disease, senile dementia and multi-infarct dementia. Acta Psychiatr Scand 78:730-736 Golbe LI, Farrell TM, Davis PH (1990) Follow-up study of early life protective and risk factors in Parkinson's disease. Mov Disord 5:66-70 Hamet P, Fraysse T, Franks DL (1978) Cyclic nucleotides and aggregation of platelets of spontaneously hypertensive rats. Circ Res 43:365-374 Hertzman C, Wiens M, Bowering D, Snow B, Calne D (1990) Parkinson's disease: a casecontrol study of occupational and environmental risk factors. Am J Ind Med 17: 349355 Humfrey CDN, Steventon GB, Sturman SG, Waring RH, Griffiths B, Williams AC (1990) Monoamine oxidase substrates in Parkinson's disease. Biochem Pharmacol 40: 25622564 Johnson JP (1968) Some observations upon a new inhibitor of monoamine oxidase in brain tissue. Biochem Pharmacol 17:1285-1297 Langston JW, Ballard P, Tetrud JW, Irwin I (1983) Chronic parkinsonism in humans due to a product of meperidine-analog synthesis. Science 219:979-980 Langston JW, Irwin I, Langston EB, Forno LS (1984) 1-Methyl-4-phenylpyridinium ion (MPP +): identification of a metabolite of MPTP, a toxin selective to the substantia nigra. Neurosci Lett 48:87-92 Lee DH, Mendoza M, Dvorozniak MT, Chung E, van Woert MH, Yahr MD (1989) Platelet monoamine oxidase in Parkinson patients: effect of L-deprenyl therapy. J Neural Transm [P-D Sect] 1:189-194 Lunec J (1990) Free radicals: their involvement in disease processes. Ann Clin Biochem 27:173-182 Mann JJ, Stanley M, Kaplan RD, Sweeney J, Neophytides A (1983) Central catecholamine metabolism in vivo and the cognitive and motor deficits in Parkinson's disease. J Neurol Neurosurg Psychiatry 46:905-910 Maret G, Testa B, Jenner P, EI Tayar N, Carrupt P-A (1990) The MPTP story: MAO activates tetrahydropyridine derivatives to toxins causing parkinsonism. Drug Metab Rev 22:291-332 Martilla R J, Lorentz H, Rinne UK (1988) Oxygen toxicity protecting enzymes in Parkinson's disease: Increase of superoxide dismutase-like activity in the substantia nigra and basal nucleus. J Neurol Sci 86:321-331 McCord JM, Fridovitch I (1969) Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244:6049-6055 Murphy D1, Wright C, Buschbaum M, Nichols A, Costa JL, Wyatt RJ (1976) Platelet and plasma amine oxidase activity in 680 normals: sex and age differences and stability over time. Biochem Med 16:254-265 Nakan o M, Kimura H, Hara M, Kuroiwa M, Kato M, Totsune K, Yoshikawa T (1990) A highly sensitive method for determining both Mn- and Cu-Zn superoxide dismutase activities in tissues and blood cells. Anal Biochem 187:277-280 Ngim C-H, Devathasan G (1989) Epidemiologic study of the association between body
290 H. Checkoway etal.: MAO-B and superoxide dismutase in Parkinson's disease burden mercury level and idiopathic Parkinson's disease. Neuroepidemiolgy 8: 128141 Ohno H, Yahara O, Ishikawa M, etal (1990) Serum manganese-superoxide dismutase in individuals with various neuromuscular diseases. Free Rad Biol Med 9:137 Olanow CW (1990) Oxidative reactions in Parkinson's disease. Neurology 40 [Suppl3]: 32-37 Riederer P, Konradi C, Youdim MBH (1990) The role of MAO in dopaminergic transmission. In: Streifler MB, Korczyn AD, Melamed E, Youdim MBH (eds) Parkinson's disease: anatomy, pathology, and therapy, Raven Press, New York, pp 149-153 (Adv neurol 53) Rosetti ZL, Sotgio A, Sharp DE, Hadjiconstantinou M, Neff NH (1988) 1-Methyl-4-phenyl1, 2, 3, 6-tetrahydropyridine (MPTP) and free radicals in vitro. Biochem Pharmacol 37:4573-4574 Saggu H, Cooksey J, Dexter D, Wells FR, Less A, Jenner P, Marsden CD (1989) A selective increase in particulate superoxide dismutase activity in Parksinsonian substantia nigra. J Neurochem 53:692-697 Schapira AHV, Cooper M, Dexter D, Clark JB, Jenner P, Marsden CD (1990) Mitochondrial complex I deficiency in Parkinson's disease. J Neurochem 54:823-827 Sinha BK, Singh Y, Krishna G (1986) Formation of superoxide and hydroxyl radicals from 1-methyl-4-phenylpyridinium ion (MPP +): reductive activation by NADPH cytochrome P 450 reductase. Biochem Biophys Res Commun 135:583-588 Snyder SH, D'Amato RJ (1986) MPTP: a neurotoxin relevant to the pathophysiology of Parkinson's disease. Neurology 36:350-358 Steventon G, Hunfrey C, Sturman S, Waring RH, Williams AC (1990) Monoamine oxidase B and Parkinson's disease. Lancet 335:180 Sturman SG, Williams AC, Waring RH, Steventon GB (1991) Is monamine oxidase B metabolism of MPTP different in Parkinson's disease? Neurology 41 [Suppl 1]: 175 (Abstract) Tanner CM (1989) The role of environmental toxins in the etiology of Parkinson's disease. Trends Neurosci 12:49-54 Uitti RJ, Rajput AH, Rozdilsky B, Bickis M, Wollin T, Yuen WK (1989) Regional metal concentrations in Parkinson's disease, other chronic neurological disease, and control brains. Can J Neurol Sci 16:310-314 Williams A, Steventon G, Sturman S, Waring R (1991) Xenobiotic enzyme profiles and Parkinson's disease. Neurology 41 [Suppl 2]: 29-32 Yong VW, Perry TL (1986) Monoamine oxidase B, smoking and Parkinson's disease. J Neurol Sci 72:265-272 Young WF, Laws ER, Sharbrough FW, Weinshilboum RM (1986) Human monoamine oxidase- lack of brain and platelet correlation. Arch Gen Psychiatry 43:604-609 Zeller EA, Boshes B, Arbit J, Bieber M, Blonsky ER, Dolkart M, Hupiler SV (1976) Molecular biology of neurological and psychiatric disorders. I. Effects of Parkinsonism, age, sex, and L-Dopa on platelet monoamine oxidase. J Neural Transm 39:63-77 Authors' address: Dr. H. Checkoway, Department of Environmental Health, School of Public Health and Community Medicine, University of Washington, Seattle, WA 98195, U.S.A. Received January 13, 1992
__ Journal o f Neural Transmission 9 Springer-Verlag 1992 Printed in Austria
Peripheral blood cell activities of m o n o a m i n e oxidase B and superoxide dismutase in Parkinson's disease H. Checkoway l, L. G. Costa 1, J. S. Woods 1, A. F. Castoldi 1, B. O. Lund1, and P. D. Swanson2 1Department of Environmental Health, School of Public Health and Community Medicine, and 2Department of Medicine, Neurology Division, School of Medicine, University of Washington, Seattle, WA, U.S.A. Accepted March 23, 1992
Summary. Monoamine oxidase type B (MAO-B) and superoxide dismutase (SOD) are two enzyme stystems that are potentially relevant to an oxidative stress model of Parkinson's disease (PD) causation. Activities of MAO-B in platelets (nmol/108 cells/hr) and total SOD in lymphocytes (U/mg protein) were assayed among 28 cases of idiopathic PD and 22 controls. As anticipated, MAO-B was lowest in PD cases on selegiline (L-deprenyl) therapy (mean 1.10). There was a slight deficit of MAO-B among male cases not taking selegiline compared to controls (3.78 vs. 4.15), but the opposite trend was observed for females (6.18 vs. 4.16). SOD was slightly higher in cases (7.40), than controls (6.81). Excess SOD among PD cases was seen irrespective of gender, age, or selegiline treatment, although none of the differences was statistically significant. Future research on SOD should take advantage of the availability of assays specific for the cytosolic and mitochondrial forms of the enzyme. Keywords: Parkinson's disease, monoamine oxidase B, superoxide dismutase, biomarkers. Introduction The identification of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) as a cause of Parkinsonism in intravenous drug abusers (Langston et al., 1983) has greatly stimulated experimental and epidemiologic research on exogenous etiologic agents (Snyder and D'Amato, 1986; Tanner, 1989). Environmental toxicants, including some pesticides (Golbe etal., 1990; Hertzman et al., 1990) and toxic metals (Uitti etal., 1989; Ngim and Devathasan, 1989), have been examined as risk factors for Parkinson's disease (PD). The interaction between
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age-related loss of dopaminergic cells and toxic environmental exposures (Calne and Langston, 1983), especially exogenous agents that induce oxidative stress (Olanow, 1990) is a plausible model of PD induction. There is an increasing emphasis on the characterization of biochemical profiles that confer particular vulnerabilities to insults from environmental toxicants. This approach has generally relied on measurements of bioactivating or detoxifying enzyme activities in accessible tissues (e.g., peripheral blood cells) in PD cases and controls. Monoamine oxidase is a mitochondrial enzyme that exists in neuronal and glial cells in two forms, A and B, with different substrate specificities (Johnson, 1968). MAO-B is an important catabolic enzyme for a number of biogenic amines, including dopamine (Cohen, 1986). Moreover, it has been established that the metabolism of MPTP to the ultimate neurotoxicant, 1-methyl-4-phenylpyridinium ion (MPP +), requires MAO-B (Langston et al., 1984). Thus, MAO-B may be predicted to be a relevant activating enzyme for other pro-neurotoxicants with MPTP-like effects (Maret et al., 1990). The platelet is a convenient source of MAO-B, and platelet MAO-B activity appears to be a reliable indicator of activity in nervous tissue (Lee et al., 1989). There have been several previous studies of platelet MAO-B activity among PD cases, some showing relative excesses compared to controls (Danielwicz et al., 1988; Bonucelli etal., 1990), and others indicating either relative deficits (Zeller etal., 1976) or no discernible differences (Mann etal., 1983; Yong and Perry, 1986). The direction of differences appears to be related to the substrate used to quantify MAO-B activity. For example, excess MAO-B activity in platelets of PD cases was observed with phenylethylamine as substrate (Humfrey et al., 1990); however, deficits of platelet activity were found among PD cases when either 4-hydroxy- or 3, 4-dihydroxyphenylethylamine (Humfrey etal., 1990) or dopmaine (Steventon et al., 1990) was used as substrate. The number of hydroxyl groups on the substrate (dopamine has two) may therefore be a critical determinant of peripheral blood cell MAO-B activity. Free radical formation and detoxification processes have been postulated to be significant contributors to PD induction. The catabolism of dopamine by MAO-B generates hydrogen peroxide (Cohen, 1986) which, on reaction with transition metals (e.g., Fe), is converted to the highly reactive hydroxyl radical (Olanow, 1990). MAO-B activity may be enhanced in the presence of hydrogen peroxide, thus leading to an increased activation of MPTP-like toxicants (Riederer etal., 1990). Support for a free radical mechanism derives from observations of relative deficiencies of mitochondrial NADH-ubiquinone reductase and NADPH cytochrome c reductase in post-mortem substantia nigra of PD cases (Schapira et al., 1990). Moreover, the toxicity of MPP + may be mediated b y free radical formation (Sinha et al., 1986). The potential relevance of free radicals to PD induction provides the rationale for exploring biomarkers of susceptibility that relate to their formation and detoxification. Superoxide dismutase (SOD) is an enzyme system that converts superoxide radicals to hydrogen peroxide, which may promote oxi-
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dative destruction to lipids, proteins and D N A (Lunec, 1990). SOD in brain exists in two forms, Cu, Z n - d e p e n d e n t (cytosolic) and M n - d e p e n d e n t (mitochondrial). Rosetti etal. (1988) reported that b o t h the M A O - B inhibitor, selegiline (L-deprenyl), and SOD inhibited free radical f o r m a t i o n in M P T P - t r e a t e d m o u s e m i t o c h o n d r i a l preparations. In h u m a n P D cases, elevated cytosolic and m i t o c h o n d r i a l SOD activities have been f o u n d in various brain regions, including substantia nigra (Saggu et al., 1989; A d a m s and Odunze, 1991). Preferentially elevated gene expression for cytosolic SOD has been detected in the n e u r o m e l a n i n - p i g m e n t e d cells of the substantia nigra, which m a y be m o s t affected by P D degenerative processes (Ceballos etal., 1990). The widespread distribution of SOD a m o n g m a n y cell types and in serum (Ohno etal., 1990) indicates that activity in peripheral blood cells m a y reflect an adaptative response to oxidative stress in target tissue. Martilla etal. (1988) detected significant excesses of cytosolic SOD in substantia nigra and basal nuclei of P D cases, but no discernible difference in peripheral lymphocytes. We u n d e r t o o k a pilot study c o m p a r i n g M A O - B activity in platelets and SOD activity in lymphocytes a m o n g P D cases and a control group of other neurological service patients. The goal of this study was to evaluate case-control differences of M A O - B and SOD, and to explore for possible indications that SOD m a y be a useful b i o m a r k e r of risk.
Subjects and methods Twenty-eight (28) idiopathic PD cases (19 males and 9 females) aged 39 to 83 yr (mean 66) were identified from the University of Washington Neurology Clinic patient population. The control group consisted of 22 Neurology Clinic patients free of PD. Control patient diagnoses included cerebral infarct (4), headaches (3), seizures (2), non-PD essential tremors or movement disorders (3), Meige's Sndrome (1), epilepsy (1), parasthesias (1), progressive supranuclear palsy (1), spino-cerebellar degeneration (1), peripheral neuropathy (1), and 4 patients for whom no definitive diagnoses could be determined. The control group included 10 males and 12 females aged 35 to 79 yr (mean 61). All cases and controls were Caucasians. Study subjects were volunteers who were informed of the purposes of the study according to approved Institutional Review Board criteria for research on human subjects. Information on MAO inhibitor treatment (selegiline) and other medications was obtained by asking study subjects themselves and by reviewing patient charts. Ten of the PD cases were on selegiline therapy, 14 were taking carbidopa-levodopa, and 4 were not taking any specific PD medication. All except 4 controls were receiving some form of medication, including, anti-depressants, anti-hypertensives, and analgesics. Ten (10)mL of venous blood was drawn into EDTA-treated tubes. Platelets were isolated and prepared for the MAO-B assay by method of Young etal. (1986). Blood samples were centrifuged for 10rain at 200 x g to obtain the platelet rich plasma (PRP). The platelets were counted and the PRP was centrifuged at 16,000 x g for 10min at 4 ~ The supernatant was discarded, and the pellet was homogenized for 15 sec in NA +/K + buffer containing 10raM dithiothreitol 2.5 mM EDTA, and 0.5 mg/mL bo~ne serum albumin. The homogenates were frozen and stored at - 80 ~ until final use. a4C-phenylethylamine (New England Nuclear, Boston, MA) was used to assay MAO-B. Lymphocytes for the SOD activity assay were separated from venous blood according to the method of Boyum (1968). Total SOD (Cu, Zn- plus Mn-dependent) activity in
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lymphocytes was measured by monitoring the rate of ferricytochrome c reduction at 550 nm in reaction mixtures containing a superoxide-generating system (hypoxanthine and xanthine oxidase) as described by McCord and Fridovitch (1969). The technicians who performed the MAO-B and SOD assays were blinded as to the case or control status of study subjects. Statistical comparisons between cases and controls were made using t-test for unpaired samples.
Results
T h e M A O - B activity (nmol/10 s platelets/hour) d a t a are s u m m a r i z e d in Table 1. M e a n activity (1.10) was m a r k e d l y lower a m o n g the 10 P D cases on selegiline therapy; thus, statistical c o m p a r i s o n s were restricted to cases n o t t a k i n g selegiline at the time o f the study a n d controls. Platelet M A O - B activity was n o t m e a s u r e d for 5 subjects (4 cases a n d 1 control) because o f difficulties e n c o u n t e r e d in platelet separation. Male cases d e m o n s t r a t e d a small deficit o f activity relative to controls (3.78 vs. 4.15); however, the reverse t r e n d was seen for females a m o n g w h o m activity was higher in cases t h a n controls (6.18 vs. 4.16). A n anticipated relative excess o f M A O - B in females ( M u r p h y etal., 1976) was f o u n d only for the cases. S O D activity in l y m p h o c y t e s was d e t e r m i n e d for 27 cases a n d 20 controls (problems with l y m p h o c y t e p r e p a r a t i o n p r e v e n t e d assays for o t h e r 3 subjects, 1 case a n d 2 controls). A relative excess o f S O D activity was seen in cases c o m p a r e d to controls (7.40 vs. 6.81), a n d the difference persisted o n stratification by gender a n d age (Table 2). Selegiline has been s h o w n to increase S O D activity in rat brain (Carillo et al., 1989), a n d the selegiline-treated cases h a d a higher m e a n l y m p h o c y t e S O D activity t h a n the o t h e r P D cases (7.62 vs. 7.26). N o n e o f the differences was statistically significant, however.
Table
1. Monoamine oxidase B activity (nmolq0 8 hr) in platelets of PD-cases and controls
Group
Mean
(S.E.M.)t
P-Value**
PD cases on selegiline (N = 10) All subjects - cases* (N = 14) - controls (N = 21) Males -cases* (N = 10) -controls (N = 10) Females -cases* (N = 4) -controls (N = 11)
1.10
(0.64)
4.46 4.15
(0.58) (0.29)
0.64
3.78 4.15
(0.50) (0.32)
0.54
6.18 4.16
(1.37) (0.48)
0.24
* Cases not on selegiline; -~ standard error of mean; ** P-value for t-test, cases vs. controls
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287
Table 2. Superoxide dismutase activity (U/mg protein) in lymphocytes of PD cases and controls Group All subjects - cases ((N = 27) - controls (N = 20) PD cases - taking selegiline (N -- 10) - n o t taking selegiline (N = 17) Males - cases (N = 19) -controls (N = 8) Females - cases (N = 8) -controls (N = 12) Age < 50 - cases (N = 2) - controls (N = 6) Age ~> 50 -cases (N = 25) - controls (N = 14)
Mean
(S.E.M.)t
P-Value**
7.40 6.81
(0.21) (0.29)
0.11
7.62 7.26
(0.30) (0.28)
0.40
7.42 6.96
(0.28) (0.62)
0.52
7.35 6.71
(0.27) (0.26)
0.11
7.20 6.72
(0.50) (0.31)
- *
7.41 6.85
(0.39) (0.22)
0.23
t Standard error of mean, ** P-value for t-test, cases vs. controls, * too few cases for t-test result
Discussion Inferences d r a w n f r o m the d a t a o b t a i n e d in this study are limited to some extent by the use o f o t h e r neurological service patients as controls a n d by the relatively small n u m b e r s of subjects. (Other neurological service patients were selected as controls primarily for convenience.) It is possible t h a t b l o o d cell M A O - B a n d S O D activities m a y be related p a t h o p h y s i o l o g i c a l l y to other neurological conditions a m o n g the controls. F u r t h e r m o r e , like nearly all p r i o r studies o f this type, we m a d e only a single d e t e r m i n a t i o n o f e n z y m e activities; thus, the stability o f the d a t a remains in d o u b t , a n d we c a n n o t d i s c o u n t the possibility t h a t i n d u c t i o n o f M A O - B or S O D m a y be influenced by e n v i r o n m e n t a l factors. In fact, there is evidence t h a t cigarette s m o k i n g ( Y o n g a n d Perry, 1986) a n d the industrial solvent, styrene, ( C h e c k o w a y etal., 1992) m a y lower platelet M A O B activity. L o n g i t u d i n a l studies with r e p e a t e d samplings o f b i o m a r k e r s , ideally before a n d after disease onset, are n e e d e d to resolve these tissues, a l t h o u g h such studies m a y suffer f r o m severe feasibility constraints. A n o t h e r limitation is the possibility o f c o n f o u n d i n g effects o f m e d i c a t i o n s o n the M A O - B a n d S O D assays a m o n g cases a n d controls. Ideally, only u n m e d i c a t e d subjects s h o u l d be studied. Previous studies o f p l a t e l e t M A O - B activity assayed using p h e n y l e t h y l a m i n e
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as substrate have yielded mixed results; thus, the failure to detect consistent differences in this study was not surprising. The markedly lower activity seen in platelets of PD cases on selegiline therapy supports the utility of the platelet as a valuable indicator cell. However, the interpretation of peripheral MAOB activity as a possible susceptibility biomarker remains an open question. Alternative explanations for relative excesses or deficits in PD case platelets have been suggested. In view of the roles of MAO-B in dopamine catabolism and subsequent hydrogen peroxide generation, and in MPTP activation, it might be predicted that excessive activity might confer increase risk. On the other hand, the active metabolite of MPTP, MPP +, does not cross the blood-brain barrier readily; hence, a relative deficit of MAO-B activity in peripheral tissues may represent the high risk profile for MPTP analogs (Sturman etal., 1991). Other possible explanations for lower MAO-B activity among cases are that the failure to metabolize dopamine efficiently may either result in toxic dopamine concentrations in nervous tissue or may stimulate other catabolic pathways for dopamine that generate toxic metabolities (Williams et al., 1991). The dependence of MAO-B activity on substrate is a further complicating factor, as relative activity in PD case platelets appears to vary inversely with the number of hydroxyl groups on the substrate (Humfrey et al., 1990). An oxidative stress model of PD causation has gained favor in recent years. Increased SOD may suggest an enhanced adaptation to superoxide free radicals with an accelerated rate of hydrogen peroxide formation. We observed only slight, and statistically non-significant, excesses of total SOD activity in lymphocytes of PD cases. Nonetheless, the excess persisted irrespective of gender, age, or selegiline treatment. The availability of specific probes for the mitochondrial and cytosolic forms of SOD (Nakano et al., 1990) should be useful for clarifying the role of SOD as a biomarker of PD risk in future research.
Acknowledgements This research was supported by Grant ES-04696 from NIEHS. Dr. Castoldi received support from the Clinica del Lavoro (Pavia, Italy). The authors are grateful to M. Linde for enrolling study subjects and data collection, to M. Finley for manuscript preparation, and to J. Picciano for data management.
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