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Brief Reports

Cardiovascular Effects ofMethamphetamine in Parkinson’s

Disease Patients

Nicola Pavese, MD, Ornella Rimoldi, MD,Alexander Gerhard, MD, David J. Brooks, MD, DSc,

and Paola Piccini, MD*

MRC Clinical Science Center and Division of Neurosciences,Faculty of Medicine, Imperial College. Hammersmith

Hospital Campus, London, United Kingdom

Abstract: Cardiovascular responses after intravenous meth-amphetamine were assessed in 11 Parkinson’s disease (PD)patients. Systolic blood pressure (SBP), diastolic bloodpressure (DBP), heart rate (HR), and electrocardiogram(ECG) were monitored for 103 minutes. After methamphet-amine administration, SBP and DBP increased significantlyin both PD and normal controls whereas placebo had noeffect. In PD patients, however, the duration of SBP andDBP responses to methamphetamine and the maximumincrease from baseline was attenuated compared with thecontrols. A significant correlation was found between indi-vidual BP responsiveness and the Unified Parkinson’s Dis-ease Rating Scale (UPDRS) motor score. These findingssuggest that in PD there is impairment of catecholaminerelease from peripheral sympathetic presynaptic terminals,which correlates with motor impairment. © 2003 Move-ment Disorder Society

Key words: Parkinson’s disease; methamphetamine; bloodpressure; postganglionic sympathetic neurons

Methamphetamine (MA) is one of the most potentindirectly acting sympathomimetic amines known.1 Inthe central nervous system (CNS), MA effects typicallyinclude psychomotor activation, anorexia, and in humanslessened fatigue and a euphoric sense of well-being.2 Inthe cardiovascular system, MA produces a dose-depen-dent increase in blood pressure (BP) and a more complex

response of heart rate (HR), where lower doses produceincreases and higher doses produce decreases in HR.3,4

These effects are mediated mainly via � 1- and �-adre-noceptor mechanisms,5 although dopaminergic mecha-nisms are also involved,4 consistent with the mechanismof MA action, which releases norepinephrine at sympa-thetic nerve synapses.6

It has been shown recently that amphetamine-inducedendogenous release of brain dopamine can be detected invivo using 11C-raclopride positron emission tomography(PET).7–9 Competition between endogenous dopamineand 11C-raclopride for D2 receptors enables assessmentof changes in synaptic dopamine after amphetamine ad-ministration.10 Using this procedure, we have demon-strated recently synaptic dopamine release from a trans-plant of fetal mesencephalic tissue in the striatum of aParkinson’s disease (PD) patient.11

The cardiovascular response to acute intravenous MAhas not been documented in PD patients. As the diseaseadvances, most patients develop impaired cardiovascularreflexes due to autonomic dysfunction leading to im-paired BP and HR regulation.12 This, in part, reflectsimpaired sympathetic innervation of the heart13 but car-diovascular reflex tests, such as a Valsalva maneuver andtilting, demonstrate both sympathetic and parasympa-thetic failure in PD patients.12,14,15 Goldstein and col-leagues16 have suggested that orthostatic hypotension inPD patients reflects primarily loss of generalized sympa-thetic denervation, including cardiac catecholamine in-nervation. We describe cardiovascular responses (BP andHR changes) to intravenous MA administered to PDpatients and normal subjects before 11C-raclopride PETstudies.

SUBJECTS AND METHODS

In total, 11 patients with idiopathic PD (1 woman, 10men; mean age, 57.5 � 7.6 years; mean disease duration,15.8 � 5.4 years), and 6 healthy male right-handedsubjects (mean age, 42 � 4.5 years) consented to partic-ipate in a PET study of MA-induced dopamine release.Ethical approval and permission was given by the Ham-mersmith Hospital Trusts Ethics Committee and the Ad-ministration of Radioactive Substances Advisory Com-mittee of the Department of Health (ARSAC), UK.Informed consent was obtained from all PD patients andhealthy volunteers.

*Correspondence to: Dr. Paola Piccini, Cyclotron Building, Ham-mersmith Hospital, DuCane Road, London W12 00N United Kingdom.E-mail: [email protected]

Received 20 December 2002; Revised 16 May 2003; Accepted 12August 2003

DOI 10.1002/mds.10651

Movement DisordersVol. 19, No. 3, 2004, pp. 298–330© 2003 Movement Disorder Society

298

A clinical diagnosis of Parkinson’s disease was madeaccording to the criteria proposed by the United King-dom Parkinson’s Disease Society Brain Bank.17 Patientswith other central or peripheral nervous system disordersand patients with a history of previous exposure to psy-chostimulants were excluded. Clinical disability of thePD was graded when withdrawn from medication for 12hours using the Hoehn and Yahr staging (HY)18 and theUnified Parkinson’s Disease Rating Scale (UPDRS).19

None of the patients had a history of autonomic dysfunc-tion. Clinical characteristics of the patients are given inTable 1.

All patients were treated with levodopa (L-dopa) eitheralone (36%) or in combination with dopamine agonists(64%). Therefore, we calculated for each patient a dailyL-dopa equivalent dose according to the following con-version factors: 100 L-dopa equivalents � 100 mg ofstandard L-dopa � 133 mg of controlled-releaseL-dopa � 10 mg of bromocriptine � 1 mg of pergolide �5 mg of ropinirole � 2 mg of apomorphine.20 Treatmentwas discontinued 12 hours before the study.

All patients and controls were studied twice, beinggiven an intravenous dose of saline on one occasion andmethamphetamine (0.3 mg/kg) on the other in a blindedrandomly selected fashion. Saline or MA was adminis-tered as a bolus over 30 seconds.

Cardiovascular Assessment

Systolic (SBP) and diastolic (DBP) blood pressure,and HR were recorded automatically by Dinamap (Cri-tikon, Tampa, FL). The subjects were in a comfortablesupine position inside the PET scanner. SBP, DBP, andHR were assessed at 2-minute intervals in the first 15minutes before the scan, in the first 16 minutes afterMA/placebo administration, and then at 10-minute inter-vals until the end of the PET scan (103 minutes afterMA/placebo injection). A 12-lead electrocardiogram(ECG) was recorded at baseline and was monitored con-tinuously throughout the procedure.

Statistical Analysis

Repeated measure analysis of variance (ANOVA)with a Bonferroni correction was used to assess theeffects of treatments. Between-group comparisons werecarried out with nonparametric Mann Whitney statistics.Spearman Rank statistics were used to assess the signif-icance of clinical correlations.

RESULTS

Effects on Heart Rate and Systemic Blood Pressure

MA administration produced mild, nonsignificant in-crease in HR in both PD and control groups whereas HRwas unchanged after placebo (Fig. 1).

After MA administration, SBP and DBP increased inboth PD (SBP, P � 0.0059; DBP, P � 0.0143;ANOVA), and control (SBP and DBP, P � 0.0001;ANOVA) groups, whereas placebo had no effect (Figs.2,3). In no case was pharmacologic treatment for acutehypertension required.

In PD patients the maximum increase from baselinewas lower (SBP, P � 0.0308; DBP, P � 0.0394) and theresponse duration shorter than that in the control group(SBP, P � 0.001; DBP P � 0.001). In the normalsubjects, we observed a mean 28.1% SBP increase frombaseline (range, 16.5–36.8%) and 37.7% DBP increase(range, 19.2–55.1%) 4 minutes and 2 minutes, respec-tively, after MA administration. PD patients showed amean 12.8% SBP increase (range, 9.8–38.3%) and 15%DBP increase (range, 8.6–43%) 4 minutes after MA.

SBP and DBP remained increased significantly 103minutes after the MA infusion in normal subjects,whereas in the PD patients the significant increases inSBP and DBP lasted for only 35 minutes and 10 minutes,respectively.

We calculated a cardiovascular responsiveness index(CRI) for each patient defined as the ratio between max-imum increase from baseline in SBP and time to reach it.A significant inverse correlation between individual re-sponsiveness index and UPDRS motor score in off(Spearman r � �0.6, P � 0.04), that is lower CRIs werecorrelated with higher UPDRS scores (Fig. 4), wasfound. There were no correlations between CRI and age(Spearman r � 0.42, P � 0.15) or daily equivalentL-dopa dose (Spearman r � 0.39, P � 0.23) (data notshown).

ECG Evaluation

No ECG changes were observed after either MA orplacebo.

TABLE 1. Clinical characteristics of the patient group

Patient characteristic (n � 11) Value

Gender (F/M) 1/10Age (year) (mean � SD) 57.5 � 7.6Disease duration (year) (mean � SD) 15.8 � 5.4Hoehn & Yahr Stage (mean � SD) 3.2 � 0.4Daily dose L-dopa equivalenta (mean � SD) 848.6 � 465.9“off” UPDRS motor score (mean � SD) 28.5 � 5.3

a100 L-dopa equivalents � 100mg of standard L-dopa � 133 mg ofcontrolled-release L-dopa � 10 mg of bromocriptine � 1 mg ofpergolide � 5 mg of ropiniriole � 2 mg of apomorphine.20

UPDRS, Unified Parkinson’s disease rating scale.

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Movement Disorders, Vol. 19, No. 3, 2004

Other Side Effects

All subjects tolerated the experimental procedurewell and no serious adverse events were observed orreported by the subjects. One subject experienced drymouth and all experienced insomnia the night afterMA administration.

DISCUSSION

After MA administration SBP and DBP increasedsignificantly in both PD and control groups. In PD pa-tients, however, the duration of response was shorter andmaximum increase from baseline lower than that in the

control group, suggesting both cardiac and vascular hy-posensitivity to MA. We believe that this results fromimpairment of MA-induced catecholamine release fromcardiac and vascular peripheral sympathetic presynapticterminals.

Recent studies have reported that patients with PDhave a high prevalence of loss of sympathetic innerva-tion of the heart and other organs.13,16,21–26 It has beensuggested that this contributes to symptoms of auto-nomic failure such as orthostatic hypotension.16 In fact,

FIG. 1. A: Time course of percentage change from baseline in heartrate (HR) of normal subjects after placebo or methamphetamine (MA).Data are expressed as mean � SE. Differences from baseline were notsignificant after either placebo or MA (Bonferroni statistics). B: Timecourse of percentage change from baseline in HR of PD patients afterplacebo or MA. Data are expressed as mean � SE. Differences frombaseline were not significant after either placebo or MA (Bonferronistatistics). Solid circles, amphetamine; open circles, placebo.

FIG. 2. A: Time course of percentage change from baseline in systolicblood pressure (SBP) of normal subjects after placebo or methamphet-amine (MA). Data are expressed as mean � SE. Significant differencesfrom baseline (P � 0.001) were found at 2, 6, 8, 10, 12, 15, 25, 35, 45,55, 65, 75, 85, 95, and 103 minutes after MA administration. Differ-ences after placebo were not significant (Bonferroni statistics). B: Timecourse of percentage change from baseline in SBP of PD patients afterplacebo or MA. Data are expressed as mean � SE. Significant differ-ences from baseline (P � 0.001) were found at 2, 6, 8, 10, 12, and (P �0.05) at 15, 25, and 35 minutes after MA administration. Differencesafter placebo were not significant (Bonferroni statistics). Solid circles,amphetamine; open circles, placebo.

300 N. PAVESE ET AL.


decreased myocardial uptake of the sympathetic markers123I-metaiodobenzylguanidine21–26 and 18F-fluorodopam-ine13,16 has been reported in PD patients. Both tracersfollow the same metabolic pathway as norepinephrineand their myocardial uptake reflects not only the densityof postganglionic sympathetic neurons but also theirfunctional integrity. In addition, reduced mean plasmanorepinephrine resting levels16 and presence of denerva-tion hypersensitivity to norepinephrine infusion27 have

also been reported and would support the notion of ageneralized sympathetic denervation.

Our findings, in line with the above studies, suggestthat decreased release of catecholamines from peripheralsympathetic presynaptic terminals occurs in PD afteracute pharmacologic challenge. The differences wefound in cardiovascular responses of PD patients can beexplained by decreased MA-induced catecholamine re-lease from impaired postganglionic sympathetic cardiacand vascular neurons. In fact, due to the absence of polarhydroxyl groups in their chemical structures, which re-sults in a loss of direct sympathomimetic activity, MAand other amphetamines exert their effects indirectlythrough release of catecholamine (norepinephrine anddopamine) rather than by acting directly on catechol-amine receptors.28

Reduced release of these neurotransmitters in otherareas of the CNS involved in the regulation of the car-diovascular system, such as the posterior hypothalamus,the ventral tegmental area, and the basal forebrain,29–31

might also be a factor. Central mechanisms underlyingshort-term regulation of the cardiovascular system, how-ever, are mediated generally via glutamatergic andGABA-ergic receptors.32 Therefore, peripheral ratherthan central catecholamine release impairment is likelyresponsible for the impaired cardiovascular responses inPD.

Reduced density of catecholamine postsynaptic recep-tors in PD patients could also explain our findings. Thisis unlikely to be the case, however, because denervationhypersensitivity to intravenous norepinephrine, similar tothat found in lesions of postganglionic sympathetic ef-FIG. 3. A: Time course of percentage change from baseline in dia-

stolic blood pressure (DBP) of normal subjects after placebo or meth-amphetamine (MA). Data are expressed as mean � SE. Significantdifferences from baseline (P � 0.001) were found at 2, 6, 8 and (P �0.01) at 10, 12, 15, 25, 35, 45, 55, 65, 75, 85, 95, and 103 minutes afterMA administration. Differences after placebo were not significant(Bonferroni statistics). B: Time course of percentage change frombaseline in DBP of PD patients after placebo or methamphetamine(MA). Data are expressed as mean � SE. Significant differences frombaseline (P � 0.001) were found at 2, 6, 8, and 10 minutes after MAadministration. Differences after placebo were not significant (Bonfer-roni statistics). Solid circles, amphetamine; open circles, placebo.

FIG. 4. Correlation between individual Cardiovascular Responsive-ness indices (CRI) defined for each patient as the ratio between max-imum increase from baseline in systolic blood pressure and time toreach and UPDRS motor scores off medication.

CARDIOVASCULAR EFFECT OF METHAMPHETAMINE IN PD 301


ferents,33 can be present in PD.27,34 No cardiac condi-tions associated with reduced postsynaptic catechol-amine receptor density were present in our patients andnone had abnormal baseline ECGs.

None of our PD patients had symptoms or signs ofautonomic system involvement, suggesting that the sym-pathetic dysfunction present was subclinical. This viewis in agreement with the observation of Taki and associ-ates26 that reduction of cardiac uptake of 123I-metaiodo-benzylguanidine occurred in PD patients without overtautonomic failure. The significant correlation betweenindividual cardiovascular responsiveness and UPDRSmotor score in PD and the absence of correlation withtreatment suggest that this sympathetic dysfunction isdisease specific.

Future studies could investigate the correlation be-tween cardiovascular response to MA and imaging datafrom PET or single-photon emission computed tomog-raphy (SPECT), not only in PD, but also in other neu-rodegenerative diseases involving primary autonomicfailure, such as multiple system atrophy where post-ganglionic cardiac sympathetic denervation is notfound.22,26,35,36 Our study has shown that intravenousbolus MA acute challenge studies in PD are generallywell tolerated and clinically safe.

Acknowledgments: We thank H. McDevitt and S. Ahier forexpert help with scanning and carrying out cardiovascularexaminations.

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1. Seiden LS, Ricaurte GA. Neurotoxicity of methamphetamine andrelated drugs. In: Meltzer HY, editor. Psychopharmacology: thethird generation of progress. New York: Raven Press; 1987. p359–366.

2. Martin WR, editor. Drug addiction II. New York: Springer Verlag;1977.

3. Angrist B, Sanfilipo M, Wolkin A. Cardiovascular effects of 0.5milligrams per kilogram oral D-amphetamine and possible atten-uation by haloperidol. Clin Neuropharmacol 2001;24:139–144.

4. Schindler CW, Zheng JW, Tella SR, Goldberg SR. Pharmacolog-ical mechanisms in the cardiovascular effects of methamphetaminein conscious squirrel monkeys. Pharmacol Biochem Behav 1992;42:791–796.

5. Hoffman BB, Lefkowitz RJ. Adrenergic receptor antagonists. In:Gilman AG, Rall AG, Nies AS, Taylor P, editors. Goodman andGilaman’s pharmacological basis of therapeutics. New York: Per-gamon Press; 1990. p 221–243.

6. Cho AK. Ice: a new dosage form of an old drug. Science 1990;249:631–634.

7. Carson RE, Breier A, de Bartolomeis A, et al. Quantification ofamphetamine-induced changes in [11C]raclopride binding withcontinuous infusion. J Cereb Blood Flow Metab 1997;17:437–447.

8. Breier A, Su TP, Saunders R, et al. Schizophrenia is associatedwith elevated amphetamine-induced synaptic dopamine concentra-tions: evidence from a novel positron emission tomographymethod. Proc Natl Acad Sci USA 1997;94:2569–2574.

9. Ginovart N, Farde L, Halldin C, Swahn CG. Changes in striatalD2-receptor density following chronic treatment with amphet-

amine as assessed with PET in nonhuman primates. Synapse1999;31:154–162.

10. Laruelle M. Imaging synaptic neurotransmission with in vivobinding competition techniques: a critical review. J Cereb BloodFlow Metab 2000;20:423–451.

11. Piccini P, Brooks DJ, Bjorklund A, et al. Dopamine release fromnigral transplants visualized in vivo in a Parkinson’s patient. NatNeurosci 1999;2:1137–1140.

12. Mathias CJ. Cardiovascular autonomic dysfunction in parkinso-nian patients. Clin Neurosci 1998;5:153–166.

13. Goldstein DS, Holmes C, Li ST, Bruce S, Metman LV, CannonRO 3rd. Cardiac sympathetic denervation in Parkinson disease.Ann Intern Med 2000;133:338–347.

14. Turkka JT, Tolonen U, Myllyla VV. Cardiovascular reflexes inParkinson’s disease. Eur Neurol 1987;26:104–112.

15. Meco G, Pratesi L, Bonifati V. Cardiovascular reflex and auto-nomic dysfunction in Parkinson’s disease J Neurol 1991;238:195–199.

16. Goldstein DS, Holmes CS, Dendi R, Bruce SR, Li ST. Orthostatichypotension from sympathetic denervation in Parkinson’s disease.Neurology 2002;23;58:1247–1255.

17. Daniel SE, Lees AJ. Parkinson’s Disease Society Brain Bank,London: overview and research. J Neural Transm Suppl 1993;39:165–172.

18. Hoehn MM, Yahr MD. Parkinsonism: onset, progression, andmortality. Neurology 1967;17:427–442.

19. Fahn S, Elton RL, Members of the UPDRS Development Com-mittee. The Unified Parkinson’s Disease Rating Scale. In: Fahn S,Marsden CD, Calne DB, Goldstein M, editors. Recent develop-ments in Parkinson’s disease, Vol. 2. Florham Park, NJ: MacmillanHealthcare Information; 1987.

20. Hagell P, Piccini P, Bjorklund A, et al. Dyskinesias followingneural transplantation in Parkinson’s disease. Nat Neurosci 2002;5:627–628.

21. Iwasa K, Nakajima K, Yoshikawa H, Tada A, Taki J, Takamori M.Decreased myocardial 123 I-MIBG uptake in Parkinson’s disease.Acta Neurol Scand 1998;97:303–306.

22. Yoshita M, Hayashi M, Hirai S. Decreased myocardial accumula-tion of 123I-meta-iodobenzyl guanidine in Parkinson’s disease.Nucl Med Commun 1998;19:137–142.

23. Satoh A, Serita T, Seto M, et al. Loss of 123I-MIBG uptake bythe heart in Parkinson’s disease: assessment of cardiac sympa-thetic denervation and diagnostic value. J Nucl Med 1999;40:371–375.

24. Takatsu H, Nishida H, Matsuo H, et al. Cardiac sympatheticdenervation from the early stage of Parkinson’s disease: clinicaland experimental studies with radiolabeled MIBG. J Nucl Med2000;41:71–77.

25. Ohmura M. Loss of 123I-MIBG uptake by the heart in Parkinson’sdisease: assessment of cardiac sympathetic denervation and diag-nostic value. J Nucl Med 2000;41:1594–1595.

26. Taki J, Nakajima K, Hwang EH, et al. Peripheral sympatheticdysfunction in patients with Parkinson’s disease without auto-nomic failure is heart selective and disease specific. Eur J NuclMed 2000;27:566–573.

27. Braune S, Reinhardt M, Bathmann J, Krause T, Lehmann M,Lucking CH. Impaired cardiac uptake of meta-[123I]iodobenzyl-guanidine in Parkinson’s disease with autonomic failure. ActaNeurol Scand 1998;97:307–314.

28. Hoffman BB. Catecholamines, sympathomimetic drugs and adren-ergic receptor agonists. In: Hardman JG, Limbird LE, Gilman AG,editors. Goodman and Gilaman’s pharmacological basis of thera-peutics. New York: McGraw Hill; 2001. p 215–268

29. Haeusler G. Cardiovascular regulation by central adrenergic mech-anisms and its alteration by hypotensive drugs. Circ Res 1975;36(Suppl.):223–232.

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31. van den Buuse M. Pressor responses to brain dopaminergic stim-ulation. Clin Exp Pharmacol Physiol 1997;24:764–769.

32. Dampney RA, Coleman MJ, Fontes MA, et al. Central mechanismsunderlying short- and long-term regulation of the cardiovascularsystem. Clin Exp Pharmacol Physiol 2002;29:261–268.

33. Bannister R, Davies B, Holly E, Rosenthal T, Sever P. Defectivecardiovascular reflexes and supersensitivity to sympathomimeticdrugs in autonomic failure. Brain 1979;102:163–176.

34. Senard JM, Valet P, Durrieu G, et al. Adrenergic supersensitivityin parkinsonians with orthostatic hypotension. Eur J Clin Invest1990;20:613–619.

35. Goldstein DS, Holmes C, Cannon RO 3rd, Eisenhofer G, Kopin IJ.Sympathetic cardioneuropathy in dysautonomias. N Engl J Med1997;336:696–702.

36. Druschky A, Hilz MJ, Platsch G, Radespiel-Troger M, DruschkyK, Kuwert T, Neundorfer B. Differentiation of Parkinson’s diseaseand multiple system atrophy in early disease stages by means ofI-123-MIBG-SPECT. J Neurol Sci 2000;175:3–12.

Long-Term Follow-Up of CervicalDystonia Patients Treated With

Botulinum Toxin A

Peter Haussermann, MD,1 Stefanie Marczoch, MD,1

Christiane Klinger, MD,1 Michael Landgrebe, MD,1

Bastian Conrad, MD,1

and Andres Ceballos-Baumann, MD*1

1Department of Neurology, Klinkum rechts der Isar,Technische Universitat Munchen, Germany

Abstract: We followed the course in 100 consecutive patientswith cervical dystonia (CD) after they were initially treatedwith botulinum toxin (BTX) in the form of Dysport 10 to 12years ago. A total of 4 patients had died, and 6 were lost tofollow-up. Of the remaining 90 patients, 57 (63%) were stilltreated with BTX. In the patients treated at one centre overthe whole period with Dysport, mean dose used during eachtreatment session was 833 (SD � 339) units Dysport with acumulative dose of 20,943 (SD � 9462) units Dysport overa mean of 26.8 (SD � 8.6) treatment cycles. Secondarynonresponse was detected in 3 of the 90 patients. Duringfollow-up, 12 patients developed blepharospasm, 13 oro-mandibular dystonia, and 17 patients writer’s cramp. Weconclude that BTX remains effective and safe for approxi-

mately 60% of CD patients for more than 10 years. © 2004Movement Disorder Society

Key words: cervical dystonia; botulinum toxin A; long-termoutcome

Cervical dystonia (CD) is the most common form ofadult-onset focal dystonia.1 The aetiology of CD is un-known, but genetic and environmental factors are impli-cated.2 Botulinum toxin (BTX) is nowadays regarded asfirst-line treatment for CD and has dramatically im-proved the quality of life of CD patients.3–5 BTX wasintroduced in the early 1980s as treatment for CD6,7;most studies, however, provided only short-term fol-low-up ranging from months to 2 years.8–11 In CD,roughly 75% of patients initially improve, and a responseis generally seen within the first week to last for anaverage of 12 weeks.12,13 CD represents a chronic diseaseoften starting in early adulthood with a mean age of onsetaround 40 years. Thus, long-term efficacy and safety ofBTX is an important question for health care provid-ers.14,15 For hemifacial spasm, a 10-year follow-up studyhas been published recently16 but until now, there is onlyone long-term study using BTX A in the form of BO-TOX in a variety of conditions, including 106 patientswith CD, but only 62 of 106 of them were followed upfor more than 5 years.17 We present the course of 100consecutive patients with CD who started injections withBTX Type A in the form of Dysport 10 to 12 years ago.We evaluated the adherence to BTX therapy, the percep-tion of improvement, and reasons for discontinuation ofBTX treatment.

PATIENTS AND METHODS

We performed a longitudinal cohort study of the 100consecutive CD patients who received their initial BTXinjections between February 1989 and October 1992.The inclusion criteria were (1) cervical dystonia, eitheridiopathic or symptomatic; (2) BTX injections, with atleast one follow-up evaluation; (3) age at CD onset �18;(4) and duration of disease �1 year before onset oftreatment.

By using a standardised questionnaire, these 100 pa-tients were re-contacted in 2001 and 2002 and eitherinterviewed when attending for a reinjection appoint-ment or contacted by telephone if they were no longertreated at our institution. We inquired family doctors,health insurers, or local authorities for those patients whocould not be contacted by phone. Patients still receivingBTX at our centre were re-examined, and those claimingto have no effect or had lost their initial treatment effectto BTX were re-evaluated. Data on mean BTX dose, siteand number of BTX injections, as well as number of

*Correspondence to: Andres Ceballos-Baumann, M.D., Departmentof Neurology, Klinikum rechts der Isar, Technische UniversitatMunchen, Mohlstr. 28, D-81675 Munchen, Germany,E-mail: [email protected]

Received 7 March 2003; Revised 6 August 2003; Accepted 20August 2003

DOI 10.1002/mds.10659

LONG-TERM BOTULINUM TOXIN TREATMENT IN CD 303


treatment visits were derived from our clinical case re-port charts. We received informed consent from all pa-tients, except for those who died or were lost tofollow-up.

We injected cervical muscles responsible for abnormalposture; each treatment cycle consisted of several injec-tions into affected dystonic muscles. Electromyographicguidance was used for injections in patients with anunusual activation pattern or with severe obesity.

We collected demographic and clinical data (aetiologyof CD; associated dystonia in other body parts; site andnumber of BTX injections; number of treatment visits inour clinic; mean BTX dose supplied during injections atour centre; type, duration, and severity of complications;initial severity of dystonia; alternative treatment options;change of treatment centre; interruption of therapy; rea-sons to stop BTX injections). At interview, patients werealso asked to determine their global subjective BTXeffect over the entire treatment period. We classifiedpatients into two categories: primary nonresponders, pa-tients who claimed not to have responded to the firsttreatment of BTX; and secondary nonresponders, pa-tients who were initially responsive and subsequentlybecame refractory to injections.

In the statistical analysis, we used �2 and Mann–Whitney U test for comparisons. All values are expressedas mean � standard deviation (SD). Adherence to ther-apy was analysed using the Kaplan-Meier method.

RESULTS

The majority of patients (79 of 100) had idiopathicprimary cervical dystonia; symptomatic cervical dysto-nia was seen in 21 of 100 patients, with a tardive aeti-ology attributed to 13 patients. Stroke (1 of 21), hypoxicencephalopathy (3 of 21), encephalitis (encephalomyeli-tis disseminata; 2 of 21), and head trauma (2 of 21)accounted for the other secondary cases. Within up to 12years, 6 patients were lost to follow-up and 4 patientsdied. Of the remaining 90 patients, 57 are still beingtreated with BTX (63%): 32 of them in our clinic (Fig.1), and 25 patients in other neurological departments.These 25 patients still on BTX in other centres werecontacted by telephone; therefore, exact data on BTXdose and frequency of injections can only be given forthe period when they were treated at our clinic.

Before BTX treatment was initiated, 89 of 100 patientsunderwent other treatments. Pharmacotherapy (mainlytiapride, anticholinergic drugs, and benzodiazepines)was used in 64 of 100 patients, psychotherapy in 29 of100, surgery (mainly selective neurectomy) in 6 of 100,physiotherapy in 58 of 100, and alternative medicinesuch as acupuncture in 57 of 100 patients. Of the 33

patients who stopped BTX injections, 16 remained with-out further treatment and 17 searched for other treatmentapproaches (pharmacotherapy 8 of 17, physiotherapy 9of 17, and alternative medicine 4 of 17).

Mean age at symptom onset was 41.16 � 13.97 years,and mean age at onset of therapy was 47.23 � 14.28years. The 100 patients had a total of 1,382 treatmentvisits in our clinic (mean 13.82 � 14.33/patient; median,13.0; range, 1–44). Mean treatment follow-up of all 100CD patients in our clinic was 61.02 � 54.53 months(median, 49; range, 3–143). All patients received injec-tions of Dysport. A mean dose of 800.79 � 241 mouseunits (MU) of Dysport was administered during eachtreatment session. Demographic and treatment data aresummarised in Table 1, including a comparison betweenprimary and secondary CD patients.

The 32 patients who were continuously treated withDysport at our centre over the whole period had a meanage at symptom onset of 41.26 � 12 years, and theirmean age at onset of therapy was 46.0 � 12.49 years.Their mean follow-up was 120 � 12.27 months (median,118; range, 101–143 months), and they received a meanof 26.8 � 8.57 injections (median, 25; range, 12–44injections). Their mean dose/injection was 833 � 339(median, 823; range, 437–2,298) MU, whereas theirmean cumulative dose was 20,943 � 9,462 (median,19,010; range, 7,660–41,370) MU of Dysport.

To assess patient’s satisfaction with BTX treatment,the 90 patients followed up were asked to determine theirglobal subjective BTX effect over the whole treatmentperiod on a rating scale going from �4 (very bad effectin all sessions) to 4 (very good effect in all treatmentsessions). The mean score was 1.93 � 1.18, which dem-onstrates a pronounced effect. Thirty-three patientsstopped BTX therapy for various reasons (Table 2). Ofthese 33 patients, 18 dropped out after only one injection;subsequently, the number of patients stopping BTX in-jections decreased constantly (Fig. 2). During follow-up,another 15 patients choose to cope with their diseaserather than to receive additional injections. Main reasonsto abstain from further injections were side effects ofBTX therapy and travel inconveniences (Table 2). Al-though 34 of the patients reported some sort of adverseevents on at least one treatment visit, most of these weretransient and not severe. The most common adverseevents reported in any treatment visit were weakness ofcervical muscles (13 patients), mild dysphagia for solidfood (12 patients), and general weakness (5 patients).Dysphagia for fluids was reported by 3 patients; 1 ofthese patients was admitted to the hospital and requirednasogastric tube feeding for several days. Side effects led11 patients to discontinue BTX.

304 P. HAUSSERMANN ET AL.


Segmental or multisegmental spread of dystonia de-veloped in 30 of 90 patients (33%) during follow-up. Atotal of 12 patients developed further blepharospasm(BL), 13 patients oromandibular dystonia (OMD), and,in 17 patients, additional writer’s cramp was encoun-tered. Of the patients experiencing spread, 5 developedcoincident BL and OMD, i.e., a Meige syndrome.

Male patients had a significantly lower age at symp-tom and therapy onset; otherwise, there were no furthersignificant differences between male and female patientsconcerning demographic and treatment characteristics.We also compared primary and secondary CD patients.Secondary CD patients were significantly younger atdisease and therapy onset. They also reported signifi-cantly more adverse events, although neither BTX dose/visit nor severity of disease were statistically differentfrom primary CD patients (Table 1).

Secondary failure to respond may occur because of achange in the pattern of muscle activity or the develop-

ment of neutralising antibodies. Neutralising antibodiesoccur in 3 to 10% of CD patients.18 BTX-resistant pa-tients no longer develop atrophy in injected muscles, aswas the case in our secondary nonresponders. Postulatedrisk factors for BTX resistance are more frequent injec-tions, “booster injections” 2 to 3 weeks after an initialinjection, and higher doses of BTX per treatment cy-cle.19,20 Three patients were initially responsive and sub-sequently became refractory to injections; these wereclassified as secondary nonresponders. Complete remis-sion was reported by 1 patient with cervical dystonia, andsignificant improvement of dystonia was seen in 5patients.

DISCUSSION

In this long-term cohort study, CD patients showedhigh adherence to BTX treatment over time. More than60% (57 of 90) of the CD patients continue with BTXinjections after up to 12 years. Main reasons to stop BTX

FIG. 1. Long-term follow-up of 100 cervical dystonia patients after 12 years. BTX, botulinum toxin. [Color figure can be viewed in the online issue,which is available at www.interscience.wiley.com.]



therapy were side effects and inconveniences (long traveldistances, costs; see Table 2). The subjectively experi-enced treatment effect was, overall, positive. Spread ofdystonia was seen in one third of the patients duringfollow-up. Other studies evaluating spread of dystoniaduring BTX therapy showed an expansion of dystonia toother body parts to occur in 2 to 30% of CD patients.21–23

The large proportion of segmental spread in our study ismost likely the result of the long-term follow-up periodcompared to other studies evaluating spread22,23 and thehigh rate of symptomatic patients known to be at risk fordystonic involvement of other body parts.2,24

That 6 patients had spontaneous and lasting improve-ment of CD is of interest. While remissions are notexceptional in CD,25 they are often not complete orprolonged.2 In idiopathic CD, remission rates of 10 to30% are found and remission typically occurs within thefirst years25; very often, patients relapse within 5 years.Younger age at symptom onset seems to be the onlyconsistent clinical characteristic that appears to predict

remission. Further analysis of our 6 patients with remis-sions showed that only 1 CD patient had complete re-mission and required no further treatment. Subjectiveimprovement of dystonia, defined as sufficient to abstainfrom further injections, was the case in the remaining 5patients. This number is less compared to the studyconducted by Lowenstein and Aminoff, where completeremission was reported in 3 of 24 patients and partialremission was reported in 8 of 24 patients.25 It is difficultto ascertain whether BTX itself influences the naturalhistory of CD. Our data suggest that BTX does notinfluence the course of CD; BTX apparently does notaffect progression of dystonia, as segmental spread was

TABLE 1. Comparison of demographic and treatment variables in CD patients, according to aetiology

All patients Primary CD Secondary CDLevel of

significance

Sex (M:F) 100 (43:57) 79 (32:47) 21 (11:10) nsa

Age at disease onset 41.16 � 13.97 42.92 � 13.31 34.29 � 14.45 P � .02b

Time to therapy onset (months) 73.32 � 79.06 70.2 � 77.89 85.05 � 84.25 nsb

Age at therapy onset 47.23 � 14.28 48.76 � 14.15 41.48 � 13.59 P � 0.03b

Treatment visits 13.82 � 14.33 14.2 � 11.75 11.43 � 10.61 nsb

Severity at therapy onset, Tsui scale12 8.98 � 3.66 9.09 � 3.76 8.63 � 3.27 nsb

Follow-up treatment in our clinic (months) 61.02 � 54.53 60.80 � 52.15 61.25 � 53.48 nsb

Patients suffering from adverse events (n) 34 20 14 P � 0.01a

Cumulative BTX A (Dysport) dose/patientgiven at our clinic (MU) 10154.45 � 10202.96 10394.87 � 10239.91 9250 � 10260.19 nsb

BTX A (Dysport) dose per visit at our clinic(MU) 800.79 � 241.0 795.60 � 232.2 849.93 � 395.2 nsb

Injections/year (excluding patients whodropped out after 1 one visit, n � 18) atour clinic 2.65 � 1.02 2.7 � 1.08 2.43 � 0.74 nsb

Incidence of spread to other body sides(affected patients/patients recontacted) 30/90 24/73 6/17 na

Values are expressed as mean � SD, unless otherwise indicated.Severity was measured using the Tsui Rating Scale.7 Level of significance indicates when comparing primary and secondary CD patients using

achi-square test, and bMann–Whitney U test.ns, not significant; CD, cervical dystonia; BTX, botulinum toxin; MU, mouse unit.

FIG. 2. Kaplan–Meier graph: proportion of cervical dystonia patientsunder botulinum toxin (BTX) therapy. Shown are the proportion ofpatients who adhere to BTX therapy after up to 143 months follow-up.

TABLE 2. Reasons for patients with cervical dystonia tostop botulinum toxin therapy (n � 33)

Patients (n)

Primary nonresponse 1Secondary nonresponse 3Side effects 11Complete remission 1Significant improvement of dystonia 5Inconvenience (long travel distances, costs) 12

306 P. HAUSSERMANN ET AL.


seen in one third of our CD patients and only 6 of 94patients reported remission, which is less compared tothe only study in the pre-BTX era assessing the naturalcourse of CD without BTX treatment over a medianfollow-up period of 5.8 years.25

Tolerance to BTX was a minor problem in our series:1 patient was a primary nonresponder and 3 patientswere secondary nonresponders, which is less than ob-served in the study performed by Hsiung and colleagues,in which 20 of 106 of the CD patients stopped injectionswith BTX Type A in the form of BOTOX due to primaryor secondary resistance during a follow-up period of upto 10 years.17

We determined antibodies to BTX in the secondarynonresponders, using the phrenic–hemidiaphragma invitro mouse bioassay test26 and did not find evidence ofantibodies in these patients. Either, there were no anti-bodies present at all or the antibody concentration wasbelow the detection level (0.4 mU/ml).26

Thirty-four patients reported some form of side effectson at least one visit. This rate is not surprising consid-ering the long follow-up period. Additionally, a mean of800 MU of Dysport may have been a relatively high dosecompared to other studies. Other studies used lowerDysport doses ranging from 300 to 400 MU and a doseranging study found that, although magnitude and dura-tion of improvement was greatest after injections of1,000 MU of Dysport; there were significantly moreadverse events compared to the lower starting dose of500 units of Dysport.8,13,27,28

Neck muscle weakness and dysphagia were the mostcommon problems encountered in our study, but sideeffects were local and transient. Only 1 patient experi-enced dysphagia needing nasogastric tube feeding forseveral days. Eleven patients opted to stop further BTXinjections because of side effects, i.e., even minor sideeffects were perceived sufficiently inconvenient to stopBTX therapy. Adverse events are difficult to comparebetween studies as some authors report the frequency ofcomplications per treatment visit,29 whereas we deter-mined side effects during a telephone interview. Ourmethod is probably more prone to recall bias than thedirect assessment of side effects after each treatmentvisit. Jankovic and Schwartz reported complications in153 of 575 treatment visits (26.6%), including dyspha-gia, neck weakness, and neck pain, resolving within daysor weeks and rarely being disabling.29 Another study bythe same authors reported side effects in 28% (58 of 205)of patients at 15% (76 of 505) of the treatment visits intheir 205 CD patients followed-up for up to 4 years.30

Mild dysphagia was reported in 35 patients, whereasneck weakness was seen in 17 patients. There was no

difference in dosage between patients who suffered fromside-effects compared to patients who did not have ad-verse events.

Twenty-five patients discontinued BTX injections atour centre and sought further BTX injections elsewhere.Eighteen patients stopped BTX injections after one treat-ment session. This is a higher rate compared to a studyfocusing on patients’ reasons to stop BTX therapy, where11 of 133 patients received only one injection.31 How-ever, when our 100 patients were first treated, BTX wasregarded as experimental and had no approval by Ger-man health authorities. This may have predisposed tohigher skepticism toward BTX, and could explain ourhigher discontinuation rate after the first treatment session.However, side effects and travel inconveniences were givenas main reasons to abstain from further BTX injections.

Other long-term studies in CD had markedly shorteraverage follow-up periods ranging from 19 of 38 months,and few patients received continuous treatment through-out these studies.18,27,29 The average number of treatmentsessions ranged from 4 to 10, the maximum number oftreatment cycles ranged from 10 to 21.8,18,27,30 Our 100CD patients had a mean number of 13 treatment sessionswith a maximum of 44 injections, and the mean treat-ment follow-up was 61 months with a maximum fol-low-up of 143 months. Ninety CD patients could bere-evaluated. Thirty-two CD patients were constantlytreated with Dysport over a mean follow-up period of120 months at our institution. Until the present cohortstudy of 100 CD patients who received their first BTXinjections between 1989 and 1992, data concerning long-term outcome, prognosis, efficacy, and adherence toBTX therapy were sparse, in particular regarding Dys-port. The only other study evaluating long-term BTXeffects in 106 CD patients over up to 10 years analysed1,267 treatment cycles and showed sustained benefit in72 of 106 patients at 2 years and in 39 of 62 patients at5 years. But this study differs both in its retrospectivedesign and in the BTX preparation analysed (BOTOX).17

Our study provides evidence that BTX Type A is asafe and effective treatment approach, leading to sus-tained relief from symptoms of CD in the long-term.More than 60% of patients adhere to therapy after 10 to12 years. Immunoresistance was not a major problem inour patients. Longitudinal follow-up demonstrated mul-tisegmental spread of dystonia in one third of our CDpatients.

REFERENCES

1. Nutt JG, Muenter MD, Melton LJ III, Aronson A, Kurland LT.Epidemiology of dystonia in Rochester, Minnesota. Adv Neurol1988;50:361–365.



2. Dauer WT, Burke RE, Greene P, Fahn S. Current concepts on theclinical features, aetiology and management of idiopathic cervicaldystonia. Brain 1998;121:547–560.

3. Hilker R, Schischniaschvili M, Ghaemi M, Jacobs A, RudolfJ. Health related quality of life is improved by botulinum neuro-toxin type A in long term treated patients with focal dystonia.J Neurol Neurosurg Psychiatry 2001;71:193–199.

4. Camfield L, Ben Shlomo Y, Warner TT. Impact of cervical dys-tonia on quality of life. Mov Disord 2002;17:838–841.

5. Ben Shlomo Y, Camfield L, Warner T. What are the determinantsof quality of life in people with cervical dystonia? J NeurolNeurosurg Psychiatry 2002;72:608–614.

6. Tsui JK, Eisen A, Mak E, Carruthers J, Scott A, Calne DB. A pilotstudy on the use of botulinum toxin in spasmodic torticollis. CanJ Neurol Sci 1985;12:314–316.

7. Tsui JK, Eisen A, Stoessl AJ, Calne S, Calne DB. Double-blindstudy of botulinum toxin in spasmodic torticollis. Lancet 1986;2:245–247.

8. Brans JW, Lindeboom R, Aramideh M, Speelman JD. Long-termeffect of botulinum toxin on impairment and functional health incervical dystonia. Neurology 1998;50:1461–1463.

9. Blackie JD, Lees AJ. Botulinum toxin treatment in spasmodictorticollis. J Neurol Neurosurg Psychiatry 1990;53:640–643.

10. Greene P, Kang U, Fahn S, Brin M, Moskowitz C, Flaster E.Double-blind, placebo-controlled trial of botulinum toxin injec-tions for the treatment of spasmodic torticollis. Neurology 1990;40:1213–1218.

11. Jankovic J, Orman J. Botulinum A toxin for cranial-cervical dys-tonia: a double-blind, placebo-controlled study. Neurology 1987;37:616–623.

12. Poewe W, Wissel J. Use of botulinum toxin in the treatment ofcervical dystonia. Baillieres Clin Neurol 1993;2:179–185.

13. Poewe W, Deuschl G, Nebe A, Feifel E, Wissel J, Benecke R, etal. What is the optimal dose of botulinum toxin A in the treatmentof cervical dystonia? Results of a double blind, placebo controlled,dose ranging study using Dysport. German Dystonia Study Group.J Neurol Neurosurg Psychiatry 1998;64:13–17.

14. Castelon KE, Trender-Gerhard I, Kamm C, Warner T, Ben ShlomoY, Gasser T, et al. Service-based survey of dystonia in Munich.Neuroepidemiology 2002;21:202–206.

15. Ceballos-Baumann AO. Evidence-based medicine in botulinumtoxin therapy for cervical dystonia. J Neurol 2001;248(Suppl.1):14–20.

16. Defazio G, Abbruzzese G, Girlanda P, Vacca L, Curra A, DeSalvia R, et al. Botulinum toxin A treatment for primary hemifacialspasm: a 10-year multicenter study. Arch Neurol 2002;59:418–420.

17. Hsiung GY, Das SK, Ranawaya R, Lafontaine AL, SuchowerskyO. Long-term efficacy of botulinum toxin A in treatment of variousmovement disorders over a 10-year period. Mov Disord 2002;17:1288–1293.

18. Kessler KR, Skutta M, Benecke R. Long-term treatment of cervicaldystonia with botulinum toxin A: efficacy, safety, and antibodyfrequency. German Dystonia Study Group. J Neurol 1999;246:265–274.

19. Goschel H, Wohlfarth K, Frevert J, Dengler R, Bigalke H. Botu-linum A toxin therapy: neutralizing and nonneutralizing antibod-ies--therapeutic consequences. Exp Neurol 1997;147:96–102.

20. Greene P, Fahn S, Diamond B. Development of resistance tobotulinum toxin type A in patients with torticollis. Mov Disord1994;9:213–217.

21. Greene P, Kang UJ, Fahn S. Spread of symptoms in idiopathictorsion dystonia. Mov Disord 1995;10:143–152.

22. Jankovic J, Leder S, Warner D, Schwartz K. Cervical dystonia:clinical findings and associated movement disorders. Neurology1991;41:1088–1091.

23. Jahanshahi M, Marion MH, Marsden CD. Natural history of adult-onset idiopathic torticollis. Arch Neurol 1990;47:548–552.

24. Defazio G, Berardelli A, Abbruzzese G, Coviello V, Carella F, DeBerardinis MT, et al. Risk factors for spread of primary adult onsetblepharospasm: a multicentre investigation of the Italian move-ment disorders study group. J Neurol Neurosurg Psychiatry 1999;67:613–619.

25. Lowenstein DH, Aminoff MJ. The clinical course of spasmodictorticollis. Neurology 1988;38:530–532.

26. Goschel H, Wohlfarth K, Frevert J, Dengler R, Bigalke H. Botu-linum A toxin therapy: neutralizing and nonneutralizing antibod-ies-therapeutic consequences. Exp Neurol 1997;147:96–102.

27. Van den Bergh P, Francart J, Mourin S, Kollmann P, Laterre EC.Five-year experience in the treatment of focal movement disorderswith low-dose Dysport botulinum toxin. Muscle Nerve 1995;18:720–729.

28. Krack P, Deuschl G, Benecke R, Ceballos-Baumann AO, MarionMH, Oertel WH, et al. Dose standardization of botulinum toxin.Mov Disord 1998;13:749–751.

29. Jankovic J, Schwartz KS. Longitudinal experience with botulinumtoxin injections for treatment of blepharospasm and cervical dys-tonia. Neurology 1993;43:834–836.

30. Jankovic J, Schwartz K. Botulinum toxin injections for cervicaldystonia. Neurology 1990;40:277–280.

31. Brashear A, Bergan K, Wojcieszek J, Siemers ER, Ambrosius W.Patients’ perception of stopping or continuing treatment of cervicaldystonia with botulinum toxin type A. Mov Disord 2000;15:150–153.

Assessing Quality of Life inParkinson’s Disease: Can a Short-Form Questionnaire Be Useful?

Zoe Katsarou, MD,1* Sevasti Bostantjopoulou, MD,1

Viv Peto, BA,2 Anna Kafantari, MD,1

Elizabeth Apostolidou, MD,1 and Eleni Peitsidou, MD1

1Third University Department of Neurology,Thessaloniki, Greece

2Department of Public Health, University of Oxford,Oxford, United Kingdom

Abstract: Various instruments with good psychometricproperties have been developed for the assessment ofhealth-related quality of life (HRQoL) in Parkinson’s dis-ease, (PD); however, in everyday practice a brief question-naire is needed for quick screening of patients. We presentthe process of development and validation of the Greekversion of PD questionnaire-8 (PDQ-8GrV), which is an8-item scale derived from a well-known measure for the

*Correspondence to: Dr. Zoe Katsarou, 3 Ipsilantou St, GR 55337Thessaloniki, Greece. E-mail: [email protected]

Received 30 January 2003; Revised 4 July 2003, 26 August 2003;Accepted 9 September 2003

DOI 10.1002/mds.10678

308 Z. KATSAROU ET AL.


evaluation of HRQoL in PD, the PD questionnaire (PDQ-39). PDQ-8 GrV was applied to 228 nondemented Greek PDpatients. Data from PDQ-39 were also collected from thesepatients for comparisons between the total scores of the twoscales. Detailed statistical analysis showed that PD-8GrV haspsychometric properties analogous to its parent question-naire. © 2004 Movement Disorder Society

Key words: Parkinson’s disease; health-related quality oflife; PDQ-8; PDQ-39; depression

Patients with Parkinson’s disease (PD) are particularlyvulnerable to deterioration of quality of life due to thesignificant motor disability imposed on them by theircondition; thus, assessment of their health-related qualityof life (HRQoL) is of paramount importance.1–4 Variousinstruments have been developed for the assessment ofHRQoL in PD patients5–7; however, in everyday practicea short form questionnaire is needed for quick screeningof patients.

The PD questionnaire (PDQ-39),8 a well-validatedquestionnaire, originally English, has been translatedinto Greek (designated PDQ-39GrV) and validated in apopulation of Greek PD patients.9 A short form of theEnglish PDQ-39, the PDQ-8, has been produced bymeans of detailed statistical analysis and was validated inEnglish PD patients.8 We present the process of devel-opment and validation of the Greek version of PDQ-8.


Scale Formation

The original PDQ-39 consists of 39 items grouped ineight scales: (1) mobility; (2) activities of daily living;(3) emotional well being; (4) stigma; (5) social support;(6) cognitions; (7) communication; and (8) bodily dis-comfort. This questionnaire also yields a total score,known as the PDQ-39 Summary Index (PDQ-39 SI),which ranges from 0 to 100. The eight PDQ-8 items werederived from the original scale by means of item corre-lation. Within each of the eight scales, the item that mosthighly correlated to the scale was selected. These wereitems 7, 12, 17, 25, 27, 31, 35, and 37. Each item isscored from 0 (never or not at all) to 4 (always or can notdo at all). A total score known as the PDQ-8 SummaryIndex (PDQ-8 SI) was calculated as follows:

Sum of scores of each question 100

4(max.score per question) 8

The PDQ-8 SI ranges also from 0 (normal) to 100(worst disability).8

The same procedure was applied to the Greek versionof PDQ-39 and because the above items had high corre-

lation coefficients comparable to those of the Englishscale, they were also selected to comprise the GreekPDQ-8 scale, designated the PDQ-8GrV.

Patient Selection and Scale Application

The study was carried out in the Outpatient Unit forParkinson’s Disease and other Movement Disorders ofthe 2nd and 3rd University departments of Neurology inThessaloniki, Greece. In total, 228 nondemented (MiniMental State Examination Score �24) Greek patients(131 men, 97 women) with idiopathic PD and with amean educational level of 8.6 � 2.6 years were selectedfor the study. Diagnosis of idiopathic PD was based onthe criteria proposed by Hughes and colleagues.10 Allpatients were classified in stages according to the mod-ified Hoehn and Yahr scale (H&Y).11 Patients in Stage 5were not included because of cognitive impairment.They were evaluated clinically by means of the motorscore (Part III) of the Unified Parkinson’s Disease RatingScale (UPDRS)11 and the Schwab and England Activitiesof Daily Living Scale (ADL).11 Motor disability wasevaluated during each patients’ optimal response to treat-ment (on phase) as well as during the wearing off oflevodopa (L-dopa) effect (off phase). PD patients werealso assessed for depression by the Greek version of theBeck Depression Inventory (BDI).12 Their individualcharacteristics are shown in Table 1.

PD patients were asked to read the PDQ-39GrV at theirown speed and check the most appropriate answer. Firstthe PDQ-39GrV SI was calculated. Then the eight chosenitems of the PDQ-8GrV were subtracted from the scaleand the PDQ-8GrV SI score was also calculated.

In total, 91 stable PD patients who were willing to visitthe hospital 5 days later were asked to repeat the PDQ-8GrV testing, and 81 patients also completed a genericinstrument for HRQoL, the Short Form-36 Health Sur-vey (SF-36).13

TABLE 1. Mean scores of clinical characteristics of PD for228 patients

Mean SD Minimum Maximum

Age (yr) 59.3 8.7 37 79Duration (yr) 6.7 4.3 1 20Stage-off 2.2 0.6 1 4ADL

On 84.7 9.9 50 100Off 73.1 16.2 30 100

UPDRSOn 17.5 8.6 4 40Off 26.5 13.1 4 65

BDI 8.1 6.1 1 25

ADL, activities of daily living; UPDRS, Unified Parkinson’s DiseaseRating Scale; BDI, Beck Depression Inventory.

HRQOL ASSESSMENT IN PD 309


The study received institutional ethical approval andall patients gave informed consent.


Student’s t test was employed for comparisons ofmean PDQ-39GrV SI and PDQ-8GrV SI . Mean PDQ-8GrV

SI scores in different stages of the disease were com-pared by means of the Kruskal-Wallis test. Correlationbetween descriptive data such as age, duration of thedisease, scores of ADL, UPDRS, BDI, and the PDQ-8GrV SI, was calculated by means of the Spearman’s rhocorrelation coefficient, to determine the criterion validityof the PDQ-8GrV scale. Cronbach’s � coefficient wasused to establish the internal consistency of the scale.Intraclass correlation coefficients (ICC) were calculatedfor comparisons between PDQ-39GrV PDQ-8GrV SIscores and PDQ-8GrV test–retest findings. Correlationbetween PDQ-8GrV and SF-36 scales was determinedusing Pearson’s correlation coefficient.14,15 Calculationswere carried out using SPSS v. 10 for Windows (SAS,Chicago, IL).

RESULTS

All PD patients responded to the questionnaire withease. There were no missing data (100% response rate).Reliability analysis for the PDQ-8GrV scale yielded asignificant � coefficient of 0.72, according to Nunally’scriterion.16 Test–retest PDQ-8GrV SI ICC was 0.72 (95%confidence interval [CI] � 0.60–0.80, P � 0.0001).

The mean PDQ-8GrV SI and PDQ-39GrV SI, which arepresented in Table 2, were almost identical. They werealso highly correlated, with an ICC of 0.90 (95% CI �0.87–0.92, P � 0.0001). To compare the spread of thetwo distributions, the 25th, 50th, and 75th percentile SIscores were calculated for both scales . These percentileSI scores were 12.50, 21.88, and 37.50, respectively, forthe PDQ-8GrV and 13.58, 23.69, and 33.79 for the PDQ-39GrV, respectively.

Correlation between PDQ-8GrV SI and SF-36 scalesshowed significant negative Pearson’s r correlation co-efficients in all scales except the role physical. As ex-pected, the highest correlation was observed betweenPDQ-8GrV and those SF-36 dimensions that involve

physical disability, pain, and energy/vitality.(r ��0.495, �0.419, and �0.464 , respectively, P � 0.000).

Correlation between PDQ-8GrV SI score and variouspertinent parameters of the disease proved significant forduration, UPDRS, ADL, and BDI scores (Table 3). Ageand gender correlations were not significant. Comparisonof PDQ-8GrV and PDQ-39GrV SI scores at various stagesof the disease showed significant differences betweengroups, with PD patients at advanced stages of the dis-ease yielding higher scores in both scales (Table 4).

DISCUSSION

The Greek version of PDQ-8 was applied to a largegroup of Greek PD patients with mild to moderate dis-ability, and proved to be easy to administer, reliable, andvalid. Its internal consistency, as determined by Cron-bach’s � coefficient, was very good.16 Additional supportfor the reliability of PDQ-8GrV was provided by thesignificant correlation of test–retest data. The criterionvalidity of a scale is defined by the extent of agreementof the scale studied with a gold standard.17 In our study,because a gold standard, a disease-specific scale forHRQoL assessment in PD, is lacking, we used the parentPDQ-39 scale as a gold standard. Mean SI scores of thetwo scales were almost identical, showing that the shortquestionnaire could mimic the overall properties of thelarge one. In addition, PDQ-8GrV correlated significantlywith the well-established generic HRQoL instrument, theSF-36.

TABLE 2. Scores for the PDQ-39GRV and PDQ-8GRV Summary Indexfor 228 patients

Mean SD Minimum Maximum 95% CI

PDQ-39GrV SI 25.29 14.51 0.52 71.25 23.40–27.19PDQ-8GrV SI 25.86 16.98 0.00 78.12 23.64–28.07

PDQ, Parkinson’s Disease Questionnaire, SI, summary index; CI, confidence interval.

TABLE 3. Spearman’s correlations between PDQ-8GrV

Summary Index score and various parameters of Parkinson’sdisease for 228 patients

Clinical parameter Spearman’s rho Significance

Age �0.106 0.112Disease duration 0.269 �0.0001UPDRS

Off 0.510 �0.0001On 0.493 �0.0001

ADLOff �0.528 �0.0001On �0.457 �0.0001

BDI 0.577 �0.0001

ADL, activities of daily living; UPDRS, Unified Parkinson’s DiseaseRating Scale; BDI, Beck Depression Inventory.

310 Z. KATSAROU ET AL.


Quality of life issues in Parkinson’s disease are linkedclosely to motor disability; therefore, a comparison of PDQ-8GrV and the pertinent clinical parameters of the disease wasnecessary to prove construct validity.17 A comparison ofmean scores of PDQ-8GrV scales at different stages of thedisease showed that significantly higher scores were ob-tained in cases at more advanced stages.

In addition, the PDQ-8GrV correlated to a standardizedmeasure of motor disability (the motor score of theUPDRS), and to a measure of dependence (the Schwab-England ADL score), in both on and off phases. This isin agreement with the original PDQ-8 validation study,which showed a tendency for poorer PDQ-8 scores to beassociated with severe symptoms of the disease.8 Depres-sion is quite frequent in PD patients, exacerbating thedistress caused by disability18 and it has been linkedconsistently to poor HRQoL.3,4 Depression as measuredby the BDI was found to correlate well with PDQ-8GrV

SI score.Clinical assessment of PD patients complemented by

reliable disease-specific HRQoL measurements can pro-vide a more accurate evaluation of both the course ofdisease and the clinical response to drug treatment. Thisis particularly important for the development of newmedications. Because large clinical trials for drug effi-cacy may be multinational, there is a pressing need fordevelopment of reliable HRQoL questionnaires, whichcan be used cross-culturally or cross-nationally.11,19 Ourstudy resulted in the generation of a validated Greekversion of PDQ-8, which was proved to have disease-specific psychometric properties analogous to the origi-nal British version.

PDQ-8GrV, as any other short form measure ofHRQoL, can be used as a general indicator of HRQoLassessment only. Although PDQ-8GrV yields a total scoreequivalent to the score derived by its parent instrumentthe PDQ-39GrV, it cannot provide measures of differentcomponents of the HRQoL concept. Detailed studies inthis field require longer questionnaires, e.g., PDQ-39, incombination with a generic instrument such as SF-36 orthe Sickness Impact Profile. The main advantage ofPDQ-8GrV is that it is quick and easy to administer.

Larger questionnaires pose a problem for patients withsevere disability, who either disregard or cannot com-plete them. This short instrument was accepted well byall patients studied, and no missing data were reported.Therefore, its diminished information capacity is an ac-ceptable trade-off with practicality for screening studiesor evaluations of elderly patients, as well as for those toofrail to respond to PDQ-39. In conclusion PDQ-8GrV is apractical and informative instrument with acceptablepsychometric properties for HRQoL screening of PDpatients in everyday clinical practice and research.

Acknowledgments: We thank Professor G. Kiosseoglou,PhD, for statistical advice.

REFERENCES

1. Levin BE, Weiner WJ. Psychosocial aspects. In: Koller WC,editor. Handbook of Parkinson’s disease. New York: Marcel Dek-ker; 1992:579–588.

2. Martinez-Martin P. An introduction to the concept of “quality oflife” in Parkinson’s disease. J Neurol 1998;245(Suppl.):2–6.

3. Schrag A, Jahanshahi M, Quinn N. How does Parkinson’s diseaseaffect quality of life? A comparison with quality of life in thegeneral population. Mov Disord 2000;15:1112–1118.

4. Global Parkinson’s Disease Survey Steering Committee. Factorsimpacting on quality of life in Parkinson’s disease: results from aninternational survey. Mov Disord 2002;17:60–67.

5. Longstreth WT Jr, Nelson L, Linde M, Munoz D. Utility of theSickness Impact Profile in Parkinson’s disease. J Geriatr Psychia-try Neurol 1992;5:142–148.

6. Damiano AM, Snyder C, Strausser B, Willian MK. A review ofhealth-related quality of life concepts and measures for Parkinson’sdisease. Qual Life Res 1999;8:235–243.

7. Marinus J, Ramaker C, van Hilten JJ, Stiggelbout AM. Healthrelated quality of life in Parkinson’s disease: a systematic review ofdisease specific instruments. J Neurol Neurosurg Psychiatry 2002;72:241–248.

8. Jenkinson C, Fitzpatrick R, PetoV. The Parkinson’s Disease Ques-tionnaire. User manual for the PDQ-39, PDQ-8 and PDQ SummaryIndex. Oxford: Health Services Research Unit; 1998.

9. Katsarou Z, Bostantjopoulou S, Peto V, Alevriadou A, Kiosseo-glou G. Quality of life in Parkinson’s disease: Greek translationand validation of the Parkinson’s Disease Questionnaire (PDQ-39).Qual Life Res 2001;10:159–163.

10. Hughes, AJ, Daniel SE, Kilford I, Lees AJ. Accuracy of clinicaldiagnosis of idiopathic Parkinson’s disease: a clinicopathologicalstudy of 100 cases. J Neurol Neurosurg Psychiatry 1992;55:181–184.

11. Fahn S, Elton RL, and members of the UPDRS DevelopmentCommittee. Unified Parkinson’s Disease Rating Scale. In: Fahn S,Marsden CD, Goldstein M, editors. Recent developments in Par-

TABLE 4. Mean scores, standard deviations and significance of the PDQ-39GrV and PDQ-8GrV

Summary Index scores in all stages of PD

SI Score

Stage

PI (n � 22) II (n � 103) III (n � 61) IV (n � 42)

PDQ-39GrV 14.95 � 12.51 24.37 � 15.21 29.37 � 13.81 36.14 � 13.10 �0.0001PDQ-8GrV 14.20 � 12.50 24.90 � 18.10 28.38 � 16.71 25.86 � 16.98 �0.0001

PDQ, Parkinson’s Disease Questionnaire, SI, summary index.

HRQOL ASSESSMENT IN PD 311


kinson’s disease, II. Florham Park: MacMillan Healthcare Infor-mation; 1987. p 293–304.

12. Donias S, Demertzis I. [Standardized assessment of depression bymeans of the Beck Depression Inventory.] Proceedings of the 5thGreek Conference for Neurology and Psychiatry 1983;1:486–492.

13. Ware JE, Sherbourne CD. The MOS 36-Item Short-Form HealthSurvey (SF-36) 1: Conceptual framework and item selection. MedCare 1992;30:473–483.

14. Cronbach L. Coefficient � and the internal structure of tests.Psychometrika 1951:16:297–334.

15. Minium EW, King BM, Bear G. Statistical reasoning in psychol-ogy and education, 3rd ed. New York: Wiley; 1993.

16. Nunally JC. Psychometric theory, 2nd ed. New York: McGraw-Hill; 1978.

17. Spector WD. Functional disability scales. In: Spilker B, editor.Quality of life and pharmacoeconomics in clinical trials, 2nd ed.New York: Lippincott-Raven; 1996. p 133–143.

18. Poewe W, Luginger E. Depression in Parkinson’s disease. Imped-iments to recognition and treatment options. Neurology 1999;52(Suppl.):2–6.

19. Wasielewski PG, Koller WC. Quality of life and Parkinson’sdisease: the CR FIRST study. J Neurol 1998;245(Suppl.):28–30.

Health-Related Quality of LifeEvaluation by Proxy in

Parkinson’s Disease: ApproachUsing PDQ-8 and EuroQol-5D

Pablo Martınez-Martın, MD, PhD,1*Julian Benito-Leon, MD, PhD,2

Fernando Alonso, MD,3 Maria Jose Catalan, MD,4

Margarita Pondal, MD, PhD,5

and Ivana Zamarbide, MD3

1Neuroepidemiology Unit, National Centre for Epidemiology,Carlos III Institute of Public Health, Madrid, Spain;

2Neurology Department, Mostoles Hospital, Madrid, Spain;3Neurology Department, Pamplona University Teaching

Clinic, Navarre, Spain; 4Neurology Department, San CarlosUniversity Teaching Hospital, Madrid, Spain; 5NeurologyDepartment, Severo Ochoa Hospital (Leganes, Madrid),

Madrid, Spain

Abstract: Patient- and caregiver-based scores were com-pared and agreement levels ascertained to determine thereliability of proxy evaluation of Parkinson’s Disease (PD)

patients’ health-related quality of life (HRQoL) using theEuroQoL and PD questionnaire (PDQ)-8. Of 72 patient-caregiver pairs, 64 (88.88%) returned the questionnaires.The degree of agreement varied for individual dimensions.Proxy evaluation of PD patients’ HRQoL showed limita-tions mainly with assessments using the EuroQoL and es-pecially in patients with severe disease and depression.© 2004 Movement Disorder Society

Key words: Parkinson’s disease; health-related quality oflife; proxy evaluation; EuroQoL; PDQ-8

Health-related quality of life (HRQoL) assessment isgaining acceptance in the field of clinical research, aswell as in public health and health policy, due to theassociation between perceived state of health and de-mand for resources, prediction of risks and mortality, andother relevant applications for management and deci-sion-making in health.1,2

In Parkinson’s disease (PD), outcomes have been eval-uated traditionally by means of clinical rating scales.3–5

Recently, there has been a rapid growth in the use ofHRQoL measures, both generic and specific, in clinicaltrials and research studies addressing the impact ofPD.6–13

In certain circumstances, e.g., when cognitive impair-ment or physical disability impedes patient self-evalua-tion of health, surrogate assessment may be obtainedfrom a close contact. Nevertheless, the reliability of thistype of measurement in different settings remains to bedetermined. Although information from proxies on ob-jective aspects such as functional status may be accept-able,14–17 uncertainty surrounds the quality of proxy as-sessment in more subjective or complex aspects ofhealth, such as HRQoL.18

We explored the reliability of proxy evaluation of PDpatients’ HRQoL using both generic (EuroQoL) andspecific questionnaires (PD questionnaire [PDQ]-8).

PATIENTS AND METHODS

Our objective was to test the following working hy-potheses: (1) the HRQoL of PD patients may be assessedreliably by their caregivers; (2) the agreement betweenpatient and proxy will be satisfactory solely for observa-tional functioning; and (3) the agreement will be betterwith the PD-specific HRQoL instrument.

The design was for an observational, cross-sectional,one-point-in-time evaluation, multicenter study. Eligiblesubjects were consecutive patients with clinical diagno-sis of PD and a stable caregiver.

Patients and Caregivers

Patients were included in the study on condition thatthey were literate (able to read, understand, and answer

*Correspondence to: Dr. P. Martınez-Martın, Unidad de Neuroepi-demiologıa, Centro Nacional de Epidemiologıa, Instituto de SaludCarlos III, C/Sinesio Delgado 6, 28029-Madrid, Spain.E-mail: [email protected]

Received 19 February 2003; Revised 6 August 2003; Accepted 21August 2003

DOI 10.1002/mds.10656

312 P. MARTINEZ-MARTIN ET AL.


the questionnaires), had a stable and literate caregiver,and presented with no co-morbidity that might in anyway impede HRQoL assessment. Appropriate verbal in-structions were given to patients and caregivers. Addi-tionally, written instructions were suitably included onall forms, conveniently enclosed in stamped envelopes,and designed to be completed independently during thevisit returned by mail within 8 days of the clinicalevaluation.

Of 72 consecutive patient-caregiver pairs, 64(88.88%) returned the questionnaire. There were missingor faulty data in some questionnaires from both patients(n � 2) and caregivers (n � 5), with the result thatcomplete evaluation was obtained ultimately for 57 of 64patient-caregiver pairs (89.06%).

Evaluation by Neurologists

Neurologists informed patients and caregivers aboutthe study and made their evaluation at a regular fol-low-up visit in clinics (4 neurologists, 65% of patients)or during a visit to a community PD Association (1neurologist, 35% of patients). They applied the UnifiedPD Rating Scale (UPDRS-3.0, including the modifiedHoehn and Yahr Staging [HY] and Schwab and EnglandScale [SES] but excluding Section 4-Complications).5

Additional measures were the Motor Complications sec-tion of the Intermediate Scale for Assessment of PD(ISAPD)19 and the Pfeiffer’s Short Portable Mental Sta-tus Questionnaire (SPMSQ).20 Neurologists also helpedcaregivers complete the scale of Quality of Life of Care-Givers (SQLC)21 in a private setting, with no patientspresent.

Patient Self-Evaluation

Patients completed the validated Spanish versions ofthe EuroQoL (EQ-5D),9,22 PDQ-8,10,13 and the HospitalAnxiety and Depression Scale (HADS).23–25

Evaluation by Caregivers

Caregivers provided proxy evaluations of patients’HRQoL using the EQ-5D (except for the visual analoguescale [VAS] rating, “health state today”) and the PDQ-8.In addition, they completed the SQLC21 during the visitto the neurologist.

HRQoL Instruments

The EQ-5D9,22,26 is a preference-based HRQoL mea-sure. The descriptive part comprises five dimensionswith three possible levels of severity, which generate 243health state profiles. Using the EQ-5D VAS and “timetrade-off methods” (EQ-5D TTO), a preference value foreach health state generated by the EQ-5D profile may be

calculated.22,26 These values range from 1.0 to �1.0,where 1.0 is assigned to “perfect health state” and 0 todeath. Other components are the visual analogue scale(“health state today”, from 100, best imaginable to 0,worst imaginable) and a question about evolution ofsubjects’ state of health in the previous year.

The PDQ-8 is a short-form questionnaire composed ofeight items, each of which represents one domain of thePDQ-39.10,11 Scores for each item run from 0 (no prob-lem) to 4 (continuous problem/unable to do it) and aPDQ-8 Summary Index (PDQ-8 SI) may be calculated.

Glozman’s SQLC quantifies the impact of disease oncaregiver activities.21,27–29 It consists of 16 items, with avariable ranking of scores. Four degrees of caregiverdistress, ranging from 141 to 149 points (normal) to lessthan 85 (severe strain) may be determined.

Data Analysis

Descriptive statistics (mean, standard deviation, range,95% confidence [CI] interval) were obtained as required.Distribution of patient versus caregiver ratings for di-mensions were compared using the Wilcoxon test.EQ-5D Index values (EQ-5D VAS and EQ-5D TTO) andthe PDQ-8 SI were compared using the paired t test.

Cronbach’s � statistic was applied by group of ratersto determine the internal consistency of the scores.Agreement beyond chance for dimensions comprisingthe EQ-5D and the PDQ-8 was analyzed using weighted� with quadratic weights. Level of agreement for EQ-5Dpreference values and the PDQ-8 SI was determinedusing the intraclass correlation coefficient (ICC). Asso-ciation between measures was tested using Spearman’srank correlation coefficient.

RESULTS

Mean age of patients was 67.09 � 7.88 years. Severityof disease was mild (HY stages 1 and 2) in 29 (45.31%),moderate (HY stages 2.5 and 3) in 27 (42.18%), andsevere (HY stages 4 and 5) in 8 patients (12.50%).Patients had been followed up for 5.70 � 5.04 years byneurologists with competence in the field of movementdisorders. Of 64 patients, 27 (41.54%) were men, 51(81%) were married, and 11 (17.5%) were widows. Afull breakdown of clinical characteristics of the studypopulation is given in Table 1. According to the SPMSQ,2 patients presented moderate cognitive impairment, butthey were not considered demented by their neurologistsand caregivers. Mild cognitive impairment could be saidto be present in 3 additional patients.

Caregiver age (mean � SD, range) was 58.84 � 14.90years (18–89), and 21 (33%) were men. Caregivers weremainly spouses (73%) and sons or daughters (22%) look-

QOL EVALUATION BY PROXY IN PD 313


ing after patients on a permanent (86%) or alternatingscheduled basis (12.5%).

Table 2 shows the descriptive statistics of scores as-signed by patients and caregivers to the EQ-5D compo-nents. The only significant statistical difference (P �0.011, Wilcoxon test) was found for the dimension“usual activities”, which was scored higher (worse) bypatients. The difference between patient and proxyEQ-5D Index proved statistically significant for bothmethods (EQ-5D VAS TTO; paired t test, P � 0.028 and0.036, respectively).

There was no statistical difference between patient andcaregiver scoring for any domain of the PDQ-8 or for thePDQ-8 SI (Table 3).

Cronbach’s � for ratings generated by patients andcaregivers proved almost identical (EQ-5D � � 0.708

and 0.709 for VAS and TTO values, and PDQ-8 � �0.842 and 0.843, respectively). In the scoring ofEQ-5D dimensions (Table 4), agreement between pa-tients and caregivers was not homogeneous, in thatwhereas two domains (“mobility” and “pain”), regis-tered � values of over 0.60 (substantial agreement),the remaining three items yielded � values of 0.48 to0.58 (moderate agreement).30 Furthermore, substantialagreement between raters was observed for the EQ-5DVAS and TTO indexes (ICC; 0.80 and 0.77, respec-tively). With regard to the question concerning sub-jects’ state of the health in the previous year, there wassignificant correlation between patients and caregivers(r � 0.61; P � 0.0001).

As far as the PDQ-8 was concerned, dimensions otherthan “concentration” (0.56) obtained � values of 0.61 to

TABLE 1. Characteristics of patients

Characteristic Mean SD Min. Max. 95% CI

Age at onset (yr) 56.33 9.34 39 75 —Age at evaluation (yr) 67.09 7.88 45 79 —Duration of disease (yr) 10.47 6.16 1 30 —Follow-up (yr) 5.70 5.04 0 21 —HY score 2.60 0.74 1 5 2.41, 2.78SES score 69.68 21.23 0 100 64.38, 75.00UPDRS score

Section 1 (Mental) 3.57 2.46 0 9 2.96, 4.19Section 2 (ADL) 16.29 8.50 3 45 14.17, 18.42Section 3 (Motor) 18.10 10.51 5 56 15.05, 21.15

ISAPD scoreComplications 6.11 6.65 0 23 4.44, 7.77

SPMSQ score 9.51 1.16 5 10 9.22, 9.80

Total n � 64.Min., minimum; Max., maximum; 95% CI, 95% confidence interval; HY, Hoehn and Yahr

staging; SES, Schwab and England Scale; UPDRS, Unified Parkinson’s Disease Rating Scale;ISAPD, Intermediate Scale for Assessment of Parkinson’s Disease; SPMSQ, Pfeiffer’s ShortPortable Mental Status Questionnaire.

TABLE 2. Descriptive statistics of the EuroQoL scorings

Items

Scoring by patients(n � 64)

Scoring by caregivers(n � 59)

Mean(SD) 95% CI

Mean(SD) 95% CI

Mobility 1.67 (0.50) 1.54, 1.80 1.62 (0.55) 1.48, 1.77Self-care 1.54 (0.53) 1.41, 1.68 1.45 (0.53) 1.31, 1.60Usual activitiesa 1.82 (0.60) 1.67, 1.98 1.64 (0.61) 1.48, 1.80Pain 1.73 (0.57) 1.60, 1.87 1.72 (0.58) 1.57, 1.88Anxiety/depression 1.61 (0.55) 1.47, 1.74 1.54 (0.65) 1.37, 1.71EQ-5D-VASb 0.58 (0.21) 0.53, 0.63 0.62 (0.21) 0.56, 0.68EQ-5D-TTOc 0.60 (0.28) 0.53, 0.67 0.64 (0.30) 0.56, 0.72Health in previous yr 2.36 (0.67) 2.20, 2.52 2.35 (0.58) 2.20, 2.51Health state today (VAS) 55.81 (21.65) 50.35, 61.26 — —

Significant difference only for aP � 0.011 (Wilcoxon test), bP � 0.028 and cP � 0.036 (paired t test).95% CI, 95% confidence interval; EQ-5D-VAS, health state value of EuroQoL by visual analogue scale

method; EQ-5D-TTO, health state value of EuroQoL by time trade-off method; health state today (VAS),health state today according to EuroQoL visual analogue scale (“thermometer”).



0.80, indicative of substantial agreement. Agreement forthe PDQ-8 SI values was similarly high (ICC � 0.83).

Correlation between patient-rated PDQ-8 SI andEQ-5D measures yielded the following coefficients: withEQ-5D VAS, r � �0.50 (P � 0.0001), and with EQ-5DTTO, r � �0.47 (P � 0.001).

Agreement (ICC) between index and proxy evalua-tions with the PDQ-8 SI proved lower at advanced stagesof PD (HY � 3), when patients were dependent (SES �50), had motor complications (dyskinesias and fluctua-tions), or were depressed (HADS-Depression � 11).Equivalent analysis with the EQ-5D showed a rise inagreement when depression was present (Table 5). Al-though there was a significant difference in SQLC scores

for patients with mild to moderate (HY � 3; SQLC,123.55 � 16.66) versus severe PD (HY � 3; SQLC,100.34 � 19.26) (P � 0.0001), patient-caregiver agree-ment in regard to patients’ HRQoL was scarcely modi-fied by level of caregiver burden (Table 5).

DISCUSSION

The EQ-5D and PDQ-8 were chosen for the presentstudy because of their simplicity and usefulness in thecontext of PD, and the proven validity of the Spanishversions.10,12,13,22,31,32 Of 72 patient-caregiver pairs, 8(11.12%) failed to return the questionnaire, and 7 of 64(10.93%) that were returned contained faulty or wrong data

TABLE 3. Descriptive statistics of the PDQ-8 scorings

Item

Scoring by patients (n � 64) Scoring by caregivers (n � 63)

Mean (SD) 95% CI Mean (SD) 95% CI

1. Getting around in public 1.67 (1.37) 1.33, 2.01 1.69 (1.25) 1.38, 2.012. Dressing 1.46 (1.34) 1.12, 1.80 1.42 (1.31) 1.09, 1.763. Feeling of depression 1.38 (1.15) 1.10, 1.67 1.46 (1.16) 1.16, 1.754. Embarrassment in public 1.33 (1.36) 1.00, 1.67 1.17 (1.29) 0.85, 1.505. Problems with relationships 0.84 (1.05) 0.58, 1.10 0.85 (1.06) 0.60, 1.126. Concentration 1.27 (1.25) 1.00, 1.55 1.36 (1.18) 1.06, 1.667. Communication with people 1.20 (1.21) 0.90, 1.50 1.11 (1.16) 0.81, 1.408. Painful cramps or spasms 1.60 (1.13) 1.32, 1.88 1.62 (1.12) 1.33, 1.90PDQ-8 Summary Index 33.17 (20.94) 28.00, 38.36 33.77 (20.25) 28.67, 38.88

There were no significant differences between scorings.PDQ-8, Parkinson’s Disease Questionnaire 8 items; 95% CI, 95% confidence interval.

TABLE 4. EQ-5D and PDQ-8 agreement between patients andcaregivers

Item Prevalence* Kappa ICC

EuroQoL (n � 59)Mobility 65.62 0.74 —Self-care 53.12 0.48 —Usual activities 71.88 0.50 —Pain 67.19 0.65 —Anxiety/depression 57.81 0.58 —

EQ-5D-VAS — 0.80EQ-5D-TTO — 0.77PDQ-8 (n � 63)

1. Getting around in public 72.31 0.78 —2. Dressing 64.62 0.80 —3. Feeling of depression 69.23 0.64 —4. Embarrassment in public 60.00 0.71 —5. Problems with relationships 44.62 0.66 —6. Concentration 69.23 0.56 —7. Communication with people 60.00 0.61 —8. Painful cramps or spasms 76.93 0.73 —

PDQ-8 Summary Index — 0.83

*Expressed in percentage (%), patients scoring �1 (EQ-5D) and �0 (PDQ-8)in each dimension.

ICC, intraclass correlation coefficient; EQ-5D-VAS, health state value of Eu-roQoL by visual analogue scale method; EQ-5D-TTO, health state value ofEuroQoL by time trade-off method.



in questionnaires that, for the most part, were completed bycaregivers, a pattern reported in other studies.33,34

Differences between patient and proxy ratings ofEQ-5D dimensions were not significant, except for“usual activities,” which was scored higher by patientsthan by caregivers (P � 0.011; Table 2). Concordance ofscores proved satisfactory (�, 0.65 and 0.74) only for twoitems (“pain” and “mobility”; Table 4). EQ-5D prefer-ence values (VAS and TTO) displayed a slightly signif-icant difference between patients and caregivers (P �0.028 and 0.036, respectively), but substantial agreement(ICC � 0.80 and 0.77, respectively). In the PDQ-8 wasconcerned, there was no statistically significant differ-ence between index and proxy ratings for dimensions orfor the PDQ-8 SI (Table 3). Seven PDQ-8 dimensionsand the summary index yielded coefficients indicative ofsubstantial agreement (Table 4).

Cronbach � values pointed to adequate internal con-sistency in the case of both instruments whether appliedby patients or caregivers,35 with the values obtainedbeing higher with the PDQ-8 (0.84) than with the EQ-5D(0.70). As a whole, findings indicate that proxy evalua-tion of patient HRQoL was more reliable with the spe-cific (PDQ-8) than with the generic questionnaire(EQ-5D).

Earlier studies found a trend among lay proxies tooverestimate patients’ disability, whereas patients them-selves tended to overestimate their functionalstate.14,34,36–38 Findings, however, are not homogeneous.Proxies of subjects under the age of 65 years may evincea tendency to underestimate patients’ disability.39 Somestudies reported a positive association between closeness

of the relationship with the patient and validity of theproxy evaluation,40,41 but Shaw and colleagues34 re-ported a finding that was quite the contrary. Hence, it hasto be said that 25% of our patients were younger than 65years and 95% of their caregivers were very close rela-tives (spouse or offspring), and these are circumstancesthat, in the light of the above-mentioned studies, mighthave conditioned our results.

As measured by the EQ-5D in the present study, someobservable functional aspects (“self-care” and “usual ac-tivities”) showed a lower degree of concordance than didmore intimate domains (“anxiety/depression”; see Table4). Results with the PDQ-8 were indicative of substantialagreement for observable functioning (“getting around inpublic”, “dressing”) as well as for more inner domains(“embarrassment in public”). It is accepted generally thata higher level of agreement between proxies and indexsubjects is obtained for observable aspects, such as func-tional ability for basic activities of daily living,15,40,41

than is obtained for more complex or private aspects (forinstance, those causing stigma or shame).18,34 The resultsof the present study fit only partially with this statement,because the level of agreement was not distributed sys-tematically according to the objectivity or intimacy ofthe domains.

Depression, disease severity, and disability impair theHRQoL of PD patients6,7,42,43 and may equally increasecaregiver burden.44–46 On the other hand, patients’ phys-ical functioning, mood, or cognitive state, and caregiv-ers’ psychological distress and burden may influence thelevel of agreement.15,47–49 This observation would sug-gest that in the advanced stages, when patient disability

TABLE 5. Agreement between patient and caregiver for HRQoL values inrelation to disease-derived variables

Assessment EuroQoL-VAS EuroQoL-TTO PDQ-8 SI

Hoehn and Yahr Stage�3 0.72 0.73 0.76�3 0.57 0.67 0.70

Schwab and England Scale�50 0.78 0.70 0.85�50 0.70 0.76 0.53

Motor fluctuationsNo 0.84 0.77 0.83Yes 0.69 0.73 0.79

HADS-Depression�11 0.74 0.68 0.85�11 0.85 0.85 0.68

Glozman’s SQOLC�99 0.82 0.78 0.81�99 0.76 0.80 0.82

Agreement determined by intraclass correlation coefficient. EuroQoL health state values byvisual analogue scale (VAS) and by time trade-off (TTO) methods. PDQ-8 SI, Parkinson’sDisease Questionnaire 8 items, Summary Index; HADS, Hospital Anxiety and DepressionScale; Glozman’s SQLC, Glozman’s Scale of Quality of Life of Caregivers.



becomes severe and caregivers are suffering from signif-icant stress, proxy evaluation of patient HRQoL may beless reliable than at the earlier stages. This is a relevantassumption because it is precisely in such severe disabilityscenarios where proxy evaluation may be really needed.

Our results (Table 5) only partially support this hy-pothesis. Agreement between patient and proxy re-sponses decreased at advanced stages of the disease(HY � 3 and with motor fluctuations). In cases wheredepression was present in patients, the level of agreementas to their HRQoL decreased when measured with thePDQ-8 but increased when measured with the EQ-5D, adiscrepancy difficult to explain and probably related tothe intrinsic characteristics of the instruments. Patientfunctional dependency and caregiver strain showed anirregular trend to decrease the level of agreement. Only 5patients (8.77%) showed mild to moderate cognitiveimpairment, the reason why the effect of this factor wasnot tested.

This study suffers from limitations linked to size andcharacteristics of the sample, yet a balanced representa-tion of disease severity adds consistency to the results. Inrelation to the objectives of this study, our conclusionsare: (1) global HRQoL of PD patients may be assessedacceptably by their caregivers; however, the degree ofagreement proved quite variable for the individual di-mensions making up the questionnaires; (2) there was noclear difference in the degree of agreement depending onobjectivity or intimacy of the assessed domains; (3)index/proxy agreement was better with the specific(PDQ-8) than with the generic instrument (EQ-5D).

Proxy evaluation of PD patients’ HRQoL showed lim-itations, mainly with assessments using the EQ-5D andespecially in patients with severe disease and depression.

Acknowledgements: We thank Dr. J. Alonso, (IMIM, Bar-celona, Spain) and A. Tobıas (National Center for Epidemiol-ogy, Madrid, Spain) for helpful comments and criticism of themanuscript.

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25. Marinus J, Leentjens AFG, Visser M, Stiggelbout AM, van Hilten JJ.Evaluation of the Hospital Anxiety and Depression Scale in patientswith Parkinson’s disease. Clin Neuropharmacol 2002;25:318–324.

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28. Martınez-Martın P, Guerrero MT, Frades B, Fontan C, GlozmanJM. Quality of life of caregivers: a new perspective on the conse-quences of Parkinson’s disease. Mov Disord 1998;13 (Suppl.):63.

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validity of disability questions for US Census 2000. Am J PublicHealth 2000;90:1297–1299.

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Parkinson’s Prevalence Estimatedby a State Registry

Daniel Strickland, PhD.1

and John M. Bertoni, MD, PhD2

1Epidemiology Section, Department of Preventive andSocietal Medicine, University of Nebraska Medical Center,

Omaha, Nebraska, USA2Department of Neurology, Creighton University College of

Medicine, Omaha, Nebraska, USA

Abstract: A solid understanding of the descriptive epidemi-ology of a disease is essential in etiologic investigations; thisincludes prevalence and incidence, as well as groups withinthe larger community who may have noticeably lower orhigher rates. We ascertained the usefulness of a non-tradi-tional registry in describing Parkinson’s disease (PD) pat-terns in a community. A passive surveillance PD registry inNebraska began data collection on 1 January 1997. Allphysicians were required to report PD diagnosis, pharma-cists reported new prescriptions of anti-PD drugs (PD caseswere confirmed later with the prescribing physician), andthere was a patient self-report mechanism. The overlap ofreporting by the sources allowed estimation of the numbernot reported by any source, using the statistical technique“capture–recapture.” As of January 2000, the Nebraska PDRegistry had reports of 5,062 PD patients. The number notreported by any Registry reporting source was calculated tobe 117, leading to an estimated total of 5,179 cases and aprevalence of 329.3 per 100,000 population. Tabulations ofage- and gender-specific prevalence rates, as well as county-level estimates, allow examination of areas of elevated orlowered prevalence. The combination of a passive surveil-lance system and capture–recapture technique presents auseful method for epidemiologic description, and more tra-ditional survey methods could benefit by including capture–recapture capability. © 2004 Movement Disorder Society

Key words: Parkinson’s disease; epidemiology; registry;capture–recapture

A solid understanding of the descriptive epidemiologyof a disease is essential before undertaking etiologic

Earlier results of this study were presented in part at the Society forEpidemiologic Research Annual Meeting, June 1999, Baltimore, MD,USA.

Daniel S. Strickland is currently at the Department of Research andEvaluation, Kaiser Permanente Southern California, Pasadena, CA,USA.

*Correspondence to: Daniel S. Strickland Department of Researchand Evaluation, Kaiser Permanente Southern California, 393 East Wal-nut St./991, Pasadena, CA, 91188. E-mail: [email protected]

Received 25 February 2003; Revised 28 June 2003; Accepted 22July 2003

DOI 10.1002/mds.10619

318 D.S. STRICKLAND AND J.M. BERTONI


investigations. Descriptive epidemiology embraces char-acteristics of prevalence, incidence, and groups withinthe larger community that may have noticeably lower orhigher rates. A typical initial approach is to examine vitalrecords for deaths over time and by location for thedisease in question. However, because Parkinson’s dis-ease (PD) is of long duration it is possible that PDpatients could die from unrelated causes and, thus, thosewith PD as the recorded cause of death would representonly a subgroup of PD patients.1,2

A second avenue could involve the aid of neurologistsin the community, to gather information on their currentpatient load (prevalence) and how many new patientsthey see annually (incidence). PD presents obstacles hereas well because patients can go to their primary physi-cian and be diagnosed and treated for their illness with-out seeing a neurologist. In the state of Nebraska, dis-tances in the sparsely populated areas of the state form anadditional barrier to visiting specialists, most of whompractice in a few larger communities.

A third possibility, a full-state survey for prevalence,would be logistically impractical, although in selectedareas estimates of prevalence could be made from exam-ination of samples of the population. At present, there areno known biological markers that would amenable tolaboratory testing and no screening instruments that aresufficiently reliable. Accurate diagnosis of PD thus re-quires a neurologist’s exam, and such an effort couldrapidly consume resources.

An alternative to the traditional epidemiologic ap-proaches comes from wildlife ecologists. Complete enu-meration is obviously impractical. It is practical to esti-mate the total population without complete sampling bya technique called capture–recapture.3 Although it isplainly impossible to count all fish in a pond, by catch-ing, tagging, and releasing from several fishing spots,while noting the number and identification, as it were, ofthe fish caught, one can collect several different samples(lists) and counting tags (duplicates). Thus, the fishermancould calculate the degree of undercounting and howmany fish are in the pond.

It is often necessary to consider problems with lack ofindependence among the reporting sources as well aspermit migration in and out. This is addressed typicallywith log-linear modeling.3,4

Animal ecologists argue that although complete cen-sus is a lofty ideal, it often is impractical both in logisticterms and in problems with missing data and refusals.5

Often duplicates in lists from multiple sources are dis-carded, but by instead using that overlap, capture–recap-ture techniques give the researcher information on themagnitude of underassessment in the reporting sources

by the degree of undercounting in the individual sources.Once an estimate of underassessment has been derivedfor each source, the total count becomes a much betterestimate of the actual population, in some instancessurpassing attempts at complete census.6

Although capture–recapture requires care in applica-tion and awareness of the assumptions, those assump-tions have been applied in human populations withenough validated success that they provide a workablecomplement to current methods.6–8 In this study, thistechnique is applied for the first time to estimating theprevalence of Parkinson’s disease (PD), a condition thatis made difficult to count by its non-fatal nature, itsdiagnosis and treatment by non-specialists, and the lackof a pathological test that might allow counting throughlabs.


The Nebraska Parkinson’s Disease Registry was es-tablished by state legislation in 1996, and data collectionbegan 1 January 1997. By December 1997, there were1,400 registrants, and by December 1999, 5,062 peoplewere in the Registry. Reporting to the Registry is re-quired of physicians and pharmacists, and a mechanismis provided for patients to self-report. Patients are madeaware of the Registry most frequently through Parkin-son’s support groups. The primary intent of the Registryis to provide a mechanism for identifying study subjectsfor PD research and for allowing descriptive epidemiol-ogy; clinical and diagnostic information is therefore min-imal. Collected information includes standard demo-graphics and identifying information: name, socialsecurity number, date and place of birth, gender, andaddress. This is essential for a registry but also allowsunique identification of subjects for capture–recapturecalculations. Illness-related information includes date ofdiagnosis, address when diagnosed, physician, and re-sponse to medication. Furthermore, the sources reportingthe patient are recorded.

Pharmacists are required to report when a patient isprescribed medication from a list of Parkinson’s medi-cations developed and updated by the Registry advisoryboard, which comprises the Registry manager, a neurol-ogist (J.M.B.), a pharmacist, support group leaders, thestate epidemiologist, and an academic epidemiologist(D.S.). A pharmacist report includes the patient’s name,the name of the prescribing physician, and the medica-tion prescribed. The Registry manager then contacts theprescribing physician to ascertain the diagnosis, becausesome medications prescribed for Parkinson’s may alsobe used for other conditions (e.g., Restless Leg Syn-drome). If the physician does not report on the patient

PD PREVALENCE 319


independently of the pharmacist’s report, it is recordedthat the patient was reported by the pharmacist alone. Afinal element of the registry includes an annual compar-ison of registrants with the Nebraska death certificates;records of deceased registrants are not retained.

The gathered data are stored with other registriesmaintained by the state (cancer and birth defects) andmanaged by the Nebraska Health and Human ServicesSystem. The Registry is open for use by any researcherafter submission and acceptance of a research proposal tothe advisory board.

Analytical Methods

These data resources above enable the application ofcapture–recapture method to the combined sets. Withidentifying information in hand, the degree of overlap ofall the datasets can be calculated, and one can thenestimate the number of PD patients who are not includedin any data resource, and hence an estimate of totalprevalence can be made. This technique is currentlyapplied in many settings to estimate prevalence of suchthings as opioid use,7 fetal alcohol syndrome preva-lence,8 and other public health problems where no singlecomprehensive data resource exists, such as registries forcancer.6

The basic calculation of capture–recapture is simpleand is similar to the traditional �2 calculation in that ituses contingency table marginals to calculate expectedcell values. For example, Table 1 outlines reporting fromtwo of the data sources in the Nebraska Parkinson’sDisease Registry (cells are labeled a–d by usualconvention).

To estimate the number expected in Cell a, for exam-ple, the marginal totals are multiplied and then dividedby the overall total. The calculation would thus be:

Expected a � � a � b� � a � c��/

�a � b � c � d�

Applying that same formula and using those data tocalculate the missing patients, i.e., those not reported byeither source the calculation would be:

a/�a � c� � �a � b�/N, or

3,740/�3,740 � 650� � �3,740 � 672�/N,

where N � � 3,740 � 650�

� 3,740 � 672��/3,740 � 5,178

Thus, the missing number in the table is 5,178 �3740 � 672 � 650 � 116.

Registry data were tabulated by reporting source (self,physician, and pharmacist) after first being stratified bythe variables of interest (see Table 2). Log-linear modelswere fitted (using STATA v.6; Stata Corp, College Sta-tion, TX) and the structural zero cells estimated. Thestructural zero expected numbers (i.e., those not reportedby any of the three sources) were added to the reportednumbers for an estimate of total prevalence.

Limitations of the capture–recapture technique are re-quirements of the independence of reporting sources, inthat the chance of being reported by a pharmacist mustnot be influenced by whether or not the physician reports.The second main assumption is that of population sta-bility, which in this case is that changes in Parkinson’sprevalence are not a result of migration in or out. It isnecessary to use the log-linear model to test the assump-tion of independence, and if sources are not independent,a correction must be made by including an interactionterm in the final model, hence the need for at least threereporting sources. The question of migration can best beaddressed by checking census statistics and vital recordsfor the stability of the population most at risk. Accordingto the U.S. Census, the population of Nebraska, espe-cially populations of the elderly and those living in ruralregions, are unusually stable, likely because of the cul-

TABLE 2. Reporting sources:Nebraska Parkinson’s Disease Registry

Physician Pharmacist Patient Number

No No No ?a

No No Yes 0No Yes No 650Yes No No 664Yes Yes No 3,646No Yes Yes 0Yes No Yes 8Yes Yes Yes 94

Results are as of January, 2000.aThe number missing estimated by capture-recapture is 116.8.

TABLE 1. Basic calculation of capture-recapture:reporting from two data sources in theNebraska Parkinson’s Disease Registry

List 1: pharmacist report

Yes No

List 2: physician reportYes 3470 672

a bNo 650 ??

c d

Cells are labeled a–d by usual convention.



tural importance in maintaining close familyconnections.

RESULTS

As of January, 2000, a total of 5,062 PD patients wereregistered in the Nebraska PD Registry. Table 2 showscases reported according to source. The number notreported by any of the three sources can be estimatedfrom those numbers to be 117, resulting in a total esti-mated prevalence of 5,179. This results in a prevalenceof 329.3 per 100,000, with the denominator being thetotal population.

Given that total population includes many not at im-mediate risk of PD, we calculated rates for those over age60 years in different areas of Nebraska and mapped themin Figure 1. Small numbers do not allow capture–recap-ture estimates for individual counties (some counties inthe central region have populations of around 2,000people) so we have used “Health Service Areas”, whichare broad divisions used by the Nebraska state healthdepartment for administrative purposes. There was con-siderable variation by age and gender, as shown in Table3. In particular, prevalence is consistently higher in men,

usually about double the prevalence in women.

DISCUSSION

Several methods have been used to count Parkinson’spatients in a community. A complete count has alwaysbeen the ideal, but the logistics and expense of such anundertaking are daunting. Those investigators who livein areas with unified health records have made use of thataccess to estimate prevalence of diagnosed PD. Some ofthe more frequently cited prevalence surveys have beenoutlined in Table 4. The range of survey techniquesranges from medical record reviews through attempts atdoor-to-door surveys of entire communities. Obviously,each of these methods presents problems and there is noreal “gold standard”9,10 but the technique described herehas not been used before to our knowledge, and presentsanother tool that is applicable for all types of PD surveys.

It is interesting that as methods improve, estimates ofprevalence increase, suggesting that undercounting is themajor problem in counting Parkinson’s patients. Al-though studies like the Copiah County study9 set thestandard for their time, there are undeniable weaknessesin such an approach, and perhaps the most difficult

FIG. 1. Parkinson’s disease prevalence per 1,000 for those of age 60 years or more. Regions are Health Service Areas used by state government.Western Nebraska has much lower population and population density, often �6 people per square mile. Results are as of January 2000.

TABLE 3. Prevalence of Parkinson’s disease by age and gender

Age(yr)

PD Cases Population Rates per 100,000 (95% CI)

Men Women Men Women Men Women

60–70 254.6 207.0 62,280 69,511 406 (382, 434) 298 (277, 324)70–80 721.0 571.1 40,191 57,614 1,794 (1,764, 1,833) 991 (963, 1,019)80 821.8 865.8 19,345 41,855 4,248 (4,164, 4,331) 2,069 (2,030, 2,107)

Results are as of January, 2000. Values are expressed as number of cases estimated by capture-recapture method.PD, Parkinson’s disease; CI, confidence interval.

PD PREVALENCE 321


hurdle to overcome is the participation rate.10 It is im-possible even in a small community to count and diag-nose everyone in a community, and often the participa-tion rate is much lower than ideal. In larger, less coherentcommunities, the task of enrollment of subjects becomesoverwhelming. One cannot imagine doing the CopiahCounty study in even a moderately sized town in Mis-sissippi, let alone in a big city. Yet comparison of suchrates is a basic tool of epidemiology, fundamental to thedevelopment of hypotheses. The capture–recapture tech-nique avoids this weakness because it uses several dif-ferent resources to allow estimation of the number thatcounting methods miss. The undercount can be estimatedwith strong assurance of accuracy, and this adds a majortool to the task of describing PD in communities. Ourresults compare well with previous estimates, and followthe trend of increasing estimates as counting techniquesare refined. The most obvious limitation is that ourtechnique counts only diagnosed cases of PD, which is alimitation difficult to overcome because counts of undi-agnosed PD have been attempted only rarely. Even es-timates of prevalence based on diagnosed disease aredifficult to arrive at in well-defined populations, andoutside of an environment such as an HMO, the Ne-braska Parkinson’s Disease Registry offers to date one ofthe best estimates of prevalence of PD. A weakness ofthe Registry that should be addressed by research is theproblem of overcounting, that is, inclusion of patientswith conditions mimicking PD such as progressive su-pranuclear palsy, multiple system atrophy, and somesequelae of multiple strokes.

A second weakness is the low number of self-reportsin this dataset, which in effect results in only two datasources being used to calculate the capture–recapture

estimates. This can be problematic because an assump-tion of the technique is independence of reportingsources. When only two sources are used, any depen-dence between sources can distort estimates. Neurolog-ical assessments have been carried out on a randomsample of 300 patients from the Registry as part of acase-control study conducted by two of the authors (D.S.and J.B.). Those assessments revealed11 that 78% ofregistrants were confirmed to have PD, including 83% ofthe patients diagnosed previously by a neurologist.Tremor was an initial symptom in 72% of confirmedcases versus 39% of excluded cases, and resting tremorwas present in 86% of those that were confirmed. Themost frequent reasons for exclusion were drug-inducedparkinsonism, multiple system atrophy, vascular disease,and essential tremor. Our estimated prevalence was notcorrected for this misdiagnosis rate, which should bekept in mind.

A final weakness when estimating counts of disease ina population over time stems from population lossthrough death or migration. Although the Registry doescheck against vital records for deaths and excludes thosewho have died, this is done annually, and at that time themost recent year of deaths is not available; accordingly,the Registry includes a year’s worth of deceased regis-trants at a given time. Migration is less measurable, butaccording to the U.S. Census, Nebraska has a very lowmigration rate even among the elderly, making it usefulfor this sort of survey.

In the future as more patients are registered withtheir date of diagnosis recorded, it is hoped that it willbe possible to estimate rates of occurrence of new PDcases (i.e., incidence). The Registry records date ofdiagnosis, but as of January 2000, only about one-third

TABLE 4. Parkinson’s disease prevalence findings from previous studies

Reference Population base Reporting method Results

Snow, 198913 British Columbia, Canada: rural communityof 80,000. Medical record review

Age-adjusted prevalence 86.25/100,000

Caradoc-Davies16 Wellington, New Zealand and Aberdeen,Scotland: review of national healthservice records

Age-adjusted prevalence, andPD prevalence in ageographically-definedpopulation

76.0/100,000 ???99.6/100,000 (1962, Wellington, NZ)102.7/100,000 (Aberdeen, Scotland)

Schoenberg 198517 Copiah County, Mississippi: study of ruralpopulation

Age-adjusted prevalence 130/100,000

Mayeux, 199215 Case study: population-based case-ascertainment survey of PD (completeascertainment)

Age-adjusted PD prevalencewithout dementia

191.2/100,000 (�1/100 �80 yr ofage)

Morgante 199218 Sicily, Italy Age-adjusted total prevalencein 1987

257.2/100,000

Svenson, 199315 Alberta, Canada: gender-based study Age-adjusted prevalence 239.8/100,000Sutcliffe and Meara, 199519 Northampton, UK Crude prevalence 121/100,000Tandberg et al., 199520 Rogaland, Norway Crude prevalence 111/100,000Wermuth et al., 199721 Faroe Islands Crude prevalence 187.6/100,000



of subjects had reported that date (it is required onlyfor those diagnosed date after 1 January 1997). Cap-ture–recapture analysis of those reporting diagnosis in1997 estimated 304 cases, which would be an inci-dence rate of 2.03 new cases per 10,000 per year. Thisis likely a serious underestimate that should be re-garded as an absolute minimum and will be revised asthe Registry develops further

This is currently the only Parkinson’s disease Reg-istry. Prevalence estimates based upon this capture–recapture registry compare favorably with previouswork and the higher rates are consistent with betterascertainment. These rates represent diagnosed andreported PD, although use of capture–recapture allowsestimation of unascertained cases. The registry is be-ing used in several research projects. For example, itserves as the case resource for a case-control study,“Epidemiology of Parkinson’s disease and farm riskfactors” (D.S., Principal Investigator) funded by theNational Institute of Neurologic Diseases andStroke.12 This technique deserves consideration inother settings, especially community surveys usingmultiple reporting settings such those undertaken bySnow and colleagues,13 Svensen and associates,14 andMayeaux and coworkers,15 but also in the classictwo-stage prevalence studies, where the degree ofunder-ascertainment could be calculated with moreaccuracy.

REFERENCES

1. De Pedro-Cuesta J. Parkinson’s disease occurrence in Europe. ActaNeurol Scand 1991:84;357–365.

2. Strickland D, Miller M. Mortality of Parkinson’s disease fromdeath certificates. Presented at World Federation of Neurology/Research Group on Neuroepidemiology. May 1996.

3. Seber GA. The estimation of animal abundance and related param-eters. 2nd ed. London: Charles Griffin; 1982.

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8. Egeland GM, Perham-Hester KA, Hook EB. Use of capture–recapture analysis in fetal alcohol syndrome surveillance inAlaska. Am J Epidemiol 1995;141:335–341.

9. Anderson DW, Schoenberg BS, Haerer AF. Prevalence surveys ofneurologic disorders: methodologic implications of the Copiahcounty study. J Clin Epidemiol 1988;41:339–345.

10. Bermejo F, Gabriel R, Vega S, Morales JM, Rocca WA, AndersonGW. Problems and issues with door-to-door, two-phase surveys:an illustration from central Spain. Neuroepidemiology 2001;20:225–231.

11. Bertoni JM, Sprenkle PM, Strickland D, Noedel N. Evaluation ofParkinson’s disease in entrants in the Nebraska State Parkinson’sDisease Registry. Presented at 55th Annual Meeting of the Amer-ican Academy of Neurology. April 2003.

12. Strickland D, Bertoni JM, Lynch JA. Epidemiology of Parkinson’sdisease and farm risk factors. Funded by the National Institute ofNeurologic Diseases and Stroke. Grant number 5 R01 NS 35206-03.

13. Snow B, Wiens M, Hertzman C, Caine D. A community survey ofParkinson’s disease. CMAJ 1989;141:418–422.

14. Svenson LW, Platt GH, Woodhead SE. Geographic variations inthe prevalence rates of Parkinson’s disease in Alberta. Can J Neu-rol Sci 1993;20:307–311.

15. Mayeux R, Denaro J, Hemenegildo N, Marder K, Tang MX, CoteLJ, Stern Y. A population-based investigation of Parkinson’s dis-ease with and without dementia. Arch Neurol 1992;49:494–497.

16. Caradoc-Davies TH, Weatherall M, Dixon GS, Caradoc-Davies G,Hantz P. Is the prevalence of Parkinson’s disease in New Zealandreally changing? Acta Neurol Scand 1992;86:40–44.

17. Schoenberg BS, Anderson DW, Haerer AF. Prevalence of Parkin-son’s disease in the biracial population of Copiah County, Missis-sippi. Neurology 1985;35:841–845.

18. Morgante L, Rocca WA, DiRosa AE, et al. Prevalence of Parkin-son’s disease and other types of parkinsonism: a door-to-doorsurvey in three Sicilian municipalities. Neurology 1992;42:1901–1907.

19. Sutcliffe RG, Meara JR. Parkinson’s disease epidemiology in theNorthampton District, England, 1992. Acta Neurol Scand 1995;92:443–450.

20. Tandberg E, Larsen JP, Nessler EG, Riise T, Aarli JA. The epi-demiology of Parkinson’s disease in the county of Rogaland,Norway. Mov Disord 1995;10:541–549.

21. Wermuth L, Joensen P, Bunger N, Jeune B. High prevalence of Parkin-son’s disease in the Faroe Islands. Neurology 1997;49:426–432.

PD PREVALENCE 323


Autosomal Recessive JuvenileParkinsonism Cys212Tyr Mutation

in Parkin Renders LymphocytesSusceptible to Dopamine- and

Iron-Mediated Apoptosis

Marlene Jimenez Del Rio, PhD,* Sonia Moreno, MSc,Gloria Garcia-Ospina, MSc, Omar Buritica, MD,

Carlos S. Uribe, MD, Francisco Lopera, MD,and Carlos Velez-Pardo, PhD

School of Medicine, Department of Internal Medicine,Neurology Service, Neuroscience Research Programme,

University of Antioquia, Medellin, Colombia

Abstract: Mutations in parkin are implicated in the patho-genesis of autosomal recessive juvenile parkinsonism (AR-JP) disease. We show that homozygote Cys212Tyr parkinmutation in AR-JP patients renders lymphocytes sensitiveto dopamine, iron and hydrogen peroxide stimuli. Indeed,dopamine-induced apoptosis by four alternative mecha-nisms converging on caspase-3 activation and apoptoticmorphology: (1) NF-�B-dependent pathway; mitochondrialdysfunction either by (2) H2O2 or (3) hydroxyl exposureand (4) increase of unfolded–protein stress. We also dem-onstrate that 17�-estradiol and testosterone prevent ho-mozygote lymphocytes from oxidative stressors-evoked ap-optosis. These results may contribute to understanding therelationship between genetic and environmental factors andiron in AR-JP. © 2004 Movement Disorder Society

Key words: apoptosis; dopamine; juvenile parkinsonism;parkin; stress

Autosomal recessive juvenile parkinsonism (AR-JP) isa clinically distinct group of hereditary neurodegenera-tive disorders typically characterized by early onset ofdisease (age �40 years), sustained response to levodopa(L-dopa), and early occurrence of L-dopa-induced dyski-nesias. Neuropathologically, AR-JP is characterized byselective loss of pigmented neurons, the absence of Lewybodies (LB), iron accumulation in the substantia nigra(SN), and is differentiated genetically by mutations in theparkin gene (PARK2).1–3 This gene encodes a 465-aminoacid (aa) protein, parkin. Importantly, a recessive

Cys212Tyr (C212Y) mutation in parkin (exon 6) hasbeen reported from 2 juvenile parkinsonism families(PJF1, PJF3) with typical AR-JP features from an iso-lated population in North West Colombia.4

During the last years, it has been demonstrated thatparkin has an E3 ubiquitin ligase function,5,6 linkingparkin-associated Parkinson’s disease (PD) to the ubiq-uitin-proteasome pathway (UPP).7 Thus, mutations inparkin result in loss of E3 ligase function, leading toabnormal accumulation of its substrate proteins. Insightinto the physiological role of parkin comes from theprominent demonstration that it is upregulated in re-sponse to unfolded protein stress and suppresses un-folded protein stress-induced cell death via its E3 activ-ity.8,9 Despite these findings, the mechanism of neuronaldeath in AR-PJ remains unknown.

Because parkin deals with unfolded protein stress, andapoptosis (programmed cell death) is relevant to PD,10 itis reasonable to assume that if the E3 ubiquitin-mediatedprotein degradation is impaired in AR-JP patients, thenexposure of cells to stressful conditions could shed newlight on the molecular association between genetic fac-tors, oxidative stress, and cell demise in PD. To addressthis, we have exposed peripheral blood lymphocyte(PBL) cells, which express parkin isoform 3,11 from 3homozygote patients bearing the C212Y mutation inparkin,4 1 heterozygote (i.e., C212Y/C), and 4 controlsubjects, to H2O2/DA/(Fe2) stressors. We provide evi-dence that the homozygotic parkin C212Y mutationfrom AR-JP increases susceptibility to dopamine andiron-mediated apoptosis in lymphocytes, probably due toits failure to dispose of unfolded proteins provoked byoxidative stress.

PATIENTS AND METHODS1

Patients

A group of 4 healthy adults (2 men, 2 women; meanage, 42 � 4 years) with no history of primary neurolog-ical or psychiatric disorders and a group of 3 patients (2men, 1 woman; mean age, 46 � 7 years) diagnosedclinically and genetically with autosomal recessive (i.e.,homozygote C212Y) juvenile parkinsonism (AR-JP) be-longing to the PJF1 family (Cases 755 and 758) and the

*Correspondence to: Dr. Marlene Jimenez Del Rio, Calle 62 #52-72,University of Antioquia, Medellin, Colombia.E-mail: [email protected]

Received 10 February 2003; Revised 13 June 2003, 25 July 2003;Accepted 28 August 2003

DOI 10.1002/mds.10670

1Abbreviations used in this article: Ac-DEVD-CHO, Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Glu-Val-Asp-CHO; AO, acridine orange; DA, dopamine; DiOc6(3),3,3�-dihexyloxacarbocyanine iodide; E2, 17�-estradiol; EB, ethidiumbromide; 6-OHDA, 6-hydroxydopamine; H2O2, hydrogen peroxide;NF-�B, nuclear factor-�-B; PBL, peripheral blood lymphocytes;PDTC, ammonium pyrrolidinedithiocarbamate; UCHL1, ubiquitin car-boxyl-terminal hydroxylase 1.

324 M. JIMENEZ DEL RIO ET AL.


PJF3 family (Case 1439), and a healthy heterozygotefrom the PJF1 family (Case 1435, aged 53 years)4 con-sented to participate in this study.

Materials

Substances were purchased from Sigma (St. Louis, MO),unless otherwise indicated, and were of analytical grade orbetter. Caspase-3 inhibitor was from Alexis Biochemicals(Ac-DEVD-CHO, 260-046-M001). DiOC6(3) was fromMolecular Probes (D-273).

Lymphocyte Isolation

PBL cells from venous blood of AR-JP patients andhealthy adults were obtained by Ficoll-Hypaque gradientcentrifugation. Isolated PBL cells were then purified,cultured, and plated on 24-well plates (1 106 cells/ml/well) as described previously.12,13

PBL Experiments

Assessment of Apoptotic Index by Acridine Orange/Ethidium Bromide.

PBLs were preincubated for 30 minutes at 37°C inculture medium containing 1 mM pargyline and thenfor 24 hours with (25 �M, 50 �M) H2O2, 150 �M6-hydroxydopamine (6-OHDA), 1 mM dopamine(DA) alone or in combination of 50 �M Fe2, respec-tively, in the presence or absence of different productsof interest. PBLs were then used for microscopic ex-aminations. Viability studies, and quantification ofapoptosis and necrosis were analyzed as describedelsewhere.12,13

Immunocytochemical Detection of NF-�BTranscription Factor Protein.

Immunocytochemical staining was carried out accord-ing to the manufacturer’s protocol (goat ABC stainingsystem, sc-2023; Santa Cruz Biotechnology, Santa Cruz,CA) and primary goat polyclonal antibodies NF-�B p65(C-20)-G (sc-372-G; Santa Cruz Biotechnology). Quan-tification of nuclei stained positively with diaminoben-zidine (DAB; dark-brown color) were evaluated from 1to 2 experimental settings as described previously.13

Inhibition of Caspase-3 Activity.

The caspase-3 inhibitor Ac-DEVD-CHO (20 �M) wasadded to PBLs at the time of incubation with DA/Fe2

and other products of interest and incubated for 24 hours.Assessment of apoptotic indexes was determined as de-

scribed elsewhere.12,13 Experiments were carried out in 2separate and independent settings.

Assessment of Mitochondrial TransmembranePotential.

PBL cells were exposed for 24 hours to 1 mM DA/50�M Fe2 and to other compounds of interest. After thistime, treated PBLs were incubated for 15 minutes withthe cationic lipophilic dye DiOC6(3) (3 �M, final con-centration) to evaluate mitochondrial transmembrane po-tential (��m). Quantification of nonfluorescent cells (re-flecting low membrane potentials) under the fluorescencemicroscope was carried out by counting a minimum of250 cells as follows: % nonfluorescent cells � 100 (number of nonfluorescent cells)/total cells counted(nonfluorescent cells fluorescent cells [reflecting highnormal membrane potentials]) compared with untreatedcontrols. In parallel, we used acridine orange/ethidiumbromide (AO/EB) staining to assess the apoptotic index.


Data are representative of at least 3 times in indepen-dent experiments and are presented as the mean � SD.One-way analysis of variance (ANOVA) was used todetermine statistical significance.

RESULTS

As a first approach, we evaluated cell death inlymphocytes by morphological changes typical of ap-optosis (chromatin condensation/fragmentation; apo-ptotic bodies) in controls (CON) and in heterozygoteand homozygote Cys212Tyr parkin mutations in aresponse to 150 �M 6-OHDA, 250, 500, and 1,000�M DA, and 25 and 50 �M H2O2 alone, or in thepresence of 25 and 50 �M Fe2 oxidative stress stim-uli. As shown in Figure 1, all stressors alone or incombination with Fe2 induced apoptosis in CON andC212Y (Fig. 2A) in a concentration-dependent fash-ion. Notably, homozygote C212Y parkin lymphocytestreated with oxidative noxious substances alone weretwo- to threefold more susceptible when comparedwith CON or heterozygotes, and when treated withstressors in combination with Fe2, exhibited a three-to fourfold increase in apoptosis compared with apo-ptosis in CON or heterozygotes. Because it has beendemonstrated that parkin suppresses unfolded proteinstress-induced cell death,8 we incubated CON, heterozy-gote, homozygote C212Y PBLs with urea14 and dithiothre-itol15 (DTT) to cause protein unfolding by denaturation. Asillustrated, 10 mM DTT or urea induced a significant in-crease in the percentage of apoptosis (three- to sixfold,

SUSCEPTIBILITY TO STRESS AND C212Y MUTATION 325


respectively) when compared with CON (assessed byAO/EB staining). Noticeably, heterozygote casesshowed similar responses to stressors as those shownby CON cells.

DA Evokes Apoptosis via NF-�B-Dependent and-Independent Pathways in Cys212Tyr

Parkin LymphocytesTo determine whether DA induces NF-�B activation,

homozygote C212Y lymphocytes were exposed to 1,000�M DA alone or in combination with 50 �M Fe2. Asexpected, DA promoted activation of NF-�B either alone(Fig. 2C) or in combination with Fe2 (data not shown).This activation was almost completely inhibited by the

potent NF-�B inhibitor ammonium pyrrolidinedithiocar-bamate (PDTC)16 (Fig. 2D) when compared with un-treated cells (Fig. 2B). In parallel, we quantified thepercentage of NF-�B activation, apoptosis (AO/EBstaining), and mitochondria function (DiOC6[3] dye). Asdescribed in Table 1, there is a correlation betweenNF-�B activation, DA-induced apoptosis, and mitochon-drial damage. Notably, PDTC did not reduce the toxiceffect of DA-evoked apoptosis and mitochondrial dys-function. Strikingly, in the presence of Fe2, DA in-creased its apoptotic effect and mitochondrial impair-ment; this increase was not affected by PDTC. Similarobservations were established with CON lymphocytesunder PDTC exposure (Table 1).

FIG. 1. DA/H2O2/(Fe2) induce apoptosis in (CON) and in heterozygote and homozygote Cys212Tyr lymphocytes. The data shown for apoptoticpercentage induced by the oxidant compounds such as DA, H2O2 alone or in combination with iron, urea and DTT, are mean values of 3 separateexperiments with error bars representing SD. *P � 0.05; **P � 0.01 vs. respective control as determined by one-way ANOVA. White bars, controllymphocytes; black bars, homozygote Cys212Tyr; gray bars, heterozygote lymphocytes.



DA and Fe2� Induce Apoptosis by HydroxylRadicals and Caspase-3-Dependent Pathways

in Lymphocytes

To characterize further the molecular mechanism bywhich DA/Fe2 caused apoptosis, CON lymphocytes wereincubated with dimethylsulfoxide (DMSO; a specific hy-droxyl radical scavenger17) and Ac-DEVD-CHO. As illus-trated in Table 2, DMSO (500 �M) did not reduce the toxiceffect of DA and H2O2 alone, whereas it reduced apoptosisand mitochondrial dysfunction when Fe2 was added (bytwo- and threefold, respectively). The caspase-3 inhibitor,20 �M Ac-DEV-CHO, completely diminishes DA/H2O2/(Fe2) cell death morphology but mitochondrial impair-ment remains detectable. Interestingly, Ac-DEVD-CHOalso reduces urea-induced apoptosis to untreated levels.

Steroid Hormones and Caspase-3 Inhibitor ProtectCys212Tyr Parkin Lymphocytes from Apoptosis

Estradiol (E212) and testosterone (TESTO18) have beendemonstrated to function as antioxidant agents. Thus, homozy-gote C212Y parkin lymphocytes were incubated with E2 andTESTO in the presence of DA alone or DA/Fe2. E2 andTESTO (4 �M) significantly decreased apoptosis (three- tosevenfold) and mitochondrial dysfunction (four- to eightfold)phenomena when compared with results of DA/(Fe2) treat-ments (Table 3). Additionally, we assessed the effect ofcaspase-3 inhibitor. In agreement with the above data on con-trol lymphocytes (Table 2), Ac-DEVD-CHO completely re-duced morphological changes typical of apoptosis induced byDA, DA/Fe2, and urea, but mitochondria were not sparedfrom their noxious effect (Table 3).

FIG. 2. Dopamine induces apoptosis andNF-�B activation in Cys212Tyr lymphocytes.A: DA-treated lymphocytes (1,000 �M) usingAO/EB staining, show highly condensed chro-matin (arrows), nuclear fragmentation (ring-likeforms, arrowheads), and apoptotic bodies (dou-ble arrows), typical nuclear apoptotic morphol-ogy. B: Untreated lymphocytes, treated withDA (1,000 �M) alone (C) or (D) treated incombination with (100 nM) PDTC for 24 hr.Cells were then stained with anti-NF�B-p65(B–D). NF�B-positive nuclei (dark browncolor) reflect nuclear translocation and seem tocorrelate with apoptotic morphology (con-densed/fragmented nuclei) when compared withuntreated cells (B) or cells treated with PDTC(D). Magnification 400 (A–D). [Color figurecan be viewed in the online issue, which isavailable at www.interscience.wiley.com.]

TABLE 1. Dopamine-induced apoptosis through NF-�B activation and mitochondrial damage in homozygoteCys212Tyr and control lymphocytes

Treatment

Homozygote Cys212Tyr CON

NF-�B(activation)

Apoptosis(AO/EB)

Mitochondria(D1Oc6[3])

NF-�B(activation)

Apoptosis(AO/EB)

Mitochondria(D1Oc6[3])

Untreated �1 � 0 3 � 2 �1 � 0 �1 � 0 �1 � 0 �1 � 01,000 �M DA 18 � 2b 40 � 4b 33 � 3b 5 � 2 13 � 3 11 � 2DA Fe2 28 � 3b 61 � 5b 70 � 4b 11 � 2 30 � 3 27 � 2100 nM PDTC �1 � 0 �1 � 0 �1 � 0 �1 � 0 �1 � 0 �1 � 0DA PDTC 3 � 1b 36 � 3b 28 � 4b 1 � 1 11 � 2 12 � 3DA Fe2 PDTC 5 � 1a 56 � 3b 85 � 3b 4 � 1 28 � 3 45 � 3

Results express the percentage of apoptosis (morphological changes), NF-�B activation, and mitochondrial dysfunction. Mean valuesof 3 separate experiments � SD are shown.

aP � 0.05; bP � 0.01 versus respective control as determined by one-way ANOVA.CON, control; AO/EB, acridine orange/ethidium bromide; DA, dopamine; PDTC, ammonium pyrrolidinedithio-carbamate.



DISCUSSION

Although recent genetic and molecular studies haveconfirmed parkin mutation as a specific genetic defect inAR-JP,3 no plausible explanation has been given untilnow linking mutations found in this gene to oxidativestress and the cascade of molecular events in neuronaldemise in this subtype of parkinsonism. On the otherhand, a large body of evidence suggests that oxidativestress-mediated mechanisms19,20 may be at least partiallyresponsible for the progressive, selective, neuronal de-generation observed in PD. Interestingly, the relationshipbetween genetic factors (e.g., mutations in �-synuclein,UCHL-1, and the parkin gene21) and environmental fac-tors such as exogenous (e.g., MPTP, and 6-OHDA) neu-rotoxins, endogenous (e.g., dopamine) neurotoxins22,and iron23 has not been established fully in AR-JP.Lymphocytes have been demonstrated to express parkinisoform 3 comprising a transcript of 9 exons11 (exons 1,2, and 6–12), and DA and iron seem to be a valuableexperimental model to study oxidative stress and itsregulation in lymphocytes.12,13,24 Therefore, it is of majorinterest to clarify the mechanisms by which DA, iron,and the parkin mutation C212Y in exon 64 lead to celldeath, to help discover potential therapeutic targets.

In this article, we show dopamine-induced apoptosisby four alternative mechanisms: (1) NF-�B-dependentpathway; (2) mitochondrial dysfunction by H2O2 or by

(3) hydroxyl radical exposure; and (4) increase of un-folded protein stress. These mechanisms activatecaspase-3, as a common pivotal executor protein. Wehave demonstrated that increasing concentrations of DA,the DA-related toxin 6-OHDA, and H2O2 significantlyaugment the percentage of apoptosis in mutated homozy-gote parkin PBLs, compared with CON and heterozygotePBLs (Fig. 1), as evidenced by the highly reliableAO/EB technique25 (Fig. 2A). This apoptotic effect wasenhanced by Fe2. This is in agreement with previousdata showing that iron potentiates DA- and 6-OHDA-evoked apoptosis in neural-like PC12 cells26 and non-neuronal PBL cells via H2O2-mediated oxidative stressmechanisms.12,13,24 Most importantly, homozygoteC212Y PBL were considerably more susceptible (three-to sixfold) to both urea and DTT protein unfoldingagents than were CON and heterozygote lymphocytes(Fig. 1). Therefore, it is tempting to conclude that theparticular increased susceptibility of homozygotesC212Y parkin lymphocytes to DA/H2O2/(Fe2)-pro-voked apoptosis is related to a functional impairment ofthe E3 ubiquitin ligase parkin involved in regulation ofunfolded-protein stress degradation. These findings sug-gest that parkin might protect cells from unfolded proteinstress as suggested previously by Imai and coworkers.8,9

Previous studies indicate that NF-�B (p65/p50) is anessential transcription factor involved in apoptosis.13,24 Wehave demonstrated that DA activates NF-�B in both ho-

TABLE 3. Steroid hormones and caspase-3 inhibitor protecthomozygote Cys212Tyr lymphocytes from apoptosis and

mitochondrial dysfunction induced by DA/(Fe2) and urea

TreatmentApoptosis

(AO/EB) %Mitochondria(D1Oc6[3]) %

Untreated 3 � 2 2 � 1DA (1,000 �M) 40 � 4 33 � 3DA Fe2 (50 �M) 61 � 5 70 � 4E2 (4 �M) 1 � 1 1 � 1E2 DA 12 � 2b 8 � 2b

E2 DA Fe2 18 � 3b 12 � 3b

TESTO (4 �M) 1 � 1 1 � 1TESTO DA 6 � 2b 4 � 2b

TESTO DA Fe2 23 � 3b 18 � 3DEVD (20 �M) �1 � 0 �1 � 0DEVD DA �1 � 0 35 � 4DEVD DA Fe2 �1 � 0 55 � 5Urea (10 mM) 28 � 2 30 � 4Urea DEVD 1 � 1b 25 � 1c

Cys 212Tyr lymphocytes were incubated for 24 hr with stressoragents with or without 17� estradiol and testosterone for evaluation ofapoptotic morphology and mitochondrial dysfunction. Percentage isexpressed as a mean of 3 independent experiments � SD.

aP � 0.05; bP � 0.01 versus respective control (one-way ANOVA);cno significance AO/EB, acridine orange/ethidium bromide; DA, do-pamine; E2, 17� estradiol; TESTO, testosterone; DEVD, Ac-DEVD-CHO (caspase-3 inhibitor).

TABLE 2. DA/(Fe2)-induced apoptosis is dependent onhydroxyl radical production, caspase-3 activation, and

mitochondrial damage in CON lymphocytes

TreatmentApoptosis

(AO/EB) %Mitochondria(D1Oc6[3]) %

Control �1 � 0 �1 � 0Fe2 (50 �M) 2 � 1 1 � 1DA (1,000 �M) 13 � 3 11 � 2DA Fe2 30 � 3 27 � 2H2O2 (50 �M) 8 � 2 15 � 2H2O2 Fe2 18 � 2 28 � 3500 �M DMSO �1 � 0 �1 � 0DA DMSO 14 � 2c 16 � 2b

DA Fe2 DMSO 15 � 3b 10 � 2b

H2O2 DMSO 6 � 2a 16 � 2c

H2O2 Fe2 DMSO 5 � 2b 7 � 2b

DEVD (20 �M) �1 � 0 �1 � 0DA DEVD �1 � 0 16 � 2b

DA Fe2 DEVD �1 � 0 25 � 3c

H2O2 DEVD �1 � 0 13 � 2a

H2O2 Fe2 DEVD �1 � 0 32 � 3a

Urea (10 mM) 5 � 1 2 � 1Urea DEVD �1 � 0 �1 � 0

Mean values of 3 separate experiments � standard deviations are shown.aP � 0.05; bP � 0.01 versus respective control (one-way ANOVA);

cno significance.AO/EB, acridine orange/ethidium bromide; DA, dopamine; DMSO,

dimethyl sulfoxide; DEVD, Ac-DEVD-CHO (caspase-3 inhibitor).



mozygote C212Y (Fig. 2C) and CON cells when comparedwith the untreated homozygote C212Y (Fig. 2B) cells.These results are consistent with observations of DA-in-duced NF-�B activation in PC12 cells and rat intrastriatalneurons by Panet and colleagues27 and Luo and associ-ates,28 respectively. Interestingly, Lee and coworkers29 havealso demonstrated auto-oxidized DA-activated NF-�B inPC12 cells, but failed to demonstrate a proapoptotic role ofNF-�B. Although the nature of this discrepancy is not yetestablished, our data show clearly that NF-�B is activatedby DA/(Fe2) stimuli and NF-�B is involved in apoptosis.This assumption is supported by direct observation ofNF-�B DAB-positive nuclei (Fig. 2C), which correlateswith typical morphology of apoptosis (see Fig. 2A vs. Fig.2C) and a positive correlation is observed between thepercentage of apoptosis and active transcription factor (Ta-ble 1). Taken together, these results suggest that NF-�Bubiquitinization is not impaired by the parkin C212Y mu-tation and activated NF-�B occurs concomitantly with cellapoptotic morphology. To confirm further, C212Y/CONcells were treated with DA/(Fe2) in the presence of thepotent NF-�B inhibitor PDTC. We found that PDTC inac-tivates NF-�B (Fig. 2D). Surprisingly, however, this inhib-itor did not diminish completely the percentage of apoptoticmorphology, as determined by the AO/EB technique (Table1). We therefore conclude that DA/(Fe2) may activateother pathway(s) involved in apoptosis. This viewprompted us to examine the effect of DA/(Fe2) on mito-chondrial function, given that this organelle is critical in cellfate decisions30 and may be implicated in etiology andpathogenesis of PD.31 By using the DiOC6(3) compound,we observed that DA/(Fe2) co-incubated with PDTC pro-vokes mitochondrial damage concurrently with apoptosis(Table 1). These results suggest that DA/(Fe2) provokesmitochondrial dysfunction independent of NF-�B activa-tion, which may be explained by the fact that NF-�B is anupstream effector molecule in the molecular cascade of celldeath signalization.13,24 In addition, we demonstrated thatH2O2 and hydroxyl radicals, resulting from the Fentonreaction (Fe2 H2O2 H � Fe3 �OH H2O),32

are also implicated in mitochondrial disturbance. Theseobservations strengthen the view that �OH represents asignificant complementary source of oxidative stress thatcontributes to cell death. This last assumption is supportedfurther by the specific hydroxyl radical scavenger DMSO,17

which significantly reduces the percentage of morphologi-cal apoptotic features and mitochondrial dysfunction under(DA/Fe2)/(H2O2/Fe2) exposure (Table 2). These obser-vations concur with the notion that mitochondria can bedamaged either by DA,33 H2O2,34 or hydroxyl radicals35

when iron is present (present study). Taken together, ourfindings raise the possibility that in addition to NF-�B

activation, �OH/H2O2 might also contribute to enhance ap-optosis in homozygote mutated parkin cells by readily at-tacking cellular proteins (compare C212Y vs. CON apopto-tic percentages, Fig. 1).

Recent data have demonstrated that caspase-3 is a finaleffector in apoptotic death of dopaminergic neurons inPD.36 We have demonstrated that Ac-DEVD-CHO com-pletely blocked apoptotic morphological features in lym-phocytes under DA/H2O2/Fe2 and urea stressors (Table2). These results imply that caspase-3 is required forapoptosis-associated morphological changes.37 Strik-ingly, Ac-DEVD-CHO was unable to reduce mitochon-drial damage. These findings are in agreement with pre-vious findings that caspase-3 activation acts as adownstream executer after mitochondrial depolarization,escape of mitochondrial apoptogenic protein cytochromec, assembly of the apoptosome.38

Our data shows that DA is a source of four main noxiouscompounds in the presence of iron: DA-o-quinones, redoxcycling DA-S-adducts, H2O2, and hydroxyl radicals.19,39

Based on this, one might predict that either DA antioxidantsor H2O2 could be potential targets to either retard or bringan end to cellular death. We show here that E2 and TESTO(4 �M) protect homozygote C212Y lymphocytes from DA/(Fe2)-evoked cell death (Table 3). The remarkable cyto-protection afforded by E2 and TESTO confirms the in-volvement of H2O2 produced by DA autoxidation as themajor messenger molecule underlying toxicity of this com-pound.12,13 Our present findings suggest that, despite thefact that estrogen has been recently added to the carcinogenlist,40 molecules with steroidal molecular structure (e.g.,flavonoids41) may offer potential first-line pharmacologicintervention to retard progression and/or deterioration ofneurons in early onset AR-JP patients.

Acknowledgments: This research was supported by Col-ciencias (1115-04-10231 to M.J.D.R.).

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