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Antioxidant and inflammatory biomarkers for the identification of prodromal Parkinson's disease
Journal of the Neurological Sciences 370 (2016) 167–172
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Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Antioxidant and inflammatory biomarkers for the identification of prodromal Parkinson's disease Jonica Campolo a, Renata De Maria a,⁎, Lorena Cozzi a, Marina Parolini a, Stefano Bernardi a, Paola Proserpio b, Lino Nobili b, Giorgio Gelosa c, Immacolata Piccolo d, Elio C Agostoni c, Maria G Trivella a, Paolo Marraccini a a
CNR Institute of Clinical Physiology, ASST- Great Metropolitan Hospital Niguarda, Milan, Italy Epilepsy Surgery Centre, Centre of Sleep Medicine, ASST- Great Metropolitan Hospital Niguarda, Milan, Italy c Neurology and Stoke Unit, Department of Neuroscience, ASST-Great Metropolitan Hospital Niguarda, Milan, Italy d Spinal Unit, ASST- Great Metropolitan Hospital Niguarda, Milan, Italy b
a r t i c l e
i n f o
Article history: Received 14 July 2016 Received in revised form 9 September 2016 Accepted 25 September 2016 Available online 26 September 2016 Keywords: Parkinson's disease Idiopathic REM behavior disorder Olfactory dysfunction Cardiac dysautonomia Glutathione
a b s t r a c t Objectives: We explored the role of oxidative stress and inflammatory molecules as potential Parkinson (PD) biomarkers and correlated biological with non-motor abnormalities (olfactory impairment and dysautonomia), in patients with idiopathic REM behavior disorder (iRBD) (prodromal PD) and established PD. Methods: We recruited 11 iRBD and 15 patients with idiopathic PD (Hohen&Yahr 1–3, on L-DOPA and dopamine agonists combination therapy) and 12 age- and sex-matched controls (CTRL). We measured total olfactory score (TOS), autonomic function [deep breathing (DB), lying to standing (LS) and Valsalva manoeuvre (VM) ratios], blood reduced glutathione (Br-GSH), oxidative stress and inflammatory markers (neopterin). Results: Anosmia was similarly prevalent in iRBD (36%) and PD (33%) patients, but absent in CTRL. Orthostatic hypotension was more common among iRBD (73%) and PD (60%) than in CTRL (25%). By univariable ordinal logistic regression, TOS, Br-GSH, LS and VM ratio worsened from CTRL to iRBD and PD groups. Only reduced Br-GSH levels (p = 0.037, OR = 0.994; 95%CI 0.988–1.000) were independently associated to PD. TOS correlated with Br-GSH (R = 0.34, p = 0.037), VM ratio (R = 0.43, p = 0.015), and neopterin (rho = 0.39, p = 0.016). Conclusions: Reduced systemic antioxidant capacity is found in prodromal and overt PD and may represent, in association with olfactory loss and cardiovascular dysautonomia, a useful biomarker for an integrative, early diagnosis of PD. © 2016 Published by Elsevier B.V.
1. Introduction Early identification of Parkinson disease (PD) is a main challenge in neurological research. To date, the clinical diagnosis of PD is primarily based on the late onset of motor disorders, which manifest when 60– 70% of the dopaminergic neurons are degenerated in the substantia nigra [1]. PD is now recognized as a more complex illness, encompassing non motor symptoms (NMS), such as sleep disturbances, sensory abnormalities, autonomic dysfunction, depression and cognitive decline [2], that may predate the onset of motor symptoms by many years and are currently being studied as features of prodromal PD [3]. Neuropathological changes related to NMS include findings of α-synuclein-containing Lewy bodies and Lewy neurites in the peripheral autonomic nervous system and neuronal loss in the dorsal motor nucleus of the vagal nerve, the olfactory bulb, and the lower brainstem nuclei that regulate ⁎ Corresponding author at: CNR Institute of Clinical Physiology Milan, ASST-Great Metropolitan Hospital Niguarda, Milan, Italy. E-mail address: [email protected] (R. De Maria).
http://dx.doi.org/10.1016/j.jns.2016.09.050 0022-510X/© 2016 Published by Elsevier B.V.
REM sleep atonia [4]. These abnormalities might be responsible for the occurrence before the onset of parkinsonism of NMS such as dysautonomia, hyposmia, and REM sleep behavior disorders (iRBD), that have been prospectively linked to PD in population [5] and cohort studies [6], as recently reviewed [7,8]. Oxidative stress causes tissue injury and inflammation [9] and may play a role in PD. The autoxidation of dopamine in the dopaminergic neurons may produce reactive oxygen species (ROS). Mitochondrial dysfunction in nervous cells may increase oxidative stress by alteration of the respiratory chain, the activated microglia, responsible for chronic neuroiflammation, but also for ROS release after activation of specific enzyme, such as NADPH oxidase and inducible nitric oxide synthetase [10]. Elevated oxidative stress and a pro-inflammatory response occur early in the course of PD and contribute to exacerbate nigro-striatal degeneration [11]. Aims of our study were to explore the potential role of antioxidant and inflammatory biomarkers as novel tools for prodromal diagnostic evaluation of PD and to correlate biological findings with functional alterations, such as olfactory impairment and cardiovascular dysautonomia in patients with prodromal and established PD.
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2. Methods 2.1. Participants We recruited eligible subjects aged ≥50 e ≤ 80 years consecutively referred between May 2014 and May 2015 by the Centre of Sleep Medicine (iRBD) and the Neurology outpatient clinic (PD) of our Hospital and age and gender-matched controls (CTRL) who attended our Institute for laboratory assessment. Subjects presented none of the exclusion criteria listed below: - other neurological disorder, major mental disorder, cognitive impairment (MMSE b26); - previous myocardial infarction, heart failure, pacemaker, atrial fibrillation, beta-blockers; - glomerular filtration rate b 15 mL/min; - hepatic insufficiency - long standing (N10 years) diabetes; subjects with hyperglycemia (serum glucose N100 and b126 mg/dL) without antidiabetic treatment could be enrolled
iRBD [12] had to meet the following criteria: - repeated episodes of sleep-related vocalization and/or complex motor behaviors; - video-polysomnographic (PSG) documentation of occurrence during REM sleep or history suggestive of dream enactment; - polysomnographic observation of REM sleep without atonia; - exclusion of other sleep disorder, medication, or substance use.
Patients with idiopathic PD, confirmed by a neurologist with expertise in movement disorders (EFNS/MDS-ES 2013 guidelines), had to be in Hohen & Yahr stage ≥1 and ≤3 and on L-DOPA and dopamine agonists combination therapy, titrated to maintenance doses since at least 1 month. We excluded PD patients with any of the following: -
genetic etiology or familial clustering; atypical or secondary parkinsonism; history of cerebrovascular events; treatment with COMT inhibitors.
The study was approved by Niguarda Hospital Ethics Committee. All patients expressed their written informed consent to participate. 2.2. Study measurements Eligible subjects underwent: medical and drug history taking; physical and neurological examination; iRBD screening question [13] olfactory and autonomic function testing; fasting blood and urine sampling. PD subjects were administered the Movement Disorder Society-UPDRS Parts I-II (nMEDL and MEDL scores) and underwent functional testing on medications. 2.3. Biochemical assessments Glutathione (GSH), the most important endogenous scavenger, was assessed in total and reduced form and in plasma and blood samples by high performance liquid chromatographic (HPLC) [14]. Plasma malondialdehyde (MDA), a marker of lipid peroxidation, was assayed by a commercial kit (Chromsystems, Germany) in HPLC with fluorescence detection. Commercial ELISA kits were used for plasma assay of 8-hydroxy-2-deoxyguanosine (8-OHdG), index of oxidative DNA damage (Trevigen, Gaithersburg, MD, USA); 3-nitrotyrosine (3-NT), a stable end product of peroxynitrite oxidation (Hycult Biotech (Uden, The
Nederland)); the inflammatory cytokines tumor necrosis factor alpha (TNFα) (Cayman Chemical Company, Ann Arbor, MI, USA) and interleukin 1-beta (IL1β), Boster Immunoleader (Pleasanton, CA, USA). Urine neopterin levels, a sensitive marker of cellular-mediated inflammation, were measured by an isocratic HPLC method and normalized by urine creatinine concentrations [15].
2.4. Functional testing Olfactory function was assessed by the Sniffin' Sticks Extended Test (Burghart, Medizintechnik, GmbH, Wedel, Germany) [16]. The olfactory threshold is the minimum concentration of an odorant (n-butanol) that can be detected by a subject when presented with 16 different dilutions in felt tip pens. Olfactory discrimination assesses the ability to discriminate between different odorants in 16 different triplets. Olfactory identification evaluates the ability to correctly identify an odorant among four possible odors for each of 16 trials. The total olfactory score (TOS) was calculated as sum of the 3 sub-scores for olfactory threshold, discrimination and identification and reclassified as normal olfaction (between 31 and 48), hyposmia (between 16 and 30) and anosmia (≤15). Autonomic function [17] was tested in the supine position at a comfortable ambient temperature. Heart rate was recorded via standard 12lead electrocardiogram (Norav PC ECG-1002). Blood pressure was measured non-invasively by a manual sphygmomanometer.
2.4.1. Deep breathing (DB) After 10-minute rest, subjects performed 1 minute DB (6 inspiratory and expiratory cycles of 5 sec each) during continuous ECG recording. The DB expiration/inspiration ratio was calculated as ratio of averages of the three longest RR intervals during expiration and the three shortest RR intervals during inspiration.
2.4.2. Lying to standing (LS) After 10-minute rest, patients were instructed to stand up and remain standing for 5 min. Changes in systolic/diastolic blood pressure and heart rate were assessed after 1 and 5 minutes standing from the supine position. Orthostatic hypotension (OH) was defined as a drop ≥ 20 mm Hg in systolic and/or ≥ 10 mm Hg in diastolic blood pressure. The LS 30/15 ratio was the ratio between the longest RR interval measured between the 25th and 35th beat after active standing and the shortest RR interval between the 10th and 20th beat.
2.4.3. Valsalva manoeuvre (VM) Patients in the sitting position were instructed to blow into a tube connected to a manometer to maintain the pressure at 40 mm Hg for 15 s, during continuous heart rate and blood pressure monitoring. VM ratio was the ratio of the shortest RR interval (tachycardia) during expiration to the longest RR interval (bradycardia) after expiration.
2.5. Statistical analysis Data are presented as median (interquartile range) or frequency (percentage). Categorical variables were compared by the chi-square test. Pearson's R correlation coefficient or Spearman's rho index were used to correlate continuous clinical, biochemical and functional variables (age, symptom duration, nMEDL, MEDL, GSH, Neopt, MDA, 3-NT, 8-OHdG, TNFα, IL1β, TOS, LS 30:15 ratio, VM ratio, DB-ratio). Associations with the dependent variable “group” were tested by univariable ordinal logistic regression; variables with p b 0.10 were entered in a multivariable model. A p value b 0.05 was considered significant. Statistical analyses were carried out with the Statistical Package for the Social Sciences (SPSS Inc., Chicago, Illinois, USA), version 17.0 for Windows.
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3. Results
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Table 2 Clinical characteristics of disease severity in PD group.
We enrolled 15 patients with PD, 11 subjects with PSG-confirmed iRBD and 12 CTRL (Table 1).
Median (I;III) Hohen & Yahr score Years onset motor symptoms Years from diagnosis UPDRS Part I UPDRS Part II, nMEDL UPDRS Part II, MEDL UPDRS Part II total DOPA dose (mg/day) Rotigotine dose (mg/day) Ropirinole dose (mg/day) Pramipexole dose (mg/day)
3.1. Findings in PD patients PD patients had long standing disease with mild to moderate motor and NMS burden (Table 2). Only 2 patients presented on-off phenomena and dyskinesia. Disease duration correlated with L-DOPA dose (R = 0.650, p = 0.012) and with the DB E/I ratio (rho = − 0.642, p = 0.021), but not with nMEDL or TOS. nMEDL score was inversely related to VM ratio (R = − 0.726, p = 0.017) and to TOS (R = − 0.873, p b 0.001). Conversely, MEDL correlated with age (R = 0.523, p = 0.045) but not with any biochemical or functional variable. Six PD patients (40%) reported sleep behaviors compatible with RBD at the screening question; no differences were observed between patients with and those without suspect RBD either in clinical findings or in functional and biochemical characteristics. 3.2. Functional and biochemical differences among PD, iRBD and CTRL Among clinical characteristics, CTRL had more commonly a history of hypertension than iRBD and PD. A significant worsening trend was observed in total olfactory score, blood reduced GSH, LS and VM ratio and neopterin from CTRL to iRBD and PD groups (Table 1). By multivariable ordinal logistic regression, only low blood reduced GSH levels (p = 0.037, OR = 0.994; 95% CI 0.988–1.000), adjusted by history of hypertension, total olfactory score, LS 30:15 ratio and VM ratio and urinary neopterin, was associated to PD.
2.5 (2.0;2.5) 10.0 (6.0;12.8) 6.0 (3.5;9.5) 2.0 (1.0;6.0) 9.0 (5.0;11.0) 12.0 (7.0;14.0) 19 (16;26) 500 (300;850) 9.0 (6.5;11.5) 6.0 (6.0;6.0) 2.10 (1.05;2.10)
Data are expressed as median and interquartile range (I;III). P for between-group differences by unpaired Student's t-test or Mann-Whitney test*. MEDL, motor experiences of daily living; nMEDL, non-motor experiences of daily living.
The cumulative effect of olfactory impairment, dysautonomia, depressed mood and reduced antioxidant capacity (blood GSH ≤ 468 μmol/L 10th percentile of CTRL distribution) for group discrimination was assessed as number of risk factors: over half of both iRBD and PD presented at least two features combined (fig. 1). 3.4. Correlations among clinical, biochemical and functional variables We tested in the overall population the relationship among clinical, biochemical and functional variables. Significant correlations were observed only between TOS and blood reduced GSH concentrations (panel A), VM ratio (panel B), and urine neopterin levels (panel C) (Fig. 2). 4. Discussion
3.3. Group comparison with respect to functional category Functional anosmia was similarly prevalent in iRBD (36%) and PD (33%) patients, but was absent in CTRL (p = 0.097). OH was more common among iRBD (73%) and PD (60%) than in controls (25%) (p = 0.055), independently of antihypertensive treatment.
The main findings of the present study are that reduced systemic antioxidant capacity is independently associated to overt PD and iRBD, a condition now established as prodromal PD, and correlates with olfactory and sympathetic dysfunction. Moreover progressive cardiovascular autonomic dysfunction, expressed as altered sympathetic (VM ratio,
Table 1 Univariable ordinal logistic regression analysis of clinical, biochemical and functional characteristics of the study groups.
Age, years Gender (M), n% Smoking habit, n% Hypercholesterolemia, n(%) Hyperglycemia n(%) Hypertension, n(%) Depressive mood, n(%) MMSE Blood reduced GSH, μmol/L Blood total GSH, μmol/L Plasma reduced GSH, μmol/L Plasma total GSH, μmol/L Plasma MDA, μmol/L Plasma 3-NT, nmol/L Plasma 8-OHdG, nmol/L Urine neopterin, (μmol/mol creat) Plasma TNFα, pg/mL Plasma IL1β, pg/mL Total olfactory score LS 30:15 ratio SBP drop at 1 minute standing DBP drop at 1 minute standing VM ratio DB E:I ratio
CTRL (n = 12)
IRBD (n = 11)
PD (n = 15)
p
OR
95% CI
71 (59;74) 7 (58%) 2 (17%) 4 (33%) 3 (25%) 7 (58%) 3 (25%) 28 (27;28) 759 (650;833) 1364 (1205;1521) 1.13 (0.88;1.57) 6.95 (6.35;8.66) 0.16 (0.14;0.17) 12.7 (6.1;29.3) 279 (229;348) 127 (93;188) 26 (21;46) 10.9 (8.9;13.6) 28.7 (22.2;33.0) 1.22 (1.05;1.37) −2.5 (−16.2;−12.5) 0 (−7.5;5.0) 1.50 (1.31;1.73) 1.17 (1.11;1.27)
71 (64;73) 10 (91%) 4 (36%) 2 (20%) 4 (36%) 4 (36%) 3(27%) 28 (27;29) 582 (380;715) 1091 (923;1563) 1.05 (0.72;1.61) 5.60 (4.25;10.90) 0.15 (0.14;0.16) 19.7 (17.1;48.6) 227 (180;307) 141 (114;185) 34 (22;66) 15.0 (12.0;16.1) 17.5 (11.0;21.5) 1.13 (1.07;1.19) −15.0 (−20.0;−10.0) −10.0 (−15.0; −5.0) 1.38 (1.26;1.45) 1.14 (1.09;1.21)
70 (58;76) 7 (47%) 1 (7%) 2 (13%) 3 (21%) 3 (20%) 8 (53%) 29 (27;30) 519 (467;639) 1070 (860;1743) 0.86 (0.69;1.01) 6.10 (4.57;7.60) 0.14 (0.13;0.16) 18.2 (10.0;34.5) 280 (209;357) 164 (125;226) 28 (22;60) 10.9 (8.4;14.5) 18.7 (15.0;24.0) 1.03 (1.01;1.18) −20.0 (−30.0;−5.0) −5.0 (−15.0;0) 1.27 (1.14;1.53) 1.15 (1.11;1.26)
0.575 0.365 0.473 0.216 0.805 0.043 0.110 0.620 0.009 0.892 0.181 0.132 0.229 0.585 0.696 0.083 0.421 0.582 0.009 0.047 0.100 0.116 0.047 0.958
0.981 0.566 0.573 0.392 0.845 0.267 0.355 1.147 0.995 1.000 0.736 0.852 0.000 0.998 1.001 1.107 1.004 1.043 0.890 0.004 0.977 0.952 0.045 0.879
0.920–1.047 0.165–1.944 0.125–2.622 0.089–1.726 0.223–3.212 0.075–0.960 0.100–1.261 0.668–1.970 0.991–0.999 0.998–1.002 0.469–1.155 0.692–1.049 0.001–8323 0.991–1.005 0.995–1.007 0.987–1.242 0.992–1.016 0.897–1.213 0.815–0.973 0.001–0.919 0.951–1.004 0.896–1.012 0.002–0.961 0.007–111
Data are expressed as median and interquartile range (I;III) or as frequency (%). DB, deep-breathing; E:I expiration/inspiration; IL, interleukin; LS, lying to standing; GSH, glutathione, MDA, malondialdehyde; TNF, tumor necrosis factor; VM, Valsalva manoeuvre. Bold values indicate significance at p value b 0.05.
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p=0.050 100%
80%
60%
40%
20%
Number of risk factors 3 2 1 0
0% CTRL
iRBD
PD
Fig. 1. Comparison among CTRL, iRBD and PD groups for number of the following risk factors: anosmia, orthostatic hypotension, depression or low blood GSH.
OH) or parasympathetic (LS 30:15 ratio) response to testing, is found from prodromal state to overt disease and correlates with olfactory dysfunction. The combination of two or more features was distinctly different between CTRL vs iRBD and PD. GSH, the most abundant aminothiol in cells, acts in the brain as antioxidant and redox regulator [18]. Experimental results in PD models and analyses of postmortem brain tissue from PD patients point to a profound loss of GSH in the substantia nigra, that parallels the severity of disease and occurs prior to other hallmarks of tissue damage [19]. GSH depletion may affect mitochondrial function through selective inhibition of mitochondrial complex I enzyme in the respiratory chain [20], with excess ROS production and decreased ATP levels. The low GSH concentrations found in blood cells of patients with both overt and prodromal PD suggest antioxidant depletion to be present not only in brain tissue, but also in systemic cellular districts, already in the early stages of disease. This perturbation might be confined to the intracellular space, since we did not find any difference in plasma GSH content and oxidative stress biomarkers. Blood reduced GSH concentrations paralleled the progression from prodromal to overt disease, as expressed by olfactory impairment and autonomic dysfunction, and were directly related to TOS in the overall population. The long established observation of olfactory dysfunction as a common early feature of PD, which often precedes motor symptoms by several months or even years [21] is grounded in Braak's hypothesis [22]. The accumulation of Lewy bodies follows a predictable sequence from the anterior olfactory nucleus and bulb and the dorsal motor nuclear complex of the glossopharyngeal and vagal nerves, through the brain stem toward the midbrain. In our cohort, olfactory dysfunction worsened from CTRL to iRBD and PD, with functional anosmia being present only in both iRBD and PD. Olfactory dysfunction did not correlate with disease duration in our PD group, consistently with previous reports of lack of deterioration in odor identification, discrimination, and threshold detection in the course of PD [23,24]. Olfactory loss may be an important preclinical marker, detectable by non-invasive low-cost methods. Conversely, hyposmia, which is strongly influenced by age, was unable to discriminate iRBD or PD patients from CTRL. On the same line, worse olfactory dysfunction correlated with increased neopterin levels, which were not independently predictive of PD, as aging per se is a low-grade
inflammatory state. Olfactory dysfunction in iRBD has been suggested to portend conversion to PD [25,26]. Cardiovascular dysautonomia in PD affects both the sympathetic and parasympathetic autonomic nervous system [27,28], with OH present in up to 52% of PD patients [29]. Supposed OH mechanisms in PD include baroreflex failure due to central lesions in the upper brainstem and post-ganglionic impairment, with loss of cardiac and peripheral vascular sympathetic innervation. While the severity of parasympathetic dysfunction worsens with disease progression [23], as confirmed in our patients by the inverse relation between PD duration and the E/I ratio, OH may predate PD development [30] and is associated with neuroimaging evidence of cardiac and extra-cardiac sympathetic denervation. In a large series, iRBD had significantly more often postural dizziness than controls [31]. iRBD who converted to a neurodegenerative disease had a higher prevalence of cardiovascular and gastrointestinal symptoms [32] and OH, with a larger systolic blood pressure drop from lying to standing than non converters [33]. In our cohort, OH prevalence overlapped among iRBD and PD patients at approximately 60%, almost 3-fold higher than in CTRL. We likewise observed a lower Valsalva ratio, a progressive decline in the LS ratio and larger systolic and diastolic blood pressure drop in iRBD, of intermediate severity with PD, than in CTRL. The combination of features, such as reduced TOS, OH and depression, has been previously shown to improve prediction of PD in community studies [25,26,34,35]. These prodromal markers have been included in the recent research criteria for prodromal PD with high positive likelihood ratios for PD development [36]. Our finding of improved discrimination of both iRBD and PD from CTRL with low GSH concentrations suggests that reduced antioxidant capacity may be a promising blood biomarker [37] and should be tested in larger series to assess its value as predictor of conversion from iRBD to PD or dementia. Our study presents several limitations. As a consequence of stringent eligibility criteria, that aimed to limit the impact of comorbid conditions or treatment on cardiac dysautonomia, biochemical variables and olfactory function, our series was relatively small. However the sample size was similar to previous studies comparing iRBD and PD for multiple domains [38]. The gender imbalance between PD and iRBD groups is in accordance with the known male prevalence among iRBD [33]. We specifically excluded patients with a family history of PD, which is known to increase the risk of PD development by 3–5 fold [7,8].
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A)
concurrent therapy might have mitigated orthostatic blood pressure changes. The frequency of RBD in PD patients has been reported to range from 15 to 59%. Older age, higher prevalence of male gender, orthostatic hypotension and cognitive impairment and lower frequency of tremorpredominant PD have been reported in the literature among PD patients with than in those without RBD [40]. Within our PD group, 40% of patients gave an affirmative answer to the RBD screening question but, possibly because of the small sample size, we found no significant differences between groups.
Blood reduced GSH (mmol/L)
1400 R=0.34 p=0.037
1200 1000 800 600 400
5. Conclusions
200
Reduced systemic antioxidant capacity is found in prodromal and overt PD and may represent, in association with olfactory loss and cardiovascular autonomic dysfunction, a useful additive biomarker of disease. Our pilot findings need to be confirmed in a larger population to establish their actual clinical value for an early diagnosis of PD.
0 0
B)
171
5
2.5
10 15 20 25 30 Total olfactory score
35
40
R=0.43 p=0.015
Conflict of interest
2 VM ratio
The authors declare that they have no conflict of interest.
1.5
Acknowledgments This study was supported by Regione Lombardia-CNR 2013–2015 Framework Agreement, grant 18089/RCC, MbMM Project. We thank Elisabetta Spagnolo for expert secretarial support.
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References
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C)
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rho=-0.39 p=0.016
300 250 200 150 100 50 0
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Fig. 2. Correlation between total olfactory score and blood reduced GSH (A), Valsalva ratio (B), and urinary neopterin (C).
Reduced antioxidant capacity alone or combined with other prodromal features may have a different predictive value for PD in this population subset. We did not study drug-naive subjects, which is important to trace the progression from prodromal to early disease, but a homogeneous PD group for disease stage and treatment. PD medications have shown differential effects on the autonomic responses, with chronic dopaminergic agonists increasing and levodopa reducing the orthostatic drop in blood pressure [39]. In our PD patients
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Antioxidant and inflammatory biomarkers for the identification of prodromal Parkinson's disease Jonica Campolo a, Renata De Maria a,⁎, Lorena Cozzi a, Marina Parolini a, Stefano Bernardi a, Paola Proserpio b, Lino Nobili b, Giorgio Gelosa c, Immacolata Piccolo d, Elio C Agostoni c, Maria G Trivella a, Paolo Marraccini a a
CNR Institute of Clinical Physiology, ASST- Great Metropolitan Hospital Niguarda, Milan, Italy Epilepsy Surgery Centre, Centre of Sleep Medicine, ASST- Great Metropolitan Hospital Niguarda, Milan, Italy c Neurology and Stoke Unit, Department of Neuroscience, ASST-Great Metropolitan Hospital Niguarda, Milan, Italy d Spinal Unit, ASST- Great Metropolitan Hospital Niguarda, Milan, Italy b
a r t i c l e
i n f o
Article history: Received 14 July 2016 Received in revised form 9 September 2016 Accepted 25 September 2016 Available online 26 September 2016 Keywords: Parkinson's disease Idiopathic REM behavior disorder Olfactory dysfunction Cardiac dysautonomia Glutathione
a b s t r a c t Objectives: We explored the role of oxidative stress and inflammatory molecules as potential Parkinson (PD) biomarkers and correlated biological with non-motor abnormalities (olfactory impairment and dysautonomia), in patients with idiopathic REM behavior disorder (iRBD) (prodromal PD) and established PD. Methods: We recruited 11 iRBD and 15 patients with idiopathic PD (Hohen&Yahr 1–3, on L-DOPA and dopamine agonists combination therapy) and 12 age- and sex-matched controls (CTRL). We measured total olfactory score (TOS), autonomic function [deep breathing (DB), lying to standing (LS) and Valsalva manoeuvre (VM) ratios], blood reduced glutathione (Br-GSH), oxidative stress and inflammatory markers (neopterin). Results: Anosmia was similarly prevalent in iRBD (36%) and PD (33%) patients, but absent in CTRL. Orthostatic hypotension was more common among iRBD (73%) and PD (60%) than in CTRL (25%). By univariable ordinal logistic regression, TOS, Br-GSH, LS and VM ratio worsened from CTRL to iRBD and PD groups. Only reduced Br-GSH levels (p = 0.037, OR = 0.994; 95%CI 0.988–1.000) were independently associated to PD. TOS correlated with Br-GSH (R = 0.34, p = 0.037), VM ratio (R = 0.43, p = 0.015), and neopterin (rho = 0.39, p = 0.016). Conclusions: Reduced systemic antioxidant capacity is found in prodromal and overt PD and may represent, in association with olfactory loss and cardiovascular dysautonomia, a useful biomarker for an integrative, early diagnosis of PD. © 2016 Published by Elsevier B.V.
1. Introduction Early identification of Parkinson disease (PD) is a main challenge in neurological research. To date, the clinical diagnosis of PD is primarily based on the late onset of motor disorders, which manifest when 60– 70% of the dopaminergic neurons are degenerated in the substantia nigra [1]. PD is now recognized as a more complex illness, encompassing non motor symptoms (NMS), such as sleep disturbances, sensory abnormalities, autonomic dysfunction, depression and cognitive decline [2], that may predate the onset of motor symptoms by many years and are currently being studied as features of prodromal PD [3]. Neuropathological changes related to NMS include findings of α-synuclein-containing Lewy bodies and Lewy neurites in the peripheral autonomic nervous system and neuronal loss in the dorsal motor nucleus of the vagal nerve, the olfactory bulb, and the lower brainstem nuclei that regulate ⁎ Corresponding author at: CNR Institute of Clinical Physiology Milan, ASST-Great Metropolitan Hospital Niguarda, Milan, Italy. E-mail address: [email protected] (R. De Maria).
http://dx.doi.org/10.1016/j.jns.2016.09.050 0022-510X/© 2016 Published by Elsevier B.V.
REM sleep atonia [4]. These abnormalities might be responsible for the occurrence before the onset of parkinsonism of NMS such as dysautonomia, hyposmia, and REM sleep behavior disorders (iRBD), that have been prospectively linked to PD in population [5] and cohort studies [6], as recently reviewed [7,8]. Oxidative stress causes tissue injury and inflammation [9] and may play a role in PD. The autoxidation of dopamine in the dopaminergic neurons may produce reactive oxygen species (ROS). Mitochondrial dysfunction in nervous cells may increase oxidative stress by alteration of the respiratory chain, the activated microglia, responsible for chronic neuroiflammation, but also for ROS release after activation of specific enzyme, such as NADPH oxidase and inducible nitric oxide synthetase [10]. Elevated oxidative stress and a pro-inflammatory response occur early in the course of PD and contribute to exacerbate nigro-striatal degeneration [11]. Aims of our study were to explore the potential role of antioxidant and inflammatory biomarkers as novel tools for prodromal diagnostic evaluation of PD and to correlate biological findings with functional alterations, such as olfactory impairment and cardiovascular dysautonomia in patients with prodromal and established PD.
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2. Methods 2.1. Participants We recruited eligible subjects aged ≥50 e ≤ 80 years consecutively referred between May 2014 and May 2015 by the Centre of Sleep Medicine (iRBD) and the Neurology outpatient clinic (PD) of our Hospital and age and gender-matched controls (CTRL) who attended our Institute for laboratory assessment. Subjects presented none of the exclusion criteria listed below: - other neurological disorder, major mental disorder, cognitive impairment (MMSE b26); - previous myocardial infarction, heart failure, pacemaker, atrial fibrillation, beta-blockers; - glomerular filtration rate b 15 mL/min; - hepatic insufficiency - long standing (N10 years) diabetes; subjects with hyperglycemia (serum glucose N100 and b126 mg/dL) without antidiabetic treatment could be enrolled
iRBD [12] had to meet the following criteria: - repeated episodes of sleep-related vocalization and/or complex motor behaviors; - video-polysomnographic (PSG) documentation of occurrence during REM sleep or history suggestive of dream enactment; - polysomnographic observation of REM sleep without atonia; - exclusion of other sleep disorder, medication, or substance use.
Patients with idiopathic PD, confirmed by a neurologist with expertise in movement disorders (EFNS/MDS-ES 2013 guidelines), had to be in Hohen & Yahr stage ≥1 and ≤3 and on L-DOPA and dopamine agonists combination therapy, titrated to maintenance doses since at least 1 month. We excluded PD patients with any of the following: -
genetic etiology or familial clustering; atypical or secondary parkinsonism; history of cerebrovascular events; treatment with COMT inhibitors.
The study was approved by Niguarda Hospital Ethics Committee. All patients expressed their written informed consent to participate. 2.2. Study measurements Eligible subjects underwent: medical and drug history taking; physical and neurological examination; iRBD screening question [13] olfactory and autonomic function testing; fasting blood and urine sampling. PD subjects were administered the Movement Disorder Society-UPDRS Parts I-II (nMEDL and MEDL scores) and underwent functional testing on medications. 2.3. Biochemical assessments Glutathione (GSH), the most important endogenous scavenger, was assessed in total and reduced form and in plasma and blood samples by high performance liquid chromatographic (HPLC) [14]. Plasma malondialdehyde (MDA), a marker of lipid peroxidation, was assayed by a commercial kit (Chromsystems, Germany) in HPLC with fluorescence detection. Commercial ELISA kits were used for plasma assay of 8-hydroxy-2-deoxyguanosine (8-OHdG), index of oxidative DNA damage (Trevigen, Gaithersburg, MD, USA); 3-nitrotyrosine (3-NT), a stable end product of peroxynitrite oxidation (Hycult Biotech (Uden, The
Nederland)); the inflammatory cytokines tumor necrosis factor alpha (TNFα) (Cayman Chemical Company, Ann Arbor, MI, USA) and interleukin 1-beta (IL1β), Boster Immunoleader (Pleasanton, CA, USA). Urine neopterin levels, a sensitive marker of cellular-mediated inflammation, were measured by an isocratic HPLC method and normalized by urine creatinine concentrations [15].
2.4. Functional testing Olfactory function was assessed by the Sniffin' Sticks Extended Test (Burghart, Medizintechnik, GmbH, Wedel, Germany) [16]. The olfactory threshold is the minimum concentration of an odorant (n-butanol) that can be detected by a subject when presented with 16 different dilutions in felt tip pens. Olfactory discrimination assesses the ability to discriminate between different odorants in 16 different triplets. Olfactory identification evaluates the ability to correctly identify an odorant among four possible odors for each of 16 trials. The total olfactory score (TOS) was calculated as sum of the 3 sub-scores for olfactory threshold, discrimination and identification and reclassified as normal olfaction (between 31 and 48), hyposmia (between 16 and 30) and anosmia (≤15). Autonomic function [17] was tested in the supine position at a comfortable ambient temperature. Heart rate was recorded via standard 12lead electrocardiogram (Norav PC ECG-1002). Blood pressure was measured non-invasively by a manual sphygmomanometer.
2.4.1. Deep breathing (DB) After 10-minute rest, subjects performed 1 minute DB (6 inspiratory and expiratory cycles of 5 sec each) during continuous ECG recording. The DB expiration/inspiration ratio was calculated as ratio of averages of the three longest RR intervals during expiration and the three shortest RR intervals during inspiration.
2.4.2. Lying to standing (LS) After 10-minute rest, patients were instructed to stand up and remain standing for 5 min. Changes in systolic/diastolic blood pressure and heart rate were assessed after 1 and 5 minutes standing from the supine position. Orthostatic hypotension (OH) was defined as a drop ≥ 20 mm Hg in systolic and/or ≥ 10 mm Hg in diastolic blood pressure. The LS 30/15 ratio was the ratio between the longest RR interval measured between the 25th and 35th beat after active standing and the shortest RR interval between the 10th and 20th beat.
2.4.3. Valsalva manoeuvre (VM) Patients in the sitting position were instructed to blow into a tube connected to a manometer to maintain the pressure at 40 mm Hg for 15 s, during continuous heart rate and blood pressure monitoring. VM ratio was the ratio of the shortest RR interval (tachycardia) during expiration to the longest RR interval (bradycardia) after expiration.
2.5. Statistical analysis Data are presented as median (interquartile range) or frequency (percentage). Categorical variables were compared by the chi-square test. Pearson's R correlation coefficient or Spearman's rho index were used to correlate continuous clinical, biochemical and functional variables (age, symptom duration, nMEDL, MEDL, GSH, Neopt, MDA, 3-NT, 8-OHdG, TNFα, IL1β, TOS, LS 30:15 ratio, VM ratio, DB-ratio). Associations with the dependent variable “group” were tested by univariable ordinal logistic regression; variables with p b 0.10 were entered in a multivariable model. A p value b 0.05 was considered significant. Statistical analyses were carried out with the Statistical Package for the Social Sciences (SPSS Inc., Chicago, Illinois, USA), version 17.0 for Windows.
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3. Results
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Table 2 Clinical characteristics of disease severity in PD group.
We enrolled 15 patients with PD, 11 subjects with PSG-confirmed iRBD and 12 CTRL (Table 1).
Median (I;III) Hohen & Yahr score Years onset motor symptoms Years from diagnosis UPDRS Part I UPDRS Part II, nMEDL UPDRS Part II, MEDL UPDRS Part II total DOPA dose (mg/day) Rotigotine dose (mg/day) Ropirinole dose (mg/day) Pramipexole dose (mg/day)
3.1. Findings in PD patients PD patients had long standing disease with mild to moderate motor and NMS burden (Table 2). Only 2 patients presented on-off phenomena and dyskinesia. Disease duration correlated with L-DOPA dose (R = 0.650, p = 0.012) and with the DB E/I ratio (rho = − 0.642, p = 0.021), but not with nMEDL or TOS. nMEDL score was inversely related to VM ratio (R = − 0.726, p = 0.017) and to TOS (R = − 0.873, p b 0.001). Conversely, MEDL correlated with age (R = 0.523, p = 0.045) but not with any biochemical or functional variable. Six PD patients (40%) reported sleep behaviors compatible with RBD at the screening question; no differences were observed between patients with and those without suspect RBD either in clinical findings or in functional and biochemical characteristics. 3.2. Functional and biochemical differences among PD, iRBD and CTRL Among clinical characteristics, CTRL had more commonly a history of hypertension than iRBD and PD. A significant worsening trend was observed in total olfactory score, blood reduced GSH, LS and VM ratio and neopterin from CTRL to iRBD and PD groups (Table 1). By multivariable ordinal logistic regression, only low blood reduced GSH levels (p = 0.037, OR = 0.994; 95% CI 0.988–1.000), adjusted by history of hypertension, total olfactory score, LS 30:15 ratio and VM ratio and urinary neopterin, was associated to PD.
2.5 (2.0;2.5) 10.0 (6.0;12.8) 6.0 (3.5;9.5) 2.0 (1.0;6.0) 9.0 (5.0;11.0) 12.0 (7.0;14.0) 19 (16;26) 500 (300;850) 9.0 (6.5;11.5) 6.0 (6.0;6.0) 2.10 (1.05;2.10)
Data are expressed as median and interquartile range (I;III). P for between-group differences by unpaired Student's t-test or Mann-Whitney test*. MEDL, motor experiences of daily living; nMEDL, non-motor experiences of daily living.
The cumulative effect of olfactory impairment, dysautonomia, depressed mood and reduced antioxidant capacity (blood GSH ≤ 468 μmol/L 10th percentile of CTRL distribution) for group discrimination was assessed as number of risk factors: over half of both iRBD and PD presented at least two features combined (fig. 1). 3.4. Correlations among clinical, biochemical and functional variables We tested in the overall population the relationship among clinical, biochemical and functional variables. Significant correlations were observed only between TOS and blood reduced GSH concentrations (panel A), VM ratio (panel B), and urine neopterin levels (panel C) (Fig. 2). 4. Discussion
3.3. Group comparison with respect to functional category Functional anosmia was similarly prevalent in iRBD (36%) and PD (33%) patients, but was absent in CTRL (p = 0.097). OH was more common among iRBD (73%) and PD (60%) than in controls (25%) (p = 0.055), independently of antihypertensive treatment.
The main findings of the present study are that reduced systemic antioxidant capacity is independently associated to overt PD and iRBD, a condition now established as prodromal PD, and correlates with olfactory and sympathetic dysfunction. Moreover progressive cardiovascular autonomic dysfunction, expressed as altered sympathetic (VM ratio,
Table 1 Univariable ordinal logistic regression analysis of clinical, biochemical and functional characteristics of the study groups.
Age, years Gender (M), n% Smoking habit, n% Hypercholesterolemia, n(%) Hyperglycemia n(%) Hypertension, n(%) Depressive mood, n(%) MMSE Blood reduced GSH, μmol/L Blood total GSH, μmol/L Plasma reduced GSH, μmol/L Plasma total GSH, μmol/L Plasma MDA, μmol/L Plasma 3-NT, nmol/L Plasma 8-OHdG, nmol/L Urine neopterin, (μmol/mol creat) Plasma TNFα, pg/mL Plasma IL1β, pg/mL Total olfactory score LS 30:15 ratio SBP drop at 1 minute standing DBP drop at 1 minute standing VM ratio DB E:I ratio
CTRL (n = 12)
IRBD (n = 11)
PD (n = 15)
p
OR
95% CI
71 (59;74) 7 (58%) 2 (17%) 4 (33%) 3 (25%) 7 (58%) 3 (25%) 28 (27;28) 759 (650;833) 1364 (1205;1521) 1.13 (0.88;1.57) 6.95 (6.35;8.66) 0.16 (0.14;0.17) 12.7 (6.1;29.3) 279 (229;348) 127 (93;188) 26 (21;46) 10.9 (8.9;13.6) 28.7 (22.2;33.0) 1.22 (1.05;1.37) −2.5 (−16.2;−12.5) 0 (−7.5;5.0) 1.50 (1.31;1.73) 1.17 (1.11;1.27)
71 (64;73) 10 (91%) 4 (36%) 2 (20%) 4 (36%) 4 (36%) 3(27%) 28 (27;29) 582 (380;715) 1091 (923;1563) 1.05 (0.72;1.61) 5.60 (4.25;10.90) 0.15 (0.14;0.16) 19.7 (17.1;48.6) 227 (180;307) 141 (114;185) 34 (22;66) 15.0 (12.0;16.1) 17.5 (11.0;21.5) 1.13 (1.07;1.19) −15.0 (−20.0;−10.0) −10.0 (−15.0; −5.0) 1.38 (1.26;1.45) 1.14 (1.09;1.21)
70 (58;76) 7 (47%) 1 (7%) 2 (13%) 3 (21%) 3 (20%) 8 (53%) 29 (27;30) 519 (467;639) 1070 (860;1743) 0.86 (0.69;1.01) 6.10 (4.57;7.60) 0.14 (0.13;0.16) 18.2 (10.0;34.5) 280 (209;357) 164 (125;226) 28 (22;60) 10.9 (8.4;14.5) 18.7 (15.0;24.0) 1.03 (1.01;1.18) −20.0 (−30.0;−5.0) −5.0 (−15.0;0) 1.27 (1.14;1.53) 1.15 (1.11;1.26)
0.575 0.365 0.473 0.216 0.805 0.043 0.110 0.620 0.009 0.892 0.181 0.132 0.229 0.585 0.696 0.083 0.421 0.582 0.009 0.047 0.100 0.116 0.047 0.958
0.981 0.566 0.573 0.392 0.845 0.267 0.355 1.147 0.995 1.000 0.736 0.852 0.000 0.998 1.001 1.107 1.004 1.043 0.890 0.004 0.977 0.952 0.045 0.879
0.920–1.047 0.165–1.944 0.125–2.622 0.089–1.726 0.223–3.212 0.075–0.960 0.100–1.261 0.668–1.970 0.991–0.999 0.998–1.002 0.469–1.155 0.692–1.049 0.001–8323 0.991–1.005 0.995–1.007 0.987–1.242 0.992–1.016 0.897–1.213 0.815–0.973 0.001–0.919 0.951–1.004 0.896–1.012 0.002–0.961 0.007–111
Data are expressed as median and interquartile range (I;III) or as frequency (%). DB, deep-breathing; E:I expiration/inspiration; IL, interleukin; LS, lying to standing; GSH, glutathione, MDA, malondialdehyde; TNF, tumor necrosis factor; VM, Valsalva manoeuvre. Bold values indicate significance at p value b 0.05.
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p=0.050 100%
80%
60%
40%
20%
Number of risk factors 3 2 1 0
0% CTRL
iRBD
PD
Fig. 1. Comparison among CTRL, iRBD and PD groups for number of the following risk factors: anosmia, orthostatic hypotension, depression or low blood GSH.
OH) or parasympathetic (LS 30:15 ratio) response to testing, is found from prodromal state to overt disease and correlates with olfactory dysfunction. The combination of two or more features was distinctly different between CTRL vs iRBD and PD. GSH, the most abundant aminothiol in cells, acts in the brain as antioxidant and redox regulator [18]. Experimental results in PD models and analyses of postmortem brain tissue from PD patients point to a profound loss of GSH in the substantia nigra, that parallels the severity of disease and occurs prior to other hallmarks of tissue damage [19]. GSH depletion may affect mitochondrial function through selective inhibition of mitochondrial complex I enzyme in the respiratory chain [20], with excess ROS production and decreased ATP levels. The low GSH concentrations found in blood cells of patients with both overt and prodromal PD suggest antioxidant depletion to be present not only in brain tissue, but also in systemic cellular districts, already in the early stages of disease. This perturbation might be confined to the intracellular space, since we did not find any difference in plasma GSH content and oxidative stress biomarkers. Blood reduced GSH concentrations paralleled the progression from prodromal to overt disease, as expressed by olfactory impairment and autonomic dysfunction, and were directly related to TOS in the overall population. The long established observation of olfactory dysfunction as a common early feature of PD, which often precedes motor symptoms by several months or even years [21] is grounded in Braak's hypothesis [22]. The accumulation of Lewy bodies follows a predictable sequence from the anterior olfactory nucleus and bulb and the dorsal motor nuclear complex of the glossopharyngeal and vagal nerves, through the brain stem toward the midbrain. In our cohort, olfactory dysfunction worsened from CTRL to iRBD and PD, with functional anosmia being present only in both iRBD and PD. Olfactory dysfunction did not correlate with disease duration in our PD group, consistently with previous reports of lack of deterioration in odor identification, discrimination, and threshold detection in the course of PD [23,24]. Olfactory loss may be an important preclinical marker, detectable by non-invasive low-cost methods. Conversely, hyposmia, which is strongly influenced by age, was unable to discriminate iRBD or PD patients from CTRL. On the same line, worse olfactory dysfunction correlated with increased neopterin levels, which were not independently predictive of PD, as aging per se is a low-grade
inflammatory state. Olfactory dysfunction in iRBD has been suggested to portend conversion to PD [25,26]. Cardiovascular dysautonomia in PD affects both the sympathetic and parasympathetic autonomic nervous system [27,28], with OH present in up to 52% of PD patients [29]. Supposed OH mechanisms in PD include baroreflex failure due to central lesions in the upper brainstem and post-ganglionic impairment, with loss of cardiac and peripheral vascular sympathetic innervation. While the severity of parasympathetic dysfunction worsens with disease progression [23], as confirmed in our patients by the inverse relation between PD duration and the E/I ratio, OH may predate PD development [30] and is associated with neuroimaging evidence of cardiac and extra-cardiac sympathetic denervation. In a large series, iRBD had significantly more often postural dizziness than controls [31]. iRBD who converted to a neurodegenerative disease had a higher prevalence of cardiovascular and gastrointestinal symptoms [32] and OH, with a larger systolic blood pressure drop from lying to standing than non converters [33]. In our cohort, OH prevalence overlapped among iRBD and PD patients at approximately 60%, almost 3-fold higher than in CTRL. We likewise observed a lower Valsalva ratio, a progressive decline in the LS ratio and larger systolic and diastolic blood pressure drop in iRBD, of intermediate severity with PD, than in CTRL. The combination of features, such as reduced TOS, OH and depression, has been previously shown to improve prediction of PD in community studies [25,26,34,35]. These prodromal markers have been included in the recent research criteria for prodromal PD with high positive likelihood ratios for PD development [36]. Our finding of improved discrimination of both iRBD and PD from CTRL with low GSH concentrations suggests that reduced antioxidant capacity may be a promising blood biomarker [37] and should be tested in larger series to assess its value as predictor of conversion from iRBD to PD or dementia. Our study presents several limitations. As a consequence of stringent eligibility criteria, that aimed to limit the impact of comorbid conditions or treatment on cardiac dysautonomia, biochemical variables and olfactory function, our series was relatively small. However the sample size was similar to previous studies comparing iRBD and PD for multiple domains [38]. The gender imbalance between PD and iRBD groups is in accordance with the known male prevalence among iRBD [33]. We specifically excluded patients with a family history of PD, which is known to increase the risk of PD development by 3–5 fold [7,8].
J. Campolo et al. / Journal of the Neurological Sciences 370 (2016) 167–172
A)
concurrent therapy might have mitigated orthostatic blood pressure changes. The frequency of RBD in PD patients has been reported to range from 15 to 59%. Older age, higher prevalence of male gender, orthostatic hypotension and cognitive impairment and lower frequency of tremorpredominant PD have been reported in the literature among PD patients with than in those without RBD [40]. Within our PD group, 40% of patients gave an affirmative answer to the RBD screening question but, possibly because of the small sample size, we found no significant differences between groups.
Blood reduced GSH (mmol/L)
1400 R=0.34 p=0.037
1200 1000 800 600 400
5. Conclusions
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Reduced systemic antioxidant capacity is found in prodromal and overt PD and may represent, in association with olfactory loss and cardiovascular autonomic dysfunction, a useful additive biomarker of disease. Our pilot findings need to be confirmed in a larger population to establish their actual clinical value for an early diagnosis of PD.
0 0
B)
171
5
2.5
10 15 20 25 30 Total olfactory score
35
40
R=0.43 p=0.015
Conflict of interest
2 VM ratio
The authors declare that they have no conflict of interest.
1.5
Acknowledgments This study was supported by Regione Lombardia-CNR 2013–2015 Framework Agreement, grant 18089/RCC, MbMM Project. We thank Elisabetta Spagnolo for expert secretarial support.
1 0.5
References
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0
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40
C)
Neopterin (mmol/molcreat)
350
rho=-0.39 p=0.016
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Fig. 2. Correlation between total olfactory score and blood reduced GSH (A), Valsalva ratio (B), and urinary neopterin (C).
Reduced antioxidant capacity alone or combined with other prodromal features may have a different predictive value for PD in this population subset. We did not study drug-naive subjects, which is important to trace the progression from prodromal to early disease, but a homogeneous PD group for disease stage and treatment. PD medications have shown differential effects on the autonomic responses, with chronic dopaminergic agonists increasing and levodopa reducing the orthostatic drop in blood pressure [39]. In our PD patients
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