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Increased plasma levels of phospholipid in Parkinson’s disease with mild cognitive impairment
Journal of Clinical Neuroscience 22 (2015) 1268–1271
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
Increased plasma levels of phospholipid in Parkinson’s disease with mild cognitive impairment Zhenguang Li, Jinbiao Zhang ⇑, Hairong Sun Department of Neurology, Weihai Municipal Hospital, The Affiliated Hospital of Binzhou Medical College, 70 Heping Street, Weihai, Shandong Province 264200, China
a r t i c l e
i n f o
Article history: Received 28 July 2014 Accepted 3 February 2015
Keywords: Mild cognitive impairment MoCA scores Parkinson’s disease Phospholipids
a b s t r a c t This study aimed to observe the clinical characteristics and changes in plasma phospholipid (PL) concentrations in Parkinson’s disease patients with mild cognitive impairment (PD-MCI), and to investigate the association between these changes and cognitive function to provide a basis for early diagnosis and intervention for PD-MCI patients. Oxidative stress plays an important role in the development and progression of Parkinson’s disease (PD). PL, important components in cellular membranes, are critical for the maintenance of cell integrity and function. Lipid peroxidation products are significantly increased in the brains of PD patients. In the present study, plasma PL levels were significantly increased in PD-MCI patients or in PD patients with no cognitive impairment (PD-NCI) compared with controls (p < 0.01 and p < 0.05, respectively). PL levels were significantly increased in the PD-MCI group compared with PD-NCI patients (p < 0.01). There was a negative correlation between plasma PL levels and Montreal cognitive assessment scores (r = 0.542; p < 0.001). These findings support the relationship between mild cognitive impairment and membrane injury. The measurement of PL reflects membrane injury in vivo and may be a new useful biomarker for the prognosis of cognitive states in patients with PD. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Parkinson’s disease (PD) is a degenerative disease of the central nervous system that involves many other systems. In addition to motor symptoms, non-motor symptoms are important components of PD clinical manifestation [1]. The cognitive impairment caused by the involvement of the cortex in PD patients is one of the major non-motor PD symptoms with an incidence of approximately 20–50% [2]. Mild cognitive impairment (MCI) is common in non-demented PD patients [3], also called PD-MCI. A proportion of PD patients already have MCI before motor symptoms manifest. Early identification and timely intervention for PD-MCI are important for improving the prognosis and quality of life of PD patients. Molecular pathological studies have shown that [4] oxidative stress plays an important role in the development and progression of PD. Phospholipids (PL) are important components in cellular membranes, and are critical for the maintenance of their integrity and function. However, studies on the changes in plasma PL in PD-MCI patients are lacking. This study aimed to observe the clinical characteristics and changes of plasma PL concentrations in PD-MCI patients and to investigate the association between these ⇑ Corresponding author. Tel.: +86 063 1528 7598; fax: +86 063 1589 0955. E-mail address: [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.jocn.2015.02.013 0967-5868/Ó 2015 Elsevier Ltd. All rights reserved.
changes and cognitive function in order to provide a basis for early diagnosis and intervention for PD-MCI patients.
2. Materials and methods 2.1. Patients The diagnosis of PD was made according to the UK Parkinson’s Disease Society Brain Bank Clinical Diagnostic Criteria. The inclusion criteria were the following: patients with idiopathic PD diagnosed by neurologists with clinical experience and according to the diagnostic criteria; and patients treated with conventional medications (levodopa or dopamine receptor agonists). The diagnostic criteria for PD-MDI were based on the diagnostic criteria formulated by the Movement Disorder Society (MDS) in the USA [3]. Further inclusion criteria were: in patients with a confirmed diagnosis of PD, gradual decrease in cognitive function was reported by patients/informants or observed by physicians; patients with cognitive impairment confirmed by formal neuropsychological testing or cognitive rating scales; and the cognitive impairment did not significantly affect functional independence, though minor difficulties in the execution of complex tasks were permitted. The exclusion criteria were the following: patients with Parkinsonian syndrome secondary to stroke, trauma, or other
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neurological and psychiatric disorders; patients with Parkinsonism plus syndrome; patients with malignant tumors, disabilities, or other severe physical diseases; patients who underwent surgical treatment, deep brain stimulation, stem cell transplantations, or other medical treatment. Other exclusion criteria based on the MDS were: patients who met the Parkinson’s disease dementia diagnosis proposed by the MDS specialty group; other causes of cognitive impairment (such as delirium, stroke, severe depression, metabolic disorders, drug side effects and head trauma); patients with PD comorbidities (such as movement disorders, severe anxiety, depression, excessive daytime sleepiness, and mental disorders). According to the above inclusion and exclusion criteria, 85 PD patients who were treated in the neurology clinic or were admitted to our hospital from May 2006 to June 2011 were enrolled. A total of 75 healthy individuals older than 55 years of age who received physical examinations comprised the control group. The control individuals had no mental disorders such as anxiety and depression, no history of severe physical disorders in the previous month, normal thyroid gland function, no physical disability or hearing or visual impairment, and no history of alcohol abuse. The severity of patients’ motor symptoms was evaluated using Part III of the unified Parkinson’s disease rating scale and the Hoehn and Yahr scale. MCI was evaluated using the Chinese version of the Montreal Cognitive Assessment (MoCA) including visuospatial ability, executive function, naming tasks, attention tasks, abstraction, language, delayed memory recall and orientation. The range of total scores on this scale is 0–30 points. If the subject’s years of education were 612 years then 1 point was added to correct the bias for educational level. A score P26 points was considered normal and MCI was indicated by a score between 15–25 points. The testing time was approximately 10 min. Two assessors, who received standardised training, completed all of the evaluation tests. 2.2. Measurement of plasma PL levels Patients were asked to avoid fatty food and alcohol for 7 days before fasting blood samples were obtained in the morning. We did not draw blood from females if they were in their menstrual period. Venous blood (4 mL) was drawn from each participant into commercially available anticoagulant tubes. Plasma was either processed immediately or stored at 70°C before lipid extraction. Lipid extraction was performed at 0–4°C to minimize damage to ester bonds, using a slight modification of published methods [5]. Whole blood was centrifuged at 3500g-force for 10 min. The supernatant was transferred to a microcentrifuge tube and centrifuged at 8000 g-force for 10 minutes. Then 1 mL suspension (platelet-poor plasma) was used for the remaining procedure. Lipids were extracted with 4 mL of 1-butanol and 2 mL of water-saturated 1-butanol. The resulting organic extracts were pooled and dried in vacuo. Each sample was resuspended in 0.3 mL of chloroform/methanol/water/28% ammonium hydroxide (NH4OH; 250:100:15:0.3, v/v) and was immediately filtered by an Econosphere 3 lm, 50 4.6 mm silica column (Alltech Associates, Deerfield, IL, USA). Compounds were eluted with a mobile phase of chloroform/methanol/water/28% NH4OH (250:100:15:0.3, v/v) at 0.5 mL/minute. The source was maintained at 250°C with a drying gas flow of 10 L/hour. The concentration of PL was quantified by measuring its inorganic phosphorus component using colorimetric assays. 2.3. Statistical analyses All of the statistical analyses were performed using SPSS statistics (version 13.0; IBM Corporation, Armonk, NY, USA). The Kolmogorov–Smirnov test was performed to ascertain the
normality of the distribution of continuous variables. Tests on the homogeneity of variances were also performed. Continuous variables were analyzed by one-way analysis of variance (ANOVA). Categorical variables were analyzed using the chi-squared test. The correlation between PL levels and MoCA scores were analyzed by Spearman’s linear correlation. All analyses were two-tailed. Results were expressed as mean ± standard deviation. Values were considered significant at p < 0.05. 3. Results 3.1. Demographic characteristics of study subjects Among 85 PD patients, 44 (51.8%) had normal cognitive function (PD-NCI; MoCA P26 points) and 41 had MCI (PD-MCI; 15 points < MoCA < 26 points). PD-NCI patient (32 male and 12 female) had a mean age of 63.5 years (range 50–76 years). PD-MCI patients (30 male and 11 female) had a mean age of 65.4 years (range 51–75 years). 75 controls (54 male and 21 female) with mean age 64.1 years (range 55–75 years), were also included in the study. There was no significant difference in sex, age, education, disease duration and use of levodopa or receptor agonists (p > 0.05; Table 1). 3.2. Comparison of MoCA scores The scores of each cognitive domain in the MoCA scale of subjects in the different groups are shown in Table 2. The results of one-way ANOVA and pairwise comparisons indicated that the scores of all items in the PD-MCI group were lower than those in the PD-NCI group (p < 0.01) and the control group (p < 0.01; Table 2). 3.3. Comparison of plasma PL concentration Plasma PL concentration in the PD-NCI and PD-MCI groups were significantly higher compared with the control group (p < 0.05 and p < 0.01, respectively). Furthermore, the concentration of plasma PL in the PD-MCI group was significantly increased compared with that in the PD-NCI group (p < 0.01; Table 3). 3.4. Correlation between plasma PL levels and MoCA scores There was a negative correlation between plasma PL levels and MoCA scores (r = 0.542; p < 0.001; Fig. 1). 4. Discussion In this study, we investigated the plasma PL levels in PD patients with mild cognitive impairment. Additionally, we
Table 1 Baseline characteristics of healthy controls and PD-NCI and PD-MCI patients Characteristic
Control (n = 75)
PD-NCI (n = 44)
PD-MCI (n = 41)
Age, years (mean ± SD) Sex, male/female Education, years (mean ± SD) DD, months (mean ± SD) UPDRS III score Use of levodopa (%) Use of receptor agonists (%)
64.1 ± 6.7 54/21 9.9 ± 7.9
63.5 ± 6.4 32/12 10.8 ± 7. 7
65.4 ± 7.1 30/11 11.6 ± 8.2
– – – –
87.6 ± 21.3 18.6 ± 6.7 56 39
90.2 ± 22.9 20.4 ± 7.8 57 45
– = not applicable, DD = disease duration, PD-MCI = Parkinson’s disease with mild cognitive impairment, PD-NCI = Parkinson’s disease with no cognitive impairment, SD = standard deviation, UPDRS = Unified Parkinson’s disease rating scale.
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Table 2 Comparison of scores of all items in the MoCA scale for healthy controls, PD-NCI and PD-MCI patients MoCA variable
Control (n = 75)
PD-NCI (n = 44)
PD-MCI (n = 41)
Visuospatial ability Naming task Attention Language Abstraction Delayed memory recall Orientation
4.15 ± 0.57 3.00 ± 0.00 5.93 ± 0.78 3.17 ± 0.29 2.10 ± 0.48 3.59 ± 0.66
4.10 ± 0.65 2.88 ± 0.37 5.88 ± 1.03 2.90 ± 0.54 1.81 ± 0.62 3.22 ± 0.98
2.54 ± 0.87* 2.33 ± 0.50* 4.99 ± 0.91* 2.20 ± 0.39* 1.07 ± 0.55* 1.18 ± 1.02*
6.00 ± 0.00
5.95 ± 0.88
5.26 ± 0.63*
All values are reported as the mean ± standard deviation. MoCA = Montreal cognitive assessment, PD-MCI = Parkinson’s disease with mild cognitive impairment, PD-NCI = Parkinson’s disease with no cognitive impairment. * Compared with the control and PD-NCI group, p < 0.01.
Table 3 Comparison of plasma PL concentration and MoCA score in healthy controls, PD-NCI and PD-MCI patients Variable
Control (n = 75)
PD-NCI(44)
PD-MC(41)
PL (U) MoCA score
4.52 ± 0.27 27.36 ± 1.72
5.56 ± 0.65N 27.11 ± 1.78
7.27 ± 0.68*,NN 20.32 ± 2. 61**,NN
All values are reported as the mean ± standard deviation. MoCA = Montreal cognitive assessment, PD-MCI = Parkinson’s disease with mild cognitive impairment, PD-NCI = Parkinson’s disease with no cognitive impairment, PL = phospholipid. * Compared with the PD-NCI group, p < 0.05. ** Compared with the PD-NCI group, p < 0.01. N Compared with the control group, p < 0.05. NN Compared with the control group, p < 0.01.
analyzed the correlation between plasma PL levels and the severity of cognitive impairment. PL are important components of the biomembrane. In the central nervous system, PL are rich in polyunsaturated fatty acids (PUFA). The metabolism of PUFA is mainly regulated by phospholipase A2 (PLA2) and acyltransferase in a process called the
deacylation-reacylation cycle. Under normal conditions, the released free fatty acids (FFA) under the action of PLA2 will be rapidly uptaken by membrane PL. PLA2 mainly recognizes the sn-2 acyl bond of glycerophospholipids in lipoproteins and stimulates the cell membrane to release FFA and lysophospholipids [5]. This study showed that the concentrations of plasma PL in the PD-NCI and PD-MCI groups were significantly higher than that in the control group, suggesting that PD patients have membrane damage. Compared with PD-NCI patients, the increase in the plasma PL concentration in PD-MCI patients was even more significant, suggesting that metabolic disorders of PL play an important role in the pathology of cognitive impairments in PD patients and that the membrane damage in PD-MCI patients is more severe. Furthermore, there was a negative correlation between plasma PL levels and MoCA scores. Therefore, PL have the potential to become a plasma biomarker of cognitive impairment in PD. The possible mechanisms underlying the increase of PL in PD patients include oxidative stress and accumulation of free radicals [6,7]. This activates relevant phospholipases, thus causing lysis of membrane PL. Many studies have indicated that lipid peroxidation products are elevated in the brains of PD patients [8,9]. In the substantia nigra and the striatum, 8-hydroxydeoxyguanosine, nitrosyl groups, protein carbonyl groups, iron ions and total iron concentration increase significantly, and the level of reduced glutathione decreases. Therefore, cells are in a state of oxidative stress. Many potential factors can cause a state of oxidative stress in PD including mitochondrial dysfunction, increased free ion levels and reductions in antioxidant defense system functions [10,11]. Another possible mechanism is endogenous metabolic products, such as nitric oxide, reactive oxygen species, active ions, and toxicophores, along with deregulation of the ubiquitin-proteasome system, affecting the function of the respiratory chain in mitochondria and causing disorders of mitochondria and energy metabolism [12,13]. Also, decreased glucose uptake by the cerebral cortex and decreased blood perfusion in the frontal, temporal and parietal lobes can activate phospholipases, causing lysis of membrane PL [14,15]. Molecules such as hydrogen peroxide change the activity of certain phospholipases, such as phospholipase D and PLA2, to
Fig. 1. Correlation between plasma phospholipid (PL) levels and Montreal Cognitive Assessment (MoCA) scores. Analysis was by Spearman linear correlation (r = p < 0.001).
0.542;
Z. Li et al. / Journal of Clinical Neuroscience 22 (2015) 1268–1271
activate cytosolic PLA2 and calcium-independent phospholipase A2, causing the release of PL [16]. Because of the rapid hydrolysis of PL in the cell membrane, the lipid complex cannot supplement the lost PL in the cell membrane in a timely manner, causing neuronal degeneration or loss [14]. A study by Aarsland et al. [17] in 1346 PD patients from eight centers showed that 25.8% (range: 23.5–28.2%) of PD patients had MCI. The results of our study showed that among the 85 patients, 41 (48.2%) had MCI, which is higher compared to the above reports from other countries. This difference is thought to be associated with other factors such as ethnic differences and educational levels. The evaluation of the MoCA scale results indicated that the cognitive levels of PD-MCI patients were significantly lower than those of PD-NCI patients. This study compared the evaluation scores of each cognitive domain including visuospatial and executive functions, naming tasks, attention and calculation abilities, language, abstract thinking, delayed memory recall, and orientation, and demonstrated that the scores of all cognitive domains in the PD-MCI group were significantly lower compared with those in the PD-NCI and control groups. The scores for calculation and attention abilities, delayed memory recall, visuospatial and executive functions were the lowest, suggesting that the damage in these three cognitive domains in the PD-MCI group was more severe, similar to the report of Sollinger et al. [18]. In summary, the incidence of PD-MCI is high and includes impairment in attention, calculation, and executive tasks, delayed memory recall, and decreased visuospatial ability. Interventions for PD patients should pay close attention to PD-MCI for early diagnosis and timely treatment to increase the quality of life and reduce care and society burden. Metabolic disorders of PL play an important role in the pathology of cognitive impairment in PD patients, with membrane damage in PD-MCI patients found to be more severe. PL have the potential to become a novel plasma biomarker for cognitive impairment in PD patients. Conflicts of Interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.
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References [1] Goldman JG, Litvan I. Mild cognitive impairment in Parkinson’s disease. Minerva Med 2011;102:441–59. [2] Petersen RC, Smith GE, Waring SC, et al. Mild cognitive impairment: clinical characterization and outcome. Arch Neurol 1999;56:303–8. [3] Litvan I, Goldman JG, Tröster AI, et al. Diagnostic criteria for mild cognitive impairment in Parkinson’s disease: Movement Disorder Society Task Force guidelines. Mov Disord 2012;27:349–56. [4] Lee J, Giordano S, Zhang J. Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling. Biochem J 2012;441:523–40. [5] Kosicek M, Hecimovic S. Phospholipids and Alzheimer’s disease: alterations, mechanisms and potential biomarkers. Int J Mol Sci 2013;14:1310–22. [6] Peterson B, Knotts T, Cummings BS. Involvement of Ca2+-independent phospholipase A2 isoforms in oxidant-induced neural cell death. Neurotoxicology 2007;28:150–60. [7] Mathisen GH, Thorkildsen IH, Paulsen RE. Secretory PLA2-IIA and ROS generation in peripheral mitochondria are critical for neuronal death. Brain Res 2007;1153:43–51. [8] Onyango IG. Mitochondrial dysfunction and oxidative stress in Parkinson’s disease. Neurochem Res 2008;33:589–97. [9] Wood-Kaczmar A, Gandhi S, Wood NW. Understanding the molecular causes of Parkinson’s disease. Trends Mol Med 2006;12:521–8. [10] Fessel JP, Hulette C, Powell S, et al. Isofurans, but not F2-isoprostanes, are increased in the substantia nigra of patients with Parkinson’s disease and with dementia with Lewy body disease. J Neurochem 2003;85:645–50. [11] Seet RC, Lee CY, Lim EC, et al. Oxidative damage in Parkinson disease: measurement using accurate biomarkers. Free Radic Biol Med 2010;48:560–6. [12] Elkharaz J, Ugun-Klusek A, Constantin-Teodosiu D, et al. Implications for oxidative stress and astrocytes following 26S proteasomal depletion in mouse forebrain neurones. Biochim Biophys Acta 2013;1832:1930–8. [13] Dasuri K, Zhang L, Keller JN. Oxidative stress, neurodegeneration, and the balance of protein degradation and protein synthesis. Free Radic Biol Med 2013;62:170–85. [14] Schaeffer EL, da Silva ER, Novaes Bde A, et al. Differential roles of phospholipases A2 in neuronal death and neurogenesis: implications for Alzheimer disease. Prog Neuropsychopharmacol Biol Psychiatry 2010;34:1381–9. [15] Lovell MA, Xie C, Markesbery WR. Acrolein, a product of lipid peroxidation, inhibits glucose and glutamate uptake in primary neuronal cultures. Free Radic Biol Med 2000;29:714–20. [16] Schaeffer EL, Gattaz WF. Inhibition of calcium-independent phospholipase A2 activity in rat hippocampus impairs acquisition of short- and long-term memory. Psychopharmacology 2005;181:392–400. [17] Aarsland D, Bronnick K, Williams-Gray C, et al. Mild cognitive impairment in Parkinson disease: a multicenter pooled analysis. Neurology 2010;75:1062–9. [18] Sollinger AB, Goldstein FC, Lah JJ, et al. Mild cognitive impairment in Parkinson’s disease: subtypes and motor characteristics. Parkinsonism Relat Disord 2010;16:177–80.
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
Increased plasma levels of phospholipid in Parkinson’s disease with mild cognitive impairment Zhenguang Li, Jinbiao Zhang ⇑, Hairong Sun Department of Neurology, Weihai Municipal Hospital, The Affiliated Hospital of Binzhou Medical College, 70 Heping Street, Weihai, Shandong Province 264200, China
a r t i c l e
i n f o
Article history: Received 28 July 2014 Accepted 3 February 2015
Keywords: Mild cognitive impairment MoCA scores Parkinson’s disease Phospholipids
a b s t r a c t This study aimed to observe the clinical characteristics and changes in plasma phospholipid (PL) concentrations in Parkinson’s disease patients with mild cognitive impairment (PD-MCI), and to investigate the association between these changes and cognitive function to provide a basis for early diagnosis and intervention for PD-MCI patients. Oxidative stress plays an important role in the development and progression of Parkinson’s disease (PD). PL, important components in cellular membranes, are critical for the maintenance of cell integrity and function. Lipid peroxidation products are significantly increased in the brains of PD patients. In the present study, plasma PL levels were significantly increased in PD-MCI patients or in PD patients with no cognitive impairment (PD-NCI) compared with controls (p < 0.01 and p < 0.05, respectively). PL levels were significantly increased in the PD-MCI group compared with PD-NCI patients (p < 0.01). There was a negative correlation between plasma PL levels and Montreal cognitive assessment scores (r = 0.542; p < 0.001). These findings support the relationship between mild cognitive impairment and membrane injury. The measurement of PL reflects membrane injury in vivo and may be a new useful biomarker for the prognosis of cognitive states in patients with PD. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Parkinson’s disease (PD) is a degenerative disease of the central nervous system that involves many other systems. In addition to motor symptoms, non-motor symptoms are important components of PD clinical manifestation [1]. The cognitive impairment caused by the involvement of the cortex in PD patients is one of the major non-motor PD symptoms with an incidence of approximately 20–50% [2]. Mild cognitive impairment (MCI) is common in non-demented PD patients [3], also called PD-MCI. A proportion of PD patients already have MCI before motor symptoms manifest. Early identification and timely intervention for PD-MCI are important for improving the prognosis and quality of life of PD patients. Molecular pathological studies have shown that [4] oxidative stress plays an important role in the development and progression of PD. Phospholipids (PL) are important components in cellular membranes, and are critical for the maintenance of their integrity and function. However, studies on the changes in plasma PL in PD-MCI patients are lacking. This study aimed to observe the clinical characteristics and changes of plasma PL concentrations in PD-MCI patients and to investigate the association between these ⇑ Corresponding author. Tel.: +86 063 1528 7598; fax: +86 063 1589 0955. E-mail address: [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.jocn.2015.02.013 0967-5868/Ó 2015 Elsevier Ltd. All rights reserved.
changes and cognitive function in order to provide a basis for early diagnosis and intervention for PD-MCI patients.
2. Materials and methods 2.1. Patients The diagnosis of PD was made according to the UK Parkinson’s Disease Society Brain Bank Clinical Diagnostic Criteria. The inclusion criteria were the following: patients with idiopathic PD diagnosed by neurologists with clinical experience and according to the diagnostic criteria; and patients treated with conventional medications (levodopa or dopamine receptor agonists). The diagnostic criteria for PD-MDI were based on the diagnostic criteria formulated by the Movement Disorder Society (MDS) in the USA [3]. Further inclusion criteria were: in patients with a confirmed diagnosis of PD, gradual decrease in cognitive function was reported by patients/informants or observed by physicians; patients with cognitive impairment confirmed by formal neuropsychological testing or cognitive rating scales; and the cognitive impairment did not significantly affect functional independence, though minor difficulties in the execution of complex tasks were permitted. The exclusion criteria were the following: patients with Parkinsonian syndrome secondary to stroke, trauma, or other
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neurological and psychiatric disorders; patients with Parkinsonism plus syndrome; patients with malignant tumors, disabilities, or other severe physical diseases; patients who underwent surgical treatment, deep brain stimulation, stem cell transplantations, or other medical treatment. Other exclusion criteria based on the MDS were: patients who met the Parkinson’s disease dementia diagnosis proposed by the MDS specialty group; other causes of cognitive impairment (such as delirium, stroke, severe depression, metabolic disorders, drug side effects and head trauma); patients with PD comorbidities (such as movement disorders, severe anxiety, depression, excessive daytime sleepiness, and mental disorders). According to the above inclusion and exclusion criteria, 85 PD patients who were treated in the neurology clinic or were admitted to our hospital from May 2006 to June 2011 were enrolled. A total of 75 healthy individuals older than 55 years of age who received physical examinations comprised the control group. The control individuals had no mental disorders such as anxiety and depression, no history of severe physical disorders in the previous month, normal thyroid gland function, no physical disability or hearing or visual impairment, and no history of alcohol abuse. The severity of patients’ motor symptoms was evaluated using Part III of the unified Parkinson’s disease rating scale and the Hoehn and Yahr scale. MCI was evaluated using the Chinese version of the Montreal Cognitive Assessment (MoCA) including visuospatial ability, executive function, naming tasks, attention tasks, abstraction, language, delayed memory recall and orientation. The range of total scores on this scale is 0–30 points. If the subject’s years of education were 612 years then 1 point was added to correct the bias for educational level. A score P26 points was considered normal and MCI was indicated by a score between 15–25 points. The testing time was approximately 10 min. Two assessors, who received standardised training, completed all of the evaluation tests. 2.2. Measurement of plasma PL levels Patients were asked to avoid fatty food and alcohol for 7 days before fasting blood samples were obtained in the morning. We did not draw blood from females if they were in their menstrual period. Venous blood (4 mL) was drawn from each participant into commercially available anticoagulant tubes. Plasma was either processed immediately or stored at 70°C before lipid extraction. Lipid extraction was performed at 0–4°C to minimize damage to ester bonds, using a slight modification of published methods [5]. Whole blood was centrifuged at 3500g-force for 10 min. The supernatant was transferred to a microcentrifuge tube and centrifuged at 8000 g-force for 10 minutes. Then 1 mL suspension (platelet-poor plasma) was used for the remaining procedure. Lipids were extracted with 4 mL of 1-butanol and 2 mL of water-saturated 1-butanol. The resulting organic extracts were pooled and dried in vacuo. Each sample was resuspended in 0.3 mL of chloroform/methanol/water/28% ammonium hydroxide (NH4OH; 250:100:15:0.3, v/v) and was immediately filtered by an Econosphere 3 lm, 50 4.6 mm silica column (Alltech Associates, Deerfield, IL, USA). Compounds were eluted with a mobile phase of chloroform/methanol/water/28% NH4OH (250:100:15:0.3, v/v) at 0.5 mL/minute. The source was maintained at 250°C with a drying gas flow of 10 L/hour. The concentration of PL was quantified by measuring its inorganic phosphorus component using colorimetric assays. 2.3. Statistical analyses All of the statistical analyses were performed using SPSS statistics (version 13.0; IBM Corporation, Armonk, NY, USA). The Kolmogorov–Smirnov test was performed to ascertain the
normality of the distribution of continuous variables. Tests on the homogeneity of variances were also performed. Continuous variables were analyzed by one-way analysis of variance (ANOVA). Categorical variables were analyzed using the chi-squared test. The correlation between PL levels and MoCA scores were analyzed by Spearman’s linear correlation. All analyses were two-tailed. Results were expressed as mean ± standard deviation. Values were considered significant at p < 0.05. 3. Results 3.1. Demographic characteristics of study subjects Among 85 PD patients, 44 (51.8%) had normal cognitive function (PD-NCI; MoCA P26 points) and 41 had MCI (PD-MCI; 15 points < MoCA < 26 points). PD-NCI patient (32 male and 12 female) had a mean age of 63.5 years (range 50–76 years). PD-MCI patients (30 male and 11 female) had a mean age of 65.4 years (range 51–75 years). 75 controls (54 male and 21 female) with mean age 64.1 years (range 55–75 years), were also included in the study. There was no significant difference in sex, age, education, disease duration and use of levodopa or receptor agonists (p > 0.05; Table 1). 3.2. Comparison of MoCA scores The scores of each cognitive domain in the MoCA scale of subjects in the different groups are shown in Table 2. The results of one-way ANOVA and pairwise comparisons indicated that the scores of all items in the PD-MCI group were lower than those in the PD-NCI group (p < 0.01) and the control group (p < 0.01; Table 2). 3.3. Comparison of plasma PL concentration Plasma PL concentration in the PD-NCI and PD-MCI groups were significantly higher compared with the control group (p < 0.05 and p < 0.01, respectively). Furthermore, the concentration of plasma PL in the PD-MCI group was significantly increased compared with that in the PD-NCI group (p < 0.01; Table 3). 3.4. Correlation between plasma PL levels and MoCA scores There was a negative correlation between plasma PL levels and MoCA scores (r = 0.542; p < 0.001; Fig. 1). 4. Discussion In this study, we investigated the plasma PL levels in PD patients with mild cognitive impairment. Additionally, we
Table 1 Baseline characteristics of healthy controls and PD-NCI and PD-MCI patients Characteristic
Control (n = 75)
PD-NCI (n = 44)
PD-MCI (n = 41)
Age, years (mean ± SD) Sex, male/female Education, years (mean ± SD) DD, months (mean ± SD) UPDRS III score Use of levodopa (%) Use of receptor agonists (%)
64.1 ± 6.7 54/21 9.9 ± 7.9
63.5 ± 6.4 32/12 10.8 ± 7. 7
65.4 ± 7.1 30/11 11.6 ± 8.2
– – – –
87.6 ± 21.3 18.6 ± 6.7 56 39
90.2 ± 22.9 20.4 ± 7.8 57 45
– = not applicable, DD = disease duration, PD-MCI = Parkinson’s disease with mild cognitive impairment, PD-NCI = Parkinson’s disease with no cognitive impairment, SD = standard deviation, UPDRS = Unified Parkinson’s disease rating scale.
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Table 2 Comparison of scores of all items in the MoCA scale for healthy controls, PD-NCI and PD-MCI patients MoCA variable
Control (n = 75)
PD-NCI (n = 44)
PD-MCI (n = 41)
Visuospatial ability Naming task Attention Language Abstraction Delayed memory recall Orientation
4.15 ± 0.57 3.00 ± 0.00 5.93 ± 0.78 3.17 ± 0.29 2.10 ± 0.48 3.59 ± 0.66
4.10 ± 0.65 2.88 ± 0.37 5.88 ± 1.03 2.90 ± 0.54 1.81 ± 0.62 3.22 ± 0.98
2.54 ± 0.87* 2.33 ± 0.50* 4.99 ± 0.91* 2.20 ± 0.39* 1.07 ± 0.55* 1.18 ± 1.02*
6.00 ± 0.00
5.95 ± 0.88
5.26 ± 0.63*
All values are reported as the mean ± standard deviation. MoCA = Montreal cognitive assessment, PD-MCI = Parkinson’s disease with mild cognitive impairment, PD-NCI = Parkinson’s disease with no cognitive impairment. * Compared with the control and PD-NCI group, p < 0.01.
Table 3 Comparison of plasma PL concentration and MoCA score in healthy controls, PD-NCI and PD-MCI patients Variable
Control (n = 75)
PD-NCI(44)
PD-MC(41)
PL (U) MoCA score
4.52 ± 0.27 27.36 ± 1.72
5.56 ± 0.65N 27.11 ± 1.78
7.27 ± 0.68*,NN 20.32 ± 2. 61**,NN
All values are reported as the mean ± standard deviation. MoCA = Montreal cognitive assessment, PD-MCI = Parkinson’s disease with mild cognitive impairment, PD-NCI = Parkinson’s disease with no cognitive impairment, PL = phospholipid. * Compared with the PD-NCI group, p < 0.05. ** Compared with the PD-NCI group, p < 0.01. N Compared with the control group, p < 0.05. NN Compared with the control group, p < 0.01.
analyzed the correlation between plasma PL levels and the severity of cognitive impairment. PL are important components of the biomembrane. In the central nervous system, PL are rich in polyunsaturated fatty acids (PUFA). The metabolism of PUFA is mainly regulated by phospholipase A2 (PLA2) and acyltransferase in a process called the
deacylation-reacylation cycle. Under normal conditions, the released free fatty acids (FFA) under the action of PLA2 will be rapidly uptaken by membrane PL. PLA2 mainly recognizes the sn-2 acyl bond of glycerophospholipids in lipoproteins and stimulates the cell membrane to release FFA and lysophospholipids [5]. This study showed that the concentrations of plasma PL in the PD-NCI and PD-MCI groups were significantly higher than that in the control group, suggesting that PD patients have membrane damage. Compared with PD-NCI patients, the increase in the plasma PL concentration in PD-MCI patients was even more significant, suggesting that metabolic disorders of PL play an important role in the pathology of cognitive impairments in PD patients and that the membrane damage in PD-MCI patients is more severe. Furthermore, there was a negative correlation between plasma PL levels and MoCA scores. Therefore, PL have the potential to become a plasma biomarker of cognitive impairment in PD. The possible mechanisms underlying the increase of PL in PD patients include oxidative stress and accumulation of free radicals [6,7]. This activates relevant phospholipases, thus causing lysis of membrane PL. Many studies have indicated that lipid peroxidation products are elevated in the brains of PD patients [8,9]. In the substantia nigra and the striatum, 8-hydroxydeoxyguanosine, nitrosyl groups, protein carbonyl groups, iron ions and total iron concentration increase significantly, and the level of reduced glutathione decreases. Therefore, cells are in a state of oxidative stress. Many potential factors can cause a state of oxidative stress in PD including mitochondrial dysfunction, increased free ion levels and reductions in antioxidant defense system functions [10,11]. Another possible mechanism is endogenous metabolic products, such as nitric oxide, reactive oxygen species, active ions, and toxicophores, along with deregulation of the ubiquitin-proteasome system, affecting the function of the respiratory chain in mitochondria and causing disorders of mitochondria and energy metabolism [12,13]. Also, decreased glucose uptake by the cerebral cortex and decreased blood perfusion in the frontal, temporal and parietal lobes can activate phospholipases, causing lysis of membrane PL [14,15]. Molecules such as hydrogen peroxide change the activity of certain phospholipases, such as phospholipase D and PLA2, to
Fig. 1. Correlation between plasma phospholipid (PL) levels and Montreal Cognitive Assessment (MoCA) scores. Analysis was by Spearman linear correlation (r = p < 0.001).
0.542;
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activate cytosolic PLA2 and calcium-independent phospholipase A2, causing the release of PL [16]. Because of the rapid hydrolysis of PL in the cell membrane, the lipid complex cannot supplement the lost PL in the cell membrane in a timely manner, causing neuronal degeneration or loss [14]. A study by Aarsland et al. [17] in 1346 PD patients from eight centers showed that 25.8% (range: 23.5–28.2%) of PD patients had MCI. The results of our study showed that among the 85 patients, 41 (48.2%) had MCI, which is higher compared to the above reports from other countries. This difference is thought to be associated with other factors such as ethnic differences and educational levels. The evaluation of the MoCA scale results indicated that the cognitive levels of PD-MCI patients were significantly lower than those of PD-NCI patients. This study compared the evaluation scores of each cognitive domain including visuospatial and executive functions, naming tasks, attention and calculation abilities, language, abstract thinking, delayed memory recall, and orientation, and demonstrated that the scores of all cognitive domains in the PD-MCI group were significantly lower compared with those in the PD-NCI and control groups. The scores for calculation and attention abilities, delayed memory recall, visuospatial and executive functions were the lowest, suggesting that the damage in these three cognitive domains in the PD-MCI group was more severe, similar to the report of Sollinger et al. [18]. In summary, the incidence of PD-MCI is high and includes impairment in attention, calculation, and executive tasks, delayed memory recall, and decreased visuospatial ability. Interventions for PD patients should pay close attention to PD-MCI for early diagnosis and timely treatment to increase the quality of life and reduce care and society burden. Metabolic disorders of PL play an important role in the pathology of cognitive impairment in PD patients, with membrane damage in PD-MCI patients found to be more severe. PL have the potential to become a novel plasma biomarker for cognitive impairment in PD patients. Conflicts of Interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.
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References [1] Goldman JG, Litvan I. Mild cognitive impairment in Parkinson’s disease. Minerva Med 2011;102:441–59. [2] Petersen RC, Smith GE, Waring SC, et al. Mild cognitive impairment: clinical characterization and outcome. Arch Neurol 1999;56:303–8. [3] Litvan I, Goldman JG, Tröster AI, et al. Diagnostic criteria for mild cognitive impairment in Parkinson’s disease: Movement Disorder Society Task Force guidelines. Mov Disord 2012;27:349–56. [4] Lee J, Giordano S, Zhang J. Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling. Biochem J 2012;441:523–40. [5] Kosicek M, Hecimovic S. Phospholipids and Alzheimer’s disease: alterations, mechanisms and potential biomarkers. Int J Mol Sci 2013;14:1310–22. [6] Peterson B, Knotts T, Cummings BS. Involvement of Ca2+-independent phospholipase A2 isoforms in oxidant-induced neural cell death. Neurotoxicology 2007;28:150–60. [7] Mathisen GH, Thorkildsen IH, Paulsen RE. Secretory PLA2-IIA and ROS generation in peripheral mitochondria are critical for neuronal death. Brain Res 2007;1153:43–51. [8] Onyango IG. Mitochondrial dysfunction and oxidative stress in Parkinson’s disease. Neurochem Res 2008;33:589–97. [9] Wood-Kaczmar A, Gandhi S, Wood NW. Understanding the molecular causes of Parkinson’s disease. Trends Mol Med 2006;12:521–8. [10] Fessel JP, Hulette C, Powell S, et al. Isofurans, but not F2-isoprostanes, are increased in the substantia nigra of patients with Parkinson’s disease and with dementia with Lewy body disease. J Neurochem 2003;85:645–50. [11] Seet RC, Lee CY, Lim EC, et al. Oxidative damage in Parkinson disease: measurement using accurate biomarkers. Free Radic Biol Med 2010;48:560–6. [12] Elkharaz J, Ugun-Klusek A, Constantin-Teodosiu D, et al. Implications for oxidative stress and astrocytes following 26S proteasomal depletion in mouse forebrain neurones. Biochim Biophys Acta 2013;1832:1930–8. [13] Dasuri K, Zhang L, Keller JN. Oxidative stress, neurodegeneration, and the balance of protein degradation and protein synthesis. Free Radic Biol Med 2013;62:170–85. [14] Schaeffer EL, da Silva ER, Novaes Bde A, et al. Differential roles of phospholipases A2 in neuronal death and neurogenesis: implications for Alzheimer disease. Prog Neuropsychopharmacol Biol Psychiatry 2010;34:1381–9. [15] Lovell MA, Xie C, Markesbery WR. Acrolein, a product of lipid peroxidation, inhibits glucose and glutamate uptake in primary neuronal cultures. Free Radic Biol Med 2000;29:714–20. [16] Schaeffer EL, Gattaz WF. Inhibition of calcium-independent phospholipase A2 activity in rat hippocampus impairs acquisition of short- and long-term memory. Psychopharmacology 2005;181:392–400. [17] Aarsland D, Bronnick K, Williams-Gray C, et al. Mild cognitive impairment in Parkinson disease: a multicenter pooled analysis. Neurology 2010;75:1062–9. [18] Sollinger AB, Goldstein FC, Lah JJ, et al. Mild cognitive impairment in Parkinson’s disease: subtypes and motor characteristics. Parkinsonism Relat Disord 2010;16:177–80.