J Neural Transm (2015) 122:411–417 DOI 10.1007/s00702-014-1280-5
NEUROLOGY AND PRECLINICAL NEUROLOGICAL STUDIES - ORIGINAL ARTICLE
Correlations between plasma levels of amino acids and nonmotor symptoms in Parkinson’s disease Qing Tong • Qinrong Xu • Qiang Xia • Yongsheng Yuan • Li Zhang • Hongbin Sun Han Shan • Kezhong Zhang
•
Received: 5 June 2014 / Accepted: 17 July 2014 / Published online: 25 July 2014 Ó Springer-Verlag Wien 2014
Abstract Converging evidence suggests that changes in plasma levels of amino acids are involved in Parkinson’s disease (PD), but their roles in nonmotor symptoms (NMS) of PD remain unclear. The aim of this study was to evaluate the correlations between plasma amino acids and NMS of PD. Plasma levels of aspartate (Asp), glutamate (Glu), glycine (Gly) and c-aminobutyric acid (GABA) were measured in 92 PD patients and 60 healthy controls. Four NMS, including depression, pain, sleep disturbances and autonomic dysfunction were assessed in enrolled subjects using the Hamilton Depression Scale, the short form of the McGill Pain Questionnaire, the Pittsburgh Sleep Quality Index and the Scale for Outcomes in Parkinson’s disease for Autonomic Symptoms, respectively. Hierarchical multiple regression analysis was used to evaluate the correlations between plasma levels of amino acids and NMS. PD patients exhibited significantly higher scores of NMS scales and lower plasma levels of amino acids compared to healthy controls. Within the PD group,
Q. Tong, Q. Xu and Q. Xia contributed equally to this work. Q. Tong Q. Xu Y. Yuan L. Zhang K. Zhang (&) Department of Neurology, The First Affiliated Hospital of Nanjing Medical University, No. 300 Guangzhou Road, Nanjing 210029, China e-mail: [email protected] Q. Xia Department of Neurology, Changzhou NO. 2 People’s Hospital, The Affiliated Hospital of Nanjing Medical University, No. 29 Xinglong Alley, Changzhou 213003, China H. Sun H. Shan Department of Pharmacology, Jiangsu Key Laboratory of Neurodegeneration, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China
plasma levels of Asp and Glu were negatively associated with the severity of depression and sleep disturbances. Moreover, decreased plasma level of GABA was correlated with more severe symptoms of sleep disturbances. After controlling for gender, disease duration, severity of motor symptoms and anti-parkinsonian medications, Glu but not Asp remained significantly associated with depression, along with Asp, GABA but not Glu remained negatively associated with sleep disturbances. The altered plasma levels of amino acids may be implicated in the pathogenesis of NMS of PD. Keywords Parkinson’s disease Amino acids Nonmotor symptoms Plasma
Introduction Parkinson’s disease (PD), the second most common neurodegenerative disorder in the elderly, is characterized by motor symptoms, including tremor, bradykinesia, rigidity and postural instability. However, a spectrum of nonmotor symptoms (NMS), such as neuropsychiatric, sensory disorders, sleep disturbances and autonomic dysfunction, has gained a greater awareness in recent years (Park and Stacy 2009). The UK National Institute for Clinical Excellence has recognized the identification and treatment of NMS as a critical unmet need in PD (Conditions 2006). Although NMS negatively contribute to the quality of life (QOL) in PD patients (Santos-Garcia and de la Fuente-Fernandez 2013), the pathogenesis of these NMS is still poorly understood. It is well known that dopamine plays an essential role in the pathogenesis of PD. There is also increasing evidence to suggest that the other amino acids in brain, such as
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glutamate (Glu) and c-aminobutyric acid (GABA), were involved in the pathogenesis of PD via disturbing balance of the basal ganglia circuit (Emir et al. 2012; Sun et al. 2012). Recently, Yuan et al. (2013) reported that the plasma levels of aspartate (Asp) and Glu in PD patients were significantly lower than in controls. Previous studies have shown that some NMS, such as depression, pain, sleep, and bladder function, are only partly alleviated by dopamine replacement therapy (Chaudhuri and Schapira 2009; Kim et al. 2009; Park and Stacy 2009; Seppi et al. 2011), suggesting that NMS of PD is driven by not only depletion of dopamine in the nigrostriatal system, but also alterations of other neurotransmitter systems. Moreover, there are data to suggest that amino acids are implicated in depression, pain, sleep disturbances and autonomic dysfunction. For example, both abnormal plasma and cerebrospinal fluid (CSF) levels of Glu were observed in depression patients (Frye et al. 2007; Mitani et al. 2006), suggesting that Glu may play a critical role in depression. Prior work also showed that Asp was implicated in pain by influencing on N-methyl-D-aspartate receptors (Alexander et al. 2013). Brooks and Peever (2011) observed that deficits in Glyand GABA-mediated inhibition were correlated with sleep disturbances. It has been also reported that microinjections of GABA into the rostral ventrolateral medulla caused inhibitory effects on sympathetic nerve activity in rats (Huber and Schreihofer 2011). However, none of the previously published studies attempted to examine whether NMS occurred in PD is associated with the altered plasma amino acids. Thus, in this study, we sought to assess the plasma levels of excitatory amino acids (Asp and Glu) and inhibitory amino acids (Gly and GABA) in PD patients, and evaluated their correlations with NMS such as depression, pain, sleep disturbances and autonomic dysfunction.
Patients and methods
Q. Tong et al.
State Examination (MMSE) score \24 (Folstein et al. 1975), were also excluded due to a consideration that their self-reporting on the questionnaire was likely unreliable. Age- and gender-matched healthy individuals were enrolled as controls, devoid of neurological disease, dementia or a family history of PD. This study was approved by the ethics committee of the first affiliated hospital of Nanjing medical university and informed consent was obtained from each participant. Assessments of NMS Standard published measures validated and widely used in Chinese populations were adopted. The Hamilton Depression Scale (HAMD) (Hamilton 1960) was used to evaluate the depressive symptoms, with a total score [17 indicating clinically significant depression. The symptoms of pain were assessed using the short form of the McGill Pain Questionnaire (SF-MPQ) (Melzack 1987), and responses were summed and transformed to a 0–10 scale (0 = no pain, 10 = worst pain) according to the standard procedure for the SF-MPQ. Sleep quality was assessed with the Pittsburgh Sleep Quality Index (PSQI) (Buysse et al. 1991) and a score[5 indicate obvious sleep disturbances. Finally, we evaluated the autonomic dysfunction using the Scale for Outcomes in Parkinson’s disease for Autonomic Symptoms (SCOPA-AUT), a scale developed and validated for use in PD patients (Visser et al. 2004). Collection and storage of plasma samples Venous blood samples from PD patients and controls were collected into EDTA-containing tubes after an overnight stop of food and medication and immediately centrifuged at a temperature lower than 4 °C. Separated plasma samples were kept at -80 °C (less than 4 months of storage time) until analysis.
Patients Measurements of plasma amino acids Consecutive PD patients, of all age groups and disease severity, satisfying the UK PD Brain Bank criteria for the diagnosis of idiopathic PD were recruited between August 2012 and June 2013 (Hughes et al. 1992). During the visit, a complete medical history was collected and the Unified Parkinson’s Disease Rating Scale (UPDRS) (Fahn et al. 1987) scores were used to assess the severity of each patient. Levodopa-equivalent daily dose (LEDD) was calculated using conversion factors as described previously (Wenzelburger et al. 2002). Patients with corticobasal degeneration, progressive supranuclear palsy, dementia with Lewy bodies, vascular parkinsonism, and other forms of parkinsonism were excluded. Moreover, patients with cognitive impairments, as evaluated using the Mini Mental
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Plasma levels of Asp, Glu, Gly and GABA were measured by high performance liquid chromatography (HPLC-RF) as previously described (Yuan et al. 2013). The system consisted of LC-10AD pump, SIL-20AC plus auto-sampler and RF-10AXL fluorometric detector (excitation 260 nm, emission 450 nm). Agilent ZORBAX XDB-C18 column (4.6 9 150 mm, 5 lm) was used at 40 °C. The homogenates of plasma samples and trichloroacetic acid (1: 0.2 V/ V) were centrifuged at 20,000g for 30 min at 4 °C. Amino acids determination was performed using precolumn derivatization procedure, with mobile phase A (methanol) and mobile phase B (70 mmol/L Na2HPO4, 0.4 mmol/L EDTA2Na, 0.1 % TEA) at a flow rate of 1.0 mL/min. The
Correlations between plasma levels of amino acids and nonmotor symptoms in Parkinson’s disease
derivatives consisted of 9 mL 0.2 mol/L H3BO3 (pH10.0), 1 mL methanol, 10 mg OPA, 10 mg IBC. Absolute concentrations of those amino acids were determined using computer analysis of peak height with external standards. Statistical analysis All data for the continuous variables were shown as mean ± standard deviation and the categorical variables were shown as a percentage. Prevalence of each NMS was calculated according to the case number of each NMS divided by the total sample and converted to a percentage. Pearson’s Chi-squared test was used to compare proportions. Group comparisons were made using Independentsamples T test or Mann–Whitney U test, as appropriate. Spearman’s Rho was employed to evaluate the associations amongst demographic and clinical variables, NMS, and plasma levels of amino acids. To further explore significant correlations, hierarchical multiple regression analysis was conducted, controlling for potential confounders as described below. The respective NMS score was entered as dependent variable in each model. Gender, disease duration, UPDRS III score and LEDD were entered into the first block as independent variables, and plasma levels of amino acids were subsequently entered into the second block. There were two outliers on HAMD scores and no outlier on PSQI scores. Hierarchical multiple regression analysis was conducted both with and without outliers. The SPSS 13.0 software (Chicago, IL) was used for statistical analyses and p values of less than 0.05 were regarded as statistically significant.
Results
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Table 1 Demographic characteristics and plasma levels of amino acids of PD patients and controls Control (n = 60)
PD (n = 92)
p value
Age (years, mean ± SD)
64.1 ± 13.0
61.6 ± 10.7
0.109
Male, n (%)
29 (48.3)
37 (40.2)
0.324
Disease duration (years, mean ± SD)
NA
3.2 ± 3.5
Hoehn and Yahr stage (mean ± SD)
NA
1.9 ± 0.8
LEDD (mg/day, mean ± SD)
NA
182.3 ± 319.7
UPDRS III score (mean ± SD)
NA
15.3 ± 9.8
HAMD score (mean ± SD)
0.6 ± 0.8
8.8 ± 7.7
0.000
SF-MPQ score (mean ± SD)
0.1 ± 0.2
3.2 ± 2.5
0.000
PSQI score (mean ± SD) SCOPA-AUT score (mean ± SD)
0.1 ± 0.3 0.4 ± 0.6
8.7 ± 2.3 11.9 ± 9.0
0.000 0.000
Asp (lmol/L, mean ± SD)
42.7 ± 14.5
9.6 ± 4.9
0.000
Glu (lmol/L, mean ± SD) Gly (lmol/L, mean ± SD)
148.9 ± 50.8
49.5 ± 22.5
0.000
288.8 ± 83.6
264.5 ± 120.0
0.005
274.8 ± 88.0
250.8 ± 63.5
0.01
GABA (lmol/L, mean ± SD)
PD Parkinson’s disease, SD standard deviation, LEDD levodopaequivalent daily dose, UPDRS Unified Parkinson’s disease rating scale, HAMD Hamilton Depression Scale, SF-MPQ short form of the McGill Pain Questionnaire, PSQI Pittsburgh Sleep Quality Index, SCOPA-AUT Scale for Outcomes in Parkinson’s disease for Autonomic Symptoms, Asp aspartate, Glu glutamate, Gly glycine, GABA c-aminobutyric acid
Demographic and clinical characteristics Demographic and clinical characteristics of the PD patients and healthy controls are shown in Table 1. A total of 92 PD patients and 60 age- and gender-matched healthy controls were enrolled in this study. In the entire group, female exhibited significantly higher levels of Gly (p = 0.019). Moreover, male PD patients displayed markedly higher level of Asp (p = 0.010) compared to female patients. In the PD group, UPDRS III score correlated positively with the plasma Asp level (r = 0.225, p = 0.031). There were significant correlations of HAMD score with disease duration (r = 0.236, p = 0.024), UPDRS III score (r = 0.288, p = 0.005), H-Y stage (r = 0.299, p = 0.004) and LEDD (r = 0.247, p = 0.018). SF-MPQ score was markedly associated with disease duration and LEDD (r = 0.347, p = 0.001; r = 0.354, p = 0.001, respectively), while weakly correlated with H-Y stage (r = 0.214, p = 0.041). Furthermore, the PSQI
score was weakly associated with UPDRS III score (r = 0.232, p = 0.026) and LEDD (r = 0.260, p = 0.012). The correlation of SCOPA-AUT score with the demographic and clinical characteristics of patients was as follows: disease duration (r = 0.287, p = 0.006), UPDRS III (r = 0.262, p = 0.012), H-Y stage (r = 0.249, p = 0.017) and LEDD (r = 0.342, p = 0.001). Hence, gender, disease duration, motor severity (UPRDS III), and dopaminergic medication (LEDD) were considered as potential confounders in the subsequent hierarchical multiple regressions. NMS and plasma levels of amino acids in patients and controls As shown in Table 1, the plasma levels of Asp (p = 0.000), Glu (p = 0.000) and Gly (p = 0.005), as well as GABA (p = 0.01) were significantly lower in PD patients than in
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Q. Tong et al. Table 2 Correlations between nonmotor symptom scores and plasma levels of amino acids in PD patients Variable
Asp r
Glu r
HAMD
-0.326*
-0.344*
SF-MPQ
-0.202
-0.164
PSQI
-0.348*
-0.271*
SCOPA-AUT
-0.065
-0.005
Gly r 0.077 0.030 -0.164 0.062
GABA r -0.176 0.064 -0.486** -0.112
r Spearman’s rho * p \ 0.01, ** p \ 0.001
Fig. 1 The percentage of PD patients with each NMS. The percentage of PD patients suffering from autonomic dysfunction was 81.5 % (75/92 patients) and pain was 69.6 % (64/92 patients), along with sleep disturbances was 46.7 % (43/92 patients). Only 29 (31.5 %) PD patients exhibited depression in our study. PD Parkinson’s disease, NMS nonmotor symptoms
healthy controls. In contrast, the scores of entire assessed NMS were markedly higher in PD patients compared to healthy controls (p = 0.000). Furthermore, PD patients suffered from a wide variety of NMS. In this enrollment, we observed that nearly all patients (96.7 %) suffered at least one kind of NMS, with a much higher prevalence in autonomic dysfunction (81.5 %) and pain (69.6 %) (Fig. 1). Interestingly, the overwhelming majority of PD patients suffered from more than two types of NMS (two NMS, 40.2 %; three NMS, 30.4 %; four NMS, 14.1 %), whereas the small minority of PD patients (12.0 %) showed one NMS.
HAMD and PSQI scores entered as dependent variable in each respective model. Gender, disease duration, UPDRS III score and LEDD were entered into the first block as independent variables, following by the natural logarithms of Asp and Glu were entered into the second block in depression model (excluding two outliers) (Table 3) and the natural logarithms of Asp, Glu and GABA in sleep disturbances model (Table 4). After controlling for confounders, Glu but not Asp remained significantly associated with depression (b = -0.32, p = 0.009), and Asp, GABA but not Glu remained negatively associated with sleep disturbances (b = -0.28, p = 0.016; b = -0.44, p = 0.000, respectively). UPDRS III score significantly correlated with depression (b = 0.24, p = 0.021), as well as LEDD was significantly associated with sleep disturbances (b = 0.27, p = 0.012). Moreover, the main results of hierarchical multiple regressions with the outliers included were similar, excepted that LEDD also became significant in depression model (b = 0.24, p = 0.041).
Correlations between plasma levels of amino acids and NMS
Discussion
Finally, we assessed the correlations of plasma levels of amino acids with NMS. In the patient group, as illustrated in Table 2, the severity of depression as rated by the HAMD was negatively correlated with plasma levels of Asp and Glu (r = -0.326, p = 0.002; r = -0.344, p = 0.001, respectively). Moreover, the increasing PSQI score, used to assess the sleep quality, was associated with the declining plasma level of Asp (r = -0.348, p = 0.001), Glu (r = -0.271, p = 0.009) and GABA (r = -0.486, p = 0.000). Univariate correlations between Glu and HAMD scores, GABA and PSQI scores are shown in Fig. 2. Nevertheless, we failed to observe the severity of pain and autonomic dysfunction significantly associated with plasma levels of any amino acids measured in this study. In the control group, there was no significant correlation between plasma levels of amino acids and NMS. In order to further explore significant correlations, we conducted two hierarchical multiple regressions with
In the past few years, NMS of PD has attracted considerable attention, and growing amount of research has devoted to the underlying biochemical and pathological mechanisms of the NMS (Park and Stacy 2009). Accumulating preclinical and clinical evidence suggests that brain amino acids, such as Asp, Glu, Gly and GABA, are involved in the pathogenesis of depression, pain, sleep disturbances and autonomic dysfunction (Alexander et al. 2013; Brooks and Peever 2011; Frye et al. 2007; Huber and Schreihofer 2011; Mitani et al. 2006). However, less systematic work has been performed to examine whether these amino acids are implicated in the NMS in PD patients. In addition, empirical and theoretical evidence suggests that the altered levels of amino acids in plasma partially reflected their alterations in central nervous system (CNS) of murid rodents (Bongiovanni et al. 2010; Murakami et al. 2009). Prior work also showed that amino acids in plasma were positively correlated with those in CSF of humans, which is
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Correlations between plasma levels of amino acids and nonmotor symptoms in Parkinson’s disease
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Fig. 2 Correlations between plasma levels of amino acids and NMS. a The plasma Glu level significantly correlated with HAMD scores (r = 0.344, r2 = 0.13, p = 0.001), b the plasma GABA level significantly correlated with PSQI scores (r = -0.486, r2 = 0.22, p = 0.000)
Table 3 Standardized b-coefficients of hierarchical multiple regression with HAMD as dependent variable Depression (HAMD) Block 1 Gender Disease duration
Block 2
0.10
0.04
-0.01
-0.03
UPDRS III score
0.16
0.24*
LEDD
0.17
0.17
Asp
-0.12
Glu Adjusted R2
-0.32** 0.02
0.15
* p \ 0.05, ** p \ 0.01
Table 4 Standardized b-coefficients of hierarchical multiple regression with PSQI as dependent variable Sleep disturbances (PSQI) Block 1
Block 2
Gender
-0.06
-0.09
Disease duration
-0.08
-0.09
UPDRS III score LEDD
0.14 0.25
Asp
0.15 0.27* -0.28*
Glu
-0.04
GABA
-0.44***
Adjusted R2
0.03
0.33
* p \ 0.05, *** p \ 0.001
suggested to be more relevant to brain disorders (Hagenfeldt et al. 1984; Jimenez-Jimenez et al. 1998; Uhlhaas et al. 1986). Again, a few of the studies indirectly examined the CNS changes in PD by detecting plasma amino acids levels (Iwasaki et al. 1992; Molina et al. 1997). On the basis of these data, we addressed the role of these amino acids in NMS of PD by detecting their plasma levels and to further address their correlations with NMS. In our study, we showed that PD patients displayed significantly more severe symptoms of depression, pain,
sleep disturbances and autonomic dysfunction than healthy controls. The prevalence of NMS in the Chinese PD patients similar to a previous report (Li et al. 2010), as 96.7 % of patients suffered from at least one type of NMS and 84.7 % of patients showed over two types of NMS. In agreement with Santos-Garcı´a et al.’s work (Santos-Garcia and de la Fuente-Fernandez 2013), pain and autonomic dysfunction were relatively common symptoms. Depression, one of the most frequently reported psychological disturbances in PD patients, was previously regarded as ‘‘reaction’’ to psychosocial stress and the associated disability of PD. However, accumulating data suggest that neurochemical changes, resulting from the neurodegeneration of PD, might contribute to the depression in PD. A recent study revealed that higher UPDRS scores in PD patients are correlated with the reduced Glu level in the bilateral lentiform nucleus using 1H magnetic resonance spectroscopy (Modrego et al. 2011). The decreased Glu level in the cerebral cortex of PD patients was also observed in another study (Griffith et al. 2008). Furthermore, the depressed subjects without PD exhibited a markedly lower CSF level of Glu (Frye et al. 2007) and reduced Glx (glutamate and glutamine) levels in the anterior cingulate cortex (Sanacora et al. 2012). In line with these previous findings, we observed that the plasma level of Glu in PD patients significantly decreased and negatively correlated with the HAMD score, possibly suggesting that altered glutamatergic functions may be involved in the pathogenesis of depression in PD patients and Glu might be potential indicator for evaluating the severity of depression of PD. Nevertheless, Mitani et al. (2006) showed that the plasma Glu level positively correlated with HAMD score in non-PD depressive patients. We speculated that the inconsistency may be attributed to participants, sample size and measurements. In their study, they enrolled a smaller sample size of younger patients, and focused on non-PD depressive patients. There is increasing body of evidence to suggest that the dysregulation of GABA, the main inhibitory amino acid in brain, might be expected to play an important role in mechanisms underlying sleep disturbances. For example, Watson et al. (2007) provided evidence that microinjection
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of morphine in the oral part of rat pontine reticular formation (PnO) resulted in a striking reduction in rapid eye movement (REM) sleep, whereas a significant augment in wakefulness by decreasing GABA level in PnO. It was also reported that deficits in Gly- and GABA-mediated inhibition were a potential mechanism for REM sleep behavior disorder (Brooks and Peever 2011). Moreover, Tohgi et al. (1991) found that GABA level in CSF of PD patients was lower when compared to healthy controls. Consistently, in our study, there was a much more reduction in plasma level of GABA in PD patients, and it was negatively associated with the severity of sleep disturbances, suggesting that GABA deficiency may be an underlying mechanism and a potential marker of sleep disturbances in PD. Furthermore, the plasma Asp level was slightly related with the severity of sleep disorder, possibly suggesting that excitatory amino acids also be partially involved in the genesis of sleep disturbances of PD. Of note, in our study, the r-values of correlations analysis were relatively low. Thus, the results from our correlation analysis were limited, only providing some information regarding the pathogenesis and severity of NMS in PD patients coupled with clinical features. Notably, the validity and specificity of these amino acids served as clinical indicator for NMS of PD remain to be confirmed in future CSF study with a larger sample size. However, previous studies showed different results (Araki et al. 1986; Jime´nez-Jime´nez et al. 1996; Perschak et al. 1987). For example, Jime´nez-Jime´nez et al. (1996) found that PD patients, compared with controls, had similar CSF Glu and higher GABA levels. Some authors also reported increased CSF Gly levels (Araki et al. 1986), or normal CSF GABA levels in PD patients (Perschak et al. 1987). The discrepancies between studies might be related with the sample size, disease status, measurements, or differences in samples. Compared with the previous reports, we enrolled a relatively larger sample size of mild PD patients, and used a more sensitive and stable measurement to detect amino acids in plasma samples. Taken together, these findings suggest that the roles of the altered amino acids levels in PD remain to be further investigated. It is noteworthy that our study has several limitations. First, we recruited a relative small sample size of patients, especially for correlation analysis; second, we only focused on four NMS of PD, and as a result probably ignored other NMS, such as sensory disorders and cognitive deficits. Third, our study was restricted to the alterations of amino acids in plasma samples. Therefore, larger scale studies are needed to determine common changes in plasma and CSF levels of amino acids in PD patients with more NMS. In addition, non-PD patients with depression, pain, sleep disturbances, and autonomic dysfunction should be included in order to obtain more precise alterations of these amino acids in NMS of PD.
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In summary, our results showed that PD patients exhibited significantly higher scores of NMS scales and lower plasma levels of amino acids compared to healthy controls. Hierarchical regression analysis revealed that plasma level of Glu was negatively associated with the severity of depression, and Asp and GABA negatively associated with sleep disturbances. Combined with the data from previous studies, our findings suggest that the altered plasma levels of amino acids in PD may participate in the pathogenesis of NMS of PD. Acknowledgments This work was supported by the University Natural Science Research Project in Jiangsu Province (No. 13KJB32009), the Natural science foundation of Jiangsu Province (No. BK20141494), the Opening Project of Jiangsu Key Laboratory of Neurodegeneration (No. SJ11KF01) and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Conflict of interest of interest.
The authors declare that they have no conflict
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NEUROLOGY AND PRECLINICAL NEUROLOGICAL STUDIES - ORIGINAL ARTICLE
Correlations between plasma levels of amino acids and nonmotor symptoms in Parkinson’s disease Qing Tong • Qinrong Xu • Qiang Xia • Yongsheng Yuan • Li Zhang • Hongbin Sun Han Shan • Kezhong Zhang
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Received: 5 June 2014 / Accepted: 17 July 2014 / Published online: 25 July 2014 Ó Springer-Verlag Wien 2014
Abstract Converging evidence suggests that changes in plasma levels of amino acids are involved in Parkinson’s disease (PD), but their roles in nonmotor symptoms (NMS) of PD remain unclear. The aim of this study was to evaluate the correlations between plasma amino acids and NMS of PD. Plasma levels of aspartate (Asp), glutamate (Glu), glycine (Gly) and c-aminobutyric acid (GABA) were measured in 92 PD patients and 60 healthy controls. Four NMS, including depression, pain, sleep disturbances and autonomic dysfunction were assessed in enrolled subjects using the Hamilton Depression Scale, the short form of the McGill Pain Questionnaire, the Pittsburgh Sleep Quality Index and the Scale for Outcomes in Parkinson’s disease for Autonomic Symptoms, respectively. Hierarchical multiple regression analysis was used to evaluate the correlations between plasma levels of amino acids and NMS. PD patients exhibited significantly higher scores of NMS scales and lower plasma levels of amino acids compared to healthy controls. Within the PD group,
Q. Tong, Q. Xu and Q. Xia contributed equally to this work. Q. Tong Q. Xu Y. Yuan L. Zhang K. Zhang (&) Department of Neurology, The First Affiliated Hospital of Nanjing Medical University, No. 300 Guangzhou Road, Nanjing 210029, China e-mail: [email protected] Q. Xia Department of Neurology, Changzhou NO. 2 People’s Hospital, The Affiliated Hospital of Nanjing Medical University, No. 29 Xinglong Alley, Changzhou 213003, China H. Sun H. Shan Department of Pharmacology, Jiangsu Key Laboratory of Neurodegeneration, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China
plasma levels of Asp and Glu were negatively associated with the severity of depression and sleep disturbances. Moreover, decreased plasma level of GABA was correlated with more severe symptoms of sleep disturbances. After controlling for gender, disease duration, severity of motor symptoms and anti-parkinsonian medications, Glu but not Asp remained significantly associated with depression, along with Asp, GABA but not Glu remained negatively associated with sleep disturbances. The altered plasma levels of amino acids may be implicated in the pathogenesis of NMS of PD. Keywords Parkinson’s disease Amino acids Nonmotor symptoms Plasma
Introduction Parkinson’s disease (PD), the second most common neurodegenerative disorder in the elderly, is characterized by motor symptoms, including tremor, bradykinesia, rigidity and postural instability. However, a spectrum of nonmotor symptoms (NMS), such as neuropsychiatric, sensory disorders, sleep disturbances and autonomic dysfunction, has gained a greater awareness in recent years (Park and Stacy 2009). The UK National Institute for Clinical Excellence has recognized the identification and treatment of NMS as a critical unmet need in PD (Conditions 2006). Although NMS negatively contribute to the quality of life (QOL) in PD patients (Santos-Garcia and de la Fuente-Fernandez 2013), the pathogenesis of these NMS is still poorly understood. It is well known that dopamine plays an essential role in the pathogenesis of PD. There is also increasing evidence to suggest that the other amino acids in brain, such as
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glutamate (Glu) and c-aminobutyric acid (GABA), were involved in the pathogenesis of PD via disturbing balance of the basal ganglia circuit (Emir et al. 2012; Sun et al. 2012). Recently, Yuan et al. (2013) reported that the plasma levels of aspartate (Asp) and Glu in PD patients were significantly lower than in controls. Previous studies have shown that some NMS, such as depression, pain, sleep, and bladder function, are only partly alleviated by dopamine replacement therapy (Chaudhuri and Schapira 2009; Kim et al. 2009; Park and Stacy 2009; Seppi et al. 2011), suggesting that NMS of PD is driven by not only depletion of dopamine in the nigrostriatal system, but also alterations of other neurotransmitter systems. Moreover, there are data to suggest that amino acids are implicated in depression, pain, sleep disturbances and autonomic dysfunction. For example, both abnormal plasma and cerebrospinal fluid (CSF) levels of Glu were observed in depression patients (Frye et al. 2007; Mitani et al. 2006), suggesting that Glu may play a critical role in depression. Prior work also showed that Asp was implicated in pain by influencing on N-methyl-D-aspartate receptors (Alexander et al. 2013). Brooks and Peever (2011) observed that deficits in Glyand GABA-mediated inhibition were correlated with sleep disturbances. It has been also reported that microinjections of GABA into the rostral ventrolateral medulla caused inhibitory effects on sympathetic nerve activity in rats (Huber and Schreihofer 2011). However, none of the previously published studies attempted to examine whether NMS occurred in PD is associated with the altered plasma amino acids. Thus, in this study, we sought to assess the plasma levels of excitatory amino acids (Asp and Glu) and inhibitory amino acids (Gly and GABA) in PD patients, and evaluated their correlations with NMS such as depression, pain, sleep disturbances and autonomic dysfunction.
Patients and methods
Q. Tong et al.
State Examination (MMSE) score \24 (Folstein et al. 1975), were also excluded due to a consideration that their self-reporting on the questionnaire was likely unreliable. Age- and gender-matched healthy individuals were enrolled as controls, devoid of neurological disease, dementia or a family history of PD. This study was approved by the ethics committee of the first affiliated hospital of Nanjing medical university and informed consent was obtained from each participant. Assessments of NMS Standard published measures validated and widely used in Chinese populations were adopted. The Hamilton Depression Scale (HAMD) (Hamilton 1960) was used to evaluate the depressive symptoms, with a total score [17 indicating clinically significant depression. The symptoms of pain were assessed using the short form of the McGill Pain Questionnaire (SF-MPQ) (Melzack 1987), and responses were summed and transformed to a 0–10 scale (0 = no pain, 10 = worst pain) according to the standard procedure for the SF-MPQ. Sleep quality was assessed with the Pittsburgh Sleep Quality Index (PSQI) (Buysse et al. 1991) and a score[5 indicate obvious sleep disturbances. Finally, we evaluated the autonomic dysfunction using the Scale for Outcomes in Parkinson’s disease for Autonomic Symptoms (SCOPA-AUT), a scale developed and validated for use in PD patients (Visser et al. 2004). Collection and storage of plasma samples Venous blood samples from PD patients and controls were collected into EDTA-containing tubes after an overnight stop of food and medication and immediately centrifuged at a temperature lower than 4 °C. Separated plasma samples were kept at -80 °C (less than 4 months of storage time) until analysis.
Patients Measurements of plasma amino acids Consecutive PD patients, of all age groups and disease severity, satisfying the UK PD Brain Bank criteria for the diagnosis of idiopathic PD were recruited between August 2012 and June 2013 (Hughes et al. 1992). During the visit, a complete medical history was collected and the Unified Parkinson’s Disease Rating Scale (UPDRS) (Fahn et al. 1987) scores were used to assess the severity of each patient. Levodopa-equivalent daily dose (LEDD) was calculated using conversion factors as described previously (Wenzelburger et al. 2002). Patients with corticobasal degeneration, progressive supranuclear palsy, dementia with Lewy bodies, vascular parkinsonism, and other forms of parkinsonism were excluded. Moreover, patients with cognitive impairments, as evaluated using the Mini Mental
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Plasma levels of Asp, Glu, Gly and GABA were measured by high performance liquid chromatography (HPLC-RF) as previously described (Yuan et al. 2013). The system consisted of LC-10AD pump, SIL-20AC plus auto-sampler and RF-10AXL fluorometric detector (excitation 260 nm, emission 450 nm). Agilent ZORBAX XDB-C18 column (4.6 9 150 mm, 5 lm) was used at 40 °C. The homogenates of plasma samples and trichloroacetic acid (1: 0.2 V/ V) were centrifuged at 20,000g for 30 min at 4 °C. Amino acids determination was performed using precolumn derivatization procedure, with mobile phase A (methanol) and mobile phase B (70 mmol/L Na2HPO4, 0.4 mmol/L EDTA2Na, 0.1 % TEA) at a flow rate of 1.0 mL/min. The
Correlations between plasma levels of amino acids and nonmotor symptoms in Parkinson’s disease
derivatives consisted of 9 mL 0.2 mol/L H3BO3 (pH10.0), 1 mL methanol, 10 mg OPA, 10 mg IBC. Absolute concentrations of those amino acids were determined using computer analysis of peak height with external standards. Statistical analysis All data for the continuous variables were shown as mean ± standard deviation and the categorical variables were shown as a percentage. Prevalence of each NMS was calculated according to the case number of each NMS divided by the total sample and converted to a percentage. Pearson’s Chi-squared test was used to compare proportions. Group comparisons were made using Independentsamples T test or Mann–Whitney U test, as appropriate. Spearman’s Rho was employed to evaluate the associations amongst demographic and clinical variables, NMS, and plasma levels of amino acids. To further explore significant correlations, hierarchical multiple regression analysis was conducted, controlling for potential confounders as described below. The respective NMS score was entered as dependent variable in each model. Gender, disease duration, UPDRS III score and LEDD were entered into the first block as independent variables, and plasma levels of amino acids were subsequently entered into the second block. There were two outliers on HAMD scores and no outlier on PSQI scores. Hierarchical multiple regression analysis was conducted both with and without outliers. The SPSS 13.0 software (Chicago, IL) was used for statistical analyses and p values of less than 0.05 were regarded as statistically significant.
Results
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Table 1 Demographic characteristics and plasma levels of amino acids of PD patients and controls Control (n = 60)
PD (n = 92)
p value
Age (years, mean ± SD)
64.1 ± 13.0
61.6 ± 10.7
0.109
Male, n (%)
29 (48.3)
37 (40.2)
0.324
Disease duration (years, mean ± SD)
NA
3.2 ± 3.5
Hoehn and Yahr stage (mean ± SD)
NA
1.9 ± 0.8
LEDD (mg/day, mean ± SD)
NA
182.3 ± 319.7
UPDRS III score (mean ± SD)
NA
15.3 ± 9.8
HAMD score (mean ± SD)
0.6 ± 0.8
8.8 ± 7.7
0.000
SF-MPQ score (mean ± SD)
0.1 ± 0.2
3.2 ± 2.5
0.000
PSQI score (mean ± SD) SCOPA-AUT score (mean ± SD)
0.1 ± 0.3 0.4 ± 0.6
8.7 ± 2.3 11.9 ± 9.0
0.000 0.000
Asp (lmol/L, mean ± SD)
42.7 ± 14.5
9.6 ± 4.9
0.000
Glu (lmol/L, mean ± SD) Gly (lmol/L, mean ± SD)
148.9 ± 50.8
49.5 ± 22.5
0.000
288.8 ± 83.6
264.5 ± 120.0
0.005
274.8 ± 88.0
250.8 ± 63.5
0.01
GABA (lmol/L, mean ± SD)
PD Parkinson’s disease, SD standard deviation, LEDD levodopaequivalent daily dose, UPDRS Unified Parkinson’s disease rating scale, HAMD Hamilton Depression Scale, SF-MPQ short form of the McGill Pain Questionnaire, PSQI Pittsburgh Sleep Quality Index, SCOPA-AUT Scale for Outcomes in Parkinson’s disease for Autonomic Symptoms, Asp aspartate, Glu glutamate, Gly glycine, GABA c-aminobutyric acid
Demographic and clinical characteristics Demographic and clinical characteristics of the PD patients and healthy controls are shown in Table 1. A total of 92 PD patients and 60 age- and gender-matched healthy controls were enrolled in this study. In the entire group, female exhibited significantly higher levels of Gly (p = 0.019). Moreover, male PD patients displayed markedly higher level of Asp (p = 0.010) compared to female patients. In the PD group, UPDRS III score correlated positively with the plasma Asp level (r = 0.225, p = 0.031). There were significant correlations of HAMD score with disease duration (r = 0.236, p = 0.024), UPDRS III score (r = 0.288, p = 0.005), H-Y stage (r = 0.299, p = 0.004) and LEDD (r = 0.247, p = 0.018). SF-MPQ score was markedly associated with disease duration and LEDD (r = 0.347, p = 0.001; r = 0.354, p = 0.001, respectively), while weakly correlated with H-Y stage (r = 0.214, p = 0.041). Furthermore, the PSQI
score was weakly associated with UPDRS III score (r = 0.232, p = 0.026) and LEDD (r = 0.260, p = 0.012). The correlation of SCOPA-AUT score with the demographic and clinical characteristics of patients was as follows: disease duration (r = 0.287, p = 0.006), UPDRS III (r = 0.262, p = 0.012), H-Y stage (r = 0.249, p = 0.017) and LEDD (r = 0.342, p = 0.001). Hence, gender, disease duration, motor severity (UPRDS III), and dopaminergic medication (LEDD) were considered as potential confounders in the subsequent hierarchical multiple regressions. NMS and plasma levels of amino acids in patients and controls As shown in Table 1, the plasma levels of Asp (p = 0.000), Glu (p = 0.000) and Gly (p = 0.005), as well as GABA (p = 0.01) were significantly lower in PD patients than in
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Q. Tong et al. Table 2 Correlations between nonmotor symptom scores and plasma levels of amino acids in PD patients Variable
Asp r
Glu r
HAMD
-0.326*
-0.344*
SF-MPQ
-0.202
-0.164
PSQI
-0.348*
-0.271*
SCOPA-AUT
-0.065
-0.005
Gly r 0.077 0.030 -0.164 0.062
GABA r -0.176 0.064 -0.486** -0.112
r Spearman’s rho * p \ 0.01, ** p \ 0.001
Fig. 1 The percentage of PD patients with each NMS. The percentage of PD patients suffering from autonomic dysfunction was 81.5 % (75/92 patients) and pain was 69.6 % (64/92 patients), along with sleep disturbances was 46.7 % (43/92 patients). Only 29 (31.5 %) PD patients exhibited depression in our study. PD Parkinson’s disease, NMS nonmotor symptoms
healthy controls. In contrast, the scores of entire assessed NMS were markedly higher in PD patients compared to healthy controls (p = 0.000). Furthermore, PD patients suffered from a wide variety of NMS. In this enrollment, we observed that nearly all patients (96.7 %) suffered at least one kind of NMS, with a much higher prevalence in autonomic dysfunction (81.5 %) and pain (69.6 %) (Fig. 1). Interestingly, the overwhelming majority of PD patients suffered from more than two types of NMS (two NMS, 40.2 %; three NMS, 30.4 %; four NMS, 14.1 %), whereas the small minority of PD patients (12.0 %) showed one NMS.
HAMD and PSQI scores entered as dependent variable in each respective model. Gender, disease duration, UPDRS III score and LEDD were entered into the first block as independent variables, following by the natural logarithms of Asp and Glu were entered into the second block in depression model (excluding two outliers) (Table 3) and the natural logarithms of Asp, Glu and GABA in sleep disturbances model (Table 4). After controlling for confounders, Glu but not Asp remained significantly associated with depression (b = -0.32, p = 0.009), and Asp, GABA but not Glu remained negatively associated with sleep disturbances (b = -0.28, p = 0.016; b = -0.44, p = 0.000, respectively). UPDRS III score significantly correlated with depression (b = 0.24, p = 0.021), as well as LEDD was significantly associated with sleep disturbances (b = 0.27, p = 0.012). Moreover, the main results of hierarchical multiple regressions with the outliers included were similar, excepted that LEDD also became significant in depression model (b = 0.24, p = 0.041).
Correlations between plasma levels of amino acids and NMS
Discussion
Finally, we assessed the correlations of plasma levels of amino acids with NMS. In the patient group, as illustrated in Table 2, the severity of depression as rated by the HAMD was negatively correlated with plasma levels of Asp and Glu (r = -0.326, p = 0.002; r = -0.344, p = 0.001, respectively). Moreover, the increasing PSQI score, used to assess the sleep quality, was associated with the declining plasma level of Asp (r = -0.348, p = 0.001), Glu (r = -0.271, p = 0.009) and GABA (r = -0.486, p = 0.000). Univariate correlations between Glu and HAMD scores, GABA and PSQI scores are shown in Fig. 2. Nevertheless, we failed to observe the severity of pain and autonomic dysfunction significantly associated with plasma levels of any amino acids measured in this study. In the control group, there was no significant correlation between plasma levels of amino acids and NMS. In order to further explore significant correlations, we conducted two hierarchical multiple regressions with
In the past few years, NMS of PD has attracted considerable attention, and growing amount of research has devoted to the underlying biochemical and pathological mechanisms of the NMS (Park and Stacy 2009). Accumulating preclinical and clinical evidence suggests that brain amino acids, such as Asp, Glu, Gly and GABA, are involved in the pathogenesis of depression, pain, sleep disturbances and autonomic dysfunction (Alexander et al. 2013; Brooks and Peever 2011; Frye et al. 2007; Huber and Schreihofer 2011; Mitani et al. 2006). However, less systematic work has been performed to examine whether these amino acids are implicated in the NMS in PD patients. In addition, empirical and theoretical evidence suggests that the altered levels of amino acids in plasma partially reflected their alterations in central nervous system (CNS) of murid rodents (Bongiovanni et al. 2010; Murakami et al. 2009). Prior work also showed that amino acids in plasma were positively correlated with those in CSF of humans, which is
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Correlations between plasma levels of amino acids and nonmotor symptoms in Parkinson’s disease
415
Fig. 2 Correlations between plasma levels of amino acids and NMS. a The plasma Glu level significantly correlated with HAMD scores (r = 0.344, r2 = 0.13, p = 0.001), b the plasma GABA level significantly correlated with PSQI scores (r = -0.486, r2 = 0.22, p = 0.000)
Table 3 Standardized b-coefficients of hierarchical multiple regression with HAMD as dependent variable Depression (HAMD) Block 1 Gender Disease duration
Block 2
0.10
0.04
-0.01
-0.03
UPDRS III score
0.16
0.24*
LEDD
0.17
0.17
Asp
-0.12
Glu Adjusted R2
-0.32** 0.02
0.15
* p \ 0.05, ** p \ 0.01
Table 4 Standardized b-coefficients of hierarchical multiple regression with PSQI as dependent variable Sleep disturbances (PSQI) Block 1
Block 2
Gender
-0.06
-0.09
Disease duration
-0.08
-0.09
UPDRS III score LEDD
0.14 0.25
Asp
0.15 0.27* -0.28*
Glu
-0.04
GABA
-0.44***
Adjusted R2
0.03
0.33
* p \ 0.05, *** p \ 0.001
suggested to be more relevant to brain disorders (Hagenfeldt et al. 1984; Jimenez-Jimenez et al. 1998; Uhlhaas et al. 1986). Again, a few of the studies indirectly examined the CNS changes in PD by detecting plasma amino acids levels (Iwasaki et al. 1992; Molina et al. 1997). On the basis of these data, we addressed the role of these amino acids in NMS of PD by detecting their plasma levels and to further address their correlations with NMS. In our study, we showed that PD patients displayed significantly more severe symptoms of depression, pain,
sleep disturbances and autonomic dysfunction than healthy controls. The prevalence of NMS in the Chinese PD patients similar to a previous report (Li et al. 2010), as 96.7 % of patients suffered from at least one type of NMS and 84.7 % of patients showed over two types of NMS. In agreement with Santos-Garcı´a et al.’s work (Santos-Garcia and de la Fuente-Fernandez 2013), pain and autonomic dysfunction were relatively common symptoms. Depression, one of the most frequently reported psychological disturbances in PD patients, was previously regarded as ‘‘reaction’’ to psychosocial stress and the associated disability of PD. However, accumulating data suggest that neurochemical changes, resulting from the neurodegeneration of PD, might contribute to the depression in PD. A recent study revealed that higher UPDRS scores in PD patients are correlated with the reduced Glu level in the bilateral lentiform nucleus using 1H magnetic resonance spectroscopy (Modrego et al. 2011). The decreased Glu level in the cerebral cortex of PD patients was also observed in another study (Griffith et al. 2008). Furthermore, the depressed subjects without PD exhibited a markedly lower CSF level of Glu (Frye et al. 2007) and reduced Glx (glutamate and glutamine) levels in the anterior cingulate cortex (Sanacora et al. 2012). In line with these previous findings, we observed that the plasma level of Glu in PD patients significantly decreased and negatively correlated with the HAMD score, possibly suggesting that altered glutamatergic functions may be involved in the pathogenesis of depression in PD patients and Glu might be potential indicator for evaluating the severity of depression of PD. Nevertheless, Mitani et al. (2006) showed that the plasma Glu level positively correlated with HAMD score in non-PD depressive patients. We speculated that the inconsistency may be attributed to participants, sample size and measurements. In their study, they enrolled a smaller sample size of younger patients, and focused on non-PD depressive patients. There is increasing body of evidence to suggest that the dysregulation of GABA, the main inhibitory amino acid in brain, might be expected to play an important role in mechanisms underlying sleep disturbances. For example, Watson et al. (2007) provided evidence that microinjection
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of morphine in the oral part of rat pontine reticular formation (PnO) resulted in a striking reduction in rapid eye movement (REM) sleep, whereas a significant augment in wakefulness by decreasing GABA level in PnO. It was also reported that deficits in Gly- and GABA-mediated inhibition were a potential mechanism for REM sleep behavior disorder (Brooks and Peever 2011). Moreover, Tohgi et al. (1991) found that GABA level in CSF of PD patients was lower when compared to healthy controls. Consistently, in our study, there was a much more reduction in plasma level of GABA in PD patients, and it was negatively associated with the severity of sleep disturbances, suggesting that GABA deficiency may be an underlying mechanism and a potential marker of sleep disturbances in PD. Furthermore, the plasma Asp level was slightly related with the severity of sleep disorder, possibly suggesting that excitatory amino acids also be partially involved in the genesis of sleep disturbances of PD. Of note, in our study, the r-values of correlations analysis were relatively low. Thus, the results from our correlation analysis were limited, only providing some information regarding the pathogenesis and severity of NMS in PD patients coupled with clinical features. Notably, the validity and specificity of these amino acids served as clinical indicator for NMS of PD remain to be confirmed in future CSF study with a larger sample size. However, previous studies showed different results (Araki et al. 1986; Jime´nez-Jime´nez et al. 1996; Perschak et al. 1987). For example, Jime´nez-Jime´nez et al. (1996) found that PD patients, compared with controls, had similar CSF Glu and higher GABA levels. Some authors also reported increased CSF Gly levels (Araki et al. 1986), or normal CSF GABA levels in PD patients (Perschak et al. 1987). The discrepancies between studies might be related with the sample size, disease status, measurements, or differences in samples. Compared with the previous reports, we enrolled a relatively larger sample size of mild PD patients, and used a more sensitive and stable measurement to detect amino acids in plasma samples. Taken together, these findings suggest that the roles of the altered amino acids levels in PD remain to be further investigated. It is noteworthy that our study has several limitations. First, we recruited a relative small sample size of patients, especially for correlation analysis; second, we only focused on four NMS of PD, and as a result probably ignored other NMS, such as sensory disorders and cognitive deficits. Third, our study was restricted to the alterations of amino acids in plasma samples. Therefore, larger scale studies are needed to determine common changes in plasma and CSF levels of amino acids in PD patients with more NMS. In addition, non-PD patients with depression, pain, sleep disturbances, and autonomic dysfunction should be included in order to obtain more precise alterations of these amino acids in NMS of PD.
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In summary, our results showed that PD patients exhibited significantly higher scores of NMS scales and lower plasma levels of amino acids compared to healthy controls. Hierarchical regression analysis revealed that plasma level of Glu was negatively associated with the severity of depression, and Asp and GABA negatively associated with sleep disturbances. Combined with the data from previous studies, our findings suggest that the altered plasma levels of amino acids in PD may participate in the pathogenesis of NMS of PD. Acknowledgments This work was supported by the University Natural Science Research Project in Jiangsu Province (No. 13KJB32009), the Natural science foundation of Jiangsu Province (No. BK20141494), the Opening Project of Jiangsu Key Laboratory of Neurodegeneration (No. SJ11KF01) and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Conflict of interest of interest.
The authors declare that they have no conflict
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