Higher cerebrospinal fluid to plasma ratio of p-cresol sulfate and indoxyl sulfate in patients with Parkinson’s disease

Journal Pre-proofs Higher cerebrospinal fluid to plasma ratio of p-cresol sulfate and indoxyl sulfate in patients with Parkinson’s disease Bartłomiej Sankowski, Karolina Księ żarczyk, Emilia Raćkowska, Stanisław Szlufik, Dariusz Koziorowski, Joanna Giebułtowicz PII: DOI: Reference:

S0009-8981(19)32102-3 https://doi.org/10.1016/j.cca.2019.10.038 CCA 15899

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Clinica Chimica Acta

Received Date: Revised Date: Accepted Date:

9 August 2019 24 October 2019 24 October 2019

Please cite this article as: B. Sankowski, K. Księ żarczyk, E. Raćkowska, S. Szlufik, D. Koziorowski, J. Giebułtowicz, Higher cerebrospinal fluid to plasma ratio of p-cresol sulfate and indoxyl sulfate in patients with Parkinson’s disease, Clinica Chimica Acta (2019), doi: https://doi.org/10.1016/j.cca.2019.10.038

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Higher cerebrospinal fluid to plasma ratio of p-cresol sulfate and indoxyl sulfate in patients with Parkinson’s disease Bartłomiej Sankowskia, Karolina Księżarczyka, Emilia Raćkowskaa, Stanisław Szlufikb, Dariusz Koziorowskib, Joanna Giebułtowicza* a

Department of Bioanalysis and Drug Analysis, Faculty of Pharmacy, Medical University of Warsaw, 1 Banacha Street, 02–097 Warsaw, Poland b

Department of Neurology, Faculty of Health Sciences, Medical University of Warsaw, 8 Kondratowicza Street,

03–242 Warsaw, Poland *Corresponding author: Joanna Giebułtowicz, Department of Bioanalysis and Drugs Analysis, Faculty of Pharmacy, Medical University of Warsaw, 1 Banacha Street, 02–097 Warsaw, Poland, [email protected], phone +48 22 572 09 49, fax +48 22 572 09 76

Abstract Background: In Parkinson’s disease (PD), impairment of brain to blood barrier and/or bloodcerebrospinal fluid (CSF) barrier is described. It can increase the level of uremic toxins in CSF. So far, role of these compounds in neurological disorders has not been completely understood. However, a link has been observed between chronic kidney disease and neurological disorders. We measured the concentrations of uremic toxins (i.e. indoxyl sulfate (IS), p-cresol sulfate (pCS), symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA), and trimethylamine N-oxide (TMAO)) in CSF and plasma, and correlated them with inflammation and oxidative stress biomarkers. Methods: Plasma and CSF samples were collected from 27 volunteers (18 with PD and 9 controls). The level of toxins was determined using liquid chromatography coupled with tandem mass spectrometry. Results: In PD, for IS and pCS, CSF-plasma ratio was higher. Concentration of pCS in CSF was higher in PD compared to controls. TMAO level was also higher in plasma of that group. Patients with motor fluctuations had higher level of uremic toxins in CSF, but not in plasma. Conclusions: The level of pCS and IS in CSF of PD is higher than expected, based on their blood level. It can influence pathogenesis and progression of PD. Keywords: asymmetric dimethylarginine; indoxyl sulfate; mass spectrometry; Parkinson’s disease; p-cresol sulfate; trimethylamine N-oxide Abbreviations: ADMA—asymmetric dimethylarginine; Arg—arginine; CSF—cerebrospinal fluid; CNS—central nervous system; eGFR—estimated glomerular filtration rate; IS—indoxyl sulfate; PD— Parkinson’s disease; pCS—p-cresol sulfate; SDMA—symmetric dimethylarginine; TAC—total antioxidant capacity; TMAO—trimethylamine N-oxide; UPDRS— unified Parkinson's disease rating scale; 8-OHdG—8-hydroxy-2'–deoxyguanosine;

1

Introduction

Uremic-retention solutes are the compounds whose concentration in an organism increases with decreasing kidney function. Indoxyl sulfate (IS), p-cresol sulfate (pCS), asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and trimethylamine Noxide (TMAO) are the examples of such molecules. At uremic concentrations, they play a crucial role in the progression of chronic kidney diseases (CKD) and have negative outcomes. Until now, the role of the compounds in pathogenesis of neurological disorders is not completely understood. However, a link between CKD and neurological disorders has been observed [1]. Uremic toxins increase the risk of cognitive disorders and dementia in patients with kidney disease [2]. Causes of cognitive disorders in CKD are diversified; generally, they include accumulation of uremic toxins and high prevalence of ischemic cerebrovascular lesions [3]. It was also shown that uremic patients have a 1.81-fold higher risk of developing Parkinsonism than the nonuremic group. Intensive dialysis can slow down the progression of the disease and alleviate symptoms [4,5]. Uremic toxins are divided mainly into three classes: the small water-soluble compounds, the middle molecules, and the protein-bound solutes. Small water-soluble compounds (molecular weight <500Da) are ADMA and SDMA [6]. These compounds are not found naturally in human genetic code. Thus, they are called nonproteinogenic amino acids. ADMA is synthesized in the process of posttranslational methylation by replacing the terminal nitrogen atom of guanidino group in arginine (Arg) from protein arginine methyltransferases by two methyl groups. SDMA is a structural isomer of ADMA. To understand the functions of ADMA and SDMA in human body, the crucial biochemical pathway they are included in should be known. Arginine:glycine amidinotransferase is the catalyst of L-homoarginine (hArg) synthesis from free Arg and L-lysine. Nitric oxide synthase (NOS) catalyzes the conversion of Arg and hArg to nitric oxide (NO) which is a main signaling molecule; whereas, ADMA and SDMA are the inhibitors of NOS activity [7]. Slightly increased ADMA and SDMA concentrations in biofluids are associated with many diseases including neurological conditions and disorders, for example, migraine [8], acute ischemic stroke [9], multiple sclerosis, neuromielitys optica [10], and depressive symptoms [11]. Gut microbiota produces IS and pCS. Their precursors are synthesized from dietary tryptophan and tyrosine, respectively. Both IS and pCS are majorly bound to albumin in plasma [12]. In literature, studying pathogenic actions of IS and pCS on stem cells and

animals at uremia concentration include the induction of reactive oxygen species formation. These species activate the nuclear factor-kappaB (NF-kB) pathway, resulting in both oxidative stress and pro-inflammatory cytokine production [13,14]. IS causes nephrotoxicity in CKD patients and is involved in a neurodegenerative diseases, such as Alzheimer’s disease (AD) [6,15,16], and neurological disorders [17–19]. Just like IS and pCS, gut bacteria also produces TMAO precursor. Thus, its blood levels are associated with the condition of intestines microbiome. Gut microbiome metabolizes dietary carnitine and choline. It produces an intermediate compound known as trimethylamine that is oxidized in the liver to TMAO [20]. TMAO downregulates the reverse cholesterol transport and enhances foam cell formation and pellet activation. This is associated with an increased risk of thrombosis and major adverse cardiovascular events [21,22]. Moreover, TMAO revealed pro-inflammatory effect on animal and in vitro models. It also causes blood-brain barrier disruption by reducing the expression of tight junction proteins like claudin-5 and tight junction protein-1 [23]. However, there is no available information about the link between TMAO and neurodegeneration on human study. Uremic toxins affect various processes or systems in the organism. They may be involved in pathogenesis of different diseases. However, their role in PD has not been recognized yet. In this paper, we compared the concentration of uremic toxins (IS, pCS, TMAO, SDMA, ADMA, ratio ADMA/Arg) with the ratio of the substances in cerebrospinal fluid (CSF) to blood in PD and controls. Moreover, we analyzed whether the presence of motor fluctuations/dyskinesis is related to the level of toxins. Finally, we correlated the level of the toxins with markers of oxidative stress (8-hydroxy-2’-deoxyguanosine (8-OHdG), total antioxidant capacity (TAC)) and inflammation (C-reactive protein (CRP), hepcidin, and its prohormone), which play a significant role in pathogenesis of PD. SDMA, ADMA, and ADMA/Arg were chosen since NO pathway dysregulation was shown to be associated with some neurodegenerative disorders [24] and oxidative stress [25]. IS, pCS, and TMAO are also related to increased oxidative stress [13,14,26] that is recognized as a central event contributing to the degeneration of dopaminergic neurons in PD pathogenesis [27].

2 2.1

Materials and methods Chemicals

The gradient-grade high-performance liquid chromatography (HPLC) solvents, such as acetonitrile (ACN), formic acid (FA), ammonium acetate, and methanol, were purchased from

Merck (Darmstadt, Deutschland). Purity of all standards were was higher than 95%. pCS, IS, ADMA, SDMA, and TMAO as well as their internal standards, that is, p-CS-D7, IS-D4, ADMA-D6, and TMAO-D9, were obtained from Toronto Research Chemicals (TRC) (Toronto, Canada). Arg was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, US), while its internal standard, Arg-13C6, was purchased from TRC. Stock standard solutions were prepared at the concentration of 1 mg/mL in methanol (pCS, IS, and TMAO), 1 mg/mL in water (SDMA), 2 mg/mL in water (ADMA), and 435 µg/mL in 0.1 mol/L HCl (Arg). All stock solutions were stored at –40°C. Prior to use, the working standard solutions were prepared by dilution of the appropriate stock solutions with water to obtain the required concentrations. Acetic acid and sodium acetate were purchased from Chempur (Piekary Slaskie, Poland). Hydrogen peroxide 30% was obtained from Avantor Performance Materials Poland S.A. (Gliwice, Poland). 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma Aldrich (Saint Louis, Missouri, US). Ultrapure water was obtained from the Millipore water purification system (Milli-Q, Billerica, US). The levels of 8-OHdG, hepcidin and hepcidin prohormone were measured using the commercially available enzyme-linked immunosorbent assays (ELISA): Highly Sensitive 8OHdG Check (Japan Institute for the Control of Aging, Fukuroi, Shizuoka, Japan), Hepcidin 25 (bioactive) HS ELISA (DRG Instruments GmbH, Marburg, Germany) and Hepcidin Prohormone ELISA (DRG Instruments GmbH, Marburg, Germany), respectively. 2.2

Patients and controls

A group of 18 PD patients were selected for the study from the Movement Disorder Clinic. Neurological examination was performed by neurologists experienced in diagnosis of movement disorders. The clinical diagnosis of PD patients was established according to UK Parkinson’s Disease Brain Bank criteria [28]. The clinical characteristics of patients with PD are presented in Table 1. Table 1: Selected clinical characteristics of patients with Parkinson’s disease

Age of onset (<60) [%]

Parkinson’s disease 89

Tremor [%]

78

Rigidity [%]

89

Body Bradykinesia and Hypokinesia [%] Time since beginning of symptoms [years] Dyskinesia [%] Time after dyskinesia appeared [years] Motor fluctuations [%] Hoehn and Yahr (HY) disease rating scale [points] Unified Parkinson's disease rating scale (UPDRS) [points]

100 8.1 ± 4.4 (0.5–16) 100 4.0 ± 3.8 (0–12) 78 2.9 ± 0.6 (2.5–4.0) 40 ± 18 (19–77)

The controls consisted of unrelated healthy volunteers with no evidence of the history of anemia and inflammatory or neurological/neurodegenerative disorder in their family. The group includes only nine individuals since CSF is collected due to lumbar puncture, which is highly invasive and is associated with such risk factors as headache, infection, brain herniation, or damage to the nerves in the spinal cord. Thus, CSF collection from healthy people without medical indication raises an ethical concern. In control group, we included patients diagnosed with headache, whose CSF analysis results were within the range. The principles of the Declaration of Helsinki were followed. Ethic committees of the Warsaw Medical University approved the study. All participants signed an informed consent. Venous blood samples were collected into a tube with EDTA and without anticoagulants, and centrifuged according to standard operating procedures to obtain plasma and serum, respectively. CSF samples were collected via lumber puncture and centrifuged to remove blood cells contamination, before freezing. These materials were stored at –30°C, until further analysis. Fresh serum was subjected to routine analyses including the measurement of CRP and creatinine. 2.3

Biochemical analysis

2.3.1 Determination of uremic solutes The analyses of pCS and IS were performed in one run; whereas analyses of TMAO, ADMA, SDMA, and Arg in the second run. Due to difference in hydrophilicity, separation of the compound from the matrix was performed on octadecyl carbon chain (C18)-bonded silica column and hydrophilic interaction liquid chromatography column (HILIC), respectively. pCS and IS Fifty µL of plasma or CSF was mixed with the internal standards solution (p-CS-D7 and ISD4) to obtain the final concentration of 600 and 80 ng/mL, respectively. Then, the samples

were deproteinized with methanol (1:4, v/v), incubated at –20°C for 20 min, and centrifuged at 9,300 × g at 4°C for 10 min. The supernatant was diluted six times (plasma) or two times (CSF) with water before being injected into HPLC. Curtain gas, ion source gas 1, ion source gas 2, and collision gas (all high-purity nitrogen) were set at 241, 414, 276 kPa, and “medium” instrument units, respectively. Ion spray voltage and source temperature were set at 4,500 V and 600°C, respectively. Chromatographic separation was achieved with a Kinetex C-18 column (100 mm × 4.6 mm, particle size 2.6 µm) supplied by Phenomenex (Torrance, CA, US). The column was maintained at 40°C, at a flow rate of 0.5 mL/min. Mobile phases consisted of HPLC-grade water with 0.1% FA as eluent A and methanol with 0.1% FA as eluent B. Gradient (%B) was as follows: 0 min 10%; 0.5 min 10%; 4.5 min 95%; and 8.5 min 95%. Volume of injection was 10 µL. Target compounds were analyzed in multiple reaction monitoring mode. Transitions used for quantitation were m/z 212>80 and m/z 216>80 for IS and IS-D4 and m/z 187>107 and m/z 194>114 for p-CS and p-CS-D7, respectively. Compound parameters, viz. declustering potential (DP), collision energy (CE), entrance potential (EP), and collision exit potential (CXP), were (−60), (−38), (−10), (−5) and (−65), (−46), (−10), (−1) V IS and IS-D4, respectively; and (−65), (−28), (−10), (−7) and (−60), (−30), (−10), (−7) V for p-CS and pCS-D7, respectively. ADMA, SDMA, Arg, TMAO Fifty µl of plasma or CSF was mixed with the internal standards solution (Arg-13C6, TMAOD9, and ADMA-D6) to obtain the final concentration of 7.5 µg/mL for Arg-13C6, 0.75 µg/mL for TMAO-D9, and 155 ng/mL for ADMA-D6. Next, the samples were mixed on vortex for 5 min, incubated at –20°C for 20 min, and centrifuged at 9,300 × g at 4°C for 10 min. The supernatant was transferred to a new test tube, centrifuged at 9,300 × g at 4°C for 10 min, and analyzed. Curtain gas, ion source gas 1, ion source gas 2, and collision gas (all high-purity nitrogen) were set at 241, 207, 345 kPa, and “high” instrument units, respectively. Ion spray voltage and source temperature were set at 5,500 V and 600°C, respectively. Chromatographic separation was achieved with a SeQuant ZIC-HILIC column (50 mm × 2.1 mm, particle size 5 µm) supplied by Merck. Column was maintained at 40°C, at a flow rate of 0.5 mL/min. Mobile phases consisted of water solution of 20 mM ammonium acetate as eluent A and ACN with 0.2% FA as eluent B. Gradient (%B) was as follows: 0 min 90%; 1

min 90%; 7 min 50%; and 9 min 90%. Volume of injection was 5 µL. Target compounds were analyzed in multiple reaction monitoring mode. Transitions used for quantitation were m/z 203>46 and m/z 203>172 and m/z 209>116 for ADMA, SDMA, and ADMA-D6, m/z 76>42 and m/z 85>66 for TMAO and TMAO-D9, and m/z 175>116 and m/z 181>121 for Arg and Arg-13C6, respectively. Compound parameters, viz. DP, CE, EP, and CXP, were 61, 41, 10, 0 and 61, 19, 10, 10 and 66, 23, 10, 8 for ADMA, SDMA, and ADMA-D6; 66, 53, 10, 6 and 61, 39, 10, 4 for TMAO and TMAO-D9; and 61, 21, 10, 8 and 31, 21, 10, 10 for Arg and Arg-13C6, respectively. 2.3.2 Determination of 8-OHdG, hepcidin, prohepcidin Hepcidin and prohepcidin levels in plasma were determined using commercially available ELISA kits produced by DRG Instruments, that is, Hepcidin 25 (bioactive) HS ELISA and Hepcidin Prohormone ELISA, respectively. Levels of 8-OHdG were measured using the ELISA kit Highly Sensitive 8-OHdG Check. The absorbance for ELISA kits was measured using a Synergy MX plate reader (BioTek Instruments, Vermont, US). ELISA assays were carried out in accordance with the procedures specified by the manufacturers. 2.3.3 Determination of TAC Following a modified method developed by Erel, ABTS assay was used to measure antioxidant capacity of CSF [29]. Basic principle of this method was the capacity of antioxidants, present in the sample, to reduce the 2,2'-azino-bis-3-ethylbenzothiazoline-6sulfonic acid cation radical (ABTS.+) to ABTS. As a result of the reaction, characteristic blue green ABTS.+ was bleached to colorless ABTS molecules. The change of the color was proportional to the concentration of antioxidant in the sample and measured spectrophotometrically as a change in absorbance. The absorbance was measured at 660 nm with the Synergy MX plate reader using 96-well plates. In short, the ABTS.+ radical cation was generated by the ABTS reaction with hydrogen peroxide (H2O2) in acetate buffer (30 mmol/L (pH 3.6)). For this, ABTS was dissolved in 2 mmol/L H2O2 in an acidic medium to achieve the final concentration of 10 mmol/L and incubated at room temperature for 1 h. Next, for the purpose of examining of CSF, 10 µL of each sample or antioxidant standards was added to 200 µL of 0.4 mmol/L acetate buffer (pH 5.8). Then, the first absorbance reading at 660 nm was taken. Following the addition of 20 µL of the solution of ABTS.+ cation radical to each well, the absorbance was measured again, 5 min after the mixing.

Results of change in absorbance were calibrated using the Trolox as antioxidant standard. The values of TAC were expressed in µmol Trolox equivalent/mL. 2.3.4 Others At the hospital laboratory, CRP and creatinine concentrations in serum were determined. CRP level was measured through an immunoturbidimetric method, using latex-particle enhanced assay. Concentration of creatinine was estimated by Jaffe’s method. Estimated glomerular filtration rate (eGFR) was calculated with abbreviated Modification of Diet in Renal Disease (MDRD) equation. 2.4

Statistical analysis

The normality of continuous variable distribution was tested with the Shapiro-Wilk test. Parameters measured as continuous variables were compared using Student’s t-test or MannWhitney test as appropriate. Categorical variables were compared using the Chi-square test. Strength of association between variables was measured using Spearman’s rank correlation. All statistical analyses were performed using Statistica 12.0 and a p-value < 0.05 was considered statistically significant. Principal component analysis (PCA) was performed using MetaboAnalyst 2.0, a web-based metabolomics tool. Data were log-transformed before the analysis. All results were presented as means and standard deviations or median and interquartile range. For each patient, CSF-plasma ratio of a particular toxin was calculated by dividing the concentration of the toxin in CSF by its level in plasma. The result was expressed in percentage. Higher CSF-plasma ratio indicates higher concentration of the toxin in CSF than expected, based on its plasma level.

3 3.1

Results Characteristic of study population

The study involved 27 participants. Eighteen of these participants were diagnosed with PD and the remaining nine represented the general population of healthy people. Clinical characteristic of the study groups is presented in Table 2. There was no statistically significant difference between the study groups regarding the percentage of males, creatinine, CRP, TAC, and marker of oxidative stress: 8-OHdG. PD patients were slightly older and had lower eGFR (all values were in the reference range regarding the age). They had elevated

hepcidin level and lower prohepcidin concentration in plasma than the control group. Table 2: Selected clinical characteristics of examined and control group

Number

Parkinson’s disease 18

Controls 9

44%

44%

Number of males [percent]

*

59 ± 10 0.7 ± 2.3 0.8 ± 0.2 89 ± 14* 16.7 ± 7.1* 71 ± 45* 0.48 ± 0.29 0.27 ± 0.14

Age [years] CRP [mg/L] Creatine level [mg/dL] eGFR [mL/min 1.73 m2] Hepcidin in plasma [ng/mL] Prohepcidin in plasma [ng/mL] 8-OHdG in CSF [ng/mL] TAC in CSF [µmol Trolox equiv./mL] *statistically

42 ± 10 0.2 ± 4.9 0.7 ± 0.2 101 ± 19 9.4 ± 2.9 97 ± 12 0.55 ± 0.14 0.24 ± 0.10

significant with p-value lower than 0.05

PD patients were treated using L-dopa (94%, 400–1800 mg/day), dopamine agonists (67%, 2– 16 mg/day), and amantadine (17%, 200–400 mg/day). None of them obtained monoamine oxidase B inhibitors or catechol-O-methyl transferase inhibitors. 3.2

Biochemical analysis

Concentrations of uremic solutes—pCS, IS, ADMA, SDMA, TMAO, and ADMA/Arg—are presented in Table 3. Despite of dissimilarity in eGFR between groups in plasma, there were no significant differences in the levels of almost all uremic toxins (beside TMAO). In CSF only, the level of pCS was higher in PD. Interestingly, the ratio of toxins in CSF and plasma (CSF/plasma) was four and eight times higher in PD than in control group, for IS and pCS, respectively. It means that concentrations of the toxins in CSF in PD are higher than expected, based on their plasma level. The level of IS in CSF correlated with hepcidin (r = 0.68, p = 0.02084). No other correlations of the toxins with markers of oxidative stress (8-OHdG, TAC) and inflammation (CRP, hepcidin, and its prohormone) were observed. Table 3: Concentration of uremic toxins in plasma and CSF of study groups

CSF

Controls (n = 9)

Parkinson’s disease (n = 18)

Parkinson’s Parkinson’s disease with disease without motor fluctuation motor fluctuation (n = 14) (n = 4)

pCS [µg/mL] IS [µg/mL] ADMA [ng/mL] SDMA [ng/mL] ADMA/Arg [-](×10–3) TMAO [ng/mL]

0.022 ± 0.023 0.0085 ± 0.0082 26 ± 15 55 ± 74 7.0 ± 1.5 76 ± 51

0.06 ± 0.1a 0.0092 ± 0.0027 23 ± 10 74 ± 51 6.9 ± 1.2 108 ± 74

0.112 ± 0.095ab 0.0108 ± 0.0060b 24.9 ± 6.6b 96 ± 56b 7.25 ± 0.96b 122 ± 78b

0.031 ± 0.020 0.0074 ± 0.0015 14.6 ± 3.9a 53 ± 27 5.91 ± 0.96 61 ± 33

5.1 ± 2.5 0.81 ± 0.64 124 ± 26 207 ± 60 7.4 ± 1.5 136 ± 120

6.7 ± 4.2 0.81 ± 0.52 122 ± 29 217 ± 47 8.4 ± 2.5 300 ± 220a

7.7 ± 4.3b 0.81 ± 0.47 122 ± 28 228 ± 89 8.8 ± 2.2 410 ± 360a

4.1 ± 1.4 0.81 ± 0.57 122 ± 29 216 ± 29 7.9 ± 2.5 278 ± 71

0.19 ± 0.27 0.42 ± 0.21 15 ± 23 51 ± 43 85 ± 24 39 ± 29

1.5 ± 1.0a 1.6 ± 0.9a 19 ± 10 35 ± 15 86 ± 25 33 ± 21

1.7 ± 1.1a 1.71 ± 0.94a 20.3 ± 6.1 39 ± 14b 88 ± 22 42 ± 24b

0.93 ± 0.60 1.38 ± 0.69a 13.5 ± 6.3 24 ± 12 80 ± 22 22 ± 11

Plasma pCS [µg/mL] IS [µg/mL] ADMA [ng/mL] SDMA [ng/mL] ADMA/Arg [-](×10–3) TMAO [ng/mL]

CSF/Plasma pCS [%] IS [%] ADMA [%] SDMA [%] ADMA/Arg [%] TMAO [%] a

statistically significant comparing to control group (p-value lower than 0.05) b statistically significant (p-value lower than

0.05) compared to PD patients without motor fluctuations—the comparison between PD patients with and without motor fluctuations

Patients with motor fluctuations had higher pCS (p = 0.0043), IS (p = 0.0361), ADMA (p = 0.0017), SDMA (p = 0.02614), and TMAO levels (p = 0.0179) in CSF than other PD patients. It was not observed for plasma. Patients with motor fluctuations were also older (61.9 ± 8.9 years vs. 50 ± 12 years; p = 0.0158) and had a higher hepcidin level (18.2 ± 7.9 ng/mL vs. 8.6 ± 6.5 ng/mL, p = 0.0199). In pooled group (n = 27), we observed a strong correlation between eGFR and CSF level of pCS (r = –0.41, p = 0.0316), IS (r = –0.52, p = 0.00493), SDMA (r = –0.40, p = 0.03841), and TMAO (r = –0.59, p = 0.00123). We also observed a relation between toxins level in plasma and CSF in case of pCS (r = 0.50, p = 0.03332), IS (r = 0.42, p = 0.04997), and TMAO (r = 0.76, p = 0.00024). Moreover, the lower eGFR resulted in higher pCS csf/plasma (r = –0.51, p = 0.03166). The concentration of ADMA in CSF correlated with SDMA (r = 0.81, p<0.00001) and TMAO (r = 0.47, p = 0.01307), but not with IS and pCS. Weaker correlation was observed for pCS and IS in CSF (r = 0.37, p = 0.04880). The correlation pattern of uremic toxins in CSF, plasma, and eGFR is shown in Fig. 1.

PCA (Fig. 2) was performed to visualize the relation between the variables and variation within study groups. The statistical procedure converts a set of observations of the variables into a set of values of linearly uncorrelated variables called principal components (PC). The first PC explains the largest possible variance. In our study, the first, second, and third PCs (PC1, PC2, and PC3) accounted for 64, 14, and 9% of total variation, respectively. The groups of controls and PD patients were separated the best by PC2 and PC5. PC2 was correlated mainly with concentration of pCS in CSF (r = 0.66) and pCS csf/plasma (r = 0.60). PC5 was correlated with IS in CSF (r = –0.68) and plasma (r = –0.50). The component was negatively correlated with unified Parkinson's disease rating scale (UPDRS) (r = –0.46) and PD duration (r = –0.46). In Fig. 2, the distribution of the subjects on score plot (PC2 vs. PC5) is shown. Regarding PD, we observed that five patients were not separated with controls (marked by arrows). These five patients differed from other PD patients by lower pCScsf/plasma (0.10 ± 0.14% vs. 1.73 ± 0.97%; p<0.00001), lower pCS in CSF (0.036 ± 0.048 µg/mL vs. 0.117 ± 0.094 µg/mL; p = 0.0152), lower UPDRS (27.2 ± 5.7 vs. 46 ± 18; p = 0.0218), but higher 8OHdG (0.71 ± 0.20 ng/mL vs 0.43 ± 0.26 ng/mL; p = 0.00021). Other parameters concerning the diseases progression did not differ.

4

Discussion

There are many methods to determine the concentration of target compounds in brain. However, due to limited access to human brain tissue, the methodological problems of extracting from brain homogenates, and ethical problems of microdialysis in human, CSF still represents the closest approximation of extracellular space of the central nervous system (CNS) regarding the concentration of the target compound [30]. This approach is the most frequently used for xenobiotics [31], but can be also useful for peripheral metabolites [32]. In our work, concentrations of five uremic toxins, that is, pCS, IS, ADMA, SDMA, and TMAO, were measured both in plasma and CSF. pCS was the only one with significantly elevated concentration in CSF (almost three times higher in PD), but not in plasma of PD. Moreover, the toxin level was also three times higher in PD with motor fluctuation than in other patients. The presence of motor fluctuation is mainly associated with long-term use of L-dopa and appears mainly in more advanced disease stage [33]. Similarly, for pCS in CSF (but not plasma) of these patients (with motor fluctuations), the elevated concentrations of other toxins like IS, ADMA, TMAO, and SDMA were observed. It indicates the role of molecules in the progression of PD.

Higher concentration of TMAO was observed in plasma of not only motor fluctuations subgroup but also in whole PD group. It might be the result of lower eGFR in that group [34]. However, the eGFR, although lower in PD, was within the reference range and none of the patients had symptoms of CKD (negative dipstick urine analysis). Moreover, the level of other uremic solutes in plasma, viz. pCS, IS, ADMA, and SDMA, was comparable in PD and controls. The concentration of TMAO in plasma depends on several factors including diet, composition of gut microflora, and activity of flavin monooxygenase enzymes in the liver [22]. A diet rich in major TMAO precursors (such as choline and carnitine), that is, red meat [35] and eggs [36] and seafood increases the TMAO level in plasma [37]. Upon absorption in the gut, TMAO precursors are converted to trimethylamine (TMA) by the various enzymes of microbiota. Then, in the liver, TMA is oxidized to TMAO by enzyme flavin-containing monooxygenase 3 FMO3 [22]. Additionally, several fishes and marine invertebrates contain free TMAO; thus, their consumption may increase the toxin concentration in plasma as well [37,38]. TMAO level depends not only on diet but also on unique gut microbiota profile [22,38]. High consumption of protein alters the composition of saccharolytic and proteolytic gut bacteria and increases the level of uremic toxins [39]. So, different dietary habits of PD and control group or changes in gut microbiota composition can be another explanation of higher TMAO level in plasma of PD. However, no differences in plasma concentration of other toxins associated with gut microbiota and diet, that is, IS and pCS, were observed. Their possible causes are different precursors (tryptophan and tyrosine), which can be found in high concentration in poultry, pork, soybeans, whole grains, eggs, dairy [40,41], and different bacteria community responsible for the production of toxins [42,43]. Gut microbiome may produce signaling molecules that interact with the host nervous system, causing the dysregulation of immune activity and neuroinflammation [44]. Shift in microbiome composition has been associated with many neurological and neurodevelopmental disorders, including PD [45,46]. In addition, PD patients, also in early stages, suffer from many different gastrointestinal symptoms (e.g. constipation) [46,47], which may increase the level of the toxin produced in intestine in circulation [48]. We have no data on gastrointestinal symptoms in our cohort group. No differences in TMAO level between groups were observed in CSF probably due to large variation of average values. However, a significantly higher TMAO level in CSF was

observed in patients with motor fluctuations compared to other PD patients. It indicates the role of the molecule in progression of PD. Recently, Del Rio et al., for the first time, described the measurable TMAO level in CSF [49]. But, due to lack of data on plasma TMAO level of the examined patients, they could not verify the hypothesis whether TMAO in CSF originates from a small fraction of liver-derived TMAO that crosses the blood-brain barrier or TMAO detected in CSF derive from de novo synthesis, as the expression of FMO3 has been detected in the adult brain. Our results clearly indicate that TMAO in CSF originates mainly from blood, since a strong correlation of its level in CSF and plasma occurred (r = 0.76). Also, moderate correlation between level of toxins in CSF and plasma was observed for pCS (r = 0.50) and IS (r = 0.42). According to our best knowledge, it is for the first time that such a relation was analyzed and described. No correlations (CSF vs. plasma) were observed regarding ADMA and SDMA levels. Similar results were obtained by Arlt et al., who observed lower ADMA level in CSF, but higher in plasma of AD patients compared to controls [24], suggesting no correlation between both matrices. ADMA is proposed to be involved in AD pathogenesis. It is an endogenous competitive inhibitor of NOS that induces NO synthesis. NO is a signal molecule in the nervous system. It affects the control of dopamine release and plays a role in emotional, behavioral, and cognitive processes [50]. Our research is the first one to assess of the levels of ADMA and ADMA/Arg in PD. We found comparable concentrations of the compounds in plasma and CSF of PD patients and controls. Similar results were obtained for SDMA. SDMA have proinflammatory properties and were shown to be in higher concentrations in CSF of AD. Both ADMA and SDMA result in neuronal damage [50]. As mentioned previously, no differences in ADMA, ADMA/Arg, and SDMA in PD and controls were observed. But, significantly higher concentrations of ADMA and SDMA were found in PD patients with motor fluctuation compared to other PDs. Thus, molecules seem to be important in PD progression. Elevated CSF concentrations of ADMA may be associated with reduced blood flow and decreases brain microperfusion [24]. Patients with motor fluctuations were also older and had higher hepcidin level. PD patients, in our experiment in general, had a higher hepcidin level in circulation that control group. Hepcidin is a small peptide produced in response to iron-load and inflammation in brain iron homeostasis. Accumulation of iron in cells due to high hepcidin level can induce oxidative stress by producing free radicals via the Fenton reaction. Oxidative stress plays an important role in pathogenesis of many neurological disorders, such as PD and AD [51]. Increase in the

pathological hallmarks of AD and PD with age is also partly related to age-dependent increase in iron contents in the brain due to the age-induced increase in the expression of hepcidin [52]. Peptide level in brain depends not only on local production but also on its level in circulation. Similar our results, higher peptide level in blood of PD, compared to controls, was observed by Manolov et al. [53]. We also observed correlation of IS in CSF and hepcidin level in plasma of PD. Previously, IS was shown to affect glial function, thus increasing oxidative stress and neuroinflammation, as well as inducing hepcidin expression [54]. IS induces hepcidin production via a pathway that not only involves oxidative stress but also AhR activation [55]. All these reasons explain the higher hepcidin level in PD and the correlation between the IS in CSF and hepcidin in plasma. Prohepcidin is a hepcidin prohormone. Surpassingly, in our study, PD group had lower prohepcidin level, suggesting no correlation with hepcidin level. Such observation was already made by other scientists. They showed that prohepcidin in blood neither correlates with hepcidin level, nor with other iron parameters and inflammatory markers [56,57]. Opposite results of higher prohepcidin level in PD were observed by Kwiatek-Majkusiak et al. [58]. The differences might be related to different clinical characteristics of the groups (The group of Kwiatek-Majkusiak included patients with deep brain stimulations.) However, prohepcidin level determination is not recommended while assessing iron homeostasis, and is biased due to low stability of prohormone in plasma [59]. One of the most interesting observations was the ratio of the pCS and IS in CSF to plasma in PD. It was shown to be four and eight times higher in PD, compared to control group, for IS and pCS, respectively. It means that both PD patients and healthy men with the same level of the toxins in plasma would have different concentrations of the IS and pCS in CSF. PD patient would have higher levels. Thus, lower concentrations of the toxins in plasma of PD would cause adverse effects in nervous system compared to the case of people without PD. One of the explanations of the phenomenon can be impairment of brain to blood barrier (BBB) and/or blood-cerebrospinal fluid barrier (BCSFB) in PD. BBB is composed of CNS microvascular endothelial cells, whereas BCSFB is composed of the choroid plexus epithelial cells [30]. Their undisturbed function is crucial in brain homeostasis and was observed to be disturbed in neurodegeneration [60]. Gray et al., using histologic markers of serum protein, iron, and erythrocyte, have shown an increased permeability of the BBB in PD patients [61]. Also, functionality of some active transporters was shown to be affected [62]. However, the function of organic anion transporters (OATs), which transport pCS and IS from CSF to the

blood and from the brain to the blood in PD-affected brain, has not been studied yet. OATs play an integral role in maintaining proper concentration of the compounds in tissues. The most important transporters involved in the renal excretion and uptake of substances mentioned above are classified as OAT1 and its close homolog OAT3, both expressed mostly on the proximal tubule cells. OAT3 is also expressed in choroid plexus and luminal and abluminal membranes of brain. The transporter mediates the transport of IS and pCS from CSF to the blood and from the brain to the blood [63]. The transporter is also involved in efflux of neurotransmitters metabolites like 5-hydroxyindole acetic acid and homovanillic acid (HVA) [64]. In PD treated with L-dopa, higher level of HVA in CSF was observed both regarding PD not treated with L-dopa and healthy controls [65]. But, our observation on higher ratio of pCS and IS in CSF to plasma is rather not associated with interaction of HVA and the uremic toxins in efflux by OAT3. It is due to lack of correlation of pCScsf/plasma and IScsf/plasma with the L-dopa dosage. However, it can be expected that high level of pCS and IS in blood can result in increased level of HVA that can affect neurotransmitter metabolic pathway and accumulation of neurotoxic intermediate metabolites such as 3,4dihydroxyphenylacetaldehyde [64]. Increased concentration of uremic toxins in brain can also promote neurodegeneration. IS was shown to glial function by increasing oxidative stress and neuroinflammation [54]. It is very likely that the decreasing kidney function may cause the accumulation of uremic solutes in brain, as confirmed by our study. This research indicates correlations between eGFR and pCS (r = –0.41), IS (r = –0.52), SDMA (r = –0.40), and TMAO (r = –0.59) in CSF. No data in the literature exist to compare with our results. PCA analysis allowed us to separate the PD group from controls. However, five PD patients were grouped in a different cluster than other patients. These five patients differed from the other PD patients by lower pCS in CSF, ratio of pCSCSF/blood, but higher 8-OHdG level, an important marker for DNA oxidative damage. Other parameters concerning the diseases progression did not differ. It suggests other mechanism of the disease pathogenesis in these groups compared to other PD patients. The mechanisms underlying the pathophysiology of PD have been far from fully understood. Increasing evidence suggests that inflammation and oxidative stress play critical roles in the cascade of events leading to the degeneration of dopaminergic neurons. However, all elements that potentially cause oxidative stress in PD are still unknown. Moreover, it should be mentioned that diverse and, at times, contradictory range of pathogenic mechanisms of PD was reported [66]. We cannot exclude that more than one mechanism occurs; for that reason, we can see two clusters in PCA. Other explanation is

nonclinically homogenous group with the features not evaluated routinely. To sum up, in PD, higher concentration of pCS in CSF was observed. The CSF-plasma ratio of pCS and IS was four and eight times higher in PD compared to the control group, respectively. It indicates their higher than expected concentration in CSF, compared to their levels in blood. Toxins were higher in CSF, but not in plasma of patients with motor fluctuations. In plasma of patients with motor fluctuation, higher hepcidin level was observed as well. eGFR correlated with pCS, IS, SDMA, and TMAO in CSF. TMAO in CSF originates mainly from blood, since a strong correlation of its level in CSF and plasma occurred. To conclude, uremic toxins like pCS, IS, ADMA, SDMA, and TMAO can be associated with pathogenesis and progression of PD. Further research on molecular background of this finding should be conducted.

Funding source LC-MS/MS analyses were carried out with the use of the CePT infrastructure financed by the European Union—the European Regional Development Fund within the Operational Program “Innovative economy” for 2007–2013. The research was founded by the Medical University of Warsaw.

Figure 1: Correlation pattern of uremic toxins in CSF, plasma, and eGFR. *—statistically significant with p-value lower than 0.05; ADMA—asymmetric dimethylarginine; CSF— cerebrospinal fluid; eGFR—estimated glomerular filtration rate; IS—indoxyl sulfate; pCS— p-cresol sulfate; SDMA—symmetric dimethylarginine, TMAO—trimethylamine N-oxide.

Figure 2: The distribution of the study group (left) and variables (right) on the score plot (principal component 2 vs. principal component 5) obtained by principal component analysis (PCA). Arrows mark five PD patients that are gathered in different clusters than the rest of the group. C—control group; P—group with Parkinson’s disease. ADMA—asymmetric dimethylarginine; Arg—arginine; CSF—cerebrospinal fluid; IS—indoxyl sulfate; pCS—pcresol sulfate; SDMA—symmetric dimethylarginine; TMAO—trimethylamine N-oxide.

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·

First report on uremic toxins level in plasma and CSF of Parkinson’s diseases (PD)

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Correlation of uremic toxins with eGFR, hepcidin and oxidative stress was tested

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Patients with motor fluctuations had higher toxins level

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In PD higher ratio CSF/plasma for indoxyl sulfate and p-cresol sulfate was seen

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Uremic toxins can be associated with pathogenesis and progression of PD