**3.1 Metabolism of biothiols**

In the body, Hcy is a point of intersection of two main metabolic pathways: transsulfuration and remethylation. Under physiological conditions, around 50% of Hcy is catabolized by transsulfuration and undergoes transformation to cystathionine and then to Cys. The remaining 50% of Hcy undergoes methylation to Met (Fig. 1).

Fig. 1. Synthesis and metabolic pathways of homocysteine, CBS- cystationine β-synthase, MTHFR- 5,10-methylenetetrahydrofolate reductase, MTR- methionine synthase, MTHFD1 methylenetetrahydrofolate dehydrogenase/ methenyltetrahydrofolate

cyclohydrolase/formyltetrahydrofolate synthetase, SAH- S-adenosylhomocysteine, SAM- Sadenosylmethionine.

Methionine is supplied with food and its transformation to Hcy involves several steps. At the first step, Met is transformed to SAM and is then demethylated to SAH (Sadenosylhomocysteine) and hydrolyzed to Hcy. SAM is the main donor of methyl groups in many reactions. A decreased content of SAM was demonstrated in the course of PD (Cheng et al., 1997).

The level of Hcy undergoes control, depending upon concentration of its metabolites: Cys and Met. In the case of Met deficit and low concentration of SAM, most Hcy undergoes remethylation to Met, catalyzed by methionine synthase (MTR). MTR is a vitamin B12 dependent enzyme responsible for transfer of methyl groups from Nmethyltetrahydrofolate to Hcy, leading to formation of Met (Jarrett et al., 1996). Mutations in the MTR gene are responsible for decreased methylcobalamine level, and result in homocysteinuria, hyperhomocysteinemia and hypomethioninemia (Watkins et al., 2002). The tri-functional enzyme, methylenetetrahydrofolate dehydrogenase/ methenyltetrahydrofolate cyclohydrolase/formyltetrahydrofolate synthetase (MTHFD1) represents another enzyme linked to transformation of Hcy to Met. Homozygotes of both *MTHFR* and *MTHFD1* are at risk of cardiovascular diseases connected with elevated levels of Hcy, or folate level-related hypoplasia of neural tube (Hol et al., 1998). However, in the literature, less numerous data are available on the involvement of MTHFD1 in the pathogenesis of degenerative diseases (Dorszewska et al., 2007).

the risk of their occurrence as much as possible. Gorell et al. (1994) indicated that patients with PD have shown an increased risk for cardiovascular disease and stroke. In PD not only Hcy, but also cysteine (Cys), product of Hcy metabolism may promote pathological

In the body, Hcy is a point of intersection of two main metabolic pathways: transsulfuration and remethylation. Under physiological conditions, around 50% of Hcy is catabolized by transsulfuration and undergoes transformation to cystathionine and then to Cys. The

Fig. 1. Synthesis and metabolic pathways of homocysteine, CBS- cystationine β-synthase, MTHFR- 5,10-methylenetetrahydrofolate reductase, MTR- methionine synthase, MTHFD1-

Homocysteine acid

Cysteine **Homocysteine** Methionine

MTHFR MTR MTHFD1

Vitamin B12 Folate

SAM, SAH

cyclohydrolase/formyltetrahydrofolate synthetase, SAH- S-adenosylhomocysteine, SAM- S-

Methionine is supplied with food and its transformation to Hcy involves several steps. At the first step, Met is transformed to SAM and is then demethylated to SAH (Sadenosylhomocysteine) and hydrolyzed to Hcy. SAM is the main donor of methyl groups in many reactions. A decreased content of SAM was demonstrated in the course of PD (Cheng

The level of Hcy undergoes control, depending upon concentration of its metabolites: Cys and Met. In the case of Met deficit and low concentration of SAM, most Hcy undergoes remethylation to Met, catalyzed by methionine synthase (MTR). MTR is a vitamin B12 dependent enzyme responsible for transfer of methyl groups from Nmethyltetrahydrofolate to Hcy, leading to formation of Met (Jarrett et al., 1996). Mutations in the MTR gene are responsible for decreased methylcobalamine level, and result in homocysteinuria, hyperhomocysteinemia and hypomethioninemia (Watkins et al., 2002). The tri-functional enzyme, methylenetetrahydrofolate dehydrogenase/ methenyltetrahydrofolate cyclohydrolase/formyltetrahydrofolate synthetase (MTHFD1) represents another enzyme linked to transformation of Hcy to Met. Homozygotes of both *MTHFR* and *MTHFD1* are at risk of cardiovascular diseases connected with elevated levels of Hcy, or folate level-related hypoplasia of neural tube (Hol et al., 1998). However, in the literature, less numerous data are available on the involvement of MTHFD1 in the

methylenetetrahydrofolate dehydrogenase/ methenyltetrahydrofolate

pathogenesis of degenerative diseases (Dorszewska et al., 2007).

alterations such as: atherosclerosis and thrombogenesis (Muller, 2008).

remaining 50% of Hcy undergoes methylation to Met (Fig. 1).

CBS

Vitamin B6

**3.1 Metabolism of biothiols** 

adenosylmethionine.

et al., 1997).

Under normal conditions, in the presence of a positive Met balance, most of Hcy undergoes transsulfuration catalyzed by cystathionine β-synthetase (CBS), which requires derivative of vitamin B6, pyridoxal phosphate.

Homocysteine or it oxidative product, homocysteine acid are thought to exhibit its prooxidative activity most probably through its direct interaction with NMDA receptors (it represents an agonist of NMDA receptor). Agnati et al. (2005) have shown that Hcy may pass the blood/brain barrier and that level of plasma Hcy corresponds to Hcy concentration in the brain.

#### **3.2 Influence of L-dopa treatment on the plasma level of biothiols in Parkinson's disease**

In PD, the high Hcy concentration may augment risk of the disease through its direct toxic effect on dopaminergic neurons. Studies *in vitro* on human dopaminergic neurons have documented a significant increase in neurotoxicity accompanying high Hcy levels (Duan et al., 2002). In parallel, elevated Hcy levels in PD have been shown to carry potential for deterioration of cognitive and motoric functions, for depression and elevated risk to develop vascular diseases (Kuhn et al., 1998).

Reports of the literature (Florczak et al., 2008; Miller et al., 2003) indicate that plasma Hcy levels in PD have been affected also by pharmacotherapy with L-dopa. It is indicated that in PD patients who are initiating L-dopa therapy, Hcy elevates within six weeks to a few months after L-dopa initiation (O'Suilleabhain et al., 2004a). Study Florczak et al., 2008 indicated that the sulfuric amino acids were also affected by duration of the L-dopa pharmacotherapy. The most exposed to neurotoxic effects of Hcy have seemed to be the patients during the first 5 years L-dopa treatment while its continued administration has resulted in stably elevated Hcy level. The study of Miller et al. (1997) indicates that L-dopa may induce elevated levels of Hcy during its methylation to 3-O-methyldopa (3-OMD) with involvement of COMT (catechol Omethyltransferase) both in peripheral blood leukocytes and in nigrostriatal neurons. In the course of the reaction, COMT in presence of magnesium ions induces in parallel transition of SAM to SAH and further hydrolysis of SAH to Hcy (Fig. 2).

Elevated level of Hcy in *substantia nigra* of PD patients has been demonstrated already after 3 months of L-dopa treatment (Yasui et al., 2003). Long-term administration of L-dopa is thought to promote benign vascular lesions in patients with PD and may result in the patients in cognitive disturbances or dementia, particular at late stages of treatment with the preparation (Muller et al., 1999). On the other hand, COMT has a broad detoxification potential in human. Two compounds are currently available, entacapone peripheral and tolcapone central blocking of COMT. COMT inhibition is also under suspicion to prevent motor complications and seems that has beneficial effect on the L-dopa-related hyper-Hcy as well (Muller, 2009a; Nevrly et al., 2010). Some animal studies shown that COMT inhibition can eliminate L-dopa-induced hyper-Hcy but not all previous studies confirm it. Study Dorszewska et al. (2007) have shown that augmented plasma levels of Hcy in PD possibly could have developed due to altered processes of Hcy remethylation to Met and transsulfuration to Cys. Simultaneuosly, in the PD patients a decreased concentration of Met has been observed, paralleled by elevated levels of Cys and lowered ratio of Met and Cys to

Hcy. The demonstrated at present decrease in Met to Hcy ratio may be linked to transformation of Hcy to thiolactone in endothelial cells. According to one of more recent hypothesis, sulfonic sulfur of thiol compounds may be involved in development of Hcyinduced arteriosclerotic lesions (Toohey, 2001). At the same time, the demonstrated at

Oxidative DNA Damage and the Level of Biothiols, and L-Dopa Therapy in Parkinson's Disease 359

The system was controlled, and the data were collected and processed using Chromeleon

Pharmacotherapy with L-dopa of PD patients (Tables 7 and 8) leads to increase of the concentrations of metabolic product of Hcy, Cys (p<0.01) as compared to the controls, in the patients treated (p<0.05) as well as untreated (p<0.01) with L-dopa. Consequently, the ratio of Cys/Hcy in PD patients decreased, as compared to the controls (p<0.05) and to the

Cys 220.7 ± 46.6 250.6 ± 49.6\*\* Cys/Hcy 19.3 ± 6.7 16.3 ± 6.5\* Table 7. Cysteine (µM) concentrations in the patients with PD and in control group. Results are expressed as a means ± SD. The nonparametric test of Mann-Whitney was used.

Cys 220.7 ± 46.6 263.6 ± 42.9\*\* 244.7 ± 51.9\* Cys/Hcy 19.3 ± 6.7 20.7 ± 6.9 14.4 ± 5.4\*\*(\*\*) Table 8. Cysteine (µM) concentrations as related to pharmacotherapy with L-dopa (+) in the patients with PD. Results are expressed as means ± SD. The nonparametric test of Mann-Whitney was used. Differences significant at \*p<0.05, \*\*p<0.01 as compared to the controls. Differences significant at (\*\*)p<0.01, as compared to patients not treated with L-dopa (-).

Muller & Kohn (2009) indicated that only PD patients with an elevated level of Hcy above 15 µM showed an increase of Cys plasma level and elevated concentration of both risk factors (Hcy, Cys) may intervene in the neurodegenerative process. Present study indication that especially PD patients before L-dopa treatment showed increased level of Cys and Ldopa treatment only little decreased higher level of Cys. Increased plasma Cys level in PD may result from intensified release of the amino acid from proteins, due to substitution by the circulating Hcy or due to diminished transformation of Cys to glutathione, important for maintenance of redox homeostasis in the body. In culture of human hepatocytes 50% Cys has been demonstrated to transform into GSH (Mosharov et al., 2000). It seems also that intensity of dementive disease in particular disturbs transsulfuration of Hcy and leads to decreased levels of the agent (Cys), which provides the natural antioxidant, GSH. Homocysteine as well as Cys may serve as biomarkers for severity or progression of PD.

**3.3 Influence of L-dopa treatment on the plasma level of Hcy and ADMA in** 

In the body Hcy is metabolized along two metabolic pathways, by the way of trassulfution and remethylation, involvement of SAM and SAH (Fig. 1). SAM is thought to provide the principal donor of methyl groups in numerous metabolic reactions, leading to formation of

**Patients with PD L-dopa (-) (34-79 years)** 

**Patients with PD (34-81 years)** 

> **Patients with PD L-dopa (+) (35-81 years)**

untreated patients (p<0.01) as compared to treated PD patients with L-dopa.

Differences significant at \*p<0.05, \*\*p<0.01 as compared to the controls.

**(22-76 years)** 

**Parameter Controls** 

**(22-76 years)** 

**Parameter Controls** 

**Parkinson's disease patients** 

software (Dionex, Germany).

**3.2.3 Results** 

present increased plasma Cys level in PD may result from intensified release of the amino acid from proteins, due to substitution by the circulating Hcy or due to diminished transformation of Cys to glutathione, important for maintenance of redox homeostasis in the body.

Fig. 2. COMT-mediated O-methylation of L-dopa, which results in formation of 3 methyldopa product, COMT- catecholamine-O-methyltrasferase, 3-OMD- 3-O-methyldopa.

In the literature there are studies of plasma Cys concentrations in PD patients (Dorszewska et al., 2007; Muller & Kuhn, 2009b) but there are no reported about relation between L-dope treatment and Cys status.

#### **3.2.1 Patients**

The studies were conducted on 98 patients with PD, including 37 women and 61 men aging 34-81 years (mean age: 60.8±10.7 years). Among the patients with PD, 27 patients (9 women and 18 men) awaited L-dopa treatment (patients' age: 34-79 years) and the remaining 71 individuals, 28 women and 43 men (patients' age: 35-81 years) were treated with L-dopa preparations in daily doses (up to 5 years treatment to 500 mg/day, 5-10 year treatment 500- 800 mg/day, and over 10 year treatment 800-1500 mg/day).

Control group included 50 individuals, 34 women and 16 men, 22-76 years of age (mean age: 44.6±16.2 years).

Patients with PD were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003), however stage of disease according to the scale of Hoehn and Yahr.

None of the control subjects had verifiable symptoms of dementia or any other neurological disorders and smoking, and drinking habits.

A Local Ethical Committee approved the study and the written consent of all patients or their caregivers was obtained.

#### **3.2.2 Analysis of Cys concentrations**

*Preparation of samples.* The analyzed plasma thiol compounds (Cys, Sigma, USA) were diluted with water at 2:1 ratio and reduced using 1% TCEP (Tris-(2-carboxyethyl)-phosphinhydrochloride; Applichem, Germany) at 1:9 ratio. Subsequently, the sample was deproteinized using 1M HClO4 (at 2:1 ratio) and applied to the HPLC/EC system.

*Determination of thiol concentration.* The samples were fed to the HPLC system (P580A; Dionex, Germany) coupled to an electrochemical detector (CoulArray 5600; ESA, USA). The analysis was performed in Termo Hypersil BDS C18 column (250 x 4.6 x 5µm) (Germany) in isocratic conditions, using the mobile phase of 0.15 M phosphate buffer, pH 2.8 supplemented with 8-10% acetonitrile for estimation of Cys (Accinni et al., 2000).

The system was controlled, and the data were collected and processed using Chromeleon software (Dionex, Germany).

#### **3.2.3 Results**

358 Etiology and Pathophysiology of Parkinson's Disease

present increased plasma Cys level in PD may result from intensified release of the amino acid from proteins, due to substitution by the circulating Hcy or due to diminished transformation of Cys to glutathione, important for maintenance of redox homeostasis in the

S-adenosylmethionine S-adenosylhomocysteine Homocysteine

Fig. 2. COMT-mediated O-methylation of L-dopa, which results in formation of 3-

**COMT L-dopa** → 3-OMD

800 mg/day, and over 10 year treatment 800-1500 mg/day).

disorders and smoking, and drinking habits.

**3.2.2 Analysis of Cys concentrations** 

their caregivers was obtained.

methyldopa product, COMT- catecholamine-O-methyltrasferase, 3-OMD- 3-O-methyldopa. In the literature there are studies of plasma Cys concentrations in PD patients (Dorszewska et al., 2007; Muller & Kuhn, 2009b) but there are no reported about relation between L-dope

The studies were conducted on 98 patients with PD, including 37 women and 61 men aging 34-81 years (mean age: 60.8±10.7 years). Among the patients with PD, 27 patients (9 women and 18 men) awaited L-dopa treatment (patients' age: 34-79 years) and the remaining 71 individuals, 28 women and 43 men (patients' age: 35-81 years) were treated with L-dopa preparations in daily doses (up to 5 years treatment to 500 mg/day, 5-10 year treatment 500-

Control group included 50 individuals, 34 women and 16 men, 22-76 years of age (mean age:

Patients with PD were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003), however stage of disease according to the scale of Hoehn and Yahr. None of the control subjects had verifiable symptoms of dementia or any other neurological

A Local Ethical Committee approved the study and the written consent of all patients or

*Preparation of samples.* The analyzed plasma thiol compounds (Cys, Sigma, USA) were diluted with water at 2:1 ratio and reduced using 1% TCEP (Tris-(2-carboxyethyl)-phosphinhydrochloride; Applichem, Germany) at 1:9 ratio. Subsequently, the sample was

*Determination of thiol concentration.* The samples were fed to the HPLC system (P580A; Dionex, Germany) coupled to an electrochemical detector (CoulArray 5600; ESA, USA). The analysis was performed in Termo Hypersil BDS C18 column (250 x 4.6 x 5µm) (Germany) in isocratic conditions, using the mobile phase of 0.15 M phosphate buffer, pH 2.8

deproteinized using 1M HClO4 (at 2:1 ratio) and applied to the HPLC/EC system.

supplemented with 8-10% acetonitrile for estimation of Cys (Accinni et al., 2000).

body.

treatment and Cys status.

**3.2.1 Patients** 

44.6±16.2 years).

Pharmacotherapy with L-dopa of PD patients (Tables 7 and 8) leads to increase of the concentrations of metabolic product of Hcy, Cys (p<0.01) as compared to the controls, in the patients treated (p<0.05) as well as untreated (p<0.01) with L-dopa. Consequently, the ratio of Cys/Hcy in PD patients decreased, as compared to the controls (p<0.05) and to the untreated patients (p<0.01) as compared to treated PD patients with L-dopa.


Table 7. Cysteine (µM) concentrations in the patients with PD and in control group. Results are expressed as a means ± SD. The nonparametric test of Mann-Whitney was used. Differences significant at \*p<0.05, \*\*p<0.01 as compared to the controls.


Table 8. Cysteine (µM) concentrations as related to pharmacotherapy with L-dopa (+) in the patients with PD. Results are expressed as means ± SD. The nonparametric test of Mann-Whitney was used. Differences significant at \*p<0.05, \*\*p<0.01 as compared to the controls. Differences significant at (\*\*)p<0.01, as compared to patients not treated with L-dopa (-).

Muller & Kohn (2009) indicated that only PD patients with an elevated level of Hcy above 15 µM showed an increase of Cys plasma level and elevated concentration of both risk factors (Hcy, Cys) may intervene in the neurodegenerative process. Present study indication that especially PD patients before L-dopa treatment showed increased level of Cys and Ldopa treatment only little decreased higher level of Cys. Increased plasma Cys level in PD may result from intensified release of the amino acid from proteins, due to substitution by the circulating Hcy or due to diminished transformation of Cys to glutathione, important for maintenance of redox homeostasis in the body. In culture of human hepatocytes 50% Cys has been demonstrated to transform into GSH (Mosharov et al., 2000). It seems also that intensity of dementive disease in particular disturbs transsulfuration of Hcy and leads to decreased levels of the agent (Cys), which provides the natural antioxidant, GSH. Homocysteine as well as Cys may serve as biomarkers for severity or progression of PD.

#### **3.3 Influence of L-dopa treatment on the plasma level of Hcy and ADMA in Parkinson's disease patients**

In the body Hcy is metabolized along two metabolic pathways, by the way of trassulfution and remethylation, involvement of SAM and SAH (Fig. 1). SAM is thought to provide the principal donor of methyl groups in numerous metabolic reactions, leading to formation of

Oxidative DNA Damage and the Level of Biothiols, and L-Dopa Therapy in Parkinson's Disease 361

individuals, 18 women and 16 men (patients' age: 46-86 years) were treated with L-dopa preparations in daily doses (up to 5 years treatment to 500 mg/day, 5-10 year treatment 500-

The control group included 35 individuals, 20 women and 15 men, 22-76 years of age (mean

Patients with PD, on the other hand, were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003). The stage of evolution of Parkinson's disease was defined according to the scale of Hoehn and Yahr. The tested patients represented

None of the control subjects had verifiable symptoms of dementia or any other neurological

A Local Ethical Committee approved the study and the written consent of all patients or

*Preparation of samples.* The analyzed plasma thiol compounds (Hcy, Fluka Germany; Met, Sigma, USA) were diluted with water at 2:1 ratio and reduced using 1% TCEP (Tris-(2 carboxyethyl)-phosphin-hydrochloride; Applichem, Germany) at 1:9 ratio. Subsequently, the sample was deproteinized using 1M HClO4 (at 2:1 ratio) and applied to the HPLC/EC system. *Determination of thiol concentration.* The samples were fed to the HPLC system (P580A; Dionex, Germany) coupled to an electrochemical detector (CoulArray 5600; ESA, USA). The analysis was performed in Termo Hypersil BDS C18 column (250 x 4.6 x 5µm) (Germany) in isocratic conditions, using the mobile phase of 0.15 M phosphate buffer, pH 2.9, supplemented with 12.5-17% acetonitrile for estimation of Hcy and Met and 0.15 M

The system was controlled and the data were collected and processed using Chromeleon

*Preparation of samples and derivatization.* Plasma and the standard, containing solution of Arg and ADMA (Sigma, USA) were diluted with water at the ratio of 1.5:1.0 and, then, they were deproteinised using 8M HCLO4 at the ratio of 5:1. Directly before HPLC analysis the samples were subjected to derivatization in a solution containing 10 mg OPA per 100 µl methanol supplemented with 900 µl 0.4 M borate buffer (pH 8.5) and 5µl 2-mercaptoethanol

*Analysis of Arg and ADMA.* The samples were fed to the HPLC system (P580A; Dionex, Germany) coupled to a fluorescence detector (RF2000; Dionex, Germany). The analysis was performed in a Termo Hypersil BDS C18 column (250 x 4.6 x 5µm) (Germany) in an isocratic conditions using 0.1 M phosphate buffer, pH 6.75 with 25 % methanol as the mobile phase. Arg and its methylated metabolites were measured fluorimetrically at excitation and

The system was controlled and the data were collected and processed using Chromeleon

The study indicated that in patients with the diagnosed PD (Table 9) the augmented export of Hcy to plasma, (p<0.001 as compared to the controls), was accompanied by increased

800 mg/day, and over 10 year treatment 800-1500 mg/day).

age: 45.1±16.0 years).

disorders.

stages I to IV of the disease evolution.

phosphate buffer (Accinni et al., 2000).

**3.3.3 Analysis of Arg and ADMA concentrations** 

emission wavelengths of 340 nm and 455nm, respectively.

software (Dionex, Germany).

at the ratio of 1:1 (Pi et al., 2000).

software (Dionex, Germany).

**3.3.4 Results** 

**3.3.2 Analysis of Hcy and Met concentrations** 

their caregivers was obtained.

methyl derivatives. One of the products of SAM methylation is thought to be asymmetric dimethylarginine (ADMA) (Gary & Clarke, 1998).

ADMA is an endogenous inhibitor of nitrogen oxide synthase (NOS) (Vallace et al., 1992). It arises from Arg contained in body proteins and may undergo hydrolysis to L-citruline and dimethylamine with involvement of dimethylaminohydrolase (DDAH). Homocysteine is thought to inhibit activity of DDAH (Stuhlinger et al., 2001) and might promote accumulation of ADMA that leads to a decreased production of nitrogen oxide (NO) and Lcitruline from Arg with participation of NOS (Fig. 3).

NO plays an important role in control of vascular tone, in neurotransmission and in body protective mechanisms as well as in memory processes. Literature reports indicate that in PD the augmented activity of glia results in increased production of NO (McGeer et al., 1988).

ADMA is regarded to act as a risk factor for vascular diseases (Yoo & Lee, 2001). Its elevated levels were demonstrated in patients with hypercholesterolemia, hypertension, chronic heart failure and in atherosclerotic processes and during physiological aging (Kielstein et al., 2003). Role of ADMA in pathogenesis of PD is less known. Until now, in PD the levels on non-methylated substrate in biosynthesis of ADMA were examined only, and the elevated levels of Arg in cerebrospinal fluid were shown in PD patients with the decrease after Ldopa administration (Qureshi et al., 1995). Literature reports indicate also that long-term administration of L-dopa in PD patients may not only lead to increased concentrations of Hcy but may also alter Arg levels (Muller et al., 1999).

Fig. 3. The roles of methionine, homocysteine and arginine in metabolism of asymmetric dimethylarginine, Hcy- homocysteine, Met- methionine, ADMA- asymmetric dimethylarginine, Arg- arginine, NO- nitric oxide, NOS- NO synthase, DDAHdimethylarginine dimethylaminohydrolase, SAM- S-adenosylmethionine, SAH- Sadenosylhomocysteine.

The present study was aimed at the estimation of plasma levels of Hcy and ADMA together with Met and Arg in patients with PD. The attention was also paid to developmental stages of the analyzed degenerative diseases and to L-dopa pharmacotherapy in PD.

#### **3.3.1 Patients**

The studies were conducted on 47 patients with PD, including 21 women and 26 men aging 41-86 years (mean age: 63.0±11.1 years). Among the patients with PD, 13 patients (3 women and 10 men) awaited L-dopa treatment (patients' age: 41-78 years) and the remaining 34 individuals, 18 women and 16 men (patients' age: 46-86 years) were treated with L-dopa preparations in daily doses (up to 5 years treatment to 500 mg/day, 5-10 year treatment 500- 800 mg/day, and over 10 year treatment 800-1500 mg/day).

The control group included 35 individuals, 20 women and 15 men, 22-76 years of age (mean age: 45.1±16.0 years).

Patients with PD, on the other hand, were diagnosed using the criteria of UK Parkinson's Disease Society Brain Bank (Litvan et al., 2003). The stage of evolution of Parkinson's disease was defined according to the scale of Hoehn and Yahr. The tested patients represented stages I to IV of the disease evolution.

None of the control subjects had verifiable symptoms of dementia or any other neurological disorders.

A Local Ethical Committee approved the study and the written consent of all patients or their caregivers was obtained.

#### **3.3.2 Analysis of Hcy and Met concentrations**

*Preparation of samples.* The analyzed plasma thiol compounds (Hcy, Fluka Germany; Met, Sigma, USA) were diluted with water at 2:1 ratio and reduced using 1% TCEP (Tris-(2 carboxyethyl)-phosphin-hydrochloride; Applichem, Germany) at 1:9 ratio. Subsequently, the sample was deproteinized using 1M HClO4 (at 2:1 ratio) and applied to the HPLC/EC system.

*Determination of thiol concentration.* The samples were fed to the HPLC system (P580A; Dionex, Germany) coupled to an electrochemical detector (CoulArray 5600; ESA, USA). The analysis was performed in Termo Hypersil BDS C18 column (250 x 4.6 x 5µm) (Germany) in isocratic conditions, using the mobile phase of 0.15 M phosphate buffer, pH 2.9, supplemented with 12.5-17% acetonitrile for estimation of Hcy and Met and 0.15 M phosphate buffer (Accinni et al., 2000).

The system was controlled and the data were collected and processed using Chromeleon software (Dionex, Germany).

### **3.3.3 Analysis of Arg and ADMA concentrations**

*Preparation of samples and derivatization.* Plasma and the standard, containing solution of Arg and ADMA (Sigma, USA) were diluted with water at the ratio of 1.5:1.0 and, then, they were deproteinised using 8M HCLO4 at the ratio of 5:1. Directly before HPLC analysis the samples were subjected to derivatization in a solution containing 10 mg OPA per 100 µl methanol supplemented with 900 µl 0.4 M borate buffer (pH 8.5) and 5µl 2-mercaptoethanol at the ratio of 1:1 (Pi et al., 2000).

*Analysis of Arg and ADMA.* The samples were fed to the HPLC system (P580A; Dionex, Germany) coupled to a fluorescence detector (RF2000; Dionex, Germany). The analysis was performed in a Termo Hypersil BDS C18 column (250 x 4.6 x 5µm) (Germany) in an isocratic conditions using 0.1 M phosphate buffer, pH 6.75 with 25 % methanol as the mobile phase. Arg and its methylated metabolites were measured fluorimetrically at excitation and emission wavelengths of 340 nm and 455nm, respectively.

The system was controlled and the data were collected and processed using Chromeleon software (Dionex, Germany).

#### **3.3.4 Results**

360 Etiology and Pathophysiology of Parkinson's Disease

methyl derivatives. One of the products of SAM methylation is thought to be asymmetric

ADMA is an endogenous inhibitor of nitrogen oxide synthase (NOS) (Vallace et al., 1992). It arises from Arg contained in body proteins and may undergo hydrolysis to L-citruline and dimethylamine with involvement of dimethylaminohydrolase (DDAH). Homocysteine is thought to inhibit activity of DDAH (Stuhlinger et al., 2001) and might promote accumulation of ADMA that leads to a decreased production of nitrogen oxide (NO) and L-

NO plays an important role in control of vascular tone, in neurotransmission and in body protective mechanisms as well as in memory processes. Literature reports indicate that in PD the augmented activity of glia results in increased production of NO (McGeer et al., 1988). ADMA is regarded to act as a risk factor for vascular diseases (Yoo & Lee, 2001). Its elevated levels were demonstrated in patients with hypercholesterolemia, hypertension, chronic heart failure and in atherosclerotic processes and during physiological aging (Kielstein et al., 2003). Role of ADMA in pathogenesis of PD is less known. Until now, in PD the levels on non-methylated substrate in biosynthesis of ADMA were examined only, and the elevated levels of Arg in cerebrospinal fluid were shown in PD patients with the decrease after Ldopa administration (Qureshi et al., 1995). Literature reports indicate also that long-term administration of L-dopa in PD patients may not only lead to increased concentrations of

Fig. 3. The roles of methionine, homocysteine and arginine in metabolism of asymmetric

**NOS** 

**ADMA** 

**Citruline + dimethylamine**

**DDAH** 

**methyl groups donor SAM Met** 

**SAH** 

**Hcy** 

The present study was aimed at the estimation of plasma levels of Hcy and ADMA together with Met and Arg in patients with PD. The attention was also paid to developmental stages

The studies were conducted on 47 patients with PD, including 21 women and 26 men aging 41-86 years (mean age: 63.0±11.1 years). Among the patients with PD, 13 patients (3 women and 10 men) awaited L-dopa treatment (patients' age: 41-78 years) and the remaining 34

dimethylarginine, Hcy- homocysteine, Met- methionine, ADMA- asymmetric dimethylarginine, Arg- arginine, NO- nitric oxide, NOS- NO synthase, DDAHdimethylarginine dimethylaminohydrolase, SAM- S-adenosylmethionine, SAH- S-

**Citruline + NO** 

**Arg** 

of the analyzed degenerative diseases and to L-dopa pharmacotherapy in PD.

dimethylarginine (ADMA) (Gary & Clarke, 1998).

citruline from Arg with participation of NOS (Fig. 3).

Hcy but may also alter Arg levels (Muller et al., 1999).

adenosylhomocysteine.

**3.3.1 Patients** 

The study indicated that in patients with the diagnosed PD (Table 9) the augmented export of Hcy to plasma, (p<0.001 as compared to the controls), was accompanied by increased

Oxidative DNA Damage and the Level of Biothiols, and L-Dopa Therapy in Parkinson's Disease 363

Pharmacotherapy with L-dopa preparations was demonstrated also to increase levels of both factors of vascular disease risk (Table 10), Hcy (p<0.001 as compared to the controls and p<0.05 as compared to patients not treated with L-dopa) and ADMA (p<0.001 as compared to the controls), although levels of ADMA increased also as a result of development of the degenerative disease (p<0.01 as compared to the controls). In parallel, in patients treated with L-dopa preparations levels of Met decreased (p<0.001 as compared to the controls) and so did concentrations of Arg (p<0.05 as compared to the controls and to Ldopa untreated patients), Met/Hcy ratios (p<0.001 as compared to the controls and p<0.05 as compared to patients not treated with L-dopa) and Arg/ADMA ratios (p<0.001, only as

Hcy 13.0 ± 4.3 15.1 ± 5.1 22.0 ± 15.9\*\*\*(\*) Met 24.0 ± 6.9 21.0 ± 7.8 17.8 ± 8.8\*\*\* Met/Hcy 2.1 ± 0.9 1.6 ± 1.0\* 1.0 ± 0.6\*\*\*(\*) Arg 79.7 ± 24.4 77.5 ± 19.7 64.4 ± 22.0\*(\*) ADMA 2.0 ± 1.0 3.3 ± 1.1\*\* 4.1 ± 2.1\*\*\* Arg/ADMA 55.3 ± 40.5 22.8 ± 7.8\*\*\* 26.7 ± 23.6\*\*\* Table 10. Homocysteine (µM), methionine (µM), asymmetric dimethylarginine (µM) and arginine (µM) concentrations as related to pharmacotherapy with L-dopa (+) in the patients with PD. The nonparametric test of Mann-Whitney was used. Results are expressed as means ± SD. Differences significant at \*p<0.05, \*\*p<0.01, \*\*\*p<0.001, as compared to the controls. Differences significant at (\*)p<0.05, as compared to patients not treated with L-

Present study indicated that ADMA may be involved in pathogenesis of PD. In development of PD the principal role is thought to be played by peroxinitrates (Padovan-Neto et al., 2009). This seems consistent with the demonstrated in present study ADMA level not elevated till the IInd stage of the disease development and the lower level of Arg, particularly accentuated in the IVth stage of PD evolution. Thus, a probability exists for involvement of reactive NO

In present study we have observed particularly in PD patients an evident decrease in Arg/ADMA ratio. The lowered ratio in blood is thought (Matsuoka et al., 1997) to be linked to development of hypercholesterolemia, congestive heart failure, arterial occlusive disease,

The levels of ADMA in PD have probably been affected also by pharmacotherapy with Ldopa. Both in the studies of Qureshi et al. (1995), and in present study decreased levels of Arg have been shown in patients treated with the drug. In the study of Qureshi et al. (1995) pharmacotherapy with L-dopa has been shown to generate nitrites, agents of neurotoxic activity, but in present studies seem that ADMA has not been shown to participate in their generation. Increased NO levels in PD have seemed to result rather from elevated activity of

It seems that ADMA may be regarded to represent a risk factor for PD and may be involved in pathogenesis of this neurodegenerative disease. Present results indicate also that

**Patients with PD L-dopa (-) (41-78 years)** 

**Patients with PD L-dopa (+) (46-86 years)** 

compared to the controls).

dopa (-).

heart failure and hypertension.

**Parameter Controls** 

**(22-76 years)** 

derivatives in induction of toxic damage to *substantia nigra* in PD.

the glutaminergic system and altered neuronal metabolism.

levels of circulating ADMA in analyzed neurodegenerative disease (p<0.001 as compared to the controls). In parallel, in the patients lower levels were observed of both Met (p<0.01 as compared to the controls), and Arg (p<0.05 as compared to the controls) expressed also by the lowered Met/Hcy and Arg/ADMA ratio (p<0.001 as compared to the controls).


Table 9. Homocysteine (µM), methionine (µM), asymmetric dimethylarginine (µM) and arginine (µM) concentrations in the patients with PD and in control group. Results are expressed as a means ± SD. The nonparametric test of Mann-Whitney was used. Differences significant at \*p<0.05, \*\*p<0.01, \*\*\*p<0.001, as compared to the controls.

Moreover, in the patients with PD (Fig. 4) development of the degenerative disease resulted in increased levels the risk factor for vascular diseases (Hcy), particularly pronounced in IVth stage of PD development (p<0.05 between Ist and IVth stage and between IInd and IVth stage of PD evolution). On the other hand, the Hcy remethylation product demonstrated a decreasing tendency only in the stage III of the disease, as compared to stage I of PD. In parallel, levels of the other analyzed risk factor of vascular diseases (ADMA) manifested higher correlation with concentration of its precursor. In parallel to the development of PD from stage I to stage IV of the disease evolution augmented levels of ADMA were accompanied by a decrease in the level of Arg (as compared to the Ist stage of PD). Also at the IInd stage of the degenerative disease evolution the highest levels of Met and ADMA and practically unaltered levels of Arg were accompanied by the lowest value of Arg/ADMA ratio (p<0.01 between stages I and II of PD evolution). In the IVth stage of PD development, however, both Met/Hcy ratio and Arg/ADMA ratio behaved in a similar manner demonstrating practically the lowest level (p<0.05, as compared to the Ist stage of the disease development).

Fig. 4. Hcy, Met, Arg and ADMA concentrations as related to the stage of the PD according to the scale of Hoehn and Yahr.

levels of circulating ADMA in analyzed neurodegenerative disease (p<0.001 as compared to the controls). In parallel, in the patients lower levels were observed of both Met (p<0.01 as compared to the controls), and Arg (p<0.05 as compared to the controls) expressed also by

**(22-76 years)** 

Hcy 13.0 ± 4.3 20.1 ± 14.1\*\*\* Met 24.0 ± 6.9 18.7 ± 8.6\*\* Met/Hcy 2.1 ± 0.9 1.2 ± 0.8\*\*\* Arg 79.7 ± 24.4 68.4 ± 21.9\* ADMA 2.0 ± 1.0 3.8 ± 1.9\*\*\* Arg/ADMA 55.3 ± 40.5 25.8 ± 20.9\*\*\* Table 9. Homocysteine (µM), methionine (µM), asymmetric dimethylarginine (µM) and arginine (µM) concentrations in the patients with PD and in control group. Results are expressed as a means ± SD. The nonparametric test of Mann-Whitney was used. Differences

Moreover, in the patients with PD (Fig. 4) development of the degenerative disease resulted in increased levels the risk factor for vascular diseases (Hcy), particularly pronounced in IVth stage of PD development (p<0.05 between Ist and IVth stage and between IInd and IVth stage of PD evolution). On the other hand, the Hcy remethylation product demonstrated a decreasing tendency only in the stage III of the disease, as compared to stage I of PD. In parallel, levels of the other analyzed risk factor of vascular diseases (ADMA) manifested higher correlation with concentration of its precursor. In parallel to the development of PD from stage I to stage IV of the disease evolution augmented levels of ADMA were accompanied by a decrease in the level of Arg (as compared to the Ist stage of PD). Also at the IInd stage of the degenerative disease evolution the highest levels of Met and ADMA and practically unaltered levels of Arg were accompanied by the lowest value of Arg/ADMA ratio (p<0.01 between stages I and II of PD evolution). In the IVth stage of PD development, however, both Met/Hcy ratio and Arg/ADMA ratio behaved in a similar manner demonstrating practically the lowest level (p<0.05, as compared to the Ist stage of

Fig. 4. Hcy, Met, Arg and ADMA concentrations as related to the stage of the PD according

**Patients with PD (41-86 years)** 

the lowered Met/Hcy and Arg/ADMA ratio (p<0.001 as compared to the controls).

**Parameter Controls** 

significant at \*p<0.05, \*\*p<0.01, \*\*\*p<0.001, as compared to the controls.

the disease development).

to the scale of Hoehn and Yahr.

Pharmacotherapy with L-dopa preparations was demonstrated also to increase levels of both factors of vascular disease risk (Table 10), Hcy (p<0.001 as compared to the controls and p<0.05 as compared to patients not treated with L-dopa) and ADMA (p<0.001 as compared to the controls), although levels of ADMA increased also as a result of development of the degenerative disease (p<0.01 as compared to the controls). In parallel, in patients treated with L-dopa preparations levels of Met decreased (p<0.001 as compared to the controls) and so did concentrations of Arg (p<0.05 as compared to the controls and to Ldopa untreated patients), Met/Hcy ratios (p<0.001 as compared to the controls and p<0.05 as compared to patients not treated with L-dopa) and Arg/ADMA ratios (p<0.001, only as compared to the controls).


Table 10. Homocysteine (µM), methionine (µM), asymmetric dimethylarginine (µM) and arginine (µM) concentrations as related to pharmacotherapy with L-dopa (+) in the patients with PD. The nonparametric test of Mann-Whitney was used. Results are expressed as means ± SD. Differences significant at \*p<0.05, \*\*p<0.01, \*\*\*p<0.001, as compared to the controls. Differences significant at (\*)p<0.05, as compared to patients not treated with Ldopa (-).

Present study indicated that ADMA may be involved in pathogenesis of PD. In development of PD the principal role is thought to be played by peroxinitrates (Padovan-Neto et al., 2009). This seems consistent with the demonstrated in present study ADMA level not elevated till the IInd stage of the disease development and the lower level of Arg, particularly accentuated in the IVth stage of PD evolution. Thus, a probability exists for involvement of reactive NO derivatives in induction of toxic damage to *substantia nigra* in PD.

In present study we have observed particularly in PD patients an evident decrease in Arg/ADMA ratio. The lowered ratio in blood is thought (Matsuoka et al., 1997) to be linked to development of hypercholesterolemia, congestive heart failure, arterial occlusive disease, heart failure and hypertension.

The levels of ADMA in PD have probably been affected also by pharmacotherapy with Ldopa. Both in the studies of Qureshi et al. (1995), and in present study decreased levels of Arg have been shown in patients treated with the drug. In the study of Qureshi et al. (1995) pharmacotherapy with L-dopa has been shown to generate nitrites, agents of neurotoxic activity, but in present studies seem that ADMA has not been shown to participate in their generation. Increased NO levels in PD have seemed to result rather from elevated activity of the glutaminergic system and altered neuronal metabolism.

It seems that ADMA may be regarded to represent a risk factor for PD and may be involved in pathogenesis of this neurodegenerative disease. Present results indicate also that

Oxidative DNA Damage and the Level of Biothiols, and L-Dopa Therapy in Parkinson's Disease 365

**5. Influence of L-dopa treatment duration on the level of oxidative damage to DNA and thiols compounds concentration in patients with Parkinson's** 

The discussion about value of the L-dopa treatment in PD concerning on: toxicity, biochemical effects, clinical motor complications, especially after long-term its administrations (Belcastro et al., 2010; Muller, 2009a). Long-term treatment with L-dopa in PD patients may be promotes Hcy levels increase. Moreover, only PD patients with hyper-Hcy (Hcy above 15 µM) may have disturbed metabolism Hcy to Cys. As showed, hyper-Hcy

**5.2 Analysis of Hcy and Met (see point 3.3.2), and Cys (see point 3.2.2) concentrations,** 

During the initial five years and within the following 10 years of treatment with L-dopa (Table 11), the levels of 8-oxo2dG were augmented (p<0.05, as compared to the controls). Similarly to 8-oxo2dG, the levels of Hcy were highest after the initial five years of L-dopa administration (p<0.05, as compared to the controls). Subsequent treatment for another five to ten years resulted in the elevated levels of Hcy (p<0.01, as compared to the controls) which were even more significant if the treatment was extended over ten years (p<0.001, as compared to the controls). Moreover, the initial five years of L-dopa treatment were accompanied by relatively low levels of Met (p<0.05, as compared to the controls) and a slight increase in concentration of Cys. After ten years of treatment, similar levels of Hcy and Met were detected (Met, p<0.01), as compared to the controls, and Cys, (p<0.05), as

> **up to 5 year treatment (34-78 years)**

8-oxo2dG 13.7 ± 7.6 21.5 ± 15.1\* 17.5 ± 11.1 27.8 ± 23.0\* Hcy 12.6 ± 4.3 28.5 ± 33.6\* 19.7 ± 9.0\*\* 18.3 ± 6.9\*\*\* Met 24.2 ± 6.7 19.2 ± 6.2\* 20.4 ± 9.1 18.2 ± 8.2\*\* Met/Hcy 2.2 ± 0.9 1.1 ± 0.5\*\* 1.2 ± 0.6\*\* 1.1 ± 0.5\*\*\* Cys 220.7 ± 46.6 232.3 ± 52.5 267.9 ± 47.1\*\* 238.2 ± 53.3(\*) Cys/Hcy 19.3 ± 6.7 12.8 ± 5.3\* 15.4 ± 5.1 14.4 ± 5.8\*\* Table 11. Levels of oxidative DNA damage (8-oxo2dG/dG x 10-5), and homocysteine (µM), methionine (µM) and cysteine (µM) concentrations as related to duration of L-dopa administration to patients with PD. Results are expressed as means ± SD. Differences significant at \*p<0.05, \*\*p<0.01, \*\*\*p<0.001, as compared to the controls. Differences

significant at (\*)p<0.05 between patients treated with L-dopa for 5-10 year and those treated

As shown by the literature (Spencer et al., 1995) and by our studies, the elevated level of oxidized guanine in DNA (8-oxo2dG) in PD reflects also pharmacotherapy with L-dopa preparations. In present study, levels of 8-oxo2dG in the patients treated with L-dopa

**Patients with PD** 

**over 10 year treatment (46-81 years)** 

**5-10 year treatment (46-81 years)** 

in PD patients has been correlated with duration of disease and L-dopa dose.

**disease** 

**5.3 Results** 

**5.1 Patients (see point 2.1.1)** 

**Parameter Controls** 

for over 10 year.

**and 8-oxo2dG level (see point 2.1.2)** 

compared to the group treated for five to ten years.

**(22-76 years)** 

developing neurodegenerative diseases are accompanied by disturbed metabolism of Hcy and ADMA and administration of L-arginine, in line with vitamins B6, B12 and folates, to PD patients may offer a modern therapy in this neurodegenerative disease.
