Is Cognitive Impairment in Cirrhotic Patients Due to Increased Peroxynitrite and Oxidative Stress?

Abstract

Cirrhotic patients may suffer minimal hepatic encephalopathy (MHE), with mild cognitive impairment. 3-Nitro-tyrosine levels are a good biomarker for diagnosis of the cognitive impairment and MHE in cirrhotic patients. This suggests that oxidative stress could be involved in the induction of cognitive and motor alterations in MHE. We have observed that patients with MHE show increased oxidative stress in blood compared with cirrhotic patients without MHE, with increased lipid peroxidation, DNA oxidation, protein carbonylation, 3-nitrotyrosine, oxidized glutathione (GSSG)/reduced glutathione (GSH) ratio, and GSH levels. The activities of antioxidant enzymes are enhanced in erythrocytes and mononuclear cells from patients with and without MHE compared with control subjects. Only glutathione peroxidase activity was increased in MHE patients compared with patients without MHE. Oxidative stress markers in blood, especially GSSG/GSH ratio, GSH, malondialdehyde, and 3-nitrotyrosine, correlate with deficits in attention and motor coordination. The increase in antioxidant activities in patients would be an adaptive mechanism to cope with enhanced oxidative stress, although it is not effective enough to normalize it. Our observations lead to the hypothesis that oxidative stress and increased peroxynitrite formation would mediate the synergistic effects of hyperammonemia and inflammation on cognitive and motor impairment in MHE. Antioxid. Redox Signal. 22, 871–877.

Introduction

Around 40% of the patients with liver cirrhosis have minimal hepatic encephalopathy (MHE), with mild cognitive impairment, attention deficits, psychomotor slowing, and impaired visuomotor and bimanual coordination, which affect more than 2 million people in the United States and a similar number in the European Union. MHE cannot be detected in routine analysis but can be unveiled using psychometric tests or neurophysiological assessment. MHE impairs quality of life, reduces the ability to perform some daily tasks, including driving, increases the risk of suffering work and driving accidents, and is associated with shortened lifespan. The two main factors responsible for the pathogenesis of the neurological alterations in MHE are hyperammonemia and inflammation, which act synergistically to induce the cognitive and motor deficits (3, 4, 7, 9). However, the mechanisms by which hyperammonemia and inflammation cooperate to induce these deficits remain unclear.

Innovation

Cirrhotic patients with minimal hepatic encephalopathy (MHE) show increased oxidative stress in blood compared with patients without MHE, with increased levels of 3-nitrotyrosine (3-NTyr), oxidized glutathione (GSSG)/reduced glutathione (GSH) ratio, 8-hydroxydeoxyguanosine (8-OHdG), and malondialdehyde (MDA). Oxidative stress would be due to enhanced formation of reactive oxygen species and not to reduced antioxidant capacity, as suggested by the increased activity of antioxidant enzymes. Oxidative stress markers in blood, especially 3-NTyr, GSSG/GSH ratio, and GSH, correlate with deficits in attention and motor coordination. These results suggest a relevant role of oxidative stress in the cognitive and motor alterations in patients with MHE. Increased peroxynitrite formation could mediate the synergistic effects of hyperammonemia and inflammation on cognitive and motor impairment in MHE.

Both hyperammonemia and inflammation may induce oxidative stress, which could contribute to the neurological alterations in MHE. Oxidative stress has been reported in blood of patients with liver disease and in some patients with overt HE. However, it has not been assessed whether oxidative stress is increased in cirrhotic patients with MHE, the mildest first stage of HE, or if oxidative stress could contribute to the early neurological alterations in MHE.

3-Nitrotyrosine (3-NTyr) is strongly increased in serum of cirrhotic patients with MHE compared with those without MHE, and 3-NTyr levels in serum are useful to diagnose MHE in patients with liver cirrhosis (6). 3-NTyr is mainly formed by reaction of tyrosine with peroxynitrite, which in turn is formed by the reaction of nitric oxide (NO) with superoxide. 3-NTyr increases therefore when oxidative stress increases. The utility of serum 3-NTyr to diagnose the neurological alterations in MHE suggests that oxidative stress may be a main contributor to the induction of cognitive and motor alterations in MHE. We have therefore assessed if oxidative stress is increased in patients with MHE compared with patients without MHE and if oxidative stress correlates with the neurological alterations in MHE.

Results

Table 1 shows the characteristics of the groups studied. Patients with MHE show impaired performance in the bimanual and visuomotor coordination tests, in the individual tests of the psychometric hepatic encephalopathy score (PHES) battery, and reduced performance in the Stroop test, reflecting impaired selective attention (Table 2).

Table 1.

Composition of the Different Groups and Etiology of Liver Disease

	Controls	Patients without MHE	Patients with MHE
Total individuals	34	46	34
Gender (M/F)	15/19	41/5	22/12
Age^a	56±10	58±10	63±10
Alcohol	—	35	24
HBV/HCV/HBV+HCV	—	1/4/1	4/5/0
Alcohol+HBV/HCV	—	2/1	1/2
Others		2	1
Ascites	—	7	10
Child Pugh A/B/C	—	33/13/0	21/11/2
MELD^a	—	9±1	10±1

Values are expressed as mean±standard deviation. The Child Pugh Score is derived from a score of 1–3 given for severity of ascites, hepatic encephalopathy, INR, albumin, and bilirubin. The higher the score is, the more severe the liver disease.

HBV, hepatitis B virus; HCV, hepatitis C virus; MHE, minimal hepatic encephalopathy; MELD, model end-stage liver disease.

Table 2.

Performance in the Psychometric Hepatic Encephalopathy Score and in Attention and Coordination Tests is Reduced in Cirrhotic Patients with MHE

		Patients without MHE	Patients with MHE	Patients with MHE	Global ANOVA
Test	Controls	p vs. control	p vs. control	p vs. without MHE	p-Values
CFF (Hz)	43.2±0.7	41.3±0.5	38.5±0.7	p<0.01	<0.001
			p<0.001
PHES Global score	−0.10±0.16	−0.46±0.16	−6.7±0.4	p<0.001	<0.001
		p<0.05	p<0.001
Symbol Digit Test	0.16±0.08	−0.17±0.06	−0.71±0.11	p<0.001	<0.001
			p<0.001
NCT-A	0.05±0.05	0.11±0.06	−1.09±0.19	p<0.001	<0.001
			p<0.001
NCT-B	0.11±0.07	−0.11±0.07	−1.81±0.22	p<0.001	<0.001
			p<0.001
Serial Dotting Test	0.00±0.00	−0.13±0.05	−0.94±0.18	p<0.001	<0.001
			p=0.001
Line Tracing Test	−0.10±0.07	−0.15±0.09	−2.21±0.16	p<0.001	<0.001
			p<0.001
Stroop
Congruent task (number of words)	117±5	105±3	83±3	p<0.001	<0.001
			p<0.001
Neutral task (number of colors)	82±3	77±2	60±2	p<0.001	<0.001
			p<0.001
Incongruent task (number of items)	48±2	41±2	31±2	p<0.001	<0.001
			p<0.001
Bimanual coordination (min)	1.67±0.04	1.96±0.04	2.65±0.08	p<0.001	<0.001
			p<0.001
Visuomotor coordination (min)	2.04±0.09	2.50±0.06	3.36±0.09	p<0.001	<0.001
			p<0.001

Values are expressed as mean±SEM. Stroop test—congruent task: number of words read in 45 s; neutral task: number of colors read in 45 s; incongruent task: number of items completed in 45 s. Bimanual and visuomotor coordination tests: score in minutes. One-way ANOVA followed by post hoc Bonferroni test were performed, except for bimanual and visuomotor coordination tests, in which univariate ANCOVA was performed, with age included as covariate, followed by post hoc Bonferroni.

ANCOVA, analysis of covariance; ANOVA, analysis of variance; CFF, critical flicker frequency; NCT-A, NCT-B, number connection tests A and B, respectively; PHES, psychometric hepatic encephalopathy score.

Patients with MHE show higher levels of interleukin-6 (IL-6) and interleukin-18 (IL-18) in serum and of cGMP in plasma compared with patients without MHE. Blood ammonia and nitrites+nitrates (stable metabolites of nitric oxide) were increased similarly in patients with and without MHE (Table 3).

Table 3.

Patients with MHE Show Increased Oxidative Stress and Inflammation

		Patients without MHE	Patients with MHE	Patients with MHE	Global ANOVA
Parameter	Controls	p vs. control	p vs. control	p vs. without MHE	p-Values
Biochemical parameters
Plasma cGMP (pmol/ml)	3±0.3	6±0.5	8.5±0.5	p<0.01	<0.001
		p<0.001	p<0.001
Nitrates+nitrites (μM)	20±1	25±1	26±1	ns	0.01
		p<0.01	p<0.01
IL-6 (pg/ml)	1±0.1	2.2±0.2	4.5±0.5	p<0.001	<0.001
		p<0.001	p<0.001
IL-18 (pg/ml)	133±10	225±20	403±44	p<0.01	<0.001
		p<0.001	p<0.001
Ammonia (μM)	13±1	27±3	35±4	ns	<0.001
		p<0.001	p<0.001
Oxidative stress parameters
MDA (nmol/ml)	0.20±0.01	0.46±0.02	0.66±0.04	p<0.001	<0.001
		p<0.001	p<0.001
Carbonylated proteins (nmol/mg protein)	0.150±0.008	0.174±0.008	0.186±0.013	ns	<0.05
		p<0.05	p<0.05
3-NTyr (nM)	7±0.6	10±1	24±4	p<0.001	<0.001
			p<0.001
8-OHdG (ng/ml)	9.2±0.4	9.9±0.4	12±0.7	p<0.05	<0.001
			p<0.001
GSH (μM)	794±49	588±55	427±36	p<0.05	<0.001
		p<0.01	p<0.001
GSSG (μM)	27±3	24±2	25±2	ns	ns
GSSG/GSH ratio (%)	4.7±0.5	7.9±1.3	12±1.5	p<0.05	<0.001
		p<0.05	p<0.001

Values are expressed as mean±SEM. Differences between groups were analyzed using one-way ANOVA followed by post hoc Bonferroni.

cGMP, cyclic GMP; GSH, reduced glutathione; GSSG, oxidized glutathione; GSSG/GSH, ratio of GSSG/GSH in %; IL-6, IL-18, interleukin-6, interleukin-18; MDA, malondialdehyde; 3-NTyr, 3-nitrotyrosine; ns, difference not significant; 8-OHdG, 8-hydroxydeoxyguanosine.

Patients with MHE show increased oxidative stress in blood (Table 3), with increased oxidized glutathione (GSSG)/reduced glutathione (GSH) ratio (12±1.5; p<0.05), compared with cirrhotic patients without MHE (7.9±1.3). This is due to a reduction in GSH levels (427±36 μM vs. 588±55 μM in patients without MHE, p<0.05) while GSSG levels were similar in all groups. Malondialdehyde (MDA), an indicator of oxidative damage to lipids, was also increased in patients with MHE (0.66±0.04 μM; p<0.001) compared with patients without MHE (0.46±0.02 μM). The levels of 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative damage to DNA, were increased in patients with MHE (12±0.7 ng/ml, p<0.001) compared with cirrhotic patients without MHE (9.9±0.4 ng/ml). In addition, serum 3-NTyr was strongly increased (p<0.001) in patients with MHE compared with patients without MHE (Table 3).

The increased oxidative stress in patients with MHE would not be due to reduced activity of antioxidant enzymes (Table 4). Catalase and glutathione S-transferase (GST) activities are increased in erythrocytes and in peripheral blood mononuclear cells (PBMCs) from cirrhotic patients with or without MHE compared with control subjects, but they are not different in patients with or without MHE. Superoxide dismutase (SOD) activity is not altered in erythrocytes but is increased similarly in PBMCs from cirrhotic patients with or without MHE.

Table 4.

Activities of Antioxidant Enzymes in Erythrocytes and Peripheral Blood Mononuclear Cells

		Patients without MHE	Patients with MHE	Patients with MHE	Global ANOVA
Antioxidant activity	Controls	p vs. control	p vs. control	p vs. without MHE	p-Values
Erythrocytes
Catalase (sec⁻¹/gHb)	71±3	83±3	84±3	ns	<0.05
		p<0.05	p<0.05
SOD (U/min×gHb)	1513±14	1510±63	1446±85	ns	ns
Glutathione peroxidase (μmol/min×gHb)	18±1	22±1	27±2	p<0.05	<0.01
		p<0.05	p<0.01
Glutathione reductase (μmol/min×gHb)	2.02±0.09	2.0±0.08	2.51±0.15	p<0.01	<0.01
			p<0.01
Glutathione S-transferase (μmol/min×gHb)	2.8±0.1	3.7±0.2	3.5±0.2	ns	<0.01
		p<0.01	p<0.05
Peripheral blood mononuclear cells
Catalase (nmol/min×mg prot)	63±4	91±5	89±8	ns	<0.01
		p<0.01	p<0.05
SOD (U/mg prot)	9.8±0.7	13.8±1	13.8±0.9	ns	<0.01
		p<0.01	p<0.05
Glutathione peroxidase (μmol/min×g prot)	84±14	104±3	133±14	p<0.05	<0.001
		p<0.01	p<0.001
Glutathione reductase (μmol/min×g prot)	28±2	35±2	38±2	ns	<0.01
		p<0.05	p<0.01
Glutathione S-transferase (μmol/min×g prot)	55±2	72±4	71±7	ns	<0.01
		p<0.01	p<0.05

Values are expressed as mean±SEM.

Differences between groups were analyzed using one-way ANOVA followed by post hoc Bonferroni.

Hb, hemoglobin; SOD, superoxide dismutase.

Patients with MHE show even higher activities of glutathione peroxidase (GPx) and glutathione reductase (GR) compared with patients without MHE (Table 4). Glutathione peroxidase activity is significantly (p<0.05) increased in erythrocytes (by 23%) and PBMCs (by 22%) of cirrhotic patients with MHE compared with patients without MHE. Glutathione reductase activity in erythrocytes was increased by 25% in patients with MHE compared with patients without MHE (Table 4).

Markers of oxidative stress correlate with neurological alterations. Serum levels of 3-NTyr and MDA correlate with PHES, mental processing speed, and attention measured by number connection tests A and B (NCT-A and NCT-B, respectively), with cognitive flexibility evaluated with the Stroop test and with scores in bimanual and visuomotor coordination tests (Table 5).

Table 5.

Increased Oxidative Stress in Patients with MHE Correlates with Neurological Alterations

	3-NTyr	GSH	GSSG/GSH	MDA
Neurological tests
PHES Global score	r=−0.518	r=0.428	r=−0.419	r=−0.566
	p<0.001	p=0.001	p=0.001	p<0.001
NCT-A	ns	ns	r=−0.396	r=−0.453
			p=0.002	p<0.001
NCT-B	r=−0.421	ns	ns	r=−0.539
	p<0.001			p<0.001
Line Tracing Test	r=−0.559	r=0.438	r=−0.374	r=−0.419
	p<0.001	p<0.001	p=0.003	p<0.001
Stroop
Congruent task (Number of words)	r=−0.358	r=0.437	r=−0.510	r=−0.493
	p=0.002	p=0.001	p<0.001	p<0.001
Incongruent task (Number of items)	ns	ns	ns	r=−0.405
				p<0.001
Bimanual coordination (min)	r=0.362	ns	ns	r=0.633
	p=0.002			p<0.001
Visuomotor coordination (min)	r=0.340	ns	ns	r=0.706
	p=0.004			p<0.001

The p and r values of the Pearson correlation test are shown.

The GSSG/GSH ratio correlated with PHES, NCT-A, line tracing test (LTT), and with the score for the congruent task in the Stroop test. GSH levels in blood correlated with the performance in the PHES, LTT, and Stroop (congruent task).

Discussion

The results reported show that patients with MHE have increased oxidative stress, which correlates with the grade of attention deficits, cognitive impairment, and of motor in-coordination. These data suggest that oxidative stress contributes to the pathogenesis of the cognitive and motor alterations in MHE. Patients with liver cirrhosis can cope with a mild level of oxidative stress, but when a certain threshold of oxidative stress is overcome, the cognitive and motor functions begin to be impaired, leading to MHE.

The increase in oxidative stress in MHE would be due to increased production of reactive oxygen species (ROS) and not to reduced antioxidant capacity. The activity of all the antioxidant enzymes analyzed is increased in cirrhotic patients compared with control subjects. The increases in glutathione peroxidase and glutathione reductase are higher in patients with MHE compared with patients without MHE. This suggests that the increased oxidative stress is not due to reduced antioxidant activity but due to increased formation of ROS.

The enhanced activities of the antioxidant enzymes would reflect an increased need to remove ROS. Increased SOD activity would indicate increased production of superoxide, which when detoxified by SOD will lead to increased formation of hydrogen peroxide, which, in turn, would lead to the induction of the enzymes that detoxify it: catalase and glutathione peroxidase. The increased formation of ROS will increase the formation of lipid hydroperoxides and MDA, which are eliminated by glutathione S-transferase by conjugation with GSH.

The increased activities of glutathione peroxidase and of glutathione S-transferase will increase the GSH consumption, leading to reduced levels of blood GSH.

The two main factors responsible for the pathogenesis of the neurological alterations in MHE are hyperammonemia and inflammation (3, 4, 7, 9), which act synergistically to induce the cognitive and motor deficits. The mechanisms by which hyperammonemia and inflammation cooperate to induce these deficits remain unclear. The data reported suggest that enhanced formation of peroxynitrite, which will modify tyrosine both free and in proteins to form 3-NTyr, is a good candidate to explain the synergistic effects of hyperammonemia and inflammation in the induction of the neurological alterations in MHE.

3-NTyr is mainly formed by reaction of tyrosine with peroxynitrite, which in turn is formed by the reaction of NO with superoxide (8). 3-NTyr is not significantly increased in patients without MHE but is strongly increased in patients with MHE, and determination of 3-NTyr in serum is useful to identify the presence of MHE in patients with liver cirrhosis, with good sensitivity, specificity, and positive and negative predictive values (6).

Increased formation of 3-NTyr in patients with MHE would be due to higher levels of peroxynitrite due to increased formation of NO and superoxide. Inflammation would be a main contributor to the increase in nitric oxide in cirrhotic patients. Systemic inflammation increases the expression of inducible nitric oxide synthase both in peripheral tissues and in the brain. On the other hand, hyperammonemia induces oxidative stress, with increased formation of superoxide (5). The increase in NO induced by inflammation together with increased oxidative stress and superoxide formation induced by hyperammonemia would result in increased peroxynitrite formation. The synergistic effects of both would increase peroxynitrite and enhance neurological impairment.

Peroxynitrite not only increases 3-NTyr but also contributes to the increase in lipid peroxidation and in MDA levels and to oxidative damage to DNA and to increased levels of 8-OHdG. Enhanced formation of peroxynitrite in MHE would explain the increased levels of 3-NTyr, MDA, and 8-OHdG.

Peroxynitrite-mediated oxidative damage seems to play a role in the pathogenesis of both sporadic and familial amyotrophic lateral sclerosis, of neurodegenerative diseases, and of other situations leading to neurological alterations, such as chronic fatigue syndrome and post-traumatic stress disorder (1, 2).

The results reported here suggest that peroxynitrite could be a main contributor to the mechanisms leading to cognitive and motor impairment in patients with MHE. Those patients showing higher inflammation, hyperammonemia, and oxidative stress would produce more peroxynitrite, which would affect more strongly their cerebral function, including mental processing speed, attention, and motor coordination.

Notes

We evaluated in 34 cirrhotic patients with MHE, 46 patients without MHE, and 34 healthy controls: (a) cognitive and motor function using psychometric tests; (b) markers of oxidative stress in blood: 3-NTyr, GSH, and GSSG/GSH ratio; (c) markers of oxidative damage of biomolecules: lipid peroxidation, DNA oxidation, and proteins carbonylation; (d) activity of antioxidant enzymes in erythrocytes and mononuclear cells; (e) inflammatory factors and hyperammonemia. We also assessed whether increased oxidative stress in MHE correlates with the neurological alterations in patients with MHE.

Patients and methods

Patients and controls

Eighty patients with liver cirrhosis were consecutively recruited from the outpatient clinics in Hospital Clínico and Hospital Arnau de Vilanova of Valencia, Spain. The diagnosis of cirrhosis was based on clinical, biochemical, and ultrasonographic data. Exclusion criteria were overt HE or history of overt HE, recent (<6 months) alcohol intake, infection, recent (<6 weeks) antibiotic use or gastrointestinal bleeding, recent (<6 weeks) use of drugs affecting cognitive function (e.g., benzodiazepines, anti-epileptic, and/or psychotropics), shunt surgery, or transjugular intrahepatic portosystemic shunt for portal hypertension, electrolyte imbalance, renal impairment (serum creatinine >1.5 mg/dl), presence of hepatocellular carcinoma, or severe medical problems (e.g., congestive heart failure, pulmonary disease, neurological or psychiatric disorder). Thirty-four healthy volunteers were also enrolled in the study once liver disease was discarded by clinical, analytical, and serologic tests. Physical examination, standard laboratory tests, and a standardized battery of neuropsychological tests were administered to all participants after signing a written informed consent. Study protocols were approved by the Scientific and Ethical Committees of both hospitals. The procedures followed were in accordance with the ethical guidelines of the Declaration of Helsinki. After a standard history and physical examination, blood was drawn for biochemical measurements. Psychometric tests, critical flicker frequency (CFF) determination, attention and coordination tests, and blood collection were carried out on the same day. After performing the psychometric tests, patients were classified as without MHE or with MHE (see Neuropsychological assessment section). Therefore, the study includes three groups: (a) control subjects, (b) patients without MHE, and (c) patients with MHE.

Neuropsychological assessment

MHE was diagnosed using the PHES. PHES comprises five psychometric tests: digit symbol test evaluates processing speed and working memory, NCT-A and NCT-B mental processing speed and attention, serial dotting test, and LTT visuospatial coordination. The scores were adjusted for age and education level using Spanish normality tables (www.redeh.org). Patients were classified as having MHE when the score was less than −4 points.

A color-word version of the Stroop task was used to assess selective attention. Bimanual and visuomotor coordination tests were assessed.

Critical flicker frequency

CFF has been proposed as an alternative procedure for detection of MHE in cirrhotic patients. The CFF was measured.

Blood extraction and isolation of blood cells

For serum and plasma obtention, venous blood (5 ml) was taken in BD Vacutainer tubes with or without EDTA (plasma and serum, respectively) and centrifuged at 500 g for 10 min. The supernatant was collected and stored frozen at −80°C.

Blood erythrocytes (red blood cells [RBCs]) were isolated from 2.7 ml of blood collected in BD Vacutainer tubes with CTAD solution as anticoagulant. Isolation was carried out by a method based on cellulose columns, which retain lymphocytes and platelets and let through plasma and erythrocytes. In brief, columns were prepared in a syringe containing a 2.5-cm Whatman filter and a mix of 1:1 (w/w) α-cellulose (Sigma) and Sigmacell cellulose (Sigma) in 0.9% NaCl. Two volumes of blood were diluted in 1 volume of a buffer containing 150 mM NaCl, 10 mM KH₂PO₄, 0.05 mM EDTA, and 0.02 mM PMSF (phenylmethylsulfonyl fluoride), pH 7.4, and applied to the column. The fraction containing RBCs was collected in a tube containing a solution of 150 mM NaCl, 10 mM KH₂PO₄, and 0.05 mM EDTA, pH 7.4. After centrifugation at 1000 g for 10 min at 4°C, RBCs were collected and supernatant was discarded. RBCs were washed three times (10 min, 1000 g, 4°C) in phosphate-buffered saline (PBS)–glucose (10 mM KH₂PO₄, 3.5 mM KCl, 145 mM NaCl, 6 mM glucose, pH 7.4), diluted 1:6 in PBS-glucose, and aliquots were frozen at −80°C until their use. To prepare lysates for measuring antioxidant activities, 0.25 ml of RBC suspension was incubated in 0.5 ml of 50 mM triethanolamine buffer, pH 7.4, containing 0.2% saponin for 10 min. Hemoglobin (Hb) concentration was determined by Drabkin's Reagent (Sigma).

PBMCs were obtained as previously. In brief, blood (10 ml) was diluted with 1 volume of PBS and layered onto Lympho separation medium (MP Biomedicals). After centrifugation at 800 g for 20 min at 18°C–20°C, the layer containing the PBMCs was collected and washed with 3 volumes of PBS and centrifuged at 1000 g for 10 min. Pellets were resuspended in PBS and distributed in several aliquots, which were centrifuged again, discarding the supernatant, and dry pellets of mononuclear cells were stored frozen at −80°C until use. For determination of glutathione peroxidase, glutathione reductase, and glutathione S-transferase activities, PBMC lysates were prepared in 0.15 ml of 50 mM triethanolamine buffer, pH 7.4, containing 0.2% saponin, were sonicated, and centrifuged at 14,000 g for 15 min at 4°C. The supernatant was used immediately to measure activities, and protein was determined by the BCA method. For determination of catalase and SOD activities, PBMCs were resuspended in 0.15 ml of 50 mM phosphate buffer, pH 7.0, containing 1 mM EDTA, and after sonication, lysates were centrifuged and supernatant was immediately used.

Biochemical determinations in blood

Nitrates+nitrites in plasma were measured. Interleukins (IL-6 and IL-18) were measured in serum using Human IL-6 ELISA kit from Thermo Scientific (Pierce Biotechnology), and Human IL-18 ELISA kit from Bender MedSystems GmbH. Blood ammonia was measured immediately after blood extraction with the Ammonia Test Kit II for the PocketChemBA system (Arkay, Inc.) following the manufacturer's specifications. cGMP in plasma was determined using the BIOTRAK cGMP enzyme immunoassay kit from Amersham (GE Healthcare, Life Sciences).

Determination of activities of antioxidant enzymes in RBCs and in PBMCs

Catalase in RBCs was assayed with hydrogen peroxide by measuring the decrease in absorption at 240 nm. The enzyme activity was expressed in terms of the first-order reaction rate constant, that is, sec⁻¹ per g of Hb.

The SOD activity in RBCs was determined by the Beauchamp and Fridovich's method by the inhibition of the reduction of nitrotetrazolium blue (NTB) in the presence of xanthine–xanthine oxidase system. One unit of SOD activity was defined as the amount of SOD required for 50% inhibition of the rate of NTB reduction. The SOD activity in RBCs was expressed as U/min×g Hb.

Catalase and SOD activities in PBMCs were measured with kits from Cayman Chemical Co. Catalase activity was expressed as nmol/min×mg protein, and SOD as U/ml×mg protein.

GR activity was measured spectrophotometrically at 340 nm by the rate of oxidation of NADPH by GSSG, and GPx activity by the rate of oxidation of NADPH in the coupled GR reaction. These activities were expressed as μmol/min×g Hb for RBCs and μmol/min×g protein for PBMCs.

GST activity was determined and was expressed as μmol/min×g Hb for RBCs and μmol/min×g protein for PBMCs.

Determination of oxidative stress markers

GSH and GSSG levels were measured in whole blood using a Glutathione Fluorescent kit (Arbor Assays).

Protein carbonyls in serum were measured by a protein carbonyl enzyme immunoassay kit (Biocell PC test; from Biocell Corporation Ltd.).

8-OHdG, one of the oxidative DNA damage byproducts, is physiologically formed. 8-OHdG in serum was quantified by the OxiSelect™ Oxidative DNA damage ELISA kit (Cell Biolabs, Inc.).

As a marker of lipid oxidation, we measured the levels of MDA in serum from controls and patients by HPLC.

3-NTyr in serum was determined by HPLC as described in (6).

Statistical analysis

Values are given as mean±SEM. Results were analyzed by one-way ANOVA followed by post hoc Bonferroni's multiple comparison test. Variables that were not previously age-adjusted (scores in bimanual and visuomotor coordination tests) were analyzed using univariate analysis of covariance (ANCOVA) with age included as covariate, followed by post hoc Bonferroni. Bivariate correlations among variables were evaluated using the Pearson correlation test. Analyses were performed using GraphPad Prism version 6.0 and SPSS version 19.0 (SPSS, Inc.), and two-sided p-values<0.05 were considered significant.

Footnotes

Acknowledgments

This work was supported by grants from Ministerio de Ciencia e Innovación (SAF2011-23051, CSD2008-00005 to V.F. and FIS PI12/00884, co-financing by the European Fund for Regional Development [FEDER] to C.M.), Consellería Educación Generalitat Valenciana (PROMETEO-2009-027; PROMETEOII/2014/033; ACOMP/2012/066, ACOMP/2013/101 to V.F. and ACOMP/2012/056, ACOMP/2014/026 to C.M.), and Fundación ERESA (BF13007) to C.M.

Abbreviations Used

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