Dynamic of oxidative and nitrosative stress markers during the convalescent period of stroke patients undergoing rehabilitation

Abstract

Background

Stroke patients have a redox imbalance, a consequence of both the cerebrovascular event and the associated pathological conditions. Our study was aimed to investigate the dynamic of some oxidative and nitrosative markers during the convalescent phase of postacute stroke patients undergoing rehabilitation.

Methods

We assessed thiol, advanced oxidation protein product, protein carbonyl, 3-nitro-l-tyrosine, ceruloplasmin and oxidized LDL concentrations, as well as gamma-glutamyltranspeptidase (GGT) activity in 20 patients at the beginning of the hospitalization and at the discharge moment, respectively, and 24 apparently healthy controls.

Results

We found significantly increased values for GGT (P = 0.04), ceruloplasmin (P = 0.01) and protein carbonyl (P = 0.04) in stroke patients at the hospitalization moment when compared with healthy controls, while total thiols were significantly decreased (P = 0.002). Rehabilitation was associated with a significant decrease of protein carbonyl (P = 0.03) and oxidized LDL particle concentrations (P = 0.03), as well as GGT activity (P = 0.02). At the hospitalization moment, both GGT and ceruloplasmin were significantly negatively correlated with non-proteic thiols (r = −0.44, P = 0.049, and r = −0.53, P = 0.015, respectively) and significantly positively with protein carbonyls (r = +0.80, P < 0.001, and r = +0.69, P < 0.001, respectively) suggesting putative roles of GGT and ceruloplasmin in the redox imbalance.

Conclusions

These results highlight the existence of a redox imbalance in postacute stroke patients, and the possible benefits of an antioxidant-based therapy for the recovery of these patients.

Introduction

The nervous system has a high susceptibility to free radicals mainly due to its high content of polyunsaturated fatty acids, relative low content of antioxidants, increased oxygen consumption, high concentration of iron that can act as a pro-oxidant and auto-oxidation of some neurotransmitters (i.e. dopamine).¹ During a cerebrovascular event (i.e. stroke), the disruption of blood flow is the first step in the generation of free radicals. Energy failure as a consequence of oxygen deprivation leads to acidosis and alteration of ion homeostasis.² Intracellular calcium accumulation inside neurons and glial cells triggers the activation of a plethora of enzymes (i.e. calpains, phospholipase A₂, neuronal nitric oxide synthase). Activation of the Ca²⁺-dependent phospholipase A₂ and cyclooxygenase is followed by the production of free radicals during both the ischaemic period and the reperfusion stage.³ Other important sources of reactive species are nicotinamide adenine dinucleotide phosphate oxidase and xanthine oxidase, both producing the superoxide anion (O₂ ^•−),⁴ while both neuronal and endothelial nitric oxid synthases generate nitric oxide (NO^•). The interaction between O₂ ^•− and NO^• gives rise to peroxynitrite anion (ONOO⁻) which is responsible for covalent modification of different proteins on tyrosil residues. Moreover, this reaction renders NO^• unavailable for endothelial tonus regulation.

Recently it was proposed that the accumulation of gamma-glutamyltranspeptidase (EC 2.3.2.2) (GGT) inside atherosclerotic plaques could be another important source of free radicals in the vasculature.⁵ GGT is the only extracelullar enzyme responsible for the catabolism of reduced glutathione.⁶ There is evidence of GGT ability to catalyse GSH-dependent lipid peroxidation in the presence of Fe³⁺.^7–9 Thus, GGT became one of the culprits responsible for the formation of pro-atherogenic oxidized low-density lipoprotein (LDLox) particles inside the atherosclerotic plaques.¹⁰ The reactive oxygen species generated through the function of GGT enable the destabilization of the plaque, leading finally to its rupture.¹¹ Consistently, recent studies reported significantly positive correlations between GGT activity and the risk of major stroke events.^12–14

Our study was aimed to investigate the dynamics of proteic blood markers of oxidative and nitrosative stress during the rehabilitation of patients with a previously diagnosed stroke. The markers of oxidative stress (total and non-protein plasma thiol content, advanced oxidation protein product [AOPP] and protein carbonyl [PC] concentrations), and nitrosative stress (3-nitrotyrosine [3NT] concentration) were evaluated. We also focused on GGT, oxidized LDL particles (LDLox), due to their central position in the aetiology of atherosclerosis, and ceruloplasmin which is well recognized for its capacity to oxidize LDL particles, with high concentrations of this protein being associated with increased risk of cardio- and cerebrovascular disease, including stroke.^15,16

Materials and methods

Study design

A total of 20 patients hospitalized for specific rehabilitation procedures were enrolled. The hospitalization period was of 13 ± 1 days (range 11–15 days). The inclusion criterion for our study was an ischaemic or haemorrhagic stroke diagnosed in the previous 90 days (39.7 ± 5.25, range 12–87 days). Exclusion criteria were as follows: cancer, chronic renal failure, chronic inflammatory, autoimmune and haematological disorders, smoking and chronic alcohol consumption. Also, patients who were under vitamins and anti-inflammatory drugs during the two months preceding the beginning of the study were excluded. A control group (Controls) was created by the enrolment of 24 apparently healthy volunteers matching for age and sex distribution. They were recruited from persons presented for routine medical control. Neither the controls nor the patients had a previous cerebrovascular event (cerebral haemorrhage, haemorrhagic infarct, transient ischaemic attack). The medication used by both patients and controls was recorded. In stroke patients (Cases), the biochemical parameters were evaluated at the hospitalization moment (t ₀) and at the discharge (t ₁).

The Barthel index (BI) was used to evaluate the patients' abilities to perform activities of daily living.¹⁷ Scoring ranged from a minimal value of 0 points (total dependency of the subject) to 100 points (total independency of the subject).

The study was approved by the ethics review board of the Elias Emergency Hospital. Informed consent was obtained from all the subjects enrolled in the study or from their relatives.

Blood collection

Blood samples were collected in tubes with (ethylenediaminetetraacetic acid [EDTA]) and without anticoagulant, after 8–10 h overnight fasting. From each patient, two samples were taken, at t ₀ and t ₁, while from controls only one sample was taken. The blood collected with an anticoagulant was immediately centrifuged at 4000 g for 15 min at 4 °C. Serum and plasma were aliquoted in 1.5 mL tubes and stored at −80 °C until the moment of analysis. All biochemical tests were done in duplicate and were expressed as mean values.

Routine analysis

Among the routine parameters, total proteins, albumin, glycaemia (Spinreact, Girona Spain) and GGT (DyaSys, Holzheim, Germany) were assessed using commercially available kits. In the case of total cholesterol, triglycerides and phospholipids, we used enzymatic methods (Spinreact). Total lipids were assessed with a phosphovanilline reagent-based method (Spinreact). After acidic hydrolysis of the samples with H₂SO₄, the solution that resulted reacted with a phosphovanilline reagent and the absorbance was recorded at 520 nm relative to a blank. The results were calculated using a standard with a known concentration of total lipids. HDL cholesterol (HDL-C) was evaluated after precipitation with phosphotungstic acid and MgCl₂ (Spinreact). The Friedewald formula was used for the estimation of the LDL cholesterol (LDL-C) concentration.

Reagents

All the solvents and reagents were purchased from Sigma-Aldrich (St Louis, MO, USA): ethanol, ethyl acetate were for high-performance liquid chromatography (99.9%), reduced l-glutathione (≥98%), 2,4-dinitrophenylhydrazine (DNPH), guanidine hydrochloride, acetic acid, potassium iodide (KI), chloramine-T, TrisHCl, EDTA, 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), sodium dodecyl sulphate (SDS), metaphosphoric acid, p-phenylenediamine, 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), potassium persulphate and sodium azide. All the reagents were reagents Ph. Eur. (p.a.). For spectrophotometric determinations, a Shimadzu UV–vis mini 120 (Shimadzu Corporation, Kyoto, Japan) spectrophotometer was used. Immunoassays were carried out using an enzyme-linked immunosorbent assay reader (Sunrise Absorbance Reader, Tecan, Männedorf, Switzerland).

Laboratory procedures

Serum protein carbonyl concentration was assessed through a previously described method based on the reaction with DNPH in acidic solution.¹⁸ Plasma AOPP concentration was evaluated using the method described by Witko-Sarsat et al. ¹⁹ with minor modifications. To avoid false increased results due to the lipid-induced turbidity, plasma was delipidated using a phosphotungstic acid reagent, before the evaluation of AOPPs. The concentration of total thiols (TTs) and non-proteic thiols (NPTs) were assessed through previously described methods.²⁰ Plasma ceruloplasmin concentration was evaluated through a method based on the ability of this protein to oxidize p-phenylendiamine at pH = 5.5.²¹ The total antioxidant capacity (TAC) was assessed using a previously described method.²² The cationic radical ABTS^•+ was generated through the reaction between ABTS and potassium persulphate during 12 h at room temperature in the dark. The working reagent had an absorbance of 0.70 ± 0.02 at 734 nm. After adding the sample, the decrease of absorbance was recorded for one minute at 734 nm and the results were expressed as mmol/L of Trolox equivalents.

For the evaluation of the concentrations of LDLox and 3NT, respectively, commercially available kits were used (Mercodia, Uppsala, Sweden; Nitrotyrosine HK501, Hycult Biotech, Uden, The Netherlands). The detection limit of the kit used to evaluate the concentration of LDLox was less than 1 mU/L, while the detection range for the 3NT kit was 2–1500 nmol/L, as specified by the producers.

Statistical analysis

All results are expressed as mean ± SEM (standard error of the mean). Due to the low number of subjects in both control and stroke patients groups, we used the Mann-Whitney test to compare the means between groups, while the Wilcoxon test was used to evaluate the effect of the rehabilitation on the dynamic of biochemical parameters. The evaluation of the correlations between variables was made with Pearson's correlation coefficient. Statistical analysis was performed with Statistical Package for Social Science 15.0 (SPSS 15.0) for Windows. The differences were considered to be significant when P < 0.05.

Results

Demographic, vascular risk factors and medication of all the subjects enrolled in our study are presented in Table 1. Our study group comprised a population of 20 stroke patients (women/men, 12/8) and 24 apparently healthy controls (women/men, 20/4). The two major stroke subtypes had an incidence of 75% for the ischaemic events and 15% for the haemorrhagic events.

Table 1

Demographic, vascular risk factors and medication in control subjects and stroke patients at the hospitalization moment

	Controls (n = 24)	Cases (t ₀) (n = 20)	P value*
Demographic characteristics
Age (years)	65.83 ± 2.03	69.60 ± 2.51	>0.05
Sex ratio (men/women)	4/20	8/12	>0.05

Vascular risk factors
Hypertension no. (%)	10 (41.66%)	17 (85%)	>0.05
Diabetes mellitus no. (%)	–	3 (15%)	–
Dyslipidemia no. (%)	4 (16.66%)	11 (55%)	>0.05
Coronary ischaemic disease no. (%)	2 (8.33%)	7 (35%)	>0.05
Carotid atherosclerosis no. (%)	–	9 (45%)	–
Cardiac arrhythmia no. (%)	–	3 (15%)	–

Medications
Statins no. (%)	–	13 (65%)	–
Beta-blockers no. (%)	8 (33.33%)	11 (55%)	>0.05
Anticoagulants no. (%)	–	5 (25%)	–
Oral hypoglycaemic agents no. (%)	–	2 (10%)	–
Insulin no. (%)	–	1 (5%)	–
ACE inhibitors no. (%)	10 (41.66%)	9 (45%)	>0.05
Antihypertensives no. (%)	8 (33.33%)	13 (65%)	>0.05
Acetylsalicylic acids no. (%)	4 (16.66%)	13 (65%)	<0.05
Antiplatelet agents no. (%)	–	4 (20%)	–
Antiacids no. (%)	14 (58.33%)	11 (55%)	>0.05
Diuretics no. (%)	–	4 (20%)	–

*Independent-samples t-test

Routine biochemical analysis

The dynamic of routine biochemical parameters is summarized in Table 2. The assessed parameters were significantly modified in stroke patients at the admission moment (Cases, t ₀) when compared with Controls: glycaemia, triacylglycerols and total proteins were significantly increased, all the other parameters were significantly decreased. When comparing these parameters at the hospitalization moment (Cases, t ₀) with the discharge moment (Cases, t ₁), it was found that only total lipids were significantly decreased (t ₀ versus t ₁, 5.65 ± 0.16 versus 4.73 ± 0.22, P < 0.001).

Table 2

Routine laboratory measurements

		Cases
	Controls	t ₀	t ₁
Glycaemia (mmol/L)	5.92 ± 0.09	6.91 ± 0.28^†,**	6.59 ± 0.24
Total lipids (g/L)	6.91 ± 0.17	5.65 ± 0.16^†,**	4.73 ± 0.22^‡,**
Total cholesterol (mmol/L)	5.16 ± 0.18	3.81 ± 0.21^†,**	3.69 ± 0.21
HDL-cholesterol (mmol/L)	1.79 ± 0.12	1.46 ± 0.09^†,*	1.58 ± 0.16
LDL-cholesterol (mmol/L)	2.83 ± 0.18	1.89 ± 0.17^†,**	1.57 ± 0.18
Triacylglycerols (mmol/L)	1.18 ± 0.06	1.40 ± 0.08^†,*	1.34 ± 0.08
Phospholipids (mmol/L)	3.22 ± 0.06	2.63 ± 0.08^†,**	2.56 ± 0.11
Albumin (mmol/L)	0.71 ± 0.05	0.61 ± 0.01^†,*	0.60 ± 0.02
Total proteins (g/L)	76.00 ± 4.48	65.52 ± 2.19^†,*	69.93 ± 2.02

^†Cases (t ₀) versus Controls, Mann-Whitney test

^‡Cases (t ₁) versus Cases (t ₀), Wilcoxon test

*P < 0.05, **P < 0.001

Baseline and dynamics of oxidative and nitrosative stress markers

Table 3 presents the baseline as well as the dynamic of all parameters of oxidative and nitrosative stress that were assessed in this study. When compared with controls, stroke patients (Cases, t ₀) had significantly increased values for GGT activity (2.33 ± 0.40 versus 4.50 ± 0.94, P = 0.04) and the concentrations of both ceruloplasmin (0.27 ± 0.01 versus 0.36 ± 0.02, P = 0.01) and PCs (0.70 ± 0.12 versus 1.21 ± 0.24, P = 0.04). On the other hand, the concentration of TTs was significantly decreased in stroke patients (Cases, t ₀) when compared with controls (494.72 ± 30.67 versus 372.66 ± 15.74, P < 0.001). During the rehabilitation period, a statistically significant decrease for GGT activity was found (4.50 ± 0.94 versus 3.17 ± 0.57, P = 0.02), as well as for the concentrations of PCs (1.21 ± 0.24 versus 0.79 ± 0.16, P = 0.03), LDLox (64.06 ± 4.10 versus 56.70 ± 4.76, P = 0.03) and TAC (2.22 ± 0.11 versus 1.94 ± 0.13, P = 0.04).

Table 3

Concentrations of oxidative and nitrosative stress markers in control subjects and stroke patients undergoing rehabilitation

	Controls	Cases (t ₀)	Cases (t ₁)
Total thiols (μmol/L)	494.72 ± 30.67	372.66 ± 15.74^†,*	395.06 ± 19.51
Non-proteic thiols (μmol/L)	62.89 ± 9.25	45.02 ± 4.95	46.90 ± 3.94
GGT (U/L)	2.33 ± 0.40	4.50 ± 0.94^†,*	3.17 ± 0.57^‡,*
Ceruloplasmin (g/L)	0.27 ± 0.01	0.36 ± 0.02^†,*	0.34 ± 0.01
AOPPs (μmol/L)	65.28 ± 4.79	67.76 ± 8.65	56.31 ± 5.05
PCs (nmol/mg proteins)	0.70 ± 0.12	1.21 ± 0.24^†,*	0.79 ± 0.16^‡,*
LDLox (U/L)	63.95 ± 2.74	64.06 ± 4.10	56.70 ± 4.76^‡,*
TAC (mmol/L)	2.26 ± 0.08	2.22 ± 0.11	1.94 ± 0.13^‡,*
3NT (nmol/L)	10.21 ± 1.72	13.89 ± 2.96	11.95 ± 2.83

AOPPs, advanced oxidation protein products; GGT, gamma-glutamyltranspeptidase; LDLox, oxidized LDL particles; PCs, protein carbonyls; TAC, total antioxidant capacity; 3NT, 3-nitro-l-tyrosine

*P < 0.05, **P < 0.001

^†Cases (t ₀) versus Controls, Mann-Whitney test

^‡Cases (t ₁) versus Cases (t ₀), Wilcoxon test

Correlations among oxidative and nitrosative stress markers at t ₀ and t ₁

The statistically significant correlations between the assessed markers in both t ₀ and t ₁ moments are depicted in Table 4. At t ₀, TAC was significantly positive correlated with NPTs (r = +0.52, P = 0.018) and negative correlated to GGT activity (r = −0.45, P = 0.048). Moreover, we found a very similar pattern of correlations for GGT activity and ceruloplasmin. Both are significantly negatively correlated with the concentration of NPTs (r = −0.44, P = 0.049, and r = −0.53, P = 0.015, respectively), on the one hand, and positively correlated with the concentration of PCs (r = +0.80, P < 0.001, and r = +0.69, P < 0.001). Also, ceruloplasmin was significantly negative correlated with TAC (r = −0.53, P = 0.017).

Table 4

Correlations among oxidative and nitrosative stress markers in stroke patients (r and P value)

	NPTs	GGT	TAC	PCs	LDLox
Hospitalization moment (Cases, t ₀)
GGT	−0.44 (0.049)
TAC	+0.52 (0.018)	−0.45 (0.048)
PCs	−0.54 (0.014)	+0.80 (<0.001)
Ceruloplasmin	−0.53 (0.015)		−0.53 (0.017)	+0.69 (<0.001)
TTs					+0.47 (0.035)
Discharge moment (Cases, t ₁)
GGT				+0.59 (0.006)	+0.76 (<0.001)
TAC		−0.64 (0.002)

GGT, gamma-glutamyltranspeptidase; LDLox, oxidized LDL particles; NPTs, non-proteic thiols; PCs, protein carbonyls; TAC, total antioxidant capacity; TTs, total thiols

Evaluation of the correlations between variables was made with Pearson's correlation coefficient

At the discharge moment (Cases, t ₁), there was a significantly negative correlation between TAC and the GGT activity (r = −0.64, P = 0.002). On the other hand, there were significantly positive correlations between GGT activity and PC and LDLox concentrations (r = +0.59, P = 0.006, and r = +0.76, P < 0.001, respectively).

Relations among plasma concentration of oxidative and nitrosative stress markers and Barthel index

We evaluated the correlations among all the markers that were assessed in our study in both t ₀ and t ₁ moments and BI values. During the rehabilitation period the value of the BI significantly increased (t ₀ versus t ₁, 54.75 ± 2.91 versus 58.50 ± 2.99, P < 0.001), suggesting an improvement of patients' abilities to perform activities of daily living. Nevertheless, four patients failed to improve their BI, having the same BI values at both discharge and hospitalization moments. At t ₀, the BI values correlated with the GGT activity (r = +0.46, P = 0.039) while at t ₁, the BI values correlated with both NPTs and LDLox particles (r = +0.63, P = 0.003, and r = +0.48, P = 0.031).

Discussion

This study was aimed to investigate the dynamic of some oxidative and nitrosative markers in postacute stroke patients undergoing rehabilitation. Most of the studies were focused on the investigation of how the redox status changes during the acute phase after stroke, and how these changes influence the patients' outcome. There is a lack of data regarding the dynamic of the redox status during the postacute phase after a cerebrovascular event.

Both TTs and NPTs (reduced glutathione, l-cysteine, cysteinylglycine and free reduced homocysteine) are important components of the blood antioxidant defence system.²³ The existence of a redox imbalance in our patients was supported by the significantly decreased concentration of TTs in cases (t ₀) when compared with healthy controls. Another indicator of the redox imbalance in our patients was the significantly increased concentration of PCs when compared with apparently healthy controls. 2-Aminoadipic semialdehyde and glutamic semialdehyde are the most important PCs resulting through oxidative modifications of l-Lys and l-Pro and l-Arg, respectively.²⁴ These markers were found to be significantly increased in other pathological conditions associated with redox disregulation (diabetes, renal failure and sepsis).

Stroke patients at the hospitalization moment (Cases, t ₀) had significantly increased values of GGT activity. GGT is able to generate reactive oxygen species (H₂O₂) during the reaction of glutathione hydrolysis to cysteinylglycine in the presence of transitional metals.^7,9,25,26 On the other hand, there is evidence suggesting that ceruloplasmin, an acute phase reactant, could have pro-oxidant properties.²⁷ In our study, ceruloplasmin had also significantly increased concentrations in stroke patients (Cases, t ₀) when compared with healthy controls.

The involvement of both GGT and ceruloplasmin in redox imbalance in the postacute phase after the cerebrovascular event is highlighted by the significantly negative correlations between these parameters and the concentration of NPTs. Moreover, we found strong positive correlations between the concentration of PCs on one hand, and the activity of GGT and the concentration of ceruloplasmin on the other. In the same context, finding a significantly negative correlation between ceruloplasmin concentration and TAC further suggests that ceruloplasmin, probably due to its ferroxidase activity, consumes NPTs, decreasing the antioxidant capacity of the blood. In our study, the concentration of NPTs is decreased in Cases (t ₀) when compared with Controls, but without statistical significance. A pitfall of the present study was the inability to assess individual chemical species belonging to the NPT group (reduced glutathione, l-cysteine, cysteinylglycine and free reduced homocysteine), and as a consequence, we cannot specify exactly which one of the chemical species is altered in our study group.

The rehabilitation period was associated with a significant decrease of GGT's activity, while the concentration of ceruloplasmin remained almost unchanged. We also found a significant decrease for both PCs and LDLox particles. Thus, it could be supposed that, even during a short period of rehabilitation, there is a correction of the blood redox status. At the discharge moment (Cases, t ₁), we found significantly positive correlations between GGT activity and the concentration of both PCs and LDLox particles, suggesting that the reduction of the activity of this enzyme had a beneficial impact upon the production of reactive oxygen species.

Despite the redox improvement suggested by the significant decrease of PC and LDLox particle concentrations, there was still a redox imbalance highlighted by the significant decrease of the TAC during the rehabilitation period. As a consequence, it could be supposed that there are other sources of free radicals that continue to generate reactive oxygen species. One possible source is represented by the leukocytes that remain chronically activated during the postacute phase after stroke, contributing to further enhancement of oxidative stress.²⁸ This is supported by a previous study made in our laboratory that indicated an inflammatory response in the same study group and blood cell activation.²⁹

AOPPs were used to assess the intensity of oxidative stress in some other pathological conditions (i.e. uraemia, sepsis, heart failure).^30,31 To our knowledge, there is only one study that investigated the concentration of AOPPs in stroke patients.³² Despite the fact that in our study AOPP concentration was increased in Cases (t ₀) when compared with healthy controls, the difference did not attain statistical significance. Moreover, the decrease of AOPP levels during the rehabilitation period did not have statistical significance. This could be due to the shortness of the hospitalization period. A similar situation was found in the case of 3NT, a marker of nitrosative stress. Despite the decreasing trend observed after the rehabilitation, we failed to detect any statistical significance. This situation could be explained by the short period of rehabilitation and also by the low number of participants enrolled in our study.

In our study, BI values were significantly positively correlated with GGT activity at the hospitalization moment (Cases, t ₀); at the discharge moment (Cases, t ₁), BI values were significantly positive correlated with both NPTs and LDLox particles. These results may suggest a relationship between redox status in postacute stroke patients undergoing rehabilitation and their recovery during a short time period of hospitalization. As previously mentioned, interpretation of these data should be made with caution due to the short period of rehabilitation and the low number of patients.

In summary, our study indicates the persistency of a redox imbalance in postacute stroke-patients. GGT activity and ceruloplasmin concentration, both affecting the TAC of the blood, seem to be important factors in the aetiology of this redox imbalance. Beside these two factors, there are probably some others, like chronically activated leukocytes. Our results suggest that a well-controlled antioxidant-based therapy could be beneficial for the recovery of postacute stroke patients undergoing rehabilitation. These are in agreement with the results obtained by other clinical trials that used antioxidants in the treatment of acute stroke patients. Ebselen, a glutathione peroxidase-like compound, and edaravone are synthetic antioxidants that proved to be efficient in the correction of redox status in acute stroke patients facilitating their recovery.^33,34 Moreover, the administration of a lipoic acid-based nutritional supplement during a period of two months to postacute stroke patients undergoing rehabilitation was beneficial for the correction of their redox status (unpublished results).

DECLARATIONS

Competing interests: None.

Funding: None.

Ethical approval: The ethics committee of Elias Emergency Hospital approved this study (1289/10.06.2009).

Guarantor: BNM.

Contributorship: BNM and EO made experimental assays. MB and LD carried out patient recruitment and obtained their consent. SV and OP assessed oxidized LDL particles and 3-nitro-l-tyrosine. NC carried out the statistical analysis. BNM and OI wrote the paper.

Acknowledgement: The kit for the assessment of 3-nitrotyrosine was brought through a donation by AR-Med Foundation.

References

Butler

, Bahr

. Oxidative stress and lysosomes: CNS-related consequences and implications for lysosomal enhancement strategies and induction of autophagy. Antioxid Redox Signal 2006;8:185–96

Kontos

. Oxygen radicals in cerebral ischemia: the 2001 Willis lecture. Stroke 2001;32:2712–16

Lipton

. Ischemic cell death in brain neurons. Physiol Rev 1999;79:1431–1586

Yenari

. Pathophysiology of acute ischemic stroke. Clev Clin J Med 2004;71:S25–S27

Franzini

, Corti

, Martinelli

, γ-Glutamyltransferase activity in human atherosclerotic plaques – biochemical similarities with the circulating enzyme. Atherosclerosis 2009;202:119–27

Forman

, Liu

, Tian

. Glutathione cycling in oxidative stress. Lung Biol Health Dis 1997;105:99–121

Stark

, Zeiger

, Pagano

. Glutathione metabolism by γ-glutamyl transpeptidase leads to lipid peroxidation: characterization of the system and relevance to hepatocarcinogenesis. Carcinogenesis 1993;14:183–89

Paolichi

, Tongiani

, Tonarelli

, Comporti

, Pompella

. Gamma-glutamyl transpeptidase-dependent lipid peroxidation in isolated hepatocytes and HepG2 hepatoma cells. Free Radic Biol Med 1997;22:853–60

Drozdz

, Parmentier

, Hachad

, Leroy

, Siest

, Wellman

. γ-Glutamyl transferase dependent generation of reactive oxygen species from a glutathione/transfering system. Free Radic Biol Med 1998;25:786–92

10.

Paolicchi

, Minotti

, Tonarelli

, Gamma-glutamyl transpeptidase-dependent iron reduction and low density lipoprotein oxidation: a potential mechanism in atherosclerosis. J Invest Med 1999;47:151–60

11.

Emdin

, Pompella

, Paolicchi

. Gamma-glutamyltransferase, atherosclerosis, and cardiovascular disease: triggering oxidative stress within the plaque. Circulation 2005;112:2078–80

12.

Ruttmann

, Brant

, Concin

, Diem

, Rapp

, Ulmer

. Gamma-glutamyltransferase as a risk factor for cardiovascular disease mortality: an epidemiological investigation in a cohort of 163 944 Austrian adults. Circulation 2005;112:2130–37

13.

Wannamethee

, Lennon

, Shaper

. The value of gamma-glutamyltransferase in cardiovascular risk prediction in men without diagnosed cardiovascular disease or diabetes. Atherosclerosis 2008;201:168–75

14.

Korantzopoulos

, Tzimas

, Kalantzi

, Association between serum gamma-glutamyltransferase and acute ischemic nonembolic stroke in elderly subjects. Arch Med Res 2009;40:582–89

15.

Ehrenwald

, Chisolm

, Fox

. Intact human ceruloplasmin oxidatively modifies low density lipoprotein. J Clin Invest 1994;93:1493–501

16.

Shukla

, Maher

, Masters

, Angelini

, Jeremy

. Does oxidative change ceruloplasmin from a protective to a vasculopathic factor? Atherosclerosis 2006;187:238–50

17.

Mahoney

, Barthel

. Functional evaluation: the Barthel index. Md State Med J 1965;24:61–9

18.

Hawkins

, Morgan

, Davies

. Quantification of protein modification by oxidants. Free Radical Biol Med 2009;46:965–88

19.

Witko-Sarsat

, Friedlander

, Capeillere-Blandin

, Advanced oxidation protein products as a novel marker oxidative stress in uremia. Kidney Int 1996;49:1304–13

20.

Himmelfarb

, McMonagle

, McMenamin

. Plasma protein thiol oxidation and carbonyl formation in chronic renal failure. Kidney Int 2000;58:2571–8

21.

Sunderman

, Nomoto

. Measurement of human serum ceruloplasmin by its p-phenylenediamine oxidase activity. Clin Chem 1970;16:903–10

22.

, Pellegrini

, Proteggente

, Pannala

, Yang

, Rice-Evans

. Antioxidant activity applying an improved ABTS radical cationic decolorization assay. Free Radic Biol Med 1999;26:1231–37

23.

Williams

, Maggiore

, Reynolds

, Helgason

. Novel approach for the determination of the redox status of homocysteine and other aminothiols in plasma from healthy subjects and patients with ischemic stroke. Clin Chem 2001;47:1031–39

24.

Sell

, Strauch

, Shen

, Monnier

. 2-Aminoadipic acid is a marker of protein carbonyl oxidation in the aging human skin: effects of diabetes, renal failure and sepsis. Biochem J 2007;404:269–77

25.

Dominici

, Valentini

, Maellaro

, Redox modulation of cell surface protein thiols in U937 lymphoma cells: the role of gamma-glutamyl transpeptidase-dependent H₂O₂ production and S-thiolation. Free Radic Biol Med 1999;27:623–35

26.

Del Bello

, Paolicchi

, Comporti

, Pompella

, Maellaro

. Hydrogen peroxide produced during γ-glutamyl transpeptidase activity is involved in prevention of apoptosis and maintenance of proliferation in U937 cells. FASEB J 1999;13:69–79

27.

Awadallah

, Hamad

, Jbarah

, Salem

, Mubarak

. Autoantibodies against oxidized LDL correlate with serum concentrations of ceruloplasmin in patients with cardiovascular disease. Clin Chim Acta 2006;365:330–36

28.

McCabe

, Harrison

, Mackie

, Platelet degranulation and monocyte-platelet complex formation are increased in the acute and convalescent phases after ischaemic stroke or transient ischaemic attack. Br J Haematol 2004;125:777–87

29.

Manolescu

, Berteanu

, Dumitru

, Dynamics of inflammatory markers in post-acute stroke patients undergoing rehabilitation. Inflammation 2010, DOI: 10.1007/s10753-010-9262-8

30.

Selmeci

, Seres

, Antal

, Lukacs

, Regoly-Merei

, Acsady

. Advanced oxidation protein products (AOPPs) for monitoring oxidative stress in critically ill patients: a simple, fast and inexpensive automated technique. Clin Chem Lab Med 2005;43:294–7

31.

Rabbani

, Thornalley

. Quantitation of markers of protein damage by glycation, oxidation, and nitration in peritoneal dialysis. Perit Dial Int 2009;29:S51–6

32.

Dominguez

, Delgado

, Vilches

, Oxidative stress after thrombolysis-induced reperfusion in human stroke. Stroke 2010;41:653–60

33.

Yamaguchi

, Sano

, Takakura

, Ebselen in acute ischemic stroke: a placebo-controlled, double-blind clinical trial. Stroke 1998;29:12–7

34.

Unno

, Katayama

, Shimizu

. Does functional outcome in acute ischaemic stroke patients correlate with the amount of free-radical scavenger treatment? A retrospective study of edaravone therapy. Clin Drug Investig 2010;30:143–55

Dynamic of oxidative and nitrosative stress markers during the convalescent period of stroke patients undergoing rehabilitation

Abstract

Background

Methods

Results

Conclusions

Introduction

Materials and methods

Study design

Blood collection

Routine analysis

Reagents

Laboratory procedures

Statistical analysis

Results

Routine biochemical analysis

Baseline and dynamics of oxidative and nitrosative stress markers

Correlations among oxidative and nitrosative stress markers at t 0 and t 1

Relations among plasma concentration of oxidative and nitrosative stress markers and Barthel index

Discussion

DECLARATIONS

References

Correlations among oxidative and nitrosative stress markers at t ₀ and t ₁