Protein oxidation status in patients with ankylosing spondylitis

C. Yazici, K. Köse, M. Calis1, S. Kuzugüden and M. Kirnap1

Departments of Biochemistry and 1 Physical Medicine and Rehabilitation, Faculty of Medicine, Erciyes University, Kayseri, Turkey.

Correspondence to: C. Yazici, Erciyes Üniversitesi Tip Fakültesi, Biyokimya Anabilim Dali, 38039 Kayseri, Turkey. E-mail: yazici{at}erciyes.edu.tr


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. Ankylosing spondylitis (AS) is a chronic inflammatory disease with unknown aetiology. Since various functions of neutrophils are increased in AS, neutrophil activation-mediated oxidative stress may have an important role in the pathogenesis of AS. Therefore, the importance of neutrophil activation as the main source of oxidative stress was investigated in patients with AS.

Methods. Forty-one patients with AS, divided into active and inactive groups according to their CRP and ESR, and 30 healthy volunteers were entered into the study. The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and the visual analogue scale (VAS) were also used in all patients before the study. In addition, the patients were evaluated according to spinal involvement and peripheral involvement. Plasma myeloperoxidase (MPO) activity, representing neutrophil activation, and oxidative stress biomarkers, such as advanced oxidation protein products (AOPP) and thiol levels, were measured.

Results. There were significant increases in plasma MPO activity and AOPP but decreases in thiol levels in the total AS patient group and in the subgroups, compared with controls. All parameters, except thiol, were higher in active and peripheral involvement groups than in the inactive and spinal patients, respectively; but the highest levels were observed in the peripheral subgroup. In addition, AOPP was found to be positively correlated with ESR, CRP and BASDAI in the total patient group and with white blood cell and neutrophil count in the peripheral involvement subgroup. BASDAI was positively correlated with VAS, ESR and CRP; but MPO was negatively correlated with thiol/albumin ratio, both in total and active AS patients. Negative correlations were also observed between albumin and ESR, and between CRP and neutrophil counts in the peripheral subgroup.

Conclusions. This is the first study suggesting neutrophil-MPO-hypochlorous acid-mediated AOPP formation in AS. Therefore, active neutrophils and chlorinated oxidants of neutrophil origin may be considered to be important factors in the pathogenesis of AS which are related to oxidative stress, notably protein oxidation.

KEY WORDS: Advanced oxidation protein products, Ankylosing spondylitis, Myeloperoxidase, Neutrophil activation, Protein oxidation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ankylosing spondylitis (AS) is a chronic inflammatory disease involving primarily the skeletal system, the peripheral joints and extra-articular structures [1]. Although the pathogenesis of AS is understood adequately, immunomediated mechanisms have been implicated [2, 3]. Furthermore, various functions of neutrophils, such as chemotaxis, phagocytosis and superoxide radical anion generation, have been shown to increase in AS. Consequently, these stimulated neutrophil functions have been considered to be correlated to the pathogenesis of AS [4, 5]. Especially, reactive oxygen species (ROS) have been proposed as possible mediators of tissue damage associated with AS [6, 7].

Increased ROS production depending on an imbalance of oxidant/antioxidant systems, defined as oxidative stress [8], may be related to the pathogenesis of AS. However, the data on AS patients related to oxidant/antioxidant systems are controversial. It has been shown that catalase is higher in active patients but normal in the inactive stage [9], and that there is unchanged superoxide dismutase [9] or catalase activity [10] in all patients. On the other hand, all antioxidant enzymes measured were found to be lower in the study of Mitrofanova et al. [11]. Similarly, levels of malondialdehyde (MDA), one of the lipid peroxidation products, have been reported to be higher [11] or normal [10] in AS patients, or there were increases depending on disease activity [9]. The variability in the MDA results of studies may be due to the short plasma half-lives of lipid peroxidation products.

Because of their relatively early formation, their greater stability and longer lifespans, protein oxidation products have increasingly been used as markers in place of lipid peroxidation products in demonstrating oxidative stress [12, 13]. A novel oxidative stress marker of protein, referred to as advanced oxidation protein products (AOPP), was recently developed in plasma [14]. Beside AOPP, oxidation of plasma thiol groups (termed ‘thiol stress’) located on albumin [8] is quantitatively the major manifestation of protein oxidation [15].

To our knowledge, changes in myeloperoxidase (MPO) activity representing neutrophil activation, AOPP and thiol levels have not been investigated in patients with AS. This study investigated the importance of neutrophil activation as the main source of oxidative stress in the pathogenesis of AS and also whether AOPP may be used to assess disease activity. Therefore MPO activity, AOPP and thiol levels were measured in the plasma of AS patients classified both, as active/inactive, their spinal/peripheral involvement.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
The local ethics committee approved the study protocol. Patients with AS who attended the Physical Medicine and Rehabilitation Department of Erciyes University Medical Faculty were included in the study after they had given informed consent. There were 41 patients, aged 17–76 yr, with mean disease duration of 7.6±5.9 yr. All patients met the modified New York Criteria for the diagnosis of AS [16] and they were in a stable clinical condition. In all patients a complete blood count, routine biochemical analyses, CRP and ESR were performed before the study and none of them had any systemic disease. Other exclusion criteria were excess alcohol consumption, regular vitamin use, a history of intercurrent infection, and systemic corticosteroid treatment. All patients received non-steroidal anti-inflammatory drugs, and treatment with these drugs was stopped 3–4 days before the study commenced.

At the time of the study all patients were divided into two groups, active and inactive, with respect to CRP and ESR. A CRP titre >8 mg/l and ESR >20 mm/h were accepted as indicating the active stage of the disease. The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and a visual analogue scale (VAS) were used for patient and physician assessment of disease activity [17]. The patients were also divided into two groups: those with spinal involvement only (n = 32) and those who also had peripheral arthritis (n = 9). The demographic features of the study groups are presented in Table 1.


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TABLE 1. Demographic features of the patient subgroups

 
A group of 30 healthy controls (24 males and six females), mean age 37.8±8.2 (range 24–58 yr), were also included in the study. None had any systemic disease or received either mineral or vitamin drugs.

Biochemical procedure
Heparinized/EDTA blood obtained from patients and controls was centrifuged at 2000 r.p.m. for 15 min at 4°C. After separating plasma, samples were subdivided into aliquots, which were kept at –70°C until analysed.

Plasma MPO activity was determined by the method of Bradley et al. [18], and was based on kinetic measurement of the formation rate of the yellowish–orange product of the oxidation of o-dianisidine with MPO in the presence of H2O2, at 460 nm. One unit of MPO was defined as that degrading 1 µmol of H2O2 per minute at 25°C. A molar extinction coefficient of 1.13 x 104 M–1 cm–1 of oxidized o-dianisidine was used for the calculation [18]. MPO activity was expressed in units per litre of plasma (U/l).

Determination of AOPP was based on a spectrophotometric assay according to Witko-Sarsat et al. [14]. One millilitre of EDTA–plasma diluted 1:5 with phosphate-buffered saline (PBS, pH 7.4), 1 ml of chloramine-T (0–100 µmol/l) for calibration and 1 ml of PBS as blank were placed on corresponding tubes; 50 µl of 1.16 mol/l potassium iodide (KI, Sigma) was then added to each tube, and 2 min later 200 µl of glacial acetic acid was added. The absorbance of the reaction mixture was read immediately at 340 nm. AOPP levels were expressed in micromoles of chloramine-T equivalents per litre of plasma (µmol/l).

Thiol levels in plasma were determined by the method of Hu et al. [19], based on the thiol disulphide interchange reaction between thiols and 5,5'-dithio bis(2-nitrobenzoic acid) (DTNB). The evaluation was performed using a standard curve for glutathione. Thiol levels were expressed in micromoles per litre of plasma (µmol/l) and also per gram of albumin (µmol/g).

Statistical analysis
Statistical evaluation was carried out with SPSS 10.0 (Statistical Packages for Social Sciences; SPSS, Chicago, IL, USA). The total patient group and the control group were compared with Student's t test. Analysis of variance (ANOVA) and post-ANOVA tests were used in statistical comparisons of the data from subgroups (active/inactive and spinal/peripheral) and controls. In all subgroups, parameters were subjected to Pearson correlation analysis. P<0.05 was considered significant. Data are presented as mean and S.D. in the tables.


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 Materials and methods
 Results
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There was no statistically significant difference in age and sex distribution between control and patients, and also among AS subgroups (Table 1) (P>0.05).

When total patients were divided into two groups, according to their ESR and CRP levels there were 23 active and 18 inactive patients. But when they were divided according to their involvement symptoms, there were 32 spinal and 9 peripheral patients. Therefore, the nine peripheral patients were also included in the active group. Patients in the active and peripheral subgroups had significantly higher ESR, CRP, BASDAI and VAS values than those in the corresponding inactive and spinal patients. Moreover, the peripheral group had the highest ESR, CRP, BASDAI and VAS values (Table 1).

Laboratory data are presented in Table 2. White blood cell (WBC) and neutrophil counts were significantly higher in the total AS patient group and in the active and peripheral subgroups than in controls. When subgroups were compared with each other, only the peripheral involvement group had higher WBC and neutrophil counts. No difference in albumin concentration was observed between the total patient group and the controls or among the patients subgroups (Table 2).


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TABLE 2. Laboratory data of controls and all patients with AS

 
MPO activity and AOPP levels were significantly higher, but plasma thiol levels and thiol/albumin ratios were lower, in the total AS patients and all subgroups, active/inactive and spinal/peripheral, than in the healthy controls. When the patient subgroups were compared with each other, MPO and AOPP levels were higher in the active and peripheral groups than in the inactive and spinal groups, respectively (Table 2). The highest WBC, neutrophil count, MPO and AOPP levels were observed in the peripheral group.

When correlation analyses were performed in the total AS patient group and all subgroups, only the significant correlations between parameters were shown in Table 3. AOPP was positively correlated with ESR, CRP, BASDAI in total patient group, and WBC and neutrophil count in peripheral subgroup. BASDAI was positively correlated with VAS, ESR and CRP; but MPO was negatively correlated with thiol/albumin ratio, both in total and active AS patients. Negative correlations were also observed between albumin and ESR, CRP and neutrophil counts in the peripheral subgroup (Table 3).


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TABLE 3. Correlations between parameters measured in the AS patients

 

    Discussion
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 Materials and methods
 Results
 Discussion
 References
 
Beside BASDAI and VAS, ESR and CRP used as indicators of inflammatory activity in AS have been reported to be increased in patients with active AS and especially in cases of peripheral involvement [20], as seen in the present study. On the other hand radicals are regarded as a major cause of inflammation. Patients with AS have been shown to have increased rates of production in their whole blood either in the resting/stimulating state, suggesting that the priming of phagocytes in the bloodstream is likely to be a causative factor in the onset of AS [5]. An increased neutrophil respiratory burst has also found to be decreased in AS patients by combined radon–hyperthermia treatment [21], or by treatment with phenolic compounds from oak leaves, as used in folk medicine [7], which prevent inflammation. Judging from these findings, phagocytic cell activation and increased production in patients with AS [4–7, 22], one may suggest that such cells, capable of producing ROS, may be responsible for the presence of oxidative stress in AS.

It is well known that activated neutrophils produce ROS and release enzymes such as collagenase, elastase and also MPO, a neutrophil-specific enzyme that catalyses the formation of hypochlorous acid/hypochloride (HOCl/OCl) from hydrogen peroxide (H2O2) and Cl. Because of its greater toxicity and longer lifespan, HOCl reacts with biological targets and leads to oxidative stress and thus can cause tissue damage [23]. In addition, since HOCl activates collagenase released in a latent form [24], activated collagenase may also contribute to tissue damage related to oxidative stress.

Increased plasma MPO activity, along with neutrophil counts in AS patients, especially higher values in active and peripheral groups, were observed in the present study. Although we have not met any study in the literature that deals with plasma MPO activity in AS patients, increased phagocytic neutrophil activity may reflect increased MPO activity, which supports the early studies suggesting the importance of neutrophil activation in AS [4–7, 22, 24], which may lead to oxidative stress. A proposed mechanism is shown schematically in Fig. 1.



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FIG. 1. Neutrophil-MPO-HOCl mediated oxidative stress mechanism in AS.

 
Since the nature of the ROS will play a significant role, it is important to determine which marker to use i.e. lipids, DNA or proteins, in assessing oxidative stress. HOCl induces oxidation of proteins but causes little or no modification of DNA or lipids. Hence, when HOCl, the only in vivo source of which is neutrophils, is the predominant ROS, proteins must be used as the marker [13]. As in the present study, it may be more meaningful to determine protein oxidation in identifying HOCl-mediated oxidative stress in AS patients.

The albumin aggregates with a high molecular mass, 670 kDa (detected by SDS–PAGE), resulting from intermolecular covalent bonds such as disulphide bridges and/or dityrosine cross-linking, undergoing oxidative modification in uraemic patients plasma were defined as AOPP for the first time by Witko-Sarsat et al. [14]. The spectral characteristics of AOPP correspond to several chromophores, which include dityrosine, carbonyls and pentosidine but not nitrotyrosine [25]. Since it has been demonstrated that the principal oxidant responsible for AOPP formation is HOCl [26], Capeillère-Blandin et al. [25] have recently suggested that AOPP, determined routinely using a simple protocol, could be used as a marker to investigate MPO–HOCl-induced oxidative stress.

Like MPO, there is no study in the literature regarding the determination of protein oxidation in patients with AS. AOPP levels were higher in all patients, but the highest level was in the peripheral group in the present study. Furthermore, AOPP was found to be positively correlated with ESR, CRP, BASDAI in the total patient group, and WBC and neutrophil count in the peripheral subgroup. These findings may reflect the presence of a cause-and-effect relationship between the extent of inflammation and neutrophil activation and also HOCl-mediated protein oxidation and thus of oxidative stress in patients with AS (Fig. 1).

The thiol groups, which were found to be low in all subgroups in the present study, are known to be the primary target of HOCl of MPO origin [15] and the most important component of the plasma antioxidant system [27]. Since albumin is the principal protein undergoing MPO-mediated oxidative stress [15], the decrease in thiol levels can be ascribed to the consumption of thiol and indicates not only the insufficiency of the plasma oxidant defence system but also the presence of protein oxidation in patients with AS (Fig. 1).

On the other hand, when it is considered that albumin is the main source of free thiol in the plasma [8], it may be expected that the low in thiols depends on decreased albumin levels. Opposite to this general concept, plasma albumin levels were found to be normal and the changes in plasma thiol and thiol/albumin ratio were similar in all AS patients. Additionally, albumin was negatively correlated with ESR, CRP and neutrophils in the peripheral group, and between MPO and thiol/albumin ratios in the total and active groups may show the extent of inflammation activity in these patients.

In addition, it has been reported that the antioxidant capacity of rheumatoid arthritis synovial fluid is low [28]. A similar mechanism may be suggested for AS: since the protein content of synovial fluid is mainly composed of albumin [29], it may be the major source of free thiols for synovial fluid as well as plasma, and thiols may be the most important component of the antioxidant system in synovial fluid. Therefore, HOCl-mediated thiol oxidation may lead not only to AOPP formation but also to the decrease in antioxidant defence of synovial fluid in AS patients, as seen in Fig. 1.

Apart from these, HOCl has been shown to inhibit some antiproteinases [30], found in normal synovial fluid, and MPO enhances the binding of leucocytes, including monocytes and neutrophils, to endothelium [31]. When these are considered, the importance of the neutrophil–MPO–HOCl system showing multi-action may be better clarified in AS (Fig. 1).

Taking the present findings along with current literature knowledge, it may be suggested that there may be an inflammation and neutrophil activation, leading to oxidative stress in AS patients, as reflected by increased MPO and AOPP and decreased thiol levels. However, it might be said that the extent of inflammation and neutrophil activation may be greater in active and peripheral patients who have higher MPO and AOPP levels.

In conclusion, based on this first study, in which MPO-HOCl-mediated AOPP formation has been demonstrated, it may be suggested that active neutrophils may play an important role in the pathogenesis of AS and that chlorinated oxidants of neutrophil origin may lead to oxidative stress, especially protein oxidation.

The authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 12 March 2004; revised version accepted 18 June 2004.



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