Systemic lupus erythematosus (SLE) is the prototypical systemic chronic autoimmune disease, characterized by diverse clinical manifestations and production of multiple autoantibodies. Common long-term complications of SLE include damage to the musculoskeletal, neuropsychiatric, renal, and cardiovascular systems (for review, see ref. 1). In recent years, it has been widely appreciated that premature atherosclerosis is a particularly striking feature of the disease (2), especially in women (3); furthermore, traditional risk factors are not believed to fully account for the increased atherosclerosis (2).
Although the cause of SLE is unknown, evidence of a complex genetic contribution has been reported, with an increased incidence in families in which one or more members already has the disease or another autoimmune disease (for review, see ref. 4). Other factors implicated in the development or progression of SLE include altered cytokine levels (5), modulated sex hormone metabolism (6), increased apoptosis (7), and elevated levels of oxidative stress. With respect to oxidative stress, evidence of increased levels of phospholipid oxidation products, particularly in patients with antiphospholipid antibodies (8) has been reported, as well as elevated plasma DNA oxidation products, such as 8-hydroxydeoxyguanosine (8-oxodG), a product that also appears to be processed abnormally (9). Many of the autoantibodies produced in SLE exhibit a preference for oxidized substrates, including oxidized double-stranded DNA (dsDNA) (10) and phospholipids (11). Oxidative stress and antiphospholipid antibodies have also been implicated in the increased atherosclerosis seen in patients with SLE (8). Elevated levels of the oxy radical–producing enzyme xanthine oxidase (12) and decreased levels of the protective enzyme superoxide dismutase and endogenous antioxidants (13) have also been reported; the latter have been suggested to be predictive of SLE onset (14).
Despite this evidence for a role for oxidative stress in SLE, there are few data on the occurrence of protein oxidation. In addition, there is no information on whether the extent of oxidation correlates with disease activity or cumulative organ damage. Such data are required for an assessment of the importance of oxidative damage as a causal agent in disease development, since enhanced oxidative stress may merely be a secondary consequence of chronic inflammation. Proteins would be expected to be major targets for oxidative damage since they are major components of most tissues, cells, and plasma (15) and exhibit rapid rates of reaction with many oxidants (15). Oxidized proteins are known to cause major physiologic perturbations, including loss of structure or function (for review, see ref. 16). The long-lived nature and slow rates of removal of many oxidized proteins (see, for example, refs. 17 and18) may make these materials valuable quantitative markers of oxidative stress. Previous studies have shown elevated levels of protein oxidation products in a number of pathologic conditions in humans, including atherosclerosis, lens cataracts, diabetes mellitus, and neurodegenerative syndromes (for review, see refs. 19 and20). Protein oxidation has been implicated as a cause of the pathology in at least some of these conditions.
In this study, the oxidation of serum proteins was quantified in SLE patients and healthy control subjects. Loss of parent, protein-bound amino acids was examined, as well as generic markers of oxidation (protein carbonyls) and specific side-chain oxidation products (methionine sulfoxide [MetSO], 3,4-dihydroxyphenylalanine [DOPA], dityrosine [di-Tyr], 3-chlorotyrosine [3Cl-Tyr], 3-nitrotyrosine [3NO2-Tyr], and o-tyrosine [o-Tyr]). Levels of the pro-oxidant heme enzyme myeloperoxidase (MPO), which generates the potent oxidant hypochlorous acid among others (21), were also examined, since recent studies found elevated levels in patients with atherosclerosis (22), an important complication of SLE and a major cause of death in people with this disease. We also examined whether the extent of protein oxidation, assessed by this battery of assays and the levels of MPO, correlate with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) and with elevated levels of antibodies to dsDNA, an SLE-specific autoantibody.
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- MATERIALS AND METHODS
The results presented here are consistent with an increased extent of protein oxidation in the serum of patients with SLE as compared with controls, as evidenced by decreased serum protein thiol levels, increased protein-bound carbonyl levels, decreased Met and total Met plus MetSO levels, and increased MetSO levels. The change in concentration of some of these species correlated with disease activity and with anti-dsDNA antibody positivity, with enhanced oxidation occurring in the presence of more severe disease. This may indicate direct causality. Analysis of protein-bound amino acids also revealed significant losses of 2 other amino acids, Arg and Phe, and an increase in Gly residues.
The sulfur-containing amino acids Cys and Met are particularly susceptible to oxidation (for review, see refs. 29 and30), making them sensitive markers of protein modification. Oxidation of Cys and, to a lesser extent, Met may be of particular significance, since these residues play an important role in the catalytic activity of many enzymes (31). Unlike other protein oxidation products, the oxidation of Cys and Met residues can be at least partly reversed. The formation of cystine from Cys can be readily reversed by reductase and isomerase enzymes (15, 30), although there is no evidence for the repair of oxyacids, such as RSO2H and RSO3H, which are formed in competition with cystine (15, 30). MetSO can be reduced to Met by methionine sulfoxide reductase enzymes (15, 32), although such repair does not occur in serum or plasma. Further oxidation of MetSO to the sulfone MetSO2 is irreversible (15, 32), with the formation of this species probably accounting for the observed decrease in the total Met plus MetSO.
It has been hypothesized that the oxidation of Met to MetSO and its subsequent reduction by methionine sulfoxide reductases may be important in the regulation of the biologic activity of proteins and an important antioxidant defense mechanism (32, 33). Oxidation of Met residues in proteins can bring about changes in hydrophobicity and protein unfolding, with resulting loss of function (15, 34). Although free methionine can spontaneously oxidize to MetSO (35), this is unlikely to be a significant problem in the current study, since both the control and the patient samples were treated in the same manner, and this process is known to be modulated by plasma proteins (35). Even if selective artifactual oxidation of Met to MetSO was occurring in the SLE patient samples, the decrease in total levels of Met plus MetSO and the increased loss seen with increased disease activity are still consistent with an enhanced level of oxidative stress in the SLE patients.
Oxidation of Cys residues has been observed in other diseases in which oxidative stress has been implicated, including adult respiratory distress syndrome (36) and coronary artery disease (37). Previous studies have reported decreased serum thiols in SLE patients compared with controls. In early studies (38, 39) a significant decrease in serum thiols was detected in SLE patients, with the latter study also reporting a correlation with increasing disease activity. These studies, however, were conducted before the development of a uniform classification system for SLE or systematic disease activity scales; thus, comparison with the results of the current study is difficult. A later study (12) also reported a loss of thiols, with the values in SLE patients being only one-third those in healthy controls. However, the expression of thiol levels in micromolar concentrations in this study makes the determination of protein thiol levels problematic, owing to variations in serum protein concentrations between patients. An inverse correlation between xanthine oxidase levels and thiols was also detected (12), which is consistent with this enzyme being the cause of the observed oxidative stress.
Low molecular mass thiols are unstable over short periods at physiologic temperatures, as well as over longer periods when frozen (40). For this reason low molecular mass thiols were not assessed in the current study, and the minor contribution from these species to the total serum levels is assumed to be zero. Only the more stable high molecular mass protein thiols that make up the majority of the total thiols in serum were analyzed here. Thiol-conserving agents (41) were not used in the current study because they are not consistent with the other assays we used. Similarly, total thiol analysis after reduction (40) was not considered because this methodology does not provide information on the extent of oxidation.
Elevated levels of protein-bound carbonyls have previously been detected in patients with diabetes mellitus (42) and in plasma and tracheal aspirates from preterm infants (43, 44). The increase in carbonyls detected in SLE patients in the current study is small when compared with the findings of some previous studies, possibly as a result of the absence of fibrinogen in these serum samples, since this protein has previously been reported to be highly susceptible to carbonyl formation in in vitro studies (45). A correlation between protein carbonyl levels and MPO concentrations in preterm infants has been reported previously (44), suggesting that this enzyme plays a role in the observed oxidation. In contrast, in the current study, a small, but significant, decrease in MPO levels was observed in the SLE patients, suggesting that the activity of this enzyme is not the cause of the observed enhanced oxidation. A previous study of patients with vasculitis and patients with autoimmune diseases associated with vasculitis including SLE, also reported a decrease in serum MPO levels in SLE patients compared with controls, although no statistical analysis was performed (46).
The increase in protein-bound carbonyls reported here may be an underestimation of the total yield of carbonyls formed, since it is known that protein oxidation yields both protein-bound and low molecular mass released carbonyls (47, 48); only the former were quantified here. The ratio of bound to released carbonyls is dependent on the oxidant (48), and so, the total carbonyl yield cannot be extrapolated from previous data, since the oxidants involved in SLE are not known. The released carbonyls arise from fragmentation reactions of alkoxyl radicals generated on aliphatic side chains on proteins (47, 48). When such reactions occur at the β-carbon (the first carbon of the side chain), an α-carbon radical is formed. Subsequent reaction of this species with a hydrogen atom donor results in the formation of an additional Gly residue. This type of reaction may account for the increase in protein-bound Gly residues detected in the SLE patients.
The lack of significant increases in the Tyr oxidation products DOPA, di-Tyr, and 3Cl-Tyr are consistent with the total amino acid analysis data (Table 2), where no significant loss of parent tyrosine (p-Tyr) was observed. Levels of serum 3NO2-Tyr have been reported to be significantly increased in SLE patients compared with controls (49, 50). In the current study, the observed increase in this product did not correlate with increasing disease activity. The nonsignificant increase in the Phe oxidation product o-Tyr is in contrast to the significant loss of the parent amino acid, as determined by total amino acid analysis. This is consistent with previous studies that have shown that o-Tyr is not the sole oxidation product from this species, with multiple hydroxylated isomers and dimeric species being detected (30). Although Arg residues react rapidly with some oxidants (e.g., hydroxyl radicals ), it is likely that the observed decrease in this side chain arises via other mechanisms, since other residues that also react rapidly with hydroxyl radicals (e.g., Tyr, Trp, and His) (51) did not decrease in concentration. One potential route to loss of Arg residues is via reaction with carbonyl compounds generated by glycation/glycoxidation reactions or lipid oxidation (19); some of these species react rapidly with Arg residues (52). This has not been explored further.
The correlation between the various markers of protein oxidation examined and anti-dsDNA antibody positivity is not as strong or as significant as the correlation between these markers and the SLEDAI scores. This is not surprising, given that only 50–80% of SLE patients have elevated levels of these autoantibodies (for review, see ref. 53). However, levels of these antibodies have been reported to be a good predictor of disease exacerbation over time (54), a factor that was not examined in the present study. Use of the SLEDAI score, in contrast, enables the disease activity to be assessed at a fixed point in time and allows superior comparison between patients. Future studies should include correlations of SLEDAI scores and anti-dsDNA antibody levels with protein oxidation markers in a longitudinal followup.
The changes in protein-bound amino acids detected in this study suggest either that oxidation is occurring continuously in SLE patients at an elevated level compared with controls or that oxidation occurs in acute bursts, with inefficient repair of these lesions once they are formed. Since the major protein in serum is albumin and since this has a rapid turnover (15–20 days ), the current data are consistent with a chronically elevated level of oxidative stress in patients with SLE. This suggestion is consistent with the enhanced levels of oxidation detected in patients with higher levels of disease activity and suggests that measurement of protein oxidation parameters may be a useful surrogate marker of disease activity. Whether such measurements can be used in a prognostic manner remains to be established. Longitudinal studies of levels of protein oxidation with cumulative end-organ damage are essential to determine if protein oxidation is a major pathogenic mechanism in SLE.