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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

To examine protein oxidation in systemic lupus erythematosus (SLE) and to correlate levels of protein oxidation products with disease activity.

Methods

Serum was collected from SLE patients and healthy control subjects. Protein-bound carbonyls and the pro-oxidant enzyme myeloperoxidase (MPO) were quantified by enzyme-linked immunosorbent assay. Protein thiols were quantified using 5,5′-dithionitrobenzoic acid. Protein-bound amino acids and methionine, tyrosine, and phenylalanine oxidation products were quantified by acid hydrolysis and high-performance liquid chromatography. Disease activity was assessed by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Levels of anti–double-stranded DNA (anti-dsDNA) antibodies were measured by radioimmunoassay.

Results

Compared with control subjects, SLE patients exhibited elevated levels of protein carbonyls (0.108 ± 0.078 versus 0.064 ± 0.028 nmoles/mg of protein; P = 0.046), decreased levels of protein thiols (3.9 ± 1.1 versus 4.9 ± 0.7 nmoles/mg of protein; P = 0.003), decreased levels of protein-bound methionine (P = 0.0007), and increased levels of protein-bound methionine sulfoxide (P = 0.0043) and 3-nitrotyrosine (P = 0.0477). SLE patients with high SLEDAI scores or elevated anti-dsDNA antibody levels exhibited increased oxidation compared with patients with low SLEDAI scores or low antibody levels. Serum MPO levels were decreased in SLE patients (P = 0.03), suggesting that this enzyme is not responsible for the enhanced protein oxidation.

Conclusion

We found elevated levels of multiple markers of protein oxidation in sera from SLE patients compared with controls, and these levels correlated with disease activity. The findings suggest that protein oxidation may play a role in the pathogenesis of chronic organ damage in SLE.

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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Materials.

Chemicals were obtained from Sigma-Aldrich (Castle Hill, New South Wales, Australia) unless noted otherwise. High-performance liquid chromatography (HPLC) solvents were purchased from EMD Chemicals (Merck, Kilsyth, Victoria, Australia) and were filtered before use through VacuCap 90 filter units with 0.2-μm Supor membranes (Pall, Cheltenham, Victoria, Australia). Water used in all experiments was passed through a 4-stage Milli Q system. Phosphate buffer solutions were pretreated with Chelex 100 resin (Bio-Rad, Hercules, CA) to remove trace metal ions.

Patients and controls.

This study was approved by the Human Ethics Committee of The St. George Hospital. All SLE patients were outpatients of the hospital Rheumatology Department, and all were classified as having SLE, as defined by the American College of Rheumatology 1997 revised criteria (23). Controls were Rheumatology Department outpatients with miscellaneous (nonautoimmune) complaints and staff of the Rheumatology Department or the Heart Research Institute. The demographics of the controls and SLE patients, including renal status and drug therapy in the SLE patients, are given in Table 1.

Table 1. Demographics of the SLE patients and controls, categorized by protein marker*
Study group, marker of interestNo. of women/ menAge, mean ± SDRace/ethnicity, %SLEDAI scoreAnti- dsDNA, IU ml−1Medications, %No. with impaired renal function
WhiteAsianArabicOtherImmuno- suppressivesCortico- steroidsAnti- malarialsCOX-2 inhibitorsAnti- coagulants
  • *

    SLE = systemic lupus erythematosus; SLEDAI = Systemic Lupus Erythematosus Disease Activity Index; anti-dsDNA = anti–double-stranded DNA; COX-2 = cyclooxygenase 2.

SLE patients              
 Protein thiols24/142.6 ± 15.356.032.04.08.03.6 ± 2.828.4 ± 43.624.036.036.016.016.01
 Protein carbonyls24/241.7 ± 13.850.038.53.87.74.5 ± 3.840.2 ± 100.826.942.334.615.411.51
 Amino acid analysis25/142.8 ± 15.053.834.63.87.73.7 ± 2.828.3 ± 42.723.138.538.515.415.41
 Tyr oxidation products17/245.1 ± 15.952.642.15.305.1 ± 4.946.0 ± 121.321.136.821.1015.82
 Myeloperoxidase19/042.5 ± 15.052.636.85.35.33.6 ± 2.927.5 ± 41.721.136.836.815.815.81
Control patients              
 Protein thiols12/043.1 ± 10.883.38.38.30        
 Protein carbonyls10/040.8 ± 7.980.010.010.00        
 Amino acid analysis8/049.1 ± 15.975.012.512.50        
 Tyr oxidation products14/140.3 ± 9.686.76.76.70        
 Myeloperoxidase11/043.7 ± 12.272.79.118.20        

Patient and control groups were matched as closely as possible with regard to age, sex, and race. There were no statistically significant differences in the ages of the subjects, either between or within the SLE patient and control groups, by two-way analysis of variance (ANOVA), or in the disease activity markers in the various SLE patient groups (categorized by SLEDAI score and anti-dsDNA antibodies) by one-way ANOVA.

Blood collection and serum preparation.

Venous blood was collected into plain clot tubes, and after ∼30 minutes, serum was prepared by centrifugation at 3,500 revolutions per minute for 10–15 minutes. Since only small volumes of serum were available, not all experiments were performed on all samples/subjects.

Measurement of disease activity.

Disease activity was assessed, at the time of blood sampling, using the SLEDAI. This instrument, which has been validated extensively (24), examines 9 organ systems, and weighted scores are assigned according to disease severity. All patients were assessed by the same clinician (ADS), who has extensive experience in the use of this scale. A SLEDAI score of ≥6 was taken as an indicator of high levels of disease activity (25).

Measurement of anti-dsDNA antibody.

Anti-dsDNA autoantibodies were determined as part of the routine assessment of all patients with SLE. A commercially available anti-dsDNA kit in which serum antibodies to dsDNA are bound to 125I-labeled recombinant DNA (Diagnostic Products, Los Angeles, CA) was used according to the manufacturer's instructions. The upper limit of the 95th percentile of anti-dsDNA antibody levels in a reference population of healthy adults is 4.2 IU ml–1. Patients positive for anti-dsDNA autoantibodies (>4.2 IU ml–1) were compared with those who were negative for these antibodies (≤4.2 IU ml–1).

Determination of protein concentrations.

Protein concentrations were determined by the bicinchoninic acid assay (Pierce, Rockford, IL) using 96-well plates, with incubation at 60°C for 30 minutes. Absorbance was measured at 562 nm using a 96-well plate reader (Tecan, Grödig, Austria) and was converted to absolute values using bovine serum albumin (BSA) standards (0–1 mg ml–1).

Determination of serum protein thiol levels.

Protein thiols were quantified spectrophotometrically using 5,5′-dithionitrobenzoic acid (DTNB) (26). Briefly, using 96-well plates, 1:2 and 1:4 dilutions of freshly thawed serum were prepared to a final volume of 10 μl. To these preparations was added either 200 μl of freshly prepared 500 μM DTNB in 100 mM phosphate buffer, pH 7.4, or 200 μl of buffer alone. Following incubation in the dark for 30 minutes at 21°C, release of 5-thiobenzoic acid was quantified by absorbance at 412 nm and converted to absolute values using reduced glutathione standards (0–0.5 mM). The absorbance of samples without added DTNB was subtracted to account for background absorbance at 412 nm. Samples were analyzed in triplicate.

Determination of serum protein carbonyl levels.

Carbonyl concentrations were determined by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Zenith Technology, Dunedin, New Zealand), according to the manufacturer's instructions. Proteins treated with 2,4-dinitrophenylhydrazine were adsorbed onto the ELISA plate overnight at 4°C, and the acidic stop solution was applied for 10 minutes. Absorbance at 450 nm was then measured. Samples and standards were assayed in duplicate.

Determination of serum MPO levels.

MPO was quantified using a commercial sandwich ELISA (Oxis Research, Portland, OR), according to the manufacturer's instructions. Samples standards were prepared in duplicate and analyzed at the same dilution (1:20).

Preparation of samples for protein hydrolysis.

Protein samples (free of free amino acids) were prepared for hydrolysis as described previously (for review, see ref. 20). Two methods using methanesulfonic acid (MSA) and HCl were employed that used different amounts of protein, which are shown here as MSA hydrolysis volume/HCl hydrolysis volume. Briefly, 5/60-μl serum samples were aliquotted into 8 × 40–mm clear vials (Alltech, Baulkham Hills, New South Wales, Australia) in triplicate. These were diluted with 100/560 μl of 100 mM phosphate buffer, pH 7.4, and 5/10 μl of freshly prepared 10 mg ml–1 sodium borohydride (Merck) was added to remove peroxides. MetSO is not affected by this treatment (Hawkins CL: unpublished observations). After incubation for 5–10 minutes at 21°C, samples were delipidated by adding 5/50 μl of 0.3% (weight/volume) sodium deoxycholate, and proteins were precipitated with 10/100 μl of 10% trichloroacetic acid, followed by centrifugation at 4,300g for 2 minutes. The protein pellets were then washed twice with 500 μl of ice-cold acetone, repelleted by centrifugation at 7,000g for 2 minutes, and dried by vacuum centrifugation for 10–15 minutes.

Protein hydrolysis with MSA and amino acid analysis.

Protein pellets were hydrolyzed using 150 μl of 4M MSA (27) containing 0.2% (w/v) tryptamine per vial. Eighty nanomoles of L-homoarginine (Fluka, Buchs, Switzerland) was added as an internal standard. The vials were placed in Pico-Tag reaction vessels (Alltech) and subjected to 3 cycles of purging with nitrogen gas and evacuation. The samples were then incubated for 17 hours at 110°C, neutralized with 150 μl of 4M sodium hydroxide (ICN Biomedicals, Aurora, OH), and filtered through 0.45-μm Nanosep MF GHP centrifugal devices (500-μl sample capacity; Pall). Samples were then diluted 50-fold in water, stored at 4°C, and analyzed by HPLC within ∼24 hours.

Analysis was performed by reverse-phase HPLC using a Zorbax ODS 5-μm, 4.6 × 250–mm column (Agilent, Forest Hill, Victoria, Australia) and a Supelco Pelliguard LC-18 2-cm guard cartridge (Sigma-Aldrich). Samples were derivatized using an autoinjector system (Shimadzu Oceania, Rydalmere, New South Wales, Australia), which added 20 μl of an o-phthaldialdehyde (1 ml)/β-mercaptoethanol (5 μl) solution to 40 μl of sample. Fifteen microliters of the derivatized sample was injected into the column after 1 minute.

Samples were separated (flow rate 1 ml · minute–1) using a gradient consisting of buffer A with 5% buffer B for 7 minutes, 5–25% buffer B over 10 minutes, 25–45% buffer B over 2 minutes, 45–50% buffer B over 8 minutes, 50–58% buffer B over 8 minutes, 58–100% buffer B over 5 minutes, 100% buffer B for 5 minutes, 100–5% buffer B over 1 minute, and reequilibration at 5% buffer B for 9 minutes. Buffer A consisted of 20% methanol and 2.5% tetrahydrofuran (THF) in 20 mM sodium acetate (BDH-Merck, Sydney, New South Wales, Australia), pH 5.4. Buffer B consisted of 80% methanol and 2.5% THF in 20 mM sodium acetate, pH 5.4. Buffers were degassed by sonication under vacuum and sparged with helium during chromatography. Derivatized amino acids were detected by fluorescence (excitation and emission spectra λex 340 nm and λem 440 nm) and quantified using standards containing added L-homoarginine and L-methionine sulfoxide.

Validation of the methanesulfonic acid protein hydrolysis method.

Validation experiments were conducted with BSA (owing to its sequence homology with human serum albumin), lysozyme, and human plasma. Hydrolysis at 130°C for 16 hours resulted in significant loss of Met and Trp residues (∼33% and 20% respectively), whereas hydrolysis at 110°C for 17 hours gave good recovery (>75% for Met and >85% for all other amino acids except Cys and cystine). Spiking experiments with added MetSO revealed slightly greater Met concentrations than expected, which is consistent with a low extent of conversion of MetSO to Met. Spiking also demonstrated a low extent of artifactual oxidation of Met to MetSO; this was quantified as 8–13% when Met was hydrolyzed alone, with a further 2–10% unaccounted for. Approximately 20% of MetSO was converted back to Met when MetSO was hydrolyzed alone, with a further 10% unaccounted for (data not shown). These data suggest that under the conditions used, the proportion of total Met plus MetSO that is present as MetSO is slightly underestimated.

Protein hydrolysis with hydrochloric acid and product analysis.

Hydrolysis using HCl was performed essentially as described previously (20); this method does not produce significant artifactual chlorination in our laboratory (28). Vials containing pretreated serum were placed in Pico-Tag reaction vessels containing 1 ml of 6M HCl (BDH-Merck) and 50 μl of mercaptoacetic acid, evacuated, then incubated at 110°C for 18 hours. The samples were then dried by vacuum centrifugation for 1 hour, redissolved in 200 μl of water, and filtered through 0.45-μm Nanosep MF GHP centrifugal devices. Samples were maintained at 4°C prior to HPLC analysis, which was performed within ∼24 hours.

Parent amino acids and Tyr and Phe oxidation products were separated by reverse-phase HPLC as described above. Samples (20 μl) were injected and eluted using a gradient consisting of buffer A with 2% buffer B for 20 minutes, 2–50% buffer B over 30 minutes, 50% buffer B for 5 minutes, 50–2% buffer B over 1 minute, and reequilibration at 2% buffer B for 4 minutes, where buffer A was 100 mM sodium perchlorate in 10 mM orthophosphoric acid and buffer B was 80% methanol in water. Buffers were degassed before and during use.

Compounds were identified by serial ultraviolet (280 nm) and fluorescence (λex 280 nm and λem 320 nm for 0–31 minutes; λex 280 nm and λem 410 nm for the remainder) detection. DOPA, o-Tyr, and di-Tyr were quantified by fluorescence; p-Tyr, 3Cl-Tyr, and 3NO2-Tyr were quantified by ultraviolet absorption. Data are expressed as micromoles of product per mole of p-Tyr.

Statistical analysis.

Statistical analysis was performed using GraphPad Prism version 4.0b for Macintosh software (GraphPad Software, San Diego, CA). Student's t-test was used to compare one condition with its corresponding control. For multiple conditions, one-way ANOVA was used, with Newman-Keuls post hoc test. Where multiple conditions in different groups were compared, a two-way ANOVA was used, with Bonferroni posttests. In all cases, P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Serum levels of protein thiols.

Protein thiol levels were significantly decreased in SLE patients compared with controls (3.9 ± 1.1 versus 4.9 ± 0.7 nmoles/mg protein; P = 0.003) (Figure 1A). Thiol concentrations correlated with the activity of disease, as measured the SLEDAI (Figure 1B). Lower levels of thiols were found in patients with high levels of disease activity (SLEDAI score ≥6) as compared with patients with low levels of disease activity (SLEDAI score <6). No statistically significant difference was detected between thiol levels in patients who were negative (≤4.2 IU ml–1) and those who were positive (>4.2 IU ml–1) for anti-dsDNA antibodies (Figure 1C).

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Figure 1. Serum protein thiol levels in individual control subjects and patients with systemic lupus erythematosus (SLE) and correlation with disease activity, as determined by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score, and anti–double-stranded DNA (anti-dsDNA) antibody levels. A, Thiol levels in controls versus SLE patients. B, Thiol levels in controls versus SLE patients with low (SLEDAI score <6) and high (SLEDAI score ≥6) levels of disease activity. C, Thiol levels in controls versus SLE patients with (>4.2 IU ml–1) and without (≤4.2 IU ml–1) anti-dsDNA antibodies. = P < 0.05; ∗∗ = P < 0.01, by 1-tailed t-test in A and by one-way analysis of variance with Newman-Keuls post hoc test in B and C.

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Serum levels of protein carbonyls.

Carbonyl levels were significantly increased in SLE patients compared with controls (0.108 ± 0.078 nmoles/mg of protein versus 0.064 ± 0.028 nmoles/mg of protein; P = 0.046) (Figure 2A). When these data were stratified according to low and high levels of disease activity as measured by the SLEDAI, the statistical significance was lost because of the small sample sizes (Figure 2B). Nevertheless, a trend toward increasing protein carbonyl concentrations with increasing disease activity was evident. We found no difference for SLE patients who were positive or negative for anti-dsDNA antibodies (data not shown).

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Figure 2. Serum protein carbonyl levels in individual control subjects and patients with systemic lupus erythematosus (SLE) and correlation with disease activity, as determined by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score. A, Carbonyl levels in controls versus SLE patients. B, Carbonyl levels in controls versus SLE patients with low (SLEDAI score <6) and high (SLEDAI score ≥6) levels of disease activity. = P < 0.05 by 1-tailed t-test.

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Serum levels of MPO.

MPO levels were significantly decreased in SLE patients compared with controls (86 ± 46 ng/ml of serum versus 140 ± 84 ng/ml of serum; P = 0.028). A trend toward decreased MPO levels with increasing disease activity, as indicated by low versus high SLEDAI scores and by negative versus positive anti-dsDNA antibody levels, was observed, but these differences were not statistically significant (data not shown).

Analysis of protein-bound amino acids and specific side-chain oxidation products.

Chromatograms from the protein-bound amino acid analysis experiments, showing a typical SLE patient and a typical healthy control subject, are presented in Figure 3. As can be seen, some of the amino acid peaks were off the scale and, therefore, not all amino acids could be analyzed. This was done in order to maximize the size of the smaller peaks of interest, notably, Met, MetSO, and Trp.

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Figure 3. Representative chromatograms of hydrolyzed and derivatized amino acids from serum proteins obtained from A, a patient with systemic lupus erythematosus (SLE) and B, a healthy control subject, as determined by high-performance liquid chromatography (HPLC). Serum proteins were hydrolyzed with methanesulfonic acid, with subsequent amino acid analysis by HPLC and fluorometric detection of derivatized amino acids (see Materials and Methods for details). The amino acids analyzed in this study (see Table 2) are labeled using the single-letter amino acid codes, except for M(O), which represents methionine sulfoxide, and hR, which represents the homoarginine internal standard. Short thick arrows indicate changes in methionine and methionine sulfoxide levels.

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Statistically significant decreases in levels of Met, increases in MetSO and 3NO2-Tyr, and decreases in total Met plus MetSO were detected in SLE patients compared with controls (Table 2). Correlation of the Met oxidation data with disease activity (SLEDAI score) and anti-dsDNA antibody levels revealed increasing oxidation with increasing disease activity and anti-dsDNA positivity (Figure 4). With the exception of 3NO2-Tyr, no statistically significant changes were observed for any of the Tyr (DOPA, di-Tyr, 3Cl-Tyr) or Phe (o-Tyr) oxidation products quantified. A small increase in o-Tyr levels in SLE patients failed to achieve significance, although a significant loss of the parent amino acid (Phe) was detected (Table 2). In addition, statistically significant increases in Gly levels and significant decreases Arg levels were observed in SLE patients compared with controls. The changes in Arg and Phe levels also correlated with disease activity and anti-dsDNA antibody levels.

Table 2. Amino acid analysis of serum proteins from SLE and control patients by HPLC*
Amino acid/oxidation productSLEControlPIncrease or decrease in SLE
  • *

    Methanesulfonic acid hydrolysis was used for amino acid and methionine sulfoxide analyses, with detection by derivatization with o-phthaldialdehyde and fluorometric detection. Amino acid and methionine sulfoxide concentrations are expressed as the mean ± SD moles of amino acid/mole of isoleucine. Tyr and Phe oxidation products are expressed as the mean ± SD μmoles of product/mole of p-Tyr. Statistical analyses were performed using a 1-tailed t-test when an increase or decrease was predicted before analysis was performed; otherwise, a 2-tailed t-test was used. SLE = systemic lupus erythematosus; HPLC = high-performance liquid chromatography.

  • By 1-tailed t-test.

  • HCl hydrolysis was used for analysis of tyrosine (3-chlorotyrosine, dityrosine, 3,4-dihydroxyphenylalanine, and 3-nitrotyrosine) and phenylalanine (o-tyrosine) oxidation products, with ultraviolet and fluorescence detection as described in Materials and Methods.

Arginine2.169 ± 0.1182.286 ± 0.0740.0133Decrease
Glycine2.433 ± 0.1092.297 ± 0.1280.0056Increase
Histidine1.456 ± 0.0731.514 ± 0.0690.0550No change
Lysine4.608 ± 0.3354.795 ± 0.3530.1816No change
Methionine0.348 ± 0.0610.431 ± 0.0480.0007Decrease
Methionine sulfoxide0.321 ± 0.0480.262 ± 0.0620.0043Increase
Methionine + methionine sulfoxide0.668 ± 0.0310.693 ± 0.0230.0234Decrease
Phenylalanine2.373 ± 0.1372.513 ± 0.1120.0135Decrease
 o-tyrosine65.9 ± 29.560.1 ± 18.00.5152No change
Threonine3.651 ± 0.1413.597 ± 0.1010.3239No change
Tryptophan0.405 ± 0.0310.384 ± 0.0250.0926No change
Tyrosine1.903 ± 0.0731.913 ± 0.0540.7222No change
 3-chlorotyrosine49.1 ± 17.743.3 ± 13.80.3083No change
 Dityrosine1.378 ± 0.8431.423 ± 0.5940.8644No change
 3,4-dihydroxyphenylalanine84.2 ± 29.274.6 ± 16.80.2643No change
 3-nitrotyrosine80.8 ± 16.067.5 ± 21.90.0477Increase
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Figure 4. Serum methionine oxidation levels in individual control subjects and patients with systemic lupus erythematosus (SLE) and correlation with disease activity, as determined by A, C, and E, the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score (<6 indicates low disease activity; ≥6 indicates high disease activity) and by B, D, and F, anti–double-stranded DNA (anti-dsDNA) antibody levels (≤4.2 IU ml–1 indicates negative and >4.2 IU ml–1 indicates positive anti-dsDNA antibodies). Serum proteins were hydrolyzed with methanesulfonic acid, with subsequent amino acid analysis by high-performance liquid chromatography and fluorometric detection of derivatized amino acids (see Materials and Methods for details). Concentrations of amino acids (methionine [Met], methionine sulfoxide [MetSO], and Met plus MetSO combined) are expressed as moles of the given amino acid per mole of Ile. = P < 0.05; ∗∗ = P < 0.01, by one-way analysis of variance with Newman-Keuls post hoc test.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

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 [51]), 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 [55]), 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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors thank the study patients, the staff of the Rheumatology Department laboratory, St. George Hospital, for their assistance, and Dr. Clare Hawkins for helpful discussion.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES