Background/Purpose:

Sjögren’s syndrome (SS) is a chronic inflammatory, autoimmune disorder characterized by diminished lacrimal and salivary gland secretion resulting in keratoconjunctivitis sicca and xerostomia. Autoantibodies, directed against 60 kD Ro protein is found in up to 90% of patients with SS. Free radical mediated oxidative damage has not been well characterized previously in SS. Therefore, we studied oxidative damage (conjugate diene formation) and modification of serum proteins by the lipid peroxidation by-product 4-hydroxy-2-nonenal (HNE) or malondialdehyde (MDA) in SS and age-and sex matched controls.

Methods:

Sixty nine primary SS subjects, 25 age and sex matched subjects that do not meet criteria (incomplete SS), and 18 normal controls were studied. We studied indices of oxidative damage, namely conjugate diene formation, and HNE or MDA-protein adducts in the sera of SS, incomplete SS and normal controls. Sera from SS subjects or normal controls were coated on ELISA plates as antigen. HNE or MDA adducts in serum proteins was determined with rabbit anti-HNE or anti-MDA antibodies purchased commercially. Sera from SS or normal controls were electrophoresed, transferred to nitrocellulose by electroblotting and subjected to immunoblotting with rabbit anti-HNE antibody. For determination of conjugate diene, 25 µl of SS or incomplete SS sera were extracted with chloroform:methanol (2:1) and the samples were centrifuged. Two ml of the clear supernatant was evaporated to dryness at 45˚C and reconstituted in one ml methanol. The spectra ranging from 200 to 360 nm was read using a spectrophotometer.

Results:

We found significantly increased oxidative damage in the sera of primary SS subjects compared to normal controls by ELISA and immunoblotting. Serum proteins from SS subjects were found to contain HNE adducts. There was significantly more HNE-modified proteins in SS sera (n=10) compared to controls (n=10; age and sex matched) by ELISA (0.074 ± 0.017 versus 0.046 ± 0.007; p=0.00015; average OD±SD). However, there was no significant difference in MDA-modified proteins between SS and controls by ELISA. When SS sera (n=34) were analyzed by immunoblotting, we found HNE adducts in several serum proteins, and significantly in a 18 kD protein. Control sera did not show significant HNE-modification (n=8). Our preliminary results for conjugate diene formation show that there is no significant difference between conjugate diene levels in the Sjogrens’s syndrome patients (n=25) and incomplete SS subjects (n=25). We are pursuing HNE-modification in the sera of incomplete SS subjects and also identifying the protein bands in SS subjects with HNE adducts by matrix assisted time of flight mass spectrometry.

Conclusion:

Significantly elevated HNE- but not MDA-protein adducts occur in the sera of SS subjects compared to normal controls, showing that oxidative damage occurs in SS.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/significantly-elevated-serum-protein-adduction-with-4-hydroxy-2-nonenal-but-not-malondialdehyde-in-sjogrens-syndrome/