title: Measurement of S-nitrosothiols in extracellular fluids from healthy human volunteers and rheumatoid arthritis patients, using electron paramagnetic resonance spectrometry.
abstract: In human tissues, S-nitrosothiols (RSNOs) are generated by the nitric oxide (NO.)-dependent S-nitrosation of thiol-containing species. Here, a novel electron paramagnetic resonance spectrometry assay for RSNOs is described, together with its application to studies of human health and disease. The assay involves degrading RSNOs using N-methyl-d-glucamine dithiocarbamate (MGD) at high pH and spin trapping the NO. released using (MGD)2-Fe2+. Because dietary nitrate might contribute to tissue RSNOs, the assay was used to monitor the effect of Na15NO3 ingestion on plasma and gastric juice RSNOs in healthy human volunteers. Na15NO3 ingestion (2 mmol) increased gastric RS15NO concentrations (p<0.01), but there was no significant effect on plasma RS15NO concentrations. Having established that dietary nitrate was not a confounding factor, we applied the RSNO assay to matched plasma and knee-joint synovial fluid (SF) from rheumatoid arthritis (RA) patients, with healthy subjects as controls. Clinical markers of RA inflammatory disease activity were quantified, as were plasma and SF NO2- and NO3-. Median RSNO concentrations were 0 (interquartile range 68) nM, 109 (282) nM, and 309 (470) nM in normal plasma, RA plasma, and SF, respectively. The median RSNO concentration was significantly elevated in RA SF compared with RA plasma (p<0.05) and in RA plasma compared with normal plasma (p<0.05). SF RSNO concentrations correlated positively with SF neutrophil counts (rs=0.55, p<0.05) and inversely with blood hemoglobin concentrations (rs=-0.52, p<0.05), but not with NO2- or NO3-. Thus the raised levels of RSNOs in RA SF correlate with some established markers of inflammation, suggesting the described RSNO assay may have applications in rapid clinical monitoring of NO metabolism in human inflammatory conditions.
authors.affiliation: Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London, Queen Mary's School of Medicine and Dentistry, London, UK.
authors.forename: Sophie A
authors.initials: SA
authors.lastname: Rocks
authors.forename: Claire A
authors.initials: CA
authors.lastname: Davies
authors.forename: Siobhán L
authors.initials: SL
authors.lastname: Hicks
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Webb
authors.forename: Rainer
authors.initials: R
authors.lastname: Klocke
authors.forename: Graham S
authors.initials: GS
authors.lastname: Timmins
authors.forename: Atholl
authors.initials: A
authors.lastname: Johnston
authors.forename: Ali S M
authors.initials: AS
authors.lastname: Jawad
authors.forename: David R
authors.initials: DR
authors.lastname: Blake
authors.forename: Nigel
authors.initials: N
authors.lastname: Benjamin
authors.forename: Paul G
authors.initials: PG
authors.lastname: Winyard
mesh_terms: D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D045604:Extracellular Fluid / Q000737:chemistry*; D005260:Female; D005750:Gastric Juice / Q000737:chemistry; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods; D008297:Male; D008875:Middle Aged; D009566:Nitrates / Q000008:administration & dosage / Q000097:blood / Q000378:metabolism; D009573:Nitrites / Q000097:blood; D009587:Nitrogen Isotopes; D026403:S-Nitrosothiols / Q000032:analysis* / Q000097:blood; D013012:Sorbitol / Q000031:analogs & derivatives / Q000737:chemistry; D013113:Spin Labels; D013582:Synovial Fluid / Q000737:chemistry; D013859:Thiocarbamates / Q000737:chemistry
chemical_list: D009566:Nitrates; D009573:Nitrites; D009587:Nitrogen Isotopes; D026403:S-Nitrosothiols; D013113:Spin Labels; D013859:Thiocarbamates; D013012:Sorbitol; C041262:N-methyl-D-glucamine dithiocarbamate; C031618:sodium nitrate