Reductive assays for S-nitrosothiols: implications for measurements in biological systems

Area of Science:

• Biochemistry
• Analytical Chemistry
• Physiology

Background:

• S-nitrosothiols (SNOs) are biologically active molecules found in various tissues.
• Conventional assays for nitric oxide metabolites may lead to misidentification of SNOs, particularly with nitrate (NO-3) reduction methods.

Purpose of the Study:

• To investigate the potential misidentification of S-nitrosothiols in conventional assays.
• To develop and validate a sensitive and specific method for SNO detection using chemiluminescence.

Main Methods:

• S-nitrosothiols were subjected to different chemical reduction conditions: VCl3 in HCl, KI in acetic acid, photolysis, and CuCl/cysteine.
• Nitric oxide (NO) released was quantified using chemiluminescence detection.
• Assay specificity was tested against nitrite (NO-2), nitrate (NO-3), and 3-nitrotyrosine.

Main Results:

• S-nitrosothiols were readily detected using the VCl3 reduction method but not with KI.
• The CuCl/cysteine reduction method demonstrated high linearity (r2 = 1.0), sensitivity (10 pmol), and specificity, as it did not detect NO-2, NO-3, or 3-nitrotyrosine.
• S-nitrosothiols constituted approximately 2.9% of total NOx in human serum, with less than 5% being low-mass species.

Conclusions:

• Conventional assays may erroneously identify nitrate (NO-3) as SNOs, while nitrite (NO-2) is not similarly misidentified.
• Chemiluminescence-based reduction systems provide a sensitive and specific method for quantifying S-nitrosothiols.
• Assessing the SNO fraction within biological nitric oxide (NOx) is a relevant and feasible approach for future research.