Establishment and application of a fully automated serum total free sulfhydryl assay based on 2,2'-DTDP

Serum free thiols (FTs) are sensitive biomarkers of oxidative stress, closely associated with aging and chronic disease risk. Existing DTNB-based detection methods suffer from product instability, affecting analytical reliability. A more stable and high-throughput method is needed.

We developed an automated FT assay using 2,2'-dithiodipyridine (2,2'-DTDP) on a clinical chemistry analyzer. Serum samples reacted with 2,2'-DTDP under optimized conditions, and absorbance at 340 nm was used to quantify FT levels. Method performance, stability, and associations with cardiovascular disease (CVD) risk factors were assessed in 341 healthy individuals.

FTs reacted with 2,2'-DTDP to generate 2-TP, a stable product with maximum absorbance at 340 nm. The method demonstrated excellent linearity (0-1200 μmol/L), low imprecision (1.8 %-3.3 %), and reasonable recovery (≥80 %). Serum FTs were stable for at least one year at -70 °C, 12 h at 2-8 °C, and 2 h at room temperature. In the study population, FT levels were positively correlated with serum albumin, a major thiol carrier, and negatively correlated with age, triglyceride, glucose and creatinine, suggesting links to redox status and CVD risks. These associations support the potential of FTs as indicators of systemic oxidative balance and aging-related health decline.

This simple and robust 2,2'-DTDP-based method enables accurate, high-throughput measurement of serum FTs. It is well suited for exploring thiol antioxidant function and evaluating age-related and cardiovascular risk in clinical and research settings.