High levels of free radicals together with low antioxidant capacity detected in obese adults indicate elevated oxidative stress, which is--together with systemic inflammation--further potentiated in the case of obese patients with metabolic syndrome. This imbalance in oxidative/antioxidative status and subclinical inflammatory state leads to higher risk of atherosclerotic and diabetic complications.

Therefore, storage at -80°C is advised to maintain a reliable assay outcome when serum samples have to be stored for longer periods.

The storage time and storage temperature might affect stability of oxidative stress biomarkers, therefore, they have to be analyzed after long-term storage of serum samples. The stability of three biomarkers reflecting oxidative stress: reactive oxygen metabolites (ROM) for hydroperoxides, total thiol levels (TTL) for the redox status and biological antioxidant potency (BAP) for the antioxidant status, was investigated at several time points during 60 months of storage at -20 and -80 °C. Biomarkers ROM and BAP showed a very good stability during storage for 60 months at both temperatures. In addition, the correlation of the data after 60 months of storage compared with the starting data was very good with correlation coefficients >0.9. The TTL assay showed good results in serum samples stored at -80 °C, but not in samples stored at -20 °C. Serum samples for analysis of the set of oxidative stress biomarkers ROM, BAP and TTL can be stored up to 60 months at -80 °C. ROM and BAP can also be stored at -20 °C during this period. The present results are very important for the biomarker-related epidemiological studies that make use of biobanks with samples stored for many years and for new project planning, including sample storage conditions.

The oxidation and antioxidant status of serum are often determined in serum samples which have been frozen for some time. The oxidative stress process is prone to fast alterations in the sample because of the possible instability of the reactants. Here one oxidation assay (ROM) and three antioxidant assays (FRAP, TAS, and BAP) have been tested on their performance and stability at short-time storage. The most commonly used temperatures for storage and handling of serum samples (+4 and +20 ∘ C) were selected. In three short-term studies in which the storage time varied between 3 and 48 hrs the performance of these assays were tested on human serum samples. The general conclusion is that most assays performed well and gave stable results during 2 days of storage of the samples at both temperatures. Only the FRAP and TAS assays showed a small deviation at some storage conditions. In conclusion, handling of serum samples at +4 and +20 ∘ C during short-time periods did not affect the quality and performance of the oxidation and antioxidant assays during day-to-day analyses.

The antioxidant status of serum or plasma can be determined using several commercially available assays. Here, four different assays, total antioxidant status (TAS), its second-generation assay (TAS2), biological antioxidant potential (BAP), and enzymatic assay using horseradish peroxidase (EAOC), were applied on human serum samples to test the temperature stability of antioxidants, upon storage of serum for 12 months. The two or three most commonly used temperatures for storage, that is, - 20, - 70 (or - 80), and - 196°C, were selected. The general conclusion is that all assays were stable at the temperatures tested. In addition, there were almost no statistically significant differences between the samples stored at different temperatures. Only the rank order of the EAOC assay was not very good in samples stored at - 20°C. Also three components contributing to the total antioxidant capacity, uric acid, creatinine and bilirubin, showed no statistically significant differences between the temperatures. Therefore, storage at - 20°C is sufficient to maintain a proper assay outcome of most of the total antioxidant assays, although storage at - 70/80°C is to be preferred for longer storage times.