Optimizing protein precipitation efficiency for assessing the contribution of low molecular weight compounds to serum antioxidant capacity

Abstract

Antioxidant capacity testing is commonly used in clinical investigations to provide an estimate of in vitro antioxidant capacity of biosamples. Although beneficial to measure the synergistic contribution of all compounds with antioxidant functionality, assessing the capacity of non-protein fractions or small molecules like ascorbic acid with primary antioxidant functionality may be more beneficial in specific populations. Thus, efficacy of solvent/s to precipitate serum proteins is critical to assessing the antioxidant contribution of these compounds.

Objectives: To compare protein precipitation efficiency of a validated precipitating solvent system to acetone, the commonly utilized precipitating solvent in the oxygen radical absorbance capacity (ORAC) assay, and to evaluate antioxidant contribution of small molecular weight compounds in serum from 20 adults aged 65 and older with stage I or stage II obesity, yet who were otherwise healthy.

Design and methods: Precipitating solvent/s included acetone (1:8 (v/v)) or methanol/acetonitrile/acetone (MAA) (1:1:1, v/v/v) in a ratio of 1:4 (v/v). Protein concentration and antioxidant capacity were measured by the Biuret and ORAC assay, respectively.

Results: Significant differences (p<0.001) were observed in protein precipitation efficiency such that the protein content of serum remaining after acetone deproteination was 2.30±0.76mg/mL compared to 0.85±0.60mg/mL with MAA. Antioxidant capacity of whole serum was significantly greater (p<0.001) than that of serum deproteinated with MAA or acetone. Small molecular weight compounds contributed 6.18±2.46% to antioxidant capacity of whole serum.

Conclusions: Precipitation by MAA is more effective than acetone alone in precipitating high molecular weight proteins, thus allowing for assessment of antioxidant capacity of small molecules in serum.

Keywords: Antioxidant capacity; Antioxidants; ORAC; Protein precipitation; Serum.