Protocol for assessing total antioxidant capacity in minimal volumes of varying clinical human samples

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TL;DR

This protocol describes a colorimetric method to measure total antioxidant capacity (TAC) in minimal volumes (10 μL) of clinical samples such as aqueous humor, tears, and plasma. It uses the reduction of Cu²⁺ to Cu¹⁺ and chelation with bicinchoninic acid, with a limit of detection of 0.016 mM and intra-assay CV of 4.25%. The method is simple, cost-effective, stable across pH 4–10, and correlates strongly with commercial FRAP assays (Spearman r = 0.891, p < 0.001), enabling TAC analysis in volume-constrained ocular fluids for research on oxidative stress in eye diseases.

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**Background:** Oxidative stress plays a crucial role in various ocular degenerative diseases, and measuring total antioxidant capacity (TAC) in small-volume body fluids (e.g., aqueous humor, tears, vitreous humor) is clinically important. Commercial TAC kits typically require larger sample volumes, limiting their use for such fluids. This protocol presents a step-by-step method for TAC analysis in minimal sample volumes (10 μL) using a colorimetric assay based on the reduction of Cu²⁺ to Cu¹⁺ by antioxidants, followed by chelation with bicinchoninic acid (BCA) to form a violet complex measured at 570 nm.

**Methods:** The protocol was developed and validated using aqueous humor samples from patients undergoing cataract surgery, with IRB approval (Chang Gung Memorial Hospital, IRB number 201900017B0). Sample collection methods are described for aqueous humor (paracentesis with 27-gauge needle), vitreous humor (at start of vitrectomy with closed infusion), tears (Schirmer test paper, 10-fold dilution, centrifugation at 12800 rcf, -4°C, 5 min), urine (midstream, stored at -80°C), and plasma (lithium heparin tubes, centrifugation at 3000 rpm, -4°C, 10 min). The working solution is 0.0016% CuSO₄ in BCA. Standard solutions are prepared from ascorbic acid (AA) via serial dilutions to create a calibration curve (2.5, 1.25, 0.625, 0.3125 mM AA, and blank). For the colorimetric reaction, 10 μL of sample or standard is loaded into a 96-well plate, 200 μL of working solution is added, incubated for 20 min in the dark, and absorbance is read at 570 nm using a microplate reader (Tecan Sunrise) with 10 s vortex before analysis. TAC is expressed as AA equivalent antioxidant capacity (AAEAC) using linear interpolation from the standard curve.

**Key Results:** The protocol achieved a limit of detection (LOD) of 0.016 mM and limit of quantitation (LOQ) of 0.053 mM. Intra-assay coefficient of variability (CV) was 4.25% (N = 8), and inter-assay CV was 4.13% (N = 19). The calibration curve showed an R² value of 0.9996 at 570 nm. The protocol demonstrated strong stability across a wide pH range (pH 4 to pH 10) and at room temperature. It correlated strongly with a commercial FRAP assay (Spearman correlation coefficient = 0.891, p < 0.001), with no significant concentration-dependent bias (Bland-Altman mean difference 0.015 mM AAEAC, 95% CI 0.368 to -0.338 AAEAC). The protocol was applied to analyze TAC in 164 human aqueous humor samples, showing a left-skewed distribution. An example of raw data from eight aqueous humor samples is provided, with TAC values ranging from 0.963 to 1.885 mM AAEAC.

**Clinical Implications:** This protocol enables reliable TAC measurement in minimal volumes (10 μL) of clinically relevant fluids, particularly aqueous humor, facilitating biochemical analyses in ocular diseases where oxidative stress is implicated. Its simplicity, cost-effectiveness, rapid results, pH stability, and accuracy make it suitable for clinical research and potential diagnostic applications. Limitations include that it measures only total antioxidant capacity (not individual components), relies on Cu²⁺ reduction (not direct free radical scavenging), and primarily targets hydrophilic antioxidants. The protocol is recommended for liquid solutions only. Proper sample storage at -80°C is critical to prevent degradation of antioxidants.