ABSTRACT
(1) Background: Much effort has been expended to investigate the antioxidant capacity of human plasma, attempting to clarify the roles of both metabolic and food substances in determining defenses against oxidative stress. The relationship between the total antioxidant capacity (TAC) and the concentrations of redox-active biomolecules in the human plasma of healthy and cardiopathic individuals was investigated in the present study to develop a chemical speciation model. (2) Methods: Plasma was collected from 85 blood donors and from 25 cardiovascular surgery patients. The TAC was measured using the CUPRAC-BCS (CUPric Reducing Antioxidant Capacity - Bathocuproinedisulfonic acid) method. Biomolecule concentrations were determined via visible spectrophotometry or HPLC/RP techniques. The relationship between the TAC and the concentrations was defined by applying a multiple regression analysis. The significance of the variables was first tested, and chemical models were proposed for the two datasets. The model equation is ßTAC=∑ißi·Ai, where ßi and [Ai] are the electronic exchange and the molar concentrations of the ith antioxidant component, respectively. (3) Results: The major contributions to the TAC, ~80%, come from endogenous compounds in both healthy and cardiopathic individuals, whereas the contributions from exogenous compounds were different between the two datasets. In particular, γ-tocopherol showed a different role in the chemical models developed for the two groups.
ABSTRACT
The total antioxidant capacity (TAC) of human plasma is an index of the redox buffer capacity of this biological fluid and could be a biomarker for those disorders affecting redox status. Distinguishing physiological from pathological conditions needs a reference. Therefore, this work aims to define the reference intervals for TAC of human plasma of apparently healthy adult individuals. TAC was measured using the CUPRAC-BCS (CUPric reducing antioxidant capacity-bathocuproinedisulfonic acid) method previously optimized and tested in a clinical laboratory. A population of 500 blood donors was selected, plus an additional 222 pathological patients carrying specific defective metabolisms, namely, hyperuricemia, hyperbilirubinemia, and type 2 diabetic mellitus. The reference intervals of TAC were calculated according to international guidelines. Due to the response of a partitioning test, the reference intervals for healthy population were separately defined for male (258) and female (151) groups. The reference intervals (µmol L-1) resulted: 727-1248 for the male subgroup and 637-1048 for the female subgroup. The absence of an age effect on TAC values was verified. The reference intervals evaluated allow a discussion on some pathological conditions overloading the plasma with redox-active waste substances.
ABSTRACT
BACKGROUND: Quantification of Total Antioxidant Capacity (TAC) of human plasma is an important clinical target, since many diseases are suspected to be related with oxidative stress. The CUPRAC-BCS (BCS=Bathocuproinedisulfonic acid) method was chosen since it works using the photometric principle, with stable and inexpensive reagents and at physiological pH. METHODS: The method is based on the complex equilibria between Cu(II)-BCS (reagent) and Cu(I)-BCS. Cu(I)-BCS complex is formed by reducing ability of the plasma redox active substances. The photometric signal is achieved at 478 nm and calibration is performed using urate as a reference substance. RESULTS: Linearity, linear working range, sensitivity, precision, LoD, LoQ, selectivity and robustness have been considered to validate the method. Absorbance at 478 nm was found linear from 0.0025 up to 2.0 mmol L(-1) of urate reference solution. Precision was evaluated as within-day repeatability, Sr=4 µmol L(-1), and intermediate-precision, SI(T)=15 µmol L(-1). LoD and LoQ, resulted equal to 7.0 µmol L(-1) and 21 µmol L(-1) respectively while robustness was tested having care for pH variation during PBS buffer preparation. Tests on plasma (80 samples) and on human cerebrospinal fluid (30 samples) were conducted and discussed. CONCLUSIONS: By the analytical point of view, the photometric method was found to be simple, rapid, widely linear and reliable for the routine analysis of a clinical laboratory. By the clinical point of view, the method response is suitable for the study of chemical plasma quantities related to redox reactivity.
