Elsevier

Nutrition

Volume 25, Issues 7–8, July–August 2009, Pages 839-846
Nutrition

Basic nutritional investigation
Synergistic interactions of antioxidant nutrients in a biological model system

https://doi.org/10.1016/j.nut.2009.01.011Get rights and content

Abstract

Objective

The antioxidant activity of fat- and water-soluble antioxidant nutrients and their interactions in physiologic concentrations were determined in an in vitro biological model system.

Methods

Reconstituted human serum consisting of delipidized human serum (DHS) combined with phosphatidylcholine liposomes (PCL) was used to determine antioxidant activities of physiologic concentrations of antioxidant nutrients. Radicals were initiated with 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (2 mmol/L), and oxidation was monitored by 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid. Fat-soluble antioxidant nutrients were incorporated into the PCL prepared by sonication and suspended in DHS to avoid any interference by the endogenous fat-soluble antioxidants. Water-soluble antioxidants were added directly into the DHS. The oxidation kinetics were monitored every 5 min up to 210 min using a microplate reader (excitation wavelength 500 nm, emission wavelength 520 nm).

Results

We confirmed the synergistic protective effect of the combination of ascorbic acid (1–5 μM) and α-tocopherol (1–5 μM) against the oxidation of DHS with PCL. Furthermore, physiologic concentrations of 1) β-carotene (0.1, 0.5 μM) and α-tocopherol (2.5, 5.0 μM), 2) β-carotene (0.1, 0.5 μM) and ascorbic acid (2.5 μM), and 3) uric acid (10 uM) and α-tocopherol (2.5, 5.0 μM) synergistically protected oxidation of reconstituted human serum.

Conclusion

The present study results suggest a wide antioxidant network between water- and fat-soluble antioxidant nutrients in a biological system, although their actions in vivo warrant further study.

Introduction

Excessive production of reactive oxygen species can cause oxidative damage in vulnerable targets such as lipids, proteins, and DNA, perhaps resulting in an increased risk for chronic disease [1], [2], [3].Conversely, excessive amounts of antioxidants in a biological system could alter the redox balance to the detriment of the organism because a certain amount of reactive oxygen species is necessary for normal cellular functions such as inducing regulatory mediators in signaling processing [4] and for killing viruses and bacteria [5].

In an attempt to gain a better understanding of the biological actions of antioxidant nutrients in vitro, the activity of single antioxidants or various combinations of antioxidants have been studied over the previous decade [6], [7], [8]. Most of these studies were carried out in homogeneous solvent systems (i.e., aqueous or lipid) [9], artificial membranes (liposomes, micelles) in buffer solutions [10], [11], or by using isolated low-density lipoproteins [12]. However, these types of model systems are far different from an actual biological system such as human serum/plasma because plasma is a heterogeneous entity consisting of hydrophilic and lipophilic compartments and contains high concentrations of other components such as protein (∼4 g/dL) and uric acid. Therefore, it is of particular interest to determine not only the actions of individual hydrophilic or lipophilic antioxidants but also their interactions as part of a biological network.

In an effort to understand the true antioxidant properties of the major human serum antioxidant nutrients and any interactions that may take place among antioxidants in vivo, a reconstituted human serum (prepared using delipidized human serum (DHS) combined with phosphatidylcholine liposomes [PCL]) was used in the present study. Further, 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) [13] was utilized as a highly active lipophilic radical initiator and the lipophilic fluorescent probe 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY 581/591) [14] was used to monitor the oxidation kinetics to avoid the interference of protein in human serum. The model system was first validated by demonstrating the synergistic interactions between ascorbic acid and α-tocopherol and then extended to the other major antioxidant nutrients to elucidate the “true” antioxidant activities of individual versus various combinations of antioxidants located in the hydrophilic and lipophilic compartments.

Section snippets

Chemicals and reagents

All-trans-β-carotene (type II, synthetic, ≥95% high-performance liquid chromatographic), α-tocopherol (synthetic, ≥95% high-performance liquid chromatographic), l-glutathione (GSH, ≥99%), l-ascorbic acid (≥99%), uric acid (≥99%), human serum albumin (96–99%), and l-α-phosphatidylcholine (type XVI-E, ∼99% thin-layer chromatographic) were purchased from Sigma Chemical Co. (St Louis, MO, USA). The fatty acid analogue BODIPY 581/591 was purchased from Molecular Probes (Eugene, OR, USA). The

Total antioxidant performance assay using lipophilic radical initiator and lipophilic probe

Figure 1 shows representative oxidation kinetics of DHS-PCL (control) in the presence of MeO-AMVN and BODIPY 581/591. After a brief lag period, the kinetics is characterized by an almost linear rate of oxidation up to 60 min, reaching a plateau after 90 min of incubation. When the radical initiator, MeO-AMVN, was not added to the reaction mixture, no significant oxidation of the fluorescence probe BODIPY 581/591 was observed, indicating its stability under the experimental conditions used. A

Discussion

The present study utilized reconstituted human serum consisting of DHS, which mimics the biological matrix of serum except for the absence of its lipid components, and PCL, to provide a lipophilic compartment. A lipophilic radical initiator (MeO-AMVN) and a lipophilic fluorescent probe were used to specifically induce and monitor the lipid peroxidation cascade. We first demonstrated the specificity of the fluorescent method by determining the greater antioxidant effect (approximately

Conclusion

The present in vitro study clearly indicates that physiologic concentrations of antioxidants located in the aqueous and lipid compartments synergistically interact to protect against oxidation of reconstituted human serum. It is possible that physiologic doses of water- and fat-soluble antioxidant nutrients are required to establish an effective antioxidant network in vivo.

Acknowledgments

The authors thank Dr. R. Cozzi at Tossicologia dell'Ambiente, Faculty of Pharmacy, University of Milan, for his review of the Nernst equation.

References (37)

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This research was supported in part by grant R03EY015674 from the National Eye Institute, BioGreen 21 Program (Code 20070301034009), the Rural Development Administration, Korea, and the U.S. Department of Agriculture, under agreement 581950-9-001.

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