Physiological microassay of plasma total antioxidant status in a model of endothelial dysfunction in the rat following experimental oxidant stress in vivo

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Abstract

We have developed a photometric microassay for the assessment of total antioxidant status in plasma at physiological pH and temperature and applied it to evaluate experimental oxidant stress in vivo associated with endothelial dysfunction in vitro. Rat plasma or l-ascorbic acid inhibited the peroxidase-mediated accumulation after 6 min at pH 7.4 and 37°C of ABTS+ (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical), measured at 405 nm, in a concentration-dependent manner. Plasma total antioxidant status, expressed as the ascorbate equivalent antioxidant concentration, was subsequently found to be significantly reduced in rats treated daily for 7 days in vivo with the oxidant compounds hydroquinone (50 mg/kg i.p.) and triethylenetetramine (100 mg/kg i.p.), either alone or in combination with the glutathione-depleting agent l-buthionine sulfoximine (50 mg/kg i.p). Furthermore, basal endothelial function in isolated aorta was impaired after hydroquinone or triethylenetetramine in a manner aggravated by l-buthionine sulfoximine.

Introduction

Assays of plasma total antioxidant status involving the inhibition of radical formation in vitro by a broad range of endogenous antioxidants, including ascorbate, vitamin E, urate, glutathione and albumin (Arnao et al., 1996), are widely used to clinically assess antioxidant depletion and oxidant stress (Rice-Evans and Miller, 1994, Gavan et al., 1997, Lantos et al., 1997, Vucic et al., 1997, McLemore et al., 1998). The principal aim of the present study was to develop a microplate reader-based, microassay of plasma total antioxidant status based on the suppression by antioxidants of ABTS+ (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical) formation catalysed by peroxidase (Miller et al., 1993, Rice-Evans and Miller, 1994, Arnao et al., 1996). This microassay was then employed as an indirect marker of oxidant stress in rats following 7-day treatment in vivo with the redox cycling agent hydroquinone (Stenius et al., 1989), or the Cu/Zn superoxide dismutase inhibitor triethylenetetramine (Deman et al., 1996, Greenman et al., 1996), either alone or in combination with the glutathione-depleting agent l-buthionine sulfoximine (Pappas et al., 1994, Baruchel et al., 1995, Meister, 1995). Since reactive oxygen species are thought to contribute to endothelial dysfunction in diseases associated with oxidant stress, such as atherosclerosis and diabetes mellitus (Halliwell, 1991), it was of additional interest to examine the ability of basal, endothelium-derived nitric oxide to modulate contractile tone in isolated aortic smooth muscle after oxidant treatment in the same animals (Laight et al., 1996, Laight et al., 1998).

Section snippets

Experimental oxidant stress in vivo

Male, 8-week-old Wistar rats were treated daily for 7 days with hydroquinone (50 mg/kg i.p.) or triethylenetetramine (100 mg/kg i.p.), either alone or in combination with l-buthionine sulfoximine (50 mg/kg i.p.). Control animals received sham injections of normal saline (1 ml/kg i.p.). Rats were subsequently anaesthetised with pentabarbitone sodium (60 mg/kg i.p.) and an arterial blood sample collected in ethylendiaminetetraacetic acid. This was centrifuged to derive plasma which was stored at

Effects of rat plasma and l-ascorbic acid on ABTS+ accumulation in vitro

The accumulation of ABTS+ read at 405 nm, catalysed by horseradish peroxidase (30 mU/ml) at pH 7.4 and 37°C, was linear with time over at least the first 8 min (r=0.981, n=3, P<0.0001) (Fig. 1). In a preliminary study, plasma derived from healthy rats reduced the rate of ABTS+ accumulation (Fig. 2), as has been previously reported for human serum (Romay et al., 1996). The addition of 6 μl plasma resulted in an optimal, stable reduction in accumulated ABTS+ after 6 min (Fig. 1, Fig. 2) and

Discussion

Antioxidants such as l-ascorbic acid and trolox, delay the onset of ABTS+ accumulation without affecting the initial rate, reflecting the rapid reduction of ABTS+ back to ABTS until all the antioxidant has been consumed (Arnao et al., 1996). This gives rise to a lag time which is linearly related to the concentration of antioxidant. However, blood products such as serum show more complex behaviour and reduce the initial rate of ABTS+ accumulation without any appreciable lag time; all of which

Acknowledgements

This work was supported by Lipha s.a., Lyon, France.

References (23)

  • N. Gavan et al.

    Effect of percutaneous absorption of flucinolone acetonide on the activity of superoxide dismutase and total antioxidant status in patients with psoriasis

    Skin Pharmacol.

    (1997)

    • Cited by (0)

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