A novel method for measuring the antioxidant activity using N, N-dimethyl-p-phenylenediamine (DMPD) was developed. The radical cation of this compound gives a stable colored solution and a linear inhibition of color formation can be observed in the presence of 0. 2-11 microg of TROLOX. The experimental protocol, which is rapid and inexpensive, ensures sensitivity and reproducibility in the measure of antioxidant activity of hydrophilic compounds. The effectiveness of the DMPD method on real foods was verified by evaluating the antioxidant ability of wine samples coming from different areas of Campania, Italy. Antioxidant capacity of wines is strictly related to the amount of phenolic compounds. The results obtained by the DMPD method are very similar to those obtained on the same samples when the radical cation of 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Miller et al., 1996) was used.

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Oxysterols are common components of oxidized low-density lipoprotein and accumulate in the core of fibrotic plaques as a mixture of cholesterol and cholesteryl ester oxidation products. The proapoptotic effects of a biologically representative mixture of oxysterols was compared with equimolar amounts of 7-ketocholesterol and unoxidized cholesterol. The oxysterol mixture in a concentration range actually detectable in hypercholesterolemic patients did not stimulate programmed cell death in cultivated murine macrophages. Unoxidized cholesterol also produced no effect. By contrast, when given alone, 7-ketocholesterol strongly stimulated the mitochondrial pathway of apoptosis with cytochrome c release, caspase-9 activation, and eventually caspase-3 activation. Subsequent experiments showed that when 7-ketocholesterol was administered to cells together with another oxysterol, namely 7betaOH-cholesterol, the strong proapoptotic effect of 7-ketocholesterol was markedly attenuated. As regards the mechanism underlying this quenching, we found that the combined oxysterol treatment counteracted the ability of 7-ketocholesterol, when administered alone, to strongly up-regulate the steady-state levels of reactive oxygen species (ROS) without interfering with sterol uptake. Furthermore, this increase in intracellular ROS appeared to be responsible for the up-regulation of proapoptotic factor, p21, after treatment with 7-ketocholesterol but not in cells challenged with the oxysterol mixture. Competition among oxysterols, apparently at the level of NADPH oxidase, diminishes the ROS induction and direct toxicity that is evoked by specific oxysterols. As a consequence, a more subtle gene modulation by oxysterols becomes facilitated in vascular cells.

SUMMARYBackground: The consumption of unfiltered coffee, containing bioactive diterpenes, causes an increase in plasma homocysteine concentration. A slight increase in plasma homocysteine is also caused by large quantities of filtered coffee. Coffee terpenes also raise plasma glutathione in mice. Aim: To verify the effect of Italian-style coffee consumption on the plasma concentration of glutathione and homocysteine in healthy subjects. Methods: Twenty-two volunteers consumed five cups of coffee per day for 1 week and maintained their usual diet. Five subjects were enrolled as controls. The intervention trial was preceded and followed by seven coffee-free days.

Oxidative stress has been clearly implicated in human disease by a growing body of scientific evidences. There is no ideal method for the measurement of this parameter. A possible strategy would be to measure simultaneously several biomarkers representing damage to different cellular components or, alternatively, a method able to evaluate the hydroperoxides, intermediate products of oxidation originating from different classes of molecules, such as lipids, peptides, amino acids, etc. can be used. We are introducing a simple, rapid and inexpensive assay to measure the oxidative status of human plasma. It is based on the properties of N,N-dimethyl-p-phenylenediamine (DMPD), a compound able to produce a fairly long-lived radical cation. The absorbance at 505 nm of a DMPD solution in the presence of plasma, which is proportional to the amount of hydroperoxyl compounds, is related to the oxidative status of the sample and could be expressed as hydrogen peroxide equivalents (HPE). This assay was not influenced by freezing-thawing and storage time of the plasma samples. The assay can be automated, performed in a kinetic mode, and used for routine analyses. The DMPD assay alone or in combination with analytical methods for assessing antioxidant capacity is suggested as a reliable tool to obtain information in pathologies related to oxidative stress.