Quantitative Studies on the Peroxidation of Human Low-Density Lipoprotein Initiated by Superoxide and by Charged and Neutral Alkylperoxyl Radicals¹

Abstract

Rates of peroxidation of human LDL and rates of consumption of the LDL's alpha-tocopherol (TocH) have been measured at 37 degrees C. Peroxidation was initiated by radicals generated in the aerated aqueous phase at known rates by thermal decomposition of appropriate precursors: superoxide (O2(*-)/HOO(*)) from a hyponitrite and alkylperoxyls (ROO(*), two positively charged, one negatively charged and one neutral) from azo compounds. The efficiencies of escape from the solvent cage of the geminate pair of neutral carbon-centered radicals was found to be 0.1, but it was 0.5 for the three charged radicals, a result attributed to radical/radical Coulombic repulsion within the cage. All four alkylperoxyls initiated and terminated tocopherol-mediated peroxidation (TMP) with about equal efficiency and essentially all of these radicals that were generated were consumed in these two reactions. TMP is a radical chain process, and when initiated by the alkylperoxyls, the rate of LDL peroxidation was faster in the early stages while TocH was present than later, after all of this "antioxidant" had been consumed. In contrast, only about 3-4% of the generated superoxide radicals reacted in any measurable fashion with TocH-containing LDL at pH's from 7.6 to 6.5 and peroxidation was much slower than with a similar rate of generation of alkylperoxyls. After all the TocH had been consumed, LDL peroxidation was negligible at pH 7.6 and 7.4, but at pH 6.8 and 6.5, the peroxidation rates showed a large increase over the rates while the TocH had been present. That is, endogenous TocH behaves as an antioxidant in LDL subjected to attack by the physiologically relevant superoxide radical, whereas TocH behaves as a prooxidant in LDL subjected to attack by the probably far less physiologically important alkylperoxyls. Rates of LDL peroxidation initiated by superoxide increased as the pH was decreased, and the results are consistent with the initiation of peroxidation of fresh LDL occurring via H-atom abstraction from TocH by HOO(*) to form the Toc(*) radical and termination by reaction of O2(*-) with Toc(*), a process that occurs partly by addition leading to TocH consumption and partly by electron plus proton transfer leading to the regeneration of TocH.

Cleavage of Supercoiled DNA by Horseradish Peroxidase plus tert-Butyl Hydroperoxide Is Not Due to tert-Butylperoxyl Radicals

“…When two phases no longer separated, 90% of the remaining material was distilled, with refluxing, at the same reduced pressure and collected. This material was 100% pure by iodiometric titration and HPLC with chemiluminescence detection (3). Further purification by recrystalization from hexane at -20°C gave BOOH which showed the same behavior toward the DNA/HRP couple as the distilled material.…”

“…In other work (3,10), we have employed HPLC with chemiluminescence detection to measure hydroperoxide concentrations reliably and quantitatively. We therefore employed this same procedure to study the reaction of 9 µM HRP (the highest concentration employed in our DNA experiments) with 60 µM BOOH (the lowest concentration employed) in our standard, metal-ion-free, buffer at 37°C.…”

“…Strand cleavage by a different positively changed peroxyl occurred with a similar efficiency (2). In contrast, the neutral, water-soluble peroxyl radical, HOCH 2 CH 2 NHC(O)C(CH 3 ) 2 OO • , generally produced no detectable strand-scission (i.e., no detectable R DNA) at a ROO • /bp ratio as high as 5:1 (2, 3), 3 and the negatively charged peroxyl radical, -O 3 SCH 2 CH 2 C(CH 3 )(CN)OO • , generally produced no strand-scission even at a ROO • / bp ratio of 24:1 (2). Essentially identical results were obtained with another SC DNA, pUC 19 (2).…”

“…1 For a succinct, general review of DNA damage by oxygen-centered radicals, see (1). 2 Abbreviations: SC, supercoiled DNA; R, relaxed DNA; L, linear DNA; + ROO • , (H2N)2 + CC(CH3)2OO • ; HRP, horseradish peroxidase; BOOH, tert-butyl hydroperoxide; CIP, Coprinus peroxidase; 3HB, 3-hydroperoxy-1-butene; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; bp, base pair. 3 For the neutral R • geminate radical pair, e was initially assumed [and stated to be an assumption (2)] to be equal to the value of 0.5 already known for one of the + R • geminate radical pairs (2). Later work (3) showed that e ) 0.1 for this neutral R • pair, and therefore the bp/ ROO • ratios given originally (2) must be reduced by a factor of 5.…”

“…3 For the neutral R • geminate radical pair, e was initially assumed [and stated to be an assumption (2)] to be equal to the value of 0.5 already known for one of the + R • geminate radical pairs (2). Later work (3) showed that e ) 0.1 for this neutral R • pair, and therefore the bp/ ROO • ratios given originally (2) must be reduced by a factor of 5. 4 From Figure 7 in ref (4), 0.74 mM Coprinus peroxidase (CIP) incubated in phosphate buffer at pH 7.0 and 20°C with 28 mM 3-hydroperoxy-1-butene (3HB) but no DNA or other added organic substrate gave ca.…”