Detlef Mohr

Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence.

Publisher Summary Peroxidation of polyunsaturated lipids proceeds via a free radical chain reaction with consumption of molecular oxygen and concomitant formation of lipid hydroperoxides as the primary reaction products. These lipid hydroperoxides may undergo a variety of secondary reactions that result in the formation of reactive carbonyl products. Lipid peroxidation is commonly assessed by measurement of lipid hydroperoxides, diene conjugates, oxygen consumption, or secondary reaction products (example, malonylaldehyde and 4-hydroxynonenal. The different methods available vary greatly in their requirement for specialized (and expensive) equipment and, at least for biological systems, in their limitations and number of potential interferences and/or artifacts. This chapter discusses developments in the use of the high-performance liquid chromatography (HPLC) postcolumn chemiluminescence (CL) method 5 for the assessment of lipoprotein lipid peroxidation. Lipoprotein oxidation, particularly that of low-density lipoproteins (LDL), as oxidative LDL modification is thought to represent an early and important step in the development of atherosclerosis. In the HPLC postcolumn CL method, the different classes of unoxidized and oxidized lipids are extracted, separated (from one another and from lipophilic antioxidants) by HPLC, and detected sequentially by UV absorption and CL, respectively.

The participation of nitric oxide in cell free- and its restriction of macrophage-mediated oxidation of low-density lipoprotein

The potential role of nitric oxide radical (NO .) in macrophage-mediated oxidation and conversion of human low density lipoprotein (LDL) to a high-uptake form was examined by exposing LDL to aerobic solutions of either NO . or 3-morpholino-sydnonimine-hydrochloride (SIN-1, a compound that spontaneously forms NO . and superoxide anion radical) or to mouse peritoneal macrophages in the presence and absence of modulators of cellular NO . synthesis. Incubation with NO . alone caused oxidation of LDLs ubiquinol-10 and accumulation of small amounts of lipid hydroperoxides, but failed to form any high-uptake ligand for endocytosis by macrophages and did not alter the LDL particle charge or the integrity of apoB. Exposure of LDL to SIN-1 resulted in complete consumption of all antioxidants and substantial formation of lipid hydroperoxides, but again had little effect on the lipoprotein particle charge or generation of high-uptake form. Preincubation of macrophages with interferon-gamma increased the cells ability to generate reactive nitrogen metabolites. The extent of cell-mediated oxidation of LDL and the generation of high-uptake LDL was substantial in resident cells in which NO . synthesis was barely detectable, depressed in cells active in NO . synthesis and restored when NO . synthesis was suppressed by the arginine analogue, NMMA. These results suggest that, while together with superoxide anion radical, NO . can oxidize LDL, its synthesis is not required for macrophage-mediated oxidation of LDL in vitro; rather it exerts a protective role in preventing oxidative LDL modification by macrophages.

Coantioxidants make alpha-tocopherol an efficient antioxidant for low-density lipoprotein.

The oxidation of low-density lipoproteins (LDLs) is now commonly implicated as an important early event in atherogenesis. The resulting interest in LDL antioxidation has focused on alpha-tocopherol, the biologically and chemically most active form of vitamin E and quantitatively the major lipid-soluble antioxidant in extracts prepared from human LDL. We review advances made in our understanding of the molecular action of alpha-tocopherol in radical-mediated oxidation of isolated human LDL and how the vitamins antioxidant activity is enhanced or even dependent on the presence of suitable reducing species, which are referred to as coantioxidants.

Radical-mediated oxidation of isolated human very-low-density lipoprotein.

Oxidative modification of human low-density lipoprotein (LDL) has received much attention because of its suggested involvement in the early events of atherogenesis. In contrast, little data exist concerning the oxidation of human very-low-density lipoprotein (VLDL), although such modification promotes foam cell formation by these lipoproteins. We therefore investigated the radical-mediated oxidation of VLDL by using controlled oxidizing conditions and sensitive and specific methods to assess lipoprotein lipid oxidation and antioxidation. We observed that the ratio of alpha-tocopherol to coenzyme Q10 in VLDL was close to that of LDL, suggesting that these lipoproteins may transport some coenzyme Q10 to extrahepatic tissues, as they do tocopherol. Most of the coenzyme Q10 associated with VLDL was present in its reduced, antioxidant active form, ubiquinol-10. The small amounts of ubiquinol-10 in VLDL provided the lipoprotein lipids with a highly efficient antioxidant protection. Also, the kinetics of radical-mediated lipid peroxidation in VLDL resembled that in LDL and therefore also probably proceeded via the recently described tocopherol-mediated peroxidation mechanism. Oxidation competition experiments using aqueous radicals and physiological concentrations and molar ratios of LDL and VLDL indicated that in contrast to the situation with high-density lipoproteins, lipid peroxidation was initiated and detected simultaneously in the former two lipoprotein particles. However, once initiated, peroxidation propagated at an approximately twofold higher rate in VLDL than LDL. Our studies suggest that radical-mediated lipid (per)oxidation proceeds via similar mechanisms in isolated LDL and VLDL. We conclude that efficient LDL antioxidants are also likely to be effective protective agents for VLDL.

Assessment of prooxidant activity of vitamin E in human low-density lipoprotein and plasma.

Publisher Summary This chapter discusses the assessment of pro-oxidant activity of vitamin E in human low-density lipoprotein and plasma. The chapter discusses a series of simple tests that manipulate both the phase- and the chain-transfer activity of α-tocopherol (α-TOH) in low-density lipoprotein (LDL). While these tests can provide useful information on the mechanism of action of α -TOH in vitro , they do not give information on the action of vitamin E in vivo , nor propose that α -TOH acts as a prooxidant in vivo . The chapter presents various procedures for testing the pro-oxidant activity of α-TOH in lipoproteins and lipid emulsions. The methods described in the chapter allow investigations on the mechanism of in vitro lipoprotein lipid peroxidation and the way this process is affected by various (co-)antioxidants surrounding or associated with lipoproteins. The methods discussed also allow investigation of a possible role of tocopherol-mediated peroxidation (TMP) in cellular lipid peroxidation and antioxidation, particularly once analytical tools comparable to those for lipoproteins are available for the assessment of cellular lipid peroxidation.

Antioxidant defenses in rat intestine and mesenteric lymph

Dietary oxysterols can reach the circulation and this may contribute to atherosclerosis, where lipid oxidation is thought to be important. There is also evidence that, in rats, peroxidized lipids are absorbed and transported into lymph [Aw TY, Williams MW, Gray L. Absorption and lymphatic transport of peroxidized lipids by rat small intestine in vivo: role of mucosal GSH. Am J Physiol 1992; 262: G99-G106], although the method used to detect lipid peroxides lacked specificity. We tested whether intragastric administration of vegetable oils containing triglyceride hydroperoxides (TG-OOH) to rats resulted in detectable lipid hydroperoxides in mesenteric lymph. Using sensitive HPLC with postcolumn chemiluminescence detection, we were unable to detect hydroperoxides of triglycerides, cholesterylesters or phospholipids during the course of lipid absorption, and lymph levels of ascorbate, urate, alpha-tocopherol and ubiquinol-9 did not change significantly. By contrast, we observed a striking reducing activity judged by the efficient reduction of administered ubiquinones-9 and -10 to the corresponding ubiquinols. Exposure of rat lymph and isolated chylomicrons to aqueous peroxyl radicals revealed patterns of antioxidant consumption and lipid hydroperoxide formation similar to those described previously for human extravascular fluids and isolated lipoproteins, respectively. In particular, rates of TG-OOH formation in lymph and chylomicrons were very low to undetectable as long as ascorbate and/or ubiquinols were present, but subsequently proceeded in a chain reaction despite the presence of alpha-tocopherol. These studies demonstrate that rat intestine and mesenteric lymph possess efficient antioxidant defenses against preformed lipid hydroperoxides and (peroxyl) radical mediated lipid oxidation. We conclude that dietary lipid hydroperoxides or postprandial oxidation of lipids are not likely to contribute to these particular forms of oxidized lipids in circulation and aortic tissue.

Plasma and LDL Levels of Major Lipophilic Antioxidants are Similar in Patients with Advanced Atherosclerosis and Age-Matched Controls

Oxidative modification of low-density lipoprotein (LDL), regarded an early event in atherogenesis, is associated with the depletion of the lipoproteins antioxidants. We tested whether the levels of major lipophilic antioxidants in the blood of patients with advanced atherosclerosis are different to those in age-matched controls. On average, plasma ubiquinol-10, total coenzyme Q and coenzyme Q redox status were slightly lower whereas the levels of alpha-tocopherol were slightly higher in patients (63 +/- 11 years, n = 32) than controls (64 +/- 10 years, n = 24). However, these differences were not statistically significant (p > 0.05). The levels of antioxidants in LDL isolated from a subset of patients (n = 20) and controls (n = 15) were also indifferent, and hydroperoxides of cholesteryl esters were undetectable (detection limit 10 nM) in plasma of patients (n = 11) and controls (n = 10). The data suggests that plasma and LDL levels of lipophilic antioxidants are not depleted in patients suffering from severe atherosclerosis, and that neither parameter serves as a useful diagnostic indicator for this disease.

Reduction of ubiquinone-1 by ascorbic acid is a catalytic and reversible process controlled by the concentration of molecular oxygen.

To address whether reduction by vitamin C may contribute to the in vivo maintenance of coenzyme Q in the reduced form, we studied the reduction of ubiquinone-1 by ascorbate at pH 7.4. Addition of ascorbate to ubiquinone-1 resulted in rapid O2 consumption and an increase in the steady-state concentration of ascorbyl radical. The initial rate of O2 consumption was proportional to the product of [ubiquinone-1] and [ascorbate] whereas [ascorbyl radical] was proportional to the square root of this parameter; both dependencies were in quantitative agreement with each other. The extent of O2 consumption greatly exceeded the amounts of ubiquinone-1 initially present. Formation of ubiquinol-1 from ubiquinone-1 by ascorbate was reversible, moderate under aerobic conditions, but substantial in the absence or near absence of oxygen. At high O2 concentration, ascorbate promoted the oxidation of ubiquinol-1 to ubiquinone-1. Addition of sodium dodecyl sulphate dramatically decreased the rate of reaction between ubiquinone-1 and ascorbate, most likely as a result of phase separation of the reagents. A preliminary reaction scheme with putative rate constants for the relevant reactions is presented that quantitatively describes the kinetic behaviour of the process studied. The key reactions in the scheme are electron transfer from ascorbate to ubiquinone-1 with formation of the ascorbyl and ubisemiquinone radical. The reaction of the latter with O2 is postulated to be responsible for O2 consumption, with ubiquinone-1 acting as a catalyst. Together, the results demonstrate that the extent of reduction of ubiquinone-1 by ascorbate was controlled by the O2 concentration and the physical availability of the reactants. As the O2 concentration in human blood is relatively high and ubiquinone-10 is located exclusively within the lipid phase of lipoproteins where negatively charged ascorbate has little access, our results suggest that direct reduction by ascorbate is unlikely to be responsible for the high reduction percentage observed for plasma coenzyme Q.