Gamma radiolysis as a tool to study lipoprotein oxidation mechanisms.

Well-defined quantities of *OH, O2*-,HO2* or RO2*)radicals (reactive oxygen species) can be specifically produced by radiolysis of water or ethanol. Such radical species can initiate one-electron oxidation or one-electron reduction reactions on numerous biological systems. The oxidative hypothesis of atherosclerosis classically admits the involvement of the oxidation of low density lipoproteins (LDLs) but also of high density lipoproteins (HDLs) in the development of the atherosclerotic process. The initiation mechanisms of this oxidation are still incompletely defined, although free radicals are likely involved. Therefore, gamma-radiolysis appears as a method of choice for the in vitro study of the mechanisms of oxidation of LDLs and HDLs by oxygen-centred free radicals (*OH, O2*-,HO2* and RO2*). Radiolytically oxidized lipoproteins exhibited a very well defined oxidation status (radiation dose-dependent quantification of vitamin E, beta-carotene, lipid peroxidation, protein carbonylation ...). gamma-Radiolysis is a less drastic method than other oxidation procedures such as for example copper ions. Moreover, gamma-radiolysis is also especially suitable for studying the reducing properties of antioxidant compounds with regard to their scavenging capacity.

[Oxidation of lipoproteins and mechanism of action of antioxidants : contribution of gamma radiolysis]. / Oxydation des lipoprotéines et méchanisme d'action des anti-oxydants: apport de la radiolyse gamma.

Water gamma radiolysis leads to a selective and quantitative production of radical species, which allows the study of the one-electron oxidation or reduction of several biological systems, especially lipoproteins. Well defined quantities of*OH, O2*-/HO2* or RO2*. free radicals can thus be specifically produced by radiolysis of water or of ethanol. Such radical species can initiate one-electron oxidation reactions on compounds dissolved in water or in ethanol. Given the oxidative hypothesis of atherosclerosis, it is classically admitted that the oxidation of low density lipoproteins (LDL) but also of high density lipoproteins (HDL) is involved in the development of the atherosclerotic process. Nevertheless, the initiation mechanisms of this oxidation are still poorly defined. Therefore, gamma radiolysis is a method of choice for the study of the mechanisms of oxidation of LDL and HDL by oxygenated free radicals (*OH, O2*-, HO2*, RO2*). Gamma radiolysis allows to obtain oxidized lipoproteins with a very well defined oxidation status, and is a less drastic method than other currently used oxidation procedures such as those using for example copper ions. Finally, gamma radiolysis is also especially appropriate to study the mechanisms of action of antioxidant molecules, either in pure solutions or as inhibitors of lipid peroxidation in lipoproteins in vitro.

Age-related increased susceptibility of high-density lipoproteins (HDL) to in vitro oxidation induced by gamma-radiolysis of water.

In the present study, we investigated the age-related susceptibility of high-density lipoproteins (HDL) to oxidation. HDL were obtained from healthy, normolipidemic young, middle-aged and elderly subjects. Oxidation of HDL was induced in vitro by oxygen free radicals generated by water gamma-radiolysis, and followed by the decrease of endogenous vitamin E and the formation of conjugated dienes and thiobarbituric acid-reactive substances, as well as the alterations of apolipoproteins A-I/A-II. The resistance of HDL to oxidation, evaluated by the length of the lag phase, decreased with aging. This increased oxidizability of HDL with aging could have a dramatic impact on the development of atherosclerosis in the elderly population.

Reciprocal protection of LDL and HDL oxidised by .OH free radicals in the presence of oxygen.

The aim of this work was to compare the behaviour of HDL oxidised by .OH or .OH/O2.- free radicals produced by gamma radiolysis in the absence or in the presence of LDL at the same concentration of 3 g x l(-1), in order to specify the possibility of reciprocal protection of HDL and LDL towards lipid peroxidation. This oxidation was quantitatively evaluated by the decrease of endogenous alpha-tocopherol and the formation of oxidation products (thiobarbituric acid-reactive substances and conjugated dienes) and by the determination of initial radiation yields. Our results demonstrated that HDL could be protected by LDL against in vitro radical oxidation only in the presence of oxygen (action of .OH/O2.- free radicals). This observation addresses new questions about the interaction between HDL and LDL, especially the possibility of a reciprocal protection.

Peroxidation of model lipoprotein solutions sensitized by photoreduction of ferritin by 365 nm radiation.

A mechanistic study involving the 365 nm irradiation of aerated, phosphate-buffered solutions of human high-density lipoproteins (HDL3 fraction) and ferritin was undertaken. The 365 nm irradiation of phosphate-buffered horse spleen ferritin solutions induces the release of Fe2+ in the medium. The initial quantum yield of Fe2+ release on irradiation is 0.002. This quantum yield is oxygen independent. The 365 nm irradiation of mixtures of HDL and ferritin leads to alterations in apolipoproteins as revealed by tryptophan (Trp) oxidation and electrophoretic pattern modification. In parallel with protein damage, lipid peroxidation is induced as shown by hydroperoxide and thiobarbituric acid reactive substances (TBARS) formation. These peroxidations are strongly reduced in 0.1 M formate solution, which suggests chain initiation by .OH radicals or subsequent radicals produced by .OH. They are completely inhibited by desferrioxamine, consistent with propagation by Fe2+ ion. By contrast incubation of HDL in the presence of ferritin and FeSO4 induces only poor auto-oxidation. The biological relevance of this study is discussed.

Modified apolipoprotein pattern after irradiation of human high-density lipoproteins by ultraviolet B.

The ultraviolet B-induced destruction of tryptophan residues and lipid peroxidation of high-density lipoproteins is accompanied by the immediate and marked structural modification of the apolipoproteins, as revealed by SDS-polyacrylamide gel electrophoresis and immunoblot with specific monoclonal antibodies. Formation of several polymers of apolipoprotein A-I, apolipoprotein A-II or both apolipoproteins occurred, although apolipoprotein A-II did not contain any Trp residue. These results suggest that initial photochemical damage can be transferred via intramacromolecular processes to other sites within the same apolipoprotein and by intermacromolecular reactions from apolipoprotein A-I to other apolipoproteins. In both cases, lipid peroxidation enhances the propagation of the initial photochemical damage. The physiological significance of this work is discussed with respect to the low-light doses required for the alterations of the high-density lipoproteins.

A mechanistic study of the interaction of UVB radiations with human serum lipoproteins.

The tryptophan residues of HDL are important chromophores of the interstitial fluid feeding the epidermal cells. The UVB light readily photoionizes tryptophan residues as demonstrated by the increase in their photolysis yield in presence of N2O. Saturation of the HDL solution with a N2O/O2 mixture (80%:20%, v/v) decreases the peroxidation of HDL lipids, thereby implying that lipid peroxidation is at least partly induced by tryptophan photolysis. Addition of EDTA and desferrioxamine to HDL solutions loaded with either Cu2+ or Fe2+ ions suggests that Fe2+ could be the 'contaminating' trace metal ions that are required to explain the occurrence of lipid photoperoxidation induced by photons absorbed by tryptophan residues.

UVB-induced photoperoxidation of lipids of human low and high density lipoproteins. A possible role of tryptophan residues.

Ultraviolet radiation of the UVB region readily destroy tryptophan (Trp) residues of low (LDL) and high (HDL) density lipoproteins. The photooxidation of tryptophan residues is accompanied by the peroxidation of low and high density lipoproteins unsaturated fatty acids, as measured by the thiobarbituric acid assay. Moreover, low and high density lipoproteins are natural carriers of vitamin E and carotenoids. These two antioxidants are also rapidly bleached by UVB. The UVA radiation promotes neither tryptophan residue destruction nor lipid photoperoxidation. The redox cycling Cu2+ ions considerably increase lipid photoperoxidation. The synergistic action of photo and auto (Cu2(+)-induced) peroxidation induces marked post-irradiation modifications of apolipoproteins as illustrated by the degradation of most tryptophan residues after overnight incubation in the dark of pre-irradiated samples.

Chemical studies on the structure of human serum high-density lipoprotein (HDL). Photochemical crosslinking of azido-labelled lipids in HDL.

The lipid classes of native human serum high-density lipoprotein (HDL) were exchanged against phosphatidylcholine, sphingomyelin and cholesteryl ester species substituted with photosensitive fatty acyl residues and against 25-azido-27-norcholesterol. The photosensitive fatty acyl residues were 5-and 16-azidopalmitic, 12-azidooleic and 18-azidolinoleic acid, all labelled with high tritium radioactivity. The lipid exchange method previously described was used. After UV irradiation and delipidation the apoproteins AI and AII, photocrosslinked with the radioactive lipids, were separated. The yield of covalently crosslinked lipid molecules amounted to 30% of the photosensitive lipid molecules incorporated into the HDL particle. ApoAI and apoAII were labelled by each of the photosensitive lipid classes, intergrated in the HDL particle although in very different stoichiometry. The regiospecific photochemical labelling of the lipopolypeptides AI and AII was established by cyanogen bromide cleavage and the separation of the four apoAI and two apoAII CNBr fragments. The analytical data prove the close steric relation and interaction of defined protein regions with hydrophobic regions of the lipid molecules by chemical means. These results are discussed with respect of the HDL structure.