Antioxidants inhibit low density lipoprotein oxidation less at lysosomal pH: A possible explanation as to why the clinical trials of antioxidants might have failed.

Oxidised low density lipoprotein (LDL) was considered to be important in the pathogenesis of atherosclerosis, but the large clinical trials of antioxidants, including the first one using probucol (the PQRST Trial), failed to show benefit and have cast doubt on the importance of oxidised LDL. We have shown previously that LDL oxidation can be catalysed by iron in the lysosomes of macrophages. The aim of this study was therefore to investigate the effectiveness of antioxidants in preventing LDL oxidation at lysosomal pH and also establish the possible mechanism of oxidation. Probucol did not effectively inhibit the oxidation of LDL at lysosomal pH, as measured by conjugated dienes or oxidised cholesteryl esters or tryptophan residues in isolated LDL or by ceroid formation in the lysosomes of macrophage-like cells, in marked contrast to its highly effective inhibition of LDL oxidation at pH 7.4. LDL oxidation at lysosomal pH was inhibited very effectively for long periods by N,N'-diphenyl-1,4-phenylenediamine, which is more hydrophobic than probucol and has been shown by others to inhibit atherosclerosis in rabbits, and by cysteamine, which is a hydrophilic antioxidant that accumulates in lysosomes. Iron-induced LDL oxidation might be due to the formation of the superoxide radical, which protonates at lysosomal pH to form the much more reactive, hydrophobic hydroperoxyl radical, which can enter LDL and reach its core. Probucol resides mainly in the surface monolayer of LDL and would not effectively scavenge hydroperoxyl radicals in the core of LDL. This might explain why probucol failed to protect against atherosclerosis in various clinical trials. The oxidised LDL hypothesis of atherosclerosis now needs to be re-evaluated using different and more effective antioxidants that protect against the lysosomal oxidation of LDL.

A retinyl palmitate model of the phenomenon of the intrinsic fluorescence increase in ceroid-lipofuscin cytosomes.

We report here on a retinoid model of the phenomenon discovered in 1980, in which ceroid-lipofuscin cytosomes (CLC) increased their intrinsic fluorescence when exposed to fluorescence-exciting (lambda(ex)=365nm) ultraviolet (UV) light. We modeled this effect in vitro, irradiating a methanolic solution of retinyl palmitate (RP) either synthetic or extracted from the aged rat liver retinoid. Following our model, the mechanism of this phenomenon can be explained by the photodecomposition of RP during fluorescence. Palmitic acid (PA) that quenched fluorescence was separated by photohydrolysis and some products of RP photodegradation shifted absorption spectra towards the fluorescence-exciting UV band compared with the absorption spectrum of the initial RP. The total intensity of intrinsic fluorescence at 490nm of RP photodegradation products increased several times, because relative absorption at 365 nm wavelength was higher. We report here also the established chemical formulae of two retinoids, derivatives of RP photohydrolysis tentatively called R(368) and R(346). R(368) was determined as anhydroretinol, a naturally occurring intracellular messenger in the transduction pathway of retinoids regulating growth inhibition in lymphoid line cells. Retinoid R(346) was determined to be a methanolic intermediate of RP photodegradation (4,5-dihydro-5-methoxy-Anhydroretinol).

Morphological study on pigmented cells in the horse testis.

One of the most attractive characteristics of a horse testis is the change of the weight during development. As the testicular weight changes and the number of Leydig cells decreases, pigments appear in interstitial tissues. In the present study, the characteristics of the pigments found in the interstitial tissues were examined histochemically and ultrastructurally. Specific stainings indicated that the pigmented granules showed almost all of the histological and histochemical characteristics of ceroid or ceroid-like pigment. The cells showed positive reaction for acid phosphatase while the pigmented cells contained a lot of lysosomes ultrastructurally. These results suggest that macrophages might phagocytize Leydig cells, and store their digested materials as ceroid-like pigment.

Biochemical basis of lipofuscin, ceroid, and age pigment-like fluorophores.

Serious studies of the formation mechanisms of age-related pigments and their possible cellular influence have been hampered for a long time by discrepancies and controversies over the definition, fluorescence emission, origin, and composition of these pigments. This review discusses several critical controversies in this field and lay special emphasis on the cellular and biochemical reactions related to the formation mechanisms of lipofuscin, ceroid, advanced glycation end-products (AGEs), and age pigment like fluorophores (APFs). Various amino compounds and their reaction with secondary aldehydic products of oxygen free radical-induced oxidation, particularly lipid peroxidation, are important sources of the fluorophores of ceroid/lipofuscin, which progressively accumulate as a result of phagocytosis and autophagocytosis of modified biomaterials within secondary lysosomes of postmitotic and other cells. Lipofuscin is the classical age pigment of postmitotic cells, while ceroid accumulates due to pathologic and experimental processes. There are good reasons to consider both ceroid and lipofuscin as materials of the same principal origin. The age-related intracellular fluorophores of retinal pigment epithelium (RPE) seems to represent a special class of lipofuscin, which partly contains derivatives of retinoids and carotenoids. Saccharide-originated fluorophores, principally AGEs formed during glycation/Maillard reactions, may be mainly responsible for the extracellular fluorescence of long-lived proteins, such as collagen, elastin, and lens crystalline. Although lipofuscin, ceroid, AGEs, and APFs can be produced from different types of biological materials due to different side reactions of essential biology, the crosslinking of carbonyl-amino compounds is recognized as a common process during their formation.

Characterization of ultraviolet laser-induced autofluorescence of ceroid deposits and other structures in atherosclerotic plaques as a potential diagnostic for laser angiosurgery.

Unstained frozen sections of normal and atherosclerotic human aorta and coronary artery were examined using histochemical and fluorescence microscopic techniques to identify the structures responsible for autofluorescence under 351 to 364 nm laser excitation. These structures included elastin and collagen in normal and atherosclerotic specimens, calcium deposits in calcified plaques, and granular or ring-shaped deposits histochemically identified as ceroid found in both calcified and non-calcified plaques. Qualitatively, both the color and intensity of ceroid autofluorescence differed greatly from that of elastin or collagen. The emission spectra of elastin, collagen, and ceroid were examined by microscopic spectrofluorimetry, and were found to differ significantly as well. When compared with spectra of elastin and collagen, spectra of ceroid were broader, shifted to the red, and were somewhat resistant to bleaching. We conclude that detection of laser-induced ceroid autofluorescence may aid in identifying plaques for laser ablation.