Aldehyde dehydrogenase-1a1 induces oncogene suppressor genes in B cell populations.

The deregulation of B cell differentiation has been shown to contribute to autoimmune disorders, hematological cancers, and aging. We provide evidence that the retinoic acid-producing enzyme aldehyde dehydrogenase 1a1 (Aldh1a1) is an oncogene suppressor in specific splenic IgG1(+)/CD19(-) and IgG1(+)/CD19(+) B cell populations. Aldh1a1 regulated transcription factors during B cell differentiation in a sequential manner: 1) retinoic acid receptor alpha (Rara) in IgG1(+)/CD19(-) and 2) zinc finger protein Zfp423 and peroxisome proliferator-activated receptor gamma (Pparg) in IgG1(+)/CD19(+) splenocytes. In Aldh1a1(-/-) mice, splenic IgG1(+)/CD19(-) and IgG1(+)/CD19(+) B cells acquired expression of proto-oncogenic genes c-Fos, c-Jun, and Hoxa10 that resulted in splenomegaly. Human multiple myeloma B cell lines also lack Aldh1a1 expression; however, ectopic Aldh1a1 expression rescued Rara and Znf423 expressions in these cells. Our data highlight a mechanism by which an enzyme involved in vitamin A metabolism can improve B cell resistance to oncogenesis.

PPARs and their metabolic modulation: new mechanisms for transcriptional regulation?

Peroxisome proliferator-activated receptors (PPARs) as ligand-activated nuclear receptors involved in the transcriptional regulation of lipid metabolism, energy balance, inflammation, and atherosclerosis are at the intersection of key pathways involved in the pathogenesis of diabetes and cardiovascular disease. Synthetic PPAR agonists like fibrates (PPAR-alpha) and thiazolidinediones (PPAR-gamma) are in therapeutic use to treat dyslipidaemia and diabetes. Despite strong encouraging in vitro, animal model, and human surrogate marker studies with these agents, recent prospective clinical cardiovascular trials have yielded mixed results, perhaps explained by concomitant drug use, study design, or a lack of efficacy of these agents on cardiovascular disease (independent of their current metabolic indications). The use of PPAR agents has also been limited by untoward effects. An alternative strategy to PPAR therapeutics is better understanding PPAR biology, the nature of natural PPAR agonists, and how these molecules are generated. Such insight might also provide valuable information about pathways that protect against the metabolic problems for which PPAR agents are currently indicated. This approach underscores the important distinction between the effects of synthetic PPAR agonists and the unequivocal biologic role of PPARs as key transcriptional regulators of metabolic and inflammatory pathways relevant to diabetes and atherosclerosis.

Oxidative modification of low-density lipoprotein (LDL) in HD patients: role in electronegative LDL formation.

High cardiovascular mortality in patients on hemodialysis (HD) is largely attributed to oxidative stress and altered lipoprotein profiles. Markedly increased levels of mildly modified LDL subfractions, such as dense LDL and electronegatively charged LDL (LDL(-)), are present in the blood of HD patients and may be markers of atherosclerosis risk. LDL(-), characterized by modified protein content and elevated levels of lipid peroxidation products, is representative of multiple oxidative processes acting on plasma lipoproteins that prevail during HD. In this review, we discussed known mechanisms leading to that may account for oxidative protein modification and/or LDL(-) formation in the context of specific conditions associated with HD.

Low density lipoprotein (LDL) modification: basic concepts and relationship to atherosclerosis.

A large number of clinical studies support the hypothesis that the risk for atherosclerosis is associated with the proportion of different LDL subfractions in blood. Electronegatively modified forms of LDL (LDL(-)) isolated using different chromatographic techniques are characterised by significant differences in the protein and lipid content as compared to the native LDL subfraction. LDL(-) composition appears to influence its atherogenic properties as well as its high susceptibility to oxidation and impaired metabolism. Increased LDL(-) levels are found in subjects with coronary artery disease, particularly in diabetics and patients undergoing haemodialysis (HD). Whether elevated LDL(-) levels are due to the LDL oxidation in blood remains disputed despite the oxidative character of LDL(-) modification. Plausible means for LDL(-) formation in blood include glycation and protein-radical interactions with ApoB 100. The latter can prevail during HD as observed in in vitro studies using a model HD system. The rapid and progressive formation of LDL(-) during standard HD can be significantly reduced employing haemolipodialysis (HLD), which provides local delivery of specific antioxidants (vitamin E and C) to blood at concentrations above normal physiologic levels. This procedure appears to be more effective than oral supplementation with antioxidants and may be a promising approach to reducing the rapid progression of atherosclerosis in HD patients.

Oxidative cross-linking of ApoB100 and hemoglobin results in low density lipoprotein modification in blood. Relevance to atherogenesis caused by hemodialysis.

Human blood contains a form of minimally modified low density lipoprotein (LDL), termed LDL-, whose origin remains unknown. Exploring the mechanism of formation, we found that LDL- can be produced in plasma in the absence of oxygen following LDL incubation with oxidized hemoglobin species. A high degree of apolipoprotein B100 modification results from covalent association of hemoglobin with LDL involving dityrosine formation but not due to the malonaldehyde epitope formation. This was evidenced by the cross-reactivity of oxidized LDL with antibodies against hemoglobin that was accompanied by a 60-fold increase in dityrosine levels. In this study we found significantly higher LDL- levels in the blood of hemodialysis patients, perhaps contributing to their greatly increased risk of atherosclerosis. The mechanism of LDL- formation was studied during ex vivo blood circulation using a model system resembling clinical hemodialysis in terms of the induction of inflammatory responses. This circulation increased free hemoglobin and LDL- levels compared with non-circulated blood without appreciable lipid peroxidation. Pronounced increases in LDL- were found also during circulation of plasma supplemented with nanomolar hemoglobin levels. The increase in dityrosine content and presence of heme in LDL after blood circulation suggest that LDL is modified, in part, by hemoglobin-LDL conjugates containing heme. Thus, hemoglobin-mediated reactions leading to LDL oxidation in plasma can account for high LDL- levels in hemodialysis patients.

Oxidative stress resulting from hemolysis and formation of catalytically active hemoglobin: protective strategies.

OBJECTIVES: The possible oxidative complications induced by free hemoglobin (Hb) released during the blood storage are discussed together with therapeutic strategies using vitamin E and specific inhibitor haptoglobin. Prooxidative properties of Hb in blood have been examined using LDL as a marker for oxidative stress, which contribute to toxicity observed in a number of pathologies aggravated by hemolysis or hemorrhagic lesions as well as after the transfusion of stored blood. MATERIALS: Experiments were performed using fresh blood or stored blood that was obtained from a blood bank on the day corresponding to the identified expiration date. METHODS: Oxidation of LDL was determined by means of the formation of mildly oxidized LDL (LDL-) using anion exchange chromatography. Concentrations of Hb were determined spectrophotometrically. RESULTS: Hb-mediated oxidative processes in cellular membranes have been well documented over the past decade. We showed that catalytic activity of Hb released during blood storage was sufficient to increase the proportion of LDL- fraction in blood after 4 h incubation at 37 degrees C. The intensity of this oxidative process as well as the release of Hb varied in different donors and may depend on the antioxidant capacity of blood. Accumulation of Hb during storage was significantly decreased in range of 15 - 32% in blood supplemented with low concentrations of alpha-, gamma-tocopherols. Similar effects were observed in the presence of low concentrations of haptoglobin, which has been reported as a specific inhibitor of hemoglobin-mediated oxidation. CONCLUSIONS: The specific inhibition of hemoglobin-mediated oxidation in lipoproteins and cellular membranes may improve the quality of stored blood and help to decrease complications arising from oxidative stress after transfusions or during hemolytic events. Based on growing evidence for a role of oxidatively modified LDL in atherosclerosis, hemolytic pathologies should receive further consideration as risk factors for cardiovascular disease.

Copper can promote oxidation of LDL by markedly different mechanisms.

Oxidation of LDL (0.1 microM) in PBS with copper concentrations ranging from 0.03 to 10 microM, equal to 0.3-100 Cu2+/LDL, was investigated by monitoring the formation of conjugated dienes at 234 nm. With all 8 LDL samples examined, the kinetics changed strongly at submicromolar Cu2+ concentrations. Based on time-course of the formation of conjugated dienes, cholesteryl linoleate hydroxides and hydroperoxides as well as the antioxidant consumption, two oxidation types were distinguished. Type A oxidations, observed at relatively high Cu2+ concentrations of 10-100 Cu2+/ LDL, represented the conventional kinetics of LDL oxidation with an inhibition period (= lag-time) followed by a propagation phase. In contrast, type C oxidations proceeded after a negligibly short lag time followed by a distinct propagation phase. The rate of this propagation increased rapidly to 0.5 mol diene/mol LDL and then slowed down in the presence of alpha-,gamma-tocopherols and carotenoids, which were consumed faster than tocopherols. The increase in diene absorption was due to the formation of both hydroxides and hydroperoxides suggesting a high initial decomposition of hydroperoxides. At submicromolar concentrations of about 0.1 to 0.5 microM, type C and type A oxidation can be combined resulting in 4 consecutive oxidation phases, i.e. 1st inhibition and 1st propagation (belonging to type C), followed by 2nd inhibition and 2nd propagation (belonging to type A). Increasing copper concentrations lowered the 1st propagation and shortened the 2nd inhibition periods until they melted into one apparent kinetic phase. Decreasing [Cu2+] increased the 1st propagation and 2nd inhibition but lowered the 2nd propagation phase until it completely disappeared. A threshold copper concentration, denoted as Cu(lim), can be calculated as a kinetic constant based on the Cu2+-dependence for the rate of 2nd propagation. Below Cu(lim), LDL oxidation proceeds only via type C kinetics. The Cu2+-dependence of the oxidation kinetics suggests that LDL contains two different Cu2+ biding sites. Cu2+ at the low-affinity binding sites, with half-saturation at 5-50 Cu2+/LDL, initiates and accelerates the 2nd propagation by decomposing lipid hydroperoxides. Cu2+ bound to the high-affinity binding sites, with half-saturation at 0.3-2.0 Cu2+/LDL, is responsible for the 1st propagation. Arguments in favor and against this propagation being due to tocopherol mediated peroxidation (TMP) are discussed. If the lag-time concept is extended to the conjugated diene curves seen for combined oxidation profiles, then a true inhibition phase does not apply to this time interval, but instead represents the time elapsed before the onset of the 2nd propagation phase.

Enhanced plasma level of lipid peroxidation in Iranians could be improved by antioxidants supplementation.

OBJECTIVE: To study the influence of supplementation with antioxidants on factors, which might increase the risk of coronary heart disease (CHD) in Iranians. DESIGN: Twenty-one male volunteers enter the prospective, single-blind, randomized study. SETTING: The supplementation was conducted at the Cardiovascular Center, University of Tehran, the biochemical analysis were carried out in the University of Graz. SUBJECTS: Twenty-one male medical students were recruited by advertisement. Five subjects were dropped out due to lack of the compliance. METHODS: One group of Iranians received 30 mg/d beta-carotene and placebo for alpha-tocopherol; the other received beta-carotene plus 400 IU alpha-tocopherol for ten weeks. Concentrations of antioxidants in plasma and low density lipoproteins (LDL), plasma lipid profile, autoantibody against oxidized LDL (oLAb) and malondialdehyde (MDA) concentrations in plasma were measured. Oxidative resistance of LDL was estimated using conjugated diene assay. RESULTS: Iranians had a significantly lower plasma levels of total cholesterol (P < 0.002), LDL-cholesterol (P < 0.01) and high density lipoprotein-cholesterol (P < 0.002), compared to healthy Austrian subjects (n = 13). Although the baseline concentrations of alpha-tocopherol and beta-carotene were comparable with Austrians, lycopene, canthaxanthin and lutein were significantly higher in Iranians (P < 0.03-0.001). In vitro oxidative resistance of LDL, measured as lag-time, was slightly higher (P < 0.01) in Iranians comparing with Austrians. Plasma MDA and oLAb concentrations were significantly higher in Iranians (P < 0.001). Both dietary supplementations reduced plasma MDA concentrations (P < 0.001-0.001). A key finding was that a supplement combined with alpha-tocopherol caused also a significant increase of oLAb concentration (P > 0.01) as well as the significant increase of lag-time (P > 0.005). CONCLUSIONS: This study shows that high plasma MDA level of Iranians can be decreased by beta-carotene supplementation with or without alpha-tocopherol. However, alpha-tocopherol is a more powerful antioxidant, which can increase the resistance of LDL to oxidation, reduce the MDA concentrations in plasma and increase autoantibodies to oLDL.

Lack of correlation between the alpha-tocopherol content of plasma and LDL, but high correlations for gamma-tocopherol and carotenoids.

In 59 healthy human subjects (37 male and 22 female) the concentrations of the lipid-soluble antioxidants alpha- and gamma-tocopherol, alpha- and beta-carotene, lycopene, cryptoxanthin, canthaxanthin, and lutein + zeaxanthin were determined in plasma (mumol/L) and in isolated low density lipoproteins (LDL) (mumol/mmol cholesterol). Plasma alpha-tocopherol concentrations were significantly correlated with plasma total cholesterol concentrations (r2 = 0.51, P < 0.0001) yet not with the LDL alpha-tocopherol content (r2 = 0.05, ns). Plasma gamma-tocopherol concentrations were weakly correlated with plasma total cholesterol (r2 = 0.12, P < 0.003) and both absolute and cholesterol standardized plasma gamma-tocopherol concentrations correlated strongly with the LDL gamma-tocopherol content (r2 = 0.58 and r2 = 0.72, respectively). In contrast, carotenoid concentrations did not correlate with cholesterol concentrations, but their LDL content correlated significantly with the respective plasma concentrations (r2 = 0.67 to 0.92, all P < 0.0001). In a subgroup of study subjects (n = 13) the distribution of vitamin E and carotenoids among LDL was calculated. The proportion of plasma alpha- and gamma-tocopherol found in LDL was 48 +/- 7 (range, 36-61%) and 41 +/- 7%, respectively, suggesting that LDL was in most of these subjects not the main carrier for these antioxidants. The lipophilic carotenoids, however, were predominantly carried by LDL (e.g., beta-carotene: 87 +/- 10%), whereas the proportion of the more polar ones carried by LDL was much smaller (e.g., lutein + zeaxanthin: 36 +/- 6%). The results of this study show that plasma alpha-tocopherol concentrations are not predictive for the alpha-tocopherol content of LDL in nonsupplemented individuals. This finding could have implications in interpreting the cause of the inverse relationship between plasma alpha-tocopherol and risk of atherosclerosis.

Factors affecting resistance of low density lipoproteins to oxidation.

Oxidation resistance (OR) of low density lipoproteins (LDL) is frequently determined by the conjugated diene (CD) assay, in which isolated LDL is exposed to CU2+ as prooxidant in the range of 1-10 microM. A brief review on major findings obtained with this assay will be given. A consistent observation is that vitamin E supplements or oleic acid-rich diets increase OR. Oxidation indices measured by the CD assay and effects of antioxidants very significantly depended on the Cu2+ concentration used for LDL oxidation. For medium and high Cu2+ concentrations, the relationship between lag time and propagation rate can be described by a simple hyperbolic saturation function, which has the same mathematical form as the Michaelis-Menten equation. At medium and high Cu2+ concentrations (0.5 to 5 microM), vitamin E increases lag time in a dose-dependent manner. The increase is higher for 0.5 microM Cu2+ as compared to 5 microM. At low Cu2+ concentrations (0.5 microM or less), the mechanism of LDL oxidation changes. Significant oxidation occurs in a preoxidation phase, which commences shortly after addition of Cu2+. Preoxidation is not inhibited by vitamin E. It is concluded that much additional work is needed to validate the importance of oxidation indices derived from CD and similar assays.

Impaired resistance to oxidation of low density lipoprotein in cystic fibrosis: improvement during vitamin E supplementation.

Antioxidants such as vitamin E protect unsaturated fatty acids of LDL against oxidation. In the ex vivo model used, LDL was exposed to Cu2+ ions, a potent prooxidant capable of initiating the oxidation of LDL. The lag time, indicating the delay of conjugated diene formation in LDL due to antioxidant protection, was measured in 54 cystic fibrosis (CF) patients with plasma alpha-tocopherol levels below (Group A, n = 30) or above (Group B, n = 24) 15.9 mumol/L (mean - 2 SD of Swiss population). Patients were reevaluated after 2 months on 400 IU/d of oral RRR-alpha-tocopherol. In group A, alpha-tocopherol concentrations in LDL increased significantly from 3.2 +/- 1.6 mol/mol LDL to 8.2 +/- 2.8 mol/mol (P < 0.001) and lag times increased from 79 +/- 33 min to 126 +/- 48 min (P < 0.001), whereas in the vitamin E sufficient group B no further increase neither in LDL alpha-tocopherol concentrations or in lag times was observed. LDL oleic acid concentrations were higher, and linoleic acid concentrations were lower in patients than in controls. After efficient vitamin E supplementation, lag times were positively related to LDL alpha-tocopherol (P < 0.01) and negatively to LDL linoleic and arachidonic acid content (P < 0.001). The maximum rate of oxidation correlated positively with linoleic and arachidonic acid concentrations, as did the maximum conjugated diene absorbance. These results indicate that LDL resistance to oxidation is impaired in vitamin E deficient CF patients but can be normalized within 2 months when alpha-tocopherol is given in sufficient amounts. Linoleic and arachidonic acid content exhibit a major influence on the LDL resistance to oxidation.