RESUMO
BACKGROUND: We have recently shown positive effects in the quality of life in autism and amyloid lateral sclerosis patients using a newly developed 25-OH vitamin D deglycosylated vitamin D binding protein complex (VitD~dgVDBP) by reducing oxidative stress. The question arises whether this reduction of oxidative stress was due to a synergistic effect of the dimer in the recognition and activation of phagocytosis on macrophages combined with a lower oxidative burst compared to the VitD free proteins, namely vitamin D binding protein (VDBP: Gc Protein) and deglycosylated dgVDBP (GcMAF). METHODS: VDBP sandwich ELISA of equal protein concentration of VDBP, dgVDBP, and VitD~dgVDBP (1 µg/ mL by BCA protein technique) was used to identify immune affinity to polyclonal antibodies raised against human VDBP. The 25(OH) vitamin D levels of VDBP, dgVDBP and VitD~dgVDBP were estimated by a competitive immune assay using a monoclonal antibody. Macrophage phagocytosis and oxidative burst in absence or presence of 400 pg/mL VDBP, 400 pg/mL dgVDBP, and 400 pg/mL VitD~dgVDBP was measured. RESULTS: The recognition of the antibody against VDBP protein was significantly more than 4-fold higher for VitD~dgVDBP (769.2 +/- 35.1%) compared to dgVDBP (186.5 +/- 16.8 %; p < 0.01) and 7-fold higher to VDBP (100 +/- 11.4 %; p < 0.001). 25(OH) vitamin D levels of VDBP (20.7 nmol/mg; p < 0.001) and dgVDBP (28.8 +/- 3.9 nmol/mL; p < 0.001) was significantly lower than of VitD~dgVDBP (324.0 +/- 12.8 nmol/mL). The calculated VitD/ protein ratio showed significantly higher results in favor of VitD~dgVDBP (1.01 +/- 0.12) compared to dgVDBP (0.06 +/- 0.03; p < 0.001) and VDBP (0.05 +/- 0.01; p < 0.001). The estimation of macrophage phagocytosis rate of VitD~dgVDBP (5,864.3 +/- 742.2 cps) was significantly higher compared to dgVDBP (2,789.6 +/- 102.7 cps; p < 0.01) and VDBP (1,134.3 +/- 135.9 cps) whereas the production of macrophage superoxide anion radicals showed significantly higher levels of dgVDBP (255.3 +/- 14.5 cps) in comparison to VDBP (148.6 +/- 24.7 cps, p < 0.01) and VitD~dgVDBP (142.3 +/- 20.0 cps; p < 0.001). Linear regression between VDBP antibody affinity and macrophage phagocytosis of VDBP, dgVDBP and VitD~dgVDBP resulted in a correlation coefficient of r = 0.95 in favor of VitD~dgVDBP. CONCLUSIONS: VitD~dgVDBP (Il-42) showed higher macrophage activation and lower oxidative burst than VitD free dgVDBP (GcMaf) and VDBP (Gc) which may result from a synergistic effect by presenting protein bound Vitamin D better to macrophages.
Assuntos
Ativação de Macrófagos/efeitos dos fármacos , Macrófagos , Proteína de Ligação a Vitamina D , Vitamina D , Células Cultivadas , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Ligação Proteica , Vitamina D/química , Vitamina D/metabolismo , Vitamina D/farmacologia , Proteína de Ligação a Vitamina D/química , Proteína de Ligação a Vitamina D/metabolismo , Proteína de Ligação a Vitamina D/farmacologiaRESUMO
Reactive oxygen species are thought to partly be responsible for the hypoxia induced performance decrease. The present study evaluated the effects of a broad based antioxidant supplementation or the combined intake of alpha-ketoglutaric acid (α-KG) and 5-hydroxymethylfurfural (5-HMF) on the performance decrease at altitude. 18 healthy, well-trained males (age: 25±3 years; height: 179±6 cm; weight: 76.4±6.8 kg) were randomly assigned in a double-blind fashion to a placebo group (PL), a α-KG and 5-HMF supplementation group (AO1) or a broad based antioxidant supplementation group (AO2). Participants performed 2 incremental exercise tests to exhaustion on a cycle ergometer; the first test under normoxia and the second under hypoxia conditions (simulated altitude, FiO2=13% ~ 4 300 m). Supplementation started 48 h before the hypoxia test. Maximal oxygen uptake, maximal power output, power output at the ventilatory and lactate threshold and the tissue oxygenation index (NIRS) were measured under both conditions. Oxidative stress markers were measured before the supplementation and after the hypoxia test. Under hypoxia conditions all performance parameters decreased in the range of 19-39% with no differences between groups. A significant change from normoxia to hypoxia (p<0.001) and between groups (p=0.038) were found for the tissue oxygenation index. Post hoc test revealed significant differences between the PL and both, the AO1 and the AO2 group. The oxidative stress parameter carbonyl protein changed from normoxia to hypoxia in all participants and 4-hydroxynonenal decreased in the AO1 group only. In conclusion the results suggest that short-term supplementation with an antioxidant does not prevent the performance decrease at altitude. However, positive effects on muscle oxygen extraction, as indicated by the tissue oxygenation index, might indicate that mitochondrial functioning was actually influenced by the supplementation.
Assuntos
Antioxidantes/farmacologia , Furaldeído/análogos & derivados , Ácidos Cetoglutáricos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Adulto , Aldeídos/metabolismo , Altitude , Antioxidantes/administração & dosagem , Desempenho Atlético/fisiologia , Método Duplo-Cego , Exercício Físico/fisiologia , Teste de Esforço , Furaldeído/administração & dosagem , Furaldeído/farmacologia , Humanos , Hipóxia/fisiopatologia , Ácidos Cetoglutáricos/administração & dosagem , Ácido Láctico/sangue , Masculino , Consumo de Oxigênio , Adulto JovemAssuntos
Acidentes de Trabalho , Sulfeto de Hidrogênio/intoxicação , Oxigenoterapia Hiperbárica , Câmaras de Exposição Atmosférica , Edema Encefálico/induzido quimicamente , Edema Encefálico/mortalidade , Edema Encefálico/prevenção & controle , Humanos , Oxigenoterapia Hiperbárica/métodos , Intubação Intratraqueal , Metemoglobina/análise , Intoxicação/sangue , Intoxicação/mortalidade , Intoxicação/terapia , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/mortalidade , Edema Pulmonar/terapiaRESUMO
INTRODUCTION: Several studies have been carried out investigating different preservation methods and preservation solutions for the pancreata of various species. Attention has to be drawn to the extreme vulnerability of porcine pancreata (PP) to oxidative stress due to the lack of endogenous antioxidants. This study sought to evaluate the influence of cannulation and infusion of different volumes of University of Wisconsin (UW) solution immediately after organ retrieval on PP organ quality. METHODS: PP from 24 slaughterhouse pigs were harvested with immediate cannulation of the pancreatic duct for infusion of 10 mL, 20 mL, 50 mL, or 100 mL UW solution. The organs were stored in cold UW solution. Control organs were only stored in UW. After 6 hours of cold ischemia, tissue and supernate samples were analyzed for markers of oxidative cell damage, adenosine triphosphate (ATP) levels, and occurrence of apoptosis. RESULTS: The fewest apoptotic cells were detected in the PP infused with 50 mL UW via the pancreatic duct (PP 50) as compared with all other groups. Oxidative cell damage was lowest and ATP levels were highest in the PP 50 group. DISCUSSION: Because PP 50 showed significantly better results when compared with all other groups, we suggest that infusion of 50 mL UW via the pancreatic duct immediately after organ retrieval may be useful to minimize oxidative cell damage and cell death in PP.
Assuntos
Soluções para Preservação de Órgãos/farmacologia , Preservação de Órgãos/métodos , Pâncreas/citologia , Traumatismo por Reperfusão/prevenção & controle , Adenosina/farmacologia , Alopurinol/farmacologia , Animais , Glutationa/farmacologia , Insulina/farmacologia , Lipase/metabolismo , Modelos Animais , Estresse Oxidativo/efeitos dos fármacos , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Pâncreas/fisiologia , Transplante de Pâncreas/fisiologia , Rafinose/farmacologia , Suínos , Coleta de Tecidos e Órgãos/métodosRESUMO
The question arises as to whether oxidative stress has a primary role in neurodegeneration or is a secondary end-stage epiphenomenon. The aim of the present study was to determine oxidative stress parameters like malondialdehyde (MDA), carbonyl proteins (CP) and Albumin-disulphide (Alb-SSR) and relate these parameters to the immune parameter neopterin, folic acid and vitamin B12 as vitamins and homocysteine in patients with neuro-degenerative diseases (NDD), namely mild cognitive impairment (MCI) and Alzheimer's disease (AD) compared to an aged matched control group. MDA, CP and Alb-SSR were significantly increased in the NDD group compared to controls, but not vitamin B12, folic acid and neopterin. Significant correlations were found between CP and Alb-SSR, CP and MDA and between MDA and Alb-SSR including patients with NDD and the control group. These results support the hypothesis that oxidative damage to lipids and proteins is an important early event in the pathogenesis of neurodegenerative diseases.
Assuntos
Doença de Alzheimer/metabolismo , Transtornos Cognitivos/metabolismo , Dissulfetos/sangue , Malondialdeído/sangue , Degeneração Neural/metabolismo , Estresse Oxidativo , Carbonilação Proteica , Albumina Sérica/metabolismo , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Ácido Fólico/sangue , Homocisteína/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Neopterina/sangue , Índice de Gravidade de Doença , Vitamina B 12/sangueRESUMO
Hydroxymethylfurfural (HMF) and alpha-ketoglutaric acid (KG) have been recently investigated as potential cancer cell damaging agents. We herein report for the first time a validated quantitative assay for their simultaneous determination in human plasma which is amenable to be applied in the future screening of the target compounds in human probands in order to properly design a targeted chemotherapeutic regimen for certain types of malignant tumors. A simple liquid chromatographic method in conjunction to derivatization after a two-step optimized solid phase clean-up procedure is described. The method is based on the reaction of HMF and KG with 2-nitrophenylhydrazine or 2,4-dinitrophenylhydrazine in an aqueous environment. Reaction conditions were studied with respect to pH, reagent volume, reaction temperature and time. Exact testing of such parameters beside careful selection of the mobile phase composition rendered feasible the quantification of the chemically significantly differing analytes along a single chromatographic run. The formed derivatives could be separated isocratically by reversed-phase LC on a C(8)-column. Detection in the UV and in the visible range is possible. Results showed good recovery and reproducibility with detection limits (S/N=3) down to 2 picomoles analyte on column. Resolution of the syn and anti geometric isomers of the HMF and KG derivatives is possible. The isomeric ratio in relation to the reaction pH is discussed.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Furaldeído/análogos & derivados , Ácidos Cetoglutáricos/sangue , Furaldeído/sangue , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrofotometria UltravioletaRESUMO
Hydroxymethylfurfural (HMF), a well-known heterocyclic Maillard reaction product, has often been studied for its potential toxic, mutagenic, and carcinogenic effects. Recent clinical studies, however, have strongly suggested that HMF might have exciting antitumor potential. We report on the development and validation of a bioanalytical assay for HMF that could be suitable as a basis for pharmacokinetic models in cancer patients. Two strategies were tested, i.e., direct and indirect methodologies. A direct isocratic LC determination at 283 nm was designed. Two indirect attempts involved derivatization coupled to HPLC-UV. It was possible to resolve the stereoisomers of the HMF derivative, and factors influencing their equilibrium ratio are discussed. HMF was extracted from the biomatrix by solid-phase extraction using different cartridges. A comparative study was made of the implemented methods as well as the extraction protocols. Both indirect assays proved to be more sensitive and were used to assess HMF quantitatively in human plasma. However, the newly introduced derivatization conditions led to the highest sensitivity with a LOD (S/N ratio = 3) of at least 2 pmol analyte on column. The assay selectivity was satisfactory in pre- and post-dose real samples. The mean recoveries of the assays were 79% and 89%, with acceptable accuracies and reproducibilities. Figure Schematic representation of hydroxymethylfurfural (HMF) in human plasma.
Assuntos
Furaldeído/análogos & derivados , Cromatografia Líquida de Alta Pressão , Furaldeído/sangue , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrofotometria UltravioletaRESUMO
Retention of atherogenic lipoproteins in the arterial intima by extracellular matrix (ECM) is assumed to occur during early atherogenesis and its further development. Low density lipoprotein (LDL) trapped in the intima may undergo oxidative modifications, which initiate a chain reaction in atherogenesis. Lipoprotein lipase (LPL) has been found to mediate the binding of native and oxidized LDL to ECM produced by cultured cells and to contribute to foam cell formation by mildly oxidized LDL. In this study ECM, isolated from human aortic intima with different atherosclerotic lesions, was used for the first time to measure the binding to it in vitro of native and differently oxidized 125I-LDL. Oxidation of 125I-LDL increased its binding to the ECM, which was most prominent with the material isolated from intima at the early stage of atherogenesis. With the progression of atherosclerosis, the ability of the isolated intimal ECM to bind native and oxidized 125I-LDL decreased, and strongly oxidized 125I-LDL decreased more than native and moderately oxidized 125I-LDL. LPL increased the binding of moderately oxidized 125I-LDL to the ECM more than native 125I-LDL, while it had only a small effect on strongly oxidized 125I-LDL. LPL-mediated binding of native and oxidized 125I-LDL decreased with the development of atherosclerotic lesions. Calcium ions also increased the binding of LDL to the ECM. This enhanced binding increased with the extent of LDL oxidation, especially at the early stage of atherogenesis, and decreased with lesion progression. These data suggest that the ability of ECM to retain LDL in arterial intima depends on LDL oxidation status and changes with the progression of atherogenesis. In addition, LPL and calcium ions may participate in the retention of LDL in vivo.
Assuntos
Doenças da Aorta/metabolismo , Arteriosclerose/metabolismo , Matriz Extracelular/metabolismo , Lipoproteínas LDL/metabolismo , Túnica Íntima/metabolismo , Adulto , Idoso , Aorta Abdominal/metabolismo , Cálcio/fisiologia , Técnicas de Cultura , Progressão da Doença , Humanos , Lipase Lipoproteica/fisiologia , Pessoa de Meia-Idade , OxirreduçãoRESUMO
Carvedilol, an antihypertensive agent, has been in clinical use for several years. In addition to its function as a beta-blocker, carvedilol has been shown to act as an antioxidant. However, there is some controversy as to how carvedilol achieves its antioxidative ability: by radical scavenging or ion chelation? We therefore used a method of radical generation independent of metal ions to investigate the antioxidative properties of carvedilol. We showed that carvedilol decreased low-density lipoprotein (LDL) oxidation induced by a peroxyl radical-generating system [2,2'-azobis(2-amidinopropane)hydrochloride]. Formation of thiobarbituric acid-reactive substances, lipid hydroperoxides, and newly generated epitopes on oxidised LDL was used to monitor LDL oxidation. We further showed that carvedilol was consumed during reaction with peroxyl radicals. However, carvedilol showed no reaction with nitrogen-centered radicals (1,1-diphenyl-2-picrylhydrazyl and 2,2'-azino-di-[3-ethylbenzthiazoline sulphonate]), which are often used in assays for determining antioxidative properties. On the other hand, we found that carvedilol acted as a chelator of ferric ions. Using mass spectrometry and NMR spectroscopy, we observed complex formation with free and acetylacetonate-complexed ferric ions. The binding constant with Fe(3+) was in the range of 10(5) L/mol. From our data, we concluded that carvedilol acts as both a metal chelator and a radical scavenger in vitro. However, it is selective in reacting with different radicals and is not an electron-donating radical scavenger as is alpha-tocopherol. Therefore, taking into account the low physiological concentration, the antioxidative properties reported earlier may not solely be explained by its radical-scavenging activity.
Assuntos
Anti-Hipertensivos/farmacologia , Antioxidantes/farmacologia , Carbazóis/farmacologia , Sequestradores de Radicais Livres/farmacologia , Quelantes de Ferro/farmacologia , Propanolaminas/farmacologia , Carvedilol , Humanos , Técnicas In Vitro , Lipoproteínas LDL/metabolismo , Oxirredução/efeitos dos fármacosRESUMO
Oxidatively modified low-density lipoprotein (LDL) has been found in vivo, and oxidized LDL (oxLDL) could bind to scavenger receptors, leading to foam cell formation. Macrophages bear a number of different scavenger receptors for oxLDL, and macrophages of different origins may have a different scavenger receptor repertoire. In addition, LDL oxidized to different degrees may differ in the ability to bind macrophage scavenger receptors. In this study, we characterized the patterns of the binding and uptake of differently oxidized LDL in mouse peritoneal macrophages (MPM) and human THP-1 macrophages, and the influence of negative charge and oxidation-specific epitopes in oxLDL on these processes. Thresholds of increased binding and uptake in MPM were found when LDL was oxidized to the degrees with a relative electrophoretic mobility (REM) of 2.6 (minor threshold) and 3.0 (major threshold), corresponding to 49 and 57%, respectively, of the loss of free amino groups in these oxLDL. There was no threshold for the binding of oxLDL to THP-1 macrophages, while for uptake, a major threshold with REM of 3.0 (57% free amino groups lost) was found. The presence of the F(ab')(2) fragments of the monoclonal antibody OB/04, which was raised against copper-oxidized LDL, led to the reduction of the binding and uptake, respectively, of Eu(3+)-oxLDL (REM:3.6) in MPM by 31 and 29%, and by 19 and 22% in THP-1 macrophages. It is concluded that LDL oxidized to different degrees binds differently to macrophages, and the patterns of binding and uptake are different for MPM and human THP-1 macrophages. Both, the negative charge and the oxidation-specific epitopes of oxLDL are involved in these processes.
Assuntos
Epitopos/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneais/metabolismo , Proteínas de Membrana , Receptores de Lipoproteínas , Animais , Anticorpos Monoclonais/metabolismo , Linhagem Celular , Európio/química , Humanos , Imunoglobulina G/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Oxirredução , Poli I/farmacologia , Polissacarídeos/farmacologia , Receptores Imunológicos/metabolismo , Receptores Depuradores , Receptores Depuradores Classe BRESUMO
We compared several "new" risk factors (autoantibodies to oxidatively modified low density lipoprotein (LDL), sialic acid content of LDL, bilirubin and C-reactive protein) with "conventional" risk factors (apolipoprotein (apo) AI, AII and B, lipoprotein(a), triglycerides, and total, LDL and high density lipoprotein (HDL) cholesterol) for the presence and the extent of coronary or carotid atherosclerosis. Forty male patients with angiographically proven coronary atherosclerosis and 31 male patients with ultrasound-proven extracranial carotid atherosclerosis were compared to 40 age matched (53+/-5 years) healthy males as control subjects, with negative parental history of atherosclerosis, no clinical signs of systemic or organ-related ischemic disease and normal extracranial carotid arteries. The apo B/apo All ratio most powerfully indicated the presence and the extent of coronary or carotid atherosclerosis. Elevated lipoprotein(a) contributed significant additional information in the assessment of the atherosclerotic risk. Increase in C-reactive protein indicated the presence (but not the extent) of coronary or carotid atherosclerosis with a similar power as lipoprotein(a). Decreased values of total bilirubin indicated the presence of atherosclerosis only in smokers. Autoantibodies to oxidatively modified LDL additionally described the atherosclerotic process, but were less important than apolipoproteins, lipoprotein(a), C-reactive protein or bilirubin. Sialic acid content of LDL added no information to the parameters discussed above. We demonstrated that in male patients apolipoproteins, especially the apo B/apo All ratio, were better indicators of the presence and the extent of coronary or carotid atherosclerosis than C-reactive protein, bilirubin, autoantibodies to oxidatively modified LDL or sialic acid content of LDL.
Assuntos
Apolipoproteína A-II/sangue , Apolipoproteínas B/sangue , Doenças das Artérias Carótidas/sangue , Doença da Artéria Coronariana/sangue , Adulto , Idoso , Doenças das Artérias Carótidas/etiologia , Doença da Artéria Coronariana/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
The influence of Ca(2+) and Mg(2+), together with lipoprotein lipase (LPL), on the binding and uptake of Eu(3+)-labeled native and oxidized low density lipoprotein (LDL) to mouse peritoneal macrophages (MPM), and on the deposition of esterified cholesterol in these macrophages, were studied. We found that both LPL and Ca(2+) (but not Mg(2+)) increased the binding and uptake of native and mildly or moderately oxidized LDL, and the subsequent deposition of cholesterol esters in MPM. When added together, LPL and Ca(2+) synergistically increased the binding and uptake of native and oxidized LDL, and the deposition of esterified cholesterol derived from native and mildly or moderately oxidized LDL, in MPM. Since both calcium and LPL are found in the atherosclerotic lesions, our results suggest that Ca(2+) and LPL may synergistically promote foam cell formation and atherogenesis. Furthermore, future research in the metabolism of lipoproteins should take into account the calcium levels in the experimental conditions.
Assuntos
Cálcio/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Lipase Lipoproteica/farmacologia , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneais/metabolismo , Animais , Arteriosclerose/etiologia , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Sítios de Ligação/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Colesterol/metabolismo , Meios de Cultura/farmacologia , Sinergismo Farmacológico , Európio , Lipoproteínas LDL/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Magnésio/farmacologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
It has been well established that purified lipoprotein lipase (LPL) can facilitate the cellular uptake of various native and modified lipoproteins when added exogenously to macrophages. Because activated macrophages express LPL endogenously, it was the aim of this study to investigate the effect of macrophage-produced LPL on the uptake of native low-density lipoprotein (LDL) and LDL that has been modified to various degrees by Cu(2+)-mediated oxidation. Cell binding and uptake of Eu(3+)-labelled native and oxidized LDL was determined in mouse peritoneal macrophages (MPM) from normal mice and induced mutant mice that lack LPL expression in MPM. We found that LPL expressed by MPM was able to increase cell binding and association of native LDL (by 121% and 101% respectively), mildly oxidized LDL (by 47% and 43%) and moderately oxidized LDL (by 30% and 22%). With increased levels of lipoprotein oxidation, the relative proportion of LPL-mediated LDL uptake decreased. This decrease was not due to weakened binding of LPL to oxidized LDL. The drastically increased uptake of highly oxidized LDL in MPM by scavenger-receptor-mediated pathways might dominate the simultaneous exogenous or endogenous LPL-mediated uptake of this lipoprotein. Competition experiments with positively charged poly(amino acids) furthermore suggested that histidine, arginine and lysine residues in LPL are important for the interaction between LDL and LPL. Our results imply that physiological levels of LPL produced by macrophages facilitate the uptake of native LDL as well as mildly and moderately oxidized LDL. This process might, in the micro-environment of arteries, contribute to the accumulation of macrophage lipids and the formation of foam cells.
Assuntos
Lipase Lipoproteica/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneais/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Aminoácidos/farmacologia , Animais , Ligação Competitiva , Bovinos , Células Cultivadas , Creatina Quinase/genética , Deleção de Genes , Heparina Liase/metabolismo , Humanos , Cinética , Lipase Lipoproteica/genética , Lipase Lipoproteica/isolamento & purificação , Lipase Lipoproteica/farmacologia , Macrófagos Peritoneais/enzimologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas do Leite/genética , Proteínas do Leite/isolamento & purificação , Proteínas do Leite/metabolismo , Proteínas do Leite/farmacologia , Músculos/enzimologia , Especificidade de Órgãos , Oxirredução , Ligação Proteica/efeitos dos fármacosRESUMO
A more negatively charged low-density lipoprotein (LDL), named minor LDL (mi-LDL), was separated by ionic exchange chromatography and further characterized. This mi-LDL contained lower amounts of polyunsaturated fatty acids, alpha- or gamma- tocopherol, but higher amounts of lipid hydroperoxides than the major LDL fraction (ma-LDL). We show here for the first time that apoB of mi-LDL is modified by lipid peroxidation products, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). Using polyclonal antibodies, generated against 4-HNE- or MDA-LDL and apoB, the ratio of 4-HNE- or MDA-derived epitopes to apoB of mi-LDL and ma-LDL was estimated by means of a solid phase fluorescence immunoassay. The ratio of 4-HNE-derived epitopes to apoB on mi-LDL was fourfold higher, while the ratio of MDA-derived epitopes to apoB was twofold higher, compared with the ratios obtained with ma-LDL. In a competition assay with mi- and ma-LDL, only mi-LDL was an effective competitor to inhibit the immunoreaction of anti-4-HNE-LDL with 4-HNE-LDL (by 24%) and of anti-MDA-LDL with MDA-LDL (by 10%).
Assuntos
Aldeídos/imunologia , Epitopos , Lipoproteínas LDL/imunologia , Antioxidantes/metabolismo , Doadores de Sangue , Fracionamento Químico , Cromatografia por Troca Iônica , Eletroquímica , Ácidos Graxos/sangue , Fluorometria , Radicais Livres , Humanos , Imunoensaio/métodos , Peróxidos Lipídicos/sangue , Malondialdeído/imunologiaRESUMO
Europium-labeled native and oxidized low-density lipoprotein (LDL) were used to measure their binding and cell association to mouse peritoneal macrophages, to suspended human monocyte cell line THP-1 cells, and to differentiated THP-1 macrophages. Cell binding and association were concentration dependent and saturable and showed the characteristics of ligand-receptor interaction. The validity of this assay was also supported by comparison with the method using 125iodine-labeled LDL. This nonradioactive assay proved to be specific, sensitive and simple and avoided any potential lipid peroxidation of LDL brought about by labeling lipoproteins with the widely used radioactive iodine. The latter fact is very important in studying lipoprotein-receptor interactions.
Assuntos
Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Animais , Células Cultivadas , Fluorometria , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/metabolismoRESUMO
The changes in the immunological properties of apolipoprotein AI (apo-AI) and AII (apo-AII) during the oxidation of the high-density lipoprotein HDL3 and its influence on the binding of heavily oxidized low-density lipoprotein (LDL) to type I and III collagen were investigated. Oxidation of HDL3 or Eu3+-labelled HDL3 was performed with CuSO4, varying the time of oxidation. Oxidation of HDL3 resulted in an increase in lipid hydroperoxides and enhanced the negative charge of this lipoprotein. Immunological studies with a solid-phase sandwich immunoassay revealed a strong increase in binding of Eu3+-labelled HDL3 to polyclonal antibodies against apo-AI and apo-AII within the first 4 h of oxidation. Neo-epitopes were also formed by interaction of the apolipoproteins with degradation products from the lipid peroxidation of polyunsaturated fatty acids, as evidenced by an immunoreaction of oxidized Eu3+-labelled HDL3 with antibodies to 4-hydroxynonenal (4-HNE)- and malondialdehyde (MDA)-protein adducts. Western blot analysis of oxidized HDL3 samples showed, as well as apo-AI and apo-AII bands, larger aggregated apolipoproteins, occurring after 0.5-2.5 h of oxidation. These aggregates were recognized by antibodies to apo-AI and apo-AII as well as by antibodies to 4-HNE- and MDA-protein adducts. Furthermore the original apo-AI monomers and apo-AII dimers decreased during the oxidation. The ability of native and oxidized HDL3 to prevent the binding of Eu3+-labelled 24 h-oxidized LDL to collagen on microtitration plates was estimated. Interestingly, 2 h-oxidized HDL3 competed most with the binding of 24 h-oxidized LDL on collagen type I and type III, followed by native HDL3. However, 24 h-oxidized HDL3 was a weaker competitor. Thus oxidative modification of HDL3 strongly alters the immunological properties of this lipoprotein and its binding affinity for collagen.
Assuntos
Apolipoproteína A-II/imunologia , Apolipoproteína A-I/imunologia , Colágeno/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Aldeídos , Anticorpos , Ligação Competitiva , Epitopos/imunologia , Humanos , Peroxidação de Lipídeos , Lipoproteínas HDL/imunologia , Lipoproteínas HDL3 , MasculinoRESUMO
The accumulation of LDL in the arterial intima is considered a key event in atherogenesis. We investigated the binding of oxidized LDL (ox-LDL) to microtiter plates coated with type I or II collagen, laminin, fibronectin, or poly-D-lysine. Oxidation of LDL, 125I-LDL, or Eu(3+)-LDL was performed with CuCl2, varying the time of oxidation. Bound lipoprotein was assessed by counting radioactivity or fluorescence in the wells. Binding of highly ox-LDL in PBS followed the order: type I collagen > poly-D-lysine > type II collagen > laminin > fibronectin. Comparing various collagen types, the binding of ox-LDL followed the order: type I > type V and, type III > type IV > type II collagen. Binding of ox-LDL in PBS was dependent on an increase in negative charge of ox-LDL. Testing certain amino acids as competitors for binding of highly ox-LDL to type I collagen put lysine first, followed by arginine and histidine. On laminin, histidine competed most, followed by lysine and arginine. When studying the influence of Na+, K+, Ca2+, Mg2+ (equivalent to their concentrations in the interstitial fluid), native LDL, moderately ox-LDL, and highly ox-LDL showed the same affinity to type I collagen. However, a fivefold dilution of the buffer increased the affinity of moderately and highly ox-LDL 3.9- and 10-fold compared with native LDL. Application of the F(ab')2 from a monoclonal antibody to ox-LDL revealed a strong competition of the binding of highly ox-LDL to type II collagen (60%), laminin (35%), type I collagen (20%), and poly-D-lysine (15%), whereas the binding to fibronectin was not affected.
Assuntos
Colágeno/metabolismo , Fibronectinas/metabolismo , Laminina/metabolismo , Lipoproteínas LDL/metabolismo , Polilisina/metabolismo , Aldeídos/imunologia , Aldeídos/farmacologia , Aminoácidos/metabolismo , Animais , Anticorpos/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Ligação Competitiva , Cátions/metabolismo , Bovinos , Cromograninas/imunologia , Colágeno/classificação , Epitopos/imunologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunoglobulina G/imunologia , Cinética , Peroxidação de Lipídeos , Lipoproteínas LDL/efeitos dos fármacos , Lipoproteínas LDL/imunologia , Malondialdeído/imunologia , Malondialdeído/farmacologia , Camundongos , Ligação Proteica , Coelhos , RadioimunoensaioRESUMO
BACKGROUND AND PURPOSE: Serum lipoprotein(a) [Lp(a)] levels are genetically determined and considered to be an independent risk factor for atherosclerosis. The aim of this study was to provide a complete analysis of Lp(a) serum levels, apolipoprotein(a) phenotypes, and other lipid parameters for different forms of severity of symptomatic ischemic cerebrovascular disorders as well as for different stages of carotid atherosclerosis. METHODS: Lp(a) concentration, apolipoprotein(a) phenotype, triglyceride, low-density lipoprotein, high-density lipoprotein, and total cholesterol levels of blind-coded specimens as well as degree of carotid artery stenosis were assessed in a consecutive series of patients with ischemic cerebrovascular disease. We evaluated 265 male (34%) and female (66%) patients (mean age, 51 +/- 7.4 years) with transient ischemic attack (55.8%), prolonged reversible ischemic neurological deficits (28.3%), and cerebral infarction (15.9%) as well as 288 male (30%) and female (70%) control subjects (mean age, 51 +/- 7.1 years). All subjects were white. RESULTS: Lp(a), total, and low-density lipoprotein cholesterol were statistically significantly elevated in all patients compared with control subjects. Lp(a) correlated with the severity of symptomatic cerebrovascular disease and the degree of carotid stenosis. Logistic regression analysis revealed Lp(a) as the best single marker for the presence of cerebrovascular disease (P < .001) followed by high-density lipoprotein cholesterol (P = .003) and triglycerides (P = .049). With a cutoff of 20 mg/dL of Lp(a), the odds ratio for a subject to have had ischemic stroke with elevated Lp(a) was 20.3 and 23.7 depending on the method of the Lp(a) estimation, whereas the odds ratio when the sonography score was > 0 was 15.4. The investigation of the distribution of the apo(a) phenotypes revealed that 16.73% of the control subjects had major isoforms < or = 580 kD molecular weight (B, F, S1, S2) versus 42.65% of the patients' group (P < .001). These isoforms were also present in 14.71% of all individuals with a sonography score of 0 but in 52.30% of all individuals with a sonography score > 0 (P < .001). CONCLUSIONS: This case-control study shows that an elevated Lp(a) level is the primary factor associated with the presence of ischemic cerebrovascular disease and that the increased portion of the smaller-molecular-weight apo(a) isoforms in patients and individuals with a sonography score > 0 points toward an inherited predisposition for this disease.