RESUMEN
Although plasma carbohydrate-deficient transferrin (CDT) is considered a viable biochemical marker for chronic alcohol consumption, it is valid only when an individual's daily alcohol consumption exceeds 60 g. In addition, it is less sensitive in women drinkers than in men drinkers. We have established that chronic alcohol consumption impairs the hepatic sialylation of a number of glycoproteins by specifically down-regulating Gal-beta-1,4GlcNAc alpha2,6-sialyltransferase mRNA. Significantly, we found that chronic ethanol consumption markedly inhibits hepatic sialylation of apolipoprotein J (Apo J), a 70-kDa N-glycosylated protein of plasma HDL. Because the sialic-acid index of Apo J (SIJ; moles of sialic acid per mole of Apo J protein) is approximately seven times more than that for transferrin (28 vs. 4), we have evaluated whether plasma SIJ would be an even more sensitive marker for chronic ethanol consumption than CDT in both rats and human subjects. The method involves immunoaffinity purification of plasma HDL-Apo J, followed by its sialic acid determination. We have found that chronic ethanol feeding resulted in loss of sialic acid residues of plasma HDL-Apo J in rats. This loss of sialic acid was positively correlated with both amount and duration of ethanol treatment. In human subjects, an intake of about 60 g of alcohol for 30 days led to almost 50% (P <.01) depletion of sialic acid from plasma HDL-Apo J. Further, we established that there was a positive correlation of alteration in SIJ with alcohol consumption, detoxification, abstinence, and relapse in human alcohol-dependent patients (sensitivity, 90%-92%). In addition, plasma SIJ was decreased by 50%-57% (P <.01) in both male and female alcohol-dependent subjects. We suggest that plasma SIJ can be used as a viable marker for early detection of chronic alcohol consumption in human beings.
Asunto(s)
Consumo de Bebidas Alcohólicas/sangre , Alcoholismo/sangre , Glicoproteínas/sangre , Chaperonas Moleculares/sangre , Ácido N-Acetilneuramínico/sangre , Alcoholismo/diagnóstico , Análisis de Varianza , Animales , Biomarcadores/sangre , Clusterina , Femenino , Humanos , Masculino , Ratas , Ratas Wistar , Templanza/estadística & datos numéricosRESUMEN
BACKGROUND: Plasma apolipoprotein (apo) E, a sialoprotein, plays an important role in reverse cholesterol transport. Previously, we showed that chronic alcohol consumption impairs glycosylation of apo E in rat liver. Peritoneal macrophages are another significant apo E synthesis site. OBJECTIVE: The main purpose of this study was to determine the effects of chronic alcohol feeding of rats on the synthesis, sialylation, and sialic acid content of macrophage apo E and its ability to bind to the HDL(3) molecule in vitro. DESIGN: Rats were fed an alcoholic diet or an isoenergetic control diet for 8 wk, after which peritoneal macrophages isolated from them were cultured and analyzed for apo E metabolism. RESULTS: Macrophages from alcohol-fed rats accumulated 33.3% more (P < 0.05) cholesterol than did those from control rats when incubated with acetylated LDL. These macrophages showed a 51-57% lower relative sialylation rate of apo E (P < 0.001) but no significant difference in relative protein synthetic rate. The sialic acid content of the intracellular and secreted forms of apo E was reduced by 41.8% (P < 0.001) and 50.3% (P < 0.001), respectively, with chronic alcohol treatment. Secretion of newly synthesized apo E was impaired by 53.7% (P < 0.001) and 26. 1% (P < 0.001) in the absence and presence of HDL in the medium, respectively. Macrophages of alcohol-treated rats secreted apo E with 47.6-67.2% lower (P < 0.001) HDL(3) binding ability; binding ability was restored completely by resialylation of the desialylated apo E. CONCLUSION: In rats, an alcohol-mediated decrease in sialylation rate resulting in loss of sialic acid residues in apo E impairs the ability of apo E to bind to HDL and consequently in defective reverse cholesterol transport.
Asunto(s)
Apolipoproteínas E/biosíntesis , Apolipoproteínas E/química , Dieta , Etanol/administración & dosificación , Macrófagos Peritoneales/metabolismo , Animales , Apolipoproteínas E/sangre , Fraccionamiento Celular , Células Cultivadas , Etanol/farmacología , Glicosilación , Aparato de Golgi/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/ultraestructura , Masculino , Microsomas/metabolismo , Ácido N-Acetilneuramínico/análisis , Ácido N-Acetilneuramínico/metabolismo , Ratas , Ratas WistarRESUMEN
OBJECTIVE: We sought to determine whether placental cytokine expression is altered in patients with preeclampsia. STUDY DESIGN: Whole placental tissue was collected at cesarean delivery, and total ribonucleic acid was extracted. Reverse transcriptase-polymerase chain reaction was performed to determine cytokine expression. Product bands were quantitated by scanning densitometry, and results were expressed as a ratio of cytokine/housekeeping gene (cytokine expression index). Statistical analysis was performed by the Student t test and the Mann-Whitney U test. RESULTS: Placentas from 6 patients with preeclampsia and 4 normotensive patients were analyzed. Placental expression of interleukin 1beta and interleukin 10 was greater in preeclamptic women than in normotensive subjects (median interleukin 1beta cytokine expression index, 0.675; range, 0.394-0. 953; vs 0.106; range, 0.084-0.166; P =.011; median interleukin 10 cytokine expression index, 1.042; range, 0.672-1.192; vs 0.126; range, 0.062-0.398; P <.011). Tumor necrosis factor alpha messenger ribonucleic acid was detected in placentas of preeclamptic subjects but not in normotensive control subjects. CONCLUSION: Placentas from preeclamptic patients demonstrated increased expression of interleukin 1beta, interleukin 10, and tumor necrosis factor alpha. This may be in association with placental hypoxia and may contribute to the global endothelial dysfunction observed in preeclampsia.
Asunto(s)
Expresión Génica , Interleucina-10/genética , Interleucina-1/genética , Placenta/metabolismo , Preeclampsia/metabolismo , Factor de Necrosis Tumoral alfa/genética , Adulto , Presión Sanguínea , Femenino , Retardo del Crecimiento Fetal/complicaciones , Humanos , Interleucina-2/genética , Interleucina-6/genética , Oligohidramnios/complicaciones , Preeclampsia/complicaciones , Embarazo , ARN Mensajero/análisisRESUMEN
We have previously shown that chronic ethanol treatment impairs the glycosylation of proteins in the rat liver. Changes in the microheterogeneity of transferrin, a N-sialoprotein under chronic alcohol consumption are well established. Apolipoprotein J, another N-glycoprotein, is a normal component of plasma high-density lipoproteins in the rat and human. Apo J is also highly sialylated and, thus, may be vulnerable to the deleterious actions of ethanol. Therefore, to understand the specific nature of alterations of Apo J sialylation as a consequence of chronic ethanol treatment, we have determined: (1) the sialylation index of Apo J (moles sialic acid per mole Apo J protein) in rats administered ethanol for 4, 6, and 8 weeks and a gradual withdrawal and a follow-up abstinence for 1, 2, and 4 weeks; and (2) enzymatic activities of hepatic sialyltransferase and plasma sialidase during the same periods of alcohol treatment and abstinence in rats. Although no significant differences in the Apo J sialylation index between rats of the control and ethanol groups were found at the 4th week of alcohol treatment, a highly significant loss of 24% (p < 0.001) and 44% (p < 0.001) was found after 6 and 8 weeks, respectively, of alcohol feeding of these animals. Furthermore, a significant recovery of 38% (p < 0.001), 78% (p < 0.001), 84% (p < 0.001) and 96% (p < 0.001) in the sialylation index of Apo J were found, respectively, during withdrawal and 1, 2, and 4 weeks of subsequent alcohol abstinence in these animals. These changes in the sialic acid content of Apo J were accompanied by a similar pattern of changes in the enzyme activities of hepatic sialyltransferase and plasma sialidase in animals undergoing chronic ethanol treatment, withdrawal, and abstinence periods. The analysis of the sialylation index of Apo J seems to be a simple and feasible method to use to evaluate the extent of ethanol exposure.
Asunto(s)
Consumo de Bebidas Alcohólicas/metabolismo , Etanol/farmacología , Glicoproteínas/sangre , Chaperonas Moleculares , Proteínas y Péptidos Salivales/sangre , Animales , Clusterina , Glicoproteínas/química , Masculino , Ácido N-Acetilneuramínico/análisis , Ratas , Ratas Wistar , Proteínas y Péptidos Salivales/químicaRESUMEN
Gangliosides are sialo-glycosphingolipids that play important roles in the interaction of cells with their environment and are thus involved in the regulation of many cellular events. Sialic acid residues are important for the conformation of a glycomolecule, their structural stability and their functions. Although decreased brain ganglioside sialic acid has been previously reported as a result of chronic ethanol treatment in rats, no reports are available on the sialylation of specific gangliosides and/or the mechanism leading to depletion of their sialic acid residues. Therefore, in this investigation, we have examined the effects of chronic ethanol treatment on (1) incorporation of [4,5-3H]N-acetylmannosamine (ManNAc) into specific rat brain gangliosides, GD3, GD1a, GT1a, and GT1b; and (2) enzymatic activities of brain sialyltransferase and sialidase at specific subcellular levels. The experiments were done in male Wistar rats pair-fed with either ethanol or control liquid diets for a period of 8 weeks. The rats were intracerebroventricularly injected with labeled ManNAc (30 microCi/rat) and killed after 90 min. Radioactivity was determined in respective ganglioside bands separated on a thin layer chromatography system. Specific activities of sialyltransferase and sialidase were assessed using GM3 and GD3 as substrates, respectively. The results showed significant decreases of 57.7% (p < 0.001) and 68.9% (p < 0.001), respectively, in the labeled ManNAc incorporation into GD3 and GD1a fractions in rats of the ethanol group, compared with rats of the control group. No significant changes were noted in the incorporation of labeled ManNAc into GT1a or GT1b ganglioside fractions between the ethanol and control groups. Concomitantly, compared with control rats, a decrease of 18.9% (p < 0.05), 20.6% (p < 0.05), and 15.8% (p < 0.001) was found in the sialyltransferase activity, respectively, at the whole brain, and brain Golgi and synaptosomal levels. However, dramatic increases of 32.4% (p < 0.05), 105% (p < 0.001), and 150% (p < 0.001) in sialidase activity were found, respectively, at the whole brain and brain cytosol and synaptosomal fractions of rat treated chronically with ethanol. Thus, we conclude that the deleterious actions of ethanol on the sialylation of rat brain gangliosides is specific, and the reduced sialic acid label found in GD3 and GD1a in this study is mainly due to increased activity of brain sialidase. Furthermore, the study reaffirms our tenet that, regardless of whether it is the liver or the brain, glycosylation cascade is one of the main target of the deleterious attacks of ethanol.
Asunto(s)
Alcoholismo/enzimología , Encéfalo/efectos de los fármacos , Etanol/toxicidad , Gangliósidos/metabolismo , Neuraminidasa/fisiología , Sialiltransferasas/fisiología , Animales , Encéfalo/enzimología , Glicosilación/efectos de los fármacos , Hexosaminas/metabolismo , Masculino , Ácido N-Acetilneuramínico/metabolismo , Ratas , Ratas Wistar , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimologíaRESUMEN
Clusterin is a N-glycosylated sialoglycoprotein present in rat brain cells. Clusterin, which elicits aggregation in a wide variety of cells, has been suggested to play an important role in synaptic remodeling through its cell adhesion property or lipid transport capacity in the brain. Sialic acid residues in clusterin may be responsible for its structural conformation, stability and functional ability. Maturation of clusterin is governed by the relative actions of sialyltransferases and sialidases that are present in brain microsomes, golgi bodies, cytosol and plasma membranes. We have earlier reported that chronic ethanol treatment in rats has a damaging effect on the hepatic glycosylation machinery. Others have reported increased hydrolysis of brain sialoconjugates in rats following chronic ethanol administration. Specificity of the effects of chronic ethanol treatment in the brain in relation to the glycosylation process, is still obscure. Therefore, in this investigation, we have studied the specific effects of chronic ethanol treatment on the glycosylation of rat brain clusterin and the causes that may lead to any possible defects in the glycosylation process. We have determined the effects of chronic ethanol treatment on (i) the incorporation of labeled leucine and N-acetylmannosamine into immunoprecipitable clusterin in whole brain homogenate, microsomes, golgi, cytosol, plasma membrane and synaptosomes, (ii) enzymatic activities of sialyltransferases in golgi and synaptosomes, and sialidase in brain cytosol and plasma membranes, and (iii) de novo synthetic rate of rat brain cytosolic sialidase. Our results showed that chronic ethanol treatment in rats resulted in (1) a decreased sialation index of brain clusterin by 47. 2% (p<0.001), 56.7% (p<0.05), 51.7% (p<0.05), 64.8% (p<0.001), and 54.5% (p<0.05), respectively, in whole brain homogenate, golgi, cytosol, plasma membranes, and synaptosomes; (2) a 46.1% (p<0.05) and 12.5% (p<0.05) decreased activities of brain sialyltransferases, respectively, in the golgi and the synaptosomal fractions; (3) a 70. 1% (p<0.05) and 42.6% (p<0.05) increased activities of sialidases, respectively, in the cytosol and plasma membrane fractions; and (4) a 22.2%-64.3% (p<0.001) increased incorporation of labeled leucine into brain cytosolic sialidase. Our findings have clearly established that long-term ethanol treatment in rats leads to a marked impairment in the glycosylation of rat brain clusterin as a result of altered activities of brain sialation and desialation enzymes. In particular, the specific increase noted in brain sialidase activity was due to concomitant increases in its synthetic rate. These defects in the glycosylation of brain clusterin may lead to changes in the molecular conformation of clusterin, and thus, may result in its structural instability and/or functional impairment.