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PURPOSE: To evaluate at 11-13 weeks' gestation biochemical markers that may predict complications of pregnancy such as pre-eclampsia, proteinuria, and hypertension. METHODS: Analyses were performed on first-morning urine and plasma samples from first trimester pregnant women with increased risk of developing pre-eclampsia such as positive personal or family history of cardiovascular disease and diabetes mellitus. A total of 62 women were enrolled, 24 of them presented complications such as pre-eclampsia, proteinuria, and hypertension during pregnancy. The remaining 38 women had a physiological course of pregnancy and formed the reference group. Urine glycosaminoglycans/proteoglycans (GAGs/PGs) distribution was determined by electrophoresis on cellulose acetate strips. Urinary N-acetyl-ß-glucosaminidase was estimated kinetically. Plasma levels of placental protein 13 (PP13) were measured by enzyme-linked immunosorbent assay. RESULTS: No significant differences in total GAG excretion and N-acetyl-ß-glucosaminidase (NAG) concentration were observed between the two groups of pregnant women, whereas we detected increased relative content of total urinary trypsin inhibitor (UTI plus low-sulfated chondroitin sulfate) (p = 0.001) and reduced excretion of heparan sulfate (p = 0.007) and chondroitin sulfate (p = 0.011) in women presenting with pregnancy complications respect to controls. Plasma levels of PP13 were significantly reduced in the group of women who went on to develop complications compared with controls (p = 0.022). CONCLUSIONS: The reduced plasma levels of PP13 and the alteration of the relative content of urinary GAGs and PGs observed in our study could be a promising tool for the prediction of pre-eclampsia in an early stage of pregnancy.
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Galectinas/urina , Glicosaminoglicanos/urina , Pré-Eclâmpsia/urina , Proteínas da Gravidez/urina , Proteoglicanas/urina , Adulto , Biomarcadores , Feminino , Humanos , GravidezRESUMO
AIM: The aim of this study was to assess the influence of glucose metabolism on the expression of glycosaminoglycans (GAGs) and proteoglycans (PGs) in pregnant women. MATERIAL AND METHODS: Seventy-six women in the first trimester of pregnancy (10-13 weeks) attending the Gynecologic and Obstetric Clinic, University of Sassari, were enrolled and gave early morning urine samples. Groups I, II and III included women with serum glucose values of 65-89 mg/dL, 90-99 mg/dL and 100-125 mg/dL, respectively. Urine GAGs/PGs distribution was determined by electrophoresis on cellulose acetate strips. Urinary N-Acetyl-ß-glucosaminidase was estimated kinetically. RESULTS: Analysis of urinary GAGs/PGs electrophoretic profiles showed a significant increase in heparan sulfate (HS) excretion (P = 0.017) as well as a reduced chondroitin sulfate (CS) excretion (P = 0.048) in the group II pregnant women compared with the group I, and higher values of the HS/CS ratio in groups II and III compared to group I. Furthermore, we observed a positive correlation among fasting blood glucose levels and the relative content of HS, the HS/CS and urinary trypsin inhibitor/CS ratios, and the N-Acetyl-ß-glucosaminidase levels. CONCLUSIONS: The assessment of risk factors for gestational diabetes mellitus should also take into account fasting blood glucose values of 90-99 mg/dL, as the findings of our study indicated an alteration in the metabolism of GAGs during the early stages of pregnancy.
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Glicemia/metabolismo , Diabetes Gestacional/urina , Glicosaminoglicanos/urina , Primeiro Trimestre da Gravidez/urina , Proteoglicanas/urina , Adulto , Diabetes Gestacional/sangue , Jejum/sangue , Jejum/urina , Feminino , Glicoproteínas/urina , Humanos , Gravidez , Primeiro Trimestre da Gravidez/sangue , Estudos ProspectivosRESUMO
Bikunin is a plasma proteinase inhibitor often associated with inflammatory conditions. It has a half-life of few minutes and it is rapidly excreted into urine as urinary trypsin inhibitor (UTI). UTI levels are usually low in healthy individuals but they can increase up to tenfold in both acute and chronic inflammatory diseases. This article describes a sensitive method for both direct UTI quantitation and structural characterization. UTI purification was performed by anion exchange micro-chromatography followed by SDS-PAGE. A calibration curve for protein quantitation was set up by using a purified UTI fraction. UTI identification and structural characterization was performed by Nano-LC-MS/MS analysis. The method was applied on urine samples from 9 patients with type 1 diabetes, 11 patients with type 2 diabetes, and 28 healthy controls, matched for age and sex with patients, evidencing higher UTI levels in both groups of patients with respect to controls (p < 0.001 and p = 0.001, respectively). Spearman's correlation tests highlighted no association between UTI levels and age in each group tested. Owing to the elevated sensitivity and specificity, the described method allows UTI quantitation from very low quantities of specimen. Furthermore, as UTI concentration is normalized for creatinine level, the analysis could be also performed on randomly collected urine samples. Finally, MS/MS analysis prospects the possibility of characterizing PTM sites potentially able to affect UTI localization, function, and pathophysiological activity. Preliminary results suggest that UTI levels could represent a useful marker of chronic inflammatory condition in type 1 and 2 diabetes.
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alfa-Globulinas/química , alfa-Globulinas/urina , Diabetes Mellitus Tipo 1/urina , Diabetes Mellitus Tipo 2/urina , Adulto , Idoso , Sequência de Aminoácidos , Biomarcadores/urina , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Humanos , Modelos Lineares , Pessoa de Meia-Idade , Dados de Sequência Molecular , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
We describe a new method for the quantification of low molecular weight thiols, as homocysteine, cysteine, cysteinylglycine, glutamylcysteine and glutathione bound to human plasma albumin. After albumin isolation and purification by SDS-PAGE, thiols were freed from protein with tri-n-butylphosphine and successively derivatized with 5-iodoacetamidofluorescein. Samples were then injected and quantified in about 18 min by CE with laser induced fluorescence detection. Precision tests indicate a good repeatability of the method both for migration times (RSD<0.63%) and areas (RSD<2.98%). The method allows to measure all five low molecular weight thiols released from just 3 microg of albumin thus improving the other described methods in which only three or four thiols were detected. Due to the elevated sensitivity (LOD of 0.3 pM for all thiols), also low molecular weight thiols bound to albumin filtered in tissues could be quantified.
Assuntos
Albuminas/metabolismo , Estenose das Carótidas , Eletroforese Capilar/métodos , Compostos de Sulfidrila/sangue , Albuminas/química , Eletroforese em Gel de Poliacrilamida/métodos , Fluoresceínas/química , Humanos , Limite de Detecção , Peso Molecular , Compostos de Sulfidrila/químicaRESUMO
PURPOSE: To assess the plasma levels of the inflammatory markers such as C-reactive protein (CRP), leptin, and glycosaminoglycans (GAGs) during the menstrual cycle. METHODS: Eighteen healthy volunteers were divided into two groups according to the presence of ovulatory or anovulatory menstrual cycles. Blood samples were collected at different time points: at the menstrual phase (days 2-3), periovulatory phase (days 12-13), and luteal phase (days 23-24). CRP and leptin concentrations were measured by enzyme immunoassay. GAGs were isolated using ion-exchange chromatography on DEAE-Sephacel and quantified as hexuronate. The structural characterization of chondroitin sulfate (CS) isomers was performed by fluorophore-assisted carbohydrate electrophoresis (FACE). RESULTS: In the women with ovulatory cycles, plasma GAG levels differed significantly during menstrual cycle, with increased values at the periovulatory with respect to the menstrual phase. No significant differences in CRP and leptin concentrations were observed through the menstrual cycle in both the examined cycles, but inter-group analysis revealed significant differences of CRP and leptin levels between the ovulatory and anovulatory cycles with higher values at periovulatory phase in the ovulatory cycles. CONCLUSIONS: There are no fluctuations of both total GAG concentration and CS isomer content during menstrual cycle in the anovulatory cycles. A significant correlation between CRP and gonadotrophins was found. There is no significant difference in CRP across the menstrual cycle among ovulatory cycles, but there is a trend toward higher CRP at the periovulatory than the other phases, consistent with the significant difference in CRP between ovulatory and anovulatory cycles at the periovulatory phase. Both the trend and the significant result suggest an elevation in CRP with ovulation. These observations provide additional evidences to the hypothesis that the ovulation is an inflammatory-like phenomenon.
Assuntos
Anovulação/sangue , Proteína C-Reativa/análise , Glicosaminoglicanos/sangue , Leptina/sangue , Ciclo Menstrual/sangue , Ovulação/sangue , Adulto , Estradiol/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Humanos , Hormônio Luteinizante/sangue , Progesterona/sangue , Adulto JovemRESUMO
PURPOSE: Female reproductive events, including ovulation, menstruation, implantation, and delivery, are physiologically characterized by deep tissue remodeling and display hallmark signs of inflammation. This review discusses the pleiotropic roles played by bikunin in human reproduction. METHODS: A comprehensive literature search of the Medline/PubMed database was performed on the following topics: bikunin structure, roles in pathophysiological conditions and involvement in human reproduction, and usefulness as a marker of gestational complications or as a drug to improve pregnancy outcomes. RESULTS: Bikunin is a small chondroitin sulfate proteoglycan found in blood, urine, and amniotic and cerebrospinal fluids, known for its anti-inflammatory and anti-proteolytic activities. Its levels are usually low, but they can increase several-fold in both acute and chronic inflammatory diseases. Bikunin plays key roles in reproductive events, such as cumulus-oocyte complex formation, pregnancy, and delivery. Its levels have been associated with the most common pregnancy complications such as preterm delivery, pre-eclampsia, and gestational diabetes mellitus. Finally, its intravaginal administration has been reported to reduce the risk of preterm delivery and to improve neonatal outcomes. CONCLUSIONS: Because of its pleiotropic roles in several reproductive events and its association with some life-threatening pathological conditions of pregnancy, bikunin may represent a non-invasive marker for improving follow-up and early diagnosis. Studies showing its usefulness as a drug for reducing the risk of preterm delivery and improving neonatal outcomes have yielded interesting results that deserve to be investigated through further research.
Assuntos
alfa-Globulinas/metabolismo , alfa-Globulinas/uso terapêutico , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Trabalho de Parto Prematuro/tratamento farmacológico , Complicações na Gravidez/metabolismo , Inibidores de Proteases/uso terapêutico , Fenômenos Reprodutivos Fisiológicos , Feminino , Humanos , GravidezRESUMO
Myasthenia gravis (MG) is an autoimmune disease leading to varying degrees of skeletal muscle weakness. It is caused by specific antibodies directed against definite components in the postsynaptic membrane at the neuromuscular junction (NMJ), such as the acetylcholine receptor (AChR) and the muscle-specific kinase (MUSK) receptor. In clinical practice, MG patients may be classified into three main subgroups based on the occurrence of serum autoantibodies directed against AChR or MUSK receptor or antibody-negative. As the MG subgroups differ in terms of clinical characteristics, disease pathogenesis, prognosis, and response to therapies, they could benefit from targeted treatment as well as the detection of other possible disease biomarkers. We performed proteomics on plasma fractions enriched in low-abundance proteins to identify potential biomarkers according to different autoimmune responses. By this approach, we evidenced a significant reduction of vitronectin in MG patients compared to healthy controls, irrespective of the autoantibodies NMJ target. The obtained results were validated by mono- and two-dimensional Western blotting analysis. Vitronectin is a multifunctional glycoprotein involved in the regulation of several pathophysiological processes, including complement-dependent immune response, coagulation, fibrinolysis, pericellular proteolysis, cell attachment, and spreading. The pathophysiological significance of the reduction of plasma vitronectin in MG patients has yet to be fully elucidated. It could be related either to a possible deposition of vitronectin at NMJ to counteract the complement-mediated muscle damage at this level or to a parallel variation of this glycoprotein in the muscle extracellular matrix with secondary induced alteration in clustering of AChRs at NMJ, as it occurs with variation in concentrations of agrin, another extracellular matrix component. The clinical value of measuring plasma vitronectin has yet to be defined. According to present findings, significantly lower plasma values of this glycoprotein might be indicative of an impaired complement-dependent immune response.
RESUMO
BACKGROUND: Diabetes mellitus is a global health problem representing the fifth leading cause of mortality and a major risk factor for cardiovascular diseases. In the last years, we reported an association among urinary trypsin inhibitor (UTI), a small proteoglycan that plays pleiotropic roles in many inflammatory processes, and both type 1 and 2 diabetes and developed a method for its direct quantitation and structural characterization. METHODS: Urine from 39 patients affected by type 1 diabetes, 32 patients with type 2 diabetes, and 52 controls were analysed. UTI was separated from the main glycosaminoglycans physiologically present in urine by anion exchange chromatography, treated for chondroitin sulphate (CS) chain complete depolymerisation, and analysed for both UTI content and CS structure. UTI identification was performed by nano-LC-MS/MS analysis. RESULTS: We evidenced increased UTI levels, as well as reduced sulphation of its CS moiety in association with diabetes, regardless of both age and medium-term glycaemic control. Furthermore, no association between UTI and albumin excretion rate was found. CONCLUSIONS: Evidences suggest that UTI levels are not directly correlated with renal function or, otherwise, that they may increase before the onset of renal impairment in diabetes, representing a potential marker for the underlying inflammatory condition.
Assuntos
Sulfatos de Condroitina/urina , Diabetes Mellitus Tipo 1/urina , Diabetes Mellitus Tipo 2/urina , Glicoproteínas/urina , Adolescente , Adulto , Idoso , Biomarcadores/urina , Carboidratos/análise , Carboidratos/urina , Estudos de Casos e Controles , Sulfatos de Condroitina/química , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/diagnóstico , Nefropatias Diabéticas/urina , Eletroforese/métodos , Feminino , Glicoproteínas/química , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência Renal/complicações , Insuficiência Renal/urina , Urinálise/métodos , Adulto JovemRESUMO
Because of the high incidence of kidney disease in diabetic patients, the early diagnosis of renal impairment is a key point for intervention and management. Although urinary albumin excretion currently represents the accepted standard to assess both diabetic nephropathy and cardiovascular risk, it has some limitations as structural changes in the glomerular basement membrane may occur before the onset of microalbuminuria. It is therefore important to identify urinary markers that may provide greater sensitivity, earlier detection, and greater predictive power for diabetes complications. In this respect, urinary glycosaminoglycans/proteoglycans (GAGs/PGs) have been long associated with several kidney diseases as well as diabetic nephropathies as their levels increase more readily than albuminuria. In particular, heparan sulfate, a key component of the glomerular basement membrane responsible for its charge-dependent permeability, is excreted into urine at higher concentrations during the early kidney remodeling events caused by the altered glucose metabolism in diabetes. Over the past few years, also urinary trypsin inhibitor has been linked to a chronic inflammatory condition in both type 1 and 2 diabetes. The underlying mechanisms of such increase are not completely known since either a systemic inflammatory condition or a more localized early renal impairment could play a role. Nevertheless, the association with other inflammatory markers and a detailed urinary trypsin inhibitor structural characterization in diabetes remain to be elucidated. This review will discuss a great deal of information on the association between urinary GAGs/PGs and type 1 and 2 diabetes, with particular emphasis on renal involvement, and their potential as markers useful in screening, diagnosis and follow up to be associated with the current standard tests.
Assuntos
Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/urina , Glicosaminoglicanos/urina , Rim/fisiopatologia , Proteoglicanas/urina , Insuficiência Renal/urina , Animais , Biomarcadores/urina , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/fisiopatologia , Progressão da Doença , Glicosaminoglicanos/sangue , Humanos , Proteoglicanas/sangue , Insuficiência Renal/sangue , Insuficiência Renal/fisiopatologiaRESUMO
Besides hyperglycaemia and insulin resistance, several factors are associated with a higher cardiovascular risk in type 2 diabetes mellitus (T2DM), many of them being closely related to each other owing to common origins or pathways. The pathophysiological mechanisms underlying vascular dysfunctions in diabetes include reduced bioavailability of nitric oxide, increased ROS and prothrombotic factors production, as well as activation of receptors for advanced glycation end-products. These alterations contribute to create a pro-inflammatory/thrombotic state that ultimately leads to plaque formation and complication. This study aimed at identifying differentially expressed plasma proteins between T2DM and non-diabetic patients undergoing carotid endarterectomy, by means of two-dimensional electrophoresis coupled with LC-MS/MS. Before analysis, plasma samples were enriched in low-expression proteins through combinatorial hexapeptide ligand libraries. Both mono- and two-dimensional western blotting were performed for data validation. Differentially expressed proteins were mapped onto STRING v10 to build a protein-protein interaction network. Sixteen differentially expressed spots were identified with a high score. Among them, there were fibrinogen beta and gamma chains, complement C1r, C3 and C4-B subcomponents, alpha-1-antitrypsin (AAT), vitronectin and CD5 antigen-like. Protein-Protein interaction analysis evidenced a network among differentially expressed proteins in which vitronectin seems to represent a potentially pivotal node among fibrinolysis, complement dependent immune responses and inflammation in accordance with a number of in vitro and in vivo evidences for a contributory role of these proteins to the development of diabetic atherosclerosis.
Assuntos
Aterosclerose/sangue , Proteínas Sanguíneas/análise , Diabetes Mellitus Tipo 2/complicações , Angiopatias Diabéticas/sangue , Idoso , Aterosclerose/complicações , Aterosclerose/epidemiologia , Aterosclerose/cirurgia , Biomarcadores/sangue , Proteínas Sanguíneas/química , Western Blotting , Cromatografia Líquida de Alta Pressão , Angiopatias Diabéticas/epidemiologia , Angiopatias Diabéticas/cirurgia , Endarterectomia das Carótidas , Feminino , Humanos , Itália/epidemiologia , Masculino , Mapeamento de Peptídeos , Proteômica/métodos , Fatores de Risco , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Eletroforese em Gel Diferencial Bidimensional , Vitronectina/sangue , Vitronectina/químicaRESUMO
Aims. T2DM often remains undiagnosed for many years because hyperglycemia develops gradually and may not produce any symptoms. As patients with T2DM are at increased risk of microvascular and macrovascular complications, the preclinical diagnosis of the state is the key point of the disease management. Methods. We evaluated parameters such as GAGs/PGs, NAG, and NGAL in urine samples from 43 normoalbuminuric T2DM patients and 31 apparently healthy control subjects. Results. The total urinary GAG excretion showed no significant differences between patients and controls. The electrophoretic analysis evidenced the presence of UTI and its degradation products (LSC and SM-LSC), CS, and HS. We observed modifications of HS and total UTI (including UTI and its degradation products) relative contents in T2DM patients compared with controls whereas no differences in CS percentage were found. NGAL levels were significantly increased in T2DM patients and were positively correlated with both NAG (r = 0.606, p < 0.0001) and the presence of hypertension (r = 0.352, p < 0.05). Conclusions. These data suggest that the assessed molecules could represent useful markers to detect early renal impairment in patients with T2DM.
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BACKGROUND: Glycosaminoglycans (GAG) play an important role in regulating glomerular permeability, and a reduction in their plasmatic concentration or urinary loss has been implicated in the pathogenesis of diseases associated with increased albumin permeability. The purpose of this study was to evaluate GAG excretion in renal pathology by analyzing the composition of urinary GAG in antibody mediated glomerular injury, such as mesangial glomerulonephritis (IgAGN) and primitive membranous glomerulonephritis (MGN), to verify the effects of glomerular capillary wall lesion with and without mesangial cell injury. METHODS: Urinary GAG excretion was analyzed in 20 patients with IgAGN, 18 patients with MGN, and in 18 healthy subjects (controls). GAG were isolated from 24-hr urine using ion-exchange chromatography on DEAE-Sephacel, and the results are expressed as mg hexuronate/g creatinine (Cr). GAG composition was determined by cellulose acetate electrophoresis and expressed as relative percentages by densitometric scanning of Alcian Blue stained strips. RESULTS: We found total GAG levels significantly higher in the urine of patients with MGN in comparison with controls and patients with IgAGN. The electrophoretic pattern analysis demonstrated low sulfated chondroitin sulfate proteoglycan (LSC-PG) in all patients compared to 44% in controls (8/18), but also low sulfated chondroitin sulfate (LSC) in 18.4% of patients (7/38) and slow migrating LSC (SM-LSC) in 8% of patients (3/38), only in the MGN group. Moreover, in patients with MGN, the LSC-PG relative content was significantly higher when compared to that observed in controls. Finally, in IgAGN and MGN patients, a significant reduction in chondroitin sulfate (CS) relative content was observed. CONCLUSIONS: It seems likely that an increase in total GAG levels takes place when a reduction in renal function occurs, but the alteration of CS and herapan sulfate (HS) relative contents, and the presence of LSC-PG and free LSC also in the urine of IgAGN patients, allows us to suggest that the GAG distribution pattern becomes abnormal before an increase in total urine GAG excretion. In addition, the quali-quantitative determination of urinary GAG and GAGprotein complex could constitute an additional non-invasive marker to appraise the metabolism of renal connective tissue in some renal diseases.
Assuntos
Glomerulonefrite Membranoproliferativa/urina , Glomerulonefrite Membranosa/urina , Glicosaminoglicanos/urina , Adulto , Idoso , Albuminúria , Estudos de Casos e Controles , Doença Crônica , Creatinina/sangue , Feminino , Taxa de Filtração Glomerular , Mesângio Glomerular/patologia , Glomerulonefrite Membranoproliferativa/sangue , Glomerulonefrite Membranoproliferativa/patologia , Glomerulonefrite Membranosa/sangue , Glomerulonefrite Membranosa/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteoglicanas/urinaRESUMO
BACKGROUND: Diabetic nephropathy may be related to an abnormal metabolism of glycosaminoglycans (GAG) in the glomerular basement membrane (GBM). The first manifestation of nephropathy is microalbuminuria, whose appearance indicates a loss of GBM selectivity. The present study evaluated whether GAG excretion becomes abnormal in parallel with microalbuminuria, and whether such abnormalities are also present in normoalbuminuric diabetic patients. METHODS: We measured urinary GAG excretion in 60 patients with type 1 (insulin-dependent) diabetes mellitus and in 22 healthy subjects. GAG were isolated from 24-h urine using ion-exchange chromatography on DEAE Sephacel. GAG composition was determined by cellulose acetate electrophoresis and expressed as percentages by densitometric scanning of Alcian Blue stained strips. RESULTS: On subgrouping for albuminuric status and glyco-metabolic control, we found high urinary GAG concentrations in all except the normoalbuminuric patients with good glyco-metabolic control. The urinary GAG electrophoretic pattern showed alterations in chondroitin sulphate (CS) and heparan sulphate (HS) relative contents. A higher frequency of low sulphated chondroitin sulphate-proteoglycan (LSC-PG) was observed in all patients, including those with normoalbuminuria and good glyco-metabolic control. CONCLUSIONS: This urinary pattern may be indicative of an abnormal GBM metabolism. Since GAG play an important role in GBM permeability, these anomalies might consequently represent a first step towards selective changes of GBM in type 1 diabetes mellitus.
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Albuminúria/complicações , Albuminúria/urina , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/urina , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/urina , Glicosaminoglicanos/urina , Proteoglicanas/urina , Adulto , Albuminúria/fisiopatologia , Membrana Basal/fisiopatologia , Creatinina/urina , Diabetes Mellitus Tipo 1/fisiopatologia , Nefropatias Diabéticas/fisiopatologia , Feminino , Seguimentos , Humanos , Masculino , Avaliação de Resultados em Cuidados de Saúde , Valor Preditivo dos Testes , Fatores de Risco , Índice de Gravidade de Doença , Fatores de TempoRESUMO
OBJECTIVES: To evaluate if the prooxidant environment present in atherosclerotic plaque may oxidatively modify filtered albumin. METHODS: Fluorescein-5-maleimide labelled plasma samples and plaque extracts from 27 patients who had undergone carotid endarterectomy were analysed through nonreducing SDS-PAGE for albumin-Cys(34) oxidation. Furthermore, degree and pattern of S-thiolation in both circulating and plaque-filtered albumin were assayed. RESULTS: Albumin filtered in the atherosclerotic plaque showed higher levels of Cys(34) oxidative modifications than the corresponding circulating form as well as different patterns of S-thiolation. CONCLUSIONS: Data indicate that the circulating albumin, once filtered in plaque, undergoes Cys(34) oxidative modifications and demonstrate for the first time that albumin is a homocysteine and cysteinylglycine vehicle inside the plaque environment.
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Artérias Carótidas/patologia , Cisteína/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Albumina Sérica/metabolismo , Calibragem , Fluoresceínas/metabolismo , Humanos , Marcação por Isótopo , Peso Molecular , Oxirredução , Compostos de Sulfidrila/sangueRESUMO
Apolipoproteins are very heterogeneous protein family, implicated in plasma lipoprotein structural stabilization, lipid metabolism, inflammation, or immunity. Obtaining detailed information on apolipoprotein composition and structure may contribute to elucidating lipoprotein roles in atherogenesis and to developing new therapeutic strategies for the treatment of lipoprotein-associated disorders. This study aimed at developing a comprehensive method for characterizing the apolipoprotein component of plasma VLDL, LDL, and HDL fractions from patients undergoing carotid endarterectomy, by means of two-dimensional electrophoresis (2-DE) coupled with Mass Spectrometry analysis, useful for identifying potential markers of plaque presence and vulnerability. The adopted method allowed obtaining reproducible 2-DE maps of exchangeable apolipoproteins from VLDL, LDL, and HDL. Twenty-three protein isoforms were identified by peptide mass fingerprinting analysis. Differential proteomic analysis allowed for identifying increased levels of acute-phase serum amyloid A protein (AP SAA) in all lipoprotein fractions, especially in LDL from atherosclerotic patients. Results have been confirmed by western blotting analysis on each lipoprotein fraction using apo AI levels for data normalization. The higher levels of AP SAA found in patients suggest a role of LDL as AP SAA carrier into the subendothelial space of artery wall, where AP SAA accumulates and may exert noxious effects.
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Aterosclerose/sangue , Endarterectomia das Carótidas , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Lipoproteínas VLDL/sangue , Proteômica/métodos , Proteína Amiloide A Sérica/metabolismo , Apolipoproteínas/sangue , Apolipoproteínas/química , Aterosclerose/cirurgia , Biomarcadores/sangue , Western Blotting , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frações Subcelulares/metabolismoRESUMO
OBJECTIVES: Oxidative stress has been implicated in the outcome of atherosclerotic plaques. However, at present, no data are available neither on the degree of plaque protein sulfhydryl groups oxidation nor on its relationship with plaque vulnerability. We investigated the entity of protein-SH oxidative modifications, focusing on low molecular weight thiols adduction, in human carotid plaque extracts in relation to plaque stability/instability. METHODS: Plaque stability/instability was histologically assessed. The extent of protein-SH oxidative modifications was established by a differential proteomic approach on fluorescein-5-maleimide-labeled plaque extracts and corresponding plasma samples from 48 endarterectomized patients. The analysis on protein thiolation was performed by capillary zone electrophoresis. RESULTS: We observed a higher protein-SH oxidation of both plasma-derived and topically expressed proteins in unstable plaques, partly due to higher levels of S-thiolation. Conversely, in plasma, none of the investigated parameters discriminated among patients with stable and unstable plaques. CONCLUSIONS: Our results suggest the presence of a more pronounced oxidative environment in unstable plaques. Identifying specific oxidative modifications and understanding their effects on protein function could provide further insight into the relevance of oxidative stress in atherosclerosis.
Assuntos
Proteínas Sanguíneas/química , Compostos de Sulfidrila/química , Proteínas Sanguíneas/metabolismo , Artérias Carótidas , Eletroforese Capilar , Humanos , Peso Molecular , Oxirredução , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Proteômica , Compostos de Sulfidrila/metabolismoRESUMO
Allograft rejection during the first year after renal transplantation can lead to persistent allograft dysfunction and reduced long-term graft survival. Thus, it is important to define early predictors of kidney damage, less invasive than allograft biopsy. Urinary glycosaminoglycan/proteoglycan concentration and distribution, N-acetyl-ß-(D)-glucosaminidase (NAG), and monokine induced by IFN-γ (MIG) levels were evaluated in the immediate post-transplant and during a 1-year follow-up. We observed increased urinary levels of MIG, urinary trypsin inhibitor and its degradation products, the lack of urinary heparan sulfate excretion, and the decreased chondroitin sulfate relative content at day 1 post-transplant in most patients who developed complications in the postoperative period. Moreover, urinary MIG levels showed significant correlations with NAG, C-reactive protein, and GFR at day 1 post-transplant. The monitoring of glycosaminoglycan/proteoglycan urinary pattern and the levels of urine MIG could serve as useful markers for predicting possible complications of transplantation, unraveling an early inflammatory state, on whose basis the immunosuppressive therapy could be appropriately modified.
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Glicosaminoglicanos/análise , Rejeição de Enxerto/diagnóstico , Interferon gama/imunologia , Transplante de Rim , Monocinas/análise , Proteoglicanas/análise , Urina/química , Adulto , Idoso , Biomarcadores/urina , Diagnóstico Precoce , Humanos , Pessoa de Meia-Idade , Prognóstico , TransplanteRESUMO
Fabry's disease is a rare lysosomal storage disorder caused by the deficiency of α -galactosidase A that leads to the accumulation of neutral glycosphingolipids in many organs including kidney, heart, and brain. Since end-stage renal disease represents a major complication of this pathology, the aim of the present work was to evaluate if urinary proteoglycan/glycosaminoglycan excretion could represent a useful marker for monitoring kidney function in these patients at high risk. Quali-quantitative and structural analyses were conducted on plasma and urine from 24 Fabry's patients and 43 control subjects. Patients were sorted for presence and degree of renal impairment (proteinuria/renal damage). Results showed that levels of urine bikunin, also known as urinary trypsin inhibitor (UTI), are significantly higher in patients with renal impairment than in controls. In this respect, no differences were evidenced in plasma chondroitin sulfate isomers level/structure indicating a likely direct kidney involvement. Noteworthy, urine bikunin levels are higher in patients since early symptoms of renal impairment occur (proteinuria). Overall, our findings suggest that urine bikunin level, as well as proteinuria, could represent a useful parameter for monitoring renal function in those patients that do not present any symptoms of renal insufficiency.
Assuntos
alfa-Globulinas/urina , Doença de Fabry/urina , Rim/patologia , Insuficiência Renal/urina , Adulto , Biomarcadores/urina , Doença de Fabry/complicações , Doença de Fabry/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteinúria/diagnóstico , Insuficiência Renal/complicações , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismoRESUMO
Cardiac valves are dynamic structures, exhibiting a highly specialized architecture consisting of cells and extracellular matrix with a relevant proteoglycan and glycosaminoglycan content, collagen and elastic fibers. Biological valve substitutes are obtained from xenogenic cardiac and pericardial tissues. To overcome the limits of such non viable substitutes, tissue engineering approaches emerged to create cell repopulated decellularized scaffolds. This study was performed to determine the glycosaminoglycans content, distribution, and disaccharides composition in porcine aortic and pulmonary valves and in pericardium before and after a detergent-based decellularization procedure. The fine structural characteristics of galactosaminoglycans chondroitin sulfate and dermatan sulfate were examined by FACE. Furthermore, the mechanical properties of decellularized pericardium and its propensity to be repopulated by in vitro seeded fibroblasts were investigated. Results show that galactosaminoglycans and hyaluronan are differently distributed between pericardium and valves and within heart valves themselves before and after decellularization. The distribution of glycosaminoglycans is also dependent from the vascular district and topographic localization. The decellularization protocol adopted resulted in a relevant but not selective depletion of galactosaminoglycans. As a whole, data suggest that both decellularized porcine heart valves and bovine pericardium represent promising materials bearing the potential for future development of tissue engineered heart valve scaffolds.
RESUMO
OBJECTIVE: To evaluate transforming growth factor beta1 (TGF-beta1) and glycosaminoglycans (GAG) changes in human plasma and urine during the menstrual cycle, IVF-ET, and pregnancy. DESIGN: Prospective clinical study. SETTING: University hospital. PATIENT(S): Thirteen women with apparently normal menstrual cycle (group 1); 18 women undergoing IVF-ET (group 2); and 14 low-risk pregnant women (group 3). INTERVENTION(S): We assayed plasma and urine concentrations of TGF-beta1, urine content, and distribution of GAG. Blood and urine samples were collected during days 2 to 3, 12 to 13, and 23 to 24 in group 1; in group 2, samples were obtained at menstrual phase, oocyte pick-up day, and 15 days after ET; in group 3, samples were obtained during gestational weeks 10-12, 22-24, and 30-32 and 1 month after delivery. MAIN OUTCOME MEASURE(S): Changes in TGF-beta1 and GAG content. RESULT(S): The mean value of total urinary trypsin inhibitor/chondroitin sulfate (UTI/CS) showed a distinct peak at day 12 of the menstrual cycle in the fertile women in whom we monitored the ovulatory period. In the IVF-ET group, GAG distribution and TGF-beta1 levels showed significant differences during the cycle. We observed increased levels of plasma TGF-beta1 15 days after ET. A significant increase of total UTI/CS value with increasing gestation was detected. CONCLUSION(S): Transforming growth factor beta1 and GAG levels could represent an additional tool to monitor reproductive events and could be useful, noninvasive markers of ovulation and ongoing pregnancy.