Altered glycosylation of alpha1-acid glycoprotein in patients with inflammation and diabetes mellitus

BACKGROUND: In certain pathophysiological conditions, such as inflammation rheumatoid arthritis and diabetes mellitus (DM), alterations in asparagine-linked glycan (N-glycan) patterns of the acute-phase protein, alpha(1)-acid glycoprotein (AGP), have been reported. In this study, we investigated N-glycan structures of AGP purified from the sera of patients with acute inflammation (n=5), type 2 diabetes mellitus (n=5), and healthy individuals (n=5). METHODS: N-Glycans were released with peptide N-glycosidase F (PNGase F) from denatured AGP and purified with cellulose cartridge. N-glycans were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS) in combination with exoglycosidase digestion. RESULTS: We revealed increases in bi-antennary complex glycans and in alpha1-3 fucosylated bi-, tri-, and tetra-antennary glycans and a decrease in tri-antennary glycans in inflammation patients. These results support increases in bindings to concanavalin A (ConA) and Aleuria aurantia lectins (AALs). In diabetic patients, the pathogenesis-specific change in N-glycan patterns of AGP was not significant. CONCLUSIONS: The MALDI-TOFMS method is sensitive and suitable for profiling analysis of N-glycans in clinical samples.     

Differential isoform profiles of alpha 2-macroglobulin from plasma of patients with chronic-progressive or relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is a human neurological disease for which no clinically useful marker has been identified in blood. This study examined alpha 2-macroglobulin (alpha 2M) from the plasma of six patients with chronic-progressive MS and six with relapsing-remitting disease. The alpha 2M trypsin-binding activity in the plasma from both groups of patients did not differ from normal controls. However, after column isoelectric focusing, consistently less alpha 2M activity was recovered from the MS samples: those from the chronic-progressive and relapsing-remitting disease groups were an average of 43% and 68%, respectively, of controls. The number and isoelectric point (pI) values of the isoforms of the alpha 2M from patients with chronic-progressive disease were similar to controls. The average pI of the major form for both groups was 6.6. By contrast, the average pI of the major form from the patients with relapsing-remitting MS was significantly elevated to 7.1, and this group displayed a significantly higher percentage of total recovered activity above pH 7.0. In eleven of the twelve cases examined, the pI of the major form of alpha 2M correctly correlated with the clinical status of the patient. The original clinical diagnosis of the patients was reassessed by a 9-year retrospective interview which verified that 9 of the 10 patients in the follow-up group retained their original clinical diagnosis. These studies demonstrate differential isoform profiles of native alpha 2M from MS patients with progressive versus remitting disease which may be useful in subclassifying MS patients.     

Apolipoprotein E phenotyping from plasma by isoelectric focusing and immunoblotting

A method for apolipoprotein (apo) E phenotyping directly from plasma by isoelectric focusing (IEF) and immunoblotting was confirmed. Ten microliters plasma were delipidated. IEF in 5% polyacrylamide flat gel with 6.4 mol/l urea and 2.8% pharmalyte (PH 4-6.5) was carried out at 3,000 V for 1 hr. Seventeen samples were applied per one flat gel, and IEF of two flat gels was made. Then, Western blotting on nitrocellulose membrane was done at 75 V for 3 hr. Immunostaining was performed using goat-anti-human apo E as first antibody and biotinylated anti-goat IgG as second antibody, and 4-chlorodel-1-naphthol as a substrate. In approximately 5% of the samples, we had difficulty in discriminating between homozygotes and heterozygotes (i.e., apo E3/3 and apo E3/2, or apo E4/4 and apo E4/3) because of equally strong sialated band, but this problem was solved by sialidase treatment of plasma before delipidation. As a result, six apo E phenotypes were clearly demonstrated. Apo E phenotyping of 34 samples could be made simultaneously in 2 days. It is concluded that the polyacrylamide gel IEF and immunoblotting method is useful for apo E phenotyping if it is made up for by sialidase treatment.     

Reduced trypsin binding capacity of alpha 2-macroglobulin in diabetes

The plasma proteinase inhibitor, alpha 2-macroglobulin, is usually elevated in diabetes. The trypsin binding capacity and the concentration of alpha 2-macroglobulin in 90 diabetics sera were compared with 30 age- and sex-matched normal sera. The mean alpha 2-macroglobulin concentration determined by radial immunodiffusion was 313 mg/dl for the diabetics as compared to 240 mg/dl for the healthy subjects (significantly higher, p less than 0.01). The mean of the ratio, mol trypsin bound/mol alpha 2-macroglobulin (molar binding ratio) for the Type I diabetics (n = 54), 0.82, was significantly lower than the mean of the healthy subjects, 0.87, or the mean of the Type II diabetics, 0.87. No relationship between the molar binding ratios and the levels of glycosylated hemoglobin was found. The alpha 2-macroglobulin was isolated from the plasma of 11 Type I diabetics and 7 normals. The maximum molar trypsin binding capacities of the diabetic alpha 2-macroglobulin were significantly lower. The mean for the diabetic alpha 2-macroglobulin was 1.72 vs. 1.97 for the normal alpha 2-macroglobulin. These results indicate that the trypsin binding function of alpha 2-macroglobulin is moderately impaired in diabetes. No differences were found in the extent of proteolytic cleavage of the 'bait region' of diabetic alpha 2-macroglobulin, autolytic cleavage or the methylamine reaction at the thiolester site between diabetic and normal alpha 2-macroglobulin. Nonenzymatic glucosylation of normal alpha 2-macroglobulin did not lower the trypsin binding capacity. The nature of the modification of alpha 2-macroglobulin leading to reduced trypsin binding capacity or the physiological significance is not yet known.     

Aberrant forms of alpha(2)-macroglobulin purified from patients with multiple sclerosis

The biochemical properties of alpha(2)-macroglobulin were investigated in four patients with multiple sclerosis and compared to alpha(2)-macroglobulin from healthy controls. An impaired stability of alpha(2)-macroglobulin from the multiple sclerosis patients was demonstrated as a spontaneous conversion to an electrophoretic"fast" form of alpha(2)-macroglobulin upon purification and storage, with a concomitant decrease in functional capacity to inhibit proteinases. The ability to form complexes with proteinases was significantly reduced in alpha(2)-macroglobulin purified from the multiple sclerosis patients. The aberrant molecular arrangements of the protein were not due to proteinase cleavages in the bait regions of alpha(2)-macroglobulin, as demonstrated by gel electrophoresis and protein sequencing. The number of functional thiol esters, however, was reduced in alpha(2)-macroglobulin purified from the multiple sclerosis patients, an observation compatible with the impaired proteinase binding property. Furthermore, differences in isoelectric points were observed between alpha(2)-macroglobulin from the multiple sclerosis patients and alpha(2)-macroglobulin from healthy controls. The results suggest that aberrant forms of alpha(2)-macroglobulin may be present in patients with multiple sclerosis.     

Structural evaluation of plasma alpha2-macroglobulin in acute pancreatitis.

In this work we evaluate the proteolytic state of plasma alpha2-macroglobulin in acute pancreatitis. In addition, the plasma activity of matrix metalloproteinase-2 (MMP-2), MMP-9 and serine proteinases were analyzed. A total of 33 patients with acute pancreatitis were studied, of whom 16 were diagnosed as having mild and 17 as having severe acute pancreatitis. In the latter group, three patients progressed to multi-organ failure and died as a consequence of these complications. The proteolytic fragmentation of alpha2-macroglobulin was evaluated by Western blotting, whereas the plasma activity of MMP-2, MMP-9 and serine proteinases was evaluated by gelatin zymography. Enhanced fragmentation of alpha2-macroglobulin was detected in severe acute pancreatitis patients with multiple organ failure and lethal complications. In this same patient group, increased plasma activity of the active forms of MMP-2 and MMP-9, as well as serine proteinases, was apparent. In addition, we demonstrate that chymotrypsin-like proteinases could be the principal cause of alpha2-macroglobulin degradation in this group of patients. Our results indicate that secondary proteolysis of alpha2-macroglobulin promotes impaired control of extracellular proteolytic activity, leading to local and distant tissue injuries during severe acute pancreatitis. Finally, the structural evaluation of plasma alpha2-macroglobulin could be used as a prognostic marker of the severity of acute pancreatitis.     

Application of isoelectric focusing in alpha1-antitrypsin phenotyping.

alpha1-Antitrypsin, the major protease inhibitor in human serum, occurs in a considerable number of variant forms, some of which are associated with lung and liver diseases. The identification of these genetic variants, generally called Pi-types, by means of isoelectric focusing is described, as well as investigations concerning the practical application of isoelectric focusing as a routine procedure for typing the variants of alpha1-antitrypsin. Finally, isoelectric focusing is compared with the most widely used Pi-phenotyping technique, namely acid starch-gel electrophoresis followed by immunoelectrophoresis in antibody-containing agarose gel.     

糖尿病患者血浆中半乳糖凝集素-3水平的表达

目的：探讨半乳糖凝集素-3（Galectin-3）在糖尿病患者血浆中的表达情况,以及与其他因素的相关性分析。方法对30例糖尿病患者、22例非糖尿病患者（对照组）血浆中的 Galectin-3进行测定,比较两组间 Galectin-3水平变化,分析 Galectin-3与其他因素的相关性。结果糖尿病患者血浆中的 Galectin-3水平[（17．95±2．61）μg／L]显著高于对照组[（15．59±2．39）μg／L],差异有统计学意义（P ＜0．01）。Galectin-3与空腹血糖（FBG）有显著地相关性（P ＜0．01）,与糖尿病病程（DD）、钠尿肽（BNP）有明显相关性（P ＜0．05）。结论Galectin-3作为重要的炎症调节因子,可能在糖尿病的发生发展中起着重要的调节作用。     

Functional levels of alpha 2-macroglobulin in plasma in relation to emphysema

In most studies, concentrations of alpha 2-macroglobulin are determined by immunological techniques. In this study, the amidolytic activity of porcine pancreatic elastase complexed with alpha 2-macroglobulin was measured using an elastase-specific substrate, succinyl-trialanyl-p-nitroanilide. The activities of plasmas from 47 emphysema cases were compared with 39 normal subjects. The age ranges of both groups were from 50 to 84 years. The mean activity of bound elastase in emphysema cases was 2.48 +/- 0.03 kU/l of plasma. The mean for normal subjects was 1.48 +/- 0.11 kU/l of plasma. The difference was very significant (2P less than 0.001). All except 2 of the emphysema cases had smoked. The same results were obtained when only people who had smoked for 25 years or more were included in the analysis. All the plasma samples of people included in the study were assayed by an immunological method for absolute level (in g/l) of alpha 1-proteinase inhibitor. The levels of alpha 1-proteinase inhibitor for all the persons studied fell within the normal range for MM-phenotypes (2 to 4 g/l).     

Immunoreactive precipitation of C1 inhibitor protein from plasma of normal subjects and of patients with hereditary angioedema after isoelectric focusing

C1-inhibitor is an acid glycoprotein, isoelectric point 3.5-3.6. Plasma of some patients with a variant form of hereditary angioedema contains high levels of functionless C1-inhibitor-albumin complex with an isoelectric point at 4.5-4.6. Therapy with Danazol, which increases C1-inhibitor levels, does not modify the isoelectric focusing pattern of such protein in patients with hereditary angioedema.     

2型糖尿病患者视网膜病变与血浆氧化三甲胺水平的相关性分析

目的探讨血浆氧化三甲胺(TMAO)水平与2型糖尿病(T2DM)患者糖尿病视网膜病变(DR)的关系。方法选取102例T2DM患者,根据有无DR和DR分期标准将患者分为无DR(NDR)组38例、非增殖性DR(NPDR)组35例、增殖性DR(PDR)组29例,另选取40名同期健康体检者作为对照组。采用同位素稀释液相色谱-串联质谱(LC-MS/MS)法检测4组血浆TMAO浓度,比较4组一般资料和实验室指标,分析血浆TMAO浓度与各临床参数的相关性,采用多因素Logistic回归分析筛选DR的主要危险因素,并采用受试者工作特征(ROC)曲线分析血浆TMAO诊断DR的效能。结果 PDR组的糖尿病病程长于NDR组、NPDR组,差异有统计学意义(P 0.05)。4组空腹血糖(FPG)、糖化血红蛋白(Hb A1c)、C反应蛋白(CRP)和TMAO水平比较,差异有统计学意义(P 0.05)。Spearman相关分析显示,TMAO与糖尿病病程、FPG、HbA1c、CRP呈显著正相关(P 0.05)。Logistic回归分析显示,糖尿病病程长和FPG、HbA1c、CRP、TMAO水平高均是DR的危险因素(P 0.05)。ROC曲线分析显示,TMAO诊断DR的曲线下面积(AUC)为0.833,糖尿病病程、FPG、HbA1c、CRP联合诊断DR的AUC为0.802,糖尿病病程、FPG、Hb A1c、CRP、TMAO联合诊断DR的AUC为0.879。结论血浆TMAO水平升高与T2DM患者DR的发生及其严重程度有关,是DR的主要危险因素之一,TMAO联合糖尿病病程、FPG、Hb A1c、CRP对DR有较高的诊断效能。     

The determination of antithrombin III, alpha 2-macroglobulin and alpha 2-antiplasmin in plasma by laser nephelometry

Specific assay procedures were developed to determine antithrombin III, alpha 2-macroglobulin, and alpha 2-antiplasmin in plasma with the aid of a Laser nephelometer. About 100 normal and pathological plasmas were examined and the results compared with those obtained by conventional immunological methods, such as radial immunodiffusion or rocket electrophoresis. Nephelometry proved to be a very rapid, precise, and reproducible method for the determination of antithrombin III and alpha 2-macroglobulin. The reproducibility of the alpha 2-antiplasmin assay was poor, probably as a consequence of the low concentration of this inhibitor in normal plasma.     

血浆Apelin水平与2型糖尿病合并冠心病风险的相关性分析

目的探讨血浆脂肪因子Apelin水平与2型糖尿病合并冠心病风险的关系。方法 2型糖尿病患者138例,依据是否合并冠心病分为合并冠心病组59例与未合并冠心病组79例,比较2组血浆Apelin水平,采用多因素Logistic回归分析评价血浆Apelin水平与冠心病发生的相关性。结果合并冠心病组血浆Apelin水平为(2.38±0.63)ng/mL,明显低于未合并冠心病组(4.82±1.06)ng/mL(P<0.01);多因素Logistic回归分析结果显示,低血浆Apelin水平是2型糖尿病患者罹患冠心病的独立危险因子(OR=0.694,95%CI:0.428～0.873,P<0.01)。结论检测血浆Apelin水平可用于评估2型糖尿病患者罹患冠心病的风险。     

Clearance of alpha 1-antitrypsin isoelectric focusing patterns in blood samples treated with heparin.

Plasma samples, whose typing for alpha 1-AT is made difficult or impossible because of an excess of heparin used as anticoagulant, can be treated very effectively with protamine sulphate. The addition of this heparin antagonist results in complete clearance of the isoelectric focusing pattern.     

Semi-automated rapid isoelectric focusing of apolipoproteins C from human plasma using Phastsystem and immunofixation.

Apolipoproteins (apo) C-I, C-II, and C-III play crucial roles in intravascular lipid metabolism. Whereas apo C-II is an obligate cofactor for lipoprotein lipase, apo C-III was shown to inhibit its action. Apo C-I can be a potent cofactor of human lecithin:cholesterol acyltransferase. Structural mutants and deficiencies of apo C-II lead to hypertriglyceridemia. A similar phenotype is associated with apo C-III mutants and is inducible by overexpression of human apo C-III in transgenic animals. No structural variant has so far been reported for apo C-I. The present paper describes a rapid semi-automated procedure for isoelectric focusing analysis of these C-apolipoproteins from whole plasma or serum and their visualization by immunofixation and silver staining. The procedure allows detection of charged variants of C-apolipoproteins. As applied to 295 patients with coronary heart disease and 85 controls, it also serves to detect deficiency syndromes of these apolipoproteins. The procedure provides reliable, easy and quick analysis of C-apolipoproteins applicable as a routine or screening procedure not restricted to specialized laboratories.     

Elastase binding capacity of alpha 2-macroglobulin in plasma of patients with asthma or chronic obstructive pulmonary disease, without alpha 1-protease inhibitor deficiency

The amidolytic activity of alpha 2-macroglobulin complexed with porcine pancreatic elastase (EC 3.4.21.11) was assayed using succinyl-trialanyl-p-nitroanilide. The levels of activity were compared in chronic obstructive pulmonary disease patients, asthma patients, and in healthy subjects with no record of lung disease. Levels of alpha 1-protease inhibitor were also determined and only those cases within the normal range for alpha 1-protease inhibitor were selected. Both the asthma cases and those with chronic obstructive pulmonary disease had levels of elastase-binding capacity related to alpha 2-macroglobulin which were significantly higher than the control groups.