Influence of blood proteins on biomedical analysis. XII. Effects of glycation on gliclazide (oral hypoglycemic drug)-binding with serum albumin in diabetics.

The sera of diabetic patients showed an inverse correlation (r = -0.67, n = 57) between free gliclazide (oral hypoglycemic drug) level and the fructosamine value. The binding capacity of the primary binding site for gliclazide in the albumin molecule was increased from 4.5 x 10(-4) to 8.0 x 10(-4) M-1 by glycation of albumin, but not that of the secondary binding site (1.2 x 10(-4) M-1). This suggests that the glycation of albumin increases its total binding capacity for gliclazide, resulting in a low free gliclazide level. Therefore, a low hypoglycemic activity of the drug is observed when it is administered to diabetic patients with hyperglycemia.     

On the search for glycated lipoprotein ApoA-I in the plasma of diabetic and nephropathic patients

The analysis of plasma samples from healthy, diabetic and nephropathic subjects was carried out by 2D gel electrophoresis. This approach shows clear differences among the three classes of subjects. In the case of diabetic and nephropathic patients intense spots appear. Their enzymatic digestion followed by matrix assisted laser desorption ionization/mass spectrometry (MALDI/MS) analysis shows that an overexpression of unglycated and glycated ApoA-I is present in both pathological states. Interestingly, this trend is also observed for the retinol-binding protein (RBP). The data obtained can be relevant to assess possible risks associated either with the glycation level of ApoA-I or with the overexpression of RBP. In fact, in the former case possibly a different functionality of the glycated protein is to be expected, reflecting a different efficiency in cholesterol transport. In the latter case, the increase of RBP level can be related to the overweight of the diabetic subjects under investigation: it is known that obesity leads to RBP overexpression. In the case of nephropathic patients, the RBP level increases in parallel with serum creatinin.     

Effect of glycation on the heterogeneity of human serum albumin analysed by reversed-phase high-performance liquid chromatography in a solvent containing formic acid.

Non-enzymic glycation of human serum albumin (HSA) induces a change in its charge heterogeneity that may account for its particular renal clearance in patients with early diabetic nephropathy. A new high-performance liquid chromatographic analysis for the study of HSA heterogeneity is described based on a high content of formic acid in the mobile phase combined with a concave gradient of isopropanol. Under these conditions, native HSA was separated into three individual components (I, II and III). When glycated HSA was analysed, it was found that although the present method is not suitable for the separation of glycated from non-glycated HSA, it shows the effect of glycation in producing changes in HSA heterogeneity that are different from those reported on surface change. This finding suggests an additional factor (probably conformational changes) that is contributing to the heterogeneity of glycated HSA.     

Comprehensive analysis of glycated human serum albumin tryptic peptides by off-line liquid chromatography followed by MALDI analysis on a time-of-flight/curved field reflectron tandem mass spectrometer

Glycated peptides arising from in vivo digestion of glycated proteins, usually called advanced glycation end (AGE) product peptides, are biologically relevant compounds due to their reactivity towards circulating and tissue proteins. To investigate their structures, in vitro glycation of human serum albumin (HSA) has been performed and followed by enzymatic digestion. Using different MALDI based approaches the digestion products obtained have been compared with those arising from enzymatic digestion of the protein. Results obtained using 2,5-dihydroxybenzoic acid (DHB) indicate this as the most effective matrix, leading to an increase in the coverage of the glycated protein. Off-line microbore liquid chromatography prior to MALDI analysis reveals that 63% of the free amino groups amenable to glycation are modified. In addition, the same approach shows the co-presence of underivatised peptides. This indicates that, regardless of the high glucose concentration employed for HSA incubation, glycation does not go to completion. Tandem mass spectrometric data suggest that the collision induced dissociation of singly charged glycated peptides leads to specific fragmentation pathways related to the condensed glucose molecule. The specific neutral losses derived from the activated glycated peptides can be used as signature for establishing the occurrence of glycation processes.     

血镁水平与糖尿病肾病

为探讨糖尿病肾病 (DN)患者血镁水平 ,以 1 5 9例 2型糖尿病 (T2DM)患者为研究对象 ,用xylidylblue比色法测定了其正常白蛋白尿期、微量白蛋白尿期 (早期DN)、临床DN及晚期DN血镁水平 ,与 2 0例正常对照组相比较 ;同时将 1 1 0例DN患者分为肾功能不全代偿组、失代偿组和肾功能衰竭组 ,将其血镁水平与 1 0 2例慢性肾小球肾炎 (CGN)患者相比较 ,组间比较采用t检验 ;对 79例T2DM正常白蛋白尿期及微量白蛋白尿期患者尿白蛋白排泄率 (UAER)与血镁水平进行了等级相关分析。结果表明 ,T2DM正常白蛋白尿期、早期DN及临床DN血镁水平降低 ,晚期DN血镁水平升高 ,各组与正常对照组相比存在显著性差异 (P <0 0 5或P <0 0 1 ) ;T2DMDN及CGN肾功能不全代偿期、失代偿期及肾功能衰竭期血镁水平渐升高 ,且肾功能处于同一期的DN和CGN相比 ,前者血镁水平均较后者显著降低 (P <0 0 1或P <0 0 5 ) ;T2DM正常白蛋白尿期和微量白蛋白尿期血镁水平与UAER呈负相关 (r=0 5 47,P <0 0 1 )。提示T2DM患者肾功能正常时存在低镁倾向 ,但晚期DN血镁水平升高 ;随着肾功能不全进展 ,DN和CGN患者血镁水平升高 ,但前者血镁水平仍较后者低 ,镁代谢紊乱与DM及其并发症的相互关系有待进一步研究 ;血镁水平测定可否作为DN的早期诊断指标亦     

Enzymatic digestion and mass spectrometry in the study of advanced glycation end products/peptides

An extensive study was carried out on HSA and non-enzymatically glycated HSA by enzymatic digestion with trypsin and endoproteinase Lys-C, with the aim of identifying specific glycated peptides deriving from enzymatic digestion of glycated HSA. They may be considered, in pectore, as advanced glycation end products/peptides. These compounds, important at a systemic level in diabetic and nephropathic subjects, are produced by enzymatic digestion of in vivo glycated proteins: They are related to the pathological state of patients and have been invoked as responsible for tissue modifications. The digested mixtures obtained by the two enzymes were analyzed by MALDI/MS and LC/ESI/MSn, and clear cut differences were found. First of all, the digestion products of glycated HSA are generally less abundant than those observed in the case of unglycated HSA, accounting for the lower proclivity of the former to enzymatic digestion. MS/MS experiments on doubly charged ions, comparisons with a protein database, and molecular modeling to identify the lysine NH2 groups most exposed to glycation, identified some glycated peptides in digestion mixtures obtained from both types of enzymatic digestion. Residues 233K, 276K, 378K, 545K, and 525K seem to be privileged glycation sites, in agreement with the fractional solvent accessible surface values calculated by molecular modeling.     

Investigation of non-enzymatic glycosylation of human serum albumin using ion trap-time of flight mass spectrometry

Non-enzymatic glycosylation or glycation involves covalent attachment of reducing sugar residues to proteins without enzyme participation. Glycation of glucose to human serum albumin in vivo is related to diabetes and many other diseases. We present an approach using liquid chromatography coupled to an electrospray ionization source of a hybrid ion trap-time of flight (IT-TOF-MS/MS) tandem mass spectrometer to identify the glycation sites on serum albumin from both a healthy person and a diabetic patient. The MetID software, which is commonly used for screening metabolites, is adapted for peptide fingerprinting based on both m/z values and isotopic distribution profiles. A total of 21 glycation sites from the healthy person and 16 glycation sites from the diabetic patient were identified successfully. We also demonstrate the use of matrix assisted laser desorption ionization-time of flight mass spectrometry to estimate the incorporation ratio of glucose to albumin during glycation. Results from this study show that the glycation in healthy person is more complicated than previously thought. Further analysis of incorporation ratio distribution may be necessary to accurately reflect the change of serum albumin glycation in diabetic patients.     

Matrix-assisted laser desorption/ionization mass spectrometry, enzymatic digestion, and molecular modeling in the study of nonenzymatic glycation of IgG

The glycation-induced functional change of immunoglobulins is of particular interest. The glycation levels of IgG in 10 healthy subjects and 20 diabetic patients with different degrees of metabolic control were studied by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. It reveals the number of glucose molecules that have condensed on the protein, which range from 1 to 5 for healthy subjects, from 5 to 9 for well controlled diabetic patients, and from 10 to 25 for poorly controlled ones. The identification of the most favored glycation sites has been obtained by MALDI analysis of standard and in vitro glycated IgG and plasma protein fraction of a healthy subject after digestion with papain, releasing Fab and Fc fragments of the molecule. Both experiments, as well as molecular modeling of the whole protein, confirm that the most of glucose molecules have condensed on the Fab fragment of IgG, suggesting that the immune deficiency observed in diabetic patients may be explained at the molecular level by a more effective glycation of the Fab fragment, thus inhibiting the process of molecular recognition between antibody and antigen.     

Study on the interactions of sulfonylurea antidiabetic drugs with normal and glycated human serum albumin by capillary electrophoresis‐frontal analysis

Diabetes is one of the most widespread diseases characterized by a deficiency in the production of insulin or its ineffectiveness. As a result, the increased concentrations of glucose in the blood lead not only to damage to many of the body's systems but also cause the nonenzymatic glycation of plasma proteins affecting their drug binding. Since the binding ability influences its pharmacokinetics and pharmacodynamics, this is a very important issue in the development of new drugs and personalized medicine. In this study, capillary electrophoresis‐frontal analysis was used to evaluate the affinities between human serum albumin or its glycated form and the first generation of sulfonylurea antidiabetics, since their inadequate concentration may induce hypoglycaemia or on the contrary hyperglycaemia. The binding constants decrease in the sequence acetohexamide > tolbutamide > chlorpropamide > carbutamide both for normal and glycated human serum albumins, with glycated giving lower values. These results provide a more quantitative picture of how these drugs bind with normal and modified human serum albumin and indicate capillary electrophoresis‐frontal analysis to be another tool for examining the changes arising from modifications of albumin, or any other protein, with all its benefits like short analysis time, small sample requirement, and automation.     

Study of Hemoglobin and Human Serum Albumin Glycation with Electrochemical Techniques

High concentration glucose in diabetes mellitus may stimulate nonenzymatic glycation of proteins. Hemoglobin (Hb) and human serum albumin (HSA) are among the most sensitive proteins for the modification by glucose. In this paper, we report our study of Hb and HSA modification by glucose using electrochemical methods. Compared with native Hb, it is found that highly glycated Hb presents lower electron transfer reactivity and electrocatalytic activity toward O₂ and H₂O₂, and the glycation is glucose concentration and time dependent. Meanwhile, the changes of the electrochemical signal of heme after binding with HSA and glycated HSA have also suggested that proteins modified by high concentration glucose lasting for months and years in diabetes mellitus might be the reason for diabetes mellitus complication.     

The structure of the sugar residue in glycated human serum albumin and its molecular recognition by phenylboronate

Quantification of the extent of glycation of human serum albumin (HSA) and of haemoglobin provides a record of average mid- and long-term blood-sugar concentrations, respectively; this is very useful for the management of diabetes. The reaction of D-glucose with propylamine affords the corresponding Schiff base, N-propylamino-D-glucoside, in the cyclic form. This compound is not stable: upon standing or treatment with acid it is converted, by an Amadori rearrangement, into N-propylfructosamine. Both amino sugars occur predominantly in the beta-pyranose form. Phenylboronate forms highly stable boronate esters through binding of the cis 1,2-diol moiety in the furanose form of N-propylfructosamine. Between pH 5 and 10, an electrostatic interaction between the protonated amino group and the negatively charged boronate moiety affords an additional stabilisation of the ester. The Schiff base, however, has no observable interaction with phenylboronate. In aqueous solution the Schiff base is in equilibrium with propylamine and glucose. Upon addition of phenylboronate, this equilibrium shifts to the side of glucose due to the formation of highly stable phenylboronate esters of the beta-furanose form of this compound. After Amadori rearrangement, the sugar moieties in glycated human serum albumin have a similar structure, they occur as an equilibrium of the beta-pyranose (59%), alpha-furanose (19%) and beta-furanose (24%) anomers. The open form was not observed. The beta-furanose anomer is selectively recognised by phenylboronate.     

Coupling of m-aminophenylboronic acid to s-triazine-activated Sephacryl: use in the affinity chromatography of glycated hemoglobins.

An improved process is described for covalent coupling of m-aminobenzeneboric acid to s-triazine-activated Sephacryl matrices. The derivatized Sephacryl gel contained up to 150-200 mumol boronate per ml. It has been applied to the separation of glycated and non-glycated hemoglobins (Hbs) present in red-cell hemolysate. The new bioaffinity support was evaluated by the analysis of 67 diabetic patients and 20 normal adults. The mean value for glycated Hb was 6.6 +/- 0.8% for non-diabetics and 11.2 +/- 2.9% for diabetics. The method effects group-specific separation between glycated and non-glycated Hbs even in presence of foetal Hb and abnormal Hb variants. There is an excellent correlation between the glycated Hb levels obtained by the new method and two established procedures, namely high-performance liquid chromatography (r = 0.933) and affinity Merckotest (r = 0.991). The inter-assay and intra-assay coefficient of variations of less than 3.0% suggest that the method is reproducible. The results indicate that the method may serve as an alternative procedure for the study of glycated proteins. The s-triazine-activated Sephacryl could also be used for immobilizing enzymes and for preparing biospecific absorbents.     

Oxidation of glycated phosphatidylethanolamines: evidence of oxidation in glycated polar head identified by LC-MS/MS

Phosphatidylethanolamine glycation occurs in diabetic patients and was found to be related with oxidative stress and with diabetic complications. Glycated phosphatidylethanolamines seem to increase oxidation of other molecules; however, the reason why is not understood. In this work, we have studied the oxidation of glycated phosphatidylethanolamines (1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylethanolamine (PLPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (dPPE)) using a Fenton system. Liquid chromatography–electrospray ionization (ESI)–mass spectrometry and ESI–tandem mass spectrometry in both positive and negative modes were used for detecting and identifying the oxidation products. We were able to identify several oxidation products with oxidation in unsaturated sn-2 acyl chain of PLPE, as long- and short-chain products with main oxidation sites on C-7, C-8, C-9, and C-12 carbons. Other products were identified in both glycated PLPE and glycated dPPE, revealing that oxidation also occurs in the glycated polar head. This fact has not been reported before. These products may be generated from oxidation of glycated phosphatidylethanolamines (PE) as Schiff base, leading to short-chain product without the amine moiety, due to cleavage of glycated polar head and long-chain product with two keto groups linked to the glycated polar head or from glycated PE as Amadori product, short-chain products with –NHCHO and –NHCHOHCHO terminal in polar head. Oxidation of glycated phosphatidylethanolamines occurred more quickly than the oxidation of non-glycated phosphatidylethanolamines probably because of the existence of more oxidation sites derived from glycation of polar head group. Monitoring glycated polar head oxidation could be important to evaluate oxidative stress modifications that occur in diabetic patients.     

Identification and relative quantification of specific glycation sites in human serum albumin

Glycation (or non-enzymatic glycosylation) is a common non-enzymatic covalent modification of human proteins. Glucose, the highest concentrated monosaccharide in blood, can reversibly react with amino groups of proteins to form Schiff bases that can rearrange to form relatively stable Amadori products. These can be further oxidized to advanced glycation end products (AGEs). Here, we analyzed the glycation patterns of human serum albumin (HSA) in plasma samples obtained from five patients with type 2 diabetes mellitus. Therefore, glycated peptides from a tryptic digest of plasma were enriched with m-aminophenylboronic acid (mAPBA) affinity chromatography. The glycated peptides were then further separated in the second dimension by RP-HPLC coupled on-line to an electrospray ionization (ESI) tandem mass spectrometer (MS/MS). Altogether, 18 Amadori peptides, encompassing 40% of the HSA sequence, were identified. The majority of the peptides were detected and relatively quantified in all five samples with a high reproducibility among the replicas. Eleven Lys-residues were glycated at similar quantities in all samples, with glycation site Lys₅₄₉ (K_Am(Glc)QTALVELVK) being the most abundant. In conclusion, the established mAPBA/nanoRP-HPLC-ESI-MS/MS approach could reproducibly identify and quantify glycation sites in plasma samples, potentially useful in diagnosis and therapeutic control.     

HPLC同时检测血清和尿样中肌酐、假尿苷、尿酸

用反相高效液相色谱法同时测定了３９例糖尿病患者的血清和尿样中的假尿嘧啶核苷、肌酐和尿酸,并与２４ｈ尿白蛋白排泄量进行了比较分析。发现血清假尿嘧啶核苷是糖尿病肾病早期诊断的一种新颖而敏感的指标,有助于连续监测肾脏的功能状态以了解病程的转归。     

Two-dimensional liquid chromatographic methods to examine phenylboronate interactions with recombinant antibodies

A two-dimensional liquid chromatography (2D-LC) system incorporating Agilent 1100 components was set up and 2D-LC methods were developed to determine how recombinant antibodies (rAbs) interact with a commercial boronate column. Analyses of ten rAbs demonstrated that a simple boronate affinity chromatography method cannot be generalized to separate or detect the glycation level for all of rAbs as the aggregates were also retained on the boronate column. A quantitative boronate affinity chromatography method was developed to examine the extent of glycation of a recombinant humanized monoclonal antibody (rhuMAb). With the method, only the glycated components of intact or papain-treated rhuMAb were bound to the boronate column. When denatured, rhuMAb heavy chain was also bound. However, enzymatic removal of the N-oligosaccharides did not delete the binding, indicating that the interaction between the heavy chain and the phenylboronate functionality is not mediated through the N-oligosaccharides. Two-stage 2D-LC analysis with boronate affinity chromatography in the first dimension and reversed-phase chromatography in the second demonstrated that the interaction between the antibody heavy chain and boronate column is not specific, as urea is able to selectively elute retained heavy chain without disrupting the retention of glycated light chain. Quantum mechanic calculations show that the vicinal cis-diols in β-pyranose conformations in N-glycosylated antibodies need to overcome at least 7.172 kcal/mol to become co-planar (zero-dihedral angle). However, the cis-diols on β-furanose rings of glycated species only have to overcome a 0.885 kcal/mol energy barrier to achieve the co-planar configuration that facilitates its complex formation with the phenylboronate groups. This minimal energy barrier, coupled with the stabilizing effect from the electrostatic interaction between the oppositely charged phenylboronate group on the column and the fructosamine group of glycated species, explains why the boronate column exhibits high binding selectivity towards the glycated proteins.     

Advanced glycation end products: a highly complex set of biologically relevant compounds detected by mass spectrometry

Structural information on 'AGE-peptides,' a class of substances belonging to advanced glycation end products (AGE) and originating by proteolysis of glycated proteins, was gained through various analytical approaches on the mixture produced by proteinase K digestion of in vitro glycated bovine serum albumin. Both matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) were employed, and the results were compared with those from conventional spectroscopic methods (UV, fluorescence, gel permeation). The data acquired by the various techniques all depict the digestion mixtures as highly complex, with components exhibiting molecular mass in the range 300-3500 Da. In the analysis of HPLC/ESI-MS data, identification of AGE-peptides was facilitated by 3D mapping. Structural information was gained by means of multiple mass spectrometric experiments.     

Separation of Amadori peptides from their unmodified analogs by ion-pairing RP-HPLC with heptafluorobutyric acid as ion-pair reagent

Glycation is a common class of nonenzymatic posttranslational modifications relevant for several diseases and cell aging in general, such as D-glucose-derived modifications at the ɛ-amino groups of lysine residues in blood proteins, especially albumin, immunoglobulin, and hemoglobin, for diabetic patients. These Amadori compounds are identified on the peptide level after enzymatic digestion and chromatographic separation by mass spectrometry. Their syntheses usually rely on a global glycation approach. Both areas require the reliable separation of glycated peptides from their unmodified congeners present in different ratios, which is typically not achieved by standard eluent systems in ion-pairing RP-HPLC (IP-RPLC). Here, we compare aqueous acetonitrile and methanol gradients containing either trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) as ion-pairing agents to separate such peptide pairs. TFA-containing eluents resulted in rather low resolutions, and the glycated and unglycated peptides often coeluted. HFBA increased the retention times of the unmodified peptide more than for the glycated peptide thereby improving the separation of all eight studied peptide pairs, even achieving baseline separations for some sequences. Thus the use of HFBA as ion-pair reagent provides a universally applicable eluent system in IP-RPLC to separate glycated peptides from their unmodified counterparts, even at the preparative scale required for synthetic peptides.     

Urinary peptides as a diagnostic tool for renal failure detected by matrix-assisted laser desorption/ionisation mass spectrometry: an evaluation of their clinical significance

The development of new analytical methodologies related to the proteome for the evaluation of renal physiology and pathology is surely of wide interest for physicians, giving them new tools for monitoring complications associated with diabetes, such as end-stage renal disease. In the present study, the clinical significance of the urinary abundance of two peptides, SGSVIDQSRVLNLGPITR (the uromodulin precursor, m/z 1912) and IGPHypGPHypGLMGPP [present in the collagen-α-5(IV) chain precursor, m/z 1219], detected by matrix- assisted laser desorption/ionisation mass spectrometry (MALDI/MS) in microalbuminuric or nephropathic diabetic patients and in non-diabetic nephropathic patients was evaluated. A progressive increase in the abundance of the ion at m/z 1219 and a decrease in the abundance of the ion at m/z 1912 have been found in diabetic microalbuminuric, diabetic-nephropathic and nephropathic patients. Linear correlations are present between serum creatinine values and the abundances of the ions at m/z 1219 (positive correlation, r=0.3645, P<0.0001) and at m/z 1912 (negative correlation, r=-0.3053, P<0.0005). Correlations between the MALDI data and the estimated glomerular filtration rate were also found, while relationships with urinary albumin excretion were found only in sub-sets of patients. Analysis of receiver operating characteristic curves showed a sensitivity up to 96% and a specificity of up to 84% for the two ionic species, or their ratio, for distinguishing diabetic patients with different degrees of nephropathy from healthy subjects, proving that the urinary abundance of the two peptides at m/z 1219 and m/z 1912, determined with MALDI/MS, may be considered as a possible diagnostic tool for the determination of progression toward renal failure, also with the aim of monitoring kidney function, in diabetic patients.     

Modification of aluminum chips for LDI mass spectrometry of proteins

Matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI TOFMS) combined with affinity chromatography on immobilized phenylboronic acid agarose gels was used for selective enrichment and detection of specifically modified proteins such as glycated proteins in complex biological samples. Physicochemical grafting of hydrophilic polymers on aluminum surface was developed to reduce nonspecific protein sorption and to create a proper support layer for a three-dimensional affinity hydrogel. Grafted agarose allowed the fixation of three-dimensional agarose hydrogel on the chip surface. Both pinched polymers and hydrogels were effectively derivatized. 3-Aminophenylboronic acid (mPBA) was covalently immobilized as an affinity ligand to achieve specific binding of glycated plasma proteins. Alternatively, the affinity sorbent was immersed into the hydrogel to increase binding capacity. MALDI TOFMS was used to evaluate binding efficiency and molecular mass changes of human serum albumin due to glycation. Glycated proteins were captured directly on the chip with high selectivity and efficacy, and low nonspecific binding. Thus they could easily be characterized by MALDI TOFMS.