Glycation Of Human Serum Albumin Research Articles

Glycation of human serum albumin by acylglucuronides of nonsteroidal anti-inflammatory drugs of the series of phenylpropionates

The covalent binding to human serum albumin (HSA), of acylglucuronides from carboxylic nonsteroidal anti-inflammatory drugs (NSAIDs) was investigated. The adduct formation was followed and quantitated by HPLC and by radiometric detection. Three types of albumin adducts were evidenced. The acylglucuronide or the drug itself was bound to 0.2 up to 9 % of the albumin molecules, depending on the drug, whereas the majority of adducts (23–49 % of albumin molecules) retained the glucuronic acid moiety. The possible involvement of specific Lys located in site I of albumin in the formation of these main adducts was demonstrated, using a series of HSA whose specific Lys residues have been modified chemically. This study shows that acylglucuronides from NSAIDs can significantly contribute to the glycation of proteins, such as albumin.

Characterization of the glycation of albumin in freeze-dried and frozen human serum.

Human serum albumin (HSA) in fresh frozen and freeze-dried serum reference materials was examined by mass spectrometry and a variety of affinity chromatography techniques. The relative molecular mass distribution of HSA in fresh frozen serum was found to be identical to that of an HSA standard. However, the HSA in the freeze-dried reference serum exhibited a relative molecular mass distribution that was shifted to higher mass, broader, and substantially more heterogeneous than that of HSA in fresh frozen serum. A proteolytic cyanogen bromide digestion of the HSA from freeze-dried serum contained adducts approximately 162 u higher in mass than digest fragments 124-298 and 447-548, suggesting glycation. The presence of glycation on fragments 124-298 and 447-548 correlates with the known sites of HSA glycation. Glycation was further confirmed by the mass spectral analysis of the retained and unretained fractions from glycoaffinity chromatography of HSA from freeze-dried serum. The relative molecular weight of the HSA in the retained fraction indicated the presence of a doubly glycated species. The chemical heterogeneity of Cys-34, the site of the only free thiol in HSA, was examined and found not to be a substantial source of molecular mass heterogeneity for HSA from either fresh frozen of freeze-dried serum.

Glycation of Human Serum Albumin by DL-Glyceraldehyde: A Fluorescence Quenching Study

Process of glycation of human serum albumin (HSA) by DL-glyceraldehyde was studied using steady-state and time-resolved fluorescence spectroscopy in the course of 100 h. During this period, measurements of steady-state tryptophan (Trp) and non-tryptophan (non-Trp) fluorescence of the glycated HSA were carried out, together with measurement of the non-Trp fluorescence decay and steady-state quenching using potassium iodide as a quencher. Observed changes in both Trp and non-Trp fluorescence intensity, as well as changes in non-Trp fluorescence lifetimes and quenching efficiency are explained with respect to a probable mechanism of glycation.

Glucose Modification of Human Serum Albumin: A Structural Study

Structural changes associated with the exposure of human serum albumin (HSA) to glucose with or without the presence of Cu (II) have been characterized using a bank of methods for structural analysis including circular dichroism (CD), amino acid analysis (AAA), fluorescence measurements, SDS-PAGE, and boronate binding (which is a measure of Amadori product formation). We show that in the short-term (10 d) incubation mixtures, HSA is resistant to Cu (II)-mediated oxidative damage and that the early products of glycation of HSA had minimal effects on the folded structure. Amino acid analysis showed that there was no formation of advanced glycation endproducts (AGE), which can be measured by loss of lysine. This remained the case in longer term incubation of HSA (56 d) in the hyperglycemic concentration range (5–25 mM glucose) despite increased levels of Amadori product (60% boronate binding) and the formation of glycophore (Excitation 350, Emission 425). At high, nonphysiological concentrations (100 mM and 500 mM) of glucose, glycophore formation increased and 3 and 11 mol Lysine-glucose adduct/mol HSA were converted to AGE, respectively. This was accompanied by increased damage to tryptophan and protein-protein crosslinking but only minor tertiary structural change. In the presence of Cu (II), however, AGE formation was accompanied by extensive damage to histidine and tryptophan side chains, main chain fragmentation, and loss of both secondary and tertiary structure. Thus, changes in structure appear to be the result of oxidation as opposed to glycation, per se. © 1997 Elsevier Science Inc.

Matrix‐assisted laser desorption/ionization capabilities in the study of non‐enzymatic protein glycation

Matrix-assisted laser desorption/ionization mass spectrometry has been successfully applied in the study of non-enzymatic glycation of different proteins. In the case of bovine serum albumin, glycated by in vitro experiments performed under pseudophysiological conditions, a clear increase in molecular weight is observed with respect to both glucose concentrations and incubation time. The in vitro glycation of ribonuclease with glucose and fructose shows some peculiar differences either in terms of the number of condensed sugar molecules or in terms of the reaction kinetics. The same approach, applied to plasma proteins of healthy and diabetic subjects, provides evidence for the occurrence of glycation of human serum albumin for the latter subjects.

The adverse effect of glycation of human serum albumin on its preservative activity in the freeze-drying and accelerated degradation of alkaline phosphatase.

The effects of a glycated protein additive on the stability of freeze-dried biological standards were studied using alkaline phosphatase as a model. Alkaline phosphatase was formulated with artificially glycated albumin, freeze-dried, sealed into glass ampoules and subjected to accelerated degradation studies at temperatures from -20 to 56 degrees C. Alkaline phosphatase, freeze-dried without an additive in neutral buffer, lost over 95% of its activity, but when freeze-dried with human serum albumin it retained approximately 70% of the initial activity. Both deliberately glycated and native albumin protected approximately 70% of the initial activity on freeze-drying and this protection was maintained during storage of the freeze-dried product at temperatures of 20 degrees C or below for up to 16 weeks. At 37 degrees C or above, alkaline phosphatase activity was lost in a time- and temperature-related manner with changes appearing in the SDS-PAGE gels and FPLC chromatograms but, with the artificially glycated albumin formulations, this loss of activity and the changes in the gels and chromatograms happened earlier and at lower temperatures. Formulations with trehalose at 1% w/v and 15% w/v, but without albumin, preserved some 40% of alkaline phosphatase activity following freeze-drying. Further, approximately half of that activity was maintained after 16 weeks' storage at all temperatures up to 56 degrees C by the 15% trehalose without albumin.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycation of human serum albumin: inhibition by Diclofenac.

Glycation is a non-enzymatic modification of proteins by sugars, probably responsible for the initiation of complications in diabetes patients and aging individuals. Our in vitro experiments show an inhibition of sugar attachment in the presence of Diclofenac. The levels of advanced glycation products, measured as specific fluorescent groups, were also lowered due to Diclofenac. These results were compared with inhibition by Aspirin (acetylsalicylic acid), a known inhibitor of the glycation process. The protection by Diclofenac is based on a non-covalent interaction of the drug with serum albumin. There is evidence that Diclofenac specifically blocks at least one of the major glycation sites of human serum albumin.

Paramagnetic water proton relaxation enhancement: From contrast agents in MRI to reagents for quantitative “in vitro” assays

The use of stable complexes of paramagnetic metal ions as probes in analytical applications of the NMR technique is discussed. Two examples are given that involve the determination of the total Fe 3+ content of human serum transferrin (Tf) and the extent of glycation of human serum albumin (HSA). The method is based on the measurement of the longitudinal water proton relaxation times, conveniently altered following a selective chemical interaction between the substrate and the paramagnetic probe.

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.

Glycation of human serum albumin in long-term incubation with low and high concentrations of glucose.

Glycated albumin levels of incubation mixtures with high (25.8 mM) and low (8.2 mM) concentrations of glucose when measured by the nitroblue tetrazolium (NBT) reducing method, showed similar values at day 20-25. But when tested by thiobarbituric acid (TBA) colorimetry, the levels with high concentrations of glucose were about twice that with low concentration of glucose.

A microassay for protein glycation based on the periodate method

The periodate assay for glycated protein has been adapted for use with a microplate reader. Up to 94 samples can be read in 40 s, and sensitivity has been improved so that only 2–40 nmol of protein-bound sugar are needed per well. Yields have been increased to over 90%, allowing in vitro glycation of human serum albumin to be assayed on 0.1-mg aliquots.

High-performance liquid chromatofocusing and column affinity chromatography of in vitro14C-glycated human serum albumin : Demonstration of a glycation-induced anionic heterogeneity

High-performance liquid chromatofocusing of human serum albumin (HSA) after in vitro glycation with purified [ 14C]glucose has shown that with increasing glycation time a progressive increase in two major anionic fractions (p I4.8 and 4.65) occurs, while the p I 4.9 fraction decreases in parallel. As early as after 5 days of glycation time, the [ 14C]glucose content in the anionic fractions was markedly higher than in the p I 4.9 fraction. After 10 and 15 days of glycation, a considerable heterogeneity of 10–15 components could be demonstrated. In addition, phenylboronic acid (PBA) affinity chromatography was applied and an enrichment of the more glycated species could be obtained using this method. We conclude that, in contrast to previous reports, glycation of HSA induces anionic heterogeneity (in accordance with the theoretically expected loss of positively charged amino groups) and, although the efficiency in separating non-glycated from monoglycated HSA was found to be very low, an enrichment of these anionic species can be achieved using PBA affinity chromatography.