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.     

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.     

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.     

Use of fullerene‐, octadecyl‐, and triaconthyl silica for solid phase extraction of tryptic peptides obtained from unmodified and in vitro glycated human serum albumin and fibrinogen

SPE plays a crucial role in bioanalytical research. In the present work a novel fullerene(C60)‐derivatised silica material is compared with octadecyl(C18) – and triaconthyl(C30)‐silicas regarding recoveries of peptides and sequence coverage of HSA and fibrinogen digests. C30‐ and C60(30 nm)‐SPE materials were found to be the two most prominent SPE materials. At low peptide concentrations C60‐material prepared from a silica gel with a pore size of 30 nm has proven to be the best material with regards to recoveries. By increasing the amount of loaded peptides recoveries decrease due to its relative low binding capacity in contrast to C30‐silica particles, showing no changes. The best sequence coverages of Aα‐ and Bβ‐chains of 20 pmol fibrinogen digest can also be achieved using these two SPE materials, C60 (30 nm) demonstrates an outstanding value of sequence coverage (62.15%) achieved for the γ‐chain. After nonenzymatic glycation the digests of fibrinogen and HSA were also separated. This makes the detection of a considerably higher number of glycated peptides possible compared to the unfractionated digests and the use of boronate affinity chromatography in the case of fibrinogen. For HSA, ten new sites of glycation at lysine and arginine residues have been explored. Using the detailed SPE/off‐line MALDI method the glycation sites on fibrinogen are first described in this paper.     

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.     

Accurate mass measurements by Fourier transform mass spectrometry in the study of advanced glycation end products/peptides

The Maillard reaction occurring between sugars and amino groups is important in living systems. When amino groups belonging to protein chains are involved, the Maillard reaction has been invoked as responsible for protein cross-linking and the production of 'toxic' compounds. The reaction leads to the production of a heterogeneous group of substances, usually called advanced glycation end products (AGEs). Classical analytical approaches, such as spectroscopic (ultraviolet, fluorescence) and mass spectrometric (matrix-assisted laser desorption/ionization, liquid chromatography/electrospray ionization mass spectrometry) methods, have shown that the digestion mixture is highly complex. However, there are clear differences between the digestion mixtures of glycated and unglycated human serum albumin (HSA). In the former case, possible glycated peptides belonging to the AGE peptide class may be identified. Tandem mass spectrometric experiments on selected species seemed to be promising as regards structural information, but it was thought of interest to undertake the present investigation, based on liquid chromatography/electrospray ionization Fourier transform mass spectrometry, in order to obtain definitive results on their elemental composition. Using this approach, about 20 glycated peptides were detected and their possible structures were postulated by examining the known sequence of HSA.     

High-performance affinity chromatography and the analysis of drug interactions with modified proteins: binding of gliclazide with glycated human serum albumin

This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (K _a) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclazide according to a two-site model, with a class of high-affinity sites (average K _a, 7.1–10 × 10⁴ M⁻¹) and a group of lower-affinity sites (average K _a, 5.7–8.9 × 10³ M⁻¹) at pH 7.4 and 37 °C. Competition experiments indicated that Sudlow sites I and II of HSA were both involved in these interactions, with the K _a values for gliclazide at these sites being 1.9 × 10⁴ and 6.0 × 10⁴ M⁻¹, respectively, for normal HSA. Two samples of glycated HSA had similar affinities to normal HSA for gliclazide at Sudlow site I, but one sample had a 1.9-fold increase in affinity at this site. All three glycated HSA samples differed from normal HSA in their affinity for gliclazide at Sudlow site II. This work illustrated how HPAC can be used to examine both the overall binding of a drug with normal or modified proteins and the site-specific changes that can occur in these interactions as a result of protein modification.     

Glycation and oxidation of histones H2B and H1: in vitro study and characterization by mass spectrometry

Among the post-translational modifications, oxidation and glycation are of special interest, especially in diseases such as diabetes, and in aging. The synergistic interaction between glycation and oxidation, also known as “glycoxidation” is highly relevant due to its involvement in the production of deleterious changes at the molecular level. Non-enzymatic damage to nuclear proteins has potentially severe consequences for the maintenance of genomic integrity [54]. In this report, we study glycated histones and its in vitro oxidation. Data concerning the modifications that occurred in the histones were obtained by analysis of enzymatic digests (Glu-C and Arg-C) of unmodified and glycated histones, obtained before and after oxidation. Analysis was then performed using a MALDI-MS/MS-based approach combined with nano liquid chromatography. This approach allowed us to identify histone H2B and H1 specific-sites of oxidation and to distinguish the most affected residues for each histone. The results showed the occurrence of a cumulative effect of oxidative damage in the glycated histones when subjected to in vitro oxidation, suggesting that structural changes caused by glycation induces histones to a pro-oxidant state. Comparing the data of oxidized glycated histones with data from unmodified oxidized histones, using the same model of oxidation, the results clearly show that these oxidative modifications occur earlier and more extensively in glycated histones. Furthermore, the results pointed to an increased oxidative damage in the vicinity of the glycated residues.     

Identification of the carboxymethyllysine residue in the advanced stage of glycated human serum albumin.

As an advanced stage of glycation, glycated human serum albumin (G-HSA; glucose content, 2 mol of 5-hydroxymethylfurfural equivalent/mol of HSA) was incubated at 37 degrees C up to 30 d in 0.2 M phosphate buffer, pH 7.4, with 100 microM Fe3+. G-HSA incubated for 30 d (G-HSA-30(Fe)) was subsequently hydrolyzed at 110 degrees C for 24 h in 6 N HCl. In the hydrolysate, N epsilon-carboxymethyllysine (CML) was identified by cochromatography with synthesized CML on an amino acid analyzer. pI of HSA (4.8) shifted to 4.5 in G-HSA. A more acidic fraction, pI 4.3, appeared in G-HSA-30(Fe). CML content (mol of CML/mol of HSA) of HSA and G-HSA was as follows; 0 in HSA, 0.2 in HSA-30(Fe), 0.4 in G-HSA and 1.5 in G-HSA-30(Fe) pI 4.3. The amino acid compositions also changed in lysine, arginine and tyrosine at the advanced stage of the reaction.     

Glycation pattern of peptides condensed with maltose,lactose and glucose determined by ultraviolet matrix‐assisted laser desorption/ionization tandem mass spectrometry

Protein glycation is the non‐enzymatic condensation of sugars with proteins. Although commonly occurring in both the therapeutic and food/beverage industries, protein glycation has not been the focus of many proteomic investigations. This study aims to establish a reliable mass spectrometric method for screening large tandem mass spectrometric (MSMS) datasets for protein glycation with glucose, lactose and maltose. Control experiments using a standard peptide containing a single glycation site led to the discovery of characteristic neutral loss fragmentation patterns in MSMS analysis for glucose, lactose and maltose condensed with peptides. Valid in both tandem time‐of‐flight (TOFTOF) and quadrupole ion trap time‐of‐flight matrix‐assisted laser desorption/ionization (QIT TOF MALDI) mass spectrometers, these neutral loss signatures were then applied to elucidation of modified peptides from a complex human serum albumin (HSA) digest glycated with each of the proposed sugars. Screening of these large datasets was made possible by specifically designed software solutions that enable the input of detailed user‐defined post‐translational modifications that are not included in the universally available databases such as Unimod. Copyright © 2010 John Wiley & Sons, Ltd.     

Structural Analysis and Aggregation Propensity of Reduced and Nonreduced Glycated Insulin Adducts

The milieu within pancreatic β cells represents a favorable environment for glycation of insulin. Therefore, in this study, insulin samples were individually subjected to glycation under reducing and nonreducing conditions. As monitored by ortho-phthalaldehyde and fluorescamine assays, the reduced glycated insulin adduct demonstrates extensively higher level of glycation than the nonreduced glycated counterpart. Also, gel electrophoresis experiments suggest a significant impact of glycation under a reducing system on the level of insulin oligomerization. Furthermore, reduced and nonreduced glycated insulin adducts respectively exhibit full and partial resistance against dithiothreitol-induced aggregation. The results of thioflavin T and Congo red assays suggest the existence of a significant quantity of amyloid-like entities in the sample of reduced glycated insulin adduct. Both fluorescence and far-ultraviolet circular dichroism studies respectively suggest that the extents of unfolding and secondary structural alteration were closely correlated to the level of insulin glycation. Moreover, the surface tension of two glycated insulin adducts was inversely correlated to their glycation extents and to the quantity of exposed hydrophobic patches. Overall, the glucose-modified insulin molecules under reducing and nonreducing systems display different structural features having significant consequences on aggregation behaviors and surface tension properties. The particular structural constraints of glycated insulin may reduce the binding interaction of this hormone to its receptor which is important for both insulin function and clearance.     

Synergistic and inhibitory effects of propolis and aspirin on structural changes of human hemoglobin resulting from glycation: an in vitro study

This study investigated the effects of the ethanolic extract of propolis (EEP) in the absence and presence of aspirin on the glycation-induced structural changes of human hemoglobin (Hb). Hb samples were incubated in the presence and absence of glucose (40 mM) for 5 weeks in the absence and presence of various concentrations of EEP and aspirin. Sampling was conducted at the end of each week, and the extent of glycation was evaluated by spectrophotometry, Thioflavin-T test, and by measuring free amino group contents and heme degradation products. Results showed that Hb was glycated by glucose after various incubation times reduced free amino group content, downturn absorption of and induced blue shifting in the UV–visible spectrum of Hb as well as increased amyloid structures and heme degradation. EEP prevented these changes and decreased the extent of glycation in a concentration-dependent manner. Aspirin also prevented glycation with the same efficacy as EEP (20 μg/ml). Surprisingly, the simultaneous presence of aspirin and EEP resulted in the extensive inhibition of glycation; in the 40 μg/ml concentration of EEP and aspirin, glycation was fully prevented in a synergic way. It is probable that EEP exerts its anti-glycation effect via its potent antioxidant and radical scavenging properties which decrease glycation-produced oxidative stress. In conclusion, EEP is to be introduced as an anti-glycation agent for the first time in this study. It could be developed as a natural agent for the design of new anti-glycation drugs aimed at reducing protein glycation as well as diabetic complications.     

Mass spectrometry characterization of the glycation sites of bovine insulin by tandem mass spectrometry

Bovine insulin was glycated under hyperglycemic reducing conditions and in nonreducing conditions. Purification through HPLC allowed isolating glycated forms of insulin and a novel triglycated form (6224.5 Da) was purified. Endoproteinase Glu-C digestion combined with mass spectrometry (MALDI-TOF/TOF) allowed determining the exact location of the glycation sites in each of the isolated glycated insulins. For the first time, a triglycated form of insulin was isolated and characterized accordingly to its glycation sites. These glucose binding sites were identified as the N-terminals of both chains (Gly1 and Phe1) and residue Lys29 of B-chain. Moreover, in diglycated insulin we found the coexistence of one specie glycated at the N-terminals of both chains (Gly1 and Phe1) and another specie containing the two glucitol adducts in B-chain (Phe1 and Lys29). Also, in monoglycated insulin generated in reducing and nonreducing conditions, one specie glycated at Phe1 and another specie glycated at Lys29, both B-chain residues coexist.     

Biophysical,Biochemical, and Molecular Docking Investigations of Anti-Glycating,Antioxidant, and Protein Structural Stability Potential of Garlic

Garlic has been reported to inhibit protein glycation, a process that underlies several disease processes, including chronic complications of diabetes mellitus. Biophysical, biochemical, and molecular docking investigations were conducted to assess anti-glycating, antioxidant, and protein structural protection activities of garlic. Results from spectral (UV and fluorescence) and circular dichroism (CD) analysis helped ascertain protein conformation and secondary structure protection against glycation to a significant extent. Further, garlic showed heat-induced protein denaturation inhibition activity (52.17%). It also inhibited glycation, advanced glycation end products (AGEs) formation as well as lent human serum albumin (HSA) protein structural stability, as revealed by reduction in browning intensity (65.23%), decrease in protein aggregation index (67.77%), and overall reduction in cross amyloid structure formation (33.26%) compared with positive controls (100%). The significant antioxidant nature of garlic was revealed by FRAP assay (58.23%) and DPPH assay (66.18%). Using molecular docking analysis, some of the important garlic metabolites were investigated for their interactions with the HSA molecule. Molecular docking analysis showed quercetin, a phenolic compound present in garlic, appears to be the most promising inhibitor of glucose interaction with the HSA molecule. Our findings show that garlic can prevent oxidative stress and glycation-induced biomolecular damage and that it can potentially be used in the treatment of several health conditions, including diabetes and other inflammatory diseases.     

Modification of bovine beta-lactoglobulin by glycation in a powdered state or in aqueous solution: adsorption at the air-water interface

The adsorption at the air-water interface of native and various glycated forms of beta-Lactoglobulin B (beta-LG), prepared under two different experimental conditions, was investigated by ellipsometry, surface tension and shear elastic constant measurements. The measurements were performed in 0.1 M phosphate buffer, 0.1 M NaCl, pH 6.8. It was found that the interfacial properties of beta-LG were more affected when the glycation was performed in solution than in the dry-way system. Dry-way glycated beta-LG, despite a higher glycation extent, affected slightly its interfacial behaviour. Solution glycated beta-LG exhibited a higher adsorption and more rigid interface as expressed by shear elastic constant measurement at saturation (16.5 mN/m against 8.7 and 11.5 mN/m for native and control treated beta-LG, respectively). These results were attributed to the specific molecular species induced during glycation in solution, which includes monomers and unfolded covalent homodimers of beta-LG molecules with a high tendency to self-association via non-covalent interactions.     

In vitro nonenzymatic glycation of guanosine 5′-triphosphate by dihydroxyacetone phosphate

Dihydroxyacetone phosphate (DHAP) is a glycolytic intermediate that has been found to be significantly elevated in the erythrocytes of diabetic patients and patients with triosephosphate isomerase deficiency. DHAP spontaneously breaks down to methylglyoxal, a potent glycating agent that reacts with proteins and nucleic acids in vivo to form advanced glycation endproducts (AGEs). Like methylglyoxal, DHAP itself is also a glycating metabolite, capable of condensing with proteins and altering their structure or function. The objective of this investigation was to evaluate the susceptibility of nucleotides to nonenzymatic attack by DHAP, and to determine the factors influencing the rate and extent of nucleotide glycation by this sugar. Of the four nucleotide triphosphates (ATP, CTP, GTP and UTP) that were studied, only GTP was reactive, forming a wide range of UV and fluorescent products with DHAP. Increases in temperature and nucleotide concentration enhanced the rate and extent of GTP glycation by DHAP and promoted the heterogeneity of AGEs. Capillary electrophoresis, HPLC, and mass spectrometry allowed for a thorough analysis of the glycated products and demonstrated that the reaction of DHAP with GTP occurred via the classical Amadori pathway.     

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.     

Unravelling the effect of N(ε)-(carboxyethyl)lysine on the conformation,dynamics and aggregation propensity of α-synuclein

α-Synuclein (αS) aggregation is a hallmark in several neurodegenerative diseases. Among them, Parkinson''s disease is highlighted, characterized by the intraneuronal deposition of Lewy bodies (LBs) which causes the loss of dopaminergic neurons. αS is the main component of LBs and in them, it usually contains post-translational modifications. One of them is the formation of advanced glycation end-products (mainly CEL and MOLD) arising from its reaction with methylglyoxal. Despite its biological relevance, there are no data available proving the effect of glycation on the conformation of αS, nor on its aggregation mechanism. This has been hampered by the formation of a heterogeneous set of compounds that precluded conformational studies. To overcome this issue, we have here produced αS homogeneously glycated with CEL. Its use, together with different biophysical techniques and molecular dynamics simulations, allowed us to study for the first time the effect of glycation on the conformation of a protein. CEL extended the conformation of the N-terminal domain as a result of the loss of transient N-/C-terminal long-range contacts while increasing the heterogeneity of the conformational population. CEL also inhibited the αS aggregation, but it was not able to disassemble preexisting amyloid fibrils, thus proving that CEL found on LBs must be formed in a later event after aggregation.

We study the effect of an advanced glycation end product (N(ε)-(carboxyethyl)lysine), found on the Lewy bodies of people suffering from Parkinson’s disease, on the conformational and aggregation features of alpha-synuclein.     

Helical peptide models for protein glycation: proximity effects in catalysis of the Amadori rearrangement.

INTRODUCTION: Non-enzymatic glycation of proteins has been implicated in various diabetic complications and age-related disorders. Proteins undergo glycation at the N-terminus or at the epsilon-amino group of lysine residues. The observation that only a fraction of all lysine residues undergo glycation indicates the role of the immediate chemical environment in the glycation reaction. Here we have constructed helical peptide models, which juxtapose lysine with potentially catalytic residues in order to probe their roles in the individual steps of the glycation reaction. RESULTS: The peptides investigated in this study are constrained to adopt helical conformations allowing residues in the i and i+4 positions to come into spatial proximity, while residues i and i+2 are far apart. The placing of aspartic acid and histidine residues at interacting positions with lysine modulates the steps involved in early peptide glycation (reversible Schiff base formation and its subsequent irreversible conversion to a ketoamine product, the Amadori rearrangement). Proximal positioning of aspartic acid or histidine with respect to the reactive lysine residue retards initial Schiff base formation. On the contrary, aspartic acid promotes catalysis of the Amadori rearrangement. Presence of the strongly basic residue arginine proximate to lysine favorably affects the pK(a) of both the lysine epsilon-amino group and the singly glycated lysine, aiding in the formation of doubly glycated species. The Amadori product also formed carboxymethyl lysine, an advanced glycation endproduct (AGE), in a time-dependent manner. CONCLUSIONS: Stereochemically defined peptide scaffolds are convenient tools for studying near neighbor effects on the reactivity of functional amino acid sidechains. The present study utilizes stereochemically defined peptide helices to effectively demonstrate that aspartic acid is an efficient catalytic residue in the Amadori arrangement. The results emphasize the structural determinants of Schiff base and Amadori product formation in the final accumulation of glycated peptides.     

Correlation between glycated lipoproteins and fructosamine level in serum.

The correlation between the level of fructosamine and glycated proteins, including glycated lipoproteins, in serum from diabetic and nondiabetic subjects was studied. Assay of glycated proteins in serum was performed using an agarose gel film electrophoresis with nitroblue tetrazolium coloration. Glycated albumin correlated well with the fructosamine level in the diabetics (r = 0.83-0.92, p less than 0.01) but showed no correlation with the nondiabetics (r = 0.25-0.26). Also, a high correlation between the glycated beta-lipoprotein and fructosamine levels was observed in diabetic patients with hyperglycemia and in nondiabetic subjects with a high risk of atherogenesis (atherogenic index, low-density lipoprotein-cholesterol/high-density lipoprotein-cholesterol greater than 2.8) (r = 0.51-0.66, p less than 0.01). Nondiabetics with a high level of beta-lipoprotein, which is well known to cause high atherogenesity, showed a high level of glycated beta-lipoprotein similar to that in the diabetic groups with hyperglycemia; therefore, the high level of glycated beta-lipoprotein seems to be attributable not only to the hyperglycemia-accelerated glycation of beta-lipoprotein but also to an increase in the level of beta-lipoprotein in serum. Consequently, the present results show that the fructosamine level in serum reflects not only the glycation of albumin but also that of lipoproteins which are known to increase in diabetes mellitus.