The Neoepitopes on Methylglyoxal- (MG-) Glycated Fibrinogen Generate Autoimmune Response: Its Role in Diabetes, Atherosclerosis, and Diabetic Atherosclerosis Subjects

Objectives In diabetes mellitus, hyperglycemia-mediated nonenzymatic glycosylation of fibrinogen protein plays a crucial role in the pathogenesis of micro- and macrovascular complications especially atherosclerosis via the generation of advanced glycation end products (AGEs). Methylglyoxal (MG) induces glycation of fibrinogen, resulting in structural alterations that lead to autoimmune response via the generation of neoepitopes on protein molecules. The present study was designed to probe the prevalence of autoantibodies against MG-glycated fibrinogen (MG-Fib) in type 2 diabetes mellitus (T2DM), atherosclerosis (ATH), and diabetic atherosclerosis (T2DM-ATH) patients. Design and Methods. The binding affinity of autoantibodies in patients' sera (T2DM, n = 100; ATH, n = 100; and T2DM-ATH, n = 100) and isolated immunoglobulin G (IgG) against native fibrinogen (N-Fib) and MG-Fib to healthy subjects (HS, n = 50) was accessed by direct binding ELISA. The results of direct binding were further validated by competitive/inhibition ELISA. Moreover, AGE detection, ketoamines, protein carbonyls, hydroxymethylfurfural (HMF), thiobarbituric acid reactive substances (TBARS), and carboxymethyllysine (CML) concentrations in patients' sera were also determined. Furthermore, free lysine and free arginine residues were also estimated. Results The high binding affinity was observed in 54% of T2DM, 33% of ATH, and 65% of T2DM-ATH patients' samples with respect to healthy subjects against MG-Fib antigen in comparison to N-Fib (p < 0.05 to p < 0.0001). HS sera showed nonsignificant binding (p > 0.05) with N-Fib and MG-Fib. Other biochemical parameters were also found to be significant (p < 0.05) in the patient groups with respect to the HS group. Conclusions These findings in the future might pave a way to authenticate fibrinogen as a biomarker for the early detection of diabetes-associated micro- and macrovascular complications.


Introduction
In diabetes mellitus, hyperglycemia-mediated nonenzymatic glycosylation or glycation of biological macromolecules (proteins, lipids, and DNA) plays a critical role in the pathogenesis of secondary complications especially micro-and macrovascular complications [1,2]. During glycation, free carbonyl groups (>C=O) of reducing sugars bind with free amino (-NH 2 ) groups at the N-terminal of proteins, ε-amino group of lysine, guanidino group of arginine, and imidazole group of histidine to form reversible aldimines or Schiff's bases [3,4]. Schiff's bases via keto-enol tautomerism and acid-base catalysis convert to irreversible ketoamines or Amadori products, and the latter undergo rearrangements, dehydrations, and cyclizations to form heterogeneous molecular adducts, known as advanced glycation end products (AGEs) [5,6].
Glycoxidation of glycation-adducts and autoxidation of glucose lead to the generation of reactive oxygen species (ROS) such as superoxide radicals (O 2 • ) via transition metal-catalyzed reaction and hydroxyl radicals ( • OH) via Fenton's reaction, thereby inducing oxidative stress [7,8]. Besides ROS generation, both autoxidation and glycoxidation give rise to aldehyde and/or ketone groups possessing highly electronegative dicarbonyls, called reactive carbonyl species (RCS), i.e., glyoxal (GO), methylglyoxal (MG), and 3deoxyglucosone (3-DG) [9,10]. Moreover, ROS-mediated oxidative stress induces lipid peroxidation and oxidation of amino acids, which result in the generation of RCS, especially MG. Furthermore, channelization of excess glucose in glycolysis and the polyol pathway leads to the generation of MG [11,12]. In human plasma, the physiological concentration of MG is 50-150 nM [13,14]. When this concentration goes beyond the normal and remains unchecked, the cellular system suffers carbonyl stress [15]. Furthermore, MG similarly glycates proteins as that of reducing sugars, but this time more violently, and induces a glycation-oxidation vicious cycle, thus accelerating glycative, oxidative, and carbonyl stress, the necessary outcome of which is AGE formation [16][17][18].
Fibrinogen (340 kDa; half-life~4 days; concentration 2-4 mg mL -1 ) is an independent marker for vascular complications especially in atherosclerosis [19,20]. Elevated fibrinogen levels and glycative insult suffered by fibrinogen are among the factors that are responsible for atherosclerosis-mediated CVD risk in diabetic patients [21,22]. The glycemic control influences the survival of fibrinogen in circulation that reduces during hyperglycemia and normalizes during euglycemia [23]. This correlation results due to the extent of fibrinogen glycation, as the glycated fibrinogen, clear more rapidly than nonglycated fibrinogen [24]. It has been reported that glycated fibrinogen usually resides extravascularly whereas the nonglycated one is distributed intravascularly. The extravascular accumulation (in the endothelial cells) of fibrinogen-AGEs (Fib-AGEs) results in their binding to receptors, called receptor for AGEs (RAGE), thereby establishing an AGE-RAGE axis. This axis triggers a signaling cascade, which in turn promotes atherosclerosis via the activation of ERK, JNK, PI3K, p38, NF-κB, and cAMP pathways. Thus, the increased uptake of glycated fibrinogen into vascular walls might contribute to the progression of atherosclerosis in diabetic patients [25]. Glycation causes structural modifications and functional alterations in fibrinogen molecule that ultimately results in the formation of a lysis-resistant dense and less porous fibrin network [26].
The lysine residues of fibrinogen play a central role in the formation and degradation (fibrinolysis) of the fibrin network. During blood coagulation, fibrinopeptides A and B cleave from the fibrinogen molecule by the action of thrombin to form fibrin monomers. These monomers polymerize to form protofibrils and get interwoven to give rise to a fibrin network or clot. The tensile strength of the clot and its resistance to lysis are imparted by peptide bonds between glutamine and lysine residues. These peptide bonds between adjacent fibrin molecules are formed by the action of activation factor XIII [25]. The lysis of the fibrin network (clot) is carried either by activation factor XIII-induced cross-linkage at the lysine residue or by direct binding of plasminogen, tissue plasminogen activator (tPA), and plasminogen activator inhibitor-1 (PAI-1) to lysine residues in fibrin molecules [27,28]. Thus, the glycation at lysine residues affects factor XIII cross-linkage, fibrin clot structure, and regulation of fibrinolysis. Glycated fibrinogen possesses higher clotting activity than nonglycated fibrinogen [29]. It has been reported that resistance to fibrinolysis increases with an increase in fibrinogen glycation [30]. The plasminolysis system that comprises plasminogen, tPA, and PIA-1 requires to bind at free lysine residues for lysis. However, lysine residues are engaged due to glycation and are not available for the process of fibrinolysis [31,32].
Moreover, AGE accumulation results in the monocytemacrophage-mediated production of cytokines (IL-1 and IL-6), which in turn activates hepatocytes for the overproduction of fibrinogen [33,34]. Besides the expression of adhesion molecules on endothelial cells, AGEs also stimulate monocyte migration and cytokine secretion from macrophages [35]. Furthermore, AGEs increase the levels of endothelin-1 and decrease the levels of nitric oxide, thereby resulting in overall vasoconstriction [36]. Additionally, AGEs facilitate clot formation by suppressing fibrinolysis via the activation of plasminogen activator inhibitor-1 (PAI-1) [37].
Glycation of fibrinogen with MG results in structural modifications and exposure of neoepitopes that elicit immune responses in experimental animals [38]. Previous studies by our research group have revealed the presence of autoantibodies against d-ribose-glycated LDL, d-riboseglycated hemoglobin (Hb), and MG-glycated LDL in type 2 diabetes mellitus (T2DM) and its associated secondary complications [39][40][41][42][43][44]. Therefore, we hypothesized that MGmediated glycation of fibrinogen leads to the generation of autoantibodies (anti-MG-Fib-IgG) in a hyperglycemic state. The present study was designed to probe the prevalence of autoantibodies against MG-glycated fibrinogen (MG-Fib) in patients suffering from T2DM, atherosclerosis (ATH), and diabetic atherosclerosis (T2DM-ATH).

Materials and Methods
2.1. Materials. Fibrinogen, methylglyoxal, oxalic acid, thiobarbituric acid (TBA), trichloroacetic acid (TCA), sodium chloride (NaCl), sodium hydroxide (NaOH), phosphotungstic acid, bovine serum albumin (BSA), ammonium persulphate (APS), bis-acrylamide, and tetramethylethylenediamine (TEMED) were purchased from Hi-Media. Sodium dodecyl sulfate (SDS) and 2,4,-trinitrobenzene-1-sulphonic acid (TNBS) were obtained from G Biosciences, whereas ethyl acetate and dinitrophenylhydrazine (DNPH) were purchased from Rankem. Guanidium hydrochloride was obtained from S. D. Fine-Chem Limited, and hydrochloric acid (HCl) was purchased from Fisher Scientific. A protein A agarose column was purchased from Sigma, and polystyrene plates were obtained from Nunc (Denmark). Analytical grade chemicals complexes (N-Fib+N-Fib-IgG) and glycated immune complexes (MG-Fib+MG-Fib-IgG) were used as inhibitors. All steps were the same as discussed earlier in the direct binding ELISA except that immune complexes were used as primary antibodies to be coated in place of sera or IgG [48]. Percent inhibition was calculated as 2.2.9. Hydroxymethylfurfural (HMF) Estimation. Hydroxymethylfurfural (HMF) estimation in the sera of HS and T2DM, ATH, and T2DM-ATH patients was performed by using thiobarbituric acid (TBA). The 0.1 mL of serum was mixed with 0.1 mL of 1 M oxalic acid and was left for 1 hour in the water bath. After cooling at room temperature, trichloroacetic acid (40%) was mixed in the solutions. The pre-cipitate was discarded, and the thiobarbituric acid (TBA) was added to the supernatant and was incubated at 37°C for half an hour. After the appearance of color, HMF concentration (nM mL -1 ) was evaluated by using the molar extinction coefficient of 4 × 104 M −1 cm −1 at 443 nm wavelength [55].

Thiobarbituric Acid Reactive Substance (TBARS)
Estimation. TBARS estimation in the sera of HS and T2DM, ATH, and T2DM-ATH patients was performed by adding H 2 SO 4 and phosphotungstic acid. The 0.1 mL of serum was mixed with 0.4 mL of 0.083 N H 2 SO 4 and 0.2 mL of 10% phosphotungstic acid. The solutions were left at room temperature for 5 minutes and then centrifuged at 3000 rpm for 10 minutes. The pellets were mixed with 2 mL of distilled water, and 0.5 mL of TBA reagent (a mixture containing 0.37% aqueous TBA, 15% aqueous TCA, and 0.25 N HCl) was added. The solutions were heated for 1 hour at 95°C, and centrifugation at 3000 rpm for 10 minutes was performed to collect the supernatant. The supernatant was left to cool at room temperature. TBARS concentration (μM mL -1 ) was evaluated by using the molar extinction coefficient of 1:56 × 10 5 M −1 cm −1 at 532 nm wavelength [56].

Free Lysine Residue Estimation.
Free lysine residue estimation in the sera of HS and T2DM, ATH, and T2DM-ATH patients was performed by using 2,4,6-trinitrobenzene sulphonic acid (TNBS). The 0.1 mL serum was mixed with 0.1 mL of NaHCO 3 buffer (4% w/v, pH 8.5). The 0.1 mL of 0.1% TNBS was added to the solutions, and the latter were incubated at 40°C for 2 hours. After incubation, 0.45 mL of HCL was added and again the solutions were incubated at 110°C for 90 minutes. After cooling the solutions, centrifugation was done at 3000 rpm for 10 minutes. The supernatant was mixed with 1 mL ether to remove the α-TNP amino complex, and finally the solutions were kept on a water bath till the evaporation of excess ether. The absorbance was taken at 420 nm [57,58].

Free Arginine Residue Estimation.
Free arginine residue estimation in the sera of HS and T2DM, ATH, and T2DM-ATH patients was performed by using 9,10-phenanthrenequinone. The 0.1 mL serum was mixed with 0.3 mL of 200 μM of phenanthrenequinone (dissolved in ethanol). After mixing, 0.5 mL of 2 N NaOH was added and the solutions were left for 60 min at 30°C. After cooling at room temperature, 0.5 mL of 1.2 M HCl was added and the fluorescence was taken on the Agilent Cary Eclipse fluorescence spectrophotometer at 25 ± 0:2°C in a 1 cm path length quartz cuvette. The samples were excited at 312 nm, and the emission was recorded at 395 nm [59].
2.2.13. Detection of Nonfluorogenic AGE: Carboxymethyllysine (CML). Carboxymethyllysine (CML) is a nonfluorogenic AGE that was detected in the sera of HS and T2DM, ATH, and T2DM-ATH patients by using a sandwich ELISA kit from Bioassay Technology Laboratory (Korain Biotech Co. Ltd.). The serum was diluted to 1 : 100 ratios with PBS (100 mM; pH 7.4). The diluted samples were added to the wells that were already coated with monoclonal antibodies against CML.

Oxidative Medicine and Cellular Longevity
After incubating for 15 minutes, the anti-CML antibody was added. The excess anti-CML antibody was removed during the washing step. Later on, streptavidin-HRP was added to the wells that bound to the anti-CML antibodies. After incubating for 15 minutes, washing was done to remove unbound streptavidin-HRP. Finally, the substrate was added that imparted color in the proportion of CML present in the serum samples. After the appearance of color, the reaction was terminated by adding an acidic stop solution and the absorbance was read at 450 nm [60].
2.2.14. Statistical Analysis. The data for the present study was presented as mean ± SD, and the statistical significance was determined by one-way ANOVA followed by the post hoc test, Tukey's multiple comparison test, and Dunnett t -test using the Statistical Package for the Social Sciences (SPSS) version 20.0. The coefficient of variation within groups (HS, T2DM, ATH, and T2DM-ATH) was analyzed by an unpaired t-test. The value of p < 0:05 was considered statistically significant. The scattered plots were plotted using GraphPad Prism version 5.0.1.

Biophysical and Biochemical Characterization of
Methylglyoxal-Glycated Fibrinogen. The in vitro methylglyoxal-(MG-) mediated glycation of fibrinogen protein results in secondary and tertiary level structural modifications as illustrated in our previous study [38]. The results of physicochemical characterization of native fibrinogen (N-Fib) and MG-glycated fibrinogen (MG-Fib) are summarized in Table 1.
Note that after the screening, all the biophysical and biochemical experimentations were performed on 54 T2DM, 33 ATH, and 65 T2DM-ATH serum samples.
3.5. Affinity of Isolated IgG to N-Fib and MG-Fib. The IgG concentration increases in T2DM and T2DM-ATH samples with respect to IgG isolated from HS. However, the concentration of IgG from ATH was higher than that from HS but was much lower in comparison to those from T2DM and T2DM-ATH subjects. To evaluate the concentration of IgG needed for the saturation of N-Fib and MG-Fib, direct binding ELISA was performed by isolated IgG from T2DM, ATH, and T2DM-ATH patients (Figure 3). The results revealed that the average saturation concentration of MG-Fib is 50 μg mL -1 of IgG from T2DM and T2DM-ATH patients. However, for the ATH group, the saturation concentration was 60 μg mL -1 .
3.6. Competitive ELISA. Competitive/inhibition ELISA was performed to ensure antigen-binding specificity of purified a5utoantibodies that were generated against MG-Fib in patients' sera. The inhibition ELISA was done for 54 IgG samples from the T2DM group, 33 IgG samples from the ATH group, and 65 IgG samples from the T2DM-ATH group, which were screened for greater affinity towards MG-Fib in the direct binding ELISA with sera (Section 3.3). The IgG samples from the HS group were used as a control.
3.12. Estimation of Free Lysine in HS and T2DM, ATH, and T2DM-ATH Patients. In the HS group, the mean absorbance at 420 nm was found to be 0:181 ± 0:007 (0.017-0.194), whereas in T2DM, ATH, and T2DM-ATH, the absorbance was lower up to 0:107 ± 0:012 (0.083-0.125), 0:116 ± 0:010 (0.100-0.139), and 0:088 ± 0:014 (0.061-0.130), respectively. The percent decrease in free lysine in T2DM, ATH, and T2DM-ATH groups with respect to healthy subjects was 40%, 35%, and 51%, respectively ( Figure 10). All the three patient groups exhibit statistical significance (p < 0:0001) with the HS group. The coefficient of variation in HS, T2DM, ATH, and T2DM-ATH groups was 4.07%, 12.08%, 8.77%, and 16.85%, respectively. The statistical data are summarized in Table 4.  Figure 11). The percent decrease in free arginine in T2DM, ATH, and T2DM-ATH groups with respect to healthy subjects was 35%, 27%, and 40%, respectively. All the three patient groups exhibit statistical significance (p < 0:0001) with respect to the HS group. The coefficient of variation in HS, T2DM, ATH, and T2DM-ATH groups was 7.95%, 7.72%, 9.94%, and 18.71%, respectively. The statistical data are summarized in Table 4. 3.14. CML Estimation in Sera of HS and T2DM, ATH, and T2DM-ATH Patients. In the HS group, the mean concentration of CML was found to be 0:215 ± 0:026 nM mL −1 (0.170-  , and diabetic atherosclerosis (T2DM-ATH, n = 65) patients. The HS group was used as a control. All the three patient groups showed statistical significance with respect to the HS group and among themselves ( * * p < 0:001, * * * p < 0:0001). Data are presented as mean ± SD of three independent experiments. The data was presented as mean ± SD, and the statistical significance level (p < 0:05) between HS, T2DM, ATH, and T2DM-ATH groups was determined by one-way ANOVA. All the three patient groups exhibit statistical significance (p < 0:0001) with the HS group. "N" is the no. of subjects in each group, "Min." is the minimum value, "Max." is the maximum value, "SD" is the standard deviation, "CV" is the coefficient of variation, and "p" is the level of significance. The upward arrows (↑) represent percent increase whereas downward arrows (↓) represent percent decrease in parameters with respect to the HS group.  Figure 12). The percent increase in CML concentration in T2DM, ATH, and T2DM-ATH groups with respect to healthy subjects was 73%, 67%, and 75%, respectively. All the three patient groups exhibit statistical significance (p < 0:0001) with respect to the HS group. The coefficient of variation in HS, T2DM, ATH, and T2DM-ATH groups was 12.31%, 17.36%, 12.95%, and 21.34%, respectively. The statistical data are summarized in Table 4.

Discussion
Reducing sugars along with dicarbonyls or reactive carbonyl species (RCS) such as methylglyoxal (MG) can instigate in vivo nonenzymatic glycosylation [18]. This reaction is a slow process but increases several folds during prolonged and persistent hyperglycemia [4]. It has been investigated that MG is a highly reactive dicarbonyl that leads to cellular dysfunction by perturbing native structure and normal function of proteins. Moreover, in the persistent hyperglycemic state, the concentration of dicarbonyls was significantly elevated [11]. Furthermore, exposure and generation of neoepitopes on glycated proteins render them highly immunogenic which leads to the production of autoantibodies [44].
The direct binding ELISA of patients' sera has revealed a higher prevalence of autoantibodies in type 2 diabetes mellitus (T2DM, 54%), atherosclerosis (ATH, 33%), and diabetic atherosclerosis (T2DM-ATH, 65%) against MG-Fib. However, healthy subject (HS) sera did not show significant binding with both the N-Fib and MG-Fib. From our in vitro characterization study, it was implied that MG-mediated    patients. The HS group was used as a control. All the three patient groups showed statistical significance with respect to the HS group and among themselves ( * * p < 0:001, * * * p < 0:0001). Data are presented as mean ± SD of three independent experiments.

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Oxidative Medicine and Cellular Longevity glycation of fibrinogen leads to the formation of fibrinogen-AGEs (Fib-AGEs), which might elicit immune response via neoepitope generation [38]. The presence of highly specific anti-MG-Fib autoantibodies or anti-Fib-AGEs autoantibodies in the sera of T2DM (54%), ATH (33%), and T2DM-ATH (65%) patients is in agreement with our in vitro study. Moreover, direct binding ELISA of sera against N-Fib also showed significant binding in some samples of T2DM and T2DM-ATH patient groups. The reason might be the denaturation and exposure of neoepitopes on N-Fib during incubation at 37°C for 7 days. The direct binding of isolated IgG from those patients' sera that showed higher affinity towards MG-Fib further validates the generation of autoantibodies (anti-MG-Fib-IgG) in the patient groups. Moreover, autoantibodies (anti-N-Fib-IgG) against N-Fib were also found in all the three patient groups and showed significant binding with respect to the HS group. However, the absorbance at 410 nm was much lower in N-Fib with respect to MG-Fib. The results are in agreement with direct binding ELISA of sera against N-Fib.
In competitive/inhibition ELISA, the maximum inhibition of isolated IgG against N-Fib and MG-Fib follows the same descending order: T2DM − ATH > T2DM > ATH. However, in the case of N-Fib, the maximum percent inhibition among T2DM and T2DM-ATH patient groups with respect to the HS group was found to be significant (p < 0:05), whereas maximum percent inhibition against MG-Fib in the patient groups with respect to the HS group was significant (p < 0:05). Thus, the results suggest that the high specificity of serum autoantibodies or isolated IgG towards MG-Fib is due to the generation of neoepitopes on MG-modified fibrinogen as compared to N-Fib. Moreover, percent inhibition towards N-Fib reveals that N-Fib gets denatured during incubation and neoepitopes are exposed, against which autoantibodies (anti-N-Fib-IgG) are generated.
The NBT reduction assay of patients' sera suggests a 5-6fold increase in ketoamine concentration in T2DM and T2DM-ATH patients with respect to healthy subjects. However, in ATH patients,~3-fold increase in ketoamines was noticed in comparison to that in healthy individuals. The early glycation products reduce the yellow NBT to purple monoformazan. It has been suggested that NBT is reduced by the action of superoxide radicals that are formed during the degradation of ketoamines [61,62]. Furthermore, the superoxide radicals via the Fenton reaction give rise to hydroxyl radicals. Thus, it is hypothesized that both superoxide and hydroxyl radicals might cause damage to protein molecules besides glycation [63,64].
The fluorescence emission profiles at 460 nm confirmed the presence of AGEs in T2DM, ATH, and T2DM-ATH patients' sera. In comparison to healthy individuals, the patient groups exhibit a 2-4-fold increase in fluorescence intensities (F.I.). However, in the ATH group, there is ã 10% decline in F.I. with respect to T2DM and T2DM-ATH , atherosclerosis (ATH, n = 33), and diabetic atherosclerosis (T2DM-ATH, n = 65) patients. The HS group was used as a control. All the three patient groups showed statistical significance with respect to the HS group and among themselves ( * p < 0:05, * * p < 0:001, and * * * p < 0:0001). Data are presented as mean ± SD of three independent experiments.  Figure 12: Carboxymethyllysine concentration (nM mL -1 ) in the sera of healthy subjects (HS, n = 50) and diabetic (T2DM, n = 54), atherosclerosis (ATH, n = 33), and diabetic atherosclerosis (T2DM-ATH, n = 65) patients. The HS group was used as a control. All the three patient groups showed statistical significance with respect to the HS group and among themselves ( * p < 0:05, * * p < 0:001, and * * * p < 0:0001). Data are presented as mean ± SD of three independent experiments.
13 Oxidative Medicine and Cellular Longevity groups which indicates more generation of fluorescent AGEs under a hyperglycemic state. Moreover, it has been reported that the fluorescence is exhibited only by those AGEs that have heterocyclic structures [65,66].
Protein carbonyls are the markers of oxidative stress formed via enediol reaction from ketoamines (67,68). During enediol reaction, superoxide radicals are formed that give rise to hydroxyl radicals via the Fenton reaction that further contributes to oxidative stress (69,70). In our study, 3-5-fold increase in carbonyl contents in T2DM, ATH, and T2DM-ATH patients confirms higher oxidative stress. However, in the ATH group, the carbonyl contents were lower in concentration than those in T2DM and T2DM-ATH further authenticating hyperglycemia-induced oxidative stress in diabetic subjects. Earlier studies on diabetic patients suggest that increased accumulation of carbonyl contents exerts an adverse effect on physiological systems (71,72). Both hydroxymethylfurfural (HMF) and malondialdehyde (MDA) are thiobarbituric acid (TBA) reactive species (TBARS) (38). HMF is released upon hydrolysis of ketoamines by weak acids, whereas MDA is an indicator of oxidative stress generated during oxidation of proteins to protein carbonyls via enediol reaction (60,73). In our study, we found a 3-8-fold increase in HMF content and a 6-8-fold increase in MDA concentration in T2DM, ATH, and T2DM-ATH patients with respect to healthy subjects. However, in the ATH group, the HMF contents and MDA concentration were lesser than those in T2DM and T2DM-ATH. This indicates that more oxidative stress is generated during diabetesinduced prolonged and persistent hyperglycemia. The results indicate enhanced oxidative stress in diabetic and diabetes atherosclerosis patients due to hyperglycemiamediated accelerated protein glycation. The ketoamines, carbonyl contents, HMF, and TBARS results are in agreement with each other.
The lysine (Lys) and arginine (Arg) amino acids are more susceptible to glycation because of the presence of free amino group (-NH 2 ) and guanidino group in their respective side chains (59). In our study, the significant drop in free Lys and free Arg in T2DM and T2DM-ATH patients with respect to the HS group portrays the extent of glycation and AGE formation. The percentage of reacted lysine and arginine residues in T2DM and T2DM-ATH patients is much higher with respect to healthy individuals.
CML is a nonfluorogenic AGE formed in hyperglycemic individuals (74)(75)(76). In our study, a 3-4-fold increase in CML levels in the patient groups suggests increased glycative stress in T2DM and T2DM-ATH patients in comparison to healthy individuals.

Conclusion
The present study explored methylglyoxal-mediated structural perturbations induced in fibrinogen protein that results in the generation of neoepitopes, which in turn provokes an immune response in the form of autoantibodies (anti-MG-Fib antibodies) in T2DM, ATH, and T2DM-ATH patients. However, the precise role of glycated fibrinogen in the pathogenesis of diabetes-associated micro-and macrovascular complications is still in veil and needs further investigation. We hope that our findings may provide an approach to predict diabetes-related atherosclerosis before its onset by validating glycated fibrinogen as a new biomarker. Moreover, this study might facilitate the scientific fraternity to minimize the menace of glycation-induced oxidative and carbonyl stress, by inhibiting and scavenging the intermediates of nonenzymatic glycosylation.

Data Availability
Data is available within the text of the manuscript.

Conflicts of Interest
The authors declare that they have no conflict of interest.