aAna Aslan - National Institute of Gerontology and Geriatrics, Bucharest, RomaniabCarol Davila - University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Biochemistry, Bucharest, Romania
A R T I C L E I N F O
☆ This article is part of a Special Issue entit⁎ Corresponding author at: Department of Bio
We aimed to explore the association of advanced oxidation and advanced glycation ofproteins, and their interrelations with endothelial nitric oxide synthesis, oxidative stress,metabolic profile as well as other atherosclerotic risk markers in prediabetic and diabeticelderly subjects. Advanced glycation end products (AGEs), advanced oxidation proteinproducts (AOPPs), low-density lipoprotein susceptibility to oxidation (oxLDL) and nitricoxide metabolic pathway products (NOx) were assessed in subjects with impaired fastingglucose (prediabetes, IFG; n = 90), and type 2 diabetes mellitus (T2DM, n = 95) versus controlsubjects (n = 88). Higher levels of AOPPs, AGEs, oxLDL, NOx, atherosclerosis risk markers,and insulin resistance were pointed out in IFG and T2DM groups compared with control.Strong positive associations (p < 0.01) of AGEs with fasting glucose and HbA1c were foundin both hyperglycemic groups, whereas AOPPs were significantly correlated (p < 0.01) onlyin T2DM. In T2DM, AGEs and AOPPs significantly (p < 0.01) correlated with insulinresistance index HOMA-IR, oxLDL and small LDL particle size (TG/HDL-C), and positivelywith NOx. Direct associations of AGEs and AOPPs with TC/HDL-C and oxLDL/HDL-C, andAGEs-AOPPs interrelations (p < 0.01) were identified in IFG and T2DM groups. AGEs andAOPPs in combination with oxLDL and NOx could be important biomarkers for evaluatingthe association between diabetes and atherosclerotic disorders in aging diabetic patients.
Biological significanceIn the present study we have made an attempt to approach the biological and clinicalsignificance of the oxidative and glycoxidative protein damage, in subjects with prediabetesand type-2 diabetes mellitus. AGEs and AOPPs in combination with oxLDL and NOx appear tobe important biomarkers for evaluating the association between diabetes and atheroscleroticdisorders in aging diabetic patients. More importantly, this cluster of biomarkers that links theshort term, “real time” metabolic impairment parameters (NOx, serum glucose, HOMA-IR,serum lipid profile) and the “metabolic memory” markers resulting from the long-termhyperglycemia and hyperlipidemia-induced oxidative stress (HbA1c, AGEs, AOPPs and oxLDL),could be valuable in predicting not only vascular complications in T2DM, but also the onset ofdiabetes, hence enabling therapeutic interventions from the early stages of diabetes.This article is part of a Special Issue entitled: Protein Modifications.
Type 2 diabetesPrediabetesElderlyOxidative stressAdvanced oxidation proteinproducts (AOPPs)Advanced glycationend products (AGEs)
led: Protein Modifications.chemistry, Faculty of Pharmacy, Carol Davila - University of Medicine and Pharmacy, 6 Taianania. Tel.: +40 748697399.(D. Gradinaru).
r B.V. All rights reserved.
al, Advanced oxidative and glycoxidative protein damage markers in the elderly with.org/10.1016/j.jprot.2013.03.034
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1. Introduction diabetes mellitus, but also to experience an adverse cardiovas-
Diabetes mellitus is a complex disease affecting almost everytissue and organ system, withmetabolic ramifications extendingfar beyond impaired glucosemetabolism. Biomarkersmay reflectthe presence and severity of hyperglycemia or the presence andseverity of the vascular complications of diabetes [1]. At systemiclevel, glycationandoxidative stress resulting fromhyperglycemiaand dyslipidemia lead to accelerated non-enzymatic modifica-tionof essential biomolecules, particularly of proteins [2]. Someofthese damaged molecules may serve as biomarkers, whereasothers may lie in the causal pathway for vascular damage [3].
Aging raises the risk of developing an impaired glucoseregulation and metabolism; therefore diabetes might be con-sidered as an “age-related” disease [4,5]. Risk prediction for type2 diabetes mellitus (T2DM) and cardiovascular disease (CVD)remains suboptimal even after the introduction of global riskassessment by various scores. A variety of blood biomarkersrepresenting various pathophysiological pathways of insulinresistance and atherosclerosis, aswell asmarkers of subclinicaldisease and geneticmarkers, have been investigated [6]. Severalmethods for determining biomarkers of cellular oxidative stresshave been developed, and some have been proposed forsensitive assessment of antioxidant defense and oxidativedamage in diabetes and its complications [3,7]. However, theirclinical utility is limited by less than optimal standardizationtechniques and the lack of sufficient large-sized, multi-markerprospective trials [8]. Proteomic methods hold special promisefor the identification of novel biomarkers that might formthe foundation for new clinical tests. Indeed, from more than300 proteins found differently modulated in body fluidsfrom diabetic patients, approximately 50 were validated with“classic” approaches like ELISA or Western blotting [9–11].
A large number of clinical studies have shown that diabetesmellitus is associated with enhanced oxidative stress throughproduction and long-term accumulation of lipids, proteins andDNA oxidation products, glycated biomolecules, advancedglycation end products (AGEs), and advanced oxidation proteinproducts (AOPPs) [12–16]. AGEs and AOPPs known as pro-inflammatory and pro-oxidative compounds that accumulatein aging patients with diabetes may play a major role inincreasing prevalence of endothelial dysfunction and subse-quent CVD [17–19]. Several studies pointed out that AGEs,AOPPs and oxidative stress markers increase in adult subjectswith type 2 diabetes with and without micro-/macro-vascularcomplications [12,13,15,20]. Also, in a recent study, Chang et al.found in an ethnic Han Chinese elderly population, thatpatients with higher AGEs had more adverse atheroscleroticparameters and indexes, suggesting that AGEs could be used asa marker to predict atherosclerotic lesions. However, thisresearch suggested that further studies are necessary toevaluate the clinical relevance and clinical application of AGEtesting, especially for the prevention of diabetes [21].
Hyperglycemia has been reported to decrease the endo-thelial nitric oxide (NO) synthesis and bioavailability, whichmay contribute to the endothelial dysfunction in impairedglucose metabolism [22–24].
The state of prediabetes – characterized by impaired fastingglucose (IFG) and/or impaired glucose tolerance (IGT) – isassociated with individuals' increased risk, not only to develop
Please cite this article as: Gradinaru D, et al, Advanced oxidative atype 2 diabetes, J Prot (2013), http://dx.doi.org/10.1016/j.jprot.2013.
cular event (myocardial infarction, stroke) later in life [25]. Sofar, the role and the involvement of advanced oxidative andglycoxidative markers in the development of endothelialdysfunction in aging and in prediabetes have been less explored.
The purpose of this study was to evaluate the advancedoxidation and advanced glycation of proteins, and theirinterrelations with endothelial nitric oxide synthesis, oxidativestress markers, metabolic profiles as well as other atheroscle-rotic risk markers in elderly subjects with type 2 diabetesmellitus, and impaired fasting glucose.
2. Methods
2.1. Study design and participants
The study population included 273 subjects (85 men and 188women) aged 60–75 years, selected from the patients admitted toAna Aslan - National Institute of Gerontology and Geriatrics(NIGG), Bucharest, Romania. Subjects had their periodic medicalcheck-up which included clinical check-up and investigations ofroutine geriatric assessment. Subjects were enrolled in threestudy groups: control, impaired fasting glucose (IFG) and type 2diabetes mellitus (T2DM), according to clinical and biochemicalcriteria of the American Diabetes Association [26]. Diabeticpatients were either on oral hypoglycemic medication (metfor-min and/or sulfonylurea) or had diet controlled diabetes. In thestudy were included normal-weight subjects with history ofT2DM <6 year duration, and with a good or moderate glycemiccontrol (glycated hemoglobin, HbA1c ≥ 6.4% and < 8.0%). The IFGgroup included90 subjectswith fastingbloodglucose ≥100 mg/dLand <126 mg/dL, and HbA1c ≥5.7% and <6.4%. More than 67% ofsubjects with IFG and T2DMwere under the usual antihyperten-sivemedications (combinations ofβ-blockers andACE inhibitors,or β-blockers and sartans). The control group included 88apparently healthy subjects recruited from the NIGG outpatientclinical department, where elderly subjects have their visits tothe geriatrician and routine laboratory tests performed. Controlsubjects had normal glucose levels and normal lipid profiles(total cholesterol < 200 mg/dL, LDL-cholesterol < 130 mg/dL,HDL-cholesterol > 50 mg/dL and triglycerides < 150 mg/dL).
Exclusion criteria were: body mass index (BMI) >25 kg/m2,prior history of myocardial infarction, heart failure, cerebrovas-cular disease, renal impairment, active liver disease or liverdysfunction, hematological and malignant overt diseases. Wealso excluded patients using insulin treatment, lipid-loweringtherapy or antioxidant vitamin supplements.
Anthropometric and clinical characteristics were collectedafter a complete clinical examination. Venous blood sampleswere drawn after an overnight fast and 12-hours refraining fromsmoking, caffeinated foods and beverages. The serum andplasma were obtained after blood centrifugation at 3500 rpm,for 15 min at 4 °C, according to standard procedures. Serum andplasma aliquotswere immediately stored at −70 °C until analysisfor oxidative and glycoxidative stress and endothelial nitric oxidesynthesis markers. The biochemical parameters were deter-mined within the same day.
The study protocol was approved by the Ana Aslan - NIGGethics committee and was carried out in accordance with the
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Declaration of Helsinki. All the participants gave their writteninformed consent prior to entering the study.
2.2. Biochemical parameters
Fasting glucose (G), total cholesterol (TC), low-density lipo-protein cholesterol (LDL-C), high-density lipoprotein choles-terol (HDL-C) and triglyceride (TG) levels were measured bystandard methods (Diagnostic Systems, Germany) on Olym-pus AU400 Autoanalyzer. HbA1c was assessed by ion-exchange HPLC (Biorad Variant II). Fasting insulin levelswere determined using a chemiluminescence immunoassay,on Immulite 1000 (Diagnostic Products Corporation, USA)with specific kit (LKIN-2500, Siemens). Insulin resistance wasevaluated using the homeostasis model assessment-insulinresistance (HOMA-IR): fasting insulin (mU/L) × fasting glu-cose (mg/dL) divided by 405 [27].
As an indirect marker of LDL particle size we used the TG/HDL-C ratio, a validated parameter previously used for assessingthe presence of small LDL particles [28,29]. According to data byMuruyama et al., we considered subjects with a TG/HDL-C ratiocut-off >2.0 as having small LDL particles [28].
2.3. Oxidative and glycoxidative markers
LDL oxidation susceptibility (oxLDL) was assessed usingserum LDL isolated by selective precipitation with heparin atpH 5.12, following the incubation in presence of a pro-oxidantsystem (Fe2+ and ascorbic acid, 1:1 molar ratio). The extent ofLDL oxidation was measured by the thiobarbituric acidreactivity assay (TBARS) and expressed as malondialdehydeequivalent content, in nmol MDA/mL serum, using a calibra-tion curve with 1,1,3,3-tetramethoxypropane [30,31]. Theintra-assay CV was 5.4% and the inter-assay CV was 6.5%.
The serum Advanced Oxidation Protein Products (AOPPs)were measured spectrophotometrically at 340 nm using acommercially available assay kit (OxySelect STA-318, Cell Biolabs,USA). Concentration of AOPP was expressed in chloramine Tequivalents (μmol/L). The inter-assay and intra-assay coefficientsof variation were 13 and 10%, respectively.
The Advanced Glycation End Products (AGEs) were deter-mined spectrofluorimetically as described by Kalousova et al.,and Bartling et al., [12,32]. The serum was diluted 1:25 withphosphate buffer saline (PBS), pH 7.4 and fluorescence inten-sity was recorded at 440 nm emission wavelength uponexcitation at 350 nm on Perkin Elmer Spectrofluorimeter (LS50 B, Germany). Fluorescence intensity was expressed inrelative fluorescence units (RFU). The coefficient of variation(CV) of replicate measurements was <5%.
The total amount of plasma stable metabolic pathwayproducts of NO, [NOx, the sum of nitrites and nitrates (NO2
− +NO3
−)] was determined using the Griess reagent, following thequantitative conversion of nitrates (NO3
−) to nitrites (NO2−),
with nitrate reductase (Nitrite/Nitrate Assay Kit, colorimetric234791-KT-F, SIGMA-ALDRICH, USA) [33]. Results were ex-pressed in μmol NOx/L plasma. Intra- and inter-assay CVwerebelow 7% and 9%, respectively.
Please cite this article as: Gradinaru D, et al, Advanced oxidative atype 2 diabetes, J Prot (2013), http://dx.doi.org/10.1016/j.jprot.2013.0
The study markers were assessed on ChemWell 2190Analyzer (Awareness Technology, USA) and Lambda Bio10Perkin-Elmer Spectrophotometer.
2.5. Statistical analysis
Statistical analysis was performed with the Statistical Packagefor Social Sciences software (SPSS Inc., Chicago, IL, USA)version 15. Data were expressed as means ± standard devia-tion (SD). After evaluating whether the variables werenormally distributed (Kolmogorov–Smirnov test), clinical andbiochemical parameters of subjects were compared amongthe three studied groups (control, IFG and T2DM) usingone-way analysis of variance (ANOVA) and Bonferroni t-test.The influence of potential confounders – age, sex, BMI, andblood pressure – on oxidative and glycoxidative stressmarkers was evaluated by using one-way analysis of covari-ance (ANCOVA). Pearson's correlation test was used in allstudy groups to observe a simple relationship of AGEs, AOPPs,oxLDL, or NOx levels with all of the other metabolic variables,after adjustment for sex, age, BMI and blood pressure. The pvalue < 0.05 was considered statistically significant.
3. Results
3.1. Clinical characteristics of the study subjects
The study included 273 elderly subjects, 70% women and 30%men, with the mean age of 67 ± 7 years. Baseline character-istics of the three study groups: impaired fasting glucose (IFG,prediabetes), type 2 diabetes mellitus (T2DM), and the controlgroup are summarized in Table 1. There was no significantdifference in the age or sex distribution between the studygroups. The body mass index (BMI) as well as systolic anddiastolic blood pressure was higher in subjects with abnormalglucose metabolism.
HbA1c and fasting glucose were significantly higher insubjects with IFG and T2DM, as compared with the controlgroup. T2DM and IFG groups had significant hyperinsulinemiaand significant increases in insulin resistance index HOMA-IR.All the lipid profile parameters were significantly different ingroups with abnormal glucose metabolism, as compared withhealthy individuals. Higher significant levels of total cholesterol,LDL-cholesterol and triglycerides, and lower significant HDL-cholesterol levels were found in T2DM and IFG groups than inthe control subjects. The total cholesterol/HDL-cholesterol andtriglyceride/HDL-cholesterol ratios used as traditional cardio-vascular risk markers were significantly higher in both hyper-glycemic groups versus control.
3.2. Serum oxidative, glycoxidative stress and endothelialnitric oxide synthesis markers
Results showed relevant changes in serum oxidative andglycoxidative stress parameters and nitric oxide metabolicpathway products: AGEs, AOPPs, oxLDL and NOx, the levels ofwhich were significantly higher in both IFG and T2DM groupscompared with controls (Table 2).
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SD = standard deviation.p values represent statistical significance versus control group. NS — non-significant.BP, blood pressure; BMI, body mass index; HbA1c, glycated hemoglobin; TC, total cholesterol; TG, triglycerides; LDL-C, low-density lipoproteincholesterol; HDL-C, high-density lipoprotein cholesterol; HOMA-IR, homeostasis model assessment of insulin resistance.
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Serum AGEs were about 30% higher in diabetic subjectsand 15% higher in prediabetes compared with healthy elderly.Similarly, serum AOPPs levels were 50% higher in diabeticsubjects and more than 40% higher in prediabetic group. Aprogressive, significant increase in the mean values of plasmanitric oxide metabolites NOx could be noticed in hyperglyce-mic subjects (IFG and T2DM groups).
The susceptibility of LDL to in vitro oxidation (oxLDL) wassignificantly higher in T2DM and IFG patients as comparedwith control group. To further estimate the extent of oxLDLinvolvement in endothelial dysfunction, the ratios of oxLDL toHDL-cholesterol and the ratio of oxLDL to NOx, were calculated;these ratios were significantly higher in subjects with impairedglucose metabolism than in controls. The LDL oxidizability(oxLDL) values were corrected by LDL-cholesterol, so the ratiosoxLDL/LDL-C showed slight, nonsignificant increases in IGFand T2DM groups.
3.3. Relationships of AGEs and AOPPs with oxidativestress and atherosclerotic risk markers in subjects with IFGand T2DM
To find out the involvement of AGEs and AOPPs on develop-ment of oxidative stress and atherosclerotic risk in prediabetesand diabetes, correlation analysis was carried out betweenlevels of AGEs and AOPPs with various metabolic, oxidativestress and atherosclerotic risk markers in IFG and T2DM elderlypatients. Pearson's correlation coefficients were calculated,after adjustment for potential confounding factors – sex, age,BMI and blood pressure – as presented in Table 3. Statisticalsignificance of correlations was different among the three
Please cite this article as: Gradinaru D, et al, Advanced oxidative atype 2 diabetes, J Prot (2013), http://dx.doi.org/10.1016/j.jprot.2013.
study groups. In healthy subjects only AGEs showed asignificant positive correlation with fasting glucose levels. Inprediabetes group (IFG) we found a significant positive corre-lation between AGEs, HbA1c, and the TG/HDL-C ratio. Also,AOPPs significantly correlated with both ratios: TG/HDL-C andTC/HDL-C, and positively associated with oxLDL/HDL-C ratio.Despite the elevated susceptibility of LDL to lipid peroxidation(oxLDL) evaluated in prediabetes subjects, its associations withAGEs or AOPPs were not significant. In T2DM group we foundpositive significant correlations for both AGEs and AOPPs withinsulin resistance index HOMA-IR and oxLDL. Even though notsignificant, a slight positive association between glycoxidativemarkers and NO metabolic pathway products (NOx), wasfound, in elderly diabetes subjects.
In the groups of subjectswith abnormal glucosemetabolism –IFG and T2DM – the LDL in vitro oxidizability was significantlyassociated with the traditional parameters of the serummetabolic profile: fasting glucose (r = 0.293, p < 0.01 in IFG andr = 0.434, p < 0.01 in T2DM), total cholesterol (r = 0.317, p < 0.01in IFG and r = 0.216, p < 0.05 in T2DM), triglycerides (r = 0.522,p < 0.01 in IFG and r = 0.485, p < 0.01 in T2DM) andHDL-cholesterol (r = −0.208, p < 0.05 in IFG and r = −0.267,p < 0.01 in T2DM) as well as with the atherosclerosis riskmarkers (total cholesterol/HDL-C and triglyceride/HDL-C ratios)(data not shown). The oxLDL levels were positively associatedwith LDL-cholesterol concentrations, this correlation beingsignificant in healthy elderly subjects (r = 0.149 in IFG group,r = 0.128 in T2DM, and r = 0.216, p < 0.05 in control group).
As regards the association between oxLDL and NO meta-bolic end products, the correlation analysis identified a positiverelationship between oxLDL and NOx in all the study groups,
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SD = standard deviation.p-Values represent statistical significance versus control group. NS — non-significant.oxLDL, LDL susceptibility to oxidation; AOPPs, advanced oxidation protein products; AGEs, advanced glycation end products; NOx, nitric oxidemetabolic pathway products.
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but significant only in the IFG group: r = 0.288 (p < 0.01),whereas it was non-significant in control (r = 0.158) and T2DM(r = 0.114).
An important finding of this study was the fact that serumAGEs were positively significantly (p < 0.01) associated withAOPPs in both hyperglycemic groups — IFG and T2DMsubjects (Fig. 1a and b).
4. Discussion
Oxidative and glycoxidative stress are postulated to be theprincipal events in the pathogenesis of type 2 diabetes mellitusand its vascular complications, therefore AGEs and AOPPs arespecific end products derived from non-enzymatic reactionsand considered potentially useful biomarkers for these dis-eases [12,13,16]. Moreover, protein glycation and increasedoxidative stress are the two main mechanisms involved inbiological aging [4]. For this reason, elderly subjects are more
Table 3 – Pearson's correlation coefficients (r) of AGEs and AOPoxidative stress markers, in control, impaired fasting glucose (I
Variable Control group(n = 88)
Imp
AGEs AOPP AGE
Glucose, mg/dL 0.221* 0.193 0.476HbA1c, % 0.131 0.018 0.315HOMA-IR 0.072 0.049 0.171TC/HDL-C ratio 0.092 0.138 0.162TG/HDL-C ratio 0.174 0.147 0.303oxLDL 0.010 0.121 0.143NOx 0.112 0.096 0.105oxLDL/HDL-C ratio 0.049 0.165 0.146oxLDL/LDL-C ratio 0.031 0.012 0.002oxLDL/NOx ratio 0.057 0.054 0.002
Statistical significance: *p < 0.05; **p < 0.01; TC, total cholesterol; TG,high-density lipoprotein cholesterol; HOMA-IR, homeostasis model assesAOPPs, advanced oxidation protein products, oxLDL, LDL susceptibility to
Please cite this article as: Gradinaru D, et al, Advanced oxidative atype 2 diabetes, J Prot (2013), http://dx.doi.org/10.1016/j.jprot.2013.0
prone to develop type 2 diabetes mellitus and indeed, clinicalphenotypes associated with insulin resistance possibly repre-sent true clinical models for systemic aging [34].
At present, prevalence of intermediate glycemic control,termed “prediabetes” or “impaired glucose regulation”, isincreasing worldwide and experts have estimated that morethan 470 million people will have prediabetes by 2030 [35,36].Prediabetes confers significant risks for developing type 2diabetes mellitus (T2DM) and cardiovascular comorbidities[37,38]. Thus, early screening and diagnosis of prediabetes,along with subsequent recommendations on preventivemeasures, are crucial in preventing or delaying progressionto T2DM [11,36].
In this study we aimed to explore the association ofadvanced oxidation and advanced glycation of proteins, andtheir interrelations with endothelial nitric oxide synthesis,oxidative stress markers, and metabolic profile as well asother atherosclerotic risk markers in prediabetic and diabeticelderly subjects. In view of this purpose, we evaluated the
Ps levels with glucose metabolism, atherosclerotic risk andFG) and type 2 diabetes mellitus (T2DM) elderly subjects.
Fig. 1 – Correlations between serum levels of AGEs andAOPPs in impaired fasting glucose (IFG) (a) and type 2diabetes mellitus (T2DM) (b) elderly subjects.
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relationships between the circulating levels of advancedoxidation protein products (AOPPs) and advanced glycationend products (AGEs) in elderly subjects with type 2 diabetes(T2DM) and impaired fasting glucose (IFG, prediabetes). Also,we investigated the strength of their interrelationships withlow-density lipoprotein susceptibility to oxidation (oxLDL).Attempts were also made to see whether systemic AGEs andAOPPs levels were related to the nitric oxide (NO) metabolicpathway products – NOx (NO2
− + NO3−) – used as a biochemical
marker of endothelial dysfunction. The aim was to assess thedegree of relevance of this combination of oxidative andglycoxidative markers across the three different fastingglucose categories – normal fasting glucose, IFG and T2DM –in the elderly as a category of risk population.
In the present study all the tested oxidative and glycoxidativestress markers – AGEs, AOPPs, oxLDL, and NOx – were signi-ficantly higher in both prediabetic and diabetic subjects com-pared with healthy elderly individuals, their levels being morepronounced in the patients with diabetes. However, the increasepattern was clearly different among these two study groups ofsubjects with impaired glucose metabolism — IGF and T2DM. Inthis respect, we could notice a gradual increase in AGEs and NOxlevels togetherwith fasting hyperglycemia and insulin resistanceindex. Therefore AGEs and NOx could represent novel progres-sive risk markers, the measurement of which appears to bevaluable in predicting possible vascular dysfunction in diabetesmellitus. In a different way the extent of increase in AOPPs and
Please cite this article as: Gradinaru D, et al, Advanced oxidative atype 2 diabetes, J Prot (2013), http://dx.doi.org/10.1016/j.jprot.2013.
oxLDL levels was much more noticeable even in the prediabeticstate.
These findings are in agreement with our recent study onthe elderly with IFG and T2DM, in which we pointed outhigher levels of circulating markers of oxidative andglycoxidative stress, inversely associated with vitamin Dstatus [39].
It has been suggested that atherosclerotic changes, includ-ing an increased arterial stiffness, are early evident in subjectswith impaired fasting glucose (IFG) as well as in those withdiabetes, and subsequent risk of cardiovascular disease (CVD)is greater in subjects with IFG than in those with normalfasting glucose [25,37,38,40]. Therefore we examined therelationships between the glycoxidative markers and thecardiovascular risk biochemical parameters in prediabetesand diabetes. After adjusting for potential confounders, sex,age, BMI, and blood pressure, we found that AGEs and AOPPslevels correlated positively with serum glucose, insulinresistance, lipid profiles and other atherosclerotic riskmarkers, but less relevantly in prediabetes than in diabetes.Of all studymarkers, only AGEs correlated with serum glucoselevels in all study groups — control, IFG and T2DM. In IFGgroup AGEs were positively associated with markers indicat-ing the persistent and moderate fasting hyperglycemia,whereas AOPPs were associated only with the atheroscleroticrisk markers TC/HDL-C and TG/HDL-C, as related to anabnormal lipid metabolism. The same statistical analysisperformed for T2DM group pointed out stronger relationshipsof AGEs and AOPPs with the metabolic profile parameters andatherosclerotic risk markers. For the diabetes group, twoadditional significant positive correlations of AGEs andAOPPs with HOMA-IR and oxLDL were revealed.
These results suggest that in prediabetes, AGEs and AOPPscould have a different pathophysiological significance inregard to prooxidative mechanisms derived from hyperglyce-mia and dyslipidemia.
Consistent with previous studies regarding the deleteriouseffects of chronic hyperglycemia and oxidative stress inT2DM, the present study additionally highlights that athero-sclerosis risk markers and risk of CVD are significantlyincreased in subjects with IFG compared with those withnormal fasting glucose. In this sense, a very interestingfinding concerns the positive association between two bio-chemical markers of endothelial dysfunction – oxLDL andNOx – identified as being significant only for the prediabetesgroup.
Oxidized LDL and NO are recognized to exert contradictoryactionswithin the vascular endotheliummicroenvironment andto influence the key events in the development of atherosclero-sis such as leukocyte adhesion, platelet aggregation andvascularsmooth muscle cell proliferation and migration. While oxidizedLDL – a biomarker of lipoprotein-associated oxidative stress –was identified as a non-traditional proatherogenic emergingcardiovascular risk factor, NO is a free radical signal-transducingmolecule that modulates the vascular tone, modulates in vitrolipid peroxidation reactions and alters proinflammatory geneexpression [41]. In subjects with prediabetes and diabeteswe found a two fold increase in LDL susceptibility tolipid peroxidation compared with control subjects. oxLDLsignificantly correlated with the cardiovascular risk biochemical
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parameters. A significant positive association between oxLDLand LDL-C was pointed out in normoglycemic and nor-molipidemic subjects (control group). This finding supports thehypothesis that chronic hyperglycemia might significantly alterthe “quality” of LDL particles and their resistance to in vitrooxidation. In IGT and T2DM patients the extent of low-densitylipoprotein oxidation seems to depend mainly on the levels ofoxidative stress exerted at endothelial level, and less on the“quantity” of substrate. In IFG and T2DM groups triglyceridelevels were also positively associated with oxLDLs levels. It isknown that high TG values are strongly associated with thepresence of small, dense LDL particles, the “pattern B” lowdensity lipoproteins, which are highly oxidizable [42]. Accordingto these specifications on pattern B low density lipoproteins, inour patients with abnormal glucose metabolism we found thatthe TG/HDL-C ratios >2.0, a validated indicator of small LDLs sizewere associated with high oxLDL levels. Our results are inaccordance with specialized literature, with regard to thedamaging effects of hyperglycemia, mediated or stimulated byoxidative stress [13,21,23,43].
The positive association of AGEs with oxLDL and small LDLparticles size (TG/HDL-C) pointed out in elderly subjects withabnormal glucose metabolism may reflect the implication ofadvanced glycation in enhancement of lipoxidative stress andatherogenesis. There are established several mechanisms bywhich AGEs may contribute to atherogenesis. First, AGEspromote protein cross-linking leading to covalent trapping ofpro-atherogenic LDL particles in the arterial wall [44]. Second,AGEs enhanced the generation of reactive oxygen species(ROS) by increasing neutrophil respiratory burst activity viaNADPH oxidase [45].
Also, the role of AGEs in endothelial dysfunction has beenclearly demonstrated in human T2DM, their levels beingnegatively associated with the extent of endothelium-dependent and endothelium-independent vasodilatation. Theproposed mechanisms to explain this association consist of:AGEs-associated induction of oxidative stress may quench andinactivate endothelium-derived NO and uncouple nitric oxidesynthase (NOS) activity. Also, AGEs may directly reduce theendothelial NOS (eNOS) activity through receptor-mediatedphosphorylation of serine residues in eNOS [46,47].
The strong interactive effect between AOPPs and pro-atherogenic oxLDL particles, as well as with atherosclerosisrisk markers (TC/HDL-C, TG/HDL-C, oxLDL/HDL-C) found inelderly T2DM patients suggest that AOPPs might be not only amarker of oxidant-mediated protein damage, but also apotential inducer of oxidative stress, LDL oxidation andatherosclerosis. Therefore, it has been shown that AOPPsinduced monocyte/macrophage activation in vitro and trig-gered oxidative burst in human monocytes. In vivo, in uremicpatients, AOPPs were linked to monocyte activation markers[48].
An important finding of this study is also the directassociation between AGE and AOPP levels pointed out inelderly subjects with IFG and T2DM, in line with recent reports[20]. This strong interrelation could support the implication ofintensive oxidative stress not only in advanced proteinoxidation, but also in AGE formation [13,49].
AOPPs and AGEs accumulated in the elderly with abnor-mal glucose metabolism could increase oxidative stress and
Please cite this article as: Gradinaru D, et al, Advanced oxidative atype 2 diabetes, J Prot (2013), http://dx.doi.org/10.1016/j.jprot.2013.0
inflammation; and this enhancement can further increaseadvanced oxidation and advanced glycoxidation of proteinsthrough stimulation of more oxidant generation. This posi-tive feedback loop could amplify or maintain the oxidativestress and thus contribute to atherosclerosis and diabeticcomplications.
In the recent decades, most biomedical areas and clinicalresearch focused interest on oxidative stress. Based on freeradical research, the emerging concept is that oxidative stressis the “final common pathway”, through which risk factors ofseveral age-related diseases exert their deleterious effects.However, it is difficult to demonstrate a direct relationbetween hyperglycemia, hyperlipidemia, oxidative stress,and beta-cell dysfunction in humans, as there is no “goldstandard” procedure to test this relation [22].
Actually, the oxidatively-modified proteins could be morerelevant than other oxidative stress biomarkers which aredeveloped to monitor disease progression and outcome, sinceproteins are the key molecules that play the pivotal structuraland functional role in living organisms. It is generallyacknowledged that AGEs and AOPPs – measured at systemiclevel – are among the effectively utilized biological markers ofoxidative and glycoxidative stress associated with diabetesand its complications [4].
In particular AGEs are represented by a heterogenous groupof bioactive compounds (e.g. pentosidine, carboxymethyllysine,and imidazolone) that are formed by nonenzymatic glycation ofmacromolecules; someof themhave characteristic fluorescence,ability of crosslinking of protein and reaction with AGE-specificreceptor RAGE [50]. Meerewald et al. showed that AGE accumu-lation in tissues reflects cumulative metabolic stress, or “meta-bolic memory” rather than short term glycemic control (HbA1C),thus pointing to protein tissue damage resulting from effects ofexposure tomany CVD risk factors [19]. Protein glycation leadingto AGE formation is enhanced in diabetes by increases in bloodglucose per se, and collaterally, by the production of smallreactive carbonyl compounds such as methylglyoxal, glyoxaland 3-deoxyglucosone [51]. Similarly, reactive aldehydessuch as 4-hydroxynonenal (HNE), 2-propenal (acrolein), andmalondialdehyde (MDA) are lipid peroxidation products whichcan covalentlymodify proteins through carbonylation and causethe formation of protein adducts and cross-links on proteins(protein aggregates) [52,53]. Most of the biological effects of theseintermediate reactive carbonyl compounds (RCCs) are attributedto their capacity to react with the nucleophilic side chain of Cys,His, or Lys protein residues, forming advanced lipoxidation endproducts (ALEs) and AGEs. In this sense, the “carbonyl stress”state could be defined as an imbalance between RCC generationfrom both oxidative and non-oxidative reactions, and theirdecreased renal detoxification and excretion from plasma [54].Therefore “carbonyl stress” might be regarded as a linkingelement between lipid peroxidation and glycoxidation indiabetes.
AOPPs are proteins, predominantly albumin and itsaggregates damaged by oxidative stress. They containabundant dityrosines which allow crosslinking, disulfidebridges and carbonyl groups and are mainly formed bychlorinated oxidants — hypochloric acid and chloraminesresulting from myeloperoxidase activity [16,55]. Apart from acommon formation mechanism (oxidative stress) leading
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to macromolecule damage, AOPPs have several similarbiological effects to AGEs. In particular AOPPs are consideredas proinflammatory mediators that can damage biologicalmembranes and endothelium and impair HDL metabolism,thus being potential key players in the development ofcardiovascular disease and immune disregulation [56].
The spectrofluorimetric and spectrophotometric techniquesused in our study for the assessment of AGEs and AOPPs aresimple, fast and inexpensive so they can be applicable on largesample size analysis in current laboratory practice, for evaluat-ing oxidative stress-linked damage in different conditions.However, the transfer of proteomic sensitive technologies suchas LC–MS/MS and MALDI/MS in clinical chemistry laboratoriescould represent a major step forward in the incorporation ofthese markers into routine use [4,57].
The current study was designed to investigate the complexrelationships between chronic hyperglycemia, hyperlipidemia,advanced oxidative and glycoxidative proteinmarkers evaluatedat systemic level, LDL oxidizability and endothelial nitric oxidesynthesis. This evaluation was restricted to a non-overweightand non-obese elderly population, in order to further eliminatethe influence of confounders such as age and BMI, onglycoxidative and lipoxidative stress biomarkers. Our results arein line with several recent studies which pointed out positiveassociation of AGEs with AOPP, HbA1c, lipid peroxidationproducts (MDA), triglycerides, cardiovascular risk markers (LDL-C/HDL-C and TG/HDL-C ratios) and atherosclerosis index [12,20,21].Some oxidative and glycoxidative stress parameters, takenseparately, were explored in various studies, especially in adultsubjects with type 2 diabetes mellitus with or without vascularcomplications [12,20,21,58]. To our knowledge, this cluster of fourdifferent biomarkers, namely AGEs, AOPPs, oxLDL and NOx andtheir interrelations were not approached, comparatively, in theelderly with prediabetes and type 2 diabetes mellitus.
There are several valuable findings in this study, but alsosome limitations. First, thediet, a source of exogenousAGEs,wasnot well controlled in this study [59]. Second, inflammationmaybe a potential confounder for the relation between AGEs, AOPPs,oxLDL and NOx and atherosclerotic status, but inflammatorymarkers were not assessed in this study. Third, the antioxidantaction of antidiabetic medication and the possible antioxidanteffect of antihypertensive medication were not taken intoaccount. As reported by recent findings, antihypertensivemedication can exert an antioxidant effect by enhancing theglutathione-related antioxidant defense system [60]. The factthat activation of the renin–angiotensin system contributes invariousways to the formation of AGEs, is well known. Moreover,ACE inhibitors and angiotensin II receptor blockers have beenshown to decrease the production of reactive carbonyl pre-cursors, particularly in animal models. Recent research pointedout that there have been conflicting data on effects of angioten-sin II receptor blockers on AGE accumulation [61]. Also, recentstudies pointed out a significant reduction in AOPPs and AGEs,and an increase in antioxidant reserve in patients with newlydiagnosed type 2, after three month treatment with metformin[62]. Finally, the evaluated lipid profiles and biochemicalatherosclerotic indexes were traditional, indirect biomarkersfor atherosclerosis. Therefore, the gold standard methodsof diagnosis for coronary artery lesions and endothelial dys-function, such as coronary angiography and brachial artery flow
Please cite this article as: Gradinaru D, et al, Advanced oxidative atype 2 diabetes, J Prot (2013), http://dx.doi.org/10.1016/j.jprot.2013.
mediated dilation together with the intima-media thicknessindex, should be further considered to evaluate these complexrelationships, in biochemical and clinical approaches.
5. Conclusion
Higher advanced oxidation and advanced glycoxidation of pro-teins, and their strong interrelations were pointed out in elderlysubjects with abnormal glucose metabolism. The strong associ-ations of advanced oxidative–glycoxidative stress markers withsmall LDL particles size and their oxidizability could reflect theirimplications in the enhanced oxidative damages of the vascularendothelium. AGEs and AOPPs in combination with oxLDL andNOx appear to be important biomarkers for evaluating theassociation between diabetes and atherosclerotic disorders inaging diabetic patients. More importantly, this combination ofbiomarkers that links the short term, “real time” metabolicimpairment parameters (NOx, serum glucose, HOMA-IR, serumlipid profile) and the “metabolicmemory”markers resulting fromthe long-term hyperglycemia and hyperlipidemia-induced oxi-dative stress (HbA1c, AGEs, AOPPs and oxLDL), could be valuablein predicting not only vascular complications in T2DM, but alsothe onset of diabetes, hence enabling therapeutic interventionsfrom the early stages of diabetes.
6. Conflict of interest
None of the authors has any financial or any other kind ofconflicts with this article.
Acknowledgments
Daniela Gradinaru acknowledges the scientific support of theEU-COST Actions on Chemistry of Non-Enzymatic ProteinModifications (CM1001 Action) and on Lipid PeroxidationAssociated Disorders (B35 Action).
The authors are grateful for the support consisting of theopportunity to make use of the equipment obtained under theframework of the EU-FP7 Project “MARK-AGE, European Studyto Establish Biomarkers of Human Ageing”.
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