research article increased dna dicarbonyl glycation and...

Research ArticleIncreased DNA Dicarbonyl Glycation and Oxidation Markers inPatients with Type 2 Diabetes and Link to Diabetic Nephropathy

Sahar Waris1 Brigitte M Winklhofer-Roob2 Johannes M Roob3 Sebastian Fuchs2

Harald Sourij4 Naila Rabbani15 and Paul J Thornalley15

1Warwick Medical School Clinical Sciences Research Laboratories University of Warwick University HospitalCoventry CV2 2DX UK2Human Nutrition amp Metabolism Research and Training Center Graz Institute of Molecular BiosciencesKarl Franzens University 8010 Graz Austria3Clinical Division of Nephrology Department of Internal Medicine Medical University of Graz 8036 Graz Austria4Clinical Division of Endocrinology and Nuclear Medicine Department of Internal Medicine Medical University of Graz8036 Graz Austria5Warwick Systems Biology Centre Senate House University of Warwick Coventry CV4 7AL UK

Correspondence should be addressed to Naila Rabbani nrabbaniwarwickacuk

Received 1 January 2015 Revised 14 March 2015 Accepted 23 March 2015

Academic Editor Patrizio Tatti

Copyright copy 2015 Sahar Waris et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Aim The aim of this study was to assess the changes of markers of DNA damage by glycation and oxidation in patients with type2 diabetes and the association with diabetic nephropathy Methodology DNA oxidation and glycation adducts were analysed inplasma and urine by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry DNA markers analysedwere as follows the oxidation adduct 78-dihydro-8-oxo-21015840-deoxyguanosine (8-OxodG) and glycation adducts of glyoxal andmethylglyoxalmdashimidazopurinones GdG MGdG and N

2-(1RS-carboxyethyl)deoxyguanosine (CEdG) Results Plasma 8-OxodG

and GdGwere increased 2-fold and 6-fold respectively in patients with type 2 diabetes with respect to healthy volunteers Medianurinary excretion rates of 8-OxodG GdG MGdG and CEdG were increased 28-fold 10-fold 2-fold and 2-fold respectively inpatients with type 2 diabetes with respect to healthy controls In patients with type 2 diabetes nephropathy was associated withincreased plasma 8-OxodG and increased urinary GdG andCEdG In amultiple logistic regressionmodel for diabetic nephropathydiabetic nephropathy was linked to systolic blood pressure and urinary CEdG Conclusion DNA oxidative and glycation damage-derived nucleoside adducts are increased in plasma and urine of patients with type 2 diabetes and further increased in patients withdiabetic nephropathy

1 Introduction

Reactive oxygen species (ROS) and dicarbonyl metabolitesglyoxal andmethylglyoxal are some of themost reactivemet-abolites present in human metabolism They are increasedin diabetes and diabetic nephropathy [1ndash3] ROS react withdeoxyguanosine residues of DNA to form 8-dihydro-8-oxo-21015840-deoxyguanosine (8-OxodG) The DNA content of8-OxodG residues was increased in lymphocyte DNA inclinical diabetes [4] Reactive dicarbonyl intermediates ofendogenous glycation glyoxal and methylglyoxal also reactwith deoxyguanosine residues of DNA to form mainly

imidazopurinone adducts Glyoxal forms 3-(21015840-deox-yribosyl)-6 7-dihydro-6 7-dihydroxyimidazo-[23-b] purin-9(8)one (GdG) and methylglyoxal forms two imidazopu-rinone structural isomers 3-(21015840-deoxyribosyl)-67-dihydro-6 7-dihydroxy-67-methylimidazo-[23-b]purine-9(8)one(MGdG) DNA glycation by methylglyoxal also forms twostereoisomers N

2-modified derivatives N

2-(1RS-carboxy-

ethyl)-deoxyguanosine (CEdG) [5] Figure 1(a) GdGMGdG and CEdG are nucleotide advanced glycationendproducts (AGEs) Methylglyoxal-derived MGdG was aquantitativelymajor adduct of endogenous damage in healthyvolunteers [5] Damage to DNA by endogenous glyoxal and

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2015 Article ID 915486 10 pageshttpdxdoiorg1011552015915486

2 Journal of Diabetes Research

OxidationGdG

8-OxodG

MGdG CEdG

N

NHN

N

N

O

H

H

NHNH

N

N

N

O

dG

N

N

N

O

HO

N

NHN

N

N

O

H

N

NHN

N

N

OOH

OHOH

OHOH

OH

H

NH

NHN

N

N

O

CH3

CH3

NH2NH2

CH CH3

CO2

minus

++

(a)

00

01

02

03

04

05

Control T2DM00

01

02

03

04

05

Control T2DM

000

005

010

015

020

025

Control T2DM000

005

010

015

020

025

Control T2DM

[GdG

] Pla

sma

(nM

)[C

EdG

] Pla

sma

(nM

)

[MdG

] Pla

sma

(nM

)[8

-Oxo

dG] P

lasm

a(n

M)

(b) (c)

(d) (e)

+ Methylglyoxal

+ Glyoxal

lowastlowastlowast

lowastlowast

Figure 1 Continued

Journal of Diabetes Research 3

0

1

2

3

4

Control T2DM0

2

4

6

8

Control T2DM

0

1

2

3

4

Control T2DM0

10

20

30

40

Control T2DM

0

1

2

3

4

00

01

02

03

Urin

ary

GdG

(nm

ol24

h)

Urin

ary

MG

dG (n

mol

24

h)

Urin

ary

CEdG

(nm

ol24

h)

Urin

ary

GdG

(nm

ol24

h)U

rinar

y8

-Oxo

dG (n

mol

24

h)

[8-O

xodG

] Pla

sma

(nM

)

(f) (g)

(h) (i)

(j) (k)T2DM minus DN T2DM + DNT2DM minus DN T2DM + DN

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowast

Figure 1 Continued


0

1

2

3

4

Urin

ary

CEdG

(nm

ol24

h)

(l)

T2DM minus DN T2DM + DN

lowast

Figure 1 (a) Formation of glycation and oxidation adducts of deoxyguanosine The common 21015840-deoxyribosyl group has been omitted forclarity (b)ndash(i) Nucleotide glycation and oxidation adducts in plasma and urine of healthy controls subjects and patients with type 2 diabetesPlasma (b)GdG (c)MGdG (d)CEdG and (e) 8-OxodGUrine (f)GdG (g)MGdG (h)CEdG and (i) 8-OxodG (j)ndash(l)Nucleotide glycationand oxidation adducts in plasma and urine in patients with type 2 diabetes with and without nephropathy (j) Plasma 8-OxodG (k) urinaryGdG and (l) urinary CE-dG Data are median with the upper and lower quartile as error bars Significance lowast119875 lt 005 lowastlowast119875 lt 001 andlowastlowastlowast

119875 lt 0001 Mann-Whitney 119880

methylglyoxal is suppressed by glyoxalase 1 (Glo1) of thecytoplasmic glyoxalase system [6] The expression andactivity of Glo1 in the kidney were decreased in experimentalmodels of diabetic nephropathymdashstreptozotocin-induceddiabetes inmice and rats and the dbdb diabetic mouse [7ndash9]This is associated with increased dicarbonyl glycation ofproteins linked to the development of diabetic nephropathy[10] Increased urinary 8-OxodG excretion has been found inpatients with diabetes type 2 diabetes (T2DM) with furtherincreases associated with the presence of microvascularcomplications and was a predictor of diabetic nephropathy[11 12]

Nucleotide AGEs are associated with DNA single-strandbreaks and increasedmutation frequencies [13] Oxidised andglycated nucleosides are removed from DNA by nucleotideexcision repair of DNA damaged by oxidation and gly-cation Recent functional genomic studies of Glo1 havelinked dicarbonyl glycation to the development of diabeticnephropathy [14 15] The plasma concentration and urinaryfluxes of glyoxal andmethylglyoxal-derived nucleoside AGEsin diabetes and diabetic nephropathy are unknown

Reliable quantitation of oxidised and glycated nucleosideshas proven difficult because of inadequate sensitivity andspecificity of detection responses and poor adduct stabilityand recovery during preanalytic processing Initial stud-ies involved 32P-labelling studies of DNA digests [16] andimmunoassay [17] Stable isotopic dilution analysis liquidchromatography with tandem mass spectrometric detection(LC-MSMS) has been used for assay of 8-OxodG [18] Forestimation of both DNA glycation and oxidation adductsthe high specificity and sensitivity of LC-MSMS makes thisthe preferred method for DNA damage marker analysis

We developed a stable isotopic dilution analysis LC-MSMSmethod to concurrently quantify GdGMGdG CEdG and 8-OxodG in plasma and urinary ultrafiltrate [5] 8-OxodG hasbeen studied extensively in plasma and urine as a biomarkerof oxidative damage [18] whereas the diagnostic utility ofDNA glycation adducts is unknown

Herein we analysed DNA oxidation and glycation mark-ers in plasma and urine of patients with T2DM with andwithout diabetic nephropathy Healthy volunteers served ascontrols The outcome revealed marked increases in DNAdamage in T2DM and further increase of selected markersin diabetic nephropathy Increased urinary excretion ofglycation adducts GdG and CEdG was indicative of diabeticnephropathy

2 Methods

21 Participants Patients with type 2 diabetes and overtnephropathy (T2DM + DN) and patients with T2DM andwithout overt nephropathy (T2DM minus DN) were recruitedin the EU-funded PREDICTIONS project [19] Healthyvolunteers were recruited in the EU-funded VITAGE project[20] Both studies were conducted at the Human NutritionandMetabolismResearch andTrainingCenter Karl FranzensUniversity of Graz Austria and the Clinical Division ofNephrology and the Clinical Division of Endocrinologyand Nuclear Medicine Department of Internal MedicineMedical University of Graz Austria The study protocolswere approved by the Ethics Committee of the MedicalUniversity of Graz Austria and written informed consentwas obtained from all study subjects Diagnosis of dia-betes was established in accordance with the WHO criteria


Table 1 Characteristics of human subjects recruited for this study

Subject group Control T2DM-DN T2DM+DN119899 28 28 28Age (years) 61 plusmn 8 63 plusmn 6 60 plusmn 10Gender (MF) 280 208 208BMI (kgm2) 261 plusmn 18 285 plusmn 50 332 plusmn 50OOO

Fasting plasma glucose (mM) 56 plusmn 05 94 plusmn 30 93 plusmn 38

A1C () ND 76 plusmn 12 75 plusmn 15(mmolmol) 60 plusmn 13 58 plusmn 17Systolic BP (mmHg) 135 plusmn 13 136 plusmn 13 153 plusmn 22

Diastolic BP (mmHg) 84 plusmn 6 81 plusmn 10 84 plusmn 10Total cholesterol (mM) 552 plusmn 094 504 plusmn 113 517 plusmn 114LDL cholesterol (mM) 331 plusmn 069 292 plusmn 096 258 plusmn 092

HDL cholesterol (mM) 149 plusmn 032 146 plusmn 038 140 plusmn 029Urinary albumin (mg24 h) ND 13 plusmn 10 2437 (371ndash9000)

eGFR (mlmin) 69 plusmn 13 73 plusmn 13 317 (200ndash453)OOO

ND = not determined Data are mean plusmn SD or median (minimumndashmaximum) Significance and 119875 lt 005 and 119875 lt 0001 with respect to healthyvolunteers and OOO 119875 lt 0001 with respect to patients with type 2 diabetes without nephropathy

fasting plasma glucose ge70mmolL a two-hour value in anoral glucose tolerance test 111mmolL or random plasmaglucose 111mmolL in the presence of symptoms aged 35ndash75 with a documented duration of diabetes of ge5 yearsbeing eligible T2DM was diagnosed by lack of criteria fortype 1 diabetes Inclusion criteria for cases were albuminuriagt300mgd and known overt diabetic retinopathy Retinopa-thy was requested to be present to ensure that albuminuriais the consequence of diabetic nephropathy rather than anondiabetic glomerulopathy A renal biopsy would be thegold standard to discriminate between diabetic nephropathyand a nondiabetic glomerulopathy but a renal biopsy israrely taken in patients with T2DM Several studies haveindicated that presence of retinopathy is a good alterna-tive inclusion criterion to discriminate between diabeticnephropathy and nondiabetic glomerulopathy in patientswith T2DM with albuminuria [21ndash23] Exclusion criteriawere chronic renal failure known causes of renal failure otherthan diabetes and non-Caucasian ethnic origin Cases andcontrols were matched for gender and diabetes durationExclusion criteria for T2DM-DN were microalbuminurianon-Caucasian ethnic origin and in case of use of RAAS-blocking medication unknown albuminuria status prior tostart of treatment Estimated GFR (eGFR) was determinedby the Chronic Kidney Disease Epidemiology Collaborationequations for females (for whom [Creatinine]plasma wasgt62120583M) eGFR = 144 times ([Creatinine]plasma62)

minus1209times

(0993)Age and for males when [Creatinine]plasma le

80 120583M eGFR = 141 times ([Creatinine]plasma80)minus0411times

(0993)Age and when [Creatinine]plasma gt 80 120583M eGFR =

141 times ([Creatinine]plasma80)minus1209times (0993)

Age [24] Glycatedhaemoglobin (A1C) was determined by a validated ionexchange high pressure chromatography method (Menarini

ARKRAY ADAMS A1C HA-8180 analyser Menarini Diag-nostics Florence Italy) [25] Subject characteristics are givenin Table 1

22 Methods Venous blood samples were collected from allstudy subjects after overnight fast and plasma samples wereprepared immediately and stored at minus80∘C until analysis24 h urines collections were made and aliquots prepared andstored at minus80∘C until analysis Ultrafiltrates were prepared bymicrospin ultrafiltration (10 kDa cut-off) of plasma and urine(100 120583L) collecting ca 50 120583L ultrafiltrate Sample processingand storage validation were published previously [5]

Nucleotide markers of glycation and oxidation weredetermined by stable isotopic dilution analysis LC-MSMSGdG MGdG CEdG and 8-OxodG and related stable iso-topic [13C

10 15N5] substituted standards were prepared as

described [5] For LC-MSMS plasma and urine ultrafiltrates(40 120583L) were spiked with 10 120583L isotopic standard mixturecontaining 01 nmol [13C

10 15N5] dG 1 pmol [13C

10 15N5] 8-

OxodG 1 pmol [13C10 15N5] MGdG and CEdG and 1 pmol

[13C10 15N5] GdG LC-MSMS was performed using an

Acquity UPLC-Quattro Premier tandem mass spectrometerwith a BEH C18 17 120583m particle size 21 times 100mm columnThemobile phase (025mLmin) was 01 formic acid with alinear gradient of 0ndash10 acetonitrile from 2 to 10 min andisocratic 10 acetonitrile from 10 to 15min After analysisthe column was washed with 50 acetonitrile 01 formicacid for 10min and thereafter reequilibrated with initialmobile phase for 10minThe column temperature was variedfrom 10∘C For GdG MGdG CEdG and 8-OxodG limits ofdetection were 08 25 22 and 07 fmol analytical recoverieswere 104 97 98 and 99 respectively and coefficients ofvariation 2ndash7 [5]


23 Statistical Analysis Data are median (minimumndashmaxi-mum) or median (lowerndashupper quartile) values Significanceof differences betweenmeanswas assessed byMann-Whitney119880 test Bivariate regression was nonparametric (Spearman)and logistic regression was performed of DN on continuousvariables excluding the recruitment qualifier of urinaryalbumin solving for regression coefficient 119861 Statistical anal-ysis was performed by the SPSS software v21

3 Results

31 DNA Glycation Adducts in Plasma and Urine Biomark-ers of nucleotide glycation and oxidation are convenientlydetermined by assay of glycated and oxidised nucleosidesin plasma and urine ultrafiltrates This was performed withsamples from healthy controls and patients with T2DM withand without diabetic nephropathy Median plasma concen-trations of GdG MGdG CEdG and 8-OxodG in healthysubjects were 007 034 014 and 008 nM respectivelyPlasma GdG was increased 6-fold and plasma 8-OxodGincreased 2-fold in patients with T2DM Median urinaryexcretion rates of GdG MGdG CEdG and 8-OxodG inhealthy subjects were 023 263 090 and 090 nmol24 hrespectively In patients with T2DM urinary GdG MGdGCEdG and 8-OxodG were increased 10-fold 2-fold 2-foldand 28-fold respectively Figures 1(b)ndash1(i)

We also assessed the effect of diabetic nephropathy (DN)in patients with T2DM on plasma and urinary nucleotideglycation and oxidation damage markers In plasma median8-OxodG concentration was increased 30 in DN and inurine GdG was increased 24 and CEdG increased 60 indiabetic nephropathy Figures 1(j)ndash1(l)

32 Correlation Analysis Bivariate correlation analysis ofclinical chemistry variables of renal function with nucleosideglycation and oxidation analytes in patients with T2DM isgiven in Table 2 As expected there were a strong negativecorrelation of plasma creatinine with eGFR (119903 = minus095 119875 lt0001) a strong positive correction of plasma creatinine withurinary albumin excretion UAE (119903 = 061 119875 lt 0001) anda strong negative correction of eGFR with UAE (119903 = minus055119875 lt 0001) There were also positive correlations of urinaryexcretions of GdG and CEdG with UAE (119903 = 038 and 042resp 119875 lt 001) and a positive correlation of plasma 8-OxodG with plasma creatinine (119903 = 038 119875 lt 001) Forcorrelation between nucleotide glycation and oxidation ana-lytes in plasma there were a strong positive correlation ofMGdG with CEdG (119903 = 046 119875 lt 0001) and also positivecorrelation of MGdG with 8-OxodG (119903 = 045 119875 lt 001) andGdG with CEdG (119903 = 047 119875 lt 001) For urinary excretionthere was a positive correlation of MGdG excretion with 8-OxodG excretion (119903 = 037 119875 lt 001) There was alsoa positive correlation of plasma MGdG with urinary MGdG(119903 = 041 119875 lt 001) but there were no similar cor-relations of plasma and urinary levels of other nucleotideglycation and oxidation analytes There was no correlationof clinical chemistry variables of glycemic control (fastingplasma glucoseA1C) total cholesterol LDL cholesterolHDL

cholesterol or systolic and diastolic blood pressure withnucleotide glycation and oxidation markers

In a multiple logistic regression analysis of diabeticnephropathy on clinical and clinical chemistry variables thefollowing variables were included clinical variables previ-ously associatedwith diabetic nephropathy (age gender A1Csystolic and diastolic blood pressure and total cholesterol)[21 26] related markers of metabolic control (fasting plasmaglucose LDL and HDL) other factors linked to increasedglyoxal and methylglyoxal (duration of diabetes BMI) [122] and markers of DNA glycation and oxidation measurednoninvasively (urinary excretions of GdG MGdG CEdGand 8-OxodG) Plasma creatinine eGFR and UAE wereexcluded from the model as established biomarkers of dia-betic nephropathy Forward stepwise selection of variablesgave a multiple logistic regression model linking diabeticnephropathy to systolic blood pressure (119861 = 005 plusmn 002exponent 105 119875 = 0009) and urinary excretion of CEdG(119861 = 100 plusmn 041 exponent 270 119875 = 0016 119899 = 56)This is consistent with the increased systolic blood pressurefound in patients with diabetic nephropathy and the positivecorrelation of urinary CEdG with UAE Tables 1 and 2

4 Discussion

Herein we found for the first time increased plasma levelsand urinary excretion of DNA glycation adducts in patientswith T2DM and a link to diabetic nephropathy Hence mea-surement of DNA glycation adducts and nucleosides in bodyfluids may be valuable biomarkers of quantitative and func-tional important DNA damage in vivo Glyoxal is formed inphysiological systems by lipid peroxidation and degradationof glycated proteins and monosaccharides Methylglyoxalis formed mainly by degradation of triosephosphates andalso by ketone body metabolism and threonine catabolism[23] Both dicarbonyls are metabolised by the glutathione-dependent Glo1 [6] recently linked mechanistically to thedevelopment of diabetic nephropathy [14] Glycation of DNAby glyoxal and methylglyoxal has been linked to DNA strandbreaks and mutagenesis [27 28] and cellular dicarbonylconcentrations increase in oxidative stress [29] Formationof MGdG and CEdG by glycation of DNA with endogenousmethylglyoxal may explain the previously reported enhance-ment of DNA modification by glucose metabolites underconditions of glutathione depletion [30] increased DNAunwinding and single-strand breaks of DNA in vascularendothelial cell in hyperglycemia in vitro [31] and increasedsingle-strand breaks of DNA of patients with diabetes[32]

In previous studies we found levels of GdG and CEdGresidues in peripheral lymphocyte DNA of healthy peopleto be similar to those of 8-OxodG and the DNA contentof MGdG exceeded that of the widely studied 8-OxodG[5] Nucleotide AGE content of DNA was also markedlyhigher than that of other physiological aldehydesmdashsuch as4-hydroxynonenal and malondialdehyde [33] Modificationof DNA by physiological dicarbonyls therefore gives rise toquantitatively important steady-state levels of deoxyguano-sine-derived adducts in cellular DNA in vivo


Table2Correlationanalysisof

nucle

otideg

lycatio

nandoxidationanalytes

with

clinicalchemicalvaria

bles

ofrenalfun

ction

Creatin

ine P

lasm

amdash

eGFR

minus095

mdashUA

E061

minus055

mdashGdG

Plasma

mdashMGdG

Plasma

mdashCE

dGPlasma

047

046

mdash8-OxodG

Plasma

038

045

mdash

GdG

Urin

e038

mdash

MGdG

Urin

e041

039

mdash

CEdG

Urin

e042

033

mdash

8-OxodG

Urin

e037

mdash

Parameter

Creatin

ine P

lasm

aeG

FRUA

EGdG

Plasma

MGdG

Plasma

CEdG

Plasma

8-OxodG

Plasma

GdG

Urin

eMGdG

Urin

eCE

dGUrin

e8-OxodG

Urin

e

Bivaria

teregressio

nwas

byno

nparam

etric

Spearman

metho

dSign

ificance

and119875lt001and119875lt0001


In patients with T2DM there were particularly largeincreases in glyoxal-derived imidazopurinoneGdG inplasmaand urine and 8-OxodG in urine We cannot discriminatebetween the contributions to glycated and oxidised nucleo-sides formed by repair of glycated and oxidised DNA andformed by direct glycation and oxidation of deoxyguanosineFormation of GdG in patients with T2DM appears to beparticularly favoured For methylglyoxal-derived metabo-lites CEdG is more stable chemically than MGdG andmay be a more robust biomarker when released from cells[5] LC-MSMS analysis of urinary excretion of CEdGin streptozotocin-induced diabetic rats indicated a 4-foldincrease inCEdGexcretion in diabeteswith respect to normalhealthy control rats [34] Increased formation of glyoxaland methylglyoxal and oxidative stress in hyperglycemiaassociated with diabetes has been linked to microvascu-lar complicationsmdashincluding diabetic nephropathy [1 35]Related damage to proteins [36] and herein DNA providespotential markers of nephropathy development

We assessed the link of urinary excretion of nucleosideglycation and oxidation adducts as potential noninvasivebiomarkers of diabetic nephropathy In multiple logisticregression analysis urinary CEdG emerged as a positivecorrelate for diabetic nephropathy Urinary CEdG also cor-related positively with UAE and hence is linked to anestablished biomarker of diabetic nephropathy It also likelyreflects increased exposure to methylglyoxal CEdG hashigher chemical stability than MGdG and higher biologicalstability than methylglyoxal in cells and physiological fluidswhich may explain its greater diagnostic value than theserelated metabolites [5 22]

Plasma and urinary 8-OxodG were also increased inpatients with T2DM Urinary oxidation adducts of RNArather thanDNAwere associatedwithmortality in a prospec-tive study of patient with T2DM [37] Increased urinary 8-OxodG in patients with T2DM was found previously but thetechnique used liquid chromatography with electrochemicaldetection appears to have overestimated urinary levels of8-OxodG by ca 10-fold [11 12] The greater sensitivity andspecificity for analyte detection provided bymultiple reactionmonitoring detection in LC-MSMS accounts for this Thetwo major analytical approaches that have been used forthe measurement of urinary 8-OxodG prior to applicationof LC-MSMS were HPLC combined with electrochemicaldetection and immunoassay Approaches other than LC-MSMS have overestimated 8-OxodG [38ndash40] Estimatesof urinary 8-OxodG herein are similar to independentestimates by stable isotopic dilution analysis LC-MSMSfor healthy controls [41] and patients with T2DM [42]Similar improvements have been made in the detection ofCEdG by LC-MSMS CEdG was determined in urine ofnormal healthy human subjects previously by immunoassaywith estimates in the range of 34ndash344 pmolmg creatinineand median of ca 30 pmolmg creatinine [43] Estimationherein by LC-MSMS gave median (minimumndashmaximum)values of 055 (017ndash158) pmolmg creatinine suggesting thatimmunoassay procedures overestimated urinary CEdG byca 50-fold as was found for similar ELISA and LC-MSMSmeasurement of 8-OxodG [39] Overestimation is likely

caused by interference due to imperfect epitope specificity ofthe monoclonal antibody used and formation of CEdG fromMGdG and other sample components during the high pH ofpreanalytic sample processing

Aweakness of this studywas lack of females in the healthycontrol study group but there was no indication in the T2DMgroup that DNA damage marker levels were linked to gender

5 Conclusion

From the quantitative amount and link to functional end-points GdG MGdG and CEdG adducts are of likely path-ogenic and diagnostic significance Recent further evidencelinking dicarbonyl glycation with development of DN [14]suggests that DNA dicarbonyl adducts may emerge asbiomarkers of development of DN This study suggests thatdicarbonyl adducts are not surrogate measures of metaboliccontrol and some are linked to DN

Abbreviations

A1C Glycated haemoglobinAGE Advanced glycation endproductCEdG andCEdGACEdGB

N2-(1RS-Carboxyethyl)-deoxyguanosine and RSepimers

dG DeoxyguanosineGdG 3-(21015840-Deoxyribosyl)-67-dihydro-67-

dihydroxyimidazo-[23-b]purin-9(8)oneeGFR Estimated glomerular filtration rateGlo1 Glyoxalase 18-OxodG 78-Dihydro-8-oxo-21015840-deoxyguanosine4HNE 4-HydroxynonenalMGdG 3-(21015840-Deoxyribosyl)-67-dihydro-67-

dihydroxy-67-methylimidazo-[23-b]purine-9(8)one

LC-MSMS Liquid chromatography with tandemmass spectrometric detection

UAE Urinary albumin excretionDN Diabetic nephropathy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Sahar Waris researched the data Brigitte M Winklhofer-Roob Johannes M Roob Sebastian Fuchs and HaraldSourij performed subject recruitment and sample collectionBrigitteMWinklhofer-Roob also edited the paper and NailaRabbani and Paul JThornalley researched the data and wrotethe paper


Acknowledgments

Sahar Waris thanks the Commonwealth Scholarship Com-mission for a split-site PhD studentship and was on second-ment fromDepartment of Biochemistry Faculty ofMedicineJawaharlal Nehru Medical College Aligarh Muslim Univer-sity Aligarh PIN 202002 India The project was also fundedby the Commission of the European Communities 6thFramework Programme Priority 1 Life Sciences Genomicsand Biotechnology forHealth LSHM-CT-2005-018733 PRE-DICTIONS (Prevention of Diabetic Complications) and 5thFramework Programme Key Action 1 Food Nutrition andHealthQLK1-CT-1999-00830 VITAGE (VitaminAVitaminE and Carotenoid Status and Metabolism during AgeingFunctional and Nutritional Consequences) The authorsthank Astrid Knopf and Sandra Wuga Human NutritionAND Metabolism Research and Training Center Instituteof Molecular Biosciences Karl Franzens University of GrazAustria for excellent technical assistance

References

[1] A C McLellan P J Thornalley J Benn and P H SonksenldquoGlyoxalase system in clinical diabetes mellitus and correlationwith diabetic complicationsrdquo Clinical Science vol 87 no 1 pp21ndash29 1994

[2] N Rabbani and P J Thornalley ldquoDicarbonyls (glyoxal methyl-glyoxal and 3-deoxyglucosone)rdquo inUremic Toxins pp 177ndash192John Wiley amp Sons 2012

[3] J W Baynes ldquoRole of oxidative stress in development of com-plications in diabetesrdquoDiabetes vol 40 no 4 pp 405ndash412 1991

[4] PDandona KThusu S Cook et al ldquoOxidative damage toDNAin diabetesmellitusrdquoTheLancet vol 347 no 8999 pp 444ndash4451996

[5] P J Thornalley S Waris T Fleming et al ldquoImidazopurinonesare markers of physiological genomic damage linked to DNAinstability and glyoxalase 1-associated tumour multidrug resis-tancerdquoNucleic Acids Research vol 38 no 16 Article ID gkq306pp 5432ndash5442 2010

[6] M Xue N Rabbani and P JThornalley ldquoGlyoxalase in ageingrdquoSeminars in Cell and Developmental Biology vol 22 no 3 pp293ndash301 2011

[7] A Bierhaus T Fleming S Stoyanov et al ldquoMethylglyoxal mod-ification of Na

11990718 facilitates nociceptive neuron firing and

causes hyperalgesia in diabetic neuropathyrdquo Nature Medicinevol 18 no 6 pp 926ndash933 2012

[8] P Palsamy and S Subramanian ldquoResveratrol protects diabetickidney by attenuating hyperglycemia-mediated oxidative stressand renal inflammatory cytokines via Nrf2-Keap1 signalingrdquoBiochimica et Biophysica Acta vol 1812 no 7 pp 719ndash731 2011

[9] M T Barati M L Merchant A B Kain A W Jevans K RMcLeish and J B Klein ldquoProteomic analysis defines alteredcellular redox pathways and advanced glycation end-productmetabolism in glomeruli of dbdb diabetic micerdquo AmericanJournal of Physiology Renal Physiology vol 293 no 4 pp F1157ndashF1165 2007

[10] N Karachalias R Babaei-Jadidi N Rabbani and P J Thor-nalley ldquoIncreased protein damage in renal glomeruli retinanerve plasma and urine and its prevention by thiamine andbenfotiamine therapy in a rat model of diabetesrdquo Diabetologiavol 53 no 7 pp 1506ndash1516 2010

[11] Y Hinokio S Suzuki M Hirai M Chiba A Hirai andT Toyota ldquoOxidative DNA damage in diabetes mellitus itsassociation with diabetic complicationsrdquo Diabetologia vol 42no 8 pp 995ndash998 1999

[12] Y Hinokio S Suzuki M Hirai C Suzuki M Suzuki and TToyota ldquoUrinary excretion of 8-oxo-7 8-dihydro-21015840-deoxygua-nosine as a predictor of the development of diabetic nephropa-thyrdquo Diabetologia vol 45 no 6 pp 877ndash882 2002

[13] W Seidel and M Pischetsrieder ldquoDNA-glycation leads todepurination by the loss of N2-carboxyethylguanine in vitrordquoCellular andMolecular Biology vol 44 no 7 pp 1165ndash1170 1998

[14] F Giacco X Du V D DrsquoAgati et al ldquoKnockdown of glyoxalase1 mimics diabetic nephropathy in nondiabetic micerdquo Diabetesvol 63 no 1 pp 291ndash299 2014

[15] N Rabbani and P J Thornalley ldquoThe critical role of methylgly-oxal and glyoxalase 1 in diabetic nephropathyrdquoDiabetes vol 63no 1 pp 50ndash52 2014

[16] C E Vaca J L Fang M Conradi and S-M Hou ldquoDevel-opment of a 32P-postlabelling method for the analysis of21015840-deoxyguanosine-31015840-monophosphate and DNA adducts ofmethylglyoxalrdquo Carcinogenesis vol 15 no 9 pp 1887ndash18941994

[17] H Li S Nakamura S Miyazaki et al ldquoN 2-carboxyethyl-21015840-deoxyguanosine a DNA glycation marker in kidneys andaortas of diabetic and uremic patientsrdquo Kidney Internationalvol 69 no 2 pp 388ndash392 2006

[18] M S Cooke R Olinski S Loft and European StandardsCommittee on Urinary Lesion ldquoMeasurement and meaning ofoxidatively modified DNA lesions in urinerdquo Cancer EpidemiolBiomarkers Prevention vol 17 no 1 pp 3ndash14 2008

[19] K Rossing H Mischak M Dakna et al ldquoUrinary proteomicsin diabetes and CKDrdquo Journal of the American Society of Neph-rology vol 19 no 7 pp 1283ndash1290 2008

[20] E Rock B M Winklhofer-Roob J Ribalta et al ldquoVitamin Avitamin E and carotenoid status and metabolism during age-ing functional and nutritional consequences (Vitage Project)rdquoNutrition Metabolism and Cardiovascular Diseases vol 11 no4 supplement pp 70ndash73 2001

[21] American-Diabetes-Association ldquoStandards of Medical Care inDiabetesmdash2014rdquo Diabetes Care vol 37 supplement 1 pp S14ndashS80 2013

[22] N Rabbani and P J Thornalley ldquoDicarbonyl stress in celland tissue dysfunction contributing to ageing and diseaserdquoBiochemical andBiophysical ResearchCommunications vol 458no 2 pp 221ndash226 2015

[23] P J Thornalley ldquoDicarbonyl intermediates in the MaillardreactionrdquoAnnals of the New York Academy of Sciences vol 1043pp 111ndash117 2005

[24] F Iliadis T Didangelos A Ntemka et al ldquoGlomerular filtrationrate estimation in patients with type 2 diabetes creatinine- orcystatin C-based equationsrdquo Diabetologia vol 54 no 12 pp2987ndash2994 2011

[25] E Urrechaga E Bereciartua E Crespo and C Izcara ldquoEval-uation of AdamsTM A1C Menarini HA-8180 HPLC analyserfor HbA1c determinationrdquo Clinical Chemistry and LaboratoryMedicine vol 49 p S406 2011

[26] M-A Gall P Hougaard K Borch-Johnsen and H-H ParvingldquoRisk factors for development of incipient and overt diabeticnephropathy in patients with non-insulin dependent diabetesmellitus prospective observational studyrdquo BritishMedical Jour-nal vol 314 no 7083 pp 783ndash788 1997


[27] N Murata-Kamiya H Kamiya H Kaji and H Kasai ldquoGlyoxala major product of DNA oxidation induces mutations at GCsites on a shuttle vector plasmid replicated inmammalian cellsrdquoNucleic Acids Research vol 25 no 10 pp 1897ndash1902 1997

[28] N Murata-Kamiya H Kamiya H Kaji and H Kasai ldquoMethyl-glyoxal induces GC to CG and GC to TA transversions in thesupF gene on a shuttle vector plasmid replicated in mammaliancellsrdquoMutation Research vol 468 no 2 pp 173ndash182 2000

[29] E A Abordo H S Minhas and P JThornalley ldquoAccumulationof120572-oxoaldehydes during oxidative stress a role in cytotoxicityrdquoBiochemical Pharmacology vol 58 no 4 pp 641ndash648 1999

[30] T K Shires J Tresnak M Kaminsky S L Herzog and B Truc-Pham ldquoDNA modification in vivo by derivatives of glucoseenhancement by glutathione depletionrdquoTheFASEB Journal vol4 no 15 pp 3340ndash3346 1990

[31] M Lorenzi D F Montisano S Toledo and A Barrieux ldquoHighglucose induces DNA damage in cultured human endothelialcellsrdquoThe Journal of Clinical Investigation vol 77 no 1 pp 322ndash325 1986

[32] M Lorenzi D F Montisano S Toledo and H-C H WongldquoIncreased single strand breaks in DNA of lymphocytes fromdiabetic subjectsrdquo Journal of Clinical Investigation vol 79 no 2pp 653ndash656 1987

[33] R de Bont and N van Larebeke ldquoEndogenous DNA damage inhumans a review of quantitative datardquoMutagenesis vol 19 no3 pp 169ndash185 2004

[34] T Synold B Xi G EWuenschell et al ldquoAdvanced glycation endproducts of DNA quantification of N 2-(1-carboxyethyl)-21015840-deoxyguanosine in biological samples by liquid chromatogra-phy electrospray ionization tandemmass spectrometryrdquo Chem-ical Research in Toxicology vol 21 no 11 pp 2148ndash2155 2008

[35] M Brownlee ldquoBiochemistry and molecular cell biology ofdiabetic complicationsrdquo Nature vol 414 no 6865 pp 813ndash8202001

[36] N Rabbani A Adaikalakoteswari K Rossing et al ldquoEffect ofIrbesartan treatment on plasma and urinary markers of proteindamage in patients with type 2 diabetes andmicroalbuminuriardquoAmino Acids vol 42 no 5 pp 1627ndash1639 2012

[37] K Broedbaek V Siersma T Henriksen et al ldquoAssociationbetween urinary markers of nucleic acid oxidation and mortal-ity in type 2 diabetes a population-based cohort studyrdquoDiabetesCare vol 36 no 3 pp 669ndash676 2013

[38] K Broedbaek A Weimann E S Stovgaard and H E PoulsenldquoUrinary 8-oxo-78-dihydro-21015840-deoxyguanosine as a biomarkerin type 2 diabetesrdquo Free Radical Biology and Medicine vol 51no 8 pp 1473ndash1479 2011

[39] M S Cooke R Singh G K Hall et al ldquoEvaluation of enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry methodology for the analysis of 8-oxo-78-dihydro-21015840-deoxyguanosine in saliva and urinerdquo FreeRadical Biology and Medicine vol 41 no 12 pp 1829ndash18362006

[40] L Barregard P Moslashller T Henriksen et al ldquoHuman andmethodological sources of variability in the measurementof urinary 8-oxo-78-dihydro-21015840-deoxyguanosinerdquoAntioxidantsand Redox Signaling vol 18 no 18 pp 2377ndash2391 2013

[41] K Broedbaek R Ribel-Madsen T Henriksen et al ldquoGeneticand environmental influences on oxidative damage assessed inelderly Danish twinsrdquo Free Radical Biology and Medicine vol50 no 11 pp 1488ndash1491 2011

[42] K Broedbaek T Henriksen A Weimann et al ldquoLong-termeffects of Irbesartan treatment and smoking on nucleic acid oxi-dation in patients with type 2 diabetes and microalbuminuriaan irbesartan in patients with type 2 diabetes and microalbu-minuria (IRMA 2) substudyrdquo Diabetes Care vol 34 no 5 pp1192ndash1198 2011

[43] M Schneider G Thoss C Hubner-Parajsz R Kientsch-EngelP Stahl and M Pischetsrieder ldquoDetermination of glycatednucleobases in human urine by a new monoclonal antibodyspecific for 1198732-carboxyethyl-21015840-deoxyguanosinerdquo ChemicalResearch in Toxicology vol 17 no 10 pp 1385ndash1390 2004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


OxidationGdG

8-OxodG

MGdG CEdG

N

NHN

N

N

O

H

H

NHNH

N

N

N

O

dG

N

N

N

O

HO

N

NHN

N

N

O

H

N

NHN

N

N

OOH

OHOH

OHOH

OH

H

NH

NHN

N

N

O

CH3

CH3

NH2NH2

CH CH3

CO2

minus

++

(a)

00

01

02

03

04

05

Control T2DM00

01

02

03

04

05

Control T2DM

000

005

010

015

020

025

Control T2DM000

005

010

015

020

025

Control T2DM

[GdG

] Pla

sma

(nM

)[C

EdG

] Pla

sma

(nM

)

[MdG

] Pla

sma

(nM

)[8

-Oxo

dG] P

lasm

a(n

M)

(b) (c)

(d) (e)

+ Methylglyoxal

+ Glyoxal

lowastlowastlowast

lowastlowast

Figure 1 Continued


0

1

2

3

4

Control T2DM0

2

4

6

8

Control T2DM

0

1

2

3

4

Control T2DM0

10

20

30

40

Control T2DM

0

1

2

3

4

00

01

02

03

Urin

ary

GdG

(nm

ol24

h)

Urin

ary

MG

dG (n

mol

24

h)

Urin

ary

CEdG

(nm

ol24

h)

Urin

ary

GdG

(nm

ol24

h)U

rinar

y8

-Oxo

dG (n

mol

24

h)

[8-O

xodG

] Pla

sma

(nM

)

(f) (g)

(h) (i)



lowastlowastlowast

lowastlowastlowast

lowast

lowast

Figure 1 Continued


0

1

2

3

4

Urin

ary

CEdG

(nm

ol24

h)

(l)


lowast


119875 lt 0001 Mann-Whitney 119880






2 Methods












minus1209times











10 15N5] dG 1 pmol [13C

10 15N5] 8-






3 Results






4 Discussion





nucle

otideg

lycatio


with


bles

ofrenalfun

ction

Creatin

ine P

lasm

amdash

eGFR

minus095

mdashUA

E061

minus055

mdashGdG

Plasma

mdashMGdG

Plasma

mdashCE

dGPlasma

047

046

mdash8-OxodG

Plasma

038

045

mdash

GdG

Urin

e038

mdash

MGdG

Urin

e041

039

mdash

CEdG

Urin

e042

033

mdash

8-OxodG

Urin

e037

mdash

Parameter

Creatin

ine P

lasm

aeG

FRUA

EGdG

Plasma

MGdG

Plasma

CEdG

Plasma

8-OxodG

Plasma

GdG

Urin

eMGdG

Urin

eCE

dGUrin

e8-OxodG

Urin

e

Bivaria

teregressio

nwas

byno

nparam

etric

Spearman

metho

dSign

ificance

and119875lt001and119875lt0001







5 Conclusion


Abbreviations













Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

Control T2DM0

2

4

6

8

Control T2DM

0

1

2

3

4

Control T2DM0

10

20

30

40

Control T2DM

0

1

2

3

4

00

01

02

03

Urin

ary

GdG

(nm

ol24

h)

Urin

ary

MG

dG (n

mol

24

h)

Urin

ary

CEdG

(nm

ol24

h)

Urin

ary

GdG

(nm

ol24

h)U

rinar

y8

-Oxo

dG (n

mol

24

h)

[8-O

xodG

] Pla

sma

(nM

)

(f) (g)

(h) (i)



lowastlowastlowast

lowastlowastlowast

lowast

lowast

Figure 1 Continued


0

1

2

3

4

Urin

ary

CEdG

(nm

ol24

h)

(l)


lowast


119875 lt 0001 Mann-Whitney 119880






2 Methods












minus1209times











10 15N5] dG 1 pmol [13C

10 15N5] 8-






3 Results






4 Discussion





nucle

otideg

lycatio


with


bles

ofrenalfun

ction

Creatin

ine P

lasm

amdash

eGFR

minus095

mdashUA

E061

minus055

mdashGdG

Plasma

mdashMGdG

Plasma

mdashCE

dGPlasma

047

046

mdash8-OxodG

Plasma

038

045

mdash

GdG

Urin

e038

mdash

MGdG

Urin

e041

039

mdash

CEdG

Urin

e042

033

mdash

8-OxodG

Urin

e037

mdash

Parameter

Creatin

ine P

lasm

aeG

FRUA

EGdG

Plasma

MGdG

Plasma

CEdG

Plasma

8-OxodG

Plasma

GdG

Urin

eMGdG

Urin

eCE

dGUrin

e8-OxodG

Urin

e

Bivaria

teregressio

nwas

byno

nparam

etric

Spearman

metho

dSign

ificance

and119875lt001and119875lt0001







5 Conclusion


Abbreviations













Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

Urin

ary

CEdG

(nm

ol24

h)

(l)


lowast


119875 lt 0001 Mann-Whitney 119880






2 Methods












minus1209times











10 15N5] dG 1 pmol [13C

10 15N5] 8-






3 Results






4 Discussion





nucle

otideg

lycatio


with


bles

ofrenalfun

ction

Creatin

ine P

lasm

amdash

eGFR

minus095

mdashUA

E061

minus055

mdashGdG

Plasma

mdashMGdG

Plasma

mdashCE

dGPlasma

047

046

mdash8-OxodG

Plasma

038

045

mdash

GdG

Urin

e038

mdash

MGdG

Urin

e041

039

mdash

CEdG

Urin

e042

033

mdash

8-OxodG

Urin

e037

mdash

Parameter

Creatin

ine P

lasm

aeG

FRUA

EGdG

Plasma

MGdG

Plasma

CEdG

Plasma

8-OxodG

Plasma

GdG

Urin

eMGdG

Urin

eCE

dGUrin

e8-OxodG

Urin

e

Bivaria

teregressio

nwas

byno

nparam

etric

Spearman

metho

dSign

ificance

and119875lt001and119875lt0001







5 Conclusion


Abbreviations













Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























minus1209times











10 15N5] dG 1 pmol [13C

10 15N5] 8-






3 Results






4 Discussion





nucle

otideg

lycatio


with


bles

ofrenalfun

ction

Creatin

ine P

lasm

amdash

eGFR

minus095

mdashUA

E061

minus055

mdashGdG

Plasma

mdashMGdG

Plasma

mdashCE

dGPlasma

047

046

mdash8-OxodG

Plasma

038

045

mdash

GdG

Urin

e038

mdash

MGdG

Urin

e041

039

mdash

CEdG

Urin

e042

033

mdash

8-OxodG

Urin

e037

mdash

Parameter

Creatin

ine P

lasm

aeG

FRUA

EGdG

Plasma

MGdG

Plasma

CEdG

Plasma

8-OxodG

Plasma

GdG

Urin

eMGdG

Urin

eCE

dGUrin

e8-OxodG

Urin

e

Bivaria

teregressio

nwas

byno

nparam

etric

Spearman

metho

dSign

ificance

and119875lt001and119875lt0001







5 Conclusion


Abbreviations













Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















3 Results






4 Discussion





nucle

otideg

lycatio


with


bles

ofrenalfun

ction

Creatin

ine P

lasm

amdash

eGFR

minus095

mdashUA

E061

minus055

mdashGdG

Plasma

mdashMGdG

Plasma

mdashCE

dGPlasma

047

046

mdash8-OxodG

Plasma

038

045

mdash

GdG

Urin

e038

mdash

MGdG

Urin

e041

039

mdash

CEdG

Urin

e042

033

mdash

8-OxodG

Urin

e037

mdash

Parameter

Creatin

ine P

lasm

aeG

FRUA

EGdG

Plasma

MGdG

Plasma

CEdG

Plasma

8-OxodG

Plasma

GdG

Urin

eMGdG

Urin

eCE

dGUrin

e8-OxodG

Urin

e

Bivaria

teregressio

nwas

byno

nparam

etric

Spearman

metho

dSign

ificance

and119875lt001and119875lt0001







5 Conclusion


Abbreviations













Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















nucle

otideg

lycatio


with


bles

ofrenalfun

ction

Creatin

ine P

lasm

amdash

eGFR

minus095

mdashUA

E061

minus055

mdashGdG

Plasma

mdashMGdG

Plasma

mdashCE

dGPlasma

047

046

mdash8-OxodG

Plasma

038

045

mdash

GdG

Urin

e038

mdash

MGdG

Urin

e041

039

mdash

CEdG

Urin

e042

033

mdash

8-OxodG

Urin

e037

mdash

Parameter

Creatin

ine P

lasm

aeG

FRUA

EGdG

Plasma

MGdG

Plasma

CEdG

Plasma

8-OxodG

Plasma

GdG

Urin

eMGdG

Urin

eCE

dGUrin

e8-OxodG

Urin

e

Bivaria

teregressio

nwas

byno

nparam

etric

Spearman

metho

dSign

ificance

and119875lt001and119875lt0001







5 Conclusion


Abbreviations













Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















5 Conclusion


Abbreviations













Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article increased dna dicarbonyl glycation and...

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