Ci a ion: Pia ulli, F.; Ban i, C.;
B ioschi, M.; Al oma e, A.; Ragazzi,
E.; Cosma, C.; Sa o e, G.; Lapolla, A.
The Bu den o Impai ed Se um
Albumin An ioxidan P ope ies and
Glyco-Oxida ion in Co ona y Hea
Disease Pa ien s wi h and wi hou
Type 2 Diabe es Melli us.
An ioxidan s 2022,11, 1501. h ps://
doi.o g/10.3390/an iox11081501
Academic Edi o : S anley Omaye
Recei ed: 1 July 2022
Accep ed: 28 July 2022
Published: 30 July 2022
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an ioxidan s
A icle
The Bu den o Impai ed Se um Albumin An ioxidan
P ope ies and Glyco-Oxida ion in Co ona y Hea Disease
Pa ien s wi h and wi hou Type 2 Diabe es Melli us
F ancesco Pia ulli 1, C is ina Ban i 2,* , Mau a B ioschi 2, Alessand a Al oma e 3, Eugenio Ragazzi 4,
Chia a Cosma 1, Gio anni Sa o e 1and Annunzia a Lapolla 1,*
1Depa men o Medicine, Uni e si y o Pado a, 35128 Pado a, I aly; [email p o ec ed] (F.P.);
[email p o ec ed] (C.C.); [email p o ec ed] (G.S.)
2Cen o Ca diologico Monzino IRCCS, 20138 Milan, I aly; [email p o ec ed]
3
Depa men o Pha maceu ical Sciences, Uni e si y o Milan, 20133 Milan, I aly; [email p o ec ed]
4
Depa men o Pha maceu ical and Pha macological Sciences (DSF), Uni e si y o Pado a School o Medicine
and Su ge y, 35131 Pado a, I aly; [email p o ec ed]
*Co espondence: [email p o ec ed] (C.B.); [email p o ec ed] (A.L.)
Abs ac :
Human se um albumin (HSA) has an impo an an ioxidan ac i i y due o he p esence o
he educed cys eine a posi ion 34, which ep esen s he mos abundan ee hiol in he plasma. In
oxida i e-based diseases, HSA unde goes S- hiola ion (THIO-HSA) wi h changes in he an ioxidan
unc ion o albumin ha could con ibu e o he p og ession o he disease. The aim o his s udy
was o e i y, o he i s ime, he di e en bu dens o THIO-HSA, glyca ed HSA (GLY-HSA), and
ad anced glyca ion end p oduc s (AGE) accumula ion bo h in ype 2 diabe es melli us (T2DM)
pa ien s and in non-diabe ic pa ien s, wi h o wi hou co ona y hea disease (CHD). In his s udy,
we assessed he p esence o modi ied o ms o HSA, THIO-HSA, and GLY-HSA by means o mass
spec ome y in 33 pa ien s wi h bo h T2DM and CHD, in 31 pa ien s wi h T2DM and wi hou CHD,
in 30 pa ien s wi hou diabe es wi h a his o y o CHD, and 27 subjec s wi hou diabe es and CHD. All
he pa ien s’ an h opome ic and clinical da a we e eco ded including age, sex, du a ion o diabe es,
body mass index (BMI), blood p essu e, and his o y o CHD de ined wi h anamnes ic da a. Me abolic
pa ame e s, such as as ing plasma glucose (FPG), glyca ed hemoglobin (HbA1c), lipids, pen osidine,
AGE, ecep o o ad anced glyca ion end-p oduc s (RAGE) and i s soluble o m (sRAGE), we e
measu ed. AGE and pen osidine a e signi ican ly highe in T2DM pa ien s wi h and wi hou CHD
wi h espec o non-diabe ic pa ien s wi h CHD and con ol subjec s. RAGE le els a e signi ican ly
highe in T2DM pa ien s wi h espec o non-diabe ic pa ien s, and among T2DM pa ien s, he g oup
wi h CHD showed signi ican ly highe RAGE le els han hose wi hou CHD (217
±
171 pg/mL and
140
±
61 pg/mL, espec i ely). Albumin iso o ms disc imina e be ween non-diabe ic pa ien s wi h
CHD and T2DM pa ien s wi h and wi hou CHD and con ol subjec s, wi h GLY-HSA le els highe
in T2DM wi h and wi hou CHD, and THIO-HSA highe in CHD pa ien s wi hou T2DM. Finally,
we demons a ed ha he oxidized o ms o HSA can inc ease he exp ession o he in lamma o y
cy okine Tumo Nec osis Fac o -alpha (TNF
α
) in monocy ic cells. In pa ien s wi h CHD, GLY-HSA
and THIO-HSA ha e a di e en p e alen dis ibu ion, he i s one p e ailing in pa ien s wi h T2DM
and he second one in pa ien s wi hou T2DM. These indings sugges ha albumin quali y and
homeos asis balance be ween glyco-oxida ion and hiola ion migh ha e an impac on he an ioxidan
de ense sys em in ca dio ascula diseases.
Keywo ds: diabe es; S- hiola ion; oxida i e s ess; albumin; co ona y hea disease
1. In oduc ion
Type 2 diabe es melli us (T2DM) is associa ed wi h a wo- o ou - old inc ease in he
isk o co ona y, ce eb al, and pe iphe al a e y disease [
1
]. A se ies o biochemical al e -
An ioxidan s 2022,11, 1501. h ps://doi.o g/10.3390/an iox11081501 h ps://www.mdpi.com/jou nal/an ioxidan s
An ioxidan s 2022,11, 1501 2 o 11
a ions due o hype glycemia, including p o ein kinase C ac i a ion, an inc ease in polyol
and hexosamine pa hway lux, and non-enzyma ic p o ein glyca ion a e also in ol ed in
a he oscle o ic disease de elopmen [
2
,
3
]. All o hese molecula mechanisms e lec a
single hype glycemia-induced p ocess o supe oxide o e p oduc ion by he mi ochond ial
elec on anspo chain [
4
]. Hype glycemia and inc eased oxida i e s ess ac h ough com-
mon pa hways leading o issue damage [
4
]. As o p o ein glyca ion, his p ocess is he i s
o a se ies o s eps, leading o he p oduc ion o ad anced glyca ion end p oduc s (AGE).
Pen osidine is one o he mos in es iga ed AGE, which esul s om he non-enzyma ic
eac ions be ween p o eins and pen oses o hexoses. I is a e y sensi i e ma ke o ch onic
diseases, and i is di ec ly associa ed wi h ea ly and ad anced a he oscle osis in pa ien s
wi h diabe es melli us [
5
]. AGE accumula ion is able o de e mine endo helial damage
h ough a se ies o mechanisms, including in acellula p o ein modi ica ions, o ma ion o
c oss-links in he ex acellula ma ix, and in e ac ion wi h he AGE ecep o , RAGE [
6
].
RAGE is a mul iligand membe o he Ig supe amily o cell su ace molecules ha binds
AGE and induces cellula signaling, including ac i a ion o he nuclea ac o κB (NF-κB),
an inc ease in cy okine and adhesion molecule exp ession, and induc ion o oxida i e s ess,
wi h an inc ease in cy osolic eac i e oxygen species [
7
,
8
]. AGE and hei ecep o s may
be use ul bioma ke s o he p esence and se e i y o co ona y hea disease (CHD) in
pa ien s wi h and wi hou diabe es melli us; he AGE/RAGE axis has been implica ed in
con ibu ing o ca dio ascula mo ali y independen ly o diabe es [
9
]. The soluble RAGE
o m (sRAGE) e lec s he o al concen a ion o RAGE p esen in cells and plasma [
3
]. In
pa ien s wi h CHD, some clinical s udies ha e demons a ed ha lowe le els o plasma
sRAGE a e in e sely associa ed wi h he se e i y o CHD [
10
]. Howe e , o he epo s
ha e shown ha se um sRAGE le els a e signi ican ly highe in pa ien s wi h T2DM han
in non-diabe ic subjec s, and a e posi i ely associa ed wi h he p esence o CHD [11].
Human se um albumin (HSA) is he mos abundan ci cula o y p o ein, and has an
impo an an ioxidan ac i i y by apping ee adicals [
12
]. This p o ein exe s his ac i i y
h ough he p esence o he educed Cys 34 [
13
]; he Cys 34 sulphyd yl g oup p esen in
he albumin is he la ges ac ion o all ee hiols in plasma, and his con e s o HSA a
majo ole in he an ioxidan capaci y o he plasma [
14
]. In a p e ious pape , B ioschi e al.
e idenced ha S- hiola ion o albumin (THIO-HSA) is inc eased in he plasma o pa ien s
wi h hea ailu e (HF) and induces changes in he an ioxidan unc ion o albumin ha
could con ibu e o he p og ession o HF [
15
]. An enhanced THIO-HSA was obse ed
also in he plasma o hemodialysis pa ien s [
16
], sugges ing i s possible ole in inducing
changes in biological esponses in he in e -cellula and in e -o gan a ic, leading o he
de elopmen o ch onic complica ions. Mo eo e , low HSA le els a e independen ly and
in e sely associa ed wi h he occu ence o ca dio ascula diseases (CVD) including HF,
CHD, a ial ib illa ion, s oke, and enous h omboembolism, ep esen ing a po en ial
isk ac o o a ious CVD [
12
]. On he o he side, as demons a ed by s udies made
wi h mass spec ome y (MS) [
17
,
18
], HSA can unde go glyca ion (GLY-HSA), bo h in
diabe es melli us and in non-diabe ic pa ien s a ec ed by HF [
15
,
19
]. GLY-HSA is now
conside ed a clinical bioma ke o glycemic con ol, e en i i s use is es ic ed o hose
clinical condi ions in which he le els o glyca ed hemoglobin (HbA1c) do no e lec
glycemic s a us (i.e., p egnancy, ch onic enal ailu e, o ans usion), o o he e alua ion o
sho - e m glycemic luc ua ions in pa ien s wi h poo me abolic con ol. Indeed, GLY-HSA
e lec s mean glycemia o e app oxima ely 2–3 weeks and, compa ed wi h HbA1c is no
in luenced by ed blood cell li espan [
20
]. Howe e , as s a ed by he In e na ional and
Na ional Recommenda ions, i canno be used ye o he diagnosis o diabe es [21,22].
The aim o his s udy was o e i y, o he i s ime, he di e en bu dens o THIO-
HSA, GLY-HSA, and AGE accumula ion bo h in T2DM pa ien s and in non-diabe ic pa ien s,
wi h o wi hou CHD.
An ioxidan s 2022,11, 1501 3 o 11
2. Ma e ials and Me hods
2.1. Pa ien s Rec ui men and Clinical Pa ame e s
Thi y- h ee pa ien s wi h bo h T2DM and CHD (g oup A), 31 pa ien s wi h T2DM and
wi hou CHD (g oup B), 30 pa ien s wi hou diabe es wi h a diagnosis o CHD (g oup C),
and 27 subjec s wi hou diabe es and CHD (g oup D) we e examined. All pa ien s a ec ed
by diabe es (g oups A and B) ollowed an isocalo ic Medi e anean-s yle die a y pa e n
(cha ac e ized by a lowe in ake o p o eins om animal ood sou ces, sa u a ed a and
choles e ol, added suga s, a highe in ake o ibe , and a lowe glycemic index and glycemic
load), and had compa able hypoglycemic he apy. In pa icula , no di e ence occu ed wi h
he use o me o min, which was adminis e ed in a small numbe o pa ien s (5 pa ien s in
g oup A, and 4 pa ien s in g oup B), and in low doses (500–750 mg/day). On he day o he
s udy, all he pa ien s’ an h opome ic and clinical da a we e eco ded including age, sex,
body mass index (BMI), blood p essu e, and diagnosis o CHD de ined wi h anamnes ic
da a om elec onic medical eco ds. S anda d c i e ia o disease diagnosis we e ollowed
o he inclusion o pa ien s in o he g oups in es iga ed. CHD was assessed om he
hospi al eco ds; 90% o he pa ien s we e aking an ihype ensi e d ugs. The s udy
was conduc ed in acco dance wi h he Decla a ion o Helsinki and i s la e amendmen s,
and was app o ed by he E hics Commi ee o Clinical T ials in he P o ince o Pado a,
e e ence s udy No. 97610. In o med w i en consen was ob ained om all subjec s
pa icipa ing in he s udy.
Blood samples we e collec ed in ubes con aining ci a e as he an icoagulan
(0.129 mmol/L). Plasma was immedia ely p epa ed by means o cen i uga ion a 1500
×
g
o 15 min a 4 ◦C, di ided in o aliquo s, and ozen a −80 ◦C un il assayed.
Me abolic pa ame e s, such as as ing plasma glucose (FPG), HbA1c, o al choles e ol,
HDL and LDL choles e ol, and iglyce ides we e e alua ed, and glyca ion and oxida ion
pa ame e s such as GLY-HSA, THIO-HSA, pen osidine, AGE, sRAGE we e measu ed.
FPG was measu ed using he glucose oxidase me hod [
23
]. HbA1c was measu ed using
an In e na ional Fede a ion o Clinical Chemis y (IFCC)-aligned s anda dized HPLC
me hod [24]. Plasma lipids we e measu ed as p oposed by Das ych e al. [25].
2.2. Immunoassays
Plasma le els o sRAGE, AGE [
26
], Pen osidine, and RAGE we e measu ed by enzyme-
linked immunoso ben assays (ELISA), acco ding o he manu ac u e ’s ins uc ion.
Soluble RAGE was measu ed wi h an ELISA ki p oduced by BioVendo Resea ch and
Diagnos ic on plasma samples. A calib a ion cu e (50–3200 pg/mL) was used o calcula e
sRAGE plasma concen a ions. The AGE ELISA ki was pu chased om Cusabio and was
used o measu e AGE le els using he p o ided calib a ion cu e (0.78
µ
g/mL–50
µ
g/mL)
o calcula ion o he concen a ion. Human pen osidine plasma le els, measu ed wi h
he ELISA ki p oduced by Cusabio, we e calcula ed using he p o ided s anda d cu e
(31.25 pmol/mL–2000 pmol/mL). The Human RAGE ELISA ki p oduced by RayBio ech
was used o measu e plasma RAGE le els, and concen a ion was calcula ed om he
calib a ion cu e (3 pg/mL–1500 pg/mL). Abso bance was measu ed wi h he Tecan
in ini e M200 spec opho ome e .
2.3. Albumin Analysis by Mass Spec ome y
Liquid ch oma og aphy-mass spec ome y (LC-MS) analysis o in ac p o eins in
plasma was used o measu e he ela i e abundance (%) o GLY-HSA and THIO-HSA, as
p e iously de ailed [
27
]. B ie ly, plasma p o eins we e dilu ed 1:40 in a dena u ing mobile
phase (30% ace oni ile:0.1% o mic acid, FA) be o e LC sepa a ion on a e e sed-phase
column Jupi e C4 (150
×
2 mm, i.d. 5
µ
m, 300 Å, Phenomenex, Milan, I aly) by means o a
20 min g adien using 0.1 % FA in wa e as he mobile phase A and 0.1% FA in ace oni ile
as he mobile phase B (30% B o 2 min; 30–50% B in 11 min; 50–95% B in 1 min; 95%B o
3 min; and hen 3 min a 30% B). An LC sys em coupled wi h a iple-quad upole mass
analyze (Finnigan TSQ Quan um Ul a, The moQues , Milan, I aly) equipped wi h an
An ioxidan s 2022,11, 1501 4 o 11
elec osp ay sou ce was se as epo ed by Al oma e e al. [
27
]. Xcalibu so wa e was
used o MS spec a acquisi ion. The ela i e abundance o HSA iso o ms, based on hei
co esponding peak a ea, was compu a ionally calcula ed by using MagT an so wa e,
designed o pe o m decon olu ion o he acqui ed mul i-cha ge spec a [27].
2.4. Regene a ion o Me cap oalbumin (HSA-SH)
Regene a ion o na i e albumin (me cap oalbumin, HSA-SH) was pe o med by ea -
ing human se um albumin (Albu ein
®
, G i ols I alia, Milan, I aly) wi h N-ace yl-cys eine
(NAC) (100
µ
g/mL) as p e iously desc ibed [
27
]. The eac ion mix u es we e incuba ed
o 1 h a 37
◦
C unde s i ing (85 pm) in a he momixe and hen dialyzed agains saline
solu ion (NaCl, 0.9%, pH 7) o emo e NAC. The amoun o HSA-HS was e alua ed by
mass spec ome y as desc ibed abo e.
2.5. Albumin Cys einyla ion
THIO-HSA was ob ained ollowing incuba ion o HSA (Albu ein
®
, G i ols I alia,
Milan, I aly) wi h 17 mmol/L o cys eine a 37
◦
C o 2 h. The solu ion was hen dialyzed
agains saline solu ion (NaCl, 0.9%, pH 7), and immedia ely used o incuba ion wi h cells.
The le el o S- hiola ion was checked by mass spec ome y as desc ibed abo e.
2.6. Cell T ea men wi h Albumin Iso o ms
Monocy ic U937 cells (Ame ican Type Tissue Cul u e Collec ion, Manassas, VA, USA)
we e cul u ed in RPMI (Eu oclone, Milan, I aly) and supplemen ed wi h 10% e al cal
se um (Eu oclone, Milan, I aly) a 37
◦
C in a humidi ied a mosphe e con aining 95% ai /5%
CO
2
. Be o e ea men , cells we e incuba ed in a se um- ee medium o 4 h and hen o
2 h wi h egene a ed me cap oalbumin (HSA-SH), S- hiola ed albumin (THIO-HSA), and
glyca ed HSA (GLY-HSA, Sigma-Ald ich, Milan, I aly, cod A8301).
2.7. Real-Time Quan i a i e Re e se T ansc ip ase PCR
To al cellula RNA was ex ac ed wi h he To al RNA Pu i ica ion Ki (No gen Bio ek
Co p.) and e e se ansc ibed (1
µ
g) as p e iously desc ibed [
28
]. The quali y o RNA
was checked by he Agilen 2100 Bioanalyze sys em. 18S ibosomal RNA was used as a
housekeeping gene o co ec o RNA le els inpu and e iciency o he eac ions. Real-
ime qRT-PCR was pe o med in iplica e wi h 2.5
µ
L o cDNA incuba ed in 22.5
µ
L IQ
Supe mix con aining p ime s and SYBRG een luo escence dye (Bio-Rad Labo a o ies,
Milan, I aly) using he iCycle Op ical Sys em (Bio-Rad Labo a o ies, Milan, I aly). The
sequences o p ime s used as no malize s we e: human 18S o wa d: 5
0
-CGG CTA CCA
CAT CCA AGG AA-3
0
; human 18S e e se: 5
0
-CCT GTA TTG TTA TTT TTC GTC ACT
ACC T-3
0
; TNF alpha Quan i ec P ime Assay om Qiagen (QT00029162). Exp ession
le els we e calcula ed by C alues no malized o 18S RNA.
2.8. TRAP Assay
Plasma an ioxidan ac i i y was e alua ed by means o he luo ime ic TRAP as-
say moni o ing he oxida ion o he subs a e 2
0
,7
0
-dichlo odihyd o luo escein diace a e
(DCFH2-DA) o 2,7 dichlo o luo escein (DCF), in he p esence o he adical ini ia o , AAPH,
as p e iously desc ibed [
15
]. The an ioxidan ac i i y was exp essed as he lag-phase (min)
induced be o e he subs a e oxida ion.
2.9. S a is ical Analysis
Con inuous a iables a e exp essed as he mean
±
s anda d de ia ion (SD). Compa i-
son among g oups was pe o med by one-way analysis o a iance (ANOVA) ollowed by
pos hoc Tukey’s hones signi icance es (HSD). The co ela ion be ween pa ame e s was
e alua ed by means o Pea son’s co ela ion coe icien . S a is ical signi icance was se a
p< 0.05.
An ioxidan s 2022,11, 1501 5 o 11
The sample size was chosen o gua an ee a powe o 80% on he de e mina ion o
signi ican di e ences equal o one s anda d de ia ion wi h an alpha alue o 0.05.
3. Resul s
The clinical and me abolic cha ac e is ics o he pa ien s unde s udy a e shown in
Figu e 1and Supplemen a y Table S1, e idencing in T2DM signi ican ly highe alues o
FPG and HbA1c wi h espec o non-diabe ic pa ien s wi h CHD and con ol subjec s. No
signi ican di e ences we e ound conce ning he LDL pa ame e be ween diabe ic and
non-diabe ic pa ien s wi h co ona y hea disease.
An ioxidan s 2022, 11, 1501 5 o 12
Con inuous a iables a e exp essed as he mean ± s anda d de ia ion (SD). Compa -
ison among g oups was pe o med by one-way analysis o a iance (ANOVA) ollowed
by pos hoc Tukey’s hones signi icance es (HSD). The co ela ion be ween pa ame e s
was e alua ed by means o Pea son’s co ela ion coe icien . S a is ical signi icance was
se a p < 0.05.
The sample size was chosen o gua an ee a powe o 80% on he de e mina ion o
signi ican di e ences equal o one s anda d de ia ion wi h an alpha alue o 0.05.
3. Resul s
The clinical and me abolic cha ac e is ics o he pa ien s unde s udy a e shown in
Figu e 1 and Supplemen a y Table S1, e idencing in T2DM signi ican ly highe alues o
FPG and HbA1c wi h espec o non-diabe ic pa ien s wi h CHD and con ol subjec s. No
signi ican di e ences we e ound conce ning he LDL pa ame e be ween diabe ic and
non-diabe ic pa ien s wi h co ona y hea disease.
Figu e 1. Clinical and me abolic da a e alua ed in he ou g oups o subjec s. In he box plo , he
ends o he box ep esen he 1s and 3 d qua ile, espec i ely. Each box is di ided by a line, which
ep esen s he median alue. The whiske s show he alues co esponding o [1s qua ile−1.5 ×
(in e qua ile ange)], and [3 d qua ile + 1.5 × (in e qua ile ange)]. Ho izon al blue lines on he
op pa o he g aph indica e pos hoc compa isons be ween g oups by Tukey’s HSD es , ollowing
signi ican ANOVA. BMI, body mass index; CHD, co ona y hea disease; FPG, as ing plasma glu-
cose; HbA1c, glyca ed hemoglobin. *** p < 0.001; ** p < 0.01; * p < 0.05.
In Table 1 and Figu e 2, he ad anced glyca ion and he oxida ion pa ame e s e alu-
a ed a e epo ed, showing ha mean alues o AGE and pen osidine a e signi ican ly
highe in T2DM pa ien s wi h and wi hou CHD wi h espec o non-diabe ic pa ien s wi h
CHD and con ol subjec s; no di e ences among he wo classes o diabe ic pa ien s we e
ound. RAGE le els a e signi ican ly highe in T2DM pa ien s wi h espec o non-diabe ic
pa ien s. Among T2DM pa ien s, he g oup wi h CHD showed signi ican ly highe RAGE
Figu e 1.
Clinical and me abolic da a e alua ed in he ou g oups o subjec s. In he box plo ,
he ends o he box ep esen he 1s and 3 d qua ile, espec i ely. Each box is di ided by a
line, which ep esen s he median alue. The whiske s show he alues co esponding o [1s
qua ile
−
1.5
×
(in e qua ile ange)], and [3 d qua ile + 1.5
×
(in e qua ile ange)]. Ho izon al blue
lines on he op pa o he g aph indica e pos hoc compa isons be ween g oups by Tukey’s HSD
es , ollowing signi ican ANOVA. BMI, body mass index; CHD, co ona y hea disease; FPG, as ing
plasma glucose; HbA1c, glyca ed hemoglobin. *** p< 0.001; ** p< 0.01; * p< 0.05.
In Table 1and Figu e 2, he ad anced glyca ion and he oxida ion pa ame e s e alua ed
a e epo ed, showing ha mean alues o AGE and pen osidine a e signi ican ly highe
in T2DM pa ien s wi h and wi hou CHD wi h espec o non-diabe ic pa ien s wi h CHD
and con ol subjec s; no di e ences among he wo classes o diabe ic pa ien s we e ound.
RAGE le els a e signi ican ly highe in T2DM pa ien s wi h espec o non-diabe ic pa ien s.
Among T2DM pa ien s, he g oup wi h CHD showed signi ican ly highe RAGE le els han
ha wi hou CHD. Fu he mo e, sRAGE le els we e simila in co ona opa hic pa ien s
wi h and wi hou diabe es melli us, and highe han in con ol subjec s.
An ioxidan s 2022,11, 1501 6 o 11
Table 1. Glyca ion and oxida ion pa ame e s e alua ed in he ou g oups o subjec s.
Pa ame e
A: Diabe ics
wi h CHD
(n= 33)
B: Diabe ics
wi hou CHD
(n= 31)
C: Non-diabe ics
wi h CHD
(n= 30)
D: Con ols
(n= 27)
AGE (µg/mL) 7.8 ±4.8 a6.7 ±5.8 a2.6 ±2.8 b2.3 ±0.8 b
Pen osidine
(nmol/mL) 0.84 ±0.24 a0.78 ±0.23 a0.62 ±0.22 b0.47 ±0.08 c
sRAGE (ng/mL) 0.95 ±0.50 a0.62 ±0.22 b0.79 ±0.42 ab 0.31 ±0.07 c
RAGE (pg/mL) 217 ±171 a140 ±61 b54 ±33 c51 ±13 c
GLY-HSA (%) 13.2 ±1.6 a13.1 ±1.7 a12.1 ±0.7 b12.02 ±0.9 b
THIO-HSA (%) 21.8 ±2.9 a22.4 ±2.9 a25.3 ±3.7 b20.98 ±3.4 a
AGE, ad anced glyca ion end p oduc s; GLY-HSA, Glyca ed albumin; RAGE, ecep o o ad anced glyca ion
end-p oduc s; sRAGE, soluble RAGE; THIO-HSA, hiola ed albumin; CHD, co ona y hea disease. Da a a e
exp essed as mean
±
SD. Fo each a iable, means o g oups no sha ing any supe sc ip le e a e signi ican ly
di e en by Tukey’s HSD es a he 5% le el o signi icance. Figu e 2indica es g aphically he co esponding
pai ed compa isons.
An ioxidan s 2022, 11, 1501 7 o 12
Figu e 2. Glyca ion and oxida ion pa ame e s e alua ed in he ou g oups o subjec s. In he box
plo , he ends o he box ep esen he 1s and 3 d qua ile, espec i ely. Each box is di ided by a
line, which ep esen s he median alue. The whiske s show he alues co esponding o [1s qua -
ile − 1.5 × (in e qua ile ange)], and [3 d qua ile + 1.5 × (in e qua ile ange)]. Ho izon al blue lines
on he op pa o he g aph indica e pos hoc compa isons be ween g oups by Tukey’s HSD es ,
ollowing signi ican ANOVA. AGE, ad anced glyca ion end p oduc s; GLY-HSA, glyca ed albu-
min; RAGE, ecep o o ad anced glyca ion end-p oduc s; sRAGE, soluble RAGE; THIO-HSA, hi-
ola ed albumin; CHD, co ona y hea disease *** p < 0.001; ** p < 0.01; * p < 0.05.
When conside ing all he pa ien s, posi i e signi ican co ela ions we e ound be-
ween AGE and FPG, HbA1c, RAGE ( 0.29, p = 0.004; 0.28, p = 0.007; 0.38, p = 0.0002;
espec i ely), bu no o sRAGE. A posi i e co ela ion was also ound be ween RAGE
and sRAGE ( 0.59, p < 0.0001), and be ween GLY-HSA and FPG and HbA1c ( 0.37, p =
0.0002; and 0.49, p < 0.0001, espec i ely) (Figu e 3). Fu he mo e, signi ican nega i e
Figu e 2.
Glyca ion and oxida ion pa ame e s e alua ed in he ou g oups o subjec s. In he box
plo , he ends o he box ep esen he 1s and 3 d qua ile, espec i ely. Each box is di ided by a line,
An ioxidan s 2022,11, 1501 7 o 11
which ep esen s he median alue. The whiske s show he alues co esponding o [1s qua -
ile
−
1.5
×
(in e qua ile ange)], and [3 d qua ile + 1.5
×
(in e qua ile ange)]. Ho izon al blue
lines on he op pa o he g aph indica e pos hoc compa isons be ween g oups by Tukey’s HSD es ,
ollowing signi ican ANOVA. AGE, ad anced glyca ion end p oduc s; GLY-HSA, glyca ed albumin;
RAGE, ecep o o ad anced glyca ion end-p oduc s; sRAGE, soluble RAGE; THIO-HSA, hiola ed
albumin; CHD, co ona y hea disease *** p< 0.001; ** p< 0.01; * p< 0.05.
GLY-HSA le els we e highe in T2DM wi h and wi hou CHD wi h espec o non-
diabe ic pa ien s wi h CHD and con ol subjec s. Finally, THIO-HSA le els we e highe
in non-diabe ic pa ien s wi h CHD wi h espec o T2DM wi h and wi hou CHD and
con ol subjec s.
When conside ing all he pa ien s, posi i e signi ican co ela ions we e ound be-
ween AGE and FPG, HbA1c, RAGE ( 0.29, p= 0.004; 0.28, p= 0.007; 0.38, p= 0.0002;
espec i ely), bu no o sRAGE. A posi i e co ela ion was also ound be ween RAGE and
sRAGE ( 0.59, p< 0.0001), and be ween GLY-HSA and FPG and HbA1c ( 0.37, p= 0.0002;
and 0.49, p< 0.0001, espec i ely) (Figu e 3). Fu he mo e, signi ican nega i e co ela ions
we e ound be ween THIO-HSA and FPG, HbA1c, and GLY-HSA (Supplemen a y Table S2).
Conside ing sepa a ely each g oup a posi i e co ela ion was ound be ween AGE and
THIO-HSA only in T2DM wi h CHD ( 0.38, p= 0.029) (Supplemen a y Table S2).
An ioxidan s 2022, 11, 1501 8 o 12
co ela ions we e ound be ween THIO-HSA and FPG, HbA1c, and GLY-HSA (Supple-
men a y Table S2). Conside ing sepa a ely each g oup a posi i e co ela ion was ound
be ween AGE and THIO-HSA only in T2DM wi h CHD ( 0.38, p = 0.029) (Supplemen a y
Table S2).
Figu e 3. Sca e plo o a iables p esen ing he mos ele an co ela ions among hose epo ed
in Supplemen a y Table S2. Each plo shows a i ed linea eg ession line wi h a shaded 95% con i-
dence in e al. GLY-HSA, glyca ed albumin; RAGE, ecep o o ad anced glyca ion end-p oduc s;
sRAGE, soluble RAGE; HbA1c, glyca ed hemoglobin.
To e i y i he age di e ence may ha e somehow in luenced he esul s, we ha e
s a i ied he heal hy con ol g oup D by age, acco ding o younge /olde subjec s wi h a
cu -o o 50 yea s. Compa able alues o all he pa ame e s o glyco-oxida ion (no signi -
ican di e ence) a e ound, con i ming ha age by i sel is no a con ounding ac o in ou
model.
In o de o assess he po en ial con ibu ion o he di e en albumin iso o ms in he
cell in lamma o y s a e, he monocy ic cell line U937 was incuba ed wi h HSA-SH, THIO-
HSA, and GLY-HSA, and he exp ession le els o he p o-in lamma o y cy okine umo
nec osis ac o -alpha (TNFα) was e alua ed by eal- ime qRT-PCR. Mass spec ome y
analysis o HSA a e egene a ion wi h NAC, and ea men wi h cys eine, e ealed ha
in he egene a ed albumin HSA-SH was he p edominan o m (88.3%), cys eine- ea ed
HSA con ained 31.1% o THIO-HSA, and GLY-HSA was cha ac e ized by a highe pe -
cen age o glyca ed albumin (49.7%) (Figu e 4A). The incuba ion o U937 cells wi h HSA
edox iso o ms, THIO-HSA and GLY-HSA, signi ican ly induced he mRNA le els o
TNFα wi h espec o HSA-SH (Figu e 4B).
In addi ion, we e idenced a signi ican nega i e co ela ion be ween he pe cen age
o THIO-HSA and he o al an ioxidan ac i i y in plasma, measu ed by means o he
TRAP assay in e ms o lag ime be o e subs a e oxida ion (Figu e 4C).
Figu e 3.
Sca e plo o a iables p esen ing he mos ele an co ela ions among hose epo ed in
Supplemen a y Table S2. Each plo shows a i ed linea eg ession line wi h a shaded 95% con idence
in e al. GLY-HSA, glyca ed albumin; RAGE, ecep o o ad anced glyca ion end-p oduc s; sRAGE,
soluble RAGE; HbA1c, glyca ed hemoglobin.
To e i y i he age di e ence may ha e somehow in luenced he esul s, we ha e
s a i ied he heal hy con ol g oup D by age, acco ding o younge /olde subjec s wi h
a cu -o o 50 yea s. Compa able alues o all he pa ame e s o glyco-oxida ion (no
signi ican di e ence) a e ound, con i ming ha age by i sel is no a con ounding ac o in
ou model.
In o de o assess he po en ial con ibu ion o he di e en albumin iso o ms in he cell
in lamma o y s a e, he monocy ic cell line U937 was incuba ed wi h HSA-SH, THIO-HSA,
and GLY-HSA, and he exp ession le els o he p o-in lamma o y cy okine umo nec osis
ac o -alpha (TNF
α
) was e alua ed by eal- ime qRT-PCR. Mass spec ome y analysis
o HSA a e egene a ion wi h NAC, and ea men wi h cys eine, e ealed ha in he
egene a ed albumin HSA-SH was he p edominan o m (88.3%), cys eine- ea ed HSA
con ained 31.1% o THIO-HSA, and GLY-HSA was cha ac e ized by a highe pe cen age
o glyca ed albumin (49.7%) (Figu e 4A). The incuba ion o U937 cells wi h HSA edox
iso o ms, THIO-HSA and GLY-HSA, signi ican ly induced he mRNA le els o TNF
α
wi h
espec o HSA-SH (Figu e 4B).
An ioxidan s 2022,11, 1501 8 o 11
An ioxidan s 2022, 11, 1501 9 o 12
Figu e 4. Albumin iso o ms and unc ional e ec s. (A) Mass spec ome y analysis o egene a ed
HSA (na i e HSA-SH), highly glyca ed albumin (GLY-HSA), and highly hiola ed albumin (THIO-
HSA). (B) TNFα mRNA le els, no malized o he housekeeping gene 18S RNA, in U937 cells incu-
ba ed wi h na i e HSA-SH, GLY-HSA, and THIO-HSA 100µg/mL o 2 h. ** p < 0.01 s HSA-SH. (C)
Co ela ion o THIO-HSA and plasma an ioxidan ac i i y measu ed by means o TRAP assay. Plo
shows i ed linea eg ession line wi h shaded 95% con idence in e al.
4. Discussion
Bo h glyco-oxida ion pa ame e s, AGE, and pen osidine, co ela e wi h FPG and
HbA1c and a e ele a ed only in T2DM pa ien s wi h and wi hou CHD, and appea o
induce g ea e plaque RAGE exp ession in diabe ic pa ien s wi h CHD, as p e iously de-
sc ibed in a pos -mo em s udy [29]. Fu he mo e, he posi i e co ela ion o THIO-HSA
wi h AGE in T2DM wi h CHD would sugges a possible syne gic e ec o glyco-oxida i e
damage wi h a educed HSA an ioxidan ac i i y on he ascula wall.
The pool o sRAGE was ound una ec ed by he p esence o diabe es and AGE le els,
while i esul ed o be ele a ed in bo h T2DM and non-diabe ics wi h CHD, sugges ing
ha plasma le els o sRAGE may be associa ed wi h plaque ulne abili y, in pa ien s wi h
CHD, independen ly om RAGE le els, acco ding o p e ious s udies [30,31].
In ou s udy, we e idenced highe le els o GLY-HSA in T2DM in compa ison o
non-diabe ics, ega dless o he p esence o absence o ch onic ca dio ascula complica-
ions, in line wi h i s po en ial ole as a clinical bioma ke o glycemic con ol. An in-
c eased GLY-HSA con en has been ecen ly de ec ed in HF pa ien s wi h espec o age-
ma ched con ol subjec s, by Ma inez Fe nandez e al. wi h highe le els o GLY-HSA in
subjec s wi h he mos se e e HF [19]. The au ho s also demons a ed he p o-in lamma-
o y and p o-oxidan e ec s o GLY-HSA on mu ine HL-1 ca diomyocy es. Based on hese
ecen indings, GLY-HSA migh be conside ed no only a po en ial bioma ke bu also a
unc ional media o o disease p og ession.
In a p e ious s udy [15], we ound ha THIO-HSA, de i ed om he binding o bo h
cys eine and homocys eine o HSA, is inc eased in he plasma o pa ien s wi h HF. The
inding ha he le els o THIO-HSA co ela e wi h an al e a ion o he plasma an ioxidan
ac i i y, sugges s ha S- hiola ion induced s uc u al and unc ional changes in HSA
which likely con ibu e o he p og ession o he disease. Indeed, we ha e also p o ided
Figu e 4.
Albumin iso o ms and unc ional e ec s. (
A
) Mass spec ome y analysis o egene a ed
HSA (na i e HSA-SH), highly glyca ed albumin (GLY-HSA), and highly hiola ed albumin (THIO-
HSA). (
B
) TNF
α
mRNA le els, no malized o he housekeeping gene 18S RNA, in U937 cells
incuba ed wi h na i e HSA-SH, GLY-HSA, and THIO-HSA 100
µ
g/mL o 2 h. ** p< 0.01 s HSA-SH.
(
C
) Co ela ion o THIO-HSA and plasma an ioxidan ac i i y measu ed by means o TRAP assay.
Plo shows i ed linea eg ession line wi h shaded 95% con idence in e al.
In addi ion, we e idenced a signi ican nega i e co ela ion be ween he pe cen age o
THIO-HSA and he o al an ioxidan ac i i y in plasma, measu ed by means o he TRAP
assay in e ms o lag ime be o e subs a e oxida ion (Figu e 4C).
4. Discussion
Bo h glyco-oxida ion pa ame e s, AGE, and pen osidine, co ela e wi h FPG and
HbA1c and a e ele a ed only in T2DM pa ien s wi h and wi hou CHD, and appea o
induce g ea e plaque RAGE exp ession in diabe ic pa ien s wi h CHD, as p e iously
desc ibed in a pos -mo em s udy [
29
]. Fu he mo e, he posi i e co ela ion o THIO-HSA
wi h AGE in T2DM wi h CHD would sugges a possible syne gic e ec o glyco-oxida i e
damage wi h a educed HSA an ioxidan ac i i y on he ascula wall.
The pool o sRAGE was ound una ec ed by he p esence o diabe es and AGE le els,
while i esul ed o be ele a ed in bo h T2DM and non-diabe ics wi h CHD, sugges ing
ha plasma le els o sRAGE may be associa ed wi h plaque ulne abili y, in pa ien s wi h
CHD, independen ly om RAGE le els, acco ding o p e ious s udies [30,31].
In ou s udy, we e idenced highe le els o GLY-HSA in T2DM in compa ison o non-
diabe ics, ega dless o he p esence o absence o ch onic ca dio ascula complica ions,
in line wi h i s po en ial ole as a clinical bioma ke o glycemic con ol. An inc eased
GLY-HSA con en has been ecen ly de ec ed in HF pa ien s wi h espec o age-ma ched
con ol subjec s, by Ma inez Fe nandez e al. wi h highe le els o GLY-HSA in subjec s
wi h he mos se e e HF [
19
]. The au ho s also demons a ed he p o-in lamma o y and
p o-oxidan e ec s o GLY-HSA on mu ine HL-1 ca diomyocy es. Based on hese ecen
indings, GLY-HSA migh be conside ed no only a po en ial bioma ke bu also a unc ional
media o o disease p og ession.
An ioxidan s 2022,11, 1501 9 o 11
In a p e ious s udy [
15
], we ound ha THIO-HSA, de i ed om he binding o bo h
cys eine and homocys eine o HSA, is inc eased in he plasma o pa ien s wi h HF. The
inding ha he le els o THIO-HSA co ela e wi h an al e a ion o he plasma an ioxidan
ac i i y, sugges s ha S- hiola ion induced s uc u al and unc ional changes in HSA which
likely con ibu e o he p og ession o he disease. Indeed, we ha e also p o ided e idence
ha THIO-HSA is no p o ec i e agains he cy o oxic e ec o oxidan hyd ogen pe oxide
in ca diomyocy es HL-1. Thus, we hypo hesized ha THIO-HSA migh also ep esen a
po en ial causal ac o in HF p og ession.
The esul s o his s udy sugges ed ha THIO-HSA in non-diabe ic pa ien s wi h
CHD, and no in T2DM wi h/wi hou CHD, could ep esen a disease ma ke , simila ly
o HF p e iously desc ibed. In T2DM, he e idence ha THIO-HSA le els we e in e sely
co ela ed wi h sho (FPG, GLY-HSA), and long- e m (HbA1c) me abolic compensa ion
may mask he THIO-HSA ole as a ma ke o CHD in his popula ion. On he o he hand,
he posi i e co ela ion o THIO-HSA wi h AGE in T2DM wi h CHD would sugges a
possible syne gic e ec o glyco-oxida i e damage on he ascula wall.
Finally, we demons a ed ha bo h THIO-HSA and GLY-HSA can inc ease he exp es-
sion o TNF
α
, one o he mos impo an p o-in lamma o y cy okines, in he monocy ic cell
line U937, hus suppo ing he hypo hesis ha pos - ansla ional modi ica ions occu ing
on HSA can con ibu e o he p og ession o he disease. Al hough his inding is con i -
ma o y o p e ious s udies on he e ec s o GLY-HSA [
32
], he p o-in lamma o y ac i i y
o THIO-HSA is o no el y. Indeed, besides ou p e ious s udy on HF [
15
] and a s udy
on hype lipidemic subjec s [
33
], he le els o THIO-HSA in T2DM wi h and wi hou CHD
ha e ne e been add essed. Reg e ably, he comme cial GLY-HSA con ains a signi ican
amoun o THIO-HSA; hus, we canno ule ou he speci ic con ibu ion o he GLY-HSA
alone. On he o he hand, we canno demons a e ha he
in i o
p oposed mechanism
is ope a i e
in i o
, e en i we used HSA wi h a pe cen age o na i e HSA simila o ha
measu ed in pa ien s and associa ed wi h a educed o al plasma an ioxidan ac i i y.
A limi a ion o he p esen in es iga ion is he obse a ional na u e o he da a ha
de i e om subjec s a ending he local ou pa ien diabe es clinic, and he e o e is ep esen-
a i e o a limi ed geog aphical a ea. The coho o conside ed subjec s, howe e , e alua ed
wi h a p io i powe analysis, is cha ac e ized by a small sample size, sugges ing ha he
ob ained esul s should be add essed as p elimina y obse a ions; indeed, he signi icance
ob ained wi h he p esen da a appea s clea , al hough i may dese e a con i ma ion wi h
la ge sample size. Ano he limi a ion is he lack o some subjec cha ac e is ics such as he
du a ion o disease in pa ien s wi h diabe es. As ega ds he co ela ions be ween me abolic
and glyco-oxida ion pa ame e s, in pa icula hose linked o HSA glyca ion and hiola ion,
a la ge sample size would be needed o be e de ine he ype o ela ionships.
In conclusion, in pa ien s wi h CHD, HSA glyca ion and S- hiola ion ha e a di e en
p e alen bu den, wi h GLY-HSA p e ailing in pa ien s wi h T2DM and THIO-HSA in
pa ien s wi hou T2DM. In pa icula , in T2DM pa ien s, dele e ious ascula e ec s could
be o igina ed om a complemen a y mechanism o ac ion ha includes highe le els o
oxida i e s ess bioma ke s, such as AGE, and he loss o albumin an ioxidan capaci y,
sugges ing how impo an is o ake in o conside a ion also albumin quali y o he main e-
nance o homeos asis balance be ween glyco-oxida ion and an ioxidan de ense sys em.
Supplemen a y Ma e ials:
The ollowing suppo ing in o ma ion can be downloaded a : h ps://
www.mdpi.com/a icle/10.3390/an iox11081501/s1, Table S1. Clinical and me abolic da a e alua ed
in he ou g oups o subjec s. Table S2. Signi ican co ela ions be ween he me abolic and glyco-
oxida ion pa ame e s e alua ed conside ing all he pa ien s (g oups A, B, and C; n= 94).
Au ho Con ibu ions:
Concep ualiza ion, A.L., C.B. and G.S.; in es iga ion, F.P., A.A., C.C., E.R. and
M.B.; esou ces, A.L., A.A. and C.B.; da a cu a ion, E.R., A.L. and M.B.; o mal analysis, A.A., E.R.;
w i ing—o iginal d a p epa a ion, A.L., F.P., C.B., G.S. and E.R.; w i ing— e iew and edi ing, all
au ho s; isualiza ion, F.P., A.L. and E.R.; supe ision, A.L.; p ojec adminis a ion, A.L., F.P., G.S. and
