Resea ch A icle
Associa ions be ween Speci ic Redox Bioma ke s and Age in a
La ge Eu opean Coho : The MARK-AGE P ojec
Daniela Webe ,
1,2
Wol gang S ue z,
3
Oli ie Toussain ,
4
Flo ence Debacq-Chainiaux,
4
Ma ijn E. T. Dollé,
5
Eugène Jansen,
5
E s a hios S. Gonos,
6
Claudio F anceschi,
7
Ewa Siko a,
8
An i He onen,
9
Nicolle B eusing,
3,10
Thilo Sindlinge ,
11
Ma ía Mo eno-Villanue a,
11
Alexande Bü kle,
11
and Tilman G une
1,2,12,13
1
Depa men o Molecula Toxicology, Ge man Ins i u e o Human Nu i ion Po sdam-Rehb uecke (DI E), 14558 Nu he al, Ge many
2
Nu iAc -Compe ence Clus e Nu i ion Resea ch Be lin-Po sdam, 14458 Nu he al, Ge many
3
Ins i u e o Biological Chemis y and Nu i ion, Uni e si y o Hohenheim, 70599 S u ga , Ge many
4
URBC-NARILIS, Uni e si y o Namu , 5000 Namu , Belgium
5
Na ional Ins i u e o Public Heal h and he En i onmen (RIVM), 3720 BA Bil ho en, Ne he lands
6
Ins i u e o Biological Resea ch and Bio echnology, Na ional Hellenic Resea ch Founda ion (NHRF), 11635 A hens, G eece
7
Depa men o Expe imen al Pa hology, Uni e si y o Bologna, 40126 Bologna, I aly
8
Nencki Ins i u e o Expe imen al Biology, Polish Academy o Sciences, 02-093 Wa saw, Poland
9
School o Medicine, Uni e si y o Tampe e, 33014 Tampe e, Finland
10
Depa men o Applied Nu i ional Science/Die e ics, Ins i u e o Nu i ional Medicine, Uni e si y o Hohenheim,
70599 S u ga , Ge many
11
Molecula Toxicology, Depa men o Biology, Uni e si y o Kons anz, 78457 Kons anz, Ge many
12
Ge man Cen e o Diabe es Resea ch (DZD), 85764 Munich-Neuhe be g, Ge many
13
Ge man Cen e o Ca dio ascula Resea ch (DZHK), 13357 Be lin, Ge many
Co espondence should be add essed o Daniela Webe ; [email p o ec ed]
Recei ed 24 Feb ua y 2017; Re ised 29 May 2017; Accep ed 15 June 2017; Published 19 July 2017
Academic Edi o : Daniela Gius a ini
Copy igh © 2017 Daniela Webe e al. This is an open access a icle dis ibu ed unde he C ea i e Commons A ibu ion License,
which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Oxida i e s ess and an ioxidan s play a ole in age- ela ed diseases and in he aging p ocess. We he e p esen da a on p o ein
ca bonyls, 3-ni o y osine, malondialdehyde, and cellula and plasma an ioxidan s (glu a hione, cys eine, asco bic acid, u ic acid,
α- ocophe ol, and lycopene) and hei ela ion wi h age in he Eu opean mul icen e s udy MARK-AGE. To a oid con ounding,
only da a om coun ies which ec ui ed subjec s om all h ee s udy g oups (fi e o eigh cen e s) and only pa icipan s aged
≥55 yea s we e selec ed esul ing in da a om 1559 pa icipan s. These included subjec s om (1) he gene al popula ion, (2)
membe s om long-li ing amilies, and (3) hei spouses. In addi ion, 683 middle-aged e e ence pa icipan s (35–54 yea s)
se ed as a con ol. A e adjus men o age, BMI, smoking s a us, gende , and coun y, he e we e diffe ences in p o ein
ca bonyls, malondialdehyde, 3-ni o y osine, α- ocophe ol, cys eine, and glu a hione be ween he 3 s udy g oups. P o ein
ca bonyls and 3-ni o y osine as well as cys eine, u ic acid, and lycopene we e iden ified as independen bioma ke s wi h he
highes co ela ion wi h age. In e es ingly, om all an ioxidan s measu ed, only lycopene was lowe in all aged g oups and om
he oxida i e s ess bioma ke s, only 3-ni o y osine was inc eased in he descendan s om long-li ing amilies compa ed o he
middle-aged con ol g oup. We conclude ha bo h li es yle and gene ics may be impo an con ibu o s o edox bioma ke s in
an aging popula ion.
Hindawi
Oxida i e Medicine and Cellula Longe i y
Volume 2017, A icle ID 1401452, 12 pages
h ps://doi.o g/10.1155/2017/1401452
1. In oduc ion
Oxida i e s ess and an ioxidan s ha e been in he ocus o
esea ch o decades due o hei associa ion wi h nume ous
age- ela ed diseases [1–4]. In humans, ele a ed le els o
oxida i e s ess ha e been epo ed in se e al medical condi-
ions, including neu odegene a i e diseases [2], obesi y,
diabe es melli us, and he aging p ocess i sel [3–5]. The e
is some epidemiological e idence on he ole o oxida i e
s ess in aging, age- ela ed diseases, and mo ali y [6–10].
Howe e , hese s udies ocused mos ly on single bioma ke s
o on bioma ke s which a e no widely used, hus making i
difficul o compa e esul .
When a emp ing o assess edox bioma ke s, i is
impo an o no e ha he e is no one single bioma ke
which is conside ed a “gold s anda d.”In ac , i is ecom-
mended o measu e a se o diffe en bioma ke s [11].
Thus, ou a emp was o analyze ma ke s o p o ein oxida-
ion, ni a ion, lipid pe oxida ion, and cellula and plasma
an ioxidan s and s udy hei ela ion wi h age in pa icipan s
o he MARK-AGE p ojec . The MARK-AGE p ojec was a
Eu opean mul icen e s udy, suppo ed by he Eu opean
Commission, aiming o iden i y bioma ke s o human aging
and o model a obus se o ma ke s o biological age and
heal hy aging [12].
Fo his pu pose, women and men we e ec ui ed om
he gene al popula ion om eigh Eu opean coun ies as
age-s a ified subg oups, as well as subjec s belonging o a
amily wi h long-li ing membe s oge he wi h hei spouses.
In diffe en biological ma ices (whole blood, se um, plasma,
u ine, buccal mucosa cells, and pe iphe al blood mononu-
clea cells), a la ge numbe o candida e bioma ke s o aging,
including DNA-based ma ke s, ma ke s based on p o eins
and hei modifica ions, immunological ma ke s, clinical
chemis y, ho mones and ma ke s o me abolism, oxida i e
s ess ma ke s, and an ioxidan mic onu ien s we e
assessed. The p ojec design including de ails o he s udy
popula ion and s anda d ope a ing p ocedu es ha e been
published ecen ly [12–15].
The objec i es o he p esen wo k we e o assess and o
compa e le els o edox bioma ke s wi hin he h ee diffe en
s udy g oups o he MARK-AGE p ojec . Ou hypo heses
we e (1) ha oxida i e s ess is ele a ed in highe age g oups
as i has been shown in diffe en a he small popula ion-
based s udies in diffe en biological ma ices and (2) ha
subjec s om amilies wi h long-li ing membe s would be
gene ically be e equipped o handle oxida i e s ess han
he gene al popula ion. The esul s o he spouses migh
show whe he (3) a sha ed li es yle may be able o influence
bioma ke concen a ions.
2. Ma e ials and Me hods
This s udy was conduc ed in acco dance wi h he Decla a ion
o Helsinki (1964) and wi h in o med w i en consen o each
pa icipan . E hical clea ance had been gi en by he e hics
commi ee o each o he ec ui ing cen e s. This s udy has
been egis e ed e ospec i ely a he Ge man Clinical T ials
Regis e (DRKS00007713).
2.1. S udy Popula ion and Sample Collec ion. Fo he whole
MARK-AGE p ojec , 3158 pa icipan s we e ec ui ed in 8
ec ui ing cen e s and he de ails o he h ee s udy g oups
will be desc ibed b iefly below.
The fi s s udy g oup was ec ui ed h ough a ious
public pla o ms such as adio and newspape ad e ising.
These pa icipan s we e included in he RASIG g oup
( ec ui ed om he age-s a ified gene al popula ion). Fo
his g oup, he main inclusion c i e ia we e he abili y o gi e
in o med consen and being in he age- ange om 35 o 75
yea s (bo h gende s).
The second s udy g oup consis ed o 537 descendan s
om long-li ing subjec s (nonagena ians; pe sons who
eached he age o 90) who had been ec ui ed as a ollow-
up om he GEHA s udy (Gene ics o Heal hy Aging;
2004–2009, o de ails, see [16]); his g oup is abb e ia ed
as GO (GEHA offsp ing). The hi d s udy g oup consis ed
o he spouses o he GEHA offsp ing (n= 311) and se ed
as a li es yle con ol g oup, he so-called SGO (spouses o
GO). The GO and SGO pa icipan s we e be ween he age
o 55 and 75 yea s [12] and we e ec ui ed in Belgium,
Finland, G eece, I aly, The Ne he lands, and Poland.
Collec ion o an h opome ic da a, ques ionnai e da a,
and da a on cogni i e unc ion was ca ied ou by ained
nu ses o physicians be ween No embe 2008 and June
2012 a he ollowing ec ui ing cen e s: Hall in Ti ol/Inns-
b uck (Aus ia), Namu (Belgium), Esslingen (Ge many),
A hens, and o he nea by egions (G eece), Bologna (I aly),
Wa saw (Poland), Tampe e (Finland), and Leiden (The
Ne he lands). Fu he mo e, pa icipan s we e asked o
comple e ques ionnai es on li es yle cha ac e is ic (nu i ion,
smoking habi s), amily his o y, and li ing en i onmen .
To a oid possible con ounding, we used only da a om
hose coun ies which ec ui ed subjec s om all h ee s udy
g oups (RASIG, GO, and SGO). This was he case in fi e o
he eigh cen e s (Belgium, G eece, I aly, Poland, and
Finland). Ge many and Aus ia did no p o ide GO o
SGO da a while The Ne he lands did no ec ui RASIG
pa icipan s; he e o e, da a om hese h ee coun ies we e
excluded in he p esen analyses.
Since he age anges o pa icipan s in he GO and SGO
g oups we e diffe en o he RASIG g oup in gene al (55–
75 e sus 35–75 yea s, esp.), only pa icipan s who we e
≥55 yea s o age we e selec ed. This esul ed in a o al o
1559 pa icipan s in all h ee s udy g oups o be included in
he p esen wo k. Addi ionally, o compa e hese s udy
g oups o a younge (middle-aged) e e ence g oup, we
selec ed all RASIG pa icipan s aged 35–54 (“middle aged”)
om Belgium, G eece, I aly, Poland, and Finland, which
esul ed in a o al o 683 e e ence pa icipan s.
2.2. De e mina ion o To al Glu a hione and To al F ee
Cys eine in Whole Blood. To al glu a hione and o al ee
cys eine in whole blood we e measu ed as p e iously
desc ibed by Chen e al. [17] by using Ellman’s eagen
(5,5′
-di hiobis-[2-ni obenzoic acid]) a e he educ ion
o any disulfides p esen . The modifica ions ega ding
he educ ion agen DTT (1,4-di hio h ei ol), he adap ion
o whole blood samples, and HPLC condi ions we e as
2 Oxida i e Medicine and Cellula Longe i y
ollows: Whole blood (100 μL) was o ex-mixed wi h
DTT (12.5 mM, 100 μL) and incuba ed o 3 min; cold i-
chlo oace ic acid solu ion (10% w/ , 200 μL) was added;
and samples we e ho oughly mixed and cen i uged a
19,500 ×g o 5 min a 4
°
C. The clea supe na an (200 μL)
was hen added o Ellman’s eagen (30 mM, 50 μL) oge he
wi h di-po assium hyd ogen phospha e buffe (2 M, 100 μL),
and o ex-mixed. Twen y μL was analyzed on a Shimadzu
P ominence HPLC (LC-20A) equipped wi h an UV-Vis
de ec o (SPD-20AV se a 326 nm). The sepa a ion o cys e-
ine and glu a hione was achie ed by using a Rep osil-Pu 120
C18 AQ column (5 μm, 250 mm ×4.6 mm; D . Maisch,
Ge many) se o 40
°
C and a mobile phase consis ing o me h-
anol (15% / ) and ace a e buffe (0.05 M, pH 5) a a flow a e
o 1 mL/min. S anda ds dilu ed o physiological concen a-
ions (62.5–250 μM o cys eine and 500–2000 μM o glu a-
hione) and ea ed as a sample we e used o quan ifica ion.
2.3. De e mina ion o Asco bic Acid and U ic Acid in Plasma.
Plasma asco bic acid and u ic acid we e analyzed by
RP-HPLC and UV de ec ion a e educ ion wi h is-
(2-ca boxye hyl)-phosphine [18]. B iefly, plasma (100 μL)
was mixed wi h is-(2-ca boxye hyl)-phosphine (20% w/w,
25 μL) and incuba ed o 5 min on ice; hen, eshly p epa ed
me aphospho ic acid solu ion (10% w/w,75μL) was added
and o ex-mixed; and samples we e cen i uged a 19,500 ×g
and 4
°
C o 10 min. Twen y μL o clea supe na an was
analyzed on a Shimadzu P ominence HPLC and using a
5μm analy ical column (Rep osil-Pu 120 C18 AQ,
250 mm ×4.6 mm; D . Maisch, Ge many) se o 40
°
C, a
mobile phase consis ing o 0.05 M sodium phospha e buffe
(pH 2.5) a a flow a e o 1 mL/min and UV-Vis de ec o
(SPD-20AV) se o 245 nm. Pu e s anda ds dilu ed o
physiological concen a ions (2.5–20 mg/L o asco bic acid
and 20–100 mg/L o u ic acid) and ea ed as a sample we e
used o quan ifica ion.
2.4. Malondialdehyde. Plasma malondialdehyde was de e -
mined by RP-HPLC coupled wi h fluo escence de ec ion
a e de i a iza ion wi h hioba bi u ic acid as desc ibed by
Wong e al. [19] wi h modifica ions [20].
2.5. Analysis o P o ein Ca bonyls and 3-Ni o y osine.
The analyses o p o ein ca bonyls [21] and 3-ni o y osine
in plasma by in-house ELISA ha e been desc ibed
elsewhe e [20].
2.6. Analysis o α-Tocophe ol and Lycopene. Plasma lycopene
and α- ocophe ol we e analyzed by RP-HPLC coupled
wi h UV- is and fluo escence de ec ion as p e iously
desc ibed [22].
2.7. S a is ical Analysis. Demog aphic cha ac e is ics a e
desc ibed by using means ±s anda d de ia ion (SD) o
con inuous a iables (age, weigh , and BMI) and equencies
(%) o ca ego ical a iables (gende , smoking s a us, age
g oups, and coun y). Diffe ences in cha ac e is ics be ween
age g oups and s udy g oups we e compa ed by one-way
ANOVA (con inuous a iables) wi h Tukey’s pos hoc es
and Pea son’s chi-squa ed es (p e alence; o ca ego ical
a iables). Da a o plasma bioma ke s we e ans o med
app op ia ely o achie e no mal dis ibu ion using squa e
oo (SR) o loga i hmic (LN) ans o ma ion and a e
desc ibed by geome ic means wi h 95% confidence in e als
(CI). Co ela ions among bioma ke s and be ween bio-
ma ke s and age a e shown as Pea son p oduc -momen
co ela ion o ans o med da a. Mean alues o plasma bio-
ma ke s be ween s udy g oups we e compa ed using one-
way ANOVA and gene al linea models wi h Fishe ’s leas
significan diffe ence es . The models we e adjus ed o
age, BMI, gende , smoking s a us (co a ia es), and coun y
( ac o ). In addi ion, a mul iple linea eg ession analysis
wi h all bioma ke s in he ini ial model and a o wa d s ep-
wise app oach was applied o iden i y independen plasma
bioma ke s wi h he highes co ela ion wi h age. Diffe ences
o concen a ions in bioma ke s be ween RASIG, GO, SGO,
and age g oups (5-yea in e als) a e p esen ed as box plo s.
All s a is ical analyses we e ca ied ou using SPSS so wa e
(SPSS Inc., Chicago, IL; Ve sion 19); s a is ical significance
o all es s was conside ed a P<005.
3. Resul s
Cha ac e is ics o he s udy g oups a e shown in Table 1. The
mean age o pa icipan s was 64.3 ±5.4 (55–75) yea s wi h no
significan diffe ence be ween he ma ched s udy g oups.
Men ep esen ed 47.7% o pa icipan s. The mean BMI was
27.3 ±4.5 kg/m
2
wi h no s a is ical significan diffe ence
among he s udy g oups. Only 14.8% o pa icipan s we e
cu en smoke s. The p e alence o smoking was diffe en
among he h ee s udy g oups being 9.9% in SGO, 12.6% in
GO, and 17.9% in RASIG. The numbe o pa icipan s om
each s udy cen e was significan ly diffe en as shown in
Table 1.
A o al o 1559 pa icipan s we e dis ibu ed in o he
g oups as ollows: RASIG (n= 794), GO (n= 493), and
SGO (n= 272).
Bioma ke concen a ions diffe ed significan ly be ween
s udy g oups (Table 2 and Figu es 1, 2, and 3). I is no ewo -
hy ha GO and SGO diffe ed only in u ic acid wi h signifi-
can ly highe concen a ions in he SGO g oup. Cys eine
was highes in GO, glu a hione in SGO, α- ocophe ol was
highes in bo h GO and SGO while lycopene was lowe in
GO and SGO han in he RASIG g oup. In e es ingly, GO
diffe ed in all bioma ke s om he RASIG g oup excep in
u ic acid and o al glu a hione (Table 2). In de ail, GO had
significan ly highe concen a ions o asco bic acid, o al ee
cys eine, α- ocophe ol, and 3-ni o y osine and lowe con-
cen a ions o lycopene, p o ein ca bonyls, and malondialde-
hyde. Fu he mo e, SGO we e diffe en om RASIG in all
bioma ke s excep o u ic acid, 3-ni o y osine, and malon-
dialdehyde wi h highe concen a ions o asco bic acid, o al
ee cys eine, o al glu a hione, and α- ocophe ol and lowe
concen a ions o p o ein ca bonyls and lycopene.
We pe o med a uni a ia e gene al linea model adjus ed
o age, BMI, gende , smoking s a us, and coun y o assess
whe he hese diffe ences we e s ill p esen a e adjus men .
This was ue o glu a hione and α- ocophe ol, as well as o
all h ee oxida i e s ess bioma ke s (p o ein ca bonyls, 3-
3Oxida i e Medicine and Cellula Longe i y
ni o y osine, and malondialdehyde), al hough lycopene
(P<0069) and cys eine (P<0072) also eached he
bo de line o significance.
The compa ison o bioma ke concen a ions o ou
s udy g oups wi h a middle-aged con ol g oup is shown in
Table 3. In gene al, he olde g oups e ealed significan ly
Table 2: Bioma ke concen a ions in he h ee s udy popula ions
1
.
All (n= 1559) RASIG (n= 794)GO(n= 493) SGO (n= 272)P
An ioxidan s
Asco bic acid (mg/L) 4.41 (4.26; 4.58) 4.13 (3.91; 4.36) 4.73 (4.44; 5.02)
‡
4.70 (4.33; 5.08)
‡
0.002
Adjus ed GLM
2
4.32 (4.13; 4.52) 4.17 (3.87; 4.49) 3.85 (3.32; 4.41) 0.258
U ic acid (mg/L) 45.9 (45.3; 46.5) 45.7 (44.9; 46.5) 45.3 (44.2; 46.3) 47.4 (46.0; 48.8)
◊
0.047
Adjus ed GLM
2
45.4 (44.6; 46.1) 46.0 (44.7; 47.2) 47.0 (44.7; 49.3) 0.341
To al cys eine (μmol/L) 148.2 (146.6; 149.8) 144.8 (142.5; 147.1) 150.8 (147.9; 153.6)
‡
153.3 (149.3; 157.3)
‡
<0.001
Adjus ed GLM
2
144.2 (141.8; 146.6) 149.6 (145.7; 153.4)
‡
145.5 (138.4; 152.6) 0.072
To al glu a hione (μmol/L) 1100 (1091; 1110) 1088 (1074; 1102) 1110 (1093; 1127) 1120 (1099; 1141)
‡
0.024
Adjus ed GLM
2
1092 (1077; 1106) 1112 (1089; 1135) 1149 (1106; 1193)
‡
0.025
Lycopene (μmol/L) 0.574 (0.556; 0.592) 0.631 (0.604; 0.659) 0.525 (0.497; 0.554)
‡
0.503 (0.464; 0.543)
‡
<0.001
Adjus ed GLM
2
0.608 (0.583; 0.635) 0.566 (0.526; 0.606) 0.539 (0.470; 0.613) 0.069
α-Tocophe ol (μmol/L) 28.8 (28.4; 29.2) 27.8 (27.3; 28.3) 30.1 (29.4; 30.8)
‡
29.6 (28.6; 30.5)
‡
<0.001
Adjus ed GLM
2
27.9 (27.4; 28.5) 29.3 (28.4; 30.3)
‡
29.9 (28.2; 31.7)
‡
0.005
Oxida i e s ess bioma ke s
P o ein Ca bonyls (nmol/mg) 0.577 (0.573; 0.582) 0.595 (0.590; 0.601) 0.558 (0.551; 0.566)
‡
0.561 (0.552; 0.571)
‡
<0.001
Adjus ed GLM
2
0.591 (0.585; 0.597) 0.566 (0.557; 0.575)
‡
0.564 (0.547; 0.581)
‡
<0.001
3-Ni o y osine (pmol/mg) 4.11 (3.97; 4.25) 3.94 (3.75; 4.13) 4.40 (4.14; 4.67)
‡
4.10 (3.77; 4.44) 0.018
Adjus ed GLM
2
3.90 (3.70; 4.11) 4.38 (4.03; 4.74)
‡
3.76 (3.18; 4.38) 0.048
Malondialdehyde (μmol/L) 0.316 (0.306; 0.326) 0.336 (0.322; 0.350) 0.284 (0.266; 0.302)
‡
0.317 (0.290; 0.346) <0.001
Adjus ed GLM
2
0.334 (0.319; 0.348) 0.288 (0.267; 0.311)
‡
0.314 (0.274; 0.357) 0.004
1
Geome ic mean (95% CI).
2
Adjus ed gene al linea model (GLM): uni a ia e gene al linea model adjus ed o age, BMI, smoking s a us, gende , and coun y
(cen e ).
‡
S a is ically significan diffe ence o RASIG.
◊
S a is ically significan diffe ence o GO. P alues: s a is ically significan diffe ences we e de e mined by
one-way ANOVA wi h Tukey’s pos hoc es and by Fishe ’s leas significan diffe ence pos hoc es in he GLM.
Table 1: Cha ac e is ics o he h ee s udy g oups.
All (n= 1559) RASIG (n= 794)GO(n= 493) SGO (n= 272)P
Age (yea s) 64.3 ±5.4 64.5 ±5.8 64.3 ±4.9 63.9 ±4.7 0.230
55–59 yea s (n, (%)) 345 (22.1) 193 (24.3) 94 (19.1) 58 (21.3)
<0.001
60–64 yea s (n, (%)) 448 (28.7) 210 (26.4) 149 (30.2) 89 (32.7)
65–69 yea s (n, (%)) 465 (29.8) 204 (25.7) 166 (33.7) 95 (34.9)
70–75 yea s (n, (%)) 301 (19.3) 187 (23.6) 84 (17.0) 30 (11.0)
Gende , male (n, (%)) 743 (47.7) 386 (48.6) 213 (43.2) 144 (52.9) 0.027
Smoke , cu en (n, (%)) 231 (14.8) 142 (17.9) 63 (12.6) 27 (9.9) 0.001
BMI (kg/m
2
)27.3 ±4.5 27.6 ±4.7 26.6 ±4.4 27.3 ±4.2 0.692
<25 (n, (%)) 516 (33.1) 249 (31.4) 188 (38.1) 79 (29.0)
0.03625 o <30 (n, (%)) 673 (43.2) 343 (43.3) 199 (40.4) 131 (48.2)
≥30 (n, (%)) 369 (23.7) 201 (25.3) 106 (21.5) 62 (22.8)
Coun y
Belgium (n, (%)) 472 (30.3) 155 (19.5) 190 (38.5) 127 (46.7)
<0.001
Finland (n, (%)) 253 (16.2) 69 (8.7) 132 (26.8) 52 (19.1)
G eece (n, (%)) 209 (13.4) 187 (23.6) 18 (3.7) 4 (1.5)
I aly (n, (%)) 323 (20.7) 187 (23.6) 87 (17.6) 49 (18.0)
Poland (n, (%)) 302 (19.4) 196 (24.7) 66 (13.4) 40 (14.7)
Values a e means ±SD; P alue: one-way ANOVA (con inuous a iables) and Pea son’s chi-squa ed es (p e alence).
4 Oxida i e Medicine and Cellula Longe i y
diffe en an ioxidan concen a ions compa ed o he e e -
ence g oup. The RASIG, GO, and SGO g oups had signifi-
can ly highe mean u ic acid, cys eine, and α- ocophe ol and
lowe lycopene compa ed o he e e ence g oup. The e we e
no diffe ences in e ms o oxida i e s ess bioma ke s be ween
he e e ence g oup and RASIG aged 55–75 yea s. In e es -
ingly, while he GO and SGO g oups had significan ly lowe
p o ein ca bonyl concen a ions compa ed o he e e ence
g oup, only he GO g oup had highe 3-ni o y osine
compa ed o he e e ence g oup.
Table 4 shows he co ela ion coefficien s among he
assessed bioma ke s in all pa icipan s aged ≥55 yea s.
The highes co ela ion coefficien s we e ound be ween
malondialdehyde and p o ein ca bonyls ( =0322), malon-
dialdehyde and asco bic acid ( =−0240), ollowed by
p o ein ca bonyls and α- ocophe ol ( =−0193). A posi i e
co ela ion was obse ed be ween he an ioxidan s asco bic
acid and α- ocophe ol ( =0164). When hese co ela ions
we e pe o med only in he RASIG g oup, he di ec ion and
s eng h o co ela ions we e simila ( esul s no shown).
The co ela ions o he indi idual bioma ke s wi h age a e
shown in Table 5. Significan posi i e co ela ion coefficien s
we e obse ed o u ic acid, cys eine, and 3-ni o y osine
while a significan in e se associa ion was only seen o
lycopene. A weak significan posi i e co ela ion be ween
p o ein ca bonyls and age was only obse ed in he RASIG
g oup ( =0098;P<001, da a no shown).
In a final mul iple eg ession model wi h a o wa d
app oach, we aimed o iden i y independen bioma ke s
wi h he highes co ela ion wi h age (Table 6). The e o e,
all bioma ke s we e assessed as co a ia es. Confi ming he
co ela ions om Table 5, u ic acid, cys eine, 3-ni o y osine,
P o ein ca bonyls (nmol/mg)
(A) (B)
RASIG SGOGO RASIG SGOGO
0.6
0.8
0.4
0.7
0.9
0.5
55‒59
60‒64
65‒69
70‒75
⁎⁎⁎ ⁎
(a)
RASIG SGOGO
3-Ni o y osine (pmol/mg)
14
8
4
2
0
10
6
12
RASIG SGOGO
(A) (B)
55‒59
60‒64
65‒69
70‒75
⁎⁎
(b)
RASIG SGOGO
Malondialdehyde (mol/L)
0.8
0.4
0.2
0
1.0
0.6
1.2
RASIG SGOGO
(A) (B)
55‒59
60‒64
65‒69
70‒75
⁎⁎⁎
(c)
Figu e 1: Classical bioma ke s o oxida i e s ess: p o ein ca bonyls (a), 3-ni o y osine (b), and malondialdehyde (c). Bioma ke
concen a ions a e displayed acco ding o s udy g oups (A) and age g oups (B), espec i ely. RASIG (n= 794); GO (n= 493);
SGO (n= 272). Ou lie s and ex eme alues a e included in he analyses bu no shown in he figu e. S a is ically significan diffe ences a e
indica ed by as e isks: ∗P<005 and ∗∗∗P<0001.
5Oxida i e Medicine and Cellula Longe i y

and lycopene we e significan ly associa ed wi h age, and
also, p o ein ca bonyls emained in he model wi h a
posi i e associa ion.
4. Discussion
In gene al, he olde g oups e ealed highe an ioxidan (u ic
acid, cys eine, and α- ocophe ol) and lowe lycopene concen-
a ions compa ed o he e e ence g oup. Lycopene, o al
cys eine, u ic acid, p o ein ca bonyls, and 3-ni o y osine
we e significan ly and independen ly associa ed wi h age in
he mul iple linea eg ession model among pa icipan s aged
55–75 yea s. Highe cys eine and α- ocophe ol, bu lowe
lycopene in bo h GO and SGO compa ed o RASIG seem
o be associa ed wi h a “beneficial”li es yle, while he signi -
ican ly lowe malondialdehyde and highe 3-ni o y osine
only in he GO compa ed o he RASIG g oup may indica e
ha amilies wi h long-li ing membe s a e gene ically be e
equipped o handle oxida i e s ess, ye he cause and impac
o he highe 3-ni o y osine le els emain unclea .
We assume ha (1) i he e a e diffe ences be ween
RASIG and he o he wo g oups bu no diffe ences be ween
GO and SGO, he eason may be li es yle- ela ed and (2) di -
e ences be ween GO and SGO (i espec i e o he RASIG
esul s) indica e gene ic easons.
Applying hese c i e ia o he bioma ke s assessed in he
p esen s udy leads o he conclusion ha a e adjus men
o age, BMI, smoking s a us, gende , and coun y, he diffe -
ences in p o ein ca bonyls, malondialdehyde, 3-ni o y o-
sine, α- ocophe ol, cys eine, and glu a hione be ween s udy
g oups seem o be li es yle- ela ed whe eas gene ics seem o
play a mino ole. Fo he case whe e bo h GO and SGO
diffe o he con ol g oup (p o ein ca bonyls, lycopene,
and α- ocophe ol), his migh indica e he influence o li e-
s yle. Thus, ou esul s show ha li es yle is an impo an
con ibu o o edox bioma ke s.
One migh hypo hesize ha when GO diffe o he o he
wo g oups and he e e ence g oup, his diffe ence may be
a ibu ed o gene ics. This was he case o 3-ni o y osine,
cys eine (bo h highe han he o he g oups), and malondial-
dehyde (lowe han he o he g oups). In ou s udy, he e
was no significan diffe ence be ween GO and SGO g oups
o hese h ee bioma ke s, ye a clea endency exis s
acco ding o he da a in Tables 2 and 3. Concen a ions o
malondialdehyde, 3-ni o y osine, and o al cys eine we e
s ill diffe en be ween he GO and he RASIG g oups a e
he GLM adjus men , which may be an indica ion o a gene ic
con ibu ion in age-associa ed handling o oxida i e s ess.
Di ec compa ison o he s udy g oups e ealed ha he
GO g oup had significan ly lowe concen a ions o p o ein
ca bonyls and malondialdehyde accompanied by highe
concen a ions o cys eine, asco bic acid, and α- ocophe ol
compa ed o RASIG. Con a ily, GO had lowe lycopene
and highe 3-ni o y osine han he RASIG g oup. These di -
e ences may be due o be e me abolic p ofiles o due o a
gene ally heal hie nu ien in ake, despi e less p ocessed
oma o p oduc s which a e especially ich in lycopene.
I is widely accep ed ha he e is a ela ionship
be ween he aging p ocess and oxida i e s ess; howe e ,
mos s udies leading o his heo y ha e been ca ied ou
in model sys ems and only ew s udies ha e analyzed di -
e en bioma ke s o oxida i e s ess in heal hy humans o
a ious age g oups [3, 6].
P o ein ca bonyls a e conside ed o be ela i ely s able
[23] and ea ly ma ke s [24] o oxida i e s ess. Measu ing
ca bonyla ion o plasma p o eins enables e alua ing he
global oxida ion s a us in plasma. P o ein ca bonyls ha e
been analyzed in a mul i ude o s udies anging om cell cul-
u e, animal, o human s udies and a e ela ed o he aging
RASIG SGOGO
Lycopene (mol/L)
1.5
0.5
0
2.0
1.0
RASIG SGOGO
(a) S udy g oups (b) Age g oups
55‒59
60‒64
65‒69
70‒75
⁎⁎⁎ ⁎⁎⁎
⁎⁎⁎
⁎⁎ ⁎⁎
⁎
Figu e 3: Lycopene concen a ion by s udy g oups (a) and age
g oups (b). RASIG (n= 794); GO (n= 493); SGO (n= 272).
Ou lie s and ex eme alues a e included in he analyses bu no
shown in he figu e. S a is ically significan diffe ences a e
indica ed by as e isks: ∗P<005,∗∗P<001, and ∗∗∗P<0001.
RASIG SGOGO
Cys eine (mol/L)
300
200
100
50
250
150
RASIG SGOGO
(a) S udy g oups (b) Age g oups
55‒59
60‒64
65‒69
70‒75
⁎⁎⁎
⁎⁎ ⁎⁎
⁎⁎
Figu e 2: Cys eine concen a ion by s udy g oups (a) and age
g oups (b). RASIG (n= 794); GO (n= 493); SGO (n= 272).
Ou lie s and ex eme alues a e included in he analyses bu no
shown in he figu e. S a is ically significan diffe ences a e
indica ed by as e isks: ∗∗P<001 and ∗∗∗P<0001.
6 Oxida i e Medicine and Cellula Longe i y
p ocess. They ha e been shown o be a p edic o o mo ali y
in mode a ely o se e ely disabled olde women [10].
Komosinska-Vasse e al. showed a posi i e co ela ion
o p o ein ca bonyls wi h age in a s udy o 56 men and
women aged ≥55 yea s ( =052) [25]. O he au ho s also
ound a s ong posi i e co ela ion o p o ein ca bonyls wi h
age ( =0786) in 80 pa icipan s be ween he age o 18 and
85 [6], whe eas in he p esen s udy, he co ela ion o he
age ange o 55–75 was e y weak ( =0098) bu s ill
s a is ically significan . Caka ay e al. demons a ed ha
plasma p o ein ca bonyl le els o elde ly pa icipan s we e
significan ly highe in compa ison o hose o middle-aged
and young pa icipan s [26]. In he p esen s udy, p o-
ein ca bonyls only co ela ed wi h age in he RASIG
g oup ( =0098,P<001; da a no shown) bu no when
all h ee s udy g oups we e combined. Mos in e es ingly,
he RASIG g oup had s a is ically significan ly highe p o ein
ca bonyl concen a ions han he GO as well as he SGO
while he e was no diffe ence be ween he GO and SGO.
A e adjus ing o co a ia es (including smoking s a us), his
diffe ence emained.
Fo 3-ni o y osine, we obse ed a weak, posi i e
associa ion wi h age. In con as o p o ein ca bonyls, 3-
ni o y osine was significan ly highe in GO compa ed o
RASIG. 3-Ni o y osine has been desc ibed o be a s able
ma ke o oxida i e/ni a i e s ess in some inflamma o y
diseases [27]. I a ises om ni a ion in ol ing eac i e
Table 4: Co ela ions be ween bioma ke s in all h ee s udy g oups in pa icipan s aged ≥55 yea s
1
.
Asco bic
acid U ic acid To al
cys eine
To al
glu a hione Lycopene α-Tocophe ol P o ein
ca bonyls
3-Ni o-
y osine Malondialdehyde
Asco bic acid −0.083∗∗∗ 0.140∗∗∗ 0.030 −0.103∗∗∗ 0.164∗∗∗
−0.157∗∗∗ −0.027 −0.240∗∗∗
U ic acid 0.067∗∗ −0.021 −0.099∗∗∗ 0.059∗−0.020 −0.054∗0.019
To al cys eine 0.047 −0.053∗0.103∗∗∗ 0.062∗0.043 −0.034
To al glu a hione 0.032 −0.033 −0.007 −0.015 0.030
Lycopene 0.073∗∗ 0.088∗∗∗
−0.092∗∗∗ 0.159∗∗∗
α-Tocophe ol −0.193∗∗∗ 0.062∗
−0.132∗∗∗
P o ein
ca bonyls 0.001 0.322∗∗∗
3-Ni o y osine 0.019
Malondialdehyde
1
Pea son co ela ion coefficien . S a is ically significan co ela ions a e ma ked by ∗P<005,∗∗ P<001, and ∗∗∗ P<0001.(n= 1559).
Table 3: Compa ison o s udy g oups wi h a e e ence g oup.
Re e ence G oup
(RASIG; 35–54 yea s)
(n= 683)
RASIG
(55–75 yea s)
(n= 794)
GO
(55–75 yea s)
(n= 493)
SGO
(55–75 yea s)
(n= 272)
An ioxidan s
Asco bic acid (mg/L) 4.37 (4.12; 4.63) 4.13 (3.91; 4.36) 4.73 (4.44; 5.02) 4.70 (4.33; 5.08)
U ic acid (mg/L) 42.4 (41.6; 43.3) 45.7 (44.9; 46.5)∗∗∗ 45.3 (44.2; 46.3)∗∗∗ 47.4 (46.0; 48.8)∗∗∗
To al cys eine (μmol/L) 130.3 (128.1; 132.5) 143.0 (140.7; 145.3)∗∗∗ 149.0 (146.1; 151.9)∗∗∗ 151.4 (147.4; 155.5)∗∗∗
To al glu a hione (μmol/L) 1107 (1092; 1121) 1088 (1074; 1101) 1110 (1093; 1127) 1120 (1099; 1142)
Lycopene (μmol/L) 0.83 (0.80; 0.86) 0.63 (0.60; 0.66)∗∗∗ 0.53 (0.50; 0.55)∗∗∗ 0.50 (0.46; 0.54)∗∗∗
α-Tocophe ol (μmol/L) 25.7 (10.4; 10.6) 27.8 (10.9; 11.2)∗∗∗ 30.1 (11.4; 11.7)∗∗∗ 29.6 (11.2; 11.7)∗∗∗
Oxida i e s ess bioma ke s
P o ein ca bonyls (nmol/mg) 0.603 (0.598; 0.609) 0.595 (0.590; 0.601) 0.558 (0.551; 0.566)∗∗∗ 0.561 (0.552; 0.571)∗∗∗
3-Ni o y osine (pmol/mg) 3.8 (3.6; 4.0) 3.9 (3.8; 4.1) 4.4 (4.1; 4.7)∗∗ 4.1 (3.8; 4.4)
Malondialdehyde (μmol/L) 0.31 (0.30; 0.32) 0.34 (0.32; 0.35) 0.28 (0.27; 0.30) 0.32 (0.29; 0.35)
∗∗∗P<0001 and ∗∗ P<001 by one-way ANOVA wi h Tukey’s pos hoc es .
Table 5: Co ela ions be ween bioma ke s and age among all
pa icipan s (aged ≥55 yea s)
1
.
P
Asco bic acid 0.026 0.297
U ic acid 0.092 <0.001
To al cys eine 0.152 <0.001
To al glu a hione −0.035 0.167
Lycopene −0.224 <0.001
α-Tocophe ol −0.005 0.847
P o ein ca bonyls 0.036 0.158
3-Ni o y osine 0.066 0.009
Malondialdehyde −0.037 0.146
1
Pea son co ela ion coefficien (n= 1559).
7Oxida i e Medicine and Cellula Longe i y
ni ogen species (RNS) and pe oxidase-media ed ni i e
oxida ion. Today, myelope oxidase is conside ed o be
in ol ed in his pa hway. Thus, 3-ni o y osine may play a
ole in inflamma ion a he han he aging p ocess i sel
[28]. F ijhoffe al. ques ion whe he 3-ni o y osine is clini-
cally use ul, in compa ison o he al eady es ablished ma ke s
o inflamma o y p ocesses such as C- eac i e p o ein [29].
Plasma 3-ni o y osine le els in pa ien s ea ed wi h an i-
inflamma o y d ugs ha e been shown o dec ease [30, 31].
This is one mo e hin why 3-ni o y osine may be a be e
ma ke o inflamma ion han o he aging p ocess in gen-
e al. Al hough he ni a ion o y osine esidues on p o eins
can esul in a loss o unc ion [32], some au ho s ha e
epo ed a gain o unc ion [33]. Pe haps si e-specific ni a-
ion may ha e a p o ec i e unc ion o a ole in longe i y.
In ou s udy popula ion, he e migh ha e been no d ama ic
diffe ence o 3-ni o y osine in he diffe en age g oups
because he pa icipan s we e gene ally heal hy. Only 4.0%
o ou pa icipan s had CRP concen a ions ≥10 mg/L.
Ne e heless, we checked he co ela ion be ween CRP and
3-ni o y osine bu he e was no co ela ion, nei he in all
pa icipan s no in hose wi h a high CRP le el. Thus, he
use ulness o 3-ni o y osine as a bioma ke o e alua e
oxida i e s ess emains o be elucida ed.
Few la ge epidemiological s udies ha e analyzed malon-
dialdehyde. Block e al. sugges plasma malondialdehyde
should be conside ed o mos epidemiologic esea ch on
edox bioma ke s [34] since hey obse ed in a alida ion
s udy ha his bioma ke had a good day- o-day s abili y.
They p opose malondialdehyde o be an effec i e ma ke o
oxida i e s ess and s a e ha he use o a single measu e o
malondialdehyde esul ed in li le a enua ion [34]. In
ano he s udy, he same au ho s ound ha malondialdehyde
was no associa ed wi h age in 298 pa icipan s aged 18–78
yea s [35]. These esul s a e suppo ed by ou finding ha
malondialdehyde was no associa ed wi h age in any o he
h ee s udy g oups. Howe e , he GO g oup had significan ly
lowe plasma malondialdehyde concen a ions han RASIG.
This diffe ence emained e en a e adjus ing o age, BMI,
smoking s a us, gende , and coun y.
To coun e ac oxida i e s ess, he e exis a ne wo k o
an ioxidan de ense mechanisms. One o hese an ioxi-
dan s is he ipep ide glu a hione (γ-glu amylcys einylgly-
cine). A decline o glu a hione wi h age has p e iously
been sugges ed in humans [36], and i has been
demons a ed ha he co ela ion o hiol g oups in
plasma wi h age was in e se ( =−0718) [6].
Concen a ions o glu a hione and cys eine we e lowe in
heal hy old (mean age 70.3 yea s) han in middle-aged (mean
age 39.8 yea s) pa icipan s (n=8each) [37].
We did no obse e any co ela ion be ween glu a hione
and age. One explana ion may be ha we measu ed o al
glu a hione ins ead o GSH/GSSH which is conside ed a be -
e ma ke o he edox s a e. Gius a ini e al. ound an
in e se co ela ion o glu a hione wi h age bu no co ela ion
o cys eine wi h age in 41 pa icipan s [38]. Simila ly, Jones
e al. used plasma o 122 heal hy indi iduals aged 19–85 yea s
o analyze hiol-based edox changes [39]. They sugges ha
he capaci y o he glu a hione an ioxidan sys em is main-
ained un il 45 yea s and hen declines apidly. Fo he
p esen analyses, pa icipan s we e selec ed which we e ≥55
yea s, ye we we e unable o show a co ela ion be ween glu-
a hione and age. In con as o p e ious findings, cys eine is
highe in he highe age g oups in he RASIG and GO s udy
g oups and co ela es posi i ely wi h age among all pa ici-
pan s ( =0152,P<0001).
I is assumed ha lowe glu a hione concen a ions
occu ing du ing aging and in diffe en diseases may mo e
likely esul om low cys eine concen a ions a he han
due o oxida ion since cys eine is he a e-limi ing p ecu so
o glu a hione. F ee cys eine is one o he main nonp o ein
hiols in plasma [40, 41] and conside ed a semiessen ial
amino acid, since i mus be aken up o syn hesized om
he essen ial amino acid me hionine [42]. I is able o egula e
nu ien me abolism, oxida i e s ess, physiologic signaling
pa hways, and associa ed diseases h ough he p oduc ion
o glu a hione, hyd ogen sulfide, and au ine [43]. An o al
in e en ion wi h cys eine (as N-ace ylcys eine) and glycine
o 14 days esul ed in significan ly inc eased glu a hione
concen a ions [37]. This was also ue when only cys eine
was supplemen ed, esul ing in an inc ease in hepa ic
glu a hione syn hesis [43]. I is likely ha he glu a hione
concen a ions in ou c oss-sec ional s udy we e simila in
he diffe en age g oups because cys eine may no ha e been
limi ed. P e ious esul s also sugges ha he pa icipan s in
ou s udy had sufficien cys eine and hus glu a hione le els
[38]. Ne e heless, he equi emen o cys eine may be
ele a ed a e oxida i e e en s due o he consump ion o glu-
a hione. Some u u e esea ch should cla i y he ole o cys-
eine in aging and as a die a y p ecu so o glu a hione.
Table 6: Associa ions o oxida i e s ess ma ke s and an ioxidan s wi h age
1
.
Compound B95% CI
2
P
(Cons an ) 59.13 56.79, 61.48 <0.001
Lycopene (μmol/L) −2.783 −3.440, −2.126 −0.207 0.047 <0.001
To al cys eine (mol/L) 0.021 0.013, 0.029 0.133 0.020 <0.001
U ic acid (mg/L) 0.031 0.009, 0.054 0.071 0.004 0.005
P o ein ca bonyls (nmol/mg) 3.274 0.252, 6.296 0.054 0.003 0.034
3-Ni o y osine (pmol/mg) 0.094 0.004, 0.183 0.052 0.003 0.040
1
Mul iple linea eg ession analysis wi h a o wa d app oach o iden i y independen blood bioma ke s wi h highes co ela ion o age; all bioma ke s
including asco bic acid, glu a hione, malondialdehyde, and α- ocophe ol we e assessed as co a ia es in he ini ial model; pa ial and
2
;R=0277,
R2=0077 (n= 1545).
8 Oxida i e Medicine and Cellula Longe i y
Fu he mo e, i should be no ed ha some au ho s measu ed
glu a hione/ hiol g oups in plasma [6]. Glu a hione is known
as he mos powe ul cellula an ioxidan , ha is, p esen in
ed blood cells, and consequen ly, we analyzed glu a hione
in whole blood.
Conce ning u ic acid, ou esul s show ha he e was no
diffe ence be ween RASIG and GO bu highe concen a ions
in he SGO g oup han in he GO. Howe e , adjus ing o
age, BMI, gende , smoking s a us, and coun y, hese diffe -
ences did no emain. U ic acid is de i ed om he deg ada-
ion o pu ine nucleo ides which can be o die a y o
endogenous o igin. The e o e, an inc eased in ake o animal
p oduc s o legumes/pulses in he SGO g oup is possible.
The e is a con o e sial discussion whe he he posi i e
effec s o u ic acid as an an ioxidan a e ou weighed by i s
ad e se effec s conce ning gou , co ona y a e y disease,
hype ension, and s oke ( o e iew, see [44]).
Besides endogenous an ioxidan s such as glu a hione,
cys eine, and u ic acid, exogenous an ioxidan s a e also
equi ed o coun e ac oxida i e s ess. Some o he mos
powe ul an ioxidan s a e o die a y o igin such as asco bic
acid, α- ocophe ol, lycopene, and o he ca o enoids. I is
known ha a high in ake o ui s and ege ables is associa ed
wi h a high plasma concen a ion o asco bic acid [45].
Simul aneously, a die ich in ui s and ege ables is associ-
a ed wi h a educed isk o some diseases such as CVD and
cance s [46]. α-Tocophe ol and asco bic acid ac syne gis i-
cally in coun e ac ing ee adicals. α-Tocophe ol is able o
quench ee adicals in a hyd ophobic en i onmen , o
example, o e mina e lipid pe oxida ion and he esul ing
ocophe ol adical is hen ecycled by asco bic acid [47].
We ound an in e se co ela ion be ween α- ocophe ol and
malondialdehyde, an in e se associa ion be ween asco bic
acid and malondialdehyde (all P<0001), and a posi i e
co ela ion be ween α- ocophe ol and asco bic acid. These
esul s a e in acco dance wi h he assump ion ha hese
an ioxidan s ac syne gis ically.
Since hese a e die -de i ed an ioxidan s and we did no
adjus ou models o die a y in ake o ui and ege ables,
we canno exclude ha he die o he season had an effec
on he plasma concen a ion o hese mic onu ien s. In a
ecen s udy, we ha e shown o e he whole age- ange o
he MARK-AGE p ojec om 35 o 75 (RASIG g oup) ha
lycopene was lowe in highe age g oups and his effec was
independen o season [22]. I emains unclea i hese diffe -
ences obse ed in he olde age g oups esul om a educed
in ake, educed abso p ion, inc eased s o age in adipose
issue, o ele a ed deg ada ion o lycopene.
The s eng hs o he p esen s udy include he la ge
sample size and ha all bioma ke s desc ibed he e we e
measu ed in one single labo a o y in blinded o m (samples
om s udy g oups and coun ies we e in mixed andom
o de by he biobank). Fo lipid-soluble mic onu ien anal-
ysis, we ha e ecen ly published he in e ba ch coefficien s
o a ia ion which we e 7.6% o lycopene and 6.3% o α-
ocophe ol in he MARK-AGE coho [22]. Conce ning he
alidi y o he me hods used o 3-ni o y osine analysis, we
ha e p e iously epo ed good specifici y, ep oducibili y,
and accu acy o his in-house ELISA [48]. The ELISA
me hod used he e o analyze p o ein ca bonyls has been al-
ida ed in a mul icen e ing s udy [49]. I was shown ha
ca bonyl concen a ions om h ee ou o ou labo a o ies
pa icipa ing in he ing s udy ell wi hin 95% confidence
in e als. Addi ionally, p o ein concen a ion was measu ed
be o e bo h ELISAs and plasma samples we e dilu ed o he
same p o ein concen a ion. S anda ds o p o ein ca bonyl
ELISA we e p epa ed acco ding o Buss e al. [21] and un
on e e y 96-well pla e.
In e ms o compa abili y o o he s udies, he e is an
excellen e iew om Gius a ini e al. [50]. As he au ho s
demons a e, he e exis nume ous analy ical me hods o
analyze p o ein ca bonyls, malondialdehyde, GSH, ocoph-
e ols, and lycopene, among o he s. An o e iew is gi en o
diffe en de i a iza ion and de ec ion me hods, heal h condi-
ions, and uni s, demons a ing huge a ie y. Fo ins ance o
malondialdehyde, Gius a ini e al. showed ha au ho s using
me hods compa able o ou s (TBA de i a iza ion and HPLC
sepa a ion) published concen a ions be ween 0.44 and
6.8 μmol/L o plasma samples [50]. One eason o his a -
ia ion may be ha hepa in plasma was used in some cases.
Especially o malondialdehyde, i is impo an o measu e
his ma ke in EDTA plasma since lipid pe oxida ion can
con inue in se um and hepa in plasma, hus a ificially con-
ibu ing o ele a ed malondialdehyde concen a ions. EDTA
and ci a e can complex i on hus p e en ing Fen on eac ion
leading o lipid pe oxida ion.
Fo GSH and cys eine, he au ho s also epo diffe en
me hods (e.g., enzyma ic, HPLC coupled wi h UV o fluo es-
cence de ec ion) and mean concen a ions in whole blood,
plasma, and e y h ocy es o heal hy and diseased indi iduals,
which span o e one o wo o de s o magni ude wi hin he
kind o used specimen; howe e , o al GSH le els in he p es-
en s udy a e in he uppe ange o p e iously epo ed mean
whole blood alues o heal hy indi iduals measu ed by HPLC
o ecycling spec opho ome y (using Ellman’s eagen ).
This s udy has some limi a ions which mus be men-
ioned. Since his s udy was obse a ional, we canno make
s a emen s on he changes o hese bioma ke s wi h age;
he e o e s udies wi h epea ed measu emen s and/o
ollow-up a e needed. Fu he mo e ou esul s may be spe-
cific o Eu opean/Caucasian subjec s and hus no ans e -
able o o he coun ies.
The inclusion o h ee diffe en s udy g oups wi h a la ge
sample size (n= 1559) is one ea u e ha dis inguishes his
s udy om o he s. Samples we e collec ed and dis ibu ed
in a blinded o m o gua an ee unbiased measu emen and
in e p e a ion, and all analyses o bioma ke s desc ibed he e
we e ca ied ou in one single labo a o y, which significan ly
educes in e labo a o y a ia ions. The assessmen o diffe -
en specific cellula and plasma bioma ke s, ha is, ma ke s
o oxida i e damage oge he wi h an ioxidan s ha a e
no analyzed by comme cial ki s bu by in-house me hods
is ce ainly an impo an s eng h.
5. Conclusion
He e, we ha e p o ided an o e iew o he le els o he diffe -
en edox bioma ke s in human plasma and whole blood in
9Oxida i e Medicine and Cellula Longe i y