Oxida i e s ess s a us in an ins i u ionalised elde ly g oup a e
he in ake o a phenolic- ich desse
M. Ca men Rami ez-To osa
1
*, Ja ie Ga cı
´a-Alonso
2
, M. Luisa Vidal-Gue a a
3
,
Jose
´L. Quiles
4
, M. Jesu
´s Pe iago
2
, Ja ie Linde
1
, M. Dolo es Mesa
1
, Gaspa Ros
2
,
Ped o Abella
´n
3
and A
´ngel Gil
1
1
Depa men o Biochemis y and Molecula Biology, Ins i u e o Nu i ion and Food Technology,
Uni e si y o G anada, Campus de Ca uja, 18071 G anada, Spain
2
Depa men o Food Science and Nu i ion, Ve e ina y Facul y, Uni e si y o Mu cia, Mu cia, Spain
3
Depa men o Resea ch and De elopmen , He o Espan
˜a S.A., Alcan a illa, Mu cia, Spain
4
Depa men o Physiology, Ins i u e o Nu i ion and Food Technology, Uni e si y o G anada, Campus de Ca uja,
18071 G anada, Spain
(Recei ed 3 Sep embe 2003 – Re ised 20 Janua y 2004 – Accep ed 16 Feb ua y 2004)
The elde ly popula ion unde goes a se ies o physiological and sociological changes common o old age wi h a high p obabili y o su e -
ing degene a i e illness and malnu i ion. A desse ich in phenolic compounds has been designed by using concen a ed juices o g ape,
che y, blackbe y, blackcu an and aspbe y wi h he aim o i being used as a complemen a y ood in adul hood. In he p esen s udy,
we in es iga ed he e ec o he in ake o his desse (a ja o 200 g daily o a pe iod o 2 weeks), wi h an an ioxidan ac i i y equi alen
o en se ings o ui s and ege ables, on se e al ma ke s o oxida i e and an ioxidan s a us in DNA and plasma in a g oup o elde ly
indi iduals. Non-smoking ins i u ionalised elde ly subjec s we e ec ui ed om a pool o olun ee s in an old-age home in Mu cia (Spain).
Twen y- wo subjec s (six men and six een women) pa icipa ed in he s udy. The s udy was designed as a andomised in e en ion ial
wi h a pe iod o 2 weeks. A days 1 and 15, blood samples we e collec ed o analyse o al an ioxidan capaci y, biochemical pa ame e s,
an ioxidan i amins, LDL pe oxida ion, and DNA damage in pe iphe al blood lymphocy es. The conclusion o he p esen s udy is ha a
2-week in e en ion wi h ou desse en iched wi h na u al polyphenol compounds in elde ly indi iduals does no gi e enough ime o ind
changes in he an ioxidan and oxida i e s a us. Also, he iew ha he ma ked an ioxidan abili y o polyphenols in i o does no ansla e
o analogous e ec s in i o was con i med. Mo eo e , a highly oxida i e s ess s a us du ing ageing was con i med, oge he wi h he need
o pe o m ollow-up nu i ional s udies o imp o e his si ua ion.
Phenolic- ich desse : Ageing: An ioxidan s a us: DNA damage
The an ioxidan p ope ies o oods in ela ion o heal h and
pa icula ly on he main enance and p o ec ion om degen-
e a i e diseases a e o g owing in e es among scien is s,
ood manu ac u e s, consume s and heal h o ganisa ions.
Since he 1990s, se e al in e na ional o ganisa ions ha e
ecommended inc easing he consump ion o ui s and
ege ables o i e o mo e daily se ings, in o de o p o-
ide a desi able in ake o an ioxidan s and o imp o e
human heal h (Wo ld Heal h O ganiza ion, 1990; Wo ld
Cance Resea ch Founda ion & Ame ican Ins i u e o
Cance Resea ch, 1997).
Howe e , o ce ain g oups o he popula ion, such as he
elde ly, i is di icul o consume hose daily amoun s o
ui s and ege ables. The elde ly popula ion unde goes a
se ies o physiological and sociological changes common
o old age wi h a high p obabili y o su e ing degene a i e
illness and malnu i ion (Tucke & Bu anapin, 2001).
Du ing he ageing p ocess, a mo e seden a y li es yle,
esul ing in less ene gy expendi u e, poo appe i e, den al
disease, al e a ions in abso p ion and me abolism o se e al
nu ien s, medica ion, e c. may lead o a decline in he in ake
o mac o- and mic onu ien s. Consequen ly, elde ly indi-
iduals a e a isk o a sub-op imal nu i ional s a e o mul-
iple mic onu ien de iciencies (De Jong, 1999). In a ecen
s udy ca ied ou wi h 10 208 pa icipan s om eigh
andom popula ion s udies and pa icipan s in i y-se en
*Co esponding au ho : D M. Ca men Rami ez-To osa, ax þ34 958248326, email [email p o ec ed]
Abb e ia ions: ABTS, 2,2’-azinobis(3-e hylbenzo hiazoline 6-sul ona e); FRAP, e ic- educing abili y o plasma; TBARS, hioba bi u ic-acid- eac i e
subs ances; TEAC, T olox-equi alen an ioxidan capaci y.
B i ish Jou nal o Nu i ion (2004), 91, 943–950 DOI: 10.1079/BJN20041146
qThe Au ho s 2004
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s udies included in a me a-analysis, i has been shown ha
he i amin s a us o he Spanish popula ion clea ly shows
oom o imp o emen , especially wi h ega d o i amins
A, E, D, B
2
,B
6
, and ola es (O ega e al. 2003). Fo his
eason, he e is a end in he ood indus y owa ds unc-
ional oods wi h heal hy e ec s based, among o he s, on
hei an ioxidan p ope ies (Ka akaya e al. 2001).
An ioxidan i amins, including i amin C, i amin E
and a a ie y o phy ochemicals, a e impo an in main ain-
ing e ec i e an ioxidan de ences agains oxidan s ess-
ela ed diseases, including cance , ca a ac s and
Alzheime ’s disease. Be ies, g apes and che ies a e
ecognised as ui s wi h a high con en o an ioxidan s.
The an ioxidan p ope ies o hese ui s a e belie ed o
be due o he con en o an hocyanins and o he phenolic
compounds. Simila ly, concen a ed juices wi h a high con-
en o phenolics should p obably exhibi an e ec (Ga cı
´a-
Alonso e al. 2002). In his basis, a desse has been
designed by using concen a ed juices o g ape, che y,
blackbe y, blackcu an and aspbe y wi h he aim o i
being used as a complemen a y ood in adul hood. The
an ioxidan ac i i y o his p oduc is conside ed simila
o ha o ed wine and highe han ha o many ui s
and ege ables. In p e ious s udies, we ha e obse ed
ha he an ioxidan capaci y o his desse du ing s o age
emained p ac ically in a iable o 1 yea a di e en em-
pe a u es (8, 21 and 308C; Ga cı
´a-Alonso e al. 2003). In
he p esen s udy, we in es iga ed he e ec o he in ake
o his desse (a ja o 200 g daily o a pe iod o 2
weeks), wi h an an ioxidan ac i i y equi alen o en se -
ings o ui s and ege ables, on se e al ma ke s o oxi-
da i e and an ioxidan s a us in DNA and plasma in a
g oup o elde ly indi iduals.
Subjec s and me hods
Tes p oduc
The es p oduc was an expe imen al desse p epa ed by
he Depa men o Resea ch and De elopmen o He o
Spain S.A. (Alcan a illa, Mu cia, Spain). This p oduc
was o mula ed and designed using he da a o o al an iox-
idan ac i i y a ailable in he scien i ic li e a u e o he
ui s used (Cao e al. 1996; Wang e al. 1996). The objec-
i e was o each, pe se ing (a ja o 200 g), an a e age
an ioxidan capaci y equi alen o en se ings o ui s
and ege ables, 2- old highe han he ‘ i e a day’ ec-
ommended by se e al in e na ional o ganisa ions (Wo ld
Heal h O ganiza ion, 1990; Wo ld Cance Resea ch Foun-
da ion and Ame ican Ins i u e o Cance Resea ch, 1997).
The majo ing edien was wa e , which was mixed wi h
comme cially a ailable concen a ed juices o g ape
(26 %), che y (2 %), blackbe y (0·6 %), blackcu an
(0·6 %) and aspbe y (1 %). Pec in was added o jelli y
he p oduc . The esul ing p oduc was pas eu ised in
o de o ob ain a mic obiologically s able oods u and
he p oduc was bo led ho in ja s o ensu e headspace
acuum.
Fo he cha ac e isa ion o he desse , se e al physical–
chemical pa ame e s and nu i ional composi ion we e
analysed. To al i a able acidi y, pH and soluble solids
we e measu ed ollowing he p ocedu es desc ibed by
Shams & Thompson (1987). P oxima e composi ion o
he desse (mois u e, ash, o al p o ein, o al a and o al
die a y ib e) was analysed by he o icial me hods o he
AOAC In e na ional (1999) and ene gy was calcula ed
based on he mac onu ien composi ion. To al phenols in
he desse we e analysed spec opho ome ically using a
Folin–Denis eagen ollowing he AOAC In e na ional
(1999) me hod. The majo phenolic compounds we e ana-
lysed by HPLC acco ding o he me hod desc ibed by
Can os e al. (2000) and i amin C con en was measu ed
by HPLC, as desc ibed by Es e e e al. (1995). The in
i o o al an ioxidan ac i i y was assessed by he
T olox-equi alen an ioxidan capaci y (TEAC) assay and
by he e ic- educing abili y o plasma (FRAP) assay, as
desc ibed la e (p. 945). All analyses we e made in 3o
p oo s and he esul s a e shown in Table 1.
S udy design
Non-smoking ins i u ionalised elde ly subjec s we e
ec ui ed om a pool o olun ee s in an old-age home
in Mu cia (Spain). The p o ocol was ca e ully explained
o he olun ee s and hei w i en in o med consen was
Table 1. A e age daily in ake o ene gy and nu ien s in a g oup o
elde ly indi iduals*
(Mean alues and s anda d de ia ions)
RDA†
A e age daily
in ake (n17)
Nu ien Men Women Mean SD
Ene gy (kJ) 9614 7942 7610 1009
Ene gy (kcal) 2300 1900 1821 242
P o eins (g) 63 50 76 13
Ca bohyd a es (g) – – 213 37
Lipids (g) – – 76 14
Sa u a ed a y acids (g) – – 19 4
MUFA (g) – – 29 8
PUFA (g) – – 10 4
Choles e ol (mg) ,310 ,310 264 50
Die a y ib e (g) 25 25 18 5
Vi amin A (mg) 1000 800 1526 1183
Vi amin B
1
(mg) 1·2 1 1·4 1·1
Vi amin B
2
(mg) 1·4 1·2 1·57 0·61
Vi amin B
6
(mg) 2 1·6 1·8 0·2
Vi amin B
12
(mg) 2 2 5·3 2·8
Vi amin C (mg) 60 60 157·6 85
Vi amin D (mg) 5 5 3·5 2·2
Vi amin E (mg) 10 8 5·7 1·6
Niacin (mg) 15 13 20·1 3·9
Folic acid (mg) 200 180 248·3 76·1
Na (mg) – – 1443 342
K (mg) – – 2955 524
Ca (mg) 800 800 859 151
P (mg) 800 800 1144 168
Ca:P a io 1 1 0·76 0·14
Mg (mg) 350 280 272·5 51·6
Fe (mg) 10 10 12·4 2·5
Zn (mg) 15 12 6·3 1·5
I(mg) 150 150 59·3 17·1
* Fo de ails o subjec s and p ocedu es, see p. 944.
† Acco ding o he Na ional Resea ch Council (1989) o men and women
o e 50 yea s old.
M. C. Ramı
´ ez-To osa e al.944
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ob ained. Twen y- wo subjec s (six men and six een
women) pa icipa ed in he s udy. The a e age age was
78·88 ( ange 65–92) yea s and a e age BMI was 30·98
( ange 23·83–37·58) kg/m
2
. A con ol g oup o elde ly ol-
un ee s (n8) who did no ecei e he ea men was
included in o de o es ablish ha any changes in pa a-
me e s we e no me ely due o he e ec s o ime. A
non-smoking, heal hy g oup o wel e subjec s (six men
and six women; age ange 25–50 yea s), ec ui ed om
a pool o olun ee s o He o Spain S.A. (Alcan a illa,
Mu cia, Spain), we e also s udied o se e as a heal hy
e e ence g oup.
The p esen s udy was app o ed by he local esea ch
e hics commi ee o Mu cia Uni e si y and by he local
go e nmen (ISSORM, Mu cia, Spain) and complied wi h
Helsinki guidelines o clinical s udies.
The p esen s udy was designed as a andomised in e -
en ion ial wi h a pe iod o 2 weeks. The subjec s
ecei ed he desse and we e ins uc ed o main ain hei
usual die and o consume one se ing (a ja o 200 g) o
es p oduc daily o 2 weeks, a a sel -selec ed ime bu
no eplacing a meal. The subjec s we e also ins uc ed o
s o e he desse in a e ige a o . The habi ual die o he
subjec s was checked daily wi h 24 h die a y ecalls and
he con en o mac onu ien s and selec ed mic onu ien s
in he die was calcula ed using he compu e p og am
ALIMENTACIO
´N Y SALUD 0698.046 (Bi ASDE Gen-
e al Me
´dica Fa mace
´u ica, Valencia, Spain). A days 1
and 15, blood samples we e collec ed o analyse o al an i-
oxidan capaci y, biochemical pa ame e s, an ioxidan i a-
mins, LDL pe oxida ion, and DNA damage o pe iphe al
blood lymphocy es. Fi e women wi hd ew du ing he
s udy due o hei dislike o he es p oduc and did no
inish all he expe imen s. Da a o wen y- wo subjec s
we e a ailable on day 1 (elde ly baseline g oup) and da a
o se en een subjec s we e a ailable a he end o he
s udy (elde ly ea ed g oup). To compa e he esul s o
he analysed pa ame e s, blood samples we e also collec ed
om he non-smoking, heal hy e e ence g oup.
Blood sample collec ion
A baseline and a he end o he s udy pe iod, blood
samples we e collec ed by enepunc u e om as ing sub-
jec s. Fo he analysis o o al an ioxidan capaci y,
asco bic acid, Fe, albumin, bili ubin and u ic acid in
se um, 10 ml blood we e collec ed in o e acua ed glass
ubes (Venojec ; Te umo, Leu en, Belgium) and allowed
o clo a oom empe a u e o 25 min. Samples we e
immedia ely cen i uged a 1000 g o 15 min a 48C o
eco e he se um. Se um samples we e dep o einised o
asco ba e de e mina ion be o e eezing and s o ed a
2808C un il analysed. Fo analysis o e inol, a- oco-
phe ol, b-ca o ene and ubiquinol in plasma, lipid pe oxi-
da ion ma ke s and DNA damage o pe iphe al blood
lymphocy es, 10 ml blood we e collec ed in o e acua ed
glass ubes con aining K
3
–EDTA (Venojec ). Samples
we e e ige a ed and anspo ed wi hin 4 h o he Ins i u e
o Nu i ion and Food Technology (G anada, Spain).
Se um o al an ioxidan capaci y
In o de o o e come p oblems and elimina e he edious
de e mina ion o indi idual an ioxidan s, me hods capable
o measu ing he an ioxidan ac i i y o all he compounds
p esen in a sample wi h one simple de e mina ion ha e
been de eloped, including he TEAC and he FRAP
assays. These o al an ioxidan assays a e use ul in ge ing
a global pic u e o ela i e an ioxidan ac i i ies in oods,
body luids and issues, and how hey change in clinical,
physiological and pa hological condi ions.
Se um TEAC was measu ed by using he me hod o
Mille e al. (1993) wi h comme cially a ailable ki s
(To al An ioxidan S a us, NX 2332; Randox Labo a o ies
L d, C umlin, Co. An im, UK). This me hod is based on
he inhibi ion by an ioxidan s o he abso bance o he ad-
ical ca ions o 2,20-azinobis(3-e hylbenzo hiazoline 6-sul-
ona e) (ABTS) a 600 nm. ABTS adical ca ions a e
o med by he incuba ion o ABTS wi h me myoglobin
and H
2
O
2
. The inal esul s a e exp essed as mmol
T olox equi alen s/l. The inhibi ion o 1 T olox equi al-
en /l equals he inhibi ion p oduced by 1 mmol T olox/l.
Se um FRAP was de e mined by he me hod o Benzie
& S ain (1996). The FRAP assay measu es he e ic- edu-
cing abili y o plasma o se um. A low pH, when a e ic
(Fe
3þ
)– ipy idyl iazine complex is educed by an ioxi-
dan s o he e ous (Fe
2þ
) o m, an in ense blue colou
wi h an abso p ion maximum a 593 nm de elops. In he
FRAP assay, Fe
2þ
was used as a s anda d. The inal esul s
we e exp essed as mmol Fe
2þ
equi alen s/l. The uni o 1
Fe
2þ
equi alen /l equals he amoun o Fe
2þ
/l equi ed o
gi e he same abso bance change.
Biochemical pa ame e s
Fe, albumin, bili ubin and u ic acid we e measu ed in
se um by using a Cobas Mi a Plus Chemis y Analyse
(ABX Diagnos ics, Mon pellie , F ance) wi h eagen ki s
pu chased om ABX Diagnos ics.
Co-enzyme Q
10
and an ioxidan i amin de e mina ion
Fo asco bic acid ( i amin C) de e mina ion, se um samples
we e added o one olume o 10 % (w/ ) me aphos-
pho ic acid con aining 0·54 mmol Na
2
–EDTA, agi a ed in
a o ex mixe and cen i uged a 7200 g o 5 min o pelle
he p ecipi a ed p o eins. The supe na an ac ion was
emo ed and s o ed a 2808C un il analysed. Se um
asco bic acid was assayed by using eagen ki s o colo i-
me ic de e mina ions (Bo
¨eh inge -Mannheim, 409677,
Mannheim, Ge many).
Analyses o co-enzyme Q
10
, e inol, b-ca o ene and a-
ocophe ol we e assayed acco ding o MacC ehan (1990)
by e e sed-phase HPLC using a Sphe iso b S5 ODS1
(Me ck, Da ms ad , Ge many) column and e hanol–pu i-
ied wa e (97:3, / ) as he mobile phase. The HPLC
sys em was a Beckman in-line diode a ay de ec o ;
model 168 (Fulle on, CA, USA) connec ed o a Wa e s
717 plus au osample (Mil o d, MA, USA). The column
was main ained a a cons an empe a u e o 228C. Co-
enzyme Q
10
, e inol, b-ca o ene and a- ocophe ol we e
Oxida i e s ess s a us in he elde ly 945
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iden i ied by p ede e mining he e en ion imes o indi id-
ual pu e s anda ds.
De e mina ion o low-densi y lipop o ein oxida ion
suscep ibili y
LDL was isola ed as desc ibed by Chung e al. (1981).
LDL p o ein was measu ed by he B ad o d (1979)
me hod. To s udy he suscep ibili y o oxida ion o LDL,
wo de e mina ions we e pe o med; hioba bi u ic-acid-
eac i e subs ances (TBARS) and conjuga ed dienes.
LDL p o ein (100 mg/l) was oxidised in he p esence o
Cu
2þ
(10 and 20 mmol/l) in PBS o 6 h a 378C (Jialal &
G undy, 1991). The lipid pe oxide con en o oxidised
LDL was de e mined as TBARS acco ding o Buege &
Aus (1978). Conjuga ed dienes in LDL we e ca ied ou
acco ding o Puhl e al. (1994) in a Pe kin Elme UV-
VIS Lambda 40 spec ome e (F emon , CA, USA)
equipped wi h an au o-cell holde and con olled by a Pel-
ie elemen a he empe a u e o 378C. The lag phase and
slope we e calcula ed using he Pe kin Elme UV-
WINLAB so wa e.
DNA oxida i e damage (come assay)
Pe iphe al blood was collec ed and he ‘bu y coa ’,
en iched in e y h ocy es, was emo ed, dilu ed 1:1 wi h
RPMI-1640 medium, laye ed on o an equi alen olume
o His opaque o ob ain pe iphe al blood lymphocy es.
The come assay was used o measu e DNA s and
b eaks in he cells (Collins e al. 1996).
S a is ical analyses
Be o e any s a is ical analysis, all a iables we e checked
o no mali y and homogeneous a iance using he Kolmo-
go o –Smi no and he Le ene es s, espec i ely. When a
a iable was ound no o ollow no mali y, i was log- ans-
o med and eanalysed. A S uden ’s es was pe o med o
e alua e di e ences be ween baseline and a e - ea men
pa ame e s in he elde ly indi iduals. All pa ame e s o
he elde ly baseline, elde ly ea ed and heal hy e e ence
g oups we e analysed by a one-way ANOVA; o e alua e
mean di e ences om elde ly baseline and elde ly ea ed
g oups . heal hy e e ence g oup a mul iple compa ison
es adjus ed by Bon e oni co ec ions was pe o med. A
P alue o less han 0·05 was conside ed signi ican . Da a
we e analysed using a s a is ical so wa e package (SPSS
o Windows, 11.0.1.; SPSS Inc., Chicago, IL, USA).
Resul s
Composi ional indices, o al an ioxidan capaci y, phenolic
p o ile and i amin C con en in he desse made o
g apes, che ies and be ies a e shown in Table 2.
The p oduc showed a high wa e con en , whils he con-
cen a ions o he nu ien s such as p o ein, ib e and min-
e als we e e y low, wi h le els unde 0·5 % o o al
weigh . Fa was no de ec ed in he composi ional analysis.
To al phenol con en was 1904·21 mg/kg, whe eas he main
phenolics we e an hocyanins, ollowed by hyd oxycinnamic
acids, s ilbenoids, la onols and ellagic acids. This p oduc
showed a high an ioxidan capaci y wi h a mean ac i i y o
18·22 mmol TEAC/l and 23·65 mmol Fe
2þ
/l, o bo h
me hods assayed.
The a e age daily in ake o ene gy and nu ien s
(Table 1) was compa ed wi h he RDA acco ding o he
Na ional Resea ch Council (1989). To al ene gy in akes
o women and men we e below he RDA, bu he subjec s
showed a s able weigh (body-weigh changes we e less
han 1 kg) du ing he in e en ion pe iod. As ega ds he
mac onu ien s, he e we e high in akes o p o ein and
a , which ep esen ed 16 and 36 % o he o al ene gy
in ake, espec i ely. The in ake o mic onu ien s showed
a high a iabili y, depending on he subjec s. Vi amin A
and i amin C in akes me he RDA bu showed a la ge
s anda d de ia ion. The die p o ided only i amins D
and E in amoun s below he RDA, showing he same pa -
e n as he o al in ake o Zn and I.
No s a is ical di e ences we e ound in all pa ame e s
analysed be ween elde ly subjec s a e he in ake o he
high an ioxidan desse and he elde ly con ol g oup a
he end o he s udy.
Se um an ioxidan capaci y and biochemical measu es
ob ained om he subjec s a e ea men compa ed wi h
he baseline and he heal hy e e ence g oup a e shown
in Table 3. The e we e no signi ican di e ences in he
an ioxidan ac i i y and he biochemical pa ame e s
measu ed in he se um o elde ly indi iduals a e he in e -
en ion s udy pe iod. In addi ion, he da a o TEAC and
FRAP o he elde ly indi iduals we e no di e en han
hose ob ained in he heal hy e e ence g oup. Only u ic
acid was signi ican ly lowe (P,0·05) in he heal hy
e e ence g oup compa ed wi h da a om he elde ly
indi iduals.
Table 2. Composi ional indices, o al an ioxidan ac i i y, phenolics
p o ile and i amin C con en o he desse *
(Mean alues and s anda d de ia ions)
Mean SD
Ene gy (kJ/100 g)† 338·00 2·10
Ene gy (kcal/100 g)† 81·00 0·50
Mois u e (%) 79·18 0·10
To al p o ein (%) 0·50 0·02
To al a (%) nd
To al die a y ib e (%) 0·28 0·00
Ash (%) 0·34 0·02
To al soluble solids (8B ix) 21·00 1·12
pH 3·65 0·01
To al i a able acidi y (% ci ic acid) 0·62 0·02
TEAC (mmol/l) 18·22 1·35
FRAP (mmol/l) 23·65 0·85
To al phenols (mg/kg) 1904·21 89·58
An hocyanins (mg/kg) 224·50 1·10
Hyd oxycinnamic acids (mg/kg) 71·97 2·97
S ilbenoids (mg/kg) 4·70 0·45
Fla onols (mg/kg) 30·73 1·40
Ellagic acids (mg/kg) 3·24 0·14
Vi amin C (mg/kg) 138·80 0·50
nd, No de ec ed; 8B ix, be e age such as juice; TEAC, T olox-equi alen
an ioxidan capaci y; FRAP, e ic- educing abili y o plasma.
* Fo de ails o p ocedu es, see p. 945.
† Calcula ed based on he mac onu ien composi ion.
M. C. Ramı
´ ez-To osa e al.946
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No di e ences we e ound in plasma lipid an ioxidan
i amins (a- ocophe ol, b-ca o ene and e inol) and co-
enzyme Q
10
alues in elde ly subjec s a e he in ake o
he high an ioxidan desse (Table 4). I is impo an o
emphasise ha plasma an ioxidan i amin le els (a- oco-
phe ol, e inol, b-ca o ene) and co-enzyme Q
10
in he
elde ly subjec s we e below hose conside ed as no mal
alues. (Human heal hy a e ages a e: e inol, 2·27 mmol/
l; a- ocophe ol, 23·5 mmol/l; b-ca o ene, 0·35 mmol/l; co-
enzyme Q
10
, 0·60 mmol/l; Cu le & Ma son, 2003.) How-
e e , he e we e signi ican di e ences o hese i amins
be ween he elde ly and heal hy g oups. Suscep ibili y o
LDL o oxida ion did no change wi h he in ake o he des-
se in he elde ly olun ee s bu he LDL TBARS le els
and LDL lag phase we e signi ican ly di e en om he
heal hy g oup, showing mo e LDL oxida ion in he elde ly
subjec s (Table 4).
DNA s and b eakage did no dec ease in he elde ly
ins i u ionalised indi iduals a e he in ake o he high
an ioxidan desse bu hei alues we e signi ican ly
highe compa ed wi h he heal hy e e ence g oup
(Fig. 1).
Discussion
Ageing is usually de ined as he p og essi e loss o unc-
ion accompanied by dec easing e ili y and inc easing
mo ali y wi h ad ancing age (Ki kwood & Aus ad,
2000). The impo ance o ageing is based, i s , in he
high pe cen age o indi iduals o e 65 yea s (close o
20 %) and he ise in he numbe o indi iduals o e 80
yea s and, second, in he g owing incidence o ageing-
ela ed ch onic diseases such as Alzheime ’s disease, Pa -
kinson’s disease, diabe es and cance . Mo eo e , a leading
cause o dea h among olde indi iduals wo ldwide is as-
cula disease and associa ed ch onic condi ions. The
impac o he die and die a y componen s on ageing and
age-associa ed degene a i e diseases has been widely
ecognised in ecen yea s (Ames e al. 1993; Meydani,
2001). Acco dingly, he e is g ea po en ial o he
p e en ion o hese diseases h ough heal hy li es yles
ha include physical ac i i y and well-balanced die s
(Tucke & Bu anapin, 2001).
The e is inc easing e idence ha he oxida ion o bio-
molecules (DNA, p o eins and lipids) may play a ole in
Table 3. Se um o al an ioxidan capaci y and biochemical measu es in elde ly olun ee s consuming a phenolic- ich desse *
(Mean alues and s anda d de ia ions)
Va iable EB (n22) ET (n17) EC (n8) HR (n12)
Mean SD Mean SD Mean SD Mean SD
TEAC (mmol/l) 0·62
a
0·14 0·66
a
0·16 0·58
a
0·21 0·65
a
0·14
FRAP (mmol/) 0·96
a
0·16 1·01
a
0·17 0·82
a
0·09 0·95
a
0·19
Albumin (g/l) 39·4
a
5·7 40·1
a
3·9 36·0
a
4·4 46·2
b
3·1
Bili ubin (mmol/l) 11·11
a
5·59 12·73
a
5·93 11·12
a
5·5 10·7
a
3·9
Fe (mmol/l) 12·9
a
6·8 15·30
a
6·2 13·1
a
3·1 15·0
a
5·9
U ic acid (mmol/l) 281
ab
72 300
b
77 240
a
70 251
a
88
Asco bic acid (mmol/l) 51
a
26 53
a
15 38
a
11 55
a
22
EB, elde ly baseline g oup; ET, elde ly a e ea men g oup; EC, elde ly con ol g oup; HR, heal hy e e ence g oup; TEAC, T olox-equi alen
an ioxidan capaci y; FRAP, e ic- educing abili y o plasma.
a,b
Wi hin a ow, mean alues wi h unlike supe sc ip le e s we e signi ican ly di e en (P,0·05).
* Fo de ails o subjec s and p ocedu es, see p. 944.
Table 4. Plasma lipid an ioxidan i amins and co-enzyme Q
10
alues, and suscep ibili y o low-densi y lipop o eins o oxida ion in elde ly ol-
un ee s consuming a phenolic- ich desse *
(Mean alues and s anda d de ia ions)
EB (n22) ET (n17) EC (n8) HR (n12)
Va iable Mean SD Mean SD Mean SD Mean SD
Plasma
a-Tocophe ol (mmol/l) 19·0
a
6·3 16·7
a
4·36 17·7
a
5·4 20·5
a
3·8
Re inol (mmol/l) 0·85
a
0·21 0·74
a
0·21 0·80
a
0·30 1·5
b
0·4
b-Ca o ene (mmol/l) 0·29
a
0·1 0·29
a
0·1 0·41
a
0·2 0·8
b
0·4
Co-enzyme Q
10
(mmol/l) 0·18
a
0·08 0·26
a
0·15 0·15
a
0·03 0·52
b
0·19
LDL
Conjuga e dienes
Phase-lag (min) 53·8
a
12 55·1
a
11 53·3
a
13·3 81·3
b
27
Slope 0·02
a
0·09 0·02
a
0·009 0·014
a
0·004 0·02
a
0·01
TBARS (nmol/mg LDL p o ein)
10 mM(Cu
2þ
)27·7
b
10·8 34·16
b
10·5 27·4
b
11·5 19·2
a
4·7
20 mM(Cu
2þ
)33·9
b
12·0 30·05
b
11·1 30·5
b
11 20·3
a
3·0
EB, elde ly baseline g oup; ET, elde ly a e ea men g oup; EC, elde ly con ol g oup; HR, heal hy e e ence g oup; TBARS, hioba bi u ic-acid, subs ances.
a,b
Wi hin a ow, mean alues wi h unlike supe sc ip le e s we e signi ican ly di e en (P,0·05).
* Fo de ails o subjec s and p ocedu es, see p. 944.
Oxida i e s ess s a us in he elde ly 947
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he suscep ibili y o disease, especially in ageing- ela ed
condi ions such as cance and hea disease, and in he
ageing p ocess i sel (P yo , 1987; Halliwell & Chi ico,
1993). Animal da a ha e shown ha DNA damage
accumula es wi h age (Rich e e al. 1988) and, as
Ha man (1956) sugges ed almos hal a cen u y ago in
his ee adical heo y o ageing, oxida i e damage is
ela ed o he debili ies associa ed wi h ageing. Conse-
quen ly, i would seem ha he oxida i e s ess s a us
and an ioxidan s a us o he elde ly popula ion should be
o conside able in e es and impo ance. The p esen esul s
a e in ag eemen wi h hose o T e isan e al. (2001).
These au ho s demons a ed ha ageing is associa ed
wi h inc eased oxida i e s ess and educed an ioxidan
po en ials, as has been epo ed in he p esen s udy o
he low TEAC, FRAP, plasma an ioxidan i amin
alues and he highe suscep ibili y o LDL o Cu-induced
oxida ion in elde ly olun ee s a he beginning o he
s udy compa ed wi h he heal hy e e ence g oup.
In he las ew yea s, much a en ion has been ocused on
he an ioxidan p ope ies o la onoids, a la ge class o
polyphenolic compounds de i ed om plan s. E idence
sugges s ha hese compounds may p o ec issues agains
damage caused by oxygen ee adicals and lipid pe oxi-
da ion (Bub e al. 2003). The an ioxidan capaci y o he
desse used in he p esen s udy was highe han ha
obse ed in many ui s and ege ables, being simila o
ha o an ioxidan be e ages such as ea and ed wine
(Ga cı
´a-Alonso e al. 2003). The an ioxidan ac i i y o
he p oduc seems o be ela ed o i s phenolic compounds,
since he e was a posi i e and signi ican co ela ion
be ween an hocyanin con en and o al an ioxidan ac i i y
as assessed by he TEAC and FRAP assays.
We hough ha maybe supplemen a ion wi h la onoids
in ou elde ly olun ee s would help o imp o e hei an i-
oxidan s a us and consequen ly a enua e hei oxida i e
s a us.
The main la onoid used in ou desse was an hocyanin
(224 mg/kg). We ha e p e iously desc ibed an hocyanin as
endowed wi h an ioxidan e ec s because i dec eases indi-
ces o lipid pe oxida ion and DNA damage in i amin
E-deple ed a s (Rami ez-To osa e al. 2001). Fu he -
mo e, o he s udies ha e epo ed ha an hocyanins a e
abso bed in elde ly women, inding a high le el o hese
compounds in plasma and u ine a e hei in ake (Cao
e al. 2001). Howe e , we did no ind a signi ican e ec
a e supplemen a ion wi h ou desse on biological ma -
ke s o oxida i e s ess, plasma an ioxidan de ence and
LDL oxidisabili y in he elde ly olun ee s and, maybe,
o ge e ec s as an ioxidan s equi es doses a in excess
o ha which is nu i ionally ele an . The e o e, he p e-
sen esul s ha e con i med he iew ha he ma ked an i-
oxidan abili y o polyphenols in i o does no ansla e o
analogous e ec s in i o.
The p esen esul s a e in acco dance wi h Young e al.
(2002), who ound ha an in e en ion in heal hy human
subjec s wi h 18·6 mg ca echin/d o 6 weeks did no
a ec ma ke s o oxida i e s ess and an ioxidan s a us,
including plasma o haemoglobin p o ein oxida ion,
plasma oxida ion and plasma lipid an ioxidan i amins.
Fu he mo e, Hininge e al. (2001) did no ind any
e ec o lu ein (15 mg), lycopene (15 mg) o b-ca o ene
(15 mg) supplemen a ion on biochemical indices o oxi-
da i e s a us in heal hy adul males a e 3 mon hs o in e -
en ion. In all hese s udies he in e en ion pe iod was
longe han in he p esen esea ch. Howe e , i is eason-
able o hink ha since he p oduc es ed he e is much
mo e ich in an ioxidan s han he amoun s used in hose
men ioned s udies, a s udy pe iod o 2 weeks should be
enough o ind an imp o emen in he oxida i e s ess
s a us o he subjec s.
Oxida i e DNA damage accumula es wi h age and is
ela ed o he li espan o he pa icula o ganism, being
also associa ed wi h p ema u e ageing (Beckman &
Ames, 1998). An ioxidan s p o ec he cellula sys em
om oxida i e damage (K insky, 1992) and he consump-
ion o oods ich in an ioxidan s such as i amin E, i a-
min C and polyphenols is associa ed wi h a dec eased
isk o cance and co ona y disease (Bye s & Pe y,
1992; He og e al. 1995). The s udy o bioma ke s o
DNA damage ( o example, he come assay) and bio-
chemical ma ke s ( o example, plasma an ioxidan s) as
pu a i e indica o s o ageing is used inc easingly o p o-
ide a ocused and mechanis ic app oach o he s udy o
die , heal h and disease. Using his app oach, we decided
o measu e he po en ial e ec o ou desse ich in la o-
noids on DNA damage. Un o una ely, we did no ind any
e ec on DNA s and b eakage. The eason o his,
maybe, is he sho in e en ion pe iod (2 weeks) and he
poo an ioxidan s a us o he elde ly olun ee s con i med
by he high endogenous DNA damage a he beginning o
he s udy. Howe e , Pool-Zobel e al. (1997) showed ha
endogenous DNA s and b eakage was educed in human
lymphocy es isola ed om subjec s gi en supplemen al
ege able juice ( oma o juice wi h lycopene, ca o juice
wi h b-ca o ene and spinach wi h lu ein in wa e ) o 2
weeks each. O he s udies showed ha p olonged sup-
plemen a ion (80 d) wi h a comme cially a ailable ui
and ege able ex ac also dec eased DNA s and b eakage
in elde ly olun ee s (Smi h e al. 1999). Lymphocy es
isola ed om human olun ee s ed a die supplemen ed
wi h lycopene (16·5 mg) o 21 d we e mo e esis an o
ex i o H
2
O
2
ea men compa ed wi h lymphocy es om
he un ea ed con ols (Riso e al. 1999).
Fig. 1. Pe iphe al lymphocy e DNA s and b eakage a e he in ake
o a high an ioxidan desse . Values a e means, wi h s anda d de i-
a ions ep esen ed by e ical ba s. EB, elde ly baseline g oup
(n22); ET, elde ly a e ea men g oup (n17); EC, elde ly con ol
g oup (n8); HR, heal hy e e ence g oup (n12). Mean alues wi h
unlike supe sc ip le e s we e signi ican ly di e en (P,0·05).
M. C. Ramı
´ ez-To osa e al.948
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I can be conside ed ha any changes in he pa ame e s
we e no due o he e ec o ime because no di e ences
be ween elde ly subjec s a e he in ake o he high-an iox-
idan desse and he elde ly con ol g oup a he end o he
s udy we e ound. Finally, i is impo an o keep in mind
ha elde ly subjec s ha e a isk o ma ginal de iciency
o lipid an ioxidan i amins such as a- ocophe ol, e inol
and b-ca o ene. Die a y componen s wi h a high an ioxi-
dan ac i i y ha e o ecei e pa icula a en ion because
o hei po en ial ole in modula ing oxida i e s ess associ-
a ed wi h ageing and ch onic condi ions (Meydani, 2001).
The conclusion o he p esen s udy is ha an in e en-
ion o 2 weeks wi h ou desse en iched wi h na u al poly-
phenol compounds in elde ly indi iduals does no p o ide
enough ime o ind changes in he an ioxidan and oxi-
da i e s a us. We ha e con i med he iew ha he
ma ked an ioxidan abili y o polyphenols in i o does
no ansla e o analogous e ec s in i o. Mo eo e , a
highly oxida i e s ess s a us du ing ageing was con i med,
oge he wi h he need o conduc ollow-up nu i ional
s udies o imp o e his si ua ion.
Acknowledgemen s
The p esen s udy was suppo ed by a g an om He o
Espan
˜a S.A. M. C. R.-T. and J. L. Q. a e ecipien s o a
‘Ramo
´n y Cajal’ con ac om he Minis y o Science
and Technology and he Uni e si y o G anada, Spain.
We also hank he Fundacio
´nSe
´neca o he C.A.R.M.
(Mu cia, Spain) o he ellowship o J. G.-A.
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