Research in Developmental Disabilities 45–46 (2015) 14–20

Contents lists available at ScienceDirect

Research in Developmental Disabilities

Persistence of the benefit of an antioxidant therapy in children

and teenagers with Down syndrome
Eduardo Benedetti Parisotto a, Andréia Gonçalves Giaretta b, Ariane Zamoner c,
Emilia Addison Machado Moreira d, Tânia Silvia Fröde e,
Rozangela Curi Pedrosa c, Danilo Wilhelm Filho a,*
a
Department of Ecology and Zoology, Federal University of Santa Catarina, Florianópolis, Brazil
b
Department of Nutrition, Federal Institute of Education of Santa Catarina, Florianópolis, Brazil
c
Department of Biochemistry, Federal University of Santa Catarina, Florianópolis, Brazil
d
Department of Nutrition, Federal University of Santa Catarina, Florianópolis, Brazil
e
Department of Clinical Analysis, Federal University of Santa Catarina, Florianópolis, Brazil

A R T I C L E I N F O A B S T R A C T

Article history: This study examined the effect of an antioxidant intervention in biomarkers of
Received 20 March 2015 inflammation and oxidative stress (OS) in the blood of Down syndrome (DS) children
Received in revised form 8 July 2015 and teenagers during four different stages. A control group was composed by healthy
Accepted 14 July 2015
children (n = 18), assessed once, and a Down group composed by DS patients (n = 21)
Available online
assessed at the basal period (t0), as well as after 6 months of antioxidant supplementation
(t1), after 12 months (after interruption of the antioxidant intervention for 6 months) (t2),
Keywords:
and again after further 6 months of antioxidant supplementation (t3). Biomarkers of
Down syndrome
Reactive oxygen species inflammation (myeloperoxidase activity – MPO and levels of IL-1b and TNF-a) and OS
Oxidative stress (thiobarbituric acid reactive substances – TBARS, protein carbonyls – PC), reduced
Antioxidant therapy glutathione (GSH), uric acid (UA) and vitamin E levels, as well as antioxidant enzymes such
Persistence effect as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GPx), glutathione
reductase (GR), glutathione-S-transferase (GST) and gamma-glutamyltransferase (GGT)
activities, were measured after each period. After the antioxidant supplementation, the
activities of SOD, CAT, GPx, GR, GGT and MPO were downregulated, while TBARS contents
were strongly decreased, the contents of GSH and vitamin E were significantly increased,
and no changes in G6PD and GST activity as well as in UA and PC levels were detected. After
the interruption of the antioxidant therapy for 6 months, DS patients showed elevated GPx
and GGT activities and also elevated UA and TBARS levels. No changes in SOD, CAT, GR, GST,
G6PD and MPO activities as well as in GSH, vitamin E, PC, TNF-a and IL-1b levels were
detected. The results showed that the antioxidant intervention persistently attenuated the
systemic oxidative damage in DS patients even after a relatively long period of cessation of
the antioxidant intervention.
ß 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Down syndrome (DS) or Trisomy 21 is the most common chromosome abnormality in humans and oxidative stress (OS)
is part of the fundamental biology of DS (Pagano & Castello, 2012; Parisotto et al., 2014; Pueschel, 1990). This increased OS

* Corresponding author at: Departamento de Ecologia e Zoologia, CCB, UFSC, Trindade, Florianópolis 88040-900, Brazil.
E-mail address: dawifi@ccb.ufsc.br (D.W. Filho).

http://dx.doi.org/10.1016/j.ridd.2015.07.010
0891-4222/ß 2015 Elsevier Ltd. All rights reserved.
 E.B. Parisotto et al. / Research in Developmental Disabilities 45–46 (2015) 14–20 15

involves one of the key enzymes associated with the detoxification of reactive oxygen species (ROS), Cu–Zn superoxide
dismutase (SOD-1), which is located on chromosome 21. There is consequently a gene dosage effect in DS leading to 50%
greater levels of SOD-1 and elevated endogenous production of hydrogen peroxide (H2O2), and potentially of hydroxyl
radical (OH) (Garlet et al., 2013; Lott, 2012), which is the most reactive and deleterious ROS inducing damage in proteins,
lipids and DNA (Halliwell & Gutteridge, 2007).
We previously demonstrated that DS individuals presented depleted levels of GSH (Parisotto et al., 2014). The induction
of GGT and G6PD activity found in the present study is probably counteracting the GSH depletion. The complex redox system
of GSH involves GPx, GR, GST, as well as GGT. The redox cycling of oxidized glutathione is catalyzed by GR, whereas the
supply of NADPH, the major reducing agent for this redox cycling, is provided by G6PD activity (Halliwell & Gutteridge,
2007). Identification and characterization of early events that contribute to and or regulate the expression of chromosome
21 gene products that are triplicated in DS is crucial to understand the ways in which neurodegenerative cycles and the
mechanisms implicated in promotion of the neuropathophysiological progression in DS. In the brain, three early events,
neuritic amyloid-b plaques (Ab), neurofibrillary tangles and glial activation, have been reported in DS fetuses and each
one is related to the others as they induce, and are induced by each other and also by cytokines subsequent to
neuroinflammatory changes (Wilcock & Griffin, 2013).
We recently demonstrated the efficacy of an antioxidant supplementation during 6 months in attenuating a systemic OS
in DS patients (Parisotto et al., 2014). This is a prospective study carried out to verify the persistence effect of the antioxidant
intervention in 1.5 years of follow-up in biomarkers of inflammation and OS in the blood of DS children and teenagers.

2. Subjects and methods

2.1. Subjects

The control group was constituted of 18 healthy children (average age of 6.7  3.0 years, all showing inflammatory markers
within normal ranges) without DS (10 males and 8 females; 3–12 years), who were recruited from the Joana de Gusmão Children’s
Hospital in Florianópolis, State of Santa Catarina, South Brazil. The DS participants were between 3 and 14 years old, with an
average age of 7.7  3.18 years (n = 21; 12 males and 9 females). Most of the patients were concentrated in the age group 6–10
years (65%). The inclusion criteria were: age between 3 and 14 years without illnesses associated with systemic diseases and not
participating in other studies. Patients taking medication or food supplements were excluded. The 21 DS individuals were
recruited from two local organizations for DS children (‘‘Associação Amigos Down’’ and ‘‘Associação de Pais e Amigos dos
Excepcionais – APAE’’). The study protocol was previously approved by the Ethics Committee of UFSC, according to the national
and international guidelines for research involving human subjects (Resolution No. 1996 of the National Health Council), which
regulate experiments involving human subjects (local Protocol CEP N. 2112/2011). All patients received information about the
study and their parents signed a free and informed consent form.

2.2. Study design

In two recent accompanied papers by Garlet et al. (2013) and Parisotto et al. (2014), both from our laboratory, biomarkers
of OS were evaluated in blood samples collected from children and teenagers with DS, using the same protocol and
experimental design of the present study. However, now the biomarkers of OS were evaluated in the blood of DS patients
(n = 21) before (t0) and after a daily antioxidant intervention (vitamin E 400 mg, C 500 mg, E-TABS1 and Energil C1,
respectively) during 6 months (t1), as well as after an interruption of the supplementation (also after 6 months) (t2) and after
a new supplementation of further 6 months (t3). The dose was lower (less than half) of that recommended by Nathens et al.
(2002) for adults, which is based in the upper intake level (UL), showing that it is safe and presenting no adverse effect
(Kappus & Diplock, 1992).

2.3. Sample preparation

Whole blood was obtained from the antecubital vein in chilled tubes containing EDTA as anticoagulant, or without EDTA to
obtain serum. Immediately after blood collection a blood fraction (200 mL) was precipitated in trichloroacetic acid (TCA 12%, 1:4
v/v) for reduced glutathione (GSH) assays. The remaining blood was centrifuged at 1500  g for 10 min to separate red cells from
plasma. For enzymatic assays, red cells were diluted in distilled water (1:4) and hemolysis was carried out by freezing and thaw
procedure. After this, plasma, serum and the acid extracts were stored in liquid nitrogen (170 8C) until analysis of the
parameters. Enzymatic evaluations were carried out in hemolysates, while the contents of GSH were obtained in whole blood
acid extracts. Thiobarbituric acid reactive substances (TBARS), protein carbonyl and vitamin E contents were examined in
plasma. The myeloperoxidase (MPO) activity and the uric acid (UA), TNF-a e IL-1b contents were analyzed in serum.

2.4. Reduced glutathione (GSH) assay

Reduced glutathione (GSH) was measured at 412 nm (Beutler, Duron, & Kelly, 1963) by using the reagent DTNB
(2-dithionitrobenzoic acid). After centrifugation at 3000  g for 5 min, the supernatants from the acid extracts were added
16 E.B. Parisotto et al. / Research in Developmental Disabilities 45–46 (2015) 14–20

to 0.25 mM DTNB in 0.1 M sodium phosphate buffer, pH 8.0, and the formation of the corresponding thiolate anion was
immediately determined. GSH contents were expressed in mmol mL1 whole blood, by using e = 14.1 mmol1 L cm1.

2.5. Determination of serum uric acid (UA)

Serum concentration of uric acid (UA) was determined spectrophotometrically by using Analisa1 kit based on the
oxidation of UA by uricase producing a red chromophore measured at 520 nm.

2.6. Vitamin E assay

Determination of vitamin E in plasma was carried out by high performance liquid chromatography (HPLC) with UV
detection at 292 nm (Nicoletti et al., 2001). An aliquot of 100 mL of plasma was added to 100 mL of ethanol and vortexed
for 10 s and added to 100 mL of hexane, and again vortexed for 45 s. After centrifugation (8000  g) for 5 min, 75 mL of
the supernatant (hexane) was transferred to a microtube and the hexane was evaporated by nitrogen flow. Then 125 mL
diethylether and 375 mL methanol were added and this mixture was injected in HPLC. Isocratical elution was carried
out with methanol using a flow rate of 1 mL min1. Samples were measured in duplicate and plasma concentrations
of a-tocopherol were determined through a standard curve and expressed as mmol vitamin E mL1.

2.7. Antioxidant enzyme assays

Catalase (CAT) activity was determined by measuring the decrease in a freshly prepared 10 mM hydrogen peroxide
solution at 240 nm (Aebi, 1984). Superoxide dismutase (SOD) activity was measured at 480 nm according to the method of
epinephrine autoxidation, with modifications (Boveris, Fraga, Varsavsky, & Koch, 1983; Misra & Fridovich, 1972).
Glutathione reductase (GR) activity was determined by measuring the rate of NADPH oxidation at 340 nm (Calberg &
Mannervik, 1985). Glutathione peroxidase (GPx) activity was assayed at 340 nm, through the glutathione/NADPH/
glutathione reductase system, by the reduction of tert-butylhydroperoxide (Flohé & Gunzler, 1984). Glutathione-S-
transferase (GST) activity was determined at 340 nm using CDNB (1-chloro-2,4-dinitrobenzene) as substrate and a 0.15 M
GSH concentration (Habig, Pabst, & Jacoby, 1976). Gamma-glutamyltransferase (GGT) and glucose-6-phosphate
dehydrogenase (G6PD) activity were measured spectrophotometrically at 530 nm using diagnostic kits (Doles1), and
Neolisa G6PD kit (Intercientı́fica1), respectively.

2.8. Lipid peroxidation assessment (TBARS levels)

Lipid peroxidation in plasma was determined spectrophotometrically at 535 nm by the thiobarbituric acid method
(TBARS) (Bird & Draper, 1984). Briefly, plasma was precipitated with trichloroacetic acid (TCA) followed by the incubation
with TBA 0.73% for 60 min in a buffer (60 mM Tris–HCl and 0.1 mM diethylenetriaminepentaacetic acid, DTPA) at pH 7.4 at
high temperature (100 8C). The samples were then centrifuged (5 min, 1500  g) and the absorbance of the pink
chromophore was measured in triplicate, using e = 1.56  105 M1 cm1. The values were expressed in nmol mL1.

2.9. Protein carbonyls (PC)

Oxidative damage promoted by protein carbonylation was determined using 100 mL of plasma, which were added to
600 mL DPNH (2,4-dinitrophenyl-hydrazine). Ethanol/ethyl acetate (1:1; v:v) was used to remove hydrazine excess. After
this, incubation with guanidine chloride (600 mL) was performed and the maximum carbonyl absorbance in the range of
360–370 nm was recorded (Levine, Garland, & Oliver, 1990). The total protein concentration was determined by the method
of Lowry, Rosenbrough, Farr, and Randall (1951), using albumin as standard. The PC concentration was expressed in
nmol mg1. All measurements were carried out in triplicate.

2.10. Myeloperoxidase assay (MPO)

Myeloperoxidase activity was determined as described by Rao, Currie, Shaffer, and Isakson (1993). Samples were thawed
at room temperature and 20 mL were transferred to buckets and the biochemical reaction initiated with the addition of
150 mL of solution (165 mL of o-dianisidine 2-HCl, 50 mL of H2O2 30%, distilled H2O and NaH2PO4 50 mM). After 15 min of
incubation at room temperature, the enzymatic reaction was stopped by the addition of 15 mL of sodium 1% azide. After
incubation for 10 min at room temperature, the optical density was measured at 450 nm in a ELISA reader (Organon-
Tecknica, Roseland, New Jersey, USA), and compared to standard curves with known activities of MPO (0.7–140 mU mL1).
MPO values were expressed as mU mL1.

2.11. TNF-a e IL-1b contends

TNF-a and IL-1b serum concentration was determined from duplicate samples using enzyme-likes immunosorbent
assay (ELISA) kits (BD Biosciences1) according to the manufacturer’s instructions.
 E.B. Parisotto et al. / Research in Developmental Disabilities 45–46 (2015) 14–20 17

2.12. Statistical analysis

Data are obtained from control and DS individuals before and after antioxidant supplementation. Statistical analysis was
performed by analysis of variance (ANOVA) for repeated measures by followed by Tukey–Kramer multiple comparisons test,
assuming a minimum level of significance of p < 0.05. All analyses were performed using the GraphPAD Prism Software
version 5.0.

3. Results

The enzymatic activity of SOD, which was higher (47%) in DS patients before antioxidant supplementation, reached
values close to controls (46% decrease) after administration of the antioxidant vitamins (Parisotto et al., 2014). Interestingly,
even after cessation of the antioxidant intervention, this value remained relatively constant. After a further period of six
months of antioxidant supplementation, these values again remained fairly constant (Table 1). The enzymatic CAT activity
was high (ffi25%) in individuals with DS before antioxidant supplementation. After the vitamin administration, this activity
decreased significantly (ffi15%). Moreover, even after cessation of therapy the values did not significantly change, even after a
further new supplementation (Table 1). However, GPx activity was not significantly different between controls and DS
individuals before the administration of vitamins. However, after the supplementation, a significant decrease of the values of
this analyte (ffi46%) was detected. After withdrawal of supplementation, the values were again close (ffi13%), to the pre-
supplementation values, as well as after a new supplementation intervention, when no statistical differences were found
(Table 1). GR was higher (ffi98%) before the antioxidant therapy, while after 6 months of such therapy there was a significant
decrease (36%) in the activity of this enzyme, and remained almost constant either after stopping the supplementation, or
after the resumption of a further supplementation period (Table 1). Like CAT, SOD and GR, the GGT activity showed higher
values (ffi97%) in DS patients not supplemented. After the antioxidant therapy, there was a significant decrease (ffi37%) in
the activity, which was again increased after the withdrawal of the supplements (ffi163%), while after the interruption of
the supplementation its activity was again significantly decreased (ffi53%) (Table 1). GST activity in DS patients was
significantly decreased (ffi61%) compared to controls before supplementation, but revealed a significant increase (ffi44%)
after the antioxidant action, while no statistical difference was found after resuming the supplementation (Table 1). GSH
concentrations in whole blood were decreased (ffi27%) in DS patients compared to controls, while the two antioxidant
interventions (t1 and t3) were able to restore its levels (Table 1). UA levels were increased before supplementation (ffi19%),
while after supplementation no statistical difference was found, neither when compared to controls nor to the DS
group before supplementation. Compared to controls plasma vitamin E levels were not altered in the group of DS
children before the vitamin supplementation, however, after supplementation a significant increase was detected
(ffi260% and ffi811%, t1 and t3, respectively) (Table 1).
No significant changes were observed in plasma levels of TBARS before the antioxidant supplementation (Table 1). After
discontinuation of the antioxidant therapy TBARS levels were increased (ffi32%). When the supplementation was resumed,
the values tended to decrease, but there was no statistical difference when compared to discontinuation of the
supplementation. PC contents showed decreased values (ffi40%) in the DS group before the antioxidant therapy compared to
the control group, while no statistical difference was observed in the other intervals (Table 1).
The activity of MPO showed a marked increase (ffi420%) before supplementation of vitamin E and C, and a ffi28%
decrease after supplementation was found, while after interruption of the antioxidant intervention and after a further

Table 1
Oxidative stress markers. Enzymatic activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR),
glucose-6-phosphate dehydrogenase (G6PD), gamma-glutamyltransferase (GGT) and glutathione S-transferase (GST) in hemolysates, levels of reduced
glutathione (GSH) in whole blood extracts, contents of uric acid (UA) in serum, levels of vitamin E, thiobarbituric acid reactive substances (TBARS) and
protein carbonyls (PC) in plasma, in Down Syndrome subjects during four different stages (n = 21).

Oxidative stress markers Control Time 0 (t0) Time 1 (t1) Time 2 (t2) Time 3 (t3)
1 ##
SOD (USOD mL ) 63.44  4.43 120.21  8.19*** 56.49  4.91 62.34  10.57 71.57  7.66
CAT (mmol min1 mL1) 118.24  6.39 159.29  3.51*** 135.45  2.14## 142.18  3.25 148.68  5.56
GPx (mmol min1 mL1) 58.81  0.77 56.50  1.87 30.54  2.92## 40.15  1.51 40.05  4.79
GR (mmol min1 mL1) 0.55  0.047 1.09  0.07*** 0.70  0.05## 0.72  0.05 0.68  0.07
G6PD (U gHb1) 9.09  0.55 9.59  1.43 8.27  1.04 8.14  0.59 8.58  1.16
aaa bbb
GGT (UI L1) 7.00  1.01 13.87  2.60* 8.74  1.54# 22.90  1.70 10.79  2.10
b
GST (mmol min1 mL1) 110.56  11.43 42.86  3.74* 61.58  5.52# 85.22  12.98 67.89  8.90
GSH (mmol mL1) 1.31  0.04 0.96  0.14* 1.24  0.21# 1.73  0.54 1.32  0.07
bbb
Vitamin E (mmol mL1) 4.48  0.82 4.90  0.36 16.14  0.51### 17.62  0.40 41.33  14.00
aaa
Uric acid (mg dL1) 3.20  0.19 3.81  0.25* 2.89  0.70 5.63  0.24 4.60  0.46
aaa
TBARS (nmol mL1) 13.62  0.27 12.25  0.98 4.42  0.86### 14.31  2.00 9.37  2.55
PC (nmol mg1) 0.04  0.01 0.02  0.01*** 0.02  0.01 0.02  0.01 0.03  0.01

Data are means  SEM. (*) (***) mean statistical differences for p < 0.05 and p < 0.001 (Time 0, t0 = before a daily antioxidant intervention) compared to controls,
respectively. (#) (###) mean statistical differences for p < 0.05 and p < 0.001 of Time 1 (t1 = after a daily antioxidant intervention) compared to Time 0,
aaa b bbb
respectively. ( ) mean statistical differences for p < 0.001 of Time 2 (t2 = after interruption of the supplementation) compared to Time 1, respectively. ( ) ( )
mean statistical differences for p < 0.05 and p < 0.001 of Time 3 (t3 = new supplementation) compared to Time 2, respectively.
[(Fig._1)TD$IG]
18 E.B. Parisotto et al. / Research in Developmental Disabilities 45–46 (2015) 14–20

Fig. 1. Proinflammatory markers. (A) Enzymatic activity of myeloperoxidase (MPO), (B) contents of tumor necrosis factor alpha (TNF-a), (C) contents of
interleukine 1b (IL-1 b) in serum samples. Data are means  SEM. Asterisks (***) mean statistical differences for p < 0.001 (Time 0, t0 = before a daily
antioxidant intervention) compared to controls. (###) mean statistical differences for p < 0.001 compared to Time 0 (t0 = before a daily antioxidant intervention).

supplementation during six months, the values remained relatively constant (Fig. 1A). TNF-a showed extremely high values
(ffi1626%) in the DS group before supplementation compared to the control group (Fig. 1B). However, after t0, t1 and t2 no
statistical differences were found among these groups (Fig. 1B). Serum contents of IL-1b showed increased (ffi293%) in the DS
group before supplementation compared to the control group, while no statistical difference were detected neither after
supplementation, nor after the interruption of a new vitamin supplementation (Fig. 1C).

4. Discussion

Interestingly, even after cessation of the vitamin supplementation for six months, a relative maintenance or persistence of
systemic antioxidant capacity was observed in the blood of DS patients. More specifically, the majority of the enzymatic
activities (SOD, CAT, GR and GGT), as well as MPO, which were previously decreased after the antioxidant supplementation,
maintained values similar to those displayed just after the supplementation period, while GPx retained intermediate levels,
and GST showed increased activity, equaling that detected in the control group. Moreover, after a further period of 6 months
of vitamin supplementation values again remained fairly constant, except the GGT activity, which was decreased.
In addition and according to the findings related to the antioxidant enzymes, levels of non-enzymatic antioxidant
defenses such as GSH and vitamin E were also very similar to those found after six months supplementation period, whereas
the levels of UA were significantly increased. This indicates the important antioxidant property of UA (Simão, Dichi, Barbosa,
Cecchini, & Dichi, 2008; Sousa et al., 2015; Tiano et al., 2008) in these individuals, because even after cessation of the
supplementation, there was a restoration (increase of 95%) of UA levels in DS patients.
On the other hand, lipid oxidation (TBARS) and protein oxidation (PC) were restored to baseline values. The activity of
G6PD remained unchanged in all groups, suggesting the likely absence of its direct participation in the antioxidant process in
these patients.
As already mentioned, the imbalance between SOD and other antioxidants enzymes such as CAT and GPx induce
oxidative damage in DS patients. More specifically, a three- to four fold increase in intracellular ROS and elevated levels
of lipid peroxidation markers have been detected in the brain of DS patients while vitamin E was able to decrease such
markers (e.g. Shichiri, 2014). This effect was previously demonstrated by Lockrow et al. (2009) showing that vitamin E
supplementation decreased markers of OS in the brain and improved cognitive performance in mice. A similar beneficial
effect was described by Shichiri et al. (2011) using an animal SD model (Ts65Dn mice), in which a-tocopherol was
 E.B. Parisotto et al. / Research in Developmental Disabilities 45–46 (2015) 14–20 19

administered to pregnant Ts65Dn females from the day of conception throughout the pregnancy, as well as from birth until
the end of a behavioral testing period, in which the cognitive performance was also improved.
There are relatively few studies regarding protein damage in the plasma of DS population, and none of them have
reported protein damage after an antioxidant intervention. Zitnanová et al. (2006) evaluated OS biomarkers in the plasma of
DS children, including measurement of PC levels, but they did not find differences between DS population and controls.
According to our result, a recent study in saliva of DS patients revealed no differences in PC levels compared to controls
(Sousa et al., 2015). On the other hand, regarding DNA damage, Jovanovic, Clements, and Macleod (1998) showed significant
changes in biomarkers of OS in the urine of DS patients compared to healthy subjects, including levels of 8-hydroxy-20 -
deoxyguanosine (8-OHdG).
Although the results of t2 showed a small increase in the activity of SOD, CAT, GPx, GR and G6PD, they did not return
to baseline values (t0), showing no statistical differences. In the case of GGT, a large increase (ffi163%) in the activity of
this enzyme was detected. A possible explanation for this result might be a consequence of the increased GSH levels
(ffi39%), since GGT promotes the turnover of GSH levels (Halliwell & Gutteridge, 2007). The vitamin E levels remained
fairly constant in t0 compared to controls, thus suggesting that the increased levels of GSH and UA are indeed playing
their important role as ROS scavengers, since UA is responsible for approximately 60% of plasma ROS elimination,
therefore preserving vitamin E plasma levels (Drozdz et al., 1998; Onur et al., 2014).
Concerning the inflammatory markers, MPO activity as well as the proinflammatory cytokines TNF-a and IL-1b were
elevated in the group of DS children before the intervention with vitamins E and C. After the antioxidant supplementation,
there was a decrease in MPO activity, suggesting that the antioxidant therapy not only decreased ROS production, but also
exhibited an indirect anti-inflammatory action by regulating the expression of proinflammatory cytokines. MPO is an
enzyme present in neutrophils and monocytes that utilizes H2O2 as a co-substrate to generate other ROS (for example HOCl),
with possesses microbicide activity (Halliwell & Gutteridge, 2007).
Concerning inflammatory markers, high levels of IL-1b in DS patients were already reported (Griffin et al., 1989). According
to these authors, the interaction between the precursor protein (APP) of Ab, S100B (which are also tripled) and IL-1b are
increased, leading to multiple neural insults, each one is characterized by glyosis-related neuroinflammation and risk for
development of the characteristic neuropathological changes of Alzheimer’s disease. Genes encoding the receptors for IFN-a1,
IFN-a2 and IFN-g are located on chromosome 21. These receptors activate signaling pathways to induce proinflammatory
cytokines, including expression of IL-1b and TNF-a (Wilcock & Griffin, 2013). However, after supplementation with vitamins E
and C no changes in the levels of these cytokines were detected in the present study. These responses suggest that apparently,
vitamins E and C do not alter the expression of these cytokines. Nevertheless, a study carried out with goat blood showed that
vitamin E was able to control the effect of the OS modulation of SOD and interleukin-2 (IL-2) in animal leukocytes by decreasing
the expression of this enzyme and cytokine levels, respectively (Das et al., 2012).
In the present study a general persistence in the systemic antioxidant capacity of DS patients was observed, even after
6 months of discontinuation of the antioxidant therapy, indicating the prolonged and beneficial effect of this antioxidant
intervention. This persistent attenuation of the systemic OS can probably be attributed to the cumulative effect related to
the lipophilicity of a-tocopherol (Sies, 1991), which enables this important and nutritional antioxidant to penetrate in
different tissues and migrating through the membranes for hydrophobic domains (Wang & Quinn, 1999).
In this sense, the effectiveness of the antioxidant supplementation with vitamins E and C on the depletion of OS markers
had already been seen in previous studies from our research group. In patients with chronic Chagas’ disease (Maçao et al.,
2007; Ribeiro et al., 2010), as well as in patients with hepatitis C (Farias et al., 2012), a significant improvement in the
antioxidant defenses present in the blood of these patients was also found, using the same protocol and experimental design.
Moreover, patients with Chagas’ disease associated with chronic cardiopathy, after an interval of 6 months without
supplementation, also maintained intermediate levels of the same OS markers, not returning to baseline values (Budni,
2011; Budni et al., 2013), in a similar pattern found in the present study.
Despite many controversies regarding the use of antioxidant interventions, there are several consistent studies that
recommend the combined supplementation with vitamins E and C, both preventively as well as therapeutically. Currently,
more than 200 diseases have been reported in experimental studies confirming the occurrence of inherent oxidative
imbalance, such as cardiovascular, neurological, endocrine, respiratory, immunological, cancer, among others (Halliwell &
Gutteridge, 2007; Sellés, 2011). The efficiency of antioxidant interventions might be associated to different approaches
and experimental designs reported in the literature and certainly more information is needed in this polemic subject.

5. Conclusion

The daily intervention with antioxidant vitamins E and C for six months consistently attenuated the systemic OS that
affect children and adolescents with DS. Furthermore, the effect of the antioxidant intervention persisted even after the
interruption of such antioxidant supplementation, but was unable to significantly alter the inflammatory markers evaluated
in the present study.

Conflict of interest

The authors declare that are no conflicts of interest regarding this study.
20 E.B. Parisotto et al. / Research in Developmental Disabilities 45–46 (2015) 14–20

Acknowledgements

This work was supported in part by grants from CNPq (Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico/
MCT/MSSCTIE-DECIT, proc. 409266/2006-0). DWF is a recipient of a research fellowship from CNPq (proc. 300353/2012-0).
EBP is recipient is a fellowship of CAPES (proc. 2606/14-13). We also thank the Brazilian Group EMS Sigma Pharma, who
gently provided vitamins E and C for the antioxidant supplementation and the associations ‘‘Amigos Down’’ and ‘‘APAE’’, as
well the parents of the patients for collaboration in this study.

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