Pol. J. Food Nutr. Sci., 2014, Vol. 64, No. 4, pp.

277–281
DOI: 10.2478/pjfns-2013-0025
http://journal.pan.olsztyn.pl
Original article
Section: Nutritional Research

Antioxidant Vitamins as Oxidative Stress Markers in Rat

Plasma After Physical Exercise – a Short Report

Agata Wawrzyniak1*, Jadwiga Hamułka1, Małgorzata Drywień1, Magdalena Górnicka1,

Jolanta Pierzynowska1, Malwina Wojtaś1, Małgorzata Gajewska2, Joanna Frąckiewicz1, Anna Gronowska–Senger1

1
Chair of Nutritional Assessment, Department of Human Nutrition, Warsaw University of Life Sciences (WULS–SGGW),
Nowoursynowska 159C, 02–776 Warsaw, Poland
2
Chair of Biochemistry, Department of Physiological Sciences,Warsaw University of Life Sciences (WULS–SGGW),
Nowoursynowska 159, 02–776 Warsaw, Poland

Key words: β–carotene, retinol, vitamin E, vitamin C, exercise, rats

The aim of the study was to verify the hypothesis that β–carotene, vitamin E, vitamin C administered individually or in combination may differ-
ently modify their levels in blood plasma being also markers of the oxidative stress. Male Wistar rats were supplemented antioxidants per os (α–to-
copherol – 2 mg/d, ascorbic acid – 12 mg/d, β–carotene – 1 mg/d), both individually or in combination of 2 or 3, for 14 days. During experiment, half
of the animals in each group (n=8) were subjected to treadmill exercise for 15 min at the speed of 20 m/min, to induce oxidative stress. Vitamins in rat
plasma were assessed by the high–performance liquid chromatography (HPLC). Results suggest that vitamin E and C supplemented simultaneously
may provide some benefit during physical exercise. The significant influence of administered α–tocopherol acetate and physical exercise on the level
of α–tocopherol in the plasma was observed. Thus only the concentration of α–tocopherol in blood may be treated as a marker of oxidative stress.

INTRODUCTION be reduced to their original forms by other antioxidants’ (vi-

tamin C, coenzyme Q10) effects. Ascorbate has the ability to
Results of  recent research indicated that intense exer- reduce tocopheryl radicals. This reaction is possible because
cise may enhance the  need for oxygen, increasing produc- of  the  chroman rings in  tocopherol molecules, which face
tion of its reactive forms [Martins et al., 2011; Powers et al., the  outside of  the  cell membrane to form ascorbyl radicals
2011]. The  oxidative damage of  cell components observed [Yeum et al., 2004; Krinsky & Johnson, 2005]. The ascorbic
during physical activity and/or during resting may be an in- radical, in turn, may return to ascorbate form by reacting with
dication for supplementing the diet with antioxidants, which NADPH reductases [Schneider, 2005].
may strengthen the  organism’s antioxidant defence or ac- The present study was undertaken because of a lack of un-
celerate a  return to prooxidative–antioxidative balance. One ambiguous data regarding the benefits of antioxidant vitamin
of  the  biomarkers of  homeostatic disruption in  the  organ- supplementation in defending against oxidative stress induced
ism is  the  level of  low–molecular–weight antioxidants, such physical activity [Lamprecht et  al., 2009; Gomez–Cabrera
as vitamins E and  C and  β–carotene, in  the  plasma [Lam- et al., 2012]. The aim of the study was to verify the hypoth-
precht et  al., 2009; Zeinab et  al., 2010; Böhm et  al., 2012]. esis that β–carotene, vitamin E, vitamin C administered indi-
The  aforementioned studies indicate mutual interaction vidually or in combination may differently modify their levels
of  lipophilic compounds and  their reciprocal regeneration in blood plasma being also markers of the oxidative stress.
from free radical forms as well as effective interaction of vi-
tamins C and E, i.e. ascorbic acid and α–tocopherol acetate, MATERIAL AND METHODS
at the border between the cell’s lipo– and hydrophilic phase
[Yeum et al., 2004; Traber & Stevens, 2011]. β–Carotene’s an- Animals and diet
tioxidant function, as an electron–rich molecule, is  stronger For these experiments, 144 male outbred Wistar rats
with smaller concentrations of oxygen in the cell, but it also from certified breeding (Medical University of Białystok, Po-
complements the antioxidant effect of vitamin E, which works land) with an initial body weight of 120–140 g were involved.
with higher oxygen concentrations. Tocopheryl radicals may The animals were fed a diet (Table 1) according to AIN–93M
[Reeves, 1997], and  had free access to water. Male Wistar
*  Corresponding Author: agata_wawrzyniak@sggw.pl
rats were supplemented antioxidants per os (α–tocopherol
(prof. A. Wawrzyniak) – 2  mg/d, ascorbic acid – 12 mg/d, β–carotene – 1 mg/d),

© Copyright by Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences
©2 014 Author(s). This is an open access article licensed under the Creative Commons Attribution-NonCommercial-NoDerivs License
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
278 Antioxidant Vitamins in Rat Plasma After Exercise

TABLE 1. Composition of experimental diet according to AIN–93M.

after previously reducing the  dehydroascorbic acid with hy-
drochloride tri–carboxyethyl phosphine. The  test samples
Ingredients Content (%)
were separated by a Discovery C18 RP (4.6 x 150 mm; 5 µm)
Caseine 14.0
column from the Supelco Company (Supelco Analytical cat.
Cornstarch 46.5
Dextrose 15.5 no. 504955, Bellafonte, PA, USA) with a  precolumn from
Sucrose 9.0 the  same company. A  2% solution of  water–phase acetoni-
Cellulose 5.0 trile was applied as eluent, which was formed from a  solu-
Soybean oil 4.0
tion of  NaH2PO4, Na2EDTA and  lautrimonium chloride
t–Butylhydroquinone 0.0008
Satl mix 3.5 in  the  following concentrations: 2.5 mmol/L, 1.25 mmol/L,
Vitamin mix 1.0 2.5 mmol/L. The flow rate was 0.75 mL/min. All of the results
L–Cystine 0.18 were applied to standard curves plotted in  accordance with
Choline bitertrate 0.25
Sigma Company (St. Louis, MO, USA) standards.
Supplement (#410950) 1.0

Statistical analysis
both individually or in  combination of  2 or 3, for 14 days. Analysis of  the  variance (ANOVA) was carried out with
During experiment, half of the animals in each group (n=8) the SPSS program (version 17; SPSS Inc, Chicago IL, USA).
were subjected to treadmill exercise for 15 min at the speed Normality and  homogeneity of  the  data distribution were
of 20 m/min, to induce oxidative stress. Design of experiment checked by Shapiro–Wilk test. Data (Table 2) consistent with
was described in  Wawrzyniak et  al. [2013]. The  experiment a  normal distribution were subjected to one–way analysis
was approved by  the  III Local Animal Experiment Ethics of  variance (ANOVA) to assess potential statistical signifi-
Committee at the Warsaw University of Life Sciences (Reso- cance for antioxidant vitamins. When significant differences
lution no. 33/2008). were observed in ANOVA tests, Tukey’s post hoc test was ap-
plied to locate the source of the significant difference. The in-
Blood sampling teraction between physical exercise and vitamins (T × V) was
After the experiment, the rats were anesthetized (intramus- assessed by 2–way analysis of variance. Pearson’s linear cor-
cular injection of ketamine and xylazine) at the same time af- relation coefficient (r) was calculated to analyse the strength
ter the end of the training [Wawrzyniak et al., 2013] and blood of the relationship between individual variables. For all analy-
was taken from the left ventricle of the heart. Heparin sodium ses, a=0.05 was the  level of  statistical significance. The  re-
was used as an anticoagulant. The blood was centrifuged at sults are expressed as means and SDs.
4000 rpm for 10 min at 4°C. The plasma was frozen in liquid
nitrogen and preserved at –80°C until analysis. RESULTS

Analysis of vitamins in blood plasma No statistically significant differences among all experi-
Vitamins in rat plasma were assessed using the high–per- mental groups were observed in initial (154±14 g) and final
formance liquid chromatography (HPLC) (Gilson Company, body mass (248±22 g) or dietary intake (307±35g) during
Middleton, WI, USA) with a UV–VIS detector. The concen- the study.
trations of  retinol and  β–carotene were measured accord- After β–carotene was supplemented, this molecule was
ing to Gackowski et  al. [2001]. Hexane solutions of  retinol not detected in  rat plasma; therefore, further analysis test-
or β–carotene were separated by  a  C18 RP (4.6 x 250 mm; ed for retinol. Retinol level in  nonexercised rat plasma was
5mm) chromatographic column from the  Vydac Company significantly increased after β–carotene supplementation,
(Vydac 201TP54, Hesperia, CA, USA) with a  precolumn while the levels remained unchanged in the exercised groups,
from the  same company. Methanol/acetonitrile (96:4; v/v) compared to the  control groups that did not receive this
was used as a  developing mixture for the  determination compound (Table 2). Concentration of  plasma retinol was
of  retinol, and  hexane/dichloromethane/methanol (15:5:80; significantly decreased in  exercised animals supplemented
v/v/v) was used as a developing mixture in the determination β–carotene (20%), as well as β–carotene and  ascorbic acid
of β–carotene, each at a flow rate of 1 mL/min. The plasma (15.2%) or all three of  the  compounds (14.3%). In  the  ex-
concentration of α–tocopherol was measured after deprotein- periment, no statistically significant interaction was noted
ising the sample with ethanol and after a two–step extraction between supplementation of the tested compounds, physical
of  α–tocopherol with hexane following a  method modified exercise on the  concentration of  plasma retinol. The  results
by Schneider et al. [2003], Gronowska–Senger et al. [2009]. showed a  significant influence both due to administered α–
LiChroCART®250–4 RP–18 (4 x 250 mm; 5mm) with a pre- tocopherol acetate and physical exercise on the level of α–to-
column (Merc, col. no. 841071, Darmstadt, Germany) was copherol in the plasma. Supplementation of α–tocopherol ac-
used. Acetonitrile/hexane/isopropanol mixture (65:14:21; etate significantly increased the level of plasma α–tocopherol
v/v/v) was applied as an eluent. The flow rate was 0.8 mL/min. in exercised and nonexercised animals compared to the cor-
The amount of vitamin C (L‑ascorbic acid and L–dehydro- responding control group. Administering vitamin E together
ascorbic acid) in  rat plasma was measured using method with β–carotene or vitamin C or both significantly increased
modified by Karlsen et al. [2005]. Samples were deproteinised the level of α–tocopherol only in the exercised groups. Physi-
with 10% metaphosphoric acid and centrifuged. The overall cal exercise, in turn, caused a significant increase in the con-
amount of  ascorbic acid in  the  supernatant was measured centration of  α–tocopherol in  plasma regardless of  whether
A. Wawrzyniak et al. 279

TABLE 2. The concentration of vitamins (retinol, vitamin E, vitamin C) in rat plasma in dependence on physical exercise and/or administered vitamins.

Groups
Exercise Retinol (µmol/L) P–value
control I BC control II BC + E BC + C E+C BC + E + C
NT 1.05±0.11a 1.25±0.16Ab 1.02±0.09a 1.05±0.14a 1.12±0.15Aab – 1.12±0.10Aa 0.01
T 1.01±0.13 1.00±0.12 B
0.96 0.11 0.94±0.10 0.95±0.10 B
– 0.96±0.07 B

0.73
P–value 0.53 0.04 0.30 0.07 0.02 – 0.001
Vitamin E (µmol/L)
control I E control II BC + E BC + C E+C BC + E + C
NT 10.2±0.8 a
12.7±3.0 Ac
10.4±1.0 ab
12.1±1.3 Abc
– 11.3±1.5 Aabc
11.6±1.1Aabc 0.04
T 10.1±1.6a 17.2±0.7Bc 9.6±1.5a 15.2±0.9Bb – 20.9±1.7Bd 21.1±1.6Bd
< 0.001
P–value 0.92 0.004 0.28 < 0.001 – < 0.001 < 0.001
Vitamin C (µmol/L)
control I C control II BC + E BC + C E+C BC + E + C
NT 23.2±3.8 a
24.2±7.8 a
21.6±6.6 a
– 24.5±4.2 a
30.9±4.6 b
22.3±3.1a 0.04
T 21.8±2.4 a
24.8±3.4 ab
21.2±3.7 a
– 23.8±4.2 a
28.7±4.3 b
24.2±5.7a
0.02
P–value 0.40 0.84 0.91 – 0.76 0.39 0.43
2–Way ANOVA, P–value Retinol Vitamin E Vitamin C
Exercise (T) < 0.001 < 0.001 0.73
Vitamins (V) 0.02 < 0.001 < 0.001
T x V 0.12 < 0.001 0.86
Administered vitamins: BC, β–carotene; C, ascorbic acid; E, α–tocopherol; exercise: NT, nonexercised; T, exercised; significantly different means bearing
different letters: small letters (a–d) in row, capital letters (A–B) in column (p<0.05).

TABLE 3. Correlation (r) between vitamins concentration in plasma.

Vitamin E Vitamin C
Pearson correlation
nonexercised exercised nonexercised exercised
Retinol 0.07 0.02 0.05 0.30
Vitamin E – – 0.14 0.64*
*statistically significant correlation (p<0.001).

it was supplemented individually or in combination. Plasma DISCUSSION

α–tocopherol of  animals in  exercised groups supplemented
with vitamins E and  C, or with β–carotene and  vitamins E Among the  nonexercised rats, a  (statistically signifi-
and  C together, was significantly higher, compared to non– cant) lower concentration of retinol was observed in plasma
–exercised groups, their level of  α–tocopherol was 81.9% to of the rats given β–carotene together with α–tocopherol ace-
85.0%. We observed a  strong interaction between physical tate compared to the groups supplemented only with β–caro-
exercise and vitamins administration on vitamin E concentra- tene. The concentration was comparable to plasma concentra-
tion in plasma. The concentration of vitamin C in rat plasma, tion of retinol in the control group when β–carotene was not
in turn, remained at similar levels regardless of whether it was given. This result is probably due to the fact that carotenoids
administered per os or not, and whether the animals were exer- and  vitamin E are transported in  the  blood in  combination
cised or not. On the other hand, a significantly higher plasma with lipoproteins and may compete with each other for bind-
concentration of vitamin C was noted in the animals which ing sides on the lipoproteins [Jenkins et al., 2000]. Thus, vi-
were given these vitamins together with vitamin E, regardless tamin E may have an inhibitory effect on β–carotene metabo-
of physical exercise. lism. Meanwhile, no change in plasma retinol concentrations
The  tests conducted confirmed a  statistically signifi- was observed in exercised animals regardless of whether they
cant correlation between the amounts of vitamins E and C received vitamins. This may attest to the  controlled release
in the plasma (r=0.64; p<0.001) but only for exercised rats of vitamin A from the liver, where it is accumulated [Weber
(Table 3). No correlation was found, in  contrast, between & Grune, 2012]. In fact, vitamin A concentration in plasma
the  concentration of  tested vitamins in  blood of  nonexer- of the exercised rats was always lower than that of nonexer-
cised rats. cised rats (from 3.8% to 20.0%).
280 Antioxidant Vitamins in Rat Plasma After Exercise

Biosynthesis of vitamin C in rat liver meets its daily needs, ACKNOWLEDGEMENTS

yet supplemental amounts given orally can improve antioxi-
dant defences in this animal [Djurašević et al., 2008], and in- The authors acknowledge financial support from the Min-
fluence blood plasma concentrations of the vitamin [Tsao & istry of Science and Higher Education (grant N N312 410037).
Young, 1989]. A study by Banhegyi et al. [1997] proved that
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