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Effect of nucleotide supplementation on lymphocyte DNA damage induced by dietary oxidative stress in pigs

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The aim of the present study was to evaluate the effect of nucleotide supplementation on the oxidative stress induced by a high proportion of dietary polyunsaturated fatty acids ( PUFAs) in pigs. Twenty-four male growing pigs were penned individually and after an adaptation period divided into three groups. All groups received isocaloric daily rations composed of a basal diet supplemented with either: starch (CONT), linseed oil (LIN) and LIN and nucleotides (LIN + NUC). The experimental period lasted 21 days. Oxidative stress was evaluated by measuring the degree of lymphocyte nuclear DNA damage, the urine malondialdehyde ( MDA) excretion rate, erythrocyte glutathione peroxidase concentration and the total anti-oxidant status of plasma. Malondialdehyde concentrations in the blood and MDA urinary excretion rates were higher (P < 0·01) in animals supplemented with LIN and LIN + NUC compared with CONT animals. The degree of DNA damage in the LIN-supplemented animals was also higher (P < 0·01). Compared with the LIN-supplemented animals, nucleotide supplementation reduced (P < 0·01) the degree of DNA damage in lymphocytes to the level of the CONT group. Erythrocyte glutathione peroxidase concentration and plasma total anti-oxidant status were similar across treatments. The results of this experiment indicate that nucleotide supplementation effectively eliminates the genotoxic effects of high PUFA intakes on blood lymphocytes and demonstrates new evidence for the immunonutritive effect of nucleotides.
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Nucleotide supplements and DNA damage from high-fat diets
135
Animal Science 2005, 81: 135-140 1357-7298/05/42290135$20.00
© 2005 British Society of Animal Science
Effect of nucleotide supplementation on lymphocyte DNA damage induced
by dietary oxidative stress in pigs
J. Salobir †, V. Rezar, T. Pajk and A. Levart
Institute of Nutrition, Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domžale,
Slovenia
†E-mail: janez.salobir@bfro.uni-lj.si
Abstract
The aim of the present study was to evaluate the effect of nucleotide supplementation on the oxidative stress induced
by a high proportion of dietary polyunsaturated fatty acids ( PUFAs) in pigs. Twenty-four male growing pigs were penned
individually and after an adaptation period divided into three groups. All groups received isocaloric daily rations composed
of a basal diet supplemented with either : starch (CONT), linseed oil (LIN) and LIN and nucleotides (LIN + NUC). The
experimental period lasted 21 days. Oxidative stress was evaluated by measuring the degree of lymphocyte nuclear DNA
damage, the urine malondialdehyde ( MDA) excretion rate, erythrocyte glutathione peroxidase concentration and the total
anti-oxidant status of plasma. Malondialdehyde concentrations in the blood and MDA urinary excretion rates were higher
( P < 0·01) in animals supplemented with LIN and LIN + NUC compared with CONT animals. The degree of DNA damage
in the LIN-supplemented animals was also higher ( P < 0·01). Compared with the LIN-supplemented animals, nucleotide
supplementation reduced ( P < 0·01) the degree of DNA damage in lymphocytes to the level of the CONT group. Erythrocyte
glutathione peroxidase concentration and plasma total anti-oxidant status were similar across treatments. The results of
this experiment indicate that nucleotide supplementation effectively eliminates the genotoxic effects of high PUFA intakes
on blood lymphocytes and demonstrates new evidence for the immunonutritive effect of nucleotides.
Keywords: damage, malondialdehyde, nucleotides, oxidative stress.
Diet has a great influence on the formation of free radicals
in animals and man. Free radicals can cause oxidative
stress, a disturbance in the pro-oxidant-anti-oxidant
balance potentially leading to tissue damage that can
play an important rôle in the etiology of many diseases.
Previous studies have shown that oxidative stress also has
detrimental effects on the immune system ( Vider et al., 2001).
Additionally, our own study in pigs has shown that oxidative
stress induced by a high intake of dietary polyunsaturated
fatty acids ( PUFAs) also increases damage to leukocyte
DNA (Rezar et al., 2003).
Nucleotides and nucleosides are not known to be anti-
oxidative substances per se, but they could nevertheless
play an important rôle in the prevention of DNA damage
induced by an elevated oxidative load. They could be
involved in the mechanisms of excision and repair of
fragmented DNA molecules. In some studies nucleotide
supplementation has been revealed to enhance T-cell
function including accelerated T-cell-dependent antibody
production, to increase in vitro proliferative response of
blood mononuclear cells and to promote the immune
response after vaccination (Jyonouchi, 1994; Yamamoto et
Introduction
Generally, it has been assumed that all living cells are capable
of meeting their requirements for nucleotides via de novo
synthesis. However, recent studies revealed that in many
tissues, except the liver, the requirements for nucleotides
are covered not only by de novo synthesis but also by the
salvage pathway (Godderis et al., 2002). There is increasing
evidence indicating that especially under various conditions
such as certain disease states, limited nutrient intake, rapid
growth etc. the endogenous supply is not sufficient for
normal cell function and that dietary sources are required
(Uauy et al., 1994; Lopez-Navarro et al., 1996; Carver,
1999). Numerous reports have demonstrated that, in these
circumstances, dietary supplementation with nucleotides,
nucleosides, or nucleic acids improves the efficacy of the
immune system, gastro-intestinal tract, liver, brain and
metabolism of lipids not only in humans but also in animals
( Martinez-Augustin et al., 1997; Yamauchi et al., 1998;
Cameron et al., 2001; Bastian and Weimann, 2002; Carver
et al., 2004). For instance Yu et al. (2002) demonstrated that
nucleotides in combination with glutamine improve food
intake, intestinal villous height and promote the immune
responses and foot and mouth disease neutralizing antibody
titres of weaned pigs.
Salobir, Rezar, Pajk and Levart
136
al., 1997; Cameron et al., 2001). Therefore nucleotides are
classified as immunomodulatory nutrients ( McCowen and
Bistrian, 2003).
Since oxidative stress is known to induce genotoxic effects on
immune cells and since nucleotides are known to play a rôle
as immunomodulatory nutrients, their rôle in the prevention of
the genotoxic effects of oxidative stress on immune cells could
be of interest. Dietary supplementation with nucleotides may
increase the repair mechanism and/or cellular proliferation
and thus optimize immune system function.
The aim of the present study was to evaluate the effect
of nucleotide supplementation on the oxidative stress
induced by a high proportion of dietary polyunsaturated
fat in the diet. In order to evaluate the oxidative stress, the
following parameters were measured in pigs : the degree
of the lymphocyte nuclear DNA damage, the plasma
malondialdehyde ( MDA) concentration, the 24-h urine
MDA excretion rate, erythrocyte glutathione peroxidase
concentration and the total anti-oxidant status of plasma.
Material and methods
Animals and diets
Twenty-four young growing castrated male crossbreed
pigs (Duroc × Landrace × Large White, live weight 11·9
±0·5 kg) were utilized in this experiment. The animals were
penned individually in balance cages that allowed separate
collection of urine and faeces. The room temperature was
maintained at 22°C. Animals were housed under 12 h
light/dark conditions. The experiment was divided into
adaptation and experimental periods that lasted for 8 and
21 days, respectively. The animals were given food at
2·5 times the maintenance requirement for growing pigs
(National Research Council, 1998). At the beginning of the
experimental period, the animals were randomly assigned to
three treatment groups. All groups received isocaloric daily
rations ( Table 1) composed of an equal amount of the basal
diet which was supplemented according to the different
dietary treatments with : starch (control; CONT), linseed
oil (LIN) and LIN and nucleotides (LIN + NUC). Nucleotides
were supplied with preparation of Ascogen (Chemoforma
Ltd, Switzerland) containing RNA extracted from yeast,
nucleotides, precursors of nucleotides, organic acids and
thermolysed yeast. The proportion of energy requirements
which were met by fat in the CONT group and in both linseed
oil supplemented groups was 0·05 and 0·30, respectively.
The composition and analysis of daily rations in different
groups are presented in Table 1. The food was given in the
form of a mix. All ingredients of the mixture, except linseed
oil, were mixed together weekly. The linseed oil was added
and mixed to the diet of individual animals before every
feeding.
During the adaptation phase all pigs received the CONT diet
and had ad libitum access to water by automatic waterer.
The animals were given food twice daily. At the beginning
and at the end of the experiment, the pigs were weighed.
Food analysis
The content of protein and fat was determined by standard
procedures published by Naumann and Bassler (1997).
The content of total dietary fibre was analysed by the
enzymatic-gravimetric method ( Prosky et al., 1992). The
fatty acid compositions of diets were analysed via gas
chromatographic method after transesterification of lipids
as described by Fidler et al. (2000). In brief, fatty acid methyl
esters (FAME) were prepared by the procedures described
by Park and Goins (1994). FAME were separated on an
Omegawax 320 Capillary column (Supelco, 30 m × 0·32
mm i.d.) by using an Agilent GC 6890 Series GC System
equipped with Agilent 7683 Series Injector and 7683 Series
Auto sampler. A 30 to 1 split ratio was used for injection of
1 µl of hexane containing FAME. The carrier gas was Ar-
CH4 (5%), and inlet pressure was maintained at 13·3 p.s.i.,
which corresponds to a nominal initial flow of 1·5 ml/min.
Table 1 Ingredient, food, energy and nutrient intakes of experimental pigs on the different diets (estimated for a 12-kg pig)
Diet
CONT LIN LIN+NUC
Ingredient intake
Linseed oil (g/day) 0·0 53·1 53·1
Wheat starch (g/day) 227·8 111·6 107·2
Maize (g/day) 59·8 59·8 59·8
Soya-bean meal (g/day) 117·6 117·6 117·6
Skimmed milk powder (g/day) 91·1 91·1 91·1
Mineral-vitamin-amino acid supplement (g/day)† 9·9 9·9 9·9
Nucleotide preparation (g/day)‡ 0·0 0·0 4·4
Total food intake (g/day) 506·2 443·1 443·1
Nutrient intake
Metabolizable energy (kJ/day)§ 7423 7423 7423
Proportion of energy from fat§ 0·050 0·300 0·300
Proportion of energy from PUF 0·029 0·209 0·209
Protein (g/day) 86·5 88·7 87·9
Fat (g/day) 8·7 56·0 57·1
Total dietary fibre (g/day) 44·0 45·2 45·9
† Calculated to meet nutritional requirements according to NRC (1998). Mineral-vitamin-amino acid supplement provided daily: 2·0 g Ca, 3·4 g
P, 0·15 g Na, 1·65 mg retinol, 5·09 mg alpha-tocopherol, 0·03 g lysine and 0·6 g methionine.
‡ Ascogen; Chemoforma Ltd, Switzerland.
§ Proportions of energy from fat and from polyunsaturated fatty acids (PUFA) were estimated. The energy values of foodstuffs and diets were
according to Gesellschaft für Ernährungsphysiologie (1988).
Nucleotide supplements and DNA damage from high-fat diets
137
Injector temperature was maintained at 250ºC, and FID
detector temperature was maintained at 290ºC. During the
chromatographic run the oven temperature was raised from
170ºC to 215ºC at a rate of 1ºC/min.
Blood and urine samples
At the beginning (before division into experimental groups)
and at the end of the 21-day experimental period fasting
blood samples were taken from the jugular vein into
evacuated tubes and 48-h urine was collected.
Lymphocyte nuclear DNA damage – comet assay
Blood samples for single cell gel electrophoresis (comet
assay) were collected in evacuated tubes containing EDTAK3
anticoagulant (367654, Brand, Plymouth, UK). Lymphocytes
were isolated from the fresh blood samples according to a
modified procedure described by Singh (1997); 2·5 ml of
Histopaque-1077 (Sigma H-8889, Sigma-Aldrich, Steinheim,
Germany) was added to 14-ml Eppendorfer centrifuge tube
and overlaid with 8 ml of a mixture of blood and RPMI-
1640 (Sigma R-8758, Sigma-Aldrich, Steinheim, Germany)
medium, which had been gently mixed at a ratio of 1 to 1.
Centrifugation (300 × g, 35 min, room temperature) followed.
Lymphocytes, white pellet approximately 3 mm under red-
cell pellet, were transferred to another centrifuge tube and
washed twice in 5 ml sterile RPMI-1640 medium, followed
by centrifugation (300 × g, 5 min, room temperature). The
pellet of lymphocytes was finally mixed with 0·5 ml of RPMI-
1640 medium and used as lymphocyte isolate. A partially
modified procedure by Singh et al. (1988) was implemented
for the comet assay. Rough microscopic slides were used
for the microgel preparation. The isolated lymphocytes were
suspended briefly in low melting point agarose. The first
layer of agarose was left to dry at normal room temperature,
the others were left on ice for 10 min. The gels were then
immersed in a lysing solution (0·03 mol/l NaOH, 1·2 mol/l NaCl,
0·5% laurylsarcosine, 1% Triton X-100, 10% DMSO) at 4ºC
for 1 h. The gels were then transferred to an electrophoresis
buffer (0·03 mol/l NaOH, 2 mmol/l ethylene diamine tetra-
acetic acid, pH 13) for 40 min before electrophoresis at 25 V
for 20 min. Following electrophoresis, the gels were washed
three times for 5 min with 0·4 mol/l tris-HCl, pH 7·5 at 4ºC
before staining with ethidium bromide (2 µg/ml) for 20 min,
and then rinsed in 400 mmol/l tris-HCl at 4ºC. An Olympus
CH 50 epifluorescent microscope at 200× magnification
was used for the examination of lymphocyte nuclei in the
microgels (100 W Hg lamp, excitation filter of 480 to 550
nm and barrier filter of 590 nm). The images were captured
by a Hamamatsu Orca 1 CCD camera, analysed and the
nuclear DNA damage estimated by the Comet 5 dedicated
computer program (Single Cell Gel Electrophoresis; Kinetic
Imaging Ltd, UK).
Plasma and urine malondialdehyde ( MDA) concentration
Blood samples for MDA concentration analysis were
collected in 10-ml evacuated tubes containing EDTAK3
anticoagulant (368457, Brand, Plymouth, UK). The blood
was centrifuged for 10 min at 400 × g and 4ºC. Plasma
supernatants were transferred to microcentrifuge tubes and
stored at -70ºC. To increase the accuracy of determination
of the 24-h urine MDA excretion rate a 48-h urine collection
was applied instead of standard 24-h urine collection.
Urine was filtered into test tubes (10 to 15 ml) through filter
paper (520 A, Schleicher and Schuell, Dassel, Germany),
transferred to microcentrifuge tubes and stored at –70ºC.
The methodology of Wong et al. (1987) modified by Chirico
(1994) and Fukunaga et al. (1995) was used to measure
the concentrations of MDA in blood plasma and urine by
high-performance liquid chromatography using a Waters
Symmetry C18 chromatography column (5 µm, 4·6 × 150 mm)
and a Waters Symmetry C18
guard
column (5 µm, 3·9 × 20 mm).
A Waters Alliance 2690 apparatus equipped with a Waters
474 scanning fluorescence detector was applied ( Waters
Corporation, Milford, MA). The mobile phase consisted of
50 mmol/l KH2PO4 buffer (pH 6·8) and methanol in a gradient
mode. Flow rate of the mobile phase was 1 ml/min at an
ambient temperature and the resulting backpressure was
2000 p.s.i. The results of the analysis were evaluated by the
Millenium32 Chromatography Manager program.
Glutathione peroxidase (GPx) and total antioxidant status
( TAS)
Blood samples for GPx and TAS concentration analysis
were collected in evacuated tubes containing heparin
(367685, Brand, Plymouth, UK). Fifty µl of whole blood for
GPx analysis was transferred to microcentrifuge tubes and
stored at –70ºC. Blood for TAS was centrifuged for 10 min at
3000 × g and 4ºC. Plasma supernatants were transferred to
microcentrifuge tubes and stored at –70ºC. The methodology
of Paglia and Valentine (1967) was used for measurements
of GPx and the methodology of Miller and Rice-Evans (1996)
was used for measurements of TAS. Samples were assayed
with commercially available glutathione peroxidase and TAS
kits (Randox, Crumlin, UK), following the instructions of the
kits.
Statistical analysis
The data were analysed by the ANOVA procedure of Statistical
Analysis Systems Institute (2000). Monofactorial analysis
of variance (effect of dietary treatment) was used. When
ANOVA revealed a significant effect, the differences among
treatment groups were tested using Scheffe’s test. The level
of significance was set at P < 0·05.
Results
The animals adapted well to the experimental conditions.
During the experiment, the animals in all groups had no
health or other problems, consumed food continuously
and normal live-weight gain for this level of feeding was
observed (315±35 g/day). The live-weight gain was at the
same level ( P > 0·05) in all groups (CONT 306 g/day, LIN 310
g/day, LIN + NUC 329 g/day).
While at the beginning of the experimental period no
statistical differences among treatments in any of the
measured parameters could be observed, at the end of the
experiment some important differences were found.
Nuclear DNA damage to lymphocytes
The rate of DNA damage ( Table 2) is presented as the
proportion of DNA in the head of the comet and as the Olive
tail moment (Olive et al., 1992), defined as the product of
the amount of DNA in the tail and the mean distance of
migration in the tail (higher values for Olive tail moment
Salobir, Rezar, Pajk and Levart
138
represent higher rates of DNA damage). The results showed
that isocaloric replacement of starch in the diet with linseed
oil increased the rate of lymphocyte DNA damage in the LIN
group by reducing ( P < 0·01) the percentage of DNA in the
head and by increasing ( P < 0·01) the Olive tail moment. In
comparison with the LIN group, nucleotide supplementation
in the LIN + NUC group improved ( P < 0·01) both parameters
of the degree of lymphocyte DNA damage to the level of the
CONT group.
Plasma MDA concentration and MDA excretion rate in urine
At the end of the experimental period, the MDA concentration
in plasma and the MDA excretion rate in urine (per 24 h) in
both linseed oil supplemented groups were higher ( P < 0·01)
than in the CONT group ( Table 3). In comparison with the
LIN group, the nucleotide supplementation in the LIN + NUC
group did not influence either MDA concentration in plasma
or the MDA excretion rate in urine.
Erythrocyte glutathione peroxidase and total anti-oxidant
status
The concentration of erythrocyte glutathione peroxidase and
total anti-oxidant status at the end of the experiment were
similar across all three experimental groups ( Table 4).
Discussion
The effect of nucleotide supplementation on oxidative stress
induced by high polyunsaturated fat intake is currently
not known. In order to measure the effect of nucleotide
supplementation on oxidative stress in our experiment,
nutritive oxidative stress was firstly induced by increasing
the proportion of energy supply from fats from 0·05 to 0·30.
Since the recommendations for the upper limit of fat intake
in pigs is not known, the recommendations for humans were
used ( World Health Organization ( WHO), 2003) Secondly,
oxidative stress was additionally induced by selection of
linseed oil, which contains 0·73 g/g of polyunsaturated
fatty acids ( PUFAs) as indicated by analysis. The energy
supply from PUFAs was approximately 0·21of total energy,
which is significantly higher than the proportion 0·06 to 0·10
proposed by WHO (2003). Polyunsaturated fatty acids are
lipids that are highly susceptible to peroxidation. The latter
was demonstrated by Dhanakoti and Draper (1987) who
found significantly higher 24-h urinary MDA excretion levels
in rats given a PUFA-rich diet than in rats given a diet rich in
saturated fatty acids. Thirdly, linseed oil contains absolutely
and in relation to its PUFA content, a very low amount of α-
tocopherol. It is known that a high intake of PUFAs increases
the nutritive requirements for anti-oxidative vitamins (Duthie
et al., 1996). The oxidative stress in both groups given
Table 2 Rate of lymphocyte DNA damage measured by comet assay
Diet
CONT LIN LIN+NUC s.e. Significance
Proportion of DNA in head of comets
Day 0 0·958 0·958 0·957 0·0044
Day 21 0·964a 0·853b 0·954a 0·0060 ***
Olive tail moment†
Day 0 0·57 0·59 0·63 0·088
Day 21 0·71a 5·09b 0·86a 0·583 ***
a,b Means without the same superscripts in the same row differ significantly (P < 0·05).
† Defi‟
Table 3 Plasma malondialdehyde (MDA) concentration and 24-h MDA excretion in urine
Diet
CONT LIN LIN+NUC s.e. Significance
Plasma MDA (nmol/ml)
Day 0 0·354 0·347 0·434 0·034
Day 21 0·179a 0·855b 0·809b 0·051 ***
MDA excretion rate in urine (nmol per 24 h)
Day 0 1889 1815 1804 190
Day 21 1646a 6110b 6751b 522 ***
a,b Means without the same superscripts in the same row differ significantly (P < 0·05).
Table 4 The concentration of glutathione peroxidase (GPx) in erythrocytes and total antioxidant status ( TAS) in plasma
Diet
CONT LIN LIN+NUC s.e. Significance
Erythrocyte GPx (U/l)
Day 0 25027 26496 22939 1367
Day 21 24425 22295 23040 2425
Plasma TAS (mmol/l)
Day 0 0·633 0·592 0·609 0·029
Day 21 0·643 0·656 0·661 0·014
Nucleotide supplements and DNA damage from high-fat diets
139
linseed oil (LIN and LIN + NUC) was additionally increased
by the fact that the supply of supplemented anti-oxidative
vitamins was not increased, i.e. it remained at the level of the
CONT group ( Table 1).
As expected, 21-day linseed oil feeding in the LIN group
increased the oxidative stress by increasing ( P < 0·01) not
only the formation of lipid peroxidation products measured
as plasma MDA concentration and the urinary MDA excretion
rate, but also by inducing genotoxic changes of lymphocyte
on the basis of the studies of Dhanakoti and Draper (1987)
and our own study (Rezar et al., 2003).
By confirming the oxidative stress induced by of high fat
intake, the basis for studying the effect of nucleotides was
achieved. The positive effect of nucleotides on oxidative
status can be seen from the results of the comet assay. The
results show that the degree of lymphocyte DNA damage
in the nucleotide-supplemented group was lower ( P < 0·01)
than in the LIN group. Moreover, the degree of lymphocyte
DNA damage in this group was at the same level as in the
CONT group. It is evident that nucleotide supplementation
can prevent the genotoxic effect of high fat intake on DNA of
lymphocytes. Previously, protection of DNA integrity under
increased oxidative stress has been demonstrated for other
nutrients like vitamin E, vitamin C, β-carotene, polyphenols
(Duthie et al., 1996; Brennan et al., 2000; Pajk et al., 2002;
Salobir et al., 2002; Bub et al., 2003).
Nucleotide supplementation had no effect ( P > 0·05) on the
plasma MDA concentration, urinary MDA excretion rate,
erythrocyte glutathione peroxidase concentration and on
the total antioxidant status of the plasma. Since nucleotides
are not known to have anti-oxidative properties per se, a
sparing effect on GPx synthesis or increased plasma TAS
because of lower expenditure of anti-oxidative substances
already present could not be expected. A possible mode of
action of nucleotides to prevent lipid oxidation could be via
improved synthesis of RNA responsible for the synthesis
of enzymes required to cope with oxidative stress. But at
least in this experiment this was not the case. Possibly also
the absence of an effect of linseed oil supplementation
on erythrocyte GPx concentration and on the TAS of the
plasma in LIN group was a inappropriate basis from which
to measure such an effect.
The results indicate that nucleotide supplementation is not
able to prevent increased lipid oxidation in the body. Thus the
mode of action of supplemented nucleotides in prevention
of DNA damage induced by high oxidative load is most likely
the improved supply of nucleotides for the mechanisms of
excision and repair of damaged parts of DNA molecules
of immune and possibly other cells. The results indicate
also that in the case of oxidative stress the demand for
nucleotides increases over the endogenous supply and that
dietary sources are required. Consequently, the results show
that beside an increased cellular (Carver, 1994; Zomborszky-
Kovacs et al., 2000; Cameron et al., 2001; Yu et al., 2002)
and humoral immune response (Jyonouchi, 1994; Cordle et
al., 2002; Yu et al., 2002), the repair of damaged parts of
DNA molecules could most likely be an important step in the
optimization of immune system function as a consequence
of nucleotide supplementation.
Immune cells play a central rôle in the immune response.
They are susceptible to the DNA-damaging effects of a wide
variety of agents (Brennan et al., 2000). Alteration in T cells
may have implications for subsequent immune response,
when for example T memory cells are required to undergo
rapid proliferation in response to rechallenge with a specific
antigen. A decline in T-cell function is thought to play a
critical part in the age-related decline in immune function
( Pawelec et al., 1998) and genetic damage that accumulates
over time in vivo within lymphocytes is thought to contribute
to age-related decline in T-cell function (Barnett and Barnett,
1998). The question of whether nucleotide supplementation
is able to protect immune cells against genetic damage in
other conditions like certain disease states, food and other
toxins, limited nutrient intake, ageing as effectively as in the
case of oxidative stress remains to be established.
In conclusion, the results of this study demonstrated for
the first time that nucleotide supplementation effectively
eliminates the genotoxic effect of high PUFA intake on
blood lymphocytes. Nucleotide supplementation in these
experimental conditions may be an important component for
the repair mechanism of immune cells and thus underlines
the immunomodulatory rôle of nucleotides.
Acknowledgements
This work was supported by a grant from the Ministry of Agriculture,
Food and Forestry and the Ministry of Education, Science and
Sports of the Republic of Slovenia.
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(Received 29 December 2004 – Accepted 5 March 2005)
... Immune-enhancing effects of nucleotides previously described in humans and nonruminant animals include lymphocyte stimulation, macrophage activation, and immunoglobulin release (Gil, 2002;Mc Naughton et al., 2007;Sauer et al., 2011). Additionally, the role of nucleotides in inflammatory status and oxidative stress responses were also demonstrated in the same animal models (Salobir et al., 2005;Frankič et al., 2006;Weaver and Kim, 2014). To the best of our knowledge, information regarding the antioxidant and antiinflammatory potential of dietary nucleotides in calves is limited, and findings on immune system effects are contradictory. ...
... Although there are differences in physiology and metabolism that make a direct comparison of our findings impossible with those recorded in fish, most studies suggest that dietary nucleotides ameliorate the oxidative status by reinforcing the antioxidant capacity and upregulating genes encoding for antioxidant enzymes (Tie et al., 2019;de Lima et al., 2020). Indeed, there is a growing body of evidence on the correlation between exogenous nucleotides and high mRNA levels associated with antioxidant enzymes (Salobir et al., 2005;Frankič et al., 2006;Tie et al., 2019). ...
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The present work aimed to investigate the effects of nucleotide oral administration on oxidative stress biomarkers, immune responses, gut morphology, serum biochemical parameters, and growth performance in calves from birth to 25 d of life. A total of 40 male Hol-stein Friesian calves were randomly divided in 2 groups. All the calves were born and reared on the same commercial dairy farm. They were fed the same colostrum, milk replacer, and calf starter. Five grams/head of an additive were orally administered with a syringe directly in the mouth to calves of the nucleotide group (NG). The additive contained 74.12 g/100 g of nucleic acids from hydrolyzed yeast, and 75.38% was free nucleotide sodium salt. The other group represented the negative control (CG). At 25 d of life all of the calves were slaughtered. Calves supplemented with nucleotides had a higher final live weight and improved average daily gain, which was associated with better efficiency of nutrient use. Oral nucleotide administration did not affect IgG absorption efficiency; however, NG calves showed greater duodenum villi length and higher crypt depth compared with CG. Oral nucleotide administration increased the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) and the antioxidant capacity [ferric reducing antioxidant power and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) scavenging activity] both in plasma and in liver. An enhanced ability of cells to counter reactive oxygen species-and reactive nitrogen species-mediated damage was also observed in peripheral blood mononuclear cells from NG. The findings highlight the effectiveness of oral nucleotide administration, and potentially dietary supplementation of nucleotides, in boosting oxidative and immune status in newborn calves.
... Hence, there is a raised requirement for nucleotides amid times of immunological difficulties. Supplementation of eating regimens with nucleotides may expand the quantity of lymphocytes and macrophages of the intra-epithelium of the piglet ileum (Domeneghini et al., 2004;Speranda et al., 2008), decline harm of the blood lymphocyte DNA (Salobir et al., 2005), decline the concentration of TNF-a and IL-6 in blood serum 2 and 4 h after an E. coli infusion (Hung, 2015), and increment plasma and serum concentration of IgA ...
... In this context the balance between oxidant and antioxidant levels is more important; if the antioxidant levels are low, the secondary effect can lead to inflammation and tissue damage. In this case, nucleotides are able to repair damaged DNA [27]. In addition, during the increase of muscle tone, nucleotides are essential for protein synthesis, and to reduce cortisol levels and increase secretory IgA [28]. ...
... Our study demonstrated that weaning pigs that were fed with HY could defend against free radicals through increased CAT activity. In agreement with this, Salobir et al. [31] showed that yeast-derived nucleotides can alleviate oxidative stress in piglets. In addition, the HY used in this study had a relatively greater quantity of glutamine that was included as an active precursor for glutathione biosynthesis. ...
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Background and aim: Weaning pigs normally suffer from many stressors which have impaired growth performance and immunity. Hydrolyzed yeast has been proposed as an alternative feed additive. The aim of this study was to investigate the effects of various levels of hydrolyzed yeast (HY) supplementation in the feed of weaning pigs on growth performance, diarrhea incidence, immunity, antioxidant capacity, and microbial populations. Materials and methods: A total of 144 crossbred weaning pigs (Duroc × Landrace × Large White) with a mean body weight (BW) of 7.46 kg were randomly assigned to one of four treatments during a 5-week feeding trial. Treatments consisted of a basal diet without HY inclusion (control), or the basal diet supplemented with HY at 0.5, 1.0, and 1.5 g/kg of diet, respectively. Results: Piglets fed with 1.0 or 1.5 g/kg HY presented significantly increased BW (p=0.009) and decreased incidence of diarrhea (p=0.001). The final BW (p=0.012), average daily gain (p=0.094), and average daily feed intake (p=0.091) showed a linear improvement with the level of HY inclusion. However, the gain-to-feed ratio was unaffected by dietary treatments. Linear responses to the HY supplementation levels were also observed for blood urea nitrogen (p=0.030), total protein (p=0.017), lymphocyte percentage (p=0.064), catalase activity (p=0.089), malondialdehyde (MDA) level (p=0.001), Salmonella spp. (p=0.024), Escherichia coli (p=0.021), and Lactobacillus spp. (p=0.048). Dietary inclusion of HY at 1.0 and 1.5 g/kg resulted in increased immunoglobulin A and G secretions (p=0.042 and p=0.022, respectively) and decreased MDA concentration (p<0.01) and Salmonella spp. (p=0.026) and E. coli (p=0.050). Conclusion: It was concluded that HY inclusion at 1.0 and 1.5 g/kg in the diet of weaning pigs improve BW, immunoglobulin secretion, and antioxidant enzyme activity, whereas it lowers diarrhea occurrence, lipid peroxidation, and pathogenic bacteria in weaning pigs.
... In the current study red sea bream fed diet CMP0.2 exhibited best condition in terms of oxidative stress resistance until 100 day and in 150-day fish were relatively low resistance against oxidative stress but the ranges were still in acceptable condition. In pigs reduced oxidative stress condition due to NT administration was also stated by Salobir et al. (2005). Although NT and their related products are not known to be antioxidative substances, but they play a significant role in the prevention of DNA damage due to higher oxidative load (Frankič et al. 2006). ...
Article
There is no definitive evidence associated with administration duration of nucleotide on aquaculture organisms. This study evaluates the long-term feeding effects of cytidine monophosphate (CMP) nucleotide as functional supplement on growth and health conditions of red sea bream, Pagrus major. A formulated basal diet added with 0% (control), 0.2% (CMP-0.2) and 0.4% (CMP-0.4) individual CMP nucleotide to prepare three experimental feeds. Each experimental feed was fed to triplicate tanks of red sea bream (3.0 ± 0.01 g) for 150 days. At day 50th, growth performances were influenced significantly by dietary CMP inclusion and fish fed CMP-0.4 diet showed significantly higher value. In day 100 and 150 growth performances also non-significantly enhanced by CMP supplementation. Feed utilization performances also showed improved values in nucleotide included diets compared to the non-supplemented control diet (P > 0.05). No significant changes observed on measured hematological parameters by nucleotide administration duration. Lysozyme activity (LA) and total serum protein (TSP) significantly rose to their highest level on 50 days in CMP-0.4 diet group. In 100 and 150th day supplementation also improved TSP and LA values and it reaches maximum in CMP-0.2 diet group (P > 0.05). Oxidative radical production (nitro-blue-tetrazolium assay) and the activity of peroxidase and catalase were not affected significantly with feeding nucleotide in all sampling days. Supplementation of nucleotide improved oxidative stress tolerance capacity. Remarkably, CMP-0.2 diet group showed the least oxidative stress condition in 50th and 100th day. In 150th day fish showed acceptable but reduced oxidative stress resistances. On the basis of the overall findings, finally we concluded that, CMP nucleotide administration regime influences the optimum growth, health performance and immune response of Pagrus major and its mostly after feeding for 50 days, but not after 100 days.
... Combined effects of d-ROM and BAP showed that fish fed diet groups D2 and D4 were located in zone-A (best condition) which was categorized as having a lower intensity of oxidative stress and higher tolerance ability against oxidative stress. In pigs, reduced oxidative stress condition due to NT supplementation was also reported by Salobir et al. (2005). ...
Article
Purine nucleotides regulate the cellular functions in the animal body. The current study evaluates the comparative efficacy of dietary purine nucleotides, that is, adenosine monophosphate (AMP), guanosine monophosphate (GMP) and inosine monophosphate (IMP) in red sea bream Pagrus major. Semi-purified basal diet was formulated (Control, D1) containing 550 g/kg protein, supplemented with purine nucleotides AMP, GMP and IMP at their optimum supplementation level (2, 4 and 4 g/ kg) to formulate the experimental diet groups D2, D3 and D4, respectively. Initial weight of 3.5 ± 0.01 g fish was randomly fed test diets in triplicate. After 56 days, % weight gain (p = .003), specific growth rate (p = .003) and apparent lipid digestibility (p = .04) were significantly higher in fish fed diet group D4 followed by D3 and D2. Supplemented groups showed significantly higher feed intake in comparison to control (p = .001). Supplemented groups showed significantly increased and decreased NBT (p = .003) and CAT (p = .003) activity, respectively. Fish fed IMP supplemented diets had significantly lower blood urea nitrogen (p = .04), glutamyl oxaloacetic transaminase (p = .04) and glutamic-pyruvate transaminase (p = .001) followed by other supplemented groups. Supplemented diet groups showed enhanced stress resistance. Interestingly, D2 and D4 groups showed best oxidative stress status of fish. Therefore, these results indicated that among purine nucleotides, supplementation of IMP could be a more effective nucleotide as functional supplement in red sea bream diet
... The concentration of MDA could have been broadly used as indicator of lipid peroxidation (Shen et al., 2012;. Salobir et al. (2005) suggested that dietary nucleotides could improve synthesis of RNA involved in the enzymes related to reduced oxidative stress. Some studies showed that dietary nucleotides could reduce cellular damage in newly weaned pigs (Godlewski et al., 2009;. ...
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Intestinal challenges upon weaning would increase the needs of nucleotides for enterocyte proliferation whereas de novo synthesis maybe insufficient. This study aimed to evaluate supplemental effects of dietary nucleotides on intestinal health and growth performance in newly weaned pigs. Fifty newly weaned pigs (19-d-old, 25 barrows and 25 gilts, 4.76 ± 0.42 kg BW) were individually housed and allotted to 5 treatments with increasing nucleotide supplementation (0, 50, 150, 250, and 500 mg/kg) based on a randomized complete block design with the initial BW and sex as blocks. Dietary nucleotides were provided from YT500 (Hinabiotech, Guangzhou, China). Pigs were fed for 21 d based on 2 phases (Phase 1: 11 d and Phase 2: 10 d) and experimental diets were formulated to meet or exceed nutrient requirements suggested by NRC (2012). Feed intake and BW were recorded. Titanium oxide (0.4%) was added as an indigestible marker from d 17. Plasma collected on d 18 was used to measure tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), and malondialdehyde (MDA). Pigs were euthanized on d 21 to collect tissues to evaluate TNF-α, IL-6, MDA, morphology, and crypt cell proliferation rate in the jejunum. Ileal digesta were collected to measure ileal nutrient digestibility. Data were analyzed using contrasts in the MIXED procedure of SAS. Nucleotide supplementation increased (P < 0.05) ADFI in phase 1. Nucleotide supplementation at 50 and 150 mg/kg increased (P < 0.05) ADG in Phase 1, whereas increased (P < 0.05) ADFI and tended to increase (P = 0.082) ADG in overall. Increasing nucleotide supplementation changed (quadratic, P < 0.05) villus height-crypt ratio (at 247 mg/kg) and decreased (linear, P < 0.05) crypt cell proliferation rate in the jejunum. Increasing nucleotide supplementation reduced (P < 0.05) jejunal IL-6 (at 50 and 150 mg/kg) and tended to change (quadratic, P = 0.074) plasma MDA (at 231 mg/kg). Nucleotide supplementation at 50 and 150 mg/kg increased (P < 0.05) ileal digestibility of energy and ether extract. In conclusion, nucleotide supplementation at a range of 50 to 250 mg/kg in the diets seems to be beneficial to newly weaned pigs by enhancing growth performance possibly due to reduced intestinal inflammation and oxidative stress as well as improved intestinal villi structure and energy digestibility.
... Accordingly, certain ROS expression is correlated with aging, inflammatory response, and the occurrence of chronic diseases. Dietary nucleotide supplements are indentified as an effective means to alleviate the inflammatory process, oxidative damage, carcinogenic activity, and aging process, along with restoring energy from fatigue [16,17,18]. Nucleotides involve rudimentary biological functions regarding encoding and deciphering genetic information, modulating metabolism and cell signaling, and as cofactors within enzyme reactions [19]. ...
... They observed increased levels of plasma immunoglobulins in weaned piglets fed 1 g/kg nucleotides. In addition, it has recently been suggested that dietary nucleotides might eliminate the genotoxic effect of high polyunsaturated fatty acids intake on blood lymphocytes (Salobir et al., 2005), which indicates its potential immunomodulation properties (Sauer et al., 2011). In this study, orally ETEC challenged pigs fed 1 g/kg or 2 g/kg NRYE diets showed higher ADG up to day 14 after weaning and tended to lower mortality rate than challenged NC pigs (Tables 3 and 4), suggesting that nucleotides intake might help sustain growth and enhance immune response during an ETEC infection. ...
Article
A study was conducted to investigate the effect of a nucleotide rich yeast extract (NRYE) on the growth performance and the colibacillosis of weaned pigs. In Experiment. 1, a total of 168 mixed-sex piglets weaned at 17 ± 2 days of age were fed diets formulated to meet or exceed nutrient requirements for 28 days. Diets consisted of i) Positive control (PC) containing antibiotics (110 mg/kg of chlortetracycline HCl and 31.2 mg/kg of tiamulin), ii) Negative control (NC) without antibiotics, iii) NC with the addition of 1 g/kg of NRYE (NRYE1), and iv) 2 g/kg of NRYE (NRYE2). No differences (P > 0.10) were found among treatments for average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR), whilst pigs fed NRYE1 weighed more (P < 0.05) than those fed NC on day 28. Pigs fed the NRYE2 diet had greater (P < 0.05) ADG and ADFI than those fed the NC diet from day 7 to 14. No differences (P > 0.10) were observed in ADFI among dietary treatments. In Experiment 2, a total of 144 barrows were randomly assigned to 4 treatments groups to give 6 replicates (6 pigs per pen) per group. Four treatment diets were similar to previous experiment. Pigs in PC fed a control diet without NYRE supplementation and also did not received an oral challenge. PC treatment was housed in a separate room within the same facility with a similar environment. Pigs in NC, NRYE1 and NRYE2 were fed the non-medicated control diet with 0g/kg, 1 g/kg or 2 g/kg NYRE, respectively and were orally challenged with enterotoxigenic Escherichia coli K88+ (ETEC) after the 3 days of weaning. From day 3 to 28 post-challenge, pigs fed diets NRYE1 or NRYE2 had greater (P < 0.05) ADG compared with those fed a NC diet. Average daily feed intake and FCR were not affected by dietary treatment throughout the study. Challenged pigs consuming NRYE1 or NRYE2 diets had less (P < 0.05) diarrhea compared with those fed NC diet. The mortality due to coliform infection tended to be higher (P < 0.10) in the challenged NC and NRYE2 pigs compared with PC and NRYE1 pigs. In conclusion, diets supplemented with NRYE enhanced BW, ADG and ADFI of weaned pigs and reduced the incidence of diarrhea in ETEC challenged pigs suggesting that NRYE could be effective in controlling post-weaning diarrhea in piglets, which could represent a major economic advantage to the swine industry.
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Increased metabolic burdens in breeding sows, which are induced by elevated systemic oxidative stress, could increase the need for nucleotides to repair lymphocyte DNA damage; however, de novo synthesis of nucleotides may be insufficient to cover this increased need. This study investigated the effects of dietary nucleotides on milk composition, oxidative stress status, and the reproductive and lactational performance of sows. Forty multiparous sows were assigned to 2 dietary treatments (Control group, and 1 g/kg Nucleotides group) based on a randomized complete block design using their BW at 85 d of gestation as a block. Sows from 2 groups were fed a restricted diet during gestation and ad libitum during lactation. The experiment lasted from 85 d of gestation to 21 d of lactation. The reproductive performance of sows and the growth performance of suckling piglets were measured. Oxidative stress parameters and milk components were also analysed. Data were analyzed using contrasts in the MIXED procedure of SAS. Sows in the Nucleotides group consumed more feed during the first week (P
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The fatty acid composition of human colostrum lipids is influenced by maternal diet. We measured the fatty acid composition of colostrum lipids in Slovenia. Milk samples were collected on the 3rd day postpartum from 41 women recruited in three Slovenian regions (Celje, Ljubljana and Koper). Dietary habits were examined by a food-frequency questionnaire. In situ transesterification and capillary gas chromatography were performed to determine the fatty acid composition. The coefficients of variation of the determinations of individual fatty acids accounting for >0.09 wt% were 1.87-5.91%. Between regional areas there were differences in the contents of linoleic (12.62-17.35 wt%; p < 0.001) and alpha-linolenic acid (0.70-1.07 wt%; p < 0.01), whereas the linoleic/alpha-linolenic acid ratios were similar (15.8 in Celje, 16.3 in Ljubljana and 17.9 in Koper). The highest proportion of docosahexaenoic acid (C22:6n-3) was in Celje (0.49 wt%), the lowest in Ljubljana (0.35 wt%; p < 0.05). The long-chain polyunsaturated fatty acid (LCP) content did not correlate with linoleic and alpha-linolenic acid, but there was a correlation between the sum of n-3 and n-6 LCPs. The colostrum linoleic/alpha-linolenic acid ratio was positively correlated with the body mass index during pregnancy. The fatty acid composition of colostrum lipids in Slovenia shows some variation according to geographical regions.
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The relationship between exhaustive exercise, oxidative stress, the protective capacity of the antioxidant defense system and cellular immune response has been determined. Exhaustive exercise in well-trained young men (n =19)-induced leukocytosis, decreased proportion of activated-lymphocyte subsets (CD4 + and CD8+) expressing CD69, decreased lymphocyte mitogenic response to concanavalin A (ConA) and phytohemagglutinin (PHA), increased lipid peroxidation, increased total antioxidant status (TAS) and catalase activity, immediately after exercise. Suppressed blood concentration of T-lymphocyte subsets (CD3 +, CD4+, CD8+, NK), increased TAS and blood total glutathione (TGSH) in early recovery period (30 min after exercise) were found. Strong positive correlation was observed between TGSH and lymphocyte mitogenic response to ConA and PHA (r=0.85 and 0.85, respectively) immediately after exercise. Moderate positive correlation was observed between TAS and lymphocyte mitogenic response to PHA (r=0.59) immediately after exercise as well as between TAS and lymphocyte mitogenic response to PHA and ConA (r=0.69 and 0.54, respectively). Moderate to weak correlation was observed between TAS and conjugated dienes with exercise (r =0.66) as well as in 30-min recovery (r =0.50). After a short-term bout of exhaustive exercise, immune system was characterized by acute phase response, which was accompanied with oxidative stress. Suppression of the cellular immunity 30 min after exercise shows that this period is not enough for recovery after exhaustive exercise. The results suggest the interactions between exercise-induced oxidative stress and immune response.
Chapter
This proceedings of a symposium on the digestive physiology of pigs contains 107 papers. Topic covered are: gut development and function; the gastrointestinal immune system; nutrient absoption and utilization by the gut; digestive processes; econutrition and health maintenance and free communications.
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The spectrophotometric technique for total antioxidant activity (TAA)1,2 measures the relative abilities of a.ntioxidants to scavenge the 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS*+) radical cation (ABTS*+) in comparison with the antioxidant potency of standard amounts of Trolox, the water-soluble vitamin E analogue. This method is based on the progressive consumption of antioxidant activity by ABTS*+ as it is generated in the reaction cuvette and can be automated with a spectrophotometric analyzer. Several different analytical strategies are possible using the same reagents, enabling the assay system to be used to determine the antioxidant activity of plasma, saliva, lipoprotein fractions, foods and beverages. To determine the activity of pure antioxidant substances, a hydrogen peroxide concentration of 75 μM is used, together with a 6 min measuring time. For biological samples with endogenous peroxidase activity the hydrogen peroxide concentration is increased fivefold and the measuring time shortened to 3.25 min. Assays with improved sensitivity are described for low-density lipoprotein (LDL) preparations and saliva. Use of a spectrophotometric endpoint makes the assay simple to carry out without special laboratory equipment. Measurement at 734 nm avoids a range of potential interfering factors, such as sample turbidity and non-specific absorbance by sample constituents. Current applications of the ABTS antioxidant assay are described and discussed.
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This study examined the effects of glutamine and nucleotides on growth, intestinal villous height and immune responses of weaned pigs. Eighty weaned pigs (Landrace ✕ Yorkshire ✕ Duroc) were randomly assigned to five treatments, including a control diet and a 2 (glutamine 1·0 and 1·5%) by 2 (nucleotides 500 and 1000 p.p.m.) factorial design. There were no differences among the five treatments in weight gain from weeks 0 to 4 and 0 to 8 of the experiment. However, the food intake of treatment 3 (glutamine 1·0% and nucleotides 1000 p. p. m. ) for weeks 4 to 8 was higher ( P < 0·05) than that of the control group. Intestinal villous height, and serum immunoglobulin-G concentration 2 h post lipopolysaccharide injection, were higher ( P < 0·05) for pigs administered treatment 3 than for controls. Furthermore, foot and mouth disease (FMD) neutralizing antibody titres were also higher ( P < 0·05) than the control group for pigs given treatment 3 when measured 3 weeks following vaccination with FMD antigen. These observations suggest that a combination of 10% of glutamine and 1000 p. p. m. of nucleotides in the diet could improve food intake and intestinal villous height and promote the immune responses and FMD antibody titres of weaned pigs.