Efficacy of Selected Treadmill Training Program on Oxidative Stress in Adolescent Patients with Down Syndrome

ABSTRACT
Introduction: Down syndrome (DS) is the most common genetic cause of mental retardation. There are several lines of evidence showing that individuals with Down syndrome are under unusual oxidative stress. Aim of work:This study was designed to assess the efficacy of electronic treadmill exercise training program on serum malondialdhyde (MDA) as a marker for lipid peroxidation and the antioxidant enzyme glutathione peroxidase (GPx) in adolescents with Down syndrome . Subjects and Methods: The study was carried out on 30 adolescents with Down's syndrome of both sexes their age ranged from 15-18 years; they were selected from the outpatient clinic of children with special need, National Research Center Cairo, Egypt. Thirty clinically healthy subjects, age and sex matched to DS patients were included in the study as control group. Treadmill training program was performed for 12 weeks in Physical Therapy College Cairo University. Results: Our results revealed significant increase in GPx activity and decrease in serum level of MDA in DS patients after exercise. Conclusion: We concluded that exercise promotion is very important for people with DS and that it requires attention to motivators and facilitators of exercise adherence. We suggested that exercise at this intensity may decrease risk factors for diseases associated with oxidative stress in those population for better quality of life.

Introduction:
Individuals with Down syndrome have been described as having high levels of oxidative stress (Carratelli et al., 2001). Accordingly, research on this topic may be of great interest since oxidative damage has been proposed as a pathogenic mechanism of atherosclerosis, cell aging, neurodegeneration, carcinogenic events and immunological disorders in this population (Pastore et al., 2003). Oxidative Stress (OS) is a term used to describe the effect of oxidation in which an abnormal level of reactive oxygen species (ROS), such as the free radicals (e.g. hydroxyl, nitric oxide, super oxide) or the non-radicals (e.g. hydrogen peroxide, lipid peroxide) lead to damage of specific molecules with consequential injury to cells or tissue (Mazza., et al 2007). It has been suggested that an increase in oxidative stress in DS patients may cause adverse effects in the cell membranes through the oxidation of polyunsaturated fatty acids (PUFAs) (Garcez et al., 2005). Increased lipid peroxidation has been observed in the brain of DS patients which may be the cause of neurotransmitter impairment (Keiichi et al., 2009). Glutathione peroxidase; plays an important role in preventing peroxide accumulation in cells, and in subsequently prevents lipid peroxide formation; it may have an important role in protection from Progression of neurobiological abnormalities within the cells of patient with DS (Garcez et al., 2005). Fortunately, regular exercise may improve redox metabolism in general population. However, far less information is available on handicapped populations such as Down syndrome individuals (Elosua et al., 2003). The aim of this study was to determine the efficacy of treadmill training program on oxidative stress in adolescent patients with Down syndrome.

SUBJECTS AND METHODS: Thirty clinically healthy subjects, age and sex matched to DS patients were included in the study and They did not receive any program of training. Thirty DS patients of both sexes their age ranged from 15-18 years were selected from the outpatient clinic of children with special need, National research center. All patients were trainable and walk freely without support. They were able to understand and follow verbal commands and instructions. 1-Investigations: a.Clinical investigations: Full history taking, Clinical examination, Anthropometric measurements included (weight, height, and body mass index). BMI is a simple index of weight-for-height that is commonly used as a measure of overweight and obesity (BMI= kg/m2) b) Biochemical investigations: Blood was withdrawn and collected in heparinized tubes for control group, For patients group blood samples were withdrawn before starting and at the end of the training program for. (1)Determination of glutathione peroxidase activity by (ELISA). (2)Determination of malondialdehyde as a marker of lipid peroxidation by using quantitative Colorimetric Microplate. Intervention: -Treadmill training program for 12 weeks (for DS patients only). 2-Training Program: Patients performed exercise on an Electronic treadmill for 12 week (three sessions per week). The duration of each session was increased gradually according to the training program from 10 minutes at the beginning of training program until reach 40 minutes at the end of the program, divided into warming- up, active phase training and cooling down. The following sequence was done:

First week: the goal was to perform exercises three days for 1st week of the program. Table (1): treadmill parameters in the first week of training: Fig (8). Week 1 Speed(mph) 1.5 - 2 Incline 3% Time(min) 10

Second week: During this week, strive to increase the time that the patient s were able to walk on the treadmill. Also higher speeds and inclinations were introduced.

Table (2): Treadmill parameters in the second week of training: Fig (9). Week 2 Speed 2 2.5 Incline 5% Time(min) 16

Next weeks Fitness of patients increased, so we continued in increasing the time, inclination, and speed as follow: Third week: Table (3): Treadmill parameters in the third week of training: Fig (10). Week 3 Fourth week: Table (4): Treadmill parameters in fourth week of training: Fig (11). Week 4 Speed 3 - 3.5 Incline 9% Time(min) 25 Speed 2.5 - 3 Incline 7% Time(min) 22

Second eight weeks of training: Table (5) Treadmill parameters in second eight weeks of training: Fig (12). Week 5-12 Speed 4 5 Incline 14% Time(min) 40

RESULTS:

This study was conducted to determine the efficacy of using selected treadmill training program on oxidative stress in adolescent patient with Down syndrome. The subject of this study was divided into two groups of equal number: Control group and DS group.

The results of this study were represented as follow: A- Sample description for available ages, weights, height, BMI , GPx and MDA. B- Effect of training procedure on the parameter of the study for DS group before and after training. C- Correlations between parameters for DS group. The results of this study are illustrated in the following tables and figures. A-Sample descripation: Table 6 shows that there was significant statistical difference between both groups as regards their weight, height and BMI. [Table (6), Fig (13, 14, 15)].

Table (6): Descriptive data of control and DS groups. Control group Items (n=30) Mean SD Age (years) Weight (Kg) Height (m) BMI(Kg/m2) 16.731.01 66.66.59 1.640.05 24.032.17 DS group (n=30) Mean SD 16.561.07 69.934.6 1.580.04 27.721.92 tvalue 0.61 2.27 3.98 7.09 P 0.53 10.0 0.00 0.00

P> 0.05 Non-significant P 0.05 Significant BMI: Body mass index

80 70 60
W h (K ) eig t g

50 40 30 20 10 0 Control group DS before exercise

Fig (13): Mean SD of the weight for Control and DS groups

1.5
Height (m)

0.5

0 Control group DS before exercise

Fig (14): Mean SD of the height for Control and DS groups

30

25

*
BMI (Kg/m )
2

20

15

10

0 Control group DS before exercise

Fig (15): Mean SD of the BMI for Control and DS groups.

GPx and MDA for control and DS group before training: The table shows low mean level of GPx in DS group compared to control group, On the other hand mean level of MDA was higher than that of control group. [Table (7), Fig (16, 17)]. Table (7): Glutathione peroxidase and Malondialdehyde levels for control and DS groups. Control group Items (n=30) Mean SD GPx (U/gHb) MDA(umol/L) 23.895.3 0.870.28 DS group (n=30) Mean SD 18.395.29 1.520.76 tvalue 5.41 7.68 P 0.00 0.00

P> 0.05 Non-significant GPx: Glutathione peroxidase

P 0.05 Significant MDA: Malondialdehyde

30

25

GPx (U/gHb)

20

15

*
10

0 Control group DS before exercise

Fig (16): Mean SD of GPx level for Control and DS groups

2.5

MDA (u mol/L)

1.5

*
0.5

0 Control Group DS before exercise

Fig (17): Mean SD of MDA level for Control and DS groups

B- Effect of training procedure on the parameter of the study for DS group before and after training

The table shows that there was no significant difference between weight, height and BMI in DS patients before and after exercise. [Table (8) figures (18, 19, and 20)]. Table (8): Anthropometric data of DS patients before and after exercise. DS before DS after exercise (n=30) exercise (n=30) Items Mean SD Weight (Kg) Height (m) BMI(Kg/m2) 69.934.6 1.580.04 27.721.92 Mean SD 69.24.18 1.580.04 27.431.8 tvalue 0.64 0.001 0.59 P 0.26 0.50 0.27

P> 0.05 Non-significant BMI: Body mass index

P 0.05 Significan

80 70 60
Weight (Kg)

50 40 30 20 10 0 DS before exercise DS after exercise

Fig (18): Mean SD of the weight for DS patients before and after exercise.

1.5
Height (m)

0.5

0 DS before exercise DS after exercise

Fig (19): Mean SD of height for DS patients before and after exercise.

35 30 25 20 15 10 5 0 DS before exercise DS after exercise

BMI (Kg/m2)

Fig (20): Mean SD of BMI for DS patients before and after exercise.

GPx and MDA for DS group before and after training: The table shows significant increase mean GPx level after exercise training program for three months, On the other hand mean MDA level showed significant decrease after exercise [Table (9), fig (21, 22)]. Table (9): Glutathione peroxidase and Malondialdehyde levels for DS patients before and after exercise. DS before DS after exercise (n=30) exercise (n=30) Items Mean SD GPx (U/gHb) MDA(umol/L) 18.395.29 1.520.76 Mean SD t-value 20.925.07 1.130.52 2.3 4.10 P 0.01 0.00

P> 0.05 Non-significant GPx: Glutathione peroxidase

P 0.05 Significant MDA: Malondialdehyd

30

25

G (U b) Px /gH

20

15

10

0 DS before exercise DS after exercise

Fig (21): Mean SD of GPx level for DS patients

before and after exercise.

2.5

2
M A(um l/L D o )

1.5

0.5

0 DS before exercise DS after exercise

Fig (22): Mean SD of MDA level for DS patients before and after exercise.

Correlations between parameters for DS group: Before training:

30

25
G x (U H ) P /g b

r = 0.522 * P = 0.001
20

15

10

5 20 25
BMI (Kg/m )
2

30

35

Fig (23): Correlation between GPx and BMI before exercise P<0.05: Significant r: Correlation Coefficient

This figure shows correlation between GPx level and BMI in DS. Significant and negative correlation was shown in DS patients before exercise training program with r value -0.0522 and P=0.001. Fig (24).
2.5

2
M A uml/ ) D ( oL

1.5

r = 0.290 * P = 0.012

0.5

0 20 25
BMI (Kg/m )
2

30

35

Fig (24): Correlation between MDA and BMI before exercise P<0.05: Significant r: Correlation Coefficient

This figure shows correlation between MDA level and BMI in DS patients. Significant and positive correlation was shown in DS patients before exercise training program with r value 0.290 and P= 0.012. Fig (25).

30

25
G x (U H ) P /g b

20

r = 0.269 * P = 0.019

15

10

5 0 0.5 1 1.5
MDA (u mol/L)

2.5

Fig (25): Correlation between GPx and MDA before exercise P<0.05: Significant r: Correlation Coefficient

This figure shows correlation between MDA and GPx levels and in DS patients before exercise. Significant and negative correlation was shown in DS patients with r value 0.269 and P= 0.019. Fig (26).

After training: After exercise training program for three months using electronic treadmill, there was significant and negative correlation between GPx level and BMI r= -0.434 & p=0.003 fig (27) , significant and positive correlation between MDA and BMI r= 0.318 & p= 0.006 and significant and negative correlation was shown between MDA and GPx levels r= 0.340 & p=0.003 fig (28).

30

r = 0.434 * P = 0.003
25
GPx (U/gHb)

20

15

10 20 25
BMI (Kg/m2)

30

35

Fig (26): Correlation between GPx and BMI after exercise P<0.05: Significant r: Correlation Coefficient

2.5

2
MDA (u mol/L)

1.5

r = 0.318 * P = 0.006

0.5

0 20 25
BMI (Kg/m )
2

30

35

Fig (27): Correlation between MDA and BMI after exercise P<0.05: Significant r: Correlation Coefficient

30

25
GPx (U/gHb)

r = 0.340 * P = 0.003
20

15

10 0 0.5 1 1.5
MDA (u mol/L)

2.5

Fig (28): Correlation between GPx and MDA after exercise P<0.05: Significant r: Correlation Coefficient

DISCUSSION
Down Syndrome (DS) is the most common chromosomal abnormality that is compatible with life, contributing to about 30% of all moderate to severe cases of mental retardation (Rachidia and Lopes, 2008). The incidence of DS is influenced by maternal age and differs between populations , between 1 in 319 and 1 in 1000 live births Human (Wiseman et al., 2009). It is considered a major cause of mental retardation in Egypt affecting 1to 750 live births (Meguid et al., 2004) Therefore it has been hypothesized that an increase in oxidative stress in patients with DS would account for the appearance of different complications, such as atherosclerosis, accelerated cell aging, cellular mutagenicity, and neurologic disorders that often occur in these patients (Ordonez and Rodriguez., 2007). This study was conducted to assess the effect of using electronic treadmill exercise program on some oxidative stress biomarkers in adolescent with Down syndrome. The study was carried out on 30 Down syndrome patients of both sexes their age ranged from 15-18 years; they were selected from the outpatient clinic of children with special need, National center of

research Cairo, Egypt. Thirty clinically healthy subjects, age and sex matched to DS patients were included in the study as control group. Regular physical activity or exercise has other important benefits rather than weight loss, that include atherogenic lipid molecules, improving glucose intolerance, increased flexibility and motor coordination(Elentz et al., 2009), such benefits in addition to psychological effects including improvement of self esteem, and social interaction and confidence. Previous studies had successfully explored the relationship of anthropometrical parameters such as body mass index, and diet, exercise, disability status and degree of social integration in individuals with Down syndrome (Fujiura et al., 1997). However, far less information is available on handicapped populations such as DS regarding association between anthropometrical parameters and antioxidant system. In the present study mean weight and BMI of DS group was significantly high as compared with control group (p> 0.01 and p> 0.00 respectively), there was no significant decrease in mean weight and BMI in DS patient after 12 week treadmill training program in comparison to the beginning of the exercise program. Similar results were observed by Aguiar et al., 2008 who reported no significant weight changes and slight decrease in BMI in DS adolescents at the end of the 16-week of supervised judo training of controlled intensity in comparison to the beginning of the exercise program. The results of the majority of studies that evaluated the anthropometric indices of DS persons with mental retardation have reported that the prevalence for obesity in this population is high, and in fact may be twice as high as their peers without mental retardation. (Wilmore and Costill., 1999. Baynard et al., 2004).This may be due to an increased prevalence of thyroid disease, particularly subclinical hypothyroidism (M h et al., 2010), also Andriolo et al., 2005 theorized that a lower resting metabolic rate was a cause of increased rates of obesity in individuals with DS. Regarding Gpx and BMI significant and negative correlation was found in DS patients before and after exercise training program (P=0.001 & 0.003 respectively).Similarly Olusi, 2002 and Roussel et al., 2009 reported inverse relationship between the erythrocyte cytoprotective enzyme GPX and BMI in healthy obese and overweight impaired fasting blood glucose, on the other hand Ordonez and Rodrigues, 2007

reported that BMI was not significantly correlated to GPX in study dealing with glutathione peroxidase activity and anthropometrical parameters in adolescents with Down syndrome. In the present study BMI also was positively significant correlated with MDA level, these results in agreement with Mohn et al., 2005 who demonstrated association between BMI and lipid peroxidation expressed as malondialdehyde (MDA) content in obese prepubertal children. It is generally accepted that antioxidant enzyme superoxide dismutase (SOD) catalyzes the dismutation of superoxide anion (O2_) to H2O2 and then, in a second step, glutathione peroxidase (GPX) and catalase (CAT) convert hydrogen peroxide (H2O2) to water. Consequently, the activity of the first-step (SOD) and second step (GPX, CAT) antioxidant enzymes, expressed as the quotient SOD/GPX+CAT, must be balanced to prevent cell damage by oxidative stress. (Crosti et al; 1989). The gene for SOD lies in humans on chromosome 21 and consequently it is conceivable that its activity is increased, whereas the above-mentioned quotient got disbalanced in individuals with Down syndrome. A pathogenesis termed SOD-catalyzed hydroxyl radical formation is proposed by Kowald and Klipp., 2004 to explain this and states the excess of un-neutralized hydrogen peroxide is converted to the highly damaging hydroxyl radical that may finally lead to increase oxidative damage in this population. In the present study there was significant decrease in GPX activity in DS group compared to control group (p<0.00) and no significant differences were found in GPx levels between the sexes in the DS group and the control group, similar results was reported by Muchova et al., 2001 he demonstrated a significant lowering of reduced glutathione concentration in DS patients that lead to decease activity of GPx. Our results revealed that there was increase in GPX activity after 12 week treadmill training program as compared with its activity before exercise (p<0.01) This agree with Ordoez et al.,2006 who found that a 12-week training program increased erythrocyte antioxidant enzyme activities such as glutathione peroxidase in adolescents with Down's syndrome, also it was reported that the levels of reduced glutathione were increased after a 16-week training program in adults with Down's syndrome (Monteiro et al., 1997). Also in a study by Elosua et al., 2003, it was revealed that regular exercise activity program for 16-week may

enhance the blood antioxidant system in general and GPX activity in Down syndrome. The mechanisms regarding the regulation of some antioxidant enzymes through exercise have been described (Ji, 2008). Carvalho et al. 2010 reported an increase in the plasma activities of both glutathione reductase and GPx may be due to the up-regulation of antioxidant enzyme mRNA, protein concentrations and activity during and after the exercise is closely related to redox changes, due to the role of reactive oxygen and nitrogen species (RONS) as important signaling molecules, the chronic increase in RONS production during multi component training would certainly result in the increased activity of these two enzymes involved in the redox cycle of glutathione. Similar results were reported in many studies as regarding the effect of regular exercise training 12 weeks of moderate intensity aerobic exercise performed three days a week on antioxidant defense mechanism references. The authors reported an upregulation in antioxidant enzymes, evident by an increase in the activity of superoxide dismutase, glutathione peroxidase and catalase. (Ennezat et al., 2001, Edwards et al., 2004 Linke et al., 2005). ROS and oxidative stress have been shown to have a major role in the manifestation of some of the neuropathological features of DS like AD (Zana et al., 2007). It was suggested that the brain may be particularly vulnerable to oxidative stress as it consumes a fifth of the total oxygen inspired and carries out the turn over of large quantities of ATP at a high rate. Since approximately 5% of oxygen consumed by cells is estimated to be reduced to ROS, relatively higher amounts of ROS may be generated in the brain as compared to other tissues that use less oxygen (Lee et al., 2001). Neurons are non replicating cells and any damage to brain tissues by the ROS tends to be cumulative over time. Brain is rich in PUFAs, which are particularly susceptible to ROS damage. It has been shown that the Down s cell membranes have PUFA composition different from normal cells and that they are more prone to lipoperoxidation (Cui et al., 2004). Oxidative stress generally causes damage to the membrane polyunsaturated fatty acids leading to the generation of MDA, a thiobarbituric acid reacting substance (TBARS). It is considered a major end-product of oxidation of polyunsaturated fatty acids and has been frequently measured as an indicator of lipid peroxidation and oxidative

stress in vivo. Increased lipid peroxidation products in DS patients have been reported (Casado et al., 2007). Recent studies have demonstrated higher MDA and/or TBARS levels in Alzheimer disease (GustawRothenberg et al., & Sinem et al., 2010). In the present study there were significant increase in serum MDA level in DS group compared to control group (p>0 .001) which means an increased rate of oxidative damage in individuals with DS no significant differences were found in MDA levels between the sexes in the DS group and the control group. These results agreed with Pincheira et al., 1999 who reported elevated the MDA and TBARS concentrations in the serum in DS patients, also Jovanovic et al., 1998 revealed that increase MDA levels in the urine in DS patients could be one of the consequences of an imbalance of the activities of the antioxidant enzymes. Our data revealed that treadmill regular exercise for 3 months reduced significantly serum MDA levels in DS group in comparison to the beginning of the exercise program (p< 0.00). Similar results have been reported by Ordonez and Rodriguez, 2007,they observed that a 12-week exercise program significantly reduced lipid peroxidation in terms of plasmatic MDA content in male adolescents with Down's syndrome. This finding may be due to improved serum lipid profile in adolescents with DS, as HDL-Cholesterol increased where as LDLCholesterol decreased (Ordoez et al., 2005), and may contribute, at least in part, to improve lipoperoxidation in exercised individuals since low-density lipoprotein (LDL) oxidation can be inhibited by high-density lipoprotein (HDL) through its oxidable components or associated enzymes like paraoxonase and platelet-activating factor acetylhydrolase (Brites et al; 2006), A similar study in obese individuals reported attenuation in exercise-induced lipid peroxidation following 24 weeks of a moderate intensity, total body, resistance training protocol (Vincent et al., 2006). Zambrano et al., 2009 also reported a decrease in salivary lipid hydroperoxides in persons with DS after aerobic exercise and suggested that aerobic exercise can be considered as a way to reduce the oxidative stress in those patients. Reviewing the literatures, previous studies addressing the role of physical exercise in oxidative stress and antioxidant status in people with DS have shown conflicting results. An extensive literature has indicated that moderate exercise (similar to that used in the present study) exerts low stress without

oxidative damage consequences in non-DS subjects (Ji 2001., Apor and Radi 2006).Interestingly, Aguiar et al., 2008 revealed that the blood markers of oxidative damage to lipids (TBARS and lipid peroxides) and proteins (carbonyls) were increased by the moderate judo training extended over a period of 16 weeks and consisted of three sessions per week. Adults with DS however, GPx activity remained unaltered in the serum of DS subjects after the moderate judo training. These results contrast with previous studies and our results that have demonstrated increased GPx activity (Ordonez et al., 2006b), This inconsistency data may be explained by differences in the age of the subjects (23.3 2.1 vs. 16 1.1 years old) and/or the duration of the training program (16 vs. 12 weeks). Taken together, these previous findings and our present results lead us to speculate that young adults with DS may be more susceptible to physical training-induced oxidative stress than adolescents with DS, which should be taken into account in physical training programs for this population .Perhaps the metabolism for equilibrium between the initial oxidative stress of physical training and its compensation is slower in the DS population, since motor performance and lactate indices were improved, but oxidative stress was not completely compensated, despite increased SOD and CAT. Also judo is may considered to be an explosive sport which demands great anaerobic strength and capacity, accompanied by a well developed aerobic system (Radovanovic., 2009).

CONCLUSION
According to the results of this study supported by the relevant literature, it can be concluded that regular exercise program for 12 week using treadmill is highly recommended to be used for adolescent with Down syndrome as it improves the level of antioxidant enzyme GPX and decrease lipid peroxidation marker MDA level, so that exercise at this intensity may decrease risk factors for diseases associated with oxidative stress in these population. Finally, this study support the efficacy of treadmill training for promoting the development of independent walking and for advancing other quantitative and qualitative aspects of brain performance.

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