Role of adenosine deaminase in alteration of cortisol and tumor necrosis factor α concentration following exhaustive exercise sessions

Behnam Bobani; Mohammad Abdi; Dariush Sheikholeslami-Vatani; Eidy Alijani; Hamid Mohammadi; Alireza Gharib

doi:10.4103/1319-6308.142369

ORIGINAL ARTICLE

Year : 2014 | Volume : 14 | Issue : 2 | Page : 128-133

Role of adenosine deaminase in alteration of cortisol and tumor necrosis factor α concentration following exhaustive exercise sessions

Behnam Bobani¹, Mohammad Abdi², Dariush Sheikholeslami-Vatani³, Eidy Alijani¹, Hamid Mohammadi⁴, Alireza Gharib⁵
¹ Sport Sciences Research Center, Physical Education College, Islamic Azad university-Karaj branch, Karaj, Iran
² Cellular and Molecular Research Center; Department of Clinical Biochemistry, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran
³ Department of Physical Education, University of Kurdistan, Sanandaj, Iran
⁴ Laboratory of Health Center, State Prisons and Security and Corrective Measures Organization, Sanandaj, Iran
⁵ Deputy of Research and Technology, Kurdistan University of Medical Sciences, Sanandaj, Iran

Date of Web Publication

9-Oct-2014

Correspondence Address:
Mohammad Abdi
Department of Clinical Biochemistry, Faculty of Medicin, Kurdistan University of Medical Sciences, Pasdaran Boulevard, Sanandaj
Iran

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/1319-6308.142369

Abstract

Context: Cortisol and tumor necrosis factor-α (TNF-α) has a significant role on the immune system. Previous studies have shown a significant increase in cortisol and TNF-α in athlete; however, the alteration of adenosine deaminase (ADA) activity in athletes has not been previously reported. Aims: This study was aimed to investigate the effect of exhaustive exercise on cortisol, TNF-α and serum ADA activity in endurance runners. Settings and Design: All endurance male athletes were enrolled in this case control study. Subjects and Methods: The participants were submitted to the same experimental protocol used in the exercise trials, they run on a treadmill until exhausting state. Saliva and blood sample were collected in resting and immediately after exercise; serums were separated and stored at −70°C pending assays. Serum was analyzed for ADA activity and TNF-α. Salivary cortisol measured by enzyme-linked immunosorbent assay method. Statistical Analysis Used: Data was analyzed using the statistical package for the social sciences 16 (SPSS Inc., Chicago) and one-sample Kolmogorov-Smirnov test. Results were presented as mean ± standard deviation and independent samples t-test used to compare mean differences. In addition, linear regression analysis was performed and means of the Pearson's correlation coefficient (r) were determined to show the correlation between variables. Results: Cortisol and TNF-α elevated following intensified training compared with the resting state. There was a significant increase in serum ADA activity between athletes and non-athletes groups. In addition, our results showed a strong direct correlation between serum total ADA activity and TNF-α. Conclusions: Based on the obtained data, an acute period of intensiﬁed training can induce an increase of ADA activity accompanies with the increase of cortisol and TNF-α. ADA is involved in the immune system development. Our results hypothesized that ADA can be associated with an increase in fatigue which lends to reduced physical activity. We showed that excessive exercise can induce an inflammatory response resulting in elevated levels of cortisol and TNF-α and perhaps by increasing activity of adenosin deaminase.

Abstract in Arabic

دور نازعة أمين الأدينوزين في التغييرات التى تحدث فى تركيز الكورتيزول وعامل نخر الورم بعد جلسات ممارسة بدنية مرهقة
خلفية الدراسة: من المعروف ان الكورتيزول وعامل نخر الورم لهما ارتباطا مع خواص الجهاز المناعة. وقد أظهرت الدراسات السابقة زيادة ملحوظة فى تركير العاملين لدى ممارسى الرياضة. ومع ذلك، لم تتطرق الدراسات لمستوى نازعة أمين الأدينوزين في الرياضيين
هدف الدراسة: هدفت هذه الدراسة إلى التعرف على اثر المجهود البدنى المرهق على تركيز الكورتيزول وعامل نخر الورم ونازعة امين الأدينوزين
المشاركون والطرق البحثية: تم اخضاع كل المشاركون على نفس البروتوكول التجريبي التى استخدمت في هذه الدراسة وان يواصل الجرى على سير متحرك حتى الإرهاق وتم جمع اللعاب وعينة من الدم في الراحة وعلى الفور بعد ممارسة المجهود البدنى المرهق. تم فصل الأمصال وتخزينها في -70 درجة مئوية وتم تحليل نازعة أمين الأدينوزين وعامل نخر الورم بالاضافة الى تركيز الكورتيزول فى اللعاب.
التحليلات الإحصائية المستخدمة: تم تحليل البيانات باستخدام الحزمة الإحصائية للعلوم الاجتماعية 16 (شركة SPSS، شيكاغو) بالاضافة الى وعينة واحدة كولموجوروف-سميرنوف الاختبار. وعرضت النتائج كعينات يعني ± الانحراف المعياري واختبار t الذى استخدم لمقارنة الفروق. بالإضافة إلى ذلك، تم إجراء تحليل الانحدار الخطي وتم تحديد وسائل معامل ارتباط بيرسون (r) لإظهار العلاقة بين المتغيرات
النتائج: سجل ارتفاعا فى تركيز الكورتيزول وعامل نخر الورم بعد المجهود المرهق مقارنة مع حالة الراحة. كانت هناك زيادة كبيرة في نشاط نازعة امين الأدينوزين فى الرياضيين وغير الرياضيين. بالإضافة إلى ذلك، أظهرت النتائج علاقة مباشرة قوية بين مصل مجمل النشاط نازعة أمين الأدينوزين وعامل نخر الورم .
الاستنتاجات: استنادا إلى البيانات التي تم الحصول عليها، وهي الفترة الحادة من التدريب المكثف يمكن أن تحدث زيادة لنشاط نازعة أمين الأدينوزين مترافقا مع زيادة الكورتيزول وعامل نخر الورم وبذلك قد يشارك نازع أمين الأدينوزين في تطوير نظام المناعة. تفترض نتائجنا أن نازعة أمين الأدينوزين يمكن ان ترتبط مع زيادة في التعب الذي يؤدى إلى انخفاض النشاط البدني. أظهرنا أن التمارين المفرطة يمكن أن تحدث استجابة التهابية مما يؤدي إلى ارتفاع مستويات هرمون الكورتيزول و وعامل نخر الورم وذلك عن طريق زيادة نشاط نازعة أمين الأدينوزين.

Keywords: Adenosine deaminase, cortisol, exercise, tumor necrosis factor-α

How to cite this article:
Bobani B, Abdi M, Sheikholeslami-Vatani D, Alijani E, Mohammadi H, Gharib A. Role of adenosine deaminase in alteration of cortisol and tumor necrosis factor α concentration following exhaustive exercise sessions . Saudi J Sports Med 2014;14:128-33

How to cite this URL:
Bobani B, Abdi M, Sheikholeslami-Vatani D, Alijani E, Mohammadi H, Gharib A. Role of adenosine deaminase in alteration of cortisol and tumor necrosis factor α concentration following exhaustive exercise sessions . Saudi J Sports Med [serial online] 2014 [cited 2023 Dec 8];14:128-33. Available from: https://www.sjosm.org/text.asp?2014/14/2/128/142369

Introduction

Moderate exercise has been shown to enhance immune activity; however, an increased load in exercise can also lead to an overall depression in certain immune responses. The main goal of these exhaustive programs is to prepare athletes for participate in competitions. The effects of intensified exercise on the immune system, inflammatory factors, ^[1] and secretion rate of stress hormones have been carried out on many studies. ^[2],[3],[4] It was established that because of severe endurance exercise, athlete's body suffered to a physiological inflammation resulting from immune response. ^[5] Many of the studies have shown that lack of complete recovery between training sessions may causes chronic fatigue and suppression of the immune system functioning. ^[6] These conditions have known as the unexplained underperformance syndrome. ^[6] As it was noted above, a systemic inflammation developed in the athlete's body result from the immune system response after extreme endurance exercise.

Many investigations proved that cytokines have a significant role in the creation of chronic fatigue symptoms. ^[7],[8] Cytokines involved in muscle and tissue proteolysis following injury or inflammation resulting from the intensified exercise. ^[7]

Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1 are the most important cytokines in acute inflammatory response during exercise. ^[7],[8],[9] TNF-α product by natural killer cells and macrophages and it is one of the most important mediators of host defense against viral infections and bacteria. ^[6]

On the other hand, severe physical or psychological stress activates the pituitary and adrenal axis and increased cortisol levels. ^[8],[10] Change in stress hormones such as cortisol and epinephrine initiate alteration in the immune system functions. Cortisol is the most important adrenal corticosteroid and has anti-inflammatory and immune suppressive effects. ^[10]

Besides, many studies have shown that adenosine concentration in plasma is involved in development of the inflammatory response and cytokine production. ^[11],[12] Adenosine (a nucleoside) regulates the stress response and cellular damage during inflammation and tissue hypoxia increases the rate of its synthesis. ^{[13],[14],[15]} Adenosine has an anti-inflammatory activity and inhibitory effect on TNF-a production. ^{[11],[16],[17]}

Adenosine levels also rise in cell damage, cell stress, hypoxia and reduced adenosine deaminase (ADA). ^[13],[18] ADA is the main regulator of adenosine and catalyzes the conversion of adenosine to inosine in purines metabolism. ^[19] Two major isoforms of ADA have been isolated with different characteristics. ADA1 exists in all human tissues and accounts for the main ADA activity in most of the tissues. ADA2 on the other hand, is the main ADA isoenzyme in serum originated mainly from monocyte to macrophage system. ^[19],[20] Reduction in ADA activity gives rise to the concentration of adenosine. ^[19]

There are not any reports on serum ADA activity in athletes; thus, we planned the present study to determine the possible effects of ADA activity on TNF-a and cortisol for better understanding the molecular mechanism of alteration these factors in athletes, particularly endurance runners.

Subjects and methods

Adenosine and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) were purchased from Sigma-Aldrich (Sigma, Missouri, USA) and enzyme-linked immunosorbent assay (ELISA) kit for determination of TNF-α was obtained from ID labs biotechnology Inc., USA, whereas DiaMetra Company (DiMetra Ltd., Foligno, Italy) supplied cortisol saliva ELISA kit.

Subjects

All members of Kurdistan province endurance running team (nine male athletes) were enrolled in this study. Physical characteristics of subjects are mentioned in [Table 1]. All subjects had at least one provincial or national championship. They are not being affected by inflammatory diseases and flu, not to take and to use the drug or complement and not to smoke. In addition, 40 age and lifestyle-matched subjects (22.9 ± 4.1 years) were enrolled as non-athletes control. The controls were tested in the absence of an exercise intervention in order to determine changes in ADA activity, TNF-α and cortisol concentration in a time dependent manner. This procedure applied on athletes after 1 week recovery and in the absence of an exercise. All subjects were informed of the aims of the study and gave written consent for participation in the project. The project was approved by the Research Ethics Committee of Kurdistan University of Medical Sciences (Iran) and conformed to the declaration of Helsinki.

Table 1: Physical characteristics of runners

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Exercise protocol

The considerable test was running on treadmill (fixed slope and increasing speed). In the first step, treadmill speed was 6 km/h and its slope was 0.01. Following appropriate warm-up procedures, treadmill speed has picked up 1 km/min until athletes would be exhaustion. Exhaustion conditions have been considered as according to athlete self-expression, pressure test and achievement of their maximal heart rate (by formula age-220 and polar pulsimeter). Exercise interventions took place at 3-4 pm. Each participant was exercised at both time and their individual samples were taken.

Sample collection and analysis

Blood and saliva samples collected pre- and post-exercise for athletes and in a time dependent manner for controls. To prepare serum, blood samples were centrifuged for 10 min at 3000 rpm and the supernatants were stored at −70°C pending assay.

Determination of ADA activity

Adenosine and EHNA were purchased from Sigma-Aldrich (Sigma, Missouri, USA). Serum total ADA (tADA) activity was determined according to the Giusti method. ^[21] In brief, based on the Bertholet reaction, the colored indophenol complex formed from ammonia liberated from adenosine was quantified spectrophotometrically. To determine ADA2 isoenzyme activity, serum ADA1 activity was selectively inhibited by the addition of EHNA into the serum samples and estimated ADA1 activity was calculated by subtracting of ADA2 and tADAactivities. Finally, all activities were expressed in unit/L. ^[21]

Determination of TNF-α and cortisol

Serum TNF-α and salivary cortisol was determined based on standard sandwich ELISA technology. Briefly, specific monoclonal antibody against human TNF-α and cortisol was pre-coated onto plates. Serum TNF-α, salivary cortisol and biotinylated human specific detection polyclonal antibodies were then added to the wells subsequently followed by the addition of avidin-biotin-peroxidase complex. Finally, horse radish peroxidase (HRP) substrate was used to visualize HRP enzymatic reaction and the concentration of serum TNF-α and salivary cortisol was expressed as pg/ml and ng/ml, respectively.

Statistical analysis

Data was analyzed by statistical package for the social sciences 16 (SPSS Inc., Chicago) and one-sample Kolmogorov-Smirnov test was applied to determine normal distribution of data. Results were presented as mean ± standard deviation (SD) and independent samples t-test used to compare mean differences. Moreover, one-way ANOVA followed by post-hoc, Tukey and Dunnett tests was used to analyze differences between groups and the P < 0.05 was considered as significant. In addition, linear regression analysis was performed and means of the Pearson's correlation coefficient (r) were determined to show the correlation between variables.

Results

Serum TNF-α concentration in post-exercise athletes was significantly higher than pre-exercise (P < 0.001). TNF-α in non-training athlete was significantly lower than athletes in post-training condition. In addition, the concentration of TNF-α in non-athlete group was 23.89 ± 8.71 pg/ml, that was remarkably lower than athletes test and control groups [Table 2].

Table 2: Adenosine deaminase activity, TNF-α in serum and salivary Cortisol of endurance runners and Non-athletes subjects

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The mean ± SD concentration of salivary cortisol in pre-exercise was 3.02 ± 0.76 ng/ml and post-exercise was 4.26 ± 0.99 ng/ml (P < 0.002). After 1 week recovery, there was not any significant difference between the mean ± SD pre- and post-exercise salivary cortisol in non-training conditions (2.43 ± 0.72 and 2.41 ± 0.65 ng/ml, respectively) and pre-exercise training condition. The mean ± SD cortisol in non-athletes was 1.59 ± 0.64 ng/ml, that was remarkably lower than athletes test and control groups [Table 2].

In the case of serum tADA activity, although it was increased in all athletes in post-exercise training condition compare to pre-exercise training and non-training day, but there was no significant difference between pre- and post-exercise. However, tADA activity was remarkably higher than non-athletes in athletes test and control conditions (P < 0.0001) [Table 2].

We also studied the possible relationship between serum tADA activity and TNF-α in response to strenuous exercise. Linear regression analysis confirmed that serum TNF-α directly increased by elevation of ADA activity (R² = 0.512, P < 0.05) [Figure 1]. Similar noticeable direct correlation (R² = 0.382, P < 0.05) was also observed between serum TNF-α and salivary cortisol [Figure 2]. Direct correlation of salivary cortisol and tADA activity (R² = 0.29, P < 0.05) was not as strong as that observed for TNF-α [Figure 3].

Figure 1: Correlation of serum tumor necrosis factor-α (TNFα) concentration with ADA activity in endurance runners and non-athletes subjects

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Figure 2: Correlation of serum tumor necrosis factor-α (TNFα) concentration with salivary Cortisol in endurance runners and nonathletes subjects

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Figure 3: Correlation of serum Cortisol concentration with ADA activity in endurance runners and non-athletes subjects

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Discussion

Based on the obtained data, an acute period of intensified training could increase TNF-α, cortisol and tADA activity.

Many of the studies have demonstrated that athletes with exhaustive exercise sessions have increased levels of serum TNF-α which is directly correlated with a systemic inflammation and increase the risk of infection. ^{[7],[8],[9],[22]} Similarly, we showed a significant increase in serum TNF-α levels in post-exercise athletes compared to pre-exercise and non-athletes groups.

TNF-α is one of the most important pro-inflammatory cytokines that increased in response of acute exercise. TNF-α is accountable for acute inflammatory response after exhaustive training. Previous study revealed that one of the most important inhibitory molecule for TNF-α is adenosine. ^{[13],[14],[17]} The importance of ADA, as the main regulator of adenosine concentration in plasma, has previously been documented. ^{[12],[19],[20]} ADA is a widely distributed enzyme which catalyzes the conversion of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively and involved in the immune system development. ^[20]

This is the first report on ADA activity in athletes. The increase of serum tADA activity, as observed here in endurance runners, heightens degradation of adenosine nucleotides and drops the concentration of adenosine. The decline in the concentration of adenosine leads to the enhanced production of TNF-α. The main source of ADA activity in serum is from monocyte to macrophage system which regulates the exact amounts of adenosine in immune cells system. ^[20] It was established that in athletes, rather than non-athletes people, monocytes count (the main source of ADA) particularly after training is elevated. ^[6],[23]

Therefore, it can be concluded that serum tADA activity rises with increasing monocytes count and elevation of ADA activity may cause decrease adenosine concentration in serum. With reduction of adenosine, the inhibitory effects of this molecule on TNF-α production removed and the next consequences will follow.

In addition, we showed that salivary cortisol in post-exercise athletes compared with pre-exercise athletes and non-athletes significantly increased. These results were parallel with other investigations that have proved the elevation of cortisol in athletes. ^[24] Stress induced by abnormal inflammatory responses such as the over-expression of pro-inflammatory cytokine TNF-α may be playing a role in production of cortisol by stimulating of hypothalamus-pituitary-adrenal axis. ^{[24],[25],[26]} This stimulation accounts for the increase of cortisol secretion in the adrenal gland. On the other hand, many studies have showed that rising cortisol concentration after intensified exercise in athletes, effect on white blood cells counts and increase the neutrophils and monocyte counts, the main source of ADA. ^[6]

Conclusion

We showed significantly higher activity of tADA in athletes compared with non-athletes and a direct correlation was observed between ADA activity, TNF-α and cortisol concentration. Athletes also showed higher serum TNF-α and salivary cortisol concentration than non-athletes subjects. It can be concluded that increasing ADA activity, may play significant roles in the progression of inflammation in athletes. May be supplementing an adenosine complements improves the inflammatory condition in endurance athletes.

Acknowledgments

The authors would like to thank the runner athletes for participating in this study.

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