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JEPonline
Effects of Training Rigor on BMI, Cardiovascular Function, and Salivary Concentration of Glutathione
Carl Bolyard1, Carissa Dunn2, Jeffrey Adams 2, Shawn Stover2
1Emergency Department, Davis Health System, Elkins, West Virginia, USA, 2Department of Biology and Environmental Science, Davis & Elkins College, Elkins, West Virginia, USA
ABSTRACT
Bolyard C, Dunn C, Adams J, Stover S. Effects of Training Rigor on BMI, Cardiovascular Function, and Salivary Concentration of Glutathione. JEPonline 2013;16(5):28-37. It has been demonstrated that regular aerobic exercise can have positive effects on body mass index (BMI) and cardiovascular (CV) function. However, increasing exercise duration and frequency can also result in excessive production of reactive oxygen species and subsequent oxidation of reduced glutathione (GSH). It was hypothesized that GSH is upregulated in response to increasing exercise rigor to neutralize the effects of reactive oxygen. Based on their reported aerobic exercise routines, 24 subjects (10 males, 14 females) were placed into 1 of 3 experimental groups: (a) minimum rigor (MIN); (b) moderate rigor (MOD); and (c) maximum rigor (MAX). Over the course of a 3-month study, the subjects maintained their normal training routines, and BMI, heart rate (HR), blood pressure, and salivary GSH concentration were assessed regularly. The average BMI decreased as training rigor increased. The average HR of the MAX group was significantly lower than that of the other two groups. The average blood pressure values of the MOD and MAX groups were significantly lower than those of the MIN group. Finally, there was no significant difference between the average salivary GSH concentrations in the 3 groups. In summary, while increasing duration and frequency of aerobic exercise training improved BMI, HR, and blood pressure, the increased efficiency of the CV system was accompanied by an upregulation of GSH that protects the tissues from exercise-induced oxidative stress.
Key Words: Aerobic exercise, Oxidative stress
INTRODUCTION
The generation of reactive oxygen species (ROS) occurs as a consequence of normal cellular metabolism (38). Oxidative stress may be defined as a condition in which the cellular production of ROS, including singlet oxygen, superoxide radical, and hydroxyl radical exceeds the body’s physiological capacity to render them inactive (6). ROS-associated molecular damage includes DNA strand breaks and single base modifications (15), oxidation of amino acid side chains and fragmentation of polypeptides (23), and the degradation of polyunsaturated fatty acids and phospholipids by lipid peroxidation (6). Neutralization of ROS is carried out by the body’s endogenous antioxidant defense system, which includes enzymatic activity of glutathione peroxidase (GPx), glutathione reductase (GR), and superoxide dismutase (SOD) in conjunction with exogenous antioxidants consumed through the diet (38).
Recent studies (27,31) report significant increases in cardiovascular function, as indicated by lowered heart rate (HR) and blood pressure, as well as significant decreases in body mass index (BMI) in response to regular aerobic exercise. Furthermore, the increase in oxygen uptake during aerobic exercise is accompanied by an elevation in ROS. Acute aerobic exercise generates ROS by creating a disturbance in electron transport that leads to excessive leakage of superoxide radicals (6). However, long-term endurance training effectively reduces the damage associated with increased oxygen uptake by enhancing the body’s antioxidant defenses. It has been demonstrated that GPx (8,39), GR (9,39), and SOD (10,22) activities increase in response to endurance training.
Reduced glutathione (GSH) plays a prominent role in the cellular defense against oxidative stress by scavenging ROS, both directly and as a substrate for GPx (12). Previous research (17,25,37) has demonstrated that strenuous physical exercise can affect GSH homeostasis by decreasing its tissue concentration, disturbing its cellular redox status, and interfering with its synthesis and transport (19,20). Furthermore, studies suggest that endogenous cellular GSH is not sufficient to withstand the increased oxidation associated with vigorous exercise. Therefore, it would be beneficial to increase levels of cellular GSH to provide protection against exercise-induced oxidative stress. Such an increase in GSH concentration has been noted in blood (18), liver (8), and skeletal muscle (16) in response to regular aerobic exercise.
GSH is also present in saliva at detectable concentrations. It has been demonstrated that salivary concentrations of GSH decrease significantly in response to asthma (11), dental caries (34), and type II diabetes (2). The present study investigated the hypothesis that an exercise-induced upregulation of GSH can be maintained during aerobic exercise training, despite the increase in oxygen uptake and potential for increased oxidative stress. If the hypothesis is supported, BMI and cardiovascular function (as determined by HR and blood pressure) will improve, and salivary GSH concentration will remain stable as exercise duration and frequency increase.
METHODS
Subjects
The Institutional Review Board of Davis & Elkins College approved this study. A total of 10 males (age 20 to 66 yrs) and 14 females (age 20 to 53 yrs) participated. Based on their reported aerobic exercise routines, the subjects were placed into 1 of 3 experimental groups: (a) minimum rigor (MIN); (b) moderate rigor (MOD); and (c) maximum rigor (MAX). Subjects in the MIN group (n=6) engaged in aerobic exercise an average of 42 min·d-1, 3 d·wk-1, and had been doing so for less than a year. Subjects in the MOD group (n=10) exercised an average of 48 min·d-1, 4 d·wk-1, and had maintained their current level of aerobic training for at least 2 yrs. Subjects who were placed into the MAX group (n=8) exercised an average of 60 min·d-1, 5 d·wk-1, and had maintained their current level of aerobic training for at least 5 yrs. Running was the primary form of aerobic training for each group, with other modes of aerobic exercise used sparingly.
Saliva Collection
At the onset of the study, all subjects signed consent forms and agreed to fast (and drink only water) for at least 8 hrs prior to each sample collection. During the initial sample collection, subjects provided the following information: age, height, current exercise routine, servings of fruits and vegetables/day, daily supplements (if any), and number of hours slept at night. After rinsing with deionized water, each subject provided a 2 to 3 ml saliva sample, which was stored in a polypropylene centrifuge tube at -20°C until analyzed. Body weight, heart rate, and blood pressure for each subject were measured. BMI was determined by entering height and weight values into an online BMI calculator provided by the National Heart, Lung, and Blood Institute (32).
Over the 3-month course of the study, the subjects engaged in their own individual aerobic exercise regimens. Subsequent fasting saliva samples were collected at approximately 1-month intervals. All sample collections (including the initial one) took place between 8:00 a.m. and 10:00 a.m. Changes (if any) to exercise routine, diet, sleep schedule, etc., were recorded, subjects were weighed, and cardiovascular function was assessed via HR and blood pressure at the time of each sample collection.
Glutathione Assessment
The subjects’ saliva samples were centrifuged at 3000 x g for 10 min. Supernatants were deproteinated with 5% metaphosphoric acid and centrifuged again at 1000 x g for 10 min. Resulting supernatants were reacted with 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) at room temperature. DTNB combines with glutathione to generate a product with maximal absorbance at 412 nm (Cuvette Assay for GSH/GSSG, Oxford Biomedical Research, Oxford, MI). All samples were analyzed spectrophotometrically, and GSH concentrations were calculated from the absorbance values using the following formula: [GSH] = [(A – B) – b] ÷ a × df, where [GSH] is the mM concentration of GSH in the sample, A is the change in absorbance of the sample at 412 nm over 10 min, B is the change in absorbance of the blank (deionized H2O) at 412 nm over 10 min, a is the slope of the GSH standard curve (standards were included in the assay kit), b is the intercept of the standard curve, and df is the dilution factor of the sample.
Statistical Analysis
Data were subjected to a multiple comparison analysis of variance (ANOVA). Fisher’s least significant difference test was employed to compare specific groups in the ANOVA. An alpha level of P<0.05 was regarded as statistically significant. Data are expressed as mean ± standard error.
RESULTS
Over the course of the study, the MAX group maintained a very stable BMI. Participants in the MOD group lost an average of 1 lb, while individuals in the MIN group lost an average of 3 lbs during the study. Average BMI was not significantly affected by the weight loss. The average HR of the MAX group was significantly lower than that of the other two groups.The average systolic and diastolic blood pressure values of the MOD and MAX groups were significantly lower than those of the MIN group (Table 1). Within each group, there were no gender- or age-related effects.
Table 1. Training Effect on BMI and Cardiovascular Function.
Training Group / BMI / Heart Rate(beats·min-1) / Systolic
Blood Pressure
(mmHg) / Diastolic
Blood Pressure
(mmHg)
MIN (n=6) / 28 ± 3.4 / 73.2 ± 3.6 / 123.8 ± 2.7 / 81.7 ± 2.5
MOD (n=10) / 24.7 ± 1 / 70 ± 4.1 / 117.1 ± 1.4a / 75.8 ± 1.4a
MAX (n=8) / 22.9 ± 0.9 / 59.5 ± 3.1b / 117.6 ± 2.7a / 74.8 ± 1.4a
BMI, Body Mass Index; Data are presented as mean ± standard error. aSignificantly different from MIN values (P<0.05). bSignificantly different from MIN and MOD values (P<0.05).
Average salivary GSH concentration for MIN, MOD, and MAX groups was 6.2 ± 1.5, 4.5 ± 1, and 3.8 ± 1.1 mM, respectively (Figure 1).One subject in the MAX group exhibited salivary GSH values that were considerably higher (an average of 12.9 mM) than those exhibited by other members of the group. Those outlying values were not included in the GSH analysis. Even though GSH numbers decreased slightly with increasing exercise duration and frequency, statistical analysis indicated that the concentration did not decrease significantly. Within each group, there were no gender- or age-related effects.
Figure 1. Training Effect on Salivary Concentration of Glutathione (GSH). Data are presented as mean ± standard error. There was no statistically significant difference between the three groups.
DISCUSSION
BMI and Cardiovascular Function
Recent studies (27,31) report significant increases in cardiovascular function (as indicated by lowered HR and blood pressure), as well as significant decreases in BMI in response to regular aerobic exercise. Results of the current study are in agreement. The average HR of the MAX group was significantly lower than that of the other two groups.The average blood pressure values (systolic and diastolic) of the MOD and MAX groups were significantly lower than those of the MIN group. The average BMI of the MAX, MOD, and MIN groups was 22.9, 24.7, and 28, respectively (Table 1). According to the National Heart, Lung, and Blood Institute (32), a BMI less than 18.5 indicates that an individual is underweight, a BMI between 18.5 and 24.9 represents a normal weight, a BMI between 25 and 29.9 indicates that an individual is overweight, and a BMI of 30 or higher represents obesity. On average, the subjects in the MOD and MAX groups exhibited healthy body weights, while those in the MIN group exhibited an average BMI in the overweight range.
Salivary Glutathione
GSH plays a prominent role in the cellular defense against oxidative stress. Conversion of GSH to the oxidized form of glutathione (GSSG) is catalyzed by GPx during the reductive detoxification of hydrogen peroxide (29). GSH is regenerated from GSSG by activity of GR (36). Although some epithelial cells have the capacity to take up intact GSH (14), most cells rely on de novo synthesis to
maintain cellular stores. GSH is synthesized by a two-step process involving the enzymes g-glutamate-cysteine ligase (GCL) and glutathione synthetase (GS). GCL catalyzes the intracellular reaction that results in attachment of a sulfhydryl-containing cysteine residue to a glutamate residue. GS activity adds a glycine residue to complete the tripeptide (12). Results of the current study indicate no significant difference between the average salivary GSH concentrations exhibited by members of the MIN, MOD, and MAX groups (Figure 1). Because oxidation of GSH will increase as exercise duration and frequency increases, these results suggest an increase in GSH synthesis as a response to the rigorous training. An analysis of GCL activity, the limiting factor in GSH synthesis, will be necessary to confirm this hypothesis.
Neutral Factors
A previous study (35) found no difference between the redox status and antioxidant defenses of subjects undergoing aerobic training concurrent with strength training and those undergoing aerobic training alone. In the current study, 11 of the 24 participants incorporated at least 30 min of strength training into their weekly workouts. Within each group, there were no significant training-related differences between BMI, cardiovascular function, or salivary GSH.
Previous research (26) indicates no significant difference between males and females in terms of HR and systolic blood pressure, post-exercise. In the current study, there were 3 males and 5 females in the MAX group, 4 males and 6 females in the MOD group, and 3 of each in the MIN group. Within each group, there were no significant gender-related differences between BMI, cardiovascular function, or salivary GSH.
The aging process leads to an increased production of reactive oxygen by mitochondria that become progressively less efficient at transporting electrons within cells that become progressively less efficient at expressing antioxidant enzymes (3,33). Consequently, oxidative stress is implicated in many physiological disorders associated with aging. For example, oxidation of low-density lipoprotein (LDL) in blood vessels is now considered a primary factor in the development of atherosclerosis (28), and the brains of patients with Alzheimer’s disease exhibit elevated concentrations of malondialdehyde (MDA), an indicator of lipid peroxidation (40). Endurance-trained muscles have higher levels of antioxidant enzyme activity and GSH content, allowing more efficient removal of ROS (20,22,39). Consequently, endurance training reduces the impact of age-related oxidative stress. Following an endurance training regimen, soleus muscles from aged rats express decreased concentrations of MDA, relative to sedentary controls (21). In the current study, the average age of the subjects in the MAX, MOD, and MIN groups was 44.6, 42.9, and 46, respectively. Within each group, there were no significant age-related differences between BMI, cardiovascular function, or salivary GSH.