Nucleotides are essential to nearly all biological processes including DNA and RNA synthesis, coenzyme synthesis, energy metabolism, cellular signaling, and protein homeostasis (8,14,17). Nucleotides produced through de novo synthesis are often insufficient to meet the needs of rapidly proliferating tissues, and therefore, the salvage pathway is required to synthesize nucleotides from exogenous sources (12). As such, dietary nucleotides are necessary to maintain immune function, tissue growth, and cellular repair (8,12,17). Exercise is a stressful and damaging stimulus, significantly increasing demands on the immune system and temporarily attenuating immune cell function. Consequently, there has been a growing interest in the implications of exogenous nucleotide supplementation on exercise-induced immune responses.
Recently, nucleotide supplementation demonstrated the ability to attenuate the transient immunosuppression that occurs after exercise (24,25,29,30). After acute exercise bouts, serum and salivary immunoglobulin levels were greater with nucleotide supplementation. Ostojic et al. (30) also demonstrated an enhanced natural killer cell count and cytotoxicity, which indicates that nucleotide supplementation can impact the adaptive and innate immune systems. Enhanced innate immune activity could aid recovery from exercise, as neutrophils contribute to the removal of debris from damaged tissue (35). The innate immune system can also induce secondary muscle damage through the generation of reactive oxygen species, further attenuating athletic performance (2,3,10). Consequently, the impact of nucleotide supplementation on innate immune system activity is of interest to athletes and active individuals.
Aside from increases in salivary immunoglobulins, McNaughton et al. (24,25) have observed a decreased cortisol response to exercise, which would partially explain the reduced immunosuppression. Animal models have observed similarly attenuated cortisol responses to stressful stimuli (31,34). Given the roles of cortisol in gluconeogenesis and glycogenolysis, a reduced cortisol response may indicate a reduction in the metabolic stress of the exercise bout. In the days after stressful exercise, elevated cortisol levels could impair recovery, as cortisol can increase protein degradation and inhibit protein synthesis (18,22). Unfortunately, McNaughton et al. (24,25) did not analyze cortisol values beyond the immediate postexercise time point.
Previously, Ostojic et al. (29,30) and McNaughton et al. (24,25) demonstrated the effects of nucleotide supplementation after acute exercise, but not recovery in the days following. Additionally, the previous investigations used cycling and running exercises, which differ in their recruitment and metabolic demands when compared with heavy resistance exercise. The effects of nucleotide supplementation on the response patterns to resistance exercise are currently unknown. Therefore, the primary purpose of this investigation was to determine whether nucleotide supplementation affects the acute hypothalamic-pituitary axis (HPA) and immune response to resistance exercise. Additionally, we sought to determine whether nucleotide supplementation could improve physical performance capabilities during recovery from such strenuous exercise.
Experimental Approach to the Problem
To evaluate the effects of a dietary nucleotide supplement on resistance exercise stress and recovery, a double-blinded, placebo-controlled, mixed methods crossover design was used. Each subject completed a familiarization visit followed by 2 supplementation and testing cycles, which were separated by a 1-week washout period. The 2 treatment cycles consisted of a nucleotide or placebo supplement, with the cycle order randomized and balanced. Each cycle began with a 2-week loading phase in which subjects took the supplement while maintaining their normal exercise routines. At the beginning of the third week, an acute heavy resistance exercise protocol (AHREP) was completed. On this day, blood draws were taken before (PRE), immediately after (IP), and at 15, 30, 60, and 120 minutes after the AHREP. Blood samples were analyzed for changes in markers of stress, muscle damage, and immune system activation. Performance measures were taken before and after the AHREP to examine changes in peak force and power. To assess effects on recovery, subjects reported to the laboratory 24, 48, and 72 hours after the AHREP for additional blood draws and performance testing.
Ten men (mean ± SD; age = 23.6 ± 4.5 years; height = 176.3 ± 5.5 cm; weight = 86.0 ± 12.7 kg) and 10 women (mean ± SD; age = 22.2 ± 2.3 years; height = 160.4 ± 4.7 cm; weight = 62.0 ± 8.5 kg) completed the protocol. All subjects were resistance trained with a Smith machine back squat 1 repetition maximum (1RM) of at least 150% of their body weight and were cleared by a physician for any musculoskeletal or pathological conditions that could affect the results of the investigation. All subjects completed a written informed consent form before participation after having the benefits and risks of the investigation explained to them. The University of Connecticut Institutional Review Board for use of human subjects approved this investigation.
The nuBound (Nu Science Laboratories, Inc., Boston, MA, USA) supplement contains dietary nucleotides, which are extracted from yeast (Saccharomyces cerevisiae). During the supplement treatment cycle, subjects took 2 capsules of nuBound daily, 2 upon waking, and 2 after exercise. Each dose of 2 capsules (1,000 mg) contained 278 mg of dietary nucleotides, 375 mg amino acids (L-glutamine, L-methionine, and L-lysine), riboflavin (4.5 mg), folate (400 μg), biotin (188 mg), and pantothenic acid (12 μg). Other ingredients included fructooligosaccharides (chicory root), inositol, and sodium citrate.
During the placebo cycle, subjects followed an identical dosing schedule. The placebo capsules were the same size, shape, and color of the nucleotide supplement but contained lactose and magnesium stearate. During the first treatment cycle, subjects recorded their daily dietary intake on a diet log. The log was then used to help subjects replicate their diet during the second treatment cycle. Subjects also replicated their physical activity during each cycle.
Before beginning the supplement or placebo cycle subjects reported to the laboratory for familiarization with the study procedures. The proper technique for the warm-up procedures, the isometric squat, and countermovement jumps were demonstrated for the subjects, which they then practiced. Subsequently, a Smith machine back squat 1RM was determined. Briefly, subjects performed 8–10 repetitions at 50%, followed by 3–5 repetitions at 85% of their estimated 1RM. Two to 4 maximal attempts were then performed to determine the individual's 1RM. After 1RM testing, subjects performed a full AHREP as described below, starting with a load equal to 75% of the previously determined 1RM.
Acute Heavy Resistance Exercise Protocol Visit
On the day of the AHREP, subjects reported to the laboratory between 0530 and 0930 hours, after a 12-hour fast. Starting times for the AHREP were matched between cycles. Subject hydration was assessed by urine specific gravity (USG) using a handheld refractometer (Reichert, Lincolnshire, IL, USA) with a USG ≤ 1.025 considered adequate. An indwelling cannula was inserted into a superficial forearm vein and the PRE blood draw was taken 10 minutes later. Next, subjects completed a standardized warm-up procedure consisting of 5 minutes on a cycle ergometer and a series of dynamic stretches. After the warm-up, subjects completed the pre-AHREP performance tests followed by the AHREP, as detailed below. Immediately after the AHREP, the IP blood draw was obtained, and post-AHREP performance tests were conducted. Subjects remained in the laboratory for 2 hours to allow for blood collection at 15, 30, 60, and 120 minutes after AHREP.
At 24, 48, and 72 hours after the completion of the AHREP, subjects reported to the laboratory for recovery testing. Adequate hydration was confirmed, and subjects sat for 10 minutes before a single blood draw. Subjects then performed an isometric squat maximum voluntary contraction (MVC) and a set of three maximal effort countermovement jumps.
Acute Heavy Resistance Exercise Protocol
The AHREP consisted of 6 sets of 10 repetitions of back squats to parallel on a Smith machine, allowing only vertical translation of the bar. The starting weight was approximately 75% of the 1RM, as determined on the familiarization day. If a subject was unable to complete all 10 repetitions, the weight was reduced at the discretion of the testers, and the remaining repetitions were completed. The weight was reduced to allow for completion of the required repetitions, with the goal of achieving the highest possible load volume. After the first and second sets, 2 minutes of rest were given; 3 minutes were given after each of the remaining sets as pilot testing indicated the additional rest allowed subjects to complete the AHREP with a higher load.
Peak force was obtained during a maximal isometric squat, and peak power was obtained during a series of 3 maximal effort countermovement jumps. The maximal isometric squat was performed on a Smith machine, with force production measured and analyzed using a force plate (Fitness Technology 400 series performance force plate, Australia) and Ballistic Measurement System software (Software Version 2009.0.0). The height of the Smith machine bar was adjusted so that subjects were positioned with an approximate 135° knee joint angle. Subjects were instructed to push into the bar as if they were performing a squat, gradually increasing force until they reached maximal effort, holding the maximal effort, and then a slow reduction of force to resting levels (a trapezoidal force development curve). The total length of time for the isometric squat test was 10 seconds. Countermovement jumps were also performed on a force plate. Subjects kept their hands on their hips and performed 3 consecutive maximal-effort countermovement jumps.
On the day of the AHREP, blood was collected at PRE, IP, and 15, 30, 60, and 120 minutes after AHREP through an indwelling cannula kept patent with sterile saline. Before each blood draw, 3 ml of fluid was extracted and discarded. Single blood draws were taken at 24, 48, and 72 hours after AHREP. Whole blood was collected and placed in serum tubes or plasma tubes. The samples were then centrifuged, aliquoted, and stored at −80° C until subsequent analyses.
The creatine kinase-SL assay (SEKISUI, Charlottetown, Canada) was performed in duplicate using serum samples. A Thermo Scientific BioMate 3 Spectrophotometer (Pittsburgh, PA, USA) was used to read the assays at a wavelength of 340 nm. The coefficient of variation (CV) was 4.2%. Lactate was measured in EDTA-plasma samples using a liquid lactate reagent (Pointe Scientific, Canton, MI, USA) and assayed according to Gutmann et al. (15) and Noll et al. (27). Serum uric acid was measured using uric acid reagents purchased from Pointe Scientific (Canton, MI, USA) and performed according to the manufacturer's instructions. Serum cortisol was measured using an enzyme-linked immunosorbent assay (ELISA) (CALBiotech, Spring Valley, CA, USA), with a sensitivity of 11.1 nmol·L−1. Myeloperoxidase (MPO) was measured in EDTA-plasma samples using an ELISA (ALPCO, NH, USA) with a sensitivity of 1.08 ng·mL−1. Lactate, uric acid, cortisol, and MPO were analyzed in duplicate on a VersaMax tunable microplate reader (Molecular Devices, Sunnyvale, CA, USA) at the appropriate wavelength for the given assay. Intra- and inter-assay CVs for lactate, uric acid, cortisol and MPO were below 3.9%, 4.7%, 7.2%, and 6.3%, respectively.
Whole blood was analyzed for absolute neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts by Quest Diagnostics (Madison, NJ, USA) using an automated hematology analyzer.
Data are presented as mean ± SD. Normality and homogeneity of variance were confirmed for the selected dependent variables. Data were analyzed using 1 between (sex) by 2 within (treatment and time point) mixed methods analyses of variance. When significant differences were detected, Fisher's least significant difference post hoc analyses were performed to make pairwise comparisons. Statistical significance was set at p ≤ 0.05.
The primary finding of this investigation was that nucleotide supplementation significantly altered resistance exercise-induced changes in cortisol, MPO, creatine kinase (CK) concentrations, and isometric force production. As expected, the stress of the AHREP perturbed all variables measured. Sex differences were observed in terms of lactate, MPO, CK, isometric force, and countermovement jump power.
The AHREP induced significant acute increases in cortisol values in male and female placebo groups, which returned to baseline by 60 and 30 minutes after AHREP, respectively. In both sexes, nucleotide supplementation resulted in significantly lower cortisol values at IP and after 15 and 30 minutes when compared with the corresponding time points under placebo. No sex-specific differences were observed. Cortisol values are presented in Figure 1.
After the AHREP, MPO increases were observed in both sexes under both treatment conditions. Additionally, men demonstrated elevated MPO levels at 72 hours after AHREP under the placebo treatment only. Acutely, nucleotide supplementation resulted in significantly reduced MPO levels after the AHREP and during recovery time points in both men and women. Women demonstrated reduced MPO values at rest after nucleotide supplementation. The MPO values are presented in Figure 2.
Acute elevations in lactate were observed after the AHREP under both treatment conditions in both sexes. All groups demonstrated elevations at IP and after 15, 30, and 60 minutes, but returned to baseline values by 120 minutes. Nucleotide supplementation had no distinct effect on lactate values in men or women. When sexes were compared, women demonstrated significantly lower lactate values at IP in both treatments. Lactate values are presented in Figure 3.
During the recovery period, CK values were significantly elevated above baseline values in men in women, regardless of the treatment condition. The CK values 24 hours after AHREP were significantly lower in nucleotide-supplemented groups than in the placebo treatment groups. Sex differences in CK were observed at baseline and recovery in the nucleotide-supplemented groups, but only during recovery in the placebo groups. The CK values are presented in Figure 4.
Significant uric acid increases were observed in men under both treatments at 60 and 120 minutes; however, no significant changes were observed in women. When compared with men, women demonstrated significantly lower uric acid values at all time points. No significant effects for nucleotide supplementation were observed in either sex. Uric acid values are presented in Figure 5.
A similar post-AHREP increase in the neutrophil count was observed after both treatments in men and women. Lymphocytes also exhibited a significant time effect, in which all groups experienced elevated counts at IP, and a subsequent decrease below baseline within 30 minutes. Monocytes were elevated at IP regardless of treatment or sex. Men and women in the placebo treatment demonstrated a decrease in the monocytes below baseline values during the acute post-AHREP period, but this treatment effect was not statistically significant. Women exhibited significantly lower monocyte counts at IP in both treatments and at 48 hours post-AHREP in the placebo condition. The examined leukocyte populations did not seem to respond to nucleotide supplementation. Absolute leukocyte counts are shown in Tables 1 and 2.
The AHREP had a significant detrimental effect on isometric force generation in men and women under placebo treatment. Isometric force generation returned to baseline values in nucleotide-supplemented women within 24 hours of the AHREP; however, men and women under placebo treatment required 48 hours for full recovery. Men receiving the nucleotide supplementation demonstrated no impairment of isometric force immediately after the protocol or during the recovery days. Compared with men, women demonstrated lower isometric force values at all time points for both treatments. Isometric force values are presented in Figure 6.
Decrements in countermovement jump peak power were observed immediately after the AHREP in all subjects; however, values returned to baseline within 24 hours. No effects of nucleotide supplementation were observed. Similar to isometric force, women demonstrated lower peak power values at all times and treatments when compared with men. Countermovement jump peak power values are presented in Figure 7.
The primary finding of this investigation is that a dietary nucleotide supplement reduced markers of HPA and inflammatory activity, and these changes corresponded with reductions in tissue damage and the preservation of force production capabilities. Sex-specific differences in the response to nucleotide supplementation included the absence of improvement of physical recovery in women, who tended to demonstrate less inflammatory activity (lower MPO), and lower lactate, CK, and uric acid generally. These sexually dimorphic observations indicate that nucleotide supplementation produced modest positive effects primarily in men, who might have differed from women in terms of absolute exertion, normal or stress-related nucleotide metabolism, or immune/inflammatory responses to heavy resistance exercise.
Acute resistance exercise consistently results in transient increases in cortisol values (20,21,33). In accordance with previous findings, post-AHREP increases in cortisol were observed in both men and women with placebo. In contrast, no post-AHREP cortisol increases were observed in the male or female nucleotide groups. Nucleotide supplementation resulted in similar reductions in the cortisol response after aerobic exercise (24,25). Furthermore, nucleotide supplementation has been shown to attenuate the cortisol response to a variety of stressful stimuli in multiple species (31,34). The mechanism(s) through which nucleotides regulate the cortisol response is currently unknown; however, it may be due to the ability of adenosine and uracil to increase hepatic glucose output (14,16). During exercise, low blood glucose stimulates cortisol release to increase glycogenolysis. If nucleotide supplementation increased hepatic glucose output, blood glucose levels may have been better preserved during the AHREP, attenuating a key signal for cortisol secretion.
The oxidative enzyme, MPO, is released from activated neutrophils and serves as an indirect measure of neutrophil activation and innate immune activity. As in previous aerobic exercise investigations, MPO increased acutely after the AHREP (5–7,11). Although acute increases were observed in both placebo and nucleotide supplemented groups, MPO values were significantly lower after nucleotide supplementation. Additionally, decreased pre-AHREP MPO values were observed in women. Glucocorticoids, such as cortisol, have been suggested to stimulate neutrophil degranulation, increasing serum concentrations of oxidative enzymes including MPO (26). Therefore, the attenuated MPO increase observed with nucleotide supplementation might reflect reduced innate immune activation subsequent to a reduced cortisol response.
Although beyond the scope of this investigation, from a physiological viewpoint, we suggest that an attenuation of endotoxemia could explain the observed differences in the cortisol and MPO response. During strenuous exercise, slight ischemia in the gut can disrupt the mucosal barrier, resulting in increased permeability, and the resultant passage of pathogens, such as lipopolysaccharide, into the circulation (4,23). As a potent inflammatory agent, LPS can stimulate the activation of neutrophils, production of inflammatory cytokines, and an HPA response that would include increased glucocorticoid secretion (13,32,36). Animal models have demonstrated the ability of dietary nucleotide intake to promote recovery of damaged gut mucosa (28) and to improve mucosal barrier function (1,19). Therefore, nucleotide supplementation might have reduced or prevented the influx of inflammatory pathogens into circulation, thereby attenuating the cortisol and MPO response.
The AHREP induced significant muscle damage in all groups. Although elevated above baseline, CK values at were significantly lower in the nucleotide-supplemented groups at the 24-hour post-AHREP time point. Reduced CK values 24 hours but not 48 or 72 hours after AHREP suggests that the nucleotide supplement reduced exercise-induced muscle damage, rather than improving recovery. Generally, because neutrophil activation is associated with the production of reactive oxygen species (9,11), which promote secondary (inflammation-induced) muscle damage, reductions in MPO may partially explain reductions in muscle damage. Previously, McNaughton et al. (24) reported an absence of effect of nucleotide supplementation on postexercise CK values, although differences in the timing of assessment likely explain the conflicting results. In the current investigation, CK measurements were extended to 72 hours, whereas McNaughton et al. (24) measured CK only immediately after exercise.
In accordance with the attenuated cortisol response, neutrophil activation, and muscle damage, nucleotide supplementation attenuated performance decrements. After nucleotide supplementation, men produced greater isometric force immediately after the AHREP and 24 and 48 hours later. Women also demonstrated greater isometric force, but only 24 hours after exercise. As structural muscle damage can impair force generation (3,9), these observations are likely explained by reductions in muscle damage.
Regardless of sex or treatment, all leukocyte populations were affected by the AHREP. The lack of differences was surprising, given the differences in immune function, as well as previous investigations. Our observations support the findings of Ostojic et al. (29), who reported no difference in the total leukocyte counts as a result of nucleotide supplementation. Together, these findings suggest the effects of nucleotide supplementation on immune cell activity are independent of changes in absolute leukocyte counts. Alternatively, offsetting changes in immune cell phenotypes are possible, but would require additional immunological phenotyping beyond the scope of this study. However, this represents a promising area in immunology with important implications for our understanding of responses and adaptations to resistance exercise.
The results of this investigation generally support the previous studies in that nucleotide supplementation may attenuate the stress response, reduce muscle damage, and preserve force production capabilities after intense resistance exercise. These effects could improve recovery from strenuous exercise. Despite these promising findings, more research is needed to determine the mechanisms through which nucleotide supplementation exerts its effects.
Dietary nucleotide supplementation reduces the stress response to resistance exercise as evidenced by lower cortisol and MPO values. Furthermore, dietary nucleotide supplementation seems to reduce resistance exercise-induced muscle damage, resulting in a greater preservation of force production capability.
Note: Brian R. Kupchak is now with Consortium for Health and Military Performance (CHAMP), Center of Excellence, Uniformed Services University, Bethesda, MD 20877.
This study was supported in part by a grant from Nu Science Labs, Inc., Boston, MA, and the makers of the nuBound nucleotide supplement. We would like to thank all subjects who volunteered for this study and worked so hard to make it all work. We also thank our medical monitor Dr Jeffrey Anderson.
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