Eight weeks of resistance training in conjunction with glutathione and L-Citrulline supplementation increases lean mass and has no adverse effects on blood clinical safety markers in resistance-trained males

This study sought to determine the effects of an 8-week RT program in conjunction with daily, orally-delivered GSH + CIT, CIT-malate, or placebo supplementation on body composition, the association between lean mass and muscle strength, and whole blood and serum clinical chemistry markers in resistance-trained men. We found that none of the three supplement interventions had any significant effect on fat mass, total body water, and blood clinical chemistry variables. However, we did observe GSH + CIT to undergo significant increases in lean mass compared to placebo after 4 weeks. Additionally, a significant correlation between lean mass and muscle strength was observed in GSH + CIT, but not PLC and CIT-malate.

We did observe GSH + CIT to undergo significant increases in lean mass compared to placebo after 4 weeks. Additionally, a significant correlation between lean mass and muscle strength was observed in GSH + CIT, but not PLC and CIT-malate.

Based on the results of previous studies showing increases in muscle performance [911, 17] in response to a single 8-g dose of L-citrulline malate, it can be assumed that longer durations of L-citrulline supplementation might bestow ergogenic effects. Case in point, 7 days of L-citrulline supplementation at a daily dose of 6 g significantly increase plasma citrulline, arginine, and nitrite levels, VO2 kinetics, in response to moderate intensity (70% VO2 peak) exercise performance [18]. Another study showed that 7 days of L-citrulline supplementation at a daily dose of 2.4 g significantly improved cycling time trial performance [14]. However, while 16 days of L-citrulline supplementation at a daily dose of 3.4 g significantly increased plasma citrulline, arginine, and nitrite levels, and increased muscle oxygenation during moderate-intensity (70% VO2 peak) exercise, there were no superior effects on time-to-exhaustion during high-intensity (90% VO2 peak) exercise. Although, the lack of impact on exercise performance in this study may have been due to the lower dose of L-citrulline compared to other studies [15].

In response to 8 weeks of L-citrulline supplementation and RT for body composition, our present results indicate that neither GSH + CIT or CIT-malate had any preferential and significant effect on total body mass, fat mass, and total body water; any changes that occurred were most likely due to the RT program. However, the data suggest that GSH + CIT increased lean mass over placebo after 4 weeks, and that a similar increasing tendency compared to PLC existed after 8 weeks. As indicated in Fig. 1, for GSH + CIT it should be noted that we observed a modest decrease in lean mass at 8 weeks compared to 4 weeks. Additionally, Fig. 3 shows some participants undergoing decreases in lean mass in all three groups during the study. Unfortunately, we are not able to provide a specific explanation for this response. However, it is possible this may have occurred since this group was eating less at week 8 than at baseline regarding kcal/kg and protein/kg. In lieu of within-participant differences that inherently exist in all studies with humans, this result may have been due to issues with effort exerted during the resistance training sessions, perhaps related to over-reaching and fatigue, differences in resistance training volume, supplementation compliance, and differences in dietary intake. However, our results show no significant differences between groups for any of these variables.

Excluding the inherent limitations that are known to exist with dietary self-reports, we expressed caloric intake relative to body mass, and based on the fact that there were no significant changes over the course of the 8-week study for the dietary variables, and that GSH + CIT did not consume more total calories or protein than the other two groups, dietary intake can likely be ruled out as a possible confounding variable for the increased lean mass. However, following baseline we only assessed dietary intake for 4 consecutive days prior to each of the two testing sessions at weeks 4 and 8 (8 out of 56 days) which constitutes a small portion of the dietary intake, approximately 86%, not accounted for during the study. Therefore, the role that dietary impact may have played on lean mass should be interpreted with caution.

We attempted to equate training volume and expressed it relative to body mass so that we could better determine any preferential effects provided by the supplements relative to the association between lean mass and muscle strength and showed there to be no differences between groups for RT volume. Interestingly, we did show that a significant relationship existed between lean mass and strength at week 4 and 8 for the bench press exercise. Regarding lean mass and leg press strength, however, there was a significant relationship observed only at week 4. More specifically, we observed that, only for GSH + CIT, the increase in muscle strength was significantly correlated to the increase in lean mass for this group.

Based on previous studies [8, 19, 20] it is conceivable that the increases in lean mass we observed for GSH + CIT in the present study could have occurred due to increases in muscle protein synthesis, and this could be linked to NO-induced increases in cGMP [19]. Even though we have yet to generate any specific data to support this statement, with our previous study [7] we did show that in response to a single bout of resistance exercise, the plasma NO metabolites, nitrate and nitrite, and cGMP were elevated 30 min following exercise when taking GSH + CIT. Regarding a possible sustained release of NO due to GSH + CIT, there are possible physiological benefits for having high NO levels at 30 min post-exercise relative to its impact on muscle protein metabolism and possible muscle performance in response to RT. For instance, it has been shown that NOS activity is necessary for calcium-induced activation of the Akt pathway (involved in translation initiation and subsequent muscle protein synthesis). Nitric oxide appears to influence Akt signaling though a cGMP/PI3K-dependent pathway [8], which is the primary pathway for up-regulating translation initiation and MPS. Similarly, NO seems to influence skeletal muscle function through effects on excitation-contraction coupling, myofibrillar function, perfusion, and metabolism. Another study showed that by using an agent to inhibit phosphodiesterase-5, that the augmentation of NO-cGMP signaling increased protein synthesis and reduced fatigue in human skeletal muscle [20]. In our previous study [7], GSH + CIT showed an improvement in cGMP activity suggesting that if this outcome was prevalent in the present study it could likely play a role in MPS and muscle performance when combined with longer-term RT. This suggests that a resistance exercise-related mechanism of inducing plasma NO, perhaps due to increased shear stress that triggered an up-regulation in NO-cGMP signaling along with a slow, sustained release of NO from GSH + CIT, may be a conceivable candidate for this response.

Considering the longer-term L-citrulline supplementation employed, we observed none of the whole blood and serum clinical chemistry markers to be negatively impacted by any of the three supplements. By the variables assessed, these results indicate that the oral ingestion of these supplements for a period of 8 weeks appears to be safe. In addition, none of the participants reported any adverse events associated with ingestion of the supplements.

Considering the typical sample size of 8–12 participants in each group for most studies with a similar experimental design, in the present study our sample size of 25 in each group can be considered a strength, rather than a limitation. Considering this, however, our study does possess several possible obvious limitations. The first limitation is a major issue and involves only using four-day dietary recalls for determining nutritional intakes prior to each of the three testing sessions, as it is possible that the information provided from the dietary intakes were not reflective of the nutritional intakes over the course of the study since we only assessed 8 out of the 56 days. Secondly, supplement compliance is a potential limitation. Even though participants returned the empty containers and self- reported their compliance to be 100%, it is possible that the information provided to study personnel was not accurate and reflective of the actual supplement compliance. Despite our confidence in the reliability and validity of our data, in lieu of these limitations, the results presented herein should be interpreted with some caution.