Abstract
Creatine (CR) is an ergogenic agent used to enhance anaerobic exercise performance by increasing phosphocreatine stores and free CR to increase adenosine triphosphate (ATP) turnover in the muscles, therefore increasing muscle mass and reducing muscle recovery time following a strenuous workout. CR hydrolyzes into creatinine (CRN) rapidly. Previous work from the laboratory has shown in vitro that CRN has some immunomodulatory properties. To further delineate the immunomodulatory properties of CRN, we used real time-polymerase chain reaction and immunohistochemical staining to determine the mRNA and protein expression of tumor necrosis factor-α (TNF-α), a potent pro-inflammatory mediator, following exposure to CRN in mouse and human macrophages and human T cells. These studies demonstrated a significant reduction in TNF-α mRNA and protein in CRN-treated cells compared to control-treated cells. The majority of TNF-α is generated by the nucelar factor-kappa B pathway (NF-κB), the translocation of the p65 subunit of NF-κB was reduced in CRN-treated cells. We extended these studies in vivo using human subjects. A double-blind study was performed using CR monohydrate and placebo. Subjects consumed the supplement in a manner consistent with standard loading (4 x 5 g for 5 d) and maintenance (5 g for 28 d) regimens. Blood was collected at the end of the loading and maintenance periods as well as prior to supplementation. Analysis of TNF-α mRNA levels revealed that subjects supplementing with CR had reduced levels of TNF-α mRNA in their white blood cells compared to placebo supplemented subjects after the loading phase. Given the results of the in vitro study and the fast hydrolysis of CR to CRN, we postulate that the observed results in vivo are attributed to the increased CRN levels in the CR-supplemented subjects. Finally, the antioxidant properties of CRN were assessed in mouse macrophages against three oxidative agents – hydrogen peroxide, tert-butyl hydroperoxide (tert-BuOOH), and 3-morpholinosydnonimine chloride (SIN-1). CRN was observed to provide cellular protection against cell death mediated by hydrogen peroxide and SIN-1, but not tert-BuOOH. Together, these data suggest that both CR and CRN act as immune modulators by inhibiting pro-inflammatory cytokine TNF-α, and it has been hypothesized that CR supplementation may decrease the ability of the host to respond rapidly to pathogens due to increased CRN levels. Furthermore, the data suggest that many of the properties attributed to CR may actually be due the effects of CRN. The anti-inflammatory and antioxidant qualities of CRN provide novel potential therapeutic roles for CRN which is largely thought to be a waste product.