Included participants were in good health and between the ages of 21 and 45; all participants were recruited from the registrant pool of the Portland Half Marathon. Participants were excluded if they had taken MSM in the past month, warfarin, corticosteroids or statins in the past three months, used tobacco in the past six months, were pregnant or nursing, or if they were peri- or postmenopausal. In order to reduce variation in training regimens, competition in a race of greater than a 5 K within the month prior to the race was considered a basis for exclusion. Potential participants were excluded if they had ever participated in a full marathon, participated in a half-marathon during previous six months, or participated in more than three half-marathons during previous three years. Previous race history was used to exclude highly trained individuals and provide a study population of participants who could be considered “weekend warriors.”
Potential participants were screened for initial eligibility via a telephone call and then scheduled for a baseline visit. At the baseline visits, participants gave informed consent and then completed an eligibility and health questionnaire, a training log capturing training information for three prior days, and listed medication and supplement use. This study was approved by the National University of Natural Medicine Institutional Review Board.
This study was a randomized, double-blind, placebo-controlled trial evaluating the effects of MSM on oxidative stress, cell damage and pain caused by exhaustive exercise. Twenty-four participants were randomized to receive either MSM or placebo at a dose of 3 g/day for three weeks preceding the Portland Half-Marathon. Blood serum and pain-scale questionnaires were collected approximately one month prior to the race day (baseline), and again 15 min, 90 min, 1 day and 2 days post-race. Serum samples were analyzed for the primary outcome measure 8-hydroxy-2′-deoxyguanosine (8-OHdG), and malondialdehyde (MDA); both are markers of oxidative stress. Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH) were measured as markers of muscle damage. A 100 mm Visual Analog Scale (VAS) was used to measure muscle pain and joint pain at each time point.
Participants ingested either 3 g/day of MSM or 3 g/day of rice flour placebo. This study used OptiMSM® provided by Bergstrom Nutrition (Vancouver, WA) as the active supplement. Placebo capsules contained rice flour, a harmless and inert substance used in food products, and not known to affect RONS. Placebo capsules, also provided by Bergstrom Nutrition, were matched for size and color to MSM capsules.
All study participants were registered for, and completed the Portland Half-Marathon (13.1 miles) held on October 5th, 2014. The start and finish lines for the race were both in downtown Portland. Decisions on training regimens, diet, and competitive goals were left to individual participants. The study did not collect pre-race training or dietary information. Participants completed a 24 h recall form for alcohol consumption at each time point, but were only asked to abstain from alcohol between the 15 min and 90 min post-race visits.
Participants took MSM or placebo starting three weeks prior to the race and continued until two days afterward. Study participants were provided with printed supplementation instructions and a capsule intake log to help track and promote supplement adherence. Participants received a reminder phone call the day before beginning supplementation and mid-study phone calls to check for adherence and adverse reactions. All participants were asked to return the remaining capsules and intake log at the final visit, as well as to complete an exit questionnaire to track any changes in participant weight or medication and supplement use.
Participants provided blood samples and completed a pain questionnaire at each study visit. Baseline visits (T0) occurred approximately one month prior to race day, and were conducted at Helfgott Research Institute (HRI) at NUNM. The 15 min (T1) and 90 min (T2) post-race visits occurred near the finish line of the Portland Half-Marathon in fully equipped study tents. The 1 day (T3) and 2 day (T4) post-race visits occurred at HRI at various times between 8:00 am and 7:00 pm. All blood draws were performed by a licensed phlebotomist, physician, or registered nurse. Blood collection at each visit consisted of a venous blood draw of approximately 8 to 11 mL into two separate serum separator tubes (SST): a 3.5 mL yellow top BD Vacutainer®, and an 8.5 mL “tiger top” BD Vacutainer®. Tubes were labeled with stickers containing the participant ID number and the visit number.
Blood samples were allowed to clot for 30 min at room temperature and then centrifuged at 2500 rpm for 30 min. Serum from the 8.5 mL tubes was transferred to 1.5 mL microcentrifuge tubes in 500 μL aliquots and stored at −80 °C in the HRI Laboratory for analysis of 8OHdG and MDA. The 3.5 mL tubes were stored at 4 °C overnight and transported the following day to the NUNM Clinic Laboratory where they were submitted to MESA Labs (Portland, OR) for analysis of Creatine Kinase and Lactate Dehydrogenase.
Serum 8-OHdG levels, the primary outcome measure, were measured through competitive ELISA kits purchased through Cayman Chemical (DNA/RNA Oxidative Damage EIA Kit: Item Number 589320). Assays were performed in the Helfgott Research Center Laboratory using serum aliquots from 8.5 mL SST stored at −80 °C. ELISAs were performed according to manufacturer’s instructions and absorbance was measured at 450 nm. Serum MDA levels were measured through a colorimetric assay purchased through Cayman Chemical (TBARS Assay Kit: Item Number 10009055). Assays were performed in the Helfgott Research Center Laboratory using serum aliquots from the 8.5 mL SST stored at −80 °C. Assays were conducted according to manufacturer’s instructions and absorbance was measured at 530 nm. Serum CK and LDH levels were measured by spectrophotometry at MESA Labs using serum from the 3.5 mL SST.
A 100 mm Visual Analog Scale was used to quantify perceived joint pain and perceived muscle pain by measuring, in millimeters, the distance between the left end of the scale (no pain) to the participant’s mark. Lowest possible score was 0, the highest was 100. For measurements of pain it is important to determine a clinically significant difference, which is the difference or change in scores for which a patient or participant is able to distinguish between pain levels. Clinical significance for the 100 mm VAS pain scale has been determined to be a difference of 10 mm or more (>Δ10mm) [25, 26].
SPSS statistical software was used for all statistical analyses . Initial analysis included independent t-tests for between group differences in BMI, age, gender, and race time, to check for possible differences between groups. Repeated ANOVAs were used to assess time and time*group effect. Data was assessed for normal distribution and corrections were made when appropriate. Correlations were analyzed for “Hours from Race Finish” and 1-Day and 2-Day outcomes. Multivariate ANOVAs were used to assess differences between groups at each time point. We tested unadjusted differences in means, group effects adjusted for baseline levels, and effects adjusted for baseline, BMI, Age, and Race Time, in a three-tiered analysis. Summary estimates are reported as mean ± standard deviation (SD) for timepoint analysis, and for between-group analysis, means are reported with 95% confidence intervals (CI). Comparison data for both timepoint and group analysis are reported with 95% confidence intervals.
Power calculations were completed for 8-OHdG as the primary outcome measure. Although little prior research had been conducted on serum 8-OHdG measured through ELISA, the number of necessary participants for each group was calculated at 16 (32 total) for the study to be powered at 0.80 with α = 0.05 [28, 29]. To account for a 25% dropout rate, the study set a recruitment goal of 44 participants. Data are presented as mean (SD) or mean change (95% CI) when appropriate.