Vitamin D2 Supplementation Amplifies Eccentric Exercise-Induced Muscle Damage in NASCAR Pit Crew Athletes

This study determined if 6-weeks vitamin D2 supplementation (vitD2, 3800 IU/day) had an influence on muscle function, eccentric exercise-induced muscle damage (EIMD), and delayed onset of muscle soreness (DOMS) in National Association for Stock Car Auto Racing (NASCAR) NASCAR pit crew athletes. Subjects were randomized to vitD2 (n = 13) and placebo (n = 15), and ingested supplements (double-blind) for six weeks. Blood samples were collected and muscle function tests conducted pre- and post-study (leg-back and hand grip dynamometer strength tests, body weight bench press to exhaustion, vertical jump, 30-s Wingate test). Post-study, subjects engaged in 90 min eccentric-based exercise, with blood samples and DOMS ratings obtained immediately after and 1- and 2-days post-exercise. Six weeks vitD2 increased serum 25(OH)D2 456% and decreased 25(OH)D3 21% versus placebo (p < 0.001, p = 0.036, respectively), with no influence on muscle function test scores. The post-study eccentric exercise bout induced EIMD and DOMS, with higher muscle damage biomarkers measured in vitD2 compared to placebo (myoglobin 252%, 122% increase, respectively, p = 0.001; creatine phosphokinase 24 h post-exercise, 169%, 32%, p < 0.001), with no differences for DOMS. In summary, 6-weeks vitD2 (3800 IU/day) significantly increased 25(OH)D2 and decreased 25(OH)D3, had no effect on muscle function tests, and amplified muscle damage markers in NASCAR pit crew athletes following eccentric exercise.


Introduction
Vitamin D deficiency is defined as a serum 25-hydroxyvitamin D (25(OH)D) concentration of 20 ng/mL or less, with vitamin D insufficiency established as 21-29 ng/mL [1]. Recent evidence suggests that optimal vitamin D status, defined by estimated maximum parathyroid (PTH) suppression, occurs at 25(OH)D levels of 40 ng/mL and higher [2,3]. Estimates from the National Health and Nutrition Examination Survey (NHANES) are that three in four individuals in the U.S. population have 25(OH)D levels less than 30 ng/mL [3].
A high proportion of athletes are also vitamin D insufficient, with prevalence rates varying according to sun exposure, time of the year, and residential latitude [4][5][6][7][8]. Early 20th century studies suggested that ultraviolet (UV) irradiation improved physical performance, and that physical training responses peaked in late summer [4]. More recent studies report that vitamin D receptors (VDR) are present in skeletal muscle, and that vitamin D treatment of deficient individuals improves muscular strength and Type II muscle fiber size [2,[9][10][11]. Epidemiologic studies of elderly individuals support direct associations between 25(OH)D levels and physical performance, with some support in randomized clinical trials, especially among vitamin D deficient adults [12][13][14][15][16]. A few epidemiologic studies support relationships between 25(OH)D and performance across all ages in adults [17][18][19].
Limited evidence suggests that treatment of vitamin D insufficient athletes may improve performance [20,21]. In the UK, 5000 IU/day vitamin D 3 supplementation for eight weeks improved 10 m sprint times and vertical jump performance in athletes who started the study with a mean serum 25(OH)D level of 12 ng/mL [5]. Maintaining adequate vitamin D status may also reduce inflammation and aid in recovery from injury or intensive workouts, but data in humans are inconsistent [4,17]. One study showed that vitamin D 3 -treated rats experienced attenuation in plasma creatine kinase (CK) and inflammation biomarkers following high-intensity exercise, with an increase in muscle VDR protein expression [22]. No previous human study has been published regarding the effect of vitamin D supplementation in countering eccentric exercise-induced muscle damage (EIMD) and delayed onset of muscle soreness (DOMS). We hypothesized that 6-weeks supplementation with vitamin D (3800 IU/day) using vitamin D 2 Portobello mushroom powder would improve muscle function and strength, and attenuate EIMD and DOMS in NASCAR pit crew athletes during their off-season in December and January.

Subjects
NASCAR pit crew athletes (n = 30) from Hendrick Motorsports (Concord, North Carolina, NC, USA) were recruited and invited to join the study if they agreed to avoid: (1) food and supplement sources (during the 6-week supplementation period) that were high in vitamin D (specifically canned fish, cod liver oil, salmon, and supplements with high-dose vitamin D); (2) large dose vitamin/mineral supplements (above 100% recommended dietary allowances); (3) anti-inflammatory medications; (4) tanning beds and prolonged sun exposure. The Appalachian State University institutional review board approved all experimental procedures.

Research Design
Pit crew members provided blood samples in mid-October (fall baseline for serum vitamin D status) and then again during baseline testing (first week of December). Baseline testing consisted of the leg-back dynamometer strength test, hand-grip dynamometer strength test, body weight bench press to exhaustion, vertical jump, and 30-s Wingate anaerobic power cycling test. Height, weight, and percent body fat (three skinfolds) were also obtained.
1 Leg-back dynamometer strength test: With arms straight and knees slightly bent, subjects grasped a bar that was attached to a platform via a chain and dynamometer (Lafayette Instruments, Lafayette, IN, USA), and then lifted up with maximal effort for several seconds. The test was repeated three times, with the highest score recorded; 2 Hand-grip dynamometer strength test: The hand-grip dynamometer (Lafayette Instruments, Lafayette, IN, USA) was adjusted to hand size (with the middle of the fingers on the handle). The subject assumed a slightly bent forward position with the right hand hanging down and forward, and then gripped maximally for 2-3 s. The best of three trials was recorded; 3 Body weight bench press to exhaustion: Subjects bench pressed a weighted bar equal to body weight as many times as possible (to a metronome set at 60 beats/min or 30 lifts/min) until fatigue. The bar touched a small foam block on the chest lightly in the down position, and lifted upwards until the arms were straight in the up position; 4 Vertical jump: Subjects first stood erect with the feet flat on the floor and reached as high as possible with both arms and hands (standing reach height). Subjects then squatted down and jumped as high as possible with one arm and hand, and tapped the measuring device (jump height) (Vertec vertical jump apparatus, Questtek Corp, Northridge, CA, USA). This was repeated three times, with the best score recorded as the difference between the jump and standing reach heights; 5 Wingate anaerobic power cycling test: The Lode cycle ergometer (Lode B.V., Groningen, The Netherlands) was adjusted to the body mass of the subject (7 W per kilogram), and then subjects cycled at maximal speed for 30 s. The peak and total wattage power output was recorded and adjusted to body mass.
Subjects were randomized to vitamin D or placebo groups, and ingested the supplement for six weeks. Following supplementation, blood samples were collected and subjects repeated the muscle function tests. Subjects then engaged in 90 min of eccentric-based exercise. Blood samples were obtained immediately following exercise, and then 24-h-and 48-h-post-exercise. The blood samples were analyzed for serum vitamin D and muscle damage biomarkers. DOMS was measured using a Likert-scale questionnaire [23] pre-and post-supplementation, and immediately post-and 24-h and 48-h after the 90 min of eccentric exercise bout.

Eccentric Exercise
The 90 min eccentric exercise bout consisted of 17 different exercises:

Supplement
Fresh Portobello mushrooms (Agaricus bisporus) were air dried at 71-76 °C for 48-72 h in a convection dryer [24]. Once dried, the mushrooms pieces were milled to approximately 35 mesh, or 500 µm. The powder was then treated on a vibrating conveyor with pulsed light (UVB) from a Xenon broad spectrum lamp (100-800 nm) operating at 3 pulses per second for a total of thirty 2 ms pulses which converted ergosterol to ergocalciferol (vitamin D 2 ). Subjects were given Portobello mushroom powder with or without vitamin D 2 mixed in soymilk powder (non-vitamin D fortified) in six plastic containers (one for each week of the study). Subjects ingested one level teaspoon of the product each day (with or without 3800 IU vitamin D 2 ) during breakfast.

Mushroom Vitamin D 2 Supplement Analysis
The mushroom vitamin D 2 analysis has been described fully [24,25]. Briefly, mushroom powder samples underwent 3 h saponification at room temperature. The final extract was dried and reconstituted in absolute ethanol for LC-MS/MS analysis. Vitamin D 2 was quantitatively determined through comparison to internal standard responses. An Accela HPLC system coupled with a PDA and a LTQ Velos tandem mass spectrometer system (Thermo Scientific) was used for liquid chromatographic separation and quantitation of vitamin D 2 in samples.

Analytical Measures
Blood samples were drawn from the antecubital vein by standard venipuncture by a trained phlebotomist. All samples were drawn into vacuum blood collection tubes without additives, allowed to coagulate for 20 min at room temperature, and centrifuged. Serum myoglobin was measured with the Elecsys Myoglobin electrochemiluminescence immunoassay kit (Roche Diagnostics, Indianapolis, IN, USA) using the Modular Analytics E170 (Roche Diagnostics, Indianapolis, IN, USA). The sensitivity of the myoglobin assay is 1 ng/mL and the coefficient of inter-assay variation is 3.1%. Serum samples were individually assessed for lactate dehydrogenase (LDH) and creatine phosphokinase (CK) with reagent specific enzymatic assays using the SYNCHRON LX ® System (Beckman Coulter, Brea, CA, USA). The sensitivity of the LDH and CK assays was 5 IU/L and the coefficient of inter-assay variation was 5.3% and 4.5%, respectively.
Analysis of serum 25-hydroxyvitamin D 2 and D 3 was measured by HPLC-MS/MS, as previously described [24]. Serum samples as well as calibration standards, water blanks, serum blanks and QCs were prepared as previously described [26], and analyzed on the same LC-MS system described above.

Statistics
Data are expressed as mean ± SE. Subject characteristics were compared between groups using independent t-tests. Muscle function, serum vitamin D, DOMS, and muscle damage data were analyzed using 2 (group) × 2 to 5 (time) repeated-measures ANOVAs. When interaction effects were significant (p ≤ 0.05), changes between time points within groups were compared across time points using independent t-tests.

Results
Subject characteristics for the placebo (n = 15) and vitamin D (n = 13) groups are compared in Table 1, with no differences noted for age, height, body mass, and body composition. Total serum 25(OH)D was 43.7 ± 2.7 and 39.6 ± 1.6 ng/mL in the placebo and vitamin D groups, respectively, during October, 40.7 ± 2.1 and 36.6 ± 1.7 ng/mL in December (pre-supplementation), and 38.6 ± 1.8 and 37.4 ± 1.9 ng/mL in January (post-supplementation) (time effect p = 0.001, interaction effect p = 0.238). Supplementation with mushroom vitamin D 2 powder for 6 weeks caused no significant change in 25(OH)D (p = 0.127), a significant increase in serum 25(OH)D 2 (8.14 ± 1.96 ng/mL, p < 0.001) and a significant decrease in serum 25(OH)D 3 (−7.48 ± 2.28 ng/mL, p = 0.036) compared to placebo (0.076 ± 1.19 ng/mL and −2.11 ± 1.09 ng/mL, respectively) ( Figure 1A,B). Serum 25(OH)D 3 was highest in October and then decreased to levels measured in December and January in the placebo group (within group contrasts, p < 0.01). Pre-to-post-study measurements for muscle function, including leg-back and hand grip dynamometer tests, the body mass bench press test, vertical jump, and the 30 s Wingate test) did not differ between groups (all interaction effects, p > 0.05) ( Table 2). The post-study eccentric training bout caused significant increases in serum myoglobin (Figure 2), LDH (Figure 3), CK (Figure 4), and DOMS ( Figure 5) (all time effects, p < 0.001). Significantly higher post-exercise serum levels for myoglobin and CK were measured in the vitamin D 2 compared to placebo group (both interaction effects, p < 0.001), with a trend for higher serum LDH levels (interaction effect, p = 0.065). The pattern of change in DOMS did not differ between groups (interaction effect, p = 0.490). For the whole group, change in serum 25(OH)D 2 correlated significantly with change in post-exercise serum myoglobin (r = 0.57, p = 0.001).

Discussion
Contrary to our hypothesis, high-dose vitamin D 2 supplementation in NASCAR pit-crew athletes during a 6-week period in December and January amplified EIMD and had no effect on muscle function. Vitamin D 2 supplementation increased serum 25(OH)D 2 ~8 ng/mL but decreased serum 25(OH)D 3 ~7.5 ng/mL, with no significant change in total 25(OH)D.
These results differ from those of Choi et al. [22] who showed that large-dose vitamin D 3 supplementation in rats (i.p. 1000 IU/kg body weight) countered muscle damage and inflammation induced by high-intensity exercise. Vitamin D 3 -treated rats had highly increased protein expression of VDR in muscles, lower post-exercise levels of plasma CK and LDH, and reduced phosphorylation of AMPK and gene expression for IL-6 and TNF-α compared to controls. A major difference in the current study was the use of mushroom vitamin D 2 powder. Aside from oily fish, few foods contain natural vitamin D, and thus most treatments utilize vitamin D supplements containing ergocalciferol (vitamin D 2 ) and cholecalciferol (vitamin D 3 ). Vitamin D 2 is the artificial form of vitamin D derived from irradiation of the plant sterol, ergosterol, and is often used in food fortification, dietary supplements, and pharmaceutical preparations. Mushrooms are abundant in ergosterol, which can be converted into vitamin D 2 by ultraviolet (UV) illumination [27]. After ingestion, vitamin D 2 undergoes a series of activation steps to give 1α,25-(OH) 2 D 2 , which is believed to be equipotent to 1α,25-(OH) 2 D 3 (calcitrol) in the prevention and cure of rickets and other vitamin D actions in the body through utilization of the same VDR-mediated regulation of gene expression. Thus vitamins D 3 and D 2 have been used interchangeably in supplements, but recent evidence suggests that vitamin D 2 should not be regarded as equivalent to vitamin D 3 [10,28,29].
In agreement with other studies, supplementation with vitamin D 2 increased serum 25(OH)D 2 but decreased serum 25(OH)D 3 [30][31][32]. Little information is available on potential functional consequences of this metabolic response, but findings from the current study showing that muscle damage was heightened after eccentric exercise in the vitamin D 2 supplemented NASCAR pit crew athletes indicate further research on functional outcomes is needed. High serum 25(OH)D 2 is not a normal occurrence in humans except after the use of vitamin D 2 supplements. Entry of vitamin D 2 into the total body pool of vitamin D dilutes the relative amount of vitamin D 3 , resulting in a gradual replacement within the total pool of 25(OH)D and 1α,25-(OH) 2 D. Early evidence suggested that vitamin D-related cytochrome P450 enzymes including CYP2R1 and CYP27B1 (vitamin D activation) and CYP24A1 (inactivation) could not discriminate between vitamins D 2 and D 3 . More recent evidence indicates that the vitamin D-dependent intestinal form of the drug-metabolizing cytochrome P450 enzyme, CYP3A4 (vitamin D inactivation), may discriminate against vitamin D 2 [10]. CYP3A4 breaks down 1α,25-(OH) 2 D 2 at a significantly faster rate than 1α,25-(OH) 2 D 3 , suggesting that this nonspecific cytochrome P450 enzyme might limit vitamin D 2 action in target cells where it is expressed [10]. Thus, one explanation for the discrimination against vitamin D 2 could be the selective catabolism of vitamin D 2 by nonspecific cytochrome P450 enzymes in the liver and intestine. Despite a 3800 IU daily dose of vitamin D 2 , the athletes in the current study experienced a relatively low serum elevation in 25(OH)D 2 (~8 ng/mL). How this may influence levels of muscle damage after eccentric exercise has yet to be determined. This is the first report in the literature that athletes experienced more EIMD when supplemented with high doses of vitamin D 2 (3800 IU/day) for six weeks. Whether or not the post-exercise elevations in CK and myoglobin were due to the combined physiological influence of elevated 25(OH)D 2 and decreased 25(OH)D 3 remains to be determined. The NASCAR pit crew athletes were not vitamin D deficient, with only one in each group below a serum 25(OH)D level of 30 ng/mL, the minimal threshold deemed necessary to overcome vitamin D deficiency. Thus further research is warranted to confirm whether or not vitamin D 2 supplementation amplifies EIMD in athletes who are vitamin D deficient, especially when compared with vitamin D 3 supplementation. Animal studies indicate that vitamin D 3 supplementation promotes muscle regeneration and accelerated recovery of skeletal muscle strength after crush injury, with augmented cell proliferation and inhibition of apoptosis [33]. Thus vitamin D 3 but perhaps not vitamin D 2 supplementation in vitamin D deficient athletes, especially during periods of training with limited sun exposure, has the potential to improve recovery from intense exercise with an eccentric component.
In this study, muscle function test scores did not differ between the vitamin D 2 and placebo groups after 6-weeks supplementation of pit crew athletes during their off season (December and January). A limitation of this study, given the heterogeneity of the athletes tested, was that group sample sizes were too low to detect significant differences unless large improvements in muscle function test scores were achieved. Data are limited, but other studies show varying performance responses to vitamin D 3 supplementation, with results perhaps dependent on the degree of vitamin D deficiency in the athletic subjects [20,21]. In one study, Close et al. [34] showed no performance effect of 20,000 (n = 10) or 40,000 (n = 10) IU/week vitamin D 3 versus placebo (n = 10) over a 12-week period in 30 club-level athletes, 57% of whom were vitamin D deficient. In another study by this research group, 8-weeks vitamin D 3 supplementation (5000 IU/day) in vitamin D deficient athletes improved sprint and vertical jump performance compared to placebo, but subject number in each group was low (n = 5) [5]. Wyon et al. [21] showed that vitamin D 3 supplementation (2000 IU/day) by vitamin D insufficient/deficient classical ballet dancers during the winter months improved isometric strength and vertical jump scores relative to controls. However, this study was non-randomized, and did not use placebo control methods.

Conclusions
In summary, the novel and unexpected finding of this randomized, double-blinded, placebo controlled study of 28 NASCAR pit crew athletes was that 6-weeks supplementation with vitamin D 2 increased serum 25(OH)D 2 , decreased serum 25(OH)D 3 , and amplified EIMD. Vitamin D 2 supplemented athletes experienced no change in muscle function test scores compared to the placebo control group. If these results are confirmed by others, underlying mechanisms explaining the negative effects of vitamin D 2 supplementation on EIMD need to be explored, with a focus on VDR and cytochrome P450 enzyme interactions.