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Abstract

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.

1. Introduction

Limited evidence suggests that treatment of vitamin D insufficient athletes may improve performance [,]. In the UK, 5000 IU/day vitamin D3 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 []. Maintaining adequate vitamin D status may also reduce inflammation and aid in recovery from injury or intensive workouts, but data in humans are inconsistent [,]. One study showed that vitamin D3-treated rats experienced attenuation in plasma creatine kinase (CK) and inflammation biomarkers following high-intensity exercise, with an increase in muscle VDR protein expression []. 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 D2 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.

2.4. Supplement

Fresh Portobello mushrooms (Agaricus bisporus) were air dried at 71–76 °C for 48–72 h in a convection dryer []. 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 D2). Subjects were given Portobello mushroom powder with or without vitamin D2 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 D2) during breakfast.

3. Results

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 D2 powder for 6 weeks caused no significant change in 25(OH)D (p = 0.127), a significant increase in serum 25(OH)D2 (8.14 ± 1.96 ng/mL, p < 0.001) and a significant decrease in serum 25(OH)D3 (−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) (A,B). Serum 25(OH)D3 was highest in October and then decreased to levels measured in December and January in the placebo group (within group contrasts, p < 0.01).

3. Results

The post-study eccentric training bout caused significant increases in serum myoglobin (), LDH (), CK (), and DOMS () (all time effects, p < 0.001). Significantly higher post-exercise serum levels for myoglobin and CK were measured in the vitamin D2 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)D2 correlated significantly with change in post-exercise serum myoglobin (r = 0.57, p = 0.001).

4. Discussion

Contrary to our hypothesis, high-dose vitamin D2 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 D2 supplementation increased serum 25(OH)D2 ~8 ng/mL but decreased serum 25(OH)D3 ~7.5 ng/mL, with no significant change in total 25(OH)D.

Abstract

Vitamin D2 deficiency is one of the most common micronutrient insufficiencies among children. Few foods, mainly those derived from animal sources, naturally contain this vitamin. The basidiomycete mushroom Pleurotus ostreatus could be used as an innovative and sustainable ingredient for food fortification with vitamin D2. This study was aimed at exploring children’s acceptance of a whole-cereal-based product (breadsticks) combined with increasing concentrations of P. ostreatus powder rich in vitamin D2. The food neophobia trait (fear of trying unfamiliar and new food) on sample acceptability was also investigated. One hundred and three children (47 girls and 56 boys, aged 9–11 years) were recruited, and breadstick-liking was studied in relation to gender and neophobic traits. Results showed that the samples enriched in vitamin D2 were well accepted by children even if liking decreased with increasing concentration of mushroom powder. Generally, neophilic subjects gave higher liking scores compared with the neophobic ones, especially for the modified samples. New, well-accepted fortified products could be developed using an adequate concentration of mushroom powder to deal with the increasing vitamin D2 deficiency among children.

2.1. Participants

One hundred and three children (47 girls and 56 boys, aged 9–11 years) were recruited in the Milan area (Italy) via primary schools. Parents received full information about the research study and provided written informed consent for their children’s participation. Children without a signed informed consent were not involved in the study. Children who suffered from food allergies or did not like the tested samples, which could negatively affect hedonic responses, performed alternative activities so that they did not feel excluded from the test activities. The study was performed in adherence with the principles established by the Declaration of Helsinki. The protocol was approved by the Ethics Committee of the University of Milan (protocol n°19/18).

2.2. Stimuli

A commercial strain of P. ostreatus deposited at the mycotheca of the Società Agricola IoBoscoVivo (Vergiate, Varese, Italy) was used. This strain was cultivated on wood of Populus tremula in a greenhouse at room temperature. After one year of incubation, about 10 kg of body fruits were collected, cut into 4-mm slices, spread on an oven rack in a single layer, and air-dried at 37 °C for 48 h. At the end of the process, about 1 kg of dried mushroom was obtained and all the product was ground to a fine powder using a Thermomix TM 31 (Vorwerk Contempora S.r.l., Italy). The production of breadsticks added with P. ostreatus was performed at the Società Agricola IoBoscoVivo (Vergiate, Varese, Italy), according to the addition levels decided on by the sensory group at the University of Milan, based on preliminary trials. The breadstick ingredients were whole-wheat flour, sunflower oil, yeast, and salt. The experimental samples were prepared by adding different increasing concentrations of P. ostreatus powder (B2 = 2%, B4 = 4%, and B6 = 6%) to the standard formulation (B0). A hidden control of the unmodified sample (Bhc = 0%) was also included to verify that children were able to assign a comparable score to the two samples without mushroom powder addition (B0 and Bhc). The nutritional composition and the energy content of the developed samples, which were provided by Società Agricola IoBoscoVivo (Vergiate, Varese, Italy), are reported in .

2.4. Experimental Procedure

The liking evaluation was performed at school in the classroom in the presence of a teacher and an experimenter in a single day. During the evaluation, each child was seated at their own table and received the questionnaire, which was extensively explained by the experimenters. The questionnaire consisted of an evaluation of food neophobia followed by the evaluation of breadstick liking. The questionnaire was self-completed by the children. To increase ecological validity, the breadsticks were offered as a snack and liking was assessed during the mid-morning break. Each of the five formulations, coded with three-digit numbers, were randomly presented to each child in blind condition. Children were instructed to drink a sip of water between each sample. The experimenters monitored the children to ensure that they did not influence each other during the evaluation. The session took approximately 30 min and children received a small award (e.g., pencil case) for their participation.

3.2. Liking Assessment

A significant sample effect was found on liking scores (F(4; 404) = 12.38, p < 0.0001). As reported in , B0 and Bhc samples obtained comparable scores (MB0 = 5.5 ± 0.2; MBhc = 5.3 ± 0.2). Children equally liked these samples, which were significantly preferred to samples with increasing added concentrations of P. ostreatus powder. Samples enriched with mushroom powder at 2% and 4% obtained comparable liking scores to each other (MB2 = 4.9 ± 0.2; MB4 = 4.9 ± 0.1) while the sample with the highest concentration of P. ostreatus powder received the lowest liking scores (MB6 = 4.3 ± 0.2). However, all the vitamin D2-enriched samples were well accepted by the children (liking scores were still up the middle of the scale).

2.3. Determination of Vitamin D2 Content

Vitamin D2 was extracted according to the procedure of Sławinska and collaborators [] and identified by the HPLC procedure by Huang and colleagues []. Analysis was performed on breadsticks enriched with 2%, 4%, and 6% of P. ostreatus powder and on the control breadstick after processing and on the breadstick enriched with 6% of P. ostreatus after storage at room temperature for six months.

3.1. Vitamin D2 Content and Product Design

As shown in , the addition of P. ostreatus from 2% to 6% significantly increased the level of vitamin D2 from not detectable to 28.4 µg in 100 g. Breadsticks enriched with 2%, 4%, and 6% can provide 32%, 54%, and 95% of the recommended daily amount of vitamin D2 per single dose (50 g). The 6% enriched breadsticks were also analyzed after being stored for six months at room temperature and found to have the same vitamin D2 content as just after production. According to the USDA database, milk and orange juice fortified with vitamin D provide 30% of the Recommended Dietary Allowance (RDA), while 20% and 10% of RDA is provided by fortified yogurt and cereal breakfast, respectively (). Hence, all the enriched breadsticks could be of interest to improve the dietary intake of vitamin D.

Methods

We measured baseline and 4-month serum concentrations of vitamin D3, 25-hydroxyvitamin D3 [25(OH)D3], 25-hydroxyvitamin D2, 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3], 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], and 4β,25-dihydroxyvitamin D3 [4β,25(OH)2D3] in 52 adults randomized to receive a total of four oral bolus doses of 2.5 mg vitamin D2 (n = 28) or vitamin D3 (n = 24) over four months. Metabolite-to-parent compound ratios were calculated to estimate hydroxylase activity. Pairwise before vs after comparisons were made to evaluate effects of vitamin D2 and vitamin D3 on metabolism of vitamin D. Mean postsupplementation metabolite-to-parent ratios were then compared between groups.

Trial design and participants

As previously described, we conducted a double-blind, randomized placebo-controlled trial that enrolled a total of 340 men and women aged 30 to 75 years who had been identified as being at increased risk of developing type 2 diabetes mellitus in London and Cambridge, United Kingdom (). Full details of inclusion and exclusion criteria are described in the published protocol (). Eligible participants were randomly allocated to one of three groups on a 1:1:1 basis within four strata defined by age (30 to 50 or 51 to 75 years) and sex, with a block size of six within each stratum. One group received four monthly oral bolus doses of 2.5 mg vitamin D2: each dose was presented as 5 mL Sterogyl solution (Desma Pharma, Paris, France) containing 0.5 mg vitamin D2 per milliliter in ethanol. The second group received four monthly oral bolus doses of 2.5 mg vitamin D3: each dose was presented as 5 mL Vigantol oil (Merck Serono, Darmstadt, Germany) containing 0.5 mg vitamin D3 per milliliter in Miglyol oil (Caesar & Loretz, Hilden, Germany). The third group received four monthly oral doses of placebo (Miglyol oil). The order of treatments within each block was determined by a computer-generated pseudo-random sequence, generated by the study medication manufacturer (Nova Laboratories, Leicester, UK). Neither the participants, the investigators, nor the laboratory staff knew the treatment allocation. Each participant was followed-up for a total of four months from their first visit; serum samples were collected at baseline and at the end of the study. Baseline and four-month serum samples taken from a subset of 28 participants and allocated to vitamin D2 and 25 participants allocated to vitamin D3 were sent for determination of concentrations of vitamin D3 and its metabolites as detailed below. The subset of participants contributing samples to the current study were selected on the basis that they were all recruited in London; that they had each received four directly observed doses of vitamin D2 or vitamin D3; and that they were the 28 samples in each group having the greatest volume of serum available at both baseline and four-month follow-up to be sent for further analysis. For the vitamin D3 group, only 25 samples had sufficient sample volume for analysis. Ethical approval for the trial was provided by the Charing Cross Medical Ethics Committee (ref 09/H0711/85) and the Cambridge Local Research Ethics Committee (ref 04/Q0108/19), and written informed consent was obtained from all participants. The trial was registered under the numbers EudraCT 2009-011264-11 and ISRCTN86515510 on 23 October 2009.

Influence of vitamin D2 on metabolism of vitamin D3

To characterize effects of vitamin D2 on metabolism of D3, we conducted pairwise statistical analyses comparing circulating concentrations of vitamin D3 and its metabolites in 28 individuals before vs after oral administration of four monthly doses of 2.5 mg vitamin D2. Results are presented in . Administration of vitamin D2 had no statistically significant effect on serum concentrations of vitamin D3 (P = 0.07), but it did reduce mean serum concentrations of 25(OH)D3 (43.2% decrease, P < 0.001; ), 24R,25(OH)2D3 (37.5% decrease, P = 0.007), 1α,25(OH)2D3 (53.4% decrease, P < 0.001; ) and 4β,25(OH)2D3 (42.0% decrease, P = 0.03). Administration of vitamin D2 reduced molar ratios of 25(OH)D3 to vitamin D3 (76.2% decrease, P = 0.003) and 1α,25(OH)2D3 to 25(OH)D3 (52.2% decrease, P = 0.02), but increased the molar ratio of 24R,25(OH)2D3 to 25(OH)D3 (24.5% increase, P = 0.04). No statistically significant effect of vitamin D2 on the molar ratio of 4β,25(OH)2D3 to 25(OH)D3 was seen (P = 0.97).

Discussion

Our findings are consistent with reports that administration of vitamin D2 reduces serum concentrations of 25(OH)D3 (–), and that this phenomenon is associated with an increase in the ratio of 24R,25(OH)2D3 to 25(OH)D3 and a decrease in the ratio of 1α,25(OH)2D3 to 25(OH)D3 in the circulation. Interestingly, serum 25(OH)D2 concentrations were markedly elevated in all study participants receiving vitamin D2, consistent with induction of the vitamin D2 25-hydroxylation pathway, although there was an opposite effect on 25(OH)D3 and rate of formation in the same treated individuals (), raising the possibility that different enzymes might catalyze 25-hydroxylation of the two forms of vitamin D. Alternatively, the decline of 25(OH)D3 following administration of vitamin D2 may reflect competition of vitamin D2 for the same 25-hydroxylation pathway as vitamin D3. Although changes in metabolite-to-parent ratios may reflect alteration in rates of conversion of one metabolite to another, they could also be explained by removal of vitamin D and its metabolites from the circulation (e.g., via direct excretion or disposition into depots such as adipose tissue and muscle). Further investigations to compare the effects of different forms of vitamin D on expression and activity of the enzymes responsible for metabolizing vitamin D3 are needed to resolve the question of whether changes in metabolite-to-parent ratios truly reflect changes in activity of cytochrome P450 enzymes. Such studies would potentially require liver and renal biopsies: both are invasive procedures and their inclusion in a study protocol could raise issues relating to ethics and acceptability to participants.

Abstract

Mushroom irradiation has been considered a sustainable process to generate high amounts of vitamin D2 due to the role of this vitamin for human health and of the global concerns regarding its deficient or inadequate intake. Mushrooms are also receiving increasing interest due to their nutritional and medicinal properties. However, there is still a knowledge gap regarding the effect of UV irradiation on mushroom bioactive compounds. In this study, two of the most cultivated mushroom species worldwide, Agaricus bisporus and Pleurotus ostreatus, were irradiated with UV-B, and the effect of processing was investigated on the contents of vitamin D2 as well as on antioxidant and antiglycation activities. UV irradiation increased vitamin D2 up to 57 µg/g d.w, which is an adequate level for the fortification of a number of target foods. UV irradiation decreased the antioxidant activity when measured by the Folin–Ciocalteu reagent, the 2,2-diphenyl-1-(2,4,6 trinitrophenyl) hydrazyl radical assay and the ferric ion-reducing antioxidant power assay, but did not decrease the mushroom’s ability to inhibit glycation of a target protein. These results open up a new area of investigation aimed at selecting mushroom species with high nutraceutical benefits for irradiation in order to maintain their potential properties to inhibit oxidative and glycation processes responsible for human diseases.

2.2. Mushrooms

P. ostreatus and A. bisporus were purchased from the market. The irradiation treatment was performed as described previously []. In brief, about 10 kg of body fruits were used for each mushroom type, cut into 4 mm slices, spread on an oven rack in a single layer and air-dried at 37 °C for 48 h. During the first 24 h of air-drying, the mushrooms were irradiated using two fluorescent lamps installed at 30 cm above the mushroom layer. In accordance to most of the previous studies so far performed [,], a UV-B source was chosen for the irradiation. The lamps delivered a UV-B (280–315 nm) irradiance of 0.4 W/m2. At the end of the process, about 1 kg of dried mushroom was obtained from each mushroom type and the product was grinded to a fine powder using a Thermomix TM 31 (Vorwerk Contempora S.r.l., Milan, Italy). Control P. ostreatus and A. bisporus powders were obtained by processing mushrooms with the same drying and grinding procedure without UV irradiation.

3.1. The Effect of Mushroom Irradiation on Vitamin D2 Content

Initial content of vitamin D2 was low in both commercial mushrooms considered. This result is consistent with previous studies and could be associated with the conditions used for industrial cultivation of mushrooms that generally occurs with low exposure to sunlight [,]. UV irradiation led to an increase in vitamin D2 content from 3.1 to 37 µg/g d.w. and from 0.3 to 57 µg/g d.w. in P. ostreatus and A. bisporus, respectively (). The initial contents of ergosterol were 3600 and 5700 µg/g d.w. in P. ostreatus and A. bisporus, respectively (). These values fall in the range previously reported [] and remained high after the generation of significant amounts of vitamin D2. This result may suggest that upon UV irradiation. the residual amount of ergosterol is still relevant for drug development and could contribute to the health-promoting effects of mushrooms [].