Tyler Barker

Higher Serum 25-Hydroxyvitamin D Concentrations Associate with a Faster Recovery of Skeletal Muscle Strength after Muscular Injury

The primary purpose of this study was to identify if serum 25-hydroxyvitamin D (25(OH)D) concentrations predict muscular weakness after intense exercise. We hypothesized that pre-exercise serum 25(OH)D concentrations inversely predict exercise-induced muscular weakness. Fourteen recreationally active adults participated in this study. Each subject had one leg randomly assigned as a control. The other leg performed an intense exercise protocol. Single-leg peak isometric force and blood 25(OH)D, aspartate and alanine aminotransferases, albumin, interferon (IFN)-γ, and interleukin-4 were measured prior to and following intense exercise. Following exercise, serum 25(OH)D concentrations increased (p < 0.05) immediately, but within minutes, subsequently decreased (p < 0.05). Circulating albumin increases predicted (p < 0.005) serum 25(OH)D increases, while IFN-γ increases predicted (p < 0.001) serum 25(OH)D decreases. Muscular weakness persisted within the exercise leg (p < 0.05) and compared to the control leg (p < 0.05) after the exercise protocol. Serum 25(OH)D concentrations inversely predicted (p < 0.05) muscular weakness (i.e., control leg vs. exercise leg peak isometric force) immediately and days (i.e., 48-h and 72-h) after exercise, suggesting the attenuation of exercise-induced muscular weakness with increasing serum 25(OH)D prior to exercise. Based on these data, we conclude that pre-exercise serum 25(OH)D concentrations could influence the recovery of skeletal muscle strength after an acute bout of intense exercise.

Positive correlation between circulating cathelicidin antimicrobial peptide (hCAP18/LL-37) and 25-hydroxyvitamin D levels in healthy adults.

BackgroundTranscription of the cathelicidin antimicrobial peptide (CAMP) gene is induced by binding of the bioactive form of vitamin D, 1,25-dihydroxyvitamin D, to the vitamin D receptor. Significant levels of the protein hCAP18/LL-37 are found in the blood and may protect against infection and/or sepsis. We hypothesized that serum vitamin D levels may modulate the circulating levels of hCAP18. Only three studies have shown a positive correlation between circulating 25-hydroxyvitamin D and hCAP18 levels. Here we provide additional evidence for such a correlation in healthy, middle-aged adults.FindingsSerum levels of 25-hydroxyvitamin D [25(OH)D] and plasma levels of hCAP18 were determined in 19 healthy middle-aged (mean of 50.1 years) adult men and women. Plasma hCAP18 concentrations correlated with serum 25(OH)D concentrations in subjects with 25(OH)D levels ≤ 32 ng/ml (r = 0.81, p < 0.005) but not in subjects with concentrations > 32 ng/ml (r = 0.19, p = 0.63).ConclusionsWe conclude that plasma hCAP18 levels correlate with serum 25(OH)D levels in subjects with concentrations of 25(OH)D ≤ 32 ng/ml as opposed to those with concentrations > 32 ng/ml and that vitamin D status may regulate systemic levels of hCAP18/LL-37.

From animals to humans: evidence linking oxidative stress as a causative factor in muscle atrophy

In a recent issue of The Journal of Physiology, Urso et al. (2007) demonstrated that there was a significant increase in the gene expression of various components of a major proteolytic pathway (i.e. ubiquitin proteasome pathway) in human skeletal muscle following spinal cord injury, and thus limb disuse. This finding is important because extended limb disuse causes a disruption in the muscle homeostatic balance between protein synthesis and protein degradation. This imbalance in favour of protein degradation is characterized by both a decrease in protein synthesis and an increase in protein degradation, which subsequently, manifests as muscle atrophy. The mechanisms of protein degradation and the specific proteolytic pathways are relatively well known compared with those of protein synthesis during limb disuse. Of these proteolytic pathways, Ca2+ activated proteases (calpains) and the ubiquitin proteasome pathway have received considerable attention as significant contributors to the catabolic state leading to skeletal muscle atrophy. Additionally, these pathways appear to be markedly influenced by oxidative stress. While various studies in experimental animals and cell culture demonstrate that oxidative stress is an important cell regulator of these proteolytic pathways, the prevalence and physiological significance of oxidative stress on limb disuse muscle atrophy in humans remains unknown. It is clear that proteolytic pathways are stimulated during limb disuse in humans (Urso et al. 2006, 2007; Jones et al. 2004), which raises the questions, is there also an increase in oxidative stress during limb disuse in humans? and is this oxidative stress a causative factor that potentiates the degree of muscle atrophy? Hypothetically, calpain activation precedes or functions ‘up-stream’ from the ubiquitin proteasome pathway. It is well-known that calpains are calcium-activated proteases; however, various calpain isoforms (i.e. μ- and m-calpains) are also sensitive to oxidative stress. Moreover, calpain activation in skeletal muscle may promote the infiltration of inflammatory cells (i.e. neutrophils) that also produce reactive oxygen species (i.e. hypochlorous acid from myeloperoxidase), and thus potentiate oxidative stress. Smith & Dodd (2007) found that calpain activation increased total protein degradation, as well as specifically proteasome-dependent proteolysis. Importantly, when proteasomes were inhibited, the increase in proteolysis following calpain activation was ameliorated. Thus, calpain activation appears to be associated with increased levels of oxidative stress and increased ubiquitin proteasome protein degradation. In attempts to identify the genes that regulate muscle atrophy in humans, Urso et al. (2006) and Jones et al. (2004) examined alterations in mRNA and protein expression following limb immobilization. These studies compliment existing experimental data because they identify various proteolytic enzymes in humans that regulate muscle atrophy and are in agreement with previous experimental animal studies (Bodine et al. 2001; Gomes et al. 2001). Urso et al. (2006) found increased gene expression of various components of the ubiquitin proteasome pathway following only 48 h of limb immobilization; however, they did not find changes in protein expression of these components. Furthermore, gene expression of major regulatory enzymes of the ubiquitin proteasome pathway, especially various E3 ligases (MuRF1 and MAFbx/Atrogin-1), was unchanged. In contrast to these findings, an earlier investigation in healthy, young men reported increased gene expression of MAFbx (P < 0.01) and a trend toward increased MuRF1 (P = 0.1) following 2 weeks of limb immobilization (Jones et al. 2004). These studies collectively suggest that there is an increase in various components of the ubiquitin proteasome pathway following limb immobilization in humans. However, the up-regulation of some of the key regulatory components associated with the ubiquitin proteasome pathway may be time dependent since 48 h of limb immobilization (Urso et al. 2006) did not display the observed increase in E3 ligases that was observed after 2 weeks of limb immobilization (Jones et al. 2004). However, it should be noted that very rarely does an individual experience limb immobilization without a preceding injury or illness, and therefore, this observation raises the question whether the aforementioned findings translate to humans following injury or illness. Skeletal muscle atrophy occurs rapidly following a variety of injuries, which include but are not limited to spinal cord and anterior cruciate ligament injuries. Following their limb immobilization study reported in 2006 (Urso et al. 2006), Urso et al. (2007) examined the influence of limb disuse as a result of a spinal cord injury on various components of the ubiquitin proteasome pathway. Interestingly, increased gene expression was observed at 2 and 5 days post-spinal cord injury; specifically various E3 ligases (UBE3C, atrogin-1 (MAFbx/FBXO32), and MuRF1) were up-regulated. Furthermore, at 5 days but not at 2 days post-spinal cord injury, atrogin-1 protein expression increased (Urso et al. 2007). Thus, the temporal up-regulation of key genes involved in the regulation of protein degradation by the ubiquitin proteasome pathway was faster following an initiating event (e.g. spinal cord injury). The data provided by Urso et al. (2006, 2007) and Jones et al. (2004) are in agreement with experimental animal studies that demonstrate that the ubiquitin proteasome pathway is up-regulated during various forms of muscular disuse in humans. However, the role of oxidative stress in triggering these mechanisms has yet to be clearly defined in human skeletal muscle. Over the past 15 years oxidative stress has received increasing recognition as an intracellular mediator of the signalling pathways that regulate muscle atrophy, especially in the experimental animal literature. Servais et al. (2007) examined the influence of vitamin E supplementation on oxidative stress and skeletal muscle proteolysis in rats exposed to 14 days of muscular disuse (e.g. hindlimb suspension). Vitamin E supplementation to rats for 21 days prior to and during the 14 days of hindlimb suspension reduced soleus muscle atrophy by ∼17%, ameliorated the increase in muscle thiobarbituric acid-reactive substance (TBARS) content and MuRF1 mRNA, and showed a tendency to prevent the up-regulation of MAFbx and μ-calpain mRNA (Servais et al. 2007). Thus, antioxidant supplementation prior to experimental manipulations appears to minimize muscle atrophy in rats, and therefore suggests in humans that longterm antioxidant supplementation or routine consumption of high antioxidant-containing diets may play an important role in the protection against muscle atrophy and oxidative stress during periods of skeletal muscle disuse. Currently, it remains unknown if dietary antioxidant status or supplementation in humans provides protection against muscle atrophy during periods of limb disuse or reduced activity. A novel class of oxidative stress-related factors has been identified in muscle atrophy studies. Metallothioneins are low molecular weight metal-binding (i.e. zinc and copper) proteins that contain cysteine residues and are induced by oxidative stress, among other stressors (i.e. glucocorticoids, catecholamines, etc.). Metallothioneins have also been suggested to display antioxidant properties because they can scavenge reactive oxygen species, and therefore may provide the protection against the elevation in oxidative stress that was responsible for their increased expression. Both metallothionein gene (Lecker et al. 2004) and protein (Kondo et al. 1992) expression increase in atrophying muscle in experimental animals. Importantly, metallothionein gene expression also increases in human muscle following limb immobilization (Urso et al. 2006) and spinal cord injury (Urso et al. 2007). Metallothioneins have been implicated in the atrophy programme gene (Lecker et al. 2004) and protein (Kondo et al. 1992) response as a result of oxidative stress in experimental animal studies. However, it is currently unknown if the observed increase in metallothioneins in atrophying muscle seen in humans is due to an increase in oxidative stress. Therefore, regulation of metallothioneins and their role in muscle atrophy requires further investigation. Oxidative stress may be the link tying together various aspects of muscle atrophy. The gene expression of ubiquitin proteasome pathway components as well as various metallothioneins, increase during limb disuse as a result of a spinal cord injury in human skeletal muscle (Urso et al. 2007). These findings are important because they identify a specific proteolytic pathway and a component involved in antioxidant protection in human skeletal muscle following an initiating event where both the former and latter have been demonstrated to be induced by oxidative stress in experimental animal studies. These studies also emphasize that data obtained in various experimental animal limb disuse and cell culture studies potentially apply to human disuse muscle atrophy. However, it is currently unknown if there is an increase in oxidative stress in atrophying muscle in humans. Applying the acquired scientific knowledge gained from experimental animal studies pertaining to the physiological interaction between the proteolytic pathways and oxidative stress to humans could have a significant impact on the rehabilitation from various muscular disuse atrophy conditions.

γ-Tocopherol-rich supplementation additively improves vascular endothelial function during smoking cessation☆

Oxidative stress and inflammation persist years after smoking cessation thereby limiting the restoration of vascular endothelial function (VEF). Although short-term smoking cessation improves VEF, no studies have examined co-therapy of antioxidants in combination with smoking cessation to improve VEF. We hypothesized that improvements in γ-tocopherol (γ-T) status during smoking cessation would improve VEF beyond that from smoking cessation alone by decreasing oxidative stress and proinflammatory responses. A randomized, double-blind, placebo-controlled study was conducted in otherwise healthy smokers (22 ± 1 years; mean ± SEM) who quit smoking for 7 days with placebo (n=14) or γ-T-rich supplementation (n=16; 500 mg γ-T/day). Brachial artery flow-mediated dilation (FMD), cotinine, and biomarkers of antioxidant status, oxidative stress, and inflammation were measured before and after 7 days of smoking cessation. Smoking cessation regardless of supplementation similarly decreased plasma cotinine, whereas γ-T-rich supplementation increased plasma γ-T by seven times and its urinary metabolite γ-carboxyethyl hydroxychroman by nine times (P<0.05). Smoking cessation with γ-T-rich supplementation increased FMD responses by 1.3% (P<0.05) beyond smoking cessation alone (4.1 ± 0.6% vs 2.8 ± 0.3%; mean ± SEM). Although plasma malondialdehyde decreased similarly in both groups (P<0.05), plasma oxidized LDL and urinary F2-isoprostanes were unaffected by smoking cessation or γ-T-rich supplementation. Plasma TNF-α and myeloperoxidase decreased (P<0.05) only in those receiving γ-T-rich supplements and these were inversely related to FMD (P<0.05; R=-0.46 and -0.37, respectively). These findings demonstrate that short-term γ-T-rich supplementation in combination with smoking cessation improved VEF beyond that from smoking cessation alone in young smokers, probably by decreasing the proinflammatory mediators TNF-α and myeloperoxidase.

Low Vitamin D Impairs Strength Recovery After Anterior Cruciate Ligament Surgery

The purpose of this study was to identify strength gains after an anterior cruciate ligament injury and surgery and during inflammatory challenge in participants with disparate vitamin D levels. Plasma samples were obtained from those who had not previously experienced an anterior cruciate ligament injury and from injured patients 2 weeks before and 3 months after anterior cruciate ligament surgery. Plasma 25-hydroxyvitamin D and cytokine concentrations were measured in each blood sample. Single-leg peak isometric forces were measured 2 weeks presurgery and 3 months postsurgery. Compared with noninjured participants, inflammatory cytokines were elevated prior to and following anterior cruciate ligament reconstruction. During this inflammatory challenge, the peak isometric force increases after surgery were significantly lower in those with plasma 25-hydroxyvitamin D concentrations <30 ng/mL compared with those with concentrations ≥30 ng/mL. The authors conclude that low vitamin D appears to hinder strength recovery after anterior cruciate ligament surgery and during inflammatory insult.

Different doses of supplemental vitamin D maintain interleukin-5 without altering skeletal muscle strength: a randomized, double-blind, placebo-controlled study in vitamin D sufficient adults

BackgroundSupplemental vitamin D modulates inflammatory cytokines and skeletal muscle function, but results are inconsistent. It is unknown if these inconsistencies are dependent on the supplemental dose of vitamin D. Therefore, the purpose of this study was to identify the influence of different doses of supplemental vitamin D on inflammatory cytokines and muscular strength in young adults.MethodsMen (n = 15) and women (n = 15) received a daily placebo or vitamin D supplement (200 or 4000 IU) for 28-d during the winter. Serum 25-hydroxyvitamin D (25(OH)D), cytokine concentrations and muscular (leg) strength measurements were performed prior to and during supplementation. Statistical significance of data were assessed with a two-way (time, treatment) analysis of variance (ANOVA) with repeated measures, followed by a Tukeys Honestly Significant Difference to test multiple pairwise comparisons.ResultsUpon enrollment, 63% of the subjects were vitamin D sufficient (serum 25(OH)D ≥ 30 ng/ml). Serum 25(OH)D and interleukin (IL)-5 decreased (P < 0.05) across time in the placebo group. Supplemental vitamin D at 200 IU maintained serum 25(OH)D concentrations and increased IL-5 (P < 0.05). Supplemental vitamin D at 4000 IU increased (P < 0.05) serum 25(OH)D without altering IL-5 concentrations. Although serum 25(OH)D concentrations correlated (P < 0.05) with muscle strength, muscle strength was not changed by supplemental vitamin D.ConclusionIn young adults who were vitamin D sufficient prior to supplementation, we conclude that a low-daily dose of supplemental vitamin D prevents serum 25(OH)D and IL-5 concentration decreases, and that muscular strength does not parallel the 25(OH)D increase induced by a high-daily dose of supplemental vitamin D. Considering that IL-5 protects against viruses and bacterial infections, these findings could have a broad physiological importance regarding the ability of vitamin D sufficiency to mediate the immune systems protection against infection.

Vitamin E and C supplementation does not ameliorate muscle dysfunction after anterior cruciate ligament surgery

Muscle atrophy and weakness are predominant impairments after anterior cruciate ligament (ACL) surgical repair. We tested the hypothesis that vitamin E and C supplementation will improve recovery from ACL injury. Men undergoing elective ACL surgery were randomly assigned to twice-daily supplements of either antioxidants (AO; vitamins E and C, n=10) or matching placebos (n=10) from 2 weeks before until 3 months after surgery. Each subject provided several fasting blood draws, two muscle biopsies from the thigh muscle of the injured limb, and strength and thigh circumference measurements of the lower limbs. Muscle atrophy was apparent in both groups before and several days after surgery. Compared with baseline measurements, peak isometric force of the injured limb increased significantly (P<0.05) by 3 months postsurgery in both treatment groups; however, AO supplementation did not augment these strength gains. By contrast, baseline plasma ascorbic acid concentrations correlated (r=0.59, P=0.006) with subsequent improvement in the strength of the injured limb. In summary, vitamin E and C supplementation was ineffective in potentiating the improvement in force production by the injured limb; however, baseline vitamin C status was associated with beneficial outcomes in strength, suggesting that long-term dietary habits are more effective than short-term supplements.

Vitamin D deficiency associates with γ-tocopherol and quadriceps weakness but not inflammatory cytokines in subjects with knee osteoarthritis.

Knee osteoarthritis (OA) is a degenerative joint condition and a leading cause of physical disability in the United States. Quadriceps weakness and inflammatory cytokines contribute to the pathogenesis of knee OA, and both of which, increase with vitamin D deficiency. Other micronutrients, such as vitamins C and E and β-carotene, modulate inflammatory cytokines and decrease during inflammation. The purpose of this study was to test the hypothesis that vitamin D deficiency associates with quadriceps weakness, an increase in serum cytokines, and a decrease in circulating micronutrients in subjects with knee OA. Subjects (age, 48±1 y; serum 25(OH)D, 25.8±1.1 ng/mL) with knee OA were categorized as vitamin D deficient (n=17; serum 25(OH)D≤20 ng/mL), insufficient (n=21; serum 25(OH)D 20–29 ng/mL), or sufficient (n=18; serum 25(OH)D≥30 ng/mL). Single-leg strength (concentric knee extension–flexion contraction cycles at 60 °/s) and blood cytokine, carotene (α and β), ascorbic acid, and tocopherol (α and γ) concentrations were measured. Quadriceps peak torque, average power, total work, and deceleration were significantly (all p<0.05) impaired with vitamin D deficiency. Serum γ-tocopherol concentrations were significantly (p<0.05) increased with vitamin D deficiency. In the vitamin D sufficient group, γ-tocopherol inversely correlated (r=−0.47, p<0.05) with TNF-α, suggesting a pro-inflammatory increase with a γ-tocopherol decrease despite a sufficient serum 25(OH)D concentration. We conclude that vitamin D deficiency is detrimental to quadriceps function, and in subjects with vitamin D sufficiency, γ-tocopherol could have an important anti-inflammatory role in a pathophysiological condition mediated by inflammation.

Modulation of inflammation by vitamin E and C supplementation prior to anterior cruciate ligament surgery.

Muscle atrophy commonly follows anterior cruciate ligament (ACL) injury and surgery. Proinflammatory cytokines can induce and exacerbate oxidative stress, potentiating muscle atrophy. The purpose of this study was to evaluate the influence of prior antioxidant (AO) supplementation on circulating cytokines following ACL surgery. A randomized, double-blind, placebo-controlled trial was conducted in men undergoing ACL surgery, who were randomly assigned to either: (1) AO (200 IU of vitamin E (50% d-alpha-tocopheryl acetate and 50% d-alpha-tocopherol) and 500 mg ascorbic acid), or (2) matching placebos (PL). Subjects took supplements twice daily for 2 weeks prior to and up to 12 weeks after surgery. Each subject provided five blood samples: (1) baseline (Bsl, prior to supplementation and approximately 2 weeks prior to surgery), (2) presurgery (Pre), (3) 90 min, (4) 72 h, and (5) 7 days postsurgery. Following surgery, inflammation and muscle damage increased in both groups, as assessed by increased circulating IL-6, C-reactive protein, and creatine kinase. During AO supplementation, plasma alpha-T and AA increased while gamma-T concentrations decreased significantly (P< 0.05). At 90 min the AO group displayed a significant decrease in AA, an inverse correlation between AA and (interleukin) IL-8 (r(2)= 0.50, P< 0.05), and a significantly lower IL-10 response than that of the PL group. IL-10 was significantly elevated at 90 min and 72 h in the PL group. In summary, our findings show that circulating inflammatory cytokines increase and AO supplementation attenuated the increase in IL-10 in patients post-ACL surgery.

Vitamin D sufficiency associates with an increase in anti-inflammatory cytokines after intense exercise in humans.

The purpose of this study was to identify the influence of vitamin D status (insufficient vs. sufficient) on circulating cytokines and skeletal muscle strength after muscular injury. To induce muscular injury, one randomly selected leg (SSC) performed exercise consisting of repetitive eccentric-concentric contractions. The other leg served as the control. An averaged serum 25(OH)D concentration from two blood samples collected before exercise and on separate occasions was used to establish vitamin D insufficiency (<30ng/mL, n=6) and sufficiency (>30ng/mL, n=7) in young, adult males. Serum cytokine concentrations, single-leg peak isometric force, and single-leg peak power output were measured before and during the days following the exercise protocol. The serum IL-10 and IL-13 responses to muscular injury were significantly (both p<0.05) increased in the vitamin D sufficient group. The immediate and persistent (days) peak isometric force (p<0.05) and peak power output (p<0.05) deficits in the SSC leg after the exercise protocol were not ameliorated with vitamin D sufficiency. We conclude that vitamin D sufficiency increases the anti-inflammatory cytokine response to muscular injury.