Muscle glycogen utilisation during Rugby match play: Effects of pre-game carbohydrate.

OBJECTIVES: Although the physical demands of Rugby League (RL) match-play are well-known, the fuel sources supporting energy-production are poorly understood. We therefore assessed muscle glycogen utilisation and plasma metabolite responses to RL match-play after a relatively high (HCHO) or relatively low CHO (LCHO) diet. DESIGN: Sixteen (mean±SD age; 18±1 years, body-mass; 88±12kg, height 180±8cm) professional players completed a RL match after 36-h consuming a non-isocaloric high carbohydrate (n=8; 6gkgday-1) or low carbohydrate (n=8; 3gkgday-1) diet. METHODS: Muscle biopsies and blood samples were obtained pre- and post-match, alongside external and internal loads quantified using Global Positioning System technology and heart rate, respectively. Data were analysed using effects sizes ±90% CI and magnitude-based inferences. RESULTS: Differences in pre-match muscle glycogen between high and low carbohydrate conditions (449±51 and 444±81mmolkg-1d.w.) were unclear. High (243±43mmolkg-1d.w.) and low carbohydrate groups (298±130mmolkg-1d.w.) were most and very likely reduced post-match, respectively. For both groups, differences in pre-match NEFA and glycerol were unclear, with a most likely increase in NEFA and glycerol post-match. NEFA was likely lower in the high compared with low carbohydrate group post-match (0.95±0.39mmoll-1 and 1.45±0.51mmoll-1, respectively), whereas differences between the 2 groups for glycerol were unclear (98.1±33.6mmoll-1 and 123.1±39.6mmoll-1) in the high and low carbohydrate groups, respectively. CONCLUSIONS: Professional RL players can utilise ∼40% of their muscle glycogen during a competitive match regardless of their carbohydrate consumption in the preceding 36-h.

Season-long increases in perceived muscle soreness in professional rugby league players: role of player position, match characteristics and playing surface.

Rugby League (RL) is a high-impact collision sport characterised by repeated sprints and numerous high-speed impacts and consequently players often report immediate and prolonged muscle soreness in the days after a match. We examined muscle soreness after matches during a full season to understand the extent to which match characteristics influence soreness. Thirty-one elite Super League players provided daily measures of muscle soreness after each of the 26 competitive fixtures of the 2012 season. Playing position, phase of the season, playing surface and match characteristics were recorded from each match. Muscle soreness peaked at day 1 and was still apparent at day 4 post-game with no attenuation in the magnitude of muscle soreness over the course of the season. Neither playing position, phase of season or playing surface had any effects on the extent of muscle soreness. Playing time and total number of collisions were significantly correlated with higher ratings of muscle soreness, especially in the forwards. These data indicate the absence "contact adaptations" in elite rugby players with soreness present throughout the entire season. Strategies must now be implemented to deal with the physical and psychological consequences of prolonged feeling of pain.

Coupling fast water exchange to slow molecular tumbling in Gd3+ chelates: why faster is not always better.

The influence of dynamics on solution state structure is a widely overlooked consideration in chemistry. Variations in Gd(3+) chelate hydration with changing coordination geometry and dissociative water exchange kinetics substantially impact the effectiveness (or relaxivity) of monohydrated Gd(3+) chelates as T1-shortening contrast agents for MRI. Theory shows that relaxivity is highly dependent upon the Gd(3+)-water proton distance (rGdH), and yet this distance is almost never considered as a variable in assessing the relaxivity of a Gd(3+) chelate as a potential contrast agent. The consequence of this omission can be seen when considering the relaxivity of isomeric Gd(3+) chelates that exhibit different dissociative water exchange kinetics. The results described herein show that the relaxivity of a chelate with "optimal" dissociative water exchange kinetics is actually lower than that of an isomeric chelate with "suboptimal" dissociative water exchange. When the rate of molecular tumbling of these chelates is slowed, an approach that has long been understood to increase relaxivity, the observed difference in relaxivity is increased with the more rapidly exchanging ("optimal") chelate exhibiting lower relaxivity than the "suboptimally" exchanging isomer. The difference between the chelates arises from a non-field-dependent parameter: either the hydration number (q) or rGdH. For solution state Gd(3+) chelates, changes in the values of q and rGdH are indistinguishable. These parametric expressions simply describe the hydration state of the chelate--i.e., the number and position of closely associating water molecules. The hydration state (q/rGdH(6)) of a chelate is intrinsically linked to its dissociative water exchange rate kex, and the interrelation of these parameters must be considered when examining the relaxivity of Gd(3+) chelates. The data presented herein indicate that the changes in the hydration parameter (q/rGdH(6)) associated with changing dissociative water exchange kinetics has a profound effect on relaxivity and suggest that achieving the highest relaxivities in monohydrated Gd(3+) chelates is more complicated than simply "optimizing" dissociative water exchange kinetics.

Protection by pyridostigmine bromide of marmoset hemi-diaphragm acetylcholinesterase activity after soman exposure.

Pyridostigmine bromide (PB) was approved by the U.S. Food and Drug Administration (FDA) in 2003 as a pretreatment in humans against the lethal effects of the irreversible nerve agent soman (GD). Organophosphate (OP) chemical warfare agents such as GD exert their toxic effects by inhibiting acetylcholinesterase (AChE) from terminating the action of acetylcholine at postsynaptic sites in cholinergic nerve terminals (including crucial peripheral muscle such as diaphragm). As part of the post-marketing approval of PB, the FDA required (under 21CFR314, the "two animal rule") the study of a non-human primate model (the common marmoset Callithrix jacchus jacchus) to demonstrate increased survival against lethal GD poisoning, and protection of physiological hemi-diaphragm function after PB pretreatment and subsequent GD exposure. Marmosets (male and female) were placed in the following experimental groups: (i) control (saline pretreatment only), (ii) low dose PB (12.5 microg/kg), or (iii) high dose (39.5 microg/kg) PB. Thirty minutes after the PB dose, animals were challenged with either saline (control) or soman (GD, 45 microg/kg), followed 1 min later by atropine (2mg/kg) and 2-PAM (25mg/kg). After a further 16 min, animals were euthanized and the complete diaphragm removed; the right hemi-diaphragm was frozen immediately at -80 degrees C, and the left hemi-diaphragm was placed in a tissue bath for 4h (to allow for decarbamylation to occur), then frozen. AChE activities were determined using the automated WRAIR cholinesterase assay. Blood samples were collected for AChE activities prior to PB, before GD challenge, and after sacrifice. RBC-AChE was inhibited by approximately 18% and 50% at the low and high doses of PB, respectively, compared to control (baseline) activity. In the absence of PB pretreatment, the inhibition of RBC-AChE by GD was 98%. The recovery of hemi-diaphragm AChE activity after the 4h wash period (decarbamylation) was approximately 8% and 17%, at the low and high PB doses, respectively, compared with the baseline (control) AChE activity prior to PB pretreatment or soman exposure. The results suggest that PB pretreatment protects a critical fraction of AChE activity in the marmoset diaphragm, which is sufficient to allow the animal to breathe despite exposure to a dose of soman that is lethal in unprotected animals.

Advantages of the WRAIR whole blood cholinesterase assay: comparative analysis to the micro-Ellman, Test-mate ChE, and Michel (DeltapH) assays.

Red blood cell AChE (RBC-AChE) and plasma BChE can be used as sensitive biomarkers to detect exposure to OP nerve agents, pesticides, and cholinergic drugs. In a comparative study, RBC-AChE and serum BChE activities in whole blood was obtained from forty seven healthy male and female human volunteers, and then exposed separately ex vivo to three OP nerve agents (soman (GD), sarin (GB) and VX) to generate a wide range of inhibition of AChE and BChE activity (up to 90% of control). These samples were measured using four different ChE assays: (i) colorimetric microEllman (using DTNB at 412 nm), (ii) Test-mate ChE field kit (also based on the Ellman assay), (iii) Michel (delta pH), and (iv) the Walter Reed Army Institute of Research Whole Blood (WRAIR WB) cholinesterase assay. The WRAIR assay is a modified Ellman method using DTP at 324 nm (which minimizes hemoglobin interference and improves sensitivity), and determines AChE and BChE in a small whole blood sample simultaneously. Scatter plots of RBC-AChE activities were determined using the WRAIR ChE assay versus the micro-Ellman, Test-mate and Michel after exposure to varying concentrations of soman, sarin and VX. Regression analyses yielded mostly linear relationships with high correlations (r2 = 0.83-0.93) for RBC-AChE values in the WRAIR assay compared to the alternate methods. For the plasma BChE measurements, individual human values were significantly more variable (as expected), resulting in lower correlations using WRAIR ChE versus the alternate assays (r2 values 0.5 - 0.6). To circumvent the limitations of simple correlation analysis, Bland and Altman analysis for comparing two independent measurement techniques was performed. For example, a Bland and Altman plot of the ratio of the WRAIR whole blood AChE and Michel AChE (plotted on the y-axis) vs. the average of the two methods (x-axis) shows that the majority of the individual AChE values are within +/- 1.96 S.D. of the mean difference, indicating that the two methods may be used interchangeably with a high degree of confidence. The WRAIR ChE assay can be thus be used as a reliable inter-conversion assay when comparing results from laboratory-based (Michel) and field-based (Test-mate ChE kit), which use different methodology and report in different units of AChE activity.

Protection of red blood cell acetylcholinesterase by oral huperzine A against ex vivo soman exposure: next generation prophylaxis and sequestering of acetylcholinesterase over butyrylcholinesterase.

As part of a phase Ib clinical trial to determine the tolerability and safety of the highly specific acetylcholinesterase (AChE) inhibitor huperzine A, twelve (12) healthy elderly individuals received an escalating dose regimen of huperzine A (100, 200, 300, and 400 microg doses, twice daily for a week at each dose), with three (3) individuals as controls receiving a placebo. Using the WRAIR whole blood cholinesterase assay, red blood cell AChE and plasma butyrylcholinesterase (BChE) were measured in unprocessed whole blood samples from the volunteers following each dose, and then for up to 48h following the final and highest (400 microg) dose to monitor the profile of inhibition and recovery of AChE. Significant inhibition of AChE was observed, ranging from 30-40% after 100 microg to >50% at 400 microg, and peaking 1.5h after the last dose. Gradual recovery of AChE activity then occurs, but even 48 h after the last dose red blood cell AChE was about 10% below control (pre-dose) values. Huperzine A levels in plasma peaked 1.5h after the final 400 microg dose (5.47+/-2.15 ng/mL). Plasma BChE was unaffected by huperzine A treatment (as expected). Aliquots of huperzine A-containing (from three individuals) and placebo blood samples were exposed ex vivo to the irreversible nerve agent soman (GD) for 10 min, followed by removal of unbound huperzine and soman from the blood by passing through a small C(18) reverse phase spin column. Eluted blood was diluted in buffer, and aliquots taken at various time intervals for AChE and BChE activity measurement to determine the time taken to achieve full return in activity of the free enzyme (dissociation from the active site of AChE by huperzine A), and thus the proportion of AChE that can be protected from soman exposure. Huperzine A-inhibited red blood cell (RBC) AChE activity was restored almost to the level that was initially inhibited by the drug. The increased doses of huperzine A used were well tolerated by these patients and in this ex vivo study sequestered more red blood cell AChE than has been previously demonstrated for pyridostigmine bromide (PB), indicating the potential improved prophylaxis against organophosphate (OP) poisoning.

Oral administration of pyridostigmine bromide and huperzine A protects human whole blood cholinesterases from ex vivo exposure to soman.

Cholinesterases (ChEs) are classified as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) according to their substrate specificity and sensitivity to selected inhibitors. The activities of AChE in red blood cells (RBC-AChE) and BChE in serum can be used as potential biomarkers of suppressed and/or heightened activity in the central and peripheral nervous systems. Exposure to organophosphate (OP) chemical warfare agents (CWAs), pesticides, anesthetics, and a variety of drugs such as cocaine, as well as some neurodegenerative and liver disease states, selectively reduces AChE or BChE activity. In humans, the toxicity of pesticides is well documented. Therefore, blood cholinesterase activity can be exploited as a tool for confirming exposure to these agents and possible treatments. Current assays for measurement of RBC-AChE and serum BChE require several labor-intensive processing steps, suffer from wide statistical variation, and there is no inter-laboratory conversion between methods. These methods, which determine only the serum BChE or RBC-AChE but not both, include the Ellman, radiometric, and deltapH (modified Michel) methods. In contrast, the Walter Reed Army Institute of Research Whole Blood (WRAIR WB, US Patent #6,746,850) cholinesterase assay rapidly determines the activity of both AChE and BChE in unprocessed (uncentrifuged) whole blood, uses a minimally invasive blood sampling technique (e.g., blood from a finger prick), and is semi-automated for high-throughput using the Biomek 2000 robotic system. To date, the WRAIR whole blood assay was used to measure AChE and BChE activities in human blood from volunteers in FDA clinical trials. In the first FDA study, 24 human subjects were given either 30 mg PB orally (n = 19) or placebo (n = 5). Blood samples were obtained pre-dosing and 2.5, 5, 8, and 24 h post-dosing. The samples were analyzed for AChE and BChE activity using the WRAIR WB robotic system, and for PB concentration by HPLC. We found that maximal inhibition of AChE (26.2%) and concentration of PB (17.1 ng/mL) occurred at 2.5 h post-PB dosing. AChE activity returned to almost 100% of pre-dose values by 6 h. A dose-dependent linear correlation was found between the amount of PB measured in the blood and the inhibition of AChE. Following soman (GD) exposure, recovered AChE activity was similar to levels that were reversibly protected by the PB administration. Therefore, the WRAIR ChE WB data clearly supports the conclusion that PB is an effective pre-treatment drug for nerve agent exposure (GD). In the second FDA human study for the treatment of Alzheimer's disease, the WRAIR ChE WB assay was used to determine the RBC-AChE and serum BChE profile of healthy elderly volunteers receiving Huperzine A. Huperzine A is a plant-derived reversible and selective AChE inhibitor compared to BChE, and is a more potent inhibitor of AChE than PB. Huperzine A is available as a nutraceutical, a natural supplement reported to improve memory, and has a variety of neuroprotective effects. Individuals received an increasing dose regimen of huperzine A (final dose 200 microg after 4 weeks), which produced more than 50% inhibition of RBC-AChE. Huperzine A was well tolerated by these patients at doses that sequestered more RBC-AChE than PB, and thus warrants further study as a prophylaxis for OP poisoning in addition to Alzheimer's therapy. Due to the documented use of OPs by terrorists and in warfare around the globe, Federal, State, and local authorities need a reliable, fast, inexpensive, and standard method for confirming such an assault in order to initiate appropriate containment, decontamination, and treatment measures. This assay is ideal for prescreening military personnel for atypical ChE activities that would preclude their deployment to areas of potential CWA exposure. The WRAIR WB ChE assay will fulfill the requirement for rapid and reliable monitoring of such exposure in military and civilian populations.