Modelling of amino acid turnover in the horse during training and racing: A basis for developing a novel supplementation strategy.

Horses in heavy training in preparation for racing and competition have increased metabolic demands to support the more intensive levels of exercise and recovery. However, little is known at the metabolic level about amino acid turnover and the specific alterations of demand caused by high intensity exercise. During exercise, certain amino acids are required in greater quantities due to disproportionate losses via excretory systems and usage in biosynthetic pathways. This investigation has built a theoretical computer model in an attempt to bring together the published rates of protein intake and utilisation to try to understand how some amino acids might be in higher demand than others. The model indicated that after evaluation of the daily amino acid turnover, glutamine/glutamic acid (Glx), serine and ornithine were in negative nitrogen balance which identified these amino acids as critical limiting factors for anabolism. Adjustment of the modelling conditions to cater for high intensity training indicated that an additional demand was placed on eight amino acids, including GLx, valine, lysine, histidine and phenylalanine which could thus become limiting under these conditions. The modelling results indicated that an amino acid supplement with the correct amino acids to match demand could theoretically be beneficial to a 500Kg horse in quantities of 20-80g/day. These results open new avenues of research for specifically tailoring amino acid supplementation to meet demands for sports horses in heavy training and improving general well-being, especially in hotter climates.

Relationships between electrolyte and amino acid compositions in sweat during exercise suggest a role for amino acids and K+ in reabsorption of Na+ and Cl- from sweat.

Concentrations of free amino acids and [K+] in human sweat can be many times higher than in plasma. Conversely, [Na+] and [Cl-] in sweat are hypotonic to plasma. It was hypothesised that the amino acids and K+ were directly or indirectly associated with the resorption of Na+ and Cl- in the sweat duct. The implication would be that, as resources of these components became limiting during prolonged exercise then the capacity to resorb [Na+] and [Cl-] would diminish, resulting in progressively higher levels in sweat. If this were the case, then [Na+] and [Cl-] in sweat would have inverse relationships with [K+] and the amino acids during exercise. Forearm sweat was collected from 11 recreational athletes at regular intervals during a prolonged period of cycling exercise after 15, 25, 35, 45, 55 and 65 minutes. The subjects also provided passive sweat samples via 15 minutes of thermal stimulation. The sweat samples were analysed for concentrations of amino acids, Na+, Cl-, K+, Mg2+ and Ca2+. The exercise sweat had a total amino acid concentration of 6.4 ± 1.2mM after 15 minutes which was lower than the passive sweat concentration at 11.6 ± 0.8mM (p<0.05) and showed an altered array of electrolytes, indicating that exercise stimulated a change in sweat composition. During the exercise period, [Na+] in sweat increased from 23.3 ± 3.0mM to 34.6 ± 2.4mM (p<0.01) over 65 minutes whilst the total concentrations of amino acids in sweat decreased from 6.4 ± 1.2mM to 3.6 ± 0.5mM. [Na+] showed significant negative correlations with the concentrations of total amino acids (r = -0.97, p<0.05), K+ (r = -0.93, p<0.05) and Ca2+ (r = -0.83, p<0.05) in sweat. The results supported the hypothesis that amino acids and K+, as well as Ca2+, were associated with resorption of Na+ and Cl-.

Amino acids and proteomic acclimation of Staphylococcus aureus when incubated in a defined minimal medium supplemented with 5% sodium chloride.

Staphylococcus aureus is a versatile bacterium that can adapt to survive and grow in a wide range of salt concentrations. This study investigated whether the cells could mount a response to survive a challenge of 5% NaCl in a minimal incubation medium that would not support cell replication. Cells were grown in liquid culture, washed and then incubated for 90 min at 37°C in a medium that contained only glycine and glucose as substrates in PBS plus trace elements. The control cells were compared with a treatment group which was incubated with an additional 5% NaCl. Significantly more glycine was taken up by the cells exposed to 5% NaCl compared with control cells, and both groups consumed 99% of the glucose supplied. The NaCl treated cells had significantly higher cytoplasmic levels of proline and glutamic acid as well as lower levels of alanine and methionine compared with the controls (p < 0.05). The levels of the two major cytoplasmic amino acids, aspartic acid and glycine, remained constant in control and treated cells. Proteomic analyses revealed that 10 proteins showed differential responses between the control and treatment groups. The reductions in proteins were primarily associated with processes of protein biosynthesis, pathogenicity, and cell adhesion. Since cell numbers remained constant during the incubation period in minimal medium, it was concluded that there was no cell division to support population growth. The results provided evidence that the cells in the minimal medium exposed to the NaCl treatment underwent in situ homeostatic changes to adjust to the new environmental conditions. It was proposed that this represented a phenotypic shift to form cells akin to small colony variants, with lower metabolic rates and lower levels of key proteins associated with pathogenicity.

The Uptake and Release of Amino Acids by Staphylococcus aureus at Mid-Exponential and Stationary Phases and Their Corresponding Responses to Changes in Temperature, pH and Osmolality.

Staphylococcus aureus is an important pathogen that is associated with nosocomial infections, as well as food poisoning. This bacterium is resistant to antimicrobial agents and can survive in a wide range of environmental conditions. The aim of this study was to measure the uptake and release of amino acids by S. aureus at mid-exponential and stationary phases of growth following exposure to a combination of conditions including variations in temperature, pH and NaCl. Bacterial cells were grown up to mid-exponential and stationary phases in tryptic soy broth (TSB), where the supernatants were collected for analyses of amino acids to determine the uptake and release characteristics. The uptake/release of amino acids was estimated by subtracting the initial levels of the free amino acids in the media from those measured at mid-exponential and stationary phases of growth. When cells were grown at ideal conditions, the analyses revealed that significant uptake of amino acids had occurred by stationary phase compared with the mid-exponential phase. A substantial release of valine and tyrosine into the external media was observed by cells at stationary phase. At both phases, the uptake and release patterns were significantly different between cells grown under ideal control conditions, when compared with those grown under various combinations of sub-optimal environmental conditions. The analyses of the supernatants harvested from controls and treatment groups at exponential phase indicated that the total uptake of amino acids was reduced approximately five times by cells grown with addition of 2.5% NaCl or with pH6 at 35°C, and 2-fold by cells grown at pH8 at 35°C. However, the final quantities of amino acids taken up by cells grown to stationary phase did not significantly alter between control and treated samples. Valine was found to be the most abundant amino acid that was significantly released into the media at stationary phase by both control and treated samples. It was evident that diverse environmental conditions resulted in differential patterns of amino acid uptake and release during adaptation to designated conditions.

Alterations in amino acid metabolism during growth by Staphylococcus aureus following exposure to H2O2 - A multifactorial approach.

Temperature and pH are known to vary in a wound site due to the immune response and subsequent healing processes. This study used a multifactorial design to examine the cellular responses of Staphylococcus aureus to hydrogen peroxide (0-100 mM) when bacteria were grown in temperatures of 37 ± 2 °C and pH 7 ± 1, conditions potentially encountered in wound sites. A centroid sample was included in the design which represented the mid-point values of all three environmental parameters (37 °C, pH 7, 50 mM H2O2). Cytoplasmic extracts and corresponding medium supernatants were analysed for amino acid composition by gas chromatography. Exposures of S. aureus to H2O2 during the inoculation process resulted in extended lag phases lasting well after the peroxide had been neutralised by the bacterium's antioxidant systems, after which the bacteria eventually resumed growth at equivalent rates to the controls. Even though the subsequent growth rates appeared normal, the cells exhibited a variant metabolic regime at the mid-exponential phase of growth as a result of the initial exposure to peroxide. The alterations in metabolism were reflected by the differential amino acid profiles measured in the cytoplasmic extracts (P < 0.0001). The data indicated that the metabolic responses to H2O2 challenge were uniquely different depending on the variations of temperature and pH. The uptake patterns of amino acids from the media also altered depending on prevailing environmental conditions. From these results, it was proposed that a specific reproducible homeostasis could be induced under a specific set of defined environmental conditions. It was also evident that early toxic insults on the bacterial culture could have lasting impacts on cellular homeostasis after successive generations, even after the offending chemical had been removed and initial cell integrity restored. It was concluded that metabolic homeostasis would be continually adjusting and responding to changing environmental conditions to deploy defensive proteins as well as optimising processes for survival. The powerful ability to continually and rapidly adapt to the environment may represent the key feature supporting the virulence of S. aureus as an opportunistic pathogen invading the wound site.

Sweat Facilitated Amino Acid Losses in Male Athletes during Exercise at 32-34°C.

Sweat contains amino acids and electrolytes derived from plasma and athletes can lose 1-2L of sweat per hour during exercise. Sweat may also contain contributions of amino acids as well as urea, sodium and potassium from the natural moisturizing factors (NMF) produced in the stratum corneum. In preliminary experiments, one participant was tested on three separate occasions to compare sweat composition with surface water washings from the same area of skin to assess contributions from NMF. Two participants performed a 40 minute self-paced cycle session with sweat collected from cleansed skin at regular intervals to assess the contributions to the sweat load from NMF over the period of exercise. The main study investigated sweat amino acid composition collected from nineteen male athletes following standardised endurance exercise regimes at 32-34°C and 20-30% RH. Plasma was also collected from ten of the athletes to compare sweat and plasma composition of amino acids. The amino acid profiles of the skin washings were similar to the sweat, suggesting that the NMF could contribute certain amino acids into sweat. Since the sweat collected from athletes contained some amino acid contributions from the skin, this fluid was subsequently referred to as "faux" sweat. Samples taken over 40 minutes of exercise showed that these contributions diminished over time and were minimal at 35 minutes. In the main study, the faux sweat samples collected from the athletes with minimal NMF contributions, were characterised by relatively high levels of serine, histidine, ornithine, glycine and alanine compared with the corresponding levels measured in the plasma. Aspartic acid was detected in faux sweat but not in the plasma. Glutamine and proline were lower in the faux sweat than plasma in all the athletes. Three phenotypic groups of athletes were defined based on faux sweat volumes and composition profiles of amino acids with varying relative abundances of histidine, serine, glycine and ornithine. It was concluded that for some individuals, faux sweat resulting from exercise at 32-34°C and 20-30% RH posed a potentially significant source of amino acid loss.

Changes in the Cytoplasmic Composition of Amino Acids and Proteins Observed in Staphylococcus aureus during Growth under Variable Growth Conditions Representative of the Human Wound Site.

Staphylococcus aureus is an opportunistic pathogen responsible for a high proportion of nosocomial infections. This study was conducted to assess the bacterial responses in the cytoplasmic composition of amino acids and ribosomal proteins under various environmental conditions designed to mimic those on the human skin or within a wound site: pH6-8, temperature 35-37°C, and additional 0-5% NaCl. It was found that each set of environmental conditions elicited substantial adjustments in cytoplasmic levels of glutamic acid, aspartic acid, proline, alanine and glycine (P< 0.05). These alterations generated characteristic amino acid profiles assessed by principle component analysis (PCA). Substantial alterations in cytoplasmic amino acid and protein composition occurred during growth under conditions of higher salinity stress implemented via additional levels of NaCl in the growth medium. The cells responded to additional NaCl at pH 6 by reducing levels of ribosomal proteins, whereas at pH 8 there was an upregulation of ribosomal proteins compared with the reference control. The levels of two ribosomal proteins, L32 and S19, remained constant across all experimental conditions. The data supported the hypothesis that the bacterium was continually responding to the dynamic environment by modifying the proteome and optimising metabolic homeostasis.

Metabolomic and proteomic responses of Staphylococcus aureus to prolonged cold stress.

The high pathogenicity of Staphylococcus aureus is thought to be due to its extraordinary capacity to rapidly adapt to changes in environmental conditions. This study was carried out to investigate whether the cytoplasmic profiles of metabolites and proteins of S. aureus were altered in response to prolonged exposure to cold stress. Metabolic profiling and proteomics were used to characterise alterations in cytoplasmic proteins and metabolites in cells from the mid-exponential phase of growth under ideal conditions at 37°C and compared with equivalent cells exposed to prolonged cold stress for 2 weeks at 4°C. Principle component analysis (PCA) of the metabolomic and proteomic data indicated that, at the mid-exponential phase of growth, prolonged cold stress conditions generated cells with different metabolite and protein profiles compared with those grown at 37°C. Nine ribosomal proteins and citric acid were substantially elevated in the cytoplasmic fractions from the cells adapted to cold-stress but most amino acids showed a reduction in their concentration in cold-stressed samples. The data provided strong evidence supporting the hypothesis that specific changes in metabolic homeostasis and protein composition were critical to the adaptive processes required for survival under cold stress. BIOLOGICAL SIGNIFICANCE: Work in our laboratory has shown that prolonged exposure of S. aureus to cold stress can result in the formation of small colony variants (SCVs) associated with significant alterations in the cell wall composition. Further studies revealed that S. aureus altered cell size and cell wall thickness in response to exposure to cold temperatures, alterations in pH and exposure to antibiotics. The current study has utilised the prolonged exposure to cold stress as a model system to explore changes in the proteome and associated metabolic homeostasis following environmental challenges. The study provides an improved understanding of how S. aureus adapts to the changing environment whilst in transition between human hosts. The results indicated an unexpected production of 9 ribosomal proteins and citric acid in response to cold stress suggesting specific survival roles for these proteins and citric acid as an adaptation mechanism for empowering survival under these conditions.

Small changes in environmental parameters lead to alterations in antibiotic resistance, cell morphology and membrane fatty acid composition in Staphylococcus lugdunensis.

Staphylococcus lugdunensis has emerged as a major cause of community-acquired and nosocomial infections. This bacterium can rapidly adapt to changing environmental conditions to survive and capitalize on opportunities to colonize and infect through wound surfaces. It was proposed that S. lugdunensis would have underlying alterations in metabolic homeostasis to provide the necessary levels of adaptive protection. The aims of this project were to examine the impacts of subtle variations in environmental conditions on growth characteristics, cell size and membrane fatty acid composition in S. lugdunensis. Liquid broth cultures of S. lugdunensis were grown under varying combinations of pH (6-8), temperature (35-39°C) and osmotic pressure (0-5% sodium chloride w/w) to reflect potential ranges of conditions encountered during transition from skin surfaces to invasion of wound sites. The cells were harvested at the mid-exponential phase of growth and assessed for antibiotic minimal inhibitory concentration (MIC), generation time, formation of small colony variants, cell size (by scanning electron microscopy) and membrane fatty acid composition. Stress regimes with elevated NaCl concentrations resulted in significantly higher antibiotic resistance (MIC) and three of the combinations with 5% NaCl had increased generation times (P<0.05). It was found that all ten experimental growth regimes, including the control and centroid cultures, yielded significantly different profiles of plasma membrane fatty acid composition (P<0.0001). Alterations in cell size (P<0.01) were also observed under the range of conditions with the most substantial reduction occurring when cells were grown at 39°C, pH 8 (514±52 nm, mean ± Standard Deviation) compared with cells grown under control conditions at 37°C with pH 7 (702±76 nm, P<0.01). It was concluded that S. lugdunensis responded to slight changes in environmental conditions by altering plasma membrane fatty acid composition, growth rates and morphology to achieve optimal adaptations for survival in changing environments.

Phenotypic variants of staphylococci and their underlying population distributions following exposure to stress.

This study investigated whether alterations in environmental conditions would induce the formation of small colony variant phenotypes (SCV) with associated changes in cell morphology and ultra-structure in S. aureus, s. epidermidis, and S. lugdunensis. Wild-type clinical isolates were exposed to low temperature (4 °C), antibiotic stress (penicillin G and vancomycin; 0-10,000 µg mL(-1)), pH stress (pH 3-9) and osmotic challenge (NaCl concentrations of 0-20%). Changes in cell diameter, cell-wall thickness, and population distribution changes (n ≥ 300) were assessed via scanning and transmission electron microscopy (SEM and TEM), and compared to control populations. Our analyses found that prolonged exposure to all treatments resulted in the subsequent formation of SCV phenotypes. Observed SCVs manifested as minute colonies with reduced haemolysis and pigmentation (NaCl, pH and 4°C treatments), or complete lack thereof (antibiotic treatments). SEM comparison analyses revealed significantly smaller cell sizes for SCV populations except in S. aureus and S. epidermidis 10% NaCl, and S. epidermidis 4 °C (p<0.05). Shifts in population distribution patterns were also observed with distinct sub-populations of smaller cells appearing for S. epidermidis, and S. lugdunensis. TEM analyses revealed significantly thicker cell-walls in all treatments and species except S. lugdunensis exposed to 4 °C. These findings suggest that staphylococci adapted to environmental stresses by altering their cell size and wall thickness which could represent the formation of altered phenotypes which facilitate survival under harsh conditions. The phenotypic response was governed by the type of prevailing environmental stress regime leading to appropriate alterations in ultra-structure and size, suggesting downstream changes in gene expression, the proteome, and metabolome.

Altered amino acid homeostasis and the development of fatigue by breast cancer radiotherapy patients: A pilot study.

OBJECTIVES: To examine altered amino acid homeostasis as a predisposing factor of fatigue in female radiotherapy breast cancer patients. DESIGN AND METHODS: Participants underwent breast-conserving surgery and adjuvant breast irradiation and were free from significant fatigue pre-radiotherapy. The Functional Assessment of Cancer Therapy fatigue subscale was used to assess fatigue pre- and post-radiotherapy. Blood biochemistry factors and urinary and plasma amino acid levels were measured. RESULTS: One third of 27 patients developed fatigue and were designated as the fatigued cohort. It was possible to differentiate between fatigued subjects pre- and post-radiotherapy based upon their urinary amino acid profiles. Univariate analysis supported altered amino acid homeostasis within the fatigued cohort. Urinary levels of histidine and alanine were increased pre-radiotherapy whilst threonine, methionine, alanine, serine, asparagine and glutamine levels were higher after 5weeks of radiotherapy for the fatigued cohort. CONCLUSIONS: Fatigue was accompanied by altered amino acid homeostasis with increased amino acid excretion suggestive of a catabolic response.