Development of a complex amino acid supplement, Fatigue Reviva™, for oral ingestion: initial evaluations of product concept and impact on symptoms of sub-health in a group of males.

BACKGROUND: A new dietary supplement, Fatigue Reviva™, has been recently developed to address issues related to amino acid depletion following illness or in conditions of sub-health where altered amino acid homeostasis has been associated with fatigue. Complex formulations of amino acids present significant challenges due to solubility and taste constraints. This initial study sets out to provide an initial appraisal of product palatability and to gather pilot evidence for efficacy. METHODS: Males reporting symptoms of sub-health were recruited on the basis of being free from any significant medical or psychological condition. Each participant took an amino acid based dietary supplement (Fatigue Reviva™) daily for 30 days. Comparisons were then made between pre- and post-supplement general health symptoms and urinary amino acid profiles. RESULTS: Seventeen men took part in the study. Following amino acid supplementation the total Chalder fatigue score improved significantly (mean ± SEM, 12.5 ± 0.9 versus 10.0 ± 1.0, P<0.03). When asked whether they thought that the supplement had improved their health, 65% of participants responded positively. A subgroup of participants reported gastrointestinal symptoms which were attributed to the supplement and which were believed to result from the component fructooligosaccharide. Analysis of urinary amino acids revealed significant alterations in the relative abundances of a number of amino acids after supplementation including an increase in valine, isoleucine and glutamic acid and reduced levels of glutamine and ornithine. Discriminant function analysis of the urinary amino acid data revealed significant differences between the pre- and post-supplement urine excretion profiles. CONCLUSIONS: The results indicated that Fatigue Reviva™ was palatable and that 65% of the study group reported that they felt the product had improved their health. The product could provide an effective tool for the management of unexplained fatigue and symptoms of sub-health. Further product development may yield additional options for those patients susceptible to fructooligosaccharide.

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.

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.

Altered amino acid excretion in children with autism.

Autism is a complex and life-long behavioural disorder of unknown aetiology. Recent reports have indicated the involvement of digestive tract dysfunction and possible complications from inadequate nutrition. In this study, 34 autistic children (12 untreated and 22 receiving therapeutic treatments related to digestive function and nutritional uptake) and 29 control subjects (all 5-15 years of age) were investigated to determine whether there were any anomalies in the urinary excretion of amino acids, glucose, sucrose, arabinose and tartaric acid using GC/FID and GC/MS analysis techniques. Significantly lower relative urinary levels of essential amino acids were revealed for both the untreated (mean +/- SEM, 32.53 +/- 3.09%) and treated (31.98 +/- 2.87%) autistic children compared with the controls (37.87 +/- 1.50%). There were no significant differences in measured excretions of sugars or tartaric acid. It was concluded that the untreated autistic children had evidence of altered metabolic homeostasis.

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.

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.

Does milk cause constipation? A crossover dietary trial.

The aims of this study were to: (1) determine whether replacement of cow's milk protein with soy resolves Chronic Functional Constipation (CFC); and (2) investigate the effects of cow's milk ß casein A1 and cow's milk ß casein A2 on CFC. Children diagnosed with CFC were recruited to one of two crossover trials: Trial 1 compared the effects of cow's milk and soy milk; Trial 2 compared the effects of cow's milk ß casein A1 and cow's milk ß casein A2. Resolution of constipation was defined as greater than eight bowel motions during a two week intervention. Thirteen children (18 to 144 months) participated in Trial 1 (6 boys, 7 girls). Nine participants who completed the soy epoch all experienced resolution (p < 0.05). Thirty-nine children (21 to 144 months) participated in Trial 2 (25 boys, 14 girls). Resolution of constipation was highest during the washout epoch, 81%; followed by cow's milk ß casein A2, 79%; and cow's milk ß casein A1, 57%; however, the proportions did not differ statistically. The results of Trial 1 demonstrate an association between CFC and cow's milk consumption but Trial 2 failed to show an effect from type of casein. Some other component in cow's milk common to both A1 and A2 milk may be causing a problem in these susceptible children.

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.