Abstract
β-alanine is the rate-limiting point for the endogenous synthesis of carnosine in skeletal muscle. Carnosine has a wide range of implications for health, normal function and exercise performance. Whilst the physiological relevance of carnosine to different tissues remains enigmatic, β-alanine administration is a useful strategy to investigate the physiological roles of carnosine in humans. Intravenous administration of β-alanine is an interesting approach to study carnosine metabolism. However, sterilisation is mandatory due to the nature of the administration route. We evaluated whether sterilising doses of gamma radiation damages the molecular structure and leads to the loss of functional characteristics of β-alanine. Pure β-alanine was sterilised by gamma radiation in sealed glass vials using a 60Co multipurpose irradiator at a dose rate of 8.5 kGy.hour-1 totalising 10, 20, 25 30 and 40 kGy. The molecular integrity was assessed by X-ray Diffraction and changes in content were determined by High Performance Liquid Chromatography (UV-HPLC) and Triple Quadrupole Mass Spectrometer (HPLC/MS-MS). Sterility assurance was evaluated by inoculation assay. To examine whether functional properties were preserved, β-alanine was infused in one participant, who rated the level of paraesthesia on the skin using a 0–3 scale. Urinary β-alanine was quantified before and 24-h following β-alanine infusion using HPLC-ESI+-MS/MS. Irradiation resulted in no change in the crystal structure of β-alanine, no degradation, and no new peaks were identified in the dose range assayed. The inoculation assay showed the absence of viable microorganisms in all β-alanine samples, including those that did not undergo irradiation. Intravenous infusion of β-alanine resulted in paraesthesia and it detected in the urine as per normal. We conclude that gamma radiation is a suitable technique for the sterilisation of β-alanine. It does not lead to degradation, damage to the β-alanine structure, content or loss of function within the evaluated irradiation conditions.
Highlights
High Performance Liquid Chromatography (HPLC) was used to determine β-alanine concentration in solutions prepared with pure β-alanine samples that were submitted to doses of radiation of up to 40 kGy, as well as with non-irradiated β-alanine
In addition to the HPLC measurements, we evaluated the structural integrity of β-alanine
Liquid Chromatography coupled to Mass Spectrometry (HPLC/MS-M) was used in the same samples to identify the presence of any non-β-alanine-related peaks, as well as to confirm HPLC findings
Summary
Available β-alanine in powder form (99%, molecular weight 89.09 g.Mol-1 obtained from Sigma-Aldrich, USA) were transferred to glass vials (1 g per vial), which were hermetically sealed and sterilised by gamma radiation. Β-alanine samples and controls were quantified by HPLC (Hitachi, Hitachi Ltd., Tokyo, Japan) according to the method described by Mora et al [42]. Β-alanine samples and controls were analysed by HPLC (Agilent series 1100, Agilent, USA) using Ascentis express column C18 (75 mm x 2.1mm, 2.7 μm) from. Derivatisation of β-alanine samples was performed using 50 μl phenyl isothiocyanate reagent 50 μl (0.1 M) and 50 μl triethylamine (1.0 M) followed by vortexing for 10 seconds and incubation at 40 ̊C under stirring (350 rpm) for 20 minutes. Urine samples and standards were quantified in duplicates and analysed by on-line HPLC-ESI+-MS/MS, as described by Carvalho et al [45] using CAR-d4 as internal standard.
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