Ashrafi, Nadia (2017) Detection of human salivary α-Defensins by LC-ESI-MS and evaluation of levels in athletes after physical stress. PhD thesis, University of Greenwich.

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Abstract

The aim of this study was to optimise and validate an LC-ESI-MS method to determine the level of individual human neutrophil alpha defensins (HNP1-4) after physical stress. A method for the analysis of individual (HNP1-4) in a saliva matrix by LC-ESI-MS has been established. An Agilent 1200 series HPLC, coupled with a Synapt G1 (Q-TOF-ESI-MS), was used for the separation and detection of HNP1-4. The need for solid phase extraction (SPE) was established. An LC-MS optimisation, focusing on mobile phases, column performances, and ion intensities, was carried out in order to enhance the detection of individual HNPs in human saliva. During LC-MS optimisation, two different mobile phase compositions (methanol with water; acetonitrile with water) and three different additives (formic acid, acetic acid, ammonium format with formic acid) were compared in response to electrospray ionisation (ESI) ion intensity of salivary HNP1-4. Kinetex® column separation efficiency was evaluated using two different column dimensions (50 x 2.1 mm and 50 x 3 mm) and two different stationary phases (C18 and C8). The performance of the core shell Kinetex® column (homogenous porous shell) was also compared to a new ultra ACE® (encapsulated bonded phase) column. The results showed that a SPE method prior to the analysis resulted in cleaner mass spectra compared to a method without SPE; whereby co-elution of proline rich proteins, peptides and other unknown peptides were observed to cause ion suppression. The peak area reduction between the HNPs in the SPE and without SPE treated samples ranged from 17-65%. HNPs were extracted by SPE with a recovery of 80-91%. Aqueous 0.1% acetic acid: methanol (50:50) provided enhanced ion intensities for individual HNPs. The Kinetex® C8 (50 x 3.0 mm, 2.6 μm) column facilitated a better separation of individual HNPs compared to the Ultra Core Super C18 ACE® (50 x 3.0 mm, 25 μm), Kinetex® C18 (50 x 3.0 mm, 2.6 μm) and Kinetex® C18 (50 x 3.0 mm, 5 μm) columns. The LC-MS method was linear for concentrations of HNP2 between 0.05 and 1 ng/μL with a detection limit of 0.05 ng/μL and quantification limit of 0.1 ng/μL (R2 = 0.99). Inter-intra assay precision was 0.3–15%, respectively. As part of the method validation, HNP1-3 salivary levels were determined by ELISA and the data compared with that obtained by LC-MS. No significant correlation was found between the two methods (R2= 0.96) for the detection of these potential antimicrobial peptides. The optimised, validated LC-MS method was applied to examine the relative levels of individual HNP1-4 in participants undertaking a rigorous physical activity intervention. The levels of individual HNP1-4 were found to be higher after exercise (at both 30 and 60 min intervals) compared to baseline assessments, where participants consumed either placebo (PL) or carbohydrate (CHO) supplements. In addition, the level of individual HNPs at 30 mins post exercise increased by around 80-134% and 103-132% for participants who consumed either PL or CHO supplements. A further increase in the levels of individual HNPs was observed around 185-415% and 90-264% at 60 mins post exercise. These levels differed significantly (HNP1, p=0.00; HNP2, p=0.05; HNP3, p=0.03; HNP4, p=0.01) in athletes that consumed either PL or CHO supplements. Furthermore, the levels of individual HNPs were investigated after supplementing with a commercially available multi-ingredient (MTN) and compared with a CHO supplement. A similar trend was observed for individual HNPs at both 30 and 60 min intervals but they did not significantly differ at any time at the intervals (post 30 mins HNP1, p=0.85; HNP2, p=0.16; HNP3, p=0.07; HNP4, p=0.40 and post 60 mins HNP1, p=0.17; HNP2, p=0.20; HNP3, p=0.09; HNP4, p=0.30). The above findings provide evidence that the level of individual HNPs varies and is sensitive to physical stress. Physical stress may induce air-inflammation and damage to the airway epithelial cells, which could be part of a normal stress response. A possible correlation between the average response of HNP1-3 and cortisol was investigated by ELISA. The findings suggest that there is no correlation between the changes in the levels of salivary HNP1-3 and cortisol. In conclusion, the optimised and validated LC-MS method revealed that individual levels of HNPs can be determined accurately and precisely, which makes a novel contribution to sports and clinical research.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Saliva; α-Defensins and cortisol levels; physical stress; athletes; exercise performance; antimicrobial peptides; electrospray ionisation (ESI);
Subjects:	Q Science > QD Chemistry
Faculty / School / Research Centre / Research Group:	Faculty of Engineering & Science Faculty of Engineering & Science > School of Science (SCI)
Last Modified:	24 Apr 2019 09:59
URI:	http://gala.gre.ac.uk/id/eprint/23692

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