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Biochemistry of Salivary Nucleotide Metabolites and Their Role in the Innate Immunity of Neonates

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Biochemistry of Salivary Nucleotide Metabolites and Their Role in the Innate Immunity of Neonates Biochemistry of Salivary Nucleotide Metabolites and their Role in the Innate Immunity of Neonates Saad Saeed Abdulwahab Al-Shehri (MSc, Clinical Biochemistry, Griffith University) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2013 The School of Pharmacy ABSTRACT Saliva contains a variety of biochemical components, some of which may be used for the diagnosis of metabolic disorders, as cancer and heart disease markers, and the drug monitoring purposes. Saliva collection is particularly attractive as a non-invasive sampling method for infants and elderly patients. However, the use of saliva to evaluate metabolic status has not been widely studied; in particular, nucleotide metabolites in saliva have received little attention. Nucleotides are divided mainly into purines and pyrimidines, and they play an essential role in many cellular processes involving biosynthesis, energy supply, DNA/RNA synthesis, essential coenzymes, and regulatory mechanisms. Nucleotides are synthesised in mammalian cells by de novo pathways, which are energy-consuming; alternatively they are recycled by energy-saving pathways from nucleosides and bases that act as nucleotide salvage metabolites. This project was initiated by the discovery of a wide variation in salivary concentrations of nucleosides and bases between individuals, raising questions about their biological function. A method was developed and validated for the collection of small volumes of saliva as well as the extraction of purines and pyrimidines from oral swabs. These were then analysed by HPLC with tandem mass spectrometry. A survey of salivary nucleotide metabolites in 77 adults was performed, followed by a study of 60 neonates. Concentrations of the major salivary nucleobases and nucleosides were significantly higher in neonatal compared to adult saliva. The median concentrations (µM) in neonates\adults respectively were: Uracil 7.3\<1.5, Uridine 12\0.4, Hypoxanthine 27\2.1, Xanthine 19\1.7, Adenosine 12\0.1, Inosine 11\0.2, Guanosine 6.8\0.1. These nucleotide metabolites were not simply in equilibrium with plasma, but appeared to be actively secreted into saliva. The high levels of xanthine and hypoxanthine in neonates were particularly interesting, because these are substrates for xanthine oxidase (XO), which occurs with high activity in breast milk. These observations led to a general hypothesis that the role of these nucleotide metabolites in neonatal saliva is concerned with regulation of the oral microbiota of infants. XO is involved in the final stage of degradation of purine nucleotides, and it has been proposed to have a role in the innate immunity of babies arising from its ability to generate antibacterial ‘reactive oxygen species’. The hypothesis proposed that high concentrations of nucleotide metabolites in neonatal saliva, together with stimulation of antibacterial breast milk XO activity, may play significant roles in early innate immunity in neonates. As part of the project, it was decided to also screen saliva from a selection of domestic mammals, which were found to have large differences in metabolite concentrations. ii Investigation of the generation of hydrogen peroxide (H2O2) by breast milk XO was conducted in three stages: (1) fresh breast milk samples were collected and the endogenous H2O2 concentration was determined to be 27.3±12.2 µM; (2) XO activity was then assayed in the same samples using a horseradish peroxidase-Ampliflu™ Red based reaction; the activity of breast milk XO was found to be 8.0±5.3 Units/L; (3) The ability of breast milk to generate H2O2 (40 µM) when mixed with neonatal saliva was successfully demonstrated. XO was confirmed to be inactive in milk formula and pasteurized bovine milk and human breast milk. Investigation of the effect of exogenous H2O2 on the in vitro growth of 4 bacterial species was undertaken. The peroxide mechanism had a strong inhibitory effect on Staphylococcus aureus at low, physiologically relevant concentrations (>25 µM H2O2). Lactobacillus plantarum and Escherichia coli required significantly higher concentrations of peroxide (~200 µM) to inhibit growth, while the effect of peroxide on the growth of Salmonella species was intermediate. As a proof-of-principle experiment, the effects of a saliva and breast milk mixture on bacterial growth in vitro were then tested. The reaction between salivary xanthine/hypoxanthine and breast milk XO was shown to be bactericidal against S. aureus and Salmonella spp., (p=0.001, p=0.007 respectively) whereas the effects on L. plantarum and E. coli were not statistically significant. A dose-dependent bactericidal effect of hypoxanthine/xanthine with milk on the 4 bacterial species was also demonstrated. Finally, to test whether breast-feeding (i.e. active XO mechanism), compared with formula-feeding (inactive XO mechanism), produces an in vivo effect on the oral microbiota, a survey of oral microbiota in infants was undertaken. Oral swabs were collected from both groups and the 16S rRNA bacterial gene was ‘deep sequenced’ using Roche 454 platform. Bioinformatic analysis software was used to determine bacterial phyla and families (and is continuing to species level). Significant variation between the oral microbiota of breast-fed and formula-fed infants was found. Although other components of breast milk such as antibodies are presumed to have an effect on the infant’s microbiota, it was considered essential to show that an effect existed, and that it could be measured in the mouth – this has not previously been demonstrated. Teasing apart the ‘innate’ (peroxidative mechanism) immune system from the specific (antibody mechanism) system is a task for future studies. iii DECLARATION BY AUTHOR This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iv PUBLICATIONS DURING CANDIDATURE Journal publications: Al-Shehri, S., M. Henman, B.G. Charles, D. Cowley, P.N. Shaw, H. Liley, A. Tomarchio, C. Punyadeera, and J.A. Duley, Collection and determination of nucleotide metabolites in neonatal and adult saliva by high performance liquid chromatography with tandem mass spectrometry. J Chromatogr B, 2013. 931C: p. 140-147, (see Appendix 9) In preparation: 1- Role of purines and pyrimidines and xanthine oxidase in regulating oral microbiota 2- Oral Bacterial 16S rRNA deep sequencing in breast-fed versus formula-fed infants Published abstracts and posters: Al-Shehri S., Shaw PN, Cowley D, Liley H, Henman M, Tomarchio A, Charles B, Duley JA. Nucleotide precursors in neonatal and adult saliva. J. Inherit. Metab. Dis. 2011, 34:S73 (Poster was presented at SSIEM, 2011, Geneva), (see Appendix 10 & 11) Al-Shehri S., Henman M, Cowley D, Charles B , Shaw PN, Liley H, Duley JA. Longitudinal study of nucleotide metabolites in infants’ saliva compared to adults and mammals. (Poster at ComBio, 2012, Adelaide), (see Appendix 12 & 13) Al-Shehri , SS., Liley, H., Knox, CL., Henman, M., Cowley, DM., Charles, B G., and Duley, JA. Human milk xanthine oxidase and neonatal salivary nucleotide precursors generate hydrogen peroxide; a novel pathway with potential role in regulating oral microflora. Journal of Paediatrics and Child Health. 2013, 49 (S2) p. 121. ISSN 1034-4810 ERA C, IF 1.281. (Poster was presented at PSANZ Annual Congress, 2013, Adelaide), (see Appendix 14 & 15) Seminar oral presentations: Al-Shehri S & Duley J. Baby Saliva and Breast Milk. Dept. of Pathology, Mater Hospital, Brisbane 25/11/2011 Al-Shehri S & Duley J. Baby Saliva and Breast Milk – What’s in the Mix? PSANZ meeting, Brisbane 15/12/2011 Al-Shehri S. Salivary nucleotide metabolites: their role in the innate immunity of babies, UQ 3MT competition, 2012 v PUBLICATIONS INCLUDED IN THIS THESIS No publications included vi CONTRIBUTIONS BY OTHERS TO THE THESIS My principal advisor, Dr John Duley, as well as my associate supervisors; A/Prof. Bruce Charles, Prof. Nick Shaw, and Dr. David Cowley contributed to this thesis by providing input on experimental design, data analysis and critically revising and editing this document. Dr Christine Knox introduced me to the field of microbiology and 16S rRNA sequencing, arranged
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