HPLC ANALYSIS OF NUCLEOTIDES Vicrant Narayan Bachelor of Applied Science Major in Microbiology Supervisors: Associate Professor Christine Knox; Dr Mark Wellard; Dr John Duley; Dr Amitha Hewavitharana; Dr Saad Al Shehri. Submitted in fulfilment of the requirements for the degree of Master of Applied Science (Research) Institute of Health and Biomedical Innovation (IHBI) Faculty of Health Queensland University of Technology 2017 hplc analysis of nucleotides i Keywords HILIC Saliva HPLC-UV Nucleotides ii hplc analysis of nucleotides Abstract Nucleotides play central roles in cellular metabolism, for synthesis of nucleic acids (DNA and RNA), as carriers of chemical energy, and as secondary messengers. Inherited defects in nucleotide pathways thus cause a range of metabolic diseases, presenting as anaemia, immunodeficiency, renal disease or severe neuropathologies. Nucleotides are normally found only intracellularly, so cell death causing the release of nucleotides into the extracellular space results in ‘alarm signals’ that are widely recognised in the body. The importance of nucleotides in energy metabolism also makes them the metabolic focus of sports performance studies. The structure of nucleotides comprises three main components: a heterocyclic nitrogenous purine or pyrimidine base, a sugar moiety (ribose in nucleotides and RNA, deoxyribose sugar in DNA), and one or more phosphate groups. Purine and pyrimidine nucleotides are formed in cells by either de novo synthesis pathways or by ‘salvage’ (or ‘recycling’) of bases and nucleosides. Since the role of nucleotides is so important for cellular metabolism scientists have been encouraged to develop methods for their detection, identification and quantification in biological specimens. However, the highly-charged and polar characteristics of nucleotides make them unsuitable for reversed-phase HPLC. Current methods available for nucleotide analysis are ion-pair and ion-exchange HPLC, but there are problems associated with these methods, particularly as they are unsuitable for mass spectrometry (MS), which uses extremes of pH and high concentrations of salts or ion-pairing reagents. Hydrophilic-interaction-HPLC (HILIC) is a relatively unexplored method, theoretically capable of separating highly polar molecules such as nucleotides. Nucleotides have been reported to be present at very low concentrations in adult saliva. The possibility of extracellular nucleotides occurring in a secretory fluid presented a biological ‘anomaly’ that was worthwhile investigating. Nucleosides, e.g. adenosine, have been shown to be present in high levels in neonatal saliva but not in adult saliva. We asked; are nucleotides present in neonatal saliva? hplc analysis of nucleotides iii Therefore the aim of this research project was to develop and validate a MS compatible HILIC method for the analysis of these very polar molecules, i.e. nucleotides, and to test the method with biological specimens, e.g. blood cells and saliva. The project aimed initially to separate adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP), but this was later expanded to encompass twelve different nucleotides. Five different HILIC columns were assessed; the best performing was a Cosmosil column. A series of eluting buffers (ammonium acetate, ammonium carbonate and ammonium citrate) were tested. A simple, MS-compatible method based on elution by ammonium citrate and methanol was then developed. This method separated twelve nucleotides within 36 minutes. Futhermore, the method was applied to red blood cell extracts (RBCX), white blood cell extracts (WBCX), adult saliva and neonatal saliva. The method was not specifically optimised for these specimens, but nucleotides were separated and identified from these extracts. This method also detected and identified the unusual metabolite 4-methylpyridonecarboxamide triphosphate (‘4PY-nucleotide’). Previous research has shown that neonatal saliva contains elevated concentrations of nucleosides and bases, but nucleotides have not yet been investigated. This study, for the first time, detected highly elevated concentrations of nucleotides in neonatal saliva, when compared to the concentrations present in adult saliva For this project Dr Amitha Hewavitharana provided a novel concept; she proposed that “competing ion” within the mobile phase could be utilised to elute nucleotides. Citrate buffer proved to be the most effective at eluting all of the nucleotides, due to the strong 3-negative charge. The other elution buffers tested, acetate (1-negative ion) and carbonate (2-negative ion), were able to elute the mono- and di-phosphates respectively. The lower limits of detection and lower limits of quantification were sufficient to detect the presence of each nucleotide in biological specimens. The HILIC method, optimised for this Masters project, separated successfully twelve nucleotides in a standard and identified endogenous nucleotides in biological specimens (RBCX, WBCX, adult and neonatal saliva). iv hplc analysis of nucleotides The results indicated that the differences in nucleotide concentrations between adult and neonatal saliva are worthy of further study, as this phenomenon may have evolved as a selective mechanism to shape the oral and gut microbiota. This novel HILIC method overcomes limitations of previous nucleotide analyses. The citrate ion is theoretically MS-compatible, providing the basis for future studies of MS-based HPLC nucleotide analysis. hplc analysis of nucleotides v Table of Contents Keywords ................................................................................................................................. ii Abstract ................................................................................................................................... iii Table of Contents .................................................................................................................... vi List of Figures ....................................................................................................................... viii List of Tables ............................................................................................................................x List of Abbreviations .............................................................................................................. xi Statement of Original Authorship ......................................................................................... xiii Acknowledgements ............................................................................................................... xiv Chapter 1: Introduction ...................................................................................... 1 1.1 Nucleotides .....................................................................................................................2 1.2 Nucleotide Cellular Functions ........................................................................................3 1.3 Dietary and Saliva Nucleotides ......................................................................................5 1.4 Intracellular and Extracellular Nucleotides ....................................................................6 1.5 Gastrointestinal Tract Microbiota and Human Health ....................................................7 1.6 Neonatal gastrointestinal tract and oral microbiota in response to delivery method and feeding ............................................................................................................................9 1.7 The Role of Saliva Nucleosides and Bases on Regulating Microbiota ........................10 1.8 Nucleotide Analysis by High Performance Liquid Chromatography (HPLC) .............11 1.8.1 Reversed-phase (RP) HPLC ...............................................................................15 1.8.2 Normal-phase HPLC ..........................................................................................15 1.8.3 Ion-exchange HPLC ...........................................................................................16 1.8.4 Ion-pairing HPLC ...............................................................................................17 1.8.5 HILIC HPLC ......................................................................................................17 1.9 Hypothesis and Aims ....................................................................................................19 1.9.1 Hypothesis ..........................................................................................................19 1.9.2 Aims ...................................................................................................................19 1.9.3 Objectives ...........................................................................................................20 Chapter 2: Materials and Methods .................................................................. 21 2.1 Ethics ............................................................................................................................22 2.2 Materials .......................................................................................................................22 2.2.1 Chemicals ...........................................................................................................22 2.2.2 Columns..............................................................................................................23 2.2.3 Nucleotides .........................................................................................................23