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Analytical Method Development for Nucleotides in Candida Albicans and Its Application to Metabolomics Research

Author: WangTianZuo
Tutor: LouZiYang
School: Second Military Medical University
Course: Pharmaceutical Analysis
Keywords: Candida albicans nucleotides metabolomics caspofungin sterols LC-MS GC-MS
CLC: R917
Type: Master's thesis
Year: 2013
Downloads: 18
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Abstract

The nucleotide compounds are important metabolites components, of which thenucleotide is not only the precursor of DNA and RNA, but also playing an important role inthe metabolism, such as storing and transitting of metabolism energy and cell signal, andparticipating in the coenzyme factor reaction. Sugar nucleotide, on the other hand, is a sugargroup donor in vivo glycosylation process, and the substrate of P (1，3)-D-glucan synthesisprocess, the most common uridine diphosphate-Glucose (UDP-GLC) in these compounds. Invivo, they are widespread. And accurate determination of the nucleotide and the sugarnucleotide in the biological sample is content for understanding the energy metabolism of thecell level, also for the understanding of the cell cycle regulation and the mechanism of drugaction.The nucleotide and sugar nucleotide is a close neighbor in the metabolic pathway, theirphysical and chemical properties is similarly, so you can usually parallel analysis. As thesubstances of polar, using conventional reversed phase chromatography can not perfectseparated. At present, the main methods of determination of nucleotides substances indomestic and foreign, including the ion exchange liquid chromatography, ion chromatography,hydrophilic chromatography and capillary electrophoresis. But for some of the ingredients ofthe separation are less than ideal, can not meet the requirements of the experiment. Furtheroptimize the conditions of the experiment on the basis of previous experiments, to establishand optimize HPLC-ESI/MS method to quantitatively analyzed of the nucleotides andnucleotide sugars [triphosphate (CTP)，three uridine phosphate (UTP)，diphosphate uridine(UDP)， guanosine of triphosphate (GTP)， deoxyadenosine triphosphate (dATP)，monophosphate adenosine (AMP), uridine diphosphate-glucose (UDP-GLC)]. Thechromatographic separation is based on the porous graphite carbon column0.1%formic acidwith aqueous ammonia to adjust the pH to8.5and acetonitrile gradient elution. Negativemode electrospray ionization mass spectrometry applied to improve the sensitivity of themethod. Quantitative methods by validated and successfully applied to the determination bythe changes in the nucleotide and nucleotide sugar after administration of caspofungin and5-fluorocytosine in Candida albicans.Candida albicans is a common opportunistic pathogen in invasive fungal infections in vitro. A high degree of resistance was found when it produce extracellular matrix to theformation of the bioiflm. In this study, by means of four drugs of different mechanismincluding caspofungin (CAS)，5-lfuorocytosine (5-FC)，baicalein (BE) and the sodiumhouttuyfonate [Sodium Houttuyfonate (SH)] to study on the mechanism of resistance andanti-fungal effect of the drug, mechanisms, and to explore the toxicity. Recently, researches offungi mainly on the level of genomics and proteomics, the researches on metabolomics isrelatively small, since the metabolites in cell are the ifnal products of regulation process, Thechange of the type and quantity is regarded as the ultimate response of biological systems togenetic or environmental changes, compared with genomics，proteomics, metabolomics hascertain advantages, it is worth further study.The subject using LC-MS，GC-MS analysis of high-throughput instrumentation andmathematical analysis combined metabolomics technology, Candida albicans bioiflmresistance mechanisms and antifungal metabolomics studies. Caspofungin as an echinocandinantifungal drug, administration of which to the bioiflm and planktonic Candida albicans,looking for the metabolic characteristics spectrum differences in Candida albicans combinedwith metabolic pathway database and its associated drug mechanism, to ifnd of drugmechanisms related metabolites and metabolic networks. Designed to open up new ideas, newantifungal contribution to research complex pathogenic mechanisms of Candida albicans andresistance mechanisms.Ergosterol is an important component of the membrane of fungal cell，and it is involvedin a variety of biological functions in cell，including the integrity of the cell，membranefluidity and the role of the enzyme bonded on the cell membrane. Squalene and lanosterol arethe main precursors of ergosterol in biosynthesis pathway. Ergosterol and its synthesispathway are the targets of clinical first-line drugs, such as polyene drugs, includingamphotericin and its derivatives, nystatin. Therefore，it is necessary to measure the sterols tostudy the mechanism of antifungal.In order to study the impact of different drugs on sterol content of Candida albicans, weestablish an accurate and sensitive GC-SIM-MS method to determine the lanosterol andergosterol. The sterols are extracted from Candida albicans by centrifugation, saponiifcation,extracted sterol portion was dissolved in cyclohexane，analyzed using a HP-5(30m x0.25mmx0.25(am) capillary column by gas chromatographic analysis coupled with single quadrupolemass under selected ion monitoring (SIM) for lanosterol, ergosterol and cholesterol (internal standard), retention time and fragmentation information are using for qualitative andquantitative analysis. The volatility of sterols are better and suitable for GC analysis,eliminating complex derivative process. This method combines the high resolution of GC andthe high sensitivity of the mass spectrometry, so the measured components and theendogenous components can be better separated, avoiding interference, and improving theaccuracy of the measured result. This method is accurate and effective, repeatable, highrecovery. It provides a powerful tool for the study of lanosterol and ergosterol in Candidaalbicans.

Analytical Method Development for Nucleotides in Candida Albicans and Its Application to Metabolomics Research

Abstract

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