Molecular diagnostics for public and environmental health

Abstract

Both human and environmental health are born out of extraordinarily complex systems and no thesis could ever satisfactorily explore both, instead the focus of this document lies in both trying to get the most out of existing analytical techniques and how to apply these techniques to study new challenges. Currently, information about public health is obtained by looking at the health of a few individuals and then building this up to an understanding of the whole population such as public health England’s health survey for England or the European Monitoring Centre for Drugs and Drug Abuse’s (EMCDDA) country drug report. The flaws in this technique are that it relies on obtaining a representative sample of individuals in order to understand the community as a whole. Wastewater based epidemiology (WBE) is a methodology that examines influent wastewater to explore what is being consumed by the population and uses this to build up a picture of public health, relying on the assumption that wastewater is equivalent to a pooled urine sample for a population. Likewise, analysis of river water can be used to gain an understanding of environmental health by measuring the concentration of anthropogenic compounds in the environment and assessing their environmental risk. However, current assessments of human and environmental health do not allow for the collection of all available data, as collected analytical measurements are either incomplete or are lacking in context. This thesis seeks to address these limitations by exploring new analytical methodologies and techniques for collecting more comprehensive data and was focused into three main areas of research: 1) the uses of chirality in determining analyte origin and its impact on assessments of public and environmental health, 2) the development of new methodologies to maximise the information obtainable from current health biomarkers and 3) the exploration of proteins as potential new biomarkers of public health.

The importance of assessing chirality was first demonstrated in chapter one, where chiral chromatography was used to measure changes in the concentration of several drugs of abuse and human pharmaceuticals in wastewater over a period of five years. By monitoring changes in the enantiomeric fraction (EF) of chiral analytes it was possible to identify that these biomarkers originated from human consumption due to stereospecific excretion. Additionally, the EF of methamphetamine changed throughout the study, which appeared to indicate a change in how and where it was manufactured. However, the main focus of chapter one was in how WBE can be used to assess public health strategies, including addressing a need for assessing the impact of drug policy on changes in drug consumption. The novel use of local pharmaceutical prescription data alongside wastewater analysis allowed for a more thorough assessment of consumption within the population, including the observation that prescription and wastewater data showed good agreement for painkillers that are at risk of being abused, suggesting that there was no significant illicit usage in contrast to expectations from ongoing opioid epidemics in other countries. Additionally, by comparing trends in UK drug consumption with trends in European drug consumption it was possible to identify UK specific trends, such as a rapid increase in cocaine consumption, and well as those that were European wide. Alongside this, the observation that mephedrone, a drug first banned in 2010, was no longer consumed after 2015 showed that WBE can be used to assess the efficacy of drug policy. Likewise, the identification of an increasing trend in UK drug consumption from 2016 onwards were postulated to be related to significant changes in UK drug policy that occurred in 2016. Overall, a combination of chiral analysis and local prescription data were used to verify the source of analytes in wastewater, whilst comparison with long term trends in prescription rates and European wide trends lead to the identification of a trend of increasing illicit drug use in the UK, as well as a strong correlation between prescription and wastewater concentrations of painkillers that were at risk of being abused.

In a similar vein, the importance of chiral analysis for the assessment of environmental health was explored in chapter two, where river simulating microcosms and ecotoxicity tests were used to assess the environmental risk posed by ephedrine. From microcosm testing two isomers of ephedrine were observed to decay significantly more, under environmental conditions, than the remaining isomers. Following this, the compounds with the greatest environmental persistence were also observed to be, generally, the most toxic to a range of important river organisms. River simulating microcosms also revealed that one of the less persistent and less toxic isomers of ephedrine could be converted under environmental conditions to a more persistent and toxic isomer. This observation was confirmed by a combination of retrospective data analysis and human liver microcosm experimentation to rule out other potential metabolic sources of the toxic isomer. What this showed was that it was important for environmental studies to consider individual isomers as discrete compounds when considering their environmental impact. Additionally, studies should also consider how human pharmaceuticals enter the environment, the initial composition of any isomers when they enter the environment, and how they are changed, or not, after excretion or by wastewater treatment. Chapter three showcased the utility of supercritical fluid chromatography (SFC) for developing new chiral methodologies for use in wastewater and river water for monitoring compounds of environmental concern (CECs). Initial method development was able to qualitatively analyse 140 CECs, with the final method being able to fully or semi-quantify 95 CECs and detect 75% of them in grab samples of influent wastewater, effluent wastewater or river water. Initial analysis of wastewater and river water showed that the
enantiomeric fractions of several analytes changed depending on the matrix they were detected in, which highlighted the importance of assessing chirality in not just environmental samples but also in the sources of CECs. The power of SFC lies in its analytical efficiency compared to contemporary chiral liquid chromatography. By developing new chiral methods that can rapidly analyse CECs it becomes increasingly easier for routine environmental and public health monitoring to measure chirality without sacrificing analytical power or sample throughput.

The need to explore new biomarkers was brought about by the realisation that most current WBE biomarkers are used to measure consumption, rather than directly measuring public health, although biomarkers of oxidative stress have begun to be explored. Chapter four lays out the current landscape of WBE analysis and showcases the range of biomarkers currently used, as well as an estimation of the population they’ve been used to study, which for drugs of abuse represented roughly 1.5 % of the total human population. The use of urinary proteomics for assessing individual health in clinical analysis was discussed and compared with how drugs of abuse were used 20 years previously before the development of WBE. The case was then made for using urinary disease proteins to assess public health via their analysis in wastewater and several potential urinary biomarkers were proposed based on a set of criteria outlined in the chapter, including: urinary excretion, a known disease-biomarker relationship and disease specificity. Chapter five then took the first tentative steps towards developing a method for the analysis of human protein biomarkers in wastewater, by developing an enzymatic digestion method for five potential biomarkers in buffer, which was then adapted to work in wastewater for the most promising two proteins. The chapter also built upon the biomarker selection criteria discussed in the previous chapter to include the selection of human specific peptides, the ability to include peptides shared with other primates as pseudo-human specific and efforts to estimate the wastewater concentration of proteins from their excreted urinary concentrations in healthy adults. Initial analysis indicated the possible detection of one peptide of the inflammation biomarker C-reactive protein (CRP) in 100 mL wastewater digests using hydrophilic interaction liquid chromatography coupled with triple quadrupole mass spectrometry. This showed that protein biomarkers of disease could be detected in wastewater using largely the same techniques as current WBE analysis, which would enable its uptake by the larger WBE community as well as facilitating direct measurements of public health.

Date of Award	24 Jun 2020
Original language	English
Awarding Institution	University of Bath
Supervisor	Barbara Kasprzyk-Hordern (Supervisor) & Anneke Lubben (Supervisor)