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An author’s perspective: George Jaskiw on quantification of phenolic acid metabolites in humans by LC–MS

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To help provide insight into the recent article published in Bioanalysis: Quantification of phenolic acid metabolites in humans by LC–MS: a structural and targeted metabolomics approach, we spoke with author George Jaskiw. He explains why he felt this was an important area of neurology and bioanalysis, worthy of publication. With over 30 years of experience in clinical psychiatry and neuroscience, Dr Jaskiw’s research has rested on bioanalytical techniques, with a particular focus on metabolomics for the past 2 years.

George Jaskiw “I completed my undergraduate, medical education and medical internship at the University of Toronto, Canada. After a year as a family physician, I entered a residency program in psychiatry affiliated with Harvard University (MA, USA). That kindled my lifelong interest in serious mental illness, particularly schizophrenia and led to a research fellowship at the Clinical Brain Disorders Branch of the National Institutes of Mental Health in Washington, DC. My goal was to explore the influence of limbic cortical regions on dopamine-mediated neurochemistry and behavior in rat models. Since 1991 I have worked in Cleveland, Ohio, USA at the Louis Stokes Cleveland Veterans Affairs Medical Center (LSCVAMC) and the School of Medicine at Case Western Reserve University. In addition to having clinical and administrative duties, I continued clinical and preclinical studies related to schizophrenia with a particular focus on the relationship between L-tyrosine availability and catecholaminergic indices. More recently, I have become interested in the potential role that gut microbiota-generated phenolic and other metabolites may have in neuropsychiatric disorders. I am currently a Professor of Psychiatry and teach at the School of Medicine, whilst continuing to care for patients with serious mental disorders, primarily schizophrenia.”

1. What inspired you to work in this field of bioanalysis?
Many laboratories, including my own, had demonstrated that schizophrenia and certain other neuropsychiatric disorders were associated with aberrant kinetics of aromatic amino acids such as L-tyrosine. However, there was no clear path to exploiting these findings for clinical purposes. It just so happened that my colleague in Infectious Disease Service at the LSCVAMC, Dr Curtis Donskey had assembled a team of investigators to identify possible biomarkers for colonization resistance to pathogenic bacteria such as C. difficile. Several of the potentially relevant chemicals were small phenolic molecules. The team included Dr Mark Obrenovich, who has an extensive background in analytic chemistry, in addition to an interest in brain disorders such as autism and Alzheimer’s Disease. Aware of my interest in aromatic amino acids, Curtis and Mark approached me about a potential collaboration. We were all aware of the burgeoning interest in gut microbiota (GMB)–brain interactions. A remarkable convergence of interests emerged. Several chemicals of interest to my colleagues were metabolites of dietary polyphenols and/or aromatic amino acids such as L-tyrosine and L-phenylalanine. C. difficile was implicated in both types of processes. While associations between various small phenolic molecules and neuropsychiatric conditions, such as schizophrenia and autism, had been intermittently appearing for over 60 years, the available body of data raised more questions than it answered. The majority of studies were conducted at a time when analytic techniques were limited and the full diversity of the human metabolome was incompletely appreciated. The early investigations did, however, point to the presence in human samples of multiple small phenolic and other molecules. For the most part, the kinetics and possible bioactivity of these molecules remained poorly characterized. Thus, the first order of business had to be development of analytic techniques that could efficiently separate, identify and quantify numerous, small and often highly similar molecules. I was propelled into this field of bioanalysis by the demands of neurobiological research.

2. What impact would you like to see/expect to see as a result of your publication?
I would hope that our paper achieves two goals, one specific and one general. The specific goal is to provide an appreciation of the richness of the small molecules, in part derived from the GMB, that access the human central nervous system. The possibility that these molecules directly affect the brain must be systematically examined. Given the diversity of chemicals and mechanisms of neuroactivity, such a project will require the participation of many multidisciplinary groups.

The general goal is to underscore the importance of methodology. While the number of publications on the GMB–brain axis has grown exponentially in recent years, there is a risk that enthusiasm for new data and speculations will compromise rigor. Without standardization of methodologies and scrupulous attention to potential confounds, we run the risk of generating a cacophony of data in which noise overwhelms possible signal. A cautionary tale is provided by the ill-fated quest for the ‘pink-spot’ in schizophrenia, some 50 years ago.

In our publication, we shared the challenges we encountered and our efforts to resolve them. For instance, 3-hydroxy-3-(3-hydroxyphenyl)propanoic acid is one small phenolic molecule of considerable interest to neuropsychiatry. As we discuss in our report, even some major databases contain incorrect information about this molecule’s nomenclature and structure. That is hardly surprising, given that the molecule has at least 12 structural isomers (MW 182.17 Da) that, in most cases, differ only in the position of a single hydroxyl group. Several of these isomers are available only through custom synthesis. The possible existence of optical isomers has not, to our knowledge, been yet examined.

The absence of authentic standards is only one of several difficulties that investigators will face as they seek to separate, identify and quantify multiple, highly structurally similar molecules in their laboratories. Given such challenges and the declining cost of commercially available metabolomic assays, it will be increasingly tempting to outsource the analytic work. Nonetheless, it will remain incumbent on investigators to partner with analytic chemists who can at the very least critically evaluate methodologies and the resultant data. Progress will depend on multidisciplinary teams that include experts in bioanalysis.

3. What are the next steps for your research and this field of bioanalysis?
We are continuing to refine our analytic techniques, adding new targets of interest as we remove those previously targeted chemicals that appear to be absent from our biological samples. At the same time, we are conducting pilot studies in bacterial cultures, in animal models and in patient populations. The proximate goal is to secure sufficient funding that would allow application of the bioanalytic techniques to important questions in neuroscience and clinical research.

4. Are there any researchers/projects/technologies that you are watching at the moment, and any you think we should be keeping an eye on?
I am entering the field of GMB-mediated small phenolic metabolites late in the day. There is a large body of foundational work from several teams of researchers led by Gary Williamson and Mike Clifford from the United Kingdom, Francisco Tomás-Barberán and Juan Carlos Espin from Spain, and Alan Crozier from the USA.

5. Do you have any advice for anyone who may be interested in working in this field?
Be part of a great team. Be scrupulous about methodology. Evaluate the resulting data with Richard Feynman’s injunction in mind: “The first principle is that you must not fool yourself, and you are the easiest person to fool.”

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Reference: Obrenovich ME, Donskey CJ, Schiefer IT, Bongiovanni R, Li L & Jaskiw GE. Quantification of phenolic acid metabolites in humans by LC–MS: a structural and targeted metabolomics approach. Bioanalysis. 10(19), 1591–1608 (2018)

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