Periodic Reporting for period 1 - FaecMet (Broadening the coverage of metabolites in faeces through advanced analytical strategies)
Okres sprawozdawczy: 2023-02-01 do 2024-02-29
Anorexia Nervosa (AN) is an eating disorder that presents a high mortality rate. The vast majority of individuals affected by this disorder are women. AN etiopathogenesis, though not fully understood, involves genetics and environmental factors. Despite the active research on AN, its syndrome and evolution/prognosis are not well-defined, varying greatly between individuals. Neurological implications associated with mental health in AN have been linked to an altered gut in these individuals. Indeed, the gut microbiota produces a variety of active biomolecules capable of causing neurological effects, and thus, the emerging idea that the intestinal microbiota could contribute to the development of AN. The understanding of the influence of gut-brain axis, i.e. gut health state on these individuals might shed light on individualized treatments and supplementation to be added to the multidisciplinary treatment strategies currently being employed for AN. In this context, this present MSCA-PF titled “Broadening the coverage of metabolites in faeces through advanced analytical strategies” aimed to develop new analytical strategies to add to the toolbox of strategies employed towards assessing gut-related metabolites to assess in understanding the gut health of individuals suffering from AN.
To this end, within FaecMet a facile, green (no solvent employed in the sample preparation) protocol was developed for profiling a broad range of volatile organic compounds (VOCs) in faeces, including determination of SCFAs. Through multivariate design of experiments the HS-SPME method was carefully optimized with the aim to detected a broad range of VOCs, including highly volatile compounds associated with gut-bacteria metabolism, often not detected by other sample preparation methods, while successfully being able to simultaneously determine short-chain fatty acids (SCFAs) in the same method. The optimized and validated protocol was applied to faecal samples from AN patients and controls, and at least 209 VOCs comprising various chemical functionalities could be annotated in faecal samples.
To broaden the scope of gut-related metabolites investigated on AN patients, we also employed a method for the determination of 25 bile acids (BAs) in faeces.
Moreover, it is noteworthy the importance of the gut-liver on SCFAs metabolism axis, since the liver serves as a major site for the metabolic processing of gut-derived SCFAs. Despite that, the number of studies conducted on this matrix is limited. Therefore, an ultrasensitive method for quantitation of trace-levels of SCFAs in liver tissue (mice) was developed and validated.
Information gathered by these tools developed within this MSCA can help to get an overview of gut-related metabolites in AN individuals which could lead to a deeper understanding on the role of gut-related metabolites and dysbiosis on AN patients, prompting studies towards individualized interventions and treatments aiming to investigate whether correction of gut dysbiosis may have therapeutic effects in AN.
To this end, within FaecMet a facile, green (no solvent employed in the sample preparation) protocol was developed for profiling a broad range of volatile organic compounds (VOCs) in faeces, including determination of SCFAs. Through multivariate design of experiments the HS-SPME method was carefully optimized with the aim to detected a broad range of VOCs, including highly volatile compounds associated with gut-bacteria metabolism, often not detected by other sample preparation methods, while successfully being able to simultaneously determine short-chain fatty acids (SCFAs) in the same method. The optimized and validated protocol was applied to faecal samples from AN patients and controls, and at least 209 VOCs comprising various chemical functionalities could be annotated in faecal samples.
To broaden the scope of gut-related metabolites investigated on AN patients, we also employed a method for the determination of 25 bile acids (BAs) in faeces.
Moreover, it is noteworthy the importance of the gut-liver on SCFAs metabolism axis, since the liver serves as a major site for the metabolic processing of gut-derived SCFAs. Despite that, the number of studies conducted on this matrix is limited. Therefore, an ultrasensitive method for quantitation of trace-levels of SCFAs in liver tissue (mice) was developed and validated.
Information gathered by these tools developed within this MSCA can help to get an overview of gut-related metabolites in AN individuals which could lead to a deeper understanding on the role of gut-related metabolites and dysbiosis on AN patients, prompting studies towards individualized interventions and treatments aiming to investigate whether correction of gut dysbiosis may have therapeutic effects in AN.
We initiated FaecMet by developing a standardized HS-SPME-GC/MS protocol for analysis of fecal VOCs. Factors such as faecal sample mass, storage conditions, vial volume, sample modifications and SPME coating type were assessed in an OFAT manner to ensure broad VOC coverage while rendering precise and accurate acquisition of data. Next, a DOE approach was used to perform the screening of important variables, followed by a RSM for optimization of HS-SPME factors. Once the method was optimized, stability of prepared sample vials on autosampler tray was assessed, as well as precision with QC samples prepared by three different analysts. Study samples and QC samples were employed to construct an initial VOC library (GC/MS) employing this method. The protocol optimized herein, completely organic solvent-free, was capable of detecting at least 306 VOCs pertaining to a variety of chemical classes, and after data filtering, 209 VOCs were annotated.
It is important to mention that while the method herein developed is quite proficient in the profiling of a broad variety of VOCs in faecal samples, the concomitant quantitation of SCFAs has been a major goal. For this, a comprehensive investigation of quantitation strategies for SCFAs was conducted and the pros and cons of each quantitation strategy discussed. For example, in metabolomics applied to clinical studies employing tens to hundreds of samples, standard addition strategy, though extremely accurate, is not feasible, as one calibration must be carried out per sample. Similarly, surrogate matrix (QC) employing background subtraction, a strategy commonly employed for quantitation of endogenous compounds in complex matrix, may also not be appropriate since the high endogenous levels of SCFAs in faecal samples will yield high limits of quantitation. Therefore, we present an alternative strategy, employing QC samples as surrogate matrix and isotopically-labelled SCFAs as surrogate analytes, which rendered accurate results (% error varying from 14 to 2% as compared to standard addition method). The optimized and validated protocol was applied to 57 samples, being 44 of AN patients and 13 controls. Next, we employed a LC-QQQMS method to analyze 25 BAs from the same fecal samples investigated above.
Moreover, during the course of this fellowship, CEMBIO received a request by a collaborator interested in the determination of SCFAs in liver tissue due to the importance of the gut-liver axis involvement on the SCFAs metabolic processing. For this, multivariate design of experiments were employed to optimized conditions for derivatization with isobutyl chloroformate as well as the HS-SPME conditions. Method validation was carried out employing different types of liver samples (mice, rat, chicken) to ensure the robustness of the method. In a similar fashion to VOCs profiling method, a comprehensive investigation of quantitation strategies for SCFAs was conducted and the pros and cons of each quantitation strategy discussed. The optimized and validated method was usefully applied to the analysis of mice liver, employing a study of control versus systemic lupus erythematosus (SLE) animals, as a proof of concept.
It is important to mention that while the method herein developed is quite proficient in the profiling of a broad variety of VOCs in faecal samples, the concomitant quantitation of SCFAs has been a major goal. For this, a comprehensive investigation of quantitation strategies for SCFAs was conducted and the pros and cons of each quantitation strategy discussed. For example, in metabolomics applied to clinical studies employing tens to hundreds of samples, standard addition strategy, though extremely accurate, is not feasible, as one calibration must be carried out per sample. Similarly, surrogate matrix (QC) employing background subtraction, a strategy commonly employed for quantitation of endogenous compounds in complex matrix, may also not be appropriate since the high endogenous levels of SCFAs in faecal samples will yield high limits of quantitation. Therefore, we present an alternative strategy, employing QC samples as surrogate matrix and isotopically-labelled SCFAs as surrogate analytes, which rendered accurate results (% error varying from 14 to 2% as compared to standard addition method). The optimized and validated protocol was applied to 57 samples, being 44 of AN patients and 13 controls. Next, we employed a LC-QQQMS method to analyze 25 BAs from the same fecal samples investigated above.
Moreover, during the course of this fellowship, CEMBIO received a request by a collaborator interested in the determination of SCFAs in liver tissue due to the importance of the gut-liver axis involvement on the SCFAs metabolic processing. For this, multivariate design of experiments were employed to optimized conditions for derivatization with isobutyl chloroformate as well as the HS-SPME conditions. Method validation was carried out employing different types of liver samples (mice, rat, chicken) to ensure the robustness of the method. In a similar fashion to VOCs profiling method, a comprehensive investigation of quantitation strategies for SCFAs was conducted and the pros and cons of each quantitation strategy discussed. The optimized and validated method was usefully applied to the analysis of mice liver, employing a study of control versus systemic lupus erythematosus (SLE) animals, as a proof of concept.
FaecMet has been built on a translational project, combining both clinical and basic research.
As such, scientifically, FaecMet has delivered: a novel protocol for profiling a broad range of VOCs related to gut-microbiota. All the features of this protocol developed within this project, such as being environmentally friendly (does not use organic solvents), minimizing sample amount required, working with lyophilized samples, and minimizing sample handling, and high throughput due to the automated HS-SPME-GC/MS analysis, makes it ideal for clinical applications where numerous samples need to be analyzed. In addition, the balanced VOC coverage permits the detection of quite volatile compounds (that are not easily lost by similar method, yet quite important gut-related metabolites, such as methanethiol), while being capable of detecting also larger, less volatile species like indole and its derivatives.
Even though the application selected here is AN, the methodology developed will be applicable to get deeper knowledge in the role of gut microbiota in different pathophysiological situations through faeces.
Being able to analyze such a broad variety of VOCs metabolites in such a facile manner represents a game-changer in gut-related metabolomics studies: for example, where GC-MS employing traditional derivatization is used for analysis of polar metabolites, this protocol can now add a new dimension to these studies, providing a profiling of gut-bacteria related volatile metabolites.
In addition, the high concentration of SCFAs in faeces, derivatization is not needed, therefore, the simple, green protocol presents itself quite competent in the analysis of SCFAs simultaneously to a broad variety of other gut-related VOCs. However, in case of other tissues or biofluids in which SCFAs are present in quite low concentration the ultrasensitive method for determination of SCFAs in liver herein developed may be implemented to provide a complementary view to those results obtained in faeces.
From an economic point of view, the protocols (as well as the results) achieved within FaecMet are simpler to execute method (decreasing time to train analyst) and of feasible implementation from research to clinical studies. Additionally, individuals with eating disorders generate greater annual health care costs, therefore treatment and prevention of eating disorders may have broad economic benefits in terms of heath care savings and gains in work productivity.
In terms of societal impact, FaecMet is aligned with UN Sustainable Development Goal 33: “Ensure healthy lives and promote well-being for all at all ages”, and more specifically, contributes to target 3.4: “By 2030, reduce by one third premature mortality from non-communicable diseases through prevention and treatment and promote mental health and well-being”. Furthermore, given the diminished usage of organic solvents and the reusability of the extraction devices (more environmentally-friendly method), this project also aligns with UN Sustainable Development Goal 124, more specifically target 12.5: “By 2030, substantially reduce waste generation though prevention, reduction, recycling and reuse.
As such, scientifically, FaecMet has delivered: a novel protocol for profiling a broad range of VOCs related to gut-microbiota. All the features of this protocol developed within this project, such as being environmentally friendly (does not use organic solvents), minimizing sample amount required, working with lyophilized samples, and minimizing sample handling, and high throughput due to the automated HS-SPME-GC/MS analysis, makes it ideal for clinical applications where numerous samples need to be analyzed. In addition, the balanced VOC coverage permits the detection of quite volatile compounds (that are not easily lost by similar method, yet quite important gut-related metabolites, such as methanethiol), while being capable of detecting also larger, less volatile species like indole and its derivatives.
Even though the application selected here is AN, the methodology developed will be applicable to get deeper knowledge in the role of gut microbiota in different pathophysiological situations through faeces.
Being able to analyze such a broad variety of VOCs metabolites in such a facile manner represents a game-changer in gut-related metabolomics studies: for example, where GC-MS employing traditional derivatization is used for analysis of polar metabolites, this protocol can now add a new dimension to these studies, providing a profiling of gut-bacteria related volatile metabolites.
In addition, the high concentration of SCFAs in faeces, derivatization is not needed, therefore, the simple, green protocol presents itself quite competent in the analysis of SCFAs simultaneously to a broad variety of other gut-related VOCs. However, in case of other tissues or biofluids in which SCFAs are present in quite low concentration the ultrasensitive method for determination of SCFAs in liver herein developed may be implemented to provide a complementary view to those results obtained in faeces.
From an economic point of view, the protocols (as well as the results) achieved within FaecMet are simpler to execute method (decreasing time to train analyst) and of feasible implementation from research to clinical studies. Additionally, individuals with eating disorders generate greater annual health care costs, therefore treatment and prevention of eating disorders may have broad economic benefits in terms of heath care savings and gains in work productivity.
In terms of societal impact, FaecMet is aligned with UN Sustainable Development Goal 33: “Ensure healthy lives and promote well-being for all at all ages”, and more specifically, contributes to target 3.4: “By 2030, reduce by one third premature mortality from non-communicable diseases through prevention and treatment and promote mental health and well-being”. Furthermore, given the diminished usage of organic solvents and the reusability of the extraction devices (more environmentally-friendly method), this project also aligns with UN Sustainable Development Goal 124, more specifically target 12.5: “By 2030, substantially reduce waste generation though prevention, reduction, recycling and reuse.