Development and Validation of a Human Blood Acyl-Specific Lipidomic Profiling Method for Clinical Applications

Blood fatty acid levels have been used to determine dietary intakes of omega-3 highly unsaturated fatty acids, but also to examine the link between blood fatty acids, health and disease. However, these lipid analyses are gas chromatography-based and are dependent on derivatization of lipids to fatty acid methyl esters and do not provide information about the fatty acyl parent lipid species. The use of ultra high-performance liquid chromatography combined with tandem mass spectrometry (UHPLC-MS/MS) has the potential to do so. This thesis adapted a lipidomic approach to traditional blood fatty acid profiling to characterize lipids in their naturally-occurring state in human blood. Semi-quantitative methods using UHPLC-MS/MS were developed using a high-resolution Quadrupole-Orbitrap Hybrid Mass Spectrometer (Thermo Q-Exactive), and were validated for the determination of lipids in whole blood. Molecular species were confirmed for over 500 lipids, including phospholipids, sphingolipids, triacylglycerols, cholesteryl esters, free cholesterol and free fatty acids. Over 150 acyl-specific species were confirmed from all lipid classes. Following this, the method was used to validate dried blood spots for lipidomic profiling as this sampling technique offers several advantages, including minimally invasive sample collection, stability, and facilitated handling. Additionally, the UHPLC-MS/MS method was used to examine whole blood lipid remodeling that follows fish oil supplementation. Specifically, the blood lipidomic profiles of samples from a previous intervention trial comprising 20 participants that received eicosapentaenoic acid + docosahexaenoic acid (EPA+DHA) at 150 mg/d, 500 mg/d, and 1000 mg/d over a twelve-week period were determined. Notably, lipidomic profiling of whole blood suggests that three specific lipids (16:0/DHA phosphatidylcholine, 16:0/EPA phosphatidylcholine, and plasmenyl-phosphatidylethanolamine P-16:0/EPA) increase in a dose-dependent manner with increasing doses of EPA+DHA supplementation. Finally, lipidomic analyses of whole blood, plasma, and erythrocytes of individuals that consumed low, intermediate, and high amounts of EPA+DHA were performed. These results support previous observations on the rapid responsiveness of 16:0/DHA phosphatidylcholine and 16:0/EPA phosphatidylcholine to varying levels of EPA+DHA intake. The mechanisms through which lipid remodeling occurs remain to be elucidated. The work presented in this thesis provides the foundation for the use of acyl-specific complex lipids as biomarkers for omega-3 highly unsaturated fatty acid status and the lipidomic profiling method developed may be extrapolated for the characterization of acyl-specific lipids in other biological matrices

Fatty Acyl-Specific Macrolipidomics and Microlipidomics for Nutritional Research

The field of lipidomics can further our understanding of the biochemical processes of human health and disease. Generally, lipidomics methods utilize high-performance liquid chromatography (HPLC) for lipid separation, followed by detection using tandem mass spectrometry (MS/MS). The joint use of HPLC and MS/MS has increased dramatically over the past few years, and novel technologies continue to increase the versatility and practical usability of various lipidomics methods. However, a lack of harmonized language, nomenclature and standardized analytical strategies can result in lipid misannotations, improper analyte identifications, and incorrect quantitative results. In this thesis, the importance of adopting appropriate analytical strategies to answer research hypothesis(es) will be highlighted. Specifically, this entails a comparison between analytical platforms and four HPLC-MS/MS data acquisition strategies for untargeted/global lipidomic profiling of highly-abundant lipids including phospholipids, triacylglycerols and cholesteryl esters in human whole blood. In addition, the advantages of targeted analytical approaches for the measurement of specific lipid classes will be examined through the development of a tailored method for the determination of regioisomers of lysophosphatidic acid in plasma (mouse), the acyl species of triacylglycerols in cooking oil (sunflower), and the acyl species of phospholipids in brain tissue (mouse). Finally, comprehensive profiling of various lipid classes in whole blood using a novel retention time-based negative/positive ion mode switching method will be used to screen for potential blood biomarkers of omega-3 polyunsaturated fatty acid intake. This will include samples from a cross-sectional dietary assessment study in humans, and an acute/chronic docosahexaenoic acid supplementation study in rats. The methods presented in this thesis have the potential to be expanded for use in agriculture, nutrition, research and clinical settings

Concentrations of docosahexaenoic acid are reduced in maternal liver, adipose, and heart in rats fed high-fat diets without docosahexaenoic acid throughout pregnancy

The final publication is available at Elsevier via https://dx.doi.org/10.1016/j.plefa.2018.10.003 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/Fetal accretion for DHA is high during late pregnancy due to the brain growth spurt. Prior evidence suggests that DHA is mobilized from maternal liver and adipose to meet fetal accretion and physiological requirements. However, changes in the DHA levels of various maternal tissues throughout pregnancy and into lactation of mothers on diets with and without dietary DHA, and with a background dietary fatty acid profile that resembles human intake has not been examined. Sprague Dawley rats were fed a total western diet with (TWD + ) or without DHA (TWD-) along with a commercial rodent chow control (Chow) throughout pregnancy and postpartum. The fatty acid compositions of adipose, brain, heart, liver, erythrocytes, and plasma were determined before pregnancy, at 15 and 20 days of pregnancy, and 7 days postpartum. The placenta, fetuses, and pups were also examined when available. Maternal DHA concentrations were increased in plasma at 20 days pregnancy in all the diets with TWD + > Chow > TWD-. Maternal DHA concentrations in the TWD- group were lower in adipose throughout pregnancy as compared with the other diets. At postpartum, DHA concentrations decreased below baseline levels in the heart of the TWD- and Chow dams and the liver of the TWD- dams. Whole body DHA concentrations of the fetuses did not differ but there was evidence of decreased DHA in the whole body and tissues of the TWD- and Chow 7d old pups. In conclusion, it appears that in this rodent model of pregnancy, maternal adaptations were made to meet fetal DHA requirements, but they may compromise maternal DHA status and the ability to deliver DHA during lactation.Natural Sciences and Engineering Research Council of Canada [327149-2013]Canada Research Chairs program as a Chair in Nutritional Lipidomics [950-228125

Assessment of buccal mucosa genotoxicity in insecticide-exposed urban fumigators in Cali, Colombia

Objectives This study aimed to evaluate cytogenetic damage in the buccal mucosa of non-exposed subjects (N = 33) and insecticide-exposed fumigators (N = 31) in the urban area of Cali, Colombia. Material and Methods Through a questionnaire sociodemographic data, anthropometric measurements, state of health, and lifestyle were collected. Buccal micronucleus cytome (BMCyt) assay was using for evaluate cytogenetic damage. Results The study showed that all fumigators used adequate personal protective equipment (PPE) and had low alcohol consumption. The authors did not find significant differences in BMCyt biomarkers between the groups (p > 0.05). Multivariate analysis showed a 13% increase in micronucleus (MN) frequency for every year of increasing age (OR = 1.13, p = 0.029), and higher MN with the decrease in daily fruit consumption (OR = 4.71, p = 0.084), without statistical significance. Conclusions The results between groups could be related to healthy habits and PPE use among the subjects. Int J Occup Med Environ Health. 2024;37(1):128–3

Implementation of human biomonitoring in the Dehcho region of the Northwest Territories, Canada (2016–2017)

Abstract Background Human biomonitoring represents an important tool for health risk assessment, supporting the characterization of contaminant exposure and nutrient status. In communities where country foods (locally harvested foods: land animals, fish, birds, plants) are integrated in the daily diet, as is the case in remote northern regions where food security is a challenge, such foods can potentially be a significant route of contaminant exposure. To assess this issue, a biomonitoring project was implemented among Dene/Métis communities of the Dehcho region of the Northwest Territories, Canada. Methods Participants completed dietary surveys (i.e., a food frequency questionnaire and 24-h recall) to estimate food consumption patterns as well as a Health Messages Survey to evaluate the awareness and perception of contaminants and consumption notices. Biological sampling of hair, urine and blood was conducted. Toxic metals (e.g., mercury, lead, cadmium), essential metals (e.g., copper, nickel, zinc), fatty acids, and persistent organic pollutants (POPs) were measured in samples. Results The levels of contaminants in blood, hair and urine for the majority of participants were below the available guidance values for mercury, cadmium, lead and uranium. However, from the 279 participants, approximately 2% were invited to provide follow up samples, mainly for elevated mercury level. Also, at the population level, blood lead (GM: 11 μg/L) and blood cadmium (GM: 0.53 μg/L) were slightly above the Canadian Health Measures Survey data. Therefore, although country foods occasionally contain elevated levels of particular contaminants, human exposures to these metals remained similar to those seen in the Canadian general population. In addition, dietary data showed the importance and diversity of country foods across participating communities, with the consumption of an average of 5.1% of total calories from wild-harvested country foods. Conclusion This project completed in the Mackenzie Valley of the Northwest Territories fills a data gap across other biomonitoring studies in Canada as it integrates community results, will support stakeholders in the development of public health strategies, and will inform environmental health issue prioritization