Multisegment Injection-Nonaqueous Capillary Electrophoresis-Mass Spectrometry Based Metabolomics for Rapid Analysis of Fatty Acids for Dietary and Cardiometabolic Risk Assessment

Biomarkers play a key role in human health for early disease screening for public health, improved diagnosis of human diseases, and monitoring of treatment responses on an individual level. However, there is urgent need for high throughput technologies to accelerate biomarker discovery based on comprehensive analyses of metabolites and lipids in complex human biofluids. This thesis contributes to new advances in metabolomics research and biomarker discovery in three major areas. In specific, this thesis describes (1) the development and validation of a multiplexed separation platform based on multisegment injection-nonaqueous capillary electrophoresis-mass spectrometry (MSI-NACE-MS) for the rapid and accurate determination of fatty acids and synthetic environmental lipids in blood specimens, (2) the application of this technique in support of nutritional epidemiology for objective assessment of dietary fat intake in women that can be correlated with self-reported food frequency questionnaires, and (3) the discovery of a panel of serum biomarkers for differentiation of peripheral artery disease (PAD) in older persons at high risk for limb amputation and death. Chapter I provides an introduction in biomarkers and metabolomics, including an overview of the data workflow when performing comprehensive metabolite profiling with emphasis on methods applicable to comprehensive fatty acid analysis. Chapter II introduces a novel assay based on MSI-NACE-MS for high throughput analyses of nonesterified or total hydrolyzed fatty acids in serum/plasma extracts following a rigorous method optimization and an inter-method comparison to conventional gas chromatography (GC) to demonstrate good mutual agreement. MSI-NACE-MS enables multiplexed analyses of seven samples within a single run with stringent quality control, robust inter-batch correction, and accurate electromigration modeling for lipid identification without pre-column chemical derivatization and complicated sample workup procedures. Chapter III further expands concentration sensitivity when using MSI-NACE-MS/MS for rapid biomonitoring of low nanomolar levels of perfluoroalkyl substance (PFAS) exposures as they are a class of ubiquitous environmental pollutants with endocrine-disruption functions. Perfluorooctanoic acid and perfluorooctanesulfonic acid were quantified in a subset of serum extracts from pregnant women before and after 2009 with lower exposures measured in accordance with enforced PFAS regulation. Chapter IV demonstrates the clinical utility of circulating NEFA for accurate nutritional assessment of fat intake in women. MSI-NACE-MS was applied in observational and intervention studies to demonstrate that polyunsaturated omega-3 and saturated odd-chain NEFAs serve as promising dietary biomarkers to monitor intake of oily fish/fish oil supplement and full-fat dairy intake, respectively. Chapter V describes an untargeted characterization of the serum metabolome of nondiabetic PAD patients for differentiation of chronic limb-threatening ischemia from intermittent claudication as compared to the ankle brachial index. A panel of serum metabolites, including several amino acids and fatty acids were identified as promising clinical biomarkers for early diagnosis and/or prognostication of PAD that also provides insights into its underlying pathophysiology. Lastly, Chapter VI provides an overview of the major contributions derived from this thesis, as well as a perspective on future research initiatives.ThesisDoctor of Philosophy (PhD

Effects of ambient air pollution on obesity and ectopic fat deposition:a protocol for a systematic review and meta-analysis

Introduction - Globally, the prevalence of obesity tripled from 1975 to 2016. There is evidence that air pollution may contribute to the obesity epidemic through an increase in oxidative stress and inflammation of adipose tissue. However, the impact of air pollution on body weight at a population level remains inconclusive. This systematic review and meta-analysis will estimate the association of ambient air pollution with obesity, distribution of ectopic adipose tissue, and the incidence and prevalence of non-alcoholic fatty liver disease among adults. Methods and analysis.The study will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines for conduct and reporting. The search will include the following databases: Ovid Medline, Embase, PubMed, Web of Science and Latin America and the Caribbean Literature on Health Sciences, and will be supplemented by a grey literature search. Each article will be independently screened by two reviewers, and relevant data will be extracted independently and in duplicate. Study-specific estimates of associations and their 95% Confidence Intervals will be pooled using a DerSimonian and Laird random-effects model, implemented using the RevMan software. The I2 statistic will be used to assess interstudy heterogeneity. The confidence in the body of evidence will be assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach.Ethics and disseminationAs per institutional policy, ethical approval is not required for secondary data analysis. In addition to being published in a peer-reviewed journal and presented at conferences, the results of the meta-analysis will be shared with key stakeholders, health policymakers and healthcare professionals.Prospero registration numberCRD42023423955

Metabolic Trajectories Following Contrasting Prudent and Western Diets from Food Provisions: Identifying Robust Biomarkers of Short-Term Changes in Habitual Diet

A large body of evidence has linked unhealthy eating with an alarming increase in obesity and chronic disease worldwide. However, existing methods of assessing dietary intake rely on food frequency questionnaires or dietary records that are prone to bias and selective reporting. Herein, metabolic phenotyping was performed on 42 healthy participants from the Diet and Gene Intervention (DIGEST) pilot study, a parallel two-arm randomized clinical trial that provided complete diets to all participants. Matching urine and plasma specimens were collected at baseline and following 2 weeks of provision of either a Prudent or Western diet with a weight-maintaining menu plan designed by a dietician. Targeted and nontargeted metabolite profiling was conducted using three complementary analytical platforms, where 80 serum metabolites and 84 creatinine-normalized urinary metabolites were reliably measured (CV 75%) after implementing a rigorous data workflow for metabolite authentication with stringent quality control. We classified a panel of metabolites with distinctive trajectories following 2 weeks of food provisions when using complementary univariate and multivariate statistical models. Unknown metabolites associated with contrasting dietary patterns were identified with high resolution MS/MS and/or co-elution after spiking with authentic standards. Overall, 3-methylhistidine and proline betaine concentrations increased consistently when participants were assigned a Prudent diet (q ± 0.30, p < 0.05) to changes in average intake of specific nutrients from self-reported diet records reflecting good adherence to food provisions. This study revealed robust biomarkers sensitive to short-term changes in habitual diet that can be used to reliably monitor healthy eating patterns for new advances in nutritional epidemiology, as well as the design of evidence-based public health policies for chronic disease prevention

Serum Metabolic Signatures of Chronic Limb-Threatening Ischemia in Patients with Peripheral Artery Disease

Peripheral artery disease (PAD) is characterized by the atherosclerotic narrowing of lower limb vessels, leading to ischemic muscle pain in older persons. Some patients experience progression to advanced chronic limb-threatening ischemia (CLTI) with poor long-term survivorship. Herein, we performed serum metabolomics to reveal the mechanisms of PAD pathophysiology that may improve its diagnosis and prognosis to CLTI complementary to the ankle–brachial index (ABI) and clinical presentations. Non-targeted metabolite profiling of serum was performed by multisegment injection–capillary electrophoresis–mass spectrometry (MSI–CE–MS) from age and sex-matched, non-diabetic, PAD participants who were recruited and clinically stratified based on the Rutherford classification into CLTI (n = 18) and intermittent claudication (IC, n = 20). Compared to the non-PAD controls (n = 20), PAD patients had lower serum concentrations of creatine, histidine, lysine, oxoproline, monomethylarginine, as well as higher circulating phenylacetylglutamine (p &lt; 0.05). Importantly, CLTI cases exhibited higher serum concentrations of carnitine, creatinine, cystine and trimethylamine-N-oxide along with lower circulating fatty acids relative to well matched IC patients. Most serum metabolites associated with PAD progression were also correlated with ABI (r = ±0.24−0.59, p &lt; 0.05), whereas the ratio of stearic acid to carnitine, and arginine to propionylcarnitine differentiated CLTI from IC with good accuracy (AUC = 0.87, p = 4.0 × 10−5). This work provides new biochemical insights into PAD progression for the early detection and surveillance of high-risk patients who may require peripheral vascular intervention to prevent amputation and premature death.</jats:p

Serum Metabolic Signatures of Chronic Limb-Threatening Ischemia in Patients with Peripheral Artery Disease

Peripheral artery disease (PAD) is characterized by the atherosclerotic narrowing of lower limb vessels, leading to ischemic muscle pain in older persons. Some patients experience progression to advanced chronic limb-threatening ischemia (CLTI) with poor long-term survivorship. Herein, we performed serum metabolomics to reveal the mechanisms of PAD pathophysiology that may improve its diagnosis and prognosis to CLTI complementary to the ankle&ndash;brachial index (ABI) and clinical presentations. Non-targeted metabolite profiling of serum was performed by multisegment injection&ndash;capillary electrophoresis&ndash;mass spectrometry (MSI&ndash;CE&ndash;MS) from age and sex-matched, non-diabetic, PAD participants who were recruited and clinically stratified based on the Rutherford classification into CLTI (n = 18) and intermittent claudication (IC, n = 20). Compared to the non-PAD controls (n = 20), PAD patients had lower serum concentrations of creatine, histidine, lysine, oxoproline, monomethylarginine, as well as higher circulating phenylacetylglutamine (p &lt; 0.05). Importantly, CLTI cases exhibited higher serum concentrations of carnitine, creatinine, cystine and trimethylamine-N-oxide along with lower circulating fatty acids relative to well matched IC patients. Most serum metabolites associated with PAD progression were also correlated with ABI (r = &plusmn;0.24&minus;0.59, p &lt; 0.05), whereas the ratio of stearic acid to carnitine, and arginine to propionylcarnitine differentiated CLTI from IC with good accuracy (AUC = 0.87, p = 4.0 &times; 10&minus;5). This work provides new biochemical insights into PAD progression for the early detection and surveillance of high-risk patients who may require peripheral vascular intervention to prevent amputation and premature death