Statistical methods and resources for biomarker discovery using metabolomics.

Metabolomics is a dynamic tool for elucidating biochemical changes in human health and disease. Metabolic profiles provide a close insight into physiological states and are highly volatile to genetic and environmental perturbations. Variation in metabolic profiles can inform mechanisms of pathology, providing potential biomarkers for diagnosis and assessment of the risk of contracting a disease. With the advancement of high-throughput technologies, large-scale metabolomics data sources have become abundant. As such, careful statistical analysis of intricate metabolomics data is essential for deriving relevant and robust results that can be deployed in real-life clinical settings. Multiple tools have been developed for both data analysis and interpretations. In this review, we survey statistical approaches and corresponding statistical tools that are available for discovery of biomarkers using metabolomics.Open Access funding provided by the Qatar National Library. This research was funded by the Qatar National Research Fund (QNRF), grant number NPRP13S-1230-190008

Serum NMR-Based Metabolomics Profiling Identifies Lipoprotein Subfraction Variables and Amino Acid Reshuffling in Myeloma Development and Progression

Multiple myeloma (MM) is an incurable hematological cancer. It is preceded by monoclonal gammopathy of uncertain significance (MGUS)—an asymptomatic phase. It has been demonstrated that early detection increases the 5-year survival rate. However, blood-based biomarkers that enable early disease detection are lacking. Metabolomic and lipoprotein subfraction variable profiling is gaining traction to expand our understanding of disease states and, more specifically, for identifying diagnostic markers in patients with hematological cancers. This study aims to enhance our understanding of multiple myeloma (MM) and identify candidate metabolites, allowing for a more effective preventative treatment. Serum was collected from 25 healthy controls, 20 patients with MGUS, and 30 patients with MM. 1H-NMR (Nuclear Magnetic Resonance) spectroscopy was utilized to evaluate serum samples. The metabolite concentrations were examined using multivariate, univariate, and pathway analysis. Metabolic profiles of the MGUS patients revealed lower levels of alanine, lysine, leucine but higher levels of formic acid when compared to controls. However, metabolic profiling of MM patients, compared to controls, exhibited decreased levels of total Apolipoprotein-A1, HDL-4 Apolipoprotein-A1, HDL-4 Apolipoprotein-A2, HDL Free Cholesterol, HDL-3 Cholesterol and HDL-4 Cholesterol. Lastly, metabolic comparison between MGUS to MM patients primarily indicated alterations in lipoproteins levels: Total Cholesterol, HDL Cholesterol, HDL Free Cholesterol, Total Apolipoprotein-A1, HDL Apolipoprotein-A1, HDL-4 Apolipoprotein-A1 and HDL-4 Phospholipids. This study provides novel insights into the serum metabolic and lipoprotein subfraction changes in patients as they progress from a healthy state to MGUS to MM, which may allow for earlier clinical detection and treatment.This work was funded by grants from the Danish Research Council for Independent Research (4183-00268) and the Obel Family Foundation (26145).Scopu

The Metabolic Switch of Physical Activity in Non-Obese Insulin Resistant Individuals

Healthy non-obese insulin resistant (IR) individuals are at higher risk of metabolic syndrome. The metabolic signature of the increased risk was previously determined. Physical activity can lower the risk of insulin resistance, but the underlying metabolic pathways remain to be determined. In this study, the common and unique metabolic signatures of insulin sensitive (IS) and IR individuals in active and sedentary individuals were determined. Data from 305 young, aged 20–30, non-obese participants from Qatar biobank, were analyzed. The homeostatic model assessment of insulin resistance (HOMA-IR) and physical activity questionnaires were utilized to classify participants into four groups: Active Insulin Sensitive (ISA, n = 30), Active Insulin Resistant (IRA, n = 20), Sedentary Insulin Sensitive (ISS, n = 21) and Sedentary Insulin Resistant (SIR, n = 23). Differences in the levels of 1000 metabolites between insulin sensitive and insulin resistant individuals in both active and sedentary groups were compared using orthogonal partial least square discriminate analysis (OPLS-DA) and linear models. The study indicated significant differences in fatty acids between individuals with insulin sensitivity and insulin resistance who engaged in physical activity, including monohydroxy, dicarboxylate, medium and long chain, mono and polyunsaturated fatty acids. On the other hand, the sedentary group showed changes in carbohydrates, specifically glucose and pyruvate. Both groups exhibited alterations in 1-carboxyethylphenylalanine. The study revealed different metabolic signature in insulin resistant individuals depending on their physical activity status. Specifically, the active group showed changes in lipid metabolism, while the sedentary group showed alterations in glucose metabolism. These metabolic discrepancies demonstrate the beneficial impact of moderate physical activity on high risk insulin resistant healthy non-obese individuals by flipping their metabolic pathways from glucose based to fat based, ultimately leading to improved health outcomes. The results of this study carry significant implications for the prevention and treatment of metabolic syndrome in non-obese individuals.</p

Comparing the Metabolic Profiles Associated with Fitness Status between Insulin-Sensitive and Insulin-Resistant Non-Obese Individuals

(1) Background: Young non-obese insulin-resistant (IR) individuals could be at risk of developing metabolic diseases including type 2 diabetes mellitus. The protective effect of physical activity in this apparently healthy group is expected but not well characterized. In this study, clinically relevant metabolic profiles were determined and compared among active and sedentary insulin-sensitive (IS) and IR young non-obese individuals. (2) Methods: Data obtained from Qatar Biobank for 2110 young (20-30 years old) non-obese (BMI ≤ 30) healthy participants were divided into four groups, insulin-sensitive active (ISA, 30.7%), insulin-sensitive sedentary (ISS, 21.4%), insulin-resistant active (IRA, 20%), and insulin-resistant sedentary (IRS, 23.3%), using the homeostatic model assessment of insulin resistance (HOMA-IR) and physical activity questionnaires. The effect of physical activity on 66 clinically relevant biochemical tests was compared among the four groups using linear models. (3) Results: Overall, non-obese IR participants had significantly ( ≤ 0.001) worse vital signs, blood sugar profiles, inflammatory markers, liver function, lipid profiles, and vitamin D levels than their IS counterparts. Physical activity was positively associated with left handgrip ( ≤ 0.01) and levels of creatine kinase ( ≤ 0.001) and creatine kinase-2 ( ≤ 0.001) in both IS and IR subjects. Furthermore, physical activity was positively associated with levels of creatinine ( ≤ 0.01) and total vitamin D ( = 0.006) in the IR group and AST ( = 0.001), folate ( = 0.001), and hematocrit ( = 0.007) in the IS group. Conversely, physical inactivity was negatively associated with the white blood cell count ( = 0.001) and an absolute number of lymphocytes ( = 0.003) in the IR subjects and with triglycerides ( = 0.005) and GGT-2 ( ≤ 0.001) in the IS counterparts. (4) Conclusions: An independent effect of moderate physical activity was observed in non-obese apparently healthy individuals a with different HOMA-IR index. The effect was marked by an improved health profile including higher vitamin D and lower inflammatory markers in IRA compared to IRS, and a higher oxygen carrying capacity and lipid profile in ISA compared to the ISS counterparts.QatarUniversity grant number IRCC-2022-467 (M.A.E.,M.S.,A.A.)

Metabolic Signatures of Type 2 Diabetes Mellitus and Hypertension in COVID-19 Patients With Different Disease Severity.

Increased COVID-19 disease severity is higher among patients with type 2 diabetes mellitus and hypertension. However, the metabolic pathways underlying this association are not fully characterized. This study aims to identify the metabolic signature associated with increased COVID-19 severity in patients with diabetes mellitus and hypertension. One hundred and fifteen COVID-19 patients were divided based on disease severity, diabetes status, and hypertension status. Targeted metabolomics of serum samples from all patients was performed using tandem mass spectrometry followed by multivariate and univariate models. Reduced levels of various triacylglycerols were observed with increased disease severity in the diabetic patients, including those containing palmitic (C16:0), docosapentaenoic (C22:5, DPA), and docosahexaenoic (C22:6, DHA) acids (FDR < 0.01). Functional enrichment analysis revealed triacylglycerols as the pathway exhibiting the most significant changes in severe COVID-19 in diabetic patients (FDR = 7.1 × 10). Similarly, reduced levels of various triacylglycerols were also observed in hypertensive patients corresponding with increased disease severity, including those containing palmitic, oleic (C18:1), and docosahexaenoic acids. Functional enrichment analysis revealed long-chain polyunsaturated fatty acids (n-3 and n-6) as the pathway exhibiting the most significant changes with increased disease severity in hypertensive patients (FDR = 0.07). Reduced levels of triacylglycerols containing specific long-chain unsaturated, monounsaturated, and polyunsaturated fatty acids are associated with increased COVID-19 severity in diabetic and hypertensive patients, offering potential novel diagnostic and therapeutic targets.This research was funded by the Qatar National Research Fund, Grant Number NPRP11S-1212-170092

The retrospective study of the metabolic patterns of BCG-vaccination in type-2 diabetic individuals in COVID-19 infection

Background: The cross-protective nature of Bacillus Calmette-Guerin (BCG) vaccine against SARS-CoV-2 virus was previously suggested, however its effect in COVID-19 patients with type 2 diabetes (T2D) and the underlying metabolic pathways has not been addressed. This study aims to investigate the difference in the metabolomic patterns of type 2 diabetic patients with BCG vaccination showing different severity levels of COVID-19 infection. Methods: Sixty-seven COVID-19 patients were categorized into diabetic and non-diabetic individuals who had been previously vaccinated or not with BCG vaccination. Targeted metabolomics were performed from serum samples from all patients using tandem mass spectrometry. Statistical analysis included multivariate and univariate models. Results: Data suggested that while BCG vaccination may provide protection for individuals who do not have diabetes, it appears to be linked to more severe COVID-19 symptoms in T2D patients (p = 0.02). Comparing the metabolic signature of BCG vaccinated T2D individuals to non-vaccinated counterparts revealed that amino acid (sarcosine), cholesterol esters (CE 20:0, 20:1, 22:2), carboxylic acid (Aconitic acid) were enriched in BCG vaccinated T2D patients, whereas spermidine, glycosylceramides (Hex3Cer(d18:1_22:0), Hex2Cer(d18:1/22:0), HexCer(d18:1/26:1), Hex2Cer(d18:1/24:0), HexCer(d18:1/22:0) were higher in BCG vaccinated non- T2D patients. Furthermore, data indicated a decrease in sarcosine synthesis from glycine and choline and increase in spermidine synthesis in the BCG vaccinated cohort in T2D and non-T2D groups, respectively. Conclusion: This pilot study suggests increased severity of COVID-19 in BCG vaccinated T2D patients, which was marked by decreased sarcosine synthesis, perhaps via lower sarcosine-mediated removal of viral antigens.This research was funded by the Qatar National Research Fund, Grant Number NPRP11S-1212-170092

The retrospective study of the metabolic patterns of BCG-vaccination in type-2 diabetic individuals in COVID-19 infection

BackgroundThe cross-protective nature of Bacillus Calmette-Guerin (BCG) vaccine against SARS-CoV-2 virus was previously suggested, however its effect in COVID-19 patients with type 2 diabetes (T2D) and the underlying metabolic pathways has not been addressed. This study aims to investigate the difference in the metabolomic patterns of type 2 diabetic patients with BCG vaccination showing different severity levels of COVID-19 infection.MethodsSixty-seven COVID-19 patients were categorized into diabetic and non-diabetic individuals who had been previously vaccinated or not with BCG vaccination. Targeted metabolomics were performed from serum samples from all patients using tandem mass spectrometry. Statistical analysis included multivariate and univariate models.ResultsData suggested that while BCG vaccination may provide protection for individuals who do not have diabetes, it appears to be linked to more severe COVID-19 symptoms in T2D patients (p = 0.02). Comparing the metabolic signature of BCG vaccinated T2D individuals to non-vaccinated counterparts revealed that amino acid (sarcosine), cholesterol esters (CE 20:0, 20:1, 22:2), carboxylic acid (Aconitic acid) were enriched in BCG vaccinated T2D patients, whereas spermidine, glycosylceramides (Hex3Cer(d18:1_22:0), Hex2Cer(d18:1/22:0), HexCer(d18:1/26:1), Hex2Cer(d18:1/24:0), HexCer(d18:1/22:0) were higher in BCG vaccinated non- T2D patients. Furthermore, data indicated a decrease in sarcosine synthesis from glycine and choline and increase in spermidine synthesis in the BCG vaccinated cohort in T2D and non-T2D groups, respectively.ConclusionThis pilot study suggests increased severity of COVID-19 in BCG vaccinated T2D patients, which was marked by decreased sarcosine synthesis, perhaps via lower sarcosine-mediated removal of viral antigens

In Vitro and In Vivo Validation of GATA-3 Suppression for Induction of Adipogenesis and Improving Insulin Sensitivity

Impaired adipogenesis is associated with the development of insulin resistance and an increased risk of type 2 diabetes (T2D). GATA Binding Protein 3 (GATA3) is implicated in impaired adipogenesis and the onset of insulin resistance. Therefore, we hypothesize that inhibition of GATA3 could promote adipogenesis, restore healthy fat distribution, and enhance insulin signaling. Primary human preadipocytes were treated with GATA3 inhibitor (DNAzyme hgd40). Cell proliferation, adipogenic capacity, gene expression, and insulin signaling were measured following well-established protocols. BALB/c mice were treated with DNAzyme hgd40 over a period of 2 weeks. Liposomes loaded with DNAzyme hgd40, pioglitazone (positive), or vehicle (negative) controls were administered subcutaneously every 2 days at the right thigh. At the end of the study, adipose tissues were collected and weighed from the site of injection, the opposite side, and the omental depot. Antioxidant enzyme (superoxide dismutase and catalase) activities were assessed in animals’ sera, and gene expression was measured using well-established protocols. In vitro GATA3 inhibition induced the adipogenesis of primary human preadipocytes and enhanced insulin signaling through the reduced expression of p70S6K. In vivo GATA3 inhibition promoted adipogenesis at the site of injection and reduced MCP-1 expression. GATA3 inhibition also reduced omental tissue size and PPARγ expression. These findings suggest that modulating GATA3 expression offers a potential therapeutic benefit by correcting impaired adipogenesis, promoting healthy fat distribution, improving insulin sensitivity, and potentially lowering the risk of T2D.Qatar University H3P grant number QPH3P-BRC-2021-451 (MAE, HN, LM)

Identification of Prognostic Metabolomic Biomarkers at the Interface of Mortality and Morbidity in Pre-Existing TB Cases Infected With SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection currently remains one of the biggest global challenges that can lead to acute respiratory distress syndrome (CARDS) in severe cases. In line with this, prior pulmonary tuberculosis (TB) is a risk factor for long-term respiratory impairment. Post-TB lung dysfunction often goes unrecognized, despite its relatively high prevalence and its association with reduced quality of life. In this study, we used a metabolomics analysis to identify potential biomarkers that aid in the prognosis of COVID-19 morbidity and mortality in post-TB infected patients. This analysis involved blood samples from 155 SARS-CoV-2 infected adults, of which 23 had a previous diagnosis of TB (post-TB), while 132 did not have a prior or current TB infection. Our analysis indicated that the vast majority (~92%) of post-TB individuals showed severe SARS-CoV-2 infection, required intensive oxygen support with a significantly high mortality rate (52.2%). Amongst individuals with severe COVID-19 symptoms, we report a significant decline in the levels of amino acids, notably the branched chains amino acids (BCAAs), more so in the post-TB cohort (FDR <= 0.05) in comparison to mild and asymptomatic cases. Indeed, we identified betaine and BCAAs as potential prognostic metabolic biomarkers of severity and mortality, respectively, in COVID-19 patients who have been exposed to TB. Moreover, we identified serum alanine as an important metabolite at the interface of severity and mortality. Hence, our data associated COVID-19 mortality and morbidity with a long-term metabolically driven consequence of TB infection. In summary, our study provides evidence for a higher mortality rate among COVID-19 infection patients who have history of prior TB infection diagnosis, which mandates validation in larger population cohorts

Metformin increases 3-hydroxy medium chain fatty acids in patients with type 2 diabetes: a cross-sectional pharmacometabolomic study

Background: Metformin is a drug with a long history of providing benefits in diabetes management and beyond. The mechanisms of action of metformin are complex, and continue to be actively debated and investigated. The aim of this study is to identify metabolic signatures associated with metformin treatment, which may explain the pleiotropic mechanisms by which metformin works, and could lead to an improved treatment and expanded use. Methods: This is a cross-sectional study, in which clinical and metabolomic data for 146 patients with type 2 diabetes were retrieved from Qatar Biobank. Patients were categorized into: Metformin-treated, treatment naïve, and non-metformin treated. Orthogonal partial least square discriminate analysis and linear models were used to analyze differences in the level of metabolites between the metformin treated group with each of the other two groups. Results: Patients on metformin therapy showed, among other metabolites, a significant increase in 3-hydroxyoctanoate and 3-hydroxydecanoate, which may have substantial effects on metabolism. Conclusions: This is the first study to report an association between 3-hydroxy medium chain fatty acids with metformin therapy in patients with type 2 diabetes. This opens up new directions towards repurposing metformin by comprehensively understanding the role of these metabolites.The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by Qatar National Research Fund (QNRF), grant number NPRP13S-1230-190008