Serum Orotidine: A Novel Biomarker of Increased CVD Risk in Type 2 Diabetes Discovered Through Metabolomics Studies.

OBJECTIVE: To identify novel biomarkers of cardiovascular disease (CVD) risk in type 2 diabetes (T2D) via a hypothesis-free global metabolomics study, while taking into account renal function, an important confounder often overlooked in previous metabolomics studies of CVD. RESEARCH DESIGN AND METHODS: We conducted a global serum metabolomics analysis using the Metabolon platform in a discovery set from the Joslin Kidney Study having a nested case-control design comprising 409 individuals with T2D. Logistic regression was applied to evaluate the association between incident CVD events and each of the 671 metabolites detected by the Metabolon platform, before and after adjustment for renal function and other CVD risk factors. Significant metabolites were followed up with absolute quantification assays in a validation set from the Joslin Heart Study including 599 individuals with T2D with and without clinical evidence of significant coronary heart disease (CHD). RESULTS: In the discovery set, serum orotidine and 2-piperidinone were significantly associated with increased odds of incident CVD after adjustment for glomerular filtration rate (GFR) (odds ratio [OR] per SD increment 1.94 [95% CI 1.39-2.72], P = 0.0001, and 1.62 [1.26-2.08], P = 0.0001, respectively). Orotidine was also associated with increased odds of CHD in the validation set (OR 1.39 [1.11-1.75]), while 2-piperidinone did not replicate. Furthermore, orotidine, being inversely associated with GFR, mediated 60% of the effects of declining renal function on CVD risk. Addition of orotidine to established clinical predictors improved (P < 0.05) C statistics and discrimination indices for CVD risk (ΔAUC 0.053, rIDI 0.48, NRI 0.42) compared with the clinical predictors alone. CONCLUSIONS: Through a robust metabolomics approach, with independent validation, we have discovered serum orotidine as a novel biomarker of increased odds of CVD in T2D, independent of renal function. Additionally, orotidine may be a biological mediator of the increased CVD risk associated with poor kidney function and may help improve CVD risk prediction in T2D.

Quantitative analysis of γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline in HeLa cells using UHPLC-MS/MS.

γ-Glutamylpeptides have been identified as potential biomarkers for a number of diseases including cancer, diabetes, and liver disease. In this study, we developed and validated a novel quantitative analytical strategy for measuring γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline, all of which have been previously reported as potential biomarkers for prostate cancer in HeLa cells using ultra-high-performance liquid chromatography-tandem mass spectrometry. A BEH C18 column was used as the stationary phase. Mobile phase A was 99:1 water:formic acid and mobile phase B was acetonitrile. Chemical isotope labeling using benzoyl chloride was used as the internal standardization strategy. Sample preparation consisted of the addition of water to a frozen cell pellet, sonication, derivatization, centrifugation, and subsequent addition of an internal standard solution. The method was validated for selectivity, accuracy, precision, linearity, and stability. The determined concentrations of γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline in HeLa cells were 1.92 ± 0.06, 10.8 ± 0.4, and 1.96 ± 0.04 pmol/mg protein, respectively. In addition, the qualitative analysis of these analytes in human serum was achieved using a modified sample preparation strategy. To the best of our knowledge, this is the first report of the use of benzoyl chloride for chemical isotope labeling for metabolite quantitation in cells.

Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis.

The analysis of proteins in biological samples is highly desirable, given their connection to myriad biological functions and disease states, as well as the growing interest in the development of protein-based pharmaceuticals. The introduction and maturation of "soft" ionization methods, such as electrospray ionization and matrix-assisted laser desorption/ionization, have made mass spectrometry an indispensable tool for the analysis of proteins. Despite the availability of powerful instrumentation, sample preparation and fractionation remain among the most challenging aspects of protein analysis. This review summarizes these challenges and provides an overview of the state-of-the-art in sample preparation and fractionation of proteins for mass spectrometric analysis, with an emphasis on those used for top-down proteomic approaches. Biological fluids, particularly important for clinical and pharmaceutical applications and their characteristics are also discussed. While immunoaffinity-based methods are addressed, more attention is given to non-immunoaffinity-based methods, such as precipitation, coacervation, size exclusion, dialysis, solid-phase extraction, and electrophoresis. These techniques are presented in the context of a significant number of studies where they have been developed and utilized.

Use of a Continuous Stirred Tank Reactor for the Determination of Electrospray Response Factors and Its Application to Underivatized Sugars Under Various Solvent Parameters.

The relationship between the electrospray ionization (ESI)-mass spectrometric (MS) response of an analyte and its concentration has been well studied for permanently charged and basic analytes in the positive ionization mode, but there has been a lack of research effort for other analytes, and for the negative ionization mode, in general. In this study, this relationship was investigated for various adducts and deprotonated species of glucose, sucrose, and raffinose using a continuous stirred tank reactor (CSTR) coupled with ESI-tandem MS to obtain a continuum of response factors across a wide concentration range in both the positive and negative ionization modes with a single injection under 18 different combinations of solvents and additives. Profiles of response factors vs. concentrations varied widely and were dependent upon the analyte and solvent parameters. The use of ammonium trifluoroacetate resulted in the highest response factors for methanol-based and acetonitrile-based solvents in the positive and negative ionization modes, respectively. Ammonium acetate, ammonium formate, and sodium chloride in 80:20 acetonitrile:water in the negative ionization mode resulted in good linearities, useful for quantitative analysis. In the positive ionization mode, response factors tended to increase with an increase in the molecular weight of the analyte, and acetonitrile was generally found to decrease response factors. We have also demonstrated the ability of CSTR-ESI-MS to visualize ionization suppression in the presence of co-analytes. These data should be useful for liquid chromatography-ESI-MS method development for sugar analysis, to help guide the choice of mobile phase that will result in high sensitivity and linearity. Graphical Abstract.

Effects of solvent parameters on the electrospray ionization tandem mass spectrometry response of glucose.

RATIONALE: The importance of saccharides, the most abundant biomolecules on Earth, extends beyond their biological roles and to consumer products and industrial processes. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) is an attractive tool for the analysis of underivatized saccharides (US), but they tend to have relatively low sensitivities due to their low surface activities and lack of easily protonable or deprotonable chemical groups. An understanding of the influences that solvent parameters have on their signal intensities would enhance the usefulness of ESI-MS/MS for their analysis. METHODS: Solutions of glucose, a model analyte for US, in various combinations of solvent, additive, additive concentration, and pH were analyzed by flow injection analysis ESI-MS/MS in both the positive and negative ionization mode. The blank-corrected signal intensities of the solvent parameter combinations were then compared. RESULTS: The addition of acetonitrile led to severe ionization suppression in the positive ionization mode through its competition with glucose for cation adduction. High signal intensity was achieved under wide pH and concentration ranges for methanol: water solutions containing ammonium trifluoroacetate in the positive ionization mode. The highest signal intensities for acetonitrile: water solutions were those containing ammonium formate or lithium fluoride in the negative ionization mode. CONCLUSIONS: An understanding of the influence of solvent parameters on the signal intensity of a given analyte is useful for guiding the selection process of mobile phases/flow solvents that lead to low limits of detection or the minimization of matrix effects by allowing its detection at high dilution factors.

A reconnaissance analysis of groundwater quality in the Eagle Ford shale region reveals two distinct bromide/chloride populations.

The extraction of oil and natural gas from unconventional shale formations has prompted a series of investigations to examine the quality of the groundwater in the overlying aquifers. Here we present a reconnaissance analysis of groundwater quality in the Eagle Ford region of southern Texas. These data reveal two distinct sample populations that are differentiable by bromide/chloride ratios. Elevated levels of fluoride, nitrate, sulfate, various metal ions, and the detection of exotic volatile organic compounds highlight a high bromide group of samples, which is geographically clustered, while encompassing multiple hydrogeological strata. Samples with bromide/chloride ratios representative of connate water displayed elevated levels of total organic carbon, while revealing the detection of alcohols and chlorinated compounds. These findings suggest that groundwater quality in the Western Gulf Basin is, for the most part, controlled by a series of natural processes; however, there is also evidence of episodic contamination events potentially attributed to unconventional oil and gas development or other anthropogenic activities. Collectively, this characterization of natural groundwater constituents and exogenous compounds will guide targeted remediation efforts and provides insight for agricultural entities, industrial operators, and rural communities that rely on groundwater in southern Texas.

Temporal variation in groundwater quality in the Permian Basin of Texas, a region of increasing unconventional oil and gas development.

The recent expansion of natural gas and oil extraction using unconventional oil and gas development (UD) practices such as horizontal drilling and hydraulic fracturing has raised questions about the potential for environmental impacts. Prior research has focused on evaluations of air and water quality in particular regions without explicitly considering temporal variation; thus, little is known about the potential effects of UD activity on the environment over longer periods of time. Here, we present an assessment of private well water quality in an area of increasing UD activity over a period of 13months. We analyzed samples from 42 private water wells located in three contiguous counties on the Eastern Shelf of the Permian Basin in Texas. This area has experienced a rise in UD activity in the last few years, and we analyzed samples in four separate time points to assess variation in groundwater quality over time as UD activities increased. We monitored general water quality parameters as well as several compounds used in UD activities. We found that some constituents remained stable over time, but others experienced significant variation over the period of study. Notable findings include significant changes in total organic carbon and pH along with ephemeral detections of ethanol, bromide, and dichloromethane after the initial sampling phase. These data provide insight into the potentially transient nature of compounds associated with groundwater contamination in areas experiencing UD activity.

A Comprehensive Analysis of Groundwater Quality in The Barnett Shale Region.

The exploration of unconventional shale energy reserves and the extensive use of hydraulic fracturing during well stimulation have raised concerns about the potential effects of unconventional oil and gas extraction (UOG) on the environment. Most accounts of groundwater contamination have focused primarily on the compositional analysis of dissolved gases to address whether UOG activities have had deleterious effects on overlying aquifers. Here, we present an analysis of 550 groundwater samples collected from private and public supply water wells drawing from aquifers overlying the Barnett shale formation of Texas. We detected multiple volatile organic carbon compounds throughout the region, including various alcohols, the BTEX family of compounds, and several chlorinated compounds. These data do not necessarily identify UOG activities as the source of contamination; however, they do provide a strong impetus for further monitoring and analysis of groundwater quality in this region as many of the compounds we detected are known to be associated with UOG techniques.