Casey Burton

Metabolomics Approach Reveals Integrated Metabolic Network Associated with Serotonin Deficiency.

Serotonin is an important neurotransmitter that broadly participates in various biological processes. While serotonin deficiency has been associated with multiple pathological conditions such as depression, schizophrenia, Alzheimer’s disease and Parkinson’s disease, the serotonin-dependent mechanisms remain poorly understood. This study therefore aimed to identify novel biomarkers and metabolic pathways perturbed by serotonin deficiency using metabolomics approach in order to gain new metabolic insights into the serotonin deficiency-related molecular mechanisms. Serotonin deficiency was achieved through pharmacological inhibition of tryptophan hydroxylase (Tph) using p-chlorophenylalanine (pCPA) or genetic knockout of the neuronal specific Tph2 isoform. This dual approach improved specificity for the serotonin deficiency-associated biomarkers while minimizing nonspecific effects of pCPA treatment or Tph2 knockout (Tph2-/-). Non-targeted metabolic profiling and a targeted pCPA dose-response study identified 21 biomarkers in the pCPA-treated mice while 17 metabolites in the Tph2-/- mice were found to be significantly altered compared with the control mice. These newly identified biomarkers were associated with amino acid, energy, purine, lipid and gut microflora metabolisms. Oxidative stress was also found to be significantly increased in the serotonin deficient mice. These new biomarkers and the overall metabolic pathways may provide new understanding for the serotonin deficiency-associated mechanisms under multiple pathological states.

Simultaneous Detection of Six Urinary Pteridines and Creatinine by High-Performance Liquid Chromatography-Tandem Mass Spectrometry for Clinical Breast Cancer Detection

Recent preliminary studies have implicated urinary pteridines as candidate biomarkers in a growing number of malignancies including breast cancer. While the developments of capillary electrophoresis-laser induced fluorescence (CE-LIF), high performance liquid chromatography (HPLC), and liquid chromatography-mass spectroscopy (LC-MS) pteridine urinalyses among others have helped to enable these findings, limitations including poor pteridine specificity, asynchronous or nonexistent renal dilution normalization, and a lack of information regarding adduct formation in mass spectrometry techniques utilizing electrospray ionization (ESI) have prevented application of these techniques to a larger clinical setting. In this study, a simple, rapid, specific, and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and optimized for simultaneous detection of six pteridines previously implicated in breast cancer and creatinine as a renal dilution factor in urine. In addition, this study reports cationic adduct formation of urinary pteridines under ESI-positive ionization for the first time. This newly developed technique separates and detects the following six urinary pteridines: 6-biopterin, 6-hydroxymethylpterin, d-neopterin, pterin, isoxanthopterin, and xanthopterin, as well as creatinine. The method detection limit for the pteridines is between 0.025 and 0.5 μg/L, and for creatinine, it is 0.15 μg/L. The method was also validated by spiked recoveries (81-105%), reproducibility (RSD: 1-6%), and application to 25 real urine samples from breast cancer positive and negative samples through a double-blind study. The proposed technique was finally compared directly with a previously reported CE-LIF technique, concluding that additional or alternative renal dilution factors are needed for proper investigation of urinary pteridines as breast cancer biomarkers.

A novel enzymatic technique for determination of sarcosine in urine samples

Metabolites impart a significant importance to the understanding of biological reactions and consequently to the development of diagnostic and therapeutic techniques for specific diseases. Furthermore, there has been recent interest in metabolite concentrations present in urine for potential noninvasive disease diagnosis. The detection of specific metabolites, however, presents certain analytical difficulties such as low or ambiguous specificity of the techniques. This study developed a new technique, utilizing oxidative, enzymatic production of formaldehyde from the metabolite to produce a pH-induced change observed by fluorescein in acetone. This probe displays high sensitivity towards pH imbalances and, coupled with high enzymatic specificity, forms an accurate method to measure metabolite concentrations. Sarcosine was used as a model analyte in this study due to its potential for serving as a prostate cancer biomarker. Sarcosine was treated with sarcosine oxidase to generate formaldehyde, which was further oxidized to formic acid, and subsequently measured by the corresponding change in fluorescein. A good linearity was revealed with a correlation coefficient of 0.9961 and a detection limit of 20 nmol L−1. This method was applied to sarcosine analysis in nine urine samples. The results suggest that this is a viable, cost-effective technique for determination of sarcosine in urine samples without interferences such as alanine.

Normalization of urinary pteridines by urine specific gravity for early cancer detection

BACKGROUND Urinary biomarkers, such as pteridines, require normalization with respect to an individuals hydration status and time since last urination. Conventional creatinine-based corrections are affected by a multitude of patient factors whereas urine specific gravity (USG) is a bulk specimen property that may better resist those same factors. We examined the performance of traditional creatinine adjustments relative to USG to six urinary pteridines in aggressive and benign breast cancers. METHODS 6-Biopterin, neopterin, pterin, 6-hydroxymethylpterin, isoxanthopterin, xanthopterin, and creatinine were analyzed in 50 urine specimens with a previously developed liquid chromatography-tandem mass spectrometry technique. Creatinine and USG performance were evaluated with non-parametric Mann-Whitney hypothesis testing. RESULTS USG and creatinine were moderately correlated (r=0.857) with deviations occurring in dilute and concentrated specimens. In 48 aggressive and benign breast cancers, normalization by USG significantly outperformed creatinine adjustments which marginally outperformed uncorrected pteridines in predicting pathological status. In addition, isoxanthopterin and xanthopterin were significantly higher in pathological specimens when normalized by USG. CONCLUSION USG, as a bulk property, can provide better performance over creatinine-based normalizations for urinary pteridines in cancer detection applications.

Lipidomic profiling of tryptophan hydroxylase 2 knockout mice reveals novel lipid biomarkers associated with serotonin deficiency

Serotonin is an important neurotransmitter that regulates a wide range of physiological, neuropsychological, and behavioral processes. Consequently, serotonin deficiency is involved in a wide variety of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, schizophrenia, and depression. The pathophysiological mechanisms underlying serotonin deficiency, particularly from a lipidomics perspective, remain poorly understood. This study therefore aimed to identify novel lipid biomarkers associated with serotonin deficiency by lipidomic profiling of tryptophan hydroxylase 2 knockout (Tph2−/−) mice. Using a high-throughput normal-/reversed-phase two-dimensional liquid chromatography–quadrupole time-of-flight mass spectrometry (NP/RP 2D LC–QToF-MS) method, 59 lipid biomarkers encompassing glycerophospholipids (glycerophosphocholines, lysoglycerophosphocholines, glycerophosphoethanolamines, lysoglycerophosphoethanolamines glycerophosphoinositols, and lysoglycerophosphoinositols), sphingolipids (sphingomyelins, ceramides, galactosylceramides, glucosylceramides, and lactosylceramides) and free fatty acids were identified. Systemic oxidative stress in the Tph2−/− mice was significantly elevated, and a corresponding mechanism that relates the lipidomic findings has been proposed. In summary, this work provides preliminary findings that lipid metabolism is implicated in serotonin deficiency.

High-throughput intracellular pteridinic profiling by liquid chromatography–quadrupole time-of-flight mass spectrometry

Pteridines are a diverse family of endogenous metabolites that may serve as useful diagnostic biomarkers for disease. While many preparative and analytical techniques have been described for analysis of selected pteridines in biological fluids, broad intracellular pteridine detection remains a significant analytical challenge. In this study, a novel, specific and sensitive extraction and high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) method was developed to simultaneously quantify seven intracellular pteridines and monitor 18 additional, naturally-occurring intracellular pteridines. The newly developed method was validated through evaluation of spiked recoveries (84.5-109.4%), reproducibility (2.1-5.4% RSD), method detection limits (0.1-3.0 μg L(-1)) and limits of quantitation (0.1-1 μg L(-1)), and finally application to non-small cell lung cancer A549 cells. Twenty-three pteridine derivatives were successfully detected from cell lysates with an average RSD of 12% among culture replicates. Quantified intracellular pteridine levels ranged from 1 to 1000 nM in good agreement with previous studies. Finally, this technique may be applied to cellular studies to generate new biological hypotheses concerning pteridine physiological and pathological functions as well as to discovery new pteridine-based biomarkers.

Simultaneous determination of urinary quinolinate, gentisate, 4-hydroxybenzoate, and α-ketoglutarate by high-performance liquid chromatography-tandem mass spectrometry

Renal cell carcinoma (RCC) remains a difficult-to-detect cancer for which new detection methodologies are urgently needed to improve screening efficacy. Urinary metabolomic profiling has identified several potential RCC biomarkers, including quinolinic acid, 4-hydroxybenzoic acid, gentisic acid and α-ketoglutaric acid. These four endogenous metabolites have unique pathophysiological mechanisms associated with RCC, suggesting that they may be promising biomarkers for earlier RCC detection. However, the lack of targeted analytical methods for their simultaneous determination in urine has hindered efforts to evaluate their clinical applicability. In this study, a robust high-performance liquid chromatography -tandem mass spectrometry (HPLC-MS/MS) technique using multiple reaction monitoring was developed to simultaneously monitor these four urinary biomarkers. The method was validated by multiple figures of merit including method detection limits (0.05 ng mL−1 to 1 ng mL−1), spiked recovery accuracy (88.6–114.8%) and reproducibility (0.4–9.3% RSD). The applicability of the method was further demonstrated in clinically relevant 100-fold diluted urine specimens. All four potential biomarkers were successfully quantified with urinary concentrations that were in agreement with previous reports. In conclusion, an accurate and sensitive method has been described for the simultaneous determination of quinolinic acid, 4-hydroxybenzoic acid, gentisic acid and α-ketoglutaric acid. We anticipate this method to significantly benefit clinical translational research aiming to evaluate the clinical applicability of these four potential biomarkers.

Current Trends in Cancer Biomarker Discovery Using Urinary Metabolomics: Achievements and New Challenges

BACKGROUND The development of effective screening methods for early cancer detection is one of the foremost challenges facing modern cancer research. Urinary metabolomics has recently emerged as a potentially transformative approach to cancer biomarker discovery owing to its noninvasive sampling characteristics and robust analytical feasibility. OBJECTIVE To provide an overview of new developments in urinary metabolomics, cover the most promising aspects of hyphenated techniques in untargeted and targeted metabolomics, and to discuss technical and clinical limitations in addition to the emerging challenges in the field of urinary metabolomics and its application to cancer biomarker discovery. METHODS A systematic review of research conducted in the past five years on the application of urinary metabolomics to cancer biomarker discovery was performed. Given the breadth of this topic, our review focused on the five most widely studied cancers employing urinary metabolomics approaches, including lung, breast, bladder, prostate, and ovarian cancers. RESULTS As an extension of conventional metabolomics, urinary metabolomics has benefitted from recent technological developments in nuclear magnetic resonance, mass spectrometry, gas and liquid chromatography, and capillary electrophoresis that have improved urine metabolome coverage and analytical reproducibility. Extensive metabolic profiling in urine has revealed a significant number of altered metabolic pathways and putative biomarkers, including pteridines, modified nucleosides, and acylcarnitines, that have been associated with cancer development and progression. CONCLUSION Urinary metabolomics presents a transformative new approach toward cancer biomarker discovery with high translational capacity to early cancer screening.