Multivariate data analysis of metabolites in the caffeinated and decaffeinated coffee samples.

<p>Panel A shows the PCA scores plot for caffeinated and decaffeinated coffee samples, panel B displays the model validation plot for PLS-DA model between caffeinated and decaffeinated coffee samples, and panel C illustrates the OPLS-DA model between caffeinated and decaffeinated coffee samples (1 predictive component, 1 orthogonal component, R²(Y) = 1, Q²(cum) = 0.998).</p

Gas Chromatography Time-Of-Flight Mass Spectrometry (GC-TOF-MS)-Based Metabolomics for Comparison of Caffeinated and Decaffeinated Coffee and Its Implications for Alzheimer’s Disease

<div><p>Findings from epidemiology, preclinical and clinical studies indicate that consumption of coffee could have beneficial effects against dementia and Alzheimer’s disease (AD). The benefits appear to come from caffeinated coffee, but not decaffeinated coffee or pure caffeine itself. Therefore, the objective of this study was to use metabolomics approach to delineate the discriminant metabolites between caffeinated and decaffeinated coffee, which could have contributed to the observed therapeutic benefits. Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS)-based metabolomics approach was employed to characterize the metabolic differences between caffeinated and decaffeinated coffee. Orthogonal partial least squares discriminant analysis (OPLS-DA) showed distinct separation between the two types of coffee (cumulative Q² = 0.998). A total of 69 discriminant metabolites were identified based on the OPLS-DA model, with 37 and 32 metabolites detected to be higher in caffeinated and decaffeinated coffee, respectively. These metabolites include several benzoate and cinnamate-derived phenolic compounds, organic acids, sugar, fatty acids, and amino acids. Our study successfully established GC-TOF-MS based metabolomics approach as a highly robust tool in discriminant analysis between caffeinated and decaffeinated coffee samples. Discriminant metabolites identified in this study are biologically relevant and provide valuable insights into therapeutic research of coffee against AD. Our data also hint at possible involvement of gut microbial metabolism to enhance therapeutic potential of coffee components, which represents an interesting area for future research.</p></div

Metabolomic profiling of coffee samples using GC-TOF-MS.

<p>Panel A shows the representative GC-TOF-MS chromatogram of caffeinated coffee sample, and panel B shows the representative GC-TOF-MS chromatogram of decaffeinated coffee sample.</p

Urinary Metabotyping of Bladder Cancer Using Two-Dimensional Gas Chromatography Time-of-Flight Mass Spectrometry

Cystoscopy is the gold standard clinical diagnosis of human bladder cancer (BC). As cystoscopy is expensive and invasive, it compromises patients’ compliance toward surveillance screening and challenges the detection of recurrent BC. Therefore, the development of a noninvasive method for the diagnosis and surveillance of BC and the elucidation of BC progression become pertinent. In this study, urine samples from 38 BC patients and 61 non-BC controls were subjected to urinary metabotyping using two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC–TOFMS). Subsequent to data preprocessing and chemometric analysis, the orthogonal partial least-squares discriminant analysis (OPLS-DA, R²X = 0.278, R²Y = 0.904 and Q²Y (cumulative) = 0.398) model was validated using permutation tests and receiver operating characteristic (ROC) analysis. Marker metabolites were further screened from the OPLS-DA model using statistical tests. GC×GC–TOFMS urinary metabotyping demonstrated 100% specificity and 71% sensitivity in detecting BC, while 100% specificity and 46% sensitivity were observed via cytology. In addition, the model revealed 46 metabolites that characterize human BC. Among the perturbed metabolic pathways, our clinical finding on the alteration of the tryptophan-quinolinic metabolic axis in BC suggested the potential roles of kynurenine in the malignancy and therapy of BC. In conclusion, global urinary metabotyping holds potential for the noninvasive diagnosis and surveillance of BC in clinics. In addition, perturbed metabolic pathways gleaned from urinary metabotyping shed new and established insights on the biology of human BC

Metabonomic Profiling of Bladder Cancer

Early diagnosis and life-long surveillance are clinically important to improve the long-term survival of bladder cancer patients. Currently, a noninvasive biomarker that is as sensitive and specific as cystoscopy in detecting bladder tumors is lacking. Metabonomics is a complementary approach for identifying perturbed metabolic pathways in bladder cancer. Significant progress has been made using modern metabonomic techniques to characterize and distinguish bladder cancer patients from control subjects, identify marker metabolites, and shed insights on the disease biology and potential therapeutic targets. With its rapid development, metabonomics has the potential to impact the clinical management of bladder cancer patients in the future by revolutionizing the diagnosis and life-long surveillance strategies and stratifying patients for diagnostic, surgical, and therapeutic clinical trials. An introduction to metabonomics, typical metabonomic workflow, and critical evaluation of metabonomic investigations in identifying biomarkers for the diagnosis of bladder cancer are presented

UA inhibits tumor growth in Group 3 TRAMP mice.

<p><b>A,</b> Mice were euthanized at 36 weeks and the tumor volume was measured as described in Material and Methods. Error bars represent SEM. * Statistical significance (P<0.05). <b>B,</b> Body weight of Groups 1–3 TRAMP mice and their controls. <b>C,</b> The effect of UA-treatment on the long term survival of TRAMP mice. Kaplan-Meier survival analysis was performed for the control and UA-treated TRAMP mice. Median survival time was 55 weeks for control (n = 5). Median survival time for group 1, group 2, group 3 and group 4 was 75, 75, 68, and 72 weeks, respectively. Overall significance was determined using log-rank (Mantel-Cox) test. UA enriched diet significantly prolonged survival.</p

Hematoxylin and Eosin stained dorso lateral prostate of TRAMP mice.

<p><b>A</b>, H&E stained section of DLP of Group 2 control (i) and UA-treated (ii) TRAMP prostate. Arrow indicates LG-PIN, notched right arrow indicates HG-PIN, and block right arrow indicates WDC. (iii) Bar chart shows the incidence of LG-PIN, HG-PIN, WDC, MDC and PDC in DLP of control and Group 2 mice. Reductions in PIN, WDC and MDC were observed after UA treatment. <b>B</b>, H&E stained section of DLP of Group 3 control (i) and UA-treated (ii) TRAMP prostate. As described in A; Arrow head indicates MDC and chevron indicates PDC. <b>C</b>, Western blot of pSTAT3, pAKT, pIKKα/β, and p65 proteins in DLP in Groups 1, 2 and 3 mice. DLP lysates was prepared and immunoblotting was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032476#s4" target="_blank">Materials and Methods</a>. Equal protein loading for pAKT and pIKKα/β was determined by stripping and probing for total AKT and IKKα, p65 and STAT3 blots were stripped and reprobed for β-actin to determine equal protein loading. The band density was quantitated using Image J software. NT = non-TRAMP-mice; T = TRAMP controls; T+UA = UA-treated TRAMP-mice.</p

Enzyme linked immunosorbent assay.

<p><b>A.</b> Suppression of serum TNF-α (i) and IL-6 (ii) by UA-treatment. Groups 1–3 mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032476#s4" target="_blank">Materials and Methods</a>. Sandwich ELISA assay was performed as per manufacturers' instruction (R&D systems, USA) to determine the levels of TNF-α and IL-6. *Statistical significance (P<0.05). <b>B,</b> Immunohistochemical analysis of cyclin D1, caspase 3 and COX-2 in tumor tissues in Group 3 TRAMP mice. DLP tumor tissues, embedded in paraffin blocks, were cut into 5 µM tissue sections and probed for cyclin D1, caspase 3 and COX-2 immunoreactivities as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032476#s4" target="_blank">Materials and Methods</a>. UA-treatment (1% w/w) decreased the expression of cyclin D1 and COX-2 but increased caspase-3 expression when compared to controls. Images were taken using a Olympus BX51 microscope (magnification, 20×). Positive cells (brown) were quantitated using The Image-Pro plus v6.3 software package (Media Cybernetics, Inc.).</p

Mass spectrometric analysis of UA in serum.

<p><b>A,</b> (i) Ion chromatograms of serum extracts from mice that had not received UA (blank). (ii) Full scan spectrum showing the m/z peak with a molecular mass of 469 corresponds to internal standard (IS) glycyrrhetinic acid. (iii) Full scan spectrum showing the m/z peak with a molecular mass of standard UA at 455 and IS at 469 and (iv) Full scan spectrum of sample, retention time was 1.7 and 1.96 min respectively for both IS and sample. The chromatograms are representative of 3 independent experiments. <b>B,</b> Serum concentrations of UA was calculated using Analyst software 1.4.2 from the linear regression equation of the peak area ratio against the concentration of the calibration curve. Error bars represent SEM.</p

Agreement between the plasma concentrations of carbamazepine and its dried blood spot concentration; and between the plasma concentrations of phenytoin and valproic acid and the theoretical plasma concentrations derived from their dried blood spot concentrations.

<p>Bland Altman plots for plasma concentrations of (<b>top</b>) carbamazepine, (<b>middle left</b>) phenytoin, K = 0.43, (<b>middle right</b>) phenytoin, K = 0.29, (<b>bottom</b><b>left</b>) valproic acid, K = 0.20 and (<b>bottom right</b>) valproic acid, K = 0.042. The broken lines represent the 95% CI (±1.96 SD) and the continuous line is the mean.</p