Evaluation of the Performance of Lipidyzer Platform and Its Application in the Lipidomics Analysis in Mouse Heart and Liver.

Lipids play important roles in cell signaling, energy storage, and as major structural components of cell membranes. To date, little work has been conducted to show the extent of tissue specificity of lipid compositions. Here, the recently acquired Lipidyzer platform was employed in this pilot study: (i) to assess the performance of the Lipidyzer platform, (ii) to explore lipid profiles in liver and cardiac tissue in mice, (iii) to examine sex-specific differences in lipids in the liver tissue, and (iv) to evaluate biological variances in lipidomes present in animals. In total, 787 lipid species from 13 lipid classes were measured in the liver and heart. Lipidomics data from the Lipidyzer platform were very reproducible with the coefficient of variations of the quality control (QC) samples, ∼10%. The total concentration of the cholesterol esters (CE) lipid class, and specifically CE(16:1) and CE(18:1) species, showed sex differences in the liver. Cardiac tissue had higher levels of phospholipids containing docosahexaenoic acid, which could be related to heart health status and function. Our results demonstrate the usefulness of the Lipidyzer platform in identifying differences in lipid profile at the tissue level and between male and female mice in specific tissues.

An Integrated Analysis of Metabolites, Peptides, and Inflammation Biomarkers for Assessment of Preanalytical Variability of Human Plasma.

Discrepancies in blood sample collection and processing could have a significant impact on levels of metabolites, peptides, and protein biomarkers of inflammation in the blood; thus, sample quality control is critical for successful biomarker identification and validation. In this study, we analyzed the effects of several preanalytical processing conditions, including different storage times and temperatures for blood or plasma samples and different centrifugation forces on the levels of metabolites, peptides, and inflammation biomarkers in human plasma samples using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Temperature was found to be the major factor for metabolite variation, and both time and temperature were identified as major factors for peptide variation. For inflammation biomarkers, temperature played different roles depending on the sample type (blood or plasma). Low temperature affected inflammation biomarkers in blood, while room temperature impacted inflammation biomarkers in plasma.

Stability of the Human Plasma Proteome to Pre-analytical Variability as Assessed by an Aptamer-Based Approach.

Variable processing and storage of whole blood and/or plasma are potential confounders in biomarker development and clinical assays. The goal of the study was to investigate how pre-analytical variables impact the human plasma proteome. Whole blood obtained from 16 apparently healthy individuals was collected in six EDTA tubes and processed randomly under six pre-analytical variable conditions including blood storage at 0 °C or RT for 6 h (B6h0C or B6hRT) before processing to plasma, plasma storage at 4 °C or RT for 24 h (P24h4C or P24hRT), low centrifugal force at 1300 × g, (Low×g), and immediate processing to plasma under 2500 × g (control) followed by plasma storage at -80 °C. An aptamer-based proteomic assay was performed to identify significantly changed proteins (fold change ≥1.2, P < 0.05, and false discovery rate < 0.05) relative to the control from a total of 1305 proteins assayed. Pre-analytical conditions Low×g and B6h0C resulted in the most plasma proteome changes with 200 and 148 proteins significantly changed, respectively. Only 36 proteins were changed under B6hRT. Conditions P24h4C and P24hRT yielded changes of 28 and 75 proteins, respectively. The complement system was activated in vitro under the conditions B6hRT, P24h4C, and P24hRT. The results suggest that particular pre-analytical variables should be controlled for clinical measurement of specific biomarkers.

Testicular function in cultured postnatal mouse testis fragments is similar to that of animals during the first wave of spermatogenesis.

BACKGROUND: Previously, we evaluated optimal organ culture conditions to produce elongated spermatids in an in vitro mouse testis culture system. However, differences in testicular function between the cultured testis fragments and animal testis have not been determined. METHODS: To examine how closely cultured testis fragments in vitro approximates what typically occurs during the first wave of spermatogenesis in vivo, C57BL/6J mouse testis fragments obtained on postnatal day (PND) 5 were cultured in AlbuMAX™ I/ α-Minimal Essential Medium for 15, 23, 30, 35, 42, and 49 days, and compared to mouse testes obtained at PND 5, 14, 20, 24, 28, 30, 35, and 40. At the specified days of culture or PND of mice, the following analyses were conducted: histology, flow cytometry for haploid cell detection, qPCR for spermatid markers, and liquid chromatography/mass spectrometry for testosterone levels. RESULTS: Round spermatids were initially observed at 23 days, and their percentage of the total number of cells continued to increase with culture time, as did gene expression of the spermatid markers and haploid cell percentage in the cultured testis fragments. These results were similar in temporal sequence to those in animals. Testosterone levels in the testis fragments reached a maximum at Day 49. CONCLUSION: These findings show this in vitro mouse testis organ culture model may be a useful and convenient tool for mechanistic studies. However, because germ cell differentiation in all seminiferous tubules was not observed, improvements in the system/methods are needed to more closely replicate spermatogenesis as observed in animals.

Metabonomics evaluation of urine from rats given acute and chronic doses of acetaminophen using NMR and UPLC/MS.

Urinary metabolic perturbations associated with acute and chronic acetaminophen-induced hepatotoxicity were investigated using nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography/mass spectrometry (UPLC/MS) metabonomics approaches to determine biomarkers of hepatotoxicity. Acute and chronic doses of acetaminophen (APAP) were administered to male Sprague-Dawley rats. NMR and UPLC/MS were able to detect both drug metabolites and endogenous metabolites simultaneously. The principal component analysis (PCA) of NMR or UPLC/MS spectra showed that metabolic changes observed in both acute and chronic dosing of acetaminophen were similar. Histopathology and clinical chemistry studies were performed and correlated well with the PCA analysis and magnitude of metabolite changes. Depletion of antioxidants (e.g. ferulic acid), trigonelline, S-adenosyl-L-methionine, and energy-related metabolites indicated that oxidative stress was caused by acute and chronic acetaminophen administration. Similar patterns of metabolic changes in response to acute or chronic dosing suggest similar detoxification and recovery mechanisms following APAP administration.

Determination of chloral hydrate and its metabolites in blood plasma by capillary gas chromatography with electron capture detection.

A sensitive, accurate, and reliable method is described for the quantitative determination of chloral hydrate (CH) and its metabolites in blood plasma of mice and rats. Metabolites of CH include trichloroacetic acid (TCA), trichloroethanol (TCE), and trichloroethanol glucuronide (TCE-Glu). This new method uses capillary gas chromatography with electron-capture detection (GC/ECD). Procedures for improving sample stability and quality assurance are also described that were not mentioned in previous literature. Rat or mouse plasma (50 microl) is acidified (or treated enzymatically for TCE-Glu determination) and extracted with peroxide free methyl t-butyl ether. Distilled diazomethane (CH(2)N(2)) is added to derivatize TCA to its methyl ester. Detection limits were estimated at 0.2 microg/ml for CH and TCE, and 0.1 microg/ml for TCA. Detector response to TCA and TCE were shown to be linear in the range of 3.125-200 microg/ml (r> or =0.9996). For CH, the response fits a second-order equation in this same range (r=0.99994)

Assessment of usnic acid toxicity in rat primary hepatocytes using ¹³C isotopomer distribution analysis of lactate, glutamate and glucose.

The lichen metabolite usnic acid (UA) has been promoted as a dietary supplement for weight loss, although cases of hepatotoxicity have been reported. Here we evaluated UA-associated hepatotoxicity in vitro using isolated rat hepatocytes. We measured cell viability and ATP content to evaluate UA induced cytotoxicity and applied (13)C isotopomer distribution measuring techniques to gain a better understanding of glucose metabolism during cytotoxicity. The cells were exposed to 0, 1, 5 or 10 µM UA concentrations for 2, 6 or 24h. Aliquots of media were collected at the end of these time periods and the (13)C mass isotopomer distribution determined for CO(2), lactate, glucose and glutamate. The 1 µM UA exposure did not appear to cause significant change in cell viability compared to controls. However, the 5 and 10 µM UA concentrations significantly reduced cell viability as exposure time increased. Similar results were obtained for ATP depletion experiments. The 1 and 5 µM UA doses suggest increased oxidative phosphorylation. Conversely, oxidative phosphorylation and gluconeogenesis were dramatically inhibited by 10 µM UA. Augmented oxidative phosphorylation at the lower UA concentrations may be an adaptive response by the cells to compensate for diminished mitochondrial function.