Identification of Mid-Polar and Polar AhR Agonists in Cetaceans from Korean Coastal Waters: Application of Effect-Directed Analysis with Full-Scan Screening.

Major aryl hydrocarbon receptor (AhR) agonists were identified in extracts of blubber, liver, and muscle from six long-beaked common dolphins (Delphinus capensis) and one fin whale (Balaenoptera physalus) collected from Korean coastal waters using effect-directed analysis. Results of the H4IIE-luc bioassay indicated that the polar fractions of blubber and liver extracts from the fin whale exhibited relatively high AhR-mediated potencies. Based on full-scan screening with high-resolution mass spectrometry, 37 AhR agonist candidates, spanning four use categories: pharmaceuticals, pesticides, cosmetics, and natural products, were selected. Among these, five polar AhR agonists were newly identified through toxicological confirmation. Concentrations of polar AhR agonists in cetaceans were tissue-specific, with extracts of blubber and liver containing greater concentrations than muscle extracts. Polar AhR agonists with great log KOA values (>5) were found to biomagnify in the marine food chain potentially. Polar AhR agonists contributed 8.9% of the observed AhR-mediated potencies in blubber and 49% in liver. Rutaecarpine and alantolactone contributed significantly to the total AhR-mediated potencies of blubber, whereas hydrocortisone was a major AhR contributor in the liver of the fin whale. This study is the first to identify the tissue-specific accumulation of polar AhR agonists in blubber and liver extracts of cetaceans.

Shotgun Lipidomics Approach to Stabilize the Regiospecificity of Monoglycerides Using a Facile Low-Temperature Derivatization Enabling Their Definitive Identification and Quantitation.

Monoglycerides play a central role in lipid metabolism and are important signaling metabolites. Quantitative analysis of monoglyceride molecular species has remained challenging due to rapid isomerization via α-hydroxy acyl migration. Herein, we describe a shotgun lipidomics approach that utilizes a single-phase methyl tert-butyl ether extraction to minimize acyl migration, a facile low temperature diacetyl derivatization to stabilize regiospecificity, and tandem mass spectrometric analysis to identify and quantify regioisomers of monoglycerides in biological samples. The rapid and robust diacetyl derivatization at low temperatures (e.g., -20 °C, 30 min) prevents postextraction acyl migration and preserves regiospecificity of monoglyceride structural isomers. Furthermore, ionization of ammonium adducts of diacetyl monoglyceride derivatives in positive-ion mode markedly increases analytic sensitivity (low fmol/µL). Critically, diacetyl derivatization enables the differentiation of discrete monoglyceride regioisomers without chromatography through their distinct signature fragmentation patterns during collision induced dissociation. The application of this approach in the analysis of monoglycerides in multiple biologic tissues demonstrated diverse profiles of molecular species. Remarkably, the regiospecificity of individual monoglyceride molecular species is also diverse from tissue to tissue. Collectively, this developed approach enables the profiling, identification and quantitation of monoglyceride regioisomers directly from tissue extracts.

Toxic Identification and Evaluation of Androgen Receptor Antagonistic Activities in Acid-Treated Liver Extracts of High-Trophic Level Wild Animals from Japan.

Sulfuric acid-treated liver extracts of representative high-trophic level Japanese animals were analyzed by toxic identification and evaluation (TIE) with chemically activated luciferase expression (CALUX) and chemical analysis to elucidate androgen receptor (AR) antagonistic activities and potential contributions of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs). The activities were detected in striped dolphins (n = 5), Stejneger's beaked whales (n = 6), golden eagle (n = 1), and Steller's sea eagle (n = 1) with CALUX-flutamide equivalents (FluEQs) as follow: 38 (20-52), 47 (21-96), 5.0, and 80 µg FluEQ/g-lipid, respectively. The AR antagonism was detected in limited number of specimens at lower levels for finless porpoise, raccoon dog, and common cormorant. Theoretical activities (Theo-FluEQs) were calculated using the concentration of OCPs and PCBs and their IC25-based relative potency (REP) values. These total contribution to CALUX-FluEQ was 126%, 84%, 53%, 55%, and 44% for striped dolphin, Steller's sea eagle, Stejneger's beaked whale, finless porpoise, and golden eagle, respectively, and the main contributor was p,p'-DDE. However, most of the activities for raccoon dog (7.6%) and common cormorant (17%) could not be explained by OCPs and PCBs. This suggests other unknown compounds could function as AR antagonists in these terrestrial species.

Characterization and quantification of diacylglycerol species in biological extracts after one-step derivatization: a shotgun lipidomics approach.

Diacylglycerols (DAGs) are important intermediates of lipid metabolism and cellular signaling. It is well-known that the mass levels of DAG are altered under disease states. Therefore, quantitative analysis of DAGs in biological samples can provide critical information to uncover underlying mechanisms of various cellular functional disorders. Although great efforts on the analysis of individual DAG species have recently been made by utilizing mass spectrometry with or without derivatization, cost-effective and high throughput methodologies for identification and quantification of all DAG species including regioisomers, particularly in an approach of shotgun lipidomics, are still missing. Herein, we described a novel method for directly identifying and quantifying DAG species including regioisomers present in lipid extracts of biological samples after facile one-step derivatization with dimethylglycine based on the principles of multidimensional mass spectrometry-based shotgun lipidomics. The established method provided substantial sensitivity (low limit of quantification at amol/µL), high specificity, and broad linear dynamics range (2500-fold) without matrix effects. By exploiting this novel method, we revealed a 16-fold increase of total DAG mass in the livers of ob/ob mice compared to their wild type controls at 4 months of age (an insulin-resistant state) versus a 5-fold difference between 3 month old mice (with normal insulin). These results demonstrated the importance and power of the method for studying biochemical mechanisms underpinning disease states.

Data dependent peak model based spectrum deconvolution for analysis of high resolution LC-MS data.

A data dependent peak model (DDPM) based spectrum deconvolution method was developed for analysis of high resolution LC-MS data. To construct the selected ion chromatogram (XIC), a clustering method, the density based spatial clustering of applications with noise (DBSCAN), is applied to all m/z values of an LC-MS data set to group the m/z values into each XIC. The DBSCAN constructs XICs without the need for a user defined m/z variation window. After the XIC construction, the peaks of molecular ions in each XIC are detected using both the first and the second derivative tests, followed by an optimized chromatographic peak model selection method for peak deconvolution. A total of six chromatographic peak models are considered, including Gaussian, log-normal, Poisson, gamma, exponentially modified Gaussian, and hybrid of exponential and Gaussian models. The abundant nonoverlapping peaks are chosen to find the optimal peak models that are both data- and retention-time-dependent. Analysis of 18 spiked-in LC-MS data demonstrates that the proposed DDPM spectrum deconvolution method outperforms the traditional method. On average, the DDPM approach not only detected 58 more chromatographic peaks from each of the testing LC-MS data but also improved the retention time and peak area 3% and 6%, respectively.

Endogenous and xenobiotic metabolite profiling of liver extracts from SCID and chimeric humanized mice following repeated oral administration of troglitazone.

1. Metabonomic analysis, via a combination of untargeted and targeted liquid chromatography-mass spectrometry (LC-MS) and untargeted (1)H NMR spectroscopy-based metabolite profiling, was performed on aqueous (AQ) and organic liver extracts from control (SCID) and chimeric humanized (PXB) mice dosed with troglitazone at 0, 300 and 600 mg/kg/day for seven days. 2. LC-MS analysis of AQ liver extracts showed a more "human-like" profile for troglitazone metabolites for PXB, compared with SCID, mice. 3. LC-MS detected differences in endogenous metabolites, particularly lipid species in dosed mice, including elevated triacylglycerols and 1-alkyl,2-acylglycerophosphates as well as lowered diacylglycerophosphocholines and 1-alkyl,2-acylglycerophosphocholines for PXB compared with SCID mouse liver extracts. Following drug administration changes in the relative proportions of the ions for various unsaturated fatty acids were observed for both types of mouse, some of which were specific to PXB or SCID mice. 4. (1)H NMR spectroscopy revealed that AQ PXB mouse liver extracts had elevated amounts of inosine, fumarate, creatine, aspartate, trimethylamine N-oxide, glycerophosphocholine, phosphocholine, choline, glutamine, glutamate, acetate, alanine and lactate relative to SCID mice and decreased histidine, glycogen, α- and ß-glucose, taurine, and glutathione. Increased uracil and tyrosine concentrations were detected for PXB mice on troglitazone administration. 5. Metabonomic profiling thus showed clear differences between humanized and SCID mice, including after administration of troglitazone.

Simultaneous determination of rosiglitazone and its metabolites in rat liver microsomal fraction using hollow-fiber liquid-phase microextraction for sample preparation.

A three-phase hollow-fiber liquid-phase microextraction method for the analysis of rosiglitazone and its metabolites N-desmethyl rosiglitazone and ρ-hydroxy rosiglitazone in microsomal preparations is described for the first time. The drug and metabolites HPLC determination was carried out using an X-Terra RP-18 column, at 22°C. The mobile phase was composed of water, acetonitrile and acetic acid (85:15:0.5, v/v/v) and the detection was performed at 245 nm. The hollow-fiber liquid-phase microextraction procedure was optimized using multifactorial experiments and the following optimal condition was established: sample agitation at 1750 rpm, extraction for 30 min, hydrochloric acid 0.01 mol/L as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 8.0. The recovery rates, obtained by using 1 mL of microsomal preparation, were 47-70%. The method presented LOQs of 50 ng/mL and it was linear over the concentration range of 50-6000 ng/mL, with correlation coefficients (r) higher than 0.9960, for all analytes. The validated method was employed to study the in vitro biotransformation of rosiglitazone using rat liver microsomal fraction.

Isolation of a Golgi apparatus-rich fraction from rat liver. IV. Thiamine pyrophosphatase.

The thiamine pyrophosphatase (the enzyme [s] catalyzing the release of inorganic phosphate with thiamine pyrophosphate as the substrate) activities of Golgi apparatus-, plasma membrane-, endoplasmic reticulum-, and mitochondria-rich fractions from rat liver were compared at pH 8. Activity was concentrated in the Golgi apparatus fractions, which, on a protein basis, had a specific activity six to eight times that of the total homogenates or purified endoplasmic reticulum fractions. However, only 1-3% of the total activity was recovered in the Golgi apparatus fractions under conditions where 30-50% of the UDPgalactose:N-acetylglucosamine-galactosyl transferase activity was recovered. Considering both recovery of galactosyl transferase and fraction purity, we estimate that approximately 10% of the total thiamine pyrophosphatase activity of the liver was localized within the Golgi apparatus, with a specific activity of about ten times that of the total homogenate. Cytochemically, reaction product was found in the cisternae of the endoplasmic reticulum as well as in the Golgi apparatus. This is in contrast to results obtained in most other tissues, where reaction product was restricted to the Golgi apparatus. Thus, enzymes of rat liver catalyzing the hydrolysis of thiamine pyrophosphate, although concentrated in the Golgi apparatus, are widely distributed among other cell components in this tissue.

Enzyme-catalyzed reactions of the carcinogen N-hydroxy-2-fluorenylacetamide with nucleic acid.

A protein fraction obtained by gel filtration of a 105,000g supernatant of rat liver catalyzes three reactions of the hepatocarcinogen N-hydroxy-2-fluorenylacetamide with nucleic acid. Cofactor requirements and isotopic studies suggest that the reactive intermediates involved may be N-2-fluorenylhydroxylamine, and phosphate and sulfate esters of N-hydroxy-2-fluorenylacetamide.

DNA polymerase activities associated with smooth membranes and ribosomes from rat liver and hepatoma cytoplasm.

Deoxyribonucleic acid polymerase activity that prefers denatured DNA primer concentrates with the smooth membranes during sucrose gradient centrifugation of rat liver and hepatoma cytoplasmic extracts. The activity in this fraction is eightfold higher in hepatoma tissue. Deoxyribonucleic acid polymerase activity that prefers native DNA primer concentrates with the free ribosome fraction from both tissues.

Histological progression of non-alcoholic fatty liver disease: a critical reassessment based on liver sampling variability.

BACKGROUND: In non-alcoholic fatty liver disease, histological lesions display a significant sampling variability that is ignored when interpreting histological progression during natural history or therapeutic interventions. AIM: To provide a method taking into account sampling variability when interpreting crude histological data, and to investigate how this alters the conclusions of available studies. METHODS: Natural history studies detailing histological progression and therapeutic trials were compared with the results of a previously published sampling variability study. RESULTS: Natural history studies showed an improvement in steatosis, which was significantly higher than expected from sampling variability (47% vs. 8%, P < 0.0001). In contrast, no study showed a change in activity grade or ballooning higher than that of sampling variability. There was only a marginal effect on fibrosis with no convincing demonstration of a worsening of fibrosis, a conclusion contrary to what individual studies have claimed. Some insulin sensitizing drugs and anti-obesity surgery significantly improved steatosis, while most did not significantly impact on fibrosis or activity. CONCLUSIONS: Sampling variability of liver biopsy is an overlooked confounding factor that should be considered systematically when interpreting histological progression in patients with non-alcoholic fatty liver disease.

Determination of five 4-hydroxycoumarin rodenticides in animal liver tissues by ion chromatography with fluorescence detection.

A novel analytical method is proposed for rapid simultaneous determination of five 4-hydroxycoumarin rodenticides in animal liver tissues by eluent generator reagent free ion chromatography (RFIC) with fluorescence detection. Rodenticides were initially extracted from homogenized animal liver tissues with ethyl acetate and the extracts subjected to a solid-phase extraction process using Oasis HLB cartridges. The IC separation was carried out on an IonPac AS11 analytical column (250 mm x 4.0 mm) using gradient KOH containing 10% acetonitrile as organic modifier at a constant flow rate of 1.0 mL/min. The analytes were detected by fluorescence at an excitation wavelength of 270 nm and an emission wavelength of 380 nm. The average recoveries of the objective compounds spiked in animal liver tissues were between 81% and 98%. The limits of quantification (LOQs) were 0.004-0.010 mg/kg for them. Within-day and day-to-day relative standard deviations (RSD) were less than 8.5% and 9.7%, respectively. It was confirmed that this method could be used in a toxicological analysis.

Evaluation of strategy for analyzing mouse liver plasma membrane proteome.

Plasma membrane (PM) proteome is one of the major subproteomes present in the cell, and is very important in liver function. In the present work, C57 mouse liver PM was purified by density-gradient centrifugation. The purified PM was verified by electron microscope analysis and Western blotting. The results showed that the PM was enriched by more than 20-fold and the contamination of mitochondria was reduced by 2-fold compared with the homogenization fraction. Proteins were separated by 2DE and 1DE, trypsin-digested and submitted to ESI-Q-TOF and MALDI-TOF-TOF mass spectrometry or directly digested in solution and analyzed by LC-ESI ion trap mass spectrometry. In all, 547 non-redundant mouse liver PM proteins were identified, of which 34% contributed to plasma membrane or plasma membrane-related proteins. This study optimized and evaluated the HLPP plasma membrane proteome analysis method and made a systematic analysis on PM proteome.

Effect of coenzyme Q10 on catalase activity and other antioxidant parameters in streptozotocin-induced diabetic rats.

Although coenzyme Q10 (CoQ10) is a component of the oxidative phosphorylation process in mitochondria that converts the energy in carbohydrates and fatty acids into ATP to drive cellular machinery and synthesis, its effect in type I diabetes is not clear. We have studied the effect of 4 wk of treatment with CoQ10 (10 mg/kg, ip, daily) in streptozotocin (STZ)-induced (40 mg/kg, iv in adult rats) type I diabetes rat models. Treatment with CoQ10 produced a significant decrease in elevated levels of glucose, cholesterol, triglycerides, very-low-density lipoprotein, low-density lipoprotein, and atherogenic index and increased high-density lipoprotein cholesterol levels in diabetic rats. CoQ10 treatment significantly decreased the area under the curve over 120 min for glucose in diabetic rats, without affecting serum insulin levels and the area under the curve over 120 min for insulin in diabetic rats. CoQ10 treatment also reduced lipid peroxidation and increased antioxidant parameters like superoxide dismutase, catalase, and glutathione in the liver homogenates of diabetic rats. CoQ10 also lowered the elevated blood pressure in diabetic rats. In conclusion, CoQ10 treatment significantly improved deranged carbohydrate and lipid metabolism of experimental chemically induced diabetes in rats. The mechanism of its beneficial effect appears to be its antioxidant property.

Comparison of Golgi apparatus and endoplasmic reticulum proteins from livers of juvenile and aged rats using a novel technique for separation and enrichment of organelles.

The broad dynamic range of protein abundances, which can vary from about 10(6) for cells to 10(10) for tissues in complex proteomes, continues to challenge proteomics research. Proteome analysis, in particular organelle proteomics, using current approaches, requires extensive fractionation, separation, and enrichment. Over the years, organelle separation was achieved through the use of differential and density-gradient ultracentrifugation. However, the traditional fixed-volume process is a time-consuming and labor-intensive method, especially with large quantities of sample. Here, we present a novel tool for subcellular fractionation of biologically complex mixtures: continuous-flow ultracentrifugation of tissue homogenates to obtain both organelle separation and extensive organelle enrichment at the same time. In this study, rat liver tissues from two different age groups (3-8 wk and greater than 1 y old) were homogenized by blending. After removing nuclei, the resulting homogenates were further fractionated at the subcellular level by the use of sucrose gradient continuous-flow ultracentrifugation. Each organelle's enriched fractions were identified by Western blot analysis. To study the possible effects of aging on the endoplasmic reticulum and Golgi apparatus, we compared the organelle protein profiles of the two groups of rat liver tissues using two-dimensional gel electrophoresis, digitized imaging of two-dimensional gel electrophoresis, and mass spectrometry. Significant differences in the protein profiles of both organelles were observed between the two groups of rat tissues. The technique described here for fractionation and enrichment of organelles demonstrated a useful tool for proteomics research, including identification of low-abundance proteins and post-translational modifications.

Effects of human fetal liver extract on the growth of HL-60 cells.

Two kinds of HL-60 cell growth suppressor present in human fetal liver were studied. One is the already known arginase, which showed nonspecific suppression on the growth of HL-60 cells and human granulocyte-macrophage progenitors (CFU-GM), and the other, a new species of suppressor with the characteristics of lower molecular weight (less than 10,000 daltons), shows preferential suppression on HL-60 cell growth, but less effect on the growth of CFU-GM.

Absorption and metabolism of delphinidin 3-O-beta-D-glucopyranoside in rats.

The absorption and metabolism of delphinidin 3-O-beta-d-glucopyranoside (Dp3G), which is the most potent antioxidant among the blueberry anthocyanins, were studied in rats. Dp3G rapidly appeared in the blood plasma within 15 min of oral administration (100 mg/kg body wt). The plasma level of absorbed Dp3G showed two peaks at 15 and 60 min after ingestion and then decreased time-dependently. However, the plasma level was maintained at approximately 30 nmol/l even after 4 h. Besides the Dp3G peak, a single major metabolite peak was detected by HPLC in the blood plasma obtained at 15 min. MS and NMR spectroscopy clarified that the chemical structure of the metabolite was 4'-O-methyl delphinidin 3-O-beta-d-glucopyranoside (methylation of the 4'-OH on the delphinidin B-ring). The present finding of this unique metabolite in anthocyanin metabolism strongly suggests that methylation of the 4'-OH on the flavonoid B-ring is a common metabolic pathway for flavonoids that carry the pyrogallol structure on the B-ring, as the same type of metabolite has been reported for other flavonoids such as epigallocatechin, but not for flavonoids carrying the catechol structure.

Determination of the molecular weight of DNA-binding proteins using UV-crosslinking and SDS-PAGE.

We describe the use of UV-crosslinking in combination with SDS-PAGE to determine the approximate molecular weight of DNA-binding proteins. A 5-bromo-2'-deoxyuridine (5-BrdU)-substituted, radioactively labeled double-stranded oligonucleotide representing the protein binding site is incubated with a crude nuclear extract containing the protein of interest. Following irradiation with a UV light source, the DNA/protein complex is subjected to SDS-PAGE and its molecular weight determined by comparison with appropriate protein standards.

HPLC/atmospheric pressure chemical ionization-mass spectroscopy of eight regulated sulfonamides.

Reversed phase high performance liquid chromatography coupled with on-line atmospheric pressure chemical ionization mass spectrometry, HPLC,APCI-MS, has been applied to a mixture of eight sulfonamides. In full scan mode, extracted ion chromatograms produced minimum detectable quantities (MDQ) of 0.8 ng on column, for six of the eight regulated sulfonamides investigated. Selected ion monitoring yielded a 50 pg MDQ for sulfamerazine, sulfadiazine and sulfamethazine, while, the other compounds presented higher values. Analysis of supercritical fluid extracts of chicken liver containing sulfadimethoxine were found to be easily detected by HPLC/APCI-MS. In extracts of chicken liver spiked with 25 microg/kg(-1) (25 ppb) of sulfadimethoxine this compound could be detected in selected ion mode, while 100 pg/microl(-1) was detectable in either full scan or single ion modes. The analysis method for extracted sulfadimethoxine also demonstrated good linearity and reproducibility in both single ion and scan mode.