Characterization of Oxidized Glycerophosphoethanolamines via Radical-Directed Dissociation Tandem Mass Spectrometry and the Paternò-Büchi Derivatization.

Oxidized glycerophosphoethanolamines (oxPEs) represent a subclass of bioactive lipids that have intricate roles in various physiological and pathological events. Conventional mass spectrometric methods cannot provide unambiguous information to locate the OH group and the sites of unsaturation. Herein, we report a combined strategy for in-depth structural characterization of oxPEs, including radical-directed dissociation tandem mass spectrometry (RDD-MS/MS) for localizing the OH group and the Paternò-Büchi derivatization coupled with tandem mass spectrometry for pinpointing carbon-carbon double-bond locations. The RDD-MS/MS method has been integrated on a reversed-phase liquid chromatography-mass spectrometry workflow. It enables the profiling of 24 distinct oxPE molecules with unequivocal assignment of the OH sites at nM sensitivity in bovine liver lipid extract treated by soybean 15-lipoxygenase. These findings showcase that the developed method has a good potential in analyzing biological systems where oxPEs may play important roles.

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.

Improved Annotation of Untargeted Metabolomics Data through Buffer Modifications That Shift Adduct Mass and Intensity.

Annotation of untargeted high-resolution full-scan LC-MS metabolomics data remains challenging due to individual metabolites generating multiple LC-MS peaks arising from isotopes, adducts, and fragments. Adduct annotation is a particular challenge, as the same mass difference between peaks can arise from adduct formation, fragmentation, or different biological species. To address this, here we describe a buffer modification workflow (BMW) in which the same sample is run by LC-MS in both liquid chromatography solvent with 14NH3-acetate buffer and in solvent with the buffer modified with 15NH3-formate. Buffer switching results in characteristic mass and signal intensity changes for adduct peaks, facilitating their annotation. This relatively simple and convenient chromatography modification annotated yeast metabolomics data with similar effectiveness to growing the yeast in isotope-labeled media. Application to mouse liver data annotated both known metabolite and known adduct peaks with 95% accuracy. Overall, it identified 26% of ∼27 000 liver LC-MS features as putative metabolites, of which ∼2600 showed HMDB or KEGG database formula match. This workflow is well suited to biological samples that cannot be readily isotope labeled, including plants, mammalian tissues, and tumors.

Application of a Novel Mass Spectral Data Acquisition Approach to Lipidomic Analysis of Liver Extracts from Sitaxentan-Treated Liver-Humanized PXB Mice.

The application of a data-independent acquisition (DIA) method ("SONAR") that employs a rapidly scanning quadrupole is described for the lipidomic analysis of complex biological extracts. Using this approach, the MS acquisition window can be varied between 1 and 25 Da, enabling the isolation of ions prior to their entering the collision cell. By rapidly scanning the resolving quadrupole window over a specified mass range, co-eluting precursor ions are transmitted sequentially into the collision cell, where collision energies are cycled between low and elevated levels to induce fragmentation. This method of data generation provides both precursor and fragment ion information at high specificity, allowing for greater accuracy of compound identification, whether using a database, spectral libraries, or comparison to authentic standards. The value of the approach in simplifying and "de-cluttering" the spectra of co-eluting lipids is shown with examples from lipidomic profiles obtained in investigations of the composition of organic extracts of livers obtained from SCID and chimeric liver-humanized mice administered under various experimental conditions.

Analysis of cytokine immune response profile in response to inflammatory stimuli in mice with genetic defects in fetal and adult hemoglobin chain expression.

Injections of a crude fetal sheep liver extract (FSLE) containing fetal hemoglobin, MPLA, and glutathione (GSSH) reversed cytokine changes in aged mice. To investigate the role of fetal hemoglobin we derived mice with homzygous deletions for either of the two major ßchains, HgbßmaKO or HgbßmiKO. Hgbßmi is the most prominent fetal Hgbß chain, with Hgbßma more prominent in adult mice. Mice lacking another fetal Hgb chain, HgbεKO, died in utero. CHO cells transfected with cloned Hgb chains were used to produce proteins for preparation of rabbit heteroantibodes. Splenocytes from HgbßmaKO mice stimulated in vitro with Conconavalin A showed a higher IL-2:IL-4 ratio than cells from HgbßmiKO mice. Following immunization in vivo with ovalbumin in alum, HgbßmaKO mice produced less IgE than HgbßmiKO mice, suggesting that in the absence of HgbßmiKO mice had a predeliction to heightened allergic-type responses. Using CHO cells transfected with cloned Hgb chains, we found that only the fetal Hgb chain, Hgbε, was secreted at high levels. Secretion of Hgbßma or Hgbßmi chains was seen only after genetic mutation to introduce the two N-linked glycosylation sites present in Hgbε, but absent in the Hgbß chains. We speculated that a previously unanticipated biological function of a naturally secreted fetal Hgb chain may be partly responsible for the effects reported following injection of animals with fetal, not adult, Hgb. Mice receiving injections of rabbit anti-Hgbε but not either anti-Hgbßma or anti-Hgbßmi from day 14 gestation also showed a bias towards the higher IL-2:IL-4 ratios seen in HgbßmiKO mice.

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.

Which Molecular Features Affect the Intrinsic Hepatic Clearance Rate of Ionizable Organic Chemicals in Fish?

Greater knowledge of biotransformation rates for ionizable organic compounds (IOCs) in fish is required to properly assess the bioaccumulation potential of many environmentally relevant contaminants. In this study, we measured in vitro hepatic clearance rates for 50 IOCs using a pooled batch of liver S9 fractions isolated from rainbow trout (Oncorhynchus mykiss). The IOCs included four types of strongly ionized acids (carboxylates, phenolates, sulfonates, and sulfates), three types of strongly ionized bases (primary, secondary, tertiary amines), and a pair of quaternary ammonium compounds (QACs). Included in this test set were several surfactants and a series of beta-blockers. For linear alkyl chain IOC analogues, biotransformation enzymes appeared to act directly on the charged terminal group, with the highest clearance rates for tertiary amines and sulfates and no clearance of QACs. Clearance rates for C12-IOCs were higher than those for C8-IOC analogues. Several analogue series with multiple alkyl chains, branched alkyl chains, aromatic rings, and nonaromatic rings were evaluated. The likelihood of multiple reaction pathways made it difficult to relate all differences in clearance to specific molecular features the tested IOCs. Future analysis of primary metabolites in the S9 assay is recommended to further elucidate biotransformation pathways for IOCs in fish.

Developmental Stage-Specific Embryonic Induction of HepG2 Cell Differentiation.

BACKGROUND: Although hepatocellular carcinoma cells can sometimes undergo differentiation in an embryonic microenvironment, the mechanism is poorly understood. AIM: The developmental stage-specific embryonic induction of tumor cell differentiation was investigated. METHODS: Both chick and mouse liver extracts and hepatoblast-enriched cells at different developmental stages were used to treat human hepatoma HepG2 cells, and the effects on the induction of differentiation were evaluated. The nuclear factors controlling differentiation, hepatocyte nuclear factor (HNF)-4α, HNF-1α, HNF-6 and upstream stimulatory factor-1 (USF-1), and the oncogene Myc and alpha-fetoprotein (AFP) were measured. HNF-4α RNA interference was used to verify the role of HNF-4α. Embryonic induction effects were further tested in vivo by injecting HepG2 tumor cells into immunodeficient nude mice. RESULTS: The 9-11-days chick liver extracts and 13.5-14.5-days mouse hepatoblast-enriched cells could inhibit proliferation and induce differentiation of HepG2 cells, leading to either death or maturation to hepatocytes. The maturation of surviving HepG2 cells was confirmed by increases in the expressions of HNF-4α, HNF-1α, HNF-6, and USF-1, and decreases in Myc and AFP. The embryonic induction of HepG2 cell maturation could be attenuated by HNF-4α RNA interference. Furthermore, the 13.5-days mouse hepatoblast culture completely eliminated HepG2 tumors with inhibited Myc and induced HNF-4α, confirming this embryonic induction effect in vivo. CONCLUSIONS: This study demonstrated that developmental stage-specific embryonic induction of HepG2 cell differentiation might help in understanding embryonic differentiation and oncogenesis.

Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: activity enhancement by alamethicin, a pore-forming peptide.

1. An existing assay for UDP-glucuronosyltransferase (UGT) activity in trout liver microsomes was optimized using trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5'-diphosphoglucuronic acid (UDPGA), substrate (p-nitrophenol) and alamethicin, a pore-forming agent added to eliminate latency. 2. Addition of Mg2+ (to 1 mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow. 3. When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout. 4. These results clearly demonstrate the advantages of using alamethicin for the removal of latency in UGT activity studies with trout and may have broad implications for the study of UGTs in other fish species.

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.

Homeopathic Oscillococcinum® for preventing and treating influenza and influenza-like illness.

BACKGROUND: Influenza is a highly infectious viral disease that is particularly common in the winter months. Oscillococcinum® is a patented homeopathic medicine that is made from a 1% solution of wild duck heart and liver extract, which is then serially diluted 200 times with water and alcohol. OBJECTIVES: To determine whether homeopathic Oscillococcinum® is more effective than placebo in the prevention and/or treatment of influenza and influenza-like illness in adults or children. SEARCH METHODS: We searched CENTRAL (2014, Issue 8), MEDLINE (1966 to August week 4, 2014), MEDLINE In-Process & Other Non-Indexed Citations (4 September 2014), AMED (2006 to September 2014), Web of Science (1985 to September 2014), LILACS (1985 to September 2014) and EMBASE (1980 to September 2014). We contacted the manufacturers of Oscillococcinum® for information on further trials. SELECTION CRITERIA: Randomised, placebo-controlled trials of Oscillococcinum® in the prevention and/or treatment of influenza and influenza-like illness in adults or children. DATA COLLECTION AND ANALYSIS: Three review authors independently extracted data and assessed risk of bias in the eligible trials. MAIN RESULTS: No new trials were included in this 2014 update. We included six studies: two prophylaxis trials (327 young to middle-aged adults in Russia) and four treatment trials (1196 teenagers and adults in France and Germany). The overall standard of trial reporting was poor and hence many important methodological aspects of the trials had unclear risk of bias. There was no statistically significant difference between the effects of Oscillococcinum® and placebo in the prevention of influenza-like illness: risk ratio (RR) 0.48, 95% confidence interval (CI) 0.17 to 1.34, P value = 0.16. Two treatment trials (judged as 'low quality') reported sufficient information to allow full data extraction: 48 hours after commencing treatment, there was an absolute risk reduction of 7.7% in the frequency of symptom relief with Oscillococcinum® compared with that of placebo (risk difference (RD) 0.077, 95% CI 0.03 to 0.12); the RR was 1.86 (95% CI 1.27 to 2.73; P value = 0.001). A significant but lesser effect was observed at three days (RR 1.27, 95% CI 1.03 to 1.56; P value = 0.03), and no significant difference between the groups was noted at four days (RR 1.11, 95% CI 0.98 to 1.27; P value = 0.10) or at five days (RR 1.06, 95% CI 0.96 to 1.16; P value = 0.25). One of the six studies reported one patient who suffered an adverse effect (headache) from taking Oscillococcinum®. AUTHORS' CONCLUSIONS: There is insufficient good evidence to enable robust conclusions to be made about Oscillococcinum® in the prevention or treatment of influenza and influenza-like illness. Our findings do not rule out the possibility that Oscillococcinum® could have a clinically useful treatment effect but, given the low quality of the eligible studies, the evidence is not compelling. There was no evidence of clinically important harms due to Oscillococcinum®.

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.

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.

Efficacy and safety of human placental extract for alcoholic and nonalcoholic steatohepatitis: an open-label, randomized, comparative study.

Human placental extract (HPE) is a traditional medicine that has been used for the symptomatic treatment of liver disease without any verifying clinical evidence. This study aimed to evaluate the efficacy and safety of HPE in patients with alcoholic or nonalcoholic steatohepatitis (ASH or NASH). We designed this clinical trial as a multicenter, open-label, randomized, comparative noninferiority study to improve the reliability of analyses. The enrollment criteria were limited to ASH or NASH patients with serum alanine aminotransferase (ALT) 1.5-fold higher than the normal level. Patients in the control group were treated with a commercially available mixture of liver extract and flavin adenine dinucleotide (LE­FAD). Intention-to-treat (ITT) analysis was applied to 194 patients, and per-protocol (PP) analysis was available for 154 patients. The rate of primary goal achievement of treatment efficacy was arbitrarily defined as 20% or greater improvement in ALT level compared with the pretreatment level and did not differ significantly between the HPE and control groups [62.9% (44/70) vs. 48.8% (41/84); p=0.0772]. ITT and modified ITT analysis showed results similar to those of PP analysis. Adverse drug reactions (ADRs) of minimal to moderate degree occurred in 3.1% of patients. The ADR and treatment compliance rates were similar in both groups. In conclusion, the clinical value of HPE in the treatment of ASH and NASH is equivalent to that of LE­FAD.

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.

Identification of prolyl oligopeptidase as a cyclosporine-sensitive protease by screening of mouse liver extracts.

Cyclosporine A (CsA) is a cyclic undecapeptide well known for its ability to prevent rejection episodes after organ transplantation via gain-of-function. Therefore, biomedical studies on CsA have been focused on both immunosuppressive properties and binding to the biocatalytically-active immune receptors, the cyclophilins. Much less attention has been spent on effects of cyclosporines on the biological function of other proteins. We used a 9-mer fluorescence-quenched peptide library with defined sequences to identify cyclosporine-sensitive proteolysis in mouse liver extracts. A highly soluble [d-Ser]8-CsA derivative was utilized to avoid drug precipitation at extended incubation times. Analysis of the time courses of proteolysis revealed 15 out of 360 peptide sequences where proteolysis exhibited marked sensitivity to the cyclosporine derivative. As a first example, a collagen-derived substrate was selected from those hits to identify the targeted proteolytic pathway. After purification from mouse liver extracts, prolyl oligopeptidase (EC 3.4.21.26) could be identified as a protease sensitive to submicromolar concentrations of cyclosporines. Surprisingly, in a series of cyclosporine derivatives an inverse relationship was found between the inhibition of prolyl oligopeptidase and inhibition of cyclophilin A.

Liver hydrolysate assists in the recovery from physical fatigue in a mouse model.

It is reported that liver hydrolysate (LH) enhances liver function. However, the effects of LH on physical fatigue are unknown. The aim of this study was to investigate the effect of LH on alterations in locomotor activity and energy metabolism such as 5'-AMP-activated protein kinase (AMPK), glycogen content, and blood lactic acid, after forced walking. Adult male ddY mice were used. Locomotor activity, AMPK phosphorylation, and glycogen content in the liver and soleus muscle, as well as blood lactic acid were determined following LH treatment before and/or after forced walking. The locomotor activity significantly decreased after forced walking for 3 h. Two administrations of LH (30 or 100 mg/kg) significantly increased the locomotor activity, while a single administration either before or after forced walking did not show any specific effect. Administering LH twice activated AMPK in the liver and soleus muscle. Glycogen levels significantly decreased in both the liver and soleus muscle after forced walking, whereas the blood lactate level significantly increased. In contrast, administering LH twice increased muscle glycogen and decreased blood lactic acid. These findings indicate that LH produced an anti-fatigue effect and that this effect appears to involve the efficient glycogen utilization through activation of AMPK.

The phytochemical curcumin inhibits steroid sulfatase activity in rat liver tissue and NIH-3T3 mouse fibroblast cells.

Curcumin is a phytochemical derived from the spice turmeric that is reported to have therapeutic effects. We are studying the enzyme steroid sulfatase (STS), which removes the sulfate group from inactive steroid hormones in peripheral tissues and we were interested in the effect of curcumin on STS activity due to its structural composition (polyphenolic). We sought to determine if curcumin affects STS activity in two model systems, rat liver and NIH-3T3 mouse fibroblast cells. STS assays were performed on tissue extracts of rat liver, and on NIH-3T3 microsomes and cells, with and without curcumin. Male and female rat liver extracts contained substantial amounts of STS activity, with males averaging higher (4-11 %) levels. Estradiol inhibited STS activity in livers of both sexes at 20 and 10 µM. Curcumin acted as a competitive inhibitor of STS activity in rat liver extracts, with a Ki of 19.8 µM in males and 9.3 µM in females. Curcumin also inhibited STS activity in NIH-3T3 microsomes at both 20 µM and 10 µM, and in whole NIH-3T3 cells at 20 µM. These data are the first to demonstrate STS inhibition by curcumin. Inhibition of STS results in lower active steroid hormone (estrogens and androgens) levels in tissues, possibly altering modulation of immune responses by these steroids.

Homeopathic Oscillococcinum(®) for preventing and treating influenza and influenza-like illness.

BACKGROUND: Influenza is a highly infectious viral disease that is particularly common in the winter months. Oscillococcinum(®) is a patented homeopathic medicine that is made from a 1% solution of wild duck heart and liver extract, which is then serially diluted 200 times with water and alcohol. OBJECTIVES: To determine whether homeopathic Oscillococcinum(®) is more effective than placebo in the prevention and/or treatment of influenza and influenza-like illness in adults or children. SEARCH METHODS: We searched CENTRAL (2012, Issue 7), MEDLINE (1966 to July week 4, 2012), MEDLINE In-Process & Other Non-Indexed Citations (6 August 2012), AMED (2006 to August 2012), Web of Science (1985 to August 2012), LILACS (1985 to August 2012) and EMBASE (1980 to August 2012). We contacted the manufacturers of Oscillococcinum(®) for information of more trials. SELECTION CRITERIA: Randomised, placebo-controlled trials of Oscillococcinum(®) in the prevention and/or treatment of influenza and influenza-like illness in adults or children. DATA COLLECTION AND ANALYSIS: Three review authors independently extracted data and assessed risk of bias in the eligible trials. MAIN RESULTS: We included six studies: two prophylaxis trials (327 young to middle-aged adults in Russia) and four treatment trials (1196 teenagers and adults in France and Germany). The overall standard of trial reporting was poor and hence many important methodological aspects of the trials had unclear risk of bias. There was no statistically significant difference between the effects of Oscillococcinum(®) and placebo in the prevention of influenza-like illness: risk ratio (RR) 0.48, 95% confidence interval (CI) 0.17 to 1.34, P = 0.16. Two treatment trials (judged as 'low quality') reported sufficient information to allow full data extraction: 48 hours after commencing treatment, there was an absolute risk reduction of 7.7% in the frequency of symptom relief with Oscillococcinum(®) compared with that of placebo (risk difference (RD) 0.077; 95% CI 0.03 to 0.12); the RR was 1.86 (95% CI 1.27 to 2.73; P = 0.001). A significant but lesser effect was observed at three days (RR 1.27, 95% CI 1.03 to 1.56; P = 0.03), and no significant difference between the groups was noted at four days (RR 1.11, 95% CI 0.98 to 1.27; P = 0.10) or at five days (RR 1.06; 95% CI 0.96 to 1.16; P = 0.25). One of the six studies reported one patient who suffered an adverse effect (headache) from taking Oscillococcinum(®). AUTHORS' CONCLUSIONS: There is insufficient good evidence to enable robust conclusions to be made about Oscillococcinum(®) in the prevention or treatment of influenza and influenza-like illness. Our findings do not rule out the possibility that Oscillococcinum(®) could have a clinically useful treatment effect but, given the low quality of the eligible studies, the evidence is not compelling. There was no evidence of clinically important harms due to Oscillococcinum(®).

Proton nuclear magnetic resonance and pattern recognition analysis of liver extracts from rats under different anesthetics.

BACKGROUND: Although general anesthesia is widely used in the surgical arena, the mechanisms by which general anesthetics act remain unclear. We previously described alterations in gene expression ratios in hepatic tissue taken from rats treated with anesthetics. Consequently, it is considered that anesthetics influence liver metabolism. Thus, the goal of this study was to use pattern recognition analysis of proton nuclear magnetic resonance spectra to visualize changes in liver metabolic phenotypes in response to widely used intravenous anesthetics (propofol and dexmedetomidine) and inhalational anesthetics (sevoflurane and isoflurane). METHODS: Rats were randomized into 13 groups (n = 6 in each group), and each group received one of following agents: propofol, dexmedetomidine, sevoflurane, isoflurane, or no anesthetic (control group). The liver was directly removed from rats immediately after or 24 h or 48 h after a 6-h period of anesthesia. Hydrophilic compounds were extracted from the liver and were analyzed with proton nuclear magnetic resonance spectroscopy. All spectral data were processed and analyzed by principal component analysis for comparison of metabolite profiles. RESULTS: Data were visualized by plotting principal component (PC) scores. In the plots, each point represents an individual sample. Each group was clustered separately on the plots, and the PC scores of the propofol group were clearly distinct from those of the control group and other anesthetic groups. The difference in PC scores was more pronounced immediately after completion of anesthesia when compared with 24 or 48 h after completion of anesthesia. Although the effect of intravenous anesthetics on the liver dissipated over time, the effect of inhalational anesthetics persisted. CONCLUSIONS: Propofol, dexmedetomidine, sevoflurane and isoflurane exert different effects on liver metabolism. In particular, liver metabolism was markedly altered after exposure to propofol. The effect of anesthesia on the liver under propofol or dexmedetomidine resolved rapidly when compared with the effect under sevoflurane or isoflurane.