Si Mi

Oxidative stress contributes to the tamoxifen-induced killing of breast cancer cells: implications for tamoxifen therapy and resistance.

Tamoxifen is the accepted therapy for patients with estrogen receptor-α (ERα)-positive breast cancer. However, clinical resistance to tamoxifen, as demonstrated by recurrence or progression on therapy, is frequent and precedes death from metastases. To improve breast cancer treatment it is vital to understand the mechanisms that result in tamoxifen resistance. This study shows that concentrations of tamoxifen and its metabolites, which accumulate in tumors of patients, killed both ERα-positive and ERα-negative breast cancer cells. This depended on oxidative damage and anti-oxidants rescued the cancer cells from tamoxifen-induced apoptosis. Breast cancer cells responded to tamoxifen-induced oxidation by increasing Nrf2 expression and subsequent activation of the anti-oxidant response element (ARE). This increased the transcription of anti-oxidant genes and multidrug resistance transporters. As a result, breast cancer cells are able to destroy or export toxic oxidation products leading to increased survival from tamoxifen-induced oxidative damage. These responses in cancer cells also occur in breast tumors of tamoxifen-treated mice. Additionally, high levels of expression of Nrf2, ABCC1, ABCC3 plus NAD(P)H dehydrogenase quinone-1 in breast tumors of patients at the time of diagnosis were prognostic of poor survival after tamoxifen therapy. Therefore, overcoming tamoxifen-induced activation of the ARE could increase the efficacy of tamoxifen in treating breast cancer.

Glucagon-Like Peptide-2 Alters Bile Acid Metabolism in Parenteral Nutrition–Associated Liver Disease

BACKGROUND We aim to study the mechanisms underlying our previous finding that exogenous glucagon-like peptide-2 (GLP-2) treatment in a preclinical model of neonatal parenteral nutrition-associated liver disease (PNALD) improves cholestasis. METHODS Neonatal piglets received 17 days of parenteral nutrition (PN) therapy and either saline control (PN/Saline n = 8) or GLP-2 treatment at 11 nmol/kg/d (PN/GLP-2, n = 7). At terminal laparotomy, bile and liver samples were collected. The relative gene expression of enzymes involved in bile acid synthesis, regulation, and transport was measured in liver by reverse-transcriptase quantitative polymerase chain reaction. Bile acid composition in bile was determined using tandem mass spectrometry. Data were analyzed using 1-way analysis of variance (ANOVA) or Kruskal-Wallis ANOVA. RESULTS GLP-2 increased the expression of bile acid export genes: multidrug resistance-associated proteins 2 (MRP2) (P = .002) and 3 (MRP3) (P = .037) over saline control. GLP-2 increased expression of Farnesoid X receptor (FXR) (P < .001) and CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1) (P = .03). GLP-2 treatment was associated with decreased concentrations of taurohyocholic acid and conjugates of toxic lithocholic acid (P < .01). GLP-2 treatment increased the liver bile acid content. CONCLUSIONS GLP-2 treatment was associated with alterations in the hepatic expression of genes involved in bile acid metabolism. The transcriptomic results indicate the mechanisms at the transcriptional level acting to regulate bile acid synthesis and increase bile acid export. Differences in bile acid profiles further support a beneficial role for GLP-2 therapy in PNALD.

Simultaneous determination of trimethylamine and trimethylamine N-oxide in mouse plasma samples by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry

A method was developed that applies hydrophilic interaction liquid chromatography with tandem mass spectrometry in the multiple reaction monitoring mode to separate and accurately quantify trimethylamine and trimethylamine N-oxide in a single chromatographic run. This was achieved by converting trimethylamine to ethyl betaine, which is less volatile and hence results in greatly improved quantitation. Ethyl betaine also gives a similar response to trimethylamine N-oxide using positive-ion electrospray ionization mass spectrometry. It is readily separated from trimethylamine N-oxide by hydrophilic liquid chromatography in a 5 min run and with improved peak shape compared to underivatized trimethylamine. Validation of the method yielded a limit of detection (S/N ≥ 3) of 0.5 ng/mL for trimethylamine and 0.25 ng/mL for trimethylamine N-oxide. Method accuracies of 91.4-105.3% with precisions of 0.4-5.5% were obtained for standard mixtures over the range of 2.5-500 ng/mL. Recoveries measured for the extraction of trimethylamine and trimethylamine N-oxide spikes into mouse plasma were both >90%. The method, which simultaneously measures trimethylamine and trimethylamine N-oxide, was successfully applied to mouse plasma samples and could be adapted for use with other biological fluids.

Supplemental Parenteral Vitamin E Into Conventional Soybean Lipid Emulsion Does Not Prevent Parenteral Nutrition–Associated Liver Disease in Full-Term Neonatal Piglets

Background: Parenteral nutrition–associated liver disease (PNALD) continues to cause morbidity and mortality for neonates with intestinal failure. Lipid peroxidation is one potential etiological factor. This study was designed to test if supplementing vitamin E into conventional soy-based lipid would reduce the risk of PNALD. Methods: Sixteen piglets, aged 2–5 days and weighing 1.8–2.5 kg, were randomized to parenteral nutrition (PN) with soy lipid (SO, n = 8) or the same lipid plus &agr;-tocopherol, the most bioactive form of vitamin E (SO+E, n = 8). After 17 days, bile flow, liver chemistry, gene expression associated with bile acid metabolism, and bile acid composition were assessed. C-reactive protein (CRP) and oxidative stress markers, including plasma 8-isoprostane, were measured. All results were compared with a sow-reared control group (CON). Results: Comparing PN-treated groups, SO vs SO+E mean bile flow (5.91 vs 5.54 µL/g liver; P = .83), serum bile acid concentration (39.2 vs 26.6 µmol/L; P = .12), and total bilirubin (35.2 vs 26.9 µmol/L; P = .56) were not different. Gene expression related to bile acid metabolism and bile composition was not different between PN groups. There was no difference in CRP (41.8 vs 36.8 µg/mL; P = .22) or in plasma 8-isoprostane (27.9 vs 26.1 pg/mL; P = .77). Conclusions: In term neonatal piglets, supplemental vitamin E did not prevent cholestasis. Additional vitamin E was not associated with reduced inflammation or oxidative stress. The benefit of supplementing vitamin E into conventional lipid, vs adding fish oil, to prevent early onset of PNALD requires further clarification.

An LC/MS/MS method for the simultaneous determination of individual sphingolipid species in B cells

Comprehensive profiling of sphingolipids is of great importance for clinical and pharmaceutical studies. An LC/MS/MS method was established for the simultaneous separation and quantification of individual sphingolipid species including ceramides, dihydroceramides, glucosylceramides, sphingosine, sphingosine-1-phosphate, sphinganine and sphinganine-1-phosphate. All target individual sphingolipid species were separated and quantified in a single chromatographic run of <20min. Method validation results indicated that calibration curves were linear in the range of 2.5-10,000nM for ceramides and glucosylceramides, 10-10,000nM for dihydroceramides, 5-10,000nM for sphingosine, sphingosine-1-phosphate, sphinganine and sphinganine-1-phosphate, respectively. The limits of detection ranged from 0.5nM to 5nM. Accuracies of 92.5-113% with precisions of 0.3-8.0% RSD were obtained for all of the standards over a wide range of concentrations. The application of this method was demonstrated using B cells collected from Chronic Lymphocytic Leukemia patients (n=5) and healthy donors (n=4). 17 sphingolipid species were successfully characterized and quantified in the lipid extract. This is a rapid method that could be readily adapted to lipidomic investigations of sphingolipids in other bio-fluids and tissues.

Impact of Clinical Use of Parenteral Lipid Emulsions on Bile Acid Metabolism and Composition in Neonatal Piglets

BACKGROUND Neonates with intestinal failure dependent on parenteral nutrition (PN) are at risk of intestinal failure-associated liver disease (IFALD). PN lipid composition relates to the risk of IFALD, but the mechanisms are poorly understood. We investigated the effects of soybean oil (SO), a mixed-lipid (ML) emulsion containing fish oil (FO), and a pure FO. We hypothesized FO-containing PN lipids would result in increased gene expression of canalicular bile acid transporters and a larger, more hydrophilic bile acid pool, predictive of increased bile flow. METHODS Neonatal piglets were allocated to receive 1 of SO, ML, or FO throughout 14 days of PN feeding. Relative expression of genes involved in bile acid synthesis and transport were determined through quantitative polymerase chain reaction. Bile secreted from the liver was collected and measured. Bile acid composition was determined using tandem mass spectrometry. Regression analysis was used to determine predictors of bile flow. RESULTS PN reduced bile acid secretion (P < .001). FO-containing PN lipids were associated with greater expression of bile acid and organic solute transport genes (P < .05) and greater secretion of hydrophobic bile acids (P < .001). Farnesoid X receptor (P = .01), bile salt export pump (P < .01), multidrug resistant protein 2 (P < .01), and unconjugated hyocholic acid (P < .001) independently predicted bile flow. CONCLUSIONS PN lipid modulation altered bile acid metabolism and composition. These alterations may explain the hepatoprotective effects of FO-containing PN lipids and support their use in the prevention and treatment of IFALD.

Hepatic Expression of PEMT, but Not Dietary Choline Supplementation, Reverses the Protection against Atherosclerosis in Pemt−/−/Ldlr−/− Mice

Background Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine to phosphatidylcholine. Pemt-/-/low density lipoprotein receptor (Ldlr)-/- mice have significantly reduced plasma lipids and are protected against atherosclerosis. Recent studies have shown that choline can be metabolized by the gut flora into trimethylamine-N-oxide (TMAO), which is an emerging risk factor for atherosclerosis. Objective The objective of this study was to determine whether ectopic hepatic PEMT expression or choline supplementation would promote atherosclerosis in Pemt-/-/Ldlr-/- mice. Methods Male 8- to 10-wk-old Pemt+/+/Ldlr-/- (SKO) and Pemt-/-/Ldlr-/- (DKO) mice were injected with an adeno-associated virus (AAV) expressing green fluorescent protein (GFP) or human PEMT and fed a Western diet (40% of calories from fat, 0.5% cholesterol) for 8 wk. In a separate experiment, 8- to 10-wk-old SKO and half of the DKO male mice were fed a Western diet with normal (3 g/kg) choline for 12 wk. The remaining DKO mice [choline-supplemented (CS) DKO] were fed a CS Western diet (10 g choline/kg). Plasma lipid concentrations, choline metabolites, and aortic atherosclerosis were measured. Results Plasma cholesterol, plasma TMAO, and aortic atherosclerosis were reduced by 60%, 40%, and 80%, respectively, in DKO mice compared with SKO mice. AAV-PEMT administration increased plasma cholesterol and TMAO by 30% and 40%, respectively, in DKO mice compared with AAV-GFP-treated DKO mice. Furthermore, AAV-PEMT-injected DKO mice developed atherosclerotic lesions similar to SKO mice. In the second study, there was no difference in atherosclerosis or plasma cholesterol between DKO and CS-DKO mice. However, plasma TMAO concentrations were increased 2.5-fold in CS-DKO mice compared with DKO mice. Conclusions Reintroducing hepatic PEMT reversed the atheroprotective phenotype of DKO mice. Choline supplementation did not increase atherosclerosis or plasma cholesterol in DKO mice. Our data suggest that plasma TMAO does not induce atherosclerosis when plasma cholesterol is low. Furthermore, this is the first report to our knowledge that suggests that de novo choline synthesis alters TMAO status.

CHAPTER 10:Hydrophilic Interaction Liquid Chromatography for Determination of Betaine

Betaine is a naturally occurring zwitterionic compound at neutral pH possessing quaternary ammonium and carboxylate groups. Hydrophilic interaction liquid chromatography (HILIC) has been developed as a powerful alternative to conventional liquid chromatographic (LC) techniques for the analysis of highly polar and hydrophilic compounds. HILIC is therefore an appropriate technique for the measurement of betaine in a wide variety of sample matrices. In this chapter, the separation principles and types of mobile and stationary phases employed in HILIC are discussed. These are followed by a comparison of analytical methods employing HILIC for betaine analysis with other methods such as normal phase chromatography. Finally, an overview of published results on the determination of betaine in various matrices by HILIC is presented.