Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite

Denitrifying bacteria in soil generate nitric oxide (NO) from nitrite as a part of the nitrogen cycle, but little is known about NO production by commensal bacteria. We used a chemiluminescence assay to explore if human faeces and different representative gut bacteria are able to generate NO. Bacteria were incubated anaerobically in gas-tight bags, with or without nitrate or nitrite in the growth medium. In addition, luminal NO levels were measured in vivo in the intestines in germ-free and conventional rats, and in rats mono-associated with lactobacilli. We show that human faeces can generate NO after nitrate or nitrite supplementation. Lactobacilli and bifidobacteria generated much NO from nitrite, but only a few of the tested strains produced NO from nitrate and at much lower levels. In contrast, Escherichia coli, Bacteroides thetaiotaomicron, and Clostridium difficile did not produce significant amounts of NO either with nitrate or nitrite. NO generation in the gut lumen was also observed in vivo in conventional rats but not in germ-free rats or in rats mono-associated with lactobacilli. We conclude that NO can be generated by the anaerobic gut flora in the presence of nitrate or nitrite. Future studies will reveal its biological significance in regulation of gastrointestinal integrity.     

Antioxidant activity and hepatoprotective potential of Hammada scoparia against ethanol-induced liver injury in rats

The present work was aimed at studying the antioxidative activity and hepatoprotective effects of methanolic extract (ME) of Hammada scoparia leaves against ethanol-induced liver injury in male rats. The animals were treated daily with 35 % ethanol solution (4 g?kg^?1?day^?1) during 4 weeks. This treatment led to an increase in the lipid peroxidation, a decrease in antioxidative enzymes (catalase, superoxide dismutase, and glutathione peroxidase) in liver, and a considerable increase in the serum levels of aspartate and alanine aminotransferase and alkaline phospahatase. However, treatment with ME protects efficiently the hepatic function of alcoholic rats by the considerable decrease in aminotransferase contents in serum of ethanol-treated rats. The glycogen synthase kinase-3 β was inhibited after ME administration, which leads to an enhancement of glutathione peroxidase activity in the liver and a decrease in lipid peroxidation rate by 76 %. These biochemical changes were consistent with histopathological observations, suggesting marked hepatoprotective effect of ME. These results strongly suggest that treatment with methanolic extract normalizes various biochemical parameters and protects the liver against ethanol induced oxidative damage in rats.     

Acute ethanol intake induces mitogen-activated protein kinase activation,platelet-derived growth factor receptor phosphorylation,and oxidative stress in resistance arteries

In the present study, we investigated the role of angiotensin type I (AT₁) receptor in reactive oxygen species (ROS) generation and mitogen-activated protein kinases (MAPK) activation induced by acute ethanol intake in resistance arteries. We also evaluated the effect of ethanol on platelet-derived growth factor receptors (PDGF-R) phosphorylation and the role of this receptor on ROS generation by ethanol. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. Acute ethanol intake did not alter angiotensin I or angiotensin II levels in the rat mesenteric arterial bed (MAB). Ethanol induced vascular oxidative stress, and this response was not prevented by losartan (10 mg/kg; p.o. gavage), a selective AT₁ receptor antagonist. MAB from ethanol-treated rats displayed increased SAPK/JNK and PDGF-R phosphorylation, responses that were not prevented by losartan. The phosphorylation levels of protein kinase B (Akt) and eNOS were not affected by acute ethanol intake. MAB nitrate levels and the reactivity of this tissue to acetylcholine, phenylephrine, and sodium nitroprusside were not affected by ethanol intake. Ethanol did not alter plasma antioxidant capacity, the levels of reduced glutathione, or the activities of superoxide dismutase and catalase in the rat MAB. Short-term effects of ethanol (50 mmol/l) were evaluated in vascular smooth muscle cells (VSMC) isolated from rat MAB. Ethanol increased ROS generation, and this response was not affected by AG1296, a PDGF-R inhibitor, or losartan. Finally, ethanol did not alter MAPK or PDGF-R phosphorylation in cultured VSMC. Our study provides novel evidence that acute ethanol intake induces ROS generation, PDGF-R phosphorylation, and MAPK activation through AT(1)-independent mechanisms in resistance arteries in vivo. MAPK and PDGF-R play a role in vascular signaling and cardiovascular diseases and may contribute to the vascular pathobiology of ethanol.     

Generation of NO by probiotic bacteria in the gastrointestinal tract

Probiotic bacteria elicit a number of beneficial effects in the gut but the mechanisms for these health promoting effects are not entirely understood. Recent in vitro data suggest that lactobacilli can utilise nitrate and nitrite to generate nitric oxide, a gas with immunomodulating and antibacterial properties. Here we further characterised intestinal NO generation by bacteria. In rats, dietary supplementation with lactobacilli and nitrate resulted in a 3-8 fold NO increase in the small intestine and caecum, but not in colon. Caecal NO levels correlated to nitrite concentration in luminal contents. In neonates, colonic NO levels correlated to the nitrite content of breast milk and faeces. Lactobacilli and bifidobacteria isolated from the stools of two neonates, generated NO from nitrite in vitro, whereas S. aureus and E. coli rapidly consumed NO. We here show that commensal bacteria can be a significant source of NO in the gut in addition to the mucosal NO production. Intestinal NO generation can be stimulated by dietary supplementation with substrate and lactobacilli. The generation of NO by some probiotic bacteria can be counteracted by rapid NO consumption by other strains. Future studies will clarify the biological role of the bacteria-derived intestinal NO in health and disease.     

Role of nitric oxide in the oxidant stress during ischemia/reperfusion injury of the liver.

The potential role of nitric oxide (NO) and its reaction product with superoxide, peroxynitrite, was investigated in a model of hepatic ischemia-reperfusion injury in male Fischer rats in vivo. Pretreatment with the NO synthase inhibitor nitro-L-arginine (10 mg/kg) did neither affect the post-ischemic oxidant stress and liver injury during the initial reperfusion phase nor the subsequent infiltration of neutrophils into the liver and the later, neutrophil-induced injury phase. Furthermore, no evidence was found for a postischemic increase of the urinary excretion of nitrite, a stable oxidation metabolite of NO. In contrast, the administration of Salmonella enteritidis endotoxin (1 mg/kg) induced a significant diuresis in Fischer rats and an 800-fold enhancement of the urinary nitrite excretion. Nitro-L-arginine pretreatment inhibited the endotoxin-induced nitrite formation by 97%. Hepatic cGMP levels, as index of NO formation in the liver, were only increased significantly after endotoxin administration but not after ischemia and reperfusion. Our results provide no evidence for any enhanced generation of NO or peroxynitrite either systemically or locally during reperfusion and therefore it is unlikely that any of these metabolites are involved in the oxidant stress and liver injury during reperfusion after hepatic ischemia.     

Characterization of the mechanism of cytochrome P450 reductase-cytochrome P450-mediated nitric oxide and nitrosothiol generation from organic nitrates

Mammalian cytochrome P450 reductase (CPR) and cytochrome P450 (CP) play important roles in organic nitrate bioactivation; however, the mechanism by which they convert organic nitrate to NO remains unknown. Questions remain regarding the initial precursor of NO that serves to link organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of CPR-CP-mediated organic nitrate bioactivation, EPR, chemiluminescence NO analyzer, NO electrode, and immunoassay studies were performed. With rat hepatic microsomes or purified CPR, the presence of NADPH triggered organic nitrate reduction to NO2(-). The CPR flavin site inhibitor diphenyleneiodonium inhibited this NO2(-) generation, whereas the CP inhibitor clotrimazole did not. However, clotrimazole greatly inhibited NO2(-)-dependent NO generation. Therefore, CPR catalyzes organic nitrate reduction, producing nitrite, whereas CP can mediate further nitrite reduction to NO. Nitrite-dependent NO generation contributed <10% of the CPR-CP-mediated NO generation from organic nitrates; thus, NO2(-) is not the main precursor of NO. CPR-CP-mediated NO generation was largely thiol-dependent. Studies suggested that organic nitrite (R-O-NO) was produced from organic nitrate reduction by CPR. Further reaction of organic nitrite with free or microsome-associated thiols led to NO or nitrosothiol generation and thus stimulated the activation of sGC. Thus, organic nitrite is the initial product in the process of CRP-CP-mediated organic nitrate activation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.     

Effect of Blood Nitrite and Nitrate Levels on Murine Platelet Function

Nitric oxide (NO) appears to play an important role in the regulation of thrombosis and hemostasis by inhibiting platelet function. The discovery of NO generation by reduction of nitrite (NO₂ ⁻) and nitrate (NO₃ ⁻) in mammals has led to increased attention to these anions with respect to potential beneficial effects in cardiovascular diseases. We have previously shown that nitrite anions at 0.1 µM inhibit aggregation and activation of human platelet preparations in vitro in the presence of red blood cells and this effect was enhanced by deoxygenation, an effect likely due to NO generation. In the present study, we hypothesized that nitrite and nitrate derived from the diet could also alter platelet function upon their conversion to NO in vivo. To manipulate the levels of nitrite and nitrate in mouse blood, we used antibiotics, NOS inhibitors, low nitrite/nitrate (NOx) diets, endothelial NOS knock-out mice and also supplementation with high levels of nitrite or nitrate in the drinking water. We found that all of these perturbations affected nitrite and nitrate levels but that the lowest whole blood values were obtained by dietary restriction. Platelet aggregation and ATP release were measured in whole blood and the results show an inverse correlation between nitrite/nitrate levels and platelet activity in aggregation and ATP release. Furthermore, we demonstrated that nitrite-supplemented group has a prolonged bleeding time compared with control or low NOx diet group. These results show that diet restriction contributes greatly to blood nitrite and nitrate levels and that platelet reactivity can be significantly affected by these manipulations. Our study suggests that endogenous levels of nitrite and nitrate may be used as a biomarker for predicting platelet function and that dietary manipulation may affect thrombotic processes.     

Oxidative and nitrosative mediators in hepatic injury caused by whole body hyperthermia in rats

The involvement of oxidative and nitrosative mediators in liver injury caused by heat stress remains unclear. This study aimed to elucidate the role of endothelial nitric oxide synthase (eNOS), and inducible NOS (iNOS)-derived NO and nitrotyrosine in the whole-body hyperthermia (WBH)-induced liver injury. Rats were anesthetized with intraperitoneal pentobarbital, and were exposed to a heating lamp for 60 min to raise the core temperature to 42.5 degrees C. The rats were maintained at the hyperthermic state for an additional 50 min. Blood urea nitrogen, creatinine, aspartate aminotransferase, alanine aminotransferase, lactic dehydrogenase, creatine phosphokinase, amylase, lipase, nitrate/nitrite, methyl guanidine, and proinflammatory cytokines (tumor necrosis factoralpha, interleukin-1beta and interleukin-10) were measured before and 14 h after hyperthermia. Immunohistochemical staining was employed to detect the eNOS, iNOS and nitrotyrosine levels. Western blotting was used to examine the expression of heatshock protein 70 (HSP 70). Histopathological examination of the liver tissue was performed. WBH caused liver injury accompanied with significant increases in biochemical factors, nitrate/nitrite, methyl guanidine, and proinflammatory cytokines. In addition, WBH enhanced the eNOS, iNOS, nitrotyrosine and HSP 70 levels. WBH caused hepatic injury. The pathogenetic mechanism is likely mediated through the NOS-derived NO, free radical, proinflammatory cytokines and nitrotyrosine. The enhanced expression of HSP 70 may play a protective role.     

Inorganic nitrate is a possible source for systemic generation of nitric oxide

Nitrate and nitrite have been considered stable inactive end products of nitric oxide (NO). While several recent studies now imply that nitrite can be reduced to bioactive NO again, the more stable anion nitrate is still considered to be biologically inert. Nitrate is concentrated in saliva, where a part of it is reduced to nitrite by bacterial nitrate reductases. We tested if ingestion of inorganic nitrate would affect the salivary and systemic levels of nitrite and S-nitrosothiols, both considered to be circulating storage pools for NO. Levels of nitrate, nitrite, and S-nitrosothiols were measured in plasma, saliva, and urine before and after ingestion of sodium nitrate (10 mg/kg). Nitrate levels increased greatly in saliva, plasma, and urine after the nitrate load. Salivary S-nitrosothiols also increased, but plasma levels remained unchanged. A 4-fold increase in plasma nitrite was observed after nitrate ingestion. If, however, the test persons avoided swallowing after the nitrate load, the increase in plasma nitrite was prevented, thereby illustrating its salivary origin. We show that nitrate is a substrate for systemic generation of nitrite. There are several pathways to further reduce this nitrite to NO. These results challenge the dogma that nitrate is biologically inert and instead suggest that a complete reverse pathway for generation of NO from nitrate exists.     

Binge Alcohol Consumption Aggravates Oxidative Stress and Promotes Pathogenesis of NASH from Obesity-Induced Simple Steatosis

The pathogenesis of nonalcoholic steatohepatitis (NASH) is a two-stage process in which steatosis is the “first hit” and an unknown “second hit.” We hypothesized that “a binge” could be a “second hit” to develop NASH from obesity-induced simple steatosis. Thirty-week-old male Otsuka Long-Evans Tokushima fatty (OLETF) rats were administered 10 mL of 10% ethanol orally for 5, 3, 2, and 1 d/wk for 3 consecutive weeks. As control, male Otsuka Long-Evans Tokushima (OLET) rats were administered the same amount of alcohol. Various biochemical parameters of obesity, steatosis and NASH were monitored in serum and liver specimens in untreated and ethanol-treated rats. The liver sections were evaluated for histopathological alterations of NASH and stained for cytochrome P-4502E1 (CYP2E1) and 4-hydroxy-nonenal (4-HNE). Simple steatosis, hyperinsulinemia, hyperglycemia, insulin resistance, hypertriglycemia and marked increases in hepatic CYP2E1 and 4-HNE were present in 30-wk-old untreated OLETF rats. Massive steatohepatitis with hepatocyte ballooning was observed in the livers of all OLETF rats treated with ethanol. Serum and hepatic triglyceride levels as well as tumor necrosis factor (TNF)-α mRNA were markedly increased in all ethanol-treated OLETF rats. Staining for CYP2E1 and 4-NHE demonstrated marked increases in the hepatic tissue of all the groups of OLETF rats treated with ethanol compared with OLET rats. Our data demonstrated that “a binge” serves as a “second hit” for development of NASH from obesity-induced simple steatosis through aggravation of oxidative stress. The enhanced levels of CYP2E1 and increased oxidative stress in obesity play a significant role in this process.     

TEMPOL enhances the antihypertensive effects of sodium nitrite by mechanisms facilitating nitrite-derived gastric nitric oxide formation

Orally administered nitrite exerts antihypertensive effects associated with increased gastric nitric oxide (NO) formation. While reducing agents facilitate NO formation from nitrite, no previous study has examined whether antioxidants with reducing properties improve the antihypertensive responses to orally administered nitrite. We hypothesized that TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) could enhance the hypotensive effects of nitrite in hypertensive rats by exerting antioxidant effects (and enhancing NO bioavailability) and by promoting gastric nitrite-derived NO generation. The hypotensive effects of intravenous and oral sodium nitrite were assessed in unanesthetized freely moving rats with L-NAME (N^ω-nitro-L-arginine methyl ester; 100 mg/kg; po)-induced hypertension treated with TEMPOL (18 mg/kg; po) or vehicle. While TEMPOL exerted antioxidant effects in hypertensive rats, as revealed by lower plasma 8-isoprostane and vascular reactive oxygen species levels, this antioxidant did not affect the hypotensive responses to intravenous nitrite. Conversely, TEMPOL enhanced the dose-dependent hypotensive responses to orally administered nitrite, and this effect was associated with higher increases in plasma nitrite and lower increases in plasma nitrate concentrations. In vitro experiments using electrochemical and chemiluminescence NO detection under variable pH conditions showed that TEMPOL enhanced nitrite-derived NO formation, especially at low pH (2.0 to 4.0). TEMPOL signal evaluated by electron paramagnetic resonance decreased when nitrite was reduced to NO under acidic conditions. Consistent with these findings, increasing gastric pH with omeprazole (30 mg/kg; po) attenuated the hypotensive responses to nitrite and blunted the enhancement in plasma nitrite concentrations and hypotensive effects induced by TEMPOL. Nitrite-derived NO formation in vivo was confirmed by using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), which blunted the responses to oral nitrite. Our results showed that TEMPOL promotes nitrite reduction to NO in the stomach and enhanced plasma nitrite concentrations and the hypotensive effects of oral sodium nitrite through mechanisms critically dependent on gastric pH. Interestingly, the effects of TEMPOL on nitrite-mediated hypotension cannot be explained by increased NO formation in the stomach alone, but rather appear more directly related to increased plasma nitrite levels and reduced nitrate levels during TEMPOL treatment. This may relate to enhanced nitrite uptake or reduced nitrate formation from NO or nitrite.     

Role of naringin supplement in regulation of lipid and ethanol metabolism in rats

The current study was performed to investigate the effect of naringin supplements on the alcohol, lipid, and antioxidant metabolism in ethanol-treated rats. Male Sprague-Dawley rats were randomly divided into six groups (n = 10) based on six dietary categories: ethanol and naringin-free, ethanol (50 g/L) plus low-naringin (0.05 g/L), ethanol plus high-naringin (0.125 g/L), and three corresponding pair-fed groups. The pair-fed control rats received an isocaloric diet containing dextrin-maltose instead of ethanol for 5 wks. Among the ethanol treated groups, the naringin supplements significantly lowered the plasma ethanol concentration with a simultaneous increase in the ADH and/or ALDH activities. However, among the ethanol-treated groups, naringin supplementation resulted in a significant decrease in the hepatic triglycerides and plasma and hepatic total cholesterol compared to that in the naringin-free group. Naringin supplementation significantly increased the HDL-cholesterol and HDL-C/total-C ratio, while lowering the AI value among the ethanol-treated groups. Hepatic lipid accumulation was also significantly reduced in the naringin-supplemented groups compared to the naringin-free group among the ethanol-treated groups, while no differences were found among the pair-fed groups. Among the ethanol-treated groups, the low-naringin supplementation resulted in a significant decrease in the levels of plasma and hepatic TBARS, whereas it resulted in higher SOD and GSH-Px activities and gluthathion levels in the liver. Accordingly, naringin would appear to contribute to alleviating the adverse effect of ethanol ingestion by enhancing the ethanol and lipid metabolism as well as the hepatic antioxidant defense system.     

The influence of chronic ethanol consumption on the distribution of thiopental in rats.

Ethanol was administered chronically for 14 days to male Sprague-Dawley rats. Day 15 was ethanol-free. On day 16 the rats received 25 mg of thiopental per kilogram (intravenously). One minute after the injection, the ethanol-treated rats showed lower blood levels of thiopental and higher liver levels of the drug than control rats given sucrose in place of ethanol. Samples of blood drawn 5 and 10 min after injection showed no significant difference in thiopental levels between the ethanol and control groups. The ethanol-treated group slept for a significantly shorter period of time. It is concluded that chronic ethanol consumption for 14 days decreases the pharmacological effects of thiopental and alters its initial distribution in the body.     

Increased loss and decreased synthesis of hepatic glutathione after acute ethanol administration. Turnover studies.

The effect of acute ethanol administration on rates of synthesis and utilization of hepatic glutathione (GSH) was studied in rats after a pulse of [35S]cysteine. A 35% decrease in hepatic GSH content 5h after administration of 4 g of ethanol/kg body wt. was accompanied by a 33% increase in the rate of GSH utilization. The decrease occurred without increases in hepatic oxidized glutathione (GSSG) or in the GSH/GSSG ratio. The rate of non-enzymic condensation of GSH with acetaldehyde could account for only 6% of the rate of hepatic GSH disappearance. The increased loss of [35S]GSH induced by ethanol was not accompanied by an increased turnover; rather, a 30% inhibition of GSH synthesis balanced the increased rate of loss, leaving the turnover rate unchanged. The rate of acetaldehyde condensation with cysteine in vitro occurred at about one-third of the rate of GSH loss in ethanol-treated animals. However, ethanol induced only a minor decrease in liver cysteine content, which did not precede, but followed, the decrease in GSH. The characteristics of 2-methylthiazolidine-4-carboxylic acid, the condensation product between acetaldehyde and cysteine, were studied and methodologies were developed to determine its presence in tissues. It was not found in the liver of ethanol-treated animals. Ethanol administration led to a marked increase (47%) in plasma GSH in the post-hepatic inferior vena cava, but not in its pre-hepatic segment. Data suggest that an increased loss of GSH from the liver constitutes an important mechanism for the decrease in GSH induced by ethanol. In addition, an inhibition of GSH synthesis is observed.     

Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrate reduction: evaluation of its role in nitrite and nitric oxide generation in anoxic tissues

In addition to nitric oxide (NO) generation from specific NO synthases, NO is also formed during anoxia from nitrite reduction, and xanthine oxidase (XO) catalyzes this process. While in tissues and blood high nitrate levels are present, questions remain regarding whether nitrate is also a source of NO and if XO-mediated nitrate reduction can be an important source of NO in biological systems. To characterize the kinetics, magnitude, and mechanism of XO-mediated nitrate reduction under anaerobic conditions, EPR, chemiluminescence NO-analyzer, and NO-electrode studies were performed. Typical XO reducing substrates, xanthine, NADH, and 2,3-dihydroxybenz-aldehyde, triggered nitrate reduction to nitrite and NO. The rate of nitrite production followed Michaelis-Menten kinetics, while NO generation rates increased linearly following the accumulation of nitrite, suggesting stepwise-reduction of nitrate to nitrite then to NO. The molybdenum-binding XO inhibitor, oxypurinol, inhibited both nitrite and NO production, indicating that nitrate reduction occurs at the molybdenum site. At higher xanthine concentrations, partial inhibition was seen, suggesting formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. The pH dependence of nitrite and NO formation indicate that XO-mediated nitrate reduction occurs via an acid-catalyzed mechanism. With conditions occurring during ischemia, myocardial xanthine oxidoreductase and nitrate levels were determined to generate up to 20 microM nitrite within 10-20 min that can be further reduced to NO with rates comparable to those of maximally activated NOS. Thus, XOR catalyzed nitrate reduction to nitrite and NO occurs and can be an important source of NO production in ischemic tissues.     

Chronic ethanol treatment potentiates ethanol-induced increases in interstitial nucleus accumbens endocannabinoid levels in rats

We employed in vivo microdialysis to characterize the effect of an ethanol challenge injection on endocannabinoid levels in the nucleus accumbens of ethanol-naïve and chronic ethanol-treated rats. Ethanol (0.75 and 2 g/kg, i.p.) dose-dependently increased dialysate 2-arachidonoylglycerol (to a maximum 157 ± 20% of baseline) and decreased anandamide (to a minimum 52 ± 9% of baseline) in ethanol-naïve rats. The endocannabinoid clearance inhibitor N -(4-hydrophenyl) arachidonoylamide (AM404; 3 mg/kg) potentiated ethanol effects on 2-arachidonoylglycerol levels but did not alter ethanol-induced decreases in anandamide. AM404 alone did not alter dialysate levels of either endocannabinoid. Then, we characterized the effect of ethanol challenge on nucleus accumbens endocannabinoid levels in rats previously maintained on an ethanol-containing liquid diet. Ethanol challenge produced a greater and more prolonged increase in 2-arachidonoylglycerol (to a maximum 394 ± 135% of baseline) in ethanol-experienced than in ethanol-naïve rats. The profile in ethanol-experienced rats was similar to that produced by AM404 pre-treatment in ethanol-naïve rats. AM404 in ethanol-experienced rats led to a further enhancement in the 2-arachidonoylglycerol response to ethanol challenge (to a maximum 704 ± 174% of baseline). Our findings demonstrate that ethanol-induced increases in nucleus accumbens 2-arachidonoylglycerol are potentiated in animals with a history of ethanol consumption.     

Betaine administration corrects ethanol-induced defective VLDL secretion

Our previous studies, demonstrating ethanol-induced alterations in phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine methyltransferase (PEMT) pathway, implicated a defect in very low-density lipoprotein (VLDL) secretion in the pathogenesis of hepatic steatosis. The objective of this study was to determine whether VLDL secretion was reduced by chronic ethanol consumption and whether betaine supplementation, that restores PEMT activity and prevents the development of alcoholic steatosis, could normalize VLDL secretion. The VLDL secretion in rats fed with control, ethanol and the betaine supplemented diets was determined using Triton WR-1339 to inhibit plasma VLDL metabolism. We observed reduced VLDL production rates in chronic alcohol-fed rats compared to control animals. Supplementation of betaine in the ethanol diet increased VLDL production rate to values significantly higher than those observed in the control diet-fed rats. To conclude, chronic ethanol consumption impairs PC generation via the PEMT pathway resulting in diminished VLDL secretion which contributes to the development of hepatic steatosis. By increasing PEMT-mediated PC generation, betaine results in increased fat export from the liver and attenuates the development of alcoholic fatty liver.     

Organic nitrate tolerance is induced by degradation of some cytochrome P450 isoforms

The mechanism of nitrate tolerance is poorly defined. We studied the rat P450 (CYP)-catalyzed conversion of organic nitrate to nitric oxide (NO) by purified CYP isoforms and the relationship between P450 expression and nitrate tolerance following continuous infusion of organic nitrates in rats. CYP1A2 effectively formed NO from isosorbide dinitrate and nitroglycerine (NTG). The hypotensive effect of an NTG bolus injection was abolished in rats which had been previously given a continuous 48 h infusion of NTG. Nitrate tolerance was reversible to control levels 2 days after cessation of the continuous infusion. At 48 h after infusion, NTG-induced NO generation of the vessels increased in acetone (a P450 inducer)-pretreated rats, and nitrite and nitrate levels were markedly greater than in normal rats. The appearance and disappearance of P450 isoforms paralleled the conversion of organic nitrates to NO as assessed by immunohistochemistry and Western blotting. Our observations indicate that nitrate tolerance is in large part the result of decreased P450 expression and activity. Interventions that maintain or increase P450 activity may be a useful strategy to provide sustained relief from ischemic conditions in humans.