1H and 31P NMR Lipidome of Ethanol‐Induced Fatty Liver

Background: Hepatic steatosis (fatty liver), an early and reversible stage of alcoholic liver disease, is characterized by triglyceride deposition in hepatocytes, which can advance to steatohepatitis, fibrosis, cirrhosis, and ultimately to hepatocellular carcinoma. In the present work, we studied altered plasma and hepatic lipid metabolome (lipidome) to understand the mechanisms and lipid pattern of early‐stage alcohol‐induced‐fatty liver. Methods: Male Fischer 344 rats were fed 5% alcohol in a Lieber‐DeCarli diet. Control rats were pair‐fed an equivalent amount of maltose‐dextrin. After 1 month, animals were killed and plasma collected. Livers were excised for morphological, immunohistochemical, and biochemical studies. The lipids from plasma and livers were extracted with methyl‐tert‐butyl ether and analyzed by 750/800 MHz proton nuclear magnetic resonance (¹H NMR) and phosphorus (³¹P) NMR spectroscopy on a 600 MHz spectrometer. The NMR data were then subjected to multivariate statistical analysis. Results: Hematoxylin and Eosin and Oil Red O stained liver sections showed significant fatty infiltration. Immunohistochemical analysis of liver sections from ethanol‐fed rats showed no inflammation (absence of CD3 positive cells) or oxidative stress (absence of malondialdehyde reactivity or 4‐hydroxynonenal positive staining). Cluster analysis and principal component analysis of ¹H NMR data of lipid extracts of both plasma and livers showed a significant difference in the lipid metabolome of ethanol‐fed versus control rats. ³¹P NMR data of liver lipid extracts showed significant changes in phospholipids similar to ¹H NMR data. ¹H NMR data of plasma and liver reflected several changes, while comparison of ¹H NMR and ³¹P NMR data offered a correlation among the phospholipids. Conclusions: Our results show that alcohol consumption alters metabolism of cholesterol, triglycerides, and phospholipids that could contribute to the development of fatty liver. These studies also indicate that fatty liver precedes oxidative stress and inflammation. The similarities observed in plasma and liver lipid profiles offer a potential methodology for detecting early‐stage alcohol‐induced fatty liver disease by analyzing the plasma lipid profile.     

Effects of Ethanol, MK-801, and Chlordiazepoxide on Locomotor Activity in Different Rat Lines: Dissociation of Locomotor Stimulation from Ethanol Preference

Several lines of research have suggested a link between the reward value of a drug and its ability to stimulate locomotion. One goal of the present study was to determine whether ethanol preferentially stimulates locomotor activity in lines of rat that show a preference for ethanol. A secondary goal was to determine the extent to which the benzodiazepine-like and NMDA antagonistic action of ethanol accounted for its effect on locomotor activity. To meet these goals, the effects of varying doses of ethanol (0.125-1.0 g/kg), MK-801 (0.1-0.3 mg/kg), and chlordiazepoxide (0.3-3 mg/kg) on locomotor activity were studied in several lines of rats that had been habituated to the testing procedure. The effect of low doses of ethanol on motor activity in the Alcohol-Preferring (P) and Fawn-Hooded rats, which show a strong ethanol preference, were similar to those of the alcohol-nonpreferring (NP), Flinders Sensitive Line, and Flinders Resistant Line rats. Only the Flinder Resistant Line rats showed a small, but significant increase in locomotor activity after the administration of ethanol. The highest dose of ethanol (1.0 g/kg) produced locomotor depression in all lines except the P and NP lines, which were not tested at this dose. These findings do not support a link between locomotor stimulation by ethanol and ethanol preference. In contrast, all lines exhibited locomotor stimulation after moderate (0.1-0.3 mg/kg) doses of MK-801, but did not exhibit increases in activity following any dose of chlordiazepoxide. These data indicate that the profiles of activity after MK-801 and chlordiazepoxide were distinct from that of ethanol in the various rat lines. Therefore, the effects of ethanol on locomotor activity cannot be accounted for by reference solely to its antagonist-like action at NMDA receptors and/or its agonist-like action at GABA/benzodiazepine receptors.     

Comparison of the Effects of ORG 30029, Dobutamine and High Perfusate Calcium on Function and Metabolism in Rat Heart

Cardiac contractility may be enhanced via multiple cellular mechanisms resulting in varied effects on cardiac energetics. The mechanisms that account for the varied energetic responses are not well understood. The purpose of this investigation was to compare the effects of the calcium sensitizing agent ORG 30029 (N-hydroxy-5,6-dimethoxy-benzo[b]thiophene-2-carboximidamide hydrochloride, a calcium sensitizing agent which increases contractility without increasing calcium transients significantly), dobutamine and high perfusate calcium on contractility and energetics. Langendorff-perfused rat hearts were stimulated with ORG 30029, dobutamine and high perfusate calcium in graduated concentrations while myocardial oxygen consumption (MVO₂) and force-time integral were measured. ORG 30029, dobutamine and high perfusate calcium increased contractility in a dose-dependent manner. Despite an increase of 50% in systolic pressure and a 17% increase in force-time integral from control, ORG 30029 had no significant effect on MVO₂at the lower concentrations (n=6). However, dobutamine (n=4) and high perfusate calcium (n=4) caused a 65% increase in systolic pressure and a 17% increase in force-time integral and a 50% and 41% increase in MVO₂respectively (P<0.05). High energy phosphates (by³¹P NMR), and lactate production were unaltered by these agents, suggesting that metabolism was steady state. Basal metabolism tended to increase slightly with dobutamine but not with ORG 30029 or high perfusate calcium. ORG 30029, dobutamine, and high perfusate calcium increase contractility in perfused rat hearts with disparate effects on energetics. These differences may be accounted for, in part, by differences in energy expenditure for calcium handling.     

Effect of Pyrazole and Dexamethasone Administration on Cytochrome P450 2E1 and 3A Isoforms in Rat Liver and Kidney: Lack of Specificity of p-Nitrophenol as a Substrate of P450 2E1

The induction effects of pyrazole and dexamethasone (known to be specific to P450 2E1 and 3A enzymes, respectively), given alone or simultaneously, were studied in rat liver and kidney microsomes. Pyrarole treatment induced the catalytic activity and the amount of P450 2E1 enzyme in both organs. Immunoreactive P450 2E1 and 4-nitrophenol 2-hydroxylation increased 8- and 13-fold, respectively (versus control), in the kidney, but only 2.4- and 2.7-fold (versus control) in the liver after pyrazole treatment. As assessed by nifedipine oxidation activity, dexamethasone treatment increased the P450 3A catalytic activity approximately 4-fold (versus control) in the liver, but not in the kidney, suggesting that P450 3A was not inducible in the kidney. Pyrazole decreased P450 3A activity in the liver but did not modify it in the kidney. A combination of both chemicals induced both enzymes, but to a lesser extent than treatment with each single chemical compound. Furthermore, the 2-hydroxylation of p -nitro-phenol, considered one of the most specific substrates for monitoring the level of P450 2E1, was mediated also by P450 3A, at least in dexamethasone-treated rats. Finally, this experimental work demonstrated that P450 3A induction is organ-specific, and it also demonstrated the lack of specificity of p -nitrophenol as a P450 2E1 substrate.     

Human Gastric Alcohol Dehydrogenase: Its Inhibition by H2-Receptor Antagonists, and Its Effect on the Bioavailability of Ethanol

Two types of alcohol dehydrogenase isoenzymes (differing in their affinity for ethanol, sensitivity to 4-methylpyrazole, and electrophoretic migration) have been identified in the human stomach. At the high ethanol concentrations prevailing in the gastric lumen during alcohol consumption, the sum of their activities could account for significant oxidation of ethanol. In vitro, these activities were inhibited by cimetidine and ranitidine, but not by famotidine. In vivo, therapeutic doses of cimetidine (but not of famotidine) increased blood ethanol levels when ethanol was given orally, but not when it was given intravenously, indicating a significant contribution of the gastric ADH to the bioavailability and thereby the potential toxicity of ethanol.     

Effect of Maternal Ethanol Consumption on in Vitro Tumor Necrosis Factor, Interleukin-6 and Interleukin-2 Production by Rat Milk and Blood Leukocytes

It has been shown that the transfer of immunity via lactation plays an important role in providing eariy protection to the neonate. Maternal ethanol consumption also results in a reduced transfer of immunity to their neonates against a Trichinella spiralis infection. Because of the known presence of cytokines in milk and their important role in inflammation, we tested the effects of maternal ethanol consumption on cytokine production by milk and blood cells from T. spiralis - infected rats. With T. spiralis antigen, Concanavalin A (Con A) or lipopolysaccharide stimulation, milk cells from both ethanol-treated and pair-fed groups were capable of producing tumor necrosis factor (TNF), interleukin (IL)-6 and IL-2. There were no differences between groups for TNF or IL-6 production by milk cells. Milk cells from the ethanol group produced a significantly higher amount of IL-2 upon Con A stimulation, as compared with that from the pair-fed group (16 ± 4 units/10⁶ cells vs. 4 ± 1 units/10⁶ cells, p < 0.05). After stimulation with Con A, blood cells from the ethanol group produced significantly lower amounts of TNF (17 ± 15 units/10⁶ cells) than that from the pair-fed group (102 ± 64 units/10⁶ cells, p < 0.05). The amount of TNF and IL-6 produced by milk cells was significantly lower, as compared with that produced by blood cells. This study suggests that ethanol consumption has some modulatory effects on cytokine production by milk and blood cells.     

Comparative study of the effects of acebutolol,atenolol, d-propranolol and dl,-propranolol on the alterations in energy metabolism caused by ischemia and reperfusion: A 31P NMR study on the isolated rat heart

Summary 31-P NMR spectroscopy data recorded for the isolated heart were analyzed, in conjunction with functional and biochemical variables, in order to investigate the effect observed for several different beta-adrenoceptor antagonists on the alterations provoked by global partial ischemia (37°C, 24 minutes, 1% residual coronary flow) and reperfusion in the metabolism of the myocardium. During ischemia: intracellular acidosis, adenosine triphosphate (ATP) degradation, and inorganic phosphate (Pi) accumulation were found to be reduced whether the perfusion fluid contained: acebutolol 2.7×10^-5 M, atenolol 10^-5 M, d-propranolol 10^-5 M, or dl-propranolol 10^-5 M. On reperfusion metabolic and functional variables were variously affected by the different drugs, except the Pi level which was, in all series, significantly lower compared with control hearts. The adenylate charge and the glycogen stores were protected in the acebutolol, dl-propranolol, and d-propranolol groups. The ATP level was higher than in controls only in the acebutolol and atenolol groups. The intracellular pH recovered to values non-significantly different from preischemic values in the acebutolol and dl-propranol-treated hearts only. The mechanical performance, expressed as the rate-pressure product, was unaltered by the ischemia-reperfusion sequence in the acebutolol and d-propranolol series, while decreasing significantly in controls and in the atenolol group. In dl-propranolol-treated hearts the mechanical activity, which in normoxic conditions was already halved during the effect of the drug, remained at this same level after ischemia. From these observations, it appears that the nonspecific properties of the drugs, as distinct from beta-blockade, play an important part in attenuating the ischemia-induced alteration in myocardial metabolism. Thus, it can be postulated that (1) the metabolic effects of dl-propranolol probably result largely from the reduction of heart work induced by this drug; (2) the maintenance of energy metabolism associated with the preservation of the myocardial activity, as observed in the case of acebutolol and d-propranolol, is possibly a consequence of the existence of a membrane-stabilizing activity.