Increased urinary F2‐isoprostanes in systemic sclerosis,but not in primary Raynaud's phenomenon: Effect of cold exposure

Objective

F₂‐isoprostanes are free radical‐dependent arachidonic acid metabolites that are used as clinical markers of lipid peroxidation in systemic sclerosis (SSc) and other microvascular diseases. The objectives of this study were to determine whether the basal urinary levels of F₂‐isoprostane in SSc patients differ from those in patients with primary Raynaud's phenomenon (RP) and to investigate whether F₂‐isoprostane formation correlates with the cutaneous microvascular perfusion decrease following cold exposure in SSc patients, patients with primary RP, and healthy controls.

Methods

Eleven women with RP secondary to SSc, 11 women with primary RP, and 11 healthy women were exposed to decreasing room temperature, from 25°C to 15°C, for 40 minutes. Urine samples were obtained before and after the test for gas chromatography/electronic impact mass spectrometry quantification of 15‐F_2t‐isoprostane (15‐F_2t‐IsoP; also called isoprostaglandin F_2α type III). Cutaneous blood flow was monitored using a laser Doppler perfusion imager.

Results

The mean ± SEM urinary 15‐F_2t‐IsoP levels at baseline in SSc patients (178 ± 32 pmoles/mmole of creatinine) were 1.9 times higher than those in healthy controls (95 ± 11 pmoles/mmole of creatinine) and 1.7 times higher than those in patients with primary RP (107 ± 19 pmoles/mmole of creatinine) (P < 0.05 for controls and patients with primary RP versus SSc patients). No significant correlation was found between basal urinary 15‐F_2t‐IsoP levels and the temperature or cutaneous blood flow decrease in response to the whole‐body cooling. Furthermore, the 15‐F_2t‐IsoP response to the cooling test was not correlated with the cutaneous blood flow decrease.

Conclusion

Lipid peroxidation is increased in SSc patients, but not in patients with primary RP. Cold exposure leads to a significant but small increase in 15‐F_2t‐IsoP levels that is independent of the cutaneous blood flow decrease. F₂‐isoprostane quantification may be an interesting pharmacologic tool for monitoring responses to antioxidant treatment in SSc patients.

     

Pathogenesis of diquat-induced liver necrosis in selenium-deficient rats: Assessment of the roles of lipid peroxidation and selenoprotein P

A dose of diquat below the amount injurious to selenium-replete animals causes lipid peroxidation and massive liver necrosis in selenium-deficient rats. The current study was undertaken to characterize the lipid peroxidation with respect to the liver injury and to correlate the presence of several selenoproteins with the protective effect of selenium. Lipid peroxidation was assessed by measurement of F₂ isoprostanes. Diquat caused an increase in liver and plasma F₂ isoprostanes. A gradient of these compounds was detected across the liver in some animals, indicating that this organ was a source of some of the plasma F₂ isoprostanes. A time-course experiment showed that liver F₂ isoprostane concentration increased before plasma alanine transaminase (ALT) levels rose. Selenium-deficient rats were injected with selenium doses from 2 to 50 μg/kg and studied 12 hours later. A dose of 10 μg/kg or more prevented diquat-induced lipid peroxidation and liver injury. This dose increased plasma selenoprotein P substantially, and a dose-response was present. Liver cellular and plasma glutathione peroxidase activities remained below 2% of their values in control rats for all selenium doses. In selenium-deficient rats given diquat, hepatic lipid peroxidation precedes hepatic necrosis and could therefore be an important mechanism of the necrosis. Selenoprotein P levels were increased by selenium injections, which protected against diquat injury, but glutathione peroxidase activity was not increased. This is consistent with selenoprotein P being the mediator of the selenium effect.     

Polyenylphosphatidylcholine Decreases Alcohol-Induced Oxidative Stress in the Baboon

Diets supplemented with polyunsaturated fatty acids or triglycerides exacerbate alcohol-induced liver injury in rats, whereas, in baboons, polyenylphosphatidylcholine (PPC) protects against alcohol-induced fibrosis and cirrhosis. Because the aggravation in rats was attributed to enhanced lipid peroxidation, the present study was undertaken to assess parameters of oxidative stress in percutaneous liver biopsies of baboons fed alcohol, with or without PPC (2.8 g per 1000 calories). F₂-isoprostanes and 4-hydroxynonenal, breakdown products of lipid peroxidation, were determined by gas chromatog-raphy/mass spectrometry, and α-tocopherol was measured by HPLC with electrochemical detection. Hepatic 4-hydroxynonenal was significantly increased in animals fed alcohol, but this was fully prevented by PPC. F₂-isoprostanes were also significantly lower after PPC and ethanol than after ethanol alone, and the alcohol-induced glutathione decrease was attenuated. All of these parameters were normal in the animals withdrawn from alcohol, even with persistence of significant liver disease. Because peroxidation products are fibrogenic, their decrease could contribute to the antifibro-genic property of the phospholipids. In conclusion, PPC significantly attenuates ethanol-induced oxidative stress, which may explain, at least in part, its protective effect against alcoholic liver injury.     

Oestrogen metabolites in relation to isoprostanes as a measure of oxidative stress

Objective Oestradiol (E2) and its metabolites 2‐hydroxyoestrone (2‐OHE1) and 16α‐hydroxyoestrone (16α‐OHE1) are thought to curtail the greater oxidative stress found in the development and progression of disease conditions including atherosclerosis. We related oestrogen levels to F_2a‐isoprostane levels, a biomarker of oxidative stress. Design and participants Data were obtained from 1647 women, aged 47–57 years, participating in the fifth annual follow‐up of the Study of Women's Health Across the Nation (SWAN), a study of the menopausal transition. Measurements Serum E2 and urinary 2‐OHE1 and 16α‐OHE1 concentrations were determined by enzyme‐linked immunosorbent assay (ELISA) and urinary F_2a‐isoprostanes were measured by enzyme immunoassay (EIA). Results F_2a‐isoprostane concentrations were elevated in women who smoked, a behaviour associated with increased oxidative stress, but not in stages of the natural menopause. Mean F_2a‐isoprostane concentrations among pre‐ and postmenopausal women who smoked were 1082 and 1064 pg/ml, respectively, values double those in pre‐ (343 pg/ml) and postmenopausal (379 pg/ml) nonsmoking women. 2‐OHE1 and F_2a‐isoprostane concentrations were positively and highly correlated (partial correlations ρ_Y|X = 0·44 and ρ_Y|X = 0·43 in pre‐ and postmenopausal women, respectively). Similarly, 16α‐OHE1 concentrations were positively and highly correlated with F_2a‐isoprostane concentrations (ρ_Y|X = 0·52 and ρ_Y|X = 0·59 in pre‐ and postmenopausal women, respectively). E2 was significantly correlated with F_2a‐isoprostanes only in postmenopausal women (ρ_Y|X = 0·20). Associations were adjusted for age, body mass index (BMI), race/ethnicity, lipids, physical activity level and alcohol consumption. Conclusions This study does not support the commonly held hypothesis that levels of endogenous E2 or its oestrone metabolites favourably modify oxidative stress by decreasing F2_a‐isoprostane levels.     

Is alcohol hepatotoxic in the baboon?

The baboon is the only animal in which alcoholic fibrosis and cirrhosis of the liver has been produced with a nutritionally adequate diet. Zinc deficiency is associated with alcoholic liver disease and may contribute to liver damage. We have therefore investigated whether zinc supplementation would reduce liver damage in ten baboons receiving ethanol and an adequate diet. Eight received ethanol at up to 25 g/kg/day (70% of calories) for up to 60 months (four were supplemented with 50 mg zinc/day). All animals gained weight, and blood concentrations of ethanol were 63-342 mg/dl. Changes in liver blood tests were slight. Liver histology only showed fatty change in six animals, severe in two, and minor inflammatory changes but no significant fibrosis or cirrhosis. In one of the animals with severe fatty change there were also degenerative changes in parenchymal cells. There was thus no significant hepatic fibrosis or cirrhosis in baboons given large amounts of ethanol and an adequate diet for up to 5 years.     

Adiponectin negatively correlates with alcoholic and non‐alcoholic liver dysfunction: Health check‐up study of Japanese men

Aim: Central obesity, insulin resistance and alcohol consumption are thought to be major risk factors for fatty liver formation. Adiponectin (APN) prevents fatty liver formation, and its serum levels are lower in subjects with central obesity and/or insulin resistance. The aim of this study was to explore the association among serum APN levels, central obesity, insulin resistance and liver dysfunction with or without fatty liver classified by alcohol consumption in healthy subjects. Methods: A total of 5588 Japanese male subjects who underwent a health check‐up were classified into three groups according to alcohol consumption: non‐ or light drinkers (15 g/day ≥ ethanol); mild drinkers (15 g/day < ethanol ≤ 30 g/day); and moderate‐ or heavy drinkers (30 g/day < ethanol). Central obesity and insulin resistance were assessed by waist circumference (WC) and Homeostasis Model of Assessment – Insulin Resistance (HOMA‐IR), respectively. Results: WC was significantly increased, while HOMA‐IR was significantly decreased according to the extent of alcohol consumption. Serum alanine aminotransferase levels were significantly lower and serum APN levels were significantly higher in mild drinkers than in the other two groups. Multiple linear regression analysis showed that serum APN level served as the significant and independent determinant for liver dysfunction in the subjects with fatty liver, irrespective of alcohol consumption. However, WC became a non‐significant determinant of liver dysfunction as alcohol consumption increased. Conclusion: Hypoadiponectinemia is a significant determinant for steatotic dysfunction for all levels of alcohol consumption, but central obesity was not a significant determinant for alcoholic fatty liver‐induced liver dysfunction.     

Development of alcoholic fatty liver and fibrosis in rhesus monkeys fed a low n-3 fatty acid diet

BACKGROUND: The amount and type of dietary fat seem to be important factors that modulate the development of alcohol-induced liver steatosis and fibrosis. Various alcohol-feeding studies in animals have been used to model some of the symptoms that occur in liver disease in humans. METHODS: Rhesus monkeys (Macaca mulatta) were maintained on a diet that had a very low concentration of alpha-linolenic acid and were given free access to an artificially sweetened 7% ethanol solution. Control and ethanol-consuming animals were maintained on a diet in which the linoleate content was adequate (1.4% of energy); however, alpha-linoleate represented only 0.08% of energy. Liver specimens were obtained, and the fatty acid composition of the liver phospholipids, cholesterol esters, and triglycerides of the two groups were compared at 5 years and histopathology of tissue samples were compared at 3 and 5 years. RESULTS: The mean consumption of ethanol for this group over a 5-year period was 2.4 g.kg.day. As a consequence of the ethanol-dietary treatment, there were significantly lower concentrations of several polyunsaturated fatty acids in the liver phospholipids of the alcohol-treated group, including arachidonic acid and most of the n-3 fatty acids and particularly docosahexaenoic acid, when compared with dietary controls. Liver specimens from animals in the ethanol group at 5 years showed a marked degree of steatosis, both focal and diffuse cellular necrosis, and an increase in the development of fibrosis compared with specimens obtained at 3 years and with those from dietary controls, in which there was no evidence of fibrotic lesions. CONCLUSION: These findings suggest that the advancement of ethanol-induced liver disease in rhesus monkeys may be modulated by the amount and type of dietary essential fatty acids and that a marginal intake of n-3 fatty acids may be a permissive factor in the development of liver disease in primates.     

Differential Effects of Ethanol on Serum GABAergic 3α,5α/3α,5β Neuroactive Steroids in Mice,Rats, Cynomolgus Monkeys,and Humans

Background: Acute ethanol administration increases plasma and brain levels of progesterone and deoxycorticosterone‐derived neuroactive steroids (3α,5α)‐3‐hydroxypregnan‐20‐one (3α,5α‐THP) and (3α,5α)‐3,21‐dihydroxypregnan‐20‐one (3α,5α‐THDOC) in rats. However, little is known about ethanol effects on GABAergic neuroactive steroids in mice, nonhuman primates, or humans. We investigated the effects of ethanol on plasma levels of 3α,5α‐ and 3α,5β‐reduced GABAergic neuroactive steroids derived from progesterone, deoxycorticosterone, dehydroepiandrosterone, and testosterone using gas chromatography‐mass spectrometry. Methods: Serum levels of GABAergic neuroactive steroids and pregnenolone were measured in male rats, C57BL/6J and DBA/2J mice, cynomolgus monkeys, and humans following ethanol administration. Rats and mice were injected with ethanol (0.8 to 2.0 g/kg), cynomolgus monkeys received ethanol (1.5 g/kg) intragastrically, and healthy men consumed a beverage containing 0.8 g/kg ethanol. Steroids were measured after 60 minutes in all species and also after 120 minutes in monkeys and humans. Results: Ethanol administration to rats increased levels of 3α,5α‐THP, 3α,5α‐THDOC, and pregnenolone at the doses of 1.5 g/kg (+228, +134, and +860%, respectively, p < 0.001) and 2.0 g/kg (+399, +174, and +1125%, respectively, p < 0.001), but not at the dose of 0.8 g/kg. Ethanol did not alter levels of the other neuroactive steroids. In contrast, C57BL/6J mice exhibited a 27% decrease in serum 3α,5α‐THP levels (p < 0.01), while DBA/2J mice showed no significant effect of ethanol, although both mouse strains exhibited substantial increases in precursor steroids. Ethanol did not alter any of the neuroactive steroids in cynomolgus monkeys at doses comparable to those studied in rats. Finally, no effect of ethanol (0.8 g/kg) was observed in men. Conclusions: These studies show clear species differences among rats, mice, and cynomolgus monkeys in the effects of ethanol administration on circulating neuroactive steroids. Rats are unique in their pronounced elevation of GABAergic neuroactive steroids, while this effect was not observed in mice or cynomolgus monkeys at comparable ethanol doses.     

4-hydroxynonenal levels are enhanced in fetal liver mitochondria by in utero ethanol exposure

Lipid peroxidation has been implicated in ethanol-induced liver injury and observed in fetal liver and brain after maternal ethanol consumption with mitochondria being the target organelles. This process generates a highly reactive and toxic product, 4-hydroxynonenal (HNE). In the present study, HNE levels and metabolism were assessed in mitochondria of fetal and maternal liver after in vivo ethanol exposure. Female Sprague-Dawley rats received five doses of ethanol (4 g/kg orally at 12-hour intervals) and were killed on day 19 of gestation. The results showed that HNE levels were enhanced in hepatic mitochondria of fetal rats exposed to ethanol, far in excess of that in adult liver mitochondria. Measurement of HNE metabolism showed that fetal mitochondria had a lower capacity for HNE catabolism than adult mitochondria. In adult mitochondria, HNE could be metabolized by nicotine adenine dinucleotide-dependent oxidation, reduced glutathione conjugation, and reduced nicotine adenine dinucleotide-dependent reduction, whereas in fetal liver only the former two pathways were active, but to a lesser degree than in adult mitochondria. On the other hand, mitochondria from fetal liver showed a higher production of HNE when oxidative stress was induced with t-butyl hydroperoxide. Prior in vivo ethanol exposure further potentiated HNE formation in t-butyl hydroperoxide-stimulated fetal liver mitochondria, but not in adult mitochondria. These findings indicate that increased levels of HNE in fetal liver mitochondria after maternal ethanol consumption reflect a higher susceptibility to HNE formation in addition to a lesser capacity to metabolize it. The enhanced accumulation of this toxic aldehyde may contribute to oxidative damage observed in fetal tissues after in utero ethanol exposure.(Hepatology 1997 Jan;25(1):142-7)     

Alcoholic liver disease in the elderly

Although per capita alcohol consumption, and thus the prevalence of alcoholic liver disease, decreases generally with age in Europe and in the United States, recently an increase in alcohol consumption has been reported in individuals over 65 years. Reasons explaining this observation may include an increase in life expectancy or a loss of life partners and, thus, loneliness and depression. Although ethanol metabolism and ethanol distribution change with age, and an elderly person's liver is more susceptible to the toxic effect of ethanol, the spectrum of alcoholic liver diseases and their symptoms and signs is similar to that seen in patients of all ages. However, prognosis of alcoholic liver disease in the elderly is poor. In addition, chronic alcohol consumption may enhance drug associated liver disease and may also act as a cofactor in other liver diseases, such as viral hepatitis and nonalcoholic fatty liver disease.     

A voluntary oral ethanol-feeding rat model associated with necroinflammatory liver injury

Background: The intragastric (IG) ethanol infusion model results in fatty liver, necrosis, inflammation and fibrosis. This model was utilized to study the pathogenesis of alcoholic liver disease (ALD). Disadvantages of the IG model include maintenance of the animals and equipment expense. To develop a voluntary feeding model for ALD, we took advantage of two important observations in the IG model: (i) female rats demonstrate greater severity of alcohol‐induced liver injury than males and (ii) rats fed fish oil as a source of fatty acids develop more severe alcoholic liver injury than rats fed other fatty acids with ethanol. Methods: Female Wistar rats (205 to 220 g) were fed for 8 weeks a diet containing 8% ethanol, fish oil (30% of calories), protein, and dextrose. Pair‐fed controls (FD) received dextrose in amounts isocaloric to ethanol. The following measurements were made: liver pathology [fatty liver (0 to 4), necrosis, inflammation and fibrosis by Sirius Red], endotoxin and alanine aminotransferase (ALT) in plasma, urine ethanol, lipid peroxidation, nuclear factor kappa‐B (NF‐κB) and mRNA levels for tumor necrosis factor‐alpha (TNF‐α), cyclooxygenase‐2 (COX‐2), and inducible nitric oxide synthase (iNOS). Protein levels for iNOS and nitrotyrosine were evaluated by immunohistochemistry and Western Blot analysis. Liver proteasome and cytochrome P450 2E1 activity and protein levels of asialoglycoprotein receptor (ASGPR) were also evaluated. In addition, mRNA levels of fibrogenic markers were assessed. Results: All animals lost weight for the initial 2 to 3 weeks but then gained weight until killing at 8 weeks. There was, however, a significant difference (p < 0.05) in weight between the ethanol‐fed (Etoh) and (FD) groups at the end of the experiment. The mean urine ethanol levels ranged between 190 and 240 mg/dl. The severity of pathological changes was greater (p < 0.01) in Etoh vs. FD: fatty liver, 3.0 ± 1.2 vs. 1.2 ± 0.4; necrosis (foci/mm²), 3.9 ± 2.3 vs. 0.4 ± 0.3; inflammation (cells/mm²), 19.0 ± 6.3 vs. 1.8 ± 0.6. Centrilobular collagen deposition (% area), assessed by Sirius Red staining, was greater in Etoh vs. FD. Levels of endotoxin, ALT, CYP2E1 and lipid peroxidation markers were also higher (p < 0.01) in Etoh vs. FD. Levels of NF‐κB and mRNA of pro‐inflammatory mediators (TNF‐α, COX‐2, iNOS) and procollagen‐I were increased (p < 0.05) in ethanol‐fed rats. Immunohistochemical analysis showed more intense staining for both iNOS and nitrotyrosine in the centrilobular areas in the Etoh vs. FD groups. The greater area of positive staining for iNOS and nitrotyrosine in Etoh vs. FD was confirmed by Western Blot analysis. An increase in the expression of mRNA for profibrogenic genes (p < 0.05) was seen in ethanol‐fed rats. Conclusions: A voluntary feeding regimen consisting of fish oil and ethanol in female rats is technically less demanding yet produces pathological and biochemical changes similar to those observed with the IG model. Pathological changes include fatty liver, necrosis and inflammation. Increased NF‐κB and mRNA and protein levels of the pro‐inflammatory mediators TNF‐α, COX‐2 and iNOS, coincided with the presence of necroinflammatory changes. The voluntary feeding regimen is proposed as an alternative to the IG model in the study of alcoholic liver injury.     

Chronic Alcohol Consumption Disrupted Cholesterol Homeostasis in Rats: Down‐Regulation of Low‐Density Lipoprotein Receptor and Enhancement of Cholesterol Biosynthesis Pathway in the Liver

Background: Chronic alcohol consumption causes alcoholic liver disease, which is associated, or initiated, with dysregulated lipid metabolism. Very recent evidence suggested that dysregulated cholesterol metabolism plays an important role in the pathogenesis of alcoholic fatty liver diseases, however, the effects of chronic alcohol exposure on cholesterol homeostasis have not been well studied and underlying mechanisms behind are still elusive. Methods: Male Sprague–Dawley rats weighing 250 ± 5.5 g (mean ± SEM) divided into 2 groups (8 rats per group) and pair‐fed with liquid diets containing (in percent of energy intake) 18% protein, 35% fat, 12% carbohydrate, and 35% either ethanol (ethanol diet) or an isocaloric maltose‐dextrin mixture (control diet), according to Lieber and De Carli, for 4 weeks. Results: Long‐term excessive alcohol feeding to rats caused fatty liver and liver injury, which was associated with disrupted cholesterol homeostasis, characterized by increased hepatic cholesterol levels and hypercholesterolemia. Hepatic cholesterol increases were concomitant with constantly activated sterol regulatory element‐binding protein‐2 (SREBP‐2) in the liver and increased expression of 3‐hydroxy‐3‐methyl‐glutaryl‐CoA (HMG‐CoA) reductase, a rate‐limiting enzyme for cholesterol de novo synthesis, indicating enhanced cholesterol biosynthesis. Alcohol‐induced hypercholesterolemia was accompanied by decreased LDL receptor (LDLr) levels in the liver. Further investigations revealed that chronic alcohol exposure increased hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) contents to down‐regulate LDLr via a post‐translational mechanism. Moreover, alcohol feeding suppressed extracellular signal‐regulated kinase (ERK) activation in the liver. In vitro studies showed that inhibition of ERK activation was associated with decreased LDLr expression in HepG2 cells. Conclusions: Our study provides the first evidence that both increased PCSK9 expression and suppressed ERK activation in the liver contributes to alcohol‐induced hypercholesterolemia in rats.     

Effects of Chronic Ethanol Administration on 02 Consumption in Whole Body and Perfused Liver of the Rat

The effects of chronic ethanol consumption on thyroid hormone levels and the rates of whole animal and perfused liver oxygen consumption were determined to test the hypothesis that alcoholic liver damage is a result of thyroid mediated liver hypermetabolism (L. Videla, J. Bernstein, and Y. Israel: Biochem. J, 134: 507–514, 1973). Whole animal minimal oxygen consumption, a sensitive indicator of the effects of thyroid hormone (W. D. Denckla: J. Clin. Invest, 53:572–581, 1974) was unchanged in rats maintained 3 wk on a liquid diet containing 34% of the calories as ethanol (2.49 ± 0.06 ml of O₂/min/1O0 g of fat-free body weight) when compared to animals fed an equicaloric sucrose containing liquid diet (2.61 ± 0.20 ml of 0₂/min/100 g of fat-free body weight) or Purina chow (2.50 ± 0.12 ml of O₂/min/100 g of fat-free body weight). Ethanol treatment lowered serum thyroxine (5.09 ± 0.20 pg/100 ml) compared to sucrose-fed control rats (7.66 ± 0.40 pg/100 ml), while serum triiodothyronine was unaffected (59.3 ± 4.0 compared to 66.9 ± 3.1 ng/100 ml for controls). Measurement of O₂ consumption in the isolated perfused rat liver showed no significant difference after chronic treatment with the ethanol diet compared to either the sucrose or chow control diets. Infusion of 10^-7 M norepinephrine into the perfusion medium resulted in an approximately 22% increase in O₂ consumption in ethanol-fed animal and sucrose controls, while a 31 % increase was observed for sucrose-treated animals given 10 μg of T₃/kg of body weight/day for 3 wk. These data indicate that T₃ potentiates the ability of norepinephrine to increase O₂ consumption. The data presented give no support to the concept that chronic ethanol ingestion results in hyperthyroidism or liver hypermetabolism and, consequently, the rationale for treatment of alcoholic hepatitis with the antithyroid drug, propylthiouracil, is incorrect (H. Orrego, H. Ka-lant, Y. Israel, et al.: Gastroenterology, 76:105–115, 1979).     

Cytochrome P450 2E1 contributes to ethanol-induced fatty liver in mice

Cytochrome P450 2E1 (CYP2E1) is suggested to play a role in alcoholic liver disease, which includes alcoholic fatty liver, alcoholic hepatitis, and alcoholic cirrhosis. In this study, we investigated whether CYP2E1 plays a role in experimental alcoholic fatty liver in an oral ethanol-feeding model. After 4 weeks of ethanol feeding, macrovesicular fat accumulation and accumulation of triglyceride in liver were observed in wild-type mice but not in CYP2E1-knockout mice. In contrast, free fatty acids (FFAs) were increased in CYP2E1-knockout mice but not in wild-type mice. CYP2E1 was induced by ethanol in wild-type mice, and oxidative stress induced by ethanol was higher in wild-type mice than in CYP2E1-knockout mice. Peroxisome proliferator-activated receptor alpha (PPARalpha), a regulator of fatty acid oxidation, was up-regulated in CYP2E1-knockout mice fed ethanol but not in wild-type mice. A PPARalpha target gene, acyl CoA oxidase, was decreased by ethanol in wild-type but not in CYP2E1-knockout mice. Chlormethiazole, an inhibitor of CYP2E1, lowered macrovesicular fat accumulation, inhibited oxidative stress, and up-regulated PPARalpha protein level in wild-type mice fed ethanol. The introduction of CYP2E1 to CYP2E1-knockout mice via an adenovirus restored macrovesicular fat accumulation. These results indicate that CYP2E1 contributes to experimental alcoholic fatty liver in this model and suggest that CYP2E1-derived oxidative stress may inhibit oxidation of fatty acids by preventing up-regulation of PPARalpha by ethanol, resulting in fatty liver.     

Interaction between oxidative stress and high‐density lipoprotein cholesterol is associated with severity of coronary artery calcification in rheumatoid arthritis

Objective

To test the hypothesis that oxidative stress is increased in patients with rheumatoid arthritis (RA) due to increased inflammation and contributes to the pathogenesis of atherosclerosis.

Methods

The independent association between urinary F₂‐isoprostane excretion, a measure of oxidative stress, and RA was tested using multiple linear regression models in 169 patients with RA and 92 control subjects, frequency matched for age, race, and sex. The relationship between F₂‐isoprostane excretion and coronary calcium, a marker of atherosclerosis, was examined in multivariable proportional odds logistic regression models that also assessed the interactions between oxidative stress and low‐density lipoprotein and high‐density lipoprotein (HDL) cholesterol.

Results

F₂‐isoprostane excretion was significantly higher in patients with RA (median 2.75 [interquartile range (IQR) 1.60–4.06] ng/mg creatinine) than in control subjects (median 1.86 [IQR 1.25–2.62] ng/mg creatinine; adjusted P = 0.006). In patients with RA, F₂‐isoprostanes were positively correlated with body mass index (P < 0.001), but not with disease activity or mediators of inflammation such as the Disease Activity Score in 28 joints or serum tumor necrosis factor α, interleukin‐6, and C‐reactive protein concentrations in adjusted multivariable models (P > 0.05 for all). In patients with RA, F₂‐isoprostanes significantly modified the effect of HDL cholesterol on coronary calcification (P = 0.02 for interaction) after adjustment for age, sex, and race. As F₂‐isoprostane levels increased, HDL lost its protective effect against coronary calcification.

Conclusion

Oxidative stress measured as F₂‐isoprostane excretion was higher in patients with RA than in control subjects. Among patients with RA, higher F₂‐isoprostane excretion and HDL cholesterol concentrations interacted significantly and were positively associated with the severity of coronary calcification.     

4-hydroxynonenal detoxification by mitochondrial glutathione S-transferase is compromised by short-term ethanol consumption in rats

BACKGROUND: 4-Hydroxynonenal (HNE), a toxic lipid peroxidation product, has been implicated in mitochondrial damage in rat liver by ethanol consumption. The present study assessed the effects of short-term in vivo ethanol exposure on HNE detoxification by mitochondrial glutathione S-transferase (GST). METHODS: Male Sprague Dawley rats were administered 5 doses of ethanol (4 g/kg) at 12 hr intervals by gavage. Pair-fed rats that received isocaloric dextrose instead of ethanol served as controls. Mitochondrial and submitochondrial fractions were prepared from the livers. Mitochondrial contents of HNE and HNE-glutathione conjugate were measured by high-performance liquid chromatography. GST isoforms were identified by Western blots in submitochondrial fractions. RESULTS: Whereas there was an 80% increase in mitochondrial HNE content after ethanol consumption, there was a 42% decrease in the content of HNE-glutathione conjugate, compared with controls (p< 0.05). After ethanol exposure, the GST activities toward HNE in intact mitochondria and in the membranous fraction were decreased by 37% and 45% (p< 0.05), respectively, whereas that in the aqueous fraction was unchanged. Kinetic analysis of HNE conjugation by the membrane-associated GST showed that ethanol decreased the V(max) nearly by half (p< 0.05), whereas it did not affect the K(m). HNE conjugation by the aqueous GST demonstrated a higher K(m) than that of the membrane-associated GST, although its kinetics were not significantly altered by ethanol. Immunochemical analysis with Western blots demonstrated that both the membranous and the aqueous fractions of mitochondria contain GST-alpha and GST-mu isoforms, whereas GST-pi was absent. CONCLUSIONS: HNE detoxification by mitochondrial GST is compromised by short-term ethanol consumption, which may contribute to elevated mitochondrial HNE content and hence its toxicity in the ethanol-exposed liver.     

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.     

Formation of malondialdehyde adducts in livers of rats exposed to ethanol: role in ethanol-mediated inhibition of cytochrome c oxidase

BACKGROUND: Previous studies in our laboratory demonstrated that short-term ethanol consumption by maternal rats increased the hepatic levels of 4-hydroxynonenal (HNE) in both the adult and the fetus. Additionally, HNE inhibited cytochrome c oxidase (COX) by forming adducts with the enzyme subunits. The present study examined modification of COX by another major aldehydic lipid peroxidation product, malondialdehyde (MDA), and its role in COX inhibition by ethanol. METHODS AND RESULTS: It is demonstrated in vitro that MDA inhibits the activity of purified COX while forming adducts with the enzyme. Compared with HNE, MDA is a more potent inhibitor of COX. Overnight incubation at room temperature caused an 80% decrease in COX activity by MDA versus a 67% decrease by HNE. MDA produced marked inhibition of COX activity at physiologically relevant concentrations, e.g., 43% inhibition at 10 microM. Although our previous studies documented that HNE formed adducts primarily with subunit IV of COX via histidine residues, the current report showed that MDA forms adducts with both subunit IV and subunit V via lysine residues. Furthermore, both aldehydes induce carbonyl formation in subunit IV. The in vivo role of MDA in the impairment of COX by ethanol is assessed in both adult and fetal liver after maternal ethanol consumption. CONCLUSIONS: The results showed that: (1) there are significant increases in MDA levels in liver homogenate as well as mitochondria in both adult and fetal livers after ethanol exposure; (2) these MDA levels are in the nanomole/mg protein range, in contrast to picomole/mg protein range of HNE in identical setting; and (3) ethanol-induced production of MDA is accompanied by enhanced formation of MDA adducts with COX. These findings suggest that MDA may play at least as equally an important role as HNE in ethanol-induced inhibition of COX.     

Hybrid bioartificial liver support in cynomolgus monkeys with D-galactosamine-induced acute liver failure

AIM: To evaluate a hybrid bioartificial liver support system (HBALSS) in cynomolgus monkeys with acute liver failure.METHODS: To establish a model of acute liver failure, 0.3 g/kg of D-galactosamine was injected intravenously into cynomolgus monkeys. Chinese human liver cells were introduced into a perfusion bioreactor to carry out hybrid bioartificial liver support treatment. Forty-eight hours after the injection, one group of cynomolgus monkeys received HBALSS care, and a second experimental group received no treatment. Clinical manifestations of all animals, survival time, liver and kidney functions and serum biochemistry changes were recorded. Simultaneous detection of the number, viability and function of hepatocytes in the hybrid bioartificial liver were also performed.RESULTS: Forty-eight hours after the injection of D-galactosamine, serum biochemistry levels were significantly increased, whereas albumin levels and the Fischer index were significantly reduced compared to baseline (all Ps < 0.05). Of the ten monkeys in the HBALSS treatment group, five survived, with an average duration of survival of 128 ± 3 h. All cynomolgus monkeys in the control group died, with a duration of survival of 112 ± 2 h. Survival time was significantly longer with HBALSS treatment (P < 0.05). Moreover, the number, viability and function of hepatocytes were maintained at a high level with HBALSS.CONCLUSION: The novel hybrid bioartificial liver plays a significant role in liver support by significantly reducing serum biochemistry levels and extending animal survival time.