1 Department of Gastroenterology, University Hospital of Crete, Crete and 2 Liver Research Laboratory, University of Crete, Medical School, Crete, Greece
* Author to whom correspondence should be addressed at: Department of Gastroenterology, University Hospital of Crete, PO Box 1352, Heraklion 71110, Crete, Greece. Tel.: +30 28 1039 2356; Fax: +30 28 1054 2085; E-mail: kouroum{at}med.uoc.gr
(Received 22 February 2003; first review notified 3 April 2003; in revised form 22 October 2003; accepted 17 November 2003)
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ABSTRACT |
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BACKGROUND |
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Nitric oxide (NO) plays an important role in the regulation of portal vein pressure and influences splachnic vasodilatation in cirrhotic patients (Vallance et al., 1991). Moreover, NO overproduction precedes hyperdynamic splachnic circulation in rats with experimental portal hypertension (Wiest et al., 1999
). It is known that in rats' Kupffer cells excessive production of NO is diminished by chronic alcohol administration (Kimura et al., 1996
). The effect of binge drinking on the production of NO metabolites in patients with chronic liver disease has not been studied before.
We therefore studied the effect of acute alcohol administration on NOx serum levels in patients with viral and alcoholic cirrhosis and compared them with healthy controls and patients with chronic viral hepatitis.
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PATIENTS AND METHODS |
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In all patients diagnosis had been confirmed by liver biopsy. Nine patients were diagnosed with viral cirrhosis (five male, seven HCV, two HBV, six Child's A, three Child's B, four smokers). Fifteen patients had alcoholic cirrhosis, (12 male, 10 Child's A, five Child's B, 10 smokers). Thirteen patients had chronic viral hepatitis (nine male, nine HCV, four HBV, six smokers).
No patient was receiving any diuretic or corticoid treatment. In the group of chronic viral hepatitis patients, eight of the 13 patients (six HCV, two HBV) had received interferon treatment that was concluded 6 months or more before the study began. Healthy controls were recruited from the personnel of the Gastroenterology Department. Patients and controls remained on ward during 24 h following binge drinking and consumed a standard hospital diet of 18002200 kcal. Permission from the Ethics Committee of the Hospital was obtained and all patients and controls gave an informed consent for participation in the study.
Study design
Ethanol at a total of 80 g as a 40% solution was ingested in the morning after an over-night fast. Ethanol administration was completed within 30 min. Whole blood was collected before drinking and at 2, 12 and 24 h after completion of alcohol consumption. Serum was separated by centrifugation and kept at 70°C until NOx measurements. Patients were kept in a normal hospital diet after ethanol administration.
Nitric oxide measurements
Total serum nitrite (NO3) and nitrate (NO2) concentration was measured by a modification of the Griess reaction as previously described (Matrella et al., 2001). Briefly, 100 µl serum sample was incubated for 30 min at 37°C in the presence of 0.2 U/ml Aspergilus nitrate reductase, 5 µmol/l FAD and 0.1 mmol/l NADPH in 50 mmol/l HEPES buffer (total volume 500 ml) for the conversion of nitrate to nitrite. Following the incubation, 5 µl lactate dehydrogenase (1500 U/ml) and 50 µl 100 mmol/l pyruvic acid were added to each tube to oxidize any unreacted NADPH (reduced pyridine nucleotides strongly inhibit the Griess reaction). Samples were then incubated for 10 min at 37°C. Finally, 1 ml pre-mixed Griess reagent was added to each tube. After a 10-min incubation time at room temperature, the absorbance of each sample was determined at 543 nm on a Hitachi U-2000 spectrophotometer (Moriyama et al., 1997
).
Statistical analysis
Differences in NOx levels within each group at 2, 12 and 24 h were evaluated by the Student's test for paired data, using levels before alcohol intake as reference values. Comparisons among groups were performed by the Student's t-test for unpaired data and P < 0.05 was considered as statistically significant. All statistical comparisons were performed using the SPSS for Windows statistical package (SPSS, Chicago, IL).
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RESULTS |
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A similar but steeper increase at 12 h was observed for patients with chronic hepatitis (012 h P < 0.01, 1224 h P < 0.01). By contrast, in cirrhotic patients irrespective of aetiology this increase at 12 h was not observed (Fig. 2).
In general, serum levels of NOx in cirrhotics at any given time were similar to those obtained in healthy controls after binge drinking.
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DISCUSSION |
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Ethanol has been experimentally shown to influence nitric oxide production in the rat (Kato et al., 1996). It has been proposed that NO has a possible protective effect by attenuating the detrimental effects of ethanol on liver microcirculation (Oshita et al., 1994
; McCuskey et al., 1995
; Nanji et al., 1995
). However, chronic ethanol administration seems to lead to NO-induced liver injury (Chamulitrat et al., 1996
). Moreover, chronic ethanol feeding in the rat has been found to reduce both nitric oxide production and anti-tumour activity of Kupffer cells (Kato et al., 1996
).
In human alcoholic liver disease there is evidence that the high haemodynamic state of patients with advanced cirrhosis is possibly due to either increased endothelial synthesis of NO (Calver et al., 1994; Campillo et al., 1995
) or smooth muscle derived NO (Ryan et al., 1996
). Peripheral blood monocytes from patients with alcoholic hepatitis secrete more nitric oxide than monocytes from patients without alcoholic hepatitis, (Hunt et al., 1992
) a finding not confirmed in another study (Criado-Jimenez et al., 1995
). Peripheral blood monocytes from patients with alcohol induced cirrhosis produce higher levels of NO compared to monocytes from healthy controls (Criado-Jimenez et al., 1995
; Sanchez-Rodriguez et al., 1998
).
Binge drinking has been experimentally shown to influence metabolic pathways in liver cells (Arteel et al., 1996; Rivera et al., 1998
). Gluconeogenesis was found to be delayed in isolated perfused rat liver (Deaciuc et al., 1992
) and F-actin content was found to be upregulated in leucocytes of female rats (Zhang et al., 1997
). Acute ethanol intoxication has been reported to stimulate superoxide anion production in the perfused rat liver (Bautista et al., 1992
) and ethanol induced oxidative stress has been shown to cause massive mitochondrial DNA degradation (Mansouri et al., 1999
). Acute alcohol intoxication may increase free radical release due to activation of Kupffer endothelial cells (Bautista et al., 1994
, 1996
, 1999
; Yokoyama et al., 1999
) and hepatocytes (Bailey et al., 1998
). Acute ethanol intoxication also influences expression of interleukin-6 surface receptors in both Kupffer cells and hepatocytes (Deaciuc et al., 1994
). Chemokine production by Kupffer cells is also modulated by ethanol binge (Bukara et al., 2000
; Bautista, 2001
).
The effect of binge drinking in serum NOx levels has not been studied before. Our findings indicate that after acute ethanol administration healthy controls and patients with histologically proven chronic viral hepatitis demonstrate an initial significant increase of serum NOx levels that is evident at 12 h after ethanol ingestion. In view of the experimental evidence mentioned before (Oshita et al., 1994; McCuskey et al., 1995
), this effect might protect liver microcirculation from a deleterious ethanol effect.
The initial increase of serum NOx levels was abolished in our cirrhotic patients both of alcoholic and viral aetiology. This might be due to the high basal levels observed in cirrhotics compared to healthy controls.
It is possible that the source organ of nitric oxide overproduction is already maximally stimulated and cannot respond with a further increase of NOx production. Higher NOx levels after binge drinking in patients with chronic hepatitis compared to cirrhotics are in disagreement to this hypothesis. However, another interpretation of these data would be that the source organ, although damaged, can still produce NOx to the maximum of this capacity.
In chronic viral hepatitis, basal NOx levels were increased compared to healthy controls but did not reach those found in cirrhotics. The role of nitric oxide production in chronic viral hepatitis is still unknown. In contrast to our study, lower levels of serum NOx have been reported in patients with chronic viral hepatitis (Amaro et al., 1997). On the other side, there is evidence of a virus-induced upregulation of inducible nitric oxide synthase expression (Majano et al., 1998
). HBV X protein has been reported to transactivate the promoter of the inducible isoform of nitric oxide synthase (Amaro et al., 1999
). Interestingly, in some patients with HCV infection, although liver tissue iNOs is increased, this was not accompanied by significantly elevated serum NO levels (Mihm et al., 1997
).
The origin of NO overproduction in our healthy controls and viral hepatitis patients after binge drinking is not clear. The 12 h time interval required for this increase indicates a NO synthase induction. Whether this induction takes place in the hepatocyte, the Kupffer cell, the peripheral monocytes or the peripheral vasculature requires further studies. Kupffer cell, however, is a strong candidate. In an experimental model of week-end type alcohol binging, activation of Kupffer cells was demonstrated with an increased production of TNF and superoxide anions (Abril et al., 1999
).
In conclusion, our data showed that binge drinking in patients with chronic viral hepatitis causes a significant increase of serum NOx 12 h after alcohol ingestion. This increase subsides at pre-drinking levels after 24 h. Similar behaviour of serum NOx levels was observed in healthy controls. In contrast, serum NOx levels of cirrhotic patients, irrespective of aetiology, are not influenced by binge drinking and remain persistently elevated.
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