We investigated the association between fasting serum and urinary nitric oxide metabolite (NOx) levels and fatty liver index (FLI), a non-invasive surrogate of non-alcoholic fatty liver disease (NAFLD) and liver steatosis.

This cross-sectional study included 598 adults (aged≥18 years, 48.6% men) who participated in the Tehran Lipid and Glucose Study (2015-2017). Serum and urine NOx concentrations were quantified using a spectrophotometric method following the Griess reaction. FLI values were calculated using γ-glutamyl transferase, triglycerides, body mass index, and waist circumference. The associations between urinary and serum NOx-to-creatinine (Cr) ratio [either as a categorical variable, i.e., tertiles, or as a continuous variable, i.e., per 1 SD) with NAFLD (i.e., FLI≥60) were assessed using multivariable-adjusted binary logistic regression.

The study participants' mean (SD) age was 42.5±14.6 y. The mean (SD) of serum and urinary NOx was 37.5±16.7 and 1310±751 μmol/L, respectively. The mean (SD) of FLI was 43.3±30.2, and the prevalence of NAFLD was 32.4%. Serum NOx-to-Cr ratio was not associated with the chance of having NAFLD (OR=1.66, 95% CI=0.98-2.82; P value=0.058). Higher urinary NOx-to-Cr ratio was significantly associated with a reduced probability of NAFLD (OR=0.61, 95% CI=0.38-0.95, and OR=0.54, 95% CI=0.34-0.87, in the second and third tertiles).

Higher dietary nitrate (NO3) intake, indicated by increased urinary NOx-to-Cr ratio, is associated with a reduced probability of NAFLD, highlighting the potential role of dietary NO3 in liver health.

Increases in the systemic vasodilator adrenomedullin and the renal vasoconstrictors thromboxane A₂ in cirrhotic patients are pathogenic factors for the development of functional acute kidney injury (AKI), including pre-renal azotemia (PRA) and hepatorenal syndrome (HRS), which is associated with high mortality. This study aims to find biomarkers that can diagnose HRS at an early stage, to enable treatment as soon as possible.

Acute decompensated cirrhotic patients who had been admitted to hospital were enrolled in this prospective cohort study. Blood and urinary samples were collected immediately after admission. In addition to initially categorizing AKI cases into PRA, acute tubular necrosis (ATN), and HRS groups, their final diagnosis was adjudicated by a nephrologist and a hepatologist who checked the corrected and misclassification rates for significant biomarkers.

The cut-off values for serum adrenomedullin and urinary thromboxane B₂ (TXB₂ ), when used as predictors for functional AKI (adrenomedullin >283 pg/mL, urinary TXB₂ >978 [pg/mg urinary creatinine]), for HRS (adrenomedullin >428, urinary TXB₂  >1604), and for good terlipressin plus albumin treatment responders (adrenomedullin >490, urinary TXB₂  >1863), were observed. Patients with HRS who could be treated, due to high mortality, had significantly higher serum adrenomedullin and urinary TXB₂ levels compared to HRS patients receiving standard treatment. In addition to predicting 60-day mortality, a combination of these two markers further increased diagnostic accuracy for HRS among functional AKI.

Prompt diagnosis of HRS by differentiating it from PRA and ATN can be achieved by using serum adrenomedullin and urinary TXB₂ in acute decompensated cirrhotic patients. In combination with severe clinical courses, these two markers are useful to select HRS patients who cannot be treated.

To investigate whether a novel immune function biomarker QuantiFERON-Monitor (QFM) can identify cirrhotic patients at greatest risk of infection.

Adult cirrhotic patients on the liver transplant waiting list were recruited for this observational cohort study from a tertiary liver transplant referral unit. The immune function biomarker, QFM was performed using the same method as the widely available Quantiferon-gold assay, and measures output in interferon gamma in IU/mL after dual stimulation of the innate and adaptive immune systems. Ninety-one cirrhotic patients were recruited, with 47 (52%) transplanted on the day of their QFM. The remaining 44 (48%) were monitored for infections until transplant, death, or census date of 1st February 2014.

Cirrhotic patients express a median QFM significantly lower than healthy controls (94.5 IU/mL vs 423 IU/mL), demonstrating that they are severely immunosuppressed. Several factors including model for end stage liver disease, presence of hepatocellular carcinoma, bilirubin, international normalized ratio and haemoglobin were associated with QFM on univariate analysis. Disease aetiology did not appear to impact QFM. On multivariate analysis, only Child-Pugh score and urea were significantly associated with a patient's immune function as objectively measured by QFM. In the 44 patients who were not transplanted immediately after their blood test and could be monitored for subsequent infection risk, 13 (29.5%) experienced a pre-transplant infection a median 20 d (range 2-182) post-test. QFM < 214 IU/mL was associated with HR = 4.1 (P = 0.01) for infection. A very low QFM < 30 IU/mL was significantly associated (P = 0.003) with death in three patients who died while awaiting transplantation (HR = 56.6).

QFM is lower in cirrhotics, allowing objective determinations of an individual's unique level of immune dysfunction. Low QFM was associated with increased susceptibility to infection.

To compare vascular Doppler waveform indices, particularly in the common carotid arteries, between cirrhotic and healthy subjects.

A total of 60 patients with Class-B cirrhosis and 60 healthy matched counterparts were enrolled in this prospective study. Vascular Doppler waveform parameters including resistance and/or pulsatility indices (RI and PI, respectively) were obtained from the common carotid, renal, celiac, superior mesenteric, femoral and brachial arteries.

Compared to patients, healthy subjects had significantly higher mean PI and RI obtained from the common carotid (1.53 ± 0.20 vs. 1.43 ± 0.14, p = 0.03; 0.75 ± 0.02 vs. 0.72 ± 0.02, p < 0.001, respectively) and celiac arteries (2.00 ± 0.36 vs. 1.81 ± 0.34, p = 0.03; 0.80 ± 0.03 vs. 0.78 ± 0.02, p < 0.001, respectively). Both the mean PI and RI derived from the renal arteries, in contrast, were significantly higher in patients compared to that in controls (1.05 ± 0.13 vs. 1.11 ± 0.07, p = 0.03; 0.59 ± 0.03 vs. 0.63 ± 0.03, p < 0.001, respectively). The mean vascular impedance values obtained from the remaining arteries were comparable between the two groups.

Blood flow increases in the common carotid and celiac arteries of Class-B cirrhotic patients with elevated renovascular impedance.

The term cirrhosis-associated immune dysfunction refers to the main syndromic abnormalities of immune function, immunodeficiency and systemic inflammation that are present in cirrhosis. The course of advanced cirrhosis, regardless of its aetiology, is complicated by cirrhosis-associated immune dysfunction and this constitutes the pathophysiological hallmark of an increased susceptibility to bacterial infection, distinctive of the disease. Cirrhosis impairs the homeostatic role of the liver in the systemic immune response. Damage to the reticulo-endothelial system compromises the immune surveillance function of the organ and the reduced hepatic synthesis of proteins, involved in innate immunity and pattern recognition, hinders the bactericidal ability of phagocytic cells. Systemic inflammation, in form of activated circulating immune cells and increased serum levels of pro-inflammatory cytokines, is the result of persistent episodic activation of circulating immune cells from damage-associated molecular patterns, released from necrotic liver cells and, as cirrhosis progresses, from pathogen-associated molecular patterns, released from the leaky gut. Cirrhosis-associated immune dysfunction phenotypes switch from predominantly "pro-inflammatory" to predominantly "immunodeficient" in patients with stable ascitic cirrhosis and in patients with severely decompensated cirrhosis and extra-hepatic organ failure (e.g. acute-on-chronic liver failure), respectively. These cirrhosis-associated immune dysfunction phenotypes represent the extremes of a spectrum of reversible dynamic events that take place during the course of cirrhosis. Systemic inflammation can affect the functions of tissue somatic cells and modify the clinical manifestation of cirrhosis. The best characterized example is the contribution of systemic inflammation to the haemodynamic derangement of cirrhosis, which correlates negatively with prognosis.

This study aimed to determine whether serum levels of nitric oxide metabolites (nitrates and nitrites) correlate with renal dysfunction in patients with liver cirrhosis and, moreover, to assess nitric oxide metabolite (NOx) power for predicting hepatorenal syndrome (HRS) in such patients.

Among patients admitted to the Tropical Medicine Department, Ain Shams University Hospital, a total of 60 patients with chronic hepatitis C-related liver cirrhosis were included in this study. Patients were divided into three groups. Group I included 20 patients with compensated liver cirrhosis (CLC). Group II included 20 patients with decompensated liver cirrhosis (DLC). Group III included 20 patients with decompensated liver cirrhosis and HRS. Twenty healthy subjects with no clinical or laboratory evidence of liver disease were enrolled as a control group (group IV).

Patients with HRS had a higher mean nitrite levels followed by DLC, then CLC, and then controls. The sensitivity and specificity of NO metabolites (nitrites) were 100 % and 93.3 %, respectively, with accuracy of 95 % at cutoff value of 387 μmol/L for diagnosing patients with HRS. There was a highly significant statistical difference between patients positive and negative for nitrites as regards renal profile (p = 0.000).

A strong relation between nitrite cutoff value and renal dysfunction in liver cirrhosis has been found. Also, patients with HRS had higher mean serum nitrite levels than decompensated liver cirrhosis or compensated liver cirrhosis, raising the possibility of using nitrate and nitrite levels as a predictor for HRS in HCV-related liver cirrhosis.

Nitric oxide (NO) is an important regulator of renal hemodynamics and sodium excretion. Systemic and splanchnic NO-synthesis is increased in liver cirrhosis contributing to the characteristic hyperdynamic circulation. The significance of renal NO in human cirrhosis is not clear.

In order to clarify the role of NO in the regulation of renal hemodynamics and sodium excretion in human cirrhosis, we studied the effects of N(G)-monomethyl-L-arginine (L-NMMA) - a nonselective NO-inhibitor - on blood pressure (MAP), heart rate (HR), GFR, RPF, UNa × V, FENa, FELi and plasma levels of renin, angII, aldo, ANP, BNP and cGMP in 13 patients with cirrhosis (Child gr.A: 8; Child gr.B+C: 5) and 13 healthy controls.

The study was randomized and placebo-controlled. Renal hemodynamics were assessed by measuring renal clearance of (51) Cr-EDTA and (125) I-Hippuran for GFR and RPF, respectively.

L-NMMA induced a similar, significant increase in MAP in both groups and a more pronounced relative decrease in HR in the CIR group (P = 0.0209, anova). L-NMMA did not change GFR in any group, but RPF decreased significantly in both groups, but most pronouncedly in CIR (P = 0.0478, anova). FENa decreased significantly in both groups after l-NMMA, but the response was again most pronounced in the CIR group (P = 0.0270, anova). All parameters remained stable after placebo. No significant differences were observed between the effects of L-NMMA in Child gr.A vs. Child gr. B+C patients.

The data supports the hypothesis that renal NO is enhanced in human cirrhosis.

To evaluate the participation of nitric oxide (NO) and prostaglandin (PGI2) on hyperdynamic state in endotoxemia-induced portal hypertension (EIP) induced by chronic endotoxemia.

The portal pressure (PP) and mean arterial pressure (MAP) were recorded, in vivo before and after administration of L-NAME (NOS inhibitor) and indomethacin (specific blocker of COX). The vasoactive responses to acetylcholine of thoracic rat aortic rings were studied in vitro before and after nitric oxide and cyclooxygenase blockade using multichannel organ bath. The mRNA expression for isoforms of (cyclooxygenase) COX and nitric oxide synthase (NOS) were analyzed using RT-PCR.

Administration of both L-NAME and indomethacin in EIP rabbits significantly reduced (p < 0.05) the PP and reversed the MAP to normal as compared to sham-operated (SO) rabbits. There was impaired vasodilatory response to acetylcholine in EIP rabbits. L-NAME caused a significant reduction in acetylcholine-induced vasorelaxation in SO rabbits than EIP due to preexisting hyperemia in EIP. Indomethacin partially restored vasoresponsiveness to acetylcholine in EIP group. The mRNA expression of eNOS (endothelial NOS) and COX-1 (constitutive COX) were significantly higher in SO than EIP rabbits. iNOS (inducible NOS) and COX-2 (inducible COX) mRNA expression was seen only in EIP rabbits.

A significant component of acetylcholine-mediated vasorelaxation in EIP model is modulated by eNOS. There was increased production of contractile prostaglandin in EIP rabbits. iNOS and COX-2 play an important role in the hemodynamic abnormalities of PHT. This novel model of PHT produced by chronic splanchnic endotoxemia in rabbit, mimics impaired vasodilation and vasoreactivity akin to other models of PHT.

Recent studies have reported a high prevalence of relative adrenal insufficiency in patients with liver cirrhosis. However, the effect of corticosteroid replacement on mortality in this high-risk group remains unclear. We examined the effect of low-dose hydrocortisone in patients with cirrhosis who presented with septic shock.

We enrolled patients with cirrhosis and septic shock aged 18 years or older in a randomized double-blind placebo-controlled trial. Relative adrenal insufficiency was defined as a serum cortisol increase of less than 250 nmol/L or 9 μg/dL from baseline after stimulation with 250 μg of intravenous corticotropin. Patients were assigned to receive 50 mg of intravenous hydrocortisone or placebo every six hours until hemodynamic stability was achieved, followed by steroid tapering over eight days. The primary outcome was 28-day all-cause mortality.

The trial was stopped for futility at interim analysis after 75 patients were enrolled. Relative adrenal insufficiency was diagnosed in 76% of patients. Compared with the placebo group (n = 36), patients in the hydrocortisone group (n = 39) had a significant reduction in vasopressor doses and higher rates of shock reversal (relative risk [RR] 1.58, 95% confidence interval [CI] 0.98-2.55, p = 0.05). Hydrocortisone use was not associated with a reduction in 28-day mortality (RR 1.17, 95% CI 0.92-1.49, p = 0.19) but was associated with an increase in shock relapse (RR 2.58, 95% CI 1.04-6.45, p = 0.03) and gastrointestinal bleeding (RR 3.00, 95% CI 1.08-8.36, p = 0.02).

Relative adrenal insufficiency was very common in patients with cirrhosis presenting with septic shock. Despite initial favourable effects on hemodynamic parameters, hydrocortisone therapy did not reduce mortality and was associated with an increase in adverse effects. (Current Controlled Trials registry no. ISRCTN99675218.).

Hemodynamic disorders of liver cirrhosis complicated with portal hypertension are associated with an increased angiogenesis in animal model of portal hypertension and cirrhosis which were linked to increased expression of vascular endothelial growth factor (VEGF) and nitric oxide (NO). The aim of study was to evaluate the serum concentration of VEGF and total nitric oxide (NO) in liver cirrhosis and the possible association with the degree of liver insufficiency. VEGF and NO were measured in serum of 64 patients with liver cirrhosis by ELISA and spectrophotometry respectively. The significant increase of serum VEGF was observed in liver cirrhosis compared to healthy individuals as well as serum NO (106.1 ± 66.7 vs. 41.5 ± 6 pg/mL, P < 0.05; 113.5 ± 65.8 vs. 20.8 ± 3.8 μmol/l, P< 0.001, respectively). Serum VEGF and NO showed significant associations with biochemical indices of liver function and with Child-pugh score where they were increased respectively to the degree of liver insufficiency. A significant association of raised serum NO in early stage of portal hypertension reflect its benefit in early expect of portal hypertension but, high serum VEGF in late stage may reflect its prognostic value in liver cirrhosis.

Increased levels of nitric oxide (NO) are hypothesized to contribute to renal dysfunction in patients with decompensated cirrhosis. In this study, we examined whether splanchnic and/or peripheral NO levels and L-arginine (L-Arg) correlate with progressive renal dysfunction in cirrhotics.

Serum NO metabolites (NOx) and L-Arg were measured in: controls (n = 10); organ donors (n = 12); compensated cirrhotics (n = 17), cirrhotics with ascites (n = 25), refractory ascites (n = 11) or hepatorenal syndrome type II (HRS) (n = 11) and chronic renal failure patients (n = 18).

Plasma NOx and L-Arg levels rose progressively with worsening renal function in decompensated cirrhotics. Both NOx and L-Arg levels were highest in patients with HRS (P < 0.001 and P < 0.025, respectively). While there were no differences in NOx levels related to the site of sampling, L-Arg levels were lowest in hepatic venous blood. There were significant relationships of NOx and L-Arg with Model for End-Stage Liver Disease score and Child-Pugh scores (P < 0.04 and P < 0.01, respectively). Multivariate analysis showed a significant relationship between NOx, L-Arg and HRS.

Worsening renal function in decompensated cirrhosis is accompanied by progressive elevation in plasma NOx and L-Arg. These findings support the hypothesis that NO-mediated vasodilation is probably linked with the mechanism of progressive renal failure in decompensated cirrhotics.

The aim of this study was to determine whether hyperbilirubinemia affects the association between a positive urine nitrite test and a positive urine culture.

We conducted an institutional review board-approved, retrospective review of 12 months of patient data, compiling information for patients having urinalysis, urine culture, and total serum bilirubin. Patients were divided into 3 groups according to total serum bilirubin: less than 1.5 mg/dL, 1.5 to 3.0 mg/dL, and greater than 3.0 mg/dL. The point estimates and 95% confidence intervals of the sensitivity, specificity, false-positive proportion (proportion of false positive to all positive tests), and other test characteristics of urine nitrite as an indicator of urinary tract infection were calculated and tested for trend as a function of the 3 total serum bilirubin ranges.

Three thousand one hundred seventy-four patients met our study criteria. Specificity of the nitrite test decreased as a function of increasing total serum bilirubin (0.974, 0.966, and 0.855 for the 3 total bilirubin levels, respectively) with a significant trend (P < .0001). There was no significant trend in comparable sensitivity values (0.380, 0.417, and 0.241, respectively) with P = .55. The false-positive proportion also increased as a function of total serum bilirubin (17.5%, 17.3%, and 72.0%) with P < .0001. Thus, if a patient's total serum bilirubin was elevated to the point of jaundice (>3.0 mg/dL), it was approximately 4 times more likely that a positive urine nitrite test would be a "false positive" (ie, nitrite-positive/culture-negative) compared with those with normal serum bilirubin levels.

Specificity of the urine nitrite test for urinary tract infection decreases as a function of increasing serum bilirubin. Most patients with hyperbilirubinemia and a positive nitrite test in our sample did not have an associated urinary tract infection.

Patients with advanced liver diseases tend to develop a hyperdynamic circulation which complicates cirrhosis. Impairment of nitric oxide (NO) metabolism has been implicated in the pathogenesis of portal hypertension. The aim of this study was to determine nitric oxide synthase (NOS)-dependent whole body NO production in patients with decompensated liver cirrhosis and portal hypertension.

Ten patients with decompensated alcoholic liver disease and portal hypertension (Child-Pugh Classifications B and C with no signs of infection) and 10 age- and gender-matched control subjects received an intravenous infusion of L-[15N]2-arginine (50 micromol/min for 30 min). Urine and serum nitrite and nitrate concentrations were determined using ion chromatography-mass spectrometry.

NOS-dependent whole body NO synthesis was estimated by the conversion of [15N]guanidino nitrogen of arginine to urine 15N-nitrite and 15N-nitrate. The amount of 15N-nitrite and 15N-nitrate in the urine of patients and control subjects was significantly correlated with the amount of urine nitrite and nitrate over 36 h (r=0.91 and 0.77, respectively, p<0.0001). However, neither a median of 12 h 15N-nitrite and 15N-nitrate nor nitrite and nitrate excretion in the urine was different between patients and control subjects, 46.4 (9.4-152.2) versus 98.7 (29.9-146.5) nmol/mmol creatinine and 20.6 (2.1-69.0) versus 40.0 (27.0-70.1) micromol/mmol creatinine, respectively. No differences were found in serum nitrite and nitrate concentrations and glomerular filtration rates between patients and control subjects, 111.4 (73.2-158.8) versus 109.3 (83.5-176.4) micromol/l.

Our results contraindicate a greater basal NOS-dependent whole body NO production in patients with decompensated liver disease and portal hypertension.

The aim of this investigation was to determine nitric oxide metabolite levels in saliva samples from hepatitis C virus-positive patients in an attempt to test the hypothesis if increased levels of nitric oxide metabolites correlates with the presence of HCV-RNA in saliva. Saliva of 39 HCV-positive patients and 13 HCV-negative patients, without clinical or laboratorial evidence of liver disease were tested for nitric oxide metabolites. HCV-RNA was detected in serum and saliva by a RT-PCR method and nitric oxide level was determined by evaluation of its stable degradation products, nitrate and nitrite. No differences were found between the concentration of nitrite in saliva from HCV patients and controls, in despite of the presence or not of HCV RNA in saliva. Patients with HCV and cirrhosis had higher concentrations of nitrite but not significantly different from the control group or the groups of anti-HCV patients without cirrhosis. Increased levels of nitrite were not detected in anti-HCV positive patients, an indirect indication that chronic sialoadenitis are infrequent in these patients or occurs with low intensity not sufficient to increase nitric oxide metabolite levels in saliva.

The renal effects of octreotide, used for bleeding esophageal varices in cirrhosis, are controversial.

Fourteen cirrhotic patients (Child-Pugh; A/B/C: 1/12/1) were enrolled. Plasma nitrite and endothelin (ET) levels, urinary nitrite output, free water clearance (FWC) and fractional excretion of filtered sodium (FENa) were measured and renal Doppler ultrasound was carried out. Octreotide was infused at a rate of 0.75 microg/kg/h for 3 h after a bolus of 0.75 microg/kg body weight. All the parameters were reevaluated during octreotide administration while the patients acted as their own controls.

Octreotide induced significant reductions in urinary nitrite, FENa and FWC. Plasma ET levels increased (baseline: 6.7 pg/ml, octreotide: 8.4 pg/ml), whereas the plasma nitrite level did not change significantly after octreotide infusion. Overall, no significant change in renal resistive index (RRI) could be demonstrated on Doppler after octreotide administration. However, patients with elevated baseline RRI values had significantly more deterioration in FWC and FENa compared with patients with normal RRI in response to octreotide.

A marked decrease in FENa, FWC and urinary nitrite output, together with a significant increase in plasma ET level in response to octreotide, may indicate renal dysfunction in cirrhotic patients. This deleterious renal effect of octreotide may be more enhanced in patients with elevated baseline RRI.

Endocannabinoids may participate in the homeostasis of arterial pressure. Recently, anandamide, the most extensively studied endocannabinoid, has been proposed as a key mediator in the peripheral arterial vasodilation of cirrhosis.

To determine if circulating levels of anandamide are related to the extent of the peripheral arterial vasodilation, the severity of portal hypertension and the degree of liver and renal dysfunction of patients with cirrhosis.

Plasma levels of anandamide and several systemic, portal and renal hemodynamic parameters were determined in 18 patients with cirrhosis and eight healthy subjects (control group).

Plasma levels of anandamide were elevated in patients compared to the control group (P<0.05), nevertheless, no differences between patients with ascites and well-compensated patients were found. There was no correlation between anandamide concentration and arterial pressure, cardiac output and systemic vascular resistance, Child-Pugh's score, portal pressure, renal vascular resistance, plasma renin activity or plasma aldosterone concentration.

Circulating levels of anandamide are increased in cirrhotic patients. However, this elevation was unrelated to the extent of arterial vasodilation, the severity of portal hypertension or the degree of hepatic and renal dysfunction. Although a local hormonal action cannot be excluded, our results do not support a relevant contribution of this system in the hemodynamic disturbance of cirrhosis.

The role of endothelial nitric oxide synthase 3 (NOS-3) in the hyperdynamic circulation associated with cirrhosis is established but not that of the neuronal (NOS-1) isoform. We therefore investigated aortic NOS-1 levels in NOS-3 knock-out (KO) and wildtype (WT) mice and in hepatic arteries of patients.

Mice rendered cirrhotic by bile duct ligation (BDL) were compared with sham-operated controls. Hepatic arteries of cirrhotic patients were collected during liver transplantation; donor vessels served as controls. mRNA levels were quantified by real-time PCR, protein levels by Western blotting and NO production by Nomega-nitro-L-arginine methyl ester inhibitable arginine-citrulline assay.

Aortae of NOS-3 KO mice exhibited higher NOS-1mRNA (5.6-fold, P < 0.004) and protein levels (8.8-fold) compared with WT. NO production in aortae of NOS-3 KO mice was 52% compared with WT (P = 0.002). BDL increased NOS-1 mRNA (2.4-fold, P = 0.01) and protein (7.1-fold) levels in aortae of WT, but no further in the NOS-3 KO mice. Hepatic artery NOS-1 mRNA levels in cirrhotic patients were markedly increased compared with controls (24.5-fold, P = 0.0007).

Increased NOS-1 mRNA and protein levels and partially maintained in vitro NO-production in aortae of NOS-3 KO mice suggest that NOS-1 may partially compensate for NOS-3 deficiency. BDL-induced increase in aortic NOS-1 mRNA and protein levels hint that not only NOS-3, but also NOS-1 may be involved in the regulation of systemic hyperdynamic circulation and portal hypertension. Upregulation of NOS-1 mRNA levels in hepatic arteries of portal hypertensive patients suggests possible clinical significance for these experimental findings.

In coeliac disease, inducible nitric oxide synthase activity in the duodenal mucosa is greatly increased, resulting in increased production of nitric oxide. We investigated whether this resulted in increased plasma concentrations of its stable end products (nitrate/nitrite: NOx).

Fasting plasma NOx was determined in 66 patients attending for upper gastrointestinal endoscopy. Of these, 21 had coeliac disease (nine were on a gluten-free diet). The remainder had a variety of other gastrointestinal disorders. NOx was determined using the Griess reaction. Distal duodenal biopsies for coeliac patients were graded according to the Marsh score.

Patients with untreated coeliac disease had a higher fasting NOx concentration (mean 117.5 microM) than either those with coeliac disease taking a gluten-free diet (mean 71.2 microM) or those with other diseases (mean 33.5 microM; one-way analysis of variance, P < 0.001). Coeliac patients with higher fasting NOx concentrations had more marked histological changes (P < 0.05).

Fasting plasma NOx is significantly elevated in untreated coeliac disease and correlates with histological grade. The potential clinical utility of serial NOx measurements to monitor improvement on a gluten-free diet requires further study.

Several mediators of systemic vasodilatation in liver cirrhosis have been reported. Among these is nitric oxide (NO), which has been proposed as one of the main mediators. In this study, sera and liver biopsies were analysed from 15 patients with clinically and pathologically diagnosed liver cirrhosis. In addition, sera from seven and liver biopsies from three healthy controls were used. Serum levels of nitrite (the end product of NO) were measured using the Griess reaction and the expression of the inducible nitric oxide synthase (iNOS) and constitutive nitric oxide synthase (ecNOS) proteins was investigated using immunohistochemistry. This study shows that serum nitrite levels (94 +/- 9.8 micro mol/l) in cirrhotic patients were significantly (p < 0.05) increased in comparison with the controls (36.6 +/- 11.03 micro mol/l). iNOS was completely absent from the control group but was highly expressed in the livers from the cirrhotic group. iNOS was seen mainly in the inflammatory cells infiltrating the portal tracts, blood monocyte-like cells, hepatocytes, sinusoidal cells, and endothelial cells. However, expression of ecNOS was only seen in the vascular endothelial cells of both the control and the cirrhotic groups, but was much higher in the latter. It is therefore clear that NO is augmented in cirrhotic patients and it is mainly produced by induction of iNOS. Moreover, NO up-regulation is dependent on the inflammatory stage of liver cirrhosis. ecNOS production could be a normal chronic adaptation mechanism of the endothelium to the chronically increased splanchnic blood flow secondary to portal hypertension. In the near future, the appropriate inhibition of NO activity by using NOS-active agents may provide a novel strategy for the treatment of patients with liver cirrhosis.

Measurement of cardiac output in hyperkinetic patients with cirrhosis by Doppler echocardiography is increasingly reported, but has not been validated. We have compared simultaneous measurements of cardiac output by Doppler echocardiography (CO(d)) and by the indicator dilution technique (CO(I)).

Twelve patients with cirrhosis were studied. CO(d) was measured as the spatial mean velocity of the left ventricular outflow tract, multiplied by the cross-sectional area and the heart rate. CO(I) was determined by the standard indicator dilution technique after injection of 125I albumin and 99mTc albumin into the right atrium and subsequent sampling from the femoral artery.

The mean CO(d) and CO(I) were similar (7.20 vs 7.15 l/min, NS). A highly significant correlation was present between CO(d) and CO(I) (r = 0.86, P < 0.0001; slope 0.91, Y(0) = 0.78 l/min). However, the mean squared difference between CO(d) and CO(I) was 2.3 (l/min)2. A Bland-Altman plot revealed no trend with the level of cardiac output. The standard deviation (0.79 vs 0.30 l/min, P < 0.01) and the coefficient of variation (10.5 vs 4.2%, P < 0.01) of duplicate measurements were significantly higher with the Doppler technique.

Doppler measurements of cardiac output in groups of patients with cirrhosis are accurate with respect to the group mean, but marked disagreements of over- and underestimation were seen in individual patients. The reproducibility of the Doppler technique is acceptable, although not as good as that of the indicator dilution technique.

Research on the free radical gas, nitric oxide (NO), during the past twenty years is one of the most rapid growing areas in biology. NO seems to play a part in almost every organ and tissue. However, there is considerable controversy and confusion in understanding its role. The liver is one organ that is clearly influenced by NO. Acute versus chronic exposure to NO has been associated with distinct patterns of liver disease. In this paper we review and discuss the involvement of NO in various liver diseases collated from observations by various researchers. Overall, the important factors in determining the beneficial versus harmful effects of NO are the amount, duration, and site of NO production. A low dose of NO serves to maximize blood perfusion, prevent platelet aggregation and thrombosis, and neutralize toxic oxygen radicals in the liver during acute sepsis and reperfusion events. NO also demonstrates antimicrobial and antiapoptosis properties during acute hepatitis infection and other inflammatory processes. However, in the setting of chronic liver inflammation, when a large sustained amount of NO is present, NO might become genotoxic and lead to the development of liver cancer. Additionally, during prolonged ischemia, high levels of NO may have cytotoxic effects leading to severe liver injury. In view of the various possible roles that NO plays, the pharmacologic modulation of NO synthesis is promising in the future treatment of liver diseases, especially with the emergence of selective NO synthase inhibitors and cell-specific NO donors.

It has been demonstrated that an overproduction of nitric oxide plays an important role in the pathogenesis of the hyperdynamic circulation exhibited by cirrhotic patients. Nevertheless, evidence is supported by studies performed in experimental models or by indirect measurements in humans. The purpose of this study has been to evaluate nitric oxide production in splanchnic vasculature of patients with cirrhosis and to investigate its possible relationship with systemic and splanchnic hemodynamics.

Nitric oxide synthase (NOS) activity was measured in hepatic artery and portal vein tissues of nine cirrhotic patients. Samples were obtained during liver transplantation. Control samples were obtained simultaneously from the corresponding tissues of the liver donors. Hemodynamic parameters were determined with Doppler ultrasonography.

NOS activity was significantly higher in hepatic artery of cirrhotic patients than in controls (8.17 +/- 1.30 vs 4.57 +/- 0.61 pmoles/g of tissue/min, P < 0.05). Patients with ascites showed a higher hepatic artery NOS activity than patients without ascites. Highly significant correlation was observed between cardiac output and hepatic artery NOS activity as well as between portal blood flow and hepatic artery NOS activity.

The present study demonstrates an enhanced production of nitric oxide in the splanchnic vasculature of patients with cirrhosis.

In recent years, the role of nitric oxide (NO) in the pathogenesis of liver disease and its complications has been extensively studied. There remain, however, many areas of controversy. In particular, the effect of NO on vascular function in the systemic circulation and the hepatic microcirculation has received the greatest attention. It has been proposed on the one hand that increased NO synthesis is responsible for the development of the hyperdynamic circulation in cirrhosis, while decreased production of NO within the hepatic microcirculation may be important in the development of parenchymal tissue damage and the onset of portal hypertension. The purpose of this review is to examine the available data concerning the role of NO in liver disease and to discuss some of the controversies and contradictions that surround it.

Increased nitric oxide level may play a critical role in the hemodynamic disturbances in patients with cirrhosis. There are few reports investigating the factors related to this increase and their results are controversial. The purpose of this study was to reveal the clinical importance of nitric oxide levels and the possible factors related to this increase in patients with cirrhosis.

Serum and ascites nitrate levels were studied in 50 patients with cirrhosis and 10 control subjects.

All cirrhotic patients (groups 2, 3, 4, 5, 6) showed significant increase in serum nitrate levels in comparison with that in control subjects (group 1) (p<0.001). Serum nitrate levels were significantly higher (282.4+/-111.3 micromol/l; p<0.05) in patients with spontaneous bacterial peritonitis (group 2) when compared with those in cirrhotic patients without spontaneous bacterial peritonitis (group 3) (186.4+/-87.6 micromol/l). Ascitic fluid nitrate levels were significantly higher (302.4+/-66 micromol/l; p<0.001) in patients with spontaneous bacterial peritonitis (group 2) when compared with those in cirrhotic patients without spontaneous bacterial peritonitis (group 3) (135.4+/-65.8 micromol/l). Serum nitrate levels were significantly lower in cirrhotic patients without ascites (group 5) when compared with those in cirrhotic patients with ascites (group 3) (98.8+/-52.6 vs. 186.4+/-87.6 micromol/l; p<0.05). No significant differences were found among patients with severe anemia (groups 4, 6) and other cirrhotic patients (group 3) (174.5+/-54.5, 168.8+/-63.8 vs. 186.4+/-87.6 micromol/l; p>0.05). Cirrhotic patients with Child--Pugh B and C scores showed higher serum nitrate levels (179.4+/-81.1, 222.5+/-101.7 micromol/l; p<0.001) than did cirrhotic patients with Child--Pugh A score (85.8+/-59.7 micromol/l).

Our findings suggest that overproduction of nitric oxide in cirrhotic patients may be related to the severity of liver damage and spontaneous bacterial peritonitis but not related to their anemia.

A balance between endothelins (ET) and nitric oxide (NO) might interfere with liver haemodynamics and disease progression in various liver diseases. Increased levels of endothelin 1 (ET-1) and nitrites and nitrates (NOx, the end products of NO metabolism) have been reported in hepatocellular carcinoma (HCC), but the balance has not been studied. The purpose of this study was to assess the ratio of NOx to ET-1 in patients with virus-related hepatocellular carcinoma and to investigate its correlation with the extent of the disease. Eighteen patients with virus-related HCC (six Okuda stage I, six Okuda stage II and six Okuda stage III) were included in the study and were compared with 22 patients with viral cirrhosis (14 decompensated, eight compensated) and seven normal controls. ET-1 was measured with an ELISA assay and NOx with a modification of the Griess reaction. Patients with virus-related HCC had the highest levels of circulating ET-1 and NOx (13.24 +/- 0.82 pg/ml and 112.28 +/- 18.56 micromol/l) compared to compensated cirrhosis (9.47 +/- 0.50 pg/ml, P < 0.004 and 54.47 +/- 2.36 micromol/l, P < 0.01), decompensated cirrhosis (9.57 +/- 0.32 pg/ml, P < 0.001 and 90.20 +/- 11.23 micromol/l, NS) and normal controls (8.84 +/- 0.61 pg/ml, P < 0.001 and 51.17 +/- 6.18 micromol/l, P < 0.01). There was a significant increase of ET-1 and NOx at HCC stage III compared to HCC stages I and II, cirhotics and controls. HCC stage III patients also had a NOx/ET-1 ratio that was higher than HCC stages I and II patients, normal controls and patients with compensated cirrhosis. Virus-related HCC patients have high levels of circulating ET-1, compared to compensated or decompensated cirrhosis. Highest levels of ET-1 are produced in Okuda III tumours. NOx are also increased but only in Okuda stage III tumours. The NOx/ET-1 ratio is increased in virus-related HCC and DC. This increase may account for the known increase in tumour blood flow.

Septic shock results in high mortality in patients with cirrhosis. Nitric oxide synthase 2 (NOS2) is induced by bacterial lipopolysaccharides (LPS) and plays a major role in the inflammatory response to bacterial infections. Little is known about the regulation of NOS2 in cirrhosis under septic conditions. Thus, the aim of this study was to determine tissue NOS2 activity, serum nitrate and tumor necrosis factor (TNF-alpha) levels and hepatic toxicity in cirrhotic rats after LPS administration.

Serum nitrates, TNF-alpha and transaminases were determined after LPS-administration in rats with secondary biliary cirrhosis and in sham-operated rats. Liver, lung, aortic and peritoneal macrophage NOS2 activities were determined by converting L[14C] arginine into L[14C] citrulline in a calcium free medium. Nitrate and TNF-alpha production were determined in a culture medium of peritoneal macrophages after in vivo LPS administration.

LPS (1.5 mg/kg) induced 50% mortality in cirrhotic rats and no mortality in sham-operated rats. After LPS, TNF-alpha, nitrate and transaminase levels were significantly higher in cirrhotic rats compared to sham-operated rats. After LPS administration, there were no differences in NOS2 activity in the aorta, lungs, or peritoneal macrophages of the two groups, whereas NOS2 activity was significantly higher in the cirrhotic liver compared to the normal liver.

In rats with cirrhosis, LPS administration induces higher mortality, hepatic toxicity, hepatic NOS2 activation and TNF-alpha release than in sham-operated rats. These results confirm the harmful role of septic shock in liver disease.

The last half century has witnessed great advances in the understanding of the pathogenesis and natural history of portal hypertension in cirrhotics. Several pharmacologic and endoscopic techniques have been developed for the treatment of portal hypertension. The use of these agents in a given patient must be based on an understanding of the stage in the natural history of the disease and the relative efficacy and safety of the available treatment options.

Increased serum nitrite/nitrate (NOx) levels, the stable metabolites of nitric oxide (NO), have been reported in patients with cirrhosis. NOx levels, however, are influenced not only by endogenous NO synthesis but also by urinary NOx excretion and dietary intake. We attempted to elucidate factors that influence NOx levels independently of endogenous NO production and to determine the conditions under which NOx levels reflect endogenous production in patients with cirrhosis and healthy controls.

NOx serum concentrations and urinary NOx excretion were determined by means of the Griess reaction in relation to Child-Pugh score, kidney function, fasting state, and after exposure to tap water.

Multifactor regression analysis showed inulin clearance (P = 0.0074) and Child-Pugh score (P = 0.0001) to be independent factors predicting NOx levels in patients with cirrhosis. NOx serum levels correlated negatively with the inulin clearance (P < 0.0001), which deteriorated with progressive loss of liver function. NOx levels decreased by about 30% within a 24-h fasting period. After 24 h fasting urinary NOx excretion was not significantly increased in patients with advanced cirrhosis.

NOx serum levels, taken as a surrogate for endogenous NO formation, have to be viewed with caution in patients with cirrhosis because they often have impaired kidney function. However, in steady-state conditions after an adequate fasting period NOx levels might be good prognostic markers in patients with cirrhosis since they reflect two possible sequelae of liver insufficiency-namely, increased NO formation and impaired kidney function.

The aim of this study was to evaluate the relationship between plasma levels of von Willebrand factor (vWF), a marker of endothelial cell activation, and nitric oxide, a powerful vasodilator synthesized by endothelial cells, in 27 patients with cirrhosis at different stages of the disease. These results were compared with those of age-matched normal, healthy subjects (n=10).

vWF:antigen was measured by electro-immunodiffusion test and serum nitrite and nitrate levels, the stable end products of nitric oxide metabolism, were determined by an enzymatic procedure.

vWF:antigen and nitrite/nitrate levels were significantly higher in cirrhotic patients (367+/-185% and 29.3+/-10.8 micromol/l) than in healthy subjects (92+/-20% and 19.2+/-8.3 micromol/l, p<0.05, respectively). Higher levels of vWF:antigen and nitrites/nitrates were observed in patients with more advanced degrees of liver failure, as reflected by quantitative Child-Pugh's score (516+/-154% and 38.3+/-7.8 micromol/l in Child-Pugh > or = 9 vs 227+/-61% and 21.0+/-6.1 micromol/l in Child-Pugh <9, p<0.001, respectively). Moreover, both endothelial-related factors were higher in patients with ascites than those without ascites (543+/-158% and 37.8+/-8.9 micromol/l vs 262+/-103% and 24.4+/-8.8 micromol/l, p<0.001, respectively). In the overall series, a highly significant linear correlation between nitrites/nitrates and vWF:antigen levels was observed in patients with cirrhosis (r=0.79, p<0.001).

These results support a cirrhosis-related endothelial dysfunction and suggest that plasma vWF measurement could be useful as a marker of endothelial disturbance in patients with cirrhosis.

Nitric oxide has been proposed as a mediator of hyperdynamic circulation in cirrhosis. Endotoxin and cytokines induce the synthesis of nitric oxide. The aim of this study was to investigate the relationship between endotoxemia, cytokines, and nitric oxide in patients with cirrhosis, and to correlate these findings with clinical, biochemical, and hemodynamic parameters.

Clinical, biochemical, and hemodynamic parameters were assessed in 66 patients with cirrhosis and 15 controls. Levels of antidiuretic hormone, plasma renin activity, aldosterone, interferon gamma, interleukin-1, interleukin-6, tumor necrosis factor alpha, endotoxin, and nitrates-nitrites were determined.

Mean arterial pressure was lower and interleukin-6, tumor necrosis factor alpha, nitrites-nitrates levels, and endotoxin positivity rates were higher in cirrhotics than in controls (p < 0.005). Mean arterial pressure decreased and interleukin-6 levels increased with worsening of Child score (p < 0.005). Patients with ascites had higher levels of interleukin-6, tumor necrosis factor alpha, and nitrates-nitrites than patients without ascites (p < 0.01). Elevated levels of interleukin-6 were found in patients with encephalopathy grade I, compared with patients without (p < 0.001); this association was independent of the severity of liver disease. In patients with low mean arterial pressure, interleukin-6 levels were higher than in patients with high mean arterial pressure (p = 0.001), whereas tumor necrosis factor alpha and nitrates-nitrites levels were not different. By multivariate analysis, high interleukin-6 levels showed independent associations with the presence of ascites, encephalopathy, and low mean arterial pressure. Only interleukin-6 levels had significant correlations with Child score, plasma renin activity, serum and urinary sodium, and mean arterial pressure (r > or = 0.4, p < 0.005).

Although the activity of the nitric oxide pathway is increased in patients with cirrhosis and might contribute to the hemodynamic alteration, other factors are involved. Interleukin-6, possibly through nitric oxide-independent mechanisms, also might play a role in the vasodilatation of cirrhosis and the pathogenesis of hepatic encephalopathy.

Peripheral arterial vasodilation may be the key factor in the sodium and water retention of cirrhosis. The mechanism responsible for this vasodilation remains to be fully elucidated. Adrenomedullin is a novel peptide, highly expressed in cardiovascular tissues, with potent and long-lasting vasodilating activity.

The possible implication of adrenomedullin in the hemodynamic changes of cirrhosis has been investigated. We measured the plasma concentration of adrenomedullin in 20 cirrhotic patients and 11 healthy subjects. In addition, systemic, portal and renal hemodynamics, hormonal factors and renal function parameters were evaluated in the same patients.

Circulating adrenomedullin was significantly higher in the group of patients with cirrhosis (72.1; 46-100 vs 21.6; 11-34 fmol/dl, respectively; p<0.02) and was directly correlated with the Pugh score (r: 0.6; p: 0.01), inversely correlated with the creatinine clearance (r: -0.6; p<0.01) and tended to inversely correlate with systemic vascular resistance index (r: -0.46; p: 0.07). There were no portal-peripheral differences in adrenomedullin levels. Transjugular intrahepatic portosystemic shunt insertion did not induce changes in the peripheral concentration of adrenomedullin, but baseline values of this hormone predicted the degree of hyperdynamic circulation after TIPS.

Circulating adrenomedullin is increased in cirrhosis. These levels increase with the severity of the disease, especially in patients with hepatorenal syndrome. This peptide may contribute to vasodilation of cirrhosis.

To review the assays available for measurement of nitrite and nitrate ions in body fluids and their clinical applications.

Literature searches were done of Medline and Current Contents to November 1997.

The influence of dietary nitrite and nitrate on the concentrations of these ions in various body fluids is reviewed. An overview is presented of the metabolism of nitric oxide (which is converted to nitrite and nitrate). Methods for measurement of the ions are reviewed. Reference values are summarized and the changes reported in various clinical conditions. These include: infection, gastroenterological conditions, hypertension, renal and cardiac disease, inflammatory diseases, transplant rejection, diseases of the central nervous system, and others. Possible effects of environmental nitrite and nitrate on disease incidence are reviewed.

Most studies of changes in human disease have been descriptive. Diagnostic utility is limited because the concentrations in a significant proportion of affected individuals overlap with those in controls. Changes in concentration may also be caused by diet, outside the clinical investigational setting. The role of nitrite and nitrate assays (alongside direct measurements of nitric oxide in breath) may be restricted to the monitoring of disease progression, or response to therapy in individual patients or subgroups. Associations between disease incidence and drinking water nitrate content are controversial (except for methemoglobinemia in infants).

Nitrate levels were measured in serum and in organs from Lshs BALB/c and Lshr C3H/HeN mice during the acute phase (30 d) of infection by Leishmania donovani strain LV9. Serum nitrate levels increased rapidly in BALB/c mice from a baseline level (17 +/- 4 mumol/L) to a plateau (504 +/- 129 mumol/L) at 24 d and correlated with parasite loads in the liver (r = 0.817, P < 0.01) and in the spleen (r = 0.854, P < 0.001). Liver and spleen nitrate contents were enhanced 2.7-fold and 22.8-fold, respectively, with respect to uninfected controls (2692 +/- 249 vs. 992 +/- 231 nmol, P < 0.02 and 20 +/- 1 vs. 456 +/- 43 nmol, P < 0.02). In contrast, serum nitrate increased to a lesser extent in C3H/HeN mice, from 31 +/- 5 mumol/L to 86 +/- 5 mumol/L at 20 d. Liver nitrate content did not differ significantly between infected and control mice (1093 +/- 83 vs. 867 +/- 104 nmol), whereas the former had a higher spleen nitrate content (145 +/- 22 vs. 40 +/- 2 nmol, P < 0.02). Our findings indicate that production of NO by the susceptible BALB/c strain exceeded that of the resistant C3H/HeN strain during the acute stage of infection by L. donovani. Tissue NO overproduction in organs infected by L. donovani was related to the progression of parasitic disease and contributed to high nitrate serum levels. It would be very interesting to extend this investigation to human disease with the aim of evaluating serum nitrate as a marker of parasite load in the follow-up of patients suffering from visceral leishmaniasis.