Injury of the vascular system can induce isoform II of nitric oxide synthase, resulting in excess nitric oxide [2]. In cirrhosis, intestinal bacterial overgrowth occurs [3] and portosystemic shunts may allow intestinal bacteria and endotoxin to enter the systemic circulation [4]. Endotoxemia may indirectly promote excessive nitric oxide and vasodilation through induction of nitric oxide synthase isoform II [5].

We tested the hypothesis that norfloxacin, a fluoroquinolone antibiotic, indirectly blocks the augmented effects of nitric oxide.

Methods

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We recruited 9 patients with alcohol-related liver disease (mean age, 54 ± 3 years) and 10 controls (mean age, 56 ± 4 years). All patients with cirrhosis were clinically classified as having well-compensated Pugh grade A disease; cirrhosis was proven by biopsy in 8 patients. All patients had evidence of portal hypertension and esophageal varices. Controls had no evidence of liver disease on history or on physical and laboratory examination (including liver function tests), and they tested negative for hepatitis B surface antigen and hepatitis C virus by enzyme-linked immunosorbent assay. The study protocol was approved by the Alfred Group of Hospitals Ethics Committee, which conforms to the Declaration of Helsinki, and participants gave written, informed consent.

In a double-blind, crossover protocol, patients with cirrhosis were randomly allocated to receive either norfloxacin (400 mg twice daily) or placebo for 4 weeks each. Responses to acetylcholine and the nitric oxide synthase inhibitor N^G -monomethyl-L-arginine were studied twice, once after the first 4 weeks of treatment and once after patients crossed over to the other treatment. No washout period separated the treatments. Because the half-life of norfloxacin ranges from 3.5 to 8 hours (normal half-life in end-stage renal disease, 6 hours), little carry-over was expected. We assumed that the extent of bacterial recolonization that had occurred during the 4-week placebo period was similar to that during the original infection. Control forearm vascular responses were obtained from untreated, healthy participants.

All participants refrained from drinking caffeinated beverages the night before and on the day of study. Controls refrained from drinking alcoholic beverages for 5 to 7 days before the study, and patients with cirrhosis abstained from alcohol from 5 days to more than 6 months before the study. Patients with cirrhosis stopped using vasoactive medication exactly 5 days before the study day.

Experiments were performed in a quiet room kept at 22°C. The left brachial artery was cannulated by inserting a 3-French, 5-cm catheter (Cook, Sydney, Australia) under local anesthesia with 1% lidocaine (Astra, Sydney, Australia) and full aseptic conditions. We obtained a 10-mL sample of arterial blood and measured the intra-arterial blood pressure (Biosensors International Proprietary, Ltd., Singapore, linked with Spacelabs, Inc., Redmond, Washington). A sealed, alloy-filled, double-stranded strain gauge was used with a plethysmograph (Medasonic, Mountain View, California). Recordings were done for 10 seconds every 20 seconds. The occlusion pressure was 40 to 50 mm Hg at the proximal end and 200 mm Hg at the distal end. Forearm vascular resistance was calculated as the mean arterial pressure (mm Hg) divided by the forearm blood flow (mL/100 mL per minute).

We measured responses to acetylcholine (9.25, 18.5, and 37 µg/min) and N^G -monomethyl-L-arginine (1, 2, and 4 µmol/min). After an initial equilibration period of 60 seconds, the average of three flow measurements was used as a measure of basal blood flow. Each drug was infused at 2 mL/min for at least 2 minutes (for acetylcholine) or 5 minutes (for N^G -monomethyl-L-arginine) or until the response of three flow measurements reached a plateau. The average of these measurements was used as the measure of drug-induced flow. Rest periods of 5 to 10 minutes between concentrations and 15 minutes between drug administrations were allowed. No effect on either systemic blood pressure or heart rate was observed (Spacelabs, Inc.).

Plasma was stored at –20°C until the time of analysis. When thawed, plasma was deproteinized by spinning in Microcon 10 microconcentrators (Amicon, Bedford, Massachusetts) for 150 minutes at 9000 g. Conversion of nitrate to nitrite by using nitrate reductase was tracked by adding Griess reagents (Cayman Chemical Co., Ann Arbor, Michigan). Total nitrite concentration was determined by photometric measurement of the absorbance at 540 nm.

Results are expressed as the mean ± SE or the median (25th, 75th percentiles) when the data were not normally distributed. The Student t-test (paired or unpaired as appropriate) was used to compare the two groups. Dose-response curves were compared by using two-way analysis of variance (with repeated measures where necessary, such as to compare cirrhotic norfloxacin recipients with cirrhotic placebo recipients). These tests were followed by post hoc t-tests with the appropriate corrections for multiple comparisons (Bonferroni t-test, Student-Newman-Keuls test, or Dunn test). We used only data obtained from the nine patients with cirrhosis who participated in the full study.

Results

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Basal forearm blood flow was higher in patients with cirrhosis who received placebo than in controls (3.69 ± 0.27 mL/100 mL per minute and 2.26 ± 0.39 mL/100mL per minute; P = 0.014). Mean arterial pressure did not differ between cirrhotic patients and controls. Derived basal forearm vascular resistance was lower in patients with cirrhosis who received placebo than in controls, but this difference was not statistically significant (27.2 ± 2.56 units and 52.8 ± 11.18 units; P = 0.065). Forearm blood flow returned to normal with norfloxacin (norfloxacin compared with control, 2.64 ± 0.31 mL/100 mL per minute and 2.26 ± 0.39 mL/100mL per minute [P > 0.2]; placebo compared with norfloxacin, P = 0.097).

Administration of N^G -monomethyl-L-arginine decreased forearm blood flow in all participants, but this effect was greater in patients with cirrhosis (Figure 1, top). Because values for basal forearm blood flow and vascular resistance were altered in patients with cirrhosis, responses to N^G -monomethyl-L-arginine were also expressed as the percentage of basal values (Figure 1, bottom). Responses to N^G -monomethyl-L-arginine returned to normal with norfloxacin (Figure 1).

View larger version (17K): [in this window] [in a new window]   Figure 1. Effect of NG -monomethyl-L-arginine. Top. Response of forearm blood flow to NG -monomethyl-L-arginine in controls (circles), cirrhotic patients who received placebo (squares), and cirrhotic patients who received norfloxacin (triangles). Bottom. The constricting effect of N (G) -monomethyl-L-arginine, expressed as a percentage of basal forearm vascular resistance in controls (circles), cirrhotic patients who received placebo (squares), and cirrhotic patients who received norfloxacin (triangles). * P < 0.05 for cirrhotic patients who received placebo compared with controls. P < 0.05 for cirrhotic patients who received norfloxacin compared with cirrhotic patients who received placebo. Error bars represent SEs.

Acetylcholine increased blood flow responses in all participants, but this effect was greater in patients with cirrhosis (Figure 2, top). These responses remained substantially greater in patients with cirrhosis when expressed as a percentage of basal values. Norfloxacin had no effect on response to acetylcholine (Figure 2).

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of acetylcholine. Top. Responses of forearm blood flow to acetylcholine in controls (circles), cirrhotic patients who received placebo (squares), and cirrhotic patients who received norfloxacin (triangles). Bottom. The dilatory effect of acetylcholine, expressed as a percentage of basal forearm vascular resistance in controls (circles), cirrhotic patients who received placebo (squares), and cirrhotic patients who received norfloxacin (triangles). * P < 0.05 for cirrhotic patients who received placebo compared with controls.

Plasma total nitrate values were elevated in patients with cirrhosis who received placebo (35.7 µmol/L [range, 29.8 to 37.1 µmol/L]) compared with controls (22.1 µmol/L [range, 16.5 to 23.1 µmol/L]; P = 0.015). Norfloxacin had no effect on these values (33.54 ± 5.93 µmol/L in norfloxacin recipients compared with 35.7 µmol/L in placebo recipients; P > 0.2).

A two-sample t-test on the effect of placebo in the norfloxacin-placebo sequence compared with the effect of placebo in the placebo-norfloxacin sequence found no difference between the two placebo phases. In addition, within-patient differences of the two sequences were examined by using paired t-tests; no sequence or period effect was found.

Discussion

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We found that peripheral vasodilation in patients with cirrhosis was associated with increased response to N^G -monomethyl-L-arginine. This result indicates that nitric oxide has a vasodilatory role. Our major new finding was that basal forearm blood flow and the increased N^G -monomethyl-L-arginine response in patients with cirrhosis returned to normal with the fluoroquinolone antibiotic norfloxacin.

Peripheral vasodilation in patients with cirrhosis was characterized by significantly higher basal forearm blood flow. Because N (^G) -monomethyl-L-arginine is a specific inhibitor of nitric oxide synthase [7], the increased response in these patients suggests increased nitric oxide synthesis. Given the potent vasodilatory action of nitric oxide, this effect is consistent with the increased basal flow that we saw.

Patients with cirrhosis have intestinal bacterial overgrowth [3]. This condition, along with portal hypertension and portosystemic shunts, can lead to systemic bacteremia or endotoxemia. This, in turn, may induce isoform II of nitric oxide synthase and lead to increased nitric oxide production and vasodilation [5]. To test this hypothesis, we administered norfloxacin, an agent commonly used to prevent bacterial infections in cirrhosis [8].

Our finding that cirrhotic patients had an augmented response to the endothelium-dependent muscarinic agonist acetylcholine has been reported with methacholine, a similar agonist [9]. These agonists promote release of nitric oxide through activation of endothelial-constitutive isoform III of nitric oxide synthase [2]. Because N^G -monomethyl-L-arginine blocks both isoforms of nitric oxide, our finding of enhanced vasoconstriction with N^G -monomethyl-L-arginine is consistent with the hypothesis that patients with cirrhosis have enhanced release of endothelial-constitutive isoform III of nitric oxide synthase. On the other hand, because norfloxacin had no effect on enhanced responses to acetylcholine, the nitric oxide responsible for peripheral vasodilation and enhanced response to N^G -monomethyl-L-arginine responses seems associated with isoform II of nitric oxide synthase. From this finding, we inferred that norfloxacin exerts its vascular effects indirectly (for example, by decontaminating gut flora) rather than by having a nonspecific direct vasoconstricting effect.

As in a previous study [10], total plasma nitrate levels were higher in cirrhotic patients than in controls. Because nitric oxide is rapidly converted to nitrate in vivo [11], serum nitrate levels have previously been used as an index of in vivo nitric oxide production. The finding of increased nitrate levels in cirrhotic patients is consistent with the hypothesis that nitric oxide synthesis is enhanced in these patients. However, because norfloxacin did not lead to normalization of nitrate levels in patients with cirrhosis, this effect is unlikely; the increased nitrate levels were probably caused by another mechanism, such as differences in dietary intake of nitrate or protein (which we did not control for) or diminished renal excretion of these metabolites.

The limitations of our study include 1) the unvalidated assumption that under the same circumstances, the vascular reactivity of alcoholic persons without cirrhosis is similar to that of controls; 2) the assumption that placebo has no effect on the vascular reactivity of patients with cirrhosis; 3) the lack of controlling for dietary intake of nitrate and protein; and 4) the assumption, validated by statistics, that there was no carry-over, sequence, or period effect. In addition, although we argue that norfloxacin exerts its vascular effects indirectly, a direct effect of the drug cannot be totally discounted.

We conclude that increased production of nitric oxide contributes to peripheral vasodilation in patients with cirrhosis. Our findings are consistent with the hypothesis that overgrowth or migration of bacteria is primarily responsible, because norfloxacin corrected the effects of enhanced nitric oxide production. Future studies should examine the effects of fluoroquinolone antibiotics on long-term clinical end points in cirrhosis.

Drs. Rasaratnam and Dudley: Department of Gastroenterology, Alfred Hospital, Commercial Road, Prahran 3181, Victoria, Australia.