Correspondence

It has been suggested that the measurement of metronidazole clearance is a sensitive method for evaluating liver function. The aim of this study was to evaluate the usefulness of plasma hydroxy-metronidazole/metronidazole ratios as indicators of dynamic liver function to detect changes resulting from the various forms of chronic hepatitis C virus (HCV) infection. A total of 139 individuals were studied: 14 healthy volunteers, 22 healthy, asymptomatic, consecutive anti-HCV-positive HCV-RNA negative subjects, 81 patients with chronic hepatitis C (49 with moderate/severe chronic hepatitis and 34 with mild hepatitis), and 20 patients with cirrhosis of the liver. HCV status was determined by the polymerase chain reaction. Plasma concentrations of metronidazole and its hydroxy-metabolite were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection in a blood sample collected 10 min after the end of a metronidazole infusion. Anti-HCV-positive HCV-RNA-negative individuals demonstrated a significantly reduced capacity to metabolize intravenously infused metronidazole compared to healthy individuals (0.0478 +/- 0.0044 vs 0.0742 +/- 0.0232). Liver cirrhosis patients also had a reduced plasma hydroxy-metronidazole/metronidazole ratio when compared to the other groups of anti-HCV-positive individuals (0.0300 +/- 0.0032 vs 0.0438 +/- 0.0027 (moderate/severe chronic hepatitis) vs 0.0455 +/- 0.0026 (mild chronic hepatitis) and vs 0.0478 +/- 0.0044 (anti-HCV-positive, HCV-RNA-negative individuals)). These results suggest an impairment of the metronidazole metabolizing system induced by HCV infection that lasts after viral clearance. In those patients with chronic hepatitis C, this impairment is paralleled by progression of the disease to liver cirrhosis.


Introduction
Hepatitis C virus (HCV) is a blood-borne pathogen apparently endemic in most parts of the world (1), and, therefore a major public health problem (2).HVC infection is a frequent cause of chronic liver disease, progressing in the majority of patients to persistent viremia and chronic hepatitis (3)(4)(5).Mortality associated with chronic hepatitis C results mainly from liver cirrhosis and complications such as liver carcinoma (6).The common course of chronic hepatitis C involves slow progression.However, the spectrum of liver disease is broad and progression rates are extremely variable (5,7).The long-term outcome is difficult to predict since patients with chronic hepatitis are seldom symptomatic and end-stage liver disease, when it occurs, can take more than three decades to develop (8).
Physical evaluation, the measurement of biochemical parameters, imaging procedures (such as ultrasound or computed tomography) and liver biopsy are used routinely to evaluate chronic HCV carriers and to determine the degree and extension of liver injury and the prognosis of the disease.However, the extent to which these variables estimate hepatic reserve is controversial.Parameters such as albumin and coagulation factors may also be used, but they lack sensitivity and occur late in the course of chronic liver disease (9,10).Thus, a test that could discriminate more subtle impairments of hepatic function would be extremely useful.
Dynamic liver function tests such as indocyanine green clearance (11), caffeine elimination (12) and lidocaine metabolism (13) have been used to complement standard liver assessment in patients with liver disease.Metronidazole clearance has been recently proposed as a highly sensitive method for evaluating liver function (14).This method consists of measuring plasma hydroxy-metronidazole/metronidazole ratio by high-performance liquid chromatography (HPLC) following the intravenous administration of a single dose of metronidazole.The test has been used to show that anti-HCV-positive blood donors and chronic hepatitis C patients have a decreased capacity to metabolize metronidazole compared to healthy individuals, thus demonstrating its usefulness for detecting impaired liver function in HCV-infected individuals, even in the absence of liver cirrhosis (14).These findings suggest that the evaluation of metronidazole metabolism may provide an easyto-perform, dynamic liver function test.The present study was conducted in order to determine whether the hydroxy-metronidazole/metronidazole test could discriminate between different stages of hepatic functional impairment during the various stages of chronic HCV infection as well as the impact of viral load and genotype on metronidazole metabolism.
Patients with abnormal bilirubin, albumin, prothrombin time, ascites, and encephalopathy and/or those diagnosed with liver tumors were excluded from the study.Individuals using drugs which could influence cytochrome P450 function were also excluded.
The clinical protocol was approved by the São Francisco University Ethics Committee, and the study was conducted in accordance with the Declaration of Helsinki.All individuals gave written informed consent prior to entering the study.

Anti-HCV-positive HCV-RNA-negative individuals
Asymptomatic, healthy consecutive anti-HCV-positive HCV-RNA-negative individuals, without previous treatment for HCV infection, were tested for anti-HCV antibodies using an enzyme immunoassay (Abbott HCV EIA, 2nd generation, Abbott Laboratories Diagnostics Division, Chicago, IL, USA).All positive results were confirmed using a gelatin particle agglutination test with recombinant antigens C22-3 and C200 (Serodia-HCV, Fujirebio Inc., Tokyo, Japan).Anti-HCV-positive individuals were considered to have cleared HCV if serum levels of enzymes synthesized by the liver such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, and gamma-glutamyl transpeptidase were within the normal reference range, and if no circulating HCV-RNA could be detected by the polymerase chain reaction (PCR).Each patient was tested on three different occasions with at least one month between tests.

Non-cirrhotic hepatopathy
Anti-HCV-positive HCV-RNA individuals had anti-HCV-positive serum (as measured by EIA, circulating HCV-RNA and persistently abnormal liver enzymes, but no other forms of viral hepatitis (negative serology for HBV).The absence of liver cirrhosis was assessed by clinical evaluation, ultrasonography and/or a CT scan, and was confirmed by a liver biopsy.

HCV-associated liver cirrhosis
Liver cirrhosis patients were selected for the study based on their medical history, clinical signs and the presence of diffuse alterations in the liver parenchyma detected by ultrasonography or a CT scan.When possible, the diagnosis of cirrhosis was con-firmed by a liver biopsy.The patients included were classified as A according to the Child-Pugh scale (11).Patients with liver tumors, an ongoing bacterial infection, or renal failure (as determined by serial serum creatinine determinations) were excluded from the study.The presence of an ongoing bacterial infection was assessed by clinical evaluation, chest radiology and urine analysis.

Clinical laboratory analysis
Serum chemical analysis, urinalysis, a complete blood cell and platelet count, prothrombin, albumin, serum AST, ALT, alkaline phosphatase, γ-glutamyl transpeptidase, total bilirubin, prothrombin time, hematocrit, and total/differential white cell counts were determined in all subjects.

Histology
Biopsy specimens were fixed in 10% formalin.Sections, 3 to 4 µm thick, were cut and stained with hematoxylin-eosin and Masson trichrome or silver for reticulin fibers.For each biopsy specimen, necro-inflammation was graded and the stage of fibrosis was classified according to Desmet et al. (15).The sum of the necro-inflammatory and fibrosis score was considered to be the final score and used to define mild and moderate/severe chronic hepatitis.Chronic hepatitis is defined as mild if the final score was 5 or less and moderate or severe if >5.

Plasma hydroxy-metronidazole/metronidazole ratio
The study consisted of a single intravenous administration of metronidazole (Flagyl ® , 5 mg/ml, Rhodia, São Paulo, SP, Brazil) for 20 min.At the end of the infusion, the cannula was washed with 20 ml sterile saline.A blood sample (5 ml) was collected from an antecubital vein into EDTA-containing tubes before and 10 min after the infusion.Blood samples were centrifuged at 2,000 g for 5 min and plasma was stored at -20ºC until assayed.The participants remained in the Clinical Pharmacology Unit during metronidazole administration and blood sampling.

Plasma metronidazole concentration
The plasma concentrations of metronidazole and its hydroxy-metabolite were measured by a validated reversed-phase HPLC method (14).The separation was performed on a reversed-phase Luna C18 column (250 x 4.6 mm ID, 10 µm; Phenomenex, Torrance, CA, USA) protected by a Securityguard C18 guard column (4.0 x 3.0 mm; Phenomenex).The mobile phase consisted of 85% methanol and 15% 0.01 M potassium dihydrogen phosphate buffer adjusted to pH 4.5 and was eluted at a flow rate of 1.8 ml/min.The detector wavelength was set at 324 nm and peak heights were measured.For the extraction, 50 µl of the internal standard (tinidazole, 100 µl in methanol) was added to 200 µl of plasma.After mixing, deproteinization was carried out by adding 20 µl 0.6 M ZnSO 4 and 20 µl 0.4 M NaOH.The samples were vortex mixed and the tubes were then centrifuged at 2000 g for 5 min.Twenty microliters of each supernatant was injected into the HPLC system.The plasma hydroxy-metabolite/metronidazole ratios were determined in all participants.This method has a sensitivity of 100 ng/ml and the mean intra-assay coefficient of variation (up to 25 µg/ml of both compounds) is 4%.
Viral load was evaluated by means of real-time quantitative RT-PCR, as previously described (17)(18)(19).The use of a sequence detector (ABI Prism 7700; Applied Biosystems, Foster City, CA, USA) allows measurement of the amplified product in direct proportion to the increase in fluorescence emission continuously during PCR amplification.A value above or below 800,000 IU/ ml was used to define a high or low viral load (20).

Statistical analysis
Data are reported as means ± SEM unless otherwise stated.All variables were analyzed by one-way ANOVA followed by the Student t-test for multiple comparisons among groups, with the level of significance set at P < 0.001.

Results and Discussion
The data in Table 1 show that the plasma hydroxy-metronidazole/metronidazole ratio revealed a reduced capacity of HCV-infected individuals to metabolize intravenously infused metronidazole compared to healthy individuals, regardless of their HCV-RNA status, as indicated by the lower ratio compared to volunteers.Liver cirrhosis patients also had a reduced plasma hydroxy-metronidazole/metronidazole ratio when compared to the other groups of anti HCV-positive individuals (Table 1).The HCV genotype was not related to differences in the plasma hydroxy-metronidazole/metronidazole ratios among patients with chronic hepatitis (Table 2).Similar data for viral load were found (0.0443 ± 0.017 vs 0.0449 ± 0.0163 for low and high viral load, respectively; 95% CI: Data are reported as means ± SEM.The numbers in parentheses indicate the upper and lower limits of the 95% confidence intervals of the significant differences compared to a healthy volunteers, b patients with liver cirrhosis and c anti-HCV + RNA individuals.OH-MET/MET ratio = hydroxy-metronidazole/metronidazole ratio.*P < 0.001 vs healthy volunteers, + P < 0.001 vs patients with liver cirrhosis (one-way ANOVA followed by the Student t-test).
-0.0080; 0.0070).Therefore, only two groups of patients were formed: individuals with mild hepatic histological alterations and individuals with moderate/severe hepatitis.Chronic viral hepatitis is a silently progressive disease with a heterogeneous course, Table 2. Plasma hydroxy-metronidazole/metronidazole ratios according to genotype and histological score.
making the clinical outcome difficult to predict (7).Since standard liver tests are not useful for predicting the progress of fibrosis in chronic hepatitis patients, a truly quantitative test for liver function could allow a prognostic assessment of various liver diseases (21).
A reliable quantitative liver test would be useful for clinicians and researchers when deciding on therapeutic strategies.The search for such a test has been going on for many years and is complicated by the lack of a universally accepted standard.The formation of monoethylglycinexylidide (MEGX), the main lidocaine metabolite, has been suggested as a simple and valuable liver function test (22)(23)(24).However, the administration of lidocaine may produce side effects in a high percentage of individuals tested (22), the formation of MEGX may be influenced by age and gender (24,25), and the lidocaine dose to be administered and the time point for measuring the MEGX concentration are still controversial (26)(27)(28).The MEGX test has also failed to discriminate between healthy volunteers and patients with chronic hepatitis (10,29).
The present results demonstrate the ability of the hydroxy-metronidazole/metronidazole ratio to discriminate between healthy individuals and patients with Child-Pugh class A liver cirrhosis, chronic C hepatitis, or even those with a past HCV infection (Table 2).
Regarding the potential usefulness of the hydroxy-metronidazole/metronidazole ratio in early HCV chronic liver disease, we have shown that this test can discriminate between Child-Pugh class A liver cirrhosis and chronic hepatitis C or anti-HCV-positive and RNA-negative individuals, but not between the latter patient group and those with chronic hepatitis C, regardless of the severity of the disease (Table 2).These results suggest that HCV infection, regardless of viral load or genotype, causes abnormalities in liver function, even though viral clearance may have already occurred, and that these abnormalities progress with further progression of liver disease.
Several viral and bacterial infections or interferon-inducing agents have been associated with impaired cytochrome P450-mediated drug metabolism.This effect may be partly mediated by the release of cytokines or related to autoimmune-mediated phenomena which might be triggered by HCV (30)(31)(32)(33)(34)(35)(36)(37).This immunological imbalance could lead to interference with drug metabolizing systems during the acute phase of hepatitis C and may persist, even when the infection is self-limited.
Our results indicate an impairment of the metronidazole-metabolizing system induced during the acute phase of HCV infection.This impairment may continue after viral clearance.In those patients with chronic C hepatitis, this impairment is paralleled by progression of the disease to liver cirrhosis, indicating that the mechanism of damage is still active and progressive.The hydroxymetronidazole/metronidazole ratio was sensitive enough to discriminate among normal individuals and those with a past or an ongoing HCV infection, confirming our previous report (14), but could not discriminate those individuals with a history of HCV infection from those harboring a chronic hepatitis and thus, at risk for a progressive liver disease.Therefore, the usefulness of this test in the clinical setting needs to be further evaluated.

Table 1 .
Plasma hydroxy-metronidazole/metronidazole ratios 10 min after intravenous administration of 500 mg metronidazole in patients with hepatitis C virus-induced liver disease.