plasma hydroxy-metronidazole/metronidazole ratio can detect early changes in hepatic function in...

Plasma hydroxy-metronidazole/metronidazole ratio can detect earlychanges in hepatic function in ethanol-induced liver injury

C. M. F. DA SILVA*, F. L. DAVID*, M. N. MUSCARAÂ *, S. S. SOUSA*, J . G. P. FERRAZà ,

G. DE NUCCI§, N. C. POLIMENO & J. PEDRAZZOLI JR*

*Clinical Pharmacology and Gastroenterology Unit and Hemocentre, SaÄo Francisco University Medical School, BragancËa

Paulista, SP, Brazil; àDepartment of Internal Medicine, Faculty of Medical Sciences, UNICAMP, Campinas, SP, Brazil;

§Cartesius Analytical Unit, Department of Pharmacology, Institute of Biomedical Sciences, University of SaÄo Paulo, SaÄo

Paulo, SP, Brazil

Accepted for publication 5 May 1999

INTRODUCTION

The association of alcohol with liver cirrhosis has been

recognized since the 18th century, and a strong

correlation between alcohol consumption and high

mortality rates associated with cirrhosis has been

documented over the past 20 years.1 Indeed, in a

prospective survey of individuals with alcohol-induced

liver injury, more than half of those with cirrhosis and

two-thirds of those with cirrhosis plus alcohol-induced

hepatitis died within 48 months of the follow-up.2

A 38% 5-year survival has been reported for patients

with liver steatosis associated with chronic alcohol

consumption, with a 28% mortality rate, suggesting

that chronic alcohol intake can lead to severe life

threatening liver injury.3

Alcohol-induced liver cirrhosis can present in many

forms, and the absence of a past history of alcohol-

induced hepatitis is not uncommon in clinical practice.

As a result, diagnosis of the early stages of cirrhosis can

sometimes be dif®cult because the most common

complaints are uncharacteristic.1, 4 In addition, despite

the fact that clinical and laboratory ®ndings can provide

SUMMARY

Aims: To evaluate the usefulness of plasma hydroxy-

metronidazole/metronidazole (OH-MET/MET) ratios as a

dynamic liver function test in ethanol abusers with or

without liver cirrhosis.

Methods: Metronidazole was administered intravenously

for 20 min to healthy volunteers, and to patients with

alcohol-induced, non-cirrhotic hepatopathy and liver

cirrhosis. Plasma concentrations of metronidazole and

hydroxy-metronidazole were measured by high perfor-

mance liquid chromatography in samples collected 5, 10,

20 and 30 min after the metronidazole infusion.

Results: Patients with non-cirrhotic alcoholic hep-

atopathy had signi®cantly elevated aminotransferase

levels compared to healthy volunteers and Child A

patients. Child-Pugh C patients had signi®cantly

prolonged prothrombin times when compared to

healthy volunteers and patients with non-cirrhotic

hepatopathy. Metronidazole metabolism, as measured

by the OH-MET/MET ratio following the intravenous

administration of 500 mg of the drug, was signi®-

cantly impaired in all ethanol-abusing individuals,

including patients with non-cirrhotic alcoholic hep-

atopathy.

Conclusions: Metronidazole metabolism was impaired in

ethanol abusers, even in the absence of liver cirrhosis,

indicating that ethanol was capable of affecting liver

function in the early stages of alcohol-induced liver

disease.

Correspondence to: Prof. Dr J. Pedrazzoli JuÂnior, Clinical Pharmacology and

Gastroenterology Unit, SaÄo Francisco University Medical School, Av. S.

Francisco de Assis 218, 12900-000-BragancËa Paulista, SP, Brazil.E-mail: [email protected]

Aliment Pharmacol Ther 1999; 13: 1335±1341.

Ó 1999 Blackwell Science Ltd 1335

valid information on the stage and extent of the liver

disease, their usefulness in the assessment of liver

function may be limited.5

Several dynamic liver function tests have been used to

complement standard liver tests in patients with liver

disease. Such tests include indocyanine green clear-

ance,6 caffeine elimination7 and lidocaine metabolism.8

Metronidazole, which has a low protein binding

(15%) and a large distribution volume, is completely

absorbed after oral administration, with a peak

concentration of 13±15 lg/mL occurring after an

i.v. dose of 500 mg infused during 20 min.9, 10

Metronidazole is extensively metabolized by cyto-

chrome P450 isozymes, giving rise to two principal

metabolites: a hydroxy metabolite (with about 65% of

the pharmacological activity of metronidazole), and

an inactive acetic acid metabolite.11 Glucuronidation

and renal excretion of the unchanged parent com-

pound represent a minor elimination pathway. Elim-

ination occurs mainly by renal excretion of the

metabolites (60±80% of the total dose); faecal excre-

tion accounts for only 6±15% of the total dose.11, 12

Metronidazole metabolism is not in¯uenced by aging

or gender,13±16 whereas renal excretion and hepatic

glucuronidation can be reduced in patients with

hepatic encephalopathy. The decrease in metronidaz-

ole elimination in patients with severe liver disease is

mainly caused by compromised hepatic oxidative

metabolism.13, 17

In a recent evaluation of the pharmacokinetics of

metronidazole in patients with different degrees of liver

dysfunction but normal renal function, there was no

difference in the metronidazole volume of distribution

compared to normal persons, even in patients with

ascites. Similarly, there were no differences in the area

under the curve for the main metabolite (hydroxy-

metronidazole). However, there was a decrease in

clearance and an increase in the half live of metroni-

dazole, among patients with liver cirrhosis and

schistosomiasis. These ®ndings suggested that evalua-

tion of metronidazole metabolism may provide an

easy-to-perform dynamic liver function test.18 Using

this approach, we were able to discriminate healthy

individuals from Child-Pugh A cirrhotic patients based

on their metronidazole metabolism as assessed by the

hydroxy-metronidazole/metronidazole ratio 10 min

after an intravenous infusion of 500 mg of metronidaz-

ole.18 The use of this metronidazole metabolism test has

also allowed the detection of impaired liver function in

HCV-infected individuals, even in the absence of liver

cirrhosis.19

The present study evaluated whether the above test

could detect hepatic functional changes in the early

stages of several forms of alcohol-induced liver disease.

METHODS

Three groups of subjects were studied: healthy volun-

teers, patients diagnosed with non-cirrhotic, alcoholic

hepatopathy, and patients with alcohol-associated liver

cirrhosis. Patients or volunteers using drugs which

could in¯uence cytochrome P450 function were not

included. All individuals gave their written informed

consent prior to entering the study. The clinical protocol

was approved by the SaÄo Francisco University Medical

School Ethics Committee, and was conducted in accor-

dance with the Declaration of Helsinki.

Volunteer selection

Fifteen healthy volunteers (12 males, mean age

32 years, range 19±48 years) were selected based on

their medical history, absence of alcoholism, a normal

physical examination, and normal laboratory tests,

including negative serologies for hepatitis B and C.

Non-cirrhotic alcoholic hepatopathy

Twenty-four individuals (23 males, mean age 45 years,

range 38±64 years) from SaÄo Francisco University

Hospital with clinical signs of ethanol abstinence or

alcohol-induced coma were screened for non-cirrhotic

alcoholic hepatopathy. Standard laboratory liver

screening tests, including alanine aminotransferase

(ALT), aspartate aminotransferase (AST), alkaline phos-

phatase, gamma glutamyl transpeptidase (GGT), albu-

min, bilirubin, and prothrombin time were performed.

Alcohol-induced, non-cirrhotic alcoholic hepatopathy

was de®ned as increased levels of serum aminotransf-

erases, with a clear predominance of AST over ALT

(AST:ALT ratio > 1.5), or GGT levels elevated at least

once above normal. All patients were tested for anti-

HCV antibodies using a second generation enzyme

immunoassay (Abbott HCV EIA, 2nd Generation;

Abbott Laboratories, IL) and a gelatine particle agglu-

tination test with recombinant antigens C22±3 and

C200 (Serodia-HCV, Fujirebio INC, Japan). The presence

of an active HBV infection was evaluated with two

1336 C. M. F. DA SILVA et al.

Ó 1999 Blackwell Science Ltd, Aliment Pharmacol Ther 13, 1335±1341

enzyme immunoassays for detection of HbsAg and

antibody to HbcAg (Auszyme and Corzyme, respective-

ly; Abbott Laboratories, IL). The absence of liver

cirrhosis was assessed by a clinical evaluation, ultraso-

nography and/or a CT-scan, and was con®rmed by a

liver biopsy, when possible. Patients with positive

serology for HCV or HBV, ongoing bacterial infection,

tumours, liver cirrhosis or renal failure (as determined

by serial serum creatinine determinations) were exclud-

ed. Using these criteria, 20 patients (18±64 years) were

considered to have alcohol-induced hepatitis and were

included in the study.

Alcoholic cirrhosis

Thirty-three liver cirrhosis patients (mean age

41 years, range 25±70 years) 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. These patients had a long-standing history of

alcohol abuse, and negative serology for HCV or an

active HBV infection. Whenever possible, the diagnosis

of cirrhosis was con®rmed by liver biopsy. The

patients were classi®ed according to the Child-Pugh

scale.6 According to this algorithm, nine patients (all

males, mean age 46 years, range 35±64 years) were

classi®ed as A, 15 were classi®ed as B (nine males,

mean age 43 years, range 25±70 years) and nine

were classi®ed as C (eight males, mean age 45 years,

range 38±58 years). Patients with liver tumours,

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, urine analysis and, when

indicated, by an ascitic ¯uid analysis.

Laboratory analysis and plasma hydroxy-metronidazole/

metronidazole ratio

Serum chemical analysis, urinalysis, a complete blood

cell and platelet count, prothrombin times and plasma

hydroxy-metronidazole/metronidazole ratios (OH-MET/

MET) were determined in all participants. The pres-

ence of ascites and hepatic encephalopathy was also

assessed in all participants. All patients with ascites

underwent a diagnostic abdominal punction to

exclude primary bacterial peritonitis. The study

consisted of a single intravenous administration of

metronidazole (Flagyl, 500 mg in 100 mL; Rhodia,

SaÄo Paulo, Brazil) over 20 min. At the end of the

infusion, the cannula was washed with 20 mL of

sterile saline. Blood samples (5 mL) were collected

from an antecubital vein into EDTA-containing tubes

before and 5, 10, 20 and 30 min after the infusion.

The blood samples were centrifuged at 2000 g for

5 min and the plasma stored at ±20 °C until assayed.

The participants remained in the Clinical Pharmacol-

ogy Unit during metronidazole administration and

blood sampling.

Metronidazole plasma concentration

The plasma concentrations of metronidazole and its

hydroxy-metabolite (OH-MET) were measured by

reverse-phase high performance liquid chromatography

(HPLC) and ultraviolet detection, as previously

described, with minor modi®cations.20, 21 Brie¯y,

50 lL of the internal standard (tinidazole, 25 lg/mL

in ethanol) was added to 200 lL of plasma (calibration

and subject's samples). After mixing, deproteinization

was carried out by adding 20 lL of 0.6 M ZnSO4 and

20 lL of 0.4 M NaOH. The samples were vortex mixed

after each addition and the tubes were then centrifuged

(2000 g for 5 min). Twenty microlitres of each super-

natant was injected into the liquid chromatograph.

Statistical analysis

The results were expressed as the mean � s.d., unless

stated otherwise. All the variables were analysed by

one-way ANOVA followed by the Student±Newman±

Keuls test for multiple comparisons among groups.

Statistical signi®cance was considered when P < 0.05.

Materials

Metronidazole and tinidazole for HPLC analysis were

purchased from Sigma Chemical Co. (St. Louis, MO).

Hydroxy-metronidazole was a gift from RhoÃne-Poulenc

(France). Analytical grade potassium, dihydrogen phos-

phate, sodium hydroxide, phosphoric acid and HPLC-

grade methanol were obtained from Merck S.A. Inds.

QuõÂms. (Rio de Janeiro, Brazil). Analytical grade zinc

sulphate and tetrahydrofuran were purchased from

Reagen (Rio de Janeiro, Brazil) and Riedel de Haen AG

(Seelze, Germany), respectively.

LIVER FUNCTION IN ALCOHOLIC LIVER DISEASE 1337


RESULTS

Healthy controls were younger than patients

(P < 0.05), but no difference was observed among the

groups of patients; there was no difference in gender

distribution among the groups studied. The intravenous

administration of metronidazole was well tolerated by

all volunteers and patients, with no reports of side-

effects. The liver biochemistry tests demonstrated

increased AST, ALT and GGT levels in patients with

alcohol-induced liver disease compared to healthy

volunteers (Table 1). As expected, in patients with

alcohol-induced liver disease AST levels predominated

over ALT levels (Table 1). In addition, Child-Pugh C

cirrhotic patients had signi®cantly prolonged prothrom-

bin times compared to healthy volunteers and patients

with alcohol-induced hepatitis, indicating severely

impaired liver function (Table 1).

Metronidazole metabolism, as measured by the plasma

OH-MET/MET ratio, was signi®cantly impaired in

patients with alcohol-induced liver cirrhosis throughout

the course of the study (Figure 1). The altered hepatic

metabolic capacity in these patients was detected as

early as 10 min following the administration of met-

ronidazole (Figure 1; Table 2). A signi®cantly

(P < 0.05) higher plasma OH-MET/MET ratio was

observed in patients with non-cirrhotic alcoholic hep-

atopathy compared to Child B and C cirrhotic patients

(Figure 1; Table 2). A non-signi®cant reduction in

metronidazole metabolism was also observed when

patients with non-cirrhotic, alcoholic hepatopathy were

compared to healthy volunteers (Figure 1; Table 2).

When metronidazole metabolism was evaluated

according to the Maddrey discriminant function22

(severe or moderate hepatopathy > 60; mild < 60),

individuals with severe or moderate disease showed a

non-signi®cant reduction (P � 0.0547) in OH-MET/

MET ratio (0.03762 + 0.04), when compared to con-

trols, whereas those with milder disease had a normal

OH-MET/MET ratio (0.05645 + 0.04).

DISCUSSION

Our results demonstrate that patients with alcohol-

induced liver injury have an impaired hydroxy-metron-

Table 1. Standard liver biochemistry tests in healthy volunteers and in patients with alcohol-induced liver disease

Group

AST

(IU/mL)

ALT

(IU/mL)

GGT

(IU/mL)

Bb

(mg%)

PT

(s)

Weight

(kg)

Mean age

(years; range)

Healthy volunteers 17.1 + 2.8 16.4 + 2.6 22.2 + 4.7 0.9 + 0.2 13.5 + 0.5 70 + 2.0 32 (19±48)

Child-Pugh A 39.0 + 4.6## 39.7 + 10.0# 71.3 + 9.9 1.5 + 0.2 13.6 + 0.5 68.5 + 3.9 45* (38±58)

Child-Pugh B 63.9 + 15.9 28.8 + 2.5# 70.8 + 16.3# 4.1 + 1.5 15.6 + 0.6 61.3 + 6.0 46* (35±64)

Child-Pugh C 98.2 + 36.0 49.2 + 14.7# 52.4 + 19.7 7.7 + 2.8 19.0 + 2.0###** 58.3 + 3.4 43* (25±70)

Alcoholic hepatopathy 83.5 + 11.7** 62.5 + 9.5*** 146.1 + 31.0** 2.5 + 1.0 10.2 + 1.2 68.2 + 3.0 45* (38±64)

AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma glutamyl transpeptidase; Bb, total bilirubin; PT, prothrombin time.

The results are expressed as the mean � s.e. Asterisks denote a signi®cant difference compared to healthy volunteers (* P < 0.05, ** P < 0.01,*** P < 0.001). # denote a signi®cant difference compared to patients with alcohol-induced hepatitis (# P < 0.05, ## P < 0.01,

### P < 0.001).

Figure 1. Plasma hydroxy-metronidazole/metronidazole (OH-

MET/MET) ratios following the intravenous administration of

500 mg of metronidazole in healthy volunteers (solid circles),

patients with non-cirrhotic, alcoholic hepatopathy (solid squares),

and Child-Pugh A (open circles), Child-Pugh B (open triangles)

and Child-Pugh C (open squares) cirrhotic patients. The results

are expressed as the mean � s.e. Plasma OH-MET/MET ratios

were signi®cantly reduced in patients with alcohol-induced liver

disease compared to healthy volunteers, indicating an impaired

metabolic capacity in the liver of chronic ethanol abusers. Plasma

OH-MET/MET ratios in cirrhotic patients were also signi®cantly

reduced compared to those of patients with alcohol-induced

hepatitis. Asterisks denote a signi®cant difference compared to

healthy volunteers (* P < 0.05, ** P < 0.01, *** P < 0.001).



idazole/metronidazole ratio, suggesting that hepatic

function is altered by chronic ethanol consumption.

The pathogenesis of alcohol-induced liver disease is

believed to be multifactorial. Ethanol toxicity, nutritional

de®ciency, and other noxious factors can contribute to

the development of liver injury in susceptible subjects.23

The impact of chronic alcohol consumption on liver

function has been extensively explored. However,

standard liver biochemistry tests can sometimes give

inaccurate information on the true status of liver

function.5 In such cases, a dynamic assessment of the

hepatic metabolic capacity should theoretically provide

a more direct and reliable assessment of liver function.

Ethanol oxidation by hepatocytes leads to the forma-

tion of several free radicals which can in¯ict oxidative

damage on a wide range of intracellular compounds.

These radicals can also be produced by the accumula-

tion of acetaldehyde, the metabolism of which is already

slowed in alcoholics. Alcohol consumption also reduces

antioxidant defences, and ampli®es the deleterious

effects of free radicals. Ten to 15 per cent of ethanol

ingested is metabolized by a microsomal P450 ethanol-

oxidizing system, mostly CYP 2E1, which is alcohol-

inducible.24 The induction of CYP 2E1 increases oxygen

consumption, acetaldehyde production and promotes

lipid peroxidation, with the generation of free radi-

cals.25, 26 This phenomenon results in the development

of steatosis and other lesions in zone 3 (perilobular) of

the hepatic acinus. Since the P450 enzymes are present

in greater amounts in the perilobular zone, this could, at

least in part, explain the observed impairment of

metronidazole metabolism, even in cases of non-cirrho-

tic alcoholic hepatopathy.

Immunological mechanisms may also contribute to the

pathogenesis of alcohol-induced hepatitis,27 and there is

a striking similarity between the biological effects of

certain cytokines and the clinical manifestations of acute

liver disease. It has been proposed that tumour necrosis

factor may play a role in alcohol-induced hepatitis.28, 29

Cytokines down-regulate the expression of major cyto-

chrome P450 enzymes in adult human hepatocytes in

primary culture, and interferon gamma down-regulates

the expression and catalytic activities of cytochrome

P450 enzymes and genes.30, 31 Thus, immune-mediated

alcohol-induced changes in cytochrome P450 enzyme

function could account for the impaired capacity of the

liver to promote metronidazole clearance.

In the absence of liver cirrhosis, patients diagnosed

with moderate to severe non-cirrhotic alcoholic hep-

atopathy based on the Maddrey discriminant function22

also had an altered capacity to metabolize metronidaz-

ole. This ®nding indicates that ethanol-associated

changes in liver function can occur in the absence of

liver ®brosis, and most importantly, that they can be

detected at earlier stages of liver disease using the

metronidazole metabolism test. A `better' metronidazole

metabolism was observed in subjects with non-cirrhotic

alcoholic hepatopathy compared to Child-Pugh A

cirrhotic patients, who had signi®cantly lower amino-

transferase levels than Child-Pugh C cirrhotic patients.

This suggests that altered metronidazole metabolism

occurs irrespective of hepatocyte injury, as determined

by AST and ALT levels. Such a conclusion agrees with

our previous observations of impaired metronidazole

metabolism in HCV-positive, non-cirrhotic patients or

asymptomatic carriers, and con®rms the presence of a

cytochrome P450 dysfunction in the initial stages of

liver disease; this disfunction is not related to active

hepatocyte damage.19

Metronidazole metabolism correlated well with the

Maddrey discriminant function,22 being normal in

individuals with episodes of mild non-cirrhotic alcoholic

hepatopathy (MDF < 60), and reduced in patients with

more severe non-cirrhotic alcoholic hepatopathy

(MDF > 60). The reduction in the latter was not

signi®cant when compared to healthy volunteers,

although there was a tendency towards signi®cance

(P � 0.0547). In addition, a Type II error resulting

Table 2. Mean plasma hydroxy-metronidazole/metronidazole

ratios 10 min after the intravenous administration of 500 mg of

metronidazole

Group [OH-MET]/[MET]

Healthy volunteers 0.069 � 0.029

Child-Pugh A 0.023 � 0.015**

(±0.080/±0.013)a

Child-Pugh B 0.023 � 0.022***#

(±0.073/±0.019)a

(±0.046/±0.005)b

Child-Pugh C 0.015 � 0.013***#

(±0.026/0.025)a

(±0.046/±0.005)b

Non-cirrhotic 0.049 � 0.043

Alcoholic hepatopathy (±0.041/0.0004)a

OH-MET, hydroxy-metronidazole; MET, metronidazole. ** P < 0.01,

*** P < 0.001 vs. healthy volunteers. # P < 0.05 vs. patients withalcohol-induced hepatitis. Numbers in brackets indicate the upper and

lower limits of the 95% con®dence intervals (CI) of the signi®cant

differences compared to ahealthy volunteers and bpatients with

alcohol-induced hepatitis.



from the reduced number of individuals evaluated

cannot be ruled out.

In most of our patients with non-cirrhotic, alcoholic

hepatopathy the diagnosis was based upon clinical,

laboratory and sonography data. Thus, the presence of

early stage liver cirrhosis could not be completely

excluded. It is noteworthy, however, that patients with

a diagnosis of liver cirrhosis involving moderate

impairment of hepatic function (Child B) showed a

reduction in metronidazole metabolism similar to

patients with liver cirrhosis who had a normal liver

function (Child A; Figure 1). The slight impairment in

metronidazole metabolism observed in Child B indivi-

duals compared to Child A patients was signi®cant

when compared to individuals with non-cirrhotic,

alcoholic hepatopathy (Figure 1). These ®ndings

suggest that these groups indeed represent different

stages of alcoholic liver disease. Nevertheless, these

results are preliminary and need to be con®rmed in a

larger study in which the diagnosis of non-cirrhotic,

alcoholic hepatopathy is based on histology.

Other dynamic liver tests that can be used to comple-

ment standard liver assessment in patients with liver

diseases include the clearance of lidocaine or the

formation of its main metabolite mono-ethyl-glycine-

xylidine (MEG-X) and the clearance of caffeine.7, 32 The

clearance of lidocaine is very complex, and has no

predictive value in patients with liver cirrhosis of any

Child class.33, 34 The MEG-X-test is reported to have

failed in predicting graft function and survival after liver

transplant.35, 36 The clearance of lidocaine is increased in

smokers, is age-related and appears to be in¯uenced by

gender.37±39 The administration of lidocaine at the dose

required to perform a dynamic liver test may also be

associated with side-effects, including severe cardiac

dysrhythmia.40 The clearance of caffeine is reduced in

cirrhosis and has no side-effects, although caffeine

metabolism is induced in smokers and is dependent on

diet.41±43 Metronidazole metabolism is not age-, gender-

or diet-dependent, and its administration at the dose

needed to perform the hydroxy-metronidazole/metron-

idazole ratio test is not associated with severe side-

effects.13±16

These results, obtained in patients with alcohol-induced

liver disease, con®rm our previous report demonstrating

that impairment of metronidazole metabolism can be

detected in the early stages of liver disease such as in

Child-Pugh A cirrhosis, and that this ®nding parallels the

clinical assessment of liver disease.11

We conclude that metronidazole metabolism is

reduced in alcohol-induced liver cirrhosis and in

patients with non-cirrhotic, alcoholic hepatopathy.

The OH-MET/MET ratio proved to be useful in the early

detection of ethanol-induced liver injury. We are

currently studying the applicability of this test to the

long-term prognosis of alcohol-induced hepatitis and

liver cirrhosis.

ACKNOWLEDGEMENTS

Dr Silva is supported by FundacËaÄo de Amparo aÁ

Pesquisa do Estado de SaÄo Paulo (FAPESP), Brazil. This

work was supported by a grant from IPPEX ± SaÄo

Francisco University (to J. P. Jr).

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