EFFECTS OF ALCOHOL CONSUMPTION ON EIGHT CIRCULATING MARKERS OF LIVER FIBROSIS
Yelena Ponomarenko,
Maria Anna Leo,
Werner Kroll1 and
Charles S. Lieber,*
Liver Disease & Nutrition Section, Veterans Affairs Medical Center & Mount Sinai School of Medicine, Bronx, NY 10468-3992, USA and
1 Bayer AG, Wuppertal, Germany
Received 30 March 2001;
in revised form 12 September 2001;
accepted 19 October 2001
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ABSTRACT
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A number of circulating breakdown products of collagen or other components of extracellular matrix, matrix degrading metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) have been proposed as markers of hepatic fibrosis. However, the published results lack consistency. Since many of the patients with fibrosis studied were alcoholics, the question was raised whether recent alcohol consumption may affect the results obtained. Using sandwich-type assays of radioimmunoassay technology with corresponding antibodies, we studied eight markers of liver fibrosis: laminin, tenascin, undulin, TIMP-1, collagen VI, procollagen type III (PIIINP), hyaluronic acid (HA) and MMP-2. A group of 10 alcoholics was studied after significant alcohol consumption and following 2 weeks of abstinence, verified with repeated breath alcohol measurement. Laminin was significantly reduced at 1 week (22%) and at 2 weeks (30%). Similarly, tenascin and undulin were also significantly decreased. By contrast, TIMP-1, collagen VI, PIIINP, HA and MMP-2 did not significantly change. The mode of action of alcohol on these tests is unknown. These differences must be considered when using those measurements to assess liver fibrosis.
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INTRODUCTION
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Because of an expanding epidemic of hepatitis B and C in recent years and a large number of patients with alcoholic liver disease with evolving liver fibrosis and cirrhosis, the diagnosis of the stage of liver fibrosis in these conditions has become an increasingly important task. Liver biopsy is the most accurate way to assess liver fibrosis or cirrhosis but it has its limitations, namely an unavoidable sampling error and a lack of practicality for screening purposes because of its invasive nature.
In recent years, several serum markers have been developed to assess fibrogenesis. Efforts have been made to replace liver biopsy with non-invasive markers of liver fibrosis which have been assessed in multiple studies (Savolainen et al., 1984
; Sato et al., 1986
; Li et al., 1994
; Tanikawa, 1994
; Oberti et al., 1997
), but questions remain on how their sensitivity and significance are affected by various factors. In a preliminary study by Nouchi et al. (1987), it was shown that the duration of abstinence from alcohol affects one of the fibrosis markers, namely laminin. However, the question of the influence of alcohol consumption on blood concentration of other markers of liver fibrosis is still largely uncharted. The aim of this project was to investigate to what extent alcohol withdrawal affects the following markers of liver fibrosis: laminin, undulin, tenascin, tissue inhibitors of metalloproteinases (TIMP-1), collagen VI (CVI), the procollagen type III N-terminal propeptide (PIIINP), hyaluronic acid (HA), and matrix degrading metalloproteinases (MMP-2), and whether some of their variability reflects the degree of drinking at the time of blood sampling.
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MATERIALS AND METHODS
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Subjects
A group of 10 alcoholics who were drinking heavily (Table 1
) for the preceding 30 days and were acutely intoxicated or had their last drink within 24 h of 0 time were followed for a period of 2 weeks from the day of detoxification.
Criteria of exclusion were: tumours, human immunodeficiency virus, severe chronic disease (chronic heart disease, chronic renal failure, endocrine diseases). Only two patients were found to be hepatitis C-positive, but none was hepatitis B surface antigen-positive. The patient characteristics are summarized in Table 1
. They were all heavy drinkers, with a drinking history of 333 years (mean ± SEM: 24.6 ± 11.2). Their age varied from 44 to 62 years (mean ± SEM: 49.3 ± 1.8) and the average body mass index was 26.4 ± 1.7 kg/m2. All patients were male. Alcohol level at 0 time is also shown in Table 1
.
A blood sample for the evaluation of markers of liver fibrosis was taken upon admission, when some alcohol was still present in the blood, and 1 and 2 weeks later. Blood-alcohol levels were verified with repeated breath-alcohol measurements before detoxification started and after 1 and 2 weeks of abstinence.
Methods
As part of the patients' work-up, the liver function tests listed in Table 2
were performed by the hospital clinical laboratory using standard methods.
CVI assay. This test was performed with the Bayer Immuno 1© immunoassay system as follows: 65 µl of serum sample volume were dispensed together with 65 µl of Reagent R1 (fluorescein-labelled monoclonal anti-CVI antibodies) and 65 µl of Reagent R2 (alkaline phosphatase-labelled monoclonal anti-CVI antibodies) into the reaction cuvette. The mixture was incubated for 20 min at 37°C to allow the formation of the sandwich immune complex. The 10 µl of magnetic particles coated with monoclonal antifluorescein antibodies were added into the cuvette. After washing the magnetic particles to remove excess reagent and sample, p-nitrophenolphosphate was added and converted by the enzyme to the yellow dye p-nitrophenoxide, the absorption of which was measured at 405 nm. The calibration curve covered an assay range of 050 ng/ml using six calibrators (0, 2, 5, 10, 20 and 50 ng/ml). Calibrators were prepared by spiking CVI isolated from human placenta. The minimum detectable value for the assay was determined as the concentration corresponding to a reaction rate 2 SD above the lowest calibrator (0 ng/ml) and calculated to be <1 ng/ml. Comparable assays were carried out for the other fibrosis markers.
The normal values (mean ± SEM) for TIMP-1, tenascin, collagen VI, PIIINP, laminin, undulin and MMP-2 are 648 ± 0.61, 457 ± 11.79, 3.04 ± 0.13, 5.84 ± 0.23, 15.5 ± 0.52, 21.4 ± 0.65, 542 ± 9.26 ng/ml, respectively. For hyaluronic acid, the values varied depending on age.
Statistics
Results are expressed as means ± SEM, and the significance of the differences among individual groups was calculated by one-way analysis of variance (ANOVA) followed by the NewmanKeuls post hoc test; for two-group comparison, the unpaired Student's group t-test was used. P < 0.05 was considered significant (Snedecor and Cochran, 1980
).
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RESULTS
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Serum transaminases [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)] and
-glutamyltransferase (GGT) were increased, reflecting alcohol abuse and associated liver damage. However, albumin, bilirubin, and prothrombin time (PT) were normal or near normal, indicating that the patients did not have decompensated liver disease (Table 2
).
The three following markers decreased with alcohol withdrawal. For laminin, the mean values changed from 47.34 ng/ml at 0 time to 37.38 ng/ml after 1 week (P < 0.01) and to 33.06 ng/ml (P < 0.02) after 2 weeks (Fig. 1
). With undulin, its mean values changed from 19.16 ng/ml to 18.04 ng/ml after 1 week and to 16.59 ng/ml (P < 0.01) after 2 weeks (Fig. 2
). The mean value for tenascin was decreased from 624.10 ng/ml to 553.39 ng/ml (P < 0.02) after 1 week and to 510.68 ng/ml (P < 0.02) after 2 weeks (Fig. 3
).

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Fig. 1. Effect of alcohol withdrawal on serum laminin concentrations. Each point indicates an individual patient value. Mean values were significantly lower at 1 week (P < 0.01) and at 2 weeks (P < 0.02) than at 0 time. The line illustrates the decrease with time of the mean value.
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Fig. 2. Effect of alcohol withdrawal on serum undulin concentrations. The values decreased significantly (P < 0.01) with time.
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Fig. 3. Effect of alcohol withdrawal on serum tenascin concentrations. Both at 1 and at 2 weeks, the values were significantly (P < 0.02) lower than at 0 time.
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There was no correlation between the alcohol level at 0 time and the laminin, undulin and tenascin values.
The following five markers were unaffected by withdrawal from alcohol. Thus, TIMP-1 was 834.64 ng/ml at week 0, 839.19 ng/ml after 1 week and 780.94 ng/ml after 2 weeks (P > 0.1). Collagen VI changed from 5.46 ng/ml at time 0 to 4.75 ng/ml after week 1, and to 5.03 ng/ml at week 2 (P > 0.1). The mean value of hyaluronic acid changed from 72.84 ng/ml at 0 time to 78.82 ng/ml after 1 week and to 59.58 ng/ml after 2 weeks (P > 0.1). MMP-2 changed from 940.91 ng/ml at 0 time to 887.70 ng/ml at 1 week and to 922.15 ng/ml at 2 weeks (P > 0.1). Finally, the PIIINP mean value was 6.88 ng/ml at 0 time, 7.22 ng/ml at week 1 (P > 0.1) and 6.92 ng/ml at week 2 (P > 0.1).
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DISCUSSION
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Only a minority of heavy drinkers eventually develop major complications such as liver cirrhosis (Lieber, 1992
), and there is an obvious need for arresting or reversing such serious alcohol-induced organ damage at an earlier stage. Individual differences in the rate of ethanol metabolism and in the severity of alcohol-induced liver disease (Lumeng and Crabb, 1994
) are, in part, genetically determined, but no such genetic markers are available. In the absence of practical or accepted genetic markers, individuals vulnerable to developing the more severe complications can be recognized empirically through the early detection of precursor lesions. We have now learned to recognize lesions in the liver, such as perivenular fibrosis, which, already at an early pre-cirrhotic stage, enables us to predict the rapid progression to cirrhosis upon the continuation of drinking (Worner and Lieber, 1985
). At present, liver biopsies are required to detect these pre-cirrhotic lesions. Of course, liver biopsy is not a practical tool for disease control on a large scale. It is not unreasonable to anticipate, however, that eventually blood tests might be developed that serve the same purpose of screening individuals who, for some reason, show a greater propensity to rapidly develop cirrhosis when exposed to the offending agent alcohol. To validate such tests, one has to define how much they actually indicate the chronic effect of alcohol on fibrosis and reflect its future progression, and how much they are due to the acute effects of alcohol on the marker.
The present study showed that, of the eight circulating markers of liver fibrosis assessed here, three (laminin, undulin and tenascin) decreased upon withdrawal from significant alcohol consumption, whereas the five other markers were predominantly unaffected (TIMP-1, collagen VI, PIIINP and MMP-2) or variable (HA). Thus, our results are consistent with those of a preliminary study by Nouchi et al. (1987), which revealed that serum values of laminin, the major non-collagenous glycoprotein of basement membranes, decreased with abstinence. Our current study showed that there was also a significant decrease of other markers of fibrosis, such as tenascin, a molecule expressed in proliferating extracellular matrix, and undulin, a glycoprotein associated with the surface of mature fibrillar collagen. Both significantly decreased after 2 weeks of abstinence from alcohol. By contrast, collagen VI, a molecule that forms filaments between large collagen fibrils, and PIIINP, the N-terminal propeptide of procollagen type III, considered a marker of fibrogenesis, as well as TIMP-1 and MMP-2, did not significantly change after 2 weeks of sobriety and, therefore, deserve further studies as possible substitutes for liver biopsy. By contrast, for serum hyaluronic acid, a ubiquitous glycosaminoglycan with high extraction by the liver sinusoidal endothelium, the values were highly variable.
The mode of action of alcohol on these markers is unknown but, regardless of the mechanisms involved, the presence of alcohol should be taken into account in assessing the significance of a given test result, and greater consideration should be given to those markers which reflect fibrosis and are unaffected by prolonged alcohol consumption.
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ACKNOWLEDGEMENTS
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These studies were supported by NIH grant AA 05934, AA11115, the Department of Veterans Affairs and the Kingsbridge Research Foundation.
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FOOTNOTES
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* Author to whom correspondence should be addressed. 
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