Serum paraoxonase-1 in chronic alcoholics: Relationship with liver disease

Clinical Biochemistry 40 (2007) 645 – 650

Serum paraoxonase-1 in chronic alcoholics: Relationship with liver disease Judit Marsillach a , Natàlia Ferré b , Maria C. Vila c , Anna Lligoña b , Bharti Mackness d , Michael Mackness d , Ramon Deulofeu b , Ricard Solá c , Albert Parés b , Juan Pedro-Botet c , Jorge Joven a , Joan Caballeria b , Jordi Camps a,⁎ a

Centre de Recerca Biomèdica, Hospital Universitari de Sant Joan, Institut de Recerca en Ciències de la Salut, Reus, Spain b Department of Clinical Biochemistry and Molecular Genetics, Institute of Psichiatry and Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques Agustí Pi i Sunyer, Barcelona, Spain c Department of Medicine, Hospital del Mar, Barcelona, Spain d Department of Medicine, Manchester Royal Infirmary, Manchester, UK Received 30 October 2006; received in revised form 21 December 2006; accepted 29 January 2007 Available online 3 February 2007

Abstract Objectives: To investigate the relationship between serum paraoxonase-1 and liver damage in chronic alcoholic patients. To assess the diagnostic accuracy of paraoxonase-1 plus standard biochemical tests in the assessment of liver damage in alcoholics. Design and methods: We studied 328 chronic alcoholics and 368 healthy individuals. Results: Paraoxonase-1 activity was decreased and the concentration was increased in alcoholics (P < 0.001). The enzyme activity was correlated with albumin (r = 0.45; P < 0.001) and prothrombin time (r = 0.49; P < 0.001). Addition of paraoxonase-1 activity measurement to a battery of biochemical tests increased the sensitivity in differentiating between patients and controls up to 96.6% but did not improve the sensitivity in differentiating between subgroups of alcoholics. Conclusions: Paraoxonase-1 was related to the severity of alcoholic liver disease. Its measurement was useful in discriminating between patients and healthy subjects, but did not add any valuable information in subgroups of alcoholics. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Alcoholism; Diagnostic accuracy; Liver disease; Paraoxonase-1

Introduction Paraoxonase-1 (PON1) is an enzyme that catalyzes the hydrolysis of organophosphorates and other xenobiotics and circulates in plasma tightly bound to high-density lipoproteins (HDL). It has been postulated to hydrolyze lipid peroxides and homocysteine-thiolactone and may play a role in the organism’s antioxidant system [1–4]. The liver plays a key role in the synthesis of PON1, the enzyme’s gene expression having been observed to occur mainly in this organ [5,6]. We have previously reported that serum PON1 activity is significantly decreased in patients with chronic hepatitis or liver cirrhosis [7,8]. We also suggested that serum PON1 activity measurement may significantly improve the evaluation of liver function ⁎ Corresponding author. Fax: +34 977 312569. E-mail address: [email protected] (J. Camps).

in these patients [7]. However, these results should be considered preliminary because the number of participants enrolled was relatively low and the etiology of their liver disease was not homogeneous. The effects of alcohol intake on serum PON1 levels are not well known. Moderate alcohol consumption has been found to be associated with slight increases in serum PON1 activity and HDL cholesterol in normal volunteers [9], but the alterations in serum PON1 status in chronic alcoholics, and how this is modulated by the concomitant presence of liver disease, remain to be elucidated. The aims of the current study were: (a) to determine serum activity and concentration of PON1 and to assess the relationship with liver damage in a large series patients with chronic alcoholic abuse and (b) to assess the diagnostic accuracy of the measurement of serum PON1 activity and concentration in combination with the standard liver function tests in evaluating liver damage in these patients.

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Subjects and methods Selection of volunteers This was a prospective study involving 328 chronic alcoholic patients (227 men and 101 women; mean age of 48 years, range 24–82 years) who, between February 2002 and March 2004, were consecutively admitted to the Hospital Clínic de Barcelona and the Hospital del Mar de Barcelona to undergo detoxification. The daily alcohol intake was ≥ 80 g for men and ≥ 60 g for women at the time of admission to a hospital. A blood sample was drawn within 24 h of admission for biochemical analyses. For the purpose of the study, patients were categorized as those with normal liver who were asymptomatic as defined by physical examination and liver function tests (group 1; n = 110; 64 men and 46 women); those with mild-to-moderate liver disease with mild hepatomegaly and/or a moderate increase in serum aminotransferase or γ-glutamyltransferase activities, but with no ultrasound changes that would suggest cirrhosis (group 2; n = 114; 87 men and 27 women); patients with liver cirrhosis confirmed by histology examination (group 3; n = 100; 74 men and 26 women). The control group was composed of individuals (n = 368; 182 men and 186 women; mean age of 42 years; range 19–75 years) selected from among a study population the characteristics of which have been reported previously [10]. Briefly, these participants were ostensibly healthy with no clinical or analytical evidence of renal insufficiency, liver damage, neoplasia or neurological disorders. Of the study sample, 54% reported a mean alcohol consumption of alcohol of 11 ± 19 g/day; 26 participants (6.7%) reportedly stopped consuming alcohol between 1 and 38 years previously and, for the purposes of the present study, were considered as non-imbibers. For comparison, we also studied a group of 41 patients (24 men and 17 women) with non-alcoholic liver cirrhosis. The etiology of their disease was viral in 29 patients and cryptogenic in 12 patients. Cirrhotic patients (alcoholic and non-alcoholic) were classified according to the Child–Pugh scale [11] into three classes: A (n = 86), B (n = 46), and C (n = 12). The study was approved by the Ethics Committees of the Hospitals involved and all patients and control subjects gave written consent to participation in the study. Biochemical measurements Blood samples were collected after an overnight fast into tubes with no anticoagulants to obtain serum, or into tubes with K2-EDTA or Na2-citrate to obtain plasma. The analyses were performed immediately, or aliquots were stored − 80 °C for subsequent batched analyses. Serum PON1 activity was determined by measuring the rate of hydrolysis of paraoxon at 410 nm and 37 °C in a 0.05 mmol L− 1 glycine buffer (pH 10.5) with 1 mmol L− 1 CaCl2 and no NaCl added [7] Serum PON1 concentration was determined by an in-house ELISA method [12]. PON1 specific activity was expressed as PON1 activity per unit concentration. Serum alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT) activities, albumin, bilirubin,

apolipoprotein A–I, and HDL cholesterol concentrations were measured with reagents obtained from Beckman-Coulter in a Synchron Lxi automated analyzer (Beckman-Coulter, Fullerton, CA). Prothrombin time was measured in an ACL 9000 analyzer (Instrumentation Laboratories, Milan, Italy). Plasma lipid peroxidation was estimated by measuring malondialdehyde concentrations using the thiobarbituric acid reaction followed by purification by high-performance liquid chromatography [13]. PON1 genotyping has not been possible in the present study because DNA and/or cells from peripheral blood from the alcoholic patients were not available. Previous studies suggested that PON1 genetic polymorphisms are not a major factor in the modulation of serum PON1 levels by alcohol intake [9,14] or liver disease [7]. Statistical analysis Differences between groups were assessed with the Student’s t-test (parametric) or the Mann–Whitney U test (nonparametric). Pearson or Spearman correlation coefficients, or curvilinear regression, were used to evaluate the degree of association between variables. Since most of the measured variables had non-Gaussian distributions, these results are expressed as medians and 95% CI (in parenthesis). Diagnostic accuracy for serum PON1 activity and other biochemical tests was calculated with ROC analysis [15–17]. Multiple logistic regression models were fitted to estimate the ability of groups of variables to predict the presence or absence of disease. Statistical significance was set at P ≤ 0.05. Statistical analyses were performed by an experienced biostatistician who was blinded with respect to the diagnostic groups. The SPSS 13.0 package was employed for all statistical calculations. Results Serum PON1 activity and concentration, and the relationship with liver damage in alcoholic patients The main demographic characteristics of the study participants, the history of alcohol abuse, and the biochemical determinations are summarized in Table 1. As expected, liver function tests were more altered in those patients with a more advanced liver disease. Serum PON1 activity was significantly decreased in alcoholic patients compared with controls. PON1 activity decreased according to the severity of liver damage; the lowest values being observed in cirrhotic patients. By contrast, serum PON1 concentrations were significantly increased in alcoholic patients, especially in those with cirrhosis. PON1 specific activity was significantly decreased in alcoholic patients compared with controls. Serum PON1 activity showed a direct exponential relationship with serum albumin concentration and plasma prothrombin time (Fig. 1) in all the study subjects. Furthermore, there were weak direct linear relationships between PON1 activity and HDL cholesterol concentration (r = 0.17; P < 0.001) and apolipoprotein A–I (r = 0.13; P = 0.003) and an inverse relationship with the years-of-alcohol consumption (r = − 0.15; P < 0.001).

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Table 1 PON1 status, biochemical, and demographic variables in the control group and in alcoholic patients Control group (n = 368) Men (%) Age (years) Ethanol consumption (g day− 1) Ethanol consumption (years) Albumin (g L− 1) Alanine aminotransferase (μkat L− 1) γ-Glutamyltransferase (μkat L− 1) Bilirubin (μmol L− 1) Prothrombin time (%) HDL cholesterol (mmol L− 1) Apolipoprotein A–I (g/L) Malondialdehyde (μmol L− 1) PON1 activity (U L− 1) PON1 concentration (mg L− 1) PON1 specific activity (U mg− 1)

49.4 42 (19–75) – – 42 (38–46) 0.33 (0.18–0.67) 0.22 (0.11–0.85) 9.0 (4.0–16.6) 98 (86–118) 1.50 (1.01–2.19) 1.64 (1.21–2.17) 51 (12–90) 383.5 (220.0–699.0) 78.2 (46.9–237.9) 4.82 (1.47–11.51)

Alcoholic patients Normal liver (n = 110) b

58.1 46 (29–67) 120 (65–250) 20 (5–30) 39 (32–46)a 0.15 (0.08–0.37)a 0.57 (0.23–1.36)a 7.0 (4.0–12.5) 100 (83–100) 1.47 (0.92–2.65) 1.73 (1.26–2.38)a 126 (24–731)a 180.5 (76.0–378.5)a 91.6 (42.8–178.9)a 1.72 (0.66–6.05)a

Moderate changes (n = 114) b

75.4 45 (33–66) 150 (80–265)c 20 (5–30) 39 (31–49)a 0.18 (0.10–0.93)a 3.85 (1.34–13.83)a 9.0 (4.0–22.0) 100 (74–100) 1.71 (1.01–3.63)a 1.95 (1.27–2.85)b 198 (24–659)a 152.2 (68.9–389.2)a 104.5 (49.6–185.8)a 1.60 (0.41–4.94)a

Cirrhosis (n = 100) 69.3b 53 (40–66) 150 (100–250)c 20 (15–30) 36 (21–48)a 0.20 (0.09–0.85) 1.17 (0.27–12.12)a 13.0 (4.1–66.9)a 71 (42–100)a 1.10 (0.37–2.50)a 1.32 (0.46–1.98)b,c 162 (23–499)a 106.4 (37.9–274.8)a 169.4 (46.8–1122.8)a 0.60 (0.04–3.50)a

All values are presented as medians (95% CI). : P < 0.01; b: P < 0.001, with respect to the control group. c: P < 0.001, with respect to alcoholics with normal liver.

a

There was a significant inverse relationship between serum PON1 activity and plasma malondialdehyde concentration in alcoholic patients with normal liver (ρ = − 0.26; P = 0.008), but not in the mild-to-moderate and cirrhosis groups. Serum PON1 concentrations showed weak but statistically significant (P < 0.001) inverse relationships with plasma prothrombin time (r = − 0.22), with albumin concentration (r = −0.14), HDL cholesterol (r = − 0.17), and apolipoprotein A–I (r = − 0.20). Patients with non-alcoholic cirrhosis had a similar serum PON1 activity [median: 113.1 U L (95% CI: 18.4–271.9)] and concentration [237.9 mg L (40.9–1693.4)] as that of alcoholic cirrhotics. Hence, these two groups of patients were combined for this analysis. We observed a significantly lower PON1 activity and a higher PON1 concentration in cirrhotic patients belonging to Child–Pugh class C, compared to those belonging to classes A and B (Table 2). PON1 measurement as a liver function test in alcoholic patients The results of the ROC analysis for serum PON1 activity and concentration, as well as for the standard biochemical tests of liver function, are summarized in Table 3. The area-underthe curve (AUC) for PON1 activity was ≥ 0.90 in the different groups of patients when compared to controls. The values were similar to those for GGT and higher than the AUC values for prothrombin time, bilirubin, ALT, albumin, and PON1 concentration. We investigated the ability of the biochemical tests to discriminate between subgroups of alcoholics having different degrees of liver disease. When patients with a normal liver (group 1) were compared with those with hepatic alterations (mild-to-moderate changes, group 2 and cirrhosis, group 3), the AUC for PON1 activity was 0.63. Conversely, when alcoholic cirrhotic patients (group 3) were compared with those with normal liver (group 1) and the moderate change group (group 2)

the AUC for PON1 was 0.76. In both situations, the AUCs for PON1 activity were similar to those obtained with the standard liver function tests. The usefulness of adding serum PON1 activity measurement to the standard panel of liver function tests was evaluated by multiple regression analysis. We compared the ability of two different models to correctly, and accurately, classify patients and controls. To predict the status of any one individual to cosegregate with any of the disease groups, the biochemical terms in the equations (Table 4) need to be substituted by their corresponding measured values. For example, if the result (x) calculated from the equation is < 0, this would classify the individual as a patient. Overall, the addition of serum PON1 activity measurement to the predictive models increased the diagnostic sensitivity, without any impairment of specificity. PON1 activity was mainly useful in distinguishing between the alcoholics with normal liver and the controls (sensitivity increased from 75.7 to 83.5%), and between the alcoholic cirrhotics and the controls (sensitivity increased from 84.7 to 96.6%). However, the inclusion of PON1 activity measurement did not improve the sensitivity and specificity of the standard battery of test usually used to assess the degree of liver disease in the different subgroups of alcoholic patients. Discussion The present study showed that serum PON1 activity was decreased in patients who were chronic alcohol abusers relative to control subjects and that PON1 activity was significantly related to the degree of liver damage; the lowest values being in patients with cirrhosis, especially those in the Child–Pugh class C. Serum PON1 activity was also correlated with serum albumin concentration and plasma prothrombin time. The decrease in serum PON1 activity may involve several factors. A considerable degree of inhibition is observed in alcoholic patients with a normal liver in which hepatic function,

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Fig. 1. Relationship between serum PON1 activity, albumin, and prothrombin time in all the study participants (n = 737).

according to standard biochemical analyses, is preserved. In these patients, serum PON1 activity is inversely related to plasma malondialdehyde concentration, a marker of oxidative stress. The PON1 active site for lipid peroxide hydrolysis requires a free sulfhydryl group at cysteine 284, and lipid peroxides react covalently with this site leading to enzyme inactivation [18]. Hence, the net result of increased oxidative stress would be decreased PON1 activity. A further decrease in PON1 activity is observed in alcoholic patients with liver impairment. A decreased synthesis of PON1 protein can be discarded as an explanation for this observation since serum PON1 concentrations are not low but are, on the contrary, increased in patients with liver disease. An alternative explanation is that alterations in HDL structure and metabolism may affect PON1 activity [19]. Patients with liver disease often exhibit impaired synthesis of several enzymes regulating HDL synthesis [20]. As a consequence, cholesterol ester synthesis is impaired and HDL particles show considerable changes in shape and structure [21,22]. HDL particles from patients with liver disease have relative increases in the content of free cholesterol, phospholipids, and also a decreased content of esterified cholesterol and apolipoprotein A–I. The resulting

abnormal structure and content of the HDL molecule could affect serum PON1 activity. For example, Deakin et al. [23] showed that the presence of apolipoprotein A–I in HDL was necessary to maintain the optimum PON1 activity and to stabilize the enzyme and that the addition of free cholesterol to HDL lowered PON1 activity in vitro. It has been shown, as well, that phospholipids deplete the HDL molecule of its PON1 content [24]. It is of note that there were weak, but statistically significant, direct associations between PON1 activity, apolipoprotein A–I, and HDL cholesterol concentrations in our patients. Patient’s treatment may also influence PON1 activity in that some antibiotics are known to inhibit PON1 activity in vitro [25]. Of considerable interest was the observation that PON1 concentrations were increased in the patient groups despite the enzyme’s activity being decreased. In a previous study in patients with liver disease, mostly secondary to hepatitis C, we had observed an increased serum PON1 concentration which was directly correlated with an enhanced PON1 expression in liver biopsies as well as with serological markers of fibrogenesis [26]. Since PON1 and collagen share nuclear transcription factors such as Sp1 and the sterol regulatory element protein and since both genes are linked on chromosome 7q [27–32], it is possible that chronic stimuli that enhance collagen expression would also induce elevated expression of PON1 as well. The consequence of these alterations would be that circulating PON1 becomes increasingly inactive in parallel with liver impairment. This is illustrated by the decrease in PON1 specific activity that is much lower in cirrhotic patients than in the healthy subjects, and is also lower than the values previously reported for ostensibly normal subjects or for patients with cardiovascular disease [33,34]. Non-invasive approaches for the diagnosis of alcohol-related liver disease include clinical evaluation, laboratory testing and radiological imaging. However, in most cases biopsy material from the liver is still necessary to confirm the diagnosis and to establish the degree of liver damage. It is for this reason that one of the objectives of the present study was to ascertain whether the measurement of PON1 (concentration and/or activity) adds a significant predictive value to that obtained from the standard battery of biochemical tests currently available for the assessment of the extent of liver damage in alcoholic patients. Our results showed that a combination of five basic biochemical Table 2 Serum PON1 activity and concentration in alcoholic and non-alcoholic cirrhotic patients segregated according to Child–Pugh classification

PON1 activity (U L− 1) PON1 concentration (mg L− 1) PON1 specific activity (U mg− 1)

Class A (n = 86)

Class B (n = 46)

Class C (n = 12)

108.3 (44.4–278.4) 158.8 (45.1–647.5) 0.63 (0.13–3.40)

111.8 (19.6–277.7) 244.1 (47.7–1206.8) 0.50 (0.04–4.01)

81.1 (10.1–208.9)a 891.2 (23.2–3225.0)a,b 0.09 (0.01–4.65)a,b

All values are presented as medians (95% CI). : P < 0.05 with respect to Child–Pugh class B; b: P < 0.01, with respect to Child–Pugh class A.

a

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Table 3 AUC of ROC plots for serum PON1 and the standard liver function tests in alcoholic patients and the control group Parameter

AUC (95% CI)

Albumin Alanine aminotransferase γ-Glutamyltransferase Bilirubin Prothrombin time PON1 activity PON1 concentration PON1 specific activity

NL vs. CG

MC vs. CG

LC vs. CG

(MC + LC) vs. NL

(NL + MC) vs. LC

0.71 (0.64–0.77) 0.88 (0.84–0.92) 0.84 (0.80–0.88) 0.67 (0.61–0.73) 0.54 (0.49–0.59) 0.90 (0.86–0.93) 0.62 (0.56–0.68) 0.84 (0.79–0.88)

0.67 (0.60–0.74) 0.75 (0.69–0.81) 0.99 (0.99–1.00) 0.51 (0.45–0.58) 0.56 (0.51–0.61) 0.90 (0.86–0.93) 0.65 (0.59–0.72) 0.87 (0.83–0.91)

0.75 (0.69–0.81) 0.55 (0.49–0.62) 0.91 (0.88–0.94) 0.73 (0.65–0.81) 0.91 (0.88–0.94) 0.98 (0.97–0.99) 0.78 (0.71–0.84) 0.96 (0.93–0.98)

0.57 (0.50–0.63) 0.37 (0.30–0.43) 0.88 (0.84–0.91) 0.73 (0.67–0.78) 0.72 (0.67–0.78) 0.63 (0.57–0.70) 0.64 (0.58–0.70) 0.68 (0.62–0.74)

0.65 (0.56–0.75) 0.57 (0.49–0.65) 0.55 (0.47–0.63) 0.76 (0.68–0.84) 0.95 (0.91–0.98) 0.76 (0.69–0.82) 0.73 (0.64–0.81) 0.79 (0.74–0.85)

CG: control group; LC: liver cirrhosis; MC: moderate changes; NL: alcoholics with normal liver.

tests + PON1 activity measurement would be most useful in discriminating between alcoholic patients and healthy subjects i.e. the AUC’s of ROC curves were ≥ 0.90 and the multiple regression analysis consistently showed that the inclusion of PON1 activity, but not concentration, to the standard battery increased the sensitivity up to 83.5–96.6% and the specificity up to 95.8–99.6%. An evident utility of serum PON1 activity measurement is the differentiation between control subjects and alcoholics with normal liver. This is, under some circumstances, an important clinical task for which several biochemical tests have been proposed with often controversial results [35,36]. Conversely, PON1 measurement did not make any significant contribution towards classifying subgroups of alcoholic patients based on the different stages of liver impairment. These observations indicate that PON1 measurement has limited clinical application in the evaluation of the degree of alcoholassociated liver impairment. A caveat on the present study is that PON1 activity was determined by measuring the hydrolysis of paraoxon. The hydrolysis of this substrate and other organophosphorous compounds is strongly influenced by PON1 genetic polymorphisms [37–39]. Hence, it may have been better to measure the PON1 arylesterase activity by using phenyl acetate which does not appear to be so influenced. However, this reaction is monitored at 270 nm, and this precludes its adaptation to most high-throughput automated analyzers and, consequently, its use as a routine measurement in clinical chemistry laboratories

would be limited. Further studies in large groups of patients with liver disease from different etiologies and measuring PON1 activity with substrates other than paraoxon are necessary to fully ascertain the utility of PON1 as a marker of liver impairment. In summary, our results indicated that: (1) serum PON1 concentration is significantly increased in alcoholic patients with liver disease, probably as a response to enhanced oxidative stress and/or collagen co-expression; (2) PON1 enzymatic activity is decreased, probably as a consequence of inactivation by lipid peroxides and/or to HDL structural alterations coexisting in alcoholic liver disease; (3) the measurement of PON1 status is useful in discriminating between alcoholic patients and healthy subjects, but does not add significant information for evaluating the extent of alcohol-induced liver damage. Acknowledgments This study was supported by grants from Fondo de Investigación Sanitaria (FIS 98/1082, 00/0232, 02/0430, 05/ 1607) and Redes de Centros y de Grupos from the Instituto de Salud Carlos III (C03/02, C03/08, G03/015). Natàlia Ferré is a researcher funded by the Juan de la Cierva programme of the Ministerio de Educación y Ciencia, Madrid, Spain. Judit Marsillach is the recipient of a grant from the Generalitat de Catalunya (FI 05/00068). The authors are indebted to Alberto

Table 4 Summary of the multiple logistic regression models for the standard liver function tests plus PON1 activity Group comparison

Equation

Sensitivity (%)

Specificity (%)

NL vs. CG

x = −0.260 (ALB) − 17.326 (ALT) + 2.989 (GGT) − 0.009 (BIL) − 0.006 (PT) + 12.156 x = −0.302 (ALB) − 13.937 (ALT) + 3.012 (GGT) − 0.022 (BIL) + 0.010 (PT) − 0.015 (PON1) + 17.538 x = −0.225 (ALB) − 2.772 (ALT) + 4.693 (GGT) + 0.061 (BIL) − 0.076 (PT) + 10.332 x = −0.106 (ALB) − 2.215 (ALT) + 5.325 (GGT) + 0.038 (BIL) − 0.094 (PT) − 0.015 (PON1) + 11.151 x = 0.009 (ALB) − 13.376 (ALT) + 2.574 (GGT) + 0.045 (BIL) − 0.130 (PT) + 10.698 x = −0.030 (ALB) − 10.539 (ALT) + 2.689 (GGT) + 0.001 (BIL) − 0.052 (PT) − 0.045 (PON1) + 15.594 x = 0.092 (ALB) + 1.043 (ALT) + 3.832 (GGT) + 0.109 (BIL) − 0.156 (PT) + 5.613 x = 0.100 (ALB) + 1.974 (ALT) + 3.931 (GGT) + 0.105 (BIL) − 0.152 (PT) − 0.004 (PON1) + 5.316 x = −0.010 (ALB) − 0.087 (ALT) − 0.051 (GGT) + 0.013 (BIL)−0.144 (PT) + 11.804 x = −0.011 (ALB) + 0.489 (ALT) − 0.064 (GGT) + 0.007 (BIL) − 0.140 (PT) − 0.008 (PON1) + 11.890

75.7 83.5 94.2 97.1 84.7 96.6 91.4 91.3 71.2 71.2

92.8 95.8 98.1 99.2 99.2 99.6 90.3 90.3 95.1 96.1

MC vs. CG LC vs. CG (MC + LC) vs. NL (NL + MC) vs. LC

ALB: albumin; ALT: alanine aminotransferase; BIL: bilirubin; CG: control group; GGT: γ-glutamyltransferase; LC: liver cirrhosis; MC: moderate changes; NL: alcoholics with normal liver; PT: prothrombin time. P < 0.001 for all the models.

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