Alcohol and Alcoholism Advance Access published online on June 15, 2007
Alcohol and Alcoholism, doi:10.1093/alcalc/agm048
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The Author 2007. Published by Oxford University Press on behalf of the Medical Council on Alcohol.
Relationship between serum sialic acid and sialylated glycoproteins in alcoholics
1 Department of Biochemical Diagnostics, Medical University, Bialystok
2 Department of Detoxification, Psychiatric Hospital, Choroszcz, Poland
* Author to whom correspondence should be addressed at: Department of Biochemical Diagnostics, Medical University, Waszyngtona 15A, Bialystok, Poland. Fax: 048 85 7468 585 E-mail: chrostek{at}amb.edu.pl
Received 4 January 2007; first review notified 8 March 2007; in revised form 11 May 2007; accepted 14 May 2007
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ABSTRACT |
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Aims: Total sialic acid (TSA) has been suggested as a marker for chronic alcohol abuse. It seems that the elevation of TSA during excessive alcohol consumption reflects the changes in sialylated glycoproteins in the sera. On the other hand, chronic ethanol consumption increases the desialylation rate of many serum glycoproteins. The aim of this study was to evaluate the relationship between the total and free form of sialic acid levels (FSA), and the concentration of sialylated glycoproteins in alcoholics. Methods: We determined the serum concentration of many glycoproteins (
1-antitrypsin, 1-acid glycoprotein, haptoglobin, ceruloplasmin, transferrin, complement C3 protein, fibrinogen and immunoglobulin G) in a sample of 100 alcoholics and 30 healthy controls. Total sialic acid was determined by the enzymatic method and its free form by using a modification of the thiobarbituric acid method. Results Among alcoholics, we found increased concentrations of 1-antitrypsin and 1-acid glycoprotein but decreased levels of transferrin. The concentrations of TSA and FSA were significantly higher in alcoholics than in healthy controls. The tested glycoproteins, except for transferrin and immunoglobulin G, positively correlated with TSA and FSA. The correlations with TSA were higher than that with FSA. Conclusions Chronic alcohol abuse alters the concentrations of some sialylated glycoproteins in the sera. The 1-antitrypsin, 1-acid glycoprotein, and transferrin are the only affected glycoproteins. The serum level of total and free form of sialic acid in the sera of alcoholics depends on the concentration of the most sialylated glycoproteins. |
Introduction |
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Blood glycoproteins contain N-linked complex glycan chains that arrange themselves into bi-, tri- or tetraantenary structures (Montreuil, 1980
; Bezouska et al., 1985). In mammals, the sialic acid occurs as a terminal sugar of glycoprotein glycans (Schauer, 2004). Therefore, all plasma glycoproteins, at physiological blood pH, are ionized and have a negative charge. By splitting the terminal sialic acid residues, the glycoproteins catabolism in the blood is initiated. The proportion of sialic acid in human glycoproteins varies between 3 and 7% (Schauer et al., 1995). Most of these glycoproteins are the acute phase proteins.
It has been reported that chronic alcohol consumption stimulates the hepatic synthesis of acute phase proteins (Perier et al., 1983; Harada et al., 1985; Lippi et al., 1992). Their serum increase is independent from the existence of liver damage. Secretory proteins such as transferrin, fibrinogen and ceruloplasmin, occur among glycoproteins. Earlier study reported that in alcoholic liver injury, their glycosylation and secretion are impaired (Tuma and Sorrell, 1984; Matsuda et al., 1991). Based on the decreased levels of transferrin and ceruloplasmin during liver diseases, they are called the negative-proteins of acute phase reaction. The production of their desialylated isoforms is the result of the impaired glycosylation of serum glycoproteins (Tsutsumi et al., 1994; Henry et al., 1999). Desialylation of glycoproteins generates free sialic acid. As the form of sialic acid (FSA) is a minor part of the total, it has been speculated that it has no effect on the total concentration of sialic acid. Recent studies have shown that serum total sialic acid (TSA) concentration is increased in alcoholics but the mechanisms that generate this increase are still unknown (Sillanaukee et al., 1999a; Romppanen et al., 2002). In addition, the increase in serum TSA concentration has been reported in cancer, diabetes, cardiovascular and inflammatory diseases, but in a free form of sialic acid, especially in urine samples, in inherited disorders such as sialidosis, Salla's disease, infantile SA storage disease, and neuraminidase deficiency (Sillanaukee et al., 1999b).
Based on the information about impaired glycosylation of glycoproteins during chronic alcohol abuse, we expect that these abnormalities may influence the serum level of FSA. Taking into consideration the altered hepatic secretion of glycoproteins in alcoholics we suspect the change in serum concentration of these glycoproteins. If altered secretion of glycoproteins is related to the impaired glycosylation the relation between serum concentration of sialic acid (total and free form) and glycoproteins should be evident. Therefore, in the present study, the relationship between serum TSA, FSA levels, and the concentration of sialylated glycoproteins in the sera of alcoholics, was evaluated.
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Methods |
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Subjects
The tested group consisted of 100 male alcohol-dependent patients (mean age: 44 years; range: 24–72) who were admitted for treatment in the detoxification ward (Department of Detoxification, Psychiatric Hospital in Choroszcz). Male sex was selected because men were more frequently admitted to treatment wards than women. Patients were initially examined and interviewed regarding their use of alcohol and drugs, smoking and history of diseases. The self-reported mean alcohol consumption was 1302 g per week (range: 168–4676) and mean time of dependency was 18 years (range 2–45). The diagnosis of dependency was established according to ICD-10 criteria (World Health Organization, 1992
). None of the alcoholics had a history of liver disease or showed clinical signs of liver disease at the time of the interview, nor were they intoxicated during the examination. The exclusion criteria were the severe liver diseases such as acute hepatitis, cirrhosis and other severe disease such as cancer, diabetes, and inflammatory disorders that may affect the level of acute-phase proteins. The study was approved by the local bioethics committee. The patients gave their assent to participate in the experiment. Blood alcohol concentration (BAC) had not been measured on admission because when we compared the results of FSA and TSA and tested glycoproteins between alcoholics drinking just before admission and patients with abstinence lasting at least 2 days, there was a lack of significant differences. Therefore, it may be speculated that acute alcohol consumption by alcohol dependent men does not influence levels of FSA, TSA and glycoproteins. The control group consisted of 30 healthy social drinkers (males; mean age: 40 years; range: 21–59) who self-reported that their consumption of ethanol was less than 30 g/day on any occasion.
Blood samples were taken from all patients by vein puncture once in the morning after admittance. The sera was separated by centrifugation at 1500x g for 10 min at room temperature. All samples were stored at –86 ° C until analysis. Besides serum, a portion of each blood sample was collected into tubes containing anticoagulants (sodium citrate) for measurement of fibrinogen.
Biochemical methods
TSA was measured with a colorimetric, an enzymatic method, adapted for use on the Konelab 60i analyzer (Thermo Clinical Labsystems, Finland) with commercial kits (Sialic Acid Test-Combination, Roche Diagnostics GmbH, Mannheim, Germany) (normal range: 58–79 mg/dl; mean: 69 mg/dl; intra- and inter-assay imprecision –%CV = 1.0% and 1.57% at 69.1 mg/dl, N = 20).
FSA was determined according to the modification of the method of Skoza and Mohos (1976). It was performed as follows. The sample (100 µl of serum) was treated with 250 µl of periodate reagent (0.025 N periodic acid in 0.125 N sulphuric acid) (Sigma-Aldrich, Chemie GmbH, Steinheim, Germany), mixed, and the tubes were incubated at 37 ° C for 30 min. The reaction was terminated through the addition of 200µl of sodium arsenite (2% sodium arsenite in 0.22 M hydrochloric acid) (Sigma-Aldrich, Chemie GmbH, Steinheim, Germany). Next, 1.5 ml of thiobarbituric acid (0.1 M, pH 9.0) (Sigma-Aldrich, Chemie GmbH, Steinheim, Germany) was added and the tubes were heated in boiling water for 7.5 min and then cooled in ice water. Finally, dimethyl sulphoxide (1.5 ml) (Sigma-Aldrich, Chemie GmbH, Steinheim, Germany) was added and the solution was measured at 549 nm using quartz cuvettes on the spectrophotometer (Shimadzu UV-1202, Shimadzu Europa GmbH, Duisburg, Germany). The amount of FSA was calculated using the following formula:
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). The intra- and inter-assay imprecision –%CV = 2.12 and 6.28% at 4.36 mg/dl, N = 20. CDT, as the percentage of transferrin (% CDT), was assayed by immunoturbidimetric method (Tina-quant % CDT 2nd generation test, Roche Diagnostics GmbH Mannheim, Norway) on the Hitachi 912 analyser (Roche, USA) after a separation with the use of anion-exchange chromatography (95th percentile for the social drinking control population is below 2.6%; intra- and interassay imprecision % CV = 3.1 at 2.8% and 4.5 at 2.9%, N = 21).
The tested glycoproteins represented different serum fractions of globulin (1-antitrypsin and 1-acid glycoprotein—1-globulin; haptoglobin and ceruloplasmin—2-globulin; transferrin—ß1-globulin; complement C3 protein—ß2-globulin; immunoglobulin G—
-globulin and fibrinogen—situated between ß2 and -globulin). Haptoglobin (normal range: 0.3–2.0 g/l, intra- and interassay imprecision % CV = 1.7 and 4.1 at 1,17 g/l; N = 20), 1-antitrypsin (normal range: 0.9–2.0 g/l, intra- and interassay imprecision % CV = 2.1 and 2.5 at 1.46 g/l; N = 20), 1-acid glycoprotein (normal range: 0.5–1.2 g/l, intra- and interassay imprecision % CV = 2.15 and 2.5 at 0.67 g/l; N = 20), transferrin (normal range: 2.0–3.6 g/l, intra- and interassay imprecision % CV = 1.75 and 5.25 at 2.33 g/l; N = 20) were determined by methods based on the measurement of immunoprecipitation enhanced by polyethylene glycol (PEG) at 340 nm on the Konelab 60i analyser with commercial kits (Thermo Clinical Labsystems, Finland). Complement C3 protein (normal range: 0.82–1.85 g/l, intra- and interassay imprecision % CV = 1.6 and 1.8 at 1.36 g/l; N = 20), ceruloplasmin (normal range: 0.20–0.60 g/l, intra- and interassay imprecision % CV = 2.2 at 0.30 g/l and 3.0 at 0.50 g/l; N = 20) and immunoglobulin G (normal range: 5.40–18.22 g/l, intra- and interassay imprecision % CV = 1.7 and 0.8 at 18.60 g/l; N = 20) were determined by an immunoturbidimetric procedures that measure increasing sample turbidity caused by the formation of insoluble immune complexes when antibodies are added to the sample. Total serum protein concentration was estimated by the biuret method on the Architect ci8200 analyser (Abbott, USA) (normal range: 60–80 g/l, intra- and interassay imprecision % CV = 0.55 and 0.7 at 53 g/l; N = 20). Serum GGT (normal range: 10–75 U/I, intra- and interassay imprecision % CV = 2.9 and 4.7 at 74,3 U/I; N = 20) was determined by using routine method (with bioMerieux reagents, France) on the Konelab 60i analyser. The quantitative determination of fibrinogen (normal range: 2.0–4.0 g/l, intra- and interassay imprecision % CV = 2.8 at 2.15 g/l, N = 21, and 2.9 at 2.05 g/l, N = 10) was carried out in citrated plasma with a modified clot-rate assay using the STA Fibrinogen kit (Roche, Germany) and Diagnostica STAGO ST4 analyser (Roche, Germany).
Statistical analysis
Results were expressed as means and standard deviations (SD). Shapiro-Wilk test confirmed that the variables were not normally distributed (P < 0.001). The differences between tested and control group were estimated by Mann-Whitney U-test (P < 0.05). Spearman rank correlation test was used to calculate correlations between FSA, TSA and sialylated glycoprotein inside of the tested group. The differences were considered statistically significant at P < 0.05. Diagnostic sensitivity and specificity were calculated using the value of 95th percentile from the control group as a cut-off. For TSA it was 77.62 mg/dl, 6.02 mg/dl for FSA and 2.47% for CDT. The GraphROC for Windows was used to calculate the diagnostic accuracy (area under ROC curve).
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Results |
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Mean serum FSA (5.95 ± 0.98 mg/dl) and TSA (85.03 ± 20.89 mg/dl) concentrations in the alcoholic group were significantly increased as compared to the control group (4.81 ± 0.65 mg/dl; 64.83 ± 6.63 mg/dl, respectively) (Fig. 1). The level of FSA was elevated by about 24% and that of TSA by about 31%. The ratio of FSA/TSA in alcoholics (7.17 ± 1.02%) was not significantly different when compared to that of the control group (7.51 ± 0.87%).
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In alcoholics, the serum concentrations of
1-antitrypsin (AAT), 1-acid glycoprotein (AGP) and total protein were found to be significantly increased, but the concentration of transferrin (TRF) was significantly lower than that of the control group (Table 1). The levels of haptoglobin, ceruloplasmin, complement C3 protein, fibrinogen, and immunoglobulin G did not differ between chronic alcohol abusers and the controls. CDT level was elevated in alcohol dependent men, which is typical in such cases.
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The correlation study showed the significant positive relationship between FSA, TSA and
1-antithrombin, 1-acid glycoprotein, haptoglobin, ceruloplasmin, complement C3 protein and fibrinogen (Table 2). FSA and TSA also correlated positively with total protein. In all cases, the correlation with TSA was higher than that with free form of sialic acid. FSA and TSA did not correlate with IgG and transferrin. Only FSA correlated negatively with CDT. There was also good correlation between FSA and TSA levels (r = 0.720, P < 0.001) (Fig. 2).
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Diagnostic sensitivity, specificity and area under ROC curve for TSA were 55.6%, 93.3% and 0.856, and for FSA 40.0%, 96.4% and 0.854, and for CDT 92.2%, 93.3% and 0.968, respectively.
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Discussion |
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The effects of ethanol administration on levels of the proteins synthesized in the liver are well known phenomenon. Acute ethanol consumption has been shown to inhibit protein secretion by the liver, resulting in their hepatocellular retention (Volentine et al., 1984
; Volentine et al., 1986; Tuma et al., 1986). The reason of this ethanol effect is the impaired movement of the secretory proteins along the secretory pathway (Tuma and Sorrell, 1984; Volentine et al., 1986). Others reported that both acute and chronic ethanol administration inhibited the secretion of glycoproteins from the liver (Tuma and Sorrell, 1984). Chronic ethanol feeding of rats also decreases the synthesis of glycoproteins in the liver and this effect is further potentiated by acute ethanol administration (Sorrell et al., 1983). The maturation and secretion of the hepatic secretory glycoproteins is dependent on the glycosylation of these in the Golgi apparatus (Kawahara et al., 1993). On the other hand, chronic ethanol consumption has been shown to stimulate the hepatic synthesis of acute phase proteins (Perier et al., 1983; Harada et al., 1985; Lippi et al., 1992). In our study, most of the tested glycoproteins are the acute-phase proteins. Among them, we found altered concentrations of 1-antitrypsin, 1-acid glycoprotein (the representatives of 1-globulins) and transferrin (the representative of ß1-globulins). The levels of 1-antitrypsin and 1-acid glycoprotein are elevated in alcoholics. They react as positive acute-phase proteins. In contrast, the level of transferrin is decreased, because it is a negative acute-phase protein. The concentrations of other tested glycoproteins do not differ between alcoholics and controls. It can be explained by the interference of acute alcohol drinking (inhibition of protein synthesis and secretion) with the effect of chronic ethanol consumption (stimulation of proteins synthesis). Most of the patients participating in our study drank alcohol just before sampling (94% in the last week). Another explanation of these results is the dependence of serum glycoproteins level on the stage of hepatocellular deficiency. It was documented that serum acute-phase proteins are increased in moderate hepatocellular deficiency (Perier et al., 1983). In contrast, in severe hepatocellular deficiency, the acute-phase proteins are decreased. The 1- acid glycoprotein is the most sensitive protein. In our study, not all acute-phase sialylated proteins are sensitive to chronic alcohol drinking, e.g. the representatives of 2-, ß2-, -globulins and fibrinogen.
The appearance of carbohydrate-deficient isoforms of glycoproteins in the sera of alcoholics has been proved as a marker of chronic alcohol consumption (Wang et al., 1993; Tsutsumi and Takase, 2001). The presence of these isoforms is the effect of impaired glycosylation/sialylation of proteins in the liver by chronic ethanol drinking. A decreased activity of cellular glycosyltransferases (i.e. mannosyltransferase, galactosyltransferase, N-acetyl-glucosaminyltransferase, sialyltransferase) and increased serum activity of sialidase have been proposed as two mechanisms responsible for the increase of desialylated isoforms of glycoproteins in the sera of alcoholics (Stibler and Borg, 1991; Xin et al., 1995). In turn, the result of sialic acid releasing from oligosaccharide chains should be its increased level in the serum. This hypothesis is confirmed in our study. Exactly, we show the elevated serum concentrations of FSA in the sera of alcoholics. The diagnostic accuracy was high and reached the value of 0.854, although the sensitivity was low (40.0%). We cannot compare our results with others because FSA had not been previously investigated. There are only a few reports concerning the effect of chronic alcohol abuse on the serum TSA level (Sillanaukee et al., 1999a; Ponnio et al., 1999; Romppanen et al., 2002). According to these papers, chronic alcohol drinking increases the concentration of serum TSA. The effect of acute alcohol drinking on the TSA levels was not previously studied. In our study, the diagnostic accuracy for TSA was also high (0.856) but sensitivity rather low (55.6%). Because the sialic acid constitutes only a minor part of total serum glycoproteins, its increase cannot influence the TSA concentration in the serum. However, the level of FSA should correspond to the concentration of sialylated glycoproteins and to the total level of sialic acid. In our study, the concentration of FSA correlated positively with TSA, which in turn significantly correlated with the levels of tested glycoproteins. In our opinion, the correlation of serum glycoproteins concentration with TSA is better than that with free form, because the changes in TSA concentration are greater than in FSA. The increased levels of FSA and TSA positively correlated with tested sialylated glycoproteins except for transferrin and immunoglobulin G. It means that all glycoproteins concentrations have an influence on the level of total and free form of sialic acid in the sera of alcoholics. Immunoglobulin G is produced by B lymphocytes and therefore its metabolism is not connected with the protein synthesis and glycosylation in the liver. An interesting result of this study is the lack of correlation between FSA, TSA and transferrin. This shows that the alteration in transferrin concentration (decrease) does not affect the level of TSA and FSA (increased levels). We also found that CDT concentration significantly correlated with the level of FSA. Unexpectedly, this correlation was negative and in contrast to that between FSA and other glycoproteins. It means that the decreased sialylation of transferrin (reflected by increased CDT) depresses the levels of FSA. The unchanged value of the ratio of FSA: TSA shows that the disturbances in glycosylation/sialylation of glycoproteins and synthesis of these acute-phase proteins are parallel. Another explanation for the unchanged ratio of FSA: TSA could be the same biological half-lives for FSA and TSA, but at the present time, these are still unknown.
In conclusion, chronic alcohol abuse alters the concentration of some sialylated glycoproteins. The 1-antitrypsin, 1-acid glycoprotein, and transferrin occur as the affected glycoproteins. The serum level of total and free form of sialic acid in the sera of alcoholics depends on the sialylated glycoproteins concentration except for immunoglobulin G and transferrin.
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