Most of the serum proteins are glycoproteins, in which glycans are terminated with sialic acid residues [
The aim of this study was to assess the changes in the sialylation of serum glycoproteins by measuring total (TSA) and free sialic acid (FSA) concentration in the sera of patients suffering from acute and chronic liver diseases.
The tested group consisted of 278 patients (99 females and 179 males) who were admitted to the Department of Infectious Diseases and Hepatology of University Hospital of Bialystok. The patients were divided into subgroups according to the diagnosis of liver diseases: 54 had alcoholic cirrhosis (AC), 34 nonalcoholic cirrhosis (NAC), 23 chronic nonviral hepatitis (CH), 32 toxic hepatitis (TH), 20 chronic viral hepatitis C (HCV), 17 chronic viral hepatitis B (HBV), 14 autoimmune hepatitis (AIH), 15 acute hepatitis B (AHB), 16 primary biliary cirrhosis (PBC), 14 fatty liver (FL), 24 primary liver cancer (HCC), and 15 primary liver cancer and cirrhosis (HCC + C). The diagnosis was performed on the basis of signs and symptoms of the disease, physical and clinical exam (ultrasonography and fine-needle biopsy in justified cases), and biochemical liver panel known as liver function tests (AST, ALT, and GGT). The diagnosis of viral hepatitis was supported by serological tests (HBsAg and anti-HCV). The causes of nonalcoholic liver cirrhosis were as follows: HBV-14, HCV-9, and unidentified factors-12. The toxic hepatitis was caused by alcohol abuse in 22 cases and drugs abuse in 10 cases. To confirm the diagnosis of primary biliary cirrhosis we performed the mitochondrial antibody test (AMA).
The control group consisted of 50 healthy subjects (18 females and 32 males) recruited from hospital workers. All subjects (healthy and sick) gave their consent to participate in the studies. The study was approved by the Bioethical Committee of the Medical University of Bialystok.
Blood samples were taken by peripheral vein puncture once after admission and before treatment. The sera were separated by centrifugation at 1500
TSA concentration in the serum was measured on the Microplate Fluorescence Reader FL600 (Bio-Tek, USA) according to the enzymatic method (EnzyChrom Sialic Acid Assay Kit, BioAssay System, Hayward, USA) using the colorimetric procedure. Each determination in a single sample was performed three times. The samples should be pretreated in hydrolysis procedure, in which neuraminidase released the N-acetylneuraminic acid (NANA) from glycolinkages. In the next step the NANA is decomposed into N-acetylmannosamine and pyruvic acid in the presence of aldolase. Then the pyruvate is oxidized by pyruvate oxidase to acetyl phosphate, carbon dioxide, and hydrogen peroxide. In the last step, peroxidases and hydrogen peroxide convert 4-aminoantipyrine and N-ethyl-N-2-hydroxyethyl-3-toluidine to red coloured derivative. Briefly, to 20
FSA was determined using the thiobarbituric method of Skoza and Mohos [
For characteristics of patients the following tests were performed: alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT), carbohydrate-deficient transferrin (CDT), prothrombin time (PT), and mean corpuscular volume (MCV). Almost all of biochemical tests (ALT, AST, and GGT) were done on the Architect c8000 system (Abbott Laboratories, Abbott Park, IL, USA) using the kits from Abbott Diagnostics (Wiesbaden, Germany). The CDT values were assayed by immunonephelometry using N-Latex CDT test (Siemens Healthcare Diagnostics, Marburg, Germany) on BN II System (Siemens Healthcare Diagnostics, USA). MCV was measured using a hematological analyzer ADVIA 120 (Bayer, Tarrytown, USA) and prothrombin time on the STA Compact Hemostasis Analyzer (Diagnostica Stago, France).
Significance of differences between groups (tested and control) was evaluated by Mann-Whitney
Table
The laboratory characteristics of patients with liver diseases and controls.
MCV |
PT |
ALT |
AST |
GGT |
CDT | |
---|---|---|---|---|---|---|
C | 88 |
12.45 |
16.5 |
23 |
21.5 |
43.3 |
|
||||||
FL | 89.3 |
10.85* |
75.5* |
43* |
63* |
— |
|
||||||
AC | 98.2* |
15* |
32* |
82* |
206* |
37.9 |
|
||||||
NAC | 92* |
14.45* |
29.5* |
46 |
58* |
38.75 |
|
||||||
PBC | 94* |
14.2 |
31* |
73* |
142* |
43.8 |
|
||||||
TH | 99.1* |
14.7 |
50* |
60* |
216* |
44.3 |
|
||||||
CH | 89.6 |
12 |
72* |
52* |
44* |
41.9 |
|
||||||
HCV | 89.5* |
12.2 |
60* |
48* |
75* |
48.7 |
|
||||||
HBV | 94.8* |
13.1 |
62* |
52* |
53* |
37.2 |
|
||||||
AIH | 92* |
13.2 |
141* |
160* |
232* |
50.5 |
|
||||||
HCC | 90.9 |
12.65 |
43* |
84* |
182* |
39.7 |
|
||||||
HCC + C | 96.8* |
17.6* |
20.5 |
79* |
141* |
46 |
|
||||||
AHB | 86.6 |
13.8 |
831.5* |
680.5* |
231* |
— |
Data are median and ranges. MCV: mean corpuscular volume, PT: prothrombin time, ALT: alanine aminotransferase, AST: aspartate aminotransferase, GGT: gamma glutamyltransferase, CDT: carbohydrate-deficient transferrin, C: controls, FL: fatty liver, AC: alcoholic cirrhosis, NAC: nonalcoholic cirrhosis, PBC: primary biliary cirrhosis, TH: toxic hepatitis, CH: chronic nonviral hepatitis, HCV: chronic viral hepatitis C, HBV: chronic viral hepatitis B, AIH: autoimmune hepatitis, HCC: primary liver cancer, HCC + C: primary liver cancer and cirrhosis, and AHB: acute hepatitis B.
*Significant differences in comparison to the control group.
There were no significant differences in the serum TSA concentration between liver diseases of different etiologies (
TSA (a) and FSA (b) concentrations in the sera of patients with liver diseases of different etiology. Results are presented as median and range.
The mean serum concentration of FSA appears to be different between liver diseases of different etiologies (
In our study we have measured the serum concentrations of TSA and FSA in liver diseases. Generally, we detected decreased levels of TSA in hepatitis of different etiology, cirrhosis, and liver cancer. Our results are similar to the results of Matsuzaki and coworkers which indicated that the serum level of TSA in patients with compensated cirrhosis was significantly lower than that in the control group and it was decreased further in those with decompensated cirrhosis [
Interestingly, we did not observe the changes in TSA concentrations in patients with alcoholic cirrhosis. In our opinion, this fact may be the result of two opposing mechanisms. At first, the cirrhosis (nonalcoholic) causes the decrease of TSA concentration; secondly, alcohol abuse causes the increase of TSA level [
Similarly to the patients with nonalcoholic cirrhosis the total sialic concentration was also significantly decreased in patients suffering from liver cancer accompanied by cirrhosis in comparison to the control group. However, in patients with liver cancers without cirrhosis the TSA concentrations were slightly decreased, near normal. This can be explained by intensity of glycosylation disturbances during malignant diseases. There are many reports certifying the changes of sialic acid concentration in the course of malignant transformation [
In our study we have also shown that FSA concentrations were significantly higher in toxic hepatitis than in nonalcoholic cirrhosis. This difference can be explained by the pathogenesis of toxic hepatitis due to excessive consumption of alcohol in about 70% of patients. We suggest that differences in FSA concentration between these diseases are the result of aberrant glycosylation in alcohol abusers. Additional confirmation of this hypothesis is the comparisons of FSA concentrations in patients with alcoholic and nonalcoholic cirrhosis. The concentration of FSA in alcoholic cirrhosis was significantly higher than in controls while in nonalcoholic cirrhosis the FSA level was the same as in the control group. Therefore, in the current literature there are reports showing the aberrations of glycosylations in liver diseases, but the exact mechanisms of these changes in each liver disease are not known or they are not clearly explained. In our study we tried to explain these mechanisms by the measurements of total and free serum sialic acid concentrations. Also, in the present literature there is no information about behavior of the FSA concentrations in nonalcoholic liver disease or whether there are differences in relation to alcoholic liver disease. We suggest that the FSA serum concentration can be useful in the differential diagnosis of liver diseases, especially between toxic hepatitis and nonalcoholic cirrhosis. The causes of the changes in FSA concentration may be the alterations in glycosylation of glycoproteins in the liver diseases. These can rely on the increased desialylation, fucosylation, and branching and increased amounts of bisecting N-acetylglucosamine (GlcNAc) [
In our previous study, we have described TSA and FSA in alcoholic and nonalcoholic cirrhosis and chronic viral hepatitis [
We suggest that the changes in the concentration of TSA and FSA in some liver diseases confirm the presence of significant aberrations in the sialylation of serum glycoproteins in these diseases.
The authors declare that there is no conflict of interests regarding the publication of this paper.