General clinical data in the patient groups and the controls
Data were obtained from 80 (41 men, 39 women) participants. General characteristics of the three hepatic disease groups and the controls are summarized in Table 1. In terms of liver function, AST and ALT levels were elevated significantly in the patients groups compared with the control group (all P < 0.001). Similarly, GGT, TBA and CG levels were significantly increased in the hepatic disease groups (GGT index: P = 0.033 in CHB, P < 0.001 in LC and HCC; TBA index: P = 0.001 in CHB, P < 0.001 in LC and HCC; CG index: P < 0.001 for all). In addition, among three hepatic disease groups, GGT in HCC group was significantly higher than that in CHB group (P = 0.009), but there was no significant difference between HCC and LC groups as well as between LC and CHB (P > 0.05 for both); TBA in LC group was significantly higher than that in CHB and HCC groups (P = 0.017 and P = 0.001, respectively); CG in LC group was significantly increased than that in CHB and HCC groups (P = 0.001 and P = 0.037, respectively). Although the levels of DBIL, ALP, and GLU in LC and HCC group were significantly higher than those in control group and CHB group (P < 0.05 for all), no difference existed between CHB group and the controls (P > 0.05 for all). Except that TBIL in LC group and HCC group was significantly higher than that in control group (P < 0.001 and P = 0.001, respectively), it was found that LC group was higher than CHB group (P = 0.01). AFU in CHB and HCC group was higher than that in control group (P = 0.042 and P = 0.005, respectively), but there was no difference between LC group and control group (P > 0.05) as well as among hepatic disease groups (P > 0.05 for all). Interestingly, ALB, CHE and RBP in LC group and HCC group were significantly lower than those in CHB group and control group (P < 0.05 for all), while no significant difference existed between CHB group and control group (P > 0.05 for all). In addition, no difference in BUN and CRE levels (renal function index) were found between the hepatic disease patients and the controls. Although WBC in LC group was significantly decreased than that in CHB, HCC and control group (P < 0.01 for all groups), no differences were found among CHB, HCC and the controls. RBC in LC and HCC groups were lower than that in CHB and the controls (P < 0.05 for all groups), no differences were found between CHB and the controls as well as LC and HCC groups (P > 0.05 for both). Although Hb levels in CHB group was significantly increased than that in LC and HCC groups (P = 0.005 and P = 0.003, respectively), no differences were found among LC, HCC and the controls. PLT in LC group was significantly lower than that in HCC, CHB and control group (P < 0.001 for all groups), while PLT in HCC group was lower than that in CHB and control group (P < 0.001 for both), but there was no difference between CHB and control group. PT in LC and HCC groups were higher than that in CHB and control group (P < 0.001 for all), while there was no difference between LC and HCC as well as CHB and control group. APTT in patients groups were higher than in the control (P = 0.028 for CHB, P = 0.002 for LC, P = 0.007 for HCC, respectively). In addition, the examination of tumor markers showed that there were no significant differences in CEA among the patients groups and the controls. In particular, CA199 and CA125 in the patients groups were higher than those in the control group (P < 0.01 for all), except that there was no difference in CA199 among the patients groups, while CA125 in the LC and HCC groups was higher than that in the CHB group (P = 0.002 and P = 0.009, respectively).
Table 1
Clinical and laboratory data in the hepatitis B virus associated liver disease groups and the controls.
|
CHB
|
LC
|
HCC
|
Control
|
n (M/F)
|
20(11/9)
|
20(10/10)
|
20(12/8)
|
20(8/12)
|
Age (years)
|
43.55 ± 10.34f
|
48.90 ± 12.57
|
54.90 ± 9.31a
|
47.15 ± 10.99
|
ALB (g/L)
|
44.27 ± 5.09df
|
36.21 ± 7.19 b
|
35.70 ± 6.12b
|
42.39 ± 3.99
|
TBIL (umol/L)
|
14.05 (11.17–17.89)c
|
22.50 (15.24-39.00)b
|
18.09 (14.45–20.85)b
|
11.87 (9.15–14.01)
|
DBIL (umol/L)
|
2.78 (1.93–3.75)de
|
5.22 (3.92–13.78)b
|
3.66 (2.91–5.61)b
|
2.18 (1.36–2.67)
|
AST (U/L)
|
39.39 (30.52–74.93)b
|
55.36 (39.59–76.92)b
|
48.81 (36.55–84.68)b
|
18.95 (17.76–21.55)
|
ALT (U/L)
|
48.87 (24.29-113.19)b
|
42.44 (25.89–57.13)b
|
41.58 (31.94–62.19)b
|
16.86 (11.29–24.34)
|
ALP (U/L)
|
90.13 (73.43–99.26)ce
|
114.87 (89.01-157.54)b
|
109.66 (81.71-175.17)b
|
70.82 (58.99–83.40)
|
GGT (U/L)
|
29.16 (17.24–61.90)af
|
43.86 (28.24–96.01)b
|
67.98 (34.40-136.46)b
|
19.77 (13.96–28.09)
|
TBA (umol/L)
|
7.77 (5.16–22.34)bc
|
26.62 (18.74–38.33)bf
|
5.80 (3.45–12.65)b
|
2.96 (1.93–2.96)
|
CHE (U/L)
|
8601.27 (7135.69-10563.73)df
|
5159.38 (4064.57-5502.13)b
|
5198.48 (5168.74-5198.48)b
|
8969.05 (8890.09-8969.05)
|
AFU (U/L)
|
28.37 (19.49–31.85)a
|
29.30 (22.36–39.67)
|
30.45 (23.25–33.30)b
|
22.75 (21.69–24.04)
|
CG (ug/ml)
|
4.70 (1.69–5.71)bd
|
13.11 (5.93–16.40)be
|
5.66 (2.25–10.70)b
|
1.27 (1.16–1.27)
|
GLU (mmol/L)
|
5.14 (4.66–5.39)ce
|
6.61 (5.03–7.54)a
|
5.99 (5.51–6.83)a
|
5.36 (4.68–5.51)
|
BUN (mmol/L)
|
4.71 (3.81–5.44)
|
5.11 (4.23–6.59)
|
5.10 (3.03–6.79)
|
4.96 (3.35–5.54)
|
CRE (mmol/L)
|
66.60 (59.13–71.97)
|
64.14 (56.54–69.65)
|
65.62 (55.02–77.44)
|
61.83 (54.28–67.41)
|
RBP (mg/L)
|
35.13 (21.34–45.33)ce
|
26.59 (20.49–34.77)a
|
26.60 (23.16–26.60)b
|
39.70 (35.64–41.61)
|
WBC (× 109/L)
|
5.89 ± 1.32d
|
3.59 ± 1.61bf
|
5.53 ± 2.04
|
6.13 ± 2.03
|
RBC (× 1012/L)
|
4.60 ± 0.56df
|
3.85 ± 0.93a
|
3.81 ± 0.74b
|
4.43 ± 0.50
|
Hb (g/L)
|
142.05 ± 17.60df
|
121.55 ± 29.24
|
120.05 ± 23.17
|
129.60 ± 18.47
|
PLT (× 109/L)
|
220.25 ± 55.00df
|
89.35 ± 55.87bf
|
158.35 ± 60.28b
|
222.60 ± 42.62
|
PT (sec)
|
11.55 (11.17–11.90)df
|
12.80 (12.25–15.45)b
|
13.25 (12.28–14.45)b
|
11.50 (11.33–11.93)
|
APTT (sec)
|
27.25 (25.90-27.88)a
|
28.50 (26.50-30.62)b
|
28.05 (26.22–30.40)b
|
26.00 (25.53–26.25)
|
CEA (ng/mL)
|
1.40 (0.87–1.50)
|
1.61 (0.54–2.20)
|
1.75 (0.84–2.09)
|
1.24 (0.73–1.31)
|
CA199 (U/mL)
|
16.84 (9.02–28.19)b
|
23.23 (15.73–31.61)b
|
17.03 (10.95–22.33)b
|
6.93 (4.38–7.18)
|
CA125 (U/mL)
|
15.06 (14.37–15.06)bdf
|
40.01 (18.38–40.01)b
|
89.05 (12.78-154.59)b
|
9.10 (9.10–9.10)
|
Data are mean ± SD values or median (25th − 75th percentile) values as indicated. |
aP < 0.05, bP < 0.01 when patients groups (chronic hepatitis B [CHB], liver cirrhosis [LC], and hepatocellular carcinoma [HCC]) were compared with control subjects. |
cP < 0.05, dP < 0.01 when the CHB group was compared with the LC group. |
eP < 0.05, fP < 0.01 when the CHB and LC groups were compared with the HCC group. |
M/F, males/females; ALB, albumin; TBIL, total bilirubin; DBIL, direct bilirubin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; GGT, γ-glutamyl transferase; TBA, total bile acid; CHE, cholinesterase; AFU, alpha-L-fucosidase; CG, cholyglycine; GLU, glucose; BUN, urea nitrogen; CRE, creatinine; RBP, Retinol binding protein; WBC, white blood cell. RBC, red blood cell; Hb, hemoglobin; PLT, platelets; PT, prothrombin time; APTT, activated partial thromboplastin time; CEA, carcinoembryonic antigen; CA199, glucoprotein antigen 19 − 9; CA125, glucoprotein antigen 125. |
Levels of AFP and N-glycan in the patient groups and the controls
Serum AFP concentrations were higher in the hepatic disease groups than in the controls (CHB, P = 0.011; LC, P = 0.029; HCC, P < 0.001). A significant difference was found between the CHB and HCC groups (P = 0.024), as well as between the LC and HCC groups (P = 0.048), but no marked difference was found between the CHB and LC groups (P = 0.892) (Fig. 1a).
Serum N-glycan levels were elevated significantly in LC and HCC groups compared with the controls (P = 0.013 for LC and P < 0.001 for HCC). The N-glycan level in the HCC group were significantly higher than that in the CHB group (P < 0.001) and LC group (P = 0.005), but there was no difference between the CHB and control groups (P = 0.534), as well as between the CHB and LC groups (P = 0.055) (Fig. 1b).
The influencing factors on AFP and N-glycan expression in the hepatic disease patient groups and the controls
To analyze the association that existed between AFP and N-glycan in hepatic disease patients, the correlation between AFP and N-glycan was studied, as well as the associations among N-glycan / AFP with other parameters in the hepatic disease patients and the controls. To minimize the statistical error caused by small samples, we put all hepatic disease patients into one group. Spearman rank correlations were used to evaluate these associations in this study. The levels of N-glycan / AFP in the combined hepatic disease patients group (CHB, LC, and HCC) and the controls are shown in Figs. 2–8. In the combined hepatic disease patients groups, a significant positive correlation was found between N-glycan and AFP (R = 0.351, P = 0.006), as well as N-glycan and age (R = 0.273, P = 0.034), N-glycan and AST (R = 0.303, P = 0.019), N-glycan and GGT (R = 0.429, P = 0.001), N-glycan and PT (R = 0.378, P = 0.003), N-glycan and CA125 (R = 0.349, P = 0.006) (Fig. 2a-f). Meanwhile, negative correlation was found between N-glycan and ALB (R = -0.319, P = 0.013), as well as N-glycan and CHE (R = -0.459, P < 0.001), N-glycan and RBC (R = -0.288, P = 0.026) (Fig. 2g-i). In the CHB group, positive correlation existed between N-glycan and ALT (R = 0.513, P = 0.021), N-glycan and TBA (R = 0.547, P = 0.012), N-glycan and APTT (R = 0.450, P = 0.047), N-glycan and CRE (R = 0.455, P = 0.044), while negative correlations between N-glycan and BUN (R = -0.460, P = 0.041) was found (Fig. 4a-e). In the LC group, a positive correlation between N-glycan and GGT (R = 0.456, P = 0.043) was found (Fig. 6). Although positive correlation existed between N-glycan and AFU (R = 0.489, P = 0.029) and negative correlation was found between N-glycan and WBC (R = -0.589, P = 0.006), No relationship was found between N-glycan and AFP in the controls (R = 0.170, P = 0.407) (Fig. 8a-b).
In the combined hepatic disease patients groups, a significant positive correlation was found between AFP and HBS DNA (R = 0.315, P = 0.014), as well as AFP and TBIL (R = 0.257, P = 0.047), AFP and DBIL (R = 0.314, P = 0.014), AFP and AST (R = 0.482, P < 0.001), AFP and ALT (R = 0.465, P < 0.001), AFP and ALP (R = 0.327, P = 0.011), AFP and GGT (R = 0.471, P < 0.001), while a negative correlation existed between AFP and ALB (R = -0.295, P = 0.022), as well as AFP and CHE (R = -0.293, P = 0.023) (Fig. 3a-i). In the CHB group, positive correlation existed between AFP and AST (R = 0.579, P = 0.007) and between AFP and ALT (R = 0.496, P = 0.026), while negative correlations between AFP and CHE (R = -0.624, P = 0.003), AFP and BUN (R = -0.457, P = 0.043), AFP and GLU (R = -0.662, P = 0.001) were found (Fig. 5a-e). In the LC group, positive correlation between AFP and HBS DNA (R = 0.686, P = 0.001) was found as well as AFP and TBIL (R = 0.515, P = 0.020), AFP and DBIL (R = 0.487, P = 0.030), AFP and AST (R = 0.820, P < 0.001), AFP and ALT (R = 0.773, P < 0.001), AFP and GGT (R = 0.533, P = 0.016), AFP and AFU (R = 0.567, P = 0.009), AFP and RBC (R = 0.579, P = 0.007), AFP and Hb (R = 0.529, P = 0.017) (Fig. 7a-f). In addition, negative relationship were found between AFP and Age (R = -0.756, P < 0.001), AFP and BUN (R = -0.544, P = 0.013), AFP and CEA (R = -0.489, P = 0.029) in the LC group (Fig. 7g-l). In the control group, a negative correlation existed between AFP and DBIL (R = -0.477, P = 0.034) (Fig. 8c). In addition, no correlation was found between N-glycan / AFP level and those of the laboratory parameters above in the HCC group.
Effect of AFP and N-glycan expression in the diabetes patients and the controls
To study whether there is a dose–response relationship between AFP and N-glycan, the 60 hepatic disease patients were redivided into three groups according to AFP level (Group A, AFP levels < 10 ng/mL; Group B, AFP levels 10–200 ng/mL; and Group C, AFP levels > 200 ng/mL) (Table 2). AST, ALT, GGT and HBV DNA in Group A were lower than in Groups B and C (P < 0.05 for all), and no difference were found between Groups B and C (P > 0.05 for all). In addition, AFU level in group A was decreased than in Group C (P = 0.002), while no difference were found between group A and group B (P = 0.052), group B and group C (P = 0.207).
Table 2
Clinical and laboratory data in the hepatitis B virus associated liver disease patients with different levels of AFP.
|
AFP groups
|
|
Group A(< 10 ng/mL)
|
Group B(10–200 ng/mL)
|
Group C(> 200 ng/mL)
|
n (M/F)
|
36(18/18)
|
14(11/3)
|
10(4/6)
|
Age (years)
|
50.97 ± 11.84
|
47.29 ± 11.67
|
45.00 ± 10.30
|
ALB (g/L)
|
41.37 (35.74–46.02)a
|
37.98 (35.91–41.30)
|
32.58 (30.97–33.53)
|
TBIL (umol/L)
|
15.71 (12.52–20.32)
|
15.71 (12.52–20.32)
|
19.27 (16.66–32.63)
|
DBIL (umol/L)
|
3.29 (2.35–4.36)
|
5.54 (2.57–7.44)
|
5.78 (3.57–10.31)
|
AST (U/L)
|
39.38 (30.20-54.34)bd
|
92.81 (47.68-123.34)
|
69.50 (42.94-180.43)
|
ALT (U/L)
|
34.19 (20.55–49.52)ad
|
90.35 (47.62-181.27)
|
80.64 (36.93-183.11)
|
ALP (U/L)
|
90.78 (75.80-119.97)
|
107.03 (96.98-151.38)
|
148.57 (87.49-345.52)
|
GGT (U/L)
|
29.16 (19.58–53.77)bc
|
67.30 (34.27-100.66)
|
123.23 (68.31-148.53)
|
TBA (umol/L)
|
9.95 (4.56–32.41)
|
19.71 (8.68–28.62)
|
5.80 (4.96–12.25)
|
CHE (U/L)
|
5975.98 (5198.48-9139.79)
|
5595.95 (5225.55–7630.00)
|
5198.48 (4902.99-5198.48)
|
AFU (U/L)
|
26.38 ± 7.55b
|
34.04 ± 13.42
|
40.51 ± 21.61
|
CG (ug/ml)
|
5.71 (2.32–11.27)
|
5.88 (3.38–12.25)
|
5.85 (2.55–10.70)
|
GLU (mmol/L)
|
5.66 (5.10–6.60)
|
4.95 (4.32–6.61)
|
5.91 (5.79–7.05)
|
BUN (mmol/L)
|
5.27 (4.19–6.32)
|
4.26 (3.29–4.85)
|
5.45 (2.81–6.95)
|
CRE (mmol/L)
|
64.90 (55.79–74.63)
|
67.23 (62.47–72.07)
|
59.88 (50.18–74.98)
|
RBP (mg/L)
|
31.39 ± 17.44
|
29.49 ± 9.13
|
24.26 ± 6.11
|
WBC (× 109/L)
|
5.09 ± 2.00
|
4.52 ± 1.51
|
5.38 ± 2.30
|
RBC (× 1012/L)
|
4.05 ± 0.85
|
4.21 ± 0.77
|
4.02 ± 0.90
|
Hb (g/L)
|
127.72 ± 26.28
|
133.50 ± 25.10
|
120.60 ± 23.66
|
PLT (× 109/L)
|
157.11 ± 84.32
|
159.43 ± 79.50
|
147.10 ± 53.01
|
PT (sec)
|
11.95 (11.38–13.30)
|
12.55 (12.07–13.47)
|
13.40 (12.65–16.82)
|
APTT (sec)
|
27.40 (25.98–28.60)
|
27.50 (26.10-32.15)
|
29.60 (27.33–35.85)
|
CEA (ng/mL)
|
1.50 (0.62–1.85)
|
0.94 (0.50–1.73)
|
1.52 (0.58–2.06)
|
CA199 (U/mL)
|
19.48 (8.69–26.55)
|
19.73 (14.89–30.66)
|
17.77 (15.29–28.96)
|
CA125 (U/mL)
|
18.08 (10.73–40.01)
|
30.45 (15.06–40.01)
|
24.25 (14.30-125.94)
|
HBV DNA (log10IU/mL)
|
2.70 (2.70–3.34)ac
|
4.34 (2.70–6.93)
|
4.39 (2.86–6.09)
|
Data are mean ± SD values or median (25th − 75th percentile) values as indicated. |
aP < 0.05, bP < 0.01 when Group A, Group B were compared with Group C. |
cP < 0.05, dP < 0.01 when when Group A was compared with Group B. |
M/F, males/females; ALB, albumin; TBIL, total bilirubin; DBIL, direct bilirubin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; GGT, γ-glutamyl transferase; TBA, total bile acid; CHE, cholinesterase; AFU, alpha-L-fucosidase; CG, cholyglycine; GLU, glucose; BUN, urea nitrogen; CRE, creatinine; RBP, Retinol binding protein; WBC, white blood cell. RBC, red blood cell; Hb, hemoglobin; PLT, platelets; PT, prothrombin time; APTT, activated partial thromboplastin time; CEA, carcinoembryonic antigen; CA199, glucoprotein antigen 19 − 9; CA125, glucoprotein antigen 125; HBV, hepatitis B virus. |
Specifically, N-glycan levels were elevated significantly in group C compared with the group A (P = 0.026), while no differences existed between group A and group B (P = 0.050) and between group B and group C (P = 0.653) (Fig. 9). There was a trend of increasing N-glycan with elevated AFP level in the combined hepatic disease group (Fig. 2a).
Levels of N-glycan and AFP in patients with the different developmental stages of LC and HCC
According to the staging criteria of liver function Child-Pugh (CP) score, we divided the LC and HCC patients into two groups respectively: the compensatory group and decompensatory group. In the patients with LC, The levels of N-glycan in the decompensatory was significant higher than in the control (P = 0.007), but no differences were found between the decompensatory and the compensatory groups (P = 0.389) as well as between the compensatory and the controls (P = 0.178). Although there was a significant difference in AFP levels between the compensatory and the controls (P = 0.003), no differences were found between the decompensatory and the compensatory groups (P = 0.092) and between the decompensatory and the controls (P = 0.109) (Fig. 10a). The results indicated that the levels of N-glycan were more valuable in reflecting the disease severity in patients with LC compared with the levels of AFP. In the patients with HCC, Levels of both N-glycan and AFP in the compensatory and decompensatory groups were significant higher than those in the control (P < 0.001 and P = 0.004 for N-glycan and P < 0.001 for both in AFP, respectively), but no difference were found between the compensatory and decompensatory groups (P = 0.155 for N-glycan and P = 0.422 for AFP) (Fig. 10b). Our data suggested that levels of N-glycan and AFP in patients with HCC were closely related to the stage of disease development.