Methodological evaluation and quality control
A good linear relationship of 15 kinds of bile acids is shown in the linear range of 1.0 to 6000 nmol/L, and the linear correlation coefficients (R2) are all over 0.993; the limit of quantification of the target is from 1.0 to 10.0 nmol/L. The low, medium and high concentration recovery rates ranged from 95.5% to 114.1%, the intra-day and inter-day RSDs were less than 11.4%, and the quality control coefficients of variation of 15 bile acids were less than15%.
Analysis of serum bile acid profile in ICP group, normal pregnant women group, cholelithiasis group and hepatitis B virus group
The serum bile acid profile of the ICP group was different from normal pregnant group, the cholelithiasis group and the hepatitis B virus group. The bile acid spectrum characteristics of the ICP1 group, ICP2 group, and ICP3 group were similar, and the serum concentrations of GCDCA, GCA, GLCA, TDCA, TCA, GUDCA, and TUDCA were significantly higher than normal pregnant group (P <0.008). The serum bile acid profile of ICP4 group was different from normal pregnant group, ICP1 group, ICP2 group, ICP3 group, cholelithiasis group and hepatitis B virus group, and the serum concentrations of UDCA, GUDCA, TUDCA, GCA and GLCA in ICP4 group were significantly higher than normal pregnant group (P <0.01 ). (See Figure 1, Table 3)
Analysis of serum bile acid profile in each subgroup of ICP
Based on the data of 15 known serum bile acids by mass spectrometry, PLS-DA was used to analyze the serum bile acid profiles of each subgroup of ICP and normal pregnant women. The parameters of the constructed PLS-DA model variables R2Y represents the explanatory ability of the model, and Q2 represents the ability of the model to predict new data. The VIP value of various cholic acids is the contribution value of the subgroups on the PLS-DA score chart. Cholic acid with a VIP value >1 could be used as the significantly differential cholic acids between two comparision groups.
PLS-DA models were established with ICP subgroups and normal pregnant women group (R2Y=0.156, Q2=0.126). The ICP subgroups and normal group can be differentiated very well based on the data of bile acid profile under the three-dimensional score chart than two-dimensional score chart (see Figure 2a and 2b). Figure 2c and Table 4 showed the VIP values of differential cholic acid for the five groups (see Figure 2c and Table 4). The differential bile acids in the ICP subgroups (ICP1 group, ICP2 group, ICP3 group and ICP4 group) and normal pregnant women group were TCDCA, DCA, TCA, GDCA and GLCA.
Table 4. Analysis of serum differential bile acid profiles of group ICP1, group ICP2, group ICP3, group ICP4 and normal pregnant women
Differential bile acid
|
VIP value
|
Comparison of concentrations in five groups
|
TCDCA
|
1.463
|
ICP1>ICP2>ICP3>normal pregnant>ICP4
|
DCA
|
1.380
|
ICP3>ICP4>normal pregnant>ICP2>ICP1
|
TCA
|
1.309
|
ICP1>ICP2>ICP3>ICP4>normal pregnant
|
GDCA
|
1.226
|
ICP3>ICP2>ICP1>normal pregnant>ICP4
|
GLCA
|
1.006
|
ICP3>ICP2>ICP1>ICP4>normal pregnant
|
Analysis of serum bile acid profile of ICP1 group (ICP with jaundice)
The differential bile acids between ICP1 group and normal pregnant group were TCDCA, TCA, GCA, GCDCA, TUDCA and GUDCA, and the concentration of these bile acids in ICP1 group were significantly higher than normal pregnant group (P<0.05). The differential bile acids between ICP1 group and ICP2 group were TCA, TCDCA, GDCA, CA, GCDCA, TLCA and DCA, and the concentration of TCA, TDCA, CA and GCDCA in ICP1 group were higher than ICP2 group (P<0.05), while the concentration of GDCA, TLCA and DCA in ICP1 group were lower than ICP2 group (P<0.05). The differential bile acids between ICP1 group and cholelithiasis group were TUDCA, UDCA, TCA, CDCA, GUDCA, DCA, CA, and GCDCA, and the concentration of TUDCA, UDCA, TCA, GUDCA, and CA in ICP1 group were higher than cholelithiasis group (P <0.05), while the concentration of DCA and GCDCA were lower than cholelithiasis group (P <0.05). The differential bile acids between ICP1 group and hepatitis B group were GCDCA, GCA, CDCA, TCDCA, and TDCA, and the concentration of TDCA in ICP1 group was higher than hepatitis B group, while the concentration of GCDCA, GCA, CDCA, and TDCA were lower than hepatitis B group (P <0.05). (See Figure 3, Table 5).
Table 5
Analysis of Bile acids profile between ICP1 and other groups
Differential bile acid
|
VIP value
|
Comparison of concentrations in five groups
|
TCDCA
|
1.463
|
ICP1༞ICP2༞ICP3༞normal pregnant༞ICP4
|
DCA
|
1.380
|
ICP3༞ICP4༞normal pregnant༞ICP2༞ICP1
|
TCA
|
1.309
|
ICP1༞ICP2༞ICP3༞ICP4༞normal pregnant
|
GDCA
|
1.226
|
ICP3༞ICP2༞ICP1༞normal pregnant༞ICP4
|
GLCA
|
1.006
|
ICP3༞ICP2༞ICP1༞ICP4༞normal pregnant
|
Analysis of serum bile acid profile in ICP3 group (Hyperchoicemia of pregnancy)
The differential bile acids between ICP3 group and normal pregnant group were GUDCA, LCA, GLCA, UDCA, TUDCA, CDCA and TLCA, and the concentration of these bile acids in ICP3 group were higher than normal pregnant group (P <0.05). The differential bile acids between ICP3 group and ICP1 group were DCA, GDCA, LCA, GLCA, GCDCA, and TDCCA, and the concentration of DCA, GDCA, LCA, GLCA, and GCDCA in ICP3 group were higher than ICP1 group (P < 0.05), while the concentration of TDCCA were lower than ICP1 group (P < 0.05). The differential bile acids between ICP3 group and ICP2 group were DCA, GDCA, TDCA, CDCA and LCA, and the concentration of these bile acids in ICP3 group were higher than ICP2 group (P <0.05). The differential bile acids between ICP3 group and cholelithiasis group were UDCA, GUDCA, TUDCA and LCA, and the concentration of these bile acids in ICP3 group were higher than cholelithiasis group (P <0.05). The differential bile acids between ICP3 group and hepatitis B group were DCA, TDCA, TLCA, GCDCA, TCDCA, TCA, CDCA, and GDCA, and the concentration of DCA, GDCA, TDCA, and TLCA in ICP3 group were higher than hepatitis B group, while the concentration of TCA, CDCA, GCDCA, TDCCA was lower than hepatitis B group (P <0.05). (See Figure 4, Table 6).
Table 6
Analysis of Bile acids profile between ICP3 and other groups
Comparation Groups
|
Bile acids profile
|
Comparation of bile acids concentration between groups
|
CA
GCA
TCA
|
CDCA
GCDCA
TCDCA
|
DCA
GDCA
TDCA
|
LCA
GLCA
TLCA
|
UDCA
GUDCA
TUDCA
|
ICP1 vs
normal group
|
GCA
TCA
|
GCDCA
TCDCA
|
DCA
|
GLCA
|
GUDCA
|
ICP1 > normal group
|
ICP1 vs
ICP2
|
CA
TCA
|
GCDCA
TCDCA
|
|
|
|
ICP1 > ICP2
|
|
DCA
|
GDCA
|
TLCA
|
|
ICP1 < ICP2
|
ICP1 vs
Cholelithiasis
|
CA
TCA
|
|
|
|
UDCA
GUDCA
TUDCA
|
ICP1 > Cholelithiasis
|
|
CDCA
GCDCA
|
DCA
|
|
|
ICP1 < Cholelithiasis
|
ICP1 vs
Hepatitis B
|
|
|
TDCA
|
|
|
ICP1 > Hepatitis B
|
GCA
|
CDCA
GCDCA
TCDCA
|
|
|
|
ICP1 < Hepatitis B
|
Analysis of serum bile acid profile in ICP4 group (idiopathic aminotransferase abnormality during pregnancy)
The differential bile acids between ICP4 group and normal pregnant group were GCA, UDCA, GUDCA, TUDCA and GLCA, and the concentration of these bile acids in ICP4 group were significantly higher than normal pregnant group (P<0.05). The differential bile acids between ICP4 group and ICP1 group were TCDCA, TCA, DCA, GCA, GCDCA, TUDCA, TDCA, and GUDCA. The concentration of TCDCA, TCA, GCA, GCDCA, TUDCA, TDCA, and GUDCA in ICP4 group were lower than ICP1 group (P<0.05), while the concentration of DCA in ICP4 group was higher than ICP1 group (P<0.05). The differential bile acids between ICP4 group and ICP2 group were TCDCA, TDCA, GCDCA, DCA, GCA, GUDCA, TUDCA, and TLCA. The concentration of TCDCA, TDCA, GCDCA, GCA, GUDCA, TUDCA, and TLCA in ICP4 group were lower than ICP2 group, while the concentration of DCA in ICP4 group was higher than ICP2 group (P<0.05). The differential bile acids between ICP4 group and ICP3 group were TLCA, LCA, TDCA, GUDCA, GDCA, GLCA, and TUDCA, and the concentration of these bile acids in ICP4 group were lower than ICP3 group (P<0.05). The differential bile acids between ICP4 group and cholelithiasis group were UDCA, CDCA, TUDCA, and GCDCA. The concentration of UDCA and TUDCA in ICP4 group were higher than cholelithiasis group, while the concentration of CDCA and GCDCA were lower than cholelithiasis group (P<0.05). The differential bile acids between ICP4 group and hepatitis B group were GCDCA, CDCA, TCDCA, TCA, GCA, GUDCA, UDCA, and TUDCA, and the concentration of these bile acids in ICP4 group were lower than hepatitis B group (P <0.05). (See Figure 5, Table 7).
Table 7
Analysis of Bile acids profile between ICP4 and other groups
Comparation Groups
|
Bile acids profile
|
Comparation of bile acids concentration between groups
|
CA
GCA
TCA
|
CDCA
GCDCA
TCDCA
|
DCA
GDCA
TDCA
|
LCA
GLCA
TLCA
|
UDCA
GUDCA
TUDCA
|
ICP3 vs
normal group
|
|
CDCA
|
|
LCA
GLCA
TLCA
|
UDCA
GUDCA
TUDCA
|
ICP3 > normal group
|
ICP3 vs
ICP1
|
|
GCDCA
|
DCA
GDCA
|
LCA
GLCA
|
|
ICP3 > ICP1
|
|
TCDCA
|
|
|
|
ICP3 < ICP1
|
ICP3 vs
ICP2
|
|
CDCA
|
DCA
GDCA
TDCA
|
LCA
|
|
ICP3 > ICP2
|
ICP3 vs
Cholelithiasis
|
|
|
|
LCA
|
UDCA
GUDCA
TUDCA
|
ICP3 > Cholelithiasis
|
ICP3 vs
Hepatitis B
|
|
|
DCA
GDCA
TDCA
|
TLCA
|
|
ICP3 > Hepatitis B
|
TCA
|
CDCA
GCDCA
TCDCA
|
|
|
|
ICP3 < Hepatitis B
|
Analysis of pregnancy outcomes in ICP subgroups
The maternal TBA level of ICP subgroups (ICP1, ICP2 and ICP3) were significantly higher than normal pregnant group (P<0.05). The TBA level in ICP4 group was at normal level, but the concentration of UDCA, GUDCA, TUDCA, GCA and GLCA in ICP4 group were significantly higher than normal pregnant group (P<0.01). The occurence of meconium-stained amniotic fluid, preterm delivery, and cesarean section in ICP1 group were significantly higher than ICP2 group, ICP3 group and ICP4 group, respectively (P<0.05). The occurence of meconium-stained amniotic fluid, preterm delivery, and cesarean section in ICP2 group, ICP3 group and ICP4 group were significantly higher than normal pregnant group (P <0.05), and no statistical differences were found among ICP2 group, ICP3 group and ICP4 group (P> 0.05). (see table 8)
Table 8 Clinical information and liver function test results of normal pregnant women and patients in every subgroup of ICP
Subjects
|
Normal pregnant(n=50)
|
ICP1(n=51)
|
ICP2(n=27)
|
ICP3(n=22)
|
ICP4(n=44)
|
H/χ2
|
P
|
Age(year)
|
28(26,32)
|
30(27,31)
|
29(23,33)
|
28(27,31)
|
29(27,33)
|
1.441
|
0.837
|
Gravidity
|
2(1,3)
|
2(1,3)
|
2(1,3)
|
1(1,2)
|
2(1,2)
|
8.069
|
0.089
|
Delivery pregnancy week
|
39(38,40)
|
37(35,39)a
|
37(36,38)a
|
38(37,39.0)a
|
37(36,39)a
|
36.587
|
0.000
|
Previous history of ICP(n,%)
|
0(0%)
|
2(3.9%)
|
5(18.5%)a
|
2(9.1%)
|
4(9.1%)
|
10.855
|
0.028
|
pregnancy outcome(n,%)
|
Cesarean section
|
4(8%)
|
42(82.4%)a
|
18(66.7%)a,b
|
8(36.7%)b
|
18(40.9%)a,b
|
53.982
|
0.000
|
Premature (<37w)
|
0(0%)
|
39(76.5%)a
|
2(7.4%)a,b
|
2(9.1%)a,b
|
5(11.4%)a,b
|
30.81
|
0.000
|
Meconium-stained amniotic fluid
|
1(2%)
|
19(37.3%)a
|
4(14.8%)a,b
|
3(13.6%)a,b
|
8(18.2%)a,b
|
27.324
|
0.000
|
Neonatal ICU hospitalization rate
|
1(2%)
|
17(33.3%)a
|
5(18.5%)a
|
0(0%)
|
5(11.4%)
|
25.422
|
0.000
|
1 min Apgar scores≤7
|
1(2%)
|
12(23.5%)a
|
2(7.4%)
|
2(9.1%)
|
3(6.8%)b
|
21.185
|
0.000
|
Newborn weight(g)
|
3305(2980,3592)
|
3180(2720,3370)a
|
2900(2720,3360)a
|
3185(2837,3320)
|
2960(2565,3870)
|
10.774
|
0.029
|
Postpartum hemorrhage(ml)
|
225(200,300)
|
200(200,300)
|
300(200,300)
|
200(200,285)
|
300(200,300) a
|
11.077
|
0.026
|
Liver function test results
|
TBA(umol/L)
|
3.3(2.4,4.5)
|
32.0(16.5,48.1)a
|
18.8(12.0,28.8)a,b
|
19.9(16.9,29.4) a
|
6.2(4.4,7.4) a,b,c
|
149.387
|
0.000
|
TBIL(umol/L)
|
11.0(8.8,12.9)
|
18.0(13.4,26.6)a
|
8.4(5.4,12.1)a,b
|
6.5(4.5,10.1) a,b
|
8.2(6.3,10.4) a,b
|
94.044
|
0.000
|
DBIL(umol/L)
|
3.4(2.6,4.8)
|
12.6(8.3,16.8)a
|
3.7(2.2,5.1)a,b
|
3(2.4,3.6) a,b
|
3.8(3.0,4.5) a,b,d
|
123.664
|
0.000
|
ALT(U/L)
|
19.0(17.0,25.3)
|
216.0(106.0,336.0)a
|
176.0(95.0,309.0) a
|
10.0(7.8,13.5) a,b
|
104.5(70.0,174.8) a,b,d
|
142.102
|
0.000
|
AST(U/L)
|
18.0(15.8,22.0)
|
146.0(73.0,205.0)a
|
116.8(60.0,232.0) a
|
15.5(12.0,18.0)a,b
|
51.5(35.3,76.5) a,b,d
|
141.737
|
0.000
|
Note: The data in the table is expressed by M (Q1, Q3). a, P <0.05 compared with normal pregnant women. b. Compared with ICP1 group, P <0.05.
c. Compared with the ICP2 group, P <0.05. d. Comparing the ICP3 group with the ICP4 group, P <0.05.