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Serum bile acid levels in pregnancy with pruritus (bii.e acids and steroids 158)
SERUM (BILE
BILE ACIDS
ACID AND
LEVELS STEROIDS
IN PREGNANCY
WITH
PRI~KTTUS
158)
SUMMARY
The serum bile acid levels in 22 healthy pregnant women have been studied with gas-liquid ~hromatograpl~y, The mean con~elltration of total bile acids was o.oS7 mgq’, (not corrected for rg-doT$ loss of bile acids in the purification of the serum extracts). The mean ratios between deoxycholic, chenodeoxycholic and cholic acids were o.g:r.o:r.o and only conjugated bile acids could be found. In six cases with pruritus in the last trimester, the mean concentration of total serum bile acids was 2.48 rngy$. The mean ratios between deoxycholic, chenodcoxycholic and cholic acids were 0.1: r.o:+j. Except for traces of free bile acids in one subject, all bile acids were conjugated. Significant amounts of bile acids other than those mentioned were not found.
A disorder occurring during pregnancy, particularly in the last trimester has been called for example “idiophathic jaundice of pregnancy”i-3, “recurrent jaundice of pregnancy” J+, “endogenous hepatotoxemia of pregnancy”6, and “hepatosis of pregnancy ” ‘. Pruritus is a common symptom and usually occurs before jaundice is noted. Some patients with pruritus may not become jaundiced at all. It has been suggested that itching (in biliary cirrhosis for example) is related to elevated serum bile acids but definitive proof for this hypothesis is lacking. The subject has been reviewed recently*. In a previous paper we have described a method for the determination of individual bile acids in serum by gas chromatography 9.This method has now been used to study the serum bile acids in normal pregnancy and in pregnancy- accompanied by pruritus. KSTERIALS
AND METHODS
The control group consisted of 22 healthy pregnant women. The patient group consisted of six women having generalized pruritus in the last trimester. One patient (G S.) had jaundice. Blood samples were taken in the morning and were allowed to coagulate. After centrifugation the serum was pipetted off, frozen and kept at -20~ until analyzed.
208
Ii. .lNU
J. SJijVALL
Since the blood samples had been left to coagulate for somewhat different periods of time, the influence of this on the analysis of bile acids was tested. A large sample taken from one subject,
was divided into four portions two of which were heparinized.
One serum and one plasma sample were immediately processed, the two remaining samples were left at room temperature for five hours before being centrifuged. The bile acid contents of the four samples were essentially the same. Serum bile acids were determined
as previously
described”.
The procedure
in
outline was as follows: Serum, 3-5 ml, was diluted with an equal volume of water and passed through a column of the anion exchanger Amberlyst XN-1006. Bile acids were eluted from the column and then hydrolyzed in IS:,, NaOH in 50:{, ethanol for IO 11 at IIO--120". The bile acids were extracted, mina column.
They were then determined
the trifluoracetates
using
(see 9) as the stationary,
columns phase
with
methylated 0.5’:;
on Gas Chrom
losses during workup of the samples.
and then purified on an alu-
quantitatively
Recoveries
QF-r
by gas chromatography or 0.5:;
P. Corrections
of a nitrile
of
silicone
were not made for
of added bile acids ranged between
60 and 850/i, (ref. 9). For the analysis of free bile acids the hydrolysis step was omitted. The eluate from the ion exchange column was evaporated, tire residue was dissolved in water and after acidification the bile acids were directly extracted and methylated. In some instances serum transaminases, alkaline phosphatase and serum bilirubin were determined. made at the Department
1Vc wish to thank
Dr. S. Lindstedt
for these determinations
of Clinical Chemistry.
RESULTS
Table I shows the results of the serum bile acid analyses in the subjects without pruritus at different weeks of gestation. Three cases (I.G., K.S. and M.S.) were followed for several weeks to study individual variations in the bile acid levels. The mean values in 25 analyses of samples from 22 women were : deoxycholic
acid
0.28 pg/ml (range 0.08%0.93), chenodeoxycholic acid 0.30 ,ug/ml (0.08%0.66), cholic acid 0.29 pg/ml (< 0.0550.83) and total bile acids 0.87 pg/rnl (0.24-1.65). Three analyses of samples taken after delivery gave similar values. Two samples taken one hour after lunch showed somewhat higher values (Table I). There was no significant correlation between the serum bile acid levels and the time of gestation. Two subjects (K.S. and K.F.) with vomiting during the entire pregnancy. and one subject (G.B.) who delivered an Rh-immunized child had serum bile acid levels which did not differ from those of the other control subjects. Free bile acids were not found in the serum of the seven subjects analyzed, i.e. less than 0.03-0.05 pg/ml of the individual bile acid was present in an unconjugated form. No bile acids other than the three common ones were seen on the gas chromatograms. In all six cases with generalized pruritus the levels of cholic and chenodeoxycholic acids were markedly ele\Tated (Table II). The deoxvcholic acid level vvas normal or low except in one case where an increase was observed. The following mean values were found in IO analyses from the six patients: deoxycholic acid 0.43 (i 0.0552.72), chenodeoxycholic acid 4.44 (I.+-12.x0), cholic acid 19.92 (4.OIFj9.70) and total bile acids 24.79 pg/ml (6.4%74,72). The ratios among the three bile acids
SERC’M ISILE .1CID IN PREGNANCY
SERUM
BILE
?,CID
Il’eek
LEVELS
of
IS
PRISGNA.NT
209
WOXEN
Bile mid concmtratiorz 11%
CD*
14 1-t
0.33 “.“K
0.24
O.24
0.81
0.08
“.I1
0.27
I.5
0.10
0.08
10
0.93 0.37
0.3”
._
0.62
o.r.+ “A+
1.63
0.q
o.gj
19 21
0.09
“.I2
0.12
0.33
".Ij o.25
0.08
0.12
"-35
30
0.32
o.24
0.21
0.22
36 36 37
O.-H 0.11 0.33 0.21
30
37 __.~_
G.S. I.J. I%I%. C.C. l.G. 1.G. KS. KS. KS. 1i.s. M.S. KS. KS. iv1.s. 1I.S.
I.37
0.42
0.18
Patierll
0.q
0.24
0.66
I.09
“. 16 0.3.; 0.45
0.18 0.43 0.q “,53 0.47
0.74 0.X6 0.84 I.10
0.10
0.23
O.Gj
l.2.j
+ D = tlcoxycholic acid, CD = clwnotleoxycholic ** Samples taken “nc hour after lunch. i After delivery.
TXliLl<
FROX
Total ____-
J7
26
SUFFERING
pg/d
gestation
16
c+
NOT
acid,
PRUKITUS
12;eek of
Bile acid concenfvatiw
gestation
D
CD
C
Total
pglml
1.12
33
0.25
0.
38
0.18
.&O “.57
o.qr “-45
38 39
0.36 0.17
0.46 0.3 ’
0.8.3
1.05
0.16
O.G.+
32 39 28 29**
29 36
38 5 tiZlySf 3
0.2.#
0.29
0. ‘.+
0.49
0.48
0.45
0.07 1.42
o.rg
0.48
0.16
30** 4 daysf
months?
I.20
0.x.3
0.49
0. j0
".77
I.82
0.80
1.1”
1.83
3.73
0.16
0.24
0.15
0.10
0.28
0.68
0.27
0.22
0.30
0.79
0.25
0.17
0.11
0.53
0.16
0.18
0.11
0.19
0.~4
0.2”
0.75
“.‘+h
<0.05
0.33
C = cholic acid.
II _..____~_
Patient
12’eek &vStati0tk
G.0.
of
Rileacid iP
cmzce,rtvation Cl1
//g/w1 ?‘otaE
c
r
U.N.
30
I<.nT.
37 37
2.72 0.37 <0.05
Il.30 3.K.! I.42
G.S.
$3
0.08
6.10
G.K.
3-1_
0.‘25
2.22
C.K.
38
0.19
G.K. G.K. u.i\. I’. 4.
39 4” 38 39
52.7” “9.70
9.64 22.4”
mgy;
plkosp~kntase**
0.5
Il.“(i
-
~8.64
-
-
~-
14.93 8.96
-
16.52 27.80
ro.38 34.00
0.5 ---
24 -
20.90
rg.1.j
I.7
I8
3.33
I.58
11.4” 7.08
0.15 0.32
3.71 5.88
0.24
4.0’
.-____.
c;ort
CPTT
-
-
210
21”
-
-
st?
“-5i: --
6.$l
:i: I;or ubbrcviations set Table i ** Buch and 13uch units. liarnlcn units. Values obtained in the 3rd wcrk of gestation
L:
Alkaline
__.-
71.7’ 13.59
.+.“I
<“.I0
IN/S
2rit
2rlt --
34 155
-
42 196
at onset of pruritus.
were different from those found in the control subjects the predominant bile acid in the serum of the patients
in that cholic acid was by far with pruritus.
Free bile acids were not found in the sample analyzed from patients K.N., G.K. and U.A. except in one sample from patient G.K. where 0.09 @g/ml of cholic acid was present. Significant amounts of bile acids other than cholic, chenodeoxycholic and deoxycholic acids were not found. Serum bile acids were determined in two cases (G.K. and U.A.) after delivery. As shown in Fig. I, there was a decrease in the serum bile acid levels towards normal during tLt. week following delivery. This decrease was most evident for cholic acid resulting in a normal ratio between cholic and chenodeoxycholic acids. Deoxycholic
40.0-
G-K. 20.0-
__ “.*.
35
36
Week
37
38
39
40
5 IO
of gestation
9,
38
Days post partum De&very
acid increased after delivery
slightly
in patient
6 lb
39 4(
Days post partum
Week of gestation De
U.A. The pruritus
disappeared
during
the week
in all cases.
DISCUSSIOK
The serum bile acid levels in pregnant to those previously centration
reported
for healthy
women without
pruritus
male and female subjects”.
of total bile acids was 0.087 rngqh as compared
were similar
The mean con-
with o.oSz mgS,, for the
15 subjects
previously studied. The ratios among the mean values of deoxycholic, thcnodeoxycholic and cholic acids were 0.9 : LO : 1.0 which are approximately the same as those found for the bile acids in the bile of healthy il~di~-~duals (0.6: 1.0: 1.1)lo. In patients with pruritus there was a 10-100 fold increase in the total bile acids (mean value z.qS’~/o) and a difference in the proportions among the means of the three bile acids. Thus, the ratios for deoxycholic, chenodeoxycholic and cholic acids were 0.1 : 1.0:q.s which is similar to the ratios for the bile acids in the bile of patients having obstructive hepatobiliary disorders lo. These results mav be compared with the tindings of Carey” and of Kudman and Kendall’” that there is an elevated trihydroxy/dihydroxy bile acid ratio in the serum of patients with obstructive jaundice. The serum bile acid pattern in pregnant women with pruritus is therefore in agreement with the results of other liver function tests and histologic findings which indicate that this condition is associated with cllolestasis2-7. It is also of interest to note that Arfwedson and ~‘on Studnitz ‘3 found elevated levels of phospholipids and cholesterol c~~ncomitant with an increased level of ~-lipoproteins in pregnant women with pruritus. It has been suggested that pruritus associated with biliary obstruction is due CYill. C’him. .+tcia, 1.3 (1006) 207 -2~ I
SERUM
BILE ACID
IN PREGNANCY
211
a retention of bile acids (see 8). However, this possibility has not yet been proved (ref. II, 14). The present study has demonstrated elevated levels of bile acids in pregnant women with pruritus in the last trimester. Although this finding is a sign of liver dysfunction it does not necessarily explain the pruritus. The ion exchange resin cholestyramine which has been given to relieve itching in patients with biliary cirrhosis15-17 has also been used successfullv in a few cases of pruritus associated with pregnancy2s3. Although this resin increases the fecal excretion of biie acids and lowers serum bile acid levels, it should be mentioned that it is also effective in binding conjugates of steroid hormones like dehydroepiandrosterone sulfatelR. The idiopathic jaundice of pregnancy has been compared with the jaundice that can be induced by certain steroids (especially r@kylated steroids) and it might therefore be speculated that the relief of itching that is obtained with cholestyramine could in addition be due to the binding of some biliary steroid conjugate other than the bile acids. Work is in progress to test this hypothesis.
to
ACKNOWLEDGEMENT
The technical assistance of Miss &laud Frankesten is gratefully ackowledged. This work was supported by a grant (I$%-z19-01) from the Swedish Medical Research Council. RE1’lZ RENCES
BILE ACIDS
ACID AND
LEVELS STEROIDS
IN PREGNANCY
WITH
PRI~KTTUS
158)
SUMMARY
The serum bile acid levels in 22 healthy pregnant women have been studied with gas-liquid ~hromatograpl~y, The mean con~elltration of total bile acids was o.oS7 mgq’, (not corrected for rg-doT$ loss of bile acids in the purification of the serum extracts). The mean ratios between deoxycholic, chenodeoxycholic and cholic acids were o.g:r.o:r.o and only conjugated bile acids could be found. In six cases with pruritus in the last trimester, the mean concentration of total serum bile acids was 2.48 rngy$. The mean ratios between deoxycholic, chenodcoxycholic and cholic acids were 0.1: r.o:+j. Except for traces of free bile acids in one subject, all bile acids were conjugated. Significant amounts of bile acids other than those mentioned were not found.
A disorder occurring during pregnancy, particularly in the last trimester has been called for example “idiophathic jaundice of pregnancy”i-3, “recurrent jaundice of pregnancy” J+, “endogenous hepatotoxemia of pregnancy”6, and “hepatosis of pregnancy ” ‘. Pruritus is a common symptom and usually occurs before jaundice is noted. Some patients with pruritus may not become jaundiced at all. It has been suggested that itching (in biliary cirrhosis for example) is related to elevated serum bile acids but definitive proof for this hypothesis is lacking. The subject has been reviewed recently*. In a previous paper we have described a method for the determination of individual bile acids in serum by gas chromatography 9.This method has now been used to study the serum bile acids in normal pregnancy and in pregnancy- accompanied by pruritus. KSTERIALS
AND METHODS
The control group consisted of 22 healthy pregnant women. The patient group consisted of six women having generalized pruritus in the last trimester. One patient (G S.) had jaundice. Blood samples were taken in the morning and were allowed to coagulate. After centrifugation the serum was pipetted off, frozen and kept at -20~ until analyzed.
208
Ii. .lNU
J. SJijVALL
Since the blood samples had been left to coagulate for somewhat different periods of time, the influence of this on the analysis of bile acids was tested. A large sample taken from one subject,
was divided into four portions two of which were heparinized.
One serum and one plasma sample were immediately processed, the two remaining samples were left at room temperature for five hours before being centrifuged. The bile acid contents of the four samples were essentially the same. Serum bile acids were determined
as previously
described”.
The procedure
in
outline was as follows: Serum, 3-5 ml, was diluted with an equal volume of water and passed through a column of the anion exchanger Amberlyst XN-1006. Bile acids were eluted from the column and then hydrolyzed in IS:,, NaOH in 50:{, ethanol for IO 11 at IIO--120". The bile acids were extracted, mina column.
They were then determined
the trifluoracetates
using
(see 9) as the stationary,
columns phase
with
methylated 0.5’:;
on Gas Chrom
losses during workup of the samples.
and then purified on an alu-
quantitatively
Recoveries
QF-r
by gas chromatography or 0.5:;
P. Corrections
of a nitrile
of
silicone
were not made for
of added bile acids ranged between
60 and 850/i, (ref. 9). For the analysis of free bile acids the hydrolysis step was omitted. The eluate from the ion exchange column was evaporated, tire residue was dissolved in water and after acidification the bile acids were directly extracted and methylated. In some instances serum transaminases, alkaline phosphatase and serum bilirubin were determined. made at the Department
1Vc wish to thank
Dr. S. Lindstedt
for these determinations
of Clinical Chemistry.
RESULTS
Table I shows the results of the serum bile acid analyses in the subjects without pruritus at different weeks of gestation. Three cases (I.G., K.S. and M.S.) were followed for several weeks to study individual variations in the bile acid levels. The mean values in 25 analyses of samples from 22 women were : deoxycholic
acid
0.28 pg/ml (range 0.08%0.93), chenodeoxycholic acid 0.30 ,ug/ml (0.08%0.66), cholic acid 0.29 pg/ml (< 0.0550.83) and total bile acids 0.87 pg/rnl (0.24-1.65). Three analyses of samples taken after delivery gave similar values. Two samples taken one hour after lunch showed somewhat higher values (Table I). There was no significant correlation between the serum bile acid levels and the time of gestation. Two subjects (K.S. and K.F.) with vomiting during the entire pregnancy. and one subject (G.B.) who delivered an Rh-immunized child had serum bile acid levels which did not differ from those of the other control subjects. Free bile acids were not found in the serum of the seven subjects analyzed, i.e. less than 0.03-0.05 pg/ml of the individual bile acid was present in an unconjugated form. No bile acids other than the three common ones were seen on the gas chromatograms. In all six cases with generalized pruritus the levels of cholic and chenodeoxycholic acids were markedly ele\Tated (Table II). The deoxvcholic acid level vvas normal or low except in one case where an increase was observed. The following mean values were found in IO analyses from the six patients: deoxycholic acid 0.43 (i 0.0552.72), chenodeoxycholic acid 4.44 (I.+-12.x0), cholic acid 19.92 (4.OIFj9.70) and total bile acids 24.79 pg/ml (6.4%74,72). The ratios among the three bile acids
SERC’M ISILE .1CID IN PREGNANCY
SERUM
BILE
?,CID
Il’eek
LEVELS
of
IS
PRISGNA.NT
209
WOXEN
Bile mid concmtratiorz 11%
CD*
14 1-t
0.33 “.“K
0.24
O.24
0.81
0.08
“.I1
0.27
I.5
0.10
0.08
10
0.93 0.37
0.3”
._
0.62
o.r.+ “A+
1.63
0.q
o.gj
19 21
0.09
“.I2
0.12
0.33
".Ij o.25
0.08
0.12
"-35
30
0.32
o.24
0.21
0.22
36 36 37
O.-H 0.11 0.33 0.21
30
37 __.~_
G.S. I.J. I%I%. C.C. l.G. 1.G. KS. KS. KS. 1i.s. M.S. KS. KS. iv1.s. 1I.S.
I.37
0.42
0.18
Patierll
0.q
0.24
0.66
I.09
“. 16 0.3.; 0.45
0.18 0.43 0.q “,53 0.47
0.74 0.X6 0.84 I.10
0.10
0.23
O.Gj
l.2.j
+ D = tlcoxycholic acid, CD = clwnotleoxycholic ** Samples taken “nc hour after lunch. i After delivery.
TXliLl<
FROX
Total ____-
J7
26
SUFFERING
pg/d
gestation
16
c+
NOT
acid,
PRUKITUS
12;eek of
Bile acid concenfvatiw
gestation
D
CD
C
Total
pglml
1.12
33
0.25
0.
38
0.18
.&O “.57
o.qr “-45
38 39
0.36 0.17
0.46 0.3 ’
0.8.3
1.05
0.16
O.G.+
32 39 28 29**
29 36
38 5 tiZlySf 3
0.2.#
0.29
0. ‘.+
0.49
0.48
0.45
0.07 1.42
o.rg
0.48
0.16
30** 4 daysf
months?
I.20
0.x.3
0.49
0. j0
".77
I.82
0.80
1.1”
1.83
3.73
0.16
0.24
0.15
0.10
0.28
0.68
0.27
0.22
0.30
0.79
0.25
0.17
0.11
0.53
0.16
0.18
0.11
0.19
0.~4
0.2”
0.75
“.‘+h
<0.05
0.33
C = cholic acid.
II _..____~_
Patient
12’eek &vStati0tk
G.0.
of
Rileacid iP
cmzce,rtvation Cl1
//g/w1 ?‘otaE
c
r
U.N.
30
I<.nT.
37 37
2.72 0.37 <0.05
Il.30 3.K.! I.42
G.S.
$3
0.08
6.10
G.K.
3-1_
0.‘25
2.22
C.K.
38
0.19
G.K. G.K. u.i\. I’. 4.
39 4” 38 39
52.7” “9.70
9.64 22.4”
mgy;
plkosp~kntase**
0.5
Il.“(i
-
~8.64
-
-
~-
14.93 8.96
-
16.52 27.80
ro.38 34.00
0.5 ---
24 -
20.90
rg.1.j
I.7
I8
3.33
I.58
11.4” 7.08
0.15 0.32
3.71 5.88
0.24
4.0’
.-____.
c;ort
CPTT
-
-
210
21”
-
-
st?
“-5i: --
6.$l
:i: I;or ubbrcviations set Table i ** Buch and 13uch units. liarnlcn units. Values obtained in the 3rd wcrk of gestation
L:
Alkaline
__.-
71.7’ 13.59
.+.“I
<“.I0
IN/S
2rit
2rlt --
34 155
-
42 196
at onset of pruritus.
were different from those found in the control subjects the predominant bile acid in the serum of the patients
in that cholic acid was by far with pruritus.
Free bile acids were not found in the sample analyzed from patients K.N., G.K. and U.A. except in one sample from patient G.K. where 0.09 @g/ml of cholic acid was present. Significant amounts of bile acids other than cholic, chenodeoxycholic and deoxycholic acids were not found. Serum bile acids were determined in two cases (G.K. and U.A.) after delivery. As shown in Fig. I, there was a decrease in the serum bile acid levels towards normal during tLt. week following delivery. This decrease was most evident for cholic acid resulting in a normal ratio between cholic and chenodeoxycholic acids. Deoxycholic
40.0-
G-K. 20.0-
__ “.*.
35
36
Week
37
38
39
40
5 IO
of gestation
9,
38
Days post partum De&very
acid increased after delivery
slightly
in patient
6 lb
39 4(
Days post partum
Week of gestation De
U.A. The pruritus
disappeared
during
the week
in all cases.
DISCUSSIOK
The serum bile acid levels in pregnant to those previously centration
reported
for healthy
women without
pruritus
male and female subjects”.
of total bile acids was 0.087 rngqh as compared
were similar
The mean con-
with o.oSz mgS,, for the
15 subjects
previously studied. The ratios among the mean values of deoxycholic, thcnodeoxycholic and cholic acids were 0.9 : LO : 1.0 which are approximately the same as those found for the bile acids in the bile of healthy il~di~-~duals (0.6: 1.0: 1.1)lo. In patients with pruritus there was a 10-100 fold increase in the total bile acids (mean value z.qS’~/o) and a difference in the proportions among the means of the three bile acids. Thus, the ratios for deoxycholic, chenodeoxycholic and cholic acids were 0.1 : 1.0:q.s which is similar to the ratios for the bile acids in the bile of patients having obstructive hepatobiliary disorders lo. These results mav be compared with the tindings of Carey” and of Kudman and Kendall’” that there is an elevated trihydroxy/dihydroxy bile acid ratio in the serum of patients with obstructive jaundice. The serum bile acid pattern in pregnant women with pruritus is therefore in agreement with the results of other liver function tests and histologic findings which indicate that this condition is associated with cllolestasis2-7. It is also of interest to note that Arfwedson and ~‘on Studnitz ‘3 found elevated levels of phospholipids and cholesterol c~~ncomitant with an increased level of ~-lipoproteins in pregnant women with pruritus. It has been suggested that pruritus associated with biliary obstruction is due CYill. C’him. .+tcia, 1.3 (1006) 207 -2~ I
SERUM
BILE ACID
IN PREGNANCY
211
a retention of bile acids (see 8). However, this possibility has not yet been proved (ref. II, 14). The present study has demonstrated elevated levels of bile acids in pregnant women with pruritus in the last trimester. Although this finding is a sign of liver dysfunction it does not necessarily explain the pruritus. The ion exchange resin cholestyramine which has been given to relieve itching in patients with biliary cirrhosis15-17 has also been used successfullv in a few cases of pruritus associated with pregnancy2s3. Although this resin increases the fecal excretion of biie acids and lowers serum bile acid levels, it should be mentioned that it is also effective in binding conjugates of steroid hormones like dehydroepiandrosterone sulfatelR. The idiopathic jaundice of pregnancy has been compared with the jaundice that can be induced by certain steroids (especially r@kylated steroids) and it might therefore be speculated that the relief of itching that is obtained with cholestyramine could in addition be due to the binding of some biliary steroid conjugate other than the bile acids. Work is in progress to test this hypothesis.
to
ACKNOWLEDGEMENT
The technical assistance of Miss &laud Frankesten is gratefully ackowledged. This work was supported by a grant (I$%-z19-01) from the Swedish Medical Research Council. RE1’lZ RENCES