THROMBOSIS RESEARCH 60; 377-384,199O 0049-3848/90 $3.00 + .OOPrinted in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.
INCREASED PLACENTAL PRODUCTION OF LEUKOTRIENE B4 IN GESTATIONAL HYPERTENSION.
G.
Biagi,
V.
De Rosa, G. Sani
G. Pelusi *, G. * and S. Coccheri.
Scagliarini
Dept. Angiology and Blood Coagulation * 1st Clinic of Obstetrics and Gynecology. University Hospital S. Orsola - 40138 Bologna
*,
-
Italy.
(Received 29.5.1990; accepted in revised form 26.7.1990 by Editor G.G. Neri Seneri) (Received by Executive Editorial Office 24.9.1990) ABSTRACT
Production of some lipoxyqenase and cyclooxyqenase derivatives of arachidonic acid was measured in placental tissue obtained from women with gestational hypertension and with normal pregnancies. The levels of leukotriene B, were about five times higher in placentas from hypertensive women and also raised thromboxane AZ and reduced prostaqlandin E2 levels were Prostacyclin production was lowered observed. only in with more severe hypertension, In women association with the highest measured levels of leukotriene B4 and thromboxane AZ_ It is suggested that increased levels of leukotriene placental Bq and thromboxane A2 appear already in mild gestation41 hypertension, while depression of prostacyclin may occur only at more severe stages of gestational hypertensive disease. INTRODUCTION
Gestational hypertension has been reported to be associated with changes in arachidonate metabolites of the cyclooxyqenase pathway (1,Z). Increased biosynthesis of thromboxane A2 has been demonstrated in pregnancy induced hypertension and attributed to platelet activation in the placental circulation (3). Both thromboxane AZ and PG12 have been measured also in placental imbalance between these products in favour tissue (4-10) and an of thromboxane A2 has been found in placentas from women with gestational hypertension (4,8,10). As involvement of lipoxyqenase products has been recently suq:zsted (ll-14), we --__--___---___--Key words: Gestational hypertensior, placenta, leukotriene B4, prostanoids. 377
30
31
35
26
27
25
26
28
35
2
3
4
5
6
7
8
9
Age (years)
1
Patient
>llO
>90
>90
>90
>90
>120
>120
>120
>llO
Diastolic pressure(mmHg)
3.0
traces
traces
traces
absent
3.0
0.5
2-3
2.5
Proteinuria (g/l/24h)
37
39
31
38
39
29
32
32
36
Gestation week at delivery
2920
3240
2900
3250
3100
920
1170
1200
1700
390
350
380
600
400
180
150
330
300
Placental weight (gm)
hypertension.
Infant birth weight (Kg)
of our patients with gestational
~_--_------~~~--~~---~-~~~~~_~~~-~~-~-~--~~~~~---_-~~-----~__-______--_______________
Some characteristics
TABLE 1
Vol. 60, No. 5 measured metabolites obtained
LTB*AND PREGNANCY HYPERTENSION
379
in
this study both cyclooxygenase and lipoxygenase of arachidonate in samples of placental tissue from women with gestational hypertension. PATIENTS AND METHODS
Patient5 Ten women with gestational hypertension and thirteen with normal referred to the 1st Clinic of Obstetrics and pregnancy, Gynecology of our University Hospital , were investigated. Gestational hypertensive women were aged 26 to 35 years and women with physiological pregnancies 20 to 38 years. Diagnosis according to the following of gestational hypertension was made one measurement of criteria (15): diastolic blood pressure 2110 consecutive measurements of diastolic mm Hg or two blood pressure of 9Omm Hg4 or more hours apart with or without proteinuria (i.e. urinary protein > 300 mg/1/24 hours urine collection). “Severe” hypertension was classified according to following criteria (15): diastolic blood pressure 2120 mm Hg on any occasion or diastolic blood pressure 1110 mm Hg on two or L4 more consecutive occasions hours apart with or without proteinuria. Clinical details of the hypertensive patients are shown in table 1. Hypertensive women delivered by caesarean section whereas normal pregnant women either by vaginal route or caesarean section. There were two perinatal deaths in the hypertensive group. Normal pregnant women had no overt medical complications such as diabetes, chronic infection, metabolic disease, nor obstetric complications as neonatal abnormalities or 01 igoand polyhydramnios. Placental fragments Fragments of placental tissue (ZOO-300 mg wet weight) were dissected from placentas obtained immediately after delivery and washed three times with ice-cold saline to remove contaminating blood and tissue fluids. The tissue then was chopped and incubated in Tris-HCl buffer pH=7.5 at 25°C for 60 min. Incubation was cooling of stopped by the test tube on ice followed by addition of 0.1 mM acetylsalicylic acid. Thereafter, tubes were centrifuged at 4°C at 1500xg for 5 min to sediment the tissue. The supernatant, collected and frozen at -80°C was processed within one month. A fragment of placental tissue after saline washing was fixed in a buffered 10% formaldehyde solution for microscopic checking of presence of leukocytes. Laboratorv investisations Prostacyclin as the stable metabolite 6-keto-PGF1,, thromboxane and prostaAi! as the stable metabolite thromboxane Bt (TXBz) glandin E2 were radioimmunological assays (16) measured by using commercial kits (New England Nuclear, Boston, U.S.). Intra and interassay variation coefficients were respectively 8.5% and 11.4 % for 6-keto-PGF1,, 9.0% and 9.2% and 10.7% for TXBz and 11.5% for PGEz measurements.
LTBhAND PREGNANCY HYPERTENSION
380
Vol. 60, No. 5
Leukotriene B1 was also assessed by a radioimmunological assay according to Salmon et al. (17) using a commercial antibody (TRK 980 Amersham International, Buckinghamshire, U.K.) and tritiated LTBI (Amersham). Specificity of this antibody for LTBI has by high-performance liquid chromatorecently been validated coefficients were graphy (18). Intra and interassay variation respectively 9.2% and 11.3 % . . Possible contribution of residual platelets to the measured levels of prostanoids was checked by measuring the levels of Pthromboglobulin in the supernatant of placental tissue, by an immunoenzimatic method (ELISA) using a commercial test kit (Boehringer Mannheim, Milan). Possible contamination of tissue fragment by circulating polymorphonuclear leukocytes as a source of LTBr WFiS examination of placental checked by microscopic fragments. Statistical difference between the groups was evaluated by a non parametric test (Wilcoxon Rank Sum Test). RESULTS Significant differences (~(0.05) between hypertensive and normal observed pregnancies were for placental weight (341k118 vs 536+87 gm, mean 'SD), fetal weight (2.25+0.90 vs 3.28+0.39 Kg), weeks of gestation at delivery (3524 vs 39+1). Production of the arachidonic acid metabolites related both to and lipoxygenase the cyclooxygenase pathways in placentas of normal and hypertensive pregnancies is reported in table 2. TABLE 2 Production of eicosanoids (pg/mg wet tissue) by placental fragments obtained from normal and hypertensive pregnant women.
Normal pregnancies
6-k-PGF1, LTBa PGEz TXBz ---_-----_-----_--------~-~-----~-------~~-~-~---~ 158t52 6.621.8 40.8t14.6 4.421.9 (n=13)
133*57 All hypertensive pregnancies (n=lO) Severe subgroup
70*7##
28.1?9.7**
17.4+7.6*X
21.4'14.7**
38.70+6.2#
10.2+-3.3#
39.3'3.16##
(n=4)
175231 21.0t2.3 9.5tO.6 Mild 22.2k5.6 subgroup (n=6) _---------_~----~~---~~~-~~~~---~~~-~-~~~---~~---~~~--~~~~--~~~~ one symbol: p< 0.05; two symbols: p< 0.01. all hypertensive vs normal pregnant women. severe vs mild gestational hypertensive subgroup.
Vol. 60, No. 5
381
LTBdAND PREGNANCY HYPERTENSION
the group of hyper6-keto-PGF1, in Placental production of not significantly different from that of tensive patients was in 4 patients with severe hypernormal pregnant women. However, 6-keto-PGFI, were significantly tension the placental levels of Similarly PGEt production was signicantly lower than normal. in the in the hypertensive than normal lower (p < 0.01) lowest values being recorded in the 4 pregnant group, the severely hypertensive subjects. The values of TXBz and LTB4 were significantly higher in the group of hypertensive than in the normal pregnant women (p
INCREASED PLACENTAL PRODUCTION OF LEUKOTRIENE B4 IN GESTATIONAL HYPERTENSION.
G.
Biagi,
V.
De Rosa, G. Sani
G. Pelusi *, G. * and S. Coccheri.
Scagliarini
Dept. Angiology and Blood Coagulation * 1st Clinic of Obstetrics and Gynecology. University Hospital S. Orsola - 40138 Bologna
*,
-
Italy.
(Received 29.5.1990; accepted in revised form 26.7.1990 by Editor G.G. Neri Seneri) (Received by Executive Editorial Office 24.9.1990) ABSTRACT
Production of some lipoxyqenase and cyclooxyqenase derivatives of arachidonic acid was measured in placental tissue obtained from women with gestational hypertension and with normal pregnancies. The levels of leukotriene B, were about five times higher in placentas from hypertensive women and also raised thromboxane AZ and reduced prostaqlandin E2 levels were Prostacyclin production was lowered observed. only in with more severe hypertension, In women association with the highest measured levels of leukotriene B4 and thromboxane AZ_ It is suggested that increased levels of leukotriene placental Bq and thromboxane A2 appear already in mild gestation41 hypertension, while depression of prostacyclin may occur only at more severe stages of gestational hypertensive disease. INTRODUCTION
Gestational hypertension has been reported to be associated with changes in arachidonate metabolites of the cyclooxyqenase pathway (1,Z). Increased biosynthesis of thromboxane A2 has been demonstrated in pregnancy induced hypertension and attributed to platelet activation in the placental circulation (3). Both thromboxane AZ and PG12 have been measured also in placental imbalance between these products in favour tissue (4-10) and an of thromboxane A2 has been found in placentas from women with gestational hypertension (4,8,10). As involvement of lipoxyqenase products has been recently suq:zsted (ll-14), we --__--___---___--Key words: Gestational hypertensior, placenta, leukotriene B4, prostanoids. 377
30
31
35
26
27
25
26
28
35
2
3
4
5
6
7
8
9
Age (years)
1
Patient
>llO
>90
>90
>90
>90
>120
>120
>120
>llO
Diastolic pressure(mmHg)
3.0
traces
traces
traces
absent
3.0
0.5
2-3
2.5
Proteinuria (g/l/24h)
37
39
31
38
39
29
32
32
36
Gestation week at delivery
2920
3240
2900
3250
3100
920
1170
1200
1700
390
350
380
600
400
180
150
330
300
Placental weight (gm)
hypertension.
Infant birth weight (Kg)
of our patients with gestational
~_--_------~~~--~~---~-~~~~~_~~~-~~-~-~--~~~~~---_-~~-----~__-______--_______________
Some characteristics
TABLE 1
Vol. 60, No. 5 measured metabolites obtained
LTB*AND PREGNANCY HYPERTENSION
379
in
this study both cyclooxygenase and lipoxygenase of arachidonate in samples of placental tissue from women with gestational hypertension. PATIENTS AND METHODS
Patient5 Ten women with gestational hypertension and thirteen with normal referred to the 1st Clinic of Obstetrics and pregnancy, Gynecology of our University Hospital , were investigated. Gestational hypertensive women were aged 26 to 35 years and women with physiological pregnancies 20 to 38 years. Diagnosis according to the following of gestational hypertension was made one measurement of criteria (15): diastolic blood pressure 2110 consecutive measurements of diastolic mm Hg or two blood pressure of 9Omm Hg4 or more hours apart with or without proteinuria (i.e. urinary protein > 300 mg/1/24 hours urine collection). “Severe” hypertension was classified according to following criteria (15): diastolic blood pressure 2120 mm Hg on any occasion or diastolic blood pressure 1110 mm Hg on two or L4 more consecutive occasions hours apart with or without proteinuria. Clinical details of the hypertensive patients are shown in table 1. Hypertensive women delivered by caesarean section whereas normal pregnant women either by vaginal route or caesarean section. There were two perinatal deaths in the hypertensive group. Normal pregnant women had no overt medical complications such as diabetes, chronic infection, metabolic disease, nor obstetric complications as neonatal abnormalities or 01 igoand polyhydramnios. Placental fragments Fragments of placental tissue (ZOO-300 mg wet weight) were dissected from placentas obtained immediately after delivery and washed three times with ice-cold saline to remove contaminating blood and tissue fluids. The tissue then was chopped and incubated in Tris-HCl buffer pH=7.5 at 25°C for 60 min. Incubation was cooling of stopped by the test tube on ice followed by addition of 0.1 mM acetylsalicylic acid. Thereafter, tubes were centrifuged at 4°C at 1500xg for 5 min to sediment the tissue. The supernatant, collected and frozen at -80°C was processed within one month. A fragment of placental tissue after saline washing was fixed in a buffered 10% formaldehyde solution for microscopic checking of presence of leukocytes. Laboratorv investisations Prostacyclin as the stable metabolite 6-keto-PGF1,, thromboxane and prostaAi! as the stable metabolite thromboxane Bt (TXBz) glandin E2 were radioimmunological assays (16) measured by using commercial kits (New England Nuclear, Boston, U.S.). Intra and interassay variation coefficients were respectively 8.5% and 11.4 % for 6-keto-PGF1,, 9.0% and 9.2% and 10.7% for TXBz and 11.5% for PGEz measurements.
LTBhAND PREGNANCY HYPERTENSION
380
Vol. 60, No. 5
Leukotriene B1 was also assessed by a radioimmunological assay according to Salmon et al. (17) using a commercial antibody (TRK 980 Amersham International, Buckinghamshire, U.K.) and tritiated LTBI (Amersham). Specificity of this antibody for LTBI has by high-performance liquid chromatorecently been validated coefficients were graphy (18). Intra and interassay variation respectively 9.2% and 11.3 % . . Possible contribution of residual platelets to the measured levels of prostanoids was checked by measuring the levels of Pthromboglobulin in the supernatant of placental tissue, by an immunoenzimatic method (ELISA) using a commercial test kit (Boehringer Mannheim, Milan). Possible contamination of tissue fragment by circulating polymorphonuclear leukocytes as a source of LTBr WFiS examination of placental checked by microscopic fragments. Statistical difference between the groups was evaluated by a non parametric test (Wilcoxon Rank Sum Test). RESULTS Significant differences (~(0.05) between hypertensive and normal observed pregnancies were for placental weight (341k118 vs 536+87 gm, mean 'SD), fetal weight (2.25+0.90 vs 3.28+0.39 Kg), weeks of gestation at delivery (3524 vs 39+1). Production of the arachidonic acid metabolites related both to and lipoxygenase the cyclooxygenase pathways in placentas of normal and hypertensive pregnancies is reported in table 2. TABLE 2 Production of eicosanoids (pg/mg wet tissue) by placental fragments obtained from normal and hypertensive pregnant women.
Normal pregnancies
6-k-PGF1, LTBa PGEz TXBz ---_-----_-----_--------~-~-----~-------~~-~-~---~ 158t52 6.621.8 40.8t14.6 4.421.9 (n=13)
133*57 All hypertensive pregnancies (n=lO) Severe subgroup
70*7##
28.1?9.7**
17.4+7.6*X
21.4'14.7**
38.70+6.2#
10.2+-3.3#
39.3'3.16##
(n=4)
175231 21.0t2.3 9.5tO.6 Mild 22.2k5.6 subgroup (n=6) _---------_~----~~---~~~-~~~~---~~~-~-~~~---~~---~~~--~~~~--~~~~ one symbol: p< 0.05; two symbols: p< 0.01. all hypertensive vs normal pregnant women. severe vs mild gestational hypertensive subgroup.
Vol. 60, No. 5
381
LTBdAND PREGNANCY HYPERTENSION
the group of hyper6-keto-PGF1, in Placental production of not significantly different from that of tensive patients was in 4 patients with severe hypernormal pregnant women. However, 6-keto-PGFI, were significantly tension the placental levels of Similarly PGEt production was signicantly lower than normal. in the in the hypertensive than normal lower (p < 0.01) lowest values being recorded in the 4 pregnant group, the severely hypertensive subjects. The values of TXBz and LTB4 were significantly higher in the group of hypertensive than in the normal pregnant women (p
