British Journal of Obstetrics and Gynaecology November 1976. Vol83. pp 879-884
PLACENTAL TRANSFER AND FETAL UPTAKE OF 3H-DTGOXIN IN HUMANS BY
S. SAARIKOSKI Department I1 of Obstetrics and Gynaecology, University Central Hospital 00290 Helsinki 29, Finland
Summary Placental transmission and fetal distribution of 3H-digoxin were studied in seven pregnant women undergoing legal termination of pregnancy during the first half of gestation. The radioactivity in fetal and maternal plasma and in fetal tissues was estimated using the oxidation method, and the integrity of the labelled drug by thin layer chromatography. The 3H-digoxin activity was clearly demonstrated in the umbilical cord blood five minutes after injection of the drug into the maternal blood, and the fetal plasma concentrations of 3H-digoxin approximated to the maternal value 30 minutes after drug administration. The distribution of 3H-digoxin in the fetal tissues was relatively even, with the highest 3H concentrations found in the heart and placenta, the lowest in the brain. The results suggest that the capacity of human fetal heart to bind digoxin during the first half of gestation is limited.
METHODS Seven pregnant women, admitted for legal termination of pregnancy, volunteered for the experiment. Their pregnancies had proceeded without medical complications and were terminated on social grounds. The number of weeks of pregnancy at the time of surgical interruption ranged from 15 to 22, according to obstetric history and fetal size. 3H-digoxin (Radiochemical Centre, Amersham, England, specific activity 6 * 2 Ci/mmol) was injected without further carrier into the maternal antecubital vein in a single dose of 5 pCi/lO kg or 0.6 pg/lO kg of maternal body weight. According to the International Commission on Radiological Protection the maximum dosage of 3H is 1 mCi; in the present experiments only about three per cent of this dosage was used. The patients received promethazine, pethidine and atropine as premedication. Anaesthesia was induced with thiopentone and after endotracheal
ALTHOUGH the number of pregnant women using digitalis glycosides has increased since the advent of open heart surgery and the availability of prosthetic valves, reports on the transplacental passage of digitalis glycosides in man and experimental animals are few (Okita et al, 1952 and 1956; Sherman and Locke, 1960; Rogers et al, 1972; Fouron, 1973; Mirkin, 1973; Hernandez et al, 1973 and 1975). From these it is obvious that the placenta does not function as a barrier protecting the fetus against digitalis. If digoxin is able to reach the fetus easily, it may be possible in some cases to treat the fetus in utero by injecting the drug into the maternal blood stream, or the amniotic fluid, when fetal paroxysmal tachycardia, cardiac disorders or possibly fetal asphyxia are involved. However, the information available about the kinetics of placental transfer of digitalis glycosides has been too scanty for the normal clinical use of digoxin or other heart glycosides in fetal medical care. 879
880
SAARIKOSKI
intubation the lungs were ventilated automatically, anaesthesia being maintained with a nitrous oxide-oxygen mixture ; suxamethonium was used as a muscle relaxant. Abortion was performed by hysterotomy 5 minutes to 24 hours after the i njection of the drug. At the time of hysterotomy, specimens were collected of maternal venous blood, amniotic fluid, fetal umbilical arterial and venous blood and fetal tissues. The placenta and the fetus were weighed, and the fetal tissues were dissected out. The organs, and an aliquot of the plasma and amniotic fluid, were dried at 4 "C. After drying, the samples were combusted using the sample oxidizer of Kaartinen (1969), and the quantity of 3H,O produced was determined by liquid scintillation counting. The scintillation liquid used was dioxane : toluene : naphthalene : PPO : dimethyl-POPOP (800 ml : 200 ml : 95 g : 5 . 0 g : 0 - 3 g). The liquid scintillation counter was an LKB-Wallac 81000 and the counting time 20 minutes. The counting efficiency was determined by means of external standardization based on a quenching curve obtained by adding water and ethanol. The counting efficiency was between 35 and 45 per cent. The lowest count per minute (cpm) values (in the brain samples) were about twice the background values of between 20 and 40 cpm. The recovery of this method for 3H-activity is 9 9 . 0 4 ~ 2 . 4per cent (Meanf SD) (Saarikoski, 1974). In order to verify the integrity of the sH-digoxin, thin layer chromatography was
carried out on aliquots of fetal and maternal blood samples and fetal heart tissue, The proteins were precipitated with ethanol after homogenization of the tissue or centrifugation of the blood. Eighty to 85 per cent of radioactivity was recovered in the supernatants after precipitation with ethanol. Supernatants were used for further analyses. After centrifugation, 25 pg of unlabelled digoxin was added to a 2 ml aliquot of the supernatant as a carrier substance. This mixture was spotted on the silica gel-plates (Kieselgel-F,,,, Merck) in a 10 cm broad track, and the chromatography was carried out using ethylacetate-methanol-water (90 : 12 : 9), and, if the material was sufficient, also using ethylacetate-pyridine-water (75 : 15 : 60) as a solvent system. The digoxin was demonstrated with anisaldehyde reagent. The Rf-value for digoxin with the first solvent system was 0.51 and with the second 0.53. The percentage distribution of 3H-digoxin and its possible metabolites were calculated by dividing the plate into 10 fractions, scraping off the silica gel and estimating the 3H-activity of every band by liquid scintillation counting after elution in ethanol. RESULTS The 3H-activity in maternal and fetal plasma is expressed as nanocuries (nCi)/ml and in the tissues as nCi/g or nCi/organ. Plasma concentrations Table I shows the maternal and fetal 3H-digoxin concentrations with respective ratios
TABLE I 3H-concentrations in the maternal and fetal plasma afrer administration of $H-digoxin to seven women
3H-concentrations (nCi/ml) Time after administration of 3H-digoxin (minutes) 5 15 30
60 90 240
1440
Ratios
Maternal vein
Umbilical vein
Umbilical artery
0.96 0.70 0.61 0.42 0.24 0.25 0.35
0.50 0.63 0.57 0.36 0.20 0.27 0.44
0.46 0.47 0.58 0.43 0.22 0.18 0,38
Umbilical vein
Umbilical artery
Maternal vein
Maternal vein
0.52 0.90 0 .9 3 0.86 0.83 1.08 1.22
0.48 0.67 0.95 1.02 0.92 0.72 1.09
DIGOXIN TRANSFER AND UPTAKE
in each of the seven patients, ranked by the times after the administration of the drug. The maternal plasma concentration diminished from 0.96 nCi/ml to 0.24 at 90 minutes. The
881
3H-concentration in the umbilical vein within 5 minutes of the administration of the drug was 0.50 nCi/ml or 52 per cent of the maternal value and in the umbilical artery nearly the same.
TABLE TI Comparison of the 3H-activity found in the umbilical vetwus plasma, amniotic Puid and fetal urine after adminisiration of 3H-digoxin to seven women ________ 3H-activit~ _ (nCi/ml) . . , Time after Gestation Fetal weight Urine volume (weeks) administration of (ml) 3H-digoxin (minutes) ()' Umbilical vein Amniotic fluid Fetal urine ______
5 15
30 60 90 240 1440
19 21 15 16 22 18 19
299 384 66 113 496 165 246
0.05
0.50 0.63 0.57 0.36 0.20 0.27 0.44
0.00 0.16 0.80 0.25 0.76 0.04 0.03
0.04
0.05 0.05 0.11 0.12 0.03
0.55 0.40 0.17 0.45 0.85 0.50 0.60
TABLE 111 3H-concentrationsand total contents in placenta and fetal tissues after administration of3H-digoxinto the mother. Individual values are given in nCi/g and nCi/organ
__
Time after administration of 3H-digoxin (minutes)
5
15
30
60
90
240
19
21
15
16
22
18
19
299
384
66
113
496
165
264
0.60 104.3
0.34 60.9
0.22 14.8
0.18 21.9
0.30 51.3
0.22 28.9
0.08 11.9
0.20 0.29
0.24 0.51
0.11 0.04
0.09 0.12
0.30 0.44
0.49 0.41
0.58 0.81
0.19 2.26
0.15 2.23
0.12 0.28
0.09 0.43
0.15 2.79
0.14 0.89
0.15 1.42
0.09 0.99
0.06 0.70
0.06 0.17
0.03 0.14
0.08 0.91
0.09 0.43
0.08 0.79
0.09 0.42
0.11 0.77
0.08 0.11
0.08 0.12
0.18 2.20
0.16 0.42
0.12 0.57
0.10 0.29
0.10 0.36
0.08 0.05
0.11 0.11
0.20 1.10
0.07 0.09
0.17 0.42
0.03 0.92
0.02 1* l o
0.04
0.03 0.41
0.02 1.40
0.05 1.29
0.02 0-63
~~
1440 ~~
Gestation (weeks) Fetal weight (g) Placenta nCi/g nCi/organ Heart nCi/g nCi/organ Liver nCi/g nCi/organ Lungs nCi/g nCi/organ Intestine nCi/g nCi/organ Kidneys nCi/g nCi/organ Brain nCi/g nCi/organ 33
0.33
882
SAARIKOSKI
Thirty minutes after the injection of the drug, and thereafter, the 3H-activity was about the same in the maternal and fetal blood. Table I1 shows the 3H-concentrations found in the fetal plasma, amniotic fluid and in the fetal urine. The3H-concentrations in the amniotic fluid were quite low in comparison with the fetal umbilical plasma. The highest values, 0.11 and 0.12 nCi/ml, were about 50 per cent of the corresponding umbilical plasma value. 3H-activity was found in fetal urine 15 minutes after the drug administration.
Distribution of 3H-digoxin in the placenta and fetaI tissues Tissue concentrations of "-digoxin (nCi/g) and total 3H-content per organ (nCi) are shown in the Table TIT. 3H-concentration was highest in the placenta during the first 90 minutes after the drug administration, and in the fetal tissues the highest concentration was seen in the heart,
except in the two smallest fetuses. The activity was relatively evenly distributed between the various tissues, although the brain was con sistently lower than the rest. The placenta was the organ showing the highest total content of 3H. By conipaiison, the sum total "-activity in all six fetal organs examined was only 5 to 17 per cent of that found in the corresponding placenta at 5 to 240 minutes after the administration of the drug, and 39 per cent in the samples taken after 24 hours.
The integrity of "H-digoxin Tt was found (Figs 1 and 2) that the activity observed in the fetus moved mainly to the same point in the chromatogram as the carrierdigoxin; 77-2f 7. 7 per cent of the total activity in the heart and 6 2 . 4 h 3 . 9 per cent in the fetal plasma. In the fetus a rather higher proportion of the activity stayed on the starting point than in the maternal blood, in which the activity,
5 min o 15 n i n
4
*
240 nin
*
Heart 0 Plasma a
T
80
60
I
>
-..->
c
0
PLACENTAL TRANSFER AND FETAL UPTAKE OF 3H-DTGOXIN IN HUMANS BY
S. SAARIKOSKI Department I1 of Obstetrics and Gynaecology, University Central Hospital 00290 Helsinki 29, Finland
Summary Placental transmission and fetal distribution of 3H-digoxin were studied in seven pregnant women undergoing legal termination of pregnancy during the first half of gestation. The radioactivity in fetal and maternal plasma and in fetal tissues was estimated using the oxidation method, and the integrity of the labelled drug by thin layer chromatography. The 3H-digoxin activity was clearly demonstrated in the umbilical cord blood five minutes after injection of the drug into the maternal blood, and the fetal plasma concentrations of 3H-digoxin approximated to the maternal value 30 minutes after drug administration. The distribution of 3H-digoxin in the fetal tissues was relatively even, with the highest 3H concentrations found in the heart and placenta, the lowest in the brain. The results suggest that the capacity of human fetal heart to bind digoxin during the first half of gestation is limited.
METHODS Seven pregnant women, admitted for legal termination of pregnancy, volunteered for the experiment. Their pregnancies had proceeded without medical complications and were terminated on social grounds. The number of weeks of pregnancy at the time of surgical interruption ranged from 15 to 22, according to obstetric history and fetal size. 3H-digoxin (Radiochemical Centre, Amersham, England, specific activity 6 * 2 Ci/mmol) was injected without further carrier into the maternal antecubital vein in a single dose of 5 pCi/lO kg or 0.6 pg/lO kg of maternal body weight. According to the International Commission on Radiological Protection the maximum dosage of 3H is 1 mCi; in the present experiments only about three per cent of this dosage was used. The patients received promethazine, pethidine and atropine as premedication. Anaesthesia was induced with thiopentone and after endotracheal
ALTHOUGH the number of pregnant women using digitalis glycosides has increased since the advent of open heart surgery and the availability of prosthetic valves, reports on the transplacental passage of digitalis glycosides in man and experimental animals are few (Okita et al, 1952 and 1956; Sherman and Locke, 1960; Rogers et al, 1972; Fouron, 1973; Mirkin, 1973; Hernandez et al, 1973 and 1975). From these it is obvious that the placenta does not function as a barrier protecting the fetus against digitalis. If digoxin is able to reach the fetus easily, it may be possible in some cases to treat the fetus in utero by injecting the drug into the maternal blood stream, or the amniotic fluid, when fetal paroxysmal tachycardia, cardiac disorders or possibly fetal asphyxia are involved. However, the information available about the kinetics of placental transfer of digitalis glycosides has been too scanty for the normal clinical use of digoxin or other heart glycosides in fetal medical care. 879
880
SAARIKOSKI
intubation the lungs were ventilated automatically, anaesthesia being maintained with a nitrous oxide-oxygen mixture ; suxamethonium was used as a muscle relaxant. Abortion was performed by hysterotomy 5 minutes to 24 hours after the i njection of the drug. At the time of hysterotomy, specimens were collected of maternal venous blood, amniotic fluid, fetal umbilical arterial and venous blood and fetal tissues. The placenta and the fetus were weighed, and the fetal tissues were dissected out. The organs, and an aliquot of the plasma and amniotic fluid, were dried at 4 "C. After drying, the samples were combusted using the sample oxidizer of Kaartinen (1969), and the quantity of 3H,O produced was determined by liquid scintillation counting. The scintillation liquid used was dioxane : toluene : naphthalene : PPO : dimethyl-POPOP (800 ml : 200 ml : 95 g : 5 . 0 g : 0 - 3 g). The liquid scintillation counter was an LKB-Wallac 81000 and the counting time 20 minutes. The counting efficiency was determined by means of external standardization based on a quenching curve obtained by adding water and ethanol. The counting efficiency was between 35 and 45 per cent. The lowest count per minute (cpm) values (in the brain samples) were about twice the background values of between 20 and 40 cpm. The recovery of this method for 3H-activity is 9 9 . 0 4 ~ 2 . 4per cent (Meanf SD) (Saarikoski, 1974). In order to verify the integrity of the sH-digoxin, thin layer chromatography was
carried out on aliquots of fetal and maternal blood samples and fetal heart tissue, The proteins were precipitated with ethanol after homogenization of the tissue or centrifugation of the blood. Eighty to 85 per cent of radioactivity was recovered in the supernatants after precipitation with ethanol. Supernatants were used for further analyses. After centrifugation, 25 pg of unlabelled digoxin was added to a 2 ml aliquot of the supernatant as a carrier substance. This mixture was spotted on the silica gel-plates (Kieselgel-F,,,, Merck) in a 10 cm broad track, and the chromatography was carried out using ethylacetate-methanol-water (90 : 12 : 9), and, if the material was sufficient, also using ethylacetate-pyridine-water (75 : 15 : 60) as a solvent system. The digoxin was demonstrated with anisaldehyde reagent. The Rf-value for digoxin with the first solvent system was 0.51 and with the second 0.53. The percentage distribution of 3H-digoxin and its possible metabolites were calculated by dividing the plate into 10 fractions, scraping off the silica gel and estimating the 3H-activity of every band by liquid scintillation counting after elution in ethanol. RESULTS The 3H-activity in maternal and fetal plasma is expressed as nanocuries (nCi)/ml and in the tissues as nCi/g or nCi/organ. Plasma concentrations Table I shows the maternal and fetal 3H-digoxin concentrations with respective ratios
TABLE I 3H-concentrations in the maternal and fetal plasma afrer administration of $H-digoxin to seven women
3H-concentrations (nCi/ml) Time after administration of 3H-digoxin (minutes) 5 15 30
60 90 240
1440
Ratios
Maternal vein
Umbilical vein
Umbilical artery
0.96 0.70 0.61 0.42 0.24 0.25 0.35
0.50 0.63 0.57 0.36 0.20 0.27 0.44
0.46 0.47 0.58 0.43 0.22 0.18 0,38
Umbilical vein
Umbilical artery
Maternal vein
Maternal vein
0.52 0.90 0 .9 3 0.86 0.83 1.08 1.22
0.48 0.67 0.95 1.02 0.92 0.72 1.09
DIGOXIN TRANSFER AND UPTAKE
in each of the seven patients, ranked by the times after the administration of the drug. The maternal plasma concentration diminished from 0.96 nCi/ml to 0.24 at 90 minutes. The
881
3H-concentration in the umbilical vein within 5 minutes of the administration of the drug was 0.50 nCi/ml or 52 per cent of the maternal value and in the umbilical artery nearly the same.
TABLE TI Comparison of the 3H-activity found in the umbilical vetwus plasma, amniotic Puid and fetal urine after adminisiration of 3H-digoxin to seven women ________ 3H-activit~ _ (nCi/ml) . . , Time after Gestation Fetal weight Urine volume (weeks) administration of (ml) 3H-digoxin (minutes) ()' Umbilical vein Amniotic fluid Fetal urine ______
5 15
30 60 90 240 1440
19 21 15 16 22 18 19
299 384 66 113 496 165 246
0.05
0.50 0.63 0.57 0.36 0.20 0.27 0.44
0.00 0.16 0.80 0.25 0.76 0.04 0.03
0.04
0.05 0.05 0.11 0.12 0.03
0.55 0.40 0.17 0.45 0.85 0.50 0.60
TABLE 111 3H-concentrationsand total contents in placenta and fetal tissues after administration of3H-digoxinto the mother. Individual values are given in nCi/g and nCi/organ
__
Time after administration of 3H-digoxin (minutes)
5
15
30
60
90
240
19
21
15
16
22
18
19
299
384
66
113
496
165
264
0.60 104.3
0.34 60.9
0.22 14.8
0.18 21.9
0.30 51.3
0.22 28.9
0.08 11.9
0.20 0.29
0.24 0.51
0.11 0.04
0.09 0.12
0.30 0.44
0.49 0.41
0.58 0.81
0.19 2.26
0.15 2.23
0.12 0.28
0.09 0.43
0.15 2.79
0.14 0.89
0.15 1.42
0.09 0.99
0.06 0.70
0.06 0.17
0.03 0.14
0.08 0.91
0.09 0.43
0.08 0.79
0.09 0.42
0.11 0.77
0.08 0.11
0.08 0.12
0.18 2.20
0.16 0.42
0.12 0.57
0.10 0.29
0.10 0.36
0.08 0.05
0.11 0.11
0.20 1.10
0.07 0.09
0.17 0.42
0.03 0.92
0.02 1* l o
0.04
0.03 0.41
0.02 1.40
0.05 1.29
0.02 0-63
~~
1440 ~~
Gestation (weeks) Fetal weight (g) Placenta nCi/g nCi/organ Heart nCi/g nCi/organ Liver nCi/g nCi/organ Lungs nCi/g nCi/organ Intestine nCi/g nCi/organ Kidneys nCi/g nCi/organ Brain nCi/g nCi/organ 33
0.33
882
SAARIKOSKI
Thirty minutes after the injection of the drug, and thereafter, the 3H-activity was about the same in the maternal and fetal blood. Table I1 shows the 3H-concentrations found in the fetal plasma, amniotic fluid and in the fetal urine. The3H-concentrations in the amniotic fluid were quite low in comparison with the fetal umbilical plasma. The highest values, 0.11 and 0.12 nCi/ml, were about 50 per cent of the corresponding umbilical plasma value. 3H-activity was found in fetal urine 15 minutes after the drug administration.
Distribution of 3H-digoxin in the placenta and fetaI tissues Tissue concentrations of "-digoxin (nCi/g) and total 3H-content per organ (nCi) are shown in the Table TIT. 3H-concentration was highest in the placenta during the first 90 minutes after the drug administration, and in the fetal tissues the highest concentration was seen in the heart,
except in the two smallest fetuses. The activity was relatively evenly distributed between the various tissues, although the brain was con sistently lower than the rest. The placenta was the organ showing the highest total content of 3H. By conipaiison, the sum total "-activity in all six fetal organs examined was only 5 to 17 per cent of that found in the corresponding placenta at 5 to 240 minutes after the administration of the drug, and 39 per cent in the samples taken after 24 hours.
The integrity of "H-digoxin Tt was found (Figs 1 and 2) that the activity observed in the fetus moved mainly to the same point in the chromatogram as the carrierdigoxin; 77-2f 7. 7 per cent of the total activity in the heart and 6 2 . 4 h 3 . 9 per cent in the fetal plasma. In the fetus a rather higher proportion of the activity stayed on the starting point than in the maternal blood, in which the activity,
5 min o 15 n i n
4
*
240 nin
*
Heart 0 Plasma a
T
80
60
I
>
-..->
c
0
