PRENATAL DIAGNOSIS

Prenat Diagn 2002; 22: 111–113.

DOI: 10.1002 / pd.254

Fetal, neonatal cord, and maternal plasma concentrations of

angiotensin-converting enzyme (ACE)
Thomas Walther1*, Renaldo Faber2, Björn Maul3, Heinz-Peter Schultheiss1, Wolf-E. Siems3 and Holger Stepan2
1
Department of Cardiology and Pneumology, Free University of Berlin, Berlin, Germany
2
Department of Obstetrics and Gynaecology, University of Leipzig, Leipzig, Germany
3
Institute of Molecular Pharmacology, Berlin, Germany

Objectives Angiotensin-converting enzyme (ACE), a component of the renin–angiotensin system (RAS),

catalyses the degradation of angiotensin I to angiotensin II. It was the aim of the present study to measure
ACE activity in human fetal blood and to determine its changes with advancing gestational age.

Methods Fetal blood was sampled by cordocentesis from six control fetuses and six fetuses with Rh
isoimmunisation. Cord blood was sampled from six preterm neonates, 15 neonates after spontaneous
delivery at term and six neonates at term after caesarean section. In addition, maternal ACE values were
determined. ACE activity was measured using the miniaturised fluorimetric method.

Results In normal fetuses (13.31t1.41 nmol HL/min/ml) and fetuses with Rh isoimmunisation
(13.08t2.00 nmol HL/min/ml) ACE activity was significantly higher than in corresponding maternal
plasma (10.39t0.93 nmol HL/min/ml, p<0.05). Neonatal cord blood of preterm newborns
(10.43t0.69 nmol HL/min/ml) and term newborns (8.99t0.49 nmol HL/min/ml) showed a significantly
decreased ACE activity compared to the fetal controls.

Conclusion We conclude that the high fetal ACE activity and the stringent regulation with advancing
gestational age indicate the physiological importance of the enzyme during prenatal development.
Copyright # 2002 John Wiley & Sons, Ltd.
KEY WORDS: angiotensin-converting enzyme (ACE); Rh isoimmunisation; peplidase activity; cordocentesis

INTRODUCTION RAS, including ACE, in the fetoplacental unit that is

The role of endogenous vasoactive peptides as
regulators of cardiac function in health and disease is
an emerging area of fetal cardiovascular research.
Components of the renin–angiotensin system (RAS)
have been found to change during ontogenesis in
humans and animals. Since these variations occur in
the prenatal and postnatal period, this system has a
relevant adaptive function during fetal development
and after birth.
Angiotensin-converting enzyme (ACE), an impor-
tant component of the RAS, is a dipeptidyl carbo-
xypeptidase and catalyses the degradation of the
decapeptide angiotensin I (A I) to the octapeptide
angiotensin II (A II), a potent vasopressor, at the
histidyl-leucine (His-Leu) dipeptide. Although the lung
is the major site for the conversion of A I to A II,
plasma ACE activity can be measured and reflects the
production of ACE in vascular endothelial cells and
the catalytic activity of this enzyme in the peripheral
circulation.
A number of studies provide evidence for an active
*Correspondence to: T. Walther, University Hospital Benjamin
Franklin, Department of Cardiology and Pneumology, Free Uni-
versity of Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany.
E-mail: thomas.walther@ukbf.fu-berlin.de
involved in the modulation of placental perfusion and
fetal haemodynamics.
Rat fetuses showed higher ACE plasma concentra-
tions compared to maternal values (Peleg et al., 1988),
whereas previous reports on fetal ACE concentration
during gestation are conflicting. In humans, premature
infants show a higher ACE activity compared to term
infants (Bender et al., 1978). At present, there are no
data available on ACE plasma concentration in
human fetal blood and its changes during gestation
and under pathological conditions. The aim of the
present study was the measurement of plasma ACE
activity in normal and Rh isoimmunised fetuses as well
as in neonatal cord blood of preterm and term
newborns and maternal plasma.
METHODS
Fetal blood samples (1 ml) were collected from six
anaemic fetuses with Rh isoimmunisation without
hydrops (mean gestational age 30 weeks, range
27–32 weeks; mean haematocrit<0.30). Blood samples
were also obtained from six fetuses undergoing
cordocentesis for maternal infection but in whom
fetal infection had been excluded (mean gestational
age 21 weeks, range 20–23 weeks); these were used as a

Copyright # 2002 John Wiley & Sons, Ltd. Received: 3 May 2001
Revised: 3 August 2001
Accepted: 14 August 2001
112 T. WALTHER ET AL.
reference group (control). In addition, blood of all
pregnant women was collected (n=12).
Neonatal cord blood was sampled from six preterm
neonates (mean gestational age 34 weeks, range
30–35 weeks) after spontaneous preterm delivery, 15
neonates at term after normal spontaneous delivery
(mean gestational age 39 weeks, range 37–41 weeks).
Additionally, cord blood was taken from six neonates
after elective caesarean section (mean gestational age
38 weeks, range 37–40 weeks). Cord blood was sampled
from the umbilical vein immediately after separation
from the placenta. In parallel, blood of the corres-
ponding mothers was taken. The maternal groups did
not differ in age or parity. Pregnant women with
cardiovascular disorders, diabetes mellitus or anti-
hypertensive treatment were excluded. Informed
consent was obtained. Approval of the study was
obtained from the medical, scientific and ethical
committees of the University of Leipzig.
All blood samples were drawn into tubes containing
citrate solution. Immediately after sampling, plasma
was separated by centrifugation at 4000 g for 10 min
and frozen at x80uC. ACE was measured according
to a miniaturised fluorimetric method (Friedland and
Silverstein, 1976). In brief, 25 ml plasma were incu-
bated for 180 min with 20 ml (0.025 M) Hip-His-Leu
as substrate at 37uC in a chloride-containing phos-
phate buffer (pH 8.3). To prevent the degradation of
His-Leu by aminopeptidases, 10 ml bestatin (10x3 M)
were added. The final volume was 250 ml per tube. The
reaction was stopped by addition of 1 ml 0.4 M NaOH.
The fluorescence was developed by reaction of
produced His-Leu with freshly prepared 2% meth-
anolic o-phthalaldehyde solution (100 ml/tube). Exactly
10 min later, the formation of fluorescence was fixed
by acidification with 300 ml 2M HCl. The suspension
was centrifuged at 10 000 g and the fluorescence was
measured at 500/365 nm (Perkin-Elmer fluorimeter).
As standardisation reagent His-Leu was used. Con-
trols of specificity were carried out for each probe
with 10x6 M of the specific ACE inhibitor, lisinopril.
Results are expressed as meantstandard error of
mean (SEM). Statistical comparisons were made by
one-way analysis of the variance and Tukey HSD test.
Statistical significance was considered as p<0.05.
RESULTS
ACE activity was detectable in all blood samples and
was significantly higher in normal fetuses than in
corresponding maternal plasma (13.31t1.41 nmol HL/
min/ml vs 10.39t0.93 nmol HL/min/ml, p<0.05;
Figure 1). Rh isoimmunisation did not affect ACE
activity (13.08t2.00 nmol HL/min/ml).
Neonatal cord blood of preterm newborns showed
a significantly decreased ACE activity compared to
the fetal controls (10.43t0.69 nmol HL/min/ml), but
no significant difference in maternal ACE activity
(9.88t0.52 nmol HL/min/ml). A further significant
decrease of ACE activity was measured in term
Copyright # 2002 John Wiley & Sons, Ltd.
Figure 1 — ACE activity (in nmol HL/min/ml) is shown as meant
SEM for fetal (black bars) and corresponding maternal (gray bars)
plasma at different time points of gestation. *p<0.05 vs fetal
control; # vs corresponding maternal control; § vs preterm
newborns (8.99t0.49 nmol HL/min/ml, p<0.05 vs
preterm newborns).
Plasma ACE activity of cord blood of neonates
at term (spontaneous delivery 9.08t0.57 nmol HL/
min/ml vs caesarean section 8.59t0.98 nmol HL/min/
ml) and of the mothers (spontaneous delivery 9.96t
0.75 nmol HL/min/ml vs caesarean section 9.52t
0.83 nmol HL/min/ml) did not depend on the mode
of delivery.
Maternal ACE activity was equal between pregnant
women and women after delivery.
DISCUSSION
The ontogeny of the fetal RAS has been investigated
in experimental animals. At present, there is a great
number of studies suggesting that the RAS is
important in cardiovascular control before birth. For
example, cord blood concentrations of angiotensin II
after delivery exceed maternal concentrations of this
effector molecule (Pipkin and Symonds, 1977) indicat-
ing an activated fetal RAS.
The present study is the first to show ACE activity
in human fetal blood. In human fetuses, ACE activity
is significantly higher compared to the maternal
circulation. A higher ACE activity in the fetus has
been reported previously from other species (Peleg
et al., 1988; Forhead et al., 1998) and indicates the
functional importance of the RAS in the fetal
organism. Since the fetal metabolic clearance rate of
angiotensin II (ANG II) is about ten-fold higher than
maternal (Rosenfeld et al., 1995), reflecting a great
placental capacity of ANG II removal, the high fetal
ACE activity contributes to the early functioning of
the RAS prior to birth.
Fetuses with Rh isoimmunisation that are charac-
terised by a hyperdynamic circulation due to fetal
anaemia show no differences in ACE activity to
control fetuses. Since hypotension is relatively
Prenat Diagn 2002; 22: 111–113.
 FETAL, NEONATAL CORD, AND MATERNAL PLASMA ACE CONCENTRATIONS
common in anaemia secondary to Rh alloimmunisa-
tion (Phibbs et al., 1976), a possible stimulation of the
RAS system is obviously not achieved by a higher
ACE activity. Moreover, it has been documented that
hypoxaemia also stimulates the RAS in human
neonates at delivery (Tetlow and Broughton Pipkin,
1983). However, the fetuses investigated in the present
study showed anaemia but not hypoxaemia. Thus, the
blood gas tension in fetuses with Rh isoimmunisation
should not trigger the RAS system.
Furthermore, the present study shows a dependency
of the fetal ACE activity on gestational age with a
decline during fetal maturation. Data on ontogenic
changes of the ACE activity during fetal development
are conflicting. In the sheep fetus, plasma ACE
increases towards term (Forhead et al., 1998). This
also occurs in the fetal guinea pig, with values at term
greater than those of the adult animals (Raimbach and
Thomas, 1990). An unchanged ACE activity during
gestation has been reported in the fetal rat (Peleg et al.,
1988).
However, the present finding that preterm newborns
show a higher ACE activity than term newborns in
cord blood corresponds with previous results: Bender
et al. (1978) reported similar values between preterm
infants and maternal controls but lower ACE activity
in term infants compared to preterm infants. Further-
more, the lack of significant differences between
neonatal and maternal ACE values at term confirms
the data of previous studies (Oparil et al., 1978; Oats
et al., 1981).
Since plasma ACE is mainly generated by the
peripheral endothelium, the observed decline in the
present study could result from the maturation of the
fetal lung that is taking over ACE production.
However, the placenta also has an ACE synthetic
capacity (Schutz et al., 1996) and shows decreasing
ACE-mRNA concentrations in humans with advan-
cing gestational age (Yagami et al., 1994). Therefore, it
must also be considered that placental ACE produc-
tion may contribute partly to the ACE activity in
circulation.
We conclude that the decline of plasma ACE
activity in fetal blood values, cord blood ACE activity
of preterm newborns and lowest values in term
newborns reflects the shift of the source of ACE
production from the peripheral vascular endothelial
cells and the placenta to the lung with advancing
gestational age. The hypothesis that a high fetal ACE
activity reflects pulmonary immaturity is supported by
the fact that premature infants that develop idiopathic
respiratory distress syndrome have significant eleva-
tions in serum ACE activity (Mattioli et al., 1975).
Thus, a decrease in circulating ACE during the second
half of gestation should be an indicator of fetal lung
development and maturation.
Moreover, the mode of delivery did not influence
Copyright # 2002 John Wiley & Sons, Ltd.
113
plasma ACE activity either in maternal or in cord
blood. Since cardiovascular and endocrine factors
during normal labour or obstetric analgesia have no
effect on fetal and maternal plasma ACE activity, the
enzyme seems to be well controlled in the peripheral
circulation. Therefore, abnormal maternal ACE activ-
ity could be a clear indicator of maternal or fetal
pathology.
ACKNOWLEDGEMENT
This research was supported by a grant (WA 1441/1)
from the Deutsche Forschungsgemeinschaft (DFG).
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Prenat Diagn 2002; 22: 111–113.