scispace - formally typeset
SciSpace - Your AI assistant to discover and understand research papers | Product Hunt

Journal ArticleDOI

A reliable radiometric assay for the determination of angiotensin I-converting enzyme activity in urine.

01 Jan 1990-Clinical Chemistry and Laboratory Medicine (J Clin Chem Clin Biochem)-Vol. 28, Iss: 11, pp 857-861

TL;DR: Comparison of dialysis and ultrafiltration for concentration of urine showed the existence of angiotensin-converting enzyme inhibitors in human urine, and the kidney tubular epithelial origin of the enzyme is confirmed.

AbstractWe present a radiometric assay for the determination of urinary angiotensin-converting enzyme activity, using benzoyl-[1-14C]glycyl-L-histidyl-L-leucine as the substrate. An optimal pH of 8.3, an optimal chloride concentration of 0.375 mol/l and complete inhibition by EDTA-Na2, captopril and enalaprilat confirm the specificity of the assay. Comparison of dialysis and ultrafiltration for concentration of urine showed the existence of angiotensin-converting enzyme inhibitors in human urine. Dialysis against water was the more effective method for avoiding enzyme inhibition. After dialysis of urine, the assay was linear with time and with enzyme concentration; it was highly sensitive (60 mU/l) and showed good reproducibility. Under our technical conditions, we found angiotensin-converting enzyme activity in urine samples with quantitatively abnormal protein contents, but not in normal urine. Urinary angiotensin-converting enzyme did not correlate with proteinuria nor with water-salt parameters or creatinine. We confirm the kidney tubular epithelial origin of the enzyme, and propose the use of our assay to study urinary angiotensin-converting enzyme as a marker of renal tubular damage.

Topics: Enzyme assay (62%), Urine (53%), Proteinuria (52%), Kidney (51%)

Summary (2 min read)

Introduction

  • The brush border of kidney tubules is one of the extravascular sites richest in angiotensin-converting activity (2) .
  • Because of this location some authors have suggested the potential use of urinary angiotensin-converting activity as an index of renal tubular damage (3, 4) .
  • Studies on urinary angiotensin-converting enzyme are, however, rare, possibly because its activity is low in urine and the accuracy of the ! ).
  • Concentration of urine before the measurement must be avoided.

Reagents

  • Potassium phosphates, sodium chloride and ethyl acetate were from Merck (Darmstadt, FRG).
  • EDTA-Na 2 and 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid were provided by Sigma Chemical Co. (Saint Louis, Mo., USA).
  • Instafluor was from Packard Instruments (Warrenville, 111., USA).
  • Captopril and enalaprilat were gifts from Squibb & Sons (Princeton, NJ., USA) and Merck Sharp & Dohme (West Point, Pa., USA), respectively.

Angiotensin-converting enzymatic assay

  • All urinary analyses were performed on 24-hour, fresh urine containing no preservatives, which was centrifuged 10 min at 2000 g, +4°C.
  • After vigorous mixing and centrifugation (10 min at 1000g, +4°C), 250 μΐ of the upper ethyl acetate layer were directly dropped into 5 ml of Instafluor and counted (Mini-Maxi-Tricarb Packard -Packard Instruments).
  • Results are expressed as angiotensin-converting enzyme units per litre of urine, one unit (U) corresponding to the release of one μηιοί of benzoylglycine in one minute at 37 °C.
  • The formula used is derived from that described in 1. c. (6) after calculation of the specific activity of the isotopic dilution (SA): [Bq -Bq ] '.

Data analysis

  • Results are expressed as mean ± standard deviation.
  • Coefficients of variation (CVs) for reproducibility studies were obtained from 20 determinations.
  • Statistical comparisons were performed with the non-parametric Mann-Whitney U-test.

Results

  • As the authors did not find any angiotensin-converting activity in urines from normal subjects, they performed further experiments on urines with high protein contents.
  • When the urines were not dialysed, the activity curves fell rapidly, suggesting an inhibitory agent in the urines which can be eliminated by dialysis (not shown).
  • There was no apparent difference between men and women.
  • These data were the same when enzyme activity was expressed as U/g of protein or as U/mmol of creatinine.

Discussion

  • The authors modified the radiometric method of Rohrbach (6) for the determination of urinary angiotensin-converting enzyme activity.
  • Such low molecular weight inhibitors have been detected also in urine.
  • Pitotti et al. (15) described a HPLC method but, once again, with difficult pretreatment of the urinary samples, and with no indication of the reliability or precision.
  • On the other hand, the radiochemical assay of Rohrbach (6) has poor sensitivity, detecting 500 mU/1, which is about 10-fold the limit of detection of their assay; thus Rohrbach's assay cannot be directly adapted to urine determination.
  • Under their technical conditions the authors could not detect angiotensin-converting activity in the urines of normal subjects, but they were able to measure the enzyme activity in urines with a quantitatively abnormal protein content.

Did you find this useful? Give us your feedback

...read more

Content maybe subject to copyright    Report

Baudin
et
al.:
Angiotensin
I-converting
enzyme assay
in
urine
857
J.
Clin.
Chem.
Clin.
Biochem.
Vol.28,
1990,
pp.
857-861
©
1990 Walter
de
Gruyter
& Co.
Berlin
· New
York
A
Reliable Radiometrie Assay
for the
Determination
of
Angiotensin
I-Converting
Enzyme Activity
in
Urine
By
B.
Baudin,
B.
Beneteau-Burnat,
F. Ch.
Baumann*)
and J.
Giboudeau
Laboratoire
de
Biochimie
A,
Höpital
Saint-Antoine,
Paris
Laboratoire
de
Chimie Biologique,
UFR
Pharmacie,
Paris
V,
France
(Received
November
28,
1989/June
11,
1990)
Summary:
We
present
a
radiometric assay
for the
determination
of
urinary angiotensin-converting enzyme
activity,
using
benzoyl-[l-
14
C]glycyl-L-histidyl-L-leucine
as the
substrate.
An
optimal
pH of
8.3,
an
optimal
chloride concentration
of
0.375
mol/1
and
complete inhibition
by
EDTA-Na
2
,
captopril
and
enalaprilat
confirm
the
specificity
of the
assay. Comparison
of
dialysis
and
ultrafiltration
for
concentration
of
urine showed
the
existence
of
angiotensin-converting enzyme inhibitors
in
human urine. Dialysis against water
was the
more
effective
method
for
avoiding enzyme inhibition. After dialysis
of
urine,
the
assay
was
linear with time
and
with
enzyme concentration;
it was
highly sensitive
(60
mU/1)
and
showed good reproducibility.
Under
our
technical conditions,
we
found angiotensin-converting enzyme activity
in
urine samples with quantitatively
abnormal protein contents,
but not in
normal urine. Urinary angiotensin-converting enzyme
did not
correlate
with
proteinuria
nor
with water-salt parameters
or
creatinine.
We
confirm
the
kidney
tubular epithelial origin
of
the
enzyme,
and
propose
the use of our
assay
to
study urinary angiotensin-converting enzyme
as a
marker
of
renal tubular damage.
Introduction
Angiotensin
I-converting
enzyme
is the
peptidyldi-
peptide hydrolase (dipeptidylcarboxypeptidase
EC
3.4.15.1)
which cleaves angiotensin
I to the
potent
vasopressor angiotensin
II and
inactivates
the
vaso-
dilator bradykinin. Angiotensin-converting enzyme
activity
is
measurable
in
plasma
and in
most organs;
the
enzyme
is
located
on the
luminal surface
of the
endothelial
cells
as an
ecto-glycoprotein
(1).
The
brush border
of
kidney tubules
is one of the
extravascular
sites richest
in
angiotensin-converting
activity
(2). Because
of
this location some authors
have
suggested
the
potential
use of
urinary angioten-
sin-converting
activity
as an
index
of
renal tubular
damage
(3, 4).
Studies
on
urinary angiotensin-con-
verting
enzyme are, however,
rare,
possibly because
its
activity
is low in
urine
and the
accuracy
of the
!
)
Deceased
on
September 1988
present assays
not
proved;
in
particular,
no
criteria
of
reliability have been
specified
(3
5). In
addition,
mammalian urine
may
contain angiotensin-convert-
ing
enzyme inhibitors (5).
In
this study,
we
describe
a
sensitive
and
specific
radiometric assay
for
determination
of
urinary angio-
tensin-converting activity.
We
draw attention
to the
presence
of
inhibitors
of the
enzyme
in
human
urine,
and
propose
a
prior dialysis
of
urine
for an
accurate
determination
of the
enzyme. Concentration
of
urine
before
the
measurement must
be
avoided.
Materials
and
Methods
Reagents
All
reagents were
of
analytical grade. Potassium
phosphates,
sodium chloride
and
ethyl acetate were
from
Merck
(Darm-
stadt, FRG).
EDTA-Na
2
and
4-(2-hydroxyethyl)-l-piperazine-
ethanesulphonic acid (HEPES) were provided
by
Sigma Chem-
ical
Co.
(Saint Louis,
Mo.,
USA).
Benzoylglycyl-L-histidyl-L-
J.
Clin. Chem. Clin. Biochem.
/
Vol.
28,1990
/ No. 11

858
Baudin
et ah:
Angiotensin
I-converting
enzyme assay
in
urine
leucine
was
from
Calbiochem (San Diego,
CaL,
USA)
and
benzoyl-[l-
14
C]glycyl-L-histidyl-L-leucine
was
purchased from
New
England Nuclear (Boston,
Ma.,
USA).
Instafluor
was
from
Packard Instruments
(Warrenville,
111.,
USA).
Captopril
and
enalaprilat
were
gifts
from
Squibb
&
Sons (Prin-
ceton, NJ., USA)
and
Merck Sharp
&
Dohme
(West
Point,
Pa.,
USA), respectively.
PM-10
(cut-off
M
r
10000)
and
XM-50 (cut-
off
M
r
50000)
ultrafiltration
membranes were from
Amicon
Ltd. (Lexington,
Ma.,
USA), dialysis membranes
from
Union
Carbide (Chicago,
111.,
USA)
(cut-off
M
r
1000).
Angiotensin-converting
enzymatic assay
All
urinary analyses were performed
on
24-hour,
fresh
urine
containing
no
preservatives, which
was
centrifuged
10 min at
2000
g,
+4°C.
Extensive dialysis
and
concentration
by
ultra-
filtration
were performed
at
4-
4 °C
with constant agitation
on
10
ml
aliquots
of
centrifuged
urine.
The
buffered
substrate contained 0.165 mmol/1 radiolabelled
substrate
and
2.175
mmol/1 cold substrate
in 0.5
mol/1 potas-
sium phosphate
0.75 mol/1 sodium chloride
pH = 8.3
buffer.
Incubation
was for one
hour
at 37 °C in 100 χ 8 mm
glass tubes, started
by the
addition
of 25
μΐ
of
urine
to 25
μΐ
of
buffered
substrate.
The
hydrolysis
was
stopped
by the
addition
of
50
μΐ
HC1 1
mol/1.
Benzoyl-[l-
14
C]glycine
was
then extracted
with
375
μΐ
of
ethyl acetate. After vigorous mixing
and
centrif-
ugation
(10 min at
1000g,
+4°C),
250
μΐ
of the
upper
ethyl
acetate layer were directly dropped into
5 ml of
Instafluor
and
counted
(Mini-Maxi-Tricarb
Packard
Packard Instruments).
Angiotensin-converting enzyme activity
was
determined
in du-
plicate
and a
blank
was run
with
0.15
mol/1 sodium chloride
replacing
the
urine.
Results
are
expressed
as
angiotensin-converting enzyme units
per
litre
of
urine,
one
unit
(U)
corresponding
to the
release
of
one
μηιοί
of
benzoylglycine
in one
minute
at 37 °C.
The
formula used
is
derived
from
that described
in 1. c. (6)
after
calculation
of the
specific
activity
of the
isotopic dilution
(SA):
[Bq
(dosage)
-
Bq
(blank)]
' SA χ
0.91
χ
0.67
χ 60 χ 25 χ
10~
6
_
ABq
~ SA χ 915 χ
ΙΟ'
6
'
where
0.91
is the
benzoylglycine fraction extracted
by
ethyl
acetate; 0.67
is the
counted organic fraction;
60 is
time
in
minutes
and 25 χ
10~
6
the
volume
of the
sample
in
litres.
For
example,
with
radiolabelled substrate
of
activity 88000
Bq/μιηοΐ,
SA
becomes 7000
Bq/μιηοΐ,
thus
Other
determinations
Proteinuria,
creatininuria,
urinary sodium (Na)
and
potassium
(K)
were measured
on the
multiparametric discrete analysers
Greiner G300 (Langenthal,
CH) and
Astra
8
(Beckman
Instru-
ments Inc.
-
Fullerton, Ca., USA).
Albumin/globulins
ratio
was
determined
with
a
Cliniscan den-
sitometer (Helena Laboratories
Beaumont, Te., USA)
after
electrophoretic separation
on
agarose
gel
(Paragon
SPE-II
Kit,
Beckman).
Urines
Normal urines were
from
15
apparently healthy people
from
the
laboratory
staff
(8
women, mean
age
27.0
±
10.8 years,
and 7
men, mean
age
30.1
±10.7
years) without
any
renal
disorder
as
judged
by a
normal diuresis
and
normal urinary
biological
criteria:
proteins
<
0.15
g/1,
Na =
131.1
±49.0
mmol/1,
K =
48.5
±
15.8 mmol/1, creatinine
=
12.2
± 4.7
mmol/1.
Urines with
an
abnormally high protein content were selected
from
44
urinary samples sent
to the
laboratory
for
electropho-
retic analysis
of
quantitatively abnormal proteinuria (i.e.
>0.15g/day).
Data
analysis
Results
are
expressed
as
mean
±
standard deviation.
Coeffi-
cients
of
variation (CVs)
for
reproducibility studies were
ob-
tained
from
20
determinations. Statistical comparisons were
performed
with
the
non-parametric Mann-Whitney U-test.
Results
As
we did not find any
angiotensin-converting activity
in
urines from
normal
subjects,
we
performed further
experiments
on
urines with high protein contents.
A
direct
determination
showed
the
presence
of
angio-
tensin-converting
activity
in
16
of the 44
urine samples
tested.
Concentration
of
these
urines
by
ultrafiltration
on
Amicon
PM-10
did not
increase angiotensin-con-
verting
activity,
but
24-hour dialysis against pure
water
increased
the
enzymatic activity
by 85 ±
42%.
Dialysis
against
water
was
more
effective than dialysis
against
the
phosphate
buffer
of the
enzymatic assay,
or
against
0.375
mol/1 sodium
chloride,
or a 25
mmol/1
HEPES
-
0.375 mol/1
NaCl
- pH = 8.3
buffer.
Concentration
of the
dialysates
on
Amicon PM-10
never
increased
the
enzymic
activity,
and
more often
led
to a
substantial
loss
of
activity. Dialysis against
water
did not
reveal
any
angiotensin-converting
ac-
tivity
in the
other
28
urines with
a
high protein content
or
in the
normal
urines.
For the
experiments reported
below,
all
urines
were dialysed
for 24
hours
against
pure
water.
Under
these conditions
we
determined
the
main
analytical
parameters
of the
radiochemical
as-
say.
The
specificity
was
assessed
by
(i)
an
optimal
pH of 8.3
(fig.
1 b) and
activation
by
an
increase
in the
chloride
concentration
(fig.
1 a),
both
of
which
are
characteristic
of
angiotensin-con-
verting enzyme activity determined with benzoylgly-
cine-histidyl-leucine
as the
substrate;
(ii)
a
complete
inhibition
of
urinary angiotensin-con-
verting activity
by 60
mmol/1
EDTA-Na
2
(angioten-
sin-converting enzyme
is a
zinc
metallopeptidase)
and
100
μηιοΐ/ΐ
captopril
or 40
μηιοΐ/l
enalaprilat (two
specific
synthetic
inhibitors
of the
enzyme).
As
phos-
phates
have
been
implicated
as
inhibitors
of
angio-
tensin-converting activity,
we
verified
that
the
con-
centration
used
(250 mmol/1)
was not
inhibitory.
In
J.
Clin.
Chem.
Clin.
Biochem.
/
Vol.
28,
1990
/ No. 11

Baudin
et
al.:
Angiotensin
I-converting
enzyme assay
in
urine
859
^
ΟΛ
0.3
0.2
0.1
g
"
0.125
0.25
0.375
NaCI[mol/l]
0.5
Fig.
1.
Urinary
angiotensin-converting
enzyme activity;
pH
and
Chloride activation
curves.
Each
point
is the
mean
of
three determinations
of a
pool
of
urinary
samples
dialysed against water.
particular, substitution
of the
phosphate
buffer
by a
50
mmol/1
HEPES
-
0.75
mol/1
NaCl
- pH = 8.3
buffer
did not
increase
the
urinary angiotensin-con-
verting
activity.
The
assay
was
linear
as a
function
of
enzyme concen-
tration
up to 4.0
U/l
and as a
function
of
time
up to
120
minutes (fig.
2).
When
the
urines were
not
dia-
lysed,
the
activity curves
fell
rapidly, suggesting
an
inhibitory
agent
in the
urines which
can be
eliminated
by
dialysis (not shown). When,
after
dialysis,
the
urines
were concentrated
on
PM-10,
the
activity curve
swiftly
decreased
but not
when
a
XM-50 membrane
was
used instead
of the
PM-10 membrane (fig. 2a).
These data suggest
the
existence
of a
second inhibitory
substance
in
human urines eliminated neither
by di-
alysis
nor by
ultrafiltration
on
PM-10 membrane,
but
excluded
by
ultrafiltration
on
XM-50.
The
sensitivity
of the
assay
was
determined
by
meas-
uring
the
radioactivity
of 30
blanks
in the
same
set
of
experiments;
the
precision
of the
counts corre-
sponded
to an
assay sensitivity
of 44
mU/1.
Linear
dilution
of
urines, previously dialysed against water,
showed
the
limit
of
detection
to be 66
mU/1 (fig. 2b).
Within-day reproducibility
CVs
were less than
1% for
two
pools
of
urines with activities
of
1.44
U/l and
3.21
U/l. Their between-days
CVs
were 10.3
and
4.4%
respectively.
CVs
were between
10 and 20% for a
urine pool containing
124
mU/1 angiotensin-convert-
ing
activity.
In the 16
urines containing angiotensin-converting
activity
the
mean activity
was 2.6 ± 4.7 U/l
with
a
range
of 0.1 to
14.5
U/l and a
median value
of 440
mU/1,
but the
distribution
of the
values
was not
unimodal. There
was no
apparent
difference
between
men
and
women.
For
these
16
urines, proteinuria
was
0.5
ΟΛ
0.3
0.2
0.1
15
30 45 60
t[min]
90
120
0.4
0.3
0.2
0.1
0
0.1
0.25
0.5 1
Factor
of
dilution
Fig.
2.
Radioassay
of
angiotensin-converting enzyme
in
urines,
showing activity with time (a),
and
linearity (b). Each
point
is the
mean
of
three determinations
of a
pool
of
urinary
samples,
dialysed only
) or
concentrated
on
Amicon PM-10
after
dialysis
(*).
6.7
±15.1
g/1
(range:
0.35-62.4
g/1).
Urinary angi-
otensin-converting activity
did not
correlate with pro-
teinuria (fig.
3), or
with
the
albumin/globulins ratio
(1.79
±
1.06),
or
with
any
kind
of
abnormality
on
the
agarose electrophoresis pattern, such
as a
peak
in
the
γ-globulins
or
patterns
of
selective
or
non-selective
proteinuria (not shown).
z>
13
σ)10
5 10
Proteinuria
[g/l]
60
Fig.
3.
Lack
of
correlation
(r =
0.29) between angiotensin-
converting activity
and
proteinuria measured
in 16
urine
samples with detectable enzyme activity.
J.
Clin.
Chem.
Clin.
Biochem.
/
Vol.
28,1990
/ No. 11

860
Baudin
et
al.:
Angiotensin
I-converting
enzyme
assay
in
urine
Angiotensin-converting
activity
in
urines
was not re-
lated
to
water-salt excretion; thus,
no
statistical link
appeared
between
the
enzyme activity
and
urinary
Na
(39.7
±
35.1
mmol/1),
K
(34.0
±
13.9 mmol/1),
or
Na/K
(1.45
±
1.49). Moreover, angiotensin-convert-
ing
activity
was not
linked
to
creatininuria
(8.16
±
7.05 mmol/1). These
data
were
the
same when
enzyme activity
was
expressed
as U/g of
protein
or
as
U/mmol
of
creatinine.
Discussion
We
modified
the
radiometric method
of
Rohrbach
(6)
for
the
determination
of
urinary angiotensin-convert-
ing
enzyme activity. Dilution
of the
radiolabelled sub-
strate,
benzoyl-[l
-
14
C]glycyl-L-histidyl-L-leucine,
with
cold
substrate gives
a
sensitivity suitable
for a
direct
determination
on
urine samples,
i.e.
without prior
concentration.
Dialysis
of the
urines
is
necessary
to
eliminate
at
least
one
inhibitor, which,
as
previously suspected (5),
is of
low
molecular weight. According
to the
results
ob-
tained
with
PM-10
and
XM-50
ultrafiltration
mem-
branes,
another inhibitor
may
also
be
present; this
inhibitor, relative molecular mass
10000
50000,
is
less
inhibitory
than
the
dialysable inhibitor since
we
could
not
reveal
its
inhibitory
effect
on
non-concen-
trated
urines. Some ions
and
metals have been impli-
cated
as
physiological angiotensin-converting enzyme
inhibitors
(5, 7);
Lieberman described
inhibition
by
human plasma
but did not
identify
the
agent respon-
sible (8).
Hazato
&
Kase
found
an
inhibitor
in pig
plasma
and
identified
it as an
oligopeptide
(9). Such
low
molecular weight inhibitors have been detected
also
in
urine. Potential physiological inhibitors
of
higher molecular weight have also been reported,
i. e.
albumin
and
some
of its
fragments (10),
or fibrinogen
fragments
(11).
In our
study,
it was
difficult
to
cor-
relate
the
inhibitory power
of the
urines with their
protein
concentration because
we had
only
a few
samples with both
low
angiotensin-converting activity
and
high protein content.
Further
investigations
are
necessary
to
characterize these urinary inhibitors
of
angiotensin-converting enzyme activity.
The
reliability
of our
radiochemical enzyme assay
after
dialysis
of
urines
is
assessed
by its
specificity,
linearity
and
reproducibility.
The
sensitivity
is
higher
than
that
of
most
the
methods described
for the
determination
of
angiotensin-converting activity
in
plasma.
Only
fluorimetric
assays attain this sensitivity
(12),
but
radioassays avoid
the
interferences fre-
quently encountered with
fluorimetric as
well
as
pho-
tometric
methods (13).
Kokubu
et al.
(14) adapted
Cushmarfs
spectrophotometric
assay (13)
to
urines
but
their method involves partial purification
of the
enzyme
from
the
urine. Pitotti
et al.
(15)
described
a
HPLC method but, once again, with
difficult
pretreat-
ment
of the
urinary samples,
and
with
no
indication
of
the
reliability
or
precision.
A
colorimetric assay
was
also proposed
but
without evidence
of
specificity
for
angiotensin-converting enzyme (3). Ryan
et al.
(5),
like
the
present authors (personal
data),
were unable
to
adapt
Cushman's
assay
for the
determination
of
angiotensin-converting
activity
in
mammalian urines,
since
the
latter contain high concentrations
of
pig-
ments.
On the
other hand,
the
radiochemical assay
of
Rohrbach
(6) has
poor sensitivity, detecting
500
mU/1,
which
is
about
10-fold
the
limit
of
detection
of our
assay; thus
Rohrbach's
assay cannot
be
directly
adapted
to
urine determination.
Under
our
technical conditions
we
could
not
detect
angiotensin-converting activity
in the
urines
of
normal
subjects,
but we
were able
to
measure
the
enzyme
activity
in
urines with
a
quantitatively abnormal pro-
tein content. Some authors
(14,15)
found angiotensin-
converting activity
in
normal, 24-hour urines; possibly
they
were measuring
the
physiological replacement
of
the
renal tubular brush-border whose epithelial cells
are
sedimentable.
In
view
of the
high specific angio-
tensin-converting activities
in the
male genital
tract
(16, 17),
a
gonadic
or
prostatic
origin
of
urinary
angiotensin-converting
enzyme might have been
ex-
pected,
but we
found
no
difference
between males
and
females;
in
particular, normal
men did not
excrete
angiotensin-converting
activity
in the
urine.
The ab-
sence
of a
correlation between this urinary enzymatic
activity
and
proteinuria,
and
even more
the
lack
of
dependence
of
this activity
on the
indices
of
glomer-
ular
function,
such
as
creatininuria
or
albuminuria,
eliminate
the
possibility
of a
passage
of the
plasmatic
enzyme
throughout
the
glomerular
filter.
Damage
of
renal glomeruli cannot explain
a
significant enzymu-
ria, since glomerular epithelial
and
endothelial cells
contain only
a
little angiotensin-converting enzyme
(18, 19). Thus,
in
agreement with other authors
(3,
4),
we
conclude that urinary angiotensin-converting
enzyme
is of
tubular origin,
as in the
case
of
most
enzymurias
(20).
We
also showed
that
urinary angio-
tensin-converting
enzyme excretion
is
independent
of
sodium
and
potassium excretion,
so
that
physiological
or
pathological variations
of
this enzyme
in
response
to
haemodynamic
or
dietary factors would
not be
expected.
Our
specific,
sensitive
and
reproducible radioassay
might enable
the
comparison
of
angiotensin-convert-
ing
enzyme with other materials
of
tubular origin
for
their suitability
as
specific,
early
and
precise markers
J.
Clin.
Chem.
Clin.
Biochem.
/
Vol.
28,
1990
/
No.
11

Baudin
et
al.:
Angiotensin I-converting enzyme assay
in
urine
861
of
tubular damage during renal disorders.
The
assay
will
also
be of use in
investigating putative inhibitors
of
angiotensin-converting
enzyme
in
mammalian
bi-
ological
fluids.
Acknowledgement
We
wish
to
express
our
gratitude
to Dr.
Georges
Morgant
for
statistical consultation
and
Yolande
Tilly
for
typing
the
manu-
script.
References
1.
Caldwell,
P. R. B.,
Seegal,
B.
C,
Hsu,
K.
C,
Das,
M. &
Soffer,
R. L.
(1976) Angiotensin-converting enzyme
vas-
cular
endothelial localization. Science
797,
1050—1051.
2.
Oshima,
G.,
Geese,
A. &
Erdös,
E. G.
(1974) Angiotensin
I-converting
enzyme
of the
kidney
cortex. Biochim.
Bio-
phys.
Acta
350,
26-37.
3.
Baggio,
B.,
Favaro,
S.,
Cantaro,
S.,
Bertazzo,
L.,
Frunzio,
A.
&
Borsatti,
A.
(1981) Increased urine angiotensin
I-
converting
enzyme activity
in
patients with upper urinary
tract
infection.
Clin.
Chim.
Acta
109,
211-218.
4.
Kato,
I.,
Takada,
Y.,
Nishimura,
K.,
Hiwada,
K. & Ko-
kubu,
T.
(1982) Increased urinary excretion
of
angiotensin-
converting
enzyme
in
patients with renal diseases.
J.
Clin.
Chem.
Clin. Biochem.
20,
473-476.
5.
Ryan,
J. W.,
Martin,
L.
C.,
Chung,
A. &
Pena,
G. A.
(1979)
Mammalian
inhibitors
of
angiotensin-converting enzyme
(kininase
II). Adv. Exp. Med.
Biol.
120
B,
599-606.
6.
Rohrbach,
M. S.
(1978)
[Glycine-l-
14
C]hippuryl-L-histidyl-
L-leucine:
a
substrate
for the
radiochemical assay
of
angi-
otensin-converting
enzyme. Anal. Biochem.
84,
272
276.
7.
Galardy,
R. E.
(1982) Inhibition
of
angiotensin-converting
enzyme
by
phosphoramidates
and
polyphosphates.
Bio-
chemistry
27,
5777-5781.
8.
Lieberman,
J. &
Sastre,
A.
(1986)
An
angiotensin-convert-
ing
enzyme
(ACE)
inhibitor
in
human serum. Increased
sensitivity
of the
serum
ACE
assay
for
detecting active
sarcoidosis. Chest
90,
869-873.
9.
Hazato,
T. &
Käse,
R.
(1986) Isolation
of
angiotensin-
converting
enzyme inhibitor
from
porcine plasma.
Biochem.
Biophys.
Res.
Commun.
739,
52
55.
10.
Klauser,
R.
J.,
Robinson,
C. J.
G.,
Marinkovic,
D. V. &
Erdös,
E. G.
(1979) Inhibition
of
human peptidyldipepti-
dase
(angiotensin I-converting
enzyme:
kininase
II) by hu-
man
serum-albumin
and its
fragments. Hypertension
7,
281-286.
11.
Saldeen,
T.,
Ryan,
J. W. &
Berryer,
P.
(1981)
A
peptide
derived
from
fibrinogen
inhibits angiotensin-converting
en-
zyme
and
potentiates
the
effects
of
bradykinin.
Thromb.
Res.
23,
465-470.
12.
Friedland,
J. &
Silverstein,
E.
(1976)
A
sensitive
fluorime-
tric assay
for
serum angiotensin-converting enzyme.
Am.
J.
Clin. Pathol.
66,
416-424.
13.
Cushman,
D. W. &
Cheung,
H. S.
(1971)
Spectrophoto-
metric assay
and
properties
of the
angiotensin-converting
enzyme
of
rabbit lung. Biochem.
Pharmacol.
20,
1637
1648.
14.
Kokubu,
T.,
Kato,
L,
Nishimura,
K.,
Hiwada,
K. &
Ueda,
E.
(1978) Angiotensin I-converting enzyme
in
human urine.
Clin.
Chim.
Acta
89,
375-379.
15.
Pitotti,
A.,
Maurich,
V.,
Moneghini,
M. &
Vianello,
S.
(1986)
HPLC method
for
evaluation
of
urinary angiotensin-
converting enzyme: some examples
of
normal subjects
and
patients
with renal transplantation.
J.
Pharmac.
Biomed.
Anal.
4,
677-683.
16.
Berg,
T.,
Sulner,
J.,
Lai,
C. V. &
Soffer,
R. L.
(1986)
Immunohistochemical
localization
of two
angiotensin
I-
converting isoenzymes
in the
reproductive
tract
of the
male
rabbit.
J.
Histochem. Cytochem.
34,
753-760.
17.
Yokoyama,
M.,
Hiwada,
K.,
Kokubu,
T.,
Takada,
M. &
Takeuchi,
M.
(1980)
Angiotensin-converting enzyme
in hu-
man
prostate.
Clin. Chim. Acta
700,
253-258.
18.
Bruneval,
P.,
Hinglais,
N.,
Alhenc-Gelas,
F.,'Tricottet,
V.,
Corvol,
P.,
Menard,
J.,
Camillieri,
J. P. &
Barietti,
J.
(1986)
Angiotensin
I-converting enzyme
in
human intestine
and
kidney.
Ultrastructural
and
immuno-histochemical locali-
zation. Histochemistry
85,
73-80.
19.
Chanse,
L.
D.,
Morin,
J. P.,
Borghi,
H.,
Ardaillou,
N. &
Ardaillou,
R.
(1987) Angiotensin I-converting enzyme
in
isolated human glomeruli. FEBS Letters
220,
247-252.
20.
Flandrois,
C.,
Gravagna,
B.,
Maire,
I. &
Mathieu,
M.
(1986)
Enzymurie.
Ann.
Biol. Clin.
44,
486-490.
Bruno
Baudin
Ph. D.
Laboratoire
de
Biochimie
A
Hopital
Saint Antoine
184,
rue du Fbg
St-Antoine
F-75571
Paris Cedex
J.
Clin. Chem.
Clin.
Biochem.
/
Vol.
28,
1990
/ No. 11

Citations
More filters

Journal ArticleDOI
TL;DR: A protocol for easy isolation and culture of human umbilical vein endothelial cells (HUVECs) is described to supply every researcher with a method that can be applied in cell biology laboratories with minimum equipment.
Abstract: We describe a protocol for easy isolation and culture of human umbilical vein endothelial cells (HUVECs) to supply every researcher with a method that can be applied in cell biology laboratories with minimum equipment. Endothelial cells (ECs) are isolated from umbilical vein vascular wall by a collagenase treatment, then seeded on fibronectin-coated plates and cultured in a medium with Earles' salts and fetal calf serum (FCS), but without growth factor supplementation, for 7 days in a 37 degrees C-5% CO2 incubator. Cell confluency can be monitored by phase-contrast microscopy; ECs can be characterized using cell surface or intracellular markers and checked for contamination. Various protocols can be applied to HUVECs, from simple harvesting to a particular solubilization of proteins for proteomic analysis.

364 citations


Journal ArticleDOI
TL;DR: Monitoring sarcoidosis obviates the measurement of ACE activity in other biological fluids, e.g., broncho-alveolar and cerebrospinal fluids, in the search of a locoregional dissemination or dis-simulation of the disease.
Abstract: Angiotensin I-converting enzyme (ACE) is a peptidyldipeptide hydrolase that is located mainly on the luminal surface of vascular endothelial cells but also in cells derived from the monocyte-macrophage system. Physiologically, ACE is a key enzyme in the renin-angiotensin system, converting angiotensin I into the potent vasopressor angiotensin II and also inactivating the vasodilator bradykinin. Increased serum ACE activity (SACE) has been reported in pathologies involving a stimulation of the monocytic cell line, primarily granulomatous diseases. Sarcoidosis is the most frequent and the better studied of these diseases; high SACE is not only a well-established marker for the diagnosis but is also a useful tool for following its course and evaluating the effect of therapy. SACE can also be increased in nonsarcoidotic pulmonary granulomatous diseases such as silicosis and asbestosis, in extrathoracic granulomatous pathologies such as Gauchers disease and leprosis, and, to a lesser extent, in nongranulomatous disorders such as hyperthyroidism or cholestasis. On the other hand, monitoring sarcoidosis obviates the measurement of ACE activity in other biological fluids, e.g., broncho-alveolar and cerebrospinal fluids, in the search of a locoregional dissemination or dis-simulation of the disease. Decreased SACE has been reported in vascular pathologies involving an endothelial abnormality, e.g., deep vein thrombosis, and in endothelium dysfunctions related to the toxicity of chemo- and radiotherapy used in cancers, leukemias, and hematopoietic or organ transplantations. SACE is also of interest for monitoring arterial hypertension treated with specific synthetic ACE inhibitors. These various reasons for determining ACE activity have led to the development of numerous methods. The most widely used is the spectrophotometric assay using hippuryl-histidyl-leucine as substrate. Fluorimetric and radiochemical assays using both classic and novel substrates have been proposed, but they are time consuming, require special apparatus, and are not suited to automation. Kinetic spectrophotometry of furylacryloyl-phenylalanyl-glycyl-glycine hydrolysis is now used extensively because it is easy to automatize. Efforts are now required to standardize one or more of these assays. Indeed, "normal" plasma values differ not only according to the substrate, but also to the method of determination and to sex and age.

85 citations


Journal ArticleDOI
Abstract: We have determined serum activity and kinetic constants of angiotensin I-converting enzyme (ACE), parallel to an insertion/deletion (I/D) polymorphism in its gene, in French centenarians and controls 20-70 years of age because this enzyme could have an impact on cardiovascular risk, and thus on longevity. Both the ACE D allele and ACE D/D genotype were more frequent in centenarians in comparison with controls, without sex-related differences nor significant correlation with a cardiovascular pathology. In centenarians, I/D polymorphism was correlated with circulating ACE activity (D/D genotype, 89.0 +/- 36.8 U/L; I/D genotype, 63.5 +/- 26.0 U/L; and I/I genotype, 55.1 +/- 39.4 U/L). The Michaelis constants for two substrates were identical whatever the genotype and were not different between centenarians and controls, i.e., 0.30 +/- 0.03 mmol/L for furylacryloyl-phenylalanyl-glycyl-glycine and 1.35 +/- 0.05 mmol/L for hippuryl-histidyl-leucine; for the latter, the optimal pH and activating concentration of chloride did not depend on I/D polymorphism. The maximal velocities with both substrates reflected the distribution of serum ACE activity as a function of the genotypes, in centenarians and in controls. In conclusion, plasma ACE activity is subject to a similar genotypic influence in centenarians as in adults 20-70 years of age; however, ACE itself appears to be functionally similar for each genotype. Furthermore, the D allele as well as the higher serum ACE activities associated with the D/D genotype cannot discriminate individuals at high risk for cardiovascular diseases, major causes of mortality before the age of 100 years.

57 citations


Cites methods from "A reliable radiometric assay for th..."

  • ...The ACE Km was also measured with hippuryl-histidyl-leucine (HHL) in a radiometric assay that we described previously (9)....

    [...]