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1 Faculté de Pharmacie, 2 Faculté des Arts et des Sciences, Département de Mathématiques et Statistique, Université de Montréal, Montréal, Canada H3C 3J7; 3 Department of Internal Medicine, University of Milan, Milano 20122 Italy; and 4 University Health Network, Toronto General Hospital, Toronto, Canada, M5G 2C4
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the serum of 116 healthy individuals, exogenous bradykinin (BK) half-life (27 ± 10 s) was lower than that of des-Arg9-BK (643 ± 436 s) and was statistically different in men compared with
women. The potentiating effect of an angiotensin-converting enzyme
(ACE) inhibitor was, however, more extensive for BK (9.0-fold) than for
des-Arg9-BK (2.2- fold). The activities of ACE,
aminopeptidase P (APP), and kininase I were respectively 44 ± 12, 22 ± 9, and 62 ± 10 nmol · min
1 · ml1. A
mathematical model (y = kt
e
t,
t > 0), applied to the BK kinetically released from
endogenous high-molecular-weight kininogen (HK) during plasma
activation in the presence of an ACE inhibitor, revealed a significant
difference in the rate of formation of BK between men and women. For
des-Arg9-BK, the active metabolite of BK, the rate of
degradation was higher in women compared with men, correlating
significantly with serum APP activity (r2 = 0.6485, P < 0.001). In conclusion, these results
constitute a basis for future pathophysiological studies of
inflammatory processes where activation of the contact system of plasma
and the kinins is involved.
kinins; angiotensin-converting enzyme inhibitors; contact system activation
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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BRADYKININ (BK) is a nonapeptide released from high-molecular-weight kininogen (HK) under hydrolysis by plasma kallikrein. Plasma kallikrein originates from prekallikrein during the activation of the contact system of plasma by a negatively charged surface (3). This activation has been considered for a long time as the unique mechanism leading to BK release. Recently, however, three different laboratories (10, 23, 35) have described HK activation by plasma kallikrein at the endothelial level.
BK exerts its pharmacological effects, mainly vasodilation, by activating constitutively expressed B2 receptors. However, the peptide is short-lived in various biological environments due to proteolytic cleavage. The different purified peptidases capable of degrading BK have been reviewed extensively (15). The importance of their participation in BK metabolism depends, however, on the nature of the biological milieu as well as the pathophysiological or experimental conditions (5, 15). Previously, the laboratory of Adam (13) developed an analytic approach that allowed the metabolism of exogenous BK to be measured in a limited number of human serum samples using the substrate at a nanomolar concentration. Adam and co-workers showed that BK is metabolized by three metallopeptidases. Angiotensin-converting enzyme (ACE) constitutes the main degradation pathway that transforms BK into its final inactive metabolite BK(1-5) (13, 15). The next most important enzyme is aminopeptidase P (APP), which transforms BK into the inactive peptide BK(2-9) (6). This metabolite is further degraded by dipeptidyl peptidase IV into BK(4-9) (6). Finally, kininase I, a generic name for different carboxypeptidases that transform BK into des-Arg9-BK (15), constitutes a minor metabolic pathway unless ACE is inhibited. Des-Arg9-BK, the active metabolite of BK, stimulates B1 receptors when expressed during experimental inflammatory conditions (4, 25, 26). In its turn, des-Arg9-BK is metabolized by ACE and APP, already involved in BK inactivation. However, in the case of des-Arg9-BK, APP represents the main metabolic pathway (6).
Because ACE has a higher affinity for BK than for angiotensin I (21, 22), it is now considered as a kininase rather than an angiotensinase (9, 21, 22). These kinetic characteristics and numerous experimental pieces of evidence, mainly of a pharmacological nature, plead for a role of BK in the cardiovascular and metabolic effects of ACE inhibitors (18, 24, 40). Besides these beneficial effects, ACE inhibitors are also endowed with acute and chronic side effects, among which are angioedema (AE), hypersensitivity reactions (HSR) during hemodialysis, severe hypotensive reaction (SHR) associated with some blood product transfusions, and cough (16, 20, 42). Although BK has been claimed to be responsible for these side effects, its participation has been scarcely documented, and only poor experimental evidence, if any, suggests such a pathophysiological role for the B2 agonist. To try to elucidate a potential role of kinins in the pathophysiology and symptoms of these acute and chronic side effects of ACE inhibitors, we (6, 7, 11) investigated BK metabolism in the serum of a limited number of patients who presented such side effects. Patients with a history of an HSR or a SHR associated with an ACE inhibitor exhibited, at the serum level, an increased half-life (t1/2) of des-Arg9-BK, this kinetic parameter correlating negatively with the APP activity.
The fact that the decreased activity of APP was not attributed to a circulating inhibitor but rather to an enzyme defect (6) required definition of the metabolism of kinins in normal individuals before extending these preliminary studies to larger groups of patients and their relatives who developed ACE inhibitor complications such as AE, HSR, or SHR.
In the present paper, we defined the metabolic profile of BK and its active metabolite, des-Arg9-BK, in a large number of healthy individuals (men and women) to obtain reference values. To accomplish these goals, we first applied to these samples the experimental approach we developed previously for a small number of men (n = 7) (13). The calculated kinetic parameters were verified by the measurement of metallopeptidase activities (ACE, APP, and kininase I) responsible for the serum metabolism of kinins. In a second part, the kinetics of generation of endogenous BK and des-Arg9-BK from HK were assessed when the contact system was activated by glass beads in the presence of an ACE inhibitor. Finally, comparisons were made between the results obtained by the different analytical approaches.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Blood Samples
Twenty milliliters of blood were obtained by venipuncture from the forearm of 116 healthy Caucasian volunteers (70 women and 46 men: 78 with were <40 yr old and 38 were
40 yr old) who were not under
any medication (mean age: 33 ± 14 yr, range 19-77 yr). Total
blood was collected half into dry tubes and the other half into tubes
containing 0.1 mol/l sodium citrate as anticoagulant (1 volume of
sodium citrate to 9 volumes of blood). After centrifugation (22°C, 15 min, 2,500 g), the serum and plasma samples were decanted and stored at 
20°C until biochemical investigation.
This study was reviewed and approved by the ethics committee for research on human subjects of the Université de Montréal.
Drugs, Peptides, and Reagents
BK and des-Arg9-BK were obtained from Peninsula Laboratories (Belmont, CA). The ACE inhibitor enalaprilat was from Merck Frosst Canada (Kirkland, QC, Canada). L-Arginine, dansyl-Ala, and o-phthalaldehyde were from Sigma-Aldrich (Oakville, ON, Canada). Arg-Pro-Pro was from Bachem (Torrance, CA). Dansyl-Ala-Arg was a generous gift from Dr. R. A. Skidgel (Dept. of Pharmacology, University of Illinois, College of Medicine, Chicago, IL).
-Mercaptoethanol and chloroform were purchased from Fisher
Scientific (Montréal, QC, Canada). HPLC grade ethanol was
procured from American Chemicals (Montréal, QC, Canada).
Metabolism of Exogenous BK and Des-Arg9-BK
Incubation procedure.
A first set of incubations was performed to assess the metabolic
profile of exogenous BK in the absence of in vitro ACE inhibition. Synthetic BK was added to the serum samples (final concentration of the
peptide = 471 nM) (13). After various incubation
periods at 37°C, ranging from 30 s to 120 min, the reaction was
stopped by adding cold anhydrous ethanol at a final concentration of
80% vol/vol. The samples were then incubated at 4°C for 1 h and
centrifuged (4°C, 15 min, 3,000 g) for the complete
precipitation of kinin precursors. The supernatant was decanted and
evaporated to dryness in a Speed Vac Concentrator (Savant; Farmingdale,
NY). The residues were stored at 20°C until quantification of the
immunoreactive peptides BK and des-Arg9-BK was performed.
Quantification of BK and des-Arg9-BK. The residues of the evaporated ethanolic extracts were resuspended in 50 mM Tris · HCl buffer, pH 7.4, containing 100 mM NaCl and 0.05% Tween 20. After resuspension, residual BK and formed des-Arg9-BK were quantified by two specific competitive chemiluminescent enzyme immunoassays, as previously described (12, 32). To detect and quantify immune complexes, both assays used highly specific polyclonal rabbit immunoglobulins raised against the carboxy-terminal end of BK and des-Arg10-kallidin and the digoxigenin-anti-digoxigenin system (Boehringer Mannheim; Laval, QC, Canada). These methods have been validated, and their analytic performances were reported (4, 8, 12, 32).
Kinetic parameters.
The kinin (S) hydrolysis rate constant (k) was
obtained with a first-order equation S = So × ekt. t1/2 was
calculated as t1/2 = ln(2)/k (28).
)] or with
[k(+)] enalaprilat served to estimate ACE relative
activity (ACE%) = 100 × [1k(+)/k()]. The relative participation of
kininase I in BK metabolism in the absence or the presence of an ACE
inhibitor was expressed as the percentage of added BK transformed into
des-Arg9-BK.
Serum Metallopeptidase Activity Measurement
Serum ACE activity.
ACE activity in serum samples was determined by Buhlmann ACE
radioenzymatic assay (Angiotensin-Converting Enzyme 3H-REA,
ALPCO; Windham, NH) according to the manufacturer's instructions. This
assay measured the hydrolysis of the synthetic substrate [3H]hippuryl-glycyl-glycine into
[3H]hippuric acid and a dipeptide, solvent extraction
separated tritiated hippuric acid from the unreacted substrate, and
radioactivity was measured in a beta counter. For each sample, tests
were performed in duplicate. ACE activity was expressed as ACE units,
as described by the manufacturer (1 unit ACE = 1 nmol of formed
[3H]hippuric
acid · min1 · ml1).
Serum APP activity.
APP activity was measured in serum samples by a modified version of the
assay described earlier by Blais et al. (6) and Simmons
and Orawski (36). Briefly, 20 µl of serum were incubated with Arg-Pro-Pro (final concentration = 0.5 mM) in 0.1 M HEPES buffer, pH 7, for a final volume of 200 µl. After an incubation period of 6 h at 37°C, the reaction was stopped by adding cold anhydrous ethanol (800 µl). The samples were then centrifuged (4°C,
15 min, 3,000 g), and the supernatant was decanted and
incubated at room temperature (between 10 and 30 min) with 3 ml of the
revelation buffer (0.05 M borate buffer, pH 9.5, containing 150 mg/l
o-phthalaldehyde and 50 µl/l -mercaptoethanol).
Fluorescence emission was read on an Aminco-Bowman Luminescence
Spectrometer (Rochester, NY). The excitation and emission wavelengths
were 310 and 455 nm, respectively. The emission intensity of a
calibration curve with L-arginine (0 to 5 mM) was obtained
under the same conditions. The results are expressed as nanomoles of
arginine released per minute per milliliter of serum sample.
Serum kininase I activity. Kininase I activity was measured by a modification of the method described by R. A. Skidgel (37). Briefly, 20 µl of serum were incubated at 37°C, for 70 min, with dansyl-Ala-Arg (final concentration: 0.2 mM) in 0.1 M HEPES buffer, pH 7 (total volume: 250 µl). After the incubation period, the reaction was stopped by adding citric acid 1 M (150 µl). Dansyl-Ala was extracted (15 s) by 3 ml of chloroform. After centrifugation (4°C, 10 min, 1,000 g) and removal of the aqueous phase, the fluorescence of the organic phase was measured, as described for APP, using 340 and 495 nm, respectively, as excitation and emission wavelengths. The emission intensity of a calibration curve (dansyl-Ala, 0 to 1 mM) was determined under the same conditions. The results are expressed as nanomoles of dansyl-Ala released per minute per milliliter of serum sample.
Kinetics of Release of Endogenous BK and Des-Arg9-BK During Plasma Activation
Contact system activation. After 1 ml of plasma was preincubated with enalaprilat (final concentration = 130 nM) for 20 min at 37°C in polypropylene tubes, the contact system was activated by incubating the plasma with glass beads (37°C, with agitation), using a modification of the method of Renaux et al. (33). The reaction was stopped after various incubation periods (0, 1, 2, 3, 4, 6, 12, 24, 36, 60 min for BK and 0, 3, 6, 12, 18, 24, 36, 48, 60, 120 min for des-Arg9-BK) by adding cold anhydrous ethanol at a final concentration of 80% vol/vol. The samples were then treated as described above for the metabolism of exogenous kinins before the quantification of endogenous BK and des-Arg9-BK.
Mathematical treatment.
For the endogenous measurements obtained at different times
t, the mathematical model y = ktet,
t > 0, was fitted to each subjects. This model with
three parameters k, 
, and (k > 0, 0, 0) corresponds to a form similar to gamma
distribution (34). Parameter k represents a
scale parameter: large (small) k values are for large
(small) values of the endogenous measurements. When is small
(large), the speed in obtaining the maximum is fast (slow), whereas for
small (large) values of , the speed at which the curve decreases to
0 is slow (fast). Parameter is therefore related to the shape of
the first part of the curve (the part that corresponds to formation),
whereas parameter is related to the second part of the curve,
corresponding to the degradation.
/; 2) maximum: the value of the
maximum of the curve that corresponds to the value of the curve for
t = /; 3) the area: area under the
curve that is mathematically given by k
( + 1)/ + 1, where ( + 1) is the gamma function; 4) half-life of formation (tf): the value tf in the
interval 0 to / for which
tet = (0.5) (/)e;
5) half-life of degradation (td): the
value td in the interval / to 
for
which
tet = (0.5) (/)e;
6) slope of the half-life of formation: the value of the
slope of the curve at half-life formation = ke
tf); and 7) slope of the
half-life of degradation: the value of the slope of the curve at
half-life degradation: ket
t
td).
Statistical Analysis
All data are expressed as means ± SD. Two-way analysis of variance was performed to study the effect of sex (men and women) and age (<40 yr and40 yr) on the variables and the parameters of the
mathematical model (29). When significant results were observed, a Satterwaite-Welch approach (30) was used to
test the possible effect of the heterogeneity of variances on the
P value. A P value < 0.05 was considered
significant. Reference limits were determined nonparametrically by
using the conventional empirical 2.5 and 97.5 percentiles. Pearson
correlation and simple linear regression were used to study the linear
relationship between certain variables (31).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Metabolism of Exogenous BK and Des-Arg9-BK
Half-life of BK and des-Arg9-BK.
The t1/2 of BK and des-Arg9-BK, in the
absence and presence of enalaprilat, in 116 healthy individuals is
presented in Table 1. In the absence of
ACE inhibition, BK was rapidly degraded (t1/2 = 27 ± 10 s). This value contrasted with the
t1/2 of des-Arg9-BK, which was 24 times higher (mean value 643 ± 436 s). When serum samples
were preincubated with enalaprilat at a concentration that completely
inhibited ACE activity, the t1/2 of BK and des-Arg9-BK was increased (244 ± 83 s and
1,410 ± 1,149 s, respectively). The potentiating effect of
enalaprilat was more pronounced in the case of BK,
t1/2 being increased by 9.0-fold compared with
2.2-fold for des-Arg9-BK. The mean values for
des-Arg9-BK t1/2 (in the absence and
presence of enalaprilat) presented elevated SD due to the wide ranges
of t1/2 without (196 to 2,878 s) and with (330 to
6,704 s) enalaprilat.
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Relative participation of ACE and kininase I in the metabolism of kinins. The relative contributions of the various metallopeptidases in the degradation of BK and des-Arg9-BK in human serum are shown in Table 1. ACE constituted the main enzyme responsible for the BK inactivation accounting for 88 ± 6% of total BK metabolic transformation. Also, this relative participation of ACE in BK metabolism showed no significant sex-to-age interaction and age effect but was statistically higher in men than in women (90 ± 3%, n = 46 vs. 87 ± 7%, n = 70, P = 0.027). Kininase I participation in BK metabolism is minor (11 ± 5%) but became more pronounced (46 ± 23%) when ACE was inhibited. In the case of des-Arg9-BK, ACE represented only 50 ± 12% of total degrading activity. The relative participation of both metallopeptidases in the serum metabolism of kinins showed no significant sex-to-age interaction and was not statistically different between both sexes and both categories of age.
Serum metallopeptidase activities.
The values obtained for serum ACE, APP, and kininase I are presented in
Table 2. For each of these
serum activities, no significant interaction sex-to-age was observed,
and the factor age was not significant. The difference in ACE activity
was highly significant between men (n = 46) and women
(n = 70) (49 ± 13 vs. 41 ± 11 nmol · min1 · ml1,
P = 0.0097). Similarly, significantly higher serum APP
activity was measured in women (24 ± 9 nmol · min1 · ml1,
n = 70) compared with men (19 ± 7 nmol · min1 · ml1,
n = 46) (P = 0.0292). In contrast,
kininase I exhibited similar values in both groups of men and women
(60 ± 10 vs. 63 ± 11 nmol · min1 · ml1 for men
and women, respectively).
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Reference intervals.
Reference intervals for all variables (t1/2 of BK
with and without enalaprilat, t1/2 of
des-Arg9-BK in the presence or absence of enalaprilat,
relative participation of ACE and kininase I in the serum metabolism of
BK and des-Arg9-BK and serum ACE, APP, and kininase I
activities) were determined in 116 healthy individuals and are shown in
Table 3. When significant differences
between men and women were observed, that is, for the variables
t1/2 of BK in the absence of enalaprilat, the relative participation of ACE in the metabolism of BK, ACE, and APP
activities (see Tables 1 and 2). Separate reference intervals for men
and women are presented in Table 3.
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Correlation between kinetic parameters and enzyme activities.
Table 4 shows the correlation between
serum enzymatic activities (ACE and APP) and the
t1/2 of BK and des-Arg9-BK in the
absence and presence of enalaprilat. As expected, a significant inverse
relationship could be calculated between the measured
t1/2 of BK in the absence of ACE inhibition and ACE activity (r = 0.6677, P = 0.0001). Also, significant correlations could be calculated between
serum APP activity and the calculated t1/2 of BK
in the presence and absence of enalaprilat (r = 0.5206, P = 0.0001 and r = 0.2433,
P = 0.0085, respectively) and the
t1/2 of des-Arg9-BK in the absence
(r = 0.6924, P = 0.0001) and presence
(r = 0.6840, P = 0.0001) of
enalaprilat. In addition, the relationship between serum APP activity
and the relative contribution of ACE in BK metabolism was also
significant (r = 0.6403, P = 0.0001) (data not shown).
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Metabolism of Endogenous BK and Des-Arg9-BK
Analysis of the influence of the factors sex and age on the different parameters characterizing the gamma model fitted to endogenous measures in time are showed in Table 5 with means ± SD. We observe no significant interaction between sex and age. No significant result was obtained for the factor age. Some significant results were obtained between men and women for the parameter (P = 0.0005), the constant k (P = 0.0001), time
of the maximum (P = 0.0065), and slope of formation
(P = 0.0002) for BK, whereas for
des-Arg9-BK, the difference in the means between men and
women was significant only for the constant k
(P = 0.0080).
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Table 6 represents the reference
intervals calculated for the different kinetic parameters. In this case
to, no influence of age could be calculated for the synthesis or the
degradation of BK and des-Arg9-BK. Figure
1 shows the mean kinetic profiles of
formation and degradation of bradykinin and
des-Arg9-bradykinin for men and women after activation of
the contact system, in the presence of enalaprilat, with glass beads in
normal human plasma.
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Comparison Between Exogenous and Endogenous BK Metabolism
Correlations were calculated between the metabolic parameters obtained from different experimental approaches. A highly significant correlation could be measured between the t1/2 of des-Arg9-BK generated from both exogenous (x) and endogenous (y) BK in the presence of ACE inhibition (y = 1004.20 + 1.27x, r2 = 0.8269, P < 0.001). Moreover, a highly significant correlation could also be assessed between the t1/2 of endogenous des-Arg9-BK (in the presence of enalaprilat) (y) and APP activity (x) (y = 6005.46 146.43x, r2 = 0.6485, P < 0.001).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In this paper, we present for the first time a global view of kinetic activation of the contact system of plasma and the characteristics of both BK and des-Arg9-BK metabolic pathways in the blood of a large number of healthy people. We used three different analytic approaches. First, we determined the t1/2 of BK and des-Arg9-BK in the absence and presence of an ACE inhibitor when BK was added to serum (13). Second, we measured the enzyme activities of metallopeptidases responsible for the BK metabolism in serum (13). Third, we defined the kinetics of release and degradation of endogenous BK and des-Arg9-BK when the contact system of plasma was activated with a negatively charged surface (23, 33). Although sex influences the values of several kinetic parameters, no effect of age could be evidenced.
The first experimental approach extends the results obtained previously on a limited number of healthy male individuals (n = 7) (13). The kinetics of exogenous kinins studied were performed in serum rather than in plasma. In fact, we have shown previously that the t1/2 of BK, measured in plasma anticoagulated with citrate, does not follow first-order kinetics, although the t1/2 of BK and des-Arg9-BK is similar in both conditions. With this approach (13), we demonstrated that BK t1/2 is significantly higher in women than in men. When sera were preincubated with enalaprilat, this difference was not further observed. In the case of des-Arg9-BK, the t1/2 was much higher than that of BK. The preincubation of serum with an ACE inhibitor also increased des-Arg9-BK t1/2, but the potentiating effect was lower than for BK. These results confirm our previous observations showing that ACE, the main degrading pathway of BK, is a secondary pathway for des-Arg9-BK (13). Under the same conditions, kininase I is a minor pathway, but its importance increases when ACE is inhibited.
We have previously used this approach to define the metabolism of exogenous kinins in the sera of patients who presented an acute side effect when treated with an ACE inhibitor. In patients who manifested HSR while on an ACE inhibitor and hemodialysis with a negatively charged membrane, we found a significantly higher t1/2 of des-Arg9-BK but not of BK compared with patients who had never developed such an acute side effect while dialyzed under the same conditions (6). This difference affecting des-Arg9-BK degradation was exaggerated when serum samples were preincubated with an ACE inhibitor. A similar anomaly affecting des-Arg9-BK metabolism could be observed in patients who presented SHR while treated with an ACE inhibitor and transfused with platelets or red blood cells that had been depleted of leukocytes by passage through a negatively charged membrane (11). When applied to the sera of patients who manifested AE associated with an ACE inhibitor, we could not, however, find a difference compared with healthy subjects (7).
We have reported previously that BK is mainly metabolized in serum by three metallopeptidases, ACE, APP, and kininase I, although des-Arg9-BK in its turn is degraded by only ACE and APP. In this study, we observed lower ACE activity in women. This finding must be related to the BK t1/2 and the calculated relative participation of ACE. Recently, it was reported that estrogen treatment modulates ACE mRNA concentration in rat tissues, with a resultant attenuation of tissue and serum ACE activity (17). Moreover, a decreased plasma level of ACE activity with a concomitant increase of plasma BK was observed in both hypertensive and normotensive postmenopausal women under hormone replacement therapy (39).
We have previously shown that APP is significantly lower in patients
with a history of HSR (HSR+) dialyzed with a negatively charged
membrane when compared with patients who did not present such reactions
(HSR), these activities being negatively correlated with
des-Arg9-BK t1/2 calculated in the
presence of an ACE inhibitor (6). In this study, we
confirmed the highly significant correlation between both parameters.
Moreover, the higher activity of APP in women can be related to the
fact that this metallopeptidase is encoded by chromosome X (38,
41).
Activation of plasma by its contact with a negatively charged surface is classically used to trigger the intrinsic coagulation pathway (23). An early study measured the kinetic release and degradation of BK (19) but did not consider des-Arg9-BK metabolism. This new approach, which reflects the activation of HK by plasma kallikrein, is important for at least two reasons. First, it allows a more physiological understanding of the plasma metabolism of BK and des-Arg9-BK than that obtained by spiking serum with exogenous BK, even at a nanomolar concentration. Second, it could allow new insights into the role of in vivo activation of the contact system in different pathophysiological conditions where its involvement is suspected (14, 27). To do that we activated the plasma with glass beads, a process that has been reported to be efficient in triggering the contact system (23). This activation is obtained in the presence of an ACE inhibitor to increase the importance of the BK/des-Arg9-BK metabolic pathway. The development of a mathematical model permits the analysis of different parameters characterizing the genesis of both endogenous B1 and B2 agonists and their degradation. We could observe, for the first time, that men and women differ in their formation of BK, whereas a difference is seen in the degradation of des-Arg9-BK. Further studies are necessary to explore the sex difference in the generation of endogenous BK. The difference cannot be attributed to the concentration of HK. In fact, in a precedent study (1), Adam et al. could find no evident difference between men and women for the precursor of BK, and they could not detect a significant difference of plasma prekallikrein activity when assessed by a chromogenic substrate in men and women (2).
In comparison to the results obtained for the exogenous peptide, the t1/2 of endogenous des-Arg9-BK could be correlated not only with the t1/2 of des-Arg9-BK generated from exogenous BK but also with APP activity. These observations show for the first time that this metallopeptidase and its sex-related differences are physiologically relevant in the degradation of the endogenous B1 agonist.
The results presented in this study constitute a new basis for further investigations not only of the BK and des-Arg9-BK metabolic pathways but also of the contact system of plasma. First, studies will be needed in the future to examine the pharmacogenetic aspect of side effects of ACE inhibitors. In fact, because APP has been shown to be encoded by chromosome X (38, 41) and because we have previously measured a decreased activity of that enzyme in HSR+ patients (6), we are working on defining the genetic aspects of this side effect. These investigations will also be extended to SHR. For ACE inhibitor-associated AE, in which we previously failed to evidence an anomaly of the exogenous BK/des-Arg9-BK metabolic pathways, we cannot exclude a dissociation between the metabolism of exogenous and that of endogenous BK, BK being generated and degraded locally. The kinetic measurement of endogenous BK and of des-Arg9-BK during plasma activation will be particularly interesting in this latter case. Finally, the kinetic release of BK from endogenous HK and its metabolism to des-Arg9-BK could open a new area of research to reassess the role of endogenous kinins in different pathological states (14, 27), where activation of the contact system of plasma has been documented.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. R. A. Skidgel for providing the substrate dansyl-Ala-Arg for measurement of serum kininase I activity. We are also grateful to Dr. R. W. Colman for constructive comments that improved our paper, to Dr. F. Péronnet for contribution to this study, and to M. D. Geadah for providing some serum and plasma samples. We also thank all the people who participated in this study by giving their blood.
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FOOTNOTES |
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C. Blais, Jr. is the recipient of a scholarship from the Fonds de la Recherche en Santé du Québec.
Address for reprint requests and other correspondence: A. Adam, Faculté de pharmacie, Université de Montréal, 2900, boul. Édouard-Montpetit, C. P. 6128, Succursale Centreville, Montréal, Québec H3C 3J7 Canada (E-mail: adama{at}pharm.umontreal.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 25 September 2000; accepted in final form 7 March 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Adam, A,
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Human kininogens of low and high molecular mass: quantification by radioimmunoassay and determination of reference values.
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Adam, A,
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Albert A,
Damas J,
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Faymonville ME.
Optimized determination of plasma prokallikrein on a Hitachi 705 analyser.
J Clin Chem Clin Biochem
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203-207,
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Bhoola, KD,
Figueroa CD,
and
Worthy K.
Bioregulation of kinins: kallikreins, kininogens, and kininases.
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