Peptides, Vol. 11, pp. 501-506. © Pergamon Press plc, 1990. Printed in the U.S.A. 0196-9781/90 $3.00 + .00

Effect of Prolonged High Salt Intake on Atrial Natriuretic Factor's

Kinetics in Rats 1'2

J IRI W I D I M S K Y , W A L D E M A R D E B I N S K I , O T T O K U C H E L , 3 N G U Y E N T. B U U A N D P A T R I C K D U S O U I C H *

Laboratory of the Autonomic Nervous System, Clinical Research Institute of Montreal, Httel-Dieu Hospital and *Department of Pharmacology, Universit~ de Montreal, Montrgal, Quebec H2W 1R7, Canada

Rece ived 15 Sep tember 1989

WIDIMSKY, J., W. DEBINSKI, O. KUCHEL, N. T. BUU AND P. DU SOUICH. Effect of prolonged high salt intake on atrial natriureticfactor's kinetics in rats. PEPTIDES 11(3) 501-506, 1990.--Plasma atrial natriuretic factor (ANF) paradoxically decreases after 5 weeks (but not after 3 weeks) of 8% NaCI intake in normotensive rats. As this phenomenon remains unaccounted for by changes in ANF production, we studied the disappearance of [125I]ANF(99-126) from the circulation as an alternative explanation of plasma ANF decline. Following 5 weeks (but not 3 weeks) of an 8% NaC1 diet, plasma concentrations of [125I]ANF were significantly decreased and metabolic clearance rate and volume of distribution were increased compared to control rats fed a 0.8% NaC1 diet. By studying [~25I]ANF tissue uptake we noted significantly greater peptide uptake after 5 weeks (but not after 3 weeks) of high salt consumption in several tissues. We hypothesize that prolonged (at least 5 weeks) 8% NaC1 ingestion increases the density and/or affinity of ANF binding sites. These changes may be responsible for the previously observed decline in plasma ANF concentrations after a prolonged high salt intake.

High salt intake ANF disappearance Metabolic clearance rate Volume of distribution Tissue uptake

ATRIAL natriuretic factor (ANF), a peptide with natriuretic, diuretic, vasorelaxant and neuromodulatory properties (9, 10, 15), is released to the circulation in response to atrial distention (16). The presence of specific ANF binding sites in renal tissue (5) suggests that the peptide may be involved in the control of sodium and water balance. A role for ANF in the regulation of volume homeostasis during acute conditions has been proposed (2,3), but the peptide's significance in the adaptation to prolonged salt intake is dubious and controversial (12, 13, 23).

Recent study in our laboratory has demonstrated that after 5 weeks of high salt intake (8% NaC1 diet), rat plasma ANF (COOH- as well as NH2-terminal ANF) does not increase, as one might expect, but is in fact significantly decreased (8). The concomitant finding of normal atrial and high left ventricular ANF concentrations without changes in atrial ANF mRNA makes it less likely that the reduction in plasma ANF is due to diminished or exhausted peptide synthesis. Therefore, in the present study, we analyzed the alternative possibility that alterations in ANF disposal could be responsible for this phenomenon. We investigated ANF

kinetics after an [125I]ANF(99-126) bolus injection in control rats kept on a standard diet and its possible modification induced by prolonged high salt diet for 3 or 5 weeks. In addition, we examined possible changes in ANF uptake by various tissues after salt loading since regional alterations may contribute to the overall disappearance rate.

METHOD

Radioiodination

ANF iodination was performed according to the lactoperoxi- dase technique (21). Monoiodinated ANF(99-126) was purified on an HPLC system, as previously described (7).

Plasma Radioactivity Measurements

After arrival, male Sprague-Dawley rats (Charles River, St. Constant, Quebec, Canada) were housed in our animal facilities under controlled temperature and humidity (4-5 rats per cage) for

~This work was supported by a grant from the Medical Research Council of Canada to the Multidisciplinary Group on Hypertension Research and by the Canadian Heart Foundation.

2This paper was presented in part at the AAP/ASCI/AFCR annual meeting in Washington, April 28-May 1, 1989. 3Requests for reprints should be addressed to Otto Kuchel, M.D., Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec

H2W 1R7, Canada.

501

502 WIDIMSKY ET AL.

10 4

~ 1° 3 u

.4 ~ CONTROLS B c---.--£] HIGH SALT- 3 WEEKS C ~'~ 75 HIGH SALT- 5 WEEKS

10 2 I I I I I I I I I 2 3 4 5 6 7 8

T IME (ram) .,~ / C VS .4

P<O.05 <,,,, C VS B

FIG. 1. Disappearance of plasma TCA-precipitable radioactivity after [~2SI]ANF(99-126) injection in controls (0.8% NaCI, n=7) and salt- loaded rats (8% NaCI) for 3 (n=7) and 5 weeks (n=6). TCA-- trichloroacetic acid. *Based on the trend of the curves: C vs. A p<0.05, F(8,128)=3.4; C vs. B p<0.05, F(8,128)=3.2. All points of curve C were significantly different compared to A and B curves except 6 and 8 min interval.

a 1-week acclimatization per iod and then divided into 3 groups: 1) controls maintained on standard rat chow (0.8% NaC1), 2) rats fed a high salt diet (8% NaC1) (Ralston Purina, Richmond, NJ) for 3 weeks, and 3) animals given a high salt diet (8% NaC1) for 5 weeks. All groups had access to drinking water ad lib.

On the final day of the experiment, both carotid arteries and right jugular vein of all animals were cannulated with PE-50 tubing under Somnotol (sodium pentobarbital, 50 mg/1 kg weight) anesthesia for blood pressure monitoring, blood collection and iodinated ANF administration. [~2SI]ANF(99-126) (~0 .4 ixCi/rat) was injected into the jugular vein in a total volume of 0.3 ml saline. After each injection, the catheter was flushed with 0.15 ml of heparinized saline (150 units heparin/ml 0.9% NaC1 containing 0.01% bovine serum albumin as vehicle). The remaining radioac- tivity in dead space (syringe, catheter) was calculated and sub- tracted from the injected values. Several 0.2 ml arterial blood samples were collected during 8 min after isotope injection (see Results) and each sample was replaced by the same volume of saline through jugular vein. After the centrifugation of blood, 0.1 ml plasma samples were precipitated with 0.5 ml of 10% trichlo- roacetic acid (TCA) as described elsewhere (1). The pellets, corresponding to the intact labeled ANF molecule (1), were counted in a gamma counter.

HPLC Study

To further evaluate the nature of measured radioactivity, 2 ml of arterial blood replaced by the same volume of saline were collected between 0.5 and 1, 2, and 2.5 and 5.5 and 6 min after [12SI]ANF injection from the rats of each group. One ml of obtained plasma was processed on Sep-Pak C18 cartridges as described elsewhere (16). The eluates were evaporated in Speed- Vac, reconstituted in 0.1 M acetic acid and loaded on Vydac C18 column (0.46 x 25 cm) of Varian 5000 HPLC system. Linear gradient from 10 to 50% CH3CN over 40 min was used and 1 ml

5 0 0 - 0 .5 - Imm

250

[ f25T]-ANF (SER 99-TYR P26)

t

500 1 2-2.5mm

500 5.5-6 rain

250

50

30

I0

? 5o

I-- 30

c5 TO

( J

! ~ - 50

10 20 30 40

TIME (rnin)

FIG. 2. HPLC profile of plasma radioactivity in different time intervals after [~2SI]ANF(99-126) injection in salt-loaded rats for 5 weeks. Time (min)--elution time. Flow rate 1 ml/min. The arrows indicate the elution position of iodinated ANF standard.

collected fractions were counted for radioactivity in a gamma counter.

Urine Radioactivity

Specimens of urine (0.5 ml) were collected 15 min after the []25I]ANF(99-126) injection and measured in a gamma counter.

Tissue Distribution

To investigate possible changes in the tissue distribution and uptake of [12SI]ANF other rats divided into the above indicated 3 groups were decapitated 30 sec after isotope administration. All tissues were quickly removed, washed in 0.9% NaC1, wiped dry and kept frozen until weighed, dissected and counted in a gamma counter no later than 3 days after sacrifice.

Intravascular Volume and Sodium Excretion

Measurement of blood and plasma volume in other animals

A N F P H A R M A C O K I N E T I C S AFTER NaC1 INTAKE 503

T A B L E 1

PHARMACOKINETIC DATA ON [125I]ANF(99-126) IN RATS AND CHANGES AFTER COLD ANF PREINJECTION

[125I]ANF + [1251]ANF Cold ANF (N = 13) (N = 13)

C o (% inj. dose/ml blood) 5.40 ___ 0.9 6.53 --- 1.2 AUC (% ml/min) 2.74 +-- 0.5 3.94 +-- 0.3* MCR (ml/min) 51.13 --- 10.2 27.60 +-- 2.5* ADJ. MCR (ml/min) 50.42 +-- 3.5 31.89 --- 3.5* VDI (ml) 31.07 ----- 8.4 22.76 -- 4.6 VD2 (ml) 234.2 ----- 47.8 120.9 ----- 10.4" tv2 (I) (sec) 8.96 ----- 0.9 9.79 ----- 1.1 tv2 (II) (sec) 261.7 ----- 15.5 241.92 - 16.7

All values are means --- SEM; *p<0.05. Co=extrapolated radioactivity concentration at time 0; AUC=area

under the disappearance curve; MCR=metabolic clearance rate; ADJ. MCR=adjusted MCR for VDt; VD~=initial volume of distribution; VD2=Volume of distribution in the second phase of disappearance; tv2 (I),(II) = half-life in circulation in the first or second phase of disappear- ance.

(controls and rats maintained on 8% NaC1 for 5 weeks) was performed by the dye-dilution method (4). Sodium excretion was measured in all 3 groups by f lame photometry from the samples, collected for 24 hr.

Pharmacokinetic Analysis

Visual inspection o f the decay of circulating labeled A N F

concentrations as a function of t ime showed two well-delineated phases, the fast first distribution and slow second elimination phase. Thus plasma concentrat ion-t ime curves were fitted to a two-compar tment open model , assuming first order distribution and elimination using a nonlinear regression curve-fitting program [PC-NONLIN, (19)]. Initial estimates for the computer program and kinetic parameters not est imated by the NONLIN program were calculated using model independent equations (11). Briefly, extrapolated [125I]ANF concentration at t ime zero (Co) was calculated f rom the sum A + B, where A and B are the coefficients o f the distribution and elimination terms, respectively. Area under the curve f rom 0 to 8 min (AUC 0-8) was calculated using the trapezoidal method, then extrapolated to infinity (AUC 8-infinity) using C 8 min/beta. AUC 0-inf ini ty (AUC) was calculated as the sum o f these terms. Metabolic clearance rate (MCR) was calculated dividing the administered dose by the AUC. Volume of distribution o f the central compartment (Vc) was calculated using relationship V c = dose/(A + B). Volume of distribution o f the beta phase (Vb~t,) was calculated using equation Vb~ta = dose/ (AUC × beta), where beta is the slope (times 2.303) of the elimination phase o f the curve. Vss was calculated according the relationship Vss = V c × [(k12 + k21)/k21], where k12 and k21 are distribution rate constants. The Vss can in fact only be predicted by this method, since steady-state conditions were not achieved following a single IV dose.

Statistics

Intergroup differences were evaluated by one- or two-way analysis o f variance and by unpaired Student ' s t-test (blood and plasma volumes). Differences were considered to be statistically significant at p < 0 . 0 5

TABLE 2

CHANGES IN [125I]ANF UPTAKE IN TISSUES AFTER 8% NaC1 INTAKE

Controls High Salt--3 Weeks High Salt--5 Weeks (n = 5) (n = 5) (n = 5)

Rel. Rad./100 mg Rel. Rad./100 mg Rel. Rad./100 mg (1) (2) (3)

Liver 4.7 +-- 0.2 3.8 _+ 0.9 4.7 --- 0.3 Lungs 31.4 --- 2.7 38.2 -+ 12.2 44.9 _.+ 4.3

(*vs. 1) Kidney 15.6 - 0.9 12.8 --- 1.1 24.9 --+ 2.1

(*vs. 1) (*vs. 1,2) Small intestine 2.4 --- 0.2 2.7 --- 0.5 4.2 --- 0.3

(*vs. 1,2) Spleen 2.7 -+ 0.2 2.7 ~ 0.3 3.6 --- 0.2

(*vs. 1,2) L. ventricle 3.8 --- 0.4 3.5 - 0.4 4.6 _ 0.4

(*vs. 2) R. ventricle 5.8 - 0.1 4.9 --- 0.5 8.0 --- 1.2

(*vs. 1,2) R. atrium 11.8 __ 2 11.8 +-- 2.3 21.3 - 6.0

(*vs. 1,2) L. atrium 4.5 ___ 0.4 3.3 --- 0.1 5.9 --- 1.1

(*vs. 2)

All values are means +_ SEM, *p<0.05; rel. rad./100 mg is relative radioactivity per 100 mg tissue = (cpm/100 mg tissue)/(cpm/100 mg weight of rat); F values for intergroup differences: Liver, F(2,12) = 3; lungs, F(2,12) = 4.6; kidneys, F(2,12) = 34.4; small intestine, F(2,12) = 28.7; spleen, F(2,12) = 11.3; 1. ventricle, F(2,12) = 4.2; r. ventricle, F(2,12) = 7.4; r. atrium, F(2,12) = 3.3; 1. atrium, F(2,12)=6.6.

504 WIDIMSKY ET AL.

TABLE 3

TISSUE [IzSI]ANF(99-126) UPTAKE AFTER COLD ANF PREINJECTION (% INJ. DOSE/t00 mg TISSUE)

Tissue

1 2 3 Controls 8% NaC1--3 Weeks 8% NaC1--5 Weeks

n=5 n=5 n=5

Kidney 0.19 ± 0.01 0.20 - 0.03 0.31 ± 0.01 (*vs. 3) (*vs. l/*vs. 2)

Adrenals 0.15 --- 0.02 0.18 ± 0.05 0.27 -- 0.02 (*vs. 3) (*vs. 1/*vs. 2)

Liver 0.11 ± 0.004 0.13 ± 0.01 0.15 _+ 0.004 (*vs. 1)

Lung 0.10 _+ 0.02 0.13 ± 0.01 0.25 --+ 0.02 (*vs. 3) (*vs. 1/*vs. 2)

R. atrium 0.09 ± 0.02 0.10 -+ 0.01 0.15 ± 0.004 (*vs. 1)

L. atrium 0.06 --- 0.01 0.07 ± 0.01 0.09 _+ 0.001 Spleen 0.06 __. 0.004 -- 0.07 ± 0.003 Small intestine 0.06 ± 0.02 0.15 - 0.01 0.17 - 0.01

(*vs. 1) (*vs. 1) R. ventricle 0.05 --- 0.01 0.07 ± 0.01 0.09 - 0.001

(*vs. 1) Mesenterium 0.03 ± 0.002 -- 0.04 - 0.001

(*vs. 1) Aorta 0.02 ± 0.002 0.05 ± 0.01 0.03 ± 0.003

(*vs. 1/*vs. 3) (*vs. 2) Skin 0.01 _+ 0.001 0.01 _+ 0.002 0.01 _+ 0.0003 Thyr. gland 0.01 ± 0.001 -- 0.02 ± 0.002 Striat. muscle 0.006 _+ 0.001 0.01 _+ 0.003 0.01 ± 0.001

(*vs. 1) (*vs. 1) Fat 0.005 ± 0.001 -- 0.012 -+ 0.001

(*vs. 1) Brain 0.002 +__ 0.0002 -- 0.004 _+ 0.001

(*vs. 1)

All values area means ± SEM; *p<0.05. Inj. dose - [125I]ANF(99-126) = 3 × 106 cpm. Cold ANF(99-126) = 5 p~g.

RESULTS

On the final day of the experiment, the weight of the rats ranged from 290 to 350 g, and no significant intergroup differ- ences were noted [controls 321---10 (SEM), 3 weeks 8% NaC1 315 -+ 7, 5 weeks 8% NaC1 305 --- 8 g]. The same applies for blood pressure levels (data not shown), that were not significantly changed by iodinated ANF injection. High salt intake for 5 weeks did not expand blood volume compared to control rats (8.7---0.5 vs. 9.4---0.7 ml/100 g body weight) as well as plasma volume (4.9---0.3 vs. 5.4---0.4 ml/100 g body weight). High salt intake for 3 and 5 weeks substantially increased sodium excretion compared to controls ( 3 3 _ + 1 vs. 2.5---0.1 and 25---2 vs. 2-+0.2 mmol/24 hr, respectively).

The decay of plasma TCA-precipitable (ppt.) radioactivity after [12SI]ANF injection in all groups is depicted in Fig. 1. It reveals that TCA-ppt. radioactivity was markedly lower after 5 weeks of salt intake compared to controls and 3 weeks salt-treated rats. There were no differences between the two latter groups. This figure also shows that the slope of the decay of plasma concen- trations is apparent after 2 half-lifes so that sampling for a longer

interval cannot, with the sensitivity of determination, change the result.

Figure 2 demonstrates the HPLC profile of plasma radioactivity in samples collected in different time intervals after [12SI]ANF injection in rats treated by salt diet for 5 weeks. At the early time interval (0.5-1 min) 70% of total radioactivity appeared between 26-30 min, where the [125I]ANF standard was eluted. This proportion was very similar to 72% of ppt. radioactivity found in the original sample. There were also 2 small peaks at 12 and 18 min, which may represent iodinated ANF degradation products, as indicated by their increase in later time periods. The [12SI]ANF peak was diminishing with time progressing after isotope injec- tion, so that between 5 .5-6 min it made up only 15% of total radioactivity, that corresponded to 13% of ppt. radioactivity in the original sample. The HPLC profile in controls or salt-treated rats for 3 weeks was similar with good correlation between proportion of radioactivity coeluted with [~25I]ANF and that of plasma ppt. radioactivity (data not shown). These findings enabled us to use postinjection plasma ppt. radioactivity as a marker of the intact [125I]ANF molecule.

ANF PHARMACOKINETICS AFI 'ER NaC1 INTAKE 505

TABLE 4

DEGREE OF INHIBITION (%) OF [12Sl]ANF(99-126) TISSUE UPTAKE AFTER COLD ANF PREINJECTION IN RATS ON PROLONGED 8% NaCI INTAKE

Tissue

1 2 3 Controls 8% NaC1--3 Weeks 8% NaC1--5 Weeks

n=5 n=5 n=5

Lung 88.5--- 2.3 89 --- 2.5

R. atrium 71.8 --- 8 78.7 --- 4.9 R. ventricle 70.5 _-. 2.6 63.3 --- 3.8 Small intestine 59.7 _ 7.2 40.3 ___ 5.8 Kidney 59.5 -+ 3.5 52 _+ 6.4 L. ventricle 57.3 _-, 2.7 47.3 +-- 7.3

Fat 57.3 -+ 6.9 -- Adrenals 55.3 _ 13.1 29.7 --- 13 Aorta 38.3 -+ 5.7 0

Spleen 29.8 - 6.2 -- Mesentefium 28.7 -+ 3.5 Thyr. gland 24.3 _+ 9 -- Liver 17.5 _+ 6.2 0 Skin 0 -- Striat. muscle 0

79.5 --- 1.3 (*vs. 1/*vs. 2) 68.3 --- 6.2 52.5 --- 8.3 51.7 +-- 5.2 53 - 3.9 35.8 --- 6.5

(*vs. 1) 0

31.3 +-- 9.6 9 + 5.3

(*vs. 1) 27.5 --- 4.7 12.7 +- 8.2 13.3 +-- 10

0 0 0

Values are means --- SEM, *p<0.05. Inj. dose - [1251]ANF(99-126) = 3 x 106 cpm. Cold ANF(99-126) = 5 jxg.

Table 1 summarizes the pharmacokinetic data calculated for [125I]ANF in all groups. Co and AUC values were significantly lower after 5 weeks of high salt diet compared to 2 other groups. Lower AUC corresponded to the markedly higher MCR levels in this group. Five weeks of salt diet also substantially enhanced all volumes of distribution (Vc, V~t a, Vss) in comparison with 2 other groups. No differences in all these kinetic parameters were found between controls and salt-treated rats for 3 weeks. High salt treatment for 3 or 5 weeks did not change the tl/2 levels for both disappearance phases.

Urinary radioactivity concentrations 15 min after isotope ad- ministration were negligible in all groups and ranged between 0.01% and 1,3% of the total injected activity per ml of sample with no intergroup differences.

Table 2 describes the radioactivity in various tissues of all groups 30 sec after isotope injection, when the majority of the [125I]ANF still circulates as an intact molecule. After 5 weeks of high salt diet, higher [~25I]ANF uptake was found in several tissues compared to controls and salt-treated rats for 3 weeks.

DISCUSSION

In the present study, we found a rapid disappearance of plasma ppt. radioactivity, which, as shown by its comparison with our HPLC data, represents intact labeled ANF, in agreement with a previous study (1). It has been shown that the disappearance of iodinated ANF is similar to nonlabeled ANT (20) and that [125I]ANF maintains its biological activity in bioassays (22). Our data may thus also apply to nonlabeled peptide.

Prolonged high salt intake for 5 weeks markedly decreased circulating [125I]ANF levels in plasma compared to controls or rats

treated by NaCI for 3 weeks. This decline corresponded well to the kinetic parameters, where we found after 5 weeks of salt intake lower AUC with corresponding increase in MCR. MCR and volume of distribution increased in parallel, resulting in un- changed levels of tu2 compared to controls or salt-treated rats for 3 weeks. Furthermore, all these changes following 5 weeks of salt

~25 intake were consistent with our data on tissue [ I]ANF uptake, where we found significantly higher levels in multiple organs in comparison to two other groups. Since, at the time of the tissue removal (30 sec after isotope injection) the majority of circulating radioactivity represents an intact labeled ANF molecule, tissue radioactivity should correspond to the binding of the intact iodinated peptide.

Taken together, all these high salt-induced changes could explain the previously observed (8) paradoxical decline of plasma ANF following 5 weeks of high salt diet. Furthermore, no apparent changes in [125I]ANF kinetics and uptake after the shorter duration of high salt diet for 3 weeks are consistent with our data of lack of differences in plasma ANF levels between this group (30 -+ 5 fmol/ml) and their controls (31 -+ 4 fmol/ml). Our findings do not permit evaluating the possible contribution of different baseline values of endogenous ANT to the changes of ANF binding sites and thus distinguishing primary and secondary mechanisms involved. However, from our finding of unchanged immunoreactive atrial ANF (8), as well as ANF mRNA with an even increase of atrial pressure after NaC1 intake (24), a decreased ANF secretion as a cause of plasma ANF decline with secondary upregulation of ANF binding sites seems to be unlikely.

The plasma decay of ANF is believed to imply 2 main processes: receptor-mediated binding to tissues and/or degradation by proteolytic enzymes (6, 14, 20). Cross-linking study revealed

506 WIDIMSKY ET AL.

that there are at least 2 types of ANF binding sites (17). One of them, a more abundant type called C-ANF (C for clearance) receptor, appears to be the major determinant of the MCR and Vss of ANF (1). Our present findings of an increase of MCR and Vss without salt-induced changes in intravascular volume are consis- tent with the possibility of an increased density and/or affinity of ANF binding sites, possibly of C-ANF receptors. Since the design of our study does not permit distinguishing between biologically active ANF receptors and C-ANF receptors, we have further studies using a specific C-ANF receptor ligand (1) presently under way. A high degree of tissue uptake inhibition by cold ANF (25) discounts a substantial contribution of nonspecific binding sites in the present results.

Negligible urinary radioactivity in all groups, involving prob- ably also [~25I]ANF hydrolytic products, discounted the role of urinary ANF excretion in its plasma clearance.

Since metabolism of ANF by degradative enzymes (14) may play a role in ANF clearance in vivo, one might argue that our data could be due to high salt-induced changes in proteolytic activity. However, it has been shown that proteolysis of ANF in the blood

plays a minor role in the peptide's clearance (20). Thus, ANF uptake by its binding sites makes up the major mechanism of the peptide's elimination from the circulation (1). Indeed, enzymatic cleavage products appear in the circulation after the majority of the hormone is cleared (6), which is in agreement with our data.

We hypothesize that prolonged high salt intake causes modifi- cations of ANF binding sites with subsequent changes of ANF's kinetics. These findings may be responsible for the previously observed marked plasma ANF decline, following prolonged high salt intake. Interestingly, all these changes have not been detected after a shorter period (3 weeks) of salt intake, so that the time factor may also play an important role in the regulation of ANF binding sites density and/or affinity in response to salt intake.

ACKNOWLEDGEMENTS

The authors thank Mr. Franqois Bellavance, M.Sc., from the Depart- ment of Mathematics and Statistics of the Universit6 de Montr6al for his statistical advice. Gratitude is also extended to Mrs. Louise Th6roux for her expert technical assistance and to Mrs. Sylvie Ouellet for her excellent secretarial help.

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