Combined effects of hyperaminoacidemia and oxandrolone on
skeletal muscle protein synthesis
Melinda
Sheffield-Moore1,
Robert R.
Wolfe1,2,4,
Dennis C.
Gore1,
Steven E.
Wolf1,
Dennis M.
Ferrer3, and
Arny A.
Ferrando1
Departments of 1 Surgery,
2 Anesthesiology, and
3 Internal Medicine, University of Texas Medical
Branch, and 4 Shriners Burns Hospital for Children,
Galveston, Texas 77550
 |
ABSTRACT |
We
investigated whether the normal anabolic effects of acute
hyperaminoacidemia were maintained after 5 days of oxandrolone (Oxandrin, Ox)-induced anabolism. Five healthy men [22 ± 3 (SD) yr] were studied before and after 5 days of oral
Ox (15 mg/day). In each study, a 5-h basal period was followed by a 3-h
primed-continuous infusion of a commercial amino acid mixture (10%
Travasol). Stable isotopic data from blood and muscle sampling were
analyzed using a three-compartment model to calculate muscle protein
synthesis and breakdown. Model-derived muscle protein synthesis
increased after amino acid infusion in both the control [basal
control (BC) vs. control + amino acids (C+AA); P < 0.001] and Ox study [basal Ox (BOx) vs. Ox + amino acids
(Ox+AA); P < 0.01], whereas protein breakdown was
unchanged. Fractional synthetic rates of muscle protein increased 94%
(BC vs. C+AA; P = 0.01) and 53% (BOx vs. Ox+AA;
P < 0.01), respectively. We conclude that the normal anabolic effects of acute hyperaminoacidemia are maintained in skeletal muscle
undergoing oxandrolone-induced anabolism.
anabolic agent; amino acids; stable isotopes
 |
INTRODUCTION |
IN MOST CASES, THE MAINTENANCE of chronic protein
homeostasis after injury, chronic illness, or trauma has been managed
by providing nutritional support alone. However, results of prospective clinical trials using nutritional support as an adjunct therapy to
primary treatment of cancer (9) and burns (14) indicate that muscle
catabolism persists despite aggressive nutritional therapy. The
difficulty in maintaining adequate nutritional status and lean muscle
mass by simply providing nutrients to patients has led to the study of
pharmacological agents such as oxandrolone (Oxandrin, Ox). Ox is a
synthetic analog of testosterone currently used as an adjunctive
therapy to promote weight gain in patients after surgery, chronic
infections, and severe trauma. Although anabolic agents such as Ox
offer clinicians viable treatment alternatives to managing disease- or
trauma-associated protein loss, few studies have directly measured
protein synthesis and protein breakdown after androgen therapy.
In a companion study (15), we recently showed that Ox, given at a
moderate dose of 15 mg/day for 5 days, increased the fractional synthetic rate (FSR) of skeletal muscle protein in normal healthy males
by 44% in the postabsorptive state, with no change in fractional breakdown rate. Furthermore, a recent study from our laboratory showed
that 5 days after a single intramuscular injection of testosterone enanthate (200 mg), FSR and model-derived protein synthesis increased twofold, with no change in fractional breakdown rate (11). Although these studies support the use of Ox or testosterone as anabolic hormones, the response during the postabsorptive state represents only
part of the day. It is thus pertinent to assess whether the normal
anabolic response to amino acids is retained after stimulation of the
basal rate of muscle FSR by an anabolic hormone.
Data from our laboratory in normal resting males showed that amino acid
infusion (10% Travasol) increased protein synthesis by ~150% after
an overnight fast (8). More recently, Volpi et al. (17) showed that
exogenous amino acids stimulate net muscle protein synthesis in the
postabsorptive elderly patient. Furthermore, orally ingested amino
acids also stimulate net muscle protein synthesis (16, 18). These data,
along with studies in animals (19) and humans (2, 3, 12), indicate that amino acid availability is an essential component in regulating muscle
protein metabolism. However, this may no longer be the case when the
basal rate of protein synthesis has been elevated by administration of
an anabolic hormone (i.e., oxandrolone). Therefore, using an
established protein kinetic model (5, 7), we investigated whether the
normal stimulatory effect of amino acids on muscle protein synthesis is
maintained in skeletal muscle after Ox-induced anabolism.
 |
METHODS |
Subjects
Five healthy males [22 ± 3 (SD) yr; 76 ± 15 kg; 176 ± 5 cm] were studied in the postabsorptive state and after amino acid infusion both before and after taking Ox for 5 days. All subjects gave
informed, written consent according to the guidelines established by
the Institutional Review Board at the University of Texas Medical Branch at Galveston. Subjects were given a thorough medical screening, and eligibility was assessed by the following tests: electrocardiogram, blood count, plasma electrolytes, blood glucose concentration, and
liver and renal function tests. Subjects presenting with heart or liver
disease, hypo- or hypercoagulation disorders, vascular diseases,
hypertension, diabetes, or an allergy to iodides were excluded from participation.
Experimental Protocol
Studies were performed at the General Clinical Research Center (GCRC)
at the University of Texas Medical Branch in Galveston. Subjects were
admitted the night before each study and were fasted from 2200 until
the completion of the 8-h study. At ~0630 the following morning
(day 0), a 20-gauge polyethylene catheter (Insyte-W, Becton-Dickinson, Sandy, UT) was inserted into the antecubital vein of
one arm for purposes of infusion of amino acids. A second 20-gauge
polyethylene catheter was placed in the contralateral wrist for
purposes of blood sampling for measurement of systemic indocyanine
green (ICG). A heating pad was placed around the arm and wrist to
maintain a temperature of ~65°C during blood flow measurements.
Baseline blood samples were drawn at 0700 for the analysis of
background amino acid enrichment, ICG concentration, and peak testosterone and oxandrolone concentrations. A primed (PD) continuous infusion of 99% pure labeled phenylalanine (Cambridge Isotope Laboratories, Andover, MA) was initiated at an
L-[ring-2H5]phenylalanine
infusion rate (IR) of 0.05 µmol · kg
1 · min
1
and a PD of 2 µmol/kg. A 3-Fr 8-cm polyethylene Cook catheter (Bloomington, IN) was placed into the femoral artery and vein for
purposes of arteriovenous (a-v) blood sampling and infusion of ICG
(artery) for determination of leg blood flow.
Biopsies of the vastus lateralis were obtained at 2, 5, and 8 h of
tracer infusion using a 5-mm Bergström needle (Fig.
1). Tissue was immediately frozen in liquid
nitrogen and stored at
80°C until analysis. The FSR of
skeletal muscle protein was determined by the incorporation of
L-[ring-2H5]phenylalanine
into protein from 2 to 5 h (values averaged) and from 5 to 8 h.
A continuous infusion (IR = 0.5 mg/min) of 100% pure ICG (Akron,
Buffalo Grove, IL) was initiated 15 min before each sampling hour
(4-5 h and 7-8 h) and allowed to reach systemic equilibrium (10-15 min) for purposes of measuring leg blood flow. Subsequent blood sampling was performed simultaneously from the femoral vein and
heated wrist vein throughout each sampling hour. Arteriovenous blood
samples were obtained at 20-min intervals from 4 to 5 h and again from
7 to 8 h to determine amino acid kinetics. To avoid disrupting blood
flow measurements, all a-v blood samples for amino acid kinetics were
obtained after blood flow measures were taken and the ICG was stopped.
After the basal period (0-5 h), a primed (PD = 0.45 ml/kg)
continuous infusion of unlabeled amino acids (10% Travasol, Clintec Nutrition, Deerfield, IL; total amino acids = 100 mg/ml) was initiated and maintained at the rate of 1.35 ml · kg
1 · h
1
until 8 h. The concentrations of the amino acids in the 10% Travasol mixture were as follows (µmol/l): alanine 232.3, arginine 66.0, glycine 137.2, histidine 30.9, isoleucine 45.7, leucine 55.6, lysine
39.7, methionine 26.8, phenyalanine 33.9, proline 59.1, serine, 47.6, threonine 35.3, tryptophan 8.8, tyrosine 2.2, and valine 49.5.
Peripheral and femoral catheters were removed at the end of each 8-h
infusion study. On the evening after the first infusion study (day
0 at 2100), all subjects began taking 15 mg of oxandrolone (BTG
Pharmaceuticals, Iselin, NJ) orally each evening for 5 days. On day
3, subjects returned to the GCRC at 0700 for venous blood sampling
to determine total testosterone and oxandrolone concentrations. This
experimental protocol was repeated again on day 5.
Analytical Methods
Blood.
Blood samples for the measurement of amino acid concentration and
enrichment were collected as previously described (7). Briefly, a-v
blood samples were collected in preweighed tubes containing 15%
sulfosalicylic acid. A known internal standard was added to the blood
samples (100 µl/ml of blood) for the measurement of blood amino acid
concentrations. The composition of this standard mixture was 50.3 µmol/l of
L-[ring-13C6]phenylalanine.
After the tubes had been reweighed to determine final blood volume, the
contents were centrifuged, and the supernatant was collected and stored
at
20°C until analysis. Blood amino acids were separated
using cation exchange chromatography (21). The enrichments and the
concentrations of phenylalanine in arterial and venous blood samples
were determined on their tert-butyldimethylsilyl (t-BDMS) derivatives by use of gas chromatography-mass
spectrometry (GC-MS) (21). The isotopic enrichment of free amino acids
in blood was determined by GC-MS in electron impact mode with selected ion monitoring (model 5973, Hewlett-Packard, Palo Alto, CA). Finally, serum concentration of ICG was determined by means of a
spectrophotometer at
= 805 nm.
Muscle.
Muscle samples were weighed and protein precipitated with 500 µl of
14% perchloric acid. A known internal standard solution (2 µl/mg of
muscle tissue) was added to measure the intracellular concentrations of
phenylalanine. The solution contained 2.4 µmol/l of
L-[ring-13C6]phenylalanine.
The supernatant was collected after homogenization of the tissue and
centrifugation. This procedure was repeated three times. The amino
acids in the pooled supernatant were then separated using cation
exchange chromatography (21). The isotopic enrichment of the
intracellular amino acids was determined on their t-BDMS
derivatives (21) by GC-MS in electron impact mode. Intracellular
enrichment was determined by correction for extracellular fluid on the
basis of the chloride method (4). The remaining pellet was washed
several times with 0.9% saline and again with absolute ethanol, dried
at 50°C overnight, and hydrolyzed in 6 N HCl at 110°C for 24 h.
The hydrolysate was then passed over a cation exchange column in the
same manner as the blood was processed. Phenylalanine enrichment was
measured by GC-MS (model 8000, MD 800, Fisons Instruments, Manchester,
UK) in electron impact mode and the standard curve approach (13).
Calculations
Kinetic model.
The mathematical derivation of the three-compartmental model of amino
acid kinetics has been described in detail (7) and again in the
companion study previously mentioned (15). However, we will briefly
detail the kinetic parameters that make up the three-pool model of leg
amino acid kinetics (Fig. 2). The
assumptions and limitations of the kinetic model have also been
described elsewhere (5, 8). However, it is important to note that the
underlying assumptions of the kinetic model hold true even when the
overall pool size of phenylalanine is increased after a primed,
continuous infusion of amino acids. Because calculation of the model
parameters depends on the maintenance of steady-state physiological
conditions, we primed the amino acids and infused at a constant
infusion rate. Finally, a-v sampling took place in the last hour of the
3-h amino acid infusion once blood amino acid concentrations and
enrichments achieved steady state.

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Fig. 2.
Three-compartment model of leg amino acid kinetics. Free amino acid
pools in femoral artery (A), femoral vein (V), and muscle (M) are
connected by arrows indicating unidirectional amino acid flow between
compartments. Amino acids enter the leg via the femoral artery
(Fin) and leave via the femoral vein (Fout).
FV,A is the direct flow from artery to vein of amino acids
that do not enter the intracellular fluid. FM,A and
FV,M are the inward and outward transport from artery to
muscle and from muscle to vein, respectively. FM,O is the
intracellular amino acid appearance from proteolysis for phenylalanine.
FO,M is the rate of disappearance of intracellular amino
acids for protein synthesis for phenylalanine.
|
|
The protocol used in this study was designed to simultaneously assess
both the kinetics of intracellular free amino acids and the FSR of
muscle proteins by the incorporation of labeled phenylalanine.
Enrichment data are expressed as tracer-to-tracee ratio. The model
enables the calculation of the rate of amino acid delivery by the
femoral artery (Fin) to the leg and also those amino acids
leaving the leg via the femoral vein (Fout). Therefore,
intercompartmental movement of amino acids is considered to occur
between the artery (A), vein (V), and muscle (M). Inward amino acid
transport from A to M (FM,A) and outward amino transport from M to V (FV,M) occur via the femoral artery and vein,
respectively. Thus inward (Fin) and outward
(Fout) tissue transport was calculated as follows
|
(1)
|
|
(2)
|
|
(3)
|
|
(4)
|
where
CA and CV and EA and EV
are amino acid concentrations and tracer enrichments in the femoral
artery and vein, and EM is enrichment in the muscle. BF
represents leg blood flow. Amino acids that bypass the muscle via the
femoral artery can be calculated by the following expression
|
(5)
|
The model also enables the calculation of the rate of
intracellular appearance (FM,O) of amino acids from protein
breakdown and the rate of amino acid utilization (FO,M) for
protein synthesis. Amino acid appearance and utilization are calculated
by the following formulas, respectively
|
(6)
|
|
(7)
|
The following expression represents the total rate of
appearance (RaM) of the intracellular amino acids, which is
a function of protein breakdown (FM,O) and inward tissue
transport (FM,A)
|
(8)
|
Protein synthesis efficiency.
Using phenylalanine, we calculated the relative efficiency of protein
synthesis as follows
|
(9)
|
PSE
is defined as the fraction of the intracellular amino acid rate of
appearance that is incorporated into muscle proteins, taking into
account that phenylalanine is not oxidized in the muscle. Therefore,
FO,M represents the amount of amino acid incorporated in
the muscle proteins.
FSR.
Using the traditional precursor-product method, we determined the FSR
of muscle proteins by measuring the rate of phenylalanine tracer
incorporation into protein and the enrichment of the intracellular pool
as the precursor
|
(10)
|
where
Ep1 and Ep2 are the enrichments of the
protein-bound
L-[ring-2H5]phenylalanine
at the 2- and 5-h and again at the 5- and 8-h sampling time points.
Average intracellular
L-[ring-2H5]phenylalanine
enrichment is EM, and time in minutes is represented by
t. To express FSR in percent per hour (%/h), the expression is
then multiplied by the factors 60 (min/h) and 100, respectively. The
assumptions and limitations necessary for the traditional derivation
have been outlined previously (20, 23).
Statistical Analysis
Group comparisons were performed using a one-way ANOVA. Post hoc
comparisons were accomplished using a one-tailed t-test with Bonferroni correction for multiple comparisons. Statistical
significance was established at P
0.05. Data are presented
as means ± SE.
 |
RESULTS |
Steady-state blood amino acid concentrations and enrichments were
maintained during each sampling hour (240-300 and 420-480 min) both before and after 5 days of Ox administration. All data identifying Ox's protein synthetic effect in the basal state, as well
as its hormonal effects, have been presented elsewhere (15). Only data
related to the response of skeletal muscle to amino acid infusion will
be presented.
As depicted in Table 1, free amino acid
enrichments in the femoral artery, vein, and muscle decreased
significantly after amino acid infusion, whereas amino acid infusion
significantly increased phenylalanine concentration in the femoral
artery and vein (Table 2). Leg blood flow
was unaffected by amino acid infusion alone [basal control (BC)
vs. control + amino acids (C+AA), 4.3 ± 0.9 vs. 5.3 ± 0.8 ml · min
1 · 100 ml leg
1] or with amino acids and
Ox combined [basal oxandrolone (BOx) vs. oxandrolone + amino
acids (Ox+AA); 4.0 ± 0.8 vs. 3.7 ± 0.5 ml · min
1 · 100 ml leg
1]. FSR of muscle protein
increased 94% (BC vs. C+AA; 0.0570 ± 0.004 vs. 0.1104 ± 0.013%/h,
P = 0.01) and 53% (BOx vs. Ox+AA; 0.0820 ± 0.009 vs. 0.1252 ± 0.008%/h, P < 0.01; Fig. 3).
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Table 1.
Effects of amino acid infusion on phenylalanine free amino acid
enrichments in femoral artery, femoral vein, and muscle before and
after oxandrolone administration
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Table 2.
Effects of amino acid infusion on phenylalanine free amino acid
concentrations in the femoral artery and femoral vein before
and after oxandrolone
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Fig. 3.
Muscle protein fractional synthetic rate (FSR). Muscle protein FSR in 5 young men before (basal control, open bar) and after amino acid
infusion (control + amino acids, hatched bar) and before (basal
oxandrolone, solid bar) and after 5 days of oxandrolone administration
(oxandrolone + amino acids, gray bar) and amino acid infusion. Amino
acid infusion significantly (* P 0.01) increased FSR both
before and after oxandrolone.
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|
Table 3 depicts the model-derived
parameters of leg muscle free amino acid kinetics of the five subjects
in the control and Ox study after amino acid infusion. As expected,
arterial delivery to the leg (Fin) increased significantly
in the control and Ox study after amino acid infusion (P < 0.01). However, arterial delivery to the leg (Fin) was
significantly less when amino acids were given after Ox treatment
(P = 0.02). As a consequence, model-derived muscle protein
synthesis (FO,M) was unchanged from C+AA to Ox+AA. However,
if synthesis was expressed relative to amino acid delivery to the leg
(Fin), a combined effect of Ox and amino acids was seen
compared with amino acids alone (P = 0.03; Fig.
4). The intracellular rate of appearance of
phenylalanine (FM,O), index of protein breakdown, was
unaffected by either amino acid infusion or Ox. However, consistent with the direct incorporation data, the rate of intracellular utilization of phenylalanine for protein synthesis (FO,M)
increased significantly after amino acid infusion in both the control
and Ox studies (BC vs. C+AA, P < 0.001; BOx and Ox+AA,
P < 0.01; Table 3).
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Table 3.
Effects of amino acid infusion on model-derived leg muscle amino acid
kinetics before and after oxandrolone administration
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Fig. 4.
Model-derived protein synthesis relative to arterial delivery to the
leg. Model-derived protein synthesis in 5 young men after amino acid
infusion before (control + amino acids, hatched bar) and after
oxandrolone (oxandrolone + amino acids, gray bar). Amino acid infusion
significantly (* P < 0.01) increased model-derived protein
synthesis both before and after oxandrolone.
|
|
Amino acid infusion resulted in a shift in net balance from a net
negative output to a net positive uptake both before and after Ox
(Table 3). No difference was seen in net balance from C+AA to Ox+AA (45 ± 12 vs. 30 ± 8). Finally, protein synthesis efficiency remained
unchanged after amino acid infusion (BC vs. C+AA and BOx vs. Ox+AA).
 |
DISCUSSION |
We examined the response of muscle protein kinetics to an acute amino
acid infusion before and after oxandrolone-induced anabolism. We
demonstrated that both the model-derived value for muscle protein synthesis and the traditionally derived value of the FSR of muscle protein increased with infusion of amino acids both before and after
oxandrolone-induced anabolism in young men. We did not, however, show a
statistically significant increase in the FSR of muscle protein when
comparing amino acids alone with the combination of oxandrolone and
amino acids. However, when the model-derived measure of muscle protein
synthesis (FO,M) was expressed relative to arterial
delivery (Fin) to the leg, the synthetic effect of amino
acids was maintained, despite the ongoing anabolism of oxandrolone. The
lower arterial delivery during oxandrolone and amino acids presumably
reflected accelerated amino acid uptake in other tissues in addition to
the muscle. Muscle anabolism during amino acid infusion occurred by
stimulation of protein synthesis, because protein breakdown was
unchanged. Moreover, protein synthetic efficiency was unchanged with
amino acid infusion from control to oxandrolone, indicating that no
greater fraction of the available intracellular amino acids was
incorporated into muscle proteins.
We recently reported that 5 days of oxandrolone administration
increased skeletal muscle anabolism by stimulation of protein synthesis, because protein breakdown was unchanged (15). Also, we
reported a significant decrease in outward amino acid transport (FV,M), along with a calculated increase in protein
synthetic efficiency, together indicating increased intracellular
reutilization of amino acids (15). These findings demonstrated the
anabolic potential of oxandrolone in the skeletal muscle of normal
fasted young men with only 5 days of administration. However, an
individual is only postabsorptive for part of the day. The overall
effectiveness of oxandrolone is thus dependent on the response during
food intake as well. We, as well as others, have shown that increased
availability of amino acids is a primary stimulus for muscle anabolism
in the fed state. Data from our own studies in both young (8) and elderly (17) volunteers indicate that the stimulation of inward amino
acid transport to the leg is the mechanism whereby the intravenous infusion of amino acids stimulates net muscle protein synthesis (8,
17). In agreement with our previous findings (8, 17), results from the
present study indicate that protein synthesis efficiency did not change
during amino acid infusion in the fasted state. In combination, these
results identify amino acid availability as the rate-limiting factor in
muscle protein synthesis in the fasted state. In contrast to the action
of amino acids, anabolic hormones such as insulin (6), testosterone
(11), and oxandrolone (15) increase the efficiency of protein synthesis
while not affecting amino acid availability.
Several investigations have examined the in vivo response of skeletal
muscle to insulin by utilizing the arteriovenous balance method (1, 6,
10, 12, 22). From these results, we can deduce that, whereas insulin
has the potential to stimulate muscle protein synthesis, this can only
be reflected in an increased rate of synthesis if an adequate
availability of amino acids is maintained. Thus systematically
administered insulin can only stimulate muscle protein synthesis if
amino acids are administered simultaneously. In contrast, the
stimulation of muscle protein synthesis by oxandrolone does not require
exogenous amino acids (15). Nonetheless, the current results indicate
that exogenous amino acids stimulate muscle protein synthesis in
subjects treated with oxandrolone. We can thus anticipate an overall
anabolic effect of oxandrolone, because not only is protein synthesis
increased in the postabsorptive state, but also the normal stimulatory
effect of amino acids on muscle protein synthesis is maintained.
In the present study, amino acid infusion before and after oxandrolone
administration significantly increased arterial delivery to the leg
(Fin) compared with basal (postabsorptive) arterial delivery. As a consequence, muscle protein synthesis was greatly improved in both cases. However, despite the considerable increase in
arterial delivery with amino acids combined with oxandrolone, arterial
delivery was significantly less than with amino acids alone. As a
consequence, no further increase in muscle protein synthesis was
realized. Thus, because delivery has an effect on protein synthesis,
and delivery was reduced when amino acids were given to subjects who
had been given oxandrolone, it is reasonable to examine protein
synthesis relative to delivery. Therefore, by expressing synthesis
relative to amino acid delivery to the leg, a combined effect of
oxandrolone and amino acids was seen compared with amino acids alone.
The retention of the anabolic effect of amino acids during oxandrolone
treatment, coupled with the anabolic effect of oxandrolone alone on net
muscle protein synthesis in the postabsorptive state, leads to the
expectation of an overall anabolic effect of oxandrolone treatment on
muscle protein.
 |
ACKNOWLEDGEMENTS |
The investigators thank the subjects who participated in this
study. Also, many thanks to the nurses and staff of the University of
Texas Medical Branch General Clinical Research Center (GCRC) for their
assistance with this project, Lakshmi McRae for nursing support, Zhi
Ping Dong for sample analysis, and BTG Pharmaceuticals for providing
the study drug.
 |
FOOTNOTES |
Studies were conducted at the GCRC at the University of Texas Medical
Branch at Galveston. Funding was provided by National Institutes of
Health Grants M01 RR-00073, from the National Center for Research
Resources, 5-R01-DK-38010-11 (to R. R. Wolfe), and R01-GM-57295-01 (to A. A. Ferrando).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Sheffield-Moore, Metabolism Unit, Shriners Burns Institute, 815 Market
St., Galveston, TX 77550 (E-mail: melmoore{at}utmb.edu).
Received 13 May 1999; accepted in final form 27 September 1999.
 |
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