1 Department of Medicine, McMaster University, Hamilton, Ontario L8S 3Z5; and 2 Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1
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ABSTRACT |
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The effects of carbohydrate deprivation
on the regulation of pyruvate dehydrogenase (PDH) were studied at rest
and during moderate-intensity exercise. An inhibitory effect of a
chronic low-carbohydrate diet (LCD) on the active form of PDH (PDHa)
mediated by a stable increase in PDH kinase (PDHK) activity has
recently been reported (Peters SJ, Howlett RA, St. Amand TA,
Heigenhauser GJF, and Spriet LL. Am J Physiol Endocrinol
Metab 275: E980-E986, 1998.). In the present study, seven males
cycled at 65% maximal O2 uptake for 30 min after a 6-day
LCD. Exercise was repeated 1 wk later after a mixed diet (MD). Muscle
biopsies were sampled from the vastus lateralis at rest and at 2 and 30 min of exercise. At rest, PDHa activity (0.18 ± 0.04 vs.
0.63 ± 0.18 mmol · min1 · kg wet
wt
1), muscle glycogen content (310.2 ± 36.9 vs.
563.9 ± 32.6 mmol/kg dry wt), and muscle lactate content
(2.6 ± 0.3 vs. 4.2 ± 0.6 mmol/kg dry wt) were significantly
lower after the LCD. Resting muscle acetyl-CoA (10.8 ± 1.9 vs.
7.4 ± 0.8 µmol/kg dry wt) and acetylcarnitine (5.3 ± 1.4 vs. 1.6 ± 0.3 mmol/kg dry wt) contents were significantly elevated after the LCD. During exercise, PDHa, glycogenolytic rate (LCD
5.8 ± 0.4 vs. MD 6.9 ± 0.2 mmol · min
1 · kg dry wt
1 ), and muscle
concentrations of acetylcarnitine, pyruvate, and lactate increased to
the same extent in both conditions. The results of the present study
suggest that inhibition of resting PDH by elevated PDHK activity after
a LCD may be overridden by the availability of muscle pyruvate during exercise.
carbohydrate and fat metabolism; glycogen phosphorylase
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INTRODUCTION |
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THE PYRUVATE DEHYDROGENASE complex (PDHc) is the primary regulator of whole body oxidative carbohydrate metabolism. Pyruvate dehydrogenase (PDH) or E1 is the first component of PDHc and is responsible for catalyzing the irreversible decarboxylation of pyruvate (45), with irreversibility of the entire complex being conferred by this initial reaction (34). PDH exists in both an active, unphosphorylated form (PDHa) and an inactive, phosphorylated form (25, 26), and its activity is largely controlled by the action of the following two enzymes: an inactivating kinase (PDHK) and an activating phosphatase (PDHP; see Ref. 45). Elevation of the mitochondrial acetyl-CoA-to-CoASH, NADH-to-NAD+ (31), and ATP-to-ADP (45) ratios increase PDHK activity, whereas pyruvate (11) decreases PDHK activity. PDHP is activated by increasing intramuscular calcium ion concentration, as seen at the onset of exercise (45). In this way, the acute regulation of the activation state of PDH is complex, being determined by the relative activities of PDHP and PDHK and the concentration of allosteric regulators.
For several years, our laboratory has been studying the regulation of carbohydrate and fat metabolism within human skeletal muscle (14, 15, 29, 33). Putman et al. (33) examined the effects of carbohydrate deprivation and high plasma free fatty acid concentration ([FFA]) on PDHa at rest and during exercise. Resting PDHa was significantly lower after a 3-day low-carbohydrate diet (LCD) compared with a 3-day high-carbohydrate diet. During exercise, PDHa increased at a slower rate after the LCD, despite lower accumulations of acetyl-CoA. The authors concluded that during exercise, PDHa is regulated by factors other than changes in intramuscular acetyl-CoA content. Recently, we reported that 3-6 days on a LCD produced a 3.5- to 5-fold stable increase in PDHK activity in human skeletal muscle (30). In light of this finding, the reduced resting and exercise PDHa reported by Putman and colleagues (33) may potentially be explained by a stable increase in PDHK activity. Alternatively, during exercise, there was an attenuated increase in pyruvate with the LCD that may have accounted for the lower PDHa.
The aim of the present study was to evaluate the effects of a 6-day LCD on the activation state of PDH at rest and during moderate-intensity exercise. Specifically, we examined the effects of an increase in PDHK activity, induced by an LCD, on the regulation of PDHa during exercise in untrained subjects. It is generally believed that pyruvate is one of the primary regulators of PDHK activity (25). In the present study, we wanted to determine the role of pyruvate in the regulation of PDHa at rest and during exercise after an LCD. In addition, because a previous study (33) of LCD was accompanied by reductions in muscle glycogen concentration ([glycogen]), every attempt was made to maintain a nonlimiting resting muscle glycogen content after the LCD. Our hypothesis was that the LCD would severely inhibit resting PDHa but that during exercise there would be no difference in the activation of PDHa due to the availability of intramuscular pyruvate in both conditions.
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METHODS |
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Subjects
Seven untrained healthy young males participated in this study. Their mean ± SE maximal O2 uptake (Preexperimental Protocol
Subjects completed a continuous incremental exercise test on a cycle ergometer, using a Quinton metabolic cart (Quinton, Q-plex 1; Quinton Instrument, Seattle, WA), to determine theirExperimental Protocol
Subjects visited the laboratory on two occasions, at the same time of day, separated by ~1 wk. Before each visit, subjects followed either an LCD or a mixed diet (MD) for a period of 6 days. For the MD, the composition of the subject's normal diet was analyzed for a period of 3 days. From the foodstuffs that the subjects normally consumed, diets were adjusted so that they were composed of 55-60% carbohydrate, 30% fat, and 10-15% protein. For the LCD, an outline of foods to avoid (i.e., potatoes, pasta, bread, rice, fruit), foods to eat only one time per day (i.e., broccoli, cauliflower, tomatoes), and foods to eat freely (i.e., oils, butter, mayonnaise, cheese, meats) was provided to subjects as previously described by Peters et al. (30). The LCD was tailored on an individual basis for each subject, and they were allowed to choose from the choices provided in the outline. The LCD was composed of 3-7% carbohydrate, 59-65% fat, and 30-35% protein. For compliance to the individual diets, subjects were asked to keep a dietary record listing everything that they ingested throughout the experimental period. During each diet condition, subjects were instructed to refrain from participating in any activities that would require an energy expenditure above that necessary for normal daily living (i.e., walking a flight of stairs was permitted). This restriction was placed on subjects to conserve their muscle glycogen stores during each 6-day diet. On test days, subjects were instructed to eat their last meal 2-3 h before their visit to the laboratory. The LCD condition was completed first, followed by the MD condition ~1 wk later. Conditions were completed in this order because it was hypothesized that subjects would have more trouble completing the exercise protocol after the LCD. This design allowed us the flexibility of matching exercise times for the MD if a subject was unable to complete the exercise regime after the LCD. One subject was unable to finish the protocol and stopped cycling after 24 min of exercise in the LCD condition. With this subject, exercise time was matched at 24 min in the MD condition.During each visit to the laboratory, the antecubital vein was
catheterized percutaneously with a Teflon catheter (18 gauge, 3.2 cm;
Becton-Dickinson Vascular Access). The catheter was maintained patent
with sterile nonheparinized isotonic saline. While lying in a supine
position before each exercise test, subjects had the lateral portion of
one thigh prepared for needle biopsy sampling as previously described
by Bergström (2). A small incision was made
superficially to the vastus lateralis muscle, through the deep fascia,
at three sites under local anesthesia (2% lidocaine). After a resting
biopsy had been taken, subjects moved to an electronically braked cycle
ergometer (Quinton, Q-plex 1; Quinton Instrument) and began pedaling.
In both conditions, subjects exercised for 30 min at 65%
O2 max. During exercise, muscle
biopsies were taken at 2 and 30 min on the cycle ergometer. Muscle
biopsies (80-120 mg wet wt) were immediately frozen and stored in
liquid N2.
During exercise, expired gases were collected at 8-10,
18-20, and 26-28 min. Measurements of O2 uptake
(O2) and CO2 output (
CO2) were made using a Quinton
metabolic cart (Quinton, Q-plex 1; Quinton Instrument). Respiratory
exchange ratios (RER) were calculated from the
O2 and
CO2 data.
Blood Sampling and Blood Analysis
Blood samples of ~3-4 ml were drawn from the antecubital vein in heparinized plastic syringes at rest and at 10, 20, and 30 min of exercise. One portion of whole blood (400 µl) was deproteinized by 800 µl of 6% perchloric acid and then was centrifuged at 15,900 g for 2 min. The supernatant was removed and stored atMuscle Sampling
An ~5- to 15-mg piece of frozen muscle (free of blood and connective tissue) was chipped from each biopsy under liquid N2 and was used to measure PDHa. The remaining frozen muscle was freeze-dried and was stored atAnalysis of Muscle
PDHa was determined using the method described by Constantin-Teodosiu et al. (8) and modified by Putman et al. (33). One aliquot of powdered muscle, ~4-10 mg, was alkaline extracted for glycogen determination. For the exercise biopsies, a single aliquot of ~3-4 mg was used to determine the activity of both the total (a + b) and the more active (a) forms of glycogen phosphorylase (Phos), as described by Chesley et al. (7) and Young et al. (47). By substituting these activities into the Lineweaver-Burke relationship (24), the maximal velocities (Vmax) for both Phos a + b and Phos a were calculated (4-6). The mole fraction of Phos in the a form was then determined by (Vmax a/Vmax a + b) × 100. For resting muscle samples, Phos a was not measured, because an accurate measurement requires that the sample be kept at room temperature for ~30 s before it is frozen in liquid N2 (38).The remainder of the powdered muscle was extracted in a solution of 0.5 M PCA and 1 mM EDTA, neutralized to pH 7.0 with 2.2 KHCO3,
and stored at 80°C until metabolite analysis was completed. Acetyl-CoA, CoASH, acetylcarnitine, and carnitine were determined by
radioisotopic assay (3). The muscle extracts were analyzed using enzymatic techniques for the following metabolites: glucose, glucose 6-phosphate, pyruvate, lactate, creatine, phosphocreatine (PCr), glycerol 3-phosphate, and ATP (1).
Calculations
The free concentrations of both adenosine diphosphate (ADPf) and adenosine monophosphate (AMPf) were calculated as described by Dudley et al. (13) using the reactants and equilibrium constants of the near-equilibrium reactions catalyzed by both creatine kinase and adenylate kinase. H+ concentration ([H+]) was estimated using the regression equation of Sahlin et al. (40). Free Pi content was calculated as the sum of the estimated resting free phosphate concentration (13) and theStatistics
For the exercise data, independent variables were examined using a two-way repeated-measures ANOVA (time × diet condition). An ![]() |
RESULTS |
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Respiratory Variables
Ventilation was unaffected by diet (Table 1). There were no differences in
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Blood Metabolites
There was no difference in venous glucose concentration between the LCD and MD conditions at rest and throughout exercise (Table 2).
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Venous lactate concentration ([Lac]) was significantly
lower in the LCD condition at all time points sampled (Table 2).
[Lac
] increased significantly in both conditions from
rest to exercise.
Plasma [FFA] was significantly greater in the LCD condition at rest and throughout the 30 min of exercise (Table 2). In the LCD condition, the [FFA] was 0.73 ± 0.09 mmol/l at rest, decreased significantly to 0.42 ± 0.03 mmol/l at 10 min, and remained constant at this level until increasing to 0.62 ± 0.08 mmol/l at 30 min of exercise. In the MD condition, the [FFA] was 0.20 ± 0.04 mmol/l at rest and remained at this level for the duration of the exercise period.
Plasma glycerol concentration ([glycerol]) was significantly greater in the LCD condition at rest and throughout the exercise period (Table 2). [Glycerol] increased significantly during the LCD condition from 23.6 ± 6.7 µmol/l at rest to 220.6 ± 6.7 µmol/l at 30 min of exercise.
At the onset of exercise, plasma insulin concentration was significantly lower after the LCD condition compared with the MD (MD 21.0 ± 6.7 vs. LCD 7.0 ± 1.7 mIU/l).
Plasma D--hydroxybutyrate concentration
([
-OHB]) was significantly greater in the LCD condition at rest
and throughout the 30 min of exercise (Table 2). In the LCD condition,
the [
-OHB] was 1.83 ± 0.49 mmol/l at rest, decreased
significantly to 1.11 ± 0.30 mmol/l at 10 min, and remained
relatively constant at this level for the remainder of the exercise
period. In the MD condition, the [
-OHB] was 0.17 ± 0.02 mmol/l at rest and remained at this level for the duration of the
exercise period.
Muscle Metabolites
Glycogen.
Muscle glycogen content ([glycogen]) was significantly lower in the
LCD condition at rest and after 30 min of exercise (Fig. 1). In the LCD and MD conditions, the
change in [glycogen] during the 30 min of exercise was 206.2 and
174.7 mmol glucosyl U/kg dry wt, respectively, but was not
significantly different.
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Glucose, Glucose 6-Phosphate, Lactate, Pyruvate, Glycerol 3-Phosphate, and [H+]
The muscle glucose content was significantly lower in the LCD condition at 30 min of exercise (Table 3). Glucose 6-phosphate content increased from rest to exercise and was similar in the two conditions at rest and throughout exercise (Table 3).
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The resting muscle lactate content was significantly lower in the LCD compared with the MD (Table 3). During exercise, muscle lactate content increased similarly in both conditions.
Resting muscle pyruvate content was 0.12 ± 0.02 mmol/kg dry wt in
both conditions (Fig. 2) and increased
similarly in each condition.
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Muscle glycerol 3-phosphate content increased during exercise in both conditions (Table 3). There were no differences between the two conditions with respect to glycerol 3-phosphate content at any time point.
The calculated [H+] increased throughout exercise in both conditions (Table 3).
Acetyl group accumulation.
Resting muscle acetyl-CoA content was higher after 6 days on a LCD
compared with the MD (Fig. 3). However,
by 2 min of exercise the acetyl-CoA content was greater in the MD
condition. At 30 min, the muscle acetyl-CoA content increased in the MD
condition, whereas it decreased in the LCD condition (Fig. 3). The
muscle acetyl-CoA content was significantly greater in the MD condition at 30 min of exercise.
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High energy phosphates.
There were no differences in the muscle content of ATP between
conditions (Table 5). There were no
differences in the muscle content of creatine phosphate concentration
([CP]) between conditions (Table 5). In both conditions, [CP] was
significantly greater at 2 and 30 min of exercise compared with rest.
There were no differences with respect to intramuscular Pi
concentration.
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Regulatory Enzymes
Phos. There were no differences either between or within the conditions with respect to the percentage of Phos in the more active a form. In the LCD condition, the percentage of phosphorylase in the a form was 47.6 ± 5.9% at 2 min and decreased to 38.5 ± 4.5% by 30 min of exercise. In the MD condition, the percentage of phosphorylase in the a form did not change between 2 and 30 min of exercise (42.3 ± 6.2 compared with 43.1 ± 4.6%).
PDH activity. PDHa was significantly lower in the LCD condition compared with the MD condition (Fig. 4). There were no differences in PDHa between conditions during the exercise period.
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DISCUSSION |
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The present study examined the chronic (days) and acute (minutes)
regulation of PDH activity at rest and during moderate intensity exercise after dietary manipulation. A 6-day LCD elevated plasma [FFA] and [-OHB] at rest and during exercise. Resting PDHa was significantly lower after the LCD and was accompanied by elevated intramuscular acetyl-CoA and acetylcarnitine contents. During exercise,
PDHa, glycogenolytic rate, and muscle contents of acetylcarnitine, pyruvate, and lactate were similar in both conditions. The results of
the present study suggest that the stable increase in PDHK activity
induced by the LCD may be overridden by the elevation of muscle
pyruvate during exercise, thereby allowing normal activation of PDH.
Substrate Utilization
During exercise, the changes in RER associated with the LCD were similar to those previously observed after a high-fat diet (12) and acute increases in plasma FFA availability by Intralipid infusion (21, 29, 44). A decrease in RER during exercise after the LCD indicates that oxidation of noncarbohydrate sources (i.e., FFA andPhos
Phos exists in two interconvertible forms, an a form, which is active in the absence of AMPf, and a b form, which is active only in the presence of AMPf (4). The activity of Phos is regulated by a two-step control process. The first step involves Phos b to a transformation, whereas the second step involves posttransformational modification by substrate availability (Pi) and allosteric modulation by AMPf (4).In the present study, there was no difference in the percentage of Phos in the a form at rest and during exercise after a 6-day LCD. In addition, there were no differences in the concentration of allosteric regulators AMPf and the substrate Pi at any time, which suggests that there were no differences in the posttransformational activation of Phos after the LCD. This is supported by our data, because there was no difference in the rate of muscle glycogen degradation between conditions.
Regulation of PDH
Resting PDHa. It is well accepted that elevation of plasma [FFA] reduces carbohydrate utilization within skeletal muscle at rest (35). In the early 1960s, Randle and colleagues (36, 37) proposed the glucose-fatty acid cycle as a mechanism to describe the acute regulation of carbohydrate and fat metabolism in rat heart muscle and adipose and liver tissues. Central to the operation of this cycle was the inhibition of PDH by elevations in the mitochondrial acetyl-CoA-to-CoASH and NADH-to-NAD+ ratios, secondary to increased FFA oxidation. The existence of this cycle in resting tissue has been supported by a number of researchers who have reported reduced PDHa after acute elevation of plasma [FFA] in rat tissues (19) and human skeletal muscle (16, 23, 29, 43). Similar results were found by Putman et al. (33) with chronic elevation of plasma FFA after a 3-day LCD. In that study, reduced resting PDHa coincided with an increased intramuscular acetyl-CoA-to-CoASH ratio, acetyl-CoA content, and acetylcarnitine content (33). Our results are in agreement with those of Putman and colleagues. In the present study, PDHa was significantly reduced at rest after the LCD and coincided with the intramuscular acetyl-CoA-to-CoASH ratio, acetyl-CoA content, and acetylcarnitine content, suggesting the existence of the glucose fatty acid cycle in human skeletal muscle under resting conditions.
Before attributing the reduced resting PDHa found in the present study after the LCD to the action of the glucose fatty acid cycle alone, it is important to consider other possibilities related to the chronic regulation of PDHa. Hutson and Randle (22) were the first to examine the chronic regulation of PDH by PDHK; these authors reported a stable increase in PDHK activity after a chronic LCD within rat skeletal muscle. In 1996, Zhou et al. (48) found that 48 h of fasting significantly impaired glucose metabolism within resting rat skeletal muscle; the authors concluded that the attenuated glucose metabolism was caused by decreased PDHa, secondary to a stable increase in PDHK activity. Recent findings from our laboratories indicate that as few as 3-6 days on a LCD (identical to the one used in the present study) results in a 3.5- to 5.0-fold increase in PDHK activity within human skeletal muscle (30). In the present study, resting PDHa was significantly depressed after the LCD. In light of the findings of Peters et al. (30), this observed decrease in PDHa is most likely attributable to a stable increase in PDHK activity. Taken together, the results of these studies provide strong support for the existence of a mechanism involved in the chronic regulation of PDHa by increased PDHK activity. Hence, PDHK appears to be an important regulator of whole body oxidative carbohydrate metabolism during conditions of chronic carbohydrate deprivation. A mechanism explaining increased PDHK activity in response to starvation or fat feeding has yet to be fully elucidated. Elevated plasma [FFA] (27), independent of changes in acetyl-CoA and NADH (22), has been proposed as a mechanism that increases PDHK activity. Saturated and monounsaturated fatty acids alone, but not polyunsaturated fatty acids, have been reported to elicit increased PDHK activity (17). It is important to note that, in the present study, diets were high in animal fat (in the LCD condition), which is composed primarily of saturated and monounsaturated fatty acids. In addition, insulin has been reported to reverse the effects of carbohydrate deprivation and starvation on PDHK activity (28, 41). Recently, four isoenzymes of PDHK were identified in human skeletal muscle (18, 39), but the stable increases in the individual isoenzymes after dietary manipulation in human skeletal muscle are not known.PDHa during exercise. The present study found no difference in the activation of PDHa after chronic carbohydrate deprivation. Ca2+ released from the sarcoplasmic reticulum during exercise activates PDH via the activation of PDHP (20). Because the subjects were exercising at the same power outputs after each diet, the activation of PDHP by Ca2+ would be similar under both conditions. A number of researchers have reported that the activation of PDH during exercise is independent of changes in the acetyl-CoA-to-CoASH (9, 15, 32, 33) and NADH-to-NAD+ (10) ratios. In the present study, the rate of activation of PDHa was similar between conditions despite lower accumulation of intramuscular acetyl-CoA after the LCD. These results again support the contention that the activation of PDHa during exercise is independent of changes in intramuscular acetyl-CoA concentration and the acetyl CoA-to-CoASH ratio.
Pyruvate inhibits PDHK activity (25), thus maintaining PDH in its active state. In 1997, Sugden et al. (42) reported that the inhibitory constant of PDHK for pyruvate was unaffected by a 28-day high-fat diet in a rat cardiomyocyte preparation. This finding suggests that the ability of pyruvate to inhibit PDHK is not attenuated by a stable increase in PDHK activity after a high-fat diet. Hence, in rat cardiac tissue, pyruvate is capable of overriding a stable increase in PDHK activity. Putman et al. (33) reported intramuscular pyruvate concentration to be significantly lower during exercise after the LCD associated with lower PDHa activity. The lower intramuscular pyruvate content after the LCD associated with a lower glycolytic rate was most probably due to the large difference in the preexercise glycogen content between conditions (185 ± 22 vs. 655 ± 79 mmol glucosyl U/kg dry wt for the LCD and high-carbohydrate diet, respectively). This was accomplished by using a glycogen depletion protocol in the LCD condition and a glycogen supercompensation protocol in the high-carbohydrate diet condition (33). This is in contrast to the findings of our study in which there was no difference in pyruvate concentration and PDHa, but in the present protocol, the similar glycolytic rates observed were due to less profound differences in initial muscle glycogen content before exercise. Hence, the results of the present study suggest that, similar to cardiac muscle, the activation of PDHa during exercise is determined by the availability of intramuscular pyruvate. This study used a 6-day LCD to examine the effects of chronically elevated plasma [FFA] on the regulation of PDH at rest and during exercise. The LCD diet was successful in elevating resting plasma FFA and ![]() |
ACKNOWLEDGEMENTS |
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We thank Tina Bragg and George Obminski for technical assistance.
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FOOTNOTES |
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This study was supported by operating grants from the Medical Research and Natural Sciences and Engineering Research Councils of Canada. T. A. St. Amand was supported by a Natural Sciences and Engineering Research Council scholarship. G. J. F. Heigenhauser is a Career Investigator of the Heart and Stroke Foundation of Ontario (no. I-2576).
Address for reprint requests and other correspondence: G. J. F Heigenhauser, Dept. of Medicine, McMaster Univ., 1200 Main St. West, Hamilton, ON, Canada L8N 3Z5 (E-mail: heigeng{at}fhs.mcmaster.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. §1734 solely to indicate this fact.
Received 2 August 1999; accepted in final form 24 February 2000.
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