School of Biomedical Sciences, University Medical School, Queen's Medical Center, Nottingham NG7 2UH, United Kingdom
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ABSTRACT |
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No studies have singularly investigated the relationship between pyruvate availability, pyruvate dehydrogenase complex (PDC) activation, and anaplerosis in skeletal muscle. This is surprising given the functional importance attributed to these processes in normal and disease states. We investigated the effects of changing pyruvate availability with dichloroacetate (DCA), epinephrine, and pyruvate infusions on PDC activation and accumulation of acetyl groups and tricarboxylic acid (TCA) cycle intermediates (TCAI) in human muscle. DCA increased resting PDC activity sixfold (P < 0.05) but decreased the muscle TCAI pool (mmol/kg dry muscle) from 1.174 ± 0.042 to 0.747 ± 0.055 (P < 0.05). This was probably a result of pyruvate being diverted to acetyl-CoA and acetylcarnitine after near-maximal activation of PDC by DCA. Conversely, neither epinephrine nor pyruvate activated PDC. However, both increased the TCAI pool (1.128 ± 0.076 to 1.614 ± 0.188, P < 0.05 and 1.098 ± 0.059 to 1.385 ± 0.114, P < 0.05, respectively) by providing a readily available pool of pyruvate for anaplerosis. These data support the hypothesis that TCAI pool expansion is principally a reflection of increased muscle pyruvate availability and, together with our previous work (J. A. Timmons, S. M. Poucher, D. Constantin-Teodosiu, V. Worrall, I. A. Macdonald, and P. L. Greenhaff. J. Clin. Invest. 97: 879-883, 1996), indicate that TCA cycle expansion may be of little functional significance to TCA cycle flux. It would appear therefore that the primary effect of DCA on oxidative ATP provision is to provide a readily available pool of acetyl groups to the TCA cycle at the onset of exercise rather than increasing TCA cycle flux by expanding the TCAI pool.
dichloroacetate; epinephrine; pyruvate dehydrogenase complex; acetylcarnitine
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INTRODUCTION |
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PYRUVATE FORMATION is required for carbohydrate-derived oxidative energy delivery in contracting skeletal muscle. First, pyruvate formation is essential for the entry of carbohydrate in the form of acetyl groups into the tricarboxylic acid (TCA) cycle via the pyruvate dehydrogenase complex (PDC). Increases in both mitochondrial Ca2+ and/or pyruvate are thought to be responsible for in vivo activation of PDC (11, 26). Furthermore, we have previously demonstrated that the pharmacological activation of PDC by dichloroacetate (DCA) before contraction can improve muscle isometric tension development by ~35% during subsequent fatiguing contraction (35, 36). Second, pyruvate is a substrate for the alanine amino transferase reaction, which has been reported to be quantitatively the most important anaplerotic process [replenishment of TCA cycle intermediates (TCAI)] in human skeletal muscle (18, 30, 31). Some authors have also suggested that the rate of anaplerosis may limit flux through the TCA cycle and thereby muscle function (5, 20, 30). To date, no studies have investigated concurrently the relationship between pyruvate availability, PDC activation, and anaplerosis in human skeletal muscle, which seems surprising given the functional significance attributed to these metabolic processes in normal (30) and disease states (5, 29).
As outlined above, we have previously demonstrated that the activation of PDC by infusion of DCA before muscle contraction markedly improved contractile function. We hypothesized that these findings were attributable to increased flux through the PDC reaction and/or to the accumulation of a readily available pool of acetyl groups before contraction. Minimizing the lag in the activation of oxidative metabolism at the onset of exercise would have counteracted the immediate activation of phosphocreatine (PCr) and glycogen degradation and glycolytic flux at the onset of exercise. Indeed, we were able to demonstrate glycogen and PCr sparing and lower muscle lactate accumulation, which presumably resulted in less inhibition of the contractile machinery. We also hypothesized that DCA infusion before contraction may have resulted in an expansion of TCAI, which, as stated earlier, has been suggested to be rate limiting to TCA cycle flux and to the rate of oxidative phosphorylation (5, 20, 30). The net effect of TCAI expansion at rest would presumably be to favor the activities of TCA cycle enzymes at the onset of exercise. This itself would facilitate TCA cycle flux and thereby reduce ADP-mediated activation of anaerobic metabolism, which we were able to demonstrate.
The aim of the present study therefore was to investigate simultaneously the importance of muscle pyruvate availability to PDC activation, acetyl group accumulation, and TCAI accumulation in human skeletal muscle with pharmacological approaches to alter muscle pyruvate availability, namely, intravenous infusion of DCA, epinephrine, and pyruvate.
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METHODS |
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Subjects. Six male subjects [age 27 ± 3 (means ± SE) yr; mass 80 ± 4 kg; height 178 ± 2 cm] volunteered to participate in this study, which was granted approval by the Ethics Committee of the University of Nottingham. The experimental procedures were explained in writing to all potential subjects, and written consent was obtained from each before beginning the study. Any subject wishing to withdraw from the study at any moment was free to do so. Before the study commenced, all subjects were screened by means of a standard health questionnaire and physical examination.
After screening, subjects attended three experimental sessions after an
overnight fast, with each session separated by at least 1 wk (total
duration 3-4 wk). In each session, after a period of rest (15 min), two intravenous cannulas were inserted under local anesthesia.
One cannula was inserted retrogradely into a vein on the dorsum of the
left hand, after which the hand was placed into a heated box (air
temperature 55°C) to arterialize the venous drainage of the hand
(17). This enabled measurements of blood glucose, lactate, pyruvate,
epinephrine, and norepinephrine concentrations to be made on
arterialized-venous blood. The second cannula was placed in an
antecubital vein and was used for the infusion of either DCA (25 mg/ml,
Na salt, sterilized by filtration, pH 7.0), epinephrine (13 µg/ml),
or pyruvate (150 mg/ml, Na salt, sterilized by filtration). Each
infusion lasted for 30 min, and the order of infusion was
random. The infusion rates for DCA, epinephrine, and pyruvate
were 1.67 mg · kg body
mass1 · min
1,
0.07 µg · kg body
mass
1 · min
1,
and 1.67 mg · kg body
mass
1 · min
1,
respectively. Heart rate was measured before and throughout the 30 min
of each infusion.
On each of the three experimental visits, a muscle biopsy sample was obtained from the vastus lateralis muscle immediately before and after infusion (1 biopsy from each leg) with the needle biopsy technique (4). After the second muscle biopsy, subjects were given time to recover and were allowed to leave the laboratory. Subjects were asked to maintain their normal dietary intake and activity patterns throughout the experiment.
Blood and muscle collection and
analysis. Arterialized-venous blood samples (5 ml) were
obtained immediately before and at 10-min intervals throughout
infusion. Blood samples were drawn, and an aliquot was mixed
immediately in a tube containing fluoride oxalate. This sample was used
to determine whole blood glucose with a Hemocue B-glucose analyzer
(Angelholm, Sweden). The remainder of the arterial and venous blood
samples was centrifuged, and the supernatant was collected and stored
at 80°C. Plasma samples were subsequently analyzed for
lactate and pyruvate (3) with a fluorometer (Hitachi F-2000) and for
epinephrine and norepinephrine with HPLC (16).
On removal from the muscle, each biopsy sample was immediately frozen
by plunging the needle into liquid nitrogen and was divided into parts
while under liquid nitrogen. One part was freeze-dried, dissected free
from visible connective tissue and blood, and powdered. Five to ten
milligrams of muscle powder were then extracted with 0.5 M perchloric
acid containing 1 mM EDTA, and after centrifugation, the supernatant
was neutralized with 2.2 M KHCO3.
Free carnitine and acetylcarnitine were measured in the neutralized
extract by enzymatic assays that made use of radioisotopic substrate as
previously described (8). Muscle lactate, pyruvate, citrate,
-oxoglutarate, succinate, malate, and fumarate concentrations were
assayed enzymatically (3) with a fluorometer (Hitachi F-2000).
The remainder of the frozen muscle was used to determine PDC activity, as previously described (13). Briefly, activity of PDC in its dephosphorylated active form (PDCa) was assayed in a buffer containing NaF and DCA and was expressed as a rate of acetyl group formation.
Statistics. The data were analyzed by two-way analysis of variance (ANOVA) for repeated measurements (time × treatment). When the ANOVA resulted in a significant F ratio (P < 0.05), Fisher's post hoc test was used to locate differences between means. Values in the text and Figs. 1 and 2 are means ± SE.
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RESULTS |
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Cardiovascular data. Heart rate was
similar at all time points when DCA and pyruvate infusions were
compared and was unchanged from the preinfusion value within each
treatment (Fig. 1). Epinephrine, however,
significantly increased heart rate compared with the preinfusion value
and the corresponding pyruvate and DCA infusions. These differences
were maintained throughout the infusion period.
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Blood and plasma hormones and
metabolites. The arterialized-venous blood and plasma
concentrations of glucose, lactate, pyruvate, epinephrine, and
norepinephrine are presented in Table
1. Blood glucose concentration
did not change with DCA and pyruvate infusion. However, epinephrine
infusion had raised blood glucose concentration by more than 1 mmol/l
by the end of the infusion period (P < 0.05).
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DCA significantly decreased plasma lactate concentration, whereas epinephrine had the opposite effect. Pyruvate did not affect plasma lactate concentration.
Neither DCA nor epinephrine infusion affected blood pyruvate concentration. However, as expected, pyruvate infusion increased blood pyruvate concentration by fivefold from the basal concentration.
Infusion with DCA and pyruvate did not affect plasma epinephrine and norepinephrine concentrations when compared with the preinfusion values. Administration of epinephrine achieved an increase in plasma epinephrine concentration within 10 min of the onset of infusion, and there was a drift toward higher concentrations for the remainder of the infusion period. Plasma norepinephrine concentration was not affected by epinephrine infusion.
Muscle PDCa and
metabolites. Infusion with DCA increased
PDCa sixfold, i.e., from 0.37 to
2.20 mmol
acetyl-CoA · min1 · kg
wet muscle
1 (Fig.
2). There was also a tendency toward an
increase in PDC activity after epinephrine infusion, but significance
was not reached. Pyruvate infusion had no effect on PDC
activity.
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Muscle pyruvate concentration was unchanged irrespective of the
solution infused (Table 2). DCA and
pyruvate infusion had no effect on muscle lactate concentration.
However, muscle lactate concentration almost doubled after epinephrine
infusion (P < 0.05). Pyruvate
infusion had no effect on muscle acetylcarnitine. However, muscle
acetylcarnitine was increased after the DCA infusion, and there was a
tendency for it to increase after epinephrine infusion (P = 0.15). In all three conditions,
the change in muscle acetylcarnitine concentration was mirrored by a
change in muscle free carnitine concentration such that the sum of
muscle free and acetylated carnitine remained constant at all times.
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The individual and pooled muscle concentrations of the five TCAI that
were measured (citrate, -oxoglutarate, succinate, malate, and
fumarate) are shown in Table 3. The
concentration of pooled intermediates was significantly decreased after
DCA infusion (P < 0.05). Conversely,
the concentration of pooled intermediates was increased after
epinephrine (P < 0.05) and pyruvate
infusion (P < 0.05). In the case of
DCA, the 35% decline in the TCAI pool comprised changes in citrate
(
37%), malate (
65%), and succinate (
23%). In
the case of epinephrine, the 43% increase in the TCAI pool also
comprised changes in citrate (+37%), malate (+108%), and succinate
(+29%). Similarly, the 25% increase in the TCAI pool after pyruvate
infusion resulted from increases in citrate (+11%), malate (+76%),
and succinate (+13%).
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DISCUSSION |
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The major findings of the present study were that infusion with epinephrine and pyruvate increased the muscle TCAI pool, probably by providing a readily available pool of pyruvate for anaplerosis. However, pyruvate and epinephrine did not activate PDC under the present experimental conditions. Conversely, DCA infusion decreased the muscle TCAI pool, probably by diverting pyruvate to acetyl-CoA and acetylcarnitine after the near-maximal activation of PDC by DCA. The present study therefore demonstrates that the muscle TCAI pool size principally reflects changes in muscle pyruvate availability and, together with our previous work (36), indicates that TCA cycle expansion may be of little functional significance to TCA cycle flux. On the basis of our previous (35, 36) and present findings, we believe that factors other than the expansion of the muscle TCAI pool will limit optimal oxidative energy provision and muscle function at the onset of intense contraction. We believe that the most likely candidate will be muscle acetyl group availability.
TCAI metabolism. The muscle TCAI pool has been shown to expand during contraction (1, 18, 29, 30), and this expansion has been suggested to be important to the attainment of an optimal oxidative energy production at the start of muscle contraction (20). Although anaplerosis is dependent on reactions catalyzed by alanine aminotransferase, pyruvate carboxylase, malate dehydrogenase (MDH), and phosphoenolpyruvate carboxykinase, the former has been suggested to be the principal mechanism for TCAI expansion (18, 30, 31). Clearly, pyruvate availability is essential for the alanine aminotransferase reaction to proceed and may be the reason that during incremental exercise, expansion of the TCAI pool only occurs at workloads greater than 150 W, i.e., the workload at which muscle pyruvate and lactate may start to accumulate (29). Similarly, perfusion of rat skeletal muscle with 1 mM pyruvate and 15 mM lactate has been shown to result in a fourfold increase in the content of the TCAI pool (20). These results are consistent with the idea that expansion of the muscle TCAI pool is principally a reflection of muscle pyruvate availability and may be of little functional importance to TCA cycle flux and contractile function. Conversely, it has been shown that fatigue development during prolonged exhaustive exercise was paralleled by a decline in the muscle TCAI pool, which the authors suggested may have been performance limiting (30). Equally, however, the observed decline in the TCAI pool could have been interpreted to be the direct consequence of a decrease in pyruvate availability as the muscle glycogen store became depleted. Clearly, a more appropriate way to assess the physiological role of muscle TCAI pool expansion would be to manipulate the pool size by pharmacological intervention and subsequently assess the effect of this on muscle metabolism and function.
The findings of the present study demonstrate that DCA infusion reduced the muscle TCAI pool. Similarly, a reduction in muscle TCAI after DCA infusion has been observed in vascular smooth muscle (2). The probable mechanism behind this response is the diversion of pyruvate to acetyl-CoA and acetylcarnitine after the near-maximal activation of PDC by DCA (Fig. 1). Unfortunately, we did not assess muscle function after DCA infusion in the present study. However, we have previously demonstrated that DCA infusion before contraction can markedly reduce the extent of PCR degradation and lactate accumulation and improve muscle function by ~35% in a canine hindlimb perfusion model (35, 36). Furthermore, we have also recently shown similar results, i.e., reductions in the rates of PCr degradation and lactate accumulation in humans after administration of DCA (34). Taken together, these findings suggest that there is no association between the expansion of muscle TCAI pool, oxidative energy delivery, and function during muscle contraction, i.e., that muscle oxidative energy delivery can be increased and function improved in the face of a reduced TCAI pool. Therefore, we would like to hypothesize that anaplerosis is simply a mechanism for removal of excess pyruvate and that the muscle acetyl group availability, which is dramatically increased after DCA administration, is more important to the attainment of optimal oxidative energy provision and subsequent contractile performance.
Interestingly, the changes in the TCAI pool size of the present study
were mainly mediated by changes in the muscle malate pool. Malate also
undergoes the largest increase of any measured TCAI during exercise (1,
18, 29, 30). However, there appears to be no obvious explanation for
this disproportionate malate accumulation in comparison with the rest
of the TCAI. There are two possible reactions by which malate could be
formed. One is that catalyzed by the NADP-linked malate dehydrogenase
decarboxylating enzyme (D-MDH;
pyruvate + CO2 + H+ + NADPH malate + NADP+) and the other by malate
dehydrogenase (L-MDH;
oxaloacetate + NADH + H+
malate + NAD+). The relative
contribution made by the former reaction is, however, likely to be
negligible given that it has only been shown to generate malate at
supraphysiological pyruvate concentrations (50 mM; Ref. 21). In the
case of L-MDH, the
Michaelis-Menten constant of the enzyme
(Km) for
oxaloacetate has been reported to be 10 times lower than that for
malate (3), and therefore the reaction greatly favors malate formation.
Oxaloacetate is also used by citrate synthase (CS); the
Km for
oxaloacetate of CS (16 µM) is relatively close to the
Km for
oxaloacetate of MDH (33 µM). However, the
Km of CS for its
second substrate, acetyl-CoA, can vary from 2 to 500 µM (32). It is
plausible, therefore, because of this relatively high
Km of CS for
acetyl-CoA, that when muscle oxaloacetate increases, a large fraction
is used for malate formation rather than condensed to citrate, and this
results in a disproportionate expansion of muscle malate when compared
with the rest of the TCAI pool.
Activation of PDC and acetyl group accumulation. PDC catalyzes the oxidative decarboxylation of pyruvate, forming acetyl groups, which can subsequently be channeled into the TCA cycle or can accumulate as acetyl-CoA and acetylcarnitine. PDC exists in a dephosphorylated active form (PDCa) and in a phosphorylated inactive form (23). The ratio of the active to inactive forms is regulated by the activities of the PDC phosphatase and the PDC kinase. Several positive physiological modulators of PDC transformation to PDCa have been demonstrated in vitro, e.g., pyruvate, ADP (22), calcium (15), NAD+, and CoASH (14, 25). However, muscle contraction and insulin have been shown to be the principal activators of PDC transformation in vivo (11, 24). The former is probably acting by increasing intramitochondrial Ca2+ availability (11, 26).
In the present study, DCA infusion increased
PDCa sixfold. The mechanism
responsible for DCA activation of PDC is thought to be via inhibition
of PDC kinase (37). Alternatively, however, DCA could also activate PDC
indirectly by activating PDC phosphatase, e.g., by facilitating calcium
entry into mitochondria (7). Whatever the mechanism, the net effect of
DCA is to divert more pyruvate to be oxidized to acetyl-CoA and thereby
reduce lactate formation. In the present study, we did not measure
plasma DCA concentration after DCA infusion. However, in a previous
study, administration of DCA to a group of patients at a rate similar to that used in the present study achieved a plasma concentration of
150 µg/l within 30 min (6). Plasma DCA concentrations of 100-200 µg/l have been suggested to be sufficient to stimulate transformation of PDC to PDCa in
most tissues (33). Indeed, the
PDCa measurements obtained after
DCA infusion in the present study (on average 2.20 mmol
acetyl-CoA · min
1 · kg
wet muscle
1) were in
close agreement with our previous work in which complete PDC activation
was achieved by muscle contraction (on average 2.11 mmol
acetyl-CoA · min
1 · kg
wet muscle
1; Ref. 12). It
would appear therefore that the present rate of DCA infusion (1.67 mg · kg body
mass
1 · min
1)
was sufficient to completely transform PDC to
PDCa in human skeletal muscle.
Clearly, DCA and other potential PDC activators have enormous
therapeutic potential to patients with compromised tissue blood flow
(27). However, given that chronic administration of DCA is known to
have a number of adverse side effects (10) and that we do not know the
effects of long-term administration to humans, particularly at high
doses (6, 33), the present findings indicate for the first time that
complete PDC activation can be achieved at a rate of 1.67 mg · kg body
mass
1 · min
1.
Therefore, future human studies might wish to utilize this infusion rate.
Epinephrine induces the transformation of phosphorylase
b (inactive) to phosphorylase
a, its active form, in
skeletal muscle, by a sequence of reactions, starting with
the binding of the hormone to -receptors located on the cell
membrane. This is followed by activation of adenyl cyclase and an
increase in the intracellular concentration of cAMP. The net effect of
this is an increase in glycogenolysis and thereby increased muscle
lactate concentrations. Accordingly, epinephrine infusion increased the
concentrations of plasma and muscle lactate (Tables 1 and 2) in the
present study. Pyruvate, at a concentration of 0.5 mM, has been shown to inhibit PDC kinase in cardiac muscle, kidney, and liver by ~50%
and thereby to activate PDC (22). However, the increase in muscle
PDCa after the epinephrine
infusion in the present study was negligible (Fig. 2). A possible
explanation for this finding is that, despite almost complete
transformation of phosphorylase b to
phosphorylase a during the initial
minutes of epinephrine infusion, the extent of phosphorylase
transformation, and hence the rate of muscle glycogenolysis, may have
declined markedly as infusion progressed beyond 2 min (9).
This effect has been attributed to a decline in the intracellular
Pi availability at the active site
of phosphorylase a with the
progression of time. Given that our second muscle biopsy was obtained
after 30 min of infusion, it cannot be ruled out that peak
phosphorylase a activity and hence
peak pyruvate formation occurred much earlier and therefore its action
went unnoticed. Furthermore, epinephrine infusion has been shown to
stimulate glycogenolysis in resting rat skeletal muscle containing a
high proportion of fast-twitch fibers but to have no effect on
slow-twitch muscles (28). As human muscle contains a mixture of ~50%
slow- and 50% fast-twitch fibers, any effect of epinephrine on one
fiber type may have been "diluted" by a lack of effect on the
other. Clearly, further studies at the level of individual human
skeletal muscle fibers are needed to clarify the effect of epinephrine
on PDC activation and acetyl group accumulation.
In the present study, pyruvate infusion had no effect on PDC. The quantity of pyruvate administered to each subject was ~36 mmol. However, the amount of pyruvate found in circulation after 30 min of infusion corresponded to ~4 mmol. This suggests therefore that 32 mmol of pyruvate (or ~0.67 mmol/l intracellular water) entered the tissue space, which should have favored the transformation of PDC to PDCa. However, the intracellular pyruvate concentration measured after pyruvate infusion did not reach anywhere near this concentration (Table 2). Indeed, even during conditions of high glycogenolytic flux, i.e., muscle lactate accumulation to ~30 mmol/l intracellular water, one would not expect to find muscle pyruvate to accumulate by more than 0.1-0.2 mmol/l intracellular water (19), which may not be enough to inhibit the PDC kinase and thereby increase the amount of PDCa. Given that the accumulation of muscle lactate (7.2 mmol) and TCAI (4.8 mmol) could only account for ~40% of the remaining 32 mmol of pyruvate unaccounted for, it would appear that more than one-half of the amount of pyruvate administered was sequestered by the liver, which would have been better perfused during the infusion period compared with the muscle. Finally, DCA, which has a structure very similar to that of pyruvate, clearly activated PDC, but not necessarily via a common mechanism. This perhaps occurred because DCA is unlikely to be metabolized by muscle and therefore can accumulate (33).
In conclusion, epinephrine and pyruvate increased the muscle TCAI pool, probably by providing a readily available pool of pyruvate for anaplerosis. Conversely, DCA infusion decreased the muscle TCAI pool, more than likely by diverting pyruvate to acetyl-CoA and acetylcarnitine after the near-maximal activation of PDC by DCA. These findings suggest that the expansion of the muscle TCAI pool seen during muscle contraction is principally a reflection of muscle pyruvate availability and, together with our previous work (36), indicate that TCA cycle expansion may be of little functional significance to TCA cycle flux and fatigue development. We hypothesize that the availability of muscle acetyl groups before contraction is more important to the attainment of optimal oxidative energy provision and contractile performance.
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ACKNOWLEDGEMENTS |
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We thank Drs. N. Pierce and J. Lambourne for valuable assistance in obtaining the muscle biopsy samples.
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FOOTNOTES |
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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: D. Constantin-Teodosiu, School of Biomedical Sciences, Univ. Medical School, Queen's Medical Center, Nottingham NG7 2UH, UK.
Received 17 August 1998; accepted in final form 12 November 1998.
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