A Couillard
email: annabelle_couillard@yahoo.fr

Evidence of local exercise-induced systemic oxidative stress in chronic obstructive pulmonary disease patients

Keywords:

Chronic inactivity may not be the sole factor involved in the myopathy of chronic obstructive pulmonary disease (COPD) patients. One hypothesis is that exercise-induced oxidative stress that leads to muscle alterations may also be involved. This study investigated whether exercise localised to a peripheral muscle group would induce oxidative stress in COPD patients.

Eleven COPD patients (FEV1 1.15¡0.4 L (mean¡SD)) and 12 healthy age-matched subjects with a similar low quantity of physical activity performed endurance exercise localised to a peripheral muscle group, the quadriceps of the dominant leg. The authors measured plasma levels of thiobarbituric reactive substances (TBARs) as an index of oxidative stress, the release in superoxide anion (O 2 ? -) by stimulated phagocytes as an oxidant, and blood vitamin E as one antioxidant.

Quadriceps endurance was significantly lower in the COPD patients compared with healthy subjects (136¡16 s versus 385¡69 s (mean¡SEM), respectively). A significant increase in TBARs 6 h after quadriceps exercise was only found in the COPD patients. In addition, significantly higher O 2 ? -release and lower blood vitamin E levels were found in COPD patients than in controls at rest. This blood vitamin E level was significantly correlated with the resting level of plasma TBARs in the COPD patients.

This study mainly showed that quadriceps exercise induced systemic oxidative stress in chronic obstructive pulmonary disease patients and that vitamin E levels were decreased in these patients at rest. The exact relevance of these findings to chronic obstructive pulmonary disease myopathy needs to be elucidated.

The contribution of impaired peripheral muscle function to the exercise intolerance of patients with chronic obstructive pulmonary disease (COPD) has essentially been confirmed [START_REF] Maltais | Oxidative capacity of the skeletal muscle and lactic acid kinetics during exercise in normal subjects and in patients with COPD[END_REF][START_REF] Jobin | Chronic obstructive pulmonary disease: capillarity and fibre type characteristics of skeletal muscle[END_REF][START_REF] Jakobson | Skeletal muscle metabolites and fibre types in patients with advanced chronic obstructive pulmonary disease (COPD), with and without chronic respiratory failure[END_REF][START_REF] Gosselink | Peripheral muscle weakness contributes to exercise limitation in COPD[END_REF][START_REF] Serres | Impaired skeletal muscle endurance related to physical inactivity and altered lung function in COPD patients[END_REF]. An important issue today is whether this myopathy is entirely due to chronic inactivity [START_REF] Young | Rehabilitation of patients with pulmonary disease[END_REF][START_REF] Prefaut | Pathophysiological basis of exercise training in patients with chronic obstructive pulmonary disease[END_REF] or if there is an intrinsic muscle disorder, since recent studies have suggested that other factors do contribute to this myopathy [START_REF] Maltais | Altered expression of myosin heavy chain in the vastus lateralis muscle in patients with COPD[END_REF][START_REF] Engelen | Altered glutamate metabolism is associated with reduced muscle glutathione levels in patients with emphysema[END_REF][START_REF] Engelen | Exercise-induced lactates increase in relation to muscle substrates in patients with chronic obstructive pulmonary disease[END_REF]. Because exercise-induced oxidative stress can induce substantial muscle alterations [START_REF] Barcklay | Free radicals may contribute to oxidative skeletal muscle fatigue[END_REF][START_REF] Davies | Free radicals and tissue damage produced by exercise[END_REF], it may be one of the mechanisms involved in the myopathy of patients with COPD.

The few studies on this topic have shown that strenuous incremental cycle exercise, and even light constant cycle exercise, results in oxidative stress in COPD patients [START_REF] Vina | Exercise causes blood glutathione oxidation in chronic obstructive pulmonary disease: prevention by O 2 therapy[END_REF][START_REF] Heunks | Xanthine oxidase is involved in exercise-induced oxidative stress in chronic obstructive pulmonary disease[END_REF]. However, these studies were without control groups so it was not possible to determine whether COPD patients are more susceptible to exercise-induced oxidative stress than healthy sedentary subjects. Moreover, since general exercise induces a considerable increase in such parameters as ventilation, cardiac output and so on, it was not possible to distinguish the source of oxidative stress in COPD: lung, peripheral skeletal muscle, both and/or others. To distinguish peripheral muscles as a potential source of oxidative stress, local exercise must be performed. During local exercise, work is performed almost exclusively by one muscle group, which minimises cardiac and respiratory responses, and muscle origin is more easily isolated [START_REF] Andersen | Dynamic knee extension as model for study of isolated exercising muscle in humans[END_REF].

Exercise-induced oxidative stress results from an imbalance between free radical generation, which increases during exercise, and the efficiency of antioxidant mechanisms. A deficiency in total plasma antioxidant activity has been described in COPD patients at rest, but the specific antioxidants were not determined [START_REF] Rahman | Systemic oxidative stress in asthma, COPD, and smokers[END_REF]. In particular, the lack of data on the vitamin E status of COPD patients is striking because different studies have demonstrated that muscle from vitamin E-deficient rats is more prone to exercise-induced oxidative stress and damage than muscle from normal rats [START_REF] Jackson | Isolated vitamin E deficiency[END_REF][START_REF] Quintanilha | Membrane effects of vitamin E deficiency: bioenergetic and surface charge density studies of skeletal muscle and liver mitochondria[END_REF][START_REF] Pillai | Concomitant brainstem axonal dystrophy and necrotizing myopathy in vitamin E deficient rats[END_REF].

The present authors hypothesis is that local exerciseinduced oxidative stress is present in COPD patients and may contribute to the myopathy of these patients. As a first step, this study was designed to investigate whether an exercise localised to the quadriceps would be able to generate oxidative stress. In addition, the authors investigated in vitro superoxide anion release as a marker of oxidant production and blood vitamin E as an antioxidant.

Methods

Study population

Subjects. This study included 23 male subjects. Eleven were COPD patients, all exsmokers, selected on the basis of moderate-to-severe COPD according to American Thoracic Society guidelines [START_REF] Thoracic | Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma[END_REF]. All patients had irreversible obstructive airway disease (forced expiratory volume in one second (FEV1)/forced vital capacity (FVC) v60% of predicted values and v10% improvement of FEV1 after inhalation of a bronchodilating agonist), no hypoxaemia and they were clinically stable. They had not experienced respiratory tract infection or exacerbation of their disease for o4 weeks prior to the study. None were on oral corticosteroid therapy, although a few inhaled steroids. All patients received anticholinergic or b 2 -agonists as bronchodilator therapy. To avoid potentially confounding effects, subjects with known muscle disorders, cardiac failure, diabetes mellitus, and alcoholism were excluded.

Twelve healthy nonsmokers, age-matched, with only sedentary activities at the time of the study, volunteered to serve as control subjects. These healthy subjects were recruited from among friends of members of the laboratory. All participants were questioned on their dietary habits to ensure that none were taking antioxidants or vitamin supplements. In addition, all had a body mass index v35. The study had the approval of the local ethics committee. Written consent was obtained from all participants after they had received a complete explanation of the purposes and protocol of the study.

Definition of recruitment criteria. Lung function. All patients and healthy volunteers underwent spirometry using a whole body plethysmograph (Transmural Bodybox 2800; Sensormedics, Yorba Linda, CA, USA). Measurements included FVC and FEV1. Tiffeneau9s ratio (FEV1/FVC) was then calculated. The values obtained were compared to the theoretical values of QUANJER et al. [START_REF] Quanjer | Lung volumes and forced ventilatory flows[END_REF].

Level of physical activity. Physical activity (PA) was assessed with a PA questionnaire adapted for older retired adults [START_REF] Voorips | A physical activity questionnaire for the elderly[END_REF]. The questionnaire scored the past year9s household activities, sports activities, and other physically active leisure-time activities and gave an overall PA score. The subjects were asked to describe the type of activity, hours per week spent on it, and period of the year in which the activity was normally performed. All activities were classified according to posture and movement. An intensity code based on net energetic costs of activities according to BINK et al. [START_REF] Bink | Assessment of the energy expenditure by indirect time and motion study[END_REF] was used to classify each activity. This questionnaire provides a reliable and valid method for classifying the activity level of older subjects as high, medium, or low. With this method, subjects who obtained a score of 9 were classified as having low physical activity, and thus as "sedentary".

Local muscle exercise

Maximum voluntary contraction (MVC) of the quadriceps for each leg was assessed using an exercise bench (Banc de Koch; Genin Medical, Les Angles, France). Using the same bench, the endurance of the quadriceps of the dominant leg, which was used for local muscle exercise, was then assessed. MVC and quadriceps endurance were assessed according to the technique of SCHERRER et al. [START_REF] Scherrer | Le travail musculaire local et la fatigue chez l9homme[END_REF] modified by SERRES et al. [START_REF] Serres | Impaired skeletal muscle endurance related to physical inactivity and altered lung function in COPD patients[END_REF].

Quadriceps femoris endurance exercise was performed by execution of maximal knee extensions against weights corresponding to 40% of MVC with a pace of 12 movements?min -1 imposed with an audio signal (metronome) until exhaustion. The dynamic knee extension was performed quickly, immediately followed by passive flexion and then by rest before the next extension. The subjects were instructed to release muscles just after maximal extension, without maintaining static contraction or resisting when weights were set back. The active part of the movement was thereby considered to be the load lift. The duration of rest was always greater than the duration of movement. The test was stopped when the subjects could no longer respect the required maximal extension or frequency two consecutive times despite verbal encouragement. Since a subject9s motivation may determine attainment of maximal effort, the same investigator supervised the endurance test and gave the same encouragement to all subjects.

The duration, called the "limit time" (Tlim, expressed in s), was then recorded. The dyspnoea score was measured at rest and immediately after exercise on a visual analogic scale.

Protocol

The subjects were instructed to abstain from strenuous physical activity 3 days before and on the day of local muscle exercise. After an overnight fast, and without breakfasting, the subjects arrived at the laboratory between 8:00-9:00 h and underwent spirometric testing. They then responded to the physical activity questionnaire and resting blood samples (Trest) were taken. Next, strength was evaluated and the subjects were familiarised with the endurance test procedures by performing five consecutive dynamic knee extensions. They then performed the local muscle exercise. The second blood samples were taken immediately at the end of the local exercise (Tend) and the third, 6 h later (T6h).

Assessment of oxidative stress parameters

Blood samples were drawn in heparinised tubes. Reactive oxygen species production was immediately determined on a 100 mL aliquot of blood. Plasma was obtained by centrifugation (12006g for 10 min at 4uC) and stored at -80uC until analysis.

Products of lipid peroxidation as markers of oxidative stress. Levels of lipid peroxidation were determined by assessment of thiobarbituric reactive substances (TBARs) using a fluorimetric method previously described by YAGI [START_REF] Yagi | A simple fluorometric assay for lipoperoxide in blood plasma[END_REF]. The reproducibility calculated as the coefficient of variation was 4.64%.

Determination of superoxide anion release by stimulated phagocytes. Superoxide anion (O 2 ? -) release by stimulated phagocytes was measured at Trest and Tend according to a method derived from VACHIER et al. [START_REF] Vachier | Enhancement of reactive oxygen species formation in stable and unstable asthmatic patients[END_REF]. The specific probe used was lucigenin (at a final concentration of 1.5610 -4 M) and the luminescence was recorded at 37uC by means of a 125I LKB Wallac Luminometer (Wallac Co., Turku, Finland). The reproducibility calculated as the coefficient of variation was 6.50%.

Determination of plasma vitamin E. Blood vitamin E was measured by high-performance liquid chromatography according to the method previously described by CACHIA et al. [START_REF] Cachia | Red blood cell vitamin E concentrations in fetuses are related to but lower than those in mothers during gestation: a possible association with maternal lipoprotein plasma level[END_REF]. The sum of triglycerides and cholesterol was assessed using a routine enzymatic method (Boehringer Mannheim, Meyland, France). The reproducibility calculated as the coefficient of variation was of 5.6%.

Statistical analysis

Values are reported as mean¡SEM. Unpaired t-tests were performed to compare COPD patients with controls. If the equal variance test failed, a Mann-Whitney U-test was used. Correlations between continuous variables were made using simple linear regression. Two-way analysis of variance followed by Tukey9s pairwise multiple comparison procedure was used to determine differences between COPD patients and controls in O 2 ? -production, blood vitamin E, and TBARs at rest and immediately/6 h after exercise. Significance was set at the 0.05 level.

Results

Anthropometric data

There were no significant differences between the COPD and control groups for anthropometric data (table 1). Differences between the two groups could be observed for spirometric function, with the COPD group showing moderate-to-severe airflow obstruction with an FEV1 of 41¡3.4% (mean¡SEM) pred. As expected, both groups were sedentary since they had a similar low level of PA (table 1).

Muscle performance

Analysis of peripheral muscle performance showed no significant difference in MVC between the COPD and control groups (28¡2.8 versus 29.75¡1.7 kg, respectively). In contrast, muscle endurance (Tlim) was significantly lower in the COPD patients compared with the healthy subjects, with the COPD Tlim being nearly three-fold lower than control Tlim (136¡16 versus 385¡69 s, respectively; pv0.01). At the end of exercise, dyspnoea was assessed in five subjects in each group. Between rest and immediate end of exercise, the average dyspnoea scores changed from 0.45¡0.2 to 2.08¡0.6 and from 0.1¡0.07 to 2.8¡0.7, respectively, in COPD patients and healthy subjects.

Rest oxidative stress variables

Rest levels of plasma TBARs were higher at rest in the COPD patients (fig. 1). This difference between groups was notable but did not reach statistical significance. O 2 ? -by stimulated phagocytes was significantly higher in the COPD patients at rest (pv0.05) (table 2). There was a positive and significant correlation in COPD patients between O 2 ? -release and TBARs (r=0.73, pv0.01). Absolute values of blood vitamin E at rest were significantly lower in the patients (pv0.05) (table 2). Both groups had similar levels of triglycerides (TG) and cholesterol. There was -Levels of plasma products of lipid peroxidation at rest, at the end of exercise (Tend) and 6 h after the end of exercise (T6h). h: chronic obstructive pulmonary disease (COPD) patients; p: controls. Local exercise induced a significant increase in plasma levels of lipid peroxidation products 6 h after the end of exercise in COPD patients (pv0.05). Plasma level of thiobarbituric reactive substances (TBARs) was unchanged in the healthy subjects after local exercise. only a positive and significant relationship between the sum of TGzcholesterol and vitamin E in the control group (r=0.66). There was a negative and significant correlation at rest between TBARs and blood vitamin E level in the COPD patients (r=-0.69; pv0.05) (fig. 2). No correlation was found between blood vitamin E and TBARs at rest in the healthy subjects.

Local muscle exercise

As shown in figure 1, quadriceps exercise induced a significant increase in plasma products of lipid peroxidation in the COPD patients only 6 h after exercise (pv0.05). Exercise had no significant effects on blood vitamin E level or O 2 ? -release in either group. No significant correlation was found between resting blood level of vitamin E and TBARs 6 h after exercise in either patients with COPD (r=-0.50, p=0.11) or healthy subjects (r=0.23).

Discussion

The major findings of this study were: 1) a significant increase in blood markers of oxidative stress in COPD patients performing an exercise localised to a peripheral muscle group, the quadriceps of the dominant leg; and 2) a significantly lower blood vitamin E level at rest in these patients.

Methodology

Physical activity. A specific physical activity questionnaire adapted for older retired adults with independent lifestyles was chosen. Although a questionnaire is not a pure objective tool, the chosen method provided a reliable and sufficient method to demonstrate that both groups had the same low level of physical activity.

Quadriceps endurance. To study muscle endurance, local exercise consisting of dynamic knee extensions at a regular pace was chosen, similar to the model described by ANDERSEN et al. [START_REF] Andersen | Dynamic knee extension as model for study of isolated exercising muscle in humans[END_REF] for study of isolated exercising muscle in humans. Using this model, electromyographic recordings taken from different muscles in addition to exercising quadriceps indicated that only the quadriceps was active during the local exercise. Furthermore, this local exercise minimised ventilatory response [START_REF] Andersen | Dynamic knee extension as model for study of isolated exercising muscle in humans[END_REF]. In the present study, the increase in dyspnoea as an index of ventilation after local exercise was of small amplitude. Thus, the advantage of this model is that all the measurements concerning oxidative stress markers are closely related to changes in the working muscle group. In an experiment consisting of repetitive tests with the same technique, Tlim was found to be highly reproducible [START_REF] Serres | Improved skeletal muscle performance after individualised exercise training in patients with chronic obstructive pulmonary disease[END_REF].

Markers of oxidative stress. A global and clinical method was used to assess the parameters of oxidative stress: plasma lipid peroxidation by measurement of TBARs. This is a nonspecific oxidative stress index that should be used with caution because under oxidative stress conditions, malondialaldehyde, hydroperoxides and certain carbohydrates and amino acids may yield products that could react with thiobarbituric acid [START_REF] Nielsen | Plasma malondialdehyde as biomarker for oxidative stress: reference interval and effects of life-style factors[END_REF][START_REF] Lefevre | Evaluation de la peroxydation lipidique par le dosage des substances re ´agissant avec l9acide thiobarbiturique[END_REF][START_REF] Halliwell | Lipid peroxidation: its mechanism measurement and significance[END_REF]. Determination of TBARs in biological samples thus reflects at least effective peroxidation or an index of peroxidisability. For this reason, TBARs should be used on a group rather than individual basis to assess oxidative stress [START_REF] Nielsen | Plasma malondialdehyde as biomarker for oxidative stress: reference interval and effects of life-style factors[END_REF]. Finally, the fluorimetric method used in this study is less technically difficult than others (magnetic resonance or isoprostane measurement) and sufficiently sensitive and reproducible to provide a valid estimation of oxidative stress [START_REF] Yagi | A simple fluorometric assay for lipoperoxide in blood plasma[END_REF]. Plasma TBARs

µmol•L -1 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■
Fig. 2. -Significant correlation was found at rest between blood vitamin E and markers of oxidative stress (thiobarbituric reactive substances (TBARs)) in chronic obstructive pulmonary disease patients (r=-0.69; pv0.05).

Quadriceps endurance and physical activity

As previously mentioned, an important issue concerning the peripheral muscle dysfunction in patients with COPD is whether it is entirely due to chronic inactivity and muscle deconditioning or if there is an intrinsic muscle disorder. In a previous study on this topic [START_REF] Maltais | Altered expression of myosin heavy chain in the vastus lateralis muscle in patients with COPD[END_REF], COPD patients and normal subjects were not matched for quantified physical activity levels and it was therefore necessary to confirm the multifactorial aspect of COPD myopathy. The present study showed that Tlim was nearly three-fold lower in the COPD patients compared with healthy subjects with the same level of physical activity. This result indicated clearly that in addition to chronic inactivity, other phenomena are involved in the muscle dysfunction of these patients.

Oxidative stress at rest

In the present study the resting difference in plasma levels of TBARs between the two groups was of some magnitude, but this did not reach statistical significance. This result contrasts with that of PRATICO et al. [START_REF] Pratico | Chronic obstructive pulmonary disease is associated with an increase in urinary levels of isoprostanes F2-III, an index of oxidant stress[END_REF], who described a significant increase in resting urinary isoprostane levels as markers of oxidative stress in patients with COPD compared with agematched healthy subjects. However, the discrepancy between results may be attributable to a difference in disease severity in terms of exacerbation and hypoxaemia, with patients of the present study less severe than the patients in the study by PRATICO et al [START_REF] Pratico | Chronic obstructive pulmonary disease is associated with an increase in urinary levels of isoprostanes F2-III, an index of oxidant stress[END_REF].

The present results showed that O 2 ? -release by in vitro-stimulated phagocytes was significantly higher at rest in COPD patients compared with controls. This result is in agreement with that of RAHMAN et al. [START_REF] Rahman | Systemic oxidative stress in asthma, COPD, and smokers[END_REF], who showed that the release of O 2 ? -from stimulated phagocytes was greater in COPD patients than in age-matched healthy subjects and greatest in those patients with an exacerbation. These results may suggest a continued oxidant burden originating from phagocytes in patients with COPD.

One of the main results of the present study was that the level of blood vitamin E, a nonenzymatic antioxidant, was significantly lower in the patients compared with controls. To the best of the authors9 knowledge, such a low level in COPD has never been reported. It is well known that blood vitamin E level is strongly correlated with cholesterol and TG levels [START_REF] Jordan | Vitamin E and vitamin A concentrations in plasma adjusted for cholesterol and triglycerides by multiple regression[END_REF]. Yet although a lower level was noted in the patients, the two groups had similar levels of TG and cholesterol. In addition, a significant relationship between the sum of TGzcholesterol and vitamin E was found, as expected, in the healthy subjects, but not in the COPD patients. Therefore, the lower blood vitamin E level in the COPD patients does not seem to be due to an abnormality in lipid absorption, which suggests that in these patients it may be regulated by other mechanisms. The patients9 lower blood vitamin E level is of interest in relation to COPD muscle dysfunction because a previous study in animals demonstrated that vitamin E deficiency can cause myopathy [START_REF] Jackson | Isolated vitamin E deficiency[END_REF]. As previously mentioned, vitamin E deficiency can induce skeletal muscle histological changes [START_REF] Nelson | Neuropathological studies of chronic vitamin E deficiency in mammals including humans[END_REF] associated with necrosis of type 1 muscle fibres [START_REF] Pillai | Concomitant brainstem axonal dystrophy and necrotizing myopathy in vitamin E deficient rats[END_REF]. Moreover, since vitamin E serves as a lipid soluble biological antioxidant with high specificity for loci of potential lipid peroxidation, a vitamin E deficiency increases the susceptibility of many subcellular membranes to oxidative damage [START_REF] Quintanilha | Membrane effects of vitamin E deficiency: bioenergetic and surface charge density studies of skeletal muscle and liver mitochondria[END_REF]. In agreement with this, it was observed that the lower blood vitamin E level was inversely and significantly correlated with resting plasma levels of oxidative stress measured by TBARs. This suggests that decreased blood vitamin E could be an indicator of oxidative stress in COPD patients. Finally, the results may suggest that antioxidative mechanisms are able to compensate the oxidant burden at rest in the study9s stable patients. In this sense, the lower blood vitamin E level observed in the study patients could be a response to the possible oxidant burden originating from phagocytes. However, this hypothesis remains speculative and needs further investigation.

Local exercise-induced oxidative stress

Previous studies have shown that strenuous and/or light exercise induces systemic oxidative stress in COPD patients, which suggests that these patients may be frequently exposed to lipid peroxidation and muscle damage in their daily living activities [START_REF] Vina | Exercise causes blood glutathione oxidation in chronic obstructive pulmonary disease: prevention by O 2 therapy[END_REF][START_REF] Heunks | Xanthine oxidase is involved in exercise-induced oxidative stress in chronic obstructive pulmonary disease[END_REF]. However, as mentioned, the few studies on this topic have focused on whole body exercise and did not identify the specific source of this exercise-induced oxidative stress (e.g. lungs, heart, liver, etc.). The present observation of a significant increase (18%) in plasma products of lipid peroxidation in COPD patients 6 h after local quadriceps exercise indicates exercise-induced oxidative stress in the patients, but not in the controls. The absence of oxidative stress in controls after this local exercise is not surprising since oxidative stress (12% increase in blood lipid peroxidation) occurs in healthy subjects when they perform a half-marathon run [START_REF] Gohil | Blood glutathione oxidation during human exercise[END_REF][START_REF] Sastre | Exhaustive physical exercise causes oxidation of glutathion in blood: prevention by antioxidant administration[END_REF]. The present study indicated clearly that the patients with COPD presented a greater susceptibility to local exerciseinduced oxidative stress. Moreover, as exercise localised to the quadriceps was used without notable respiratory response, the authors suggest that a part of the local exercise-induced oxidative stress assessed in the patients9 plasma originated from this muscle. However, the possibility that the COPD increase in lipid peroxidation originated from sources other than the quadriceps (heart, liver, etc.) cannot be completely ruled out. In fact, the best way to identify a specific muscle origin of exercise-induced oxidative stress would be by needle biopsy of the quadriceps. If confirmed, this result may be of clinical relevance since muscle oxidative stress generated during exercise might be one of the mechanisms of the peripheral muscle dysfunction described in patients with COPD.

Two mechanisms linking exercise and oxidative stress are increased pro-oxidant activity and inadequate antioxidant activity. The activity of O 2 ? -release in circulating phagocytes was unchanged after local exercise in the COPD patients. This result is not surprising since the circulating catecholamines generated during local exercise are not sufficient to induce an increase in phagocyte activation [START_REF] Katzuhiko | Capacity of circulating neutrophils to produce reactive oxygen species after exhaustive exercise[END_REF]. In this sense, the local exercise-induced oxidative stress in COPD patients may not be attributable to increased phagocyte O 2 ? -release in response to exercise. However, the present study did not investigate other mechanisms of free radical production, such as an altered mitochondrial respiratory chain or xanthine oxidase activity. HEUNKS et al. [START_REF] Heunks | Xanthine oxidase is involved in exercise-induced oxidative stress in chronic obstructive pulmonary disease[END_REF] showed that both exercise-induced glutathion oxidation and elevation in lipid peroxides were prevented by allopurinol treatment, a xanthine oxidase inhibitor. This strongly suggests that xanthine oxidase, mainly localised in capillary endothelium, is involved in the exercise-induced oxidative stress in COPD patients.

Apart from increased muscular oxidants, a decrease in antioxidative defences may have contributed to the oxidative stress induced by exercise. In this study, the correlation between resting blood vitamin E levels and TBARs 6 h after exercise did not reach statistical significance and local exercise had no significant effects on COPD blood vitamin E levels. These results suggest that vitamin E was not involved in the exercise-induced oxidative stress in these patients. However, it is also possible that the oxidant burden induced by exercise exceeded vitamin E capacity, which was already significantly decreased at rest. In this sense, the lower resting blood vitamin E levels in these patients, probably associated with other altered antioxidants, may have enhanced the exercise-induced oxidative stress. On this topic, ENGELEN et al. [START_REF] Engelen | Altered glutamate metabolism is associated with reduced muscle glutathion levels in patients with emphysema[END_REF] showed a significant decrease in quadriceps glutathione (GSH) level in patients with emphysema, and RABINOVICH et al. [START_REF] Rabinovich | Reduced muscle redox capacity after endurance training in patients with chronic obstructive pulmonary disease[END_REF] reported that patients with COPD had a reduced ability to adapt to endurance training, as reflected by a lower capacity to synthesise GSH. These recent studies suggest that at least another antioxidant system may be exceeded during exercise and involved in local exercise-induced oxidative stress in these patients.

In conclusion, the present study showed that local exercise induces oxidative stress in chronic obstructive pulmonary disease patients and it also revealed a significant decrease in blood vitamin E in these patients. Further studies based on needle muscle biopsies are needed to confirm the muscular origin of exerciseinduced oxidative stress. If these preliminary findings are indeed confirmed, the next step will be to determine whether and how this muscle oxidative stress is involved in the myopathy of chronic obstructive pulmonary disease patients.

  Fig. 1.-Levels of plasma products of lipid peroxidation at rest, at the end of exercise (Tend) and 6 h after the end of exercise (T6h). h: chronic obstructive pulmonary disease (COPD) patients; p: controls. Local exercise induced a significant increase in plasma levels of lipid peroxidation products 6 h after the end of exercise in COPD patients (pv0.05). Plasma level of thiobarbituric reactive substances (TBARs) was unchanged in the healthy subjects after local exercise.

Table 1 .

 1 -Characteristics of the study population

		COPD	Controls Statistical
				difference
	Subjects n	11	12
	Age yrs	65 ¡ 3.6	58 ¡ 1.5	NS
	Height cm	168 ¡ 2.9	171 ¡ 2.7	NS
	Weight kg BMI kg?m 2	73 ¡ 4.9 25.81¡1.2	77 ¡ 4 26¡0.8	NS NS
	FEV1 L	1.15¡0.11 3.29¡0.2	***
	FEV1 % pred	41¡3.4	102¡5.3	***
	Physical activity score	7.6¡1.9	6.6¡1.1	NS
	Data are presented as mean¡SEM unless otherwise indi-
	cated. BMI: body mass index; FEV1: forced expiratory
	volume in one second; COPD: chronic obstructive pulmo-
	nary disease; pred: predicted. ***: pv0.001. NS: nonsignifi-
	cant difference between groups.

Table 2 .

 2 -Values of superoxide anion (O 2 ? -) release by stimulated phagocytes at rest (Trest) and end (Tend) of exercise and blood vitamin E (Vit. E) at Trest, Tend and 6 h (T6h) postexercise in chronic obstructive pulmonary disease (COPD) patients and controls

		O 2 ? -IU			Vit. E mg?L -1
		Trest	Tend	Trest	Tend	T6h
	Controls	385.9¡66.3	407.9¡69.2	27.7¡1.28	28.9¡1.5	28.5¡1.8
	COPD	794.5¡189*	759¡179*	23.3¡1.2*	21.55¡1.09**	20.45¡1.26**
	Values are expressed as mean¡SEM. COPD patients: n=11; controls: n=12. *: pv0.05 and **: pv0.01 compared with control
	values.


A Vinet

D Le Gallais

S Bouges

P-L Bernard

M Poulain

A Varray

J-P Micallef

Prediction of VO 2peak in wheelchair-dependent athletes from the adapted Le ger and Boucher test

Keywords: paraplegics, incremental ®eld test, peak oxygen uptake, predictive equation

Purpose: The purpose of this study was to provide a predictive peak oxygen uptake (V . O 2 peak) equation in wheelchair-dependent athletes using the Adapted Le ger and Boucher test. Subjects and protocol: Fifty-six wheelchair-dependent athletes, 47 males and nine females (30.3+4 years), underwent a clinical examination to assess their anthropometric characteristics: height, mass, body mass index (BMI), lean body mass, arm length, and muscular arm volume. They performed a deceleration ®eld test to assess the subject-wheelchair resistance de®ned as a mechanical variable, and they then performed the Adapted Le ger and Boucher test to assess physiological data at maximal exercise (V .

O 2 peak, heart rate max) concomitantly with biomechanical (number of pushes) and performance variables (maximal aerobic velocity Va max and maximal distance). The V .

O 2 peak was measured directly using a portable telemetric oxygen analyzer. Subjects were then randomly assigned to an experimental group (n=49) to determine the predictive equation, and a validation group (n=7) to check the external validity of the equation. Results: A stepwise multiple regression with V . O 2 peak (l min 71 ) as the dependent variable led to the following equation: V . O 2 peak=0.22 Va max 7 0.63 log(age)+0.05 BMI 0.25 level+0.52, with r 2 =0.81 and SEE=0.01. Paraplegic subjects with high and low lesion level spinal injuries were attributed the coecient of 1 and 0, respectively. The external validity of the equation was positive since the predicted V .

O 2 peak values did not signi®cantly dier from directly measured V . O 2 peak (P40.05).

Conclusion:

We concluded that V . O 2 peak in wheelchair-dependent athletes was predictable using the equation of the present study and the described incremental test.

Introduction

Maximal oxygen uptake (V . O 2 max) has been described as an important parameter of aerobic performance. [START_REF] Saltin | Maximal oxygen uptake in athletes[END_REF][START_REF] Wyndham | Physiological requirement for world-class performance in endurance running[END_REF] Incremental exercise testing with direct laboratory measurement provides the most widely used and accurate V . O 2 max assessment. However, since equipment, expense, time and personal requirements make the laboratory procedure prohibitive for large populations, ®eld tests have been developed for able-bodied subjects. One of the most commonly used is the Universite de Montre al Track Test of Le ger and Boucher. [START_REF] Leâ Ger | An indirectly measured continuous running multistage ®eld test: the Universite de Montre al track test[END_REF] Wheelchair-dependent subjects usually per-form laboratory tests, [START_REF] Coutts | Maximal exercise responses of tetraplegics and paraplegics[END_REF][START_REF] Flandrois | Aerobic performance capacity in paraplegic subjects[END_REF][START_REF] Vinet | Aerobic metabolism and cardioventilatory responses in paraplegic athletes during an incremental wheelchair exercise[END_REF] but the growing number who participate competitively in athletic activities need regular assessments of V . O 2 peak. Thus Di Carlo 7 used a sustained 12-min propulsion task to predict the examined changes in the V . O 2 peak of wheelchairdependent subjects. This replacement of Cooper's 12 min run-walk test appeared to be a reliable tool in the assessment of wheelchair propulsion-endurance for individuals with spinal cord injury. Moreover, Rhodes et al. [START_REF] Rhodes | A ®eld test for the prediction of aerobic capacity in male paraplegics and quadriplegics[END_REF] and Franklin et al. [START_REF] Franklin | Field test estimation of maximal oxygen consumption in wheelchair users[END_REF] also used this Cooper 12 min test to predict the V . O 2 peak of wheelchairdependent subjects. They established their predictive equation of V . O 2 peak in wheelchair-dependent subjects during rectangular ®eld tests by using incremental laboratory assessment of V . O 2 peak as the dependent variable. One may suspect, however, that the use of two dierent test protocols in two dierent conditions would result in a methodological bias. In contrast, the present study assessed V . O 2 peak as the dependent variable in both an incremental protocol and ®eld conditions, and thus eliminated this methodological bias. As previously reported, [START_REF] Franklin | Field test estimation of maximal oxygen consumption in wheelchair users[END_REF][START_REF] Vinet | Validation of an incremental ®eld test for the directly measured assessment of peak oxygen uptake in wheelchair-dependent athletes[END_REF] we validated an adapted Le ger and Boucher test (ALBT) for wheelchair-dependent athletes [START_REF] Franklin | Field test estimation of maximal oxygen consumption in wheelchair users[END_REF][START_REF] Vinet | Validation of an incremental ®eld test for the directly measured assessment of peak oxygen uptake in wheelchair-dependent athletes[END_REF][START_REF] Poulain | Reproducibility of the Adapted Leger and Boucher Test for wheelchair-dependent athletes[END_REF] which allowed the subjects to reach their V . O 2 peak. As expected, the predictive V . O 2 max equation of Le ger and Boucher 3 for able-bodied subjects was inappropriate for the wheelchair-dependent athletes, as it included biomechanical and anthropometric variables that do not apply to wheelchair-dependent subjects, such as running cost and body surface area. In order to complement our previous work, this study was designed to provide a predictive V . O 2 peak equation in well-trained wheelchair-dependent athletes during the ALBT using a set of anthropometric, physiological, mechanical, bio-mechanical and performance variables.

Methods

Subjects

Fifty-six wheelchair-dependent athletes (47 males and 9 females), aged 30.3+0.4 years (range: 18 to 47 years) provided written informed consent to participate in this study, which had been approved by the institutional review board. All subjects had had lower-limb disability (39 paraplegics, 5 amputees and 12 postpolio) for at least 1 year prior to the study. The subjects were divided in two groups: high lesion level paraplegic subjects (HP) were attributed a coecient of 1, and low lesion level paraplegic subjects (LP) were attributed 0. Amputees and post-poliomyelitic subjects were classed in the LP group. The dierence between these two groups was the functionality or not of abdominal muscles. All subjects used their wheelchairs for everyday life and/or physical activities, and they were considered as having good control of their wheelchairs. All practiced sports (wheelchair-racing, tennis, fencing, swimming or basketball) for at least 6.25+4 h per week and all competed at national level. All subjects performed the two tests, and after the tests, they were randomly assigned to one of two subgroups with the number of subjects in each group adapted to the following objectives: (1) an experimental group (EG) with 49 subjects (40 males and 9 females, 15 HP and 34 LP) to determine the predictive equation for V . O 2 peak, and (2) a validation group (VG) with 7 subjects (7 males, 1 HP and 6 LP) to verify the external validity of the predictive equation.

Protocol

During a clinical examination, anthropometric variables were measured. During a deceleration ®eld test, mechanical variables were recorded. During the ALBT, physiological, biomechanical and performance variables were assessed. These three steps were conducted the same day and in the same ®eld conditions. The variables were chosen because of their relatively strong relationship with V . O 2 peak reported in the literature, and because they are easily measured.

The clinical examination, always made by the same examiner, assessed seven anthropometric variables. Height was measured with the subject in the supine position. The mass of the subject with the wheelchair was measured using a platform scale with a 5 kg wooden plank to allow wheelchair positioning. This process was repeated twice: once to weigh the subject+wheelchair ®rst and then to weigh the wheelchair alone. The dierence was the subject mass. Body mass index (BMI, kg m 72 ) was calculated as the ratio of the subject mass by his squared height. Body fat was estimated by the sum of 4 skinfolds: triceps, biceps, subscapularis and suprailiac, measured twice on the left side of the body as recommended for use of the Durnin and Womersley equation. [START_REF] Durnin | Body fat assessed from total body density and 1st estimation from skinfold thickness: measurements on 481 men and women aged from 16 to 72 years[END_REF] Lean body mass was then calculated. Lastly, the muscular volume of the arm was assessed using skinfolds, circumferences and diameters according to Shephard et al., [START_REF] Shephard | Muscle mass as a factor limiting physical work[END_REF] and arm length (AL, cm) was measured between the arm bend and the longest ®nger.

As subjects used their own wheelchair, the deceleration ®eld test [START_REF] Vinet | A ®eld deceleration test to assess total wheelchair resistance[END_REF] assessed the total wheelchair resistance (R Newton), i.e., the internal and external resistances of the subject+wheelchair system de®ned as a mechanical variable to standardize mechanical characteristics of wheelchairs. Brie¯y, the protocol required the subject to propel his wheelchair over a 20 m distance: the ®rst 10 m in acceleration and the second 10 m maintaining a constant velocity. Once the subject passed the 20 m mark, he or she stopped propulsion and remained motionless in the wheelchair. The time needed to cover the 10 m at constant velocity (To, s) and the time for the wheelchair to decelerate and stop (Td, s) were measured using a chronometer with a precision of 1/100 s. As the velocity versus time slope was linear throughout the test, the deceleration was constant and R was a constant force calculated according to the following equation: R=10m* (Mass The incremental ®eld test assessed physiological, biomechanical and performance variables. It was conducted on a 400 m tartan ®eld marked-o every 50 m with pylons. The wind speed was measured with an anemometer prior to testing and had to be less than 2 m s 71 . The ALBT protocol has been described elsewhere. [START_REF] Vinet | Validation of an incremental ®eld test for the directly measured assessment of peak oxygen uptake in wheelchair-dependent athletes[END_REF] Brie¯y, subjects propelled their wheelchairs as far as possible following the rhythm imposed by means of an audio-tape. The turning speed of the tape recorder was checked prior to the start of each test to ensure that any deviation was less than 1 s min 71 . [START_REF] Leâ Ger | Speed reliability of cassette and tape players[END_REF] The athletes adjusted their velocity when crossing a pylon at the sound signal. The initial velocity was 4 km h 71 for 1 min, thereafter it was increased by 1 km hr 71 every minute until exhaustion, which was de®ned as the inability to maintain the required velocity with a 3-m distance behind the appropriate pylon at the sound signal and the inability to catch up at the next pylon. At the end of the ALBT, performance variables were noted: the maximal distance (d max m) and the maximal velocity (Va max , km h 71 ) maintained in 1 min, de®ned as the velocity necessary to attain V . O 2 peak. A biomechanical variable was also noted, i.e., the number of pushes (NP) during the last 50 m of each 400 m lap were counted by an experimenter, which was used to assess the propulsion eciency. Throughout the ALBT, the subjects were equipped with the Cosmed K2 (Rome, Italy), a portable telemetric oxygen uptake analyzer. This system is composed of a transmitting unit, battery, face mask, heart rate chest strip, and receiving unit. The face mask contains a photoelectric turbine for recording breathing frequency, tidal volume and minute ventilation, as well as a capillary gas sampling port within the turbine's housing. A sample of expired gas enters into a mixing microchamber with a capacity of 2 ml. A miniature polarographic electrode for the oxygen concentration measurement is inside the microchamber. The V . O 2 , l min 71 and ml kg 71 min 71 were collected every 15 s, after a 30-min warm-up for the K2 unit before every testing session. The K2 analysis system was calibrated with O 2 atmospheric air, which was assumed to be 20.9% before each test. The expired gases were sampled at the rate proportional to ventilation by means of a dynamic sampling pump. Despite the lack of a CO 2 electrode, the Cosmed K2 has been validated for direct V . O 2 assessment. [START_REF] Crandall | Evaluation of the Cosmed K2 portable telemetric oxygen system[END_REF][START_REF] Lucia | Validity and reliability of the Cosmed K2 instrument[END_REF] Heart rate (HR, beat min 71 ) was measured with the Cosmed K2 every 15 s. The criteria for V . O 2 peak were HR within 15 beat min 71 of age predicted maximal HR [220 -age], and the inability for the athlete to maintain the required velocity.

Statistical analysis

The statistical analysis was done in three parts. In the ®rst part, comparisons between anthropometric, physiological, mechanical, biomechanical and performance variables of the EG and VG groups were made using the Student unpaired t-test. The second part concerned only the EG subjects and it allowed us to choose which independent variables (anthropometric, hours of training, physiologic, bio/mechanical) would enter into the stepwise regression. The ®rst step consisted of determining the Gaussian distribution (using the Skewness, Kurtosis and the Shapiro Wilk W tests) and the availability of the linear model between each regressor and the explained variables. The second step consisted of searching for a statistical link between each regressor and the variable of interest; using a correlation matrix, the threshold value of P was 0.1. instead of 0.05, thereby ensuring a wide set of regressors. The third step consisted of submitting the selected regressors to two co-linearity tests according to Besley et al. [START_REF] Besley | Regression Diagnostic[END_REF] the ®rst runs a condition index (square root of the highest Eigenvalue and each one of the variables) whose highest values make co-linearity a reliable hypothesis; the second indicates the consequence of such a supposed co-linearity on the variance in¯ation of estimates. The last step was a multiple stepwise regression to predict V . O 2 peak in the EG subjects. For this, the speci®ed signi®cance levels for entering and staying in the model were, respectively, 0.1 and 0.2, since the approach was an experimental one. In addition, a coecient of variation was calculated, and regression tests were computed (residuals analysis, studentized residuals and Cook's D test) to assess the precision of the prediction. The third part concerned the VG subjects. The external validity of the equation was veri®ed by comparing predicted V . O 2 peak and measured V . O 2 peak with a Student paired t-test. Statistical results were considered as signi®cant when P50.05. All tests were performed using the S.A.S statistical software package of S.A.S. Institute, running on Vax/VMS. [START_REF]User's Guide Version 6.08[END_REF]

Results

No signi®cant dierence between any of the variables was found between the EG and VG groups. Data on the anthropometric, physiological, mechanical, biomechanical and performance variables, for groups EG and VG, are reported in Tables 1 and2

Discussion

The present study provided a predictive V . O 2 peak equation for wheelchair-dependent athletes from an incremental ®eld test. The validity of this equation was checked on only seven subjects. The variables, anthropometric, mechanical, physiological, biomechanical and performance, were chosen very carefully.

The primary purpose of this investigation was to provide a predictive equation for V . O 2 peak in a ®eld test which would be easy to use for both wheelchairdependent athletes and their training sta. Anthropometric variables (height, mass, BMI, lean body mass, AL and muscular volume of the arm) were measured. The stepwise regression procedure needs non-correlated (and/or orthogonal) variables to avoid colinearity and thus to ensure accurate estimates of regression parameters. Muscular volume of the arm and AL were highly correlated. Because of the above requirement and because accurate measurement of muscular volume of the arm is not always possible, we retained AL as the easier variable to measure. Similarly, lean body mass and BMI were correlated and we kept BMI.

We used the functionality of abdominal muscles to divide our subjects after consideration of the results of Coutts et al. [START_REF] Coutts | Maximal exercise responses of tetraplegics and paraplegics[END_REF] These authors studied maximal exercise responses of paraplegic subjects of dierent classes. They reported a `break' in the responses between class III (lesion level T5 ± T10 without active abdominal muscles) and class IV (lesion level T11-L3 with active abdominal muscles), and then divided their subjects into only two groups: HP and LP subjects. This classi®cation has been used [START_REF] Flandrois | Aerobic performance capacity in paraplegic subjects[END_REF] and studies have shown that HP subjects O 2 peak than LP subjects. [START_REF] Coutts | Maximal exercise responses of tetraplegics and paraplegics[END_REF][START_REF] Flandrois | Aerobic performance capacity in paraplegic subjects[END_REF] In the present study, HP subjects were attributed a coecient of 1, thus the constant term in the predictive equation was 0.27 (70.25*1+0.52). The LP subjects were attributed to 0, and the constant term was 0.52 (70.25*0+0.52). This V . O 2 peak dierence of 0.25 l min 71 (0.52 ± 0.27) between LP and HP groups was close to the V . O 2 peak dierence reported by Flandrois et al. [START_REF] Flandrois | Aerobic performance capacity in paraplegic subjects[END_REF] in active LP and HP subjects (0.18 l min 71 ), and was lower than that reported by Coutts et al. [START_REF] Coutts | Maximal exercise responses of tetraplegics and paraplegics[END_REF] O 2 peak from maximal distance in wheelchair-dependent subjects. However, because of the incremental characteristic of the ALBT, Va max was retained instead of maximal distance, which is often used with rectangular tests. Moreover, Va max corresponded to the last minute of the ALBT and thus to the V . O 2 plateau reported at the ®nal stages of the test. HR max was not entered into the regression model, both because it did not enhance the predictive equation and because a HR telemetric system with a chest strip would have been necessary, which would have limited the use of the equation. Surprisingly, R did not enter into the predictive equation. One would expect to correlate Va max with R since every subject performed the ALBT with his own general purpose wheelchair but, in fact, Va max may already include R. Thus, R was not necessary to predict V . O 2 peak in wheelchair-dependent athletes. Similarly, NP was negatively correlated with V . O 2 peak (70.53) and was not retained by the stepwise procedure. NP was measured during the last 50 m of each 400 m lap, therefore it was not always measured concomitantly with the attainment of Va max and thus was not predictive of V . O 2 peak. The selected predictor variables, Va max , log(age), BMI, and lesion level, were easily measured without special equipment, therefore no material was needed to predict V . O 2 peak throughout the ALBT. Moreover, the equation was highly predictive (0.80) with a small SEE (0.01) and a quite low coecient of variation (15.3%). The residuals analysis showed the independence of these residuals against the predicted V . O 2 peak and the measured V . O 2 peak. The non-homogeneity of each variable between the groups, assessed by the standard-deviation dispersion, con®rmed the extrapolation quality of the equation. The external validity was veri®ed by comparing measured V . O 2 peak and predicted V . O 2 peak in the VG subjects. This result, in only seven randomly chosen subjects, will need to be veri®ed. Nevertheless, it indicates that the ALBT with this new equation may provide a good prediction of V . O 2 peak in these well-trained wheelchair-athletes, men and women, whose disability does not involve the upper extremities, and who are using their own general-purpose wheelchairs.

In conclusion, the ALBT allowed the determination of predictive V . O 2 peak equation in well-trained wheelchair-dependent athletes. Although the external validity of the equation was checked in only some subjects, it may have wide applicability in aerobic evaluation and during training sessions of wheelchairdependent athletes. The assessment of a much larger and heterogeneous population, particularly in terms of age and training level, using the protocol of the present study could enhance the predictive V . O 2 peak equation for all wheelchair-dependent subjects.

Table 1

 1 Anthropometric variables included in the stepwise regression for the prediction of V . O 2 peak (EG) and the external validity (VG)

	Groups		Age yr	Anthropometric H cm MS kg BMI kg m ±2	LBM kg	MVA l	AL cm
	EG	x	29.6	172.4	62.4	20.9	58.9	3.7	71.4
	(n=49)	SD	1.29	13.2	10.3	2.4	10.1	0.9	4.8
	VG	x	27.2	172.6	63.7	21.3	59.4	4.4	70.7
	(n=7)	SD	1.32	7.9	8.3	1.0	7.9	0.07	4.4
	No signi®cant dierence between EG and VG groups.
	H=height, MS=subject mass, BMI=body mass index,
	LBM=lean body mass, MVA=muscular volume of arm,
	AL=arm length

  The coecient of variation was calculated as equal to 15.3%. Figure2displays the linearity between residuals and predictors, and homoscedasticity was veri®ed by graphical analysis of residuals, which showed the independency of residuals against predicted V . O 2 peak (horizontal distribution). Moreover, studentized residuals and Cook's D test did not detect any outliers in the model. The external validity showed that measured V . O 2 peak and predicted V .O 2 peak values in VG was not statistically dierent (P=0.65).

	V . O 2 peak.
		. Values of V . O 2
	peak in women and men were not signi®cantly dierent
	that is why all results were reported as a whole group.
	According to the correlation matrix and the Gaussian
	distribution, the relevent variables (only age required
	transformation; log(age) successfully stabilized the variable) were V . O 2 peak, Va max , HR max, log(age),
	BMI, AL and NP.
	The stepwise regression model used these variables for the prediction of V . O 2 peak in l min 71 . This derived equation included Va max (km h 71 ), log(age), BMI (kg m 72 ), and lesion level (Table 3):
	V .	O 2 peak=
		0.22Va max ±0.63log(age)+0.05BMI±0.25level-0.52,
	with r 2 =0.81 (P=0.01) and SEE=0.097. Figure 1 displays the measured V . O 2 peak and the predicted

Table 2

 2 Mechanical, physiological, biomechanical and performance variables included in the stepwise regression for the prediction of V . O 2 peak (EG) and the external validity (VG)

	Groups		Physiological O 2 peak V . l min ±1 HR max b min ±1	Mechanical R N	Biomechanical NP n	d max m	Performance Va max km h ±1
	EG	x	2.05	179.9	12.5	21.1	1327.1	12.22
	(n=49) SD	0.63	11.7	3.4	2.2	424.4	1.93
	VG	x	2.32	181.4	13.3	20.9	1492.8	13.14
	(n=7) SD	0.34	14.6	5.2	5.8	329.7	1.58
	No signi®cant dierence between EG and VG groups. R=total wheelchair resistance, NP=number (n) of pushes,
	d max =maximal distance, Va max =maximal aerobic velocity

Table 3

 3 Stepwise procedure for dependent V . O 2 peak (l min ±1 ) variable

	Step Variable Partial R 2 Model R 2	F	P
	1	Vamax	±	0.67	96.08	0.0001
	2	log(age)	0.08	0.75	14.90	0.0004
	3	BMI	0.027	0.78	5.56	0.0227
	4	lesion level	0.027	0.81	6.32	0.0157
	Figure 1 Relationship between measured V . O 2 peak and predicted V . O 2 peak solid line=regression line, dashed
	line=identity line

  in sedentary LP and HP subjects (0.77 l min 71 ). The homogeneous training level of the subjects in the present study may have reduced the dierence between the HP and LP athletes. Va max and maximal distance have been reported to be highly correlated with V . O 2 peak by, respectively, Le ger and Boucher 3 and Cooper et al. 20 Di Carlo 7 used maximal distance covered of 12 min as an indicator of functional endurance in subjects with spinal cord injury. Using the same rectangular test, Rhodes et al. 8 and Franlin et al. 9 estimated V .

*Correspondence: A Vinet, Laboratoire de Physiologie des Adaptations Cardiovasculaires aÁ l'exercice, Faculte des Sciences, De partement STAPS, 33 rue Louis Pasteur, F-84000 Avignon, France

Acknowledgements

The authors would like to acknowledge the Fe de ration FrancË aise Handisport for allowing us to assess their athletes during training sessions, and all the athletes who participated in the present study.


PhD Nathalie S Ville

PhD Be ´atrice Alain Varray

PhD Maurice Mercier

MD Hayot

PhD Bernard Albat

MD Karim Chamari

PhD Christian Pre ´faut

PhD Jacques Mercier

Effects of an Enhanced Heart Rate Reserve on Aerobic Performance in Patients with a Heart Transplant

Keywords: Cardiac Transplantation, Oxygen Uptake, Chronotropic Response, Exercise

Objective:

The aim of this study was to investigate whether a high-intensity warm-up at the start of a graded, symptom-limited exercise test would enhance heart rate reserve and thus improve the aerobic performance of orthotopic heart transplant patients.

Design: Adrenal and cardiorespiratory responses were compared in 10 orthotopic heart transplant patients who performed two graded, symptom-limited exercise tests on an ergocycle.

Results: At the start of the graded, symptom-limited exercise test, high intensity increased the norepinephrine level more than usual intensity between rest and the third minute of exercise. This higher norepinephrine level was followed by a higher heart rate response from the fourth minute of exercise. Heart rate reserve was enhanced during high-intensity exercise, without any significant change in peak oxygen uptake.

Conclusions: This specific warm-up enhanced heart rate reserve during a graded, symptom-limited exercise test on an ergocycle. Mechanisms more important than limited heart rate reserve are involved in the limitation of exercise tolerance in orthotopic heart transplant patients.

Al tered resting [START_REF] Pflugfelder | Hemodynamic profiles at rest and during supine exercise after orthotopic cardiac transplantation[END_REF][START_REF] Pope | Exercise response of the denervated heart in long-term cardiac transplant recipients[END_REF] and exercise [START_REF] Pflugfelder | Hemodynamic profiles at rest and during supine exercise after orthotopic cardiac transplantation[END_REF] hemodynamics caused by cardiac denervation are a common finding after orthotopic heart transplantation (OHT). Heart rate reserve (HRR), which is peak heart rate (HR) minus the resting HR, [START_REF] Brooks | Ex-ercise Physiology[END_REF] is the most decreased cardiac parameter 4 because of a high resting HR and a low peak HR. Moreover, it has been shown that the percentage of change in HR from rest to peak exercise is correlated with peak oxygen uptake (V ˙O2 ; r ϭ 0.60). [START_REF] Kemp | Association of resting heart rate and chronotropic response[END_REF] Thus, HRR may affect aerobic power after OHT, but no causal relationship has been demonstrated to date. The factors that limit the exercise tolerance of heart transplant recipients are not yet well understood, but abnormal chronotropic response is generally cited as one of the explanations for the peak V ˙O2 limitation. [START_REF] Savin | Cardiorespiratory responses of cardiac transplant patients to graded, symptom-limited exercise[END_REF][START_REF] Ehrman | Cardiovascular responses of heart transplant recipients to graded exercise testing[END_REF] Because HR response to exercise is delayed and reduced after heart transplantation, 6,8 -10 we hypothesized that a high workload (50 W) at the start of an incremental exercise test allowing the assessment of HRR and peak V ˙O2 would increase catecholamine levels rapidly and markedly. We further speculated that after 6 min of exercising at this workload, the effects would be nearly optimal in terms of chronotropic response. The aim of this study was thus to investigate the ability of a specific warm-up (50 W) to enhance adrenal response, HR increase, and HRR during a symptom-limited exercise test and then to assess the effect on aerobic power.

METHODS

Subjects

Ten male OHT patients (age, 55 Ϯ 2 yr; weight, 74.5 Ϯ 2.8 kg; height, 173.6 Ϯ 1.2 cm; postsurgery time, 31.6 Ϯ 6.6 mo) volunteered to participate in the study. None was involved in a rehabilitation program. Before surgery, their clinical status was class IV according to the New York Heart Association, with etiologies including dilated cardiomyopathy (n ϭ 3) and ischemic cardiopathy (n ϭ 7). After surgery, all patients were considered to be class I and were receiving triple-drug immunosuppressive therapy (cyclosporine, azathioprine, and steroid [average dose, 10 mg/day]). None was receiving ␤-blocker agents or other therapies that could affect the chronotropic response. All were free of acute rejection, systemic infection, coronary artery disease, and peripheral vascular disease. Informed written consent was obtained from all patients, and the study was accepted by the hospital ethics committee (Comite ´de protection des personnes pour la recherche biologique No. 950187).

Materials

Exercise Testing. Each subject performed two different graded, symptom-limited exercise tests in random order on an electromagnetic cycle ergometer (Ergo-Metrics 900; Ergoline, Bitz, West Germany). Respiratory variables and gas exchanges were measured with a breath-by-breath automated metabolic system (CPX, Cardio2, Breeze; Medical Graphics, St. Paul, MN). [START_REF] Ville | Exercise tolerance in heart transplant patients with altered pulmonary diffusion capacity[END_REF] During exercise, oxygen saturation was continuously determined with a pulse oximeter (Datex, Helsinki, Finland), and a 12-lead electrocardiograph (CPX, Cardio2, Breeze) was monitored for the measurement of HR. Finally, systolic and diastolic blood pressure were measured with a cuff blood pressure transducer (Ergoline).

Cardiorespiratory

Measurements. We were principally interested in HR, which allowed the calculation of HRR (peak exercise HR Ϫ resting HR) and V ˙O2 . We also measured CO 2 output (V ˙CO 2 ) to determine the respiratory exchange ratio (V ˙CO 2 /O 2 ).

Circulating Catecholamine Analysis.

One of the 10 subjects did not agree to venous sampling. So in nine subjects, a venous catheter was inserted in a superficial forearm vein to allow sampling for measurement of epinephrine and norepinephrine concentrations. A three-way tap was placed on the catheter to allow rinsing with a syringe containing a mixture of heparin and physiologic saline (250 IU/ml) and blood sampling with a dry syringe after the catheter had been cleared of saline. Approximately 3 ml of blood was placed in a tube (lithium heparin) containing reduced glutathione (1.2 mg/ml) to control catecholamine oxidation, centrifuged (3000 rpm for 10 min) and stored at Ϫ80°C for catecholamine analysis. Plasma catecholamine concentrations were determined by high-performance liquid chromatography (HPLC). [START_REF] Hjemdahl | Catecholamine measurements by high-performance liquid chromatography[END_REF] The catecholamines were extracted by selective absorption to aluminum oxide (Chromsystems-HPLC-Kit; Waters Corp., Milford, MA) before the HPLC run. Aluminum oxide was briefly shaken up in extraction buffer (50 l), and then 1 ml of plasma was added with 50 l of internal standard solution (600 pg of dihydroxybenzylamine). The aluminum oxide was washed three times, with brief centrifugation between washes. The catecholamines were extracted with 120 l of elution buffer with brief shaking and then centrifuged (final centrifugation) at 2000 rpm for 1 min. Then 50 l of sample eluent was injected into the HPLC column (Resolve 5-l spherical C18 HPLC column; Waters Corp.) and eluted with mobile phase. The flow rate was 1 ml/min at 2000 psi with a potential of 0.60 V. The chromatogram was analyzed by computer integration (Baseline 815; Waters Corp.). Three assays were made for each sample, and the mean of these assays was taken for statistics. The error of measurement within sample was satisfactory (less than 5%), similar to resting value. ⌬Catecholamines was also calculated between rest and peak exercise (peak exercise Ϫ resting values).

Indications for halting the exercise test before the appearance of a plateau V ˙O2 were as follows: (1) adverse symptoms: severe dyspnea, light-headedness and faintness, confusion, severe fatigue, or inability to maintain the pedaling frequency; (2) adverse signs: facial pallor, HR or blood pressure decrease (or failure of either to increase with increasing effort), or a rapid increase in systolic or diastolic blood pressure, or both, exceeding 210/115 mm Hg for a moderate intensity of exercise (Ͻ60 W); and (3) adverse electrocardiographic changes: frequent complex ventricular extrasystoles, ventricular tachycardia, sustained supraventricular tachycardia, second-or third-degree heart block, severe ST depression (horizontal or down sloping greater than 2 mm). [START_REF] Mercier | Influence of post-surgery time after cardiac transplantation on cardiorespiratory responses during exercise[END_REF] A respiratory exchange ratio Ͼ1.1 and the inability to maintain the pedaling frequency despite the standardized encouragements of the experimenters were the criteria for verifying that the exercise tests were really symptom limited. [START_REF] Ville | Exercise tolerance in heart transplant patients with altered pulmonary diffusion capacity[END_REF][START_REF] Mercier | Influence of post-surgery time after cardiac transplantation on cardiorespiratory responses during exercise[END_REF]

Statistical Analysis

Values of all variables are expressed by the mean Ϯ SEM. Paired Student's t-tests were performed on resting HR and HRR. Resting and peak V ˙O2 were compared for statistical difference by using the same procedure. Submaximal HR time courses were compared by using a fully factorial repeated-measures design. When the F ratio was significant, the Tukey test was performed to identify differences. In addition, catecholamine values at rest and during exercise (norepinephrine and epinephrine values at 3 and 6 min) and ⌬norepinephrine and ⌬epinephrine (peak exercise Ϫ resting values) were compared with the Wilcoxon's signed rank test. SigmaStat (Jandel Scientific, Costa Madre, CA) and SYSTAT (SPSS Inc., Chicago, IL) software was used for these statistical analyses. The level of significance for all analyses was P Յ 0.05.

RESULTS

During the graded exercise tests, none of the OHT patients developed decreases in oxygen saturation of more than 4%, adverse signs or symptoms, or electrocardiogram changes such as severe ST segment depression, tachycardia, or atrial fibrillation. All stopped the exercise test because of leg fatigue and pain. The duration of the two tests was not significantly different (12.4 Ϯ 1 min and 12.3 Ϯ 0.9 min for HI and UI, respectively), and most of the patients were able to exercise longer after the HI warm-up; only two patients exercised for less than 10 min.

There was no significant difference for epinephrine response during exercise (at 3 and 6 min) or for ⌬epinephrine (112.3 Ϯ 37.9 pg/ml for HI vs. 54.9 Ϯ 43.6 pg/ml for UI). Concerning norepinephrine concentration, during HI, norepinephrine values at 3 and 6 min were significantly lower than ⌬norepinephrine (peak Ϫ resting value; P Ͻ 0.05). During UI, norepinephrine at 3 min was significantly lower than norepinephrine at 6 min (P Ͻ 0.05), but it was not significantly different from ⌬norepinephrine. Norepinephrine at 3 min was significantly higher during HI vs. UI (P Ͻ 0.05; Fig. 1). There was no significant difference for ⌬norepinephrine between HI and UI (Fig. 1).

At rest, cardiorespiratory values were similar for the two tests (Table 1). HR was significantly higher in HI than in UI from the fourth to sixth minute of exercise (Fig. 2). HRR was significantly higher during HI vs. UI (P Ͻ 0.05), in contrast to peak V ˙O2 (Fig. 3).

DISCUSSION

This study shows that, during a symptom-limited exercise test, a spe-that found in a previous study of our group. [START_REF] Hue | Catecholamine, blood lactate and ventilatory responses to multi-cycle-run blocks[END_REF]

Protocol

The order of the two exercise tests was randomized, and the subjects performed the two tests within a 2-wk period. On the days of the experiment, patients arrived at the laboratory at the same hour in the morning and received standardized instructions as to the testing procedure. On day 1, they underwent a complete physical examination and a resting electrocardiogram. Each patient then performed a graded, symptom-limited cycle exercise test. The second exercise test was performed on day 2. One of the tests had a usual intensity (UI) for warm-up (i.e., 20 W). [START_REF] Ville | Exercise tolerance in heart transplant patients with altered pulmonary diffusion capacity[END_REF] The patient rested for 3 min while seated on the cycle and then, after a warm-up at 20 W for 3 min, loads were increased 10 W each minute until exhaustion. The other test, with a high intensity (HI) warmup, was similar in that the patient rested for 3 min on the cycle. But the warm-up consisted of cycling at 50 W for 6 min, with loads then increased 10 W each minute (in such a way that at the seventh minute, all patients were exercising at 60 W) until exhaustion.

All cardiorespiratory values were recorded at rest, up to peak exercise, and during recovery, for the last 20 sec of every minute. Systolic and diastolic blood pressure were measured every 3 min up to the end of exercise.

Samples for catecholamines were taken at rest, at the third minute (which corresponded to the end of the usual starting workload), and at the sixth minute (which corresponded to the end of the HI starting workload) of the exercise test and at peak exercise. To minimize the intersubject variation, as seen in some studies, [START_REF] Ehrman | Cardiovascular responses of heart transplant recipients to graded exercise testing[END_REF][START_REF] Pe ´rini | Kinetics of heart rate and catecholamines during exercise in humans[END_REF] the concentrations of norepinephrine and epinephrine at the third and sixth minute of exercise were expressed by using the third-or sixth-minute exercise values minus cific warm-up enhances the submaximal HR and HRR in OHT patients. This enhancement of HRR is not associated with a significant enhancement of peak V ˙O2 .

The HR values at rest and during submaximal and peak exercise in the UI condition are typical of the denervated heart, i.e., a delayed and gradual increase in HR with the onset of exercise [START_REF] Savin | Cardiorespiratory responses of cardiac transplant patients to graded, symptom-limited exercise[END_REF][START_REF] Cerretelli | Gas exchange and metabolic transients in heart transplant recipients[END_REF][START_REF] Degre | Cardiorespiratory response to early exercise testing after orthotopic cardiac transplantation[END_REF] and a low peak HR, [START_REF] Savin | Cardiorespiratory responses of cardiac transplant patients to graded, symptom-limited exercise[END_REF][START_REF] Degre | Cardiorespiratory response to early exercise testing after orthotopic cardiac transplantation[END_REF] attaining only 78.1 Ϯ 3.2% of predicted maximal HR. In normal subjects, the HR increase observed at the onset of exercise is initially caused by the withdrawal of vagal tone. [START_REF] Robinson | Control of heart rate by the auto-nomic nervous system[END_REF][START_REF] Rowell | Reflex control of the circulation during exercise: chemoreflexes and mechanoreflexes[END_REF] The literature notes that during exercise in heart transplant patients, the observed HR change is closely linked to the level of circulating catecholamines, [START_REF] Pope | Exercise response of the denervated heart in long-term cardiac transplant recipients[END_REF][START_REF] Pe ´rini | Kinetics of heart rate and catecholamines during exercise in humans[END_REF] with a linear relationship during constant-load exercise between HR and norepinephrine increments. [START_REF] Pe ´rini | Kinetics of heart rate and catecholamines during exercise in humans[END_REF] Nevertheless, during HI, because of the higher and longer starting workload compared with UI, the increase in norepinephrine was significantly higher at the third minute of exercise, whereas the HR response was significantly increased from the fourth to sixth minute.

First, because norepinephrine at 3 and 6 min during HI were not different, we can speculate that the norepinephrine concentration was stable because it was adapted to the level of the intensity (50 W). In UI, in contrast, the norepinephrine level increased between the third and sixth minute of exercise proportionally to the increase in exercise intensity. Indeed, at the sixth minute of exercise, the work load was the same in HI and UI (50 W), and there was no difference in norepinephrine concentrations between the two tests at this moment. Second, concerning the chronotropic response, at the third minute of HI, HR showed only a tendency to be higher than in UI. Moreover, at the sixth minute of HI, HR was significantly higher. This result can be explained by a delay between the release of norepinephrine, its fixation on the endomyocardial ␤receptors, and the subsequent chronotropic response. This hypothesis would explain in part why, during the first phases of recovery, a rapid decrease in norepinephrine concentration was observed without the same change in HR in OHT patients. [START_REF] Pe ´rini | Kinetics of heart rate and catecholamines during exercise in humans[END_REF] Furthermore, the gain in HR at the start of exercise during HI seemed to persist until the end of exercise, as indicated by the significantly higher HRR in HI vs. UI.

This study shows that it was possible to increase HRR in heart transplant patients. This enhanced HRR was not followed by higher aerobic power, because peak V ˙O2 was not statistically different between HI and UI. However, HRR may still be linked to the limitation in aerobic performance, because the percentage of change in HR from rest to peak exercise is correlated with peak V ˙O2 , and patients with a high percentage of change in HR (Ͼ40%) have a higher peak V ˙O2 than patients with a low percentage of change (Ͻ40%). [START_REF] Kemp | Association of resting heart rate and chronotropic response[END_REF] Because OHT patients generally have a low HRR, 4 in our study the mean increase in this variable during HI (7 beats/min) was high when expressed as a relative value, i.e., as a percentage (20%). These results are in agreement with previous data from our group that were obtained during a longitudinal study, which found a peak HR increase with postsurgery time, without an increase in peak V ˙O2 . [START_REF] Mercier | Influence of post-surgery time after cardiac transplantation on cardiorespiratory responses during exercise[END_REF] This peak V ˙O2 was symptom limited in the two exercise conditions, at 77.4 Ϯ 3.2% and 76.4 Ϯ 5.7% of predicted in HI vs. UI. Because the relation between cardiac output and oxygen consumption is linear in OHT patients, [START_REF] Jensen | Exercise hemodynamics and oxygen delivery measurements using rebreathing techniques in heart transplant patients[END_REF] this indicates that whatever the HR, cardiac output remained unchanged. Some studies have suggested that the inability of the denervated heart to achieve the same HR level as the innervated heart may be responsible for the decreased oxygen uptake observed in OHT patients. [START_REF] Savin | Cardiorespiratory responses of cardiac transplant patients to graded, symptom-limited exercise[END_REF][START_REF] Ehrman | Cardiovascular responses of heart transplant recipients to graded exercise testing[END_REF][START_REF] Kao | Allograft diastolic dysfunction and chronotropic incompetence limit cardiac output response to exercise two to six years after heart transplantation[END_REF] Our data, in agreement with a study by Jensen et al., [START_REF] Jensen | Exercise hemodynamics and oxygen delivery measurements using rebreathing techniques in heart transplant patients[END_REF] indicate that factors other than the limited and sluggish increase in HR may influence the exercise limitation in OHT patients. First, we and other authors recently demonstrated that the abnormal pulmonary diffusion capacity found in many OHT patients was associated with the peak V ˙O2 . [START_REF] Ville | Exercise tolerance in heart transplant patients with altered pulmonary diffusion capacity[END_REF][START_REF] Oa | Exercise intolerance following heart transplantation: the role of pulmonary diffusing capacity impairment[END_REF] Second, the exaggerated skeletal deconditioning of OHT patients caused by extended periods of inactivity both before and after surgery 7 may be implicated. Third, a detrimental effect of immunosuppressive therapy on mitochondrial skeletal muscle respiration has been reported in rats. [START_REF] Hokanson | Cyclosporine A decreases skeletal muscle mitochondrial respiration in vitro[END_REF][START_REF] Kimberg | Cortisone-induced alterations in mitochondrial function and structure[END_REF][START_REF] Mercier | Effects of cyclosporine A on skeletal muscle mitochondrial respiration and endurance time in rats[END_REF] A detrimental effect of immunosuppressive therapy on vasodilatory capacity (already impaired in humans after heart transplantation) has also been suggested. [START_REF] Braith | Exercise following heart transplantation[END_REF][START_REF] Andreassen | Effect of heart transplantation on impaired peripheral microvascular perfusion and reactivity in congestive heart failure[END_REF][START_REF] Andreassen | Exercise capacity in transplant recipients: relation to impaired endothelium-dependent vasodilatation of the peripheral microcirculation[END_REF] Fourth, Jensen et al. [START_REF] Jensen | Exercise hemodynamics and oxygen delivery measurements using rebreathing techniques in heart transplant patients[END_REF] showed that OHT patients are able to maintain their oxygen-carrying capacity within normal range, and persistent skeletal muscle abnormalities have been found in these patients. [START_REF] Stratton | Effects of cardiac transplantation on bioenergetic abnormalities of skeletal muscle in congestive heart failure[END_REF][START_REF] Mettauer | Persistent exercise intolerance following cardiac transplantation despite normal oxygen transport[END_REF] Furthermore, the maximal arteriovenous oxygen difference seems to be low in heart transplant recipients. [START_REF] Kao | Allograft diastolic dysfunction and chronotropic incompetence limit cardiac output response to exercise two to six years after heart transplantation[END_REF] These last studies seem to indicate a peripheral limitation during exercise. In our study, all patients stopped exercise because of leg pain and fatigue. Moreover, because our results indicate a cardiac output response adapted to the level of exercise, the hypothesis of a peripheral limitation seems to be increasingly pertinent.

In conclusion, our study clearly demonstrates that a specific warm-up can enhance HRR during a graded, symptom-limited exercise test in OHT patients. This warm-up resulted in a higher increase in catecholamine levels during submaximal exercise, which was followed, after a slight delay, by an enhancement of HR. However, because aerobic power, as determined by peak V ˙O2 values, was similar between the high warm-up protocol and the usual one, whereas the HRR was 20% enhanced with the HI, more important mechanisms are probably involved in the limitation of exercise tolerance in these patients. Although it is worthwhile in itself to try to normalize HR response to exercise by using a specific warm-up, studies are needed to further investigate the role of pulmonary and especially peripheral factors that limit exercise capacity. A better understanding of these factors is essential for the prescription of physical training programs that can optimize exercise tolerance.

Figure 1 :

 1 Figure 1: Norepinephrine concentration changes from rest to the third minute of exercise ([NE] 3 ϭ third minute of exercise value Ϫ resting value) to the sixth minute of exercise ([NE] 6 ϭ sixth minute of exercise value Ϫ resting value) and to peak exercise (Delta [NE] ϭ peak exercise value Ϫ resting value) for the high-intensity warm-up test (HI warm-up ϭ 50 W for 6 min) and the usualintensity warm-up test (UI warm-up ϭ 20 W for 3 min). *P Ͻ 0.05 between HI and UI; #P Ͻ 0.05 within the group. The other compared values were not significantly different.

Figure 2 :

 2 Figure 2: Time course of heart rate during submaximal exercise. *P Ͻ 0.05 and **P Ͻ 0.01 between the high-intensity warm-up test (HI warm-up ϭ 50 W for 6 min) and the usual-intensity warm-up test (UI warm-up ϭ 20 W for 3 min).

Figure 3 :

 3 Figure 3: Peak oxygen uptake (V ˙O2 ) and heart rate reserve (peak exercise Ϫ resting heart rate) for the high-intensity warm-up test (HI warm-up ϭ 50 W for 6 min) and the usual-intensity warm-up test (UI warm-up ϭ 20 W for 3 min). *P Ͻ 0.05 between HI and UI.

TABLE 1

 1 Cardiorespiratory values for the two tests

		Rest	Peak Exercise
		HI	UI	HI	UI
	HR (beats ⅐ min Ϫ1 )	101.1 Ϯ 3	101.1 Ϯ 3	143 Ϯ 5	136 Ϯ 5
	V ˙O2 (ml ⅐ kg Ϫ1 ⅐ min Ϫ1 )	4.8 Ϯ 0.4	5 Ϯ 0.2	23.2 Ϯ 1.6	23 Ϯ 1.8
	HI, high-intensity workload; UI, usual-intensity workload; HR, heart rate;
	V ˙O2 , oxygen uptake.

ACKNOWLEDGMENTS

We thank the medical teams for their technical help and all the patients who participated in this study.

Supported by a grant from the "Centre Hospitalo-Universitaire" (Contrat de recherche clinique DGS 950187).


A Piccialli
email: arianna.piccialli@aeronomie.be

A C Vandaele

S Robert

F Daerden

S Viscardy

L Neary

S Aoki

A test case: new retrievals of ozone at the terminator on Mars

ASIMUT, the BIRA-IASB radiative transfer code, was modified in order to take into account the changes in the atmospheric composition and structure across the martian day/night terminator. Here, we will discuss the impact of this implementation on the retrievals of ozone profiles derived from SPICAM solar occultations in the ultraviolet.

Introduction

The martian atmosphere at the day-night terminator is a region of great interest characterized by gradients of density and temperature, driven by differences in the solar illumination, and by sharp transitions in the chemical regime. Ozone, in particular, displays rapid changes due to photolysis across the terminator [START_REF] Lefèvre | Heterogeneous chemistry in the atmosphere of Mars[END_REF]. Nowadays, most of the retrieval algorithms for solar and stellar occultations rely on the assumption of a spherically symmetrical atmosphere. In order to handle concentration gradients along the line of sight it is necessary therefore to improve the retrieval scheme used to analyse occultation observations.

Observations

SPICAM (SPectroscopie pour l'Investigation des

Caractéristiques Atmosphériques de Mars), on board the ESA's mission Mars Express, is a remote sensing spectrometer observing in the ultraviolet (118-320 nm) and in the near infrared (1-17 μm) [START_REF] Bertaux | SPICAM on Mars Express: Observing modes and overview of UV spectrometer data and scientific results[END_REF]. In the solar occultation mode, the UV sensor is particularly well suited to measure the vertical profiles of O 3 and aerosols of the martian atmosphere [START_REF] Määttänen | A complete climatology of the aerosol vertical distribution on Mars from MEx/SPICAM UV solar occultations[END_REF].

Retrieval technique

SPICAM-UV spectra are simulated using the line-byline radiative transfer code ASIMUT-ALVL developed at IASB-BIRA [START_REF] Vandaele | Composition of the Venus mesosphere measured by SOIR on board Venus Express[END_REF]. ASIMUT has been modified in order to take into account the atmospheric composition and structure at the daynight terminator. Three different gradients along the line of sight (LOS) can be considered: temperature, total density gradients and the variations of the concentration of specific species. As input for ASIMUT, we used gradients predicted by a 1D model with high temporal resolution [START_REF] García Muñoz | Airglow on Mars: Some model expectations for the OH Meinel bands and the O2 IR atmospheric band[END_REF] driven by the 3D GEM-Mars v4 Global Circulation Model (GCM) [START_REF] Daerden | A solar escalator on Mars: Self-lifting of dust layers by radiative heating[END_REF][START_REF] Neary | The GEM-Mars General Circulation Model for Mars: Description and Evaluation[END_REF]. Figures 2 and3 show examples of temperature and density profiles obtained around the terminator by the GEM-GCM.

Summary and Conclusions

We will apply the ASIMUT improved retrieval scheme to SPICAM-UV solar occultation data, focusing on ozone (O 3 ). The main objective is to test the impact of these gradients on ozone retrievals. Results of this study will then be used for the analysis of the data expected from the NOMAD instrument on the ExoMars 2016 Trace Gas Orbiter.

  Figure 1 displays transmission spectra obtained at different altitudes for the observation 05555A02. The signal below 200 nm has a poor S/N ratio due to the low emission of the Sun at those wavelengths. The ozone absorption band (Hartley band) is clearly visible around 250 nm.

Figure 1 :

 1 Figure 1: Example of SPICAM transmission spectra at different altitudes: (blue) low altitudes; (red) high altitudes.

Figure 2 :

 2 Figure 2: Vertical temperature profiles obtained by the GEM-GCM. The reference profile (yellow) is obtained at the terminator; the average profile (green) is the mean of the day (red) and night (blue) side profiles.

Figure 3 :

 3 Figure 3: Vertical CO 2 mixing ratio profiles obtained by GEM-GCM. Colours code as in Fig. 2

Acknowledgements

The research leading to these results has received funding from the European Union's Horizon 2020 Programme (H2020-Compet-08-2014) under grant agreement UPWARDS-633127. SA has been supported by the FNRS "CRAMIC" project under grant agreement n° T.0171.16