Muscle carnitine
after strenuous endurance exercise
JACQUES DECOMBAZ, BERNARD AND PAOLO CERRETELLI
GMUENDER,
GEORGES
SIERRO,
Nestec Limited, Research Centre, Vers-chez-les-Blanc, CH-1000 Lausanne 26; and Department of Physiology, School of Medicine, University of Geneva, CH-1200 Geneva, Switzerland DECOMBAZ, JACQUES, BERNARD GMUENDER, GEORGES SIERRO,ANDPAOLOCERRETELLI. Musclecarnitineafterstrenuous endurance exercise.J. Appl. Physiol. 72(Z): 423-427,1992.The effect of very long enduranceexercise on musclecarnitine was studied. Eighteen cross-country skiers took part in a race in the Alps (average inspired partial pressure of 0, 100-110 Torr) that lasted on average 13 h 26 min. Carnitine intake, evaluated for 2 wk before the event, was 50 t 4 (SE) mg/day. Muscle (vastus lateralis) total carnitine concentration, measuredtwice with a 2-yr interval on eight rested subjects,did not change with time (17 vs. 16 pmol/g dry wt, NS) but showed consistent interindividual differences (range 12-22, P = 0.001) with no correlation with intake. After exercise, total muscle carnitine was unaltered (from 17.9 ~fi1.0 at rest to 18.3 t 0.8 pmol/g dry wt postexercise in the 15 subjectswho completed the race, NS), but muscle free carnitine decreased20% (from 14.9 t 0.8 pmol/g, P = 0.01) and short-chain acylcarnitine increased108%(from 3.5 t 0.4 pmol/g, P = 0.01). These results suggestthat carnitine deficiency will probably not result from strenuous aerobic exercise in trained subjectswho consumea moderate amount of carnitine in their food. L-carnitine; diet; altitude
MUSCLETISSUErepre!%%h -98%
of the whole body pool of L-carnitine. Because carnitine synthesis does not occur in the muscle tissue, the latter is totally dependent on cellular uptake, release, and interorgan transport of free and acylated carnitine (29). Carnitine is essential for the translocation of activated long-chain fatty acids (FA) into mitochondria (29). This and other functions, such as the export of short-chain acyl groups out of the mitochondrial space and trapping of unphysiological acyl groups, have necessitated reconsideration of the role of carnitine in physical exercise (see Refs. 7 and 32 for review). Moreover it has been hypothesized that naturally occurring carnitine concentration in muscle could limit athletic performance (7) because I) carnitine concentration in human muscle is in the range expected to substantially limit the capacity for FA oxidation (23), 2) physical training seems to increase carnitine levels less than mitochondrial enzyme activities including carnitine-palmityltransferase activity (3O), and 3) Lcarnitine supplementation has had a positive influence in situations in which lipolytic flux is very high (29). On the other hand, Lennon et al. (22) reported a 20% decrease in carnitine concentration in human muscle after 40 min of exercise. Later studies showed that total carnitine in muscle was unaltered after 4 min (14) but was reduced after 10 min of high-intensity exercise (15);
in the latter study, however, no further decrease was observed after 20 min of exercise. Where endurance exercise is concerned, no change was observed after 60 (15), 90 (4), or 230 min (17). Except for the above study (22), a net increase in short-chain acylcarnitine in muscle and/or blood and a decrease in free carnitine during exercise have been reported in humans and animals by a majority of authors (4,13,X& l&33), even after short exercise bouts (2 min) (14). In view of the increased urinary excretion of carnitine after prolonged exercise reported by some (1,4) but not all authors (15, 33), there could still be a net loss of carnitine, and this could be detrimental to performances in extreme conditions. According to Rebouche et al. (28), the body’s capacity to synthesize carnitine is hardly sufficient to maintain tissue and fluid levels in adults. Because differences in the response pattern of muscle carnitine to exercise with different carnitine intakes have been reported in the rat (8, 26), it. is important to assess the contribution of dietary carnitine to carnitine status when energy turnover is increased by exercise. This, to our knowledge, has so far not been done on humans (4,14,15,17,22). Long strenuous exercise depends largely on lipid sources. Moreover, physical training increases reliance on intramuscular triglycerides relative to peripheral FA extraction (16), so that local transport factors may become critical. It has also been suggested that exercise under hypoxic conditions might increase the dependence of energy metabolism on the adequacy of carnitine concentration (7). For this reason, we measured muscle carnitine concentration in trained subjects before and after strenuous long-distance exercise at high altitude. We also examined the stability of the resting value over 2 yr, attempting to relate muscle carnitine with dietary carnitine intake. MATERIALS AND METHODS Subjects. Eighteen male endurance skiers [mean maximum oxygen uptake (VO 2max) 62 ml/kg body wt, range 51-721 took part in the study (Table 1). They were competing as six three-man teams in the Patrouille des Glaciers 1988 (i.e., “1988 Glaciers’ Patrol”). The protocol was approved by the Ethical Committee of the Geneva University Hospital. Endurance exercise. The Glaciers’ Patrol, which takes place in Switzerland in the spring every second year, is an alpine ski race that starts in Zermatt (1,600 m above sea level) between 10 P.M. and 2 A.M. and finishes in Verbier
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423
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424 TABLE
MUSCLE
CARNITINE,
1. Subject characteristics n
Age, Yr
Weight, kg Body mass index, kg/m2 Training level, miniday”
17 17 17 16
Mean + SE 34.1tl.4 68.5d.8 22.4t0.6 107tl5
Range 24-42 59.7-88.4 X4-26.1 12-200
n, No. of subjects. * Average of 2 wk during month before the race; the subject training an average of 12 miniday did not finish the race.
(1,500 m) the next day. The course is 53-km long at an average altitude of -2,500 m (inspired partial pressure of 0, of 100-110 Torr) with two peak elevations of up to 3,650 and 3,300 m, respectively, and the profile is very mountainous (total ascent 4,000 m, total descent 4,100 m). The equipment, including that needed for safety and survival on the glaciers, weighs between 10 and 12 kg. In 1988, two-thirds of the 600 participants finished the race. The average time of the parties participating in the study was 13 h 26 min (i.e., between 9 h 23 min and 16 h 20 min). Three subjects did not finish. With use of the linear regression between heart rate and 0, consumption (as determined in a laboratory preand postrace test) and heart rates obtained over the whole race from continuous records in 10 subjects, the energy cost of the entire event was estimated at 38 MJ (9,100 kcal) at an average fraction of 72% of vozmax. Food intake during training. During the month before the race, each subject was told how to record his total food consumption in a diary (with the help of kitchen scales) for 13.3 (range 7-16) consecutive days. The records were reviewed with the subjects to clarify incomplete information. Diet composition was calculated using manufacturers’ data and food tables (11,27). Food carnitine is not figured in standard tables and was therefore calculated with published values (2, 24) and completed with analytic values when data were unavailable (Table 2). During the race, food records (1,770 t 225 kcal, 82 t 2% carbohydrate energy) indicated a negligible intake of carnitine (range O-12 mg), mostly from milk-based products. Procedure. Percutaneous muscle biopsy was performed 4 wk before the event on the rested subjects and 30 min after the finish, after a step test (for the purpose of postrace physiological measurements), in the deep part of the vastus lateralis muscle at midfemoral length, with use of Bergstrom’s needle and the aspiration technique (10). The muscle samples were immediately frozen in liquid nitrogen. They were later cleaned of blood residues, weighed (mean wet wt 74 mg), freeze-dried, reweighed, and ground in a mortar. Particles of connective tissue were carefully removed. One sample (0.5-l mg dry wt) was hydrolyzed (2 h at 37°C) with 200 ~11.8 N KOH and then neutralized and analyzed for total carnitine radioenzymatically (31); free carnitine and short-chain (acid-soluble) acylcarnitine were determined in a separate muscle sample. All determinations were duplicated (mean error 5.9%). The sum of free carnitine and shortchain acylcarnitine was equal to total carnitine within 4%; long-chain (acid-insoluble) acylcarnitine, a very small fraction of total carnitine, was not specifically
EXERCISE,
AND DIET
measured. The accuracy of sample preparation and analysis was cross-checked with muscle samples of known carnitine concentration measured by C. Rijssle (gift from C. Pichard, University of Lausanne). Noncollagen muscle protein was analyzed by Lowry’s method. Two years earlier, before the Glaciers’ Patrol 1986, total muscle carnitine was also measured at rest, by the same procedure, in eight of the same subjects. The race was interrupted because of bad weather conditions, and postrace values are therefore not available. For a separate investigation on the role of the antioxidant status on blood indexes after a hypoxic stress, half the subjects of the present study received a placebo and the other half received 600 mg vitamin E daily for 4 wk before the event. Because there was no difference between groups in either initial muscle carnitine or in the evolution of free and short-chain acylcarnitine during exercise (lowest P value = 0.55), they were pooled for the purpose of this report. Statistics. Where appropriate, pre- and postrace data were compared using Student’s t test or paired t test. Muscle carnitine data obtained at a 2-yr interval in the rested subjects were compared by two-way (subject X yr) analysis of variance. Confidence limits were set at P = 0.05, P = 0.01, and P = 0.001. RESULTS
Energy and carnitine intake. During 2 wk of the training period, energy intake was 176 (119-242) kJ kg-l. day-l (42 kcal kg-l day-l). At the same time, carnitine intake ranged from 21 to 81 mg/day, and iron (a cofactor required for carnitine biosynthesis) intake ranged from i3 to 29 mg/day (Table 3). To a large extent, protein was derived from cereals and dairy products, and meat was not consumed every day. The interindividual variation coefficient in carnitine intake (30%) was not reduced when it was expressed either per kilogram of body weight (31%) or per gram of protein intake (35%). Day-to-day variation of carnitine intake within subjects was higher than other nutrients (Fig. 1). Effect of time and diet on muscle carnitine. Total carnitine concentration in the vastus lateralis muscle at rest was the same in a 2-yr interval among subjects having undergone both measurements (n = 8): 17.1 t 1.58 pmoll g dry wt in 1988 vs. 15.9 t 1.17 in 1986 (NS). On the other hand, the between-subject variance was large (P = l
TABLE
l
l
2. Total carnitine in selectedfood items Carnitine Content, mg/lOO g Edible
Beef (sirloin) Pork (neck) Protein bar (Nestle Trimmi, milk based) Sweetened condensed milk (Migros) Sea perch Yogurt (whole, natural) Danish blue cheese (50% fat) Shrimp Swiss Gruyere cheese Dutch Gouda cheese (Babybel) Values determined by radioenzymatic lysis (31). 1 mmol = 161.2 mg.
26.1 22.7 3.3 1.9 1.3
0.79 0.54 0.30 0.27 0.23
analysis after alkaline hydro-
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MUSCLE
TABLE
3. Daily food and carnitine
CARNITINE,
EXERCISE,
425
AND DIET
intake Energy*
MJ
Mean t SE Range
kcal
11.8t0.5
%Protein
2,817+124
%Lipid
13.8t0.2
%CHO
35.7tl.3
50.5tl.6
Iron, mg 17.ltl.O
13-29
Total Carnitine, mg
50.2t3.7 21431
Values are averages of 17 subjs for 2 wk during the month before the race. CHO, carbohydrate. * Ethanol (0.75*0.28% of energy) not included.
O.OOl),with some individuals as high as others (Fig. 2).
after 1 yr of training for a marathon (17), the present invariance of individual values over 2 yr in trained subjects as well as the lack of correlation between muscle carnitine and maximal aerobic power (or duration of training) indicate that endurance conditioning has little effect on skeletal muscle carnitine levels in humans. This is consistent with the lack of (20) or the relatively small (26, 30) training effect observed in laboratory animals. Even though after training red muscle of rats is reported to contain more carnitine than white muscle (12, 18), independent factors are likely to affect muscle carnitine more than endurance training. For instance, alcoholic patients, presumed free from carnitine-linked pathology, have been found to have 84% higher total carnitine concentration in the quadriceps femoris muscle than controls (P = 0.02) (9). Normal daily carnitine intake for individuals consuming a mixed Western diet is estimated at 50-100 mg (29) but may vary from ~0 to 500 mg depending on the diet because of the selectivity of carnitine distribution in foods. The wide variability of day-to-day carnitine intake (Fig. 1) is a reflection of this selective occurrence, as well as of the fact that meat was not consumed daily. Actual reports of intake are scarce in the literature because of the paucity of food carnitine data. However, the carnitine intake of the skiers in our study (50 mg/day) should be as representative of their customary intake as possible, because it was obtained over 2 wk and made use of an enlarged database for carnitine in foodstuffs. Nevertheless, some reservation is suggested in view of the unreliability of early analytic values for food carnitine, as raised by Mitchell (24). In agreement with results of Lennon et al. (22), the
having values nearly twice
There was no correlation between total muscle carnitine and either dietary carnitine (mg/day; r = 0.10) or dietary protein (g . kg-l day-l; r = 0.10). The correlation was not improved if carnitine intake was expressed as milligrams per kilograms per day (r = 0.04) or protein intake as grams per day (r = 0.02). Aerobic capacity (ml OJkg) did not correlate with total muscle carnitine (r = 0.00, n = 8); nor did age, duration of training, or iron intake. Effect of exercise on muscle carnitine. The effect of strenuous endurance exercise on total carnitine and carnitine fractions in the muscle is shown on Fig. 2 and Table 4. Total carnitine on average was unchanged and showed no consistent response from one individual to another. Free carnitine decreased 20% (P = 0.01) and short-chain acylcarnitine increased 108% (P = 0.01) as a result of exercise (Table 4). The correlation between the drop of the one and the rise of the other was, however, weak (r = 0.23). The ratio of short-chain acylcarnitine to total soluble carnitine rose from 19 to 37% (P = 0.001). There was no correlation between total carnitine concentration in muscle at rest and finishing time (r = 0.01). Exercise reduced muscle water content from 81.3 t 0.6% (n = 18) to 78.4 t 0.5% (n = 15; P = 0.01). l
DISCUSSION
In this group of trained skiers, total muscle carnitine at rest was in the range observed for human values by others (4,6,14,15,17,21). If anything, they were on the low side. Together with data available from Morgan et al. (25) and data showing no change in a group of subjects Coefficient of variation (percent)
.
.
.
.
-
. t
.
I
.
-8
FIG. 1. Median, middle 50% (central box), and lower and upper quartiles (whiskers) of dayto-day (within subject) variation in nutrient intake of 17 subjects. Daily carnitine intake is the most variable of major dietary constituents. Mean duration of food records was 13.3 days.
.
.
I
OL 1 WATER PROTEINS
.
I
I
I
I
I
FAT
ENERGY
flBRE
SUGARS
CARNITINE
I
CAfBOHYDRATE
IRON
BEVERAGES
I ST’ARCH
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426
MUSCLE CARNITINE,
Muscle
total carnitine
20
10
A
B
C
0 FIG. 2. Total muscle carnitine concentration in red vastus lateralis muscle of individual subjects at a %yr interval and after strenuous endurance exercise. Data common to each individual are connected by a line. A: rested, spring 1986; B: rested, spring 1988; C: postrace, 1988.
lack of correlation between carnitine intake and muscle earn .itine status (Fig. 2) supports the concept that renal conservation and/or turnover rate are important mechanisms in maintaining carnitine homeostasis in healthy individuals, whereas within large limits dietary intake is not. Thus dietary carnitine cannot explain why these trained individuals were characterized by a large variability in muscle carnitine concentration (the between-subject coefficient was 31.6%). Perhaps the most significant finding of this work is the stability of total carnitine in the muscle after such a long and demanding exercise. The energy turnover was maintained at a relatively high load (72% VO, m,) as assessed by the heart rate. The reduced 0, pressure at high altitude added-a hypoxic stress to the exertion. Skiing uphill and downhill in a difficult terrain with a backpack and at racing pace for >l3 h was bound to use up muscle glycogen practically , from the whole body. The contribution of carbohydrates to the energy expenditure of the race, adding a mean intake of 353 g (unpublished data) to an estimated utilization of 850 g of glycogen from muscles (25 kg, 3% glycogen) and liver (100 g), was 20.1 MJ (4,800 kcal). Because the estimated average cost of the exercise was 38 MJ (9,100 kcal) and accounting for -10% of the energy derived from proteins, -350 g of fat must have been used up. With the assumption that 90% (i.e., 1,100 mmol FA) of this fat was oxidized in muscle, the pool of muscle carnitine must have been acylated &fold during the race (1,100 mmol FA 25 kg muscle-l 3.33 mmol total carnitine-l -13.4 h-l), with no net loss. It is unlikely that acylcarnitine accumulated in muscle at the finish might have escaped later during recovery, because unchanged total carnitine levels have been found 60 min postexercise (15) an .d 4-5 h after a marathon (17) . Recent measuremen .ts of femoral arteriovenous differences (33) seem to exclude muscle as a potential source for the rise in plasma and urinary acylcarnitine, whe Ireas there is a release of free carnitine from muscle to plasma. These data corroborate exchange meal
l
EXERCISE, AND DIET
surements during fasting, showing a net release of free carnitine and a net uptake of short-chain acylcarnitine by muscle (3). To support this, we only found a poor correlation between the increase of acylcarnitine and the decrease of free carnitine within the muscle. This suggests that, in addition to acylation in situ, there may be significant interorgan exchange in the form of an efflux of free carnitine from the muscle and an influx of acetylcarnitine from the liver [where it may be raised after acute exercise (26)] to the muscle. Therefore the persistently elevated urinary carnitine excretion along with large energy deficits and exercise (1) would not be at the expense of muscle carnitine. Because muscle carnitine is -70-fold that in the plasma (29), part of the decrease of free carnitine in muscle may be explained by passive leakage related to membrane damage. In fact, activity of the muscle-specific creatine phosphokinase rose fivefold in blood between rest and finish (not shown), suggestive of some cell damage. However, because other studies show that plasma free carnitine is also depressed in comparable situations, active interorgan transport mechanisms are likely to override passive leakage. The increased muscle short-chain acylcarnitine and acylated-to-free ratio after exercise reported here, as well as by others, in humans (4, 15), horses (13), and rats (5, 18) contrast with other data in rats, in which exercise induced either no change (26) or a significant reduction in short-chain acylcarnitine, particularly in the deep vastus lateralis (20) and the deep red quadriceps femoris and the soleus (8). Total muscle carnitine concentration in the rat is 4- to 5-fold smaller than in humans and 7- to lo-fold smaller than in the horse, so differences in metabolism can be expected. Lennon and Mance (20) suggested that reduced short-chain forms in red muscle may reflect an increased diversion of free carnitine toward the formation of long-chain acylcarnitine, because in this type of fiber particularly, there is a great flux through the tricarboxylic acid cycle during periods of increased energy need. The present results, showing unchanged total carnitine in skeletal muscle, may not apply to the heart. The heart normally contains large amounts of carnitine; carnitine acyl transfer is of particular importance for cardiac tissue (a single beat consumes more than the intramitochondrial acetyl-CoA content existing at any given
4. Muscle carnitine endurance exercise
TABLE
Free Short-chain acylatedt SCYTASC Total+
after 13 h of strenuous
At Rest
Postexercise
Paired Increase
P*
14.9t0.81 (14.8kO.70) 3.5k0.36 (3.7kO.32) 0.19t0.02 17.9kO.98 (18.OkO.85)
12.OzkO.70
-2.9220.84
X0.01
7.5kl.03
+4.01t1.09
after strenuous endurance exercise
JACQUES DECOMBAZ, BERNARD AND PAOLO CERRETELLI
GMUENDER,
GEORGES
SIERRO,
Nestec Limited, Research Centre, Vers-chez-les-Blanc, CH-1000 Lausanne 26; and Department of Physiology, School of Medicine, University of Geneva, CH-1200 Geneva, Switzerland DECOMBAZ, JACQUES, BERNARD GMUENDER, GEORGES SIERRO,ANDPAOLOCERRETELLI. Musclecarnitineafterstrenuous endurance exercise.J. Appl. Physiol. 72(Z): 423-427,1992.The effect of very long enduranceexercise on musclecarnitine was studied. Eighteen cross-country skiers took part in a race in the Alps (average inspired partial pressure of 0, 100-110 Torr) that lasted on average 13 h 26 min. Carnitine intake, evaluated for 2 wk before the event, was 50 t 4 (SE) mg/day. Muscle (vastus lateralis) total carnitine concentration, measuredtwice with a 2-yr interval on eight rested subjects,did not change with time (17 vs. 16 pmol/g dry wt, NS) but showed consistent interindividual differences (range 12-22, P = 0.001) with no correlation with intake. After exercise, total muscle carnitine was unaltered (from 17.9 ~fi1.0 at rest to 18.3 t 0.8 pmol/g dry wt postexercise in the 15 subjectswho completed the race, NS), but muscle free carnitine decreased20% (from 14.9 t 0.8 pmol/g, P = 0.01) and short-chain acylcarnitine increased108%(from 3.5 t 0.4 pmol/g, P = 0.01). These results suggestthat carnitine deficiency will probably not result from strenuous aerobic exercise in trained subjectswho consumea moderate amount of carnitine in their food. L-carnitine; diet; altitude
MUSCLETISSUErepre!%%h -98%
of the whole body pool of L-carnitine. Because carnitine synthesis does not occur in the muscle tissue, the latter is totally dependent on cellular uptake, release, and interorgan transport of free and acylated carnitine (29). Carnitine is essential for the translocation of activated long-chain fatty acids (FA) into mitochondria (29). This and other functions, such as the export of short-chain acyl groups out of the mitochondrial space and trapping of unphysiological acyl groups, have necessitated reconsideration of the role of carnitine in physical exercise (see Refs. 7 and 32 for review). Moreover it has been hypothesized that naturally occurring carnitine concentration in muscle could limit athletic performance (7) because I) carnitine concentration in human muscle is in the range expected to substantially limit the capacity for FA oxidation (23), 2) physical training seems to increase carnitine levels less than mitochondrial enzyme activities including carnitine-palmityltransferase activity (3O), and 3) Lcarnitine supplementation has had a positive influence in situations in which lipolytic flux is very high (29). On the other hand, Lennon et al. (22) reported a 20% decrease in carnitine concentration in human muscle after 40 min of exercise. Later studies showed that total carnitine in muscle was unaltered after 4 min (14) but was reduced after 10 min of high-intensity exercise (15);
in the latter study, however, no further decrease was observed after 20 min of exercise. Where endurance exercise is concerned, no change was observed after 60 (15), 90 (4), or 230 min (17). Except for the above study (22), a net increase in short-chain acylcarnitine in muscle and/or blood and a decrease in free carnitine during exercise have been reported in humans and animals by a majority of authors (4,13,X& l&33), even after short exercise bouts (2 min) (14). In view of the increased urinary excretion of carnitine after prolonged exercise reported by some (1,4) but not all authors (15, 33), there could still be a net loss of carnitine, and this could be detrimental to performances in extreme conditions. According to Rebouche et al. (28), the body’s capacity to synthesize carnitine is hardly sufficient to maintain tissue and fluid levels in adults. Because differences in the response pattern of muscle carnitine to exercise with different carnitine intakes have been reported in the rat (8, 26), it. is important to assess the contribution of dietary carnitine to carnitine status when energy turnover is increased by exercise. This, to our knowledge, has so far not been done on humans (4,14,15,17,22). Long strenuous exercise depends largely on lipid sources. Moreover, physical training increases reliance on intramuscular triglycerides relative to peripheral FA extraction (16), so that local transport factors may become critical. It has also been suggested that exercise under hypoxic conditions might increase the dependence of energy metabolism on the adequacy of carnitine concentration (7). For this reason, we measured muscle carnitine concentration in trained subjects before and after strenuous long-distance exercise at high altitude. We also examined the stability of the resting value over 2 yr, attempting to relate muscle carnitine with dietary carnitine intake. MATERIALS AND METHODS Subjects. Eighteen male endurance skiers [mean maximum oxygen uptake (VO 2max) 62 ml/kg body wt, range 51-721 took part in the study (Table 1). They were competing as six three-man teams in the Patrouille des Glaciers 1988 (i.e., “1988 Glaciers’ Patrol”). The protocol was approved by the Ethical Committee of the Geneva University Hospital. Endurance exercise. The Glaciers’ Patrol, which takes place in Switzerland in the spring every second year, is an alpine ski race that starts in Zermatt (1,600 m above sea level) between 10 P.M. and 2 A.M. and finishes in Verbier
0161-7567192 $2.00 Copyright 0 1992 the American Physiological
Society
423
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424 TABLE
MUSCLE
CARNITINE,
1. Subject characteristics n
Age, Yr
Weight, kg Body mass index, kg/m2 Training level, miniday”
17 17 17 16
Mean + SE 34.1tl.4 68.5d.8 22.4t0.6 107tl5
Range 24-42 59.7-88.4 X4-26.1 12-200
n, No. of subjects. * Average of 2 wk during month before the race; the subject training an average of 12 miniday did not finish the race.
(1,500 m) the next day. The course is 53-km long at an average altitude of -2,500 m (inspired partial pressure of 0, of 100-110 Torr) with two peak elevations of up to 3,650 and 3,300 m, respectively, and the profile is very mountainous (total ascent 4,000 m, total descent 4,100 m). The equipment, including that needed for safety and survival on the glaciers, weighs between 10 and 12 kg. In 1988, two-thirds of the 600 participants finished the race. The average time of the parties participating in the study was 13 h 26 min (i.e., between 9 h 23 min and 16 h 20 min). Three subjects did not finish. With use of the linear regression between heart rate and 0, consumption (as determined in a laboratory preand postrace test) and heart rates obtained over the whole race from continuous records in 10 subjects, the energy cost of the entire event was estimated at 38 MJ (9,100 kcal) at an average fraction of 72% of vozmax. Food intake during training. During the month before the race, each subject was told how to record his total food consumption in a diary (with the help of kitchen scales) for 13.3 (range 7-16) consecutive days. The records were reviewed with the subjects to clarify incomplete information. Diet composition was calculated using manufacturers’ data and food tables (11,27). Food carnitine is not figured in standard tables and was therefore calculated with published values (2, 24) and completed with analytic values when data were unavailable (Table 2). During the race, food records (1,770 t 225 kcal, 82 t 2% carbohydrate energy) indicated a negligible intake of carnitine (range O-12 mg), mostly from milk-based products. Procedure. Percutaneous muscle biopsy was performed 4 wk before the event on the rested subjects and 30 min after the finish, after a step test (for the purpose of postrace physiological measurements), in the deep part of the vastus lateralis muscle at midfemoral length, with use of Bergstrom’s needle and the aspiration technique (10). The muscle samples were immediately frozen in liquid nitrogen. They were later cleaned of blood residues, weighed (mean wet wt 74 mg), freeze-dried, reweighed, and ground in a mortar. Particles of connective tissue were carefully removed. One sample (0.5-l mg dry wt) was hydrolyzed (2 h at 37°C) with 200 ~11.8 N KOH and then neutralized and analyzed for total carnitine radioenzymatically (31); free carnitine and short-chain (acid-soluble) acylcarnitine were determined in a separate muscle sample. All determinations were duplicated (mean error 5.9%). The sum of free carnitine and shortchain acylcarnitine was equal to total carnitine within 4%; long-chain (acid-insoluble) acylcarnitine, a very small fraction of total carnitine, was not specifically
EXERCISE,
AND DIET
measured. The accuracy of sample preparation and analysis was cross-checked with muscle samples of known carnitine concentration measured by C. Rijssle (gift from C. Pichard, University of Lausanne). Noncollagen muscle protein was analyzed by Lowry’s method. Two years earlier, before the Glaciers’ Patrol 1986, total muscle carnitine was also measured at rest, by the same procedure, in eight of the same subjects. The race was interrupted because of bad weather conditions, and postrace values are therefore not available. For a separate investigation on the role of the antioxidant status on blood indexes after a hypoxic stress, half the subjects of the present study received a placebo and the other half received 600 mg vitamin E daily for 4 wk before the event. Because there was no difference between groups in either initial muscle carnitine or in the evolution of free and short-chain acylcarnitine during exercise (lowest P value = 0.55), they were pooled for the purpose of this report. Statistics. Where appropriate, pre- and postrace data were compared using Student’s t test or paired t test. Muscle carnitine data obtained at a 2-yr interval in the rested subjects were compared by two-way (subject X yr) analysis of variance. Confidence limits were set at P = 0.05, P = 0.01, and P = 0.001. RESULTS
Energy and carnitine intake. During 2 wk of the training period, energy intake was 176 (119-242) kJ kg-l. day-l (42 kcal kg-l day-l). At the same time, carnitine intake ranged from 21 to 81 mg/day, and iron (a cofactor required for carnitine biosynthesis) intake ranged from i3 to 29 mg/day (Table 3). To a large extent, protein was derived from cereals and dairy products, and meat was not consumed every day. The interindividual variation coefficient in carnitine intake (30%) was not reduced when it was expressed either per kilogram of body weight (31%) or per gram of protein intake (35%). Day-to-day variation of carnitine intake within subjects was higher than other nutrients (Fig. 1). Effect of time and diet on muscle carnitine. Total carnitine concentration in the vastus lateralis muscle at rest was the same in a 2-yr interval among subjects having undergone both measurements (n = 8): 17.1 t 1.58 pmoll g dry wt in 1988 vs. 15.9 t 1.17 in 1986 (NS). On the other hand, the between-subject variance was large (P = l
TABLE
l
l
2. Total carnitine in selectedfood items Carnitine Content, mg/lOO g Edible
Beef (sirloin) Pork (neck) Protein bar (Nestle Trimmi, milk based) Sweetened condensed milk (Migros) Sea perch Yogurt (whole, natural) Danish blue cheese (50% fat) Shrimp Swiss Gruyere cheese Dutch Gouda cheese (Babybel) Values determined by radioenzymatic lysis (31). 1 mmol = 161.2 mg.
26.1 22.7 3.3 1.9 1.3
0.79 0.54 0.30 0.27 0.23
analysis after alkaline hydro-
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MUSCLE
TABLE
3. Daily food and carnitine
CARNITINE,
EXERCISE,
425
AND DIET
intake Energy*
MJ
Mean t SE Range
kcal
11.8t0.5
%Protein
2,817+124
%Lipid
13.8t0.2
%CHO
35.7tl.3
50.5tl.6
Iron, mg 17.ltl.O
13-29
Total Carnitine, mg
50.2t3.7 21431
Values are averages of 17 subjs for 2 wk during the month before the race. CHO, carbohydrate. * Ethanol (0.75*0.28% of energy) not included.
O.OOl),with some individuals as high as others (Fig. 2).
after 1 yr of training for a marathon (17), the present invariance of individual values over 2 yr in trained subjects as well as the lack of correlation between muscle carnitine and maximal aerobic power (or duration of training) indicate that endurance conditioning has little effect on skeletal muscle carnitine levels in humans. This is consistent with the lack of (20) or the relatively small (26, 30) training effect observed in laboratory animals. Even though after training red muscle of rats is reported to contain more carnitine than white muscle (12, 18), independent factors are likely to affect muscle carnitine more than endurance training. For instance, alcoholic patients, presumed free from carnitine-linked pathology, have been found to have 84% higher total carnitine concentration in the quadriceps femoris muscle than controls (P = 0.02) (9). Normal daily carnitine intake for individuals consuming a mixed Western diet is estimated at 50-100 mg (29) but may vary from ~0 to 500 mg depending on the diet because of the selectivity of carnitine distribution in foods. The wide variability of day-to-day carnitine intake (Fig. 1) is a reflection of this selective occurrence, as well as of the fact that meat was not consumed daily. Actual reports of intake are scarce in the literature because of the paucity of food carnitine data. However, the carnitine intake of the skiers in our study (50 mg/day) should be as representative of their customary intake as possible, because it was obtained over 2 wk and made use of an enlarged database for carnitine in foodstuffs. Nevertheless, some reservation is suggested in view of the unreliability of early analytic values for food carnitine, as raised by Mitchell (24). In agreement with results of Lennon et al. (22), the
having values nearly twice
There was no correlation between total muscle carnitine and either dietary carnitine (mg/day; r = 0.10) or dietary protein (g . kg-l day-l; r = 0.10). The correlation was not improved if carnitine intake was expressed as milligrams per kilograms per day (r = 0.04) or protein intake as grams per day (r = 0.02). Aerobic capacity (ml OJkg) did not correlate with total muscle carnitine (r = 0.00, n = 8); nor did age, duration of training, or iron intake. Effect of exercise on muscle carnitine. The effect of strenuous endurance exercise on total carnitine and carnitine fractions in the muscle is shown on Fig. 2 and Table 4. Total carnitine on average was unchanged and showed no consistent response from one individual to another. Free carnitine decreased 20% (P = 0.01) and short-chain acylcarnitine increased 108% (P = 0.01) as a result of exercise (Table 4). The correlation between the drop of the one and the rise of the other was, however, weak (r = 0.23). The ratio of short-chain acylcarnitine to total soluble carnitine rose from 19 to 37% (P = 0.001). There was no correlation between total carnitine concentration in muscle at rest and finishing time (r = 0.01). Exercise reduced muscle water content from 81.3 t 0.6% (n = 18) to 78.4 t 0.5% (n = 15; P = 0.01). l
DISCUSSION
In this group of trained skiers, total muscle carnitine at rest was in the range observed for human values by others (4,6,14,15,17,21). If anything, they were on the low side. Together with data available from Morgan et al. (25) and data showing no change in a group of subjects Coefficient of variation (percent)
.
.
.
.
-
. t
.
I
.
-8
FIG. 1. Median, middle 50% (central box), and lower and upper quartiles (whiskers) of dayto-day (within subject) variation in nutrient intake of 17 subjects. Daily carnitine intake is the most variable of major dietary constituents. Mean duration of food records was 13.3 days.
.
.
I
OL 1 WATER PROTEINS
.
I
I
I
I
I
FAT
ENERGY
flBRE
SUGARS
CARNITINE
I
CAfBOHYDRATE
IRON
BEVERAGES
I ST’ARCH
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426
MUSCLE CARNITINE,
Muscle
total carnitine
20
10
A
B
C
0 FIG. 2. Total muscle carnitine concentration in red vastus lateralis muscle of individual subjects at a %yr interval and after strenuous endurance exercise. Data common to each individual are connected by a line. A: rested, spring 1986; B: rested, spring 1988; C: postrace, 1988.
lack of correlation between carnitine intake and muscle earn .itine status (Fig. 2) supports the concept that renal conservation and/or turnover rate are important mechanisms in maintaining carnitine homeostasis in healthy individuals, whereas within large limits dietary intake is not. Thus dietary carnitine cannot explain why these trained individuals were characterized by a large variability in muscle carnitine concentration (the between-subject coefficient was 31.6%). Perhaps the most significant finding of this work is the stability of total carnitine in the muscle after such a long and demanding exercise. The energy turnover was maintained at a relatively high load (72% VO, m,) as assessed by the heart rate. The reduced 0, pressure at high altitude added-a hypoxic stress to the exertion. Skiing uphill and downhill in a difficult terrain with a backpack and at racing pace for >l3 h was bound to use up muscle glycogen practically , from the whole body. The contribution of carbohydrates to the energy expenditure of the race, adding a mean intake of 353 g (unpublished data) to an estimated utilization of 850 g of glycogen from muscles (25 kg, 3% glycogen) and liver (100 g), was 20.1 MJ (4,800 kcal). Because the estimated average cost of the exercise was 38 MJ (9,100 kcal) and accounting for -10% of the energy derived from proteins, -350 g of fat must have been used up. With the assumption that 90% (i.e., 1,100 mmol FA) of this fat was oxidized in muscle, the pool of muscle carnitine must have been acylated &fold during the race (1,100 mmol FA 25 kg muscle-l 3.33 mmol total carnitine-l -13.4 h-l), with no net loss. It is unlikely that acylcarnitine accumulated in muscle at the finish might have escaped later during recovery, because unchanged total carnitine levels have been found 60 min postexercise (15) an .d 4-5 h after a marathon (17) . Recent measuremen .ts of femoral arteriovenous differences (33) seem to exclude muscle as a potential source for the rise in plasma and urinary acylcarnitine, whe Ireas there is a release of free carnitine from muscle to plasma. These data corroborate exchange meal
l
EXERCISE, AND DIET
surements during fasting, showing a net release of free carnitine and a net uptake of short-chain acylcarnitine by muscle (3). To support this, we only found a poor correlation between the increase of acylcarnitine and the decrease of free carnitine within the muscle. This suggests that, in addition to acylation in situ, there may be significant interorgan exchange in the form of an efflux of free carnitine from the muscle and an influx of acetylcarnitine from the liver [where it may be raised after acute exercise (26)] to the muscle. Therefore the persistently elevated urinary carnitine excretion along with large energy deficits and exercise (1) would not be at the expense of muscle carnitine. Because muscle carnitine is -70-fold that in the plasma (29), part of the decrease of free carnitine in muscle may be explained by passive leakage related to membrane damage. In fact, activity of the muscle-specific creatine phosphokinase rose fivefold in blood between rest and finish (not shown), suggestive of some cell damage. However, because other studies show that plasma free carnitine is also depressed in comparable situations, active interorgan transport mechanisms are likely to override passive leakage. The increased muscle short-chain acylcarnitine and acylated-to-free ratio after exercise reported here, as well as by others, in humans (4, 15), horses (13), and rats (5, 18) contrast with other data in rats, in which exercise induced either no change (26) or a significant reduction in short-chain acylcarnitine, particularly in the deep vastus lateralis (20) and the deep red quadriceps femoris and the soleus (8). Total muscle carnitine concentration in the rat is 4- to 5-fold smaller than in humans and 7- to lo-fold smaller than in the horse, so differences in metabolism can be expected. Lennon and Mance (20) suggested that reduced short-chain forms in red muscle may reflect an increased diversion of free carnitine toward the formation of long-chain acylcarnitine, because in this type of fiber particularly, there is a great flux through the tricarboxylic acid cycle during periods of increased energy need. The present results, showing unchanged total carnitine in skeletal muscle, may not apply to the heart. The heart normally contains large amounts of carnitine; carnitine acyl transfer is of particular importance for cardiac tissue (a single beat consumes more than the intramitochondrial acetyl-CoA content existing at any given
4. Muscle carnitine endurance exercise
TABLE
Free Short-chain acylatedt SCYTASC Total+
after 13 h of strenuous
At Rest
Postexercise
Paired Increase
P*
14.9t0.81 (14.8kO.70) 3.5k0.36 (3.7kO.32) 0.19t0.02 17.9kO.98 (18.OkO.85)
12.OzkO.70
-2.9220.84
X0.01
7.5kl.03
+4.01t1.09
