Ammonia metabolism during intense dynamic exercise and recovery in humans T. E. GRAHAM, J. BANGSBO, P. D. GOLLNICK, August Krogh Institute, 2100 Copenhagen, Denmark
GRAHAM, T.E.,J. BANGSBO, P.D. GOLLNICK,C.JUEL,AND B. SALTIN. Ammonia metabolism during intense dynamic exercise and recovery in humans. Am. J. Physiol. 259 (Endocrinol. Metab. 22): El70-E176, 1990.-This study examined the dynamics for ammonia (NH3) metabolism in human skeletal muscle during and after intense one-legged exercise. Subjects (n = 8) performed dynamic leg extensor exercise to exhaustion (3.2 min). Muscle NH, release increased rapidly to a maximum of 314 t 42 pmol/min and declined immediately on cessation of exercise. Recovery was complete in -20 min. Arterial [NH,] increased less rapidly and reached its maximum 2-3 min into recovery. These data demonstrate that NH3 clearance is more sensitive to the cessation of exercise than is NH3 release from skeletal muscle. Muscle [NH,] increased three to fourfold during exercise and represented 74 t 8% of the total net NH3 formation. Thus the change in muscle [NH,] alone underestimates the NH3 production. There was no evidence that the muscle-to-venous blood NH3 ratio shifts in accordance with the H+ data. Thus other factors must contribute to the NH, release from active muscle. The total net NH3 formed corresponded with the intramuscular inosine 5’-monophosphate accumulation, suggesting that the NH3 was derived from AMP deamination. Changes in the known modulators of AMP deaminase (ATP, ADP, H’) were moderate, so the mechanisms initiating the deamination remain obscure. purine nucleotide cycle; inosine 5’-monophosphate; lactate; adenosine 5’-monophosphate deaminase; hydrogen ion
SEVERAL STUDIES of ammonia and ammonium (NH# metabolism in exercising humans have relied on the determination of the NH3 intramuscular concentration as a reflection of its production (11, 12, 16). The basis for this assumption is that ammonium crosses the sarcolemma predominantly as ammonia, and during strenuous exercise acidification of the intracellular environment could impede the conversion of ammonium to ammonia (pK, = 9.3). Indirect evidence supporting this theory is that the relationship between increases in muscle NH3 and inosine 5’-monophosphate (IMP) is approximately stoichiometric (4, 15, 16). However, blood NH3 concentration rises during exercise (5, 33)) suggesting that efflux from active muscle is occurring. The dynamics of NH3 efflux from active skeletal muscle are poorly understood as there have been very few direct measures of NH3 efflux for human skeletal muscle ’ In physiological systems both ammonia and the ion, ammonium, exist. In this paper NH3 represents the sum of both forms, and when a specific form is referred to the terms “ammonia” and “ammonium” will be used. El70
0193~1849/90
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C. JUEL,
AND
B. SALTIN
during intense exercise (8, 15). These studies made no more than one or two measurements during an entire exercise period, making it impossible to quantify accurately the total efflux. Furthermore, intramuscular NH3 and IMP were determined in only one of these investigations (15), and even then muscle pH was not measured. The same limitations exist in our understanding of the recovery processes. Thus, although the change in muscle NH, concentration during exercise is theoretically an accurate reflecti .on of N ‘H3 production, direct experimental evidence for human muscle is lacki .ng. In the present experiments, NH3 production in active human muscle was investigated using the single-leg extensor exercise model. This exercise mode allows the subject to achieve a very high power output for a given muscle mass. Thus one can study human skeletal muscle when its metabolic rate and ATP turnover rate are extreme. This should ensure a large NH3 production. This was examined by determining the changes in skeletal muscle [IMP], [NH31 and [H’] during exercise and making multiple deterrecovery and by simultaneously minations of NH, efflux from the muscle. MATERIALS
AND
METHODS
Subjects. Eight adult males aged 23-29 yr gave their informed consent to participate in the study, which was approved by the University of Copenhagen Ethics Committee. Exercise protocol. Subjects reported to the laboratory after a light breakfast and having refrained from strenuous physical activity the day before the experiment. Teflon catheters were inserted into the femoral artery and vein below the inguinal ligament of the leg to be exercised. The femoral arterial catheter was placed using the Seldinger technique with the tip l-2 cm proximal to the inguinal ligament. One of the two femoral vein catheters was placed -8 cm in the retrograde direction (distal to the inguinal ligament) for blood sampling and for infusion of ice-cold saline. The second venous catheter was placed in the inguinal region l-2 cm distal to the ligament. A thermistor was placed through this catheter just proximal to the tip of the original catheter to make leg blood flow measurements possible. The subject rested supine for 30 min after this procedure. The subject performed knee extension exercise with a modified Krogh cycle ergometer (1) with a power output (51.9-78.7 W) equivalent- to 139 t 9% of maximum oxygen consumption of (V~z,,,)for the leg extensors.
0 1990 the American
Physiological
Society
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AMMONIA
The exercise
was performed
FLUX
to exhaustion
(mean
IN
EXERCISE
AND
exercise
’
time was 3.20 min with a range of 2.15-4.94 min) after which the subjects rested quietly for 1 h. Measurements. The amount of active muscle mass
(quadriceps femoris) was estimated by Simpson’s rule, using measurements of thigh length, multiple circumferences of the thigh, and the skinfold thickness (I). This anthropometric approach has been shown to give values similar to those from estimations based on multiple computer-assisted tomography (CAT) scans (29). Values ranged from 2.54 to 3.51 kg with a mean value of 2.83
NH3
El71
RECOVERY flux
fumote/min)
~0
%’ 300
200
kg. Leg blood flow, determined by the constant-infusion
thermodilution technique (l), was estimated at rest four to six times during the exercise and lo-13 times during recovery (with the majority of the determinations conducted within the first 10 min of recovery). An occlusion cuff placed just below the knee was inflated (>220 mmHg) during the whole period of intense exercise and
100
during recovery. During the latter phase, the cuff was deflated for -30 s at 8, 25, 40, and 50 min. Immediately after
each blood
flow measurement,
blood
samples
were
collected from the femoral artery and vein for determination of oxygen saturation, hemoglobin, hematocrit,
C
lactate, and NH3.
pH,
Oxygen saturation was measured with an OSM-2 hemoximeter (radiometer), and the hematocrit was determined after high-speed centrifugation. Plasma
NH,
Lactate was de-
1
1
1.0
1.5
I
I
2.0
1
2.5
3.0
Time (minutes) 2. NH3
FIG.
1. Final
flux
2 data points
during
exercise.
represent
Figure
fewer
is organized
subjects;
same
as Fig.
SE is not given.
termined
on whole blood samples (al), and NH3 was measured on plasma by the method of Kun and Kearney (19). Leg oxygen consumption (VO,) and lactate and
(uM)
NH3 exchange were determined
by the Fick principle.
Although plasma rather than blood NH3 were measured,
it was used in combination with blood flow to estimate the total blood NH3 flux, since the absolute changes in plasma and whole blood NH3 concentrations are virtually identical during heavy exercise (3, 13, 15). Muscle biopsies from vastus lateralis were taken at rest, exhaustion, and at 3, 10, and 60 min of recovery.
These were analyzed for NH3 as described
previously
(12), for creatine phosphate (CrP), ATP, ADP, AMP, and IMP by high-performance liquid chromatography
(HPLC)
(22) an d muscle pH (25). Muscle samples were
analyzed for NH, before freeze drying. The remaining tissue was weighed before and after freeze drying to
determine
total water content.
The total lactate and NH, exchange was estimated during both exercise and recovery by averaging the efflux
values for every two consecutive time points, multiplying this Exercise 1
’
1
0
0.5
1
1.0 Time
FIG.
exercise.
1. Femoral
Data
1
1
1
L
1.5
2.0
2.5
3.0
&
(minutes)
arterial and venous plasma are means within SE represented
concentrations during by vertical bars. Open
circles and dashed line, femoral venous data; closed symbols and solid line, femoral arterial data. Both lines are drawn subjectively. Subjects exercised to exhaustion and their endurance times were all different. Thus the last 2 data points represent 5 and 4 subjects as indicated in parentheses. Samples were not taken at same time for all subjects. Thus SE for sampling time is represented by horizontal bars with arterial data. The same SE applies to venous data.
average release by the time span being considered,
and summing each of th .ese estimates fo r the entire period of exercise or recovery. During these time periods the amount
of accumulation
or depletion
of IMP
and NH3
in the muscle was estimated from the changes in the concentration of these metabolites multiplied by the amount of active extensor muscle. The concentration difference for II’ and NH3 between the muscle intracellular compartment and the interstitial fluid was calculated as follows. The venous concentrations were used as estimates of interstitial concentramuscle concentrations of NH3 and tions. The measured
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El72
AMMONIA NH3 flux
400
(umole/m
FLUX
IN
EXERCISE
AND
RECOVERY
in)
300
200
100
0 1 0
FIG.
L 1
3. NH3 flux
during
1 2 recovery.
1 I 3 4 Time(minutes) Figure
is organized
I 5
Recovery I 6
same as Fig. 2. Time,
H+ were calculated per liter of total muscle water. Then we used the data of Sjplgaard et al. (30) for the intra- and extracellular water distribution in muscle during leg extensor exercise and recovery to derive the intracellular muscle concentrations. Paired t tests were employed to determine at which time point there was a significant change from rest in arterial and venous [NH31 and in efflux. The paired t test was also used to determine whether there was a Plasma
NH,
1 7
IA,1 r, 0 ‘0
1
, 20
,
, , 1 , 30 40 Time (minutes)
, 50
,
, 60
0 min, end of exercise.
further increase in arterial exercise. In all comparisons significant if P C 0.05.
[NH31 after the cessation of differences were accepted as
RESULTS
During the first 30 s of exercise there were no significant changes in arterial or venous [NH31 (Fig. 1). By -60 s of exercise the venous [NH31 had risen signifi-
(ubl)
200
150
100
50
0
1 0
1
1 2 Time
FIG.
4. Femoral
arterial
and venous
1
I 4
Recovery I
I 6
1
I#& 0 10
1
(minutes) plasma
I 20
1
1 30
1
1 40
1
, 50
Time(minutes) concentrations
during
recovery.
Figure
is organized
same as Fig. 3.
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1
, 60
AMMONIA
FLUX
IN
EXERCISE
cantly, and the arterial [NH31 was also elevated significantly by -2.5 min. The release of NH3 increased significantly from rest at -1 min, and at exhaustion the NH3 efflux had risen to 314 t 42 pmol/min (Fig. 2). Immediately after cessation of exercise, the NH, efflux from the leg declined in a curvilinear manner such that after 3 min of recovery, the release was less than 50% of that measured at exhaustion (Fig. 3). In marked contrast, the arterial plasma [NH31 continued to rise during this initial recovery period (Fig. 4). This is not apparent in the mean data because the peak value occurred at various times into recovery (0.5-3.5 min). However, every subject had a further increase in arterial [NH31 in recovery (P < 0.05), and -25% of the total increase in arterial NH3 occurred during the initial recovery phase even though release of NH3 into the circulation was declining rapidly. At the end of the exercise the ventilation was 77.5 t 6.7 l/min; this fell to 55.2 t 5.9 l/min during the first 30 s, to 32.6 t 5.6 l/min during 30-60 s and was 21.5 t 2.2 l/ min by 3 min of recovery. After this initial recovery phase both NH3 efflux from the exercised muscle and [NH31 in arterial and venous plasma declined and approximated resting values by 2030 min (Figs. 3 and 4). The lactate release from the active leg was qualitatively similar to that of NH3, although it increased sooner and occurred to a much greater extent. Lactate efflux increased within 15-20 s after the onset of exercise from a resting value of 0.036 t 0.011 mmol/ min to 2.46 t 0.80 mmol/min, whereas NH, efflux showed no significant change over this time period. The total NH3 efflux from the leg during the entire exercise and the recovery period was 2.10 t 0.59 mmol (Table 2) while lactate release during exercise alone was calculated as 126.8 t 12 mmol. Skeletal muscle [NH31 rose three to fourfold during the exercise (Table 1). This represented -75% of the total net NH3 produced during the exercise (Table 2). During the recovery phase the muscle [NH31 returned to rest and the estimated NH3 efflux during recovery represented 88% of the accumulated muscle NH3. The majority of the NH3 released from the muscle occurred during the early part of the recovery process; 23 t 5% was released during exercise, 31 t 4 and 30 t 3% during O-3 and 3-10 min of recovery, respectively, whereas only 17 -+ 5% was released during the remaining 50 min. Even though 1,141 t 268 pmol NH3 were released into the bloodstream during exercise and the first 3 min of recovery, arterial [NH31 had a maximum increase of only 77 t 8 PM above that of rest. 1. Muscle ammonia, creatine phosphate, nucleotides, and IMP concentrations TABLE
Condition
Rest Exhaustion Ret-3 Ret-10 Ret-60
NH,
309t26 955t146 581t89 356t53 283&33
ATP
6.21kO.41 4.10t0.42 4.98rt0.34 5.57t0.17 5.85t0.40
ADP
1.22kO.10 1.21t0.09 1.12t0.11 1.02t0.04 1.05&0.08
IMP
l.lOt0.17 0.69kO.17 0.18t0.09
CrP
22.32t0.34 7.99t0.76 12.74t1.56 17.44t1.41 19.451t0.82
Values are means t SE in mmol/kg wet wt except for NH3, which is in pmol/kg wet wt. There was no detectable IMP at rest and at 60 min of recovery. Ret-3, -10, and -60 represent 3, 10, and 60 min of recovery.
AND
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RECOVERY
2. Summary of estimations of net NH3 production and release of quadriceps muscle during exercise and recovery TABLE
Calculation
Net
Exercise NH, released, pmol Accumulated muscle NH3, pmol Total net NH, production, pm01 Proportion NH3 production accumulated, % Recovery NH, released, pmol Exercise and recovery Total net NH, released, bmol
NH,
533&183 1,945+529 2,517+534 74t8
1,718+427 2,100&590
Values are means & SE. Calculation for accumulated muscle data is derived from product of measured increase in muscle [NH31 and estimated mass of active muscle (2.83 * 0.11 kg). Total net NH, production is sum of NH, released and accumulated muscle NHS. Similarly, proportion NH3 production accumulated is accumulated muscle NH3 divided by total net NH3 times 100.
3. Summary of calculations of intracellular-toextracellular ratio for NH, TABLE
Biopsy
Venous
Condition
Rest Exhaustion Ret-3 Ret-10 Ret-60
w-a%
pmol/l
309226 955U46 581t89 356*53 283*33
376-1-42 1,228+175 749t105 461-1-66 366&41
IC, pmol/l
NH3, pmol/l
419t49 1,418f208 850t122 545k94 427k49
38&17 165&14 158*15 lllt23 42tll
IC/Venous
23.7t7.3 8.9t2.1 5.4t0.7 7.3t2.2 16.3t4.2
Values are means & SE. Biopsy concentration (biopsy) was measured in pmol/kg wet wt and converted to r.Lrnol/l muscle water based on the wet-to-dry wt ratio. Intracellular concentration (IC) was calculated using data of Sjprgaard et al. (30) to estimate intracellular-to-extracellular water distribution and using measured femoral venous concentration (venous) as an approximation of extracellular concentration. Conditions are same as in Table 1.
At exhaustion muscle [CrP] declined to 36% of the resting value. Although [ADP] was unaltered, [ATP] declined and there was a comparable rise in [IMP] ([AMP] was undetectable). The increase in IMP predicted NH3 formation -20% greater than that which could be accounted for by the sum of net efflux and changes in muscle [NH31 during exercise. During recovery all metabolites returned toward resting concentrations, but even after 10 min of recovery none was back to basal values. Although there was an accumulation of NH3 in the muscle during exercise, the estimated ratio across the muscle membrane actually declined by >60% and fell even further 3 min postexercise before slowly returning to a preexercise level (Table 3). In marked contrast the ratio H+ increased -70% at exhaustion and rapidly returned to resting levels within 10 min of recovery (Table 4) . DISCUSSION
This study examined the time course of NH3 metabolism in human skeletal muscle and its relations to the purine nucleotide cycle during intense, dynamic exercise. Total NH3 production was underestimated by the change
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El74
AMMONIA
4. summary of calculations extracellular ratio for H+
TABLE
FLUX
IN
EXERCISE
of intracellular-to-
Condition
Biopsy, pmol/l
IC, pmol/l
Venous NH3, pmol/l
Rest Exhaustion Ret-3 Ret-10 Ret-60
74t1 211t22 134t17 82t5 76t5
79k2 235k26 145t20 87t7 82t6
4321 79t3 65t3 49s 42&l
IC/Venous
l&O.1 3.OkO.3 2.3k0.3 1.8kO.l 1.9t0.2
Values are means t SE. Data are derived in an identical fashion to that used for Table 3 except that ori ginal measure was pH, which was converted into [H’] (biopsy).
in muscle NH3 alone. These data demonstrate that the large efflux of NH3 was only qualitatively reflected by increased arterial and venous plasma [NH3]. The temporal mismatch between muscle NH, efflux and plasma [ NH31 during the exercise-recovery transition suggests that the major processes for removing NH3 from the plasma are very rapid. The intramuscular IMP-NH3 relationship supports the suggestion that the purine nucleotide cycle works in series, with the resynthesis portion of the cycle operating predominantly in recovery. Finally, the inability to demonstrate concomitant increases in the muscle-to-venous H+ and muscle -to-venous NH3 ratios suggests that the NH3 exchange across the sarcolemma is regulated by factors in addition to
w +1
Many studies of human subjects have used only venous (11, 1% 33) as a reflection of NH3 metabolism, and the present data demonstrate that such data can be quantitatively greater than arterial [NH3]. Of greater importance is the disparity between NH3 flux and plasma concentration. The NH3 flux has much greater rel.ative changes, and the time courses are different, with the NH3 efflux being maximal during the last stage of the exercise and declining at the cessation of the exercise, whereas both arterial and venous [NH31 reach peak values several minutes into recovery. This particular finding may illustrate a very important concept in the management of plasma [NH3]. The clearance system must increase rapidly with the initiation of exercise in order to minimize the rise in arterial [NH3]. Furthermore, the initial postexercise rise in arterial [NH& coupled with the rapid decline in the rate of NH3 release into the circulation, means that NH3 clearance is decreasing even more rapidly than the change in release from the muscle. The active muscle group added >l,lOO pmol NH3 to the blood in a 6-min period (3 min exercise and 3 min recovery). Dilution within the circulation could account for -375 pmol (assuming a blood volume of 5 liters). Liver NH3 uptake could account for an additional 90 pmol (8). Several studies have suggested that the distribution of NH3 in the other tissues of the body (particularly resting muscle) could be a major sink for the remaining (-60%) NH3 (3, 15, 27). Ammonia is extremely permeable and so such a clearance mechanism is possible. However, the uptake of [NH31 by resting muscle is only a few micromoles per kilogram per minute, and it is unlikely that it is a major [NH31 sink. Furthermore, the rapid decline in the NH3 clearance rate in the first 3 min of recovery suggeststhat such NH3 distribution into the [NH31
AND
RECOVERY
body water may not necessarily be the major clearance process. In both human subjects (20) and dogs (14, 26) it has been shown that the lung releases ammonia. The pulmonary ammonia concentration or its partial pressure is in equilibrium with the arterial blood [NH31 (14, 26). In addition, the large capillary bed of the lungs receives the entire cardiac output and is the first one that the NH3 is exposed to after being released by the active muscle. This combined with the increased concentration gradient and the high permeability of ammonia support the suggestion that the lungs may be releasing ammonia from the blood. Furthermore, ventilation (and pulmonary blood flow) increases and decreases rapidly in the exercise and recovery, respectively. Thus the lung has the possibility to be a prime NH3 clearance site, but it must be recognized that this is speculative, and the present study cannot confirm this hypothesis. Studies with animals have demonstrated that during heavy exercise muscle NH3 production is predominantly associated with IMP formation because of deamination of AMP (6, 9, 10). This is one reaction of the purine nucleotide cycle (PNC). When the reamination reactions of the PNC are blocked, there is little impact on the muscle [NH31 or [IMP] (9, 24, 32). This suggests that with strenuous exercise the steps of the PNC do not act in parallel and that reamination is predominantly a recovery process. In the present study there was a decline in [ATP] and a concomitant increase in [IMP] during exercise. The NH3 production that must accompany this IMP formation was -20% greater than that measured in NH3 efflux and accumulation of NH3 in the muscle. This “missing” NH3, together with any NH3 derived from amino acid catabolism, probably represents NH3 that has been used in amino acid production (e.g., glutamine production). Similarly, NH3 efflux in recovery accounted for all but 12% of the decline in muscle NH3. This probably also represents NH3 involved in amino acid metabolism within the muscle tissue. Because NH3 production did not exceed IMP formation, this supports previous suggestions that the AMP deaminase reaction is the major active part of the PNC during strenuous exercise and the reamination portion (adenylosuccinate synthase and adenylosuccinase) is dominant during recovery (9, 15, 16, 28). However, this interpretation assumes amino acid metabolism is minimal. Although the calculations of change in IMP and NH3 production and of NH3 efflux and production are in close agreement, they do not match precisely. The former disagree by 20% and the latter by 12%. It cannot be established in the present study whether this is due to small errors in the measurements and assumptions or whether it is due to other metabolic events. Examples of the former include possible errors in the estimation of quadriceps mass and the assumption that the biopsy [NH,] represents the entire mass. Similarly, if the hamstring muscles are taking up NH3 (as muscles usually do at rest) this would result in an underestimation of the actual NH3 efflux of the quadriceps. These potential errors are real considerations, but they are minimal compared with those encountered in most studies of exercising humans. These limitations do restrict the
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AMMONIA
FLUX
IN
EXERCISE
interpretation of the data, but it is highly possible that the “extra” NH, predicted from the [IMP] increase and the missing NH3 unaccounted for in the NH3 efflux are involved in amino acid metabolism as suggested above. Regardless, the data of the present study are consistent with the hypothesis that human muscle produces NH3 predominantly from AMP deamination during intense exercise. The reasons for the activation of the AMP deaminase reaction in the active muscle in the present study are uncertain. Major positive modulators are increases in [ADP], [AMP], [Pi] and [H’] (6,28). Dudley andTerjung (6) emphasized the importance of [H+] as a regulating factor and pointed out that muscle lactate formation occurred before NH, formation. Although both the present data and those of Katz et al. (15) show that lactate release increases prior to NH3 release, the significance of this remains to be established. Dudley and Terjung (6) suggested that a pH of 6.6 was necessary to activate AMP deaminase and Sahlin et al. (28) report that the optimal pH for the enzyme is 6.5-6.1 (i.e., H+ concentration of 500-900 nmol/l). In the current study muscle pH at exhaustion was only 6.69 t 0.05 ([H+] of 211 t 22 nmol/l). Those modulators that are normally postulated to activate AMP deaminase either did not change or changed less than reported in other investigations (6,15, 16, 28, 31), and yet the changes in [NH31 and in muscle [IMP] are comparable to those reported in these studies. For example, [CrP], [ATP], [ADP], and [H’] at exhaustion in the present study are similar to those reported by Spriet et al. (31) after 25 of 102 s of electrical stimulation of human muscle. However, the [IMP] in the present study is approximately twice as great as that found in their investigation at 25 s. These modulators are altered dramatically in the extreme metabolic challenges employed with animal models, but in the conditions of the present study other factors must contribute to the regulation of AMP deaminase. Although [ADP] was not changed and [AMP] was undetectable, in three subjects the free Pi, ADP, and AMP concentrations were calculated as described by Dudley et al. (7). The subjects had a rise in these compounds at exhaustion, and the ratio of [ATP] to free [ADP] and the phosphorylation state both fell. The data are preliminary, but they suggest that the shifts in the free concentrations of the nucleotides could be important in regulating AMP deamination. During the exercise it was estimated that only -25% of the NH3 formed was released by the muscle. Although this is similar to the conclusion by Katz et al. (15) that the majority of the NH3 remains in the muscle during activity, the 25% leaving the muscle is greater than the 10% estimated by Katz et al. (15) for subjects performing bipedal cycling at VO, max. It is uncertain whether this is due to differences in protocol (for example, in the present study the perfusion rate per unit of active muscle is greater than in two-legged cycling). It may also be because Katz et al. (15) did not use their directly measured NH, flux data to estimate the total NH3 released from the active muscle, but rather they indirectly estimated it from changes in arterial [NHS]. The present data demonstrate that the change in muscle [NH31 does not ac-
AND
El75
RECOVERY
curately reflect NH, production for single-leg knee extensor exercise. Although it has been speculated (13, 15, 23) that muscle NH3 accumulates during exercise because of increased intracellular acidosis inhibiting the conversion of ammonium to ammonia, the hypothesis has not been tested and the present data do not support this hypothesis. The muscle-to-venous NH3 ratio decreased from rest to exercise and only returned to resting values after 60 min. In contrast, the comparable ratio for H+ quickly rose during exercise and was back to a resting value within 10 min. Hence, the H+ and NH3 ratios did not correspond either in direction or time course. Interestingly, by examining the data of Katz et al. (15) one can calculate that the muscle-to-venous NH3 ratio was unchanged (9.3-9.5) from rest to maximal exercise. Furthermore, the data of Broberg and Sahlin (3, 4) predict similar resting ratios and a decline to approximately three during prolonged exercise. Ammonia is extremely permeable, and the data of Visek (34) suggest that the increase in [H+] in the present study would make less than a 1% shift in the ammonia-ammonium equilibrium, and thus the impact of H+ on ammonia diffusion would be minimal. In addition, the distribution of ammonium across a membrane is influenced not only by [H’] but also by the membrane potential (17, 18). If there is a decline in the membrane potential during exercise this would tend to lower the muscle-to-venous NH, ratio. Finally, it has been shown that ammonium competes with K+ for K+-channels (2,18) with a conductance ratio of -1:lO (ammonium:potassium) (2). Thus some NH3 leaves the cell as ammonium, and during exercise intracellular K+ declines; this, together with the rise in intramuscular NH3, could allow for enhanced ammonium transport out of the cell. In summary, intramuscular [NH31 and muscle efflux during intense dynamic exercise and the subsequent recovery suggest that NH3 clearance from the circulation is very rapid and may occur via the pulmonary system. The changes in [NH31 and [IMP] during exercise were approximately stoichiometric, supporting the concept that the PNC acts in series. The process by which AMP deaminase was activated in the present study is not clear. Finally, the study demonstrates that the muscle-to-venous NH3 ratio does not shift as predicted from the H+ data. The authors gratefully acknowledge the constructive discussions with P. Bennekou and the excellent technical assistance of Premila Sathasivam and Winnie Taagerup. The study was supported by a grant from Team Danmark and the Danish Science Research Council. T. Graham was supported by an National Sciences and Engineering Research Council of Canada International Collaborative Research Grant and by the Forster Fellowship, University of Guelph. Present address of P. D. Gollnick: Dept. of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, College of Veterinary Medicine, Washington State Univ., Pullman, WA 99164-6520. Address for reprint request and address of T. Graham: School of Human Biology, Univ. of Guelph, Ontario NlG 2W1, Canada. Received
17 August
1989; accepted
in final
form
21 March
Downloaded from www.physiology.org/journal/ajpendo at Tulane University (129.081.226.078) on February 12, 2019.
1990.
El76
AMMONIA
FLUX
IN
EXERCISE
REFERENCES 1. ANDERSEN, P., AND B. SALTIN. Maximal perfusion of skeletal muscle in man. J. Physiol. Lond. 366: 233-249, 1985. 2. BLATY, A. L., AND K. L. MAGLEBY. Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle. J. Gen. Physiol. 84: l-23, 1984. 3. BROBERG, S., AND K. SAHLIN. Hyperammoniemia during prolonged exercise: an effect of glycogen depletion? J. Appl. Physiol. 65: 2475-2477,1988. 4. BROBERG, S., AND K. SAHLIN. Adenine nucleotide degradation in human skeletal muscle during prolonged exercise. J. AppZ. Physiol. 67: 116-122, 1989. 5. DUDLEY, G. A., R. S. STARTON, T. F. MURRAY, F. C. HAGERMAN, AND A. LUGINBUHL. Muscle fiber composition and blood ammonia levels after intense exercise in humans. J. AppZ. Physiol. 54: 582586,1983. 6. DUDLEY, G. A., AND R. L. TERJUNG. Influence of acidosis on AMP deaminase activity in contracting fast-twitch muscle. Am. J. PhysioZ. 248 (CeZZ Physiol. 17): C43-C50, 1985. 7. DUDLEY, G. A., P. C. TULLSON, AND R. L. TERJUNG. Influence of mitochondrial content on the sensitivity of respiratory control. J. BioZ. Chem. 262: 9109-9114,1987. 8. ERIKSSON, L. S., S. BROBERG, 0. BJORKMAN, AND J. WAHREN. Ammonia metabolism during exercise in man. CZin. Physiol. Oxf. 5: 325-336, 1985. 9. FLANAGAN, W. F., E. W. HOLMES, R. L. SABINA, AND J. L. SWAIN. Importance of the purine nucleotide cycle to energy production in skeletal muscle. Am. J. Physiol. 251 (CeZZ Physiol. 20): C795-C802, 1986. 10. GOODMAN, M. N., AND J. M. LOWENSTEIN. The purine nucleotide cycle. Studies of ammonia production by skeletal muscle in situ and in perfused preparations. J. BioZ. Chem. 252: 5054-5060, 1977. 11. GRAHAM, T. E., P. K. PEDERSEN, AND B. SALTIN. Muscle and blood ammonia and lactate responses to prolonged exercise with hyperoxia. J. AppZ. Physiol. 63: 1457-1462, 1987. 12. GRAHAM, T. E., AND B. SALTIN. Estimation of the mitochondrial redox state in human skeletal muscle during exercise. J. AppZ. Physiol. 66: 561-566, 1989. 13. HARRIS, R. T., AND G. A. DUDLEY. Exercise alters the distribution of ammonia and lactate in blood. J. AppZ. Physiol. 66: 313-317, 1989. 14. JACQUEZ, J. A., J. W. POPPELL, AND R. JELTSCH. Partial pressure of ammonia in alveolar air. Science Wash. DC 129: 269-270, 1959. 15. KATZ, A., S. BROBERG, K. SAHLIN, AND J. WAHREN. Muscle ammonia and amino acid metabolism during dynamic exercise in man. CZin. Physiol. Oxf. 6: 365-379, 1986. 16. KATZ, A., K. SAHLIN, AND J. HENRIKSSON. Muscle ammonia metabolism during isometric contraction in humans. Am. J. Physiol. 250 (CeZZ Physiol. 19): C834-C840, 1986.
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RECOVERY
17. KLEINER, D. The transport of NH3 and NH4+ across biological membranes. Biochim. Biophys. Acta 639: 41-52, 1981. 18. KNEPPER, M. A., R. PACKER, AND D. W. GOOD. Ammonium transport in the kidney. Physiol. Reu. 69: 179-249, 1989. 19. KUN, E., AND E. B. KEARNEY. Ammonia. In: Methods of Enzymatic Analysis, edited by H. U. Bergmeyer. New York: Academic, 1974, p. 1802-1805. 20. LARSON, T. V., D. S. COVERT, AND R. FRANK. A method for continuous measurement of ammonia in respiratory airways. J. AppZ. Physiol. 46: 603-607, 1979. 21. LOWRY, 0. H., AND J. V. PASSONNEAU. A Flexible System of Enzymatic Analysis. New York: Academic, 1972. 22. MAGSON, N. S., AND L. PERRYMAN. Adenine nucleotides in erythrocytes of horses, Equs caballus. Comp. Biochem. Physiol. B Comp. Biochem. 67: 205-221,198O. 23. MEYER, R. A., G. A. DUDLEY, AND R. L. TERJUNG. Ammonia and IMP in different skeletal muscle fibers after exercise in rats. J. AppZ. Physiol. 49: 1037-1041, 1980. 24. MEYER, R. A., AND R. L. TERJUNG. Differences in ammonia and adenylate metabolism in contracting fast and slow muscle. Am. J. Physiol. 237 (Cell Physiol. 6): Clll-C118, 1979. 25. PARKHOUSE, W. S., D. C. MCKENZIE, P. W. HOCHACHKA, AND W. K. OVALLE. Buffer capacity of deproteinized human vastus lateralis muscle. J. AppZ. Physiol. 58: 14-17, 1985. 26. ROBIN, E. D., D. M. TRAVIS, P. A. BROMBERG, C. E. FORKNER, JR., AND J. M. TYLER. Ammonia excretion by mammalian lung. Science Wash. DC 129: 270-271,1959. 27. ROSADO, A., G. FLORES, J. MORA, AND G. SOBERON. Distribution of an ammonia load in the normal rat. Am. J. Physiol. 203: 37-42, 1962. 28. SAHLIN, K., G. PALMSKOG, AND E. HULTMAN. Adenine nucleotide and IMP contents of the quadriceps muscle in man after exercise. Pfluegers Arch. 374: 193-198, 1978. 29. SALTIN, B. Hemodynamic adaptations to exercise. Am. J. CardioZ. 55: 420-470, 1985. 30. SJOGAARD, G., R. P. ADAMS, AND B. SALTIN. Water and ionic shifts in skeletal muscle of humans with intense dynamic knee extension. Am. J. Physiol. 248 (Regulatory Integrative Comp. Physiol. 17): R190-R196, 1985. 31. SPRIET, L. L., K. SODERLUND, M. BERGSTROM, AND E. HULTMAN. Anaerobic energy release in skeletal muscle during electrical stimulation in men. J. AppZ. Physiol. 62: 611-615, 1987. 32. SWAIN, J. L., J. J. HINES, R. L. SABINA, 0. L. HARBURY, AND E. W. HOLMES. Disruption of the purine nucleotide cycle by inhibition of adenylosuccinate lyase produces skeletal muscle dysfunction. J. CZin. Invest. 74: 1422-1427, 1984. 33. WILKERSON, J. E., D. L. BATTERTON, AND S. M. HORVATH. Ammonia production following maximal exercise: treadmill vs. bicycle testing. Eur. J. AppZ. Physiol. 34: 169-172, 1975. 34. VISEK, W. J. Some aspects of ammonia toxicity in animal cells. J. Dairy Sci. 51: 286-295, 1968.
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GRAHAM, T.E.,J. BANGSBO, P.D. GOLLNICK,C.JUEL,AND B. SALTIN. Ammonia metabolism during intense dynamic exercise and recovery in humans. Am. J. Physiol. 259 (Endocrinol. Metab. 22): El70-E176, 1990.-This study examined the dynamics for ammonia (NH3) metabolism in human skeletal muscle during and after intense one-legged exercise. Subjects (n = 8) performed dynamic leg extensor exercise to exhaustion (3.2 min). Muscle NH, release increased rapidly to a maximum of 314 t 42 pmol/min and declined immediately on cessation of exercise. Recovery was complete in -20 min. Arterial [NH,] increased less rapidly and reached its maximum 2-3 min into recovery. These data demonstrate that NH3 clearance is more sensitive to the cessation of exercise than is NH3 release from skeletal muscle. Muscle [NH,] increased three to fourfold during exercise and represented 74 t 8% of the total net NH3 formation. Thus the change in muscle [NH,] alone underestimates the NH3 production. There was no evidence that the muscle-to-venous blood NH3 ratio shifts in accordance with the H+ data. Thus other factors must contribute to the NH, release from active muscle. The total net NH3 formed corresponded with the intramuscular inosine 5’-monophosphate accumulation, suggesting that the NH3 was derived from AMP deamination. Changes in the known modulators of AMP deaminase (ATP, ADP, H’) were moderate, so the mechanisms initiating the deamination remain obscure. purine nucleotide cycle; inosine 5’-monophosphate; lactate; adenosine 5’-monophosphate deaminase; hydrogen ion
SEVERAL STUDIES of ammonia and ammonium (NH# metabolism in exercising humans have relied on the determination of the NH3 intramuscular concentration as a reflection of its production (11, 12, 16). The basis for this assumption is that ammonium crosses the sarcolemma predominantly as ammonia, and during strenuous exercise acidification of the intracellular environment could impede the conversion of ammonium to ammonia (pK, = 9.3). Indirect evidence supporting this theory is that the relationship between increases in muscle NH3 and inosine 5’-monophosphate (IMP) is approximately stoichiometric (4, 15, 16). However, blood NH3 concentration rises during exercise (5, 33)) suggesting that efflux from active muscle is occurring. The dynamics of NH3 efflux from active skeletal muscle are poorly understood as there have been very few direct measures of NH3 efflux for human skeletal muscle ’ In physiological systems both ammonia and the ion, ammonium, exist. In this paper NH3 represents the sum of both forms, and when a specific form is referred to the terms “ammonia” and “ammonium” will be used. El70
0193~1849/90
$1.50 Copyright
C. JUEL,
AND
B. SALTIN
during intense exercise (8, 15). These studies made no more than one or two measurements during an entire exercise period, making it impossible to quantify accurately the total efflux. Furthermore, intramuscular NH3 and IMP were determined in only one of these investigations (15), and even then muscle pH was not measured. The same limitations exist in our understanding of the recovery processes. Thus, although the change in muscle NH, concentration during exercise is theoretically an accurate reflecti .on of N ‘H3 production, direct experimental evidence for human muscle is lacki .ng. In the present experiments, NH3 production in active human muscle was investigated using the single-leg extensor exercise model. This exercise mode allows the subject to achieve a very high power output for a given muscle mass. Thus one can study human skeletal muscle when its metabolic rate and ATP turnover rate are extreme. This should ensure a large NH3 production. This was examined by determining the changes in skeletal muscle [IMP], [NH31 and [H’] during exercise and making multiple deterrecovery and by simultaneously minations of NH, efflux from the muscle. MATERIALS
AND
METHODS
Subjects. Eight adult males aged 23-29 yr gave their informed consent to participate in the study, which was approved by the University of Copenhagen Ethics Committee. Exercise protocol. Subjects reported to the laboratory after a light breakfast and having refrained from strenuous physical activity the day before the experiment. Teflon catheters were inserted into the femoral artery and vein below the inguinal ligament of the leg to be exercised. The femoral arterial catheter was placed using the Seldinger technique with the tip l-2 cm proximal to the inguinal ligament. One of the two femoral vein catheters was placed -8 cm in the retrograde direction (distal to the inguinal ligament) for blood sampling and for infusion of ice-cold saline. The second venous catheter was placed in the inguinal region l-2 cm distal to the ligament. A thermistor was placed through this catheter just proximal to the tip of the original catheter to make leg blood flow measurements possible. The subject rested supine for 30 min after this procedure. The subject performed knee extension exercise with a modified Krogh cycle ergometer (1) with a power output (51.9-78.7 W) equivalent- to 139 t 9% of maximum oxygen consumption of (V~z,,,)for the leg extensors.
0 1990 the American
Physiological
Society
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AMMONIA
The exercise
was performed
FLUX
to exhaustion
(mean
IN
EXERCISE
AND
exercise
’
time was 3.20 min with a range of 2.15-4.94 min) after which the subjects rested quietly for 1 h. Measurements. The amount of active muscle mass
(quadriceps femoris) was estimated by Simpson’s rule, using measurements of thigh length, multiple circumferences of the thigh, and the skinfold thickness (I). This anthropometric approach has been shown to give values similar to those from estimations based on multiple computer-assisted tomography (CAT) scans (29). Values ranged from 2.54 to 3.51 kg with a mean value of 2.83
NH3
El71
RECOVERY flux
fumote/min)
~0
%’ 300
200
kg. Leg blood flow, determined by the constant-infusion
thermodilution technique (l), was estimated at rest four to six times during the exercise and lo-13 times during recovery (with the majority of the determinations conducted within the first 10 min of recovery). An occlusion cuff placed just below the knee was inflated (>220 mmHg) during the whole period of intense exercise and
100
during recovery. During the latter phase, the cuff was deflated for -30 s at 8, 25, 40, and 50 min. Immediately after
each blood
flow measurement,
blood
samples
were
collected from the femoral artery and vein for determination of oxygen saturation, hemoglobin, hematocrit,
C
lactate, and NH3.
pH,
Oxygen saturation was measured with an OSM-2 hemoximeter (radiometer), and the hematocrit was determined after high-speed centrifugation. Plasma
NH,
Lactate was de-
1
1
1.0
1.5
I
I
2.0
1
2.5
3.0
Time (minutes) 2. NH3
FIG.
1. Final
flux
2 data points
during
exercise.
represent
Figure
fewer
is organized
subjects;
same
as Fig.
SE is not given.
termined
on whole blood samples (al), and NH3 was measured on plasma by the method of Kun and Kearney (19). Leg oxygen consumption (VO,) and lactate and
(uM)
NH3 exchange were determined
by the Fick principle.
Although plasma rather than blood NH3 were measured,
it was used in combination with blood flow to estimate the total blood NH3 flux, since the absolute changes in plasma and whole blood NH3 concentrations are virtually identical during heavy exercise (3, 13, 15). Muscle biopsies from vastus lateralis were taken at rest, exhaustion, and at 3, 10, and 60 min of recovery.
These were analyzed for NH3 as described
previously
(12), for creatine phosphate (CrP), ATP, ADP, AMP, and IMP by high-performance liquid chromatography
(HPLC)
(22) an d muscle pH (25). Muscle samples were
analyzed for NH, before freeze drying. The remaining tissue was weighed before and after freeze drying to
determine
total water content.
The total lactate and NH, exchange was estimated during both exercise and recovery by averaging the efflux
values for every two consecutive time points, multiplying this Exercise 1
’
1
0
0.5
1
1.0 Time
FIG.
exercise.
1. Femoral
Data
1
1
1
L
1.5
2.0
2.5
3.0
&
(minutes)
arterial and venous plasma are means within SE represented
concentrations during by vertical bars. Open
circles and dashed line, femoral venous data; closed symbols and solid line, femoral arterial data. Both lines are drawn subjectively. Subjects exercised to exhaustion and their endurance times were all different. Thus the last 2 data points represent 5 and 4 subjects as indicated in parentheses. Samples were not taken at same time for all subjects. Thus SE for sampling time is represented by horizontal bars with arterial data. The same SE applies to venous data.
average release by the time span being considered,
and summing each of th .ese estimates fo r the entire period of exercise or recovery. During these time periods the amount
of accumulation
or depletion
of IMP
and NH3
in the muscle was estimated from the changes in the concentration of these metabolites multiplied by the amount of active extensor muscle. The concentration difference for II’ and NH3 between the muscle intracellular compartment and the interstitial fluid was calculated as follows. The venous concentrations were used as estimates of interstitial concentramuscle concentrations of NH3 and tions. The measured
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El72
AMMONIA NH3 flux
400
(umole/m
FLUX
IN
EXERCISE
AND
RECOVERY
in)
300
200
100
0 1 0
FIG.
L 1
3. NH3 flux
during
1 2 recovery.
1 I 3 4 Time(minutes) Figure
is organized
I 5
Recovery I 6
same as Fig. 2. Time,
H+ were calculated per liter of total muscle water. Then we used the data of Sjplgaard et al. (30) for the intra- and extracellular water distribution in muscle during leg extensor exercise and recovery to derive the intracellular muscle concentrations. Paired t tests were employed to determine at which time point there was a significant change from rest in arterial and venous [NH31 and in efflux. The paired t test was also used to determine whether there was a Plasma
NH,
1 7
IA,1 r, 0 ‘0
1
, 20
,
, , 1 , 30 40 Time (minutes)
, 50
,
, 60
0 min, end of exercise.
further increase in arterial exercise. In all comparisons significant if P C 0.05.
[NH31 after the cessation of differences were accepted as
RESULTS
During the first 30 s of exercise there were no significant changes in arterial or venous [NH31 (Fig. 1). By -60 s of exercise the venous [NH31 had risen signifi-
(ubl)
200
150
100
50
0
1 0
1
1 2 Time
FIG.
4. Femoral
arterial
and venous
1
I 4
Recovery I
I 6
1
I#& 0 10
1
(minutes) plasma
I 20
1
1 30
1
1 40
1
, 50
Time(minutes) concentrations
during
recovery.
Figure
is organized
same as Fig. 3.
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1
, 60
AMMONIA
FLUX
IN
EXERCISE
cantly, and the arterial [NH31 was also elevated significantly by -2.5 min. The release of NH3 increased significantly from rest at -1 min, and at exhaustion the NH3 efflux had risen to 314 t 42 pmol/min (Fig. 2). Immediately after cessation of exercise, the NH, efflux from the leg declined in a curvilinear manner such that after 3 min of recovery, the release was less than 50% of that measured at exhaustion (Fig. 3). In marked contrast, the arterial plasma [NH31 continued to rise during this initial recovery period (Fig. 4). This is not apparent in the mean data because the peak value occurred at various times into recovery (0.5-3.5 min). However, every subject had a further increase in arterial [NH31 in recovery (P < 0.05), and -25% of the total increase in arterial NH3 occurred during the initial recovery phase even though release of NH3 into the circulation was declining rapidly. At the end of the exercise the ventilation was 77.5 t 6.7 l/min; this fell to 55.2 t 5.9 l/min during the first 30 s, to 32.6 t 5.6 l/min during 30-60 s and was 21.5 t 2.2 l/ min by 3 min of recovery. After this initial recovery phase both NH3 efflux from the exercised muscle and [NH31 in arterial and venous plasma declined and approximated resting values by 2030 min (Figs. 3 and 4). The lactate release from the active leg was qualitatively similar to that of NH3, although it increased sooner and occurred to a much greater extent. Lactate efflux increased within 15-20 s after the onset of exercise from a resting value of 0.036 t 0.011 mmol/ min to 2.46 t 0.80 mmol/min, whereas NH, efflux showed no significant change over this time period. The total NH3 efflux from the leg during the entire exercise and the recovery period was 2.10 t 0.59 mmol (Table 2) while lactate release during exercise alone was calculated as 126.8 t 12 mmol. Skeletal muscle [NH31 rose three to fourfold during the exercise (Table 1). This represented -75% of the total net NH3 produced during the exercise (Table 2). During the recovery phase the muscle [NH31 returned to rest and the estimated NH3 efflux during recovery represented 88% of the accumulated muscle NH3. The majority of the NH3 released from the muscle occurred during the early part of the recovery process; 23 t 5% was released during exercise, 31 t 4 and 30 t 3% during O-3 and 3-10 min of recovery, respectively, whereas only 17 -+ 5% was released during the remaining 50 min. Even though 1,141 t 268 pmol NH3 were released into the bloodstream during exercise and the first 3 min of recovery, arterial [NH31 had a maximum increase of only 77 t 8 PM above that of rest. 1. Muscle ammonia, creatine phosphate, nucleotides, and IMP concentrations TABLE
Condition
Rest Exhaustion Ret-3 Ret-10 Ret-60
NH,
309t26 955t146 581t89 356t53 283&33
ATP
6.21kO.41 4.10t0.42 4.98rt0.34 5.57t0.17 5.85t0.40
ADP
1.22kO.10 1.21t0.09 1.12t0.11 1.02t0.04 1.05&0.08
IMP
l.lOt0.17 0.69kO.17 0.18t0.09
CrP
22.32t0.34 7.99t0.76 12.74t1.56 17.44t1.41 19.451t0.82
Values are means t SE in mmol/kg wet wt except for NH3, which is in pmol/kg wet wt. There was no detectable IMP at rest and at 60 min of recovery. Ret-3, -10, and -60 represent 3, 10, and 60 min of recovery.
AND
El73
RECOVERY
2. Summary of estimations of net NH3 production and release of quadriceps muscle during exercise and recovery TABLE
Calculation
Net
Exercise NH, released, pmol Accumulated muscle NH3, pmol Total net NH, production, pm01 Proportion NH3 production accumulated, % Recovery NH, released, pmol Exercise and recovery Total net NH, released, bmol
NH,
533&183 1,945+529 2,517+534 74t8
1,718+427 2,100&590
Values are means & SE. Calculation for accumulated muscle data is derived from product of measured increase in muscle [NH31 and estimated mass of active muscle (2.83 * 0.11 kg). Total net NH, production is sum of NH, released and accumulated muscle NHS. Similarly, proportion NH3 production accumulated is accumulated muscle NH3 divided by total net NH3 times 100.
3. Summary of calculations of intracellular-toextracellular ratio for NH, TABLE
Biopsy
Venous
Condition
Rest Exhaustion Ret-3 Ret-10 Ret-60
w-a%
pmol/l
309226 955U46 581t89 356*53 283*33
376-1-42 1,228+175 749t105 461-1-66 366&41
IC, pmol/l
NH3, pmol/l
419t49 1,418f208 850t122 545k94 427k49
38&17 165&14 158*15 lllt23 42tll
IC/Venous
23.7t7.3 8.9t2.1 5.4t0.7 7.3t2.2 16.3t4.2
Values are means & SE. Biopsy concentration (biopsy) was measured in pmol/kg wet wt and converted to r.Lrnol/l muscle water based on the wet-to-dry wt ratio. Intracellular concentration (IC) was calculated using data of Sjprgaard et al. (30) to estimate intracellular-to-extracellular water distribution and using measured femoral venous concentration (venous) as an approximation of extracellular concentration. Conditions are same as in Table 1.
At exhaustion muscle [CrP] declined to 36% of the resting value. Although [ADP] was unaltered, [ATP] declined and there was a comparable rise in [IMP] ([AMP] was undetectable). The increase in IMP predicted NH3 formation -20% greater than that which could be accounted for by the sum of net efflux and changes in muscle [NH31 during exercise. During recovery all metabolites returned toward resting concentrations, but even after 10 min of recovery none was back to basal values. Although there was an accumulation of NH3 in the muscle during exercise, the estimated ratio across the muscle membrane actually declined by >60% and fell even further 3 min postexercise before slowly returning to a preexercise level (Table 3). In marked contrast the ratio H+ increased -70% at exhaustion and rapidly returned to resting levels within 10 min of recovery (Table 4) . DISCUSSION
This study examined the time course of NH3 metabolism in human skeletal muscle and its relations to the purine nucleotide cycle during intense, dynamic exercise. Total NH3 production was underestimated by the change
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El74
AMMONIA
4. summary of calculations extracellular ratio for H+
TABLE
FLUX
IN
EXERCISE
of intracellular-to-
Condition
Biopsy, pmol/l
IC, pmol/l
Venous NH3, pmol/l
Rest Exhaustion Ret-3 Ret-10 Ret-60
74t1 211t22 134t17 82t5 76t5
79k2 235k26 145t20 87t7 82t6
4321 79t3 65t3 49s 42&l
IC/Venous
l&O.1 3.OkO.3 2.3k0.3 1.8kO.l 1.9t0.2
Values are means t SE. Data are derived in an identical fashion to that used for Table 3 except that ori ginal measure was pH, which was converted into [H’] (biopsy).
in muscle NH3 alone. These data demonstrate that the large efflux of NH3 was only qualitatively reflected by increased arterial and venous plasma [NH3]. The temporal mismatch between muscle NH, efflux and plasma [ NH31 during the exercise-recovery transition suggests that the major processes for removing NH3 from the plasma are very rapid. The intramuscular IMP-NH3 relationship supports the suggestion that the purine nucleotide cycle works in series, with the resynthesis portion of the cycle operating predominantly in recovery. Finally, the inability to demonstrate concomitant increases in the muscle-to-venous H+ and muscle -to-venous NH3 ratios suggests that the NH3 exchange across the sarcolemma is regulated by factors in addition to
w +1
Many studies of human subjects have used only venous (11, 1% 33) as a reflection of NH3 metabolism, and the present data demonstrate that such data can be quantitatively greater than arterial [NH3]. Of greater importance is the disparity between NH3 flux and plasma concentration. The NH3 flux has much greater rel.ative changes, and the time courses are different, with the NH3 efflux being maximal during the last stage of the exercise and declining at the cessation of the exercise, whereas both arterial and venous [NH31 reach peak values several minutes into recovery. This particular finding may illustrate a very important concept in the management of plasma [NH3]. The clearance system must increase rapidly with the initiation of exercise in order to minimize the rise in arterial [NH3]. Furthermore, the initial postexercise rise in arterial [NH& coupled with the rapid decline in the rate of NH3 release into the circulation, means that NH3 clearance is decreasing even more rapidly than the change in release from the muscle. The active muscle group added >l,lOO pmol NH3 to the blood in a 6-min period (3 min exercise and 3 min recovery). Dilution within the circulation could account for -375 pmol (assuming a blood volume of 5 liters). Liver NH3 uptake could account for an additional 90 pmol (8). Several studies have suggested that the distribution of NH3 in the other tissues of the body (particularly resting muscle) could be a major sink for the remaining (-60%) NH3 (3, 15, 27). Ammonia is extremely permeable and so such a clearance mechanism is possible. However, the uptake of [NH31 by resting muscle is only a few micromoles per kilogram per minute, and it is unlikely that it is a major [NH31 sink. Furthermore, the rapid decline in the NH3 clearance rate in the first 3 min of recovery suggeststhat such NH3 distribution into the [NH31
AND
RECOVERY
body water may not necessarily be the major clearance process. In both human subjects (20) and dogs (14, 26) it has been shown that the lung releases ammonia. The pulmonary ammonia concentration or its partial pressure is in equilibrium with the arterial blood [NH31 (14, 26). In addition, the large capillary bed of the lungs receives the entire cardiac output and is the first one that the NH3 is exposed to after being released by the active muscle. This combined with the increased concentration gradient and the high permeability of ammonia support the suggestion that the lungs may be releasing ammonia from the blood. Furthermore, ventilation (and pulmonary blood flow) increases and decreases rapidly in the exercise and recovery, respectively. Thus the lung has the possibility to be a prime NH3 clearance site, but it must be recognized that this is speculative, and the present study cannot confirm this hypothesis. Studies with animals have demonstrated that during heavy exercise muscle NH3 production is predominantly associated with IMP formation because of deamination of AMP (6, 9, 10). This is one reaction of the purine nucleotide cycle (PNC). When the reamination reactions of the PNC are blocked, there is little impact on the muscle [NH31 or [IMP] (9, 24, 32). This suggests that with strenuous exercise the steps of the PNC do not act in parallel and that reamination is predominantly a recovery process. In the present study there was a decline in [ATP] and a concomitant increase in [IMP] during exercise. The NH3 production that must accompany this IMP formation was -20% greater than that measured in NH3 efflux and accumulation of NH3 in the muscle. This “missing” NH3, together with any NH3 derived from amino acid catabolism, probably represents NH3 that has been used in amino acid production (e.g., glutamine production). Similarly, NH3 efflux in recovery accounted for all but 12% of the decline in muscle NH3. This probably also represents NH3 involved in amino acid metabolism within the muscle tissue. Because NH3 production did not exceed IMP formation, this supports previous suggestions that the AMP deaminase reaction is the major active part of the PNC during strenuous exercise and the reamination portion (adenylosuccinate synthase and adenylosuccinase) is dominant during recovery (9, 15, 16, 28). However, this interpretation assumes amino acid metabolism is minimal. Although the calculations of change in IMP and NH3 production and of NH3 efflux and production are in close agreement, they do not match precisely. The former disagree by 20% and the latter by 12%. It cannot be established in the present study whether this is due to small errors in the measurements and assumptions or whether it is due to other metabolic events. Examples of the former include possible errors in the estimation of quadriceps mass and the assumption that the biopsy [NH,] represents the entire mass. Similarly, if the hamstring muscles are taking up NH3 (as muscles usually do at rest) this would result in an underestimation of the actual NH3 efflux of the quadriceps. These potential errors are real considerations, but they are minimal compared with those encountered in most studies of exercising humans. These limitations do restrict the
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AMMONIA
FLUX
IN
EXERCISE
interpretation of the data, but it is highly possible that the “extra” NH, predicted from the [IMP] increase and the missing NH3 unaccounted for in the NH3 efflux are involved in amino acid metabolism as suggested above. Regardless, the data of the present study are consistent with the hypothesis that human muscle produces NH3 predominantly from AMP deamination during intense exercise. The reasons for the activation of the AMP deaminase reaction in the active muscle in the present study are uncertain. Major positive modulators are increases in [ADP], [AMP], [Pi] and [H’] (6,28). Dudley andTerjung (6) emphasized the importance of [H+] as a regulating factor and pointed out that muscle lactate formation occurred before NH, formation. Although both the present data and those of Katz et al. (15) show that lactate release increases prior to NH3 release, the significance of this remains to be established. Dudley and Terjung (6) suggested that a pH of 6.6 was necessary to activate AMP deaminase and Sahlin et al. (28) report that the optimal pH for the enzyme is 6.5-6.1 (i.e., H+ concentration of 500-900 nmol/l). In the current study muscle pH at exhaustion was only 6.69 t 0.05 ([H+] of 211 t 22 nmol/l). Those modulators that are normally postulated to activate AMP deaminase either did not change or changed less than reported in other investigations (6,15, 16, 28, 31), and yet the changes in [NH31 and in muscle [IMP] are comparable to those reported in these studies. For example, [CrP], [ATP], [ADP], and [H’] at exhaustion in the present study are similar to those reported by Spriet et al. (31) after 25 of 102 s of electrical stimulation of human muscle. However, the [IMP] in the present study is approximately twice as great as that found in their investigation at 25 s. These modulators are altered dramatically in the extreme metabolic challenges employed with animal models, but in the conditions of the present study other factors must contribute to the regulation of AMP deaminase. Although [ADP] was not changed and [AMP] was undetectable, in three subjects the free Pi, ADP, and AMP concentrations were calculated as described by Dudley et al. (7). The subjects had a rise in these compounds at exhaustion, and the ratio of [ATP] to free [ADP] and the phosphorylation state both fell. The data are preliminary, but they suggest that the shifts in the free concentrations of the nucleotides could be important in regulating AMP deamination. During the exercise it was estimated that only -25% of the NH3 formed was released by the muscle. Although this is similar to the conclusion by Katz et al. (15) that the majority of the NH3 remains in the muscle during activity, the 25% leaving the muscle is greater than the 10% estimated by Katz et al. (15) for subjects performing bipedal cycling at VO, max. It is uncertain whether this is due to differences in protocol (for example, in the present study the perfusion rate per unit of active muscle is greater than in two-legged cycling). It may also be because Katz et al. (15) did not use their directly measured NH, flux data to estimate the total NH3 released from the active muscle, but rather they indirectly estimated it from changes in arterial [NHS]. The present data demonstrate that the change in muscle [NH31 does not ac-
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curately reflect NH, production for single-leg knee extensor exercise. Although it has been speculated (13, 15, 23) that muscle NH3 accumulates during exercise because of increased intracellular acidosis inhibiting the conversion of ammonium to ammonia, the hypothesis has not been tested and the present data do not support this hypothesis. The muscle-to-venous NH3 ratio decreased from rest to exercise and only returned to resting values after 60 min. In contrast, the comparable ratio for H+ quickly rose during exercise and was back to a resting value within 10 min. Hence, the H+ and NH3 ratios did not correspond either in direction or time course. Interestingly, by examining the data of Katz et al. (15) one can calculate that the muscle-to-venous NH3 ratio was unchanged (9.3-9.5) from rest to maximal exercise. Furthermore, the data of Broberg and Sahlin (3, 4) predict similar resting ratios and a decline to approximately three during prolonged exercise. Ammonia is extremely permeable, and the data of Visek (34) suggest that the increase in [H+] in the present study would make less than a 1% shift in the ammonia-ammonium equilibrium, and thus the impact of H+ on ammonia diffusion would be minimal. In addition, the distribution of ammonium across a membrane is influenced not only by [H’] but also by the membrane potential (17, 18). If there is a decline in the membrane potential during exercise this would tend to lower the muscle-to-venous NH, ratio. Finally, it has been shown that ammonium competes with K+ for K+-channels (2,18) with a conductance ratio of -1:lO (ammonium:potassium) (2). Thus some NH3 leaves the cell as ammonium, and during exercise intracellular K+ declines; this, together with the rise in intramuscular NH3, could allow for enhanced ammonium transport out of the cell. In summary, intramuscular [NH31 and muscle efflux during intense dynamic exercise and the subsequent recovery suggest that NH3 clearance from the circulation is very rapid and may occur via the pulmonary system. The changes in [NH31 and [IMP] during exercise were approximately stoichiometric, supporting the concept that the PNC acts in series. The process by which AMP deaminase was activated in the present study is not clear. Finally, the study demonstrates that the muscle-to-venous NH3 ratio does not shift as predicted from the H+ data. The authors gratefully acknowledge the constructive discussions with P. Bennekou and the excellent technical assistance of Premila Sathasivam and Winnie Taagerup. The study was supported by a grant from Team Danmark and the Danish Science Research Council. T. Graham was supported by an National Sciences and Engineering Research Council of Canada International Collaborative Research Grant and by the Forster Fellowship, University of Guelph. Present address of P. D. Gollnick: Dept. of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, College of Veterinary Medicine, Washington State Univ., Pullman, WA 99164-6520. Address for reprint request and address of T. Graham: School of Human Biology, Univ. of Guelph, Ontario NlG 2W1, Canada. Received
17 August
1989; accepted
in final
form
21 March
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1990.
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FLUX
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