Transient expansion of plasma volume (PV) is commonly reported after endurance events (5,7,15,17,21,27) and intense exercise (9,11,18,19,28). On stopping the exercise, there is a return to control values that may take up to 5–7 d (17,27). It has been suggested that albumin retention in the vascular compartment has greater importance than sodium in mediating hypervolemia. To our knowledge, hypervolemia observed after endurance events is higher than the change in total body water (TBW) and interstitial space. Alterations in PV could not result, in fact, from sodium retention alone because, in this case, the interstitial fluid would be expanded by the same magnitude as PV. Therefore, increased protein mass should have considerable importance in the contribution to hypervolemia.
In contrast to the sodium effect, the influx of proteins into the vascular space does in fact favor water retention, specifically in circulating blood. Due to its colloid osmotic properties, albumin mass expansion is the major driving force for PV expansion. Plasma albumin retention can be observed in several types of dynamic exercise (9,11,28). A progressive increase in the intravascular mass of proteins, or more specifically albumin, was also reported after exercise training (4,13) and after endurance races such as marathon (15,21). More recently, we found that during the first 4 d of 7-d ultra-endurance racing (running, cycling, and cross-country skiing), the increase in plasma albumin mass was responsible for 20% of the observed PV expansion (unpublished observations).
A redistribution of previously existing albumin by increased lymph flow (9), reduced albumin degradation and transcapillary escape rate (11), and an increase in albumin synthesis are possible mechanisms for increase in plasma albumin mass and consequently for PV expansion. The role of albumin synthesis has been well documented within the first 24–48 h after single exposures to intense intermittent exercise (18,28). In contrast, the only study that has focused on the albumin synthetic rate after endurance exercise has failed to demonstrate an increase in albumin synthesis after a 4-h aerobic session at moderate intensity (5). Because such an endurance event is known to induce water and protein retention in the vascular compartment during the recovery period, this finding is not consistent with one of the mechanisms proposed for albumin mass increase after short and intense exercise. Moreover, it should be noted that no study has yet examined the effect of ultra-endurance trials on albumin synthesis in relation to the transient PV expansion, which is also consistently observed after such events (5,7,17,27).
Therefore, the purpose of the study was to test the hypothesis that an ultra-endurance event is a strong stimulus to increase albumin synthesis associated with transient hypervolemia. To test this hypothesis, we simulated an ultra-endurance event over 4 d that had been demonstrated to expand PV and plasma albumin mass with a maximal response on the 4th day of exercise (7). Furthermore, we extended the duration of the measurement period to the 8th day of the recovery period when PV has returned to its control level, assuming that albumin synthesis and plasma protein mass may also be normalized.
Six young men (aged 24.0 ± 0.9 yr; mean ± SEM) volunteered for this study. The study received approval from the Committee of Human Protection in Biomedical Research of Auvergne. Subjects were informed of potential risks involved with procedure and gave their written informed consent. Five participants were recreationally active (exercising 2–5 h·wk−1) and one was a well-trained cyclist (12 h training per week). All the subjects have never performed this type of workout before the experimental trial, and the relative intensity was the same for all subjects in order to perform the same challenge.
The study was divided into three periods: control (C), exercise (from day 1 to 4), and recovery: R1 (17–20 h after the end of the last exercise bout) and R8 (on the 8th day). Between R1 and R8, subjects were asked to abstain from physical exercise or physical strenuous work. Measurements of water compartments, biochemical parameters, and protein metabolism were performed on C, R1, and R8.
Four days before beginning the trial, an infra-maximal test was performed on a treadmill (Super 2500, Gymrol, Roche La Molière, France). For the 5-min warming-up, the speed was of 8.1 ± 0.2 km·h−1. Every 3 min, the speed was increased by 1.5 km·h−1 until the heart rate was close to 80% of the age-predicted maximal value. On the same day, 2 h later, maximal oxygen uptake (V̇O2max) was determined on a cycle ergometer (Ergomeca, La Bayette, France). For the 5-min warm-up, the workload was of 102.1 ± 5.2 W. Every 3 min, the load was increased by 30 W until exhaustion. The criteria for determining whether V̇O2max was obtained were a respiratory exchange ratio of at least 1.1 and HR close to the age-predicted maximal value.
During the two tests, an electrocardiogram was recorded continuously (Schiller, Villiers/Marne, France). The expired air volume was collected into Douglas bag during 30 s at the end of each stage and was measured with a Tissot spirometer. CO2 and O2 fractions in the expired air were measured by CO2 and O2 analyzers (CPX/D, MedGraphics, St. Paul, MN). The results of these tests were used to adapt the exercise intensity on the cycle ergometer and treadmill during the exercise procedure. The same relative intensity was choice to be the same for all the subjects in order to impose the same exercise challenge.
The exercise period was performed during 4 d in the laboratory under moderate temperature conditions that varied from 20 to 25°C. The exercise period consisted of 300-min exercise per day during 4 d on the cycle ergometer and treadmill alternately (Fig. 1). Daily exercise was divided into six 50-min bouts, three sessions in the morning and three sessions in the afternoon, with a 15-min rest period between each session. A 135-min break was allowed for lunch. The cycle load and the treadmill speed calculated from the data obtained during the previous tests corresponded to a moderate exercise intensity: 57.4 (SEM 1.3) %V̇O2max, i.e., 130.6 (SEM 7.1) W for cycling and 65.0 (SEM 2.1) %V̇O2max, i.e., 8.6 (SEM 0.3) km·h−1 for running. The moderate intensity was close to the mean intensity sustained in a field trial in a previous study (49–58% V̇O2max;7).
All measurements on C, R1, and R8 were made in the early morning at the same hour. The participants were asked to avoid exhaustive exercise in the 24 h preceding the control day and R8. On R1, the measurements were done at least 15 h after the last exercise bout. After an overnight fast and micturition, subjects were weighed on a precision scale (ID1, Mettler Toledo, Columbus, OH). At 07:00 a.m., they laid down, lightly dressed, on a bed at moderate temperature (20–22°C), and measurements began after they had been in the supine position for 1 h. They stayed in that position during all the measurements. Two catheters were inserted: the first in an antecubital vein of the right arm, and the second on a retrograde vein of the left hand. The protocol design is described on Figure 2.
Bioimpedance analysis (BIA) was chosen for TBW estimation because our well-controlled test conditions (describe above) make the method quite reliable in estimating changes in TBW (25). Moreover, we have demonstrated that, in the same conditions of measurement, TBW estimates by BIA were well correlated with values obtained by 18O dilution (7), which has been found to be the best probe for TBW (24). For this evaluation, Analycor 3® apparatus (Eugedia, Chambly, France) was used at the frequency of 100 kHz. The limbs were abducted during measurements and four electrodes were placed on the right side, one at the end of the third metacarpal bone and the second metatarsal bone for current emission, and detector electrodes were located on the mid-dorsum of the wrist, in the middle of the radius and ulna, and between the two malleoli of the ankle.
PV was measured using the Evans Blue dye (EB) dilution method. Two samples of 7.5-mL blood were drawn for blanks. Immediately after 2.5-mL EB (5 mg·mL−1) were injected into the opposite arm followed by 5 mL of saline solution for rinsing. Blood samples were taken 5, 10, 15, and 20 min after the injection to measure plasma EB concentration. Optical densities of plasma samples were determined using a spectrophotometer (S500, Secomam, Domont, France) at two wavelengths (620 nm (where EB has it maximal absorption) and 740 nm (at which EB has no absorption)) according to the method described by Foldager and Blomqvist (8). The dilution curve was extrapolated to time 0 concentration to calculate PV.
The PV changes were also calculated from changes of hematocrit (Hct) and hemoglobin concentration (Hb) between C, R1, and R8. The relative PV changes on R1 and R8 as compared with C value (ΔPV%) were calculated according to the equationMATH (26):
Hct was corrected (0.96) for plasma trapped with the packed red cells, and an additional correction (0.91) was made for the venous-to-total body Hct ratio. Hb was also corrected (0.92) for the venous-to-total body Hb ratio (10).
Protein metabolism kinetics and albumin synthetic rate.
To assess albumin synthesis, a stable isotopically labeled leucine was infused. The left hand was placed in a heated ventilated box during about 15 min before each blood withdrawal to arterialize venous blood. Ten mL of blood were drawn to determine basal isotopic enrichments of [13C] leucine, ketoisocaproate (KIC), and albumin bound leucine. Two breath samples were also taken and stocked in tubes (Vacutainer, Becton-Dickinson, Grenoble, France) for baseline isotopic enrichment of CO2. After an injection of 5-mL (6-mg) [13C] sodium bicarbonate (SSY-16-127), a primed continuous infusion of [1-13C] leucine (5 μmol·kg−1·h−1) was begun (T0) and was continued for 3 h. Every 20 min between time 2 and 3 h (T120, T140, T160, and T180), blood and breath samples were taken. Total carbon dioxide production was determined between T120 and T140 and between T160 and T180 by open-circuit indirect calorimetry (Deltratrac, Datex, Boblingen, Switzerland).
CO2 enrichments in expired breath samples were measured on an isotopic mass spectrometer (GC-C-IRMS, Micromass, Manchester, UK). Plasma [13C] leucine and KIC enrichments were measured on a GCMS (HP 5971, Hewlett-Packard, Palo Alto, CA), using tertiary-butyldimethylsilyl derivatives as previously described (1). Leucine concentrations were measured with the same system using norleucine as internal standard. Plasma albumin was isolated, purified from plasma with ethanol, and hydrolyzed (14). Amino acids in hydrolysate were transformed in N-acetyl-propyl derivatives. Then [13C] leucine enrichments in albumin were determined on the same GCC-IRMS.
The [13C] leucine incorporation rate into albumin was calculated between time 120 and 180 min by least square regression analysis. FSR was obtained by dividing the slopes of incorporation (corrected for 24 h) by the plasma [13C] KIC enrichment at plateau. Absolute synthetic rates (ASR, mg·kg body weight−1·24 h−1) were then calculated as FSR (%·24 h−1) times plasma albumin concentrations (g·L−1) times plasma volume (L·kg body weight−1) divided by 100. Whole body leucine flux (total leu Ra) was determined using steady-state equations and precursor pool model, as previously described (2). In this context, whole body leucine oxidation (Leu ox) was measured from 13CO2 isotopic enrichment multiplied by CO2 production, which was then divided by [13C] KIC enrichment in plasma during the same time. Whole body protein synthesis (in μmol·kg−1·min−1) was then estimated by calculating the nonoxidative leucine disposal rate (NOLD) as follows:MATH
Total volume of urine voided during 24 h was collected on C and during each day of the exercise period (from D1 to D4).
Plasma and urine osmolality were determined by means of freezing-point depression method (The AdvancedTM Osmometer 3D3, Advanced Instruments, Inc., Norwood, MA). Automatic analyzers were used for the measurements of albumin (Immage®, Beckman Coulter, Inc., Fullerton, CA) and sodium and protein (Hitachi 911, Boehringer Mannheim, Rueil Malmaison, France) concentrations. Intravascular albumin, sodium, and protein masses were calculated as the product of PV by albumin, sodium, and protein concentrations, respectively.
During the exercise period, measured food and beverages were available ad libitum. Energy intake and composition of food (carbohydrates, lipids, protein percentage, and sodium mass) were determined from dietary recording using GENI software with French (REGAL) and German (SOUCI) tables for food composition (MICRO-6, Meylan, France). The quantity of fluid intake during meals and snacks was the sum of beverages drunk and the water content of food for the 4 d. Water content of food was measured as the difference between the wet weight of a homogenized sample of the complete diet and the weight of the same sample after freeze-drying followed by drying at 103°C for 24 h. The volume of fluid consumed was obtained by weighing. During the recovery period, diet was not monitored.
All values were expressed as mean ± SEM. After having checked that our data are parametric (Graph Pad Instat, Graph Pad Software, San Diego, CA), repeated measures analyses of variance (ANOVA) were performed followed by a paired t-test to determine significant differences between the measurement periods (Statview software 5.0, SAS Institute Inc., Chicago, IL). Pearson-product correlations were also calculated. The level of statistical significance was set at P ≤ 0.05 for all analyses.
The physical characteristics of the subjects were: body weight (body weight) 73.0 ± 2.7 kg, height 176.8 ± 2.0 cm, V̇O2max 49.9 ± 2.8 mL·min−1·kg body weight−1: 43.2 mL·min−1·kg body weight−1 for the less fit subjects, and 64.0 mL·min−1·kg body weight−1 for the trained subjects. All the subjects completed all exercise sessions.
No significant differences (ANOVA P = 0.08) were found between the weight obtained at C and after the exercise period (R1 and R8), i.e., a difference of +0.16 ± 0.33 kg body weight between C and R1 and of −0.64 ± 0.29 kg body weight between C and R8 (Table 1).
After 4 d of exercise, TBW was significantly expanded by 26 ± 5 mL·g−1·kg body weight−1 (+4.2 ± 0.8%;P = 0.004;Table 1). The mean PV increased by 10 ± 1 mL·g−1·kg body weight−1 (+23.3 ± 3.2%;P = 0.001). The magnitude of the change was similar to that calculated from the changes of Hct and Hb (+19.8 ± 3.5%;P = 0.54). By R8, TBW and PV had returned to their control level (P > 0.05).
Plasma sodium, albumin, and protein concentrations and masses, osmolality, and osmotic mass are summarized in Table 2. Whereas albumin concentrations in C and R1 were not statistically different (P = 0.13), albumin mass increased significantly after 4 d of exercise by 0.30 ± 0.09 g·kg body weight−1 (+15.3 ± 4.9%;P = 0.03;Fig. 3). Whereas protein concentration was decreased by 8.1 ± 1.9% after 4 d of exercise (P = 0.02), protein mass increased by 0.41 ± 0.12 g·kg body weight−1 between C and R1 (P = 0.03). No changes were observed for plasma sodium concentration and osmolality between C and R1, but sodium and osmotic masses were increased by +22.2 ± 3.2% (+1.39 ± 0.20 mmol·kg body weight−1;P = 0.001) and +23.7 ± 2.8% (+3.04 ± 0.36 mosmol·kg body weight−1;P < 0.001), respectively, after the exercise period. All values obtained on R8 were similar to those measured on C (P > 0.05) except for albumin mass which was below control: −0.19 ± 0.06 g·kg body weight−1 (−9.5 ± 2.7%;P = 0.03;Fig. 3).
Urinary losses were higher on D1 (2.13 ± 0.25 L·24 h−1;P = 0.0004), D2 (2.67 ± 0.16 L·24 h−1;P = 0.006), and D3 (2.14 ± 0.18 L·24 h−1;P = 0.02) than value on C (1.20 ± 0.18 L·24 h−1). However, diuresis on D4 (1.98 ± 0.31 L) was not significantly different from the values obtained on C and on the other days of exercise. As compared with C (871 ± 110 mosm·kg−1), urine osmolality was lower on D1 (580 ± 64 mosm·kg−1;P = 0.01), D2 (473 ± 39 mosm·kg−1, P = 0.03), and D3 (561 ± 25 mosm·kg−1, P = 0.03). The value obtained on D4 (605 ± 68 mosm·kg−1) was not significantly different from the value obtained on C.
Albumin synthetic rate.
Albumin FSR increased from C (5.36 ± 0.46%·24 h−1) to R1 (6.86 ± 0.62%·24 h−1;P = 0.01), i.e., a difference of +29.0 ± 7.1% between C and R1 (Fig. 4). During the same period, ASR increased from C (103 ± 10 mg·kg body weight−1·24 h−1) to R1 (153 ± 19 mg·kg body weight−1·24 h−1;P = 0.007), i.e., a difference of +47.5 ± 6.8% between C and R1 (Fig. 5). On R8, albumin FSR (6.27 ± 0.62%·24 h−1;Fig. 4) and ASR (108 ± 12 mg·kg body weight−1·24 h−1) mean values were not statistically different from mean values obtained on C (Fig. 5).
As illustrated in Figure 6, all the subjects increased their ASR and PV between C and R1 but no significant correlation was found between these two parameters when expressed in percent of changes. In contrast, the variations (in percent) of PV were highly correlated with changes of sodium mass (P < 0.0001, r2 = 0.97) and osmotic mass (P = 0.0001, r2 = 0.96).
Whole body flux, leucine oxidation, and protein synthesis were not affected by 4 d of ultra-endurance exercise. When NOLD was extrapolated to whole body protein synthesis (590 μmol leucine·g protein−1), the absolute synthetic rate of albumin was found to contribute to about 4.6 ± 0.5%, 5.8 ± 1.0%, and 4.9 ± 0.6% to whole body protein synthesis on C, R1, and R8, respectively.
Food and beverages.
Energy intake during the 4 d of exercise period was 69,743 ± 2,766 kJ (i.e., 17,436 kJ·d−1) with 53.8 ± 0.5% of carbohydrates, 15.1 ± 0.6% of proteins, and 31.1 ± 0.3% of lipids. Total sodium intake (food and beverages) was 8.6 ± 0.3 g·d−1 of exercise. The total beverage intake was of 6.65 ± 0.65 L·d−1 during exercise period with a sodium content of 0.24 ± 0.06 g·d−1.
The present investigation has demonstrated for the first time that the albumin synthetic rate was significantly increased for all the subjects after 4 d of ultra-endurance trial (Figs. 4 and 5). Moreover, the trained cyclist showed the same response pattern as the five recreationally active subjects. This finding is not consistent with that obtained by Carraro et al. (3), who did not find an increase in albumin synthetic rate at the end of 4 h aerobic exercise and after 4 h of recovery. The discrepancy could be due to a difference in exercise intensity and measurement period. First, the moderate intensity applied in the study of Carraro et al. (3; 40% vs 57–65% V̇O2max in our study) may have not been a sufficient stimulus for increasing albumin synthesis. In support of this hypothesis, the significant correlation found in a previous study (7) between PV expansion and mean exercise intensity relative to V̇O2max suggest that factors such as the increase in albumin synthetic rate favoring water retention in the vascular compartment were dependent on exercise intensity. Second, the [13C] leucine infusion was performed 17–20 h after the last exercise session in the current study, whereas Carraro et al. (3) made the last measurement during the fourth hour of recovery. Because the time course of the albumin synthetic rate during the recovery period of exercise is not known, 4 h would not have been a sufficient time to detect an increase in albumin synthetic rate in the study by Carraro et al. (3). In contrast, our study confirms the results obtained after single intense exercises (18,28), but the magnitude of the response (48%) was three- to four-fold higher. Our protocol design (prolonged exercise during 5 h·d−1 and repetition over 4 d) may have amplified the mechanism. However, although the increase in albumin ASR was considerable, a significant elevation of whole body protein synthesis was not observed, which was probably due to the small contribution of albumin ASR to whole body protein synthesis (close to 6%).
The increase in plasma albumin mass (15%) observed on R1 may be considered as the result of the stimulation of albumin synthetic rate induced by the endurance trial. However, under our experimental conditions, it is too speculative to estimate the amount of albumin mass in which the increase in albumin synthetic rate was involved. The value of albumin synthetic rate measured at 17–20 h after the last exercise bout did not allow to predict the time course of additional plasma albumin content from the first day of exercise. Moreover, the contribution of plasma albumin degradation and albumin transcapillary escape rate were unknown. Nevertheless, it is suggested that the exercise repetition over 4 d may be a strong stimulus for albumin synthesis. Thus, there might be no effect on the first day as reported by Carraro et al. (3) and greater increase after the exercise protocol. This mechanism may explain the concomitant PV increase from 45 to 55 mL·kg body weight−1; i.e., an expansion of 10 mL·kg body weight−1. These values were exceptionally high. However, it is noteworthy that the methodology used in this study, the EB dilution, yields consistently higher values than radioactive isotope-labeled albumin dilution (23). Our basal PV level corresponded to the highest frequency distribution of the mean PV value usually reported by dye dilution methods (23). Consequently, the value of 55 mL·g−1·kg body weight−1 reached on R1 led to a PV expansion of 23%, which was confirmed by the indirect method using Hct and Hb changes (20%) and was consistent with those reported after such endurance events: 22% by Fellmann et al. (5,7) and Williams et al. (27), and 25% by Milledge et al. (17). This high PV extent may have been facilitated by an abundant water supply (mean 6.7 ± 0.7 L·d−1 during the trial), allowing the subjects to remain well hydrated as indicated by the values of urine osmolality.
Although all the subjects had an increased albumin synthetic rate and PV, the magnitude of the increase in ASR did not allow the prediction of PV expansion within subjects, probably because of the small sample size (Fig. 6). If 1 g of albumin is considered to bind 18-mL water, the increase in albumin mass observed in our study would explain the 5.4 mL·g body weight−1 of water retained in PV, which represents 54% of the total PV expansion. Consequently, the role of albumin mass increase on PV expansion is smaller after this endurance trial than that observed after single intense exercise (9). The remaining part of PV expansion might have been the consequence of sodium retention as supported by a significant increase in sodium mass (+1.39 mmol·kg body weight−1) and the significant correlations found between sodium and osmotic masses, and PV changes during the same period. Our laboratory has previously shown that sodium mass expansion was also one factor responsible for hypervolemia after an endurance race (7). A similar finding has recently been reported by Nagashima et al. (18), who suggested that a reduced water and sodium excretion by the kidneys 1 d after a single bout of intense exercise participated in the initial process of PV expansion. A similar mechanism could be evoked in our study. However, the precise role of this renal adaptation to such an endurance event over several days remains to be investigated.
It is noteworthy that PV and TBW returned to baseline 8 d after the last exercise bout concomitantly with the albumin synthetic rate, and protein and osmotic masses. In this regard, these findings strengthen the fact that exercise has been the stimulus for all these changes observed on R1 although diet and water balance was not controlled during the recovery period. This transient water retention after endurance trial was previously reported by Milledge et al. (17), who also observed a net water loss and PV decline on the 4th day after 5-d hill walking. To determine accurately the time course of these variations, sequential measurements should be performed during the recovery period. The fact that albumin mass was below the control level on R8 whereas albumin FSR had returned to the preexercise value suggests that other mechanisms such as an increase in albumin degradation and/or increase in the albumin transcapillary escape rate may have counterbalanced the effect of albumin synthesis.
Explanations for albumin synthesis stimulation during physical exercise as in the present study are unclear. Cell volume alterations in vitro have been reported to regulate intracellular metabolic pathways. Liver cell swelling counteracts proteolysis whereas cell shrinkage promotes protein breakdown (12). Within this context, it is noteworthy that we found, as usually reported after ultra-endurance races (7,17,27), an increase in TBW (26 ± 5 mL·kg body weight−1). The TBW assessment by BIA could be considered as reliable because adequate controls have been employed for posture and ambient temperature. Although measurements of skin temperature were lacking, it is reasonable to postulate that skin and body temperatures had returned to their basal level 15 h after the cessation of the last exercise bout. Moreover, our experimental conditions (isotonic hyperhydration) did not correspond to those that are known to diminish the resolution of the measurements such as isotonic dehydration (20). The difference in TBW and PV (10 ± 1 mL·kg body weight−1) expansions accounted for an increase in the remaining body water fluids (interstitial and/or intracellular compartments) of 15 ± 5 mL·kg body weight−1. Therefore, this result cannot rule out a possible cell swelling that could constitute a signal for albumin hepatic synthesis stimulation. However, all the methods used in vivo, including BIA, do not allow us to differentiate organs that are hydrated. Nutritional conditions have also been suggested as an influence on albumin synthesis (22). Because our subjects were fasting during each measurement, they were in the same nutritional status, and the fast therefore should not have affected comparisons of changes in albumin synthesis between the measurements made on C, R1, and R8. Albumin synthesis may also be influenced by hormonal status. Stress hormones such as catecholamines, cortisol, and glucagon induce an increase in albumin FSR in humans (16). Under our experimental conditions, it is reasonable to speculate on a potential effect of these hormones. Indeed, we found in a previous study a significant increase in cortisol, free and conjugated adrenaline, and noradrenaline in the plasma during a 6-d endurance race (6).
In conclusion, this is the first investigation to demonstrate that an ultra-endurance event during 4 d induced a dramatic increase in the albumin synthetic rate that was associated with a greater circulating protein mass. The magnitude of this increase, however, did not predict PV expansion. The impact of exercise on albumin synthesis disappeared 8 d later, as did PV and total protein mass expansion. These results have implications for a better understanding of the role of the albumin synthetic rate in plasma volume expansion during ultra-endurance exercise in young adults.
The authors thank the subjects for their contribution. They would also like to thank Xavier Leverve (Grenoble) for a critical review of the manuscript.
This study was supported by a grant from La société des eaux d’Evian-Volvic.
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Keywords:©2003The American College of Sports Medicine
STABLE ISOTOPE; WHOLE-BODY PROTEIN TURNOVER; EXERCISE-INDUCED HYPERVOLEMIA; PLASMA SODIUM RETENTION