AJP - Endo Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 294: E645-E653, 2008. First published January 22, 2008; doi:10.1152/ajpendo.00720.2007
0193-1849/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/4/E645    most recent
00720.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bergouignan, A.
Right arrow Articles by Blanc, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bergouignan, A.
Right arrow Articles by Blanc, S.

The acetate recovery factor to correct tracer-derived dietary fat oxidation in humans

Audrey Bergouignan,1 Dale A. Schoeller,2 Susanne Votruba,3 Chantal Simon,4 and Stéphane Blanc1

1Institut Pluridisciplinaire Hubert Curien-Département d'Ecologie, Physiologie, Ethologie Université Louis Pasteur Centre National de la Recherche Scientifique 7178, Strasbourg, France; 2Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, Wisconsin; 3Obesity and Diabetes Research Section-National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases, Phoenix, Arizona; and 4Département de Nutrition-Hôpital d'Hautepierre, Université Louis Pasteur, Strasbourg, France

Submitted 14 November 2007 ; accepted in final form 16 January 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
When using 13C tracer to measure plasma fat oxidation, an acetate recovery factor should be determined in every subject to correct for label sequestration. Less is known regarding the acetate recovery factor for dietary fatty acid oxidation. We compiled data from six studies to investigate the determinants of the dietary acetate recovery factor (dARF) at rest and after physical activity interventions and compared the effects of different methods of dARF calculation on both the fat oxidation and its variability. In healthy lean subjects, dARF was 50.6 ± 5.4% dose (n = 56) with an interindividual coefficient of variation of 10.6% at rest and 9.2% after physical activity modifications. The physical activity interventions did not impact dARF, and the intraindividual coefficient of variation was 4.6%. No major anthropological or physiological determinants were detected except for resting metabolic rate, which explains 7.4% of the dARF variability. Applying an individual or an average group dARF did not affect the mean and the variability of the derived dietary lipid oxidation at rest or after physical activity interventions. Using a mean dARF for a group leads to over- or underestimation of fat oxidation of less than 10% in individual subjects. Moreover, the use of a group or individual correction did not affect the significant relationship found between fasting respiratory exchange ratio and dietary fat oxidation. These data indicate that an average dARF can be applied for longitudinal and cross-sectional studies investigating dietary lipid metabolism.

exogenous fatty acid oxidation; stable isotopes; mass spectrometry

STABLE ISOTOPES ARE EMPLOYED in clinical nutrition studies to measure fatty acid oxidation. The rate of excretion of labeled CO2 following a constant infusion or ingestion of 13C-labeled fatty acids is used to measure the tracer oxidized (2, 5, 6, 11). However, early studies (3) showed that the appearance of 13CO2 in breath is very low in the first hours of infusion, especially at rest. This finding suggested that part of the labeled carbon is temporarily sequestrated in the organism due to label dilution and/or redistribution, resulting in an underestimation of the tracer fat oxidation rates. Although dilution occurs mainly in the bicarbonate pool (4, 7), Wolfe and Jahoor (22) reported that the main sequestration of labeled carbon actually occurs in the TCA cycle. Indeed, labeled carbons were found in glutamate and glutamine and in lower quantity in glucose and lactate and pyruvate (15). On the basis of the observation that acetate is converted into acetyl-CoA and directly enters the TCA cycle, Sidossis et al. (14) were the first to propose that infused [14C]acetate can be used to correct for the retention of a 13C-fatty acid (13C-FA) in the organism.

Schrauwen et al. (12) compiled data from six studies and investigated the within- and between-subject variability of the infused acetate recovery factor (iARF). They observed a high interindividual variability (12.0%) that was explained by resting metabolic rate (RMR) adjusted for fat-free mass (FFM; 13%), percent body fat (15%), and fasting respiratory exchange ratio (RER; 9%). Together with the low intraindividual coefficient of variation (CV; 4%), this suggested that although the iARF is reproducible it should be measured in every subject, except when only the group mean data are of interest. In many countries the use of radioactive isotopes is restricted for ethical reasons, and the conclusions of Schrauwen and colleagues (12, 13) imply that two separate tests, performed under identical conditions, are needed to determine the iARF required to correct the tracer oxidation data. Although this might be feasible for infusion studies given the relatively short duration required to obtain a steady state ({approx}2 h) (13), it presents operational constraints for studies investigating dietary fat oxidation that require ≥10-h tests due to digestion and trafficking processes.

Surprisingly, apart from our studies looking at the effect of physical activity on dietary fat oxidation (1, 9, 18–21), we did not find data on the use of a dietary acetate recovery factor (dARF) to correct tracer oxidation rates. Before the determinants of dARF are investigated, however, it is vital to demonstrate that the rate of 13CO2 appearance reflects the intracellular metabolism of the ingested acetate and thus is comparable with the metabolism of acetate derived from the intracellular metabolism of a fatty acid. The two studies (9, 21) conducted at the University of Wisconsin to validate the use of d31-palmitate as a tracer of dietary fat oxidation strongly support the validity of a dARF correction in that d31-palmitate oxidation rates (measured as 2H2O recovery, and thus minimally subjected to sequestration) only equal those of dietary [1-13C]palmitate when the latter is corrected for isotopic sequestration using a dARF (Fig. 1). However, the cumbersome experimental implications of the Schrauwen et al. (12) acetate infusion study for dARF have to be tested for postprandial lipid oxidation studies.


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 1. [2H]palmitate recovery vs. [1-13C]acetate recovery corrected [1-13C]palmitate recovery at 10 and 9 h postdose at rest and during exercise, respectively. When [2H]palmitate is correlated with [1-13C]palmitate recovery corrected for acetate, the correlation r2 is 0.79 (n = 19).

 
Here, we combined data from six of our studies and reported data from 69 subjects to 1) determine the interindividual variability of the dietary acetate recovery factor and examine the factors that might explain this variability, 2) determine the effect of the physical activity level and of physical activity interventions on the acetate recovery factor, and 3) determine the reproducibility of the acetate recovery factor. Indeed, in five of the six studies considered here, the subjects performed acute exercise prior to the experimental protocols or were submitted to chronic modifications of their physical activity level, e.g., detraining or training sessions of different durations. On the basis of these results, we evaluated the effects of different methods of dARF calculation to correct the fat oxidation for label sequestration and their impact on both the fat oxidation rates and their variability.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Study design. For the present analysis, we compiled dARF data from six studies performed in our two laboratories: Votruba et al. (21), Votruba et al. (18), Raman et al. (9), Bergouignan et al. (1), the Women International Space Simulation for Exploration study (WISE), and the Strasbourg Lipid Oxidation study (LIPOX). The WISE study was completed in 2006 and is currently being prepared for publication, and the LIPOX study is still in progress.

Votruba et al. (21) and Raman et al. (9) were studies designed to validate deuterium-labeled fatty acids for the measurement of dietary fat oxidation in five women and two men at rest and in six women and seven men during a light exercise [25% of peak oxygen consumption (VO2 peak)] of >2 h. The second Votruba et al. (18) study investigated the effect of prior acute exercise on dietary fat utilization. In this analysis, we used a [1-13C]acetate recovery factor in ambulatory conditions and after acute heavy-intensity exercise (85% of VO2 peak) consisting of three bouts of 10–12 min with 10-min rest periods between bouts from seven women. Bergouignan et al. (1), WISE, and LIPOX were longitudinal studies aimed at determining the effect of chronic physical activity interventions on dietary lipid oxidation. Briefly, Bergouignan et al. (1) and WISE were long-term bed rests of 3 mo in 15 men and of 2 mo in 16 women, respectively. Both were performed at the Institute of Space Medicine and Physiology in Toulouse, France, and supported by the Centre National d'Etudes Spatiales (France), the European Space Agency, the Canadian Space Agency, and NASA. During the bed rest period, the volunteers were randomly divided in two groups [Bergouignan et al. (1): n = 8 in the control group and n = 7 in the exercise group; WISE: n = 8 each]: one severely detrained group that strictly stayed in bed and one group subjected to different exercise countermeasures. The exercise group also remained supine but concomitantly performed either resistance exercise training 4 times/wk (men) or resistance exercise training every 3 days combined with an aerobic exercise training 4 days/wk (women). Details on these exercise-training protocols can be found elsewhere (16). The LIPOX study investigates the effect of training and detraining on dietary lipid partitioning in men and took place at the Hautepierre University Hospital of Strasbourg (France). Detraining was imposed on active subjects with a physical activity level (PAL >1.7) and consisted of 1 mo of voluntary reduction in structured and spontaneous physical activities. Aerobic training was emphasized for 2 mo in sedentary subjects (PAL <1.5) and consisted of four 60-min sessions per week at 50% VO2 max. Here, we present the results of the first 11 male subjects (n = 8 in the detrained group and n = 3 in the trained group). The protocols of WISE and LIPOX were approved by the Institutional Review Board of Midi-Pyrénées I (France) and of Alsace I (France), respectively. For all but the validation studies, dietary acetate and lipids recoveries were measured before and after the intervention on physical activities.

Subjects. Data from 69 participants, 34 women and 35 men, were compiled. Of these, 49 subjects were studied twice. From these six studies, we collected the following parameters: body weight and composition, RMR, VO2 peak, and fasting RER. The initial subject characteristics are shown in Table 1. Few data are missing because of difference in the protocol's objectives.


View this table:
[in this window]
[in a new window]

  		Table 1. Characteristics of the participants of each study

 
Acetate recovery factor and dietary fatty acid oxidation protocols. The experimental protocols to measure the dARF and the dietary fatty acid oxidation were similar in each of the studies included herein, and in each individual study both tests were conducted in few days apart under identical experimental conditions. Thirty-six hours before the test, all subjects were provided with microwaveable meals at breakfast, lunch, and dinner and were asked to not participate in any structured physical activities. After an overnight fast the subjects were weighed, and baseline breath and urine samples were collected. RMR and RER were measured for 1 h using indirect calorimetry under the supervision of the investigators. Then, a standard breakfast was provided to the participants, which included a homogenized liquid meal labeled with one of the following: 2 mg/kg [1-13C]acetate [>99% enriched; Cambridge Isotope Laboratories (CIL), Andover, MA], 10 mg/kg [1-13C]oleate (>99% enriched; CIL), or 10 mg/kg [1-13C]palmitate (>98% enriched; CIL), depending on the test day and the study. Breath samples were collected before the meal, every 30 min for the first 2 h, and then hourly for 7–10 h after the meal according to the study. Throughout the test, VCO2 was measured hourly by indirect calorimetry using a Deltatrac metabolic cart (Deltatrac II; GE) or a room calorimeter (respiratory chamber of the University of Wisconsin-General Clinical Research Center). The detailed experimental conditions of each study are summarized in Table 2.


View this table:
[in this window]
[in a new window]

  		Table 2. Experimental conditions of the acetate and lipid oral load tests

 
The body composition was assessed by the H218O isotope dilution method, as routinely used in our laboratories, in the Votruba et al. (21), Votruba et al. (18), Raman et al. (9), and LIPOX studies and by dual energy X-ray absorptiometry for the Bergouignan et al. (1) and WISE bed rests.

Sample analysis. The details on the isotope ratio mass spectrometry analyses can be found in previous studies from our laboratories (9, 18, 21). Briefly, the ratio of 13CO2 to 12CO2 in breath from the Votruba et al. (21), Votruba et al. (18), and Raman et al. (9) studies was measured and analyzed in triplicates on a Delta-S isotope ratio mass spectrometer (Finnigan MAT, San Jose, CA) using a continuous flow inlet system developed at the Department of Nutritional Sciences at the University of Wisconsin-Madison (10). For LIPOX and the two bed rest studies, a continuous flow system connected to GV Instruments Isoprime was used. The 18O enrichments in urinary water were measured by CO2 equilibration in quadruplicates in a continuous-flow isotope ratio mass spectrometer consisting of the above-cited Finnigan MAT Delta-S [Votruba et al. (21), Votruba et al. (18), Raman et al. (9)]. For the WISE and LIPOX studies, H218O was reduced to CO by carbon reduction at 1,400°C in an elemental analyzer (Flash HT; ThermoFisher Germany) coupled to a Delta-V isotope ratio mass spectrometer in Strasbourg, and isotopic abundances were measured in quintuplicate. All enrichments were expressed against International Atomic Energy Agency standards.

Calculations. Recovery of [1-13C]acetate, [1-13C]oleic acid, or [1-13C]palmitic acid was calculated as the instantaneous recovery of 13C in expired CO2 per hour, expressed as a percentage of the dose: %dose recovery/hour = [VCO2 x {Delta}{delta}Formulax RSTD/1,000]/[D x P x n/(MW x 100)] x 100, where {delta}Formula represents {delta}/mil = (Rsample/RSTD – 1) x 1,000, with R being the ratio 13C/12C, {Delta} represents {delta}Formula values corrected for each individual's baseline and for the meal-related natural background variation obtained from two subjects not dosed, RSTD represents 13C/12C of the standard CO2, P represents 13C isotope purity ([1-13C]acetate, 99% Na salt; 98% [1-13C]oleate; 99% [1-13C]palmitate), n represents the number of labeled atoms per molecule (1 for all tracers), MW is the molecular weight (Na[1-13C]acetate = 83; [1-13C]oleate = 282; [1-13C]palmitate = 257 g/mol), D is the dose (in g), and VCO2 is the carbon dioxide production in milliliters per hour. Cumulative recovery was calculated using the trapezoid rule. Cumulated acetate recoveries were further extrapolated to infinity according to the formula Ac({infty}) = Ac(t)/[1 – exp(–t/k)], with Ac(t) the acetate percentage recovery at time t, Ac({infty}) the acetate percentage recovery to infinity, and k the terminal time constant. Corrected fatty acid oxidation rates were calculated by dividing the cumulated fatty acid recovery by the cumulated acetate recovery extrapolated to infinity times 100.

Data organization and statistical analysis. Prior to the analysis, the normality of the data was ascertained by the Kolmogorov-Smirnov test.

We first considered the results from the subjects in resting conditions, i.e., the control (rest) tests from all the studies with interventions on physical activity. The study by Raman et al. (9) investigating the validity of deuterium-labeled fatty acid during acute exercise was therefore excluded from this first analysis. A one-way ANOVA was used on the resulting 56 subjects to test whether the dARF differs according to sex and study. The between-subject CV was calculated, and correlation analyses were used to test putative anthropological and physiological determinants of the dARF.

We then investigated the influence of different methods to apply the dARF on final fatty acid oxidation rates. The Votruba et al. (18) study was excluded from this analysis because dietary 13C-FA percentage recoveries corrected by individual dARF values were not available. The objective was to study the variability in dietary fat oxidation when individual, within-study mean, or between-study mean dARFs were used. The corresponding corrected fatty acid oxidation rates were identified as 13C-FAINDV, 13C-FASTUDY, and 13C-FAMEAN, respectively. To date, only the first two methods have been published by our teams (1, 18–20). The differences between the averages of 13C-FAINDV vs. 13C-FASTUDY and 13C-FAINDV vs. 13C-FAMEAN were compared using a paired t-test. We also tested whether or not the methods of correction influenced correlation analyses of fatty acid oxidation with the fasting RER, a predictor that displayed a negative correlation with dietary fatty acid oxidation. The slopes resulting from the different methods were compared by an analysis of covariance. We further evaluated the variability introduced by the three methods of correction calculating 13C-FAINDV, 13C-FASTUDY, and 13C-FAMEAN in our population for extreme values of fasting RER taken at the 10 (RER = 0.731) and 90% (RER = 0.930) quintiles.

In a third analysis, we determined the effect of physical activity interventions on the dARF and on the dARF-corrected dietary lipid oxidation rates. The analyses included subjects from the Bergouignan et al. (1), WISE, LIPOX, and Votruba et al. (18) studies. For the reason mentioned above, only the dARF was available for the Votruba et al. (18) study. Since no differences in cumulated acetate recoveries were noted between studies (F = 0.17, P = 0.95) or between sexes (F = 0.01, P = 0.91), we combined subjects based on the type of physical activity intervention to which they were submitted. Six groups were accordingly generated: acute heavy exercise, severe detraining, severe detraining plus resistance exercise, severe detraining plus resistance and aerobic exercise, detraining, and training. The effect of the physical activity intervention was determined by a multiple analysis of variance (MANOVA) with time as the repeated measurement (before vs. after the intervention) and physical activity group as main effect. Because we did not observe an effect of the physical activity modifications on the dARF (F = 0.01, P = 0.94), we calculated the within-subject CV.

Finally, the kinetics of the instantaneous and the cumulative acetate percentage recoveries was analyzed by a MANOVA with time as the repeated measurement and physical activity group as main effect followed by the protected leasy significant difference post hoc test.

All data are represented as means ± SD, and the level of significance was set as P < 0.05. All statistics were performed using JMP version 5.1.1 (SAS Institute).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Between-subjects CV of the acetate recovery factor. The average dARFs are summarized in Table 3. Overall, the average dARF in healthy subjects at rest was 50.6 ± 5.4% (n = 56), ranging between 37.3 and 63.8%, with a between-subject CV of 10.6%. This interindividual variability was not accounted for by differences between studies (F = 0.168, P = 0.95). Moreover, the dARF did not differ between women (50.7 ± 4.7%) and men (50.5 ± 6.0%, F = 0.012, P = 0.912). Neither age [r2 = 0.015, root mean square error (RMSE) = 5.4, P = 0.4], RER (r2 = 5.10–5, RMSE = 5.4, P = 0.96), FFM (r2 = 0.027, RMSE = 5.1, P = 0.2), nor percent body fat (r2 = 5.10–5, RMSE = 5.2, P = 0.9) explained the variability in dARF. dARF tended to have a relationship with VO2 peak in liters per minute (r2 = 0.107, RMSE = 739, P = 0.06), which disappeared when normalized for body mass (r2 = 0.047, RMSE = 7.2, P = 0.22). RMR explained 7.4% of the interindividual variability of the dARF (r2 = 0.074, RMSE = 5.2, P = 0.04; Fig. 2), but that was no longer a significant contribution when adjusted for FFM (r2 = 0.005, RMSE = 4.7, P = 0.71).


View this table:
[in this window]
[in a new window]

  		Table 3. [1-13C]Acetate recovery factor and effect of individual, within-study mean, or between-study mean acetate recovery factors on dietary fat oxidation variability in resting conditions

 

Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 2. Relationship between dietary acetate recovery factor (dARF) and resting metabolic rate (RMR) at rest in both men and women with distinction between the studies: Votruba et al. (21), Votruba et al. (18), Bergouignan et al. (1), Women International Space Simulation for Exploration study (WISE), and Lipid Oxidation study (LIPOX). The dietary fat oxidation correlates positively with RMR (y = 37.47 + 0.002x, r2 = 0.074, P = 0.04).

 
Effect of individual, within-study mean, or between-study mean acetate recovery factors on dietary fat oxidation variability. As illustrated in Fig. 3, regardless of whether we used individual, group mean, or grand mean dARF, the variability in fatty acid oxidation rates measured under resting control conditions remained the same. In all studies combined (n = 47), the CV was 36.3% for the 13C-FAINDV, 36.6% for the 13C-FASTUDY, and 36.0% for the 13C-FAMEAN. Because no within-subject or within-study variations were noted in dARF, such a large CV was explained by the between-study differences in fatty acid oxidation rates. As indicated in Table 3, we observed no differences between 13C-FAINDV and 13C-FASTUDY or 13C-FAMEAN; the mean dietary fatty acid oxidation was 35.0 ± 12.7, 35.0 ± 12.8, and 34.8 ± 12.6%, respectively. None of the average differences were different from zero. Applying the between-study acetate recovery factor to correct individual dietary fatty acid oxidation rates led to over- or underestimation of fat oxidation percentage recoveries of <10%.


Figure 3
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 3. Box plot representation of the variability of the %recoveries of [1-13C]fatty acid (13C-FA) corrected for label sequestration by individual (13C-FAINDV), within-study mean (13C-FASTUDY), or between-study mean (13C-FAMEAN) acetate recovery factor. The box plot represents the median of these data, the 25th and 95th percentiles at each side of the box, and the 10th and 90th percentiles at the extremes, and the black points are the values above and below the 90th and 10th percentiles, respectively. The mean dietary lipid oxidations are 35.0 ± 12.7, 35.0 ± 12.8, and 34.8 ± 12.6% for 13C-FAINDV, 13C-FASTUDY, and 13C-FAMEAN, respectively. No differences were noted between 13C-FAINDV vs. 13C-FASTUDY (P = 0.97) or vs. 13C-FAMEAN (P = 0.66).

 
We tested whether the method of correction affects the putative deterministic relationship of dietary fat oxidation and other metabolic parameters. We used the interesting relationship we observed in this population between the dietary fat oxidation and the fasting RER. The relationship remained significant irrespective of the type of correction for dietary fat oxidation applied (13C-FAINDV: r = –0.55, P < 0.0001; 13C-FASTUDY: r = –0.58, P < 0.0001; 13C-FAMEAN: r = –0.57, P < 0.0001; Fig. 4). More importantly, the slopes of the three regression lines were not different (type of correction-by-RER interaction: F = 0.02, P = 0.98). Using the equations of the respective regression lines, we calculated 13C-FAINDV, 13C-FASTUDY, and 13C-FAMEAN for those in our population with extreme values of fasting RER, 0.731 as the 10th percentile (13C-FAINDV = 43.8%, 13C-FASTUDY = 44.3%, and 13C-FAMEAN = 43.8%) and 0.930 as the 90th percentile (13C-FAINDV = 27.4%, 13C-FASTUDY = 26.9%, and 13C-FAMEAN = 27.0%). Such extreme extrapolation results in a 0.5% maximum difference in the estimated fat oxidation rates.


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 4. Relationship between the dietary fatty acid oxidation corrected by 13C-FAINDV, 13C-FASTUDY, or 13C-FAMEAN acetate recovery factor and the fasting respiratory exchange ratio (RER). RER has a significant negative correlation with the dietary fatty acid oxidation, whatever the method of correction (13C-FAINDV: r2 = 0.30, P < 0.0001; 13C-FASTUDY: r2 = 0.33, P < 0.0001; 13C-FAMEAN: r2 = 0.32, P < 0.0001). The slopes of the 3 regression lines are not different (type of correction-by-RER interaction: F = 0.02, P = 0.98).

 
Effect of interventions on physical activity on acetate recovery factor and the related dietary fat oxidation rates. In this subgroup of subjects, the overall initial between-subject CV for dARF was 10.1% (Table 4). After intervention, the CV was 9.2% and the average dARF 50.6 ± 4.7% (n = 49), ranging from 39.7 to 60.4%. When we added the 13 subjects from the acute light exercise validation study of Raman et al. (9), the CV was 12.2%. We observed neither a group effect (F = 0.14, P = 0.15) nor an effect of the exercise regimen (F = 0.01, P = 0.94) on the dARF value (time-by-exercise regimen interaction not significant). Consequently, the within-subject CV was 4.6%. The 13C-FA percentage recoveries corrected by either the individual dARF (40.4 ± 8.8%, F = 1.10, P = 0.30), the within-study mean dARF (40.2 ± 8.1%, F = 1.55, P = 0.22), or the between-study mean dARF (40.6 ± 8.1%, F = 0.83, P = 0.37) were not modified by the interventions on physical activity. None of the interactions were significant (time-by-exercise regimen interaction for 13C-FAINDV: F = 1.42, P = 0.25; 13C-FASTUDY: F = 0.61, P = 0.19; 13C-FAMEAN: F = 0.84, P = 0.51). Moreover, as observed in Fig. 5, the variability in dietary fat oxidation was not affected by the different calculations.


View this table:
[in this window]
[in a new window]

  		Table 4. Effect of interventions on physical activity on acetate recovery factor and the related dietary fat oxidation rates

 

Figure 5
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 5. Box plot representation of the variability of the %recoveries of 13C-FA corrected for label sequestration by 13C-FAINDV, 13C-FASTUDY, and 13C-FAMEAN acetate recovery factor before and after the following interventions on the physical activity: detraining (top left), severe detraining (bottom left), severe detraining accompanied by a resistive exercise (RE) training (top right), and severe detraining accompanied by a combined RE and aerobic exercise (AE) training (bottom right). The box plot represents the median of these data, the 25th and 95th percentiles at each side of the box, and the 10th and the 90th percentiles at the extremes, and the black points are the values above and below to the 90th and 10th percentiles, respectively.

 
Kinetic results: instantaneous and cumulative acetate recoveries. Figure 6 depicts the kinetics of instantaneous and cumulative acetate percentage recoveries of each physical activity group. For the MANOVA analyses, we took into account the data until 420 min postdose, the last common time point between all the studies. We observed a significant interaction between the kinetics of the instantaneous acetate percentage recoveries and both the physical activity levels (F = 0.18, P < 0.0001) and the studies (F = 14.3, P < 0.0001). Similar significant interactions between the kinetics of the cumulative acetate recoveries and both the physical activity level (F = 0.17, P < 0.0001) and the studies (F = 0.02, P <0.0001) were also noted. However, further analyses showed that the differences between studies and activity groups disappeared from time 240 and 300 min postdose for the instantaneous and cumulative kinetics of acetate percentage recoveries, respectively. Furthermore, cumulative acetate percentage recoveries extrapolated to the infinite did not show any study or group effects.


Figure 6
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 6. Kinetics of instantaneous (top) and cumulative (bottom) acetate %recoveries of each physical activity group. Multiple ANOVA performed until 420 min postdose, the last common point between all the studies, showed a significant interaction between the kinetics of both the instantaneous and cumulative acetate %recoveries and both the physical activity levels (instantaneous: F = 0.18, P < 0.0001; cumulative: F = 0.17, P < 0.0001) and the studies (instantaneous: F = 14.3, P < 0.0001; cumulative: F = 0.02, P < 0.0001). Post hoc analyses showed that differences in instantaneous and cumulative kinetics of acetate %recoveries and both studies and groups of physical activity disappeared from 240 and 300 min postdose, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Since the introduction of the acetate correction factor by Sidossis et al. (14), estimations of plasma fatty acid oxidation are corrected for the sequestration of the tracer in the TCA cycle and in the bicarbonate pool. Although the characteristics of the acetate recovery factor measured under infusion conditions to correct plasma fat oxidation have been widely studied, the variation and the reproducibility of the dietary acetate recovery factor have not yet been fully investigated. Such an investigation is warranted because it may lead to economies in the complex protocols used for many lipid oxidation studies.

The present study is the first to analyze the dARF under ingestion conditions to correct dietary fat oxidation. By compiling data from six distinct studies, which measured the exogenous [1-13C]acetate recovery, we found an average dARF of 50.6 ± 5.4% dose in healthy subjects at rest. This value illustrates that omission of this factor when dietary lipid metabolism is studied would result in a twofold underestimation of fatty acid oxidation. This result is in accordance with percentage recoveries of [1-13C]acetate of 49, 45, and 54% reported under infusion conditions at rest by Trimmer et al. (17) in five endurance-trained males, by Pouteau et al. (8) in five healthy volunteers, and by Sidossis et al. (14) in six healthy subjects, respectively. This similarity between iARF and dARF led us to apply the model established by Sidossis et al. under infusion conditions to predict dARF. Nevertheless, when we applied this single exponential model on WISE data, taking into account the average VO2 measured all along the duration of the acetate test, the predicted dARF (56.7%) was significantly higher than the measured dARF (49.9%) (P < 0.0001). The time delay associated with the gastrointestinal transit and absorption processes is likely responsible for this difference.

On the other hand, our average dARF is twofold higher than the average iARF of 23% measured between 90 and 120 min after the start of the acetate infusion in 89 subjects reported in the compilation study of Schrauwen et al. (12). They used the [1,2-13C]acetate, and the difference in tracer position explains the difference in the acetate recovery found between our study and the study of Schrauwen et al. Indeed, in 1990, Wolfe and Jahoor (22) studied the effect of the position of the labeled carbon atom on percentage-recovered label and showed that after a 4-h infusion period 81% of [1-13C]acetate infused was recovered, whereas only 53% of [2-13C]acetate was recovered. Therefore, the acetate-labeling pattern should be the same as the fatty acid-labeling pattern, and only the acetate tracer with the label carbon in the first position should be used to correct the dietary [1-13C]fatty acid oxidation.

Furthermore, we determined a between-subject CV of 10.6% in resting conditions, with dARF values ranging from 37.3 to 63.8%, and a within-subject CV of 4.6%. Clearly, if the protocols had been simply repeated in the same subjects without any intervention on physical activity (although not significant), the intraindividual CV would likely be lower. The between-study variability is observed in the kinetics of both instantaneous and cumulative percentage recoveries of acetate. This variability may be explained by differences in the subjects and in the protocols employed in each study. Moreover, even if the gastrointestinal processes remained up to 4 h, differences between individuals may account for the intervariability in the kinetics of instantaneous recoveries of acetate. But once the complete absorption is assumed to have been obtained, which may correspond to 240–300 min after the breakfast ingestion, the percentage values of instantaneous or cumulative acetate recovery no longer differ between subjects. Therefore, the dARF, calculated by the extrapolation to the infinite of the cumulative percentage recovery of [1-13C]acetate, is not different between studies. We suggest that the major difference between the determinants of variation between dARF and iARF reflect the shorter time interval for iARF studies and that these differences dissipate with the longer study times of most dARF studies. However, only new investigations with a longer duration of acetate infusion may distinguish the respective influences of the methods (ingestion vs. infusion) and of the duration of the protocol in the observed variability in iARF. Another point to consider is our mathematical method of extrapolation to infinite, which may decrease the variability in dARF. Indeed, this mathematical method based on the 7- to 10-h cumulated recovery percentage of 13C differs from the studies using infusion, where a standard period of 2–3 h of infusion is used to reach a steady state.

Contrary to the iARF (12), the dARF did not show differences between men and women. Moreover, we did not find a relationship between the dARF at rest and age, RER, or the percentage of body fat as observed in the Schrauwen et al. study (12), and VO2 peak tended to correlate only positively with dARF. Only RMR displayed a weak but significant, positive correlation with the acetate correction factor, explaining 7.4% of the variability. This lack of a significant relationship may be explained by low ranges in the considered parameters due to a standard recruitment of only healthy and normal-weight volunteers and by the fact that the protocols were performed at rest and not during exercise. For example, in the studies included herein, the percentage of body fat ranged from 17 to 31% and percent fat did not correlate significantly the dARF, whereas in the studies performed by Schrauwen et al. (12) the percentage of body fat ranged from 10 to >40%, and a significant negative relationship between the iARF and percent fat was found. Nevertheless, to better point out the potential role of percent fat on the dARF, identical investigations in the LIPOX study context are currently being undergone in our laboratory in obese persons. Moreover, whereas carbon in position 1 entering the TCA cycle is oxidized mostly to CO2 within the second rotation, carbon 2 of acetyl-CoA is not eliminated as CO2 before three rotations of the TCA cycle and thus is more likely involved in labeling TCA cycle metabolites such as glucose and glutamine (8). Consequently, the [1,2-13C]acetate used in the study by Schrauwen et al. presents a recovery percentage more likely influenced by the metabolic state of the subjects, which likely explains the associations between iARF and the subjects’ characteristics. Although dARF is characterized by a similar interindividual CV that has been reported for iARF (12.0%) (12), no major anthropological or physiological determinants for dARF have been elucidated.

Because of this high interindividual CV, Schrauwen et al. (12) strongly advised to determine an iARF for every subject. Nevertheless, use of an individual, an average group, or the grand mean correction factor does not affect the dietary lipid oxidation variability, which represents the most interesting finding of the present study. Indeed, the percentage recoveries of 13C-FA corrected by the individual, the within-study mean, or the between-study mean acetate recovery factor are not significantly different and have the same mean and variability, ~35 ± 13%. But more importantly, these three values have a similar between-subject CV of 36%, which suggests that this high variability is not due to the method of correction but rather by the differences inherent to the studies. Using a mean dARF for a group as a whole leads to changes in fatty acid oxidation values of <10% in individual subjects (observed range from –7.4 to 9.7%), whereas Schrauwen et al. (13) reported a difference of >30% between the plasma fatty acid oxidations corrected by the individual iARF and those corrected by an average group recovery factor. This discrepancy between our results (10 vs. 30%) is surprising since the iARF and dARF present similar CVs. Going back on, we did not find sufficient data in the Schrauwen et al. paper (13) to compare their method of fat oxidation calculations with ours. However, we can speculate that this difference may be attributed to extreme values in iARF in a larger sample size that also included a larger variability in body mass index (BMI; i.e., 19 kg/m2 < BMI < 30 kg/m2) than in our studies (i.e., 20 kg/m2 < BMI < 25 kg/m2). We can also assume that the dietary acetate and lipid metabolism present a lower variability than the metabolism of the plasma acetate and lipid, suggesting that the digestion processes likely act as a buffer to reduce interindividual differences or that the longer postdose measurement period of the dietary studies obviates the interindividual differences. Consequently, we conclude that it is valid to apply a mean dARF of 51% to correct the dietary lipid oxidation in all of the subjects. This result allows a compromise between the accuracy of the measurement, the cost of the tracers, and the intensity of the protocols.

For investigations aiming to determine the effect of one treatment on the dietary fat oxidation or to compare dietary fat oxidation between different groups of subjects, we advise the use of this average group dARF method of correction since only accurate and not precise measurements are required for having relevant results. This method has already been used in the Votruba et al. (18) study to compare the effect or prior exercise on subsequent fat utilization. In this study, the end cumulative recovery of [1-13C]oleate was corrected by a dARF measured in exactly similar conditions in seven other subjects involved in a previous study. Nevertheless, in clinical research and in particular obesity and diabetes research, correlative investigations are crucial to assess relationships between dietary fat oxidation and other metabolic parameters, since the dietary fat partitioning between storage and oxidation has major implications for the regulation of the body mass and composition and for other metabolic parameters such as insulin sensitivity or plasma lipid levels. Because such correlative studies require both accurate and precise measurements of the dietary fatty acid oxidation, a dARF determined for every subject might be preferable in light of the observation that within-subject CVs for dARF were a little smaller than between-subject CVs. Nonetheless, we show that the relationship observed between fasting RER and dietary fatty acid oxidation corrected by individual dARF remained unchanged for both average and extreme values when we applied the within-subject mean and the between-subject mean dARFs. This result indicates that an average dARF may be used to correct dietary lipid oxidation in healthy subjects. Further investigations would be required to confirm these results in diabetic and obese subjects before the acetate test in studies involving them was suppressed.

The present study also analyzes the dARF in groups of different physical activity levels. After acute or chronic interventions on physical activity the recoveries are similar to those measured at rest, with an average dARF of 50.6 ± 4.7%, ranging from 39.7 to 60.4%, and with a between-subject CV of 9.2%. The modifications of physical activity do not affect the variability of the dARF. Votruba et al. (18) reported that [1-13C]acetate recovery 7.5 h postdose measured in seven healthy women submitted to four distinct trials was 49.2 ± 2.8, 47.4 ± 3.3, and 46.4 ± 2.8% for recent periods of rest, light, and heavy exercise, respectively, and was not significantly different. Furthermore, using an individual or an average dARF for label sequestration affected neither the variability nor the mean of the dietary lipid oxidation after intervention on physical activity. Taken together, these results indicate that, for studies investigating the effect of physical activity level on dietary fat oxidation with a longitudinal or comparative design, a mean dARF of 51% may also be applied as in resting conditions.

In conclusion, we show that the dietary acetate recovery factor has a low interindividual variability both at rest and after interventions involving physical activity of different intensity levels. Using an average group acetate recovery factor does not affect the mean and the variability of the dietary lipid oxidation and is considered valid for both deterministic and comparative studies investigating lipid metabolism. Compiling data from 69 subjects, we propose an average dietary acetate recovery factor of 51%, which obviates the need for an additional separate acetate test and represents an acceptable scientific trade-off between the cost of the experiments, the cumbersome aspects of the protocols, and the accuracy of the measurements.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The WISE study was sponsored by the European Space Agency, NASA, the Canadian Space Agency, and the French "Centre National d'Etudes Spatiales," which has been the "Promoteur" of the study, according to French law. The study has been performed by MEDES. The LIPOX study was sponsored by the the Programme National de Recherche en Alimentation et Nutrition Humaine, the Fondation Couer et Artères, and the Louis Pasteur University (Strasbourg, France).

A. Bergouignan was supported by a grant cofunded by the French Space Agency and the Alsace Region. S. Blanc was funded by the Fondation Betencourt Schueller.


   ACKNOWLEDGMENTS
 
We are grateful for the outstanding contributions of all the volunteers who committed themselves to the success of these studies. We also thank the MEDES, Institute for Space Physiology and Medicine (Toulouse, France) staff for excellent support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Blanc: Institut Pluridisciplinaire Hubert Curien-Département d'Ecologie, Physiologie, Ethologie UMR CNRS Université Louis Pasteur 7178, 23 rue Becquerel 67087, Strasbourg, France (e-mail: stephane.blanc{at}c-strasbourg.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bergouignan A, Schoeller DA, Normand S, Gauquelin-Koch G, Laville M, Shriver T, Desage M, Maho YL, Ohshima H, Gharib C, Blanc S. Effect of physical inactivity on the oxidation of saturated and monounsaturated dietary fatty acids: results of a randomized trial. PLoS Clin Trials 1: e27, 2006.[CrossRef][Medline]
  2. Bessesen DH, Rupp CL, Eckel RH. Trafficking of dietary fat in lean rats. Obes Res 3: 191–203, 1995.[Web of Science][Medline]
  3. Heiling VJ, Miles JM, Jensen MD. How valid are isotopic measurements of fatty acid oxidation? Am J Physiol Endocrinol Metab 261: E572–E577, 1991.[Abstract/Free Full Text]
  4. Irving CS, Wong WW, Shulman RJ, Smith EO, Klein PD. [13C]bicarbonate kinetics in humans: intra- vs. interindividual variations. Am J Physiol Regul Integr Comp Physiol 245: R190–R202, 1983.[Abstract/Free Full Text]
  5. Jones PJ, Pencharz PB, Clandinin MT. Whole body oxidation of dietary fatty acids: implications for energy utilization. Am J Clin Nutr 42: 769–777, 1985.[Abstract/Free Full Text]
  6. Klein S, Wolfe RR. The use of isotopic tracers in studying lipid metabolism in human subjects. Baillieres Clin Endocrinol Metab 1: 797–816, 1987.[CrossRef][Web of Science][Medline]
  7. Leijssen DP, Elia M. Recovery of 13CO2 and 14CO2 in human bicarbonate studies: a critical review with original data. Clin Sci (Lond) 91: 665–677, 1996.[Medline]
  8. Pouteau E, Maugere P, Darmaun D, Marchini JS, Piloquet H, Dumon H, Nguyen P, Krempf M. Role of glucose and glutamine synthesis in the differential recovery of 13CO2 from infused [2–13C] versus [1–13C] acetate. Metabolism 47: 549–554, 1998.[CrossRef][Web of Science][Medline]
  9. Raman A, Blanc S, Adams A, Schoeller DA. Validation of deuterium-labeled fatty acids for the measurement of dietary fat oxidation during physical activity. J Lipid Res 45: 2339–2344, 2004.[Abstract/Free Full Text]
  10. Schoeller DA, Luke AH. Rapid 18O analysis of CO2 samples by continuous-flow isotope ratio mass spectrometry. J Mass Spectrom 32: 1332–1336, 1997.[CrossRef][Web of Science][Medline]
  11. Schoeller DA, Schneider JF, Solomons NW, Watkins JB, Klein PD. Clinical diagnosis with the stable isotope 13C in CO2 breath tests: methodology and fundamental considerations. J Lab Clin Med 90: 412–421, 1977.[Web of Science][Medline]
  12. Schrauwen P, Blaak EE, Van Aggel-Leijssen DP, Borghouts LB, Wagenmakers AJ. Determinants of the acetate recovery factor: implications for estimation of [13C]substrate oxidation. Clin Sci (Lond) 98: 587–592, 2000.[Medline]
  13. Schrauwen P, van Aggel-Leijssen DP, van Marken Lichtenbelt WD, van Baak MA, Gijsen AP, Wagenmakers AJ. Validation of the [1,2–13C]acetate recovery factor for correction of [U-13C]palmitate oxidation rates in humans. J Physiol 513: 215–223, 1998.[Abstract/Free Full Text]
  14. Sidossis LS, Coggan AR, Gastaldelli A, Wolfe RR. A new correction factor for use in tracer estimations of plasma fatty acid oxidation. Am J Physiol Endocrinol Metab 269: E649–E656, 1995.[Abstract/Free Full Text]
  15. Sidossis LS, Coggan AR, Gastaldelli A, Wolfe RR. Pathway of free fatty acid oxidation in human subjects. Implications for tracer studies. J Clin Invest 95: 278–284, 1995.[Web of Science][Medline]
  16. Trappe S, Creer A, Slivka D, Minchev K, Trappe T. Single muscle fiber function with concurrent exercise or nutrition countermeasures during 60 days of bed rest in women. J Appl Physiol 103: 1242–1250, 2007.[Abstract/Free Full Text]
  17. Trimmer JK, Casazza GA, Horning MA, Brooks GA. Recovery of 13CO2 during rest and exercise after [1-13C]acetate, [2-13C]acetate, and NaH13CO3 infusions. Am J Physiol Endocrinol Metab 281: E683–E692, 2001.[Abstract/Free Full Text]
  18. Votruba SB, Atkinson RL, Hirvonen MD, Schoeller DA. Prior exercise increases subsequent utilization of dietary fat. Med Sci Sports Exerc 34: 1757–1765, 2002.
  19. Votruba SB, Atkinson RL, Schoeller DA. Prior exercise increases dietary oleate, but not palmitate oxidation. Obes Res 11: 1509–1518, 2003.[Web of Science][Medline]
  20. Votruba SB, Atkinson RL, Schoeller DA. Sustained increase in dietary oleic acid oxidation following morning exercise. Int J Obes 29: 100–107, 2005.[CrossRef][Web of Science][Medline]
  21. Votruba SB, Zeddun SM, Schoeller DA. Validation of deuterium labeled fatty acids for the measurement of dietary fat oxidation: a method for measuring fat-oxidation in free-living subjects. Int J Obes Relat Metab Disord 25: 1240–1245, 2001.[CrossRef][Web of Science][Medline]
  22. Wolfe RR, Jahoor F. Recovery of labeled CO2 during the infusion of C-1- vs C-2-labeled acetate: implications for tracer studies of substrate oxidation. Am J Clin Nutr 51: 248–252, 1990.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 DiabetesHome page
A. Bergouignan, G. Trudel, C. Simon, A. Chopard, D. A. Schoeller, I. Momken, S. B. Votruba, M. Desage, G. C. Burdge, G. Gauquelin-Koch, et al.
Physical Inactivity Differentially Alters Dietary Oleate and Palmitate Trafficking
Diabetes, February 1, 2009; 58(2): 367 - 376.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/4/E645    most recent
00720.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bergouignan, A.
Right arrow Articles by Blanc, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bergouignan, A.
Right arrow Articles by Blanc, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.