Measurement of exogenous carbohydrate oxidation: a comparison of [U-14C]glucose and [U-13C]glucose tracers

L. Moseley,1 R. L. P. G. Jentjens,1 R. H. Waring,2 R. M. Harris,2 L. K. Harding,3 and A. E. Jeukendrup1

Human Performance Laboratory, 1Schools of Sport and Exercise Sciences and 2Biosciences, The University of Birmingham, Edgbaston, Birmingham, United Kingdom; and 3Physics and Nuclear Medicine Department, City Hospital, Birmingham, United Kingdom

Submitted 6 September 2004 ; accepted in final form 10 February 2005


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The purpose of this study was to assess the level of agreement between two techniques commonly used to measure exogenous carbohydrate oxidation (CHOEXO). To accomplish this, seven healthy male subjects (24 ± 3 yr, 74.8 ± 2.1 kg, O2max 62 ± 4 ml·kg–1·min–1) exercised at 50% of their peak power for 120 min on two occasions. During these exercise bouts, subjects ingested a solution containing either 144 g glucose (8.7% wt/vol glucose) or water. The glucose solution contained trace amounts of both [U-13C]glucose and [U-14C]glucose to allow CHOEXO to be quantified simultaneously. The water trial was used to correct for background 13C enrichment. 13C appearance in the expired air was measured using isotope ratio mass spectrometry, whereas 14C appearance was quantified by trapping expired CO2 in solution (using hyamine hydroxide) and adding a scintillator before counting radioactivity. CHOEXO measured with [13C]glucose ([13C]CHOEXO) was significantly greater than CHOEXO measured with [14C]glucose ([14C]CHOEXO) from 30 to 120 min. There was a 15 ± 4% difference between [13C]CHOEXO and [14C]CHOEXO such that the absolute difference increased with the magnitude of CHOEXO. Further investigations suggest that the difference is not because of losses of CO2 from the trapping solution before counting or an underestimation of the "strength" of the trapping solution. Previous research suggests that the degree of isotopic fractionation is small (S. C. Kalhan, S. M. Savin, and P. A. Adam. J Lab Clin Med89: 285–294, 1977). Therefore, the explanation for the discrepancy in calculated CHOEXO remains to be fully understood.

exercise; isotopic fractionation; enrichment; specific activity


BOTH STABLE AND RADIOACTIVE TRACERS have been used in the study of human metabolism. For example, to study the oxidation of ingested carbohydrate (CHO), it is common to use a 13C tracer (1, 8, 1315, 18, 23, 27, 29) or 14C tracer (4, 5, 911, 19, 24) to trace a CHO of interest. Whichever tracer is used, the principles and assumptions are the same, and the calculations depend on the enrichment (13C) or specific activity (SA; 14C) of tracer in the ingested CHO, the enrichment/SA of tracer in expired gas, and the rate of carbon dioxide production (CO2). Stable isotopes are measured in solids or expired gases by separation and quantification of the tracer-to-tracee ratio by mass spectrometry. Radioactive isotopes are measured after a quantity of CO2 is trapped in solution and mixed with a scintillation liquid. The scintillator absorbs the radiation and releases the energy as photons, which are subsequently detected by a photomultiplier. The 13C method is generally considered to be the gold standard, since the potential for errors would seem to be greater in the measurement of 14C SA compared with 13C enrichment (17).

Without data reporting the level of agreement between two different methods, comparisons between those data sets is difficult. In addition, data regarding the agreement between the two techniques would be useful in developing more complex experimental designs, e.g., quantifying the individual oxidation rates of two different CHO ingested simultaneously. It has been demonstrated that such experiments can be conducted using only 13C tracers but with an experimental design that requires each subject to complete three experimental trials differing in the enrichment of the ingested CHOs (1, 21). The results are then combined, and the oxidation rate of each CHO is calculated using simultaneous equations. The combination of three sets of results amplifies the effects of within-subject variation and extraneous variables, potentially increasing errors. An alternative method would be to label the ingested CHO with different tracers ([13C]fructose and [14C]glucose for example), allowing the simultaneous measurement of the oxidation rates in one exercise trial (12). However, this design depends on the assumption that both methods are in direct agreement; therefore, this study aims to directly compare exogenous carbohydrate oxidation rate (CHOEXO) measured with [13C]glucose and [14C]glucose.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects. Seven trained cyclists/triathletes whose characteristics [age, mass, and maximal oxygen uptake (O2 max)] were 24 ± 3 yr, 74.8 ± 2.1 kg, 62 ± 4 ml·kg–1·min–1 and whose maximal aerobic power (Wmax) was 370 ± 13 W participated in this study. Subjects were fully informed of the nature, purpose, and associated risks of the study before providing written consent. All subjects were healthy as deemed by a general health questionnaire. The South Birmingham Local Research Ethics Committee approved all procedures before the onset of the study.

General design. Subjects visited the laboratory for three exercise tests, a preliminary exercise test to determine the workload for the experimental trials and two experimental trials consisting of 120 min of steady-state cycling exercise. During the experimental trials, subjects ingested an experimental beverage (8.7% glucose solution or water) at regular intervals. The glucose solution was labeled with trace amounts of [U-13C]glucose and [U-14C]glucose, and samples of expired gas were collected to allow determination of CHOEXO.

Preliminary testing. All subjects attended the laboratory ~7 days before the first experimental trial for an incremental exercise test to exhaustion on an electromagnetically braked cycle ergometer (Excalibur Sport; Lode, Groningen, The Netherlands). The test began at an intensity of 95 W, and the workload was increased by 35 W every 3 min until exhaustion. Heart rate (HR) was recorded continuously during the test using radio telemetry (Polar Vantage NV; Polar Electro, Kempele, Finland). Wmax was calculated from the last completed work rate, plus the fraction of time (in s) spent in the final noncompleted work rate multiplied by the work rate increment. Breath-by-breath measurements were performed throughout exercise using an on-line automated gas analysis system (Oxycon Pro; Jaeger, Wuerzberg, Germany). The volume and gas analyzers of the system were calibrated using a 3-liter calibration syringe and calibration gas (4.95% CO2-balance N). Oxygen uptake (O2) was considered to be maximal when at least two of the following three criteria were met: 1) a leveling off of O2 with increasing work rate (increase of no more than 2 ml·kg–1·min–1), 2) an HR within 10 beats/min of the predicted maximum (220 beats/min – age), 3) a respiratory exchange ratio (RER) >1.05. O2 max was calculated as the average O2 over the last 60 s of the test.

Experimental trials. All exercise was performed in the morning with subjects having fasted for at least 10 h and having refrained from strenuous activity and alcohol for 24 h. On arrival in the laboratory, each subject's body mass was recorded (model 708; Seca, Hamburg, Germany) and a Teflon catheter (Quickcath II; Baxter, Norfolk, UK) was inserted into an antecubital vein for blood sampling. Before the onset of exercise, resting breath samples were collected. Exercise consisted of 120 min of cycling on an electromagnetically braked cycle egometer at 50% of Wmax, with breath samples collected at 15-min intervals throughout exercise. O2 and CO2 were measured for 4-min periods every 15 min, as described above. Recordings were made as the mean of eight breaths, and data were averaged every 30 s.

Beverages. Subjects consumed 600 ml liquid at the onset of exercise and 150 ml every 15 min for the remainder of exercise (1.65 liters in total). Beverages were either water (WATER) or a glucose solution delivering 1.2 g/min glucose (Glc); both beverages contained 20 mmol/l sodium in the form of sodium chloride. The CHO beverage was prepared with wheat-derived glucose (Amylum, London, UK) with a naturally low 13C enrichment [–27.5 {delta}13C{permil} vs. Pee Dee Bellemnitella (PDB)]. To this 0.0748 g/l [U-13C]glucose (Cambridge Isotope Laboratories) and 0.45 MBq/l [U-14C]glucose (Amersham Pharmacia Biotech, Little Chalfont, UK) was added, resulting in a 13C enrichment of 45.2 {delta}13C{permil} vs. PDB and a radiation dose rate of 0.37 MBq/h (58,963 ± 639 dpm/mmol ingested glucose). The 13C enrichment and 14C SA of the beverage was measured using isotope ratio mass spectrometry (EA-IRMS; Europa Scientific, Crewe, UK) and a liquid scintillation counter (LS 1800; Beckman) respectively.

Breath sample collection and analysis. Subjects exhaled via a two-way mouthpiece in a 6-liter mixing chamber for no less than 60 s before sample collection. Samples for the determination of 13C enrichment were then collected in duplicate in 10-ml evacuated tubes (Exetainers; Labco, Buckinghamshire, UK) and analyzed using continuous-flow isotope ratio mass spectrometry (IRMS; Europa Scientific). Briefly, the contents of samples and references were flushed and transported by helium carrier gas through a packed-column gas chromatograph, held at 75°C. The resultant chromatographic peak then entered the IRMS, where the isotopomers at mass-to-charge ratio 44, 45, and 46 for CO2 were measured and a {delta}13C value determined. The reference gas used during analysis was 3.3% CO2 in a helium balance with {delta}13C = –29.01 vs. PDB. The 3.3% CO2 mixture was prepared from a CO2 cylinder calibrated against calcite (NBS-19); ({delta}13C value of +1.95 vs. PDB), an isotope reference standard distributed by the International Atomic Energy Agency, Vienna.

Duplicates of 28 breath samples were analyzed in a second laboratory using a different mass spectrometer (MAT 252; Finnigan, Bremen, Germany) to verify the 13C enrichment data. The mean changes in enrichment from baseline were similar between duplicates (6.12 ± 0.37 and 6.12 ± 0.38 {delta}13C{permil} vs. PDB) with no significant difference between data sets (P = 0.234).

For the determination of breath 14C activity, a 6-liter rubber anesthetic gas bag was filled from the mixing chamber via a two-way Hans Rudolph valve. The collected air was then passed in duplicate through a solution containing 1 ml hyamine hydroxide in 1 M methanol (Zinsser Analytic, Berkshire, UK), 2 ml of 96% ethanol (BDH Laboratory Supplies, Poole, UK), and one to two drops of phenolphthalein (Riedel-de Haën, Seeize, Germany; see Ref. 10). When the breath sample was passed through this trapping solution, a color change (pink to clear) occurred when exactly 1 mmol CO2 was trapped in the solution. Scintillation cocktail (17 ml; Ready Gel, Beckman Coulter, High Wycombe, UK) was then added to this liquid before samples were mixed thoroughly, and activity (cpm) was measured using a liquid scintillation counter (LS 1800; Beckman). Counts were automatically corrected for quench and converted to disintegrations per millimole.

Calculations. By use of the measurements of enrichment/SA of the expired air and in addition to the rate of CO2, values for exogenous CHO oxidation rate were calculated.

13C calculations. The isotopic enrichment was expressed as the change ({delta}) per mil ({permil}) difference between the 12C-to-13C ratio of the sample and a known laboratory reference standard according to the formula of Craig (6):

|<|\delta|>|^|<|13|>|\mathrm|<|C|>||<|=|>| \left[\left(\frac|<|^|<|12|>|\mathrm|<|C|>|/^|<|13|>|\mathrm|<|C|>| \mathrm|<|sample|>||>||<|^|<|12|>|\mathrm|<|C|>|/^|<|13|>|\mathrm|<|C|>| \mathrm|<|standard|>||>|\right)|<|-|>|1\right]|<|\times|>|10^|<|3|>|{permil}
The {delta}13C was then related to an international standard PDB.

Exogenous glucose oxidation determined using a [13C]glucose tracer ([13C]CHOEXO) was then calculated using the formula:

where {delta}Exp is 13C enrichment of expired air during exercise ({delta}13C{permil} vs. PDB), {delta}Ing is 13C enrichment of the ingested beverage ({delta}13C{permil} vs. PDB), {delta}Expbkg is 13C enrichment of expired air in the water trial [background ({delta}13C{permil} vs. PDB)], and k is equal to 0.7467 [volume of CO2 (liters) produced by the oxidation of 1 g glucose].

14C calculations. Exogenous glucose oxidation determined using a [14C]glucose tracer ([14C]CHOEXO) was calculated using the formula:

where units for CO2 are liters per minute, SA is the radioactivity of 1 mmol expired CO2 (dpm/mmol), and SAGlc is the radioactivity of the ingested drink (dpm/mmol) (note: The radioactivity of the expired CO2 is multiplied by a factor of 6 because there are 6 carbon atoms in one glucose molecule.).

It is possible that a proportion of the CO2 produced by CHO oxidation during exercise may be temporarily trapped in the bicarbonate pool. Under resting conditions, the turnover of this pool is slow, and equilibrium may take several hours to achieve (26). As a result, the appearance of labeled CO2 may not represent the production of CO2 within the exercising musculature. During exercise, however, the turnover rate is accelerated and thus it is likely that an equilibrium will be obtained after ~60 min (20, 26). Therefore, calculations of CHOEXO in the first 60 min of exercise are likely to underestimate the actual rate of oxidation. However, both [14C]CHOEXO and [13C]CHOEXO are likely to be equally affected by CO2 trapping; therefore, comparisons between methods are valid at all time points.

Dietary controls. During exercise, the relative amounts of endogenous fat and CHO oxidized will change. Because CHO stored as glycogen generally has a higher 13C enrichment than fat, changes in substrate oxidation can affect background 13C enrichment (28). These shifts in background 13C enrichment have the potential to lead to erroneous data (22). To decrease the magnitude of the shift in 13C enrichment during exercise, steps were taken to reduce the enrichment of the endogenous CHO stores. Subjects were instructed to undertake a glycogen-exhausting exercise bout 5 days before each trial and to avoid foodstuffs with a naturally high 13C enrichment until the experimental trial. Subjects were given guidance to do this (28), and this method has previously been shown to be effective in reducing the magnitude of the background shift in 13C enrichment during exercise (25, 29).

Statistics. Data were checked to ensure parametric assumptions were not violated before an ANOVA for repeated measures was applied. In the case of significant differences, paired-samples t-tests were used to identify their location. Data analysis was performed using SPSS 10.0 for Windows software (SPSS). Statistical significance was set at P < 0.05, and all data are presented as means ± SE.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
13C enrichment. There were no differences between trials in resting breath 13C enrichment (–26.2 ± 0.2 vs. –26.0 ± 0.2 {delta}13C{permil} vs. PDB for Glc and WATER, respectively, P = 0.760). Corrected mean breath 13C enrichments rose significantly in Glc between time points 15–30, 30–45, and 45–60 min (P < 0.001). There were no significant increases between any time points in WATER. Breath 13C enrichments above resting were greater in Glc than WATER at all time points other than rest. At the cessation of exercise, breath 13C enrichment above resting was 17.9 ± 1.3 and 0.7 ± 0.3 {delta}13C{permil} vs. PDB for Glc and WATER, respectively. All breath 13C enrichment data are shown in Fig. 1. The enrichment of the breath in WATER represented 5.4 ± 0.5% (range 4–8%) of the enrichment in Glc. The coefficient of variation (CV) for the continuous-flow IRMS method was 0.2% (based on 250 measurements of 1 sample), whereas the CV for the EA-IRMS method was 0.4% (9 measurements of 1 sample).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. Mean corrected breath 14C activity (dpm/mmol) and increase in 13C breath enrichment [{delta}{permil} vs. Pee Dee Bellemnitella (PDB)]. Data are means ± SE. *Glucose (Glc) 13CO2 enrichment significantly greater than water. #Glc 14CO2 enrichment significantly greater than previous time point. {triangleup}13CO2 enrichment significantly greater than previous time point.

 
14C SA. Mean breath 14C SA increased from 44 ± 3 dpm/mmol at rest to 2,188 ± 129 dpm/mmol at 120 min (Fig. 1). There were significant increases in breath 14C SA between 0–15, 15–30, 30–45, and 45–60 min. The CV within samples was 1.8% (based on 50 duplicate samples).

[14C]CHOEXO vs. [13C]CHOEXO. A comparison of the absolute values of [14C]CHOEXO and [13C]CHOEXO over time is shown in Fig. 2. [13C]CHOEXO was significantly greater than [14C]CHOEXO between 30 and 120 min, with peak exogenous oxidation rates of 0.87 ± 0.04 g/min ([13C]CHOEXO) and 0.80 ± 0.04 g/min ([14C]CHOEXO). When all time points were collapsed, [13C]CHOEXO was 15 ± 4% greater than [14C]CHOEXO (Fig. 3). The absolute difference between data points is presented in a Bland-Altman plot (2, 3; Fig. 4) with the mean difference between measures being 0.08 ± 0.06 g/min. The magnitude of the difference between measurements was normally distributed, and 95% confidence intervals and limits of agreement are indicated in Fig. 4.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. Exogenous carbohydrate oxidation (g/min) as calculated with 13C and 14C isotopic tracers. *Significant difference between trials (P < 0.01).

 


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3. Exogenous carbohydrate (CHOEXO) measured with [13C]glucose ([13C]CHOEXO) as a percentage of CHOEXO measured with [14C]glucose ([14C]CHOEXO; %); 100% represents the theoretical 1:1 relationship of the two variables.

 


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. Bland-Altman plot showing the agreement of the two methods. For a given pair of values, the average (x) is plotted against the difference (y); also included are 95% limits of agreement (LA) and 95% confidence intervals (CI).

 
RER and O2. RER fell over time in WATER (from 0.88 ± 0.02 at time 0 to 0.81 ± 0.01 at 120 min) but not in Glc (from 0.92 ± 0.01 at time 0 to 0.88 ± 0.02 at 120 min) and was significantly different between trials at 45–120 min. There were no significant differences at any time point between WATER and Glc in O2. Mean O2 collapsed across all time points/trials was 3.01 ± 0.03 l/min.


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The purpose of this study was to directly compare the level of agreement between CHOEXO rate measured with [13C]glucose and [14C]glucose. The key finding of this study is that there is a consistent ~15% difference between [13C]CHOEXO and [14C]CHOEXO. In 51 of the 56 data points, CHOEXO determined with a [13C]glucose tracer was greater than CHOEXO determined with a [14C]glucose tracer. Overall, there was a significant difference between the calculated CHOEXO at seven of the eight time points. In addition, the relative difference between techniques is fairly constant at 15 ± 4%, with 54 of the 56 data points lying inside the 95% limits of agreement (±2 SD around the mean). If the data points corresponding to a mean CHOEXO of 0.3 g/min or below are excluded, then the relative difference between the two techniques becomes even more consistent (13 ± 1%).

Although the 13C method is often considered superior because of the lower potential for errors, the use of a background correction introduces variables not used in the calculation of [14C]CHOEXO. Therefore, although absolute agreement between the two methods is theoretically possible, it is unlikely. However, expressing the relationship between the two methods as though there was 100% agreement provides a useful reference point when attempting to identify an explanation for the discrepancy:

which can be simplified to:

From the above equations, it can be seen that an artificial increase in [13C]CHOEXO could be because of underestimations of {delta}Ing and Expbkg and/or an overestimation of {delta}Exp. The duplicate measurements of {delta}Ing and {delta}Exp using different mass spectrometers suggest that measurement of 13C enrichment is accurate (mean corrected enrichment: 6.12 ± 0.37 and 6.12 ± 0.38 {delta}13C{permil} vs. PDB). In addition, two types of evacuated tubes were compared and found to give similar values for 13C enrichment [difference = 0.1 ± 0.3% (Exetainers; Labco, Bucks, UK, and Vacutainers; Becton-Dickinson, Drogheda, Ireland)].

The remaining explanation for an artificial increase in [13C]CHOEXO is an underestimation of the background 13C enrichment, i.e., the enrichment caused by shifts in endogenous substrate metabolism. This potential problem in using 13C tracers during exercise has been discussed previously (22). However, the dietary instructions subjects were given have been shown to be effective in reducing background 13C enrichments (28). In addition, the background enrichment represented ~5.5% of the enrichment seen in the Glc trial and therefore cannot explain the observed ~15% difference seen here. The use of a water trial to calculate Expbkg is a theoretical source of error, since CHO oxidation and CO2 were greater after the ingestion of Glc. However, based on previous studies, it is reasonable to assume that CHO ingestion during cycling exercise did not alter muscle glycogen use (15) and will result in a small reduction in liver glycogen use (15). Only if there is a substantial shift in endogenous CHO use, there will be a shift in the background that could affect the calculations of exogenous CHO oxidation.

The authors are aware of the fact that the use of a CHO with a low natural 13C abundance would have been a more appropriate control trial. However, the addition of a relatively large amount of [13C]glucose tracer reduced the effect of a shift in background on [13C]CHOEXO to a minimum.

Thus it seems unlikely that errors in the measurement of {delta}Ing or {delta}Exp are responsible for an overestimation of [13C]CHOEXO.

Consequently, examination of the techniques used here suggests that methodological errors in the quantification of [13C]CHOEXO are unlikely to be the source of the discrepancy. Examination of the relationship between [13C]CHOEXO and [14C]CHOEXO shows that an overestimation of the measured SA of the ingested drink (SAGlc) or an underestimation of the SA of the expired gases (SA) would result in an overall underestimation of [14C]CHOEXO. Samples of each subjects' beverage were analyzed in duplicate to determine SAGlc, and the linearity of the scintillation counter over the range encompassed by SAGlc and SA was confirmed (the equation of the line describing the relationship was: y = 1.0022x + 50.386, R2 = 0.9997). This would suggest that the trapping of the 14CO2 in solution is the most likely source of an underestimation of [14C]CHOEXO. Errors could originate in the preparation of the solution (pipette calibration and dispensing error), the absolute ability of the solution to trap CO2, and losses of CO2 from the solution (17) or through losses of the solution itself during trapping. Significant falls in SA from a capped sample of trapping solution + scintillation fluid have been observed previously (17). These losses were large and rapid (~15% after <5 min) and increased over time (~35% after 400 min). The authors reported that inclusion of an additional 2.5 mmol hyamine hydroxide, after adding the scintillant, reduced these losses to 5% over 400 min. However, data from this laboratory suggest this has no effect on measured SA in this case; vials were prepared in duplicate as normal, and 2.5 mmol hyamine hydroxide were added to one of the vials immediately after passing of the expired gas through the trapping solution was completed. Vials were counted repeatedly over time for 24 h. There was no significant difference between the SA of the vial pairs (mean difference = 10 ± 13 dpm) and no trend for the difference between vials to increase over time. There is a slight possibility that passing the expired gases through the trapping solution at high rates caused droplets of solution to escape from the vial. However, measurement of the mass of the vials before and after CO2 trapping, correcting for the amount of CO2 in solution, suggests this error is small (2 ± 1%). The remaining methodological explanation is that the "strength" of the trapping solution (i.e., the amount of CO2 trapped in solution by 1 ml hyamine hydroxide) was less than assumed. However, titration of the hyamine hydroxide with 0.1 M hydrochloric acid (bromocresol green indicator) indicated that the strength of the trapping solution was within acceptable parameters (99 ± 1%).

The degree to which the [14C]glucose tracer used in this study was labeled should also be considered when discussing this issue, since it has the potential to cause some inconsistency between the two methods. The calculation of [14C]CHOEXO assumes that six molecules of 14CO2 are produced per ingested molecule of labeled glucose. The calculation of [13C]CHOEXO, however, makes no such assumption because the production of 13CO2 is related to the amount of labeled carbons in the ingested beverage. As a result, any shortfall in labeling the [14C]glucose will be directly reflected in the measurement of [14C]CHOEXO. In this case, the [14C]glucose tracer used was ≥95% labeled, which would therefore introduce a difference of ≤5%.

Finally, the occurrence of isotopic fractionation must be considered as an explanation. The use of tracers depends on the assumption that the tracer is metabolically indistinguishable from the tracee, and at present there is no evidence to suggest that any significant isotopic fractionation occurs when using either 13C or 14C. The metabolism of 14C involves greater mass displacement than the metabolism of 13C (30), suggesting that the tracers could be fractioned from each other during metabolism. If this were to occur then the enrichment/SA of the expired gases would give an erroneous representation of the rate of tracee oxidation. It is also possible that use of both isotopes simultaneously could magnify small variations in metabolic treatment of the isotopes. However, comparisons of 13C and 14C isotopes in the measurement of fatty acid turnover/oxidation (31) and glucose turnover (16) in dogs and CO2 recovery after HCO3 infusion in humans (7) have suggested that the level of agreement is high. For example, Kalhan et al. (16) infused both [13C]glucose and [14C]glucose tracers in dogs to obtain estimations of glucose turnover. Liquid scintillation was used to measure 14C SA, whereas 13C enrichment was measured with magnetic-deflection double-collector mass spectrometry. The authors concluded that the level of agreement between tracers was such that [13C]glucose could be substituted for [14C]glucose when measuring glucose turnover.

In summary, this study has directly compared the use of [U-14C]glucose and [U-13C]glucose tracers to measure exogenous glucose oxidation. There was a consistent and significant difference in the measured rates of exogenous oxidation between the two techniques (15 ± 4%). Although previous investigators have not found evidence of isotopic fractionation (7, 16, 31), we were unable to identify a methodological explanation for these findings.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was funded by a grant from GlaxoSmithKline Consumer Healthcare.


    ACKNOWLEDGMENTS
 
We acknowledge the contribution of the late Maarten Van-Den-Braak. A. Wagenmakers and his colleagues at the Stable Isotope Research Centre were extremely kind in analyzing the duplicate breath samples.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. E. Jeukendrup, School of Sport and Exercise Sciences, The Univ. of Birmingham, Edgbaston, Birmingham, B15 2TT, UK (e-mail: A.E.Jeukendrup{at}bham.ac.uk)

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. Adopo E, Peronnet F, Massicotte D, Brisson GR, and Hillaire-Marcel C. Respective oxidation of exogenous glucose and fructose given in the same drink during exercise. J Appl Physiol 76: 1014–1019, 1994.[Abstract/Free Full Text]
  2. Bland JM and Altman DG. Comparing methods of measurement: why plotting difference against standard method is misleading. Lancet 346: 1085–1087, 1995.[CrossRef][ISI][Medline]
  3. Bland JM and Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1: 307–310, 1986.[CrossRef][ISI][Medline]
  4. Bosch AN, Dennis SC, and Noakes TD. Influence of carbohydrate ingestion on fuel substrate turnover and oxidation during prolonged exercise. J Appl Physiol 76: 2364–2372, 1994.[Abstract/Free Full Text]
  5. Costill DL, Bennett A, Branam G, and Eddy D. Glucose ingestion at rest and during prolonged exercise. J Appl Physiol 34: 764–769, 1973.[Free Full Text]
  6. Craig H. Isotopic standards for carbon and oxygen and correction factors. Geochim Cosmochim Acta 12: 133–149, 1957.[CrossRef][ISI]
  7. Fuller NJ, Harding M, McDevitt R, Jennings G, Coward WA, and Elia M. Comparison of recoveries in breath carbon dioxide of H13CO-3 and H14CO-3 administered simultaneously by single 6 h constant unprimed intravenous infusion. Br J Nutr 84: 269–274, 2000.[ISI][Medline]
  8. Guezennec CY, Satabin P, Duforez F, Merino D, Peronnet F, and Koziet J. Oxidation of corn starch, glucose, and fructose ingested before exercise. Med Sci Sports Exerc 21: 45–50, 1989.[ISI][Medline]
  9. Hawley JA, Bosch AN, Weltan SM, Dennis SC, and Noakes TD. Effects of glucose ingestion or glucose infusion on fuel substrate kinetics during prolonged exercise. Eur J Appl Physiol 64: 381–389, 1994.
  10. Hawley JA, Bosch AN, Weltan SM, Dennis SC, and Noakes TD. Glucose kinetics during prolonged exercise in euglycemic and hyperglycemic subjects. Pflügers Arch 426: 378–386, 1994.[CrossRef][ISI][Medline]
  11. Hawley JA, Dennis SC, Laidler BJ, Bosch AN, Noakes TD, and Brouns F. High rates of exogenous carbohydrate oxidation from starch ingested during prolonged exercise. J Appl Physiol 71: 1801–1806, 1991.[Abstract/Free Full Text]
  12. Jentjens RL, Moseley L, Waring RH, Harding LK, and Jeukendrup AE. Oxidation of combined ingestion of glucose and fructose during exercise. J Appl Physiol 96: 1277–1284, 2004.[Abstract/Free Full Text]
  13. Jeukendrup AE, Borghouts LB, Saris WH, and Wagenmakers AJ. Reduced oxidation rates of ingested glucose during prolonged exercise with low endogenous CHO availability. J Appl Physiol 81: 1952–1957, 1996.[Abstract/Free Full Text]
  14. Jeukendrup AE, Mensink M, Saris WHM, and Wagenmakers AJM. Exogenous glucose oxidation during exercise in endurance-trained and untrained subjects. J Appl Physiol 82: 835–840, 1997.[Abstract/Free Full Text]
  15. Jeukendrup AE, Raben A, Gijsen A, Stegen JH, Brouns F, Saris WH, and Wagenmakers AJ. Glucose kinetics during prolonged exercise in highly trained human subjects: effect of glucose ingestion. J Physiol 515: 579–589, 1999.[Abstract/Free Full Text]
  16. Kalhan SC, Savin SM, and Adam PA. Estimation of glucose turnover with stable tracer glucose-1–13C. J Lab Clin Med 89: 285–294, 1977.[ISI][Medline]
  17. Leijssen DP and Elia M. Recovery of 13CO2 and 14CO2 in human bicarbonate studies: a critical review with original data. Clin Sci (Lond) 91: 665–677, 1996.[ISI][Medline]
  18. Massicotte D, Peronnet F, Brisson G, Bakkouch K, and Hillaire-Marcel C. Oxidation of a glucose polymer during exercise: comparison with glucose and fructose. J Appl Physiol 66: 179–183, 1989.[Abstract/Free Full Text]
  19. Moodley D, Noakes TD, Bosch AN, Hawley JH, Schall R, and Dennis SC. Oxidation of exogenous carbohydrate during prolonged exercise: the effects of the carbohydrate type and its concentration. Eur J Appl Physiol 64: 328–334, 1992.[CrossRef]
  20. Pallikarakis N, Sphiris N, and Lefebvre P. Influence of the bicarbonate pool and on the occurrence of 13CO2 in exhaled air. Eur J Appl Physiol 63: 179–183, 1991.[CrossRef]
  21. Peronnet F, Adopo E, Massicotte D, Brisson GR, and Hillaire-Marcel C. Method for computing the oxidation of two 13C-substrates ingested simultaneously during exercise. J Appl Physiol 75: 1419–1422, 1993.[Abstract]
  22. Peronnet F, Massicotte D, Brisson G, and Hillaire-Marcel C. Use of 13C substrates for metabolic studies in exercise: methodological considerations. J Appl Physiol 69: 1047–1052, 1990.[Abstract/Free Full Text]
  23. Pirnay F, Lacroix M, Mosora F, Luyckx A, and Lefebvre P. Effect of glucose ingestion on energy substrate utilization during prolonged exercise in man. Eur J Appl Physiol 36: 1620–1624, 1977.
  24. Rauch LHG, Bosch AN, Noakes TD, Dennis SC, and Hawley JA. Fuel utilization during prolonged low-to-moderate intensity exercise when ingesting water or carbohydrate. Pflügers Arch 430: 971–977, 1995.[ISI][Medline]
  25. Rehrer NJ, Wagenmakers AJ, Beckers EJ, Halliday D, Leiper JB, Brouns F, Maughan RJ, Westerterp K, and Saris WH. Gastric emptying, absorption, and carbohydrate oxidation during prolonged exercise. J Appl Physiol 72: 468–475, 1992.[Abstract/Free Full Text]
  26. Robert JJ, Koziet J, Chauvet D, Darmaun D, Desjeux JF, and Young VR. Use of 13C-labeled glucose for estimating glucose oxidation: some design considerations. J Appl Physiol 63: 1725–1732, 1987.[Abstract/Free Full Text]
  27. van Loon LJ, Jeukendrup AE, Saris WH, and Wagenmakers AJ. Effect of training status on fuel selection during submaximal exercise with glucose ingestion. J Appl Physiol 87: 1413–1420, 1999.[Abstract/Free Full Text]
  28. Wagenmakers AJ, Rehrer NJ, Brouns F, Saris WH, and Halliday D. Breath 13CO2 background enrichment during exercise: diet-related differences between Europe and America. J Appl Physiol 74: 2353–2357, 1993.[Abstract]
  29. Wagenmakers AJM, Brouns F, Saris WHM, and Halliday D. Oxidation rates of orally ingested carbohydrates during prolonged exercise in man. J Appl Physiol 75: 2774–2780, 1993.[Abstract]
  30. Wolfe RR. Isotope ratio mass spectrometry: instrumentation and calculation of isotopic enrichment. In: Radioactive and Stable Isotope Tracers in Biomedicine: Principles and Practise of Kinetic Analysis, edited by Wolfe RR. New York: Wiley, 1992, p. 23–36.
  31. Wolfe RR, Evans JE, Mullany CJ, and Burke JF. Measurement of plasma free fatty acid turnover and oxidation using [1–13C]palmitic acid. Biomed Mass Spectrom 7: 168–171, 1980.[CrossRef][ISI][Medline]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
289/2/E206    most recent
00423.2004v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Moseley, L.
Articles by Jeukendrup, A. E.
Articles citing this Article
PubMed
PubMed Citation
Articles by Moseley, L.
Articles by Jeukendrup, A. E.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.