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
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ABSTRACT |
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exercise; isotopic fractionation; enrichment; specific activity
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.
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METHODS |
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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·kg1·min1), 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 13C
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
13C
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 13C value determined. The reference gas used during analysis was 3.3% CO2 in a helium balance with
13C = 29.01 vs. PDB. The 3.3% CO2 mixture was prepared from a CO2 cylinder calibrated against calcite (NBS-19); (
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 13C
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 () per mil (
) 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|>|![]() |
Exogenous glucose oxidation determined using a [13C]glucose tracer ([13C]CHOEXO) was then calculated using the formula:
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14C calculations.
Exogenous glucose oxidation determined using a [14C]glucose tracer ([14C]CHOEXO) was calculated using the formula:
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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.
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RESULTS |
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[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.
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DISCUSSION |
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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:
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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 Ing or
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.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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