Recovery of 13CO2 from infused [1-13C]leucine and [1,2-13C2]leucine in healthy humans

Michael J. Toth, Michael J. MacCoss, Eric T. Poehlman, and Dwight E. Matthews

Departments of Medicine and Chemistry, University of Vermont, Burlington, Vermont 05405


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Carbon (C) in the 1-position of leucine is released as CO2 with the decarboxylation of alpha -ketoisocaproate (KIC). Carbon in the 2-position of leucine undergoes several additional metabolic steps before entering the tricarboxylic acid (TCA) cycle in the 1-position of acetyl-CoA, where it can be released as CO2 or be incorporated into other compounds. This study examined the metabolic fate of C in the 2-position of leucine. We infused 11 healthy subjects with [1-13C]leucine and [1,2-13C2]leucine for 3.5-4 h to measure leucine kinetics and the oxidation of the tracers from enrichments of 13C in blood and expired CO2. The fraction of leucine infused that was oxidized (fox) was used to define the degree of recovery of the 13C label(s) for each tracer. As expected, leucine appearance (means ± SE) did not differ between tracers (13C1: 92.1 ± 3.1 vs. 13C2: 89.2 ± 3.2 µmol · kg-1 · h-1) when calculated using plasma leucine enrichments as an index of intracellular enrichment. A small (3%) but significant (P = 0.048) difference between tracers was found when KIC was used to calculate leucine appearance (13C1: 118.0 ± 4.1 vs. 13C2: 114.4 ± 4.5 µmol · kg-1 · h-1). The value of fox was 14 ± 1% for [1,2-13C2]leucine and was lower than the fox for [1-13C]leucine (19 ± 1%). From the fox data, we calculated that the recovery of the 2-13C label in breath CO2 was 58 ± 6% relative to the 1-13C label. These findings show that, although a majority of the 2-13C label of leucine is recovered in breath CO2, a significant percentage (~42%) is retained in the body, presumably by transfer to other compounds, via TCA exchange reactions.

leucine oxidation; leucine kinetics


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ISOTOPE TRACERS labeled with 13C or 14C have been used to assess the oxidation of a variety of metabolic substrates. At isotopic steady state, measurements of the enrichment or specific activity of the carbon (C) label in expired CO2 and the precursor pool for oxidation (usually plasma) permit the calculation of both the turnover and oxidation rates of the given substrate. For example, the essential amino acid leucine, labeled with 13C or 14C in the carboxyl position (e.g., [1-13C]leucine), can be used to measure leucine turnover and oxidation (11). The isotope label in the carboxyl position is released as CO2 when leucine is irreversibly committed to oxidation with the decarboxylation of alpha -ketoisocaproate (KIC; see Fig. 1) (8). Thus the appearance of the 13C label in breath CO2 provides a direct measure of leucine oxidation.


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Fig. 1.   Biochemical pathway for the catabolism of leucine. TCA, tricarboxylic acid. Asterisks denote 13C labels.

Unlike a carboxyl-labeled leucine that is released directly as CO2, a C label placed in many substrates is not released as CO2 until after it has passed through the tricarboxylic acid (TCA) cycle. Passage of the tracer through the TCA cycle creates a potential problem in the interpretation of oxidation data, because some of the C label may be incorporated into other compounds (e.g., glucose, glutamine/glutamate) via TCA cycle exchange reactions instead of being released as CO2 (15). Oxidation of a C-labeled substrate will be underestimated from breath CO2 measurements according to the degree of label retention during passage through the TCA cycle.

This problem of retention of C label has been evaluated using tracers of metabolites that enter directly into the TCA cycle. For example, acetate enters directly into the TCA cycle as acetyl-CoA, and studies using labeled acetate have been performed to define the degree of label retention in TCA cycle intermediates in dogs and in humans (2). These studies showed that a substantial portion (~30%) of the 13C label of acetate was retained in the body, presumably through transfer into other compounds via TCA exchange reactions. Data from infusions of labeled acetate have been proposed to be used to correct oxidation data from substrates that have small intracellular pools and are metabolized directly to acetyl-CoA, such as fatty acids (19). In such substrates, the probability that the C label will be lost before entry into the TCA cycle is minimal.

Another limitation of all tracer oxidation studies is that the C label must pass through the body bicarbonate pool, where the C label can be retained through pathways not leading to release as exhaled CO2. Bicarbonate kinetics and retention of C have been examined in a variety of studies (1, 3). These studies have shown both constancy and variability of bicarbonate retention. None of the studies infusing labeled acetate and measuring acetate label recovery in exhaled CO2 has simultaneously measured bicarbonate C retention with a labeled bicarbonate infusion. Thus the acetate C-label retention values include retention of C from both the TCA cycle and the bicarbonate pool.

In performing studies with the use of both L-[1-13C]leucine and L-[1,2-13C2]leucine, we realized that we had two tracers with the potential to define retention of C metabolized to acetyl-CoA while simultaneously correcting for many of the other limitations mentioned above. Infusion of [1-13C]leucine results in direct decarboxylation of the 13C to produce "metabolically-derived" CO2, which then passes through the bicarbonate pool. The same is true of the carboxyl 13C of [1,2-13C2]leucine; however, the second 13C of this tracer will undergo several metabolic steps and then become [1-13C]acetyl-CoA. Thus, by measuring the differential recovery of 13CO2 from infusions of [1,2-13C2]leucine and [1-13C]leucine, we can determine the recovery of the carboxyl C of acetyl-CoA with simultaneous correction for bicarbonate retention and other factors not related to acetyl-CoA metabolism.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. L-[1-13C]leucine, L-[1,2-13C2]leucine, sodium [13C]bicarbonate (99% 13C), and [4,5,5,5,5',5',5'-2H7]ketoisocaproic acid ([2H7]KIC) were obtained from MassTrace (Woburn, MA). [4,5,5,5,5',5',5'-2H7]leucine ([2H7]leucine) was obtained from the former MSD Isotopes (St. Louis, MO; no longer in business). Only the [1-13C]leucine, [1,2-13C2]leucine, and sodium [13C]bicarbonate were administered to human subjects. Chemical, isotopic, and optical purities were determined by gas chromatography-mass spectrometry (GC-MS). Insignificant (<0.05%) amounts of the D-isomer were noted in both L-leucine tracers. Chemical purities were indistinguishable from 100%. The isotopic composition of the L-[1-13C]leucine tracer was 97.2% 13C and 2.3% 18O. The isotopic composition of the L-[1,2-13C2]leucine was 99.6% of the dilabeled (1,2-13C2 species), 99.8% average 13C in the 1- and 2-positions, and 5.9% 18O. The small amount of 18O is not uncommon in carboxyl-labeled [13C]leucine tracers.

Before each infusion study, sterile solutions of the administered isotopes were prepared using aseptic techniques. Compounds were accurately weighed and dissolved in weighed volumes of sterile, pyrogen-free, 0.45% saline and filtered through a 0.22-µm sterile filter before use. An aliquot of the sterile solution was initially verified to be pyrogen free before administration. Solutions were prepared <= 48 h before use and were kept at 4°C until use.

Subjects. Eleven healthy male (n = 8) and female (n = 3) adult volunteers having normal weights for heights [20-38 yr old (mean ± SD = 26 ± 5 yr) and 49-114 kg (mean ± SD = 74 ± 18 kg)] were studied at the University of Vermont General Clinical Research Center (CRC). Before study, a medical history was obtained, and physical examinations and biochemical laboratory tests were performed to verify that subjects were healthy and free of disease. Volunteers were informed of the purpose and risks of the study before giving consent to participate. The experimental protocol was approved by the Committee on Human Research for the Medical Sciences of the University of Vermont.

Experimental protocol. Each subject participated in two infusion studies of either protocol 1 or protocol 2, outlined in Infusion protocols. For both protocols, each subject was admitted to the CRC on the day before each infusion study. Subjects were asked to consume a weight-maintaining, liquid diet (Ensure Plus, Ross Laboratories, Columbus, OH) of adequate and known caloric and protein content that day. The last meal was consumed before 1900, and subjects drank only water until the completion of the infusion study on the following day. After an overnight stay at the CRC, subjects were awakened at 0600 and allowed to void. Catheters were placed in an antecubital vein for infusion (18 gauge) and retrogradely in a dorsal hand vein of the contralateral arm (20 gauge) for blood sampling. The hand was placed in a warming box (air temperature 55°C) to obtain arterialized venous blood. The catheters were kept patent with infusions of 0.45% saline (30 ml/h).

Infusion protocols. Two separate but related protocols were conducted (Fig. 2). Seven subjects (all men) participated in protocol 1. In protocol 1, a primed, continuous infusion of either [1-13C]leucine or [1,2-13C2]leucine was started at 0700 and continued for 3.5 h. At that point, the tracer infusion was switched to the opposite isotope ([1,2-13C2]leucine or [1-13C]leucine) for an additional 3.5 h. When [1-13C]leucine was infused first (5 µmol · kg-1 · h-1), priming doses of 4.3 µmol/kg [1-13C]leucine and 1.8 µmol/kg [13C]bicarbonate were given before the infusion. When [1,2-13C2]leucine was infused first (3 µmol · kg-1 · h-1, corresponding to a 13C infusion rate of 6 µmol equivalents of 13C · kg-1 · h-1), priming doses of 2.4 µmol/kg [1,2-13C2]leucine and 1.8 µmol/kg [13C]bicarbonate were given before the infusion. In protocol 1, the infusion rate of the [1,2-13C2]leucine was chosen to produce a 13CO2 enrichment approximately equal to that produced by the [1-13C]leucine infusion, with the assumption that 75% of the 2-13C would be recovered in breath CO2. The infusion rate of each tracer during the second 3.5-h period was the same as its infusion rate during the first 3.5-h period: 5 µmol/kg for [1-13C]leucine and 3 µmol · kg-1 · h-1 for [1,2-13C2]leucine. Blood and breath samples were obtained before the start of the tracer administration and at 15-min intervals during the last 1.5 h of the first and second 3.5-h periods. Each subject completed two infusion studies two days apart. The difference between the infusion studies was whether [1-13C]leucine or [1,2-13C2]leucine was infused first (order randomized among subjects).


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Fig. 2.   Description of infusion protocols. up-arrow , Delivery of priming doses; black-triangle, indirect calorimetry measurements; *, blood/breath sampling.

Four subjects (3 women and 1 man) participated in protocol 2. Protocol 2 was similar to protocol 1, period 1, except that the tracers were infused for 4 h. At the end of the 4 h, the tracer infusion was stopped, but blood and breath sampling continued for an additional 3 h. When [1-13C]leucine was infused (5 µmol · kg-1 · h-1), priming doses of 4.3 µmol/kg [1-13C]leucine and 1.8 µmol/kg [13C]bicarbonate were given before the infusion was started. When [1,2-13C2]leucine was infused (5 µmol · kg-1 · h-1, corresponding to a 13C infusion rate of 10 µmol equivalents of 13C · kg-1 · h-1), priming doses of 4.3 µmol/kg [1,2-13C2]leucine and 3.1 µmol/kg [13C]bicarbonate were given before the infusion was started. In protocol 2, the infusion rate of the [1,2-13C2]leucine was chosen to produce a plasma tracer enrichment approximately equal to that produced by the [1-13C]leucine infusion. Blood and breath samples were obtained before the start of tracer administration, at 15-min intervals during the last 1.5 h of the 4-h tracer infusion, at 15-min intervals for the hour after discontinuation of the infusion, and at 30-min intervals for the next 2 h after discontinuation of the tracer infusion. Each subject completed two infusion studies two days apart, where [1-13C]leucine was infused on the first day and [1,2-13C2]leucine was infused on the second (order randomized among subjects).

In every infusion study, oxygen consumption and carbon dioxide production were determined by indirect calorimetry with the use of a ventilated hood technique (DeltaTrac, Sensor Medics, Yorba Linda, CA) for 15-min intervals five or six times throughout each infusion period. Breath samples were placed into 20-ml evacuated tubes until later analysis for 13CO2 enrichment by isotope ratio mass spectrometry (IRMS). The 13C enrichment [atom % excess (ape)] of expired CO2 in the breath samples was measured by IRMS (VG SIRA II, Middlewich, Cheshire, UK). Blood samples were placed in heparinized tubes and stored on ice until the plasma was separated by centrifugation (4°C) and frozen at -70°C until later analysis.

Analytical methods. Plasma leucine concentrations and enrichments were measured by negative chemical ionization GC-MS, and KIC plasma concentrations and enrichments were measured by electron impact ionization GC-MS, both as previously described (9). In brief, samples were prepared by adding accurate aliquots of [2H7]leucine and [2H7]KIC as internal standards to 500-µl aliquots of plasma. The amino and keto acids were isolated from the plasma samples and derivatized to the N-heptafluorobutyryl,n-propyl (HFBP) amino acid esters and t-butyldimethylsilyl-quinoxalinol keto acid derivatives, respectively (9). Injections of the HFBP amino acid derivatives were made into the GC-MS (model 5988A, Hewlett-Packard, Palo Alto, CA) with selected monitoring of the [M-HF]- ion. The ions m/z = 349, 350, 351, and 356 were monitored for unlabeled, [1-13C]-, [1,2-13C2]-, and [2H7]leucine, respectively. KIC was measured by GC-MS (model 5971A, Hewlett-Packard) with selected monitoring of the [M-57]+ ion at m/z = 259, 260, 261, and 266 for unlabeled, [1-13C]-, [1,2-13C2]-, and [2H7]KIC, respectively.

For each sample, the area under the leucine and KIC peaks for the each ion monitored was determined by the GC-MS data system, and these areas were used to form ion intensity ratios, either as the labeled masses against the unlabeled for measuring tracer enrichments or as unlabeled against the mass from the 2H peak area for measuring amino and keto acid concentrations. The tracer-to-tracee mole ratios (TTR) for the 13C2 and 13C1 species of leucine and KIC were determined from the ion intensity ratios as previously described (4). Standards of known [1,2-13C2]- and [1-13C]leucine and [1,2-13C2]- and [1-13C]KIC tracer-to-tracee content were run along with all samples. These standards were used to calibrate each day's GC-MS measurements of samples when the GC-MS ion intensity ratio data were transformed into TTRs. The TTR values were then converted as required to tracer enrichments as mole fractional excess of tracer: E = 100 · [TTR/(1+TTR)], where E is expressed as mole percent excess (mpe) of the specific tracer species.

Kinetic calculations. The rate of appearance (Ra) of leucine in plasma was calculated from tracer enrichments at plateau for [1-13C]leucine and [1,2-13C2]leucine as
R<SUB>a</SUB><IT>=</IT>i<SUB><IT>x</IT></SUB><IT>·</IT>(100<IT>/</IT>E<SUB><IT>x</IT></SUB><IT>−</IT>1) (1)
where x = 1 for [1-13C]leucine and x = 2 for [1,2-13C2]leucine, ix is the molar rate of the respective leucine tracer (µmol of tracer · kg-1 · h-1), and Ex is the mean plasma enrichment (mpe) of the respective leucine tracer in plasma at isotopic steady state. The factor 100 normalizes the enrichment in percentages. Leucine Ra was calculated using plasma leucine tracer enrichments (hereafter referred to as the primary pool model) and using plasma KIC tracer enrichments (hereafter referred to as the reciprocal pool model) as proxy measures of intracellular leucine enrichment (12).

The rate of 13CO2 excretion into expired air (F13C; µmol 13C · kg-1 · h-1) was calculated as
F<SUB>13C</SUB><IT>=</IT>F<SUB>CO2</SUB><IT>·</IT>E<SUB>CO2</SUB><IT>·</IT>10<IT>/</IT>0.81 (2)
where FCO2 is the CO2 production rate (mmol · kg-1 · h-1), ECO2 is the enrichment of expired 13CO2 (ape 13C), the constant factor 10 accounts for unit changes, and the factor 0.81 accounts for the recovery in exhaled air of 13CO2 released into the body bicarbonate pool (1).

Division of F13C by the rate of tracer 13C infusion (i13Cx) gives the fraction of tracer infused that was oxidized to CO2 [fox(x)]
f<SUB>ox(<IT>x</IT>)</SUB><IT>=</IT>F<SUB>13C<IT>x</IT></SUB><IT>/</IT>i<SUB>13C<IT>x</IT></SUB> (3)
where x = 1 or 2 for [1-13C]- and [1-13C2]leucine, respectively, and i13Cx reflects the infusion rate of 13C (µmol · kg-1 · h-1). Note that the molar infusion rate of [1,2-13C2]leucine (i2) delivers twice as much 13C (i13C2) because two C atoms are labeled with 13C. Thus the tracer infusion rate in Eq. 3 must reflect the rate of 13C-atom tracer infusion, whereas the infusion rate in Eq. 1 reflects the leucine tracer-molar infusion rate.

Our primary goal was to define the recovery of the 13C label in the 2-position of leucine in breath 13CO2. The fraction of infused [1-13C]leucine oxidized to 13CO2 (fox1) defines the extent of oxidation of the 1-C of leucine. The fraction of infused [1,2-13C2]leucine tracer oxidized to 13CO2 is sum of the oxidation of the 1-C and the 2-C. Thus, for a given infusion study, the fraction of the infused 2-C oxidized to CO2 relative to the 1-C (F2/1) will be
F<SUB>2/1</SUB><IT>=</IT>(2f<SUB>ox2</SUB><IT>−</IT>f<SUB>ox1</SUB>)<IT>/</IT>f<SUB>ox1</SUB> (4)
With the assumption that the 1-C of leucine is released 100% into the CO2/bicarbonate pool, F2/1 expresses the fraction of the 2-C that is released as CO2 relative to the 1-C. Note that any uncertainty from use of a fixed value for recovery of 13C label in exhaled CO2 in Eq. 2 cancels from Eq 4, making the determination of F2/1 independent of bicarbonate kinetics.

The purpose of this study was to test our ability to measure recovery in humans of metabolic C entering as the carboxyl-C of acetyl-CoA by use of infusions of L-[1-13C]leucine and L-[1,2-13C2]leucine. Ideally, these studies would have been better conducted using L-[2-13C]leucine instead of L-[1,2-13C2]leucine, because the differential response of the 1-C and 2-C of the leucine would have been highlighted without requiring a subtraction of the 1-C response simultaneously with the 2-C response, as when [1,2-13C2]leucine is used (fox1 in the numerator of Eq. 4). However, the L-[2-13C]leucine tracer is considerably more expensive, and the [1,2-13C2]leucine tracer was used instead for practical issues of cost and availability.

Statistics. Data are presented as means ± SE. The Origin program (version 6.0, Microcal Software, Northampton, MA) was used to perform nonlinear least squares fitting of tracer data in protocol 2 from hours 4 to 7 to mono- and diexponential decay curves of the form A(t) = A1 · e<SUP>−k<SUB>1</SUB>t</SUP> + A2 · e<SUP>−k<SUB>2</SUB>t</SUP>, where A(t) is the tracer enrichment at time t after discontinuation of the tracer infusion. Tracer enrichments after cessation of the infusion were expressed as a fraction of the mean plateau enrichment. A 2 × 2 repeated-measures analysis of variance, with time (first or second 3.5-h period of study) and tracer ([1-13C]- or [1,2-13C2]leucine) as factors, was used to examine differences for protocol 1. In addition, paired t-tests were used to compare the recovery of 13C label within and between studies for protocol 1 and to compare concentration, enrichment, Ra, and oxidation data for protocol 2.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Leucine and KIC concentration and enrichment data and leucine Ra for protocol 1 are shown in Table 1. A time effect (P < 0.05) was found for plasma leucine concentrations, indicating that the plasma leucine concentration increased as the duration of the fasting period increased. No effect of time or tracer was found for KIC concentrations. There was no difference in either leucine or KIC concentrations between studies, and concentration values were typical for an overnight fast. Because the [1-13C]- and [1,2-13C2]leucine tracers were infused at different rates, leucine and KIC enrichments were higher (P < 0.01) during infusion of the [1-13C]leucine tracer in both studies 1 and 2. No differences were found with time or between tracers for Ra leucine calculated using the primary pool model. In contrast, a small tracer effect was found (P = 0.048) for Ra leucine calculated using the reciprocal pool model. Specifically, a 3% lower average Ra was found during infusion of the [1,2-13C2]leucine tracer compared with the [1-13C]leucine tracer.

                              
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Table 1.   Leucine and KIC concentration and enrichment data and leucine rates of appearance from protocol 1 

Leucine tracer oxidation data and recovery as 13CO2 for protocol 1 are shown in Table 2. CO2 production rates, measured several times per study and averaged for each individual, did not differ between studies. Differences were found among breath 13CO2 enrichment, 13CO2 excretion, and the fraction of tracer oxidized to CO2 data with respect to infusion order (first vs. second 3.5 h) and tracer-infused ([1-13C]- vs. [1,2-13C2]leucine). The fraction of infused 13C tracer oxidized to 13CO2 (fox) for [1-13C]- and [1,2-13C2]leucine was used to calculate the recovery of the 2-13C label as 13CO2 relative to the 1-13C label. When fox values were used from the same study day, there was a significant (P < 0.001) difference in 2-13C recovery between studies 1 and 2 (74 vs. 41%), indicating that the order of tracer infusion produced differences in 13CO2 recovery. These results show a carryover of the 13C label from the first 3.5-h infusion into the second 3.5-h infusion. When 2-13C recovery was calculated using 13CO2 data across infusion days for a specific infusion period (i.e., first vs. second 3.5 h), the recovery of 2-13C tracer was not significantly different (56 vs. 59%).

                              
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Table 2.   Leucine tracer oxidation data from protocol 1 

The conclusion from protocol 1 is that the order of tracer infusion ([1-13C]- vs. [1,2-13C2]leucine) significantly affects the recovery of the 2-13C label when the calculations are performed for 13CO2 data from the same infusion day. The mean of the 2-13C recovery values calculated within the same study [(74 + 41)/2 = 58%] was not different from the 2-13C recovery calculated between studies [(56 + 59)/2 = 58%]. Thus protocol 2 was performed to obtain data for plasma [13C]leucine, [13C]KIC, and breath 13CO2 enrichments after the respective [1-13C]- and [1,2-13C2]leucine tracer infusions had been stopped. Our intent in protocol 2 was to define the effect of 13C tracer carryover on the recovery of the 13C label(s) in expired 13CO2.

The plateau enrichments and time course for the decay in enrichments after cessation of the infusions are shown in Fig. 3 for protocol 2. The average plateau enrichments and concentrations for leucine and KIC for protocol 2 are shown in Table 3. In protocol 1, the leucine tracer infusion rates were set to produce similar enrichments of 13CO2 between the [1-13C]- and [1,2-13C2]leucine infusions. In protocol 2, the infusion rates of the tracers were set equal to keep the plasma tracer molar enrichments the same (Fig. 3). Thus no differences were found between studies for plasma leucine or KIC concentration or for tracer enrichments at isotopic steady state (samples taken between hours 3 and 4; Fig. 3 and Table 3). Similarly, the Ra leucine (calculated by use of both the primary and the reciprocal pool models from the tracer data from infusion hours 3-4) did not differ between studies.


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Fig. 3.   Time course of plasma and breath 13C enrichments in protocol 2. Enrichment data obtained during infusion of [1-13C]leucine are indicated by open circle ; data obtained during infusion of [1,2-13C2]leucine are indicated by . Top: enrichment for plasma leucine; middle: plasma alpha -ketoisocaproate (KIC); bottom: exhaled CO2. Enrichments are expressed as mole percent excess (mpe) for plasma leucine and KIC and as atom percent excess 13C (ape) × 103 for 13CO2. Solid lines represent the best fit of the data to the exponential decay equations shown in Table 5.


                              
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Table 3.   Plasma leucine and KIC concentration and enrichment data and leucine turnover for protocol 2 

The CO2 production rates, breath 13CO2 enrichments, and excretion rate data taken at plateau (hours 3.25-4) for protocol 2 are shown in Table 4. There was no difference in CO2 production between studies. As expected, the breath 13CO2 enrichment and excretion rates were greater in study 2 when [1,2-13C2]leucine was infused compared with study 1 when [1-13C]leucine was infused, because the rate of 13C infusion (i.e., equivalents of 13C · kg-1 · h-1) was almost double that for the [1,2-13C2]leucine tracer. The fraction of the 2-13C recovered as 13CO2 relative to the 1-13C was 61 ± 11% (Table 4).

                              
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Table 4.   Leucine tracer oxidation and recovery data for protocol 2 

We also measured plasma leucine 13C, plasma KIC 13C, and 13CO2 for 3 h after discontinuation of the [1-13C]- and [1,2-13C2]leucine tracer infusions in protocol 2. These data (Fig. 3) were fitted to one- and two-component exponential decay curves. Each component term included a normalized enrichment factor (A) and an exponential decay constant (k) of the form A · e-kt, where t is time. Components of the equations are shown in Table 5. The leucine and KIC 13C enrichment data fit best to two-component curves (significantly improved r2 for two components vs. one component). Addition of a second component did not improve the 13CO2 data fit. There were no significant differences between either k or A values from the [1-13C]- and [1,2-13C2]leucine infusions except for 13CO2. The k for disappearance of 13CO2 was slower (P < 0.05) for [1,2-13C2]leucine infusion compared with [1-13C]leucine infusion, indicating that the 2-13C passed through an additional pool prior to recovery as 13CO2.

                              
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Table 5.   Decay constants and fractional enrichment values for plasma leucine enrichment, plasma KIC enrichment, and 13CO2 excretion after discontinuing infusions of [1-13C]leucine and [1,2-13C2]leucine in protocol 2 

The decay constants from Table 5 were then used to estimate the time course of the decay in [1-13C]- and [1,2-13C2]leucine enrichments during the second 3.5 h of each infusion in protocol 1. Because the leucine and KIC enrichments are measured as discrete isotopomers in plasma, there is no interference of one tracer into the other's plasma enrichment measurement. However, that is not the case for breath 13CO2. From the kinetic fit data in Table 5, we estimate that the 13C1 leucine tracer carryover from period 1 would account for ~10% of the breath 13CO2 at the end of the second-half [1,2-13C2]leucine infusion in protocol 1. Conversely, the 13C2 leucine tracer carryover from period 1 into the second half would account for ~20% of the breath 13CO2 at the end of the second-half [1-13C]leucine infusion. Thus we can use the protocol 2 fitted data of Table 5 to correct the 13CO2 enrichment data obtained in protocol 1 during the second-half infusion for tracer carryover from the first half.

The corrected second 3.5-h infusion period data for 13CO2 excretion and the fraction of tracer oxidized to 13CO2 were then used to calculate corrected 2-13C label recovery values for protocol 1. These data are shown in Table 6. There were still significant differences for 13CO2 excretion and the fraction of tracer oxidized to CO2 between tracer types. However, there were no longer significant differences in 13CO2 excretion with time (i.e., first vs. second half). The recovery of the 2-13C relative to the 1-13C also no longer showed significant differences when calculated within each study between the first and second infusion periods. The mean "within study" recovery was (49 + 54)/2 = 51%, and the mean difference in recovery between studies was (56 + 48)/2 = 52%.

                              
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Table 6.   13CO2 excretion data for protocol 1 after correcting 13CO2 enrichment data in the second infusion period for 13CO2 carried over from the tracer infused during the first infusion

Finally, we combined the between-study values of 2-13C recovery of protocol 1, where seven subjects were studied, and 2-13C recovery values of protocol 2, where four subjects were studied. By use of these data (n = 11), the mean recovery for the 2-13C label was 58 ± 6%.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The goal of this study was to use two tracers of leucine to probe the lower reaches of intermediary metabolism. Specifically, the recovery of the 2-13C label of [1,2-13C2]leucine in expired CO2 was compared with the recovery of [1-13C]leucine in expired CO2. The recovery of the 1-13C label was used to define retention of tracer once it had entered the metabolic CO2 pool. In contrast to the 1-13C label, the 2-13C of leucine is not released as 13CO2 until it has been reduced to acetyl-CoA (Fig. 1). Using this method, we found that 58 ± 6% of the 2-13C label was recovered in breath CO2 relative to the 1-13C label of leucine.

Another point of this study was that the movement of the [1-13C]leucine and the [1,2-13C2]leucine tracers from KIC to exhaled CO2 was kinetically different between the two tracers. That is, the rate of tracer clearance up to and through the bicarbonate pool was slower when the [1,2-13C2]leucine tracer was administered than it was when the [1-13C]leucine was infused (Table 5). This difference is due to a separate pool after the conversion of the [1,2-13C2]leucine label to [1-13C]isovaleryl-CoA and before entry of the 13C into the bicarbonate pool.

The equivalence of the metabolism of the two tracers can be established up through decarboxylation of KIC by comparing the kinetics of leucine and KIC 13C disappearance from plasma after cessation of infusion in protocol 2. The disappearance of both leucine and KIC enrichments from plasma was similar between the [1-13C]- and [1,2-13C2]leucine tracers (Table 5), indicating identical kinetics up to and through KIC. Moreover, the measured leucine Ra values were the same for the [1-13C]- and [1,2-13C2]leucine in both protocols 1 and 2. The slower rate of disappearance of 13C from exhaled CO2 when the [1,2-13C2]leucine tracer was infused compared with the [1-13C]leucine tracer indicates retention of the 2-13C label after formation of [1-13C]isovaleryl-CoA .

The slower passage of the 2-13C tracer through the intermediary pool resulted in a delayed exit of tracer from the bicarbonate pool. We should have anticipated that this result might occur and would affect a study designed to infuse one tracer after the other. However, protocol 1, with back-to-back infusions, was performed before protocol 2 because we had assumed that the kinetics of bicarbonate metabolism would dampen the effect of earlier pools on exhaled 13CO2. Administering both tracers on the same day also removes day-to-day variability in metabolism. Although protocol 2 demonstrated differences in release of 13C label between the two leucine tracers even after 3 h, the results of protocol 2 could be used to correct the influence of the first- half tracer infusion on the second half in protocol 1 and remove the tracer effect upon that study.

The 2-13C label of leucine is metabolized to [1-13C]acetyl-CoA and enters the TCA cycle. The recovery of the 2-13C label of the [1,2-13C]leucine in exhaled 13CO2 should be similar to the recovery of carboxyl-labeled acetate, which also enters the TCA cycle as [1-13C]acetyl-CoA. Although several studies have examined the recovery of carboxyl-labeled acetate in breath CO2, their results cannot be directly compared with ours (15, 16, 19). The duration of the infusions and the metabolic state of the volunteers (postabsorptive) were identical to those in the present study. However, the retention of 13C label reported in these studies includes retention through the bicarbonate system. The carboxyl-labeled acetate recovery was not separated from bicarbonate label retention. In contrast, our use of a pair of leucine 13C tracers determines the recovery of the second C separately from bicarbonate retention of label. To compare our results to the literature data with the use of acetate tracer infusions, we must first correct reported values for bicarbonate retention of label.

Applying an assumed 78% bicarbonate correction factor, Pouteau et al. (16) found that 69 ± 4% of the infused [1-13C]acetate was recovered in breath CO2 during a 3-h infusion. Sidossis et al. (19) infused [1-14C]acetate and recovered 54 ± 2% as 14CO2. If we assume that bicarbonate label recovery as CO2 was 81% (1), the Sidossis et al. data indicate that 69 ± 3% of the 1-C of acetate was oxidized. A second study by Pouteau and colleagues (15) determined a 45 ± 3% recovery of [1-13C]acetate as exhaled 13CO2 for a 3-h infusion of acetate tracer. If we correct this value for an assumed bicarbonate recovery of 81%, the oxidation of the 1-C of acetate becomes 56 ± 3% in this second study. These three studies highlight the range of reported results. Our value of 58 ± 6% recovery of the 2-13C label of leucine is less than, but similar to, other reported values using acetate tracers.

Are our estimates of retention of C from acetyl-CoA by use of a pair of leucine tracers superior to the use of an acetate tracer coupled with a bicarbonate tracer? First, the measurements that we report would be simpler to interpret if we had used a [2-13C]leucine tracer instead of a [1,2-13C2]leucine tracer. Using a [2-13C]leucine would remove the subtractive term from the numerator of Eq. 4 and reduce the variance in the recovery of the 2-13C label of [1,2-13C2]leucine. For the purposes of this first study, the [1,2-13C2]leucine tracer was available to us, compared with the more expensive [2-13C]leucine tracer. Second, use of a pair of leucine tracers does have the advantage of measuring retention of label through the acetate pool while simultaneously determining bicarbonate C-label retention, but bicarbonate label retention could also be determined using an acetate-bicarbonate tracer combination. Third, there may be differences in the metabolism of leucine compared with acetate.

An infused acetate tracer enters the cell and likely enters directly into the mitochondria for oxidation. The degree of label oxidation in the TCA cycle derived from infusions of [1-13C]acetate should be a maximal estimate. In contrast, the leucine tracer forms [1-13C]acetyl-CoA from [1-13C]isovaleryl-CoA within the mitochondria after several intermediate reactions (Fig. 1). The possibility exists for some label retention during these metabolic steps. For example, in the liver and possibly other tissues, alpha -ketoisocaproate can be converted to beta -hydroxy-beta -methylbutyrate, which can then be excreted in the urine or metabolized to mevalonate and be used for cholesterol synthesis. However, only a relatively small amount of leucine oxidation is thought to proceed through this pathway (14).

There may also be organ-specific differences in metabolism between leucine and acetate tracers. The majority of whole body leucine oxidation takes place in nonsplanchnic tissues such as skeletal muscle (10), whereas a larger proportion of infused acetate is metabolized by splanchnic tissues (2). Although Mittendorfer et al. (13) showed that recovery of [1-13C]acetate was similar in the postabsorptive state between splanchnic and leg tissues, there may be some tissue-specific differences in the metabolism of leucine and acetate. If anything, the use of the leucine tracers focuses on muscle metabolism of the tracer.

In conclusion, we present data from a pair of leucine tracers that can be used to evaluate intermediary metabolism. We found that 58 ± 6% of the 2-13C label of [1,2-13C2]leucine was recovered in breath CO2 relative to the 1-13C label. This value represents the intracellular fate of acetyl-CoA and represents that fraction of acetyl-CoA that enters the TCA cycle and is oxidized. This approach provides an alternative to infusion of labeled acetate and will simultaneously provide measurement of protein kinetics via leucine.


    ACKNOWLEDGEMENTS

The authors thank all the participants who volunteered their time for this study. We are grateful to Judit Fabian for skilled technical assistance.


    FOOTNOTES

This work was supported in part by National Institutes of Health Grants DK-38429, DK-26687, AG-15821, AG-17494 AR-02125, and RR-00109.

Address for reprint requests and other correspondence: D. E. Matthews, Cook Bldg., Univ. of Vermont, Burlington, VT 05405 (E-mail: dmatthew{at}zoo.uvm.edu).

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.

Received 28 June 2000; accepted in final form 8 March 2001.


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ABSTRACT
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RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 281(2):E233-E241
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