Departments of Medicine and Chemistry, University of Vermont, Burlington, Vermont 05405
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Carbon (C) in the 1-position of leucine is released as
CO2 with the decarboxylation of -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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -ketoisocaproate
(KIC; see Fig. 1) (8). Thus
the appearance of the 13C label in breath CO2
provides a direct measure of leucine oxidation.
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 preparedSubjects. 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 · kg1 · 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).
|
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
[MHF]
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.
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
![]() |
(1) |
![]() |
(2) |
![]() |
(3) |
![]() |
(4) |
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
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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%).
|
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.
|
|
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 · kg1 · 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).
|
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 · ekt,
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.
|
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%.
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, -ketoisocaproate can be converted
to
-hydroxy-
-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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Allsop, JR,
Wolfe RR,
and
Burke JF.
Tracer priming the bicarbonate pool.
J Appl Physiol
45:
137-139,
1978
2.
Bleiberg, B,
Beers TR,
Persson M,
and
Miles JM.
Systemic and regional acetate kinetics.
Am J Physiol Endocrinol Metab
262:
E197-E202,
1992
3.
Clugston, GA,
and
Garlick PJ.
Recovery of infused [14C]bicarbonate as respiratory 14CO2 in man.
Clin Sci (Colch)
64:
231-233,
1983[ISI][Medline].
4.
Gilker, CD,
Pesola GR,
and
Matthews DE.
A mass spectrometric method for measuring glycerol levels and enrichments in plasma using 13C and 2H isotopic tracers.
Anal Biochem
205:
172-178,
1992[ISI][Medline].
5.
Irving, CS,
Wong WW,
Shulman RJ,
Smith EO,
and
Klein PD.
[13C]bicarbonate kinetics in humans: intra- vs. interindividual variations.
Am J Physiol Regulatory Integrative Comp Physiol
245:
R190-R202,
1983
6.
Issekutz, B,
Paul P,
Miller HI,
and
Bortz WM.
Oxidation of plasma FFA in lean and obese humans.
Metabolism
17:
62-73,
1968[ISI][Medline].
7.
Leijssen, DPC,
and
Elia M.
Recovery of 13CO2 and 14CO2 in human bicarbonate studies: a critical review with original data.
Clin Sci (Colch)
91:
665-677,
1996[ISI][Medline].
8.
Matthews, DE.
Stable isotope methodologies in studying human amino acid and protein metabolism.
Ital J Gastroenterol
25:
72-78,
1993[ISI][Medline].
9.
Matthews, DE,
Harkin R,
Battezzati A,
and
Brillon DJ.
Splanchnic bed utilization of enteral alpha-ketoisocaproate in humans.
Metabolism
48:
1555-1563,
1999[ISI][Medline].
10.
Matthews, DE,
Marano MA,
and
Campbell RG.
Splanchnic bed utilization of leucine and phenylalanine in humans.
Am J Physiol Endocrinol Metab
264:
E109-E118,
1993
11.
Matthews, DE,
Motil KJ,
Rohrbaugh DK,
Burke JF,
Young VR,
and
Bier DM.
Measurement of leucine metabolism in man from a primed, continuous infusion of L-[1-13C]leucine.
Am J Physiol Endocrinol Metab
238:
E473-E479,
1980
12.
Matthews, DE,
Schwarz HP,
Yang RD,
Motil KJ,
Young VR,
and
Bier DM.
Relationship of plasma leucine and alpha-ketoisocaproate during a L-[13C] leucine infusion in man: a method for measuring human intracellular leucine tracer enrichment.
Metabolism
31:
1105-1112,
1982[ISI][Medline].
13.
Mittendorfer, B,
Sidossis LS,
Walser E,
Chinkes DL,
and
Wolfe RR.
Regional acetate kinetics and oxidation in human volunteers.
Am J Physiol Endocrinol Metab
274:
E978-E983,
1998
14.
Nissen, SL,
and
Abumrad NN.
Nutritional role of the leucine metabolite beta-hydroxy beta-methylbutyrate (HMB).
J Nutr Biochem
8:
300-311,
1997[ISI].
15.
Pouteau, E,
Maugère P,
Darmaun D,
Marchini JS,
Piloquet H,
Dumon H,
Nguyen P,
and
Krempf M.
Role of glucose and glutamine synthesis in the differential recovery of 13CO2 from infused [2-13C] versus [1-13C] acetate.
Metabolism
47:
549-554,
1998[ISI][Medline].
16.
Pouteau, E,
Piloquet H,
Maugeais P,
Champ M,
Dumon H,
Nguyen P,
and
Krempf M.
Kinetic aspects of acetate metabolism in healthy humans using [1-13C]acetate.
Am J Physiol Endocrinol Metab
271:
E58-E64,
1996
17.
Saccomani, MP,
Bonadonna RC,
Caveggion E,
DeFronzo RA,
and
Cobelli C.
Bicarbonate kinetics in humans: identification and validation of a three-compartment model.
Am J Physiol Endocrinol Metab
269:
E183-E192,
1995
18.
Schwenk, WF,
Beaufrère B,
and
Haymond MW.
Use of reciprocal pool specific activities to model leucine metabolism in humans.
Am J Physiol Endocrinol Metab
249:
E646-E650,
1985
19.
Sidossis, LS,
Coggan AR,
Gastaldelli A,
and
Wolfe RR.
A new correction factor for use in tracer estimations of plasma fatty acid oxidation.
Am J Physiol Endocrinol Metab
269:
E649-E656,
1995
20.
Sidossis, LS,
Coggan AR,
Gastaldelli A,
and
Wolfe RR.
Pathway of free fatty acid oxidation in human subjects: implication for tracer studies.
J Clin Invest
95:
278-284,
1995[ISI][Medline].
21.
Strisower, EG,
Kohler GD,
and
Chaikoff IL.
Incorporation of acetate carbon into glucose by liver slices from normal and alloxan diabetic rats.
J Biol Chem
198:
115-126,
1952
22.
Wolfe, RR,
and
Jahoor F.
Recovery of labeled CO2 during infusion of C-1- vs C-2-labeled acetate: implications for tracer studies of substrate oxidation.
Am J Clin Nutr
51:
248-252,
1990[Abstract].