(Received for publication, September 30, 1994; and in revised form, November 16, 1994)
From the
The validity of the use of a carbon tracer for investigating
liver intermediary metabolism in vivo requires that the
labeling pattern of liver metabolites not be influenced by metabolism
of the tracer in other tissues. To identify such specific tracer,
livers from 48-h starved rats were perfused with recirculating buffer
containing [3-C]lactate,
[2-
C]acetate, or
-keto[3-
C]isocaproate. Conscious 48-h
starved rats were infused with the same tracers for 5 h. The labeling
patterns of liver glutamate and extracellular glucose were assayed by
gas chromatography-mass spectrometry. In vivo data were
corrected for
CO
reincorporation into C-1 of
glutamate and C-3 and C-4 of glucose, using data from control rats
infused with NaH
CO
. With
[3-
C]lactate the labeling pattern of liver
glutamate was the same in perfused organs and in vivo. In
contrast, with [2-
C]acetate and
-keto[3-
C]isocaproate the labeling pattern
of liver glutamate in vivo was clearly influenced by the
expected labeling pattern of citric acid cycle intermediates formed in
non-gluconeogenic organs, presumably glutamine made in muscle. Indeed,
the labeling pattern of plasma glutamine and liver glutamate were
similar in experiments with [3-
C]lactate but
different in experiments with [2-
C]acetate and
-keto[3-
C]isocaproate. Similar conclusions
were drawn from the labeling patterns of glucose. Therefore, labeled
lactate appears as the best tracer for studies of liver intermediary
metabolism in vivo. Our data also show that a substantial
fraction of
-ketoisocaproate metabolism occurs in peripheral
tissues.
Carbon-labeled tracers are used extensively to investigate
various aspects of liver intermediary metabolism, in particular
gluconeogenesis (GNG). ()Measurements of GNG are complicated
by isotopic exchanges at the level of oxaloacetate (OAA), leading to
underestimations of the rate of GNG calculated from the incorporation
of labeled lactate, pyruvate, or alanine into
glucose(1, 2, 3) . Various novel techniques,
using acetate tracers, have been proposed to correct for this isotopic
dilution(4, 5, 6, 7) . These
techniques were used in a number of
studies(8, 9, 10) , but their limitations
were soon recognized for in vivo studies. First, labeled
acetate is probably used mostly in peripheral tissues where it is
oxidized to labeled CO
(11, 12) . The
latter is carried by blood to the liver where it is incorporated into
C3 and C4 of glucose. Indeed, some investigators calculate an index of
GNG from the incorporation of labeled
CO
(10, 13, 14, 15) .
Second, in muscle, acetate metabolism may label glutamine, which is
transported to the liver, where it can influence the labeling pattern
of citric acid cycle (CAC) intermediates and of glucose(12) .
Third, some models use the label of mitochondrial acetyl CoA estimated
from the labeling of C-1 and C-2 of plasma acetoacetate or R-
-hydroxybutyrate(5, 6) . This
estimation is inaccurate especially in vivo because of (i)
dilution of the labeling of acetoacetate in peripheral tissues via
reversal of 3-oxoacid-CoA transferase, a process we called
pseudoketogenesis(16, 17) , and (ii) heterogeneity of
the labeling of liver mitochondria acetyl CoA, so that with some
tracers, the labeling of acetyl CoA going to acetoacetate is different
from that going to citrate(18) .
The problems raised with
the utilization of labeled acetate could be prevented, at least in
part, if one had a selective tracer for liver mitochondrial acetyl CoA.
In conceiving the present study, we hypothesized that
-keto[3-
C]isocaproate
([3-
C]KIC) could be such a tracer. This
hypothesis was based on the notion that KIC is formed from leucine in
peripheral tissues and is degraded in liver(19) . In addition,
we wanted to find out whether labeled lactate is metabolized in
peripheral tissues to compounds that influence the labeling pattern of
liver CAC intermediates. This question is important in view of the
recent development of a noninvasive technique for determining the
labeling pattern of liver CAC intermediates in
humans(12, 20, 21, 22) . One can
sample liver glutamine by its conjugation with exogenous or endogenous
phenylacetate, forming phenylacetylglutamine (PAGN), which is excreted
in urine(23) . It is assumed that the labeling pattern of the
glutamine moiety of PAGN reflects that of liver
-ketoglutarate
(
KG). This labeling pattern is used to calculate parameters of the
CAC and the degree of isotopic dilution of gluconeogenic carbon at the
OAA cross-road(12, 20, 21, 22) .
In the present study we compared the labeling patterns of liver
glutamate and extracellular glucose labeled in vivo and in
isolated organs from [3-C]lactate,
[2-
C]acetate, or
[3-
C]KIC. Our hypothesis was that a
liver-specific tracer should yield the same labeling patterns in
vivo and in perfused livers.
In experiments with [2-C]acetate
and [3-
C]KIC, the ratio of enrichments of
PEP/(mitochondrial acetyl CoA) was calculated using the glucose
-labeling ratio, R = (average enrichment of C-1, C-2,
C-5, and C-6)/(average enrichment of C-3 and C-4) and Katz
equations(6) . Then, using the MPE of C-1 and C-2 of R-
-hydroxybutyrate as reflecting that of mitochondrial
acetyl CoA, we calculated the MPE of PEP. The MPE of PEP was also
directly measured.
In orientation liver perfusion experiments, we determined the
rates of substrate infusion into the recirculating perfusate that are
necessary to maintain constant concentrations (see ``Experimental
Procedures''). With this protocol, lactate/pyruvate concentrations
ranged from 0.6 to 1.2 mM, whereas KIC and acetate
concentrations remained between 0.2 and 0.3 mM. Glucose
concentration increased from 4 to about 7 mM over 2 h. R--Hydroxybutyrate/acetoacetate concentration increased
to 2.8-3.0 mM over 2 h. The uptake of lactate plus
pyruvate was 120-150 µmol
g
, whereas
the net accumulation of glucose was 54-78
µmol
g
, corresponding to 75-100% of
the net lactate plus pyruvate uptake. There was no significant
difference in the above balance parameters between the three series of
perfusions.
During infusion of labeled substrates, their MPE
stabilized in the recirculating perfusate. The MPEs of
[3-C]lactate/[3-
C]pyruvate
(infused at 15% MPE) stabilized from 60 to 120 min at 7.7 ± 0.5
and 10.3 ± 0.4%, respectively. Since the rate of
[3-
C]lactate infusion was 5 times that of
[3-
C]pyruvate infusion, the apparent rate of
production of endogenous lactate + pyruvate was about equal to the
rate of labeled substrate infusion, i.e. 9.6
µmol
min
. This dilution of infused
[3-
C]lactate and
[3-
C]pyruvate could result from glycolysis,
isotopic exchanges, or both(34, 35, 36) . We
showed previously that livers from 2-day starved rats perfused with 4
mM glucose release very little lactate(37) .
Therefore, in the present experiments, we ascribe most of the dilution
of infused [3-
C]lactate to isotopic exchanges.
Still, we cannot exclude some substrate cycling between glucose and
pyruvate/lactate.
The MPEs of [2-C]acetate
and [3-
C]KIC (infused at 99%) stabilized at 50
± 5 and 97 ± 2%, respectively. Thus, the rate of
production of endogenous acetate, presumably from acetyl-CoA
hydrolysis(38, 39, 40) , was about 1.25
µmol
min
. This confirms the coexistence of
acetate uptake and production in perfused rat
livers(41, 42) . The negligible dilution of the MPE of
infused KIC is consistent with the low activity of branched-chain amino
acid transaminase in the liver(19) .
In in vivo experiments with [3-C]lactate (99%) infused
at 10 µmol
min
kg
,
the MPE of arterial plasma lactate stabilized at 13-18%. Thus,
the apparent turnover of plasma lactate was 55-76
µmol
min
kg
. This
apparent turnover represents a combination of lactate production and
isotopic exchanges (34, 35, 36) .
Fig. 1and Fig. 2show the labeling patterns ()of liver glutamate and glucose isolated from liver
perfusate and from rat blood, expressed as relative enrichments for
glutamate and labeling ratio, R, for glucose. In liver
perfusion experiments, there was no detectable
C
enrichment in perfusate bicarbonate and urea. This results from the
extensive gassing of the oxygenator with 95% O
, 5% CO
(1 liter
min
). Therefore, reincorporation
of
CO
via pyruvate carboxylase did not
contribute to the labeling pattern of glucose and glutamate in liver
perfusions. In contrast, in in vivo experiments, the MPE of
urea was detectable in the final blood sample: 0.63 ± 0.11%
(S.D., n = 8) from [3-
C]lactate,
1.09 ± 0.27% (n = 6) from
[2-
C]acetate, and 0.38 ± 0.08% (n = 10) from [3-
C]KIC. Therefore, it
is likely that in these experiments, reincorporation of
CO
contributed to the labeling of C-3 and C-4
of glucose and C-1 of glutamate. We therefore infused
NaH
CO
into a fourth series of catheterized
rats and measured the MPE of urea (2.06 ± 0.7%, n = 6), C-1 of glutamate (0.50 ± 0.15%), and C-3 and
C-4 of glucose (1.07 ± 0.39%, average of C-3 and C-4). Using
equations developed by Magnusson et al.(20) , we
corrected the labeling patterns of plasma glucose and liver glutamate
for reincorporation of
CO
. Corrected values
are also included in Fig. 1and Fig. 2.
Figure 1:
Labeling pattern of liver glutamate in
perfused organs and in vivo. Each panel refers to
experiments with the indicated [C]substrate.
Each set of three bars corresponds to experiments (i) in
perfused livers (open bars), (ii) in vivo, with the
labeling pattern as measured (hatched bars), and (iii) in
vivo, with the labeling pattern corrected for
CO
reincorporation on C-1 (solid
bars). All labeling patterns are corrected for natural
C enrichment. Data are presented as mean ± S.D. The
number of experiments with [3-
C]lactate,
[2-
C]acetate, and
[3-
C]KIC is 10, 6, and 9 in perfused livers and
7, 7, and 8 in in vivo, respectively. *, significantly
different from liver perfusions (p <
0.05).
Figure 2:
Labeling ratio of glucose. Other data are
shown from the same experiments as in Fig. 1. R is
defined as (average enrichment of C-1 + C-2 + C-5 +
C-6)/(average enrichment of C-3 + C-4). Each set of three bars refers to experiments with the indicated
[C]substrate and includes experiments (i) in
perfused livers (open bars), (ii) in vivo, with the
labeling pattern as measured (hatched bars), and (iii) in
vivo, with the labeling pattern corrected for
CO
reincorporation on C-3 and C-4 (solid
bars). Statistical significance is by comparison with perfused
liver data *, (p < 0.05);**, (p <
0.01).
In isolated
livers perfused with
[3-C]lactate/[3-
C]pyruvate,
most of the glutamate label is on C-2 and C-3, as shown
previously(31, 32) . In contrast, with
[2-
C]acetate and
[3-
C]KIC, most of the glutamate label is on C-4.
This was expected based on previous work with
[2-
C]acetate (31) and on the notion
that [2-
C]acetate and
[3-
C]KIC are both precursors of
[2-
C]acetyl-CoA, which labels mostly C-4 of
glutamate.
In the presence of [3-C]lactate,
the labeling patterns of glutamate are very similar in perfused livers
and in vivo, except for higher labeling of C-1 in
vivo. After correction for
CO
reincorporation the labeling on C-1 is no longer different
between isolated livers and in vivo livers. In contrast, in
the presence of [2-
C]acetate or
[3-
C]KIC, the labeling of glutamate is different
for all carbons between isolated livers and in in vivo livers,
before correction for
CO
reincorporation.
After such correction, labeling of C-2 to C-5 still remains different.
The data on glucose labeling pattern were used to
calculate the R labeling ratio in glucose (1) isolated
from liver perfusate and rat plasma (Fig. 2). For plasma
glucose, R is presented without and with correction for
CO
reincorporation into C-3 and C-4 of
glucose. R values differ between perfused liver and in vivo data uncorrected for
CO
reincorporation.
However, after such correction, R values in
[3-
C]lactate experiments are not significantly
different in perfusion and in vivo experiments. Significant
differences remain in [2-
C]acetate and
[3-
C]KIC experiments.
Fig. 3shows
comparisons between the total MPE of liver KG, glutamate, and
glutamine in isolated organs and in vivo. Fig. 3also
includes the MPE of plasma glutamine. In isolated livers, the MPEs of
KG and glutamate are the same for all tracers. The MPE of
glutamine is about one-fifth less than the MPE of glutamate. In
vivo, the MPE of glutamate is less than that of
KG. The main
difference between tracers is in the relative labeling of liver
glutamate and plasma glutamine. With
[3-
C]lactate, plasma glutamine is significantly
less labeled than liver glutamate. With
[2-
C]acetate and
[3-
C]KIC, the corresponding differences in MPE
are not significant. In addition, in experiments with
[2-
C]acetate and
[3-
C]KIC, the plasma glutamine labeling ratios
(C-1 to C-3)/(C-1 to C-5) were different from the corresponding liver
glutamate ratios (0.66 ± 0.06 versus 0.57 ± 0.06 (n = 7, p < 0.05) for
[2-
C]acetate and 0.67 ± 0.05 versus 0.59 ± 0.03 (n = 7, p < 0.05)
for [3-
C]KIC). In contrast, in experiments with
[3-
C]lactate these ratios were not significantly
different (0.87 ± 0.09 versus 0.91 ± 0.03, n = 8).
Figure 3:
C Enrichment of liver
-ketoglutarate, glutamate, and glutamine in perfused organs (left panels) and in vivo (right panels).
The labeled substrates infused were [3-
C]lactate (top panels), [2-
C]acetate (middle
panels), and [3-
C]KIC (bottom
panels). The right panels (in vivo) also include
the enrichment of plasma glutamine. *, significantly different (p < 0.05) from
KG (all panels) or from glutamate (upper right panel).
The absolute distributions of C in
glutamate labeled in perfused livers and in vivo from
[3-
C]lactate were introduced into the equations
of Magnusson et al.(20) to calculate rates shown in Table 1. These equations yield rates expressed relative to the
CAC flux taken as 10. In perfused liver experiments, these relative
rates were converted to absolute rates since (i) we measured the net
rate of glucose accumulation, and (ii) the other rates of the Magnusson et al. model are linked to the rate of glucose production
(V9). Note that the rate of lactate/pyruvate uptake calculated from the
model, i.e. 1.26
µmol
min
g
is very
close to the actually measured rate of uptake, i.e. 1.20
µmol
min
g
. Thus, in
these livers perfused with lactate/pyruvate/KIC, the uptake of
lactate/pyruvate is practically converted to glucose. Comparing the
relative rates of reactions in perfused livers and in livers in
vivo, the main differences are in the uptake of lactate/pyruvate
and in fluxes related to gluconeogenesis. All other relative rates are
almost identical. Note that in perfused livers, the concentration of
pyruvate in the perfusate when the livers were freeze-clamped was 0.18
mM. This concentration is 3-4 times that of plasma
pyruvate in starved rats. Since PC has a K
for pyruvate of about 0.4 mM(43) , GNG
in perfused livers was clearly stimulated by substrate supply. This
also explains the higher PC/PDH flux ratio in perfused livers compared
to livers in vivo.
The calculated and measured MPE ratios, lactate/PEP (Table 1) are very similar in perfused livers and in vivo provided one takes into account the dilution of the MPE of portal vein lactate, compared with arterial lactate (see ``Experimental Procedures'').
Table 2compares the
labelings of PEP and C-1, C-2, and C-3 of glutamate with one-half the
MPE of glucose, in perfused livers and in vivo. The MPEs of
PEP and of C-1, C-2, and C-3 of glutamate are very similar in perfused
livers and in vivo, except in the presence of
[2-C]acetate. The labeling of glucose in
vivo is close to that of liver PEP. Note that in vivo,
labeled substrates were infused for 5 h. Under similar conditions, the
turnover rate of glucose is about 45
µmol
min
kg
,
corresponding to a half-life of extracellular glucose of about 15
min(44) . Thus, after 5 h, the initial pool of unlabeled
extracellular glucose in live animals had been replaced by labeled
glucose synthesized during the experiment.
In contrast, in isolated livers, the labeling of perfusate glucose was still lower than that of liver PEP. During the 2 h of the experiment, glucose concentration increased from 4 to 7 mM. Thus, the initial pool of unlabeled glucose was not entirely replaced by newly synthesized labeled glucose.
Data from experiments with [2-C]acetate and
[3-
C]KIC, i.e. the R glucose
labeling ratios and the MPE of C-1 and C-2 of R-
-hydroxybutyrate were used to calculate the MPE of PEP,
using Katz equations(6) . In perfused livers, there was good
agreement between the calculated and measured MPE (Table 2). In
contrast, in in vivo experiments, the calculated and measured
MPE of PEP were very different, although R ratios had been
corrected for
CO
reincorporation.
The main finding of this report is that the labeling pattern
of liver glutamate is identical in the perfused liver and in the in
vivo liver, when [3-C]lactate is the
labeled substrate (Fig. 1). With
[3-
C]lactate, the glucose labeling ratio is also
similar in perfused liver and in vivo (Fig. 2). These
identities require correcting the labeling patterns of glutamate and
glucose for
CO
reincorporation (20) .
In contrast, with [2-C]acetate and
[3-
C]KIC, the labeling patterns of liver
glutamate and extracellular glucose are very different in isolated
livers from those in vivo, even after corrections for
CO
reincorporation ( Fig. 1and Fig. 2).
In the liver, [2-C]acetate
and [3-
C]KIC yield
[2-
C]acetyl CoA, which labels C-4 of glutamate
more than C-2 and C-3. This is because label on C-2 and C-3 leaves the
CAC on the gluconeogenic pathway. In the starved liver,
[3-
C]lactate yields a labeling pattern of
glutamate with enrichments C-2 > C-3 > C-4. This is because (i)
[3-
C]pyruvate derived from
[3-
C]lactate is both carboxylated to
[3-
C]OAA and decarboxylated to
[2-
C]acetyl CoA, (ii) the flux ratio PC/PDH is
greater than unity, and (iii) the randomization of OAA label via
reversible reactions catalyzed by malate dehydrogenase and fumarase is
incomplete (31, 45, 46, 47) . In
non-gluconeogenic tissues such as muscle, the three labeled substrates
are converted to [2-
C]acetyl CoA, which should,
at steady state, label
KG, glutamate, and glutamine almost equally
on C-2, C-3, and C-4. The randomization of label on C-2 and C-3 is
complete because all of the label passes through symmetrical succinate.
Peripheral tissues can influence the labeling pattern of liver CAC
intermediates by two mechanisms. First CO
formed in peripheral tissues is incorporated in liver into C-1 of
KG and glutamate and into C-3 and C-4 of glucose. This results
from carboxylations catalyzed by PC and possibly from the reversal of
PEP carboxykinase and malic enzyme. This incorporation of
CO
is corrected using data from control
experiments with labeled bicarbonate(20) .
Second, glutamine released by muscle is deamidated in liver to glutamate, which could influence the labeling pattern of CAC intermediates by both isotopic exchange and anaplerosis. Note that glutaminase is located in periportal hepatocytes, which are the main site of gluconeogenesis(48) . The extent to which muscle glutamine influences the labeling pattern of liver CAC intermediates depends on (i) the difference in total labeling of muscle and liver CAC intermediates, (ii) the difference in labeling pattern of muscle and liver CAC intermediates, (iii) the rate of glutamine flux from muscle to liver (this rate depends on the rate of protein catabolism and the nutritional status), (iv) the extent to which glutamine carbon is used as a gluconeogenic substrate in liver, and (v) the relative rates of label entry into liver CAC via glutamine on the one hand and acetyl CoA and/or OAA on the other hand. Based on these observations, a discussion of the observed labeling patterns of liver glutamate follows.
In the
presence of [2-C]acetate, the relative labeling
of C-2 and C-3 of glutamate is increased, and the relative labeling of
C-4 is decreased in vivo compared with the perfused liver.
This results from the superposition of liver type glutamate with C-2
= C-3 < C-4 and muscle type glutamate with C-2 = C-3
C-4. This is confirmed by the higher labeling ratio (C-1 to
C-3)/(C-1 to C-5) of plasma glutamine than liver glutamate.
In the
presence of [3-C]KIC, the labeling patterns of
glutamate are almost identical to what was observed with
[2-
C]acetate. Also, the labeling ratio of plasma
glutamine is higher than that of liver glutamate. This was surprising
given the classical concept that KIC is formed in peripheral tissues
and degraded in liver(19) . We were expecting that the relative
labeling patterns of liver glutamate would be identical in perfused
organs and in vivo. This is why we had
[3-
C]KIC synthesized as a potential specific
tracer of liver intermediary metabolism. In fact, it is clear that a
substantial fraction of [3-
C]KIC must be
metabolized in peripheral tissues, probably in muscle. This is
consistent with recent studies of the state of activation of
branched-chain ketoacid dehydrogenase (reviewed in (49) ).
Although the activity of muscle branched-chain ketoacid dehydrogenase
is low in rat muscle compared with liver(19) , the large amount
of muscle compared with liver (30 versus 4% of body weight)
can probably metabolize a large fraction of infused
[3-
C]KIC.
In the presence of
[3-C]lactate, the identity of the liver
glutamate labeling pattern in perfused organ and in vivo results probably from the much higher rate of direct entry of
lactate label via OAA/acetyl CoA, compared with indirect entry via
glutamine made in muscle. That this must be the case can also be
deduced from examination of Fig. 3. When
[3-
C]lactate or [3-
C]KIC
is infused in vivo, the MPE of plasma glutamine is two-thirds
that of liver
KG (Fig. 3, B and F).
However, when [3-
C]KIC is infused, muscle
glutamine appears to have a greater influence on the labeling pattern
of liver CAC intermediates than when
[3-
C]lactate is infused. Therefore, the direct
influx of lactate label into liver CAC intermediates is greater than
the direct influx of KIC label. Also, the labeling ratio (C-1 to
C-3)/(C-1 to C-5) of plasma glutamine is similar to the corresponding
ratio in liver glutamate. This is compatible with reports showing that
anaplerosis of the CAC via PC occurs in skeletal muscle and heart (50, 51, 52, 53) . Metabolism of
[3-
C]pyruvate via PC increases the amount of
label on C-2 and C-3 of muscle glutamine. Therefore, the relative
labeling pattern of muscle glutamine and liver
KG should be very
similar. Thus, the influence of muscle glutamine on the labeling
pattern of liver CAC intermediates must be minimal.
The data on the
labeling pattern of liver glutamate were computed using the model of
Magnusson et al.(20) , which yields relative rates of
CAC reactions. In a previous study (31) conducted in isolated
rat livers perfused with non-recirculating buffer containing
[3-C]lactate, we converted the relative rates
calculated from the model to absolute rates, using the measured uptake
of lactate/pyruvate. This yielded a rate of GNG that we could not
verify by balance measurements since the difference in glucose
concentration across the liver could not be measured with precision. In
the present liver perfusion study, conducted with recirculating buffer,
we could measure both lactate/pyruvate uptake and glucose accumulation.
We converted the relative rates to absolute rates, using the measured
glucose accumulation. Then the calculated rate of lactate/pyruvate
uptake (1.26
µmol
min
g
) was the
same as the measured rate (1.20
µmol
min
g
). Also,
the MPE ratio lactate/PEP calculated from the model was practically the
same as that measured in liver (Table 1, last two rows). These
good agreements support the applicability of the Magnusson et al. model to estimate rates of GNG in liver. However, as we showed
previously(31) , some rates are very sensitive to the amount of
label on C-5 of glutamate, i.e. pyruvate kinase (V7), pyruvate
carboxylase (V6), and PEP carboxykinase (V6). Since the absolute
enrichment on C-5 of glutamate was very low (0.1-0.2%), we do not
give much credence to these calculated rates (Table 2). However,
we also showed (31) that the other rates of the model are not
sensitive to variations in the enrichment of C-5 of glutamate.
Therefore, we feel confident about them.
Our data have a bearing on
the choice of tracer used to investigate liver intermediary metabolism in vivo. Labeled acetate has been used extensively in studies
of GNG to quantitate isotopic exchange at the OAA
crossroad(4, 5, 6, 7) . Various
authors have warned that the bulk of the acetate tracer is used in
peripheral tissues, which can influence the labeling pattern of glucose
and of liver CAC intermediates(11, 12, 20) .
Our data ( Fig. 1and Fig. 2; Table 2) confirm that
labeled acetate is suitable for tracing liver intermediary metabolism
in isolated organs but not in vivo. In addition, we show that
KIC catabolism occurs to a large extent in peripheral tissues. Thus,
[3-C]KIC is also unsuitable to trace liver
intermediary metabolism in vivo. Last, we show that labeled
lactate is the best tracer for studies of liver intermediary metabolism in vivo, at least in the fasting state. Whether or not labeled
lactate is a suitable tracer during a short fast or in the fed state
needs to be investigated.
The C- and
C-labeling patterns of liver CAC intermediates yield
kinetics parameters of the CAC and
GNG(31, 54, 55) , including rates of isotopic
exchanges with other substrates such as aspartate and
glutamate(7) . In experiments with
C tracers, this
labeling pattern has been probed noninvasively in humans by degradation
of urinary PAGN(12, 20, 21, 22) . In
humans infused with [
C]lactate(20) , the
labeling pattern of the glutamine moiety of PAGN yielded relative rates
of liver CAC reactions that are very similar to those we measured in
live rats in the present study. This supports the use of labeled
lactate and the PAGN probe in humans.
The probing of human and
primate liver CAC intermediates with urinary PAGN can be extended to
the use of stable isotopic tracers(56, 57) ,
particularly [C]lactate. This could yield useful
information on metabolic disorders, such as inborn errors of pyruvate
metabolism and diabetes.