An integrated 2H and 13C NMR study of
gluconeogenesis and TCA cycle flux in humans
John G.
Jones1,
Michael A.
Solomon2,
Suzanne
M.
Cole3,
A. Dean
Sherry1,3, and
Craig R.
Malloy1,2
1 Department of Radiology, University of Texas Southwestern
Medical Center, Dallas 75235; 2 Department of Internal
Medicine, University of Texas Southwestern Medical Center and
Department of Veterans Affairs Medical Center, Dallas 75216; and
3 Department of Chemistry, University of Dallas, Richardson,
Texas 75083
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ABSTRACT |
Hepatic glucose
synthesis from glycogen, glycerol, and the tricarboxylic acid (TCA)
cycle was measured in five overnight-fasted subjects by 1H,
2H, and 13C NMR analysis of blood glucose,
urinary acetaminophen glucuronide, and urinary phenylacetylglutamine
after administration of [1,6-13C2]glucose,
2H2O, and
[U-13C3]propionate. This combination of
tracers allows three separate elements of hepatic glucose production
(GP) to be probed simultaneously in a single study: 1)
endogenous GP, 2) the contribution of glycogen, phosphoenolpyruvate (PEP), and glycerol to GP, and
3) flux through PEP carboxykinase, pyruvate recycling,
and the TCA cycle. Isotope-dilution measurements of
[1,6-13C2] glucose by 1H
and 13C NMR indicated that GP in 16-h-fasted humans was
10.7 ± 0.9 µmol · kg
1 · min
1.
2H NMR spectra of monoacetone glucose (derived from plasma
glucose) provided the relative 2H enrichment at glucose
H-2, H-5, and H-6S, which, in turn, reflects the
contribution of glycogen, PEP, and glycerol to total GP (5.5 ± 0.7, 4.8 ± 1.0, and 0.4 ± 0.3 µmol · kg
1 · min
1,
respectively). Interestingly, 13C NMR isotopomer analysis
of phenylacetylglutamine and acetaminophen glucuronide reported
different values for PEP carboxykinase flux (68.8 ± 9.8 vs. 37.5 ± 7.9 µmol · kg
1 · min
1), PEP
recycling flux (59.1 ± 9.8 vs. 27.8 ± 6.8 µmol · kg
1 · min
1),
and TCA cycle flux (10.9 ± 1.4 vs. 5.4 ± 1.4 µmol · kg
1 · min
1).
These differences may reflect zonation of propionate metabolism in the liver.
monoacetone glucose; acetaminophen glucuronide; carbon 13; deuterium; gluconeogenesis; liver metabolism
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INTRODUCTION |
THE LIVER PLAYS A PRINCIPAL
ROLE in glucose homeostasis by regulating glucose synthesis and
storage in response to the normal changes in daily nutritional and
hormonal status. Under postabsorptive conditions, hepatic
glycogenolysis and gluconeogenesis contribute to endogenous glucose
production (GP) (6, 15-17, 23, 31, 32, 41, 44, 45).
Because the majority of gluconeogenic carbons are derived from
phosphoenolpyruvate (PEP) via the tricarboxylic acid (TCA)
cycle (1, 6, 23, 31, 32), hepatic GP is also intimately
linked to acetyl-CoA oxidation and energy production. Together, these
biochemical pathways form a metabolic network (Fig.
1) that is highly responsive to matching
the external demand for glucose with the availability of glycogen,
gluconeogenic precursors, and energy. Measurements of carbon flux
through this network typically combine an isotope dilution measurement
of endogenous GP with additional tracer measurements of the
contributing pathways (6, 8-10, 15, 18, 18, 25). As
one example, GP in fasting humans was determined by analysis of the
2H enrichment at glucose C-6 using the
hexamethylenetetramine method and mass spectrometry (MS) after infusion
of [6,6-2H2]glucose at a known rate
(6). It was possible to also give oral
2H2O and measure the contribution of
gluconeogenesis to GP from 2H enrichments at glucose C-5
vs. C-2 after parallel selective degradations of plasma
glucose and analysis by MS (6). However, the contributions
of glycerol vs. TCA cycle intermediates to gluconeogenesis could not be differentiated in this experiment, nor can 13C
tracers be combined with 2H tracers using the
hexamethylenetetramine method.

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Fig. 1.
Metabolic pathways involved in hepatic glucose synthesis and
tricarboxylic acid (TCA) cycle activity. G-6-P,
glucose 6-phosphate; G-3-P, glyceraldehyde
3-phosphate; DHAP, dihydroxyacetone phosphate; FBP, fructose
bisphosphate; PEP, phosphoenolpyruvate; OAA,
oxaloacetate; Succ-CoA, succinyl-CoA; -kg,
-ketoglutarate.
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13C tracers have also been used to measure metabolic flux
through the network. Endogenous GP was monitored with
[U-13C6]glucose by following disappearance of
the parent m+6 isotopomer from plasma glucose (23, 24,
38, 40, 41). Additional metabolic information can be derived
from partially labeled glucose molecules generated by recycling of
13C label in this experiment. However, the extent to which
the recycled label can be reliably analyzed and related to rates of
gluconeogenesis and TCA cycle flux is controversial (24, 26,
28). Others have used tracers, such as
[3-13C]lactate, that enter at the level of the TCA cycle
along with a separate tracer for measuring endogenous GP (9,
10). Neither [U-13C6]glucose nor
[3-13C]lactate tracers, however, can differentiate
glucose produced from glycogen, glycerol, or the TCA cycle.
In this report, we demonstrate that a 2H NMR spectrum of
monoacetone glucose (42) may be used to measure the
distribution of deuterium in blood glucose after ingestion of
2H2O. This information allows the direct
calculation of the contribution of glycogen, glycerol, and the TCA
cycle to GP in humans. Although inherently less sensitive, the NMR
method offers several advantages over MS analysis of glucose
2H enrichment (31, 32). First, it does not
require carbon-by-carbon degradation of glucose; rather, the relative
2H enrichment at each carbon position of glucose can be
read out in a single 2H NMR spectrum. Second, the prochiral
H-6R and H-6S resonances are well separated in
the 2H NMR spectrum of monoacetone glucose
(42); thus the normal assumptions required by MS to
quantitatively evaluate exchange at the level of fumarase in the TCA
cycle are eliminated. This allows a separate measure of gluconeogenesis
from the level of the triose phosphates (glycerol) vs. PEP
(the TCA cycle). Finally, the 2H measurement is not
compromised by the presence of 13C tracers, so experiments
can be designed to measure GP, gluconeogenic flux, pyruvate recycling
flux, and TCA cycle flux in a single experiment. We illustrate the
method here by reporting these flux values in 16-h-fasted humans using
the combined tracers [1,6-13C2]glucose,
2H2O, and
[U-13C3]propionate. Thus a rather
comprehensive picture of liver metabolism can be obtained during a
single patient visit, making the technique highly suitable for routine
clinical application.
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METHODS |
Materials.
Cambridge Isotopes (Cambridge, MA) was our source for 99%
2H2O, 99%
[1,6-13C2]glucose, and 99%
[U-13C3]propionate. Acetaminophen was derived
from regular-strength Tylenol capsules, and phenylacetate was obtained
from Sigma (St. Louis, MO).
Experimental protocol.
Five healthy, nonobese subjects [2 men and 3 women, 21-36 yr of
age, 50-86 (70 ± 14) kg body wt] were studied under a
protocol approved by the institutional human studies committee.
Subjects were admitted to the General Clinical Research Center at
Parkland Hospital and examined (history and physical) by an internist. All had blood glucose levels in the normal range (70-110 mg/dl), and none reported a history of chronic illness or use of medications on
a regular basis. All subjects began fasting at 6 PM. At 11 PM and again
at 3 AM, 99% 2H2O was taken orally (2.5 g/kg
body water, calculated as total weight times 0.6 for men or 0.5 for women). During the remainder of the study, 0.5%
2H2O was given ad libitum. At 6 AM, subjects
ingested a tablet containing 325 mg of acetaminophen. Between 7 and 8 AM, subjects ingested tablets containing phenylacetate (20 mg/kg) and
another 650 mg of acetaminophen. At 8 AM, a 3-h primed infusion of
[1,6-13C2]glucose (167 mg, 1.67 mg/min) was
initiated for each subject. The infusion medium was prepared by
dissolving 500 mg of sterile and pyrogen-free
[1,6-13C2]glucose into 150 ml of saline. The
infusion medium was passed through a 0.22-µm filter during
administration. Subjects received a 50-ml bolus of infusion medium over
2 min followed by constant infusion of 30 ml/h. Subjects also ingested
[U-13C3]propionate (10 mg/kg, packaged into 3 gel caps) between 8 and 9 AM. Also, beginning at 8 AM, 10 ml of blood
were drawn from a contralateral vein every 20 min for 2 h, with
additional blood drawn at 2.5 and 3 h. This amounted to seven
blood samples and a total of 70 ml of whole blood per subject. Urine
was also collected every hour from 8 AM to 2 PM, at which point the
study was concluded.
Analytic procedures.
For each subject, an aliquot of the
[1,6-13C2]glucose infusion medium was frozen
and enzymatically assayed for glucose. Blood samples were chilled
immediately after being drawn and centrifuged at 4°C in heparinized
tubes. The plasma was then processed for 13C and
1H spectroscopy of plasma glucose by perchloric acid
extraction, as previously described (21). For
2H NMR analysis of positional deuterium enrichment, two to
three plasma extracts from each subject were pooled and lyophilized to
complete dryness. Glucose was converted to monoacetone glucose by use
of the method of Landau et al. (31). After lyophilization, the residue containing monoacetone glucose was dissolved in 0.6 ml of
90% acetonitrile-10% 2H-depleted water plus a few grains
of sodium bicarbonate (42), and insoluble material was
centrifuged and discarded. Urine samples were treated with urease and
-glucuronidase and lyophilized as described previously
(21). The extract was then reconstituted in 10 ml of
water, and insoluble material was precipitated by centrifugation. The
supernatant was adjusted to pH 1.0 with perchloric acid, and the sample
was applied to an 8- to 10-ml cation-exchange column
(Dowex-50 × 8-H+) followed by 40 ml of water.
The column effluent was neutralized with KOH, lyophilized, and
resuspended in 600 µl of 2H2O. The pH was
then adjusted to 8.0 with NH4OH, the samples were centrifuged at 13,000 rpm with an Eppendorf centrifuge, and the supernatants were pipetted into 5-mm NMR tubes.
NMR spectroscopy.
Proton-decoupled 13C NMR spectra of blood and urine
extracts were collected using a Unity Inova 14.1-T spectrometer
operating at 150.9 MHz. Free-induction decays were multiplied by a 0.1- to 0.2-Hz exponential function before Fourier transformation. Typically, 9,000-18,000 free-induction decays were summed for each
blood extract and 6,000 for each urine extract, resulting in collection
times of 5-14 h per extract. Proton-decoupled 2H NMR
spectra of monoacetone glucose were acquired at 50°C
(42) with the same probe using a 90° pulse and a sweep
width of 920 Hz digitized into 992 points, giving an acquisition time
of 0.512 s. No additional interpulse delays were used in this pulse
sequence. Typically 90,000-100,000 scans were averaged.
The data were zero-filled to 4 K, multiplied by a 1-Hz exponential
function to increase signal-to-noise ratio, and Fourier
transformed. 2H signal intensities obtained with these
parameters were corrected for minor effects of differential saturation.
A comparison of 2H signal areas in spectra collected using
these standard pulsing conditions with those measured in spectra
collected using a 1.0-s acquisition time [sufficient for complete
relaxation (38)] were identical to within 7%.
Nevertheless, small correction factors were used to allow for partial
saturation when the 0.512-s acquisition conditions were used.
1H NMR spectra were obtained with the same spectrometer by
means of a 5-mm indirect probe. Spectra were acquired with a 90°
pulse after presaturation of the residual water signal and a 15-s
interpulse delay. Long-range couplings between 13C C-2 to
C-6 and H-1
were abolished by the application of a narrow-band WALTZ-16 13C-decoupling pulse sequence covering the 60- to
75-ppm region of the 13C NMR spectrum (18).
Two hundred fifty-six acquisitions were collected for a total
collection time of 64 min. All NMR spectra were analyzed using the
curve-fitting routine supplied with the NUTS PC-based NMR spectral
analysis program (Acorn NMR, Fremont, CA).
Metabolic flux calculations.
The 13C enrichment in the
[1,6-13C2]glucose used for the infusions was
verified by 1H NMR. The fraction of blood glucose that
contained 13C in C-1 was defined as f, the area
of the doublet due to flux (JCH) in the glucose
H-1 resonance relative to the total H-1 resonance area (Fig.
2). The fraction of blood glucose that
was [1,6-13C2]glucose relative to all glucose
containing 13C in C-1 was defined as g, the
doublet due to flux (JCC) arising from
[1,6-13C2]glucose relative to the total area
of the glucose C-1 resonance (Fig. 2). The fraction of
[1,6-13C2] glucose in plasma glucose was
calculated as fg. The values measured in spectra collected
at 120, 150, and 180 min were averaged for each subject and used in the
calculation of GP. The rate of appearance of glucose was calculated
from the known infusion rate of
[1,6-13C2]glucose (r) divided by
the average fraction found in plasma over the 120- to 180-min period.
GP is then defined as the rate of appearance of glucose minus the rate
of infusion of [1,6-13C2]glucose, or GP = (r/fg)
r.

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Fig. 2.
Plasma glucose C-1 (A) and H-1 resonance
(B) from 13C and 1H NMR spectra of a
plasma glucose extract prepared from 10 ml of blood drawn at the end of
[1,6-13C2]glucose infusion. S, singlet
component; D12, multiplet component from glucose isotopomers with
13C in positions 1 and 2; D123, multiplet component from
glucose isotopomers with 13C in positions 1, 2, and 3; D16,
multiplet component from glucose isotopomers with 13C in
positions 1 and 6.
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The fraction of glucose derived from glycogen, PEP, and gluconeogenesis
was estimated from the ratio of deuterium enrichment at positions 2, 5, and 6S as reported in the 2H NMR spectrum of
monoacetone glucose (42) by use of the following equations
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(1)
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(2)
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(3)
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Relative anaplerotic flux (OAA
PEP, where OAA is
oxaloacetate), pyruvate recycling flux (PEP
pyruvate or equivalent
pathway), and gluconeogenic flux (PEP
glucose) were calculated from
the multiplet areas measured in the 13C NMR spectrum of
urinary glucuronate or phenylacetylglutamine (PAGN), as described
previously (18, 20, 21). For urinary glucuronate C-5 (the
C-5
resonance was analyzed), the relevant equations are
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(4)
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(5)
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(6)
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For PAGN C-2, the relevant equations are
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(7)
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(8)
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(9)
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Gluconeogenic flux from PEP is the difference between
anaplerosis (OAA
PEP) and pyruvate recycling (PEP
pyruvate).
PEP recycling is indicated here by PEP
pyruvate, although it should be noted that the combined pathway OAA
PEP
pyruvate cannot be
distinguished from malate
pyruvate.
These relative fluxes were then converted to absolute values as
follows. First, absolute fluxes in hexose units from glycogen, PEP, and glycerol were defined as the product of each fractional contribution (Eqs. 1-3) times the endogenous GP in
micromoles of glucose per kilogram per minute. Second, the rate of GP
from PEP was converted to the rate of production of PEP by multiplying by 2. Finally, fluxes involved in the TCA cycle were calculated by
indexing the relative fluxes (Eqs. 4-6 or 7-9) to the rate of production of PEP. For example, if
GP is 10 µmol · kg
1 · min
1 and the
relative sources of glucose are glycerol (4%), glycogen (50%), and
PEP (46%), then the rate of GP from PEP is 4.6 µmol hexose
units · kg
1 · min
1 or 9.2 µmol triose
units · kg
1 · min
1. Flux
ratios related to the TCA cycle are defined relative to citrate
synthase. Given relative fluxes of OAA
PEP (6.5), PEP
pyruvate
(5), and PEP
glucose (1.5), then flux through OAA
PEP = 6.5 × 9.2
1.5 = 39.9 µmol · kg
1 · min
1,
and citrate synthase flux = 9.2
1.5 = 6.1 µmol · kg
1 · min
1.
Statistical analysis.
Values are means ± SD. Means were compared as noted using a
t-test assuming unequal variances.
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RESULTS |
Each of the three separate components required for analysis of
gluconeogenesis is presented individually.
Endogenous GP measurement from
[1,6-13C2]glucose.
A 13C NMR spectrum of the plasma glucose C-1
resonance
from blood taken 180 min after administration of
[U-13C3]propionate and
[1,6-13C2]glucose is shown in Fig. 2. The
resonance features well-resolved multiplets arising from
13C-13C splitting, reflecting the presence of
multiply labeled glucose molecules. These include signals from glucose
isotopomers generated from the gluconeogenic metabolism of
[U-13C3]propionate (D12 and D123) in addition
to the tracer amount of infused
[1,6-13C2]glucose (D16). As previously
demonstrated (18), the fraction of
[1,6-13C2]glucose remaining in plasma at any
time point can be quantified by measuring the contribution of
[1,6-13C2]glucose to the C-1
resonance
(13C spectrum) and the total 13C enrichment as
reported in the H-1
resonance (1H spectrum). The
1H NMR spectrum of the H-1
proton features well-resolved
13C satellites with sufficient signal-to-noise ratio for
reliable quantitation of the 2-3% excess 13C
enrichment levels from this experiment. Figure
3 summarizes the
[1,6-13C2]glucose fractional enrichment
values obtained from serial blood sampling for the five subjects.
The fractional enrichment of plasma [1,6-13C2]glucose reached steady state
well before the end of the infusion, with enrichments of
0.75-1.35%. For each individual, the steady-state enrichment was
calculated as the mean of the 120-, 150-, and 180-min enrichments. From
these data, average endogenous GP for these five individuals was
10.7 ± 0.9 µmol · kg
1 · min
1, with a
range of 9.8-12.1
µmol · kg
1 · min
1. These
values are in good agreement with other measures of endogenous GP in
healthy individuals after similar fasting times (the average of all
values reported in Refs. 4, 6,
13-15, 23, 27, and
39 is 11.2 ± 1.7 µmol · kg
1 · min
1).

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Fig. 3.
Time course of plasma [1,6-13C2]glucose
enrichment over the duration of
[1,6-13C2]glucose infusion for 5 subjects.
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Analysis of plasma glucose 2H enrichment by
2H NMR.
Figure 4 shows a 2H NMR
spectrum of monoacetone glucose derived from two pooled plasma extracts
(corresponding to 20 ml of whole blood). The area of each resonance is
proportional to 2H enrichment at that position, so the
spectrum provides a simple and direct readout of 2H
enrichment ratios (42). It is important to point out that the 2H NMR measurement is not influenced by the presence of
tracer levels of 13C in the glucose (or monoacetone
glucose) molecule. Table 1 summarizes the
relative contributions of glycogen, glycerol, and PEP to GP.

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Fig. 4.
2H NMR spectrum of the 3.0- to 6.0-ppm region
featuring the signals of monoacetone glucose that were derived from the
7 aliphatic hydrogens of plasma glucose (H-1 to H-6). The sample was
prepared from 2 pooled plasma glucose extracts (60 and 90 min).
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TCA cycle and gluconeogenic flux measurements from
[U-13C3]propionate incorporation into hexose
and PAGN.
As previously demonstrated (21), relative anaplerotic,
pyruvate recycling, and gluconeogenic fluxes can be obtained by a 13C isotopomer analysis of plasma glucose, urinary
glucuronide, or the glutamine fragment in urinary PAGN. Equations
4-9 describe these relationships. Figure
5 illustrates typical multiplets observed in the 13C NMR spectrum of plasma glucose C-2
and
urinary glucuronate C-5
of the same individual. The multiplet
pattern arises from metabolism of
[U-13C3]propionate at the level of the liver
TCA cycle and is not affected by the presence or metabolism of
[1,6-13C2]glucose (18). The
difference in signal-to-noise ratio in these two spectra is largely due
to the amount of urinary glucuronate in ~100-150 ml of urine
compared with the amount of glucose in 10 ml of blood. Given that the
multiplets in blood glucose C-2
and urinary glucuronate C-5
report identical flux values (21) and given the large
differences in signal-to-noise ratio of the spectra shown, relative
flux values as reported by the glucuronate spectra are reported here.
Estimates of flux ratios based on acetaminophen glucuronide for five
healthy, nonobese individuals were OAA
PEP = 7.1 ± 1.1, PEP
pyruvate = 5.3 ± 1.0, and PEP
glucose = 1.8 ± 0.3 (Table 1).

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Fig. 5.
13C NMR spectra of the glucose C-2
multiplet from a plasma glucose extract prepared from 10 ml of blood
drawn at 150 min after [U-13C3]propionate
ingestion (A) and the glucuronate C-5 multiplet of a
urine extract prepared from urine collected 3-4 h after
[U-13C3]propionate ingestion (B).
Both spectra were obtained from the same subject. S, singlet component;
D12, multiplet component from glucose isotopomers with 13C
in positions 1 and 2; D23, multiplet component from glucose isotopomers
with 13C in positions 2 and 3; Q, multiplet component from
glucose isotopomers with 13C in positions 1, 2, and 3 or
from glucuronate isotopomers with 13C in positions 4, 5, and 6; D56, multiplet component from glucuronate isotopomers with
13C in positions 5 and 6; D45, multiplet component from
glucuronate isotopomers with 13C in positions 4 and 5; X,
unknown signal.
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The 13C NMR spectra of urinary PAGN were also of high
quality (Fig. 6). Analysis of the
glutamine C-2 multiplets using Eqs. 7-9 provided the
following relative flux estimates (Table 1): OAA
PEP = 6.3 ± 0.4, PEP
pyruvate = 5.4 ± 0.4, and PEP
glucose = 0.9 ± 0.2. As noted in an earlier study of 24- to
28-h-fasted individuals (21), flux estimates determined by
analysis of PAGN were significantly different from those derived from
spectra of blood glucose or urinary glucuronate. A comparison of the
flux ratios derived from acetaminophen glucuronide and PAGN (Table 1)
shows a significantly lower relative PEP
glucose flux for PAGN
than for glucuronate (P < 0.01). There was no
significant difference in estimates of PEP
pyruvate or OAA
PEP
(relative to citrate synthase, Table 1).

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Fig. 6.
13C NMR spectra of phenylacetylglutamine C-2,
C-3, and C4 resonances from the 3- to 4-h urine extract. S, singlet
component; D12, multiplet component from glutamine isotopomers with
13C in positions 1 and 2; D23, multiplet component from
glutamine isotopomers with 13C in positions 2 and 3; D45,
multiplet component from glutamine isotopomers with 13C in
positions 4 and 5; Q, multiplet component from glutamine isotopomers
with 13C in positions 1, 2, and 3.
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With the methylene carbon of phenylacetate (~44 ppm, 1.1%
13C) as an internal standard, the enrichment in PAGN C-2
and C-3 was 2.13 ± 0.19 and 2.04 ± 0.16%, respectively,
while the 13C enrichment in C-4 was essentially equal to
natural abundance levels (1.00 ± 0.06%). The enrichment in C-4
reports enrichment in acetyl-CoA and suggests that C-2 of acetyl-CoA
was not enriched above natural abundance. However, the PAGN C-4
resonance did show a very small doublet (D45) characteristic of C-4 to
C-5 coupling (52 Hz) resulting from entry of
[1,2-13C2]acetyl-CoA into the TCA cycle.
Because [1,2-13C2]acetyl-CoA can only arise
from [1,2,3-13C3]- or
[2,3-13C2]pyruvate, the presence of D45
indicates that a small portion of recycled pyruvate was oxidized to
acetyl-CoA by pyruvate dehydrogenase. The intensity of the D45 signal
was ~5% of that of the C-2 multiplet, indicating that the level of
acetyl-CoA labeling was very low compared with that of OAA, pyruvate,
and PEP. However, because Eqs. 4-9 explicitly assume
zero labeling of acetyl-CoA (20), this small enrichment of
acetyl-CoA could potentially introduce errors into the flux estimates.
The possible impact of this on anaplerosis, pyruvate recycling, and
gluconeogenic flux estimates was tested by simulating (using
tcaSIM1) 13C NMR
spectra of glutamine for two metabolic situations, one with no labeling
of acetyl-CoA and another with entry of 5%
[1,2-13C2]acetyl-CoA. Identical flux values
(within 5%) were obtained by analysis of the resulting glutamate C-2
multiplet areas with the use of Eqs. 7-9. We
conclude that enrichment of glutamate C-4 at the level seen here in
PAGN does not interfere with accurate measurement of the metabolic
fluxes of interest.
Integration of metabolic data.
The relative flux values reported by the
[U-13C3]propionate and
2H2O tracers were converted to absolute flux
values by referencing them to GP as reported by turnover of
[1,6-13C2] glucose. This provides the
rather comprehensive picture of TCA cycle-related fluxes and
gluconeogenesis summarized in Table 2.
Absolute flux estimates are reported for glucuronate- and PAGN-based
13C-isotopomer analyses.
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DISCUSSION |
This study demonstrates that a comprehensive metabolic profile of
gluconeogenesis and TCA cycle activity may be obtained in humans with
three stable isotope tracers and NMR analysis of blood and urine
samples. Multiple tracers were required, because multiple experimental
determinations are necessary to describe the known pathways involved in
gluconeogenesis. These data were obtained after enrichment of total
body water with 2H2O and two 13C
tracers, [1,6-13C2] glucose to measure
endogenous GP and [U-13C3] propionate as an
index of TCA cycle activity.
2H NMR of monoacetone glucose.
The 2H NMR spectrum of monoacetone glucose
(42) derived from 20 ml of plasma glucose provided a
convenient, direct measure of 2H enrichment at each site in
the glucose molecule. Enrichment at H-2, reflecting isotopic
equilibration of plasma glucose H-2 with body water (4, 8,
39), was highest in every sample, while enrichment at H-5 was
~50% of that at H-2. This indicates that glycogenolysis and
gluconeogenesis contribute equally to endogenous GP after a 16- to 18-h
fast. These observations agree with gas chromatography (GC)-MS
measurements of H-5 and H-2 enrichments in 14- and 18.5-h-fasted
individuals, where the contribution of gluconeogenesis to endogenous GP
was reported to be 47 ± 4 and 54 ± 2%, respectively
(6, 31). Furthermore, the observation that 2H
enrichment at H-6S was ~95% of that at H-5 indicates that
the majority of gluconeogenic carbons are derived from the TCA cycle (PEP) and very few from glycerol (4, 8, 31). Most
importantly, the 2H NMR spectrum of monoacetone glucose
shows separate resonances for the prochiral H-6 hydrogens of glucose
(42), while exchange of 2H2O at
the level of fumarase in the TCA cycle specifically enriches the
H-6S position (46). Interestingly, the
prochiral H-6 protons had comparable levels of enrichment, with a
tendency toward higher enrichment in H-6S.
The capacity to measure individual enrichment of the prochiral glucose
hydrogens by 2H NMR provides additional insight into the
mechanisms that contribute to the enrichment of the methylene hydrogens
of PEP from body water. Although the exchange mechanisms for enriching
H-2 and H-5 of glucose from body water are considered to be essentially quantitative, exchange of pyruvate and water hydrogens is believed to
be only 80% complete (31). If this is true, then
enrichment of the PEP methylene hydrogens would be less than that of
body water, and the H-6-to-H-2 enrichment ratio of glucose as measured by GC-MS would underestimate the contribution of PEP to GP. However, this should only be true for glucose H-6R, because pyruvate
enrichment is reported by the pro-S hydrogen of
OAA2, while the
pro-R hydrogen of this intermediate can undergo complete exchange with body water by interconversion with malate and fumarate (Fig. 7). If randomization between OAA,
malate, and fumarate is complete, then 2H enrichment at the
pro-R hydrogen of OAA would equal that of body water. Thus,
to the extent that randomization is incomplete, enrichment of the
pro-R hydrogen of OAA will be a weighted average of pyruvate
and body water enrichments. However, given that carbon tracer data from
this and other studies have demonstrated that there is extensive
backward scrambling of OAA with fumarate as well as extensive recycling
of PEP, pyruvate, and OAA in liver (9, 10, 19, 32), it is
perhaps not surprising to find that glucose H-6R and
H-6S had near-equivalent levels of 2H enrichment
from 2H2O. This observation means that the
H-6-to-H-2 enrichment ratio reported by GC-MS (a single H-6
measurement is taken, and a 2H enrichment distributed
equally between H-6R and H-6S is assumed) should
be equivalent to the H-6S-to-H-2 ratio reported by
2H NMR.

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|
Fig. 7.
Labeling of the prochiral methylene hydrogens of PEP from
pyruvate and body water in the absence (1) and presence
(2) of exchange between OAA, malate, and fumarate. *,
Labeled hydrogen derived from labeled water as a result of fumarase
activity. , labeled hydrogen derived from pyruvate.
|
|
TCA cycle fluxes.
In this study, PEP carboxykinase and pyruvate recycling fluxes were
about seven- and fivefold higher than TCA cycle flux, while net
gluconeogenic outflow (defined as PEP
glucose flux) was about
twofold greater than TCA cycle flux. Compared with 24- to 28-h-fasted
individuals (19), gluconeogenic outflow in the present
group (fasted for 16-18 h) tended to be lower, while PEP carboxykinase and pyruvate recycling fluxes tended to be higher.
Estimates of relative PEP
glucose flux show a large variation
between different studies. In the study of Magnusson et al. (35), PEP
glucose flux was approximately three
times citrate synthase flux for 60-h-fasted individuals and
overnight-fasted subjects given intravenous glucose and glucagon. In
comparison, the values reported by Diraison et al. (9,
10) are only ~0.5 times flux through citrate synthase.
Absolute TCA cycle fluxes are derived by indexing relative fluxes to an
independent measurement of absolute PEP
glucose flux; therefore,
uncertainties in relative PEP
glucose flux propagate systematic
uncertainties in estimates of absolute TCA cycle fluxes. For example,
absolute hepatic citrate synthase fluxes estimated by Diraison et al.
range from 20 to 35 µmol · kg
1 · min
1, while
estimates3 from Magnusson et
al. range from 5 to 7 µmol · kg
1 · min
1.
Absolute citrate synthase flux estimates from our study (5.4 and 10.9 µmol · kg
1 · min
1 from
urinary glucuronate and PAGN, respectively) are similar to the
estimates of Magnusson et al. No other published estimates of human
hepatic citrate synthase flux are available for comparison.
The anaplerotic, PEP recycling, and TCA cycle fluxes reported here are
based on a simple set of equations that are valid when excess
enrichment of acetyl-CoA and C-4 and C-5 of PAGN are negligible relative to enrichment of C-1 to C-3 (18). The
advantage of such equations over computational analysis is that they
provide a simple and direct way of relating the C-2 multiplet pattern from the 13C NMR spectrum of glucose or PAGN to metabolic
fluxes through anaplerotic, recycling, and oxidative pathways
(16, 18, 19). Although the relative enrichments of PAGN
C-4 encountered in the present study were sufficiently low for the
simple equations to be valid, there may be circumstances where the
relative enrichment of C-4 is high enough to introduce significant
errors into metabolic flux estimates derived by this method. For
example, in 60-h-fasted subjects infused with
[3-14C]lactate, the specific activity of C-4 relative to
C-2 and C-3 of PAGN was substantially higher than that in subjects
infused with [2-14C]propionate or
[3-14C]propionate (27). In overnight-fasted
subjects infused with [3-13C]lactate, excess enrichment
in C-4 of PAGN was 35-42% of that in C-2 and C-3 (9,
10). On the basis of these results, it is likely that a
[U-13C3]lactate tracer might also generate
more enriched acetyl-CoA and a higher relative enrichment of PAGN C-4
than [U-13C3]propionate under the same
physiological conditions. Such conditions generate more complex
isotopomer distributions and require computational analysis for
obtaining metabolic flux estimates (12, 34). The labeling
discrepancies between propionate and lactate tracers likely reflect
heterogeneous metabolism of these substrates within the hepatic lobule.
Given that propionate is quantitatively extracted from the portal
circulation while lactate is not, it is probable that most of the
propionate tracer is metabolized by the highly gluconeogenic periportal
cells. Consequently, the perivenous cells are exposed to little
[U-13C3]propionate tracer directly but are
likely to encounter secondary labeled products of periportal
[U-13C3]propionate metabolism, such as
13C-enriched glucose or lactate. We hypothesize that this
mechanism could contribute to the different isotopomer distributions of the OAA moieties of hepatic glucose and glutamine from
[U-13C3]propionate (19). In
comparison, a lactate tracer is likely to be more evenly distributed
across the lobule; hence, the labeling pattern may reflect a different
combination of periportal and perivenous metabolism, resulting in
analyte labeling distributions that are different from those obtained
from a propionate tracer. The question of which tracer and analyte
combination best describes human hepatic TCA cycle activity remains
outstanding and will require correlation of metabolic flux estimates
with measurements of hepatic arteriovenous substrate balances and
glycogen levels.
Analytic considerations.
A minimum of 20 ml of whole blood was required for the 2H
NMR measurements of plasma glucose under conditions where total body water is enriched to ~0.5% in 2H. Although this amount
of blood can be safely collected from adults, this volume would be
excessive for small children. Thus, for an equivalent study in
children, one would be required to increase the NMR sensitivity by
using smaller-volume 2H microprobes, or perhaps cryoprobes,
or by collecting larger amounts of glucose equivalents via urinary
glucuronide (31). Although ~1.0 mmol of glucuronide
might be available in urine (compared with ~0.11 mmol from 20 ml of
blood), this must be weighed against the loss of 2H data
from the H-6 position (glucose C-6 becomes a carboxylate in the
glucuronide) and the additional synthetic steps involved in the
conversion of acetaminophen glucuronide to monoacetone glucose.
Clearly, the preference would be to perform the 2H analysis
on plasma glucose whenever possible.
In summary, any comprehensive analysis of gluconeogenesis in
humans requires multiple tracers. Existing protocols typically require
experimental studies at different times to account for the complex
pathways involving the TCA cycle. The combined
2H-13C NMR isotopomer method reported here
allows a practical clinical method for measuring gluconeogenesis in a
single study. Furthermore, all the experimental data are collected from
blood or urine, and placing humans into magnets is not required. Given
that the metabolic tracers can be administered orally and that all data
can potentially be collected from urinary metabolites, this method now
offers, for the first time, the possibility of a noninvasive
examination for outpatient studies that may prove valuable in
population-based studies of glucose metabolism.
 |
ACKNOWLEDGEMENTS |
We acknowledge the excellent technical assistance and support
provided by the staff of the General Clinical Research Center at the
University of Texas Southwestern Medical Center.
 |
FOOTNOTES |
This research was supported by National Institutes of Health Grants
RR-02584, HL-34557, and M01-RR-00633.
1
tcaSIM is an isotopomer simulation program; it is
available by e-mail (mark.jeffrey{at}utsouthwestern.edu), or it can be
downloaded from the "Available Products" page of the Rogers Center
website (www2.swmed.edu/rogersmr2). Address requests by mail to F. M. H. Jeffrey, Mary Nell and Ralph B. Rogers Center, 5801 Forest Park Rd., Dallas, TX 75235-9085.
2
R-[3-2H]OAA
(Z)-[3-2H]PEP
S-[3-2H]PEP
S-[6-2H]glucose;
S-[3-2H]OAA
(E)-[3-2H]PEP
R-[3-2H]PEP
R-[6-2H]- glucose
(46).
3
Published flux estimates in millimoles per minute
were converted to micromoles per kilogram per minute using the reported average weight of the subjects.
Address for reprint requests and other correspondence: C. R. Malloy, Mary Nell and Ralph B. Rogers Magnetic Resonance Center, 5801 Forest Park Rd., Dallas, TX 75235-9085 (E-mail:
craig.malloy{at}utsouthwestern.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 23 October 2000; accepted in final form 21 May 2001.
 |
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