Download [U-13C]propionate, phenylacetate, and acetaminophen

13C
NMR measurements of human gluconeogenic fluxes
after ingestion of [U-13C]propionate, phenylacetate,
and acetaminophen
JOHN G. JONES,1 MICHAEL A. SOLOMON,2 A. DEAN SHERRY,1,3 F. M. H. JEFFREY,1
AND CRAIG R. MALLOY1,2
1Department of Radiology, University of Texas Southwestern Medical Center, Dallas 75235;
2Department of Internal Medicine, University of Texas Southwestern Medical Center and
Department of Veterans Affairs Medical Center, Dallas 75216; and 3Department of Chemistry,
University of Dallas, Richardson, Texas 75083
glucose; glucuronide; glutamine; isotopomers; nuclear magnetic resonance
offer promise for aiding
diagnosis of abnormal glucose metabolism in the clinical setting. Although radioactive precursors can be
used to trace human hepatic metabolism (28, 30), they
are unlikely to be widely adopted in the clinical environment because of possible risks to the patient and the
impracticality of systematic radiation containment.
More recently, similar metabolic models have been
successfully adapted for use with stable isotope tracers
(3, 15), and well-established chemical biopsy methods
have been used to target carbon skeletons of hepatic
UDP-glucose and glutamine (12, 15, 27–30, 40).
TRACERS OF HEPATIC METABOLISM
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solely to indicate this fact.
Most common tracers of hepatic metabolism, such as
the gluconeogenic substrates lactate and alanine, may
be metabolized to some extent by peripheral tissues
and thus possibly confound analysis of liver metabolism. Ideally, any tracer should be infused directly into
the hepatic portal vein and extracted quantitatively by
the liver. Portal vein infusions are too invasive for
human studies, however, and most hepatic tracers are
not quantitatively extracted from portal circulation by
the mammalian liver (34). Propionate may be an important exception in that it is quantitatively extracted into
liver from portal circulation in a wide range of mammalian species, likely including humans (2, 8). It is
generated in significant quantities by colonic fermentation of a-amylase-resistant starch and nonstarch polysaccharides (2, 8). Propionate has been administered by
gastric infusion to human subjects at rates of 4 mmol/h
over a 3-h period with no ill effects and no alteration of
hepatic glucose production (26). Being a weak acid,
propionate is likely to pass rapidly and efficiently into
both splanchnic and portal vein circulation from the
stomach after ingestion, in a manner similar to aspirin.
These features suggest that oral administration of
propionate may be an effective method for noninvasive
analysis of hepatic gluconeogenesis in humans.
Although deployment of more hepatophilic tracers
has minimized labeling contributions from peripheral
metabolism (24, 30, 41), values of gluconeogenic flux
reported by these newer methods remain controversial.
Katz et al. (23) suggested that net gluconeogenic flux
values of about three times the tricarboxylic acid cycle
rate are unsustainable, because the ATP demand for
gluconeogenesis exceeds the total ATP output of the
citric acid cycle via substrate level and oxidative phosphorylation. Others have proposed that sufficient ATP
is available if a significant portion of hepatic acetyl-CoA
originates from b-oxidation (24). Because measurements of gluconeogenesis in humans to date have been
derived by using tracers that enter the citric acid cycle
via pyruvate or acetyl-CoA, it is important to verify
these observations by use of a tracer that enters the
citric acid cycle via a different metabolic pathway.
Herein, we demonstrate that such measurements are
easily derived from a single 13C NMR spectrum of human
plasma glucose, urinary glucuronide, and urinary phenylacetylglutamine after ingestion of [U-13C]propionate,
phenylacetate, and acetaminophen.
0193-1849/98 $5.00 Copyright r 1998 the American Physiological Society
E843
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Jones, John G., Michael A. Solomon, A. Dean Sherry,
F. M. H. Jeffrey, and Craig R. Malloy. 13C NMR measurements of human gluconeogenic fluxes after ingestion of
[U-13C]propionate, phenylacetate, and acetaminophen. Am.
J. Physiol. 275 (Endocrinol. Metab. 38): E843–E852, 1998.—
Anaplerotic, pyruvate recycling, and gluconeogenic fluxes
were measured by 13C isotopomer analysis of plasma glucose,
urinary phenylacetylglutamine, and urinary glucuronide in
normal, 24-h-fasted individuals after ingestion of [U-13C]propionate, phenylacetate, and acetaminophen. Plasma glucose
isotopomer analysis reported a total anaplerotic flux of 5.92 6
1.03 (SD) relative to citrate synthase. This was not significantly different from glucuronide and phenylacetylglutamine
analyses (6.08 6 1.16 and 7.14 6 1.94, respectively). Estimates of pyruvate recycling from glucose and glucuronide
isotopomer distributions were almost identical (3.55 6 0.99
and 3.66 6 1.11, respectively), whereas phenylacetylglutamine reported a significantly higher estimate (5.74 6 2.13).
As a consequence, net gluconeogenic flux reported by phenylacetylglutamine (1.41 6 0.28) was significantly less than that
reported by glucose (2.37 6 0.64) and glucuronide (2.42 6
0.76). This difference in fluxes detected by analysis of phenylacetylglutamine vs. hexose is likely due to compartmentation
of hepatic metabolism of propionate. Net gluconeogenic flux
estimates made by use of this stable isotope method are in
good agreement with recent measurements in humans with
[14C]propionate.
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MEASUREMENTS OF GLUCONEOGENESIS WITH [U-13C]PROPIONATE
METHODS
Fig. 1. Time course for administration of
[U-13C]propionate, phenylacetate, and acetaminophen and sampling of blood and urine.
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Experimental protocol. Six healthy subjects [5 male and 1
female, 23–39 yr old, and weighing 54–88 kg (76 6 12 kg)]
were studied under a protocol approved by the institutional
human studies committee. Subjects were fasted for 24 h
before the start of the experiment and allowed access to
water. Figure 1 shows a flowchart of drug ingestion and blood
and urine sampling. From 7:00–8:00 AM on the day of the
study, each subject ingested 66 mg/kg phenylacetic acid
distributed in 12 gelatin capsules (Gallipot, St. Paul, MN). At
8:00 AM, each subject ingested 2 tablets (500 mg each) of
acetaminophen, and the first blood sample was also drawn
into heparinized tubes (25 or 30 ml). Over the next hour, five
of the subjects ingested a total of 20 or 25 mg/kg sodium
[U-13C]propionate distributed in 6 gelatin capsules, and the
sixth subject received 17 mg/kg of the sodium [U-13C]propionate. After the 8:00 AM sample, blood was drawn at 20-min
intervals over the next 2 h, with additional draws at 2.5 and 3
h (total blood drawn was 225 or 270 ml). Urine was also
collected every hour over a 6-h period starting at 9:00 AM,
with the exception of one individual whose urine was collected every 2 h.
Analytic procedures. Blood samples were chilled and centrifuged at 4°C in heparinized tubes immediately after being
drawn. The plasma was deproteinized by addition of 0.5 ml of
ice-cold 70% perchloric acid, and the precipitated protein was
removed by centrifugation. The supernatant was adjusted to
pH 5.0 with potassium hydroxide, and the insoluble potassium perchlorate was removed by a second centrifugation.
The glucose content of each sample was assayed enzymatically before oxidation to gluconate with glucose oxidase (18).
The samples were deproteinized a second time, and the pH
was adjusted to 10.0 with KOH before lyophilization. Samples
were resuspended in 600 µl 2H2O containing a potassium
glycolate standard in which the glycolate carbons had natural
13C abundance (assumed to be 1.11%). For each NMR sample,
the amount of glycolate added was equal to the amount of
glucose assayed in the parent plasma extract (typically 20–40
µmol).
Our initial studies revealed that lyophilized urine samples
were unsuitable for high-resolution 13C NMR analysis of the
phenylacetylglutamine and glucuronide moieties, because 1)
they were highly viscous, 2) they had a high salt content, and
3) the glucuronide was partially converted into a mixture of
a,b-glucuronate and a,b-6,3-glucuronolactone in many of the
samples. Hence, all urine was subsequently treated with
urease and b-glucuronidase and then partially purified by
ion-exchange chromatography before 13C NMR spectroscopy.
Urine samples were adjusted to pH 7.0 and incubated
overnight with 10,000 U of urease per sample. The pH was
then readjusted to 7.0, and the urine was further incubated
for 24 h at 37°C with 10,000 Fishman units of Escherichia coli
b-glucuronidase. A trace of sodium azide was also added to
inhibit possible infection from airborne microorganisms. The
samples were deproteinized by perchloric acid and centrifuged, the supernatant pH was adjusted to 10.0, and the
sample was left at room temperature for 30 min. (This last
step hydrolyzed any glucuronolactone that might have formed
from the nascent glucuronate during incubation.) The pH was
then adjusted to 8.0, the sample was loaded onto a 5- to 7-ml
volume anion-exchange column (Dowex-138-acetate), and
the column was washed with 40 ml of water. Phenylacetylglutamine and a,b-,D-glucuronic acid were released by eluting
with 15 ml of 10 M acetic acid. This fraction was lyophilized
and resuspended in 700 µl 2H2O. The pH was then adjusted to
9.0 with NaOD, the samples were centrifuged at 13,000 rpm
with an Eppendorf centrifuge, and the supernatants were
pipetted into 5-mm NMR tubes for NMR analysis.
NMR spectroscopy. Proton-decoupled 13C NMR spectra of
blood extracts were obtained using a 5-mm probe on a 9.4 T
Bruker Omega spectrometer operating at 100.61 MHz. Freeinduction decays were digitized into 32 K points and were
routinely multiplied by a 0.5- to 1.0-Hz exponential before
Fourier transformation. Proton-decoupled 13C NMR spectra
of urine extracts were obtained with a Unity INOVA 14.5 T
spectrometer operating at 150.9 MHz (Varian Instruments,
Palo Alto, CA). Free-induction decays were digitized into 44 K
points and were multiplied by a 0.5-Hz exponential before
Fourier transformation. Spectra from both systems were
analyzed using the curve-fitting routine supplied with the
NUTS PC-based NMR spectral analysis program (Acorn NMR,
Fremont, CA). Typical numbers of acquisitions were 12,000–
18,000 for each blood extract and 6,000–12,000 for each urine
extract. Collection times per extract ranged from 4.5 to 13.5 h.
Quantitation of 13C enrichments. 13C enrichments of carbons in the glutamine moiety of phenylacetylglutamine were
quantitated directly from the 13C NMR spectrum of the
isolated phenylacetylglutamine by use of the method of
Dugelay et al. (11). Gluconate 13C enrichments were quantitated directly from the 13C NMR spectrum by comparing the
total area of each gluconate resonance with the C-2 resonance
of the glycolate standard. Correction factors to account for T1
MEASUREMENTS OF GLUCONEOGENESIS WITH [U-13C]PROPIONATE
E845
Figure 4 shows the complete 13C NMR spectrum of
gluconate from the 120-min blood sample. The nineline C-2 resonance indicates that all four possible 13C
isotopomers are present in the C-1 to C-3 triose unit,
the relative ratios of which provide quantitative information about metabolism of [U-13C]propionate to glucose (18). The key components of this metabolic network are anaplerotic inflow (y), oxaloacetate-pyruvate
(OAA-PYR) recycling (pk), net gluconeogenic outflow (g), and citrate synthase flux. Anaplerotic inflow is the total influx of both new and recycled C-4
units into the oxaloacetate pool. OAA-PYR recycling
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Fig. 2. 13C NMR spectra of gluconate carbons 1 (GL-1) and 2 (GL-2)
obtained by oxidation of glucose from a single set of plasma extracts
obtained at the stated time intervals after ingestion of [U-13C]propionate.
and nuclear Overhauser enhancement differences between
the glycolate C-2 and gluconate carbons were derived from
the time 0 blood extract, where all the gluconate carbons are
at natural abundance enrichment.
Metabolic flux calculations. Estimates of total anaplerotic
inflow into oxaloacetate (y), recycling of oxaloacetate carbons
via pyruvate (pk), and net gluconeogenic flux (g) (all relative
to citrate synthase flux) were calculated from the 13C-13C
spin-coupled multiplets of gluconate C-2, phenylacetylglutamine C-2, and glucuronate C-5b by use of methods described
previously (18).
RESULTS
13C
NMR spectra from blood samples. Figure 2 shows
NMR spectra of gluconate C-1 and C-2 from blood
drawn from an individual at various times after oral
ingestion of [U-13C]propionate. Initially, only singlets
from natural abundance 13C were detected, but by 20
and 40 min [U-13C]propionate conversion into glucose
was already evident from the appearance of 13C-13C
spin-coupled multiplets in all six gluconate resonances.
The intensity of the multiplet resonances increased
gradually for 120 min and then remained relatively
constant between 120 and 180 min. In all subjects, the
nine-line C-2 gluconate multiplet was clearly formed at
120 min, and the ratios of multiplet components remained constant between 150 and 180 min. The time
course of gluconate fractional enrichment from 0 to 180
min is shown in Fig. 3. Carbons 1, 2, 5, and 6 of
gluconate showed identical enrichment kinetics, reaching a peak enrichment of ,4.5%1, whereas carbons 3
and 4 reached peak enrichments of ,2.5%. Between
120 and 180 min, enrichment of all six gluconate
carbons remained relatively constant.
13C
1 Enrichment values include the natural abundance
tion (assumed to be 1.11%).
13C
contribu-
Fig. 3. Time course of enrichment of plasma gluconate carbons 1–6
(GL-1–GL-6) from 0–180 min after ingestion of [U-13C]propionate.
Each plot represents means 6 SD for 5 individuals given 20–25
mg/kg [U-13C]propionate and does not include the 6th individual, who
received 17 mg/kg propionate.
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MEASUREMENTS OF GLUCONEOGENESIS WITH [U-13C]PROPIONATE
flux is the combined activities of all pathways that
convert OAA to phosphoenolpyruvate (PEP) and back
to OAA via pyruvate carboxylase. These include
OAA=PEP=PYR=OAA (the pyruvate kinase pathway) and OAA=MAL=PYR=OAA (the malic enzyme
pathway) in hepatocytes, and, through peripheral catabolism and hepatic resynthesis of glucose, the Cori
and glucose-alanine cycles. Gluconeogenic flux, the
difference between total anaplerotic inflow and total
OAA-PYR recycling, represents net export of carbons
from the metabolic network. Although carbons can
leave the citric acid cycle through pathways other than
gluconeogenesis (for example as aspartate and glutamate), it is generally assumed that gluconeogenesis
accounts for the bulk of the total anaplerotic carbon
outflow in liver (30). Given certain assumptions2, simple
mathematical equations relate the glucose 13C isotopomers (as reported by the C-2 resonance) to relative fluxes
through these pathways (18).
Urine analysis. The time course of urinary phenylacetylglutamine enrichment was similar to that of
blood glucose, with peak enrichments being achieved
2–3 h after ingestion of propionate (Fig. 5). However,
the 13C fractional enrichment in C-1, C-2, and C-3 of
phenylacetylglutamine was significantly lower than
that of C-1, C-2, C-5, or C-6 of glucose or glucuronate
(compare Figs. 3 and 5). This difference is thought to be
due to dilution of the glutamine pool by inflow of
unenriched glutamine from peripheral tissues such as
skeletal muscle (24, 30). Figure 6 shows the 20- to
100-ppm region of a 13C NMR spectrum of urine
2 Key assumptions are 1) isotopic steady-state conditions, 2) negligible labeling of acetyl-CoA, and 3) complete randomization of
oxaloacetate carbons via fumarate.
Fig. 5. Time course of enrichment of carbons 1–4 from glutamine
moiety of urinary phenylacetylglutamine 30–330 min after ingestion
of [U-13C]propionate. Times denote midpoint of each hourly urine
collection. Each plot represents means 6 SD for 5 individuals given
20–25 mg/kg [U-13C]propionate and does not include the 6th individual, who received 17 mg/kg propionate. j, Carbon 1; k, carbon 2;
s, carbon 3; r, carbon 4.
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Fig. 4. Complete 13C NMR spectrum of plasma gluconate from a
120-min blood sample and isotopomer analysis of 9-line multiplet of
carbon 2 (inset). GL-1 to GL-6 refer to resonances of gluconate
carbons 1–6. In inset, S, singlet resonance of carbon 2; D12, doublet
from coupling of carbon 2 with neighboring carbon 1; D23, doublet
arising from coupling of carbon 2 with neighboring carbon 3; Q,
doublet of doublets, or quartet, arising from coupling of carbon 2 with
both its neighbors.
collected 2 h after the last propionate ingestion. The
C-2, C-3, and C-4 resonances of the glutamine moiety of
phenylacetylglutamine are prominent, as is the natural
abundance signal of the phenylacetyl methylene carbon
at 43 ppm. The phenylacetylglutamine C-2 and C-3
resonances appear as multiplets arising from 13C-13C
spin-coupling, whereas the C-4 resonance is largely a
singlet (flanked by a weak doublet) arising primarily
from natural abundance levels of 13C. This indicates
that few of the 13C-enriched gluconeogenic precursors
that leave the cycle actually reenter the cycle pools via
acetyl-CoA, thus validating one important simplification of the metabolic model (18).
Several key glucuronate multiplets are also fully
resolved in the 13C NMR urinary spectrum, including
the C-5b resonance (see Fig. 6). The C-5b carbon of
glucuronate, like that of gluconate C-2 and phenylacetylglutamine C-2, has different coupling constants from
its nearest neighboring carbons (JC-5–C-6 5 59 Hz and
JC-5–C-4 5 40 Hz) and hence appears also as nine
resolved resonances. The relative areas of the gluconate C-2, glucuronate C-5b, and glutamine C-2 multiplets are compared in Table 1. The multiplet areas of
glucuronate C-5b will be identical to those of gluconate
C-2 (from blood) if the distribution of 13C isotopomers is
identical in dihydroxyacetone phosphate (triose unit
reflected by gluconate C-2) and glyceraldehyde-3phosphate (triose unit reflected by glucuronate C-5b).
Because both species originate from PEP with no
rearrangement of the carbon skeleton, their labeling
patterns should be identical, with the assumption of
negligible pentose cycle activity (28). However, the
fractional enrichments of the two pools can differ as a
result of 1) incomplete equilibration of the label by
triose phosphate isomerase and 2) influx of unlabeled
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MEASUREMENTS OF GLUCONEOGENESIS WITH [U-13C]PROPIONATE
glycerol into the dihydroxyacetone phosphate pool.
Both of these effects dilute the 13C enrichment of
dihydroxyacetone phosphate relative to glyceraldehyde3-phosphate; hence, carbons 4, 5 and 6 of glucose-6phosphate will be more enriched than carbons 1, 2 and
3. Within both glucuronate and gluconate, carbons 4, 5
and 6 had a slight but systematic elevation of 13C
enrichment compared with the corresponding carbons
1, 2 and 3, consistent with a small dilution from this
source. Significant excesses in labeling of the bottom
Table 1. Multiplet areas from the 13C NMR spectra of gluconate C-2, glucuronate C-5b and
phenylacetylglutamine C-2 for six normal individuals following a 24-hour fast
Subject
Gluconate C-2
Glucuronate C-5b
Phenylacetylglutamine C-2
ML
S
D12
D23
Q
0.289 6 0.024
0.419 6 0.002
0.056 6 0.001
0.237 6 0.023
S
D56
D45
Q
0.262 6 0.026
0.445 6 0.004
0.060 6 0.008
0.234 6 0.023
S
D23
D12
Q
0.396 6 0.032
0.374 6 0.011
0.060 6 0.008
0.161 6 0.016
GA*
S
D12
D23
Q
0.260 6 0.032
0.420 6 0.039
0.059 6 0.008
0.244 6 0.017
S
D56
D45
Q
0.353 6 0.004
0.409 6 0.016
0.053 6 0.011
0.163 6 0.032
S
D23
D12
Q
0.469 6 0.062
0.358 6 0.063
0.050 6 0.001
0.124 6 0.001
DE
S
D12
D23
Q
0.336 6 0.030
0.390 6 0.018
0.072 6 0.016
0.202 6 0.008
S
D56
D45
Q
0.343 6 0.035
0.428 6 0.001
0.051 6 0.011
0.178 6 0.008
S
D23
D12
Q
0.513 6 0.039
0.345 6 0.031
0.047 6 0.004
0.116 6 0.020
MW
S
D12
D23
Q
0.322 6 0.063
0.403 6 0.027
0.068 6 0.009
0.205 6 0.040
S
D56
D45
Q
0.217 6 0.031
0.432 6 0.024
0.067 6 0.010
0.284 6 0.003
S
D23
D12
Q
0.527 6 0.069
0.317 6 0.034
0.043 6 0.008
0.113 6 0.028
SH †
S
D12
D23
Q
0.338 6 0.041
0.429 6 0.034
0.058 6 0.005
0.175 6 0.015
S
D56
D45
Q
0.251
0.465
0.086
0.198
S
D23
D12
Q
0.389
0.476
0.041
0.094
JJ
S
D12
D23
Q
0.478 6 0.047
0.350 6 0.034
0.043 6 0.001
0.129 6 0.011
S
D23
D12
Q
0.567 6 0.062
0.330 6 0.030
0.036 6 0.015
0.069 6 0.016
ND
ND
ND
ND
Values are means6SD of 120-, 150-, and 180-min blood samples for each individual (gluconate) or of 2- to 3-h and 3- to 4-h urine samples for each
individual (glucuronate and phenylacetylglutamine). Multiplet nomenclature is as in Fig. 6. ND, not determined. *Glucuronate multiplets
(means6SD) were obtained from 1- to 2-h and 2- to 3-h urine spectra. †Glucuronate and phenylacetylglutamine multiples obtained from a single urine
collection (2–4 h). Gluconate multiplet means were obtained from 120- and 180-min blood collections, since no blood was collected at 150 min from this
subject.
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Fig. 6. Region (20 to 100 ppm) from a 150.9MHz proton-decoupled 13C NMR spectrum of
a purified urine extract. Extract was obtained
from urine collected 2–3 h after ingestion of
[U-13C]propionate. PAG-2, PAG-3, and PAG-4
are carbons 2, 3 and 4 of the glutamine moiety
of phenylacetylglutamine; PAM is the methylene carbon of phenylacetyl moiety of phenylacetylglutamine; H-1b, H-1a, and H-5b are
carbons 1b, 1a, and 5b of glucuronate;
3-OHP-2 is carbon 2 of 3-hydroxypropionate;
3-OHP-3 is carbon 3 of 3-hydroxypropionate;
A-2 is carbon 2 of acetate. For the PAG-2 and
PAG-3 insets: S, singlet resonance; D12, doublet from coupling of carbon 2 with neighboring carbon 1; D23, doublet arising from coupling of carbon 2 with neighboring carbon 3;
Q, doublet of doublets, or quartet, arising from
coupling of carbon 2 with both its neighbors.
For the H-5b inset: S, singlet resonance; D56,
doublet arising from coupling of carbon 5 with
neighboring carbon 6; D45, doublet arising
from the coupling of carbon 5 with neighboring carbon 4; Q, doublet of doublets, or quartet, arising from coupling of carbon 5 with
both its neighbors.
E848
MEASUREMENTS OF GLUCONEOGENESIS WITH [U-13C]PROPIONATE
components of the parent hepatic glucose-6-phosphate
molecule had similar 13C isotopomer distributions.
DISCUSSION
Analytic considerations. We have shown that complex 13C isotopomer distributions of phenylacetylglutamine, glucuronate, and glucose produced by hepatic
metabolism of [U-13C]propionate can be analyzed in a
straightforward manner by use of 13C NMR spectroscopy. The analyte pools, particularly the urinary products, provide sufficient material to quantitate isotopomer distributions by 13C NMR at 13C enrichment
levels only 1–2% above natural abundance. 13C NMR
proved to be a particularly valuable tool for direct
isotopomer analysis of the glucuronide hexose skeleton
after its enzymatic hydrolysis from the parent acetaminophen b-glucuronide. There are three advantages to
the methods reported here compared with previously
published methods. First, the determination of positional 13C glucose isotopomers appears to be much
simpler by 13C NMR than by gas chromatography-mass
spectrometry. Second, others have isolated acetaminophen-b-glucuronide from urine and converted the glycone moiety to glucose by chemical reduction of the
carboxy glycone C-6 followed by enzymatic cleavage of
the b-glucoside product (27, 28). Again, the chemical
approach reported here appears simpler and provides
complete recovery of labeled urinary glycones, an important consideration given the relatively low sensitivity
of 13C NMR compared with MS methods. In situ
hydrolysis of the b-glucuronide may also have advantages in the clinical setting. A wide range of medications and drugs are cleared by glucuronidation; hence,
a patient has the potential to produce a complex
cocktail of urinary b-glucuronides even in the absence
of acetaminophen. For such an individual, collective
hydrolysis in situ should be far easier than quantitative
isolation, reduction, and hydrolysis of the complex
glucuronide mixture. Finally, our simple urine purification procedure retains both phenylacetylglutamine and
glucuronide, thereby allowing a 13C isotopomer analysis of both metabolites from a single 13C NMR spectrum.
Table 2. Estimations of total anaplerosis, oxaloacetate-pyruvate recycling, and net anaplerotic outflow from 13C
multiplets of gluconate, glucuronate, and phenylacetylglutamine from 24-h-fasted human subjects
Gluconate C-2
Subject
ML
GA
DE
MW
SH
JJ
Means 6 SD
Glucuronate C-5b
Phenylacetylglutamine C-2
y
pk
g
y
pk
g
y
pk
g
6.48
6.12
4.42
4.93
6.40
7.14
3.25
2.98
2.61
2.91
4.38
5.14
3.23
3.14
1.81
2.02
2.02
2.00
6.42
6.72
7.39
5.45
4.41
ND
3.45
4.64
4.90
2.21
3.10
ND
2.97
2.08
2.49
3.24
1.31
ND
5.23
6.16
6.34
6.37
10.60
8.16
3.55
4.68
4.87
4.74
9.32
7.25
1.68
1.48
1.47
1.63
1.28
0.91
5.92 6 1.03
3.55 6 0.99a
2.37 6 0.64c
6.08 6 1.16
3.66 6 1.11a
2.42 6 0.76c
7.14 6 1.94
5.74 6 2.13b
1.41 6 0.28d
y, Estimations of total anaplerosis; pk, oxaloacetate-pyruvate recycling; g, net anaplerotic outflow.
each other (P , 0.05); c and d are significantly different from each other (P , 0.01).
a
and
b
are significantly different from
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one-half vs. the top one-half of plasma glucose after
infusion of [14C]lactate have been reported in fed humans, but both halves were equally labeled after 60 h of
fasting (30). Our observations fall between these two
extremes, consistent with the intermediate 24-h fasting period used in our study.
The 13C NMR spectra of urine samples from three
subjects featured weak multiplet signals at 58 and 39
ppm, which on the basis of chemical shifts and coupling
constants have been tentatively assigned to carbons 2
and 3 of 3-[U-13C]hydroxypropionate. The C-1 carboxy
resonance of this metabolite was obscured by numerous
multiply labeled and natural abundance carboxy resonances that occupy a crowded region of the 13C NMR
spectrum. 3-[U-13C]hydroxypropionate is the terminal
b-oxidation product of [U-13C]propionyl-CoA, and its
formation has been associated with saturation of the
propionyl-CoA carboxylase pathway (1). The fact that
only the uniformly labeled isotopomer was observed
indicates that the carbon skeleton of [U-13C]propionate
was intact when it entered the hepatic [U-13C]propionylCoA pool.
Metabolic analysis. For calculations of y, pk, and g,
we selected blood spectra from 120 to 180 min, when
the 13C enrichment was relatively constant. These were
compared with the 2- to 3-h and 3- to 4-h urine samples,
which sampled the hepatic metabolite pools at ,1.25–
2.25 h and 2.25–3.25 h, respectively (7). The gluconate
C-2, glucuronate C-5b, and phenylacetylglutamine C-2
multiplet areas (Table 1) were substituted into equations previously presented (18) to give estimates of y,
pk, and g (Table 2). For all three metabolites, total
anaplerotic flux relative to citrate synthase was estimated at 6.0–7.0. However, the phenylacetylglutamine
analysis reported a significantly higher OAA-PYR recycling flux than either glucose and glucuronate, and this
resulted in a significantly lower estimate of net gluconeogenesis compared with the hexose analyses (1.4 vs.
2.4). These data parallel the results of Landau et al.
(24), in which net gluconeogenic fluxes reported by
glucose and phenylacetylglutamine after infusion of
[14C]propionate were ,3.0 and ,1.8, respectively (24).
With the exception of one individual, the multiplet
ratios of glucuronate C-5b and gluconate C-2 in all
subjects were similar, providing consistent values for y,
pk, and g (see Table 2). This indicates that both triose
MEASUREMENTS OF GLUCONEOGENESIS WITH [U-13C]PROPIONATE
µmol · kg21 · min21 (3.6/2.4 3 11.2). In relation to these
fluxes, metabolism of 1.5 g of sodium [U-13C]propionate
over 1–2 h represents a consumption rate of 1.6–3.2
µmol · min21 · kg21, which is 6–12% of the total anaplerotic inflow.
Metabolic flux estimates. Anaplerotic flux (y), determined here by 13C NMR analysis of glucose/glucuronate, is slightly lower (6.0 vs. 7.0) than that reported by
Magnusson et al. (30), whereas pyruvate-oxaloacetate
recycling (pk) flux was similar (,3.6 vs. 3.7). Although
estimates of pk may be inflated by flux from the Cori
cycle (23, 30), we believe that our study protocol
minimized any contribution from extrahepatic pathways. The Cori cycle has been estimated to contribute
maximally ,15% to the total hepatic glucose output in
starved rats (23). Any lactate generated in extrahepatic
tissues from 13C-enriched glucose and returned to the
liver via the Cori cycle would supplement estimates of
intrahepatic pyruvate-oxaloacetate recycling. With the
assumption of steady-state conditions and no glycogenolysis, a 15% Cori cycle contribution would result in a
15% underestimation of g, and pk would be correspondingly increased (y being unchanged). The effects of this
level of Cori cycling on pk are rather modest; 15% of g
amounts to 0.36 units or ,10% of our pk estimate.
However, it has been estimated that about one-third of
the total hepatic glucose output is derived from glycogenolysis (6a) after a 24-h fast, so dilution of blood
glucose with unlabeled glucose via glycogenolysis would
further attenuate the 10% overestimate of pk to ,3%.
Thus we conclude that the bulk of the observed pk flux
activity detected in this study originates from hepatic
metabolism.
Our estimate of net gluconeogenic flux was somewhat lower than the value (,2.4 vs. 3.3) reported by
Magnusson et al. (30). One explanation could be the
difference between the fasting periods in the two studies (24 h here vs. 60 h in Magnusson et al.). It has been
shown that 3-carbon gluconeogenic sources contribute
67% of the total hepatic glucose output after a 22-h
fasting period but .90% after a 42-h fasting period
(6a). We would expect that this increased demand for
3-carbon precursors would be fulfilled in part by an
increased ratio of gluconeogenic to citrate synthase
flux. However, the additional gluconeogenic precursors
could also be provided by a systematic increase in flux
through all the pathways without any change in flux
ratios. Hence, the modest differences between our flux
estimates and those of Magnusson et al. may simply be
methodological in origin. Conversion of relative to
absolute fluxes by indexing g to the gluconeogenic
portion of hepatic glucose output by use of additional
tracers (15, 25, 30) will better resolve this uncertainty
and is the focus of our ongoing studies. Finally, because
net anaplerotic efflux can also be directed into nongluconeogenic pathways, such as glutathione biosynthesis, g
represents the upper limit of glucose output from the
tricarboxylic acid cycle.
Effects of 13C reincorporation via bicarbonate on the
measurements. Measurements of gluconeogenesis by
use of singly labeled substrates such as [3-14C]lactate
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Ingestion of gram quantities of [U-13C]propionate.
Because 13C is a nonradioactive isotope and bulk substitution of 12C by 13C is not known to have a significant
kinetic isotopic effect, substantial quantities of 13Cenriched substrates can be safely given to humans (7,
13, 14, 35). However, such experiments can generate
supraphysiological substrate levels that could alter
basal metabolic activities. In our study, fasted individuals ingested an average of 1.5 g of propionate over 1 h.
With the assumptions of quantitative capture of propionate by portal circulation and a portal vein blood flow of
,700 ml/min (10), one can estimate that the average
portal propionate concentration is no higher than 0.36
mM over the 1-h period. Portal vein propionate concentrations averaging 0.03 mM have been reported in
patients undergoing gall-bladder surgery (9), but these
probably represent fasting levels, which are likely to be
much less than postprandial concentrations. The only
other measurements of human portal vein propionate
concentrations have been obtained by autopsy of sudden death victims, for whom levels ranging from 0.02 to
0.19 mM were reported (8). Measurements of portal
vein propionate levels in the pig, a good model for
human digestion, indicated levels of 0.08 mM under
fasting conditions and 0.25–0.40 mM after feeding (38).
These postprandial levels span the estimated portal
vein propionate levels generated during the experimental protocol. Nonetheless, the fact that urinary 3-[U13C]hydroxypropionate was detected in several subjects
suggests that the hepatic propionyl-CoA carboxylase
can become saturated (1), and physiological consequences of saturating propionyl-CoA carboxylase in
normal fasted humans are not known. In rat hepatocytes, high levels of propionate can inhibit the activities
of several enzymes by lowering the free CoA levels (4, 5,
33), including pyruvate carboxylase. Because pyruvate
carboxylase is the predominant anaplerotic pathway in
the liver, inhibition of this enzyme can result in decreased gluconeogenic flux (4, 6). However, in fasted
humans, the provision of 1.2 g of propionate over 3 h did
not alter hepatic glucose production in fasted volunteers (26), suggesting that hepatic gluconeogenesis was
maintained under these conditions. Our study featured
comparable amounts of ingested propionate (,1.5 g),
but the shorter ingestion period of our protocol probably generated higher levels of propionate in portal
circulation. Nonetheless, our estimated portal vein propionate levels of 0.36 mM are still far below the 5 to 10 mM
concentrations reported to inhibit gluconeogenesis and
citric acid cycle fluxes (4, 6, 32).
The maximal contribution of propionate carbons to
the total anaplerotic flux can also be estimated. From
the study of Chandramouli et al. (6a), gluconeogenic
flux in a 24-h-fasted individual is ,5.6 µmol · kg21 ·
min21 out of a total hepatic glucose output of 8.6 µmol ·
kg21 · min21. Setting g equal to 11.2 µmol · kg21 · min21 of
triose equivalents and applying the relative flux measurements from our study give a citrate synthase flux
value of 4.7 µmol · kg21 · min21 (1/2.4 3 11.2), a total
anaplerotic inflow value of 27.8 µmol · kg21 · min21 (5.9/
2.4 3 11.2), and a pyruvate recycling flux value of 16.6
E849
E850
MEASUREMENTS OF GLUCONEOGENESIS WITH [U-13C]PROPIONATE
3
Gluconate C-6 analysis was chosen over the C-1 carboxy terminus
because of its higher signal-to-noise ratio and the possibility of faster
relaxation of the C-1 doublet relative to the C-1 singlet.
the perivenous region, including periportal contributions (in the form of glutamate).
Why are these labeling differences revealed with
propionate but not with lactate? A key difference in the
hepatic metabolism of propionate vs. lactate is that the
former is quantitatively extracted from the circulation,
whereas the latter is not (34). Because mitochondrial
density is highest in the periportal region of the liver
(39), it is possible that a propionate tracer is preferentially consumed by the periportal cells compared with a
lactate tracer. Therefore, downstream regions could
receive little direct labeling from propionate while
receiving significant contributions from secondary labeled products of periportal propionate metabolism,
including glucose. Reutilization of hepatic glucose by
the perivenous region is consistent with the zonal
model of regulated hepatic glucose output proposed by
Jungermann and colleagues (19–22), in which periportal glucose synthesis is appropriately attenuated by
perivenal glucose reutilization. Utilization of secondary
labeled products by the perivenous region could generate quite a different oxaloacetate isotopomer distribution than that produced from periportal metabolism of
[U-13C]propionate if the two regions have different
metabolic flux characteristics. In contrast, a lactate
tracer is metabolized more uniformly across the lobule
and therefore generates an averaged labeling distribution in glucose and glutamine. The fact that glucose and
glutamate were labeled equivalently in perfused rat
livers supplied with supraphysiological and saturating
levels of [U-13C]propionate, lactate, and pyruvate (18)
suggests that intralobular substrate gradients may be
an important determinant of labeling heterogeneity
among different metabolites in the liver.
In conclusion, we have shown that a detailed analysis
of the relative rates of anaplerosis, oxaloacetatepyruvate cycling, and gluconeogenesis can be obtained
in a rather simple procedure by 13C NMR analysis of
blood and urine after ingestion of [U-13C]propionate,
phenylacetate, and acetaminophen. Our results are in
good agreement with other recently published studies
that used 14C-labeled radioisotopes. Finally, the simple
processing protocols for both blood and urine are well
within the capability of a standard medical analytic
laboratory and its personnel.
We acknowledge the excellent technical assistance and support
provided by the staff of the General Research Clinical Center at
University of Texas Southwestern.
This research was supported by National Institutes of Health
Grants RR-02584, HL-34557, and M01-RR-00633 and a Clinical
Investigator Award from the Department of Veterans Affairs.
Address for reprint requests: J. G. Jones, Mary Nell and Ralph B.
Rogers Magnetic Resonance Center, 5801 Forest Park Rd., Dallas, TX
75235–9085.
Received 29 April 1998; accepted in final form 23 July 1998.
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