MODELING IN PHYSIOLOGY
Simultaneous time-varying systemic appearance of oral and hepatic
glucose in adults monitored with stable isotopes
G.
Livesey1,
P. D. G.
Wilson2,
J. R.
Dainty2,
J. C.
Brown1,
R. M.
Faulks1,
M. A.
Roe1,
T. A.
Newman1,
J.
Eagles1,
F. A.
Mellon1, and
R. H.
Greenwood3
Departments of 1 Nutrition,
Diet, and Health and of 2 Food
Biophysics, Institute of Food Research, Norwich NR4 7UA; and
3 Department of Medicine, Norfolk
and Norwich Hospital, Norwich NR1 3SR, United Kingdom
 |
ABSTRACT |
The rates (and extent) of appearance of glucose
in arterialized plasma from an oral glucose load and from liver
(RaO,
RaH) can be estimated in humans
using radioisotopes, but estimates vary among laboratories. We
investigated the use of stable isotopes and undertook 22 primed
intravenous infusions of
D-[6,6-2H2]glucose
with an oral load including
D-[13C6]glucose
in healthy humans. The effective glucose pool volume (VS) had a lower limit of 230 ml/kg body weight (cf. 130 ml/kg commonly assumed). This
VS in Steele's one-compartment
model of glucose kinetics gave a systemic appearance from a 50-g oral
glucose load per 70 kg body weight of 96 ± 3% of that ingested,
which compared with a theoretical value of ~95%. Mari's
two-compartment model gave 100 ± 3%. The two models gave
practically identical RaO and
RaH at each point in time and a
plateau in the cumulative RaO when
absorption was complete. Less than 3% of
13C was recycled to
[13C3]glucose,
suggesting that recycling errors were practically negligible in this
study. Causes of variation among laboratories are identified. We
conclude that stable isotopes provide a reliable and safe alternative
to radioactive isotopes in these studies.
metabolism; absorption; hepatic production; modeling
 |
INTRODUCTION |
DUAL ISOTOPE MONITORING of time-varying rates of entry
into the systemic circulation of glucose from the gut and the liver in
humans generally involves a primed-continuous intravenous infusion with
labeled glucose, oral loading with differently labeled glucose molecules, and simultaneous intermittent sampling of blood plasma for
the analysis of isotopic species. In humans, this technique is growing
in popularity but has been dependent on radioisotope labels (7-9,
11, 12, 16, 17, 20, 21, 23, 26, 28, 29), although not exclusively (6).
However, stable isotopic labels offer an alternative, albeit expensive,
means of safely and ethically meeting the need for studies of glucose metabolism. Abnormalities in glucose metabolism are central to current
hypotheses on obesity and diabetes and risk factors linked with
coronary artery disease (31, 36). The abnormalities appear early in the
disease process (24), and the disease conditions are increasingly
prevalent (19, 34). We have, therefore, determined whether the use of
the mass isotopomers
D-[6,6-2H2]glucose
and
D-[13C6]glucose
in place of their radioactive 3H
and 14C counterparts in the dual
isotope method presents unexpected obstacles when used with oral
glucose loading. On the basis of our stable isotope data and
observations that we summarize from the literature (for references see
Table 1), we identify possible errors in published results. Such errors
were substantial, explaining large differences (up to 20% of expected
results) in reported estimates of oral glucose appearance in the
systemic circulation, and much larger differences (up to
300% of
expected results) in estimates for splanchnic retention of glucose
during orosystemic passage.
In assessing the usefulness of stable isotopes for this purpose, it is
prudent to examine the performance of models chosen to make rate
estimates. Previous dual radioisotope work with oral loading has been
conducted using one- and two-compartment models (see Fig. 1 and Refs.
5, 21, 30), but substantially different model- or parameter-dependent
rates of glucose entry from the liver and gut have been estimated. A
theoretical analysis indicates that different rates can arise from
either volume or structure errors or both (5). Because there has been
no experimental assessment of the models by use of dual stable
isotopes, nor any estimation of the effective glucose distribution
volume in this situation, we examine both in the present study.
One model we chose to assess was a simple one-compartment model
developed by Steele, although this has been criticized as not
representing the underlying physiology of glucose kinetics (5, 33).
Steele's model has often, and recently, been used with radioisotopes
in the dual tracer method (9, 10, 12, 13, 20, 22, 27), and it has been
employed recently in a dual tracer study involving stable isotopes and
oral loading with glucose (6). Furthermore, we have used Steele's
one-compartment model in dual isotope studies with
13C-labeled starch and
D-[6,6-2H2]glucose,
reported in early communications (14, 15). It is appropriate,
therefore, to establish whether or not Steele's model can yield
realistic quantitative results when either radioactive or stable
isotopes are used to monitor the perturbation of metabolism resulting
from an oral load.
A weakness in Steele's model to which he drew attention (33) has now
been demonstrated in situations involving rapid or step changes in
intravenous glucose loading, such as the euglycemic clamp and minimal
models applied to data from intravenous glucose tolerance tests (2, 13,
27). In these situations, glucose appearance in plasma is
underestimated by Steele's model when compared with the known
unlabeled glucose infusion rates. The weakness in the model results in
an underestimation of hepatic glucose production to an extent that
unacceptable negative rates are calculated (5, 13). This problem is
largely overcome by the use of two- or higher-order models (6, 8) and
by minimizing changes in the tracer-to-tracee ratio (2, 13, 27, 33).
Interestingly, by contrast, in dual radioisotope studies involving oral
loading, application of Steele's model can result in higher (not
lower) estimates of hepatic glucose production compared with estimates
from two- or higher-order models (5, 7), but the extent to which this
is, in practice, due to the model or to the distribution volume is
unclear. The present study determines whether a one- compared with a
two-compartment model necessarily yields substantially different
systemic appearance rates of glucose from liver and gut during
application of the dual isotope method when mass isotopes are used. In
the present study, we use oral glucose loads that yield near to
habitual postprandial plasma glucose concentrations.
Two-compartment models offer more complex and arguably more dynamically
realistic approaches than Steele's model when rates of glucose entry
into the circulation change rapidly. Of such models that have been
proposed, only those of Radziuk et al. (30) and Mari et al. (18) have
been extensively validated; these use radioisotopes under several
experimental conditions, including, importantly, the dual radioisotope
method. Both models give plausible results, so that a decision at
present to choose one in preference to the other appears to be
arbitrary. Mari et al. developed their model from the basis of work by
Radziuk et al. and, although structurally very similar, can be solved
in a simpler and more intuitive manner using a spreadsheet called SMART
(17). The only precondition for this approach is a steady-state period
before the nonsteady perturbation with oral glucose. The spreadsheet
makes Mari's model an accessible analysis tool that we chose to use
for comparison with Steele's one-compartment model. A feature of the
software is the simultaneous estimation of error bounds, which is
essential to the objective comparison of the two models.
Finally, no authors appear to have determined an effective glucose
distribution volume in humans for application specifically with
Steele's or equivalent one-compartment models when dual isotopes and
oral loading are applied. Not only is this distribution volume difficult to measure in a way that is suitable for application with
experimental data, but the effective volume depends on the time scale
over which it is measured. This is because of the longer time scale for
mixing at the "extremities" of the pool. We present a stable
theoretical approach to the derivation of the distribution volume,
together with parameter estimates derived from the primed-constant infusion of
D-[6,6-2H2]glucose.
As will be discussed, the determination and choice of correct
distribution volume are important for the estimation of the least
erroneous rates of glucose entry into the systemic circulation.
 |
METHODS |
Participants
Thirteen "healthy" volunteers with no family history of diabetes
mellitus were recruited. Eight were female and five were male, and they
were 19-59 (39 ± 14 SE) yr old, weighed 61-104 (79 ± 11) kg, and had body mass index of 22-37 (28 ± 4)
kg/m2. They ate their habitual
diets for
6 wk before investigation, were weight stable (± 2 kg),
and were nonmedicated. Investigations were repeated in nine of the
volunteers, which gave a total of 22 investigations. Informed written
consent was provided by all participants, and the study was approved by
both the Norfolk and Norwich Health District Ethics Committee and the
Institute of Food Research Human Research Ethics Committee.
Experimental Design
Volunteers were fasted for 12 h overnight before being seated for up to
10 h in a recliner chair (Parker Knoll, Chipping-Norton, UK), with ambulation limited to two toilet visits. An antecubital vein
and a subsequently heated dorsal hand vein were cannulated (18-gauge
Teflon) and kept patent with physiological saline. Pyrogen-free D-[6,6-2H2]glucose
(99 mol% enrichment, C/D/N Isotopes, K&K Greeff, Croydon, UK) was
administered as a primed (500-mg in a 10-ml aqueous
solution)-continuous (6 mg/min as a 68.5 g/l aqueous solution) infusion
into an antecubital vein, starting at ~0900, denoted
time 0 in
RESULTS. After 2 h, we administered
peroral D-glucose (50 g in 200 ml water, each per 70 kg body weight) that was labeled with
D-[13C6]glucose
(99 mol% enrichment, 10 mg/g total glucose, also C/D/N Isotopes).
Arterialized venous blood was drawn into fluoride-treated tubes from
the heated (41°C) hand at 30-min intervals for up to 7 h, and twice
as often for 1.5 h after the oral glucose. Plasma obtained by
centrifugation at 4°C was stored at
20°C.
Glucose and Isotopomer Analysis
Plasma glucose was determined on plasma without further treatment by
use of hexokinase (4) and a COBAS MIRA centrifugal analyzer (Welwyn
Garden City, UK). The coefficient of variation on replicate analyses
was 0.014 for a normal basal concentration of 5.3 mmol/l glucose in
plasma, and the percentage recovery of a doubling spike was 100.5 ± 0.8 SE. The coefficient of variation in the quality control for plasma
glucose (mean 4.5 mmol/l) analyzed on repeated occasions was 0.016. Glucose isotopomer ratios were determined on butyl boronic acid acetate
derivatives using gas chromatography electron-impact mass spectrometry
(25). Protein in plasma (200 µl = 1 vol) was precipitated with 2 volumes of 2-propanol, and after centrifugation the supernatant was
mixed with 5 volumes of n-hexane, in
glass. After centrifugation, the lower phase was washed with 5 volumes
of 2-propanol onto 1-g mixed-bed ion-exchange resin (Amberlite IRN-150L
mono-bed mixed resin, Merck) for 15 min. After filtration (Gelman
Acrodise LC13) and washing through with 5 volumes of 2-propanol, the
extract was dried at 50°C under nitrogen gas with a sample
concentrator (Techne, Cambridge, UK) and then freeze-dried after it was
first dissolved in 2 volumes of water. After derivatization (25), the
dry residue was taken into trimethylpentane containing 1% acetic
anhydride. A sample was injected into a Hewlett-Packard 5890 Series II
gas chromatograph containing a BD-5MS column (Fisons,
Loughborough, UK) under conditions previously described (25). Ions
generated by electron impact were selectively monitored in a Trio 1-S
mass spectrometer (Finnigan MASSLAB, Manchester, UK) at
mass-to-charge (m/z) 297, 299, 300, and 303 for molecular enrichments M,
M+2,
M+3, and
M+6, respectively, which correspond to
natural-,
[6,6-2H2]- + [13C2]-,
[13C3]-,
and
[13C6]glucose
molecules, respectively. Plasma isotope enrichments were determined
from linear standard curves of 5-40 mg of
D-[6,6-2H2]-
or
D-[13C6]glucose/g
glucose (Thornton and Ross, Huddersfield, UK), with residual standard
errors (SE) of 0.72 and 0.16 mg/g, respectively (or a coefficient of
variation of 0.018 at a steady-state enrichment of
[6,6-2H2]glucose
and 0.016 at a maximum plasma enrichment of
[13C6]glucose
in the present studies). Quality controls aliquoted from a single
plasma sample containing a mean of 1.925 mg of
D-[6,6-2H2]glucose
and 0.736 mg of
D-[13C6]glucose/g
total glucose had coefficients of variation of 0.048 and 0.039, respectively, when analyzed on repeated occasions. No such standards
were available for the
[13C3]glucose
produced by the recycling of 13C
back into glucose. The occurrence of
[13C3]glucose
in plasma was calculated from the relative abundance (areas) of
m/z ions at 303 (M+6), 300 (M+3), 299 (M+2), and 297 (M). To do this, the theoretical
mass spectra of each derivative were calculated from a consideration of
the derived elemental and isotopic composition of each glucose
isotopomer and the natural abundance of the elements added during
derivatization. The spectrum of the mixture of the derivatized glucose
isotopomers was a linear combination of the spectra of each of the
components. In this way, four equations (one for each of the
m/z ions) were set up with four
unknowns (the mole fraction of glucose derived from each of the
isotopomers). These equations were solved to give the mole fraction of
total glucose present as the (M+3)
isotopomer.
Rate of Appearance Calculations
The rates at which glucose appeared in arterialized plasma from the
exogenous (oral) and endogenous (hepatic) sources
(RaO and
RaH) were estimated by both
Mari's two-compartment and Steele's one-compartment models (Fig.
1) (16, 33). A term that estimates glucose
appearance rate
Ra(t) in the
steady state (Eq. 1) was present in
each model (Eqs. 2 and 3);
is the known rate of tracer
infusion. The second term in each model was essentially identical,
differing only in the chosen volume of the sampled compartment,
abbreviated to VS in Steele's
model and V1 in Mari's model. The
two-compartment model included a third term
R2(t)
(Eq. 3) to describe a rate of
(re)appearance of glucose in the sampled compartment
(V1) from the unobservable
compartment. Computation of
R2(t) used a
second pool volume, V2, and was
complex, and so for brevity we refer to Mari (16). Mean parameter
values V1 (146 ml/kg),
V2 (84 ml/kg),
k22 = k02+k12
(0.067 min
1), and sum of
volumes
(V1+V2 = 230 ml/kg) were as derived previously (18). Steele's volume
(VS) was estimated by using data
from the above protocol (see below).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
Compartmental descriptions of Steele's
(A) and Mari's
(B) equations.
C(t), tracee concentration;
C*(t), tracer
concentration;
Ra(t), tracee
rate of appearance; , tracer rate
of appearance; VS, Steele's
volume sampled via arterialized plasma;
V1 and
V2, volumes of the 2 compartments
in Mari's model;
kij, rate
parameters with compartment numbers in the to-from notation; and
kij(t),
time-varying parameters.
|
|
Measurable plasma glucose concentrations were
[6,6-2H2]+ [13C2]glucose,
[13C3]glucose,
[13C6]glucose,
and total glucose (labeled and unlabeled). To obtain [6,6-2H2]glucose,
the contribution to the M+2 peak from
[13C2]glucose
was assumed to be negligible (e.g., the present computations of
RaO and
RaH); otherwise, the
contribution was assumed to be small and equal in size to the
M+3 peak (i.e.,
M+2
M+3; Ref. 3). The latter assumption
permitted rate data to be recalculated and errors from assumed
negligible recycling to be estimated. Identical behavior of labeled and
unlabeled glucose molecules was assumed, so that the concentrations in
plasma of glucose derived from both the liver and gut could be
calculated. Endogenous or hepatic glucose (i.e., total glucose less
glucose traced with D-[13C6]glucose
and
D-[6,6-2H2]glucose)
and oral glucose (traced with
D-[13C6]glucose)
were the tracees in the computational models, and intravenous glucose
(D-[6,6-2H2]glucose)
was the tracer (Eqs. 2 and 3). Thus endogenous glucose included
recycled glucose. Recycling of 13C
label as
[13C3]glucose
was also estimated by using the latter as tracee in place of
D-[13C6]glucose.
By substituting the appropriate tracee concentration into
Eqs. 2 and 3,
RaO and
RaH were calculated using SMART
(17), a program developed for the analysis of the rate of appearance of substances in the nonsteady state. The program smoothed both tracer and
tracee concentrations and interpolated at time intervals of 5 min to
aid integration.
Steady state
|
(1)
|
Steele's equation
|
(2)
|
where
a(t) was the tracer-to-tracee
concentration ratio, i.e.,
C*(t)/C(t),
and
(t) was its derivative with
respect to time.
Mari's equation
|
(3)
|
The
function R2(t)
was complex. It gave an estimate of the contribution to
Ra of the second compartment and
was dependent on the values of
k02,
k12,
V2,
C(t) and
C*(t) (16).
Statistics, Error Analysis, and Model Superiority
There were 13 subjects, and of these, 9 subjects repeated the
investigation. Calculations using the one- and two-compartment models
were made for each of the 22 investigations, and the results were
summarized as mean values. Repeated-measures analysis of variance on
sampled data showed that between-subject variation was frequently
insignificant, particularly for rate data, and so each investigation
was treated as a separate observation, with the associated SE of the
mean being determined separately at each time point. Significance of
difference was tested using the least significant difference test.
RaO and
RaH had means that were
practically identical to their medians, and normal probability
plots indicated that distributions were approximately Gaussian.
The calculated rates of appearance were subject to error, because
models of necessity approximate the true system. A thorough analysis of
Steele's model (5) has identified volume and structure errors, which
constitute Steele's error, as
ES(t).
If the error in Mari's two-compartment model is
E(t), then Steele's error can be
written (16)
|
(4)
|
where
|
(5)
|
The
absolute value of E(t) was shown not
to be computable (16, 18); instead, an error bound was obtained at each
time point, |E(t)|, to take
into account the time-varying nature of the model parameters, as
described elsewhere (5, 16). Mari (17) derived the following
inequality, which must be satisfied to demonstrate superiority of the
two-compartment model over Steele's model
|
(6)
|
|E(t)|
and |R(t)| were calculated
at each time point using SMART.
To distinguish one error type from another in subsequent figures, SE of
means were illustrated with vertical bars, and model error bounds were
illustrated with curves.
Estimation of the Effective Glucose Distribution Volume
A new approach was used to estimate Steele's pool volume for glucose,
because existing approaches were unstable with the available data. It
has been traditional to derive Steele's pool volume as a product of
the total glucose distribution volume
(VT) and the fraction of this
pool that is apparently accessible when glucose concentration changes
(p), such that
VS = pVT.
The present approach estimates neither
VT nor
p, and so these remain unknown.
Consider a glucose pool of constant size
P, with an hepatic production rate
Ra, a constant infusion of tracer
at a rate
, an initial bolus of
size B, and a mole fraction
(x) of tracer within the pool. The
initial mole fraction of tracer
(x0) due to the
bolus is then given by
|
(7)
|
In
principle, the pool size could be determined from the bolus size and
the concentration of tracer immediately after injection of the bolus.
In practice, however, the mixing of the tracer is not rapid enough to
allow this estimate. We can, nevertheless, learn about the size of the
glucose pool by considering the time course of the tracer
concentration. Using the assumption that the pool size remains at a
steady state during priming, i.e., that the rate of glucose
disappearance equals the sum of the endogenous appearance plus the
tracer infusion rate, we may write a mass balance for the tracer
concentration, describing the approach to a steady-state enrichment as
follows
|
(8)
|
This
equation may be integrated to give the time course of tracer
|
(9)
|
where
|
(10)
|
Eq. 9 describes an exponential rise or fall to a
steady-state enrichment
(xss) given by
|
(11)
|
Whether
x rises or falls to this limit depends
on the size of the initial bolus with respect to the pool size and the
two rates of appearance. If the tracer concentration rises to the limit, i.e.,
(dx/dt)>0,
it can be shown (by use of Eqs. 8 and 10) that
|
(12)
|
If
the tracer concentration falls to the limit, then the inequality is
reversed. More generally, we may define a function, z(t)
|
(13)
|
At
t = 0, Eq.13 reduces to Eq. 7, giving
z0 equal to the
pool size as a function of the (unmeasurable in practice) initial tracer concentration. As t
, Eq. 13 reduces to
Eq. 12, giving a lower limit on the
pool size. Equation 9 already
demonstrates that x(t) changes
monotonically and with a constant direction of curvature with time, and
so from Eq. 13 we know that
z(t) also does. Hence, we can
extrapolate a plot of z(t) back to
t = 0 to obtain an estimate of the
pool size, P. A linear extrapolation will always give a lower bound for P
when the tracer rises to a limit, and an upper bound when the tracer
falls. Taking values of z(t) and
linear extrapolation to
zo was a more
stable procedure than taking the values of
x and curve fitting to
xo; this was because the linear approach, which produces a bound on the pool size,
has one less parameter to estimate.
Substituting the time course for x(t)
from Eq. 9 into Eq. 13 allows us to make an estimate of how close to the
real value our estimate will be, given a knowledge of true pool size
and hepatic output. For the experiment carried out in this study, the
worst case (extrapolating a tangent to the curve at
t = 120 min back to
t = 0) would give an underestimate of
24%. A better estimate of the error is a line passing through
z(t) at
t = 30 and
t = 120 min, which indicates an
underestimate of 3.8%. VS was
estimated at each investigation, but a pooled estimate was used every
time Steele's model was applied, because individual estimates were based on only a few sampling intervals.
 |
RESULTS |
Mean Arterialized Plasma Glucose Concentrations
The concentration of the M+2 glucose,
which is the sum of
[6,6-2H2]-
and
[13C2]glucose,
increased (Fig. 2) to approach a steady
state close to 0.18 ± 0.01 mmol/l during the 2 h immediately after
the start of the primed-continuous infusion and before the start of the nonsteady perturbation with oral glucose (50 g glucose/70 kg body weight). The perturbation decreased the tracer concentration to a nadir
at 3 h after ingestion, ~30% below that in the near steady state. By
5 h after ingestion, the near-steady-state concentration had been
reached again. A similar pattern of observations occurred in all
volunteers, which gave rise to the narrow error values. The
concentration of M+3 glucose (label
recycled into
[13C3]glucose)
began to rise immediately after the oral load of
D-[13C6]glucose
at 120 min (Fig. 2). By the end of the infusion, the M+3 peak had reached 2.8 ± 0.3%
of the M+2 peak, which was a slight fall from a maximum mean percentage of 2.9 ± 0.9 at 330 min. A reasonable assumption that recycling produced
[13C2]glucose
in quantities similar to
[13C3]glucose
(3) allows that, on average, >97% of the
M+2 glucose was the tracer
D-[6,6-2H2]glucose.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
Concentration of M+2 and
M+3 glucose in arterialized plasma of
normal adult volunteers after a bolus injection
(t = 0) and a constant infusion
(t = 0-420 min) of
D-[6,6-2H2]glucose
and an oral glucose load (50 g glucose per 70 kg body wt) including
D-[13C6]glucose
at t = 120 min.
Top: dashed line, mean approach to
steady state of M+2; solid line, mean
concentration of M+2 after oral
glucose. Bottom: solid line, mean
concentration of M+3 glucose; vertical
bars, corresponding SE (n = 22) at
each 5-min step.
|
|
After its ingestion,
D-[13C6]glucose
soon appeared in the arterialized plasma (Fig.
3) and peaked within ~1.5 h, at a
concentration of 7.0 ± 0.3 mmol/l, before returning 80% of the way
toward zero again at the close of the investigation.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
Concentrations of gut-derived (long-dashed lines), hepatic-derived
(short-dashed lines), and total (solid lines)
D-glucose after an oral load (50 g glucose per 70 kg body wt) at t = 120 min in normal adult volunteers. Vertical bars, corresponding SE
(n = 22).
|
|
The concentration of glucose that originated from the liver decreased
soon after ingestion of the glucose drink and reached a nadir of 1.1 ± 0.1 mmol/l at 3 h after ingestion. Between 3.5 and 5 h after
ingestion, the decreasing concentration of glucose derived from the
oral dose was matched by an increasing concentration of glucose from
the liver, a process that was ~50% completed 5 h after ingestion of
the glucose drink.
The total glucose concentration in the arterialized plasma was (by
definition) the sum of concentration of glucose from the liver, oral
load, and infusion. From a basal concentration of 4.8 ± 0.1 mmol/l,
it peaked at 9.8 ± 0.4 mmol/l at ~1 h after ingestion
and then returned to a new "plateau" of 3.8 ± 0.2 mmol/1 between 3.5 and 5 h after ingestion.
Estimation of Steele's Pool Volume
At each investigation, pool volume estimates were computed
(Eq. 13,
z0 when
t = 0) from the primed-continuous
infusion rate and the rise in
D-[6,6-2H2]glucose
concentration before 2 h (Fig. 2), when contribution to the
M+2 peak from
[13C2]glucose
was absent. The estimate increased with increasing body weight (Fig.
4), and linear regression indicated a mean
value of 0.22 ± 0.01 l/kg when zero pool volume was forced at zero
body weight. The results from the five male volunteers were not
distinguishable from those for the eight female volunteers
(P > 0.1).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 4.
Variation of effective glucose distribution volume with body weight in
normal adult volunteers. , Females; , males; solid line, linear
regression coefficient (0.22 ± 0.01 l/kg body wt,
n = 22) after zero distribution volume
was forced at zero body wt.
|
|
Mean Rate of Systemic Appearance of Hepatic Glucose
The mean basal steady-state RaH
was 2.2 ± 0.1 mg · min
1 · kg
body weight
1, whether it
was obtained with equations of the one- or the two-compartment model.
Oral glucose acutely suppressed
RaH by ~90% to a nadir at 1.1 h
after glucose ingestion at 0.2 ± 0.2 mg · min
1 · kg
1
(Fig. 5). Thereafter,
RaH increased, returning to 75%
of the basal value by 5 h after the oral glucose.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
Mean rates of hepatic glucose production in normal adult volunteers
after ingestion of an oral glucose load (50 g glucose per 70 kg body
wt) at t = 120 min estimated by 1- and
2-compartment models. , Steele's 1-compartment model with
VS = 230 ml/kg body wt; bold
curve, 2-compartment model; light curves, mean error bounds on
2-compartment model.
|
|
Choosing an upwardly rounded value for the lower bound of the glucose
pool volume (230 ml/kg; cf. Fig. 4) resulted in
RaH estimates by the one- and the
two-compartment models that were nearly identical throughout the
non-steady-state period (Fig. 5). Estimates using the one-compartment
model were well within the error bounds for the two-compartment model
(Fig. 5), and at no time did the inequality term
|E(t)|/|R(t)|
(Eq. 6) fall to a
significant value (<0.5), but frequently reached nearly 100. Thus the
two-compartment model was far from superior for the present application.
Mean Rate of Systemic Appearance of Oral Glucose
As with RaH, values of
RaO were similar in the
non-steady-state period when estimated by the one- and the
two-compartment models (Fig. 6). In both
models, RaO accelerated to a peak
of 6.2 ± 0.2 mg · min
1 · kg
body weight
1 at ~50 min
after glucose ingestion, peaking only slightly ahead in the
higher-order model. The rate later decelerated, over a longer time
period, to near zero by 3 h after glucose ingestion. Rates estimated
using the one-compartment model were, again, well within the error
bounds for the two-compartment model (Fig. 6), and at no time did the
inequality term
|E(t)|/|R(t)|
(Eq. 6) fall to a significant value
(<0.5). Thus the two-compartment model was again not significantly
superior for this application.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
Mean rates of glucose appearance in systemic circulation from the gut
in normal adult volunteers after ingestion of an oral glucose load (50 g glucose per 70 kg body wt) at t = 120 min estimated by 1- and 2-compartment models. , Steele's
1-compartment model with VS = 230 ml/kg body wt; bold curve, Mari's 2-compartment model; light curves,
mean error bounds on 2-compartment model.
|
|
Cumulative Appearance of Oral Glucose
The rates of oral glucose appearance in the peripheral circulation
(Fig. 6) gave a poor indication of the extent to which the glucose load
was absorbed. Cumulative absorption and peripheral appearance were calculated by integration of the
RaO data and expressed as a
fraction of the glucose ingested. Such estimates of fractional systemic
appearance indicated 1.00 ± 0.03 of the oral dose reaching the
periphery when the two-compartment model was used, and 0.96 ± 0.03 when the one-compartment model was used (Fig.
7). In both models, the systemic glucose
appearance reached a plateau ~3 h after oral glucose. SE values were
reasonably small.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 7.
Mean cumulative systemic appearance of
M+6 glucose from the gut and
M+3 glucose from glucose recycling in
normal adult volunteers after ingestion of an oral glucose load (50 g
glucose per 70 kg body wt ) including
D-[13C6]glucose
at t = 120 min. , Appearance from
the gut according to Steele's 1-compartment model with
VS = 230 ml/kg body wt; bold
curve, appearance from the gut according to Mari's 2-compartment
model; lowest curve, appearance of M+3
glucose (i.e.,
[13C3]glucose);
vertical bars, corresponding SE.
|
|
The reappearance of label from oral
D-[13C6]glucose
as M+3 glucose (i.e.,
[13C3]glucose)
was calculated using the one-compartment model (Fig. 7). After oral
glucose, the systemic appearance of
M+3 glucose rose steadily and
continued to rise even after the systemic appearance of
[13C6]glucose
reached plateau. When this plateau was reached, by 300 min, just 1.6 ± 0.3% of the label from oral
[13C6]glucose
had reappeared in M+3 glucose, and by
the end of the experimental period, at 420 min, it had reached just 2.9 ± 0.5%.
Assessment of Errors
We consider first the errors that arise from an inappropriate choice of
Steele's volume VS, and then
those that arise from assuming that measurements of
M+2 glucose are solely due to the tracer
D-[6,6-2H2]glucose.
Values of VS applied in studies
with oral glucose range from 95 to 230 ml/kg body weight (Table
1). Departure from the
VS applied at present (230 ml/kg)
by just 10 ml/kg resulted in values for
RaH,
RaO, and cumulative systemic
appearance of oral glucose that varied time dependently, and maximally
by between 1.5 and 3%. Expressing these percentage values as fractions
(0.015 and 0.030) gave the time-dependent fractional sensitivities of
these rates to VS (fraction per 10 ml/kg or per liter/100 kg), as shown in Fig.
8. RaH
decreased, whereas RaO and
cumulative systemic appearance of oral glucose increased with increase
in VS. Across the range of
VS values noted in Table 1, we
calculate maximum differences for
RaH of 30%, for
RaO of 40%, and for cumulative systemic appearance of oral glucose of 21%. Although
VS for the one-compartment model
is well known to be critically important and potentially lower than the
true glucose distribution space, values chosen (Table 1) are often well
below the value we indicate on the basis of the present results, and
this is a major source of potential error.
View this table:
[in this window]
[in a new window]
|
Table 1.
Models, isotopes, assumptions, and parameter estimates for systemic
glucose appearance before and after an oral glucose load in adult
volunteers
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 8.
Sensitivity of mean estimates of hepatic glucose production rates
( ), gut-derived glucose production rates ( ), and cumulative
systemic appearance of gut-derived glucose ( ) in normal adult
volunteers after ingestion of an oral glucose load (50 g glucose per 70 kg body wt) at t = 120 min, estimated
by Steele's 1-compartment model.
|
|
The contribution to the M+2 peak from
13C label recycled into
[13C2]glucose
was not measurable in the present circumstances because of the infusion
of
D-[6,6-2H2]glucose.
Such a contribution would have caused systemic appearance of glucose to
be underestimated because of overestimation of tracer D-[6,6-2H2]glucose
present in plasma. However, it can be assumed that the concentration of
[13C2]glucose
in plasma is approximately similar to the concentration of
[13C3]glucose;
this is because the M+2 and
M+3 glucoses are generated from
[13C6]glucose
in approximately similar quantities in humans (and other species) (3).
Making this assumption, we calculated the extent to which systemic
appearance of oral glucose would be underestimated (Fig.
9). By the time the systemic glucose
appearance had approached plateau in Fig. 7, at 300 min, the appearance
was underestimated by just 1.7 ± 0.3%, and by the end of the
experiment, at 420 min, by just 2.2 ± 0.2%. This suggests that
recycling of glucose leads to practically negligible errors, even
though the error estimates are statistically significant.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 9.
Potential error in cumulative systemic appearance of glucose from the
gut when it is assumed that all M+2
glucose in plasma is
D-[6,6-2H2]glucose
uncontaminated with
[13C2]glucose
derived from glucose recycling in the present study. Curve, course of
mean values (vertical bars, SE) at each 5-min step.
|
|
 |
DISCUSSION |
Stable isotopes have been used increasingly for monitoring both
steady-state and time-varying rates of glucose turnover in humans
during intravenous glucose loading, for example in euglycemic clamps
and labeled intravenous glucose tolerance tests (for examples and
references see Refs. 2, 27). However, there is a relative absence of
data when the glucose is administered orally, a condition which is
particularly relevant to meal-induced perturbation of metabolism. The
last situation requires dual isotope methodology, and various
combinations of isotopes have been used (Table 1), but they have been
almost exclusively radioactive isotopes. In the present study, we
investigate
D-[6,6-2H2]glucose
and
D-[13C6]glucose
as stable isotope labels to estimate the systemic appearance rates of
both oral and hepatic glucose (RaO
and RaH). While doing so, we
quantify some potential errors and identify causes of variation in
results from different laboratories, whether radioactive or stable
isotopes are used.
After perturbation of metabolism with oral glucose, considerable
variation exists among laboratories in both the cumulative and the peak
rates of systemic glucose appearance (Table 1). Such differences arise
even though variation in the results within laboratories is relatively
small, suggesting the occurrence of at least one systematic error.
Using arterio-hepatic-venous difference techniques, Ferrannini et al.
(7), Pilo et al. (26), and Mari et al. (18) have shown that only ~5%
of glucose is captured during its first pass through the splanchnic
bed. Further capture of glucose for hepatic glycogen deposition must
result from the 30-50 additional passes during the absorptive
period. Consequently, the group mean end point for the appearance of
glucose in the systemic circulation, given room for a ±5% error
due to possible bias and sampling of the population, is expected to be
between 90 and 100% by the time glucose absorption is complete. Values observed at present are 96 and 100% (Fig. 7) when the one-compartment model of Steele (33) and the two-compartment model of Mari et al. (16),
respectively, are used. These agree with the observations by Radziuk
and co-workers (28, 29), who found 93 and 92% with oral loads of 50 and 93 g glucose, respectively, when using radioactive tracers
D-[3-3H]glucose
intravenously and
D-[1-14C]glucose
orally. Other authors have also found cumulative systemic appearances
in the range of 90-100%, notably on several different groups of
volunteers in studies by Pehling et al. (23), who used mixed
radioactive and stable isotopes, and Firth et al. (9), who used
entirely radioactive isotopes (see Table 1).
Such expected systemic appearance of gut-derived glucose is not
observed universally. Pilo et al. (26), Ferrannini et al. (7), Kelley
et al. (11), Mitrakou and co-workers (20, 21), Kruszynska et al. (12),
Mari et al. (18), and Delarue et al. (6) (see Table 1) all find
cumulative systemic appearances of ~70%. Some authors have assumed
that the discrepancy from the expected 100% absorption of glucose is a
measure of first-pass splanchnic sequestration (20, 21), whereas others
have accepted incomplete glucose absorption as the explanation of their
results (7). Ferrannini et al. (7), Pilo et al. (26), and Mari et al.
(18) demonstrate that their low recovery of oral
D-[1-14C]glucose
in systemic plasma, traced with intravenous
D-[3-3H]glucose,
cannot be due to first-pass splanchnic sequestration. Direct evidence
of possibly incomplete absorption of glucose before the end of their
experiments was not available. However, this seems an unlikely
explanation for all of those studies with radioactive isotopes
extending up to 5 or 6 h, in which cumulative
RaO glucose is still estimated to
be as low as 70% of the oral dose (Table 1). Such unlikelihood is
supported by the present stable isotope study, which indicates nearly
complete absorption before 3.5 h (Fig. 7).
On the basis of observations we summarize from the literature (Table
1), no single explanation is apparent that will explain why some
authors find theoretical systemic appearances of gut-derived glucose,
whereas others find ~20% less. The glucose dose, the duration of the
postprandial phase of the study, the balance of male and female
volunteers, the existence of glucose intolerance and
non-insulin-dependent diabetes, and the degree of obesity each offer no
explanation. The analytic model, parameter assumptions, plasma sampling
frequency at critical times, and the isotope combinations chosen are
factors that might affect the precise estimate; however, individually,
none is sufficiently influential to explain those published shortfalls
in the cumulative systemic glucose appearance.
A partial explanation of the discrepancy (Table 1) is readily at hand
for those studies in which the Steele or a similar model has been used
with an assumption that the effective glucose distribution volume is in
the range of 95-150 ml/kg body weight. These volumes were a priori
assumptions taken from the early literature, where a different
experimental situation existed, one that involved higher fluxes of
intravenous glucose and insulin (to be discussed). The effective
glucose distribution volume found at present is 230 ml/kg (Fig. 4).
Should this value be reasonably accurate for other studies that use the
current experimental situation (Table 1), we would expect
underestimation of the cumulative glucose appearance by between 7 and
13%, which is approximately one-half of the 20% discrepancy noted.
Before discussing possible sources of the remaining discrepancy, we
consider in the present and subsequent paragraphs the effective glucose
distribution volumes adopted in the various studies
(VS in Table 1). In prior
publications, volumes as low as 95-150 ml/kg body weight were
adopted (rather than determined) for use with Steele's (and
equivalent) one-compartment model(s) applied to the oral loading dual
tracer experiment. Such values originate from a total distribution
space (VT), which varies from 200 ml/kg determined with mannitol and thiosulfate in dogs (10) to 260 ml/kg based on glucose in humans (33), and empirical observations that
only a part (p) of this space seems
to be accessible to glucose when metabolism is perturbed by either a
rapid or a step change in intravenous glucose load or insulin injection
(22). And so VS in Table 1 equals
pVT.
The lower values of
pVT
in Table 1 (95-150 ml/kg) aimed to take account of the slow mixing
of the infused glucose with the "extremities" of the total
glucose distribution volume, as seemed to be apparent from intravenous infusion studies. However, a higher value would apply to the slower rates of entry of glucose from the gut than generally obtained in
intravenous infusion experiments. With the change in the experimental situation, a different effective glucose pool would be expected, because the effective pool size is mass and time dependent (5, 10, 33).
In the present work, we estimate neither
VT nor
p; however, when we assume that
VT is independent of the
experimental setup, the higher value for
VS (230 ml/kg) suggests that
p must be large for the present type
of study. High values of VS due to
high values of p are advantageous,
because as p approaches unity,
VS loses time dependency.
It should be noted that the 230 ml/kg value is a study population mean
value that we chose to use for all volunteers because variation in
estimates among individuals was small (Fig. 4). Greater accuracy could
result from the use of individually determined values of
VS, but we should recommend the
use of more time intervals than used at present if this were to be
done. However, potential error due to application of a pooled value of
VS is too small to be a major
source of concern and cannot explain the large differences in results
between laboratories.
Among the differences between laboratories in the estimates of
cumulative glucose appearance (Table 1), model order appears not to be
a significant factor. Radziuk and co-workers (28, 29) find high
recoveries by using a two-compartment model, whereas Ferrannini et al.
(7) find low recoveries using the same model. Similarly, Mari et al.
(18) find low recoveries (when using radioisotopes), whereas we find
high recoveries (using stable isotopes) when the same two-compartment
model is used. Nevertheless, model order may be viewed as important.
Although Mari et al. showed that neither the one- nor the
two-compartment model was demonstrably superior over the other for the
assessment of glucose kinetics after oral glucose, it was also shown
that considerable differences occur in rate estimates between models
when VS in Steele's model is
assumed to be 150 ml/kg. At present, we find such differences between
models are largely eliminated when a value for
VS of 230 ml/kg is used; indeed,
the models yield very similar, or practically identical, information on
RaO and
RaH at each time interval. It is
noteworthy that Pilo et al. (26) report a result for just one subject,
which indicates comparable kinetics and recovery of oral glucose in
plasma when one- and two-compartment models are used and
VS is made large and possibly
close to or equal to the "total glucose distribution space." The
far greater numbers of observations in the present study now make this
particular observation robust. Clearly, marked differences in
RaO and
RaH between the one- and
two-compartment models occur only when
VS fails to balance the
"volume" and "structure" errors suggested (5). The
similarities in parameter estimates when
VS = 230 ml/kg support the
adequacy for the present experimental situation of the larger glucose
distribution volume found at present than that used previously (Table
1). Except for the confounding effects of an inappropriate
VS, model order does not explain a
significant part of the large differences in literature values (Table
1) for cumulative systemic appearance of oral glucose.
A further outcome of using the higher glucose distribution volume with
Steele's model is a plateau in the cumulative appearance of oral
glucose beyond 3 h after glucose ingestion (Fig. 7). By trial, we found
that too high a value of VS
resulted in an overestimation of cumulative
RaO initially and underestimation
eventually, as summarized by the fractional sensitivity of rate
estimates to VS in Fig. 8. The
reverse is true for values of VS
that are too small. A consequence is that an incorrect volume results
in the absence of a plateau when glucose absorption is complete. It is noteworthy that when VS is too
small, as in most cases (Table 1), absorption never appears to reach
completion. Inspection of rate of appearance curves for oral glucose in
several publications shows this to be a frequent occurrence (6, 9, 20,
21). Conversely, too high a value for
VS results in peaking of the cumulative appearance curve, reaching too high values initially and
false negative appearance rates eventually. Thus, in either case, the
estimate of the cumulative appearance is then dependent on the duration
over which glucose Ra values are
integrated, irrespective of when absorption is complete.
Both the present study and that of Delarue et al. (6) use
D-[6,6-2H2]glucose
and
D-[13C6]glucose
species, but the results appear discrepant (Table 1). The present
results cluster within studies from investigators using radioactive
isotopes who find 90-100% systemic recovery of oral glucose,
whereas those of Delarue et al. fall among those finding 70% recovery.
Even after adjustment for the use of different effective glucose
distribution volumes in the two studies, the results would still differ
by 10% or more of the ingested glucose. The similar choice of isotopes
indicates that incorrect assumptions about isotopomer metabolism are
not the cause of the discrepancy. A possible source of variation is the
frequency of plasma sampling for isotope analysis, particularly in the
early period when absorption is rapid and changing. Table 1 indicates
those published studies that sample plasma more frequently than every
30 min early after glucose ingestion (denoted F for frequent sampling)
and those that do not (denoted I for infrequent sampling). With the
studies of Ferrannini et al. (7) and Mari et al. (18) being exceptional (see below), there is direct correspondence between early sampling frequency and result (Table 1), with the infrequent sampling resulting
in an underestimation of cumulative systemic appearance of glucose.
Furthermore, by close inspection of the one-compartment RaO time curve (Fig. 6), it can be
seen that omission of a time point 15 min postingestion would result in
a lower early RaO than that
actually observed. The decrease in cumulative
RaO would have been 9% for such
an omitted sampling time. In addition, omission of the sample between
30 and 60 min would result in a further 5% error. The difference in
estimated outcomes for the previous and the present dual stable isotope
studies can therefore be explained by summation of a sampling frequency
error and an effective volume error.
The two errors identified above, although sufficient to reconcile the
majority of the study results, do not explain every difference. We have
not positively identified the cause of the remaining differences,
between Radziuk and co-workers (28, 29) and Ferrannini et al. (7) on
the one hand and between the present results and those of Mari et al.
(18) on the other, but we do not exclude sources of uncertainty, which
may include incomplete absorption associated with the lower systemic
appearances or the presence of a neutral, nonglucose radioactive
contaminant in some preparations of commercial
D-[3-3H]glucose
(1). The purification of glucose during derivatization, gas
chromotography, and selective mass spectrometry substantially reduces
any risk of error due to contamination of stable isotopes of glucose.
The discussion so far has centered on possible explanations for too low
values of oral glucose appearance in the systemic circulation. However,
there are possible reasons why too high values could be observed
because of the use of particular tracer isotopes. Thus Pehling et al.
(23) and Firth et al. (9) used D-[2-3H]glucose
intravenously in their mixed radioactive and stable dual isotope work.
The loss of label from this isotopomer occurs before either glycogen
synthesis or glycolysis (35) and so is expected to overestimate glucose
utilization to a variable extent (30), pending the combined cycling
rate of glucokinase and hexokinase. Appearance of oral
glucose labeled with this isotopomer will be overestimated on this
account, and this will offset an underestimate from use of a low
VS; therefore, a result that is
approximately correct is possible when these two errors cancel to yield
the 92-93% recovery values (Table 1).
In conclusion, provided isotope recycling is minimal, as evident in the
present study, stable isotopes are a suitable and safe alternative to
radioactive isotopes for application in the dual isotope method with
physiologically relevant oral glucose loads, at least in healthy
humans. Sources of error in the methodology can occur whether
radioactive or stable isotopes are used. Particular attention should be
given both to the use of frequent plasma sampling, whatever model is
used for data analysis, and to the larger effective glucose
distribution volume when a single-compartment model is used. After such
considerations, the differences in results for cumulative
RaO among several laboratories are
narrowed and favor the view that first-pass hepatic fractional
sequestration of oral glucose is about the same as that for one-pass
hepatic fractional sequestration of systemic glucose. In studies
involving less rapid changes in glucose
Ra values, such as with certain
starchy foods (14, 15), the use of the one-compartment model would seem even more justified than at present for glucose.
 |
ACKNOWLEDGEMENTS |
Thanks are due to Dr. Andrea Mari, University of Padua, for the
gift of SMART software and helpful discussions, and to Dr. Marinos
Elia, Dunn Clinical Nutrition Centre, Cambridge, for advice on clinical
techniques.
 |
FOOTNOTES |
This study was supported financially by the Biotechnology and
Biological Sciences Research Council, and the Ministry of Agriculture, Fisheries, and Food.
Address for reprint requests: G. Livesey, Dept. of Nutrition, Diet and
Health, Institute of Food Research, Norwich Research Park, Colney,
Norwich NR4 7UA, UK.
Received 13 May 1997; accepted in final form 28 July 1998.
 |
REFERENCES |
1.
Allsop, J. R.,
R. R. Wolfe,
and
J. F. Burk.
The reliability of glucose appearance in vivo calculated from constant tracer infusions.
Biochem. J.
172:
407-416,
1978[Medline].
2.
Avogaro, A.,
P. Vicini,
A. Valerio,
A. Caumo,
and
C. Cobelli.
The hot but not the cold minimal model allows precise assessment of insulin sensitivity in NIDDM subjects.
Am J. Physiol.
270 (Endocrinol. Metab. 33):
E532-E540,
1996[Abstract/Free Full Text].
3.
Berthold, H. K.,
L. J. Wykes,
P. D. Klein,
and
P. J. Reeds.
The use of uniformly labelled substrates and mass isotopomer analysis to study intermediary metabolism.
Proc. Nutr. Soc.
53:
345-354,
1994[Medline].
4.
Bondar, R. J. L.,
and
D. C. Mead.
Evaluation of glucose-6-phosphate dehydrogenese from Leuconostoc mesenteroids in the hexokinase method for determining glucose in serum.
Clin. Chem.
20:
586-590,
1974[Abstract/Free Full Text].
5.
Cobelli, C.,
A. Mari,
and
E. Ferrannini.
Non-steady state: error analysis of Steele's model and developments for glucose kinetics.
Am. J. Physiol.
252 (Endocrinol. Metab. 15):
E679-E689,
1987[Abstract/Free Full Text].
6.
Delarue, J.,
C. Couet,
R. Cohen,
J.-F. Bréchot,
J.-M. Antoine,
and
F. Lamisse.
Effects of fish oil on metabolic responses to oral fructose and glucose loads in healthy humans.
Am. J. Physiol.
270 (Endocrinol. Metab. 33):
E353-E362,
1996[Abstract/Free Full Text].
7.
Ferrannini, E.,
O. Bjorkman,
G. A. Reichard,
A. Pilo,
M. Olsson,
J. Wahren,
and
R. A. DeFronzo.
The disposal of an oral glucose load in healthy subjects.
Diabetes
34:
580-588,
1985[Abstract].
8.
Féry, F.,
and
E. O. Balasse.
Glucose metabolism during the starved-to-fed transition in obese patients with NIDDM.
Diabetes
43:
1418-1425,
1994[Abstract].
9.
Firth, R. G.,
P. M. Bell,
H. M. Marsh,
I. Hansen,
and
R. A. Rizza.
Postprandial hyperglycaemia in patients with non-insulin-dependent diabetes mellitus.
J. Clin. Invest.
77:
1525-1532,
1986[Medline].
10.
Insel, P. A.,
J. E. Liljenquist,
J. D. Tobin,
R. S. Sherwin,
P. Watkins,
R. Andres,
and
M. Berman.
Insulin control of glucose metabolism in man. A new kinetic analysis.
J. Clin. Invest.
55:
1057-1066,
1975.
11.
Kelley, D.,
A. Mitrakou,
H. Marsh,
F. Schwenk,
J. Benn,
G. Sonnenberg,
M. Arcangeli,
T. Aoki,
J. Sorensen,
M. Berger,
P. Sonksen,
and
J. Gerich.
Skeletal muscle glycolysis, oxidation, and storage of an oral glucose load.
J. Clin. Invest.
81:
1563-1571,
1988[Medline].
12.
Kruszynska, Y. T.,
A. Meyer-Alber,
F. Darakhshan,
P. D. Home,
and
N. McIntyre.
Metabolic handling of orally administered glucose in cirrhosis.
J. Clin. Invest.
91:
1057-1066,
1993[Medline].
13.
Levy, J. C.,
G. Brown,
D. R. Matthews,
and
R. C. Turner.
Hepatic glucose output in humans measured with labeled glucose to reduce negative errors.
Am J. Physiol.
257 (Endocrinol. Metab. 20):
E531-E540,
1989[Abstract/Free Full Text].
14.
Livesey, G.,
R. Faulks,
P. Wilson,
M. Roe,
J. Brown,
T. Newman,
F. Mellon,
F. Eagles,
J. Dennis,
I. Parker,
R. Greenwood,
and
D. Halliday.
Development of a dual stable-isotope method for determining glucose absorption from 13C-enriched starchy foods.
Proc. Nutr. Soc.
56:
39A,
1997.
15.
Livesey, G.,
R. Faulks,
P. Wilson,
M. Roe,
J. Brown,
T. Newman,
F. Mellon,
F. Eagles,
J. Dennis,
I. Parker,
R. Greenwood,
and
D. Halliday.
Individual rates of glucose absorption from 13C-enriched starch in peas eaten by healthy adults can be very different: is there a role in the aetiology of disease?
Proc. Nutr. Soc.
56:
40A,
1997.
16.
Mari, A.
Estimation of the rate of appearance in the non-steady state with a two-compartment model.
Am. J. Physiol.
263 (Endocrinol. Metab. 26):
E400-E415,
1992[Abstract/Free Full Text].
17.
Mari, A.
SMART Reference Manual. Padova, Italy: CNR, 1992. (LADSEB-CNR Int. Rep. 03/92)
18.
Mari, A.,
J. Wahren,
R. A. DeFronzo,
and
E. Ferrannini.
Glucose absorption and production following oral glucose: comparison of compartmental and arteriovenous-difference methods.
Metabolism
43:
1419-1425,
1994[Medline].
19.
Marks, L.
Counting the Cost: The Real Impact of Non-Insulin-Dependent Diabetes. London: King's Fund Policy Institute, 1996.
20.
Mitrakou, A.,
D. Kelley,
M. Mokan,
T. Veneman,
T. Pangburn,
J. Reilly,
and
J. Gerich.
Role of reduced suppression of glucose production and diminished early insulin release in impaired glucose tolerance.
N. Eng. J. Med.
326:
22-29,
1992[Abstract].
21.
Mitrakou, A.,
D. Kelley,
T. Veneman,
T. Jenssen,
T. Pangburn,
J. Reilly,
and
J. Gerich.
Contribution of abnormal muscle and liver glucose metabolism to postprandial hyperglycemia in NIDDM.
Diabetes
39:
1381-1390,
1990[Abstract].
22.
Norwich, K. H.,
J. Radziuk,
D. Lau,
and
M. Vranic.
Experimental validation of the non-steady state rate measurements using a tracer infusion method and inulin as tracer and tracee.
Can. J. Pharmacol.
52:
508-521,
1974.
23.
Pehling, G.,
P. Tessari,
J. E. Gerich,
M. W. Haymond,
F. J. Service,
and
R. A. Rizza.
Abnormal meal carbohydrate disposition in insulin-dependent diabetes.
J. Clin. Invest.
74:
985-991,
1984[Medline].
24.
Perry, I. J.,
S. G. Wannamethee,
M. K. Walker,
A. G. Thomson,
P. H. Whincup,
and
A. G. Shaper.
Prospective study of risk factors for the development of non-insulin dependent diabetes in middle aged British men.
Br. Med. J.
310:
560-564,
1995[Abstract/Free Full Text].
25.
Pickert, A.,
D. Overkamp,
W. Renn,
H. Liebich,
and
M. Eggstein.
Selected ion monitoring gas chromatography/mass spectrometry using uniformly labelled (13C)glucose for the determination of glucose turnover in man.
Biol. Mass Spectrom.
20:
203-209,
1991[Medline].
26.
Pilo, A.,
E. Ferrannini,
O. Biorkman,
J. Wahren,
G. A. Reichard,
P. Felig,
and
R. A. DeFronzo.
Analysis of glucose production and disappearance rates following an oral glucose load in normal subjects: a double tracer approach.
In: Carbohydrate Metabolism, edited by C. Cobelli,
and R. N. Bergman. London: Wiley, 1981, p. 221-238.
27.
Powrie, J. K.,
G. D. Smith,
T. R. Hennessy,
F. Shojaee-Moradie,
J. M. Kell,
P. H. Sönksen,
and
R. H. Jones.
Incomplete suppression of hepatic glucose production in non-insulin dependent diabetes mellitus measured with [6,6-2H2]glucose enriched infusion during hyperinsulinaemic euglycaemic clamps.
Eur. J. Clin. Invest.
22:
244-253,
1992[Medline].
28.
Radziuk, J.
Hepatic glycogen formation by direct uptake of glucose following oral glucose loading in man.
Can. J. Physiol. Pharmacol.
57:
1196-1199,
1979[Medline].
29.
Radziuk, J.,
T. J. McDonald,
D. Rubenstein,
and
J. Dupre.
Initial splanchnic extraction of ingested glucose in man.
Metabolism
27:
657-669,
1978[Medline].
30.
Radziuk, J.,
K. H. Norwich,
and
M. Vranic.
Experimental validation of measurements of glucose turnover in non-steady state.
Am. J. Physiol.
234 (Endocrinol. Metab. Gastrointest. Physiol. 3):
E84-E93,
1978[Abstract/Free Full Text].
31.
Reaven, G. M.
Role of insulin resistance in human disease. Banting lecture 1988.
Diabetes
7:
1595-1607,
1988.
32.
Schwartz, I. L.
Measurement of extracellular fluid by means of a constant infusion technique without collection of urine.
Am. J. Physiol.
160:
526-531,
1950.
33.
Steele, R.
Influences of glucose loading and of injected insulin on hepatic glucose output.
Ann. NY Acad. Sci.
82:
420-430,
1959.
34.
Colhoun, H.,
and
P. Prescott-Clark
(Editors).
The Health of the Nation. Health Survey for England London: HMSO, 1996, p. 235-252.
35.
Weber, J. M.,
S. Klein,
and
R. R. Wolfe.
Role of the glucose cycle in control of net glucose flux in exercising humans.
J. Appl. Physiol.
68:
1815-1819,
1990[Abstract/Free Full Text].
36.
Williams, B.
Insulin resistance: the shape of things to come.
Lancet
344:
521-524,
1994[Medline].
Am J Physiol Endocrinol Metab 275(4):E717-E728
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society