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Departments of 1 Nutrition, 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
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 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.
Participants
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
300% of
expected results) in estimates for splanchnic retention of glucose
during orosystemic passage.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
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).
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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.
|
(1) |
|
(2) |
(t) was its derivative with
respect to time.
|
(3) |
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) |
|
(5) |
|
(6) |
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) |
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(8) |
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(9) |
|
(10) |
|
(11) |
|
(12) |
|
(13) |
, 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.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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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.
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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.
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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).
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Mean Rate of Systemic Appearance of Hepatic Glucose
The mean basal steady-state RaH was 2.2 ± 0.1 mg · min1 · kg
body weight1, 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 · min1 · kg1
(Fig. 5). Thereafter,
RaH increased, returning to 75%
of the basal value by 5 h after the oral glucose.
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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 · min1 · kg
body weight1 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.
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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.
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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.
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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.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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, 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.
, 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.
), and cumulative
systemic appearance of gut-derived glucose (