SPECIAL COMMUNICATION
Rapid and accurate
13CO2
isotopic measurement in whole blood: comparison with expired
gas
Martial
Dangin1,2,
Jean Claude
Desport1,
Pierre
Gachon1, and
Bernard
Beaufrère1
1 Laboratoire de Nutrition
Humaine, Université Clermont Auvergne, Centre de Recherche en
Nutrition Humaine, 63009 Clermont-Ferrand, Cedex 1, France; and
2 Nestlé Research Center,
CH-100 Lausanne 26, Switzerland
 |
ABSTRACT |
Determination of
13CO2
enrichment on the CO2 released
from blood by acid has been used in situations in which breath sampling is difficult. This method can be improved by measuring this enrichment on the CO2 spontaneously released
from blood. Therefore, simultaneous comparisons of
13CO2
content between breath and arterialized blood added with or without
acid were performed in 51 samples from human studies, using the
statistical method of Bland and Altman (J. M. Bland and D. G. Altman.
Lancet 1: 307-310, 1986). Strong
relationships exist between the methods
(r > 0.99) expressed in atom percent excess (APE). Compared with breath, the acid method overestimates the
13CO2
enrichment (0.318 ± 0.632 APE × 1,000, P < 0.001). The acid-free method
shows similar enrichments to breath (0.003 ± 0.522 APE × 1,000, P = 0.97) with good precision
and degree of agreement (95% confidence interval 0.15 APE × 1,000). The analysis can be performed up to 5 days after sampling with
a good reproducibility. In conclusion, measuring
13CO2
enrichments on the CO2
spontaneously released from blood is feasible, gives identical results
to the breath method, and lessens operator manipulations. It allows
study of situations in which the breath sampling method is not feasible.
carbon dioxide; bicarbonate; isotopic fractionation; mass
spectrometry
 |
INTRODUCTION |
THE MEASUREMENTS OF
13CO2
enrichment are required in numerous studies to estimate either nutrient
oxidation rates (17), CO2 production (9), or assimilation of various
13C-labeled substrates (7; i.e.,
breath tests). For these purposes, 13CO2
atom percent (AP) is usually determined by isotope ratio mass spectrometry on breath samples collected at sequential time points (17). However, voluntary breath sampling can be difficult or impossible
to perform in various situations such as ventilatory abnormalities,
artificially ventilated patients, or in specific populations such as
newborns or patients with neurological disorders (8, 14). Thus two
alternatives have been proposed to solve this problem. The first one is
to sample gas from the ventilated hood of an indirect calorimeter (8).
This method is not widely used, in particular because large volumes of
gas are needed for isotope ratio mass spectrometry analysis due to
atmospheric air dilution. In addition, this technique is limited by the
mandatory utilization of the indirect calorimetry system, which is not
required for breath test or for the estimation of
CO2 production by an isotopic
dilution method. The second method (4, 6, 10, 14, 15) analyzes the
gaseous CO2 released from blood by
acid addition. Compared with the breath method, this method is more demanding due to operator manipulations. For diagnosis purposes, it is
important that blood samples can be processed rapidly, reliably, and
with minimal operator manipulations. Considering that
1) blood CO2 is spontaneously released in
the air and 2) the sensitivity of
the isotope ratio mass spectrometer has dramatically increased over the
past few years, it should be possible to improve the latter method by
the measurement of
13CO2
AP spontaneously released from blood (e.g., without acid addition). Thus the aim of the present work was to determine whether
CO2 released from the blood
without acid addition can be used to measure 13CO2
AP. The technical aspects of this methodology were validated, and
simultaneous comparisons for breath, blood with acid, and blood without
acid were made at each sampling point.
 |
SUBJECTS AND METHODS |
Subjects and experimental protocols.
Blood and gas samples were obtained from nine young healthy subjects
(age 30.6 ± 11.7 yr, weight 63.9 ± 5.4 kg, height 171 ± 7 cm, mean ± SD) who participated in two different protocols that
were previously described in detail (2, 11). The protocols were
approved by the ethical committee of Clermont-Ferrand. Briefly, five
subjects received (11) a primed continuous constant intravenous
infusion of
L-[1-13C]leucine
for 10 h after a prime dose of
[13C]bicarbonate, and
samples were collected before the tracer infusion and when the
13CO2
AP had reached a plateau (510, 540, 580, and 600 min). The four other
volunteers (2) received a single oral load of 30 g of whey protein
added with free
L-[1-13C]leucine
(13 mmol/kg). Samples were taken before and after (at 60, 120, 180, 240, and 300 min) the administration of oral tracer and were utilized
for the present study. We chose this latter study because the obtained
13CO2
AP both covered and exceeded the normal range usually observed in
classic oxidation studies at steady state. A total number of 51 time
points were obtained. For each of these, expired gas and arterialized blood samples were collected simultaneously. Breath samples were collected as previously described (6). Before blood
sampling, the hand was warmed for 15 min at 60°C in a heated ventilated box as previously described (2). Immediately after sampling,
1 ml of arterialized venous blood was injected in a 10-ml tube
(Vacutainer; Becton-Dickinson, Meylan, France) previously flushed with
helium, and 1 ml was injected in a 10-ml tube also previously flushed
and containing 0.5 ml of 0.2 M lactic acid (Merck, Darmstadt, Germany).
An additional set of experiments was carried out, with lactic acid
alone, to determine whether lactic acid in the absence of blood
generates a signal. The tubes were then immediately agitated (1 rotation/min) during 2 h at room temperature before the isotope
analysis. Measurements were performed on the following three sets of
samples: breath, CO2 spontaneously
released by blood, and CO2
released from blood by lactic acid. Each method is further referred to
as "Breath," "Blood," and "Blood+LA," respectively.
Analytical methods and calculations.
The
13CO2
analyses were performed by gas chromatography isotope ratio mass
spectrometry (µGas System; Fisons Instruments, VG Isotech,
Middlewich, UK). Briefly, a sample (40 µl) of the gaseous content of
each tube analyzed was automatically injected (Gilson automatic
sampler) in the gas chromatograph (Hewlett Packard 5890; Palo Alto, CA) in which the CO2 was separated
from the other components using a packed column (Chrompack column, type
HAYSEP Q, 2.5 m × 1/8, 60/80 mesh; Les Ulis, France) at
100°C. The CO2 was then
introduced in the isotope ratio mass spectrometer to measure the
13C-to-12C
isotopic ratio of the sample
(RSAM) versus a reference gas
(RREF). The
13C-to-12C
ratio of the samples was expressed as delta per mil (
)
versus RREF after Craigs
corrections (REF; see Ref. 3)
In
this study, the reference gas had a
13C of
31.00 relative to
the international standard reference Pee Dee Belemnite
(RPDB = 0.0112372). This
calibration was obtained by periodic comparison with other laboratories.
The results obtained in these conditions were corrected to express the
values in 
vs. PDB. The values calculated were then converted in AP using the following formula
Finally,
the
13CO2
atom percent excess (APE) was obtained by subtracting the background AP
(before tracer administration) from the sample AP (during or after
tracer administration). Results obtained were expressed in 
vs. PDB, AP, and in APE × 1,000.
Statistical analysis. Comparison
between the methods was tested using the statistical method described
by Bland and Altman (1). The statistics (Student's
t-test, ANOVA) were performed using
Statwiew 4.02 from Abacus Concepts (Berkeley, CA). Results were
expressed as means ± SD.
 |
RESULTS |
Interassay and intra-assay reproducibilities and
storage. Interassay and intra-assay reproducibilities
of the measurement on Breath, Blood, and Blood+LA were tested as shown
in Table 1. The area of the
CO2 peak derived from arterialized
venous blood decreased by 60% compared with Breath or Blood+LA.
Despite this difference, the SD of Blood in the interassay replicate
(n = 5 subjects) was similar
to Breath, whereas the SD of Blood+LA was slightly higher. Intra-assay
reproducibility was good for all three methods, indicating that up to
five replicates can be performed in the same tube. There was no
statistical difference between the Breath and the Blood methods. In
contrast, Blood+LA was significantly higher than Breath
(P <0.001,
t-test). When 1.5 ml of lactic acid were analyzed, traces of CO2 were
detected, but the very low intensity of the peak did not allow any
measurement of the 13C isotopic
content.
The storage of Blood samples at
20°C for 5 days was also
tested in another batch of blood samples. No significant change in the
13CO2
isotopic ratio was found (P = 0.69, t-test) between Blood immediately
treated (
20.28 ± 0.08 
vs. PDB) and Blood
treated after 5 days of storage (
20.30 ± 0.08 
vs. PDB). In addition, the storage did not modify the reproducibility
of the isotopic analysis (SD = 0.08 
vs. PDB in each case,
n = 5 subjects).
Relationship, precision, and agreement between the methods for
13CO2 isotopic
content.
The
13C-to-12C
isotopic ratio tested in Breath ranged from
25.66 to
6.05

vs. PDB, in Blood from
26.75 to
7.49

, and in Blood+LA from
21.5 to
2.79

vs. PDB.
As shown in Fig. 1, there were strong
positive relationships between Breath and Blood and between Breath and
Blood+LA expressed in AP (r > 0.99).
In the range of our values, Blood+LA was systematically higher than
Breath and Blood. Moreover, in both cases,
y-intercepts were not significantly
different from zero (0.30 < P < 0.49, ANOVA).

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Fig. 1.
Relationships between CO2
spontaneously released by blood (Blood) and breath (Breath) ( ) or
CO2 released from blood by lactic
acid (Blood+LA) and Breath ( ) expressed as
13CO2
atom percent (AP). Regression lines were as follows: Blood
13CO2
AP = 0.982x + 0.019, r = 0.993; Blood+LA
13CO2
AP = 1.017x + 0.019, r = 0.994.
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|
With the use of the Bland and Altman (1) plot (difference between the 2 methods of measurement vs. their mean at each time point), there was
good precision and agreement between the AP of Breath and AP of Blood
13CO2
isotopic ratios, as shown in Fig. 2.
Indeed, there was no statistical difference between the two methods
(0.10 ± 0.71 AP × 1,000, P = 0.31, t-test for difference) and no
relationship between the difference and the mean
(P = 0.56, ANOVA). However, the
scatter of the difference increased as
13CO2
enrichment increased. The 95% confidence interval ranged from
0.099 to 0.300 AP × 1,000.

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Fig. 2.
Difference between Blood ( ) or Blood+LA ( ) and Breath expressed
in AP vs. their means. Dotted lines show means of the difference for
Blood and Breath (0.10 AP × 1,000) and the difference between
Blood+LA and Breath (5.86 AP × 1,000).
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|
In contrast, the difference between AP of Blood+LA and AP of Breath was
always positive (Fig. 2) and was statistically different from zero
(5.86 ± 0.66 AP × 1,000, P < 0.001). In addition, despite the absence of a relationship between
the difference against their mean (P = 0.16), the scatter of the difference also increased as the
13CO2
abundance increased. The 95% confidence interval was always positive
despite the precision (range from 5.678 to 6.049 AP × 1,000).
Relationship, precision, and agreement between the methods for
13CO2 isotopic
enrichments.
Regression line equations for APE of Breath vs. APE of Blood and APE of
Breath versus APE of Blood+LA are given in Fig.
3. The slope for APE of Breath versus APE
of Blood (1.0003 ± 0.013, 0.2 < P < 0.1) and the
y-intercept (
0.015 ± 0.110 APE × 1,000, P = 0.89),
statistically, did not differ from the median slope and zero,
respectively. In contrast, for APE of Breath versus APE of Blood+LA,
the slope was different from the median slope (1.029 ± 0.015, P < 0.001), whereas the
y-intercept was not different from
zero (0.131 ± 0.129 APE × 1,000, P = 0.31).

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Fig. 3.
Relationships between Blood and Breath ( ) or Blood+LA and Breath
( ) expressed as
13CO2
AP. Regression lines were as follows: Blood
13CO2
atom percent excess (APE) = 1.003x + 0.015, r = 0.996; Blood+LA
13CO2
APE = 1.0297x + 0.131, r = 0.995.
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|
The difference between APE of Blood and APE of Breath (Fig.
4A, 0.003 ± 0.522 APE × 1,000) did not appear to be biased
(P = 0.97, t-test of the difference), and there
was no relationship between the difference vs. their mean
(P = 0.60). Despite this observation,
the difference increased as the
13CO2
enrichments increased. However, the 95% confidence interval (
0.144 to 0.150 APE × 1,000) was small enough to ensure
that the method based on the free
CO2 released from blood can be
used with an acceptable precision and degree of agreement.

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Fig. 4.
Difference between Blood and Breath expressed in APE × 1,000 (e.g., by subtracting the basal value) vs. their means
(A) and between Blood+LA and Breath
(B). Dotted line in
B indicates the relationship between
the difference for Blood+LA vs. Breath and the mean
(y = 0.034x + 0.095, P = 0.02)
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|
In contrast (Fig. 4B), the
difference between APE of Blood+LA and APE of Breath (0.318 ± 0.632 APE × 1,000, P < 0.001; 95% confidence interval = 0.141 to 0.496 APE × 1,000) is
significantly different from zero. In addition, there was a positive
statistical relationship between the difference (APE of Blood+LA
APE of Breath) vs. their mean
(P = 0.02), indicating a slight
overestimation of the enrichments at higher values.
 |
DISCUSSION |
Our intent was to develop an automated technique for measuring
13CO2
APE in conditions in which the breath method is difficult or impossible
to perform. For this purpose, we have compared the results obtained by
our method with the analysis performed in expired gas (considered as
the reference method) and in gaseous CO2 released from blood by acid
addition (4, 6, 10, 14, 15).
Comparison between exhaled air and gaseous
CO2 released from blood by acid
revealed higher
13CO2
AP in whole blood, which is in close agreement with previously published works (10, 14). Because lactic acid solution per se is not a
significant provider of CO2, this
difference cannot be due to lactic acid itself. Therefore, it implies
that there is an isotopic fractionation during the reaction leading to
CO2 production. This reaction is
the following
the
latter step being catalyzed in vivo by carbonic anhydrase. When lactic
acid is added to a test tube containing blood, the equilibrium of the
reaction is modified to produce gaseous CO2, and the relative contribution
of carbonic anhydrase is strongly reduced. Therefore, the
13C content measured on the
released CO2 primarily reflects
the 13C content of bicarbonate. An
isotopic effect was demonstrated when acid was added to dissolved
bicarbonate, without carbonic anhydrase (5, 12, 13). The authors found
that the 13C content of
bicarbonate was higher compared with
CO2, which is consistent with our
results. A second isotopic effect was also shown at the carbonic
anhydrase step (13). Indeed, in an in vitro system, enzymatic
dehydration of bicarbonate dissolved in water results in an additive
lower 13C content in the evolved
CO2 compared with the bicarbonate,
which is also in agreement with our findings. Therefore, the difference of 13C content that we observed
between expired CO2 and
CO2 derived from bicarbonate is
likely to be due to these two factors. Our data do not allow evaluation
of their relative contribution. In this respect, it would be of
interest to compare 13C content in
expired CO2, whole blood, and
plasma (i.e., without carbonic anhydrase) or to use carbonic anhydrase inhibitors.
When the
13CO2
content of exhaled air and the CO2
provided by acid addition were corrected by subtraction of their
respective basal value, we found a modest but persistent overestimation
of the enrichment, which increases as the
13CO2
content increases, in blood degassed by acid. Such results were also
reported by Read et al. (14) but not by Denne and Kalhan (4) and
El-Khoury et al. (6). However, in these studies, the statistical
analyses were only performed on a limited number of samples. This
uncertainty thus restricts the use of this method because such a
technique has to give similar results to the measurement in breath,
considered as the reference method.
In contrast, the measurements of the
13CO2
isotopic content spontaneously released by blood gave good precision
and agreement with similar results to exhaled air whether it was
expressed in AP or in APE. An isotopic fractionation may also exist at
the alveolar blood-breath interface (i.e., in vivo) or at the water-air interface (i.e., in the test tube; see Ref. 16). The absence of
difference in the carbon isotope analysis between the expired CO2 and the
CO2 spontaneously released by
blood suggests that the fractionation is negligible or similar under
these two circumstances.
A potential analytical drawback of the method developed is the small
quantity of CO2 dissolved in the
blood and therefore evolved in the tube compared with the two other
methods (Breath and Blood+LA). This could limit the number of
replicates of isotope analysis in the same tube. Nevertheless, in our
conditions, up to six replicates can be done with an excellent
intra-assay reproducibility. In practice, duplicate or triplicate
measurements give reliable results. In addition, the gas chromatography
isotope ratio mass spectrometry analysis can be performed up to 5 days
after blood sampling. Compared with the acid method, our technique is
also superior for manipulator ease of performance and analysis. It simply requires 1 ml of arterialized whole blood introduced in a tube
previously flushed with helium and agitated (1 rotation/min) during 2 h
at room temperature before the isotope analysis.
Such a technique can be employed to estimate whole body nutrient
oxidation rate by measuring separately
13CO2
APE in whole blood and CO2
production rate by indirect calorimetry in the situations mentioned in
the Introduction. Furthermore, when indirect calorimetry is
unavailable, it should be possible to assess whole blood flow and blood
CO2 concentrations and
13C enrichments. Finally,
applications also exist to measure nutrient oxidation rate in specific
organs and to quantify CO2
production rate.
 |
ACKNOWLEDGEMENTS |
We thank C. McCormack and Y. Boirie for help and advice. The
technical and nursing assistance of P. Rousset and L. Morin is also
gratefully acknowledged.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: B. Beaufrère, Laboratoire de
Nutrition Humaine, B.P. 321-58, rue Montalembert, 63009 Clermont-Ferrand Cedex 1, France.
Received 19 May 1998; accepted in final form 2 September 1998.
 |
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