J Physiol Volume 513, Number 1, 215-223, November 15, 1998
The Journal of Physiology (1998), 513.1, pp. 215-223
© Copyright 1998 The Physiological Society
Validation of the [1,2-13C]acetate recovery factor for correction of [U-13C]palmitate oxidation rates in humans
Patrick Schrauwen, Dorien P. C. van Aggel-Leijssen, Wouter D. van Marken Lichtenbelt, Marleen A. van Baak, Annemie P. Gijsen and Anton J. M. Wagenmakers
Department of Human Biology, Maastricht University, The Netherlands
Received 16 February 1998; accepted after revision 5 August 1998.
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ABSTRACT |
- The validity of estimations of plasma fatty acid oxidation using tracers has often been questioned. The appearance of isotopic markers in breath CO2 is delayed and incomplete. Recently suggestions have been made that substantial amounts of tracer are incorporated into products of the tricarboxylic acid cycle (e.g. glucose, glutamine and glutamate) and that an acetate correction factor can be used to correct for tracer fixation. In the present study we investigated whether the appearance of 13CO2 during a separate infusion of [1,2-13C]acetate could be used for correction of [U-13C]palmitate oxidation rates in studies lasting <2 h and we quantified the appearance of tracer in the glutamine, glutamate and glucose pools of the body.
- An infusion of either [1,2-13C]acetate (0·104 µmol min-1 kg-1) or [U-13C]palmitate (0·013 µmol min-1 kg-1) was given to eight male subjects and continued for 2 h at rest. In six subjects the infusion of [1,2-13C]acetate was repeated to determine reproducibility of the acetate recovery.
- Fractional recovery in breath from [1,2-13C]acetate gradually increased during the infusion period at rest from 14·1 ± 0·6 % at 60 min to 26·5 ± 0·5 % at 120 min after the start of the infusion. Intersubject coefficient of variance was 8·3 ± 0·6 % and intrasubject coefficient of variance of the acetate recovery tests was 4·0 ± 1·5 %. After 2 h of [1,2-13C]acetate infusion, 12·4 ± 0·8 and 10·3 ± 0·9 % of infused 13C was incorporated in the glutamine and glutamate pools, respectively.
- In conclusion, the [1,2-13C]acetate recovery factor can be used for correcting the rate of [U-13C]palmitate oxidation in infusing studies of 2 h in resting conditions. Failure to use this recovery factor leads to a substantial underestimation of the rate of plasma free fatty acid oxidation. The extent of label fixation could largely be explained by accumulation of tracer carbon in glutamine and glutamate, and the accumulation in glucose is negligible.
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INTRODUCTION |
The use of 13C- and 14C-fatty acid tracers to estimate the oxidation of plasma fatty acids has been questioned (Heiling et al. 1991). The appearance of 13CO2 (and 14CO2) in breath, coming from the oxidation of fatty acid tracers, is very low in the first hours of infusion, especially under resting conditions (Irving et al. 1983). This means that part of the label is temporarily fixed in metabolic pools in the body. When, in resting conditions, labelled NaHCO3 is infused, up to 70-90 % of the label is recovered implying that only a minor fraction of the tracer is entrapped in the bicarbonate pool and CO2 fixation reactions (carboxylation and urea production) (Leijssen & Elia, 1996). Heiling et al. (1991) suggested that the plasma tracer first had to mix with a large intracellular fatty acid pool before it would be oxidized and that this was the main reason for the delayed and incomplete appearance of 13CO2 (14CO2) in the breath. However, in contrast to Heiling et al. (1991), Sidossis et al. (1995b) recently showed that part of the tracer accumulated in products of the tricarboxylic acid (TCA) cycle (glucose, glutamate and glutamine; Fig. 1). It was claimed that the fixation of the tracer in these products, together with fixation in the bicarbonate pool, was the main reason for the delayed and incomplete appearance of 13CO2 (14CO2) in the breath. They suggested that the amount of this label fixation can be determined by measuring the 13CO2 production during infusion of 13C- (or 14C) acetate, because acetate, like palmitate, is converted to acetyl-CoA and then enters the TCA cycle before CO2 is produced. According to Sidossis et al. (1995a) a constant value was observed for the acetate recovery factor between 3 and 4 h after the start of the infusion and it was suggested that this steady state value should be used to correct palmitate oxidation rates. The first aim of the present study was to investigate whether the acetate recovery factor can be used during infusions lasting 2 h maximum, which would allow a shorter and less demanding infusion period for healthy volunteers and patients.
Sidossis et al. (1995a) used a simultaneous infusion of 14C-acetate and 13C-palmitate, so that only one experiment is needed to quantify and correct the oxidation of the palmitate tracer. However, the use of radioactive tracers is under severe limitation in many countries due to medical ethical considerations (Beckers et al. 1994). This implies that two separate experiments are needed, one to measure the 13CO2 production from [U-13C]palmitate and one to measure the 13CO2 production from [1,2-13C]acetate under identical conditions. However, in that case, the reproducibility of the acetate recovery factor should be high to obtain a valid estimate in two separate experiments. Therefore, the second aim of the present study was to investigate whether the acetate recovery factor has to be measured in each individual or whether the mean of a group is representative for individuals. So, we measured both the intra- and intersubject coefficient of variation of the acetate recovery factor in a group of healthy volunteers.
The main TCA cycle products in which label from [1,2-13C]acetate and [U-13C]palmitate may accumulate are glucose formed in the liver by gluconeogenesis (Marliss et al. 1971), glutamate released by gut and liver and glutamine formed and released by skeletal muscle (Marliss et al. 1971; Darmaun et al. 1986; Elia et al. 1989). The final aim of the present study was to quantify the appearance in time in these circulating products of the TCA cycle to judge which process and tissue are most important for tracer fixation from a quantitative point of view during a 2 h infusion of acetate.
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METHODS |
Subjects
Eight healthy, male volunteers participated: age, 35·1 ± 1·8 years; height, 1·80 ± 0·03 m; weight, 79·5 ± 5·3 kg; percentage body fat, 22·3 ± 2·6 %; with a body mass index ranging from 19 to 30 kg m-2. The nature and risks of the experimental procedure were explained to the subjects, and all subjects gave their written informed consent. The study was approved by the Ethical Committee of Maastricht University.
Experimental design
The experiment consisted of two tests separated by a minimum of 1 week to prevent carry over of tracer. Subjects came to the laboratory at 08.00 h after an overnight fast. Teflon catheters were inserted in an antecubital vein for isotope infusion and retrogradely into a contralateral dorsal hand vein for sampling of arterialized venous blood. After placement of the catheters subjects rested on a bed and the hand was placed in a hot-box where air was circulated at 60°C in order to obtain arterialized venous blood. After 30 min baseline oxygen consumption and carbon dioxide production were measured and breath and blood samples were collected. Immediately thereafter, subjects were given an intravenous dose of 0·085 mg kg-1 of NaH13CO3 to prime the bicarbonate pool. Then, at t = 0, a constant intravenous infusion of [U-13C]palmitate (0·013 µmol min-1 kg-1) or [1,2-13C]acetate (0·104 µmol min-1 kg-1) was started and continued for 120 min. These infusion rates led to an equal amount of 13C-tracer infusion with both palmitate and acetate. The concentration of palmitate in the infusate was measured for each experiment, so that the exact infusion rate could be determined on an analytical gas chromatograph using heptadecanoic acid as internal standard (see sample analysis). The palmitate tracer (60 mg of potassium salt of [U-13C]palmitate, 99 % enriched; Cambridge Isotope Laboratories, Andover, MA, USA) was dissolved in heated sterile water and passed through a 0·2 µm filter into 5 % warm human serum albumin to make a 0·670 mM solution. The acetate concentration was measured in each infusate with an enzymatic method (Boehringer, Mannheim, Germany). The acetate tracer (sodium salt of [1,2-13C]acetate, 99 % enriched; Cambridge Isotope Laboratories) was dissolved in 0·9 % saline. The chemical and isotopic purity (99 %) of palmitate and acetate tracers were checked by 1H and 13C NMR and gas chromatography-mass spectrometry (GC-MS). Blood samples were taken every 30 min, and breath samples every 10 min. Oxygen consumption (O2) and carbon dioxide production (CO2) were also measured every 10 min. In six subjects the acetate test was repeated to determine the intrasubject reproducibility of the measurements.
Oxygen consumption and carbon dioxide production were measured using open circuit spirometry (Oxycon-
, Mijnhardt, Bunnik, The Netherlands). No changes in oxygen consumption, carbon dioxide production and respiratory quotient occurred during the last hour of the resting period and therefore measurements were averaged for the last hour of the resting period (t = 60-120 min).
Diet and activity prior to testing
Subjects were asked to fill in a 3 day food intake and activity record prior to the first test. In the other tests they were asked to repeat their activity schedule and eat the same food, making sure the diet was similar in the two tests. Also, subjects were asked not to consume any products with a high natural abundance of 13C (carbohydrates derived from C4 plants: maize, sugar cane) 1 week before and during the entire experimental period.
Body composition
After an overnight fast, body density was determined by underwater weighing in the fasted state. Body weight was measured with a digital balance accurate to 0·01 kg (Sauter, type E1200, Ebungen, Germany). Lung volume was measured simultaneously with the helium dilution technique using a spirometer (Volugraph 2000, Mijnhardt). Body fat percentage was calculated using the equations of Siri (1956). Fat-free mass (FFM) in kilograms was calculated by subtracting fat mass from total body mass.
Sample analysis
Oxygen saturation (Hemoximeter OSM2, Copenhagen, Denmark) was determined immediately after sampling in heparinized blood. Fifteen millilitres of arterialized venous blood was sampled in tubes containing EDTA to prevent clotting and immediately centrifuged at 3000 r.p.m. (1000 g) for 10 min at 4°C. Plasma was frozen in liquid nitrogen and stored at -80°C until further analysis. Plasma substrates were determined using the hexokinase method for glucose (Roche, Basel, Switzerland) and the Wako NEFA C test kit for free fatty acids (Wako Chemicals, Neuss, Germany). Plasma for the analysis of glutamine and glutamate was deproteinized with 0·3 M perchloric acid (PCA) and analysed enzymatically for glutamate with glutamate dehydrogenate performed on a COBAS FARA analyser (Roche, Basel, Switzerland). Glutamine concentration was subsequently determined by converting glutamine to glutamate using glutaminase.
Breath samples were analysed for 13C/12C ratio using a gas chromatography-isotope ratio mass spectrometry system (GC- IRMS; Finnigan MAT 252, Bremen, Germany). Plasma glutamine and glutamate tracer/tracee ratio (TTR) was determined as the N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) derivative using GC-IRMS. The resulting derivative contains twenty-three carbon atoms of which five are labelled; TTR is therefore corrected by the factor 23/5. For determination of plasma palmitate, free fatty acids (FFAs) were extracted from plasma, isolated by thin-layer chromatography, and derivatized to their methyl esters. Palmitate concentration was determined on an analytical gas chromatograph with flame ionization detection using heptadecanoic acid as internal standard and on average it comprised 24 ± 1 % of total FFAs. Isotope tracer/tracee ratio of palmitate was determined using GC-IRMS and corrected for the extra methyl group in its derivative. For determination of the plasma glucose tracer/tracee ratio, the glucose was extracted with chloroform- methanol-water and derivatization occurred with butylboronic acid and acetic anhydride as described before (Pickert et al. 1991). The resulting derivative contains sixteen carbon atoms of which six are labelled; TTR is therefore corrected by the factor 16/6.
Calculations
13C enrichment of breath CO2 and plasma metabolites is given in the form of the tracer/tracee ratio (TTR). TTR was defined as (13C/12C)sa - (13C/12C)bk, in which sa indicates sample and bk indicates background.
Total carbohydrate and fat oxidations were calculated using stoichiometric equations (Péronnet & Masssicotte, 1991):
Total fat oxidation = 1·695O2 - 1·701CO2,
Total carbohydrate oxidation = 4·585CO2 - 3·226O2,
with O2 and CO2 in litres per minute and total fat and carbohydrate oxidations in grams per minute.
Total fatty acid oxidation was determined by converting the rate of total fat oxidation to its molar equivalent, with the assumption that the average molecular weight of triglyceride is 860 g mol-1, and multiplying the molar rate of triglyceride oxidation by three because each molecule contains three fatty acid residues.
Fractional recovery of label (as a percentage) in breath CO2, derived from the infusion of labelled palmitate or acetate was calculated as follows:
Fractional recovery of label = (TTRCO2 × CO2)/F × 100,
where TTRCO2 is tracer/tracee ratio (TTR) in breath CO2, CO2 is carbon dioxide production (mmol min-1) and F is infusion rate (mmol min-1).
Breath 13CO2 production, corrected for acetate recovery, during [U-13C]palmitate infusion was calculated as:
Corrected 13CO2 production = (TTRCO2 × CO2)/ar,
where TTRCO2 is tracer/tracee ratio (TTR) in breath CO2, CO2 is carbon dioxide production (mmol min-1) and ar is fractional acetate recovery.
The rate of [U-13C]palmitate oxidation (µmol min-1) was calculated as follows:
Palmitate oxidation = (TTRCO2 × CO2)/(TTRp × ar) × 1000,
where TTRp is the tracer/tracee ratio of fatty acid carbon in plasma and ar is the fractional acetate recovery.
Total plasma fatty acid oxidation was then calculated by dividing palmitate oxidation rate by the fractional contribution of palmitate to the total FFA concentration.
Rate of appearance (Ra, µmol min-1) of palmitate in plasma, which under steady state conditions is equal to the rate of disappearance (Rd) minus tracer infusion rate, was calculated as:
Ra = F(TTRi/TTRp),
where TTRi is the tracer/tracee ratio of fatty acid carbon in infusion. Percentage of plasma FFA cleared from the circulation that was oxidized (%Ra oxidized) was calculated as:
% Ra oxidized = plasma FFA oxidation/Ra FFA.
To estimate the amount of infused 13C incorporated in the glutamine, glutamate and glucose pools it was assumed that the tracer/tracee ratio in plasma at the end of the infusion period was in equilibrium with the respective body pools. Using available data on the size of the glutamine and glutamate pools, the amount of infused 13C incorporated in the glutamine and glutamate pools could then be calculated, by multiplying the plasma enrichment with the body pool size, as follows:
% 13C incorporated in glutamine =
(TTRglutamine,120 × 5·369 × BW)/(F × 120) × 100,
and
% 13C incorporated in glutamate =
(TTRglutamate,120 × 1·247 × BW)/(F × 120) × 100,
where TTRglutamine,120 and TTRglutamate,120 are tracer/tracee ratio of plasma glutamine and glutamate, respectively, at t = 120 min, 5·369 and 1·247 is the size of the glutamine and glutamate pools, respectively (mmol kg-1) (Darmaun et al. 1986), BW is body weight, and F is infusion rate (mmol min-1).
For glucose there is inconsistency about the true volume of distribution. Therefore, a minimal and maximal estimation of the amount of infused 13C incorporated in the glucose pools was calculated as follows:
% 13C incorporated in glucose =
(TTRglucose,120 × 40 × Concglucose × BW)/(F × 120) × 100,
and
% 13C incorporated in glucose =
(TTRglucose,120 × 200 × Concglucose × BW)/(F × 120) × 100,
where TTRglucose,120 is tracer/tracee ratio of plasma glucose at t = 120 min, 40 and 200 are the minimum and maximum volume of distribution of glucose (ml kg-1), Concglucose is the average glucose concentration (t = 90-120 min, mmol l-1), BW is body weight and F is infusion rate (mmol min-1).
Statistical analysis
To examine if there were individual differences in increase in acetate recovery factor, linear regression lines were determined for the period of t = 60 to t = 120 min and slopes and intercepts were tested using analysis of covariance (ANCOVA). The coefficient of variance (CV) was calculated as S.D./mean × 100. All data are presented as means ± S.E.M. and P < 0·05 is considered as significance level.
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RESULTS |
Breath 13CO2 tracer/tracee ratio gradually increased during the infusion period both when infusing palmitate and when infusing acetate (Fig. 2A). No plateau was reached at the end of the 2 h infusion period.
Fractional label recovery (as a percentage) in breath CO2 of [U-13C]palmitate increased in a linear way (slope, 0·056 % min-1; r2, 0·99) from 5·1 ± 0·2 % at t = 60 min to 8·4 ± 0·4 % at t = 120 min. Fractional label recovery in breath CO2 from [1,2-13C]acetate also increased in a linear way (slope, 0·20 % min-1; r2, 0·99) during the infusion period from 14·1 ± 0·6 % at t = 60 min to 26·5 ± 0·5 % at t = 120 min (Fig. 2B). With acetate, there were no significant differences in slopes of the linear regression lines between subjects, but the intercept was significantly different between subjects (P < 0·05).
Percentage difference (percentage of maximal value) in the fractional recovery in breath CO2 from [1,2-13C]acetate between subjects ranged from 15·1 to 32·9 % at different time points. This resulted in a mean intersubject coefficient of variation of 8·3 ± 0·6 %.
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Figure 2
Breath 13CO2 tracer/tracee ratio (A) and fractional label recovery (%) in breath CO2 (B) when infusing either [1,2-13C]acetate or [U-13C]palmitate.
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Reproducibility
In six subjects, the determination of the acetate recovery factor was repeated after 2-4 weeks. There were no significant differences in fractional acetate recovery between the first and second test at any time point. Intraclass correlation coefficient between test 1 and test 2 was 0·985 (r2 = 0·97, P < 0·0001). Percentage difference (percentage of maximal value) in the fractional recovery in breath CO2 from [1,2-13C]acetate between two tests in the same subject ranged from 0·1 to 14·0 %, with an average of 5·4 %. Mean intrasubject coefficient of variation over the whole infusion period was 4·0 ± 1·5 %.
Plasma substrate tracer/tracee ratio
Plasma palmitate tracer/tracee ratio reached a plateau after 30 min of rest (Fig. 4A) but 13CO2 production, corrected for recovery, reached a plateau during the last hour of infusion (Fig. 3). Therefore, substrate kinetics were averaged over the last hour. Plasma FFA concentration was 416·8 ± 26·4 µmol l-1 and plasma palmitate concentration was 100·5 ± 8·1 µmol l-1 (24 ± 1 % of FFA).
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Figure 3
Breath 13CO2 production corrected for recovery when infusing either [1,2-13C]acetate or [U-13C]palmitate.
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Isotopically determined plasma FFA oxidation (Fig. 4B), rate of appearance (Ra) and percentage of rate of appearance oxidized are given in Table 1, both with and without using the acetate correction factor. Using the acetate correction factor, Ra of FFA was 7·32 ± 0·31 µmol kg-1 min-1. Plasma FFA oxidation was 2·43 ± 0·17 µmol kg-1 min-1 (= 33·1 ± 1·6 % of Ra).
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Figure 4
13C-palmitate tracer/tracee ratio (A) and plasma FFA oxidation calculated using acetate correction factor (B) when infusing [U-13C]palmitate.
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Table 1. Plasma (tracers) and total (indirect calorimetry) fatty acid kinetics for the last hour of [U-13C]palmitate infusion
| Plasma fatty acid oxidation (µmol kg-1 min-1) | Ra (µmol kg-1 min-1) | Percentage Ra oxidized (%) | Total fatty acid oxidation (µmol kg-1 min-1) |
| No correction | 0·56 ± 0·05 | 7·32 ± 0·31 | 7·6 ± 0·4 | 3·83 ± 0·28 |
| Acetate correction | 2·43 ± 0·17 | 7·32 ± 0·31 | 33·1 ± 1·6 | 3·83 ± 0·28 |
Values are means ± S.E.M. for 8 volunteers.
Plasma glutamine concentration was 539 ± 13 and 531 ± 14 µmol l-1 when given palmitate and acetate, respectively. Glutamine tracer/tracee ratio gradually increased during both palmitate and acetate infusion and did not reach a plateau after the 120 min of rest. The TTR at t = 120 min was 0·000101 and 0·000544 after palmitate and acetate infusion, respectively (Fig. 5A).
Plasma glutamate concentration was 83 ± 9 and 76 ± 6 µmol l-1 after palmitate and acetate infusion, respectively. Glutamate tracer/tracee ratio gradually increased during both palmitate and acetate infusion and did not reach a plateau after the 120 min of rest. The TTR at t = 120 min was 0·000386 and 0·001946 after palmitate and acetate infusion, respectively (Fig. 5B).
Plasma glucose concentration was 4·9 ± 0·1 and 5·1 ± 0·1 mmol l-1 after palmitate and acetate infusion, respectively. Glucose tracer/tracee ratio gradually increased during both palmitate and acetate infusion and did not reach a plateau after 120 min of rest. The TTR at t = 120 min was 0·0000214 ± 0·0000017 and 0·0000928 ± 0·0000084 after palmitate and acetate infusion, respectively (Fig. 5C).
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Figure 5
13C glutamine tracer/tracee ratio (A), 13C glutamate tracer/tracee ratio (B) and 13C glucose tracer/tracee ratio (C) when infusing either [1,2-13C]acetate or [U-13C]palmitate.
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DISCUSSION |
The results of the present study demonstrate that the acetate recovery factor can be used to correct palmitate oxidation rates in infusion studies of 2 h or less. However, to correct palmitate oxidation rates, the acetate recovery factor needs to be determined for every time point, because the fractional label recovery in breath CO2 of both [U-13C]palmitate and [1,2-13C]acetate increased in a linear way.
The present study demonstrates a good reproducibility for the acetate recovery factor. Intrasubject coefficient of variation was 4·0 %. However, this does not guarantee that in any given individual the acetate recovery factor can be determined in a separate experiment. Therefore the range in percentage difference in individual subjects between the two tests at different time points is more important. The maximum difference found within one subject between the two tests at any time point was 14·0 %. The mean difference between the two tests was 5·4 %. These results show that it is valid to estimate the fractional recovery in a separate experiment, making it possible to use 13C-labelled acetate to determine the correction factor needed to correct the plasma FFA oxidation data.
To study whether it is necessary to determine the acetate correction factor in each individual the intersubject coefficient of variance was calculated. The intersubject coefficient of variance was 8·3 ± 0·6 %, with a percentage difference between subjects ranging from 15·1 to 32·9 % at different time points. This means that an individual estimation of the recovery of 13C-acetate is needed. Using one average recovery factor for a group as a whole can lead to over- or underestimation of fatty acid oxidation of more than 30 % in individual subjects.
One possible pathway for label fixation is the loss of 13C from the TCA cycle in the form of glutamate and glutamine. The label is then stored in the glutamine and glutamate pools, which are in equilibrium with plasma glutamine and glutamate. We found a gradual increase in both glutamine and glutamate tracer/tracee ratio in plasma. This implies that the tracer/tracee ratio of the intracellular glutamine and glutamate pools will also gradually increase. Once the intracellular glutamine and glutamate pools increase in tracer/tracee ratio, more tracer will be recovered after oxidation of these pools. Indeed, in our experiment, we did not reach a steady state in breath 13CO2 tracer/tracee ratio at the end of the 2 h infusion period. 13CO2 tracer/tracee ratio increased in a linear way. As long as the glutamine/glutamate tracer/tracee ratio has not reached a plateau, the amount of recycling of 13C will increase and therefore breath 13CO2 tracer/tracee ratio will increase. Considering the large glutamine/glutamate pools in muscle, it is not to be expected that during a 2 h infusion period an isotopic steady state can be derived. Furthermore, Van Acker et al. (1996) showed that no isotopic steady state in both plasma and muscle glutamine tracer/tracee ratio was obtained after 11 h of infusion of labelled glutamine. Wolfe & Jahoor (1990) also showed a gradually increasing 13CO2 production for a period of 4 h. In contrast, Sidossis et al. (1995a) reported an apparent plateau in breath 13CO2 tracer/tracee ratio at the end of 3 h of infusion at rest. However, they also reported an increase in glutamine tracer/tracee ratio which had not reached a plateau at the end of the infusion period. The extracellular glutamine pool has been estimated to be 110 µmol kg-1, the intracellular muscle glutamine pool to be 5200 µmol kg-1 and the intracellular liver glutamine pool to be 39 µmol kg-1 (Darmaun et al. 1986). Using these figures, and assuming that the plasma glutamine tracer/tracee ratio at the end of the 2 h infusion period is in equilibrium with the muscle glutamine tracer/tracee ratio, it can be estimated that 12·4 ± 0·8 % of infused 13C was incorporated in the glutamine pools after 2 h of [1,2-13C]acetate infusion. The same estimation can be performed for the glutamate pools, considering an extracellular glutamate pool of 7 µmol kg-1, an intracellular liver free glutamate pool of 40 µmol kg-1 and intracellular muscle glutamate pool of 1200 µmol kg-1 (Darmaun et al. 1986), resulting in 10·3 ± 0·9 % of infused 13C incorporated in the glutamate pools. It should be kept in mind that these calculations reflect the minimal amount of tracer that is incorporated in the glutamine and glutamate pools as it may well be possible that after 2 h of infusion the 13C tracer/tracee ratio of glutamine and glutamate at the endogenous production site is higher than the plasma tracer/tracee ratio. Thus the loss of 13C from the TCA cycle in the form of glutamate and glutamine contributes significantly to the low recovery of acetate at rest.
Another possible pathway for label fixation is the loss of 13C from the TCA cycle to glucose in gluconeogenic pathways. In our experiment the glucose tracer/tracee ratio gradually increased. When only the plasma volume (40 ml kg-1) is considered as the volume of distribution for glucose, it can be calculated that 0·06 ± 0·09 % of infused 13C is incorporated in the glucose pools. When the total extracellular fluid is assumed to be 0·2 l kg-1 (Darmaun et al. 1986) these figures become 0·32 ± 0·04 %. There is some discrepancy about the true distribution volume of glucose, but the true value will be somewhere between these two figures. Thus it can be concluded that the loss of 13C to glucose is negligible. This also means that gluconeogenesis contributes only little to label fixation and forms a minor component of the acetate recovery factor in post-absorptive subjects in resting conditions. Label fixation in glutamine and glutamate from a quantitative point of view is more important.
In our experiment, recovery of uniformly labelled acetate was ±27 % at the end of the 2 h infusion period. These figures are far less than the results obtained by Sidossis et al. (1995a). They found an acetate recovery of 56 % at the end of a 3 h infusion period at rest. In our experiment, the recovery increased in a linear way and extrapolation to 3 h results in an acetate recovery of 38 %. This is still less then the recovery found in the study of Sidossis et al. (1995a). However, Sidossis et al. (1995a) used 14C-acetate, which was labelled in the (C-1) position, while in our experiment acetate was uniformly labelled (C-1 and C-2). The effect of the position of the labelled carbon atom on percentage recovered label was studied by Wolfe & Jahoor (1990). After a 4 h infusion period 81 % of infused [1-13C]acetate was recovered, while only 53 % of [2-13C]acetate was recovered. The difference in tracer position therefore may explain a major part of the difference in acetate recovery found between our study and the study of Sidossis et al. (1995a).
The acetate correction factor had to be determined using uniformly labelled acetate because it was used to correct the oxidation rate of [U-13C]palmitate. A major reason for use of [U-13C]palmitate is that only one-sixteenth of the dose of [1-13C]palmitate tracer is required to get an equivalent amount of 13CO2 production. A major practical limitation with the use of palmitate tracers is the low solubility in aqueous solutions. For this reason palmitate is infused bound to human albumin. In case of the [1-13C]palmitate tracer the amount of albumin infused is so large that infusion periods maximally may last 1-2 h (to prevent unacceptable osmotic shifts in the plasma). This period can be substantially lengthened when a [U-13C]palmitate tracer is used. The amount of fixation of tracer in TCA cycle products will be larger with [U-13C]palmitate than with [1-13C]palmitate but can be corrected by measuring the breath 13CO2 recovery from [1,2-13C]acetate.
The impact of the acetate correction factor is shown in Table 1. In this table plasma FFA oxidation is shown with and without applying an acetate correction factor. In our study, without the correction factor plasma FFA oxidation at rest is underestimated 4-fold. Using the acetate correction factor, the calculated plasma fatty acid oxidation was approximately 66 % of total fatty acid oxidation. The contribution of other fatty acids than those derived from plasma to energy expenditure in the resting state is controversial. In their study, Sidossis et al. (1995b) suggest that, at rest, nearly all fatty acids used for oxidation are plasma-borne fatty acids. However in another study by Sidossis et al. (1996) plasma fatty acid oxidation is only responsible for 80 % of total FFA oxidation, leaving 20 % for other tissues (intramuscular oxidation, liver oxidation or very-low-density lipoprotein (VLDL) oxidation).
The need to correct oxidation for the recovery of infused label not only concerns fat metabolism. Because loss of label occurs in the TCA cycle, the fraction of label which is recovered should also be determined when studying carbohydrate and protein metabolism. Recently, it was shown that during glucose infusion exogenous glucose oxidation (determined by tracer) accounted for all glucose oxidation (determined by indirect calorimetry) when the acetate recovery factor was applied, whereas when only using a bicarbonate correction factor (i.e. 0·8; Bonadonna et al. 1990) endogenous glycogen oxidation contributed substantially to total glucose oxidation (Tounian et al. 1996). These studies indicate the importance of the recovery factor in studying oxidation rates of 13C-enriched substrates especially at rest.
In conclusion, the [1,2-13C]acetate recovery factor can be used for correcting the rate of [U-13C]palmitate oxidation in infusing studies of 2 h in resting conditions. Intrasubject coefficient of variation was small, which makes it possible to determine the acetate recovery factor in a second separate experiment using [13C]acetate in one experiment and [13C]palmitate in the other. However, the acetate recovery factor should be determined in each individual and for every time point, because the acetate recovery increases with time. From a quantitative point of view accumulation of tracer carbon in glutamine and glutamate seems much more important to explain the label fixation than accumulation in glucose released by the liver. Failure to correct for the low acetate recovery under these conditions leads to dramatic underestimation of the oxidation rate of plasma fatty acid tracers. Therefore, most previous estimates of plasma fatty acid oxidation published in the literature are major underestimations.
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[Abstract] |
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Corresponding author
P. Schrauwen: Department of Human Biology, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands.
Email: P.Schrauwen{at}HB.Unimaas.NL
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Introduction
