The Journal of Nutrition Vol. 128 No. 1 January 1998, pp. 111-115

Whole-Body, Peripheral and Intestinal Endogenous Acetate Turnover in Dogs Using Stable Isotopes^1,2

Etienne Pouteau^*, , Henri Dumon, Patrick Nguyen, Dominique Darmaun^*, Martine Champ^*, and Michel Krempf^{*, 3}

^* Human Nutrition Research Center, Metabolism Division, CHU, Nantes, France and  Laboratory of Nutrition and Alimentation, Ecole Nationale Vétérinaire de Nantes, Nantes, France

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Acetate metabolism supplies about 10% of energy requirements in food-deprived nonruminant animals. This study used a stable isotope dilution method to investigate the fate of acetate in 24-h food-deprived dogs free of colonic fermentation. Three dogs received intravenous bolus injections of 40 or 70 µmol/kg of [1-¹³C] acetate, and carotid blood was then sampled during a 15-min period to estimate the acetate distribution volume. Ten dogs received intravenous [1-¹³C] acetate infusions of 1.05 ± 0.02 or 2.10 ± 0.10 µmol/(kg·min) for 120 or 200 min after a prime of 200 or 70 µmol/kg, respectively. Cephalic venous and carotid arterial blood were sampled for all dogs, and portal blood for five. Acetate distribution volume was 0.27 ± 0.16 L/kg (mean ± SEM). The concentrations of acetate in arterial (144 ± 17 µmol/L), venous (155 ± 20 µmol/L) and portal plasma (131 ± 16 µmol/L) were not significantly different during infusion, whereas isotopic enrichments [mole percent excess (MPE): labeled acetate/all acetate molecules] in portal (1.2 ± 0.2 MPE) and venous plasma (1.7 ± 0.3 and 2.6 ± 0.7 MPE) were lower than in arterial plasma for both infusion rates (4.9 ± 0.6 and 7.6 ± 0.8 MPE, respectively, P < 0.005). Whole-body acetate turnover was 24.4 ± 2.4 µmol/(kg·min). Fractional acetate extractions for forelimb and intestine were 62 ± 7 and 72 ± 6%, respectively, and the production for each organ was 0.3 and 1.1 µmol/(kg·min) respectively, similar to that of utilization (P > 0.05). It is concluded that the forelimb and intestine produce and utilize acetate as an energy source in 24-h food-deprived dogs free of colonic fermentation.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Acetate is the main exogenous short-chain fatty acid produced by bacterial fermentation of nondigestible carbohydrate in the forestomach of ruminants and the hindgut of nonruminants (Bergman 1990). Several endogenous tissues are also capable of producing and utilizing acetate. This component can supply up to 35% of daily energy requirements in ruminants (Bergman 1990), 8-10% in humans (Pouteau et al. 1996, Skutches et al. 1979) and 25% in pigs (Bergman 1990).

An acetate-enriched solution was found to produce a trophic effect on intestinal mucosa in rats undergoing total parenteral nutrition, suggesting that acetate energy is supplied to the gut (De Michele and Karlstad 1995). However, few studies have considered acetate utilization and production in the intestine itself. A single study in dogs (Bleiberg et al. 1992) showed that infused ¹⁴C acetate can be released and taken up by the intestine in the postabsorptive state. However, because these authors did not evaluate colonic fermentation by an expired hydrogen and methane breath test, the study did not clarify whether acetate was produced by colonic fermentation or gut cells.

The purpose of this study was to assess the acetate turnover rate in dogs in the specific state of 24-h food deprivation and absence of colonic fermentation (negative breath test). The contribution of the intestine and peripheral tissue to the whole-body acetate turnover rate was evaluated by using intravenous [1-¹³C] acetate infusions. To determine the fractional turnover of acetate from the forelimb and intestine, acetate distribution volume was actually measured instead of being estimated as total extracellular fluid volume (Bleiberg et al. 1992). Our main finding was that acetate utilization and production in 24-h food-deprived dogs were quite comparable in the tissues explored.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals.  Thirteen adult dogs (11-23 kg) of both sexes (12 beagles and 1 mongrel) were supplied by the kennels of the National Veterinary School of Nantes after approval of our protocols by this institution's animal ethics committee. Three dogs were used to assess acetate distribution volume, and 10 were subjected to [1-¹³C] acetate infusion to measure acetate production and utilization in the forelimb. Five of these 10 dogs were further investigated to evaluate acetate production and utilization in intestine free of colonic bacterial fermentation. All dogs received permanent vascular access systems (DistriCath implantable infusion system, Districlass, St. Etienne, France) in the carotid artery under total anesthesia 4 d before the start of the experiment; five dogs received a second system in the portal vein. Dogs were trained for handling operations and calmed for 30 min before the start of the protocol. Two additional catheters (20 gauge, Vigon, Paris, France) were inserted into the cephalic vein of both forelimbs of each dog, one for infusion of tracer [1-¹³C] acetate and the other for collection of venous blood.

Experimental design.  To avoid any interference of endogenous acetate metabolism with exogenous acetate production from bacterial colonic fermentation of carbohydrate, the dogs were fed beef meat and no carbohydrate for 3 d before the start of the protocol. The study was then conducted in the morning after 24-h food deprivation. The hydrogen and methane breath test was performed before protocol 2 to confirm the absence of colonic fermentation.

Protocol 1: Measurement of acetate distribution volume. Three dogs received an intravenous bolus injection of 40 or 70 µmol/kg of [1-¹³C] acetate (99% ¹³C enrichment, Tracer Technologies, Somerville, MA) through a catheter into the forelimb vein. Blood samples (3 mL) were taken from the arterial catheter at regular intervals for 15 min (every 15 s for the first 4 min, and then at 4.5 and 5 min, and every 2 min thereafter). The blood was centrifuged at 3000 × g for 15 min, and plasma was stored at 80°C until analysis.

Protocol 2: Measurement of acetate production and utilization. An intravenous infusion of [1-¹³C] acetate was started in 10 dogs at a rate of 1.05 ± 0.02 or 2.10 ± 0.10 µmol/(kg·min) for 120 or 200 min after a prime of 200 or 70 µmol/kg, respectively. Blood samples (3 mL) were collected at regular intervals from 60 min to the end of the infusion from the opposite cephalic vein (4 and 8 samples, respectively) and carotid artery (6 and 10 samples) for all dogs, and from the portal vein (6 samples) for five dogs. The total blood volume taken was about 50 mL, representing <5% of the dog's total blood volume. The collected blood was centrifuged at 2200 g for 5 min at 4°C, and plasma was stored at 80°C until analysis.

Analytic procedures.  Acetate analysis. Plasma acetate enrichment was analyzed with the use of our previously published method (Simoneau et al. 1994). To measure plasma acetate concentration, [D₃] acetate (99% ¹³C enrichment, Tracer Technologies) was added (8 µL, 2.35 mmol/L), as an internal standard to plasma samples (500 µL) before processing. Briefly, plasma samples (500 µL) were deproteinized with 10 mg of sulfosalicylic acid (Sigma, St Quentin Fallavier, France) and centrifuged at 2200 × g for 10 min. The supernatant was then transferred into a vial containing 30 µL of HCl (10 mol/L) (Panreac, Barcelona, Spain), and 3 mL of diethyl ether (Merck, Darmstadt, Germany) was added. The vial was vortexed for 15 min and centrifuged for 10 min at 1200 × g for extraction. Eight microliters of tert-butyl-dimethyl-silyl-imidazole (Fluka Chemika, Buchs, Switzerland) was added to the separated organic phase. The sealed vial was then heated at 60°C for 30 min before the sample was cooled down and evaporated to 500 µL.

Two microliters were injected into a gas chromatograph (model 5890A, Hewlett Packard, Palo Alto, CA) connected to a quadrupole mass spectrometer (5971A, Hewlett Packard). Chromatographic separations were conducted on a 30 m × 0.25 mm capillary column (DB5, J/W Scientific, Folsom, CA). The temperature program was as follows: initial temperature 80°C for 1 min and then 10°C/min up to 180°C. Methane chemical ionization and selected ion monitoring were used on ions at m/z 175, 176 and 178, representing natural, [1-¹³C] and [D₃] acetate, respectively. Calibration curves for isotopic enrichments were obtained from known isotopic enrichment solutions in the range of 0.5-15% for [1-¹³C] acetate. Within (n = 5) and between (n = 5) assay coefficients of variation were both 3%. Calibration for acetate concentration was obtained from basal values to 390 µmol/L. The within (n = 5) and between (n = 5) assay coefficients of variation were 4 and 9%, respectively.

Hydrogen and methane analysis. Hydrogen and methane concentrations in breath samples were measured on a Microlyzer DP gas chromatogaph (Quintron Instruments, Milwaukee, WI).

Calculation methods.  Distribution volume. The concentration of [1-¹³C] acetate in plasma was calculated at each sampling point as the total acetate concentration multiplied by isotopic enrichment. The distribution volume (DV in L/kg) of acetate was calculated from the fit of an exponential decay of [1-¹³C] acetate concentrations in plasma by dividing the dose of tracer injected by the extrapolated [1-¹³C] acetate concentration at time zero (SAAM II, SAAM Institute, Washington, DC), as previously reported (Beylot et al. 1987).

Rate of appearance. The total rate of appearance [Ra in µmol/(kg·min)] of acetate was calculated according to the steady-state equation: where i is the infusion rate [µmol/(kg·min)] and Et and Epa the isotopic enrichment (labeled acetate/all acetate molecules) of the tracer solution ([1-¹³C] acetate) and of arterial plasma, respectively, expressed in mole percent excess (MPE).

Fractional turnover. The systemic fractional turnover (%Turn in %/min) was calculated as follows: where Ca is the arterial concentration of acetate (µmol/L).

Metabolic clearance rate. The metabolic clearance rate [mL/(kg·min)] was calculated according to the following equation:

Fractional extraction. The fractional extraction (%extrac in %) was as follows: where Cv (µmol/L) is the concentration and Epv the isotopic enrichment of acetate in the vein.

Uptake and release. Acetate utilization and production were calculated as follows: where uptake and release are expressed in µmol/(kg·min), and plasma flow is estimated from blood flow values in the literature (Bleiberg et al. 1992) and hematocrits of dogs (46%). Blood flow from the limb was 5.1 mL/(kg·min), and intestinal blood flow was 21.7 mL/(kg·min).

Statistics.  Results are reported as means ± SEM (n as indicated). Comparisons were done with the Instat statistical software package (GraphPad, San Diego, CA). Differences in concentrations and enrichments in venous, arterial and portal plasma were evaluated by ANOVA without a post-test. Unpaired t tests were used to determine the infusion rate effect on enrichment and fractional extraction between the forelimb and intestine. Differences in acetate uptake and release were evaluated by a paired t test, and differences between the forelimb and the intestine with a nonparametric test (Mann-Whitney).

	RESULTS

Abstract Introduction Methods Results Discussion References

Protocol 1.  Isotopic enrichment and plasma [1-¹³C] acetate concentrations decreased rapidly (within 3 min) when labeled acetate was injected intravenously as a bolus (Fig. 1). Arterial acetate concentration increased immediately after bolus injection of the tracer, returning to its basal level within 2 min (Fig. 1). On the basis of this drop in [1-¹³C] acetate concentration, we calculated a mean acetate distribution volume of 0.27 ± 0.16 L/kg, ranging from 0.13 to 0.53 L/kg.

View larger version (23K): [in this window] [in a new window]   Fig 1. Plasma acetate and [1-13C] acetate concentrations, and isotopic enrichment (labeled/all molecules) of plasma acetate in a dog administered 40 µmol/kg of [1-13C] acetate intravenously at time zero. MPE, mole percent excess.

Protocol 2.  The hydrogen level in breath samples was <5 ppm (~0.25 µmol/L) and methane was undetectable in all dogs.

The steady state in isotopic enrichments and acetate concentrations was reached at 60 min upon continuous infusion of [1-¹³C] acetate. As expected, the plateau enrichments (Table 1) for arterial and venous plasma were higher when 2 µmol/(kg·min) was infused compared with 1 µmol/(kg·min) (P < 0.05, unpaired t test). In all cases, arterial enrichments were significantly higher than venous and portal enrichments (P < 0.005, ANOVA), whereas no significant differences were observed in acetate concentrations between these sampling sites (Table 1). The mean arterial rate of acetate, arterial clearance and fractional turnover are reported in Table 2.

Fractional extraction of [1-¹³C] acetate for the forelimb was 62 ± 7% (Table 3). Forelimb acetate uptake did not differ from acetate release, and the contribution of the forelimb to whole-body acetate turnover was 1.1%. Thus, there was no net production or utilization of acetate in the forelimb of 24-h food-deprived dogs.

Fractional extraction for the intestine (Table 3) did not differ significantly from that for the forelimb. Even though acetate uptake and production rates did not differ for the intestine, they were about four times as active as in the forelimb (P < 0.01, unpaired Mann-Whitney test). Although no hydrogen and methane were measured in breath, acetate production was observed from the intestine of food-deprived dogs (24 h), accounting for 4.5% of whole-body acetate turnover. However, no net acetate production was observed from dog intestine because utilization and release were identical (Table 3).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

This study investigated endogenous acetate metabolism in 24-h food-deprived dogs through a combination of [1-¹³C] acetate infusion and measurement of arteriovenous gradients across one forelimb, with the gut free of fermentation. Acetate turnover was found to be about 25 µmol/(kg·min). Intestine and peripheral tissues were both able to utilize and produce acetate with no net production of acetate. The gut was able to produce acetate, even in the absence of colonic fermentation.

The isotopic dilution method with ¹³C has been applied to humans in previous work (Pouteau et al. 1996, Simoneau et al. 1994), but to our knowledge has not been used to assess endogenous acetate metabolism in dogs. Our results show that plasma acetate concentrations were higher than in previous studies in dogs (David et al. 1994, Persson et al. 1991), and that the rate of acetate appearance was about three times as high as when stable isotopes (Pouteau et al. 1996, Simoneau et al. 1994) or radioactive isotopes (Skutches et al. 1979) were used in humans or ¹⁴C acetate in dogs (Bleiberg et al. 1992). Discrepancies in concentration and turnover may depend on the methodologies used or the different nutritional states of the subjects. Dogs in this study were not fed for 24 h, whereas subjects evaluated in other studies were in the postabsorptive state (Bleiberg et al. 1992, Pouteau et al. 1996). Moreover, the dogs used here were smaller (half the weight) and of a different breed (mainly beagles) than those in Bleiberg's work.

Acetate metabolic clearance was rapid because most molecules in the pool were removed each minute. Clearance and fractional turnover showed large interindividual variations. To estimate fractional turnover, we measured distribution volume (mean ~0.27 L/kg), which gave a wide range of values because of the rapid metabolic clearance of the tracer; this raised major difficulties for extrapolation of tracer concentration at time zero from the exponential fit. Other authors have assumed a distribution volume of 0.20 L/kg as corresponding to the extracellular volume (Bleiberg et al. 1992). We found a close but higher value than that for extracellular volume, probably because the acetate molecule is readily diffused throughout the body (Ballard 1972).

Previous studies showed that different tissues use and produce acetate (Ballard 1972, Knowles et al. 1974), whereas this study indicates that no net acetate production occurred in the forelimb, which provided only 1% of the whole-body acetate turnover rate in the food-deprived state (24 h). Acetate production may be due to lipolysis because an early study showed radiolabel transfer from lipids to acetate in adipose tissues of the human forearm (Hagenfeldt and Wahren 1971). The presence of acetyl-CoA hydrolase in peripheral tissues (Ballard 1972, Knowles et al. 1974) suggests that acetyl-CoA from intermediate metabolism could convert to acetate released into the bloodstream. However, the enzyme acetyl-CoA synthetase found in most tissues could also induce acetate consumption (Knowles et al. 1974). The difference between the uptake and release of acetate may be related to variations in these enzyme activities (Ballard 1972, Knowles et al. 1974), which are probably dependent on the nutritional state of individual dogs, fat content of peripheral tissues and the rate of lipolysis. This would explain the large interindividual range of the ratio of acetate uptake to release in forelimb, from 0.2 to 1.5. Nevertheless, in the 24-h food-deprived state, the peripheral tissues showed no difference between mean uptake and release. A previous study in humans in the postabsorptive state (Pouteau et al. 1996) showed a net uptake of acetate by hand muscles but with no production, and a study of dogs in the postabsorptive state revealed that acetate utilization by total skeletal muscle (7% of acetate turnover) was greater than acetate production (5% of acetate turnover) (Bleiberg et al. 1992). The overall contribution of peripheral tissues would appear to be ~5% in 24-h food-deprived dogs in our study, which suggests that other organs are involved in whole-body acetate turnover.

In the 24-h food-deprived state, dog intestine retained acetate at a mean rate similar to that of its release, although the interindividual ratio of acetate uptake to release ranged from 0.6 to 1.2. This ratio difference was less important in intestine than in forelimb probably due to the faster effect of the 24-h food-deprived state on intestine than on peripheral tissues of the different dogs. In a previous study, acetate production from the intestine was twice as high as utilization (~9 vs. ~5% of acetate turnover) in dogs in the postabsorptive state (Bleiberg et al. 1992). Because the authors of that study did not perform a methane and hydrogen breath test to assess colonic bacterial fermentation, this net production could have been due to residual fermentation of complex carbohydrate. To reduce exogenous acetate production, our dogs were fed a diet composed exclusively of meat 3 d before the protocol. Because the hydrogen and methane breath test revealed no colonic activity of hydrogen or methane bacteria, we assumed that no fermentation or significant acetate production occurred in the gut lumen (Livesey 1995, Rumessen 1992).

Acetate from the intestine represented 4.5% of whole-body production in this study. Its release was probably not due to colonic fermentation of residual substrates such as nonabsorbed bile acids and mucus (Stephen et al. 1981) because no breath methane and hydrogen were detected. Other sources may in fact contribute to gut acetate production in the 24-h food-deprived state. Intestinal cells could release acetate into portal blood, although there are no in vitro data to support this assumption; fat in mesenteric adipose tissue could produce acetate from lipolysis and activation of acetyl-CoA hydrolase, which may be present as shown in many other tissues in mammals (Knowles et al. 1974). Thus, acetate released from the intestine could have an endogenous origin contributing to the whole-body acetate supply in the food-deprived state.

Acetate consumption was also observed in the gut. This metabolite could be activated into acetyl-CoA and later involved in free fatty acid synthesis for the building of epithelial membrane, or it could enter mitochondria and yield ketone bodies or provide energy (Livesey and Elia 1995). Rapid energy supply from acetate turnover was observed in a previous human study (Pouteau et al. 1994), suggesting that acetate could have been consumed in part by the gut. Acetate uptake by gut cells would provide further support for studies in rats on parenteral nutrition enriched in triacetin (Koruda et al. 1990), which showed trophic effects on mucosa indicative of triacetin hydrolysis and acetate utilization by the epithelium for lipid synthesis or energy supply. This trophic effect may also account for higher acetate utilization by the gut compared with the forelimb.

The forelimb and intestine showed possible acetate production and utilization in our study. The contribution of these tissues to the relatively large acetate turnover [25 µmol/(kg·min)] was about 9%, which is comparable to the ~12% contribution for skeletal muscle plus intestine reported in a previous study (Bleiberg et al. 1992). When other tissues such as the liver and kidneys were taken into consideration, these authors could account for only ~26% of acetate turnover in the postabsorptive state (Bleiberg et al. 1992). When the tracer was infused with a large amount of triacetin, these tissues accounted for 73% of acetate utilization (Bleiberg et al. 1993). Other organs may be involved in acetate metabolism because heart and brain are also capable of using and releasing acetate (Ballard 1972, Knowles et al. 1974).

Whole-body acetate circulation could contribute in part to energy supply in dogs. The recommended daily energy requirement for dogs is 552 kJ·kg BW^0.75 of metabolizable energy (Sheffy et al. 1985). If it is assumed that at least 70% of acetate turnover is oxidized (Pouteau et al. 1996), yielding 879 kJ/mol (Chioléro et al. 1993), whole-body acetate turnover could contribute ~8% of energy supply during the 24-h food-deprived state. Studies in humans (Pouteau et al. 1996, Skutches et al. 1979) have indicated that acetate is readily distributed to the whole body, providing rapid energy to many tissues. The utilization of acetate by the intestine in this study may be relevant to the issue of nutritional support for intestinal diseases (Koruda et al. 1990). Current data further suggest that short-chain triglyceride solutions such as triacetin may be useful parenteral nutrients in the intestine and whole body for clinical purposes in humans (Bleiberg et al. 1993, De Michele and Karlstad 1995).

	ACKNOWLEDGMENTS

We thank Coraline Berthelot for preparation of acetate samples and Sylvain Dufour and Philippe Bleis for sample analysis and work on the experimental protocol.

	FOOTNOTES

Manuscript received 7 February 1997. Initial reviews completed 19 March 1997. Revision accepted 4 September 1997.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• Ballard F. J. Supply and utilization of acetate in mammals. Am. J. Clin. Nutr. 1972; 25:773-779 [Medline]
• Bergman E. N. Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Physiol. Rev. 1990; 70:567-590 [Abstract/Free Full Text]
• Beylot M., Martin C., Beaufrère B., Riou J. P., Mornex R. Determination of steady state and nonsteady-state glycerol kinetics in humans using deuterium-labeled tracer. J. Lipid Res. 1987; 28:414-422 [Abstract]
• Bleiberg B., Beers T. R., Persson M., Miles J. M. Systemic and regional acetate kinetics in dogs. Am. J. Physiol. 1992; 262:E197-E202 [Abstract/Free Full Text]
• Bleiberg B., Beers T. R., Persson M., Miles J. M. Metabolism of triacetin-derived acetate in dogs. Am. J. Clin. Nutr. 1993; 58:908-911 [Abstract/Free Full Text]
• Chioléro R., Mavrocordatos P., Burnier P., Cayeux M. C., Schindler C., Jéquier E., Tappy L. Effects of infused sodium acetate, sodium lactate, and sodium B-hydroxybutyrate on energy expenditure and substrate oxidation rates in lean humans. Am. J. Clin. Nutr. 1993; 58:608-613 [Abstract/Free Full Text]
• David F., Beylot M., Reider M. W., Anderson V. E., Brunengraber H. Assay of the concentration and ¹³C enrichment of acetate and acetyl-CoA by chromatography-mass spectrometry. Anal. Biochem. 1994; 218:143-148 [Medline]
• De Michele, S. J. & Karlstad, M. D. (1995) Short-chain triglycerides in clinical nutrition. In: Physiological and Clinical Aspects of Short-Chain Fatty Acids (Cummings, J., Rombeau, J. L. & Sakata, T., eds.), pp. 537-559. Cambridge University Press, New York, NY.
• Hagenfeldt L., Wahren J. Metabolism of free fatty acids and ketone bodies in skeletal muscle. Adv. Exp. Med. Biol. 1971; 11:153-160
• Knowles S. E., Jarrett I. G., Filsell O. H., Ballard F. J. Production and utilization of acetate in mammals. Biochem. J. 1974; 142:401-411 [Medline]
• Koruda M. J., Rolandelli R. H., Bliss D. Z., Hastings J., Rombeau J. L., Settle R. G. Parenteral nutrition supplemented with short chain fatty acids: effect on the small bowel mucosa in normal rats. Am. J. Clin. Nutr. 1990; 51:685-689 [Abstract/Free Full Text]
• Livesey G. Polyols, breath hydrogen and fermentation revisited. Br. J. Nutr. 1995; 74:867-868 [Medline]
• Livesey, G. & Elia, M. (1995) Short-chain fatty acids as an energy source in the colon: metabolism and clinical implications. In: Physiological and Clinical Aspects of Short-Chain Fatty Acids (Cummings, J., Rombeau, J. L. & Sakata, T., eds.), pp. 427-481. Cambridge University Press, New York, NY.
• Persson M., Bleiberg B., Kiss D., Miles J. M. Measurement of plasma acetate kinetics using high-performance liquid chromatography. Anal. Biochem. 1991; 198:149-153 [Medline]
• Pouteau E., Piloquet H., Maugeais P., Champ M., Dumon H., Nguyen P., Krempf M. Kinetic aspects of acetate metabolism in healthy humans using [1-13^C] acetate. Am. J. Physiol. 1996; 271:E58-E64 [Abstract/Free Full Text]
• Rumessen J. J. Hydrogen and methane breath tests for evaluation of resistant carbohydrates. Eur. J. Clin. Nutr. 1992; 46:S77-S90
• Sheffy, B. E., Hayes, K. C., Knapka, J. J., Milner, J. A., Morris, J. G. & Romsos, D. R. (1985) Nutrient Requirements of Dogs. National Academy Press, Washington, DC.
• Simoneau C., Pouteau E., Maugeais P., Marks L., Ranganathan S., Champ M., Krempf M. Measurement of whole body acetate turnover in healthy subjects with stable isotopes. Biol. Mass Spectrum. 1994; 23:430-433
• Skutches C. L., Holroyde C. P., Myers R. N., Paul P., Reichard G. A. Plasma acetate turnover and oxidation. J. Clin. Invest. 1979; 64:708-713
• Stephen, A. M., Haddad, A. C. & Phillips, S. F. (1981) Dietary and endogenous sources of carbohydrate substrate in the human colon. Gut 22: A893.

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