A kinetic study of acetate metabolism in dogs using [1-13C] acetate

Acetate is the main exogenous short-chain fatty acid produced by the bacterial fermentation of nondigestible carbohydrate in the forestomach of ruminants and in the hindgut of non-ruminants (Bergman 1990). Acetate supplies ~35% of daily energy requirements in ruminants (Bergman 1990), and ~6-10% of the basal energy expenditure in non-ruminants such as humans (Pouteau et al. 1996, Skutches et al. 1979). In dogs, acetate metabolism has not been investigated extensively. The aim of the study was to investigate the whole-body acetate turnover in dogs and its exchange from the forelimb muscle and from the intestine with the use of a stable isotope technique. Materials and methods. Thirteen adult dogs [11-23 kg, 12 beagles and one mongrel (# 10)] of both sexes were supplied by the kennels of the National Veterinary School of Nantes. They were studied according to the French Ministry of Agriculture and Fisheries regulatory rules for animal welfare. All dogs were implanted with permanent vascular subcutaneous access systems (Implantable infusion system, DistriCath, Districlass, St Etienne, France) in the carotid artery; five dogs had a second system implanted in the portal vein. In all dogs, two additional catheters (20 gauge, Vigon, Paris, France) were placed in the cephalic vein of each forelimb. To avoid any interference from the bacterial colonic fermentation of carbohydrate, dogs were fed beef meat with no carbohydrate for 3 d before starting the protocol. The study was conducted in the morning after a 24-h period of food deprivation. The hydrogen breath test (Quintron instrument, Milwaukee, WI) attested to the absence of fermentation ([H.sub.2] < 5 ppm, 1 ppm [approximately equal to] 0.05 [[micro]mol]/L). Protocols. Three dogs received an intravenous bolus injection of [1-13C] acetate (99% 13C enrichment, Tracer Technologies, Somerville, MA) of either 40 or 70 [[micro]mol/kg in the forelimb vein. Blood samples (3 mL) were taken from the arterial catheter at regular intervals (every 15 s during the first 4 min, at 4 min 30 s, 5 min and then every 2 rain to t = 15 min). In 10 dogs, an intravenous infusion of [1-13C] acetate was started at a rate of either 1.05 [+ or -] 0.02 or 2.10 [+ or -] 0.10 [[micro]mol]/(kg [center dot] min) for 120 or 200 min after a prime of 200 or 70 [[micro]mol]/kg, respectively. Blood sampling (3 mL) was also performed, from 60 min to the end of the infusion at regular intervals, from the opposite cephalic vein, from the carotid artery for all dogs and from the portal vein for half of the group. The collected blood was centrifuged and plasma was stored at -80 [degrees] C until analysis. Analytical procedure. The analysis of plasma acetate enrichment was performed according to our previously published method (Simoneau et al. 1994). In addition, to be able to measure plasma acetate concentration, [[D.sub.3]] acetate (99% 13C enrichment, Tracer Technologies, Somerville, MA) was added (8 [[micro]liter], 2.35 mmol/L), as an internal standard to plasma samples (500 [[micro]liter]) before processing. Calculation methods. The volume of distribution (Vd in L/kg) of acetate was calculated as previously described (Beylot et al. 1987) with the use of the software SAAM II (SAAM II, SAAM Institute, Washington, DC). The total rate of appearance [Ra in [[micro]mol]/(kg [center dot] min)] of acetate was calculated according to the equation for steady state: Ra = i [multiply by] (Et/Epa - 1) where i is the infusion rate [[[micro]mol]/(kg [center dot] min)], Et and Epa are the isotopic enrichment of the tracer ([1-13C] acetate) and of arterial plasma, respectively, given in mole percent excess (MPE). The fractional turnover (%Turn in %/min) was calculated as follows: %Turn = 100 [multiplied by] Ra/(Ca [multiplied by] Vd) where Ca is the arterial concentration of acetate ([[micro]mol]/L). The metabolic clearance rate [mL/(kg [center dot] min)] was calculated with the following equation: Clearance = 1000 [multiplied by] Ra/Ca The fractional extraction (%) of tissues was as follows: %Extract = 100[(Epa [multiplied by] Ca) - (Epv [multiplied by] Cv)]/(Epa [multiplied by] Ca) where Cv ([[micro]mol]/L) is the concentration, and Epv is the isotopic enrichment of acetate in vein. The acetate utilization and production of tissues were calculated as follows: Uptake = Extraction [multiplied by] Ca [multiplied by] plasma flow Release = Uptake + (Cv - Ca) [multiplied by] plasma flow where uptake and release are expressed in [[micro]mol]/(kg [center dot] min), and the plasma flow was estimated from literature blood flow values and hematocrits of dogs (46%) (Bleiberg et al. 1992). The blood flow from the limb was 5.1 mL/(kg [center dot] min), and the intestinal blood flow was 21.7 mL/(kg [center dot] min). All results are reported as means [+ or -] SEM. Paired and unpaired t tests and ANOVA were performed with the Instat statistical software package (GraphPad, San Diego, CA). Results. After the intravenous bolus of the tracer, the isotopic enrichments rapidly reached 41 [+ or -] 6 MPE and then decreased to zero within 3 min. The arterial acetate concentration increased just after the bolus injection from 143 [+ or -] 3 to 234 [+ or -] 16 [[micro]mol]/L, and returned to basal level within 3 min, 146 [+ or -] 21 [[micro]mol]/L. The volume of acetate distribution was 0.27 [+ or -] 0.16 L/kg. Steady state was reached at 60 min under continuous infusion of [1-13C] acetate. In all cases, arterial enrichments were significantly higher than venous (P < 0.005) and portal enrichments (P < 0.005), whereas no significant differences were observed in acetate concentrations between those sampling sites (P > 0.05, Table 1). The mean arterial flux rate of acetate was 24.4 [+ or -] 2.4 [[micro]mol]/(kg [center dot] min) and the arterial clearance was 191 [+ or -] 30 mL/(kg [center dot] min), with 71 [+ or -] 11% of the acetate pool being replaced per minute. The mean forelimb fractional extraction of [1-13C] acetate was 62 [+ or -] 7%. Forelimb acetate uptake [0.25 [+ or -] 0.04 [[micro]mol]/(kg [center dot] min)] was not different from acetate release [0.28 [+ or -] 0.04 [[micro]mol]/(kg [center dot] min), P > 0.05]. The mean intestine fractional extraction was not significantly different (72 [+ or -] 6%, P > 0.05) from that of the forelimb. The intestinal acetate uptake and release were not different [1.06 [+ or -] 0.28 and 1.16 [+ or -] 0.24 [[micro]mol]/(kg [center dot] min), respectively, P > 0.05]. Discussion. In this study, acetate metabolism was investigated by using a combination of [1-13C] acetate infusion and measurement of arteriovenous gradients across forelimb and gut of dogs that had been food deprived for 24 h. Acetate turnover was found to be ~25 [[micro]mol]/(kg [center dot] min). Both the intestine and the peripheral tissues were able to utilize and produce acetate. The intestine was able to produce acetate even in the absence of colonic fermentation. The isotopic dilution method using 13C was first developed in humans (Pouteau et al. 1996, Simoneau et al. 1994) but has not been used as yet in dogs. From our study, plasma acetate concentrations in dogs were higher than these found in previous work with mongrel dogs (Persson et al. 1991); acetate turnover was three times higher than that found in humans (Pouteau et al. 1996, Simoneau et al. 1994, Skutches et al. 1979) and also three times higher than that found in a dog study that used 14C acetate (Bleiberg et al. 1992). The discrepancy could be due to different methodologies, different breeds or difference in physiologic state (Persson et al. 1991). Our dogs were half the weight and of a different breed than those of Bleiberg et al. 1992. They were food deprived for 24 h rather than in the post-absorptive state as in previous studies, i.e., acetate metabolism was rapid as illustrated from the high clearance and fractional turnover. The forelimb tissues were able to release and utilize acetate; the exchange rate contributed ~4% of the overall acetate turnover when considering all four legs. From previous studies (Knowles et al. 1974), cells in these tissues contain acetyl-CoA hydrolase and acetyl-CoA synthetase enzymes that are capable of releasing and utilizing acetate, respectively. The intestine was also capable of producing and utilizing acetate at the same rate. In a previous study, the intestinal production of acetate was twice as high as its utilization in dogs fed 12 h before the study (Bleiberg et al. 1992). This net production could be due to residual fermentation of complex carbohydrate because the hydrogen breath test was not performed to assess colonic bacterial fermentation in Bleiberg's study. In this study, the dogs were fed for 3 d a diet composed entirely of meat to avoid exogenous acetate production from carbohydrate fermentation, and the dogs were studied after 24 h of food deprivation. Because the hydrogen breath test did not reveal hydrogeno-bacterial colonic fermentations, we assumed that there was no acetate production from colonic bacterial activity (Livesey 1995). In this study, intestinal acetate production could originate from endogenous sources such as intracellular supply or lipolysis from adipose tissue, thus yielding acetate even in the absence of fermentation. The forelimb and the intestine showed possible production and utilization of acetate. The contribution of those tissues to the relatively large acetate turnover [25 [[micro]mol]/(kg [center dot] min)] was ~9%. Other organs may be involved in acetate metabolism. Because heart and brain are also capable of using and releasing acetate (Knowles et al. 1974), their contribution would probably complete the remaining production sources. The rapid utilization of acetate by the intestine and the forelimb further illustrates the nutritional contribution of acetate, suggesting that this substrate may play a role in energy transport and supply. KEY WORDS: * acetate * turnover * stable isotopes * dogs