Lactulose ingestion has no effect on plasma acetate in dogs studied with [1-13c] acetate

Apart from its endogenous turnover, acetate is produced mainly by the bacterial fermentation of nondigestible carbohydrate in the hindgut of non-ruminants. Acetate supplies as much as 8-10% of the basal energy expenditure in humans (Pouteau et al. 1996, Skutches et al. 1979). Bacterial colonic fermentation, in addition to its energetic gain, induces metabolic benefits for the host. This has been poorly studied in dogs. Therefore, the aim of this work was to investigate in dogs colonic fermentation of lactulose, a nondigested and entirely fermentable disaccharide, by studying the metabolism of its main product, i.e., acetate. Materials and methods. Animals. Five adult dogs (15.0 [+ or -] 2.7 kg, one mongrel and four beagles) 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. A permanent vascular access system with a subcutaneous sampling device (implantable infusion system, DistriCath, Districlass, St Etienne, France) was inserted in the carotid artery of each completely anesthetized dog 4 d before the start of the experiment; a second system was placed in the portal vein. Two additional catheters (20 gauge, Vigon, Paris, France) were inserted into the cephalic vein of each forelimb of each dog on the day of the study. Dogs were given a constant [1-13C] acetate infusion through one catheter; the other was used for sampling venous blood. Experimental design. To avoid any interference of endogenous acetate metabolism by exogenous acetate production from the bacterial colonic fermentation of carbohydrate, the dogs were fed beef meat (S.E.R., Cuiseaux, France, 20% protein, 0% carbohydrate and 15% lipids) plus a vitamin and mineral addition on the basis of 555 kJ/([kg.sup.0.75] [multiplied by] d) of energy requirement for 3 d before starting the protocol. This 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 on the day of the study ([H.sub.2] < 5 ppm, 1 ppm [approximately equal to] 0.05 [micro]mol/L). Protocol. At time 0, the dogs received intravenously a priming dose of 190 [micro]mol/kg of [1-13C] acetate followed by a constant infusion at a rate of 1.06 [+ or -] 0.02 [micro]mol/(kg [multiplied by] min) for 5 h. After 2 h, the dogs were administered orally a bolus of lactulose (10 g diluted in 15 mL of aqueous solution, Duphar, Villeurbanne, France). Blood sampling (3 mL) was performed at regular times, before (t = 1, 1.25, 1.5, 1.75 and 2 h) and after lactulose ingestion (t = 3.5, 4, 4.5 and 5 h), from the opposite cephalic vein, the carotid artery and the portal vein. The total blood volume taken was ~75 mL. The collected blood was centrifuged (3000 x g for 10 min), and plasma was stored at -80 [degrees] C until analysis. Analytical procedures. The analysis of plasma acetate enrichment was performed using our previously published method (Simoneau et al. 1994). In addition, and in order 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. The total rate of appearance [[Ra.sup.4] in [micro]mol/(kg [multiplied by] min) of acetate was calculated according to the equation for steady state: Ra = i [multiplied by] (Et/Epa - 1) where i is the infusion rate [[micro]mol/(kg [multiplied by] min)], and Et and Epa are the isotopic enrichment of the tracer solution ([1-13C] acetate) and of arterial plasma, respectively, expressed in mole percent excess (MPE). Epa was obtained from the difference between the measured isotopic enrichment at the plateau and at time zero. The fractional extraction (%extract) is as follows: %extract = 100 [multiplied by] [(Epa [multiplied by] Ca) - (Epv [multiplied by] Cv)]/(Epa [multiplied by] Ca) where Ca and Cv ([micro]mol/L) are the arterial and venous concentrations, and Epa and Epv are the arterial and venous isotopic enrichments of acetate in the forelimb and the intestine, respectively. Statistics. Results are reported as means [+ or -] SEM. Paired t tests were made with the Instat statistical software package (GraphPad, San Diego, CA). Differences in concentrations and enrichments in venous, arterial and portal plasma were evaluated using an ANOVA test with Instat software. Results. During the basal period, the concentration of acetate was steady throughout the 2 h of infusion of the tracer. Figure 1 shows the mean concentrations measured in the artery, the cephalic vein and the portal vein. No difference was found among the sample sites. The isotopic enrichment, however, rapidly reached a plateau after 1 h of intravenous infusion of the tracer. The isotopic enrichment in the artery was significantly higher than that in venous sites for all dogs (P < 0.05), whereas no significant difference was observed between venous enrichments [ILLUSTRATION FOR FIGURE 1 OMITTED]. The fractional extraction of acetate was 66 [+ or -] 7% and 67 [+ or -] 10% in the intestine and the forelimb tissues, respectively. After lactulose ingestion, the mean acetate concentration was unchanged, and no difference was noticed among the three sample sites [ILLUSTRATION FOR FIGURE 1 OMITTED]. No change was observed in the isotopic enrichment after lactulose ingestion throughout the three last hours of [1-13C] acetate infusion; the arterial isotopic enrichment was still higher than the cephalic and portal venous enrichments (P < 0.05, [ILLUSTRATION FOR FIGURE 1 OMITTED]). The fractional extraction was unchanged in the intestine (64 [+ or -] 8%) and it decreased significantly in forelimb tissues (55 [+ or -] 6%, P < 0.05). All individual and mean whole-body acetate turnover rates are shown in Table 1 before and after lactulose ingestion. Although the absolute value of the acetate turnover rate was higher after lactulose ingestion, no significant difference of acetate turnover was found throughout the study. Discussion. Acetate metabolism in dogs has been poorly investigated, especially with the use of a stable isotope dilution technique. The acetate concentration and whole-body turnover were higher than in a previous study performed in mongrel dogs in the postabsorptive state using radioisotopes (Bleiberg et al. 1992); they were also higher than those measured in humans (Pouteau et al. 1996, Simoneau et al. 1994, Skutches et al. 1979). This discrepancy may be due to different breeds in different nutritional states or to differences in methodology. Because our dogs were food deprived for 24 h, the endogenous production of acetate may have been emphasized, thereby producing a rapid whole-body acetate turnover rate and higher concentrations (Scheppach et al. 1991). Further studies are required to confirm acetate turnover rate in dogs in different nutritional states. Lactulose should have produced exogenous acetate that should have decreased the plasma isotopic enrichment and increased the concentration. In this study, no significant production of acetate from fermentation was observed in dogs that had been food deprived for 24 h. Only a slight, but not significant increase in the whole-body acetate turnover was noticed. The concentration and isotopic enrichment were monitored for 3 h, long enough for the lactulose bolus to reach the cecum as previously observed from hydrogen breath tests (unpublished results). Four hypotheses could explain these observations. Lactulose would not be significantly degraded with acetate production by the colonic microflora of dogs, resulting in no change in whole-body acetate metabolism. The second hypothesis would be that such a bolus of lactulose might have shortened the transit time and the substrate may have then rapidly moved into the distal colon w here the fermentation process is less important than in the cecum, thus yielding only a very limited amount of acetate. The third would be that the absorption of short-chain fatty acids such as acetate might be limited in the colon of dogs (Stevens et al. 1980). The fourth would be that acetate produced by lactulose colonic fermentation would be consumed mainly by the epithelial mucosa. In vitro studies performed on colonic inocula from dogs have shown that lactulose, citrus pectin and other substrates can be degraded by the bacterial activity, subsequently producing acetate (Reinhart et al. 1994, Sunvold et al. 1994). Our present work thus illustrates that in vitro studies cannot reflect accurately the in vivo effects. KEY WORDS: * acetate * flux rate * stable isotopes * lactulose * fermentation * dogs