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Toscana virus (TOSV), an arthropodborne phlebovirus transmitted by sandflies, can cause febrile illness and meningitis. The vector of TOSV in France was unknown, We detected TOSV RNA in 2 (female [...]

We previously demonstrated improvements of colonic lesions due to dextran sulfate sodium (DSS) in rats after 7 d of supplementation with resistant starch (RS) type 3, a substrate yielding high levels of butyrate ([C.sub.4]), a colonic cell fuel source. In the present study, we hypothesized that if inflammation is related to decreased [C.sub.4] utilization by the colonic mucosa, RS supplementation should restore [C.sub.4] use simultaneously with an increase in the amount of [C.sub.4] present in the digestive tract. Hence, we compared, in vivo, the cecocolonic uptake of [C.sub.4] and its oxidation into C[O.sub.2] and ketone bodies in control and DSS-treated rats fed a fiber-free basal diet (BD) or a RS-supplemented diet. Sprague-Dawley rats (n = 60) were used. DSS treatment was performed to induce acute colitis and then to maintain chronic colitis. After cecal infusion of [[1-.sup.13]C]-C4 (20 [micro]mol in 1 h), concentrations and [sup.13]C-enrichment of [C.sub.4], ketone bodies, and C[O.sub.2] were quantified in the abdominal aorta and portal vein. Portal blood flow was recorded. During acute colitis, [sup.13][C.sub.4] uptake and [sup.13][CO.sub.2] production were lower in DSS rats than in controls. During chronic colitis, DSS rats did not differ from controls. After 7 d of chronic colitis, RS-DSS rats exhibited the same [C.sub.4] uptake as BD-DSS rats in spite of higher [C.sub.4] cecocolonic disposal. After 14 d, [C.sub.4] uptake was higher in RS-DSS than in BD-DSS rats. Thus, the increased utilization of C4 by the mucosa is subsequent to evidence of healing and appears to be a consequence rather than a cause of this RS healing effect. KEY WORDS: * butyrate * metabolism * colitis * rat * resistant starch

Great variations in the postprandial glucose concentration and insulin secretory responses to different foods have been shown in dogs (Holste and al. 1989, Nguyen and al. 1994). It has been suggested that foods yielding low glycemic responses would be recommended for diabetic or obese subjects and in the prevention of many other disorders. High carbohydrate/high fiber diets enhance peripheral glucose disposal and decrease insulin requirements in insulin-dependent diabetic subjects. In overweight patients with noninsulin-dependent diabetes mellitus (NIDDM), reducing diet glycemic response improves overall blood glucose control, long-term glycemic control and lipid control. Diets with a high glycemic response that are low in fiber increase the risk of NIDDM in humans. Foods with a low glycemic response combined with a high dietary fiber content decrease free fatty acid level, which is associated with abdominal obesity and cardiovascular risk. They cause rapid intestinal absorption of glucose into the blood, leading to postprandial hyperinsulinemia, which may play a role in promoting colon carcinogenesis. A diet high in refined carbohydrates and low in water-soluble fiber causes rapid absorption of glucose with similar results. Because of the clinical implications of the glycemic index, notably in diabetes management or in dietary strategy to avoid or treat overweight or moderate obesity, the factors that affect it have been the subject of many studies (Wolever and al. 1991). The extent of postprandial hyperglycemia and insulin secretion depends on the amount of food and carbohydrate consumed per meal. However, different kinds of carbohydrate elicit different glucose and insulin concentrations, because their chemical nature, especially the ratio of amylose to amylopectin forms of starch, may affect their rate and speed of digestion. Dietary fiber slows down the rate of passage and the rate of hydrolysis of starchy polysaccharides (Wolever 1990). Dietary fat delays stomach emptying (Gulliford and al. 1989), and high intakes of rapidly digested proteins modify the glycemic response by increasing insulin secretion (Nuttall and Gannon 1990). The food processing may be of particular importance for dog food. The type of food, dry, canned or soft moist, affects the maximal postprandial glucose concentration as much as the time at which this peak occurs (Holste and al. 1989). The glycemic index methodology is based on tests of single foods and could be applied to the testing of mixed meals. Nevertheless, its practical utility is controversial because differences among foods could be partially abolished in mixed meals by the effects of protein and fat. Whatever it may be, an individual food evaluation is not realistic in dogs. Their complete foods contain many components. There are large variations in their protein and fat content and the technological processes can largely modify the intrinsic carbohydrate availability. Nevertheless, information concerning postprandial responses would be of great interest in regard to obesity. Along with a long-term excessive energy intake, food quality may play a significant role according to its humoral and metabolic effects. This information may also be of interest in the management of NIDDM (which elicits alteration of carbohydrate tolerance and insulin action) as much as insulin-dependent diabetes mellitus (IDDM; reduction of fluctuations in blood glucose, synchronization of glucose increase and insulin administration). The purpose of this study was to determine how the differences in carbohydrate (starch and dietary fiber, soluble and insoluble), protein and fat content of complete (and complex) foods given to healthy dogs in a single meal on a normoenergetic basis modify their postprandial plasma glucose and insulin responses. Material and methods. Animals. Twelve adult (older than 15 mo) beagle dogs were studied, according to the French Ministry of Agriculture and Fisheries regulatory rules for animal welfare. They were allotted to two groups; from each group, five dogs were used alternately for tests, excluding in particular dogs that did not eat the entire meal that they were offered. None of the dogs was obese (13.5 kg mean body weight) and they were clinically normal. Their basal plasma glucose (5.21 [+ or -] 0.50 mmol/L) and their response to the intravenous glucose tolerance test (performed after a 24-h period of food deprivation, using a glucose dose of 500 mg/kg body weight, infused as 50% glucose solution in 30 s) were also normal. These dogs were accustomed to the experimental procedure. They were commonly used for digestibility trials in the cages used in this study and had been previously subjected to repeated venipuncture. Therefore, their responses were due to the experimental variables and not to stress. Experimental diets. Twenty experimental foods (C1-C5 and D1-D15) were tested. Foods D1-D15 were dry foods, whereas C1-C5 were canned. These test foods were intended to be representative of foods currently used for maintenance or for clinical purposes in adult dogs. They were designed to vary in macronutrient composition [15.4-62.6% crude protein (CP), 7.9-31.0% ether extract (EE), 3.2-39.1% total dietary fiber (TDF) and 0.4-52.7% starch (ST), on a dry matter basis]; the energy content was 965-2045 kJ/100 g dry matter. The composition of test meals is shown in Table 1. The daily chromium intake was not KEY WORDS: * dog foods * analytical composition * food processing * glycemic response * insulinemic response * dogs

Variations in the blood glucose and insulin responses to different foods have been studied in dogs (Holste et al. 1989, Nguyen et al. 1994), and different trends in postprandial concentrations have been observed. These differences arose in the extent of the variations (areas under the curves and maximal increments) and the time from meal to peak increases. The main purpose of these studies was to rank foods on the basis of the incremental glucose responses that they produced and to relate these responses to foods characteristics. It is known that methodologic variables can markedly modify the interpretation of the glycemic response. In particular, this concerns the length of time of blood sampling (Gannon and Nuttall 1987), short-term (Wolever and al. 1988) and long-term (Cannon and al. 1980) remnant effects of the previous meal, blood sampling (Jackson and al. 1983) and fasting blood glucose values (Nielsen and Nielsen 1989). In dogs, the variations of the glycemic response have been evaluated with (Holste et al. 1989) or without (Nguyen et al. 1994) an adaptation period to the tested meals. As in human beings, the plasma concentrations were measured over a period of 3 or 4 h even though the gastrointestinal transit time is shorter in dogs than in humans. The purpose of our study was to examine whether an adaptation time (inducing digestive changes and modifications in basal insulin secretion and glucose tolerance) modifies the postprandial response to meal feeding in normal dogs. We also studied the effect of length of time of blood sampling on the consistency of the response value expressed by the areas under the glycemic and insulin curves, the peak incremental values and the times from meal to peaks. Materials and methods. Animals. Twelve adult (older than 15 mo) beagle dogs, allotted to two groups, were studied, according to the French Ministry of Agriculture and Fisheries regulatory rules for animal welfare. None of the dogs was obese (13.7 kg mean body weight) and all were clinically normal. Their basal plasma glucose (Experiment 1:5.41 [+ or -] 0.54 mmol/L; Experiment 2:5.27 [+ or -] 0.55 mmol/L) and their response to the intravenous glucose tolerance test (performed after a 24-h period of food deprivation), using a glucose dose of 500 mg/kg body weight, infused as 50% glucose solution in 30 s, were also normal. These dogs were accustomed to the experimental procedure. They were commonly used for digestibility trials in the cages used in this study and had been previously subjected to repeated venipuncture. Therefore, their responses were due to the experimental variables and not to stress. Experimental diets. Two experiments were performed according to the two objectives. Experiment 1. Five experimental foods (A1, A2 and B1-B3 foods) were tested. Foods A1 and A2 were canned foods, whereas B1-B3 were dry. Foods B1 and B3 were given as is, with separate water, whereas food B2 was hydrated before feeding. These test foods were intended to be representative of foods currently used for maintenance in adult dogs. They were designed to vary in macronutrient composition [27.2-62.6% crude protein (CP), 10.9-25.7% ether extract (EE), 1.1-3.6% crude fiber (CF), 4.4-50.5% nitrogen-free extract (NFE) and 3.2-12.4% total dietary fiber (TDF), on a dry matter basis]. The energy profile was as follows: 25-54% energy derived from protein, 25-51% from fat and 4-48% from nitrogen-free extract, according to standard energy conversion factors, 14.6 kJ/g CP or NFE, 35.6 kJ/g EE). The composition of the test meals is shown in Table 1. The daily chromium intake was not 90 min appears to be unnecessary to characterize the glycemic and insulin responses to meals in normal dogs. The effects of the length of time of blood sampling on the relative area under the glucose curve of foods in humans has been reviewed (Gannon and Nuttall 1987). Long-time measurements tend to reduce the differences in AUCG between foods, especially between foods resulting in high peak rises followed by a rapid decrease, and foods for which the glucose response presents a lower peak but tends to remain above the baseline for a prolonged period of time. If measured for too long a time, the area under these types of curves may be nearly the same, despite markedly different effects on insulin and counterregulatory hormone responses. These factors are related to the acute rate of carbohydrate absorption, which in turn is related to the incremental area under the early part (120 min) of the glucose response curve in normal subjects (Wolever et al. 1991). A shorter duration of blood sampling could be sufficient in normal dogs because of their high gastrointestinal transit rate. The relatively rapid gastric emptying and digestive processes allow an early expression of the acute effects of foods on the glucose response curve. Increasing the period of blood sampling beyond 90 min did not modify the discrimination between foods. We concluded that the diet consumed before the initiation of the study had not influenced the response to test foods and that an adaptation time appears to be unnecessary. The postprandial glycemic and insulin responses of normal dogs may be obtained after a single meal. We also conclude that a 90-min blood sampling period was sufficient to characterize the glycemic and insulin responses in normal dogs. KEY WORDS: * dog foods * glycemic response * insulinemic response * dogs

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 [[micro]mol]/kg of [113C] acetate, and carotid blood was then sampled during a 15-min period to estimate the acetate distribution volume. Ten dogs received intravenous [1-136] acetate infusions of 1.05 [+ or -] 0.02 or 2.10 [+ or -] 0.10 [[micro]mo]l/(kg [multiplied by] min) for 120 or 200 min after a prime of 200 or 70 [[micro]mol]/kg, respectively. Cephalic venous and carotid arterial blood were sampled for all dogs, and portal blood for five. Acetate distribution volume was 0.27 [+ or -] 0.16 L/kg (mean [+ or -] SEM). The concentrations of acetate in arterial (144 [+ or -] 17 [[micro]mol]/L), venous (155 [+ or -] 20 [[micro]mol]/L) and portal plasma (131 [+ or -] 16 [[micro]mol]/L) were not significantly different during infusion, whereas isotopic enrichments [mole percent excess (MPE): labeled acetate/all acetate molecules] in portal (1.2 [+ or -] 0.2 MPE) and venous plasma (1.7 [+ or -] 0.3 and 2.6 [+ or -] 0.7 MPE) were lower than in arterial plasma for both infusion rates (4.9 [+ or -] 0.6 and 7.6 [+ or -] 0.8 MPE, respectively, P < 0.005). Whole-body acetate turnover was 24.4 [+ or -] 2.4 [[micro]mol]/(kg [multiplied by] min). Fractional acetate extractions for forelimb and intestine were 62 [+ or -] 7 and 72 [+ or -] 6%, respectively, and the production for each organ was 0.3 and 1.1 [[micro]mol]/(kg [multiplied by] 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. KEY WORDS: acetate, turnover rate, stable isotopes, dogs

Postprandial hyperglycemia and insulin secretion depend on many factors, such as food composition, carbohydrate type and processing. The purpose of this study was to evaluate the effects of foods differing in composition in nonobese clinically normal dogs. These dogs were offered in a single meal one of the four foods. Blood samples were collected before feeding and during the 180 min after the meal. Plasma glucose and insulin concentrations were determined. The integrated areas under the postprandial glucose and insulin response curves were calculated. Times to response peaks, maximum plasma insulin elevation, area under the insulin response curve and the insulinemic index differed between dog foods. Crude fiber content had no significant effect. The time to glucose peak was higher and the time to insulin peak lower with the food highest in protein and ether extract content. The time to glucose peak was correlated to protein, fat and nitrogen-free extract content of foods, whereas the time to insulin peak and the area under the insulin curve were correlated to protein and nitrogen-free extract. INDEXING KEY WORDS: * dog foods * glycemic index * insulinemic index * dogs

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

The hindgut fermentation of undigestible carbohydrate macromolecules yields short-chain fatty acids such as acetate, propionate and butyrate, and gases (Bergman 1990). The last mentioned are mainly hydrogen and methane. These metabolites are readily absorbed from the colon and transported through the blood circulation. Hydrogen and methane pass through the pulmonary alveoles where they are both rapidly eliminated in breath. In human studies, the hydrogen breath test is currently used to assess qualitatively the fermentation process of undigestible substrates (Christi et al. 1992, Rumessen 1992). The aim of this study was to apply this approach in dogs fed a control meat-based diet to clarify whether colonic micro flora could adapt and ferment an undigestible carbohydrate (lactulose). Materials and methods. Six adult Beagle dogs (14.3 [+ or -] 1.2 kg, mean [+ or -] SEM) 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. Protocol. Dogs were fed once daily the control diet [two thirds beef (S.E.R., Cuiseaux, France, 20% protein, 0% carbohydrate and 15% lipids) plus one third extruded rice and a vitamin and mineral addition] on the basis of 555 kJ/([kg.sup.0.75] [multiplied by] d) of energy requirement for 3 wk. During wk 2, 10 g of lactulose (100% fermentable disaccharide, Duphar, Villeurbanne, France) was added to the control diet. Meals were eaten within 20 min. On four occasions, breath samples were collected at regular times for 10 h after food intake. The first breath sampling (Experiment A) was performed on d 7, after 1 wk of consuming the control diet. The next one (Experiment B) was done the following day, d 8, the first day lactulose was added. After 1 wk of lactulose adaptation, breath samples were collected on d 14 (Experiment C). A final breath sampling (Experiment D) was performed on d 21, after a final week consuming the control diet. Breath sampling technique. The day of the study, dogs were placed in individual boxes. The breath sampling was performed with a permeable bag filled through a dual-inlet valve system connected to a mask that was put on the dog's face. During the inhalation, the bag valve automatically closed, whereas during the exhalation, breath was allowed to enter the bag through the open valve. The bag was filled within 2-4 min. On d 7 and 21, samples were collected every 30 min and on d 8 and 14, they were collected every 20 min. Each breath sample (30 mL) was kept in an airtight syringe at 4 [degrees] C until analyzed within 24 h. Analysis of gases. Hydrogen and methane concentrations in breath samples were measured on a Microlyzer DP gas chromatograph (Quintron instrument, Milwaukee, WI). Results were expressed as parts per million (1 ppm [approximately equal to] 0.05 [micro]mol/L). Statistics. All results are expressed as means [+ or -] SEM (n = 6 dogs). Paired t tests were performed between the peak and the initial level of hydrogen breath response for different experiments with Instat statistical software (GraphPad, San Diego, CA). Experiments were compared by using ANOVA with Instat software. Results. The weight of the dogs was unchanged during the 3 wk of the study. The methane level in the dogs' breath was insignificant throughout all experiments (C[H.sub.4] < 2 ppm). Hydrogen level increased 5 h after the meal in all four experiments. In Experiment A [ILLUSTRATION FOR FIGURE 1 OMITTED], during the first 5 h, hydrogen level was 2 [+ or -] 1 ppm; it increased rapidly and significantly up to 24 [+ or -] 10 ppm from t = 5-8 h and returned to near basal level at 10 h (8 [+ or -] 1 ppm). The first day of lactulose addition (Experiment B), hydrogen excretion increased progressively from 4 [+ or -] 2 ppm at 2 h to 21 [+ or -] 2 at 7 h without any return to the initial level after 10 h [ILLUSTRATION FOR FIGURE 2 OMITTED]. In contrast, after 7 d of lactulose ingestion (Experiment C), the hydrogen breath concentration was low from time 0 to 5 h 20 min (4 [+ or -] 1 ppm) and, after a slight increase, remained at a low level from 5 h 40 min to 10 h (7 [+ or -] 1 ppm). This plateau was significantly lower than that in the other 3 experiments (P < 0.05, peak vs. peak, [ILLUSTRATION FOR FIGURE 2 OMITTED]). After 1 wk of again consuming the control diet (Experiment D), the hydrogen stabilized throughout the first 5 h and increased rapidly from 4 [+ or -] 1 ppm (t = 5 h) to 19 [+ or -] 8 ppm (t = 9 h, [ILLUSTRATION FOR FIGURE 3 OMITTED]). Discussion. This study focused on breath hydrogen excretion as evidence of microflora activity in the intestinal tract of dogs. The beagles fed the control diet (meat and extruded rice) exhaled hydrogen 5 h after feeding. The addition of lactulose to the food induced an increase in the area under the curve of breath hydrogen on d 1, whereas daily supplementation decreased the hydrogen exhalation considerably. This study demonstrates an adaptation of the microflora in dogs after chronic ingestion of lactulose. Experiment A. Although the rice and meat meal was a low residue diet, part of it escaped digestion and was degraded by microflora, as shown from a net increase of breath hydrogen 5-6 h after eating. Some of the ingested food may have reached the microbial activity in the colon, undergone fermentation and produced hydrogen. Recent studies in dogs offer another explanation; some of the diet can be catabolized by bacteria in the ileum, and these microorganisms are capable of degrading amino acids and of producing a large amount of hydrogen (Zentek 1995a and 1995b). Experiments B and C. To assess the fermentation process, dogs were fed a basal diet composed of rapidly and highly digestible meat and carbohydrate to which a nondigestible carbohydrate had been added (lactulose). Although the basal diet induced exhalation of hydrogen breath, which is evidence of bacterial activity, lactulose supplementation resulted in a major change in gas excretion. On d 1 of lactulose intake (Experiment B), the transit time has shortened, and the microflora produced a larger amount of hydrogen as represented by the greater area under the curve. Lactulose may have modified the upper gastrointestinal transit rate, thereby spreading the dietary substrate irregularly to the microflora. Thus, undigested substrate could rapidly reach the ileum, resulting in rapid hydrogen exhalation probably produced from amino acid degradation (Zentek 1995a). Lactulose, which is degraded by colonic bacteria in nonruminants (Bergman 1990), would be a likely source of the hydrogen produced in the large intestine 10 h after meal ingestion, compared with hydrogen exhalation in Experiment A, which returned toward its initial level at the end of the study (10 h). After 7 d of lactulose supplementation (Experiment C), hydrogen exhalation had dramatically diminished although an increase at 5 h 40 min was still apparent. This decrease in breath hydrogen could have come from a shorter transit time and a quicker flushing of the intestine, although no diarrhea was noticed. The cecal content could be rapidly emptied into the distal colon, resulting in less bacterial activity and hydrogen production. This decrease in hydrogen exhalation was also observed in humans who consumed lactulose daily for 1 wk (Florent et al. 1985). Cecal acetate concentration also increased, showing a change in the microbial behavior and metabolism probably due to lactulose supplementation. The authors concluded that the fermentation process within the microflora evolved to a lower production of hydrogen, with the metabolism of the micro flora changing and adapting to a new environment enriched in lactulose. This change may also be the result of a competition between bacteria, resulting in a flora composed of fewer hydrogen producers or more hydrogen consumers. Clearly, an adaptation of the intestinal micro flora occur red during the lactulose supplementation. Dogs chronically fed lactulose demonstrate behavior similar to that of humans who have colonic microflora capable of entirely fermenting the lactulose fraction. Our study suggests that dogs adapt and probably ferment lactulose at a low level compared with humans because breath hydrogen test values of humans are three or four times higher than those of dogs when similar amounts of lactulose are ingested (Rumessen 1992). From this study, it is clear that a meat and extruded rice diet induces hydrogen breath production probably due to partial degradation by bacteria in the intestine; adaptation to lactulose induces a change in the colonic bacterial flora that results in a decrease in hydrogen production. Dogs probably contain microflora in the intestine that spread from the ileum to the distal colon with different populations capable of adapting quickly to the nondigested substrates. Further microbial studies in the lumen of the alimentary tract would be most beneficial in understanding to what extent dogs are capable of degrading fermentable carbohydrates. KEY WORDS: * hydrogen * lactulose * fermentation * dogs

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