In vivo study of colonic fermentation of carbohydrate in infants.

Dietary carbohydrate in the colon is fermented and converted into short-chain fatty acids. We studied the fate of carbohydrate that arrives in the colon under circumstances similar to those that occur during an episode of diarrhea and determined whether a quantitative correlation exists among certain indicators of colonic fermentation of carbohydrate arriving in the large bowel. A stable-isotope method was used to estimate carbon scavenging by the colon. Fourteen infants with severe malnutrition and history of watery stools and/or increased numbers of stools in the preceding 20 days were studied. Infants underwent nasocecal intubation and a 60-min infusion of 0.5 g/kg glucose containing 5 mg/kg of 13C-glucose. Stools were assessed for carbohydrate-fermenting bacteria, acetate, glucose, and 13C abundance; blood was assessed for acetate; and breath was assessed for hydrogen. Some of the infants eliminated the infusate per anus within 30 min of the infusion (group I; n = 5), while others did so 120 min or more after the infusion (Group II; n = 9). The volume of fecal output after the intracecal infusion differed significantly between group I and group II (57 +/- 13 vs. 24 +/- 4 ml; p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Colonic acetate in the circulating acetate pool of the infant pig.

To identify potential sites of acetate utilization and synthesis, we studied the contribution of colonic acetate to the circulating acetate pool in six neonatal pigs by the simultaneous i.v. infusion of [3H]acetate and colonic infusion of [14C]acetate. In the fasting state, the mean (+/- SEM) acetate concentration was 17 +/- 1 mumol/L in peripheral venous blood, 28 +/- 4 mumol/L in the femoral artery, and 46 +/- 4 mumol/L in portal blood. This concentration gradient implies that acetate was utilized either by peripheral tissues alone or by both liver and peripheral tissues. At the end of the 2-h intracecal acetate infusion, mean acetate concentration increased in the femoral artery to 186 +/- 20 mumol/L and in the portal vein to 333 +/- 31 mumol/L. In the fasted state, mean acetate concentration in the portal vein was on average 63% higher than the acetate concentration of the femoral artery, whereas specific radioactivity of the [3H]acetate in the portal vein was only 5% of that in the femoral artery. It is possible, therefore, that a high proportion of the arterial input of acetate is utilized by the portal-drained viscera. Our study identified the gastrointestinal tract as an important site of acetate utilization in the fasted state. Further, it showed that colonic acetate was efficiently absorbed and utilized in the gastrointestinal tract of infant pigs.

Age and diet affect the composition of porcine colonic mucins.

Colonic mucins may serve as a defense mechanism by binding bacterial, viral, or dietary lectins, thereby preventing them from attaching to the intestinal epithelium. Presumably, the composition of the mucins would be responsible for this phenomenon, and the composition of mucins from mature mammals would be the most effective in binding lectins. To determine whether differences in diet and/or age affect the composition of colonic mucins, we scraped fresh colonic mucosae from pigs at 0 (n = 3), 7 (n = 3), 21 (n = 3), and 180 (n = 3) d of age and purified the mucins from these mucosal scrapings. Mucins were purified by ribonuclease and deoxyribonuclease digestion, high-performance size-exclusion chromatography, and cesium chloride density-gradient ultracentrifugation. The 180-d-old pig was considered mature. No changes were observed in any of the variables analyzed in the 7-d-old animals. No changes were observed in quantities of galactosamine and galactose. The amounts of fucose and glucosamine increased by 165 and 37%, respectively, (p < 0.05) from d 0 to d 21 in the sow-fed animals, at which time fucose and glucosamine content were 48 and 22% greater, respectively, than in the 21-d-old, artificially fed group (p < 0.05). A further significant increase in fucose content was observed in the mucins from mature animals. The sulfate content in the 21-d-old, sow-fed animals was significantly lower than in both the newborn and the 21-d-old artificially fed animals. The sulfate content in all three of these groups, however, was significantly higher than that observed in the mucins of mature animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of glucooligosaccharides and polysaccharide hydrolysates by gradient elution hydrophilic interaction chromatography with pulsed amperometric detection.

Commercial glucooligosaccharide mixtures (Polycose) and polysaccharide hydrolysates (acid and enzymatic) were fractionated by hydrophilic interaction chromatography and observed by pulsed amperometric detection. Seven peaks were observed when 625 ng of glucose oligomers in Polycose were fractionated. The between-run precision of retention times (n = 10, 100 micrograms, 15 peaks) ranged from a relative standard deviation (R.S.D.) of 0.09 to 0.40%; between-run precision of peak areas (n = 10) for the same separations had values that ranged from 2.66 to 14.4%. Injection-to-injection time was 48 min. When polysaccharide hydrolysates were fractionated using a gradient program capable of resolving all of the oligosaccharide species, dextran-derived alpha-(1-->6)-glucooligosaccharides were retained to a greater degree than amylose-derived alpha-(1-->4)-glucooligosaccharides, which were retained to a greater degree than beta-(2-->1)-fructooligosaccharides derived from inulin. Excluding the peaks that eluted before glucose or fructose, 25 to 35 peaks were observed after fractionation of the hydrolysates. Differences in elution profiles were observed between acid and enzymatic hydrolysis products of the same polysaccharide as well as between hydrolysis products of different polysaccharides. In conjunction with high-performance size-exclusion chromatography, the method demonstrated the effect of preheating starch before hydrolysis with isoamylase.

Reversed-phase chromatography of phenylthiocarbamyl amino acid derivatives of physiological amino acids: an evaluation and a comparison with analysis by ion-exchange chromatography.

Reversed-phase chromatography of phenylthiocarbamyl (PTC) amino acid derivatives of physiological amino acids was evaluated and compared with the traditional method of ion exchange. The PTC amino acid derivatives were stable for at least 32 h at ambient temperature before injection. The relationship of detector response to concentration for the PTC derivatives was linear from 39 to 1250 pmol. With few exceptions, the within- and between-run precisions of plasma amino acid retention times were less than 0.2 and 0.3%, respectively; the within- and between-run precisions of their concentrations were less than 4.0 and 5.0%, respectively. Twenty-four plasma samples were quantitated by both reversed-phase and ion-exchange chromatography; fifteen of the twenty amino acids determined had correlation coefficients in the range 0.81-1.00. Nine non-standard amino acids and ten therapeutic drugs were added to plasma; D-glucosaminic acid and alpha-amino-beta-guanidinopropionic acid co-eluted with alpha-aminoadipic acid and threonine, respectively. Of the ten drugs added, only metronidazole and theophylline co-eluted with beta-alanine and histidine, respectively. The precision, stability, and sensitivity of the method render it ideal for the quantitation of plasma amino acids.

High-performance size-exclusion chromatography of porcine colonic mucins. Comparison of Bio-Gel TSK 40XL and Sepharose 4B columns.

A high-performance size-exclusion chromatography (HPSEC) method was developed for the separation of porcine colonic mucins using a Bio-Gel TSK 40XL HPSEC column (300 mm x 75 mm). In addition, porcine gastric and bovine submaxillary mucin preparations were used to describe more fully the separation characteristics of the HPSEC column. For comparison, the same preparations were also separated using a Sepharose 4B column (100 cm x 2.6 cm). The colonic and gastric mucins eluted in the void volume (V0) of both columns. Bovine submaxillary mucin was in the elution volume (Ve) of both columns. Analytical HPSEC of fractions (V0 and Ve) of the various preparations obtained by Sepharose 4B chromatography exhibited retention times identical to those for fractions obtained by HPSEC. After separation by both methods, purified mucins were obtained by CsCl2 density gradient ultracentrifugation; analytical HPSEC profiles, protein contents, and monosaccharide compositions of both gastric and colonic mucins from either column were similar. The HPSEC method, however, is ideally suited to separate microgram to milligram quantities of colonic mucin preparations quickly: 2 to 4 h, compared with 24 to 30 h for the Sepharose 4B method.