Ion chromatographic methods for the detection of starch hydrolysis products in ruminal digesta.

Dionex high-performance ion chromatographic methods were evaluated for separation and quantitation of plant sugars and starch digestion products in the ruminal digesta of cattle. Mono- and disaccharides were eluted from a Dionex CarboPac PA1 column with sodium hydroxide used isocratically or as a pH gradient. Maltooligosaccharides which had a degree of polymerization (DP) less than 30 glucose residues were eluted in 60 min by a sodium hydroxide eluent containing a sodium acetate gradient. Carbohydrates were detected amperometrically. Responses were linear (r2 greater than 0.99) for glucose, disaccharides and maltooligosaccharides (DP less than 8). Precipitation and solid-phase extraction methods were evaluated for clean-up of samples of feedstuffs, ruminal contents, and bacterial culture fluids. Perchloric acid precipitation hydrolyzed sucrose but did not affect recoveries of cellobiose, isomaltose or maltose. Ethanol in concentrations of 79 and 86% precipitated maltooligosaccharides having chain lengths larger than 14 and 9 glucose residues, respectively. Maltooligosaccharide recoveries from solid-phase extraction columns varied with maltooligosaccharide size and column packing. Recoveries were greater than 94% for short chains (DP less than 6) eluted from phenyl-substituted columns and variable for all oligosaccharides eluted from C18 columns. Applications of these methods are presented and include: (1) detection of sugars in ruminant feed, (2) monitoring changes in ruminal sugars after feeding and (3) monitoring changes in extracellular sugars and oligosaccharides in the culture fluids of the ruminal bacterium, Bacteroides ruminicola.

Postprandial trends in estimated ruminal digesta polysaccharides and their relation to changes in bacterial groups and ruminal fluid characteristics.

In a diurnal study, feedstuff and digesta polysaccharides, ruminal bacterial carbohydrate-fermenting groups, and selected ruminal fluid characteristics (ruminal pH, ammonia and volatile fatty acids) were measured in ruminal-cannulated Holstein steers fed high- or low-forage diets at maintenance level intake once daily. A procedure for the sequential extraction of soluble sugar, starch, pectin, hemicellulose and cellulose from feedstuffs was developed to measure these carbohydrates in dietary and ruminal digesta samples. Recovery of dry matter (determined chemically) using this scheme was 60 to 70%. Data were obtained within the ranges of those in the literature for similar feedstuffs and(or) by similar methods. Dietary analysis by the sequential method yielded total recovery across all carbohydrate fractions of 87 and 81% for the high- and low-forage diets, respectively, and similar recoveries were obtained for the digesta samples. Analytical variation was small (less than or equal to 15% CV), which permitted comparison of the carbohydrate profiles of the digesta over time. From these values, total ruminal digesta polysaccharide content was calculated and, when plotted over time, indicated that the disappearance per fraction corresponded with theoretical curves for ruminal fermentation of major feedstuff components. The postprandial variation within the bacterial population carbohydrate-fermenting groups was small, but changes were consistent with digesta component fermentation. Xylan- and cellulose-fermenting groups followed a pattern compatible with the disappearance of these polysaccharides from the rumen. In contrast, soluble sugar-fermenting groups predominated at all times despite the rapid rise and fall of these components in the digesta. Ruminal fluid pH, ammonia and total carbohydrate supported the digesta and bacterial trends observed. The data are interpreted to suggest that once daily maintenance feeding of high- or low- forage diets permits detection of digesta sugar and polysaccharide changes, supports a relatively stable microbial population while specific groups increase and decrease with the availability of substrate, and results in few differences in ruminal fluid traits.

Sulfation of minoxidil by liver sulfotransferase.

The 100,000 g supernatant fraction of rat liver homogenate contains a sulfotransferase activity which catalyzes the sulfation of minoxidil. Synthetic minoxidil N-O sulfate and the enzyme synthesized product had identical chromatographic characteristics on high pressure liquid chromatography. Minoxidil sulfate, which yields minoxidil when treated with sulfatase, was slowly hydrolyzed in water. Several N-oxides of other heterocycles, including several other pyrimidines, triazines and imidazoles, were also substrates for this sulfotransferase.