Studies of stools from pseudomembranous colitis, rotaviral, and other diarrheal syndromes by frequency-pulsed electron capture gas-liquid chromatography

Abstract

Thirty-five patients with various diarrheal syndromes and 22 controls were studied. All stool samples were carefully cultured for Clostridium difficile, using selective isolation media. Cytotoxin assays with proper antitoxin neutralization were done in MRC-5 cells. The stool samples were extracted four times, three times at pH 2 and once at pH 10, using CHCl3 or ether. Derivatizations of extracts were done with trichloroethanol, heptafluorobutyric anhydride, and heptafluorobutyric anhydride-ethanol, and all derivatives were analyzed by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC). A dedicated computer was used to assist in both qualitative and quantitative data analysis. Isocaproic acid (iC6) was always found in stool from which C. difficile was isolated and was absent in C. difficile-negative specimens. p-Cresol was found frequently in both persons with pseudomembranous colitis and controls. Tryptamine was found in stool containing C. bifermentans. The FPEC-GLC profiles of persons with acute diarrhea were very different from those of normal persons. Diarrhea associated with adenovirus and rotavirus, Klebsiella spp., and Escherichia spp. showed different FPEC-GLC patterns. Stools from well persons consistently contained full-scale peaks of pyruvic, acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids. In rotavirus stools isobutyric, isovaleric, and valeric acids were reduced in quantity from those found in control stools, whereas propionic and butyric acids were increased.

SynbioticLactobacillus acidophilusNCFM and cellobiose does not affect human gut bacterial diversity but increases abundance of lactobacilli, bifidobacteria and branched-chain fatty acids: a randomized, double-blinded cross-over trial

“…The production of BCFA and SCFA is often used to gain knowledge on the metabolic activity of the gut microbiota as BCFA are produced solely through protein breakdown by the gut microbiota while SCFA are also produced from fermentation of carbohydrates (Wong et al, 2006). While SCFA often are studied due to their beneficial effects, effects of BCFA are seldom studied and largely unknown, but a role has been indicated in Na + uptake, bowel disorders, and rotavirus infections (Brooks et al, 1984;Zaharia et al, 2001;van Nuenen et al, 2004;Huda-Faujan et al, 2010;Le Gall et al, 2011). Modulation of gut microbiota and its activity is therefore a potential strategy for improving gut health and well-being and currently probiotics, prebiotics, and synbiotics receive attention for their effects on gut microbiota (Roberfroid et al, 2010;Quigley, 2011).…”

Frequency-pulsed electron capture gas-liquid chromatographic analysis of metabolites produced by Clostridium difficile in broth enriched with amino acids

“…A Perkin-Elmer programmable processor (PEP-2) equipped with a modular software system (MS-16 revision B) accumulated data from the gas chromatograph, analyzed the data according to a stored method, and prepared a report. An internal standard analysis was performed on the data, using nonanoic acid, di-n-butylamine, 2-hydroxyisovaleric acid, and nonylalcohol (which were added) as internal standards (3,8). Tentative identification was made by comparing the retention times of knowns and unknowns on packed and capillary columns.…”

“…In recent years Clostridiupn difficile has been the object of intense study (2,8,(10)(11)(12)(13)(16)(17)(18)). The main reason for this increased interest rests in the possibility that C. difficile is a causative agent of pseudoniembranous colitis.…”

“…Frequency-pulsed electron capture (FPEC) GLC, when used with specific functional-group halogenated derivatives to increase sensitivity and high-resolution columns, has been shown to offer both the sensitivity and the specificity needed to detect metabolites in body fluids and spent culture media (1,5,6,8). Methods have been established for analysis of carboxylic acids, hydroxy acids, alcohols, and amines.…”

Selective procedures for detecting femtomole quantities of tuberculostearic acid in serum and cerebrospinal fluid by frequency-pulsed electron capture gas-liquid chromatography

“…A major drawback to this approach in the analysis of body fluids is loss of the FPEC-GLC profile of short-chain carboxylic acids. Detection of the entire spectrum of carboxylic acid from C2 to C22 provides an additional diagnostic criterion that is potentially more useful than detection of C18 CH3 alone (3,4,6,10). Use of a 25-m OV-101 column is recommended over the 10-m OV-101 column for best resolution of both short-and long-chain acids.…”