Frequency-pulsed Electron Capture Gas-liquid Research Articles

Differentiation of sarcoidosis from tuberculosis by use of electron capture gas-liquid chromatography.

To explore further the possible etiologic role of mycobacteria in the development of sarcoidosis, we measured free, nonbound tuberculostearic acid (TSA, 10-methyloctadecanoic), a component of mycobacteria, in the sera of subjects with sarcoidosis or active untreated pulmonary tuberculosis and in healthy controls by use of frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC). The selective analytic system is capable of measuring as little as 15-fmol quantities of free, nonbound TSA in serum and cerebral spinal fluid. We found that TSA was present in the sera of all subjects with Mycobacterium tuberculosis (n = 10) but was undetectable in subjects with sarcoidosis (n = 15) and in healthy controls (n = 15), thereby suggesting that if sarcoidosis is caused by a mycobacterial organism, TSA is not produced or does not gain access to the systemic circulation in quantities sufficient for measurement. However, in the course of the studies we found that a peak, designated p11, was elevated in the sera of all subjects with acute sarcoidosis (n = 4). Also, a peak designated p3 was reduced significantly in all subjects with acute and chronic sarcoidosis and absent in subjects with M. tuberculosis compared with healthy controls. Both peaks were later shown by chemical analysis and mass spectral studies to be carboxylic acids not previously associated with specific disease entities. Follow-up detailed studies will be needed to determine if quantitation of these unique carboxylic acids will be useful in differentiating sarcoidosis from other disorders.

Detection of malignancy-associated metabolites in the sera of cancer patients by electron capture gas chromatography.

A reliable test that detects malignancy and indicates response to therapy is needed. Frequency-pulsed electron-capture gas-liquid chromatography (FPEC-GLC), a selective analytical technique that is sensitive to 15 fmol quantities of metabolites, was used to analyse derivatised acidic chloroform extracts of sera from patients with biopsy-proven cancer, non-malignant infectious and non-infectious disease, and healthy controls. Two peaks designated P1 and P10, not found in serum from healthy controls (n = 7) or patients with non-malignant disease (n = 85), were detected in biopsy-proven samples (n = 52) from cancer patients. P1 and P10 were later shown by chemical and mass spectral studies to be carboxylic acids. When one or both of these peaks were detected in the sera of non-treated patients they were always associated with malignancy. In patients responding to therapy, a reduction or disappearance of these peaks was observed. Further, it was noted that P10 persisted or increased in sera of patients with progressive cancer not responding to therapy. We conclude that this test has potential in diagnosis and for following the response of the disease to therapy.

Rapid diagnosis of tuberculous meningitis by frequency-pulsed electron-capture gas-liquid chromatography detection of carboxylic acids in cerebrospinal fluid

The frequency-pulsed electron-capture gas-liquid chromatography technique described previously by Brooks et al. was modified and applied to the studies of coded and routine clinical specimens. Uncentrifuged cerebrospinal fluid (2 ml) was extracted under acidic conditions, derivatized, and analyzed by frequency-pulsed electron-capture gas-liquid chromatography on large-bore fused silica polar and nonpolar capillary columns. The frequency-pulsed electron-capture gas-liquid chromatography profile of carboxylic acids (C2 through C22) along with identification of tuberculostearic acid, established by retention time comparison of derivatized tuberculostearic acid and derivatized sample extract, strongly suggests the presence of Mycobacterium tuberculosis in patients with lymphocytic meningitis. Results from 41 coded cases and 75 clinical cases showed that the frequency-pulsed electron-capture gas-liquid chromatography test had a specificity of 91% and a sensitivity of 95%.

Analyses of fermentation products of Listeria species by frequency-pulsed electron-capture gas–liquid chromatography

Aerobic fermentation broths of eight Listeria monocytogenes strains, two or more strains of the remaining six Listeria species, and one strain of Jonesia denitrificans were examined by frequency-pulsed electron-capture gas-liquid chromatography for carboxylic acids, alcohols, amines, and hydroxy acids. All species produced acetic, isobutyric, butyric, isovaleric, phenylacetic, lactic, 2-hydroxybutyric, 2-hydroxyvaleric, and 2-hydroxyisocaproic acids. Propionic acid was not formed, and traces of isocaproic acid were observed. Of the alcohol and amine derivatives observed, only acetylmethylcarbinol, butylamine, and putrecine were identified. Recognition of the products of glucose and amino acid metabolism serves to further characterize the members of the genus Listeria both taxonomically and physiologically.

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

Conditions are described for the detection of tuberculostearic acid (10-methyloctadecanoate; C18 X CH3) in cerebrospinal fluid and serum of patients with tuberculous meningitis. C18 X CH3 was found in both the cerebrospinal fluid and serum of patients with tuberculous meningitis at concentrations of 25 to 50 fmol (10(-15) mol). The necessary specificity and sensitivity for detection of C18 X CH3 were obtained by extraction under acid conditions with organic solvent, specific functional group esterification with trichloroethanol, cleanup with disposable reverse-phase sorption chromatography columns, analysis on high-resolution polar and nonpolar capillary columns, and detection by a frequency-pulsed electron capture detector. Use of an IBM 9000 computer equipped with CAP software significantly aided comparison between known C18 X CH3 standards and C18 X CH3 in clinical specimens. Scale expansion and attenuation changes were the major contributions obtained by use of the computer. The data indicate that detection of C18 X CH3 by frequency-pulsed electron capture gas-liquid chromatography may be a valuable aid for early detection of tuberculous meningitis.

Detection of metabolites by frequency-pulsed electron capture gas-liquid chromatography in serum and cerebrospinal fluid of a patient with Nocardia infection.

Serum (SR) and cerebrospinal fluid (CSF) from a patient suspected of having tuberculous meningitis were submitted to our laboratory for analysis by frequency-pulsed electron capture gas-liquid chromatography (FPEC GLC). The samples were tested for the presence of carboxylic acids, alcohols, hydroxy acids, and amines by methods described previously (C. C. Alley, J. B. Brooks, and D. S. Kellogg, Jr., J. Clin. Microbiol. 9:97-102, 1977; J. B. Brooks, C. C. Alley, and J. A. Liddle, Anal. Chem. 46:1930-1934, 1974; J. B. Brooks, D. S. Kellogg, Jr., M. E. Shepherd, and C. C. Alley, J. Clin. Microbiol. 11:45-51, 1980; J. B. Brooks, D. S. Kellogg, Jr., M. E. Shepherd, and C. C. Alley, J. Clin. Microbiol. 11:52-58, 1980). The results were different from previous FPEC GLC profiles of SR and CSF from patients with known tuberculous meningitis. Both the SR and CSF contained several unidentified compounds that were not previously detected in tuberculous meningitis or any of our other studies of body fluids. Nocardia brasiliensis was later isolated from the patient. Detection of these metabolites by FPEC GLC could prove to be useful for rapid diagnosis of Nocardia disease, and their identification will provide a better understanding of metabolites produced by Nocardia sp. in vivo.

Review of frequency-pulsed electron-capture gas-liquid chromatography studies of diarrheal diseases caused by members of the family Enterobacteriaceae, Clostridium difficile, and rotavirus.

Review of frequency-pulsed electron-capture gas-liquid chromatography studies of diarrheal diseases caused by members of the family Enterobacteriaceae, Clostridium difficile, and rotavirus.

Studies of metabolites in diarrheal stool specimens containing Shigella species by frequency-pulsed electron capture gas-liquid chromatography

Eleven diarrheal stool specimens and 10 control stool specimens from Cairo, Egypt, were studied by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC). Four cases involving Shigella sonnei, three cases involving Shigella boydii, and four cases involving Shigella flexneri were studied. The aqueous stools were centrifuged, extracted with organic solvents, and derivatized to form specific electron-capturing derivatives of carboxylic acids, alcohols, hydroxy acids, and amines. Analyses were performed on high-resolution glass columns with an instrument equipped with an extremely sensitive electron capture detector that is specific for the detection of electron-capturing compounds. The diarrheal stools studied had specific FPEC-GLC profiles and contained metabolic markers that readily distinguished between the Shigella spp. studied and Escherichia coli producing heat-stable or heat-labile enterotoxins. S. sonnei stools contained hexanoic acid, 2-hydroxy-4-methylmethiobutyric acid, and some unidentified alcohols that distinguished this organism from other enteric pathogens. S. boydii produced an acid that was unique for this species, and S. flexneri produced alcohols that could be used to distinguish between it and other enteric organisms. The FPEC-GLC profiles obtained during this study were also very different from those reported earlier for Clostridium difficile and rotavirus. This study presents further evidence that the selectivity and sensitivity of FPEC-GLC techniques can be used to rapidly identify causative agents of diarrhea and detect physiological changes that occur in the gut during the course of diarrheal illness.

Frequency-pulsed electron-capture gas—liquid chromatographic studies of chemical changes in sera of patients with schistosomiasis

Sera from well documented cases of Schistosoma mansoni and S. haematobium infections as well as controls, were studied by frequency-pulsed electron-capture gas—liquid chromatography (FPEL-GLC) and mass spectrometry for detection of carboxylic acids and amines. Many carboxylic acids and unidentified peaks were detected. In a few serum specimens from infected patients, putrescine and cadaverine were detected. Indications are that in these few patients with high egg counts enough diamines were present to possibly produce amine toxicity. Following the initial investigation, the basic chloroform extractions, which contained amines, were further studied by FPEC-GLC with the aid of splitless injection and a capillary column. Several amines were detected which seemed to be related to schistosomiasis. Mass spectra were obtained on an unidentified schistosamine peak. The possible significance of the data is discussed.

Rapid differentiation of enterotoxigenic Escherichia coli that produce heat-stable and heat-labile toxins by frequency-pulsed electron capture gas-liquid chromatography analysis of diarrheal stool specimens.

Thirty-three stool specimens from infants in the village of Tamooh near Cairo, Egypt, were studied by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC). In 13 of the diarrheal cases, the suspected causative agent isolated was Escherichia coli which produced heat-stable toxin (ST), and in 10 other cases E. coli that produced heat-labile toxin (LT) were isolated. Ten control stool samples, collected from infants from whom no pathogenic organisms were isolated, were analyzed at the same time. Comparisons also were made against healthy control stools from individuals in the United States who had been previously analyzed by FPEC-GLC (Brooks et al., J. Clin. Microbiol. 20:549-560, 1984). The stools were suspended in water and centrifuged, and the supernatant was extracted with organic solvents and derivatized to form electron-capturing derivatives of carboxylic acids, hydroxy acids, alcohols, and amines. Results from the study showed distinct differences among the FPEC-GLC profiles of E. coli ST-positive stools, of E. coli LT-positive stools, and of the control stool samples. An unidentified compound appearing in the ether-soluble hydroxy acid fraction from E. coli ST-positive stools was tentatively identified by mass spectrometry as 6-methoxy-2-hydroxyhexanoic acid. 6-Methoxy-2-hydroxyhexanoic acid was found in all stools that contained E. coli ST but was not present either in stools from which E. coli LT was isolated or in control samples. 6-Methoxy-2-hydroxyhexanoic acid may prove to be an important marker for use in the identification of E. coli ST. In addition to 6-methoxy-2-hydroxyhexanoic acid, the carboxylic acid, alcohol, and amine FPEC-GLC profiles obtained from stools were very different between these two organisms. The data indicate that FPEC-GLC analysis of diarrheal stool specimens might be a rapid way to distinguish diarrhea caused by E. coli ST, E. coli LT, Clostridium difficile, and rotavirus.

Changes in hydroxy and carboxylic acid compositions in the supernatant fluids of mosquito cell cultures infected with dengue viruses

Hydroxy and carboxylic acids in the supernatant fluids of mosquito cell cultures infected with four serotypes of dengue viruses (DEN) were analyzed by frequency-pulsed electron capture gasliquid chromatography. The hydroxy acid profiles of all virus-infected cell cultures differed qualitatively and quantitatively from the profile of normal cell culture. Furthermore, the profiles of hydroxy acids in the DEN 1- and DEN 4-infected cultures were type specific. Although quantitative differences of a few peaks could be found between the hydroxy acid profiles of DEN 2- and DEN 3-infected cultures, in the absence of clear qualitative differences the two profiles were considered to be essentially indistinguishable. The carboxylic acid profiles of virus-infected cultures differed from the profile of a normal cell culture, but none of the four serotypes of DEN viruses induced type-specific profiles. Thus, these findings contrasted to previous results with rhesus monkey kidney cell cultures (LLC-MK 2), in which serotype-specific sets of hydroxy acids and a DEN 1-specific set of carboxylic acids were released in the supernatant fluids by the infection with dengue viruses.

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

Clostridium difficile strain CDC A-567 was cultured in Trypticase (BBL Microbiology Systems)-yeast-salt broth supplemented with 0.2% L-leucine, L-norleucine, L-isoleucine, L-tyrosine, or L-tryptophan. Four extractions were done on the spent medium, three at pH 2 and one at pH 10, using CHCL3 or ether. Derivatizations were done with trichloroethanol, heptafluorobutyric anhydride, and heptafluorobutyric anhydride-ethanol. All samples were analyzed with frequency-pulsed electron capture gas-liquid chromatography. A dedicated computer was used to assist in data analysis. C. difficile produced both short-chain and aromatic acids in Trypticase-yeast-salt broth; hydroxy acids were also detected. p-Cresol, indoleacetic acid, 4-methylthio-2-hydroxybutyric acid, and some unidentified alcohols were observed. The basic chloroform extraction contained cadaverine and putrescine. Leucine, norleucine, and isoleucine influenced the production of C5 and C6 acids and alcohols. L-Tyrosine underwent successive degradation to produce p-cresol and aromatic acids as final products. Tryptophan increased the production of indoleacetic, indolepropionic, and indolebutyric acids. Isocaproic acid was produced in relatively high concentrations regardless of medium substitution. The consistent production of iC6 under various substrate conditions indicates that the production of this compound might be consistent enough in vitro to form the basis of a rapid test for detection of C. difficile in stool specimens by frequency-pulsed electron capture gas-liquid chromatography.

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

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.

Identification of diethylene glycol in sera from egyptian children by frequency-pulsed electron-capture gas—liquid chromatography

Sera taken from fifteen patients (from Kerdasa village near Cairo, Egypt) infected with Schistosoma haematobium, with eggs present in the urine, were studied by frequency-pulsed electron-capture gas—liquid chromatography (FPEC—GLC). Some of the patients were treated with metrifonate and again studied by FPEC—GLC. Diethylene glycol was detected in the sera of untreated patients infected with S. haematobium. This compound was identified by negative chemical ionization and electron-impact mass spectrometry. Initially we suspected that the build-up of diethylene glycol in these patients was caused by schistosomiasis infection. However, in a follow-up blind-coded study using FPEC—GLC, which included 37 sera from Kerdasa and Tamooh villages near Cairo, Egypt, we detected diethylene glycol in eleven samples, four of which were controls from the villages. These latter findings indicate that the source of diethylene glycol might be the environment or foodstuffs, but the specific source has not been determined. Regardless of the source, diethylene glycol could affect the health of these Egyptian children by causing a narcotic effect, increased bladder stones, and increased numbers of bladder tumours.

Studies of metabolic changes in cell cultures infected with four serotypes of dengue fever viruses by frequency-pulsed electron-capture gas—liquid chromatography

Monkey kidney cell cultures were infected with four serotypes of dengue viruses, and the supernatant fluids of the cell cultures were extracted for amines, alcohols, carboxylic acids, and hydroxy acids. The derivatized extracts were then analyzed by frequency-pulsed electron-capture gas—liquid chromatography (FPEC—GLC). FPEC—GLC profiles of the hydroxy acids showed peaks that were different for different serotypes and the FPEC-GLC carboxylic acid profiles differed from the control medium. These differences were reproducible when the same lot of medium was used. There were differences in profiles between lots of control media due apparently to different fetal bovine sera used in the growth medium. Therefore, the same lot of medium was necessary to reproduce profiles. The data obtained from the study indicate that FPEC—GLC can be used to detect changes in cellular metabolism caused by viral infection, and that these metabolic changes might be useful for detection of genetic differences in viruses as reflected by detectable changes in the metabolism of the infected cell.

Rapid differentiation of rocky mountain spotted fever from chickenpox, measles, and enterovirus infections and bacterial meningitis by frequency-pulsed electron capture gas-liquid chromatographic analysis of sera

Normal sera and sera from patients with Rocky Mountain spotted fever, chickenpox, enterovirus infections, measles, and Neisseria meningitidis infections were extracted with organic solvents under acidic and basic conditions and then derivatized with trichloroethanol or heptafluorobutyric anhydride-ethanol to form electron-capturing derivatives of organic acids, alcohols, and amines. The derivatives were analyzed by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC). There were unique differences in the FPEC-GLC profiles of sera obtained from patients with these respective diseases. With Rocky Mountain spotted fever patients, typical profiles were detected as early as 1 day after onset of disease and before antibody could be detected in the serum. Rapid diagnosis of Rocky Mountain spotted fever by FPEC-GLC could permit early and effective therapy, thus preventing many deaths from this disease.

Metabolic analysis of serologically defined Neisseria meningitidis isolates by frequency-pulsed electron capture gas-liquid chromatography

Eight serogroups [A, B, C, D, X, Y, 29E [Z'], and W135] along with nongroupable and nontypable strains of Neisseria meningitidis were cultured in a defined liquid medium. The whole-culture spent medium was extracted, derivatized, and analyzed by frequency-pulsed electron capture gas-liquid chromatography. The frequency-pulsed electron capture gas-liquid chromatography profiles were then used to group the organisms on the basis of their metabolic profiles. First, two basic groups were formed which consisted of elevated spent-medium compounds and reduced spent-medium compounds. Then, these two basic groups were further subdivided on the basis of metabolites and cellular fatty acids detected in the spent medium to form a total of seven metabolic groups. The nongroupable strains (with the exception of two strains) fell within one of these metabolic groups. The elevated spent-medium compounds group contained all of the A, B, Y, and Z' strains, and the reduced spent-medium compounds group contained most of the C and all of the W135 and D strains. The potential usefulness of the metabolic group scheme is discussed.

Frequency-pulsed electron capture gas-liquid chromatography and the tryptophan color test for rapid diagnosis of tuberculous and other forms of lymphocytic meningitis.

A total of 260 samples of cerebrospinal fluid received from Egypt, the United States, Canada, and South America were examined by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC) for tuberculous and other forms of lymphocytic meningitis. Thirty-four of the specimens were culture positive for M. tuberculosis, and four cerebrospinal fluid specimens of herpes meningitis were established by immunological techniques. The compound, 3-(2'-ketohexyl)-indoline, was found in about 60% of the Egyptian tuberculous specimens and none of the culture-positive American specimens. the carboxylic and hydroxy acid FPEC-GLC profiles were used effectively in conjunction with other clinical data to make the diagnosis even in the absence of 3-(2'-ketohexyl)indoline. Herpes meningitis and mixed infections of Myeobacterium tuberculosis-herpes, M. tuberculosis-leptospira, and M. tuberculosis-Haemophilus influenzae produced profiles different from each other and from pure culture cases. The color test for tuberculous meningitis was evaluated, and free tryptophan was eliminated as the source of color reaction. Indications are that 3-(2'-ketohexyl)indoline, in most cases, is not responsible for the positive color reaction. Differences in the clinical and FPEC-GLC data obtained from samples from different geographical regions are discussed.

Gas Liquid Chromatography: From Taxonomy to Diagnosis

Gas Liquid Chromatography: From Taxonomy to Diagnosis

Rapid differentiation of the major causative agents of bacterial meningitis by use of frequency-pulsed electron capture gas-liquid chromatography: analysis of amines.

The major causative agents of bacterial meningitis (Haemophilus influenzae serogroup B, Neisseria meningitidis serogroups B and C, Klebsiella pneumoniae, Steptococcus pneumoniae, and two types of Escherichia coli) were cultured in a chemically defined medium, and selected strains were further studied in Todd-Hewitt medium. After acidic extraction of the spent media with chloroform, a basic extraction was made with chloroform to obtain amines. A third extraction was performed on re-acidified Todd-Hewitt medium with ethyl ether to obtain hydroxyacids. The extracts were derivatized with heptafluorobutyric anhydride-ethanol to form electron-capturing derivatives, and the derivatives were analyzed on a frequency-pulsed electron capture gas-liquid chromatograph (FPEC-GLC) equipped with a PEP-2 computer. The data obtained from the study showed that amines were produced by these organisms that formed characteristic patterns. Different serotypes of K. pneumoniae and the two serogroups of N. meningitidis produced different types of FPEC-GLC profiles within serotypes. E. coli produced several hydroxy acids on Todd-Hewitt medium that made it unique among the organisms studied. The methods used are practical and the techniques have potential for use in clinical laboratories and hospitals as a valuable aid for the rapid identification of the major causative agents of bacterial meningitis.