RESUMO
OBJECTIVES: To report the results from the Brazilian database on multiple sclerosis (MS) and pregnancy. METHODS: Retrospective data from MS patients who became pregnant at any time of their disease were sent to a Brazilian database, using a specific file for this purpose. RESULTS: Data on 128 women (142 pregnancies) from 30 neurologists working in 21 cities in Brazil were collected. Patients' average age at pregnancy was 29.8 years (range 16-42). EDSS at start of pregnancy was 1.5±1.4; and the relapse rate in the year preceding pregnancy was 1.2±1.5. Exposure to medication at any time during pregnancy was high (69.7%): 48.6% to interferon beta; 14.1% to glatiramer acetate; and 7% to other immunomodulatory and immunosuppressive drugs. There was a significant decrease in relapse rate during pregnancy. The prevalence of complications was relatively low, with 4.9% of obstetric and 1.4% neonatal unfavorable outcomes. CONCLUSIONS: Our patients had low degrees of disability, short histories of disease, high drug exposure, and relatively high relapse rate in the year previous to pregnancy. Obstetric and neonatal outcomes were successful in over 90% of our patients.
Assuntos
Esclerose Múltipla/epidemiologia , Complicações na Gravidez/epidemiologia , Adolescente , Adulto , Peso ao Nascer/efeitos dos fármacos , Brasil/epidemiologia , Interpretação Estatística de Dados , Bases de Dados Factuais , Feminino , Acetato de Glatiramer , Humanos , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Recém-Nascido , Doenças do Recém-Nascido/epidemiologia , Interferon Tipo I/efeitos adversos , Interferon Tipo I/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Peptídeos/efeitos adversos , Peptídeos/uso terapêutico , Gravidez , Resultado da Gravidez , Proteínas Recombinantes , Recidiva , Estudos Retrospectivos , Adulto JovemRESUMO
We analysed 37 clinical samples from 33 patients with bacteriologically confirmed tuberculosis, two cerebrospinal fluid samples from patients with cured tuberculous meningitis, and 14 serum samples from healthy individuals, for the presence of tuberculostearic acid (TSA) by frequency pulsed electron capture-gas chromatography (FPEC-GC) and chemical ionisation gas chromatography-mass spectrometry (CIGC-MS). TSA was detected in 36 of the 37 samples from patients with active tuberculosis and none of the patients with cured tuberculous meningitis; only one of 14 controls generated a similar chromatographic profile. Analysis of biological fluids by FPEC-GC and CIGC-MS for the presence of TSA may be a valuable method for rapid diagnosis of tuberculosis.
Assuntos
Líquidos Corporais/química , Ácidos Esteáricos/metabolismo , Tuberculose/metabolismo , Estudos de Casos e Controles , Criança , Cromatografia Gasosa/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , HumanosRESUMO
Serum samples from 26 individuals vaccinated with bacille Calmette-Guerin (BCG) and from 26 controls (10 patients with pulmonary tuberculosis and 16 non BCG-vaccinated healthy individuals) were analyzed by frequency-pulsed electron-capture gas chromatography (FPEC-GC) and chemical ionization gas chromatography-mass spectrometry (CIGC-MS) for the presence of characteristic metabolites. A distinct pattern consisted of tuberculostearic acid (TSA) and a peak, labeled peak 1, was observed in all BCG-vaccinated individuals, whereas only three of 26 controls generated this chromatography profile. TSA was detected in all patients with pulmonary tuberculosis but peak 1 was absent. Sera drawn from 12 individuals 11 to 14 days after BCG vaccination yielded three transitional FPEC-GC profiles. A permanent FPEC-GC profile consisting of TSA and of a full scale peak 1 appeared 28 days to a few months after BCG vaccination. Peak 1 was tentatively identified by CIGC-MS as 9-methyl-hexacosanol. The findings suggest that peak 1 may serve as a marker to detect Mycobacterium bovis BCG and to distinguish individuals infected with M. tuberculosis from individuals vaccinated with BCG.
Assuntos
Vacina BCG/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Vacina BCG/administração & dosagem , Elétrons , Humanos , Mycobacterium tuberculosis , Ácidos Esteáricos/sangue , Tuberculose/sangue , Tuberculose/metabolismoRESUMO
We developed a mass spectral method to verify the detection of free tuberculostearic acid (TSA) by frequency-pulsed electron-capture gas chromatography (FPEC-GC) in cerebrospinal fluid (CSF), serum, pericardial fluid, ascites fluid and pleural fluid of patients infected with Mycobacterium tuberculosis. To obtain satisfactory sensitivity and specificity for comparison of the test using mass spectrometry (MS) in the single ion monitor (SIM) mode to the FPEC-GC test, we developed a specific, sensitive, quantitative chemical ionization mass spectrometry capillary gas chromatography (QCIGC-MS) test. The procedure maximized the molecular ion (i.e., made it the base peak) for increased specificity and sensitivity, and instrument parameters for increased sensitivity. The procedure uses a computerized approach, requiring an internal standard (nonadecanoic acid) for precise measurement of the retention time and quantitation of the molecular ion of TSA. Data from this study suggest that QCIGC-MS analysis could be a valuable tool to confirm FPEC-GC identification of TSA in CSF, serum, and in pleural, ascites, and pericardial fluids.
Assuntos
Líquidos Corporais/química , Ácidos Esteáricos/análise , Líquido Ascítico/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Masculino , Mycobacterium tuberculosis/isolamento & purificação , Derrame Pericárdico/química , Derrame Pleural/química , Ácidos Esteáricos/sangue , Ácidos Esteáricos/líquido cefalorraquidiano , Tuberculose/metabolismo , Tuberculose/microbiologia , Tuberculose Meníngea/metabolismo , Tuberculose Meníngea/microbiologia , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/microbiologiaRESUMO
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.
Assuntos
Cromatografia Gasosa , Sarcoidose Pulmonar/diagnóstico , Ácidos Esteáricos/análise , Tuberculose Pulmonar/diagnóstico , Ácidos Carboxílicos/análise , Diagnóstico Diferencial , Seguimentos , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/patogenicidade , Sarcoidose Pulmonar/microbiologia , Tuberculose Pulmonar/microbiologiaRESUMO
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.
Assuntos
Biomarcadores Tumorais/sangue , Ácidos Carboxílicos/sangue , Neoplasias/sangue , Neoplasias/diagnóstico , Adenocarcinoma/sangue , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adolescente , Adulto , Idoso , Análise de Variância , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Distribuição de Qui-Quadrado , Criança , Pré-Escolar , Neoplasias do Colo/sangue , Neoplasias do Colo/diagnóstico , Feminino , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/diagnóstico , Masculino , Pessoa de Meia-Idade , Neoplasias/metabolismo , Valor Preditivo dos Testes , Reprodutibilidade dos TestesRESUMO
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%.
Assuntos
Ácidos Carboxílicos/líquido cefalorraquidiano , Cromatografia Gasosa/métodos , Tuberculose Meníngea/diagnóstico , Estudos de Avaliação como Assunto , Humanos , Ácidos Esteáricos/líquido cefalorraquidiano , Fatores de Tempo , Tuberculose Meníngea/líquido cefalorraquidianoRESUMO
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.
Assuntos
Acetoína/metabolismo , Aminas/metabolismo , Butanonas/metabolismo , Ácidos Carboxílicos/metabolismo , Fermentação , Hidroxiácidos/metabolismo , Listeria/metabolismo , Cromatografia Gasosa/métodos , Elétrons , Listeria monocytogenes/metabolismoRESUMO
Experiments, relevant to growth in milk, were done to delineate the aerobic and anaerobic growth of Listeria species on selected sugars in several media. All species grew on glucose aerobically, forming lactic acid and (or) acetic acid. Anaerobically, only lactic acid was formed; cell yields were 80% of those obtained aerobically. When incubated aerobically, small amounts (1.5 microns/mL) of isovaleric acid, 2-hydroxyisovaleric acid, and trace amounts of isobutyric acid were formed. These products were characteristically formed by 26 strains representing all the species of Listeria. Added leucine stimulated isovaleric acid formation. Anaerobic fermentations of glucose could be followed by 60 to 80% cell lysis; less lysis occurred in air. Anaerobically, only hexoses and pentoses supported growth; aerobically, maltose and lactose supported growth of some strains, but sucrose did not support growth of any strain tested. Listeria grayi and Listeria murrayi utilized the galactose and glucose moieties of lactose for growth; Listeria monocytogenes and Listeria innocua used only the glucose moiety. Glucosamine and N-acetylglucosamine supported aerobic and anaerobic growth as well as glucose, and their presence stimulated the utilization of lactose by "lactose-negative" strains. Analyses of cultures grown at 5 degrees C in sterile milk treated with glucose oxidase supported the conclusion that the glucose of the milk was the major, if not the limiting, substrate that supported growth.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias , Metabolismo dos Carboidratos , Listeria monocytogenes/crescimento & desenvolvimento , Listeria/crescimento & desenvolvimento , Leite/microbiologia , Acetatos/metabolismo , Aerobiose , Anaerobiose , Animais , Meios de Cultura , Fermentação , Galactose/metabolismo , Glucose/metabolismo , Lactatos/metabolismo , Ácido Láctico , Lactose/metabolismo , Listeria/metabolismo , Listeria monocytogenes/metabolismo , Peptídeos/farmacologia , TemperaturaRESUMO
Diarrheal stools from infants from which Klebsiella pneumoniae, Serratia liquefaciens, and Proteus mirabilis were isolated as possible causative agents of diarrhea were studied. These stools, along with control stool specimens which were collected from infants in the same village of Tamooh (near Cairo, Egypt), were analyzed by frequency-pulsed electron-capture gas chromatography (FPEC-GC). Watery stools and formed stools, to which distilled water was added, were centrifuged, and the supernatant was extracted with organic solvents and derivatized with specific functional group reagents to form electron-absorbing derivatives of carboxylic acids, hydroxy acids, alcohols, and amines. Results from the study showed distinct differences in FPEC-GC profiles of stools positive for K. pneumoniae, S. liquefaciens, and P. mirabilis. The major differences found were that diarrheal stools from which K. pneumoniae was isolated contained acetoin, a hydroxy acid-labeled peak F, and an unidentified amine, peak A. S. liquefaciens diarrheal stools had FPEC-GC profiles like the controls with the exception that an amine, peak A, was detected. The diarrhel stools containing P. mirabilis produced a distinct amine profile.
Assuntos
Diarreia/metabolismo , Fezes/microbiologia , Klebsiella/metabolismo , Proteus/metabolismo , Serratia/metabolismo , Cromatografia Gasosa , Diarreia/microbiologia , Fezes/análise , Humanos , Concentração de Íons de Hidrogênio , LactenteRESUMO
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.
Assuntos
Ácidos Esteáricos/análise , Tuberculose Meníngea/diagnóstico , Cromatografia Gasosa , Humanos , Ácidos Esteáricos/sangue , Ácidos Esteáricos/líquido cefalorraquidianoRESUMO
Disposable reversed-phase chromatography columns were tested for their effectiveness in removing unreacted trichloroethanol (TCE) from derivatized samples for gas-liquid chromatography analysis. Derivatized acidic chloroform extracts of saponified whole cells of Mycobacterium species, spent culture media, and derivatized acidic chloroform extracts of serum and cerebrospinal fluids from patients with tuberculous meningitis were tested. Samples were added to preconditioned reversed-phase chromatography columns, and various solvents and solvent mixtures were tested to determine maximum recovery of the TCE derivatives. With this procedure, we were able to quickly remove the TCE reagent and efficiently recover TCE-derivatized carboxylic acids. Use of these columns improved the reagent cleanup procedure, simplified the derivatization step, permitted increased detection of trace components, such as tuberculostearic acid, in body fluids, and improved the selectivity of the procedure for detection of carboxylic acids.
Assuntos
Líquidos Corporais/análise , Ácidos Carboxílicos/análise , Cromatografia Gasosa , HumanosRESUMO
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.
Assuntos
Líquido Cefalorraquidiano/análise , Meningite/diagnóstico , Nocardiose/diagnóstico , Nocardia/metabolismo , Aminas/sangue , Aminas/líquido cefalorraquidiano , Ácidos Carboxílicos/sangue , Ácidos Carboxílicos/líquido cefalorraquidiano , Cromatografia Gasosa , Diagnóstico Diferencial , Humanos , Hidroxiácidos/sangue , Hidroxiácidos/líquido cefalorraquidiano , Nocardiose/sangue , Nocardiose/líquido cefalorraquidiano , Ácidos Esteáricos/sangue , Ácidos Esteáricos/líquido cefalorraquidiano , Tuberculose Meníngea/sangue , Tuberculose Meníngea/líquido cefalorraquidiano , Tuberculose Meníngea/diagnósticoRESUMO
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.
Assuntos
Esquistossomose/sangue , Análise Química do Sangue , Cromatografia Gasosa , Humanos , Peso Molecular , Contagem de Ovos de Parasitas , Schistosoma haematobium , Schistosoma mansoni , Esquistossomose/parasitologiaRESUMO
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.
Assuntos
Diarreia/microbiologia , Fezes/análise , Shigella/metabolismo , Cromatografia Gasosa , Escherichia coli/metabolismo , Fezes/microbiologia , HumanosRESUMO
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.
Assuntos
Toxinas Bacterianas/análise , Diarreia/microbiologia , Enterotoxinas/análise , Proteínas de Escherichia coli , Escherichia coli/classificação , Fezes/química , Cromatografia Gasosa , Fezes/microbiologia , Humanos , LactenteRESUMO
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.
Assuntos
Aminoácidos/farmacologia , Clostridium/metabolismo , Álcoois/biossíntese , Aminas/biossíntese , Ácidos Carboxílicos/biossíntese , Cromatografia Gasosa/métodos , Meios de Cultura , Eletroquímica , Hidroxiácidos/biossíntese , Espectrometria de MassasRESUMO
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.