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

Abstract

Conditions are described for the detection of tuberculostearic acid (10-methyloctadecanoate; C18 * CH3) in cerebrospinal fluid and serum of patients with tuberculous meningitis. C18. 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 * 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. CH3 standards and C18. 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. CH3 by frequency-pulsed electron capture gas-liquid chromatography may be a valuable aid for early detection of tuberculous meningitis.

Applications of cellular fatty acid analysis.

“…For direct analysis of clinical material in which increased sensitivity may be required, i.e., requiring electron capture rather than flame ionization detection, halogenated CFAs are prepared. Many of the methods pertaining to the preparation of trichloroethanol esters have been developed by Brooks and co-workers (20,21,33) and have been most successfully applied to cerebrospinal fluid (CSF) (21) and diarrheal stool specimens (19). A drawback of these applications is that the sample preparation steps are more involved and time-consuming than those for the preparation of fatty acid methyl esters.…”

“…The sensitivity of GLC, however, can be made to exceed that of DNA probes without amplification. By using an electron capture detector, femtomole (10-15) quantities of a Mycobacterium tuberculosis CFA can be detected (20). Both methods can provide results the same day that tests are begun, both have similar requirements of sample preparation, and both potentially can be adapted to direct detection of pathogens in clinical material.…”

“…Cryptococcal (30,133), enteroviral (24,30,133), herpesvirus (30), measles virus, varicella-zoster virus, Rocky Mountain spotted fever, meningococcal (24), nocardial (22), and tuberculous (21, 30, 51) meningitis have been studied. Modification of GLC resulting in increased sensitivity is central to the direct analysis of clinical specimens (20,85). Preparation of samples appropriate for fatty acid analysis by frequency-pulsed electron capture (FPEC) GLC involves production of pentafluorobenzyl (20, 33) or trichloroethyl (21) derivatives.…”

“…Thus, any of the CFAs uniquely found among prokaryotes could serve as a marker of infection. In CSF, for example, TBSA has been studied most extensively in this regard (20,50,51,84,117). Such markers are useful for diagnosis when detected in normally sterile sites and in the setting of a clinical picture consistent with the agent represented by the CFA marker.…”

Diagnosis of tuberculosis: Available technologies, limitations, and possibilities

“…One easily detectable marker/component of M. tuberculosis is tuberculostearic acid, which can be detected in femtomole quantities by gas-liquid chromatography (7). The presence of tuberculostearic acid in cerebrospinal fluid is thought to be a diagnostic marker for tuberculous meningitis (8-10) and has been suggested to be useful in diagnosing pulmonary tuberculosis as well (11,12).…”