Metabolic profiling of aromatic compounds in cerebrospinal fluid of neurosurgical patients using microextraction by packed sorbent and liquid-liquid extraction with gas chromatography-mass spectrometry analysis.

A new approach to the quantitative analysis of aromatic metabolites in cerebrospinal fluid samples of neurosurgical patients based on microextraction by packed sorbent coupled with derivatization and GC-MS was developed. Analytical characteristics such as recoveries (40-90%), limit of detection (0.1-0.3 µm) and limit of quantitation (0.4-0.7 µm) values, accuracy (<±20%), precision (<20%) and linear correlations (R2 ≥ 0.99) over a 0.4-10 µm range of concentrations demonstrated that microextraction by packed sorbent provides results for the quantitative analysis of target compounds comparable with those for liquid-liquid extraction. Similar results were achieved using 40 µl of sample for microextraction by packed sorbent instead of 200 µl for liquid-liquid extraction. Benzoic, 3-phenylpropionic, 3-phenyllactic, 4-hydroxybenzoic, 2-(4-hydroxyphenyl)acetic, homovanillic and 3-(4-hydroxyphenyl)lactic acids were found in cerebrospinal fluid samples (n = 138) of neurosurgical patients in lower concentrations than in serum samples (n = 110) of critically ill patients. Analysis of the cerebrospinal fluid and serum samples taken at the same time from neurosurgical patients (n = 5) revealed similar results for patients without infection and multidirectional results for patients with central nervous system infection. Our preliminary results demonstrate the necessity of further evaluating the aromatic compound profile in cerebrospinal fluid for its subsequent verification for potential diagnostic markers.

Isotope Corrected Chiral and Achiral Nontargeted Metabolomics: An Approach for High Accuracy and Precision Metabolomics Based on Derivatization and Its Application to Cerebrospinal Fluid of Patients with Alzheimer's Disease.

We propose a chiral metabolomics approach based on a data-dependent MS/MS analysis (DDA) using high-resolution quadrupole-time-of-flight mass spectrometry (Q-TOFMS) and 13C-isotope coded derivatization (ICD) reagents, i.e., iDMT-( S)-A and iDMT-( S)-PO. The advantage of the method is the correction of all detected derivatives by parallel derivatization of the isotope-coded and noncoded reagents. The automatic data analysis platform using an MSDIAL and ICD discrimination program, called DINA, was also developed and used for the data analysis process. As a result, a 0.5-2.0% (d-/l-isomer) variation of the isomers was correctly recognized in the automatic data analysis step. Both the semiquantitative comparison and identification efficiency were improved as a result of the high resolution/accuracy of the MS and MS/MS spectra derived from the DDA analysis. This method was used for biomarker discovery in the cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD). Twenty-four biomarker candidates were successfully determined, including 8 chiral ones.

Amino compounds and organic acids in CSF, plasma, and urine of autistic children.

Amino compounds were measured with an amino acid analyzer in the fasting plasma of 34 patients with childhood psychoses (28 having infantile autism) and 40 control children, and in the cerebrospinal fluid (CSF) of 19 of the psychotic children and 23 control children. Organic acids were determined by gas chromatography in urine, plasma, and CSF of the psychotic patients. The mean concentration of ethanolamine in CSF was significantly higher in psychotic children than in control subjects. A subgroup of autistic children may possibly have a brain disorder involving ethanolamine metabolism. None of the known inherited diseases of organic acid metabolism was found in any of the psychotic children, but future studies utilizing sophisticated gas chromatography--mass spectrometry--computer techniques might disclose abnormal organic acid content in the CSF of such patients.

Acidic amino acid accumulation by rat choroid plexus during development.

Acidic amino acid accumulation by the choroid plexuses of the lateral ventricles was investigated using 1, 2, 3 week and adult (7-10 weeks old) rats. The accumulation from both blood and CSF sides of the choroid plexuses were investigated. The uptake from blood side was studied using the bilateral in situ brain perfusion, and time-dependent uptake profiles (2, 10, 20, and 30 min) of 14C-labelled aspartate, glutamate, and NMDA were measured. [3H]Mannitol was also included in perfusion fluid as a baseline for [14C]amino acid uptake into choroidal tissue. Uptake of [14C]aspartate and [14C]glutamate declined with age, while [14C]NMDA showed no significant uptake at any age. Twenty min [3H]mannitol uptake in the 1-week-old rat was significantly greater than the adult (P < 0.05). The K(m) for [14C]aspartate and [14C]glutamate obtained from multiple time uptake profiles also showed reduction with development but it was greater than that for mannitol. [14C]Aspartate declined from 69.8 +/- 21.1 microliters.min-1.g-1 in the neonate to 40.6 +/- 4.0 microliters.min-1.g-1 in the adult (P < 0.05), while glutamate showed a sharper decline from 78.9 +/- 24.2 microliters.min-1.g-1 to 17.7 +/- 5.4 microliters.min-1.g-1 (P < 0.01). Accumulation of 14C-labelled aspartate and glutamate by the choroid plexus from CSF side was also measured using ventriculo-cisternal perfusion. The accumulation in the adult was found to be 2-3 times greater than that in the neonatal rat (P < 0.05) for both amino acids. The uptake from either side was found to be saturable, stereospecific, not inhibited by neutral amino acid analogues, and shared by both aspartate and glutamate.

Changes in the kinetics of the acidic amino acid brain and CSF uptake during development in the rat.

Using a bilateral in situ brain perfusion technique, the rate of influx of the acidic amino acids, aspartate and glutamate, into both brain and CSF, were measured in the rat. The kinetic constants for uptake of these amino acids across the blood-brain and blood-CSF barriers in neonatal (1-week-old) and adult (7-10 weeks-old) rats were calculated; the half saturation constant (K(m)) at both barriers did not change with age, whereas the maximal transport (Vmax) at both barriers was greater in the younger age group, and reduced by more than 50% with maturity. The diffusion constant Kd at the blood-brain barrier was not different from zero at either age, although at the blood-CSF barrier there was some diffusion at both ages, which did not change with maturity. The entry of these amino acids into the neonatal brain shown in our previous study can be explained by a greater maximal transport in the neonates which, coupled with the elevated plasma amino acid concentrations of the young animal, would result in higher blood-to-brain and blood-to-CSF flux in the neonate.

Microbore HPLC method with online microdialysis for measurement of topotecan lactone and carboxylate in murine CSF.

We developed a chromatography method to measure lactone and carboxylate forms of topotecan (TPT) in mouse cerebrospinal fluid (CSF) using microdialysis sampling. The chromatography method utilized a microbore (0.8 mm) column. Analytes, which eluted in less than 5 min, were detected with a fluorescence detector. The calibration range was 0.25-100.0 ng/mL for both forms. The within-day and between-day precision was < or =16% for 0.8 ng/mL and < or =8.0% for 3, 12, and 80 ng/mL. Accuracy was +/-15% (0.8 and 3 ng/mL) and +/-10% (12 and 80 ng/mL). TPT lactone hydrolyzes to the carboxylate during sampling, so we developed an equation and parameters to describe the TPT lactone hydrolysis in artificial CSF (aCSF). After TPT administration, CSF dialysate samples (2 microL) were analyzed for lactone and carboxylate using online injection. The hydrolysis of each dialysate sample was then estimated and a correction applied. We conclude that this HPLC method coupled with online microdialysis sampling allows for the rapid measurement of both TPT forms in small volumes of murine CSF dialysate. The system allows for the determination of TPT pharmacokinetics in murine CSF and provides a tool to extend pharmacological studies in this brain compartment.

Plasma and cerebrospinal fluid pharmacokinetics of tasidotin (ILX-651) and its metabolites in non-human primates.

PURPOSE: To evaluate the plasma and cerebrospinal fluid (CSF) pharmacokinetics and CSF penetration of tasidotin and metabolites in a nonhuman primate model. METHODS: Tasidotin 0.75 mg/kg was administered intravenously. The plasma and CSF concentrations of tasidotin and its metabolites were determined. Pharmacokinetic parameters were estimated using model-independent and model-dependent methods. RESULTS: The mean (+/-SD) CSF:plasma AUC ratio for tasidotin was 1.1 +/- 0.4. For tasidotin, tasidotin-C-carboxylate and desprolyl-tasidotin-C-carboxylate the plasma AUCs (mean +/- SD) were 30 +/- 10, 54 +/- 19 and 12 +/- 2 microM min, and apparent plasma half-lives were 27 +/- 4, 229 +/- 73 and 100 +/- 29 min. The plasma clearance of tasidotin was 44 +/- 14 ml/min/kg. The CSF AUC and half-life of tasidotin was 28 +/- 10 microM min and 96 +/- 40 min. The model-dependent plasma clearance was 35 ml/min/kg for tasidotin and 2 ml/min/kg for tasidotin-C-carboxylate. CONCLUSIONS: Tasidotin penetrates into the CSF well and further evaluation of its activity in the treatment of central nervous system malignancies should be considered.

Direct sample injection for capillary electrophoretic determination of organic acids in cerebrospinal fluid.

Organic acids in cerebrospinal fluid (CSF) are potential diagnostic markers for neurological diseases and metabolic disorders. A capillary electrophoretic (CE) method for the direct analysis, i.e., without any sample preparation, of six organic acids in CSF was developed. A capillary coating consisting of a triple layer of charged polymers (polybrene-dextran sulfate-polybrene) was used in combination with a negative separation voltage, providing fast and efficient analysis of acidic compounds. Separation conditions, such as background electrolyte (BGE) concentration and pH were optimized, and the influence of albumin and sodium chloride was systematically studied using a set of test compounds. With injection volumes of ca. 44 nL, plate numbers of up to ca. 150,000 were obtained with a BGE of 200 mM sodium phosphate (pH 6.0). It appeared that high sodium chloride concentrations in the sample hardly affected the peak width and shape of the organic acids, most probably due to transient isotachophoresis effects occurring in the sample zone. Adverse effects of CSF proteins, which frequently compromise the CE performance, could be effectively minimized by the triple layer coating in combination with rinses of 0.1 M hydrochloric acid. Overall, the developed CE system allowed direct injections of CSF samples, yielding good separation efficiencies and stable migration times (RSDs<2%) for organic acids. Validation of the method with artificial and real CSF samples showed good linear responses (r>0.99), and LODs for the organic acids were in the range of 2-8 microg/mL when applying UV detection. RSDs for migration times and peak areas were <2% and <7%, respectively. The applicability of the CE system is shown for the determination of organic acids in CSF samples.

GC-MS determination of organic acids with solvent extraction after cation-exchange chromatography.

We combined column and partition chromatography to isolate, purify, and quantify biological organic acids in urine and cerebrospinal fluid (CSF). Urine and CSF samples were introduced onto a preconditioned cation-exchange column (Dowex 50 W x 8 resin) to remove the biological interferences. The effluent with water was extracted with ethyl acetate two times (pH 1 and 3) and the organic acids were quantitatively converted into their trimethylsilyl derivatives for detection by gas chromatography-mass spectrometry. The good quality-control data were obtained through precision and accuracy tests. Inter- and intraassay CVs were 0.01-10.2% and 0.02-12.2%, respectively. Analytical recoveries compared favorably with results from the commonly used solvent extraction method. This method was used for the measurement of the 14 organic acids in the urine and CSF of healthy volunteers. The values obtained were in the range of the published data.

Physiology and pathophysiology of organic acids in cerebrospinal fluid.

Concentrations of organic acids in cerebrospinal fluid (CSF) appear to be directly dependent upon their rate of production in the brain. There is evidence that the net release of short-chain monocarboxylic acids from the brain is a major route for removing these products of cerebral metabolism. Concentrations of organic acids in blood and CSF are largely independent of each other. Quantitative reference values for the concentrations of organic acids in CSF and plasma as well as ratios of individual organic acids between CSF and plasma were determined in 35 pairs of samples from paediatric patients. Over 25 organic acids were quantifiable in all or in the majority of CSF and/or plasma specimens (limit of detection 1 mumol/L). There were substantial differences in the CSF/plasma ratios between subgroups of organic acids. Metabolites related to fatty-acid oxidation were present in CSF in substantially less amounts than in plasma. Organic acids related to carbohydrate and energy metabolism and to amino acid degradation were present in CSF in the same amounts as or slightly smaller amounts than in plasma. Finally, some organic acids were found in substantially higher amounts in CSF than in plasma, e.g. glycolate, glycerate, 2,4-dihydroxybutyrate, citrate and isocitrate. Studies of organic acids in CSF and plasma samples are presented from patients with 'cerebral' lactic acidosis, disorders of propionate and methylmalonate metabolism, glutaryl-CoA dehydrogenase deficiency and L-2-hydroxy-glutaric aciduria. It became apparent that derangements of organic acids in the CSF may occur independently of the systemic metabolism. Quantitative organic acid analysis in CSF will yield new information on the pathophysiology in the central nervous system (CNS) of these disorders and may prove necessary for successful monitoring of treatment of organoacidopathies, which present mainly with neurological disease. For example, in glutaryl-CoA dehydrogenase deficiency the urinary excretion of glutarate appears to be an inadequate parameter for monitoring the effect of dietary therapy, without plasma and CSF determinations. In L-2-hydroxyglutaric aciduria the elevation of L-2-hydroxyglutarate was found to be greater in CSF than in plasma. In addition, some other organic acids, glycolate, glycerate, 2,4-dihydroxybutyrate, citrate and isocitrate, were also elevated in the CSF of the patients out of proportion to normal levels in plasma and urine. High concentrations of an unknown compound, which was tentatively identified as 2,4-dihydroxyglutarate, were found in the CSF of patients with L-2-hydroxyglutaric aciduria.(ABSTRACT TRUNCATED AT 400 WORDS)

Gas-liquid chromatographic profile of neutral and acidic metabolites in cerebrospinal fluid from newborns and infants.

A profile (chromatographic pattern) of the neutral and acidic metabolites present in cerebrospinal fluid from newborns and infants was obtained by gas-liquid chromatography. The metabolites are those extracted with ethyl acetate and diethyl ether. They are converted to trimethylsilyl ether derivatives, and chromatographed on a column containing 5% silicone OV-101 (methyl) packed on Chromosorb W. Several substances were tentatively identified from their methylene unit values. We established a control profile for infants, and compared profiles for infants and adults. Noteworthy qualitative and quantitative differences from the control were seen for cerebrospinal fluid from subjects with neurological disorders of infections. The technique may be of use in neurological diagnosis.

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%.

Beta-methylcrotonic aciduria associated with lactic acidosis.

A patient is described in whom lactic acidosis of very severe degree was found to coincide with the presence of beta-methylcrotonic acid and rho-hydroxyphenyllactic acid in urine in large amounts, while beta-hydroxyisovaleric acid was found to be a relatively minor excretion product. Beta-methylcrotonic acid is demonstrated, for the first time, to be present in blood and CSF. These findings are discussed in relation to the patients previously reported to have beta-methylcrotonylglycinuria and raise the possibility that our patient's organic aciduria may be secondary to acquired disease rather than to an inborn error of metabolism.

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.