Polar anionic metabolome analysis by nano-LC/MS with a metal chelating agent.

We have developed a practical method for the comprehensive analysis of polar anionic metabolites in biological samples with the use of a nano-LC/MS system. A polyamine-bonded polymer-based apHera NH2 column, which is compatible with ammonium carbonate buffer, effectively retained anionic polar metabolites, such as organic acids, sulfates, and phosphates, but multiply phosphorylated or carboxylated compounds showed highly distorted peak shapes on chromatograms. We found that addition of a trace amount of the metal chelating reagent ethylenediaminetetraacetic acid (EDTA) to the sample solution dramatically improved peak shapes of multiply charged anionic compounds, even though the mass spectra showed no trace of adduct ions in the absence of EDTA. The detection limits of typical polar anionic metabolites in the full-scan mode were from 0.19 to 2.81 pmol. After optimization of all the procedures from sample preparation to nano-LC/MS analysis, we applied our method to real biological samples: Hela cells, mouse brain, human cerebrospinal fluid (CSF), and human plasma. Our results indicated that phosphorylated metabolites were abundant in Hela cells and brain, while plasma and cerebrospinal fluid (CSF) mostly contained organic acids. Phosphorylated compounds might not be secreted into CSF/plasma or might be unstable in CSF/plasma. Finally, the method was used to examine the mode of action of the anticancer drug methotrexate (MTX), which inhibits purine de novo biosynthesis and thymidine biosynthesis. In addition of the expected changes of metabolite levels, we found that a previously unreported metabolite, probably a methylated uridine 5'-triphosphate (UTP), was produced by MTX-treated Hela cells.

A branched-chain organic acid linked to multiple sclerosis: first identification by NMR spectroscopy of CSF.

(1)H NMR spectroscopy of cerebrospinal fluid (CSF) is currently being used to study metabolic profiles characteristic of distinct multiple sclerosis (MS) manifestations. For select MS patient groups, we have previously detected significantly increased concentrations of several identified metabolites and one unidentified compound. We now present, for the first time, the identification of the latter molecule, beta-hydroxyisobutyrate (BHIB). A combination of dedicated 1D and 2D (1)H NMR experiments was employed for signal assignment. To our knowledge, BHIB has not previously been identified in (1)H NMR spectra of biofluids or biological tissues. Our assignment suggests new biochemical pathways involved in specific MS pathologies.

Barth's syndrome-like disorder: a new phenotype with a maternally inherited A3243G substitution of mitochondrial DNA (MELAS mutation).

An Argentine male child died at 4.5 years of age of a lethal mitochondrial disease associated with a MELAS mutation and a Barth syndrome-like presentation. The child had severe failure to thrive from the early months and for approximately two years thereafter. In addition, the patient had severely delayed gross motor milestones, marked muscle weakness, and dilated cardiomyopathy that progressed to congestive heart failure. He also had persistently elevated urinary levels of 3-methylglutaconic and 2-ethylhydracrylic acids and low blood levels of cholesterol. Detailed histopathologic evaluation of the skeletal muscle biopsy showed high activity of succinate dehydrogenase, a generalized decrease of COX activity, and abundant ragged-red fibers. Electron microscopic studies revealed multiple mitochondrial abnormalities in lymphocytes and monocytes, in the striated muscle, and in the postmortem samples (muscle, heart, liver, and brain). Biochemical analysis showed a pronounced and constant lactic acidosis, and abnormal urinary organic acid excretion (unchanged in the fasting and postprandial states). In addition, in CSF there was a marked increase of lactate and beta-hydroxybutyrate (beta-HOB) and also a high systemic ratio beta-HOB/acetoacetate. Enzymatic assay of the respiratory chain in biopsied muscle showed 10% of complex I activity and 24% of complex IV activity compared with controls. Molecular studies of the mitochondrial genome revealed an A to G mutation at nucleotide pair 3243 in mitochondrial DNA, a well-known pathogenetic mutation (MELAS mutation) in all the patient's tissues and also in the blood specimens of the probands mother and sibs (4 of 5). The diagnosis of MELAS mutation was reinforced by the absence of an identifiable mutation in the X-linked G4.5 gene of the propositus. The present observation gives additional evidence of the variable clinical expression of mtDNA mutations in humans and demonstrates that all clinical variants deserve adequate investigation to establish a primary defect. It also suggests adding Barth-like syndrome to the list of phenotypes with the MELAS mutation.

[Gas chromatography of organic acids: methodological and diagnostic aspects]. / Gazovaia khromatografiia organicheskikh kislot: metodicheskie i diagnosticheskie aspekty.

Gas liquid chromatography (GLC) remains the most efficient method for analysis of organic acids in biological fluids. This paper presents a GLC method for measuring organic acids in the urine. Our method of extraction and GLC of trimethylsilyl derivatives of organic acids is a specific and highly sensitive procedure which permits investigation of the complex spectrum of organic acids, including aliphatic oxocarbonic, dicarbonic, hydroxycarbonic, and aromatic acids and glycine conjugates in the urine and other physiological fluids. It can be used for the diagnosis and prenatal screening of hereditary and acquired metabolic disorders.

[The acid-base balance of cerebrospinal fluid]. / Równowaga kwasowo-zasadowa plynu mózgowo-rdzeniowego.

On the basis of literature the mechanisms of acid-base balance in cerebrospinal fluid (CSF) in physiological and pathological conditions were discussed. The author focused on acid-base balance in CSF in the course of meningitis and meningoencephalitis and the influence of its disorders in the pathogenesis of those illnesses of central nervous system.

Quantitative organic acid analysis in cerebrospinal fluid and plasma: reference values in a pediatric population.

Quantitative reference values for the concentrations of organic acids in cerebrospinal fluid (CSF) and plasma, as well as ratios of individual organic acids between CSF and plasma, were determined in twenty-three pairs of samples from pediatric patients. Twenty-six organic acids were present and quantifiable in all or the majority of plasma and CSF specimens (limit of detection 1 mumol/l). There were substantial differences between subgroups of organic acids, best reflected by the ratios of individual acids between CSF and plasma. Metabolites related to fatty acid oxidation were present in CSF in substantially lower amounts than in plasma. Organic acids related to carbohydrate and energy metabolism and to amino acid degradation were present in CSF in equal or slightly lower 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. Quantitation of organic acids in CSF and plasma should aid diagnosis and monitoring of treatment of patients with organic acid disorders.

Use of a thick-film capillary column for the analysis of organic acids in body fluids.

An improved method for the identification and quantification of organic acids in body fluids employing capillary gas chromatography-mass spectrometry has been developed. A thick-film capillary column, that combines the properties of a capillary column with those of a megabore column, has been successfully introduced into an existing method. Analysis over a concentration range from 1 mumol/l to 500 mumol/l body fluid is possible. This permits the assay of samples that are usually obtained in small volumes, e.g. cerebrospinal fluid.

Organic acids in post-mortem cerebrospinal fluid.

Organic acids of cerebrospinal fluid (CSF) have been determined by gas chromatography-mass spectrometry in 29 post-mortem samples obtained from infants and 10 samples obtained from hospitalized children, as controls. Though the organic acids profile was similar in the two groups, eight organic acids not observed in CSF from live infants were inconstantly found in post-mortem CSF and for three of them, malic acid, lactyllactic acid and uracile, concentrations were correlated with the delay in sampling.

Isotachophoretic separation of organic acids in biological fluids.

The operating conditions for the isotachophoretic separation of organic acids were evaluated. At pH values ranging from 2.90 to 4.25 both relative step heights and molar flow-rates were determined experimentally for 26 anions. Comparing the observed values with simulated data, highly significant (p = 0.0001) correlation coefficients of 0.993 and 0.920, respectively, were found at pH 3.50. Whereas the concentration of the leading electrolyte did not affect the relative step heights, it increased the molar flow-rates significantly. The same applied to the detection current. The time of analysis was observed to be a function of the concentration of the leading electrolyte. However, the time elapsed between injection of the analyte and its detection depended solely on the volume and not on the amount of analyte injected. In isotachophoresis, incomplete separation of two compounds is indicated by the occurrence of a mixed zone which can hardly be distinguished from a pure zone. Thus, knowledge of the separation capacity is a prime prerequisite in optimizing the system for the analysis of biological fluids. The separability of nine equimolar pairs of anions was determined at pH values ranging from 2.90 to 4.25. Although two ionogenic constituents would separate only when their migration rates in the mixed state were different, no clear correlation was observed between separation capacity and difference in relative mobility. Separability, however, was found to increase with increasing concentration of the leading electrolyte. While the separation capacity was not influenced by the electric current, it was significantly affected by the volume injected. In subsequent analyses of serum, cerebrospinal fluid, seminal plasma and prostatic fluid, a variety of organic acids could be detected. Calibration graphs for the detected anions revealed a detection limit of 1 nmol and linearity over their biological concentration ranges. Further, the isotachophoretic results correlated well with high-performance liquid chromatographic and enzymatic analyses of citric acid and lactic acid in human seminal plasma and cerebrospinal fluid, respectively.