Manganese speciation in human cerebrospinal fluid using CZE coupled to inductively coupled plasma MS.

The neurotoxic effects of manganese (Mn) at elevated concentrations are well known. This raises the question, which of the Mn species can cross neural barriers and appear in cerebrospinal fluid (CSF). CSF is the last matrix in a living human organism available for analysis before a compound reaches the brain cells and therefore it is assumed to reflect best the internal exposure of brain tissue to Mn species. A previously developed CE method was modified for separation of albumin, histidine, tyrosine, cystine, fumarate, malate, inorganic Mn, oxalacetate, alpha-keto-glutarate, nicotinamide-dinucleotide (NAD), citrate, adenosine, glutathione, and glutamine. These compounds are supposed in the literature to act as potential Mn carriers. In a first attempt, these compounds were analyzed by CZE-UV to check whether they are present in CSF. The CZE-UV method was simpler than the coupled CZE-inductively coupled plasma (ICP)-dynamic reaction cell (DRC)-MS method and it was therefore chosen to obtain a first overview information. In a second step, the coupled method (CZE-ICP-DRC-MS) was used to analyze, in detail, which of the compounds found in CSF by CZE-UV were actually bound to Mn. Finally, 13 Mn species were monitored in CSF samples, most of them being identified: Mn-histidine, Mn-fumarate, Mn-malate, inorganic Mn, Mn-oxalacetate, Mn-alpha-keto glutarate, Mn-carrying NAD, Mn-citrate and Mn-adenosine. By far the most abundant Mn species was Mn-citrate showing a concentration of 0.7 +/- 0.13 microg Mn/L. Interestingly, several other Mn species can be related to the citric acid cycle.

Elevation of 2-methylcitric acid I and II levels in serum, urine, and cerebrospinal fluid of patients with cobalamin deficiency.

Citrate synthase catalyzes the condensation of acetyl-coenzyme A (CoA) and oxaloacetic acid to form citric acid. The enzyme also catalyzes the condensation of propionyl-CoA and oxaloacetic acid with a maximal reaction velocity (Vmax) approximately 10(-4) times that of acetyl-CoA to form 2-methylcitric acid, which contains two asymmetric carbon atoms and exists as two pairs of related enantiomers designated as 2-methylcitric acid I and II. Cobalamin (Cbl) deficiency can lead to increases in intracellular levels of propionyl-CoA. To assess the magnitude of increased synthesis of 2-methylcitric acid in Cbl deficiency, we developed a new capillary gas chromatographic-mass spectrometric assay and measured 2-methylcitric acid levels in serum and cerebrospinal fluid (CSF) of normal subjects and patients with clinically confirmed Cbl deficiency. The normal range for 2-methylcitric acid level was 60 to 228 nmol/L for serum in 50 normal blood donors and 323 to 1,070 nmol/L for CSF in 19 normal subjects. In 50 patients with clinically confirmed Cbl deficiency, values for 2-methylcitric acid in serum ranged from 93 to 13,500 nmol/L; 44 (88%) had values above the normal range. In five patients with clinically confirmed Cbl deficiency, levels of the sum of 2-methylcitric acid I and II ranged from 1,370 to 16,300 nmol/L in CSF, and all five (100%) patients had levels above the normal range. We conclude that levels of 2-methylcitric acid are elevated in serum and CSF of most patients with Cbl deficiency.

NMR spectroscopy of human post mortem cerebrospinal fluid: distinction of Alzheimer's disease from control using pattern recognition and statistics.

1H NMR spectra have been measured at 500 and 600 MHz on 23 human cerebrospinal fluid samples obtained at autopsy from Alzheimer's disease patients and controls. The spectra at 500 MHz were quantified using 42 descriptors based on NMR peak heights and it was shown that differences between the two classes were apparent in the delta 2.4-2.9 region. Remeasured at 600 MHz a detailed examination of this chemical shift range identified citrate, aspartate, N-acetyl aspartate, methionine and glutamate in this region of the spectra. Principal components analysis showed that a separation of the two classes was possible and detailed statistics indicated that citrate level was the principal marker. Patient age and the interval between death and autopsy (parameters not closely matched between the two groups) were examined statistically to establish whether these might account for the citrate differences. Although they could possibly account for them to some extent, the relationship between citrate levels and disease state remained significant at p < 0.05. The data invite a test of the importance of citrate levels in Alzheimer's disease using samples taken ex vivo.

The Rett syndrome and CSF lactic acid patterns.

We investigated both blood and cerebrospinal fluid (CSF) lactate and pyruvate levels in seven girls with the Rett syndrome (RS) and evaluated the relationship between CSF lactate and pyruvate levels and the clinical manifestations, particularly seizures, anticonvulsant medication, and breathing dysfunction including breath holding, apnea and hyperventilation. Elevated lactate and pyruvate levels in CSF with normal serum lactate were found in two RS patients. Elevated CSF lactate correlated significantly with the clinical occurrence of hyperventilation (P0 = 0.048, Fisher exact probability). We measured native and dichloroacetate (DCA)-activated pyruvate dehydrogenase (PDH) complex activities in two patients (#1 and 2) using cultured lymphoblastoid cell lines which were transformed by EB virus and the results were normal. We also analyzed CSF citric acid intermediates from 7 RS patients including citric acid, cis-aconitate, alpha-ketoglutarate, succinate, fumarate, malate and oxaloacetate. These concentrations were not significantly different from those control patients (N = 21). An elevated lactate level may be a clue to clarify the etiology of RS.

High resolution proton nuclear magnetic resonance studies of human cerebrospinal fluid.

One- and two-dimensional (correlated shift spectroscopy) high resolution proton n.m.r. spectra of human cerebrospinal fluid (CSF) are reported. The merits of water suppression by freeze drying or irradiation, and spectral simplification by spin-echo methods, are discussed. Well-resolved resonances for a range of low molecular weight metabolites such as lactate, 3-D-hydroxybutyrate, alanine, acetate, citrate, glucose, valine and formate were observed. Resonances for glutamine were observed only from freeze dried samples. Concentrations determined by n.m.r. were in reasonable agreement with those from conventional methods. The n.m.r. spectra of CSF were related to the clinical conditions of the subjects. No resonances for citrate were present in spectra of CSF from subjects (three infants) with bacterial meningitis; high lactate and lowered glucose levels were observed. Strong resonances for glucose and glycine were observed for mildly diabetic subjects. Both the aromatic and the aliphatic regions of the CSF spectra from subjects suffering from liver failure contained distinctive features characteristic for hepatic coma: Intense resonances for lactate, alanine, valine, methionine, tyrosine, phenylalanine and histidine. In some cases guanine was also present, which does not appear to have been reported previously. The two-dimensional spectrum suggested the presence of abnormally high levels of a number of endogenous metabolites. Such assignments were not possible using one-dimensional spectra alone because of signal overlap.

Depression and cerebrospinal fluid citrate.

In relation to studies of calcium metabolism in manic-melancholic patients, the calcium binding metabolite citrate was analyzed in cerebrospinal fluid from 163 psychiatric and non-psychiatric patients. Patients with symptoms of depression, irrespective of whether the origin of the depression was endogenous or exogenous, had significantly higher cerebrospinal fluid citrate concentrations than non-psychiatric patients. Manic patients and other psychiatric patients without symptoms of depression were not significantly different from the non-psychiatric patients. When the mood was normalized after various treatments, cerebrospinal fluid citrate decreased in all groups of depressed patients.

Carbohydrate metabolism in motor neurone disease.

Eight patients with classical motor neurone disease, and eight control patients with neurological disease and matched for degree of wasting, were studied. Both groups had abnormal glucose tolerance, but the patients with motor neurone disease had a significantly lower insulin response both to oral glucose loading and to intravenous tolbutamide. These results suggest that in patients with motor neurone disease there is an impaired synthesis or release of insulin due to islet cell damage. Blood pyruvate and lactate, and cerebrospinal fluid pyruvate, lactate, and citrate, did not differ significantly from the control group. Blood citrate levels were significantly higher in patients with motor neurone disease compared with the controls. Triglyceride levels were raised in patients with motor neurone disease compared to the control patients. This may be secondary to the increased citrate levels.

Hyperglycinemia and propionyl coA carboxylase deficiency and episodic severe illness without consistent ketosis.

Propionyl CoA carboxylase deficiency was found in a 7-month-old boy who presented with attacks of vomiting, anorexia, weight loss, weakness, and hypotonia. He failed to thrive and had generalized seizures. He had propionic acidemia and hyperglycinemia; these are the manifestations of the ketotic hyperglycinemia syndrome. However, ketonuria was not a consistent part of his clinical picture, and he had at least two episodes of acute overwhelming illness, the latter one fatal, in which ketones were never found in the urine. Large amounts of pyrrolidone carboxylic acid were found in body fluids.