Metabolomic profiling of CSF in multiple sclerosis and neuromyelitis optica spectrum disorder by nuclear magnetic resonance.

Multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) are inflammatory diseases of the central nervous system. Although several studies have characterized the metabolome in the cerebrospinal fluid (CSF) from MS and NMOSD patients, comparative analyses between them and between the relapse and the remission of each disease have not been performed. Both univariate and multivariate analyses were used to compare 1H-NMR spectra of CSF from MS, NMOSD, and healthy controls (HCs). The statistical analysis showed alterations of eight metabolites that were dependent on the disease. Levels of 2-hydroxybutyrate, acetone, formate, and pyroglutamate were higher and levels of acetate and glucose were lower in both MS and NMOSD. Citrate was lower in MS patients, whereas lactate was higher in only NMOSD specifically. The shared feature of metabolic changes between MS and NMOSD may be related to altered energy metabolism and fatty acid biosynthesis in the brain. Another analysis to characterize relapse and remission status showed that isoleucine and valine were down-regulated in MS relapse compared to MS remission. The other metabolites identified in the disease comparison showed the same alterations regardless of disease activity. These findings would be helpful in understanding the biological background of these diseases, and distinguishing between MS and NMOSD, as well as determining the disease activity.

Phase 1 dose-escalation, pharmacokinetic, and cerebrospinal fluid distribution study of TAK-285, an investigational inhibitor of EGFR and HER2.

INTRODUCTION: This phase 1 study assessed safety, maximum tolerated dose (MTD), pharmacokinetics, cerebrospinal fluid (CSF) distribution, and preliminary clinical activity of the receptor tyrosine kinase inhibitor TAK-285. METHODS: Patients with advanced, histologically confirmed solid tumors and Eastern Cooperative Oncology Group performance status ≤2 received daily oral TAK-285; daily dose was escalated within defined cohorts until MTD and recommended phase 2 dose (RP2D) were determined. Eleven patients were enrolled into an RP2D cohort. Blood samples were collected from all cohorts; CSF was collected at pharmacokinetic steady-state from RP2D patients. Tumor responses were assessed every 8 weeks per Response Evaluation Criteria in Solid Tumors. RESULTS: Fifty-four patients were enrolled (median age 60; range, 35-76 years). The most common diagnoses were cancers of the colon (28 %), breast (17 %), and pancreas (9 %). Escalation cohorts evaluated doses from 50 mg daily to 500 mg twice daily; the MTD/RP2D was 400 mg twice daily. Dose-limiting toxicities included diarrhea, hypokalemia, and fatigue. Drug absorption was fast (median time of maximum concentration was 2-3 h), and mean half-life was 9 h. Steady-state average unbound CSF concentration (geometric mean 1.54 [range, 0.51-4.27] ng/mL; n = 5) at the RP2D was below the 50 % inhibitory concentration (9.3 ng/mL) for inhibition of tyrosine kinase activity in cells expressing recombinant HER2. Best response was stable disease (12 weeks of nonprogression) in 13 patients. CONCLUSIONS: TAK-285 was generally well tolerated at the RP2D. Distribution in human CSF was confirmed, but the free concentration of the drug was below that associated with biologically relevant target inhibition.

A new method for the HPLC determination of gamma-hydroxybutyric acid (GHB) following derivatization with a coumarin analogue and fluorescence detection: application in the analysis of biological fluids.

A new method of the determination of gamma-hydroxybutyric acid (GHB) in human biological fluids cerebrospinal fluid (CSF) and saliva after off-line derivatization is described. The proposed method was based on the reaction of 4-bromomethyl-7-methoxy coumarin (Br-MMC) with GHB in the presence of dibenzo-18-crown-6-ether (acting as reaction catalyst) to produce a fluorescent derivative. The formed derivative was monitored fluorimetrically at lambda(ext.)=330 nm and lambda(em.)=390 nm. The effect of derivatization parameters such as the concentration of Br-MMC, reaction time and the temperature was investigated in order to achieve the maximum method's sensitivity. The separation was achieved by use of a C(18) analytical column (Kromasil 250 mm x 4 mm i.d., 5 microm) while the injected sample volume was set to 25 microL. A binary gradient elution program of methanol versus phosphate buffer (40 mM, pH 3) was selected for the quantitative analysis of GHB. The method showed satisfactory linearity (R(2)=0.9979) in a linear range from 2.4 x 10(-6) to 7.2 x 10(-5) M. Ultrafiltration method was employed for the pre-treatment of the cerebrospinal fluid (CSF) prior to the analysis of GHB. The limit of detection (LOD) of the method was 3 x 10(-7) M in saliva and 2 x 10(-7) M in CSF samples, respectively, while the limit of quantitation (LOQ) was 1 x 10(-6) M for both specimens. The proposed protocol offers sensitivity comparing with the existing HPLC analytical methods or the CE indirect UV methods and can function as an attractive alternative to be used in clinical and toxicological analysis.

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.

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.

D-2-hydroxyglutaric aciduria in three patients with proven SSADH deficiency: genetic coincidence or a related biochemical epiphenomenon?

Succinic semialdehyde dehydrogenase (SSADH) deficiency and D-2-hydroxyglutaric aciduria (D-2-HGA) are rare inborn errors of metabolism primarily revealed by urinary organic acid screening. Three patients with proven SSADH deficiency excreted, in addition to GHB considerable amounts of D-2-HG. We examined whether these patients suffered from two inborn errors of metabolism by measuring D-2-HG concentrations in the culture medium of cells from these patients. In addition, mutation analysis of the D-2-hydroxyglutarate dehydrogenase gene was performed. Normal concentrations of D-2-HG were measured in the culture media of fibroblasts or lymphoblasts derived from the three patients. In one patient, we found a heterozygous likely pathogenic mutation in the D-2-hydroxyglutarate dehydrogenase gene. These combined results argue against the hypothesis that the patients are affected with "primary" D-2-HGA in combination with their SSADH deficiency. Moderately increased levels of D-2-HG were also found in urine, plasma, and cerebrospinal fluid samples derived from 12 other patients with SSADH deficiency, revealing that D-2-HG is a common metabolite in this disease. The increase of D-2-HG in SSADH deficiency can be explained by the action of hydroxyacid-oxoacid transhydrogenase, a reversible enzyme that oxidases GHB in the presence of 2-ketoglutarate yielding SSA and D-2-HG.

Sedation with 4-hydroxybutyric acid: a potential pitfall in the diagnosis of SSADH deficiency.

Deficiency of succinic semialdehyde dehydrogenase (SSADH) is a rare neurometabolic disorder with accumulation of 4-hydroxybutyric acid (4-HBA) as a biochemical hallmark. We present a boy with an unresolved severe neurological disorder and intermittent elevation of 4-HBA in serum and CSF which was later shown to result from iatrogenic administration of 4-HBA for sedation purposes.

Increased cerebrospinal fluid glutamine levels in depressed patients.

BACKGROUND: There is increasing evidence for an association between alterations of brain glutamatergic neurotransmission and the pathophysiology of affective disorders. METHODS: We studied the association between cerebrospinal fluid (CSF) metabolites, including glutamine, in unipolar and bipolar depressed patients versus control subjects using a proton magnetic resonance spectroscopy technique. Cerebrospinal fluid samples were obtained from 18 hospitalized patients with acute unmedicated severe depression without medical problems and compared with those of 22 control subjects. RESULTS: Compared with the control group, the depressed patient group had significantly higher CSF glutamine concentrations, which correlated positively with CSF magnesium levels. CONCLUSIONS: These findings suggest an abnormality of the brain glial-neuronal glutamine/glutamate cycle associated with N-methyl-D-aspartate receptor systems in patients with depression.

beta-Hydroxybutyrate fuels synaptic function during development. Histological and physiological evidence in rat hippocampal slices.

To determine whether ketone bodies sustain neuronal function as energy substrates, we examined the effects of beta-hydroxybutyrate (betaHB) on synaptic transmission and morphological integrity during glucose deprivation in rat hippocampal slices. After the depression of excitatory postsynaptic potentials (EPSPs) by 60 min of glucose deprivation, administration of 0.5-10 mM D-betaHB restored EPSPs in slices from postnatal day (PND) 15 rats but not in slices from PND 30 or 120 rats. At PND 15, adding D-betaHB to the media allowed robust long-term potentiation of EPSPs triggered by high frequency stimulation, and prevented the EPSP-spike facilitation that suggests hyperexcitability of neurons. Even after PND 15,D-betaHB blocked morphological changes produced by either glucose deprivation or glycolytic inhibition. These results indicate that D-betaHB is not only able to substitute for glucose as an energy substrate but is also able to preserve neuronal integrity and stability, particularly during early development.

ICV beta-hydroxybutyrate: effects on food intake, body composition, and body weight in rats.

This study examined the effect of long-term intracerebroventricular (ICV) infusion of beta-hydroxybutyrate (beta HB) on food intake, diet selection, body weight, and body composition in rats. Female rats were divided into 2 groups and implanted with a 28-day osmotic pump connected to a lateral cerebroventricular cannula. One group was infused with artificial cerebrospinal fluid (aCSF) and the other with beta HB for 28 days. The rats had free access to both a high-fat/low-carbohydrate and a low-fat/high-carbohydrate diet (isocaloric) for the 28-day infusion period. The group infused with beta HB had a significantly lower body weight gain during the infusion period. Cumulative food intake increased in the same manner in both groups. Fat pad weights and carcass lipid content were significantly higher in beta HB rats, despite the equivalent caloric intake in both groups and the decreased body weight. Our observations are in accord with earlier studies indicating that beta HB infused ICV reduces body weight, but not necessarily food intake. Increased adiposity in association with decreased body weight change in beta HB-infused rats strongly suggests that energy is being partitioned to fat deposition at the expense of lean tissue growth when ketone bodies are infused into the cerebroventricles.

CSF concentration and CSF/blood ratio of fuel related components in children after prolonged fasting.

In order to obtain information about blood and cerebrospinal fluid (CSF) concentrations, and CSF/blood ratio data of fuel related substrates at the end of a prolonged fast in children, we have selected biochemical data from fasting test procedures in 11 control children aged 3-5 yr, fasted 24 h, and 58 control children aged 6-15 yr, fasted 40 h. There was a good correlation between blood and CSF concentrations for glucose, acetoacetate and beta-hydroxybutyrate. The relation with age and sex has been analyzed only in the older children. CSF and blood values for glucose are positively related with age, and both ketones are negatively related with age. Lactate, pyruvate and alanine concentrations in blood and CSF are not related with age, except for CSF pyruvate. With respect to the CSF/blood ratio for the above mentioned components, only the value for acetoacetate is sex and age related. The calculated median caloric values for the sum of glucose, lactate, pyruvate and ketones in CSF are independent of age at the end of a 40-h fast. The diminished glucose contribution on the CSF caloric homeostasis in younger children is fully compensated by the ketone bodies.

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.

Plasma and central nervous system kinetics of gamma-hydroxybutyrate.

Sodium gamma-hydroxybutyrate was administered to dogs in dosages of 200 to 1000 mg per kg with continuous EEG and temperature monitoring. Based on these studies, pulsed doses of 500 mg/kg of GHB were given and timed serum, cerebrospinal fluid (CSF) and brain samples obtained. In addition a group of animals were sacrificed 60 minutes after GHB administration and the regional distribution of GHB determined. The CSF kinetics of GBH suggests a passive diffusionof GHB into CSF but brain concentration peaked very early. Highest concentrations of GHB were found in cortical white matter with lower concentrations in subcortical areas.

Gamma hydroxybutyrate. Correlation of serum and cerebrospinal fluid levels with electroencephalographic and behavioral effects.

Gamma hydroxybutyrate was administered to adult cats by intravenous infusion at varying dosages while an electroencephalogram (EEG) was recorded from electrodes placed stereotactically in the right and left hippocampus and thalamic intralaminar nucleus and from cortical electrodes. Blood cerebrospinal fluid samples were assayed for gamma hydroxybutyrate. The first EEG change, slowing was occasional spikes, was seen at serum levels of 75 mug per milliliter. Changes in the recordings progressed through a number of stages, culminating in bursts of poly spiking interspersed among periods of electrical silence first seen at 350 mug per milliliter. Behavior was characterized by a progressively deepening trancelike state punctuated at higher serum levels by spontaneous and stimulus-induced myoclonic jerks. These changes were correlated with serum levels and are more similar to petit mal stupor than any kind of natural sleep-like state previously used to describe them.