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.

Androgen metabolites impact CSF amines and axonal serotonin via MAO-A and -B in male macaques.

A number of studies have shown that mutations or deletions of the monoamine oxidase-A (MAO-A) gene cause elevated CNS serotonin and elevated impulsive aggression in humans and animal models. In addition, low cerebrospinal fluid (CSF) 5-hydroxyindole acetic acid (5HIAA) has been documented in a limited number of violent criminal populations and in macaques that exhibit impulsive aggression. To reconcile these different analyses, we hypothesized that CSF 5HIAA reflected degradation of serotonin by the activity of MAO-A; and that low MAO-A activity would result in lower CSF 5HIAA, but overall higher serotonin in the CNS. To test this hypothesis, male Japanese macaques (Macaca fuscata) were castrated, rested for 5-7months, and then treated for 3months with [1] placebo, [2] testosterone (T), [3] dihydrotestosterone (DHT; non-aromatizable androgen) and 1,4,6-androstatriene-3,17-dione (ATD) (steroidal aromatase inhibitor), or [4] flutamide (FLUT; androgen antagonist) and ATD (n=5/group). These treatments enable isolation of androgen and estrogen activities. In the dorsal raphe, MAO-A and MAO-B expressions were determined with in situ hybridization (ISH) and protein expression of aromatase was determined with immunohistochemistry (IHC). CSF concentrations of 5HIAA, 3-methoxy-4-hydroxyphenylglycol (MHPG), and homovanillic acid (HVA) were determined with liquid chromatography/mass spectrometry (LC/MS). From the same animals, previously published data on serotonin axon density were used as a proxy for CNS serotonin. Aromatase conversion of T to estrogen (E) suppressed MAO-A (positive pixel area, p=0.0045), but androgens increased MAO-B (positive pixel area, p=0.014). CSF 5HIAA was suppressed by conversion of T to E (Cohen's d=0.6). CSF 5HIAA was positively correlated with MAO-A-positive pixel area (r(2)=0.78). CSF 5HIAA was inversely correlated with serotonin axon-positive pixel area (r(2)=0.69). In summary, CSF 5HIAA reflects MAO-A activity rather than global serotonin. Low CSF 5HIAA may, in this paradigm, reflect higher serotonin activity. Androgens lower MAO-A activity via metabolism to E, thus elevating CNS serotonin and decreasing CSF 5HIAA. Since androgens increase certain types of aggression, these data are consistent with studies demonstrating that lower MAO-A activity is associated with elevated serotonin and increased aggression.

XP13512 [(+/-)-1-([(alpha-isobutanoyloxyethoxy)carbonyl] aminomethyl)-1-cyclohexane acetic acid], a novel gabapentin prodrug: II. Improved oral bioavailability, dose proportionality, and colonic absorption compared with gabapentin in rats and monkeys.

The absorption of gabapentin (Neurontin) is dose-dependent and variable between patients. Rapid clearance of the drug necessitates dosing three or more times per day to maintain therapeutic levels. These deficiencies appear to result from the low capacity, limited intestinal distribution, and variable expression of the solute transporter responsible for gabapentin absorption. Saturation of this transporter at doses used clinically leads to unpredictable drug exposure and potentially ineffective therapy in some patients. XP13512 [(+/-)-1-([(alpha-isobutanoyloxyethoxy)carbonyl]aminomethyl)-1-cyclohexane acetic acid] is a novel prodrug of gabapentin designed to be absorbed by high-capacity nutrient transporters located throughout the intestine. XP13512 was efficiently absorbed and rapidly converted to gabapentin after oral dosing in rats and monkeys. Exposure to gabapentin was proportional to prodrug dose, whereas exposure to intact XP13512 was low. In rats, >95% of an oral dose of (14)C-XP13512 was excreted in urine in 24 h as gabapentin. In monkeys, oral bioavailability of gabapentin from XP13512 capsules was 84.2% compared with 25.4% after a similar oral Neurontin dose. Compared with intracolonic gabapentin, intracolonic XP13512 gave a 17-fold higher gabapentin exposure in rats and 34-fold higher in monkeys. XP13512 may therefore be incorporated into a sustained release formulation to provide extended gabapentin exposure. XP13512 demonstrated improved gabapentin bioavailability, increased dose proportionality, and enhanced colonic absorption. In clinical use, XP13512 may improve the treatment of neuropathic pain, epilepsy, and numerous other conditions by increasing efficacy, reducing interpatient variability, and decreasing frequency of dosing.

Delusional disorder in a boy with phenylketonuria and amine metabolites in the cerebrospinal fluid after treatment with neuroleptics.

A case report of a 16-year-old boy with the classical form of phenylketonuria (PKU) and suffering from a delusional disorder is presented. He did not respond to a traditional antipsychotic medication but improved on an atypical antipsychotic, risperidone. The cerebrospinal fluid metabolites of the biogenic amines were measured and are also reported in the context of a recent interest in the ratios of these amine metabolites in schizophrenia. It is proposed that such dually diagnosed patients with PKU may provide a model in which to test the prevalent biogenic amine theories in psychiatric disorders.

Advances in the analysis of cerebrospinal fluid.

The laboratory examination of cerebrospinal fluid (CSF) continues to play an important role in the clinical diagnosis and treatment of various disorders of the central nervous system (CNS). The major conditions currently include, as they have in the past, infectious diseases, neoplastic processes, multiple sclerosis, other demyelinating disorders, and intracerebral hemorrhage. Recent publications suggest a variety of new laboratory tests that may be useful in the evaluation of patients with both primary and metastatic malignancies, Alzheimer's disease, Creutzfeld-Jacob disease, global ischemia, various psychiatric disorders, CSF otorrhea and rhinorrhea, and in the differential diagnosis of cortical vs lacunar stroke, among others. Examples of these recent developments and their possible clinical usefulness are discussed.

Child cerebrospinal fluid analysis by capillary electrophoresis and laser-induced fluorescence detection.

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to analyze a 50-microliters sample of cerebrospinal fluid from leukaemic children treated with high doses of methotrexate. Free amino acids and primary amines are labelled with fluorescein isothiocyanate prior to analysis. Electropherograms containing more than 50 peaks were obtained in less than 22 min. Twenty-one peaks were identified, and 19 were quantitated. Observed differences in individual amino acid levels are compared with healthy reference values. The results indicate that CE-LIF is useful as a selective, rapid and sensitive tool for the determination of free amino acids and amines in clinical biology studies.

The trace amines and their acidic metabolites in depression--an overview.

1. Investigations of the role of the trace amines (phenylethylamine, tryptamine, m- and p-tyramine) and their acidic metabolites (phenylacetic, indoleacetic, m- and p-hydroxyphenylacetic acids) in depression are reviewed. 2. The evidence for the phenylethylamine hypothesis of depression is mixed. 3. Reduced phenylacetic acid levels in urine, plasma and CSF and changes in those levels during treatment with antidepressants show potential as state markers for depression. 4. Impaired p-tyramine conjugation following a tyramine challenge may be a good trait marker for depression.

Comparison of the behavioral and neurochemical effects of the two optical enantiomers of medetomidine, a selective alpha-2-adrenoceptor agonist.

Medetomidine (MED) is a veterinary sedative whose mode of action is activation of alpha-2 adrenoceptors. Because the carbon atom which separates the two ring systems is methylated, there is a center of stereochemical asymmetry in the molecule. The resulting enantiomers, d-MED and l-MED have recently become available for study. The biological activity of MED, as is now demonstrated in rats in vivo, seems to reside almost exclusively in the d-MED form. Only at extremely high doses (e.g., 10 mg/kg) does l-MED exert any effects, interpreted as alpha-2 adrenoceptor antagonism. In contrast, doses of d-MED as low as 30 micrograms/kg cause sedation, hypothermia and induce neurochemical changes in norepinephrine and 5-hydroxytryptamine metabolism in brain characteristic of alpha-2-agonists (decreases in concentrations of biogenic amine metabolites, turnover and increases in concentration of parent amine). The most sensitive neurochemical indicator of the alpha-2-agonist action of d-MED was the concentration of unconjugated 3-methoxy-4-hydroxy-phenyethylene glycol in rat cerebral spinal fluid, doses of d-MED as low as 10 micrograms/kg caused a significant reduction in this norepinephrine metabolite. Simultaneous administration of the specific alpha-2 adrenoceptor antagonist, atipamezole (1 mg/kg), effectively inhibited the behavioral and most of the neurochemical actions of d-MED (100 micrograms/kg). It is concluded that the enantiomers of MED may be extremely useful in elucidating structure action relationships at alpha-2 adrenoceptors.

Cerebrospinal fluid monoamines in Down's syndrome adults at different ages.

Markers of monoamine metabolism in lumbar cerebrospinal fluid (CSF) and plasma were determined in nine young, healthy adults with trisomy 21 Down's syndrome (DS), 21-34-years-old, and in three DS subjects over 45 years, two of whom were demented, as well as in two groups of age-matched controls. Test scores of general intelligence, visuospatial ability, visual discrimination and verbal intelligence were reduced significantly in the old as compared to the young DS subjects. Dementia in DS was evident from a history of mental deterioration, disorientation and hallucinations. In the young DS adults, as compared to the controls, CSF 5-HIAA and norepinephrine were significantly elevated but plasma levels were unchanged. HVA, MHPG and biopterin did not differ between the DS groups and age-matched controls, or with relation to age in the DS or control subjects. These results suggest an increased turnover of monoamines in young adults with DS but that alterations in monoamine metabolism are unrelated to the cognitive decline with age in DS.

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.

Effects of yohimbine and idazoxan on monoamine metabolites in rat cerebrospinal fluid.

Effects of two alpha 2-adrenoceptor antagonists, idazoxan and yohimbine, on the concentrations of monoamine metabolites in cisternal cerebrospinal fluid (CSF) of freely moving rats were investigated. Both drugs caused a dose-dependent, up to 250% increase in the concentration of 3-methoxy-4-hydroxyphenylglycol (MHPG) in CSF indicating enhanced release, metabolism and turnover of noradrenaline in the central nervous system (CNS). In addition, a similar increase in homovanillic acid (HVA) in CSF was observed, while the level of 5-hydroxyindoleacetic acid was unchanged. The present results demonstrate the usefulness of monitoring drug-induced alterations in noradrenergic activity in the CNS by measurement of free MHPG in repeatedly collected cisternal CSF samples from awake rats. The possibility that the observed increase in the concentration of HVA after the highly specific alpha 2-antagonist idazoxan reflects increased noradrenergic rather than dopaminergic neuronal activity is discussed.

Free and conjugated amines in human lumbar cerebrospinal fluid.

Significant amounts of acid-hydrolyzable conjugates of 3,4-dihydroxyphenylethylamine, norepinephrine, and 5-hydroxytryptamine were detected in lumbar CSF from 22 awake unpremedicated healthy individuals. In the CSF samples, the amounts of conjugated amines almost always exceeded the amounts of free amines, but were less than the amounts of the acid metabolites 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid.

Brain amine metabolism is reflected in cerebral ventricular CSF.

The choroid plexuses are suspended within the ventricles and account for approximately 75% of CSF production. The sodium-potassium ATPase operates within the choroidal epithelial cells and moves sodium ions towards the ventricular surface and potassium ions in the direction of the stroma. Water flows into CSF along osmotic gradient produced by sodium pump. The existence of extracellular channels by which brain metabolites could passively diffuse into the ventriculosubarachnoid space suggests an excretory role for CSF. Removal of solutes from the CSF could occur across the choroidal epithelium or arachnoid membrane into the blood. Systematically administered monoamine metabolites do not cross the blood-brain or the blood-CSF barrier. The regional concentrations of amine metabolites in the CSF is in part a reflection of the concentration of catecholamines and indoleamines in the immediately adjacent neuronal parenchyma. In order to illustrate the validity of monoamine metabolite determinations in cerebral ventricular CSF we developed a device which allowed for a continuous third ventricular CSF withdrawal in freely moving (or anesthetized) rats at a constant flow of 1 microliter/min. The elevation of biogenic amine metabolites in CSF by probenecid or their decline by monoamine oxidase inhibition was used to assess the rate of turnover of amines. Pharmacological manipulations (yohimbine, haloperidol, ouabain) resulted in mono-amine metabolite fluctuations in CSF similar to those previously described in brain tissue. Insulin administration caused an abrupt decrease in CSF glucose and elevated dopamine and serotonin metabolites in rats which had no access to food. These studies demonstrate the adaptation of in vivo analysis of CSF in rats but also exemplify the usefulness of monoamine metabolite determination in the CSF as indicators of brain function.

Estimation of monoamine and cyclic-AMP turnover and amino acid concentrations of spinal fluid in autistic children.

A group of autistic children had the concentrations of spinal fluid 5-HIAA, HVA and cAMP studied before and twenty-four hours after afflux blockade with probenecid. Spinal fluid aminoacids were studied before probenecid administration and found to be normal. Three children lacked an increase in the concentration of 5-HIAA after probenecid administration, and two had only modest increases in the concentration of spinal fluid HVA. The concentration of spinal fluid cAMP was increased by probenecid administration in all eight children. These findings suggest an abnormality in monoamine metabolism in a small group of autistic children.

Alterations in plasma and CSF amino acids, amines and metabolites in hepatic coma.

The dog with an end-to-side portacaval shunt (PCS) has been extensively used as a model to investigate hepatic encephalopathy (HE) as it demonstrates a plasma amino acid pattern similar to patients with chronic liver disease. In adult mongrel dogs, the effect of PCS on plasma and CSF amino acids, octopamine (OCT), phenylethanolamine (PEA) and CSF 5-hydroxyindolacetic acid (5-HIAA), were studied. Moreover, the effect of correction of plasma amino acids by infusional techniques was investigated.Tyrosine, tryptophan and phenylalanine levels increased dramatically during the development of HE in plasma and CSF, while valine, leucine and isoleucine decreased in plasma only, but CSF levels remained stable. Plasma and CSF octopamine and phenylethanolamine and CSF 5-HIAA increased markedly as clinical features in the dogs' behavior, characteristic of hepatic encephalopathy occurred, including hypersalivation, ataxia, flapping tremor, somnolence and finally coma. Once in coma, the dogs were infused with an amino acid mixture (F080) calculated to normalize the plasma amino acid pattern. After one to eight hours, the dogs began to awake. Simultaneously, blood, and CSF aromatic amino acids returned to their control values, as did OCT, PEA and CSF 5-HIAA. If F080 infusion was stopped, biochemical alterations would appear within one week, again accompanied by clinical hepatic encephalopathy.The results indicate that the altered levels of aromatic and branched chain amino acids, octopamine and PEA in plasma and CSF correlate well with the development of HE and that correction of the plasma amino acid abnormalities improves encephalopathy simultaneously with correction of neurotransmitter derangements in CSF.

Cerebrospinal fluid amine metabolites in acute schizophrenia.

The metabolites of serotonin, dopamine, and norepinephrine, 5-hydroxyindoleacetic acid (5HIAA), homovanillic acid (HVA), and 3-methoxy-4-hydroxy-phenylethylene glycol (MHPG), respectively, were studied in cerebrospinal fluid of patients with acute schizophrenia. Base line levels of these metabolites were not significantly different from those in normal, neurological, and affectively ill controls. Accumulations of 5HIAA and HVA following probenecid administration, which provide a measure of serotonin and dopamine turnover, were also not significantly different in patients with acute schizophrenia and affective illness. After patients had recovered from their acute schizophrenic illness, HVA accumulations were significantly reduced. We discuss results in relation to amine hypotheses of schizophrenia and the suggestion that altered dopamine metabolism may reflect a biological change predisposing to acute schizophrenia.