Metabolites In Rat Brain Research Articles

Liquid chromatography-mass spectrometry utilizing multi-stage fragmentation for the identification of oxysterols

In humans, the brain accounts for about 20% of the body's free cholesterol, most of which is synthesized de novo in brain. To maintain cholesterol balance throughout life, cholesterol becomes metabolized to 24S-hydroxycholesterol, principally in neurons. In mouse, rat, and probably human, metabolism to 24S-hydroxycholesterol accounts for about 50% of cholesterol turnover; however, the route by which the remainder is turned over has yet to be elucidated. Here, we describe a novel liquid chromatography (LC) multi-stage fragmentation mass spectrometry (MS(n)) methodology for the identification, with high sensitivity (low pg), of cholesterol metabolites in rat brain. The methodology includes derivatization to enhance ionization, exact mass analysis at high resolution to identify potential metabolites, and LC-MS(n) (n=3) to allow their characterization. 24S-hydroxycholesterol was confirmed as a major oxysterol in rat brain, and other oxysterols identified for the first time in brain included 24,25-, 24,27-, 25,27-, 6,24,- 7alpha,25-, and 7alpha,27-dihydroxycholesterols. In addition, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol, two molecules linked to amyloidogenesis of proteins, were characterized in rat brain.

Effects of subchronic exposure to styrene on the extracellular and tissue levels of dopamine, serotonin and their metabolites in rat brain

At present, there is controversy over the neurotoxic potential of styrene. Several epidemiological and clinical studies have shown that styrene exposure causes alterations of central nervous system functions in humans. Neurotransmitters have been implicated in the pathogenesis of styrene neurotoxicity in rodents. Several studies carried out on postmortem brain tissue suggest that styrene may alter dopaminergic neurotransmission in rabbit or rat brain. Moreover, in vitro studies suggest that both styrene and styrene oxide inhibit the uptake of dopamine (DA) in purified synaptic vesicles prepared from rat brain striata. To date, biochemical studies on animals have explored global tissue levels of neurotransmitters with sub-acute exposures to styrene. However, extracellular levels of neurotransmitters are more closely related to behaviour than are global tissue levels. The present study determined changes in the extracellular concentrations of DA, serotonin (5-HT) and their acid metabolites, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA), in striatal dialysates from freely moving adult male rats after exposure to 750 and 1,000 ppm styrene, 6 h per day, 5 days per week for 4 weeks. We also determined the concentrations of DA, 5-HT and their acid metabolites in striatum, nucleus accumbens and prefrontal cortex obtained postmortem from similarly exposed rats. Exposure to 1,000 ppm of styrene caused a significant decrease in extracellular acid metabolite concentrations. Tissue levels of acid metabolites were also decreased to a lesser extent. The effects were observed 72 h after discontinuing exposure but had vanished 17 days later. There was no change in DA or 5-HT concentrations either in the dialysates or tissues. Exposure to 750 ppm styrene caused no changes in the concentrations of DA, 5-HT and their acid metabolites either in the dialysates or tissues. The possibility that the effect of styrene is mediated by monoamine oxidase (MAO) inhibition is discussed.

The effects of repeated endotoxin exposure on rat brain metabolites as measured by ex vivo 1HMRS

Abnormalities in brain chemistry induced by acute or chronic treatment with LPS were studied in the rat model. Ex vivo brain metabolites were measured using proton magnetic resonance spectroscopy, whereas serum corticosterone levels were determined using radioimmunoassay. We observed increased lactate levels in all measured brain regions and decreased choline in the hypothalamus after chronic LPS treatment. Acute LPS treatment led to an elevation of corticosterone, whereas chronic LPS treatment led to attenuation of the HPA response. These findings suggest that chronic inflammation induced by LPS could lead to cell loss/dysfunction, and hence, desensitization of the HPA axis, particularly in the hypothalamus.

Effects of chronic lithium administration on rat brain phosphatidylinositol cycle constituents, membrane phospholipids and amino acids

There is evidence linking affective disorders and their treatment to alterations in membrane phospholipid metabolism, the phosphatidylinositol (PtdIns) second messenger cycle and brain excitatory and inhibitory amino acids. This study examines lithium effects on rat brain metabolites associated with the above systems and their reversal by myo-inositol. Thirty rats were treated for 14 days with i.p. lithium, saline or lithium plus myo-inositol. 1H, 31P and 7Li NMR were used to measure brain metabolites. Lithium, administered alone or with myo-inositol, resulted in brain lithium concentrations of approximately 0.6 microM/gram brain tissue. Brain myo-inositol was unchanged when lithium was co-administered with myo-inositol. Lithium increased brain inositol-1-phosphate (I1P) by 98% compared with saline and this effect was not attenuated by the addition of myo-inositol. Lithium treatment decreased phosphatidylserine (PtdSer) and PtdIns by 3% and 8%, respectively. Lithium also decreased taurine levels by 8% and increased aspartate levels by 9%. The above effects of lithium on PtdSer, PtdIns and taurine were attenuated or abolished by the co-administration of myo-inositol. Lithium alters levels of key membrane phospholipids and appears to affect the balance between inhibitory and excitatory amino acids in rat brain. Co-administration of myo-inositol attenuates some of these lithium effects on brain metabolites.

Changes in apparent diffusion coefficients of metabolites in rat brain after middle cerebral artery occlusion measured by proton magnetic resonance spectroscopy.

Diffusion-weighted proton MR spectroscopy and imaging have been applied to a rat brain model of unilateral middle cerebral artery occlusion between 1 and 4 hr post occlusion. Similar apparent diffusion coefficients (ADC) of most metabolites were observed within each hemisphere. In the ischemic ipsilateral hemisphere, the ADCs were (0.083--0.116). 10(-3) mm(2)/sec for lactate (Lac), alanine (Ala), gamma-amino butyric acid (GABA), N-acetyl aspartate (NAA), glutamine (Gln), glutamate (Glu), total creatine (tCr), choline-containing compounds (Cho), and myo-inositol (Ins), in the contralateral hemisphere (0.138--0.158). 10(-3) mm(2)/sec for NAA, Glu, tCr, Cho, and Ins. Higher ADCs was determined for taurine (Tau) in the ipsilateral (0.144. 10(-3) mm(2)/sec) and contralateral (0.198. 10(-3) mm(2)/sec) hemisphere. In the ischemic hemisphere, a relative ADC decrease to 65--75% was observed for NAA, Glu, tCr, Cho, Ins and Tau, which was similar to the decrease of the water ADC (to 67%). The results suggest a common cause of the observed ADC changes and provide a broader experimental basis to evaluate theories of water and metabolite diffusion. Magn Reson Med 45:383-389, 2001.

Pharmacokinetics of the New Potential Dipeptide Nootrope GVS-111 and Related Metabolites in Rat Brain

Pharmacokinetics of the New Potential Dipeptide Nootrope GVS-111 and Related Metabolites in Rat Brain

Pharmacokinetics of aniracetam and its metabolites in rat brain.

The pharmacokinetics of aniracetam (AP) and its main metabolites, 4-p-anisamidobutyric acid (ABA), 2-pyrrolidinone (PD) and p-anisic acid (AA), in 3 brain regions (cerebral cortex, hippocampus and thalamus) was investigated after single intravenous (i.v.) and oral administrations of AP to rats. AP, AA and PD were rapidly distributed into the 3 brain regions after i.v. administration of AP, but the amounts of AP were low. The concentrations of AP and AA in brain regions rapidly declined, whereas PD levels were higher and more sustained than those of AP and AA. ABA levels in the regions were below the detection limit. There were no significant differences in the distribution of these compounds in the 3 brain regions. The AUCbrain/AUCplasma ratio of PD was 53--55%, in contrast to the low ratio of AP (2.4--3.2%) and AA (3.9--4.2%). On oral administration of AP, the AUCbrain/AUCplasma ratio of PD was also higher than that of AA. When the transport of PD was tested using the in situ brain perfusion technique, it was clarified that PD was not transported across the blood-brain barrier (BBB) by a neutral amino acid carrier system. The high brain levels of PD and the low levels of AP suggest that the clinical efficacy of dosed AP may partly result from PD penetrating into the brain.

Pharmacological screen for activities of 12-hydroxyibogamine: a primary metabolite of the indole alkaloid ibogaine.

The purported efficacy of ibogaine for the treatment of drug dependence may be due in part to an active metabolite. Ibogaine undergoes first pass metabolism and is O-demethylated to 12-hydroxyibogamine (12-OH ibogamine). Radioligand binding assays were conducted to identify the potency and selectivity profiles for ibogaine and 12-OH ibogamine. A comparison of 12-OH ibogamine to the primary molecular targets identified previously for ibogaine demonstrates that the metabolite has a binding profile that is similar, but not identical to the parent drug. Both ibogaine and 12-OH ibogamine demonstrated the highest potency values at the cocaine recognition site on the 5-HT transporter. The same rank order (12-OH ibogamine > ibogaine), but lower potencies were observed for the [3H]paroxetine binding sites on the 5-HT transporter. Ibogaine and 12-OH ibogamine were equipotent at vesicular monoamine and dopamine transporters. The metabolite demonstrated higher affinity at the kappa-1 receptor and lower affinity at the NMDA receptor complex compared to the parent drug. Quantitation of the regional brain levels of ibogaine and 12-OH ibogamine demonstrated micromolar concentrations of both the parent drug and metabolite in rat brain. Drug dependence results from distinct, but inter-related neurochemical adaptations, which underlie tolerance, sensitization and withdrawal. Ibogaine's ability to alter drug-seeking behavior may be due to combined actions of the parent drug and metabolite at key pharmacological targets that modulate the activity of drug reward circuits.

Quantitative microdialysis for the in vivo measurement of carbamazepine, oxcarbazepine and their major metabolites in rat brain- and liver tissue and in blood using the internal standard technique

Microdialysis as in vivo sampling technique in pharmacokinetic studies requires the estimation of the in vivo relative recovery in order to calculate unbound blood- and extracellular tissue concentrations of the compounds of interest. Therefore, the internal standard technique was evaluated in this study, as in vivo calibration method to determine true concentrations of carbamazepine, oxcarbazepine and their major metabolites in the extracellular fluid of brain- and liver tissue and in blood. As internal standard, 2-methyl-5H-dibenz(b,f)azepine-5-carboxamide (m-CBZ) was used. The compounds, metabolites and the internal standard were added to the perfusate and their in vivo relative loss was determined. The in vivo relative recovery of the compounds and metabolites was established using the slow perfusion method. It was found that the ratios of the in vivo relative loss or relative recovery of the compounds and metabolites to the relative loss of the internal standard remain constant in time for both tissues and for blood. It is concluded that with m-CBZ as internal standard, the internal standard technique is a useful in vivo calibration method to determine the true concentrations of carbamazepine, oxcarbazepine and their metabolites in the extracellular space of brain-and liver tissue and in blood.

Alteration of Intracellular Metabolite Diffusion in Rat Brain In Vivo During Ischemia and Reperfusion

Diffusion-weighted MRI can demonstrate decreases of the apparent diffusion coefficient (ADC) of brain tissue water shortly after the onset of ischemia. To further elucidate underlying mechanisms, this study extended diffusion assessment to intracellular metabolites in rat brain in vivo before, during, and after ischemia. Changes in molecular mobility were studied in a rat model of global forebrain ischemia (n = 8, 20-minute occlusion, 120-minute reperfusion) with the use of diffusion-weighted localized proton MR spectroscopy. During ischemia and early reperfusion the time course of ADC changes was monitored by strongly diffusion-weighted spectra. ADC values of N-acetylaspartate, creatines, cholines, and myo-inositol were evaluated from series of differently diffusion-weighted spectra before ischemia, 90 minutes after reperfusion, and 60 minutes postmortem. Parallel to a rise in diffusion-weighted water signal (133 +/- 20%), pertinent intensities of all brain metabolites increased during ischemia. Changes were most pronounced for myo-inositol (46 +/- 9%) and smallest for N-acetylaspartate (12 +/- 4%). During reperfusion water ADC values returned to basal values, whereas metabolite ADC values were decreased by 22% (after 40 minutes). Postmortem ADC values (after 60 minutes) were reduced by 46% for water and 38% for metabolites. The present findings indicate that water ADC changes during ischemic stroke are accompanied by significant alterations in intracellular mobility in both neuronal and glial cell populations as reflected by N-acetylaspartate and myo-inositol, respectively. Altered metabolite ADC values during reperfusion are consistent with irreversible tissue damage in this model and offer new means to assess circulatory and metabolic compromise.

T1 and T2 relaxation times of the major 1H‐containing metabolites in rat brain after focal ischemia

The relaxation properties of water and metabolites were measured in rat brain following the occlusion of the middle cerebral artery (MCA) with localized 1H MRS. The PRESS sequence was employed to select volumes of 39 microL in the ischemic and the contralateral hemisphere. T1 and T2 relaxation times and peak intensities of water, choline containing compounds (Cho), creatine and phosphocreatine (Cre) and N-acetyl aspartate (NAA) in both hemispheres were determined at 3-6 h, 1 day and 3 or 4 days after occlusion. Lactate in the ischemic hemisphere was also quantified. The relaxation properties and peak intensities of NAA, Cre and Cho remained unchanged in the ischemic volume during the first 3-6 h of ischemia as compared to the contralateral volume. Water T2 was slightly increased in the ischemic volume. After 24 h the T1 and T2 of water and Cre and the T1 of Cho had increased significantly in the ischemic volume, while the peak intensities of Cho, Cre and NAA were reduced. It appears therefore that tissue changes which occur in the early phase of ischemia have no significant effects on the relaxation behaviour of the metabolites. However, ischemic brain damage affects the relaxation behaviour and concentration of the metabolites and water at later stages.

Regional alterations of dopamine and its metabolites in rat brain following portacaval anastomosis

Hyperammonemia and changes in brain monoamine metabolism have been proposed to contribute to the pathogenesis of the neuropsychiatric symptoms characteristic of human portal-systemic encephalopathy (PSE) resulting from chronic liver disease. Portacaval anastomosis (PCA) in the rat leads to sustained hyperammonemia and mild encephalopathy. In order to evaluate the role of dopamine (DA) metabolism in PSE, levels of DA and its metabolites were measured by HPLC with electrochemical detection in brain regions of rats with PCA at various stages of encephalopathy precipitated by ammonium acetate administration. Following ammonium acetate administration, rats with PCA rapidly develop severe neurological signs of encephalopathy progressing through loss of righting reflex to coma; sham-operated control animals administered ammonium acetate showed no such neurological deterioration. Concentrations of the DA metabolites DOPAC and HVA as well as [DA metabolites]/[DA] ratios, an indirect measure of DA turnover in brain, were increased in caudate-putamen, in cingulate and pyriform entorhinal cortices as well as in raphe nucleus and locus coeruleus. Increased DA metabolites, however, did not worsen at coma stages of PSE. Increased DA turnover thus appears to relate to early neuropsychiatric and extrapyramidal symptoms of PSE.

Resolution, EPC-syntheses, absolute stereochemistry, and pharmacology of the (S)-(+)- and (R)-(-)-isomers of the MAO-A inhibitor tetrindole hydrochloride.

Resolution of (RS)-tetrindole (3) and enantioselective reductions of the imine 7 yielded (S)-(+)-(4) and (R)-(-)-tetrindole (5). The absolute stereochemistry of 4 was established by X-ray analysis of the corresponding Mosher amide 6. From in vitro as well as in vivo data (MAO-inhibition, levels of monoamines and their respective metabolites in rat brain), 4 was identified as the eutomer.

In vivo31P NMR studies of orientation effects upon rat brain metabolism during mild hypoxia

Energy metabolites in rat brain under the same level of hypoxia were monitored by 31P NMR in both horizontal and vertical magnets. The changes in PCr, Pi, and pHi in the vertical setting toward the end of hypoxia were significantly larger and the recovery in the horizontally held animals was more complete. The results demonstrated quantitatively that the stress of the alignment is superimposed on the stress of hypoxia in the vertical magnet.

Narrow-bore liquid chromatographic assay for oxcarbazepine and its major metabolite in rat brain, liver and blood microdialysates

An isocratic narrow-bore high-performance liquid chromatographic assay with UV detection is described for the detection of oxcarbazepine and its major metabolite, 10,11-dihydro-10-hydroxycarbamazepine, using carbamazepine as the internal standard. This method is sufficiently sensitive to allow quantification of oxcarbazepine and its metabolite in rat brain, liver and blood microdialysates.

Regional differences in the in vivo regulation of the extracellular levels of noradrenaline and its metabolites in rat brain

Microdialysis was used to determine extracellular levels of both noradrenaline and its metabolites in several brain regions of rats under basal conditions and in response to drugs selective for theα 2-adrenoceptor to study regional differences in the regulation of noradrenaline overflow. Basal overflow of noradrenaline was about 1.3 fmol/min in frontoparietal cortex, amygdala and hippocampus and in the medial prefrontal cortex 2.4 fmol/min was measured, whereas the overflow of the noradrenaline metabolites 3,4-dihydroxyphenylglycol and 3-methoxy-4-hydroxyphenylglycol was 10-fold higher. After correction for recovery and membrane length no regional differences in the basal overflow of noradrenaline (NA) were found. There were, however, regional differences in the drug-induced effects: locally applied moxonidine decreased extracellular noradrenaline stronger in the frontoparietal cortex than in the medial prefrontal cortex. The increase in noradrenaline overflow caused by idazoxan (10 −4M) was stronger in frontoparietal cortex than in amygdala and hippocampus. The metabolites were also generally decreased by moxonidine and increased by idazoxan, although less markedly. The present study shows that the regulation of noradrenaline overflow by the presynapticα 2-autoreceptor was stronger in cortical regions than in amygdala and hippocampus. In those latter regions the uptake mechanisms probably plays a relatively more important role in the regulation of noradrenaline overflow.

Decreases of plasma tryptophan concentrations following restricted feeding do not decrease serotonin and its metabolite in rat brain

Rats trained on a restricted feeding (RF) schedule of 4 h/day were killed before (starved) and after (fed) the presentation of food to compare hepatic and brain tryptophan metabolism with feeding (FF) controls. The RF rats exhibited greater holo-tryptophan pyrrolase activity in the liver than FF controls. Holoenzyme activity of starved rats was greater than fed animals. Plasma levels of tryptophan higher in the FF rats were comparable in the starved and fed groups of RF rats. Plasma levels of glucose comparable in the FF and RF fed rats were smaller in the starved animals. Brain levels of tryptophan comparable in the RF fed rats and FF controls were smaller in RF starved rats. Brain concentrations of serotonin (5-hydroxytryptamine) and its metabolite 5-hydroxyindoleacetic acid comparable in the RF starved rats and FF controls were higher in the RF fed rats. Possible implication of the findings in the pathogenesis of food deprivation/starvation related disease, anorexia nervosa is discussed.

Concentrations of remoxipride and its phenolic metabolites in rat brain and plasma. Relationship to extrapyramidal side effects and atypical antipsychotic profile.

The cataleptic effect of remoxipride was examined in the horizontal bar test after i.v.,i.p. and s.c. administration to male rats. Remoxipride induced immediate catalepsy after high i.v. doses (ED50 = 49 mumol/kg) while peak effects were seen 60-90 min after i.p. administration (ED50 = 38 mumol/kg). Following s.c. administration remoxipride failed to produce a statistically significant catalepsy in the 20-100 mumol/kg dose range (ED50 > 100 mumol/kg). In contrast, haloperidol was found to be more effective in inducing catalepsy after i.v. (ED50 = 0.4 mumol/kg) than after i.p. or s.c. administration (ED50 = 0.9 mumol/kg). The atypical antipsychotic profile of remoxipride was more pronounced when the compound was given i.v. or s.c. as compared with the i.p. route. Plasma and brain (striatum and nucleus accumbens) concentrations of remoxipride and its active phenolic metabolites FLA 797(-) and FLA 908(-) were measured by high performance liquid chromatography. The 40 mumol/kg dose of remoxipride resulted in plasma and brain concentrations of remoxipride which were 300-1000-fold higher (depending on the route of administration) than the most potent of the phenolic metabolites, e.g., FLA 797(-). The plasma and brain concentrations of remoxipride and its phenolic metabolites were related to DA D2 receptor blocking potency and to the temporal course and effectiveness to induce catalepsy. This analysis suggested that the unbound concentrations of the phenolic metabolites were too low to play a major role in the DA blocking action of remoxipride. However, FLA 797(-) may contribute marginally to the cataleptic effects following high (i.p.) doses of remoxipride.

Free choline and choline metabolites in rat brain and body fluids: sensitive determination and implications for choline supply to the brain

In the central nervous system, choline is an essential precursor of choline-containing phospholipids in neurons and glial cells and of acetylcholine in cholinergic neurons. In order to study choline transport and metabolism in the brain, we developed a comprehensive methodical procedure for the analysis of choline and its major metabolites which involves a separation step, selective hydrolysis and subsequent determination of free choline by HPLC and electrochemical detection. In the present paper, we report the levels of choline, acetylcholine, phosphocholine, glycerophosphocholine and choline-containing phospholipids in brain tissue, cerebrospinal fluid and blood plasma of the untreated rat. The levels of free choline in blood plasma (11.4 μM), CSF (6.7 μM) and brain intracellular space (64.0 μM) were sufficiently similar to be compatible with an exchange of choline between these compartments. In contrast, the intracellular levels of glycerophosphocholine (1.15 mM) and phosphocholine (0.59 mM) in the brain were considerably higher than their CSF concentrations of 2.83 and 1.70 μM, respectively. In blood plasma, glycerophosphocholine was present in a concentration of 4.58 μM while phosphocholine levels were very low or absent (<0.1 μM). The levels of phosphatidylcholine and lyso-phosphatidylcholine were high in blood plasma (1267 and 268 μM) but very low in cerebrospinal fluid (<10 μM). We concluded that the transport of free choline is the only likely mechanism which contributes to the supply of choline to the brain under physiological conditions.

Regional differences in the levels of biogenic amines and their metabolites in rat brain after tricyclic antidepressant treatments.

Changes in the levels of biogenic amines in different brain regions and the cerebrospinal fluid in rats were measured after acute or chronic treatment with tricyclic antidepressants. After single or 3 weeks' treatment with imipramine or desipramine, blocks of tissues were obtained from seven regions of the brain (frontal cortex, corpus striatum, hippocampus, thalamus, hypothalamus, substantia nigra and cerebellum) immediately after collection of the cerebrospinal fluid (CSF) from the cisterna magna. The concentrations of biogenic amines and their metabolites (norepinephrine, epinephrine, dopamine, 5-hydroxytryptamine (5-HT), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA)) in brain tissues and the CSF were measured using the high performance liquid chromatography-electrochemical detection system (HPLC-ECD). Treatment with desipramine or imipramine caused major alterations in the concentrations of central norepinephrine or 5-HT and its metabolite, respectively. Brain regional responses were variable according to the kind of tricyclic antidepressants and the duration of treatment. It is noteworthy that chronic treatment with both desipramine and imipramine caused altered hippocampal concentrations of norepinephrine and/or 5-HT and its metabolites. Striatal DOPAC concentrations were also changed after acute or chronic treatment with both drugs. These results suggest that tricyclic antidepressants altered neurotransmission according to the brain region, and the hippocampal norepinephrine and 5-HT and/or the striatal dopamine may have a significant role for the expression of antidepressant action of tricyclic antidepressants.