Acid In Rat Brain Research Articles

P.2.006 - Changes in composition of phospholipids and fatty acids in rat brain during aging

P.2.006 - Changes in composition of phospholipids and fatty acids in rat brain during aging

Properties of the omega-hydroxylation system of docosahexaenoic or arachidonic acid in brain or liver homogenate of rat

The homogenate of a brain or liver obtained from a 1–55-day-old rat was incubated with NADPH and docosahexaenoic or arachidonic acid as the substrate. ω-Hydroxydocosahexaenoic or ω-hydroxyeicosatetraenoic acid from an incubation mixture of the homogenate was detected on a selected-ion monitoring chromatogram of reversed phase-HPLC-thermospray-mass spectrometry. ω-Hydroxylation activity in the brain homogenate considerably increased with growth up to 55 days. Activity in the liver homogenate decreased much with growth up to 55 days. ω-Hydroxylation activity in homogenates of rat brain gray matter, white matter, medula oblongata and cerebellum was much the same. ω-Hydroxylation activity of docosahexaenoic acid in rat brain homogenate was maximal at pH 7.5–8.0 in 50 mM Tris-HCL buffer and was inhibited by CO gas, metyrapone, ADP-Fe 3+, heat treatment at 100°C for 5 min and without NADPH. Based on these results, it is suggested that ω-hydroxylation activity is associated with cytochrome P-450 without NADPH-ADP-Fe 3+-dependent lipid peroxidation, and the ω-hydroxylation system may be a metabolic pathway of the fatty acids in adult rat brain or neonatal rat liver. Since ω-hydroxyeicosatetraenoic acid produces relaxation of artery, it is suggested that blood flow changes in rat brain or liver with growth are caused by ω-hydroxylation activity changes in these organs with growth.

In situ transblot and immunocytochemical comparisons of astrocytic connexin-43 responses to NMDA and kainic acid in rat brain

Intracerebral injection of kainic acid (KA) in rat brain was previously found to cause altered immunohistochemical recognition of connexin-43 (Cx43) epitopes (epitope masking) with different sequence-specific antibodies against this gap junction protein. We demonstrate here that similar alterations occur when nitrocellulose membranes containing protein transferred from fresh cryostat sections of KA-injected brain are probed with these antibodies (in situ transblotting), indicating that epitope masking is not a result of epitope alteration due to fixation conditions used in earlier studies. Alterations in immuno-recognition of astrocytic Cx43 subsequent to injections of NMDA were also observed and were similar to those seen with KA in some, but not all respects. The results provide further indications of Cx43 molecular modification in excitotoxin-lesioned tissue and suggest that the sequelae of reactions by astrocytes and their gap junctions in these tissues is dependent on cell-type susceptibility to excitotoxin action.

The release and uptake of excitatory amino acids in rat brain: Effect of aging and oxidative stress

The excitatory amino acids (EAAs) l-aspartate and l-glutamate constitute the major neurotransmitters in the mammalian brain. This study established the influence of aging and oxidative stress on the release and uptake of EAAs. The high affinity uptake of d-[ 3H]aspartate in synaptosomal fractions of the neostriatum, hippocampus, and neocortex was not significantly different in Fisher 344/Norwegian Brown hybrid rats aged 3, 12, 24, and 37 months. Similarly, the K 1-evoked efflux of endogenous aspartate and glutamate from neocortical minislices was also unaffected by age. To examine the possibility that EAA nerve terminals become more vulnerable to oxidative stress with age, the influence of an inhibitor of the electron transport chain (sodium cyanide) on EAA uptake and release was determined. Although cyanide inhibited d-[ 3H]aspartate uptake and potentiated the potassium-evoked efflux of aspartate and glutamate in a Ca 2+-independent fashion, neither of these changes were influenced by age. Thus, the functional integrity of EAA nerve terminals and their vulnerability to oxidative stress are both preserved in normal aging. The potency of cyanide to inhibit d-[ 3H]aspartate uptake did, however, display regional variability: hippocampus > neocortex > neostriatum (IC 50 = 1.2 ± 0.2 mM, 1.9 ± 0.1 mM and 2.7 ± 0.2 mM, respectively), suggesting that EAA nerve terminals in the hippocampus may be selectively vulnerable to oxidative stress.

Purification of peroxisomes and subcellular distribution of enzyme activities for activation and oxidation of very-long-chain fatty acids in rat brain

Brain contains high amounts of very-long-chain (VLC) fatty acids (> C22). Since mitochondria from liver and skin fibroblasts lack lignoceroyl-CoA ligase, in liver and skin fibroblasts fatty acids are exclusively oxidized in peroxisomes. Findings by Poulos and associates [9]suggested that contrary to liver and cultured skin fibroblasts brain mitochondria contain lignoceroyl-CoA ligase and can oxidize lignoceric acid. The present study was undertaken to develop a procedure for the isolation of subcellular organelles of higher purity from brain and to get a better understanding of the subcellular localization of the oxidation of VLC fatty acids in brain. The enzyme activities for activation and oxidation of palmitic and lignoceric acids were determined in peroxisomes, mitochondria, microsomes and a myelin fraction from rat brain and peroxisomes, mitochondria and microsomes purified from rat liver. Like in liver, brain lignoceroyl-CoA ligase activity in microsomes and peroxisomes was approx. 9 times higher than in mitochondria. In addition to palmitoyl-CoA ligase the antibodies against palmitoyl-CoA ligase inhibited the residual mitochondrial lignoceroyl-CoA ligase activity, meaning that lignoceroyl-CoA ligase activity in mitochondria was derived from palmitoyl-CoA ligase. Accordingly, in peroxisomes lignoceric acid was oxidized at 7 times higher rate than in mitochondria. Mitochondria were able to oxidize lignoceric acid efficiently when supplemented with lignoceroyl-CoA ligase activity from microsomes or myelin. These results show that in brain lignoceric acid is oxidized in peroxisomes and that lignoceroyl-CoA ligase activity is localized in peroxisomes and microsomes, but not in mitochondria. Peroxisomes and microsomes contain both lignoceroyl-CoA and palmitoyl-CoA ligases. Similar to peroxisomes and microsomes, the antibodies against palmitoyl-CoA ligase inhibited only the palmitoyl-CoA ligase activity in myelin but not the lignoceroyl-CoA ligase activity. These results suggest that in addition to palmitoyl-CoA ligase, myelin also contaies lignoceroyl-CoA ligase.

Effect of L-thyroxine and carbimazole on brain biogenic amines and amino acids in rats.

Effect of L-thyroxine and carbimazole on brain biogenic amines and amino acids content and circulating levels of thyroid hormones has been investigated in rats. L-thyroxine treatment caused marked elevation of 5-HT, histamine and glutamate along with the decline in the level of GABA whereas administration of carbimazole had a contrary effect on rat hypothalamus. Further, L-thyroxine administration also raised histamine as well as glutamate content and decreased GABA level in thalamic region of the rat brain but carbimazole treatment reduced 5-HT and glutamate content in this area of the rat brain. Similarly, cortical content of 5-HT and histamine also increased following L-thyroxine administration whereas carbimazole treatment lowered 5-HT, histamine and glutamate levels. Simultaneously, there was a considerable rise in the circulating levels of T3 and T4 in L-thyroxine-treated rats and a marked reduction in their levels in carbimazole-treated rats. There is a positive correlation between these amines and the thyroid hormone level. Thus, the results suggest that L-thyroxine and carbimazole administration cause marked alteration in biogenic amines and amino acids in rat brain, which may have an important role in the functioning of thyroid gland.

On the production and disposition of quinolinic acid in rat brain and liver slices.

The de novo production and subsequent disposition of the endogenous excitotoxin quinolinic acid (QUIN) was investigated in vitro in tissue slices from rat brain and liver. Incubation of tissue with QUIN's immediate bioprecursor 3-hydroxyanthranilic acid (3-HANA) in oxygenated Krebs-Ringer buffer yielded measurable amounts of QUIN both in the tissue and in the incubation medium. Saturation was reached between 16 and 64 microM 3-HANA (166 pmol of QUIN formed per milligram of protein after a 60-min incubation with 64 microM 3-HANA). In the brain, more QUIN was recovered from the tissue than from the incubation medium at all time points examined (5 min to 4 h). In contrast, the tissue-to-medium ratio for QUIN in parallel experiments with hepatic slices was << 1. The disposition of newly synthesized QUIN was further elaborated in tissue slices that had been preincubated for 60 min with 64 microM 3-HANA. Subsequent incubation of brain tissue in fresh buffer revealed a steady but relatively slow efflux of QUIN from the cellular compartment, with > 30% remaining in the tissue after a 90-min incubation. Analogous experiments with liver slices showed that > 93% of newly synthesized QUIN had entered the extracellular compartment within 30 min. Striatal and nigral slices obtained 7 days after an intrastriatal ibotenic acid injection showed severalfold increases in QUIN production compared with control tissues, in all likelihood due to astrogliosis and associated large increases in 3-hydroxyanthranilic acid oxygenase activity. In addition, the apparent tissue-to-medium ratio was markedly reduced in striatal slices from lesioned animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of free amino acid enantiomers in rat brain and serum by high-performance liquid chromatography after derivatization with N- tert.-butyloxycarbonyl- l-cysteine and o-phthaldialdehyde

The concurrent determination of free amino acid enantiomers and non-chiral amino acids in rat brain and serum was accomplished by high-performance liquid chromatography with fluorimetric detection after derivatization with N- tert.-butyloxycarbonyl- l-cysteine and o-phthaldialdehyde. The method revealed the presence of a large amount of free d-serine (0.22 μmol/g of tissue; d/d + l ratio = 0.25) in the brain whereas d-aspartate and d-alanine were established to be at trace levels. These results further support the presence of d-serine in adult brain tissues as demonstrated by recent work using gas chromatography.

Effects of nutritional status on contents of tryptophan, serotonin and 5-hydroxyindoleacetic acid in rat brain

Effects of nutritional status on the levels of tryptophan (Trp), serotonin (5HT), and its metabolite, 5-hydroxyindoleacetic acid (5HIAA) in six brain regions of rats were investigated. 1) A low-protein high-carbohydrate diet decreased Trp, 5HT and 5HIAA levels in the cortex and hippocampus, and those of 5HT and 5HIAA in the hypothalamus. This diet did not affect the contents of Trp and 5HT in the midbrain, but decreased 5HIAA. No significant changes of Trp, 5HT and 5HIAA were observed in the pons and medulla and striatum. 2) A low-carbohydrate high-protein diet increased the levels of Trp, 5HT and 5HIAA in the striatum, and 5HT and 5HIAA in the cortex, but showed no effect on the contents of Trp, 5HT and 5HIAA in the hippocampus, midbrain, pons and medulla and hypothalamus. 3) An energy-restriction low-carbohydrate diet increased Trp, 5HT and 5HIAA contents in the striatum, and 5HT and 5HIAA in the cortex and pons and medulla. In the hypothalamus, only 5HIAA was increased by this diet. The diet did not influence Trp, 5HT and 5HIAA contents of the midbrain and hippocampus. These results suggest that i) lowered fat and carbohydrate intakes enhance 5HT synthesis and metabolism in the cortex and that lowered carbohydrate intake enhances them in the striatum, ii) energy restriction enhances the 5HT metabolism in the cortex, pons and medulla and hypothalamus, and 5HT synthesis in the cortex and pons and medulla, iii) lowered protein intake inhibits 5HT metabolism in the cortex, midbrain, hippocampus and hypothalamus, and 5HT synthesis in the cortex, hippocampus and hypothalamus.

O-020 - In situ hybridization study of IL-1β mRNA induced by kainic acid in rat brain

O-020 - In situ hybridization study of IL-1β mRNA induced by kainic acid in rat brain

Effects of ageing on the content in sulfur-containing amino acids in rat brain

Concentrations of the sulfur-containing amino acids methionine, homocysteic acid, cysteic acid and taurine were measured in brain structures of young and old Wistar rats in an attempt to establish a possible link between the increase in oxidative stress with ageing and changes in tissue levels of these amino acids. Contrary to data reported by others, in all brain structures of young and old rats homocysteic acid levels could not be quantified. Compared with young rats, in old animals taurine and methionine concentrations significantly decreased in striatum and cortex; decreased taurine levels were also found in nucleus accumbens and cerebellum and lower concentrations of methionine were found in midbrain, hippocampus and pons-medulla. Cysteic acid levels either did not change or significantly increased in cortex and hippocampus. These results are discussed taking into account the biosynthesis of sulfur-containing amino acids in rat brain and the decrease in glutathione in relation to oxidative stress with ageing. Changes in aspartic acid, glutamic acid, serine, glutamine, glycine and GABA concentrations with ageing were also determined in the same brain structures and were in good agreement with those previously reported (Strolin Benedetti et al., 1990 a, b).

Oxidation of very-long-chain fatty acids in rat brain: cerotic acid is β-oxidized exclusively in rat brain peroxisomes

We studied the effect of sodium 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA), a potent inhibitor of mitochondrial carnitine palmitoyltransferase I, on fatty acid oxidation by rat brain cells. In cultured glial cells as well as in dissociated brain cells from adult rats palmitic acid (16:0) oxidation was inhibited by about 85% of control values when 25 microM POCA was added to the medium, whereas no inhibition of cerotic acid (26:0) oxidation was observed. Furthermore, omission of carnitine from the culture medium resulted in a 57.7% decrease in palmitic acid oxidation in cultured glial cells, whereas cerotic acid oxidation was not influenced. These results indicate that rat brain peroxisomes contribute only little (about 15%) to palmitic acid oxidation and provide conclusive evidence that cerotic acid is oxidized exclusively in rat brain peroxisomes.

Simultaneous measurement of serotonin and 5-hydroxyindoleacetic acid in rat brain using a liquid chromatographic method with electrochemical detection

A simple and sensitive method for the simultaneous measurement of 5-hydroxytryptamine and its main metabolite 5-hydroxyindoleacetic acid in rat brain is described. Brain tissue samples were only deproteinated and, without further extraction, were injected directly onto a high-performance liquid chromatography column and detected electrochemically. The detection limit for 5-hydroxytryptamine and 5-hydroxyindoleacetic acid was 16 and 8 g per injection, respectively. Within 8 min, the total separation of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid is achieved. With this method over 100 analyses can be performed in a single working day. Brain samples from young and old, male and female Brown Norway rats were analysed for indoles by this method. The ratio 5-hydroxytryptamine 5-hydroxyindoleacetic acid, an index for serotonin turnover, showed significant differences between age groups and genders in the cortex, and a significant difference between genders in the hypothalamus.

The origin of free brain malonate

Rat brain contains substantial concentrations of free malonate (192 nmol/g wet weight) but origin and biological importance of the dicarboxylic acid are poorly understood. A dietary source has been excluded. A recently described malonyl-CoA decarboxylase deficiency is associated with malonic aciduria and clinical manifestations, including mental retardation. In an effort to study the metabolic origin of free malonate, several labeled acetyl-CoA precursors were administered by intracerebral injection. [2-14C]pyruvate or [1,5-14C]citrate produced radioactive glutamate but failed to label malonate. In contrast, [1-14C]acetate, [2-14C]acetate, and [1-14C]butyrate were converted to labeled glutamate and malonate after the same route of administration. The intracerebral injection of [1-14C]-beta-alanine as a precursor of malonic semialdehyde and possibly free malonate did not give rise to radioactivity in the dicarboxylate. The labeling pattern of malonic acid is compatible with the reaction sequence: acetyl-CoA----malonyl-CoA----malonate. The final step is thought to occur by transfer of the CoA-group from malonyl-CoA to succinate and/or acetoacetate. Labeling of malonate from acetate is most effective at the age of 7 days when the net concentration of the dicarboxylic acid in rat brain is still very low. At this age, butyrate was a better precursor of malonate than acetate. It is proposed that fatty acid oxidation provides the acetyl-CoA which functions as the precursor of free brain malonate. Compartmentation of malonate biosynthesis is likely because the acetyl-CoA precursors citrate and pyruvate are ineffective.

Dietary linoleic acid and polyunsaturated fatty acids in rat brain and other organs. Minimal requirements of linoleic acid

Starting three weeks before mating, 12 groups of female rats were fed different amounts of linoleic acid (18:2n-6). Their male pups were killed when 21-days-old. Varying the dietary 18:2n-6 content between 150 and 6200 mg/100 g food intake had the following results. Linoleic acid levels remained very low in brain, myelin, synaptosomes, and retina. In contrast, 18:2n-6 levels increased in sciatic nerve. In heart, linoleic acid levels were high, but were not related to dietary linoleic acid intake. Levels of 18:2n-6 were significantly increased in liver, lung, kidney, and testicle and were even higher in muscle and adipose tissue. On the other hand, in heart a constant amount of 18:2n-6 was found at a low level of dietary 18:2n-6. Constant levels of arachidonic acid (20:4n-6) were reached at 150 mg/100 g diet in all nerve structures, and at 300 mg/100 g diet in testicle and muscle, at 800 mg/100 g diet in kidney, and at 1200 mg/100 g diet in liver, lung, and heart. Constant adrenic acid (22:4n-6) levels were obtained at 150, 900, and 1200 mg/100 g diet in myelin, sciatic nerve, and brain, respectively. Minimal levels were difficult to determine. In all fractions examined accumulation of docosapentaenoic acid (22:5n-6) was the most direct and specific consequence of increasing amounts of dietary 18:2n-6. Tissue eicosapentaenoic acid (20:5n-3) and 22:5n-3 levels were relatively independent of dietary 18:2n-6 intake, except in lung, liver, and kidney. In several organs (muscle, lung, kidney, liver, heart) as well as in myelin, very low levels of dietary linoleic acid led to an increase in 20:5n-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Unique molecular species of phosphatidylcholine containing very-long-chain (C24-C38) polyenoic fatty acids in rat brain

Rat brain has been shown to contain polyenoic very-long-chain fatty acids (VLCFA) belonging to the n-3 and n-6 series with four, five and six double bonds and even-carbon chain lengths from 24 to 38. These fatty acids are almost exclusively located in unusual molecular species of phosphatidylcholine at the sn-1 position of the glycerol backbone, whereas saturated, monoenoic and polyenoic fatty acids with less than 24 carbon atoms are present at the sn-2 position. Polyenoic VLCFA phosphatidylcholine in neonatal rat brain is enriched with n-6 pentaenoic and n-3 hexaenoic VLCFA with up to 36 carbon atoms, whereas the corresponding phospholipid in adult rat brain mainly contains n-6 tetraenoic and n-3 pentaenoic VLCFA with up to 38 carbon atoms. The total amount of polyenoic VLCFA associated with phosphatidylcholine is highest in the brain of immature animals. Polyenoic VLCFA phosphatidylcholine appears to be predominantly confined to nervous tissue in rats, and it is envisaged that this phospholipid is of physiological significance.

High-performance liquid chromatographic determination of selected amino acids in rat brain by precolumn derivatization with phenylisothiocyanate

We describe here a simple, sensitive, selective and reproducible assay method for quantitative determination of aspartate, glutamate, serine, glutamine, glycine and γ-aminobutyric acid in rat brain using reversed-phase high-performance liquid chromatography. The method is based upon formation of phenylthiocarbamyl derivatives of the amino acids. Good resolution of the six amino acids and the internal standard norvaline is achieved within 40 min. Other amino acids which have been reported to be present in rat brain do not interfere with the analysis. Standard curves for each of the amino acids exhibited good linearity ( r > 0.9993) over the range 0.5–20 nmol. The coefficient of variation for the intra-day and inter-day determinations ranged from 0.4% at the highest to 11% at the lowest concentration limit. Storage of whole brains at — 0°C for up to 8 weeks did not affect mean concentrations of the six amino acids.

O-235 - Effects of water-immersion restraint stress on amines and amino acids in rat brain and plasma

O-235 - Effects of water-immersion restraint stress on amines and amino acids in rat brain and plasma

In vivo conversion of γ-aminobutyric acid and 1,4-butanediol to γ-hydroxybutyric acid in rat brain : Studies using stable isotopes

The formation of 4-[1,4- 13C]hydroxybutyric acid ([ 13C]γ-hydroxybutyric acid; [ 13C]GHB) in rat brain was studied following intracerebroventricular (i.c.v.) administration of either 4-[1,4- 13C]aminobutyric acid ([ 13C]GABA or 1,4-[1,4- 13C]butanediol ([ 13C]1,4-BD) to awake, freely moving animals. GHB and [ 13C]GHB were measured with a gas Chromatographic mass spectrometric (GC/MS) technique designed to detect the lactone derivative of GHB with the acid or lactone being determined by conditions of tissue extraction. [ 13C]GHB was detected following i.c.v. administration of [ 13C]GABA with a turnover rate of 2.04nmol/g tissue/hr and [ 13C]1,4-BD with a turnover rate of 1.4 nmol/g/hr. The formation of [ 13C]GHB from [ 13C]GABA was blocked by an inhibitor of GABA-transaminase, but this drug had no effect on the formation of [ 13C]GHB from [ 13C]1,4-BD. The latter pathway was also unaffected by alcohol dehydrogenase inhibitors, compounds which block this pathway in the periphery. Further, in the course of these experiments, naturally occurring endogenous γ-butyrolactone (GBL) was detected in rat brain in a concentration of 200 pmol/g tissue weight, but lactonization in vivo of [ 13C]GHB formed from either labeled GABA or 1,4-BD was not demonstrated. These data confirm two separate pathways of synthesis for GHB in brain, demonstrate the presence of GBL in brain, and illustrate the utility of a new GC/MS technique for analysis of GHB and for GBL which does not involve extensive derivitization.

Cerebral uric acid, xanthine, and hypoxanthine after ischemia: the effect of allopurinol.

The existence of uric acid in mammalian brain was recently reported, but it has not yet become a consensus. The mammalian brain has been thought to lack xanthine oxidase, which catalyzes hypoxanthine to xanthine and xanthine to uric acid as the last steps of ATP degradation in other tissue. Using high-performance liquid chromatography, we performed assays for hypoxanthine, xanthine, and uric acid in rat brain after cerebral ischemia. It was confirmed that all three substances showed significant augmentation in the removed brains and that the chronological order of those increases corresponded to the order in the metabolic pathway. Allopurinol, a specific inhibitor of xanthine oxidase, significantly suppressed the increases in uric acid and xanthine, and a compensatory accumulation of hypoxanthine was observed. From these results, it was concluded that uric acid does exist in the brain, increases after ischemia, and is possibly the end product of purine degradation in the brain. Furthermore, it is suggested that xanthine oxidase exists in the brain and catalyzes the reaction from hypoxanthine to xanthine and then to uric acid. These reactions catalyzed by xanthine oxidase are considered to be a source of free radicals and may play important roles in the pathogenesis of cerebral ischemic injury.