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PubMed1984

Malonate, malonyl-coenzyme A, and acetyl-coenzyme A in developing rat brain.

Free malonate, malonyl-coenzyme A (malonyl-CoA), and acetyl-CoA were assayed in rat brain at developmental ages from the 20th day of gestation to 60 days of postnatal life. The determination of malonate was based on its conversion to malonyl-CoA and decarboxylation to acetyl-CoA by enzyme extracts from Pseudomonas fluorescens. The resulting acetyl-CoA reacted with [4-14C]oxaloacetate to form [5-14C]citrate, which was isolated by TLC. Malonyl-CoA in perchloric acid extracts from brain was converted to acetyl-CoA by rat liver mitochondrial malonyl-CoA decarboxylase (EC 4.1.1.9). Acetyl-CoA derived from this step was assayed by a modified CoA-cycling procedure. Brain acetyl-CoA was also assayed by CoA cycling. Prenatal brain contained no free malonate but malonyl-CoA was present. The acetyl-CoA level was relatively high just prior to birth and declined slightly with growth. Malonate concentrations after birth rose rapidly to reach 192 nmol/g wet weight at 60 days. Adult levels for malonyl-CoA and acetyl-CoA were 1.83 and 1.90 nmol/g wet weight, respectively. The origin and natural role of free malonate in brain are not known but deacylation of malonyl-CoA by reversal of the malonyl-CoA synthetase reaction is postulated. Rat liver and kidney also contain substantial concentrations of free malonate.

Acetyl Coenzyme A↗
PubMed1985

Composite technique for regional neurochemical studies: measurement of energy and neurotransmitter metabolites in single tissue sample.

A combined method is described for the determination of various metabolites from a single tissue sample of the brain. It comprises a quick inactivation of cerebral enzymes by microwave irradiation, easy separation of the desired brain regions, and perchloric acid extraction of tissue substances, which are assayed either by specific enzymatic techniques or by HPLC with electrochemical detection. The obtained values of most energy and neurotransmitter metabolites in the brain are in agreement with those reported using other methods. However, this technique, in contrast to the brain freezing in vitro or freeze-blowing, provides a more efficient procedure for rapid arrest of cerebral metabolism even in the deep brain structures and is therefore suitable for detection of early changes particularly those occurring in experimental pathological conditions such as ischemia.

Adenosine Triphosphate↗
PubMed1988

Brain slice glucose utilization.

The metabolism of 2-deoxyglucose has been studied in 540 micron and 1,000 micron hypothalamic brain slices. Slice 2-deoxyglucose (2DG) and 2-deoxyglucose-6-phosphate (2DG6P) levels were measured after tissue homogenization and perchloric acid extraction. By analyzing the uptake and washout kinetics with nonlinear least-squares methods, we have determined the rate constants for three-, four-, or five-parameter kinetic models and obtained a value for the in vitro lumped constant (LC). The kinetic analysis reveals a small, slowly decaying, 2DG component that is not predicted by any of the models. If this component is treated as a separate, parallel compartment, then the four- and five-parameter models are essentially equivalent. To compare our data to prior in vivo data, we combined 2DG and 2DG6P to produce Ci*, the total slice radioactivity, and analyzed the first 45 min of uptake. These data were fit best by a three-parameter model and the slowly decaying pool was not identified. Calculation of glucose utilization from total tissue radioactivity, measured by whole slice homogenization and by image analysis of autoradiograms, showed excellent correlation between the two methods. Image analysis of radioactivity in the suprachiasmatic nucleus, which is present in these slices, revealed a spontaneous diurnal variation in in vitro glucose utilization in close quantitative agreement with prior in vivo measurements. The kinetic analysis of the 1,000 micron slice was qualitatively similar to that of the 540 micron slice but revealed an increase in the LC and a large decrease in k1 as well as the expected large increase in the hexokinase rate constant, k3. Overall, in vitro glucose utilization increased by about 60%. These results are consistent with our prior studies of the 1,000 micron slice and support our interpretation that the 1,000 micron slice is an excellent in vitro model for brain ischemia without infarction.

Animals↗
PubMed1990

In vivo measurements of ethanol concentration in rabbit brain by 1H magnetic resonance spectroscopy.

In vivo 1H magnetic resonance spectroscopy was used to measure the cerebral ethanol concentration in the rabbit after both intraarterial and intragastric administration. There was good agreement between cerebral and blood ethanol concentrations at all times after administration by either route. Cerebral ethanol levels, measured using in vivo 1H spectroscopy, agreed well with those measured in perchloric acid extracts of brain, analyzed by both high-resolution 1H spectroscopy and gas chromatography. Ethanol may be useful as an indicator to measure cerebral blood flow by 1H spectroscopy and chemical shift-selective magnetic resonance imaging.

Animals↗
PubMed1991

Characterization of histamine release from the rat hypothalamus as measured by in vivo microdialysis.

The release of endogenous histamine (HA) from the hypothalamus of anesthetized rats was measured by in vivo microdialysis coupled with HPLC with fluorescence detection. Freshly prepared Ringer's solution was perfused at a rate of 1 microliter/min immediately after insertion of a dialysis probe into the medial hypothalamus, and brain perfusates were collected every 30 min into microtubes containing 0.2 M perchloric acid. The basal HA output was almost constant between 30 min and 7 h after the start of perfusion, with the mean value being 7.1 pg/30 min. Thus, the extracellular HA concentration was assumed to be 7.8 nM, by a calculation from in vitro recovery through the dialysis membrane. Perfusion with a high K+ (100 mM)-containing medium increased the HA output by 170% in the presence of Ca2+. Systemic administration of either thioperamide (5 mg/kg, i.p.), a selective H3 receptor antagonist, or metoprine (10 mg/kg, i.p.), an inhibitor of HA-N-methyltransferase, caused an approximately twofold increase in the HA output 30-60 min after treatment. The combined treatment with thioperamide and metoprine produced a marked increase (650%) in the HA output. The HA output decreased by approximately 70% 4-5 h after treatment with alpha-fluoromethylhistidine (alpha-FMH; 100 mg/kg, i.p.), an inhibitor of histidine decarboxylase. Furthermore, the effect of combined treatment with thioperamide and metoprine was no longer observed in alpha-FMH-treated rats. These results suggest that both HA-N-methyltransferase and H3 autoreceptors are involved in maintaining a constant level of extracellular HA and that their blockade effectively results in a higher activity level of the endogenous histaminergic system in the CNS.

Animals↗
PubMed1991

Phosphorylation of nuclear proteins in myelinating oligodendrocytes and its control by cyclic AMP.

Oligodendroglial nuclei isolated from rat brains at different stages of myelinogenesis (10, 18, and 30 days of age) were incubated with [gamma-32P]ATP and extracted with 0.75 M perchloric acid to yield a fraction of nonacidic chromatin proteins. The protein extracts were then analyzed by polyacrylamide gel electrophoresis. The phosphorylation pattern of these proteins was found to be different for different age groups. In 10-day-old rat oligodendrocytes the most extensive phosphorylation occurred in low molecular mass species (less than 30 kDa), in contrast to fractions obtained from 18- and 30-day-old rat oligodendrocytes which showed a significantly higher labeling of the proteins with molecular masses greater than 30 kDa. The phosphorylation of the latter species was greatly stimulated by the presence of cyclic AMP in the incubation media. The results suggest that the phosphorylation of specific nuclear proteins, which may play a regulatory role at different stages of oligodendroglial maturation and myelinogenesis, may be at least partially modulated by intracellular cyclic AMP.

1-Methyl-3-isobutylxanthine↗
PubMed1993

1H nuclear magnetic resonance spectroscopy study of cerebral glutamate in an ex vivo brain preparation of guinea pig.

Cerebral glutamate was monitored in a superfused cerebral cortical preparation by 1H NMR spectroscopy using a semiselective spin-echo sequence N-acetyl aspartate (NAA) as an internal concentration reference. During controlled metabolic conditions, the cerebral 1H NMR-detected glutamate-to-NAA ratio was approximately 20-30% lower than expected from the ratio of neutralized perchloric acid extracts of the preparations. Inhibition of respiration in the presence of glucose did not change the 1H NMR glutamate-to-NAA ratio in brain slice preparation. In contrast, either complete depletion of ATP during cyanide poisoning together with 0 mM glucose, anoxia in the absence of glucose, or treatment with nigericin or with a protonophore, carbonyl cyanide-m-fluorophenylhydrazone, increased 1H NMR-detected glutamate/NAA in the cerebral preparations without a change in the relative and absolute concentration ratios determined from the tissue acid extracts. Spin-spin relaxation times of glutamate and NAA peaks in anoxic slices were 749 +/- 89 and 729 +/- 94 ms, respectively, and thus, the portion of glutamate that could not be detected by 1H NMR was quantified in absolute terms. It was calculated that an increase in the glutamate-to-NAA ratio from 0.55 +/- 0.02 to 0.67 +/- 0.02 during aglycemic anoxia corresponded to some 6 mmol/kg of tissue dry weight of glutamate from the total concentration of 28 mmol/kg dry weight. It is suggested that this 22% of total glutamate pool is present in a noncytoplasmic compartment during controlled metabolic state.

Animals↗
PubMed1982

Acute effect of ethanol on metabolite concentrations of dog pancreas in vivo.

Sections of dog pancreas were freeze-clamped before and 1 hr after oral administration of 1 g/kg of ethanol in anesthetized, respirated, 24-hr-fasted animals, and multiple metabolites were determined in the perchloric acid extract of the frozen tissue. In spite of the fact that the pancreas contained little or no alcohol dehydrogenase activity (less than 0.01 IU/g of tissue), significant metabolite changes did occur. Although arterial oxygenation was constant and adequate (pO2 = 100 +/- 7 mM Hg) there was a significant 1.7-fold rise in L-lactate and fall in the cytoplasmic free [NAD+]/[NADH] ratio from 719 +/- 87 to 453 +/- 88 after ethanol. Except for a tendency for L-malate and L-alpha-glycerolphosphate to parallel the rise in L-lactate, there was little consistent disturbance in the remainder of the glycolytic metabolites (glucose, glucose 6-phosphate, pyruvate, 2-ketoglutarate, citrate, 3-phosphoglycerate, etc.) or in high energy intermediates and related compounds (ATP, creatine phosphate, ADP, Pi, creatine). There was, however, an unexpected and significant fall in the levels of the major transaminating amino acids, L-glutamate, L-aspartate, and L-alanine. The results can be only partially explained by an influence of blood-borne metabolites and imply significant effects of ethanol on the metabolism of the pancreas in vivo not directly mediated through alcohol dehydrogenase. Evidence is presented suggesting that both L-alanine and L-aspartate aminotransferases are functioning near equilibrium in the pancreas in vivo.

Amino Acids↗
PubMed1992

Enzymatic production of acetaldehyde from ethanol in rat brain tissue.

The capacity for the brain to produce acetaldehyde (AcHO) from ethanol was determined in rat brain homogenates. Rat brains were perfused with saline-heparin solution and homogenized in a phosphate buffer. Varying amounts of tissue were incubated with ethanol (0-100 mM) for periods of up to 60 min. The reaction was stopped by the addition of desferrioxamine and ice-cold perchloric acid. Supernatants were treated with dinitrophenylhydrazine reagent, extracted with isooctane in the presence of an internal standard, and the derivatives were separated by HPLC. The addition of 4-methyl pyrazole (an alcohol dehydrogenase inhibitor) or metyrapone (a cytochrome P450 inhibitor) had no effect on the amount of recovered AcHO. On the other hand, treatment with the catalase inhibitors sodium azide, cyanamide, or 3-amino-1,2,4-triazole blocked the production of AcHO while the addition of exogenous peroxide or a peroxide-generating system enhanced the production of AcHO. Overall, these results suggest that AcHO may be produced in the brain during alcohol intoxication, through the action of the enzyme catalase.

Acetaldehyde↗
PubMedDate not supplied

Analysis of lysine-rich histones from the unicellular green alga Chlamydomonas reinhardtii.

The H1 histones of the unicellular green alga Chlamydomonas reinhardtii were extracted from isolated nuclei, fractionated by high performance liquid chromatography, and analyzed by two-dimensional electrophoresis, peptide mapping, and N-terminal sequencing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 5% perchloric acid extracts of isolated C. reinhardtii nuclei revealed two H1 proteins (H1A and H1B). Two-dimensional gel analysis did not reveal heterogeneity of either algal H1 protein, but did detect differences in the hydrophobic amino acid content of the C. reinhardtii H1A and H1B. Digestion of H1A and H1B with V8 protease revealed two distinctly different peptide maps. C. reinhardtii H1 peptide maps were not at all similar to those of Pisum H1, but algal and pea H2B peptide maps did show some peptides in common. Seventeen amino acid residues were obtained from C. reinhardtii H1A amino terminal sequencing, while the H1B N-terminus was blocked. A search of protein data bases revealed no sequence homology of the H1A N-terminus with any known protein. Chlamydomonas histones fractionated by high performance liquid chromatography revealed minor components (histone variants) for H2A and H2B. The amino acid composition of Chlamydomonas lysine-rich histones was compared to those of various other unicellular algae.

Amino Acid Sequence↗
PubMed1975

A field biopsy method for fluoride determinations in human surface enamel.

An enamel biopsy method was developed that is especially intended for use under primitive clinical conditions in field studies. Three successive enamel layers, each of 1 mum or less, were etched off the same delimited area of 3 mm2 by means of perchloric acid, using a microsampler with disposable tips. The biopsy technique was applicable to the vestibular surface of almost all teeth, causing no perceptible damage to the enamel surface. Based on cumulated data from the successive enamel layers, curves were drawn from which F levels at various depths in the outer 3 mum of the enamel could be interpolated. F levels in surface enamel of maxillary first premolars in 40 12-13-year-old children in a locale with 0.5 mg F/l in the drinking water were found to be 3,800, 2,400 and 2,000 parts/10(6) in three successive enamel layers of 0.6, 1.0 and 1.1 mum, respectively, demonstrating a decrease in F concentration of about 1,800 parts/10(6) within this depth interval. Identical F concentrations were found in bilateral premolars.

Adolescent↗
PubMed1976

A comparison between the release of fluoride from sodium fluoride lozenges and bone meal tablets.

The release of fluoride from a fluoride-containing bone meal tablet and a chewable sodium fluoride lozenge to 0.5 M perchloric acid, deionized water, and saliva was compared. In acid, all the fluoride was released from the bone meal tablet (0.25 mg F), while there was a poor release in water and saliva (2-10 %). The release from the sodium fluoride lozenge (0.25 mg F) was essentially complete in water and saliva as well as in acid. Following sucking and chewing on a sodium fluoride lozenge, the mean salivary fluoride concentration increased from 0.04 to 36 parts/10(6) 5 min after intake. Concentrations exceeding the preintake level were still recorded after 20 min. With the bone meal tablet, only a slight increase (to 0.2 parts/10(6)) in the salivary fluoride level was obtained.

Calcium Phosphates↗
PubMed1985

Relationship between enamel fluoride concentration and dental caries in a selected population.

The relationship between dental caries experience (DMFT) and enamel fluoride concentration (EnF) was determined in 100 freshman dental students aged between 20 and 26 yr. DMFT was determined by clinical examination and supplemented by bitewing radiographs. Biopsies were performed on the maxillary right canine of each student by etching with 1 M perchloric acid for 5 s. A regression analysis of DMFT and EnF and of square root DMFT and 1nEnF indicated that the correlation coefficients were not significant. Students exposed to fluoridated drinking water had significantly lower DMFT and significantly greater EnF than the students reared in areas with non-fluoridated drinking water.

Acid Etching, Dental↗
PubMedDate not supplied

Determination of the concentration of complex boronated compounds in biological tissues by inductively coupled plasma atomic emission spectrometry.

The application of inductively coupled plasma atomic emission spectrometry (ICP-AES) to the determination of the concentration of complex boron-containing compounds in biological tissue samples is described. Tissue digestion is achieved with perchloric acid and hydrogen peroxide in 1 hr at 75 degrees C. The ICP-AES method gave a linear response for elemental boron concentration in the range 0.05 to 100 ppm and does not require the reduction of the boron to a simple species, such as boric acid. Complete recovery of boron in complex boron cluster compounds was obtained. The procedure has been applied to the determination of the boron content in compounds synthesised for neutron capture therapy and is suitable for use in biodistribution studies of such compounds.

Animals↗
PubMed1980

Purification of GABA on small columns of Dowex 50W; Combination with a method for separation of biogenic amines.

Elution of GABA has been included in the method for separation of biogenic amines on small Dowex 50W columns described by Atack & Magnusson (1978). After extraction with perchloric acid and neutralization to pH 3.0, the sample is passed through the column. 5-HIAA might thereafter be eluted as described by Lindqvist (1971). After washes with water and 8 ml of a 0.025 M sodium citrate buffer pH 4.5, GABA is eluted in 3 ml of a 0.05 M sodium citrate buffer pH 5.3-5.4. The elution of tryptophane and the above mentioned amines can thereafter be performed. The recovery of GABA from the column is 100%. Silica thin-layer chromatography of the GABA eluate gives only one ninhydrine positive spot with a Rf value identical to GABA standard. When the GABA eluate from a whole brain and spinal cord is run on an automatic amino acid analyser, only one peak is detectable, eluted in the same position as GABA standard. Fluorescence analysis of the GABA eluate gives similar values if a( ninhydrine, b) acetylacetone plus formaldehyde or c) fluorescamine is used to form the flurophore. The GABA values are identical whether the samples reacted with ninhydrine are diluted with water or copper tartrate. The GABA concentrations found in some parts of the rat brain are of the same order of magnitude as reported by other authors.

5-Hydroxytryptophan↗
PubMed1994

Detection and quantification of melatonin in a dinoflagellate, Gonyaulax polyedra: solutions to the problem of methoxyindole destruction in non-vertebrate material.

Preservative procedures are described for the extraction and quantification of melatonin in cell material from the dinoflagellate Gonyaulax polyedra, an organism in which this indoleamine is rapidly degraded due to interaction with free oxygen radicals and photooxidation. Cells were shock-frozen in liquid nitrogen and pulverized. Various extraction methods were applied to the powder, and rates of recovery were compared. For the determination of melatonin by high performance liquid chromatography (HPLC), extractions with acetone or perchloric acid were more suitable than the use of other solvents. For purposes of radioimmunoassay (RIA), extraction with acetone gave the best results. Several other inorganic solvents, which are often applied in melatonin research, such as chloroform, dichloromethane, and diethyl ether, led to considerable losses of the indoleamine. The procedures developed for HPLC with either electrochemical or fluorescence detection also allow the quantification of other indolic compounds, in particular, tryptophan and 5-methoxytryptamine. The methods described may be of value in the further search for melatonin and related indoleamines in non-vertebrate material, especially, from unicells, multicellular plants, and invertebrates.

Animals↗
PubMed2006

Quantification of melatonin in phototrophic organisms.

Melatonin, the chief secretory product of the vertebrate pineal gland is also known to occur in numerous photoautotrophic organisms. The indoleamine is suspected to act as a transducer of photoperiodic information and/or to participate in antioxidative protection. In higher plants and other photoautotrophic organisms, contradictory results for melatonin content for samples from the same species show that further improvement of methods for reliable quantification is required. In the present study, melatonin was quantified in tomatoes, ginger and the marine green macroalga, Ulva lactuca, after extraction with three different extraction methods based on ether, acetone or perchloric acid. Melatonin was determined by enzyme-linked immunosorbent assay (ELISA) in high-performance liquid chromatography (HPLC)-purified extracts. The same HPLC system used for purification of extracts was used for parallel quantifications after derivatization of melatonin under alkaline conditions in the presence of hydrogen peroxide (HPLC-PD). Both quantification methods gave similar results with a high correlation [f(x) = 0.99x + 3.01; R(2) = 0.99]. In ginger, the melatonin concentration was below 5 pg/g (fresh weight, f.w.), whereas in tomatoes about 1200 pg/g (f.w.) were found, and in the green alga, U. lactuca, approximately 12 pg/g (f.w.). Taking into account the recovery rates for synthetic melatonin added prior to extraction, no substantial differences were observed in melatonin quantification between different extraction methods. The demonstrated methods based on HPLC purification and subsequent quantification by ELISA and HPLC-PD allow highly sensitive melatonin determinations in diverse photoautotrophic organisms with a low risk of overestimations by false-positive results.

Chromatography, High Pressure Liquid↗
PubMed1993

Detection of radical ion intermediates in flavin-photosensitized pyrimidine dimer splitting.

Photosensitized splitting of cis-syn- and trans-syn-1,3-dimethyluracil dimers by 2',3',4',5'-tetraacetylriboflavin in acetonitrile containing a trace of perchloric acid was studied by laser flash photolysis. Protonation of the flavin prior to excitation resulted in excited singlet and triplet states that abstracted an electron from the dimers and yielded the protonated flavin radical (FlH2.+), which was detected by absorption spectroscopy. Electron abstraction by the excited singlet state predominated over abstraction by the triplet state. Approximately one-third to one-half of the excited states quenched by the trans-syn dimer yielded FlH2.+, the balance presumably undergoing back electron transfer within the geminate radical ion pair generated by the initial electron transfer. A covalently linked dimer-flavin exhibited very inefficient flavin radical ion formation, consistent with the known low efficiency of dimer splitting in this system. These results constitute the first identification of a flavin radical ion intermediate in photosensitized pyrimidine dimer splitting.

Free Radicals↗