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PubMed1998

The nu5 Band of HClO4

The high-resolution infrared spectrum of perchloric acid has been observed in the 700-750 cm-1 region using the infrared beamline at the MAX-I electron storage ring in Lund, Sweden. The spectrum displays extensive rotational structure due to a type a band and is assigned to nu5, the HO-ClO3 stretch. Approximately 1100 transitions in H35ClO4 and ca. 300 in H37ClO4 have been fitted using single subband analysis, generating constants for transitions having the same K. The origin of H35ClO4 K = 3t series is found to be 726.9971(4) cm-1. Rotationally resolved infrared line positions are now available for the identification of HClO4 in the atmosphere, which may be produced by the heterogeneous oxidation of chlorine containing species in the stratosphere. Copyright 1998 Academic Press.

Journal Article↗
PubMed1980

Low acetaldehyde levels in blood, breath and cerebrospinal fluid of intoxicated humans as assayed by improved methods.

A sensitive method for determination of human blood acetaldehyde (AcH), which avoids artefactual ethanol-derived Ach formation, was developed. AcH was trapped by collecting blood directly into isotonic semicarbazide, the plasma separated, AcH liberated by perchloric acid and analyzed by gas chromatography. Breath AcH was also trapped in semicarbazide and analyzed similarly. Using an experimentally determined blood:breath partition ratio of 190, calculated pulmonary blood and measured antecubital blood AcH were very similar at various concentrations. Blood AcH was found generally to be very low (< 10 microM) at moderate ethanol levels. Calcium carbimide (0.25 mg/kg) caused moderate flushing reactions and elevated AcH to 25-188 microM. 4-Methylpyrazole (5 mg/kg i.v.) rapidly attenuated AcH levels and symptoms, indicating its potential use in the treatment of disulfiram-ethanol reactions. AcH in the cerebrospinal fluid of 5 highly intoxicated patients was almost absent (0-5 microM). Blood AcH in occasional or chronic alcohol abusers were generally low (< 10 microM), elevated AcH levels being observed only in association with clinical abnormalities. The results indicate that in general, previously reported human blood AcH levels are erroneously high and that breath levels reflect blood levels. Blood AcH may play a lesser role in the actions of ethanol in humans than is often assumed.

Acetaldehyde↗
PubMed2007

Revealing the metabolome of animal tissues using 1H nuclear magnetic resonance spectroscopy.

The measurement of tissue-specific metabolic fingerprints can be of particular interest when investigating disease processes, mechanisms of toxicity, or when knowledge of the metabolic interactions between different organs is required. This chapter presents several optimized protocols for the extraction of metabolites from animal tissues, their analysis by 1H nuclear magnetic resonance (NMR) spectroscopy, and the subsequent spectral preprocessing required for an NMR-based metabolomics experiment. First, the three critical steps in the preparation of tissue extracts for NMR analysis are described, including both a perchloric acid protocol for the extraction of polar metabolites, and a methanol:chloroform protocol for extraction of polar and lipophilic metabolites. Then a series of NMR experiments are described including a standard one-dimensional (1D) 1H NMR study, a 1D 1H Carr-Purcell-Meiboom-Gill spin-echo experiment, and a two-dimensional 1H-1H J-resolved NMR experiment. The advantages and limitations of each experiment for metabolomics research are discussed. Analysis of the resulting NMR datasets is typically conducted in two phases comprising "low level" spectral preprocessing and "high level" multivariate analysis. NMR spectral preprocessing is a critical step that converts raw NMR spectra into an appropriate data format for multivariate analysis. A detailed protocol for preprocessing NMR data, using ProMetab software, is presented. Because a plethora of algorithms exist for multivariate analyses, which can be used to construct classification models or for biomarker discovery, this is beyond the scope of the current chapter.

Animals↗
PubMed2003

Regional N-acetyl-aspartate level and immunohistochemical damage in the hippocampus after transient forebrain ischemia in gerbils.

We investigated changes in regional N-acetyl-aspartate (NAA) levels in the vulnerable CA1 and resistant CA3 areas of the hippocampus after transient forebrain ischemia in gerbils. Under light ether anesthesia, bilateral common carotid arteries of adult male Mongolian gerbils (60-80 g) were occluded for 5 min and reperfused for 7 days. Brains from experimental and control gerbils (n = 4 each) were frozen in situ, and frozen sections (20 microm) were prepared using cryostat (-20 degrees C). After overnight lyophilization, the CA1 and CA3 areas were dissected out separately, weighed (50-200 microg), and the supernatant of the perchloric acid extract was used for assay of NAA using HPLC. Adjacent 10 microm-thick sections were used for immunohistochemical analysis using antiserum against microtubule-associated protein I and II. The preischemic NAA levels were not significantly different between CA1 and CA3 areas. After transient ischemia, a significant (P < 0.01) decrease in the NAA level was observed in the CAI area, but not in the CA3 area of the hippocampus. Immunohistochemical ischemic damage evolved only in the CA1 area. Thus, the decrease of the regional NAA level was associated with development of immunohistochemical neuronal damage.

Animals↗
PubMed2000

Malignancy of brain tumors evaluated by proton magnetic resonance spectroscopy (1H-MRS) in vitro.

Biopsies of 6 malignant gliomas (grade 3 or 4) and 11 low-grade meningiomas were extracted with perchloric acid or methanol/water, and the fully-relaxed 1H-MRS spectra of the extracts containing water-soluble metabolites and a concentration and chemical shift standard were recorded at 11.4 T. The resonance signals assigned to inositol (Ino), glycerophospho- and phosphocholine (GPC + PC), choline (Cho), creatine and phosphocreatine (Cr + PCr), glutamate (Glu), acetate (Ac), alanine (Ala) and lactate (Lac) were integrated, and analyzed by two methods. First, the concentrations of the aforementioned substances in the bioptates were estimated from their resonance signals in the extracts. Second, these signals were normalized to the Cr + PCr resonance signal. The Mann-Whitney U-test was used to verify statistical significance between the data sets obtained for gliomas and meningiomas. When the first method of analysis was used, the only difference was in the Ala concentration, which in meningiomas was on average 4 times higher than in gliomas (P < 0.01). However, when the second method of analysis was applied, gliomas expressed lower normalized resonance signals of Ala and Glu (P < 0.001, ranges not overlapping), Lac (P < 0.005), as well as Ino and GPC + PC (P < 0.05). In proton MR spectra of brain tumor tissue extracts containing water soluble metabolites, the resonance signals normalized to that of total creatine may provide a very good discrimination between malignant gliomas and low-grade meningiomas.

Biopsy↗
PubMed1982

Kinetics of the reactivity of subpopulations of spleen cells of mice bearing virus-induced mammary tumors to syngeneic antigenic extracts in vitro. Supportive and suppressive effects of macrophages.

This study was designed to investigate the nature of lymphocyte reactivity to soluble tumor antigens with respect to the kinetics of the reactivity, the responding cell type, and the role of accessory cells, within a syngeneic system. BALB/c mice were inoculated with 1 X 10(6) viable cells of syngeneic MTV-induced mammary tumors. Assessment of proliferative activity of spleen cells of these animals by DNA synthesis (3H-thymidine incorporation in vitro) indicated a biphasic response to stimulation by 200 micrograms of a syngeneic perchloric acid (PCA)-soluble extract (AMMT) of the tumor over a 25-day period, with peak activities at days 13 and 19 post inoculation. The response was predominantly T-cell-mediated. Splenic macrophage population rose from less than 2% of total spleen cells by day 25 without any appreciable change in the T or B cell population. Depletion of spleen cells of macrophages abolished the first peak activity (at day 13) but significantly enhanced the second (at day 19). Reconstitution of the depleted cells with macrophages prepared from peritoneal exudates of tumor-bearing or normal mice restored the responses to undepleted values, thus indicating an accessory role for macrophages in these responses. These results provide new data which should contribute to a better understanding of the tumor-host relationship.

Animals↗
PubMed1988

Determination of fluoride in urinary calculi using a quantitative microdiffusion method.

A microdiffusion method for the separation of fluoride from other ions in urinary calculi has been developed, tested and assessed. The procedure involves digestion at 75 degrees C of samples with silicone-impregnated mixtures of nitric and perchloric acids in a specially designed diffusion cell and determination of the diffused fluoride with an ion-sensitive electrode. Several test samples were used to assess the recovery, accuracy and reproducibility of the procedure. Results for 20 stones of Indian origin are presented and discussed.

Fluorides↗
PubMed1992

Effects of fasting and food restriction on sympathetic activity in brown adipose tissue in mice.

The activity of the sympathetic nervous system in mice that were either fed ad libitum, food restricted or fasted was estimated by measuring the accumulation of dopamine following the inhibition of dopamine beta-hydroxylase activity. Mice in each group were injected with the dopamine beta-hydroxylase inhibitor 1-cyclohexyl-2-mercaptoimidazole and were exposed to either 30 degrees C (warm) or 4 degrees C (cold). Mice were killed 1 h after the injection. Both heart and brown adipose tissue were then quickly removed and homogenized in ice-cold perchloric acid. Dopamine and noradrenaline were determined using high performance liquid chromatography. Regardless of whether mice were warm or cold exposed, both content and concentration of brown adipose tissue and dopamine were predictably higher in 1-cyclohexyl-2-mercaptoimidazole-injected mice than in non-injected animals. In mice fed ad libitum, post-injection content and concentration of dopamine in both brown adipose tissue and heart were higher in cold-exposed mice than in warm-exposed animals. In food-restricted and fasted mice, post-injection concentrations of dopamine in brown adipose tissue were higher in cold-exposed mice than in warm-exposed animals. In food-restricted and fasted mice there was no difference between warm- and cold-exposed animals with respect to post-injection contents and concentrations of dopamine in heart tissue. In fasted mice there was no difference between warm- and cold-exposed animals in post-injection content of dopamine in brown adipose tissue. This study provides further evidence that fasting, in contrast to food restriction, may blunt the tissue sympathetic nervous system response in brown adipose tissue of cold-exposed mice.

Adipose Tissue, Brown↗
PubMed1979

Nephrotoxicity of aminophenols: effects of 4-dimethylaminophenol on isolated rat kidney tubules.

In isolated rat kidney tubules DMAP was found to inhibit the gluconeogenesis from lactate, pyruvate, or dihydroxyacetone. The ratio DMAP/protein rather than the calculated concentration of DMAP determined the strength of the effect, 20--25 nmoles DMAP/mg protein inhibiting the rate of gluconeogenesis by about 50%. The inhibition was not reversible. Phenacetin, 4-aminophenol and 4-acetamidophenol were much less effective than DMAP in inhibiting gluconeogenesis in isolated rat kidney tubules. DMAP 14C-labeled in the ring was quickly bound to proteins in kidney tubules. A portion of DMAP which did not exceed about 4 nmoles/mg protein, was bound in compounds soluble in perchloric acid. From this portion tris-GS-DMAP was isolated. DMAP diminished the glutathione content of isolated rat kidney tubules. Reduced glutathione added before DMAP prevented the inhibition of gluconeogenesis and diminished the binding of DMAP to proteins. The binding of DMAP required oxygen and was inhibited by carbon monoxide or cyanide. Several enzymes from isolated kidney tubules were found to be inhibited by DMAP doses which inhibited gluconeogenesis. Large DMAP doses also diminished the sums of ATP + ADP + AMP as well as NAD + NADH and NADP + NADPH. This effect corresponded to an increase in nucleotide degradation products and to increased activity of extracellular LDH. The results indicate that the inhibition of gluconeogenesis by DMAP is not due to a specific effect on one enzyme or on membranes but to unspecific reactions with many substances.

Aminophenols↗
PubMed1979

Oxidized and reduced glutathione levels of the cornea in vivo.

A new method for preparing the rabbit cornea is described for the assay of metabolite levels in the in vivo state. The preparation includes in vivo rapid freezing of the tissue by liquid nitrogen, freeze sawing, lyophilization, and extraction with 0.5 N perchloric acid. Reduced (GSH) and oxidized (GSSG) glutathione levels and the GSH/GSSG ratios were compared with adenosine phosphate levels and ATP/ADP ratios. The most important results of this investigation seemed to be a significant difference in the redox state of the glutathione between the epithelium and the endothelium of the corea and the different reducing capacity of these tissues as indicated by the steady-state levels of the GSH and the GSSG. These metabolic processes may be used to eliminate toxic peroxides from the transparent ocular tissues produced by light or other chemical compounds.

Adenine Nucleotides↗
PubMed1979

The effect of the combination of lithium and haloperidol on brain intermediary metabolism in vivo.

The effect of the chronic administration of the combination of lithium and haloperidol has been studied in rat brain in vivo. Lithium was administered in food in amounts sufficient to maintain serum lithium levels of 1.0 +/- 0.1 mEq/l; haloperidol (1.5 mg/kg) was given i.p. once daily. Control animals pair-fed with the lithium/haloperidol alone, or neither drug. Fifteen days after the beginning of the experiments the brains were instantaneously frozen with a rapid brain-freezing device and multiple metabolites were measured in the perchloric acid extract of the tissue. Intermediates examined included selected metabolites of the glycolytic pathway and the tricarboxylic acid cycle, N-acetylaspartate and cofactors such as ATP, CoA, and acetyl-CoA. Estimates of the effects of the treatments on cytoplasmic and mitochondrial redox states were also made. The results showed only minor effects of any of the treatments on any of the parameters studied and little or nothing to distinguish the combination of lithium and haloperidol from either treatment alone.

Animals↗
PubMed1976

Ultrastructure of tubular inclusions in endothelial cells of pituitary tumors associated with acromegaly.

Tubular inclusions were present in 13 out of 43 pituitary adenomas of acromegalic patients and in a single chromophobe pituitary adenoma. There were none in 76 other pituitary adenomas with differing endocrinological symptomatology. The arrays were usually located in the perinuclear cistern of capillary endothelial cells. The tubule diameter in osmium fixed material measured 19-26 nm and the light core averaged 6-11 nm. A longitudinal period of about 4.5 nm could be demonstrated with PTA block staining. Fixation with glutaraldehyde and block staining with ethidium bromide as well as permanganate fixation followed by RNAse treatment showed only the core of the tubules consisting of globular subunits. Several histochemical reactions (perchloric acid extraction, methenamine-silver staining, trypsin and DNAse digestion of frozen sections) suggested that the particles consist of a core of DNA coated with protein. No virus multiplication could be detected in cell cultures or in mice innoculated with fresh tumor material. No significant antibody titers against several virus antigens could be demonstrated.

Acromegaly↗
PubMedDate not supplied

Identification of minor tightly bound H1 histone subfractions which fail to cleave their initiator methionine.

Groups of CBA mice were administered [35S] methionine (1 mCi/mouse). Non-histone proteins, H1 and H1(0) histones and nucleosomal core histones were isolated from different issues by selective extractions. The measurements of radioactivity of individual bands and autoradiography of dry gels were used to identify methionine-containing and methionine-free histone variants. H1A and H1B histone variants extracted with 5% perchloric acid were methionine-free. However, minor sub-fractions of these histones which are more tightly bound to DNA (and which can be extracted only with 0.25 N HCl) contained [35S] methionine and did show a higher specific activity than methionine-containing nucleosomal hitones. Cyanogen Bromide reaction which destroys non-histone proteins and methionine-containing nucleosomal histones removes radioactivity but does not alter the position of methionine-containing H1 minor bands. This indicates that the radioactive methionine occupies only the N-terminus of the H1 molecules. It is suggested that this methionine is an uncleaved initiator methionine. The presence of these methionine-containing minor H1 subfractions varies in different tissues.

Age Factors↗
PubMed1992

Qualitative differences in nuclear proteins correlate with neuronal terminal differentiation.

1. Protein composition of neuronal nuclei was studied at two stages of brain maturation, i.e., before (embryonic day 16; E16) and after (postnatal day 10; P10) shortening of the nucleosomal repeat length. Glial nuclei were analyzed in parallel as a control. 2. Total nuclear or HCl- and 5% perchloric acid (PCA)-soluble proteins were analyzed by different electrophoretic techniques. 3. Our results show an increase in the concentration of histone H1 zero with differentiation, although the H1 class undergoes an overall decrease. 4. The chromatin of mature neurons is also enriched in the ubiquinated form of histone H2A (A24), while the high-mobility group (HMG) proteins 1 and 2 seem to decrease slightly relative to core histones. 5. Both quantitative and qualitative differences in the abundance of nonhistone proteins relative to histones accompany neuronal terminal differentiation.

Animals↗
PubMed1995

Influence of sample preparation on cellular glutathione recovery from adherent cells in culture.

During the last decade, the unbound glutathione content of cultured adherent cells has become a very important biological marker for many pharmacological and toxicological in vitro studies with regard to the protective role of the tripeptide in its reduced form (GSH). However, the literature does not provide extensive information on the influence of sample preparation on cellular GSH and thiol analyses. Using the fibroblast-like V79 cell line as model, we undertook a comparative study of the efficiency of different procedures reported in the literature with respect to GSH recovery. Depending on the preanalytical step, up to 10-fold discrepancies could be observed in the recovery of intracellular GSH. Different parameters that must be controlled in order to maximize GSH recovery are discussed. The optimal strategy consisted in rapid perchloric acid deproteinization performed directly in the dish, which was extremely valuable for preparing GSH samples from adherent cells, and especially from cells expressing elevated gamma-glutamyl transferase activity.

Animals↗
PubMedDate not supplied

Exogenous fructose-1,6-bisphosphate is a metabolizable substrate for the isolated normoxic rat heart.

Isolated rat hearts were perfused by the recirculating Langendorff mode under normoxic conditions for 60 min. The Krebs-Ringer buffer was supplemented with 10 mM glucose + 12 IU/l insulin and either [U-14C]-fructose-1,6-bisphosphate (together with 5 mM cold fructose-1,6-bisphosphate) or [U-14C]-fructose (together with 5 mM cold fructose). At the end of perfusion, gaseous 14CO2, 14CO2 trapped in the perfusates, 14C-lactate output and tissue 14C-lactate were assayed in both groups of hearts. Analysis of high-energy compounds, glycogen, lactate, and pyruvate was also performed on the neutralized perchloric acid extracts of the freeze-clamped hearts. Data obtained from the 14C catabolites, originating from the metabolism of the radiolabeled substrates, indicated that the isolated normoxic rat heart metabolizes an 8.5 times higher amount of fructose-1,6-bisphosphate (7.07 mumoles/min/g d.w.) than of fructose (0.83 mumoles/min/g d.w.). CrP, CrP/Cr, glycogen, and total lactate in both tissue and perfusate were significantly higher in fructose-1,6-bisphosphate-perfused hearts. The overall indication is that fructose-1,6-bisphosphate can be taken up in its intact form by myocytes and successively metabolized to support their energy demand, and that its effects on myocardial performance and metabolism should be attributed to the molecule itself rather than to its eventual degradation products.

Animals↗
PubMed1980

Studies on distribution and metabolism of valproate in rat brain, liver, and kidney.

Time-course studies on the distribution and metabolism of valproate (VPA) in rat brain, liver, and kidney, after intraperitoneal injection of a mixture of [14C]VPA and [3H]VPA, showed that: (1) maximal amount of radioactivity in the various tissues was observed after 30 min from the time the drug was administered; (2) at 30 min the distribution of labeled VPA in brain, liver, and kidney was 17%, 64%, and 19% of the total radioactivity, respectively; (3) at 24 hr more than 97% of the total radioactivity was lost from the tissues and the 14C/3H ratios increased significantly with time. Studies on the regional distribution of the drug showed that it is relatively homogeneously distributed. Studies on the subcellular distribution of the drug showed that it is associated mostly with the soluble and mitochondrial fractions, with little radioactivity in the myelin and symaptosomal fractions. Radiochromatography of VPA metabolites in perchloric acid extracts from brain, liver, and kidney revealed the presence of four metabolites. VPA was not incorporated into phospholipids of the neuronal membranes. Furthermore, it had no significant effects on Mg2+-ATPase and (Na+ + K+)-ATPase in synaptosomes and microsomes obtained either from control or from rats injected with VPA. It was concluded that this antiepileptic drug does not appear to act through its incorporation into neuronal membrane or through its action of the Na+ pump.

Adenosine Triphosphatases↗
PubMed1984

P-31 nuclear magnetic resonance analysis of brain: normoxic and anoxic brain slices.

Perchloric acid extracts were prepared from liquid-N2-frozen gerbil and guinea pig brain slices studied under one of three conditions: O2-incubated, N2-incubated or O2-incubated recovery following N2 incubation. Mole percentages of the various phosphatic components contained in the extracts were determined by phosphorus-31 nuclear magnetic resonance spectroscopy. The brain slice extract spectrum revealed a previously unreported group of brain phosphodiesters at -0.73 delta relative to 85% orthophosphoric acid. Although the phosphatic profiles from O2-incubated slices from gerbils and guinea pigs revealed only minor species variations, which differed quantitatively rather than qualitatively, species-specific differences were made readily apparent and amplified by incubating brain slices under oxygen-deficient conditions. Despite these differences which were most prevalent during the recovery phase, the overall metabolic changes described herein in response to N2-incubation were in accord with the results obtained by other analytical techniques. Inorganic orthophosphate (2.63 delta) was increased, while nucleoside (principally, adenosine) triphosphate (alpha-, -10.92 delta, beta-, -21.45 delta, and gamma-, -5.80 delta) and phosphocreatine (-3.12 delta) levels were decreased in response to N2 incubation. In addition, inosine monophosphate (3.78 delta) was increased and the levels of a partially characterized acid-labile phosphate (0.85 delta, guinea pig) were decreased upon N2 incubation. Phosphoglyceride metabolism also appeared to be altered by oxygen deprivation (gerbil). These latter findings provide additional information concerning the metabolic responses of cerebral tissue to oxygen deficient conditions.

Adenine Nucleotides↗