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PubMed1991

Biochemical events occurring during the respiratory burst of macrophages: A 31P and 13C NMR study.

In a previous study, 31P NMR revealed that an intracellular acidification occurred during the respiratory burst of P388 D1 macrophages, but this NMR technique could not provide information about the localization of this event in cells. However, using a fluorescent pH-dependent probe, it was confirmed that this transient pH decrease does not occur in the cytosol but more probably occurs in relation to the function of endocytic vesicles. A 31P NMR study allowed us to evidence a transient increase in ADP phosphorylation at the beginning of the respiratory burst, possibly in connection with the initiation of the oxidase complex involved in superoxide anion production. A 13C NMR study of perchloric acid extracts from in vivo primed cells revealed an increase in glucose consumption due to Con A triggering. Sugar phosphates, which must be considered markers of the hexose monophosphate shunt involved in the respiratory burst, were also observed upon this activation process.

Adenosine Diphosphate↗
PubMed1985

Distribution of phosphatic metabolites in the porcine cornea using phosphorus-31 nuclear magnetic resonance.

This study describes the phosphatic metabolite contribution of the porcine corneal stroma [by phosphorus-31 nuclear magnetic resonance (P-31 NMR)] and the corneal epithelium (by difference calculation) to the total phosphatic metabolic profile of the intact (i.e. whole, undissected) cornea. In order to provide such a regional analysis, the epithelium and endothelium were surgically removed from three sets of 12 corneas. Three additional sets of 12 intact corneas were used as controls. Histological examination of two randomly selected stromata from each set of corneas were analyzed to verify that epithelium and endothelium were removed. Perchloric acid extracts were prepared from the 10 remaining stromata for P-31 NMR analysis. Twenty phosphatic metabolites were measured including six unidentified (unknown) signals. Components with diminished relative concentrations in the stromal profile compared to that of the intact cornea profile are: the triose and hexose phosphates associated with glycolysis (including alpha-glycerophosphate), choline phosphate, ATP, uridine diphosphohexoses and the unknown compound resonating at 0.93 delta. The relative level of inorganic phosphate in the stroma is nearly doubled. The remaining measured phosphates are not significantly changed relative to the total extractable phosphorus content. Consistent with previous biochemical findings, phosphates associated with energy metabolism are found primarily in the epithelial fraction. The metabolic status of the endothelial monolayer cannot be assessed because of low tissue mass and the relatively low sensitivity of P-31 NMR spectroscopy. This study serves as baseline data for the interpretation of experimental and clinical phosphorus NMR data from intact cornea by providing the contribution to the whole corneal spectrum of metabolites of the individual corneal layers.

Animals↗
PubMed1988

Methionine adenosyltransferase and S-adenosylmethionine in the developing rat lens.

Methionine adenosyltransferase (MAT) activity, and the concentration of its reaction product, S-adenosylmethionine (AdoMet), were measured in the lenses of rats of different ages; ranging from 1 day of age to over 1 yr. The highest specific activity of MAT was found in the lenses of the one day old rats (sp. ac. 0.327 units/mg-1 protein). After 1 week the specific activity had dropped to 0.067, and by 6 weeks had declined to adult levels (0.02 units/mg-1 protein). AdoMet concentrations were measured by HPLC in perchloric acid extracts. The highest concentration of AdoMet was found in the lenses of day-old rats (48.2 microM), and gradually declined with increasing age, reaching 5.5 microM in the oldest rats. In addition, the specific activity of MAT was found to be higher in the lens epithelium than in the cortex plus nucleus. The specific activity of MAT is almost an order of magnitude higher in the lens epithelial fraction (0.099 units mg-1 protein) than in the combined cortex plus nucleus fraction (0.011 units mg-1 protein).

Aging↗
PubMed1991

The effects of age on phosphatic metabolites of the human crystalline lens.

Paired human lenses ranging from 2 to 96 yr old (n = 17) were harvested within 2 hr post-mortem and immersed in liquid nitrogen. Phosphorus containing metabolites from perchloric acid extracts were quantitated using phosphorus-31 nuclear magnetic resonance spectroscopy. Low-energy metabolites detected include the phosphomonoesters (PME) hexose-6 phosphate, alpha-glycerol phosphate, fructose 1,6-diphosphate, beta-glycerol phosphate (beta-GP), uncharacterized phosphomonoesters at 4.63 delta, 4.34 delta and 3.05 delta, phosphorylethanolamine (PE), inosine and adenosine monophosphates, and phosphorylcholine (PC). Inorganic orthophosphate (Pi), glucose 1-phosphate and glycerol 3-phosphorylcholine were the other low-energy metabolites detected. High-energy metabolites phosphocreatine (PCr), adenosine tri- and di-phosphates (ATP, ADP), nucleoside diphosphorylsugars (NS) and the dinucleotides were also detected. The following metabolic indices were calculated: PCr/Pi, PME/Pi; ATP/Pi, PCr/ATP, ATP/ADP, PE + PC, PC/PE, energy charge, phosphorylation potential, and the energy modulus. A significant linear increase with age (P less than 0.05) occurred only in the uncharacterized resonance at 3.05 delta (r = 0.4593). Significant linear decreases with age (P less than 0.05) occurred in the uncharacterized resonance at 4.63 delta (r = -0.5950), beta-GP (r = -0.5018), PCr (r = -0.4495), N.S. (r = -0.4882). There was a significant (P less than 0.05) linear decrease in the energy modulus (ratio of high-energy to low-energy metabolites).

Adolescent↗
PubMed1984

Phosphorylethanolamine--the major constituent of the phosphomonoester peak observed by 31P-NMR on developing dog brain.

31P-NMR spectra of newborn dog brains exhibit a prominent phosphomonoester (PME) peak (6.78 +/- SD 0.05 ppm from phosphocreatine peak), similar to those of human neonates. Studies were undertaken to identify the chemical constituents of this peak. Brains of puppies were funnel frozen for methanol-HCl-perchloric acid extraction after in vivo 31P-NMR spectra were taken. The pK of the major component of the PME region in the NMR spectrum of extract was 5.4, corresponding to that of phosphorylethanolamine (PEt). Addition of PEt increased the major peak on the PME region over a wide range of pH, while addition of phosphorylcholine or ribose 5-phosphate yielded distinct peaks. We suggest that the major constituent of phosphomonoester peak of 31P-NMR spectra of newborn dog brain is phosphorylethanolamine. Biochemical mechanisms relevant to changes of phosphorylethanolamine during brain development are discussed.

Animals↗
PubMed1990

Tightly bound pyrophosphate in Escherichia coli inorganic pyrophosphatase.

Hexameric inorganic pyrophosphatase of Escherichia coli contains about 1 mol/mol of 'structural' pyrophosphate, which survives gel filtration and prolonged incubation with Mg2+, does not exchange with medium phosphate and pyrophosphate but is removed with 0.8 M perchloric acid. The site of pyrophosphate binding seems to be another than the active site. An additional 0.9 mol of enzyme-bound pyrophosphate is formed in the presence of phosphate and Mg2+ but this pyrophosphate is in fast equilibrium with medium phosphate and appears to be bound to the active site.

Animals↗
PubMed1991

A rapid purification method for neurogranin, a brain specific calmodulin-binding protein kinase C substrate.

A rapid purification method is reported for bovine brain neurogranin, a calmodulin-binding protein kinase C (PKC) substrate. This method takes advantage of the fact that the protein remains soluble in 2.5% perchloric acid (PCA) and that it binds to a calmodulin-Sepharose column in the absence of calcium: Other PKC substrate proteins that remain to be identified were also found to share these two properties, suggesting that a class of calmodulin-binding PKC substrates may exist in the brain.

Amino Acid Sequence↗
PubMed1994

Glycolysis is operative in amphibian oocytes.

It is generally accepted that in frog full-grown oocytes glycolysis is absent and that carbon metabolic flux is largely directed to glycogen synthesis. Use of an anion exchange pellicular resin for analytical resolution of intermediates in perchloric acid extracts of oocytes has allowed us to observe the formation of labelled lactate after microinjection of [U-14C]glucose. Further, formation of [32P]ATP was observed after microinjection of 32P-labelled glucose-6-P, fructose-6-P or fructose-1,6-bis-P, either in the presence or absence of 0.1 mM cyanide. The presence of phosphofructokinase activity, previously thought to be absent in oocytes, is also reported. These findings indicate that glycolysis to lactate is operative in frog oocytes.

Adenosine Triphosphate↗
PubMed1984

The degradation of [125I]iodoinsulin by cytosol from rat liver and the formation of high-molecular-weight products. A study by gel chromatography.

Degradation of [125I]iodoinsulin by cytosol from rat liver (as judged by the appearance of perchloric acid soluble counts) occurs rapidly and generates both high- and low-molecular-weight labelled products on chromatography on AcA54 or AcA202. One of the low-molecular-weight products can be converted to a high-molecular-weight product by further incubation with fresh cytosol. Degradation is not inhibited by chloroquine, but is affected by bacitracin. In the presence of bacitracin most of the radioactivity elutes in the void volume and possibly represents interaction of undegraded insulin with other cytoplasmic components.

Animals↗
PubMed1990

31P NMR study of phosphorus metabolites in fast and slow muscles.

1. 31P NMR was used to characterize phosphate pools in perchloric acid extracts of muscles with various composition of muscle fibre types. 2. The white m. pectoralis major (MPM) of chickens 15 min post mortem is characterized by 1.6-times higher relative content of phosphocreatine (PCr) in comparison with mixed leg muscle (LM) of this species. The glycerophosphorylcholine (GPC) does not occur in MPM at NMR detectable level in contrast to the leg muscles. Relative amounts of other phosphates are similar in both muscles. 3. The intermediate MPM of pigeons as well as mixed LM of this species contain 15 min post mortem a very small amount of PCr and ATP but a large amount of inorganic phosphate. Relative content of GPC is higher in leg muscles than in intermediate MPM. 4. Muscles with higher occurrence of white fibres contain relatively more PCr than muscles with lower occurrence of white fibres. 5. The occurrence of GPC seems to be connected with metabolism of red muscle fibres.

Adenosine Triphosphate↗
PubMed1996

A NMR study of parasitized Tenebrio molitor and Hymenolepis diminuta cysticercoids.

In vivo NMR spectra of uninfected and Hymenolepis diminuta-infected Tenebrio molitor fed D-(1-13C)glucose showed that infected beetles of both sexes had a significantly higher ratio for (glycogen C1/lipid (CH2)n) than the corresponding controls. Quantitative metabolic profiles and the per cent 13C-label in metabolites, based on NMR of perchloric acid extracts, are presented for control and infected beetles fed D-(1-13C)glucose and for H. diminuta cysticercoids. Female beetles, both control and infected, contained more glycogen than their male counterparts and infected beetles of both sexes possessed less glycerophos-phocholine, but more glycogen and a higher percentage label in glucose and trehalose than their respective controls. Label was also incorporated into glycogen, succinate, acetate, alanine and lactate. Extracts of cysticercoids from beetles fed D-(1-13C)glucose contained the following labelled compounds, in order of decreasing per cent 13C label: glucose, trehalose, alanine, succinate, lactate, glycogen and acetate. In vitro cultivation experiments, employing D-(1-13C)glucose, revealed that trehalose found in cysticercoids was of parasite, and not beetle, origin.

Amino Acids↗
PubMed1986

31P-nuclear magnetic resonance analysis of extracts of vascular smooth muscle.

31P-nuclear magnetic resonance spectroscopy was used to assess phosphate metabolites in perchloric acid extracts of rabbit aorta. In addition to the high energy phosphates, several other phosphorus compounds were detected and quantified. Most notable was the presence of a prominent phosphomonoester compound appearing at a chemical shift of 3.86 delta. This compound constituted 26% of the total extractable tissue phosphorus and is tentatively identified as ribose-5-phosphate, a pentose phosphate pathway intermediate. While ATP and phosphocreatine did not change during glucose and oxygen deprivation or during prolonged muscle contraction, the 3.86 delta phosphate decreased significantly. Furthermore, theophylline, an agent that increases intracellular cAMP, also decreased the level of the 3.86 delta phosphate. These results are consistent with the concept that intermediate metabolism sustains high energy phosphate pools in vascular smooth muscle in the steady state under various conditions. The pentose phosphate pathway may play an important role in vascular smooth muscle metabolism.

Animals↗
PubMed1996

Sample preparation for the determination of purine nucleotide analogues in tissues.

A sample treatment procedure for the determination of thiopurine and ganciclovir nucleotides in human tissues was developed. Owing to the lack of suitable standards for most of the active nucleotide analogues, the procedure was based on two steps: (1) perchloric acid homogenization and deproteinization of the tissue specimen and (2) conversion of purine nucleotides into parent drug or free bases by enzymatic or acid hydrolysis. The parent drug or purine bases formed were then analyzed on a Hypersil ODS column using isocratic elution with dihydrogenphosphate buffer for ganciclovir nucleotides or the gradient elution mode with dihydrogenphosphate buffermethanol for thiopurine nucleotides. The sample treatment procedure was evaluated using guanosine triphosphate (GTP), 6-thioinosinic acid (6TIMP) and 6-thioguanosine monophosphate (6TGMP) as standards. Mean analytical recoveries determined by adding known concentrations of standards to the tissue specimen before sampling processing were higher than 97%. The sample preparation described is simple and represents a suitable method for the investigation of active nucleotide pool in tissues.

Antimetabolites↗
PubMed1979

Radioimmunoassay of carcinoembryonic antigen (CEA) without extraction and dialysis using solid-phase antibody.

A modified radioimmunoassay has been developed for plasma CEA levels. This procedure used immobilized, solid-state antibodies, does not require perchloric acid extraction of the plasma samples and purification of the extract by dialysis. The accuracy, sensitivity and precision are equal to those of the most widely used procedure and the laboratory effort is greatly reduced.

Carcinoembryonic Antigen↗
PubMed1982

Modification of the serum bromide assay for the measurement of extracellular fluid volume in small subjects.

Serum bromide has been used in the past to measure extracellular fluid volume in infants. In this report, a modification and refinement of a previously described spectrophotometric assay suitable for determining bromide levels in blood is presented. The assay involves sample deproteinization with perchloric acid followed by spectrophotometric analysis of the complexes formed by the reaction between sodium bromide and gold chloride. Linear standard curves were obtained by diluting 1:2 with pooled serum at the following bromide concentrations: 10, 20, 30 and 40 mg%. Intra- and interassay variation using this technique was extremely small (n = 6, correlation coefficient, 0.995, and n = 5, correlation coefficient, 0.988, respectively). For toxicological analysis, a linear standard curve was derived from sodium bromide standards at the following concentrations: 40, 60, 80, and 100 mg%. The reproducibility and reliability of this assay have been excellent and the correlation with neutron activation analysis was excellent. The assay is easily adapted for the analysis of large numbers of samples, is easy to perform, and requires only small sample volumes.

Age Factors↗
PubMed1992

31P nuclear magnetic resonance study of phospholipid metabolites in ischemic liver.

To assess the metabolic alterations induced by normothermic hepatic ischemia, 31P nuclear magnetic resonance analysis was performed on liver samples using perchloric acid extraction. In particular, phosphomonoesters and phosphodiesters, the intermediary metabolites of membrane phospholipid turnover, were characterized precisely and quantitated. Phosphocholine and phosphoethanolamine, the precursors of phospholipid anabolism, did not change, while the phosphodiesters decreased. In contrast, alpha-glycerophosphate, which is both a precursor of phospholipid synthesis and the intermediary product of phospholipid degradation, markedly increased following 30 min of normothermic ischemia. These findings suggest that cellular phospholipids are actively degraded during normothermic hepatic ischemia.

Adenosine Triphosphate↗
PubMed1983

The plasma pharmacokinetics and tissue distribution of dimethyl sulfoxide in mice.

We defined the plasma and tissue concentrations and pharmacokinetics of dimethyl sulfoxide (DMSO) in 22-34 g male Swiss Webster mice injected i.v. with 15% DMSO at a dosage of 1.5 mg per g. Concentrations of DMSO in alkalinized, perchloric acid extracts of tissue and plasma were determined by gas-liquid chromatography. Plasma concentrations of DMSO declined in a biexponential fashion that was well described by the equation Ct = 2.36 exp(-0.449 t) + 1.28 exp(-0.00768 t), indicating a t 1/2 (alpha) of 1.5 min and t 1/2 (beta) of 90 min. DMSO was rapidly and extensively distributed through tissues and was not concentrated in any particular tissue, although at 1 min after injection, the brain contained the lowest concentration of DMSO of any tissue studied. By 8 hr after injection, there was little DMSO in plasma or any tissue. Intravenous injection of DMSO produced neuro-muscular disturbances, hemolysis, and hemoglobinuria in all animals. Intravenous injection of DMSO produced little increase in plasma osmolality and did not produce any histological evidence of central nervous system or renal tubular damage.

Animals↗
PubMed1985

Dopamine and norepinephrine in the alimentary tract changes after chemical sympathectomy and surgical vagotomy.

The aim of this study was to examine the distribution of dopamine and norepinephrine in the proximal alimentary tract of the rat and to assess the contributions of sympathetic and vagal fibers to the tissue concentrations of both catecholamines. Tissues were extracted in perchloric acid and the catecholamines were separated by high pressure liquid chromatography and detected electrochemically. In untreated rats (controls) both catecholamines were concentrated in the gastric muscle but norepinephrine levels were 6-8 times higher (corpus, dopamine 35 +/- 7 ng . g-1, norepinephrine 265 +/- 50 ng . g-1, mean +/- SE, n = 6). In the mucosa norepinephrine concentrations were 10-12 times higher (corpus, dopamine 12 +/- 3 ng . g-1, norepinephrine 140 +/- 26 ng . g-1). Chemical sympathectomy (6 hydroxydopamine, 100 mg . kg-1 ip 3 days) significantly reduced dopamine concentrations in muscle and norepinephrine in muscle, mucosa, pylorus and duodenum. In all tissues the effects on norepinephrine were greater. Surgical vagotomy significantly reduced dopamine concentrations in the gastric muscle, but not the mucosa. Norepinephrine concentrations in the stomach of vagotomized rats were significantly reduced only in the pylorus. Differences in the relative concentrations of dopamine and norepinephrine in gastric tissues of the normal rat and differences in the effects of sympathectomy and vagotomy suggest that dopamine and norepinephrine exist, to an extent, in separate populations of cells and that dopamine is not merely a precursor of norepinephrine. Gastric mucosal dopamine, which was mainly unaffected by either treatment, may exist in APUD cells.

Animals↗