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The thermolysis of various substituted ammonium salts of nitric and perchloric acids has been reviewed in the present communication. The mechanistic aspects of thermal decomposition of these salts have been discussed critically. It has been observed that the proton transfer process do play a major role during thermolysis of these salts. The plausible decomposition pathways have also been described.
Guanylate cyclase in cultured neuroblastoma N1E 115 cells was readily solubilized. MgCl2 as well as MnCl2 served as a metal cofactor of the guanylate cyclase. The maximal guanylate cyclase activity obtained with MgC12 was 80% of that with MnCl2. When the supernatant of cell homogenate was adjusted to pH 5.2, all of enzyme activity was precipitated. The guanylate cyclase activity recovered in the pH 5.2 precipitate was reduced to about 10% of the original supernatant. Combination of the pH 5.2 supernatant and precipitate fractions, however, restored guanylate cyclase activity, indicating that the pH 5.2 supernatant contains an endogenous activator for guanylate cyclase. The activating factor in the pH 5.2 supernatant remained in the aqueous phase after proteins were removed by perchloric acid. The factor was filterable through Diaflo ultrafilter membranes UM 2 and UM 10 indicating that the factor is a small molecule. The activation by the endogenous activator was prevented by N-methylhydroxylamine and lysolecithin.
A method for determining N-ethyldiethanolamine (EDEA), N-methyldiethanolamine (MDEA) and triethanolamine (TEA), hydrolysis products of nitrogen mustards, in water, urine and blood samples using gas chromatography-mass spectrometry (GC-MS) after derivatization by tert-butyldimethylsilylation (TBDMS) is described. The sample solution was evaporated to dryness, and reacted with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) at 60 degrees C for 1h. The TBDMS derivatives were separated on a DB-5 column and detected by electron-ionization MS. The quantitation of EDEA, MDEA and TEA was performed by measuring the respective peak areas on the extracted ion chromatograms of m/z 216, m/z 202 and m/z 346, respectively, using nonadecane (C19), the peak area of which was measured at m/z 268, as an internal standard. When the water sample was initially analyzed, considerable loss of EDEA, MDEA and TEA occurred by evaporation. The addition of hydrochloric acid (HCl) to the water sample (final 1 mM), however, permitted quantitative recoveries to be achieved (88%, 88% and 79% for EDEA-(TBDMS)2, MDEA-(TBDMS)2 and TEA-(TBDMS)3, respectively). The limits of detections (LODs, scan mode, S/N = 3) were 2.5, 2.5 and 10 ng/ml for EDEA, MDEA and TEA, respectively. Ethanolamines could be also determined in urine samples (volume 0.1 ml), with reasonable recoveries of 72-100% by the addition of HCl (final 1 mM). For the analysis of serum samples, the sample was precipitated by the addition of perchloric acid (final 3.2%), and the resulting supernatant was neutralized with potassium carbonate, and then acidified by the addition of HCl. The recovery of TBDMS derivatives of ethanolamines was found to rather low (7-31%).
Nucleosomal chains of various repeat unit lengths were generated by a mild micrococcal nuclease digestion of purified pancreatic nuclei. Maximal nucleosome associated poly(ADP-ribose) polymerase activity was recovered in trimeric to tetrameric chromatin fragments, after which the enzyme activity gradually decreased and stabilized towards oligomeric periodicities of 11 to 16 nucleosomes. Electrophoresis of [32P]ADP-ribosylated histones on first-dimension acid-urea or acid-urea-Triton gels and on second-dimension acid--urea--cetyltriammonium bromide gels revealed that, of all histones, only histone H1 could be significantly poly(ADP-ribosyl)ated while only minimal modification could be recovered with histone H1(0). Furthermore, the extent of ADP-ribosylation present on pancreatic histone H1 is shown to proportionally retard this protein's electrophoretic mobility in all gel systems and to consist of a distinct series of at least 12 modification intermediates which can be evidenced, in nuclei or nucleosomes, and fully recovered along with histone H1 upon its selective extraction with 5% perchloric acid. The generation of these increasingly ADP-ribosylated forms of histone H1 is also demonstrated to be time dependent and the more complex ADP-ribosylated forms of this histone are favored at high NAD+ concentrations. Moreover, the electrophoretic mobilities of all intermediates are unaffected by the presence of the nonionic detergent Triton X-100.
BACKGROUND: Alpha-1-acid glycoprotein (AGP) is an acute phase protein that increases in concentration in infectious and inflammatory conditions. The serum and peritoneal fluid concentrations of AGP may be useful in the diagnosis of feline infectious peritonitis (FIP), a lethal disease of cats. Currently AGP can be measured by radioimmunodiffusion (RID) assays, which are time consuming and difficult. OBJECTIVES: The objectives of this study were to develop a rapid immunoturbidimetric assay for measurement of AGP in feline serum and peritoneal fluid and to compare the results with those obtained by RID. METHODS: AGP was purified by perchloric acid precipitation and ion-exchange chromatography from a pool of peritoneal fluid obtained from cats with FIP, as determined by a panel of laboratory tests, including serum AGP concentration, albumin: globulin ratio, and total protein concentration, anti-coronavirus antibody titers, and effusion analysis. The purified AGP in a complete Freund's adjuvant and Tween 20 mixture was injected into a sheep and blood was collected at monthly intervals. Anti-AGP antiserum, as confirmed by ELISA and Western blot techniques, and a pool of peritoneal fluid from cats with FIP were used to prepare standards. Clinical samples of feline peritoneal fluid (n=55) and serum (n=59) were assayed for AGP and results from the immunoturbidimetric and RID methods were compared. RESULTS: Significant correlation (P < .001) was obtained between methods for both peritoneal fluid (R2=.9259) and serum (R2=.9448) samples. Coefficients of variation for the immunoturbidimetric method were <5%. CONCLUSIONS: This rapid immunoturbidimetric assay for measurement of feline AGP in serum and peritoneal fluid may be of value in the diagnosis of FIP and possibly other inflammatory diseases in cats.
Although it is beyond doubt that mammary cells accumulate iodide via a Na+-dependent transport mechanism, it is not clear if this is the only pathway for iodide transport in mammary tissue. In view of this, experiments were designed to test for the presence of an anion-exchange pathway which could mediate the transport of iodide into mammary cells; thus, the effect of external iodide on sulfate efflux from rat mammary tissue has been investigated. Iodide trans-stimulated sulfate efflux from mammary tissue explants in a dose-dependent manner: 0.1, 1.0 and 10.0 mM iodide stimulated the fractional release of iodide by 56 +/- 2.2, 166.5 +/- 17.5, and 302.9 +/- 29.8%, respectively. The stimulation of sulfate efflux by external iodide was completely inhibited by DIDS (4.4'-diisothiocyanatostilbene 2,2'-disulfonic acid). Perchlorate (1 mM) also trans-stimulated sulfate efflux in a manner that was inhibited by DIDS. Furthermore, iodide trans-accelerated sulfate efflux from rat mammary acini via a DIDS-sensitive mechanism. The results are consistent with the presence of a DIDS-sensitive anion-exchange mechanism which can accept iodide as a substrate. It appears that the iodide-sulfate exchange mechanism is independent from the sodium-dependent iodide transporter given that sulfate is not a substrate of the latter system. The iodide-sulfate exchanger may operate in parallel with the sodium-dependent iodide transporter to mediate iodide uptake into mammary cells.
We have investigated free-solution capillary electrophoresis (FSCE) and micellar electrokinetic capillary chromatography (MECC) separations of metallothionein (MT) isoforms conducted in uncoated and surface-modified fused-silica capillaries. At alkaline pH, FSCE rapidly resolves isoforms belonging to the MT-1 and MT-2 charge classes. At acidic pH, additional resolution of MT isoforms is achieved. The use of high-ionic-strength (0.5 M) phosphate buffers can result in high peak efficiencies and increased resolution for some MT isoforms. Interior capillary surface coatings such as polyamine and linear polyacrylamide polymers permit separation of MT isoforms with enhanced resolution through their effects on electroosmotic flow (EOF) and protein-wall interactions. Improvements in MT isoform resolution can also be achieved by MECC using 100 mM borate buffer pH 8.4 containing 75 mM SDS. Deproteinization of tissue cytosol samples with acetonitrile (60-80%) or perchloric acid (7%) produces extracts that can be subjected to direct analysis of MT by FSCE or MECC. We conclude that optimal separation of MT isoforms by capillary electrophoresis (CE) can be achieved with the appropriate combination of different capillaries, buffers and sample preparation techniques.
1H-nuclear magnetic resonance (NMR) has been applied to the study of postmortem biochemical changes in perchloric acid extracts of rat skeletal muscle. Several metabolites have been detected and the dependence upon the postmortem time has been considered. The simultaneous quantitative determination of metabolites showing up at very low and very high fields has been suggested to yield a satisfactory delineation of the thanatochronology.
Radioactively labeled carbon monoxide (CO) dehydrogenase has been obtained in good yield and purity from Pseudomonas carboxydoflava grown in the presence of [32P]phosphate. One enzyme molecule contained an average of 8.32 molecules of phosphate. The entire phosphate content was confined to 2 molecules of FAD and 2 molecules of a pterin. These were noncovalently bound. Molybdoenzyme cofactors could be extracted into N-methyl formamide; pterins were isolated by thin-layer chromatography. CO dehydrogenase contained a novel pterin, different from molybdopterin, which was also resolved in other bacterial molybdoenzymes. Therefore, it was tentatively named bactopterin. The characteristic features of bactopterin were as follows. A relative molecular mass, Mr, of 730 which was much greater than that of molybdopterin (330) (Mr values refer to molybdenum-free forms of the cofactors; presumably, the latter were also devoid of the sulfhydryl groups contained in the native compounds). A content of 2 molecules of phosphate/molecule compared to only 1 phosphate in molybdopterin. Bactopterin was three times less susceptible to air oxidation than molybdopterin. Native bactopterin was cleaved by perchloric acid into two phosphorous-containing fragments with Mr of 330 and 420. The smaller one is believed to be very similar to molybdopterin, the larger one was not a pterin but probably contained an aromatic structure.
Creatine and phosphocreatine are substrates for creatine kinase which is a key enzyme involved in energy transfer within the cell. Analogues of creatine have been fed to animals to determine the role this enzyme plays in energy metabolism, but progress in interpretation has been hampered by the lack of quantitative techniques to determine tissue content of these compounds. We describe the separation and quantitation of substituted guanidino compounds and their phosphorylated forms by high-performance liquid chromatography. First, a cation-exchange column is used to assay free creatine and its unphosphorylated analogues, and then phosphocreatine and its phosphorylated analogues as well as adenylate content (AMP, ADP, ATP) are assayed on an anion-exchange column. These methods have proven successful in measuring the chemical contents of these compounds in neutralized perchloric acid extracts of mammalian skeletal muscles. The sensitivity of this method ranges from 50 to 200 pmol, which is adequate to provide information from tissue extracts of 5- to 10-mg samples.
BACKGROUND: Little data exist wherein both the 31P nuclear magnetic resonance (NMR) signals and biochemical changes associated with hepatic regeneration after a 70% hepatic resection have been assessed simultaneously. EXPERIMENTAL DESIGN: Two groups of rats were used: one group underwent a 70% partial hepatectomy and the second underwent a sham operation. Both groups were followed sequentially for 192 hours by in vivo serial 31P-NMR spectroscopy of the liver and its phospholipid extracts. Liver injury and function were assessed by biochemical means. RESULTS: After surgery, a significant reduction in ATP and an increase in the phosphomonoester signal for the hepatectomized animals were noted as compared with the controls (p < 0.05). The phosphodiester content of the liver in the hepatectomized rats declined to nonmeasurable amounts in vivo. The nadir of ATP occurred 72 hours after surgery. The area of the phosphomonoester relative to an external reference of methylenediphosphonic acid peak increased steadily over the first 96 hours, whereas that of the area ratio of the inorganic phosphate/methylenediphosphonic increased over the first 72 hours posthepatectomy. The intracellular pH declined sharply in the first 3 days, followed by a gradual recovery over the next 5 days. Little change in the intracellular pH was observed for the control animals. A significant increase in the area of the phosphorylethanolamine relative to an internal reference of methylenediphosphonic and a reduction in the glycerophosphorylethanolamine and glycerophosphorylcholine peaks were noted during the first four post-hepatectomy days as measured by 31P-NMR of perchloric acid liver extracts (p < 0.05). CONCLUSIONS: It has been found that a concerted reduction in the intracellular ATP and intracellular pH coupled with an increase in inorganic phosphate and high levels of phosphorylethanolamine occur as a result of hepatic regeneration and the physiologic changes induced. These data demonstrated that a coordinated pattern of biochemical changes occur with and after hepatic regeneration. Moreover, NMR spectroscopy demonstrates an increase in phosphomonoesters and a decline in phosphodiesters during hepatic regeneration. These measures and, more specifically, the ratio of these two lipid classes may provide a biochemical snapshot of the regeneration status of the liver.
Six extraction media (acetic acid, EDTA, tetrabutylammonium hydroxide, NaOH, MeOH/H2O, acetonitrile/H2O) were tested for their ability to extract antimony (Sb) and arsenic (As) from freeze-dried poplar leaves, pine shoots and spruce shoots, as well as from a peat matrix. Additionally, the extraction efficiency of Sb and As in fresh and freeze-dried elder leaves and poplar leaves was compared. Total concentrations of Sb and As of aliquots (approximately 220 mg) of the freeze-dried samples were analysed by flow injection hydride generation atomic absorption spectrometry (FI-HG-AAS) after open vessel digestion with adequate mixtures of nitric, sulfuric, hydrochloric, and perchloric acid. Three reference materials GBW 07602 Bush Branches and Leaves, GBW 07604 Poplar Leaves, and SRM 1575 Pine Needles were analysed with every batch of samples to ensure the accuracy and precision of the applied analytical procedures. The use of hydrofluoric acid in the digestion mixture leads to distinctly lower As values (down to 40%) than actual concentrations in the investigated plant materials. Extraction efficiencies were generally low and lower for Sb than for As. Solutions of 0.66 mol L(-1) NaOH liberated highest amounts of Sb with approximately 10% for poplar leaves, and approximately 19% each for pine shoots and spruce shoots. Distinctly higher concentrations of As in NaOH extracts of poplar leaves (22%), pine shoots (32%), and spruce shoots (36%) were quantified. Extraction experiments resulted in yields of 7-9% from fresh elder and poplar leaves, respectively, and 8-13% for freeze-dried samples for Sb. The corresponding values for As were 10-35% for the fresh material and 7-37% for the freeze-dried samples.
A novel acid-soluble protein has been extracted from nuclei of developing embryos of H. dromedarii ticks and purified to homogeneity. This tick embryo basic protein (TEBP) was predominant during the cleavage stage of tick embryogenesis, whereas the complete set of histones was detectable at the late cleavage stage. The amount of TEBP reaches a maximum value at day 9 after oviposition. Thereafter, the original N-terminal dipeptide (leucine-serine) is eliminated. This coincides with the start of organogenesis. In spite of its low molecular mass, TEBP seems to be related to histone H1 in some properties such as solubility in perchloric acid and binding affinity to DNA. A task for the future will be to define the role of this protein as a counterpart of the histones for the genome organization during embryogenesis.
The test-combination from Fa. Boehringer Mannheim GmbH for measuring citric acid present in foodstuffs is also suitable for use in human pancreatic juice. Accuracy, within-run and between-day imprecision are satisfactory. The detection limit of the method is at least 10 mumol citrate/l pancreatic juice. The limit can be reduced when exact additions of citrate are given to the pancreatic juice and then evaluated using regression analysis. The presence of Ca-ions and possibly also trypsin in the material to be tested does not interfere with the reaction. It is, however, necessary to remove proteins from the sample. Deproteinization can be performed either by ultrafiltration or with perchloric acid.
A fast, simple and accurate method to determine the concentration of E-1100, a novel beta-lactam antibiotic, in rat plasma has been developed. This method involves deproteinization by methanol with 0.2% perchloric acid and reversed-phase high-performance liquid chromatography with ultraviolet detection. Regression analysis showed that the method was linear over the standard-curve range from 0.191 to 191.0 micrograms/ml. This newly developed method has been applied for the analysis of plasma samples in a preliminary pharmacokinetic study in rats.
Twenty-three pairs of normal mucosa and colonic adenocarcinoma biopsy specimens have been examined in this pilot study by 1H NMR spectroscopy at 9.4 T to determine whether it was possible to find spectral malignancy markers. The 3.2 ppm (trimethylamine-containing compounds)/0.9 ppm (methyl of fatty acids) resonance intensity ratio in water suppressed spectra, proposed by other authors as a malignancy marker, results in our hands, using resonance areas, in partial overlap between tumor and mucosa values, which reduces its diagnostic value. Furthermore, we have found that submucosa contamination could mask the normal mucosa pattern and artifactually decrease the 3.2/0.9 ppm, ratio value by increasing the 0.9 ppm resonance due to the known triglyceride content of normal submucosa. On the other hand, we have observed in the Hahn spin-echo spectra of intact biopsies resonances arising from taurine and exogenous polyethyleneglycol (PEG). Their assignment and quantification has been carried out in perchloric acid extracts of the tissue biopsies. The taurine (3.4 ppm)/creatine (3.0 ppm) area ratio produced an excellent discrimination between normal mucosa and tumour groups while the PEG (3.7 ppm)/creatine (3.0 ppm) area ratio presented a large overlap, although it was clearly higher in the mucosae than in the tumors for paired samples. These two NMR observable parameters are in our hands highly discriminating and are accordingly proposed as malignancy markers in tissue biopsies although their possible utility for in vivo studies remains to be demonstrated.
meso-Alanopine and D-strombine are separated by high pressure liquid chromatography using a cation exchange resin and 2.5 X 10(-5) M sulfuric acid as eluant, at a flow rate of 1.0 ml/min, 20 degrees C column temperature and a pressure of 4 500 kPa. Both opines were detected by conductivity. Separation and quantitation was possible in the range of 0.05 to 25 nmol of meso-alanopine and D-strombine. Chemically or enzymatically synthesized opines were quantitated using alanopine/strombine dehydrogenase from Crassostrea angulata. The enzyme was purified by ammonium sulfate precipitation, Sephadex G-100 filtration and fast-protein-liquid chromatography. Specific activity of the final preparation was 500 U/mg protein with glycine as substrate. The formation of meso-alanopine and D-strombine was demonstrated in neutralized perchloric acid extracts from muscle tissue of Arenicola marina L. following enhanced muscular activity and in Mytilus edulis L., Nucula nitida and Crassostrea angulata after 24 h of anoxia.
The lymphokine activity, macrophage aggregation factor (MAgF) has been investigated further. Activity was consistently found in 24 hr test, but not control, spleen cell culture supernatants. This was higher after dialysis against water, than in the original culture supernatants. MAgF was heat-stable, inactivated by alpha-chymotrypsin, partially inactivated by trypsin and not affected by neuraminidase. Activity was recovered from the supernatant after protein precipitation with 1 M perchloric acid, leading to a modest purification. Activity was only marginally reduced after treatment with periodate, and was not absorbed by Concanavalin A-Sepharose. Polyacrylamide gel electrophoresis showed that MAgF migrated cathodally to albumin. Aggregation, as measured in a batch centrifugation assay, was an expression both of cell-substrate and cell--cell adhesion.