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PubMed1997

Use of spin-traps during warm ischemia-reperfusion in rat liver: comparative effect on energetic metabolism studied using 31P nuclear magnetic resonance.

Detection of free radicals by electron spin resonance (ESR) proves the involvement of reactive oxygen species (ROS) in reperfused organ injuries. Spin-traps are known to ameliorate hemodynamic parameters in an isolated postischemic heart. The effects of 5 mmol/L DMPO (5,5-dimethyl-1-pyrroline-N-oxide) or DEPMPO (5-(diethlphosphoryl)-5-methyl-1-pyrroline N-oxide) on intracellular pH (pHin) and ATP level were evaluated by 31P nuclear magnetic resonance on isolated rat liver submitted to 1 hour of warm ischemia and reperfusion. At the end of the reperfusion period, during which pHin recovered to its initial value (7.16 +/- 0.03) in all groups, the ATP recovery level (expressed in percentage of initial value) was similar in controls and DEPMPO (60% +/- 5%, n = 6 and 54% +/- 4%, n = 6, respectively), but only 37% +/- 1% in DMPO-treated livers (n = 6) (p < 0.05 versus controls and p < 0.05 versus DEPMPO). Oxidative phosphorylation was not affected by an addition of nitrones on isolated mitochondria extracted from livers not submitted to ischemia-reperfusion. In contrast, mitochondria extracted at the end of the ischemia-reperfusion showed an impairment in the phosphorylation parameters, particularly in the presence of DMPO. Mass spectrum of ischemic liver perchloric acid extracts evidenced probable catabolites in treated groups. The differences in the effect of the two nitrones on energetic metabolism may be explained by the production of deleterious catabolites by DMPO as compared to DEPMPO. Even though a specific radical scavenging effect could be operative in the liver, our results indicate that catabolic effects were predominant. The absence of deleterious effects of DEPMPO in contrast to DMPO on the liver energetic metabolism was evidenced, allowing the use of DEPMPO for ESR detection.

Animals↗
PubMed1984

Effect of serum on organogenesis of the rat testis in vitro.

It was observed previously that primordia of fetal rat testes when explanted in vitro in a synthetic medium at the outset of sexual differentiation differentiate seminiferous cords during the following days, but that the addition of 15% fetal bovine serum prevents this morphogenesis. In the present study, human, horse, bovine calf, and rat sera were shown to exert the same effect. Very low concentrations of human or fetal bovine serum (0.5 or 1%) were sufficient to produce the serum effect, which was only slightly reduced when the serum was heated. The serum activity was not removed by dialysis (membrane cut-off 15 000), but it disappeared after treatment with trichloroacetic or perchloric acids or after trypsin digestion. Partial purification of the active factor(s) from human serum was achieved by successive gel filtration, affinity chromatography, and ion exchange chromatography. Analysis of the active fractions by electrofocusing and immunoelectrophoresis placed the activity within the alpha globulin group. Among a series of purified serum proteins tested, alpha 2-HS-glycoprotein was found to exhibit the serum effect, though this activity was heat labile.

Animals↗
PubMed2003

Bioluminometric assay of ATP in mouse brain: Determinant factors for enhanced test sensitivity.

Firefly luciferase bioluminescence (FLB) is a highly sensitive and specific method for the analysis of adenosine-5-triphosphate (ATP) in biological samples. Earlier attempts to modify the FLB test for enhanced sensitivity have been typically based on in vitro cell systems. This study reports an optimized FLB procedure for the analysis of ATP in small tissue samples. The results showed that the sensitivity of the FLB test can be enhanced several fold by using ultraturax homogenizer, perchloric acid extraction, neutralization of acid extract and its optimal dilution, before performing the assay reaction.

Adenosine Diphosphate↗
PubMed1998

Serum zinc levels in healthy subjects from southeastern Spain.

The serum zinc (Zn) concentrations of 80 healthy subjects (48 male, 32 female) from southeastern Spain were determined by atomic absorption spectrometry. The samples were digested by heating in a 4:1 mixture of nitric and perchloric acids. The concentration of Zn was determined against a Contox Trace Metal Serum Control Panel A standard reference. Zn concentrations in the standard were found to be 2.332+/-0.489 mg/L, with a mean recovery of 102.7%. In the serum samples, the relative standard deviation was <6% for the range of concentrations determined: 0.420-1.540 mg/L for women (mean value 0.947+/-0.265 mg/L) and 0.490-1.480 mg/L for men (mean value 0.951+/-0.243 mg/L). In healthy subjects, no statistically significant differences were observed in the Zn levels with respect to their sex (p > 0.05) or the location where they lived (mountainous vs coastal zones). It is concluded that the dietary Zn intake and Zn status for healthy adults in this region of Spain are within normal values.

Adolescent↗
PubMed1990

Microdetermination of selenium in protein fractions isolated by analytical methods.

A method has been developed for the determination of selenium associated with proteins in chromatographic fractions, in polyacrylamide gels, and on nitrocellulose membranes after transfer. This method involves the complete digestion of samples in the purest nitric and perchloric acids and takes advantage of the specificity afforded by the 99% pure 2,3-diaminonaphthalene. The use of these and other reagents of highest purity produces very low blank values and allows a detection limit as low as 0.76 picomoles (60 picograms) of selenium. Quantitative recoveries of selenium in glutathione peroxidase and low coefficients of variation were obtained.

Blotting, Western↗
PubMed1991

Creatine phosphate as energy source in the cerulein-stimulated rat pancreas study by 31P nuclear magnetic resonance.

Stimulation of the rat exocrine pancreas by cerulein induces a variety of cellular processes, some of which require the expenditure of energy. In this study, changes in the amounts of various energy metabolites, including creatine phosphate (PCr), ATP, and ADP were determined by high-resolution 31P NMR spectroscopy. The spectrum of a perchloric acid extract of pancreas from the 48 h fasted rat was taken as a reference for comparison of 31P NMR spectra recorded after stimulation by cerulein. The NMR results obtained from rat pancreas stimulated in vivo by cerulein (3, 5, 10, 20, 40 min) were compared to those determined by HPLC. We show that during hormonal stimulation, the relative concentrations of PCr in the pancreas of the fasted rat rise significantly (p less than 0.02), reach a maximum at 10 min, fall between the 10th and 20th min, and then return to the relatively low levels observed in controls. On the other hand, the relative concentrations of ATP fall during the first 10 min after stimulation by cerulein, then rise significantly between the 10th and 20th min, whereas the levels of ADP rise during the first 10 min and fall between the 10th and 20th min. The energy required for exocytosis was assumed to be supplied by ATP synthesized in acinar cells. The 31P NMR results indicated that this ATP was derived from phosphorylation of ADP by PCr, and that large amounts of PCr are synthesized during the first minutes after cerulein stimulation. In addition, a significant rise in glycerophosphocholine was observed after cerulein stimulation, which was attributed to an enhanced catabolism of membranes and an increase in phospholipid turnover. Injection of cerulein antagonists, such as asperlicin or lorglumide, inhibited the effects of cerulein stimulation on energy metabolites. Furthermore, no changes were observed after injection of secretin, a hormone that stimulates secretion of bicarbonate. However, the analog of cerulein, pentagastrin, produced the same effects as cerulein, although to a lesser extent.

Adenine Nucleotides↗
PubMed2006

Endogenous coenzyme A glutathione disulfide in human myocardial tissue.

Besides its role as a mechanical pump, the human heart serves as an endocrine organ, where known and as yet unknown hormones are produced. It is very likely that these hormones play an important role in cardiovascular regulation. In this study, a new endogenous vasoactive substance, coenzyme A glutathione disulfide (CoASSG), was isolated and identified in myocardial tissue. Human myocardial tissue was extracted with perchloric acid and fractionated by size exclusion-, displacement-, anion-exchange- and reversed-phase chromatography. In one fraction purified to homogeneity, CoASSG was identified by matrix assisted laser desorption/ionization (MALDI) mass-spectrometry, post-source decay MALDI-mass spectrometry and enzymatic structure analysis. Furthermore, CoASSG was also isolated from human cardiac specific granules. CoASSG has potent vasoconstrictive and proliferative effects. Therefore, CoASSG may affect myocardial function as an endocrine or autocrine substance after being released from myocardial specific granules.

Coenzyme A↗
PubMed1997

Contribution of proteolysis and de novo synthesis to alanine production in diabetic rat skeletal muscle: a 15N/1H nuclear magnetic resonance study.

To assess the role of leucine as a precursor of alanine alpha-amino nitrogen in skeletal muscle during diabetes, extensor digitorum longus muscles from control (n = 7 experiments) and streptozotocin-diabetic rats (n = 8 experiments) were isolated and superfused with [15N]leucine (3 mmol/l) in the presence of glucose (10 mmol/l) for 2 h. Muscle perchloric acid extraction was performed at the end of superfusion in order to quantify newly synthesized alanine by 15N/1H nuclear magnetic resonance. Release of [15N]alanine in the superfusion medium was also measured. The pool of newly synthesized [15N]alanine was significantly increased (approximately 40%) in extensor digitorum longus muscles from streptozotocin-diabetic rats. Whereas a significant enhancement of total alanine release from muscle was induced by diabetes (20%), only a slight increase in [15N]alanine release was detectable under our experimental conditions. Consequently, we conclude that streptozotocin-diabetes in growing rats induces in skeletal muscle: 1) an increase in nitrogen exchange between leucine and alanine leading to newly synthesized [15N]alanine; and 2) an increase of total alanine release from muscle originating from both proteolysis and de novo synthesis.

Alanine↗
PubMed2002

Determination of 41Ca in biological-shield concrete by low-energy X-ray spectrometry.

An X-ray spectrometric method has been developed for the determination of 41Ca in the biological-shield concrete of nuclear reactors. The concrete sample was first decomposed with nitric, hydrofluoric, and perchloric acids. Calcium was then separated from other radionuclides by ion-exchange chromatography and recovered as an oxalate precipitate. X-rays at 3.3 keV from 41Ca in the calcium oxalate pellet were measured. The detection efficiency of the X-ray measurement at 3.3 keV was calculated from those obtained by measuring 55Fe standard pellets at 5.9 keV using mass-absorption coefficients of the calcium oxalate pellet at each X-ray energy. A lower limit of determination of 8 Bq g(-1) was obtained for a sample weight of 1 g.

Journal Article↗
PubMed2002

HPLC-ICP-MS determination of selenium distribution and speciation in different types of nut.

In addition to determination of total selenium in nuts, the element distribution among different fractions (lipid extract, low molecular weight, and protein fractions), and speciation analysis were studied. Improved precision for total selenium determination was observed after elimination of lipids. Because selenium was not detected in any of the lipid extracts obtained from the different types of nuts (ICP-MS), in each determination and/or speciation procedure used in this work lipids were extracted (chloroform-methanol, 2:1) and discarded before analysis. In agreement with previously reported data, high selenium levels were found in Brazil nuts (those purchased without shells contained approximately a quarter the content than those purchased with shells) and significantly lower levels in walnuts, cashews, and pecans nuts. Low-molecular-weight compounds were extracted with perchloric acid (0.4 mol L(-1)) to furnish a fraction containing 3 to 15% of the total selenium in different types of nuts. The proteins were isolated from nut samples by dissolution in 0.1 mol L(-1) sodium hydroxide and subsequent precipitation with acetone. They were then dissolved in phosphate buffer pH 7.5. Analysis of protein fractions focused on selenium in two possible states - weakly and firmly bound to proteins. Results obtained for Brazil nuts by size-exclusion chromatography with on-line ICP-MS detection, in the absence and in the presence of beta-mercaptoethanol, showed that approximately 12% of total selenium was weakly bound to proteins. To obtain information about firmly bound selenium, the protein extracts were hydrolyzed enzymatically with proteinase K. Speciation was performed by means of ion-pairing HPLC-ICP-MS. The primary species found in all types of nuts was Se-methionine (19-25% of total selenium for different types of nuts).

Chromatography, High Pressure Liquid↗
PubMed2003

Morin applied in speciation of aluminium in natural waters and biological samples by reversed-phase high-performance liquid chromatography with fluorescence detection.

A reversed-phase high-performance liquid chromatographic method with fluorescence detection for the determination of labile monomeric aluminium has been developed through pre-column complexation using morin as the analytical reagent. The highly fluorescent aluminium-morin complex (excitation wavelength 418 nm, emission wavelength 490 nm) was separated on a Spherisorb ODS 2 column with an eluent consisting of 30% methanol and 70% water (pH 1.0 with perchloric acid). The most remarkable point of this protocol was that only the most toxic aluminium species, that is, free aqua-aluminium ion and its monomeric hydroxo complex ions, selectively respond among various aluminium complexes. This strategy has been successfully applied to direct fractionation of the toxic aluminium in natural waters and biological samples without any pretreatment.

Aluminum↗
PubMed2004

Determination of the thiol redox state of organisms: new oxidative stress indicators.

This study describes a new methodology by which the concentrations of non-protein (NP) thiols glutathione (GSH), cysteine (CSH), N-acetylcysteine (AcCSH), and protein (P) thiols (PSH), as well as the contribution of these components to symmetric and mixed disulfides (NPSSR, NPSSC, NPSSCAc, PSSR, PSSC, PSSCAc, PSSP) can reliably be measured. The methodology consists of a strict sequence of methods which are applied to every sample. Free thiols at any given state of the procedure are measured by Ellman's assay, the CSH fraction is measured by its unique response in the ninhydrin assay, AcCSH is selectively measured with ninhydrin after enzymatic deacylation, proteins are separated from non-protein thiols/disulfides by precipitation with trichloroacetic or perchloric acid, disulfides are reduced into free thiols with borohydride, mixed disulfides between a protein and a non-protein component are measured by extracting the non-protein thiol from the protein pellet after borohydride treatment, and protein thiols/disulfides are measured after resolubilization of the protein pellet. When this method was applied to animal and fungal tissue, new molecular indicators of the thiol redox state of living cells were identified. The findings of the present study clearly show that the new parameters are very sensitive indicators of redox state, while at the same time the traditional parameters GSH and GSSG often remain constant even upon dramatic changes in the overall redox state of biological tissue. Therefore, unbiased assessment of the redox state also requires explicit measurement of its most sensitive thiol indicators.

Animals↗
PubMed2001

Study of an Au colloid self-assembled electrode and its application to the determination of carbon monoxide.

A novel electrochemical sensor has been developed for the detection of carbon monoxide. The chemically modified electrode, prepared by reaction of cysteine and then an Au colloid of size approximately 15 nm with a platinum microelectrode, has excellent catalytic activity toward carbon monoxide, with an oxidation potential of +600 mV relative to the Ag/AgCl electrode. The CO gas sensor is based on an Au colloid self-assembled modified electrode as working electrode, an Ag/AgCl electrode as reference electrode, a Pt electrode as counter electrode, and a porous film which is in direct contact with the gas-containing atmosphere. The effects on the determination of CO of different internal electrolyte solutions of perchloric acid, hydrochloric acid, sulfuric acid, nitric acid, and phosphate buffer of different concentrations were also studied. The sensor is characterized by a short response time and highly reproducible detection of CO. This sensor can be used in the field of environmental monitoring and control.

Calibration↗
PubMed2006

Bacterial stress enrichment enhances anaerobic hydrogen production in cattle manure sludge.

Methodology was evaluated to selectively enrich hydrogen-producing species present in biological sludge produced during organic wastewater treatment. The influence of bacterial stress enrichment on anaerobic hydrogen-producing microorganisms was investigated in batch tests using serum bottles. Enrichment conditions investigated included application of acute physical and chemical stresses: wet heat, dry heat and desiccation, use of a methanogen inhibitor, freezing and thawing, and chemical acidification with and without preacidification of the sludge at pH 3. For each enrichment sample, cultivation pH value was set at an initial value of 7. After application of selective enrichment (by bacterial stress), hydrogen production was significantly higher than that of untreated original sludge. Hydrogen production from the inocula with bacterial stress enrichment was 1.9-9.8 times greater when compared with control sludge. Chemical acidification using perchloric acid showed the best hydrogen production potential, irrespective of preacidification. Enhancement is due to the selective capture of hydrogen-producing sporeformers, which induces altered anaerobic fermentative metabolism.

Anaerobiosis↗
PubMed2002

Plasma and cellular pharmacology of 8-chloro-adenosine in mice and rats.

PURPOSE: The nucleoside 8-chloro-adenosine (8-Cl-Ado) is currently being developed for treatment of multiple myeloma and leukemias. Although accumulation of the phosphorylated drug product is known to occur within cell lines, its metabolic fate in plasma or circulating cells in animals is unclear. The purpose of the present study was to determine the pharmacology of 8-Cl-Ado in rodents through examination of plasma and cellular levels of parent drug and metabolites. In addition, we sought to determine whether an inhibitor of adenosine deaminase, 2'-deoxycoformycin (dCF), could enhance intracellular formation of 8-Cl-ATP by preventing degradation of 8-Cl-Ado to 8-Cl-inosine (8-Cl-Ino). METHODS: A validated HPLC assay permitted simultaneous determination of 8-Cl-Ado, 8-Cl-adenine (8-Cl-Ade), dCF, and 8-Cl-Ino. Radiolabeled cellular nucleotides were obtained from peripheral blood mononuclear cells (PBMC) of both mice and rats using a perchloric acid extraction procedure and were separated by HPLC. RESULTS: Stability of 8-Cl-Ado in the presence or absence of dCF was examined in fresh plasma from mice, rats and humans. Conversion of 8-Cl-Ado to 8-Cl-Ino was only marginally affected by coincubation with dCF. In CD(2)F(1) mice given 8-Cl-Ado i.p. at 100 mg/kg, there was rapid appearance in plasma of both 8-Cl-Ade and 8-Cl-Ino. The identities of the metabolites were confirmed by mass spectrometry. The plasma [(3)H]8-Cl-Ado concentration 1 h after drug injection was 1.3 micro M in mice while the intracellular levels of [(3)H]8-Cl-AMP and [(3)H]8-Cl-ATP were 1 m M and 350 micro M, respectively. Mice that had received dCF (2 mg/ml) 30 min prior to [(3)H]8-Cl-Ado had 27% less intracellular [(3)H]8-Cl-ATP in PBMC compared to mice without dCF pretreatment. The pharmacokinetics of 8-Cl-Ado were examined in greater detail in Sprague-Dawley rats. Animals were given [(3)H]8-Cl-Ado (42.5 mg/kg, i.v.) by itself or 30 min following injection of dCF (4 mg/kg). Mononuclear cells in mice accumulated 350 or 1200 micro M [(3)H]8-Cl-ATP 1 h after injection of either 50 or 100 mg [(3)H]8-Cl-Ado, respectively. The major metabolite in these cells was the monophosphate, which was four- to sevenfold higher in concentration than the triphosphate metabolite. In rats, [(3)H]8-Cl-AMP concentrations in PBMC were similar to those of the triphosphate metabolite which achieved a peak of 90 micro M 2 h after a bolus injection of 8-Cl-Ado (40 mg/kg). Cellular clearance of 8-Cl-ATP appeared to be slow: 24 h after injection of 8-Cl-Ado the cellular concentration of 8-Cl-ATP was still 40 micro M. CONCLUSIONS: The use of dCF did not significantly alter 8-Cl-ATP levels in PBMC and is not considered to be a useful therapeutic strategy. Even though a portion of 8-Cl-Ado is metabolically inactivated in plasma, high levels of cytotoxic 8-Cl-ATP accumulated intracellularly in these animals and were retained for a considerable length of time. Further development of 8-Cl-Ado is recommended.

2-Chloroadenosine↗
PubMed2000

A new diquat derivative appropriate for colourimetric measurements of biological materials in the presence of paraquat.

A new colourimetric method is described for the quantification of diquat using a yellow-coloured derivative produced by heating diquat in alkaline solution at 80 degrees C. The absorption maximum of the yellow derivative is 420 nm and the molar absorption coefficient is 2.76 x 10(4) (0.15 in 1 microgram diquat/ml with 1 cm light path). The absorption at 420 nm shows a linear concentration dependence in the range 0.1-10 micrograms/ml and fading of the colour is about 5% after 1 h. Under the same conditions, paraquat does not produce any coloured products. The concentration of diquat in the solution containing both diquat and paraquat can be determined by the absorption of diquat derivative at 420 nm without interference from paraquat. By adding sodium dithionite to the solution the concentration of paraquat can be determined by the absorption of paraquat radicals at 600 nm without interference from diquat, because the yellow derivative does not react with dithionite. This yellow diquat derivative can be extracted completely with cyclohexanol by saturating the solution with Na2SO4. The absorption maximum in cyclohexanol shifts to 440 nm with the same molar absorbance and the same half-band width as in water. Fading of the colour is less than 5% after 24 h in cyclohexanol. Perchloric acid (3%) and trichloroacetic acid (4.5%) which are often used for deproteinization of tissue homogenates, do not inhibit production of the coloured derivative at pH 13.5 or extraction of the derivative with cyclohexanol. This method is suitable for a quick determination of small amounts of diquat in tissues, since the extraction with cyclohexanol not only concentrates the derivative rapidly but also quite efficiently eliminates the coloured substances in tissue homogenates. The detection limit of diquat is 0.02 microgram/ml for blood and 0.05 microgram/g for liver when 1 ml or 1 g is used for analysis. In three human cases of fatal intoxication, both paraquat and diquat were quantified using 50 microliters of serum. In non-toxic dosing of diquat to rats for 14 days, the diquat level was highest in the spleen followed by the kidneys.

Animals↗
PubMed2002

Analysis of immediate changes of water-soluble metabolites in alkali-burned rabbit cornea, aqueous humor and lens by high-resolution 1H-NMR spectroscopy.

PURPOSE: To investigate immediate changes in water-soluble metabolites of ocular tissue in alkali-burned eyes by using high-resolution 1H-NMR spectroscopy. METHODS: Adult New Zealand rabbit eyes were burned with 1 M NaOH for 1 min. Normal eyes were used as control. Samples from aqueous humor and perchloric acid extracts of the cornea and lens were analyzed on a NMR spectrometer operating at 500 MHz for protons. Metabolites were quantified by comparing peak area with an added internal standard, TSP (3'-trimethylsilylpropinate-2,2,3,3-d4). RESULTS: Alkali burn of corneal surface causes immediate changes in concentration of many water-soluble metabolites in the anterior segment. Even as far away as the lens a significant increase in lactate was found. Cornea showed a significant increase in glucose and a significant decrease in hypo-taurine concentration. Most changes were observed in aqueous humor, with significant increases in succinate, creatine, scyllo- and myo-inositol and a significant decrease in citrate concentration. Furthermore, a small decrease in ascorbate concentration in aqueous humor was observed. CONCLUSIONS: The present study provides a valuable contribution to the knowledge of metabolic alterations in alkali-burned eyes. It shows that 1H-NMR spectroscopy is well suited for simultaneous qualitative and quantitative analysis of changes of metabolite concentrations in damaged tissues. This can help us to better evaluate and understand the biological alterations due to alkali burn.

Amino Acids↗
PubMed1998

High-pressure liquid chromatographic determination of toluene in urine as a marker of occupational exposure to toluene.

OBJECTIVE: To establish a convenient method by high-pressure liquid chromatography (HPLC) to measure toluene in urine as a marker of occupational exposure to toluene. METHODS: As soon after sampling as possible, 1 ml of urine was mixed with an equal volume of acetonitrile in a 2.2-ml HPLC glass bottle, and the bottle was tightly sealed and stored at 4 degrees C. Immediately before HPLC determination, 100 microl methanol was added to the mixture to prevent confounding effects of glycosuria, and the bottle was spun to remove any suspended matter. An aliquot of the supernate was introduced into the HPLC system and analyzed on a PRODIGY column, with an acetonitrile - perchloric acid phosphoric acid - water mixture serving as the mobile phase. The effluent was monitored at 191 nm. RESULTS: The method can measure toluene in urine every 20 min, the detection limit was 2 microg/l, the coefficient of variation was less than 5%, and the recovery rate was 100%. No significant reduction in toluene concentration was observed for 1 week after storage at 4 degrees C. When the method was applied to end-of-shift urine samples from 13 male workers exposed to toluene at 18-140 ppm and also to urine samples from 10 nonexposed male controls, toluene in urine was linearly related to toluene exposure concentration, with a regression line passing close to the origin. The correlation coefficient was as high as 0.97 (n=23). No toluene was detected in control urine samples. Calculations suggest that urinary toluene accounts for as little as less than 0.01% of the toluene absorbed via inhalation and that the absorbed toluene is converted almost quantitatively to hippuric acid and, by less than 0.1%, to o-cresol.

Adult↗