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PubMed1999

Changes of cerebral energy metabolism and lipid peroxidation in rats leading to mitochondrial dysfunction after diffuse brain injury.

The effect of mild closed head trauma, induced by the weight-drop method (450 g from a 1-m height), on lipid peroxidation and energy metabolism of brain tissue was determined at various times after cerebral injury in spontaneously breathing rats (1, 10, 30 minutes and 2, 6, 15, 24, 48, and 120 hours). Animals were continuously monitored for the evaluation of blood pressure, blood gases, heart rate, and intracranial pressure. Analysis of malondialdehyde (MDA) as an index of lipid peroxidation, ascorbic acid, high-energy phosphates, nicotinic coenzymes, oxypurines, and nucleosides was performed by high-performance liquid chromatography (HPLC) on neutralized perchloric acid extract of the whole brain. Data showed that MDA, undetectable in control, sham-operated rats, was already present within 1 minute of trauma (1.77 nmol/g wet weight; SD = 0.29) and reached maximal values by 2 hours (72.26 nmol/g w.w.; SD = 11.26), showing a progressive slow decrease thereafter. In contrast, ATP, GTP, and nicotinic coenzyme (NAD and NADP) concentrations showed significant reduction only by the second hour postinjury. Maximal decrease of the ATP and GTP concentrations were seen at 6 hours postinjury, whereas NAD and NADP concentrations showed maximum decline by 15 hours. Values recorded in mechanically ventilated rats did not differ significantly from those obtained in spontaneously breathing animals. These findings, supported by the absence of blood gas and blood pressure changes in the spontaneously breathing rats, strongly support the premise that biochemical changes (primarily lipid peroxidation) are not caused by secondary ischemic-hypoxic phenomena but rather are triggered by these forces acting on the brain at the time of impact. In addition, these results suggest that depression of energy metabolism might be caused by peroxidation of the mitochondrial membrane with a consequent alteration of the main mitochondrial function-that is, the energy supply.

Adenine Nucleotides↗

PubMed2001

Radiation effects on soluble metabolites in cultured HeLa cells examined by 1H MRS: changes in concentration of glutathione and of lipid catabolites induced by gamma rays and proton beams.

Cultured HeLa cells were irradiated with a single acute dose of either gamma rays (40 Gy) or low-energy proton beams (20 Gy). (1)H magnetic resonance spectra of intact cells harvested at different times after irradiation and of the correspondent perchloric acid (PCA) extracts prepared at different times after irradiation were run. Selected signals from glutathione and lactate were examined with the aim of investigating effects of irradiation on antioxidative stores and on mitochondrial activity. An increase of signal intensity of glutathione (GSH) takes place at 15 and 24 hr after irradiation, while a decrease of its signal intensity, accompanied by an increase of that of free glutamate, starts appearing 48 hr after irradiation. Lactate signal increases 48 hr after irradiation. Signals from lipid catabolites were also examined to explore their sensitivity in predicting the response to radiotherapy. Intensity ratios of signals of glycerophosphorylcholine and choline to that of phosphorylcholine increase with time after irradiation. Irradiating cells with gamma rays or proton beams at half a dose produces effects comparable to the metabolic variations presented here. The present experiments allow more insight into the complex pattern of the changes of GSH by irradiation and indicate that magnetic resonance spectroscopy signals from GSH, glutamate, lactate, and lipid catabolites are affected by irradiation. Finally, these data represent a first indication that the relative biological efficiency for some metabolic damage of low-energy proton beams with respect to gamma rays can reach a value of 2.

Flow Cytometry↗

PubMed1994

Regulation of intracellular pH in neuronal and glial tumour cells, studied by multinuclear NMR spectroscopy.

The effect of extracellular pH (pHe) on intracellular pH (pHi) and cellular metabolism was examined by multinuclear NMR spectroscopy of cells in vivo and in vitro. A decrease in pHe from 7.4 to 6.4 led to a significant drop in pHi, in both neuronal and glial tumour cells, as detected by in vivo 31P NMR of cells embedded in basement membrane gel threads. A more than 50% decrease in both the phosphocreatine (PCr) level and derivatives of glycolysis (i.e., glycerol 3-phosphate) was observed, concomitantly to the fall in pHi. A 50% decrease in intracellular lactate levels was seen in in vivo 1H NMR spectra under these conditions. Reperfusion with fresh medium (pHe 7.4) resulted in the full recovery of pHi, simultaneously with an increase in both PCr and intracellular lactate back to their control levels. Perchloric acid and lipid extract measurements confirmed the observations made by in vivo 31P and 1H NMR spectroscopy and further showed a decrease both in tricarboxylic acid cycle activity and phospholipid synthesis. The data revealed no significant differences between the neuronal and glial tumour cells investigated. pHi measurements in the presence of inhibitors of the various pH regulatory mechanisms showed that the Na+/H+ exchanger, the carbonic anhydrase and at least one of the bicarbonate-transport systems are involved in pH regulation of both cell types. The results suggest that Na+/H+ exchange is the preferred mechanism by which both neuronal and glial cells regulate their pHi after extracellular acidification.

Animals↗

PubMed1990

Evidence for reactivity of serine-74 with trans-4-(N,N-dimethylamino)cinnamaldehyde during oxidation by the cytoplasmic aldehyde dehydrogenase from sheep liver.

A nucleophilic group in the active site of aldehyde dehydrogenase, which covalently binds the aldehyde moiety during the enzyme-catalyzed oxidation of aldehydes to acids, was acylated with the chromophoric aldehyde trans-4-(N,N-dimethylamino)cinnamaldehyde (DACA). Acyl-enzyme trapped by precipitation with perchloric acid was digested with trypsin, and the peptide associated with the chromophoric group was isolated and shown to be Gln-Ala-Phe-Gln-Ile-Gly-Ser-Pro-Trp-Arg. After redigestion with thermolysin, the chromophore was associated with the C-terminal hexaresidue part. If the chromophore is attached to this peptide, serine would be expected to bind the aldehyde and lead to the required acylated derivative. Differential labeling experiments were performed in which all free thiol groups on the acylated enzyme were blocked by carboxymethylation. The acyl chromophore was then removed by controlled hydrolysis and the protein reacted with [14C]iodoacetamide. No 14C-labeled tryptic peptides were isolated, suggesting that the sulfur of a cysteine cannot be the acylated residue in the precipitated acyl-enzyme.

Aldehyde Dehydrogenase↗

PubMed1991

Direct measurement of the lambda of the lumped constant of the deoxyglucose method in rat brain: determination of lambda and lumped constant from tissue glucose concentration or equilibrium brain/plasma distribution ratio for methylglucose.

Steady-state distribution spaces of 2-[14C]deoxyglucose ([14C]DG), glucose, and 3-O-[14C]methylglucose at various concentrations of glucose in brain and plasma ranging from hypoglycemic to hyperglycemic levels have been determined by direct chemical analyses in the brains of conscious rats. The hexose concentrations were measured chemically in freeze-blown brain extracted with ethanol to avoid the degradation of acid-labile products of [14C]DG back to free [14C]DG that has been found to occur with the more commonly used perchloric acid extraction of brain. Corrections were also made for nonphosphorylatable, labeled products of [14C]DG found in the nonacidic fractions of the brain extracts, which were previously included with the assayed [14C]DG, and for the contribution of the hexose contents in the blood in the brain, which was found to be particularly critical for the determination of the glucose distribution space, especially in hypoglycemic states. From the measured contents of the hexoses in brain and plasma, the relationships of the tissue concentrations and distribution spaces of each of the hexoses and of the lambda (i.e., ratio of tissue distribution space of DG to that of glucose) of the DG method to the tissue glucose concentration were derived. The lambda was then quantitatively related to the measured equilibrium ratio for [14C]methylglucose over the full range of brain and plasma glucose levels. By combining these new data with the values for the lumped constant, the factor that converts the rate of DG phosphorylation to glucose phosphorylation, previously determined in rats over the same range of plasma glucose levels, the phosphorylation coefficient was calculated and the lumped constant graphed as a function of the measured distribution space in brain for [14C]methylglucose.

3-O-Methylglucose↗

PubMed1976

Incorporation of tritiated uridine into DNA of Ehrlich ascites tumor cells.

Ehrlich ascites tumor cells were labeled with [5,6-3H]uridine in vivo during the exponential growth phase of the tumor in the mouse. Hydroxyapatite column chromatography of the total cell nucleic acid revealed a level of activity in the DNA approaching 50% of the incorporated activity of the RNA after 24 hours. After perchloric acid hydrolysis, the constituent bases of the DNA, separated by paper chromatography, contained more than 90% of the tritium radioactivity in the cytosine and thymine, at a ratio of approximately 2:1. Prior to digestion of the polymer, the level of label in the DNA was not sensitive to RNase, alkaline, or heat denaturation. Equilibrium density gradient centrifugation produced a single peak, coincidental for radioactivity and optical density at 260 nm. Our results indicate that tumor cells under replicative stress incorporated more than one-third of the tritium radioactivity of uridine into the DNA, whereas those at a growth plateau had less than 10% of the label in the DNA. This exogenous uridine radioactivity observed in the DNA represented neither a DNA-RNA hybrid, RNA primer pieces in DNA synthesis, nor any other RNase-sensitive species, but was apparently the consequence of amination and methylation of the tritium-labeled uracil moiety to satisfy the metabolic needs of the replicating cells for cytosine and thymine bases.

Animals↗

PubMed1989

Determination of antileukemic diimidazolinyl compounds in plasma of experimental animals by high-performance liquid chromatography.

A method is reported for the determination of the fluorescent, antileukemic diimidazolinyl compounds 261/96(1). 253/152(2) and 272/131(3) in plasma. HPLC is performed on a RP-2 10 microns column with a mobile phase of methanol/water (1:1, v/v), to which 0.005 mol/l octanesulfonic acid sodium and 0.003 mol/l dimethyloctylamine are added. Samples are prepared by precipitation of plasma proteins with a mixture of methanol-70% perchloric acid. The assay is linear up to 750 ng/ml for all compounds with limits of determination of 4 ng/ml, 2 ng/ml and 0.5 ng/ml for compounds 1, 2 and 3, respectively. Coefficients of variation are below 10 percent at all concentrations studied.

Antineoplastic Agents↗

PubMed1977

Rapid digestion and flameless atomic absorption spectroscopy of mercury in fish: collaborative study.

A collaborative study of the determination of mercury in fish has been completed in which wet oxidation of fish tissue in nitric acid, using vanadium as a catalyst, is compared with the AOAC official final action digestion technique, 25.103-25.105, involving a nitric-perchloric acid mixture. The study used tuna fish samples of known mercury content and included spike recovery studies in which methyl mercury solutions of known composition were provided to each laboratory. The study was designed to provide recovery information that bracketed the regulatory level of mercury in fish. The results indicate that the proposed digestion method is at least as precise and accurate as the AOAC method. The proposed method is also more rapid and less hazardous. It has been adopted as official first action.

Animals↗

PubMed1983

Liquid-chromatographic assay of cefmenoxime in serum and urine.

This is a simple, precise liquid-chromatographic procedure for determining cefmenoxime in patients' serum and urine. p-Anisic acid is used as the internal standard. Protein is precipitated from 0.5 mL of serum or dilute urine with 100 microL of perchloric acid. The clear supernate is injected directly onto a mu-Bondapak CN reversed-phase column. The mobile phase is acetate buffer, pH 3.8 (25 degrees C). The flow rate is 2.5 mL/min. Column effluent is monitored at 254 nm. Extraction recovery from serum averaged 74.6%. Calibration curves were linear from 0.5 mg/L, the lower limit of quantification, to 100 mg/L. We present cefmenoxime concentrations in serum from a patient being treated for pneumonia. The procedure was evaluated in the clinical setting to determine its applicability to the study of cefmenoxime pharmacokinetics in critically ill patients.

Aged↗

PubMed1984

Structures of the oligosaccharide chains of two forms of alpha 1-acid glycoprotein purified from liver metastases of lung, colon, and breast tumors.

Two forms of alpha 1-acid glycoprotein with common immunological determinants and almost identical amino acid compositions but different amounts of carbohydrate were isolated from liver metastases of primary colon, lung, and breast tumors by extraction with perchloric acid, gel filtration on Sepharose CL-6B and Sephadex G-200, and affinity chromatography on concanavalin A:agarose and Ricinus communis agglutinin l:agarose. Both forms of the antigen yielded single bands which stained for protein and carbohydrate when examined by disc gel electrophoresis and immunodiffusion. The molecular weights of the two forms were 45,000 and 37,000 respectively. The larger form contained about five to six oligosaccharide chains, whereas the smaller form had only three to four chains. The composition and structures of the oligosaccharide chains in the two forms of this glycoprotein were very similar. Each contained di-, tri-, and tetraantennary complex-type oligosaccharide chains. The diantennary oligosaccharide chains caused both forms of alpha 1-acid glycoprotein to be retained by concanavalin A-agarose columns. The lower-molecular-weight form contained fewer chains and correspondingly fewer terminal galactosyl residues. This resulted in the separation of this species from the higher-molecular-weight form on columns containing R. communis agglutinin I. Three types of reduced oligosaccharides were released from the light and heavy forms of alpha 1-acid glycoprotein by treatment with alkaline borohydride or by hydrazinolysis. These chains were isolated by chromatography on concanavalin A:agarose and Bio-Gel P-6 columns. The arrangement and linkage of sugars in the purified oligosaccharides were determined by periodate oxidation, sequential hydrolysis with glycosidases, and methylation analysis. The major oligosaccharide chain, comprising 50 to 55% of the carbohydrate, had a triantennary structure as shown in the structure: (formula; see text) in which NeuNAc is N-acetylneuraminic acid, Gal is galactose, GlcNAc is N-acetylglucosamine, Man is mannose, GlcNAcol is N-acetylglucosaminitol, and Fuc is fucose. Tetraantennary chains comprised about 25 to 30% of the carbohydrate, and the additional outer chain was attached to the alpha 1,6-mannosyl residue through a beta 1,6-linked GlcNAc unit. The remaining 15 to 20% of the oligosaccharide chains had a diantennary structure. The extent of sialylation of these chains varied in samples isolated from tumors of the same histological type from different individuals. However, a relatively constant proportion of the three types of chains was present in different forms of the glycoprotein isolated from liver metastases.

Amino Acids↗

PubMed1983

Determination of lead in bonemeal by differential pulse anodic stripping voltammetry using a hydrochloric acid solubilization.

A safe, rapid method is described for the determination of lead in bonemeal. This method uses a hydrochloric acid solubilization performed under pressure followed by determination by differential pulse anodic stripping voltammetry. This provides an alternative to a nitric-perchloric acid wet ash. Data obtained using both methods are compared. The mean recovery of a standard Pb spike was 99.2 +/- 7%. The concentration of Pb in bonemeal ranged from 1.0 to 15.6 micrograms/g.

Biological Products↗

PubMed1987

Functional heterogeneity correlates with structural heterogeneity of breast carcinoma acid-soluble glycoproteins.

Patients with malignant tumors, specifically with metastatic breast carcinoma (BCa), are immunosuppressed and have defective lymphocyte responsiveness to antigenic and mitogenic stimulation. The present study examined the role of perchloric acid (PCA)-soluble glycoproteins and their oligosaccharide moieties from tumor cells and from sera of patients with metastatic BCa. Sera from 15 patients and from age-matched healthy adults were examined for immunoregulatory glycoproteins. BCa tissue was obtained from 9 of the 15 patients. The PCA extracts from tumor tissue were resolved into six (GP-I to GP-VI), and from serum into three (GP-II, GP-IV, and GP-V), BCa-associated glycoproteins. After alkaline borohydride treatment, six groups of BCa-associated oligosaccharides were obtained. Peripheral blood mononuclear (MNC) and natural killer (NK) cells were obtained from patients with metastatic BCa and from age-matched healthy adults. These were used as effector cells in the well-established 4-hr cytotoxicity assay. The results indicated that interleukin 2 (IL-2) significantly (P less than 0.001) enhanced the cytotoxic activities of MNC and NK cells from healthy adults, but it had a nonsignificant effect on MNC and NK cells from patients with metastatic BCa. The BCa-associated glycoproteins and their oligosaccharides varied in their effects on MNC and NK cells from both the healthy adults and the patients with metastatic BCa. IL-2 activated MNC cytotoxic activity against BCa cells. GP-I, GP-II, GP-III, and GP-IV inhibited MNC inherent cytotoxicity and blocked MNC stimulation by IL-2, GP-IV fraction had a statistically nonsignificant effect, whereas GP-V enhanced both MNC inherent and IL-2-activated cytotoxic activities. Oligosaccharides obtained from PCA extracts of BCa tissue and by alkaline borohydride treatment differentially bound and inhibited a series of monoclonal antibodies raised against BCa-associated glycoproteins. These results indicated that the oligosaccharide moieties of the BCa-associated glycoproteins modulate recognition of the BCa cells by the effector cells.

Adult↗

PubMed1994

A high-performance liquid chromatography method for measurement of oxidized glutathione in biological samples.

A high-performance liquid chromatography method to determine oxidized glutathione (GSSG) in biological samples with ultraviolet-visible detection using N-ethylmaleimide to prevent reduced glutathione (GSH) oxidation is described. Previous methods based on high-performance liquid chromatography to quantitative GSH and GSSG are unsuitable for determining GSSG in biological samples. This is due to GSH oxidation during sample processing. N-Ethylmaleimide, but not iodacetic acid, prevents this oxidation. Blood GSH oxidation measured by the widely used method of Reed et al. (Anal. Biochem. 106, 55-62, 1980) can be as high as 24 +/- 6% (n = 6). When blood samples were assayed by our procedure, GSH oxidation was only 0.13 +/- 0.28% (n = 5). GSH can be determined enzymatically, i.e., with glutathione-S-transferase, but perchloric acid should not be used to deproteinize samples. Trichloroacetic acid (15% final concentration) may be used. This method allows an accurate calculation of the GSH/GSSG ratio, which is important for determining oxidative stress in tissues in various pathophysiological situations.

Animals↗

PubMed1982

Analysis of creams. II. Quantitative determination of drugs in creams by titration in non-aqueous solvents.

The possibilities for the determination of active components in creams by acid-base titrations in non-aqueous solvents were investigated. Interference by cream-base components with the titration of weak organic bases and their halides with perchloric acid in acetic acid, and with the titration of weak acids with tetrabutylammonium hydroxide in N,N-dimethylformamide were studied. It appeared to be possible to determine alkaloid halides, salicylic acid, hexachlorophene and methyl salicylate without previous clean-up of the cream samples.

Ointments↗

PubMed2005

The zeta potential of cyclo-olefin polymer microchannels and its effects on insulative (electrodeless) dielectrophoresis particle trapping devices.

While cyclo-olefin polymer microchannels have the potential to improve both the optical detection sensitivity and the chemical resistance of polymer microanalytical systems, their surface properties are to date not thoroughly characterized. These surface properties dictate, among other things, electrokinetic effects when electric fields are present. Here, we report the measurement of the zeta potential of cyclo-olefin polymers (injection-molded and hot-embossed Zeonor 1060R and 1020R) microchannels as a function of pH, counter-ion concentration, storage conditions, and chemical treatment in aqueous solutions both with and without EOF-suppressing additives. In contrast with previous reports, significant surface charge is measured, consistent with titration of charged sites with pK(a) = 4.8. Storage in air, acetonitrile, or aqueous solutions has relatively minor effects. While the source of the surface charge is unclear, chemical functionalization has shown that carboxylic acid groups are not present at the surface, consistent with the chemical structure of Zeonor. EOF-suppressing additives (hydroxypropylmethylcellulose) and conditioning in perchloric acid allow the surface charge to be suppressed. We demonstrate dielectrophoretic particle trapping devices in Zeonor 1060R substrates that show reduced trapping voltage thresholds as compared to previous implementations in glass.

Cycloparaffins↗

PubMed1990

Rapid non-enzymatic HPLC determination of total MHPG in human plasma.

We have previously reported a method for the determination of total 3-methoxy-4-hydroxy phenylethylene glycol (MHPG) in brain, based on a simple acid-catalyzed hydrolysis. Now we extend this procedure to the determination of plasma total MHPG. The method involves the deproteinization of plasma with perchloric acid, followed by 3 minutes of an acid-catalyzed step. The hydrolysates are injected into the HPLC system, using a formic acid/methanol eluent with fluorimetric detection. Sample detection limit is below 1 ng MHPG/mL of plasma. This procedure has been used for the determination of plasma total MHPG from 109 healthy individuals of both sexes. Mean value was: 5.4 + 2.3 ng total MHPG/mL of plasma (means +/- S.D., N = 109). No sex differences were observed, and a slight correlation with age (r = 0.24, p less than 0.02) has been found. Plasma-free MHPG was also determined in a subgroup of 15 randomly chosen individuals (3.0 +/- 1.2 ng free MHPG/mL plasma, means +/- S.D.). A significant correlation was obtained with plasma total MHPG (r = 0.77, p less than 0.001, N = 15). The main advantage of the present method lays in its simplicity, since no enzymatic hydrolysis or extraction procedures are needed, being its reliability fully proven through 109 plasma total MHPG determinations.

Chromatography, High Pressure Liquid↗

PubMed2004

Microanalysis of DNA by stripping transfer voltammetry.

A cathodic stripping transfer voltammetric procedure for trace determination of DNA and its components is described. The method is based on the DNA acid hydrolysis with subsequent electrochemical determination of released purine bases. In the first step, DNA is hydrolyzed for 30 min in 0.5 M perchloric acid at 75 degrees C. The electrochemical step involves generation of Cu(I)-purine base complex on a mercury electrode surface, transfer of electrode with accumulated complex into supporting electrolyte where voltammetric measurement is performed. Analysis is carried out in 14-microl drop volume (two-electrode connection) or in 30-microl drop (three-electrode connection) on a platinum plate, which is used as a counter electrode. Blank electrolyte contains 0.05 M borate buffer, pH 9.2 with 6.3 microM Cu(II). We could observe voltammetric signal at hydrolyzed nucleosides, nucleotides, ODN, and DNA containing purine bases. We are able to accumulate under the controlled potential and determine subnanomolar concentration of DNA corresponding to the amount of 200 pg of DNA.

Biosensing Techniques↗

PubMed2002

Quantitation of tryptophan, kynurenine and kynurenic acid in human plasma by capillary liquid chromatography-electrospray ionization tandem mass spectrometry.

Concentrations of tryptophan and its metabolites in plasma are of great interest in determining proper diagnosis and medication of several neurological diseases like, for example, Alzheimer's disease. A method of standard addition was developed to determine total level of tryptophan and two of its metabolites, kynurenine and kynurenic acid, in human plasma by capillary liquid chromatography-electrospray ionization tandem mass spectrometry. Plasma samples were simply deproteinized by addition of diluted perchloric acid. Samples were then mixed with trichloroacetic acid and injected onto a capillary column. Analytes were separated by a fast gradient elution of the injected samples. Detection was performed by sheathless electrospray tandem mass spectrometry in the multiple reaction monitoring mode. Linear calibration curves were obtained for spiked plasma sample with up to 100% of the expected analytes concentrations. The determined concentrations were well within ranges previously reported (i.e., 6 nM-95 microM) and limit of detections were around 3 nM for each analyte.

Calibration↗