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PubMed1989

Factors influencing erythrocyte choline concentrations.

Choline concentrations in human erythrocytes increase after freezing and thawing, during incubation in Krebs-phosphate for 30 min or on storage at 0 degrees C for 3-24 hr. The increase is prevented by protein precipitation by 10% perchloric acid, 10% zinc hydroxide, 10% sodium tungstate or boiling in water. It is not prevented by EDTA (10 mM) and is increased by oleate (5 mM). We suggest that the increase is due to the action of phospholipase D on erythrocyte phospholipids.

Calcium↗
PubMed1990

Impairment of histone H1 DNA binding by adduct formation with acetaldehyde.

Incubation of histone H1 with pharmacologically relevant concentrations of acetaldehyde resulted in the formation of spontaneously stable acetaldehyde-protein linkages. The reaction of acetaldehyde and H1 purified from rat liver either by a DNA recognition site affinity chromatography or by perchloric acid extraction occurred primarily at the lysine residues in the carboxyterminal tail of H1, which is crucial for its function as a eukaryotic repressor. It was further shown using an H1-lacZ fusion protein produced in E. coli and the protein isolated from rat liver that the formation of acetaldehyde adducts with H1 impair its DNA binding properties. We propose that such a reaction may occur in vivo and lead to an inability to repress genes in the liver upon excessive alcohol consumption. This mechanism may play a role in acetaldehyde-induced collagen synthesis in alcoholics.

Acetaldehyde↗
PubMed1995

In vivo effects of lipopolysaccharide on hepatic free-NAD(P)(+)-linked redox states and cytosolic phosphorylation potential in 48-hour-fasted rats.

This study was performed to determine the magnitude and time of onset of in vivo changes in hepatic bioenergetics in response to a sublethal dose of lipopolysaccharide (LPS), a bacterial endotoxin. Male rats (48-hour-fasted) were administered an intraperitoneal injection of LPS (5 mg/kg body weight) or vehicle alone, and the livers were freeze-clamped 5, 30, or 180 minutes or 24 hours later. Liver tissue was extracted with perchloric acid, and the metabolites necessary to calculate NAD(+)- and NADP(+)-linked redox states and the cytosolic phosphorylation potential were measured. There was no significant difference in hepatic cytosolic phosphorylation potential between LPS and control groups at any of the times investigated. This indicated that the ability of the liver to synthesize adenosine triphosphate (ATP) was not compromised under the conditions of the study. No changes in hepatic redox states were observed 5 or 30 minutes after LPS treatment. Three hours after LPS treatment, hepatic cytosolic and mitochondrial free-[NAD+]/[NADH] redox states and the cytosolic free-[NADP+]/[NADPH] redox state were more oxidized. By 24 hours, only NAD(+)-linked redox states were more oxidized than the time-matched controls. Hepatic urea content was elevated at both 3 and 24 hours, compatible with an increased rate of urea synthesis as a consequence of increased amino acid metabolism, whereas hepatic beta-hydroxybutyrate and total ketone bodies were decreased 24 hours after LPS treatment, indicating decreased hepatic ketogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxybutyric Acid↗
PubMed1995

Characterization of the effect of cocaine on catecholamine uptake by pregnant myometrium.

OBJECTIVE: To characterize the effect of cocaine on catecholamine uptake by myometrium from pregnant women. METHODS: Slices of myometrium obtained from nine women during elective cesarean delivery at term were incubated with [3H]-norepinephrine and various uptake inhibitors for 30 minutes. The radiolabeled material was extracted with perchloric acid, expressed as percent control (+/- standard error of the mean), and compared by one-factor analysis of variance and Fisher multiple range test. RESULTS: Myometrial uptake was inhibited by cocaine (42 +/- 9%) by neuronal (type 1) uptake inhibitors (desipramine 41 +/- 14%; N-ethylmaleimide 53 +/- 8%), and by extraneuronal (type 2) inhibitors (normetanephrine 56 +/- 19%; corticosterone 73 +/- 9%). When uptake inhibitors were used in combination with cocaine, uptake was not decreased further in the presence of neuronal inhibitors (desipramine plus cocaine 40 +/- 20%; N-ethylmaleimide plus cocaine 42 +/- 4%). However, the effect of cocaine appeared to be added to that of extraneuronal inhibitors (normetanephrine plus cocaine 25 +/- 14%; corticosterone plus cocaine 32 +/- 1%). CONCLUSION: Catecholamine uptake by myometrium in pregnant women appears to be both extraneuronal and neuronal in nature, and cocaine inhibits the neuronal portion of this uptake. This mechanism may play a role in the increased rate of premature delivery associated with cocaine abuse.

Analysis of Variance↗
PubMed1983

Vascular intracardiac catheterization technique for multiphasic evaluation of rat heart in vivo.

Extensive in vivo classification of the latent or acute cardiotoxic manifestations associated with exposure to chemicals has been limited in the past to larger laboratory animals. To facilitate the conduct of similar studies in smaller experimental animals, a cardiologic model with the rat is introduced that permits determination of in vivo cardiac contractility (e.g., left intraventricular pressure, dP/dt) and excitability (e.g., atrioventricular node conductivity, His-Purkinje system conduction time) with subsequent biochemical analysis of the same heart. To evaluate the influence of possible surgically induced artifacts on the metabolic parameters measured, three surgical protocols were compared: invasive intracardiac catheterization, sham surgery, and nonsurgical control. Male Sprague-Dawley rats were weighed and anesthetized with sodium pentobarbital. Venous blood hematocrit and serum osmolalities were determined in rats subjected to sham and intracardiac catheterization procedures. Surface limb-lead ECG recordings were obtained from rats in these two groups throughout the surgical and catheter manipulation procedures. In rats subjected to the intracardiac catheterization procedure, an electrode was inserted into the right jugular vein and advanced into the right atrium for recording the His bundle electrogram. An ultraminiature solid-state cathetertip pressure transducer was inserted into the right carotid artery and advanced into the left ventricle for recording left ventricular contractility characteristics. Upon completion of the electrode and catheter manipulations, myocardial contractility and excitability indices were assessed for time-dependent changes during a 20-min stabilization period. Subsequently, heart perchloric acid extracts were prepared from hearts frozen in liquid N2, and metabolite (e.g., ATP, ADP, AMP, IMP, phosphocreatine, inorganic phosphate) concentrations were analyzed. Cardiac contractility and excitability characteristics, including peak left intraventricular and isovolumic pressures, peak dP/dt, time to peak dP/dt, heart conduction intervals, and heart rate, stabilized within 10 min after surgery. Thereafter, no surgically induced physiologic or metabolic changes were detected relative to either sham-treated or nonsurgical controls. These findings demonstrate the feasibility and integrity of this method and suggest its potential utility for investigating the in vivo cardiotoxicity of various chemical agents using the rat as the animal model.

Animals↗
PubMed1985

Cardiotoxicity of lead at various perfusate calcium concentrations: functional and metabolic responses of the perfused rat heart.

Equilibrated rat hearts were perfused for 60 min with a standard crystalloid buffer containing either 0.9, 1.8, 3.5, or 5.0 mM Ca with or without added lead (0.3 and 30 microM). Contractile tension (T), rate of tension development (dT/dt), electrocardiographic (EKG), His bundle electrographic (HBE) indices, heart rate (HR), preejection period (PEP), and coronary flow rate (CFR) were recorded as a function of perfusion time. Endpoint analyses of myocardial phosphatic metabolites were performed on heart perchloric acid extracts by standard phosphorus-31 nuclear magnetic resonance (P-31 NMR) spectroscopic techniques. The contractile activity and glycerol 3-phosphorylcholine content of the myocardium were found to vary directly as a function of the perfusate calcium concentration; however, except for a prolonged atrioventricular-His bundle conduction time detected in hearts treated with 0.9 mM Ca, the variable perfusate calcium concentrations were devoid of any other significant physiologic and metabolic effects. In contrast, perfusion of control equilibrated hearts with 30 microM lead significantly attenuated the positive, and exacerbated the negative, inotropic responses to elevated, and low perfusate calcium concentrations, respectively. Moreover, a secondary, time-dependent decline in myocardial contractile strength was also observed in response to this lead concentration, which was progressively more pronounced with each increment in the perfusate calcium concentration. The preejection period of ventricular systole was also prolonged in response to 30 microM lead; however, this effect was less pronounced at higher perfusate calcium concentrations. Hearts perfused with 30 microM lead were also characterized by significant prolongation in atrioventricular node and His bundle conduction time, reduced coronary flow rate, and decreased heart rate, irrespective of the perfusate calcium concentration. Hearts treated with 0.3 microM lead exhibited functional properties that were diminished, but still comparable to control hearts. Analysis of myocardial phosphatic metabolite amounts following 60 min of perfusion revealed a significant lead-induced reduction in the energy status of the heart. The combination of 5.0 mM calcium with either 0.3 or 30 microM lead resulted in significant disturbances in phosphoglyceride, glycolytic, and high-energy phosphate pathways. These findings suggest that the cardiotoxic actions of lead are linked to complex mechanisms that are partially related to an interference with calcium-dependent cellular processes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Cadmium and lead contents of cigarettes produced in various areas of the world.

Cigarette packs, 331 in total, were purchased in 20 areas including nine in Asia between 1982 and 1984, and analyzed for cadmium and lead by flameless atomic absorption spectrophotometry after wet digestion with nitric, sulfuric and perchloric acids. The arithmetic mean (geometric mean in parentheses) for Cd was 1.15 (1.06) micrograms/cigarette or 1.45 (1.31) micrograms/g, and the values for Pb were 1.70 (1.31) micrograms/cigarette or 1.76 (1.67) micrograms/g. The Cd content of cigarettes was distributed over a wide range from 0.29 to 3.38 micrograms/g, and the range was even wider for Pb, 0.46 to 43.66 micrograms/g. The mean values varied markedly depending on the area of production. It was not possible, however, to obtain any evidence to suggest that the differences in Cd and Pb contents are related to the area of production or the extent of industrial development of the area.

Cadmium↗
PubMed1990

Iron, copper, zinc and lead in hair from Sudanese populations of different age groups.

Energy-dispersive X-ray fluorescence was used for the analysis of hair samples from three different age groups of the Sudanese population. Hair samples were digested in a mixture of nitric and perchloric acids and the metals were then precipitated with ammonium pyrrolidine dithiocarbamate. The variations of the Fe, Cu, Zn and Pb content of hair with age were investigated. The averages of the elemental concentrations in each age group were compared with the other age groups and with literature values. The correlation of each pair of elements in the hair samples was also investigated.

Adolescent↗
PubMed1983

Platelet adenine nucleotide levels in patients with Dacron vascular prostheses.

Platelets adenine nucleotide levels were determined in 11 patients with Dacron bifemoral aortic prostheses using high performance liquid chromatography. Total platelet and dense granule adenine nucleotide levels were measured in neutralized perchloric acid extracts prepared from gel-filtered platelet suspensions and from platelet supernatants following thrombin-induced release of granule constituents respectively. Dense granule adenine nucleotide levels were significantly decreased in the patient group compared with age and sex-matched controls (p less than 0.01), whereas no differences in cytoplasmic adenine nucleotide levels were observed (p greater than 0.2). Platelet survival measurements were made on 3 patients; when grouped with control subjects, a positive correlation was observed between platelet survival and platelet dense granule ADP (r = 0.96; p less than 0.01). These results suggest that platelet dense granule adenine nucleotide content may be a clinically significant indicator of vascular graft thrombogenesis.

Adenine Nucleotides↗
PubMed1987

An immunosorbent assay for blood group I antigens in breast carcinoma.

Tumor cells elaborate and release into the circulation a variety of glycoproteins. An enzyme-linked immunosorbent assay (ELISA) was developed to monitor carbohydrate structures secreted into the circulation. Among these antigens are the structures specific for the blood group I antigens, which are incompletely converted to ABH antigens on the membranes of tumor cells. The I antigens in the sera of 67 women with breast carcinoma (BCa), 58 with benign breast disease (BBD), and 47 controls were measured by the ELISA. In this assay, I antigen from ovarian cyst mucin was bound to the wells of polystyrene microtiter plates. The monoclonal human anti-I antibody (Hy) was added to the wells along with perchloric acid extracts of patient and control sera at five different dilutions. The anti-I binding to the solid-phase I antigen was determined after incubation steps with peroxidase-labeled anti-human IgM and substrate. The amount of sera extracts giving 50% inhibition of anti-I (Hy) binding was determined from the inhibition curves which were corrected by integrating the slope values into that of the standard curve obtained with extracts of normal sera. The I antigens were significantly higher in pathologic stage (PS) IV sera (P less than 0.001), and comparable in PS I, PS II, and PS III and BBD sera to those in control sera. The anti-I (Hy) binds strongly Gal 1,4 GlcNAc 1,6 Gal (alpha GalNAc); Gal 1,4 GlcNAc 1,6 (Gal 1,4 GlcNAc 1,3) Gal; and to a lesser extent Gal 1,4 GlcNAc 1,3 Gal 1,4 GlcNAc (0.06, 0.09, and 0.35 mM, giving 50% inhibition, respectively). It was concluded that similar or related structures may be expressed on the membrane of metastatic BCa cells.

Antibodies, Neoplasm↗
PubMed1991

A rapid method for the estimation of prostaglandin E2 in intestinal tissues using fluorescence derivatization.

A sensitive spectrofluorimetric method is described to determine small quantities of prostaglandin E2 in complex biological systems as intestinal tissues. The method is based on a solid phase extraction combined with a coupling with a fluorescent marker and measuring the derivatization product by fluorescence densitometry. After mixing the tissue with an ice-cold perchloric acid solution, adjusting the pH, centrifugation and filtration steps, the prostaglandins are retained on a solid phase extraction C18 disposable column. They are eluted with diethylether, derivatized with 4-bromomethyl-7-methoxy-coumarin using potassium carbonate as condensating agent and finally analysed using fluorescence densitometry on silica gel TLC plates. Applying this method, amounts down to 5 ng (per gram wet tissue) could be measured in intestinal tissues, the s.e.m. for replicated total analysis being less than 15%. The foregoing method is applied for the determination of PGE2 released in the intestinal wall under the influence of laxatives.

Animals↗
PubMed1983

Affinity chromatography separation of tumor associated antigens from ascitic fluid of ovarian cancer patients.

Tumor associated immunoglobulins previously isolated from ovarian cancer ascitic fluid have been used in an affinity chromatography step to purify putative tumor associated antigens from a perchloric acid extract of ascitic fluid. Examination of the affinity chromatography purified proteins revealed 4 bands on electrophoresis. Antisera raised to the affinity chromatography products have been examined by Ouchterlony diffusion as well as a solid-phase radioimmunoassay. Two of thirteen ovarian tumor extracts investigated reacted in Ouchterlony diffusion analysis, whereas all thirteen extracts gave positive results in the more sensitive solid-phase radioimmunoassay.

Antigens, Neoplasm↗
PubMed1988

A rapid and simple HPLC microassay for biogenic amines in discrete brain regions.

A rapid microassay is described for the measurement of biogenic amines using an isocratic HPLC system with electrochemical detection. Catecholamines, indoleamines and their major metabolites were extracted with 150 microliters of perchloric acid from brain tissue punches (less than 250 micrograms) using a simple one-step sample preparation method. These compounds were separated on a short (80 mm) column with 3 microns particle size packing, and electrochemically detected within a total run time of less than 6 minutes. Detection limit sensitivity was approximately 2-5 pg. This method, detailed in an easy-to-follow description, reduces assay time, minimizes the possibility for errors, maximizes efficiency, and requires only standard HPLC equipment and supplies.

Animals↗
PubMed1985

Distribution and characterization of cyclo (His-Pro)-like immunoreactivity in the human gastrointestinal tract.

Cyclo (His-Pro) [C(HP)] has been measured by radioimmunoassay in perchloric acid extracts of human gastrointestinal (GI) tract structures derived from autopsy sources and fresh colonic biopsies. C(HP) was identified in all regions of the human GI tract, ranging in concentrations from 599 +/- 102 pg/mg protein in stomach, to 127 +/- 26 pg/mg protein in esophagus. The mean concentration of C(HP) from colonic biopsies was 335 +/- 30 pg/mg protein, statistically similar to values derived from postmortem sources. Since C(HP) concentrations are within the range of other gut peptide modulators, cyclo (His-Pro) is speculated to play a role as a new paracrine modulator of human GI tract function(s).

Animals↗
PubMed1980

Modification of the 4-(p-nitrobenzyl) pyridine assay for measurement of melphalan and mechlorethamine in plasma.

A sensitive, reproducible spectrophotometric assay for mechlorethamine and melphalan was developed through modifications in the use of 4-(p-nitrobenzyl) pyridine as the chromogenic reagent. Key factors that led to enhanced color development and consistency of results were: (1) protein precipitation with 4 degrees C perchloric acid; (2) control of the pH of the reaction with acetate buffer; (3) precise timing of color development in alkali; and (4) sample reading at the appropriate wavelength. The modifications allowed measurement of mechlorethamine and melphalan levels in perfusate samples obtained from isolated rat liver perfusions. Half-lives calculated from perfusate decay curves after addition of mechlorethamine at 50 microgram/ml and 10 microgram/ml were 15.9 min and 15.0 min respectively. The perfusate half-life after addition of melphalan at 5 microgram/ml was 44.2 min. Plasma levels of mechlorethamine were not detectable by this assay after intrapleural administration of 15 mg to one patient but were detectable after intravenous administration of 20 mg to a second patient.

Animals↗
PubMed1985

Gas-liquid chromatographic analysis of ethanol and acetaldehyde in blood with minimal artifactual acetaldehyde formation.

A gas-liquid chromatographic procedure utilizing headspace gas analysis is described for the determination of ethanol and its metabolite, acetaldehyde, in a 100-microliters sample of blood from the rat, guinea pig, sheep, or human. Artifactual formation of ethanol-derived acetaldehyde is minimized during sample preparation by using a chemical solution containing perchloric acid and sodium azide in saline, and thiourea. Aqueous standards of ethanol and acetaldehyde are used to calibrate the procedure, and 1-propanol is used as the internal standard of the method. The recovery of ethanol and acetaldehyde from spiked blood samples is quantitative and reproducible, with a within-day coefficient of variation of less than 7% for ethanol and less than 9% for acetaldehyde. The lower limit of quantitative sensitivity is 0.006 mg/ml ethanol and 0.10 microgram/ml acetaldehyde. The instrumental analysis time is less than 3 min, which enables high sample throughput.

Acetaldehyde↗
PubMed1985

HPLC-ECD procedure for the measurement of O-methylation of catechol estrogens by vascular tissue.

A precolumn extraction procedure combined with high-performance liquid chromatography with electrochemical detection (HPLC-ECD) has been developed for the identification and quantitation of catechol estrogens, their immediate precursors (estrogens), and their 0-methylated metabolites. These compounds were isolated from Krebs solutions and from perchloric acid tissue extracts or whole tissues by simple solvent extraction prior to quantitation. The optimal conditions for detection by HPLC-ECD were those employing a radial compression module, a C18 Bondapak cartridge, and an isocratic mobile phase of ammonium phosphate (pH 3.0) combined with 30-70% acetonitrile. A biological application of this procedure includes the investigation of the 0-methylation of catechol estrogens by vascular tissue. In the present study, the 0-methylation of 2-hydroxyestradiol by rabbit aorta has been examined. The 0-methylation in segments of thoracic aorta was significantly less than that in abdominal aortic segments, but was reproducible along the length of the thoracic aorta.

Animals↗
PubMed1985

Delineation of four carcinoembryonic antigen (CEA) related antigens in normal plasma by transblot studies using monoclonal anti-CEA antibodies with different epitope specificities.

Antigens related to the carcinoembryonic antigen (CEA) were isolated from normal human plasma by perchloric acid extraction, gel permeation chromatography and immunoaffinity chromatography using a monoclonal antibody with broad specificity and high affinity. The antigens were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. The binding of five monoclonal anti-CEA antibodies with different epitope specificities to the immobilized antigens was analyzed. Two antigens with mol. wts of greater than 200,000 and 177,000 bound all five antibodies, and two antigens with mol. wts of 114,000 and 85,000 bound three of the five antibodies. The findings reported indicate that even monoclonal antibodies with high specificity for colonic cancer CEA detect CEA-related antigens in normal human plasma.

Antibodies, Monoclonal↗