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Prolonged relief of neuralgia after regional anesthetic blocks. A call for further experimental and systematic clinical studies.

Thirty-eight consecutive patients with neuralgia after peripheral nerve injury were treated with one or two series of peripheral local anesthetic blocks. All patients experienced an initial total relief of ongoing pain for 4-12 h. Evoked pain (hyperalgesia or allodynia), which occurred in 17 patients, was blocked simultaneously with the spontaneous pain. In 18 patients the analgesia outlasted the conduction block and there was a period of complete pain relief of 12-48 h in 13 patients and of 2-6 days in the other 5. In 8 patients there was a second phase of analgesia of 4 h to 6 days duration occurring within 12 h of pain recurrence. Thus, mono- or biphasic prolonged complete analgesia occurred in 25 out of 38 patients. A prolonged analgesia may be the result of a central action of the local anesthetic at the spinal level after intra-axonal incorporation and centripetal axoplasmic transport. To test this hypothesis, an experimental study with [3H]lidocaine was performed in 6 rats. The radioactive local anesthetic was injected into one hind limb foot with the other side serving as a control. Tissue samples from the peripheral nerve, nerve root and the lumbosacral spinal cord segment were analyzed for radioactivity using a scintillation counter technique at various time intervals after the [3H]lidocaine injection. There was a low grade of activity in all samples and no difference between the test side and the control side. Thus these experiments provided no evidence in support of this hypothesis. Various alternative peripheral and central mechanisms are discussed. Further studies specifically directed to these alternatives and with longitudinal controls are prompted.

Adolescent↗

Rhodococcus equi: equine neutrophil chemiluminescent and bactericidal responses to opsonizing antibody.

The opsonic capacity of serum containing R. equi-specific antibody was compared with antibody-deficient sera using luminol-dependent chemilumenscence (LDCL) and bactericidal assays. These assays incorporated peripheral blood polymorphonuclear neutrophilic leukocytes (PMNL) exposed to R. equi opsonized with neonatal equine pre-colostral serum (control) or serum from foals with R. equi infections (principal). All sera were complement inactivated at 56 degrees C for 30 min. Bacteria were obtained from the lung of a foal with R. equi pneumonia. Neutrophils were obtained from one adult horse for LDCL and another for bactericidal assays. Chemiluminescence of PMNL exposed to R. equi opsonized with control or principal sera was measured in a liquid scintillation counter. Mean peak LDCL within 1 h was significantly (P less than 0.01) higher with principal sera (2.4 X 10(5) cpm) than with control sera (0.018 X 10(5) cpm). A radioisotope bactericidal assay was used to determine the effect of control or principal sera on PMNL capacity to kill R. equi. Mean peak percent kill of R. equi by PMNL within 2 h was significantly (P less than 0.01) higher with principal sera (95.2%) than with control sera (54.6%). Enzyme-linked immunosorbent assay (ELISA) values for R. equi-specific antibody were determined on all sera. Mean ELISA values were significantly (P less than 0.01) higher for principal sera (71.8) than for controls (0.0). This investigation documents the presence and biological effectiveness of opsonic activity in complement-inactivated sera from foals with R. equi infections and R. equi-specific antibody.

Actinomycetales Infections↗

Influence of dietary ingredients on the body retention of strontium, cadmium and mercury in suckling rats.

Six-day-old suckling rats were artificially fed over 8 h with cow's milk or a mixture of rat food ingredients (fish meal, sunflower meal, alfalfa, cane molasses and premix) labelled with 85Sr, 115mCd or 203Hg. Whole-body radioactivity was determined in a double crystal scintillation counter every 24 h over a 6-day period. Carcass, gut, liver and kidney retention were determined at the end of the experiment. Relative to pups that were fed on cow's milk, administration of the mixture of the solid dietary ingredients caused a significant reduction in whole body, carcass and organ retention of all metals and was highest for 203Hg. This indicates that high absorption of toxic metals in sucklings can be reduced by dietary means. Rat food ingredients deserve further consideration because they are non-toxic dietary constituents and might be useful for decreasing the body burden of some metals in conditions of increased environmental exposure.

Animal Population Groups↗

High-pressure liquid chromatographic assay for the determination of thyrotropin-releasing hormone and its common metabolites in a physiological salt solution circulated through the isolated perfused rat lung.

A specific and sensitive assay for TRH (L-pyroglutamyl-L-histidyl-L-proline amide), 3H-TRH (L-proline 3,4-3H(N), histidyl-3-3H(N)), and four possible metabolites of TRH, present in the recirculated perfusate of an isolated perfused rat lung preparation, was developed. Unlike previous methods, the method developed does not require extraction of the analytes from the biological matrix. The crude sample was adjusted to a pH of 3.2 with concentrated trifluoroacetic acid and injected on to a PRP-1 (polystyrene divinylbenzene) column (10 microns, 25 cm x 4.6 mm i.d.). The mobile phase was 10% v/v acetonitrile and 90% v/v 0.75 g l-1 1-hepantanesulfonic acid in 0.004 M trifluoroacetic acid, adjusted to a pH of 2.4 with concentrated NaOH. The flow rate was 0.5 ml min-1 and the analytes were detected by UV absorption at a wavelength of 26 nm and by radiochemical detection utilizing a liquid scintillation counter. The nominal retention times for L-PRO, L-PRO-NH2, TRH, cyclo(HIS-PRO) and TRH-OH were 4.0 +/- 0.9, 10.0 +/- 0.2, 15.5 +/- 0.4, 19.2 +/- 0.5 and 25.3 +/- 0.5 min respectively. The assay performs well in terms of precision and accuracy as indicated by linear regression and intra-assay variability analysis.

Animals↗

Lack of hormonal stimulation of pyridoxine metabolism in isolated rat hepatocytes.

Isolated hepatocytes obtained from Sprague-Dawley rats (145-175 g) were incubated for 15 min at 30 degrees C in Krebs-Henseleit bicarbonate buffer, pH 7.4, containing 0.5 mM concentration of each of the 20 natural amino acids and either 4.5 or 23 microM [U-14C]pyridoxine. Pyridoxine, pyridoxal, pyridoxal phosphate, and pyridoxic acid separated by an anion-exchange chromatographic technique were quantified using a phosphate analyzer and a liquid scintillation counter. The conversion of [U-14C]pyridoxine to its metabolites was more than doubled by increasing the amount of pyridoxine (4.5 to 23 microM) in the incubation medium. Insulin (10 mU/ml), glucagon (1 nM), or epinephrine (10 microM) did not have any significant effect on the conversion of [14C]-pyridoxine to pyridoxal, pyridoxal phosphate, or pyridoxic acid. Our earlier observations of a large decrease in serum pyridoxal phosphate in the diabetic rat cannot be explained by any direct hormonal effects on pyridoxine metabolism.

Animals↗

Hydroperoxide-initiated chemiluminescence: an assay for oxidative stress in biopsies of heart, liver, and muscle.

Hydroperoxide-initiated chemiluminescence was standardized as a microassay to evaluate the occurrence of oxidative stress in human biopsies. Samples of 10 to 50 mg of rat liver or heart were homogenized, diluted in reaction medium, added with tert-butyl hydroperoxide, and assayed for chemiluminescence in a liquid scintillation counter in the out-of-coincidence mode. Optimal conditions for the assay were: 0.3 to 1.2 mg/mL of homogenate protein in 120 mM KCl, 30 mM phosphate buffer (pH 7.4), and 3 mM tert-butyl hydroperoxide at 30 degrees C. In these conditions, maximal chemiluminescence values were 550 +/- 30 and 1100 +/- 40 cps/mg protein, for liver and heart homogenates, respectively. Liver and heart homogenates were subjected to in vitro oxidative stresses such as supplementation with organic hydroperoxide or with enzymatic systems generating superoxide anion or hydrogen peroxide. Chemiluminescence was higher in the poststress samples than in the control ones. The ratio: poststress chemiluminescence/control chemiluminescence (B/A) was about 1.4 or higher for both tissues. Human heart biopsies were utilized to investigate the occurrence of oxidative stress after clinical situations associated to ischemia-reperfusion. B/A ratios were 2.1 +/- 0.4, 1.4 +/- 0.1, and 2.8 +/- 0.4 for human heart, liver, and skeletal muscle, respectively.

Animals↗

Cellular immunity to human insulin in individuals at high risk for the development of type I diabetes mellitus.

In order to investigate the role of insulin as a potential target autoantigen of cellular immunity in the prediabetic period, proliferative responses of T lymphocytes to human insulin were studied in nine islet-cell antibody (ICA) + first-degree relatives of patients with Type I diabetes (individuals at high risk for the development of Type I diabetes, or the 'prediabetic' group, which was never treated with insulin) and in 12 control individuals. Insulin autoantibodies were present in 6/9 (67%) of the prediabetic subjects and none of the controls. Peripheral blood lymphocytes were collected on Ficoll and incubated with human insulin, control antigens, or media alone for 5-6-day and 9-10-day incubation periods. Cells were pulsed with 3H-thymidine, harvested, and analysed in a scintillation counter. Results are expressed as stimulation index (SI = cpm with antigen/cpm without antigen), with a SI greater than or equal to 1.5 considered a positive response. Eight of nine (89%) prediabetic individuals responded positively to insulin after a 9-10-day incubation period, in contrast to four of 12 (33%) control subjects, P less than 0.05. The mean proliferative response to insulin after 9-10 days' incubation was 2.1 +/- 0.4 and 1.2 +/- 0.1, for the prediabetic and control groups, respectively. The proliferative response to insulin was not directly correlated with levels of insulin autoantibodies (r = -0.05, NS). These data suggest that most individuals at high risk for the development of Type I diabetes display a cellular immune response to insulin, and a subset of these individuals does not display a concomitant humoral immune response to insulin based on the presence or absence of insulin autoantibodies.

Adolescent↗

Leukotriene B4 omega-hydroxylase activity in polymorphonuclear leukocytes from patients with inflammatory bowel disease.

omega-oxidation is regarded as the major pathway for the catabolism of leukotriene B4 (LTB4). To determine how LTB4 omega-hydroxylase is modulated in inflammatory bowel disease, we investigated the kinetic characteristics of this enzyme in 10 patients with Crohn's disease (CD), nine with ulcerative colitis (UC) and eight healthy control subjects. After incubating polymorphonuclear leukocytes with various concentrations of 3H-labeled LTB4, omega-oxidation products were separated by high performance liquid chromatography (HPLC) and the radioactivity was measured by a liquid scintillation counter. The apparent Vmax values were significantly higher in both disease than in healthy control subjects, although the difference between CD and UC was insignificant. There was no difference in the apparent Km values. And the Vmax/Km ratios of patients with CD were significantly higher than that of healthy control subjects. It is suggested that LTB4 metabolism is activated in inflammatory bowel disease (IBD) and that the modulation of this enzyme activity has an important role in the pathogenesis of inflammatory bowel disease.

Adolescent↗

5-Aminosalicylic acid inhibits leukotriene B4 omega-hydroxylase activity in human polymorphonuclear leukocytes.

omega-Oxidation is regarded as the major pathway for the metabolism and inactivation of leukotriene B4 (LTB4). To investigate the action of 5-aminosalicylic acid (5-ASA) on LTB4 omega-hydroxylase activity, we incubated human polymorphonuclear leukocytes (PMNLs) with 3H-labeled LTB4 after pre-incubation with various concentrations of 5-ASA. omega-oxidation metabolites were separated by high performance liquid chromatography and each radioactivity was measured by a liquid scintilation counter. LTB4 omega-hydroxylase activity was inhibited by 5-ASA in a concentration-dependent fashion. The 50% inhibitory dose was about 50 mumol/l, which is within the concentration range found in the colonic mucosa. Our findings may be valuable in elucidating the potentially critical aspect of 5-ASA treatment in ulcerative colitis (UC).

Aminosalicylic Acids↗

Erythrocyte adenosine transport. A rapid screening test for cardiovascular drugs.

An erythrocyte (RBC) model based on whole blood was used to investigate the effect of cardiovascular drugs on the uptake of adenosine in vitro. Fresh whole blood obtained from healthy volunteers was allowed to equilibrate with various concentrations (5-1000 microM) of a tested agent. (2-3H)-Adenosine was used as a substrate, and the reaction was terminated after 2 sec of incubation at room temperature by rapid addition of a "Stopping Solution" which was a mixture of erythro-9-(2-hydroxy-3-nonyl)adenine, dipyridamole, and EDTA. The mixture was centrifuged (1760 g, 4 degrees C, 10 min), and the radioactivity of an aliquot of the supernatant was determined by a scintillation counter. The results showed that dipyridamole was the most potent agent tested (IC50 = 0.2 microM). Amongst the calcium antagonists studied, isradipine was most potent, followed by verapamil, clentiazem, diltiazem, and then nifedipine. The racemates of two metabolites of diltiazem, MX and MB, were more potent than the parent drug. The antiarrhythmic agents, amiodarone and sotalol, the two new lipid peroxidation inhibitors, U-74389F and U-78517F, and the anxiolytic agent, alprazolam, were as active as verapamil. The beta-receptor antagonist propranolol and the angiotensin converting enzyme (ACE) inhibitor, enalapril, were practically inactive. In addition, the model was stereoselective such that the S(-)-enantiomer of verapamil was considerably more potent than the R(+)-antipote, whereas d(+)-sotalol was practically inactive compared to racemic sotalol.

Adenosine↗

An assay for adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase.

Adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5'-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S(0.5) determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.

Adenosine Diphosphate Glucose↗

Determination of coenzyme Q biosynthesis in cultured cells without the necessity for lipid extraction.

Ubiquinone (coenzyme Q; CoQ) is the only lipophilic antioxidant that is endogenously synthesized by all organisms. CoQ biosynthesis is determined in vitro by supplying a radiolabeled precursor and, after lipid extraction and CoQ separation by thin-layer chromatography or high-performance liquid chromatography, the radioactivity present in the sample is quantified. In the rapid and simple method described here, we avoid the use of organic solvents by supplying 4-hydroxy-[U-14C]benzoate as radiolabeled precursor and precipitating CoQ with trichloroacetic acid (TCA). After TCA precipitation, all radioactivity was present in the precipitate and CoQ was the only radiolabeled molecule detected. The radioactive material was then solubilized with NaOH and quantified in a scintillation counter.

Carbon Radioisotopes↗

Sequential isotopic determination of plutonium, thorium, americium, strontium and uranium in environmental and bioassay samples.

A procedure has been developed to provide sequential analysis of 238Pu, 230Th, 241Am, 238U, and 90Sr in environmental and bioassay samples. Tracers and/or carriers (242Pu, 243Am, 232U, and stable strontium) are added into the sample as chemical yield monitors, and then, plutonium, thorium, strontium, americium, and uranium are sequentially separated and purified by Dowex ion-exchange resin, EiChroM Sr-resin, EiChroM TRU-resin, and Chelate-100 resin, respectively. The radioactivities of 90Sr and the actinides are measured using the liquid scintillation counter and alpha-particle spectrometer, respectively. Acidified water, glass-fiber air filter, soil, synthetic urine and synthetic feces samples of US National Institute of Standard and Technology Radiochemical Intercomparison Program(NRIP) were analyzed to verify this method. All the analytical results of 238Pu, 230Th, 90Sr, 241Am and 238U meet the traceability limit per ANSI N42.22, and when appropriate, evaluation of radiobioassay measurement bias and precision per ANSI N13.30.

Chromatography, Ion Exchange↗

Natural radiation doses for cosmic and terrestrial components in Costa Rica.

A study of external natural radiation, cosmic and terrestrial components, was carried out with in situ measurements using NaI scintillation counters while driving along the roads in Costa Rica for the period July 2003-July 2005. The geographical distribution of the terrestrial air-absorbed dose rates and the total effective dose rates (including cosmic) are represented on contour maps. Information on the population density of the country permitted the calculation of the per capita doses. The average effective dose for the total cosmic component was 46.88+/-18.06 nSvh(-1) and the average air-absorbed dose for the terrestrial component was 29.52+/-14.46 nGyh(-1). The average total effective dose rate (cosmic plus terrestrial components) was 0.60+/-0.18 mSv per year. The effective dose rate per capita was found to be 83.97 nSvh(-1) which gives an annual dose of 0.74 mSv. Assuming the world average for the internal radiation component, the natural radiation dose for Costa Rica will be 2.29 mSv annually.

Background Radiation↗

Gamma irradiation of human dendritic cells influences proliferation and cytokine profile of T cells in autologous mixed lymphocyte reaction.

Dendritic cells (DC) are the most potent antigen-presenting cells (APC); their ability to induce proliferation of T cells in a mixed lymphocyte reaction (MLR) assay is commonly used for the evaluation of their function. It is a general thought that gamma irradiation of APC does not influence their ability to activate T-cell proliferation, but the data from several studies are controversial. To further determine the mechanisms involved in DC-induced T-cell activation in MLR assay, human DC induced from peripheral blood mononuclear cells (PBMC) were gamma-irradiated and determine their effects on the proliferation and cytokine profiles of T cells in an autologous MLR. DC were induced from the PBMC of 11 multiple sclerosis (MS) patients with RMPI 640 medium containing recombinant human GM-CSF (rhGM-CSF; 800 U/ml) and recombinant human IL-4 (rhIL-4; 500 U/ml). DC harvested on day 7 were divided into two equal parts. One part was not irradiated (naive DC); the other was gamma-irradiated at a dose of 30 Gy. Cell surface molecules were analyzed by flow cytometry. T-cell proliferation was determined using a beta-scintillation counter. The levels of IL-2, IL-4, IL-6 and IL-10 in co-culture supernatants were measured by ELISA. The results indicated that gamma irradiation reduced expression of CD86, CD80 and HLA-DR molecules on DC, especially CD86 (P=0.0072). DC, irradiated or non-irradiated, effectively stimulated autologous T-cell proliferation. Compared to naive DC, irradiated DC showed a markedly lower capacity to promote T-cell proliferation (P=0.0073), and strikingly up-regulated secretion of IL-4 (P=0.0145) and IL-2 (P=0.0323) by autologous T cells. No significant differences were noted in IL-6 and IL-10 production between T cells co-cultured with naive DC and irradiated DC (P>0.05). It is concluded that gamma irradiation of DC not only influences the phenotype of DC but also alters their capacity to stimulate the proliferation and the cytokine profiles of autologous T cells in a MLR.

Antigens, CD↗

TopCount coupled to ultra-performance liquid chromatography for the profiling of radiolabeled drug metabolites in complex biological samples.

The recent commercial availability of small particle packed columns (<2microm) and associated instrumentation capable of withstanding the high pressures of such columns, has lead to an increase in the application of so called ultra-performance liquid chromatography (UPLC). It has recently been shown that the improved efficiency, resolution and peak capacity of these columns, when coupled to mass spectrometry, provides particular benefit for the identification of drug metabolites in complex biological samples. In this work, the ability of TopCount, a microplate scintillation counter, to act as a suitable radiodetection system for ultra-performance liquid chromatography methods is tested. TopCount, has innumerable benefits over more traditional on-line radioactivity flow detection methods, when dealing with the narrow peak widths and small peak volumes associated with the enhanced efficiency of sub-2microm columns. The system is tested for robustness and sensitivity, and then used to undertake successful metabolite profiling of actual samples, and the data compared to traditional HPLC with on-line radioactivity flow detector.

Animals↗

Bacterially synthesized folate and supplemental folic acid are absorbed across the large intestine of piglets.

A large pool of folate exists in the large intestine of humans. Preliminary evidence, primarily in vitro, suggests that this folate may be bioavailable. The purpose of this study was to test the hypothesis that supplemental folic acid and bacterially synthesized folate are absorbed across the large intestine of piglets. The pig was used as an animal model because it resembles the human in terms of folate absorption, at least in the small intestine. A tracer of [3H]-folic acid or [3H]-para-aminobenzoic acid ([3H]-PABA), a precursor of bacterially synthesized folate, was injected into the cecum of 11-day-old piglets. Feces and urine were collected for 3 days. Thereafter, piglets were killed, and livers and kidneys harvested. [3H]-Folate was isolated from biological samples by affinity chromatography using immobilized milk folate binding proteins and counted using a scintillation counter. In piglets injected with [3H]-folic acid, the feces, liver, urine and kidneys accounted for 82.1%, 12.3%, 3.9% and 1.7% of recovered [3H]-folate, respectively. In piglets injected with [3H]-PABA, the amount of recovered bacterially synthesized folate in the feces, liver and urine was 85.1%, 0.4% and 14.6%, respectively. Twenty-three percent and 13% of tritium were recovered in samples examined (liver, kidney, fecal and urine) from piglets injected with [3H]-folic acid and [3H]-PABA, respectively. Using our estimates of [3H]-folic acid absorption and the total and percent monoglutamyl folate content of piglet feces, we predict that at least 18% of the dietary folate requirement for the piglet could be met by folate absorption across the large intestine.

4-Aminobenzoic Acid↗

Influence of vitamin C on cadmium absorption and distribution in rats.

The purpose of the present study was to evaluate the effect of a dietary vitamin C supplement on cadmium absorption and distribution in an animal model. An aqueous solution of cadmium chloride (labelled with cadmium-109) was given by gavage to male Wistar rats for 28 days at a daily dose corresponding to 10 mg Cd/kg diet (1.0-1.2 mg Cd/kg b.w.). The animals assigned to groups 1 and 2 (45 animals per group) received a standard laboratory diet LSM, and tap water or tap water supplemented with ascorbic acid (1.5 mg/l), respectively. The radioactivity of the samples was measured using a liquid scintillation counter (tissue samples) and a gas-flow automatic counter (ashed carcasses). The fractional uptake of cadmium-109 in the carcass and organs was evaluated within 32 days after treatment by dividing the cadmium-109 activity in the whole sample by the total activity of cadmium-109 administered for 28 days. Results were compared using AUC (areas under the concentration time curve) values. The vitamin C supplement decreased the carcass cadmium burden and the cadmium content in the liver, kidneys, testicles and muscles; the highest decreases were found in the testicles, the lowest ones in the muscles. In addition, the rats supplemented with vitamin C revealed an improved body weight gain during the experimental period.

Animals↗