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PubMed2005

Acute effects of exogenous nitric oxide on glucose uptake in skeletal muscle of normoglycaemic and diabetic rats.

BACKGROUND: The role of nitric oxide (NO) in the modulation of basal and insulin-stimulated glucose uptake remains controversial. The aim of this study was to investigate the role of NO released from its donors, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP), on glucose uptake in skeletal muscle of normoglyceamic and type 2 diabetic rats. MATERIAL/METHODS: Skeletal muscle strips of type 2 diabetic and normoglycaemic Sprague-Dawley rats were incubated with or without various concentrations (200 microM, 500 microM, 1000 microM, 10 mM & 20 mM) of SNAP or GSNO in the presence or absence of insulin (100 nM or 10 microM). The associated radioactivity was determined by liquid scintillation counting in a Beckman LS6000 scintillation counter programmed for dual-channel counting. RESULTS: SNAP and GSNO at 1000 microM significantly elevated basal and insulin-stimulated 2-deoxyglucose uptake (P<0.05) in normoglycaemic and diabetic rats. Millimolar concentrations (10 & 20 mM) of GSNO and SNAP significantly decreased basal and insulin-stimulated glucose uptake in skeletal muscle strips of normoglycaemic and type 2 diabetic rats in a concentration-dependent manner (P<0.05). The inhibition of insulin-stimulated glucose uptake was greater in the diabetic rats using both NO donors compared with normoglycaemic rats (P<0.05). CONCLUSIONS: The stimulatory effect of micromolar concentrations of GSNO and SNAP enhances basal and insulin-stimulated glucose uptake in normoglycaemic and type 2 diabetic rats, however higher concentrations elicited an inhibitory effect in normoglycaemic and diabetic rats. This highlights the NO-glucose uptake mechanism as a possible potential therapeutic target in the treatment of type 2 diabetes.

Animals↗
PubMed1989

Effect of counting errors on immunoassay precision.

Using mathematical analysis and computer simulation, we studied the effect of gamma scintillation counting error on two radioimmunoassays (RIAs) and an immunoradiometric assay (IRMA). To analyze the propagation of the counting errors into the estimation of analyte concentration, we empirically derived parameters for logit-log data-reduction models for assays of digoxin and triiodothyronine (RIAs) and ferritin (IRMA). The component of the analytical error attributable to counting variability, when expressed as a CV of the analyte concentration, decreased approximately linearly with the inverse of the square root of the maximum counts bound. Larger counting-error CVs were found at lower concentrations for both RIAs and the IRMA. Substantially smaller CVs for overall assay were found when the maximum counts bound progressively increased from 500 to 10,000 counts, but further increases in maximum bound counts resulted in little decrease in overall assay CV except when very low concentrations of analyte were being measured. Therefore, RIA and IRMA systems based in duplicate determinations having at least 10,000 maximum counts bound should have adequate precision, except possibly at very low concentrations.

Computer Simulation↗
PubMed1998

The suitability of five liquid scintillation cocktails for use with a continuous flow-through mucosal perfusion system.

Continuous flow-through perfusion systems can be successfully used to study the permeability of small mucosal samples to various permeants. The latter often need to be radiolabelled to enable detection by means of liquid scintillation counting. However, the efficacy of the latter is dependent on the liquid scintillation cocktail used. The present study was conducted to evaluate five liquid scintillation cocktails commercially available in South Africa. Although four out of the five cocktails had similar efficacies in the concentration range 0.05 to 50 nCi, this was not the case in the low range (0.01 to 0.5 nCi). Overall Ready Protein+ was the most efficacious and has the added advantage that this cocktail has protein solubilising properties. The latter is important when we extend our permeability studies to include small proteins.

Linear Models↗
PubMed1999

Biomedical applications of accelerator mass spectrometry-isotope measurements at the level of the atom.

Accelerator mass spectrometry (AMS) is a nuclear physics technique developed about twenty years ago, that uses the high energy (several MeV) of a tandem Van de Graaff accelerator to measure very small quantities of rare and long-lived isotopes. Elements that are of interest in biomedicine and environmental sciences can be measured, often to parts per quadrillion sensitivity, i.e. zeptomole to attomole levels (10(-21)-10(-18) mole) from milligram samples. This is several orders of magnitude lower than that achievable by conventional decay counting techniques, such as liquid scintillation counting (LSC). AMS was first applied to geochemical, climatological and archaeological areas, such as for radiocarbon dating (Shroud of Turin), but more recently this technology has been used for bioanalytical applications. In this sphere, most work has been conducted using aluminium, calcium and carbon isotopes. The latter is of special interest in drug metabolism studies, where a Phase 1 adsorption, distribution, metabolism and excretion (ADME) study can be conducted using only 10 nanoCurie (37 Bq or ca. 0.9 microSv) amounts or less of 14C-labelled drugs. In the UK, these amounts of radioactivity are below those necessary to request specific regulatory approval from the Department of Health's Administration of Radioactive Substances Advisory Committee (ARSAC), thus saving on valuable development time and resources. In addition, the disposal of these amounts is much less an environmental issue than that associated with microCurie quantities, which are currently used. Also, AMS should bring an opportunity to conduct "first into man" studies without the need for widespread use of animals. Centre for Biomedical Accelerator Mass Spectrometry (CBAMS) Ltd. is the first fully commercial company in the world to offer analytical services using AMS. With its high throughput and relatively low costs per sample analysis, AMS should be of great benefit to the pharmaceutical and biotechnology industries as well as other life science areas.

Biotechnology↗
PubMedDate not supplied

The development and evaluation of a 125I radioimmunoassay for the measurement of LSD in body fluids.

A radioimmunoassay (RIA) has been developed for the direct detection of LSD in biological fluids. The radiotracer, (+)-2-[125I]iodo-LSD, allows the use of gamma-counting rather than the liquid scintillation counting currently used for existing 3H radioimmunoassays. The assay is specific for LSD and very closely related compounds. It is inexpensive, sensitive, simple to use and small volumes of samples (50 microliter) can be assayed directly without the need for any time-consuming extraction procedures. The cut-off levels are 1.2 ng/ml in blood and 3.0 ng/ml in urine. The results obtained using the 125I assay described in this work compare very favourably with those obtained using the 3H assay currently used by Home Office Forensic Science Laboratories. The advantages of the assay make it a most appropriate method for the routine screening of LSD in biological samples of forensic interest.

Cross Reactions↗
PubMed2003

Effects of cerulenin on the endogenous fatty acid synthetic activity in squamous cell carcinoma of the oral cavity.

PURPOSES: Inhibition of cerulenin on the endogenous fatty acid synthetic activities of oral squamous cell carcinoma (OSCC) and normal oral mucosa was assayed. METHODS: Squamous cell carcinoma and normal oral mucosa were collected fresh from surgical specimens. The collected tissues were minced in RPMI 1640 and divided into 3 groups: cerulenin treated, dimethylsulfoxide treated, and control. The tissues were incubated in [1(2)-(14)C]acetic acid, sodium salt for the last 2.5 hours of the treatment at 37 degrees C in 5% CO(2). After labeling, total lipids were extracted and counted for (14)C by scintillation counting. RESULTS: Endogenous fatty acid synthetic activities of oral squamous cell caranoma in the cerulenin-treated group decreased by 19% at 1 hour, 64% at 2 hours, and 87% at 4 hours; remained nearly unchanged in the dimethylsulfoxide-treated group; and increased slightly in the control group. The oral mucosa tissues were only mildly affected by cerulenin in fatty acid synthesis. CONCLUSIONS: Cerulenin significantly inhibits fatty acid synthetic activity in squamous cell carcinoma and only mildly affected the oral mucosa, indicating that the fatty acid synthetic pathway may be exploited as a target for developing anticancer drugs.

Aged↗
PubMed1989

Biocompatibility of wound management products: standardization of and determination of cell growth rate in L929 fibroblast cultures.

To facilitate the development of a bioassay procedure by which the biocompatibilities of materials used in wound management may be assessed and compared, those environmental factors affecting cell growth in mouse L929 fibroblast cultures have been identified. Standardization of the initial cell number and frequency of change of medium resulted in the virtual elimination of variation of growth curves of L929 cells cultured in flasks of specified surface area. In addition, three methods for assessing fibroblast growth rate in the presence of alginate products used in wound management were evaluated. These were the haemacytometer counting chamber method, the Coulter counting method, and a liquid scintillation counting method. The first two methods determine the number of cells in a given volume of a cell suspension, whereas the third method determines the rate of synthesis of deoxyribonucleic acid (DNA), and hence cell growth, by measuring the incorporation of [3H]thymidine. The haemacytometer method had significant advantages over the other two procedures in providing both qualitative and quantitative data on culture morphology and cell growth response.

Alginates↗
PubMed1997

[Reproducibility of regional cerebral blood flow measurement at rest with autoradiography method using 123I-IMP and SPECT].

Reproducibility of regional cerebral blood flow (rCBF) values obtained by the autoradiography (ARG) method with one SPECT scan and one point arterial blood sampling at 10 min after 123I-N-isopropyl-p-iodoamphetamine (IMP) infusion was evaluated. The variation in the cross calibration factor between SPECT counts and well-type scintillation counts remained 2.2% for 13 months. The error in rCBF by sampling time difference was less than 3% when sampling time was within 10 min +/- 30 sec after infusion. There was a favorable correlation between the rCBF values in two separate resting scans measured by the ARG method in 10 patients (r = 0.907). And the mean ratio of rCBF values by the second study to those by the first study in the cerebellum and cerebral lobes in 10 patients was 0.989 +/- 0.056 (mean +/- SD). There was no significant difference in rCBF values between two separate studies. Reproducibility of rCBF with the ARG method was proved to be good. The ARG method was thought to be useful for the assessment of treatment effects and clinical courses.

Aged↗
PubMed2002

Chicken lines differ in production of interferon-like activity by peripheral white blood cells stimulated with phytohemagglutinin.

Interferon (INF) activity was evaluated in the supernatants from peripheral white blood cells (WBC) of chickens from six lines. The WBC were cultured in flasks or 24-well plates with medium or medium and phytohemagglutinin (PHA). After 2 to 5 d, duplicate supernatant samples were tested for INF activity, i.e., the log2 titer inhibiting 50% destruction of the cytopathic effect of vesicular stomatitis virus on primary chick embryo fibroblasts. Also triplicate WBC samples were tested for proliferation by [H3]-thymidine labeling and scintillation counting. In the absence of PHA, INF was significant for only two lines, i.e., 7(2) (two trials) and C (one trial). With PHA the level of INF produced was similar if flasks were sampled daily or on successive days. The INF levels were highest using 10 or 20 microg/mL PHA, but line differences were best distinguished using 5 or 10 microg/mL. In three trials there was a low correlation between PHA-stimulated WBC proliferation and INF titer (r > or = 0.30; P < 0.05). It was concluded that supernatants from chicken WBC stimulated with 10 microg/mL PHA contain INF, and inbred Lines 72 and C repeatedly produce more INF than inbred Lines 63 and 15I(5). This is the first evidence for line differences in INF production in chickens, and these lines may be useful for characterization of the relevant genes and their importance in immune response(s) and disease resistance.

Animals↗
PubMed2006

Metabolism of a 14C/3H-labeled GABAA receptor partial agonist in rat, dog and human liver microsomes: evaluation of a dual-radiolabel strategy.

The metabolism of 2-{[2-(3-fluoropyrid-2-yl)-1H-imidazol-1-yl]methyl}-1-propyl-5-cyano-1H-benzimidazole (1), a potent subtype-selective GABA(A) receptor partial agonist, was investigated in rat, dog and human liver microsomes. Due to its significant metabolic cleavage at C(8) observed in preliminary biotransformation studies with non-radiolabeled 1, both [(14)C]1 and [(3)H]1 were synthesized with respective radioisotopes placed on either side of C(8) to determine if all microsomal metabolites formed after C(8)N-dealkylation of 1 (or its core-intact metabolites) could be detected and quantified adequately. Both radiolabeled forms of 1, used separately in mono-radiolabel studies in cross-species microsomes and concomitantly in dual-radiolabel studies in rat microsomes, permitted the detection and quantification of all metabolites of 1, and a combination of radioactive and mass spectral data allowed structural elucidation of its Phase I metabolites. As expected, the sum of (14)C-only metabolites equaled that of (3)H-only metabolites in all incubations. In-line radiometric analysis worked extremely well (and was very reproducible) for quantifying either (14)C- or (3)H-compounds within separate incubations when using mono-radiolabeled 1. However, although the in-line radiodetector provided a comprehensive qualitative metabolic profile using dual-radiolabled 1, its inability to exclude completely (14)C- from (3)H-generated counts caused a degree of ambiguity pertaining to metabolite quantification. Thus, off-line liquid scintillation counting of collected dual-radiolabeled incubation LC-fractions was employed to quantify both (14)C- and (3)H-metabolites simultaneously, while in-line radiodetection was only used for qualitative analyses accompanying MS and MS/MS experiments. These studies demonstrated the analytical feasibility of using a dual-radiolabel approach for subsequent in vivo ADME studies with 1.

Animals↗
PubMed1996

Direct comparison of three methods for the determination of radon in well water.

Radon concentrations obtained using a bubbler device developed to collect and bubble water samples in the laboratory and field were compared with results from conventional liquid scintillation counting. Measurements from standard solutions with a wide concentration range showed excellent agreement between liquid scintillation and results obtained using the bubbler device in conjunction with alpha-scintillation cells. Measurements of waterborne radon concentrations in 110 community and private wells in New York State ranged from 1 to 4,100 Bq L-1, with arithmetic and geometric means of 200 and 30 Bq L-1, respectively. Excellent agreement between the analytical techniques was obtained for each field site.

Evaluation Studies as Topic↗
PubMed2001

In vitro adherence properties of Lactobacillus rhamnosus DR20 and Bifidobacterium lactis DR10 strains and their antagonistic activity against an enterotoxigenic Escherichia coli.

Adhesion and colonisation properties of three probiotic strains namely, Lactobacillus rhamnosus DR20, L. acidophilus HN017, and Bifidobacterium lactis DR10, were determined in vitro using the differentiated human intestinal cell-lines including HT-29, Caco-2, and HT29-MTX, and compared with properties of L. acidophilus LA-1 and L. rhamnosus GG (two commercial probiotic strains). Two independent methods were employed to quantitate the "adhesiveness" of each strain. In the first method, the bacteria adhered to human cells were detected by Gram staining and counted in different fields under a microscope. Bacteria were also radio-labelled and extent of adhesion determined by scintillation counting. All three strains showed strong adhesion with the human intestinal cell lines in vitro. Adhesion indices of the three strains to two cell lines, i.e. HT-29, and Caco-2 varied between 99 +/- 17 and 219 +/- 36. With mucus-secreting cell-line HT29-MTX, the adhesion indices of all the strains were 2-3 times higher. The adhesion indices of L. acidophilus LA-1 and L. rhamnosus GG were comparable to the other three probiotic strains. We also investigated the inhibitory effect of adhering strains against the intestinal cell monolayer colonization by a known enterotoxigenic strain of Escherichia coli (strain O157:H7). Pre-treatment of E. coli O157:H7 with 2.5-fold concentrated cell-free culture supernatants from L. acidophilus HN017, L. rhamnosus DR20 and B. lactis DR10 reduced the culturable E. coli numbers on TSB plates and also reduced the invasiveness and cell association characteristics of this toxic strain. The inhibitory molecules secreted into the spent media by these strains were partially affected by treatments with lactate dehydrogenase, trypsin and proteinase K suggesting that overall inhibition may be due to a synergistic action of lactic acid and proteinaceous substances.

Bacterial Adhesion↗
PubMed1990

Radiochromatography in pharmaceutical and biomedical analysis.

Recent advances in the chromatographic analysis of radiolabelled compounds are discussed, with emphasis on the optimization of radioactivity detection in modern chromatographic techniques. Applications are mentioned only infrequently. The state-of-the-art of common radioactivity detection methods is reviewed, as well as promising new detection principles. The latter include flow-through liquid scintillation counting of reversed-phase column eluates based on post-column extraction and solvent segmentation, flow-through heterogeneous counting using a flow cell constructed from axially aligned scintillation fibres, flow cells especially constructed for the detection of beta-emitting radioisotopes in capillary zone electrophoresis, position-sensitive analysers employed in radio planar chromatography and, finally 14C atom counting (as opposed to 14C decay counting) by accelerator mass spectrometry.

Autoradiography↗
PubMed1987

Radiometric assay of bacterial growth: analysis of factors determining system performance and optimization of assay technique.

A quantitative technique for the measurement of 14CO2 released from a bacterial culture was evaluated. The technique uses liquid scintillation counting to record 14CO2 accumulation on a fluor-impregnated filter paper within a double-chambered scintillation vial that also houses the bacterial growth medium. We have successfully identified and corrected the major causes for a variably low detection efficiency, and also established the optimum mixture of reagents for the detection system. Incorporation of Triton X-100 into the scintillation fluid used for the detector reduced the variability between identical assays in a single batch from 50% to 5%, and, in conjunction with an increase in the scintillator concentration, raised the counting efficiency from 30% to 70-88%. The response of the improved detector is linear over a wide range of count-rates. Another significant modification was the interchange of growth and detector chambers. Overall, a 40-fold increase in count-rate during the exponential phase of bacterial growth was obtained by improving 14CO2 detection efficiency, increasing the rate of 14CO2 transfer from liquid to gas phases and enlarging the growth supporting capacity of the detector system. The minimum detection time for bacterial growth was shortened and the exponential phase of bacterial proliferation was lengthened by at least 2 hr. High counting efficiency, precision, and linearity make the improved detector a sensitive and reliable tool for radiometry of bacterial growth and metabolism.

Bacteria↗
PubMed1977

Correlation of in vitro 32P counts with histologic features of malignant melanoma of choroid and ciliary body.

The radioactive phosphorus uptake of 30 samples fron nine uveal melanomas was determined by liquid scintillation counting. These in vitro measurements correlated positively with clinical (in vivo) 32P values and with the number of mitotic figures and percentage of epithelioid cells (PEC) within the specimens. Three possible explanations were found for the failure of previous workers to demonstrate a correlation between cell type and clinical 32P. (1) Cell type, using Callender's classification, provides only a crude estimate of the PEC within a tumor; in particular, tumors of mixed cell type can vary widely in epithelioid cell content. (2) The rate of 32P absorption is not su-ficiently sensitive to distinguish spindle cell tumors from mixed cell tumors with a low PEC. (3) Clinical 32P measurements may be affected by extraneous factors that cause large deviations from the expected values.

Choroid Neoplasms↗
PubMed1981

The potential laboratory health hazard of 210Pb and a simple procedure for separation of 210Pb from the daughters 210Bi and 210Po.

Lead 210 (Radium D) is a naturally occurring radionuclide which is frequently used in toxicological studies due to its long half-life. The use of 210Pb in tracer studies poses two problems. First, 210Pb, along with its daughters 210Bi and 210Po, presents a significant health hazard to laboratory personnel. Second, the presence of the daughter products may interfere with the detection of 210Pb, particularly by techniques which discriminate poorly between different radioactive emissions, e.g., autoradiography. The potential laboratory health hazards of 210Pb and its daughters are briefly reviewed and a simple dithiozone extraction procedure which allows quantitative separation of 210Pb frm the daughters 210Po and 210Bi is described. The purified 210Pb may then be utilized to reduce the health hazard from the daughter products and to construct calibration curves for the quantitation of 210Pb in the presence of 210Bi and 210Po by liquid scintillation counting.

Bismuth↗