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PubMed1983

Evaluation and interpretation of grain density autoradiographs by reflectance photometry.

The tritium content of nervous tissue sections is calculated from grain-density of autoradiograph without loss in anatomical information. The calculation is not based on determination of (expected) grain numbers but on photometric measurements (SKphot), interpreted as the result of the function of exposure time (T) and incorporated activity (beta): SKphot = f(T, beta). As experimental radiation standards homologous material (optic nerve preparations) with different activities determined by liquid scintillation counting has been used. Exposure times between 12 h and 28 weeks for these preparations with known radioactivities yielded a set of curves (nomogram) relating photometric values of exposure times. At any given exposure time the knowledge of photometric readings and related radioactivities within that set of curves enabled the development of a function covering all photometer readings within the range of the nomogram. Thus, any photometric reading could be correlated to the corresponding radioactivity. Such a function has been applied for the transformation of photometric values obtained from particular brain-section areas exposed for 16 weeks. Since the results are given in Bq, they may be related and, if necessary, corrected to results obtained by other tritium detection methods. Terminology used in the text: experimental radiation source = experimental standards prepared from optic nerve sections; standard radiation source = polymer reference source.

Animals↗
PubMed1996

Acute epithelial injury in the rat small intestine in vivo is associated with expanded expression of transforming growth factor alpha and beta.

BACKGROUND: Previous studies have shown the importance of transforming growth factors alpha and beta (TGF alpha and TGF beta) in modulating epithelial cell restitution after injury in vitro. AIM: To investigate the role of the growth factors TGF alpha and TGF beta after acute epithelial injury in vivo. METHODS: An in vivo model of phytohaemagglutinin (PHA) induced acute epithelial injury in rat small intestine was used. Epithelial cell turnover was assessed by autoradiography and liquid scintillation counting of thymidine uptake. Expression of TGF alpha and TGF beta was assessed by immunohistochemistry. RESULTS: An expansion of the proliferative compartment and increased turnover of intestinal epithelial cells was seen in rats with PHA induced intestinal epithelial injury. Expression of TGF alpha and TGF beta peptides was shown in both the epithelial cell and lamina propria compartment. Different patterns of TGF alpha and TGF beta expression were seen, however, within the epithelium of rats with acute intestinal injury compared with untreated controls, while the expression of these peptides within the lamina propria was not changed. CONCLUSIONS: These findings suggest that acute intestinal epithelial cell injury in vivo is associated with compensatory changes in expression of TGF alpha and TGF beta in the epithelial cell compartment, while the lamina propria does not seem to be significantly affected.

Animals↗
PubMed2006

Method to assess fatty acid ethyl ester binding to albumin.

AIMS: To develop a method to assess the relative binding of fatty acid ethyl esters (FAEE) and free fatty acids (FFA) to albumin, and to determine if binding affinity is related to fatty acid chain length and or degree of saturation. METHODS: Radiolabelled ethyl-[14C] oleate bound to albumin was challenged with various ratios of FFA to FAEE. The displacement of ethyl-[14C] oleate by FFA was visualized and quantitated through a combination of native-PAGE, autoradiography, and liquid scintillation counting (LSC). RESULTS: As the ratio of FFA to FAEE increased from 0:1 to 12:1, for all fatty acids tested (myristate, palmitate, stearate, oleate, linoleate, and arachidonate), ethyl-[14C] oleate displacement increased, when expressed as radioactivity (in DPM) as a percentage of control. In contrast, ethyl oleate did not displace stearate or oleate from albumin at molar ratios up to 5:1 (FAEE:FFA). CONCLUSIONS: The method developed gave reproducible visualization of noncovalent binding of radiolabelled FAEE to albumin. The combination of native-PAGE and autoradiography LSC works well in assessing the binding properties of albumin and radiolabelled FAEE. The data indicate preferential binding of FFA over FAEE to albumin with six different FFA displacing FAEE to an approximately equal extent.

Albumins↗
PubMed1991

Report of a comparative study of DNA damage and repair assays in primary rat hepatocytes with five coded chemicals.

We report the results of a collaborative study for the detection of chemical-induced DNA damage in primary cultures of rat hepatocytes. The methods include the detection of unscheduled DNA synthesis (UDS) with either autoradiography (5 laboratories) or liquid scintillation counting (2 laboratories) and the assessment of DNA single-strand breaks with the alkaline elution assay (1 laboratory). Interlaboratory standardization was omitted in order to prove the agreement of the assays under routine conditions. Five coded chemicals were tested. For 4 chemicals (2-acetylaminofluorene, thiourea, glycerine and potassium chloride) the UDS data were consistent in all laboratories, thus indicating a high consensus of the test systems applied in the different laboratories. Those 3 chemicals that were not expected to elicit genotoxic activity (thiourea, glycerine, and potassium chloride) yielded negative results in all laboratories. 2-Acetylaminofluorene, a known DNA-damaging agent in hepatocytes, gave strongly positive responses in all laboratories. In contrast, N-nitrosodiphenylamine led to equivocal responses.

2-Acetylaminofluorene↗
PubMed1996

A procedure to study unscheduled DNA synthesis in rat spermatogenic cells in vivo: strain differences between Fisher-344 and Sprague-Dawley rats.

A liquid scintillation counting (LSC) procedure was used to study unscheduled DNA synthesis (UDS) in pachytene spermatocytes and spermatids from Fisher-344 (F344) and Sprague-Dawley (SPD) rats treated with methyl methanesulfonate (MMS). MMS induced a large, dose-dependent, UDS response in pachytene spermatocytes from both rat strains. On average, F344 pachytene spermatocytes showed a larger UDS response than those of SPD rats. The lowest dose of MMS that elicited a significant UDS response was 1 mg/kg in F344 rats but 5 mg/kg in SPD rats. Early spermatid stages from F344 rats also showed a larger UDS response than those from SPD rats. The time interval at which spermatid stages showed the maximum UDS response was between 20 and 24 days after MMS treatment. It is concluded that UDS can be measured quantitatively in rat spermatogenic cells in vivo by using the LSC procedure.

Animals↗
PubMed2002

Negligible depletion solid-phase microextraction with radiolabeled analytes to study free concentrations and protein binding: an example with [3H]estradiol.

A new method is presented that enables sensitive measurement of free concentrations of radiolabeled ligands. Additionally, protein binding of radiochemicals in complex matrixes can be determined with this new technique that combines negligible depletion solid-phase microextraction (nd-SPME) with liquid scintillation counting (LSC) as detection. [3H]Estradiol was taken as an example compound. Possible matrix effects of protein on fiber uptake kinetics were studied. No matrix effect was found, either by fouling of the fiber, or by changed uptake kinetics. The validity of the method was shown in the determination of the affinity constant (Ka) of estradiol for human serum albumin (HSA). The Ka was estimated at 8.9 x 10(4) M(-1), which corresponds well with literature values. This study shows that nd-SPME is suitable to study the free concentration and protein binding of [3H]estradiol. The method described in this paper combines the advantages of nd-SPME with the advantages of radiolabeled analytes, creating a timesaving, simple, and sensitive analytical tool that will be particularly useful in complex matrixes containing many potential interferences for chromatographic methods.

Estradiol↗
PubMedDate not supplied

Pharmacokinetics and biodistribution of a monoclonal antibody to Cryptococcus neoformans capsular polysaccharide antigen in a rat model of cryptococcal meningitis: implications for passive immunotherapy.

Several investigators have developed monoclonal antibodies against the capsular polysaccharide of Cryptococcus neoformans which have potential therapeutic applications. Using a rat model of C. neoformans meningitis, we studied the biodistribution and pharmacokinetics of a murine anticryptococcal capsular monoclonal antibody (mAb 2H1) after intravenous and intracisternal administration. After intravenous administration of 125I-labelled 2H1 to infected rats, there was no detectable localization of 125I in the brain or cerebrospinal fluid by either gamma-camera imaging of the whole animal or organ scintillation counting. In contrast, direct intracisternal instillation of 2H1 to infected rats resulted in persistent intracranial activity. In addition, the whole body half-life of intravenously administered radio labelled mAb 2H1 was significantly reduced in infected rats compared with uninfected rats. Our observations suggest that if high central nervous system (CNS) levels of mAb are needed to achieve a therapeutic effect in human C. neoformans meningoencephalitis, direct administration of mAb into the cerebrospinal fluid or modification of the mAb to increase penetration into the CNS may be required. Furthermore, higher or more frequent dosing of mAb may be required to maintain therapeutic levels in the presence of infection. This study demonstrates the usefulness of the rat as an experimental system for studying issues related to cryptococcosis.

Animals↗
PubMedDate not supplied

Demonstration of the deposition and modification of dietary fatty acids in pinniped blubber using radiolabelled precursors.

Radioisotopes are commonly used to study the in vivo metabolism and deposition of dietary fatty acids in adipose tissue. The application of this approach to pinnipeds is problematic because of their large mass and blubber fat content. We have developed a method where labelled lipids can be fed to seals at financially feasible levels, with the radioactivity in individual fatty acids isolated from blubber detected with standard laboratory equipment. A combination of techniques including argentation thin layer chromatography, high performance liquid chromatography with ultraviolet detection, and independent liquid scintillation counting were employed. Juvenile gray seals (Halichoerus grypus) were fed either 0.5 mCi (3)H-labelled triolein (18:1n-9, n=2) or palmitic acid (16:0, n=2). Blubber samples were taken 12 h later, and the radioactivity in individual fatty acids was determined. Radioactivity was detected in only 18:1 from the animals fed (3)H-labelled triolein, indicating direct deposition without modification. Both animals fed (3)H-labelled palmitic acid showed clear peaks of radioactivity in 16:0; however, there was also significant activity (23%-29%) found in the desaturation product 16:1. Our results demonstrate that this method is sufficiently sensitive to track the deposition of labelled dietary lipids as well as modification products of ingested fatty acids and will be important in the application of fatty acid signatures to study predator diets.

Adipose Tissue↗
PubMed1993

Application of oxime formation in a radiometric assay of aminooxy compounds.

A radiometric determination of monoaminooxy analogues of naturally occurring polyamines is described in which [2-14C]acetone is employed as reagent. The reagent is volatile while the oxime product is not allowing unreacted reagent to be removed and the oxime formation to be completed by lyophilization in vacuo. The residual radioactive compound is soluble in water and proportional to the reactive aminooxy content of the reaction mixture and can be quantified by liquid scintillation counting. The assay method is inexpensive and simple and has high specificity and flexibility in sample volume enabling reliable quantification of reactive aminooxy amines in biological extracts at concentrations exceeding 0.25 microM. Optimal pH values for oxime formation of five monoaminooxy analogues of polyamines with acetone were resolved. Reactions via reversible intermediates to irreversible oximes were sped up by removal of water. Complete oxime formation and stability was confirmed by 1H NMR studies. Tested drugs readily formed oximes with pyridoxal 5'-phosphate, too. Diaminooxy analogue of cadaverine formed a volatile oxime with acetone. The method was used to monitor the stability of aminooxy analogues of putrescine and spermidine during storage and under culture conditions and to establish their scant accumulation in, but fast catabolism by, cultured baby hamster kidney cells.

Amines↗
PubMed2001

[Radon 222 in drinking water from Jelenia Gora area].

In the last decade one can observe an increasing interest in the study of 222Rn levels in water samples. In Poland, radon concentration is measured continuously and routinely only in mineral and medical waters from springs located in the area of health resorts in the Sudety Mountains. The reason for these studies is the fact that waters present in this area have a high radon concentration. Radon--222 concentration in surface water, wells water and tap water in Jelenia Góra has been quantitative determined. The measurements were performed using the alpha liquid scintillation counting method. "Grabarów", the main waterworks in Jelenia Góra is supplied with the mixed water consisting from the surface water (river Bóbr), which main characteristic is low radon concentration (below 11 Bq/l), and from ground water with the radon concentration from 179.6 Bq/l to 289.0 Bq/l in it. Also, waterworks "Ceglana", "Pod Karpaczem", "Sniezne Kotły" is supplied with the ground water have a high radon concentration: from 93.2 Bq/l to 216.4 Bq/l. The next waterworks: "Podgórzyn", "Leśniczówka", "Kamienna Wieza", "Górzyniec" and "Centrum-Jelchem" is supplied with the surface water in which the radon concentration is low: from 1.1 Bq/l to 7.5 Bq/l. The annual effective dose to an individual from an intake of radon-222 via ingestion of drinking water is calculated by the Crawford-Brown's biokinetic model. In the present study it was found to be about 0.9 mSv/y (222Rn concentration level in drinking waters about 200 Bq/l).

Environmental Monitoring↗
PubMed1988

Disposition of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice before and after monoamine oxidase and catecholamine reuptake inhibition.

The distribution of radioactivity in pigmented mice after a single intravenous injection of 1-(3H) methyl-4-phenyl-1,2,3,6-tetrahydropyridine (3H-MPTP) was studied by quantitative whole body autoradiography and liquid scintillation counting. Pretreatment with the monoamine oxidase inhibitors clorgyline, pargyline and deprenyl, or the catecholamine reuptake inhibitor nomifensine was performed 30 min. prior to the 3H-MPTP administration. A high uptake of radioactivity was observed in the striatum, nucleus accumbens, midbrain area and locus coeruleus, and also in the adrenal medulla. This uptake was inhibited by deprenyl or pargyline pretreatment, but not after clorgyline or nomifensine pretreatment. An extensive uptake which was not influenced by deprenyl or pargyline treatment was found in the melanin-containing tissues of the eye. This accumulation is due to the melanin affinity of MPTP and its metabolites. A comparatively rapid elimination from the brain of MPTP and its metabolites was observed, which may be due to the lack of neuromelanin in mice.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1984

Estimation of assimilation of simultaneously ingested 14C-triolein and 3H-oleic acid as a test of pancreatic digestive function.

The ratio between assimilation of simultaneously ingested 14C-triolein and 3H-oleic acid was investigated as a test of pancreatic digestive function in 48 consecutive patients suspected of malassimilation. Faecal excretion of 14C-triolein and 3H-oleic acid was measured by means of a combustion/liquid scintillation counting technique. Compared with the patients in whom the clinical investigation showed normal lipid assimilation (no. = 31) the assimilation of both 14C-triolein and 3H-oleic acid was significantly reduced in the patients with malabsorption (no. = 6) and maldigestion (no. = 11). However, in all except one patient with maldigestion the assimilation of 14C-triolein was more reduced than that of 3H-oleic acid, whereas there was no difference in 14C-triolein and 3H-oleic acid assimilation in the patients with malabsorption and normal lipid assimilation. Measurement of the postprandial serum radioactivities of 14C and 3H, like the faecal measurements, indicated more severely reduced assimilation of 14C-triolein than of 3H-oleic acid in the patients with maldigestion than in the other patient groups. In fact, the 2-h postprandial 3H/14C ratio at a level of 1.3 correctly classified digestive function in 47 of the 48 patients.

Digestion↗
PubMed1998

Surfactant versus saline as a vehicle for corticosteroid delivery to the lungs of ventilated rabbits.

Local administration of steroids to the lungs in ventilated newborn infants can minimize the harmful side effects that occur with systemic administration. An efficient system of drug delivery that provides uniform distribution within the lungs is essential for the treatment of bronchopulmonary dysplasia. In this study we compare surfactant with 0.9% saline solution as vehicles for the direct instillation of a steroid (budesonide) into the lungs. Twenty-two anesthetized, ventilated rabbits received [3H]budesonide in either 0.9% saline or surfactant, administered through an endotracheal tube. Before drug administration, bronchial lavage was performed on half of the animals to serve as a model for surfactant deficiency. Lung samples were analyzed by both autoradiography (alveolar versus airway distribution) and liquid scintillation counting (central versus peripheral deposition). As expected, the delivered concentration of [3H]budesonide decreased as airway size decreased and branching increased. Significantly less [3H]budesonide was deposited in the alveolar spaces of all study groups compared with that deposited in the small and large airways (p < 0.05). However, both vehicles were equally efficient in delivering [3H]budesonide to the lungs. Although the alveolar and peripheral areas received less (4-11%) of the drug than the central tissue (14-28%), this was consistent among all the groups and was not affected by altered lung compliance. Therefore, either surfactant or saline could be used to efficiently and reliably deliver budesonide to the lungs at a level greater than that reported using nebulizers. Because normal saline is currently used for tracheal toilette, it is likely to be preferred considering the increased costs associated with surfactant.

Adrenal Cortex Hormones↗
PubMed1994

Retinoid processing in retinal pigment epithelium of toad (Bufo marinus).

The formation of 11-cis-[3H]retinal in the retinal pigment epithelium (RPE) and its release to extracellular medium containing interphotoreceptor retinoid-binding protein (IRBP) were studied in the RPE eyecup of the toad (Bufo marinus). The RPE was labeled with all-trans-[3H]retinol during an initial 1-h incubation. In phase 2 of the incubation (0-2 h), the extracellular medium contained initially ligand-free IRBP (0-26 microM). Retinoids subsequently extracted from the extracellular medium and RPE were analyzed by high performance liquid chromatography and scintillation counting. IRBP increased both the molar amount and specific radioactivity of 11-cis-retinal released by the RPE during phase 2. The molar amount of 11-cis-retinal in the RPE was small relative to that of retinal released with high IRBP. With 21 microM IRBP and phase 2 incubations of > or = 10 min, the specific radioactivity of released 11-cis-retinal exceeded that of all-trans-retinyl ester in the RPE. The specific radioactivity of 11-cis-retinyl ester was less than that of all-trans-ester, independent of IRBP concentration. The results indicate that IRBP promotes the formation (from all-trans-precursor) as well as the release of 11-cis-retinal and suggest the preferred utilization of recently incorporated and esterified all-trans-retinol in 11-cis-retinal synthesis in a "last in/first out" manner.

Animals↗
PubMed1999

A new experimental determination of the dose calibrator setting for 188Re.

UNLABELLED: Accurate activity measurements of radionuclides using commercial dose calibrators requires that the correct dial setting (or calibration factor) be applied. The dose calibrator setting for the medical radionuclide 188Re (as 188ReO4-) has been determined experimentally using solution sources prepared and calibrated at the National Institute of Standards and Technology (NIST). METHODS: The specific activity of two sources (in units of MBq/g) in the standard 5-mL NIST ampoule and in a 5-mL SoloPak dose vial were calibrated using 4pibeta liquid scintillation counting with 3H-standard efficiency tracing and gamma-ray/bremmstrahlung counting in the NIST "4pi" gamma ionization chamber on gravimetrically related sources. RESULTS: The newly determined settings for the NIST Capintec CRC-12 dose calibrator are (631+/-4) x 10 and (621+/-3) x 10 for the respective ampoule and dose vial geometries with an expanded (at a presumed 95% confidence level) uncertainty of 0.4%-0.5% in the activity determination. The setting for the dose vial geometry was independently confirmed using a Capintec CRC-15R at Cedars-Sinai Medical Center using sources calibrated against a NIST standard. CONCLUSION: These new settings result in activity readings 28%-30% lower than those obtained using the previously recommended setting of 496 x 10. This discrepancy most likely results from underestimating the total radiation yield from 188Re decay when calculating the dose calibrator response. This study emphasizes the need for experimental determinations of dose calibrator settings in the geometry in which the measurements will be performed.

Calibration↗
PubMed2000

Suppression of inositol phosphate release by cardiac myocytes isolated from fish oil-fed pigs.

Fatty acid composition of cardiac myocytes and release of inositol phosphates in pigs fed a fish oil supplemented diet was examined. Two groups of female pigs were fed diets supplemented with either 50 g/kg diet beef tallow (as control) or 50 g/kg diet fish oil (MaxEPA) rich in n-3 fatty acids. After 6 weeks of supplementation, the pigs were anesthetized and hearts were removed. Cardiac myocytes were isolated, lipid extracted and separated into non-polar and polar lipids by thin-layer chromatography. Fatty acid composition of individual neutral and polar lipid classes were examined by gas chromatography. To study the effect of membrane phospholipid modification on the phospholipase C (PLC) mediated release of inositol phosphates, cardiac myocytes were labelled with 4 microCi/mL myo-[2-(3)H]inositol for 48 h. After stimulation with epinephrine and phenylephrine, the water soluble [3H]inositol products were extracted, separated from [3H]inositol and [3H]glycerophosphoinositol by chromatography on Dowex AG 1-X8 and quantitated by scintillation counting. Cardiac myocytes isolated from fish oil-fed pigs had higher levels of n-3 polyunsaturated fatty acid in the non-esterified fatty acid and phospholipid fraction. Similarly, these cardiac myocytes had increased level of n-3 fatty and decreased n-6 fatty acids in all the phospholipid fractions, PE, PC, P1 and PS (p < 0.05). After stimulation, the levels of [3H]inositol trisphosphate (IP3) and [3H]inositol tetrakisphosphate (IP4) in cardiac myocytes isolated from fish oil-fed pigs were significantly reduced (p < 0.05) compared to myocytes isolated from beef tallow fed-pigs. This study for the first time has utilised adult cardiac myocytes to demonstrate the effect of n-3 PUFA supplementation on cardiac myocyte phospholipid fatty acid composition and release of second messengers.

Animals↗
PubMed1989

Temporal and topographic changes in DNA synthesis after induced follicular atresia.

Hamsters were hypophysectomized on the morning of estrus (Day 1) and injected immediately with 30 IU pregnant mare's serum (PMS). This was followed on Day 4 by the injection of an antiserum to PMS (PMS-AS) that initiated follicular atresia (Time zero). From 0 to 72 h after PMS-AS, the animals were injected with [3H]thymidine and killed 4 h later. One ovary was saved for autoradiography and histology; from the other ovary, 5-10 large antral follicles were dissected and pooled, and incorporation into DNA was determined by scintillation counting. DNA synthesis dropped sharply between 12 and 18 h, coinciding with a fall in labeling index of the cumulus oophorus and thecal endothelial cells and a sharp fall in thecal vascularity. In contrast, for the mural granulosa cells bordering on the antral cavity, labeling index dropped sharply between 8 and 12 h when thecal vascularity was still high. The earliest sign of atresia was evident by 4 h in cumulus cells when, paradoxically, DNA synthesis was still high. It took 3 days for atresia of the antral follicles to progress to advanced stages, as evidenced by pseudo-pronuclei in the free floating ovum, further erosion of the mural granulosa, and minimal DNA/follicle. However, the theca still retained its histological integrity and contained no pyknotic cells. Although by 48 h the granulosal compartment was in disarray (DNA/follicle significantly different from earlier values), the egg was still viable, as judged by maximal fluorescence after the addition of fluoroscein diacetate.

Animals↗
PubMed1983

Pharmacokinetics of [3H]levamisole in pigs after oral and intramuscular administration.

A single oral (10 mg/kg of body weight) or IM (7.5 mg/kg) dose of [3H]levamisole was administered to pigs. Liquid scintillation counting and high performance liquid chromatography were used to determine total radioactivity and drug levels in plasma, duodenal and cecal contents, bile, and urine for 24 and 72 hours after dosing. Pharmacokinetic analysis indicated a 1-compartment open model with higher plasma bioavailability of levamisole after IM injection. Biological half-lives for elimination of the drug were 9.3 and 6.9 hours after oral and IM administration, respectively. Anthelmintic concentrations were higher in intestinal contents after oral gavage than after IM injection. The drug appeared extensively metabolized in all body fluids and particularly in bile, regardless of the route of administration. Biliary excretion of radioactivity and unchanged levamisole represented only slight percentages of the administered dose (approx 0.4% and 4.2%, respectively, in 72 hours). In contrast, about 60% and 20% of the dose were eliminated via urine as tritiated materials and unchanged drug. The choice of the most efficacious route of administration is discussed in regard to localization of helminthic disease.

Administration, Oral↗