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In vivo-synthesized radioactively labelled alpha-methyl serotonin as a selective tracer for visualization of brain serotonin neurons.

To investigate the use of alpha-[3H]methyl tryptophan (alpha-[3H]MTrp) as a tracer for the in vivo study of brain serotonergic neurons, we examined whether alpha-[3H]MTrp and its metabolite alpha-[3H]methyl serotonin (alpha-[3H]M5-HT) selectively label serotonergic neurons and whether once accumulated in these neurons, the radioactive metabolite behaves like endogenous serotonin. Rats received a systemic injection of 1-5 mCi of alpha-[3H]MTrp and 24 h later their brains were immediately removed or fixed by perfusion before removal. Tissue sections in which serotonergic neurons had been immunostained for 5-HT or its synthesizing enzyme, tryptophan hydroxylase, were processed for radioautography at the light and electron microscopic level. In another group of rats, the release of radioactivity from different brain areas was studied both under basal and depolarizing conditions. In the dorsal raphe nucleus, the light microscopic examination revealed almost complete colocalization between serotonergic neurons and those that accumulated radioactivity, with a heterogeneity in the content of alpha-[3H]M5-HT among the various cells. At the ultrastructural level, immunoidentified serotonergic perikarya and dendritic processes in the dorsal raphe nucleus, as well as nerve terminals in the cerebral cortex were also found to contain alpha-[3H]M5-HT. Under basal conditions, radioactivity was released from the brainstem raphe region and from projection areas such as the striatum and hippocampus. The basal output of alpha-[3H]M5-HT increased approximately twofold after a depolarizing 50 mM KCl solution was added to the perfusion fluid. These findings suggest that newly synthesized alpha-[3H]M5-HT can be released both at somatodendritic and terminal sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Fourier processing of liquid chromatograms using flow radioactive detection.

Flow radioactivity counters coupled to liquid chromatography devices cause a systematic bias to the separation by broadening peaks within the radiochromatogram. Such signal smearing may be evaluated on standardization runs with a single peak, using the ratio between Fourier transforms of whole chromatographic data for the measured radioactivity time series (radioactivity channel) and for the concentration time series (optical density channel). This ratio constitutes a kernel suitable to perform the deconvolution of any radiochromatogram performed under similar conditions. Through deconvolution, the signal smearing is removed, reverting to peaks with the same geometry as in the concentration chromatogram: same retention time, peak width, and shape. The deconvolution method in processing radiochromatograms allows an easier interpretation and gives more reliable radioactivity quantification (improved linearity of the measured response). Fourier transformation of the radiochromatogram also allows the removal of transitory events (noise) through the correlation procedure. This method may provide substantial gain in sensitivity, depending upon the residence time of the sample in the counting cell.

Chromatography, Liquid↗

Determination of the specific radioactivity of [14C]lactate by enzymatic decarboxylation and 14CO2 collection.

We present an enzymatic method for the determination of L-[14C]lactate specific radioactivity in complex biological samples containing other radiolabeled compounds. The method is based on the conversion of L-lactate to L-pyruvate by lactate oxidase (no EC number assigned) and the decarboxylation of L-pyruvate by pyruvate oxidase (EC 1.2.3.3). The 14CO2 produced by the enzymatic decarboxylation of pyruvate is quantitatively captured in a CO2 trap and its radioactivity is measured. The method is simple, specific, and precise (2% relative SD). It can be conveniently used for routine multiple determinations of L-[14C]lactate specific radioactivity in tracer metabolic studies. Under specified conditions, the method can also be used to determine the specific radioactivity of L-[14C]pyruvate.

Animals↗

A radioactive tracer technique to determine in vivo the number of fibers in the lungs of rats following their administration by intratracheal instillation.

A radioactive tracer technique is described which enables the total number of fibers present in the lungs of rats to be estimated following administration of the fibers by intratracheal instillation. The glass fiber used in the study was irradiated with thermal neutrons to induce radioactive 24Na. A suspension of the radioactive fiber was administered to eight rats by intratracheal instillation and to two additional rats by intraesophageal instillation. The 24Na radioactivity in the rats was counted in vivo at 24 and 48 hr after administration, after which they were killed. The amounts of fiber in the lungs, in the gastrointestinal tracts, and excreted in feces were estimated radiometrically. On average 93% of the administered fiber was accounted for. The lungs were digested with sodium hypochlorite solution and aliquots of the resulting digest filtered through membrane filters which were clear for examination by phase-contrast optical microscopy (PCOM). The numbers of fibers in the lungs, estimated by PCOM, were well correlated with the in vivo counting rates at 48 hr, indicating that the latter can be used to provide an accurate index of the number of fibers retained in the lung at that time.

Administration, Inhalation↗

Detection of microsatellite instability in human colorectal carcinomas using a non-radioactive PCR-based screening technique.

The aim of the present study was to establish a rapid, non-radioactive screening method for the detection of microsatellite instability (MIN). MIN is the primary characteristic of the mutator phenotype in tumours constituting hereditary non-polyposis colon cancers (HNPCC). We investigated 30 patients suffering from colorectal cancer using a non-radioactive PCR-based technique. MIN was present in 7 of 30 (23%) of the cases. There was a statistically significant correlation between MIN and localization of the tumour. Five of 7 (72%) tumours with MIN but only 4 of 23 (17%) tumours without MIN were localized in the proximal colon (P < 0.01). There was a tendency to higher MIN frequency in tumours of patients with familial clustering of cancers. However, this was statistically not significant (P > 0.05). In addition, no correlation between MIN and tumour grade and stage was found. For the investigations in the present study we used a non-radioactive PCR-based method followed by denaturating polyacrylamide gel electrophoresis and silver staining. This method is highly sensitive and reproducible. Thus, PCR-based analysis using a non-radioactive staining technique represents a comprehensive tool for MIN screening in diagnostic pathology.

Adult↗

Direct molecular analysis of the fragile X syndrome in a sample of Egyptian and German patients using non-radioactive PCR and Southern blot followed by chemiluminescent detection.

Molecular genetic analysis of individuals from 6 Egyptian and 33 German families with fragile X syndrome and 240 further patients with mental retardation was performed applying a completely non-radioactive system. The aim of our study was the development of a non-radioactive detection method and its implementation in molecular diagnosis of the fragile X syndrome. Furthermore, we wanted to assess differences in the mutation sizes between Egyptian and German patients and between Egyptian and German carriers of a premutation. Using non-radioactive polymerase chain reaction (PCR), agarose gel electrophoresis and blotting of the PCR products, followed by hybridisation with a digoxigenin-labelled oligonucleotide probe (CGG)5 and chemiluminescent detection, we identified the fragile X full mutation (amplification of a CGG repeat in the FMR-1 gene ranging from several hundred to several thousand repeat units) in all patients. We observed no differences in the length of the CGG repeat between the Egyptian and German patients and carriers, respectively. However, in one prenatal diagnosis, we detected only one normal sized allele in a female fetus using the PCR-agarose assay, whereas Southern blot analysis with the digoxigenin labelled probe StB 12.3 revealed presence of a full mutation. Our newly established nonradioactive genomic blotting method is based on the conventional radioactive Southern blot analysis. Labelling of the probe StB 12.3 with digoxigenin via PCR allowed the detection of normal, premutated and fully mutated alleles. For exact sizing of small premutated or large normal alleles, we separated digoxigenin labelled PCR products through denaturing polyacrylamide gelelectrophoresis (PAGE) and transfered them to a nylon membrane using a gel dryer. The blotted PCR-fragments can easily be detected with alkaline phosphate-labelled anti-digoxigenin antibody. The number of trinucleotide repeat units can be determined by scoring the detected bands against a digoxigenated M13 sequencing ladder. Our newly developed digoxigenin/chemiluminescence approach using PCR and Southern blot analysis provides reliable results for routine detection of full fragile X mutations and premutations.

Base Sequence↗

Pharmacologic studies with radiolabeled N-trifluoroacetyladriamycin-14-valerate (AD 32). Comparison of total anthracycline fluorescence and radioactivity in mouse serum and urine.

In connection with pharmacologic studies with AD 32, isotopically-labeled drug prepared from 1-[14C]-trifluoroacetic anhydride and adriamycin-14-valerate was used to determine murine serum and urine levels of radioactivity. Other studies, performed in parallel, measured serum and urinary total fluorescence. Serum fluorescence disappeared in a biphasic pattern, with an initial rapid rate of disappearance followed by a somewhat slower phase. For the first hour, serum radioactivity levels were not significantly different than those measured by fluorescence. After this, however, serum radioactivity decayed at a much slower rate than did fluorescence. Furthermore, a large fraction of the injected radioactivity was found excreted in the urine, whereas urine accounted for only a small fraction of the fluorescence. These results suggest the formation, in part, of a hitherto unrecognized nonfluorescent metabolite, most probably N-trifluoracetyldaunosamine.

Animals↗

Tissue distribution of 14C-heteronium bromide: radioactivity levels at different time intervals after oral administration in the rat.

The tissue distribution of radioactivity after oral administration to rats of 14C-heteronium bromide is measured by liquid scintillation counting and the results expressed as specific activity and percentage of administered radioactivity. From the data obtained in blood, liver, kidney. stomach, duodenum, cecum, large intestine and stool some conclusions can be drawn. Heteronium bromide undergoes a rapid systemic absorption, the radioactivity being present as early as 15 min from the administration, in all the tested organs. The blood levels show two peaks: one at 120 min and a second at 360 min. This diphasic behaviour can be explained either by the presence of an active enterohepatic circulation, as indirectly indicated by the data from liver and duodenum, or by a transient shift of the molecule from blood to other tissues, rich in polysulfuronic acids. The principal route of excretion is represented by the kidney, where consistent levels are reached at 120 min, while the intestinal route becomes evident at 240 min and reaches its maximum at 720 min. The complete metabolic cycle of the compound is long lasting, since in all the tested tissues, marked radioactivity levels are still present after 720 min. The pharmacokinetic profile obtained, suggesting a long persistence of the drug and/or of its metabolites in the organism, is in agreement with previous pharmacodynamic data showing a long lasting action for heteronium bromide.

Administration, Oral↗

Localization of radioactivity in rat organs after oral administration of tritiated 8-methoxypsoralen in therapeutic doses.

Tritiated 8-methoxypsoralen was given perorally to rats in amounts corresponding to therapeutic human doses. The rats were exposed to UVA light or kept in darkness. None of the fractions (apart from 3H2O from the lens) examined changed their level of radioactivity under the influence of UVA light. Time-radioactivity curves were recorded for the skin, lens, residual eye, and the liver. Four fractions were measured: 3H2O, soluble pool, DNA-RNA, and protein. Tritiated water appeared already 1 h after ingestion, and attained maximum value 9-24 h after ingestion, indicating the efficiency with which the liver degrades 8-MOP. 3H-8-MOP and metabolites could be detected in the soluble pool in maximum amounts 2-3 h after the administration. Pretreatment with trypsin increased the concentration of 3H-8-MOP and metabolites; the origin of this extra radioactivity was the protein fraction. The 3H-8-MOP binding to DNA or RNA was studied by pretreatments of the homogenates with DNase or RNase followed by measurement of radioactivity in the TCA extracts. This indicated that no measurable amount of 3H-8-MOP had been bound to DNA or RNA. We conclude that 8-MOP administered to rats in amounts corresponding to human therapeutic doses does not bind to DNA or RNA in measurable amounts either after UV-light or in darkness. The experiments have shown proteins to be the main binding site in rat organs.

Administration, Oral↗

The effect of cocaine on the distribution of labelled noradrenaline in rabbit aortic strips and on efflux of radioactivity from the strips.

Reserpine-pretreated or normal rabbit aortic strips (whose noradrenaline-metabolizing enzymes were inhibited by treatment with 0.5 mM pargyline and by the presence of 0.1 mM U-0521) were incubated with 1.18 muM labelled noradrenaline for 30 min. Subsequently, strips were either analysed or washed out with amine-free solution for 240 to 250 min. From the efflux curves the original distribution of radioactivity was estimated by compartmental analysis. 1. The accumulation of radioactivity in the strips was reduced by 30 muM cocaine to that observed for nerve-free strips. In reserpine-pretreated strips this was due to a partial reduction of the filling of comartment IV (characterized by a half time of efflux of 10 to 20 min; earlier evidence had already shown that part of this compartment is of neuronal origin; Hensling et al., 1976) and a pronounced decrease of the filling of compartment V (characterized by the longest half time). In normal strips the same changes were observed as well as pronounced reduction of the "bound fraction". Thus, cocaine reduced the filing of those compartments which had been identified as neuronal ones (Henseling et al., 1976). 2. When 30 muM cocaine was added to the wash out solution only, the neuronal efflux of radioactivity from reserpine-pretreated strips was accelerated, while the efflux from extraneuronal or extracellular compartments remained unaffected. This effect of cocaine was the same for (-)- and (+)noradrenaline, and its was not significaltly decreased when paired strips exposed to an inhibitor of extraneuronal uptake (86 muM corticosterone) throughout the experiment. In normal strips, cocaine had very little or no effect on the efflux of radioactivity. 3. The results are consistent with the view that cocaine impairs the influx of amine into the neurone, while block of re-uptake of unchanged amine is one of the determinants of its effect on the rate of efflux from the axoplasm. Comparison of efflux curves with corresponding relaxation curves (determined in the absence and presence of cocaine) indicates that, when both noradrenaline-metabolizing enzymes are inhibited, relaxation of the strips is accounted for by a) the efflux of unchanged amine from neuronal (and extraneuronal) stores and b) the sensitivity of the preparation to noradrenaline.

Animals↗

Effect of subconvulsive doses of bicuculline on the incorporation of radioactive precursors into glycerolipids in rat brain areas.

Bicuculline (either 25 mumol or 12.5 mumol/kg body wt) was administered to rats by intraperitoneal route. Animals treated with 25 mumol/kg experienced convulsions, whereas those receiving 12.5 mumol/kg did not. Controls received saline instead of the drug. Radioactive precursors [2-3H] glycerol and/or [1,2 14C] ethanolamine were injected into cerebral ventriculi simultaneously with bicuculline and the rats were killed 12 min afterwards. Their brains were dissected by hand into four parts (cerebellum, brain stem, hippocampus, cerebral cortex) and the labeling of lipid classes determined after extraction and separation. Although glycerol was incorporated into lipid better than ethanolamine in all areas, the fate of the injected radioactive precursors varied according both to area and treatment. The lowest uptake of radioactivity was in the cerebral cortex and the highest in the brain stem and hippocampus. Moreover, the administration of bicuculline influenced the distribution of radioactivity among lipid classes; these variations, however, were not dependent on the administered doses of bicuculline. We conclude that the effects on glycerolipid metabolism observed in convulsing animals are due to several causes including alterations of systemic parameters (hypertension, hypoxia, etc.). The distribution of glycerol label between phospholipid and neutral lipid is proposed as a biochemical model for the study of convulsive and subconvulsive states.

Animals↗

Expression of type I and IV collagen mRNAs in healing gastric ulcers--a comparative analysis using isotopic and non-radioactive in situ hybridization.

The sensitivity and practicability of in situ hybridization methods utilizing isotopic or non-radioactive labeling were compared. The aim of this study was to determine whether digoxigenin-labeled riboprobes are as sensitive as 35S-labeled probes to detect changes in type I and IV procollagen expression in an animal model of rat gastric ulcer. Both labeling and detection methods yielded similar results, with a superimposable signal distribution in the specimens. High levels of procollagen type I and IV transcripts were observed in spindle-shaped cells, presumably fibroblasts or myofibroblasts, localized in the ulcer base and rim. The increased expression of these collagen types suggests a remarkable upregulation of collagen expression during the healing of gastric ulcers. Liver tissue adhering to perforated ulcers displayed signals related to non-parenchymal cells, with hepatocytes demonstrating no detectable transcripts of type I or IV collagen genes. Due to the identical pattern of signal distribution by both hybridization techniques it is concluded that non-radioactive in situ hybridization is of value in monitoring highly expressed genes and yields results similar to those achieved with radioactive probes. In these cases, non-radioactive techniques are preferable because they are performed more rapidly and do not require handling of isotopes.

Animals↗

Incorporation of radioactive polyunsaturated fatty acids into liver and brain of developing rat.

The incorporation of radioactivity from orally administered linoleic acid-1-14C, linolenic acid-1-14C, arachidonic acid-3H8, and docosahexaenoic acid-14C into the liver and brain lipids of suckling rats was studied. In both tissues, 22 hr after dosing, 2 distinct levels of incorporation were observed: a low uptake (from 18:2-1-14C and 18:3-1-14C) and a high uptake (from 20:4-3H8 and 22:6-14C). In adult rats, the incorporation of radioactivity into brain lipids from 18:2-1-14C and 20:4-3H was considerably lower than the incorporation into the brains of the young rats. In the livers of the suckling rats, the activity from the 18 carbon acids was associated mostly with the triglyceride fraction, whereas the activity from the 20:4-3H8 and 22:6-14C was concentrated in the phospholipid fraction. In the brain lipids, the activity from the different fatty apid fatty acids, some of the activity in the 18:2-1-14C and 18:3-1-14C experiments was associated with 20 and 22 carbon polyunsaturated fatty acids; however, radioactivity from orally administered 20:4-3H8 and 22:6-14C was incorporated intact into the tissue phospholipid to a much greater extent compared with the incorporation of radioactivity into 20:4 and 22:6 in the experiments where 18:2-1-14C and 18:3-1-14C, respectively, were administered. Possible reasons for these differences are discussed. Rat milk contains a wide spectrum of polyunsaturated fatty acids, including linoleate, linolenate, arachidonate, and docosahexaenoate. During the suckling period in the rat, there is a rapid deposition of 20:4 and 22:6 in the brain. The results of the present experiments suggested that dietary 20:4 and 22:6 were important sources of brain 20:4 and 22:6 in the developing rat.

Animals↗

Radioactive labeling of proteins in cultured postimplantation mouse embryos. II. Dose and time dependency.

The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode's solution. Incubation time and concentration of [3H (or 14C)]amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 microCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions.

Amino Acids↗

Investigation of single, random, and true counts from natural radioactivity in LSO-based clinical PET.

OBJECTIVE: Lutetium oxyorthosilicate (LSO) contains natural radioactivity that emits beta particles and three gamma photons simultaneously. These beta particles and gamma photons increase the single and random rates in a positron emission tomography (PET) system while a beta particle and gamma photon produced in the same decay of Lu-176 and detected by another detector can be beta-gamma coincidence true events. The purpose of this work is to measure the single, random, and true count rates due to the natural radioactivity in LSO and determine the optimum lower energy threshold level for an energy window in an LSO-based clinical PET. METHODS: First, we measured the energy spectra of these beta particles and gamma photons in LSO using a single crystal to obtain the basic data. Then, we measured single, random, and true count rates of an LSO-based clinical PET from the natural radioactivity as a function of the lower energy threshold. RESULTS: In the PET, single and random count rates due to the natural background activity were gradually decreased as the lower energy threshold level increased. The true count rates due to the beta-gamma coincidence were more than 10 kcps below a lower energy threshold of 250 keV. However, these true count rates due to the natural radioactivity in LSO can be decreased to less than 1 kcps at a lower energy threshold level set at more than 350 keV. CONCLUSION: With these considerations, in an LSO-based clinical PET, a lower energy threshold level set at above 350 keV is recommended.

Artifacts↗

Assessment of enterohepatic circulation of radioactivity following a single dose of [14C]nimodipine in the rat.

4 male Sprague-Dawley rats with bile-duct fistulae were joined in a cascade. The first animal was given 5 mg/kg of [14C]-labelled nimodipine administered intraduodenally as a bolus. The bile of animals 1 and 2 was dynamically infused intraduodenally respectively into animals 2 and 3 and the bile of animal 3 was collected in fractions and infused i.d. into animal 4. The excretion and the course of the plasma concentration were studied radiometrically. The described investigation technique allows a detailed determination of the quantity and the kinetics of reabsorption and excretion during each observed pass of radioactivity in the enterohepatic circulation. On the basis of the balance between radioactivity administered and recovered and of the course of the plasma concentration it has been shown, on the example of nimodipine, that the experiment successfully simulates the situation in the intact animal. The [14C]Nimodipine radioactivity is subject to pronounced enterohepatic circulation: on average 43% of the amount excreted with the bile is reabsorbed with each recirculation and 57% is excreted with the faeces, 81% and 19% of the quantity reabsorbed being excreted respectively with the bile and urine. Because of the repeated reabsorption, the effective dose is increased by 54% and the drop in plasma concentration is slowed down in the middle time interval between 3 and 15 h after administration. The kinetic processes (absorption, excretion) become slower with each recirculation. An increasing fraction of the absorbed radioactivity undergoes direct hepatic excretion, by-passing the systemic circulation.

Animals↗

Distribution study of radioactivity in rats after oral administration of the lipido/sterolic extract of Serenoa repens (Permixon) supplemented with [1-14C]-lauric acid, [1-14C]-oleic acid or [4-14C]-beta-sitosterol.

The study carried out on rats given orally the n-hexane lipido/sterolic extract of Serenoa repens (LSESR), supplemented with [14C]-labelled oleic or lauric acids or beta-sitosterol, demonstrated that radioactivity uptake in prostatic tissues shows the highest level in the case of administration of LSESR supplemented with [14C]-labelled oleic acid. This was clearly demonstrated on a rat with an induced fibro-muscular hyperplasia of the prostate and by quantitative measurements of radioactivity. Ratios of radioactivity in tissues compared to plasma show an uptake of radioactivity greater in prostate as compared to other genital organs, i.e. the seminal vesicles or to other organs such as liver.

Administration, Oral↗

Gallium-67 activated charcoal: a new method for preparation of radioactive capsules for colonic transit study.

Indium-111 is currently the radionuclide of choice for colonic transit study. However, it is expensive and not available in many hospitals. Technetium-99m has been proposed for colonic transit study but the short half-life has limited its use. Gallium-67 citrate is inexpensive and available in most countries. Most importantly, it has a suitable half-life for colonic transit study. Attempts have been made in some studies to use (67)Ga citrate to label activated charcoal, but the results have not been good because of poor stability. In this study, we successfully labelled activated charcoal()with (67)Ga citrate by adding alcohol and 5% glucose solution. To evaluate the in vitro stability, the (67)Ga-activated charcoal was incubated in a milieu mimicking the intestinal content, containing lipase, trypsin and glycochenodeoxycholate at different pH values (6.0, 7.0, 7.4 and 8.0) and for different durations (0 h, 24 h, 48 h, 72 h and 96 h). For the in vivo study, the (67)Ga-activated charcoal was loaded into a commercial empty enteric capsule. Colonic transit scintigraphy was performed in five volunteers, including three healthy people and two constipated patients, after intake of the radioactive capsule. Images were obtained at 2 h, 4 h, 6 h, 8 h, 24h, 48 h, 72 h etc. until no radioactivity was detected in the bowel. Our data show that the in vitro stability of (67)Ga-activated charcoal was good. The labelling efficiency still exceeded 91% at 96 h at pH values of 6.0, 7.0 and 7.4. In the group with a pH value of 8.0, the labelling efficiency gradually fell during the 4-day incubation but was still higher than 88% at the end of the fourth day. In the in vivo study, most capsules disintegrated in the caecum/colon region, and the (67)Ga-activated charcoal mixed very well with bowel content. In addition, the radioactive charcoal could be detected clearly on the 72-h image, which is very important for the evaluation of colonic transit time in patients with constipation. In conclusion, activated charcoal labelled with (67)Ga citrate is a potential radioactive marker for colonic transit study.

Capsules↗