Development of a Quantitative Polyacrylamide Gel Electrophoresis Analysis Using a Multichannel Radioactivity Counter for the Evaluation of Oligonucleotide-Bound Drug Carrier

A quantitative polyacrylamide gel electrophoresis (PAGE) analysis using a multichannel radioactivity counter was designed for the evaluation of³³P-labeled antisense oligonucleotide associated with polymeric drug carrier (nanoparticles). The proposed analytical method was first validated. The criteria of specificity, linearity, reliability, detection and quantification limits, and resolution power were determined. Results were compared to those obtained using liquid scintillation counting of crude samples or after solubilization of gel slices. The proposed method gave a better linearity and reliability than liquid scintillation counting of solubilized gel slices. In comparison with the liquid scintillation counting of crude samples, the method presented the advantage of being able to directly separate oligonucleotides differing by only one nucleotide in length. This method was applied for the separation of free oligonucleotides and oligonucleotides bound onto nanoparticles, allowing quantification of the amount of free and bound oligonucleotides without any further separation steps. Thus, because it is easy and rapid, the quantitative PAGE analysis using a multichannel radioactivity counter offers interesting possibilities for the characterization of oligonucleotide nanoparticles.     

Measurement of the 125I and 14C radioactivity in double-labeled blood samples

A method for detection of 14C and 125I radioactivity in labeled samples with variable quenching using only liquid scintillation counter is described. Precision, sensitivity and reproduction of this method are comparable with those of the previously used ones, but this method is more suitable and adequate for computerisation.     

Iron absorption in gastric and duodenal pathology in patients with iron deficiency anaemias.

Absorption of radioactive iron was studied in 87 patients with different types of iron deficiency anaemias and in 23 healthy subjects. The subjects were given 1...2muci of radiactive iron in the form of FeSO4 together with 5 mg of nonradioactive iron as a carrier and 100 to 150 g of white bread, radioactivity on the whole body being studied with a big liquid scintillation counter 4 pi (BLSC-2). In clinical observations and in single experiments on volunteers there was no conformity of the values of absorption with the levels of acid-formation. But in the same time the gastric juice from an anaemic horse almost doubled iron absorption in healthy individuals. Marked morphological changes in the gastric mucosa inhibited the absorption in the intestine and the degree of increase of absorption in patients with anaemia depended to some extent on the morphological conditions of the gastric mucosa. When healthy subjects and patients with iron deficiency anaemia were given bread "enriched" with iron before baking instead of common bread with "external" mark there was observed similar correlation between the values of absorption but the figures were somewhat lower.     

Measuring Radioactive Methane with the Liquid Scintillation Counter

Although a gas proportional counter is the most convenient method of measuring the radioactivity of fixed gases such as methane, it cannot be used when high nonradioactive concentrations of methane are present in the gas phase, due to quenching. If only methane and carbon dioxide are present in radioactive form in the gas phase, a liquid scintillation method for measuring these substances can be used. The procedure is described in detail, and the solubility of methane in liquid scintillation cocktails is determined.     

Preparation of 14C-labeled algal samples for liquid scintillation counting

Four commonly used techniques for preparation of ¹⁴C-labeled algal samples on membranes for liquid scintillation counting were compared and a simple technique for apparent net assimilation measurement from aqueous samples was introduced. All four techniques yielded similar radioactivities from the test cultures and are thus suitable for measurements of ¹⁴C algal samples. The possibly carcinogenic solvent dioxane was not necessary with PCS scintillation cocktail for dissolving radioactivity from algae on filters.     

Reduction of 141Ce absorption in suckling rats

The influence of diet or its ingredients on 141Ce absorption and retention was investigated in six-day-old rats. Animals were fed over 8h with cow's milk, rat diet or a mixture of rat diet ingredients (fish meal, sunflower meal, alfalfa, cane molasses and premix) labelled with 141Ce. Whole-body radioactivity was determined in a double crystal scintillation counter every 24 h over a six-day period. Gut, liver, kidney and femur retention and cerium distribution in the gut was determined at the end of the experiment. Compared to milk diet, administration of rat diet or ingredients caused respectively 3 and 7.5 times lower whole body retention. Carcass retention was reduced by rat diet or ingredients 2-3 times and intestinal retention 3 and 8 times respectively. Irrespective of the dietary treatment the main site of cerium intestinal retention was the ileum. Our present results indicate that some compounds of rat diet might be considered as a means of reducing cerium absorption and intestinal retention in the very young.     

Pharmacokinetics of 125I-GST-TatdMt, a recombinant fusion protein possessing potent anti-obesity activity, after intravenous, nasal, oral, and subcutaneous administration

This study first reports the absorption kinetics of GST-TatdMt, a recombinant Tat protein possessing potent anti-obesity activity, in rats after nasal, s.c., and p.o. administration. GST-TatdMt was over-expressed in E. coli, purified, and radioiodinated using the IODO-GEN method. The radioiodinated 125I-GST-TatdMt was administered to rats by nasal, s.c., and oral routes at doses of 7.3 microg (420.7 nCi), 146.5 microg (8413.8 nCi), and 146.5 microg (8413.8 nCi), respectively. For the determination of absolute bioavailability, 125I-GST-TatdMt was also given to rats by i.v. injection (73.2 microg, 4206.9 nCi). Following administration by extravascular routes, the systemic absorption of radioactivity was prolonged, with Cmax being attained within 4.2-8.0 h. The absolute bioavailability calculated as dose-normalized AUC(extravascular)/AUC(i.v.) was 98.0, 75.8, and 87.1% after nasal, s.c., and oral administration, respectively. The majority of administered radioactivity was excreted in urine (57.5-64.7%), with fecal excretion being less (2.5-12.7%). The distribution of 125I-GST-TatdMt to various tissues was also determined at 4 and 72 h after s.c. injection. The findings of this study suggest that this protein may be absorbed into the systemic circulation when given by extravascular administration.     

Determination of myocardial high-energy phosphates using bioluminescence

The measurement of myocardial high-energy phosphates (HEP) has become essential in the evaluation of current methods of myocardial protection both in the experimental and clinical setting. Assays for high-energy phosphates have required as much as 50 mg of myocardial tissue which prevents repeated biopsies in the clinical setting as well as in the experimental laboratory. Using the reaction of bioluminescence described by McElroy W. D. and B. L. Strehler (1949, Arch. Biol. Chem.22, 420), we have developed a technique to measure both adenosine triphosphate and creatine phosphate on samples of myocardial tissue weighing less than 10 mg. A liquid scintillation counter measures the light produced by ATP when added to a firefly extract containing luciferin/luciferase. The reaction is complete in seconds and is detected in the counter during the first 10-s count. Repeated samples have demonstrated a variation of less than 4% between samples. A 25-μl sample is diluted up to 40 μl of firefly extract for detection of adenosine triphosphate. Creatine phosphate is measured by the in vitro production of adenosine triphosphate which is maximum in 10 min when adenosine diphosphate and creatine kinase are added. Again reproducibility of repeated analyses demonstrates a 4% difference in creatine phosphate values. The rapidity, reproducibility, and ability to use ultramicrosamples allows investigators to analyze high-energy phosphates during various methods of myocardial protection currently used in clinical setting.     

Determination of the specific activity of sheep plasma amino acids using high-performance liquid chromatography: comparison study between liquid scintillation counter and on-line flow-through detector

A method was developed for the determination of the specific activities of leucine and phenylalanine in plasma using a flow-through scintillation counter coupled with high-performance liquid chromatography components. Results were compared with those obtained from liquid scintillation counting. Differences in the specific activities of leucine and phenylalanine between the two methods were not statistically significant. We concluded that flow-through radioactivity detection can be used for quantitative amino acid assays. However, the minimum activity that can be detected may be prohibitively low in certain applications.     

Measurement of specific radioactivity of tryptophan labeled with carbon-14 in plasma by high-performance liquid chromatography with a synchronized accumulating radioisotope detector

Measurement of specific radioactivity by a high-performance liquid chromatograph with a synchronized accumulating radioisotope detector was conducted. Accuracy of measurement for an authentic sample containing 0.2 nCi of tryptophan labeled with carbon-14 exceeded 95%. In the case of a plasma sample obtained 120 min following intravenous administration of 15 muCi of labeled tryptophan to a rat, the coefficient of variation was 7.0%.     

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.     

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.     

Measurement of cell proliferation by enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody to bromodeoxyuridine

An ELISA was developed and optimized to measure cell proliferation using a monoclonal antibody to bromodeoxyuridine (BrdUrd). Incorporation of BrdUrd into myoblast monolayers, measured as the optical density at 492 nm, increased in response to fetal calf serum, IGF-I and EGF, the ELISA data correlated closely with data obtained by BrdUrd immunocytochemistry (r = 0.984), cell counting (r = 0.972) and tritiated thymidine uptake by liquid scintillation counting (r = 0.990). The BrdUrd ELISA is a useful alternative to measurement of tritiated thymidine uptake by scintillation counting, and has the added advantages of dispensing with the use of radioactivity and of being less labour intensive.     

Altered Uptake and Biological Half-Lives of 65Zn on Arsenic Exposure—Modulation by Zinc Treatment

The present study revealed the effects of zinc on the biokinetics of (65)Zn in rats following arsenic intoxication. The animals were segregated into four groups: group I--untreated controls, group II--arsenic treated (100 ppm as NaAsO(2) in drinking water), group III--zinc treated (227 mg ZnSO(4) per liter drinking water), and group IV--arsenic?+?zinc treated. Each rat was injected intraperitoneally with 1.85 MBq radioactivity of (65)Zn following 3 months of different treatments, and the radioactivity was determined using a suitably shielded scintillation counter. Arsenic treatment showed a significant increase in the fast component (Tb(1)) of the biological half-life of (65)Zn in liver, which remained unaltered in the whole body. Furthermore, arsenic treatment decreased significantly the slow component (Tb(2)) in the whole body, which remained unchanged in the liver. However, zinc supplementation to arsenic-treated rats normalized Tb(1) in the liver, but caused no change in Tb(2) in the whole body. Furthermore, the uptake values of (65)Zn were significantly increased in the liver, brain, kidney, and intestine following arsenic treatment, and the values in the liver and brain were decreased by zinc. Hence, zinc plays a significant role in regulating the biokinetics of (65)Zn in the liver and the whole body of arsenic-intoxicated rats.     

Chloride absorption and distribution in chlorine-deficient Pharbitis nil

Determination and distribution of radioactive chloride in Pharbitis nil was investigated by a bio-imaging analyzer. When leaves that contained various amounts of ³⁶Cl were analyzed with the imaging analyzer and then each sample was homogenized and its radioactivity measured in a liquid scintillation counter, radioactive levels recorded by the analyzer were directly proportional to the radioactivity determined by the counter. When plants that had been grown in full nutrient solution were incubated in [³⁶Cl]-containing solution, more activity was found in young leaves than in mature leaves, while little radioactivity was detected in shrivelled leaves and the nonsymptomatic cusp of young leaves of plants that had been grown in chlorine-deficient solution.     

Biotin-avidin microplate assay for the quantitative analysis of enzymatic methylation of DNA by DNA methyltransferases

An assay is described to measure methylation of biotinylated oligonucleotide substrates by DNA methyltransferases using [methyl-3H]-AdoMet. After the methylation reaction the oligonucleotides are immobilized on an avidin-coated microplate. The incorporation of [3H] into the DNA is quenched by addition of unlabeled AdoMet to the binding buffer. Unreacted AdoMet and enzyme are removed by washing. To release the radioactivity incorporated into the DNA, the wells are incubated with a non-specific endonuclease and the radioactivity determined by liquid scintillation counting. As an example, we have studied methylation of DNA by the EcoRV DNA methyltransferase. The reaction progress curves measured with this assay are linear with respect to time. Methylation rates linearly increase with enzyme concentration. The rates are comparable to results obtained with the same enzyme using a different assay. The biotin-avidin assay is inexpensive, convenient, quantitative, fast and well suited to process many samples in parallel. The accuracy of the assay is high, allowing to reproduce results within +/- 10%. The assay is very sensitive as demonstrated by the detection of incorporation of 0.8 fmol methyl groups into the DNA. Under the experimental conditions, this corresponds to methylation of only 0.03% of all target sites of the substrate. Using this assay, the DNA methylation activity of some M.EcoRV variants could be detected that was not visible by other in vitro methylation assays.     

DNA and protein synthesis in cultured lens epithelial cells. I. Test system]

An in vitro test system for measuring DNA and protein synthesis in cultivated lens epithelium cells was developed. The method is suited also for other monolayer cultures; it has the following advantages: a) Cultivation of cells, incubation with radionuclides, preparation of the samples and measurement of radioactivity are carried out in the same vessel (scintillation vial); b) The use of 3H-thymidine and 14C-phenylalanine allows simultaneous measurement of DNA and protein synthesis; c) Only small amounts of cells (10(4) to 10(5) cells) are required to measure DNA and protein synthesis. The test system is highly sensitive to synthetic effectors (cytosone arabinoside, actinomycin D, puromycin), and is thus appropriate for the detection of inhibitors of DNA and protein synthesis and for testing the toxicity of drugs.     

Cotranslational formation of active photoprotein obelin in a cell-free translation system: direct ultrahigh sensitive measure of the translation course

Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity of in vitro translation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes.     

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.     

Milk fat progesterone concentrations in goats and early pregnancy diagnosis

Blood and milk samples from foremilk during afternoon milking, were simultaneously collected from 285 dairy goats. In experiment 1, fiva cyclic goats were sampled daily for 21 days. In experiment 2, 280 females from 9 flocks were submitted to sampling 21 days after insemination. In addition, some milk samples were divided in two parts, after which one was frozen and the other kept at +4 degrees C until assay. Progesterone concentrations were measured in blood, whole milk and milk fat by radioimmunoassay. No difference in whole milk or fat progesterone levels was found between frozen and refrigerated milk samples. Milk butterfat progesterone concentrations paralleled those in plasma or whole milk throughout the estrous cycle and ranged from about 20 ng/ml at estrus to about 400 ng/ml in mid-luteal phase. The ratio of mid-luteal phase progesterone levels to those seen in the estrous period was over 20 in fat and in blood. This ratio was very much lower in whole milk. Consequently the determination of pregnant and non-pregnant goats from the samples collected 21 days after service was very much easier and accuracy was better when the progesterone content was assayed from milk fat than from whole milk. It was concluded that early pregnancy diagnosis in goats can be done routinely by determination of progesterone levels in milk fat.