Radioactivity liquid scintillation counting

In the carbon-14 expts, HMX/RDX product was isolated qualitatively, separated Into its components, and each component assayed for carbon-14 beta radioactivity using a liquid scintillation counting technique (Ref 11). DPT-l4C was isolated as an intermediate product from the reaction mixt and similarly radioassayed. For the nitrogen-15 tagged AN expts, HMX and RDX were assayed mass spectrometrically for i5N/i4N ratios from which atom %1SN contents were calcd. In die course of these expts, each tagged species was added initially and also at subsequent stages of the reaction process. The important observations and results are summarized as ... 

Since cigarette tobacco already contains several micrograms of the TSNA, we determined the transfer rate of NNN into the smoke by spiking the tobacco column with NNN-2 - C. The smoke from such radiolabeled cigarettes is then analyzed by HPLC and the amount of unchanged NNN-2 - C is determined by liquid scintillation counting. Independent of the smoke pH, about 11% of the radioactive NNN is found in the mainstream smoke thus 41-46% of mainstream smoke NNN stems from the tobacco NNN and 54-59% are pyrosynthesized (11). 

To determine the efficiency of aminoacylation of [14C]Phe-tRNA, 5 fil aliquots of the aminoacylation mixture are withdrawn before and after the reaction the samples taken from the reaction mixture at the end of the incubation are spotted onto 3-MM paper discs (Schleicher Schuell) and processed by the cold TCA precipitation method, while the sample taken before the reaction is spotted on a paper disc pretreated empty by the same cold TCA procedure. Determination of the radioactivity present on these filters by liquid scintillation counting allows one to calculate the aminoacylation efficiency of the reaction (which, for phenylalanine, should be >2% of total tRNA). The specific activity of the [14C] Phe-tRNA can be determined after one-step purification of Phe-tRNA by BD cellulose chromatography (Gillam et al., 1968), followed by determination of the radioactivity and of the A260. 

At the end of the aminoacylation reaction, a 5-fA aliquot of the reaction mixture is spotted onto a 3MM paper disc (Schleicher Schuell) that is immediately placed in 10% ice-cold TCA for 30 min. After three washes in 5% TCA at room temperature for 5 to 10 min, the filter is placed in an ethyl ether ethanol (1 1) mixture for 10 min and then in ethyl ether for 10 min before being dried under an infrared lamp. The amount of radioactivity precipitated on each filter is finally determined by liquid scintillation counting. 

For the quantitation of in vivo absorption of 5Ca2+, 200 jd 1 of radioactive solutions were administered by gastric gavage to male mice kept on a deionized water diet during the previous 18 hours. After 4 hours blood was obtained by axillary incision and Ca2+ was extracted with TCA and determined by liquid scintillation counting (26). 

Arukwe et al. [15] dosed Atlantic salmon with radioactive labeled NP. Fish were frozen immediately until gall bladder, skin, kidney, gill, liver, muscle, fat, remaining carcass and viscera were sampled and analysed. Tissue radioactivity was analysed by liquid scintillation counting after combustion of aliquots in an oxidiser apparatus. Metabolites (biliary and urinary) were separated by radio-HPLC. 

DDT Metabolism.-- The metabolism of DDT has been studied in R and S fish, following similar protocols to chlorinated cyclodiene metabolism organic extraction (acetonitrile), thin layer chromatography of organic extracts, and liquid scintillation counting of the resultant spots (4). When S and R fish were exposed to 60 yg/l of 14C-labelled , -DDT for 4 hr, radioactivity was found in the spots which co-chromatographed with... 

For Ptdins 4 kinase and Ptdins (4) P5 kinase activities, the assay can be modified with assay buffer (50 mM Tris-HCl, pH 7.2 10mM MgCh 1 mM DTT 0.4% Triton X-100), 0.5 mM of substrate Ptdins or PtdIns4P, and 1 (xCi [y- P]-ATP. Terminate the reaction with 0.6 mL chloroformimethanol (1 1, v/v). After addition of 0.5mL 12N HCl, phosphoinosi-tides are extracted into the lower chloroform phase, which are washed with 1 mL methanoLlM HCl (1 1, v/v) followed by 1 mL methanoLO.l mol/L HCl (1 1, v/v). The radioactive reaction product can be isolated by TLC and quantified by liquid scintillation counting. 

Exposure through inhalation As most of the quantity of the cyclic siloxanes evaporate shortly after application of the PCPs or volatilize from electronics, adhesives and sealants, polishes and surface cleaners, etc., the main pathway of exposure is through inhalation. Plotzke et al. [286] studied the inhalation exposure to D4 by exposing rats to C-DA. Using liquid scintillation counting, it was determined that the retention of inhaled D4 in the body of the rats was 5-6%. The radioactivity reached maximum concentrations in the fat 24 h after exposure, but in the plasma and other tissues (except for fat) in only 3 h. The fat tissue acted as a depot because the elimination of the radioactivity from it was slower than from other tissues. 

Traditionally, HAT activity is measured with a discontinuous radioactive filterbinding assay, which uses pH]acetyl-CoA as a histone acetyltransferase substrate [46]. The transfer of [ H]acetyl-groups to the histone substrate by histone acetyltransferases is detected by liquid scintillation counting of pHjacetylated histones, which are retained on a phosphocellulose disk. Due to its discontinuous character, this assay is technically problematic and not ideal for kinetic analysis. Hence, other assays that work with radiolabeled acetyl-CoA have been described that are suitable for a higher throughput. These work with streptavidin-covered beads [47] or a variant of the SPA with microtiter plates that contain a scintillant (FlashPlates) [48]. But as all these protocols are based on radioactively labeled substrates, they apparently show the same disadvantages that were described for the radioactive HDAC assay protocols. Therefore, nonradioactive assays have been developed to study histone acetyltransferase activity. 

Commonly, in vitro determination of HDAC activity is a manual assay utilizing a coupled two-step process, including enzymatic deacetylation of a substrate followed by reaction termination and readout [10]. Assays utilize nuclear extracts and substrates containing labeled (radioactive or fluorescent) acetylated histones. For the isotope-based assays, the enzymes are incubated with acetate-radiolabled histones prepared from chicken reticulocytes or chemically [ Hjacetylated peptide substrates, and the enzymatic activity is determined by liquid scintillation counting [11]. Alternatively, histones may be obtained from cells following treatment with [ H]acetyl-CoA [12]. The caveats of these approaches include the variability of prelabeled acetylated core histones within preparations, potential high costs, their labor-intensive nature and the presence of radioactive waste. 

The LPL catalytic assay measures the hydrolysis of a [14C[- or [3H]-triolein emulsion producing the 14C- or 3H -labeled free oleic acid [6]. The 14C- or 3H-labeled oleic acid is isolated by a selective extraction procedure and its radioactivity is determined by liquid scintillation counting [40]. Lipase activity is calculated as nanomoles of oleic acid released per minute per milliliter of postheparin plasma [41]. 

An HPLC system with electrospray-MS and liquid scintillation counting (LSC) was used for assaying [ l4C] NAR in chicken fat. The sample was combined with hexane and melted at 70°C, the hexane extract was reextracted with MeCN and evaporated to dryness, and the residue was resuspended in chloroform. Then it was passed through an unconditioned silica cartridge, and the retained radioactivity was eluted with MeOH. After the reconstitution and injection into the HPLC system, fractions for LSC were collected. Liver radioactivity was isolated by LLE and preparative silica LC (100). 

Reagents. Aqueous ethanol solutions of the triethylammonium salts of adenosine-5 -[a-S2P]triphosphate (a- PATP), adenosine-5 -[7-S2P]triphosphate (7-S2P-ATP), thymidine-5 -[a-S2P]triphosphate (a- p-TTP), cytidine-5 -[q-S2P]triphosphate (a-S2P-CTP), and guanosine-5 -[a-S2P]triphosphate (a-S2P-GTP) were purchased from Amersham (Arlington Heights, IL). Radioactive sample concentrations reported for detector efficiency determination were adjusted from the manufacturer s specifications after subjecting several diluted aliquots of the stock solution to liquid scintillation counting. 

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