Liquid scintillation counting assay

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 ... 

Guilmette RA, Bay AS. 1981. Radio assays of americium or curium in biological material by isoctyl acid phosphate solvent extraction and a liquid scintillation counting. Anal Chem 53 2351-2354. 

The initial mixture and each time point are then assayed for doxorubicin and lipid. Lipid concentrations can be quantified by the phosphate assay (see above) or by liquid scintillation counting of an appropriate radiolabel. Doxorubicin is quantified by an absorbance assay (see below). The percent uptake at any time point (e.g., t = 30 minutes) is determined by %-uptake = [(D/L), =30minutes] x 100/[(D/L) inuiai]. Doxorubicin can be assayed by both a fluorescence assay and an absorbance assay, but we find the latter to be more accurate. The standard curve consists of four to five cuvettes containing 0 to 150 nmol doxorubicin in a volume of 0.1 mL samples to be assayed are of the same volume. To each tube is added 0.9 mL of 1% (v/v) Triton X-100 (in water) solution. For saturated lipid systems such as DSPC/Chol, the tubes should be heated in a boiling water bath for 10 to 15 seconds, until the detergent turns cloudy. Samples are allowed to cool, and absorbance is read at 480 nm on a UV/Visible spectrophotometer. 

The initial mixture and each time point are then assayed for ciprofloxacin and lipid. Lipid can be quantified using the phosphate assay (64,65) or by liquid scintillation counting. Ciprofloxacin is quantified by an absorbance assay following removal of drug from lipid by a Bligh-Dyer extraction procedure (78) (see below). The percent uptake is determined as described in the section Remote Loading of Doxorubicin into DSPC/Cholesterol (55 45) Large Unilamellar Vesicle. ... 

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. 

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. 

Because specific activity is always an intensive variable, one need not recover all of the sample to determine the ratio mol A /mol A. To evaluate SAa, one need only obtain an amount that provides an accurate measurement of A and total A. The former is easily achieved by liquid scintillation counting, and spectrophotometry or some other enzymatic assay usually allows accurate determination of mol A in a sample. 

Table 4.6.11 Mixtures for liquid scintillation counting of amylo-1,6-glucosidase (debranching enzyme assay with [14C]-glucose) for liver and muscle, and for RBC...

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]. 

The activity of dihydropyrimidinase or /J-urcidopropionasc can only be measured in liver or kidney. The activity of dihydropyrimidinase is determined using a radiochemical assay with subsequent separation of radiolabeled dihydrouracil from radiolabeled N-carbamyl-/>-alanine with reverse-phase HPLC combined with detection of 14C02by liquid scintillation counting [11]. The activity of /1-ureidopropionase can be determined using radiolabeled N-carbamyl-/l-alanine followed by separation of radiolabeled N-carbamyl-/>-alanine from radiolabeled /1-alanine by reverse-phase HPLC [10,14]. 

An interesting set of radiotracer studies of the deposition of pesticides was made by Atkins and Eggleton (1971). The pesticides were labeled with 14C, which was assayed using liquid scintillation counting. Results of the study showed that direct... 

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). 

Isotopic dilution techniques were used to determine residual, tritium-labeled gibberellic acid in potatoes, grapes, and various products derived from barley The seeds, the young plants, or the fruit were treated with labeled gibberellic acid and analyzed at the end of the growth period by extraction of the labeled residue in the presence of carrier gibberellic acid, isolation of a pure crystalline specimen, and subsequent assay by liquid scintillation counting ... 

Nonetheless, it has been shown to elute immediately after the PC peak. LysoPC, if present, would elute between the PAF and sphingomyelin (Sph) peak. The latter component often shows a double peak, and this can be attributed to the separation of distinct fatty acyl species. It is well to emphasize again that compounds such as PAF and lysoPC have very low levels of unsaturated bonds present, and hence a detector other than an ultraviolet monitor would have to be used. An alternate approach would be to use tritiated PAF or tritiated lysoPC as examples and assay the eluates by liquid scintillation counting. Since the labeled compounds are at tracer dose levels, one could still assay for biological activity associated with the compounds. 

Measurement of Natural Radiocarbon Concentrations. By the middle of the 1950s, the original solid carbon method of assay of natural radiocarbon concentrations had been completely superseded by the development of either gas or liquid scintillation counting systems. More than 20 years of experimentation and development in the low level counting technology field has turned what was once a black art and analytical tour de... 

R. S. Hendler, Anal. Biochem.,l 10 (1964). Procedure for Simultaneous Assay of Two jS-Emitting Isotopes with the Liquid Scintillation Counting Technique. 

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