Radioactivity profiling

Fig. 4. Nucleosome relaxation, and influence of histone N-terminal tails. Example of nucleosomes on 356 bp ALk= —2.9 topoisomer from the pBR DNA minicircle series [28]. (a) Mononucleosomes (Mo) were reconstituted with control (Control) or acetylated (Acetyl) histones, incubated at 37 °C in Tris buffer [T 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 50 mM KCl, 5 mM MgC, and 0.5 mM dithiothreitol] or phosphate buffer [P same as Tris buffer with 50 mM potassium phosphate (pH 7.5) instead of 50 mM Tris-HCl] in the absence (Topo I —) or presence (Topo I +) of topoisomerase I, and electrophoresed in a native polyacrylamide gel at room temperature. Note the splitting of nucleosome relaxation products in two bands. TE starting chromatin in TE buffer, (b) Gel slices (brackets) were cut out, and eluted DNAs were electrophoresed in a chloroquine-containing native polyacrylamide gel, together with control naked topoisomers (C1-C4). Lanes were numbered as in the (a) gel. Autoradiograms are shown, (c) Radioactivity profiles of lanes 2 and 5 in the (b) gel. Topoisomers are indicated by their ALk values. (Adapted from Fig. 2 in Ref. [28].)...

Compound A produces a predominant fragment at miz 264, which corresponds to a neutral loss of 175, which in this case corresponds to the loss of 4-trifluo-romethylbenzylamine [37]. This neutral loss can be used to monitor the presence of other species sharing this common feature. Radioactively labelled compound A was incubated in rat liver microsomes, analysed by LC-MS-MS and the TIC for the neutral loss of a mass of 175 (Figure 6.20a) was obtained. When compared with the radioactivity profile in Figure 6.20b, two extra components were detected in the TIC. This was due to the loss of the radiolabel during metabolism. LC-MS-MS is very useful as a complementary detection method where the radiolabel is lost during metabolism or in situations where a radiolabel is not available. 

Figure 6.20 (a) LC-MS total ion current of neutral loss 175 and (b) HPLC-radioactivity profile of a rat liver microsomal incubation of radioactive compound A. Reproduced from [37J with permission from Elsevier. 

Conditions C - Bonded cartridge. 8 mm 10 cm (Radial pak A. Waters Associates, Inc.) was used with a mobile phase of a linear gradient ofO-tuOtfc isopropyl alcohol-water (9 1, v/v) (solvent B) in acetonitrile-water (9 1, v/v) (solvent A) over a period of60 min at a flow rate of3 m Lj min. Both solvents contained 5 mM terrabutv/ammonium phosphate. For the determination of the radioactivity profile, fractions were collected at 3(fs intervals and assayed for radioactivity. 

Fig. 2. Plasma radioactivity profiles obtained in 2 healthy volunteers after administration of123I-Parj 1 by sublingual (a) or intranasal (b) route, respectively. 123I-Parj 1 was administered at time 0 for sublingual administration the subject swallowed at 30 min. The radioactivity is expressed as percent of the administered dose. 

In these experimental conditions too, similar results were obtained no transmucosal passage of the tracer to the bloodstream before swallowing, as well as local persistence for hours after administration were observed. Accordingly, plasma radioactivity profile and radioactivity gel filtration profile at its peak were similar to the former set of experiments. Preliminary studies were also performed in subjects with allergic rhinitis due to P. judaica pollen, receiving radiolabelled Par j 1 intranasally. At variance with what is shown in normal individuals, the disappearance rate of radioactivity from the nasal... 

Figure 9. Radioactivity profile across a SDS-polyacrylamide (5%) gel in which surface-membrane proteins of human neutrophil granulocytes have been separated. Before this experiment, isolated granulocytes had been incubated 2 h at 37°C with inIn-BLEDTA in plasma. As indicated by the arrow, a single protein with molecular weight 1.3 X 10s was...

This is the two-dimensional equivalent of [1.6.5.24]. In the differential method, the broadening of the radioactivity profile with time is fitted to [3.8.3] using D" as the fitting parameter. 

Isomer interconversion due to assay manipulations could also be determined from the recovery experiment. Isomerization data was obtained from the radioactivity profile of the HPLC purification step. The amount of cis isomerizing to trans was 3%. The same amount of trans retinoic acid isomerized to cis. The assay causes a small amount of isomerization but to an equal extent for both isomers. 

Unfortunately, the OPA condensation products decompose to some extent on the HPLC column (as shown by means of 14C-labelled amino acids - the positions of emerging samples shown by their fluorescence lag behind maximum radioactivity profiles), although users favouring these OPA derivatives have learned to practise strict protocols for their use, in order to get reliable results. The OPA-TV-acetyl-L-cysteine condensation product is a diastereoisomer mixture when formed with a... 

Figure 4 Footprint of histones on the DNA of irradiated core nucleosome. A) Nucleosome core particle structure extracted from lAOl entry of PDB. The 146 base pairs DNA fragment Is In green and yellow, and the histone octamer is in blue and magenta tones. B) Experimental relative probabilities of FSB deduced from the radioactive profile (electrophoresis ) and relative probability of attack to deoxyriboses leading to FSB (calculated with RADACK). C) The Fourier transform (power) of the probabilities showing that probability variation has a period of 10.4 base pairs both for the experlmen t and for the calculated values.

FIGURE 9.3 Comparative plasma profiles of radioactivity in rat, dog, and humans showing parent and metabolites after a single oral dose of C-14-labeled compound. The table in the figure shows the percent AUC of total radioactivity for parent (P) and two major circulating metabolites in plasma samples. The AUCs for parent and metabolite were generated from radioactivity profiles generated at several time points. 

FIGURE 10.9 Representative plasma metabolite profiles of a radiolabeled drug candidate in rats (a), dogs (b), and humans (c). The pooled plasma samples from radiolabeled ADME studies after an oral administration were collected around the maximum concentration of the drug. The radioactivity profiles were determined by offline HPLC-TopCount (four functions per min and 10 min counting time). Approximately, 400 9000 DPM of radioactivity was injected. 

FIGURE 10.10 Analysis of [ H]GSH trapped reactive metabolites by HPLC with TopCount (Zhu et al., 2005b). A nonlabeled drug (50 mM) was incubated with a mixture of GSH (1 mM) and trace [ H]GSH (1-2 p,Ci/mL) in human liver microsomes. After precipitating proteins, the samples were analyzed by HPLC (1 mL/min) with TopCount (4wlls/min, 10-min counting time) (a) Radioactivity profile of the incubation. Ml and M2 were GSH-trapped reactive metabolites and (b) Radioactivity profile of a control incubation sample (without NADPH). 

Radioactivity profiling in plasma, urine, bile, and feces of appropriate animals and human subjects from mass balance studies enable a quantitative evaluation of the absorption, tissue distribution, and excretion of a drug candidate. The data can be supplemented with tissue distribution information from whole-body analysis and the further defining the sites of absorption and excretion in surgically prepared animals. 

A human serum containing a mono-clonal anti-(blood-group I) antibody has been used to investigate the distribution of blood-group I antigens on erythrocyte membrane components. Different radioactive profiles were revealed by the two radiolabels used with solubilized stroma ( H and In the immuno-precipitates, the predominant I-labelled band had molecular weight 0.9-1.0x10 , whereas the label was associated with a diffuse band (molecular... 

The first set of information generated from the ADME studies are the overall plasma profiles of total radioactivity (TRA) versus time which is compared to the plasma profile of parent versus time measured by a validated LC/MS/MS assay. For those drugs where the parent is the major component at all time points in plasma, the total radioactivity profile usually parallels the profile of the parent. Metabolie profiles of plasma samples generated at different time points by high-performance liquid chromatography (HPLC) analysis followed by radioactivity and mass spectrometric detection provide exposure-related information for parent and metabolites in humans and animal species. In addition, this profile provides information about metabolites that humans are exposed to and how this compares to exposures in animal species. 

Fig. 7. Radioactivity profile in SDS-polyacrylamide gel of acetyl-CoA carboxylase isolated from control and epinephrine-treated rats. Each of three rats in the group was injected intraperitoneally with 0.41 mCi of carrier-free P, at 3 hours and 1 hour before killing. Epinephrine (1 mg/kg of body weight) or saline was administered 30 minutes before killing. The preparation of carboxylase from epididymal adipose tissue and all subsequent procedures were as described in Table II. In this experiment, carboxylase activity of epinephrine-treated animals was 76% of the control. O, Control carboxylase , epinephrine-inactivated carboxylase. Arrow indicates the location of carboxylase subunit band f shows the location of tracking dye front. From Lee and Kim (68).

Fig. 1. Size exclusion chromatography of ADP-ribose made in nucleotide permeable SVT2 cells [1] and purified as described somewhere else [4], using one Bio Sil TSK-125 column. The running buffer used was 0.1 A/ sodium phosphate, pH 6.8 at a flow rate of 1.0 ml min". 0.5 ml fractions were collected. The arrows indicate void volume and included volume, respectively. — Radioactivity profiles -absorbance profile...

Figure 3. Radioactivity profile of rhinoviral 1A polypeptides on a preparative SDS-polyacrylamide gel. Bars indicate fractions pooled in preparing isolated proteins. In some cases further electrophoresis under altered conditions was necessary to resolve mixtures (e, 39a, 39b), (38, a) and...

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