Radioactivity labelling

A difficulty in the physicochemical study of penetration is that the amount of soluble component present in the monolayer is not an easily accessible quantity. It may be measured directly, through the use of radioactive labeling (Section III-6) [263, 266], but the technique has so far been used only to a limited extent. 

Some further details are the following. Film nonideality may be allowed for [192]. There may be a chemical activation barrier to the transfer step from monolayer to subsurface solution and hence also for monolayer formation by adsorption from solution [294-296]. Dissolving rates may be determined with the use of the radioactive labeling technique of Section III-6A, although precautions are necessary [297]. 

How many carbon atoms of citronellal would be radioactively labeled if the acetic acid used in the experiment were enriched with at C 1 instead of at C 2 Identify these carbon atoms... 

Radiochemical methods of analysis take advantage of the decay of radioactive isotopes. A direct measurement of the rate at which a radioactive isotope decays may be used to determine its concentration in a sample. For analytes that are not naturally radioactive, neutron activation often can be used to induce radioactivity. Isotope dilution, in which a radioactively labeled form of an analyte is spiked into the sample, can be used as an internal standard for quantitative work. 

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

Biosynthesis. Biochemical studies on dalbaheptides have been reviewed (92,97). Experiments with and H have shown that in vancomycin (39), D-tyrosine is the precursor of D-/> -hydroxyphenyiglycine and P-hydroxy-y -chlorotyrosine, and acetate the precursor of the two y jy -dihydroxyphenyiglycines (98). Similar results using either or radioactively labeled material have been reported for avoparcin (Table 5) (23), ristocetin (Table 2) (99,100), ardacin (Table 3) (101), and A47934 (102). 

Radioisotopes have become very important ia the practice of modem medicine, for both diagnosis and treatment. Some diagnoses are done by injecting a radionucHde ia a biochemical form such that it goes to a particular organ, and the measured radiation then allows the functional level of that organ to be determined. A common treatment is to expose a portion of the body, for example a tumor, to radiation from a radioisotope with the source either internal or external to the body. Another usage iavolves radioactively labeled antibodies (see Immunoassay). 

Eor virtually all radiopharmaceuticals, the primary safety consideration is that of radiation dosimetry. Chemical toxicity, although it must be considered, generally is a function of the nonradio active components of the injectate. These are often unreacted precursors of the intended radioactive product, present in excess to faciUtate the final labeling reaction, or intended product labeled with the daughter of the original radioactive label. 

Radioisotope dilution assays are based on the principle of competition between radioactive labeled ( Co) vitamin B 2 and cobalamins extracted from matrices for binding sites on the intrinsic factor (a glycoprotein). Binding is in proportion to the concentration of the radioactive and nonradio active B 2 with the concentration of intrinsic factor as the limiting factor. Free cobalamins are separated from those bound on the intrinsic factor by absorption... 

The biosynthetic origin of monobactams has been elucidated by fermentation experiments using radioactively labeled amino acids (qv). The... 

Physical methods Physical methods include photometric absorption and fluorescence and phosphorescence inhibition, which is wrongly referred to as fluorescence quenching [1], and the detection of radioactively labelled substances by means of autoradiographic techniques, scintillation procedures or other radiometric methods. These methods are nondestructive (Chapt. 2). 

Physical detection methods are based on inclusion of substance-specific properties. The most commonly employed are the absorption or emission of electromagnetic radiation, which is detected by suitable detectors (the eye, photomultiplier). The / -radiation of radioactively labelled substances can also be detected directly. These nondestructive detection methods allow subsequent micropreparative manipulation of the substances concerned. They can also be followed by microchemical and/or biological-physiological detection methods. 

The scintillators are a special type of fluorescence indicators they are employed for the fluorimetric detection of radioactively labelled substances. They are stimulated by ) -radiation to the emission of electromagnetic radiation and will be discussed in Volume 2. 

Radioactively labelled compounds have been employed in biology for the clarification of metabolic processes since the mid-1940s It has, thus, been necessary to prepare such substances and to check on their punty... 

Because dideoxynucleotides lack 3 -OH groups, these nucleotides cannot serve as acceptors for 5 -nucleotide addition in the polymerization reaction, and thus the chain is terminated where they become incorporated. The concentrations of the four deoxynucleotides and the single dideoxynucleotide in each reaction mixture are adjusted so that the dideoxynucleotide is incorporated infrequently. Therefore, base-specific premature chain termination is only a random, occasional event, and a population of new strands of varying length is synthesized. Four reactions are run, one for each dideoxynucleotide, so that termination, although random, can occur everywhere in the sequence. In each mixture, each newly synthesized strand has a dideoxynucleotide at its 3 -end, and its presence at that position demonstrates that a base of that particular kind was specified by the template. A radioactively labeled dNTP is included in each reaction mixture to provide a tracer for the products of the polymerization process. 

Another widely used approach to the elucidation of metabolic sequences is to feed cells a substrate or metabolic intermediate labeled with a particular isotopic form of an element that can be traced. Two sorts of isotopes are useful in this regard radioactive isotopes, such as and stable heavy isotopes, such as or (Table 18.3). Because the chemical behavior of isotopically labeled compounds is rarely distinguishable from that of their unlabeled counterparts, isotopes provide reliable tags for observing metabolic changes. The metabolic fate of a radioactively labeled substance can be traced by determining the presence and position of the radioactive atoms in intermediates derived from the labeled compound (Figure 18.13). 

In 1952, Konrad Bloch and Robert Langdon showed conclusively that labeled squalene is synthesized rapidly from labeled acetate and also that cholesterol is derived from squalene. Langdon, a graduate student of Bloch s, performed the critical experiments in Bloch s laboratory at the University of Chicago, while Bloch spent the summer in Bermuda attempting to demonstrate that radioactively labeled squalene would be converted to cholesterol in shark livers. As Bloch himself admitted, All I was able to learn was that sharks of manageable length are very difficult to catch and their oily livers impossible to slice (Bloch, 1987). 

Acetate, labeled at either the methyl or carboxyl position, was significantly incorporated into both citronellal and citral. 2-C14-Mevalonate was similarly well incorporated, but, in contrast, 1-C14-mevalonate produced only very slight radioactive labeling in the terpenes. The results of these experiments are summarized in Table I. 

In studying two-component polymerization catalysts, beginning with Feldman and Perry (161), a radioactive label was introduced into the growing polymer chain by quenching the polymerization with tritiated alcohols. The use of these quenching agents is based on the concept of the anionic coordination mechanism of olefin polymerization occurring... 

Wiekowski, A. In Situ Surface Electrochemistry Radioactive Labeling 21... 

In-vitro models can provide preliminary insights into some pharmacodynamic aspects. For example, cultured Caco 2 cell lines (derived from a human colorectal carcinoma) may be used to simulate intestinal absorption behaviour, while cultured hepatic cell lines are available for metabolic studies. However, a comprehensive understanding of the pharmacokinetic effects vfill require the use of in-vivo animal studies, where the drug levels in various tissues can be measured after different dosages and time intervals. Radioactively labelled drugs (carbon-14) may be used to facilitate detection. Animal model studies of human biopharmaceutical products may be compromised by immune responses that would not be expected when actually treating human subjects. 

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