Radiolabeled tritiation

One approach that has been used quite widely to quantitate neurotransmitter release employs radiolabeled (tritiated) neurotransmitter analogs (e.g.. Reference 67). First, tissue is incubated in a buffer solution that contains tritiated neurotransmitter. During this time, the radiolabeled transmitter is taken up into cells by endogenous plasma-membrane transporters and packaged into vesicles by vesicular transporters. The tissue preparation then is rinsed in buffer to remove extracellular radiolabeled transmitter leaving only that which was taken up into cells. This stored transmitter is then released over time by exocytosis. To quantitate its release, the tissue is continuously perfused with buffer, and time-dependent aliquots are collected. Radioactivity is measured in the aliquots with a scintillation counter and is used as an index of endogenous neurotransmitter release. Rather than estimate absolute neurotransmitter release, this method is typically used to compare the relative release between two or more conditions. 

These factors make 125I the iodine label of choice for radiolabeling biological molecules. Its commercial availability from a number of suppliers at relatively low cost further adds to its popularity. Even though it has lower specific activity than 131I, iodine-125 still provides much greater sensitivity than 14C, 32P, 35S, or 3H in labeling biomolecules. In fact, the use of a radioactive iodine label can create probes that have 150-fold more sensitivity than tritiated molecules and as much as 35,000 times the detectability of 14C-labeled molecules (Bolton and Hunter, 1986). 

Cells that exhibit high (several hundredfold) levels of resistance to vinblastine, vincristine, and vindesine have an extremely limited capacity to accumulate radiolabeled vinblastine for example, essentially no increase in radioactivity associated with human leukemic lymphoblastic cells resistant to vinblastine could be detected over a 60-min incubation period in the presence of concentrations of tritiated vinblastine that were cytotoxic to parent cells. The parent cells, highly sensitive to vinblastine, were observed to accumulate vinblastine to levels seven-fold higher than those observed for the resistant cells (76). 

Know the physical properties of the substances with which you are working. Keep in mind that some compounds (such as acetaldehyde and tritiated water) have low boiling points. Again, keep in mind that some gloves do not offer an adequate barrier to certain chemicals. Some compounds enter the body with such facility that special care must be exercised when they are in use. One example is dimethyl sulfoxide, which as a solvent facilitates the entry of many solutes into the body. There are many known cases where radiolabeled compounds contaminated individuals who failed to consider this power of DMSO as a solute vehicle. 

The use of radi olabeled nucleic acid precursors, primarily tritiated thymidine or (deoxy)uridine, however, is subject to numerous drawbacks. Nevertheless, the use of radiolabeled precursors have provided some promising clinical correlation data [174]. 

Several applications of photoreactive peptides require the presence of a radionuclide to allow specific and sensitive detection of the photo-cross-linked conjugates. In several cases, radioiodination of tyrosyl moieties and radiolabeled Bolton-Hunter reagents have been used. However, the presence of a radiolabel within the benzophenone photophore is desirable, particularly when the objective is to identify the site of photo-insertion of benzophenone. To this end some radiolabeled, benzophenone-based compounds have been developed and used in peptide synthesis, in particular tritiated Phe(4-Bz) (Scheme 24)J2161 [1-14C-carboxy]-4-benzoylbenzoic acid,1221 and 4-benzoyl-(2,3-3H2)-dihydrocinnamic acid.[154l In addition, 4-(4-hydroxybenzoyl)phenylalanine (Scheme 25) has been directly radioiodinated with Na125I and Chloramine-T)151 ... 

Radiolabelled compounds for the histamine H3 receptor have been proven to be of great value in pharmacological and biochemical research. Some iodinated and tritiated compounds haven become standard research tools. In contrast, the development of PET or SPECT ligands has been proven to be troublesome. So far there has been no radiolabelled compound which is taken up into the brain in reasonable amounts to allow for PET or SPECT studies of the H3 receptor in the brain. The search for new radiolabelled compounds is still going on however, as is research to find out why these radiolabelled compounds do not enter the brain as opposed to their non-radiolabelled equivalents. One can detect pharmacological effects which can only be caused by central nervous system activity of these non-radiolabelled compounds. In view of the... 

Investigating metabolism or stability of prostanoids, radiolabeled precursors or analytes are often used. The tritiated or -labeled compounds can easily be detected without any further derivatization using online or off-line liquid scintillation, which is not impaired by any interferences derived from matrix components. Efficient but less sensitive PG analysis is possible by UV detection (190-210 nm) of underivatized substances demanding the remove of interfering contaminants or simple sample matrices like buffers or some cell supernatants. 

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