Radiolabelling

In the human cell there are 23 pairs of chromosomes containing approximately 3000 million base pairs of DNA. Short sequences of DNA, perhaps with as few as 20 nucleotide units and sometimes radiolabeled, can be obtained either by chemical synthesis (gene machine) or from cloning. These short sequences can be used to probe for a complementary sequence by looking for the position to which they bind to any DNA sample under investigation, from blood for example. Such probes can detect as little as 100 fg of DNA and are the basis of forensic genetic fingerprinting tests. 

The actual time required for poly-L-lactide implants to be completely absorbed is relatively long, and depends on polymer purity, processing conditions, implant site, and physical dimensions of the implant. For instance, 50—90 mg samples of radiolabeled poly-DL-lactide implanted in the abdominal walls of rats had an absorption time of 1.5 years with metaboHsm resulting primarily from respiratory excretion (24). In contrast, pure poly-L-lactide bone plates attached to sheep femora showed mechanical deterioration, but Httie evidence of significant mass loss even after four years (25). 

The introduction of tritium into molecules is most commonly achieved by reductive methods, including catalytic reduction by tritium gas, PH2], of olefins, catalytic reductive replacement of halogen (Cl, Br, or I) by H2, and metal pH] hydride reduction of carbonyl compounds, eg, ketones (qv) and some esters, to tritium-labeled alcohols (5). The use of tritium-labeled building blocks, eg, pH] methyl iodide and pH]-acetic anhydride, is an alternative route to the preparation of high specific activity, tritium-labeled compounds. The use of these techniques for the synthesis of radiolabeled receptor ligands, ie, dmgs and dmg analogues, has been described ia detail ia the Hterature (6,7). 

The methods for detection and quantitation of radiolabeled tracers are deterrnined by the type of emission, ie, y-, or x-rays, the tracer affords the energy of the emission and the efficiency of the system by which it is measured. Detection of radioactivity can be achieved in all cases using the Geiger counter. However, in the case of the radionucHdes that emit low energy betas such as H, large amounts of isotopes are required for detection and accurate quantitation of a signal. This is in most cases undesirable and impractical. Thus, more sensitive and reproducible methods of detection and quantitation have been developed. 

The nonquantitative detection of radioactive emission often is required for special experimental conditions. Autoradiography, which is the exposure of photographic film to radioactive emissions, is a commonly used technique for locating radiotracers on thin-layer chromatographs, electrophoresis gels, tissue mounted on sHdes, whole-body animal sHces, and specialized membranes (13). After exposure to the radiolabeled emitters, dark or black spots or bands appear as the film develops. This technique is especially useful for tritium detection but is also widely used for P, P, and 1. 

Other methods of sensitive detection of radiotracers have been developed more recently. Eourier transform nmr can be used to detect (nuclear spin 1/2), which has an efficiency of detection - 20% greater than that of H. This technique is useful for ascertaining the position and distribution of tritium in the labeled compound (14). Eield-desorption mass spectrometry (fdms) and other mass spectral techniques can be appHed to detection of nanogram quantities of radiolabeled tracers, and are weU suited for determining the specific activity of these compounds (15). 

Radioactive tracers account for about 20% of the worldwide market for consumables and reagents for life science research. In 1994 the value was estimated at about 300 million. The principal fuU line manufacturers are Du Pont—NEN Research Products (Boston, Massachusetts) and Amersham International (Amersham, U.K.). These companies share roughly equaHy about 85% of the radiochemicals worldwide market. In addition to an extensive line of catalog products, these suppHers offer custom labeling and custom synthesis services. The rest of the market is shared by producers of a limited range of products or services, such as ICN Biomedicals (Costa Mesa, California) and American Radiolabeled Chemicals (St. Louis, Missouri). 

Radiotracers have also been used extensively for the quantitative rnicrodeterrnination of blood semm levels of hormones (qv), proteins, neurotransmitters, and other physiologically important compounds. Radioimmunoassay, which involves the competition of a known quantity of radiolabeled tracer, usually I or H, with the unknown quantity of semm component for binding to a specific antibody that has been raised against the component to be deterrnined, is used in the rnicro deterrnination of physiologically active materials in biological samples (see Immunoassay). 

The deterrnination of the presence of reverse transcriptase in vims-infected cells can be done using labeled nucleotide triphosphates. Reverse transcriptase is an enzyme capable of synthesizing DNA from RNA and it is thought to play an important role in vims-mediated cell modification. This enzyme is also a marker enzyme for HIV, the vims impHcated in causing acquired immunodeficiency syndrome (AIDS). The procedure utilizes radiolabeled nucleotides with nonlabeled substrates to synthesize tagged DNA. The degree of radioactive incorporation reflects the reverse transcriptase activity. 

In the petroleum industry, the size of an underground oil deposit is deterrnined by the injection of radiolabeled substances into a well head. The occurrence of radioactivity in the oil—water mixture, which is pumped out of adjoining wells, gives an indication of the pocket size of the oil deposit (see Petroleum). 

There are three general types of radiopharmaceuticals elemental radionucHdes or simple compounds, radionucHde complexes, and radiolabeled biologically active molecules. Among the first type are radionucHdes in their elemental form such as Kr and Xe or Xe, and simple aqueous radionucHde solutions such as or I-iodide, Tl-thaUous chloride, Rb-mbidium(I) chloride [14391-63-0] Sr-strontium(II) chloride, and Tc-pertechnetate. These radiopharmaceuticals are either used as obtained from the manufacturer in a unit dose, ie, one dose for one patient, or dispensed at the hospital from a stock solution that is obtained as needed from a chromatographic generator provided by the manufacturer. 

An important consideration for all radiopharmaceuticals and especially radiolabeled biologically active molecules is specific activity. There are two types of specific activity radionucHdic and biological. RadionucHdic specific activity refers to the ratio of the number of atoms of a particular radioisotope to the total number of atoms of the element. For Tc, the radionuchdic specific activity is the number of Tc atoms to the total number of Tc and Tc atoms. Because all isotopes of an element ate chemically identical, a low specific activity may lead to a low yield in the synthesis of a radiopharmaceutical if a significant proportion of the reagents is consumed by the undesited isotopes. 

Radiolabeled folate provides a powerful tool for folate bioavaHabiUty studies in animals and for diagnostic procedures in humans. Deuteration at the 3- and 5-positions of the central benzene ring of foHc acid (31) was accompHshed by catalytic debromination (47,48) or acid-cataly2ed exchange reaction (49). Alternatively, deuterium-labeled fohc acid (32) was prepared by condensing pteroic acid with commercially available labeled glutamic acid (50). 

EoUowing po administration moricizine is completely absorbed from the GI tract. The dmg undergoes considerable first-pass hepatic metabolism so that only 30—40% of the dose is bioavailable. Moricizine is extensively (95%) bound to plasma protein, mainly albumin and a -acid glycoprotein. The time to peak plasma concentrations is 0.42—3.90 h. Therapeutic concentrations are 0.06—3.00 ]l/niL. Using radiolabeled moricizine, more than 30 metabolites have been noted but only 12 have been identified. Eight appear in urine. The sulfoxide metabolite is equipotent to the parent compound as an antiarrhythmic. Elimination half-life is 2—6 h for the unchanged dmg and known metabolites, and 84 h for total radioactivity of the labeled dmg (1,2). 

A number of excellent studies have used a variety of radiolabeled sods to iavestigate the removal of small amounts of colorless sods such as ods (102—104). By proper use of different radiolabels (such as and " C), the preferential removal of various components ia a sod mixture can be followed. In these cases ia particular, detergency can also be calculated from measurements of the amount of radioactivity that is removed from the fabric and is found ia the wash Hquor. 

Hyphenation of HPLC with NMR combines the power of sepai ation with a maximum of stiaictural information by NMR. HPLC-NMR has been used in the detection and identification of diaig metabolites in human urine since 1992. The rapid and unambiguous determination of the major metabolites of diaigs without any pretreatment of the investigated fluid represents the main advantage of this approach. Moreover the method is non-destmctive and without the need to use radiolabelled compounds. 

The synthetic procedure has been extended to the preparation of trifluoro-acetyl hypofluorite [16, 17], long-chain perfluorinated hypofluorites [18, 19], and radiolabeled hypofluorites [II, 12],... 

Applications in agrochemicals [42, 43], pharmaceuticals [44,45], and positron emission tomography (PET) [46, 47, 48 49] have resulted in the resuscitation of the Wallach reaction The Wallach technique provides high-specific-activity F-radiolabeled aromatic fluoride for PET studies, in contrast to the low-specific-ac-tivity product by the Balz-Schiemann route... 

Aromatic fluorodenitration was first discovered in the reaction of polychloro-nitrobenzenes with potassium fluoride, when 2,3,5,6-tetrachlorofluorobenzene was prepared in 37% yield from 2,3,5,6-tetrachloronitrobenzene 105] The technique has been adapted to prepare aryl fluorides from other activated nitro aromatics for applications in pharmaceutical and polymer chemistry (equation 31) Fluorodenitration also has been applied to prepare radiolabeled ( F) fluo-roaromatics [74, 106]... 

The AMAPs (2-[ arylmethyl amino]-l,3-propanediols) are a class of planar polycyclic aromatic derivatives, which contain polar side-chains. They are known to be DNA intercalators and possess broad spectrum antitumour activity. An approach to C-radiolabelled AMAP derivative 40 used the Bucherer reaction as an initial starting reaction. 2-Naphthol was reacted with 4-bromophenylhydrazine 38 in the presence of sodium metabisulfite and HCl to afford 39. Subsequent derivatisation of 39 afforded 40. 

Finally, receptor stimulus can be measured through membrane assays directly monitoring G-protein activation (group IV assays). In these assays, radiolabeled GTP (in a stable form for example, GTPj/S) is present in the medium. As receptor activation takes place, the GDP previously bound to the inactive state of the G-protein is released and the radiolabeled GTP/S binds to the G-protein. This is quantified to yield a measure of the rate of GDP /GTP j/S exchange and hence receptor stimulus. 

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