Multiple labeled precursors

As can be concluded from the above outline, access to carotenoids isotopically labelled with and at predetermined positions is an essential part of this strategy. Labelled carotenoids can be prepared in two ways, namely by biosynthesis or by chemical synthesis. For the preparation of specifically labelled carotenoids biosynthesis is not suitable the labelled carotenoids are obtained by growing bacteria or yeasts on media containing simple isotopically labelled precursors such as sodium acetate [19]. The position of labelling can often not be predetermined and multiple labelling often occurs. Another major disadvantage is that isotopic dilution occurs for studies of carotenoid-protein complexes, carotenoids labelled at specific positions with high isotopic enrichment (preferably >99%) are needed. 

A second argument that was finnished for the hypothesis that (22) is formed via the direct condensation of C-4 of acetoacetate with (10) stennned fi-om a feeding experiment with [l,2-i C2] acetate in D. stramonium in which a small proportion of molecules of (3) carried contiguous label at C-2, C-3 and C-4. However, this is simply due to the incorporation of two labelled acetate molecules into the same molecule of (3), which could happen whichever pathway is followed. The extent to which such multiple labeling occurs is solely determined by the degree of dilution of the labeled precursor by natural abundance material... 

Reticuline is a precursor of sinomenine, via sinocutine, in Sino-menium acutum (115). It is also a precursor of berberine (LXI) in Hydrastis canadensis L. and of protopine (LXII) in Dicentra spectabilis (L.) Lem. (116). In the case of the former base the i T-methyl group of reticuline is used to form the berberine carbon (carbon atom 8) of the tetracyclic alkaloid and with the latter base it is found as the same carbon atom in the heterocyclic ring of protopine. By feeding multiple-labeled (+ )-reticuline, as indicated in LXIII, to Chelidonium majus L., it was shown that it is a precursor of chelidonine (LXIV), (— )-stylopine (LXV), and protopine (LXII), and that the labeled atoms are found in the places expected from current biogenetic theories (117). 

These hold for nucleus i of radical A, and fi, e and uy are labels indicating precursor multiplicity, c or e product, and whether i and j are in the same or different radicals, viz.. 

The resulting device has demonstrated both FDG and FLT labelling at yields of 98% and 90%, respectively, in 100 s compared to typical macro-scale labelling of 65% in 45 min for FDG and 30% in 90 min for FLT. The use of acetonitrile, DMSO and HC1 have shown no degrading effect on the system. Extremely efficient labelling illustrates the effectiveness of flow-based micro-reactors for PET biomarker synthesis. Multiple biomarkers can be produced in 1-2 min, while using only micro-litres of precursor and can revolutionise the production of radiotracers. Small reaction volumes, improved yields, and the ability to synthesise small quantities of a variety of new compounds will allow preclinical and clinical evaluation of new PET agents with potential for clinical utilisation. 

Once the RNA is prepared, it is converted to cDNA and labeled either with radioactive precursors ([33P]-dCTP or [32P]-dCTP) or with non-radioactive fluorescent labels such as Cy3-dNTP and Cy5-dNTP. Radioactive labels are commonly used for membrane approaches while fluorescent probes are the method of choice for glass slides. When using fluorescent probes, cDNA prepared from controls can be labeled with one dye while cDNA from treated samples can be labeled with the other. These are then mixed and used in competitive binding to probes on the chip. Alternatively, one can compare all control and experimental samples to the same reference RNA sample, labeled with one of the dyes. Reference RNA samples are useful for multiple sample analysis and to account for chip-to-chip variations. In the case of the Affymetrix technique, cDNAs are converted back into fluorescently labeled RNA targets, which bind more tightly than cDNA targets to the short oligonucleotides present on those chips, and the control and experimental samples are applied individually to separate chips. 

Many of the chromatographic procedures are applicable to the measurement of labeled hydroxyproline in general they do not allow for multiple simultaneous determinations. Since collagen hydroxyproline is labeled only by administering its precursor, proline, in labeled form, the difficulties in radioactive studies have arisen largely because the nonhydroxy-proline metabolic products of proline or proline itself have not been rigorously separated from hydroxyproline or hydroxyproline-derived pyrrole. This is the purpose of the more recently introduced modifications for labeled hydroxyproline assay (Jll, L12). It is too early to know whether one or both of these will suffice for general use. 

The possibUity of multiple pathways in bile acid biosynthesis in man has been discussed by Vlahcevic et al. [180-182]. A number of labelled 7 -hydroxylated intermediates in bile acid biosynthesis were administered to bile fistula patients as well as patients with an intact enterohepatic circulation. In accordance with previous work with bile fistula rats, the spedfic activity of the isolated chohc add was in general considerably lower than that of chenodeoxychohc acid. On the basis of this finding, it was suggested that a portion of chohc acid was synthesized via a route not involving initial 7a-hydroxylation of cholesterol. It must then be assumed that the administered intermediate mixes with the endogenous pool of the same steroid. However, due to compartmentation, the metabolic fate of a precursor reaching the hepatocyte might be different from that of the the same compound formed within the cell. Normally, the different precursors are present in the cells in trace amounts... 

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