Isotopic labels, randomization

In the determination of the sequence of intermediates of a specific pathway, one single probe, the use of isotopically labelled precursors, has proven of pivotal importance. With the appropriate safeguards, which we shall consider below, the administration of a compound isotopically labelled in a recognizable pattern and the subsequent isolation of a labelled metabolite in which the distribution of label can be recognized as occurring without randomization constitutes de facto evidence of a precursorial relationship. 

For HPLC MS/MS assays the use of stable isotope labeled internal standards is by far the best method to overcome any potential matrix effects and random variation in the MS/MS detector. If for any reason this stable isotope internal standard is not available, an analog compound with a mass different from the analyte can also be used. The chromatographic retention time of the internal standard, however, should be as close as possible to the retention time of the analyte. This ensures, that time dependent random variation in the ionization chamber, or whereever else in the MS/MS detector, are compensated by the internal standard. In a toxicokinetic assay described by Chi et al. (2003), for example, an internal standard was used which showed the same retention time as the analyte. 

In summary the methane fermentation of H2 (Reaction 2) is the only example so far which unequivocally uses COo as a methane precursor, and it may be the only methanogenic component in most other substrate decompositions reported as methane fermentations. There are, however, two examples of methane fermentations where the major methane precursor is never COo but an intact methyl group. In the methane fermentations of acetate and methanol, isotopic labels of the methyl groups are transferred without loss or randomization of their hydrogen substituents, to methane ... 

The effective interaction parameter (hXj/dXj.ej) in Eq. (19) is a function of two unknown parameters, %E/EE and %h/d, so these can be extracted [143] from two experimentally determined values of the overall interaction parameter for each pair of structurally identical mixtures with a swapped isotope labeled component, i.e., for dXj/hXj and hxj/dxj. In order to fit to the specific form AT-1 of the interaction parameter used in the approach described above we re-express all effective interaction parameters as [91] x(hXj/dxi,e1)=xcTc/T, where %c= (N1l/2+Njl/2)2/(2N1Nj) and Tc is given by experiment. It turns out [145] that this is a good approximation for studied random polyolefins (see Fig. 9) as the entro-pic term B of Eq. (17a,b) is small (it contributes less than 6% for most of the blends) and the weak ( -dependence of % may be neglected as it is not directly involved here. 

We have studied binary blends dxx/hx2 of random olefinic copolymers x=(Ex x EEx)n, with one blend constituent protonated (hx) and the other deuterated (dx). The blends examined were grouped in four pairs of structurally identical mixtures xx/x2 but with a swapped isotope labeled component (dxx/hx2 and hxx/dx2). For such blend pairs the bulk interaction parameter % (and hence also the critical point Tc) has been found (see Sect. 2.2.3 and references therein) to be higher when the more branched (say xx>x2) component is deuterated, i.e., X(dx /hx2)>x(hx /dx2) or Tc(dxx/hx2)>Tc(hxx/dx2) (see Fig. 9). An identical pattern is exhibited here by the force driving the segregation at the free surface. This is illustrated in Fig. 26a,b where the composition vs depth profiles of the more branched (xx) component are shown for blend pairs with swapped isotope... 

Let us consider the photolysis of DBK on NaX as a standard system (Table 3). Under a vacuum or in an argon atmosphere, the major product is 1,2-diphenyl ethane (DPE) which results from diffusional separation of the primary radical pair, followed by decarbonylation and random coupling of the benzyl radicals produced in the secondary radical pair. These results, along with isotopic labelling experiments, show that diffusional motion of primary and secondary radical pairs is fast compared to coupling or decarbonylation reactions of the primary or the secondary radical pairs. The results for... 

The isomerization of o-xylene in liquid-phase radiolysis, as well as in vapor-phase photolysis either at 2537 A. (17) or in the vacuum ultraviolet (13), gives a very high ratio of meta to para isomer. The more nearly statistical distribution of isomers observed in the vapor-phase radiolysis may again be rationalized in terms of an ionic process. Mass spectral studies (4) of isotopically labeled p-xylene indicate extensive randomization of carbon atoms in the abundant C8H9+ ion. It is not known whether such randomization ocurs in the parent ion or whether randomized ions could revert to xylenes, but the rearomatization of a randomized species has been demonstrated (19) in the radiolysis of toluene-7-14C vapor. 

We consider the situation in which a number of similar (but not necessarily identical) experiments are performed on a number of different subjects randomly selected from a larger population of individuals. Examples include a metabolic study in which isotopically labeled lipoprotein turnover is measured in a number of patients with heart disease, a nutritional study in which vitamin turnovers are examined in some randomly selected graduate students, or an agricultural study in which calcium kinetics are studied in a few dairy cattle randomly selected from a school s experimental herd. In... 

We turn, now, to the second question. A necessary condition for the application of quasiequilibrium theory is that the lifetime of the intermediate should be long enough for the internal energy to be equilibrated. There will, of course, always be a distribution of lifetimes and no method exists to measure this distribution.f Three means exist to set qualitative lower bounds on the lifetimes randomization of isotopic labels, energy equilibration (as revealed by the translational energy of the ionic products), and the angular distribution of the products. In the absence of any definite information on the time scales for these processes, we present possible order-of-magnitude values for the purpose of this discussion. The first process can be extremely rapid and requires only a few bond vibrations, i.e., 5 X 10 sec. The second process requires more vibrations, but,... 

This section provides a simplified introduction to the methods by which calibration solutions containing analytical (or reference) standards, with or without internal standards, are used in practice to measure amounts of analytes in unknown samples. The main deficiency of this brief account is its lack of any attempt to take into account experimental uncertainties (both random and systematic) and thus the level of confidence in the results thus obtained. While this book is directed towards analyses in which mass spectrometry is used as the chromatographic detection technique, most of the following discussion is applicable also to other detectors the main exception concerns use of isotope-labeled surrogate internal standards, for which only mass spectrometry can provide adequate detection. A much more complete account of this material, including a discussion of the associated random and systematic errors, is given in Section 8.5. 

Both primary and secondary carbocations with )8-phenyl substituents usually give evidence of aryl participation. For example, isotopically labeled carbons are scrambled to some extent during solvolysis of j8-phenylethyl tosylates, A bridged-ion intermediate or rapidly reversible rearrangement of a primary carbocation could account for the randomization of the label. The extent of label scrambling increases as solvent nucleophilicity decreases. The data are shown in Table 5.19. This trend can be attributed to competition between Sn2 displacement by solvent and ionization with participation of the aryl group. While substitution in more nucleophilic solvents such as ethanol proceeds almost exclusively by direct displacement, the non-nucleophilic solvent trifluoroacetic acid leads to complete randomization of the label. 

The target amino acids are usually chosen based on an a priori knowledge of some of the relevant structural details [162]. These methods require a carefijUy selected labeling scheme to counteract the effects of isotope scram-bhng and amino acid conversion, which might randomize the isotopic label within the protein [162,163]. 

---