Double-isotopic fractionation method

Is any reasonable mechanism consistent with the data The answer lies in an observation of a probable isotope effect in a coupled nonenzymic phenomenon. The double-isotope fractionation method does not enter into the analysis. The keto group of glyoxalate is actually present as a covalent hydrate to the extent of about 99% of the total glyoxalate concentration (27). However, the ketone is the form that will react in the enzymic process and the concentration of ketone determines the rate of reaction and binding to the enzyme. The equilibrium between ketone and hydrate is not catalyzed by the enzyme and as a result the isotope effect on this equilibrium will appear in the measured kinetic isotope effects. Of course, the extent of this equilibrium will not be affected by deutera-tion of the methyl group of acetyl-CoA. Therefore, the observed HVIK) is not an indication of kinetically significant carbon-carbon bond formation but of a preequilibrium hydration, a process that is independent of the enzyme. The value for HV/K) of 1.0037 is consistent with measured equilibrium isotope effects in related molecules (23). Therefore, the deuteration of acetyl-CoA has no effect on the observed kinetic because that value in fact is due to a preequilib-... 

The double-isotopic fractionation method was employed in this study. This procedure consists of the use of deuterium substitution to selectively slow down the rate of one step in a reaction and observing the changes in a second kinetic isotope effect. In this study flie aim is to obtain information from the secondary D KIE at C4 and the fluorine isotope effects, respectively. For that reason, substrates on which the deuterium has been placed at C3 to slow down the step in which the C3-H bond is being broken, have been employed. Then, the extra labels were deuterium at C4 (compound 4) and labeled fluorine (compound 6) (atoms colored red in Fig. 37.1). Compounds 3 and 4 were used to determine the effect of the deuterium at C3 on the secondary D KIE values at C4 (k lk ) (Fig. 37.1). Similarly, compounds 5 and 6 were used to study the influence of the deuterium label at C3 on the leaving group F KIEs... 

The double-isotopic fractionation method is a good tool to distinguish between... 

Only by means of primary, secondary and leaving group F KIEs it is not possible to decide whether the base-promoted HF elimination of ketone 1 follows a concerted or a stepwise mechanism. The double-isotopic fractionation method provides strong evidence for a stepwise process, more hkely via an ElcBin mechanism. 

In principle, the three isotope method may be widely applied to new isotope systems such as Mg, Ca, Cr, Fe, Zn, Se, and Mo. Unlike isotopic analysis of purified oxygen, however, isotopic analysis of metals that have been separated from complex matrices commonly involves measurement of several isotopic ratios to monitor potential isobars, evaluate the internal consistency of the data through comparison with mass-dependent fractionation relations (e.g., Eqn. 8 above), or use in double-spike corrections for instrumental mass bias (Chapter 4 Albarede and Beard 2004). For experimental data that reflect partial isotopic exchange, their isotopic compositions will not lie along a mass-dependent fractionation line, but will instead lie along a line at high angle to a mass-dependent relation (Fig. 10), which will limit the use of multiple isotopic ratios for isobar corrections, data quality checks, and double-spike corrections. 

Corrections for instrumentally-produced mass fractionation that preserve natural mass dependent fractionation can be approached in one of two ways a double-spike method, which allows for rigorous calculation of instrumental mass fractionation (e.g., Dodson 1963 Compston and Oversby 1969 Eugster et al. 1969 Gale 1970 Hamelin et al. 1985 Galer 1999 see section Double-spike analysis ), or an empirical adjustment, based on comparison with isotopic analysis of standards (Dixon et al. 1993 Taylor et al. 1992 1993). The empirical approach assumes that standards and samples fractionate to the same degree during isotopic analysis, requiring carefully controlled analysis conditions. Such approaches are commonly used for Pb isotope work. However, it is important to stress that the precision and accuracy of isotope ratios determined on unknown samples may be very difficult to evaluate because each filament load in a TIMS analysis is different. 

Figure 9. Sketch of the double spike Zn- Zn method. The surface is constructed by drawing an infinite number of straight-lines through the point representing the spike composition (supposed to be known with no error) and each point of the mass fractionation line going through the point representing the measured mixture. One of these straightlines, which is to be determined from the calculations, is the sample-spike mixing line (stippled line). Each determination of the Zn isotope composition of a sample involves only one run for the mixture of the sample with the spike. Since all natural samples plot on the same mass fractionation line, any reference composition will adequately determine isotope composition of the sample, note that, since the instrumental bias is not linear with mass, the mass discrimination lines are curved.

The limitations discussed above also apply approximately to measurements of mass dependent Ca isotope effects. The additional problem is to separate mass dependent fractionation in nature from mass dependent fractionation in the mass spectrometer. The maximum observed natural fractionation is about +0.1% per mass unit, whereas instrumental fractionation is about +0.5% per mass unit (for TIMS and much larger for ICPMS). The separation is accomplished with the use of a double spike (Russell et al. 1978b). The approach is illustrated here using the methods of Skulan et al. (1997), but other researchers have used slightly different algorithms and double spike isotopes (Zhu and MacDougall 1998 Heuser et al. 2002 Schmitt et al. 2003a). 

The double-spike technique of Rosman (1972) has been revived by Tanimizu et al. (2002), who used a Zn- Zn spike and obtained precisions in the range of a fraction of a per mil. Jackson and Gunther (2003) describe a laser-ablation technique of isotopic measurement, which provides a precision comparable to the standard solution nebulization methods. 

Another way to do the correction of Pb isotope mass fractionation is to use double spike method, which will be discussed in Chapter 11. 

The spike in most cases is single spike, but it can be double spike. Single spike method provides the information of the concentration of the sample, whereas the double-spike method can yield the concentration of the sample and the mass fractionation factor that can be used to calculate the true isotopic ratios in the sample. Take Pb isotope as an example. Natural Pb samples have 1.4% ° Pb, 24.4% ° Pb, 22.1% ° Pb and 52.4% ° Pb. A single spike is made by artificially concentrating one of the minor Pb isotopes, for example Pb. A double spike is made by artificially concentrating two minor isotopes. For example, we can concentrate Pb and b isotopes to make ° Pb- Pb double spike or concentrate Pb and Pb to make Pb- ° Pb double spike. 

A single spike is enriched in one minor isotope whereas a double spike is enriched in two minor isotopes (Fig. 11.3). Double spike method needs two runs. The first run is to measure the un-spiked sample and the second run is to measure the spiked mixture. Double spike method not only provides the concentrations of the sample but also corrects the mass fractionation in mass spectrometers (and thus giving the true isotopic ratios). 

Measurement of fecal excretion of isotopic bile acids (65) gives only the half-life of the labeled bile acid used. The isotope is injected intravenously, and the daily fecal excretion of radioactivity is measured. According to this procedure, the fractional excretion rate of cholic acid in man is normally about 12-13% per day (66,67). Disadvantages of the method are that the absolute values are not obtained, the cholic and chenodeoxycholic acid excretions must be measured separately or a double label method must be used, and the fecal flow should be regular, though an unabsorbable fecal marker can be used. The method appears to be suitable for screening of ileal dysfunction. 

The primary disadvantages of the double-spike technique are that (i) the preparation and calibration of a new double spike require significant effort and (ii) four interference-free isotope signals are needed for accurate data reduction, and this also rules out double-spike analysis of elements that feature only two or three isotopes. In many cases, however, these factors will be outweighed by the advantages of the method (i) it offers an instrumental mass bias correction that is similar in application and reliability to internal normalization and hence is even more robust towards matrix effects than external normalization (ii) the approach can correct for laboratory-induced mass fractionation effects, if the spike is added to the samples prior to the chemical processing and (iii) precise elemental concentration data are obtained as a byproduct of the double-spike method. Hence the double-spike method has recently found increasing popularity in MC-ICP-MS stable isotope analysis of non-traditional elements. 

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