Fractionation, double-isotope

The double-isotope technique is carried out as follows (DeGrazia et al., 1%5). The subject eon.sumes a test food containing caldum-45. About halfway through the absorpbve process, caJcium-47 is injected into a vein, in the form of a solution of CaCb. The absorptive process is about halfway complete at about 2 hours after a meal. The injected Ca is calcium that is 100% absorbed- A urine sample is collected 1 day after the oral aiid intravenous doses of radioactive calcium. The fraction of the oral dose absorbed is calculated by the formula ... 

Calcium absorption can be measured by the double-isotope technique. In this technique a meal containing caldum-45 is consumed and the radioactivity in the urine measured. The measurement of urinary Ca alone cannot provide the fractional absorption, because some of the Ca absorbed is taken up by cells, deposited in bone, or excreted in the bile. A second isotope of calcium, Ca, is used to correct for the fates of absorbed calcium, other than excretion in the urine. The use of the Ca is intended to eliminate cell uptake, bone deposit, and biliary losses as variables in the study of the absorption of the dose of cdcium-45. 

Based on the background I have presented on double-isotope fractionation, the observation that for malate synthase and (WA )h are identical supports... 

A comparison of the primary hydrogen isotope effect measured in two different ways for the malate synthase reaction provides the clue that permits interpretation of the unusual result in the double-isotope fractionation study. It leads to... 

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-... 

Kuo and Rose showed that the proton that is removed is retained by the enzyme (67). Stubbe and Abeles prepared an alternative substrate in which fluoride elimination competes with carboxylation 68, 69). Neither result defines the mechanism, but they do show that it is likely that the carbanion derived from the substrate is generated as an intermediate and therefore the reaction is not concerted. Definitive results come from double-isotope fraction studies by O Keefe and Knowles (70) and by Cleland and co-woricers (71). As described for Claisen enzymes, this methodology tests whether processes occur in one or two steps. Labeling of the carboxyl to be transferred with carbon-13 and the proton to be transferred as deuterium provided the means to do this test. The results indicate clearly that proton removal from the substrate to generate the carbanion and transfer of the carboxyl occurs in distinct steps. The resulting attack of the carb-... 

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 C4-secondary deuterium KIEs obtained from the double-isotopic fractionation mediod in substrates 3 and 4 were 1.009, 1.000 and 1.010, for formate, acetate and imidazole, respectively. 

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. 

For precise measurement of isotopic composition by mass spectrometry, it is also common to use either a natural, known isotopic ratio to correct for instrumental mass fractionation (e g., internal normalization) or to add a tracer for this purpose. For example for natural uranium samples, one can use the natural U/ U of 137.88 to correct for fractionation. Alternatively, one can use an added double spike of ratio -unity... 

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. 

The nucleosynthetic sources for Ti isotopes are very similar to those of the isotopes of Ca, and Ti requires a neutron-rich zone to be produced in significant amoimts. In addition to the nonlinear effects, absolute isotopic compositions have been measured in a number of samples using double spike techniques (Niederer et al. 1985). Mass dependent fractionation effects are rarely resolved and are small, below 1 %o/amu except in one sample, where it reaches 1.3 %o/amu. In general the fractionation is in favor of the heavy isotopes partial condensation or evaporation may explain of this observation. 

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.

Hamelin B, Manhes G, Albarede F, Allegre CJ (1985) Precise lead isotope measurements by the double spike technique a reconsideration. Geochim Cosmochim Acta 49 173-182 Hart SR, Zindler A (1989) Isotope fractionation laws A test using calcium. Int J Mass Spectr Ion Proc 89 287-301... 

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. 

Isotopic double spike. The most rigorous approach is to use an isotopic double spike , in which samples are doped with a known quantity of spike Mo which consists of two isotopes in a known ratio (Wetherill 1964 Siebert et al. 2001). These spike isotopes serve as an internal standard to monitor mass fractionation of the sample subsequent to spiking. The fundamental advantage over the element spike is that the spike isotopes follow exactly the same fractionation behavior as the isotopes of interest. A disadvantage is the need to carefully prepare and calibrate the double spike. 

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