Experimental design of tracer experiments

The calculation of corresponding analytical equations for realistic conditions, considering bidirectional fluxes, natural isotopes, or other tracer substrates such as multiply labeled compounds is much more complex or even impossible, with respect to the non-linearity of such systems. The same holds for alternative metabolites applied for the labeling measurement, such as glutamate or lysine, which are located in the network at a far distance to the flux node of inte- 

Obviously, the behavior of the network under realistic conditions is significantly different from the one, predicted in the simplified example in Eq. (4). Calculation of ppp from the pyruvate labeling via the simplified approach thus leads to errors in the result. 

Using this approach, general guidelines for experimental design of C-tracer studies with MS could be shown for the central metabolism of Corynebacterium glutamicum comprising various flux scenarios and tracer substrates [26]. 

Besides C, also other stable isotopes, such as H, or have been applied. An example for the use of is the quantification of the flux partitioning 

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Two formal approaches have been established to solve isotopomer balances for biochemical networks in a generally applicable way (i) the transition matrix approach by Wiechert [22] and (ii) the isotopomer mapping matrix (IMM) approach by Schmidt et al. [14]. The matrix transition approach is based on a transformation of isotopomer balances into cumomer balances exhibiting a much greater simplicity. As shown, non-linear isotopomer balances can always be analytically solved by this approach [16]. The matrix transition approach was applied for experimental design of tracer experiments and for parameter estimation from labeling data [16,23]. 

Fig. 2. Experimental design of a C tracer experiment for the determination of the flux partitioning ratio between pentose phosphate pathway and glycolysis ( ppp) C label distribution from l- C glucose through the network with C atoms (black) and C atoms (white)...

The above example demonstrates that treatment of the basic data by different numerical methods can produce distinctly different results. The discrepancy between the results in this case is, in part, due to the inadequacy of the data provided the data points are too few in number and their precision is poor. A lesson to be drawn from this example is that tracer experiments set up with the intention of measuring dispersion coefficients accurately need to be very carefully designed. As an alternative to the pulse injection method considered here, it is possible to introduce the tracer as a continuous sinusoidal concentration wave (Fig. 2.2c), the amplitude and frequency of which can be adjusted. Also there is a variety of different ways of numerically treating the data from either pulse or sinusoidal injection so that more weight is given to the most accurate and reliable of the data points. There has been extensive research to determine the best experimental method to adopt in particular circumstances 7 " . 

Baptist and Lewis (1969) designed an experiment to study the transfer of Zn and Cr through an estuarine food chain that involved transfer of assimilated and unassimilated radionuclides through four trophic levels. They described their study as that of an . . . unnatural and simple but reproducible food chain and uniform, controlled environmental conditions to facilitate comparisons between the experiments. Their stepwise experiments are illustrated in Figures 88 and 89. The use of Zn and Cr was not in the function of tracers, since the investigators were interested in these specific radionuclides that have been introduced into the marine environment by man s activities. The authors commented that limiting experimental conditions probably resulted in lower concentrations than might be expected in the natural environment. They stated ... 

The proper design and execution of radiotracer experiments requires preliminary possession of both the technical background and the basic principles of the tracer method The latter make it possible to evaluate the kinetic aspects of the system under investigation, their possible infiuence on experimental results, and the technical requirements, which should be never overlooked. Attention must emphatically be drawn to these factors, in view of the considerable misuse of radio-tracers in biological investigations which has unfortunately accompanied their growing popularity. 

The conclusions presented above can be applied in designing experiments on chemical identification and studies of element 112. There is little doubt that the element is a congener of mercury, at least equally volatile and chemically inert. Then proper chemical environment can be even simpler than in the case of HSO4. However, the experimental technique must allow for the possibility that element 112 is much more volatile and chemically inert than mercury. The problem is to guarantee the registration of the element (not to lose it) even if it resembles Rn, rather than Hg, in volatility and inertness. Atomic mercury in tracer quantities can be transported by inert gas at ambient temperature through tubes made of various materials. However, it adsorbs onto some metals, in particular, on gold. 

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