The Use of Isotopic Tracers in Estimating Rates of Metabolic Reactions

THE USE OF ISOTOPIC TRACERS IN ESTIMATING RATES OF METABOLIC REACTIONS JANE A. RUSSELL, Ph.D.* Since the introduction of practical methods for the production and measurement of substances containing isotopes of elements common in biological systems—such as hydrogen, carbon, nitrogen, and phosphorus— these have found expanding uses as "tracers" in biological systems. For the identification ofintermediate compounds and the qualitative description ofbiochemical pathways and reaction mechanisms, they have allowed unprecedented advances; and some isotopically labeled substances also have been useful in analytical measurements—by the "isotope dilution" principle—-ofthe quantities ofmaterial present or oftheir volumes ofdistribution in biological systems. A further type of use, of great potential importance theoretically, has often been attempted: by observation ofthe "movement" of labeled material to measure the absolute or relative rate ofmovement or ofchemical transformation ofunlabeled substance already present in the system. To the uninitiated in this field, this process would seem to be quite simple in principle. A substance containing a known quantity ofthe isotopic element would be introduced into the animal or surviving metabolizing system, and after a time one would find out how much ofthe isotope was present in a particular place or form. This might be feasible ifone were dealing only with a single simple transfer ofmaterialfrom one "compartment" ofknown dimensions to another. In biological systems, however, this is very seldom the case. Instead, one finds that the transfer may be reversible; that what appears to be a simple transfer is infact a series ofreactions (some ofthem reversible, perhaps); that the substance introduced is itselfbeing replaced with non-isotope material as fast as it is used; or that it can exchange with not one but many other forms. * Department ofBiochemistry, Emory University, Georgia. I38 Jane A. Russell · Isotopic Tracers Perspectives in Biology and Medicine · Winter 1 tj Hence the interpretation ofrates ofmovement ofthe isotope in terms of the rates ofmovement of unlabeled materials is seldom simple and may be deceptively complicated. The purpose ofthe present paper is to describe as simply as possible the principles of the kinetics of labeled materials, to follow these principles through some of their logical consequences, and to illustrate their application to metabolic systems. In this analysis, it may seem that the pitfalls, difficulties, and alternative interpretations have been emphasized more than the positive applications of these methods. If so, this is not because the possibility of using isotopic techniques for these purposes is seriously questioned but because the naive interpretation of data from the use of isotopes can lead to completely mistaken conclusions—and, indeed, many times has done so. In this discussion no attempt has been made at rigorous mathematical analysis of the more complex systems, which have been treated by others elsewhere. Rather, it is hoped that a more simplified discussion may assist the general reader in biological fields in understanding the nature of the evidence and the extent of the valid conclusions which may be drawn from the experimental uses ofisotopic substances in living or surviving systems. The principles upon which this discussion is based are not at all new. However, although many analyses are available, most of these have been either advanced mathematical treatments ofa theoretical nature, or applications to special situations only, or wider reviews which could not give detailed attention to metabolic systems. The papers which have served as the principal bases here are those ofSheppard and Householder (i) on the theory ofthe kinetic distribution ofisotopes and ofReiner (2) on the application of theoretical considerations to model systems. A recent and very useful review by Robertson (3) on the theory and use of isotopes in the determination oftransfer rates includes a fulllist ofpublications related to this field. I. Elementary Principles The fundamental relationships between the amounts of isotopically labeled substance present at any time and rates of movement of material, identical in form with those derived by more rigorous and general mathematical treatment (1), can be deduced from logical considerations. Let us take first the most simple situation possible. 139 Some material ofa certain molecular species is labeled by the introduction ofa small amount ofmaterial which is identical except that some of the molecules contain one or more isotopic atoms. The labeled substance is assumed to...