Method Fundamentals

Stable Isotopes as Tracers

Although radioactive tracers are familiar tools, the use of tracer elements and compounds to measure metabolic processes was developed first with stable isotopes in the late 1930s by Schoenheimer and Rittenberg soon after 2H and 15N (both stable isotopes) became available. Unlike radioactive isotopes, which are largely man-made, unstable, and decay to other elements, stable isotopes do not decay and are ubiquitous. Virtually all elements exist in nature in at least two stable isotopic forms with the same numbers of electrons and protons but with differing numbers of neutrons in the nucleus. The level of a specific isotopic form in nature is called its natural abundance. For tracer experiments, an element or a simple compound containing it, enriched with one of the isotopes, is prepared by mass-dependent separation on an industrial scale. This is then incorporated into the substrate of interest for biological experiments. In the current context, 2H2O (deuterium oxide, heavy water) is readily available from the electrolysis of water. Water enriched with 18O is prepared directly by fractional distillation or from nitric oxide after its cryogenic distillation.

No radioactivity is involved in the use of stable isotopes in human experiments; thus, the only effects that have to be considered in relation to risk to the subject are related to the physical properties of the isotopic labeled compound. There is inevitably some degree of isotopic discrimination in physical and enzymatic processes, but because stable isotopes are normally present in all biological material at natural abundance levels, the relevant consideration is only by how much and for how long amounts are changed in experimental procedures. Because highly precise measurement techniques are used, it is necessary only to increase isotopic enrichments in body water from natural abundance by very small amounts. In a typical experiment, 2H enrichment might be increased from 150 to 300 parts per million (ppm) and 18O from 2000 to 2400 ppm, and a return to natural abundance levels will occur with a biological half-life of 5-7 days. There is no evidence that amounts many times larger than these have any harmful effects.

Measuring Isotopic Enrichment

Mass spectrometry is a generic name for a family of methodologies in which compounds are ionised and separated on the basis of mass:charge ratio. The method of choice for the measurement of isotopic enrichment with sufficient precision for DLW experiments is isotope ratio mass spectrometry. This technique is applicable only to relatively simple molecules. It separates ions such as [2H—1H]+ and [1H—1H]+ (mass 3 and 2) or [12C16O18O]+ and [12C16O16O]+ (mass 46 and 44) and measures isotopic ratios (R) relative to an international standard, such as Vienna Standard Mean Ocean Water (V-SMOW; Table 1). For the DLW method, therefore, the isotopic enrichment in water from biological samples has to be measured as hydrogen or carbon dioxide. For hydrogen isotope analysis, a variety of methods have been used for the conversion including reduction by reaction with hot uranium or zinc, but these methods are difficult to automate. Currently favoured methods are the exchange of hydrogen in the water sample with gaseous hydrogen by equilibration in the presence of a platinum catalyst or reduction with hot chromium. Both of these techniques are automated in commercially available equipment. For oxygen isotopes, samples are usually equilibrated

Table 1 Typical isotopic ratios and equivalent enrichments measured in DLW experimentsa

Sample

2H

2H

18O

18O

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