An ideal tracer is chemically identical to the compound of interest (the tracee) but distinct in some characteristic that enables its precise detection. In the case of tracers labeled with stable isotopes, the principal characteristic of distinction is their difference in mass from the naturally occurring form. Thus, mass spectrometry is the most precise means of detecting the abundance of tracers labeled with stable isotopes. Consequently, the focus of this chapter will be on techniques relying on mass spectrometry to quantify the abundance of stable isotopes of carbon and hydrogen in order to gain insight into the regulation of substrate metabolism.

Conventionally, the methodology involves the infusion of a compound labeled in a specific position of the molecule with a stable isotope (e.g., [1-¹³C]-palmitic acid) in tracer doses. The infusion rate of the tracer, therefore, is trivial by comparison to the endogenous kinetics of the tracee. Blood, tissue, and/or breath samples are then obtained, and kinetic parameters are calculated using a mathematical model of varying complexity. The aim of this chapter will be to present examples of tracer methods to quantify both the plasma kinetics of glucose and free fatty acids (FFA) and also the relative contributions of the oxidation of intracellular glycogen and triglyceride to the total rate of oxidation of carbohydrate and fat, respectively. An exhaustive exposition on all possible methods of studying glucose and fat metabolism with stable isotopes is beyond the scope of this report. Because it is possible to make use of varying abundances of ¹³C that occur naturally, a method also will be presented in this chapter that enables the quantitation of total carbohydrate and fat oxidation using the measurement of the rate of total carbon dioxide excretion () and the natural abundance of ¹³C in breath, and in the glucose, fat, and protein in the body. This method will be referred to as the "breath ratio method."

The method is based on the measurement of the absolute ¹³C/¹²C ratios in expired breath and in endogenous glucose, fat, and protein (Romijn et al., 1992). Because of a small amount of fractionation of ¹³C in certain synthetic pathways such as photosynthesis, the natural abundance of ¹³C in glucose, fat, and protein varies. Whereas the macronutrients may differ in enrichment, over time in one individual, the values should be reasonably constant if the diet is stable. The differences can be further amplified by the ingestion of ¹³C-enriched cornstarch for a few days before the study.

The enrichment of the CO₂ in the breath (RB) will be the sum of the proportional contribution of the oxidation of carbohydrate (x), fat (y), and protein (z) to produce CO₂, multiplied by the respective enrichments of each substrate (R_x, R_y, and R_z). Thus,