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One reason horses are fed is to provide them with the energy necessary to perform the exercise they are being asked to do. This energy can come from various feed ingredients such as carbohydrates, fats, and fermentable fiber. Some of these energy substrates are better suited to sprint-type performance, while others are more efficient in fueling endurance exercise.

Nutrition research methods can analyze the particular energy substrate a horse is using as it performs different types of exercise. One technique used to find this information is the stable isotope tracer method. This protocol, which was the subject of a presentation at a Kentucky Equine Research conference, is summarized below.

Use of tracers labeled with stable (nonradioactive) isotopes is an important technique for studying energy substrate metabolism in horses. A wide variety of metabolic processes involving carbohydrate, lipid, or protein metabolism can be studied using stable isotopes of carbon (13C), hydrogen (2H, deuterium), nitrogen (15N), or oxygen (18O).

By definition, the term isotope refers to all forms of a given element containing different numbers of neutrons. A stable isotope is defined as the nonradioactive isotope that is less abundant than the most abundant naturally occurring isotope. For tracer studies, one or more atoms in the structure of interest are replaced with a stable isotope such as deuterium. For example, in studies of glucose kinetics, [6,6-2H] glucose is frequently used. In this form, two 1H atoms at the 6-carbon position on the glucose ring have been replaced by two deuterium (2H) atoms.

When studying the kinetics of a metabolic substrate such as glucose, we refer to the endogenous unlabeled substrate as the tracee. The tracer is the form of the trace substance containing one or more stable isotopes (e.g. [6,6-2H] glucose), and the isotopic enrichment is the ratio of tracer to tracee in blood samples. Using glucose as the example, the isotopic enrichment is the ratio of labeled to unlabeled glucose in the sample. One of the underlying principles of this method is that the tracer should not significantly change the tracee pool size (the total amount of tracee substance in the body). Therefore, a typical infusion protocol will result in about a 2-3% enrichment of the blood, so in the case of glucose studies, the tracer is 2-3% of the total glucose pool. Nonetheless, highly sensitive analytical methods (gas chromatography-mass spectrometry) allow detection of very small changes in isotopic enrichment, thus permitting study of the effects of various interventions on the kinetics or turnover of the substrate of interest.

In practical terms, the stable isotopically labeled tracer substance is administered to “trace” the kinetics of production and utilization of that substance. Two main kinetic parameters are calculated. The rate of appearance, or Ra, is the total rate of appearance of the substrate into the sampling pool. Physiologically, the Ra is the production rate of a given substrate. For example, for the glucose system, the Ra reflects hepatic glucose production; for studies of fatty acids, the Ra represents release of NEFA from adipose tissue and lipoproteins. Depending on the feeding state of the animal, a portion of the total Ra may also reflect uptake of the nutrient from the gut. The rate of disappearance, or Rd, is the rate of loss of substrate from the sampling pool. Physiologically, the Rd is the rate of irreversible tissue uptake, with perhaps a small component from excretion in urine. During exercise, it is well recognized that more than 90% of the Rd for substrates such as glucose and NEFA reflects uptake and utilization by working muscle. Taken together, the Ra and Rd provide estimates of the rates of production and utilization of a substrate. Thus, when compared to a static measure such as the blood glucose concentration, a significant advantage of the isotopic tracer method is the ability to monitor the dynamics of substrate metabolism during exercise. Furthermore, it becomes possible to obtain estimates of the sources of carbohydrate and lipid when measurements of plasma kinetics are combined with rates of substrate oxidation (from indirect calorimetry).

The tracer technique is not suitable for assessment of substrate metabolism during high-intensity exercise when the brevity of the exercise task will limit the number of samples that can be obtained and make kinetic analysis difficult. Therefore, the isotopic tracer method is also best suited for studies involving prolonged, low-to-moderate intensity exercise. A further disadvantage of this method is the expense of the instrumentation (gas chromatograph/mass spectrometer) required for measurement of isotopic enrichment. Finally, the cost of the isotope itself can limit applications in the horse. Whereas deuterated tracers are reasonably priced, 13C-labeled tracers which permit direct measurements of rates of oxidation of that substrate are currently cost-prohibitive for use in horses.

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