Application Note 30

Determination of Nitric Oxide Production and de novo Arginine Production with Stable Isotopes

Juan C. Marini, DVM, PhD

Baylor College of Medicine, Houston, TX 77030 USA

Arginine is a semi-essential amino acid involved in many physiological and pathophysiological processes. The endogenous synthesis of arginine depends on the production of its precursor, citrulline, by the small intestine. Citrulline can be utilized by many cell types to produce arginine, but quantitatively the kidney is the main site for citrulline utilization and arginine production. 

One of the products of arginine metabolism, nitric oxide (NO), is an important signaling molecule involved in the regulation of blood pressure, post-translational regulation of proteins, and in the modulation of the immune response. Because of its high reactivity and short life, the measurement of NO depends, for the most part, on indirect methods of quantitation. For each NO generated from arginine, a citrulline molecule is produced (Figure 1). Thus, citrulline can be both the precursor and a product of arginine metabolism. 

The whole body quantification of these processes can then be accomplished by utilizing stable isotopes to determine the entry rate of arginine and citrulline and their rates of interconversions (Figure 2). 

Because only a small proportion of the entry rate of arginine (~1%) is converted into NO (and citrulline), the choice of tracers and method of analysis is crucial for an optimal quantitation. The possible recycling of the tracer through ornithine, also a product of arginine and the precursor for citrulline synthesis, further limits the choice of tracers. For this reason the arginine tracer of choice is labeled in the guanidino group (although additional labeled atoms may also be present). The use of L-arginine·HCl (guanido-¹⁵N₂, 98%) (NLM-395) to determine the rate of appearance of arginine (or more appropriately of its guanidino group) and its conversion into L-citrulline (ureido-¹⁵N, 98%) (NLM-6850) has become the protocol of choice for the determination of NO production. To determine the rate of appearance of citrulline and the rate of conversion to arginine a citrulline tracer is employed (Figure 3).

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