Glutamine: precursor or nitrogen donor for citrulline synthesis?

Abstract

Although glutamine is considered the main precursor for citrulline synthesis, the current literature does not differentiate between the contribution of glutamine carbon skeleton vs. nonspecific nitrogen (i.e., ammonia) and carbon derived from glutamine oxidation. To elucidate the role of glutamine and nonspecific nitrogen in the synthesis of citrulline, l-[2-(15)N]- and l-[5-(15)N]glutamine and (15)N-ammonium acetate were infused intragastrically in mice. The amino group of glutamine labeled the three nitrogen groups of citrulline almost equally. The amido group and ammonium acetate labeled the ureido and amino groups of citrulline, but not the delta-nitrogen. D(5)-glutamine also infused in this arm of the study, which traces the carbon skeleton of glutamine, was utilized poorly, accounting for only 0.2-0.4% of the circulating citrulline. Dietary glutamine nitrogen (both N groups) incorporation was 25-fold higher than the incorporation of its carbon skeleton into citrulline. To investigate the relative contributions of the carbon skeleton and nonspecific carbon of glutamine, arginine, and proline to citrulline synthesis, U-(13)C(n) tracers of these amino acids were infused intragastrically. Dietary arginine was the main precursor for citrulline synthesis, accounting for approximately 40% of the circulating citrulline. Proline contribution was minor (3.4%), and glutamine was negligible (0.4%). However, the glutamine tracer resulted in a higher enrichment in the ureido group, indicating incorporation of nonspecific carbon from glutamine oxidation into carbamylphosphate used for citrulline synthesis. In conclusion, dietary glutamine is a poor carbon skeleton precursor for the synthesis of citrulline, although it contributes both nonspecific nitrogen and carbon to citrulline synthesis.

Fig. 1.

Fate of glutamine (Gln) and the origin of ornithine (Orn) in the enterocyte. A small portion (14%) of the Gln carbon is incorporated into protein or other acid-insoluble material, whereas most of the Gln is deamidated to glutamate (Glu) and ammonia by action of mitochondria glutaminase (1). Glutamate can be transaminated (2a) or oxidized by glutamate dehydrogenase (2b) to α-ketoglutarate (α-KG) and ammonia. Since this is a reversible reaction, glutamine may exchange its amino group. α-KG is further metabolized to ketoacids and CO₂ and accounts for 18 and 57% of the glutamine carbon, respectively. Glu is converted to glutamate-semialdehyde (GSA) by pyrroline-5-carboxylate (P5C) synthase (3), which is the substrate for Orn transaminase (OAT; 4), generating Orn. Proline (Pro) can be oxidized to P5C by action of proline oxidase (5); P5C spontaneously interconverts to GSA (6). Ornithine can be generated directly by hydrolysis of arginine (Arg) by arginase (7). Carbamyl phosphate (CP), which is synthesized from CO₂ and ammonia by action of carbomyl phosphate synthase (8), condenses with Orn by action of Orn carbomyl transferase (9) to generate citrulline (Cit). Cit accounts for 6% of glutamine carbon. Other enzymes involved in this pathway are P5C dehydrogenase (10) and P5C reductase (11). Based on Windmueller and Spaeth (56) and Smith et al. (43a).

Fig. 2.

Time to reach plateau enrichment of primed, continuously infused tracers (A) and their products (B). Four mice were infused using a lateral vein catheter and samples collected from a carotid artery catheter. Sample size was ∼20 μl/time point. Tracers infused were [U¹³C₆]arginine, [¹⁵N](ureido) citrulline, and [5,5]D₂-ornithine; their products are [U¹³C₅]ornithine, [¹⁵N](guanidino) arginine, and [5,5]D₂-citrulline, respectively. Error bars denote SE.

Fig. 3.

Fragmentation pattern of the dansyl derivative of citrulline. At low-collision energy (15 eV), the mass-to-charge ratio (m/z) 392 fragment predominates and is used to determine the enrichment of the ureido group. At higher-collision energy (26 eV) m/z 70 (carrying the α-nitrogen) and 170 fragment are more abundant.

Fig. 4.

Nitrogen arm results: plasma citrulline isotopic enrichments achieved in each of the 3 nitrogen positions of citrulline with identical infusion rates (200 μmol·kg⁻¹·h⁻¹) of [2-¹⁵N]glutamine (A), [5-¹⁵N]glutamine (B), and [¹⁵N]ammonium acetate (Ac.; C). The D₅-citrulline isotopic enrichment in was achieved with a coinfusion of D₅-glutamine (also a rate of 200 μmol·kg⁻¹·h⁻¹).

Fig. 5.

Carbon arm results. Contribution of dietary glutamine, proline, and arginine to the appearance rate of citrulline. Prec, precursor.