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Citrulline a more suitable substrate than arginine to restore NO production and the microcirculation during endotoxemia.

Wijnands KA, Vink H, Briedé JJ, van Faassen EE, Lamers WH, Buurman WA, Poeze M - PLoS ONE (2012)

Bottom Line: L-Arginine supplementation did not increase the intracellular arginine availability.Jejunal tissues in the L-Citrulline-supplemented group showed, compared to the endotoxemic and L-Arginine-supplemented endotoxemic group, an increase in degree of phosphorylation of eNOS (Ser 1177) and a decrease in iNOS protein level.In conclusion, L-Citrulline supplementation during endotoxemia and not L-Arginine reduced intestinal microcirculatory dysfunction and increased intracellular NO production, likely via increased intracellular citrulline and arginine availability.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Maastricht University Medical Center, Maastricht, The Netherlands. n.wijnands@maastrichtuniversity.nl

ABSTRACT

Background: Impaired microcirculation during endotoxemia correlates with a disturbed arginine-nitric oxide (NO) metabolism and is associated with deteriorating organ function. Improving the organ perfusion in endotoxemia, as often seen in patients with severe infection or systemic inflammatory response syndrome (SIRS) is, therefore, an important therapeutic target. We hypothesized that supplementation of the arginine precursor citrulline rather than arginine would specifically increase eNOS-induced intracellular NO production and thereby improve the microcirculation during endotoxemia.

Methodology/principal findings: To study the effects of L-Citrulline and L-Arginine supplementation on jejunal microcirculation, intracellular arginine availability and NO production in a non-lethal prolonged endotoxemia model in mice. C57/Bl6 mice received an 18 hrs intravenous infusion of endotoxin (LPS, 0.4 µg • g bodyweight(-1) • h(-1)), combined with either L-Citrulline (6.25 mg • h-1), L-Arginine (6.25 mg • h(-1)), or L-Alanine (isonitrogenous control; 12.5 mg • h(-1)) during the last 6 hrs. The control group received an 18 hrs sterile saline infusion combined with L-Alanine or L-Citrulline during the last 6 hrs. The microcirculation was evaluated at the end of the infusion period using sidestream dark-field imaging of jejunal villi. Plasma and jejunal tissue amino-acid concentrations were measured by HPLC, NO tissue concentrations by electron-spin resonance spectroscopy and NOS protein concentrations using Western blot.

Conclusion/significance: L-Citrulline supplementation during endotoxemia positively influenced the intestinal microvascular perfusion compared to L-Arginine-supplemented and control endotoxemic mice. L-Citrulline supplementation increased plasma and tissue concentrations of arginine and citrulline, and restored intracellular NO production in the intestine. L-Arginine supplementation did not increase the intracellular arginine availability. Jejunal tissues in the L-Citrulline-supplemented group showed, compared to the endotoxemic and L-Arginine-supplemented endotoxemic group, an increase in degree of phosphorylation of eNOS (Ser 1177) and a decrease in iNOS protein level. In conclusion, L-Citrulline supplementation during endotoxemia and not L-Arginine reduced intestinal microcirculatory dysfunction and increased intracellular NO production, likely via increased intracellular citrulline and arginine availability.

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Amino-acid concentrations and NO production in murine jejunal tissue.(A) L-Citrulline supplementation (LPS-Cit) increased tissue citrulline concentration compared to the LPS-Ala, LPS-Arg and control groups (P<0.001) (B) Arginine concentrations were significantly reduced in the LPS-Ala group compared to the control group (P<0.05). Interestingly, this concentration was not increased by L-Arginine supplementation (LPS-Arg), whereas this concentration was significantly increased by L-Citrulline supplementation (LPS-Cit group; P<0.01). (C) Ornithine levels increased in the LPS-Ala treated group compared to the control group (P<0.05) and increased further upon L-Citrulline supplementation (P<0.0001 relative to the control group and P<0.05 relative to the LPS group). (D) In the jejunum, prolonged endotoxemia resulted in a significant decrease in NO production (measured as pmol mono-nitrosyl-iron complexes (MNIC)/mg wet tissue) compared to control (P<0.05). NO production was significantly improved in the LPS-Arg and LPS-Cit group compared to the LPS group (P<0.0001 and P<0.05 respectively).
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pone-0037439-g005: Amino-acid concentrations and NO production in murine jejunal tissue.(A) L-Citrulline supplementation (LPS-Cit) increased tissue citrulline concentration compared to the LPS-Ala, LPS-Arg and control groups (P<0.001) (B) Arginine concentrations were significantly reduced in the LPS-Ala group compared to the control group (P<0.05). Interestingly, this concentration was not increased by L-Arginine supplementation (LPS-Arg), whereas this concentration was significantly increased by L-Citrulline supplementation (LPS-Cit group; P<0.01). (C) Ornithine levels increased in the LPS-Ala treated group compared to the control group (P<0.05) and increased further upon L-Citrulline supplementation (P<0.0001 relative to the control group and P<0.05 relative to the LPS group). (D) In the jejunum, prolonged endotoxemia resulted in a significant decrease in NO production (measured as pmol mono-nitrosyl-iron complexes (MNIC)/mg wet tissue) compared to control (P<0.05). NO production was significantly improved in the LPS-Arg and LPS-Cit group compared to the LPS group (P<0.0001 and P<0.05 respectively).

Mentions: The tissue amino-acid concentrations in all groups exhibited results that were largely similar to the changes in plasma concentrations. Endotoxemia resulted in a decreased jejunal tissue arginine concentration in the LPS-Ala group versus control (n = 13; P<0.05), which was accompanied by a significantly decreased tissue citrulline concentration (n = 13; P<0.05, Figure 5A and 5B). Interestingly, the beneficial effects of L-Arginine supplementation in the LPS-Arg group on the plasma concentrations was not observed in the jejunal tissue as the tissue arginine concentration was significantly decreased compared to control (n = 13; P<0.05, Figure 5A). However, L-Citrulline supplementation during endotoxemia also enhanced the tissue arginine concentration compared to LPS-Ala and LPS-Arg treated mice (n = 13; both P<0.01). L-Citrulline supplementation under basal conditions also enhanced the tissue arginine concentrations (see Figure S3). During endotoxemia ornithine tissue concentrations increased in the LPS-Ala and LPS-Cit treated groups compared to the control group, which was in line with the increase in the plasma concentrations (Figure 5C). In the LPS-Arg group ornithine tissue concentrations were lower than in the LPS-Ala group.


Citrulline a more suitable substrate than arginine to restore NO production and the microcirculation during endotoxemia.

Wijnands KA, Vink H, Briedé JJ, van Faassen EE, Lamers WH, Buurman WA, Poeze M - PLoS ONE (2012)

Amino-acid concentrations and NO production in murine jejunal tissue.(A) L-Citrulline supplementation (LPS-Cit) increased tissue citrulline concentration compared to the LPS-Ala, LPS-Arg and control groups (P<0.001) (B) Arginine concentrations were significantly reduced in the LPS-Ala group compared to the control group (P<0.05). Interestingly, this concentration was not increased by L-Arginine supplementation (LPS-Arg), whereas this concentration was significantly increased by L-Citrulline supplementation (LPS-Cit group; P<0.01). (C) Ornithine levels increased in the LPS-Ala treated group compared to the control group (P<0.05) and increased further upon L-Citrulline supplementation (P<0.0001 relative to the control group and P<0.05 relative to the LPS group). (D) In the jejunum, prolonged endotoxemia resulted in a significant decrease in NO production (measured as pmol mono-nitrosyl-iron complexes (MNIC)/mg wet tissue) compared to control (P<0.05). NO production was significantly improved in the LPS-Arg and LPS-Cit group compared to the LPS group (P<0.0001 and P<0.05 respectively).
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pone-0037439-g005: Amino-acid concentrations and NO production in murine jejunal tissue.(A) L-Citrulline supplementation (LPS-Cit) increased tissue citrulline concentration compared to the LPS-Ala, LPS-Arg and control groups (P<0.001) (B) Arginine concentrations were significantly reduced in the LPS-Ala group compared to the control group (P<0.05). Interestingly, this concentration was not increased by L-Arginine supplementation (LPS-Arg), whereas this concentration was significantly increased by L-Citrulline supplementation (LPS-Cit group; P<0.01). (C) Ornithine levels increased in the LPS-Ala treated group compared to the control group (P<0.05) and increased further upon L-Citrulline supplementation (P<0.0001 relative to the control group and P<0.05 relative to the LPS group). (D) In the jejunum, prolonged endotoxemia resulted in a significant decrease in NO production (measured as pmol mono-nitrosyl-iron complexes (MNIC)/mg wet tissue) compared to control (P<0.05). NO production was significantly improved in the LPS-Arg and LPS-Cit group compared to the LPS group (P<0.0001 and P<0.05 respectively).
Mentions: The tissue amino-acid concentrations in all groups exhibited results that were largely similar to the changes in plasma concentrations. Endotoxemia resulted in a decreased jejunal tissue arginine concentration in the LPS-Ala group versus control (n = 13; P<0.05), which was accompanied by a significantly decreased tissue citrulline concentration (n = 13; P<0.05, Figure 5A and 5B). Interestingly, the beneficial effects of L-Arginine supplementation in the LPS-Arg group on the plasma concentrations was not observed in the jejunal tissue as the tissue arginine concentration was significantly decreased compared to control (n = 13; P<0.05, Figure 5A). However, L-Citrulline supplementation during endotoxemia also enhanced the tissue arginine concentration compared to LPS-Ala and LPS-Arg treated mice (n = 13; both P<0.01). L-Citrulline supplementation under basal conditions also enhanced the tissue arginine concentrations (see Figure S3). During endotoxemia ornithine tissue concentrations increased in the LPS-Ala and LPS-Cit treated groups compared to the control group, which was in line with the increase in the plasma concentrations (Figure 5C). In the LPS-Arg group ornithine tissue concentrations were lower than in the LPS-Ala group.

Bottom Line: L-Arginine supplementation did not increase the intracellular arginine availability.Jejunal tissues in the L-Citrulline-supplemented group showed, compared to the endotoxemic and L-Arginine-supplemented endotoxemic group, an increase in degree of phosphorylation of eNOS (Ser 1177) and a decrease in iNOS protein level.In conclusion, L-Citrulline supplementation during endotoxemia and not L-Arginine reduced intestinal microcirculatory dysfunction and increased intracellular NO production, likely via increased intracellular citrulline and arginine availability.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Maastricht University Medical Center, Maastricht, The Netherlands. n.wijnands@maastrichtuniversity.nl

ABSTRACT

Background: Impaired microcirculation during endotoxemia correlates with a disturbed arginine-nitric oxide (NO) metabolism and is associated with deteriorating organ function. Improving the organ perfusion in endotoxemia, as often seen in patients with severe infection or systemic inflammatory response syndrome (SIRS) is, therefore, an important therapeutic target. We hypothesized that supplementation of the arginine precursor citrulline rather than arginine would specifically increase eNOS-induced intracellular NO production and thereby improve the microcirculation during endotoxemia.

Methodology/principal findings: To study the effects of L-Citrulline and L-Arginine supplementation on jejunal microcirculation, intracellular arginine availability and NO production in a non-lethal prolonged endotoxemia model in mice. C57/Bl6 mice received an 18 hrs intravenous infusion of endotoxin (LPS, 0.4 µg • g bodyweight(-1) • h(-1)), combined with either L-Citrulline (6.25 mg • h-1), L-Arginine (6.25 mg • h(-1)), or L-Alanine (isonitrogenous control; 12.5 mg • h(-1)) during the last 6 hrs. The control group received an 18 hrs sterile saline infusion combined with L-Alanine or L-Citrulline during the last 6 hrs. The microcirculation was evaluated at the end of the infusion period using sidestream dark-field imaging of jejunal villi. Plasma and jejunal tissue amino-acid concentrations were measured by HPLC, NO tissue concentrations by electron-spin resonance spectroscopy and NOS protein concentrations using Western blot.

Conclusion/significance: L-Citrulline supplementation during endotoxemia positively influenced the intestinal microvascular perfusion compared to L-Arginine-supplemented and control endotoxemic mice. L-Citrulline supplementation increased plasma and tissue concentrations of arginine and citrulline, and restored intracellular NO production in the intestine. L-Arginine supplementation did not increase the intracellular arginine availability. Jejunal tissues in the L-Citrulline-supplemented group showed, compared to the endotoxemic and L-Arginine-supplemented endotoxemic group, an increase in degree of phosphorylation of eNOS (Ser 1177) and a decrease in iNOS protein level. In conclusion, L-Citrulline supplementation during endotoxemia and not L-Arginine reduced intestinal microcirculatory dysfunction and increased intracellular NO production, likely via increased intracellular citrulline and arginine availability.

Show MeSH
Related in: MedlinePlus