Temporal relationship between ADMA and L-arginine plasma levels in patients with sepsis: potential consequences for iNOS activity in the early post diagnosis period

Introduction: In sepsis, inducible nitric oxide synthase (iNOS) is expressed in immune cells as part of the innate immune defence response and in blood vessels where, if unchecked, it can contribute to vascular collapse associated with septic shock. In this way, iNOS is both a beneficial response as well as a potential therapeutic target for inhibition in sepsis. Other studies have shown that in sepsis, depending on the time of sampling, the iNOS substrate L-arginine is reduced and the endogenous competitive iNOS inhibitor; asymmetric dimethylarginine (ADMA) is increased (1). However, two important questions remain regarding the importance of changes in the ADMA:L-arginine ratio in sepsis. Firstly, how is the ADMA:L-arginine affected in early time points after diagnosis and secondly what might the consequences be of changes in plasma ADMA:L-arginine to iNOS activity. This second point is particularly important since plasma levels of ADMA are very low relative to L-arginine. In this study we have measured L-arginine and ADMA in plasma of a well-defined cohort of patients newly diagnosed with sepsis and at each 24 hour period after diagnosis, up to 72 hours. Amouse macrophage cellular system (RAW264.7) was then used to test the consequences of subtle changes in ADMA and L-arginine concentrations on iNOS activity.

Methods: Plasma from 38 patients with sepsis and 21 healthy volunteers were assayed for ADMA and L-arginine concentrations using ELISA (DLD Diagnostika, Germany), after ethical review by Royal Brompton, NHS trust (code:01-152). In RAW264.7 cells,50μM or 80μM L-arginine was added in the presence or absence of ADMA at 0.30μM or 0.45μM and treated with LPS (1μg/ml)in L-arginine free DMEM, for 24 hours. iNOS activity was measured by the Griess assay.

Results: Mean plasma ADMA levels insepsis (0.4-0.9µM; 0-72 hours) were significantly increased at 24,48 and 72 hours post diagnosis, when compared to healthyvolunteers (≈0.30µM; Figure 1). However, there was a significant decrease in L-arginine, from ≈80μM in healthy volunteers to ≈50μMat sepsis onset. iNOS activity, measured as nitrite concentration (μM; n=9),was greater in RAW264.7 cells cultured with 80μM of L-arginine (5±0.58μM) than with 50μM of L-arginine (2±0.34μM), p<0.05;student’s t-test. Moreover, LPS stimulated iNOS activity was reduced by 47% in DMEM containing both relatively high ADMA (0.45μM) and low arginine (50μM)concentrations (n=18, p<0.05; two-way ANOVA with Bonferroni’s post-hoc test).

Figure 1: Temporal changes in plasma ADMA and L-argininein sepsis. Data is mean ± SEM for n=38 sepsis patients and n=21 healthy volunteers. Analysis was based on one-way ANOVA with Dunnett’s post-hoc test (A-C); *p<0.05.

Discussion: In sepsis, the ADMA:L-arginine ratio is elevated, at all time points (Figure1). In the presence of low levels of L-arginine, seen in patients with sepsis, even small changes in ADMA impact on iNOS activity, suggesting that ADMA is mechanistically important and that L-arginine supplementation will impact on functional iNOS activity in sepsis patients.

1Davis JS, et al. PLoS One 6: e17260 (2011).