Metabolomic profiling of amines in sepsis predicts changes in NOS canonical pathways

Rationale Nitric oxide synthase (NOS) is a biomarker/target in sepsis. NOS activity is driven by amino acids, which cycle to regulate the substrate L-arginine in parallel with cycles which regulate the endogenous inhibitors ADMA and L-NMMA. The relationship between amines and the consequence of plasma changes on iNOS activity in early sepsis is not known. Objective Our objective was to apply a metabolomics approach to determine the influence of sepsis on a full array of amines and what consequence these changes may have on predicted iNOS activity. Methods and measurements 34 amino acids were measured using ultra purification mass spectrometry in the plasma of septic patients (n = 38) taken at the time of diagnosis and 24–72 hours post diagnosis and of healthy volunteers (n = 21). L-arginine and methylarginines were measured using liquid-chromatography mass spectrometry and ELISA. A top down approach was also taken to examine the most changed metabolic pathways by Ingenuity Pathway Analysis. The iNOS supporting capacity of plasma was determined using a mouse macrophage cell-based bioassay. Main results Of all the amines measured 22, including L-arginine and ADMA, displayed significant differences in samples from patients with sepsis. The functional consequence of increased ADMA and decreased L-arginine in context of all cumulative metabolic changes in plasma resulted in reduced iNOS supporting activity associated with sepsis. Conclusions In early sepsis profound changes in amine levels were defined by dominant changes in the iNOS canonical pathway resulting in functionally meaningful changes in the ability of plasma to regulate iNOS activity ex vivo

Understanding the link between amino acids and vascular function in cardiovascular inflammation and sepsis

Sepsis is a ‘life-threatening disease caused by a dysregulated host response to an infection’. Despite advancements, effective treatments and biomarkers have remained elusive. However, nitric oxide (NO) and related pathways are emerging as biomarkers/targets. NO levels are driven by amino acids, which cycle to regulate NO synthase (NOS) substrate L-arginine and inhibitors including asymmetric dimethylarginine (ADMA). Furthermore, the consequence of changes of plasma amine-constituents in sepsis, on NOS activity ex vivo is not known. Thus, in this thesis I used a well-defined clinical cohort to address the influence of sepsis on (i) the amine metabolome, (ii) the ability of plasma to ‘support’ NOS activity in a simple mouse cell bioassay and (iii) plasma amine-constituents and correlations to clinical outcome measures. The main results reported within this thesis include: (i) LPS-activated mouse macrophages provided a viable bioassay platform to test ‘NOS supporting activity’ in the presence of 100% human plasma. In sepsis, ‘NOS supporting activity’ of plasma was reduced, which corresponded to an increased ADMA:L-arginine ratio. Both ADMA and ADMA:L-arginine ratio also positively correlated to clinical scores of sepsis. Similarly, the ADMA:L-arginine ratio was increased in an in vivo mouse model of sepsis but unlike in humans this was driven entirely by reduced L-arginine. (ii) Analysis of gene expression in spleen and kidney tissues from LPS-treated mice revealed a complex interplay between pathways that utilise L-arginine and that generate/metabolise methylarginines. (iii) Using a novel ultra-high-performance mass spectrometry platform, 34 human plasma amines were quantified from sepsis patients, of which 21 amines were significantly altered and mapped to both canonical NOS pathways and L-arginine/L-glutamine degradation pathways. Taurine emerged as an important diagnostic (reduced in sepsis) and prognostic (reduced in survivors) indicator. (iv) Using a blood vessel bioassay demonstrated taurine augmented the vasoplegia induced by LPS and interferon.Open Acces

iNOS activity in LPS-activated mouse macrophages treated in culture media.

<p>iNOS activity in macrophages was measured in cells cultured in the presence of <b>(A,B)</b> 50μM or <b>(C,D)</b> 80μM L-arginine. Data are ± SEM for n = 4–6. Data was analysed by <b>(A,C)</b> paired t-test and <b>(B,D)</b> one-way ANOVA with Dunnett’s post-hoc test. *p<0.05.</p

ADMA:L-arginine ratio in healthy donors vs patients with sepsis.

<p>ADMA and L-arginine were measured from plasma of healthy donors and patients with sepsis at diagnosis (0 hours), 24 and 72 hours post diagnosis using ELISA. Data are ± SEM for n = 21 healthy donors and n = 38 patients with sepsis. Data was analysed by one-way ANOVA with Dunnett’s post-hoc test. *p<0.05.</p

Targeted metabolic profiling of amines and methylarginines in human plasma from healthy donors and patients with sepsis.

<p>Amine and methylarginine levels were measured using <b>(A)</b> UHPLC-MS/MS, LC-MS/MS and/or ELISA in the plasma of healthy donors and patients with sepsis at diagnosis (0 hours), 24 hours and 72 hours post diagnosis. Comparisons between levels of L-arginine from <b>(B)</b> UHPLC-MS/MS, LC-MS/MS and ELISA and <b>(C)</b> ADMA from LC-MS/MS and ELISA are shown at diagnosis (0 hours), 24 hours and 72 hours, post diagnosis. <b>(D)</b> Canonical pathways extracted from IPA software were based on an input of read outs comparing plasma levels of amines in healthy donors and patients with sepsis as a ratio. Data are ± SEM for n = 21 healthy donors and n = 38 patients with sepsis. Data was analysed by one-way ANOVA with Dunnett’s post-hoc test and Benjamini-Hochberg test with a false discovery rate of 0.05; *p<0.05.</p

iNOS activity in LPS-activated mouse macrophages treated in plasma from healthy donors and patients with sepsis.

<p><b>(A)</b> iNOS activity in plasma from healthy vs sepsis patients at 0, 24 and 72 hours. Correlations in ADMA:L-arginine ratio with iNOS activity at <b>(B)</b> 0–24 hours and <b>(C)</b> 24–72 hours post diagnosis. Data are mean ± SEM for n = 21 healthy donors and n = 38 patients with sepsis. Data was analysed by <b>(A)</b> Kruskal-Wallis one-way ANOVA with Dunn’s post-hoc test and <b>(B-C)</b> linear regression. *p<0.05 when compared to healthy.</p