INTRODUCTION

The proposed pathogenesis of IBS is complex. The most prominent features of the condition include impaired GI motility, visceral hypersensitivity, abnormal secretion and absorption and an altered intestinal microbiome.^{1, 10} There is emerging evidence that low grade inflammation in the GI mucosa and immune activation may be responsible for the motor and sensory alterations of IBS in a subset possibly triggered by infection, food antigens or stress.^{11, 12} Indeed there is a clinical overlap between inflammatory bowel disease (IBD) and IBS, with IBS-like symptoms frequently reported in patients before the diagnosis of IBD, and a higher than expected prevalence of IBS symptoms in patients in remission from established IBD.¹³

Nitric oxide (NO) is a major inhibitory nonadrenergic, noncholinergic neurotransmitter in the GI tract.¹⁴ Motility of the GI tract is directly controlled by enteric inhibitory and excitatory motor neurons that innervate the smooth muscle layers. NO released in response to nerve stimulation of the myenteric plexus causes relaxation of the smooth muscle and has been shown to play an important role in esophageal, gastric, and intestinal motility regulation.¹⁵ The other major role of NO in the GI tract is the maintenance of GI mucosa integrity via modulation of gastric mucosal blood flow, epithelial mucus and fluid secretion, and barrier function. Within the myenteric plexus NO is primarily synthesized from the amino acid L-arginine by the constitutive enzyme neuronal nitric oxide synthase (nNOS) producing L-citrulline in the process (Figure 1).¹⁶ The similar process in the endothelium of GI blood vessels is catalyzed by the constitutive enzyme endothelial nitric oxide synthase (eNOS).¹⁷ An inducible nitric oxide synthase (iNOS) is also expressed in large amounts in the enteric nervous system and GI tract in response to inflammation, bacterial and viral infection, or trauma.¹⁸

Subsets of patients with IBS have inflammatory changes in their gut mucosa.¹⁹ A prominent feature is the increase in the number of mast cells and intraepithelial lymphocytes within the small and large intestines in IBS.²⁰ Furthermore, mast cell concentrations and their distance from enteric neurons are positively associated with IBS pain.²¹

Evidence from animal studies supports an inhibitory action of NO on mast cell activation and mast cell-dependent inflammatory processes in vivo ²² This is consistent with work in rodent gut^{23, 24} and blood vessels,²⁵ and pig²⁶ and rabbit lungs.²⁷ Overall, this evidence suggests that inhibition of NOS and a corresponding decrease in NO synthesis is likely to activate mast cells within the GI tract and impair NO-dependent regulatory processes resulting in disturbed GI motility, secretion, permeability, and the development of inflammatory changes within the GI tract.

Given the available evidence in support of the hypothesis that impaired NO synthesis leads to disturbed GI function and potentially the development of IBS, it is important to identify new potentially modifiable factors responsible for decreased synthesis of NO within the GI tract. Asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of all NOS isoforms is one potential factor that has received a great deal of attention in the area of cardiovascular medicine. ADMA competes with L-arginine for eNOS and nNOS and has been shown to reduce NO synthesis in isolated human blood vessels and numerous cell types.^28-32 There is substantial evidence that excessive serum ADMA concentrations inhibit NOS activity and cause sustained endothelial dysfunction and a proinflammatory state.³³ Given that L-arginine is the substrate for the synthesis of NO, a depletion of L-arginine and/or reduced intracellular bioavailability of L-arginine may also be an important factor in the development of IBS.

The primary aim of this research was to determine if serum concentrations of L-arginine and the methylarginines ADMA and Symmetric dimethylarginine (SDMA), another methylated arginine with indirect inhibitory effects on NO synthesis,³⁴ are independenly associated with IBS in a representative sample of IBS cases and controls nested within a cohort of community-dwelling adults.

METHODS

RESULTS

At the HCS 5-year follow-up in 2011 there were 228 IBS cases identified according to Rome III criteria. Of these, 156 cases had serum available for measurement of L-arginine and methylated arginines. According to Rome III definitions of IBS subtypes, 35% (N = 55) were classified as diarrhea-predominant (IBS-D), 15% (N = 24) were constipation-predominant (IBS-C), 42% (N = 65) were a mixed phenotype, and the remainder were unclassified. Demographic characteristics are described in Table 1. Significantly more IBS cases were female compared with controls (p = 0.049); however, there was no difference in age (p = 0.57). Mean K10 and CESD scores were significantly higher in IBS cases than controls (p < 0.001). The mean serum concentrations of ADMA, SDMA, L-arginine, and L-arginine/ADMA ratio were statistically significantly higher in IBS cases than controls (p < 0.001).

Unadjusted and adjusted logistic regression analyses of L-arginine, ADMA, SDMA, and L-arginine/ADMA ratio with IBS are also described in Table 1. In both unadjusted and adjusted logistic regression analyses, L-arginine, ADMA, SDMA, and L-arginine/ADMA ratio were all statistically significantly associated with IBS. The AUC and AIC values indicate the models with L-arginine and the L-arginine/ADMA ratio were the best fitting models.

The odds of IBS increased by 6% for a one unit increase (i.e., 1 μmol/L) in serum L-arginine concentration and this effect was unchanged by controlling for confounders (OR = 1.06; 95% CI: 1.05–1.07). This effect corresponds to a ∼ninefold increase in odds of IBS (OR = 9.03 [95% CI: 5.99–13.62]) for those in the 75th percentile (84 μmol/L) compared to those in the 25th percentile (46 μmol/L). For the association between L-arginine/ADMA ratio with IBS, a 10 unit increase in L-arginine/ADMA ratio is associated with a 36% increase in odds of IBS diagnosis (OR = 1.36; 95% CI: 1.28–1.44). This corresponds to a ∼sevenfold increase in odds of IBS (OR = 7.38 [95% CI: 5.01–10.86]) for those in the 75th percentile (151 μmol/L) compared to those in the 25th percentile (86 μmol/L).

For the L-arginine-IBS model a suitable serum L-arginine concentration cut-off identified using the Youden Index was 77.81 μmol/L. Using this cut-off, the model has a sensitivity of 77.6% (95% CI: 70.7–83.5) and specificity of 89.5% (95% CI: 85.7–92.6). For the L-arginine/ADMA ratio-IBS model a suitable serum L-arginine/ADMA concentration cut-off identified using the Youden Index was 143.53 μmol/L. Using this cut-off the model has a sensitivity of 71.3% (95% CI: 63.9–77.9) and specificity of 88.9% (95% CI: 85.1–92.1).

We also investigated the discriminative ability of the models using two subtypes of IBS (IBS-Diarrhoea and IBS-Constipation) as outcomes and found that the model describing the association between L-arginine/ADMA ratio and each subtype performed equally well (IBS-Diarrhoea and IBS-Constipation Adjusted AUC = 0.725; see Table 2).

DISCUSSION

This population-based nested case-control study examined the association of plasma L-arginine and the endogenous methylarginines, ADMA, and SDMA with IBS defined by Rome III criteria. In adjusted analyses L-arginine, ADMA, SDMA, L-arginine/ADMA, and K10 score were statistically significant independent predictors of IBS diagnosis. Model discrimination was strongest for the model with L-arginine (Adjusted AUC = 0.859) and L-arginine/ADMA ratio (Adjusted AUC = 0.850). At their optimal cut points, L-arginine and L-arginine/ADMA ratio showed moderate sensitivity (71%–77%) and high specificity (approximately 90%) yielding positive likelihood ratios in the range of seven to eight, suggesting that these biomarkers may be useful for IBS diagnosis in a clinical setting, This research provides evidence to support the hypothesis that higher serum concentrations of L-arginine and the endogenous methylarginines are associated with IBS in adults. Interestingly, this finding applied to all subtypes of IBS suggesting it is not bowel related but more likely represents some CNS effect. Corsetti et al. performed a small randomized crossover trial (n = 10) of the effects of the NOS inhibitor, N-monomethyl-L-arginine, on the motility and sensitivity of the distal colon in healthy humans.⁴² This study showed that inhibition of NO synthase did not affect the colonic motor responses whereas it increased the sensitivity to colonic distension. In another double-blind randomized crossover trial (n = 22), N-monomethyl-L-arginine significantly increased the threshold for pain in IBS patients compared with healthy volunteers, while rectal compliance was not affected by N-monomethyl-L-arginine.⁴³ These findings support our hypothesis that NOS inhibitors are involved in the pathophysiology of visceral hypersensitivity in IBS.

The association of increased serum ADMA concentration and IBS observed in this study is similar to that observed in a recent study that examined the association of serum concentrations of ADMA and SDMA with IBD.⁴⁴ In a case-control study involving 31 consecutive patients with ulcerative colitis (UC) and 32 with Crohn's disease, ADMA, and SDMA concentrations were increased in the IBD group as compared to an age and sex-matched control group.⁴⁴ In a preliminary study of IBS (n = 18) versus IBD (n = 100) and controls (n = 40), significant alterations of L-arginine pathway metabolites were observed in both IBS and IBD.⁴⁵ By comparison the effects of various NOS inhibitors in animal models of experimental colitis have been conflicting. Some synthetic NOS inhibitors have attenuated disease activity while others have exacerbated the disease.⁴⁶ These conflicting findings are likely explained by the fact that some NOS inhibitors lack specificity and inhibit all NOS isoforms, potentially also inhibiting NOS-mediated tissue repair.

Research suggests that iNOS may be the primary NOS implicated in the pathophysiology of IBD and there is some evidence for a corresponding role in IBS. Activation of iNOS produces potentially damaging micromolar amounts of NO compared with the constitutive nanomolar amounts produced by eNOS and nNOS. The high concentration of iNOS-derived NO contributes to an inflammatory response and leads to cellular damage by reaction with superoxide anions (O^2–), forming reactive nitrogen species such as peroxynitrite (ONOO-) which mediate this effect.⁴⁷ Research indicates that iNOS-derived NO produced within colonic epithelial cells and granulocytes participates in the inflammatory process of IBD⁴⁸ and IBS.⁴⁹

The finding of increased serum L-arginine concentration with IBS has not been seen previously. By comparison, a recent study of 14 normal controls and 22 UC patients with pancolitis of moderate or severe activity determined by histopathology score observed that L-arginine concentrations were increased in subjects with severe colitis when compared to those with moderate colitis or normal mucosa.⁵⁰ However, relative arginine availability, as calculated by the Arginine Availability Index, was not increased in these subjects suggesting a functional deficiency of intracellular L-arginine availability in UC patients.

Given the evidence for low-grade inflammation and activation of mast cells in IBS and the clinical overlap with IBD it is reasonable to speculate that the elevated serum L-arginine observed in the current investigation is the result of decreased cationic amino acid transporter (CAT)-mediated L-arginine transport into intestinal cells. Hence, as described in Figure 1, there may be a functional deficiency of intracellular L-arginine that impairs NO synthesis. Figure 2 describes the effects of increased ADMA on NO synthesis and the proposed pathophysiology of intestinal dysmotility, epithelial permeability, increased production of proinflammatory cytokines, and visceral hypersensitivity in IBS. The increased serum L-arginine observed in the DSS colitis model in mice⁵¹ (and the corresponding decreased intracellular L-arginine availability) may be the direct result of defective CAT-mediated L-arginine transport as CAT2 expression is up-regulated in UC subjects compared to control tissues, and deletion of CAT2 results in increased serum L-arginine and exacerbation of DSS colitis.⁵² As speculated by Hong et al., this may be the result of competitive inhibition of CAT-mediated L-arginine transport into cells by other amino acids such as L-ornithine and L-lysine. One further possibility is that increased serum ADMA may also compete with L-arginine for transport into intestinal cells as methylarginine transport into cells is also facilitated by CATs. Indeed one study has shown that increased ADMA and SDMA inhibited CAT1-mediated uptake of L-arginine in Human embryonic kidney cells (HEK293) stably overexpressing CAT1 (HEK-CAT1) and vector-transfected control cells (HEK-VC).⁵³

This study has a number of strengths. The research was conducted in a relatively large population-based sample of community-dwelling older adults and cases of IBS were incident cases selected from within this cohort. Second, controls are population-based controls (without IBS) selected from the same cohort. Third, serum L-arginine, ADMA, and SDMA were all measured using hydrophilic-interaction liquid chromatography and isotope dilution tandem mass spectrometry, currently the gold standard for methylarginine measurement. Fourth, the bias-adjusted AUC estimates virtually did not change from the crude estimate and this suggests that the findings are robust. Finally, IBS cases were ascertained using a well-validated self-reported questionnaire according to Rome III criteria.

Limitations of our study include the use of a subjective self-reported questionnaire tool rather than a physician diagnosis of IBS. There were also no measures of NO metabolites to determine if these levels were altered in the presence of elevated L-arginine or methylarginines. Another limitation is the absence of dietary L-arginine intake measures and the inability to control for its potential confounding effects. Although there is likely to be variation in dietary L-arginine intake in the population, it is known that less than 5% of bioavailable L-arginine comes from dietary intake. Most is derived from endogenous synthesis from citrulline. However, there is evidence that increasing L-arginine intake can reduce ADMA concentrations and hence it is possible that dietary arginine may confound the observed association. Finally, the calculated fit and discrimination of the models may be overstated given the artificial dichotomy of those with IBS and those without, rather than an undifferentiated clinical population with symptoms; nevertheless, these results are encouraging and warrant further investigation.

In conclusion, higher serum concentrations of L-arginine and endogenous methylarginines are strong predictors of IBS in adults. These findings are potentially clinically relevant as L-arginine cellular uptake may be enhanced through L-arginine supplementation and ADMA concentrations may be lowered using several available medications. Longitudinal research is needed to determine if elevated concentrations of L-arginine or ADMA are a cause or a consequence of IBS. It is important to determine this causal direction as lowering their concentrations may lead to treatment. However, if it is the result of IBS, it is unlikely to have benefit. Furthermore, serum L-arginine concentration may be a useful clinical biomarker of IBS diagnosis. Further longitudinal research is needed to validate these findings.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest. Dr. Talley reports grants from Abbott Pharmaceuticals, Commonwealth Diagnostics, Viscera USA, non-financial support from HVN National Science Challenge NZ, grants and personal fees from GI therapies, personal fees from Adelphi values, Allergens PLC, Takeda, Ampligent, Progenity Inc, Sanofi-aventis, IM Health Sciences, Napo Pharmaceutical, Outpost Medicine, Samsung Bioepis, Synergy, Theravance, Yuhan, outside the submitted work; In addition, Dr. Talley has a patent Biomarkers of IBS licensed, a patent Licensing Questionnaires Talley Bowel Disease Questionnaire licensed to Mayo/Talley, a patent Nestec European Patent licensed, and a patent Singapore Provisional Patent “Microbiota Modulation Of BDNF Tissue Repair Pathway” issued. Committees: Australian Medical Council (AMC) [Council Member]; Australian Telehealth Integration Programme; MBS Review Taskforce; NHMRC Principal Committee (Research Committee) Asia Pacific Association of Medical Journal Editors. Boards: GESA Board Member, Sax Institute, Committees of the Presidents of Medical Colleges. Community group: Advisory Board IFFGD, Avant Foundation (judging of research grants).