IBS and IBD have similar symptoms and internal relationships, but the pathologic mechanisms underlying these relationships remain elusive. Lipid metabolism plays a critical role in human function, especially in biologic signaling and the regulation of inflammation. With the emergence of new technologies, lipids can be analyzed extensively. Many studies have focused on the metabolome of IBS and IBD patients and have shown that metabolites change in different disease conditions [19, 20]. Previous studies have also shown that fatty acid metabolism plays an important role by participating in the inflammatory pathway [16, 24]. However, few studies have described lipidomic analyses of multiple biologic matrices. In addition, compared with those of the intestinal mucosa and serum, stool samples might be more useful for reflecting the state of the disease and revealing its intrinsic pathogenesis. In this study, we targeted the lipidome of IBS-D and UCR-IBS patients to describe comprehensive lipid profiles by analyzing multiple biologic samples, and found the abnormal expression of PUFAs in intestinal diseases.

In mucosa samples, the lipid analysis indicated subtle differences among IBS-D patients, UCR-IBS patients, and healthy control individuals. The significant increase in PE (24:0/22:2(13Z,16Z)) and decrease in LysoPC(16:1(9Z)) in IBS-D patients compared to UCR-IBS patients suggest that these lipids may play critical roles in the pathophysiology of these conditions. PE (24:0/22:2(13Z,16Z)) is a kind of phosphatidylethanolamine that has many different combinations of fatty acids of varying lengths and saturation. It is involved in cellular membrane structure and signaling, potentially affecting intestinal barrier function or inflammation in the intestinal mucosa [25, 26]. Diab et al. [17] reported that a kind of PE (PE (38:3)) changed the most in the mucosa of UC patients. This PE increased under active conditions and decreased in remission, suggesting that this kind of lipid could be used to monitor the development of UC. In our study, PE was increased in IBS-D patients, which may reflect the slight inflammation in IBS-D patients compared to UCR patients. Lysophosphatidylcholines (LysoPCs), such as LysoPC(16:1(9Z)), are also involved in inflammatory processes [27]. The dramatic 27.11-fold decrease in PS(O-16:0/15:1) in UCR-IBS patients compared to healthy control individuals could indicate significant disruptions in cellular signaling and membrane composition, as PS plays a key role in cell apoptosis, coagulation, and immune response regulation [28]. Such a marked reduction might reflect changes in intestinal epithelial cell turnover, barrier function, or immune responses.

The distinct lipid profiles in serum, particularly those of the five overlapping lipids, point to specific metabolic disruptions in UCR-IBS. The elevated levels of decanoyl-CoA, PE(O-20:0/15:0), and TG (12:0/12:0/18:1(9Z)) [iso3] in UCR-IBS patients suggest a distinct metabolic pattern that might influence the disease pathophysiology. Decanoyl-CoA, a kind of saturated fatty acyl CoA, is involved in fatty acid metabolism and oxidation, indicating altered energy utilization or storage [13]. PE(O-20:0/15:0) is another phosphatidylethanolamine that is similar to PE (24:0/22:2(13Z,16Z)) and is detected in mucosa samples. TG (12:0/12:0/18:1(9Z)) [iso3], a triglyceride, has been reported to be elevated in UCR patients compared to healthy control individuals [29, 30], suggesting that this difference may be due to complex interactions between inflammatory cytokines. In our study, we also found the highest TG level in the UCR-IBS group, which is consistent with previous studies. Sphingosine, a long-chain unsaturated amino alcohol, acts as an endogenous inducer of apoptosis by inhibiting cell proliferation and promoting programmed cell death [31]. The most important metabolite of sphingosine is its phosphorylation product in the first step, which is sphingosine-1-phosphate (S1P). The role of S1P in immune cell trafficking has been widely studied [32], and S1P is also a new therapeutic target for reducing inflammation in IBD patients [33]. Our study found reduced levels of sphingosine in the IBS-D and UCR-IBS groups; however, we did not directly measure S1P levels. In addition, the samples we analyzed were from patients with UC in remission. Therefore, the specific roles and associations of sphingosine and S1P in these conditions require further investigation.

Another interesting finding is the differences among different biologic samples. Among the three specimens, feces contained the most lipid species, and mucosa samples contained the fewest. Feces are very important samples in gastroenterology, not only because they are convenient to obtain but also, they are directly collected from the intestines. Thus, they would be more comprehensively reflect the metabolic activities of both microbes and the body [34]. For the mucosa, we tried to collect representative regions, but only very small biopsies could be obtained, representing more localized and specific profiles of the intestinal tissue. In addition, the sample size is also the smallest among the three samples. All of these factors could explain why the mucosa samples had the fewest differential substances. In intestinal mucosa samples, the greatest changes were observed between the IBS-D and HC groups, and the other group comparisons showed few differences. This may imply that the mucosa samples of the IBS-D and UCR-IBS groups are possibly similar. However, the opposite results were obtained for the fecal samples. Taking the characteristics of the two kinds of specimens into account, the intestinal tissue is better to reflect the local metabolic condition of the intestine, but the feces could better represent the whole gut metabolites. Considering the convenience of sample collection and its effectiveness in reflecting metabolic information, fecal samples might have advantages in lipidomic studies.

Notably, we found that the fatty acid metabolism pathways, especially alpha-linolenic acid metabolism and omega-3/omega-6 fatty acid synthesis, were significantly enriched in fecal and serum samples in the IBS-D and UCR-IBS groups, which is consistent with our previous study [16]. ALA is a polyunsaturated fatty acid that is essential in the human diet. It is the substrate for the synthesis of EPA, DHA and other anti-inflammatory omega-3 PUFAs, which are important for human functions [35]. From the untargeted lipidomics results, tetracosahexaenoic acid (THA), tetracosapentaenoic acid (24:5n-6) and tetracosapentaenoic acid (24:5n-3), very important precursors of DHA in the omega-3 PUFA biosynthetic process [36, 37], are significantly changed. The reduction of precursors affects the synthesis of downstream substances, resulting in an imbalance in the omega-6/omega-3 ratio. We also verified this by retesting the PUFAs in serum samples, showing the consistency with untargeted lipidomics results. DHA can interfere with a variety of inflammatory signaling pathways, regulating and reducing inflammatory responses. A reduction in DHA reflects an imbalance between anti-inflammatory and proinflammatory mediators (e.g., ARA). In addition, a high ratio of omega-6/omega-3 PUFAs predicts various aspects of psychologic distress, including the onset of post-partum depression and mood disorders [38, 39]. This imbalance is more pronounced in IBS patients, leading to more severe somatic disorders and depressive symptoms [40]. In our study, DHA were significantly decreased in the UCR-IBS group, while LA, ARA and the omega-6/omega-3 PUFAs ratio were significantly higher in both UCR-IBS and IBS-D group. The imbalances in the PUFAs indicated the IBS-D and UCR-IBS might share the same mechanism in psychologic tissues and the activation of inflammatory pathways.

There were some limitations in our study. First, the sample size was relatively small, and we did not match the case and control groups. Nevertheless, we adjusted for these variables during the statistical analysis process to minimize the potential impact on the results. Second, we didnot provide standard diet for participants. But we required them follow a light, low-residue diet for at least 3 days before sample collection to reduce differences from dietary factors as long as possible. Besides, only a portion of the subjects underwent endoscopic screening in this study. However, the remainder of UCR-IBS group had the colonoscopy within a year, and we used fecal calprotectin with blood inflammation markers to screen them out and ensure them in remission. Furthermore, given the varying sampling preferences for IBS and UC observed in prior studies [19, 20], with IBS in sigmoid and UC in rectum, we chose to collect mucosal samples from the rectosigmoid junction. This site could provide a relatively comprehensive analysis of the metabolic condition both in sigmoid and rectum.

In conclusion, this study is the first to comprehensively describe the distribution and differences in the lipidome of IBS-D patients, UCR-IBS patients via an integrated multisample analysis of intestinal mucosa, feces, and serum samples. We found that fatty acids, particularly PUFAs, participate in the IBS and IBD-IBS processes, suggesting that the dysregulation of omega-3 and omega-6 metabolism is associated with psychologic disorders and inflammatory activation. We also revealed that fecal samples were more likely to reflect the condition more comprehensively than serum or intestinal mucosa samples.