Differentiation of CD and UC patients, control subjects and IFX response types
The study confirms previously published studies [
17‐
21] and displays significant differential diagnostic power in models comparing active UC patients versus control subjects, active CD patients versus control subjects, and remission CD patients versus control subjects but no significant discrimination for active UC versus active CD patients, active UC versus remission UC patients, active CD versus remission CD patients, and remission UC patients versus control subjects (Table
3 and Additional file
5: Table S3 and Additional file
6: Table S4). No previous studies have actually compared the active and quiescent disease stages of CD and UC patients, although Dawiskiba et al. [
17] did compare active IBD and remission IBD patients and found significant metabolic profiles that differentiated these two congregated cohorts. They also compared remission IBD patients and control subjects, but without any significant results. This, however, contrasts with the findings of Williams et al. [
20], who were able to differentiate between remission CD patients and control subjects, as in the present study, and remission UC patients and control subjects, in contrast to the present study. The methodology and statistical procedures in the study by Williams et al. [
20] and those used herein are comparable, and no obvious explanation has been identified for the discrepancy in outcome.
The sustained metabolic changes at 14 weeks of IFX treatment in the remission CD patients versus control subjects might be an indication of the more profound inflammation compared with the relatively superficial inflammation seen in UC patients; in Table
4, the number of significant up- or down-regulated metabolites decreases as treatment proceeds from the initial IFX infusion (CD Rem(0) vs. control) to the fourth infusion (CD Rem(14) vs. control). In contrast, patients with UC have fewer significant metabolic changes at the time of treatment initiation (UC Rem(0) vs. control), and these changes are already absent after 6 weeks of treatment. This circumstance might also explain why the metabolic profiles from the successive serum samples (i.e. weeks 0, 2, 6 and 14) within each response type (i.e. Rem, Res and NRes) did not show any significant discrimination (Additional file
5: Table S3 and Additional file
6: Table S4); the metabolic changes seen from week 0 through week 14 in patients with CD entering remission are simply too few (Table
4). Future studies need to collect serum samples within an expanded time frame, at least in CD patients. In contrast, patients with UC entering remission have very few significantly deregulated metabolites at the time of treatment initiation (Table
4, UC Rem(0) vs. control), and the subsequent metabolic changes may consequently be insufficient to create significant discrimination between successive serum samples.
As could be expected, the metabolic profiles of CD Res and UC Res patients stayed significantly different from that of control subjects (Additional file
4: Table S2) throughout the induction period. Surprisingly, this was not the case for CD NRes and UC NRes patients. The obvious explanation is the relatively low number of samples in these groups (n = 9 and 10, respectively), but it might also be explained by an entirely different metabolic profile in the serum of patients who are primary non-responders to IFX. However, comparing the different response types (i.e. Rem, Res and NRes) did not produce any significant discrimination. Thus, in order to answer this question, lager cohorts are needed in future studies. Furthermore, the lack of discriminant power might also be explained by the poor correlation in general between clinical disease activity indices and the actual burden of disease, although we did incorporate biochemical parameters to substantiate the clinical scores.
An abnormal lipid metabolism was identified in both CD and UC patients compared with control subjects. LDL data could not be separated from the broad lipoprotein NMR peak, but the identified low levels of HDL and high levels of VLDL seem to be the consequence of malabsorption, increased transit times and the inflammatory environment (e.g. TNF-α, interleukin-1 and -6). TNF-α in particular has been shown to increase the release of free fatty acids from adipocytes, stimulate the production of triglycerides in the liver, and inhibit lipoprotein lipase and thereby the hydrolysis of triglycerides in VLDL, leading to an increase in VLDL and a decrease in HDL cholesterols [
37]. This proatherogenic lipid profile correlates with the increased incidence of cardiovascular morbidity seen in IBD patients, particularly in women and adolescents [
38]. As seen in this study (Table
4) and others [
39,
40], treatment with IFX ameliorates this proatherogenic lipid profile in both UC and CD patients. Thus, women and young adults with chronically active disease or frequent flares might be considered candidates for concomitant statin treatment if other atherogenic risk factors are present. However, the biobank from which the samples originated did not contain patient data on quantitative lipid measurements or other potential atherogenic risk factors except for sex, age and smoking.
This study also demonstrated decreased levels of the membrane metabolite GPC (Table
4). GPC is a derivative of the membrane metabolite choline and one of the major forms of choline storage in the cytosol. The decreased levels of GPC may be a consequence of the compromised integrity of the intestinal mucosal membrane [
15] with an inflammation-driven increased rate of apoptosis and cellular turnover [
41]. Interestingly, sustained choline deprivation stimulates cell survival through nuclear factor-kappa B activation, leading to sustained inflammation and oncogenesis [
42]. Furthermore, low levels of GPCs have been correlated with cardiovascular disease [
43], underlining the proatherogenic nature of the inflammation.
In contrast to other studies [
17,
19‐
21], low levels of glucose and lactate were found in both UC and CD patients compared with control subjects, and these changes persisted in CD patients achieving remission after 14 weeks of IFX treatment (Table
4). The low level of glucose could be explained by impaired intestinal absorption due to the inflammation, which is more profound in CD than UC. However, the concomitant low level of lactate and high level of pyruvate indicate the need for scavengers of reactive oxygen species [
44] and an inflammation-driven high energy demand with high glycolytic activity and low lactate dehydrogenase activity and hence accumulation of pyruvate but no production of lactate [
45].
Phenylalanine was persistently up-regulated and positively correlated with tyrosine in IBD patients even during induction of remission, especially in patients with CD (Table
4). This is in contrast to the other observed amino acids (i.e. valine, glutamine, glycine and histidine; Table
4). However, the pattern of increased aromatic amino acids (e.g. phenylalanine and tyrosine) and a decrease in branched chain amino acids (e.g. valine) is well known during catabolic conditions like sepsis and liver failure [
46,
47]. The inflammation-driven process causes muscle proteolysis with a subsequent decrease in branched chain amino acids due to muscle energy consumption and an increase in aromatic amino acids due to hepatic insufficiency with impairment of the hepatic enzyme phenylalanine-4-hydroxylase [
48].
Formate is an essential intermediary metabolite in virtually all living organisms, but in this study, formate was found in low levels in IBD patients (Table
4). This potentially reflects the high demand of formate locally in the inflamed intestines, where formate is tightly related to folate metabolism [
49]. A previous study [
15] has indicated an increased phospholipid metabolism (e.g. GPC and choline) and glutathione synthesis (e.g. glutamine and glycine) in inflamed colonic tissue from patients with UC, and both of these processes are linked via the methionine-homocysteine cycle, where formate and folate are essential and central elements [
49].
N-Acetyl glycoprotein is a well-known acute-phase protein, which is in accordance with the high levels seen in this study (Table
4), but due to a long half-life, it is not applicable as a useful clinical tool [
50]. Finally, three unidentified metabolites were consistently down-regulated in both active and inactive patients with IBD (Table
4).