Metabolome alterations in severe critical illness and vitamin D status

Low vitamin D status is common in the intensive care unit (ICU) [1‐3]. Several observational studies in critically ill cohorts suggest that vitamin D status is associated with important clinical outcomes [1‐3]. In particular, low vitamin D status is associated with increased risk of sepsis and with worse outcomes in patients with sepsis [4, 5]. Moreover, recent studies support vitamin D as a potential therapeutic agent in hospitalized patients [6, 7].

Vitamin D has broad biological effects on nuclear transcription, cell cycle regulation, differentiation, and apoptosis [8]. Vitamin D metabolic enzymes and vitamin D receptors have a wide tissue distribution, reflecting the involvement of vitamin D in the metabolism and function of many cell types [9]. Indeed, differential metabolic profiles are demonstrated in ambulatory patients who respond to vitamin D supplementation relative to those who do not [10, 11]. Since metabolic homeostasis is often disrupted in critical illness, substantial alterations of several intrinsic pathways can be expected in septic patients [12]. Only a limited number of metabolomic studies have been published to date in experimental sepsis models [13], pediatric sepsis [14], and critically ill adults [15].

While some existing data support anti-inflammatory and immune modulating effects related to vitamin D supplementation [16], and while metabolomic approaches are used to understand the pleiotropic effects of Vitamin D [17, 18], there is limited understanding of the metabolic alterations associated with low vitamin D status in critical illness. Therefore, we analyzed metabolite profiles with regard to vitamin D status in a prospective study of adult patients with systemic inflammatory response syndrome (SIRS) and sepsis [19]. We hypothesized that the metabolomic profile of patients with severe critical illness near the time of ICU admission is influenced by vitamin D status and that this metabolic difference in turn can illuminate important biologic pathways that may contribute to pathogenesis and prognosis.

Table 1 shows the demographic characteristics of the study cohort. Most patients were male (58%) and white (83%). The mean (SD) age at ICU admission was 55 (15) years. The mean (SD) 25(OH)D concentration was 20 (16) ng/ml, and 63% of cohort patients were diagnosed with sepsis. The mean APACHE II score was 26 (10). The 28-day mortality within the cohort was 35%. There were no significant differences between patients with 25(OH)D ≤ 15 ng/ml relative to those 25(OH)D > 15 ng/ml regarding any key baseline characteristic or for 28-day mortality.

In the present study, our goal was to investigate whether vitamin D status in the early course of severe critical illness was associated with differences in the metabolic profiles of critically ill patients. Utilizing several analytic strategies, we demonstrated that the metabolic profile of critically ill patients differs based on their vitamin D status and there is evidence that metabolites related to vitamin D status are most prominently related to glutathione and glutamate metabolism and glucuronidation.

In humans, 25(OH)D is the major circulating form of vitamin D₃. Steady-state plasma 25(OH)D concentrations represent a balance between formation and clearance activities, which are mediated by phase I and phase II oxidation and conjugation processes. Variation in the efficiency of these detoxification reactions contributes to variability in circulating plasma concentrations of 25(OH)D, thereby altering the activity of this prohormone. Based on the results of this metabolomic profiling study, we have identified two important phase II metabolism pathways for glutamate, glucuronidation and glutathione cycling that are associated with vitamin D homeostasis in critically ill ICU patients.

By MSEA, we identified the overlapping pathways for glutathione and glutamate metabolism as the most highly enriched pathways in our metabolite data. Pyroglutamine, a cyclic metabolite of glutamine and component of the glutathione cycle, was identified in PLS-DA analysis as a classifier of vitamin D status (Fig. 3). Glutathione, a major cellular thiol antioxidant, is a cofactor of the enzymatic detoxification of oxygen radicals [36, 37]. In vitro data suggest that vitamin D upregulates cellular glutathione [36]. Furthermore, in community-dwelling adults, serum 25(OH)D levels are associated with increased circulating glutathione [38]. Though the redox state of reduced/oxidized glutathione (GSH/GSSG) is closely regulated, it decreases with tissue injury, inflammation, sepsis, and toxin exposure [39‐41]. Oxidative stress is well-described in patients with sepsis, with supporting evidence for production of reactive oxygen species (ROS) and associated damage [42]. In patients with sepsis, inflammatory response initiation via oxidative stress occurs through redox pathway activation of nuclear factor κB (NFκB) and expression of a substantial number of genes involved in the immune response and cell survival [43, 44].

Glutamate, a highly concentrated intracellular amino acid is important for biosynthesis of multiple amino acids, nucleic acids, nucleotides and metabolites [45]. Though glutamate has a low concentration in plasma [46] it has an important role in peripheral organs and tissues as an extracellular signal mediator [47]. More germane to the severely ill cohort under study, ionotropic glutamate receptors are expressed on T cells and B cells. Dendritic cells and macrophages and glutamate serve as an immunomodulator in the initiation and development of T-cell-mediated immunity in peripheral tissues [48, 49]: α-ketoglutarate, a Krebs cycle intermediate, is produced in a glutamine-dependent fashion and regulates the T helper 1 cell and regulatory T cell generation balance [50].

Glucuronidation is crucial for the hepatic and renal metabolism of compounds, including bile acids, steroids, bilirubin, and fatty acids, to facilitate their elimination from the body and to improve the disposition and activity of drugs and hormones across tissues. Glucuronidation is an essential chemical reaction for rendering 1, 25(OH)₂D₃ (the most metabolically active vitamin D metabolite) to a water-soluble, biologically inactive form, but may also serve as a reservoir for enterohepatic circulation [51]. This conjugation reaction is performed by UDP glucuronosyltransferase (UGT) enzymes in the liver. In addition to glucuronate, we also identified bilirubin and biliverdin as important metabolite predictors of vitamin D status. Bilirubin, a metabolite of the heme end product biliverdin, is glucuronidated by UGT1A isoforms, chiefly UGT1A1, in addition to UGT1A4.

While UGTs catalyze the conjugation of a wide variety of endogenous substrates, recent studies have identified UGT1A4 as the primary catalyst of 25(OH)D glucuronidation in vivo [52]. Failure to recycle glucuronides could contribute to low vitamin D status through promoting the metabolism of 25(OH)D to its inactive, polar forms, which are more readily excreted, thereby reducing its levels in the systemic circulation. In addition, as UGT enzymes are highly polymorphic, and “gain-of-function” variants with high substrate clearance activity have been described in humans [52, 53], inter-individual variation in 25(OH)D levels due to variable UGT1A4 activity could contribute to lower 25(OH)D levels in circulation. Because homozygous carriers of UGT1A4*3 demonstrate enhanced 25(OH)D glucuronidation activity, patients with this genotype might be expected to have lower circulating levels of 25(OH)D and may therefore be at greater risk of low vitamin D status [52, 53]. In addition to UGT1A4, UGT1A1 was also predicted by network modeling of chemical-protein interactions for all seven metabolites, in addition to vitamin D metabolites, to co-interact with 1,25(OH)₂D₃, bilirubin, and glucuronic acid (Fig. 4). A specific role for UGT1A1 in glucuronidation of vitamin D has not been investigated but may contribute to vitamin D status in critically ill patients.

Two additional metabolites, 1-palmitoyl-glycerophosphoinositol and 2-hydroxybutyrate, were also associated with 25(OH)D plasma levels (Table 2). While little is known about the specific roles of 1-palmitoyl-glycerophosphoinositol in vitamin D metabolism, this compound belongs to the glycerophosphoinositol family and, along with pyroglutamine and 2-hydroxyisobutyrate (a derivative of 2-hydroxybutyrate), was associated with anti-hypertensive and lipid-lowering drugs in serum samples from a study of 1762 participants in the Cooperative Health Research in the Region of Augsburg (KORA) study [54].

The present study is not without potential limitations. Metabolites were measured early in the ICU course of severe critical illness, from a relatively small number of patients, at a single time point, and from a single biofluid (plasma). As the timing of plasma collection was within 72 hours of ICU admission and not at a uniform time point, the potential for variability and switches in metabolic pathways during the course of critical illness cannot be excluded. Our observational study included patients who were critically ill for various reasons, creating a heterogeneous study sample with high severity of illness. Further, selection bias may be present as we are analyzing only a subset of patients in the RoCI cohort. Without a control population of healthy vitamin-D-sufficient individuals, we do not have comparative metabolomic information on vitamin D status in the critically ill relative to the control. We are unable to account for the impact of race on metabolic profiles as our cohort was mostly white. Though we do have information on nutrition status, we do not have information related to nutrition intake, body mass index (BMI) or alcohol intake at the time metabolomic profiles were obtained. As our study was performed on a convenience sample, our results may not be generalizable to all critical care patients. Our bioinformatics approaches, while robust, are not without risk of introducing sources of bias. Although PLS-DA is well-suited for metabolomic data with much larger numbers of predictors than observations and multi-collinearity [55], it may be subject to over-fitting; to limit this, we performed cross-validation and permutation testing [56, 57]. Our measurement of 25(OH)D in a critically ill population with a mean estimated GFR of 59.6 ml/minute may not accurately account for the biologically active form of vitamin D. Our data do not allow for the distinction between metabolites that may be on a causal pathway or simply confounders of the association between vitamin D and outcome. Further, though aging is noted to be an important factor in metabolic homeostasis [25, 58] our study age range cannot account for such alterations. Finally, we cannot fully account for potential confounding, reverse causation, and the lack of a randomly-distributed exposure [59].

In summary, vitamin D status is associated with differential metabolic profiles in early severe critical illness. Glutathione and glutamate metabolism, which play principal roles in redox regulation and immunomodulation, respectively, were significantly altered with vitamin D status.