Clin Chem Lab Med 2018; 56(3): 373–385

Review

Katell Peoc’h*, Alexandre Nuzzo, Kevin Guedj, Catherine Paugam and Olivier Corcos Diagnosis biomarkers in acute intestinal ischemic injury: so close, yet so far https://doi.org/10.1515/cclm-2017-0291 Received April 3, 2017; accepted July 21, 2017; previously published Introduction online August 25, 2017 Acute intestinal ischemic injury (i3) is a life-threatening Abstract: Acute intestinal ischemic injury (i3) is a life- condition associated with a high short-term mortality threatening condition with disastrous prognosis, which rate ranging from 32% to 86%. Such a prognosis remains is currently difficult to diagnose at the early stages of unchanged through decades despite significant improve- the disease; a rapid diagnosis is mandatory to avoid irre- ments in vascular , and versible , extensive , and resuscitation. In the emergency setting, these catastrophic death. The overlapping protein expression of liver and gut outcomes are closely linked to delays in diagnosis [1] and related to the complex physiopathology of the disease, the treatment, which is of major concern since the early pres- heterogeneity of the disease and its relative rarity could entation of acute i3 is potentially fully reversible when explain the lack of a useful early biochemical marker of i3. using a specific multimodal management that includes Apart from non-specific biological markers of , revascularization [1, 2]. However, at this stage, the clinical hypoxia inflammation, and infection, several more spe- presentation is dominated by acute non-specific abdominal cific biomarkers in relation with the gut barrier dysfunc- pain without any other discriminating clinical or biologi- tion, the villi injury and the enterocyte mass have been cal characteristics [3]. As a result, early diagnosis may only used in the diagnosis of acute i3. It includes particularly be achieved by a high degree of clinical suspicion and a D-lactate, intestinal fatty acid-binding protein (FABP) prompt confirmation by an abdominal computed tomog- and citrulline. Herein, we will discuss leading publica- raphy (CT) scan identifying features of both tions concerning these historical markers that point out splanchnic vascular insufficiency and intestinal injury the main limitations reagrding their use in routine clini- [4]. However, selection of patients requiring CT evaluation cal practice. We will also introduce the first and limited remains a challenge due to the lack of an available diag- results arising from omic studies, underlying the remain- nostic sign or biomarker. As a consequence, physicians ing effort that needs to be done in the field of acute i3 bio- have long sought to identify a biomarker or a combination logical diagnosis, which remains a challenge. of markers to ensure a sensitive, specific and early diagno- sis of acute i3. In this study, we will review the basis of the Keywords: biomarker; citrulline; D-dimer; D-lactate; pathophysiology of i3, the conventional past and present intestinal fatty acid-binding protein; . strategies for biomarker discovery and their remaining gaps and introduce the new perspectives opened by the “-omics” *Corresponding author: Katell Peoc’h, Biochimie Clinique, Hôpital technologies. We focused in the first part of this review on Beaujon, Université Paris Diderot, UFR de Médecine Xavier Bichat human clinical studies, then we presented the pre-clinical and APHP, HUPNVS, DHU Unity, Clichy, France; and INSERM, UMRs research in the second part since we did not find enough 1149, CRI, Université Paris Diderot, Paris, France, Phone: +33 (0)1 40 87 54 36, E-mail: [email protected] published materials to limit our presentation to humans. Alexandre Nuzzo: SURVI, Hôpital Beaujon, APHP, HUPNVS, DHU Unity, Clichy, France; and Gastroenterologie, Hôpital Beaujon, APHP, HUPNVS, Clichy, France Pathophysiology of i3 Kevin Guedj: SURVI, Hôpital Beaujon, APHP, HUPNVS, DHU Unity, Clichy, France; and INSERM, UMRs 1148, LVTS, Paris, France Catherine Paugam: Anesthésie Réanimation, Hôpital Beaujon, Definition of i3 Université Paris Diderot, UFR de Médecine Xavier Bichat and APHP, HUPNVS, Clichy, France Olivier Corcos: SURVI, Hôpital Beaujon, APHP, HUPNVS, DHU Unity, i3 is an digestive injury related to intestinal vascular Clichy, France; Gastroenterologie, Hôpital Beaujon, APHP, HUPNVS, insufficiency, occlusion or low splanchnic-mesenteric Clichy, France; and INSERM, UMRs 1148, LVTS, Paris, France flow. The pathophysiology of i3 responds to a multi-step 374 Peoc’h et al.: Acute intestinal ischemic injury’s biomarkers process that begins with an intermittent or continuous, relate to intra-parietal arterioles, lesions of ischemia complete or incomplete decrease in digestive flow. remain superficial. Subsequent mucosal/submucosal ischemia evolves into Intestinal vascular insufficiency leads to hypoxia, first transmural ischemia, often acute, followed by intestinal with mucosal and submucosal consequences. The hypop- necrosis and death without treatment [5]. erfusion of the intestinal mucosa is responsible for an early Several theories have been proposed to explain how hypoxic cellular desquamation of the intestinal villi. Poly- this non-infectious could lead to sys- morphonuclear neutrophils are early major lesional actors temic inflammatory response syndrome (SIRS) followed that adhere and migrate to the ischemic site to ensure the by sepsis and multi-visceral failure [6]. removal of tissue debris during necrosis. Mucosal and sub- mucosal cells switch to anaerobic glycolysis with local pro- duction of lactate initially fully metabolized by the liver. Multistep pathophysiology The increase in intracellular acidosis blocks anaerobic metabolism and the membrane pumps of ionic and acid- Acute mesenteric ischemia (AMI) should be considered base regulation. This leads to a profound alteration of as one of the stages of the i3 process (Figure 1), which cellular homeostasis and, ultimately, to cell death by apop- starts from digestive vascular insufficiency to intestinal tosis [7–9]. Initially, there is a dissociation between high necrosis. Ischemia begins early and superficially and porto-mesenteric blood lactate levels and normal periph- then spreads deep and in the surface of the intestinal eral blood lactate levels due to the active liver metabolism wall. Vascular insufficiency is initially responsible for an [1]. Systemic is, therefore, a late phenom- inadequacy between inputs and requirements for energy enon, which often indicates intestinal necrosis and the substrates by overcoming the adaptive processes of a onset of a multi-visceral failure [10]. Associated endothe- digestive territory. This loss of homeostasis results from lial lesions can lead to platelet, pro- and anti-thrombotic a sudden decrease or interruption of the splanchnic- agent (protein C, S, and antithrombin) consumption, mesenteric blood flow. The decrease in splanchnic blood which causes the hemorrhagic syndrome. flow in the proximal circulation induces a deep exten- Furthermore, the intestinal neuro-hormonal regu- sion of the ischemia which then becomes transmural and lation of vasomotricity is associated with the activa- gangrenous. Conversely, when perfusion abnormalities tion of the renin-angiotensin-aldosterone system, which

Early and reversible i3 Late and irreversible i3

Vascular Mucosal/sub-mucosal Transmural Systemic injury injury injury injury

- Renin- Local Acute vascular angiotensin - Transmural - Translocation inflammatory insufficiency activation injury - Autodigestion pathway - Necrosis (low flow - Sympathic - Systemic - Polynuclear - Organ failure and/or stimulation inflammatory afflux occlusion) - Vasospasm Ischemic pathway - Hypoxia

Figure 1: Schematic representation of the time course and physiopathology of i3. i3 is the result of a multistep process that is initially limited, reversible and then became systemic and irreversible and led to death. The pathophysiological process can be divided into two main stages related to the prognosis, the early and reversible phase and the late and irreversible phase. Peoc’h et al.: Acute intestinal ischemic injury’s biomarkers 375 maintains the mucosal oxygen extraction rate. This inflammatory response. Degradation products of pancre- induces a reflex splanchnic arterial vasospasm, irrespec- atic enzymes, residues of bacterial products pass through tive of the initial vascular mechanism, that may prolong the lymphatic, hematogenous or peritoneal barrier and and worsen ischemia despite therapeutic revasculariza- are likely to induce not only a loco-regional but also a tion. This vasoconstriction accompanies, for example, sit- systemic reaction [19, 20]. In animal models, inhibition uations of hypovolemia, during which digestive ischemia of these enzymes results in a decrease in intra-parietal develops before clinical hemodynamic instability [11, 12]. micro-bleeding, systemic inflammatory response, and The disruption of the epithelial barrier resulting from even in mortality in some studies [21]. The action of these mucosal alterations leads to interactions among micro- enzymes would involve degradation of inter-enterocytic organisms, bacterial antigens, endotoxins of the intes- tight junction’s proteins such as E-cadherin. Moreover, tinal lumen with the mucosal and submucosal immune these enzymes would also induce a cleavage of the pro- system. The stimulation of innate immunity will result metalloproteinases into active metalloproteinases [22]. in local then systemic inflammatory pathways activation The systemic consequences of bowel ischemia and such as TLR, NF-κB or TNF [13, 14]. Through the blood- necrosis are lethal in most patients in the absence of stream, bacteria, endotoxins, cells degradation products curative treatment including revascularization [25, 26]. and activated immune cells translocate and promote However, reoxygenation of the digestive mucosa can also SIRS. Cytokines, chemokines, cellular and bacterial paradoxically worsen epithelial and vascular lesions, due debris can also reach the pulmonary circulation from the to an oxidative burst mechanism causing the influx and lymphatic circulation and thus cause acute respiratory death of neutrophils with the formation of neutrophil distress syndromes [15, 16]. The absence of a rapid recov- extracellular traps and the secretion of their granular ery of a sufficient digestive perfusion leads to irrevers- content [27]. ible transmural necrosis and then to . Without intestinal resection, the SIRS evolves to multiple organ syndrome and death [16]. In the model of the “gut origin of sepsis”, the gut Clinical unmet needs was considered to be the “motor” of multi-organ failure [11, 15–17]. Aside from its barrier function, the gut con- in the diagnosis workup of i3: tains growth factors, adenosine and hormones, which are urgent need for a biomarker potential mediators of the modulation of intestinal inflam- mation and repair, due to their roles in cellular prolifera- Early diagnosis of i3 requires a high degree of suspi- tion, differentiation, migration, apoptosis and autophagy cion faced with any , especially when [18–22]. Physiologically, the gut could initiate and propa- the pain is sudden or rapidly growing (“vascular-like”), gate sepsis due to the ability of bacteria, endotoxins, and unusual, intense and requiring opioids. Other clinical other antigens to translocate, along with the production of and biological associated signs (, , gas- pro-inflammatory cytokines and toxins [11]. In the “three trointestinal hemorrhage, hyperleukocytosis, lactic aci- hits model”, Deitch [23] added the phenomenon of reper- dosis) are not constant or appear too late in the course fusion injury. In the “gut-lymph” theory, bacteria, cellular of the disease and have no diagnostic value [1]. In our components, immune cells, cytokines and chemokines retrospective experience of a cohort of 221 patients, peri- generated by the injured gut travel via the lymphatics to toneal signs, organ failure and serum lactate elevation reach the pulmonary circulation, activating alveolar mac- were initially lacking in 85%, 77% and 57% of the cases, rophages and contributing to acute lung injury, acute res- respectively [28]. When unrecognized at this stage, the piratory distress syndrome and multi-organ failure related diagnosis was carried out later at the stage of necrosis to AMI [15, 16, 24]. and complications, explaining why 184/221 (83%) of the patients required intestinal resection, resulting in in 148/184 (80%). Intestinal autodigestion Improving the prognosis of i3 requires the discovery of early, sensitive and specific diagnostic biomarkers. In This quite recent concept describes the effect of pan- the last decade, some potential biomarkers have emerged creatic enzymes on the intestinal barrier altered by from the literature. Some of these markers have been ischemia. Self-digestion contributes to the worsening of studied with particular interest, because of their higher i3 lesions and the development of the related systemic presumed enterocyte specificity. 376 Peoc’h et al.: Acute intestinal ischemic injury’s biomarkers

Materials and methods phosphate, ) have frequently been observed [29, 30]. is commonly observed as pre- sented earlier. Searching strategy Regarding hematological parameters, attention has been given to platelet indices, particularly platelets In an attempt to identify all studies that evaluate markers volume, but also to the various combination of neutro- for human intestinal ischemia, two methods were used phils, lymphocytes and platelets ratios [31]. Budak et al. to retrieve information for this review. First, we con- [32] proposed in a systematic review that low platelets ducted search onto the on-line databases PubMed and volume could be used in the diagnosis, but considered Google. The search was conducted using the follow- that high platelet volume would be a poor prognosis indi- ing terms: (((“mesenteric ischemia” [MeSH terms] OR cator. As a whole, the use of such indices appears to be (“mesenteric” [all fields] AND “ischemia” [all fields]) OR difficult to translate into clinical practice. “mesenteric ischemia” [all fields]) OR AMI [all fields] OR ((“intestines” [MeSH terms] OR “intestines” [all fields] OR “intestinal” [all fields]) AND (“ischaemia” [all fields] Biological markers of thrombosis OR “ischemia” [MeSH terms] OR “ischemia” [all fields]))) AND (“­diagnosis” [subheading]­ OR “diagnosis” [all fields] D-dimer, an enzymatic degradation product of fibrin, has OR “diagnosis” [MeSH terms]) AND (“biomarkers” [MeSH been found to be the most consistent highly sensitive early terms] OR “biomarkers” [all fields] OR “marker” [all marker, but has low specificity. Moreover, D-dimers are fields]) OR (“mesenteric ischemia” [MeSH terms] AND usually increased either in arterial or venous occlusive proteomic [all fields]) OR (“mesenteric ischemia” [MeSH forms (A. Nuzzo and O. Corcos, personal communica- terms] AND “metabolomic” [all fields]) OR (“mesen- tion), although they remain in the normal range in non- teric ischemia” [MeSH terms] AND “genetics” [all fields] occlusive acute i3 [33]. In a meta-analysis, Cudnik et al. [34] OR (“mesenteric­ ischemia” [MeSH terms] AND “genetic evidenced that L-lactate and D-dimers exhibited a good variant” [all fields])). pooled sensitivity (96%), although both were not specific The second method was to examine the references of enough (40%) to be used as diagnostic markers. The inclu- the articles found by the electronic searches methods for sion of both occlusive and non-occlusive forms of i3, as dis- additional citations. cussed in the larger paper of Matsumoto et al. [33], could With the use of retrieval mentioned above method, all lead, however, to high heterogeneity in patients. However, English-language studies describing human subjects were a recent meta-analysis estimated the area under the curve considered for this review. (AUC) of the receiver operating curve (ROC) of D-dimer to 0.81, underlying its potential utility in clinical practice [35]. Selection

Two authors performed the searches. Any duplicates were Biological markers of hypoxia and oxidative removed. Two authors selected resulting articles indepen- stress dently. A first selection was made by screening the titles and abstracts of all articles. Next, full articles were read to L-lactate is a ubiquitous product of glycolysis in the context make a final selection. Where there was no consensus, the of anaerobia. In 1994, Lange and Jackel [36] qualified manuscript was discussed with all authors to make a final L-lactate as the best marker of intestinal ischemia, regard- decision about inclusion. ing its negative predictive value. However, as expected, L-lactate elevation in plasma could not differentiate intestinal ischemia from the other etiologies of abdomi- nal emergencies or intensive care diseases [37–39]. Its Non-specific biomarkers elevation better reflects the late stage of the disease, with extensive transmural necrosis, anaerobic metabolism due Common laboratory findings to systemic hypoperfusion [10]. Hence, it should not be used anymore as an early diagnostic marker of AMI [40]. Variations in common biological blood parameters (base Glutathione S-transferases (GST) are enzymes deficit, lactate dehydrogenase, aspartate aminotrans- involved in the detoxification of a wide variety of endo- ferase, creatinine phosphokinase, alkaline phosphatase, and xeno-biotics, conjugating them to glutathione. These Peoc’h et al.: Acute intestinal ischemic injury’s biomarkers 377 enzymes are sensitive biomarkers of cytolysis, with a . As a whole, the use of PCT for the diagnosis very short half-life, and are currently used in the diagno- of acute i3 lacks specificity. sis of hepatic cytolysis [41]. A-GST showed a significant Finally, none of the parameters cited above show increase in 50% of acute i3 patients, as compared with 12 enough clinical accuracy as a diagnostic marker of early, other types of unclear acute abdominal pain suspected limited and reversible i3, which is mandatory to prevent as being ischemic, with a negative predictive value of the occurrence of intestinal necrosis and reduce the mor- 100% [42]. In the analysis published by Evennett et al. tality rate. Therefore, the markers cited above, which [30], the pooled estimate of sensitivity was 68% and the can be assessed through current laboratory assays, show pooled estimate of specificity was 85%. However, A-GST acceptable sensitivities, but none of them are specific also increases in non-specific hypotensive patients with enough to be used as a diagnostic marker. multiple organ failures [43]. During acute ischemic conditions, albumin’s metal- binding capacity is reduced, leading to the appearance of a metabolic variant known as ischemia-modified albumin Promising candidate biomarkers (IMA). It is a sensitive but non-specific marker of myo- cardial and muscle ischemia, pulmonary embolism and In a candidate approach, some markers related to the stroke [44]. IMA is usually measured in the plasma or pathophysiology of ischemia, intestinal insufficiency and serum by enzyme-linked immunosorbent assay (ELISA) gut barrier failure would be of interest in the biological or using an assay based on a spectrophotometric method diagnosis. Selected clinical studies concerning these bio- that measures altered cobalt-human serum albumin markers are presented in Table 1. binding. Significantly increased plasmatic concentra- tions were found at admission time of seven patients with acute i3, as compared to healthy controls [45]. Another D-lactate, a biomarker of gut barrier small study reported 100% sensitivity and 86% specificity dysfunction in the detection of 12 intestinal ischemia in preoperative plasmas of 26 patients scheduled for exploratory laparot- D-lactate, the second stereoisomer of lactate, is a byprod- omy of mesenteric ischemia suspicion [46]. uct of bacterial fermentation, with only a small amount being produced by human cells [58]. It can be found in the circulation after ischemic injury, increased intestinal Biomarkers of inflammation permeability, or bacterial overgrowth. i3 is associated with growth of the resident bacterial microbiota that releases The C-reactive protein is commonly increased in acute i3. D-lactate into portal and systemic circulations. The analy- Acute inflammatory mediators such as interleukin-2 and sis of D-lactate concentration requires strict preanalyti- 6 and tumor necrosis factor are non-specific of intestinal cal conditions, which are comparable to the one needed injury, although interleukin-6 (IL-6) has been proposed for the assessment of L-lactate concentration. D-lactate to be both sensitive and specific in a small cohort of 10 is usually assayed using an enzymatic UV spectrophoto- AMI [47]. metric method on deproteinized plasma [59]. This latter method could be automated [60]. In the last decades, few Biomarker of infection studies were focused on the use of seric D-lactate in the biological diagnosis of i3. In 2006, in a prospective study, Acute i3 is usually associated with an increase in leuko- Collange et al. [61] compared D-lactate concentrations in cytosis that can exceed 20 G/L [48]. Procalcitonin (PCT) 29 surgical abdominal aortic aneurysms patients (AAA), has been proposed for the diagnosis of AMI. This precur- which may be associated with i3. D-lactate levels were sor of the calcitonin is currently used in clinical practice increased in AAA patients for whom the inferior mesen- for the differential diagnosis of infection of bacterial teric artery was hypoperfused during the surgery (n = 6, origin. According to a systematic review by Cosse et al. 0.13 mmol/L), as compared with AAA patients without [49], PCT’s positive and negative predictive values for hypoperfusion (n = 23, 0.03 mmol/L, p = 0.007). In 2015, the diagnosis of acute i3 ranged between 27% and 90% Shi et al. [55] showed that serum D-lactate levels were and between 81% and 100%, respectively. As underlined increased in i3 patients (52.73 ± 26.46 μg/mL in i3 vs. by the authors, PCT is usually elevated during sepsis, 15.58 ± 5.17 μg/mL in non-intestinal ischemia) and could in specific bacterial infections, and in various types of participate in the diagnosis. As a whole, according to the 378 Peoc’h et al.: Acute intestinal ischemic injury’s biomarkers Experimental Experimental procedure Spectrophotometric on protein method free plasma Spectrophotometric Spectrophotometric on protein method free plasma ELISA (Hycult (Hycult ELISA Uden, Biotech, The Nederlands) ELISA (Hycult (Hycult ELISA Uden, Biotech, The Nederlands) ELISA

– – 96 75 87 NPV NPV 71.4

– – 70 83 PPV PPV 100 93.8 − – – – – 45 11 LR + – – – – LR 23.4 81.8

% 87 77 71 89 100 93.8 Specificity, Specificity,

% 90 82 68 90 90 71.4 Sensitivity, Sensitivity,

– 6.5 ng/mL 0.09 ng/mL value Cut-off 0.20 mmol/L 0.268 ng/mL 0.551 ng/mL

– – Range 0.04–5 ng/mL 11.71–48.66 μ g/L 0.04–74.711 ng/mL 0.142–442.795 ng/mL

– 32.37 μ g/L value Mean 18.5 ng/mL 0.653 ng/mL 3.377 ng/mL 0.421 ng/mL

Plasma Serum Tissue Plasma Urine Serum Serum

D-lactate D-lactate Biomarker I-FABP I-FABP I-FABP I-FABP

Thirty-one patients Thirty-one patients laparotomy undergoing abdominal an acute for including emergency, acute with patients ischemia mesenteric bowel small (n = 9), with (n = 5) (SBO) obstruction acute only with or patient (n = 17). condition abdomen were controls healthy Ten included also Ruptured abdominal aortic (AAA) aneurysm (n = 11) or with patients (n = 13) ischemic without were complication controls with compared (n = 21) Clinical condition Clinical Forty-six patients suspected suspected patients Forty-six ischemia, intestinal for were 22 that including intestinal with diagnosed 24 patients and ischemia with diagnosed were that other diseases Thirty patients diagnosed diagnosed Thirty patients ischemia intestinal for 27 with compared were other types with patients with and abdomen acute of controls 20 healthy Thirty-seven patients patients Thirty-seven SBO diagnosed with 21 that including with diagnosed were SBO and strangulated were that 16 patients SBO simple with diagnosed

41 45 46 77 37 size Sample Sample

Murray 1994 et al. [50] et al. Poeze 1998 [51] Selected clinical studies about citrulline, I-FABP and D-lactate as biomarkers of acute i3. acute of biomarkers as D-lactate and I-FABP citrulline, about studies clinical 1: Selected Table author, First year Thuijls et al. et al. Thuijls 2011 [37] Güzel et al. et al. Güzel 2014 [52] Kittaka Kittaka 2014 et al. [53] Peoc’h et al.: Acute intestinal ischemic injury’s biomarkers 379 Experimental Experimental procedure ELISA ELISA ELISA ELISA Ion-exchange Ion-exchange chromatography coupled method LC-MS/MS with (amino acids analysis LC-MS/MS in biological, kit Technologies, Zivak Turkey) Istambul, ELISA (R&D Systems ELISA (R&D Systems DuoSet., MN, USA)

– – – 50 NPV NPV 96.3 72.6 64.1

– – – PPV PPV 100 97.6 32.1 86.3

− – – 0.1 LR 0.24 0.31 0.61 0.19

+ – – – LR 3.25 2.82 1.54 4.58

% 40 80 100 100 89.1 74.8 85.9 Specificity, Specificity,

% 90 83.3 76.2 66.7 92.3 91.7 39.13 Sensitivity, Sensitivity,

15.82 mmol/L 9.1 ng/mL 1.3 ng/mL 0.69 ng/mL 2.52 ng/mL value Cut-off 93.07 ng/mL 34.28 μ g/mL

– – – – – – Range 1.1–498.4 ng/mL

9 ng/mL 7 ng/mL 31 ng/mL 463 ng/mL value Mean 21.7 nmol/L 52.73 μ g/mL 149.74 ng/mL

Serum Tissue Arterioveinous Arterioveinous difference Serum Serum Plasma Serum Urine

I-FABP Biomarker I-FABP I-FABP D-lactate Citrulline I-FABP I-FABP

Two hundred and eight eight and hundred Two 24 including patients vascular, with patients ischemia. intestinal with patients Sixty-two and ischemia non-vascular the non-ischemic 122 with disease Clinical condition Clinical Human ischemia- Human model reperfusion Three hundred and nine and hundred Three 39 including patients ischemic with patients Two abdomen. acute thirty-three and hundred non-ischemic with patients and abdomen acute controls 37 healthy Forty-eight patients patients Forty-eight intestinal for suspected 23 including ischemia with diagnosed were that and ischemia intestinal were that 25 patients other with diagnosed diseases Eighteen patients patients Eighteen AMI among suspected diagnosed whom 13 were were left The five AMI. with used as controls

32 48 18 208 309 size Sample Sample

Matsumoto Matsumoto 2014 et al. [33] Table 1 (continued) Table patients. of n, number value; predictive negative NPV, value; predictive positive −; PPV, ratio likelihood + ; LR−, ratio LR + , likelihood harmonized. were the units simplification, For First author, author, First year Schellekens Schellekens 2014 et al. [54] Shi et al. Shi et al. 2015 [55] Kulu et al. et al. Kulu 2016 [56] Salim et al. et al. Salim 2017 [57] 380 Peoc’h et al.: Acute intestinal ischemic injury’s biomarkers chosen threshold, the sensitivity of this marker in either between the duration of ischemia and the increase of plasma or serum ranged between 67% and 90%, whereas serum I-FABP. Kittaka et al. [53] found that serum I-FABP the specificity reached 87%. The design of the studies and concentrations were significantly increased in patients their definition of intestinal ischemia were, however, het- with strangulated (n = 21; 18.5 ng/mL), erogeneous. Therefore, there is a need for larger studies as compared with patients with simple obstruction (n = 16; with well-characterized populations to confirm the poten- 1.6 ng/mL). tial use of D-lactate. This could be a promising track since Urine I-FABP could be a biomarker with high speci- the assay could be easily automated. ficity and sensitivity (area under the receiver operating curve = 0.88) for the diagnosis of acute i3 in 18 patients with suspected i3 [57]. Fatty acid-binding proteins (FABP), Large variations are observed among studies regard- ­biomarkers of villi injury ing mean values and ranges. The comparison between seric and plasmatic values has not yet been published. FABP are cytosolic proteins involved in the uptake and Moreover, different ELISA kits are used participating to intracellular transport of fatty acids. The mature entero- the variability of the results. For example, Shi et al. [55] cyte expresses three isoforms: intestinal FABP (I-FABP), described higher mean values and a higher cut-off value ileal bile acid-binding protein (I-BABP) and liver FABP than other studies. As they do not mention the use of (L-FABP). Hycult ELISA, which is used in all other studies, we can The intestine, liver and kidneys express L-FABP, reasonably speculate that this difference could be related whereas I-BABP is specific of the . I-FABP is a 15-kDa to the assay that was used. soluble protein expressed by enterocytes located at the A recent meta-analysis evidenced a pooled sensitivity tips of the intestinal mucosal villi, the anatomical region of 80% for serum I-FABP, a pooled specificity of 85%, and that is first affected by ischemic injuries. In physiological an area under the ROC curve of 0.86 in the diagnosis of conditions, I-FABP is low in peripheral circulation and is acute i3 [69]. cleared via the urine [62, 63]. After mucosal tissue injury, and especially enterocyte necrosis, the protein is quickly released into the bloodstream [64]. The liberation of the Citrulline, a biomarker of enterocyte mass biomarker has been shown in rat to be concomitant with and intestinal failure the ischemia [62] underlying its potential interest as a very early diagnosis marker [64]. Citrulline is a non-proteinogenic amino acid synthesized Many studies have reported a relationship between from glutamine by small bowel enterocytes. This amino blood I-FABP concentration and small intestinal diseases, acid is a precursor of nitrogen oxide and participates in in acute i3, critically ill patients, or post cardiac surgery, in the transformation of ammonia into urea, and in the syn- which i3 represents roughly 1% of common complications thesis of arginine. Its plasmatic concentration depends on [65]. Most of them are presented in Table 1. gut synthesis and renal elimination, decreasing in short In clinical settings, I-FABP concentrations measured bowel conditions and thus known as a functional marker in peritoneal fluid, plasma and urine are significantly of enterocyte mass, correlated with remnant small bowel higher in patients with i3 than in healthy controls and length and home parenteral nutrition dependence [70]. patients with other causes of the acute abdomen [66–68]. Citrulline is usually measured in plasma or serum, using In peritoneal fluid, whose presence reveals late and severe ELISA methods, high-performance liquide chromatogra- disease, high levels of I-FABP were detected in patients phy or mass spectrometry. Critically ill patients with with intestinal diseases [68]. may have an acute non-occlusive i3 resulting in a reduc- In the study by Kanda et al. [67], elevated levels of tion of enterocyte mass and related citrulline synthesis, serum I-FABP upon admission at the hospital were asso- leading to low plasma citrulline concentrations [71]. In a ciated with 2/8 patients presenting with a strangulated study by Piton et al. [71], plasmatic citrulline concentra- bowel obstruction and 5/5 patients suffering from acute i3, tion was shown to decrease in the first hours of shock in whereas ranges were normal for healthy subjects (n = 35) 24/55 critically ill patients and was correlated with mortal- and patients with abdominal pain of other etiologies ity within 28 days. (n = 48). In 2016, Kulu et al. [56] found that the concentration In a human experimental model of ischemia-reper- of plasmatic citrulline was significantly decreased in 23 fusion, Schellekens et al. [54] evidenced a correlation patients with acute abdominal findings preoperatively Peoc’h et al.: Acute intestinal ischemic injury’s biomarkers 381 attributed to AMI (mean: 0.72 mmol/L; range 0.57–0.84), complications and the need for surgery. The emerging use as compared to those in patients with other acute abdomi- of omics in the discovery of biomarkers opens new ways. nal conditions. Acute renal failure induces high plasmatic Regarding genomic data, no clue arises from genetic citrulline concentrations by decreasing renal clearance studies in acute i3. However, no large genetic study has and citrulline transformation into arginine [72], which been published in this disease. The genetic variants pre- may complicate the interpretation of the results in severe disposing to venous thromboembolism (i.e. factors V and patients with multiorgan failures. Moreover, post prandial II Leiden, MTHFR variants) could participate in the physi- samples are associated with a 10%–20% decrease in blood opathological process of the disease, although descrip- concentration [72]. Moreover, some inter-ethnic variations tions are rare and related to private patients [76]. have been described in reference ranges. Transcriptomic studies in models of acute i3 are These results suggest that citrulline is probably more scarce. In a pig model with induced proximal arterial promising as a prognostic than a diagnostic marker of occlusion of the superior mesenteric artery, Block et al. AMI. Moreover, the interpretation of a ratio between plas- identified a panel of up- and down-regulated mRNA (157 matic citrulline and creatinine could help minimize the and 57 transcripts, respectively). Up-regulated mRNA effect of acute renal failure leading to possible false-neg- included monocyte chemoattractant protein 1 (MCP1) and ative results. acyl CoA synthetase long-chain family member 4 [77]. In a preclinical study in swine, a microRNA signature was identified in an i3 model related to hypothermic circula- tory arrest associated with the gut barrier dysfunction. Overview on current biomarkers This signature included a noticeable decrease in mRNA- 31, which interacts with the hypoxia-inducible factor HIF Two recent systematic reviews have presented similar function [78]. data as ours. Derikx et al. [73] presented D-dimer, I-FABP Proteomic studies investigate the pool of full-length, and α-GST as the most promising biomarkers. Treskes truncated and post-traductionally modified proteins and et al. [74] calculated combined AUC for IFABP, D-lactates peptides, whereas metabolomics explores the whole met- and α-GST and α-GST appeared to be the more accurate, abolic process of peptides, saccharides and lipids. Both before IFABP and D-lactates, with only discrete differences approaches have been increasingly used for biomarker between AUC (0.876, 0.84 and 0.814), respectively. Overall, discovery in the last decade, thanks to the development of on the basis of available studies, IFABP, α-GST, D-dimers mass spectrometry and bioinformatics. and D-lactates appear to be useful in the diagnosis of acute Experimental studies for acute i3 biomarkers search i3, none of this marker being performant enough to be use using both proteomic and metabolomics approaches are solely. Moreover, a recent study by Schellekens et al. [75] summarized in Table 2. We found only five studies using proposed the smooth muscle of 22 kDa (SM22) as a poten- heterogeneous techniques and models, and no common tial marker of transmural intestinal ischemia in patients, marker was evidenced. adding to the spectra of potential biomarkers in i3. Steelman et al. [83] confirmed in the horse the interest Extensive efforts are still needed to design studies of citrulline as a biomarker. None of the proteomic studies with increased population size, well-characterized in identified in either pig or rodent FABP proteins. However, terms of phenotypic groups, and with standardized pre- this could be due to imperfect animal models and interspe- analytical and analytical conditions. On the other hand, cies variability. Regarding metabolomic studies, the only more performant biomarkers could perhaps emerge from common observation was a decrease in glucose observed omics studies. in two rodent models.

Towards promising novel Conclusions approaches for finding biomarkers Acute i3 is a highly severe condition, with a high mortality The lack of suitably validated markers for the diagnosis rate and major anatomical and functional consequences of i3 pushes the scientific community to continue their in case of survival. Acute i3 represents a gut and life-threat- investigations on the search for the molecule that will ening emergency for which the main identified prognosis help for the early diagnostic of i3 and that will prevent the factor is the precocity of the diagnosis and treatment. 382 Peoc’h et al.: Acute intestinal ischemic injury’s biomarkers Procedure Microdialysis 2D Gel electrophoresis electrophoresis 2D Gel MALDI-TOF with coupled Microdialysis GC/MS GC-LC/MS Specie Pig Rat Pig Mouse Horse Model Mesenteric ischemia-reperfusion model model ischemia-reperfusion Mesenteric artery for mesenteric the superior of (ligature reperfusion 3 h of by 60 or 120 min followed Ischemia/reperfusion model Ischemia/reperfusion Mesenteric ischemia-reperfusion model model ischemia-reperfusion Mesenteric artery for mesenteric the superior of (ligature reperfusion) 2 h of by 60 min followed Mesenteric ischemia model (ligature of the of (ligature model ischemia Mesenteric artery 2–4 h) for mesenteric superior Horse laminitis model laminitis Horse Results Increase Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Increase Decrease Decrease Increase Increase Decrease Increase Decrease Decrease Decrease Decrease Decrease Increase Increase Increase Decrease Molecule Glycerol Phosphoglycerate mutase 1 mutase Phosphoglycerate b-c1 complex Cytochrome Pyruvate kinase hydratase aconitate Cytoplasmic dehydrogenase Glutamate 1 A hydratase coenzyme Enoyl dehydrogenase Isocitrate Glyceraldehyde-3-phosphate dehydrogenase reductase Aldose dehydrogenase Aldehyde A3 isomerase disulfide Protein a-1B chain Tubulin 1 Intelectin 2 protein binding Retinol precursor Albumin A2 Annexin Glucose Lactate Arabinose Xylose Glucose Ribose acid Stearic Urea acid Threonic phosphate Inorganic Citrulline Tissue Gut lumen lumen Gut microdialysate Intestinal Intestinal mucosa Intestinal Intestinal segments Serum Serum Omic Metabolomic Proteomic/ metabolomic Metabolomic Metabolomic Metabolomic Experimental animal studies using either metabolomics or proteomic approaches. or proteomic either metabolomics using studies animal 2: Experimental Table year author, First Solligard et al. et al. Solligard 2005 [79] Li et al. 2010 [80] Li et al. Birke-Sorensen Birke-Sorensen Andersen and 2010 [81] Fahrner et al. et al. Fahrner 2012 [82] Steelman et al. et al. Steelman 2014 [83] GC/MS, Gaz chromatography/mass spectrometry. chromatography/mass Gaz GC/MS, Peoc’h et al.: Acute intestinal ischemic injury’s biomarkers 383

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