INTRODUCTION AND RATIONALE Acute intestinal ischemia remains a common clinical emergency with short-term mortality up to 80% and is often highly underestimated. , , It is a life-threatening clinical condition of diminished blood flow towards the gut, resulting in decreased supply of oxygen and nutrients to the intestinal tissue. Prolonged periods of ischemia are associated with disruption of the intestinal wall that normally functions as an important barrier. , The intestinal wall is composed of different layers and has a multitude of functions, such as the absorption of nutrients as well as the protection against harmful pathogens and carcinogens located in the intestinal lumen. When tissue damage occurs as consequence of ischemia, it progresses from the inner to the outer intestinal layer. This event results in release of a specific set of structural and functional proteins. Over the past few years many circulating biomarkers, such as D-dimer, glutathione S-transferase (GST), D-lactate and citrulline have been described as potential biomarkers for intestinal ischemia , , .

The incidence of acute intestinal ischemia increases exponentially with age, with an equal occurrence between men and women. Consequently, it is expected that there will be a strong increase in the incidence of acute intestinal ischemia due to population aging. Once there is a high index of suspicion of acute intestinal ischemia, the clinician should act fast. Early unambiguous diagnosis of the ischemic intestine is crucial to prevent further progression from reversible to irreversible intestinal injury, and henceforth decrease morbidity and mortality.

Despite the advances in medical diagnosis and treatment over the past decades, intestinal ischemia still has a poor prognosis.1 Delayed diagnosis is one of the leading causes of the high mortality rates. Delayed recognition of this condition results into the development of irreversible intestinal necrosis- and peritonitis, which is closely correlated with the high mortality in these patients. There are various causes of intestinal ischemia, which are generally divided into two categories: causes of vascular origin (mesenteric embolism/thrombosis, or non-occlusive mesenteric ischemia) and non-vascular causes (such as strangulated intestinal obstruction). Interestingly, there is a high variation of clinical presentation and features between the various causes of acute intestinal ischemia. The clinical presentation is mostly characterized by non-specific signs and symptoms for instance, abdominal pains, loss of weight, elevated blood cell count and metabolic acidosis.1-3, For example, arterial embolisms are associated with nausea and vomiting, whereas, thrombotic mesenteric arterial occlusion is more commonly associated with pain that intensifies after food ingestion .

Currently, high-tech diagnostic equipment such as compute tomography (CT) imaging is used for diagnosis. However, identification of acute intestinal ischemia by CT remains difficult, because most of the characteristic features such as occlusion of mesenteric vessels, bowel wall thickening and intestinal pneumatosis can be easily missed. , , Also, these features are highly variable and non-specific, i.e. they overlap with radiological appearances of other acute abdominal complications. It remains a challenge, even for experienced clinicians, to identify patients who are at risk of acute intestinal ischemia among patients presenting with other conditions. Hence, fast and accurate diagnosis is vital for improving the clinical outcomes of patients with acute intestinal ischemia.

In recent years, a number of studies have described new potential biomarkers, such as intestinal fatty acid binding protein (I-FABP), for the identification of acute intestinal ischemia. Previously, it has been shown in our lab that I-FABP plasma levels are increased in patients suffering from intestinal ischemia. I-FABP is a small (14-15 kDa) intracellular and water-soluble protein that is specifically expressed by mature epithelial cells in the intestinal mucosa. I-FABP has a high abundancy within the small intestine and accounts for approximately 2% of the cytosolic protein expressed. , Upon a decrease of bowel perfusion and consequent loss of enterocyte cell membrane integrity, a rapid release of I-FABP within the circulation is observed. In an earlier study, we could reveal a relationship between the release of I-FABP and the severity of intestinal ischemia mucosal damage, where I-FABP levels in the circulation were inversely correlated with the remaining villus length. The exclusive expression of I-FABP in the intestine potentially makes it an important marker for the early detection of intestinal damage as a consequence of ischemia.

Another biomarker of intestinal ischemia is the cytoskeleton protein Villin-1 (VIL-1). VIL-1 is a large (95 kDa) protein that is localized in the cytoplasm and luminal membranes where it is described to play a role in actin regulation. , It was shown in rat and human models of ischemia that VIL-1 is released into the circulation and can be measured in plasma (Ceulemans et al. submitted, 2018). As opposed to I-FABP, VIL-1 remained detectable during prolonged periods after the onset of ischemic damage in rats due to the longer half-life. Plasma protein levels of VIL-1 correlated strongly with histopathological damage scored according to Park-Chiu. , I-FABP disappears after 30 minutes of reperfusion, whereas serological VIL-1 levels persist relatively high until 120 minutes after onset of reperfusion. Thus, these data indicate that both I-FABP and VIL-1 may provide new information on the duration of the ischemic events in humans.

Ischemia can cause significant damage to the mucosal layer within a short amount of time. Fifteen minutes of ischemia results into the formation of sub-epithelial spaces (a.k.a. 'blebs'), whereas ischemic periods lasting longer than 45 minutes can lead to irreversible mucosal damage.23, , Sustained periods of ischemia result in damage to the muscle layers also known as transmural ischemia. Transmural ischemia can develop into perforation of the intestinal wall, sepsis and eventually death.2, Plasma I-FABP and VIL-1 are very sensitive biomarkers for the identification of early intestinal mucosal damage. Adding a smooth muscle marker, such as Smooth Muscle 22 (SM22) to the panel of biomarkers could aid in evaluating the severity of the transmural ischemic tissue damage. SM22 is a small (22 kDa), water-soluble protein that is abundantly expressed in intestinal smooth muscle cells. , An experimental rat model revealed that SM22 is rapidly released in plasma upon ischemic damage, and remains detectable up to 6 hours following the induction of ischemia. Histology revealed complete disintegration of the villi following 4 hours of ischemia. Furthermore, immunohistochemical staining for SM22 indicated necrosis of the longitudinal muscle layer of the intestinal wall after 4 hours of ischemia, demonstrating that there is a transition from mucosal damage into transmural damage in this period of time. In patients with proven transmural infarction, SM22 plasma levels were significantly higher compared to patients with only mucosal ischemic injury or healthy controls.33 In addition, it was demonstrated that SM22 plasma levels were significantly higher in patients suffering from ischemia compared with patients that had other acute abdominal complaints. These data indicate that SM22 is most certainly a relevant biomarker to evaluate the severity of acute intestinal ischemia.

The last part of this study focuses on the analysis of volatile organic compounds (VOCs) in exhaled air of patients suspected of acute intestinal ischemia. The many hundreds of VOCs in exhaled air can give valuable information about several processes, such as metabolism and lipid peroxidation. The analysis of VOCs in exhaled air is a non-invasive technique that can be used to differentiate between various clinical conditions. Our project group already demonstrated the capability of VOC profiling to discriminate patients with intestinal and liver diseases such as inflammatory bowel disease (IBD) and non-alcoholic steatohepatitis from healthy subjects. , , Furthermore, chemical identification of VOCs from patients with Crohn's disease revealed that VOCs are involved in inflammatory, oxidative stress, gut barrier and leaky gut processes (Smolinska A, et al. in preparation). As the pathophysiologic processes of IBD and intestinal ischemia share common mechanisms, it is expected that VOC analysis will aid in the diagnosis of intestinal ischemia in a non-invasive manner. In a pilot study, we demonstrated that systemic inflammatory responses triggered by circulating endotoxin in human volunteers could be accurately detected using VOCs (Figure 2, Lubbers et al. in preparation). As VOC analyses can detect changes in metabolic and inflammatory processes with a high sensitivity and specificity, this promising technique enables early detection of the pathophysiologic processes occurring in intestinal ischemia. Altogether, these findings support the hypothesis that VOC profiles improve the diagnosis of intestinal ischemia.