25% HUMAN SERUM ALBUMIN IMPROVES HEMODYNAMICS AND PREVENTS THE NEED FOR NEARLY ALL PREHOSPITAL RESUSCITATION IN A RAT ( RATTUS NORVEGICUS ) MODEL OF TRAUMA AND HEMORRHAGE.

ABSTRACT: Combat casualty care can be complicated by transport times exceeding the "golden hour," with intervention and resuscitation limited to what the medic can carry. Pharmaceutical albumin comes highly saturated with nonesterified fatty acids (NEFAs). We recently showed that treatment with 25% bovine serum albumin (BSA) loaded with oleic acid, but not NEFA-free BSA, improved survival for hours after severe hemorrhage and often eliminated the need for resuscitation in rats. However, it was unknown whether pharmaceutical albumin, derived from human sources and loaded with caprylic acid (CA), would have the same benefits. We compared adjunct treatment with oleic acid-saturated BSA, CA-saturated BSA, pharmaceutical human serum albumin, or a no-albumin control in a similar rat hemorrhagic shock model to determine whether the three NEFA-albumin groups provided the same benefits relative to control. We found almost no significant differences among the NEFA-albumin groups in any measure. Mortality in controls was too low to allow for detection of improvement in survival, but NEFA-albumin groups had significantly improved hemodynamics, lactate clearance, and greatly reduced fluid requirements compared with controls. Contrary to expectations of "dehydration," 25% albumins shifted little additional fluid into the vasculature. Rather, they restored protein to the autotransfusion fluid. Nonesterified fatty acids-albumin did not worsen lung permeability, but we observed a loss of circulating protein suggesting it may have increased overall vascular permeability. Our findings suggest that, though imperfect, 25% human serum albumin could be a solution for resuscitation in austere conditions requiring prolonged field care.

Albumin Saturated With Fatty Acids Prevents Decompensation in a Rat Hemorrhagic Shock Trauma Model With Tourniquet and Hypotensive Resuscitation.

ABSTRACT: Decompensation is a major prehospital threat to survival from trauma/hemorrhage shock (T/HS) after controlling bleeding. We recently showed higher than expected mortality from a combat-relevant rat model of T/HS (27 mL/kg hemorrhage) with tourniquet (TQ) and permissive hypotensive resuscitation (PHR) with Plasmalyte. Mortality and fluid requirements were reduced by resuscitation with 25% albumin presaturated with oleic acid (OA-sat) compared with fatty-acid -free albumin or Plasmalyte. The objective of this follow-up analysis was to determine the role of decompensation and individual compensatory mechanisms in those outcomes. We observed two forms of decompensation: slow (accelerating fluid volumes needed to maintain blood pressure) and acute (continuous fluid administration unable to prevent pressure drop). Combined incidence of decompensation was 71%. Nearly all deaths (21 of 22) were caused by acute decompensations that began as slow decompensations. The best hemodynamic measure for predicting acute decompensation was diastolic arterial pressure. Decompensation was due to vascular decompensation rather than loss of cardiac performance. Albumin concentration was lower in decompensating groups, suggesting decreased stressed volume, which may explain the association of low albumin on admission with poor outcomes after trauma. Our findings suggest that acute decompensation may be common after trauma and severe hemorrhage treated with TQ and PHR and OA-sat albumin may benefit early survival and reduce transfusion volume by improving venous constriction and preventing decompensation.

Fatty Acid-Saturated Albumin Reduces High Mortality and Fluid Requirements in a Rat Model of Hemorrhagic Shock Plus Tourniquet and Hypotensive Resuscitation.

Military prehospital care for hemorrhage is often characterized by use of tourniquets (TQ) and permissive hypotensive resuscitation (PHR) with crystalloids or colloids, but these treatments have not been previously combined in an animal model. Although albumin resuscitation solutions have been tested, the potential effects of nonesterified fatty acids (NEFAs) bound to albumin have not been evaluated in vivo, and few studies have investigated concentrated albumin solutions to reduce fluid requirements. We created a militarily relevant rat model of trauma and hemorrhagic shock (T/HS) (27 mL/kg hemorrhage) with TQ and PHR. We investigated the ability of resuscitation with concentrated (250 mg/mL) albumin, followed by Plasmalyte as needed to maintain PHR, to reduce fluid volumes (vs. Plasmalyte alone, N = 17). Albumin was free of nonesterified fatty acids (N = 15) or saturated with oleic acid (OA; N = 13). The model resulted in high (53%) mortality within 3 h of injury. Only OA-saturated albumin was able to significantly reduce mortality (from 47% to 8%) and fluid requirements (from 56 to 6 mL/kg) compared to Plasmalyte alone. Plasma NEFA-binding capacity was saturated earliest in the OA-saturated albumin group. Likewise, OA-saturated albumin tended to increase cell-free hemoglobin in the broncheoalveolar lavage fluid, which was significantly associated with survival. Our findings suggest incorporating TQ and PHR in T/HS models may result in high mortality and fluid requirements and that OA-saturated albumin, but not NEFA-free albumin or Plasmalyte alone, may provide a benefit to early survival and resuscitation volume, though a hemolytic mechanism may have later consequences, so caution is advised.

Changing the paradigm of defining, detecting, and diagnosing NEC: Perspectives on Bell's stages and biomarkers for NEC.

Better means to diagnose and define necrotizing enterocolitis are needed to guide clinical practice and research. Adequacy of Bell's staging system for clinical practice and clarity of cases used in NEC clinical datasets has been a topic of controversy for some time. This article provides reasons why a better global definition for NEC is needed and offers a simple alternative bedside definition for preterm NEC called the "Two out of Three" rule. Some argue that biomarkers may fill knowledge gaps and provide greater precision in defining relevant features of a clinical disease like NEC. NEC biomarkers include markers of inflammation, intestinal dysfunction, hematologic changes, and clinical features. Development and reporting of NEC biomarkers should be guided by the FDA's BEST Consensus resource, "Biomarkers, EndpointS, & other Tools" and consistently report metrics so that studies can be compared and results pooled. Current practice in the NICU would be enhanced by clinical tools that effectively inform the clinical team that a baby is at increasing risk of NEC. Ideally, these tools will incorporate both clinical information about the baby as well as molecular signals that are indicative of NEC. While meaningful biomarkers for NEC and clinical tools exist, translation into practice is mediocre.

Fatty Acid Saturation of Albumin Used in Resuscitation Fluids Modulates Cell Damage in Shock: in vitro Results Using a Novel Technique to Measure Fatty Acid Binding Capacity.

The use of albumin for resuscitation has not proven as beneficial in human trials as expected from numerous animal studies. One explanation could be the practice of adding fatty acid (FA) during manufacture of pharmaceutical albumin. During ischemia, unbound free FAs (FFA) in the circulation could potentially induce cellular damage. We hypothesized that albumins with higher available binding capacities (ABC) for FFAs may prevent that damage. Therefore, we developed a technique to measure ABC, determined if pharmaceutical human serum albumin (HSA) has decreased ABC compared with FA-free bovine serum albumin (BSA), and if binding capacity would affect hemolysis when blood is mixed with exogenous FFA at levels similar to those observed in shock. The new assay used exogenous oleic acid (OA), glass fiber filtration, and a FFA assay kit. RBC hemolysis was determined by mixing 0 to 5 mM OA with PBS, HSA, FA-free BSA, or FA-saturated BSA and measuring plasma hemoglobin after incubation with human blood. 5% HSA contained 4.7±0.2 mM FFA, leaving an ABC of 5.0 ±â€Š0.6 mM, compared with FA-free BSAs ABC of 7.0 ±â€Š1.3 mM (P < 0.024). Hemolysis after OA was reduced with FA-free BSA but increased with FA-saturated BSA. HSA provided intermediate results. 25% solutions of FA-free BSA and HSA were more protective, while 25% FA-saturated BSA was more damaging than 5% solutions. These findings suggest that increased FA saturation may reverse albumin's potential benefit to lessen cellular damage and may explain, at least in part, its failure in human trauma studies.

Heart period and blood pressure characteristics in splanchnic arterial occlusion shock-induced collapse.

The nature of hemodynamic instability typical of circulatory shock is not well understood, but an improved interpretation of its dynamic features could help in the management of critically ill patients. The objective of this work was to introduce new metrics for the analysis of arterial blood pressure (ABP) in order to characterize the risk of catastrophic outcome in splanchnic arterial occlusion (SAO) shock. Continuous ABP (fs = 1 kHz) was measured in rats during experimental SAO shock, which induced a fatal pressure drop (FPD) in ABP. The FPD could either be slow (SFPD) or fast (FFPD), with the latter causing cardiovascular collapse. Time series of mean arterial pressure, systolic blood pressure and heart period were derived from ABP. The sample asymmetry-based algorithm Heart Rate Characteristics was adapted to compute the Heart Period Characteristics (HPC) and the Blood Pressure Characteristics (BPC). Baroreflex sensitivity (BRS) was assessed by means of a bivariate model. The approach to FPD of the animals who collapsed (FFPD) was characterized by higher BRS in the low frequency band versus SFPD animals (0.36 ± 0.15 vs. 0.19 ± 0.12 ms/mmHg, p value = 0.0196), bradycardia as indicated by the HPC (0.76 ± 0.57 vs. 1.94 ± 1.27, p value = 0.0179) and higher but unstable blood pressure as indicated by BPC (3.02 ± 2.87 vs. 1.47 ± 1.29, p value = 0.0773). The HPC and BPC indices demonstrated promise as potential clinical markers of hemodynamic instability and impending cardiovascular collapse, and this animal study suggests their test in data from intensive care patients.

Breast Milk Protects Against Gastrointestinal Symptoms in Infants at High Risk for Autism During Early Development.

OBJECTIVES: Parents of children with autism spectrum disorders (ASDs) often report gastrointestinal (GI) dysfunction in their children. The objectives of the present study were to determine whether infants at high risk for developing ASD (ie, siblings of children diagnosed as having ASD) show greater prevalence of GI problems and whether this prevalence is associated with diet and age at weaning from breast milk. METHODS: Using questionnaires, diet history and GI problems were tracked prospectively and retrospectively in 57 high-risk infants and for comparison in 114 low-risk infants (infants from families without ASD history). RESULTS: In low-risk infants, prevalence of GI symptoms, in aggregate, did not vary with diet or age of weaning. By contrast, high-risk infants with GI symptoms were weaned earlier than those without symptoms (P < 0.04), and high-risk infants showed greater prevalence of GI symptoms, in aggregate, on a no breast milk diet than on an exclusive breast milk diet (P < 0.017). Constipation, in particular, was more prevalent in high-risk infants compared with low-risk infants (P = 0.01), especially on a no breast milk diet (P = 0.002). High-risk infants who completed weaning earlier than 6 months showed greater prevalence of constipation (P = 0.001) and abdominal distress (P = 0.004) than those fully weaned after 6 months. CONCLUSIONS: The greater prevalence of GI symptoms in high-risk infants suggests that GI dysfunction during early infant development may be a part of the ASD endophenotype. Late weaning and exclusive breast milk were associated with protection against GI symptoms in high-risk infants.

Effect of digestion and storage of human milk on free fatty acid concentration and cytotoxicity.

OBJECTIVES: Fat is digested in the intestine into free fatty acids (FFAs), which are detergents and therefore toxic to cells at micromolar concentration. The mucosal barrier protects cells in the adult intestine, but this barrier may not be fully developed in premature infants. Lipase-digested infant formula, but not fresh human milk, has elevated FFAs and is cytotoxic to intestinal cells, and therefore could contribute to intestinal injury in necrotizing enterocolitis (NEC), but even infants exclusively fed breast milk may develop NEC. Our objective was to determine whether stored milk and milk from donor milk (DM) banks could also become cytotoxic, especially after digestion. METHODS: We exposed cultured rat intestinal epithelial cells or human neutrophils to DM and milk collected fresh and stored at 4°C or -20°C for up to 12 weeks and then treated for 2 hours (37°C) with 0.1 or 1 mg/mL pancreatic lipase and/or trypsin and chymotrypsin. RESULTS: DM and milk stored 3 days (at 4°C or -20°C) and then digested were cytotoxic. Storage at -20°C for 8 and 12 weeks resulted in an additional increase in cytotoxicity. Protease digestion decreased, but did not eliminate cell death. CONCLUSIONS: Present storage practices may allow milk to become cytotoxic and contribute to intestinal damage in NEC.

Transmural intestinal wall permeability in severe ischemia after enteral protease inhibition.

In intestinal ischemia, inflammatory mediators in the small intestine's lumen such as food byproducts, bacteria, and digestive enzymes leak into the peritoneal space, lymph, and circulation, but the mechanisms by which the intestinal wall permeability initially increases are not well defined. We hypothesize that wall protease activity (independent of luminal proteases) and apoptosis contribute to the increased transmural permeability of the intestine's wall in an acutely ischemic small intestine. To model intestinal ischemia, the proximal jejunum to the distal ileum in the rat was excised, the lumen was rapidly flushed with saline to remove luminal contents, sectioned into equal length segments, and filled with a tracer (fluorescein) in saline, glucose, or protease inhibitors. The transmural fluorescein transport was determined over 2 hours. Villi structure and epithelial junctional proteins were analyzed. After ischemia, there was increased transmural permeability, loss of villi structure, and destruction of epithelial proteins. Supplementation with luminal glucose preserved the epithelium and significantly attenuated permeability and villi damage. Matrix metalloproteinase (MMP) inhibitors (doxycycline, GM 6001), and serine protease inhibitor (tranexamic acid) in the lumen, significantly reduced the fluorescein transport compared to saline for 90 min of ischemia. Based on these results, we tested in an in-vivo model of hemorrhagic shock (90 min 30 mmHg, 3 hours observation) for intestinal lesion formation. Single enteral interventions (saline, glucose, tranexamic acid) did not prevent intestinal lesions, while the combination of enteral glucose and tranexamic acid prevented lesion formation after hemorrhagic shock. The results suggest that apoptotic and protease mediated breakdown cause increased permeability and damage to the intestinal wall. Metabolic support in the lumen of an ischemic intestine with glucose reduces the transport from the lumen across the wall and enteral proteolytic inhibition attenuates tissue breakdown. These combined interventions ameliorate lesion formation in the small intestine after hemorrhagic shock.

Removal of luminal content protects the small intestine during hemorrhagic shock but is not sufficient to prevent lung injury.

The small intestine plays a key role in the pathogenesis of multiple organ failure following circulatory shock. Current results show that reduced perfusion of the small intestine compromises the mucosal epithelial barrier, and the intestinal contents (including pancreatic digestive enzymes and partially digested food) can enter the intestinal wall and transport through the circulation or mesenteric lymph to other organs such as the lung. The extent to which the luminal contents of the small intestine mediate tissue damage in the intestine and lung is poorly understood in shock. Therefore, rats were assigned to three groups: No-hemorrhagic shock (HS) control and HS with or without a flushed intestine. HS was induced by reducing the mean arterial pressure (30 mmHg; 90 min) followed by return of shed blood and observation (3 h). The small intestine and lung were analyzed for hemorrhage, neutrophil accumulation, and cellular membrane protein degradation. After HS, animals with luminal contents had increased neutrophil accumulation, bleeding, and destruction of E-cadherin in the intestine. Serine protease activity was elevated in mesenteric lymph fluid collected from a separate group of animals subjected to intestinal ischemia/reperfusion. Serine protease activity was elevated in the plasma after HS but was detected in lungs only in animals with nonflushed lumens. Despite removal of the luminal contents, lung injury occurred in both groups as determined by elevated neutrophil accumulation, permeability, and lung protein destruction. In conclusion, luminal contents significantly increase intestinal damage during experimental HS, suggesting transport of luminal contents across the intestinal wall should be minimized.

Digested formula but not digested fresh human milk causes death of intestinal cells in vitro: implications for necrotizing enterocolitis.

BACKGROUND: Premature infants fed formula are more likely to develop necrotizing enterocolitis (NEC) than those who are breastfed, but the mechanisms of intestinal necrosis in NEC and protection by breast milk are unknown. We hypothesized that after lipase digestion, formula, but not fresh breast milk, contains levels of unbound free fatty acids (FFAs) that are cytotoxic to intestinal cells. METHODS: We digested multiple term and preterm infant formulas or human milk with pancreatic lipase, proteases (trypsin and chymotrypsin), lipase + proteases, or luminal fluid from a rat small intestine and tested FFA levels and cytotoxicity in vitro on intestinal epithelial cells, endothelial cells, and neutrophils. RESULTS: Lipase digestion of formula, but not milk, caused significant death of neutrophils (ranging from 47 to 99% with formulas vs. 6% with milk) with similar results in endothelial and epithelial cells. FFAs were significantly elevated in digested formula vs. milk and death from formula was significantly decreased with lipase inhibitor pretreatment, or treatments to bind FFAs. Protease digestion significantly increased FFA binding capacity of formula and milk but only enough to decrease cytotoxicity from milk. CONCLUSION: FFA-induced cytotoxicity may contribute to the pathogenesis of NEC.

Protease activity increases in plasma, peritoneal fluid, and vital organs after hemorrhagic shock in rats.

Hemorrhagic shock (HS) is associated with high mortality. A severe decrease in blood pressure causes the intestine, a major site of digestive enzymes, to become permeable - possibly releasing those enzymes into the circulation and peritoneal space, where they may in turn activate other enzymes, e.g. matrix metalloproteinases (MMPs). If uncontrolled, these enzymes may result in pathophysiologic cleavage of receptors or plasma proteins. Our first objective was to determine, in compartments outside of the intestine (plasma, peritoneal fluid, brain, heart, liver, and lung) protease activities and select protease concentrations after hemorrhagic shock (2 hours ischemia, 2 hours reperfusion). Our second objective was to determine whether inhibition of proteases in the intestinal lumen with a serine protease inhibitor (ANGD), a process that improves survival after shock in rats, reduces the protease activities distant from the intestine. To determine the protease activity, plasma and peritoneal fluid were incubated with small peptide substrates for trypsin-, chymotrypsin-, and elastase-like activities or with casein, a substrate cleaved by multiple proteases. Gelatinase activities were determined by gelatin gel zymography and a specific MMP-9 substrate. Immunoblotting was used to confirm elevated pancreatic trypsin in plasma, peritoneal fluid, and lung and MMP-9 concentrations in all samples after hemorrhagic shock. Caseinolytic, trypsin-, chymotrypsin-, elastase-like, and MMP-9 activities were all significantly (p<0.05) upregulated after hemorrhagic shock regardless of enteral pretreatment with ANGD. Pancreatic trypsin was detected by immunoblot in the plasma, peritoneal space, and lungs after hemorrhagic shock. MMP-9 concentrations and activities were significantly upregulated after hemorrhagic shock in plasma, peritoneal fluid, heart, liver, and lung. These results indicate that protease activities, including that of trypsin, increase in sites distant from the intestine after hemorrhagic shock. Proteases, including pancreatic proteases, may be shock mediators and potential targets for therapy in shock.

An elementary analysis of physiologic shock and multi-organ failure: the autodigestion hypothesis.

Physiological shock and subsequent multi-organ failure is one of the most important medical problems from a mortality point of view. No agreement exists for mechanisms that lead to the relative rapid cell and organ failure during this process and no effective treatment. We postulate that the digestive enzymes synthesized in the pancreas and transported in the lumen of the small intestine as requirement of normal food digestion play a central role in multi-organ failure. These powerful enzymes are usually compartmentalized in the lumen of the intestine by the mucosal barrier, but may escape into the wall of the intestine if the permeability of the mucosal lining increases. Entry of the digestive enzymes into the wall of the intestine precipitates an autodigestion process as well as an escape of pancreatic enzymes and breakdown products generated by them into the system circulation. The consequence of autodigestion is multiorgan failure. We discuss the possibility to block the digestive enzymes in acute forms of shock as a potential therapeutic intervention.

Severe intestinal ischemia can trigger cardiovascular collapse and sudden death via a parasympathetic mechanism.

Hemorrhagic shock and splanchnic arterial occlusion (SAO) followed by reperfusion are associated with high mortality. However, rapid cardiovascular failure and death may also occur before reperfusion in hemorrhagic shock and SAO. We show in a rat SAO model that, upon gut ischemia, mean arterial blood pressure transiently elevates and then drops fatally in one of two time courses: (i) gradually over ∼1 to 3 h or (ii) rapidly (often by >80 mmHg) over a period of 1 to 6 min. We hypothesize that fast fatal pressure drops (FFPDs) are due to failure of autonomic nervous system control. To test this, we treated rats with Glucose (10%) in the small intestinal lumen and intramuscularly administered xylazine to activate the parasympathetic nervous system or with a muscarinic anticholinergic (glycopyrrolate) or by total subdiaphragmatic vagotomy to attenuate parasympathetic nervous system activity. We also tested nafamostat mesilate (ANGD [6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate]), a protease inhibitor efficacious in preventing blood pressure loss in SAO with reperfusion, in the intestinal lumen. Fifty percent of animals receiving xylazine and Glucose died by FFPD (vs. 33% with neither, not statistically significant). Total subdiaphragmatic vagotomy or glycopyrrolate treatment significantly reduced the incidence to 0% (P < 0.008), although slow fatal pressure drops still occurred. ANGD did not prevent FFPDs, but delayed onset of slow fatal pressure drops (P < 0.013). These results suggest that gut ischemia can cause sudden death via an autonomic nervous system mechanism and that SAO with Glucose and xylazine may serve as a useful model for the study of neurogenic shock or autonomic dysregulation associated with sudden death.

The intestine as source of cytotoxic mediators in shock: free fatty acids and degradation of lipid-binding proteins.

Shock and multiple organ failure remain primary causes of late-stage morbidity and mortality in victims of trauma. During shock, the intestine is subject to extensive cell death and is the source of inflammatory factors that cause multiorgan failure. We (34) showed previously that ischemic, but not nonischemic, small intestines and pancreatic protease digested homogenates of normal small intestine can generate cytotoxic factors capable of killing naive cells within minutes. Using chloroform/methanol separation of rat small intestine homogenates into lipid fractions and aqueous and sedimented protein fractions and measuring cell death caused by those fractions, we found that the cytotoxic factors are lipid in nature. Recombining the lipid fraction with protein fractions prevented cell death, except when homogenates were protease digested. Using a fluorescent substrate, we found high levels of lipase activity in intestinal homogenates and cytotoxic levels of free fatty acids. Addition of albumin, a lipid binding protein, prevented cell death, unless the albumin was previously digested with protease. Homogenization of intestinal wall in the presence of the lipase inhibitor orlistat prevented cell death after protease digestion. In vivo, orlistat plus the protease inhibitor aprotinin, administered to the intestinal lumen, significantly improved survival time compared with saline in a splanchnic arterial occlusion model of shock. These results indicate that major cytotoxic mediators derived from an intestine under in vitro conditions are free fatty acids. Breakdown of free fatty acid binding proteins by proteases causes release of free fatty acids to act as powerful cytotoxic mediators.

Pancreatic enzymes generate cytotoxic mediators in the intestine.

Recent evidence indicates that shock is accompanied by a failure of the mucosal barrier in the intestine and entry of pancreatic digestive enzymes into the wall of the intestine. To investigate the formation of cytotoxic mediators produced by enzymatic digestion of the intestine, we applied homogenates of rat small intestinal wall to human neutrophils and used flow cytometry measurements of propidium iodide uptake to determine cytotoxicity. We show that homogenates of the small intestine after ischemia by occlusion of the superior mesenteric and celiac arteries for 3 h, but not without ischemia, are cytotoxic. Digestion of homogenates of nonischemic intestinal wall with purified trypsin, chymotrypsin, or elastase, proteases normally present in the intestinal lumen, yielded cytotoxic mediators. Before cell death, we saw cell damage in the form of bleb formation and flow cytometry measurements of cell size changes due to blebbing. Cytotoxicity was prevented by serine protease inhibition with phenylmethylsulfonyl fluoride (PMSF) before, but not after proteolytic digestion of the wall homogenates, indicating that enzymatic action of proteases on the homogenate is necessary for cytotoxicity. Cytotoxicity of wall homogenates digested by enzymes in the fluid collected from the lumen of the intestine was greater than digests by the individual purified proteases. Cytotoxicity is undetectable if digestive enzymes in the luminal fluid are inhibited with a combination of enzyme inhibitors PMSF and 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate before addition of wall homogenates. Passage of digested intestinal wall homogenates across a hydrophobic glass-fiber filter reduced cytotoxicity. Furthermore, we found that luminal fluid itself may be cytotoxic, possibly because of digestion of ingested food. To test whether digested food can be cytotoxic, we homogenized rat food and digested it in vitro with chymotrypsin or endogenous enzymes in luminal fluid. Cytotoxicity was significantly increased after digestion of food by luminal fluid compared with luminal fluid or undigested food. These results indicate the presence of a previously unknown mechanism for hemorrhagic necrosis in shock.

Pancreatic digestive enzymes are potent generators of mediators for leukocyte activation and mortality.

Shock is associated with a dramatic rise in the level of inflammatory mediators found in plasma. The exact source of these mediators has remained uncertain. We recently examined a previously undescribed mechanism for production of inflammatory mediators in shock involving pancreatic digestive enzymes. The current in vitro study was designed to identify particular pancreatic enzymes and organs that may potentially produce inflammatory mediators. A selection of different organs from the rat (heart, liver, brain, spleen, pancreas, intestine, diaphragm, kidney, and lung) were homogenized and incubated with purified trypsin, chymotrypsin, elastase, lipase, nuclease, or amylase and the supernatant was incubated with fresh naïve leukocytes for 15 min. The level of leukocyte activation in the form of pseudopod formation and the fraction of cell death were measured. Without the addition of purified enzymes, only the homogenate of the pancreas yielded enhanced cell activation. Organs incubated with physiological concentrations of trypsin also stimulated significantly higher levels of pseudopod formation as compared with the undigested organs or enzymatic controls. Lipase and chymotrypsin were able to elicit cellular activation from selected organs such as the heart, intestine, liver and diaphragm. Undigested pancreatic homogenates were capable of producing substantial cell death, as compared with all other undigested organs. Intestinal digests with elastase, lipase, trypsin and chymotrypsin also stimulated significant cell mortality. Lipase-treated heart, liver, intestine, diaphragm, kidney, and lung stimulated cell death as well. We conclude that the intestine, as well as several other organs, may serve as a major source of inflammatory mediators during shock if exposed to digestive enzymes.