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Journal of Parenteral and Enteral Nutrition

http://pen.sagepub.com/ Drivers of Oxidative Stress in Acute Pancreatitis : The Role of Nutrition Therapy
Stephen A. McClave JPEN J Parenter Enteral Nutr 2012 36: 24 DOI: 10.1177/0148607111424410 The online version of this article can be found at: http://pen.sagepub.com/content/36/1/24

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2010 Presidential Address

Drivers of Oxidative Stress in Acute Pancreatitis: The Role of Nutrition Therapy

Stephen A. McClave, MD
Financial disclosure: none declared.

Journal of Parenteral and Enteral Nutrition Volume 36 Number 1 January 2012 24-35 2012 American Society for Parenteral and Enteral Nutrition 10.1177/0148607111424410 http://jpen.sagepub.com hosted at http://online.sagepub.com

Severe acute pancreatitis is a disease process distinguished by increasing oxidative stress and potential destruction of the pancreatic gland. An initial injury to the acinar cell initiates a sentinel event, which leads to a vicious cycle of inflammation and cell death by either apoptosis or necrosis. Whether the acute inflammation resolves or goes on to a pattern of chronicity may be related to genetic predisposition, failure to remove injurious agents, and innate systems for antioxidant defense. The degree to which nutrition therapy can modulate oxidative stress, main-

tain intestinal function, and preserve the structure of the acinar cell is truly amazing. Understanding the mechanisms involved in this complex disease process and the manner in which these mechanisms are influenced by dietary agents affords new and exciting therapeutic options for the future. (JPEN J Parenter Enteral Nutr. 2012;36:24-35) Keywords: oxidative stress; acinar antioxidant defense; pancreatitis cell; nutrition therapy;

here are any number of ways to design a presidential address. Recently, the Board of Directors for the American Society for Parenteral and Enteral Nutrition (A.S.P.E.N.) has been working hard to increase the level of science at our national Clinical Nutrition Week meeting. Therefore, I thought the best address at this time would be to discuss good science. Good science is that which captures our imagination, broadens our understanding, and excites us about what we are doing at the bedside for our patients. The strongest evidence in the literature for the impact of nutrition therapy on patient outcome is in the disease process of severe acute pancreatitis.1 The level of oxidative stress that builds in this disease determines the systemic inflammatory response syndrome (SIRS), the duration of the disease process, and its ultimate morbidity and mortality.2,3 The degree to which nutrition therapy can modulate oxidative stress in this situation is nothing short of phenomenal.

From the Department of Medicine, University of Louisville School of Medicine, Louisville, Kentucky. Received for publication September 13, 2010; accepted publication September 1, 2011. for

Address correspondence to: Stephen A. McClave, MD, Division of Gastroenterology/Hepatology, University of Louisville School of Medicine, Louisville, KY 40202; e-mail: Stephen.McClave@ louisville.edu.

Over the past 3 decades, our perspective on nutrition therapy for severe pancreatitis has been severely limiting. When we published the first prospective randomized trial comparing enteral with parenteral therapy in acute pancreatitis in 1997, the overriding perspective was that of pancreatic rest.4 The fear of stimulating an already inflamed pancreas prevented us from using enteral therapy at all. Once our study was published and others followed, we became comfortable with enteral feeding in pancreatitis. And then a second perspective further restricted our choices. In a patient with a significant SIRS response, there was concern of providing pharmaconutrition with arginine to a patient who was potentially hemodynamically unstable and might have infection, for whom the arginine could be converted to nitric oxide and contribute further to hemodynamic instability.5 To maintain these perspectives when treating pancreatitis is like having your house catch on fire and taking the response of running out to the sidewalk, standing there, and hoping that the fire does not destroy the entire house. A better perspective in that situation is to bring in firefighters with their hoses to quickly put out the fire, to save as much of the structure of the house as possible. In severe acute pancreatitis, the gland is on fire with oxidative stress. As nutritionists, we have the capability of bringing in our hoses and tubes to provide enteral formula with pharmaconutrient agents to put out the fire of inflammation as soon as possible to preserve the structure of the pancreatic gland.

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Drivers of Oxidative Stress in Acute Pancreatitis / McClave


As we discuss these different mechanisms, there are 3 separate layers of appreciation, understanding, and/or utilization. The outermost evident layer are those mechanisms that are identified and already used in pancreatitis, and the discussion here will simply provide better understanding on how the mechanisms work and why they are so important. A perfect example is the effect of enteral feeding on maintaining gut integrity. A slightly deeper layer includes those mechanisms that are identified to be operative in pancreatitis, but we have yet to really use them in the nutrition therapy of these patients. An example here would be the role of glutamine in stimulating heat-shocked proteins or the use of arginine to treat the vascular abnormalities seen in pancreatitis. The third and least evident layer are those mechanisms identified to be operative in oxidative stress that should be a factor in pancreatitis but have not yet been identified or used in this particular disease process. An example here would be stimulating the bodys own endogenous antioxidant response elements or manipulating epigenetics and gene expression to reduce oxidative stress.

Sentinel Acute Pancreatitis Event

Through the work of Whitcomb,6,7 we have begun to understand that although many etiologic factors can insult the pancreas, there is quickly one common pathway of pathogenesis for the disease process. The sentinel acute pancreatitis event (SAPE) hypothesis suggests that any number of events or agents may insult the pancreatic acinar cell (Figure 1).6,7 That insult leads to the sentinel event, which is characterized by an early proinflammatory response followed by a late profibrotic response mediated by stellate cells (Figure 2).6,7 The sentinel event and its characteristic inflammatory response constitute the common pathway of pathogenesis for all etiologies that initiate pancreatitis. Following the sentinel event, however, an interesting thing happens. Some patients are able to remove the stimulus and resolve the inflammation. Other patients, however, fail to remove the stimulus, the inflammation continues, and the process proceeds down a pathway of chronicity characterized by ongoing inflammation and chronic pancreatitis.6,7 What factors would cause some people to resolve the sentinel event and not others? The easy answer is that removal of an offending drug, a cholecystectomy, cessation of alcohol abuse, or control of hypertriglyceridemia would remove the insulting stimulus and promote resolution. Surprisingly, results from the recent National Acute Pancreatitis Study (NAPS-2) indicate that only 10% of heavy alcohol drinkers suffer clinically evident pancreatic disease and that most patients with chronic pancreatitis have no abuse of alcohol in their history.8 Thus, for many patients, the answer may be much more

complex, relating to genetic predisposition, epigenetics, and the patients ability to manage oxidative stress.6,7,9 The work of Pandol et al3 has focused on the initial insult to the acinar cell and the subsequent vicious cycle of events that follow. In most diseases in critical illness, the acute insult itself drives the morbidity and mortality (insults such as a gunshot wound, a motor vehicle accident, or a burn). But in severe acute pancreatitis, the insult may be fairly innocuousa small sand particle in the common bile duct, a mild elevation of triglyceride levels, or a pharmacologic agent. The sentinel event clearly refers to the cycle of inflammation that follows the insult because this insult to the acinar cell leads to the release of proinflammatory mediators such as tumor necrosis factor (TNF-), interleukin (IL)1, and platelet-activating factor (PAF).7,10 There is also an aggressive recruitment and activation of inflammatory cells. In pancreatitis, the subsequent events in this cycle of inflammation determine the morbidity and mortality we see at the bedside.3 In response to the initial insult to the acinar cell, there is release of cytokines, including TNF-and IL-8.11 These cytokines stimulate the adhesion molecules ICAM (intercellular adhesion molecule) and selectins, expressed on the surface of the vascular endothelium and on neutrophils, which subsequently lead to passage of neutrophils out of the vascular space into the extracellular space surrounding the acinar cell.10,11 As these neutrophils migrate down into the area around the acinus (the group of acinar cells in the pancreatic gland), their activation leads to 4 subsequent events.10,11 Activation of neutrophils leads to increased release of inflammatory cytokines, further raising the level of oxidative stress. There is greater expression of adhesion molecules, which increase vascular permeability further.11 And chemokines are released, which lead to even greater recruitment of neutrophils into the bed of the pancreas.10 There is also a direct stimulation of the Toll-like receptor 4 on macrophages, again leading to further increases in inflammation.11 The net effect of this process is the increased production of reactive oxygen species.2 As the level of oxidative stress builds, 2 subsequent events occur that ultimately lead to the death of the acinar cell: intra-acinar activation of pancreatic enzymes and the inhibition of their secretion (Figure 3).7 One of the main roles of the pancreatic acinar cell is to produce a number of secretory enzymes that are capable of digesting any nutrient substrate. These same caustic, lytic enzymes are also capable of dissolving any structure in the body. As a result, they are handled very carefully by the acinar cell, packaged separately in zymogen granules and kept separated from the activating agents (such as cathepsin B and other lysosomes) in the lysosomal granules.7 As the level of oxidative stress increases in the acinar cell and the cell begins to malfunction, the activating lysosomes and the

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Journal of Parenteral and Enteral Nutrition / Vol. 36, No. 1, January 2012

Figure 1. Insult to the acinar cell. An insult from any number of etiologic events or agents precedes the sentinel event, leads to cytokine release and chemotaxis of inflammatory cells, and ultimately causes death of the cell. Used with permission from Pandol et al.3

Figure 2. The sentinel acute pancreatitis event (SAPE) hypothesis. The sentinel event refers to the characteristic inflammatory response that constitutes the common pathway of pathogenesis for all etiologies of acute pancreatitis. Some patients remove the stimulus and resolve inflammation, whereas others fail to do so and go on to a pattern of fibrosis and chronic pancreatitis. Used with permission from Whitcomb.6

Figure 3. Intra-acinar activation of pancreatic enzymes and inhibition of their secretion. In response to increasing oxidative stress, these 2 adverse events occur, which leads to autolysis or autodigestion of the acinar cell. CC, cholesterol ester; FAEE, fatty acid ethyl ester; L, lysosome; ZG, zymogen granule. Used with permission from the following Web site: www.niaaa.nih.gov/ pathogenesis. Figure 4. Intracellular and extracellular processes that contribute to the systemic inflammatory response syndrome (SIRS) in acute pancreatitis. These processes ultimately lead to death of the acinar cell. Used with permission from Pandol et al.3

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Figure 5. Cell death pathways in acute pancreatitis. As the level of oxidative stress increases, the acinar cell becomes marked for death either by apoptosis or necrosis. ATP, adenosine triphosphate; ER, endoplasmic reticulum; IAP, Inhibitors of Apoptosis; PARP, poly(ADP-ribose) polymerase. Used with permission from Pandol et al.3

Figure 6. Role of heat shock proteins (HSPs) in normal protein synthesis. The small HSP-40 molecule provides a chaperone function, transporting the newly formed protein and preventing its aggregation. The larger HSP-70 and HSP-90 molecules join the smaller HSP-40 to form and fold the protein into its final 3-dimensional configuration. Used with permission from the following Web sites: www.sciam.com/media/inline/2008-07/cancer and www.assaydesigns.com/corp/images/hs.

Figure 7. Toll-like receptor 4. Activation of this receptor on macrophages, neutrophils, adipocytes, and intestinal epithelial cells leads to release of cyclooxygenase-2 cytokines, stimulation of the transcription factor NF-B, and formation of tumor necrosis factor. Many agents influence this receptor. HSP, heat shock protein; LCFA, long chain fatty acids; LPS, lipopolysaccharide. Used with permission from Seong and Matzinger.28

Figure 8. Neurogenic inflammation. Neurogenic signals arising from a bed of inflammation leads to the release of 2 neuropeptides (substance P and calcitonin gene-related peptide [CGRP]), which causes further increases in vascular permeability and neurophil infiltration. NO, nitric oxide. Used with permission from the following Web site: www.chronicprostatitis .com/images/neurogenicpain.

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Journal of Parenteral and Enteral Nutrition / Vol. 36, No. 1, January 2012

pancreatic enzymes become co-packaged or co-localized, leading to the activation of the enzymes.3 The second event involves the inhibition of secretion, which means that the activated zymogen granules are retained inside the acinar cell.3 This whole process leads to autolysis, or autodigestion, of the acinar cell. As necrosis occurs, there is release of more cytokines. Nearby stellate cells are activated, which leads to the fibrotic response (Figure 3).7

Cell Death Pathways

By focusing on the events that occur following the insult of the acinar cell, Pandol et al3 have identified both intercellular and extracellular processes that contribute to the SIRS response (Figure 4). Within the acinar cell, factors such as the extent of inhibition of secretion, the degree of activation of the digestive enzymes, the level of calcium within the cytosol, the degree to which the protective heat shock proteins are stimulated, the nature of the inflammatory signals and cytokines released, and even the manner in which the acinar cell dies all contribute to the systemic inflammatory response.3 The SIRS response is the toxicity that is seen at the bedside, which accounts for the fever, leukocytosis, organ failure, infection, prolonged duration on mechanical ventilation, and even mortality (Figure 4).3 As the level of oxidative stress increases, at some point, the acinar cell becomes marked for death,3,12 but there are 2 ways for the cell to die (Figure 5).3 The best and cleanest way to die is through apoptosis, where the cell simply shrinks, the nuclear chromatin is condensed, and eventually the mass is ingested cleanly by a macrophage.3,12 There is no spillage of intercellular content, and no inflammatory response ensues. The worse and more messy pattern of cell death is that of necrosis, where the acinar cell simply disintegrates, releasing all of its intracellular contents, which in turn initiates a significant inflammatory response.3,12 Clinical studies have shown that patients with mild to moderate acute pancreatitis tend to have a pattern of apoptosis, whereas patients with severe pancreatitis tend to have more of a pattern of necrosis.12 In animal models of acute pancreatitis, steps to induce apoptosis first, followed by the laboratory insult that causes the pancreatitis, will lead to a much milder form of injury.3 Profound injury to organs (such as the liver) has been attributed to excessive induction of apoptosis.12 Apoptosis may be converted to necrosis through the excessive recruitment of neutrophils.3,12 An alternative interpretation of these events suggests that these 2 pathways may not be distinct and separate but that apoptosis may be the match that lights the flame of necrosis. The exact mechanisms that lead to either mode of death are unclear but are definitely crucial in determining the fate of the acinar cell and establishing the course of

the disease process.3 For apoptosis to occur, the acinar cell has to retain a certain degree of function. The mitochondria have to be functioning enough to stimulate initiator and effector caspases. The caspases are the effector or executioner agents, responsible for clean cleavages and disassembling of the acinar cell leading to apoptosis.3,12 As the level of oxidative stress increases within the acinar cell, specific parts of the cell or organelles begin to malfunction as well. If the mitochondria are damaged or dysfunctional (such as from chronic ethanol ingestion), the organelle may not be able to maintain adequate levels of adenosine triphosphate (ATP) within the cell, and a pattern of necrosis would occur instead. Calcium levels are usually low in the cytosol of the acinar cell, and as a result, diffusion gradients work to raise the level of intracellular calcium. Active pumps are required to prevent this. With increasing oxidative stress, malfunction of the endoplasmic reticulum may cause displacement of calcium into the cytosol, which would in turn promote a pathway of necrosis. Malfunction of the acinar cell in packaging lysosomes separately from the proenzymes results in the activation of these enzymes, and a pattern of necrosis ensues.3,12 To understand how the endoplasmic reticulum malfunctions in oxidative stress, it is important to understand its normal function.13 Normal synthesis and folding of proteins occurs in the endoplasmic reticulum. As messenger RNA leaves the nucleus, the strip of DNA material is read by a ribosome. As the ribosome moves along the string of DNA, the newly formed protein is extruded. The new protein has to be folded into a 3-dimensional configuration to achieve a fully functioning protein (such as an enzyme or cytokine).13 Occasionally in this process, the protein becomes aggregated or misfolded. The protein aggregate then has to be marked by ubiquitin for disposal, degradation into its constituent amino acids, and the chance to start over in the synthesis process.13 Heat shock proteins (HSPs) play a large role in the synthesis of proteins (Figure 6).14 As a chaperone function, small HSP-40 molecules carry the newly formed protein string and prevent it from becoming aggregated. A larger HSP-70 or HSP-90 molecule joins the smaller molecule and, with claws or hands that look like the pincers of a lobster, form and fold the protein into its final 3-dimensional configuration. If a protein does end up unfolded, HSPs serve a rescue function and can actually refold the protein into its final 3-dimensional form.14 If it is unable to do this, the HSPs can mark the protein aggregate with ubiquitin for subsequent disposal. As mentioned earlier, increasing levels of oxidative stress in the acinar cell cause parts of the cell, or organelles, to malfunction. With respect to the endoplasmic reticulum, the oxidative stress leads to an increase in the amount of unfolded proteins.3,14-16 Even proteins that start out being

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Drivers of Oxidative Stress in Acute Pancreatitis / McClave


folded properly may be degraded or denatured to a misfolded configuration. As the unfolded proteins accumulate, they begin to stimulate apoptosis. An unfolded protein response ensues, which initiates further inflammation in itself. If the process is not truncated, it leads to necrosis and ultimately autophagy of the acinar cell.3,15,16 The reason HSPs are so important in severe acute pancreatitis is the protective role that they play for the acinar cell.3,17 HSP proteins are upregulated in the acinar cell in acute pancreatitis.17 As a result, they can aid in the management of unfolded proteins by refolding or marking the proteins for ubiquitination. An experimental model for acute pancreatitis where animals are given a toxin (such as cerulein or perchloric acid) prior to induction of HSPs by water immersion or heating the animal reduces the subsequent severity of acute pancreatitis.3 But the mechanism of the protective effects of HSPs on the acinar cell in pancreatitis is much more involved. HSPs help prevent colocalization of lysosomes with zymogen granules, as well as help prevent a rise in intracellular calcium and block activation of trypsinogen. They also promote apoptosis and are actually able to block activation of NF-B through an inhibitory effect on Toll-like receptor 4 (Figure 7).3 Diet modulation through the provision of glutamine can stimulate production of HSPs.18,19 With this myriad of beneficial effects, it is hard to imagine not including the enteral or parenteral delivery of glutamine in the treatment of patients with severe acute pancreatitis. Most clinicians are familiar with the role of fish oils in modifying inflammation. The substitution of -3 fish oils generates less inflammatory prostaglandins, leukotrienes, and thromboxanes than -6 lipids.20,21 When neutrophils or macrophages perform an oxidative burst, the cell does not automatically die.22 A single neutrophil may be responsible for up to 68 bacterial killings. The neutrophil develops channels that allow encroachment on the bacterial cell wall and the development of channels through which the reactive oxygen species may be injected into the bacteria.22 There is scant evidence that fish oil, through the generation of the alternative prostaglandin E3, leukotriene B5, and thromboxane A2, reduces the actual number of reactive oxygen species generated in an oxidative burst. The more profound effect is a reduction in the recruitment and chemotaxis of neutrophils into a particular tissue.23 But the fact that neutrophil stimulation, activation, and recruitment are such a key factor in the sentinel event of acute pancreatitis 11 indicates a tremendous potential role for diet modulation of inflammation through delivery of -3 fish oils. Animal studies would suggest that the docosahexaenoic acid (DHA) derived from fish oils has even further effects on the acinar cell itself, inhibiting intracellular signaling, reducing the amount of inflammatory cytokines generated, stabilizing potential DNA fragmentation, and inducing apoptosis instead of necrosis.24 There may be many other

mechanisms by which provision of -3 fish oils contribute to reduced inflammation. Dietary fat has an effect on the Toll-like receptor 4, which is present on macrophages, neutrophils, adipocytes, and intestinal epithelial cells (Figure 7).25-27 Activation of this receptor leads to release of cyclooxygenase-2 (COX-2) cytokines and the transcription factor NF-B (which goes on to synthesize TNF, one of the principal instigators of the inflammatory response). Researchers have described the Toll-like receptor 4 as promiscuous because it has relations with so many agents.25-28 Lipopolysaccharide (LPS) endotoxin and neutrophil elastase stimulate Toll-like receptor 4, whereas HSPs inhibit or block the receptor.28 There is a similarity between saturated long-chain fatty acid and the lipid A moiety of the LPS endotoxin.26,27 The similarity is such that the receptor cannot distinguish between the two. That means that on macrophages, saturated long-chain fat can activate the receptor to a similar degree as LPS endotoxin.26,27 Trans fat and -9 monounsaturated fatty acids have less stimulation, whereas -3 fish oils may actually block or inhibit the receptor. Pretreatment of a lab animal with -3 fish oil will actually inhibit the stimulatory effects of saturated long-chain fatty acids on the Toll-like receptor 4.26 This same receptor on adipocytes is similarly activated by saturated long-chain fat, but the effect is slightly less than that seen with LPS endotoxin.26

Extracellular Factors and Inflammation

Just when the acinar cell is struggling to survive, extracellular factors outside the acinar cell related to blood supply and nerve innervation contribute to SIRS and the likelihood for cell death.3 Neurogenic inflammation may be a contributing process to the inflammatory response seen in severe acute pancreatitis (Figure 8).3,29 In neurogenic inflammation, a body or area of inflamed tissue activates sensory afferent nerve endings. Afferent neurogenic signals return to the dorsal column of the spinal cord, which then sends efferent signals back to the area of inflammation, resulting in the release of 2 neuropeptide agents, substance P and calcitonin gene-related peptide. The release of these neuropeptides causes increased vascular permeability, leading to edema, and increased neutrophil infiltration into the pancreatic parenchyma. Thus, these neuropeptides increase inflammation further and contribute to worsening disease severity. Dietary fat intake surprisingly may alter the crosstalk between these nerve endings, such that the effect of inflammation may be reduced by ingestion of dietary -3 and -6 polyunsaturated fatty acids.30 In disease states such as diabetic Charcot joints and trigeminal neuralgia, use of polyunsaturated fatty acids downregulates this neurogenic inflammation, resulting in less pain and swelling.30 This process is clearly operative in

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Figure 9. Relationship between lumenal microbiota, gut integrity, permeability, and release of cytokines from the gastrointestinal tract. Under normal circumstances (on left), commensal flora and dietary short-chain fatty acids stimulate heat shock proteins and maintain gut integrity. In oxidative stress (on right), virulent organisms adhere to the epithelium, increasing gut permeability and causing release of a proinflammatory cytokine storm into lymphatic channels. IL, interleukin; NO, nitric oxide; TNF, tumor necrosis factor . Used with permission from Alverdy and Chang.42

Figure 11. Antioxidant response elements (ARE). Removal of the inhibitory Keap-1 protein allows the transcription factor Nrf-2 to enter the cell nucleus and encode antioxidant proteins. PKC, protein kinase C; ROS, reactive oxygen species. Used with permission from the following Web site: www.biocarta.com/pathfiles/h_arenrf2Pathway.asp.

Figure 10. Transport of endotoxin from the gut to the systemic circulation by chylomicrons. In the normal process of the formation of chylomicrons following a fatty meal, lipopolysaccharide (LPS) endotoxin may be engulfed and transported safely through lymphatics to the systemic circulation. In the absence of chylomicrons, LPS endotoxin can pass directly through a permeable intestinal epithelium, stimulate macrophages, and lead to further inflammation. HDL, high-density lipoprotein; LDL, low-density lipoprotein; LTP, long-term potentiation; MHC, major histocompatibility complex; TCR, T-cell receptor. Used with permission from Seong and Matzinger.28

Figure 12. Epigenetics and the control of gene expression by methylation or histone modification. Methylation of a gene always results in suppression of that gene, whereas histone modification may turn on or turn off gene expression. Used with permission from Qiu.60

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severe acute pancreatitis,29 but clinicians are not taking advantage of this with our nutrition therapy. The other extracellular phenomenon contributing to cell death and SIRS involves the vascular abnormalities seen in severe acute pancreatitis.31,32 The pancreatic acinus is surrounded by a rich capillary network of blood vessels. In acute pancreatitis, a systemic vascular disorder involves both the microcirculation of the pancreas and large blood vessels throughout the body.31,32 There is actually a Purtscher ischemic retinopathy described in severe pancreatitis, where vasospasm and ischemic injury damage the optic nerve.33 As oxidative stress in and around the acinar cell increases, a high incidence of vasospasm and vasoconstriction leads to reduced perfusion.32 Areas of necrosis on computerized tomography scan have been shown to correspond to these areas of vasospasm. The vascular response to acute pancreatitis sets up a classic model of ischemia/reperfusion injury. An increase in vascular permeability precedes an initial phase of arterial vasoconstriction and ischemia, which is then followed by a reversible vasodilation. Although the vasodilation restores reperfusion, it also serves to wash reactive oxygen species out into the systemic circulation. Leukocyte adherence to the vascular endothelium and stasis from capillary hypoperfusion lead to thrombosis, continued ischemic injury, and necrosis of the gland.31,32 One of the most reliable physiologic responses to enteral feeding is the increase in blood flow to the splanchnic circulation.34,35 Splanchnic blood flow increases by as much as 40% after a meal, and the intestinal microcirculation may increase by as much as 60%.34,35 Continuous feeding promotes continued increases in blood flow. An important contributor to the vascular abnormalities seen in severe acute pancreatitis may be the generation of asymmetric dimethyl arginine (ADMA).36 ADMA is considered the evil twin of L-arginine. As a precursor to nitric oxide production, arginine acts as a vasodilator promoting tissue perfusion. L-arginine protects vascular endothelium and supports the cell-mediated defense system. ADMA, on the other hand, inhibits endothelial nitric oxide elaboration and therefore leads to vasoconstriction and reduced perfusion.36 Elevation of ADMA occurs in critical illness because of increased production from muscle catabolism, as well as decreased elimination (related to dysfunction of enzymes, which normally degrade it, and reduced clearance due to renal insufficiency).36 In a study of critically ill patients, including patients with severe acute pancreatitis, elevation of ADMA levels correlated with increased intensive care unit length of stay, multiple organ failure, and mortality.36 In normal circumstances, there is a balance between these 2 agents, such that the levels of L-arginine are higher, nitric oxide is formed, and vasodilation with tissue perfusion occurs.37 In a setting of critical illness, the excess ADMA creates an imbalance that inhibits production of nitric oxide from arginine, resulting in vasoconstriction and

reduced tissue perfusion. In that situation, provision of L-arginine restores the balance by increasing arginine levels, nitric oxide production is restored, and vasodilation ensues.37 In a disease process so prone to vascular abnormalities, provision of L-arginine might stabilize perfusion of the pancreatic acinus.

Maintenance of Gut Integrity

Probably the most important protective mechanism of enteral nutrition (EN) is the physiologic response of maintaining gut integrity.38 Early EN maintains the integrity of barrier function and prevents bacterial overgrowth. In patients with severe pancreatitis, increased gut permeability has been linked directly to increasing multiple organ failure and disease severity.39 In the past, researchers focused on bacterial translocation. Studies by Nettelbladt et al40 have documented the translocation of Escherichia coli across gut epithelium to mesenteric lymph nodes, into the peritoneum, and into the blood of patients with acute pancreatitis. However, even when the bacteria pass all the way to the intravascular space, the levels are low and the effects are clinically insignificant. In this respect, the presence of bacteria translocating from the gut to the blood becomes simply a surrogate marker for increased gut permeability.40,41 Alverdy and Chang42 has shown that the dose of Pseudomonas aeruginosa, Staphylococcus aureus, or E coli needed to cause septic syndrome is much lower if the bacteria are infused into the lumen of the gut than if the same organisms are infused directly into the intravascular space. The reason for this differential effect is the inflammatory response generated by the gut. A number of factors related to intraluminal nutrients and the intestinal microbiota affect gut integrity and the risk for increased permeability (Figure 9).42 Under normal circumstances with good intake of enteral nutrients, there are an adequate number of protective probiotic commensal flora and dietary short-chain fatty acids, all of which stimulate HSPs in the gut epithelial cells. These processes stabilize barrier function and maintain gut integrity. Although potentially virulent organisms are present, their numbers are low. As long as enough nutrients are provided for all the bacteria, the virulent bacteria remain as silent colonizers.42 At some point in a disease process such as acute pancreatitis where oxidative stress is increasing, the potentially virulent microbiota perform a process of quorum sensing. Through quorum sensing, organisms such as S aureus, P aeruginosa, or E coli determine whether a critical bacterial density has been achieved. If the number of organisms is high enough, virulent genes are expressed, quorum-sensing molecules are secreted, and the bacteria establish cell-to-cell communication to synchronize their behavior and coordinate

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a toxic offensive against the host. In their virulent phase, these bacteria express adherent tentacles, which allows binding to the gut epithelium, and an immediate increase in permeability results. The virulence expression and binding of organisms to the epithelial cell lead to the formation of IL-1, TNF, and IL-8, and a veritable cytokine storm is released into the lymphatic channels.42 In previous studies comparing the fecal contents of patients with SIRS to that of normal healthy controls, the feces of those patients with SIRS have been shown to have a reduced number of protective organisms, an increase in the number of virulent organisms, and an absence of short-chain fatty acids.42 Unfortunately, a number of factors related to the routine management of acute pancreatitis upset the balance of factors in the intestinal lumen, promoting bacterial overgrowth, virulence expression, increased gut permeability, and gut-derived inflammation. Such factors that upset the balance include vasopressor agents to manage hypotension (which leads to further splanchnic vasospasm and ischemia), use of prophylactic antibiotics (which increases the number of virulent organisms), opiates to control pain (which reduce gastrointestinal motility and promote bacterial overgrowth), and use of parenteral nutrition (which creates a lumenal nutrient deficiency in the cecum). EN is a huge factor in modulating the gut/lung axis of inflammation, which occurs with increases in gut permeability. As bacteria engage the epithelial cells and macrophages in the gut, the cytokine storm releases inflammatory agents in the lymphatic channels.42 These cytokines travel up through the thoracic duct, into the left subclavian vein, and into the capillary system of the lung.43 Passage of these cytokines into the pulmonary alveoli leads to activation of neutrophils, sequestration, endothelial permeability, and cell death. Increases in gut permeability acting through this axis of inflammation lead directly to the acute respiratory distress syndrome (ARDS) and ventilator-associated pneumonia.43 Of all the enzymes released by the pancreas, elastase is particularly toxic to the lung (through activation of NF-B in pulmonary tissue).3 In animal models, elastase placed in the peritoneal space can lead directly to lung injury.4 Passage of this enzyme through the gut/lung conduit of inflammation might increase susceptibility to ARDS in a patient already critically ill from acute pancreatitis.44 Zinc has a tremendous effect on maintaining gut integrity and barrier function.45,46 The protective effects of zinc include improving mucosal function, decreasing intestinal permeability, increasing production of metallothioneins (used to form glutathione), and reducing oxidative stress.45 In critical illness and acute pancreatitis, zinc deficiency can develop from 2 mechanisms: inadequate dietary intake or mobilization of zinc into the cytoplasm of cells.46 As oxidative stress increases in the gut, the zinc falls off the zinc finger proteins and accumulates

in the cytosol of the epithelial cell.46 This contributes to the accumulation of reactive oxygen species in the cytoplasm. Zinc is particularly important in maintaining the zona occludens or tight junctions between the epithelial cells. If a zinc chelator is given, the tight junctions between the mucosal epithelial cells begin to disassemble and break down.46 Provision of exogenous zinc restores these junctions and reassembles the zona occludens. Chylomicrons may transport endotoxin from the gut to the systemic circulation through this previously mentioned gut/lung conduit (Figure 10).28,47 In the normal process of the formation of chylomicrons following a fatty meal, endotoxin may be engulfed and transported across an intact gut epithelium (where there is no increased permeability). Endotoxin is transported silently within the chylomicrons to the systemic circulation, where macrophages can ingest the chylomicrons and eradicate the endotoxin safely.28,47 Even though serum levels of endotoxin are actually higher when this process is in place, the lethality from endotoxin is reduced. The process, though, represents a classic doubleedged sword. On one side there is less toxicity from the endotoxin because engulfing the endotoxin within the chylomicron prevents stimulation and activation of macrophages.28,47 However, on the other side, oxidative stress may be increased through the delivery of saturated longchain fat, which is capable of stimulating the Toll-like receptor 4 on macrophages.26 A potential strategy to take advantage of this transport of endotoxin would be to provide -3 fish oil, which would still allow formation of chylomicrons and transport of endotoxin, without the stimulation of macrophages through the Toll-like receptor 4.

Factors That Determine Resolution of the Sentinel Event

Why do some people recover and resolve the acute inflammatory sentinel event, whereas others do not? What factors determine a path of resolution of inflammation from that of continuation of injury and a pattern of chronicity? In many patients, there may be no history of alcohol, and removal of obvious stimulatory factors (such as gallstones, high triglyceride levels, or an offending pharmacologic agent) fails to resolve the inflammatory process. The work of Whitcomb9 has focused on those genetic factors that predispose patients to ongoing inflammation and failure of resolution. Repeated activation of trypsinogen to trypsin is part of the process that leads to continuity of inflammation in acute pancreatitis. Patients with a PRSS-1 genetic defect (cationic, trypsinogen mutation) have hereditary pancreatitis and as a result have a lower threshold for forming trypsin.9 Once trypsin is formed, rapid degradation may help prevent further injury. People with a CTRC genetic defect have difficulty degrading the trypsin and are more likely to have ongoing inflamma-

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Drivers of Oxidative Stress in Acute Pancreatitis / McClave


tion.9 Secretion of the formed trypsin or flushing the trypsin out through the pancreatic ductules is an important protective mechanism that is dysfunctional in patients with the cystic fibrosis gene defect (CFTR).9 Patients with the SPINK-1 (saline protease inhibitor of Kajal) genetic defect lack the protective effect of rapid degradation of the formed trypsin.9 On the other hand, factors relating to the patients ability to mount an antioxidant defense may determine whether he or she resolves inflammation arising from the sentinel event.2 The overall balance of pro-oxidants vs antioxidants in the systemic circulation may be a factor in resolution.2 The ability to activate the bodys own endogenous antioxidant response elements may help reduce the likelihood for chronicity. Modulating epigenetics to increase the expression of genes involved in the production of antioxidant agents may determine whether patients resolve the initial inflammatory process.48 What is amazing is that dietary modulation through nutrition therapy may influence all of these antioxidant defense mechanisms. If one considers the overall balance of pro-oxidants vs antioxidants, there is a condition where excess generation of pro-oxidants leads to severe pancreatitis. A condition known as nutritional (or tropical) pancreatitis is caused by chronic ingestion of the cassava root or yucca plant.49 This plant is the main dietary staple in certain areas of South Africa and Latin America. Cassava root is eaten raw, boiled in water, or processed into flour. When the plant comes in contact with gastric acid, cyanogenic glycosides are formed, resulting in low-grade cyanide toxicity.49 Chronic ingestion leads to increased production of reactive oxygen species and depletion of antioxidant defenses. Although damage to the thyroid and injury to the nervous system are described in this disease process, the ingestion of the plant is particularly toxic to the pancreatic acinar cell.49 Children show evidence of insulin resistance early on, which progresses to severe diabetes at a young age. They develop a chronic, severe, fibrocalcific pancreatitis, which on pathology shows intense fibrosis, presence of pancreatic ductal stones, and an absolute decimation of the acinar cells.49 On the other hand, shifting the overall systemic balance by providing a cocktail of exogenous antioxidant agents may help resolve the inflammatory response and speed recovery from severe acute pancreatitis.2,50-52 In the disease process of pancreatitis, where antioxidant levels are diminished, the generation of reactive oxygen species is increased, and lipid peroxidation is activated, it should not be surprising that a concoction of antioxidant agents may replenish stores and improve recovery.2,50-52 Typical antioxidant cocktails include selenium 600 mg, -carotene 9000 IU, methionine 2 g, vitamin C 0.54 g, or vitamin E 270 IU given orally once per day.51 In animal models, pretreatment with an antioxidant cocktail reduces subsequent pancreatic tissue injury and extra-pancreatic complications com-

pared to that seen in control animals receiving placebo.2 In clinical trials of patients with recurrent acute or chronic pancreatitis, chronic ingestion of an antioxidant cocktail reduces abdominal pain, decreases the number of hospital admissions, and reduces disease recurrence and chronicity.50-53 Treatment for acute pancreatitis is less well documented, but evidence suggests that an antioxidant cocktail might reduce disease severity, duration of the disease process, and likelihood for complications.2,50 Another factor in determining recovery and resolution vs continuing inflammation and chronic disease may be the ability to turn on the bodys own endogenous antioxidant defense system or the antioxidant response elements (ARE) (Figure 11).54 Nrf-2 is a transcription factor in the cytoplasm of cells that is normally kept inactive through binding by the Keap-1 protein. Removal of the Keap-1 protein allows the Nrf-2 to enter the nucleus of the cell and encode antioxidant proteins (such as glutathione transferase).54 Interestingly, activation of Nrf-2 is stimulated through dietary modulation with agents such as sulforaphane in broccoli, resveratrol in blueberries and red wine, and polyphenols in cauliflower.55 Activating the transcription factor that encodes for these antioxidant enzymes raises the bodys own production of endogenous antioxidant agents.54,56 Clinically, a patient who had a lifelong dietary pattern of ingesting intact fruits and vegetables theoretically would be more likely to recover from an attack of gallstone pancreatitis than a patient not on such a diet. In animal models, pretreatment with broccoli extract (which provides sulforaphane) results in a milder injury (retinal damage) and a reduced inflammatory response than receipt of placebo in controls.57 Theoretically, in a patient undergoing elective endoscopic retrograde cholangiopancreatography (ERCP), where there is a 5%10% chance of getting post-ERCP acute pancreatitis, a diet high in broccoli, cauliflower, red wine, and blueberries in the week beforehand might provide greater protection than a regular Western diet without these agents over the same time period. One last issue that may influence the ability by which a patient recovers from the sentinel event involves epigenetics and variable gene expression.48 Every cell in the body is genetically homogeneous, containing the same DNA. However, cell structure and function are heterogeneous, a phenomenon that reflects epigenetics or the differences in gene expression. It was originally thought under classic Darwinian genetics, for a change to occur in adaptation to environmental stresses, a mutation of the DNA would have to occur. Such mutations are infrequent, spontaneous, and unpredictable. But adaptation is better facilitated through phenotypic expression, which can change much more readily through epigenetics. Epigenetics is defined as the heritable changes in gene expression and concomitant organization, which is independent of the DNA sequence.48 Epigenetic inheritance is an essential mechanism that allows stable propagation of gene activity states from one

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Journal of Parenteral and Enteral Nutrition / Vol. 36, No. 1, January 2012

generation of cells to the next. As a result of epigenetics, different chromatin states exist, as genes move toward being activated and turned on or being silent and turned off.48 Epigenetic changes can occur as a result of dietary or other environmental exposures and can create a new event by controlling access to the genes or by altering gene expression.48,58 MicroRNAs are one way to influence gene expression.48,59 Theoretically, a functioning TNF promoter gene would direct the production of the protein TNF. A microRNA could bind that gene, blocking its protein production. Only by adding an anti-microRNA agent or antibody to bind the microRNA could the gene be reexpressed and the TNF protein produced. It is clear that both diet and environmental exposures can alter gene expression.48,58,59 Two other ways to influence gene expression is through DNA methylation and histone modifications.48 Conceptually, a chromosome with its sequence of DNA may be teased out like a string (Figure 12).48,60 Methylation of the gene always results in suppression of that gene, whereas a histone modification may shut off or turn on gene expression.48 A tumor suppressor gene, for example, might protect the host against colon carcinoma. With normal aging, however, the gene might become hypermethylated. At an older age, the gene would be turned off and the persons risk for colon cancer would increase. External dietary factors might influence gene expression to better manage oxidative stress and reduce the likelihood for chronic pancreatitis. Folate, betaine, vitamin B12 , and methionine are all methyl donors.48,58 Theoretically, if a TNF promotor gene were methylated, it would be turned off, reducing the degree of oxidative stress. If in the course of acute pancreatitis, however, a folate deficiency developed, the gene could be demethylated, be turned on, and begin producing TNF, increasing the level of oxidative stress. Histone transferase, on the other hand, is affected by trace metals such as zinc and copper.48,58 Theoretically, if a gene for glutathione transferase were turned on by histone modification, a zinc deficiency could cause decreased histone transferase function, and the gene could ultimately be turned off. Critical illness such as severe pancreatitis certainly may affect the epigenetic environment, and dietary modification may be a big factor in gene expression.

include agents such as glutamine, arginine, fish oil, zinc, and antioxidant vitamins, should help preserve the structure of the acinar cell and extinguish the fire of oxidative stress that rages in this disease process.

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