THE ROLE OF OXIDATIVE STRESS AND MITOCHONDRIAL DYSFUNCTION IN THE PATHOGENESIS OF ACUTE PANCREATITIS

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

Irma Kuliavienė

THE ROLE OF OXIDATIVE STRESS

AND MITOCHONDRIAL DYSFUNCTION

IN THE PATHOGENESIS

OF ACUTE PANCREATITIS

Doctoral Dissertation Biomedical Sciences, Medicine (06B) Kaunas, 2014

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The dissertation was prepared in the Medical Academy of Lithuanian University of Health Sciences during the period of the 2010–2014 year.

Scientific Supervisor

Prof. Dr. Habil. Limas Kupčinskas (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B)

Dissertation is defended at the Medical Research Council of the Medi-cal Academy of Lithuanian University of Health Sciences

Chairperson

Prof. Dr. Mindaugas Kiudelis (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B)

Members:

Prof. Dr. Habil. Andrzej Dabrowski (Medical University of Bialystok, Biomedical Sciences, Medicine – 06B)

Prof. Dr Laima Ivanovienė (Lithuanian University of Health Sciences, Biomedical Sciences, Biology – 01B)

Prof. Dr. Elona Juozaitytė (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B)

Prof. Dr. Habil. Jonas Valantinas (Vilnius University, Biomedical Sciences, Medicine – 06B)

Dissertation will be defended at the open session of the Medical Research Council of Lithuanian University of Health Sciences on December 5th, 2014 at 13 p.m. in 204 auditorium at Faculty of Pharmacy of Lithuanian University of Health Sciences.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA

Irma Kuliavienė

OKSIDACINIO STRESO IR

MITOCHONDRIJŲ FUNKCIJOS

SUTRIKIMO REIKŠMĖ

ŪMINIO PANKREATITO

PATOGENEZĖJE

Daktaro disertacija Biomedicinos mokslai, medicina (06B) Kaunas, 2014

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Disertacija rengta Lietuvos sveikatos mokslų universitete Medicinos akade-mijoje 2010–2014 metais.

Mokslinis vadovas

prof. habil. dr. Limas Kupčinskas (Lietuvos sveikatos mokslų universi-tetas, biomedicinos mokslai, medicina – 06B).

Disertacija ginama Lietuvos sveikatos mokslų universiteto Medicinos akademijos medicinos mokslo krypties taryboje:

Pirmininkas

prof. dr. Mindaugas Kiudelis (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, medicina – 06B)

Nariai:

prof. habil. dr. Andrzej Dabrowski (Bialystoko medicinos universitetas, biomedicinos mokslai, medicina – 06B);

prof. dr. Laima Ivanovienė (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, biologija – 01B);

prof. dr. Elona Juozaitytė (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, medicina – 06B);

prof. habil. dr. Jonas Valantinas (Vilniaus universitetas, biomedicinos mokslai, medicina – 06B).

Disertacija ginama viešame LSMU Medicinos mokslo krypties tarybos posėdyje 2014 m. gruodžio 5 d. 13 val. LSMU Farmacijos fakulteto 204 auditorijoje.

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Skiriu savo seneliams

Dr. Aloyzui ir Janinai Pundziams

Dedicated to my grandparents Dr. Aloyzas and Janina Pundziai

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ABREVIATIONS

AA – arachidonic acid (20:4 n-6) AP – acute pancreatitis

APACHE II – Acute Physiology and Chronic Health Evaluation scale ATP – adenosine triphosphate

DHA – docosahexaenoic acid 9(22:6 n-3) DNA – deoxyribonucleic acid

DPA – docosapentaenoic acid (22:5 n-3) ΔΨ – mitochondrial membrane potential EPA – eicosapentaenoic acid (20:5 n-3) FAs – fatty acids

FRAP – Ferric Reducing Antioxidant Power GPx – glutathione peroxidase GSH – glutathione IL 1 – interleukin-1 IL 1β – interleukin-1β IL 6 – Interleukin 6 IL 10 – interleukin 10 MB – methylene blue

MODS – multiple organ dysfunction syndrome

NADPH – nicotinamide adenine dinucleotide phosphate NF-κB – nuclear factor kappa B

NO – nitric oxide OF – organ failure OS – oxidative stress

PAI 1 – plasminogen activator inhibitor type 1 PI – Peroxidation Index

PUFAs – polyunsaturated fatty acids RCI – respiratory control index RNS – reactive nitrogen species ROM – reactive oxygen metabolites ROS – reactive oxygen species

SIRS – systemic inflammatory response syndrome SOD – superoxide dismutase

TAC – total antioxidative capacity TNF α – tumor necrosis factor α TOC – total oxidative capacity TS – iron saturation of transferrin UI – Unsaturation Index

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INTRODUCTION

Acute pancreatitis (AP) is an acute inflammation of exocrine pancreas. About 20%–30% of patients develop severe forms of the disease mani-festing with local and systemic complications. AP carries an overall mor-tality rate of 5%–15%1. The main causes of death are associated with multiple organ failure and pancreatic infection2,3. Mortality of patients with OF is 30% and patients with both, OF and infection, have 43% mortality rate2. Though the initial process starts in the pancreas it does not affect mortality of AP4. Systemic inflammatory response is responsible for mul-tiple organ failure and has the most considerable impact on the severity of acute pancreatitis and mortality from this disease5.

Oxidative stress plays an important role in the pathogenesis of AP6. It is shown that ROS are important mediators of tissue injury by direct damage of cells membranes as well as by acting as inflammatory cascade media-tors7,8. On the other hand it is suggested that intracellular ROS generation during AP may be a protective response by promoting apoptosis instead of necrosis of acinar cells9. These controversies are reflected in the studies of antioxidant treatment of patients with AP and have been recently reviewed by several authors10–12.

The various pathways of ROS formation in humans generally start from the superoxide anion, the “primary” ROS, and by interacting with other molecules can generate so called “secondary” ROS13. The production of superoxide anion occurs mostly in the mitochondria of the cell in the Complexes I and III of the respiratory chain. The disturbance of the process can lead to oxidative damage and dysfunction of mitochondria that can significantly affect the existence of the cell14.

A disturbance of pancreatic mitochondrial function has been considered as one of the mechanisms for the onset of AP in various studies15–18. It has been suggested that mitochondrial dysfunction plays a pivotal role in the pathogenesis of critical conditions19. Several reviews describe the link between mitochondrial dysfunction and sepsis20–22. Recently, early dysfunc-tion of jejunum, lung and pancreatic mitochondria in rats during early (6 h) phases of cerulein- (mild) and taurocholate-induced (severe acute) pan-creatitis has been described 23. However the alterations of mitochondria in different organs within first 48 hours after onset of AP as well as and their role for the pathogenesis of AP has not been fully elucidated.

There are multiplying experimental evidence about the mitochondria tar-geted therapies during sepsis24 and other diseases that are known to be asso-ciated with mitochondrial damage25. Methylene blue reduces mitochondrial

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ROS overproduction via alternative electron transfer that bypasses mito-chondrial complexes I–III26. It is studied as neuroprotective agent27,28 as well as improves liver mitochondrial function during gut ischemia-reper-fusion experiments29. MB could be a potential therapeutic agent for treat-ment of systemic complications of AP.

A precise role of oxidative stress in the pathogenesis of AP and its systemic complications is important but still far from being clear. The mitochondrial dysfunction is associated with ROS production in the process of AP and could have a role in the systemic complications of this disease. The mitochondrial dysfunction could also be a target for future therapeutic implications for AP.

Aim and objectives of the study

The aim of the study was to evaluate the role of oxidative stress and mitochondrial respiratory function in the pathogenesis of acute pancreatitis.

The objectives of the study:

1. To evaluate the oxidative stress markers in human serum during the early phase of acute pancreatitis.

2. To find out possible interactions of oxidative stress with iron metabolism during the early phase of acute pancreatitis.

3. To study the impact of oxidative stress during early phase of acute pancreatitis by evaluating the composition of fatty acids of erythrocyte membrane of patients with severe and mild acute pancreatitis of alcoholic and nonalcoholic etiology.

4. To assess the mitochondrial respiratory chain function of pancreas and distant organs (liver, kidney and lungs) during the first 48 hours after the induction of acute pancreatitis in experimental rat model of severe acute pancreatitis.

5. To study the association of mitochondrial respiratory chain dysfunction and systemic inflammatory response syndrome as well as organ failure. 6. To evaluate the effect of intra-mitochondrial antioxidant methylene blue

for the amelioration of pancreas and kidney mitochondrial respiratory chain dysfunction in the experimental rat model of severe acute pan-creatitis.

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Originality of the study

The growing evidence suggests that the systemic processes occuring during AP are responsible for multiple organ failure and have the most considerable impact on the severity of AP and mortality from this disease. Our study adds important information to the pathogenesis of the systemic events during early phase of AP.

Various experimental data suggest that oxidative stress plays an im-portant role during AP but the lack of human studies evaluating redox status in the course of this disease makes it difficult to establish a precise role of oxidative stress in the pathogenesis of AP and its systemic complications. Moreover the complexity of the oxidative and anti-oxidative systems in human body makes the redox changes difficult to assess. In our study we evaluate the markers that reflect more primary oxidative stress products - hydroperoxides, and more complex anti-oxidative systems during early phase of AP. We hypothesize that measurement of such markers should give more information about the process of oxidative stress. To our knowledge there is no published data about the changes of most of these markers during AP.

While evaluating the redox state during AP various other players should be taken into account. One of them is iron which has many different roles in normal physiological functions and human diseases. To our knowledge our study is the first evaluating the disturbance of iron metabolism as well as its associations with oxidative stress in the early phase of AP.

The role of FAs in the pathogenesis of AP is studied by various authors but is far from being clear. The FAs of membranes are responsible for inflammatory and oxidative processes. There are studies investigating the alterations of the FAs during AP. The targets of these studies are the FA composition of serum that is greatly influenced by necrotic changes in the pancreas and peri-pancreatic tissues. The erythrocyte membrane phospholi-pids can better reflect systemic changes caused by oxidative stress and inflammatory response as well as alcohol impact in patients with AP. To our knowledge, no studies examining the FA composition of membranes during AP have been carried out. The results of our study thus provide important information about this matter.

Our experimental study’s target is to go further into the pathogenesis of oxidative damage of distant organs during AP. There are data about mitochondrial role in the primary events of pancreas, but mitochondrial disturbances of distant organs during severe AP are poorly characterized. It is known though that during SIRS of other origin the severity of organ dysfunction and eventual poor outcome is associated with mitochondrial

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dysfunction. Our experimental study presents valuable information about mitochondrial respiratory chain dysfunction within first 48 hours not only of pancreas but of distant vital organs (kidney, lungs and liver) as well. The estimation of the time point when mitochondrial dysfunction occurs in specific organs and association of these disturbances with inflammatory and organ function markers proves a gap for possible therapeutic intervention to prevent early organ failure and mortality in patients suffering from AP. Yet more, the results of our methylene blue study open the door for further investigations of this mitochondria targeted antioxidant as a therapeutic intervention for severe AP complications.

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1. REVIEW OF LITERATURE

1.1. Acute pancreatitis

AP is an acute inflammation of exocrine pancreas. It is a common disease. The incidence of AP in European countries varies from 5 to 45 per 100,000 inhabitants/year and seems to be increasing over the years30,31. The most common etiology of AP is gallstones and alcohol. They cover more than 60% of all AP. The proportion of etiology differ slightly in various countries and regions reflecting the specific differences in prevalence of risk factors1. The etiology of alcohol is more frequent in Germany, Denmark and Sweden1,32.

In clinical range AP presents itself mostly as mild self-limiting localized disease. About 20%–30% of patients develop severe forms manifesting with local and systemic complications33,34..The main causes of death are associated with multiple organ failure and pancreatic infection. Mortality of patients with OF is 30% and patients with both, OF and infection, have 43 % mortality rate2. It has been noticed that most deaths occur within the first 7–14 days35. There is a trend for decreasing of overall mortality over the years1, but the early deaths of patients with AP remain a major contributing factor36. The early phase of AP lasts usually up to seven days but may extend into the second week. It covers not only early events in pancreas, but also systemic inflammatory response syndrome.

The local injury of the pancreas involves triggering events that lead to premature activation of trypsin, disruption of the acinar cells and pancreatic auto digestion37. However, it is not entirely clear which events trigger premature activation of trypsin. The sustained calcium release is thought to be associated with premature activation of digestive enzymes. As well as high calcium concentrations can destroy the cytoskeleton and lead to va-cuole formation in acinar cells38. On the other hand it is becoming in-creasingly clear that another important thing happening in acinar cells – the activation of intense inflammatory signaling mechanisms mainly through NF-κB is crucial to the pathogenesis of pancreatitis and may explain the strong inflammatory response during AP39.

Loss of the local control of inflammatory processes results in excessive uncontrolled activation of inflammatory cells (neutrophils and macropha-ges) and mediators, a systemic response to the local inflammation develops. The two phase hypothesis is offered for the pathogenesis of systemic inflammatory response during AP. It grips systemic inflammatory and anti-inflammatory response syndromes which are usually characterized as a

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subset of cytokine storm. The main pro-inflammatory cytokines are TNF α and IL 1β whereas IL 10 and IL 1 have anti-inflammatory function5

. IL 6 is generally considered as the pro-inflammatory cytokine, and is found to be a good severity predicting marker for AP40–42. However it has been shown that it can act predominantly as an anti-inflammatory subject by down regu-lating the synthesis of pro inflammatory cytokines such as TNF α, IL 1β, macrophage inflammatory protein-2, interferon γ43. The imbalance of these processes is thought to be the major pathological implication to the severity of AP5.

The initial events in the pancreas are very important for the pathogenesis of AP. On the other hand it seems that the acinar cell damage (such as necrosis) during AP does not affect severity nor mortality of AP4,44. It is SIRS that is associated with the multiple organ dysfunction and has the most considerable impact on the severity of AP and mortality from this di-sease5,45,46 Renal and respiratory failures are of utmost importance and have a large impact on mortality rate4,47.

In clinical practice patients with AP, for the most part, seek medical attention within 12±18 h of the onset of pain. The effects of distant organ damage in patients with a severe AP attack are possibly detectable at the admission but often are not fully established, and only become apparent over the following 48 h48. Thus potentially there could be a therapeutic window for intervention between hospital presentation and the development of distant organ dysfunction.

It should be noted that there are much more things happening during AP and SIRS. In this dissertation we focused on oxidative stress with lipid peroxidation and disturbance of iron metabolism as well as mitochondrial dysfunction. These processes are associated not only with initial events in the pancreas but also systemic response during AP. Elucidation of these pathogenetic mechanisms could open new possibilities for therapeutic inter-ventions for AP complications.

1.2. Oxidative stress and acute pancreatitis

Oxidative stress plays an important role in various normal and pathological functions in human body. It is defined as a disturbance of the balance between the production of ROS/RNS (free radicals) and antioxidant defenses49. ROS can be defined as molecules or molecular fragments con-taining one or more unpaired electrons in atomic or molecular orbitals. Superoxide anion (O2•–) is considered to be the primary ROS and can further

interact with other molecules to generate “secondary” ROS. The production of peroxide anion radical occurs mainly within the mitochondria of the cell.

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The more exact mechanism is reviewed further. Another important sources of primary ROS are purine metabolism, with main enzyme xanthine oxidase, and respiratory burst of neutrophils mediated by NADPH oxi-dase13. NO• is a main RNS. It contains one unpaired electron and is ge-nerated in biological tissues by specific nitric oxide synthases50.

There are two main antioxidant defense systems in the organism: enzy-matic (SOD, GPx, catalase) and non-enzyenzy-matic (ascorbic acid – vitamin C, α tocopherol – vitamin E, GSH, carotenoids, flavonoids and others)13

. In stable state of the cell the rates of ROS production are in the balance with the rates of their removal by various antioxidants. This is the redox state of the cell51.

The ROS and RNS are recognized to play a dual role in live organisms as deleterious and beneficial species. Overproduction of ROS results in oxi-dative stress and can damage the cell structures, lipids and membranes, proteins and DNA. The moderate concentrations of ROS/RNS can par-ticipate in defense against infectious agents, in cellular signaling pathways. Various ROS mediated actions can protect cells against ROS-induced oxidative stress13. Thus the balance between ROS/RNS generating enzymes and scavenger enzyme systems is of utmost importance and the disturbance of equilibrium of this complicated system leads to various pathologic pro-cesses13.

The measurement of oxidative stress in live organism is complicated because reactive molecules are of short age. Lipid peroxidation is the major consequence of oxidative stress and cause of oxidative damage52. There are hundreds of lipid peroxidation products that have stable structure and can be measured. As well as anti-oxidative capacity markers53. The big variety of oxidative stress markers shows that there is no a perfect one.

Oxidative stress has a definite role in the pathogenesis of AP6. Several main sources of ROS production in the cell during AP are studied in dif-ferent experimental AP models54. These are xanthine oxidase, nitric oxide synthase, cytochrome P450 and NADPH oxidase. It is shown that ROS and RNS are important mediators of tissue injury by direct damage of the essential cellular components. It can react with lipids of the cell and mito-chondrial membrane leading to disintegration of membrane and pancreatic necrosis or dysfunction of mitochondria8. The oxidation of proteins can affect enzyme activity, membrane–receptor function or form nonfunctional proteins55,54. Simultaneous generation of ROS and RNS can induce DNA fragmentation and 8 hydroxylation of guanine residues56.

It should be also noted, that intracellular ROS generation during AP may have a protective response by promoting apoptosis instead of necrosis of acinar cells and restricting disastrous insult in the acute phase of

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mation9. But extensive ROS/RNS generation could induce direct damage on mitochondrial membrane leading to depletion of ATP and eventually switching apoptosis to necrosis57.

Apart of the direct impact on pancreas oxidative stress finds its place in the inflammatory response during AP. ROS can serve as a messenger in intracellular signaling involving mitogen – activated protein kinases, and apoptotic pathways54. Cytokines such as TNF α, IL 1β and interferon γ were reported to generate ROS in the cells by involving mitogen – activated protein kinases58,59. It is shown that activation of mitogen – activated protein kinase and nuclear factor kappa B stimulates pro-inflammatory gene expres-sion in acinar cells as well as inhibition of this kinase abolishes stress in-duced cytokine expression60,61. Oxidative stress can also initiate the migra-tion, adhesion62 and infiltration of inflammatory cells thus contribute to SIRS.

The association between antioxidant enzymes and pancreatic inflam-mation has been studied over the last years. The major ROS scavenger in the pancreas is GSH and GPx antioxidant systems63. The pancreatic GPx level is significantly altered in various experimental models of AP as well as in serum and erythrocytes of patients with AP. The activity of other anti-oxidant enzymes such as SOD and catalase was shown to be changed during AP as well54.

Experimental and clinical studies indicate that oxidative stress is a common pathway in the pathogenesis of AP, and the use of antioxidants for treating AP seems to be a reasonable idea. Results from animal studies show that anti- oxidative therapy could be helpful but the data from human projects are still controversial6. Some studies showed that enteral forms of antioxidants in the treatment of AP have clinical benefits and improve oxidant status64,65. While other randomized control trials show no significant benefits from antioxidant therapy in patients with established severe AP66,67. Even the higher inflammatory values are recorded in the group of anti-oxidant treatment68. As reviewed by Gu et al antioxidant supplementation shows no beneficial effect on the incidence or the severity of post-ERCP pancreatitis and there is not enough evidence to support using antioxidants for the prevention of post-ERCP pancreatitis69. These controversial findings contribute to the complexity of oxidative processes during AP and need for elucidation of the field.

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1.3. Fatty acids and acute pancreatitis

FAs have a number of physiological roles. As triacylglycerols of dietary fat they are a principle source of energy and take part in transferring and depositing important lipid soluble molecules (such as vitamins A, D , E and K). Short chain FAs represent an energy source for enterocytes and colono-cytes70. Free FAs of the cell can take part in the signaling mechanisms through protein kinase C71. FAs are structural components of the cell membranes in the form of phospholipids. The profile of FA composition influences the thickness and fluidity of the membrane as well as the activity of the membrane associated proteins. The PUFAs of cis configuration increase membrane fluidity while saturated FAs and unsaturated FAs of trans configuration have opposite effect on the cell membranes70.

The FAs of the cell membrane are precursors of various lipid mediators with eicosanoids (prostaglandins, thromboxanes, leukotrienes) being one of the most important72. These eicosanoids are important actors of inflamma-tory responses and may have various effects to the cell. Prostaglandins stimulate inflammation, regulate blood flow, control ion transport across membranes and modulate synaptic transmission. Thromboxanes are vaso-constrictors and potent hypertensive agents which also facilitate the platelet aggregation. Different PUFAs, mainly AA, EPA, DHA and DPA and others, are precursors for these eicosanoids70. Fatty acids are closely associated with oxidative stress. They can be damaged by ROS and lose their important function in human metabolism. ROS can affect the synthesis of eicosa-noids73.

Lipid peroxidation is the major reaction taking place under oxidative stress and assumed to play an important role in the pathogenesis and gression of many diseases. There are hundreds of lipid peroxidation pro-ducts. They are usually stable structures thus lipid peroxidation products despite their limitations serve as useful biomarkers of oxidative stress53.

The role of FAs in the pathogenesis of AP is important but still not clear enough. An increased total serum free FA level is observed during AP74. Unsaturated FAs, especially polyunsaturated FAs (PUFAs), are liberated from pancreatic necrotic tissues and are responsible for the disturbance of FA profile in the serum of patients with AP74,75. The increased amount of unsaturated FAs in the necrotic pancreatic tissue and serum during AP is associated with multisystem organ failure and worsen outcomes of pa-tients76. Moreover, alcohol, an important etiological factor for pancreatitis, has an impact on the FA composition of serum and erythrocyte mem-branes77–80. Surprisingly, during alcohol-induced pancreatitis, the percentage of PUFAs is decreased in the serum FA profile in mild and moderate AP as

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well as chronic pancreatitis81,82. These data suggest that alcohol could play a specific role in the pathogenesis of pancreatitis.

Experimental findings show that n-3 PUFAs may be beneficial in the prevention of oxidative stress-induced inflammation during pancreatitis83. Moreover, it influences the histological severity of AP84–87. Human studies also indicate likely clinical benefits of enteral feeding rich in n-3 PUFAs in patients with AP64. Much more research is needed in this field to have sufficient data for translating science to clinical practice.

1.4. Iron and acute pancreatitis

Iron is the second most abundant metal of the earth crust. Most of the body’s demand for iron is met through efficient recycling from senescent red blood cells by macrophages and the rest is collected from the diet. Iron status is regulated by three cell systems: the duodenal enterocytes that ab-sorb iron, splenic and hepatic macrophages and hepatocytes88. The master regulator of iron homeostasis is hepcidin, which is a peptide hormone with antimicrobial properties that is encoded in the liver (Fig. 1.4.1)89. The expression and activity of hepcidin is influenced by systemic iron status and inflammation. Several inflammatory cytokines are known to increase hep-cidin expression, e.g., IL 6, IL 1β, TNF α and interferon γ88.

Iron has dual biological function. It is essential as a part of the hemo-proteins but is toxic as it has a potency to accept and donate electrons thus forming radical species and participating in the process of oxidative stress90. Iron containing proteins carry or store oxygen (e.g. hemoglobin and myo-globin), catalyze metabolic, signaling related, and antimicrobial redox reac-tions (cytochromes, ribonucleotide reductase, nitric oxide synthase, NADPH oxidase, myeloperoxidase), and transport or store iron (e.g., transferrin, lactoferrin or ferritin)91.

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Fig. 1.4.1. Regulation of circulating iron by hepcidin

Phagocytosis and degradation of senescent red blood cells by macrophages releases Fe2+

from heme, a reaction catalyzed by heme oxygenase 1 (HO1), for storage within ferritin or export into the circulation via the transmembrane protein SLC40A1 (also known as

ferroportin 1). Oxidation into Fe3+ by the membrane-bound ferrioxidase hephaestin (also

known as HePH) enables binding to transferrin for transport. Dietary heme iron is taken

up by enterocytes and degraded by HO1 to yield Fe2+. Fe2+ is exported into the circulation

via SLC40A1. Liver-derived hepcidin, induced by iron overload or inflammation, degrades SLC40A1 and blocks iron export, thereby diminishing absorption and recycling by

macrophages (Weiss, 2010)89.

In plasma majority of iron is bound to transferrin. Unbound iron which does not have the shield in the form of protein has capacity to generate highly reactive free radicals and cause known damage of oxidative stress90. The redox potential lies in the ability of iron to slide between Fe2+ and Fe3+. These processes are characterized by Fenton and Haber – Weiss reaction:

Fe 2+ +H2O2→Fe3+ +OH• +OH–

O2•–+ H2O2→ O2+ OH• +OH–

Fe3++ O2•–→Fe 2+ + O2 92

Non transferrin bound iron is also suggested to play supporting role in growth of certain bacteria and fungi. Thus along with the increased load of ROS it makes host more susceptible against various infections93. Numerous studies have reported the role of iron in different inflammatory conditions (e.g., cardiac diseases, diabetes mellitus, renal and hematological illness-ses)90.

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The data about iron metabolism and its role during AP is limited to some experimental findings. It was shown that in the early stages of AP, the pancreatic concentrations of iron are significantly decreased and can have an impact in pathophysiology of AP94. The study of Sledzinsky et al. showed that cerulein stimulation increases labile iron pool in pancreatic acinar cells and is accompanied by a decrease in the cellular ferritin-L level and an increase in the ROS formation95. In another study it was suggested that elevated Fe levels in serum and pancreatic tissue in rats with early-stage alcohol-induced AP is associated with various hemorheological changes and with oxidative damage of the pancreas96. Moreover FeSO4 can cause

parenchymal necrosis of pancreas with intracytoplasmic formation of va-cuoles, fusion of the vacuoles and zymogen granules, and autophagosomes containing cellular organelles in experimental AP rat model97. But there are still lack of data to clarify the mechanisms of relations between iron and oxidative stress in AP pathogenesis.

1.5. Mitochondria and acute pancreatitis

Mitochondria are the organelles of the cell that are usually called the powerhouse of the cell. They consist of two membrane systems. The inner mitochondrial membrane forms cristae, which contain the membrane bound enzymes of the respiratory chain. It is an impermeable barrier between the inter membrane space and the matrix. The outer membrane is more permeable for ions and small molecules98. Mitochondria are responsible for different and important functions of the cell. The mains are (1) ATP synthesis, (2) ROS production, (3) regulation of calcium homeostasis in the cell, (4) special pathways involving heme and urea synthesis, (5) control of cell death (apoptosis and necrosis).

Oxidative phosphorylation is the major ATP synthetic pathway in eukaryotes. There are two systems – tricarboxylic acid cycle and respiratory chain – that play a crucial role in the production of ATP (Fig. 1.5.1). Tricarboxylic acid cycle degrades carbon substrates of acetyl CoA and reduces NAD+ to NADH and FAD2+ to FADH2. These are the main

subs-trates to the enzymes of the respiratory chain which consists of four complexes (Complex I–IV). The respiratory chain produces H+ gradient via the ‘downhill’ transport of electrons. Electrons are transferred from NADH to Complex I and from FADH2 to Complex II. Ubisemiquinone conveys

electrons from these complexes to Complex III from where cytochrome C carries the electrons to Complex IV. During the operation of Complexes I, II and IV, protons are transported from the matrix to the inter membrane space, which creates Δψ. ATP is synthesized by F1F0-ATP synthase and

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transported from the matrix to the cytosol by adenine nucleotide translocase. Ca2+ increases the activity of the TCA cycle enzymes and is transported via voltage-dependent anion channels and CaU into the mitochondria.98

Fig 1.5.1. Mitochondrial energy production under physiological conditions The tricarboxylic acid (TCA) cycle provides reducing substrates (NADH, FADH2) for the

respiratory chain which produces H+ gradient via the ‘downhill’ transport of electrons.

From Complexes I and II ubisemiquinone (UQ) conveys electrons to Complex III from where cytochrome C carries the electrons to Complex IV. During the operation of Complexes I, II and IV, protons are transported from the matrix to the inter membrane

space, which creates Δψ. ATP is synthesized by F1F0-ATP synthase and transported from

the matrix to the cytosol by adenine nucleotide translocase (ANT). Ca2+ increases the

activity of the TCA cycle enzymes and is transported via VDAC and CaU into the

mitochondria (Maléth et al, 201398.

The regulation of calcium homeostasis in the cell is regulated by mito-chondria. Intracellular Ca2+ signaling plays a central role in the physio-logical regulation of HCO3– secretion and trypsinogen activation in the

pancreas, however uncontrolled Ca2+ release can lead to intracellular Ca2+ overload and toxicity, including mitochondrial damage and impaired ATP production99,100. In the acinar cells there are three groups of mitochondria: the perinuclear, the perigranular and subplasmalemmal. These different subgroups have different roles in the regulation of cellular Ca2+ homeo-stasis. The activity of rate-limiting enzymes of the tricarboxylic acid cycle is Ca2+ dependent and helps mitochondria to adapt to the increased cellular ATP demand98.

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Mitochondria are an important source of ROS within most mammalian cells. Complex I and III are the main O2•– production sites in isolated

mito-chondria. Complex I produces large amounts of ROS by two main me-chanisms: when the NADH/NAD+ ratio in the matrix is high and when the mitochondria are not making ATP and consequently have a high proton motive force and a high reduced Coenzyme Q14.

The mitochondrial membrane permeabilisation determines cell fate. It is mediated by two permeability systems, the premeability transition pore and the mitochondrial outer membrane permeability. Permeability transition pore opening results in loss of the ΔΨ and, ultimately, ATP depletion and necrosis. Mitochondrial outer membrane permeability mediates the release into the cytosol of cytochrome c, triggering caspase activation cascade and subsequent apoptotic events (Fig. 1.5.2)16.

Fig. 1.5.2. Roles of mitochondria in apoptosis and necrosis

A critical event in cell death is mitochondrial membrane permeabilisation, mediated by two permeability systems, the permeability transition pore (PTP) and the mitochondrial outer

membrane permeability (MOMP). PTP opening results in loss of the mitochondrial membrane potential (ΔΨ) and, ultimately, ATP depletion and necrosis. MOMP mediates the

release into the cytosol of cytochrome c, triggering caspase activation cascade and subsequent apoptotic events. IMM – inner mitochondrial membrane; OMM – outer

mitochondrial membrane (Gukovsky et al, 2011)16.

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A disturbance of pancreatic mitochondrial function has been considered one of the mechanisms for the onset of AP in various studies. Hirano et al. described an increased fragility of mitochondria in pancreas of mild edematous (cerulein induced) AP model101. The study of Halangk et al. revealed, that supramaximal cerulein stimulation induces a drastic reduction of the capacity of mitochondrial ATP production at 5 h and 24 h in rat pancreas102. They also demonstrated the diminished respiration rates and ATP levels in intact acinar cells isolated from cerulein-treated rats103. In another study, the increase in the leak respiration and the decrease in the mitochondrial membrane potential, swelling or rupture of some pancreatic mitochondria 5 h after the cerulein injection was found as an evidence for the opening of the mitochondrial permeability transition pore (PTP)104. Later studies of others15–18 confirmed that the loss of mitochondrial mem-brane potential is an early event in various models of non-alcoholic AP. Thus, the disturbance of cellular energy metabolism in pancreas as an important factor that contributes to the dysfunction of pancreas in mild AP model has been discussed105,106.

Organ-specific mitochondrial changes in severe AP are poorly charac-terized. Recently, early organ – specific mitochondrial dysfunction of jeju-num, lung and pancreatic mitochondria in rats during early (6 h) phases of cerulein- (mild) and taurocholate-induced (severe acute) pancreatitis has been described23. However the alterations in mitochondria in different or-gans within first 48 hours after onset of AP and their role to pathogenesis of AP has not been fully elucidated.

1.6. Mitochondria targeted therapies

To our knowledge there are no studies about the mitochondria targeted antioxidants for treating AP and its complications. It has been suggested that mitochondrial dysfunction plays a pivotal role in the pathogenesis of other critical conditions that evidence with SIRS19. Several reviews describe the link between mitochondrial dysfunction and sepsis20–22. Brealey et al. found that the severity of organ dysfunction and eventual poor outcome were associated with increase in mitochondrial dysfunction (complex I inhibition and ATP depletion) in a long-term rodent model of sepsis and organ fai-lure107. Pathak et al. showed that serum from septic mice produced a time-dependent decrease in mitochondrial membrane potential of primary murine cortical renal epithelial cells108.

There is growing evidence that in MODS caused by sepsis mitochondrial function is important and mitochondria targeted therapies could be help-ful20–22. For this purpose various substances have been tested. Most popular

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are antioxidants conjugated to specific lipophilic cautions that help them to target the mitochondria of the cells109. The MitoQ is the most extensively studied and the best understood member of this family110. As well as other antioxidants like superoxide dismutase and catalase mimetic (e.g. EUK-8, EUK-134) are used and show beneficial results in the experimental models of endotoxic, hemorrhagic shock and cardiomyopathies111–113.

1.7. Methylene blue – an antioxidant targeted to mitochondria Methylene blue is heterocyclic aromatic compound (Fig. 1.7.1) that has many biological and medical applications. It is used as a die in diagnostic procedures and for the treatment of multiple disorders; including

methemoglobinemia, malaria, and cyanide and carbon monoxide poisoning as well as septic shock114,115.

.

Fig. 1.7.1. Chemical structure of methylene blue

The antioxidant capacity of MB is based on its potency to reduce mitochondrial ROS overproduction 26. Wen et all demonstrated that MB functions as an alternative electron carrier that efficiently shuttles electrons between NADH and cytochrome C. This process reroutes electron transfer upon inhibition of Complexes I and III, reduces electron leakage and atte-nuates ROS overproduction as shown in Fig. 1.7.2.27

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Fig. 1.7.2. Mechanism of electron transfer by methylene blue (MB) in the mitochondrial oxidative phosphorylation chain

MB prevents the “electron leakage” induced by complexes I–III inhibition and avoids

the massive ROS production (Wen et al, 2011)27.

These features open the door for MB to various studies of diseases with mitochondrial dysfunction. Nowadays the potential positive effect of MB on neurological diseases is studied. It retains its protective activity in animal models of stroke, Parkinson’s disease and optic neuropathy27,28. It is studied in various ischemia reperfusion experimental models of the gut and liver as well29,116. MB has never been studied for treatment of AP.

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2. METHODS

2.1. Ethics

The clinical study was approved by Kaunas Regional Ethics Committee for Biomedical Research (No. BE-2-47). All patients and healthy subjects provided written informed consent. The experimental procedures used in the study were performed according to the permission of the Lithuanian Committee of Good Laboratory Animal Use Practice (No. 0228/2012).

2.2. Patients

All consecutive patients with a diagnosis of AP and onset of the disease within the last 72 hours admitted to the Departments of Surgery and Gastroenterology at the Hospital of Lithuanian University of Health Scien-ces between June and December 2007 were included in the study. The diagnosis of AP was established based on acute abdominal pain, at least 3-fold elevated levels of serum amylase, and typical radiological findings. According to the APACHE II scale, the patients were subdivided into the mild (APACHE II score <7, n=22) and severe (APACHE II score ≥7, n=17) AP groups. Healthy subjects (n=26) without a past history of pancreatic diseases were enrolled as controls.

Peripheral blood samples were drawn from patients on admission to the hospital. Plasma and leukocytes were removed after centrifugation. Eryth-rocytes were washed and centrifuged twice. The samples were stored at – 80°C until analysis. The blood samples of the control group were subjected to the same procedure.

The FAs of erythrocyte membrane and erythrocyte antioxidant analysis as well as calculation method for PI and UI are described in the publication by Kuliaviene et al117.

2.2.2. Serum oxidative stress and iron circulation marker analysis

Blood sample analysis was uniformly performed in the Laboratory for Health Protection Research, National Institute for Public Health and the Environment (the Netherlands). Serum oxidative stress markers measured in serum included ROM and FRAP, TOC and TAC. The ROM assay was obtained from Diacron (Grosseto, Italy) and adapted on a clinical auto analyzer LX20-Pro from Beckman-Coulter (Woerden, the Netherlands). The

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ferric reducing ability of plasma (FRAP) was performed according to Ben-zie and Strain118.

Serum iron and transferrin were measured with a clinical auto analyzer LX20-Pro from Beckman-Coulter (Woerden, the Netherlands) using dedi-cated kits. The iron saturation of transferrin was calculated using the for-mula:

TS (%) = Fe (ug/dL)*70.9/transferrin (mg/dL).

Ferritin was measured with an immuno-analyzer Access-2 from Beck-man-Coulter (Woerden, the Netherlands) using a dedicated kit.

2.3. Animals

Adult male Wistar rats weighing 200–250 g were used in experiments. They were housed under standard laboratory conditions, maintained on natural light and dark cycle and had free access to food and water. Animals were acclimatized to laboratory conditions before the experiment.

The chemicals, surgical procedure of induction of AP, experimental design of first set of experiments, methods for histological, rat serum analysis, protocols for pancreatic, liver, kidney and lung mitochondria iso-lation, measurements of mitochondrial respiratory rates, measurements of Complex I activity are described in the publication by Trumbeckaite el al119.

2.3.1. Methylene blue experiments

In the second set of experiments animals were divided into three groups: 1) MB group – methylene blue 5mg/kg was injected into vena cava 10 min prior to AP induction.

2) AP group (Vehicle group) – isotonic sodium chlorine solution was infused intravenously 10 min prior to AP induction.

3) Control group – healthy rats.

Animals were sacrificed after 24 hours from the induction of AP. Blood was taken for serum marker assay; pancreas and kidney were removed for mitochondrial assay. The chemicals, surgical procedure of induction of AP, pancreatic and kidney mitochondria isolation, measurements of mitochon-drial respiratory rates, measurements of Complex I activity were described in the publication by Trumbeckaite el al119. Urea was measured with a cli-nical auto analyzer LX20-Pro from Beckman-Coulter (Woerden, the Nether-lands) using dedicated kit.

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2.4. Statistical analysis

Statistical analysis was performed using SPSS version 16.0 for Windows. The Mann-Whitney test, one-way and two-way ANOVA tests were applied for analysis of variables in human study. For associations, Spearman cor-relation coefficient was calculated.

In the study of experimental AP, data are presented as mean ± SEM of three-five separate experiments. The mean for individual rat experiment was obtained from at least three repetitive measurements. All statistical tests were two sided, and P<0.05 was considered statistically significant.

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3. RESULTS

3.1. Clinical set

The demographic and etiology characteristics of patients and controls are presented in the publication by Kuliaviene et al117.

3.1.1. Serum oxidative stress and iron metabolism markers

The serum oxidative stress parameters reflecting the lipid peroxidation process ROM and TOC showed significantly decreased levels in the AP group as well as mild and severe AP groups. TOC decreased more in mild AP patients than in severe AP patients, the difference was significant. One of the measurements of total antioxidant capacity (FRAP) was increased in all AP groups, compared with the control group (Table 3.1.1.1). Another method for antioxidant capacity, TAC, showed no significant changes in the groups.

A disturbed iron status in all pancreatitis groups was found. As shown in Table 3.1.1.1, iron, transferrin and the iron saturation of transferrin were significantly lower and ferritin was significantly higher in all AP groups.

Table 3.1.1.1. Serum oxidative stress and iron circulation markers in acute pancreatitis (AP): Reactive Oxygen Metabolites (ROM U/mL), Total Oxidative Capacity (TOC mmol/l H2O2), Ferric Reducing Antioxidant

Power (FRAP mmol/l); Total Antioxidant Capacity (TAC mmol/l trolox), Iron (Fe ug/dL), Transferrin (mg/dL), Ferritin (ug/L), Iron saturation of transferrin (TS %)

AP Severe AP Mild AP Control

ROM TOC 239.91±142.17* 0.05±0.06* 219.77±150.85# 0.06±0.06⁰ a 255.82±136.94# 0.03±0.05* 368.97±72.54 0.12±0.08 FRAP TAC 1472.94±513.83* 1.55±0.76 1637.25±559.25# 1.77±0.64 1343.23±447.80# 1.38±0.81 1012.36±187.76 1.76±0.30 Fe Transferrin 6.44±5.44* 1.69±0.45* 7.33±6.03* 1.61±0.43* 5.74±4.98* 1.76±0.46* 20.81±7.47 2.60±0.42 Ferritin TS 518.32±277.39* 16.74±18.78* 603.20±281.82* 21.51±25.87# 451.32±261.82* 12.97±9.61* 118.11±94.38 32.56±12.81 Results are presented as mean ± standard deviation.

*P<0.001, #P<0.01, ⁰P<0.05 compared to controls,

a _{p<0.05 compared mild to severe AP.}

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There was a significant correlation of ROM with transferrin and ferritin, as well as of FRAP with transferrin and ferritin (Fig. 3.1.1.1) TOC and TS correlated significantly as well as TOC and Fe (Fig. 3.1.1.2).

Fig. 3.1.1.1. Scatter plot of correlation between oxidative stress markers and iron circulation markers

There was a significant correlation between reactive oxygen metabolites (ROM U/mL) with transferrin (mg/dL) (Spearman’s correlation coefficient 0.44, p=0.00) and with ferritin

(ug/L) (Spearman’s correlation coefficient 0.32, p=0.008), between ferric Reducing Antioxidant Power (FRAP mmol/l) and transferrin (Spearman’s correlation coefficient

0.38, p=0.001) and ferritin (Spearman’s correlation coefficient 0.52, p=0.000).

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Fig. 3.1.1.2. Scatter plot of correlation of total oxidative capacity (TOC) with iron saturation of transferrin (TS) and iron (Fe)

There was a significant correlation between TOC (mmol/l/l H2O2) and TS (%)

(Spearman’s correlation coefficient 0,352, p=0.006) as well as TOC and Fe (ug/dL) (Spearman’s correlation coefficient 0.346, p=0.007).

3.1.2. Fatty acids of erythrocyte membrane

The results of FA of erythrocyte membrane and erythrocyte antioxidant as well as PI and UI analysis are described in the publication by Kuliaviene

et al 117.

3.2. Experimental set 3.2.1. Acute pancreatitis experiments

The effect of AP on the mitochondria respiration rates of pancreas, kidney, lungs and liver as well as the serum markers and histology after 1, 3, 6, 12, 24, 48 hours after the induction of AP is presented in the publication

by Trumbeckaite el al 119.

3.2.2. Methylene blue experiments

The respiration rates of pancreatic mitochondria 24 hours after AP induction were significantly decreased in AP as well as MB group with both substrates in State 2 and 3 (Fig. 3.2.2.1).

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Fig. 3.2.2.1. Effect of severe acute pancreatitis (AP) and methylene blue (MB) on respiration of rat pancreatic mitochondria

Substrates were 5 mM glutamate + 5 mM malate (Glu/mal) or 15 mM succinate (Succ)

(+2 mM amytal). V2 – the routine respiration rate in the presence of 1 mg/mL of

mitochondria and substrates; V3 – State 3 respiration rate in the presence of 1mM ADP;

V3+Cyt c , State 3 respiration rate in the presence of 32 µM cytochrome c. *p<0.05 compared to control.

The effect of methylene blue on kidney mitochondria is shown in Fig. 3.2.2.2. Our data indicate an increase in mitochondrial State 3 respiration rate (by 62%, p<0.05) and in respiratory control index (RCI, by 30% p=0.088) with mitochondrial Complex I linked substrate glutamate/malate in the group after methylene blue treatment as compared to untreated group. The Complex I activity was increased by two-fold (p<0.05) after methylene blue treatment. Methylene blue slightly increased RCI with Complex II dependent substrate succinate (by 16%, p<0.05), though had no effect on State 3 respiration rate with this substrate.

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Fig. 3.2.2.2. Effect of severe acute pancreatitis (AP) and methylene blue (MB) on respiration of rat kidney mitochondria

A. Substrates were 5 mM glutamate + 5 mM malate (A) or 15 mM succinate

(+ 2 mM amytal). V2 – the routine respiration rate in the presence of 0.5 mg/mL of

mitochondria and substrates; V3 – State 3 respiration rate in the presence of 1mM ADP;

V3+Cyt c , State 3 respiration rate in the presence of 32 µM cytochrome c. (B) Respiration control index was calculated as the ratio of state 3 and state 2. (C) The Complex I activity

was measured spectrophotometrically at 340 nm as described in Methods. *p<0.05 comparing MB to AP group.

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Fig. 3.2.2.3. Amylase and urea in the serum of experimental groups Controls – healthy rats, AP group – rats with acute pancreatitis (isotonic sodium chlorine

prior to acute pancreatitis induction) and MB group -methylene blue prior to acute pancreatitis induction.

A. Amylase (U/L) is significantly elevated in both groups after 24 hours after AP induction. B. Urea (mmol/l) is decreased in both groups after 24 hours after AP induction.

*p<0.001, #p<0.005.

The amylase was significantly increased in both groups with AP com-paring to healthy rats. Urea was significantly decreased 24 hours after the onset of AP in AP group as well as MB group as shown in Fig. 3.2.2.3.

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4. DISCUSSION

4.1. Serum oxidative stress markers

It is known that oxidative stress plays an important role in the patho-genesis of AP 6. However the precise role of changes of redox state during the pathogenesis of AP and its systemic complications is still far from being clear. There are many controversies about the possible mechanisms and value of oxidative stress in the development of pancreatic injury and the systemic complications. More importantly, the possibility to evaluate these processes is complicated. The difficulty lays in the complexity of oxidative and anti-oxidative systems. Thus in the first part of our research we aimed to evaluate the oxidative stress markers in human serum during the early phase of AP and their possible links to other confusing factors.

There are two ways to measure oxidative stress: the free radical activity and the antioxidants. The methods for detecting the free radical activity are too insensitive mainly because free radicals such as superoxide anions and hydroxyl radicals have very short half-lives in biological systems120. It is easier to measure the products arising from an attack by ROS, RNS. There are hundreds of lipid peroxidation products that have stable structure and can be measured53. The big variety of these markers makes it difficult to compare different studies in this field.

The number of different antioxidants in plasma, serum, urine, or other biological samples makes it difficult to measure each antioxidant separately. Most systems are inhibitory assays: a free radical is generated and its response to an endpoint can be observed and quantified. The addition of antioxidants (present in the biological sample) suppresses the response to a certain extent and for a certain time, depending on the antioxidant activity of the sample. In this context, e.g. the FRAP assay uses the reduction of ferric ions to ferrous ions by the reducing constituents of plasma120. The total antioxidant capacity measurements seem to be more promising even taking into account that these markers could be influenced by other factors of the serum like bilirubin, protein and uric acid121.

In our study we chose to measure serum OS parameters reflecting lipid peroxidation process with hydroperoxide markers – ROM and TOC. They reflect the primary peroxidation reactions. In our opinion they can be more accurate for evaluating redox status. For the same reason we chose the antioxidative parameters that reflect total anti-oxidative capacity (FRAP, TAC), not the single antioxidants.

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The participation of oxidative stress in the pathogenesis of AP has been widely studied in animal and in vitro (with pancreatic acinar cells) mo-dels7,122–124. However there are not much human studies evaluating redox status during AP125–128. There are many controversies even of the same OS markers in those that are carried out in AP patients. E.g. SOD have been shown to be increased129 and decreased125 in different studies. Of course the location of the studied markers (pancreatic tissue, erythrocytes or serum) should be taken into account. Thus our study was performed to retrieve additional information to the elucidation of oxidative stress in human platform.

For the antioxidant markers we found that FRAP was elevated and TAC didn’t show significant change. These findings are in accord with the observation of other groups that have clearly shown that healthy people and AP patients have different structures of antioxidant defence. The study of

Sajewicz et al.128 revealed that acute pancreatitis patients have “para-doxically” high concentrations of ascorbic acid and higher total hydroper-oxyl radical trapping potential, that could only attributed to some uniden-tified source of antioxidant protection (including bilirubin, carotenoids, flavonoids, free amino acids, glucose, lipid hydro peroxides, steroids, cho-lesterol, etc.)128,130,131. The same tendency is seen in septic patients.

Ander-sen et al121 found that more severe septic shock patients exhibited higher total hydroperoxyl radical trapping potential, FRAP, vitamin-C, uric acid and bilirubin levels. On the contrary the study of Thareja et al132 found that the antioxidant markers were decreased in AP patients comparing to con-trols and showed a tendency to decrease over time. We hypothesized that difference in the results of our studies lies partly in the difference of met-hods for oxidative damage measurement and the difference of samples that were used for markers of anti-oxidative stress (serum and plasma).

The oxidative stress markers showed unexpected behaviour. We found that ROM and TOC were significantly decreased in all AP groups. To our knowledge there are no such studies that evaluate ROM and TOC during AP. Usually it is stated that the oxidative stress parameters increase in AP patients. Park et al. found that there were higher plasma levels of lipid peroxide and myeloperoxidase among the patients with acute pancreatitis

125

. In another study thiobarbituric acid reactive substances were found to be significantly higher in AP patients as compared to controls moreover they showed a more distinct fall in mild AP and better clinical outcome132. The difference in these findings may lie in the difference of measured para-meters. All studies measured lipid peroxidation, but the chosen biomarkers were not the same. We measured hydroperoxides which are major primary products of lipid peroxidation of PUFA and cholesterol. Other methods

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measure the further products of lipid peroxidation. It should be noted that all the markers can involve artifactual oxidation during analytical processes 53. Furthermore unsaturated FAs, especially polyunsaturated FAs (PUFAs), are liberated from pancreatic necrotic tissues, and increased total serum free FA level is observed during AP74,75. Thus measurement of the lipid peroxidation products could be influenced by these processes in pancreas and may show higher lipid peroxidation in necrotic AP patients. We hypothesize that the more primary lipid peroxidation products which were measured in our study give more information about early OS events during AP and add valuable data to the understanding of pathogenesis of the disease.

The complexity of oxidative – antioxidative system of human body makes it difficult to measure and compare the results of redox state during AP. For this purpose we suggest choosing serum markers that reflect more primary OS products and more complex antioxidative systems taking in mind that even these tests could be influenced by various other players during AP. One of them possibly is iron metabolism.

4.2. Iron in association with oxidative stress

A special attention should be paid to the iron in the process of OS. It is well known that iron has redox properties133 and that the circulation of iron is changed during inflammation90. Still the data about iron and its role during AP is limited to some experimental findings94–97. There is a lack of human data about iron metabolism during AP and its role in the inflam-matory and redox processes during this disease.

We found that iron circulation was disturbed in the AP patients. The total iron was decreased as was transferrin and iron saturation of transferrin, but the ferritin was increased. It is known that iron metabolism is affected by inflammation. The mechanisms of this process are widely studied not only in the neurodegenerative diseases but also in inflammatory diseases of digestive tract, such as nonalcoholic liver disease and inflammatory bowel diseases134–136. Inflammatory interleukins such as IL 6, IL 1β, TNF α and interferon γ are supposed to activate hepcidin, the master regulator of iron homeostasis89. It is known that gene expression of these interleukins in peripheral blood leukocytes is elevated during AP40. According to these findings we hypothesize that inflammatory process could have impact on the iron circulation disturbance during AP, especially the decrease of iron concentration and TS.

As mentioned above iron is essential for heme synthesis in erythrocytes and guarantees the sufficient oxygen transport to the tissues. Thus the decrease of iron during inflammation leads to anemia and the associated

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complications. On the other hand, iron has a potency to accept and donate electrons and form radical species that are participating in the process of OS90. Majority of iron in plasma is bound to transferrin. Unbound iron which does not have the shield in the form of protein has capacity to generate ROS and cause known damage of oxidative stress90. If the iron saturation of transferrin is higher, there is a possibility for more unbound iron to exist and thus the possible ROS generation could be increased.

In our study we found that iron, transferrin and more importantly iron saturation of transferrin was decreased in AP group comparing to controls. The iron saturation of transferrin and iron concentration in the blood was significantly directly associated with the decrease of TOC. We also found that oxidative and anti-oxidative markers that are analyzed by using iron in their methods (ROM and FRAP) were associated with iron metabolism proteins. ROM had positive correlation with transferrin on the contrary anti-oxidative marker – FRAP, was negatively correlated with transferrin. Thus we think that iron is a very important OS player during pathogenesis of AP, is associated with oxidative stress and could have an influence of severity of the disease.

Ferritin is another protein in iron metabolism. In some experimental studies it is suggested that overexpression of different subunits of ferritin reduce the accumulation of ROS in response to oxidant challenge and decreases the cells' sensitivity to oxidative stress137,138. Moreover the down regulation of ferritin subunits cause the enhanced OS leading to cardio-myocyte death in experimental models139. It was shown that cerulein-in-duced AP in rats is accompanied by ferritin degradation, increases labile iron pool and ROS formation, in experimental AP study95. In our study we found that ferritin was significantly elevated in all AP groups and negatively correlated with ROM and positively with FRAP. This suggest that ferritin should be taken in mind while thinking about redox state during AP, but the exact mechanisms and further implication of these findings remain to be elucidated.

Iron metabolism is disturbed and could be associated with inflammatory as well as oxidative processes in the pathogenesis of AP. Not only iron, but iron circulation proteins – transferrin and ferritin have different roles in the redox state. The exact mechanisms and value of these changes remain to be clarified.

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4.3. Fatty acids of erythrocyte membranes

The possibility to assess the effect of oxidative stress on distant organs could add information about the actual importance of these processes during systemic response to AP. To achieve this goal we investigated the FAs of erythrocyte membranes. The phospholipids of the membranes are important target for ROS during oxidative stress damage13. Erythrocytes are supposed to be more or less inactive cells during the inflammation. On the contrary it is shown that the FAs, especially PUFAs, of serum are greatly influenced by necrotic changes in the pancreas and peri-pancreatic tissues and by them-selves can aggravate the course of the disease74–76. Thus we believe that erythrocyte membrane phospholipids can better reflect systemic changes caused by oxidative stress and inflammatory response in patients with AP and add interesting information about oxidative stress pathophysiology during AP.

To our knowledge, no studies examining the FA composition of eryth-rocyte membrane phospholipids during AP have been carried out. We chose to evaluate changes in the FA profile of erythrocyte membrane phosphor-lipids and antioxidant enzymes of erythrocytes in patients with severe and mild AP, in comparison with healthy individuals. Taking into account that alcohol is important etiological factor of AP, and knowing that alcohol consumption by itself can affect the composition of FAs in serum and membranes as well as the oxidative enzymes, we chose to look into changes of FAs of erythrocyte membranes in patients with different AP origin, al-coholic and nonalal-coholic.

In our study, we found that the FA composition of erythrocyte membrane phospholipids was significantly altered during AP compared with controls mainly because of the increased percentages of saturated and monounsa-turated acids, namely palmitic and palmitoleic, and a decreased percentage of PUFAs. Sztefko et al. found that the proportion of saturated and mo-nounsaturated acids was decreased and the proportion of PUFAs was in-creased in the serum levels of free FAs in patients with AP74. An increase in the percentage of PUFAs in the necrotic pancreatic tissue has also been reported75. On the other hand, in severe sepsis, a similar pathology with systemic inflammatory response syndrome, the lower proportions of PUFAs and the greater proportions of monounsaturated FAs in erythrocyte phospholipids have been documented140. These findings suggest that the FA composition of erythrocyte membrane phospholipids may reflect not only the direct events in the pancreas, but also the systemic response syndrome during AP.

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Alcohol consumption can be associated with the higher percentages of saturated and monounsaturated FAs, such as palmitic and oleic acids, and the lower percentages of PUFAs, especially DHAs and arachidonic acid, in serum and membranes77–80. Alcoholics have also been shown to have a disturbed oxidant status of plasma and erythrocyte enzymes141–143. In the study by Khan et al., the authors showed the increased percentages of saturated palmitic and monounsaturated FAs as well as the decreased percentages of some PUFAs in serum of patients with alcohol-induced AP comparing with alcoholic controls81. Moreover, Gabianelli et al. reported that ethanol can have a direct toxic effect on erythrocyte membranes and antioxidant systems of the cells 144. These findings indicate that alcohol may have an impact on the FA composition of erythrocyte membrane phospho-lipids. Thus, the increased percentages of saturated and monounsaturated FAs in our study could partly be explained by etiological factors, most probably alcohol.

To rule out the impact of alcohol and to study the influence of inflam-matory and oxidative processes during AP on the phospholipid composition of erythrocyte membranes, we analyzed patients with AP of nonalcoholic etiology. The PUFAs of cell membranes are precursors for prostaglandins and other lipid mediators of inflammatory process70. Arachidonic acid is the main pro-inflammatory actor. Meanwhile, EPA, DHA, and possibly DPA are precursors for products with anti-inflammatory and pro-resolving func-tions145,146. We found that in the severe AP group, the percentage of pro-inflammatory arachidonic acid was significantly decreased, and in the mild AP group, a decrease in the percentages of anti-inflammatory players (EPA, DHA, and DPA) was seen as compared with controls. It is now thought that saturated FAs could also be involved in the inflammatory process147,148. We also found a significant increase in the percentage of total saturated FAs in the mild but not severe AP group. Erythrocytes are not usually considered to be active players in the inflammatory process, but our study showed that the changes in the percentage of FAs in erythrocyte membrane phosphorlipids were different during mild and severe AP; therefore, we hypothesize that the composition of erythrocyte membrane phospholipids may reflect the inflammatory processes and the severity of the disease.

Oxidative stress plays a central role in the development of pancreatic inflammation and extra pancreatic complications54,149,150. The changes of the FA composition of erythrocyte membrane phospholipids could be affected from “the outside” as PUFAs of erythrocyte membrane phospholipids are extremely sensitive to oxidation13. SOD and GPx are important components of enzymatic antioxidant defense13. This suggests that oxidative stress might

THE ROLE OF OXIDATIVE STRESS AND MITOCHONDRIAL DYSFUNCTION IN THE PATHOGENESIS OF ACUTE PANCREATITIS

Irma Kuliavienė