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Nutrient digestion and absorption during chemotherapy-induced intestinal muscositis
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Fijlstra, Margot
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Nutrient Digestion and absorption during Chemotherapy‐induced intestinal mucositis In the rat Margot Fijlstra The research described in this thesis was conducted at the Department of Pediatrics, Beatrix Children’s Hosptial, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands, and was financially supported by unrestricted research grants from Fonds NutsOhra (onderzoeksproject SNO‐T‐0602‐49) and KiKa Kinderen Kankervrij (onderzoeksproject 88). The author gratefully acknowledges the financial support for printing this thesis of: Cover illustrations:
Hematoxylin and eosin (H&E) staining of the rat jejunum Background: after treatment with the chemotherapeutic agent methotrexate (MTX) Superimposed: under normal conditions Margot Fijlstra Design and layout:
Rob van den Sigtenhorst
Printed by: Wöhrmann Print Service, Zutphen
ISBN: 978‐90‐367‐5825‐3 (printed)
978‐90‐367‐5824‐6 (digital)
© 2012 Margot Fijlstra All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means without permission of the author and the publisher(s) holding the copyrights of the articles. Nutrient digestion and absorption during chemotherapy‐induced intestinal mucositis in the rat Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus, dr. E. Sterken, in het openbaar te verdedigen op woensdag 21 november 2012 om 16.15 uur door Margot Fijlstra geboren op 10 januari 1980 te IJlst Promotores: Copromotor: Beoordelingscommissie:
Prof. dr. E.H.H.M. Rings
Prof. dr. H.J. Verkade
Prof. dr. E.S.J.M. de Bont
Dr. W.J.E. Tissing
Prof. dr. J.B. van Goudoever
Prof. dr. A.K. Groen
Prof. dr. W.A. Kamps
We are causing animals to be born, causing them to live through a variety of unusual experiences, and causing them to die. This is a form of power that cannot be taken lightly. Today is an opportunity to acknowledge the animals’ role in what we do. To acknowledge that without them, our research and teaching would be fundamentally altered. To thank the animals seems logically inappropriate because their contribution was taken, not given. Yet, I am grateful for, and even dependent upon, their role. From ‘The Gathering to Reflect on the Use of Animals in Research and Teaching’ at the University of Guelph (Ontario, Canada) in 1993 by O’Neill, Taylor and Davis In tribute to research animals, whose contributions have saved millions of human and animal lives and reduced suffering worldwide From a plaque displayed in an animal facility at Merck Research Laboratories (Rahway, New Jersey) Voor Pa, Ma Wen, Ruud en Lex Paranimfen: Annebet van der Meulen
Mariëtte van der Wulp
CONTENTS CHAPTER 1
General introduction, aims and outline of the thesis
CHAPTER 2
Lactose maldigestion during methotrexate‐induced gastrointestinal mucositis in a rat model CHAPTER 3
Continuous enteral administration can overcome the limited capacity to absorb glucose in rats with methotrexate‐induced gastrointestinal mucositis CHAPTER 4
Reduced absorption of long‐chain fatty acids during methotrexate‐induced gastrointestinal mucositis in the rat CHAPTER 5
Continuous enteral administration can enable normal amino acid absorption in rats with methotrexate‐induced gastrointestinal mucositis CHAPTER 6
Parenteral feeding during methotrexate‐induced gastrointestinal mucositis prevents weight loss in the rat CHAPTER 7
Systematic review of agents for the management of gastrointestinal mucositis in cancer patients CHAPTER 8
Summary and general discussion, conclusions and future perspectives
CHAPTER 9
Nederlandse samenvatting
APPENDICES
Dankwoord – Acknowledgements
Affiliations of Co‐Authors
Publications
Biografie 1 15 37 53 71 93 103 127 141 155 156 166 168 169 CHAPTER 1 GENERAL INTRODUCTION, AIMS AND OUTLINE OF THE THESIS
Chapter 1 GENERAL INTRODUCTION 1 Mucositis Mucositis is a severe side effect of chemotherapy and radiotherapy, affecting both children and adults. It is defined as damage to the mucous membranes throughout the entire alimentary tract caused by anti‐cancer treatment [1]. Of all the side effects that can result from chemotherapy, mucositis is mentioned by patients as the most debilitating one [2]. Mucositis is often subdivided into ‘oral mucositis’ and ‘gastrointestinal (GI) mucositis’. Although aspects of GI mucositis have been studied in all segments of the lower alimentary tract, investigations have been focussing on the small intestine [1]. Oral mucositis is visible in the oropharynx as highly red, easily bleeding tissue, with or without the presence of ulcers. Patients with oral mucositis suffer from pain and dysphagia [1]. GI mucositis can only be visualized when a biopsy of the mucosa is taken via endoscopy, which is rather invasive. Patients with GI mucositis suffer from anorexia, nausea, vomiting, abdominal pain, diarrhea and weight loss [1, 3]. Oral and GI mucositis do not always coincide. Research conducted on mucositis mainly focuses on oral mucositis in adult patients. Less attention is paid to GI mucositis, especially in children, although GI mucositis forms a major complication in patients [4, 5]. The pathobiology of mucositis Mucositis is a transient condition, involving 5 (artificially determined) consecutive phases according to Sonis [6, 7]. Although Sonis’ model describes the development of oral mucositis, the pathobiology of GI mucositis is thought to be similar. However, local differences due to specialized differentiation cause both morphological and functional differences [1]. 1. Initiation. Upon anti‐cancer treatment, both DNA and non‐DNA damage occur. DNA strand breaks cause direct injury in cells of the basal epithelium (mucosa) as well as in the submucosa. At the same time, reactive oxygen species (ROS) are formed, starting downstream biological events. 2. Primary damage response. DNA and non‐DNA damage (including ROS) initiate a cascade of events. Several transduction pathways are activated, activating transcription factors like protein 53 (p53) and nuclear factor‐kappaB (NF‐κB). As a result, pro‐inflammatory cytokines like tumor necrosis factor alpha and interleukins IL‐1β and IL‐6 are produced, resulting in basal‐cell death and injury. Furthermore, hydrolyzation of the cell‐membrane lipid sphingomyelin can lead to an increase of ceramide, causing apoptosis. Via activation of activator protein 1 (AP‐1), the secretion of metalloproteinases (MMPs) is stimulated, thereby damaging fibroblasts within the submucosa. 2 | 169 General Introduction, Aims and Outline of the Thesis 3. Signal amplification. Upon initial activation of transcription factors, genes get upregulated and biologically active proteins accumulate and target the submucosa. Apart from tissue damage, pro‐inflammatory cytokines cause a positive feed‐back loop which amplifies the primary damage that is initiated by chemotherapy and radiation. 4. Ulceration. This is the most debilitating phase for patients, causing the symptoms of mucositis. The mucosal integrity gets lost, causing painful lesions that are prone to bacterial colonization. These portals of entry can cause bacteremia and sepsis especially in neutropenic cancer patients. Cell‐wall products from bacteria can penetrate the submucosa, thereby stimulating the formation of more pro‐inflammatory cytokines by mononuclear cells. This process is thought to promote the expression of pro‐apoptotic genes and enables tissue injury. Via chemotaxis, inflammatory cells migrate to the lesion where they cause local inflammation. 5. Healing. Mostly, mucositis is an acute disease that resolves by itself shortly after anti‐cancer treatment ends. Healing of the mucosa is thought to be roughly similar to healing of other types of mucosal injury. However, the sequence of events that leads to mucositis probably influences the healing process. Signals from the submucosa influence the rate of epithelial cell migration and proliferation, and the differentiation of healing tissue. Factors of influence on tissue behavior are probably the type of anti‐
cancer treatment (chemotherapy versus radiation), the selected agents and the dose and timing of therapy. In Sonis’ model, the resident intestinal microbiota hardly play a role in the pathogenesis of mucositis. However, recent research has shown that anti‐cancer treatment is associated with a decrease in the number of anaerobic bacteria and a decrease in microbial diversity [8, 9]. Moreover, these changes in intestinal microbiota coincide in time with an increase in the severity of mucositis. Van Vliet et al. hypothesized that the deregulation of microbial homeostasis upon chemotherapy treatment causes decreased microbial protection of enterocytes against harmful stimuli. They suggest that the intestinal commensal bacteria could influence all phases of Sonis’ mucositis model [10]. Future research on this topic is needed. In patients, mucositis presents with clinical symptoms as early as day 3‐5 after starting chemotherapy with a peak around day 7‐14. In mice and rat models, mucositis follows a similar pattern over a shorter time course [1]. Mucositis and the small intestine Since many kinds of anti‐cancer treatment kill rapidly dividing cells, the GI tract is highly sensitive to these treatments, especially the small intestine. The small intestine can be divided into 3 regions: the duodenum (proximal part), jejunum (middle part) and ileum (distal part). There are morphological and functional differences along the 3 | 169 1 Chapter 1 1 proximal‐to‐distal axis. For instance, towards the distal end of the small intestine, the length of villi as well as the proliferation rate in crypts decreases [11‐13]. The mucosa of the small intestine consists of 3 layers: the epithelium (innermost layer, facing the intestinal lumen), the lamina propria mucosae (connective tissue) and the muscularis mucosae (smooth muscle) [14]. Small intestinal epithelium is made of a monolayer of cells with numerous invaginations (crypts of Lieberkühn) and finger‐like protrusions (villi). Figure 1 shows a longitudinal section of the rat jejunal mucosa with its characteristic crypt‐villus organization under normal conditions and after treatment with the chemotherapeutic agent methotrexate (MTX). Figure 1. Hematoxylin and eosin staining of the rat jejunum (Fijlstra).
The small intestinal epithelium forms a highly folded structure (plicae of Kerckring) that separates the exterior (intestinal lumen) from the interior. It forms a highly selective barrier that prevents the entrance of toxins and noxes into the body and at the same time enables nutrient digestion and absorption. However, upon mucositis, pathogenic bacteria can invade the host and cause severe infections [8]. Among mammalian tissues, the small intestinal epithelium has one of the most rapid cell turnover rates. Stem cells in the lower half of the crypts give rise to daughter cells, thereby producing all the cells of the epithelium. Newly produced cells migrate out of the crypts up to the villus or migrate downward into the base of the crypts and reside under or between the stem cells. During migration up to the villus, most cells differentiate into functional enterocytes (±88%, facilitating digestion and absorption of nutrients), Goblet cells (±4%, production of mucus) and entero‐endocrine cells 4 | 169 General Introduction, Aims and Outline of the Thesis (±0.5%, production of hormones) [15]. Cells migrating into the crypts form Paneth cells (±7,5%, production of defensins that are important for immunity and host defense, and providing a niche for intestinal stem cells [16]). Migration takes about 3 days in rodents and 5 in humans. Within a few days, cells are deleted from the villus tips by apoptosis or shedding into the lumen [17]. Stem cells in the crypts are capable of proliferation (giving progeny to all epithelial cells) and allow regeneration of tissue after injury [18]. They are organized in a hierarchic manner and 3 distinct categories can be distinguished. The actual stem cells, that are the least differentiated ones, are very sensitive to DNA damage and can’t repair such damage. The second category is formed by clonogenic or daughter cells, derived after division of actual stem cells and becoming dividing transit cells that ultimately differentiate. However, in an early stage, they can repair their DNA damage and retain stem cell properties [19]. These cells may repopulate the crypt when actual stem cells are killed by chemotherapy or radiation. A third category is also formed by clonogenic or daughter cells, but is only recruited when the level of damage increases and the first 2 stem cell types have been killed, to ensure crypt survival [18]. Nutrient digestion and absorption One of the major functions of the intestinal mucosa is nutrient digestion and absorption [14]. Enterocytes, which are highly specialized and polarized, are responsible for the degradation and absorption of the major food constituents (i.e. carbohydrates, fats and proteins) after ingestion [14]. This process is facilitated by several enzymes and transporters that are present in the highly folded apical membrane domain of villus enterocytes, named the brush border. Intracellular binding proteins are needed for the transport of specific substrates from the apical to the basolateral membrane of enterocytes. At the basolateral side, these substrates are transported to the blood (carbohydrates and proteins) or lymph (fat). Apart from transcellular transport by enterocytes, carbohydrates and proteins can also be absorbed via paracellular transport [20]. Carbohydrates. The digestion of dietary polysaccharides starts with salivary and pancreatic amylase. Then, resulting disaccharides need to be digested by glycohydrolizing enzymes (i.e. lactase, sucrase, isomaltase and maltase) that are present on the epithelial brush border, before absorption of their derived monosaccharides takes place. Absorption of monosaccharides glucose, galactose and fructose occurs by active and passive transport across the epithelial border by Sodium‐dependent Glucose Transporter 1 (SGLT1), Glucose Transporter 2 (GLUT2) and Sodium‐dependent Glucose Transporter 5 (SGLT5) [21]. Fats. Dietary lipids undergo a number of intraluminal physicochemical alterations (emulsification of bulk fat, lipolysis of triglycerides into di‐ and monoacylglycerol and unesterified or free fatty acids (medium‐ or long‐chain), solubilization of lipolytic 5 | 169 1 Chapter 1 1 products to form mixed micelles) before lipolytic products are translocated across the enterocyte membrane [22]. After translocation, absorbed fatty acids undergo reacylation and assembly into chylomicrons before they end up in mesenteric lymph and finally in the blood [23, 24]. The exact mechanism of fatty acid uptake from the intestinal lumen by enterocytes is still a matter of debate [22, 24]. Proteins. After protein digestion by gastric (i.e. pepsin) and pancreatic (i.e. trypsin, chymotrypsin and carboxypeptidases) enzymes, the resulting oligopeptides are hydrolyzed by peptidases on the brush border membrane. Then, the resulting small peptides (tri‐ and dipeptides) and amino acids are absorbed by enterocytes via highly regulated transporter systems that are present in their apical and basolateral membrane. These systems are defined by the kinetic properties of the specific amino acids they transport; i.e. neutral, cationic or anionic. A unique feature of intestinal enterocytes is that they do not only absorb amino acids directly from the lumen by their apical membrane: they can also take up amino acids from the mesenteric arterial circulation by their basolateral membrane after such amino acids have become systemically available [25‐28]. A number of studies showed that protein‐ and mRNA expression of brush border enzymes and transporters involved in nutrient digestion and absorption are decreased during mucositis, suggesting maldigestion and malabsorption [29‐31]. Also, a few functional digestion and absorption studies during mucositis have been performed [32, 33]. However, the way mucositis functionally affects nutrient digestion and absorption is thus far not well understood, and is the focus of this thesis. The clinical burden of mucositis The incidence of GI mucositis is not exactly known because accurate evaluation by intestinal biopsies is problematic in patients. Therefore, it is scored by more subjective symptoms in clinic, which are not very accurate [1]. With chemotherapy, 40‐100% of patients report GI mucositis, depending on the chemotherapeutic agent that is used and the given dose [1]. Little is known about the incidence in children. However, mucositis seems to be observed more frequently in children with cancer than in adults, probably because of a higher mitotic rate of the GI mucosa in children [5]. In children with acute myeloid leukemia (receiving high doses of chemotherapy), mucositis was found to be present in 55% of chemotherapy cycles [34]. The complications of mucositis (i.e. anorexia, diarrhea, weight loss etc.) are associated with an increased use of injectable analgesics, nutritional problems and longer hospitalizations [1]. Moreover, since mucositis and its associated complications lead to a dose‐reduction of chemotherapy, mucositis compromises overall survival in cancer patients [35]. 6 | 169 General Introduction, Aims and Outline of the Thesis Mucositis scoring systems Mucositis is typically scored via rather subjective symptoms like pain and diarrhea as described by the ‘Common Terminology Criteria for Adverse Events’ from the National Cancer Institute (NCI) [34, 36]. However, these criteria were never designed to score mucositis on a day to day basis, and have not been validated in children. Moreover, symptoms of GI mucositis correlate poorly with the severity of mucositis, especially in young children who are less capable of localizing pain and are often incontinent for feces due to their developmental stage [34]. Thus, a more objective, easy measurable parameter to score GI mucositis is needed, in order to diagnose this (sometimes subclinical) disease and to offer patients optimal treatment. Diverse parameters reflecting inflammation, loss of enterocytes and intestinal permeability have been tested to score mucositis [34]. Of these, plasma citrulline (a nonprotein amino acid made by enterocytes [37]) was found to be a promising marker [34, 38‐40]. The value of plasma citrulline as an objective marker for the level of mucositis and the respective intestinal function is another focus of this thesis. Methotrexate (MTX) MTX is a folate antagonist which is widely used in adult and pediatric cancer patients, alone or in combination with other chemotherapeutic agents [1, 4, 41]. After entering the cell, MTX inhibits the enzyme dihydrofolate reductase (DHFR, catalyzing the conversion of dihydrofolate to tetrahydrofolate). Thereby, it interferes with folate synthesis and indirectly inhibits the synthesis of thymidine monophosphate which is a nucleotide (pyrimidine) required for DNA synthesis. In addition, MTX, its metabolites and folate byproducts that are formed during binding of MTX to DHFR can also directly inhibit folate‐dependant enzymes of nucleotide (pyrimidine and purine) synthesis. As a result, MTX leads to the inhibition of DNA synthesis, and subsequently to the inhibition of RNA and protein synthesis. MTX is active during the S‐phase of the cell cycle (the synthesis phase during which DNA is replicated), and therefore has a large toxic effect on rapidly dividing cells such as malignant cells and cells of the GI mucosa [42, 43]. The treatment of mucositis Well‐designed studies regarding treatment regimens for GI mucositis are scarce due to the relative inaccessibility of the intestine and the obvious difficulty in obtaining biopsies at multiple time points after cytotoxic therapy. Nevertheless, evidence‐based guidelines for the prevention and treatment of GI mucositis have been formed by the Mucositis Study Group of the Multinational Association of Supportive Care and Cancer/International Society for Oral Oncology (MASCC/ISOO), using symptoms of mucositis as clinical endpoints [1, 3, 44]. Guidelines for patients with chemotherapy‐induced mucositis (+/‐ radiotherapy) recommend basic bowel care, including the maintenance of adequate hydration. Also, consideration should be given to the potential for transient lactose intolerance and the presence of bacterial 7 | 169 1 Chapter 1 1 pathogens. When loperamide fails to control chemotherapy‐induced diarrhea, octreotide (>100 µg subcutaneously, twice daily) is recommended [3]. AIMS AND OUTLINE OF THE THESIS As stated earlier, patients with mucositis suffer from weight loss, which is associated with a reduced overall survival in cancer patients [35]. Weight loss during mucositis seems primarily the result of a reduced food intake [45], which suggests that (force‐) feeding might be able to prevent weight loss during mucositis. Apart from a reduced food intake, alterations in nutrient digestion and absorption and energy metabolism might also play a role. There are indications that nutritional support might not only improve the nutritional state, but also accelerate recuperation and increase survival of mucositis patients [46‐49]. It is unknown how to optimally feed patients with mucositis, because their capacity to digest and absorb nutrients is hardly known. Normally, enteral nutrition, which is the physiological way of feeding, is preferred to total parenteral nutrition (TPN) because the latter carries an increased risk of infection and, upon prolonged administration, may cause liver disease [50, 51]. However, when the absorptive function of the intestine is compromised, TPN offers a useful feeding alternative. In contrast to pediatric patients, adult patients with mucositis regularly receive TPN [52]. Both pediatric and adult patients with mucositis sometimes receive enteral tube feeding, although there is no consensus about the optimal mode of enteral feeding (bolus versus continuous), or the composition of enteral formulas. We aimed to determine the capacity to digest and absorb nutrients during GI mucositis, to ultimately design a rational feeding strategy for mucositis patients. Accurate evaluation of mucositis via intestinal biopsies is rather invasive and potentially dangerous in patients because of the risk for infection and intestinal perforation. Also, there is no objective, easy measurable parameter to score GI mucositis in patients. Therefore, we chose to determine nutrient digestion and absorption in a chemotherapy‐induced mucositis rat model. Since plasma citrulline seems to be a promising marker for mucositis in patients [34, 38‐40], we also aimed to determine the value of plasma citrulline as an objective marker for the level of GI mucositis and the respective intestinal function, in a rat model. In Chapter 2, we describe the clinical and histological characteristics of the MTX‐induced mucositis rat model that we developed in our lab. Regarding nutrient digestion and absorption in this model, we first focused on carbohydrates because of their major role in dietary energy supply. Especially lactose is an important carbohydrate in breast milk and Western pediatric diets and formulas. During mucositis, we determined lactose digestion and absorption of its derivative glucose by 8 | 169 General Introduction, Aims and Outline of the Thesis using stable isotope labeled lactose and glucose in trace amounts. Enzyme activity and/or expression of glycohydrolases (lactase, sucrose, isomaltase and maltase) and epithelial glucose transporters (SGLT1, GLUT2 and GLUT5) were also determined. Furthermore, we describe the value of plasma citrulline as an objective marker for mucositis and lactose digestion/glucose absorption during mucositis. To determine whether glucose could be a useful source of energy during mucositis, we performed a follow‐up experiment. As described in Chapter 3, we determined the quantitative capacity to absorb glucose in rats with mucositis, relative to that in controls. Therefore, we administered a physiologically relevant amount (meal size) of stable isotope labelled glucose as a bolus by oral gavage (resembling the physiological situation of consuming meals [53]) or continuously by intraduodenal infusion (improving nutrient absorption during another form of intestinal failure; short bowel syndrome [54]). We also describe the value of plasma citrulline as a marker for glucose absorption during mucositis. Since long‐chain fatty acids serve several important functions in the body, and provide twice as much energy as carbohydrates and proteins on weight basis, we next determined the absorptive capacity of long‐chain fatty acids during mucositis, as described in Chapter 4. Therefore, rats with and without mucositis received a physiologically relevant amount (meal size) of fat containing stable isotope labeled palmitic acid and linoleic acid, either as a bolus by oral gavage or continuously by intraduodenal infusion. Furthermore, we assessed whether either plasma citrulline or the presence or absence of diarrhea would be a better marker for the long‐chain fatty acid absorption capacity during mucositis. There are indications that intestinal absorption of amino acids might be intact during mucositis [33], in contrast to absorption of di‐ and tripeptides [55]. Therefore, we at last aimed to determine the capacity to absorb enterally administered amino acids during mucositis as described in Chapter 5. After rats with and without MTX‐induced mucositis received a physiologically relevant amount (meal size) of stable isotope labeled amino acids (leucine, lysine, phenylalanine, threonine and methionine) via continuous intraduodenal infusion, we determined the plasma availability of amino acids, their utilization for protein synthesis, and the preferential side of the intestine for amino acid uptake. We also describe the value of plasma citrulline as a marker for amino acid absorption during mucositis. Based on our findings on nutrient digestion and absorption during mucositis in the mucositis rat model, we finally determined the effects of 4 different (par)enteral feeding strategies during mucositis on body weight, as described in Chapter 6. Rats with MTX‐induced mucositis continued ad libitum purified diet (AIN‐93G, strategy 1), received continuous enteral force‐feeding with glucose and amino acids (Nutriflex®, strategy 2) or with standard tube‐feeding (Nutrini®, strategy 3), or received standard 9 | 169 1 Chapter 1 1 parenteral feeding (NuTRIflex® Lipid, strategy 4) for 3 days. Control rats continued ad libitum purified diet. We also describe the effects of these feeding strategies on intestinal recovery from mucositis, as measured by plasma citrulline concentration and jejunal histology. Although significant progress has been made in understanding the pathobiology of GI mucositis, progress is difficult due to the relative inaccessibility of the small and large intestine. Because of the difficulty in obtaining biopsies at multiple time points after cytotoxic therapy, new agents for the management of mucositis are being tested in patients by using clinical symptoms of mucositis as endpoints. 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Tissing CHAPTER 2 LACTOSE MALDIGESTION DURING METHOTREXATE‐INDUCED GASTROINTESTINAL MUCOSITIS IN A RAT MODEL Chapter 2 ABSTRACT 2 Background Patients with chemotherapy‐induced gastrointestinal mucositis suffer from anorexia, diarrhea and stomach pain, often causing weight loss and malnutrition. When the intestinal function during mucositis would be known, a rational feeding strategy might improve the nutritional state, accelerate recuperation and increase survival of mucositis patients. Methods We developed a methotrexate (MTX)‐induced mucositis rat model to study nutrient digestion and absorption. To determine lactose digestion and absorption of its derivative glucose during mucositis, we injected Wistar rats intravenously with MTX (60 mg/kg) or NaCl 0.9% (controls). Four days later, we orally administered trace amounts of [1‐13C]lactose and [U‐13C]glucose and quantified appearance of labeled glucose in the blood for 3 h. Finally, we determined plasma citrulline level and harvested the small intestine to assess histology, myeloperoxidase level, glycohydrolase activity, immunohistochemical protein and mRNA expression. Results MTX‐treated rats showed profound villus atrophy and epithelial damage. During the experimental period, the absorption of lactose‐derived [1‐13C]glucose was 4.2‐fold decreased in MTX‐treated rats, as compared with controls (p<0.01). Lactose‐derived [1‐13C]glucose absorption correlated strongly with villus length (rho=0.86, p<0.001) and with plasma citrulline level (rho=0.81, p<0.001). MTX treatment decreased jejunal lactase activity (19.5‐fold, p<0.01), immunohistochemical protein‐ and mRNA expression (39.7‐fold, p<0.01), as compared with controls. Interestingly, MTX treatment did not affect the absorption of [U‐13C]glucose during the experimental period. Conclusions We conclude that lactose digestion is severely decreased during mucositis while glucose absorption is still intact, when supplied in trace amounts. Plasma citrulline level might be a useful objective, noninvasive marker for lactose maldigestion during mucositis in clinic. INTRODUCTION Gastrointestinal mucositis (further referred to as ‘mucositis’) is a severe and debilitating side effect of chemotherapy, especially in children [23, 30]. Mucositis is a transient condition that consists of different stages of inflammation and loss of enterocytes, ending with spontaneous healing of the mucosa [28, 29]. Since accurate evaluation of mucositis by intestinal biopsies is problematic in patients, mucositis is primarily diagnosed by more subjective symptoms in clinic [30]. With chemotherapy, 40‐100% of patients report symptoms of mucositis, depending on the chemotherapeutic agent that is used and the given dose per cycle [30]. In children with acute myeloid leukemia, who receive multiple high doses of different chemotherapeutic agents, mucositis was found to be present in 55% of chemotherapy cycles [38]. There is a lack of objective, noninvasive markers for mucositis [30, 38], albeit recently, we and others suggested plasma citrulline level to be a good marker [3, 21, 22, 38]. Citrulline is a nonprotein amino acid, made by enterocytes. Since 16 | 169 Lactose Maldigestion during Methotrexate‐induced Mucositis plasma citrulline represents functional enterocyte mass, reduced citrulline levels during mucositis represent recduced enterocyte mass [6]. Patients with mucositis suffer from anorexia, diarrhea and stomach pain, often leading to weight loss and malnutrition [16]. These complications of mucositis are associated with an increased use of injectable analgesics, nutritional problems and longer hospitalizations [30]. Moreover, since mucositis and its associated complications lead to a dose reduction of chemotherapy, mucositis compromises overall survival in cancer patients [9]. Mucositis is histologically characterized by villus atrophy, enterocyte damage and infiltration of inflammatory cells [30‐32]. Although these histological changes suggest loss of epithelial function, the digestive and absorptive capacity of enterocytes during mucositis is still not known. A number of studies showed that protein and mRNA expression of enzymes and transporters involved in nutrient absorption are decreased during mucositis, indicating maldigestion and malabsorption [8, 31, 40]. However, only a few functional digestion and absorption studies during mucositis have been performed [14]. Up to now there is still no rational feeding strategy for mucositis patients. Directed nutritional support might actually improve the nutritional state, accelerate recuperation and increase survival of mucositis patients [2, 17, 24, 27]. We chose to determine nutrient digestion and absorption in a methotrexate (MTX) induced mucositis rat model. Our ultimate objective is to design a more rational feeding strategy for mucositis patients. We focus on carbohydrate digestion and absorption because of its major role in dietary energy supply, and started with lactose. Lactose is an important carbohydrate in Western pediatric diets and formulas [26]. It is a disaccharide that has to be digested by the glycohydrolazing enzyme lactase into the monosaccharides glucose and galactose before absorption of these monosaccharides takes place [33]. Absorption of glucose and galactose occurs by active and passive transport across the epithelial border by Sodium‐dependant Glucose Transporter 1 (SGLT1) and Glucose Transporter 2 (GLUT2) respectively [33]. Both the enzyme lactase and the transporters SGLT1 and GLUT 2 are normally present in the brush border of enterocytes. In this study, we aim to determine lactose digestion and absorption of its derivative glucose in our mucositis rat model, by using stable isotope labeled [1‐13C]lactose and [U‐13C]glucose. We hypothesize that both digestion and absorption of these carbohydrates is decreased during mucositis. 17 | 169 2 Chapter 2 MATERIALS AND METHODS 2 Rats and housing Male Wistar outbred rats (4 wk old, 95‐105 g) were obtained from Harlan (Horst, the Netherlands). Rats were individually housed in plexiglass cages (42.5 x 26.6 x 18.5 cm) on a layer of wood shavings under controlled temperature (21  1 C) with a relative humidity of 55  10% and a 12:12‐h light‐dark cycle (lights on 7 A.M.‐7 P.M.). Water and purified diet (AIN‐93G, Harlan Laboratories, Madison, WI, USA) were available ad libitum unless otherwise stated. The experimental protocol was approved by the Ethics Committee for Animal Experiments, Faculty of Medical Sciences, University of Groningen, the Netherlands. Materials Methotrexate was obtained from Pharmachemie Holding B.V. (Haarlem, the Netherlands). [1‐13C]lactose (kindly donated by Dr. R.J. Vonk) and [U‐13C]glucose of 99% isotopic purity were purchased from Isotec (Miamisburg, OH, USA). Experimental procedures The mucositis rat model. We developed an MTX‐induced mucositis rat model to determine nutrient digestion and absorption during mucositis. To find the optimal dosage of MTX and time interval to study digestion and absorption during mucositis, we did some pilot experiments. Pilot experiments were done with different dosages of MTX (30, 45, 60, 90, 120 and 150 mg/kg) via a single intravenous injection in the tail vein under general anesthesia (day 0), based upon other mucositis rat and mouse models [8, 39, 40]. Clinical findings were recorded daily and rats were sacrificed at several days post injection (days 2, 4, 6 and 10) to study small intestinal damage. Based on results from our Pilot studies (see ‘Results’), digestion and absorption experiments were performed with MTX 60 mg/kg, 4 days after injection. Jejunal histology was used as a representative for small intestinal damage. The lactose digestion and glucose absorption test. Two weeks after arrival at the animal facility, rats (6 wk old, 184‐215 g) were injected once intravenously with MTX (60 mg/kg, n=14) or NaCl 0.9% (controls, n=7). Intake of food and water and body weight was recorded daily at 8 A.M. Four days after injection, after an overnight fast (11 P.M. day 3–8 A.M. day 4), rats received a bolus with trace amounts of [1‐13C]lactose (40 mg per rat) and [U‐13C]glucose (20 mg per rat) in 600 l PBS by oral gavage to study both lactose digestion and glucose absorption. Before and at time points 7.5, 15, 30, 45, 60, 90, 120 and 180 min after bolus administration, blood samples were obtained by blood spot technique from the tail tip to measure blood glucose levels and to quantify blood enrichment of lactose‐derived [1‐13C]glucose and of [U‐13C]glucose [35]. Before and at the end of the test, we obtained additional blood samples to measure plasma insulin levels. Samples were centrifuged immediately (10 min at 3,000 rpm) and collected plasma was stored at ‐80°C until further analysis. 18 | 169 Lactose Maldigestion during Methotrexate‐induced Mucositis Euthanazation. At the end of the digestion/absorption test (3 h after bolus administration), rats were euthanized under general anesthesia by obtaining a large blood sample through cardiac puncture for determination of plasma citrulline levels. Blood samples were centrifuged immediately (10 min at 3,000 rpm) and collected plasma was stored at ‐80°C until further analysis. Then, the abdomen was opened via a midline incision and the small intestine was excised, flushed with ice‐cold PBS and divided into three segments of similar size (4.5 cm) [duodenum (proximal small intestine), jejunum (anatomical middle of the small intestine) and ileum (1/6 part proximal from the cecum)]. Smaller parts from each intestinal segment were harvested for assessment of histology and immunohistochemical protein expression (2.5 cm), myeloperoxidase (MPO) levels (0.5 cm), glycohydrolase activity (1.0 cm) and mRNA expression (0.5 cm). Small intestinal parts for histology and protein expression were fixed in formalin (1 cm) or 2% paraformaldehyde (PFA, 1 cm) dissolved in PBS, dehydrated and embedded in paraffin according to standard procedures for (immuno)histochemistry. Extra parts were frozen in isopentane (0.5 cm) and stored at ‐80°C until further use. The intestinal parts for MPO levels, glycohydrolase activity and mRNA expression were immediately frozen in liquid nitrogen and stored at ‐80°C until further analysis. Analytical methods Histological assessment. Hematoxylin and eosin (H&E) staining was done on 3‐m‐thick sections of formalin‐ and PFA‐fixed jejunal segments to assess histology, according to standard procedures. Morphometric analysis was carried out as described previously [20]. Villus and crypt length were measured manually in well‐orientated sections (10 measurements per rat) from digitized images that were evaluated at 10x magnification (1 pixel = 0.397 m) using a calibrated image analysis system (Qwin V3.0, Leica Microsystems Inc.). Goblet cell distribution was analyzed by Alcian‐Blue staining of 3‐m‐thick sections of PFA‐fixed material according to standard procedures. Mucosal MPO levels. Mucosa of frozen jejunal sections was scraped on ice to make tissue homogenates in lysis buffer (reaction tubes: Greiner Bio‐One B.V., Alphen a/d Rijn, the Netherlands). Homogenates were 5‐50 times diluted in dilution buffer before MPO levels were quantitatively measured via a solid bound antibody against MPO as described by the manufacturer (rat MPO ELISA kit, Hycult Biotech, Uden, the Netherlands). Plasma citrulline levels. Plasma citrulline levels were measured in 30 μl plasma at room temperature by using automated ion exchange column chromatography as described before [37, 38]. Blood glucose and plasma insulin levels. Blood glucose levels were measured with a Lifescan EuroFlash glucose meter (Lifescan Benelux, Beerse, Belgium). Plasma insulin 19 | 169 2 Chapter 2 levels were measured in 25 μl plasma via a solid bound antibody against insulin as described by the manufacturer (Rat Insulin Ultrasensitive EIA, Alpco Diagnostics, Salem, NH, USA). 2 [1‐13C]‐ and [U‐13C]glucose absorption. After bolus administration with trace amounts of [1‐13C]lactose and [U‐13C]glucose, blood samples were obtained to quantify blood enrichment of lactose‐derived [1‐13C]glucose and of [U‐13C]glucose. The quantification of lactose‐derived [1‐13C]glucose and [U‐13C]glucose enrichment in blood from blood spots was performed according to Van Dijk et al. [35] by gas chromatography‐mass spectometry (Agilent 5957C Series GC/MSD, Agilent Technologies, Amstelveen, the Netherlands) as has been done before [20]. The calculations for blood glucose kinetics were described recently by Van Dijk et al. [34] and Laskewitz et al. [18]. In short, a single‐pool, first‐order kinetic model was assumed for this test. The mole percent enrichments of mass isotopomers M1 and M6, due to administered [1‐13C]lactose and [U‐13C]glucose respectively, were used to calculate the first order absorption process in an one‐compartment model using SAAM‐II software (version 1.2.1; SAAM Institute, University of Washington, Seattle, WA, USA) [36]. Absorption of lactose‐derived [1‐13C]glucose and [U‐13C]glucose during the experimental period was calculated as area under the curve of [1‐13C]glucose and [U‐13C]glucose concentration (time 0‐180 min) respectively. Mucosal glycohydrolase activity. Mucosa of frozen duodenal, jejunal and ileal sections was scraped on ice to make tissue homogenates in distilled water (reaction tubes: Greiner Bio‐One B.V., Alphen a/d Rijn, the Netherlands). Homogenates were 100‐400 times diluted before glycohydrolase activity levels were measured of lactase, sucrase, isomaltase and maltase as described previously [7; 20]. Activity levels were normalized to protein levels that were measured by the BCA method as described by the manufacturer (BCA protein assay kit, Thermo Fischer Scientific, Inc., Rockford, IL, USA). Immunohistochemical protein expression. Jejunal protein expression of lactase, sucrase‐isomaltase (SI) and SGLT1 was detected using immunohistochemistry according to standard procedures. Lactase was visualized on frozen material (4‐m slides) using a monoclonal mouse anti‐rat lactase antibody (kindly donated by Dr. A. Quaroni) [25; 40], dilution 1:500, as described previously [12]. After incubation with the first antibody (30 min), endogenous peroxidase activity was blocked and slides were incubated with the peroxidase‐conjugated secondary (rabbit anti‐mouse) and tertiary (goat anti‐rabbit) antibodies (Dako North America, Carpinteria, CA, USA). SGLT1 was also visualized on frozen material (4‐m slides) using a commercially available polyclonal goat anti‐mouse antibody (Santa Cruz Biotechnology, Inc., sc‐20584, Santa Cruz, CA, USA), dilution 1:20, with a slightly adapted protocol for immunofluorescent staining. After incubation with the SGLT1 antibody (overnight), 20 | 169 Lactose Maldigestion during Methotrexate‐induced Mucositis slides were incubated with fluorescent secondary (donkey anti‐goat) antibody (Invitrogen Corporation, Alexa Fluor 488, Carlsbad, CA, USA). Slides were covered with fluorescent mounting medium (Dako North America, Carpinteria, CA, USA). SI was visualized on formalin fixed material (3‐m slides) using a polyclonal rabbit anti‐rat SI antibody (kindly donated by Prof. dr. K.Y. Yeh) [42], dilution 1:600, as described previously [40]. Mucosal mRNA expression. Mucosa of frozen duodenal, jejunal and ileal sections was scraped on ice to isolate RNA, synthesize cDNA and subsequently measure mRNA expression of glycohydrolases lactase (Lct) and SI (Si) and glucose transporters SGLT1 (Slc5a1), GLUT2 (Slc2a2) and Glucose Transporter 5 (GLUT5 or Slc2a5). mRNA expression was measured by real‐time PCR as described previously [1] (PCR plates and tubes: Greiner Bio‐One B.V., Alphen a/d Rijn, the Netherlands). Integrity of isolated RNA was checked via gel electrophoresis and disintegrated samples were not included for PCR‐analysis. PCR results were normalized to ‐actin (Actb) mRNA levels. Sequences of the primers and probes are listed in the supplementary data (Table S1). Gene GenBank Forward Primer Reverse Primer Actb NM_03114 (‐actin)
AGC CAT GTA CGT AGC CAT CCA TCT CCG GAG TCC ATC ACA ATG TGT CCC TGT ATG CCT CTG GTC GTA CCA C Lct XM_341115 (lactase)
GCT TCT GCT TCA TAC CAG GTT GA GTG GGA AAA TGT GTC CCA GAT ACT TCC TTT GCC ATC TGC TCT CCA CGC Si (sucrase‐ NM_013061.1 isomaltase)
TGT TTG GGT GAA TGA GTC AGA TG CCC ACC ACT CGA TGG TTT G ACT GTT AAT CCT GGC CAT ACC TCT CCA ATA A TCC TCC TCT CCT GCA TCC AGG TCG TaqMan probe Slc5a1
(SGLT1)
NM_013033 GCT GGA GTC TAC GTA ACA GCA CA GGG CTT CTG CAT CTA TTT CAA TG Slc2a2
(GLUT2)
NM_012879 GCA TCA GCC AGC CTG TGT ATG GCA GCA CAG AGA CCA TCG GCG TTG CAG CTG TGA GTG CCA TCA AC Slc2a5
(GLUT5)
NM_031741.1 CCT GCC TCA CAG CTT GCA TA AAT GAC ACA GAC GAT GCT GAC ATA CTG CAG AAC ACC ATC TCC TGG ATG C Table S1. The PCR primers and TaqMan probes. Depicted genes are stated in symbols according to the official gene nomenclature, their corresponding commonly used names are placed in parentheses. Statistical analysis Statistical analysis was performed using the Mann‐Whitney U‐test (SPSS 16.0 for Windows, Chicago, IL, USA). Values represent medians and first to third quartiles (Figures) or ranges (Tables) for the indicated number of rats per group. All correlations are expressed as nonparametric Spearman correlation coefficient. P values were considered statistically significant if P<0.05. NS means ‘not significant’. 21 | 169 2 Chapter 2 RESULTS 2 Pilot studies To find the optimal dosage of MTX and time interval to study nutrient digestion and absorption during mucositis, we did some pilot experiments. At day 2, MTX‐treated rats (60 mg/kg) showed crypt loss and atrophy while villi still appeared normal. Typical histological signs of mucositis like villus atrophy and blunting, enterocyte damage and infiltration of inflammatory cells were present in most MTX‐treated rats (60 mg/kg) at day 4. By this time, crypts tended to be elongated, a sign of crypt regeneration. From day 6 on, villi of MTX‐treated rats started to recover (results not shown). Histological signs of mucositis were basically the same in the duodenum, jejunum and ileum. Typical clinical signs of mucositis, such as a decreased food intake, weight loss and diarrhea, were present in most MTX‐treated rats (60 mg/kg) from day 2 until day 5, after which rats started to recover (results not shown). Histological and clinical signs of mucositis differed substantially between MTX‐treated rats, dependant on the dosage of MTX. When lower MTX dosages were used (30‐60 mg/kg), some rats developed only mild signs of mucositis. When higher dosages were used (90‐150 mg/kg), all rats developed severe signs of mucostis but mortality increased. For our experiments, we chose the MTX dosage of 60 mg/kg since this caused pronounced mucositis in most rats, without causing mortality. The mucositis rat model during the present experiment Histological findings. We analyzed jejunal sections by H&E staining to demonstrate that MTX‐treated rats developed histological signs of mucositis (Figure 1A‐B). Most MTX‐treated rats showed profound villus atrophy and blunting with irregular, sometimes even vacuolized enterocytes (Figure 1B). Furthermore, there was an influx of inflammatory cells into the stroma of villi (Figure 1B, arrows). However, individual signs of mucositis varied between MTX‐treated rats, with some of them only showing scattered cuboidal shaped enterocytes. Villus length of MTX‐treated rats was 1.8‐fold decreased (p<0.01, Figure 2A) while crypt length was 1.3‐fold increased (p<0.01, Figure 2B), as compared with controls. Goblet cells were evenly distributed along the crypt‐villus axis in controls (Alcian‐Blue staining, Figure 1C). In contrast, Goblet cells were restricted along the villi or solely present on villus tops of MTX‐treated rats (Figure 1D). Our findings indicate that MTX‐treated rats developed histological signs of mucositis, varying from mild to severe. Mucosal MPO levels. We measured MPO levels in scraped mucosa of the jejunum to quantify intestinal inflammation during mucositis (Figure 1B, arrows). MPO levels were 20.3‐fold increased in MTX‐treated rats, as compared with controls (p<0.01, Figure 2C), indicating significant infiltration of neutrophils in the small intestine during mucositis. 22 | 169 Lactose Maldigestion during Methotrexate‐induced Mucositis Figure 1. Histological findings in the mucositis rat model. Hematoxylin and eosin (H&E) staining showing morphology (A‐B), Alcian Blue staining showing Goblet cell distribution (C‐D) and immunohistochemical protein expression of lactase (E‐F), sucrase isomaltase (SI, G‐H) and Sodium dependant Glucose Transporter 1 (SGLT1, I‐J) in the jejunum of NaCl‐ (left panels) and methotrexate (MTX)‐treated rats (right panels). Arrows (B) point out infiltration of inflammatory cells in the stroma of villi. Magnification: 20x. Plasma citrulline levels. We measured plasma citrulline levels to estimate the level of functional enterocyte mass during mucositis [6] and to see whether plasma citrulline can serve as a noninvasive marker for mucositis, as has been suggested before [3, 21, 22, 38]. Citrulline levels were 3.6‐fold decreased in MTX‐treated rats, as compared with controls (p<0.01, Figure 2D), corresponding with significant loss of functional, citrulline producing small intestinal enterocytes during mucositis. Plasma citrulline level correlated with the severity of mucositis as measured by villus length (rho=0.90, p<0.001, Figure S1). Clinical findings. We recorded the intake of food and water and bodyweight daily after injection with NaCl or MTX to see if MTX‐treated rats developed clinical signs of mucositis. Food intake in MTX‐treated rats was decreased on all days post injection (day 0) with a maximum of 1.5‐fold on both day 2 and 3, as compared with controls (p<0.01, Figure 3A). On day 3, food intake of all rats was decreased since rats were fasted before the digestion and absorption test at day 4. Water intake of MTX‐treated rats was decreased from day 2 on with a maximum of 1.9‐fold on day 3, as compared with controls (p<0.01, Figure 23 | 169 2 Chapter 2 2 3B). Body weight was decreased in MTX‐treated rats from day 1 on with a maximum on day 4, as compared with controls (p<0.05, Figure 3C). Compared to the day of injection, MTX‐treated rats lost 2% of initial body weight at day 4 while, in contrast, controls gained 9% of initial body weight by this time (Figure 3C). Other clinical signs of mucositis, like a sick appearance in general and watery diarrhea were present in MTX‐treated rats from day 3 on. Our findings indicate that MTX‐treated rats developed clinical signs of mucositis. Figure 2. Morphometric analysis, myeloperoxidase (MPO) and citrulline levels in the mucositis rat model. Jejunal villus (A) and crypt length (B), mucosal MPO levels (C) and plasma citruline levels (D) in NaCl‐ (○, n=7) and MTX‐treated rats (●, n=14). Dots represent data of individual rats, horizontal lines represent medians of groups. *P<0.01 for NaCl‐ versus MTX‐treated rats. Figure S1. Correlation between plasma citrulline level and villus length in the mucositis rat model. The correlation is shown in NaCl‐ (○, n=7) and MTX‐treated rats (●, n=14). The Spearman correlation (r) and P value is indicated. 24 | 169 Lactose Maldigestion during Methotrexate‐induced Mucositis Figure 3. Clinical findings in the mucositis rat model. Intake of food (A) and water (B) and relative body weight (compared to weight at day 0 which is arbitrarily set at 100%, C) in NaCl‐ (○, n=7) and MTX‐treated rats (●, n=14). Intake is shown until day 3, since rats were fasted from 11 P.M. on because of the digestion/absorption test at day 4. Data represent medians and p25‐p75. #P<0.05 and *P<0.01 for NaCl‐ versus MTX‐
treated rats. The lactose digestion and glucose absorption test Blood glucose and plasma insulin levels. There was hardly a rise in blood glucose after bolus administration, both in MTX‐treated rats and in controls (Figure S2). Plasma insulin levels did not differ between MTX‐treated rats and controls at the start or at the end of the test (results not shown). Lactose digestion and absorption of its derivative [1‐13C]glucose. Blood appearance of lactose‐derived [1‐13C] glucose was determined over a 3‐h period after bolus administration. In controls, [1‐13C]glucose entered the blood glucose pool 11 minutes after bolus administration (tlag, Figure 4A and Table 1). Maximal [1‐13C]glucose concentration (cmax) was reached at 56 min after bolus administration (tmax), and was 0.34 mmol/l. In MTX‐treated rats, [1‐13C]glucose appearance was significantly delayed and decreased, as compared with controls (Figure 4A and Table 1). During the experimental period, the absorption of [1‐13C]glucose was 4.2‐fold decreased in MTX‐treated rats, as compared with controls (p<0.01, Table 1). Absorption correlated with villus length (rho=0.86, p<0.001, Figure 4B) and with plasma citrulline level (rho=0.81, p<0.001, Figure 4C). Our findings indicate that lactose digestion and/or absorption of its derivative glucose is severely decreased during mucositis. 25 | 169 2 Chapter 2 Figure S2.Blood glucose levels during the lactase digestion/glucose absorption test. Levels are shown
in NaCl‐ (○, n=7) and MTX‐treated rats (●, n=14). Dots represent medians and p25‐p75. *P<0.01 for NaCl‐ versus MTX‐treated rats. 2 Lactose‐derived [1‐13C]glucose appearance NaCl (n=7) MTX (n=14) tlag (min) 11 (5‐13) 31 (6‐52) * tmax (min) 56 (54‐82) 144 (63‐175) * 0.34 (0.30‐0.38) 0.09 (0.02‐0.46) * 45 (35‐50) 11 (2‐58) * cmax (mmol/l) AUC (mmol/l∙min) [U‐13C]glucose appearance tlag (min) tmax (min) cmax (mmol/l) AUC (mmol/l∙min) NaCl (n=7) MTX (n=14) 5 (3‐6) 6 (3‐20) 33 (28‐48) 45 (30‐61) # 0.33 (0.26‐0.37) 0.28 (0.19‐0.41) 29 (23‐33) 30 (23‐38) Table 1. Blood appearance of
13
lactose‐derived [1‐ C]glucose
13
and of [U‐ C]glucose during
the lactose digestion/glucose
absorption test. Appearance is
shown after administration
13
13
of the [1‐ C]lactose/[U‐ C]
glucose bolus in NaCl‐ and
MTX‐treated rats. Data belong
to the curves that are plotted in
Figure 4A and D. Tlag is the point
of time where the label enters
the glucose pool, tmax is the
point of time where the
concentration of the label
is maximal and cmax is the
concentration of the label that is
reached at tmax. Data indicate
medians of groups, ranges are
in parentheses. #P<0.05 and
*P<0.01 for NaCl‐ versus MTX‐
treated rats. [U‐13C]Glucose absorption. Blood appearance of [U‐13C]glucose was determined over a 3‐h period after bolus administration. In controls, [U‐13C]glucose entered the glucose pool 5 min after bolus administration (tlag, Figure 4D and Table 1). Maximal [1‐13C]glucose concentration (cmax) was reached at 33 min after bolus administration (tmax), and was 0.33 mmol/l. Interestingly, [U‐13C]‐glucose appearance was slightly delayed (p<0.05) but not decreased in MTX‐treated rats, as compared with controls (Figure 4D and Table 1). MTX treatment did not affect the absorption of [U‐13C]glucose during the experimental period (Table 1). Absorption correlated with the level of mucositis as measured by villus length (rho=0.48, p=0.027, Figure 4E) but did not correlate with plasma citrulline level (rho=0.36, NS, Figure 4F). Our findings indicate that glucose absorption is still intact during mucositis, when given in trace amounts. Therefore, decreased absorption of lactose‐derived [1‐13C]glucose during mucositis indicates disturbed lactose digestion instead of glucose malabsorption, since [U‐13C]glucose is absorbed normally. 26 | 169 Lactose Maldigestion during Methotrexate‐induced Mucositis 2 13
13
Figure 4. Lactose digestion and absorption of glucose (lactose‐derived [1‐ C]glucose and [U‐ C]Glucose) 13
during the lactose digestion/glucose absorption test. Blood appearance of lactose‐derived [1‐ C]glucose 13
(A) and [U‐ C]glucose (D) in NaCl‐ (○, n=7) and MTX‐treated rats (● ▬, n=14) after oral administration of 13
13
the [1‐ C]lactose/[U‐ C]glucose bolus. Dots represent medians and p25‐p75 at 7.5, 15, 30, 45, 60, 90, 120 and 180 min after bolus administration. #P<0.05 and *P<0.01 for NaCl‐ versus MTX‐treated rats. B and C: 13
Correlation between lactose derived [1‐ C]glucose absorption on the one hand and villus length (B) or plasma citrulline level (C) on the other hand, in NaCl‐ (○) and MTX‐treated rats (●). E and F: Correla on 13
between [U‐ C]glucose absorption on the one hand and villus length (E) or plasma citrulline level (F) on the other hand, in NaCl‐ (○) and MTX‐treated rats (●). Spearman correla ons (r) and P values are indicated. 13
13
AUC: area under the curve of [1‐ C]glucose and [U‐ C]glucose concentration (time 0‐180 min, Figure 4A 13
13
and D), representing [1‐ C]glucose absorption and [U‐ C]glucose absorption respectively. 27 | 169 Chapter 2 2 Mucosal glycohydrolase activity We measured mucosal glycohydrolase activity of lactase to investigate whether disturbed lactose digestion during mucositis can be explained by a decreased lactase activity. We also studied activity of glycohydrolases sucrase, isomaltase and maltase. Lactase activity was most abundant in the jejunum of controls and was 19.5‐fold decreased in MTX‐treated rats, as compared with controls (p<0.01, Table 2). As with lactase activity, a decreased jejunal activity of sucrase (13.9‐fold, p<0.05), isomaltase (17.0‐fold, p<0.01) and maltase (9.1‐fold, p<0.01) was found in MTX‐treated rats, as compared with controls (Table 2). Our findings indicate that the hydrolyzing activities of lactase, sucrase, isomaltase and maltase are all severely decreased during mucositis. Disturbed lactose digestion during mucositis can therefore be explained by a decreased lactase activity. Glycohydrolase activity (mol/mg protein/hr) Duodenum Jejunum Ileum NaCl (n=7) MTX (n=14) NaCl (n=7) MTX (n=14) NaCl (n=7) MTX (n=14) Lactase 0.1 (0.1‐0.2) 0.0 (0.0‐0.2) *
1.3 (0.9‐1.6) 0.1 (0.0‐1.1) *
0.2 (0.0‐0.4) 0.0 (0.0‐0.4) Sucrase 7 (6‐9) 0 (0‐8) * 8 (6‐10) 1 (0‐11) # 1 (1‐2) 1 (0‐3) Isomaltase 18 (15‐22) 0 (0‐20) * 41 (32‐49) 2 (0‐38) * 14 (10‐21) 11 (0‐36) Maltase 63 (51‐82) 5 (0‐73) * 102 (80‐113) 11 (2‐103) * 31 (26‐45) 30 (0‐65) Relative mRNA expression (normalized to ‐actin) Duodenum Jejunum Ileum NaCl (n=6) MTX (n=13) NaCl (n=7) MTX (n=12) NaCl (n=7) MTX (n=13) Lactase 0.0 (0.0‐0.0) 0.0 (0.0‐0.1) 8.5 (5.9‐12.6) 0.2 (0.0‐7.3) *
2.0 (0.6‐2.9) 0.4 (0.0‐2.7) # SI 0.8 (0.5‐1.1) 0.8 (0.1‐1.4) 2.2 (1.4‐2.5) 0.2 (0.0‐2.4) #
1.4 (0.9‐1.9) 0.9 (0.1‐1.6) # SGLT1 1.3 (0.7‐1.6) 1.0 (0.3‐1.9) 2.8 (2.3‐3.2) 0.3 (0.1‐2.7) *
0.7 (0.5‐1.2) 0.3 (0.1‐1.4) * GLUT2 1.8 (0.9‐2.1) 1.1 (0.3‐1.8) 2.4 (2.1‐2.8) 0.2 (0.0‐2.6) *
0.8 (0.5‐1.1) 0.4 (0.0‐1.1) # GLUT5 1.0 (0.5‐1.1) 0.9 (0.1‐1.6) 2.2 (1.7‐2.9) 0.2 (0.0‐2.4) *
1.1 (1.0‐2.0) 0.7 (0.0‐1.4) * Table 2. Glycohydrolase activity and relative mRNA expression of glycohydrolases and glucose
transporters in the mucositis rat model. Activity and/or mRNA expression profiles of lactase, sucrase,
isomaltase, maltase, SGLT1, Glucose Transporter 2 (GLUT2) and Glucose Transporter 5 (GLUT5) of NaCl‐ and MTX‐treated rats are shown. Data indicate medians of groups, ranges are in parentheses. #P<0.05 and *P<0.01 for NaCl‐ versus MTX‐treated rats. 28 | 169 Lactose Maldigestion during Methotrexate‐induced Mucositis Immunohistochemical protein expression We studied jejunal immunohistochemical protein expression of lactase and SI to investigate whether a decreased activity of these glycohydrolases during mucositis can be explained by a decreased protein expression. Immunohistochemical protein expression of SGLT1 was studied to investigate whether intact glucose absorption during mucositis can be explained by an intact protein expression of this glucose transporter. Protein expression of lactase (Figure 1 E‐F), SI (Figure 1 G‐H) and SGLT1 (Figure 1 I‐J) was normally present along the brush border of villi in control rats. In contrast, expression was merely present in the remaining villus tops of MTX‐treated rats. Our findings indicate that decreased lactase, sucrase and isomaltase activity during mucositis can be explained by a decreased lactase and SI protein expression. However, intact glucose absorption during mucositis can not be explained by the protein expression of SGLT1, since this was severely decreased. Mucosal mRNA expression We measured mRNA expression of lactase, SI and SGLT1 to investigate whether a decreased protein expression of these glycohydrolases and glucose transporter during mucositis can be explained by a decreased mRNA expression. We also studied expression of GLUT2 and GLUT5. All mRNA expression profiles were most abundant in the jejunum of controls (Table 2). Jejunal expression of lactase and SI was decreased 39.7 and 9.4‐fold respectively in MTX‐treated rats, as compared with controls (p<0.01 and p<0.05 respectively). Jejunal mRNA expression of SGLT1, GLUT2 and GLUT5 was decreased 9.6‐, 10.1‐ and 9.5‐fold respectively in MTX‐treated rats, as compared with controls (both p<0.01). Our findings indicate that a decreased lactase, SI and SGLT1 protein expression during mucositis can be explained by a decreased mRNA expression. DISCUSSION In this study, we aimed to determine lactose digestion and absorption of its derivative glucose during mucositis. We hypothesized that both digestion and absorption of these carbohydrates is decreased during mucositis. Our results show that lactose digestion is severely decreased during mucositis. Interestingly, the absorption of glucose is still intact during mucositis, at least, when supplied in trace amounts. We used an MTX‐induced mucositis rat model to determine lactose digestion and absorption of its derivative glucose. Histology and mucosal MPO level (indicating infiltration of neutrophils) were studied to show that the model really represented mucositis. MTX‐treated rats showed typical histological characteristics of mucositis like blunting of villi with irregular or even vacuolized enterocytes. Goblet cells were depleted and accumulated at villus tops. Crypts of MTX‐treated rats tended to be elongated, which is a sign of crypt regeneration via hyperproliferation and hyperplasia after initial crypt damage caused by MTX [39, 40]. Also, we saw an influx of 29 | 169 2 Chapter 2 2 inflammatory cells in villus stroma and increased mucosal MPO levels during MTX treatment. Besides typical histological characteristics of mucositis, MTX‐treated rats also showed typical clinical characteristics like a decreased intake of food and water, weight loss and diarrhea. These characteristics of mucositis were also found by others [8, 19, 31, 32, 40]. Histological and clinical signs of mucositis differed substantially between MTX‐treated rats. Out of 14 MTX‐treated rats, 3 rats showed minimal histological and clinical signs of mucositis, normal MPO and citrulline levels (Figure 2) and a normal absorption of lactose derived glucose (Figure 4B and C). The variance in observed individual signs of mucositis could be a result of genetic variability between outbred Wistar rats [30]. Also, our mucositis model is based upon a single intravenous injection of MTX, leaving the period of epithelial crypt cell susceptibility to MTX shorter than in models where multiple MTX injections are used [4, 10, 13, 28, 29]. The amount of subsequent crypt loss by apoptosis, crypt atrophy and ultimately villus atrophy [39] therefore differs per MTX‐injected rat. During the experimental period, the absorption of lactose‐derived [1‐13C]glucose was severely decreased in MTX‐treated rats, as compared with controls. In contrast, the absorption of [U‐13C]glucose was still intact in MTX‐treated rats. We therefore concluded that decreased absorption of lactose‐derived glucose during mucositis is a result of disturbed lactose digestion instead of glucose malabsorption. The hydrolysis of the disaccharide lactose into the monosaccharides glucose and galactose by the enzyme lactase must be defective during mucositis. A decreased in vitro lactase enzyme activity, as well as a decreased immunohistochemical protein and mRNA expression of lactase in MTX‐treated rats, as compared with controls, further supported this conclusion. Although others already showed a decreased lactose breath test, lactase activity and lactase protein and mRNA expression during mucositis [8, 14, 31, 40], we are the first to functionally demonstrate that lactose is indeed maldigested during mucositis. Furthermore, the fact that we could confirm lactase activity and expression profiles found in other mucositis studies, demonstrates the correct establishment of a mucositis rat model in our lab using a single injection with MTX. Here, we prove that ‘lactose malabsorption’ during mucositis is a result of defective lactose hydrolysis instead of defective absorption of its derivative glucose. Our findings imply that lactose should be omitted from the diet of mucositis patients since it can not be used as a source of energy. Lactose maldigestion might even exaggerate diarrhea and stomach pain which often is already present during mucositis [11, 26, 41]. We also found a decreased enzyme activity of other glycohydrolases such as sucrase, isomaltase and maltase in MTX‐treated rats, as compared with controls. These findings indicate that all disaccharides, as well as polysaccharides, will propably not be hydrolyzed and its derivatives not be absorbed during mucositis. It therefore 30 | 169 Lactose Maldigestion during Methotrexate‐induced Mucositis seems wise to omit disaccharides and polysaccharides from the diet of patients with mucositis. Because plasma citrulline level was earlier suggested to be a good, noninvasive marker for mucositis [3, 21, 22, 38], we measured plasma citrulline levels in NaCl‐ and MTX‐treated rats. Levels of plasma citrulline, a non‐protein amino acid, were severely decreased in MTX‐treated rats, as compared with controls, corresponding with loss of functional enterocyte mass [6]. In individual rats, plasma citrulline level strongly correlated with the level of mucositis as measured by villus length and with lactose digestion during mucositis. Plasma citrulline level might therefore not only be an objective, non‐invasive marker for the level of mucositis but, more important, for lactose maldigestion during mucositis. It could be a better alternative for the currently used, more subjective ‘National Cancer Institute Common Toxicity Criteria’ [30, 38], as a parameter for gastrointestinal mucositis. Furthermore, plasma citrulline level could be easily used in clinic to adapt the (feeding) strategy of mucositis patients. Because the absorption of [U‐13C]glucose was still intact in MTX‐treated rats, we conclude that glucose transport across the epithelial border must, at least to some extent, still be intact during mucositis. However, immunohistochemical protein and/or mRNA expression of glucose transporters SGLT1 and GLUT2 was decreased in MTX‐treated rats, as compared with controls, as was found by others [8, 31, 40]. It should be noted that the given bolus contained only trace amounts of [U‐13C]glucose and [1‐13C]lactose. Minimal glucose absorption might therefore have been possible via residual transporters on the damaged epithelial membrane, maybe in combination with leakage through damaged tight junctions since mucositis often leads to an increased gut permeability [5, 15]. Whether glucose can be an appropriate source of dietary energy for mucositis patients should be further studied by glucose absorption studies using relevant amounts of glucose. In conclusion, our study shows that lactose digestion is severely decreased during mucositis while glucose absorption is still intact, when supplied in trace amounts. We recommend to omit lactose from the diet of mucositis patients to prevent possible negative side effects of lactose maldigestion, like lactose intolerance. Plasma citrulline level might be a useful objective, noninvasive marker for lactose maldigestion during mucositis in clinic. ACKNOWLEDGEMENTS We thank Dr. R.J. Vonk for kindly providing [1‐13C]lactose. Also, Dr. A. Quaroni and Dr. K.Y. Yeh a gratefully thanked for their donation of lactase and SI antibody respectively. We are grateful to J.F.W. Baller, T. Boer, Dr. F. Stellaard, A.R.H. van der Molen and P.A. Klok for their assistance in our studies. 31 | 169 2 Chapter 2 GRANTS This study was financially supported by an unrestricted research grant from Fonds NutsOhra. 2 REFERENCES 1.
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Yeh KY, Yeh M and Holt PR (1991) Thyroxine and cortisone cooperate to modulate postnatal intestinal enzyme differentiation in the rat. Am J Physiol 260: G371‐G378 2 34 | 169 Lactose Maldigestion during Methotrexate‐induced Mucositis 2 35 | 169 Supportive Care in Cancer 2012 Sep 26; in press M. Fijlstra E.H.H.M. Rings T.H. van Dijk T. Plösch H.J. Verkade W.J.E. Tissing CHAPTER 3 CONTINUOUS ENTERAL ADMINISTRATION CAN OVERCOME THE LIMITED CAPACITY TO ABSORB GLUCOSE IN RATS WITH METHOTREXATE‐INDUCED GASTROINTESTINAL MUCOSITIS Chapter 3 ABSTRACT 3 Background Patients with chemotherapy‐induced gastrointestinal mucositis often suffer from weight loss. It is not well known how to enterally feed mucositis patients, potentially experiencing malabsorption. Recently, we showed in a rat model of methotrexate (MTX)‐induced mucositis that intestinal absorption of glucose in trace amounts is still intact. We now determined the quantitative capacity to absorb glucose in rats with mucositis, relative to controls. Methods We administered a physiologically relevant amount of [1‐13C]glucoseenriched glucose (meal size) as a bolus by oral gavage (2 g/kg once) or continuously by intraduodenal infusion (±1.9 g/(kg∙h) for 5 h) to rats with MTX‐induced mucositis and controls. Blood [1‐13C]glucose concentrations were determined during the experimental period. To calculate the quantitative absorptive capacity, Steele’s one‐compartment model, including simultaneous intravenous infusion of [6,6‐2H2]glucose, was used. After the experiment, jejunal histology and plasma citrulline concentrations were assessed. Results MTX‐induced mucositis was confirmed by a reduction in villus length and plasma citrulline (both ‐57%, relative to controls, P<0.01). When glucose was administered as a bolus, MTX‐treated rats only absorbed 15% of administered glucose, compared with 85% in controls (medians, P<0.01). Upon continuous intraduodenal glucose infusion, the median absorptive capacity for glucose in MTX‐treated rats did not differ from controls (80 versus 93% of administered glucose respectively, P=0.06). However, glucose absorption differed substantially between individual MTX‐treated rats (range 21‐95%), which correlated poorly with villus length (rho=0.54, P=0.030) and plasma citrulline (rho=0.56, P=0.024). Conclusion Continuous enteral administration can almost completely overcome the reduced absorptive capacity for glucose in rats with mucositis. INTRODUCTION Chemotherapy‐induced gastrointestinal mucositis (“mucositis”) is seen in 40‐100% of patients after chemotherapy [1, 2]. Pediatric as well as adult patients with mucositis often experience anorexia, nausea, diarrhea and weight loss [3]. Histologically, small intestinal villus atrophy and loss of enterocytes is seen [2]. Since accurate evaluation of mucositis via invasive intestinal biopsies is problematic, mucositis is often scored by more subjective symptoms like pain and diarrhea [2]. These symptoms correlate poorly with the severity of mucositis, especially in young children who are less capable of localizing pain and are often incontinent for feces due to their developmental stage [4]. Plasma citrulline (a nonprotein amino acid made by enterocytes [5]) has earlier been shown to be a useful marker for mucositis, and for lactose maldigestion during mucositis [4, 6, 7]. 38 | 169 Maintaining Glucose Absorption during Mucositis To date, there is no rational feeding strategy for mucositis patients, although nutritional support might actually improve the nutritional state, accelerate recuperation and increase survival of mucositis patients [8‐11]. Enteral nutrition, which is the physiological way of feeding, is normally preferred to total parenteral nutrition (TPN) because the latter carries a high risk of infection and, upon prolonged administration, may cause liver disease [12‐14]. However, when the absorptive function of the intestine is compromised, TPN offers a useful feeding alternative. Since the capacity to digest and absorb nutrients during mucositis is still largely unknown, we developed a methotrexate (MTX)‐induced mucositis rat model to determine it, and to ultimately design a rational feeding strategy for mucositis patients [6]. Before, using this model, we showed that the digestion of lactose was severely reduced during mucositis. In contrast, trace amounts of glucose were absorbed normally during mucositis [6]. Therefore, we propose that enteral glucose might be a useful source of energy for mucositis patients. Here, we aimed to compare the quantitative capacity to absorb glucose between rats with MTX‐induced mucositis and saline‐treated controls. We administered a physiologically relevant amount of glucose (meal size, meaning a representative percentage of average daily carbohydrate intake in controls) as a bolus by oral gavage, since intermittent bolus administration resembles the physiological situation of consuming meals [15]. We also determined absorption of a physiologically relevant amount of glucose when continuously administered by intraduodenal infusion, since continuous enteral nutrient administration has been shown to improve nutrient absorption during other forms of intestinal failure, like short bowel syndrome [13]. To address whether plasma citrulline levels could function as a surrogate parameter for the glucose absorptive capacity during MTX‐induced mucositis, we related these two parameters in individual rats. MATERIALS AND METHODS Rats and housing For both experiments, young male Wistar outbred rats (3‐4 wk old, 45‐75 g) were obtained from Harlan (Horst, the Netherlands). Rats were individually housed in Plexiglas cages (42.5 x 26.6 x 18.5 cm) on a layer of wood shavings under controlled temperature (21  1 C) with a relative humidity of 55  10% and a 12:12‐h light‐dark cycle (lights on 7:00 A.M.‐7:00 P.M.). Water and purified diet (AIN‐93G [16], Harlan Laboratories, Madison, WI, USA) were available ad libitum unless otherwise stated. The experimental protocol, which resembled the protocol that we used previously [6], was approved by the Ethics Committee for Animal Experiments, Faculty of Medical Sciences, University of Groningen, the Netherlands. 39 | 169 3 Chapter 3 Materials MTX was obtained from Pharmachemie Holding B.V. (Haarlem, the Netherlands). [1‐13C]glucose of 99% isotopic purity was purchased from Sigma Aldrich Chemie B.V. (Zwijndrecht, the Netherlands). [6,6‐2H2]glucose of 98% isotopic purity was purchased from Isotec (Miamisburg, OH, USA). 3 Experimental procedures The mucositis rat model. Rats (n=32, 5 wk old, 2 wk after arrival at the animal facility) were surgically equipped with permanent silicone catheters in the duodenum and /or jugular vein under isoflurane anesthesia, as described previously [17]. One week after surgery, rats (6 wk old, 180‐250 g) were injected once intravenously in the tail vein with MTX (n= 20, 60 mg/kg, to induce mucositis [6]) or with saline 0.9% (n=12, controls) under isoflurane anesthesia. Intake of food (in grams, calculated by the amount of administered food minus the residual food in the cage on the next day), body weight (in grams, using a weighing scale), and diarrhea (present as watery diarrhea or completely absent, as described previously [6]) were recorded daily on clinical records at 8:00 A.M. Four days after injection, when histological and clinical symptoms of MTX‐induced mucositis are most severe [6], the glucose absorption tests were performed (see below). At the end of both experiments (4h after an oral glucose bolus or 7h after starting continuous intraduodenal glucose infusion), rats were killed under isoflurane anesthesia by obtaining a large blood sample through cardiac puncture for determination of plasma citrulline concentrations, followed by cervical dislocation. Blood samples were centrifuged immediately (10 min at 2,000 x g) and collected plasma was stored at ‐80°C until further analysis. Next, the abdomen was opened via a midline incision, and the small intestine was excised and flushed with ice‐cold PBS. Small parts of the duodenum (proximal small intestine), jejunum (anatomical middle of the small intestine) and ileum (1/6 part proximal from the cecum) were collected for histology and fixed in formalin (1 cm) or 2% paraformaldehyde (PFA, 1 cm) dissolved in PBS, dehydrated and embedded in paraffin according to standard procedures for histochemistry. An extra jejunal part (0.5 cm) for myeloperoxidase (MPO) concentrations was collected, immediately frozen in liquid nitrogen and stored at ‐80°C until further analysis. The glucose absorption tests. Similar to our previous study, where absorption of glucose in trace amounts was determined [6], rats were fasted for 9 h on the day of experiment (11:00 P.M. day 3 – 8:00 A.M. day 4). Thereafter, rats received a continuous intravenous infusion of [6,6‐2H2]glucose at a rate of 12.1 µmol/(rat∙h) during the whole experiment to determine the total rate of glucose appearance (RaT), as described previously [18, 19]. To determine the rate of appearance of enterally administered glucose (RaE), one hour after the start of the experiment, rats enterally received a physiologically relevant amount of glucose (meal size, meaning a representative percentage of average daily carbohydrate intake via AIN‐93G in 40 | 169 Maintaining Glucose Absorption during Mucositis saline‐treated controls, i.e. ±12 g carbohydrates/(rat∙day) ≈ ±52 g carbohydrates/ (kg∙day) ≈ ±2.2 g carbohydrates/(kg∙h) [6, 16]). Enteral glucose (i.e. glucose monohydrate) was administered either as a bolus by oral gavage (2.0 g/kg in ±0.8 ml water as described previously [18, 19], ≈ 4% of average daily carbohydrate intake), or as continuous intraduodenal infusion (±1.3 g/(kg∙h) for the first 2h, and ±1.9 g/(kg∙h) for the last 5 h to reach a steady state, ≈ 85% of average hourly carbohydrate intake). An overview of the number of MTX‐ and saline‐treated rats per glucose absorption test (glucose as a bolus or continuously) is stated in Table 1. Continuous glucose infusion rates and doses were chosen to avoid severe hyperglycemia (blood glucose concentrations >30 mmol/L) and signs of drowsiness in MTX‐treated rats, which were experienced in pilot studies that we had executed earlier, upon higher doses of continuous intraduodenal glucose infusion (up to ±2.5 g/(kg∙h), unpublished material). Of the administered glucose, 10% was [1‐13C]glucose. Blood samples were obtained from the tail tip before the start of the experiment and at indicated time points during the experiment on filter paper to quantify the fractional contributions of [6,6‐2H2]glucose and [1‐13C]glucose [18, 19], and to measure blood glucose concentrations using a Lifescan EuroFlash glucose meter (Lifescan Benelux, Beerse, Belgium). Additionally, we obtained blood samples to measure plasma insulin concentrations. Blood samples were centrifuged immediately (10 min at 2,000 x g) and collected plasma was stored at ‐80°C until further analysis. Analytical methods Histological assessment. Hematoxylin and eosin (H&E) staining was performed on 3‐m‐thick sections of formalin‐ and PFA‐fixed small intestinal segments to confirm mucositis, according to standard procedures. Morphometric analysis was carried out on jejunal segments as described previously [6]. Villus length was measured manually by a blinded researcher (MF) in well‐orientated sections (10 measurements per rat) from digitized images that were evaluated at 10x magnification (1 pixel = 0.397 m) using a calibrated image analysis system (Qwin V3.0, Leica Microsystems). Mucosal MPO concentrations. Mucosa of frozen jejunal sections (of rats that received glucose as a bolus) was scraped on ice to make tissue homogenates in lysis buffer (reaction tubes: Greiner Bio‐One B.V., Alphen a/d Rijn, the Netherlands). Homogenates were 5‐50 times diluted in dilution buffer before MPO concentrations (indicating inflammation) were quantitatively measured via a solid bound antibody against MPO as described by the manufacturer (rat MPO ELISA kit, Hycult Biotech, Uden, the Netherlands) and as done previously [6]. Plasma citrulline concentrations. Plasma citrulline concentrations (indicating functio‐
nal enterocyte mass [5]) were measured in 30 µl plasma at room temperature by using automated ion exchange column chromatography as described previously [4, 6, 20]. 41 | 169 3 Chapter 3 Plasma insulin concentrations. Plasma insulin concentrations were measured in 5 µl plasma via a solid bound antibody against insulin as described by the manufacturer (Rat Insulin Ultrasensitive EIA, Alpco Diagnostics, Salem, NH, USA) and as done previously [6]. 3 Intestinal [1‐13C]glucose absorption. The fractional contribution of intravenously administered [6,6‐2H2]glucose and enterally administered [1‐13C]glucose in blood glucose was performed essentially according to Van Dijk et al. [6, 21]. All samples were analyzed by gas chromatography/mass spectrometry (GC/MS, Agilent 5957C Series GC/MSD, Agilent Technologies, Amstelveen, the Netherlands). The calculations of RaT and RaE were calculated according to Steele’s one compartment model, modified by Debodo et al. [22, 23] and Tissot et al. [24]. The absorption of bolus‐administered glucose during the experimental period was expressed as the fraction of administered glucose. The average absorption of continuously administered [1‐13C]glucose in steady state was expressed as the fraction of intraduodenal glucose infusion in steady state. Statistical analysis Statistical analysis was performed using the Mann‐Whitney U‐test (SPSS 16.0 for Windows, Chicago, IL, USA). Values represent medians and [ranges in text] or (ranges in Table 1), or first to third quartiles (p25‐p75) in Figures 1‐4, for the indicated number of rats (n) per group. Correlations are expressed as nonparametric Spearman correlation coefficient. P values were considered statistically significant if P<0.05. RESULTS The mucositis rat model We determined the quantitative capacity to absorb glucose in a previously established rat model of MTX‐induced mucositis. MTX‐treated rats showed typical histological and clinical symptoms of mild to severe mucositis, in contrast to controls and as seen in previous studies by us and others [6, 25‐30] (Table 1). Most MTX‐treated rats (100% of rats that received glucose as a bolus and 89% of rats that received glucose continuously) suffered from severe mucositis (i.e. villus length <300 µm and plasma citrulline concentration <30 µmol/L [6]). Glucose absorption when administered as a bolus by oral gavage Blood glucose and plasma insulin concentrations. From the start of intravenous [6,6‐2H2]glucose infusion (‐60 min) until bolus [1‐13C]glucose administration (0 min), blood glucose concentrations hardly changed and were similar in controls and MTX‐treated rats (Figure 1A). Upon administration of the glucose bolus, blood glucose concentrations increased only in control rats whereas it hardly changed in MTX‐treated rats (maxima 10.8 versus 7.3 mmol/L respectively, after 45 versus 60 min 42 | 169 Maintaining Glucose Absorption during Mucositis respectively, P<0.01). Peaks in plasma insulin concentration (indirectly indicating the amount of glucose absorption) were reduced in MTX‐treated rats, compared with controls (maxima 0.6 versus 1.1 µg/L respectively, both after 15 min, P<0.05, Figure 1B). Intestinal glucose absorption. Upon administration of the glucose bolus, control rats showed an increase in [1‐13C]glucose appearance with a maximum of 75 µmol/(kg∙min) after 60 min. MTX‐treated rats only showed a minor increase in [1‐13C]glucose appearance (maximally 10 µmol/(kg∙min) after 120 min, Figure 2A). Calculated over the experimental period, this resulted in an almost six times reduced [1‐13C]glucose appearance in MTX‐treated rats, compared with controls (15 versus 85% of the administered bolus respectively, P<0.01, Figure 2B). Values of individual rats for glucose absorption, villus length and plasma citrulline show that glucose absorption correlated strongly with villus length (rho=0.69, P=0.003, Figure 2C) and plasma citrulline (rho=0.90, P<0.001, Figure 2D). Histological and clinical symptoms of mucositis in MTX treated rats An oral bolus of glucose Continuous intraduodenal glucose infusion Control (n=5) MTX (n=11) Control (n=7) MTX (n=9) 428 (382‐444) 179 (148‐212)* 403 (375‐500) 174 (163‐448)* Citrulline (day 4, µmol/L plasma)
84 (81‐114) 13 (10‐18)* 72 (51‐80) 15 (7‐69)* MPO (day 4, ng/mg mucosa)
5 (3‐9) 227 (76‐250)* NDe ND Food intake (day 3 ,g/day)a 8 (7‐12) 0 (0‐2)* 9 (8‐11) 2 (0‐8)* Body weight change (day 4 ,% relative to day 0)b +9 (+7 to +11) ‐5 (‐1 to ‐8)* +8 (+7 to +12) ‐2 (‐10 to +9)* Presence of watery diarrhea (day 4, % of rats)c 0 73 0 44 Villus length (day 4, µm)
Table 1. Characteristics of rats with MTX‐induced mucositis and controls used in the glucose absorption
tests. Controls are saline‐treated. Glucose (meal size) was administered as a bolus by oral gavage or
continuously by intraduodenal infusion to determine glucose absorption. Data indicate medians of groups,
and ranges are in parentheses. n no. of rats, ND not determined. * P<0.01 for controls versus MTX‐treated
rats a
Intake is shown on day 3 (8:00 A.M. – 11:00 P.M.) since rats were fasted from 11.00 P.M. on because of
the absorption tests at day 4 b
Body weight change (+ indicates weight gain, ‐ indicates weight loss) is relative to weight at day 0 (day
of MTX or saline injection, see ‘Materials and Methods’) c Diarrhea was present as watery diarrhea or completely absent (see ‘Materials and Methods’) 43 | 169 3 Chapter 3 3 Figure 1. Glucose and insulin concentrations when glucose was administered as a bolus by oral gavage.
Blood glucose concentrations (A) and plasma insulin concentrations (B) in controls (○, n=5) and
13
methotrexate (MTX)‐treated rats (● ▬, n=11) before and after oral administration of the [1‐ C]glucose
bolus (at 0 min). Dots represent medians and p25‐p75 per time point. *P<0.01 and #P<0.05 for controls versus
MTX treated rats. 13
Figure 2. Glucose absorption when administered as a bolus by oral gavage. [1‐ C]Glucose appearance (A
and B) in controls (○ ‐‐‐, n=5) and methotrexate (MTX)‐treated rats (● ▬, n=11) after oral administration of
13
the [1‐ C]glucose bolus (at 0 min). Dots represent medians and p25‐p75 per time point. *P<0.01 for controls
13
versus MTX‐treated rats. C and D: correlation between [1‐ C]glucose absorption on the one hand and villus
length (C) or plasma citrulline concentration (D) on the other hand in controls (○ ‐‐‐) and MTX‐treated rats
(● ▬). Dots represent data of individual rats. Spearman correlations (r) and P values are indicated. 44 | 169 Maintaining Glucose Absorption during Mucositis Figure 3. Glucose and insulin concentrations when glucose was administered continuously by intraduodenal infusion. Blood glucose concentrations (A) and plasma insulin concentrations (B) in controls (○ ‐‐‐, n=7) and methotrexate (MTX)‐treated rats (● ▬, n=9) before and during continuous intraduodenal 13
[1‐ C]glucose administration (starting at 0 min). Dots represent medians and p25‐p75 per time point. The vertical dotted line (‐‐‐) marks the transition from 1.3 to 1.9 g/(kg∙h) continuous glucose infusion (see
‘Materials and Methods’). *P<0.01 for controls versus MTX‐treated rats. Glucose absorption when administered continuously by intraduodenal infusion Blood glucose and plasma insulin concentrations. During the basal period (‐60 ‐ 0 min), blood glucose concentrations hardly changed and were similar in controls and MTX‐treated rats (Figure 3A). After starting intraduodenal glucose infusion, blood glucose concentrations rose quickly in both groups to 8.7 mmol/L in MTX‐treated rats and 10.1 mmol/L in controls after 30 min, and remained elevated during the experimental period. From 180 min on, blood glucose concentrations were similar in both groups. Plasma insulin concentrations were elevated during the experiment and did not differ between groups (Figure 3B). Intestinal glucose absorption. After an initial increase, until 240 min after the start of intraduodenal glucose infusion, the rate of [1‐13C]glucose appearance reached a steady state in both groups (Figure 4A). In MTX‐treated rats, the average rate in steady state was not different from controls (128 [36‐173] versus 138 [135‐152] µmol/(kg∙min) respectively, P=0.61). Similarly, the average glucose absorption in steady state did not differ significantly between controls and MTX‐treated rats (93 [88‐98] versus 80 [21‐95]% of infused glucose respectively, P=0.06, Figure 4B), although glucose absorption varied substantially between individual MTX‐treated rats (Figure 4C and D). Values of individual rats for glucose absorption, villus length and plasma citrulline show that glucose absorption correlated poorly with villus length (rho=0.54, P=0.030, Figure 4C) and plasma citrulline (rho=0.56, P=0.024, Figure 4D). 45 | 169 3 Chapter 3 3 13
Figure 4. Glucose absorption when administered continuously by intraduodenal infusion. [1‐ C]Glucose
appearance (A and B) in controls (○ ‐‐‐, n=7) and methotrexate (MTX)‐treated rats (● ▬, n=9) during
13
continuous intraduodenal [1‐ C]glucose administration (starting at 0 min). Dots represent medians without
p25‐p75 per time point (because of large interindividual differences and therefore a large spread in
MTX‐treated rats as seen in C and D). The vertical dotted line (‐‐‐) marks the transition from 1.3 to 1.9
g/(kg∙h) continuous glucose infusion (see Materials and Methods). *P<0.01 for controls versus MTX‐treated
13
rats. C and D: correlation between average [1‐ C]glucose absorption in steady state (240‐420 min) on the
one hand and villus length (C) or plasma citrulline concentration (D) on the other hand in controls (○ ‐‐‐) and
MTX‐treated rats (● ▬). Dots represent data of individual rats. Spearman correlations (r) and P values are
indicated. DISCUSSION We aimed to determine the quantitative capacity to absorb glucose in rats with MTX‐induced mucositis relative to that in controls. Our data indicate that continuous enteral administration can almost completely overcome the reduced absorptive capacity for glucose in rats with mucositis, although glucose absorption differs substantially between individual rats. Glucose absorption was determined in a previously developed and characterized rat model of MTX‐induced mucositis [6]. We initially administered glucose as an oral bolus to approximate the physiological situation of consuming meals. The bolus contained a physiologically relevant amount of glucose (2.0 g/kg, ≈ 4% of average daily carbohydrate intake), corresponding with the amount of glucose used for the Oral 46 | 169 Maintaining Glucose Absorption during Mucositis Glucose Tolerance Test (OGTT) in rodents [18, 19] and in patients [31]. In controls, blood glucose concentrations increased with 5 mmol/L upon bolus glucose administration, and maximal blood glucose concentrations were preceded by a significant peak in plasma insulin concentration, together indicating physiological glucose absorption. During the experimental period, 85% of the administered glucose was absorbed, as previously described by others [18, 19]. In contrast, blood glucose concentrations hardly increased upon the glucose bolus in MTX‐treated rats, and there was only a small peak in plasma insulin concentration, indicating minimal glucose absorption. Indeed, during MTX‐induced mucositis only 15% of the administered glucose was absorbed during the experimental period. So, although intestinal absorption of glucose in trace amounts (0.10 g/kg, ≈ 0.2% of average daily carbohydrate intake) was still intact during mucositis [6], the quantitative capacity to absorb a physiologically relevant amount of glucose during mucositis was severely reduced (when administered as a bolus). This might be explained by the reduced presence of glucose transporters sodium‐dependant glucose transporter 1 (SGLT1), glucose transporter 2 (GLUT2) and glucose transporter 5 (GLUT5) during mucositis [6, 30]. We then compared absorption of a physiologically relevant amount of glucose when continuously administered by intraduodenal infusion, since continuous enteral nutrient administration has been shown to improve nutrient absorption during intestinal failure [13]. The amount of glucose infusion in steady state was set at ±1.9 g/(kg∙h), ≈ 85% of average hourly carbohydrate intake. Blood glucose and plasma insulin concentrations were elevated from the start of intraduodenal infusion on, both in controls and MTX‐treated rats, suggesting physiological glucose absorption during mucositis. Indeed, in steady state, similar amounts of intraduodenal infused glucose were absorbed in controls and in MTX‐treated rats (medians 80 and 93% respectively). However, individual glucose absorption varied from severely reduced to completely normal between individual MTX‐treated rats (range 21‐95%), despite the fact that 8 out of 9 rats suffered from severe mucositis. Villus atrophy and plasma citrulline reduction in MTX‐treated rats must have been caused primarily by the cytotoxic effects of MTX and not by a spontaneous reduction in food intake upon MTX‐injection, as shown by others using pair‐feeding of saline‐treated control rats [25]. We hypothesize that the large interindividual differences in glucose absorption during mucositis can be explained by the fact that individual MTX‐treated outbred rats might have been in different stages of mucositis (described by Sonis et al. [2]) during the absorption experiment. Absorption of continuously administered glucose could have been possible via glucose transporters on the recovered epithelial membrane, via residual transporters on damaged epithelial membrane [6] and/or via paracellular absorption [32]. Leakage of glucose through damaged tight junctions could also have been possible since mucositis can lead to increased gut permeability [33, 34]. 47 | 169 3 Chapter 3 Continuous administration of enteral nutrition during intestinal failure is thought to enhance enteral absorption by maximizing saturation of the (residual) carrier proteins, thereby increasing intestinal function [13]. This concept could provide an explanation for the observation that a glucose bolus is malabsorbed while continuous glucose administration seems to overcome this defect during MTX‐induced mucositis. 3 To address whether plasma citrulline levels could function as a surrogate parameter for the glucose absorptive capacity during MTX‐induced mucositis, we related these two parameters in individual rats. In the bolus experiment, glucose absorption correlated strongly (rho=0.90) with plasma citrulline, making citrulline a suitable marker for reduced absorption of bolus‐administered glucose during mucositis. In the continuous infusion experiment, glucose absorption correlated poorly (rho=0.56) with plasma citrulline since half of MTX‐treated rats with severe mucositis (plasma citrulline concentration <30 µmol/L) had a reduced absorption of continuously administered glucose (<40%), but the other half absorbed glucose efficiently (>80%). Citrulline is therefore excluded as a suitable marker for absorption of continuously‐administered glucose during mucositis. Apparently, MTX‐induced damage to enterocytes does not evenly affect different properties of the enterocyte; absorption of glucose seems less affected than the production of citrulline, or glucose absorption is earlier recovered from mucositis. This could explain why some rats absorb glucose quite well, when administered continuously, while the production of citrulline is still low. If we extrapolate our findings in rats with (mostly severe) mucositis to the clinic, where patients show similar histological and clinical symptoms of mucositis [2], they imply that glucose should not be enterally administered as a bolus to mucositis patients. Malabsorption of bolus‐administered glucose during mucositis could possibly be detected via reduced plasma citrulline concentrations or via an OGTT. In contrast, enteral glucose administration by continuous infusion could be useful for a substantial portion of mucositis patients, in order to improve their nutritional state, recuperation and survival [9‐11, 35]. Moreover, enteral nutrition might accelerate intestinal recovery since intraluminal nutrients have a stimulatory effect on intestinal epithelial cells and the production of trophic hormones [15, 36, 37]. We performed the glucose absorption tests at day 4 after injection with MTX or saline, while symptoms of mucositis are actually present from day 2 until day 5 after injection with MTX [6]. We did not study whether the observed glucose malabsorption in a portion of mucositis rats is structural (present on all days during mucositis) or temporal (only present on day 4). When glucose malabsorption is structural, indeed only a portion of mucositis patients would benefit from continuous enteral glucose administration. Then, a marker that distinguishes between mucositis patients with a good or poor glucose absorptive capacity is highly desirable in order to anticipate which patients would benefit the most from continuous enteral glucose administration. However, if glucose 48 | 169 Maintaining Glucose Absorption during Mucositis malabsorption is temporal, all mucositis patients might benefit from continuous enteral glucose administration during mucositis. In conclusion, we show that continuous enteral administration can almost completely overcome the reduced absorptive capacity for glucose in rats with severe chemotherapy‐induced gastrointestinal mucositis, although glucose absorption differs substantially between individual rats. Our data suggest that glucose might be an appropriate source of dietary energy for at least a substantial portion of mucositis patients, when enterally administered continuously. 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DiBaise JK, Young RJ, Vanderhoof JA (2004) Intestinal rehabilitation and the short bowel syndrome: part 1. Am J Gastroenterol 99:1386‐1395 51 | 169 3 Clinical Nutrition; in press M. Fijlstra W.J.E. Tissing F. Stellaard H.J. Verkade E.H.H.M. Rings CHAPTER 4 REDUCED ABSORPTION OF FATTY ACIDS DURING METHOTREXATE‐INDUCED GASTROINTESTINAL MUCOSITIS IN THE RAT Chapter 4 ABSTRACT 4 Background Patients with chemotherapy‐induced gastrointestinal mucositis suffer from weight loss and possibly malabsorption. Since long‐chain fatty acids serve important functions in the body, we aimed to determine the intestinal capacity of fat absorption in rats with and without methotrexate‐induced mucositis. Methods Four days after intravenous injection with methotrexate (60 mg/kg) or saline, rats received saturated ([U‐13C]palmitic acid) and unsaturated ([U‐13C]linoleic acid) fatty acids dissolved in oil, either as a single bolus by oral gavage or by continuous intraduodenal infusion. We determined plasma and liver label concentrations at specific time points. Results We confirmed methotrexate‐induced mucositis by villus atrophy using microscopy. Methotrexate treatment severely reduced the appearance of [U‐
13
C]palmitic‐ and [U‐13C]linoleic acid in plasma and liver, compared with controls, either when administered as a bolus or continuously (all at least ‐63%, P<0.05). Liver [U‐13C]palmitic acid appearance was higher than [U‐13C]linoleic acid appearance, either when administered as a bolus (2.8‐fold, P<0.01) or continuously (5.7‐fold, P<0.01). Conclusions The intestinal capacity to absorb long‐chain fatty acids is severely reduced in rats with methotrexate‐induced mucositis. Continuous administration does not overcome this impairment. The liver takes up and/or retains mainly saturated fatty acids during mucositis. INTRODUCTION Gastrointestinal mucositis (further referred to as “mucositis”) is one of the most debilitating side effects of chemotherapy and radiotherapy, especially in children [1, 2]. Patients with mucositis often experience anorexia, diarrhea and weight loss [2]. Mucositis induces small intestinal villus atrophy and loss of enterocytes, suggesting loss of epithelial function [2]. However, the digestive and absorptive capacity during mucositis is still largely unknown. We earlier showed that the digestion of lactose was reduced, but that the absorption of glucose, administered in trace amounts, was still intact in a rat model of methotrexate (MTX)‐induced mucositis [3]. We now aimed to determine the intestinal capacity and physiology of fat absorption during mucositis, when enterally administered in physiologically relevant amounts (meal size). Up to now, there is still no rational feeding strategy for mucositis patients, although nutritional support might actually improve the nutritional state, accelerate recuperation and increase survival of mucositis patients [4]. It is known that long‐
chain fatty acids (LCFA) serve several important functions in the human body [5]. First of all, LCFA provide energy, in fact more than twice as much as carbohydrates and proteins on weight basis. Secondly, LCFA are the major constituent of cell membranes in esterified form (phospholipids, glycolipids). Thirdly, dietary LCFA are the only source of the essential fatty acids linolenic acid and linoleic acid. Finally, LCFA are particularly 54 | 169 Reduced Fatty Acid Absorption during Mucositis needed during periods of growth and development [6]. Concerning the mode of nutrient administration, continuous rather than bolus administration of enteral nutrition is recommended during intestinal failure which is thought to enhance enteral absorption by maximizing saturation of the carrier proteins, thereby increasing intestinal function [7]. Slow continuous administration of enteral nutrition also reduces the risk of osmotic diarrhea during intestinal failure [7]. When tolerated, enteral nutrition is preferred to total parenteral nutrition because the latter carries a high risk of infection and, upon prolonged administration, may cause liver disease [7]. No data are available concerning the best mode of LCFA administration during mucositis. Mucositis is primarily diagnosed by subjective symptoms like pain and diarrhea [2]. These symptoms correlate poorly with the severity of mucositis, especially in young children who are less capable of localizing pain and are often incontinent for feces [8]. However, plasma citrulline (a nonprotein amino acid made by enterocytes) has been shown to be a good, objective marker for mucositis [8] and even for lactose malabsorption during mucositis in the rat [3]. Here, we aimed to determine the intestinal capacity and physiology of LCFA absorption in a previously established mucositis rat model [3] when enterally administered as a bolus (exp. 1) or continuously (exp. 2). MATERIALS AND METHODS Rats and housing For both experiments, male Wistar Unilever outbred rats (4 wk old, 70‐85 g) were obtained from Harlan (Horst, the Netherlands). Rats were individually housed in Plexiglas cages (42.5 x 26.6 x 18.5 cm) on a layer of wood shavings under controlled temperature (21  1 C) with a relative humidity of 55  10% and a 12:12‐h light‐dark cycle (lights on 7:00 A.M. – 7:00 P.M.). Water and purified diet (AIN‐93G [9], Harlan Laboratories, Madison, WI, USA) were available ad libitum unless otherwise stated. The experimental protocol was approved by the Ethics Committee for Animal Experiments, Faculty of Medical Sciences, University of Groningen, the Netherlands. Materials MTX was obtained from Pharmachemie Holding B.V. (Haarlem, the Netherlands). [U‐13C]palmitic acid was purchased from Isotec (Miamisburg, OH, USA: exp. 1) and CortecNet (Voisins‐Le‐Bretonneux, France: exp. 2), both of 99% isotopic purity and of identical composition. [U‐13C]linoleic acid was purchased from Lans Medical (Amsterdam, the Netherlands: exp. 1) and Campro Scientific GmbH (Berlin, Germany: exp. 1), both of 99% isotopic purity and of identical composition. Different suppliers were used because of limited availability of both isotopes. 55 | 169 4 Chapter 4 Experimental procedures The mucositis rat model. We determined the intestinal capacity and physiology of LCFA absorption in rats with and without MTX‐induced mucositis [3] by quantifying plasma, liver and fecal appearance of enterally administered [U‐13C]palmitic acid and [U‐13C]linoleic acid. Rats of exp. 1 (‘LCFA absorption when administered as a bolus´) did not undergo surgery, while rats of exp. 2 (‘LCFA absorption when administered continuously’) were equipped with permanent catheters in the duodenum as described previously [10]. One week later, all rats (6‐7 wk old, 180‐260 g) were injected once intravenously with MTX (60 mg/kg, to induce mucositis [3]) or NaCl 0.9% (controls) in the tail vein. Intake of food and water, body weight and the presence or absence of diarrhea were recorded daily around 8:00 A.M. Four days after injection, when symptoms of MTX‐induced mucositis are most severe [3], the LCFA absorption tests were performed. 4 The LCFA absorption tests. After an overnight fast (11:00 P.M. day 3 – 8:00 A.M. day 4), rats received a physiologically relevant amount of fat (containing 20 mg ≈ 73.5 μmol [U‐13C]palmitic acid, and 10 mg ≈ 33.5 μmol [U‐13C]linoleic acid, per ml of oil mixture, i.e. 25% [v:v] olive oil ‐ 75% [v:v] medium chain triacylglycerol oil), either as a single bolus by oral gavage (exp. 1: 400 µl/rat, i.e. 7.5% of the daily LCFA intake, based on earlier studies [11]) or by continuous intraduodenal infusion (exp. 2: 228 µl/rat/h for 9 h, representing a normal hourly LCFA intake, based on 20g AIN93G/rat/day [3], ≈ 2 ml/rat during the experiment). For practical reasons, fat administration was not corrected for body weight in individual rats. Blood samples (0.1 ml) were obtained before the start of enteral fat administration (0h) and afterwards (at 1, 2, 3, 4, 5 and 6 h after bolus administration or at 3, 6, 7.5 and 9 h during continuous infusion) by tail bleeding to quantify plasma appearance of [U‐13C]palmitic acid and [U‐13C]linoleic acid [11, 12]. Samples were centrifuged immediately (10 min at 2,000 x g) and collected plasma was stored at ‐80°C until further analysis. Tissue collection. At the end of both experiments (6 h after an oral fat bolus or 9 h after starting continuous fat infusion), rats were killed under general anesthesia by obtaining a large blood sample through cardiac puncture for determination of plasma citrulline concentrations, followed by cervical dislocation. Blood samples were centrifuged immediately (10 min at 2,000 x g) and collected plasma was stored at ‐80°C until further analysis. Immediately after rats were killed, the abdomen was opened via a midline incision and the small intestine, large intestine and liver were quickly removed. After the small intestine was flushed with ice‐cold PBS, small parts of the jejunum (anatomical middle of the small intestine) were collected for histology and fixed in formalin (1 cm) or 2% paraformaldehyde (PFA 1 cm) dissolved in PBS, dehydrated and embedded in paraffin according to standard procedures for histochemistry [3]. An extra jejunal part (0.5 cm, exp. 1) for myeloperoxidase (MPO) concentrations was collected, immediately frozen in liquid nitrogen and stored 56 | 169 Reduced Fatty Acid Absorption during Mucositis at ‐80°C until further analysis. Contents from the large intestine (further referred to as “feces”) were collected and stored at ‐20°C until further use. The liver was freeze‐clamped and stored at ‐80°C until further analysis. Analytical methods Hematoxylin and eosin staining of formalin‐ and PFA‐fixed jejunal segments to assess histology, as well as their morphometric analysis, was carried out as described previously [3]. Mucosal MPO concentrations (exp. 1) and plasma citrulline concentrations were measured as described before [3]. Plasma [U‐13C]palmitic acid and [U‐13C]linoleic appearance. The quantification of enterally administered [U‐13C]palmitic acid and [U‐13C]linoleic acid in plasma was performed according to Oosterveer et al. [13] by gas chromatography‐mass spectrometry (GC‐MS, Agilent 5957C Series GC/MSD, Agilent Technologies, Amstelveen, the Netherlands). In short, C17:0 was added to the plasma as an internal standard. Then, fatty‐acid Br‐2,3,4,5,6‐pentafluorobenzyl (PFB) derivates were made, extracted in hexane and its isotopomer patterns analyzed using GC‐MS [14]. The mole percent enrichment of mass isotopomers M16 ([U‐13C]palmitic acid) and M18 ([U‐13C]linoleic acid), relative to their M0 isotopomers (unlabeled palmitic acid and linoleic acid), and the concentrations of these unlabeled LCFA (determined via their ratios to the added C17:0), were used to calculate the [U‐13C]palmitic acid and [U‐13C]linoleic acid plasma concentrations respectively. Enrichment of labeled LCFA on all time points after fat administration was corrected for baseline enrichment at 0 h. Plasma appearance of [U‐13C]palmitic acid and [U‐13C]linoleic acid during the experimental period was expressed as AUC of plasma [U‐13C]palmitic acid and [U‐13C]linoleic acid concentration respectively (0‐6 h after bolus fat administration, 0‐9 h during continuous fat infusion). Liver and fecal [U‐13C]palmitic acid and [U‐13C]linoleic appearance. After frozen livers were crushed, liver homogenates were prepared in PBS and C17:0 was added as an internal standard. Then, lipids were extracted according to Bligh and Dyer and PFB derivates were made [13]. Collected feces was freeze‐dried and mechanically homogenized. Subsequently, liver and fecal enrichment of [U‐13C]palmitic acid and [U‐13C]linoleic acid were quantified and concentrations of labeled LCFA in liver and feces were calculated as described above. Liver LCFA appearance versus plasma citrulline and diarrhea. We calculated the sensitivity and specificity of plasma citrulline (<30 µmol/L, based on earlier studies [3]) and diarrhea (present as liquid diarrhea or absent, as seen before [3]) to detect reduced liver appearance of [U‐13C]palmitic acid and [U‐13C]linoleic acid during mucositis. Estimations of cumulative absorption of LCFA based on plasma appearance have been proven difficult [12]. Rather, we assessed the amount of LCFA present in liver at the end of the experiments (see also ‘Discussion’). Reduced liver LCFA 57 | 169 4 Chapter 4 appearance was defined as an appearance below the fifth percentile of normal appearance, which is comparable with ≥2SD in a normally distributed population. Fecal/food ratio of [U‐13C]palmitic acid and [U‐13C]linoleic acid. In both experiments, the relative amount of each labeled LCFA in the administered fat was 69% for [U‐13C]palmitic acid (≈ 73.5 μmol per ml oil) and 31% for [U‐13C]linoleic acid (≈ 33.5 μmol per ml oil). We determined the relative amount of each LCFA in the feces (GC‐MS determinations), and subsequently calculated the fecal/food ratio of each LCFA, to find out if reduced LCFA absorption during mucositis could be caused by impaired micellar solubilization [15‐17]. 4 Metabolism of [U‐13C]linoleic acid to [13C18]arachidonic acid. Linoleic acid can be metabolized into the long‐chain polyunsaturated fatty acid arachidonic acid. Theoretically, MTX could affect the metabolic conversion rate of linoleic acid, either directly, or indirectly by changing the absorption efficacy of [U‐13C]linoleic acid. Next to the quantification of [U‐13C]palmitic acid and [U‐13C]linoleic appearance, we quantified plasma and liver [13C18]arachidonic acid appearance (exp. 2, derived from metabolic conversion of absorbed [U‐13C]linoleic acid [18] after 9 h of continuous fat infusion) to further determine LCFA absorption during mucositis. After C17:0 was added as an internal standard, plasma and liver PFB derivates were made, enrichment of [13C18]arachidonic acid was quantified and concentrations of labeled LCFA were calculated as described above. Intestinal Fat distribution. Jejunal fat distribution was visualized to study whether intraluminal fat passes the enterocytes during mucositis, using Oil‐Red‐O (ORO) staining on frozen material (4‐m‐thick sections, exp. 2), according to standard procedures as has been done before [19]. Statistical analysis Statistical analysis was performed using the Mann‐Whitney U‐test (SPSS 16.0 for Windows, Chicago, IL, USA). Values represent medians and ranges (text and Table) or first to third quartiles (Figures), for the indicated number of rats (n) per group. Correlations are expressed as nonparametric Spearman correlation coefficient. P values were considered statistically significant if P<0.05. RESULTS The mucositis rat model We determined intestinal LCFA absorption in a previously characterized rat model of MTX‐induced mucositis. In agreement with our earlier observations, MTX‐treated rats showed typical symptoms of mild to severe mucositis (i.e. villus atrophy and blunting, irregular and vacuolized enterocytes, mucosal inflammation, reduced plasma citrulline 58 | 169 Reduced Fatty Acid Absorption during Mucositis concentrations, reduced food intake, loss of body weight and liquid diarrhea), in contrast to controls and as seen previously [3] (Table 1). After a fat bolus Control (n=7) Citrulline day 4, µmol/L a
Food intake day 3 , g/d b
Relative body weight day 4 , %
530 (424‐549) 148 (125‐461)* 124 (4‐250)* ND ND 77 (66‐91) 11 (7‐59)* 61 (42‐69) 11 (7‐47)* 8 (6‐11) 1 (0‐6)* 12 (9‐15) 1 (0‐12)* 109 (105‐109) 39 (35‐51) [U‐ C]linoleic acid, µmol/kg [13C18]arachidonic acid, µmol/kg 13
[U‐ C]palmitic acid, fecal/food ratio 13
[U‐ C]linoleic acid, µmol/g [U‐13C]linoleic acid, fecal/food ratio 107 (104‐110) 94 (88‐105)* 250 (240‐275) 205 (175‐240)* 27 (3‐153)* 577 (508‐658) 76 (14‐598)* 7 (0‐36)* 34 (28‐43) 3 (1‐35)* Liver appearance 100 (52‐124) 35 (13‐137)# #
717 (485‐776) 134 (33‐496)* 15 (6‐19) 6 (2‐21) 36 (20‐44) 11 (2‐29)* ND ND 1.8 (1.0‐2.3) 0.6 (0.1‐1.7)* Fecal appearanced [U‐13C]palmitic acid, µmol/g 96 (91‐105)* 185 (165‐220)* Plasma appearancec (AUC) [U‐13C]palmitic acidc, µmol/(L•6h or L•9h) 155 (116‐199) 13
MTX (n=10) 229 (109‐433)* [U‐13C]palmitic acid, µmol/kg Control (n=6) 4 (3‐4) Absolute body weight day 4, g 210 (190‐230) [U‐13C]linoleic acid, µmol/(L•6h or L•9h) MTX (n=14) Rat characteristics Villus length day 4, µm 418 (395‐467) MPO day 4, ng/mg mucosa After continuous fat infusion ND ND 5 (1‐26) 43 (7‐80)* 1.4 (1.4‐1.4) 1.4 (1.2‐1.4) 1.3 (1.3‐1.4) 1.4 (1.3‐1.4)# ND ND 0.5 (0.1‐2.1) 2.4 (0.4‐4.3)# 0.1 (0.0‐0.2) 0.2 (0.0‐0.4) 0.3 (0.2‐0.4) 0.2 (0.1‐0.2) # Table 1. Rat characteristics and long‐chain fatty acid (LCFA) appearance after an oral fat bolus or after continuous intraduodenal fat infusion. LCFA appearance was determined in contents from the large
intestine (referred to as “feces”) because of diarrhea in MTX‐treated rats. Data indicate medians (ranges) of groups. #P<0.05 and *P<0.01 for control versus MTX‐treated rats. a
Food intake is shown on day 3 (8.00 A.M. – 11.00 P.M.), since rats were fasted from 11.00 P.M. on because of the LCFA absorption test at day 4 b
Body weight is relative to weight at day 0 (day of injection) which is arbitrarily set at 100% c
Plasma appearance (AUC, area under the curve) of LCFA, 6 h after an oral fat bolus or after 9 h of continuous intraduodenal fat infusion d
Fecal LCFA concentrations could not be determined (ND) after a fat bolus because the internal standard (i.e. C17) was not detectable 59 | 169 4 Chapter 4 Exp. 1: LCFA absorption when administered as a bolus Plasma [U‐13C]palmitic acid and [U‐13C]linoleic acid appearance Figure 1A and B show that MTX treatment markedly lowered plasma concentrations of [U‐13C]palmitic acid and [U‐13C]linoleic acid at all time points after fat administration, compared with controls (P<0.01). In MTX‐treated rats, maximal concentrations of [U‐13C]palmitic acid and [U‐13C]linoleic acid (6.9 and 1.7 µmol/L, at 6 4 13
Figure 1. Long‐chain fatty acid (LCFA) appearance after an oral fat bolus. Appearance of [U‐ C]palmitic
13
acid and [U‐ C]linoleic acid in the plasma (A and B) and liver (C‐F) of control (○ ‐‐‐, n=7) and methotrexate
(MTX)‐treated rats (● ▬, n=14) until (plasma) or at (liver) 6 h after an oral fat bolus. C‐F: plasma citrulline
13
13
(C and D) versus diarrhea (E and F) as markers for liver appearance of [U‐ C]palmitic acid and [U‐ C]linoleic
acid in control (○) and MTX‐treated rats (●). Data represent medians and p25‐p75 (A and B) or data of
individual rats (C‐F). Spearman correlations (r) and P values are indicated. The dotted horizontal line (‐‐‐‐‐)
marks the cut‐off of reduced LCFA appearance (i.e. <p5 of controls, C‐F). *MTX differs from control, P<0.01. 60 | 169 Reduced Fatty Acid Absorption during Mucositis and 5 h respectively) were reached with a delay, compared with controls (33.5 and 8.8 µmol/L, both at 4 h respectively). Plasma appearance of both LCFA during the experimental period was reduced to 17% in MTX‐treated rats, compared with controls (AUC, P<0.01, Table 1). Liver [U‐13C]palmitic acid and [U‐13C]linoleic acid appearance Figure 1C and D and Table 1 show that liver concentrations of [U‐13C]palmitic acid and [U‐13C]linoleic acid were reduced to 35 and 37% respectively in MTX‐treated rats, compared with controls (P<0.01). Corrected for enteral [U‐13C]palmitic acid and [U‐13C]linoleic acid administration (molar [U‐13C]palmitic acid administration was 2.2‐fold higher than molar [U‐13C]linoleic acid administration), liver [U‐13C]palmitic acid appearance was higher than [U‐13C]linoleic acid appearance, both in MTX‐treated rats (2.8‐fold, P<0.01) and in controls (3.0‐fold, P<0.01). 4 Fecal/food ratio of [U‐13C]palmitic acid and [U‐13C]linoleic acid 13
13
The relative amount of [U‐ C]palmitic acid and [U‐ C]linoleic acid in feces was similar between MTX‐treated rats (medians 94 and 6% respectively) and controls (medians 98 and 2% respectively, P=0.15). The fecal/food ratio of each LCFA was also similar between MTX‐treated rats and controls (Table 1). LCFA absorption versus plasma citrulline and diarrhea Liver appearance of [U‐13C]palmitic acid and [U‐13C]linoleic acid correlated with plasma citrulline (rho=0.80 and 0.76 respectively, P<0.001, Figure 1C‐D) and villus length (rho=0.84 and 0.79 respectively, P<0.001, data not shown). Figure 1C‐F show that the sensitivity of plasma citrulline (<30 µmol/L) to detect reduced LCFA appearance in the liver (<p5 of controls) was higher (Se 0.91 / Sp 0.80) than that of diarrhea (Se 0.72 / Sp 1.00, i.e. liquid diarrhea), although its specificity was lower. Exp. 2: LCFA absorption when administered continuously Plasma [U‐13C]palmitic acid and [U‐13C]linoleic acid appearance Figure 2A and B show that MTX treatment caused markedly lower plasma concentrations of [U‐13C]palmitic acid and [U‐13C]linoleic acid at all time points during fat infusion, compared with controls (P<0.01). Maximal concentrations of [U‐13C]palmitic acid and [U‐13C]linoleic acid in MTX‐treated rats (15.9 and 0.7 µmol/L respectively) and in controls (135.2and 8.8 µmol/L respectively) were reached after 9 h of fat infusion (no plateau reached yet). Plasma appearance of [U‐13C]palmitic acid and [U‐13C]linoleic acid during the experimental period was reduced to 13 and 9% respectively in MTX‐treated rats, compared with controls (AUC, P<0.01, Table 1). 61 | 169 Chapter 4 4 Figure 2. Long‐chain fatty acid (LCFA) appearance during and after continuous intraduodenal fat 13
13
infusion. Appearance of [U‐ C]palmitic acid and [U‐ C]linoleic acid in the plasma (A and B) and liver (C‐F) of control (○ ‐‐‐, n=6) and methotrexate (MTX)‐treated rats (● ▬, n=10) during (plasma) or after (liver) 9 h
of continuous intraduodenal fat infusion. C‐F: plasma citrulline (C and D) versus diarrhea (E and F) as 13
13
markers for liver appearance of [U‐ C]palmitic acid and [U‐ C]linoleic acid in control (○) and methotrexate
(MTX)‐treated rats (●). Data represent medians and p25‐p75 (A and B) or data of individual rats (C‐F). Spearman correlations (r) and P values are indicated. The dotted horizontal line (‐‐‐‐‐) marks the cut‐off of reduced LCFA appearance (i.e. <p5 of controls, C‐F). *MTX differs from control, P<0.01. Liver and fecal [U‐13C]palmitic acid and [U‐13C]linoleic acid appearance Figure 2C and D and Table 1 show that liver concentrations of [U‐13C]palmitic acid and [U‐13C]linoleic acid were reduced to 19 and 30% respectively in MTX‐treated rats, compared with controls (P<0.01). Corrected for enteral [U‐13C]palmitic acid and [U‐13C]linoleic acid administration (molar [U‐13C]palmitic acid administration was 2.2‐fold higher than molar [U‐13C]linoleic acid administration), liver [U‐13C]palmitic acid appearance was higher than [U‐13C]linoleic acid appearance, both in MTX‐treated 62 | 169 Reduced Fatty Acid Absorption during Mucositis rats (median 5.7‐fold, P<0.01) and in controls (9.0‐fold, P<0.01). Fecal concentrations of [U‐13C]palmitic acid and [U‐13C]linoleic acid were increased 9.1‐fold and 4.4‐fold respectively in MTX‐treated rats, compared with controls (P<0.05, Table 1). Fecal/food ratio of [U‐13C]palmitic acid and [U‐13C]linoleic acid The relative amount of [U‐13C]palmitic acid and [U‐13C]linoleic acid in feces was comparable between MTX‐treated rats (medians 94 and 6% respectively) and controls (medians 92 and 8% respectively, P=0.012). The fecal/food ratio of both LCFA was also comparable between MTX‐treated rats and controls (Table 1). Metabolism of [U‐13C]linoleic acid to [13C18]arachidonic acid Plasma [13C18]arachidonic acid concentrations were extremely low in MTX‐treated rats and in controls (data not shown). Absolute liver [13C18]arachidonic acid concentrations were reduced to 32% in MTX‐treated rats, compared with controls (P<0.01, Table 1). The amount of [13C18]arachidonic acid that was present in the liver at the end of the experiment, expressed per amount of its parent compound [U‐13C]linoleic acid, was similar in MTX‐treated rats (median 5, range 3‐10%) and in controls (median 5%, range 5‐6%, P=0.75). Intestinal fat distribution In controls, most enterocytes contained no fat droplets while many small fat droplets were present in the villus interstitium (Figure 3A). In contrast, some vacuolized enterocytes on remaining villus tops of MTX‐treated rats contained a few fat droplets while fat droplets were completely absent in the villus interstitium (Figure 3B). Figure 3. Fat distribution after continuous intraduodenal fat infusion. Oil‐Red‐O (ORO) staining showing
fat distribution after 9 h of continuous intraduodenal LCFA infusion in the jejunum of control (A) and
methotrexate (MTX)‐treated rats (B). Magnification: 20x. 63 | 169 4 Chapter 4 LCFA absorption versus plasma citrulline and diarrhea Liver appearance of [U‐13C]palmitic acid and [U‐13C]linoleic acid correlated with plasma citrulline (rho=0.84 and 0.80 respectively, P<0.001, Figure 2C‐D), and appearance of [U‐13C]palmitic acid with villus length (rho=0.61, P=0.012, data not shown). Figure 2C‐F show that the sensitivity of plasma citrulline (<30 µmol/L) to detect reduced LCFA appearance in the liver (<p5 of controls) was higher (Se ≥ 0.80 / Sp ≥ 0.83) than that of diarrhea (Se ≥ 0.50 / Sp 1.00, i.e. liquid diarrhea), although its specificity was lower. DISCUSSION 4 We aimed to determine the intestinal capacity and physiology of LCFA absorption during mucositis. Our data indicate that the intestinal capacity to absorb LCFA is severely reduced in rats with MTX‐induced mucositis. Continuous enteral administration does not overcome this impairment. We determined LCFA absorption in a previously established rat model of MTX‐induced mucositis [3] by quantifying plasma, liver and fecal appearance of enterally administered, stable isotope labeled saturated and polyunsaturated LCFA, as have been successfully used before [11, 12, 18, 22‐26]. We could not use the gold standard for measuring fat absorption, i.e. the fecal fat balance [21], to determine LCFA absorption because of diarrhea, reduced food intake and non‐steady state conditions during mucositis. To estimate quantitative LCFA absorption, we determined the hepatic labeled LCFA content at the end of the experiments. Theoretically, MTX could have affected liver uptake and retention of systemically available LCFA, but we do not have any indication to support this possibility: we did neither see histological differences between the livers of MTX‐treated rats and controls, nor is MTX‐induced liver dysfunction after short‐term MTX exposure reported in the literature. For assessment of quantitative carbohydrate or amino acid absorption, dual isotope infusion (enteral and intravenous) of differentially labeled but identical nutrients can be applied: their plasma appearance can then be corrected for first pass splanchnic utilization and the flux of molecules out of the plasma compartment [27‐29]. However, this methodology is not usable for LCFA absorption, since one cannot reliably reconstitute and administer LCFA in the identical physical particles (chylomicrons) and biochemical nature (acylated as triglyceride of phospholipid) in which the absorbed LCFA appear in the plasma compartment. We initially administered LCFA as an oral bolus to approximate the physiological situation of consuming meals. Plasma and liver appearance of labeled LCFA was severely reduced during mucositis, compared with controls, indicating reduced LCFA absorption. Since all rats received the same absolute amount of fat, MTX‐treated rats 64 | 169 Reduced Fatty Acid Absorption during Mucositis received more fat per gram body weight than controls because of a lower body weight. We therefore cannot exclude that the observed LCFA acid uptake in MTX‐
treated rats is even overestimated. We aimed to determine the physiology of reduced LCFA absorption during mucositis. Dietary lipids undergo a number of intraluminal physicochemical alterations before lipolytic products are translocated across the enterocyte membrane [5]. After translocation, absorbed LCFA undergo reacylation and assembly into chylomicrons before they end up in mesenteric lymph and finally in the blood [15, 20]. Since we administered unesterified LCFA, we could exclude impaired emulsification or lipolysis as causes for their reduced absorption during mucositis. Impaired micellar solubilization is expected to result in a relative increase of nonabsorbed [U‐13C]palmitic acid in the feces since unsaturated LCFA have appeared to be less dependent on micellization than saturated LCFA [15‐17]. In MTX‐
treated rats, however, the fecal/food [U‐13C]palmitic acid ratio was similar compared with that in controls, excluding the possibility that impaired micellar solubilization predominantly contributes to impaired LCFA absorption during mucositis. By inference, reduced LCFA absorption during mucositis seems to be mediated at the level of the enterocytes (defective translocation and/or intracellular LCFA processing), which seems plausible regarding the observed enterocyte damage and dysfunction (i.e. reduced plasma citrulline concentrations [3], which cannot merely be explained by a reduced food intake [32]). We did not study enterocyte expression of fatty acid binding proteins since they are not essential for fat absorption [30]. Others found them to be relatively resistant to chemotherapy‐induced damage [31]. We then determined intestinal absorption of LCFA upon continuous administration, which is thought to improve nutrient absorption during other forms of intestinal failure [7]. Overall, the results were rather similar to those obtained after bolus administration (reduced plasma and liver LCFA appearance and increased fecal LCFA appearance during mucositis), indicating reduced LCFA absorption. Absolute liver appearance of [13C18]arachidonic acid was reduced during mucositis, compared with controls, while its relative amount compared with liver [U‐13C]linoleic acid was similar, indicating that MTX does not affect the metabolic conversion rate of linoleic acid directly but indirectly by changing the absorption efficacy of [U‐13C]linoleic acid. Impaired micellar solubilization due to changes in bile salt metabolism could again be excluded, since the fecal/food [U‐13C]palmitic acid ratio was in the same order of magnitude in MTX‐treated rats and in controls. The absence of fat droplets in the villus interstitium (draining on the lymphatic system [19]) of MTX‐treated rats, in contrast to controls, was in accordance with the earlier found absorptive problems at the level of the enterocytes. During continuous LCFA administration, the total amount of administered LCFA was about five times higher than in the bolus experiment. Accordingly, absolute plasma and liver appearance of labeled LCFA during mucositis was higher compared with 65 | 169 4 Chapter 4 4 bolus administration (although their relative appearance stayed low, compared with controls). In both experiments, the fecal/food ratio of [U‐13C]palmitic acid appearance was much higher than the fecal/food [U‐13C]linoleic acid appearance, both in MTX‐
treated rats and controls, confirming the physiological lower absorption efficacy of saturated LCFA relative to unsaturated LCFA [20]. We assumed that fecal appearance of labeled LCFA was only the result of nonabsorbed enterally administered LCFA. Theoretically, excretion of labeled LCFA by the intestine via the enterohepatic loop or via enterocyte shedding, after initial absorption, could have also played a role. Of note, liver [U‐13C]palmitic acid appearance was much higher than [U‐13C]linoleic acid appearance, both in MTX‐treated rats and controls, either after a fat bolus and after continuous fat administration. This might have been due to a high oxidation rate for [U‐13C]linoleic acid after absorption [33]. Apparently, the liver has a higher uptake and/or retention of saturated than of unsaturated LCFA, under normal circumstances and during mucositis. Our findings might indicate that especially saturated LCFA are important in the diet of mucositis patients. LCFA absorption correlated with plasma citrulline concentration and villus length, similar to previous observations on lactose absorption [3]. We also showed that plasma citrulline <30 µmol/L has a higher sensitivity (≥0.80 versus ≥0.50), but a lower specificity (≥0.8 versus 1.0), than diarrhea to detect reduced LCFA absorption during mucositis. In rats, the absence of diarrhea after chemotherapy apparently does not exclude nutrient malabsorption. Since plasma citrulline seems a valuable indicator of the functional status of the gastrointestinal tract in rats [3] as well as in humans [34], it might help to tailor the feeding strategy of mucositis patients. ACKNOWLEDGEMENTS The authors acknowledge Rick Havinga, Juul Baller, Theo Boer, Angelika Jurdzinski and Pieter Klok (University Medical Center Groningen, University of Groningen, the Netherlands) for excellent technical assistance in our studies. GRANTS This work was financially supported by an unrestricted research grant from Fonds NutsOhra (the Netherlands). REFERENCES 1.
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Vassileva G, Huwyler L, Poirier K, Agellon LB, Toth MJ (2000) The intestinal fatty acid binding protein is not essential for dietary fat absorption in mice. FASEB J 14:2040‐2046 31. Verburg M, Renes IB, Van Nispen DJ, Ferdinandusse S, Jorritsma M, Buller HA, Einerhand AW, Dekker J (2002) Specific responses in rat small intestinal epithelial mRNA expression and protein levels during chemotherapeutic damage and regeneration. J Histochem Cytochem 50:1525‐1536 32. Boukhettala N, Leblond J, Claeyssens S, Faure M, Le PF, Bole‐Feysot C, Hassan A, Mettraux C, Vuichoud J, Lavoinne A, Breuille D, Dechelotte P, Coeffier M (2009) Methotrexate induces intestinal mucositis and alters gut protein metabolism independently of reduced food intake. Am J Physiol Endocrinol Metab 296:E182‐E190 33. Cunnane SC (2003) Problems with essential fatty acids: time for a new paradigm? Prog Lipid Res 42:544‐568 34. Crenn P, Vahedi K, Lavergne‐Slove A, Cynober L, Matuchansky C, Messing B (2003) Plasma citrulline: A marker of enterocyte mass in villous atrophy‐associated small bowel disease. Gastroenterology 124:1210‐1219 68 | 169 Reduced Fatty Acid Absorption during Mucositis 4 69 | 169 Journal of Nutrition 2012; 142: 1983‐1990 M. Fijlstra H. Schierbeek G. Voortman K.Y. Dorst J.B. van Goudoever E.H.H.M. Rings W.J.E. Tissing CHAPTER 5 CONTINUOUS ENTERAL ADMINISTRATION CAN ENABLE NORMAL AMINO ACID ABSORPTION IN RATS WITH METHOTREXATE‐INDUCED GASTROINTESTINAL MUCOSITIS Chapter 5 ABSTRACT 5 Background It is unknown what feeding strategy to use during chemotherapy‐induced gastrointestinal mucositis, causing weight loss and possibly malabsorption. We aimed to study the absorptive capacity of amino acids during mucositis. Methods We determined the plasma availability of enterally administered amino acids (AA), their utilization for protein synthesis, and the preferential side of the intestine for AA uptake in rats with and without methotrexate (MTX)‐induced mucositis. Four days after injection with MTX (60 mg/kg) or saline (controls), rats received a primed, continuous dual‐isotope infusion (intraduodenal and intravenous) of labeled L‐leucine, L‐lysine, L‐phenylalanine, L‐threonine and L‐methionine. We took blood samples, assessed jejunal histology and determined labeled AA incorporation in proximal and distal small intestinal mucosa, plasma albumin, liver and thigh muscle. Results MTX‐induced mucositis was confirmed by histology. The median systemic availability of all AA except for leucine was similar in MTX‐treated rats and in controls. However, individual availability of all AA differed substantially within the group of MTX‐treated rats, ranging from severely reduced (<10% of intake) to not different from controls (>40% of intake in 5 of 9 rats). More AA originating from basolateral uptake than those originating from apical uptake were used for intestinal protein synthesis in MTX‐treated rats (at least 420% more, P<0.05). Conclusions We conclude that continuous enteral administration can enable normal AA absorption in rats with MTX‐induced mucositis. The intestine prefers basolateral AA uptake to meet its need for AA for protein synthesis during mucositis. INTRODUCTION Gastrointestinal mucositis (further referred to as “mucositis”) is one of the most severe and debilitating side effects of anti‐cancer treatment, causing small intestinal villus atrophy and loss of enterocytes [1]. Patients with mucositis suffer from anorexia, nausea, diarrhea and weight loss [2]. It is unknown how to optimally feed patients with mucositis, although nutritional support might improve the nutritional state, accelerate recuperation and increase survival of mucositis patients [3‐6]. Normally, enteral nutrition, which is the physiological way of feeding, is preferred to total parenteral nutrition (TPN) because the latter carries a high risk of infection and, upon prolonged administration, may cause liver disease [7, 8]. However, when the absorptive function of the intestine is compromised, TPN offers a useful feeding alternative. We developed a methotrexate (MTX)‐induced mucositis rat model to determine nutrient digestion and absorption during mucositis, and to ultimately design a rational feeding strategy for mucositis patients [9]. In this model, we showed that trace amounts of glucose are absorbed normally during mucositis [9]. Because there are 72 | 169 Normal Amino Acid Absorption during Mucositis indications that intestinal absorption of amino acids (AA) might be intact during mucositis [10], in contrast to absorption of di‐ and tripeptides [11], we here aimed to determine the capacity to absorb enterally administered AA during mucositis. AA serve several important functions in the human body [12], particularly during periods of growth [13], and play an important role in mucosal homeostasis [14, 15]. Normally, the intestine itself metabolizes a substantial part (up to 80%) of nutrients after absorption from the intestinal lumen before nutrients become systemically available; a process called ‘first‐pass splanchnic utilization’ [12, 16‐19]. When more nutrients are used for first‐pass utilization, fewer nutrients are systemically available for whole‐body energy metabolism and peripheral tissue synthesis [12]. A unique feature of intestinal enterocytes is that they do not only absorb AA directly from the lumen by their apical membrane, they can also take up AA from the mesenteric arterial circulation by their basolateral membrane after becoming systemically available [12, 20‐22]. It is not well known how mucositis affects the first pass splanchnic uptake and the resulting systemic availability of AA, or to what extent systemically available AA are used for protein synthesis in diverse tissues. Knowledge about these processes is needed in order to determine the absorptive capacity of enterally administered AA, and whether the gut can be used for uptake of AA during mucositis. Furthermore, we hypothesized that there could be a preferential side of the intestine for AA uptake, in order to synthesize proteins during mucositis. To study the absorptive capacity of amino acids during mucositis, we determined the plasma availability of five enterally administered, essential AA (to indirectly test the function of different AA transporter systems), their utilization for protein synthesis, and the preferential side of the intestine for AA uptake in rats with and without MTX‐induced mucositis. We determined absorption of a physiologically relevant amount of AA (i.e. a normal hourly AA intake, instead of a trace amount of AA) when continuously administered by intraduodenal (i.d.) infusion, since continuous enteral nutrient administration has been shown to improve absorption of another nutrient during mucositis in the rat; i.e. glucose [23]. MATERIALS AND METHODS Rats and housing Male Wistar outbred rats (4 wk old, 65‐75 g, Specific Pathogen Free) were obtained from Charles River (Sulzfeld, Germany). Rats were individually housed in Plexiglas cages (42.5 x 26.6 x 18.5 cm) on a layer of wood shavings under controlled temperature (21  1 C) with a relative humidity of 55  10% and a 12 h light ‐ 12 h dark cycle (lights on 07.00‐19.00 h). Water and purified diet (AIN‐93G [24], Research Diet Services B.V., Wijk bij Duurstede, the Netherlands) were available ad libitum unless otherwise stated. The experimental protocol was approved by the Ethics 73 | 169 5 Chapter 5 Committee for Animal Experiments, Faculty of Medical Sciences, University of Groningen, the Netherlands. Materials MTX was obtained from Pharmachemie Holding B.V. Stable isotope‐labeled AA of 88‐99% isotopic purity for i.d. and intravenous (i.v.) infusion (Table 1) were purchased from CortecNet. Unlabeled AA for i.d. infusion (Table 1) were purchased from Sigma‐Aldrich Chemie GmbH. Infusion rate, µmol∙kg‐1∙h‐1 Intraduodenal Stable isotope‐labeled L‐[1,2‐13C2]leucine 13
L‐[ C6, N2]lysine‐HCl 13
L‐[1‐ C]phenylalanine 117 107 73 Experimental procedures AA infusion protocol in the mucositis rat L‐[ C5, N]methionine 26 model. One week after arrival at the Unlabeled animal facility, rats were equipped with L‐leucine 262 permanent catheters in the duodenum L‐lysine‐HCl 218 and jugular vein as described previously L‐ phenylalanine 86 [25]. One week after surgery, rats (6 wk L‐ threonine 153 old, 205‐250 g) were injected once i.v. in L‐methionine 68 the tail vein with MTX (60 mg/kg, n=9) to Intravenous induce mucositis or with saline (0.9%, Stable isotope‐labeled controls, n=7) under general anesthesia L‐[5,5,5‐2H2]leucine 228 [9]. Assignment of rats to one of the L‐[15N2]lysine‐HCl 181 treatment groups was randomly L‐[2H5]phenylalanine 75 performed by the researcher (MF). Intake L‐[15N]threonine 146 of food and water, body weight and the L‐[2H3]methionine 56 presence of diarrhea [present as watery diarrhea or absent [9]] were recorded daily at ~08.00 h. Four days after injection, when histological and clinical symptoms of MTX‐induced mucositis were most severe [9], the AA absorption experiment was performed. A primed (once the hourly dose), continuous i.d. and i.v. infusion of AA in distilled water was started in unanesthetized rats for 5 h after an overnight feed deprivation (23.00 h on d 3 to 08.00 h on d 4) to reach a steady state [26]. Infusion rates and doses of the i.d. and i.v. infusates (Table 1) were based on results from pilot studies that we had executed earlier (unpublished material). Unlabeled AA were added to stable isotope‐labeled AA in the i.d. infusate to reach a normal intake of each amino acid during the experimental period, based on the mean daily AA intake in L‐[13C4, 15N]threonine 13
5 15
Table 1. Rates of intraduodenal and intravenous infusion of stable isotope‐labeled and unlabeled essential amino acids in control and MTX‐treated rats. Values are absolute. For individual rats, values were adjusted to their individual body weight on d 4 (range 185‐250 g), n = 16. MTX, methotrexate. 74 | 169 15
46 Normal Amino Acid Absorption during Mucositis control rats (i.e. 5/24 x mean daily AA intake in controls, calculated from their mean daily food intake which is ±20g AIN93G per 230g body weight [9]), to study physiological AA absorption instead of studying a tracer effect. Blood samples were obtained at baseline and in steady state (at 4, 4.5 and 5 h after the start of the dual‐isotope infusion, based on pilot studies and as done previously [26]) for mass spectrometry (MS) analyses. After the 5‐h AA infusion protocol, rats were killed under general anesthesia by obtaining a large blood sample through cardiac puncture for determination of plasma albumin and citrulline concentrations, followed by cervical dislocation. Blood samples were centrifuged immediately (10 min at 2,000 x g) and collected plasma was stored at ‐80°C until further analysis. Tissue collection. Immediately after rats were killed, the abdomen was opened via a midline incision and the small intestine, liver and a sample from the thigh muscle were quickly removed. After the small intestine was flushed with ice‐cold PBS, a small part of the jejunum (anatomical middle of the small intestine) was collected for histology and fixed in formalin (1 cm) or 2% paraformaldehyde (PFA, 1 cm) dissolved in PBS, dehydrated and embedded in paraffin according to standard procedures for histochemistry [9]. Mucosa of the rest of the small intestine, being a proximal part (between stomach and anatomical middle, i.e. duodenum and proximal jejunum) and a distal part (between anatomical middle and cecum, i.e. distal jejunum and ileum), was scraped on ice, weighed (wet weights) and stored at ‐80°C until further analysis. Liver and thigh muscle were weighed (wet weights), freeze‐clamped, pulverized in liquid nitrogen and stored at ‐80°C until further analysis. Analytical methods Hematoxylin‐and‐eosin (H&E) staining of formalin‐ and paraformaldehyde‐fixed jejunal segments to assess histology, as well as their morphometric analysis, was carried out as described previously [9]. Plasma citrulline concentrations [indicating functional enterocyte mass [27]] were measured as described previously [9]. Plasma albumin concentrations were measured in 150 µL plasma via the bromocresol green method, which is a calorimetric assay, as described by the manufacturer (Roche Diagnostics GmbH). Tissue samples (± 50 mg) of proximal and distal small intestinal mucosa, liver and thigh muscle were homogenized in distilled water (± 500 µL) to measure tissue isotopic enrichment of all AA (see ‘Tissue enrichment analyses’ below). An aliquot of 50 µL was taken to measure tissue protein concentrations according to Lowry et al. [28]. Mass spectrometry Plasma enrichment analysis. Plasma samples (30 µL) were prepared to determine isotopic enrichment of all AA by gas chromatography‐MS (MSD 5975C, Agilent Technologies), as described previously [26, 29‐31]. Instead of plasma leucine enrichment, plasma enrichment of its keto‐analogue α‐ketoisocaproic acid was 75 | 169 5 Chapter 5 measured to correct for intracellular transamination of leucine, as done before by us and others [10, 32]. The enrichment of all AA in steady state was calculated by using the mean enrichment between 4 and 5 h after continuous dual‐isotope infusion, corrected for the enrichment at baseline, as described previously [33]. Enrichment was expressed in mole percent excess. 5 Tissue enrichment analysis. Aliquots of 200 µL homogenized tissue (see ‘Analytical methods’ above) were taken to measure isotopic enrichment of all free (unbound) and protein‐bound AA. The protein fraction was isolated and analyzed as previously described [30]. In short, proteins were precipitated and the supernatant was collected and used for enrichment analysis of free AA. The washed, precipitated protein pellets were hydrolyzed by adding 1 mL of 6 mol/L HCl and incubated at 110°C for 20 h. An aliquot was dried at room temperature in a speedvac (GeneVac miVac, GeneVac Ltd), and the residue was dissolved in 0.2 mL milli‐Q. AA were isolated by cation exchange separation. To measure the enrichment of AA in the protein‐bound tissue pool, hydrolyzed samples were derivatized to form acetyl‐ethoxycarbonyl‐ethylesters. The 13 12
C: C ratio of AA in protein isolates were measured by using a gas chromatograph/combustion/isotope ratio MS (Delta XP, Thermo Fisher) according to the method used in previous work [26, 31]. Isotopic enrichment of protein‐bound 2
H‐ or 15N‐labeled AA, and of all free AA in the supernatants, was determined by gas chromatography/MS analysis of their acetyl‐ethoxycarbonyl‐ethylesters by using electron impact ionization, as described for the plasma samples. Enrichment was expressed in mole percent excess. Plasma albumin analysis. Albumin was isolated from collected plasma at 4, 4.5 and 5 h after the start of the dual‐isotopically labeled AA infusions. After hydrolysis, the isotopic enrichment of enterally administered AA was measured by using a gas chromatograph/combustion/isotope ratio MS to determine the synthesis rate of plasma albumin as described previously [34, 35]. Values obtained for isotopic enrichment of all AA (including α‐ketoisocaproic acid) were corrected for the contribution of natural abundance on the measured fragments, as well as for the contribution of administered tracers to the measured fragments. Calculations The equations used to obtain the results are detailed in the Supplemental Methods. Statistical analysis Statistical analysis of data in MTX‐treated rats versus controls (i.e. rat characteristics, AA kinetics, and protein synthesis) was performed by using the Mann‐Whitney U‐test (SPSS 16.0 for Windows, SPSS). Analysis of data on basolateral versus apical AA uptake for protein synthesis in MTX‐treated rats or in controls (see ‘Supplemental Methods’) 76 | 169 Normal Amino Acid Absorption during Mucositis was performed by using the Wilcoxon signed‐rank test. Data are presented as absolute values (Table 1) and median and range (Tables 2‐5) or as data for individual rats (Supplemental Figure 1) for the indicated number of rats (n) per group. Correlations are expressed as nonparametric Spearman correlation coefficient. For significant correlations, optimal curve fitting was performed by using non‐linear regression with a polynomial model (Supplemental Figure 1). P<0.05 was considered significant. RESULTS The mucositis rat model We studied the capacity to absorb enterally administered AA during mucositis in a previously established MTX‐induced mucositis rat model [9]. As seen in previous studies by us and others [9, 36‐41], MTX‐treated rats showed typical histological and clinical symptoms of mucositis (i.e. villus atrophy, a reduced plasma citrulline concentration, a reduced intake, weight loss and watery diarrhea), in contrast to controls (Table 2). Although symptoms of mucositis varied from mild to severe in individual rats, most MTX‐treated rats (7 of 9) suffered from severe mucositis [i.e. villus length <300 μm and plasma citrulline concentration <30 μmol/L [9]] (Supplemental Figure 1). Control MTX Villus length d 4, µm 408 (375‐423) 262 (198‐354) * Citrulline d 4, µmol/L plasma Food intake d 3, gr/d 69 (54‐105) 15 (10‐55) * 11 (8‐12) 0 (0‐9) * 1
Body weight d 4 , % 108 (105‐110) Diarrhea d 42, % of rats 91 (88‐104) * 0 67 Table 2. Characteristics of control and MTX‐treated rats. Values are medians and range, except for diarrhea for which values are absolute, n = 7‐9. *Different from control, P<0.01. MTX, methotrexate. 1 Body weight was relative to weight at d 0 (day of MTX or saline injection), which was arbitrarily set at 100%. 2 Diarrhea was present as watery diarrhea or completely absent. Plasma kinetics of i.v.‐ and i.d.‐infused AA As shown in Table 3, median plasma AA fluxes, based on i.v.‐infused tracers and on i.d.‐infused tracers, were similar in MTX‐treated rats and in controls for 4 out of 5 AA. However, MTX‐treated rats showed substantial interindividual differences in AA fluxes based on i.d.‐infused tracers, with maximal values 500‐1400% higher than maximal values in controls. This was due to a large variability in individual plasma enrichment of i.d.‐infused tracers in MTX‐treated rats (ranging from severely reduced to normal, data not shown). 77 | 169 5 Chapter 5 5 Supplemental Figure 1. Correlation between systemic availability of intraduodenally infused stable isotope‐
labeled amino acids on the one hand and villus length (A, C, E, G and I) or plasma citrulline concentration (B,
D, F, H and J) on the other hand in control (○) and methotrexate (MTX)‐treated (●) rats. Values are data of individual rats, n=7‐9. Spearman correlations (r) and P values are indicated. For significant correlations
(P<0.05), optimal curves were plotted using non‐linear regression with a polynomial model (A, B and H). 78 | 169 Table 3. Kinetics of intravenously‐ and/or intraduodenally infused, stable isotope‐labeled amino acids in control and MTX‐treated rats. Values are medians and range, n=7‐9. *Different from control, P<0.05. MTX, methotrexate. Normal Amino Acid Absorption during Mucositis Median first‐pass splanchnic utilization, and resulting systemic availability, of all i.d.‐infused AA except for leucine were not different between MTX‐treated rats and controls. In contrast to controls, MTX‐treated rats showed substantial inter‐
individual differences (Table 3). Maximal first‐pass splanchnic utilization of all AA was >90% of intake in MTX‐treated rats, whereas it was ≤60% of intake in controls. As a result, minimal systemic availability of all AA was <10% of intake in MTX‐treated rats, while it was ≥40% of intake in controls. Availability of all AA varied from severely reduced (<10% of intake) to not different from controls among individual MTX‐treated rats with symptoms of severe mucositis (7 of 9 rats, Supplemental Figure 1). Although the systemic availability of enterally administered leucine in all rats correlated with villus length (r=0.80, P<0.05), that of lysine, phenylalanine, threonine and methionine did not. Similarly, the systemic availability of enterally administered leucine and threonine in all rats correlated with plasma citrulline (r=0.64‐0.81, P<0.05) but that of lysine, phenylalanine and methionine did not. Protein breakdown in MTX‐
treated rats, based on i.d.‐infused tracers, varied according to AA and was higher than (leucine and 79 | 169 5 Chapter 5 methionine, P<0.05), lower than (threonine, P<0.05) or similar to controls (lysine and phenylalanine, Table 3). Tissue protein and albumin synthesis with i.d.‐infused AA The utilization of enterally administered AA for protein synthesis was expressed by the fractional and absolute synthesis rate of protein (FSR and ASR respectively), indicating the relative and absolute need for AA for protein synthesis, respectively. 5 Small intestinal mucosa. The FSR with systemically available, enterally administered AA (FSRbasolateral) was ≥ 20% higher in MTX‐treated rats than in controls, depending on the specific AA (proximal and distal mucosa, P<0.05, Table 4). However, since the total amounts of mucosa (proximal and distal mucosa ≥49% lower, P<0.05) and/or the protein concentration of mucosa (proximal mucosa 16% lower, P<0.05) were lower in MTX‐treated rats than in controls, the ASR (ASRbasolateral) was lower in MTX‐treated rats than in controls (proximal mucosa, P<0.05) or similar to controls (distal mucosa). In both MTX‐treated rats and in controls, protein synthesis in proximal and distal small intestinal mucosa was higher with enterally administered AA taken up from the systemic side (FSRbasolateral and ASRbasolateral) than with AA taken up from the luminal side (FSRapical and ASRapical, P<0.05). These differences between basolateral and apical AA uptake seemed to be more pronounced for MTX‐treated rats than for controls, both in proximal and distal mucosa. The enrichment of methionine was too low to measure. Albumin. The FSR with systemically available, enterally administered AA was ≥60% higher in MTX‐treated rats than in controls, depending on the specific AA (P<0.05, Table 5). However, since the plasma albumin concentration was lower in MTX‐treated rats than in controls (24% lower, P<0.05), the ASR was similar in MTX‐treated rats and in controls. The enrichment of threonine and methionine was too low to measure. Liver. The FSR with systemically available, enterally administered AA was 20% and 30% higher in MTX‐treated rats than in controls for leucine and lysine, respectively (P<0.05, Table 5), or similar to controls for phenylalanine. Both the total amounts of liver and the protein concentrations in liver were similar in MTX‐treated rats and in controls, and therefore the ASR was also 20% and 30% higher in MTX‐treated rats than in controls for leucine and lysine respectively (P<0.05), or similar to controls for phenylalanine. The enrichment of threonine and methionine was too low to measure. Thigh muscle. The FSR with systemically available, enterally administered AA was similar in MTX‐treated rats and in controls (Table 5). The protein concentration of thigh muscle was similar in MTX‐treated rats and in controls, and therefore the ASR (per kg muscle) was also similar in both groups. The enrichment of threonine and methionine was too low to measure. 80 | 169 Table 4. Amount, protein
concentration, FSR and ASR of
small intestinal mucosa using
intraduodenally infused, stable
isotope‐labeled amino acids in
control and MTX‐treated rats.
Values are medians and range,
n=7‐9. * Different from control,
#
P<0.05. Different from apical
FSR or apical ASR, P<0.05. ASR,
absolute synthesis rate; BW,
body weight; FSR, fractional
synthesis rate; MTX, metho‐
trexate; ND, not detectable. 1 FSR and ASR with intra‐
duodenally infused amino
acids via basolateral uptake
(from the systemic side). 2
FSR and ASR with intra‐
duodenally infused amino
acids via apical uptake
(from the luminal side). Normal Amino Acid Absorption during Mucositis 5 81 | 169 5 Table 5. Amount, protein concentration, FSR and ASR of albumin, liver and thigh muscle using intraduodenally infused, stable isotope‐labeled amino acids in control and MTX‐treated rats. * Different from control, P<0.05. ASR, absolute synthesis rate; BW, body weight; FSR, fractional synthesis rate; MTX, methotrexate. 1
The total amount of muscle per rat is unknown; therefore, the total amount of muscle protein per kilogram BW cannot be calculated. Chapter 5 82 | 169 Normal Amino Acid Absorption during Mucositis DISCUSSION We aimed to determine the absorptive capacity of AA in rats with and without MTX‐induced mucositis. Our data indicate that continuous enteral administration can enable normal AA absorption in rats with MTX‐induced mucositis. The intestine prefers basolateral AA uptake to meet its need for AA for protein synthesis during mucositis. The absorptive capacity of AA was determined in a previously established MTX‐induced mucositis rat model [9]. We chose five essential AA that are absorbed by different transporter systems, normally present on the apical and basolateral membrane of intestinal enterocytes [42, 43], to indirectly test the function of all these systems during mucositis. In earlier studies, only the absorption of leucine was studied during mucositis in children [10]. AA were administered by continuous i.d. infusion, since continuous administration of enteral nutrition during intestinal failure is thought to enhance enteral absorption by maximizing saturation of the (residual) carrier proteins, thereby increasing intestinal function [8]. Furthermore, we previously showed that continuous enteral glucose administration improved glucose absorption during mucositis in the rat, compared with an oral bolus of glucose [23]. The median systemic availability of 4 out of 5 enterally administered AA was similar in MTX‐treated rats and in controls, as found before for leucine [10], suggesting normal absorption of continuously administered AA during mucositis. However, individual availability varied from severely reduced to not different from controls among individual MTX‐treated rats, despite the fact that most rats (7 of 9) suffered from severe mucositis. We hypothesize that these large interindividual differences in AA absorption during mucositis can be explained by the fact that individual MTX‐treated outbred rats might have been in different stages of mucositis [as described by Sonis et al. [1]] during the AA absorption experiment. Absorption of continuously administered AA could have been possible via AA transporters on the recovered epithelial membrane, via residual transporters on damaged epithelial membrane [9] and/or via paracellular absorption [44]. Leakage of AA through damaged tight junctions could also have been possible since mucositis often leads to increased gut permeability [45, 46]. Individual AA availability often did not correlate with villus length or plasma citrulline concentrations. Although plasma citrulline was earlier shown to be a useful surrogate marker for mucositis and for malabsorption of lactose during mucositis [instead of intestinal histology [9]], we showed that plasma citrulline is not a useful marker for AA absorption during mucositis. Apart from the systemic availability of enterally administered AA, we measured their utilization for absolute protein synthesis in small intestinal mucosa, plasma albumin, liver and thigh muscle to learn whether a potentially reduced plasma AA availability would be the result of AA malabsorption from the intestinal lumen or of increased AA 83 | 169 5 Chapter 5 utilization (by first‐pass splanchnic utilization or by utilization after becoming systemically available). A third explanation for reduced plasma AA availability would be increased AA oxidation. Although we did not measure AA oxidation, others found whole body leucine oxidation to be similar between patients with and without mucositis [10]. Because the utilization of enterally administered leucine, lysine, and phenylalanine for tissue protein and albumin synthesis was mostly lower or similar in MTX‐treated rats, compared with controls, reduced systemic AA availability in some MTX‐treated rats (≤21% of intake for all AA in 4 of 9 rats, Supplemental Figure 1) was probably caused by AA malabsorption. The enrichment of threonine and/or methionine was too low to measure. 5 In contrast to absolute synthesis, fractional synthesis of intestinal proteins and plasma albumin with enterally administered leucine, lysine and phenylalanine was higher in MTX‐treated rats than in controls. A relatively increased AA utilization for intestinal protein synthesis during mucositis might indicate an increased renewal of the intestinal mucosa, which seems plausible after initial chemotherapy‐induced intestinal damage [47]. However, intestinal inflammation during mucositis might also cause an increased synthesis of inflammatory proteins, such as myeloperoxidase (MPO) [9]. Albumin synthesis might be increased during mucositis to compensate for the reduced albumin concentrations that we measured. Reduced albumin concentrations during mucositis were probably caused by increased losses via the intestine as seen with other gastroenteropathies like colitis ulcerosa [48]. In general, the rat seems in a catabolic state during mucositis as can be concluded from weight loss and increased protein breakdown (containing leucine and methionine), as found previously [10]. Furthermore, peripheral protein synthesis in thigh muscle per kilogram body weight might have been reduced in MTX‐treated rats, compared with controls, but could not be calculated because the total amount of muscle per rat was unknown. We hypothesized that there could be a preferential side of the intestine for AA uptake during mucositis. In MTX‐treated rats, protein synthesis in proximal and distal small intestinal mucosa with enterally administered leucine, lysine, phenylalanine and threonine was higher when taken up basolaterally than when taken up apically, indicating preferred AA uptake from the systemic side for intestinal protein synthesis during mucositis. Others have shown that enterocytes near the crypt‐villus junction prefer systemically available AA, whereas enterocytes at villus tips prefer AA at the luminal side for protein synthesis [22]. Therefore, AA absorption by the intestine during mucositis was probably mainly performed by enterocytes at the crypt‐villus junction, which is compatible with the observation that villi of MTX‐treated rats were atrophied and damaged while crypts were already regenerating [as shown in this study and seen before by us and others [9, 47, 49]]. Also in controls, AA uptake for intestinal protein synthesis was preferred with AA originating from the systemic side, as found by others [12, 17], although differences seemed less pronounced than in 84 | 169 Normal Amino Acid Absorption during Mucositis MTX‐treated rats, especially in the proximal mucosa. Normally, intestinal AA absorption is very efficient [50]: the proximal small intestine (i.e. duodenum and proximal jejenum) absorbs almost all intraluminal AA, thereby leaving few AA to be absorbed in the distal small intestine (i.e. distal jejunum and ileum). The distal small intestine is therefore mainly dependent on systemically available AA, except for some AA at the luminal side that become available by recycling (proteolysis and reabsorption) of intestinal proteins [12]. If we extrapolate our findings to the clinic, they imply that enteral AA administration by continuous infusion could be useful for a substantial portion of mucositis patients, in order to improve their nutritional state, recuperation, and survival [4‐6, 51]. Furthermore, enteral nutrition could possibly accelerate intestinal recovery since intraluminal nutrients have a stimulatory effect on intestinal epithelial cells and the production of trophic hormones [52‐54]. Our results show that AA are important for mucositis patients so that they can meet their need for AA for intestinal protein synthesis and albumin synthesis. We determined AA absorption during mucositis at d 4 after injection with MTX or saline. However, symptoms of mucositis are actually present during a longer period of time; from d 2 until 5 after injection with MTX [9]. We do not know whether the observed AA malabsorption in a portion of mucositis rats is structural (present on all days during mucositis) or temporal (only present on d 4). When AA malabsorption is structural, indeed only a portion of mucositis patients would benefit from continuous enteral AA administration. However, if AA malabsorption is temporal, all mucositis patients might benefit from continuous enteral AA administration during mucositis. Future studies should focus on studying the effects of continuous enteral AA administration in mucositis patients. If only a portion of patients benefit from continuous enteral AA, a marker that distinguishes between mucositis patients with a good or poor AA absorptive capacity would be highly desirable to anticipate which patients would benefit the most from continuous enteral AA administration. For now, parenteral AA administration might be a rational alternative for enteral AA administration to guarantee optimal AA availability in all patients with mucositis. In conclusion, we show that continuous enteral administration can enable normal AA absorption in rats with chemotherapy‐induced gastrointestinal mucositis. The intestine prefers basolateral AA uptake to meet its need for AA for protein synthesis during mucositis. So, although the gut might be usable for AA uptake in at least a portion of mucositis patients, when enterally administered continuously, parenteral AA administration might be a rational alternative to guarantee optimal AA availability in all patients with mucositis. 85 | 169 5 Chapter 5 SUPPLEMENTAL METHODS The capacity to absorb enterally administered amino acids (AA) during mucositis was studied by determining their systemic availability and utilization for (fractional and absolute) protein synthesis. To determine the systemic availability of enterally administered AA, we first determined the rate of turnover, or flux, of enterally (intraduodenally, i.d.‐) and intravenously (i.v.‐) infused tracers. Then, the first‐pass splanchnic utilization and resulting systemic availability of enterally administered AA was determined. After the systemic availability was known, the amount of protein breakdown [indicating catabolism] could also be determined. Isotopic enrichment of plasma AA (in mole percent excess) was used to calculate the flux (Q, in mmol∙kg‐1∙h‐1). The AA flux obtained with i.d.‐administered tracers (13C‐labeled, Table 1) and i.v.‐administered tracers (2H‐ or 15N‐labeled, Table 1), the determination of first‐pass splanchnic utilization (in % of AA intake) and resulting systemic availability of i.d.‐administered AA (i.e. AA intake ‐ first‐pass uptake, in %) were calculated as previously described [10, 13]. Protein breakdown (in mmol∙kg‐1∙h‐1) was calculated by using the following equation: 5 Qi.v. = Intake + Breakdown [10, 13] where Qi.v. is the flux of the i.v.‐administered tracer (mmol∙kg‐1∙h‐1) and Intake represents the systemic availability (in % of intake) of i.d.‐administered AA (in mmol∙kg‐1∙h‐1). So, breakdown was calculated by: Breakdown = Qi.v ‐ (systemic availability x infusion rate of i.d.‐administered AA) The utilization of enterally administered AA for protein synthesis was expressed by the fractional synthesis rate (FSR) and by the absolute synthesis rate (ASR) of protein. Both the FSR and ASR were determined in proximal and distal small intestinal mucosa, plasma albumin, liver and thigh muscle. The FSR (in %/d) reflects the percentage of the total protein pool that is newly synthesized per day, and therefore indicates the relative need for AA for protein synthesis. Values were calculated as previously described [30, 55]. The isotopic enrichment of AA in tissues and albumin at baseline was assumed to be 0 mole percent excess. Normally, the enrichment of the intracellular free (unbound) AA pool (for tissues, i.e. small intestinal mucosa, liver, thigh muscle) or the plasma AA enrichment (for plasma albumin) are used as precursors. However, since the enrichment of intracellular free AA turned out to be extremely low in our tissues [possibly due to proteolysis as a result of freezing and thawing], we used the plasma AA enrichment as a precursor for small intestinal mucosa, liver and thigh muscle. The ASR (in g/d) reflects the total amount of protein that is newly synthesized per day, and therefore indicates the absolute need for AA for protein synthesis. It was measured as the FSR multiplied by the protein mass of 86 | 169 Normal Amino Acid Absorption during Mucositis the organ in g/L (plasma albumin), in g/kg organ (thigh muscle, since only a sample of this muscle was collected) or in g/kg body weight (intestinal mucosa and liver). We also aimed to determine the preferential side of the intestine to take up enterally administered AA for protein synthesis during mucositis. The i.v. infusion of AA leads to exclusive intestinal uptake from the systemic side (basolaterally). However, during i.d. infusion of AA, the intestinal uptake of enterally administered AA is from the luminal side (apically) but, after transport by the enterocyte into the systemic pool, also from the systemic side (basolaterally). Thus, by the end of the dual‐tracer infusion, there are two populations of labeled AA in the small intestinal mucosa: i.d.‐administered 13
C‐labeled AA derived from both the luminal and the systemic side, and i.v.‐administered 2H‐ or 15N‐labeled AA derived directly from the systemic side (Table 1). The enrichment of 13C‐labeled AA absorbed from the systemic side (E[13C]AA‐
2
15
basolateral) was calculated by using the fraction of plasma [ H or N]AA incorporated into mucosa as precursor pool, as described before [56]: E[13C]AAbasolateral = E[13C]AAplasma x (E[2H or 15N]AAmucosa / E[2H or 15N]AAplasma) where E[13C]AAplasma is the enrichment of [13C]AA in plasma, [2H or 15N]AAmucosa is the enrichment of [2H or 15N]AA in intestinal mucosa and E[2H or 15N]AAplasma is the enrichment of [2H or 15N]AA in plasma. Then, the enrichment of [13C]AA in mucosa absorbed from the luminal side (E[13C]AAapical) was calculated by: E[13C]AAapical = E[13C]AAmucosa ‐ E[13C]AAbasolateral where E[13C]AAmucosa is the total [13C]AA enrichment in intestinal mucosa, i.e. from both the apical and the basolateral side. To calculate the intestinal FSR with i.d.‐administered AA taken up from the systemic side (FSRbasolateral), E[13C]AAplasma was used as a precursor. To calculate the intestinal FSR with i.d.‐administered AA taken up from the luminal side (FSRapical), the enrichment of [13C]AA in the i.d. infusate was used a precursor. As mentioned before, the enrichment of intracellular free AA was too low to use as a precursor. Instead, we used AA enrichment of plasma or of the intraduodenal infusate as precursors, which were probably higher than enrichment of intracellular free AA would have been, because of intracellular dilution after AA uptake from the plasma or intestinal lumen. Therefore, calculated FSR’s and ASR’s might be somewhat underestimated. However, differences between MTX‐treated rats and controls are expected to be similar, regardless of the precursor that is used. 87 | 169 5 Chapter 5 ACKNOWLEDGEMENTS The authors thank Rick Havinga, Juul Baller, and Angelika Jurdzinski for technical assistance in our studies. GRANTS This study was financially supported by an unrestricted research grant from KiKa Kinderen Kankervrij (the Netherlands). REFERENCES 1.
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Dietary modulation of innate defense (Dissertation). Erasmus MC, Rotterdam, the Netherlands 90 | 169 Normal Amino Acid Absorption during Mucositis 5 91 | 169 Clinical Nutrition; under review M. Fijlstra W.J.E. Tissing H.J. Verkade E.H.H.M. Rings CHAPTER 6 PARENTERAL FEEDING DURING METHOTREXATE‐
INDUCED GASTROINTESTINAL MUCOSITIS PREVENTS WEIGHT LOSS IN THE RAT Chapter 6 ABSTRACT Background It is unknown what feeding strategy to use to prevent weight loss in patients with chemotherapy‐induced gastrointestinal mucositis. In a mucositis rat model, we demonstrated disaccharide maldigestion and fat malabsorption, but up to normal absorption of glucose and amino acids upon their continuous enteral administration. We now determined the effects of 4 different (par)enteral feeding strategies during mucositis on body weight and intestinal recovery. Methods From day 2‐5 after injection with methotrexate (60mg/kg), rats continued ad libitum AIN‐93G (strategy 1), received continuous enteral feeding with glucose and amino acids (Nutriflex®, strategy 2) or with standard formula (Nutrini®, strategy 3), or received standard parenteral feeding (NuTRIflex® Lipid, strategy 4). Saline‐treated controls continued ad libitum AIN‐93G. Results From day 2 on, methotrexate‐treated ad libitum‐fed rats showed a reduced intake and body weight (P<0.05), while most enterally‐fed rats (88%) were terminated early (because of severe abdominal distention). Parenterally‐fed rats grew similarly like controls. On day 5, the jejunum of methotrexate‐treated ad libitum‐fed rats showed hypertrophic crypts and a normal villus length while parenterally‐fed rats showed villus atrophy, compared with controls (P<0.05). Conclusions Continuous enteral feeding during mucositis is poorly tolerated in rats. Parenteral feeding prevents weight loss, but ad libitum feeding causes accelerated intestinal recovery (both well tolerated). 6 INTRODUCTION Gastrointestinal mucositis (“mucositis”), a severe side effect of many anti‐cancer treatments, causes villus atrophy and loss of enterocytes [1]. Patients with mucositis suffer from anorexia, diarrhea and weight loss [1]. Since weight loss seems primarily the result of a reduced intake [2], (force‐) feeding might be able to prevent weight loss during mucositis. It is unknown how to optimally feed patients with mucositis although it seems reasonable to assume that nutritional support might improve the nutritional state, accelerate recuperation and increase survival of mucositis patients. Enteral feeding might be inappropriate because of potential nutrient maldigestion and malabsorption during mucositis [1]. Parenteral feeding (TPN) on the other hand bypasses potential intestinal malfunction, but this invasive approach carries an increased risk of infection [3]. To determine nutrient digestion and absorption during mucositis, we developed a methotrexate (MTX)‐induced mucositis rat model [4]. We showed that the digestion of disaccharides is compromised during mucositis [4], as is the absorption of long‐chain fatty acids [5]. Interestingly, the absorption of glucose and amino acids could be normal during mucositis, upon their continuous enteral administration [6, 7]. 94 | 169 Prevention of Weight Loss during Mucositis Here, we determined the effects of 4 different (par)enteral feeding strategies during mucositis on body weight in the rat, and compared them with body weight in saline‐treated controls. We also determined the effects of these feeding strategies on intestinal recovery by assessing plasma citrulline concentrations and intestinal histology [4]. Plasma citrulline has been shown to be a good marker for the histological severity of, and gut function during, mucositis [4]. MATERIALS AND METHODS Rats and housing Male Wistar Unilever outbred rats (3.5 wk old, 50‐65 g) were obtained from Charles River (Sulzfeld, Germany). Rats were individually housed in Plexiglas cages under controlled temperature and humidity, and a 12:12‐h light‐dark cycle [4]. Water and purified diet (AIN‐93G [4, 8]) were available ad libitum unless otherwise stated. The experimental protocol was approved by the Ethics Committee for Animal Experiments [4]. The mucositis rat model At the age of 5.5 wk, rats were either sham operated or equipped with permanent catheters in the duodenum or jugular vein [9] (Table 1). At the age of 6‐7 wk, rats (175‐210 g) were injected intravenously with MTX (60 mg/kg [4], day 0) or‐saline (0.9%, controls). Food intake and body weight were daily recorded [4]. Blood samples were daily obtained from the tail tip (75 µl) under general anesthesia to measure plasma citrulline concentrations [4]. On day 2, rats were individually housed in custom made Plexiglas cages (48.0 x 26.5 x 21.0 cm) with filter top (model 2154F, Tecniplast), modified to accommodate a swivel joint and counterbalance system [9]. Rats were attached to swivels under general anesthesia. The experimental model allows for continuous (force‐) feeding in unanesthetized rats without the interference of stress or restraint. At day 5, rats were killed [4], the abdomen was opened [4] and photographs were taken (macroscopic overview, Nokia E5, 5 MP camera). The small intestine was excised, flushed with ice‐cold PBS, and intestinal parts were collected for histology [4]. Enteral and parenteral feeding during mucositis From day 2 until 5 (± 10:00 A.M.‐10:00 A.M.) after MTX injection, the period when symptoms of mucositis are present [4], rats followed one of 4 feeding strategies. They continued ad libitum purified diet (AIN‐93G, n=9, strategy 1), received continuous enteral feeding with either glucose and amino acids (Nutriflex® plus 48/150, BBraun, Oss, the Netherlands, mixed with sterile water to reduce its high osmolarity, n=7, strategy 2), or with standard tube‐feeding (Nutrini®, Nutricia, Zoetermeer, the Netherlands, n=9, strategy 3), or received standard parenteral feeding (NuTRIflex® Lipid special, BBraun, Oss, the Netherlands, n=6, strategy 4) as summarized in Table 1. 95 | 169 6 Chapter 6 Table 1. Details of the different feeding strategies from day 2‐5
after injection with methotrexate (MTX) or saline (control). 1
Gluc/AA = glucose/amino acids 2 The i.v. catheter of one rat was lost after one day of TPN 3
147 ml sterile water/kg BW/day was added to the diet in order
to reduce the high osmolarity to 930 mOsmol/l (total
volume = 339 ml/kg BW/day) 4
MCT = medium chain triglycerides 5
From earlier experiments (ref. 4), we know that the average
intake of saline treated controls ≈ 329 kcal/kg body weight
(BW)/day (see ‘Materials and Methods’) 6 Saline‐treated controls continued ad libitum intake (AIN‐93G, n=5). Calorie admini‐
stration (infusion with pumps from Terumo STC‐521) was based on the average daily food intake in saline‐treated controls (20g AIN‐93G [3760 kcal/kg [8]] per 230g bodyweight [4] ≈ 329 kcal/kg bodyweight/day) and was daily adjusted to the body weight. Maximal enteral and parenteral glucose administration was set at 1.7 and 1.6 g anhydrous glucose/kg bodyweight/day respectively (≈ 85% of average hourly carbohydrate intake) to avoid severe hyperglycemia [6]. To test whether rats would tolerate calculated volumes of nutrition, as found by others [10, 11], we had executed a pilot study in saline‐treated rats with exactly similar feeding strategies and found that rats showed similar growth and intestinal histology upon ad 96 | 169 Prevention of Weight Loss during Mucositis libitum, continuous enteral or parenteral feeding (data not shown). Histological assessment, determination of plasma citrulline and statistical analysis were performed as described previously [4]. RESULTS Ad libitum intake during mucositis (strategy 1) MTX‐treated ad libitum‐fed rats showed a reduced food intake and body weight from day 2 and 3 on respectively, compared with saline‐treated controls, as seen before [4] (Figure 1A and B, P<0.05). From day 1 on, plasma citrulline concentrations were reduced, compared with controls (Figure 1C, P<0.05). At termination (day 5), rats showed an increased crypt length (P<0.05, indicating crypt regeneration [4]) and a normal villus length, compared with controls (Figure 1D and E). However, histology varied substantially among individual rats, ranging from signs of ‘recovery from mucositis’ (Figure 1Fd) to ‘active mucositis’ (Figure 1Fe), as seen before [4]. Continuous enteral feeding during mucositis (strategy 2 and 3) Most MTX‐treated enterally‐fed rats (all rats that received standard tube feeding and 5 out of 7 rats that received glucose and amino acids) had to be terminated within 2 days of feeding because of severe watery diarrhea, abdominal distention (Figure 1Ff), lethargy and hyperglycemia (up to 30 mmol/L). Histology confirmed severe mucositis (crypt damage and villus atrophy, Figure 1Fg) as seen before at day 3 and 4 after MTX injection [4]. The 2 rats that survived glucose and amino acids actually grew similarly as saline‐treated controls (data not shown). Moreover, at day 5, their plasma citrulline concentrations were higher (median 58, range 54‐63 µmol/L) than in ad libitum‐fed MTX‐treated rats (median 23, range 15‐33 µmol/L, P<0.05), while median crypt and villus length was similar between these groups (data not shown). Continuous parenteral feeding during mucositis (strategy 4) In contrast to MTX‐treated ad libitum‐fed rats, parenterally‐fed rats grew similarly like saline‐treated controls (Figure 1B). Plasma citrulline concentrations were similarly reduced as in MTX‐treated ad libitum‐fed rats, compared with controls (Figure 1C, P<0.05). At termination, parenterally‐fed rats showed no increase in crypt length and a reduced villus length (P<0.05), compared with controls, in contrast to ad libitum‐fed rats (Figure 1D and E). Histology varied substantially among individual rats, like in ad libitum‐fed rats, ranging from signs of ‘recovery from mucositis’ (Figure 1Fi) to ‘active mucositis’ (Figure 1Fj). 97 | 169 6 Chapter 6 6 Figure 1. The effects of (par)enteral feeding during mucositis on body weight and intestinal recovery. Food
intake (A), body weight (B), plasma citrulline concentration (C), jejunal crypt and villus length (D and E) and
macroscopic (F, upper panels) and histological (F, lower panels) overview in methotrexate (MTX)‐ or saline‐
treated (control) rats (i.v. injection at day 0) before, during or after ad libitum AIN 93 feeding (control, ○‐‐‐ ,
n=5 or MTX ad lib, ● ▬, n=9 or) or during parenteral feeding (MTX + TPN, ▲ ▬ in blue). Intake is shown
until day 4, since rats were terminated at day 5. The macroscopic and histological overview is also given for
rats that received continuous enteral feeding (MTX + EF) that had to be terminated early because of severe
abdominal distention. Data represent medians and p25‐p75 (A C) or data of individual rats (D E). The extra
vertical line (A C) marks the start of continuous (par)enteral feeding (day 2). * and ^ indicate significant
changes between ‘MTX ad lib rats’ and ‘MTX + TPN rats’ respectively on the one hand and saline treated
$
control rats on the other hand (P<0.05). indicates significant changes between ‘MTX ad lib rats’ and ‘MTX +
TPN rats’ (P<0.05). NS means ‘not significant’. 98 | 169 Prevention of Weight Loss during Mucositis DISCUSSION We determined the effects of 4 different (par)enteral feeding strategies during mucositis in the rat on body weight and intestinal recovery. Continuous enteral feeding was often not tolerated during mucositis, in contrast to ad libitum and parenteral feeding. While parenteral feeding prevented weight loss during mucositis, as present in ad libitum‐fed rats, ad libitum feeding caused accelerated intestinal recovery. Continuous enteral feeding during mucositis consisted either of glucose and amino acids (since absorption of these nutrients was up to normal upon their continuous enteral administration [6, 7]) or of standard formula in order to mimick the clinical situation in pediatric oncology. Since several complex nutrients in standard formula cannot be absorbed during mucositis [4], similar weight loss as present in ad libitum‐fed rats (seen in this study and previously [4]) was expected upon this feeding. In contrast, enteral glucose and amino acids were expected to at least reduce or even prevent weight loss during mucositis. Parenteral feeding, used in adult mucositis patients [12], should be able to allow normal growth during mucositis since nutrients are delivered directly into the blood and the damaged intestine is completely bypassed. We found that both continuous enteral feeding strategies were poorly tolerated during mucositis. In contrast, all parenterally fed rats with mucositis grew similarly as non‐mucositis controls. Intestinal recovery was studied by plasma citrulline concentrations and jejunal histology. We had previously shown that plasma citrulline concentrations are reduced at day 4 after MTX injection, indicating reduced functional enterocyte mass [4]. We now show that plasma citrulline already starts to decrease at 1 day after MTX injection. Plasma citrulline was similarly reduced in all MTX‐treated rats before feeding was started (at day 2, including rats that were going to receive enteral feeding), indicating similar levels of mucositis [4]. Therefore, differences in body weight, citrulline and histology hereafter must have been caused by the different feeding strategies. In the few rats that tolerated feeding with glucose and amino acids, plasma citrulline was higher than in ad libitum‐fed MTX‐treated rats, while histology was similar between these groups. Upon parenteral feeding, plasma citrulline did not improve and intestinal histology worsened, compared with ad libitum‐fed MTX‐treated rats. Altogether, we saw advantageous effects of enteral feeding (including minimal intake in ad libitum‐fed rats) during mucositis on intestinal citrulline synthesis (preferably glucose and amino acids) and histology, in comparison with solely parenteral nutrition. Advantageous effects of enteral nutrition were also seen during other forms of intestinal failure [13]. We conclude that further research in mucositis patients is indicated to determine their optimal feeding strategy: either parenteral feeding (quite invasive, increased risk 99 | 169 6 Chapter 6 of infection [3]) to prevent weight loss at the expense of delayed intestinal recovery, or continuous enteral feeding in tolerated amounts (preferably glucose and amino acids) to stimulate intestinal recovery at the expense of weight loss, or maybe a combination of both. ACKNOWLEDGEMENTS The authors wish to acknowledge Rick Havinga, Juul Baller, Pieter Klok, Angelika Jurdzinski, Wolter de Goede, Rick Oosterhuis, Cyril Moers, Petra Ottens and Leo Deelman (University Medical Center Groningen, the Netherlands) for excellent technical and/or facilitative assistance in our studies. Also, Ronald Jan Corbee (Faculty of Veterinary Medicine, University of Utrecht, the Netherlands) is gratefully thanked for his advice regarding enteral and parenteral feeding of rats. GRANTS This work was financially supported by an unrestricted research grant from Fonds NutsOhra (the Netherlands). REFERENCES 1.
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Blijlevens NM, Donnelly JP, Naber AH, Schattenberg AV, DePauw BE (2005) A randomised, double‐blinded, placebo‐controlled, pilot study of parenteral glutamine for allogeneic stem cell transplant patients. Support Care Cancer 13:790‐796 13. Buchman AL, Scolapio J, Fryer J (2003) AGA technical review on short bowel syndrome and intestinal transplantation. Gastroenterology 124:1111‐1134 6 101 | 169 Based on the original paper in Supportive Care in Cancer (in press) R. Gibson D. Keefe R. Lalla E. Bateman N. Blijlevens M. Fijlstra E. King A. Stringer W. van der Velden R. Yazbeck S. Elad J. Bowen CHAPTER 7 SYSTEMATIC REVIEW OF AGENTS FOR THE MANAGEMENT OF GASTROINTESTINAL MUCOSITIS IN CANCER PATIENTS Chapter 7 ABSTRACT Background The aim of this study was to review the available literature and define clinical practice guidelines for the use of agents for the prevention and treatment of gastrointestinal mucositis. Methods A systematic review was conducted by the Mucositis Study Group of the Multinational Association of Supportive Care in Cancer / International Society of Oral Oncology (MASCC/ISOO). The body of evidence for each intervention, in each cancer treatment setting, was assigned an evidence level. Based on the evidence level, one of the following three guideline determinations was possible: Recommendation, Suggestion, No guideline possible. Results A total of 251 clinical studies across 29 interventions were examined. Panel members were able to make one new evidence‐based negative recommendation; two new evidence‐based suggestions and one evidence‐based change from previous guidelines. Firstly, the panel recommends against the use of misoprostol suppositories for the prevention of acute radiation‐induced proctitis. Secondly, the panel suggests probiotic treatment containing Lactobacillus spp. may be beneficial for prevention of chemotherapy and radiotherapy‐induced diarrhea in patients with malignancies of the pelvic region. Thirdly, the panel suggests the use of hyperbaric oxygen as an effective means in treating reducing radiation‐induced proctitis. Finally, new evidence has emerged which is in conflict with our previous guideline surrounding the use of systemic glutamine, meaning that the panel is unable to form a guideline. No guideline was possible for any other agent, due to inadequate and/or conflicting evidence. Conclusion This updated review of the literature has allowed new recommendations and suggestions for clinical practice to be reached. This highlights the importance of regular updates. INTRODUCTION 7 Gastrointestinal (GI) mucositis is an extremely common toxicity occurring after chemotherapy and radiotherapy for cancer [1, 2]. Mucositis occurs in approximately 40% of patients after standard doses of chemotherapy and in up to 100% of patients undergoing high‐dose chemotherapy and hematopoietic stem cell transplantation, or radiation for head and neck cancer [3, 4], affecting over 2 million people worldwide each year. GI mucositis is associated with many symptoms of which significant pain, ulceration, abdominal bloating, nausea, vomiting, diarrhea and constipation are a few [5]. The potentially severe nature of GI mucositis can have some further devastating effects for patients including a reduction or cessation of treatment (which may decrease the chance for remission or cure), and increased stays in hospitals, leading to increased costs of treatment and use of opioids for pain management [5, 6]. In addition to the economic costs associated with GI mucositis, there is also a significant impact on the quality of life of cancer patients with increased morbidity and mortality [7]. There is currently a huge unmet market for management interventions for 104 | 169 Systematic Review of Agents for Management of Gastrointestinal Mucositis gastrointestinal symptoms of GI mucositis. There are a number of new agents which have been trialed in the clinical setting. This paper reports the findings of the most recent updated review, against the background of the previous literature, as related to the use of agents for GI mucositis. In May 2004, the Mucositis Study Group of the Multinational Association of Supportive Care and Cancer/International Society for Oral Oncology (MASCC/ISOO) published results of an evidence‐based review of the clinical literature on mucositis [8]. These results were then updated in 2007 [9]. Both of these critical reviews examined in detail the literature from January 1996 to May 2002 (Original Guidelines) and January 2002 to May 2005 (updated Guidelines). Where made possible by the literature, evidence‐based guidelines were determined both for the prevention and treatment of GI mucositis. It is well recognized that the underlying pathobiology of mucostis is the same throughout the alimentary tract [10] meaning that regardless of where mucositis occurs in the gut, it will still pose significant problems. What does make the alimentary tract beyond the oral cavity different, however is the differences in morphology, which are largely related to the specialized function that each performs [10]. Whilst we have made significant progress in our understanding of mucositis “beyond” the oral cavity, progress is difficult due to the relative inaccessibility of the small and large intestine and the obvious difficulty in obtaining biopsies at multiple time points after cytotoxic therapy. Nevertheless, evidence‐based guidelines for the prevention and treatment of GI mucositis were able to be formed using symptoms and signs as clinical endpoints. As part of a comprehensive update of the MASCC/ISOO clinical practice guidelines for mucositis, the aim of this project was to systematically review the available literature and define evidence‐based clinical practice guidelines for the use of agents for the prevention and treatment of gastrointestinal mucositis. 7 METHODS The methods, including detailed search strategies, inclusion and exclusion criteria, and rubric for assigning levels of evidence to form guidelines, are described in detail in a special mucositis issue in Supportive Care in Cancer (in press). Briefly, a literature search for relevant papers indexed in Medline on or before 31st December 2010 was conducted using OVID/MEDLINE, with papers selected for review based on defined inclusion and exclusion criteria. Papers were reviewed by two independent reviewers and data was extracted using a standard electronic form. Studies were scored for their Level of Evidence based on Somerfield criteria [11] and flaws were listed according to Hadorn criteria [12]. A well‐designed study was defined as a study with no major flaws per the Hadorn criteria. Findings from the reviewed studies were integrated into guidelines based on the overall Level of Evidence for each intervention. Guidelines were classified into 3 types: recommendation, suggestion, and no guideline possible. 105 | 169 Chapter 7 Guidelines were separated based on (1) the aim of the intervention (prevention or treatment of mucositis); (2) the treatment modality (radiotherapy, chemotherapy, chemoradiotherapy, or high dose chemotherapy for hematopoietic stem cell transplant) and (3) the route of administration of the intervention. The list of intervention keywords used for the literature search of this section was as follows; 5‐aminosalycilates, all‐trans retinoic acid, amifostine, aminoguanidine, atropine, balsalazide, basic water, bellyboard, benzydiamine, budesonide, butyrate, cefixime, celecoxib, charcoal, chlorhexidine, cholestyramine, circadian variation, endorectal balloon, exercise, glutamine, hyperbaric oxygen, indomethacin, intestinal alkalization, intra‐abdominal tissue expanders, Kampo medicine, keratinocyte growth factor, kremazin, levofloxacin, loperamide, magnesium oxide, mesalamine, mesalazine, mesh, misoprostol, neomycin, nifuroxazide, octreotide, olsalazine, palifermin, pelvic displacement prosthesis, probiotics, prostaglandin, psychoeducation, racecadotril, relaxation, sodium bicarbonate, sucralfate, sulphasalazine, superoxide dismutase, tantum rosa, thalidomide, ursodeoxycholic acid, WR‐2721. RESULTS 7 A total of 1336 papers were originally identified of which 1040 papers were excluded after evaluating the title/abstract. 296 papers were then retrieved for detailed analysis, with a further 45 papers excluded prior to being sent out to review for not meeting inclusion criteria. Finally 251 papers were sent out for assessment, with 146 included in the final review. Papers were excluded due to not meeting study inclusion criteria (for more details refer to Bowen et al. and Elad et al. in the special mucositis issue in Supportive Care in Cancer). The following interventions were included in the final review: 5‐aminosalycyclic acid (5‐ASA); acetorphan; activated charcoal; amifostine; antidiarrheal programs; balsalazide; busedinide; cefixime; celecoxib; cholestyramine/levofloxacin; chrysin; circadian rhythms; formalin instillation; glutamine; hyperbaric oxygen; heater probes; leucovorin; loperamide; mesalazine, metronidazole; misoprostol; neomycin; octreotide; olsalazine, palifermin; physical activity; probiotics; salicylazosulfapyridine; sodium butyrate, sucralfate and sulfasalazine (Table 1). Of these interventions reviewed, one had sufficient evidence to produce a new evidence‐based recommendation; two had sufficient evidence to produce new evidence‐based suggestions and one had sufficient evidence to warrant changing a guideline. All remaining interventions had insufficient evidence to form a guideline. 106 | 169 Systematic Review of Agents for Management of Gastrointestinal Mucositis Standard‐dose chemotherapy; OR standard dose radiotherapy OR concomitant chemotherapy / radiotherapy: Prevention
Probiotics: New Guideline Guideline: The panel suggests probiotic treatment containing Lactobacillus spp. may be beneficial for prevention of chemotherapy and radiotherapy‐induced diarrhea in patients with pelvic malignancies. At this stage the panel cannot recommend doses/regimen as the studies to date have investigated a wide variety of products. Nonetheless, Lactobacillus‐containing probiotics as a class of intervention are to date overwhelmingly positive. In three separate randomized controlled studies [13‐15] patients who received probiotics had significantly reduced diarrhea when compared to control patients. Delia et al randomized 490 patients to receive probiotics (VSL#3) or placebo following adjuvant postoperative radiation therapy and found that patients receiving placebo had significantly more diarrhea than probiotics patients (p<0.001). Furthermore placebo patients had significantly more grade 3 or 4 diarrhea compared with probiotics patients (p<0.001) [13]. Osterlund and colleagues randomized 150 patients receiving concomitant chemoradiotherapy for colorectal cancer to probiotics (Lactobacillus rhamnosus GG) or placebo. Patients receiving probiotics had significantly less diarrhea than placebo controls (p < 0.027) [14]. Urbancsek and colleagues found in a randomized study of 200 patients receiving pelvic radiotherapy that probiotics (Lactobacillus rhamnosus) significantly improved stool consistency (P<0.05) compared to controls. Other studies were similarly positive. Amifostine: No change Guideline: The panel suggests the use of amifostine to reduce oesophagitis induced by concomitant chemotherapy and radiotherapy in patients with nonsmall cell lung carcinoma. Updates in the literature since the publication of the 2006 guidelines have seen small studies with conflicting results that do not help to delineate the role of amifostine further [41, 42, 48, 49] and therefore the panel continues to suggest the use of amifostine in this setting. Radiotherapy: Prevention 5‐ASA, mesalazine and olsalazine: No change Guideline: The panel recommends that 5‐ASA and the related compounds mesalazine and olsalazine not be used for the prevention of GI Mucositis. There has been no updated literature since the publication of the original guidelines in 2004 [9]. Briefly, three independent randomized controlled trials [16‐18] reported that 5‐ASA, mesalazine and olsalazine offered no protection for patients receiving external radiotherapy. Of concern was that these compounds caused significantly more diarrhea than the placebo counterpart, leading to the early closure of one study [17]. Therefore the panel continues to recommend against the use of these compounds. 107 | 169 7 Chapter 7 Amifostine: No change Guideline: The panel recommends that amifostine should be administered intravenously at a dose ≥340 mg/m2 prior to radiotherapy to prevent radiation proctitis. There have been no updates in the literature since the publication of the original guidelines and therefore the panel continues to recommend the use of intravenous amifostine. Circadian rhythm: No change Guideline: No guideline possible. However in light of emerging research, administering standard radiotherapy for pelvic malignancies may be delivered using circadian rhythm to minimize toxicity in the future. A single well designed randomized controlled trial [19], reported findings of 229 patients who were randomized to receive radiotherapy in the morning (8.00‐10.00 A.M.) or the evening (6.00‐8.00 P.M.). Patients randomized to the morning arm had significantly worse mucositis overall (p<0.01) and grade 3‐4 diarrhea was significantly increased (p<0.05) compared to patients randomized to the evening arm. Importantly there was no difference in patient response to treatment between the two arms (p>0.05) [19]. This study provides novel and exciting evidence of the effect of circadian rhythm on intestinal mucosa. The panel was encouraged by these findings and look forward to more evidence in future years. 7 Misoprostol: New Guideline Guideline: The panel recommends against the use of misoprostol suppositories for the prevention of acute radiation‐induced proctitis. In two separate randomized controlled trials [20, 21] men receiving radiotherapy for prostate cancer received either misoprostol suppositories or placebo control. There was no significant difference in radiation‐induced proctitis between the two groups, however, patients receiving misoprostol suppositories experienced significantly more rectal bleeding (p<0.03) [20]. Kertesz et al however found that although misoprostol did not influence acute radiation‐induced toxicity, it had no negative impact on the patients [21]. Sucralfate: No change Guideline: The panel recommends that oral sucralfate not be used to reduce the side effects induced by radiotherapy. It does not prevent acute diarrhea in patients with pelvic malignancies undergoing external beam radiotherapy. Furtherore, compared with placebo, it is associated with increased GI side effects including rectal bleeding. There have been no updates in the literature since the publication of the original guidelines and therefore the panel continues to recommend against the use of oral sucralfate. Sulfasalazine: No change Guideline: The panel suggests the use of 500 mg of sulfasalazine administered orally twice daily to help reduce the incidence and severity or radiation‐induced 108 | 169 Systematic Review of Agents for Management of Gastrointestinal Mucositis enteropathy in patients receiving external beam radiotherapy to the pelvis. Again, there have been no updates in the literature since the publication of the original guidelines and therefore the panel continues to suggest the use of sulfasalazine. Radiotherapy: Treatment Hyperbaric Oxygen: New Guideline Guideline: The panel suggests that use of hyperbaric oxygen may be an effective means in treating radiation‐induced proctitis. Fifteen studies were reviewed [22‐34; 94, 95] and were all positive, with many patients experiencing complete resolution of their radiation‐induced proctitis. Costs may well be prohibitive, but all studies show similar results. Sucralfate: No change Guideline: The panel suggests the use of sucralfate enemas as an effective way of managing chronic radiation‐induced proctitis in patients with rectal bleeding. There have been no updates in the literature since the publication of the original guidelines and therefore the panel continues to suggest the use of sucralfate enemas. Standard‐dose and high‐dose chemotherapy: Prevention Glutamine: Changed Guideline Guideline: No Guideline possible. The previous guideline was not to use systemic glutamine because of severe toxicity. However, three new double‐blinded randomized controlled trials published since the last update, have shown effect without severe toxicity. These studies had small numbers of patients and therefore the panel is unable to make a clinical guideline based on conflicting data. Blijlevens and colleagues found that in patients receiving stem cell transplants who received glutamine via parenteral nutrition had significantly improved gut scores (p<0.001) [35]. Sornsuvit and colleagues also reported that patients who received glutamine maintained their nutritional status [36], whereas Li et al reported that glutamine prevented intestinal permeability and clinical manifestations of chemotherapy‐induced gut toxicity [37]. Standard‐dose and high‐dose chemotherapy: Treatment Octreotide: No change Guideline: When loperamide fails to control diarrhea induced by standard‐dose or high‐dose chemotherapy associated with HSCT, the panel recommends octreotide at a dose of ≥100g subcutaneously, twice daily. No guideline was possible for the remaining agents due to inadequate and/or conflicting evidence: 109 | 169 7 Chapter 7 Table 1 (this page and following pages). Agents reviewed for the management of gastrointestinal
mucositis. NSCLC = Non small cell lung carcinoma, PO = per oral, IV = intravenous, SC = subcutaneous,
IM = intramuscular, P = prevention, T = treatment. 7 110 | 169 Systematic Review of Agents for Management of Gastrointestinal Mucositis 7 111 | 169 Chapter 7 7 112 | 169 Systematic Review of Agents for Management of Gastrointestinal Mucositis 7 113 | 169 Chapter 7 7 114 | 169 Systematic Review of Agents for Management of Gastrointestinal Mucositis Table 2. Guidelines with no changes. Guidelines with no changes Agent Guideline •
Amifostine •
The panel suggests the use of amifostine to reduce oesophagitis induced by concomitant chemotherapy and radiotherapy in patients with nonsmall cell lung carcinoma The panel recommends that amifostine should be administered intravenously at a dose ≥ 340 mg/m2 prior to radiotherapy to prevent radiation proctitis 5‐ASA, meslalzine, olsalazine •
The panel recommends that 5‐ASA and the related compounds mesalazineand olsalazine not be used for the prevention of GI mucositis Octreotide •
When loperamide fails to control diarrhea induced by standard dose or high‐dose chemotherapy associated with HSCT the panel recommends octreotideat a dose of ≥ 100 μg subcutaneously twice daily Sucralfate •
The panel recommends that oral sucralfate not be used to reduce the side effects induced by radiotherapy The panel suggests the use of sucralfate enemas as an effective way of managing chronic radiation‐induced proctitis in patients with rectal bleeding •
•
Sulfasalazine The panel suggests the use of 500 mg of sulfasalazine administered orally twice daily to help reduce the incidence and severity of radiation‐induced enteropathy in patients receiving external beam radiotherapy to the pelvis Table 3. New Guidelines. New Guidelines Agent Guideline Glutamine •
No guideline possible Hyperbaric Oxygen •
The panel suggests that use of hyperbaric oxygen may be an effective means in treating radiation‐induced proctitis Misoprostol •
The panel recommends against the use of misoprostol suppositories for the prevention of acute radiation‐induced proctitis Probiotics •
The panel suggests probiotic treatment containing Lactobacillus spp. may be beneficial for prevention of chemotherapy and radiotherapy‐induced diarrhea in patients with pelvic malignancy DISCUSSION Following a thorough review of the recent clinical literature on interventions for the management of GI mucositis, the panel has found sufficient evidence to form just four new guidelines. These include one new recommendation against the use of misoprostol suppositories for the prevention of radiotherapy‐induced procititis. Two new suggestions were also made: one in favor of the use of a lactobacillus spp. containing probiotic for the prevention of chemotherapy‐ and radiotherapy‐induced diarrhea. Secondly, we were able to suggest the use of hyperbaric oxygen as an effective means of treatment for radiotherapy‐induced proctitis. The panel members 115 | 169 7 Chapter 7 also reviewed a number of new clinical studies regarding the use of glutamine, and in light of conflicting evidence, were unable to reach consensus on the formation of a guideline. This is a significant change from previous guidelines where the use of systemic glutamine was not recommended due to excessive toxicity. However, overall it is disappointing that so little has changed in the last five years. Although pre‐clinical science is evolving, there is very little in the way of new clinical studies 7 Probiotics It is well known that radiotherapy is capable of disrupting the commensal gut bacteria leading to potentially life threatening side effects [123]. Generally, probiotics are “preparations” that contain sufficient numbers of specific viable bacteria that are able to exert beneficial effects [123]. Over the past four years there have been three randomized clinical trials which have shown that probiotics have a significant effect on reducing diarrhea caused by cytotoxic therapies. Delia and colleagues enrolled 490 pelvic radiotherapy patients in a double ‐blind randomized fashion. Patients who received probiotics containing lactobacillus spp. had significantly better outcomes than the placebo patients. 31.6% of probiotic patients had diarrhea compared with 51.8% of placebo patients (p<0.001); 1.4% of probiotic patients had grade 3/4 diarrhea compared with 55.4% of placebo patients (p< 0.001); and average daily bowel motions were 5.1 in probiotic patients compared with 14.7 in placebo patients (p<0.05) [123]. In a 5‐FU‐based chemotherapy study, patients who received a lactobacillus spp. probiotic also had significantly less grade 3/4 diarrhea (p<0.027) and had less chemotherapy‐dose reductions as a result of their toxicity [14]. Finally, a randomized controlled study conducted by Giralt and colleagues reported that patients undergoing radiotherapy for pelvic malignancies, who received a lactobacillus spp. drink had a significant improvement in stool consistency as measured by the Bristol scale [119]. These findings strongly support the clinical usage of probiotics. Advantages of probiotics are that they are cheap, well tolerated by patients, and easy to administer. No studies to date, have reported any adverse reactions to the probiotics in patients treated with cytotoxic therapies for cancer. However, it is worth noting a theoretical concern of increased risk of infection in patients with mucosal barrier dysfunction, particularly if neutropenic. This level of risk is not clear yet, although rare cases of Lactobacillus bacteremia have been documented [144]. Circadian Rhythm Administering cytotoxic drugs according the body’s natural circadian rhythm is not a new idea. Unfortunately there is conflicting evidence around administering chemotherapy, and no guideline is possible. However a single institutional study was conducted by Shukla and colleagues in 2010. They conducted a large randomized controlled trial involving in excess of 200 patients, with cervical cancer. Patients were randomized to receive their radiotherapy in the morning or in the evening and those who received their radiotherapy in the morning, had significantly increased diarrhea 116 | 169 Systematic Review of Agents for Management of Gastrointestinal Mucositis (p< 0.01). Importantly there was no difference in response to treatment (p>0.05) [19]. This is in accordance with the animal studies of Ijiri and Potten (1988) [145, 146]. These studies clearly demonstrated that mice irradiated at varying times of the day displayed different levels of apoptosis. However, as there is only one study in this field, the panel is unable to make a guideline around optimal delivery of radiotherapy. Further well‐designed randomized controlled trials are now warranted. Hyperbaric Oxygen This updated critical literature review has also found enough evidence to warrant a suggestion for the treatment of chronic radiation‐induced proctitis. Hyperbaric oxygen has been reported to cause neovascularisation, reversing the effects of radiotherapy [147]. This review examined in detail 15 studies and all of them were positive. However, studies reviewed suggested that patients may need up to 50 “dives” to achieve effective treatment. With each “dive” costing many thousands of dollars it is highly likely that costs will be prohibitive in many cases. Misoprostol The panel also identified one agent which they recommended against using in the prevention of gastrointestinal toxicities. Misoprostol suppositories were found to be ineffective in two large double‐blind randomized controlled trials [20, 21], and in one instance were reported to actually increase the incidence of acute rectal bleeding [20]. Glutamine Perhaps one of the most important findings from this critical literature review was the change of guideline for the use of systemic glutamine. Previously literature suggested that this agent caused severe toxicity. However, new literature, albeit small studies, demonstrated that glutamine may be effective without severe toxicities. The panel now is unable to form a clinical guideline for the use of glutamine. It will be critically important to continue to watch for new publications of this agent to see if changes in recommendations are possible in the future. Conclusion As highlighted by these guidelines, it is important to continue to update the clinical guidelines for the prevention and treatment of GI mucositis. There were several new well‐designed studies in the published arena since the last update, allowing panel members to make evidence‐based informed guidelines which will hopefully improve clinical practice. GRANTS The Mucositis Guidelines Update was sponsored by Helsinn Healthcare S.A. (Switzerland) and BioAlliance Pharma (France). 117 | 169 7 Chapter 7 REFERENCES 1.
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It is unknown how to optimally feed patients with mucositis, because their capacity to digest and absorb nutrients is hardly known. Normally, enteral nutrition, which is the physiological way of feeding, is preferred to total parenteral nutrition (TPN) because the latter carries a high risk of infection and, upon prolonged administration, may cause liver disease [2‐4]. However, when the absorptive function of the intestine is compromised, TPN offers a useful feeding alternative. The experiments described in this thesis aimed to determine the digestive and absorptive capacity of nutrients during gastrointestinal (GI) mucositis in the rat, to ultimately design a rational feeding strategy for mucositis patients. Since accurate evaluation of mucositis via intestinal biopsies is rather invasive and potentially dangerous in patients, and there is no objective, easy measurable parameter to score GI mucositis in patients, we chose to determine nutrient digestion and absorption in a chemotherapy‐induced mucositis rat model. Because plasma citrulline seemed to be a promising marker for mucositis in patients [5‐8], we also aimed to determine the value of plasma citrulline as an objective marker for the level of GI mucositis and the respective intestinal function, in a rat model. 8 The mucositis rat model We developed a rat model with methotrexate (MTX)‐induced GI mucositis based on other rodent mucositis models [9‐11], as described in chapter 2. To find the optimal dosage of MTX (i.e. development of mucositis without causing mortality), and the best time interval to study nutrient digestion and absorption during mucositis (i.e. when histological symptoms of mucositis were most severe), we performed several pilot studies with varying dosages of MTX (30‐150 mg/kg). Rats were killed at different days after intravenous MTX injection (day 2‐10) to determine the level of mucositis by histology. Histological symptoms of mucositis were comparable between all parts of the small intestine, i.e. the duodenum, jejunum and ileum. The optimal dosage of MTX turned out to be 60 mg/kg. Based on these pilot studies, nutrient digestion and absorption experiments were performed in rats that received 60 mg/kg MTX or saline (controls), 4 days after their intravenous injection. Jejunal histology was used as a representative for small intestinal damage. In the model, rats transiently develop mucositis upon MTX injection, thereby showing the typical subsequent phases as described by Sonis [12, 13]. At day 2 after injection with MTX, crypt loss is seen while villi still appear normal. At day 4, typical histological symptoms of mucositis like villus atrophy and blunting, enterocyte damage, infiltration of inflammatory cells and accumulation of Goblet cells at villus tops is 128 | 169 Summary and General Discussion, Conclusions and Future Perspectives seen. By this time, crypts tend to be elongated, indicating crypt regeneration. From day 6 on, villi of rats start to recover. Typical clinical symptoms of mucositis like a decreased intake of food and water, diarrhea and weight loss are present from day 2 until day 5, after which rats recover. Histological and clinical symptoms of mucositis differed substantially between individual MTX‐treated rats. We hypothesized that this variance in observed individual symptoms of mucositis, as also seen in patients, could be a result of genetic variability between outbred Wistar rats [14]. Moreover, the mucositis rat model is based on a single intravenous injection of MTX, leaving the period of epithelial crypt cell susceptibility to MTX shorter than in models where multiple MTX injections are administered [12, 13, 15‐17]. The amount of subsequent crypt loss by apoptosis, crypt atrophy and ultimately villus atrophy [18] per MTX‐injected rat might therefore be explained by these factors. The variance in individual symptoms of mucositis in the rat model allowed us to determine intestinal function during mild to severe mucositis. Nutrient digestion and absorption during GI mucositis in the rat After establishing a stable and reproducible mucositis rat model, we subsequently determined the digestive and absorptive capacity of the major food constituents (i.e. carbohydrates, fats, proteins) during mucositis in the rat model. Therefore, we used a stable isotope dilution technique; a technique that has successfully been applied for many years in our lab [19‐26]. Stable isotope labeled nutrients were enterally administered as a bolus by oral gavage (since intermittent bolus administration resembles the physiological situation of consuming meals [27]) or continuously by intraduodenal infusion (since continuous enteral nutrient administration has been shown to improve nutrient absorption during other forms of intestinal failure [3]). Carbohydrates In our first experiment, as described in chapter 2, rats with and without MTX‐induced mucositis orally received trace amounts of [1‐13C]lactose and [U‐13C]glucose to determine carbohydrate digestion and absorption. The digestion of lactose (the sole disaccharide in breast milk and an important disaccharide in Western pediatric diets and formulas [28]) was severely decreased during mucositis, and we showed that this was due to a decreased jejunal lactase activity and immunohistochemical protein‐ and mRNA expression after MTX treatment. The enzyme activity of other glycohydrolases (i.e. sucrase, isomaltase and maltase) was also decreased during mucositis, indicating that all disaccharides, as well as polysaccharides, will probably not be hydrolyzed and its derivatives not be absorbed during mucositis. It therefore seems wise to omit disaccharides and polysaccharides from the diet of patients with mucositis to prevent possible negative side effects of carbohydrate maldigestion, like lactose intolerance. In contrast to lactose digestion, glucose absorption was still intact during mucositis, when supplied in trace amounts, in spite of decreased immunohistochemical 129 | 169 8 Chapter 8 protein‐ and/or mRNA expression of glucose transporters SGLT1 and GLUT2. To determine whether glucose could be a useful source of energy during mucositis, we studied the quantitative capacity to absorb [1‐13C]glucose during mucositis when enterally administered in physiologically relevant amounts (meal size) as a bolus by oral gavage or continuously by intraduodenal infusion, as described in chapter 3. When glucose was administered as a bolus, rats with mucositis only absorbed 15% of the administered glucose, compared with 85% in controls. However, upon continuous intraduodenal glucose infusion, the median absorptive capacity for glucose in rats with mucositis did not differ from controls (80 versus 93% of administered glucose respectively), although glucose absorption varied from severely reduced to completely normal between individual MTX‐treated rats (range 21‐95%). Apparently, continuous enteral administration could completely overcome the reduced absorptive capacity for glucose in about half of rats with mucositis. Our findings can be explained by the concept that by continuous enteral nutrient infusion, saturation of the (residual) carrier proteins is maximized and the intestinal function increased, when compared with bolus administration of nutrients. Apart from absorption via transporters on the epithelial membrane, glucose absorption could also have been possible via paracellular absorption [29] or via leakage through damaged tight junctions since gut permeability is increased during mucositis [30, 31]. Our findings suggest that enteral glucose is preferably administered continuously to patients with mucositis, to optimize glucose absorption. 8 Fats To determine the absorptive capacity of long‐chain fatty acids (LCFA) during mucositis, rats with and without MTX‐induced mucositis enterally received a physiologically relevant amount (meal size) of saturated ([U‐13C]palmitic acid) and unsaturated ([U‐13C]linoleic acid) fatty acids dissolved in oil, either as a bolus by oral gavage or continuously by intraduodenal infusion, as described in chapter 4. MTX treatment severely reduced the appearance of [U‐13C]palmitic‐ and [U‐13C]linoleic acid in plasma and liver, compared with controls, either when administered as a bolus or continuously (all at least ‐63%). It is remarkable that continuous enteral fat administration could not overcome the reduced absorptive capacity for LCFA during mucositis, in contrast to glucose absorption. Differences in absorption might be explained by the fact that intestinal absorption of fatty acids is much more complicated than that of carbohydrates (and proteins). In fact, the exact molecular mechanism of fatty acid translocation across the epithelial membrane is still a matter of debate [32‐34]. Apparently, the mucosal damage during mucositis is too prominent to allow for normal absorption of LCFA, even when administered continuously. Parenteral administration of LCFA might therefore be a rational alternative for enteral LCFA administration in patients with mucositis. Furthermore, plasma citrulline turned out to be a better marker than diarrhea to detect LCFA malabsorption during mucositis. 130 | 169 Summary and General Discussion, Conclusions and Future Perspectives Proteins Next, we determined the capacity to absorb amino acids during mucositis. Rats with and without MTX‐induced mucositis enterally received a physiologically relevant amount (meal size) of 13C‐ or 15N‐labeled amino acids (leucine, lysine, phenylalanine, threonine and methionine), as described in chapter 5. Since glucose absorption during mucositis improved upon continuous enteral administration, as compared with bolus administration, amino acids were enterally administered by continuous intraduodenal infusion. The median systemic availability of all amino acids except for leucine was similar in MTX‐treated rats and in controls. However, individual availability of all amino acids differed substantially within the group of MTX‐treated rats, ranging from severely reduced (<10% intake) to not different from controls (>40% intake in 5 of 9 rats). Since absolute amino acid utilization was mostly reduced or similar in MTX‐treated rats, compared with controls, we concluded that continuous enteral administration enabled normal amino acid absorption in about half of rats with mucositis. These findings were in line with our findings regarding glucose absorption. Like glucose absorption, amino acid absorption could have been possible via transporters on the epithelial membrane, via paracellular absorption [29] or via leakage through damaged tight junctions [30, 31]. Our findings suggest that enteral amino acids are preferably administered continuously to patients with mucositis, to optimize amino acid absorption. Furthermore, we showed that the intestine prefers basolateral instead of apical amino acid uptake to meet its need for amino acids for protein synthesis during mucositis. We aimed to determine the digestive and absorptive capacity of nutrients during GI mucositis in the rat. We found the digestion of disaccharides to be severely reduced during mucositis, as was the absorption of LCFA. In contrast, the absorption of glucose and amino acids could be normal during mucositis, when enterally administered continuously by intraduodenal infusion. Of note, large interindividual differences in glucose and amino acid absorption were seen during mucositis. Continuous (par)enteral feeding during mucositis in the rat Now that we concluded that the absorption of glucose and amino acids could be normal during mucositis, when enterally administered continuously, we set out to determine if continuous enteral feeding with these nutrients could prevent weight loss during mucositis. In this experiment, we determined the effects of 4 different (par)enteral feeding strategies during mucositis on body weight, as described in chapter 6. Rats with MTX‐induced mucositis continued ad libitum purified diet (AIN‐93G, strategy 1), received continuous enteral force‐feeding with glucose and amino acids (Nutriflex®, strategy 2) or with standard tube‐feeding (Nutrini®, strategy 3), or received standard parenteral feeding (NuTRIflex® Lipid, strategy 4) for 3 days. Control rats continued ad libitum purified diet. We found that both enteral feeding strategies were poorly tolerated by rats with mucositis. Most rats had to be killed 131 | 169 8 Chapter 8 early because of severe watery diarrhea, abdominal distention, lethargy and hyperglycemia after 1‐2 days of feeding. Only a few rats tolerated enteral feeding with glucose and amino acids. We hypothesized that the reduced spontaneous intake during mucositis (causing the loss of body weight), as seen in rats as well as in humans, might be a mechanism to protect the damaged intestine that apparently does not tolerate normal daily amounts of enteral nutrition. In contrast to enterally‐fed rats, all parenterally‐fed rats with mucositis grew similarly as saline‐treated controls. This could be explained by the fact that via parenteral nutrition, nutrients are delivered directly into the blood and the damaged intestine is completely bypassed. Our findings in the rat suggest that nutrition should be administered parenterally during GI mucositis to prevent weight loss, since daily amounts of enteral nutrition (even of glucose and amino acids) are badly tolerated during mucositis. Apart from the effect on body weight, we also determined the effect of the mentioned feeding strategies on intestinal recovery as measured by plasma citrulline concentrations and jejunal histology. We found advantageous effects of enteral nutrition (including minimal intake in ad libitum‐fed rats) during mucositis on intestinal citrulline synthesis (preferably glucose and amino acids) and histology, in comparison with solely parenteral nutrition. Advantageous effects of enteral nutrition have also been reported during other forms of intestinal failure [35], as may be explained by its stimulatory effect on intestinal epithelial cells and the production of trophic hormones [27, 35, 36]. Taken together, our findings in the rat indicate that the optimal feeding strategy during GI mucositis consists of a combination of parenteral nutrition to prevent weight loss, and tolerated amounts of enteral glucose and amino acids via continuous infusion to optimize intestinal recovery from mucositis. 8 Plasma citrulline as a marker for (intestinal function during) mucositis An objective, easy measurable parameter to score GI mucositis in patients is needed in order to diagnose this (sometimes subclinical) disease and to offer patients the best treatment. Since plasma citrulline (a nonprotein amino acid made by enterocytes [37]) was earlier found to be a promising marker for mucositis in patients [5‐8], we measured plasma citrulline in rats of all our previously described experiments. At day 4 after MTX injection (the day when symptoms of mucositis were most severe and nutrient digestion and absorption experiments were performed), plasma citrulline was always severely decreased in MTX‐treated rats, as compared with controls. In individual rats, plasma citrulline level strongly correlated with villus length, which is the most accurate (but rather invasive) indicator of mucositis [5, 14]. However, at day 5 after MTX injection (when intestinal villi were mostly recovered from mucositis, as described in chapter 6) plasma citrulline was still decreased in most MTX‐treated rats, as compared with controls. Our findings therefore suggest that plasma citrulline has 132 | 169 Summary and General Discussion, Conclusions and Future Perspectives limited value as a marker for the level of mucositis; low plasma citrulline concentrations post chemotherapy might indicate that individuals suffer from mucositis or that they are in an early recovery phase from mucositis. We related villus length and plasma citrulline concentrations with nutrient digestion and absorption in individual rats to determine if individual levels of mucositis correlated with individual levels of nutrient (mal)digestion and (mal)absorption. If so, plasma citrulline (as a relatively noninvasive marker for mucositis) might be used in clinic to adapt the feeding strategy of individual patients, regardless of their level of mucositis. Both villus length and plasma citrulline correlated strongly with lactose maldigestion and fat malabsorption during mucositis, but poorly with the absorption of glucose and amino acids when enterally administered continuously. About half of MTX‐treated rats with severe mucositis (villus length <300 µm and plasma citrulline <30 µmol/L) showed a rather reduced absorption of continuously administered glucose and amino acids, while the other half absorbed glucose and amino acids efficiently. The second aim of this thesis was to determine the value of plasma citrulline as an objective marker for the level of GI mucositis and the respective intestinal function, in the rat. We found plasma citrulline to be a marker with limited value for the level of GI mucositis. Since absorption of continuously administered glucose and amino acids did not correlate with the level of mucositis (as measured by villus length and plasma citrulline), citrulline seems not a usable marker to differentiate between individuals with intact or reduced glucose and amino acid absorption during mucositis. The management of mucositis in cancer patients Progress in understanding the pathobiology of mucositis is difficult due to the relative inaccessibility of the intestine and the obvious difficulty in obtaining biopsies at multiple time points after cytotoxic therapy. Nevertheless, new agents for the management of mucositis are being tested in patients using symptoms of mucositis as clinical endpoints. As part of the Mucositis Study Group of the Multinational Association of Supportive Care in Cancer/International Society of Oral Oncology (MASCC/ISOO), we reviewed the literature and updated the evidence‐based guidelines for the prevention and treatment of GI mucositis [38, 39]. For our review, a literature search for relevant papers indexed in Medline on or before December 31, 2010 was conducted (including only human studies), as described in chapter 7. One new recommendation, two new suggestions and one change from previous guidelines could be made. Firstly, the panel recommends against the use of misoprostol suppositories for the prevention of acute radiation‐induced proctitis. Secondly, the panel suggests probiotic treatment containing Lactobacillus spp. may be beneficial for prevention of chemotherapy and radiotherapy‐induced diarrhea in patients with 133 | 169 8 Chapter 8 malignancies of the pelvic region. Thirdly, the panel suggests the use of hyperbaric oxygen as an effective means in treating reducing radiation‐induced proctitis. At last, new evidence has emerged which is in conflict with the previous guideline concerning the use of systemic glutamine, meaning that the panel is unable to form a guideline. No guideline was possible for any other agent, due to inadequate and/or conflicting evidence. Our updated review of the literature has allowed new recommendations and suggestions for clinical practice to be reached, highlighting the importance of regular updates. CONCLUSIONS The major aim of this thesis was to determine the capacity of nutrient digestion and absorption during GI mucositis in a rat model, to ultimately design a rational feeding strategy for mucositis patients. In the last 4.5 years, we were able to develop and characterize a stable MTX‐induced mucositis rat model in which we determined the digestion and/or absorption capacity of carbohydrates, long‐chain fatty acids and amino acids. Of these nutrients, only the absorption of glucose and amino acids could be normal during mucositis (but differed substantially between individual rats), when enterally administered continuously. 8 We performed the nutrient digestion and absorption tests at day 4 after injection with MTX or saline, while symptoms of MTX‐induced mucositis were actually present from day 2 until day 5 in the rat. We therefore do not know when maldigestion and malabsorption of nutrients exactly starts or ends and, if we extrapolate our findings to the clinic, for how long the feeding strategy of mucositis patients should be adapted. Although plasma citrulline turned out to be to be a marker with limited value for the level of mucositis, it could still be used in clinic to partially adapt the feeding strategy (i.e. no enteral polysaccharides or LCFA), and more generally the treatment, of mucositis patients. Plasma citrulline could be useful in addition to the currently used, more subjective ‘National Cancer Institute Common Toxicity Criteria’ as a parameter for GI mucositis in patients [5, 40]. Since plasma citrulline was reduced from day 1 after MTX injection on, nutrient maldigestion and malabsorption may actually already start in an early phase of mucositis when clinical symptoms are still absent. Moreover, since plasma citrulline remained reduced until day 5 after MTX injection, when intestinal villi were mostly recovered from mucositis, nutrient maldigestion and malabsorption may even exist longer than expected upon intestinal histology. Therefore, the nutritional status of patients receiving chemotherapy should be carefully watched, and their plasma citrulline concentrations measured, from the start of anti‐cancer treatment on, at least until plasma citrulline levels start getting back to normal. 134 | 169 Summary and General Discussion, Conclusions and Future Perspectives Unfortunately, continuous enteral feeding with glucose and amino acids (as well as with standard formula) in normal daily amounts was often poorly tolerated by rats. Parenteral feeding on the other hand (which is quite invasive and carries an increased risk of infection [2‐4]) prevented weight loss during mucositis, in contrast to ad libitum feeding, although ad libitum feeding caused accelerated intestinal recovery. Future research in mucositis patients is indicated to define their optimal feeding strategy in order to prevent weight loss during mucositis and optimize intestinal recovery from mucositis. FUTURE PERSPECTIVES Our findings in the rat indicate that during GI mucositis, the optimal feeding strategy consists of a combination of parenteral nutrition to prevent weight loss, and tolerated amounts of enteral glucose and amino acids via continuous infusion to optimize intestinal recovery from mucositis. However, we do not know to what extent enteral nutrition is tolerated in patients with mucositis. Further research in mucositis patients is needed to determine whether either continuous enteral administration with glucose and amino acids or TPN is indicated to prevent weight loss during mucositis. Since both enteral tube‐feeding (in pediatric and adult mucositis patients) and parenteral feeding (only in adult mucositis patients [41]) are already commonly used in the clinic, studies with varying doses of enteral and parenteral nutrition in mucositis patients can now be undertaken. If continuous enteral administration of glucose and amino acids in normal daily amounts is well tolerated in mucositis patients, but individual glucose and amino acid absorption differs substantially between patients (like in rats), plasma citrulline will probably not be a usable discriminatory marker between their reduced or intact absorption. Future research should then focus on finding another discriminatory marker in order to select patients that would benefit most from continuous enteral glucose and amino acids. If continuous enteral administration of glucose and amino acids in normal daily amounts is poorly tolerated in mucositis patients (like in rats), additional research in patients on the role of enteral glucose and amino acids to improve intestinal recovery from mucositis seems attractive. The design should focus on titrating sufficient enteral nutrients to promote intestinal recovery from mucositis on the one hand, and on negative side effects of enteral feeding on the other hand. To date, there is a considerable need for management interventions to prevent and/or treat GI mucositis. Unfortunately, well performed human studies in which new interventions are tested are scarce, due to the difficulty in obtaining biopsies at multiple time points after cytotoxic therapy. However, now that proof of citrulline to 135 | 169 8 Chapter 8 be a useful marker for GI mucositis accumulates (in addition to the more subjective ‘National Cancer Institute Common Toxicity Criteria’), this might change. For now, the mucositis rat model seems ideal to test new strategies for the prevention and treatment of GI mucostis. The model allows for repeated scoring of mucositis (by plasma citrulline and/or intestinal histology) and absorptive function before, during and after mucositis, by using stable isotope‐labeled nutrients. Promising agents could then be examined in patients in the clinic. Theoretically, all agents that intervene in the pathobiology of GI mucositis, as described by Sonis [12, 13], could prevent or treat GI mucositis and should therefore be the focus of future research. Of these agents, especially R‐spondin1 (a novel epithelial mitogen that stimulates the growth of mucosa in the small and large intestine [42]), seems attractive to test in the rat model. Prophylactic treatment with R‐spondin1 has been shown to protect mice against chemotherapy‐ or radiation‐induced oral mucositis [43]. Since the pathobiology of oral and GI mucositis is thought to be similar [12, 13], R‐spondin1 might also prevent chemotherapy‐induced GI mucositis. 8 Another important focus of future research in the rat model could be the role of intestinal microbiota during mucositis. Recent research has shown that anti‐cancer treatment is associated with a decrease in the number of anaerobic bacteria and a decrease in microbial diversity [44, 45]. We found similar changes in the mucositis rat model (preliminary data). Although not specifically mentioned in Sonis’ model, Van Vliet et al. suggest that the intestinal commensal bacteria could influence all phases of Sonis’ mucositis model [46]. A causal relationship between the intestinal microbiota and mucositis has been proposed but has not been demonstrated so far. Further research to understand the role of bacteria in the pathogenesis of mucositis is indicated, for instance by determining the effects of broad‐spectrum antibiotics on mucositis. 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Besselink MG, van Santvoort HC, Buskens E, Boermeester MA, van Goor H, Timmerman HM, Nieuwenhuijs VB, Bollen TL, van Ramshorst B, Witteman BJ, Rosman C, Ploeg RJ, Brink MA, Schaapherder AF, Dejong CH, Wahab PJ, van Laarhoven CJ, van der Harst E, van Eijck CH, Cuesta MA, Akkermans LM, Gooszen HG, Dutch Acute Pancreatitis Study Group (2008) Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double‐blind, placebo‐controlled trial. Lancet 371:651‐659 8 139 | 169 CHAPTER 9 NEDERLANDSE SAMENVATTING Chapter 9 ALGEMENE INTRODUCTIE Mucositis Mucositis is een van de meest voorkomende en meest ernstige bijwerkingen van chemotherapie en bestraling en kan zowel kinderen als volwassenen treffen. Mucositis wordt gedefinieerd als schade aan het slijmvlies (de mucosa) van het spijsverteringskanaal ten gevolge van de behandeling van kanker. Vaak wordt mucositis onderverdeeld in orale mucositis (schade aan het mondslijmvlies) en intestinale mucositis (schade aan het darmslijmvlies). Orale mucositis is zichtbaar in de mondholte als rood, makkelijk bloedend weefsel, al dan niet in combinatie met zweren. Deze patiënten hebben pijn en moeite met eten. Intestinale mucositis kan alleen gezien worden wanneer met behulp van een endoscoop (een kijkbuis) een darmbiopt (hapje darmslijmvlies) wordt afgenomen, wat nogal ingrijpend is. Deze patiënten ervaren een verminderde eetlust, misselijkheid, braken, buikpijn, diarree en gewichtsverlies. Orale en intestinale mucositis treden niet altijd tegelijkertijd op. Onderzoek naar mucositis richt zich meestal op orale mucositis in volwassenen. Er is minder aandacht voor intestinale mucositis, vooral in kinderen, alhoewel deze aandoening ernstige gevolgen kan hebben voor patiënten. Aangezien dit proefschrift over intestinale mucositis gaat praten we vanaf nu alleen nog over ‘mucositis’, waarmee we darmslijmvliesschade bedoelen. 9 Hoe ontstaat mucositis? Mucositis is een tijdelijke aandoening waarbinnen vijf (kunstmatig ingedeelde) opeenvolgende fases kunnen worden onderscheiden, zoals beschreven door Dr. S.T. Sonis (Boston, MA, USA). Als gevolg van de behandeling van kanker ontstaat eerst schade aan het DNA van de cel, maar ook andere celschade. Hierdoor raken zowel het darmslijmvlies als de onderliggende weefsels beschadigd, waardoor er een kettingreactie van schadelijke processen op gang komt. Zo ontstaat een soort ontstekingsproces dat leidt tot verdere celschade en celdood. De hieropvolgende ulceratieve fase is de meest vervelende fase voor patiënten, waarbij alle symptomen van mucositis tot uiting komen. Het slijmvlies verliest zijn integriteit, wat pijnlijke ulcers (zweren) veroorzaakt die tevens een toegangsroute voor bacteriën vormen. Op deze manier kunnen kankerpatiënten, met toch al vaak een verminderde weerstand, ernstige infecties oplopen. Uiteindelijk geneest mucositis meestal weer vanzef, kort nadat de behandeling van kanker wordt gestaakt. Zowel de schade aan, als het herstel van, het slijmvlies zijn afhankelijk van het type behandeling (chemotherapie en/of bestraling), de specifieke soort behandeling, de dosering en de timing ervan. In Sonis’ model spelen darmbacteriën geen grote rol in het ontstaan van mucositis. Recent onderzoek heeft echter aangetoond dat de behandeling van kanker leidt tot een verminderde hoeveelheid darmbacteriën, en ook minder soorten darmbacteriën. Het microbiële evenwicht, wat belangrijk is voor een goede darmfunctie, raakt 142 | 169 Nederlandse Samenvatting hierdoor ernstig verstoord. Zodoende kunnen darmbacteriën mogelijk een rol spelen bij alle fasen van mucositis zoals beschreven door Sonis. Dit behoeft verder onderzoek. Patiënten ervaren klachten van mucositis vanaf 3 tot 5 dagen nadat zij met chemotherapie gestart zijn, met een piek van klachten op dag 7 tot 14. In knaagdiermodellen beloopt mucositis hetzelfde als bij patiënten, alleen over een korter tijdsbestek (zo’n 5 dagen in totaal). Mucositis en de dunne darm Vele soorten chemotherapie zijn erop gericht om sneldelende cellen te doden met als doel de kanker te vernietigen. Aangezien het spijsverteringskanaal een van de snelst delende weefsels in het lichaam is, is deze erg gevoelig voor chemotherapie, met name de dunne darm. De dunne darm loopt vanaf het einde van de maag tot aan de dikke darm, en kan onderverdeeld worden in drie opeenvolgende delen (het duodenum, jejunum en ileum) die onderling enigszins verschillen in vorm en functie. Het darmslijmvlies bestaat uit talloze vingervormige uitstulpingen die villi heten, of darmvlokken. Tussen de darmvlokken liggen uitsparingen, de crypten, waar de sneldelende stamcellen en haar dochtercellen zich bevinden. Figuur 1 laat een doorsnede zien van de dunne darm van de rat waarin de karakteristieke opbouw van het darmslijmvlies met de villi en de crypten duidelijk naar voren komt. Links wordt het slijmvlies onder normale omstandigheden weergegeven, rechts tijdens mucositis door behandeling met het chemotherapeuticum methotrexaat (MTX). 9 Figure 1. Hematoxyline en eosine kleuring van het jejunum van de rat (Fijlstra).
143 | 169 Chapter 9 Het slijmvlies van de dunne darm bestaat uit drie lagen. De bovenste laag, het epitheel, grenst aan de darmholte waar de voeding zich bevindt. De middelste laag bestaat uit bindweefsel waarin de lymfe‐ en bloedvaten lopen. De onderste laag wordt gevormd door een dunne spierlaag. De dunne darm scheidt de darmholte van de binnenkant van het lichaam. Enerzijds voorkomt de darm hiermee het binnendringen van schadelijke stoffen in het lichaam en anderzijds maakt het de vertering en opname van voedingsstoffen mogelijk. Tijdens mucositis kunnen schadelijke bacteriën het lichaam binnendringen via het beschadigde darmslijmvlies en ernstige infecties veroorzaken. De vertering en opname van voeding Het epitheel van de darmvlokken bestaat uit gespecialiseerde cellen, de enterocyten, die de vertering en opname van voedingsstoffen (koolhydraten, vetten en eiwitten) mogelijk maken. Hierbij wordt gebruik gemaakt van vele enzymen en transportsystemen die aanwezig zijn op het geplooide oppervlak van de enterocyten, de zogenaamde borstelzoom. De enterocten zorgen er op deze manier voor dat voedingsstoffen vanuit de darmholte worden opgenomen/getransporteerd en afgegeven aan het bloed (dat geldt voor de koolhydraten en eiwitten) of aan het lymfe (dat geldt voor de vetten). Alhoewel de meeste voeding op deze manier door de enterocyten wordt vervoerd, kunnen koolhydraten en eiwitten ook langs de enterocyten sijpelen en zo de bloedbaan bereiken. Koolhydraten. De vertering van lange‐keten koolhydraten (zoals zetmeel) start met het enzym amylase uit speeksel en de alvleesklier. Amylase breekt lange‐keten koolhydraten af tot tweevoudige koolhydraten, ofwel disacchariden. De disacchariden worden vervolgens weer verder verteerd tot enkelvoudige koolhydraten, ook wel suikers genoemd (glucose, galactose en fructose), met behulp van enzymen die aanwezig zijn op de borstelzoom van enterocyten (de glycohydrolases lactase, sucrase, isomaltase en maltase). Deze suikers worden uiteindelijk opgenomen door de enterocyten die ze via specifieke transportsystemen afgegeven aan het bloed. 9 Vetten. Vetten ondergaan een aantal biochemische processen in de darmholte voordat de producten die daarbij ontstaan (onder andere vetzuren) getransporteerd worden naar de enterocyten. Vanuit de enterocyten worden de vetproducten afgegeven aan het lymfe in de vorm van zogenaamde chylomicronen die uiteindelijk ook terechtkomen in de bloedbaan. De manier waarop vetzuren worden opgenomen vanuit het darmlumen door enterocyten is nog steeds onduidelijk. Eiwitten. De vertering van eiwitten (polypeptiden) begint met enzymen uit de maag (pepsine) en de alvleesklier (trypsine, chymotrypsine en carboxypeptidasen) die ze afbreken tot kleinere eiwitten (oligopeptiden). Deze oligopeptiden worden verder verteerd door enzymen (peptidasen) die aanwezig zijn op de borstelzoom van de 144 | 169 Nederlandse Samenvatting enterocyten. Uiteindelijk worden de kleinste eiwitten, die nu nog maar bestaan uit drie of twee ketens (tri‐ en dipeptiden) of uit enkelvoudige ketens (aminozuren), opgenomen door de enterocyten. Dit gebeurt via gespecialiseerde transportsystemen die aanwezig zijn in enterocyten. Welk transportsysteem welk eiwit opneemt hangt af van de lading van het eiwit dat neutraal, positief of negatief kan zijn. Een unieke eigenschap van enterocyten is dat deze niet alleen aminozuren kunnen opnemen vanuit de darmholte (net als de koolhydraten en vetten), maar ook direct vanuit de bloedbaan nadat deze eerst zijn opgenomen door de darm en beschikbaar zijn gekomen in het bloed. Een aantal studies heeft aangetoond dat de enzymen en transporters die belangrijk zijn voor de vertering en opname van voeding (die normaliter dus aanwezig zijn op de enterocten van het darmslijmvlies) verminderd aanwezig zijn tijdens mucositis. Dit suggereert dat er sprake kan zijn van een slechte vertering (maldigestie) en opname (malabsorptie) van bepaalde voedingsstoffen tijdens mucositis. Er zijn ook enkele studies geweest die functie van deze enzymen en transporters hebben getest tijdens mucositis. Het is echter nog steeds vrijwel onbekend hoe mucositis de vertering en opname van voeding precies beïnvloedt en dit is dan ook de focus van dit proefschrift. De impact van mucositis in de kliniek Van de patiënten met chemotherapie ervaart zo’n 40‐100% mucositis, afhankelijk van de specifieke chemotherapie die gebruikt wordt en de dosis ervan. Mucositis lijkt vaker bij kinderen dan bij volwassenen met kanker voor te komen, wat een gevolg zou kunnen zijn van de hogere delingssnelheid van het spijsverteringskanaal in kinderen. Bij kinderen met acute myeloïde leukemie (bloedkanker) die meerdere malen hoge doses chemotherapie kregen, werd mucositis vastgesteld in ongeveer 55% van alle cycli met chemotherapie. Ernstige mucositis hangt samen met een toename in het gebruik van pijnstilling, voedingsproblemen en de duur van de ziekenhuisopname. Vaak leidt mucositis tot een noodgedwongen dosis‐vermindering van chemotherapie en daarmee tot een suboptimale behandeling van kanker, wat leidt tot een slechtere overleving van kankerpatiënten. Het scoren van mucositis Het is niet precies bekend hoe vaak mucositis precies voorkomt, aangezien het nauwkeurig vaststellen van mucositis met behulp van een darmbiopt problematisch is in patiënten. Daarom wordt mucositis in de praktijk vaak vastgesteld met behulp van meer subjectieve symptomen zoals pijn en diarree, wat niet erg nauwkeurig is. De scoringscriteria voor mucositis zijn beschreven door het National Cancer Institute (NCI) in de USA. Deze criteria zijn echter nooit ontworpen om mucositis te scoren op een dagelijkse basis en zijn bovendien niet gevalideerd voor kinderen. Daarbij komt dat de symptomen van mucositis slecht samenhangen met de daadwerkelijke ernst 145 | 169 9 Chapter 9 daarvan, vooral in jonge kinderen die minder goed in staat zijn om pijn aan te geven en vaak incontinent voor ontlasting zijn ten gevolge van hun ontwikkelingsstadium. Er is daarom behoefte aan een meer objectieve, makkelijk te meten parameter voor mucositis om de aanwezigheid van deze aandoening vast te stellen en patiënten de optimale behandeling aan te bieden. Diverse parameters die ontsteking, verlies van enterocyten of doorgankelijkheid van de darm weerspiegelen zijn reeds getest om mucositis te scoren. Van deze opties is de concentratie van citrulline in het bloedplasma als veelbelovend uit de bus gekomen. Citrulline is een aminozuur dat gemaakt wordt door enterocyten, en een lage plasma citrullineconcentratie weerspiegelt dan ook een lage enterocytmassa. De waarde van plasma citrulline als een objectieve marker voor de mate van mucositis en de respectievelijke darmfunctie is een tweede focus van dit proefschrift. Methotrexate (MTX) MTX is een chemotherapeuticum dat vaak gebruikt wordt voor volwassenen en kinderen met kanker, als enige behandeling of in combinatie met andere chemotherapeutica. Nadat MTX in de cel wordt opgenomen, verhindert het de aanmaak van foliumzuur en daarmee de vorming van nucleïnezuren; essentiële bouwstenen voor het maken van ons DNA. Uiteindelijk leidt dit tot een verminderde aanmaak van diverse eiwitten. Aangezien MTX alleen actief is tijdens een bepaalde delingsfase van de cel, is het met name toxisch voor sneldelende cellen zoals kankercellen en de cellen van het spijsverteringskanaal. 9 De behandeling van mucositis Goede studies waarbij verschillende behandelingen voor mucositis getest zijn, zijn erg schaars. Dat komt omdat het afnemen van darmbiopten om mucositis vast te kunnen stellen problematisch is in patiënten. Toch zijn er op wetenschappelijke gronden gebaseerde richtlijnen tot stand gekomen voor de preventie en behandeling van mucositis. Deze zijn opgesteld door de Mucositis Studie Groep van de Multinational Association of Supportive Care and Cancer/International Society for Oral Oncology (MASCC/ISOO, een internationale vereniging die zich bezig houdt met de ondersteunende zorg voor kankerpatiënten) door gebruik te maken van symptomen van mucositis als klinische eindpunten. De MASCC/ISOO richlijnen voor patiënten met chemotherapie‐geïnduceerde mucositis (al dan niet in combinatie met bestraling) raden de standaardzorg voor darmpatiënten aan, inclusief het handhaven van een adequate vochthuishouding. Ook zou in ogenschouw moeten worden genomen dat er tijdelijk intolerantie voor het koolhydraat lactose kan zijn, net als de aanwezigheid van schadelijke bacteriën in de darm. 146 | 169 Nederlandse Samenvatting HET DOEL EN DE HOOFDLIJNEN VAN DIT PROEFSCHRIFT Zoals eerder beschreven lijden patiënten met mucositis aan gewichtsverlies wat uiteindelijk lijkt samen te hangen met een slechtere overleving van kankerpatiënten. Dit gewichtsverlies kan een gevolg kan zijn van een verminderde inname van voeding, een verminderde vertering en opname van voeding en/of een veranderd energieverbruik tijdens mucositis. Uit rattenstudies van anderen is gebleken dat gewichtsverlies tijdens mucositis voornamelijk het gevolg is van een verminderde inname van voeding, wat suggereert dat dit gewichtsverlies voorkomen zou kunnen worden door het (zonodig dwangmatig) toedienen van een normale hoeveelheid voeding. Voor het toedienen van voeding wordt enterale voeding (middels het spijsverteringskanaal), wat de natuurlijke manier van voeden is, normaal gesproken verkozen boven parenterale voeding (direct via de bloedbaan). Dit, aangezien parenterale voeding een toegenomen risico op infectie met zich meebrengt en bij langdurig gebruik leverschade kan veroorzaken. Wanneer echter de opnamecapaciteit van de darm beperkt is, biedt parenterale voeding een nuttig voedingsalternatief. Er is weinig kennis over de capaciteit van de darm om voeding te verteren en op te nemen tijdens mucositis. Mede daardoor is het moeilijk de optimale voedingsstrategie voor mucositispatiënten te formuleren. In tegenstelling tot kinderen, krijgen volwassen patiënten met mucositis al regelmatig parenterale voeding. Daarnaast krijgen zowel kinderen als volwassen patiënten met mucositis soms enterale voeding via aan sonde (middels een slangetje in de maag of darm), al bestaat er geen overeenstemming over de optimale manier van voeden (in bolussen van grotere hoeveelheden voeding in een keer, of als continue drupsonde) of over de optimale samenstelling van de voeding. Ons doel was om de vertering en opname van voeding tijdens mucositis te onderzoeken, om uiteindelijk een rationele voedingsstrategie voor patiënten met mucositis te ontwerpen. Het nauwkeurig vaststellen van mucositis middels darmbiopten is nogal ingrijpend en mogelijk gevaarlijk voor patiënten vanwege het risico op infectie en perforatie van de darm. Daarnaast is er geen objectieve, makkelijk meetbare methode om mucositis te scoren in patiënten. Daarom hebben we ervoor gekozen om de vertering en opname van voeding te onderzoeken in een rattenmodel met chemotherapie‐geïnduceerde mucositis. Aangezien plasma citrulline een veelbelovende marker voor mucositis in patiënten bleek te zijn, hadden we als tweede doel om de waarde van plasma citrulline als objectieve marker voor de mate van mucositis en de respectievelijke darmfunctie te onderzoeken, in het rattenmodel. In onze studies hebben we gebruik gemaakt van voedingsstoffen die waren gelabeld met stabiele isotopen waardoor ze na inname en opname door de darm zichtbaar 147 | 169 9 Chapter 9 waren in het bloed en de weefsels. Deze techniek wordt veel gebruikt in ons laboratorium. Het mucositis rattenmodel In Hoofdstuk 2 beschrijven we de kenmerken van het rattenmodel met MTX‐
geïnduceerde mucositis dat we in ons laboratorium hebben opgezet. Van dag twee tot vijf na behandeling met MTX vertoonden ratten, net als patiënten, typische klinische kenmerken van mucositis als een verminderde voedingsinname, diarree en gewichtsverlies. Hun darmslijmvlies liet ook typische kenmerken van mucositis zien zoals afgeplatte darmvlokken, vergrote crypten, kapotte enterocyten en ontsteking van het bindweefsel. Ratten met mucosits hadden sterk verlaagde plasma citrulline concentraties ten opzichte van controle‐ratten. De vertering en opname van koolhydraten In het mucositismodel hebben we als eerste de vertering van lactose (melksuiker, een belangrijke koolhydraat in borst‐ en flesvoeding) en de opname van glucose onderzocht wanneer deze koolhydraten in zeer kleine hoeveelheden werden gegeven via een spuitje in de keel, zoals beschreven in Hoofdstuk 2. Wij vonden dat de vertering van lactose ernstig verminderd was tijdens mucositis, terwijl de opname van glucose nog intact was. Dit kon deels worden verklaard door een verminderde werking en/of aanwezigheid van de hiervoor benodigde enzymen en transporters in het darmslijmvlies. Plasma citrulline bleek een goede, objectieve marker voor mucositis en voor de vertering van lactose tijdens mucositis te zijn. Om vast te stellen of glucose een bruikbare bron van energie tijdens mucositis zou kunnen zijn, hebben we een vervolgexperiment gedaan zoals beschreven in Hoofdstuk 3. Wij wilden de opnamecapaciteit van glucose tijdens mucositis onderzoeken en dienden ratten een normale hoeveelheid glucose toe (ter grootte van een maaltijd) als bolus via een spuitje in de keel (wat lijkt op de normale manier van een maaltijd nuttigen) of via continue drupsonde in de dunne darm (deze manier van voeden via de darm leidt tot een verbeterde opname van voeding tijdens andere vormen van darmfalen). Wij vonden dat de opname van glucose tijdens mucositis ernstig verminderd was wanneer deze als bolus werd toegediend, maar normaal kon zijn wanneer deze enteraal via continue drupsonde enteraal werd toegediend. Wel werden grote verschillen in opname tussen individuele ratten met mucositis gezien. Plasma citrulline bleek geen geschikte marker te zijn om onderscheid te maken tussen ratten met een intacte of verminderde opnamecapaciteit van glucose. 9 De opname van vetten Aangezien lange‐keten vetzuren meerdere belangrijke functies in het lichaam uitoefenen en per gram twee keer zoveel energie leveren als koolhydraten en eiwitten, hebben we vervolgens de opnamecapaciteit van lange‐keten vetzuren onderzocht tijdens mucositis, zoals beschreven in Hoofdstuk 4. Ratten met en zonder 148 | 169 Nederlandse Samenvatting mucositis kregen een normale hoeveelheid vet (ter grootte van een maaltijd) met verzadigde (palmitinezuur) en onderzadigde (linolzuur) vetzuren, als bolus via een spuitje in de keel of via continue drupsonde in de dunne darm. Wij vonden dat de opname van lange‐keten vetzuren ernstig verminderd was tijdens mucositis, ongeacht de wijze waarop deze enteraal werden toegediend. Plasma citrulline bleek een betere marker dan de aanwezigheid van diarree om vetmalabsorptie tijdens mucositis vast te kunnen stellen. De opname van eiwitten Aangezien er aanwijzingen zijn dat de opname van aminozuren tijdens mucositis intact is, in tegenstelling tot de absorptie van tri‐ en dipeptiden, hebben we hierna de opnamecapaciteit van aminozuren tijdens mucositis onderzocht, zoals beschreven in Hoofdstuk 5. Ratten met en zonder mucositis kregen een normale hoeveelheid aminozuren (ter grootte van een maaltijd, te weten leucine, lysine, fenylalanine, threonine en methionine) via continue drupsonde in de dunne darm. Wij vonden dat de opname van aminozuren tijdens mucositis normaal kon zijn wanneer deze enteraal via continue drupsonde werden toegediend. Net als bij glucose‐opname werden grote verschillen in aminozuuropname tussen individuele ratten met mucositis gezien. Plasma citrulline bleek geen geschikte marker te zijn om onderscheid te maken tussen ratten met een intacte of verminderde opnamecapaciteit van aminozuren. Ratten met mucositis bleken minder darmslijmvlies te hebben dan controleratten, waardoor hun darmen absoluut gezien minder aminozuren verbruikten dan controleratten. Relatief (per gram darm) was er echter een toegenomen aminozuurverbruik tijdens mucositis. Tijdens mucositis had de darm een voorkeur om aminozuren op te nemen vanuit het bloed, in plaats van vanuit de darmholte, om eiwitten te kunnen maken. De effecten van (par)enterale voeding tijdens mucosits op gewicht en darmherstel Gebaseerd op onze bevindingen met betrekking tot de vertering en opname van voeding tijdens mucositis in het rattenmodel hebben we uiteindelijke de effecten van vier verschillende enterale dan wel parenterale voedingsstrategieën tijdens mucositis op het gewichtsbeloop onderzocht, zoals beschreven in Hoofdstuk 6. Ratten met mucositis kregen ofwel normale pellets voer aangeboden, zoals gebruikelijk is voor ratten (AIN‐93G, strategie 1), ofwel kregen een continue drupsonde in de dunne darm met glucose en aminozuren (Nutriflex®, strategie 2) of met standaard sondevoeding uit de kliniek (Nutrini®, strategie 3), ofwel kregen alleen parenteraal standaardvoeding uit de kliniek (NuTRIflex® Lipid, strategie 4) gedurende drie dagen. Controleratten kregen normale pellets voer aangeboden. Mucositisratten die gewone pellets voer kregen aangeboden lieten een sterk verminderde intake zien en vielen ernstig af (net als in eerdere experimenten). Helaas bleek dat continue enterale voeding (met glucose en aminozuren, maar ook met standaard sondevoeding) in gebruikelijke, dagelijkse hoeveelheden vaak slecht verdragen werd door ratten met mucositis; deze ratten werden ernstig ziek met zeer opgezette darmen. Parenterale voeding (wat 149 | 169 9 Chapter 9 nogal ingrijpend is en een hoog risico op infectie met zich meebrengt) werd daarentegen wel goed verdragen en kon gewichtsverlies voorkomen tijdens mucositis. Naast het gewichtsbeloop hebben we tevens de effecten van deze verschillende voedingsstrategiën op het darmherstel van mucositis onderzocht: de darmfunctie hebben we gemeten middels plasma citrulline concentraties en de darmmorfologie (hoe het darmslijmvlies er uitziet) door te kijken naar darmvlok‐ en cryptlengte. Wij vonden dat enige enterale voeding (zelfs de minimale intake in ratten die normale pellets kregen) tijdens mucositis een gunstig effect had op plasma citrullineconcentraties (meer bij glucose en aminozuren dan bij standaard voeding) en darmvlok‐ en cryptlengte, in vergelijking met geen enterale voeding maar enkel parenterale voeding. Herziening van de richtlijnen voor de preventie en behandeling van mucositis Als onderdeel van de Mucositis Studie Groep van de MASCC/ISOO hebben we de relevante literatuur herzien en de op wetenschappelijke gronden gebaseerde richtlijnen voor de preventie en behandeling van mucositis (uit 2007) bijgewerkt, op basis van alle relevante literatuur die gepubliceerd is voor december 2010, zoals beschreven in Hoofdstuk 7. Dit heeft geleid tot een nieuwe richtlijn, twee suggesties en een verandering ten opzichte van de eerdere richtlijnen. Onze belangrijkste bevinding was dat patiënten met kanker van het bekken probiotica met Lactobacillus spp. (een melkzuurbacterie) zouden moeten gebruiken omdat dit een gunstig effect kan hebben op de preventie van chemotherapie‐ en bestraling‐geinduceerde diarree. Ook bleek er nieuwe literatuur te zijn over het intraveneus gebruik van het aminozuur glutamine die in tegenspraak is met eerdere richtlijnen; de studiegroep kan daardoor geen duidelijke richtlijn vormen. Het was niet mogelijk om een richtlijn te maken met betrekking tot alle andere middelen die getest zijn voor de preventie of behandeling van mucositis, wegens onvoldoende of tegenstrijdige data. In Hoofdstuk 8 vatten we (in het Engels) al onze bevindingen samen en bediscussiëren we de meest relevante bevindingen van dit proefschrift, trekken we onze conclusies en beschrijven we de toekomstperspectieven voor onderzoek met betrekking tot mucositis. Dit hoofdstuk bevat de Nederlandse samenvatting; hieronder formuleren wij onze conclusies en worden toekomstperspectieven gegeven. CONCLUSIES 9 Het belangrijkste doel van dit proefschrift was om de vertering en opname van voeding tijdens mucositis te onderzoeken, om uiteindelijk een rationele voedingsstrategie voor patiënten met mucositis te ontwerpen. In de afgelopen 4.5 jaar tijd hebben we een stabiel en representatief rattenmodel met MTX‐geïnduceerde intestinale mucositis opgezet en gekarakteriseerd, waarin we de vertering en opname van koolhydraten, vetten en eiwitten hebben onderzocht. Wij vonden dat de 150 | 169 Nederlandse Samenvatting vertering van koolhydraten evenals de opname van vetten ernstig verminderd is tijdens mucositis. Daarentegen bleek dat de opname van glucose en aminozuren tijdens mucositis normaal kan zijn wanneer deze voedingsstoffen enteraal via continue drupsonde worden toegediend. Belangrijk hierbij is wel dat er grote verschillen in opname werden gezien tussen individuele ratten met mucositis. Ongeveer de helft van alle ratten met ernstige mucositis had een sterk verminderde opname van continu toegediende glucose en aminozuren, terwijl de andere helft van de ratten deze voedingsstoffen goed opnam. In ons laatste experiment hebben we onderzocht of het enteraal toedienen van glucose en aminozuren via een continue drupsonde tijdens mucositis in staat was om gewichtsverlies te voorkomen. Helaas bleek dat enterale voeding in normale, dagelijkse hoeveelheden vaak slecht verdragen werd door ratten met mucositis, in tegenstelling tot parenterale voeding wat goed werd verdragen en waarmee gewichtsverlies kon worden voorkomen. Wel bleek dat enige enterale voeding tijdens mucositis een gunstig effect had op het darmherstel. Samengevat wijzen onze bevindingen in de rat uit, dat de optimale voedingsstrategie tijdens mucositis een combinatie is van parenterale voeding om gewichtsverlies te voorkomen en tolerabele hoeveelheden glucose en aminozuren via continue enterale toediening om het darmherstel te bevorderen. Wij hadden als tweede doel om de waarde van plasma citrulline als objectieve marker voor de mate van mucositis en de respectievelijke darmfunctie te onderzoeken, in het rattenmodel. Plasma citrulline bleek een marker met beperkte waarde te zijn voor de mate van mucositis in de rat. Lage plasma citrulline concentraties duiden op het aanwezig zijn van mucositis of op het begin van herstel daarvan. Citrulline zou gebruikt kunnen worden in de kliniek om de voedingsstrategie (geen enterale koolhydraten die verteerd moeten worden, of lange‐keten vetzuren) en de behandeling van mucositispatiënten aan te passen. Echter, aangezien de opname van continue, enteraal toegediende glucose en aminozuren niet bleek samen te hangen met de ernst van de mucositis, lijkt de plasma citrullineconcentratie geen geschikte marker om onderscheid te maken tussen mucositispatiënten met een intacte of verminderde opnamecapaciteit van glucose en aminozuren. TOEKOMSTPERSPECTIEVEN Onze bevindingen in de rat laten dus zien dat parenterale voeding tijdens mucositis nodig is om gewichtsverlies te voorkomen, aangezien enterale voeding in normale dagelijkse hoeveelheden vaak slecht verdragen wordt tijdens mucositis. We weten echter niet of enterale voeding goed of slecht verdragen wordt in patiënten met mucositis. Toekomstig onderzoek in mucositispatiënten moet dan ook uitwijzen of continue enterale toediening van glucose en aminozuren, al dan niet samen met 151 | 169 9 Chapter 9 parenterale voeding, is aangewezen om gewichtsverlies tijdens mucositis te voorkomen. Aangezien zowel sondevoeding (in kinderen en volwassen patiënten) als parenterale voeding (enkel in volwassen patiënten) in de kliniek reeds gebruikt wordt tijdens mucositis, kunnen relatief makkelijk vervolgstudies worden opgezet met mucositispatiënten waarbij de hoeveelheid enterale en parenterale voeding steeds varieert. Wanneer blijkt dat continue enterale toediening met glucose en aminozuren in gebruikelijke dagelijkse hoeveelheden goed verdragen wordt tijdens mucositis (in tegenstelling tot in de rat), maar de opname van glucose en aminozuren enorm verschilt tussen individuele patiënten (zoals bij de rat), dan zal plasma citrulline waarschijnlijk geen goede marker zijn om onderscheid te maken tussen patiënten met een intacte of verminderde opnamecapaciteit. Toekomstig onderzoek zou zich dan moeten richten op het vinden van een andere onderscheidende marker om die patiënten te kunnen selecteren die het meeste baat hebben bij continue enterale toediening met glucose en aminozuren. Wanneer blijkt dat continue enterale toediening met glucose en aminozuren in gebruikelijke dagelijkse hoeveelheden slecht verdragen wordt tijdens mucositis (net als in de rat), dan lijkt aanvullend onderzoek naar de rol van enterale toediening van glucose en aminozuren op het darmherstel aantrekkelijk. Daarbij moet op zoek worden gegaan naar de optimale hoeveelheid enterale voeding waarbij darmherstel van mucositis bevorderd wordt terwijl negatieve bijwerkingen van enterale voeding worden voorkomen. 9 Tot op heden is er grote behoefte aan interventies die mucositis kunnen voorkomen of behandelen. Helaas zijn er maar weinig goed uitgevoerde studies gepubliceerd waarin nieuwe interventies onderzocht worden, gezien de problematiek die gepaard gaat met het afnemen van meerdere darmbiopten tijdens mucositis. Nu plasma citrulline een bruikbare marker blijkt voor de mate van mucositis (naast de klinische scoringscriteria voor mucositis), kan dit echter snel veranderen. Op dit moment lijkt het mucositis rattenmodel ideaal om nieuwe interventies voor de preventie en behandeling van mucositis te testen. In het model kan mucositis herhaaldelijk worden gescoord (middels het meten van de plasma citrullineconcentraties of de darmvlok‐ en cryptlengte) terwijl tegelijkertijd de voedingsopnamecapaciteit kan worden getest voor, tijdens en na de aanwezigheid van mucositis. Hierna zouden veelbelovende interventies getest kunnen worden in patiënten met mucositis. Theoretisch zouden alle interventies die aangrijpen op de het ontstaansmechanisme van mucositis, mucositis kunnen voorkomen of behandelen en daarom het speerpunt kunnen zijn van nieuw onderzoek. Van de mogelijke interventies lijkt vooral de groeifactor R‐spondin1, die de groei van het darmslijmvlies stimuleert, aantrekkelijk om te testen in het rattenmodel. Een andere onderzoeksfocus zou de rol van de 152 | 169 Nederlandse Samenvatting darmflora tijdens mucositis kunnen zijn. In overeenstemming met bevindingen van anderen, vonden wij in ons rattenmodel een verminderde hoeveelheid darm‐
bacteriën, en ook minder soorten darmbacteriën (voorlopige data). Een oorzakelijk verband tussen de darmflora en mucositis wordt wel verondersteld, maar is niet bewezen. Verder onderzoek naar de rol van bacteriën op het ontstaan van mucositis is dan ook aangewezen, bijvoorbeeld door de effecten van antibiotica op mucositis te testen. Ook de rol van pre‐ en probiotica die mogelijk een gunstige invloed op mucositis hebben (zoals bepaalde melkzuurbacteriën) verdienen de aandacht. Enige voorzichtigheid met het toedienen van levende bacteriën aan patiënten met een verminderde weerstand (zoals kankerpatiënten die chemotherapie of bestraling krijgen) is echter geboden, aangezien de toediening van deze bacteriën levensbedreigend kan zijn. Alternatieven, zoals onschadelijke onderdelen van bacteriën (bijvoorbeeld hun DNA), zijn interessant. 9 153 | 169 APPENDICES APPENDICES Appendices | Dankwoord – Acknowledgements DANKWOORD – ACKNOWLEDGEMENTS Hora est! Ruim viereneenhalf jaar geleden begon ik aan mijn grote Groningse avontuur. Ik zie mezelf nog zitten in de trein vanuit Leiden om te solliciteren in het UMCG voor een positie als promovendus op het Lab Kindergeneeskunde. Hier, gezeten aan de keukentafel in New York, denk ik met enige weemoed terug aan die mooie, leerzame maar ook stressvolle tijd die (achteraf) voorbij lijkt te zijn gevlogen. Ik heb destijds niet kunnen overzien waar ik precies aan begon – en misschien is dat maar goed ook. Want promoveren doe je niet alleen ‘op je werk’, daar begin je aan op je eerste werkdag en stop je pas mee na je verdediging. Overal en altijd ben je er mee bezig. Met vallen en opstaan, met pieken en dalen, maar bovenal met heel veel andere mensen samen. Ik zou het nooit hebben kunnen, maar ook niet willen doen zonder de hulp en steun van al mijn lieve, enthousiaste collega’s, vrienden en familie. Via dit dankwoord wil ik jullie allemaal hartelijk bedanken. Vergeef me als ik je vergeet bij naam te noemen; in alle drukte en chaos rondom de afronding van mijn boekje kan dat gebeuren... Weet dan: mijn dank is er niet minder om! Wim en Edmond, mijn ‘dagelijkse begeleiders’, jullie hebben maar half een idee hoe belangrijk jullie de afgelopen jaren voor mij zijn geweest! In de loop van de tijd zijn jullie gegroeid van officiële begeleiders tot bevriende collega’s. Alhoewel we, zeker in het begin, even moesten wennen aan de ideale manier van samenwerken, denk ik dat we een heel goed ‘trio’ waren. Wim als immer enthousiaste, vrolijke, geen‐
problemen‐ziende, rasoptimistische regelaar. Edmond als weloverwegende, alles in ogenschouw nemende, kritische maar faire denker. En ikzelf die volgens mij behoorlijk in het midden van jullie karaktertrekken uitkom. Onwijs bedankt voor het vertrouwen in mij, voor het feit dat ik altijd op erg korte termijn bij jullie terecht kon voor vragen en ‘problemen’, voor de constructieve wijze waarop we alle agendapunten wekelijks langsgingen en voor de gezelligheid en lol die we hadden tijdens werkbesprekingen, congressen en daarbuiten! Mijn (co)promotoren: A Dr. Tissing, beste Wim: de keren dat we overleg hadden zonder jou, zijn op een hand te tellen. Altijd maakte jij tijd. Mailtjes werden vaak acuut, of binnen zeer korte termijn, beantwoord. Daar waar ik niet wist te kiezen uit alle mogelijke opties, of acute ‘beren op de weg’ zag, hakte jij in no time knopen door en gaf mij weer frisse moed voor de komende week. Nespressootje erbij, pen en papier op tafel, en kordaat alle punten langsgaan. Voor mij, zeker als beginnende promovendus, zijn jouw enthousiasme en positiviteit enorm belangrijk geweest om mijn (al dan niet terechte) ‘zorgen’ weg te nemen. Ook ben ik je dankbaar voor het feit dat jij je altijd als makkelijk benaderbaar hebt opgesteld: een absolute pre om succesvol te kunnen 156 | 169 Appendices | Dankwoord – Acknowledgements promoveren! Ik denk met veel plezier terug aan de BBQ bij jou en Anneke thuis in Peize, de mucositisworkshops van MASCC in Athene en NYC en gezellige diners en borrels met onze collega’s aldaar. Hopelijk houden we contact met elkaar en wie weet blijven we samenwerken als artsen en/of onderzoekers op het overlappende gebied van de kinderoncologie en de kinder‐MDL: mucositis! Prof. dr. Rings, beste Edmond: ik weet nog goed dat ik je opbelde om te vragen of ik nog kon reageren op de vacature voor een AIO. Die dag zou de reactietermijn verstrijken en jij zei (met indrukwekkend zware stem) ‘dat ik dan maar even een brief moest schrijven die avond’. Zo geschiedde en een week later zat ik in de trein naar Groningen voor een sollicitatiegesprek. Op mijn weg terug naar Leiden belde je me al op om te zeggen ‘dat jullie er eigenlijk wel uit waren’ en ik riep enthousiast “ja!”. Jij waarschuwde me nog ‘dat ik er nog maar even over na moest denken, want het hield wel heel wat in’ (achteraf gezien je eerste wijze advies). Maar ik wist genoeg en enthousiast als ik was heb ik je de volgende dag (pro forma) teruggebeld om te zeggen dat ik het écht wilde. Edmond, jij bent een wijze man. Jouw rust, kalmte en doordachtheid brachten mij steeds weer ‘op aarde’ wanneer ik (te?) wilde plannen had of veel te ver vooruit keek. Niet alleen met betrekking tot ons onderzoek maar ook met betrekking tot mijn verdere carrière binnen de kindergeneeskunde voorzag jij mij van goede, eerlijke adviezen. Nooit drong jij je mening op, maar je gaf me handvaten waarmee ik verder kon en liet me mijn eigen keuzes maken. Dansen in de disco van De Koningshof in Veldhoven tijdens NVGE‐congressen en biertjes drinken in de kroeg in New Orleans tijdens DDW, dat waren mooie momenten. Ook was het een enorm feest toen jij professor werd, en wij (Willemien, Marjan, Andrea, Mariëtte en ik) jouw Spijs‐girls waren. Via jou heb ik in 2008 een onderzoeksstage bij Richard (Dick) Grand’s lab in het Boston Children’s Hospital gedaan en ben ik in contact gekomen met Dr. John Thompson uit The Children’s Hospital at Montefiore en zijn collega (mijn huidige labhoofd) Prof. dr. Len Augenlicht in het Montefiore Hosptial, in The Bronx. De kinder‐MDL heeft mijn hart gestolen en al ga ik nu niet voor een ‘megadeal’ in Groningen, ik zou het een eer vinden om ooit als directe collega aan jouw zijde te werken :). Heel veel dank voor alles! Prof. Verkade, beste Henkjan: ook jij hebt een heel bijzondere rol vervuld in mijn promovendusleven. Jij was altijd op de achtergrond, en indien nodig op de voorgrond, aanwezig als ‘vierde man’ naast ons trio. Dat het hebben van meerdere begeleiders naast een vruchtbare samenwerking ook tot onbedoelde misverstanden kan leiden, heb jij van dichtbij gezien, én aangepakt als ‘bemiddelaar’. Ondanks jouw gillend drukke agenda maakte je steeds tijd om op heldere wijze uiteen te zetten hoe een en ander had kunnen gebeuren, hoe ratio en gevoel soms botsten. Jouw prachtige alledaagse metaforen om dergelijke misverstanden te verklaren vergeet ik niet. Ik heb veel respect voor jouw megasnelle analyses, absolute kennis, en betrokkenheid die je als afdelingshoofd, kinderarts, onderzoeker en in vele andere hoedanigheden weet te 157 | 169 A Appendices | Dankwoord – Acknowledgements combineren. Ik heb genoten van de journal clubs op jouw kamer en werkbesprekingen die we met onze Transportgroep hadden. Er was geen fysiologisch proces dat NIET met behulp van winkelwagentjes bij de Albert Heijn geanalyseerd kon worden! Heel veel dank voor alles wat je me geleerd hebt afgelopen jaren. Prof. De Bont, beste Eveline: wij kennen elkaar van de kinderoncologiebesprekingen waarin al jouw promovendi en die van Wim afwisselend hun werk presenteerden. Ik heb je daar leren kennen als een vrolijke dame die kritisch naar het gepresenteerde onderzoek kijkt open de discussie aangaat. Je geeft daarbij de ruimte aan andere aanwezigen, en probeert met een concrete oplossingen te komen. Dank je voor deze leuke, leerzame bijeenkomsten, en het feit dat je mijn promotor wilt zijn! Leden van de Beoordelingscommissie: dank voor jullie tijd en moeite om mijn proefschrift door te nemen! Prof. dr. Groen, beste Bert: al snel nadat ik als promovendus startte, volgde jij Folkert op als hoofd van het Lab. Kindergeneeskunde. Je was geen echte begeleider van mij, maar gaf als vast lid van de Transportgroep altijd wijze adviezen en inzichten over de opzet van experimenten en de interpretatie van hun uitkomsten. Daarbij wist je me te prikkelen om vooral zelf na te denken en keuzes te maken. Altijd was jij in voor een gezellig praatje bij de koffiemachine, een goed gesprek over ‘de zin des levens’ tijdens labuitjes, of een avondje swingen op de dansvloer tijdens feesten en partijen. Net als ik kende jij het traject Groningen‐Amsterdam maar al te goed en de beslommeringen die daar soms bij kwamen kijken. Veel dank voor dat alles! Prof. dr. Van Goudoever, beste Hans: toen wij de aminozuuropname in ons mucositis rattenmodel wilden testen, vertelden Wim en Edmond mij ‘dat jij wel wat van aminozuuropname’ wist en we jou dus moesten benaderen. Al snel was ik onder de indruk van jouw expertise en soepele, heldere manier van kennisoverdracht. Jij bracht me in contact met jouw lab in het Erasmus MC alwaar ik samen met Henk, Gardi en Kristien weken heb doorgebracht om ons gezamenlijke experiment uit te werken. Daar waar de formules me eerst nog voor de ogen duizelden, hebben we er inmiddels een mooie publicatie bij! Daarnaast kennen we elkaar inmiddels ook van de ESPGHAN congressen, jouw AMC‐onderzoeksgroep en in verband met mijn persoonlijk ambitie om kinderarts te worden in Amsterdam. Ik zou het erg leuk vinden om met je samen te blijven werken. Veel dank voor je tijd, geduld, gezelligheid en wijze adviezen. A Prof. dr. Kamps: toen ik als promovendus in het UMCG begon ging u, als Prof. in de Kinderoncologie, al bijna met emeritaat dus we hebben elkaar helaas niet echt goed leren kennen. Die paar keren dat ik u echter ontmoet heb, onder andere tijdens uw afscheid van het UMCG, hebben ik u leren kennen als een heel hartelijke en betrokken man. Dank u dat u alsnog mijn proefschrift heeft willen beoordelen! 158 | 169 Appendices | Dankwoord – Acknowledgements Mijn overige opponenten tijdens de verdediging van mijn proefschrift: ik zie uit naar een levendige discussie! Prof. dr. Nieuwenhuis: ik heb u leren kennen via het NVGE Voorjaarscongres waar u voorzitter was van de sessie waar ik een presentatie hield. Daarna hadden we een leuke discussie over mijn onderzoeksresultaten en de interpretatie daarvan. Ook heb ik u, samen met Dr. Frenkel, een aantal keer gesproken over de mogelijkheden tot MDL‐onderzoek en de opleiding Kindergeneeskunde in het WKZ/UMCU. Dank u voor alle tijd en moeite die u nam om alle mogelijkheden en ‘voors en tegens’ op een open, eerlijke manier met mij te bespreken. Ik vind het erg leuk dat u mijn opponent wilt zijn en hopelijk komen we elkaar ook daarna nog tegen komen binnen de kinder‐MDL, als arts en/of onderzoeker. Dr. Blijlevens, beste Nicole: ik leerde jou via Wim en Michel kennen als collega binnen het mucositisonderzoek met veel ervaring, onder andere met plasma citrulline. Naast de wetenschappelijke gedeeltes van de MASCC congressen hebben we vooral ook veel lol gehad in Athene en afgelopen zomer in New York. We kunnen supergezellig praten over alles en nog wat, onder het genot van een diner, een drankje en/of een sigaar. Ik ben benieuwd welke vragen je straks op me af gaat vuren als opponent! Dr. Renes, beste Ingrid: wat ontzettend leuk dat ‘de cirkel weer rond is’ nu jij als opponent bij mijn verdediging aanwezig bent! Toen ik in Groningen aankwam was er geen mucositismodel en bijna niemand op ons lab had überhaupt van mucositis gehoord...! Ik was dan ook superblij dat ik jou herhaaldelijk mocht bellen, mailen en zelfs bij je langskomen voor vragen en adviezen, en dat heb je geweten :). Jij hebt me laten zien hoe ik mucositis kan scoren door de lengte van darmvilli te meten, en dat heb ik vervolgens duizenden malen gedaan. Jarenlange ervaring had jij met verschillende mucositismodellen, en diverse promovendi zijn dan ook onder jouw begeleiding gepromoveerd op het onderwerp mucositis. Ik voel me vereerd dat je ook bij mijn promotie aanwezig wilt zijn en ik ben je enorm dankbaar voor al je enthousiasme, hulp, uitleg en adviezen!! En natuurlijk mijn paranimfen: Mariëtte: lieve Mjetje, het is toch werkelijk unglaublich dat er een einde aan deze ‘bevalling’ is gekomen!? En jij promoveert al 2 weken na mij! We weten beiden dat promoveren niet makkelijk is en dat je er hard voor moet werken. Dat promoveren met diepe dalen en hoge pieken gepaard gaat is dan ook geen wonder, zowel op werkvlak als op persoonlijk vlak. Maar, volgens mij delen goede vrienden lief en leed en zo hebben ook wij het nodige meegemaakt. Niet alleen de stress tijdens experimenten, de blijdschap na een publicatie en de gezellige avondjes bij jou thuis (eerst alleen en later met Steffan) of in de kroeg, maar ook de ziekte van jouw zus en van mijn vader. Ik ben erg blij dat we goede vriendinnen zijn geworden en dat je 159 | 169 A Appendices | Dankwoord – Acknowledgements langskwam in NYC, supermooi! Naast vriendinnen hoop ik natuurlijk ook dat we collega’s in de kindergeneeskunde worden! Annebet: lieve Betje, een warm thuis, dat is wat jij me afgelopen jaren in Grunn hebt gegeven! We waren al vriendinnen op de RSG in Sneek, maar daarna scheidden onze wegen zich; jij naar Grunn, ik naar Leiden. Vanaf het moment dat ik naar Groningen ging om te promoveren was alles snel weer als vanouds. Jij hielp me zoeken naar een huisje, liet me slapen op je logeerkamer, zette mijn eigen stoel, voetenbankje en dekentje klaar als ik op bezoek kwam. Samen deelden we alle ‘sores’ die bij promoveren komen kijken, want daar weet jij zelf ook alles van. Je maakte me wegwijs in het UMCG en de stad, en introduceerde me bij jouw vrienden om gezellig samen te borrelen, voetbal te kijken, Koninginnenach te vieren en uit te gaan. Je bent een supervriendin en ik hoop dat we op dezelfde voet (zij het misschien iets minder intensief) door gaan ná NYC! Maar eerst kom jij hier! A Alle (ex)collega’s van het Lab. Kindergeneeskunde en de aangrenzende laboratoria op Y2: Bert, Folkert (aanwezig bij mijn sollicitatie en nog even als labhoofd, ik zou het enorm leuk vinden als jij als decaan voorzitter kunt zijn tijdens mijn promotie), Sabina (heldin, zoveel vragen had ik toen ik vers uit de kliniek weer het lab in moest...dank voor je geduldige uitleg!), Maxi (powerrrrrrrrrr! Lekker sporten kan met jou altijd), Hilde H. (gezellig op het werk en tijdens de bieravondjes!) , Marijke (zachtaardig en behulpzaam), Jeltsje, Anniek K. (het blijft een wonder hoe jij promoveren met het krijgen van 2 kinderen combineert :)), Jaap (gezellige kinderarts), Frans C. (wat had je ’t zwaar tussen al die vrouwen :P), Jurre (een echte leraar), Aldo (helaas kwam het niet meer van een MCT‐opname‐experiment), Harmen (in gedachten), Karin G., Marc D., Janine (bench‐buur), Agnes (gezellig voor een praatje), Annelies (harde werker!), Jelena (lieverd, succes met de coschappen), Carolien (gedeelde passie voor roeien en fietsen), Anke (trots op jou!), Marije (gezellige hockey‐ster!), Gemma (Sangria...!), Marleen (promoveer ik op je verjaardag nota bene en zet je die ff opzij, topper!), Aycha (weekendrelaties zijn ons niet onbekend, op zoek naar steady state nu!), Brenda (houdt ook wel van een feestje), Anne v.Z (wat een toestand dat laatste experiment! Maar draaglijk door jouw hulp, thanks!), Anne K. (de microbiologie was niet helemaal jouw ding, maar ik vond t leuk met je samen te hebben gewerkt), Mark G. (wordt het de kindergeneeskunde?), Jan Fraerk (wanneer ging jij nou promoveren...? ben er graag bij!), Weilin (married! congrats!), Nienke (lekker pittig en gezellig!), Matthijs, Wytse, Elodie, Jolita, Hilda (ik zeg DISA! Dank voor je hulp), Gerard (in New York geniet ik nog vrijwel dagelijks van je mooie foto’s van Groningen! Keep on posting!), Wytse, Renze (reizen is de moeite waard!), Angelika (dank voor je hulp op ’t CDL en de gezellige borreltjes!), Barbara (voor kritische doordenkvragen bij jou aan ’t goede adres), Theo B. (GC‐MS is onlosmakelijk met jou verbonden, veel dank voor al je analyses en uitleg! Niet meer vallen op de fiets hoor :)), Theo v. D. (als ik glucose hoor denk ik aan jou. Dank voor al je hulp en berekeningen bij de 160 | 169 Appendices | Dankwoord – Acknowledgements lactose/glucose experimenten!), Vincent (dank voor je statistieklessen), Torsten (problemen met PCR? Torsten voor al uw vragen! Dank je!), Frans S. (wat hebben wij veel overlegd over welke isotopen te gebruiken voor welk experiment! Veel dank voor al je geduld en geregel!), Rebecca (altijd vrolijk), Klary, Albert (komt los op labuitjes), Ingrid (altijd aardig, dank voor het integreren van mijn vetpieken), Pim de B. (gezellige praatjes tijdens het maken van de PFB’s), andere Pim, Elles, Herman (inmiddels kan ik zelf enveloppen versturen :P), Hans (altijd een kritische vraag tijdens de Journal Clubs, heel goed), Dirkjan (lopende encyclopedie, dank voor je aminozuuruitleg), Uwe (daar waar koffie en koekjes zijn ben jij te vinden! Gezellig geleuter met een bakkie pleur, maar JIJ moet ook eens koffiepads kopen hoor :)), Fjodor (vlug als een hazewind...), Tineke (ren je blij), Klaas (ook al zo’n snelle haas) , Henk B. (dank voor het regelen van alle labbepalingen), Hilde R. en Gea: wat zou het secretariaat zonder jullie zijn? Een saaie boel, denk ik!), Anouk R. (gezellige meid, dank voor de lekkere etentjes en goede gesprekken, samen met Niek!), Rebecca (jij ging me voor in NYC, dank voor al je tips en hints, en kom vooral nog even langs met Annebetje!), Anouk F. (GUIDE‐genootje), Mark H. (stille wateren hebben diepe gronden), Trijnie, Nicolette, Floris (lekkere reepjes!), Tjasso (goeiesmorgens!), Golnar (vloeiend Nederlands, mét een glimlach), Atta (wilde danser), Krystof, Marjolein, Shiva (opvolgster op de Korreweg), Bojana en Manon en Mariska (lekker vrolijke MDL‐dames!), Jannes, Titia, Jannet, Axel, Sandra, Fiona, Klaas Nico (ik zeg NVGE) en Han (nog steeds denk ik aan GUIDE). Anne Margot (bijna naamgenoot, ik dacht dat IK snel praatte... duizendpootje! gaan we nog eens samen eten en/of rennen?), Petra en Cyril (beiden bedankt voor de swivel‐kooien!). Mijn opvolgster Nicoline wens ik veel plezier en success met het mucositisonderzoek! Lieve Spijs‐girls: niet alleen maakten we er een knalfeest van tijdens Edmond’s oratie, maar ook heb ik erg genoten van onze Journal Clubs, Transport meetings, en alle sociale en sportieve activiteiten daarnaast! Willemien (lieve Mientje, wat leuk dat we zoveel gedeelde ‘passies’ hebben! Sorrento was een topper, maar NYC wordt een serieuze tegenhanger :P. Super dat je mijn proefschrift voor me meeneemt, je hebt de primeur als eerste externe lezer! stiekum ben je mijn derde paranimf :)), Marjan (Mjan, lieve meid, eerlijk en oprecht, dat ben jij, dank voor de fijne tijd in Grunn, je wordt zeker een goede internist), Andrea (An, onze baby‐spijs‐girl. Ik heb genoten van samen spinnen en rennen en gezellig lunchen, met jouw motivatie en inzet komt het helemaal goed!) en Mariëtte (Mjet, paranimf, zie aldaar!). Een speciaal woord van dank voor mijn kamergenootjes die in wisselende samenstelling samen met mij op kamer Y2.163 hebben gezeten: Esther (altijd bereid om te helpen met engelengeduld, laten we weer es een koffietje pakken!) , Maaike (fel en gedreven, we lijken inderdaad meer op elkaar dan je misschien in eerste instantie zou zeggen. Welcome back!), Niels (rustig aan maar), Martijn (je magnetron doet het nog steeds), Jaana (back to Finland, nice that you were our roommate!), Henk (de rust en kalmte zelf, dank voor het bewaren van de vrede op onze kamer, je bent 161 | 169 A Appendices | Dankwoord – Acknowledgements een mooie Groninger!), Karen (handy babe, ik kom snel je nieuwe huissie bewonderen), Wytske (Wyts, als iedereen al weg was hadden wij de mooiste gesprekken :P. Veel succes verder!), Maurien (lieve Maurienepien, dank voor je blijde, rustige aanwezigheid en hulpbereidheid, en dat je je ouders hebt overtuigd dat ik een goede huisgenoot zou zijn :)), Gijs (Gijs jongen, wat hadden we ’t leuk in Yankee stadium, niet? Wanneer wordt je nou huisvader?) en Arne (Arnie, er komt weer een mooi feessie aan!). Jullie weten allemaal dat ik een onwijze stresskip kan zijn die met megasnelheid door de gangen naar het lab of de Bloedbank racet, maar daarentegen het liefst in volledige stilte met oorpluggen in achter mijn pc werk... en desondanks ook erg hou van een gezellig praatje of een goede grap, koffie of thee tussendoor (en die dan dikwijls vergeet op te drinken omdat ik ‘te druk ben’, I know Wyts.... Eten met ‘de kamer’ is superleuk en moeten we erin houden! Ontzettend veel dank dat jullie mij geaccepteerd hebben ondanks mijn ‘rare fratsen’, dat jullie mij hielpen als Excel of Word weer eens ‘gek deed’ of ik acute hulp op het CDL nodig had, of even een moeilijk experiment aan jullie voor wilde leggen zodat ik vervolgens makkelijker knopen kon doorhakken. Dank voor alle goede gesprekken over het leven in het algemeen en jullie luisterend oor en oprechte geïnteresseerdheid toen mijn vader ziek werd, naast de ‘standaard’ moeilijkheden die mijn dubbelleven toch al opleverde. A Alle medewerkers van het CDL: onwijs bedankt voor alle hulp die jullie hebben geboden bij de planning en uitvoering van mijn experimenten! Zonder jullie had ik geen experiment kunnen doen! Daarnaast heb ik bewondering voor hoe jullie je inzetten voor het welzijn van de proefdieren. Juul (een hart van goud! Altijd maakte jij tijd om te helpen op het CDL met mijn experimenten, of met histologie, of coupes snijden... Mensen als jij vindt je maar weinig, helemaal met zo’n lekker Amsterdams accent! Onwijs veel dank voor al je hulp, onze leuke gespreken en sportieve activiteiten die je organiseerde!), Rick (mannen worden knapper naarmate ze ouder worden... en wijzer! Zo leerde jij mij al heel vroeg dat koekjes helpen om iets geregeld te krijgen, maar ook ‘dat alles niet altijd zomaar gaat’. Ik wilde vaak van alles, en natuurlijk het liefst zo snel mogelijk, maar wel in goed overleg. Dank voor al je jugulariscatheters en duodenumsondes, en wijze lessen en heldere inzichten over de verschillen tussen de man en de vrouw!), Pieter (dank voor je ontelbare injecties in de staartvene, zoals jij prikt er geen een! En daarnaast natuurlijk voor de gezellige koffietjes). Alex, Flip, Arie, Wiebe, Mirjam, Annet, Hester, Katrien en Jochem: dank voor al jullie ondersteuning! Angela (run for fun), Yvonne (gusellig), Sylvia (alles wordt geregeld), Harm (goedlachs), Natascha (nuchter en oprecht, zo mag ik het!), Ramon (blije schoonmaker, hulde!), Maurice (lekker relaxed), Annemiek (betrokken bij de dieren!), Annemieke en Michel en Andre: dank voor alle ‘spoedoperaties’, ‘spoedconsulten’ en swivel‐kooien! Michel, zelfs in de weekenden kwam jij helpen, top! 162 | 169 Appendices | Dankwoord – Acknowledgements Andere collega’s van de afdeling Kinder‐MDL/vierde verdieping: Els (dank voor de gezellige gesprekjes beyond onderzoek, en alles wat je voor me geregeld hebt afgelopen jaren!), Jannie (sportieve fietser, altijd in voor een gezellig praatje), Anneke (gezellig mens, dank voor de BBQ bij jullie thuis en trui van Edmond), Johan (laat je ’t me weten als er weer een feestje is? Die racefietstocht maken we volgend jaar!), Astrid (helemaal op je plek, vindt altijd nog wel een gaatje in de agenda), Aad (alle financiën altijd vlug en netjes afgehandeld, dankjewel!), Frank B. (wanneer mogelijk aanwezig bij de JC :)), Frank v.N. (voor de @BK), Hester en Rene (ESPGHAN in Sorrento... top :), Robert (onderzoeker). Collega’s van de afdeling Kinderoncologie: Karin M. (in de tussentijd getrouwd, aangenomen voor de opleiding, en bijna gepromoveerd, super! Athene was erg gezellig, maar ook de gewone koffietjes en lunches tussendoor, thanks!), Louise (dank voor je relaxte opstelling als Wim en ik weer moesten overleggen en jij achter je bureau aan ’t werk was, gelukkig deed je zelf af en toe ook even een koffie mee), Michel (dank voor je ‘inwijding in de wereld van mucositis’, je tips en hints toen ik als frisse promovendus in het UMCG aankwam), Esther, Kim, Alida, Erik, Arja, Tiny, Aeltsje en overigen: voor de gezellige KION‐besprekingen. Carolina en de andere dames van het secretariaat: voor de ondersteuning. En verder nog uit het UMCG of elders in Groningen: de hulp van de afdeling Pathologie bij het doorvoeren, inbedden, snijden en/of kleuren van weefsel! (in het bijzonder Henk, Marco, Edwin en Jan). Fred en Monique Spijkervet (voor het overleg over orale mucositis wat helaas niet haalbaar is in ons rattenmodel... Wel leuk om elkaar te zien en spreken tijdens MASCC!). Lieve familie Pruis, onwijs bedankt dat ik mijn laatste jaar bij jullie thuis mocht wonen! Collega’s van de Bloedbank, dank voor jullie flexibiliteit en gezellige sfeer op de werkvloer!). Collega’s in den lande: Henk, Gardi en Kristien uit het Erasmus MC te Rotterdam: dank voor jullie hartelijke ontvangst in jullie lab waar we al met al toch heel wat weekjes hebben samengewerkt! Henk, dank dat je me steeds opnieuw weer wilt uitleggen hoe het ook weer zit met al die verschillende isotopen...! Superleuk dat we via het AMC contact houden). Carin (MASCC in NYC was supergaaf meid! en die review zit nog steeds in ’t vat hoor :)) en Walter (leuk dat je erbij bent!) uit het UMC Nijmegen. Jody en Henny uit het Canisius‐Wilhelmina ziekenhuis te Nijmegen: veel dank voor alle snelle citrulline bepalingen en fijne communicatie! My colleagues abroad: Dear Dr. Sonis: thank you so much for letting me visit you and your lab in Boston in 2008! It’s been an honor to talk with THE mucositis ‘founding father’ about my mucositis research in Groningen. Your opinions and advice have been a great inspiration for me and very useful. As you can see, everything turned out just fine :). Great that we met again last summer in NYC during MASCC. Whenever there is time, I look forward to visiting your lab in Boston again (much easier from NYC 163 | 169 A Appendices | Dankwoord – Acknowledgements than from the Netherlands). Dear Dr. Grand, Dr. Krasinki and Dr. Montgomery: thank you all for having me at your lab in 2008 and discussing my results with you. It’s always good to get a fresh view on your research, especially from experienced intestinal researchers. It was also very nice to meet the other Dutch students and to see the old pictures of Edmond as Sinterklaas :). Dear members from the mucositis study group of MASCC, thank you all for the fruitful presentations and discussions about mucositis in all its aspects! I’m very glad that I could learn everything about mucositis from you guys! Rachel, thanks for asking me to join the ‘GI mucositis review group’! It was the beginning of a nice friendship and will maybe lead to a sincere collaboration with Adelaide, let’s keep in touch via Skype! (13.5 h time difference, a piece of cake :)). Dear Prof. Augenlicht (Len) and Dr. Thompson (and all colleagues from the labs on the 4th and 5th floor): thank you for letting me join you at the Montefiore Hospital here in the Bronx. It’s a wonderful experience to work here, and to learn new techniques in the lab (Len, we will isolate those Paneth cells yet!) and at the same time being able to join Pediatric Gastroenterology lectures, patient rounds and to watch interesting endoscopies! Now that my thesis is finally done, I will have plenty of time :) Dank aan mijn lieve vrienden in Amsterdam, Leiden, Groningen en elders... Ik heb heel wat afgereisd afgelopen jaren, per trein en Mercedes en geprobeerd bij alle feestjes, verjaardagen, Sinterklaaspartijen, Kerstdiners, kraambezoeken en andere bijzondere gelegenheden te zijn, en veel vaker dan ik wilde heb ik af moeten zeggen... Soms al weken vantevoren omdat ik wist dat ik een experiment had, maar vaak ook op het laatste moment omdat op een lab alles nou eenmaal altijd anders loopt dan gewenst en ik toch echt maar op 1 plek tegelijk kan zijn en Amsterdam‐Groningen minstens 1 uur en 40 minuten rijden is, hoe hard ik ook rij... (en natuurlijk ook omdat ik zelf altijd teveel plan in te weinig tijd en ik er zelf voor gekozen heb een dubbelleven te leiden :)). Heel veel dank voor al jullie begrip! Ik hoop dat jullie middels dit boekje een beetje een idee krijgen waar ik me afgelopen jaren mee bezig heb gehouden. Wat zal ik straks genieten van alle tijd ‘die over is’! Ouderwets cordialeten en avondjes afspreken met mijn lieve vriendinnen van Fatum Fuit, racefietsen langs de Amstel met Marianne en Matthijs en daarna lekker koken, thee drinken bij Amardy, traditioneel Kerstdiner in april met Gijs, Yael, Egbert, Carolien, Daniël en Lisette, mijmeren over het leven met Bertolli (Robert‐Jan), zondagmiddagbrunch met Joost en Marije, borrelen en feesten met Thijs, Marloes, Chris, Tosca, Jasper, Linde, Paul, Jolanda, Rick en Liselotte, weekendje Zuidveld met mijn lieve bestuursgenootjes, reunie met mijn oude RSG‐vriendinnen, middagje bijkletsen met studievrienden Dinaat, Heida en Rolf, Oud Asopos feesten met oude roeimaatjes om maar eens wat te noemen... A En natuurlijk wil ik ook mijn lieve familie bedanken: Lieve Pa, met je hart van goud en nuchtere Friese wijsheden ben je een voorbeeld voor ons allemaal. Aan die Alzheimer doen we helaas niks, maar we maken er het beste van en we rukken straks 164 | 169 Appendices | Dankwoord – Acknowledgements zeker een mooie fles open! Lieve Ma, even mijn verhaal kwijt onder het genot van een heerlijk bakkie koffie! Ik kijk uit naar de volgende brief in NYC. Lieve Wen, jij zet altijd alles op alles om elkaar even te kunnen zien en hebt altijd een kleine attentie op zak, je bent een lieve zus! Ook met Maurice en Mereltje ben ik natuurlijk heel blij, en kleine Melissa is in mijn gedachten. Lieve Ruud en Lex, mijn helden! Onwijs veel respect heb ik voor hoe jullie samen zorgen voor Pa, iedere dag opnieuw, en dat naast een drukke baan, sport, en vrienden. Ruud hard aan het werk met je eigen huis, gelukkig heb je een heel lieve vriendin, Faye! En Lex, aan de vriendin of toch een scharrel...? We gaan ’t zien. Lieve Hugo en Christa, mijn ‘bijna’ schoonouders. Al 16 jaar staan jullie deuren voor mij open en ben ik kind aan huis. Zo’n beetje alles bespreken we met elkaar, en jullie positieve advies om de baan in Groningen aan te pakken heb ik dan ook ter harte genomen. Met ‘praktische problemen’ valt prima te dealen dus is geen reden om het niet te doen! Heel veel dank voor jullie luisterend oor, wijze adviezen, begrip en warme thuis! Jullie Rob is een held en Jos is in mijn gedachten. Last but not least: mijn allerliefste Rob... Waar moet ik in vredesnaam beginnen met jou te bedanken...?! Als geen ander weet jij hoe zwaar het af en toe is geweest en hoe moeilijk we het hebben gehad. Niet alleen de promotie an sich, maar vooral ook het elkaar moeten missen, de stress, het altijd maar alles moeten regelen en op elkaar afstemmen omdat er altijd te weinig tijd was... Al langer dan de helft van mijn leven sta jij aan mijn zijde en je bent zoveel meer dan mijn vriend. Je bent mijn partner, mijn maatje, mijn alles. Op jou kan ik altijd en onvoorwaardelijk steunen. Jij weet steeds mijn stress te temperen, mij te troosten met bloemen en chocola, te verrassen met een lekker diner in de stad, een mooie voorstelling of concert, een weekendje Parijs of gewoon een filmpje op de bank met een kopje thee. Redder in nood als de pc of laptop kuren krijgt! Dit boekje is volledig opgemaakt door jou en ik zweer dat je inmiddels bijna zelf op mucositis kunt promoveren :). Maar bovenal was jij er gewoon zelf voor mij en regelde jij dat we elkaar zo vaak mogelijk zagen en spraken, hoe lastig dat soms ook was. Wat ontzettend heerlijk hebben we het nu samen in New York!! Ik beloof je dat ik nooit meer zo’n project op afstand zal aannemen, so you better get used to me :). Ik hou van je! Je Margot En dan nu: feest! A 165 | 169 Appendices | Affiliations of Co‐Authors AFFILIATIONS OF CO‐AUTHORS Author Bateman, Emma
Blijlevens, Nicole
Bowen, Joanne
Dorst, Kristien Elad, Sharon Gibson, Rachel Kamps, Willem Keefe, Dorothy King, Emily Lalla, Rajesh Plösch, Torsten
Rings, Edmond Schierbeek, Henk
Stellaard, Frans
Stringer, Andrea
A 166 | 169 Affiliation
School of Medicine, University of Adelaide, Adelaide, Australia Department of Hematology, Radboud University Medical Center, Nijmegen, the Netherlands School of Medical Sciences, University of Adelaide, Adelaide, Australia Department of Pediatrics, Sophia Children’s Hospital, Erasmus MC, Rotterdam, the Netherlands Division of Oral Medicine, Eastman Institute for Oral Health, University of Rochester Medical Center, Rochester, NY, USA School of Medical Sciences, University of Adelaide, Adelaide, Australia Department of Pediatrics, Beatrix Children’s Hospital, University Medical Center Groningen, Groningen, the Netherlands School of Medical Sciences, University of Adelaide, Australia Royal Adelaide Hospital, Adelaide, Australia Section of Oral Medicine, University of Connecticut Health Center, Farmington, CT, USA Section of Oral Medicine, University of Connecticut Health Center, Farmington, CT, USA Department of Pediatrics, Beatrix Children’s Hospital, University Medical Center Groningen, Groningen, the Netherlands Department of Pediatrics, Beatrix Children’s Hospital, University Medical Center Groningen, Groningen, the Netherlands Department of Pediatrics, Sophia Children’s Hospital, Erasmus MC, Rotterdam, the Netherlands Department of Pediatrics, Emma Children’s Hospital, Academic Medical Center, Amsterdam, the Netherlands Department of Laboratory Medicine, University Medical Center Groningen, Groningen, the Netherlands School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, Australia Appendices | Affiliations of Co‐Authors Tissing, Wim
Van der Velden, Walter
Van Dijk, Theo
Van Goudoever, Hans
Verkade, Henkjan
Voortman, Gardi
Yazbeck, Roger
Department of Pediatrics, Beatrix Children’s Hospital, University Medical Center Groningen, Groningen, the Netherlands Department of Hematology, Radboud University Medical Center, Nijmegen, the Netherlands Department of Pediatrics, Beatrix Children’s Hospital, University Medical Center Groningen, Groningen, the Netherlands Department of Pediatrics, Emma Children’s Hospital, Academic Medical Center, Amsterdam, the Netherlands Department of Pediatrics, VU University Medical Center, Amsterdam, the Netherlands Department of Pediatrics, Beatrix Children’s Hospital, University Medical Center Groningen, Groningen, the Netherlands Department of Pediatrics, Sophia Children’s Hospital, Erasmus MC, Rotterdam, the Netherlands School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, Australia A 167 | 169 Appendices | Publications PUBLICATIONS 1.
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Spatial and temporal expression of surfactant proteins in hyperoxia‐induced neonatal rat lung injury. S.A.J. ter Horst, M. Fijlstra, S. Sengupta, F.J. Walther, G.T.M. Wagenaar. BMC Pulmonar Medicine 2006 Apr 18; 6: 8. Lactose maldigestion during methotrexate‐induced gastrointestinal mucositis in a rat model. M. Fijlstra, E.H.H.M. Rings, H.J. Verkade, T.H. van Dijk, W.A. Kamps, W.J.E. Tissing. Am. J. Physiol. Gastrointest. Liver Physiol. 2011 Feb; 300 (2): G283‐G291. Continuous enteral administration can overcome the limited capacity to absorb glucose in rats with methotrexate‐induced gastrointestinal mucositis. M. Fijlstra, E.H.H.M. Rings, T.H. van Dijk, T. Plösch, H.J. Verkade, W.J.E. Tissing. Supportive Care in Cancer 2012 Sep 26; in press. Reduced absorption of fatty acids during methotrexate‐induced gastrointestinal mucositis in the rat. M. Fijlstra, W.J.E. Tissing, F. Stellaard, H.J. Verkade, E.H.H.M. Rings. Clinical Nutrition; in press. Continuous enteral administration can enable normal amino acid absorption in rats with methotrexate‐induced gastrointestinal mucositis. M. Fijlstra, H. Schierbeek, G. Voortman, K.Y. Dorst, J.B. van Goudoever, E.H.H.M. Rings, W.J.E. Tissing. Journal of Nutrition 2012; 142: 1983‐1990. Parenteral feeding during methotrexate‐induced gastrointestinal mucositis prevents weight loss in the rat. M. Fijlstra, W.J.E. Tissing, H.J. Verkade, E.H.H.M. Rings. Clinical Nutrition; under review. Systematic review of agents for the management of gastrointestinal mucositis in cancer patients. R. Gibson, D. Keefe, R. Lalla, E. Bateman, N. Blijlevens, M. Fijlstra, E. King, A. Stringer, W. van der Velden, R. Yazbeck, S. Elad, J. Bowen. Supportive Care in Cancer; in press. A 168 | 169 Appendices | Biografie BIOGRAFIE Margot Fijlstra werd op 10 januari 1980 geboren te IJlst, een van de Friese Elfsteden, waar zij opgroeide met haar vader en moeder, zus Wendy en broertjes Rudy en Alex. In 1998 behaalde Margot haar VWO‐diploma aan de RSG Magister Alvinus te Sneek. Daar ontmoette zij tevens haar grote liefde Rob, met wie zij in 1998 naar Leiden verhuisde om te studeren. Na haar propedeuse Biologie stapte ze in 1999 over op Geneeskunde, waarvoor zij inmiddels was ingeloot (propedeuse cum laude). Margot zong als alt bij Collegium Musicum. Ook werd zij lid bij studentenvereniging L.V.V.S. Augustinus en ging ze wedstrijdroeien bij de A.L.S.R.V. Asopos de Vliet. In 2002 was zij een jaar fulltime vice‐praeses en wedstrijdcommissaris in het bestuur van Asopos. In 2003 zat Margot voor de Studenten Groepering Leiden (SGL) in de Universiteitsraad. In 2004 deed zij via het Excellente Studenten Traject in het Laboratorium Neonatologie van het Leids Universitair Medisch Centrum (LUMC) onderzoek naar experimentele broncho‐pulmonaire dysplasie, onder supervisie van Dr. Ir. G.T.M. Wagenaar en Prof. dr. F.J. Walther. Dit leidde tot haar eerste publicatie. Na een extra keuzecoschap kindergeneeskunde in Zuid Afrika deed Margot in 2007 haar semi‐artsstage op de afdeling Neonatologie van het LUMC, waarna zij haar artsexamen haalde. Na haar afstuderen werkte Margot tien maanden als artsassistent Kindergeneeskunde in het Westeinde Ziekenhuis te Den Haag. Daarna volgde zij haar interesse voor basaal wetenschappelijk onderzoek. Van januari 2008 tot januari 2012 was zij promovendus in het Laboratorium Kindergeneeskunde van het Universitair Medisch Centrum Groningen (UMCG) waar zij onderzoek deed naar de vertering en opname van voedingsstoffen tijdens chemotherapie‐geïnduceerde darmslijmvliesschade (mucositis) in een rattenmodel, onder begeleiding van kinderarts‐oncoloog Dr. W.J.E. Tissing en kinderarts‐MDL Prof. dr. E.H.H.M. Rings. De resultaten van haar onderzoek zijn beschreven in dit proefschrift. In mei 2012 won zij de Young Investigator Award van de Multinational Association of Supportive Care in Cancer (MASCC). Naast haar promotieonderzoek werkte Margot in deeltijd als donorarts bij Sanquin Bloedvoorziening. Doordeweek woonde zij in Groningen; in de weekenden samen met Rob, eerst in Leiden en vanaf 2009 in Amsterdam. Sinds mei 2012 doet Margot onderzoek als postdoctal fellow in The Bronx, New York, USA (in de Colon Group, Department of Oncology, Albert Einstein Cancer Center, Montefiore Medical Center) naar de rol van Wnt‐signalering en Panethcellen op diëet‐
geïnduceerde darmkanker, onder begeleiding van ontwikkelingsbioloog Prof. dr. L.H. Augenlicht en kinderarts‐MDL Dr. J.F. Thompson (The Children’s Hospital at Montefiore). Zij woont samen met Rob in de Upper East Side, Manhattan. Na terugkomst in Nederland in 2013 is Margot voornemens als artsassistent Kindergeneeskunde te beginnen in Amsterdam. Zij streeft ernaar om kinderarts‐MDL te worden en patiëntenzorg met wetenschappelijk onderzoek te combineren. 169 | 169 A