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Cystic Fibosis

n te Gsto-Intestinl Tct

Mjn Wotyzen-Bkke

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The research described in this thesis was carried out at the Department of Pediatrics,

Beatrix Childrens Hospital, Center for Liver, Digestive and Metabolic Diseases, University

of Groningen, University Medical Center Groningen and was inancially supported by

the Junior Scientiic Masterclass Groningen.

The author gratefully acknowledges the inancial support for printing this thesis by:

Graduate school for drug exploration University of Groningen

University Medical Center Groningen

ISBN 978-90-367-5090-5 (printed version)

ISBN 978-90-367-5091-2 (digital version)

2011 Mjn Wotyzen-Bkke

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 holding the

copyrights of the articles.

Cover & layout design: Marjan Wouthuyzen-Bakker

Printed by: Whrmann Print Service, Zutphen

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Cystic Fibosis

n te Gsto-Intestinl Tct

Proefschrift

ter verkrijging van het doctoraat in de

Medische Wetenschappen

aan de Rijksuniversiteit Groningen

op gezag van deRector Magniicus, dr. E. Sterken,

in het openbaar te verdedigen op

woensdag 12 oktober 2011

om 11.00 uur

door

Marjan Wouthuyzen-Bakker

geboren op 7 december 1978

te Delfzijl

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Promotor: Prof. dr. H.J. Verkade

Copromotor: Dr. H.R. de Jonge

Beoordelingscommissie: Prof. dr. E.J. Duiverman

Prof. dr. R.O.B. Gans

Prof. dr. A.K. Groen

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For children, the disease Cystic Fibrosis is a word that is dificult to pronounce.

In 1965, a young boy with Cystic Fibrosis overheard his mother talking about the disease

on the phone. When she said the words Cystic Fibrosis, he thought she said 65 roses.

The mother was touched, because her son saw something beautiful in a disease that canoften be quite ugly. Since that time, the term 65 Roses has been used by children of all

ages to describe their disease. The phrase is now a registered trademark of the Cystic

Fibrosis Foundation, which adopted the rose as its symbol.

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Paranimfen: Farah Klingenberg-Salachova

Willemien de Vries

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CONTENTS

CHAPTER 1General introduction to the thesis ......................................................................................................... 7

CHAPTER 2

Energy intake and growth in cystic ibrosis patients with preserved pulmonary

function .......................................................................................................................................................... 27

Submitted

CHAPTER 3

Persistent fat malabsorption in cystic ibrosis; lessons from patients and mice .......... 41Journal of Cystic Fibrosis 2011; 10(3): 150-8

CHAPTER 4

Antibiotic treatment and fat absorption in cystic ibrosis mice ............................................ 59

Pediatric Research, accepted for publication

CHAPTER 5

Ursodeoxycholic acid modulates bile low and bile salt pool independently of

the cystic ibrosis transmembrane regulatory gene in mice ................................................... 77

Am J Physiol Gastrointestinal Liver Physiol, conditionally accepted for publication

CHAPTER 6

Calcium induced anion secretion in gallbladder epithelium of pigs and mice with

cystic ibrosis ............................................................................................................................................... 93

Submitted

CHAPTER 7

General discussion and summary .....................................................................................................111

APPENDICES

Nederlandse samenvatting ..................................................................................................................126

Dankwoord .................................................................................................................................................130

Biograie .......................................................................................................................................................133

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ChapTEr 1

INTRODUCTION TO THE THESIS

M. Wouthuyzen-Bakker

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10

ChapTEr 1 INTrOduCTION

INTrOduCTION

Cystic Fibrosis (CF) is a complex, potentially fatal disease and affects multiple organs.

Respiratory failure due to irreversible destruction of the lungs is the predominant cause

of mortality in CF patients.1-2 Gastro-intestinal involvement is the second most severe

manifestation of the disease. CF-related pancreatic and intestinal disease results in

various problems, including malabsorption of fat and a suboptimal nutritional status.

Biliary involvement may contribute to the former and, in addition, may also lead to end-

stage liver disease in a subset of patients.3 The aim of this thesis was to obtain more

detailed pathophysiological insight in CF-related gastro-intestinal disease by evaluating

current treatment and by investigating potential future treatment options. First, the

normal function of the CFTR protein (Cystic Fibrosis Transmembrane conductance

Regulator) will be described. Second, the consequences of CFTR dysfunction in CF-

disease in various organs will be outlined, with a particular emphasis on CF-related

symptoms in the gastro-intestinal tract and corresponding treatment. In addition,

various CF animal models will be discussed as they greatly contribute(d) to theknowledge on CF pathophysiology and related treatments.

ThE CYSTIC FIBrOSIS TraNSMEMBraNE CONduCTaNCE rEGuLaTOr (CFTr)

The human Cftrgene is located on the long arm of chromosome 7 and encodes for the

Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CFTR is a glycoprotein

located at the apical membrane of (primarily) epithelial cells and is a member of the

ATP-binding cassette (ABC) transporter family.4 CFTR is mostly expressed in gland-rich

organs, e.g. the lungs, pancreas, intestine, gallbladder, intra- and extra-hepatic bile ducts,

reproductive tract and sweat-glands.1-2 Recent data showed that CFTR is also localizedin non-epithelial cells and other tissues, including lymphocytes, skeletal muscle,

smooth muscle, the thyroid gland and neurons.5-13 The most well deined function of

CFTRcomprises its role as a chloride and bicarbonate channel. However, the knowledge

about the function of CFTR has expanded impressively during the last decade. Apart

from its role in anion-transport, CFTR also transports hyaluronic acid, regulates the

activity of several other (transporter) proteins, is involved in fatty acid metabolism and

autophagy and is (indirectly) responsible for the proper function of defensins (proteins

with an antimicrobial function).14-20 The secretory and regulatory function of CFTR

especially underlines its importance in luid and electrolyte transport across epithelialtissues. The CFTR protein structure comprises two transmembrane spanning domains,

two nucleotide binding domains and one regulatory domain (Figure 1).4

CFTR is (indirectly) activated by an increase of cyclic AMP in the cell (caused by various

substances, including secretin). Cyclic AMP increase induces CFTR phosphorylation at

the regulatory domain by protein kinase A.The phosphorylation stimulates the binding

of ATP at the nucleotide binding domains and consequently results in the opening

of the CFTR channel. Subsequently, chloride, but also bicarbonate, can be secreted

into the lumen of the particular organ or gland. The secretion of anions generates a

transepithelial potential difference (lumen negative) which promotes paracellular

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sodium secretion and, consequently, osmotically driven luid secretion. Additionally,

CFTR may act itself as a bicarbonate channel or as a chloride shunt for a separate

chloride-bicabonate exchanger (Figure 3), resulting in active bicarbonate secretion.4

The cascade of anion and luid secretion results in a ine water-layer at the epithelial

surface which is responsible for the hydration of mucus and in the airways- allows the

cilia to clear the mucus in a coordinated manner.21 Hydration of mucus and suficient

mucus clearance prevents the harbouring of (pathogenic) bacteria and toxic/infectious

agents to accumulate in the lumen.22 Recently, the importance of bicarbonate release

for the expansion of mucins by electrostatic repulsion has become evident.23-26 Thus,

bicarbonate might be essential for the formation of a normal gel-forming protective

mucus layer. The described cascade of events after CFTR activation clearly shows the

important role of CFTR in the protection of the epithelial lining of various glands and

organs (Figure 3A). Inactivity or absence of CFTR, caused by various Cftrgene mutations,

results in the disease known as: cystic ibrosis.

Figure 1. Protein structure of the cystic ibrosis transmembrane conductance regulator(CFTR). CFTR is

mainly located in the apical membrane of epithelial cells and consists of two transmembrane spanning domains,

two nucleotide binding domains and one regulatory domain.

CYSTIC FIBrOSIS (CF)

Cystic Fibrosis (CF) is a potentially fatal, autosomal recessive disease with an incidence

of 1:3600 life births in the Caucasian population.1-2 The prevalence of the disease

increases due to screening of newborns and increased recognition of patients with a

mild(er) CF phenotype.27-28 The prognosis of CF patients greatly improved during the

last twenty years. Life expectancy increased from an average of thirty years up to forty

years of age (Figure 2). 29 CF can be detected by a positive sweat chloride test, e.g. above

30 mmol/L for infants below six months and above 60 mmol/L for children above

six months of age. A second positive chloride test or the identiication of two CFTR

mutations is conirmative for the diagnosis.30

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CF is caused by absent, reduced or aberrant synthesis of CFTR due to Cftr gene

mutations.31 Dysfunctional CFTR results in an impaired secretion of luid at the epithelial

surface and consequently results in the formation of sticky and viscous mucus (which is

the reason why CF is also referred to as mucoviscidosis).Viscous mucus obstructs and

damages the particular organ and may allow the harbouring of pathogenic bacteria.

The cascade of events can induce a chronic state of inlammation in various organs and

recurrent (primarily pulmonary) infections.1-2,4

One of the classic concepts in CF pathophysiology for the development of viscous mucus

is the low volume hypothesis. The low volume hypothesis postulates that sodium

hyper-absorption occurs by the loss of inhibition of the sodium channel ENaC, which

results in the inability to hydrate the epithelial lining and results in the development of

viscous mucus (Figure 3).32-34 This hypothesis has been recently challenged by indings in

newborn CF pigs. In the airways of these pigs, no sodium hyper absorption or reduction

Figure 2. Adapted from the Cystic

Fibrosis Foundation Registry 2007

Annual Data Report. Bethesda, Maryland.

2009 CF foundation.

Figure 3. Simpliied scheme

of the function of CFTR under

physiological and CF conditions. The

scheme represents the classic concept

of CF pathogenesis in bronchial

epithelial cells. Note: (1) In gastro-

intestinal tissues, sodium transport is

not mediated via ENaC, but via Na+/

H+ exchangers (NHEs) or Na+-coupled

glucose and aminoacid transporters.

(2) In sweat-gland ducts, CFTR serves

to absorb chloride, not to secrete it.

Median predicted survival age, 1985-2007

1985 1990 1995 2000 2005

24

26

28

30

32

34

36

38

40

42

year

mediansurvivalage

(years)

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in the volume of the periciliary liquid layer (the water-layer at the epithelial surface)

was present, but bacterial colonization and inability to clear bacteria was nevertheless

observed.35-36 These indings are supported by the fact that not all tissues affected in CF

express the epithelial sodium channel ENaC. Sodium absorption in the gastro-intestinal

tract (e.g. small intestine, pancreas, liver and gallbladder) is not mediated via ENaC, and

despite this, these tissues do exhibit obstruction by viscous mucus. This paradox led

Quinton et al. to investigate the importance of bicarbonate in the pathogenesis of viscous

mucus. 23-26 They hypothesized that bicarbonate is essential for the expansion of mucins

and demonstrated that the absence of bicarbonate, at least under ex-vivo conditions, led

to the formation of viscous mucus. Therefore, it is reasonable to speculate that the defect

in bicarbonate secretion, aside chloride, is the most important factor contributing to

CF-related disease. Unravelling the complex pathogenesis of CFTR dysfunction clearly

remains an important challenge in CF research.

At present, more than 1800 CFTR mutations have been identiied.37 In Europe, the deltaF508 mutation is the most common mutation found in CF patients. In the European

population, 90% of the CF patients are heterozygous for the delta F508 mutation, while

70% carry the mutation on both alleles.38-39 Most of the mutations can be classiied into

one of six categories, each classiication class with a distinct functional alteration of

CFTR (Figure 4).31

Figure 4. Classiication of Cftr mutations. Simpliied scheme of the different classes of Cftr mutations and their

consequences for the functionality of the CFTR protein. Class I mutations are found in ~10% of CF patients and

include non-sense and out-frame mutations. In most of these mutations, premature truncation of normal Cftr

translation results in the absence of synthesis of CFTR. Class II mutations include the delta F508 mutation (in-

frame deletion), which is found in ~90% of CF patients. The deletion of phenylalanine (located in the nucleotide

binding domain 1) causes an abnormal folding of CFTR, which is recognized by the endoplasmatic reticulum

and is subsequently followed by degradation of the protein. Part of the misfolded/immature proteins escape

degradation and still reaches the cell surface where residual anion secretion may be present. In class III and

IV mutations, the CFTR reaches the cell surface, but the function of the protein is impaired by either abnormal

gating of the ion-channel or altered conductance of ion transport respectively. In class V and VI mutations there

is a quantitative reduction of CFTR at the cell surface. In class V mutations, reduced CFTR synthesis occurs, whilein class VI mutations, there is an increased endocytosis and degradation of CFTR in the lysosomes. 31

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Although the severity of the disease tends to be highest in mutation class I III and

mildest in IV - VI, an apparent genotype-phenotype association in CF patients is

not found. Symptoms may even differ in the same family, which indicates that the

type of mutation is not solely responsible for the CF phenotype. Modiier genes and

environmental factors are suggested to contribute to the disease severity, especially

concerning the pulmonary and hepato-biliary phenotype.40-43 Depending on the

type of mutation, therapies are currently developed and tested to inhibit premature

termination of CFTR synthesis, to correct the misfolded CFTR or to stimulate residual

CFTR function in the apical membrane.44 The so called CFTR potentiators may restore

gating defects and the correctors may improve CFTR protein folding and plasma

membrane traficking. At present, VX-770, a CFTR potentiator that may be beneicial for

patients with the CftrG551D mutation (class III), is currently tested in a phase 3 study

with promising results.45

CLINICaL MaNIFESTaTIONS CF aNd COrrESpONdING TrEaTMENT

As described above, the severity and occurrence of clinical symptoms may differ among

CF patients. We will outline the most common clinical manifestations in patients with

classicor typical CF (diagnosed as described above) and corresponding treatment

options. 46

Respiratory symptoms

Respiratory involvement is the irst cause of mortality in CF patients. 47-48 The

accumulation of viscous and thick mucus in the distal airways results in obstruction,

inlammation and recurrent pulmonary infections. Chronic and productive cough,dyspnoea, malaise, anorexia and weight loss often occurs, depending on the stage of

the disease.1-2 Aggressive pulmonary intervention improved the nutritional status and

survival of CF patients.49-53 Inhalation with bronchodilators, mucolytics and hypertonic

saline may reduce the severity of symptoms and, inhalation with antibiotics may partly

prevent pulmonary infections. As the disease progresses, irreversible pulmonary

damage with a gradual decline in pulmonary functions occurs. When pulmonary

function is severely declined (FEV1

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of pancreatic insuficiency involves supplementation with pancreatic enzymes and

fat soluble vitamins and a high caloric diet. Pancreatic suficient CF patients are more

prone to the development of episodes of acute and chronic pancreatitis.56-57 In roughly

one-third of adult patients with CF, the endocrine pancreas becomes also affected and

results in CF-related diabetes mellitus.47

Gastro-intestinal abnormalities

Compared to healthy peers, symptoms of gastroesophageal relux disease are more

described in CF patients and are mostly related to transient relaxation of the lower

esophageal sphincter.58-59 Intestinal obstruction syndromes are a common problem in

CF patients. Around 10-20% of CF newborns present with meconium ileus at birth, a

condition in which the meconium (the irst stool of the infant) is thickened and can

severely obstruct the intestine, particularly at the level of the distal small intestine.

Meconium ileus is often the irst sign for CF. 60-61 Reversely, around 50% of infants with

meconium ileus are diagnosed with CF.62 In older children, episodes of bowel obstruction(also known as distal ileal obstruction syndrome) and constipation may occur. Intestinal

obstruction can be treated with oral laxatives and/or intestinal lavage, or if very severe,

with surgical intervention. 60-61 Several secondary changes in the small intestine, like

chronic intestinal inlammation, mucus accumulation, prolonged intestinal transit time

and small intestinal bacterial overgrowth are also described in the CF intestine and may

contribute to malnutrition. 63-68

Hepatobiliary involvement

CFTR is expressed in the gallbladder and intra- and extra-hepatic bile ducts. 69 A broadspectrum of hepato-biliary manifestations due to CFTR dysfunction has been identiied

and may include the occurrence of gallbladder cysts, micro-gallbladder, cholelithiasis

and cholestasis.3 Only a subset of patients (~30%) develops CF liver disease (CFLD)

characterized by focal biliary cirrhosis, from which the course is subclinical in one-third

of these patients.70-73 Autopsy data have indicated that, 70% of CF patients have evidence

of focal biliary cirrhosis. Although the deinition of CFLD may vary between CF care

centers, CFLD has been commonly deined by positive liver histology for cirrhosis or

the presence of at least two of the following parameters: hepatomegaly, two abnormal

serum liver enzymes or ultrasound abnormalities other than hepatomegaly (e.g.increased echogenicity, nodularity, irregular margins, splenomegaly).70 The cascade of

decreased bile low, bile duct plugging, inlammation and focal biliary cirrhosis may

ultimately progress to multilobar cirrhosis with portal hypertension in ~8% of CF

patients. 71 In addition, increased bile toxicity and an altered bile salt metabolism are

suggested to contribute to the bile duct damage.74-76 At present, end-stage CFLD accounts

for approximately the third leading cause of death in CF patients after pulmonary

disease and complications of lung transplantation.70 A peak incidence of CFLD occurs

around the age of ten. It is unclear why some patients do and some patients do not

develop CFLD. Unlike a clear genotype-phenotype relation for pancreatic involvement,

a relation between genotype and liver disease is not so apparent. Modiier genes and

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environmental factors are thought to play a dominant role in the development of CFLD.

The male gender, the presence of meconium ileus at birth, pancreatic insuficiency, age

of diagnosis and ethnic background have been identiied as risk factors for CFLD, but

have not been conirmed in other studies. At present, only the 1-antitrypsin Z-allele

as modiier gene has been associated with the presence CLFD, with an odds ratio of

4.2.70,77 Ursodeoxycholic acid (UDCA) is the common medical treatment for CFLD.

Although routinely applied in CF patients with increased levels of biochemical liver

function tests, the therapeutic action of UDCA in CF conditions remains unclear. It has

been hypothesized that UDCA is beneicial by its choleretic activity and/or its capacity

to correct aberrant bile salt metabolism.78-80 No beneicial effect on survival has been

documented, but it has been shown to improve biochemical abnormalities.81

Other

Since CFTR is expressed in various cell types, several other CF-related symptoms are

described.1-2 CF patients often suffer from nasal polyps and chronic sinusitis. Especiallywhen children with CF are frequently treated with corticosteroids, osteoporosis and

delayed puberty may occur. Male patients with CF are infertile due to obstruction of

the vas deferens. Female patients may have problems with fertility when the cervix is

severely obstructed with viscous mucus.

NuTrITIONaL STaTuS

Most children with CF have a suboptimal nutritional status. Since an impaired nutritional

status is correlated with morbidity and mortality, optimizing the nutritional status has

become an essential part of treatment in CF patients.82-85 The main factors contributingto the suboptimal nutritional status are malabsorption of nutrients, inadequate energy

intake and, high energy expenditures (mainly due to recurrent pulmonary infections).

Accordingly, cornerstones in the nutritional treatment of CF are Pancreatic Enzyme

Replacement Therapy (PERT) for pancreas insuficiency, dietary energy intakes of

120-150% of the Recommended Dietary Allowance (RDA) and the prevention and

early treatment of pulmonary infections.85 Most CF infants fail to thrive at birth. After

establishing the diagnosis of CF, supplementation with pancreatic enzymes will usually

induce catch-up growth, but this growth spurt is often not suficient for complete

normalization. On average, children with CF grow between the 20th

and 30th

percentileon the growth chart compared to healthy peers, and decline when pulmonary disease

progresses.86-88 The implementation of neonatal screening for CF may prevent the irst

decline in nutritional status directly after birth due to the early start of nutritional

intervention. Because fat malabsorption persists in around 25-50% of CF patients

despite optimal treatment with PERT,3 increased insight into the pathophysiology of CF

related fat malabsorption is necessary to investigate new treatment strategies. Proton

pomp inhibiters may improve the process of fat digestion and absorption in some

individuals by reducing the duodenal acidic environment.89

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CYSTIC FIBrOSIS aNIMaL MOdELS

The development of several CF animal models has led to an increased understanding

regarding the pathophysiology of CF. Until recently, CF mouse models were the only

available animal model to study CF-related disease. CF mice proved to be very suitable

to study intestinal disease and to evaluate novel therapeutic strategies to correct or

potentiate defective CFTR. An important limitation of the CF mice, however, is that they

tend to have a relatively mild CF phenotype, apart from the intestine.90-91 Most CF mouse

models are pancreas suficient and do not exhibit CF lung or liver disease. The mild

to absent phenotype in the organs of CF mice is in contrast to that in CF humans, and

this has stimulated the development of other CF models in other species. Recently, the

development of the CF pig and the CF ferret has opened a new era in CF research to

investigate CF related pancreatic, liver and lung disease.92-95

CF mice

Complete Cftrknockout mice and mice with the homozygous delta F508 mutation havebeen developed by several research groups.90-91 Intestinal disease is the most prominent

feature in CF mice. Most of the developed Cftr knockout mice have severe intestinal

obstruction at birth, comparable to meconium ileus in the CF infant. The mortality

rate in these mice is as high as 95%, and, dependent on the genetic background, Cftr

knockout mice only survive on a liquid diet. The homozygous delta F508 mice still have

residual anion secretion and therefore, exhibit a very mild intestinal phenotype. Both

CF mouse models have impairments in the absorption of fat and increased fecal loss of

bile salts compared to wild type littermates, but only the Cftrknockout mice also have a

reduced body weight.96 Because CF mice are pancreas suficient, the CF mouse modelsare especially suited to gain insight into the mechanisms of pancreatic independent fat

malabsorption.

CF pigs

Cftr knockout pigs and homozygous delta F508 pigs are born with meconium ileus,

exocrine pancreas obstruction and focal biliary cirrhosis.93-95 Due to the severe intestinal

obstruction at birth, all piglets require surgical intervention (e.g. temporarily ileostoma)

for survival beyond neonatal age. Both CF-pig models have a micro-gallbladder, a

condition which is also observed in approximately 30% of CF patients.3

Although thebody weight at birth is the same as in wild type littermates, newborn Cftrknockout

piglets fail to thrive shortly after birth, similar as seen in CF infants. No pulmonary

abnormalities (cellular inlammation, infection or dilated or plugged submucosal

glands) are described at birth, but characteristic features of lung disease do develop

spontaneously with time. 93-95

CF ferrets

Cftr knockout ferrets display many disease characteristics similar to the pathology

observed in CF patients, including a predisposition to pulmonary infections during the

postnatal period, meconium ileus at birth, pancreatic insuficiency and biochemical

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signs of liver disease.92 The biochemical parameters for liver disease (increased serum

transaminases and bilirubin) normalize in these animals after UDCA treatment. Around

50% of Cftr knockout ferrets with meconium ileus die after birth from intestinal

obstruction and subsequent perforation.Additionally, even the pups without meconium

ileus fail to thrive and die a couple of days after birth, probably because of severe

malabsorption of nutrients. Due to the high mortality rate accompanying the very

severe intestinal obstruction at birth, a gut corrected ferret model was developed by

incorporating human CftrcDNA in the intestine. Although not suficient to completely

escape the severe intestinal phenotype, one out of four of these transgenic ferrets

passed normal stools and escaped meconium ileus. This gut corrected ferret will be

used to expand the (recently developed) CF ferret line.92

ThE rOLE OF aLTErNaTIVE ChLOrIdE ChaNNELS

It has been speculated, that the marked difference in phenotypic characteristics between

CF patients, pigs, ferrets and mice (Table 1) may be due to differences in alternative

chloride transport.97-99 Various studies investigated the occurrence and relevance of

several apically located alternative chloride channels in CF conditions. 97-99 Calcium

Table 1. Organ involvement CF patients, pigs, ferrets and mice. Percentages given are estimates of the

fraction of all CF patients/CF animals in which the pathology is manifest. The degree at which organs are affected

may vary between animals of the same genotype. *Data on Cftr knockout mice are mainly based on observations

in Cftr tm1Cam and Cftrtm1Unc knockout mice. Homozygous F508 mice display a very mild phenotype with

none of the above described abnormalities, except the occurrence of diet-dependent intestinal obstruction. - : not

(explicitly) reported in literature.

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induced chloride channels have been considered as the most important candidate

for alternative chloride transport in epithelial cells.100 Because the molecular identity

of calcium induced chloride channels were unknown for quite some time, it was not

possible to further explore and characterize their functionality in different organs. Due

to the recent identiication of TMEM16 as a new family of chloride channels, alternative

chloride transport regained interest in the CF ield.101-105 Alternative chloride channels

may serve as potential rescue channel in CF conditions and could be of interest in

therapeutic strategy to prevent or mitigate the CF phenotype.100

SCOpE aNd OuTLINE OF ThE ThESIS

We ultimately aim to improve the prognosis of CF by treatment of the several intestinal

and hepato-biliary manifestations of CF disease. In this thesis we address various aspects

of the gastro-intestinal manifestations of CF disease by evaluating current treatment

and investigating potential future treatment options. In Cte 2, we evaluated the

necessity of a high caloric diet in CF patients with a preserved pulmonary function.Especially during the last decades much emphasis has been placed on a high caloric

diet in CF patients in order to improve nutritional status. We wondered whether this

approach is necessary in all CF patients, especially those who are in a relatively mild

state of disease. In retrospect, we cross-sectionally and longitudinally analyzed energy

intake in this subgroup of CF patients and its relation to nutritional status and growth.

Cte 3 provides an overview of literature on several aspects, other than pancreatic

insuficiency, that might contribute to persistent fat malabsorption in CF patients. We

reviewed intervention studies in CF patients and CF mice and their clinical implications.

As antibiotic treatment has been shown to improve body weight in CF mice with smallintestinal bacterial overgrowth, we tested the hypothesis (Cte 4), that this might

be due to improved fat absorption. We treated homozygous delta F508, Cftrknockout

mice and wild type littermates, with broad-spectrum antibiotics and evaluated fat

absorption. We analyzed small intestinal bacterial lora and bile salt composition,

as these could be inluenced by antibiotic treatment and may provide mechanistic

information on possible changes in fat absorption. In Cte 5, we addressed the

therapeutic eficacy of ursodeoxycholate (UDCA) in CF conditions. UDCA is presently the

only medical treatment option for CF-related liver disease, but the therapeutic action of

UDCA in CF conditions is unclear. The postulated effect of UDCA could be mediated viaits choleretic activity or its effect on bile salt metabolism. In Cftrknockout mice and

wild type littermates, we evaluated these options by determining several parameters

of bile salt metabolism and bile low after UDCA treatment. Finally,we hypothesized

that the severe gallbladder phenotype in CF pigs, in contrast to the near absence of a

gallbladder phenotype in CF mice, is due to the differential expression and/or activity

of alternative calcium activated chloride channels, in particular TMEM16A (Cte

6). This hypothesis was tested experimentally by measuring trans-epithelial anion

currents in gallbladder samples of CF mice, CF pigs and wild type littermates, following

stimulation with CFTR-or CaCC-speciic agonists, and by monitoring expression levels

of TMEM16A by immunocytochemistry and real-time PCR. Identifying putative rescue

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channels in CF might lead to a therapeutic strategy to prevent or mitigate the CF

phenotype in patients. In Cte 7 a summary of the most relevant indings will be

discussed, including suggestions for further research.

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4. Gadsby DC, Vergani P, Csanady L. The ABC protein turned chloride channel whose failure

causes cystic ibrosis. Nature 2006;440:477-483.

5. McDonals TV, Nghiem PT, Gardner P et al. Human lymphocytes transcribe the cystic ibrosis

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6. Lamhonwah AM, Bear CE, Huan LJ et al. Cystic ibrosis transmembrane conductance regulator

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8. Guo Y, Su M, McNutt MA et al. Expression and distribution of cystic ibrosis transmembrane

conductance regulator in neurons of the human brain. J Histochem Cytochem

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57. De Boeck, K, Weren, M, Proesmans, M, et al. Pancreatitis among patients with cystic ibrosis:

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60. Houwen RH, van der Doef HP, Sermet I et al. Intestinal obstruction syndromes in cystic ibrosis:

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62. Gorter RR, Karimi A, Sleeboom C. Clinical and genetic characteristics of meconium ileus in

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65. Bruzzese ER, Raia V, Gaudiello G et al. Intestinal inlammation is a frequent feature of

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69. Cohn JA, Strong TV, Picciotto MR, et al. Localization of the cystic ibrosis transmembrane

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70. Colombo C, Battezzati PM, Crosignani A, et al. Liver disease in cystic ibrosis: A prospective

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74. Lindblad A, Hultcrantz R, Strandvik B. Bile-duct destruction and collagen deposition: a

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75. Freudenberg F, Broderick AL, Yu BB et al. Pathophysiological basis of liver disease incystic ibrosis employing a deltaF508 mouse model. J Physiol Gastrointest Liver Physiol

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76. Smith JL, Lewindon PJ, Hoskins AC et al. Endogenous ursodeoxycholic acid and cholic acid in

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77. Wilschanski M, Rivlin J, Cohen S et al. Clinical and genetic risk factors for cystic ibrosis-

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78. Fiorotto R, Spirli C, Fabris L, et al. Ursodeoxycholic acid stimulates cholangiocyte luid

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79. Shimokura GH, McGill JM, Schlenker T, et al. Ursodeoxycholate increases cytosolic calciumconcentration and activates Cl- currents in a biliary cell line. Gastroenterology 1995;109:965-

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80. Gurantz D,.Hofmann AF. Inluence of bile acid structure on bile low and biliary lipid secretion

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81. Cheng K, Ashby D, Smyth R. Ursodeoxycholic acid for cystic ibrosis-related liver disease.

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83. Beker LT, Russek-Cohen E, Fink RJ. Stature as a prognostic factor in cystic ibrosis survival. J

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85. Sinaasappel M, Stern M, Littlewood J, et al. Nutrition in patients with cystic ibrosis: a

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86. Haeusler G, Frisch H, Waldhor T et al. Perspectives of longitudinal growth in cystic ibrosis

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87. Patel L, Dixon M, David TJ. Growth and growth charts in cystic ibrosis. J R Soc Med 2003;96

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88. Stettler N, Kawchak DA, Boyle LL et al. Prospective evaluation of growth, nutritional status,

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91. Scholte BJ, Davidson DJ, Wilke M, De Jonge HR.Animal models of cystic ibrosis. J Cyst Fibr

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92. Sun X, Sui H, Fisher JT et al. Disease phenotype of a ferret CFTR-knockout model of cystic

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93. Meyerholz DK, Stoltz DA, Pezzulo AA, Welsh MJ. Pathology of gastro-intestinal organs in a

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94. Rogers CS, Stoltz DA, Meyerholz DK et al. Disruption of the CFTR gene produces a model of

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95. Ostedgaard LS, Meyerholz DK, Chen JH et al. The F508 mutation causes CFTR misprocessing

and cystic ibrosis-like disease in pigs. Sci Transl Med 2011; in press.

96. Bijvelds MJ, Bronsveld I, Havinga R, et al. Fat absorption in cystic ibrosis mice is impeded

by defective lipolysis and post-lipolytic events. Am J Physiol Gastrointest Liver Physiol

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97. Zdebik AA, Cuffe JE, Bertog M et al. Additional disruption of the ClC-2 Cl- channel does

not exacerbate the cystic ibrosis phenotype of cystic ibrosis transmembrane conductanceregulator mouse models. J Biol Chem 2004;279(21):22276-83.

98. Bijvelds MJ, Bot AG, Escher JC et al.Activation of intestinal Cl- secretion by lubiprostone requires

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99. Jentsch TJ. CLC chloride channels and transporters: from genes to protein structure, pathology

and physiology. Crit Rev Biochem Mol Biol 2008;43(1):3-36.

100. Verkman AS, Galietta LJV. Chloride channels as drug targets. Nat Rev 2009;8:153-68.

101. Yang YD, Cho H, Koo JY et al. TMEM16A confers receptor-activated calcium-dependent

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102. Schroeder BC, Cheng T, Jan YN et al. Expression cloning of TMEM16A as a calcium-activated

chloride subunit. Cell 2008;134:1019-1029.

103. Huang F, Rock JR, Harfe BD, et al. Studies on expression and function of the TMEM16A calcium-

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104. Galietta LJV. The TMEM16A protein family: a new class of chloride channels. Biophys J2009;97:3047-3053.

105. Caputo A, Caci E, Ferrera L, et al. TMEM16A, a membrane protein associated with calcium

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ChapTEr 2

ENERGY INTAKE AND GROWTH

IN CYSTIC FIBROSIS PATIENTS WITH

PRESERVED PULMONARY FUNCTION

M. Wouthuyzen-Bakker1

C.K. van der Ent2

J.W. Woestenenk3

M. Zwolsman1

H.J. Verkade1

F.A.J.A. Bodewes1

1 Pediatric Gastroenterology, Department of Pediatrics, University Medical Center

Groningen, University of Groningen, Groningen, The Netherlands.2 Department of Pediatrics, University Medical Center Utrecht, Utrecht,

the Netherlands3 Julius Centre for Health Sciences and Primary Care, Department of Dietetics and

Nutritional Science, University Medical Center Utrecht, Utrecht, the Netherlands

Sbmitte

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ChapTEr 2 INTaKE aNd GrOWTh

aBSTraCT

Improving the nutritional status remains an important challenge in pediatric cystic

ibrosis (CF) patients. The effect of a high energy intake on the nutritional status in

relatively healthy pediatric CF patients is not well described. We investigated the

relation between energy intake, nutritional status and growth in school-age CF patients

with preserved pulmonary function. In a cohort of Dutch CF patients, we analyzed

data of 81 children with FEV1-values >80%. Patients below 6 and above 9 years were

excluded to avoid inaccurate spirometric outcomes and growth interference during

puberty respectively. Wecross-sectionally assessed the relation between energy intake,

nutritional status and growth (multiple-regression) and longitudinally evaluated the

effect of changes in energy intake on weight (paired t-test). The nutritional status

was slightly below average compared to the healthy reference population (z-score

weight:-0.61.8, BMI:-0.10.7). Upon cross-sectional analysis, we found no positive

relation between energy intake and nutritional status or growth. Upon longitudinal

analysis, 19 patients increased their energy intake (10.29.8 kcal/kg/bw) in the yearafter the initial assessment, but the z-score for weight improved (0.180.16) in only

20% of the 19 patients. The positive effect of energy intake on weight was irrespective

from the initial z-score for weight or the initial energy intake. Conclusion. The relation

between energy intake, nutritional status and growth is weak in school-age CF patients

with preserved pulmonary function. The results emphasize the need for individualized

nutritional treatment, but simultaneously underline the dificulty to design nutritional

trials with suficient statistical power in this subgroup of CF patients.

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INTrOduCTION

Cystic Fibrosis (CF) is an autosomal recessive disease and is caused by a mutation on

the Cftrgene located on chromosome 7, which encodes for a chloride and bicarbonate

channel in, particularly, epithelial cells. A defective CFTR protein results in the

formation of viscous and sticky mucus with bacterial colonization, inlammation and

eventually damage to different glands and gland rich organs. Intestinal malabsorption

of nutrients (mainly due to pancreatic insuficiency), inadequate energy intake and

increased energy expenditure due to recurrent pulmonary infections all contribute

to a suboptimal nutritional status in CF patients. 28 Since nutritional status is related

to morbidity and mortality in CF, improving the nutritional status remains one of the

important challenges in CF care.

The well known epidemiological study in Boston and Toronto of Corey et. al. in the late

80s, introduced a major shift in the nutritional treatment of CF. The formerly restricted

fat diet adviced for CF patients, was changed into a high fat diet. Another major changecomprised more aggressive pulmonary treatment and prevention of weight loss

during infections. This renewed approach resulted in an improved nutritional status

and overall survival of CF patients.2, 6, 8, 26 Consequently, the current general CF dietetic

recommendations comprises high dietary energy intakes ranging from 110-200% of

the Recommended Daily Allowance (RDA) in patients with pancreatic insuficiency, in

which energy derived from fat should be approximately 40%.28,29

However, many children with CF have dificulties to meet the dietary recommendations.

24,25 This phenomenon often creates a burden for CF children, parents and health careproviders in their attempt to comply to the general recommendation. Nowadays,

several studies report on adverse social and psychological consequences for CF patients

and their families aiming to achieve high energy intakes, like family stress and feeding

dificulties.7, 16, 23, 39

Since there are no conclusive studies which exclusively substantiated the potential

beneicial effects of a high dietary energy intake in CF patients with preserved

pulmonary function, we aimed to investigate the relation between dietary energy intake

and nutritional status and growth in this subgroup of patients.

paTIENTS aNd METhOdS

We retrospectively evaluated data of pediatric CF patients who were under medical

control on the 1st of January 2008 in the University Medical Centers in Utrecht and

Groningen in the Netherlands. Both centers are specialized regional CF care centers.

Patients visited the CF Center, at least annually, for a complete check up, consisting of a

72 hours fecal fat balance test, lung function test and a sputum culture test (if obtained).

Children with FEV1 values above 80% were included in the study. Patients below

6 and above 9 were excluded to avoid inaccurate spirometry outcomes.10, 20, 37 and to

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avoid pubertal growth effects respectively. For our cross-sectional analysis (n=81), the

irst recorded measurement for each child was used. In order to evaluate the effect of

energy intake on weight (longitudinally), children from which complete datasets of at

least two time points were available were included in the study (n=44) (Figure 1). The

excluded children (n=37) had similar patient characteristics (parameters nutritional

status, mutation class, co-morbidity etc) as the included children (data not shown).

This study was deemed exempt by the University Medical Center Groningen and Utrecht

Institutional Review Board.

Assessment of nutritional status

Weight (kg), length (cm) and BMI measurements of every child were collected. Annual

growth velocity for length and weight were calculated using the charts of Tanner and

Whitehouse. 35 Growth velocity was deined as the increment in the childs length (cm/

yr) and weight (kg/yr) divided by the time interval between the two measurements(with a minimum of 9 months and a maximum of 18 months). All growth parameters

were converted into age-corrected z-scores based on Dutch national growth studies

using Growth analyzer 3 software of the Dutch Growth Foundation.9

Assessment of energy intake

A three-day dietary diary was used to determine dietary intake of total energy. The

dietary diaries contained three day prospective recorded household measurements

with detailed information on macronutrient intake. We assumed that the dietary diary

relected the average daily dietary energy intake in the preceding year. All dietary

diaries were manually processed by experienced specialized CF dieticians. The dietary

Figure 1. Flow chart.

Flow chart for the retrospective

analysis in pediatric CF patients

between the age of 6-9 years with

preserved pulmonary function

(FEV1>80%). For the cross-sectional

analysis, the irst measurement of

each child was used (n=81). For

the longitudinal analysis, data

available with at least two time

points per child was used. The irst

two measurements per time pointwere selected (n=44). FEV1: Forced

Expiratory Volume in 1 second.

dataset 1999-2008

657 measurements

n = 268

FEV1 > 80%

440 measurements

n = 183

FEV1 < 80%

217 measurements

n = 108

6-9 years

43 measurements

n = 31

6-9 years

150 measurements

n = 81

2 measurements per child:

n = 44

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Statistical analysis

Statistical analysis was performed using SPSS 16.0 (SPSS Inc., Chicago, IL).Cross-sectional

analysis. The relation between energy intake and nutritional status and growth was

cross-sectionally assessed by linear regression analysis. A stepwise multiple regression

analysis was performed to correct for confounding effects on nutritional status and

growth. The analysis included z-scores for weight, length, BMI, weight gain and length

growth as dependent variablesand were tested for normal distribution.Total energy

intake, fat absorption (%), mutation class, age and gender were used as independent

variables. We repeated the analysis and replaced the variable total energy intake for the

variable energy intake of fat. The differentCftrmutation classes were included in the

multiple regression analysis by transforming them into dummies and by using the enter

method. Longitudinal analysis. We longitudinally evaluated the effect of an increased or

decreased energy intake (compared to the previous year) on the z-score for weight by

using the paired student t-test. All data are reported as means the standard deviation

(SD). P-values < 0.05 were considered as statistically signiicant.

rESuLTS

Characteristics study group

Table 1 shows the patient characteristics for the cross-sectional as the longitudinal

analysis for several parameters. Parameters were similar between the cross-sectional

and the longitudinal data. All patients within our cohort were pancreas insuficient and

were treated with pancreatic enzymes. The overall nutritional status was slightly belowaverage compared to the healthy reference population according to length, weight and

BMI. Growth velocity scores were above average for length as well as weight. The mean

total energy intake and the percentual energy intake derived from fat in our cohort of

pediatric CF patients was comparable to the general CF nutritional recommendations

of 120-150% [28]. Except for meconium ileus, with a higher incidence in males, gender

was equally distributed among the other parameters.

Relation between energy intake and nutritional status (cross-sectional analysis)

Although the mean z-scores for nutritional status in our cohort of patients was slightlybelow average compared to the healthy reference population (Table 1), the z-score for

weight was below zero in 78% of patients (Figure 3). We found no relation between

total energy intake or fat intake and z-scores for weight, length, BMI, weight gain and

length growth (Figure 3). A weak negative association was found between total energy

intake and the z-score for weight (R=0.27, p=0.02). The multiple regression analysis,

that included several potential confounding factors on nutritional status or growth (fat

absorption, mutation class, age and gender), showed no dependency on these results.

The z-score for BMI was positive associated with age (R= 0.22, p=0.03).

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Table 1. Patient characteristics. Baseline characteristics of cross-sectional and longitudinal data of pediatric

cystic ibrosis patients with preserved pulmonary function (deined as FEV1 values > 80%). Z-scores for weight

and length are depicted as z-scores for age. Cross-sectional and longitudinal values for all parameters were

not statistically different from each other. Ursodeoxy-cholate was prescribed because of suspicion of CF related

liver disease, based on persistent increased serum trans-aminases, hepato-megaly and/or hepatic ultrasound

abnormalities (40-41). CFTR gene mutations were divided in ive classes based on their demonstrated orpresumed molecular consequences (19).

FEV1: Forced Expiratory Volume in 1 second. RDA: Recommended Daily Allowance. Kcal/kg/bw: kilocalories per

kilogram body weight. Parameters are reported as a percentage or as mean SD.

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The effect of an increased or decreased energy intake on z-score weight (longitudinal

analysis)

In correspondence with the cross-sectional results, we found no relation between energy

intake and the z-score for weight in the longitudinal analysis (Figure 4). Out of the 44

patients, 17 increased their energy intake in the year after the initial assessment (mean

increase 10.29.8 kcal/kg/bw), but only 4 of these patients showed a corresponding

improvement in the z-score for weight (right upper quadrant in Figure 4A), while

the majority of patients showed a stabilization or decrease in the z-score for weight

(right lower quadrant in Figure 4A). A decrease in energy intake in the year after the

initial assessment (mean decrease 13.39.5 kcal/kg/bw) in 27 of patients, resulted in a

corresponding decrease in the z-score for weight in roughly 50% of these patients, while

the other 50% of patients showed an improvement (left lower and upper quadrant inFigure 4A, respectively).

Figure 3. Relation between energy intake and nutritional status (cross-sectional analysis).

Upper panel: relation between total energy intake and z-scores for weight (A), BMI (B), and weight gain (C).

Lower panel: relation between fat intake and z-scores for weight (D), BMI (E), and weight gain (F). kcal/kg/bw:

kilocalories per kilogram body weight. R: correlation coeficient.

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To examine these results in more detail, we additionally investigated whether the

effect of an increased or decreased energy intake on weight after a year of the initial

assessment, depended on the initial z-score for weight or on the initial amount of

energy intake. We subdivided patients with initial z-scores for weight above or below

0. The subdivision did not affect the longitudinal relation between energy intake and

z-score for weight (Figure 5). We found a decrease in z-score for weight in patients

with initial high z-scores for weight during increased energy intake (n=5; Figure 5B).

When subdividing patients with initial energy intakes above and below 90 kilocalories

per kilogram body weight, we neither found a difference between an increased or a

decreased energy intake concerning the effect on the z-score for weight (data not

shown).

Figure 4. Relation between delta energy intake and delta z-score for weight (longitudinal analysis).

A) Relation between the difference () in annual energy intake and the corresponding annual difference in z-score

for weight. B) Percent distribution of patients with increased or decreased energy intake after a year of the initial

assessment and the corresponding effect on the z-score for weight. kc/kg/bw: kilocalories per kilogram body

weight. R: correlation coeficient. : increased, : decreased, : not changed.

Figure 5. The effect of an increased or

decreased energy intake on z-score

weight.

In order to conclude about the effect of

an increased (A-B) or decreased (C-D)

energy intake in relation to the initial

nutritional status of CF patients, patients

were categorized according to their initial

z-score of weight (below or above 0) at

time point 0 (T0). Figures A and C depicts

the actual increase or decrease in energy

intake in kilocalories per kilogram body

weight (kcal/kg/bw) in the following year

(T1) . Figures B and D depicts the effect of

the increased or decreased energy intake

on z-score weight in the following year

(T1). Values are depicted as mean SD. *: p-value < 0.05 (comparing T0 with T1).

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dISCuSSION

Our study is the irst which exclusively investigated the relation between energy intake

and nutritional status and growth in school-age pediatric CF patients with a preserved

pulmonary function. We found that approximately 80% of CF children have a z-score for

weight below zero, despite preservation of pulmonary function. However, retrospective

evaluation, in a cross-sectional and longitudinal setting, showed no relation between

energy intake and nutritional status or growth.

Until now, most studies evaluated the relation between energy intake and either

nutritional status or growth in pediatric CF patients who already are in a more advanced

stage of disease 1, 4, 13, 15, 18-19, 22, 27, 30-33, 36, 38 (e.g. patients with impaired pulmonary function

and/or patients who are more severely malnourished). From retrospective studies we

learned that, in this group of CF patients, a weak correlation (R

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subgroup of patients; all of our patients were pre-pubertal, had preserved pulmonary

function and suffered from pancreatic insuficiency. Each of these factors might have a

major inluence on resting energy expenditure and growth40, but it seems reasonable

to expect that these parameters were comparable between patients. In addition, we

analyzed for possible inluence of several other parameters on the observed correlations

in our multiple regression analysis (e.g. gender, age, mutation class). Yet, neither of

these parameters seemed to affect our results.

Since the CF nutritional recommendations still comprises high energy intakes for all

pediatric CF patients, our data support the concept that sub-analysis of different groups

of CF patients is necessary to categorize or even individualize the dietary advices. We

believe it is important to investigate the relatively healthy CF patients with preserved

pulmonary function, to conclude whether the actual beneicial effects on nutritional

status or growth outweigh the possible adverse physiological consequences in our

strive to achieve high dietary energy intakes. Our retrospective analysis in school-ageCF patients with preserved pulmonary function indicates no straight-forward relation

between energy intake and nutritional status or growth. We do believe that our data

supports the need for prospective nutritional studies in this subgroup of CF patients,

but our results simultaneously underline the dificulty to design such nutritional trials

with suficient statistical power.

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survival. J Am Diet Assoc 101(4):438-442

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6. Corey M, McLaughlin FJ, Williams M, Levison H (1988)A comparison of survival, growth, and

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7. Duff AJ, Wolfe SP, Dickson C, Conway SP, Brownlee KG (2003) Feeding behavior problems

in children with cystic ibrosis in the UK: prevalence and comparison with healthy controls. J

Pediatr Gastroenterol Nutr 36(4):443-7

8. Durie PR, Pencharz PB (1992) Cystic ibrosis: nutrition. Br Med Bull 48(4):823-846

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9. Dutch Growth Foundation, growth and development in children. A programme for

pediatricians and researchers, by pediatricians and researchers. The Netherlands 6 A.D.;

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with cystic ibrosis in the clinical setting. Pediatr Pulmonol 45:419-433

11. Groeneweg M, Tan S, Boot AM, de Jongste JC, Bouquet J, Sinaasappel M (2002)Assessment

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12. Haeusler G, Frisch H, Waldhor T, Gtz M (1994) Perspectives of longitudinal growth in cystic

ibrosis from birth to adult age. Eur J Pediatr 153(3):158-163

13. Hanning RM, Blimkie CJR, Bar-Or O, Lands RC, Mos LA, Wilson WM (1993) Relationships

among nutritional status and skeletal and respiratory muscle function in cystic ibrosis: does

early dietary supplementation make a difference?Am J Clin Nutr 57:580-587

14. Health Council of the Netherlands (2001). Dietary Reference Intakes: Energy, Proteins, Fats,and Digestible Carbohydrates Publication No. June 2002. Council of the Netherlands: The

Hague, ISBN-10: 90-5549-384-8.

15. Jelalian E, Stark LJ, Reynolds L, Seifer R (1998) Nutrition intervention for weight gain in

cystic ibrosis: a meta analysis. J Pediatr 132(3 Pt 1):486-492.

16. Kindstedt-Arfwidson K, Strandvik B (1988) Food intake in patients with cystic ibrosis on an

ordinary diet. Scand J Gastroenterol Suppl 143:160-162

17. Lenaerts C, Lapierre C, Patriquin H, Bureau N, Lepage G, Harel F et al (2003) Surveillance for

cystic ibrosis-associated hepatobiliary disease: early ultrasound changes and predisposing

factors. J Pediatr 143(3):343-350

18. Lloyd-Still JD, Smith AE, Wessel HU (1989) Fat intake is low in cystic ibrosis despite

unrestricted dietary practices. J Parenter Enteral Nutr 13:296-298

19. Luder E, Kattan M, Thornton JC, Koehler KM, Bonforte RJ (1989) Eficacy of a nonrestricted

fat diet in patients with cystic ibrosis. Am J Dis Child 143:458-464

20. Mayer OH, Jawad AF, McDonough J, Allen J (2008) Lung function in 3-5 year old children with

cystic ibrosis. Pediatr Pulmonol 43:1214-1223

21. Patel L, Dixon M, David TJ (2003) Growth and growth charts in cystic ibrosis. J R Soc Med 96

Suppl 43:35-41

22. Powers SW, Byars KC, Mitchell MJ (2003) A randomized pilot study of behavorial treatment

to increase calorie intake in toddlers with cystic ibrosis. Child Health Care 32(4):297-311

23. Powers SW, Mitchell MJ, Patton SR, Byars KC, Jelalian E, Mulvihill MM, et al (2005) Mealtime

behaviors in families of infants and toddlers with cystic ibrosis. J Cyst Fibros 4(3):175-82

24. Powers SW, Patton SR, Byars KC, Mitchell MJ, Jelalian E, Mulvihill MM, et al (2002) Caloric

intake and eating behavior in infants and toddlers with cystic ibrosis. Pediatrics 109(5):E75

25. Powers SW, Patton SR (2003)A comparison of nutrient intake between infants and toddlers

with and without cystic ibrosis. J Am Diet Assoc 103(12):1620-1625

26. Ramsey BW, Farrell PM, Pencharz P (1992) Nutritional assessment and management incystic ibrosis: a consensus report. The Consensus Committee. Am J Clin Nutr 55(1):108-116

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27. Shepherd RW, Holt TL, Cleghorn G, Ward LC, Isles A et al (1988) Short-term nutritional

supplementation during management of pulmonary exacerbation in cystic ibrosis: a

controlled study, including effects of protein turnover. Am J Clin Nutr 48:235-239

28. Sinaasappel M, Stern M, Littlewood J, Wolfe S, Steinkamp G, Heijerman HG, et al (2002)

Nutrition in patients with cystic ibrosis: a European Consensus. J Cyst Fibros 1(2):51-75

29. Stallings VA, Stark LJ, Robinson MS, Feranchak AP, Quinton H (2008) Evidence-based practice

recommendations for nutrition-related management of children and adults with cystic ibrosis

and pancreatic insuficiency: Results of a systematic review. J Am Diet Assoc 108: 832-839

30. Stark LJ, Bowen AM, Tyc VL, Evans S, Passero MA (1990)A behavorial approach to increasing

calorie consumption in children with cystic ibrosis. J Pediatr Psychol 15(3):309-326

31. Stark LJ, Knapp LG, Bowen AM, Powers SW, Jelalian E et al (1993) Increasing calorie

consumption in children with cystic ibrosis: replication with 2-year follow up. J App Beh Anal

26: 435-450

32. Stark LJ, Opipari LC, Spieth LE, Jelalian E, Quittner AL et al (2003) Contribution of behaviour

therapy to dietary treatment in cystic ibrosis: a randomized controlled study with 2-yearfollow-up. Behavior Therapy 34:237-258

33. teinkamp G, Demmelmair H, Rhl-Bagheri I, von der Hardt, H, Koletzko, B (2000) Energy

supplements rich in linoleic acid improve body weight and essential fatty acid status of cystic

ibrosis patients. J Pediatr Gastro Enterol Nutr 31:418-423

34. Stettler N, Kawchak DA, Boyle LL, Propert KJ, Scanlin TF, Stallings VA et al (2000)Prospective

evaluation of growth, nutritional status, and body composition in children with cystic ibrosis.

Am J Clin Nutr 72(2):407-413

35. Tanner JM (1976) Clinical longitudinal standards for height, weight, height velocity, weight

velocity, and stages of puberty. Arch Dis Child 51(3):170-179

36. Vaisman N, Clarke C, Pencharz PB (1991) Nutritional rehabilitation increases resting energy

expenditure without affecting protein turnover in patients with cystic ibrosis. J Pediatr

Gastroenterol Nutr 13:383-390

37. Vilozni D, Bentur L, Efrati O, Minuskin T, Barak A, Szeinberg A, et al (2007) Spirometry in

early childhood in cystic ibrosis patients. Chest 131:356-361

38. Walkowiak J, Przyslawski J (2003) Five year prospective analysis of dietary intake and clinical

status in malnourished cystic ibrosis patients. J Hum Nutr Dietet 16:225-231

39. Ward C, Massie J, Glazner J, Sheehan J, Canterford L, Armstrong D et al (2009) Problem

behaviours and parenting in preschool children with cystic ibrosis. Arch Dis Child 94:341-347

40. Wiskin AE, Davies JH, Wootton SA, Beattie RM (2010) Energy expenditure, nutrition and

growth. Arch Dis Child 1-6

41. Zielenski J, Tsui LC (1995) Cystic Fibrosis: Genotypic and phenotypic variations. Annu Rev

Genet 29: 777-807.

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ChapTEr 3

PERSISTENT FAT MALABSORPTION IN

CYSTIC FIBROSIS;

LESSONS FROM PATIENTS AND MICE

M. Wouthuyzen-Bakker1

F.A.J.A. Bodewes1

H.J. Verkade1

1Pediatric Gastroenterology, Department of Pediatrics, University Medical Center

Groningen, University of Groningen, Groningen, The Netherlands.

Jonl of Cystic Fibosis 2011; 10(3): 150-8

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ChapTEr 3 FaT MaLaBSOrpTION IN CYSTIC FIBrOSIS

aBSTraCT

Fat malabsorption in pancreatic insuficient cystic ibrosis (CF) patients is classically

treated with pancreatic enzyme replacement therapy (PERT). Despite PERT, intestinal

fat absorption remains insuficient in most CF patients. Several factors have been

suggested to contribute to the persistent fat malabsorption in CF (CFPFM). We reviewed

the current insights concerning the proposed causes of CFPFM and the corresponding

intervention studies. Most data are obtained from studies in CF patients and CF mice.

Based on the reviewed literature, we conclude that alterations in intestinal pH and

intestinal mucosal abnormalities are most likely to contribute to CFPFM. The presently

available data indicate that acid suppressive drugs and broad spectrum antibiotics could

be helpful in individual CF patients for optimizing fat absorption and/or nutritional

status.

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INTrOduCTION

Most patients with cystic ibrosis (CF) have a suboptimal nutritional status. In order to

minimize morbidity and mortality, obtaining and maintaining a good nutritional status

remains one of the important challenges in CF.1-3 Next to preventing and controlling

pulmonary infections, a high dietary energy intake diet combined with pancreatic

enzyme replacement therapy (PERT) are classic cornerstones in CF care.

Despite optimizing and maximizing PERT, fat malabsorption in most CF patients persist

(CFPFM). Rather than the physiological absorption above 95%, most CF patients show

a decreased intestinal fat absorption of 85-90% of dietary fat intake.4,5 It is reasonable

to assume that at least part of the failure to approach a physiological degree of fat

absorption could still be attributed to ineficiency and inaccurate dosing of PERT. Also,

a non-synchronous entrance of PERT with dietary energy intake into the duodenum

may play a role in CFPFM.6 However, besides PERT related issues, other factors have

been implied to be involved in CFPFM.7,8

During the last decades, several of these potential contributing factors to CFPFM have

been addressed and investigated in patient studies as well as animal studies. In this

review, we will provide an overview of the different mechanisms that have been implied

to contribute to CFPFM and describe the results of corresponding interventional studies

on fat absorption and/or nutritional status. We will mainly describe studies performed

in CF patients and CF mice. CF mice are especially suitable to exclusively investigate

CFPFM, as most CF mouse models do not suffer from pancreatic insuficiency but do

exhibit impaired absorption of fat.9 Based on the reviewed literature, we will concludeour review with clinical implications and suggestions for further research.

In order to fully recognize and understand the CF-related factors that might be involved

in CFPFM, we start to describe the physiological process of fat digestion and absorption.

phYSIOLOGY OF FaT dIGESTION aNd aBSOrpTION

Dietary fat intake mainly consists of long-, medium- and short-chain triglycerides. The

mechanism of digestion and absorption of these lipids can be dissected into severalsequential steps. In contrast to long-chain triglycerides, medium- and short-chain

triglycerides are known to circumvent several steps in lipid digestion and absorption.

For example, unlike long-chain triglycerides, medium- and short chain triglycerides

are less dependent upon solubilization, escape the re-esteriication into triglycerides

and are directly absorbed into the portal system without being assembled into

chylomicrons.10 Since the majority of dietary fat and energy intake consists of long-

chain triglycerides (92-96%), we exclusively focused on their mechanism of intestinal

digestion and absorption. The mechanism of digestion and absorption of long-chain

triglycerides can be dissected into several sequential processes (igure 1A).

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1) Emulsiication. Emulsiication of triglycerides is a process in which (water-insoluble)

fat droplets are suspended in an aqueous environment. Emulsiication can be achieved by

mechanical as well as chemical means. Mechanical emulsiication is attained by chewing

and forcing dietary fat with high pressure through a small opening (e.g. the pylorus) and

dispersing large fat droplets into smaller droplets. Chemical emulsiication is attained

by the action of bile and prevents the emulsion from re-coalescing. Emulsiication

results in a ine, relatively stable oil-in-water emulsion with an increased surface area.

The water soluble digestive / lipolytic enzymes are active at the site of the water-oil

interface.

2) Lipolysis. During lipolysis, the breakdown (hydrolysis) of lipids into smaller particles

is continued. Around 10-30% of the triglycerides are hydrolyzed in the stomach into

diglycerides and free fatty acids, catalyzed by lingual and gastric lipase (a process also

known as biochemical/lipolytic emulsiication10). Under physiological conditions,

pancreatic lipase completes the process in the proximal part of the small intestine, byhydrolyzing the remaining triglycerides and diglycerides into monoglycerides and free

fatty acid molecules. Bile salts are amphipathic molecules, i.e. they possess a hydrophilic

and a hydrophobic site. Upon exposure to dietary lipid emulsion at the level of the

proximal small intestine, the hydrophobic site orients itself towards the hydrophobic

lipid droplets, whereas their hydrophilic site exposes itself to the aqueous (water) phase.

Lipids droplets whose surface is thus covered bile salts are not accessible to pancreatic

lipase. Pancreatic co-lipase allows the anchoring of the pancreas lipase to the oil-water

interface and is essential for lipolytic activity. Under physiological conditions, pancreatic

lipase is present in vast excess. Thus, in CF, fat maldigestion only occurs during severepancreatic malfunction.11 During exocrine pancreatic insuficiency when pancreatic

lipase is severely reduced, lipolysis is more dependent upon the activity of lingual and

gastric lipase. In CF conditions, lingual lipase remains fully active in the intestine, where

it accounts for more than 90% of lipase activity, even when the intestinal pH drop below

the optimal level for bile salt dependent lipolysis.80

3) Solubilization. The process in which fat molecules are dissolved in water in the form of

(mixed) micelles is called solubilization. Solubilization enhances the aqueous solubility

of fatty acids by several orders of magnitude (100 to 1000 fold).10

Solubilization isnecessary for monoglycerides and free fatty acids to eficiently overcome the diffusion

barrier of the so called unstirred water layer of the enterocytes.10 The unstirred water

layer separates the enterocytes from the luminal contents of the intestine. Solubilization

is achieved by the formation of mixed micelles; mainly consisting of phospholipids and

bile salts derived from biliary secretion. The diameter of the lipid droplets ranges from

100 1000 nm, whereas that of mixed micelles ranges 3-5 nm. The hydrophobic part

of the lipid molecules, s

thesis cf 4

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