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Phospholipid excretion and metabolism and thiol status in cyctic fibrosis Chen, Alice Ho-Wing 2004

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PHOSPHOLIPID EXCRETION AND METABOLISM AND THIOL STATUS IN CYSTIC FIBROSIS By Al ice Ho-Wing C h e n B . S c . (Dietetics) University of British Columbia , 1999 A T H E S I S IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F Master of Sc ience In T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Human Nutrition) W e accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A September 2004 ©Alice Chen , 2004 M a n u s c r i p t t i t le: A s s e s s m e n t of phospholipid malabsorption by quantification of fecal phospholipid S ta tement o f c o - a u t h o r s h i p A C participated in enrollment of subjects, collection of samples , experiment design, fecal analysis, data analysis and interpretation. SI contributed as the principal investigator in grant funding, study concept and design, data analysis and interpretation. Print name: /QR. .<ZM&J-A /A/A//< Date: / Ocjt ^ao^f-Print name: M\ce. CJ\f>n Date: Jf. f)r* Jnn^ M a n u s c r i p t t i t le: Phosphatidylcholine and lysophosphatidylcholine excretion is increased and related to altered plasma homocysteine and methionine in children with cystic fibrosis S ta tement o f c o - a u t h o r s h i p A C participated in enrollment of subjects, collection of samples and in fecal and p lasma analyses , data analysis and interpretation. SI contributed as the principal investigator in grant funding, in study concept and des ign, data analysis and interpretation. A G F D participated as the clinician scientist in patient selection, enrollment, and collection of clinical information. S J J provided analyses of plasma thiols. Print name:?) /? . SHP.iLfl MNl<> Print name: ftticP. C.keh Date: j l OcJr Jhuk Date: ~4 Ort D^f)(f Library Authorization In p r e s e n t i n g th is t h e s i s in par t ia l fu l f i l lment of the r e q u i r e m e n t s for a n a d v a n c e d d e g r e e at the U n i v e r s i t y of Br i t i sh C o l u m b i a , I a g r e e that the L i b r a r y sha l l m a k e it f ree ly a v a i l a b l e for r e f e r e n c e a n d s tudy . I fur ther a g r e e that p e r m i s s i o n for e x t e n s i v e c o p y i n g of th is t h e s i s for s c h o l a r l y p u r p o s e s m a y b e g r a n t e d by the h e a d of my d e p a r t m e n t or by h i s or he r r e p r e s e n t a t i v e s . It is u n d e r s t o o d that c o p y i n g or p u b l i c a t i o n of th is t h e s i s for f i nanc ia l ga i n sha l l not b e a l l o w e d w i thou t m y wr i t ten p e r m i s s i o n . N a m e of A u t h o r (please print) D a t e (dd/mm/yyyy) D e g r e e : _J^4cr_j2i_Sck^C^ Y e a r : D e p a r t m e n t of 4kohv2 n _ ALW^bn T h e U n i v e r s i t y of B r i t i sh C o l u m b i a V a n c o u v e r , B C C a n a d a ABSTRACT Chol ine is an essential nutrient and is present in the diet predominantly (>90%) in phosphatidylcholine (PC) . Large amounts of P C are also secreted into the intestine in bile. P C digestion and absorption requires pancreatic phospholipase A 2 ( P L A 2 ) , which hydrolyzes P C to l y s o P C and fatty ac id . P L A 2 is inhibited at low intraluminal p H , which is common in patients with cystic fibrosis (CF) . Chol ine deficiency results in hepatic steatosis, expla ined by the need for P C synthesis via the cytidine diphosphocholine pathway for secretion of triglyceride in very-low-density lipoprotein. In choline deficiency, the liver increases synthesis of P C via the phosphatidylethanolamine N-methylfransferase pathway in which methyl groups from methionine are transferred to phosphatidylethanolamine, forming P C and S-adenosylhomocyste ine , which is then metabolized to homocysteine. Hepatic steatosis is c o m m o n in patients with C F , but whether patients with C F malabsorb P C , which is a phospholipid (PL) , is unknown. Current methods for fecal fat extraction extract triglyceride and fatty acid but do not quantitatively recover P L . This study a imed to establish a method for quantification of P L , P C and l y s o P C excretion and to determine P L , P C and l y s o P C excretion in children with C F and their associat ions with p lasma thiols. This is a cross-sect ional study of children with C F (n=18) and children without C F (n=8, control). Al l participants provided a venous blood sample , a 72h fecal sample and 5d food record. Feca l total fat and P L were recovered by sequential extraction with ethanol, ether, hexane, chloroform and methanol and were separated and quantified using high performance liquid chromatography ( H P L C ) with evaporative light scattering detector. P l a s m a thiols and P L were analyzed using H P L C . C o m p a r e d with the controls, children with C F had significantly lower fat absorption ( m e a n ± S E M ) ( 8 6 ± 1.6%, 94+1.2%), and higher excretion of fat (12 .9±1 .7g /d , 3 .9±0 .7g /d ) , P L ( 1 3 9 ± 2 0 m g / d , 6 6 ± 1 8 m g / d ) , P C ( 3 9 ± 1 1 m g / d , 2 . 6 ± 1 . 0 m g / d ) and l y s o P C (57+8.1mg/d, 2 2 ± 6 . 2 m g / d ) , respectively. Feca l P L excretion was significantly related to plasma homocysteine (r=0.64) and methionine (r=-0.49). P L , P C and l y s o P C excretion is higher in children with C F and is associated with lower p lasma methionine and higher homocysteine. Altered choline metabol ism may occur in C F and may be important to the hepatic complications. in TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables vi List of Figures vii Lis t of Abbrev ia t ions vii i Acknowledgements xi Chapter 1 Literature Rev iew 1 1. Introduction . 2 1.1 Biochemica l and clinical features of cystic fibrosis 4 1.2 Liver d i sease in cystic fibrosis 6 1.2.1 Prevalence 6 1.2.2 Diagnosis 7 1.2.3 Hepat ic d isease in cystic fibrosis 9 1.2.4 Hepatic steatosis 10 1.2.5 Management 12 1.3 Maldigest ion and malabsorption in cystic fibrosis 13 1.3.1 Pancreat ic insufficiency 13 1.3.2 Abnormali t ies in bile acid metabolism 15 1.3.3 Intestinal abnormalities 17 1.3.4 Management 17 1.3.4.1 Microencapsula ted pancreatic e n z y m e preparations 18 1.3.4.2 Ac id reducing agents 21 1.4 Nutrition requirements for patients with cystic fibrosis 22 1.4.1 Energy requirement. 24 1.4.2 Macronutrient requirement 24 1.4.3 Micronutrient requirement 26 1.5 Cho l ine and phosphatidylcholine 27 1.5.1 Function, dietary sources, digestion and absorption of choline 27 1.5.2 Choline/phosphatidylcholine synthesis 29 1.6 Conc lus ions 34 1.7 Objectives 38 1.7.1 Specific aims 38 1.7.2 Organization of thesis 39 1.8 References 41 Chapter 2 Quantification of Feca l Phospholipids: Method Development 57 2. Introduction 58 2.1 Materials and methods 60 2.1.1 Collect ion and preparation of samples 60 iv 2.1.2 Extraction and quantification of total fat 60 2.1.3 Phosphol ipid quantification 62 2.1.4 Statistical analysis 63 2.2 Resul t s 64 2.3 Discuss ion 66 2.4 References 72 Chapter 3 Phosphat idylchol ine and Lysophosphat idylcholine Excret ion and Its Assoc ia t ion with Altered P l a s m a Homocys te ine and Methionine in Chi ldren with Cyst ic Fibrosis 75 3. Introduction 76 3.1 Subjects and methods 79 3.1.1 Feca l analysis 80 3.1.2 P l a s m a analysis 80 3.1.3 Dietary analysis 81 3.1.4 Statistical analysis 81 3.2 Resul ts 83 3.3 Discuss ion 87 3.4 References 99 Chapter 4 D i scuss ion and Conc lus ions 105 4. Introduction 106 4.1 Discuss ion 108 4.1.1 Extraction and quantification of fecal total triglyceride and phospholipids: method development and validation 108 4.1.2 Fat absorption and phospholipid excretion 114 4.1.3 Assoc ia t ion between choline phosphoglycer ide excretion, and plasma methionine, homocyste ine and S A H 122 4.2 Limitations 126 4.3 Future directions 128 4.4 Conc lus ion 131 4 .5 References 133 Appendix A Informed consent 141 Appendix B F o o d record 154 Appendix C Character is t ics of participants with cystic fibrosis 171 Appendix D U S D A database for the choline content of c o m m o n foods 174 Appendix E Es t imated average choline intake of study participants 192 v L I S T O F T A B L E S C h a p t e r 3 Tab le 3.1 Feca l fat and energy content of children with cystic fibrosis and control children 93 Tab le 3.2 Feca l phospholipid excretion in children with cystic fibrosis and control children 94 Tab le 3.3 P l a s m a thiols and phospholipids in children with C F and control children 95 Tab le 3.4 Associa t ions between p lasma thiols and fecal phospholipid excretion 96 vi LIST OF FIGURES Chapter 1 Figure 1.1 C a u s e s of malabsorption in patients with cystic fibrosis 14 Figure 1.2 C a u s e s and management of malabsorption in patients with cystic fibrosis 19 Figure 1.3 Schemat ic representation of phosphatidylcholine synthesis 30 Figure 1A Schemat ic representation of the research hypothesis of this study 35 Chapter 2 Figure 2.1 Extraction of total fat and phospholipids using a) hexane, diethyl ether and ethanol, b) chloroform, methanol, or c) hexane, diethyl ether and ethanol followed by re-extraction with chloroform, methanol 69 Figure 2.2 Validation of H P L C - E L S D method for the determination of fecal phospholipids w h e n compared with lipid soluble phosphorus quantified by phosphomolybdate colorimetric a s say 70 Figure 2.3 Distribution of phospholipids in fecal fat from children with cystic fibrosis and children without cystic fibrosis 71 Chapter 3 Figure 3.1 Correlat ion between fecal total fat, and fecal energy and fecal total phospholipids 97 Figure 3.2 Scatterplots of p lasma homocysteine versus fecal total phospholipids, phosphatidylcholine, lysophosphatidylcholine and total choline phosphoglyceride in the subgroup of children with cystic fibrosis 98 vii LIST O F ABBREVIATIONS Al adequate intake ALT alanine t ransaminase Apo B apolipoprotein B APP alkaline phosphatase AST aspartate t ransaminase B C C H British Columbia ' s Children 's Hospital BMR basal metabolic rate cAMP cyclic adenosine, 3 ' ,5 ' -monophosphate CDP-choline cytidine diphosphocholine °C degree Ce ls ius CF cystic fibrosis CFTR cystic fibrosis t ransmembrane conductance regulator d day or deci DRI dietary reference intakes EDTA e thylenediamine tetraacetic acid E F A essential fatty acids ELSD evaporative light scattering detector g gram GGT y-glutamlytransferase h hour HDL high-density lipoprotein HPLC high performance liquid chromatography IDL intermediate-density lipoprotein k kilo kcal Calor ies kJ kilo Jou les L liter L D H lactate dehydrogenase L D L low-density lipoprotein L F T liver function test L y s o P C lysophosphatidylcholine m milli or meter M molar m E q molar equivalent m i n minute m o l mole m p h miles per hour micro n nano P C phosphatidylcholine P E phosphatidylethanolamine P E M T phosphatidylethanolamine /V-methyltransferase PI phosphatidylinositol P K A protein kinase A P L phospholipid P L A 2 phosphol ipase A 2 P S phosphatidylserine R E E resting energy expenditure S A H s-adenosylhomocysteine S A M s-adenosylmethionine S E M standard error of the mean S p h sphingomyelin T G triglyceride T N F a tumor necrosis factor alpha T P N total parenteral nutrition U units U D C A ursodeoxycholic acid V L D L very-low-density lipoprotein wt weight y year ACKNOWLEDGEMENTS Specia l thanks to: • Dr She i la Innis, my research supervisor, for her inspiration, guidance, support, patience, and encouragement; • Cyst ic fibrosis team and Dr. A . G . F . Davidson at B . C . ' s Chi ldren 's Hospital for their ass is tance in facilitating this study; • The parents and children who participated in this study; • Hospital for Sick Chi ldren Foundation for funding this study; • Natural Sc iences and Engineer ing Resea rch Counc i l of C a n a d a and the Michael Smith Foundat ion for Health Resea rch for the studentships; • Mr Roger Dyer and M s Janette King for their friendship, encouragement and technical ass is tance throughout every phase of the study; • Dr S u s a n Barr for her academic advice and support; • All my friends a n d my coworkers in Dr Innis' laboratory for the encouragement and company. This work is dedicated to my family, M o m , Dad , Derek, and K e i who have supported me throughout the recent years to make all things possible . X I Chapter 1 Literature review C H A P T E R 1 L I T E R A T U R E R E V I E W Chapter 1 Literature review 1. INTRODUCTION Cyst ic fibrosis (CF) is the most common lethal genetic disorder affecting Caucas i an populations. Hepatic steatosis (i.e. fatty infiltration of the liver) is prevalent in cystic fibrosis. Despite many hypotheses, the link between the cystic fibrosis gene and the etiology of C F liver disease, particularly hepatic steatosis, is not known. Pancreat ic insufficiency is present in 8 5 % of patients with C F , resulting in decreased secretion of digestive enzymes , bicarbonate, water and electrolytes into the duodenum, which leads to malabsorption of nutrients. Although pancreatic enzyme replacement preparations are often prescribed to correct malabsorption, they are not 100% effective. Phosphatidylcholine ( P C ) represents >90% of the essential nutrient choline in the diet. Further, large amounts of P C are found in bile. The digestion of P C requires phosphol ipase A 2 , which releases l y s o P C and an unesterified fatty acid for absorption. Phosphol ipase A 2 is found in pancreatic enzyme supplements, however, its activity decl ines at pH below 5.8. Further, the intestinal pH of patients with C F is often below pH 5.8 secondary to failure of bicarbonate secretion from the pancreas. W e hypothesized that patients with C F have decreased digestion and thus, increased excretion of P C secondary to pancreatic insufficiency and failure of pancreatic enzyme replacement therapy to completely correct the malabsorption, resulting in choline depletion. Fatty infiltration of the liver is a well-known feature of choline deficiency, explained by the requirement of P C for secretion of triglyceride from the liver in 2 Chapter 1 Literature review very-low-density lipoprotein. It is conceivable that choline depletion may contribute to the pathophysiology of hepatic steatosis in C F . The purpose of this chapter is to provide a review of the pertinent literature on: i . T h e biochemical and clinical features of cystic fibrosis ii. Liver d i sease in C F iii. Maldigest ion and malabsorption in C F iv. Nutrition requirements for patients with C F v. Chol ine and phoshatidylcholine Finally, this chapter will be concluded by the research hypotheses, objectives of the study, and the organization of this thesis. 3 Chapter 1 Literature review 1.1 Biochemical and Clinical Features of Cystic Fibrosis Cyst ic fibrosis is an autosomal recessive genetic d isease that affects Caucas i an populations, particularly those of Northern European origin. The incidence of C F is about 1 in 2,500 live births in C a n a d a (1). T h e primary defect in C F is in the regulation of trans-epithelial chloride transport by a chloride channel protein, known as the cystic fibrosis t ransmembrane conductance regulator ( C F T R ) , encoded by the cystic fibrosis gene located on the long arm of chromosome 7 (2). Normal epithelial cells often possess p lasma membrane channels on the apical surface, which upon opening allow chloride to flow along an electrochemical gradient (3). Increases in intracellular cyclic adenos ine 3',5'-monophosphate ( c A M P ) and subsequent activation of protein kinase A ( P K A ) can mediate the opening of these channels (3). The movement of chloride across epithelial cell surfaces plays an important physiological role in salt and water balance (3). The C F T R mediated chloride channels in patients with C F , however, do not open in response to increased levels of c A M P / P K A . This leads to abnormally thick, viscid mucous secretions that obstruct glands and ducts in various organs. Over 1200 mutations of the cystic fibrosis gene have been identified. The most common cystic fibrosis gene abnormality in C a n a d a is the deletion of three nucleotides coding for phenylalanine at amino acid position 508 (AF508) . This deletion accounts for about 7 0 % of mutations in patients with C F (4). The diagnosis of C F is primarily based on elevated sweat sod ium and chloride concentrations (>60 mEq/L) performed using the method of G ibson and 4 . Chapter 1 Literature review C o o k e (5). M a j o r c l i n i c a l m a n i f e s t a t i o n s of C F i n c l u d e r e c u r r e n t p u l m o n a r y i n fec t i ons a n d c h r o n i c l ung d i s e a s e , p a n c r e a t i c i n su f f i c i ency , s t e a t o r r h e a , ma lnu t r i t i on , h e p a t i c c i r r h o s i s , in tes t ina l o b s t r u c t i o n , a n d infert i l i ty (6). P o s i t i v e fami l y h is to ry is a l s o a s u g g e s t i v e c l i n i ca l s i g n . S u r v i v a l in pa t i en t s w i th C F h a s b e e n s h o w n to c o r r e l a t e pos i t i ve l y wi th the i r nut r i t ional s t a t u s (7). A d d i t i o n a l l y , d e c r e a s e d p u l m o n a r y f unc t i on ( i nc lud ing p e r c e n t a g e p r e d i c t e d f o r c e d v i ta l c a p a c i t y a n d p e r c e n t a g e p r e d i c t e d f o r c e d exp i ra to ry v o l u m e in o n e s e c o n d ) , sho r t s ta tu re , h igh wh i t e ce l l c o u n t a n d c h r o n i c l iver d i s e a s e (as e v i d e n c e d by t h e p r e s e n c e of h e p a t o m e g a l y ) h a v e a l s o b e e n f o u n d to c o r r e l a t e n e g a t i v e l y w i th su r v i va l in a p r e d o m i n a t e l y adu l t C F p o p u l a t i o n (8). T h e m e d i a n s u r v i v a l a g e o f pa t i en ts wi th C F in C a n a d a is n o w a p p r o x i m a t e l y 3 7 y e a r s ( P e r s o n a l c o m m u n i c a t i o n , C a n a d i a n C y s t i c F i b r o s i s F o u n d a t i o n , M a y 2 0 0 3 ) . T h e m o s t r e c e n t repor t f r o m the U . S . C y s t i c F i b r o s i s F o u n d a t i o n h a s d o c u m e n t e d the p r e d i c t e d s u r v i v a l a g e of pa t ien ts wi th C F in the U n i t e d S t a t e s to b e o v e r 3 1 . 6 y e a r s (9). 5 Chapter 1 Literature review 1.2 Liver Disease in Cystic Fibrosis The relatively asymptomatic liver complications in CF are often eclipsed by the more obvious manifestations of pulmonary and pancreatic abnormalities (10). However, as survival has improved as a result of the more effective management of respiratory and pancreatic disease, liver disease has become an important complication in many patients with CF (11). The clinical consequences of liver disease in CF include portal hypertension, deteriorating nutrition and growth, worsening respiratory status, and eventually liver failure (12), which often proceeds at an unpredictable pace (10). Liver disease in CF is known to affect survival and quality of life. Despite many hypotheses, the etiology of liver disease in CF remains unclear. In this section, the prevalence and diagnosis of liver disease, the etiologies of hepatic cirrhosis and steatosis, and the management of liver disease in patients with CF will be reviewed. 1.2.1 Prevalence According to the 2002 United States CF Foundation patient registry, hepatic disease has been reported as the primary cause of death in 1.7% of CF patients, and was the second most common cause of death after pulmonary decompensation (9, 13). The prevalence estimates of liver disease in CF are highly variable, ranging between 4 and 30% (11, 14-16). The variability is partly accounted for by the lack of specific and sensitive diagnostic markers, and the different diagnostic criteria used by the different study groups (see below). In our clinic at British Columbia's Children Hospital (BCCH), 23% of patients with CF 6 : Chapter 1 Literature review were found to have biopsy proven liver disease, based on a program of close clinical follow-up, including clinical, biochemical and ultrasound monitoring (17). Additionally, the prevalence of liver disease appears to peak in adolescence, then level off at older ages (11, 14, 15). Tanner and Taylor (12) proposed that teenagers with CF and liver disease may have an increased mortality and the surviving adults are less vulnerable to liver disease as a possible explanation for the apparent reduced prevalence of CF liver disease in adults. Feigelson and colleagues (18) further proposed that the rare onset of cirrhosis after puberty might suggest the existence of a self-stabilising factor responsible for the initial triggering of cirrhosis. Nevertheless, as respiratory complications are being treated with greater efficiency and CF life expectancy increases, one might expect to see greater numbers of adolescents with cirrhosis who have survived respiratory or other complications of CF, and more deaths from cirrhosis. 1.2.2 Diagnosis The diagnosis of liver disease in CF has been difficult and inaccurate because of the general absence of symptoms of the developing fibrotic liver lesion (10). Clinically evident symptoms and signs tend to appear only when pathological changes are pronounced and probably are a late sign of liver disease (19). Further, a recent study suggested that some patients could develop severe fibrosis or cirrhosis without any risk factor being identified (14). To date, no diagnostic criteria with adequate sensitivity and specificity are available for 7 : Chapter 1 Literature review early diagnosis and assessment of the progression of CF-associated liver disease (19). Nevertheless, liver function tests (LFTs), ultrasonography and liver biopsy are commonly used to screen and diagnose liver disease in patients with CF. It is well recognized that LFTs including serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (APP), y-glutamlytransferase (GGT), and lactate dehydrogenase (LDH) are not specific and sensitive. Indeed, it has been shown that none of the LFTs correlates with the degree of hepatic fibrosis in CF (10), and ALT, AST and GGT are not useful in predicting the degree of fatty infiltration and cirrhosis in CF (14, 17, 20). Moreover, LFTs could periodically be normalized in CF patients who had elevated serum liver enzymes for at least two consecutive years (14). Thus, LFTs are more often used as a screening, rather than a diagnostic tool (10). Nevertheless, normal LFT results at regular check-ups were good negative predictors for advanced liver disease (14). Ultrasonography appears to be a noninvasive way to detect the presence of liver disease in patients with CF. However, it is less useful for the detection and quantification of hepatic fibrosis or cirrhosis because periportal steatosis can appear sonographically similar to focal fibrosis in the liver (10). Furthermore, Davidson and colleagues has shown that significant ultrasound changes might be absent in biopsy proven CF liver disease (17). Thus liver biopsy is the 'gold standard' for the diagnosis of most chronic liver diseases (21). However, even 8 . Chapter 1 Literature review biopsy may have sampling error in C F due to the focal nature of the early changes. Ultrasound-guided biopsy may reduce this risk of sampl ing error (22). 1.2.3 Hepatic disease in cystic fibrosis The hepatobiliary lesions of C F are unique and the pathogenesis of C F liver d isease is poorly understood (20). The characteristic hepatic lesion in C F is focal biliary fibrosis with bile duct obstruction, fibrosis, chronic inflammatory cell infiltration and bile duct proliferation (23). The initial formation of biliary obstruction has been shown to relate to the expression of C F T R at the apical membrane of epithelial cells lining the intrahepatic cholangiocytes and extrahepatic biliary tree (24). Cholangiocytes regulate bile volume, fluidity and alkalinity rapidly in response to a complex network of hormones and paracrine mediators, whose interplay results in net secretion or absorption of osmolytes, such as chloride and bicarbonate (19). Abnormalit ies of bicarbonate and chloride transport secondary to the mutated C F T R gene wil l , therefore, result in decreased hydration of bile, which will ultimately lead to increased bile viscosi ty and the tendency to form biliary plugs (22). Additionally, in the presence of a reduced biliary fluidity and alkalinity, other factors, such as bile acids and cytotoxic compounds , or an increased susceptibility to infectious agents, may damage the bile ducts (19, 25). In response to the epithelial damage, cytokines and inflammatory mediators will be released, which will lead to chronic portal inflammation. This process may 9 Chapter 1 Literature review progress with death of hepatocytes to focal biliary cirrhosis and , eventually, liver decompensat ion, depending on the immunogenetic background and a number of concurrent events (19). Elevation and changes in components of the serum bile acid pool, including the possibility of an increase in bile-related toxin have also been proposed to be involved in the development of liver cirrhosis in patients with C F (22). Although abnormal C F T R in the biliary tree provides a possible etiological basis for chronic liver d i sease in C F , it does not account for the absence of liver involvement in a s izeable proportion of patients with C F and the wide spectrum of severity among those where this occurs (22). Co lombo and col leagues (26) conducted a genetic analysis of the major mutations present in an Italian population of patients with C F and were not able to find a specific genetic marker for the development of CF-as soc ia t ed liver d isease . Soko l and Durie (10) further proposed that the variable onset and severity of liver d i sease may imply there are other modifying genetic or environmental factors involved. 1.2.4 Hepatic steatosis Hepatic steatosis is the abnormal accumulat ion of fatty ac ids and triglycerides within parenchymal cells, where they may account for up to 50% of the total weight of the liver (27). Fatty infiltration of the liver is a recognized feature in patients with C F and is often clinically silent. S o m e authors suggest hepatic steatosis may be a precursor of fibrosis/cirrhosis of the liver (22). However, proof of this is lacking. The relationship between the steatosis of C F 10 Chapter 1 Literature review and the development of focal biliary fibrosis or cirrhosis remains to be established. Hepatic steatosis has been reported to be present in 14-23% of C F patients based on clinical , biochemical and echographic assessments (20, 26). The pathogenesis of steatosis in C F remains obscure . Biochemical ly , there is no evidence that it may result from increased delivery of fatty acids and triglycerides to the liver from dietary fat, or from adipose t issue stores. Increased synthesis of fatty acids within the liver and decreased oxidation of hepatic fatty acids has not been shown to occur (27). A case report in 1966 w as among the first to propose malnutrition as a possible cause for fatty infiltration of the liver in patients with C F (28). However , subsequent studies have reported hepatic steatosis in patients with C F who were well nourished, consuming an appropriate diet, receiving vitamin supplementation, as well as pancreatic enzyme replacement (25). Carnitine deficiency has also been proposed as a possible contributing factor in hepatic steatosis in patients with C F (29). Carnit ine is derived from trimethyllysine, which is synthesized from lysine and methionine in humans . Carnitine is involved in fatty acid transport into mitochondria. However , data on the carnitine status of patients with C F are limited and inconclusive (30-32). A n earlier study at B C C H found a lower level of carnitine in children with C F , but it did not s e e m to correlate with steatosis (17). Of interest, rats fed a choline-deficient diet had reduced levels of carnitine in liver, heart and skeletal muscle (33) (refer to Sect ion 1.5). Essent ial fatty acid ( E F A ) deficiency also has been descr ibed in patients with C F (refer to section 1.4.2) and was found significantly more often in patients with marked steatosis (14). However , the link between E F A deficiency and hepatic steatosis in C F remains unclear. 1.2.5 Management Ursodeoxychol ic acid ( U D C A ) , a secondary bile ac id that is found in small concentrations in normal human bile, is commonly prescribed to C F patients with liver complicat ions. U D C A protects the hepatocytes from the toxicity of hydrophobic bile acids and improves the fluidity of bile (22). Improvement and even complete normalization of L F T s has been documented in C F patients treated with U D C A (19, 34-36). Compar i son of liver biopsies from children with C F before and after treatment with U D C A indicated statistically significant improvement in focal biliary fibrosis, but no change in steatosis (Davidson A G F 2004, oral communicat ion, 2 n d July). However, U D C A is not effective in all patients, possibly due to the different pathogenic mechan i sms involved in C F -related liver d isease (19). W h e n focal biliary fibrosis progresses to mutilobular biliary cirrhosis with portal hypertension and liver failure, liver transplantation becomes the only treatment option (26). 12 ; Chapter 1 Literature review 1.3 Maldigestion and Malabsorption in CF C F patients with untreated pancreatic insufficiency absorb about 5 0 - 6 0 % of dietary fat ( 3 7 ) . W h e n pancreatic enzyme preparations are prescribed to C F patients with pancreatic insufficiency to correct fat maldigestion and malabsorption, however, they do not completely normalize maldigestion and malabsorption ( 3 8 - 4 0 ) . Typical ly, fat absorption is about 8 5 % in children with C F and is > 9 3 % in children without C F ( 3 8 ) . The pathophysiology of maldigestion and malabsorption in patients with C F , including pancreatic insufficiency, abnormalities in bile acid metabolism and intestinal abnormalit ies, and its management are d i scussed below. 1.3.1 Pancreatic insufficiency Malabsorpt ion in cystic fibrosis occurs mainly as a result of maldigestion secondary to pancreatic insufficiency. Pancreatic insufficiency is associated with decreased pancreatic secretion of water, bicarbonate, electrolytes and digestive enzymes including lipase, col ipase and phospholipase A 2 (41) into the duodenum. The pathological changes in the C F pancreas are related to defective C F T R . Ion and fluid influx into the pancreas is impeded and leads to decreased pancreatic enzyme output (42). The concentrated pancreatic secretions lead to the typical, progressive destructive lesion of the pancreas; ductal obstruction and pancreatic exocrine insufficiency (42). Moreover , decreased bicarbonate secretion may contribute further to fat malabsorption (discussed below) (Figure 1.1). Although only 1-2% of the pancreatic capacity for secreting l ipase is thought to be 13 c o o CO JD _ro CQ T3 ._ cu >- 5 co ro o 8 1 1 =i o CD CU *? co ^ Q) CO « cu TJ ® S CO CU o CD O CO CD Q O CD C CO ro XJ TD ro 2-8 CO c c CO Q_ =0 CO CD ~co E O c JD CO "co » CO CD c S o E 8 ? c j ro ^ 8 2 c O O Q. CD £ Q ° o o ed X CL reas enal o CU o Q du _CD CD J5 c TD o ad C1VJ o o o JD se du zz* t_ -4—' CO to ro cu JD CD dis O TD O O 4— TD CD -4-* to CD o> TD C ZD TD O CO _cy JD o CJ) c TD c CQ f to to o CD CO O £ TD CU CO CO CD i_ o c CO to o ). _Q ti— O 0 0 o 5 to -*—' c CD •H ro CL c o o to JD _ro ro E CO CD to ZI co O CD i_ 3 CD i Z 14-Chapter 1 Literature review required to prevent impaired lipolysis, pancreatic exocrine abnormalit ies are present in approximately 85 to 90% of patients with C F (6, 43). O n the other hand, pancreatic-sufficient patients, which constitute approximately 15% of the C F population, retain sufficient pancreatic function to permit digestion and absorption of nutrients (44), although pancreatic fluid and bicarbonate secretion is usually decreased (45). 1.3.2 Abnormalities in bile acid metabolism Bile acids are synthesized by the liver, where they are conjugated with glycine or taurine. They are then secreted as bile salts in micelle together with cholesterol and phospholipid into the duodenum. Bile ac ids are important for solubilizing lipid and are essential in the formation of mixed micelles together with phospholipids in the intestine to provide an increased surface area for digestion of lipids and fat-soluble vitamins. Normally, the bile acid pool undergoes enterohepatic circulation about 5-10 times daily; the intestinal absorption of the pool is about 9 5 % efficient in non-CF individuals, and about 0.3-0.6 g bile acids are lost daily in the feces (46). N e w bile acids are typically synthesized in an amount equivalent to that lost in the feces (47). Bile acids readily precipitate in an acid milieu. At p H s below 5, bile acids precipitate from the aqueous phase of the intestine, leading to a reduction in the aqueous phase bile acid concentration (48). Moreover , precipitated bile salts appear to be lost from the enterohepatic circulation in greater quantities than those that are not precipitated, thus further decreasing the bile acid concentration 15 ' Chapter 1 Literature review (49). W h e n the duodenal bile acid concentration falls below 2 m M , the formation of micelles is impaired (50). Micel lar formation might be impaired in patients with C F because acidic pH values are commonly found in the duodenum (51). Thus , duodenal bile salt concentrations may fall below the critical micellar concentration. Reduced duodenal bile acid concentrations (52) and increased fecal bile acid excretion have been documented in some studies with patients with C F (49, 53). However, W e i z m a n and col leagues (52) reported that duodenal bile acid concentrations were significantly higher in C F subjects than controls. This could be explained by the markedly reduced pancreatic water output shown in C F patients (45), which would increase the apparent bile acid concentration, despite a lower absolute amount present. W e b e r and R o y (50) reported a higher glycine:taurine ratio of conjugated bile acids in patients with C F . In response to the ongoing bile acid losses in feces, newly synthesized bile acids are formed and conjugated mainly with glycine. Glycine is readily available in contrast to taurine, which may become rate-limiting when bile acid losses are large (50). Furthermore, as glycine-conjugated bile salts tended to precipitate more readily in acidic milieu (i.e. the intraduodenal pH must remain within narrow limits (at p H 6-8) to avoid precipitation of glycine-conjugated bile acid), they are lost more readily than taurine-conjugated bile salts (which is soluble at pH 2-14) when the intradoudenal pH is low (50). Thus glycine-conjugated bile acids are less efficient at micelle formation. In summary, patients with C F tend to have bile salts that are conjugated with glycine rather than taurine secondary to ileal malabsorption of bile ac ids and the differential rate 16 Chapter 1 Literature review of synthesis of glycine over taurine (42). The increased glycine: taurine ratio of bile salts will likely further increase bile acid loss by precipitation secondary to the low intraduodenal p H . S o m e research groups have also proposed other mechan i sms related to bile acids to explain fat malabsorption in patients with C F . Binding of bile acids to undigested fat, protein, fiber or carbohydrate has been suggeted to increase bile loss in feces (53, 54). A d v a n c e d liver d isease with multifocal biliary cirrhosis may result in inadequate bile salt secretion, which in turn would worsen fat malabsorption (42). Finally, any abnormality of the gall bladder, or bile-duct obstruction may also interfere with bile secretion to the duodenum. 1.3.3 Intestinal abnormal i t ies Visc id , thick intestinal mucus, with altered physical properties, may have a deleterious effect on the thickness of the intestinal unstirred layer, further limiting nutrient absorption by the microvilli (44, 55). Decreased bicarbonate secretion by the intestine may (56) also contribute to the low intraduodenal p H mentioned previously. 1.3.4 Management Microencapsulated pancreatic enzyme preparations, and somet imes acid reducing agents are prescribed to correct maldigestion and malabsorption in patients with C F . Their functions and limitations are d i scussed below. 17 Chapter 1 Literature review 1.3.4.1 Microencapsulated pancreatic enzyme preparations Microencapsula ted enzyme preparations have been prescribed to patients with C F to correct maldigestion and malabsorption since the late 1970s. However , steatorrhea and azotorrhea still occur, to varying extents, among patients using pancreatic preparations (40, 57). Both l ipase inactivation and s low dissolution of the pH-sensit ive coating may contribute to the failure of enteric-coated enzyme preparations to normalize fat absorption (Figure 1.2). The duodenal pH in patients with pancreatic insufficiency due to C F is known to be abnormally acidic because pancreatic bicarbonate production is low (45). Whether gastric acid hypersecretion occurred in C F patients has not been clearly established (58). In the absence of adequate pancreatic bicarbonate secretion, gastric acid entering the duodenum may lower intestinal p H which persists well into the jejunum. Pancreat ic l ipase activity is optimal at pH 8.0, and is irreversibly inactivated below p H 4 (59, 60). The digestive capacity of pancreatic l ipase is reduced at a lower p H . Prior to the availability of enteric-coated pancreatic enzymes , gastric inactivation of the pancreatic enzyme supplements w a s a major limitation to the efficacy of pancreatic enzyme supplements (61). Enteric-coated enzymes were eventually developed to minimize gastric inactivation of the pancreatic enzyme supplements . However, depending upon the formulation, the threshold for rapid dissolution (i.e. within 15 minutes of exposure buffered emuls ion at 18 T J CD CO CO o S ._ CD .= O _ CO CO C O ed X CL — • reas enal o T J CD o Q Z J T J T J _ 0) i - 5 co ro . 2 S "CD ro ir o c cu E o Q CD T J CD O O CD co ^ CD CO _ 9> -o K S ro CD o CD O CO CD Q c o o CD _ . CO ro o CD c co ro _j _D CD _5 c T J o ad CHJ o -*—> O ' o _ Q se du Z J i_ — CO "co CO CD _ o CD dis o T J O a T J 0) "co CD cn T J _ Z J o -*—< T J o ro _D _5 4— o CJ) c T J rz oo CO CO _o _D _5 ro o T J CD CO ro CD o c to CO o _ -Q CO o CO •+-» c CD » ro a. c o "-»—• C L _ o CO j _ ro ro E cz CD E CD C D CO £Z CO E T J CZ ro CO CD CO Z J ro O CM 0) 3 CO Chapter 1 Literature review specific pHs) of the pH-sensitive coating ranges from pH 5.6 to pH 6.2 and higher (59, 62). For example , P a n c r e a s e ® requires exposure to a pH above 5.6 for 10 minutes or a pH less than 5.2 for more than 120 minutes for the enteric coating to dissolve (59). Youngberg and col leagues (62) found lower postprandial duodenal pHs in patients with C F than in healthy subjects, especial ly in the first postprandial hour. Thus , the acid-resistant coating of the enzyme preparations may not dissolve in the proximal intestine and this would result in delayed release of active enzymes . Robinson , Smith and Sly (63) also found significantly lower postprandial duodenal pHs in patients with C F and suggested that failure of release of enzymes from the enteric coated preparations is a major factor in inefficient enzyme function. Another possible explanation for the different effectiveness of the pancreatic preparations among individuals is the varying level of residual pancreatic function. Patients with documented steatorrhea may have variable, but very limited residual pancreatic function. G a s k i n et al (64) reported positive correlation between residual pancreatic function (colipase secretion) and fat absorption (the correlation coefficient and p value were not reported in the publication). Furthermore, pancreatic enzyme preparations given to patients with C F are subjected to degradation by proteolytic enzymes in the absence of the inhibitor normally present in pancreatic juice (39), which can result in degradation 20 Chapter 1 Literature review of the supplemented enzymes thus limiting their ability to correct maldigestion and malabsorption. 1.3.4.2 Acid reducing agents In addition to the pancreatic enzyme preparations, C F patients with pancreatic insufficiency are sometimes prescribed ac id reducing agents, including antacids (e.g. sodium bicarbonate), or H 2 blockers to increase their gastric and duodenal p H , and thus improve the efficacy of the enzyme supplements and consequently digestion and absorption. Several studies have shown that patients treated with the combinat ion of microencapsulated pancreatic enzyme preparations and acid reducing agents had improved fat digestion and absorption (37, 39, 48, 63). The effect of gastric acidity reducing agents in overall C F nutrition status, lung function, quality of life and survival remains to be determined. 21 Chapter 1 Literature review 1.4 Nutrition Requirements for Patients with Cystic Fibrosis Despite dramatic improvement in the nutritional care of C F patients over the past two decades , malnutrition (based on height or weight less than 5 t h percentile in the general population) is still present in 16-17% of patients with C F (9). Malnutrition and growth failure have been shown to be closely linked with pulmonary function (65), and have been recognized as adverse prognostic factors in patients with C F (7, 66, 67). Poor nutritional state and growth failure in patients with C F have been suggested to be caused by an imbalance between energy intake and absorption, and energy requirements (68). Factors contributing to the energy imbalance can include decreased intake, increased energy loss and increased energy expenditure. Factors contributing to a poor dietary intake in C F include: recurrent vomiting from coughing, and/or gastroesophageal reflux, foul-tasting sputum, chronic respiratory infections, and psychosocial s tresses (43). In addition, patients with C F commonly have gastrointestinal dysmotility, including gastroesophageal reflux and delayed gastric emptying which lead to anorexia (42). Anorex ia is c o m m o n in C F and can become more of a problem during recurrent chest infections. Earl ier studies in this field have sugges ted that the higher than normal level of tumor necrosis factor alpha (TNF-a) in the p lasma of patients with C F may contribute to the anorexia (68, 69). Increased energy loss because of nutrient maldigestion and malabsorption is known to contribute to energy imbalance in patients with C F . T h e loss of energy in stools has been shown to be three times higher in C F patients 22 : Chapter 1 Literature review compared with controls who have similar energy intakes (38). Malabsorpt ion in cystic fibrosis occurs mainly as a result of maldigestion secondary to pancreatic insufficiency, as d i scussed in the section 1.3. Moreover, dec reased bicarbonate secretion and decreased bile acid reabsorption can further reduce digestive enzyme activity, and thus further contribute to fat malabsorption. Regurgitation and vomiting from gast roesophageal reflux may also increase energy loss. Continuous inflammation, acute exacerbation of infection, chronic obstructive pulmonary d isease , and deteriorating lung function appear to be the major factors associa ted with increased resting energy expenditure ( R E E ) (70). Furthermore, it has been proposed that the consequences of abnormal function of the C F T R gene, at the cellular level, are energy requiring, and may increase the R E E in patients with C F (71). However, not all studies support this hypothesis (72). The resting metabolic rate in patients with C F was increased by 20-25% during acute pulmonary exacerbation (67). Overal l , the goal of nutritional care in patients with C F is to maintain a zero energy balance in adults, and provide sufficient energy to support growth and maintenance requirements in children (73). Specifically, the nutritional care plan aims to control maldigestion and malabsorption, to provide adequate nutrients to promote optimal growth or maintain weight for height and pulmonary function, and to prevent nutritional deficiencies (44, 74). The following section provides a brief d iscuss ion of the current knowledge on the energy and nutrient requirements in C F . 23 Chapter 1 Literature review 1.4.1 Energy requirement A s a result of the increased energy loss and increased energy expenditure, the energy requirements for patients with cystic fibrosis are often higher than normal. W h e n calculating energy requirements, all factors contributing to the energy balance equation, such as fecal energy losses , increased basal metabolic rate ( B M R ) , catch-up growth and frequency of infection need to be taken into consideration. A detailed description for the calculation of the energy requirements for patients with C F can be found in the consensus report by R a m s e y et al . (74). However, in practice, dietitians often calculate a patient's energy requirement based on a knowledge of the dietary history and an estimation of 120-150% of that recommended for n o n - C F patients of similar age and sex (43). T h e calculation of energy requirement for patients with C F based on the Dietary Reference Intakes. (DRI) was not available at the time of this study. 1.4.2 Macronutrient requirement The requirement for dietary protein is increased in C F a s a result of malabsorption. However , when energy needs are adequately met, individuals with C F general ly can meet their protein need by following a typical North Amer ican diet, with at least 15% to 20% of the total calories consumed as protein (75). Patients with C F often require a higher fat intake (35 to 4 0 % of calories) than that r ecommended for the general population. Accord ing to the 2002 DRI for macronutrients, the acceptable macronutrient distribution range for fat is 20-35% 24 . Chapter 1 Literature review of energy for adults, 30 -40% of energy for children 1-3 years , and 25-35% of energy for children 4-18 years (76). Until the late 1970's, a high energy, low-fat diet was recommended in some C F centres. It was reasoned that reduction in dietary fat would improve bowel symptoms and reduce stool bulk. However , a retrospective study conducted by a Canad ian research group in the 1980's comparing the C F population in Toronto with that in Boston found a marked difference in median age of survival between the two centres (77). T h e median survival in Bos ton of 21 years was significantly less than that of 30 years for patients in Toronto. It was found that the general approach of treatment was mostly the same , except that the approaches to diet and pancreatic supplementation were radically different. The Toronto centre advocated a high-fat, high-caloric diet with up to 20-30 or more pancreatic enzyme capsules per meal (note that the enzyme preparations in those days were non-enteric coated and less potent compared with those that are presently used). The Boston centre advocated a low-fat, high calorie diet with less pancreatic enzyme capsules per meal (77). Thus , the authors concluded that a restriction of fat in the diet of C F patients is not desirable (77). A reduced level of p lasma essential fatty acids ( E F A s ) is a frequent feature in patients with C F (78, 79). Correction of the abnormal pattern of essential fatty ac ids has proven to be difficult (80). Further, there has been little published evidence of clinical benefit from supplementing C F patients with E F A s except in patients with E F A deficiency (81). Nevertheless, vegetable oils such as flax, canola and soy oils are rich in alpha linolenic acid and are a good source of 25 Chapter 1 Literature review energy, and fish is a source of long chain n-3 fatty ac ids such as e icosapentaenoic acid and docosahexaenoic acid and has been recommended (73). 1.4.3 Micronutrient requirement With the except ion of vitamin B - i 2 , the absorption of water-soluble vitamins and minerals appears to be normal in patients with C F (42). Pancreat ic enzymes are involved in the intrinsic-factor-mediated vitamin B 1 2 absorption. Nevertheless, vitamin B i 2 absorption can be normalized with adequate pancreatic enzyme replacement therapy in pancreatic-insufficient patients (82, 83). Deficiencies of fat-soluble vitamins (vitamins A , D, E , and K) have been demonstrated in patients with C F . Fat-soluble vitamin supplements are a necessary part of the nutritional care of C F patients with pancreatic insufficiency, or severe liver d i sease (44). Most patients with C F receive adequate supplementation of most vitamins and micronutrients from multiple-vitamin preparations (74). 26 ; Chapter 1 Literature review 1.5 Choline and Phosphatidylcholine A well-known feature of the deficiency of the essential nutrient choline is hepatic steatosis, and a reduction of plasma P C , triglyceride (TG) and very low density lipoprotein ( V L D L ) concentrations (84-87). The accumulat ion of T G in the liver and decrease in p lasma T G is believed to be explained by the absolute requirement of active P C biosynthesis for the secretion of T G and V L D L by hepatocytes (88, 89). Hepat ic steatosis is often resolved following choline (85, 90, 91) or P C supplementation (92) of patients supported by long term total parenteral nutrition (TPN) devoid of choline. To our knowledge, the choline status of patients with C F has not been reported. This sect ion will review the function, dietary sources , digestion and absorption of choline and P C , and the pathways for P C synthesis . 1.5.1 Function, dietary sources, digestion and absorption of choline Chol ine , a quaternary amine, is present in t issue predominantly as the head group of P C and sphingomyel in (Sph), which are structural constituents of cell membranes , p lasma lipoproteins and lung surfactant (93). Additionally, choline is a component of the neurotransmitter acetylcholine, platelet-activating factor, p lasmalogen, a phospholipid found in highest concentrations in cardiac muscle membranes , and is a source of labile methyl groups (94, 95). Free choline is present in small amounts in foods, but is found ubiquitously in normal diets as a component of P C and S p h . Major dietary sources of choline, P C and Sph include: egg yolks, organ meats, spinach, nuts, and wheat germ (94, 96). P C represents >90% of choline in animal tissue. T h e adequate intake (Al) for choline 27 Chapter 1 Literature review is 425 and 550 mg/d.for adult women and men, respectively (97). Normal plasma choline concentrations in healthy adult men are 10.5 u.mol/L, with a range of 7-20 u.mol/L compared to a mean of 7.5 uimol/L in men fed a choline deficient diet. P C concentrations are 1-1.5 mmol/L in healthy individuals (97). In a study published in 1980, Zeise l et al prepared a high choline diet with food with high choline content, including egg (with 2.1g choline/d) and liver (with 2.6g choline/d), which resulted in a diet with 5.6g of P C per day (98). A more representative diet with meat at both the mid day and evening meal provided about 1g of P C per day (98). Data on usual P C intakes are not available. Chol ine is absorbed though a specific non energy-dependent carrier in the gut, and no other component has been identified that competes with choline for transport (99). P C is digested by phospholipase A 2 ( P L A 2 ) in pancreatic secretions with the help of bile salts and bicarbonate, to form l y s o P C and an unesterified fatty ac id . The optimal pH for P L A 2 activity is not known, but it appears to be more sensitive to an acidic pH than pancreatic l ipase (50). The digested products of P C form mixed micelles with bile salts and other digested lipid products before being transported to the brush border for pass ive absorption. Of importance, P C is essential for normal bile synthesis, micelle formation and consequently fat absorption. P C represents about 4 % (wt/wt) of normal bile, with 8 2 % water, 12% bile acids and 0.7% cholesterol in normal bile (46). The normal enterohepatic pool has about 1g of P C and the bile pool circulates 5-10 times/d. Thus, about 5-10 g P C enters the intestine in bile lipid e a c h day. 28 Chapter 1 Literature review Maintenance of the body choline/ P C pool is, therefore, critically dependent on the absorption of dietary P C and reabsorption of biliary P C . Normally there is a c lose coupling between the secretion of bile salts, phospholipid, and cholesterol in bile (27). Thus , P C depletion could lead to poor bile synthesis and secretion, which in turn may further worsen fat malabsorption, and P C and dietary fat losses will likely result. Similarly, when the secretion rate of bile salts decreases below a certain limit because of increased fecal losses , the secretion rate of phospholipids is reduced proportionately (27). 1.5.2 Choline/phosphatidylcholine synthesis Phosphatidylcholine can be synthesized via two pathways in liver: the CDP-cho l ine pathway, which requires free choline, and the de novo phosphatidylethanolamine A/-methyltransferase ( P E M T ) pathway which includes sequential methylation of phosphatidylethanolamine (PE) , us ing S-adenosylmethionine ( S A M ) as the methyl donor (F igu re 1.3). Ava i lab le estimates suggest that in the well-nourished state, the C D P - p a t h contributes about 60-80% of hepatic P C formation (100, 101), and the P E M T pathway contributes the remainder. During experimental choline deficiency, transmethylation of P E to form P C and the hepatic concentration of S-adenosylhomocyste ine ( S A H ) is increased, and S-adenosylmethionine ( S A M ) and methionine are decreased , presumably reflecting an attempt of the liver to increase P C synthesis by the de novo P E M T pathway (84, 102). Whether or not this occurs in vivo in humans has not been directly establ ished. Increased P E M T activity has a lso been shown in 29 Chapter 1 Literature review choline deficiency as measured in vitro in hepatocytes from rats and guinea pigs fed a choline deficient diet (103-105). C u i and V a n c e (106) have further demonstrated that P E M T gene express ion was increased in rats fed with a choline deficient diet for more than three weeks . The available experimental data derived from studies with rat hepatocytes indicate that P C synthesized from either the P E M T pathway or the C D P - c h o l i n e pathway can support normal V L D L lipoprotein assembly and secretion. W h e n choline-depleted hepatocytes were supplemented with excess S A M (84), methionine (88), or betaine and homocysteine (89), normal V L D L secretion was resumed. However , these in vitro studies used concentrations that were m u c h higher than the normal physiological levels. In P E M T knockout mice, the C D P -choline pathway is capable of fulfilling the requirements for P C biosynthesis as long as sufficient choline is provided in the diet (107-108). However , very recent studies have suggested that P C formed from the C D P - c h o l i n e pathway and the P E M T pathway have distinct functions, and are not fully metabolically interchangeable. Houwel ing and col leagues (109, 110) have repeatedly shown that P C s from the C D P - c h o l i n e and P E M T pathways have opposite effects o n hepatocyte proliferation. The C D P - c h o l i n e pathway has been shown to favor faster proliferation of hepatocytes, while the P E M T pathway has been shown to strongly inhibit the growth of hepatoma cell lines, and associate negatively with the developmental growth of the liver. In addition, D e L o n g and col leagues (111) reported that the two pathways result in synthesis of different P C molecular species , which further suggests the distinctiveness and 31 Chapter 1 Literature review unique function of P C molecules originating from the two pathways. The latter group also reported that the P C synthesized from the C D P - c h o l i n e pathway were mainly compr ised of medium chain and saturated fatty acids, whereas the P C synthesized from the methylation pathway were mainly compr ised of long chain and highly unsaturated fatty acids. Additionally, Noga and col leagues (112) recently reported that P E M T activity is required for optimal apolipoprotein B (apo B), V L D L and T G secretion, and that P E M T activity cannot be fully replaced by increased C D P - c h o l i n e pathway activity even in the presence of additional choline in hepatocytes isolated from P E M T knockout mice. Similarly, another recent study showed that P E M T knockout mice developed steatosis and were unable to maintain normal concentrations of choline metabolites, even when supplemented with four times the normal choline requirement (113). This suggests the P E M T path has significance beyond its recognized role as a second pathway for P C biosynthesis, and is essential for normal liver function. B a s e d on the current literature, both the C D P - c h o l i n e path and P E M T path appear to be essential and have distinct and non-interchangeable functions. T h e link between choline and hepatic steatosis depends on whether the P E M T pathway can support the liver's requirement of P C (both functionally and quantitatively) for V L D L and T G secretion in the absence of sufficient absorbed choline for P C synthesis via the C D P - c h o l i n e pathway. In support of this, our research group has shown that p la sma homocysteine and S A H are higher and S A M is lower in children with cystic fibrosis when compared to children without cystic fibrosis (114). These findings are consistent with an increased de novo 32 Chapter 1 Literature review synthesis of P C via the P E M T pathway. Interestingly, studies examining the biochemical and nutritional status of patients with C F have shown lower levels of low-density lipoprotein (LDL) and LDL-choles tero l (40, 115-117), which is a lso consistent with reduced hepatic apo B containing lipoprotein secretion, s ince V L D L is the precursor of L D L (118). 33 Chapter 1 Literature review 1.6 Summary T o our knowledge, there are no published studies that specifically determined fecal phospholipid excretion in patients with C F . It is conceivable that choline and P C depletion occur in patients with C F due to P C maldigestion and/or malabsorption secondary to pancreatic insufficiency, decreased bile output, intestinal abnormalities and/or failure of pancreatic enzymes to normalize phospholipid digestion and absorption. Considerable research has focused on total fat and fat soluble vitamins, with no attention given to phospholipid absorption in patients with C F . B e c a u s e T G represents about 9 5 % of dietary fat (119), measures of the total fat content in stools are not sufficiently sensit ive to detect even large differences in P C excretion. This study quantified fecal P C and l y s o P C excretion with a newly developed high performance liquid chromatography ( H P L C ) method for separation of lipids and with an evaporative light scattering detector for quantification. W e hypothesized that reduced lipolysis of dietary and biliary P C , due to maldigestion and/or malabsorption, results in increased P C excretion. W e further hypothesized that increased excretion of choline containing lipids leads to hepatic choline and P C depletion in children with C F and this results in an increase in the de novo ( P E M T ) pathway of choline synthesis to support P C requirements (Figure 1.4). Because patients given choline-free T P N develop steatosis and this is corrected by choline or P C supplementation, we hypothesize that the P E M T pathway is not able to support all the requirements for normal liver 34 o 5 8-8 2 I CO T3 g CD — ro J CD J2 -E o .CD CD T3 CO CD CO O CD c CO CO > TD Ad ver co CD CO ' c "co CO E CD —' o c ab 1 ^ CD o CD i— • CO o o to o c _CD o S ro ed CL CO o CO 0) CD l_ CL o •*-» _c ro CO CD 5 "O . _ r o J2 cz o o 3 o _^ CD "5) CO -Q CO O CO CD -t^ CD w " cu CD > c — — c o — sz c o o O ]*> Q. Q-Q) "a zz CO to o co to CD sz o a. >> XZ sz o 1— ro CD to CD CD C o ro —^» c CD CO CD i— C L CD ro E CD - C o CO CD i_ 3 CO Chapter 1 Literature review function in humans, thus, P C depletion in the liver may be important in etiology of hepatic steatosis in children with C F . The objective of this study is to quantify p lasma P C , l y s o P C , total cholesterol, high-density lipoprotein (HDL) cholesterol and apo B , and fecal total phospholipid, P C and l y s o P C excretion and fecal fat absorption in children with C F in comparison to normal, healthy children. Al though p lasma (free) choline concentration decreases with dietary choline restriction, it may not be a sensitive marker of choline status because membrane phospholipids, which are a large storage pool for choline, are hydrolyzed to maintain p lasma choline concentration above this minimal level (97). P l a sma P C concentration also dec reases in choline deficiency (120). Rapid equilibration of liver and p lasma P C has been shown to occur (121). T o date, only one study has reported p lasma P C concentrations in patients with C F ; this study used separation of lipids, followed by an enzymatic colorimetric assay involving phosphol ipase D and horseradish peroxidase to quantify P C (122). T h e authors found no difference in p lasma P C concentrations between patients with C F and healthy controls. Data on p lasma P C concentrations are not available for healthy children, thus in this study, children without C F were also recruited to generate 'normal ' data. W e did not measure dietary phospholipid and P C absorption because of the lack of data on the phospholipid content of foods and the inability to quantify the amount of P C secreted into the intestine in bile. Folate and vitamin B 1 2 deficiency are known to result in elevation of plasma homocysteine (123). Thus , to address the possibility that elevated 36 - Chapter 1 Literature review homocysteine, if present, in children with C F is not due to folate and/or vitamin B 1 2 deficiency, we also measured the status of these nutrients. P l a s m a thiols including methionine, S A M , homocysteine, S A H were measured in this study and as part of a larger study and the results have been published recently (114). 37 Chapter 1 Literature review 1.7 O B J E C T I V E S The overall objective of this study was to determine the extent of total phospholipid (PL) , P C and l y s o P C excretion in children with C F , and their possible associat ions with the abnormal homocysteine and methionine status in children with cystic fibrosis (CF) . W e hypothesized that children with C F malabsorb phospholipids, particularly P C , which contributes to higher choline phosphoglycerides excretion, and therefore choline and P C depletion in the liver, leading to reduced hepatic apolipoprotein B secretion, and lipid accumulat ion in liver in patients with C F . W e also hypothesized that the higher phospholipid excretion is associated with elevated homocysteine and methionine status. Current standard methods for quantifying fat excretion involve extraction of fat from fecal samples with heptane, ether and ethanol. T h e s e solvents extract triglycerides and unesterified fatty acids but do not quantitatively recover phospholipids. The second objective of this study was to develop a quantitative method using high performance liquid chromatography with evaporated light scattering detection ( H P L C - E L S D ) for measurement of fecal phospholipid excretion, which also allows determination of the phospholipid c lasses excreted. 1.7.1 S p e c i f i c a i m s 1. To develop a quantitative method using high performance liquid chromatography with evaporated light scattering detection ( H P L C - E L S D ) for measurement of fecal phospholipid excretion. 38 . Chapter 1 Literature review 2. To validate the newly developed H P L C - E L S D method for fecal phospholipid excretion using the phosphomolybdate colorimetric a s say of lipid soluble phosphorus (124). 3. T o quantify fecal total P L , P C , l y s o P C and total fat excretion in children with C F and healthy children without C F . 4. To determine dietary fat absorption in children with C F and healthy children without C F . 5. To quantify apo B , total and high-density lipoprotein (HDL)-cholesterol in children with C F and healthy children without C F . 6. T o identify relations between fecal total fat excretion, and the excretion of total P L , P C and l y s o P C excretion in children. 7. To identify relations between dietary fat (TG) absorption ((intake-loss)/intake x 100%) and fecal total fat, P L , P C and l y s o P C excretion in children. 8. To determine potential associat ions between fecal fat, P L , P C , l y s o P C excretion, and p lasma apo B concentration, as indicators of hepatic T G and V L D L secretion in children. 9. T o determine potential associat ions between fecal P L , P C , l y s o P C excretion, and p lasma P C , l y s o P C , P C : P E ratio, methionine, homocyste ine , S-adenosylhomocyste ine concentrations in children. 1.7.2 O r g a n i z a t i o n o f t h e s i s Chapter 2 of this thesis addresses specific a ims 1 and 2 of this study. The chapter is titled 'Assessment of phospholipid malabsorption by quantification of 39 . Chapter 1 Literature review fecal phospholipid ' and has recently been published in the June 2004 issue of the Journal of Pediatric Gastroenterology and Nutrition. Chapter 3 of this thesis addresses specific aims 3-9 of this study. Chapter 3 is titled 'Phosphat idylcholine and lysophosphatidylcholine excretion is increased and related to altered plasma homocysteine and methionine in children with cystic fibrosis' and has been submitted to the Amer ican Journal of Clinical Nutrition for publication. 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Vaughan W J , Lindgren FT , W h a l e n J B , A b r a h a m S. S e r u m lipoprotein concentrations in cystic fibrosis. Sc ience 1978;199:783-6. 116. So lomons N W , Wagonfe ld J B , Rieger C , et al . S o m e biochemical indices of nutrition in treated cystic fibrosis patients. A m J Cl in Nutr 1981;34:462-74. 117. Sles inski M J , Gloninger M F , Costantino J P , Orenstein D M . Lipid levels in adults with cystic fibrosis. J A m Diet A s s o c 1994;94:402-8. 118. Ginsberg H N . Lipoprotein metabolism and its relationship to atherosclerosis. M e d Cl in North A m 1994;78:1-20. 119. Groff J L , Gropper S S , Hunt S M . The digestive system: mechan i sm for nourishing the body. In A d v a n c e d Nutrition and Human Metabol i sm. Minneapolis : W e s t Publication, 1995:26-51. 120. Ze ise l S H , D a C o s t a K, Franklin P D , et al . Chol ine , an essential nutrient for humans. F A S E B J 1991;5:2093-8. 121. Bjornstad P , Bremer J . In vivo studies on pathways for the biosynthesis of lecithin in the rat. J Lipid R e s 1966;7:38-45. 55 Chapter 1 Literature review 122. Burdge G C , G o o d a l e A J , Hill C M , et al . P l a s m a lipid concentrations in children with cystic fibrosis: the value of a high-fat diet and pancreatic supplementation. B r J Nutr 1944;71:959-64. 123. Jacques P F , Andrew G B , Wi l son P W F , Rich S, Rosenberg IH, Se lhub J . Determinants of p lasma total homocysteine concentration in the Framingham offspring cohort. A m J Clin Nutr 2001;73:613-21. 124. C h e n P S , Toribara T Y , Warner H . Microdetermination of phosphorus. A n a l C h e m 1956;28:1756-8. 56 Chapter 2 Quantification of fecal phospholipid: method development CHAPTER 2 QUANTIFICATION OF F E C A L PHOSPHOLIPID: METHOD DEVELOPMENT A version of this chapter has been published. Chen A, Innis S. Assessment of phospholipids malabsorption by quantification of fecal phospholipids. Journal of Pediatric Gastroenterology & Nutrition 2004;39:85-91. 57 Chapter 2 Quantification of fecal phospholipid: method development 2. I N T R O D U C T I O N Phosphol ipid (PL) constitutes 4-8 percent of the fat in most human diets. A major portion of dietary P L is phosphatidylcholine (PC) (1,2). Dietary P C is a source of essential fatty acids and choline, also an essential nutrient. Chol ine is required for synthesis of the P C in lung surfactant, p lasma lipoproteins and cell membranes (3-6). Large amounts of P C are also found in bile. In normal adults, the bile pool circulates 5-10 t imes per day and approximately 1 gram of P C is secreted in each cycle (7). The digestion and absorption of dietary and biliary P C requires hydrolysis by pancreatic phospholipase A 2 ( P L A 2 ) to release l y s o P C . This P L is then absorbed and reassembled as P C in the liver (6). Exocrine pancreatic insufficiency affects more than 8 5 % of patients with cystic fibrosis (CF) (8, 9). Although pancreatic enzyme replacement therapy is commonly prescribed for patients with exocrine pancreatic insufficiency, it does not completely correct malabsorption of fat (10). W e sought to determine whether increased excretion of P C occurs in C F . S o m e of the complicat ions of C F , namely hepatic steatosis and altered P L and thiol metabol ism, might be a result of P L malabsorption (11, 12). Hepatic steatosis is a characteristic feature of choline deficiency which results in part from the inability of the choline deficient liver to synthesize P C required for hepatic secretion of triglyceride in very- low-density lipoproteins ( V L D L ) (6, 13). To our knowledge, a reliable method for analysis of fecal P C excretion in C F or other d iseases characterized by impaired fat digestion has not been previously reported. The standard clinical laboratory method for a s sessment of fecal fat excretion is based on the method of V a n de Kamer et al (14). T h e V a n de Kamer 58 Chapter 2 Quantification of fecal phospholipid: method development method involves convers ion of triglycerides and fatty acids to fatty acid soaps , acidification to liberate unesterified fatty acids and extraction with petroleum ether and ethanol. Al though this method extracts long chain triglycerides and fatty acids, it does not extract medium-chain triglycerides or fatty acids. To overcome this, Jee jeebhoy et al (15) used heptane, diethyl ether and ethanol to extract medium and long chain triglycerides and fatty acids from fecal samples . B a s e d on the properties of P L , we predicted that these solvents would not quantitatively extract P L from fecal samples . Although total t issue lipids (including P L ) are efficiently extracted with chloroform and methanol (16), this solvent system does not recover the more water soluble medium chain fatty acids . Our purpose was to develop a method of extracting polar and non-polar lipids including P L and medium chain fatty acids. W e used high performance liquid chromatography with an evaporative light scattering detector ( H P L C - E L S D ) for the separation and quantification of P L , including P C and l y s o P C . Quantification of phospholipid by H P L C - E L S D was validated using the phosphomolybdate colorimetric assay of lipid soluble phosphorus (17). 59 Chapter 2 Quantification of fecal phospholipid: method development 2.1 Materials and Methods 2.1.1 Collection and preparation of samples Feca l samples were collected quantitatively for 72 h from 18 children with C F and pancreatic insufficiency requiring pancreatic enzyme replacements, and from 8 healthy children. Pancreat ic insufficiency in the children with C F was established based on subnormal fecal chymotrypsin and/or elastase. The mean ages of the children with and without cystic fibrosis were 9 . 3 ± 1 . 4 and 10 .0±0 .1 y, respectively. Three (17%) children with C F had had meconium ileus as infants. Five (28%) children with C F were taking ursodeoxycholic acid at the time of the study to treat biopsy proven liver disease, although none had clinically significant liver d isease . None of the children had any other gastrointestinal d i sease or intestinal resection. Feca l samples were frozen immediately after collection and maintained at - 7 0 ° C until analysis . The study protocol was approved by the University of British Co lumbia Clinical Research Ethics Board and the British Columbia Children 's and W o m e n ' s Health Centre of British Co lumbia Resea rch Review Commit tee . A l l participants and/or their parents or legal guardians provided written informed consent (Appendix A). 2.1.2 Extraction and quantification of total fat Feca l samples were thawed, weighed and homogenized with a known amount of saline containing 15% (weight/volume) ethylenediamine tetraacetic acid. A portion was dried to constant weight to allow calculation of fat content relative to fecal dry weight. Homogenized stool was acidified with 18M hydrochloric acid to release fatty acids from soaps. Aliquots were then extracted 60 Chapter 2 Quantification of fecal phospholipid: method development based on the methods of Jeejeebhoy et al (15), Fo lch et al (16), and by our method which involved an initial extraction to recover medium and long chain triglycerides followed by re-extraction to recover P L . Sample s extracted by the method of Jeejeebhoy were extracted with hexane:diethyl ether:95% ethanol (1:1:1, by volume) The aqueous phase was then re-extracted twice with hexane:diethyl ether (1:1, by volume). P h a s e separation at each step was facilitated by centrifugation at 500 g x 5 min, 4 °C. The supernatants were filtered, combined in pre-weighed acid washed glass tubes and the solvents evaporated under a steady stream of nitrogen. Samples extracted by the method of Folch were extracted with choloroform:methanol (2:1 by volume). The organic solvent extraction was repeated twice. The infranatants were combined and the solvents evaporated as above. To achieve extraction of medium chain triglycerides and fatty acids and P L by our method, the sample was extracted with hexane:diethyl ether:95% ethanol (1:1:1 by volume) and then extracted twice with hexane:diethyl ether (1:1 by volume). The infranatant was re-extracted with choloroform:methanol (2:1 by volume) and then re-extracted with chloroform. The organic phases were filtered, combined, and the solvents evaporated. For all methods, the quantity of total fat was determined gravimetrically. The intra- and inter-assay coefficient of variation for each method was determined by analysis of 10 stool samples in triplicate in three independent experiments (90 assays) . 61 : Chapter 2 Quantification of fecal phospholipid: method development 2.1.3 P h o s p h o l i p i d q u a n t i f i c a t i o n Total P L , P C and l y s o P C in fecal fat were determined by H P L C - E L S D (18). The total fat was resuspended in chloroform/methanol/acetone/hexane (2.0/3.0/0.5/0.5, by volume) and filtered through a 4 m m Millex-FH® filter (with hydrophobic P T F E membrane , 0.5 u.m pore size Nihon Millipore Ltd, Y o n e z a w a , Japan) to remove potential interfering substances that might lower the sensitivity of the detector. Separat ion of lipids was achieved using H P L C (Waters 2690, All iance, Milford, M A ) equipped with an autosampler, a column heater and a normal phase co lumn ( Y M C - P a c k Diol 120NP, 25 cm X 4.6 m m i.d., 5 um particle s ize and 12 n m pore size, Millford, M A ) with a flow rate of 2 ml/min using a quaternary solvent sys tem consist ing of hexane/petroleum ether (97:3 by volume); methanol/triethylamine/acetic acid (765:15:13 by volume); acetone/triethylamine/acetic acid (765:15:13 by volume) and isopropanol/acetic acid (800:40 by volume). The autosampler chamber and the column heater were kept at 18°C and 3 5 ° C , respectively. Lipid c lasses were detected and quantified using an evaporative light scattering detector (Model 2000, Al l tech , Mande l Scientific, Gue lph , O N ) with a nitrogen flow rate of 1.8 ml/min and drift tube temperature at 6 0 ° C . Ana lyse s were conducted in the linear range of the detector with calibration curves constructed using authentic standards for each lipid class . Total P L was calculated as the sum of the individual P L . Total phosphorus in the fecal lipid extract was also quantified using the colorimetric method of C h e n et al (17), with monobas ic potassium phosphate as the standard. 62 Chapter 2 Quantification of fecal phospholipid: method development 2.1.4 Statistical analysis The amounts of total fat and P L extracted were compared among the methods using repeated-measures A N O V A and least significant difference post-hoc test. Pea r son correlation coefficients were calculated to determine the strength of the correlation between the H P L C - E L S D quantification of total phospholipid and the colorimetric assay of lipid soluble phosphorous. Al l statistical analyses were performed using S P S S 9.0.0 for W i n d o w s ( S P S S Inc, Chicago) . P-va lues <0.05 were considered statistically significant. M e a n values are followed by + S E M . 63 Chapter 2 Quantification of fecal phospholipid: method development 2.2 R e s u l t s The increased extraction of total fat and P L from fecal samples achieved by sequential extraction with solvents chosen to optimize extraction of medium and long chain triglycerides and fatty acids and with solvents des igned to recover P L is shown in F i g u r e 2 .1. Although extraction with hexane:diethyl ether:95% ethanol optimizes recovery of medium and long chain triglycerides and fatty acids from human feces (15), it does not, as shown by our results, quantitatively recover P L . O n the other hand, the greater solubility of medium chain triglycerides and fatty ac ids in water results in poor recovery in solvent systems based on chloroform, methanol and saline. Thus , re-extraction of the infranatant with chloroform:methanol following extraction with hexane and diethyl ether resulted in significantly greater fat extraction than achieved with either hexane:diethyl ether or chloroform and methanol alone (Figure 2.1). W e achieved a three fold increase in recovery of P L by our method (2.01 ± 0 . 2 5 mg/g fecal dry weight) compared to the method of Jeejeebhoy ( 0 . 6 4 ± 0 . 1 3 / g fecal dry weight) or a 3 5 % increase compared to the method of Fo lch ( 1 . 4 9 ± 0 . 2 3 m g / g fecal dry weight) ( P O . 0 0 1 ) . Despite the quantitative differences in fat extraction a m o n g the methods, Pearson correlation coefficients showed that the results for the different subjects were significantly correlated. The correlation coefficients (r) for total fat excretion using the method of Jeejeebhoy or Fo lch with total fat extraction using our method were 0.98 and 0.96, respectively when express ing total fat per gram of dry fecal weight and 0.99 for both methods when express ing total fat per gram of wet fecal weight. A highly significant correlation w a s a lso present between the 64 Chapter 2 Quantification of fecal phospholipid: method development amount of P L extracted with chloroform:methanol alone and the amount extracted by our method using chloroform and methanol following extraction with hexane and diethyl, ether (r = 0.87). No significant relationship was found between the recovery of P L by the method of Jeejeebhoy and P L recovery by our method or that of Fo lch . The intra-assay and inter-assay coefficient of variation for total fat were 1.8 and 5.2, 4.0 and 2.3, and 1.1 and 4.1 for the fecal fat extraction using the method of Jeejeebhoy, Fo lch and our method, respectively. The quantification of P L by H P L C - E L S D was significantly correlated with lipid soluble phosphorous determined by the phosphomolybdate colorimetric assay (F igure 2.2). Although the phosphomolybdate method quantifies lipid soluble phosphorous, it provides no information on the nature of the lipid soluble phosphorous, or on the types or amounts of individual P L . The distribution of individual P L , including P C , l y s o P C , phosphatidylethanolamine ( P E ) and sphingomyelin quantified by H P L C - E L S D is shown in F i g u r e 2.3. Feca l phospholipids from children with C F contained a large proportion of l y s o P C . 65 • Chapter 2 Quantification of fecal phospholipid: method development 2.3 Discussion Feca l P L is made up of unabsorbed dietary and biliary P L and possibly a component contributed by s loughed intestinal cells and colonic bacteria. Current routine clinical methods for assessment of fat malabsorption are based on recovery of triglycerides (14), which may be optimized to include extraction of more water soluble medium chain fatty acids and triglycerides (15). Standard methods for quantification of P L using colorimetric a s say of lipid soluble phosphorous (17) are limited because they do not differentiate the origin of the phosphorous and give no information on the amount or types of P L present. In addition, average convers ion factors are used to convert phosphorus to P L (19), and this calculation introduces errors of varying magnitude in the analysis of biological samples containing mixtures of l y s o P L and P L . Dietary fat malabsorption secondary to pancreatic insufficiency or hepatobiliary dysfunction is likely to result in reduced digestion and absorption of P L . In healthy individuals, biliary and dietary P C together with bile acids are adsorbed to dietary triglyceride in the duodenum to provide a larger surface area for triglyceride digestion by the pancreatic l ipase-colipase complex. P L A 2 then hydrolyzes P C to release l y s o P C and an unesterfied fatty ac id , resulting in desorption of P L from the triglyceride substrate. T h e l y s o P C and unesterified fatty acid are then absorbed by enterocytes (20). However , in patients with pancreatic insufficiency, the secretion of P L A 2 , l ipase, col ipase and other digestive enzymes is decreased . Although P L A 2 is present in the pancreatic enzyme supplements provided to patients with pancreatic insufficiency, including C F patients, the activity of phosphol ipase is inhibited at an intraluminal p H below 66 Chapter 2 Quantification of fecal phospholipid: method development 5.8 (21), a pH level common in patients with CF (22). The ability to digest and absorb PL is important because PC is the major source of the essential nutrient choline (1,3). Deficiency of choline results in hepatic triglyceride accumulation secondary to failure of de novo synthesis of PC, which is needed for secretion of triglycerides from the liver in VLDL (13). In this study we sought to develop a simple method for extraction and quantification of total lipid, including PL, from fecal samples, to enable investigation of increased PC excretion in clinical settings. Dietary PL and PC absorption were not calculated because of a lack of data on the PL content of foods and the inability to quantify the amount of PC secreted into the intestine in bile. Our results show that current clinical methods for extraction of fecal fat using heptane, diethyl ether and ethanol recover only about one third of the total fecal PL. Re-extraction with chloroform:methanol is a simple method to recover PL. Our results also show that separation and analysis of PL using HPLC-ELSD provides a method to quantify PL excretion and characterize the nature of PL lost in the feces. We found that choline containing phosphoglycerides are lost in significant amounts in the stool of children with CF. Choline depletion secondary to PC malabsorption has not been reported to occur in humans. However, choline depletion is known to lead to hepatic steatosis in humans (23). We have recently reported decreased plasma methionine and increased homocysteine and S-adenosylhomocysteine, which were related to a decreased plasma PC/PE ratio in children with cystic fibrosis, a finding consistent with altered PL metabolism (11). 67 Chapter 2 Quantification of fecal phospholipid: method development The new methodology descr ibed in this paper will be helpful in elucidating the mechan i sm of lipid malabsorption in patients with pancreat ic insufficiency and may thereby lead to more appropriate diet planning and revision of current concepts regarding pancreatic enzyme replacement for these patients. Furthermore, it may affect recent attempts to develop synthetic pancreatic replacement enzymes which do not contain phosphol ipase (Davidson A G F 2004, oral communicat ion, 2nd July). It remains to be seen whether chol ine supplementation to compensa te for increased P L excretion should be part of the nutrition management of these patients. 68 Chapter 2 Quantification of fecal phospholipid: method development Figure 2.1 Extraction of total fat and phospholipid using H i hexane, diethyl ether and ethanol, I—I chloroform, methanol, or WM hexane, diethyl ether and ethanol followed by re-extraction with chloroform, methanol1. Total fat Dry stool Phospholipid 1 1.5 CT E Q. Dry stool 1The bars represent the means+SEM, n=10. Values with a different superscript are significantly different, P<0.05. 69 Chapter 2 Quantification of fecal phospholipid: method development Figure 2.2 Val idat ion of H P L C - E L S D method for the determination of fecal phospholipid when compared with lipid soluble phosphorus quantified by phosphomolybdate colorimetric assay, n=26; • cystic fibrosis, o control. r=0.774, P O . O O l Phosphorous mg/g dry stool Phosphomolybdate colorimetric assay 70 Chapter 2 Quantification of fecal phospholipid: method development Figure 2.3 Distribution of phospholipids in fecal fat from children with cystic fibrosis, n=18 and children without cystic fibrosis, n=8 1 . C y s t i c F i b r o s i s C o n t r o l Sph 14±3% P C 2 8 ± 8 % LysoPC 41 ± 6 % Sph 32+11% 10±4% PI 2 ± 1 % PC 4 ± 2 % PE 19±6% LysoPC 34±9% 1 P E , phosphatidylethonalamine; PI, phosphatidylinositol; P S , phosphatidylserine; P C , phosphatidylcholine; Sph , sphingomyelin; L y s o P C , lysophosphatidycholine. The amount of fecal phospholipid in children with and without cystic fibrosis was ( m e a n ± S E M ) 1 3 8 . 9 ± 2 0 . 0 mg/day and 65.5+18.2 mg/day, respectively. 71 Chapter 2 Quantification of fecal phospholipid: method development 2.4 References 1. Ze ise l S H , Growdon J H , Wurtman R J , et al . Normal p lasma choline responses to ingested lecithin. Neurology 1980;30:1226-9. 2. Carey M C , Hernell , O. Digestion and absorption of fat. Semin Gastrointes Dis 1992;3:189-208. 3. Ze ise l S H , D a C o s t a K, Franklin P D , et al . Chol ine , an essent ial nutrient for humans. F A S E B J 1991;5:2093-8. 4. Canty D J , Ze i se l S H . Lecithin and choline in human health and d isease . Nutr Rev 1994;52:327-39. 5. Brouwers J F H M , Gade l l a B M , van Golde L M G , et al . Quantitative analysis of phosphatidylcholine molecular species using H P L C and light scattering detection. J Lip R e s 1998;39:344-53. 6. Institute of Medic ine . Chol ine . In Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6 , folate, vitamin B12 , panthothenic acid, biotin, and choline. Washing ton , D C : National A c a d e m y Press ; 2000:390-422. 7. Greenberger N J , Isselbacher K J . D iseases of the gal lbladder and bile ducts. •In Fauc i A , Braunwald E , Isselbacher K J , Wi l son J D , Martin J B , eds . Harrison's Principles of Internal Medicine. N Y : McGraw-Hi l l ; 1998:1725-6. 8. Waters DL , Dorney S F , G a s k i n K J , et al . Pancreat ic function in infants identified as having cystic fibrosis in a neonatal screening program. N Engl J M e d 1990;322:303-8. 9. Couper R T , Corey M , Moore D J , et al . Decline of exocrine pancreatic function in cystic fibrosis patients with pancreatic sufficiency. P e d R e s 1992;32:179-82. 72 Chapter 2 Quantification of fecal phospholipid: method development 10. Borowitz D, Ba ke r R D , Stallings V . Consensus report on nutrition for pediatric patients with cystic fibrosis. J Pediatr Gastroenterol Nutr 2002;35:246-59. 11. Innis S M , Davidson A G F , C h e n A , et al . Increased p lasma homocysteine and s-adenosylhomocysteine and decreased methionine is assoc ia ted with altered phosphatidylcholine and phosphatidylethanolamine in cystic fibrosis. J Pediatr 2003;143:351-6. 12. Co lombo C , Apos to lo M G , Ferrari M , et al . Analys i s of risk factors for the development of liver d isease associa ted with cystic fibrosis. J Pediatr 1994;124:393-9. 13. Y a o Z , V a n c e D E . H e a d group specificity in the requirement of phosphatidylcholine biosynthesis for very low density lipoprotein secretion from cultured hepatocytes. J Biol C h e m 1989;264:11373-80. 14. V a n de K a m e r J H , T e n Bokke l Huinink H , W e y e r s H A . R a p i d method for the determination of fat in feces. J Biol C h e m 1949;177:347-55. 15. Jeejeebhoy H N , A h m a d S, K o z a k G . Determination of fecal fats containing both medium and long chain triglycerides and fatty acids . C l in B i o c h e m 1970;3:157-63. 16. Folch J , L e e s M , Sloane-Stanley G H . A simple method for the isolation and purification of total lipides from animal t issues. J Bio l C h e m 1957;226:497-509. 17. C h e n P S , Toribara T Y , Warne r H . Microdetermination of phosphorus. A n a l C h e m 1956;28:1756-8. Chapter 2 Quantification of fecal phospholipid: method development 18. Innis S M , Dyer R A . Brain astrocyte synthesis of docosahexaeno ic acid from n-3 fatty acids is limited at the elongation of docosapentaenoic ac id . J Lipid R e s 2002;43:1529-36. 19. Rouser G , Siakotos A N , F le ischer S. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1965;1:85-6. 20. Nouri-Sorkhabi M H , C h a p m a n B E , Kuche l P W , et al . Paral lel secretion of pancreatic phosphol ipase A 2 , phospholipase A 1 , l ipase, and col ipase in children with exocrine pancreatic dysfunction. P e d R e s 2000;48:735-40. 21. Dressman J B , Shtohryn L V , Diokno D. Effects of product formulation on in vitro activity of pancreatic enzymes . A m J Hosp Pha rm 1985;42:2502-6. 22. Barraclough M , Taylor C J . Twenty-four hour ambulatory gastric and duodenal pH profiles in cystic fibrosis: effect of duodenal hyperacidity on pancreatic enzyme function and fat absorption. J P e d Gastroenterol Nutr 1996;23:45-50. 23. Buchman A L , Amen t M E , Sohe l M , et al . Chol ine deficiency causes reversible hepatic abnormalities in patients receiving parenteral nutrition: proof of a human choline requirement: a placebo-controlled trial. J P E N 2001;25:260-8. 74 Chapter 3 Phosphatidylcholine and ^phospha t i dy l cho l i ne excretion in C F CHAPTER 3 PHOSPHATIDYLCHOLINE AND LYSOPHOSPHATIDYLCHOLINE EXCRETION AND ITS ASSOCIATION WITH A L T E R E D P L A S M A HOMOCYSTEINE AND METHIONINE IN CHILDREN WITH CYSTIC FIBROSIS A version of this chapter has been submitted for publication. Chen A H , Innis S M , Davidson A G F , James S J . Phosphatidylcholine and lysophosphatidylcholine excretion is increased and related to altered plasma homocysteine and methionine in children with cystic fibrosis. Am J Clin Nutr (submitted). 75 Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in CF 3. INTRODUCTION Cyst ic fibrosis (CF) is an autosomal recessive disorder caused by a mutation in the cystic fibrosis t ransmembrane conductance regulator, an integral membrane protein that when activated by cAMP/pro te in kinase A , opens to form a channel to allow chloride ions to enter the cell (1). Exocr ine pancreatic insufficiency resulting in malabsorption of nutrients is present in 85-90% of patients with C F (2,3). Despite pancreatic enzyme replacements, somet imes with acid reducing agents and/or proton pump inhibitors to improve nutrient absorption, patients with C F continue to show fat malabsorption (4-6). Dec reased bile acid concentrations, low intra-luminal intestinal pH and decreased mucosa l absorption have all been suggested to contribute to the decreased absorption of fat in C F (5, 7-11). Hepatic steatosis has been extensively descr ibed in C F (12,13), although the incidence is uncertain because liver biopsy, which is the only way to confirm its presence is not usually performed in C F . In addition to steatosis, significant progressive liver d isease , which may include cirrhosis and fibrosis has been estimated to affect 17-37% of patients with C F (12,13). A n estimated incidence of hepatic steatosis in children with C F based on liver biopsy has been reported as 14-23% (12,13). Although several hypotheses, including carnitine and essential fatty acid deficiency, have been proposed (14-16), the etiology of steatosis and its relation to the defective epithelial cell chloride ion transport in C F is unclear. Fatty infiltration of the liver is a characteristic feature of choline deficiency (17,18) and is believed to be explained by the requirement for de novo 76 Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F synthesis of phosphatidylcholine ( P C ) for the secretion of triglyceride from the liver in apolipoprotein (apo) B containing very-low-density lipoprotein ( V L D L ) (19). The major pathway for P C biosynthesis in humans is the cytidine diphosphocholine (CDP-chol ine) path, which requires preformed choline (17,20). In the alternate phosphatidylethanolamine-A/-methyltransferase ( P E M T ) path, methyl groups from methionine are transferred via S-adenosylmethionine ( S A M ) to phosphatidylethanolamine (PE) to form P C (19). The other product of the P E M T path is S-adenosylhomocysteine (SAH) which is subsequent ly converted to homocysteine. During choline deficiency, the liver increases the synthesis of P C via the P E M T path (21). W e recently showed an inverse relation between plasma homocysteine and the plasma phospholipid P C content in children with C F (22), providing evidence of possible insufficient choline to support P C synthesis via the C D P - c h o l i n e path. The usual intake of P C is not known, although experimental studies by Zeisel et al published in 1980 suggested that a mixed diet containing meat is likely to provide about 1g P C per day, of which about 9 0 % of choline in animal tissues is P C (23,24). However, large amounts of P C are also secreted into the intestine in bile. In normal individuals, the enterohepatic pool of P C is about 1g and this pool circulates 5-10 times/d with almost complete hydrolysis and re-absorption of P C (25). The absorption of dietary and biliary P C involves hydrolysis by pancreatic phospholipase A 2 ( P L A 2 ) , which hydrolyzes P C to l y s o P C and an unesterified fatty acid (26), but this enzyme is inhibited at pHs below 5.8 (27). Due to failure of bicarbonate secretion from the pancreas, and 77 Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F despite some buffering capaci ty in enzyme supplements, many patients with C F have a postprandial duodenal intraluminal p H below 5.8 (11,28). O u r a im was to determine if fecal chol ine phosphoglyceride excretion is increased, and if fecal choline phosphoglyceride excretion is related to p lasma homocysteine and methionine in children with C F . 78 Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F 3.1 Subjects and Methods This study involved 18 children with C F and 8 control children with no known health problems. Al l of the children with C F had pancreatic insufficiency and were taking pancreatic enzyme replacements (500-2500 U lipase/kg/meal). These children were a subset of 53 children with C F who were patients at the C F outpatient clinic at British Columbia ' s Children's Hospital ( B C C H ) and 18 control children without C F who participated in a cross-sectional study des igned to quantify p lasma lipids and thiols in C F (22). Comple te details of the study, together with the patients' characteristics have been reported (22). Chi ldren enrolled in the study reported here all provided a quantitative 72h stool sample and a 5d weighed food record, in addition to a venous blood sample . The food records were collected over 5 consecutive days, that included 3 week and 2 weekend days. The children with C F followed their usual diet and therapeutic regimen throughout the study period. The food record was commenced 2d before the start of the stool collection. This procedure was used because children in our study were unwilling to separate feces based on the presence of markers. T h e height and weight of each study participant were recorded using a stadiometer (M2B, E N M C o . , Ch icago , IL) and electronic scale (ST5005 , Scale-Tronix Inc., White Plains, N Y ) , respectively, and the z scores for each child were calculated (29). Al l study protocols and procedures were approved by the University of British Co lumbia Clinical Resea rch Ethics Board and the Children 's and W o m e n ' s Health Centre of British Co lumbia Research Review Commit tee . Al l participants and their parents provided written informed consent (Appendix A). 79 Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F 3.1.1 Fecal analysis Feca l samples were frozen in pre-weighed containers immediately following collection and stored at - 7 0 ° C until analysis. For analysis , fecal samples were weighed , homogenized and a portion dried to constant weight to allow determination of fecal water content. Total lipids were extracted and quantified gravimetrically. Feca l phospholipids including P C , l y s o P C , P E , phosphatidylserine (PS) , phosphatidylinositol (PI) and sphingomyelin (Sph) were separated and quantified using H P L C with evaporative light scattering detector ( H P L C - E L S D ) (30,31). Feca l energy was quantified using a bomb calorimeter (Model 1341, Parr, IL) according to manufacturer's instructions. 3.1.2 Plasma analysis Two 7mL venous blood samples were drawn from each subject into tubes containing E D T A as anti-coagulant (22). The plasma and red blood cells were separated by centrifugation at 2,000 g, 15 min at 4 ° C , and frozen at - 7 0 ° C within 20 min of blood collection. P l a s m a total lipids were extracted and the phospholipids separated and quantified by H P L C - E L S D (22,31). P l a s m a methionine, homocysteine and their metabolites were ana lyzed by H P L C with reversed phase ion-pairing (32). P l a s m a apo B was quantified using immunoturbidimetric reagents (Sigma Diagnostic, St. Louis , M O ) , and total cholesterol and high density lipoprotein (HDL)-cholesterol were determined using colorimetric enzymatic reagents (Diagnostic Chemica ls , Charlottetown, PEI) . 80 Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F 3.1.3 Dietary analysis All vitamin, mineral and other nutritional supplements, in addition to foods and beverages were recorded on the food records (Appendix B). The dietary records were entered into a nutrient analyses data base ( E S H A Food Processor Vers ion 7.71, E S H A Resea rch , S a l e m O R ) and the total energy, fat, carbohydrate and protein intake (g/d) was calculated. The total energy intake of each subject was also calculated as % estimated energy requirement ( E E R ) per d for healthy individuals, based on age, gender, weight, height and physical activity levels (33). A physical activity coefficient equivalent to walking 1.5-2.9 miles/d at 2-4 mph was a s sumed for each subject. Fat absorption was calculated as [(total fat intake (g/d)-fecal fat excretion (g/d))/total fat intake (g/d)] x 100%. Total choline intake and the intake of choline from P C was estimated using the U S D A database on the choline content of common foods (24). 3.1.4 Statistical analysis Data are presented as m e a n s ± S E M s . Independent 2-tailed t-testfor normally distributed data, and Mann-Whitney test for nonparametric unpaired data were used to compare the results for children with C F and the controls. Pearson correlation coefficient for normal data and Spearman ' s rank correlation coefficient for nonparametric data were calculated to determine potential associat ions between fecal phospholipid excretion and p lasma homocysteine, methionine and S A H . Al l statistical analyses were performed using S P S S 9.0.0 81 - Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F for Windows ( S P S S Inc, Chicago) . P-values <0.05 were considered statistically significant. 82 Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F 3.2 Results In this study, we determined fecal fat and phospholipid excretion and the relation of fecal choline phosphoglyceride excretion to p lasma methionine, S A H and homocysteine of children with and without C F . The age of the children with C F (n=18) and of the control children was 9 .3±1 .4 and 10 .0±0 .1y , respectively. The z-scores for height-for-age of the C F and control groups were -0.47+0.17, and 0.51+0.24, respectively, and the z-scores for weight-for-age were -0.48+0.16 and 0.24+0.16, respectively, P>0.05. Three of the 18 children with C F had meconium ileus as infants, 5 were taking ursodeoxycholic acid at the time of the study for elevated liver enzymes or abnormal liver ultrasound findings. None of the children had any other gastrointestinal d isease or resection, or clinically significant liver d isease . The children with C F had a mean energy intake of 142.0+3.5% of the E E R . The energy intake of the control children was 1 0 6 . 2 ± 4 . 7 % E E R and was significantly lower than in the children with C F , P<0.001. T h e intake of dietary fat was also significantly higher in children with C F ( 9 6 . 7 ± 6 . 8 g/d) than in the control children (70.1 ± 6 . 7 g/d, P=0.025). Fat, carbohydrate and protein contributed to 3 5 ± 1 . 6 % , 5 0 ± 1 . 7 % and 14+0.7% of the total energy intake in children with C F , and 3 0 ± 1 . 6 % , 5 6 ± 1 . 5 % and 1 4 ± 0 . 6 % of total energy intakes in the control children, respectively (P>0.05). The intake by children with and without C F for choline ( 3 3 5 ± 3 3 and 2 6 8 ± 2 5 mg/d) and P C ( 8 6 5 ± 1 1 2 and 8 1 2 ± 1 5 8 mg/d) was not different. 83 Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F The children with C F had a significantly lower absorption of dietary fat ( 8 6 . 2 ± 1 . 6 % ) than the control children ( 9 4 . 1 ± 1 . 2 % ) , P=0.004 (Table 3.1). Consistent with the reduced dietary fat absorption, daily fecal fat excretion by the children with C F was 3-4 fold higher than in the control children, P - 0 . 0 0 3 . Feca l energy excretion was about two fold higher in children with C F than in the control children, but was not statistically significant, possibly due to the wide inter-individual variability in fecal energy excretion (Table 3.1). However , fecal fat and the fecal energy content were significantly associated, r=0.89, P O . 0 0 0 1 , (Figure 3.1). There was no significant difference in fecal water content between the children with C F and the control children. Total phospholipids, P C and l y s o P C excretion were all significantly higher in children with C F than in the control children (Table 3.2). T h e lower limit of the H P L C - E L S D linear range for quantification of phospholipid was 50 mg/g dry stool. Of the 8 children in the control group, 2 had a fecal P C concentration in the detectable range, but below 0.5 mg/d P C excretion. The range of fecal P C excretion in children with C F was 0.86-140.9 mg/d, compared to <0.5-8.8 mg/d in the control children. About 9 0 % of the fecal choline phosphoglycer ides excreted by the control children was l y s o P C . In contrast, l y s o P C represented about 60% and P C represented about 4 0 % of the fecal choline phosphoglyceride excretion in children with C F (Table 3.2). P E , P S , PI and Sph excretion were not significantly different between the children with C F and the control children. However, due to the higher fecal excretion of P C and l y s o P C , P E , P S , PI and Sph represented 3 1 % of the phosphoglycerides excreted by children with C F and 84 Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F 6 2 % phosphoglycerides excreted by the control children, P<0.001. The fecal phospholipid excretion wa s also significantly associated with the fecal total fat excretion, at r=0.78, P<0.001 (Figure 3.1). A significant positive associat ion w as also found between the fecal excretion of P E , P C , l y s o P C and S p h and fecal fat excretion at r=0.55, P =0.003, r s=0.82, PO.001, r=0.60, P=0.001, and r=0.43, P=0.027, respectively, and between fecal P C , l y s o P C , P E , PI, and S p h with fecal total phospholipids excretion at rs =0.88, PO.001, A=0.86, P<0.001, r =0.55, P=0.004, r =0.44, P=0.024, and r=0.55, P=0.003, respectively. Fa t absorption (%) was also significantly inversely associa ted with fecal total fat, total phospholipid, P C , l y s o P C and P E excretion, r=-0.86, P<0.001, r=-0.82, PO.001, rs =-0.75, PO.001, r=-0.78, PO.001 and r=-0.49, P=0.011, respectively. The p lasma concentrations of methionine (PO.001) and the P C / P E ratio (P=0.03) were lower, and P E (PO.001), homocysteine (PO.001) and S A H (P=0.001) were higher in the children with C F than in the control children (Table 3.3). The p lasma P C and l y s o P C were not significantly different (P=0.1) between the children with C F and the control children. The p lasma apo B , but not the total or H D L cholesterol was a lso significantly lower (P=0.03) in the children with C F than in the control children. P l a sma apo B was inversely associa ted with fecal total phospholipids and P C at r=-0.58, P=0.008, rs =-0.43, P=0.034, respectively. W e did not find any associat ion between p lasma apo B , and fecal fat and l y s o P C . This study also found a significant positive associat ion between the fecal total phospholipid, P C and l y s o P C and the plasma homocysteine and an inverse association between the fecal phospholipid, P C and l y s o P C and the p lasma 85 Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F methionine for all subjects in the study (Table 3.4). In addition, the p lasma homocysteine w a s significantly and positively associa ted with fecal total phospholipid, fecal choline and fecal total choline phosphoglycerides within the subgroup of children with C F (Figure 3.2). The fecal total phospholipid and P C excretion were also significantly and positively associa ted with the p lasma S A H . There were no statistically significant associat ions between the fecal total phosphlipid, P C or l y s o P C excretion and p lasma P C , l y s o P C or P C : P E ratio, respectively. 86 Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F 3.3 Discussion T o the best of our knowledge, our study provides the first quantitative demonstration of increased fecal choline phosphoglyceride ( P C and l y s o P C ) excretion in cystic fibrosis. Our study also provides new data to show increased fecal choline phosphoglycer ide excretion is associated with elevated p lasma homocysteine in humans. Together with our recent report to show p lasma homocysteine and S A H are positively associated with p lasma P E and inversely related to p lasma P C (22), our work provides evidence that P C synthesis via P E M T is inter-related with the transfer of methyl groups from S A M to S A H in a manner which impacts p lasma homocysteine and p lasma P C / P E ratios in humans. Consis tent with this suggestion, recent studies have shown an approximately 5 0 % lower p lasma homocysteine concentration in P E M T -/- mice when compared with wild type mice (34), thus suggest ing that the rate of P E M T methylation of P E to form P C is a determinant of p lasma homocysteine in this species . It is well known that despite pancreatic enzyme replacement therapy, dietary fat absorption remains lower in patients with C F than in individuals without C F (7,35,36). In our study, children with C F , all of w h o m were taking pancreatic enzymes, absorbed about 8 6 % of the dietary fat intake compared with 94% fat absorption by the control children. Similarly, Murphy et al (35) and Burdge et al (36) found 8 6 % and 78% of dietary fat was absorbed, respectively, in patients with C F . To the best of our knowledge, our work is the first to descr ibe the amount and type of phospholipids excreted by children or adults. Al though 87 Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F phospholipids usually contribute 4-8% of dietary fat (37), s tandard methods for the extraction of fecal lipids do not allow quantitative recovery of phospholipids (30). To address this, we recently developed methodology for quantitative extraction of fecal phospholipids, followed by separation and quantification of phospholipid c lasses using H P L C - E L S D (30). The significant relations between fecal energy and fat excretion, and between fecal fat and phospholipid excretion suggests that fat malabsorption is accompanied by a higher excretion of phospholipids, as well a s energy. B e c a u s e P C secreted into the intestine in bile is essential for normal fat digestion (38), it is possible that reduced P C availability, secondary to increased excretion of choline phosphoglycerides could limit the digestion and absorption of dietary triglycerides. However, dec reased bile acids, reduced mucosa l absorptive function (7,8), or reduced activities of co-l ipase dependent pancreatic l ipase as well as P L A 2 could also explain our results. Whether increased excretion of choline phospholipids occurs in other disorders involving impaired pancreatic or biliary function, or intestinal absorptive dysfunction is not known. The high amounts of l y s o P C excreted by the control children and the children with C F in our study were unexpected, although we are unaware of similar data on the composit ion of fecal phospholipids. F e c a l l y s o P C could originate from dietary and biliary P C hydrolyzed lower in the intestine beyond the site of l y s o P C absorption, or from the activity of colonic microflora. L o w activity of P L A 2 due to limited bicarbonate secretion from the pancreas, and/or s low dissolution of enteric pancreatic enzymes in the upper intestine could explain the 88 Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F increased hydrolysis of P C to l y s o P C in the lower intestines, and thus increased excretion of l y s o P C in children with C F . However, l y s o P C was a lso the major choline phosphoglyceride excreted by healthy children without C F . Lecithinase-positive bacteria are present in colonic microflora, and the abundance of these organisms has been reported to be sensitive to dietary variables (39,40). Whether or not the types and amounts of phosphoglycerides present in the colon has any physiological relevance to the colonization of lecithinase-positive bacteria and intestinal function is unknown. Our studies were initiated to address the possibility that altered hepatic P C metabolism secondary to increased P C excretion could contribute to the high prevalence of hepatic steatosis among patients with C F (12,13). The estimated intake of P C in our study was 8 6 5 ± 1 1 2 mg/d for children with C F and 8 1 2 ± 1 5 8 mg/d for children without C F , which is similar to the intake of 1g/d P C in the studies of Zeise l et al (23). W e found that fecal P C and l y s o P C excretion was about 80mg/d in children with C F and about 20mg/d in the children without C F . These results suggest that P C absorption is probably as efficient as total fat absorption in children, i.e. about 92-98%. S o m e studies however, have provided evidence of increased membrane lipid turnover in C F defective cells , which could suggest that choline requirements are higher in patients with C F (41). A s s e s s m e n t of hepatic triglyceride accumulation involving liver biopsy in the absence of clinical evidence of liver d isease is not ethically acceptable. Therefore, we sought evidence of increased P C excretion through measures of fecal choline phosphoglyceride excretion and used a novel approach to address 89 Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F alteration of hepatic P C metabolism based on the intersection of the methionine-homocysteine pathway with phospholipid metabolism at the methylation of P E to form P C , with the generation of S A H and homocysteine from methionine via S A M (22). Our hypothesis that altered hepatic choline metabolism with reduced synthesis in the C D P pathway may occur in C F was based on the knowledge that hepatic triglyceride accumulat ion is a characteristic feature of choline deficiency (17,18), explained by the requirement for de novo P C biosynthesis v ia the C D P -choline path for secretion of triglyceride in apo B containing lipoproteins (19,20). In addition, patients with C F show fat malabsorption despite pancreatic replacement enzyme therapy (7,35,36). Further, many patients with C F have an intraluminal pH below 5.8 (4,11), which is the threshold for activity of P L A 2 (26). In this study, we have shown that children with C F excrete higher amounts of P C and l y s o P C than healthy control children. Total choline phosphoglyceride ( ly soPC+PC) excretion in the children with C F was about 50-200mg/day, compared with 6-70 mg in the control children. The enterophepatic pool of P C in healthy adults has been estimated to be about 1 gram (25). Whether or not the increased in fecal P C compromises P C status is not known. O n the other hand, the estimated net absorption of P C appears to be similar in both the children with C F (785mg/d) and the healthy control (792mg/d) (Total P C intake - fecal P C and fecal l y s P C ) . S o m e studies have provided evidence of increased membrane lipid turnover in C F defective cells, which could suggest that choline requirements are higher in patients with C F (41). The higher fecal phospholipids excretion, together with the possibly the increased choline requirement raise the question of whether 90 Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F or not the availability of choline to support the C D P - c h o l i n e path might be decreased , and thus increase the demand in the liver to generate P C via the P E M T path in C F . The significant positive relations between choline phosphoglyceride excretion, and p lasma S A H and homocysteine, and the inverse relation with p lasma methionine support a hypothesis that the increased homocysteine and S A H level found in children with C F is related to increased synthesis of P C via P E M T . Alternatively, elevation of S A H could result in inhibition of methyltransferase reactions, including P E M T (42,43), resulting in a decrease in the p lasma P C / P E ratio as found by us in children with C F . W e previously showed that the higher p la sma homocysteine and S A H in children with C F is not explained by deficiency of folate or vitamin B12 (22). However, alternate explanations such as oxidative reduction of methionine synthase are possible. Measures of p lasma free choline were not included in our study because p lasma free choline concentrations are low, with a range of about 7-20 u.mol/L in adult men which overlaps the mean of 7.5 nmol/L found in p lasma of adults fed a choline deficient diet (44). Dietary phospholipid and P C absorption was quantified using the recent U S D A database on the choline content of foods (24). Further details, including the U S D A database, are included in Appendices D and E. W e were not able to quantify the amount of P C secreted into the intestine in bile without invasive techniques, thus P C absorption could not be quantified. However , consistent with reduced secretion of V L D L from the liver, the p lasma apo B , although not total or H D L cholesterol concentrations, were lower in the children in our study with C F than in the control children. Reduced p lasma apo B 91 Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F and cholesterol concentrations have been reported in previous studies with patients with CF ( 45 -47 ) . In summary, children with CF excrete higher amounts of choline phosphoglycerides than children without CF. The excretion of choline phosphoglycerides is positively associated with plasma homocysteine and inversely associated with plasma methionine. These novel findings provide evidence of altered hepatic choline metabolism, and raise the question of whether the CDP choline or PEMT pathway for PL synthesis are altered in patients with CF. Our work also provides new information to support an important functional interdependence between phospholipid metabolism and the methionine-homocysteine cycle in humans. We postulate that altered phospholipid metabolism could be relevant to some of the complications associated with CF, such as hepatic steatosis. Further studies are warranted to address the importance of the PEMT cycle in PC metabolism in humans and the inter-relation of this pathway with plasma homocysteine. 92 Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F TABLE 3.1 Fecal fat and energy content of children with cystic fibrosis and control children7 CF (n=18) Control (n=8) P value Fecal total fat (g/d) 12.9+1.7 (2.2-34.8) 3.9±0.7 (2.0-7.2) 0.003 Fat absorption (% fat intake) 86.2±1.6 94.1 ±1.2 0.004 Fecal water content (% fecal weight) 69.0±2.2 68.9±2.3 NS2 Fecal energy (kJ/d) 1130±215 627±146 NS Fecal energy (kJ/g dry weight) 24.2±1.7 19.7±1.4 NS Va lues shown are mean ± SEM (range) and were analyzed using independent 2-tailed t-tests 2NS, not significant. 93 Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F TABLE 3.2 Feca l phospholipid excretion in children with cystic fibrosis and control children 1 z j o r uontrol 0 . m g / d (n=18) (n=8) P v a l u e Total phospholipids ( ^ W ? 2 9 . 7 ) (16¥i%V47.7) 0 0 3 4 Phosphatidylcholine ^ f f ^ . e ) {0.wil)> 0 0 0 5 Lysophosphatidylcholine { J f 2 ? 2 f l 0 3 ) (J^ffe.V ° ° 1 4 Phosphatidylethanolamine {0^f]t0) ( A J ^ % N S * Phosphatidylinositol (olu^z.T) N S Phosphatidylserine ^24A 5.3) (0.ll7%%.7) N S c „ h i „ „ „ _ , „ = l i „ 19.6+3.6 2 0 . 7 ± 7 . 2 Sphingomyelin (1.4-51.6,17.0) (6.7-66.1,11.0) N S 1 D a t a shown are m e a n s ± S E M (range,median) and were ana lyzed using independent 2-tailed t-test for normally distributed data, and Mann-Whi tney test for nonparametric data. 2 F e c a l excretion of phospholipid of >0 and <0.5mg/d was below the linear range of calibration, and recorded as 0.5mg/d. 3 N S , not significant. 94 Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F T A B L E 3.3 P l a sma thiols and phospholipids in children with cystic fibrosis and control chi ldren 7 C F (n=18) Control (n=8) P value Homocysteine (u.M) 9 .2±0 .4 4 . 9 ± 0 . 2 <0.001 Methionine (u.M) 19 .0±0 .9 30.5+2.6 <0.001 S-adenosylhomocysteine (u.M ) 3 2 . 6 ± 2 . 0 20.3+1.4 0.001 S-adenosylmethionine (u.M) 7 4 . 7 ± 3 . 4 8 4 . 5 ± 6 . 4 N S 2 Total Phospholipid (u.M) 7 3 2 ± 7 6 . 7 747+110 N S Phosphatidylethanolamine (PE) (%) 10 .2±1 .4 3.7+0.4 <0.001 Phosphatidylcholine ( P C ) (%) 76.7+2.1 82.3+1.8 N S Sphingomyelin (%) 9.1+0.9 11.0+1.4 N S Lysophosphatidylcholine (%) 3 .6±0 .3 2 . 8 ± 0 . 3 N S P C / P E ratio 13 .0±3 .2 2 4 . 8 ± 3 . 5 0.03 Total cholesterol (mg/dL) 1 5 8 ± 1 0 . 4 154+7.6 N S HDL-cholesterol (mg/dL) 4 2 . 1 ± 2 . 2 4 4 . 5 ± 4 . 2 N S Apolipoprotein B (mg/dL) 4 6 . 9 ± 1 . 7 5 4 . 4 ± 3 . 2 0.03 V a l u e s shown are mean ± S E M and were analyzed using independent 2-tailed t-tests 2 N S , not significant P>0.05 95 Chapter 3 Phosphatidylcholine and Ivsonhosphatidvlcholine excretion in CF T A B L E 3.4 Assoc ia t ions between plasma thiols and fecal phospholipid excret ion 7 Feca l Phosphol ipid P l a sma Total P C L y s o P C P L Methionine r=-0.49, P=0.013 r s=-0.59, P=0.002 r=-0.60, P=0.001 Homocyste ine r=0.64, P=0.001 r s=0.76, P<0.001 r=0.58, P=0.002 S A H r=0.52, P=0.008 r s=0.64, P=0.001 N S 2 V a l u e s are r = Pea r son correlation coefficient, r s =Spearman 's rank correlation coefficient. P L = phospholipids, P C = phosphatidylcholine, L y s o P C = lysophosphatidylcholine, and S A H = S-adenosylhomocysteine. 2 N S , not significant. 96 Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in CF FIGURE 3.1 Correlation between fecal total fat, and fecal energy and fecal total phospholipid 1200 ! . 97 Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F FIGURE 3.2 Scatterplots of p lasma homocysteine versus a) fecal total phospholipid, b) phosphatidylcholine, c) ^ p h o s p h a t i d y l c h o l i n e , and d) total choline phosphoglycerides in the subgroup of children with cystic fibrosis*, n=18. 50 100 150 200 250 300 350 Fecal total phospholipid (mg) 13 -12 -_ _ 11 me % 10 • o 9 -rs=0.66, P=0.00 0 20 40 60 80 100 120 140 160 Fecal phosphatidylcholine (mg) _ 10 w 8 r=0.38, P=0.14 20 40 60 80 100 120 140 Fecal ^ phosphatidylcholine (mg) 0 50 100 150 200 250 300 Fecal choline phosphoglyceride (mg) *r= Pearson correlation coefficient, r s =Spearman's rank correlation 98 Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F 3.4 References 1. 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J Pediatr Gastroenterol Nutr, 1994;19:421-4. 16. Strandvik B , Hultcrantz R. Liver function and morphology during long-term fatty acid supplementat ion in cystic fibrosis. Liver 1994;14:32-6. 100 Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in CF 17. B u c h m a n A L , A m e n t M E , S o h e l M , et al . Chol ine deficiency causes reversible hepatic abnormalit ies in patients receiving parenteral nutrition: proof of a human choline requirement: a placebo-controlled trial. J P E N 2001;25:260-8. 18. Zeise l S H , Blusztajn J K . Chol ine and human nutrition. A n n u Rev Nutr 1994;14:269-96. 19. Y a o Z , V a n c e D E . H e a d group specificity in the requirement of phosphatidylcholine biosynthesis for very low density lipoprotein secretion from cultured hepatocytes. J Biol C h e m 1989;264:11373-80. 20. V a n c e D E . Phosphat idylcholine metabolism: masochist ic enzymology, metabolic regulation, and lipoprotein assembly. 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Diseases of the gallbladder and bile ducts. In Fauci A, Braunwald E, Isselbacher KJ, Wilson JD, Martin JB, eds. Harrison's Principles of Internal Medicine. NY: McGraw-Hill, 1998:1725-6. 26. Nouri-Sorkhabi MH, Chapman BE, Kuchel PW, et al. Parallel secretion of pancreatic phospholipase A2, phospholipase A1, lipase and colipase in children with exocrine pancreatic dysfunction. Pediatr Res 2000;48:735-40. 27. Dressman JB, Shtohryn LV, Diokno D. Effects of product formulation on in vitro activity of pancreatic enzymes. Am J Hosp Pharm 1985;42:2502-6. 28. Barraclough M, Taylor CJ . Twenty-four hour ambulatory gastric and duodenal pH profiles in cystic fibrosis: effect of duodenal hyperacidity on pancreatic enzyme function and fat absorption. J Pediatr Gastroenterol Nutr 1996;23:45-50. 29. Kuczmarski RJ, Ogden CL, Grummer-Strawn LM, et al. CDC growth charts: United States. Advance data from vital and health statistics; no. 314. Hyattsville, Maryland: National Center for Health Statistics; 2000. 30. Chen A, Innis SM. Extraction and quantification of fecal phospholipid for the measurement of phospholipid malabsorption. J Pediatr Gastroenterol Nutr 2004;39:85-91. 31. Innis SM, Dyer RA. Brain astrocyte synthesis of docosahexaenoic acid from n-3 fatty acids in limited at the elongation of docosapentaenoic acid. J Lipid Res 2002;43:1524-36. 102 Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in CF 32. J a m e s S J , Pogr ibna M , Pogribny IP, et al . Abnormal folate metabol ism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome. A m J Cl in Nutr 1999;70:495-501. 33. Institute of Medic ine . Dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acid. Washing ton , D C : National A c a d e m i e s Press , 2002. 34. Noga A A , S tead L M , Z h a o Y , Brosnan M E , Brosnan JT , V a n c e D E . P l a s m a homocysteine is regulated by phospholipid methylation. J Biol C h e m 2003;278:5952-5. 35. Murphy J L , Woot ton S A , B o n d S A , J ackson A A . Energy content of stools in normal healthy controls and patients with cystic fibrosis. A r c h Dis Chi ld 1991;66:495-500. 36. Burdge G C , G o o d a l e A J , Hill C M , et al . P l a s m a lipid concentrations in children with cystic fibrosis: the value of a high-fat diet and pancreatic supplementation. B r J Nutr 1994;71:959-64. 37. Carey M C , Hernell O . Digestion and absorption of fat. S e m i n Gastrointes Dis 1992;3:189-208. 38. Roy C C , W e b e r A M , Morin C L , et a l . Hepatobiliary d i sease in cystic fibrosis: a survey of current issues and concepts . J Pediatr Gastroenterol Nutr 1982;1:469-78. 39. Benno Y , Mizu tam T, N a m b a Y , Komor i T, Mitsuoka T. C o m p a r i s o n of fecal microflora pf elderly persons in rural and urban areas of J a p a n . A p p l Environ Microbiol 1989;55:1100-5. Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F 40. Benno Y , S a w a d a K, Mitsuoka T. The intestinal microflora of infants: composit ion of fecal flora in breast-fed and bottle-fed infants. Microbiol Immunol 1984;28:975-86. 41 . Ulane M M , Butler J D , Per i A , Miele L, Ulane R E , Hubbard V S . Cys t ic fibrosis and phosphatidylcholine biosynthesis. Cl in C h i m A c t a 1994;230:109-16. 42. Hoffman D R , Haning J A , Cornatzer W E . Microsomal phosphatidylethanolamine methyltransferase: inhibition by S-adenosylhomocyste ine . Lipids 1981;16:561-7. 43. Y i P , Melnyk S, Pogr ibna M , Pogribny IP, Hine R J , J a m e s S J . Increase in plasma homocysteine associa ted with parallel increases in p lasma S-adenosylhomocyste ine and lymphocyte D N A hypomethylation. J Biol C h e m 2000;275:29318-23. 44. Zeise l S H , D a C o s t a K , Franklin P D , et a l . Chol ine , a n essential nutrient for humans. F A S E B J 1991,5:2093-8. 45. Vaughan W J , Lindgren F T , W h a l e n J B , A b r a h a m S. S e r u m lipoprotein concentrations in cystic fibrosis. Sc ience 1978;199:783-6. 46. Benabdes lam H , Garc i a I, Bel lon G , e t a l . B iochemica l a s sessment of the nutritional status of cystic fibrosis patients treated with pancreatic enzyme extracts. A m J C l in Nutr 1998;67:912-8. 47 .S les insk i M J , Gloninger M F , Constantin J P , Orenstein D M . Lipid levels in adults with cystic fibrosis. J A m Diet A s s o c 1994;94:402-8. 104 Chapter 4 Discussion and conclusions CHAPTER 4 DISCUSSION AND CONCLUSIONS 105 Chapter 4 Discussion and conclusions 4. INTRODUCTION The objective of this thesis research was to determine if phospholipid (PL) and P C excretion in children with cystic fibrosis (CF) is increased and to a s sess possible associat ions between fecal P C excretion and p lasma methionine and homocysteine. P C excretion and plasma thiols were studied because hepatic steatosis is a recognized problem among patients with C F (1, 2) and hepatic steatosis is also a characteristic feature of choline deficiency (3, 4). T h e hepatic steatosis of choline deficiency is believed to be explained by a specific requirement for active hepatic de novo P C biosynthesis via the C D P - c h o l i n e pathway to support the secretion of triglyceride from the liver in apolipoprotein B (apo B) containing very low density lipoprotein ( V L D L ) (5, 6). It is well known that despite pancreatic enzyme replacement therapy, patients with C F continue to malabsorb fat (7-9). Whether or not patients with C F also malabsorb phospholipids, particularly P C , has not been previously reported. The amount of phospholipid in typical diets is not known; some authors have sugges ted that phospholipid represent 4-8% of dietary fat (10, 11). However , in human milk, which contains fat as triglyceride in a globule surrounded by phospholipids, P L contributes only about 0.7% of total fat (12). In our study, the total dietary P C was about 870mg/d in children with C F and 810mg/d in the non C F controls, while total intake of fat was 97g/d in children with C F and 70g/d in the control children. Assuming P C represent 9 0 % of dietary P L , then in our studies, P L was 0.9% of dietary fat, P L represented 1% and 1.3% of dietary fat intake in our children with C F and in the control children, respectively. It is notable that these estimates of 106 Chapter 4 Discussion and conclusions P L intake based on our experimental data are c lose to human milk, for which the total fat and P L content have been well descr ibed. P C represents >90% of the dietary intake of the essent ial nutrient choline (10, 13). The major route of hepatic P C biosynthesis is the C D P - c h o l i n e pathway, which requires a source of preformed choline (14,15). In the alternate pathway for P C synthesis, catalyzed by phosphatidylethanolamine methyltransferase ( P E M T ) , sequential methylation of P E using methyl groups from methionine via S-adenosylmethionine ( S A M ) leads to the formation of P C , with S-adenosylhomocysteine ( S A H ) as the other product. S A H is subsequently metabolized to homocyste ine (16). During experimental choline deficiency in animals, the hepatic concentration of S A H is increased, and S A M and methionine are decreased . This has been interpreted to reflect an attempt by the liver to increase P C synthesis by the de novo P E M T pathway (17,18). In a larger study involving all of the subjects in this thesis research, we have shown that children with C F have a higher plasma homocysteine, and S A H and lower plasma methionine and P C / P E ratio when compared with children without C F . Further, we have shown an inverse relation between p lasma homocyste ine and the proportion of P C in p l a sma phospholipids (16). T h e alteration in p lasma thiols and associat ion with p l a sma P C is consistent with a hypothesis of hepatic choline depletion in C F . A n objective of this thesis research was to develop a quantitative method for extraction and quantification of fecal phospholipids. High performance liquid chromatography with evaporative light scattering detection ( H P L C - E L S D ) was 107 Chapter 4 Discussion and conclusions used for the separation and quantification of fecal phospholipids c lasses . A new lipid extraction solvent sys tem was developed for extraction and quantification of fecal phospholipids. B a s e d on the properties of phospholipids, w e predicted that current standard methods for extracting fecal fat only extract triglycerides and unesterified fatty acids , but do not quantitatively recover phospholipids. Furthermore, a reliable method for analysis of P C excretion has not been previously reported. The purpose of this chapter is to d iscuss the method developed for extraction and quantitation of fecal triglycerides and phospholipids, followed by a discussion of fat and phospholipids excretion in children with C F compared with the n o n - C F healthy control, and the implications of the associat ions between choline phosphoglyceride ( P C and ly soPC) excretion, and p lasma methionine, homocysteine and S A H . Finally, a d iscuss ion of limitations and future directions, followed by the conclus ion will be presented to summarize the major findings of this thesis research. 4.1 Discussion 4.1.1 Extraction and quantitation of fecal total triglycerides and phospholipids: method development and validation In chapter 2 of this thesis research, a new method involving sequential extraction of fecal lipids with hexane:diethyl ether followed by chloroform and methanol to quantify fecal total fat and phospholipids is reported (specific a im 1 of this thesis research). T h e combination of solvent sys tems descr ibed results in 108 Chapter 4 Discussion and conclusions about a 3 0 % increase in total fat extraction and a three fold increase in recovery of phospholipids compared with the standard clinical method used for extraction of fecal fat (19). In addition, the method gives about a 10% increase in total fat extraction and a 3 5 % increase in recovery of phospholipids compared with the extraction method of Folch et al (20), which is a standard method for t issue lipid extraction. The new method involving the combination of the two solvent systems was developed for two reasons . First, phospholipids typically represent only 4-8% of dietary fat and previously published methods for fecal fat quantitation were not capable of detecting small but meaningful differences in fecal excretion. For example, in a 30 year-old low active adult man with a height of about 1.7m and weight of 70kg, consuming 2,500 kcal/d with 20-35% of dietary energy intake from fat, 4-8% dietary fat from P L would be equivalent to an intake of 2.2-7.8 g phospholipid/d. Similarly, for a low active 12 year-old boy, of 1.5 m height and 40.5 kg body weight consuming 2100 kcal/d with 25-35% dietary energy from fat, the intake would be 2.3-6.6 g phospholipid/d (21). Typical ly, fat absorption is >93% in healthy children without C F or any gastrointestinal abnormalities while fat absorption is about 8 5 % in children with C F related pancreatic insufficiency taking enzyme supplements(22). A literature search of papers published 1966-2004 did not reveal any published studies on phospholipid absorption or excretion in humans; hence the extent of phospholipid malabsorption or excretion can only be estimated from published data on fat absorption. A s s u m i n g 8 5 % absorption of dietary phospholipid in children with C F and 9 3 % absorption in the 109 Chapter 4 Discussion and conclusions healthy control children, our study population of C F children and control children might be expected to excrete 0.35-0.98 g and 0.16-0.46 g phospholipid in feces, respectively. A s s u m i n g the standard clinical method for the quantification of fecal fat absorption has a coefficient of variation of 5%, a difference in total fat absorption of less than 0.2-0.6 g/d could not be detected. Thus , as the difference in phospholipid excretion between children with and without C F is small relative to the amount of total fat excreted and would be below the sensitivity of current standard methods for quantification of total fat excretion. T h u s in this research, a method was developed that would specifically allow the extraction and quantification of phospholipid. Of note, in addition to the unabsorbed dietary phospholipid, the fecal phospholipid pool is also likely to contain unabsorbed biliary phospholipid and phospholipids derived from sloughed intestinal cells and colonic microflora. Thus , the total amount of phospholipid excreted by the healthy subjects without malabsorption could be higher than 0.16-0.46 g/d. Secondly , based on the solubility of polar and non-polar lipids and different solvents, we predicted that the standard clinical laboratory methods, such as the method of Jeejeebhoy et al (19), which involves extraction with hexane:diethyl ether:95% ethanol (1:1:1, by volume) and hexane:diethyl ether (1:1, by volume) (19), would not allow complete extraction of phospholipids from fecal samples . However , solvent systems based on chloroform and methanol (20) while extracting phospholipids, are not expected to recover the more water soluble medium chain fatty acids from fecal samples . Therefore, a combination of lipid extraction sys tems that combined the strengths of ether and chloroform n o Chapter 4 Discussion and conclusions based solvent extraction was used to extract polar and non-polar lipids, including P L and medium chain fatty acids . Med ium chain fatty acids may be present in fecal samples as a result of malabsorption of medium chain fatty ac ids from the nutrition supplements consumed by some C F patients. In our study, two children with C F (11%) were on nutrition supplements that contained M C T oil at the time of the study. W e showed 10% higher total fat and a 3 5 % higher recovery of phospholipid using our method for fecal fat extraction when compared to the Folch method for t issue lipid extraction (20) and 30% higher total fat and a three fold higher recovery of phospholipid compared to a standard clinical method for assessment of fecal fat excretion (19). First, the increased total fat and phospholipid could be explained by the more appropriate use of solvent systems for extraction of fat and phospholipids. However, it is well recognized that repeat extraction of samples with solvents also improves the recovery of lipid (23). W e extracted the fecal samples with chloroform and methanol twice, and extracted three times with hexane and diethyl ether. Christie W W (1993) has reported that in the method by Fo lch et al (20), the proportions of chloroform, methanol and water in the combined phases should be as c lose as possible to 8:4:3 (by volume) to prevent selective losses of some lipids to the aqueous phase and for reliable results (23). Feca l samples typically consist of about 60-80% of water and 20-4 0 % undigested fiber, unabsorbed protein, lipids and intestinal microflora (24). W e calculated the volumes of chloroform and methanol required to extract fecal lipid assuming that 100% of the fecal sample was aqueous; consequently, it is i n Chapter 4 Discussion and conclusions still possible that w e failed to extract some more polar lipids from the fecal samples. However, the chloroform-methanol extraction followed the extraction of triglyceride and unesterified fatty acids using hexane:diethylether:95% ethanol (1:1:1 by volume) and hexane:diethylether (1:1 by volume) should have resulted in the removal of most of the fecal lipid and may have altered the proportions of chloroform, methanol and water in the subsequent steps, any the error in assuming 100% aqueous material in the fecal samples would have been reduced. The use of freeze dried fecal samples in future studies would allow better control of the proportions of chloroform, methanol and water, and may further improve lipid extraction. In chapter 2 of this thesis research, we also reported the use of H P L C -E L S D for separation and quantification of fecal phospholipids. Quantification of fecal phospholipids by H P L C - E L S D showed a high and significant correlation with lipid soluble phosphorous, as determined by the phosphomolybdate colorimetric assay (A>0.75, P<0.01) (specific a im 2 of this thesis research). Standard methods for quantification of phospholipids are based on colorimetric assay of lipid soluble phosphorus (25). The later method is limited because it does not provide any information on the type of phospholipids, i.e. phospholipid c lasses present. A n advantage of the H P L C - E L S D method for fecal phospholipids quantitation is its ability to separate the phospholipids into individual phospholipid c lasses for detection and quantification. Additionally, the phosphomolybdate a s say relies on average convers ion factor of 25 to covert lipid-soluble phosphorus to phospholipid (26). This conversion factor of 25 is 112 Chapter 4 Discussion and conclusions based on the average molecular weight of a phospholipid (i.e. 750), divided by the approximate atomic weight of phosphorus (i.e. 30). This calculation thus introduces errors of varying magnitude in the analysis of biological samples because typically biological samples contain a mixture of phospholipids which differ in molecular weight, which may range from about 460 to 870, due to both differences in the polar head groups and in fatty acyl components (e.g. P C , l y s o P C , phosphatidylinositol, sphingomyelin. . .etc). In the research presented in this thesis, the amount of phospholipid quantified using the colorimetric assay was two to three fold higher (after multiplying the amount of phosphorus by the conversion factor of 25) than that determined by the H P L C - E L S D method, although the results of the 2 methods were highly correlated. The overestimation of phospholipid by colorimetric a s say could be explained by the large amount of l y s o P C , which represented about 30-4 0 % of total phospholipid in the fecal samples from children with and without C F in this study. The molecular weight of l y s o P C with 16:0 as the sole fatty acyl group is 475 , compared to 758 for P C with 16:0, 18:2n-6. The higher apparent lipid soluble phosphorus in the phosphomolybdate assay might also be due to presence of other phospholipids (e.g. l y s o P E , lysoPI, lyso P S ) in the fecal samples that were not identified on the H P L C chromatogram. In summary, chapter 2 of this thesis research has reported a simple method for extraction and quantification of phospholipids from fecal samples . This new method will be helpful in providing insights regarding the mechan i sms of lipid malabsorption in patients with pancreatic insufficiency. The method has 113 Chapter 4 Discussion and conclusions clinical relevance because increased P C excretion could lead to hepatic P C depletion, which would worsen bile synthesis and secretion, which in turn may worsen fat malabsorption. The publication of the results may also affect recent attempts to develop new synthetic pancreatic replacement enzymes that, unlike current formulations, do not contain phospholipase (Davidson A G F 2004, oral communication, 2 n d July). Pancreat ic phosphol ipase A 2 ( P L A 2 ) is essential for the hydrolysis of P C to l y s o P C and a free fatty acid for absorption. Al though P L A 2 is inactivated at low intraluminal pHs , which are common in patients with C F , P L A 2 from pancreatic enzyme supplements may be important for P L digestion in some patients with C F who are pancreatic insufficient but respond well to pancreatic enzyme replacement therapy in conjunction with acid reducing agents. It remains to be seen whether supplementation with water soluble choline to compensate for increased phospholipid excretion should be part of the nutrition management of patients with cystic fibrosis. 4.1.2 Fat absorption and phospholipid excretion Chapter 3 of this thesis descr ibes a research study that used the methodology for extraction and quantification of fecal phospholipid excretion described in chapter 2 to determine if children with C F excrete higher amounts of phospholipid than children without C F . T o the best of our knowledge, this research is the first quantitative demonstration of increased fecal choline phosphoglyceride ( P C and l y s o P C ) excretion in children with C F compared to Chapter 4 Discussion and conclusions children without C F . Further, these studies are the first to describe the amount and type of phospholipid excreted by humans. The digestion and absorption of P C requires hydrolysis by P L A 2 to release l y s o P C and an unesterified fatty acid (27). However, in patients with pancreatic insufficiency due to C F , the secretion of bicarbonate, P L A 2 , l ipase, col ipase and other digestive enzymes is decreased (27). Although pancreatic enzyme supplements are provided to patients with C F who are pancreatic insufficient, it is not known if the enzymes are sufficiently active in the intestine to hydrolyze dietary and biliary P C . It has been reported that the activity of pancreatic lipase is inhibited at an intraluminal pH below 5.8 (28). In addition, as d i scussed in chapter 1, P L A 2 appears to be more sensitive to inhibition at an acidic pH than pancreatic co-lipase dependent lipase (29). The postprandial intraluminal pH in the upper small intestine of many patients with C F is below 5.8 (7, 30). Of the dietary intake of the essential nutrient choline, more than 90% is in the form of P C , with the remaining smaller portion being as water soluble free choline and sphingomyelin, and minor amount of glycerophosphochol ine and phosphocholine (10 ,13) . Chol ine as P C is an important constituent of cell membranes , lipoproteins and lung surfactant, and is also the precursor for synthesis of sphingomyelin. Chol ine functions in the neurotransmitter acetylcholine. Cho l ine is a lso present in small amounts as other derivatives of choline phosphoglycerides including 1-alk-1-enyl-, 2-acyl-glycero-3-phosphocholine, a lso named plasmalogen, which is present in the heart muscle , seminal fluid, and in smaller amounts in nervous system, platelets or red blood 115 Chapter 4 Discussion and conclusions cells and 1-alkyl-,2-acetyl-glycero-3-phosphocholine, also known as platelet activating factor which is important in inflammatory and immune responses and in platelet aggregation. The metabolite of choline, betaine is also a source of labile methyl groups (31-33). Further, P C is an important component of bile, which is important for solubilizing biliary and dietary lipids; bile lipids are essential in the formation of mixed micelles to provide an increased surface area for digestion of lipids and fat-soluble vitamins. Hepatic steatosis, and a reduction of plasma P C , triglyceride and very-low-density lipoprotein ( V L D L ) concentrations are well-known features of choline deficiency, and has been reported in patients on long-term total parenteral nutrition (TPN) lacking choline (17, 34-36). The research descr ibed in this thesis was prompted by the high prevalence of hepatic steatosis among patients with C F and was directed at investigating the possibility that increased choline excretion may occur in C F (1,2). If so, this would raise the possibility that choline depletion may be important as a contributing factor in the development of hepatic steatosis in C F . The results of the study reported in chapter 3 showed that children with C F had a three fold higher total fat excretion, two fold higher total phospholipid excretion, a 14-fold higher P C excretion and a 2.5-fold higher l y s o P C excretion, with an overall fourfold higher total choline phosphoglyceride excretion than the healthy control children without C F (specific aim 3 of this thesis research). Consistent with literature, our results also showed that despite pancreatic enzyme replacement therapy, children with C F had a lower fat absorption (86%) than children without C F (94%) (specific aim 4 of this thesis research) (22, 37, 116 Chapter 4 Discussion and conclusions 38). Several explanations have been offered for the decreased fat absorption, despite pancreatic enzyme supplementation in CF patients. These include increased bile acid loss and/or decreased duodenal bile acid concentrations resulting in decreased micellar formation, reduced activities of co-lipase dependent pancreatic lipase, decreased duodenal pH and intestinal abnormalities leading to impaired uptake of nutrients (36, 39-43). Possible explanations for the higher phospholipid excretion in children with CF than in the healthy control without CF include a lower duodenal pH (42), which would impair PLA 2 activity and reduce the ability to digest dietary and biliary phospholipids to lysophospholipids and unesterified fatty acids for absorption. Alternatively, the higher phospholipid excretion in children with CF than in the healthy control children could be explained by intestinal abnormalities in CF that lead to impaired uptake of the digested lysoPC (40). The latter explanation of intestinal absorptive abnormality would provide a mechanism for the higher fecal lysoPC excretion found in children with CF than in the control children. At this point, very little is known about the mechanisms of lysoPC absorption, although it appears to be energy independent. To the best of our knowledge, our studies provide the first quantitative information of phospholipid excretion in humans. Whether or not increased excretion of choline phosphoglycerides occurs in other disorders involving impaired pancreatic, biliary, or intestinal absorptive function is not known. However, based on the results of our studies and the possible benefit of water soluble forms of choline as a source of this nutrient for patients with gastrointestinal disorder, future studies to determine if choline phosphoglycerides 117 Chapter 4 Discussion and conclusions occur in other patients with pancreatic and gastrointestinal disorders would seem worthwhile. The finding of higher amounts of lysoPC than PC in the fecal phospholipids excreted by both the control children and the children with CF in our study was unexpected, although we are unaware of similar data on the composition of fecal phospholipids in humans. Fecal lysoPC could originate from dietary and biliary PC hydrolyzed in the lower intestine, beyond the sites of lysoPC absorption in the small intestine from the activity of colonic microflora, or from slow dissolution of enteric pancreatic enzymes in the upper intestine due to low intraluminal pH caused by limited bicarbonate secretion from the pancreas, resulting in hydrolysis of lysoPC beyond the site of lysoPC absorption in the lower intestine, and thus increase excretion of lysoPC in children with CF. However, lysoPC was also the major choline phosphoglyceride excreted by healthy children without CF. This may suggest that the absorption of choline from PC is limited at the level of lysoPC absorption and not at PC hydrolysis. Lecithinase-positive bacteria are present in colonic microflora, and the abundance of these organisms has been reported to be sensitive to dietary variables (44, 45). Whether the nature of the colonic microflora influences the types and amounts of phosphoglycerides excreted or is of any physiological relevance to intestinal function or intraluminal pH is unknown. Of relevance, patients with CF are commonly treated with antibiotics due to repeated respiratory infections. The use of antibiotics is known to affect bacterial flora of 118 Chapter 4 Discussion and conclusions the gastrointestinal tract (46); because of this, it is reasonable to speculate that the colonic microflora in patients with CF may be different than those without CF. In chapter 3 of this thesis research, we have shown that children with CF had a mean fat intake of 97g/d and the control children had a mean fat intake of 70 g/d. The amount of phospholipids in typical diets is not known; some authors have suggested that phospholipid represent 4-8% of dietary fat (10). Based on our studies, the intake of PC by the children with CF was about 870mg/d. Assuming PC represent 90% of dietary PL, then the children with CF in our study consumed about 970mg of PL per day and the control children consumed about 900mg PL/d. Phospholipid excretion (mean) was 139mg/d, representing 14% of intake and 66mg/d, representing 7% intake in the children with CF and the control children respectively and thus the efficiency for absorption for total PL seems to be the same as for total fat. Whether or not this compromises PC status is not known. On the other hand, the estimated net absorption of PC appears to be similar in both the children with CF and the healthy control (Total PC intake - (fecal PC + fecal lysoPC); 865mg-80mg = 785mg and 812mg-20mg = 792mg, respectively). Some studies have provided evidence of increased membrane lipid turnover in CF defective cells, which could suggest that choline requirements are higher in patients with CF (47). Possibly, a chronic increase in fecal phospholipid excretion, and increased choline requirement could lead to lower free choline in the liver to support the CDP choline pathway in patients with CF. Further, we did find significant positive associations between fecal PC and lysoPC excretion and the plasma homocysteine and an inverse association with 119 Chapter 4 Discussion and conclusions Although statistical associations do not establish causal relations, studies in animals have shown that choline deficiency results in increased use of methyl groups from methionine for the synthesis of PC via the PEMT path (17, 18). Of note, dietary choline deficiency has been shown to have no effect on the phospholipid composition of lung surfactant in rats fed a choline-deficient diet compared to rats fed a choline-supplemented diet for eight days (48). Whether dietary choline deficiency affects bile synthesis does not appear to have been reported. The results of the studies in chapter 3 show that fecal fat excretion was positively associated with PC and lysoPC excretion (specific aim 6 of this thesis research), and that the excretion of total phospholipid, PC and lysoPC were higher in children with lower fat absorption (specific aim 7 of this thesis research). The significant positive correlation between fecal fat and phospholipid excretion, and the significant inverse correlation between fat absorption and phospholipid excretion may be explained by reduced biliary PC secretion, leading to poor micelle formation thus impairing triglyceride and phospholipid digestion and absorption. If correct, this suggestion implies that fat malabsorption in CF may potentially be corrected by improving intraluminal PC perhaps through choline or , PC supplementation. Alternatively, insufficient activity of both pancreatic co-lipase dependent lipase and of PLA 2 , or intestinal mucosal or unstirred water layer defects that reduced the absorption of the products of fat digestion could explain our results. 120 Chapter 4 Discussion and conclusions The positive correlation between fecal fat and phospholipid excretion, and the inverse correlation between fat absorption and phospholipid excretion raise the question if the higher phospholipid excretion in children with C F than in the control children is s imply explained by increased fat malabsorption. However, closer examination of the study data shows that the distribution of the phospholipid c lasses excreted by the children with C F was significantly different from that excreted by the healthy control children without C F (Figure 2.3). Thus, the phospholipid pool excreted by the children with C F had about 2 8 % and 4 1 % P C and l y s o P C respectively, compared to 4 % and 34% P C and l y s o P C , respectively in the control children. T h e s e data suggested that the higher phospholipid excretion in children with C F does involve a specific excretion of P C , and is not simply as a result of a higher choline phosphoglyceride excretion. Dietary phospholipid was estimated using the 2004 U S D A database for choline content of c o m m o n foods (Appendices D & E ) . P C absorption could not be quantified due to the invasiveness of the procedures. In our study, p la sma apo B was lower, but p lasma total and H D L cholesterol concentration were not different between the children with C F and the control children (specific a im 5 of this thesis research). A p o B w a s measured because apo B is present in V L D L , intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) , but not in high density lipoprotein ( H D L ) . Thus , the plasma apo B was used as an indicator of hepatic V L D L secretion (49), recognizing that some p lasma apo B is also present in chylomicrons of intestinal origin. The lower p lasma apo B in children with C F than in the control children is 121 Chapter 4 Discussion and conclusions consistent with reduced secretion of apo B containing lipoprotein from the liver. Further, the inverse associat ion found between p lasma apo B and fecal total phospholipid and P C are a lso consistent with hepatic choline depletion in C F (specific a im 8 of this thesis). However, an alternate explanation is that reduced fat absorption, and thus increased fecal fat excretion, results in reduced plasma apo B containing triglyceride rich lipoproteins. For practical purposes and because of ethical considerations, the blood samples in this study were not collected under fasting conditions of studying children. However , published data are available to show that, although p lasma triglyceride is affected by fed-fasting state, total p lasma cholesterol, H D L cholesterol and apo B concentrations are not affected (50). 4.1.3 Association between choline phosphoglyceride excretion, and plasma methionine, homocysteine and S A H W e recently published the first report to show that p la sma homocysteine and S A H are higher, and p lasma methionine and the proportion of P C in p lasma P L is lower in a large group of children with C F when compared with healthy control children without C F (16). The children who participated in the research described in this thesis were a subset of the children who participated in this study. Chapter 3 of this thesis provides new data to show significant positive associations between choline phosphoglyceride excretion and p lasma S A H and homocysteine, and an inverse relation between choline phosphoglyceride excretion and p lasma methionine (specific a im 9 of this thesis research). 122 Chapter 4 Discussion and conclusions Elevated homocysteine and SAH has been shown to be associated with increased risk of cardiovascular disease (51, 52). The clinical significance of elevated homocysteine and SAH in CF remains unclear. The concentrations of plasma homocysteine in adults with CF have not been documented, thus it is not known whether the elevated homocysteine found in children is relevant to later risk of adult cardiovascular disease. The mean life span of patients with CF in Canada is currently 37 years (Personal communication, Canadian Cystic Fibrosis Foundation, May 2003); thus it is possible that with continued improvements in CF care and a larger number of CF patients living beyond the 5 t h and 6 t h decade of life, cardiovascular disease may emerge among this patient population. Although type I diabetes affects 12% of patients with CF (53), type II diabetes, obesity, smoking and hypertension, which are also risk factors for cardiovascular disease are not frequently seen among the CF population. Several factors could explain an increase in plasma homocysteine concentrations including: a reduction in the expression of methionine synthase or betaine-homocysteine methyltransferase, which are enzymes that convert homocysteine to methionine, or in cystathionine [3 synthase, which is the first enzyme in the transulfuration pathway for homocysteine catabolism, deficiency of folate, vitamins B-|2 and B 6 and impaired renal function (54). Vitamin Bi 2 and folate levels in the children with CF and the healthy control children in our study were in the normal range (16). Vitamin B 6 status was not determined, but deficiency of this vitamin seems unlikely because the children with CF in our study were taking multivitamin supplements. Similarly, renal function was not 123 Chapter 4 Discussion and conclusions measured in our study participants but clinically significant kidney disease was not present. A literature search of Medline (1966-July 2004) and its associated databases did not reveal any published studies to suggest a defect in the enzymes involved in methionine-homocysteine metabolism which could explain the elevated homocysteine and SAH and decreased methionine in CF. In a recent study, Noga and colleagues have reported an approximately 50% lower plasma homocysteine concentration in PEMT -/- mice when compared with wild type mice (55), which suggests that the increased activity of PEMT resulting in increased methylation of PE to form PC, may be an important factor influencing plasma homocysteine, at least in mice. Further, during experimental choline deficiency, the hepatic concentration of SAH is increased and SAM and methionine are decreased (17, 18), which further suggests that PEMT activity may play an important role in regulating the concentration of methionine and its metabolites. Thus, the available evidence suggests that the decreased methionine and increased homocysteine and SAH concentrations in children with CF may be related to increased PEMT activity, and thus formation of PC from PE. This is consistent with the adaptive metabolic changes that can be expected secondary to increased fecal choline loss or increased choline requriement, and would explain the positive associations found between fecal PC and lysoPC excretion and plasma homocysteine, and the inverse associations with plasma methionine. In summary, the positive associations between choline phosphoglyceride excretion and plasma SAH and homocysteine, and the inverse relation between 1 2 4 Chapter 4 Discussion and conclusions choline phosphoglyceride excretion and p lasma methionine are consistent with altered hepatic choline metabolism with reduced synthesis of P C via the C D P pathway and increased P C synthesis via the P E M T path. It is possible that patients with C F have conditional hepatic choline deficiency due to chronic increased choline loss and increased choline requirement. Additionally, it is possible that the methionine synthase pathway alone cannot fulfill the total requirement for methionine synthesis when the betaine-homocysteine methyltransferase pathway for remethylation of homocysteine is limited by the availability of betaine. Betaine is an important methyl donor and is formed from the irreversible oxidation of choline. Data to support this suggest ion has not been published. However , it is possible that the positive associat ions between choline phosphoglyceride excretion and p lasma homocysteine and S A H , and inverse association with p lasma methionine may be explained by dec reased remethylation of homocysteine to methionine possibly involving the betaine-homocysteine methyltransferase pathway or the methionine synthase pathway. Future studies are needed to confirm the causal link between increased choline phosphoglycerides excretion and p lasma homocysteine, S A H and methionine levels and to determine whether or not increased P C synthesis via the transfer of methyl groups from S A M to S A H in the P E M T pathway is a determinant of plasma homocysteine in humans. 125 Chapter 4 Discussion and conclusions 4.2 Limitations This thesis research used a cross-sectional study design to determine the extent of phospholipid excretion, and its associat ion with p lasma methionine and homocysteine in children with C F when compared with healthy control children without C F . Posi t ive correlations between choline phosphoglyceride excretion and p lasma homocyste ine and S A H concentrations, and inverse correlation between choline phosphoglyceride excretion and p lasma methionine concentration were found. However, as statistical associat ions do not imply causation, a conclus ion of.a causa l link between choline phosphoglyceride excretion and the altered p lasma thiol status in C F cannot be drawn. The number of subjects included in this research was small and the subjects included children of a wide age range. Nevertheless, the sample s ize was more than twofold the sample s ize required to detect a significant difference in fecal lipid excretion between the patients with C F and healthy controls based on power analysis using published data on fecal lipid content with = 8 0 % and a = 0.05 (21). Publ ished data on fecal phospholipid excretion in humans was not available to allow the above power calculations. The children with C F were recruited using purposive sampl ing and the control group recruited w a s a convenience sample . It is possible that sampling bias existed, and this may limit the generalizability of the study results. Future multi-centre studies with a larger sample size, possibly involving adult patients with C F and C F patients with biopsy confirmed liver d i sease may increase the generalizability of the findings and specific re levance of the results to hepatic steatosis. 126 Chapter 4 Discussion and conclusions For practical purposes and because of ethical considerat ions, blood samples were not collected under fasting conditions or with a controlled time over the last meal . S ince all the blood samples were collected in the morning or early afternoon when the patients with C F attended the outpatient laboratory for blood sampling at a routine clinic appointment, it is likely that all of the subjects in our study had eaten within 4 hours of the blood sampling. However , published data is available to show that total p lasma cholesterol, H D L cholesterol and apo B concentration are minimally affected by fasting status (50). O n the other hand, fasting status and time s ince last meal may influence levels of homocysteine and research data has shown that homocysteine level is 4 -5% higher after a 6-h fast compared with those who have eaten within 1 to 6 hours in adults (56). Nevertheless, our results showed that plasma homocysteine values with a range of 7.08-12.32 uM in children with C F compared with 4.27-5.58 u M in the control children without C F . It s e e m s unlikely that the marked difference in p lasma homocysteine between patients with and without C F could be explained by differences in time s ince the last meal . Other limitations of this thesis research include the inability to quantify dietary plus biliary excret ion of phospholipid, thus phospholipid and P C absorption. Quantification of the amount of P C secreted into the intestine in bile would require invasive techniques. Liver biopsy was not performed in the study participants to confirm the presence of steatosis and to determine the hepatic P C and choline pool, a lso because of the need for invasive procedures. T h e s e were considered inappropriate for studies with children. 127 Chapter 4 Discussion and conclusions 4.3 Future Directions Chapter 2 of this thesis research describes the development of a new solvent system for the extraction of fecal phospholipids, and the validation of an H P L C - E L S D method for separation and quantification of fecal phospholipids. The new method may be applied in future studies to determine P C excretion in patients with n o n - C F related pancreatic insufficiency, or impaired biliary or intestinal absorption function. The results will be helpful in providing insights regarding the mechan i sm of lipid malabsorption in patients with fat malabsorption, as increased P C excretion could lead to hepatic P C depletion, which could worsen bile lipid synthesis and secretion, which in turn m a y worsen fat malabsorption. In chapter 3 of this thesis research, we have shown that children with C F excrete higher amounts of choline phosphoglycerides than healthy children without C F . W e further showed that excretion of choline phosphoglycer ides is positively associa ted with p lasma homocysteine and inversely associa ted with plasma methionine. The findings provide circumstantial evidence of altered hepatic choline metabol ism in children with C F . Additionally, it is not known if patients with C F have increased choline requirements. However , choline status was not measured directly in this study. In this regard, p l a sma choline does not appear to be a sensitive indicator of choline status. Thus , it will be helpful to analyze the choline and P C concentrations of the liver biopsy spec imens collected from patients with C F (where available) and in n o n - C F patients to confirm hepatic choline depletion in C F . Animal models may be useful to address 128 . Chapter 4 Discussion and conclusions this, for example, compar ison of the hepatic P C and choline pool in A508"'" mice and wild type mice. Recent studies have indicated elevated plasma homocysteine concentrations are associa ted with an increased risk of osteoporotic fractures in older persons, and it has been proposed that a homocysteine-associated disturbance in col lagen cross-linking in bone is involved (57, 58). Osteoporosis has been reported in 9% of adult patients with C F (53). Whether the increased homocysteine level is associa ted with a risk of cardiovascular d i sease in C F patients is also uncertain, but the clinical significance of elevated homocysteine and S A H in C F may become more important as lifespan improves. Future studies involving the use of stable isotopes are also needed to elucidate the etiology of the elevated homocysteine in patients with C F . Additionally, nutrition intervention studies involving choline, betaine and strategies to increase the intraluminal pH are warranted to determine if these supplements will correct the thiol abnormalities and improve choline status. Studies to evaluate the effect of supplementation on clinical endpoints, including improving life quality in patients with C F , will be warranted in future. The high amounts of l y s o P C excreted by the control children and the children with C F in our study were unexpected, and we are unaware of similar data on the composi t ion of fecal phospholipids. Future studies are needed to determine if l y s o P C rather than P C is the primary choline phospholipids excreted in feces. 129 Chapter 4 Discussion and conclusions Finally, data on dietary intake for phospholipid is limited because data on the content of phospholipid in common foods is not available. Future studies will be needed to provide better estimation of dietary phospholipids. 130 Chapter 4 Discussion and conclusions 4.4 Conclusions In conclusion, this was a cross-sectional study aimed at determining the extent of phospholipid excretion in children with CF compared with healthy controls without CF, and the association of phospholipid excretion with plasma methionine and homocysteine. Chapter 2 of this thesis research describes the development of a new solvent system for the extraction of fecal phospholipids, and validated an HPLC-ELSD method for separation and quantification of phospholipids from fecal samples. The method described could be used in future studies to determine if increased phospholipid excretion is present in patients with non-CF related pancreatic insufficiency, or impaired biliary or intestinal absorptive function. Such research would be helpful for the development/fine-tuning of strategies designed to correct malabsorption. Chapter 3 of this thesis research applied the methodology for quantification of fecal phospholipid excretion described in chapter 2 to determine if children with CF have higher phospholipid excretion than children without CF. Our studies have shown that children with CF excrete higher amounts of choline phosphoglycerides than healthy children without CF. The excretion of choline phosphoglycerides is positively associated with plasma homocysteine and inversely associated with plasma methionine. These novel findings provide evidence to suggest altered hepatic choline metabolism in children with CF. Our work also provides new information to support an important functional interdependence between phospholipid metabolism and the methionine-homocysteine cycle in humans. We postulate that altered phospholipid 131 Chapter 4 Discussion and conclusions m e t a b o l i s m c o u l d b e r e l evan t to s o m e of the c o m p l i c a t i o n s a s s o c i a t e d w i th C F , s u c h a s h e p a t i c s t e a t o s i s . F u r t h e r s t u d i e s a r e w a r r a n t e d to a d d r e s s t he i m p o r t a n c e o f t he P E M T c y c l e in P C m e t a b o l i s m in h u m a n s a n d t h e in te r - re la t ion o f th is p a t h w a y w i th p l a s m a h o m o c y s t e i n e . T h e c l i n i ca l i m p l i c a t i o n s of e l e v a t e d h o m o c y s t e i n e in p a t i e n t s w i th C F r e m a i n s to b e d e t e r m i n e d a s i n c r e a s e d h o m o c y s t e i n e l eve l m a y b e m o r e impor tan t in C F a s pa t i en t ' s l ife e x p e c t a n c y c o n t i n u e s to i n c r e a s e . 132 Chapter 4 Discussion and conclusions 4.5 References 1. C o l o m b o C , Battezzati P , S t azzabosco M , P o d d a M . Liver and biliary problems in cystic fibrosis. S e m Liver Disease 1998;18:227-235. 2. Potter C J , Fisbein M , H a m m o n d S, M c C o y K, Qua lman S. C a n the histologic changes of cystic f ibrosis-associa ted hepatobiliary d i sease be predicted by clinical criteria? J Pediatr Gastroenterol Nutr 1997;25:32-6. 3. Buchman A L , Ament M E , Sohe l M , et al . Chol ine deficiency causes reversible hepatic abnormalities in patients receiving parenteral nutrition: proof of a human choline requirement: a placebo-controlled trial. 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Nutr R e v 1994;52:327-39. 32. Leray C [homepage on the Internet]. Cybe r Lipid Centre, [updated 2004 A u g 10; cited 2004 A u g 11]. Avai lable from: http ://www. cybe rl ipid. org/ph I ip/pgly03. htm 33. Meyer K C , S h a r m a A , Brown R, et a l . Function and composi t ion of pulmonary surfactant and surfactant-derived fatty acid profiles are altered in in young adults with cystic fibrosis. Ches t 2000; 118:164-74. 136 Chapter 4 Discussion and conclusions 34. C h a w l a R K , Wolf D C , Kutner M H , Bonkovsky H L . Chol ine may be an essential nutrient in malnourished patients with cirrhosis. Gastroenterology 1989;97:1514-20. 35 . Y a o Z , V a n c e D E . Reduct ion in V L D L , but not H D L , in p lasma of rats deficient in choline. B i o c h e m Ce l l Biol 1990;68:552-8. 36. Buchman A L , Dubin M D , Moukarze l A A , et al . Chol ine deficiency: a cause of hepatic steatosis during parenteral nutrition that can be reversed with intravenous choline supplementation. Hepatology 1995;22:1399-403. 37 . Walters M P , Littlewood J M . Feca l bile acid and dietary residue excretion in cystic fibrosis: age group variations. J Pediatr Gastroenterol Nutr 1998;27:296-300. 38. Burdge G C , G o o d a l e A J , Hill C M , et a l . P l a s m a lipid concentrations in children with cystic fibrosis: the value of a high-fat diet and pancreatic supplementation. Br J Nutr 1994;71:959-64. 39. Francisco M P , W a g n e r M H , She rman J M , Theriaque D, Bowse r E , Novak D A . Ranitidine and omeprazole as adjuvant therapy to pancrel ipase to improve fat absorption in patients with cystic fibrosis. J Pediatr Gastroenterol Nutr 2002;35:79-83. 40 . Kalivianakis M , Minich D M , Bijleveld C M , et al . Fat malabsorption in cystic, fibrosis patients receiving enzyme replacement therapy is due to impaired intestinal uptake of long-chain fatty acids . A m J Cl in Nutr 1999;69:127-34. 137 ; Chapter 4 Discussion and conclusions 41 .Dav idson A G F . Gastrointestinal and pancreatic d i sease in cystic fibrosis. 2 n d ed . In: Hodson M E , G e d d e s D M , eds. Cys t ic fibrosis. London: C h a p m a n & Hall , 2000:384-95. 42 . Gregory P C . Gastrointestinal p H , motility/transit time and permeability in cystic fibrosis. J Pediatr Gastroenterol Nutr 1996;23:513-23. 43 . Rob inson P J , Smith A L , S ly P D . Duodenal p H in cystic fibrosis and its relationship to fat malabsorption. Dig Dis S c i 1990;35:1299-304. 44. Benno Y , Mizu tam T, N a m b a Y , Komori T, Mitsuoka T. Compar i son of fecal microflora pf elderly persons in rural and urban areas of Japan . A p p l Environ Microbiol 1989;55:1100-5. 45 . Benno Y , S a w a d a K, Mi tsuoka T. The intestinal microflora of infants: composi t ion of fecal flora in breast-fed and bottle-fed infants. Microbiol Immunol 1984;28:975-86. 46. Levy J . The effects of antibiotic use on gastrointestinal function. A m J Gastroenterol 2000;95:S8-10. 47 . Ulane M M , Butler J D , Peri A , Miele L, Ulane R E , Hubbard V S . Cys t ic fibrosis and phosphatidylcholine biosynthesis. Cl in C h i m A c t a 1994;230:109-16. 48 . M c M a h o n K E , Farrell P M . Effect of choline deficiency on lung phospholipid concentrations in the rat. J Nutr 1986; 116:936-43. 49 . Durrington P N . C a n measurement of apolipoprotein B replace the lipid profile in the follow-up of patients with lipoprotein disorders? C l in C h e m 2002;48:401-2. 138 Chapter 4 Discussion and conclusions 50. Reinhart R A , G a n i K, Arndt M R , Broste S K . Apoliproteins A - l and B as predictors of angiographically defined coronary artery d isease . A r c h Intern M e d 1990;150:1629-33. 5 1 . D e Bree A , Verschuren W M , Kromhout D, Kluijtmans L A , B l o m H J . Homocys te ine determinants and the evidence to what extent homocysteine determines the risk of coronary heart d isease . Pharmacol R e v 2002;54:599-618. 52 . Kerins D M , Koury M J , Capdev i l a A , R a n a S, Wagne r C . P l a s m a S-adenosylhomocyste ine is a more sensitive indicator of cardiovascular d i sease than p l a sma homocysteine. A m J C l in Nutr 2001;74:723-9. 53 . Cyst ic Fibrosis Foundat ion. Cyst ic Fibrosis Foundation Patient Registrty Annua l Data Report. Bethesda , M D : Cyst ic Fibrosis Foundation, 2003. 54. Garcfa-Tevijano E R , Berasa in C , Rodr iguez J A , et al . Hyperhomocyste inemia in liver cirrhosis: mechan i sms and role in vascular and hepatic fibrosis. Hypertension 2001;38:1217-21. 55 . N o g a A A , S tead L M , Z h a o Y , Brosnan M E , Brosnan JT , V a n c e D E . P l a s m a homocyste ine is regulated by phospholipid methylation. J Biol C h e m 2003;278:5952-5. 56 . Nurk E , Tel l G S , Nygard O , Refsum H , Ueland P M , Vollset S E . P l a s m a total homocysteine is influenced by prandial status in humans: the Hordaland homocyste ine study. J Nutr 2001;131:1214-6. 57. van Meurs J B J , Dhonukshe-Rut ten R A M , Pluijm S M F , et a l . Homocyste ine levels and the risk of osteoporotic fracture. N Engl J M e d 2004;350:2033-41. Chapter 4 Discussion and conclusions 58. McLean RR, Jacques PF, Selhub J, et al. Homocysteine as a predictive factor for hip fracture in older persons. N Engl J Med 2004;350:2042-9. 140 A P P E N D I X A I N F O R M E D C O N S E N T 3. T o measure p l a sma folate, vitamin B-12 and homocyste ine levels in chi ldren with C F and compare them to children without C F . Folate a n d vi tamin B-12 are B vi tamins and homocys te ine is a blood marker of these vi tamins . 4. To measure dietary intake of energy and all essential nutrients from a 3 to 5-day food record. 5. T o determine the extent of fat malabsorption by analysis of 3-day stool samples . W h o C a n P a r t i c i p a t e A l l patients with C F w h o are scheduled for regular blood work. S t u d y P r o c e d u r e s If I agree to let my child participate, an additional two tubes of b lood (14ml_) will be taken. M y child 's height, weight as well as my child's current e n z y m e therapy and regimen will be obta ined from his/her medical record. Th i s study will not involve an extra needle. I will be asked if I am willing to keep a record of the foods that my child eats for 3-5 days, and if my child will collect s tool s amp le s over 3 days. R i s k s There are no known r isks to participating in this study. A certified technologist , nurse, or other qualified person will draw the small amount of b lood . Minor discomfort and s o m e temporary discoloration may occur at the site of b lood draw. Bene f i t s Deficiencies that may be corrected through supplements or c h a n g e s in diet may be identified in this study. I will be provided with a copy of my chi ld ' s results when they are avai lable. If I agree to keep a record of my child's food intake, I will be provided with a copy of his/her nutrient intakes when the results are ready. C o n f i d e n t i a l i t y A n y information resulting from this research study will be kept confidential . A l l documents will be identified by code number and kept in a locked filing cabinet . M y child will not be identified by name in any reports of the comple t ed study. C o n s e n t The objectives and procedures of the study have been expla ined to me to my satisfaction a n d I unders tand that my child may withdraw from the s tudy at any time. I have the right to refuse my child's participation or voluntari ly wi thdraw from the s tudy without consequence to continuing medica l care . M y chi ld 's n a m e will be treated confidentially by use of code numbers during the s tudy and will not be mentioned in any report or publications of the study results. I unders tand that I a m to receive a copy of this consent form. I understand that if I have any questions or desi re further information, I should contact Dr S h e i l a Innis or one of Page 2 of 3 C F Version: [CF/P/ Jan 30, 2000] 3. T o measure p l a s m a folate, v i tamin B-12 and homocysteine levels in children with C F and compare them to chi ldren without C F . Folate and vitamin B-12 are B vitamins and homocys te ine is a blood marker of these vitamins. 4. T o measure dietary intake of energy and all essential nutrients from a 3 to 5-day food record. 5. T o determine the extent of fat malabsorpt ion by analysis of 3-day stool samples . W h o C a n P a r t i c i p a t e Al l patients with C F who are schedu led for regular blood work. S t u d y P r o c e d u r e s If I agree to participate, an addit ional two tubes of blood (14mL) will be taken. M y height, weight as well as my current enzyme therapy and regimen will be obtained from my medica l record. Th i s study will not involve an extra needle. I will be asked if I a m willing to keep a record of the foods that I eat for 3-5 days, and if I will collect s tool samples over 3 days . R i s k s There are no known risks to participating in this study. A certified technologist, nurse, or other qualified person will draw the small amount of blood. Minor discomfort and some temporary discolorat ion may occur at the site of blood draw. Bene f i t s Deficiencies that may be corrected through supplements or changes in diet may be identified in this study. I will be provided with a copy of my results when they are available. If I agree to keep a record of my food intake, I will be provided with a copy of my nutrient intakes w h e n the results are ready. C o n f i d e n t i a l i t y A n y information resulting from this research study will be kept confidential. A l l documents will be identified by c o d e number and kept in a locked filing cabinet. I will not be identified by name in any reports of the completed study. C o n s e n t The objectives and procedures of the study have been expla ined to me to my satisfaction and I understand that I may withdraw from the study at any time. I have the right to refuse to participate or voluntarily withdraw from the study without consequence to continuing medica l care. M y name will be treated confidentially by use of code numbers during the study and will not be ment ioned in any report or publications of the study results. I understand that I a m to rece ive a copy of this consent form. I understand that if I have any quest ions or desi re further information, I should contact Dr She i l a Innis or one of her assoc ia tes at 875-2434 or Dr G e o r g e Dav idson at 875-2142. If I have any concerns about my CF Version: [CF/C/ Jan 30, 2000] Page 2 of 3 treatment or rights as a research subject, I may telephone Dr R . D . Sprately, Director of R e s e a r c h Serv ices at 822-8598. I have received a copy of the consent form for my own records. I vo lun ta r i l y g i v e c o n s e n t to p a r t i c i p a t e in the (Please print) s t u d y en t i t led A S t u d y of B i o c h e m i c a l Nutrit ional Parameters in C h i l d r e n with C y s t i c F i b r o s i s . S i g n e d D a t e W i t n e s s D a t e I nves t i ga to r ' s S i g n a t u r e D a t e CF Version: [CF/C/ Jan 30, 2000] Page 3 of 3 2. T o measure fatty acids levels in phosphol ipid , triglyceride, and cholesterol ester in the blood of individuals with C F and compare them to individuals without C F . 3. T o measure p lasma folate, vitamin B-12 and homocyste ine levels in children with C F and compare them to children without C F . Folate and vitamin B-12 are B vitamins and homocyste ine is a blood marker of these vitamins. 4. T o measure dietary intake of energy and all essent ial nutrients from a 3 to 5-day food record. 5. T o determine the extent of fat malabsorpt ion by analysis of 3-day stool samples . Who Can Participate A l l patients without C F , pulmonary, hepatic, gastrointestinal or inflammatory d i seases and metabolic d i seases likely to result in alterations of lipids, amino acids, folate, vitamin B-12 metabol ism, who are not taking drugs that affect fat or vitamin absorption, and are schedu led for blood-work can act as controls. S t u d y P r o c e d u r e s If I agree to let my child participate, an additional two tubes of blood (14mL) will be taken. M y child's height, weight as wel l as my child's current e n z y m e therapy and regimen will be obtained from his/her medical record. This study will not involve an extra needle. I will be a sked if I am willing to keep a record of the foods that my child eats for 3-5 days , and if my child will collect stool samples over 3 days. Risks There are no known risks to participating in this study. A certified technologist, nurse, or other qualified person will draw the small amount of b lood. Minor discomfort and some temporary discolorat ion may occur at the site of blood draw. Benefits Deficiencies that may be corrected through supplements or changes in diet may be identified in this study. I will be provided with a copy of my chi ld 's results when they are available. If I agree to keep a record of my child's food intake, I will be provided with a copy of his/her nutrient intakes when the results are ready. Confidentiality A n y information resulting from this research study will be kept confidential. A l l documents will be identified by code number and kept in a locked filing cabinet. M y child will not be identified by name in any reports of the comple ted study. Consent The objectives and procedures of the study have been explained to me to my satisfaction and I understand that my child may withdraw from the study at any Page 2 of 3 C F Version: [Controls/P/ Jan 30, 2000] 2. T o measure fatty ac ids levels in phospholipid, triglyceride, a n d cholesterol ester in the blood of individuals with C F and compare t h e m f o individuals without C F . 3. T o measure p lasma folate, vitamin B-12 and homocys te ine levels in children with C F and compare them to children without C F . Folate and vitamin B-12 are B vitamins and homocyste ine is a blood marker of these vitamins. 4. T o measure dietary intake of energy and all essent ia l nutrients from a 3 to 5-day food record. 5. T o determine the extent of fat malabsorption by analys is of 3-day stool samples . Who Can Participate A l l patients without C F , pulmonary, hepatic, gastrointestinal or inflammatory d i seases and metabolic d i seases likely to result in alterations of lipids, amino acids , folate, vitamin B-12 metabol ism, who are not taking drugs that affect fat or vitamin absorption, and are scheduled for blood-work c a n act as controls. S t u d y P r o c e d u r e s If I agree to participate, an additional two tubes of blood (14mL) will be taken. M y height, weight as well as my current enzyme therapy and regimen will be obtained from my medica l record. This study will not involve an extra needle. I will be a s k e d if I am willing to keep a record of the foods that I eat for 3-5 days, and if I will collect stool samples over 3 days. Risks There are no known risks to participating in this study. A certified technologist, nurse, or other qualified person will draw the smal l amount of blood. Minor discomfort and s o m e temporary discoloration may occur at the site of b lood draw. Benefits Deficiencies that may be corrected through supplements or changes in diet may b e identified in this study. I will be provided with a copy of my results w h e n they are avai lable . If I agree to keep a record of my food intake, I wil l be provided with a copy of my nutrient intakes when the results are ready. Confidentiality A n y information resulting from this research study will be kept confidential. A l l documen t s will be identified by code number and kept in a locked filing cabinet. I will not be identified by name in any reports of the comple ted study. Consent T h e objectives and procedures of the study have been exp la ined to me to my satisfaction and I understand that I may withdraw from the study at any time. I have the right to refuse to participate or voluntarily withdraw from the study Page 2 of 3 C F Version: [Controls/C/ Jan 30, 2000] APPENDIX B FOOD RECORD E B o o iz c _ CO c to CO (0 <U Q-TJ ro _ _ .5 o </) V _ c ._ O P (D O 1!» 2 _ 2 E — _, Q) O CM 0. UJ C0 _. • a 0) "5 o to CO _ o to co 9. 0 o 5, <D o co to £ 45 -~ _ ro ca 1 s > J3 c\i CO E o _ CO _. c <D ro _ _^  tf) -p ro £ "§1 £ _ £ o _ o co o _ _ > in 00 co HI 5 15 i _ _ Q Q _ 8 5 . 8 | H < y UJ Q. CQ Q. a. => o w to o Q < 0. 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Q-CD E i -OT CO £ % 0 Z. 0 fe to n. to 1 5 5 ' ' to t3 S ra | n o to c tu H I o 0 to CO CD o .2 >. o c CO 0 CD CO CN fe 0. A8 UJ n ST TJ c to CO TJ o Q CD x: • ~ "to CD XI b CO Q 0 O t - JS Q_ 0-LU oO b> g • o z • CD > O x> 0 to CO E CD to CO JD CO TJ C o TJ => cr a. to CD C E CD C to 0 •o b • o z • 0 o E CO 0 TJ c to 0 E to c TJ c to XI 0 CL o a . CL CO 0 JZ E 0 Q. c^  0 Q. 0 E 0 TJ 0 E c o c fe x: c CO "TJ b • • >-c 0 E 0 C l C l 3 c 3 o E ra 0 ±3 CO ±3 TJ C CO 0 E CD C TJ C to L-XI 0 £ Si £ 0 x: CO 0 CL CO 0 3 O — 0 CD CD CO CO CO <o J9> APPENDIX C CHARACTERISTICS OF PARTICIPANTS WITH CYSTIC FIBROSIS 1 7 1 Cl 3 2 OJ CO 'co 2 .Q M— O CO o CD CO -I—» c co D. 'o •c 03 CL CO O '•4—> CO -1_ CD •+-" o CO L _ CO o ns _o> .Q ns .S> E •2 </> N C i n ^ i n o i c N i t C B r o r f o ^ i n i n c n o o j n t D c o c v i c N i o i c n c v i c r o ^ ^ c N i c D c d r o ^ ^ ^ ^ ^ ^ C N i c N i N - c o c N t N c o c o i n L n ^ i n CD 00 00 00 O) CD O T - c o i O T - c D c n c N O - r - o ^ t ^ r ' T * ^ - t O C M C N C D r O t N N - L O C O S C D N ' C D o c LU CD a >» o c a> CD L U O CD TJ C a> .5 'E u * l CO 00 oo 00 oo 00 00 ECS ECS o ECS ECS ECS ECS tazym tazym Creor tazym azym azym azym Col Col Col Col Col Col 3 ° LO CN 00 O LD LO LO LL I] o < co co o o o LO LO \Ci < < u_ < CD CO CO CD L_ o c co Q_ 00 00 00 CO oo o o o o o LO LO LO LO LO LL LL LL LL LL < < < < < CO CO CO CO CO o o o o o LO LO LO LO LO IL LL 11 LL LL < < o CM CO O LU E >^  N CO o ° oo CO o LU E >> N CO O OJ co O LU E >^  N CO +-* o O oo ^ CO h-o LU E N £ TO o CD CO CO o o CM CN CO CO O O O CM LU LU Q) O CJ c CO CL E E >^  >^  N N CO CO o o O O —) CO CO CO O LU E >^  N CO o O o CM CO CJ LU E >^  N CO -*-» o O CO CO 00 CO CO o o o o o LO LO LO LO IO LL LL LL LL LL < < < < < CO 00 00 CO CO o o o o o LO LO LO LO LO < < < ] < ] < ] < ] < ] < LL LL o JUL C rs oo o LO LL < CO o LO CO o LO LL < o c c 00 o LO CO 00 o o LO LO LL LL < < CO 00 o o LO LO LL < < 00 CO CD CM OO 00 CD N- 00 00 00 Q J O O C D N - O C O C D T I - C D C O C O C M C M co oo TJ- 00 o C M or Q o CD 00 CD oo LO Tt AS or TD DK CD CD Tt CO LO CO CO < CD s m c o LO LO T— ^ LU Q ^ 2 CD < CD CM co ^ C M r o ^ m ( O N c o o ) ° T " N ( 0 , 1 ' i n ( D S ( » sz 0) z* c o ^ t c q c N O s o s CM CM co ^ m • - ' { = T - CM CN ID .O, CD •o c d> < 1. .15 E u # ° to CM O T - CD CD t to in CD CO CM IO S co co cs O O CM O LO LO LO IS-(N co m co K oo APPENDIX D USDA D A T A B S E FOR THE CHOLINE CONTENT OF COMMON FOODS [database on the Internet] Howe JC, JR Williams JR, Holden JM: US Department of Agriculture, [cited 2004 Aug 10]. Available from: http://www.nal.usda.gov/fnic/foodcomp/Data/Choline/Choline.pdf 174 o o JC IO CD o 2 1 at o a. 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OJ CO TJ ZJ OJ Ci) 13 TJ O ° TJ if I 8 g OJ 1 0 6 ZJ CO o c .•= ' to cu ^ tz ¥" >< tu E P 8, E D) o .£ CD J Z C Q - x : CO a. TJ CO £ "o ro c ro J? » o Ji 2 o I i " c5 ro "5 a — to 5 x 15 ^ a. co .£ 0- .5 - o CL C/) O - CL o 2 CL 0) OJ O tz -= CD O c JZ = o o O JZ in tu CD LL •o c > OJ _ o o E >< ZJ O "> . CD JZ c o CD rz o JZ o ro o in a> c i CL co tu T3 o o CD o c CD TJ c o a rz ro CD E c tu > CO ro M— O >. ro CL ^ ro to * CD JD E cz CD a OJ — 3 fz ~ cu CD JZ TJ LZ ro ro 15 TJ o >> —^* ro ZJ CT CU > ro tu o ro u TJ rz tu £ £ tu tu > CD CD JZ JZ L_ < (- O U . h *— IZ CM r> i? x . o o tu ro E c ZJ ro to .. CD a> £ E c O tu y •§ g Biz APPENDIX E ESTIMATED A V E R A G E CHOLINE INTAKE OF STUDY PARTICIPANTS ) 192 In t roduc t ion In M a r c h 2004, the U S D A has made available the choline content of about 400 common foods on the internet. Hence, the average choline and phosphatidylcholine ( P C ) intakes of children with C F and healthy children without C F , for w h o m food records were obtained, were estimated us ing data from A p p e n d i x D of this thesis. The adequate intake (Al) of choline for children 1-3 years, 4-8 years, 9-13 years is 200mg/d, 250mg/d and 375mg/d, respectively. The A l for boys and girls 14-18 years is 550 mg/d and 400mg/d , respectively. M e t h o d Briefly, data from Appendix D were entered into Microsoft® Exce l 2002 for Windows to form a choline database. The weight of the individual food items from the food records were entered into the same database to allow calculation of mean P C and total choline intakes. R e s u l t s The mean chol ine and phosphatidylcholine ( P C ) intakes of children with C F and healthy children without C F is summarized in the T a b l e below. P C and total choline intakes were not significantly different between children with C F and the controls. 5 0 % (8/16) of children with C F and 12 .5% (1/8) of control children had the choline intake at or above the A l for age and gender. 193 Discussion The U S D A choline database was first published in M a r c h 2004, which contained about 400 c o m m o n foods to provide researchers and consumers means to estimate choline intake from common foods. A n d our estimation showed that 5 0 % of children with C F and 12.5% of children without C F had an intake that met or above the A l . However, it is possible that the results were an underestimation of the actual choline intake as some c o m m o n foods consumed by our study participants were not included in the database, including margarine, chocolate syrup drink mix, hot chocolate, honey, waffle, puff pastry and pie shells. Further, all of the nutrition supplements, such as T o l e r e x ® , P e d i a s u r e ® and V i v o n e x ® were not included in the database. Examinat ion of the ingredient list of the above products suggested that they contained P C and choline. Although the amount of total choline w a s shown on the product label, the P C content of the formula was unknown. Thus children who were on tube feed and/or with >25% energy intake from nutrition supplements were excluded from the analysis. 194 T a b l e M e a n choline and phosphatidylcholine (PC) intakes of children with C F and healthy children without C F m g / d C F (n=16 1) C o n t r o l (n=8) P v a l u e Chol ine from P C 123.5±16.0 116.0+22.5 0.8 P C 2 864.5±112 812±158 0.8 Total C h o l i n e 3 335.3±32.9 268.U25.4 0.2 ^=16 in the C F group because 2 participants were on tube feed in addition to oral intake with >25% calories from nutrition supplements and were removed from the statistical analysis . 2 T h e amount of P C w a s calculated base on the assumption that the molecular weight of P C equals 800 and the molecular weight of 113 for choline. 3 Tota l choline refers to the sum of free choline, glycerophosphocholine, phosphocholine, Phosphat idylchol ine and sphingomyelin. 1 9 5 

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