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

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PHOSPHOLIPID E X C R E T I O N AND M E T A B O L I S M A N D THIOL S T A T U S IN C Y S T I C FIBROSIS By  Alice Ho-Wing C h e n  B . S c . (Dietetics) University of British C o l u m b i a , 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 FOR THE DEGREE OF M a s t e r of S c i e n c e In THE FACULTY OF GRADUATE STUDIES ( H u m a n Nutrition)  W e a c c e p t this thesis as conforming to the required standard  T H E UNIVERSITY O F BRITISH C O L U M B I A S e p t e m b e r 2004 ©Alice C h e n , 2004  M a n u s c r i p t t i t l e : A s s e s s m e n t of phospholipid malabsorption by quantification of fecal phospholipid  Statement of co-authorship A C participated in enrollment of subjects, collection of s a m p l e s , experiment d e s i g n , fecal analysis, data analysis a n d interpretation. SI contributed a s the principal investigator in grant funding, study c o n c e p t a n d d e s i g n , d a t a analysis and interpretation.  Print name: Date:  /QR. /  .<ZM&J-A  Ocjt ^ao^f-  /A/A//<  Print n a m e : Date:  M\ce. Jf. f)r*  CJ\f>n Jnn^  M a n u s c r i p t title: P h o s p h a t i d y l c h o l i n e and lysophosphatidylcholine excretion is increased a n d related to altered p l a s m a homocysteine a n d methionine in children with cystic fibrosis  Statement of co-authorship A C participated in enrollment of subjects, collection of s a m p l e s a n d in fecal and p l a s m a a n a l y s e s , data analysis and interpretation. SI contributed as the principal investigator in grant funding, in study c o n c e p t a n d d e s i g n , 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 a n a l y s e s of p l a s m a thiols.  Print n a m e : ? ) / ? . SHP.iLfl Date:  jl  OcJr  Jhuk  MNl<>  ftticP.  Print name: Date:  ~4  Ort  C.keh D^f)(f  Library Authorization  In p r e s e n t i n g t h i s 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 of t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e at t h e U n i v e r s i t y of B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e it f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t 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 t h i s 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 b y t h e h e a d of m y d e p a r t m e n t o r b y h i s or h e r r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n of t h i s t h e s i s for f i n a n c i a l g a i n s h a l l not b e a l l o w e d w i t h o u t my written p e r m i s s i o n .  N a m e of A u t h o r (please  Degree:  Date  print)  _J^4 _j2i_Sck^C^ cr  n _ ALW^bn  D e p a r t m e n t of 4kohv2 T h e U n i v e r s i t y of B r i t i s h C o l u m b i a Vancouver, B C  Canada  (dd/mm/yyyy)  Year:  ABSTRACT C h o l i n e is a n essential nutrient a n d is present in the diet predominantly (>90%) in p h o s p h a t i d y l c h o l i n e ( P C ) . L a r g e a m o u n t s of P C are a l s o s e c r e t e d into the intestine in bile. P C digestion a n d absorption requires p a n c r e a t i c p h o s p h o l i p a s e A ( P L A ) , w h i c h h y d r o l y z e s P C to l y s o P C a n d fatty a c i d . P L A is 2  2  2  inhibited at low intraluminal p H , w h i c h is c o m m o n in patients with cystic fibrosis ( C F ) . C h o l i n e deficiency results in hepatic steatosis, e x p l a i n e d by the n e e d for P C synthesis via the cytidine d i p h o s p h o c h o l i n e pathway for secretion of triglyceride in very-low-density lipoprotein. In choline deficiency, the liver i n c r e a s e s s y n t h e s i s of P C v i a the phosphatidylethanolamine N-methylfransferase pathway in w h i c h methyl groups from methionine are transferred to phosphatidylethanolamine, forming P C a n d S - a d e n o s y l h o m o c y s t e i n e , which is then m e t a b o l i z e d to h o m o c y s t e i n e . Hepatic steatosis is c o m m o n in patients with C F , but whether patients with C F m a l a b s o r b P C , w h i c h is a p h o s p h o l i p i d ( P L ) , is unknown. Current m e t h o d s for fecal fat extraction extract triglyceride a n d fatty acid but do not quantitatively recover P L . T h i s study a i m e d to establish a method for quantification of P L , P C a n d l y s o P C excretion a n d to d e t e r m i n e P L , P C a n d l y s o P C excretion in children with C F a n d their a s s o c i a t i o n s with p l a s m a thiols. T h i s is a c r o s s - s e c t i o n a l study of children with C F (n=18) a n d children without C F (n=8, control). All participants provided a v e n o u s blood s a m p l e , a 7 2 h fecal s a m p l e a n d 5 d food record. F e c a l total fat and P L w e r e r e c o v e r e d by sequential extraction with ethanol, ether, h e x a n e , chloroform a n d m e t h a n o l a n d w e r e separated a n d quantified using high performance liquid c h r o m a t o g r a p h y ( H P L C )  with evaporative light scattering detector. P l a s m a thiols a n d P L w e r e a n a l y z e d using H P L C . C o m p a r e d with the controls, children with C F h a d significantly lower fat absorption ( m e a n ± S E M ) ( 8 6 ± 1.6%, 94+1.2%), a n d higher excretion of fat (12.9±1.7g/d, 3.9±0.7g/d), P L (139±20mg/d, 66±18mg/d), P C (39±11mg/d, 2 . 6 ± 1 . 0 m g / d ) a n d l y s o P C (57+8.1mg/d, 2 2 ± 6 . 2 m g / d ) , respectively. F e c a l P L excretion w a s significantly related to p l a s m a h o m o c y s t e i n e (r=0.64) a n d methionine (r=-0.49). P L , P C a n d l y s o P C excretion is higher in children with C F and is a s s o c i a t e d with lower p l a s m a methionine a n d higher h o m o c y s t e i n e . Altered choline m e t a b o l i s m may o c c u r in C F a n d m a y be important to the hepatic complications.  in  TABLE OF CONTENTS Abstract T a b l e of C o n t e n t s List of T a b l e s List of F i g u r e s List o f A b b r e v i a t i o n s Acknowledgements Chapter 1  Chapter 2  Literature R e v i e w  ii iv vi vii viii xi 1  1. Introduction . 1.1 B i o c h e m i c a l a n d clinical features of cystic fibrosis 1.2 Liver d i s e a s e in cystic fibrosis 1.2.1 P r e v a l e n c e 1.2.2 D i a g n o s i s 1.2.3 H e p a t i c d i s e a s e in cystic fibrosis 1.2.4 Hepatic steatosis 1.2.5 M a n a g e m e n t 1.3 M a l d i g e s t i o n a n d malabsorption in cystic fibrosis 1.3.1 P a n c r e a t i c insufficiency 1.3.2 Abnormalities in bile acid m e t a b o l i s m 1.3.3 Intestinal abnormalities 1.3.4 M a n a g e m e n t 1.3.4.1 M i c r o e n c a p s u l a t e d p a n c r e a t i c e n z y m e preparations 1.3.4.2 A c i d reducing a g e n t s 1.4 Nutrition requirements for patients with cystic fibrosis 1.4.1 E n e r g y requirement. 1.4.2 Macronutrient requirement 1.4.3 Micronutrient requirement 1.5 C h o l i n e a n d phosphatidylcholine 1.5.1 F u n c t i o n , dietary s o u r c e s , digestion a n d absorption of choline 1.5.2 Choline/phosphatidylcholine s y n t h e s i s 1.6 C o n c l u s i o n s 1.7 Objectives 1.7.1 Specific aims 1.7.2 Organization of thesis 1.8 R e f e r e n c e s  2 4 6 6 7 9 10 12 13 13 15 17 17  27 29 34 38 38 39 41  Quantification of F e c a l P h o s p h o l i p i d s : M e t h o d D e v e l o p m e n t  57  2. Introduction 2.1 Materials a n d methods 2.1.1 C o l l e c t i o n a n d preparation of s a m p l e s  58 60 60  18 21 22 24 24 26 27  iv  Chapter 3  Chapter 4  2.1.2 Extraction and quantification of total fat 2.1.3 P h o s p h o l i p i d quantification 2.1.4 Statistical analysis 2.2 R e s u l t s 2.3 D i s c u s s i o n 2.4 R e f e r e n c e s  60 62 63 64 66 72  Phosphatidylcholine and Lysophosphatidylcholine Excretion a n d Its A s s o c i a t i o n with Altered P l a s m a H o m o c y s t e i n e a n d M e t h i o n i n e in C h i l d r e n with C y s t i c F i b r o s i s  75  3. Introduction 3.1 Subjects a n d m e t h o d s 3.1.1 F e c a l analysis 3.1.2 P l a s m a analysis 3.1.3 Dietary analysis 3.1.4 Statistical analysis 3.2 R e s u l t s 3.3 D i s c u s s i o n 3.4 R e f e r e n c e s  76 79 80 80 81 81 83 87 99  Discussion and Conclusions  105  4. Introduction 4.1 D i s c u s s i o n 4.1.1 Extraction a n d quantification of fecal total triglyceride and phospholipids: m e t h o d d e v e l o p m e n t and validation 4.1.2 Fat absorption and phospholipid excretion 4.1.3 A s s o c i a t i o n between choline p h o s p h o g l y c e r i d e excretion, and p l a s m a methionine, h o m o c y s t e i n e and S A H 4.2 Limitations 4.3 Future directions 4.4 C o n c l u s i o n  106 108  122 126 128 131  4.5 References  133  108 114  Appendix A Informed c o n s e n t  141  Appendix B F o o d record  154  Appendix C C h a r a c t e r i s t i c s of participants with cystic fibrosis  171  Appendix D U S D A d a t a b a s e for the choline content of c o m m o n foods  174  Appendix E E s t i m a t e d a v e r a g e choline intake of study participants  192  v  LIST O F T A B L E S Chapter 3 T a b l e 3.1 F e c a l fat and e n e r g y content of children with cystic fibrosis and control children  93  T a b l e 3.2 F e c a l p h o s p h o l i p i d excretion in children with cystic fibrosis and control children  94  T a b l e 3.3 P l a s m a thiols a n d phospholipids in children with C F a n d control children  95  T a b l e 3.4 A s s o c i a t i o n s b e t w e e n p l a s m a thiols and fecal p h o s p h o l i p i d excretion  96  vi  LIST O F F I G U R E S 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 a n d m a n a g e m e n t of malabsorption in patients with cystic fibrosis  19  Figure 1.3 S c h e m a t i c representation of phosphatidylcholine s y n t h e s i s  30  Figure 1 A S c h e m a t i c representation of the r e s e a r c h h y p o t h e s i s of this study  35  Chapter 2 Figure 2.1 Extraction of total fat a n d phospholipids using a) h e x a n e , diethyl ether a n d ethanol, b) chloroform, methanol, or c) h e x a n e , diethyl ether a n d ethanol followed by re-extraction with chloroform, m e t h a n o l  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 c o m p a r e d with lipid soluble p h o s p h o r u s quantified by p h o s p h o m o l y b d a t e colorimetric a s s a y  70  Figure 2.3 Distribution of phospholipids in fecal fat from children with cystic fibrosis a n d children without cystic fibrosis  71  Chapter 3 Figure 3.1 Correlation b e t w e e n fecal total fat, a n d fecal e n e r g y a n d fecal total phospholipids  97  Figure 3.2 Scatterplots of p l a s m a h o m o c y s t e i n e v e r s u s fecal total phospholipids, phosphatidylcholine, lysophosphatidylcholine a n d total choline p h o s p h o g l y c e r i d e in the s u b g r o u p of children with cystic fibrosis  98  vii  LIST O F A B B R E V I A T I O N S Al  a d e q u a t e intake  ALT  alanine transaminase  Apo B  apolipoprotein B  APP  alkaline p h o s p h a t a s e  AST  aspartate t r a n s a m i n a s e  BCCH  British C o l u m b i a ' s C h i l d r e n ' s Hospital  BMR  b a s a l metabolic rate  cAMP  cyclic a d e n o s i n e , 3 ' , 5 ' - m o n o p h o s p h a t e  CDP-choline  cytidine d i p h o s p h o c h o l i n e  °C  degree Celsius  CF  cystic fibrosis  CFTR  cystic fibrosis t r a n s m e m b r a n e c o n d u c t a n c e regulator  d  d a y or d e c i  DRI  dietary reference intakes  EDTA  e t h y l e n e d i a m i n e tetraacetic acid  EFA  e s s e n t i a l fatty a c i d s  ELSD  evaporative light scattering detector  g  gram  GGT  y-glutamlytransferase  h  hour  HDL  high-density lipoprotein  HPLC  high performance liquid c h r o m a t o g r a p h y  IDL  intermediate-density lipoprotein  k  kilo  kcal  Calories  kJ  kilo J o u l e s  L  liter  LDH  lactate  LDL  low-density lipoprotein  LFT  liver function test  LysoPC  lysophosphatidylcholine  m  milli or meter  M  molar  mEq  m o l a r equivalent  min  minute  mol  mole  mph  miles per hour  dehydrogenase  micro n  nano  PC  phosphatidylcholine  PE  phosphatidylethanolamine  PEMT  phosphatidylethanolamine /V-methyltransferase  PI  phosphatidylinositol  PKA  protein kinase A  PL  phospholipid  PLA PS  2  phospholipase A  2  phosphatidylserine  REE  resting energy expenditure  SAH  s-adenosylhomocysteine  SAM  s-adenosylmethionine  SEM  standard error of the m e a n  Sph  sphingomyelin  TG  triglyceride  TNFa  tumor n e c r o s i s factor a l p h a  TPN  total parenteral nutrition  U  units  UDCA  u r s o d e o x y c h o l i c acid  VLDL  very-low-density lipoprotein  wt  weight  y  year  ACKNOWLEDGEMENTS S p e c i a l thanks to: •  Dr S h e i l a Innis, m y r e s e a r c h supervisor, for her inspiration, g u i d a n c e , support, patience, a n d e n c o u r a g e m e n t ;  •  Cystic fibrosis t e a m a n d Dr. A . G . F . D a v i d s o n at B . C . ' s C h i l d r e n ' s Hospital for their a s s i s t a n c e in facilitating this study;  •  T h e parents a n d children w h o participated in this study;  •  Hospital for S i c k C h i l d r e n Foundation for funding this study;  •  Natural S c i e n c e s a n d E n g i n e e r i n g R e s e a r c h C o u n c i l of C a n a d a a n d the M i c h a e l Smith F o u n d a t i o n for Health R e s e a r c h for the studentships;  •  M r R o g e r Dyer a n d M s Janette King for their friendship, e n c o u r a g e m e n t a n d technical a s s i s t a n c e throughout every p h a s e of the study;  •  Dr S u s a n Barr for her a c a d e m i c a d v i c e a n d support;  •  All my friends a n d m y c o w o r k e r s in Dr Innis' laboratory for the e n c o u r a g e m e n t and c o m p a n y .  T h i s work is d e d i c a t e d to m y family, M o m , D a d , Derek, a n d K e i w h o h a v e supported m e throughout the recent years to m a k e all things p o s s i b l e .  XI  Chapter 1 Literature review  CHAPTER 1 LITERATURE REVIEW  Chapter 1 Literature review  1. INTRODUCTION C y s t i c fibrosis ( C F ) is the most c o m m o n lethal genetic d i s o r d e r affecting C a u c a s i a n populations. H e p a t i c steatosis (i.e. fatty infiltration of the liver) is prevalent in cystic fibrosis. Despite m a n y hypotheses, the link b e t w e e n the cystic fibrosis g e n e a n d the etiology of C F liver d i s e a s e , particularly hepatic steatosis, is not k n o w n . P a n c r e a t i c insufficiency is present in 8 5 % of patients with C F , resulting in d e c r e a s e d secretion of digestive e n z y m e s , bicarbonate, water a n d electrolytes into the d u o d e n u m , w h i c h l e a d s to malabsorption of nutrients. Although pancreatic e n z y m e replacement preparations are often p r e s c r i b e d to correct malabsorption, they are not 1 0 0 % effective. P h o s p h a t i d y l c h o l i n e ( P C ) represents >90% of the e s s e n t i a l nutrient choline in the diet. Further, large amounts of P C are found in bile. T h e digestion of P C requires p h o s p h o l i p a s e A , w h i c h r e l e a s e s l y s o P C a n d a n unesterified 2  fatty acid for absorption. P h o s p h o l i p a s e A is found in pancreatic e n z y m e 2  supplements, however, its activity d e c l i n e s at p H below 5.8. Further, the intestinal p H of patients with C F is often below p H 5.8 s e c o n d a r y to failure of bicarbonate secretion from the p a n c r e a s . W e h y p o t h e s i z e d that patients with C F have d e c r e a s e d digestion a n d thus, i n c r e a s e d excretion of P C s e c o n d a r y to pancreatic insufficiency a n d failure of pancreatic e n z y m e r e p l a c e m e n t therapy to completely correct the malabsorption, resulting in choline depletion. Fatty infiltration of the liver is a well-known feature of c h o l i n e 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 c o n c e i v a b l e that choline depletion m a y contribute to the pathophysiology of hepatic steatosis in C F . T h e purpose of this chapter is to provide a review of the pertinent literature on: i. T h e b i o c h e m i c a l a n d clinical features of cystic fibrosis ii. Liver d i s e a s e in C F iii. M a l d i g e s t i o n a n d malabsorption in C F iv. Nutrition requirements for patients with C F v. C h o l i n e a n d phoshatidylcholine Finally, this chapter will be c o n c l u d e d by the r e s e a r c h  hypotheses,  objectives of the study, a n d the organization of this thesis.  3  Chapter 1 Literature review  1.1 Biochemical and Clinical Features of Cystic Fibrosis C y s t i c fibrosis is a n a u t o s o m a l r e c e s s i v e genetic d i s e a s e that affects C a u c a s i a n populations, particularly t h o s e of Northern E u r o p e a n origin. T h e i n c i d e n c e 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 c h a n n e l protein, k n o w n a s the cystic fibrosis t r a n s m e m b r a n e  conductance  regulator ( C F T R ) , e n c o d e d by the cystic fibrosis g e n e located o n the long arm of c h r o m o s o m e 7 (2). N o r m a l epithelial cells often p o s s e s s p l a s m a m e m b r a n e c h a n n e l s o n the a p i c a l surface, w h i c h u p o n o p e n i n g allow chloride to flow a l o n g an e l e c t r o c h e m i c a l gradient (3). Increases in intracellular cyclic a d e n o s i n e 3',5'm o n o p h o s p h a t e ( c A M P ) a n d s u b s e q u e n t activation of protein k i n a s e A ( P K A ) c a n mediate the o p e n i n g of t h e s e c h a n n e l s (3). T h e m o v e m e n t of chloride a c r o s s epithelial cell surfaces plays a n important physiological role in salt a n d water b a l a n c e (3). T h e C F T R m e d i a t e d chloride c h a n n e l s in patients with C F , however, do not o p e n in r e s p o n s e to i n c r e a s e d levels of c A M P / P K A . T h i s l e a d s to abnormally thick, viscid m u c o u s secretions that obstruct g l a n d s a n d ducts in various o r g a n s . O v e r 1200 mutations of the cystic fibrosis g e n e h a v e b e e n identified. T h e most c o m m o n cystic fibrosis g e n e abnormality in C a n a d a is the deletion of three nucleotides c o d i n g for p h e n y l a l a n i n e at a m i n o acid position 508 ( A F 5 0 8 ) . T h i s deletion a c c o u n t s for about 7 0 % of mutations in patients with C F (4). T h e d i a g n o s i s of C F is primarily b a s e d o n elevated s w e a t s o d i u m a n d chloride concentrations (>60 m E q / L ) performed using the method of G i b s o n a n d  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 o f 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 infections a n d chronic lung d i s e a s e , pancreatic insufficiency, steatorrhea, m a l n u t r i t i o n , h e p a t i c c i r r h o s i s , i n t e s t i n a l o b s t r u c t i o n , a n d infertility (6). P o s i t i v e f a m i l y h i s t o r y is a l s o a s u g g e s t i v e c l i n i c a l s i g n . S u r v i v a l in p a t i e n t s w i t h C F h a s b e e n s h o w n to c o r r e l a t e p o s i t i v e l y w i t h t h e i r n u t r i t i o n a l 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 u n c t i o n ( i n c l u d i n g 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 vital 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 e x p i r a t o r y v o l u m e in o n e s e c o n d ) , s h o r t s t a t u r e , h i g h w h i t e c e l l c o u n t a n d c h r o n i c l i v e r d i s e a s e ( a s e v i d e n c e d b y 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 t h s u r v i v a l in a p r e d o m i n a t e l y a d u 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 p a t i e n t s w i t h 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 37 years (Personal communication, Canadian Cystic Fibrosis Foundation, M a y 2003). T h e most recent report from 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 t h e p r e d i c t e d s u r v i v a l a g e o f p a t i e n t s w i t h C F in t h e U n i t e d S t a t e s t o 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 C F 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), yglutamlytransferase (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 m a y h a v e s a m p l i n g error in C F d u e to the focal nature of the early c h a n g e s . U l t r a s o u n d - g u i d e d b i o p s y m a y reduce this risk of s a m p l i n g error (22).  1.2.3 Hepatic disease in cystic fibrosis The hepatobiliary l e s i o n s of C F are unique a n d the p a t h o g e n e s i s of C F liver d i s e a s e is poorly u n d e r s t o o d (20). T h e characteristic hepatic l e s i o n in C F is focal biliary fibrosis with bile duct obstruction, fibrosis, c h r o n i c inflammatory cell infiltration a n d bile duct proliferation (23). The initial formation of biliary obstruction has b e e n s h o w n to relate to the e x p r e s s i o n of C F T R at the a p i c a l m e m b r a n e of epithelial cells lining the intrahepatic c h o l a n g i o c y t e s a n d extrahepatic biliary tree (24). C h o l a n g i o c y t e s regulate bile v o l u m e , fluidity a n d alkalinity rapidly in r e s p o n s e to a c o m p l e x network of h o r m o n e s a n d paracrine mediators, w h o s e interplay results in net secretion or absorption of o s m o l y t e s , s u c h a s chloride a n d bicarbonate (19). Abnormalities of bicarbonate a n d chloride transport s e c o n d a r y to the mutated C F T R g e n e will, therefore, result in d e c r e a s e d hydration of bile, w h i c h will ultimately l e a d to i n c r e a s e d bile v i s c o s i t y a n d the t e n d e n c y to form biliary plugs (22). Additionally, in the p r e s e n c e of a reduced biliary fluidity a n d alkalinity, other factors, s u c h a s bile a c i d s a n d cytotoxic c o m p o u n d s , or a n i n c r e a s e d susceptibility to infectious agents, m a y d a m a g e the bile ducts (19, 25). In r e s p o n s e to the epithelial d a m a g e , cytokines a n d inflammatory mediators will be released, w h i c h will lead to chronic portal inflammation. T h i s p r o c e s s m a y  9  Chapter 1 Literature review  progress with d e a t h of hepatocytes to focal biliary cirrhosis a n d , eventually, liver d e c o m p e n s a t i o n , d e p e n d i n g o n the immunogenetic b a c k g r o u n d a n d a n u m b e r of concurrent events (19). E l e v a t i o n a n d c h a n g e s in c o m p o n e n t s of the s e r u m bile acid pool, including the possibility of a n i n c r e a s e in bile-related toxin h a v e also b e e n p r o p o s e d to b e involved in the d e v e l o p m e n t of liver cirrhosis in patients with C F (22). Although a b n o r m a l C F T R in the biliary tree provides a p o s s i b l e etiological basis for chronic liver d i s e a s e in C F , it d o e s not account for the a b s e n c e of liver involvement in a s i z e a b l e proportion of patients with C F a n d the w i d e spectrum of severity a m o n g t h o s e w h e r e this o c c u r s (22). C o l o m b o a n d c o l l e a g u e s (26) conducted a genetic a n a l y s i s of the major mutations present in an Italian population of patients with C F a n d w e r e not able to find a specific genetic marker for the d e v e l o p m e n t of C F - a s s o c i a t e d liver d i s e a s e . S o k o l a n d Durie (10) further p r o p o s e d that the variable onset a n d severity of liver d i s e a s e m a y imply there are other modifying genetic or environmental factors involved.  1.2.4 Hepatic steatosis Hepatic steatosis is the a b n o r m a l a c c u m u l a t i o n of fatty a c i d s a n d triglycerides within p a r e n c h y m a l cells, w h e r e they m a y a c c o u n t for up to 5 0 % of the total weight of the liver (27). Fatty infiltration of the liver is a r e c o g n i z e d feature in patients with C F a n d is often clinically silent. S o m e authors s u g g e s t hepatic steatosis m a y be a precursor of fibrosis/cirrhosis of the liver (22). However, proof of this is lacking. T h e relationship between the steatosis of C F  10  Chapter 1 Literature review  a n d the d e v e l o p m e n t of focal biliary fibrosis or cirrhosis r e m a i n s to be e s t a b l i s h e d . H e p a t i c steatosis has b e e n reported to be present in 1 4 - 2 3 % of C F patients b a s e d o n clinical, b i o c h e m i c a l and e c h o g r a p h i c a s s e s s m e n t s (20, 26). T h e p a t h o g e n e s i s of steatosis in C F r e m a i n s o b s c u r e . B i o c h e m i c a l l y , there is no e v i d e n c e that it m a y result from i n c r e a s e d delivery of fatty a c i d s a n d triglycerides to the liver from dietary fat, or from a d i p o s e tissue stores. Increased synthesis of fatty a c i d s within the liver a n d d e c r e a s e d oxidation of hepatic fatty acids has not b e e n s h o w n to o c c u r (27). A c a s e report in 1966 w a s a m o n g the first to p r o p o s e malnutrition a s a p o s s i b l e c a u s e for fatty infiltration of the liver in patients with C F (28). H o w e v e r , s u b s e q u e n t studies h a v e reported hepatic steatosis in patients with C F w h o w e r e well n o u r i s h e d , c o n s u m i n g a n appropriate diet, receiving vitamin s u p p l e m e n t a t i o n , as well a s pancreatic e n z y m e replacement (25). Carnitine deficiency has a l s o b e e n p r o p o s e d a s a p o s s i b l e contributing factor in hepatic steatosis in patients with C F (29). Carnitine is d e r i v e d from trimethyllysine, w h i c h is s y n t h e s i z e d from lysine a n d methionine in h u m a n s . Carnitine is involved in fatty acid transport into mitochondria. H o w e v e r , data on the carnitine status of patients with C F are limited a n d i n c o n c l u s i v e (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). O f interest, rats fed a c h o l i n e deficient diet h a d r e d u c e d levels of carnitine in liver, heart a n d skeletal m u s c l e (33) (refer to S e c t i o n 1.5).  E s s e n t i a l fatty a c i d ( E F A ) deficiency a l s o has b e e n d e s c r i b e d in patients with C F (refer to section 1.4.2) a n d w a s found significantly m o r e often in patients with m a r k e d steatosis (14). H o w e v e r , the link b e t w e e n E F A deficiency a n d hepatic steatosis in C F r e m a i n s unclear.  1.2.5 Management U r s o d e o x y c h o l i c acid ( U D C A ) , a s e c o n d a r y bile a c i d that is found in small concentrations in normal h u m a n bile, is c o m m o n l y p r e s c r i b e d to C F patients with liver c o m p l i c a t i o n s . U D C A protects the hepatocytes from the toxicity of hydrophobic bile a c i d s a n d improves the fluidity of bile (22). Improvement a n d e v e n c o m p l e t e normalization of L F T s h a s b e e n d o c u m e n t e d in C F patients treated with U D C A (19, 34-36). C o m p a r i s o n of liver b i o p s i e s from children with C F before and after treatment with U D C A indicated statistically significant improvement in focal biliary fibrosis, but no c h a n g e in steatosis ( D a v i d s o n A G F 2004, oral c o m m u n i c a t i o n , 2  n d  July). H o w e v e r , U D C A is not effective in all  patients, p o s s i b l y d u e to the different pathogenic m e c h a n i s m s involved in C F related liver d i s e a s e (19). W h e n focal biliary fibrosis p r o g r e s s e s to mutilobular biliary cirrhosis with portal hypertension a n d liver failure, liver transplantation b e c o m e s the only treatment option (26).  12  ;  Chapter 1 Literature review  1.3 Maldigestion and Malabsorption in CF C F patients with untreated pancreatic insufficiency a b s o r b about  50-60%  of dietary fat ( 3 7 ) . W h e n pancreatic e n z y m e preparations are p r e s c r i b e d to C F patients with pancreatic insufficiency to correct fat maldigestion a n d malabsorption, however, they d o not completely normalize maldigestion a n d malabsorption  (38-40).  Typically, fat absorption is about  8 5 %  in children with C F  a n d is > 9 3 % in children without C F ( 3 8 ) . T h e pathophysiology of maldigestion and malabsorption in patients with C F , including pancreatic insufficiency, abnormalities in bile a c i d m e t a b o l i s m a n d intestinal abnormalities, a n d its m a n a g e m e n t a r e d i s c u s s e d below.  1.3.1 Pancreatic insufficiency M a l a b s o r p t i o n in c y s t i c fibrosis o c c u r s mainly a s a result of maldigestion s e c o n d a r y to pancreatic insufficiency. Pancreatic insufficiency is a s s o c i a t e d with d e c r e a s e d pancreatic secretion of water, bicarbonate, electrolytes a n d digestive e n z y m e s including lipase, c o l i p a s e a n d p h o s p h o l i p a s e A (41) into the d u o d e n u m . 2  T h e pathological c h a n g e s in the C F p a n c r e a s are related to defective C F T R . Ion a n d fluid influx into the p a n c r e a s is i m p e d e d a n d l e a d s to d e c r e a s e d  pancreatic  e n z y m e output (42). T h e concentrated pancreatic secretions l e a d to the typical, progressive destructive l e s i o n of the pancreas; ductal obstruction a n d pancreatic exocrine insufficiency (42). M o r e o v e r , d e c r e a s e d bicarbonate secretion m a y contribute further to fat malabsorption ( d i s c u s s e d below)  (Figure 1.1).  Although  only 1-2% of the pancreatic capacity for secreting lipase is thought to be  13  2-8  =0  c CO CO Q_ c  TD O O  4—  TD  CD to CD  -4-*  o>  TD C  ZD  TD  CO  X  reas ed enal  CD  CL  c  E  o  O c  o CU  JD  Q  CO  o  "co  CO JD  _cy JD  o CJ) c  TD c CQ  »  _ro  o  du  ~co  O CO  CO  CD c  CQ  CO  to  o ).  _Q ti— O 00  o  f  5  to -*—'  c CD  ro CL  •H  S  T3  o E  8?  ._  cu >- 5 co ro o  j  811  to to  o  c  ro  ^  CD  8 2  c  CO O  CU  o  CD O CO CD  Q  CD J5 C1VJ  TD  ^ ® CS O D C CO XJ  ro  ro TD  ro  JD  CD  c  o o o o  se du  «CO cu T J  Q) CO  CO cu  dis  CD *? co  CU  ad  o  to  o  CO  CU  CO C i_D  c  =i  o  CO  TD  _CD  o  JD _ro ro E  £  O O Q. CD £ Q o° o  c  CD to ZI  co O  zz*  t_  -4—'  to  JD O  CD i_  3 CD  iZ 14-  Chapter 1 Literature review  required to prevent impaired lipolysis, pancreatic e x o c r i n e abnormalities are present in approximately 8 5 to 9 0 % of patients with C F (6, 43). O n the other hand, pancreatic-sufficient patients, which constitute approximately 1 5 % of the C F population, retain sufficient pancreatic function to permit d i g e s t i o n a n d absorption of nutrients (44), although pancreatic fluid a n d bicarbonate secretion is usually d e c r e a s e d (45).  1.3.2 Abnormalities in bile acid metabolism Bile a c i d s are s y n t h e s i z e d by the liver, w h e r e t h e y are conjugated with glycine or taurine. T h e y are then secreted as bile salts in micelle together with cholesterol a n d p h o s p h o l i p i d into the d u o d e n u m . Bile a c i d s are important for solubilizing lipid a n d are essential in the formation of m i x e d m i c e l l e s together with phospholipids in the intestine to provide an i n c r e a s e d surface a r e a for digestion of lipids a n d fat-soluble vitamins. Normally, the bile acid pool u n d e r g o e s enterohepatic circulation about 5-10 times daily; the intestinal absorption of the pool is about 9 5 % efficient in n o n - C F individuals, a n d about 0.3-0.6 g bile a c i d s are lost daily in the f e c e s (46). N e w bile a c i d s are typically s y n t h e s i z e d in a n amount equivalent to that lost in the f e c e s (47). Bile a c i d s readily precipitate in an acid milieu. A t p H s b e l o w 5, bile a c i d s precipitate from the a q u e o u s p h a s e of the intestine, leading to a reduction in the a q u e o u s p h a s e bile acid concentration (48). M o r e o v e r , precipitated bile salts a p p e a r to be lost from the enterohepatic circulation in greater quantities than t h o s e that are not precipitated, thus further d e c r e a s i n g the bile a c i d concentration  15  '  Chapter 1 Literature review  (49). W h e n the d u o d e n a l bile acid concentration falls b e l o w 2 m M , the formation of micelles is impaired (50). Micellar formation might be impaired in patients with C F b e c a u s e acidic p H v a l u e s are c o m m o n l y found in the d u o d e n u m (51). T h u s , d u o d e n a l bile salt concentrations m a y fall below the critical micellar concentration. R e d u c e d d u o d e n a l bile a c i d concentrations (52) a n d i n c r e a s e d f e c a l bile a c i d excretion h a v e b e e n d o c u m e n t e d in s o m e studies with patients with C F (49, 53). However, W e i z m a n a n d c o l l e a g u e s (52) reported that d u o d e n a l bile acid concentrations w e r e significantly higher in C F subjects than controls. T h i s could be explained by the m a r k e d l y r e d u c e d pancreatic water output s h o w n in C F patients (45), w h i c h w o u l d i n c r e a s e the apparent bile acid concentration, despite a lower absolute a m o u n t present. W e b e r a n d R o y (50) reported a higher glycine:taurine ratio of conjugated bile acids in patients with C F . In r e s p o n s e to the o n g o i n g bile a c i d l o s s e s in feces, newly s y n t h e s i z e d bile a c i d s are formed and conjugated mainly with glycine. Glycine is readily a v a i l a b l e in contrast to taurine, which m a y b e c o m e rate-limiting w h e n bile acid l o s s e s are large (50). Furthermore, a s glycine-conjugated bile salts t e n d e d to precipitate m o r e readily in acidic milieu (i.e. the intraduodenal p H must remain within narrow limits (at p H 6-8) to avoid precipitation of glycineconjugated bile acid), they are lost more readily than taurine-conjugated bile salts (which is soluble at p H 2-14) w h e n the intradoudenal p H is low (50). T h u s glycine-conjugated bile a c i d s are less efficient at micelle formation. In s u m m a r y , patients with C F tend to h a v e bile salts that are conjugated with g l y c i n e rather than taurine s e c o n d a r y to ileal malabsorption of bile a c i d s a n d the differential rate  16  Chapter 1 Literature review  of synthesis of g l y c i n e o v e r taurine (42). T h e i n c r e a s e d glycine: taurine ratio of bile salts will likely further i n c r e a s e bile a c i d loss by precipitation s e c o n d a r y to the low intraduodenal p H . S o m e r e s e a r c h g r o u p s have a l s o p r o p o s e d other m e c h a n i s m s related to bile acids to e x p l a i n fat malabsorption in patients with C F . B i n d i n g of bile a c i d s to undigested fat, protein, fiber or carbohydrate has been s u g g e t e d to i n c r e a s e bile loss in feces (53, 54). A d v a n c e d liver d i s e a s e with multifocal biliary cirrhosis m a y result in inadequate bile salt secretion, w h i c h in turn w o u l d w o r s e n fat malabsorption (42). Finally, a n y abnormality of the gall bladder, or bile-duct obstruction m a y a l s o interfere with bile secretion to the d u o d e n u m .  1.3.3 Intestinal a b n o r m a l i t i e s  V i s c i d , thick intestinal m u c u s , with altered p h y s i c a l properties, m a y h a v e a deleterious effect o n the t h i c k n e s s of the intestinal unstirred layer, further limiting nutrient absorption by the microvilli (44, 55). D e c r e a s e d bicarbonate secretion by the intestine m a y (56) also contribute to the low intraduodenal p H mentioned previously.  1.3.4 Management M i c r o e n c a p s u l a t e d pancreatic e n z y m e preparations, a n d s o m e t i m e s acid reducing a g e n t s are p r e s c r i b e d to correct maldigestion a n d m a l a b s o r p t i o n in patients with C F . T h e i r functions a n d limitations are d i s c u s s e d below.  17  Chapter 1 Literature review  1.3.4.1 Microencapsulated pancreatic enzyme preparations M i c r o e n c a p s u l a t e d e n z y m e preparations h a v e b e e n prescribed to patients with C F to correct maldigestion a n d malabsorption s i n c e the late 1 9 7 0 s . H o w e v e r , steatorrhea a n d azotorrhea still occur, to varying extents, a m o n g patients u s i n g pancreatic preparations (40, 57). Both lipase inactivation a n d s l o w dissolution of the pH-sensitive c o a t i n g m a y contribute to the failure of enteric-coated e n z y m e preparations to normalize fat absorption (Figure 1.2). T h e d u o d e n a l p H in patients with pancreatic insufficiency d u e to C F is k n o w n to be abnormally a c i d i c b e c a u s e pancreatic bicarbonate production is low (45). W h e t h e r gastric acid hypersecretion o c c u r r e d in C F patients has not b e e n clearly established (58). In the a b s e n c e of a d e q u a t e pancreatic bicarbonate secretion, gastric a c i d entering the d u o d e n u m m a y lower intestinal p H w h i c h persists well into the jejunum. P a n c r e a t i c lipase activity is optimal at p H 8.0, a n d is irreversibly inactivated below p H 4 (59, 60). T h e digestive c a p a c i t y of pancreatic lipase is r e d u c e d at a lower p H . Prior to the availability of enteric-coated pancreatic e n z y m e s , gastric inactivation of the pancreatic e n z y m e s u p p l e m e n t s w a s a major limitation to the efficacy of pancreatic e n z y m e supplements (61). Enteric-coated e n z y m e s w e r e eventually d e v e l o p e d to minimize gastric inactivation of the pancreatic e n z y m e s u p p l e m e n t s . However, d e p e n d i n g upon the formulation, the threshold for rapid dissolution (i.e. within 15 minutes of e x p o s u r e buffered e m u l s i o n at  18  T J  CD CO  CO  o  S ._  CD  .=  _  O  CO CO CO  TJ  O  a T J  0)  "co CD  cn T J _ ZJ  reas ed enal  X  CL  — •  o TJ CD Q  o ZJ  T J  o< *-—  to  T J  CO  o ro _D  o  _ -Q  _5 4— o  CO  o  CJ)  c  T J  rz  oo  CO •+-»  c CD »  ro  a. c o  "-»—• CL  _  o  CO  j_  CO CO T J  0) co  _o  _  i-  5  ro  . 2  _D  _5  S "CD ro ir o c  ro o  cu E o  T J  CO  ro CD o c _D  O CD CD CO  O  CD Q  CO  o CD _ . CO  ro  S  _5  CHJ  ^  ad  co  _ 9> - o K ro o co oc CD co CD CD ro _j  TJ  _Q  — CO CD  o  c  -*—> O  se du  CD  CO CD  dis  CD  O  E  CD CD  CO CO  CD  T J  cz CD  £Z  Q  CD  ro ro E  o' o ZJ i_ "co _o  o  E T J CZ  ro CO  CD CO ZJ  ro O CM  0) 3  CO  Chapter 1 Literature review  specific p H s ) of the pH-sensitive coating r a n g e s from p H 5.6 to p H 6.2 a n d higher (59, 62). F o r e x a m p l e , P a n c r e a s e ® requires e x p o s u r e to a p H a b o v e 5.6 for 10 minutes or a p H less than 5.2 for m o r e than 120 minutes for the enteric coating to d i s s o l v e (59). Y o u n g b e r g a n d c o l l e a g u e s (62) found lower postprandial d u o d e n a l p H s in patients with C F than in healthy subjects, e s p e c i a l l y in the first postprandial hour. T h u s , the acid-resistant coating of the e n z y m e preparations m a y not d i s s o l v e in the proximal intestine a n d this would result in d e l a y e d release of active e n z y m e s . R o b i n s o n , S m i t h a n d S l y (63) also found significantly lower postprandial d u o d e n a l p H s in patients with C F a n d s u g g e s t e d that failure of release of e n z y m e s from the enteric c o a t e d preparations is a major factor in inefficient e n z y m e function. A n o t h e r p o s s i b l e explanation for the different effectiveness of the pancreatic preparations a m o n g individuals is the varying level of residual pancreatic function. Patients with d o c u m e n t e d steatorrhea m a y h a v e 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) a n d fat absorption (the correlation coefficient a n d p v a l u e w e r e not reported in the publication). Furthermore, pancreatic e n z y m e preparations given to patients with C F are subjected to degradation by proteolytic e n z y m e s in the a b s e n c e of the inhibitor normally present in pancreatic juice (39), w h i c h c a n result in degradation  20  Chapter 1 Literature review  of the s u p p l e m e n t e d e n z y m e s thus limiting their ability to correct maldigestion a n d malabsorption.  1.3.4.2 Acid reducing agents In addition to the pancreatic e n z y m e preparations, C F patients with pancreatic insufficiency are s o m e t i m e s prescribed a c i d reducing agents, including a n t a c i d s (e.g. s o d i u m bicarbonate), or H b l o c k e r s to i n c r e a s e 2  their gastric a n d d u o d e n a l p H , a n d thus improve the efficacy of the e n z y m e s u p p l e m e n t s a n d consequently digestion a n d absorption. S e v e r a l studies h a v e s h o w n that patients treated with the c o m b i n a t i o n of m i c r o e n c a p s u l a t e d pancreatic e n z y m e preparations a n d acid reducing agents had i m p r o v e d fat digestion a n d absorption (37, 3 9 , 4 8 , 63). T h e effect of gastric acidity reducing a g e n t s in overall C F nutrition status, lung function, quality of life a n d survival remains to be d e t e r m i n e d .  21  Chapter 1 Literature review  1.4 Nutrition Requirements for Patients with Cystic Fibrosis D e s p i t e dramatic improvement in the nutritional c a r e of C F patients o v e r the past two d e c a d e s , malnutrition ( b a s e d on height or weight l e s s than 5  t h  percentile in the g e n e r a l population) is still present in 16-17% of patients with C F (9). Malnutrition a n d growth failure have b e e n s h o w n to be c l o s e l y linked with pulmonary function (65), a n d have b e e n recognized a s a d v e r s e prognostic factors in patients with C F (7, 66, 67). P o o r nutritional state a n d growth failure in patients with C F h a v e b e e n s u g g e s t e d to be c a u s e d by a n i m b a l a n c e between energy intake a n d absorption, a n d energy requirements (68). F a c t o r s contributing to the energy i m b a l a n c e c a n include d e c r e a s e d intake, i n c r e a s e d energy loss a n d increased energy expenditure. F a c t o r s contributing to a poor dietary intake in C F include: recurrent vomiting from c o u g h i n g , and/or g a s t r o e s o p h a g e a l reflux, foul-tasting sputum, chronic respiratory infections, a n d p s y c h o s o c i a l s t r e s s e s (43). In addition, patients with C F c o m m o n l y h a v e gastrointestinal dysmotility, including g a s t r o e s o p h a g e a l reflux a n d d e l a y e d gastric emptying which l e a d to a n o r e x i a (42). A n o r e x i a is c o m m o n in C F a n d c a n b e c o m e m o r e of a p r o b l e m during recurrent c h e s t infections. Earlier studies in this field h a v e s u g g e s t e d that the higher than normal level of tumor necrosis factor a l p h a ( T N F - a ) in the p l a s m a of patients with C F m a y contribute to the anorexia (68, 69). Increased energy l o s s b e c a u s e of nutrient maldigestion a n d m a l a b s o r p t i o n is k n o w n to contribute to e n e r g y i m b a l a n c e in patients with C F . T h e l o s s of energy in stools has b e e n s h o w n to be three times higher in C F patients  22  :  Chapter 1 Literature review  c o m p a r e d with controls w h o h a v e similar energy intakes (38). M a l a b s o r p t i o n in cystic fibrosis o c c u r s mainly a s a result of maldigestion s e c o n d a r y to pancreatic insufficiency, a s d i s c u s s e d in the section 1.3. M o r e o v e r , d e c r e a s e d  bicarbonate  secretion a n d d e c r e a s e d bile acid reabsorption c a n further r e d u c e digestive e n z y m e activity, a n d thus further contribute to fat malabsorption. Regurgitation a n d vomiting from g a s t r o e s o p h a g e a l reflux may also i n c r e a s e energy loss. C o n t i n u o u s inflammation, acute exacerbation of infection, chronic obstructive p u l m o n a r y d i s e a s e , a n d deteriorating lung function a p p e a r to be the major factors a s s o c i a t e d with i n c r e a s e d resting energy expenditure ( R E E ) (70). Furthermore, it has b e e n p r o p o s e d that the c o n s e q u e n c e s of a b n o r m a l function of the C F T R gene, at the cellular level, are energy requiring, a n d m a y increase the R E E in patients with C F (71). H o w e v e r , not all studies support this hypothesis (72). T h e resting metabolic rate in patients with C F w a s i n c r e a s e d by 2 0 - 2 5 % during acute p u l m o n a r y e x a c e r b a t i o n (67). Overall, the g o a l of nutritional care in patients with C F is to maintain a z e r o energy b a l a n c e in adults, a n d provide sufficient energy to support growth a n d m a i n t e n a n c e requirements in children (73). Specifically, the nutritional c a r e plan a i m s to control m a l d i g e s t i o n a n d malabsorption, to provide a d e q u a t e nutrients to promote optimal growth or maintain weight for height a n d pulmonary function, a n d to prevent nutritional deficiencies (44, 74). T h e following section provides a brief d i s c u s s i o n of the current k n o w l e d g e on the energy a n d nutrient requirements in C F .  23  Chapter 1 Literature review  1.4.1 Energy requirement A s a result of the i n c r e a s e d energy loss a n d i n c r e a s e d e n e r g y expenditure, the energy requirements for patients with cystic fibrosis are often higher than normal. W h e n calculating e n e r g y requirements, all factors contributing to the energy b a l a n c e equation, s u c h a s fecal energy l o s s e s , i n c r e a s e d b a s a l metabolic rate ( B M R ) , catch-up growth a n d frequency of infection n e e d to be t a k e n into consideration. A detailed description for the calculation of the e n e r g y requirements for patients with C F c a n be found in the c o n s e n s u s report by R a m s e y et a l . (74). H o w e v e r , in practice, dietitians often calculate a patient's energy requirement b a s e d o n a k n o w l e d g e of the dietary history a n d a n estimation of 1 2 0 - 1 5 0 % of that r e c o m m e n d e d for n o n - C F patients of similar a g e a n d s e x (43). T h e c a l c u l a t i o n of energy requirement for patients with C F b a s e d on the Dietary R e f e r e n c e Intakes. (DRI) w a s not available at the time of this study.  1.4.2 Macronutrient requirement T h e requirement for dietary protein is i n c r e a s e d in C F a s a result of malabsorption. H o w e v e r , w h e n energy n e e d s are adequately met, individuals with C F g e n e r a l l y c a n meet their protein n e e d by following a typical North A m e r i c a n diet, with at least 1 5 % to 2 0 % of the total calories c o n s u m e d a s protein (75). Patients with C F often require a higher fat intake (35 to 4 0 % of calories) than that r e c o m m e n d e d for the g e n e r a l population. A c c o r d i n g to the 2 0 0 2 DRI for macronutrients, the a c c e p t a b l e macronutrient distribution r a n g e for fat is 2 0 - 3 5 %  24  .  Chapter 1 Literature review  of energy for adults, 3 0 - 4 0 % of e n e r g y for children 1-3 y e a r s , a n d 2 5 - 3 5 % of energy for children 4 - 1 8 y e a r s (76). Until the late 1970's, a high energy, low-fat diet w a s r e c o m m e n d e d in s o m e C F centres. It w a s r e a s o n e d that reduction in dietary fat w o u l d i m p r o v e bowel s y m p t o m s a n d r e d u c e stool bulk. H o w e v e r , a retrospective study c o n d u c t e d by a C a n a d i a n r e s e a r c h group in the 1980's c o m p a r i n g the C F population in Toronto with that in B o s t o n found a m a r k e d difference in m e d i a n a g e of survival b e t w e e n the two centres (77). T h e m e d i a n survival in B o s t o n of 21 y e a r s w a s significantly l e s s than that of 30 y e a r s for patients in Toronto. It w a s found that the g e n e r a l a p p r o a c h of treatment w a s mostly the s a m e , e x c e p t that the a p p r o a c h e s to diet a n d p a n c r e a t i c supplementation w e r e radically different. T h e Toronto centre a d v o c a t e d a highfat, high-caloric diet with up to 20-30 or more pancreatic e n z y m e c a p s u l e s per meal (note that the e n z y m e preparations in those d a y s w e r e non-enteric c o a t e d a n d l e s s potent c o m p a r e d with t h o s e that are presently used). T h e B o s t o n centre a d v o c a t e d a low-fat, high calorie diet with l e s s pancreatic e n z y m e c a p s u l e s per meal (77). T h u s , the authors c o n c l u d e d that a restriction of fat in the diet of C F patients is not d e s i r a b l e (77). A r e d u c e d level of p l a s m a e s s e n t i a l fatty a c i d s ( E F A s ) is a frequent feature in patients with C F (78, 79). Correction of the a b n o r m a l pattern of essential fatty a c i d s h a s p r o v e n to b e difficult (80). Further, t h e r e h a s b e e n little published e v i d e n c e of clinical benefit from s u p p l e m e n t i n g C F patients with E F A s except in patients with E F A deficiency (81). N e v e r t h e l e s s , v e g e t a b l e oils s u c h as flax, c a n o l a a n d s o y oils a r e rich in a l p h a linolenic a c i d a n d a r e a g o o d s o u r c e of  25  Chapter 1 Literature review energy, a n d fish is a s o u r c e of long c h a i n n-3 fatty a c i d s s u c h a s e i c o s a p e n t a e n o i c a c i d a n d d o c o s a h e x a e n o i c acid a n d has b e e n r e c o m m e n d e d (73).  1.4.3 Micronutrient requirement With the e x c e p t i o n of vitamin B - i , the absorption of water-soluble vitamins 2  a n d minerals a p p e a r s to b e normal in patients with C F (42). P a n c r e a t i c e n z y m e s are involved in the intrinsic-factor-mediated vitamin B vitamin B i  2  1 2  absorption. N e v e r t h e l e s s ,  absorption c a n be n o r m a l i z e d with a d e q u a t e pancreatic e n z y m e  replacement therapy in pancreatic-insufficient patients (82, 83). Deficiencies of fat-soluble vitamins (vitamins A , D , E , a n d K ) h a v e b e e n demonstrated in patients with C F . Fat-soluble vitamin s u p p l e m e n t s are a n e c e s s a r y part of the nutritional care of C F patients with pancreatic insufficiency, or s e v e r e liver d i s e a s e (44). M o s t patients with C F receive a d e q u a t e supplementation of most vitamins a n d micronutrients from multiple-vitamin preparations (74).  26  ;  Chapter 1 Literature review  1.5 Choline and Phosphatidylcholine A w e l l - k n o w n feature of the deficiency of the e s s e n t i a l nutrient choline is hepatic steatosis, a n d a reduction of p l a s m a P C , triglyceride ( T G ) a n d very low density lipoprotein ( V L D L ) concentrations (84-87). T h e a c c u m u l a t i o n of T G in the liver a n d d e c r e a s e in p l a s m a T G is believed to be e x p l a i n e d b y the absolute requirement of active P C biosynthesis for the secretion of T G a n d V L D L by hepatocytes (88, 89). H e p a t i c steatosis is often resolved following c h o l i n e (85, 90, 91) or P C s u p p l e m e n t a t i o n (92) of patients supported by long term total parenteral nutrition ( T P N ) d e v o i d of choline. T o our k n o w l e d g e , the choline status of patients with C F h a s not b e e n reported. T h i s s e c t i o n will r e v i e w the function, dietary s o u r c e s , digestion a n d absorption of c h o l i n e a n d P C , a n d the pathways for P C s y n t h e s i s .  1.5.1 Function, dietary s o u r c e s , digestion and absorption of choline C h o l i n e , a quaternary a m i n e , is present in tissue predominantly a s the head group of P C a n d s p h i n g o m y e l i n (Sph), w h i c h are structural constituents of cell m e m b r a n e s , p l a s m a lipoproteins a n d lung surfactant (93). Additionally, choline is a c o m p o n e n t of the neurotransmitter acetylcholine, platelet-activating factor, p l a s m a l o g e n , a p h o s p h o l i p i d found in highest concentrations in c a r d i a c m u s c l e m e m b r a n e s , a n d is a s o u r c e of labile methyl g r o u p s (94, 95). F r e e choline is present in s m a l l a m o u n t s in foods, but is found ubiquitously in normal diets a s a c o m p o n e n t of P C a n d S p h . Major dietary s o u r c e s of choline, P C a n d S p h include: e g g yolks, o r g a n m e a t s , s p i n a c h , nuts, a n d w h e a t g e r m (94, 96). P C represents >90% of c h o l i n e in a n i m a l tissue. T h e a d e q u a t e intake (Al) for choline  27  Chapter 1 Literature review is 4 2 5 a n d 5 5 0 mg/d.for adult w o m e n a n d m e n , respectively (97). N o r m a l p l a s m a choline concentrations in healthy adult m e n are 10.5 u.mol/L, with a range of 7-20 u.mol/L c o m p a r e d to a m e a n of 7.5 uimol/L in m e n fed a c h o l i n e deficient diet. P C concentrations are 1-1.5 m m o l / L in healthy individuals (97). In a study published in 1980, Z e i s e l et al prepared a high choline diet with food with high choline content, including e g g (with 2.1g choline/d) a n d liver (with 2.6g choline/d), which resulted in a diet with 5.6g of P C per d a y (98). A more representative diet with meat at both the mid d a y a n d e v e n i n g m e a l provided about 1g of P C per d a y (98). Data on usual P C intakes are not available. C h o l i n e is a b s o r b e d though a specific non e n e r g y - d e p e n d e n t carrier in the gut, a n d no other c o m p o n e n t has b e e n identified that c o m p e t e s with choline for transport (99). P C is digested by p h o s p h o l i p a s e A ( P L A ) in pancreatic 2  2  secretions with the help of bile salts a n d bicarbonate, to form l y s o P C a n d a n unesterified fatty a c i d . T h e optimal p H for P L A activity is not k n o w n , but it 2  a p p e a r s to be m o r e sensitive to an acidic p H than pancreatic lipase (50). T h e digested products of P C form mixed micelles with bile salts a n d other digested lipid products before being transported to the brush border for p a s s i v e absorption. O f importance, P C is essential for normal bile s y n t h e s i s , micelle formation and c o n s e q u e n t l y fat absorption. P C represents about 4 % (wt/wt) of n o r m a l bile, with 8 2 % water, 1 2 % bile a c i d s a n d 0.7% cholesterol in n o r m a l bile (46). T h e normal enterohepatic pool has about 1g of P C a n d the bile pool circulates 5-10 times/d. T h u s , about 5-10 g P C enters the intestine in bile lipid e a c h day.  28  Chapter 1 Literature review  M a i n t e n a n c e of the b o d y choline/ P C pool is, therefore, critically d e p e n d e n t o n the absorption of dietary P C a n d reabsorption of biliary P C . Normally there is a c l o s e c o u p l i n g between the secretion of bile salts, phospholipid, a n d cholesterol in bile (27). T h u s , P C depletion c o u l d lead to poor bile synthesis a n d secretion, which in turn m a y further w o r s e n fat malabsorption, a n d P C a n d dietary fat l o s s e s will likely result. Similarly, w h e n the secretion rate of bile salts d e c r e a s e s b e l o w a certain limit b e c a u s e of i n c r e a s e d fecal l o s s e s , the secretion rate of p h o s p h o l i p i d s is reduced proportionately (27).  1.5.2 Choline/phosphatidylcholine synthesis P h o s p h a t i d y l c h o l i n e c a n be s y n t h e s i z e d v i a two p a t h w a y s in liver: the C D P - c h o l i n e pathway, w h i c h requires free choline, a n d the de novo phosphatidylethanolamine A/-methyltransferase ( P E M T ) p a t h w a y w h i c h i n c l u d e s sequential methylation of phosphatidylethanolamine ( P E ) , u s i n g S a d e n o s y l m e t h i o n i n e ( S A M ) a s the methyl d o n o r ( F i g u r e 1.3). A v a i l a b l e e s t i m a t e s s u g g e s t that in the w e l l - n o u r i s h e d state, the C D P - p a t h contributes about 6 0 - 8 0 % of hepatic P C formation (100, 101), a n d the P E M T pathway contributes the remainder. During experimental c h o l i n e deficiency, transmethylation of P E to form P C a n d the hepatic concentration of S - a d e n o s y l h o m o c y s t e i n e ( S A H ) is i n c r e a s e d , a n d S - a d e n o s y l m e t h i o n i n e ( S A M ) a n d methionine are d e c r e a s e d , presumably reflecting a n attempt of the liver to i n c r e a s e P C s y n t h e s i s by the de novo P E M T pathway (84, 102). W h e t h e r or not this o c c u r s in vivo in h u m a n s h a s not b e e n directly e s t a b l i s h e d . Increased P E M T activity has a l s o b e e n s h o w n in  29  Chapter 1 Literature review  choline deficiency a s m e a s u r e d in vitro in hepatocytes from rats a n d g u i n e a pigs fed a c h o l i n e deficient diet (103-105). C u i a n d V a n c e (106) h a v e further d e m o n s t r a t e d that P E M T g e n e e x p r e s s i o n w a s i n c r e a s e d in rats fed with a choline deficient diet for m o r e than three w e e k s . T h e available experimental d a t a derived from studies with rat hepatocytes indicate that P C s y n t h e s i z e d from either the P E M T pathway or the C D P - c h o l i n e pathway c a n support n o r m a l V L D L lipoprotein a s s e m b l y a n d s e c r e t i o n . W h e n c h o l i n e - d e p l e t e d hepatocytes w e r e s u p p l e m e n t e d with e x c e s s S A M (84), methionine (88), or betaine a n d h o m o c y s t e i n e (89), normal V L D L secretion w a s r e s u m e d . H o w e v e r , t h e s e in vitro studies u s e d concentrations that w e r e m u c h higher than the normal physiological levels. In P E M T knockout m i c e , the C D P choline pathway is c a p a b l e of fulfilling the requirements for P C b i o s y n t h e s i s a s long a s sufficient choline is provided in the diet (107-108). H o w e v e r , very recent studies h a v e s u g g e s t e d that P C formed from the C D P - c h o l i n e pathway a n d the P E M T pathway h a v e distinct functions, a n d are not fully metabolically interchangeable. H o u w e l i n g a n d c o l l e a g u e s (109, 110) h a v e repeatedly s h o w n that P C s from the C D P - c h o l i n e a n d P E M T p a t h w a y s h a v e o p p o s i t e effects o n hepatocyte proliferation. T h e C D P - c h o l i n e pathway h a s b e e n s h o w n to favor faster proliferation of hepatocytes, while the P E M T pathway h a s b e e n s h o w n to strongly inhibit the growth of h e p a t o m a cell lines, a n d a s s o c i a t e negatively with the d e v e l o p m e n t a l growth of the liver. In addition, D e L o n g a n d c o l l e a g u e s (111) reported that the two pathways result in s y n t h e s i s of different P C m o l e c u l a r s p e c i e s , w h i c h further s u g g e s t s the distinctiveness a n d  31  Chapter 1 Literature review unique function of P C m o l e c u l e s originating from the two pathways. T h e latter group a l s o reported that the P C s y n t h e s i z e d from the C D P - c h o l i n e pathway w e r e mainly c o m p r i s e d of m e d i u m c h a i n a n d saturated fatty a c i d s , w h e r e a s the P C s y n t h e s i z e d from the methylation pathway w e r e mainly c o m p r i s e d of long c h a i n a n d highly unsaturated fatty a c i d s . Additionally, N o g a a n d c o l l e a g u e s (112) recently reported that P E M T activity is required for optimal apolipoprotein B (apo B), V L D L a n d T G secretion, a n d that P E M T activity cannot be fully r e p l a c e d by i n c r e a s e d C D P - c h o l i n e pathway activity e v e n in the p r e s e n c e of additional choline in hepatocytes isolated from P E M T knockout m i c e . Similarly, another recent study s h o w e d that P E M T knockout mice d e v e l o p e d steatosis a n d w e r e unable to maintain normal concentrations of choline metabolites, e v e n w h e n s u p p l e m e n t e d with four times the normal choline requirement (113). T h i s s u g g e s t s the P E M T path has significance b e y o n d its r e c o g n i z e d role a s a s e c o n d pathway for P C biosynthesis, a n d 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 a n d P E M T path a p p e a r to be essential a n d have distinct a n d non-interchangeable  functions.  T h e link b e t w e e n choline a n d hepatic steatosis d e p e n d s o n whether the P E M T pathway c a n support the liver's requirement of P C (both functionally a n d quantitatively) for V L D L a n d T G secretion in the a b s e n c e of sufficient a b s o r b e d choline for P C s y n t h e s i s v i a the C D P - c h o l i n e pathway. In support of this, our r e s e a r c h group has s h o w n that p l a s m a h o m o c y s t e i n e a n d S A H are higher a n d S A M is lower in children with cystic fibrosis w h e n c o m p a r e d to children without cystic fibrosis (114). T h e s e findings are consistent with a n i n c r e a s e d de novo  32  Chapter 1 Literature review  synthesis of P C v i a the P E M T pathway. Interestingly, studies e x a m i n i n g the b i o c h e m i c a l a n d nutritional status of patients with C F h a v e s h o w n lower levels of low-density lipoprotein (LDL) a n d L D L - c h o l e s t e r o l (40, 115-117), w h i c h is a l s o consistent with r e d u c e d hepatic a p o B containing lipoprotein secretion, s i n c e V L D L is the precursor of L D L (118).  33  Chapter 1 Literature review 1.6 Summary T o our k n o w l e d g e , there are no published studies that specifically determined fecal p h o s p h o l i p i d excretion in patients with C F . It is c o n c e i v a b l e that choline a n d P C depletion o c c u r in patients with C F due to P C maldigestion and/or malabsorption s e c o n d a r y to pancreatic insufficiency, d e c r e a s e d bile output, intestinal abnormalities and/or failure of pancreatic e n z y m e s to normalize phospholipid digestion a n d absorption. C o n s i d e r a b l e r e s e a r c h h a s f o c u s e d o n total fat a n d fat s o l u b l e vitamins, with no attention given to p h o s p h o l i p i d absorption in patients with C F . B e c a u s e T G represents about 9 5 % of dietary fat (119), m e a s u r e s of the total fat content in stools are not sufficiently sensitive to detect e v e n large differences in P C excretion. T h i s study quantified fecal P C a n d l y s o P C excretion with a newly d e v e l o p e d high performance liquid chromatography ( H P L C ) method for separation of lipids a n d with an evaporative light scattering detector for quantification. W e h y p o t h e s i z e d that r e d u c e d lipolysis of dietary a n d biliary P C , d u e to maldigestion and/or m a l a b s o r p t i o n , results in i n c r e a s e d P C excretion. W e further hypothesized that i n c r e a s e d excretion of choline containing lipids l e a d s to hepatic choline a n d P C depletion in children with C F a n d this results in a n increase in the de novo ( P E M T ) pathway of choline synthesis to support P C requirements (Figure 1.4). B e c a u s e patients g i v e n choline-free T P N d e v e l o p steatosis a n d this is corrected by choline or P C supplementation, w e h y p o t h e s i z e that the P E M T pathway is not able to support all the requirements for n o r m a l liver  34  o  5  8-8  I  2  "a  zz  CO  to CD co CD  CO ' c "co CO E CD  cu CD >  CD i— • CO o  o c  o  ab  —'  o  o  c — — c o — sz c o o  O ]*> Q. QQ)  to  o  co to CD  sz  o a. >>  XZ sz  o 1— ro  CD to CD  ro J  CD J2  -E o  .CD  1 ^  ed  g  CD —  c o S ro _CD  CO  T3  CD  CL  CO  o  C  o  ro ^— » c  CO 0) CD l_  CD CO C — iD  CL o •*-» _c ro  CL  CD  CO  CD  T3 CO  CD CO O CD c CO CO TD  Ad ver  >  cz  o o 3 o _^  CD 5 "O .  D "5) J2 C CO -Q _ro  CO O CO CD -t^  CD w "  ro E  CD -C  o  CO C D  i_ 3  CO  Chapter 1 Literature review function in h u m a n s , thus, P C depletion in the liver m a y be important in etiology of hepatic steatosis in children with C F . T h e objective of this study is to quantify p l a s m a P C , l y s o P C , total cholesterol, high-density lipoprotein ( H D L ) cholesterol a n d a p o B , a n d fecal total phospholipid, P C a n d l y s o P C excretion a n d fecal fat absorption in children with C F in c o m p a r i s o n to normal, healthy children. A l t h o u g h p l a s m a (free) choline concentration d e c r e a s e s with dietary choline restriction, it m a y not be a sensitive marker of choline status b e c a u s e m e m b r a n e phospholipids, w h i c h are a large storage pool for choline, are hydrolyzed to maintain p l a s m a choline concentration above this minimal level (97). P l a s m a P C concentration also d e c r e a s e s in choline deficiency (120). R a p i d equilibration of liver and p l a s m a P C has b e e n s h o w n to o c c u r (121). T o date, only o n e study h a s reported p l a s m a P C concentrations in patients with C F ; this study u s e d separation of lipids, followed by an e n z y m a t i c colorimetric a s s a y involving p h o s p h o l i p a s e D a n d h o r s e r a d i s h p e r o x i d a s e to quantify P C (122). T h e authors found no difference in p l a s m a P C concentrations b e t w e e n patients with C F a n d healthy controls. D a t a o n p l a s m a P C concentrations are not available for healthy children, thus in this study, children without C F w e r e also recruited to g e n e r a t e 'normal' data. W e did not m e a s u r e dietary phospholipid a n d P C absorption b e c a u s e of the lack of data o n the phospholipid content of foods a n d the inability to quantify the a m o u n t of P C secreted into the intestine in bile. Folate a n d vitamin B  1 2  deficiency are known to result in elevation of  p l a s m a h o m o c y s t e i n e (123). T h u s , to a d d r e s s the possibility that elevated  36  -  Chapter 1 Literature review  homocysteine, if present, in children with C F is not d u e to folate and/or vitamin B  1 2  deficiency, w e a l s o m e a s u r e d the status of t h e s e nutrients. P l a s m a thiols  including methionine, S A M , h o m o c y s t e i n e , S A H w e r e m e a s u r e d in this study a n d as part of a larger study a n d the results h a v e b e e n published recently (114).  37  Chapter 1 Literature review  1.7 O B J E C T I V E S T h e overall objective of this study w a s to determine the extent of total phospholipid ( P L ) , P C a n d l y s o P C excretion in children with C F , a n d their possible a s s o c i a t i o n s with the a b n o r m a l h o m o c y s t e i n e a n d methionine status in children with cystic fibrosis ( C F ) . W e hypothesized that children with C F m a l a b s o r b p h o s p h o l i p i d s , particularly P C , which contributes to higher choline p h o s p h o g l y c e r i d e s excretion, a n d therefore choline a n d P C depletion in the liver, leading to reduced hepatic apolipoprotein B secretion, a n d lipid a c c u m u l a t i o n in liver in patients with C F . W e also hypothesized that the higher p h o s p h o l i p i d excretion is a s s o c i a t e d with e l e v a t e d h o m o c y s t e i n e a n d methionine status. Current standard m e t h o d s for quantifying fat excretion involve extraction of fat from fecal s a m p l e s with heptane, ether a n d ethanol. T h e s e solvents extract triglycerides a n d unesterified fatty a c i d s but do not quantitatively r e c o v e r phospholipids. T h e s e c o n d objective of this study w a s to d e v e l o p a quantitative method using high performance liquid c h r o m a t o g r a p h y with e v a p o r a t e d light scattering detection ( H P L C - E L S D ) for m e a s u r e m e n t of fecal p h o s p h o l i p i d excretion, w h i c h also allows determination of the p h o s p h o l i p i d c l a s s e s e x c r e t e d .  1.7.1 S p e c i f i c a i m s 1. T o d e v e l o p a quantitative method using high performance liquid chromatography with e v a p o r a t e d light scattering detection ( H P L C - E L S D ) for m e a s u r e m e n t of fecal phospholipid excretion.  38  .  Chapter 1 Literature review  2. T o validate the newly d e v e l o p e d H P L C - E L S D method for fecal phospholipid excretion using the p h o s p h o m o l y b d a t e colorimetric a s s a y of lipid soluble p h o s p h o r u s (124). 3. T o quantify fecal total P L , P C , l y s o P C a n d total fat excretion in children with C F a n d healthy children without C F . 4. T o determine dietary fat absorption in children with C F a n d healthy children without C F . 5. T o quantify a p o B , total a n d high-density lipoprotein ( H D L ) - c h o l e s t e r o l in children with C F a n d healthy children without C F . 6. T o identify relations between fecal total fat excretion, a n d the excretion of total P L , P C a n d l y s o P C excretion in children. 7. T o identify relations between dietary fat ( T G ) absorption ((intake-loss)/intake x 100%) a n d fecal total fat, P L , P C and l y s o P C excretion in children. 8. T o determine potential a s s o c i a t i o n s between fecal fat, P L , P C , l y s o P C excretion, a n d p l a s m a a p o B concentration, as indicators of hepatic T G a n d V L D L secretion in children. 9. T o determine potential a s s o c i a t i o n s between fecal P L , P C , l y s o P C excretion, a n d p l a s m a P C , l y s o P C , P C : P E ratio, methionine, h o m o c y s t e i n e , S a d e n o s y l h o m o c y s t e i n e 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 C h a p t e r 2 of this thesis a d d r e s s e s specific a i m s 1 a n d 2 of this study. T h e chapter is titled ' A s s e s s m e n t of phospholipid malabsorption by quantification of  39  .  Chapter 1 Literature review  fecal p h o s p h o l i p i d ' a n d h a s recently b e e n published in the J u n e 2 0 0 4 i s s u e of the Journal of Pediatric G a s t r o e n t e r o l o g y a n d Nutrition. C h a p t e r 3 of this thesis a d d r e s s e s specific a i m s 3-9 of this study. C h a p t e r 3 is titled ' P h o s p h a t i d y l c h o l i n e a n d lysophosphatidylcholine excretion is increased a n d related to altered p l a s m a h o m o c y s t e i n e a n d methionine in children with cystic fibrosis' a n d has b e e n submitted to the A m e r i c a n J o u r n a l of Clinical Nutrition for publication. C h a p t e r 4 of this thesis provides a s u m m a r y of the major findings of the study, d i s c u s s i n g the implications a n d suggesting the future directions for continuation of this r e s e a r c h .  40  Chapter 1 Literature review  1.8 1.  References C o r e y M , F a r e w e l l V . Determinants of mortality from cystic fibrosis in C a n a d a , 1970-1989. A m J E p i d e m i o l 1996;143,1007-17.  2.  R o m m e n s J M , l a n n u z z i M C , K e r e m B , et al. Identification of the cystic fibrosis g e n e : c h r o m o s o m e walking a n d jumping. S c i e n c e  1989,245:1059-  65. 3.  Sferra T J , C o l l i n s F S . T h e molecular biology of cystic fibrosis. A n n u R e v Med  4.  1993;44:133-44.  K e r e m B , R o m m e n s J M , B u c h a n a n J A , et a l . Identification of the cystic fibrosis g e n e : genetic analysis. S c i e n c e  5.  1989;245:1073-80.  G i b s o n L E , C o o k e R E . A test for concentration of electrolytes in s w e a t in cystic fibrosis of the p a n c r e a s utilizing pilocarpine by iontophoresis. Pediatrics  6.  1959;23:545-9.  Schofield D , C o t r a n R S . D i s e a s e s of infancy a n d c h i l d h o o d . In Cotran R S , K u m a n V , C o l l i n s T, eds. R o b b i n s Pathologic B a s i s of D i s e a s e . 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A r c h Dis Child 1991;66:495-500.  39.  B a r r a c l o u g h M , T a y l o r C J . Twenty-four hour ambulatory gastric a n d d u o d e n a l p H profiles in cystic fibrosis: effect of d u o d e n a l hyperacidity on pancreatic e n z y m e function and fat absorption. J P e d G a s t r o e n t e r o l Nutr 1996;23:45-50.  40.  B e n a b d e s l a m H , G a r c i a I, B e l l o n G , Gilly R, R e v o l A . B i o c h e m i c a l a s s e s s m e n t of the nutritional status of cystic fibrosis patients treated with pancreatic e n z y m e extracts. A m J C l i n Nutr 1998;67:912-8.  41.  N o u r i - S o r h k a b i M H , C h a p m a n B E , K u c h e l P W , et a l . P a r a l l e l secretion of pancreatic p h o s p h o l i p a s e A 2 , p h o s p h o l i p a s e A 1 , lipase a n d c o l i p a s e in children with e x o c r i n e pancreatic dysfunction. P e d i a t r R e s 2000;48:735-40.  42.  D a v i d s o n A G F . 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Directed modification instead of normalization of fatty acid patterns in cystic fibrosis: a n e m e r g i n g c o n c e p t . C u r r O p i n C l i n Nutr M e t a b C a r e 2001;4:111-3.  82.  W e i z m a n Z . V i t a m i n B12 absorption in cystic fibrosis. A c t a Pediatr S c a n d 1986;75:173-4.  83.  H e i d e H , H e n d r i k s H J E , Heijmans H , et al. A r e children with cystic fibrosis w h o are treated with a proton-pump inhibitor at risk for vitamin B12 deficiency? J Pediatr Gastroenterol Nutr 2001 ;33:342-5.  50  P a s c a l e R , Pirisi L , D a i n o S, et a l . R o l e of p h o s p h a t i d y l e t h a n o l a m i n e methylation in the synthesis of phosphatidylcholine by hepatocytes isolated from choline-deficient rats. F E B S Letters 1 9 8 2 ; 1 4 5 : 2 9 3 - 7 . C h a w l a R K , W o l f D C , Kutner M H , B o n k o v s k y H L . C h o l i n e m a y be a n e s s e n t i a l nutrient in m a l n o u r i s h e d patients with cirrhosis. G a s t r o e n t e r o l o g y 1989;97:1514-20. Y a o Z , V a n c e D E . R e d u c t i o n in V L D L , but not H D L , in p l a s m a of rats deficient in choline. B i o c h e m C e l l Biol 1990;68:552-8. B u c h m a n A L , D u b i n M D , M o u k a r z e l A A , et al. C h o l i n e deficiency: a c a u s e of hepatic steatosis during parenteral nutrition that c a n be r e v e r s e d with intravenous c h o l i n e supplementation. Hepatology 1 9 9 5 ; 2 2 : 1 3 9 9 - 4 0 3 . Y a o Z , V a n c e D E . T h e active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rate h e p a t o c y t e s . J Biol C h e m 1988;263:2998-3004. 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 1 9 8 9 ; 2 6 4 : 1 1 3 7 3 - 8 0 . S h e a r d N F , T a y e k J A , Bistrian B R , B l a c k b u r n G L , Z e i s e l S H . P l a s m a choline concentration in h u m a n s fed parenterally. A m J C l i n Nutr 1986;43:219-24. B u c h m a n A l , A m e n t M E , S o h e l M , et al. C h o l i n e deficiency c a u s e s reversible h e p a t i c abnormalities in patients receiving parenteral nutrition:  51  Chapter 1 Literature review proof of a h u m a n choline requirement: a placebo-controlled trial J P E N 2001;25:260-8. 92.  B u c h m a n A L , Dubin M , J e n d e n D , et a l . Lecithin i n c r e a s e s p l a s m a free choline a n d d e c r e a s e s hepatic steatosis in long-term total parental nutrition patients. G a s t e r o e n t e r o l o g y 1992;102:1363-70.  93.  Z e i s e l S H , Blusztajn J K . C h o l i n e a n d h u m a n nutrition. A n n u R e v Nutr 1994;14:269-96.  94.  C a n t y D J , Z e i s e l S H . Lecithin a n d choline in h u m a n health a n d d i s e a s e . Nutr R e v 1 9 9 4 ; 5 2 : 3 2 7 - 3 9 .  95.  M e y e r K C , S h a r m a A , B r o w n R, et al. Function a n d c o m p o s i t i o n of pulmonary surfactant a n d surfactant-derived fatty a c i d profiles a n d altered in y o u n g adults with cystic fibrosis. C h e s t 2 0 0 0 ; 1 1 8 : 1 6 4 - 7 4 .  96.  Z e i s e l S H , M a r M , H o w e J C , H o l d e r J M . C o n c e n t r a t i o n of c h o l i n e containing c o m p o u n d s a n d betaine in c o m m o n foods. J Nutr 2003;133:1302-7.  97.  F o o d a n d Nutrition B o a r d , Institute of M e d i c i n e . C h o l i n e . In Dietary R e f e r e n c e Intakes for T h i a m i n , Riboflavin, Niacin, V i t a m i n B , Folate, 6  Vitamin B , P a n t o t h e n i c A c i d , Biotin, a n d C h o l i n e . W a s h i n g t o n , D C : 12  National A c a d e m y P r e s s , 1998:12;1-20. 98.  Z e i s e l S H , G r o w d o n J H , W u r t m a n R J , Magil S G , L o g u e M . N o r m a l p l a s m a choline r e s p o n s e s to ingested lecithin. N e u r o l o g y 1 9 8 0 ; 3 0 : 1 2 2 6 - 9 .  52  : 99.  .  Chapter 1 Literature review  Z e i s e l S H . C h o l i n e a n d phosphatidylcholine. In S h i l s M E , O l s o n J A , S h i k e M , e d s . M o d e r n Nutrition in Health a n d D i s e a s e . P h i l a d e l p h i a : L e a & Febiger, 1 9 9 9 : 5 1 3 - 2 3 .  100.  S u n d l e r R, A k e s s o n B . R e g u l a t i o n of p h o s p h o l i p i d b i o s y n t h e s i s in isolated rat hepatocytes. J Biol C h e m 1975;250:3359-67.  101.  R e o N , A d i n e h a z a d e h M , F o y B . Kinetic a n a l y s e s of liver phosphatidylcholine a n d phosphatidylethanolamine biosynthesis using (13)C  102.  N M R spectroscopy. Biochim Biophys Acta 2002;1580:171-88.  Z e i s e l S H , Z o l a T, D a C o s t a K , Pomfret E A . Effect of choline deficiency o n S - a d e n s y l m e t h i o n i n e a n d methionine concentrations in rat liver. B i o c h e m J 1989;259:725-9.  103.  F a l l o n H J , G e r t m a n P M , K e m p E L . T h e effects of ethanol ingestion a n d choline deficiency o n hepatic lecithin biosynthesis in the rat. B i o c h i m B i o p h y s A c t a 1975;187:94-104.  104.  Hoffman D R , H a n i n g J A , C o r n a t z e r W E . Effects of a methyl-deficient diet on rat liver phosphatidylcholine biosynthesis. C a n J B i o c h e m 1981;59:543-50.  105.  Hoffman D R , H a n i n g J A , C o r n a t z e r W E . M i c r o s o m a l p h o s p h a t i d y l e t h a n o l a m i n e methyltransferase: inhibition by S a d e n o s y l h o m o c y s t e i n e . Lipids 1981;16:561-7.  106.  C u i Z , V a n c e D E . E x p r e s s i o n of phosphatidylethanolamine Nmethyltransferase-2 is markedly e n h a n c e d in long term choline-deficient rats. J Biol C h e m 1996;271:2839-43.  53  Chapter 1 Literature review 107.  W a l k e y C J , D o n o h u e L R , B r a n s o n R , A g e l l o n L B , V a n c e D E . Disruption of the murine g e n e e n c o d i n g phosphatidylethanolamine A/-methyltransferase. P r o c Natl A c a d S c i 1997;94:12880-5.  108.  W a i t e K A , Cabillio N R , V a n c e D E . C h o l i n e d e f i c i e n c y - i n d u c e d liver d a m a g e is reversible in P E M T - / - mice. J Nutr 2 0 0 2 ; 1 3 2 : 6 8 - 7 1 .  109.  H o u w e l i n g M , C u i Z , V a n c e D E . E x p r e s s i o n of p h o s p h a t i d y l e t h a n o l a m i n e A/-methyltransferase-2 cannot c o m p e n s a t e for a n impaired C D P - c h o l i n e pathway in mutant C h i n e s e hamster ovary cells. J Biol C h e m 1995;270: 16277-82.  110.  H o u w e l i n g M , C u i Z , T e s s i t o r e L , V a n c e D E . Induction of hepatocyte proliferation after partial h e p a t e c t o m y is a c c o m p a n i e d by a markedly reduced e x p r e s s i o n of phosphatidylethanolamine N-methyltransferase-2. B i o c h i m B i o p h y s A c t a 1997;1346:1-9.  111.  D e L o n g C J , S h e n Y J , T h o m a s M J , C u i Z . M o l e c u l a r distinction of phosphatidylcholine synthesis between the C D P - c h o l i n e pathway a n d phosphatidylethanolamine methylation pathway. J B i o l C h e m 1999;274:29683-8.  112.  N o g a A A , Z h a o Y , V a n c e D E . A n u n e x p e c t e d requirement for p h o s p h a t i d y l e t h a n o l a m i n e N-methyltransferase in the secretion of v e r y low density lipoproteins. J Biol C h e m 2 0 0 2 ; 2 7 7 : 4 2 3 5 8 - 6 5 .  113.  Zhu X , S o n g J , Mar M , Edwards L J , Zeisel S H . Phosphatidylethanolamine N-methyltransferase ( P E M T ) knockout m i c e h a v e hepatic steatosis a n d  54  Chapter 1 Literature review a b n o r m a l hepatic choline metabolite concentrations d e s p i t e ingesting a r e c o m m e n d e d dietary intake of choline. B i o c h e m J 2 0 0 3 ; 3 7 0 : 9 8 7 - 9 3 . 114.  Innis S M , D a v i d s o n A G F , C h e n A , et a l . Increased p l a s m a h o m o c y s t e i n e and s - a d e n o s y l h o m o c y s t e i n e a n d d e c r e a s e d methionine is a s s o c i a t e d with altered phosphatidylcholine a n d phosphatidylethanolamine in cystic fibrosis. J P e d i a t r 2003;143:351-6.  115.  V a u g h a n W J , L i n d g r e n 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. S c i e n c e 1978;199:783-6.  116.  S o l o m o n s N W , W a g o n f e l d J B , R i e g e r C , et a l . S o m e b i o c h e m i c a l indices of nutrition in treated cystic fibrosis patients. A m J C l i n Nutr 1981;34:46274.  117.  S l e s i n s k i M J , G l o n i n g e r M F , C o s t a n t i n o J P , O r e n s t e i n D M . Lipid levels in adults with cystic fibrosis. J A m Diet A s s o c 1994;94:402-8.  118.  G i n s b e r g H N . Lipoprotein m e t a b o l i s m and its relationship to atherosclerosis. M e d Clin North A m 1994;78:1-20.  119.  Groff J L , G r o p p e r S S , Hunt S M . T h e digestive s y s t e m : m e c h a n i s m for nourishing the body. In A d v a n c e d Nutrition a n d H u m a n M e t a b o l i s m . M i n n e a p o l i s : W e s t Publication, 1995:26-51.  120.  Z e i s e l S H , D a C o s t a K , Franklin P D , et a l . C h o l i n e , a n e s s e n t i a l nutrient for h u m a n s . F A S E B J 1991;5:2093-8.  121.  Bjornstad P , B r e m e r 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.  B u r d g e 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 a n d pancreatic supplementation. B r J Nutr 1944;71:959-64.  123.  J a c q u e s P F , A n d r e w G B , W i l s o n P W F , R i c h S, R o s e n b e r g IH, S e l h u b J . Determinants of p l a s m a total h o m o c y s t e i n e concentration in the F r a m i n g h a m offspring cohort. A m J Clin Nutr 2 0 0 1 ; 7 3 : 6 1 3 - 2 1 .  124.  C h e n P S , T o r i b a r a T Y , W a r n e r H . Microdetermination of p h o s p h o r u s . A n a l C h e m 1956;28:1756-8.  56  Chapter 2 Quantification of fecal phospholipid: method development  CHAPTER 2 QUANTIFICATION O F F E C A L PHOSPHOLIPID: M E T H O D D E V E L O P M E N T  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 P h o s p h o l i p i d ( P L ) constitutes 4-8 percent of the fat in most h u m a n diets. A major portion of dietary P L is phosphatidylcholine ( P C ) (1,2). Dietary P C is a source of essential fatty acids a n d choline, also a n essential nutrient. C h o l i n e is required for synthesis of the P C in lung surfactant, p l a s m a lipoproteins and cell m e m b r a n e s (3-6). L a r g e a m o u n t s of P C are also found in bile. In normal adults, the bile pool circulates 5-10 t i m e s per d a y a n d approximately 1 g r a m of P C is secreted in e a c h c y c l e (7). T h e digestion a n d absorption of dietary a n d biliary P C requires hydrolysis by pancreatic p h o s p h o l i p a s e A ( P L A ) to release l y s o P C . 2  2  This P L is then a b s o r b e d a n d r e a s s e m b l e d as P C in the liver (6). E x o c r i n e pancreatic insufficiency affects more than 8 5 % of patients with cystic fibrosis ( C F ) (8, 9). A l t h o u g h pancreatic e n z y m e r e p l a c e m e n t therapy is c o m m o n l y prescribed for patients with exocrine pancreatic insufficiency, it d o e s not completely correct malabsorption of fat (10). W e sought to determine whether i n c r e a s e d excretion of P C o c c u r s in C F . S o m e of the c o m p l i c a t i o n s of C F , namely hepatic steatosis a n d altered P L a n d thiol m e t a b o l i s m , might be a result of P L malabsorption (11, 12). H e p a t i c steatosis is a characteristic feature of choline deficiency w h i c h results in part from the inability of the choline deficient liver to s y n t h e s i z e P C required for hepatic secretion of triglyceride in v e r y - l o w density lipoproteins ( V L D L ) (6, 13). T o our knowledge, a reliable method for analysis of fecal P C excretion in C F or other d i s e a s e s c h a r a c t e r i z e d by impaired fat digestion has not b e e n previously reported. T h e standard clinical laboratory method for a s s e s s m e n t of fecal fat excretion is b a s e d o n the m e t h o d of V a n d e K a m e r et al (14). T h e V a n d e K a m e r  58  Chapter 2 Quantification of fecal phospholipid: method development  method involves c o n v e r s i o n of triglycerides a n d fatty a c i d s to fatty a c i d s o a p s , acidification to liberate unesterified fatty a c i d s a n d extraction with petroleum ether a n d ethanol. A l t h o u g h this method extracts long c h a i n triglycerides a n d fatty acids, it d o e s not extract m e d i u m - c h a i n triglycerides or fatty a c i d s . T o o v e r c o m e this, J e e j e e b h o y et al (15) u s e d heptane, diethyl ether a n d ethanol to extract m e d i u m a n d long c h a i n triglycerides a n d fatty a c i d s from fecal s a m p l e s . B a s e d on the properties of P L , w e predicted that t h e s e solvents w o u l d not quantitatively extract P L from fecal s a m p l e s . Although total tissue lipids (including P L ) are efficiently extracted with chloroform a n d methanol (16), this solvent s y s t e m d o e s not r e c o v e r the more water soluble m e d i u m c h a i n fatty a c i d s . O u r p u r p o s e w a s to d e v e l o p a method of extracting polar a n d non-polar lipids including P L a n d m e d i u m chain fatty a c i d s . W e u s e d high performance liquid c h r o m a t o g r a p h y with a n evaporative light scattering detector ( H P L C - E L S D ) for the separation a n d quantification of P L , including P C a n d l y s o P C . Quantification of p h o s p h o l i p i d by H P L C - E L S D w a s validated using the p h o s p h o m o l y b d a t e colorimetric a s s a y of lipid soluble p h o s p h o r u s (17).  59  Chapter 2 Quantification of fecal phospholipid: method development  2.1 Materials and Methods 2.1.1 Collection and preparation of samples F e c a l s a m p l e s w e r e collected quantitatively for 72 h from 18 children with C F a n d pancreatic insufficiency requiring pancreatic e n z y m e r e p l a c e m e n t s ,  and  from 8 healthy children. P a n c r e a t i c insufficiency in the children with C F w a s established b a s e d o n s u b n o r m a l fecal chymotrypsin and/or e l a s t a s e . T h e m e a n a g e s of the children with a n d without cystic fibrosis w e r e 9 . 3 ± 1 . 4 a n d 1 0 . 0 ± 0 . 1 y, respectively. T h r e e (17%) children with C F had had m e c o n i u m ileus a s infants. Five (28%) children with C F were taking u r s o d e o x y c h o l i c a c i d at the time of the study to treat b i o p s y p r o v e n liver d i s e a s e , although n o n e h a d clinically significant liver d i s e a s e . N o n e of the children had any other gastrointestinal d i s e a s e or intestinal resection. F e c a l s a m p l e s were frozen immediately after collection and maintained at - 7 0 ° C until a n a l y s i s . T h e study protocol w a s a p p r o v e d by the University of British C o l u m b i a Clinical R e s e a r c h E t h i c s B o a r d a n d the British C o l u m b i a C h i l d r e n ' s a n d W o m e n ' s Health Centre of British C o l u m b i a R e s e a r c h R e v i e w C o m m i t t e e . A l l participants and/or their parents or legal g u a r d i a n s provided written informed c o n s e n t (Appendix A).  2.1.2 Extraction and quantification of total fat F e c a l s a m p l e s w e r e thawed, w e i g h e d a n d h o m o g e n i z e d with a k n o w n amount of saline containing 1 5 % (weight/volume) ethylenediamine tetraacetic acid. A portion w a s dried to constant weight to allow calculation of fat content relative to fecal dry weight. H o m o g e n i z e d stool w a s acidified with 1 8 M hydrochloric a c i d to r e l e a s e fatty acids from s o a p s . A l i q u o t s w e r e then extracted 60  Chapter 2 Quantification of fecal phospholipid: method development  b a s e d on the m e t h o d s of J e e j e e b h o y et al (15), F o l c h et al (16), a n d by our method w h i c h involved a n initial extraction to recover m e d i u m a n d long c h a i n triglycerides followed by re-extraction to recover P L . S a m p l e s extracted by the method of J e e j e e b h o y w e r e extracted with hexane:diethyl ether:95% ethanol (1:1:1, by volume) T h e a q u e o u s p h a s e w a s then re-extracted twice with hexane:diethyl ether (1:1, by v o l u m e ) . P h a s e separation at e a c h step w a s facilitated by centrifugation at 5 0 0 g x 5 min, 4 ° C . T h e supernatants w e r e filtered, c o m b i n e d in pre-weighed acid w a s h e d g l a s s tubes and the solvents e v a p o r a t e d under a steady s t r e a m of nitrogen. S a m p l e s extracted by the method of F o l c h w e r e extracted with choloroform:methanol (2:1 by volume). T h e o r g a n i c solvent extraction w a s repeated twice. T h e infranatants w e r e c o m b i n e d a n d the solvents evaporated as a b o v e . T o a c h i e v e extraction of m e d i u m c h a i n triglycerides a n d fatty a c i d s and P L by our method, the s a m p l e w a s extracted with hexane:diethyl ether:95% ethanol (1:1:1 by v o l u m e ) and then extracted twice with hexane:diethyl ether (1:1 by v o l u m e ) . T h e infranatant w a s re-extracted with choloroform:methanol (2:1 by v o l u m e ) a n d then re-extracted with chloroform. T h e o r g a n i c p h a s e s w e r e filtered, c o m b i n e d , a n d the solvents e v a p o r a t e d . F o r all methods, the quantity of total fat w a s d e t e r m i n e d gravimetrically. T h e intra- a n d inter-assay coefficient of variation for e a c h method w a s d e t e r m i n e d by a n a l y s i s of 10 stool s a m p l e s in triplicate in three independent experiments (90 a s s a y s ) .  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 a n d l y s o P C in fecal fat w e r e determined by H P L C - E L S D (18). T h e total fat w a s r e s u s p e n d e d in chloroform/methanol/acetone/hexane (2.0/3.0/0.5/0.5, by v o l u m e ) a n d filtered through a 4 m m Millex-FH® filter (with hydrophobic P T F E m e m b r a n e , 0.5 u.m pore s i z e Nihon Millipore Ltd, Y o n e z a w a , J a p a n ) to r e m o v e potential interfering s u b s t a n c e s that might lower the sensitivity of the detector. S e p a r a t i o n of lipids w a s a c h i e v e d using H P L C (Waters 2 6 9 0 , Alliance, Milford, M A ) e q u i p p e d with a n autosampler, a c o l u m n heater a n d a normal p h a s e c o l u m n ( Y M C - P a c k Diol 1 2 0 N P , 2 5 c m X 4.6 m m i.d., 5 u m particle s i z e a n d 12 n m pore s i z e , Millford, M A ) with a flow rate of 2 ml/min using a quaternary solvent s y s t e m c o n s i s t i n g of hexane/petroleum ether (97:3 by volume); methanol/triethylamine/acetic acid (765:15:13 by v o l u m e ) ; acetone/triethylamine/acetic acid (765:15:13 by v o l u m e ) a n d isopropanol/acetic acid (800:40 by v o l u m e ) . T h e a u t o s a m p l e r c h a m b e r a n d the c o l u m n heater were kept at 1 8 ° C a n d 3 5 ° C , respectively. Lipid c l a s s e s w e r e detected a n d quantified using a n evaporative light scattering detector ( M o d e l 2 0 0 0 , A l l t e c h , M a n d e l Scientific, G u e l p h , O N ) with a nitrogen flow rate of 1.8 ml/min a n d drift tube temperature at 6 0 ° C . A n a l y s e s w e r e c o n d u c t e d in the linear range of the detector with calibration c u r v e s constructed using authentic standards for e a c h lipid c l a s s . Total P L w a s c a l c u l a t e d a s the s u m of the individual P L . Total p h o s p h o r u s in the fecal lipid extract w a s a l s o quantified using the colorimetric m e t h o d of C h e n et al (17), with m o n o b a s i c p o t a s s i u m p h o s p h a t e a s the standard.  62  Chapter 2 Quantification of fecal phospholipid: method development  2.1.4 Statistical analysis T h e a m o u n t s of total fat and P L extracted w e r e c o m p a r e d a m o n g the m e t h o d s using r e p e a t e d - m e a s u r e s A N O V A a n d least significant difference posth o c test. P e a r s o n correlation coefficients w e r e calculated to determine the strength of the correlation between the H P L C - E L S D quantification of total phospholipid a n d the colorimetric a s s a y of lipid soluble p h o s p h o r o u s . All statistical a n a l y s e s w e r e performed using S P S S 9.0.0 for W i n d o w s ( S P S S Inc, C h i c a g o ) . P - v a l u e s <0.05 w e r e c o n s i d e r e d statistically significant. M e a n v a l u e s are followed by + S E M .  63  Chapter 2 Quantification of fecal phospholipid: method development  2.2 R e s u l t s T h e i n c r e a s e d extraction of total fat a n d P L from fecal s a m p l e s a c h i e v e d by sequential extraction with solvents c h o s e n to optimize extraction of m e d i u m and long c h a i n triglycerides a n d fatty acids a n d with solvents d e s i g n e d to recover P L is s h o w n in F i g u r e 2.1. A l t h o u g h extraction with hexane:diethyl ether:95% ethanol optimizes recovery of m e d i u m a n d long c h a i n triglycerides a n d fatty acids from h u m a n feces (15), it d o e s not, a s s h o w n by our results, quantitatively recover P L . O n the other h a n d , the greater solubility of m e d i u m c h a i n triglycerides a n d fatty a c i d s in water results in poor recovery in solvent s y s t e m s b a s e d on chloroform, methanol a n d saline. T h u s , re-extraction of the infranatant with chloroform:methanol following extraction with h e x a n e a n d diethyl ether resulted in significantly greater fat extraction than a c h i e v e d with either hexane:diethyl ether or chloroform a n d methanol a l o n e (Figure 2.1). W e a c h i e v e d a three fold i n c r e a s e in recovery of P L by our method (2.01 ± 0 . 2 5 mg/g fecal dry weight) c o m p a r e d to the method of J e e j e e b h o y ( 0 . 6 4 ± 0 . 1 3 / g fecal dry weight) or a 3 5 % i n c r e a s e c o m p a r e d to the method of F o l c h ( 1 . 4 9 ± 0 . 2 3 m g / g fecal dry weight) ( P O . 0 0 1 ) . D e s p i t e the quantitative differences in fat extraction a m o n g the m e t h o d s , P e a r s o n correlation coefficients s h o w e d that the results for the different subjects w e r e significantly correlated. T h e correlation coefficients (r) for total fat excretion using the m e t h o d of J e e j e e b h o y or F o l c h with total fat extraction u s i n g our method w e r e 0.98 a n d 0.96, respectively w h e n e x p r e s s i n g total fat per g r a m of dry fecal weight a n d 0.99 for both m e t h o d s w h e n e x p r e s s i n g total fat p e r g r a m of wet fecal weight. A highly significant correlation w a s a l s o p r e s e n t b e t w e e n the  64  Chapter 2 Quantification of fecal phospholipid: method development  amount of P L extracted with chloroform:methanol a l o n e a n d the a m o u n t extracted by o u r m e t h o d u s i n g chloroform a n d m e t h a n o l following extraction with h e x a n e a n d diethyl, ether (r = 0.87). N o significant relationship w a s found between the recovery of P L by the method of J e e j e e b h o y a n d P L recovery by our method or that of F o l c h . T h e intra-assay a n d inter-assay coefficient of variation for total fat w e r e 1.8 a n d 5.2, 4 . 0 a n d 2.3, a n d 1.1 a n d 4.1 for the fecal fat extraction u s i n g the method of Jeejeebhoy, F o l c h a n d o u r m e t h o d , respectively. T h e quantification of P L by H P L C - E L S D w a s significantly correlated with lipid soluble p h o s p h o r o u s determined by the p h o s p h o m o l y b d a t e colorimetric a s s a y ( F i g u r e 2.2). A l t h o u g h the p h o s p h o m o l y b d a t e m e t h o d quantifies lipid soluble p h o s p h o r o u s , it provides no information on the nature of the lipid soluble p h o s p h o r o u s , or o n the types or a m o u n t s of individual P L . T h e distribution of individual P L , including P C , l y s o P C , phosphatidylethanolamine ( P E ) a n d sphingomyelin quantified by H P L C - E L S D is s h o w n in F i g u r e 2 . 3 . F e c a 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 F e c a l P L is m a d e u p of u n a b s o r b e d dietary a n d biliary P L a n d p o s s i b l y a c o m p o n e n t contributed by s l o u g h e d intestinal cells a n d c o l o n i c bacteria. Current routine clinical m e t h o d s for a s s e s s m e n t of fat malabsorption are b a s e d on recovery of triglycerides (14), which m a y be optimized to include extraction of more water soluble m e d i u m chain fatty a c i d s a n d triglycerides (15). S t a n d a r d methods for quantification of P L using colorimetric a s s a y of lipid soluble p h o s p h o r o u s (17) are limited b e c a u s e they d o not differentiate the origin of the p h o s p h o r o u s a n d give no information o n the a m o u n t or types of P L present. In addition, a v e r a g e c o n v e r s i o n factors are u s e d to convert p h o s p h o r u s to P L (19), and this calculation introduces errors of varying magnitude in the a n a l y s i s of biological s a m p l e s containing mixtures of l y s o P L a n d P L . Dietary fat malabsorption s e c o n d a r y to pancreatic insufficiency o r hepatobiliary dysfunction is likely to result in r e d u c e d digestion a n d absorption of P L . In healthy individuals, biliary a n d dietary P C together with bile a c i d s are a d s o r b e d to dietary triglyceride in the d u o d e n u m to provide a larger surface a r e a for triglyceride digestion by the pancreatic l i p a s e - c o l i p a s e c o m p l e x . P L A  2  then  hydrolyzes P C to r e l e a s e l y s o P C a n d a n unesterfied fatty a c i d , resulting in desorption of P L from the triglyceride substrate. T h e l y s o P C a n d unesterified fatty acid are t h e n a b s o r b e d b y enterocytes (20). H o w e v e r , in patients with pancreatic insufficiency, the secretion of P L A , lipase, c o l i p a s e a n d other 2  digestive e n z y m e s is d e c r e a s e d . A l t h o u g h P L A  2  is present in the pancreatic  e n z y m e s u p p l e m e n t s provided to patients with pancreatic insufficiency, including C F patients, the activity of p h o s p h o l i p a s e is inhibited at a n intraluminal p H b e l o w  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 P C 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 Sadenosylhomocysteine, 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  T h e n e w m e t h o d o l o g y d e s c r i b e d in this p a p e r will be helpful in elucidating the m e c h a n i s m of lipid m a l a b s o r p t i o n in patients with p a n c r e a t i c insufficiency a n d may thereby l e a d to more appropriate diet planning a n d revision of current concepts regarding pancreatic e n z y m e replacement for t h e s e patients. Furthermore, it m a y affect recent attempts to d e v e l o p synthetic pancreatic replacement e n z y m e s w h i c h do not contain p h o s p h o l i p a s e ( D a v i d s o n A G F 2 0 0 4 , oral c o m m u n i c a t i o n , 2 n d July). It remains to be s e e n w h e t h e r c h o l i n e supplementation to c o m p e n s a t e for i n c r e a s e d P L excretion s h o u l d be part of the nutrition m a n a g e m e n t of t h e s e patients.  68  Chapter 2 Quantification of fecal phospholipid: method development  Figure 2.1  Extraction of total fat and phospholipid using  ether and ethanol, — I I  Hi  hexane, diethyl  chloroform, methanol, or WM hexane, diethyl ether and  ethanol followed by re-extraction with chloroform, methanol . 1  Total fat  Dry stool  Phospholipid  1  1.5  CT E  Q.  Dry stool 1  The 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 V a l i d a t i o n of H P L C - E L S D method for the determination of fecal phospholipid w h e n c o m p a r e d with lipid soluble p h o s p h o r u s quantified by p h o s p h o m o l y b d a t e colorimetric a s s a y , 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  F i g u r e 2.3 Distribution of phospholipids in fecal fat from children with cystic fibrosis, n=18 and children without cystic fibrosis, n=8 . 1  Cystic Fibrosis Sph 14±3%  PC 28±8%  LysoPC 41 ± 6 % Control  PC  Sph 32+11%  10±4%  1  4±2%  PI  PE  2±1%  19±6%  P E , phosphatidylethonalamine;  LysoPC 34±9%  PI, phosphatidylinositol; P S ,  P C , phosphatidylcholine; S p h , sphingomyelin; L y s o P C ,  phosphatidylserine;  lysophosphatidycholine.  T h e a m o u n t of fecal p h o s p h o l i p i d in children with a n d without cystic fibrosis w a s ( 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. Z e i s e l S H , G r o w d o n J H , W u r t m a n R J , et al. 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J Lipid R e s 2002;43:1529-36. 19. R o u s e r G , S i a k o t o s A N , F l e i s c h e r S. Quantitative a n a l y s i s of p h o s p h o l i p i d s by thin-layer c h r o m a t o g r a p h y a n d p h o s p h o r u s a n a l y s i s of spots. Lipids 1965;1:85-6. 20. Nouri-Sorkhabi M H , C h a p m a n B E , K u c h e l P W , et a l . Parallel secretion of pancreatic p h o s p h o l i p a s e A 2 , p h o s p h o l i p a s e A 1 , lipase, a n d c o l i p a s e in children with exocrine pancreatic dysfunction. P e d R e s 2 0 0 0 ; 4 8 : 7 3 5 - 4 0 . 2 1 . D r e s s m a n J B , S h t o h r y n L V , Diokno D . Effects of product formulation on in vitro activity of pancreatic e n z y m e s . A m J H o s p P h a r m 1985;42:2502-6. 22. B a r r a c l o u g h M , T a y l o r C J . Twenty-four hour ambulatory gastric a n d d u o d e n a l p H profiles in cystic fibrosis: effect of d u o d e n a l hyperacidity o n pancreatic e n z y m e function a n d fat absorption. J P e d G a s t r o e n t e r o l Nutr 1996;23:45-50. 23. B u c h m a n A L , A m e n t M E , S o h e l M , et a l . C h o l i n e deficiency c a u s e s reversible hepatic abnormalities in patients receiving parenteral nutrition: proof of a human choline requirement: a placebo-controlled trial. J P E N 2 0 0 1 ; 2 5 : 2 6 0 - 8 .  74  Chapter 3 Phosphatidylcholine and ^ p h o s p h a t i d y l c h o l i n e excretion in C F  CHAPTER 3 PHOSPHATIDYLCHOLINE AND LYSOPHOSPHATIDYLCHOLINE E X C R E T I O N A N D ITS A S S O C I A T I O N WITH A L T E R E D P L A S M A H O M O C Y S T E I N E A N D METHIONINE IN C H I L D R E N WITH C Y S T I C 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. A m J Clin Nutr (submitted).  75  Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F  3. INTRODUCTION C y s t i c fibrosis ( C F ) is a n a u t o s o m a l r e c e s s i v e d i s o r d e r c a u s e d b y a mutation in the cystic fibrosis t r a n s m e m b r a n e c o n d u c t a n c e regulator, a n integral m e m b r a n e protein that w h e n activated by c A M P / p r o t e i n k i n a s e A , o p e n s to form a c h a n n e l to allow chloride ions to enter the cell (1). E x o c r i n e pancreatic insufficiency resulting in malabsorption of nutrients is present in 8 5 - 9 0 % of patients with C F (2,3). Despite pancreatic e n z y m e r e p l a c e m e n t s , s o m e t i m e s with a c i d reducing a g e n t s and/or proton pump inhibitors to i m p r o v e nutrient absorption, patients with C F continue to s h o w fat malabsorption (4-6). D e c r e a s e d bile acid concentrations, low intra-luminal intestinal p H a n d d e c r e a s e d m u c o s a l absorption have all b e e n s u g g e s t e d to contribute to the d e c r e a s e d absorption of fat in C F (5, 7-11). Hepatic steatosis has b e e n extensively d e s c r i b e d in C F (12,13), although the incidence is uncertain b e c a u s e liver biopsy, which is the only w a y to confirm its p r e s e n c e is not usually performed in C F . In addition to steatosis, significant progressive liver d i s e a s e , w h i c h m a y include cirrhosis a n d fibrosis h a s b e e n estimated to affect 1 7 - 3 7 % of patients with C F (12,13). A n estimated i n c i d e n c e of hepatic steatosis in children with C F b a s e d on liver b i o p s y h a s b e e n reported as 1 4 - 2 3 % (12,13). A l t h o u g h s e v e r a l hypotheses, including carnitine a n d essential fatty acid deficiency, h a v e b e e n p r o p o s e d (14-16), the etiology of steatosis a n d 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 c h o l i n e deficiency (17,18) a n d is b e l i e v e d to be e x p l a i n e d 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). T h e major pathway for P C biosynthesis in h u m a n s is the cytidine d i p h o s p h o c h o l i n e ( C D P - c h o l i n e ) path, which requires preformed c h o l i n e (17,20). In the alternate phosphatidylethanolamine-A/-methyltransferase ( P E M T ) path, methyl groups from methionine are transferred via S - a d e n o s y l m e t h i o n i n e ( S A M ) to phosphatidylethanolamine ( P E ) to form P C (19). T h e other product of the P E M T path is S - a d e n o s y l h o m o c y s t e i n e ( S A H ) w h i c h is s u b s e q u e n t l y converted to h o m o c y s t e i n e . During choline deficiency, the liver i n c r e a s e s the s y n t h e s i s of P C via the P E M T path (21). W e recently s h o w e d a n inverse relation between p l a s m a h o m o c y s t e i n e a n d the p l a s m a phospholipid P C content in children with C F (22), providing e v i d e n c e of possible insufficient choline to support P C synthesis v i a the C D P - c h o l i n e path. T h e u s u a l intake of P C is not k n o w n , although e x p e r i m e n t a l studies by Z e i s e l et al published in 1980 s u g g e s t e d that a mixed diet containing meat is likely to provide about 1g P C per day, of which about 9 0 % of choline in a n i m a l tissues is P C (23,24). H o w e v e r , large amounts of P C are a l s o s e c r e t e d into the intestine in bile. In normal individuals, the enterohepatic pool of P C is about 1g a n d this pool circulates 5-10 times/d with almost c o m p l e t e h y d r o l y s i s a n d reabsorption of P C (25). T h e absorption of dietary a n d biliary P C involves hydrolysis by pancreatic p h o s p h o l i p a s e A ( P L A ) , w h i c h h y d r o l y z e s P C to 2  2  l y s o P C a n d a n unesterified fatty acid (26), but this e n z y m e is inhibited at p H s below 5.8 (27). D u e to failure of bicarbonate secretion from the p a n c r e a s , a n d  77  Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F  despite s o m e buffering c a p a c i t y in e n z y m e s u p p l e m e n t s , m a n y patients with C F h a v e a postprandial d u o d e n a l intraluminal p H b e l o w 5.8 (11,28).  O u r a i m w a s to  determine if fecal c h o l i n e p h o s p h o g l y c e r i d e excretion is i n c r e a s e d , a n d if fecal choline p h o s p h o g l y c e r i d e excretion is related to p l a s m a h o m o c y s t e i n e a n d methionine in children with C F .  78  Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F  3.1 Subjects and Methods T h i s study involved 18 children with C F a n d 8 control children with no known health problems. All of the children with C F h a d pancreatic insufficiency a n d were taking pancreatic e n z y m e replacements (500-2500 U lipase/kg/meal). T h e s e children w e r e a s u b s e t of 53 children with C F w h o w e r e patients at the C F outpatient clinic at British C o l u m b i a ' s Children's Hospital ( B C C H ) a n d 18 control children without C F w h o participated in a c r o s s - s e c t i o n a l study d e s i g n e d to quantify p l a s m a lipids a n d thiols in C F (22). C o m p l e t e details of the study, together with the patients' characteristics have b e e n reported (22). C h i l d r e n enrolled in the study reported here all provided a quantitative 7 2 h stool s a m p l e a n d a 5d w e i g h e d food record, in addition to a v e n o u s blood s a m p l e . T h e food records w e r e collected o v e r 5 c o n s e c u t i v e days, that included 3 w e e k a n d 2 w e e k e n d d a y s . T h e children with C F followed their u s u a l diet a n d therapeutic regimen throughout the study period. T h e food record w a s c o m m e n c e d 2 d before the start of the stool collection. T h i s procedure w a s u s e d b e c a u s e children in our study w e r e unwilling to s e p a r a t e f e c e s b a s e d o n the p r e s e n c e of markers. T h e height a n d weight of e a c h study participant were r e c o r d e d u s i n g a stadiometer ( M 2 B , E N M C o . , C h i c a g o , IL) a n d electronic s c a l e ( S T 5 0 0 5 , S c a l e - T r o n i x Inc., White P l a i n s , N Y ) , respectively, a n d the z s c o r e s for e a c h child w e r e calculated (29). All study protocols a n d procedures w e r e a p p r o v e d by the University of British C o l u m b i a Clinical R e s e a r c h Ethics Board a n d the C h i l d r e n ' s a n d W o m e n ' s Health C e n t r e of British C o l u m b i a R e s e a r c h R e v i e w C o m m i t t e e . All participants a n d their parents provided written informed c o n s e n t (Appendix A).  79  Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F  3.1.1 Fecal analysis F e c a l s a m p l e s were frozen in pre-weighed containers immediately following collection a n d stored at - 7 0 ° C until analysis. F o r a n a l y s i s , fecal s a m p l e s were w e i g h e d , h o m o g e n i z e d a n d a portion dried to constant weight to allow determination of fecal water content. Total lipids were extracted and quantified gravimetrically. F e c a l phospholipids including P C , l y s o P C , P E , phosphatidylserine ( P S ) , phosphatidylinositol (PI) a n d s p h i n g o m y e l i n (Sph) were s e p a r a t e d a n d quantified using H P L C with evaporative light scattering detector ( H P L C - E L S D ) (30,31). F e c a l energy w a s quantified using a b o m b calorimeter (Model 1341, Parr, IL) a c c o r d i n g to manufacturer's instructions.  3.1.2 Plasma analysis T w o 7 m L v e n o u s blood s a m p l e s w e r e drawn from e a c h subject into tubes containing E D T A as anti-coagulant (22). T h e p l a s m a a n d red blood cells were s e p a r a t e d by centrifugation at 2 , 0 0 0 g, 15 min at 4 ° C , a n d frozen at - 7 0 ° C within 20 min of blood collection. P l a s m a total lipids were extracted a n d the phospholipids s e p a r a t e d a n d quantified by H P L C - E L S D (22,31). P l a s m a methionine, h o m o c y s t e i n e a n d their metabolites were a n a l y z e d by H P L C with reversed p h a s e ion-pairing (32). P l a s m a a p o B w a s quantified u s i n g immunoturbidimetric reagents ( S i g m a Diagnostic, St. L o u i s , M O ) , and total cholesterol and high density lipoprotein (HDL)-cholesterol w e r e determined using colorimetric e n z y m a t i c reagents (Diagnostic C h e m i c a l s , Charlottetown, P E I ) .  80  Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F  3.1.3 Dietary analysis All vitamin, mineral a n d other nutritional s u p p l e m e n t s , in addition to foods and b e v e r a g e s w e r e r e c o r d e d o n the food records (Appendix B). T h e dietary records w e r e entered into a nutrient a n a l y s e s data b a s e ( E S H A F o o d P r o c e s s o r V e r s i o n 7.71, E S H A R e s e a r c h , S a l e m O R ) and the total energy, fat, carbohydrate a n d protein intake (g/d) w a s calculated. T h e total energy intake of e a c h subject w a s a l s o calculated a s % estimated energy requirement ( E E R ) per d for healthy individuals, b a s e d o n age, gender, weight, height a n d physical activity levels (33). A physical activity coefficient equivalent to walking 1.5-2.9 miles/d at 2-4 m p h w a s a s s u m e d for e a c h subject. Fat absorption w a s calculated a s [(total fat intake (g/d)-fecal fat excretion (g/d))/total fat intake (g/d)] x 1 0 0 % . Total choline intake a n d the intake of choline from P C w a s estimated using the U S D A d a t a b a s e o n the choline content of c o m m o n foods (24).  3.1.4 Statistical analysis Data are p r e s e n t e d a s m e a n s ± S E M s . Independent 2-tailed t-testfor normally distributed data, a n d M a n n - W h i t n e y test for nonparametric unpaired data were u s e d to c o m p a r e the results for children with C F a n d the controls. P e a r s o n correlation coefficient for normal data a n d S p e a r m a n ' s rank correlation coefficient for n o n p a r a m e t r i c data w e r e calculated to determine potential a s s o c i a t i o n s b e t w e e n fecal phospholipid excretion a n d p l a s m a h o m o c y s t e i n e , methionine a n d S A H . A l l statistical a n a l y s e s w e r e performed u s i n g S P S S 9.0.0  81  -  Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F  for W i n d o w s ( S P S S Inc, C h i c a g o ) . P - v a l u e s <0.05 w e r e c o n s i d e r e d statistically significant.  82  Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F  3.2 Results In this study, w e determined fecal fat a n d p h o s p h o l i p i d excretion a n d the relation of fecal choline p h o s p h o g l y c e r i d e excretion to p l a s m a methionine, S A H a n d h o m o c y s t e i n e of children with a n d without C F . T h e age of the children with C F (n=18) a n d of the control children w a s 9 . 3 ± 1 . 4 a n d 1 0 . 0 ± 0 . 1 y , respectively. T h e z - s c o r e s for height-for-age of the C F a n d control g r o u p s w e r e -0.47+0.17, a n d 0.51+0.24, respectively, a n d the z - s c o r e s for weight-for-age w e r e 0.48+0.16 a n d 0.24+0.16, respectively, P>0.05. T h r e e of the 18 children with C F had m e c o n i u m ileus a s infants, 5 w e r e taking u r s o d e o x y c h o l i c acid at the time of the study for elevated liver e n z y m e s or a b n o r m a l liver ultrasound findings. N o n e of the children had a n y other gastrointestinal d i s e a s e or resection, or clinically significant liver d i s e a s e . T h e children with C F had a m e a n energy intake of 142.0+3.5% of the E E R . T h e e n e r g y intake of the control children w a s 1 0 6 . 2 ± 4 . 7 % E E R a n d w a s significantly lower t h a n in the children with C F , P < 0 . 0 0 1 . T h e intake of dietary fat w a s a l s o significantly higher in children with C F ( 9 6 . 7 ± 6 . 8 g/d) t h a n in the control children (70.1 ± 6 . 7 g/d, P=0.025). Fat, carbohydrate a n d protein contributed to 3 5 ± 1 . 6 % , 5 0 ± 1 . 7 % a n d 14+0.7% of the total energy intake in children with C F , a n d 3 0 ± 1 . 6 % , 5 6 ± 1 . 5 % a n d 1 4 ± 0 . 6 % of total energy intakes in the control children, respectively (P>0.05). T h e intake by children with a n d without C F for choline ( 3 3 5 ± 3 3 a n d 2 6 8 ± 2 5 mg/d) a n d P C ( 8 6 5 ± 1 1 2 a n d 8 1 2 ± 1 5 8 mg/d) w a s not different.  83  Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F  T h e 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 . 0 0 4 (Table 3.1). Consistent with the r e d u c e d dietary fat absorption, daily fecal fat excretion by the children with C F w a s 3-4 fold higher than in the control children, P - 0 . 0 0 3 . F e c a l energy excretion w a s about two fold higher in children with C F than in the control children, but w a s not statistically significant, possibly d u e to the wide interindividual variability in fecal e n e r g y excretion (Table 3.1). H o w e v e r , fecal fat and the fecal e n e r g y content w e r e significantly a s s o c i a t e d , r=0.89, P O . 0 0 0 1 , (Figure 3.1). T h e r e w a s no significant difference in fecal water content b e t w e e n the children with C F a n d the control children. Total p h o s p h o l i p i d s , P C a n d l y s o P C excretion w e r e all significantly higher in children with C F t h a n 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 w a s 5 0 mg/g dry stool. Of the 8 children in the control group, 2 h a d a fecal P C concentration in the detectable range, but below 0.5 mg/d P C excretion. T h e range of fecal P C excretion in children with C F w a s 0.86-140.9 mg/d, c o m p a r e d to <0.5-8.8 mg/d in the control children. A b o u t 9 0 % of the fecal choline p h o s p h o g l y c e r i d e s  excreted  by the control children w a s l y s o P C . In contrast, l y s o P C r e p r e s e n t e d about 6 0 % and P C represented about 4 0 % of the fecal choline p h o s p h o g l y c e r i d e  excretion  in children with C F (Table 3.2). P E , P S , PI and S p h excretion w e r e not significantly different b e t w e e n the children with C F a n d the control children. However, d u e to the higher fecal excretion of P C a n d l y s o P C , P E , P S , PI a n d S p h represented 3 1 % of the p h o s p h o g l y c e r i d e s e x c r e t e d by children with C F a n d  84  Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F  6 2 % p h o s p h o g l y c e r i d e s excreted by the control children, P<0.001. T h e fecal phospholipid excretion w a s also significantly a s s o c i a t e d with the fecal total fat excretion, at r=0.78, P<0.001 (Figure 3.1). A significant positive a s s o c i a t i o n w a s also found between the fecal excretion of P E , P C , l y s o P C a n d S p h a n d fecal fat excretion at r=0.55, P =0.003, r =0.82, P O . 0 0 1 , r=0.60, P=0.001, a n d r=0.43, s  P=0.027, respectively, a n d between fecal P C , l y s o P C , P E , PI, a n d S p h with fecal total phospholipids excretion at r =0.88, P O . 0 0 1 , A=0.86, P<0.001, r =0.55, s  P=0.004, r =0.44, P=0.024, and r=0.55, P=0.003, respectively. F a t absorption (%) w a s also significantly inversely a s s o c i a t e d with fecal total fat, total phospholipid, P C , l y s o P C a n d P E excretion, r=-0.86, P<0.001, r=-0.82, P O . 0 0 1 , r =-0.75, s  P O . 0 0 1 , r=-0.78, P O . 0 0 1 and r=-0.49, P=0.011, respectively. T h e p l a s m a concentrations of methionine (PO.001) a n d the P C / P E ratio (P=0.03) were lower, a n d P E (PO.001), h o m o c y s t e i n e (PO.001) a n d S A H (P=0.001) w e r e higher in the children with C F than in the control children (Table 3.3). T h e p l a s m a P C a n d l y s o P C were not significantly different (P=0.1) between the children with C F and the control children. T h e p l a s m a a p o B , but not the total or H D L cholesterol w a s a l s o significantly lower (P=0.03) in the children with C F than in the control children. P l a s m a a p o B w a s inversely a s s o c i a t e d with fecal total phospholipids a n d P C at r=-0.58, P=0.008, r =-0.43, P=0.034, respectively. s  W e did not find a n y a s s o c i a t i o n between p l a s m a a p o B , a n d fecal fat a n d l y s o P C . This study a l s o found a significant positive a s s o c i a t i o n b e t w e e n the fecal total phospholipid, P C a n d l y s o P C a n d the p l a s m a h o m o c y s t e i n e a n d an inverse association between the fecal phospholipid, P C a n d l y s o P C a n d the p l a s m a  85  Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F  methionine for all subjects in the study (Table 3.4). In addition, the p l a s m a h o m o c y s t e i n e w a s significantly a n d positively a s s o c i a t e d with fecal total phospholipid, fecal choline a n d fecal total choline p h o s p h o g l y c e r i d e s within the subgroup of children with C F (Figure 3.2). T h e fecal total p h o s p h o l i p i d a n d P C excretion were a l s o significantly a n d positively a s s o c i a t e d with the p l a s m a S A H . T h e r e were no statistically significant a s s o c i a t i o n s b e t w e e n the fecal total phosphlipid, P C or l y s o P C excretion and p l a s m a 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 k n o w l e d g e , our study provides the first quantitative demonstration of i n c r e a s e d fecal choline p h o s p h o g l y c e r i d e ( P C a n d l y s o P C ) excretion in cystic fibrosis. O u r study also provides n e w data to s h o w i n c r e a s e d fecal choline p h o s p h o g l y c e r i d e excretion is a s s o c i a t e d with e l e v a t e d p l a s m a h o m o c y s t e i n e in h u m a n s . T o g e t h e r with our recent report to s h o w p l a s m a homocysteine a n d S A H are positively a s s o c i a t e d with p l a s m a P E a n d inversely related to p l a s m a P C (22), our work provides e v i d e n c e that P C synthesis via P E M T is inter-related with the transfer of methyl g r o u p s from S A M to S A H in a m a n n e r which i m p a c t s p l a s m a h o m o c y s t e i n e a n d p l a s m a P C / P E ratios in h u m a n s . C o n s i s t e n t with this suggestion, recent studies h a v e s h o w n a n approximately 5 0 % lower p l a s m a h o m o c y s t e i n e concentration in P E M T -/- m i c e w h e n c o m p a r e d with wild type m i c e (34), thus s u g g e s t i n g that the rate of P E M T methylation of P E to form P C is a determinant of p l a s m a h o m o c y s t e i n e in this species. It is well k n o w n that despite pancreatic e n z y m e r e p l a c e m e n t therapy, dietary fat absorption remains lower in patients with C F t h a n in individuals without C F (7,35,36). In our study, children with C F , all of w h o m w e r e taking pancreatic e n z y m e s , a b s o r b e d about 8 6 % of the dietary fat intake c o m p a r e d with 9 4 % fat absorption by the control children. Similarly, M u r p h y et al (35) a n d B u r d g e et al (36) found 8 6 % a n d 7 8 % of dietary fat w a s a b s o r b e d , respectively, in patients with C F . T o the best of our k n o w l e d g e , our work is the first to d e s c r i b e the amount a n d type of phospholipids excreted by children or adults. A l t h o u g h  87  Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F  phospholipids usually contribute 4-8% of dietary fat (37), s t a n d a r d m e t h o d s for the extraction of fecal lipids do not allow quantitative recovery of p h o s p h o l i p i d s (30). T o a d d r e s s this, w e recently d e v e l o p e d methodology for quantitative extraction of fecal phospholipids, followed by separation a n d quantification of phospholipid c l a s s e s using H P L C - E L S D (30). T h e significant relations between fecal energy a n d fat excretion, a n d between fecal fat a n d p h o s p h o l i p i d excretion s u g g e s t s that fat malabsorption is a c c o m p a n i e d by a higher excretion of phospholipids, a s well a s energy. B e c a u s e P C s e c r e t e d into the intestine in bile is essential for normal fat digestion (38), it is possible that r e d u c e d P C availability, s e c o n d a r y to i n c r e a s e d excretion of choline p h o s p h o g l y c e r i d e s could limit the digestion a n d absorption of dietary triglycerides. H o w e v e r , d e c r e a s e d bile acids, reduced m u c o s a l absorptive function (7,8), or reduced activities of c o - l i p a s e d e p e n d e n t pancreatic lipase as well a s P L A could also e x p l a i n our results. 2  W h e t h e r i n c r e a s e d excretion of c h o l i n e phospholipids o c c u r s in other d i s o r d e r s involving impaired pancreatic or biliary function, or intestinal absorptive dysfunction is not k n o w n . T h e high a m o u n t s of l y s o P C excreted by the control children a n d the children with C F in our study w e r e u n e x p e c t e d , although w e are u n a w a r e of similar data o n the c o m p o s i t i o n of f e c a l phospholipids. F e c a l l y s o P C c o u l d originate from dietary a n d biliary P C h y d r o l y z e d lower in the intestine b e y o n d 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 d u e to limited bicarbonate secretion from the p a n c r e a s , and/or s l o w 2  dissolution of enteric pancreatic e n z y m e s in the upper intestine could e x p l a i n the  88  Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F  i n c r e a s e d hydrolysis of P C to l y s o P C in the lower intestines, a n d t h u s i n c r e a s e d excretion of l y s o P C in children with C F . H o w e v e r , l y s o P C w a s a l s o the major choline p h o s p h o g l y c e r i d e excreted by healthy children without C F . L e c i t h i n a s e positive bacteria are present in colonic microflora, a n d the a b u n d a n c e of t h e s e o r g a n i s m s has b e e n reported to be sensitive to dietary v a r i a b l e s (39,40). W h e t h e r or not the types a n d amounts of p h o s p h o g l y c e r i d e s present in the c o l o n has any p h y s i o l o g i c a l r e l e v a n c e to the colonization of lecithinase-positive bacteria and intestinal function is unknown. O u r studies w e r e initiated to a d d r e s s the possibility that altered hepatic P C metabolism s e c o n d a r y to i n c r e a s e d P C excretion could contribute to the high prevalence of hepatic steatosis a m o n g patients with C F (12,13). T h e estimated intake of P C in our study w a s 8 6 5 ± 1 1 2 mg/d for children with C F a n d 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 Z e i s e l et al (23). W e found that fecal P C a n d l y s o P C excretion w a s about 8 0 m g / d in children with C F a n d about 2 0 m g / d in the children without C F . T h e s e results s u g g e s t that P C absorption is probably a s efficient a s total fat absorption in c h i l d r e n , i.e. about 9 2 - 9 8 % . S o m e studies however, h a v e provided e v i d e n c e of i n c r e a s e d m e m b r a n e lipid turnover in C F defective cells, w h i c h could s u g g e s t that c h o l i n e requirements are higher in patients with C F (41). A s s e s s m e n t of hepatic triglyceride a c c u m u l a t i o n involving liver b i o p s y in the a b s e n c e of clinical e v i d e n c e of liver d i s e a s e is not ethically a c c e p t a b l e . Therefore, w e s o u g h t e v i d e n c e of i n c r e a s e d P C excretion through m e a s u r e s of fecal choline p h o s p h o g l y c e r i d e excretion a n d u s e d a novel a p p r o a c h to a d d r e s s  89  Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F  alteration of hepatic P C m e t a b o l i s m b a s e d on the intersection of the methionineh o m o c y s t e i n e pathway with p h o s p h o l i p i d m e t a b o l i s m at the methylation of P E to form P C , with the generation of S A H a n d h o m o c y s t e i n e from methionine v i a S A M (22). O u r hypothesis that altered hepatic choline m e t a b o l i s m with r e d u c e d synthesis in the C D P pathway m a y o c c u r in C F w a s b a s e d on the k n o w l e d g e that hepatic triglyceride a c c u m u l a t i o n is a characteristic feature of choline deficiency (17,18), e x p l a i n e d by the requirement for de novo P C biosynthesis v i a the C D P choline path for secretion of triglyceride in a p o B containing lipoproteins (19,20). In addition, patients with C F s h o w fat malabsorption despite pancreatic replacement e n z y m e therapy (7,35,36). Further, many patients with C F h a v e a n intraluminal p H b e l o w 5.8 (4,11), w h i c h is the threshold for activity of P L A  2  (26).  In this study, w e h a v e s h o w n that children with C F excrete higher a m o u n t s of P C a n d l y s o P C than healthy control children. Total choline p h o s p h o g l y c e r i d e ( l y s o P C + P C ) excretion in the children with C F w a s about 5 0 - 2 0 0 m g / d a y , c o m p a r e d with 6-70 m g in the control children. T h e enterophepatic pool of P C in healthy adults has b e e n estimated to be about 1 gram (25). W h e t h e r or not the i n c r e a s e d in fecal P C c o m p r o m i s e s P C status is not k n o w n . O n the other h a n d , the estimated net absorption of P C a p p e a r s to b e similar in both the children with C F (785mg/d) a n d the healthy control (792mg/d) (Total P C intake - fecal P C a n d fecal l y s P C ) . S o m e studies have provided e v i d e n c e of i n c r e a s e d m e m b r a n e lipid turnover in C F defective cells, which c o u l d s u g g e s t that choline requirements are higher in patients with C F (41). T h e higher fecal phospholipids excretion, together with the p o s s i b l y the i n c r e a s e d choline requirement raise the question of whether  90  Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F  or not the availability of c h o l i n e to support the C D P - c h o l i n e path might be d e c r e a s e d , a n d thus i n c r e a s e the d e m a n d in the liver to generate P C v i a the P E M T path in C F . T h e significant positive relations b e t w e e n choline p h o s p h o g l y c e r i d e excretion, a n d p l a s m a S A H a n d h o m o c y s t e i n e , a n d the inverse relation with p l a s m a methionine support a hypothesis that the i n c r e a s e d h o m o c y s t e i n e a n d S A H level found in children with C F is related to i n c r e a s e d synthesis of P C v i a 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 d e c r e a s e in the p l a s m a P C / P E ratio a s found by us in children with C F . W e previously s h o w e d that the higher p l a s m a h o m o c y s t e i n e a n d S A H in children with C F is not e x p l a i n e d by deficiency of folate or vitamin B12 (22). H o w e v e r , alternate explanations s u c h a s oxidative reduction of methionine s y n t h a s e are p o s s i b l e . M e a s u r e s of p l a s m a free choline w e r e not included in our study b e c a u s e p l a s m a free c h o l i n e concentrations are low, with a range of about 7-20 u.mol/L in adult m e n which overlaps the m e a n of 7.5 n m o l / L found in p l a s m a of adults fed a c h o l i n e deficient diet (44). Dietary p h o s p h o l i p i d a n d P C absorption w a s quantified using the recent U S D A d a t a b a s e o n the c h o l i n e content of foods (24). Further details, including the U S D A d a t a b a s e , are i n c l u d e d in Appendices D a n d E. W e w e r e not able to quantify the a m o u n t of P C s e c r e t e d into the intestine in bile without invasive techniques, thus P C absorption c o u l d not be quantified. H o w e v e r , consistent with r e d u c e d secretion of V L D L from the liver, the p l a s m a a p o 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. R e d u c e d p l a s m a a p o B  91  Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F  and cholesterol concentrations have been reported in previous studies with patients with CF ( 4 5 - 4 7 ) . In summary, children with C F 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 children  7  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  NS  Fecal energy (kJ/d)  1130±215  627±146  NS  Fecal energy (kJ/g dry weight)  24.2±1.7  19.7±1.4  NS  2  V a l u e s shown are mean ± SEM (range) and were analyzed using independent 2-tailed t-tests 2  NS, not significant.  93  Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F  TABLE 3.2 F e c a l phospholipid excretion in children with cystic fibrosis a n d control children  1  zj m  g  /  or (n=18)  d  Total phospholipids  (  ^W?29.7)  Phosphatidylcholine  ^ f f ^ . e )  Lysophosphatidylcholine  {  Phosphatidylethanolamine  Jf  ? 2  2  fl  ^24A  „  h i  „„„_,„  = l i  „  1  5.3)  19.6+3.6  0  a  0  l  3  u  4  0 0 0 5  A  J  ° ° ^  %  (0.ll7%%.7)  (1.4-51.6,17.0)  D a t a s h o w n are m e a n s ± S E M (range,median)  v  6  (  Phosphatidylserine Sphingomyelin  (1 ¥i%V47.7)  (J^ffe.V  t0)  (olu^z.T)  P  0.wil)>  ^f]  {0  .  0  {  0 3 )  Phosphatidylinositol  c  uontrol (n=8)  N  1  4  *  S  N  S  N  S  N  S  20.7±7.2  (6.7-66.1,11.0)  a n d w e r e a n a l y z e d using  independent 2-tailed t-test for normally distributed data, a n d M a n n - W h i t n e y test for nonparametric data. 2  F e c a l excretion of phospholipid of >0 a n d <0.5mg/d w a s b e l o w the linear range  of calibration, a n d recorded a s 0.5mg/d. 3  N S , not significant.  94  e  Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F  T A B L E 3.3 P l a s m a thiols and phospholipids in children with cystic fibrosis a n d control c h i l d r e n  7  CF (n=18)  Control (n=8)  P value  H o m o c y s t e i n e (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  32.6±2.0  20.3+1.4  0.001  S-adenosylmethionine (u.M)  74.7±3.4  84.5±6.4  NS  Total P h o s p h o l i p i d (u.M)  732±76.7  747+110  NS  10.2±1.4  3.7+0.4  <0.001  76.7+2.1  82.3+1.8  NS  S p h i n g o m y e l i n (%)  9.1+0.9  11.0+1.4  NS  L y s o p h o s p h a t i d y l c h o l i n e (%)  3.6±0.3  2.8±0.3  NS  P C / P E ratio  13.0±3.2  24.8±3.5  0.03  Total cholesterol (mg/dL)  158±10.4  154+7.6  NS  H D L - c h o l e s t e r o l (mg/dL)  42.1±2.2  44.5±4.2  NS  Apolipoprotein B (mg/dL)  46.9±1.7  54.4±3.2  0.03  S-adenosylhomocysteine  (u.M )  Phosphatidylethanolamine  ( P E ) (%)  Phosphatidylcholine ( P C ) (%)  2  V a l u e s s h o w n are m e a n ± S E M and w e r e a n a l y z e d u s i n g i n d e p e n d e n t 2-tailed ttests 2  N S , not significant P > 0 . 0 5  95  Chapter 3 Phosphatidylcholine and Ivsonhosphatidvlcholine excretion in CF  T A B L E 3.4 A s s o c i a t i o n s b e t w e e n p l a s m a thiols a n d fecal p h o s p h o l i p i d e x c r e t i o n  Plasma  Total PL  Fecal Phospholipid PC  LysoPC  Methionine  r=-0.49, P = 0 . 0 1 3  r =-0.59, P = 0 . 0 0 2  r=-0.60, P=0.001  Homocysteine  r=0.64, P=0.001  r =0.76, P<0.001  r=0.58, P=0.002  SAH  r=0.52, P=0.008  r =0.64, P=0.001  NS  s  s  s  2  V a l u e s are r = P e a r s o n correlation coefficient, r = S p e a r m a n ' s rank correlation s  coefficient. P L = phospholipids, P C = phosphatidylcholine, L y s o P C = lysophosphatidylcholine, a n d S A H = 2  S-adenosylhomocysteine.  N S , not significant.  96  7  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 l a s m a h o m o c y s t e i n e v e r s u s 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 , a n d d) total choline p h o s p h o g l y c e r i d e s in the subgroup of children with cystic fibrosis*, n=18.  13 -  12 -  _  11  me  _  % o  10 •  9 -  r =0.66, P=0.00 s  50  100  150  200  250  300  350  0  20  Fecal total phospholipid (mg)  _  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  Fecal ^phosphatidylcholine (mg)  140  0  50  100  150  200  Fecal choline phosphoglyceride (mg)  *r= P e a r s o n correlation coefficient, r = S p e a r m a n ' s rank correlation s  98  250  300  Chapter 3 Phosphatidylcholine and lysophosphatidylcholine excretion in C F  3.4 References 1. 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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;23:4550. 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 , P o g r i b n a M , P o g r i b n y IP, et al. A b n o r m a l folate m e t a b o l i s m a n d mutation in the methylenetetrahydrofolate  r e d u c t a s e g e n e m a y be maternal  risk factors for D o w n s y n d r o m e . A m J Clin Nutr 1 9 9 9 ; 7 0 : 4 9 5 - 5 0 1 . 33. Institute of M e d i c i n e . Dietary reference intakes for energy, carbohydrate, fiber, fat, fatty a c i d s , cholesterol, protein, and amino a c i d . W a s h i n g t o n , D C : National A c a d e m i e s P r e s s , 2 0 0 2 . 34. N o g a A A , S t e a d L M , Z h a o Y , B r o s n a n M E , B r o s n a n J T , V a n c e D E . P l a s m a h o m o c y s t e i n e is regulated by phospholipid methylation. J Biol C h e m 2003;278:5952-5. 35. M u r p h y J L , W o o t t o n S A , B o n d S A , J a c k s o n A A . E n e r g y content of stools in normal healthy controls a n d patients with cystic fibrosis. A r c h Dis Child 1991;66:495-500. 36. B u r d g e 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 a n d pancreatic supplementation. B r J Nutr 1994;71:959-64. 37. C a r e y M C , Hernell O . Digestion a n d absorption of fat. S e m i n G a s t r o i n t e s D i s 1992;3:189-208. 38. R o y C C , W e b e r A M , Morin C L , et a l . Hepatobiliary d i s e a s e in cystic fibrosis: a survey of current i s s u e s a n d c o n c e p t s . J Pediatr G a s t r o e n t e r o l Nutr 1982;1:469-78. 39. B e n n o Y , M i z u t a m T, N a m b a Y , K o m o r i T, M i t s u o k a T . C o m p a r i s o n of fecal microflora pf elderly p e r s o n s in rural a n d urban a r e a s of J a p a n . A p p l Environ Microbiol 1 9 8 9 ; 5 5 : 1 1 0 0 - 5 .  Chapter 3 Phosphatidylcholine and Ivsophosphatidvlcholine excretion in C F  40. B e n n o Y , S a w a d a K , M i t s u o k a T. T h e intestinal microflora of infants: composition of fecal flora in breast-fed a n d bottle-fed infants. M i c r o b i o l Immunol 1984;28:975-86. 4 1 . U l a n e M M , Butler J D , P e r i A , Miele L, U l a n e R E , H u b b a r d V S . C y s t i c fibrosis and phosphatidylcholine biosynthesis. Clin C h i m A c t a 1994;230:109-16. 42. Hoffman D R , H a n i n g J A , C o r n a t z e r W E .  Microsomal  phosphatidylethanolamine methyltransferase: inhibition by S a d e n o s y l h o m o c y s t e i n e . Lipids 1981;16:561-7. 43. Y i P , M e l n y k S, P o g r i b n a M , Pogribny IP, H i n e R J , J a m e s S J . Increase in p l a s m a h o m o c y s t e i n e a s s o c i a t e d with parallel i n c r e a s e s in p l a s m a S a d e n o s y l h o m o c y s t e i n e a n d lymphocyte D N A hypomethylation. J Biol C h e m 2000;275:29318-23. 44. Z e i s e l S H , D a C o s t a K , Franklin P D , et a l . C h o l i n e , a n e s s e n t i a l nutrient for h u m a n s . F A S E B J 1991,5:2093-8. 45. V a u g h a n W J , L i n d g r e n 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. S c i e n c e 1978;199:783-6. 46. B e n a b d e s l a m H , G a r c i a I, Bellon G , e t a l . B i o c h e m i c a l a s s e s s m e n t of the nutritional status of cystic fibrosis patients treated with pancreatic e n z y m e extracts. A m J C l i n Nutr 1998;67:912-8. 4 7 . S l e s i n s k i M J , G l o n i n g e r M F , Constantin J P , O r e n s t e i n 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 A N D C O N C L U S I O N S  105  Chapter 4 Discussion and conclusions 4. INTRODUCTION T h e objective of this thesis r e s e a r c h w a s to determine if phospholipid ( P L ) a n d P C excretion in children with cystic fibrosis ( C F ) is i n c r e a s e d a n d to a s s e s s possible a s s o c i a t i o n s b e t w e e n fecal P C excretion a n d p l a s m a methionine a n d homocysteine. P C excretion a n d p l a s m a thiols were studied b e c a u s e hepatic steatosis is a r e c o g n i z e d problem a m o n g patients with C F (1, 2) a n d hepatic steatosis is a l s o a characteristic feature of choline deficiency (3, 4). T h e hepatic steatosis of choline deficiency is believed to be e x p l a i n e d by a specific requirement for active hepatic de novo P C biosynthesis v i a 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 k n o w n that despite pancreatic e n z y m e replacement therapy, patients with C F continue to malabsorb fat (7-9). W h e t h e r or not patients with C F a l s o m a l a b s o r b phospholipids, particularly P C , has not been previously reported. T h e a m o u n t of phospholipid in typical diets is not known; s o m e authors h a v e s u g g e s t e d that phospholipid represent 4 - 8 % of dietary fat (10, 11). H o w e v e r , in h u m a n milk, which contains fat a s triglyceride in a globule s u r r o u n d e d by p h o s p h o l i p i d s , P L contributes only about 0 . 7 % of total fat (12). In our study, the total dietary P C w a s about 8 7 0 m g / d in children with C F a n d 810mg/d in the n o n C F controls, while total intake of fat w a s 97g/d in children with C F a n d 70g/d in the control children. A s s u m i n g P C represent 9 0 % of dietary P L , then in our studies, P L w a s 0 . 9 % of dietary fat, P L represented 1% a n d 1.3% of dietary fat intake in our children with C F a n d in the control children, respectively. It is notable that t h e s e e s t i m a t e s of  106  Chapter 4 Discussion and conclusions P L intake b a s e d o n our experimental data are c l o s e to h u m a n milk, for w h i c h the total fat a n d P L content h a v e b e e n well d e s c r i b e d . P C r e p r e s e n t s >90% of the dietary intake of the e s s e n t i a l nutrient choline (10, 13). T h e major route of hepatic P C biosynthesis is the C D P - c h o l i n e pathway, which requires a s o u r c e of preformed choline (14,15). In the alternate pathway for P C synthesis, c a t a l y z e d by phosphatidylethanolamine  methyltransferase  ( P E M T ) , sequential methylation of P E using methyl g r o u p s from methionine via S-adenosylmethionine ( S A M ) leads to the formation of P C , with Sa d e n o s y l h o m o c y s t e i n e ( S A H ) a s the other product. S A H is s u b s e q u e n t l y metabolized to h o m o c y s t e i n e (16). During experimental choline deficiency in animals, the hepatic concentration of S A H is i n c r e a s e d , a n d S A M a n d methionine are d e c r e a s e d . T h i s has b e e n interpreted to reflect a n attempt by the liver to i n c r e a s e 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 r e s e a r c h , w e h a v e s h o w n that children with C F h a v e a higher p l a s m a homocysteine, a n d S A H a n d lower p l a s m a methionine a n d P C / P E ratio w h e n c o m p a r e d with children without C F . Further, w e h a v e s h o w n a n inverse relation between p l a s m a h o m o c y s t e i n e a n d the proportion of P C in p l a s m a phospholipids (16). T h e alteration in p l a s m a thiols a n d a s s o c i a t i o n with p l a s m a P C is consistent with a h y p o t h e s i s of hepatic choline depletion in C F . A n objective of this thesis r e s e a r c h w a s to d e v e l o p a quantitative method for extraction a n d quantification of fecal phospholipids. High performance liquid chromatography with evaporative light scattering detection ( H P L C - E L S D ) w a s  107  Chapter 4 Discussion and conclusions used for the separation a n d quantification of fecal phospholipids c l a s s e s . A new lipid extraction solvent s y s t e m w a s d e v e l o p e d for extraction a n d quantification of fecal p h o s p h o l i p i d s . B a s e d o n the properties of phospholipids, w e predicted that current standard m e t h o d s for extracting fecal fat only extract triglycerides a n d unesterified fatty a c i d s , but d o not quantitatively recover p h o s p h o l i p i d s . Furthermore, a reliable method for analysis of P C excretion has not b e e n previously reported. T h e purpose of this chapter is to d i s c u s s the method d e v e l o p e d for extraction and quantitation of fecal triglycerides and p h o s p h o l i p i d s , followed by a d i s c u s s i o n of fat a n d phospholipids excretion in children with C F c o m p a r e d with the n o n - C F healthy control, a n d the implications of the a s s o c i a t i o n s b e t w e e n choline p h o s p h o g l y c e r i d e ( P C and l y s o P C ) excretion, a n d p l a s m a methionine, homocysteine a n d S A H . Finally, a d i s c u s s i o n of limitations and future directions, followed by the c o n c l u s i o n will be presented to s u m m a r i z e the major findings of this thesis r e s e a r c h .  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 s e q u e n t i a l extraction of fecal lipids with hexane:diethyl ether followed by chloroform a n d methanol to quantify fecal total fat and phospholipids is reported (specific a i m 1 of this thesis research). T h e combination of solvent s y s t e m s d e s c r i b e d results in  108  Chapter 4 Discussion and conclusions about a 3 0 % i n c r e a s e in total fat extraction a n d a three fold i n c r e a s e in recovery of phospholipids c o m p a r e d with the standard clinical method u s e d for extraction of fecal fat (19). In addition, the method gives about a 1 0 % i n c r e a s e in total fat extraction a n d a 3 5 % i n c r e a s e in recovery of p h o s p h o l i p i d s c o m p a r e d with the extraction method of F o l c h et al (20), which is a standard method for tissue lipid extraction. T h e new m e t h o d involving the combination of the two solvent s y s t e m s w a s d e v e l o p e d for two r e a s o n s . First, phospholipids typically represent o n l y 4 8% of dietary fat a n d previously published methods for fecal fat quantitation w e r e not c a p a b l e of detecting s m a l l but meaningful differences in fecal excretion. F o r e x a m p l e , in a 30 year-old low active adult m a n with a height of about 1.7m a n d weight of 7 0 k g , c o n s u m i n g 2,500 kcal/d with 2 0 - 3 5 % of dietary energy intake from fat, 4 - 8 % dietary fat from P L would be equivalent to a n intake of 2.2-7.8 g phospholipid/d. Similarly, for a low active 12 year-old boy, of 1.5 m height a n d 40.5 kg b o d y weight c o n s u m i n g 2 1 0 0 kcal/d with 2 5 - 3 5 % dietary e n e r g y from fat, the intake would be 2.3-6.6 g phospholipid/d (21). Typically, 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 e n z y m e s u p p l e m e n t s ( 2 2 ) . A literature s e a r c h of p a p e r s p u b l i s h e d 19662 0 0 4 did not reveal any p u b l i s h e d studies on p h o s p h o l i p i d absorption or excretion in h u m a n s ; h e n c e the extent of p h o s p h o l i p i d malabsorption or excretion c a n only be estimated from published data on fat absorption. A s s u m i n g 8 5 % absorption of dietary p h o s p h o l i p i d in children with C F a n d 9 3 % absorption in the  109  Chapter 4 Discussion and conclusions healthy control children, our study population of C F children a n d control children might be e x p e c t e d 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. T h u s , a s the difference in phospholipid excretion between children with and without C F is s m a l l relative to the amount of total fat excreted and would be below the sensitivity of current standard m e t h o d s for quantification of total fat excretion. T h u s in this r e s e a r c h , a method w a s d e v e l o p e d that w o u l d specifically allow the extraction a n d quantification of p h o s p h o l i p i d . O f note, in addition to the u n a b s o r b e d dietary phospholipid, the fecal phospholipid pool is also likely to contain u n a b s o r b e d biliary phospholipid a n d phospholipids derived from s l o u g h e d intestinal cells and colonic microflora. T h u s , the total amount of phospholipid e x c r e t e d by the healthy subjects without malabsorption c o u l d be higher than 0.16-0.46 g/d. S e c o n d l y , b a s e d on the solubility of polar and non-polar lipids and different solvents, w e predicted that the standard clinical laboratory methods, s u c h a s the m e t h o d of J e e j e e b h o y et al (19), w h i c h i n v o l v e s extraction with hexane:diethyl ether:95% ethanol (1:1:1, by v o l u m e ) and hexane:diethyl ether (1:1, by v o l u m e ) (19), w o u l d not allow complete extraction of phospholipids from fecal s a m p l e s . H o w e v e r , solvent s y s t e m s b a s e d on chloroform a n d methanol (20) while extracting p h o s p h o l i p i d s , are not expected to recover the more water soluble m e d i u m c h a i n fatty a c i d s from fecal s a m p l e s . Therefore, a c o m b i n a t i o n of lipid extraction s y s t e m s that c o m b i n e d the strengths of ether a n d chloroform  n o  Chapter 4 Discussion and conclusions b a s e d solvent extraction w a s u s e d to extract polar a n d non-polar lipids, including P L and m e d i u m c h a i n fatty a c i d s . M e d i u m chain fatty a c i d s m a y b e present in fecal s a m p l e s a s a result of malabsorption of m e d i u m c h a i n fatty a c i d s from the nutrition s u p p l e m e n t s c o n s u m e d by s o m e C F patients. In our study, two children with C F (11%) w e r e on nutrition s u p p l e m e n t s that contained M C T oil at the time of the study. W e s h o w e d 1 0 % higher total fat a n d a 3 5 % higher recovery of phospholipid using our method for fecal fat extraction w h e n c o m p a r e d to the F o l c h method for tissue lipid extraction (20) a n d 3 0 % higher total fat a n d a three fold higher recovery of phospholipid c o m p a r e d to a standard clinical method for a s s e s s m e n t of f e c a l fat excretion (19). First, the i n c r e a s e d total fat a n d phospholipid could be e x p l a i n e d by the more appropriate u s e of solvent s y s t e m s for extraction of fat a n d phospholipids. H o w e v e r , it is well r e c o g n i z e d that repeat extraction of s a m p l e s with solvents a l s o improves the recovery of lipid (23). W e extracted the fecal s a m p l e s with chloroform a n d methanol twice, a n d extracted three times with h e x a n e a n d diethyl ether. Christie W W (1993) h a s reported that in the method by F o l c h et al (20), the proportions of chloroform, m e t h a n o l a n d water in the c o m b i n e d p h a s e s s h o u l d b e a s c l o s e a s p o s s i b l e to 8:4:3 (by v o l u m e ) to prevent selective l o s s e s of s o m e lipids to the a q u e o u s p h a s e a n d for reliable results (23). F e c a l s a m p l e s typically consist of about 6 0 - 8 0 % of w a t e r a n d 2 0 4 0 % undigested fiber, u n a b s o r b e d protein, lipids a n d intestinal microflora (24). W e calculated the v o l u m e s of chloroform a n d methanol required to extract fecal lipid a s s u m i n g that 1 0 0 % of the fecal s a m p l e w a s a q u e o u s ; c o n s e q u e n t l y , it is  in  Chapter 4 Discussion and conclusions still p o s s i b l e that w e failed to extract s o m e more polar lipids from the fecal s a m p l e s . H o w e v e r , the chloroform-methanol extraction followed the extraction of triglyceride a n d unesterified fatty a c i d s using hexane:diethylether:95% ethanol (1:1:1 by v o l u m e ) a n d hexane:diethylether (1:1 by v o l u m e ) s h o u l d h a v e resulted in the r e m o v a l of m o s t of the fecal lipid and m a y h a v e altered the proportions of chloroform, methanol a n d water in the s u b s e q u e n t steps, any the error in a s s u m i n g 1 0 0 % a q u e o u s material in the fecal s a m p l e s w o u l d have b e e n r e d u c e d . T h e u s e of freeze dried fecal s a m p l e s in future studies w o u l d allow better control of the proportions of chloroform, methanol a n d water, and m a y further improve lipid extraction. In chapter 2 of this thesis r e s e a r c h , w e a l s o reported the u s e of H P L C E L S D for separation a n d quantification of fecal p h o s p h o l i p i d s . Quantification of fecal phospholipids by H P L C - E L S D s h o w e d a high a n d significant correlation with lipid soluble p h o s p h o r o u s , a s determined by the p h o s p h o m o l y b d a t e colorimetric a s s a y (A>0.75, P<0.01) (specific a i m 2 of this thesis research). Standard methods for quantification of phospholipids are b a s e d on colorimetric a s s a y of lipid soluble p h o s p h o r u s (25). T h e later method is limited b e c a u s e it d o e s not provide any information o n the type of p h o s p h o l i p i d s , i.e. p h o s p h o l i p i d c l a s s e s present. A n a d v a n t a g e of the H P L C - E L S D method for fecal phospholipids quantitation is its ability to s e p a r a t e the p h o s p h o l i p i d s into individual p h o s p h o l i p i d c l a s s e s for detection a n d quantification. Additionally, the p h o s p h o m o l y b d a t e a s s a y relies o n a v e r a g e c o n v e r s i o n factor of 2 5 to covert lipid-soluble p h o s p h o r u s to phospholipid (26). T h i s c o n v e r s i o n factor of 2 5 is  112  Chapter 4 Discussion and conclusions b a s e d on the a v e r a g e m o l e c u l a r weight of a phospholipid (i.e. 750), divided by the approximate atomic weight of phosphorus (i.e. 30). T h i s calculation thus introduces errors of varying magnitude in the a n a l y s i s of biological s a m p l e s b e c a u s e typically biological s a m p l e s contain a mixture of p h o s p h o l i p i d s w h i c h differ in m o l e c u l a r weight, w h i c h may range from about 4 6 0 to 8 7 0 , d u e to both differences in the polar h e a d groups a n d in fatty acyl c o m p o n e n t s (e.g. P C , l y s o P C , phosphatidylinositol, sphingomyelin...etc). In the r e s e a r c h presented in this thesis, the amount of p h o s p h o l i p i d quantified using the colorimetric a s s a y w a s two to three fold higher (after multiplying the amount of p h o s p h o r u s by the c o n v e r s i o n 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. T h e overestimation of phospholipid by colorimetric a s s a y could be e x p l a i n e d by the large amount of l y s o P C , w h i c h represented about 3 0 4 0 % of total phospholipid in the fecal s a m p l e s from children with a n d without C F in this study. T h e m o l e c u l a r weight of l y s o P C with 16:0 a s the s o l e fatty acyl group is 4 7 5 , c o m p a r e d to 7 5 8 for P C with 16:0, 18:2n-6. T h e higher apparent lipid soluble p h o s p h o r u s in the p h o s p h o m o l y b d a t e a s s a y might a l s o be due to p r e s e n c e of other p h o s p h o l i p i d s (e.g. l y s o P E , lysoPI, lyso P S ) in the fecal s a m p l e s that w e r e not identified o n the H P L C c h r o m a t o g r a m . In s u m m a r y , chapter 2 of this thesis research has reported a simple method for extraction a n d quantification of phospholipids from fecal s a m p l e s . T h i s new m e t h o d will be helpful in providing insights regarding the m e c h a n i s m s of lipid malabsorption in patients with pancreatic insufficiency. T h e method h a s  113  Chapter 4 Discussion and conclusions  clinical r e l e v a n c e b e c a u s e i n c r e a s e d P C excretion could l e a d to hepatic P C depletion, w h i c h w o u l d w o r s e n bile synthesis a n d secretion, w h i c h in turn m a y w o r s e n fat m a l a b s o r p t i o n . T h e publication of the results m a y a l s o affect recent attempts to d e v e l o p n e w synthetic pancreatic replacement e n z y m e s that, unlike current formulations, d o not contain p h o s p h o l i p a s e ( D a v i d s o n A G F 2 0 0 4 , oral communication, 2  n d  July). P a n c r e a t i c p h o s p h o l i p a s e A ( P L A ) is e s s e n t i a l for the 2  2  hydrolysis of P C to l y s o P C a n d a free fatty acid for absorption. A l t h o u g h P L A  2  is  inactivated at low intraluminal p H s , which are c o m m o n in patients with C F , P L A  2  from pancreatic e n z y m e s u p p l e m e n t s m a y be important for P L digestion in s o m e patients with C F w h o are pancreatic insufficient but respond well to pancreatic e n z y m e r e p l a c e m e n t therapy in conjunction with a c i d r e d u c i n g a g e n t s . It r e m a i n s to be s e e n whether supplementation with water soluble c h o l i n e to c o m p e n s a t e for i n c r e a s e d phospholipid excretion s h o u l d be part of the nutrition m a n a g e m e n t of patients with cystic fibrosis.  4.1.2 Fat absorption and phospholipid excretion C h a p t e r 3 of this thesis d e s c r i b e s a research study that u s e d the methodology for extraction a n d quantification of fecal p h o s p h o l i p i d excretion d e s c r i b e d in chapter 2 to determine if children with C F excrete higher a m o u n t s of phospholipid than children without C F . T o the best of our k n o w l e d g e , this research is the first quantitative demonstration of i n c r e a s e d fecal c h o l i n e p h o s p h o g l y c e r i d e ( P C a n d l y s o P C ) excretion in children with C F c o m p a r e d to  Chapter 4 Discussion and conclusions children without C F . Further, t h e s e studies are the first to d e s c r i b e the amount a n d type of phospholipid e x c r e t e d by h u m a n s . T h e digestion a n d absorption of P C requires hydrolysis by P L A to release 2  l y s o P C a n d a n unesterified fatty acid (27). However, in patients with pancreatic insufficiency d u e to C F , the secretion of bicarbonate, P L A , lipase, c o l i p a s e a n d 2  other digestive e n z y m e s is d e c r e a s e d (27). A l t h o u g h pancreatic e n z y m e supplements are provided to patients with C F who are pancreatic insufficient, it is not known if the e n z y m e s are sufficiently active in the intestine to h y d r o l y z e dietary a n d biliary P C . It has b e e n reported that the activity of pancreatic lipase is inhibited at a n intraluminal p H below 5.8 (28). In addition, a s d i s c u s s e d in chapter 1, P L A a p p e a r s to be more sensitive to inhibition at an a c i d i c p H than pancreatic 2  co-lipase d e p e n d e n t lipase (29). T h e postprandial intraluminal p H in the upper small intestine of m a n y patients with C F is below 5.8 (7, 30). Of the dietary intake of the essential nutrient choline, more than 9 0 % is in the form of P C , with the remaining smaller portion being a s water soluble free choline a n d s p h i n g o m y e l i n , a n d minor amount of g l y c e r o p h o s p h o c h o l i n e a n d p h o s p h o c h o l i n e ( 1 0 , 1 3 ) . C h o l i n e a s P C is an important constituent of cell m e m b r a n e s , lipoproteins a n d lung surfactant, a n d is a l s o the p r e c u r s o r for synthesis of s p h i n g o m y e l i n . C h o l i n e functions in the neurotransmitter acetylcholine. C h o l i n e is a l s o present in s m a l l a m o u n t s a s other derivatives of choline p h o s p h o g l y c e r i d e s including 1-alk-1-enyl-, 2-acyl-glycero-3p h o s p h o c h o l i n e , a l s o n a m e d p l a s m a l o g e n , w h i c h is p r e s e n t in the heart m u s c l e , s e m i n a l fluid, a n d in s m a l l e r amounts in nervous s y s t e m , platelets or red blood  115  Chapter 4 Discussion and conclusions cells and 1-alkyl-,2-acetyl-glycero-3-phosphocholine, a l s o k n o w n a s platelet activating factor w h i c h is important in inflammatory a n d i m m u n e r e s p o n s e s a n d in platelet aggregation. T h e metabolite of choline, betaine is a l s o a s o u r c e of labile methyl groups (31-33). Further, P C is a n important c o m p o n e n t of bile, which is important for solubilizing biliary and dietary lipids; bile lipids are essential in the formation of m i x e d micelles to provide a n i n c r e a s e d surface a r e a for digestion of lipids and fat-soluble vitamins. Hepatic steatosis, and a reduction of p l a s m a P C , triglyceride and very-low-density lipoprotein ( V L D L ) concentrations are w e l l - k n o w n features of choline deficiency, a n d h a s b e e n reported in patients on long-term total parenteral nutrition ( T P N ) lacking choline (17, 34-36). T h e research d e s c r i b e d in this thesis w a s prompted by the high p r e v a l e n c e of hepatic steatosis a m o n g patients with C F and w a s directed at investigating the possibility that i n c r e a s e d choline excretion m a y o c c u r in C F (1,2). If so, this w o u l d raise the possibility that choline depletion m a y be important a s a contributing factor in the d e v e l o p m e n t of hepatic steatosis in C F . T h e results of the study reported in chapter 3 s h o w e d 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 a n overall f o u r f o l d higher total choline p h o s p h o g l y c e r i d e excretion than the healthy control children without C F (specific a i m 3 of this thesis research). C o n s i s t e n t with literature, our results also s h o w e d that despite pancreatic e n z y m e replacement therapy, children with C F had a lower fat absorption (86%) than children without C F (94%) (specific aim 4 of this thesis r e s e a r c h ) (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 activity and reduce the ability to digest dietary and biliary phospholipids to 2  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 C F 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 C F defective cells, which could suggest that choline requirements are higher in patients with C F (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 colipase dependent lipase and of PLA , or intestinal mucosal or unstirred water 2  layer defects that reduced the absorption of the products of fat digestion could explain our results.  120  Chapter 4 Discussion and conclusions T h e positive correlation between fecal fat a n d p h o s p h o l i p i d excretion, a n d the inverse correlation b e t w e e n fat absorption a n d p h o s p h o l i p i d excretion raise the question if the higher phospholipid excretion in children with C F than in the control children is s i m p l y e x p l a i n e d by i n c r e a s e d fat malabsorption. H o w e v e r , c l o s e r examination of the study data s h o w s that the distribution of the phospholipid c l a s s e s excreted by the children with C F w a s significantly different from that excreted by the healthy control children without C F (Figure 2.3). T h u s , the phospholipid pool excreted by the children with C F h a d a b o u t 2 8 % a n d 4 1 % P C and l y s o P C respectively, c o m p a r e d to 4 % and 3 4 % P C a n d l y s o P C , respectively in the control children. T h e s e d a t a s u g g e s t e d that the higher phospholipid excretion in children with C F d o e s involve a specific excretion of P C , a n d is not simply a s a result of a higher choline p h o s p h o g l y c e r i d e excretion. Dietary phospholipid w a s estimated using the 2004 U S D A d a t a b a s e for choline content of c o m m o n foods ( A p p e n d i c e s D & E ) . P C absorption c o u l d not b e quantified d u e to the i n v a s i v e n e s s of the procedures. In our study, p l a s m a a p o B w a s lower, but p l a s m a total a n d H D L cholesterol concentration were not different between the children with C F and the control children (specific a i m 5 of this thesis research). A p o B w a s m e a s u r e d b e c a u s e apo B is present in V L D L , intermediate density lipoprotein (IDL) a n d low density lipoprotein ( L D L ) , but not in high density lipoprotein ( H D L ) . T h u s , the p l a s m a a p o B w a s u s e d as an indicator of hepatic V L D L secretion (49), recognizing that s o m e p l a s m a a p o B is a l s o present in c h y l o m i c r o n s of intestinal origin. T h e lower p l a s m a a p o B in children with C F than in the control children is  121  Chapter 4 Discussion and conclusions consistent with r e d u c e d secretion of a p o B containing lipoprotein from the liver. Further, the inverse a s s o c i a t i o n found between p l a s m a a p o B a n d fecal total phospholipid a n d P C are a l s o consistent with hepatic choline depletion in C F (specific a i m 8 o f this thesis). H o w e v e r , a n alternate e x p l a n a t i o n is that r e d u c e d fat absorption, a n d thus i n c r e a s e d fecal fat excretion, results in r e d u c e d p l a s m a a p o B containing triglyceride rich lipoproteins. F o r practical p u r p o s e s a n d b e c a u s e of ethical considerations, the blood s a m p l e s in this study w e r e not collected under fasting conditions of studying children. H o w e v e r , p u b l i s h e d data are available to s h o w that, although p l a s m a triglyceride is affected by fed-fasting state, total p l a s m a cholesterol, H D L cholesterol and a p o B concentrations are not affected (50).  4.1.3 A s s o c i a t i o n between choline phosphoglyceride excretion, and plasma methionine, homocysteine and S A H W e recently p u b l i s h e d the first report to s h o w that p l a s m a h o m o c y s t e i n e a n d S A H are higher, a n d p l a s m a methionine a n d the proportion of P C in p l a s m a P L is lower in a large g r o u p of children with C F w h e n c o m p a r e d with healthy control children without C F (16). T h e children w h o participated in the r e s e a r c h described in this thesis w e r e a s u b s e t of the children w h o participated in this study. C h a p t e r 3 of this t h e s i s provides n e w d a t a to s h o w significant positive associations b e t w e e n c h o l i n e p h o s p h o g l y c e r i d e excretion a n d p l a s m a S A H a n d homocysteine, a n d a n inverse relation between choline p h o s p h o g l y c e r i d e excretion a n d p l a s m a methionine (specific a i m 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 and 6 decade th  th  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-| and B and impaired renal function (54). Vitamin B i a n d 2  6  2  folate levels in the children with CF and the healthy control children in our study were in the normal range (16). Vitamin B status was not determined, but 6  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  124  Chapter 4 Discussion and conclusions choline p h o s p h o g l y c e r i d e excretion a n d p l a s m a methionine are consistent with altered hepatic c h o l i n e m e t a b o l i s m with r e d u c e d synthesis of P C v i a the C D P pathway a n d i n c r e a s e d P C synthesis via the P E M T path. It is p o s s i b l e that patients with C F h a v e conditional hepatic choline deficiency d u e to chronic increased choline loss a n d i n c r e a s e d choline requirement. Additionally, it is possible that the methionine s y n t h a s e pathway alone c a n n o t fulfill the total requirement for methionine synthesis w h e n the b e t a i n e - h o m o c y s t e i n e methyltransferase pathway for remethylation of h o m o c y s t e i n e is limited by the availability of betaine. B e t a i n e is an important methyl d o n o r a n d is formed from the irreversible oxidation of choline. D a t a to support this s u g g e s t i o n h a s not b e e n published. H o w e v e r , it is p o s s i b l e that the positive a s s o c i a t i o n s b e t w e e n choline phosphoglyceride excretion a n d p l a s m a h o m o c y s t e i n e a n d S A H , a n d inverse association with p l a s m a methionine may be e x p l a i n e d by d e c r e a s e d remethylation of h o m o c y s t e i n e to methionine p o s s i b l y involving the betaineh o m o c y s t e i n e methyltransferase pathway or the methionine s y n t h a s e pathway. Future studies are n e e d e d to confirm the c a u s a l link b e t w e e n i n c r e a s e d choline p h o s p h o g l y c e r i d e s excretion a n d p l a s m a h o m o c y s t e i n e , S A H a n d methionine levels a n d to determine whether or not increased P C synthesis v i a the transfer of methyl g r o u p s from S A M to S A H in the P E M T pathway is a determinant of p l a s m a h o m o c y s t e i n e in h u m a n s .  125  Chapter 4 Discussion and conclusions 4.2 Limitations This thesis r e s e a r c h u s e d a cross-sectional study d e s i g n to determine the extent of p h o s p h o l i p i d excretion, a n d its a s s o c i a t i o n with p l a s m a methionine a n d h o m o c y s t e i n e in children with C F w h e n c o m p a r e d with healthy control children without C F . P o s i t i v e correlations between choline p h o s p h o g l y c e r i d e excretion a n d p l a s m a h o m o c y s t e i n e a n d S A H concentrations, a n d inverse correlation between choline p h o s p h o g l y c e r i d e excretion a n d p l a s m a methionine concentration w e r e found. H o w e v e r , a s statistical a s s o c i a t i o n s do not imply causation, a c o n c l u s i o n of.a c a u s a l link b e t w e e n choline p h o s p h o g l y c e r i d e excretion and the altered p l a s m a thiol status in C F cannot be d r a w n . T h e n u m b e r of subjects included in this research w a s s m a l l a n d the subjects included children of a w i d e a g e range. N e v e r t h e l e s s , the s a m p l e s i z e w a s more than twofold the s a m p l e s i z e required to detect a significant difference in fecal lipid excretion b e t w e e n the patients with C F a n d healthy controls b a s e d o n power a n a l y s i s u s i n g p u b l i s h e d data o n fecal lipid content with  = 8 0 % and  a = 0.05 (21). P u b l i s h e d data o n fecal phospholipid excretion in h u m a n s w a s not available to allow the a b o v e power calculations. T h e children with C F w e r e recruited using p u r p o s i v e s a m p l i n g a n d the control group recruited w a s a c o n v e n i e n c e s a m p l e . It is p o s s i b l e that s a m p l i n g bias existed, a n d this m a y limit the generalizability of the study results. Future multi-centre studies with a larger s a m p l e s i z e , p o s s i b l y involving adult patients with C F a n d C F patients with biopsy confirmed liver d i s e a s e may increase the generalizability of the findings a n d specific r e l e v a n c e of the results to hepatic steatosis.  126  Chapter 4 Discussion and conclusions F o r practical p u r p o s e s a n d b e c a u s e of ethical c o n s i d e r a t i o n s , blood s a m p l e s w e r e not c o l l e c t e d under fasting conditions or with a controlled time o v e r the last m e a l . S i n c e all the blood s a m p l e s w e r e collected in the m o r n i n g or early afternoon w h e n the patients with C F attended the outpatient laboratory for blood s a m p l i n g at a routine clinic appointment, it is likely that all of the subjects in our study had eaten within 4 hours of the blood s a m p l i n g . H o w e v e r , p u b l i s h e d data is available to s h o w that total p l a s m a cholesterol, H D L cholesterol a n d a p o B concentration are minimally affected by fasting status (50). O n the other h a n d , fasting status a n d time s i n c e last m e a l may influence levels of h o m o c y s t e i n e a n d research data has s h o w n that h o m o c y s t e i n e level is 4 - 5 % higher after a 6-h fast c o m p a r e d with t h o s e w h o h a v e eaten within 1 to 6 hours in adults (56). Nevertheless, our results s h o w e d that p l a s m a h o m o c y s t e i n e v a l u e s with a r a n g e of 7.08-12.32 uM in children with C F c o m p a r e d with 4.27-5.58 u M in the control children without C F . It s e e m s unlikely that the m a r k e d difference in p l a s m a h o m o c y s t e i n e b e t w e e n patients with a n d without C F could be e x p l a i n e d by differences in time s i n c e the last m e a l . O t h e r limitations of this thesis research include the inability to quantify dietary plus biliary e x c r e t i o n of phospholipid, t h u s p h o s p h o l i p i d a n d P C absorption. Quantification of the amount of P C s e c r e t e d into the intestine in bile w o u l d require i n v a s i v e t e c h n i q u e s . Liver biopsy w a s not performed in the study participants to confirm the p r e s e n c e of steatosis a n d to determine the hepatic P C a n d choline pool, a l s o b e c a u s e of the n e e d for invasive p r o c e d u r e s . T h e s e w e r e c o n s i d e r e d inappropriate for studies with children.  127  Chapter 4 Discussion and conclusions 4.3 Future Directions C h a p t e r 2 of this thesis r e s e a r c h d e s c r i b e s the d e v e l o p m e n t of a n e w solvent s y s t e m for the extraction of fecal phospholipids, a n d the validation of a n H P L C - E L S D m e t h o d for separation and quantification of fecal p h o s p h o l i p i d s . T h e new method m a y be applied in future studies to determine P C excretion in patients with n o n - C F related pancreatic insufficiency, or i m p a i r e d biliary or intestinal absorption function. T h e results will be helpful in providing insights regarding the m e c h a n i s m of lipid malabsorption in patients with fat malabsorption, as i n c r e a s e d P C excretion c o u l d lead to hepatic P C depletion, w h i c h c o u l d w o r s e n bile lipid s y n t h e s i s a n d secretion, w h i c h in turn m a y w o r s e n fat malabsorption. In chapter 3 of this thesis research, w e have s h o w n that children with C F excrete higher a m o u n t s of choline p h o s p h o g l y c e r i d e s than healthy children without C F . W e further s h o w e d that excretion of choline p h o s p h o g l y c e r i d e s is positively a s s o c i a t e d with p l a s m a h o m o c y s t e i n e and inversely a s s o c i a t e d with p l a s m a methionine. T h e findings provide circumstantial e v i d e n c e of altered hepatic choline m e t a b o l i s m in children with C F . Additionally, it is not k n o w n if patients with C F h a v e i n c r e a s e d choline requirements. H o w e v e r , choline status w a s not m e a s u r e d directly in this study. In this regard, p l a s m a choline d o e s not a p p e a r to be a sensitive indicator of choline status. T h u s , it will be helpful to a n a l y z e the choline and P C concentrations of the liver b i o p s y s p e c i m e n s collected from patients with C F (where available) and in n o n - C F patients to confirm hepatic c h o l i n e depletion in C F . A n i m a l m o d e l s m a y b e useful to a d d r e s s  128  .  Chapter 4 Discussion and conclusions  this, for e x a m p l e , c o m p a r i s o n of the hepatic P C a n d choline pool in A508"'" m i c e a n d wild type m i c e . R e c e n t studies h a v e indicated elevated p l a s m a h o m o c y s t e i n e concentrations are a s s o c i a t e d with a n i n c r e a s e d risk of osteoporotic fractures in older p e r s o n s , a n d it h a s b e e n p r o p o s e d that a h o m o c y s t e i n e - a s s o c i a t e d disturbance in c o l l a g e n cross-linking in bone is involved (57, 58). O s t e o p o r o s i s has been reported in 9% of adult patients with C F (53). W h e t h e r the i n c r e a s e d h o m o c y s t e i n e level is a s s o c i a t e d with a risk of c a r d i o v a s c u l a r d i s e a s e in C F patients is a l s o uncertain, but the clinical significance of e l e v a t e d h o m o c y s t e i n e a n d S A H in C F m a y b e c o m e more important as lifespan i m p r o v e s . Future studies involving the u s e of stable isotopes are a l s o n e e d e d to e l u c i d a t e the etiology of the elevated h o m o c y s t e i n e in patients with C F . Additionally, nutrition intervention studies involving c h o l i n e , betaine a n d strategies to i n c r e a s e the intraluminal p H are warranted to d e t e r m i n e if t h e s e s u p p l e m e n t s will correct the thiol abnormalities a n d improve choline status. S t u d i e s to evaluate the effect of supplementation o n clinical endpoints, including improving life quality in patients with C F , will b e w a r r a n t e d in future. T h e high a m o u n t s of l y s o P C e x c r e t e d by the control children a n d the children with C F in our study were u n e x p e c t e d , a n d w e are u n a w a r e of similar data o n the c o m p o s i t i o n of fecal phospholipids. Future studies are n e e d e d to determine if l y s o P C rather than P C is the primary choline p h o s p h o l i p i d s 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 C o n c l u s i o n s  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/finetuning 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 methioninehomocysteine 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 e v a n 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 with 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 are w a r r a n t e d to a d d r e s s the i m p o r t a n c e o f t h e 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  inter-relation  of this p a t h w a y with p l a s m a h o m o c y s t e i n e . T h e clinical 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 t h 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 e v e l m a y b e m o r e i m p o r t a n t in C F a s p a t i e n t ' s life 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 a z z a b o s c o M , P o d d a M . Liver a n d biliary problems in cystic fibrosis. S e m Liver D i s e a s e 1998;18:227-235. 2. Potter C J , F i s b e i n M , H a m m o n d S, M c C o y K , Q u a l m a n S . C a n the histologic c h a n g e s of cystic f i b r o s i s - a s s o c i a t e d hepatobiliary d i s e a s e be predicted by clinical criteria? J Pediatr Gastroenterol Nutr 1997;25:32-6. 3. B u c h m a n A L , A m e n t M E , S o h e l M , et a l . C h o l i n e deficiency c a u s e s reversible hepatic abnormalities in patients receiving parenteral nutrition: proof of a h u m a n choline requirement: a placebo-controlled trial. J P E N 2001 ;25:260-8. 4. Z e i s e l S H , Blusztajn J K . C h o l i n e a n d h u m a n nutrition. A n n u R e v Nutr 1994;14:269-296. 5. 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. 6. V a n c e D E . Phosphatidylcholine m e t a b o l i s m : m a s o c h i s t i c e n z y m o l o g y , metabolic regulation, a n d lipoprotein a s s e m b l y .  B i o c h e m C e l l Biol  1990;68:1151-1165. 7. B a r r a c l o u g h M , T a y l o r C J . Twenty-four hour ambulatory gastric a n d d u o d e n a l p H profiles in cystic fibrosis: effect of d u o d e n a l hyperacidity o n pancreatic e n z y m e function a n d fat absorption. J P e d Gastroenterol Nutr 1996;23:45-50. 8.  Francisco M P , Wagner M H , Sherman J M , Theriaque D, Bowser E, Novak D A . Ranitidine a n d o m e p r a z o l e as adjuvant therapty to p a n c r e l i p a s e to improve  Chapter 4 Discussion and conclusions  fat absorption in patients with cystic fibrosis. J Pediatr G a s t r o e n t e r o l Nutr 2002;35:79-83. 9. P r o e s m a n s M , D e B o e c k K . O m e p r a z o l e , a proton p u m p inhibitor, improves residual steatorrhea in cystic fibrosis patients treated with high d o s e pancreatic e n z y m e s . E u r J Pediatr 2 0 0 3 ; 1 6 2 : 7 6 0 - 3 . 10. Z e i s e l S H , G r o w d o n J H , W u r t m a n R J , et a l . N o r m a l p l a s m a c h o l i n e r e s p o n s e s to ingested lecithin. N e u r o l o g y 1980;30:1226-9. 11 . C a r e y M C , Hernell, O . Digestion a n d absorption of fat. S e m i n G a s t r o i n t e s D i s 1992;3:189-208. 12. J e n s e n R G . Lipids in h u m a n milk. 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In Dietary R e f e r e n c e Intakes for Energy, C a r b o h y d r a t e , Fiber, Fat, Fatty A c i d s , C h o l e s t e r o l , Protein, a n d A m i n o A c i d s (Macronutrients). W a s h i n g t o n , D C : National A c a d e m y P r e s s , 2002. 2 2 . M u r p h y J L , W o o t t o n S A , B o n d S A , J a c k s o n A A . E n e r g y content of stools in normal healthy controls a n d patients with cystic fibrosis. A r c h Dis C h i l d 1991;66:495-500. 2 3 . Christie W W . Preparation of lipid extracts from tissues. A d v Lipid M e t h o d o l o g y 1993;2:195-213. 2 4 . N e u m e i s t e r V , H e n k e r J , Kaltenborn, G S p r o s s i g C , J a r o s s W . S i m u l t a n e o u s determination of fecal fat, nitrogen, a n d water by near-infrared s p e c t r o s c o p y . J Pediatr G a s t e r o e n t e r o l Nutr 1997;25:388-93.  reflectance  Chapter 4 Discussion and conclusions 2 5 . C h e n P S , T o r i b a r a T Y , W a r n e r H . Microdetermination of p h o s p h o r u s . A n a l C h e m 1956;28:1756-8. 2 6 . P»ouser G , S i a k o t o s A N , F l e i s c h e r S. Quantitative a n a l y s i s of phospholipids by thin-layer c h r o m a t o g r a p h y a n d phosphorus a n a l y s i s of spots. Lipids 1965;1:85-6. 2 7 . N o u r i - S o r k h a b i M H , C h a p m a n B E , K u c h e l P W , et a l . P a r a l l e l secretion of pancreatic p h o s p h o l i p a s e A 2 , p h o s p h o l i p a s e A 1 , lipase, a n d c o l i p a s e in children with exocrine pancreatic dysfunction. P e d R e s 2000;48:735-40. 2 8 . D r e s s m a n J B , S h t o h r y n L V , D i o k n o D . Effects of product formulation on in vitro activity of pancreatic e n z y m e s . A m J H o s p P h a r m 1985;42:2502-6. 2 9 . W e b e r A M , R o y C C . Intraduodenal events in cystic fibrosis. J Pediatr Gastroenterol Nutr 1 9 8 4 ; 3 : S 1 1 3 - 9 . 30. R o b i n s o n P J , S m i t h A L , S l y P D . D u o d e n a l p H in cystic fibrosis a n d its relationship to fat malabsorption. Dig Dis S c i 1990;35:1299-304. 3 1 . C a n t y D J , Z e i s e l S H . Lecithin a n d choline in h u m a n health a n d d i s e a s e . Nutr R e v 1994;52:327-39. 3 2 . L e r a y C [ h o m e p a g e on the Internet]. C y b e r Lipid C e n t r e , [updated 2 0 0 4 A u g 10; cited 2 0 0 4 A u g 11]. A v a i l a b l e from: http ://www. c y b e rl ipid. org/ph I ip/pgly03. htm 3 3 . M e y e r K C , S h a r m a A , B r o w n R, et a l . Function a n d c o m p o s i t i o n of pulmonary surfactant a n d surfactant-derived fatty a c i d profiles are altered in in y o u n g adults with cystic fibrosis. C h e s t 2 0 0 0 ; 118:164-74.  136  Chapter 4 Discussion and conclusions 3 4 . C h a w l a R K , Wolf D C , Kutner M H , B o n k o v s k y H L . C h o l i n e m a y b e a n essential nutrient in m a l n o u r i s h e d patients with cirrhosis. Gastroenterology 1989;97:1514-20. 3 5 . Y a o Z , V a n c e D E . R e d u c t i o n in V L D L , but not H D L , in p l a s m a of rats deficient in choline. B i o c h e m C e l l Biol 1990;68:552-8. 36. B u c h m a n A L , Dubin M D , M o u k a r z e l A A , et a l . C h o l i n e deficiency: a c a u s e of hepatic steatosis during parenteral nutrition that c a n be r e v e r s e d with intravenous choline supplementation. Hepatology 1995;22:1399-403. 3 7 . W a l t e r s M P , Littlewood J M . F e c a l bile a c i d a n d dietary residue excretion in cystic fibrosis: a g e group variations. J Pediatr Gastroenterol Nutr 1998;27:296-300. 38. B u r d g e 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 v a l u e of a high-fat diet a n d pancreatic supplementation. B r J Nutr 1994;71:959-64. 39. F r a n c i s c o M P , W a g n e r M H , S h e r m a n J M , T h e r i a q u e D, B o w s e r E , N o v a k D A . Ranitidine a n d o m e p r a z o l e a s adjuvant therapy to p a n c r e l i p a s e to improve fat absorption in patients with cystic fibrosis. J Pediatr Gastroenterol Nutr 2002;35:79-83. 4 0 . Kalivianakis M , M i n i c h D M , Bijleveld C M , et a l . Fat malabsorption in cystic, fibrosis patients receiving e n z y m e replacement therapy is d u e to impaired intestinal uptake of long-chain fatty a c i d s . A m J C l i n Nutr 1999;69:127-34.  137  ;  Chapter 4 Discussion and conclusions  41 . D a v i d s o n A G F . Gastrointestinal a n d pancreatic d i s e a s e in cystic fibrosis. 2  n d  e d . In: H o d s o n M E , G e d d e s D M , e d s . C y s t i c fibrosis. L o n d o n : C h a p m a n & Hall, 2 0 0 0 : 3 8 4 - 9 5 . 4 2 . G r e g o r y P C . Gastrointestinal p H , motility/transit time a n d permeability in cystic fibrosis. J Pediatr Gastroenterol Nutr 1996;23:513-23. 4 3 . R o b i n s o n P J , S m i t h A L , S l y P D . D u o d e n a l p H in cystic fibrosis a n d its relationship to fat malabsorption. Dig Dis S c i 1990;35:1299-304. 4 4 . B e n n o Y , M i z u t a m T, N a m b a Y , K o m o r i T, M i t s u o k a T . C o m p a r i s o n of fecal microflora pf elderly p e r s o n s in rural a n d urban a r e a s of J a p a n . A p p l Environ Microbiol 1989;55:1100-5. 4 5 . B e n n o Y , S a w a d a K , M i t s u o k a T. T h e intestinal microflora of infants: c o m p o s i t i o n of fecal flora in breast-fed a n d bottle-fed infants. Microbiol Immunol 1984;28:975-86. 4 6 . L e v y J . T h e effects of antibiotic use o n gastrointestinal function. A m J Gastroenterol 2 0 0 0 ; 9 5 : S 8 - 1 0 . 4 7 . U l a n e M M , Butler J D , Peri A , M i e l e L , U l a n e R E , H u b b a r d V S . C y s t i c fibrosis a n d phosphatidylcholine biosynthesis. C l i n C h i m A c t a 1994;230:109-16. 4 8 . M c M a h o n K E , Farrell P M . Effect of choline deficiency o n lung p h o s p h o l i p i d concentrations in the rat. J Nutr 1986; 116:936-43. 4 9 . Durrington P N . C a n m e a s u r e m e n t of apolipoprotein B replace the lipid profile in the follow-up of patients with lipoprotein d i s o r d e r s ? C l i n C h e m 2002;48:401-2.  138  Chapter 4 Discussion and conclusions 5 0 . Reinhart R A , G a n i K , Arndt M R , B r o s t e S K . A p o l i p r o t e i n s A - l a n d B as predictors of angiographically defined coronary artery d i s e a s e . A r c h Intern M e d 1990;150:1629-33. 5 1 . D e B r e e A , V e r s c h u r e n W M , K r o m h o u t D , Kluijtmans L A , B l o m H J . H o m o c y s t e i n e determinants a n d the e v i d e n c e to what extent h o m o c y s t e i n e d e t e r m i n e s the risk of c o r o n a r y heart d i s e a s e . P h a r m a c o l R e v 2002;54:599618. 5 2 . K e r i n s D M , Koury M J , C a p d e v i l a A , R a n a S, W a g n e r C . P l a s m a Sa d e n o s y l h o m o c y s t e i n e is a more sensitive indicator of c a r d i o v a s c u l a r d i s e a s e than p l a s m a h o m o c y s t e i n e . A m J C l i n Nutr 2001;74:723-9. 5 3 . C y s t i c Fibrosis F o u n d a t i o n . C y s t i c Fibrosis Foundation Patient Registrty A n n u a l D a t a Report. B e t h e s d a , M D : C y s t i c Fibrosis Foundation, 2 0 0 3 . 54. Garcfa-Tevijano E R , B e r a s a i n C , R o d r i g u e z J A , et al. H y p e r h o m o c y s t e i n e m i a in liver cirrhosis: m e c h a n i s m s a n d role in v a s c u l a r a n d hepatic fibrosis. Hypertension 2001;38:1217-21. 55. Noga A A , Stead L M , Zhao Y , Brosnan M E , Brosnan JT, Vance D E . Plasma h o m o c y s t e i n e is regulated by p h o s p h o l i p i d methylation. J Biol C h e m 2003;278:5952-5. 5 6 . Nurk E , Tell G S , N y g a r d O , R e f s u m H , U e l a n d P M , Vollset S E . P l a s m a total h o m o c y s t e i n e is influenced by prandial status in h u m a n s : the H o r d a l a n d h o m o c y s t e i n e study. J Nutr 2 0 0 1 ; 1 3 1 : 1 2 1 4 - 6 . 5 7 . v a n M e u r s J B J , D h o n u k s h e - R u t t e n R A M , Pluijm S M F , et a l . H o m o c y s t e i n e levels a n d the risk of osteoporotic fracture. N E n g l J M e d 2 0 0 4 ; 3 5 0 : 2 0 3 3 - 4 1 .  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  APPENDIX A INFORMED  CONSENT  3.  T o m e a s u r e p l a s m a folate, vitamin B - 1 2 a n d h o m o c y s t e i n e l e v e l s in c h i l d r e n with C F a n d c o m p a r e t h e m to children without C F . Folate a n d v i t a m i n B - 1 2 are B v i t a m i n s a n d h o m o c y s t e i n e is a blood marker of t h e s e v i t a m i n s . 4. T o m e a s u r e dietary intake of energy and all e s s e n t i a l nutrients from a 3 to 5day food r e c o r d . 5. T o determine the extent of fat malabsorption by a n a l y s i s of 3-day s t o o l samples. Who C a n Participate A l l patients with C F w h o are s c h e d u l e d for regular b l o o d work. Study Procedures If I agree to let m y child participate, a n additional two tubes of b l o o d (14ml_) will be taken. M y c h i l d ' s height, weight a s well as my child's current e n z y m e t h e r a p y and r e g i m e n will be o b t a i n e d from his/her m e d i c a l record. T h i s study will not involve a n extra n e e d l e . I will be a s k e d if I a m willing to k e e p a r e c o r d of the foods that my child e a t s for 3-5 d a y s , a n d if my child will collect s t o o l s a m p l e s over 3 d a y s . Risks T h e r e are n o k n o w n r i s k s to participating in this study. A certified t e c h n o l o g i s t , nurse, or other qualified p e r s o n will d r a w the small amount of b l o o d . Minor discomfort a n d s o m e t e m p o r a r y discoloration m a y o c c u r at the site of b l o o d draw. Benefits Deficiencies that may b e corrected through s u p p l e m e n t s or c h a n g e s in diet m a y be identified in this study. I will be p r o v i d e d with a c o p y of my c h i l d ' s results w h e n they are a v a i l a b l e . If I a g r e e to keep a record of my child's food intake, I will b e provided with a c o p y of his/her nutrient intakes w h e n the results a r e ready. Confidentiality A n y information resulting from this r e s e a r c h study will be kept confidential. A l l d o c u m e n t s will be identified by c o d e number a n d kept in a l o c k e d filing cabinet. M y child will not b e identified by n a m e in any reports of the c o m p l e t e d study. Consent T h e objectives a n d p r o c e d u r e s of the study h a v e b e e n e x p l a i n e d to m e to m y satisfaction a n d I u n d e r s t a n d that m y child m a y withdraw from t h e s t u d y at a n y time. I h a v e the right to refuse my child's participation or voluntarily w i t h d r a w from the s t u d y without c o n s e q u e n c e to continuing m e d i c a l c a r e . M y c h i l d ' s n a m e will be treated confidentially by u s e of c o d e n u m b e r s during the s t u d y a n d will not b e m e n t i o n e d in any report or publications of the study results. I u n d e r s t a n d that I a m to r e c e i v e a c o p y of this c o n s e n t form. I u n d e r s t a n d that if I h a v e a n y q u e s t i o n s or d e s i r e further information, I s h o u l d contact Dr S h e i l a Innis or o n e of Page 2 of 3 C F Version: [ C F / P / Jan 30, 2000]  3.  T o m e a s u r e p l a s m a folate, v i t a m i n B - 1 2 a n d h o m o c y s t e i n e levels in children with C F a n d c o m p a r e t h e m to c h i l d r e n without C F . F o l a t e a n d vitamin B - 1 2 are B vitamins a n d h o m o c y s t e i n e is a b l o o d marker of t h e s e vitamins. 4. T o m e a s u r e dietary intake of e n e r g y a n d all e s s e n t i a l nutrients from a 3 to 5day food r e c o r d . 5. T o determine the extent of fat m a l a b s o r p t i o n by a n a l y s i s of 3-day stool samples. Who C a n Participate All patients with C F w h o are s c h e d u l e d for regular blood work. Study Procedures If I a g r e e to participate, a n a d d i t i o n a l two t u b e s of blood ( 1 4 m L ) will be taken. M y height, weight a s well a s my current e n z y m e therapy a n d r e g i m e n will be obtained from my m e d i c a l r e c o r d . T h i s study will not involve a n extra needle. I will b e a s k e d if I a m willing to k e e p a record of the foods that I eat for 3-5 d a y s , a n d if I will collect s t o o l s a m p l e s over 3 d a y s . Risks T h e r e are no k n o w n risks to participating in this study. A certified technologist, nurse, or other qualified p e r s o n will d r a w the small amount of blood. Minor discomfort a n d s o m e temporary d i s c o l o r a t i o n m a y o c c u r at the site of blood draw. Benefits Deficiencies that m a y b e c o r r e c t e d through s u p p l e m e n t s or c h a n g e s in diet m a y be identified in this study. I will be p r o v i d e d with a c o p y of m y results w h e n they are a v a i l a b l e . If I a g r e e to k e e p a r e c o r d of m y food intake, I will be p r o v i d e d with a c o p y of my nutrient intakes w h e n the results are ready. Confidentiality A n y information resulting from this r e s e a r c h study will be kept confidential. A l l d o c u m e n t s will b e identified by c o d e n u m b e r a n d kept in a l o c k e d filing cabinet. I will not b e identified by n a m e in a n y reports of the c o m p l e t e d study. Consent T h e objectives a n d p r o c e d u r e s of the study h a v e b e e n e x p l a i n e d to m e to m y satisfaction a n d I u n d e r s t a n d that I m a y withdraw from the study at a n y time. I h a v e the right to refuse to participate or voluntarily withdraw from the study without c o n s e q u e n c e to continuing m e d i c a l care. M y n a m e will be treated confidentially by u s e of c o d e n u m b e r s during the study a n d will not b e m e n t i o n e d in a n y report o r p u b l i c a t i o n s of the s t u d y results. I u n d e r s t a n d that I a m to r e c e i v e a c o p y of this c o n s e n t form. I u n d e r s t a n d that if I h a v e any q u e s t i o n s or d e s i r e further information, I s h o u l d c o n t a c t Dr S h e i l a Innis or o n e of her a s s o c i a t e s at 8 7 5 - 2 4 3 4 or Dr G e o r g e D a v i d s o n at 8 7 5 - 2 1 4 2 . If I h a v e a n y c o n c e r n s about m y Page 2 of 3 CF Version: [CF/C/ Jan 30, 2000]  treatment or rights a s a r e s e a r c h subject, I m a y t e l e p h o n e D r R . D . S p r a t e l y , Director of R e s e a r c h S e r v i c e s at 8 2 2 - 8 5 9 8 . I h a v e r e c e i v e d a c o p y of the c o n s e n t form for m y o w n r e c o r d s .  I  v o l u n t a 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 i n t h e (Please print)  s t u d y entitled A S t u d y of B i o c h e m i c a l Nutritional P a r a m e t e r s in C h i l d r e n with Cystic F i b r o s i s .  Signed  Date  Witness  Date  Investigator's Signature  Date  Page 3 of 3 C F Version: [CF/C/ Jan 30, 2000]  2.  T o m e a s u r e fatty a c i d s l e v e l s in p h o s p h o l i p i d , triglyceride, a n d cholesterol ester in the b l o o d of individuals with C F a n d c o m p a r e t h e m to individuals without C F . 3. T o m e a s u r e p l a s m a folate, vitamin B - 1 2 a n d h o m o c y s t e i n e l e v e l s in children with C F a n d c o m p a r e t h e m to c h i l d r e n without C F . Folate a n d vitamin B - 1 2 are B vitamins a n d h o m o c y s t e i n e is a b l o o d m a r k e r of t h e s e vitamins. 4. T o m e a s u r e dietary intake of e n e r g y a n d all e s s e n t i a l nutrients from a 3 to 5day food record. 5. T o determine the extent of fat m a l a b s o r p t i o n by a n a l y s i s of 3-day stool samples. Who Can Participate A l l patients without C F , p u l m o n a r y , hepatic, gastrointestinal or inflammatory d i s e a s e s and metabolic d i s e a s e s likely to result in alterations of lipids, a m i n o a c i d s , folate, vitamin B - 1 2 m e t a b o l i s m , w h o are not taking d r u g s that affect fat or vitamin absorption, a n d are s c h e d u l e d for blood-work c a n act a s controls. Study Procedures If I a g r e e to let m y child participate, a n additional two t u b e s of b l o o d (14mL) will b e taken. M y child's height, weight a s w e l l a s m y child's current e n z y m e therapy a n d regimen will be obtained from his/her m e d i c a l record. T h i s study will not involve an extra needle. I will be a s k e d if I a m willing to k e e p a r e c o r d of the foods that my child eats for 3-5 d a y s , a n d if my child will collect stool s a m p l e s over 3 d a y s .  Risks T h e r e are no k n o w n risks to participating in this study. A certified technologist, n u r s e , or other qualified p e r s o n will d r a w the s m a l l amount of b l o o d . Minor discomfort a n d s o m e temporary d i s c o l o r a t i o n m a y o c c u r at the site of blood draw. Benefits Deficiencies that m a y b e c o r r e c t e d through s u p p l e m e n t s or c h a n g e s in diet m a y b e identified in this study. I will be p r o v i d e d with a c o p y of m y c h i l d ' s results w h e n they are a v a i l a b l e . If I a g r e e to k e e p a r e c o r d of m y child's food intake, I will b e provided with a c o p y of his/her nutrient intakes w h e n the results are ready. Confidentiality A n y information resulting from this r e s e a r c h study will b e kept confidential. A l l d o c u m e n t s will b e identified by c o d e n u m b e r a n d kept in a l o c k e d filing cabinet. M y child will not b e identified by n a m e in a n y reports of the c o m p l e t e d study. Consent T h e objectives a n d p r o c e d u r e s of the study h a v e b e e n e x p l a i n e d to m e to m y satisfaction a n d I u n d e r s t a n d that m y child m a y withdraw from the study at a n y Page 2 of 3 C F Version: [Controls/P/ Jan 30, 2000]  2.  T o m e a s u r e fatty a c i d s l e v e l s in p h o s p h o l i p i d , triglyceride, a n d c h o l e s t e r o l e s t e r in the b l o o d of individuals with C F a n d c o m p a r e t h e m f o individuals without C F .  3.  T o m e a s u r e p l a s m a folate, vitamin B - 1 2 a n d h o m o c y s t e i n e levels in children with C F a n d c o m p a r e t h e m to children without C F . F o l a t e a n d vitamin B - 1 2 are B vitamins a n d h o m o c y s t e i n e is a blood m a r k e r of t h e s e v i t a m i n s . T o m e a s u r e dietary intake of e n e r g y a n d all e s s e n t i a l nutrients from a 3 to 5d a y food r e c o r d .  4.  5. T o d e t e r m i n e the extent of fat m a l a b s o r p t i o n by a n a l y s i s of 3-day s t o o l samples. W h o C a n Participate A l l patients without C F , pulmonary, hepatic, gastrointestinal or inflammatory d i s e a s e s a n d metabolic d i s e a s e s likely to result in alterations of lipids, a m i n o a c i d s , folate, vitamin B - 1 2 m e t a b o l i s m , w h o are not taking d r u g s that affect fat or vitamin absorption, a n d are s c h e d u l e d for blood-work c a n act a s controls. Study Procedures If I a g r e e to participate, a n additional two tubes of b l o o d ( 1 4 m L ) will b e t a k e n . M y height, weight a s well a s m y current e n z y m e therapy a n d r e g i m e n will be o b t a i n e d from m y m e d i c a l record. T h i s study will not involve a n extra n e e d l e . I will b e a s k e d if I a m willing to k e e p a record of the foods that I eat for 3-5 d a y s , a n d if I will collect stool s a m p l e s over 3 days. Risks T h e r e are no k n o w n risks to participating in this study. A certified technologist, nurse, or other qualified p e r s o n will d r a w the s m a l l a m o u n t of b l o o d . M i n o r discomfort a n d s o m e temporary discoloration m a y o c c u r at the site of b l o o d draw. Benefits D e f i c i e n c i e s that m a y b e c o r r e c t e d through s u p p l e m e n t s or c h a n g e s in diet m a y b e identified in this study. I will b e p r o v i d e d with a c o p y of m y results w h e n they a r e a v a i l a b l e . If I a g r e e to k e e p a r e c o r d of m y food intake, I will b e p r o v i d e d with a c o p y of m y nutrient intakes w h e n the results are ready. Confidentiality A n y information resulting from this r e s e a r c h study will b e kept confidential. A l l d o c u m e n t s will b e identified by c o d e n u m b e r a n d kept in a l o c k e d filing cabinet. I will not be identified by n a m e in a n y reports of the c o m p l e t e d study. Consent T h e objectives a n d p r o c e d u r e s of the study h a v e b e e n e x p l a i n e d to m e to m y satisfaction a n d I u n d e r s t a n d that I m a y withdraw from t h e s t u d y at a n y time. I h a v e the right to refuse to participate or voluntarily w i t h d r a w from the s t u d y P a g e 2 of 3 C F Version: [Controls/C/ Jan 30, 2000]  APPENDIX B FOOD RECORD  E  o  5,  <D o co to  £ -~  Q) 1ro > c\i  45 _s  ca  J3  -p  "§1 £ _ ro £  H  £ o _ o co o  < y  _ _  UJ Q. 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T h e a d e q u a t e intake (Al) of choline for children 1-3 years, 4-8 years, 9-13 y e a r s is 2 0 0 m g / d , 2 5 0 m g / d a n d 3 7 5 m g / d , respectively. T h e A l for boys a n d girls 14-18 y e a r s is 550 mg/d a n d 4 0 0 m g / d , respectively.  Method Briefly, data from A p p e n d i x D were entered into Microsoft® E x c e l 2 0 0 2 for W i n d o w s to form a choline d a t a b a s e . T h e weight of the individual food items from the food records w e r e entered into the s a m e d a t a b a s e to allow calculation of m e a n P C a n d total choline intakes.  Results T h e m e a n c h o l i n e a n d phosphatidylcholine ( P C ) intakes of children with C F a n d healthy children without C F is s u m m a r i z e d in the T a b l e below. P C a n d total choline intakes w e r e not significantly different between children with C F a n d the controls. 5 0 % (8/16) of children with C F and 1 2 . 5 % (1/8) o f control children had the c h o l i n e intake at or a b o v e the A l for a g e a n d g e n d e r .  193  Discussion T h e U S D A c h o l i n e d a t a b a s e w a s first published in M a r c h 2 0 0 4 , which contained about 4 0 0 c o m m o n foods to provide r e s e a r c h e r s a n d c o n s u m e r s m e a n s to estimate choline intake from c o m m o n foods. A n d our estimation s h o w e d that 5 0 % of children with C F a n d 1 2 . 5 % of children without C F h a d a n intake that met or a b o v e the A l . H o w e v e r , it is possible that the results were a n underestimation of the a c t u a l choline intake a s s o m e c o m m o n f o o d s c o n s u m e d by our study participants w e r e not included in the d a t a b a s e , including margarine, c h o c o l a t e syrup drink mix, hot chocolate, honey, waffle, puff pastry a n d pie shells. Further, all of the nutrition supplements, s u c h a s T o l e r e x ® , P e d i a s u r e ® a n d V i v o n e x ® w e r e not i n c l u d e d in the d a t a b a s e . E x a m i n a t i o n of the ingredient list of the a b o v e products s u g g e s t e d that they contained P C a n d choline. A l t h o u g h the a m o u n t of total c h o l i n e w a s s h o w n o n the product label, the P C content of the formula w a s u n k n o w n . T h u s children w h o w e r e on tube feed and/or with >25% energy intake from nutrition s u p p l e m e n t s w e r e e x c l u d e d from the analysis.  194  T a b l e M e a n choline a n d phosphatidylcholine ( P C ) intakes of children with C F a n d healthy children without C F  CF (n=16 )  Control (n=8)  P value  C h o l i n e from P C  123.5±16.0  116.0+22.5  0.8  PC  864.5±112  812±158  0.8  335.3±32.9  268.U25.4  0.2  mg/d  1  2  Total C h o l i n e  ^=16  3  in the C F group b e c a u s e 2 participants w e r e o n tube feed in addition to  oral intake with >25%  calories from nutrition s u p p l e m e n t s a n d w e r e r e m o v e d  from the statistical a n a l y s i s . 2  T h e amount of P C w a s c a l c u l a t e d b a s e o n the a s s u m p t i o n that the m o l e c u l a r  weight of P C e q u a l s 800 a n d the molecular weight of 113 for c h o l i n e . 3  T o t a l choline refers to the s u m of free choline, g l y c e r o p h o s p h o c h o l i n e ,  phosphocholine, Phosphatidylcholine and sphingomyelin.  195  

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