The  Effectiveness  of    Modified  Fat  Breast  Milk  for  the  Treatment  of  Chylothorax  in  

Infants  Following  Cardiothoracic  Surgery        by          

Sarah  Linda  Farmer      

A  thesis  submitted  in  conformity  with  the  requirements      

   

Graduate  Department  of  Nutritional  Sciences    

University  of  Toronto        

©  Copyright  by  Sarah  Linda  Farmer  2011                        

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The  Effectiveness  of  Modified  Fat  Breast  Milk  for  the  Treatment  of  Chylothorax  in  Infants  Following  Cardiothoracic  Surgery  

   

 Sarah  Linda  Farmer  

Masters of Nutritional Sciences

Department of Nutritional Sciences University of Toronto

2011

Abstract Background: Chylothorax occurs in ~4 % of children undergoing cardiac surgery.

Treatment requires transition to a medium chain triglyceride (MCT) based formula.

Provision of breast milk (EBM) is discontinued due to the presence of long chain

triglycerides. Objective:  To determine the effectiveness of a modified fat breast milk for

the treatment of chylothorax in comparison to an MCT formula. Methods: Infants with

chylothorax were eligible. Treatment infants (n=8) received EBM that had been modified

by removing the fat layer (centrifugation) from EBM and adding MCT and nutrients to

provide 67 kcal/ml and 11 g/100 ml protein. Control infants (n=8) received an MCT

formula. Results: Volume of chest tube drainage was not different (p<0.40). Treatment

infants experienced declines in mean weight (p<0.006), length (p<0.013) and head

circumference (p<0.008) z-scores. Conclusion:  Modified fat breast milk allowed for

successful resolution of chylothorax. Strategies to address poor growth, however, need to

be tested before clinical adoption of this novel treatment.

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 TABLE  OF  CONTENTS  

   

Table of Contents............................................................................................................ .iii

i i

.viii List of Appendic CHAPTER  1  INTRODUCTION,  RATIONALE  AND  

    CHAPTER  2  REVIEW  O   2.1 7   2.1.1 Definition of C 2.1.3.1 Congenital 2.1.3.2 Non-Traumatic or Obstructive C 2.1.3.3 Traumatic 2.1.4 Incidence of Ch 2.1.5 Composition o 2.1.6 Diagnosis of 2.1.7 Management of 2.1.8 Determinants of Successful Conservati 2.1.9 Complications of 2.1.10 Breast Milk and 2.2 MEDIUM  CHAIN  TRIGLYC .27   2.2.1 Chemical Structure and Properties of 2.2.2 Food Sources of Medium Ch 2.2.3 Metabolism of Medium Ch 2.2.4 Medium Chain Triglycerides and the Neo 2.2.5 Clinical Uses of Medium Ch 2.3  BREASTFEEDING  AND  THE  PROVISION  OF  BREAST     2.3.1 Breast Milk and Di 2.3.2 Breast Milk and 2.3.3 Breast Milk and Diseas 2.3.3.1 Breast Milk and Dise

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2.3.3.2 Breast Milk and 2.3.3.3 Breast Milk and Gastrointe 2.3.3.4 Breast Milk and Cardi 2.3.3.5 Breast Feedin 2.3.3.6 Breast Milk an 2.4  NUTRITION  ISSUES  IN  CONGENITAL  HEART  DISE                    2.4.1 Congenital Heart Disease 2.4.2 Congenital Heart Disease an 2.4.3 Causes of Growth Failure in Cong CHAPTER  3  THE  EFFECTIVENESS  OF  LOW  FAT  BREAST  MILK  FOR  THE  TREATMENT  OF  CHYLOTHORAX  IN  INFANTS  FOLLOWING  CARDIOVASCULAR  SURGE    3.1  INTRODUCTION  AND  RA                    3.1.1 Research Que 3.1.2 2 3.1.3 Hypoth                      3.2  SUBJECTS  AND     3.2.1 Subje 3.2.2 Study Inclusion C 3.2.2.1 Treatment Inclusi 3.2.2.2 Control Inclusion ..... 3.2.3 Study 3.2.4 Study D 3.2.4.1 Treatme 3.2.4.3 Fat Removal Following Discharge - 3.2.4.4 Fat Removal Following Discharge - 3.2.4.5 Nutrient 3.2.4.6 Contro 3.2.5 Fat Anal 3.2.6 Protein Anal 3.2.7 Electrolyte A 3.2.9 Data Coll 3.2.11 Statistical A

  3.3.1 Subject Charact 3.3.2 Family Characteri 3.3.3 Fat Rem

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3.3.4 Chest Tube 3.3.5 Modified Fat Br 3.3.5.1 Nutrient Enric 3.3.6 Nutrient An 3.3.6.1 Protein A 3.3.6.2 Fat A 3.3.6.3 Electrolyte 3.3.6.4 Intake 3.3.6.5 Feeding In 3.3.6.6 Nasogastric Tube 3.3.7 Gro 3.3.7.1 Weight-for- 3.3.7.2 Length-for- 3.3.7.3 Weight-for- 3.3.7.4 Head Circumference-for- 3.3.8 Microbiological 3.4  DISCUSSI                    3.4.1 Chest Tube                  3.4.2 Grow 3.4.3 Provision of Br  

                   3.5.1 Conclus 3.5.2 Future Direct 15 4. REFERENCE    5.  APPENDIC  

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LIST  OF  TABLES   Table 1. Features of chyl Table 2. Recipes for the Fortification of Mo Table 3. Approximate Energy and Select Nutrient Composition of Mature Human Milk, Portagen®, Modified Fat Human Milk and Nutrient Enriched Modified Fat Human Milk Subject characteris Table 4. Subject Characteristics Table 5. Family Characteristics    Table 6.            Percentage of feeds consumed by treatment subjects as modified fat milk vs. MCT formula Table 7. Measured protein content of breast milk sample after centrifugation Table 8. Measured sodium and potassium content of modified fat breast milk Table 9. Characteristics of infants requiring a feeding tube during the study period and timing of feeding tube removal in relation to study comp Table 10. Weight-for-age, Length-for-age, Weight-for-length and Head Circumference-for-age z-scores at study enrolment and study completion for treatment and control 1 Table 11. Microbiological analysis of milk sample pre-centrifugation and post- centrifuga Table 12. Average daily weight gain over the study period and expected daily weight gain for age for treatmen

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LIST  OF  FIGURES   Figure 1. Care map flow sheet for the diagnosis and management Figure 2. Total volume of drainage for the treatment and control group 81 Figure 3. Variability of remaining fat content of samples of modified fat breast milk as measured using the creamatocrit method for all treatment subje Figure 4. Daily volume of milk intake (a), daily energy intake from milk feedings alone (b) and daily energy intake from milk feedings + solid foods (c) in the treatm Figure 5. Protein intake from milk and solids for the treatment group and contro Figure 6. Z-score for weight-for-age, length-for-age, weight-for-length and head circumference-for-age at study enrolment and study completion for subjects in the treatment and cont Figure 7. Microbiological De Figure 8. Trajectory of change in weight-for-age z-score for treatment and control subjects from study enrolment t

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ABBREVIATIONS    BCPS Bidirectional cavopulmonary shunt CHD Congenital Heart Disease CoA Coenzyme A HMF Human Milk Fortifier ICPA Infant Care Practice Study IgA Immunoglobin A IGF-1 Insulin-like Growth Factor 1 IQ Intelligence Quotient LCFA Long Chain Fatty Acids LCT Long Chain Triglycerides LDL Low Density Lipoprotein MCFA Medium Chain Fatty Acids MCT Medium Chain Triglycerides MPR Milk Preparation Room PROBIT Promotion of Breastfeeding Intervention Trial SAS Statistical Analysis System SD Standard Deviation SGA Small for Gestational Age sIgA Secretory Immunoglobin A TEE Total Energy Expenditure TDEE Total Daily Energy Expenditure WASI Weschler Abbreviated Scales of Intelligence                                    

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 APPENDICES    

 Appendix A. Consent form Treatment Group Appendix B. Consent form Control Group Appendix C. Data Collection Forms Appendix D. Food Record Treatment Group Appendix E. Food Record Control Group Appendix F. Minimal Fat Diet Guidelines Appendix G: Chest Tube Drainage Data for all Subjects                                              

1

1

1.  INTRODUCTION,  RATIONALE  AND  OBJECTIVES    1.1.  INTRODUCTION  AND  RATIONALE       Breast milk has long been recognized as the ideal food for the vast majority of

infants (1). Although rare, situations do exist in which the breastfeeding or the provision

of expressed breast milk is not possible (1). When an infant is diagnosed with

chylothorax, a post-operative complication following corrective surgery for a congenital

heart defect (CHD), breastfeeding or expressed breast milk must be temporarily replaced

with a therapeutic formula. In comparison to breast milk, which contains fat

predominantly as long chain triglycerides (LCT), the therapeutic formula contains fat

primarily as medium chain triglycerides (MCT). This raised the question of whether or

not it would be possible to modify breast milk to make it a safe and effective treatment

for infants with chylothorax. The idea of low fat breast milk was not novel. There have

been sporadic reports in scientific publications, as well as anecdotal reports accessible via

the internet, of the successful use of low fat breast milk to treat chylothorax. However, at

e effectiveness

of a modified low fat breast milk to treat chylothorax in infants following corrective

surgery for a congenital heart defect. Hamdan et al published a case study in which an

infant with chylothorax was successfully treated with a combination of medication and

low fat breast milk after a patient exhibited intolerance to the MCT based formula (2).

Chan et al published a report detailing experience with the use of fat free human milk for

the treatment of 7 infants with chylothorax that was either congenital or developed

following surgery to repair a cardiac defect (3). A more recent case study by Lessen

described successful treatment of chylothorax with skim breast milk in an infant

2

following repair of a coarctation of the aorta (4). However, none of these reports involved

a scientific comparison to one of the fat modified formulas that are the current treatment

standard for infants with chylothorax. Thus, the question was not; could we modify the

fat content of breast milk? The question was, if we remove as much fat as possible from

breast milk, will the resulting modified fat breast milk be an effective treatment for

chylothorax and if so, will it be as effective, more effective or less effective than the fat

modified formula that is currently used to treat chylothorax?

If a successful means of treatment is available, why seek out a new treatment?

Breast milk has long been recognized as the gold standard for infant feeding (5). The

World Health Organization recommends exclusive breastfeeding for the first six months

of life (6). The American Academy of Pediatrics, Health Canada and the Canadian

Paediatrics Society also promote exclusive breastfeeding through the first 6 months of

life (7-9). Properties inherent to human breast milk contribute to ease of digestibility,

improved tolerance of feeds and superior nutrient delivery (10). The list of health benefits

associated with breastfeeding is long and includes reduced risk of infections including

otitis media and gastrointestinal infections, suspected prevention of certain types of

childhood cancers and other diseas (11-16).

Breastfeeding may also prevent the development of certain allergies, reduce the risk of

obesity and aid in the prevention of cardiovascular disease (17-24). Importantly, many of

these studies supporting the role of breast milk in health also suggest that the duration of

breastfeeding is an important factor in these apparent health benefits (12-13, 18, 20).

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recognized benefits are denied to infants with chylothorax for a period of approximately

6 weeks. Six weeks is a significant portion of time in the short duration of infancy.

Facilitating the continued use of breast milk throughout treatment of chylothorax may

increase the likelihood that mothers will pump to maintain their milk supply and thus

facilitate the resumption of breastfeeding and/or breast milk provision following

completion of treatment. In turn, this allows the infant to continue to receive breast milk

further into infancy and thus providing on-going exposure to its known benefits.

Cardiac infants are a delicate population in whom weight gain is a persistent

challenge and growth failure prominent (25). Any disruption in feeding pattern can

further complicate these issues (25). Anecdotal observations at our institution suggest that

the introduction of the fat modified formula that is the current standard of treatment is

frequently accompanied by feeding intolerance such as increased episodes of vomiting,

gassiness and discomfort with feeds, refusal to orally feed and the need for nasogastric

tube feeding for the duration of treatment. In addition, there is an important psychosocial

component to the provision of breast milk that cannot be ignored. Families are often very

distressed when they are told they cannot breastfeed or provide breast milk, in any form,

families have worked hard to establish a routine of providing breast milk for their cardiac

infant that effectively supports growth. Parents are often desperate to be able to do all

they can for their infant and to ensure their well being, especially when so much of their

medical care is dependent on others. Finally, the need to purchase an expensive

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therapeutic formula can be an additional and often unexpected financial burden for

families.

This research examines the possibility of providing breast milk, in a modified

form, to infants with chylothorax, so that they may continue to receive some of its

recognized benefits, to allow parents to continue to provide an important component of

their infants care and to allow families to be consistent with the feeding and nutrition

goals that they have established for their infant. This thesis includes a comprehensive

review of the literature followed by the research data chapter providing an outline of the

methods, results and discussion of the completed research. A final chapter summarizes

the clinical significance of the research and examines the future clinical implications of

the results.

The literature review that precedes the description of the research discusses

chylothorax, the current treatment and the associated complications. Chylothorax is a

condition that occurs when there is leakage of chylous fluid into the chest cavity that, if

left untreated, can accumulate and lead to compromised respiration (26-28). Prolonged

leakage of chylous fluid, a fluid high in protein and fat, can also lead to associated

complications such as malnutrition and immunodeficiency, putting affected individuals at

increased risk of infection (29)

will be examined.

The second section of the literature review examines the properties of MCT and

how they differ from LCT thus setting the foundation for their use in the treatment of

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chylothorax. Treatment of chylothorax in infants requires the use of a modified fat

formula. This formula is high MCT and low in LCT and forms the basis of conservative

management of this condition. Other clinical uses of MCT, particularly in the paediatric

population, will be briefly examined.

The third section of the literature review examines the benefits of breastfeeding

and the provision of breast milk. The World Health Organization as well as the American

Academy of Pediatrics and the Canadian Paediatric Society recommends the provision of

breast milk as the first and exclusive food for infants, continuing through infancy with

appropriate introduction of complimentary foods at 6 months (6-7, 9). There is growing

evidence to support the role of breastfeeding and the provision of breast milk in the

prevention of certain childhood diseases as well as playing an important a role in

establishing a foundation for good health later in life (21). This evidence will be

reviewed.

The final section of the literature review examines the nutritional issues and

challenges commonly faced by the cardiac infant. Recent literature suggests that infants

with CHD often start life at a disadvantage, more often being classified as small for

gestational age or were classified as being of low birth weight in comparison to healthy

infants (30-32). Postnatal life is complicated by increased energy demands and

difficulties achieving adequate caloric intake to support growth (25). The literature

review examines the nature of the growth difficulties experience by this population and

possible causes of growth failure.

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1.2  OBJECTIVE  

The objective of this research study was to compare the effectiveness of a modified

fat breast milk for the treatment of chylothorax in infants following cardiothoracic

surgery to the high MCT-containing therapeutic formula that is the current standard of

care.

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2.  REVIEW  OF  THE  LITERATURE  

2.1  CHYLOTHORAX  

 2.1.1  Definition  of  Chylothorax  

 

Chylothorax is a condition in which there is an accumulation of chylous fluid in

the pleural cavity. Chylothorax occurs when there is injury to the thoracic duct or one of

its primary branches, causing chylous fluid to leak into the surrounding space (26-27, 33).

Chylothorax is initially asymptomatic. Associated symptoms only develop when a

significant amount of fluid has accumulated, causing the patient to experience an increase

in respiratory effort and ultimately respiratory distress (26). In previous years, mortality

rates were upwards of 50% in untreated cases (29, 33-35). Advancements in conservative

management through the use of MCT based diets or formulas or the use of parenteral

nutrition has significantly reduced associated morbidities and mortality by reducing the

amount of chylous fluid produced, facilitating resolution of the chylothorax and

subsequently improving outcomes for patients affected by chylothorax (29).

2.1.2  Anatomy  of  the  Thoracic  Duct  

The thoracic duct is the largest vessel in the lymphatic system and functions as a

drainage route for the main lymphatic vessels of the body, with the exception of the right

side of the head and neck, right upper limb, right lung, right side of the heart and the

convex surface of the liver (28). It also functions as the primary transport pathway for

intestinal chyle into main circulation (28). Embryologically, the thoracic duct presents in

8

a two-pronged configuration and therefore may grow into a wide array of anatomical

arrangements (35-36)

become one common vessel, the thoracic duct (35-36).

The origin of the thoracic duct is a triangular dilatation known as the cisterna

chyli which is located in the abdomen at the level of the second lumbar vertebra (28).

From the abdomen, the thoracic duct begins its journey through the thorax. The thoracic

duct enters the thorax through the aortic hiatus (37). It then travels upward through the

thoracic cavity in the posterior mediastinum, directly in front of the vertebral column, to

the right of the aorta and behind the oesophagus. Initially, the thoracic duct ascends on

the right side of the oesophagus but then, at the level of the fifth or sixth vertebra, it

crosses to the left side (28, 35, 37). At this point, the thoracic duct follows a route behind

the aortic arch and leaves the thoracic cavity via the superior thoracic aperture. The

thoracic duct ends its journey at the base of the neck where it empties its contents into the

subclavian veins, below the shoulders, near the clavicle, at its juncture with the left

internal jugular (28, 36). During its ascent through the thorax, the thoracic duct comes in

close proximity with the aortic arch and other key structures of the heart, making it

particularly vulnerable to traumatic injury during cardiothoracic procedures.

In adults, the thoracic duct is variable in length and can range from 37 45 cm in

length. It can be structurally inconsistent and may segregate into two or more branches in

up to 40 per cent of individuals (28). These branches can form a complex arrangement as

the duct ascends through the thorax and may terminate as multiple branches or converge

9

into one duct before emptying its contents into the main circulation (28). The thoracic

duct may transport up to 4 L of chyle each day in the healthy adult and as much as 250 ml

in a neonate (28, 38). The magnitude of chyle flow may range from 10 ml to greater than

100 ml per kg bodyweight (28, 38). The volume of chyle is influenced by diet,

medications, intestinal function and physical activity levels (28, 36-37). The rate of flow

through the thoracic duct is largely dependent on the inflow from the lacteals, the

lymphatic capillaries found on the villi of the small intestine that are responsible for

absorption of fat. Consequently, chyle flow is especially amplified following a meal that

is high in fat (35-37)

of 13-14 seconds and are responsible for movement of chyle (28). Flow of chyle may also

be stimulated in situations of increased intrathoracic pressure. For example, during

inhalation, the diaphragm and liver descend in the abdomen creating an increase in

intrathoracic pressure, compressing the cisterna chyli, and subsequently increasing flow

of chyle. A similar effect may be seen if an individual coughs (28). The Bernoulli

suction effect, or the Bernoulli vacuum, is the force that drives the drainage of the chyle

from the thoracic duct into the subclavian vein and is produced by blood flow at the

opening of the thoracic duct (28, 36).

The anatomy of the thoracic duct can determine the nature of the chylothorax

(34). If the site of damage is the lower thoracic duct, the chylothorax is likely to occur on

the right side. Accordingly, damage to the upper thoracic duct results in a left-sided

chylothorax (34).

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2.1.3  Classifications  of  Chylothorax  

Chylothorax has a variety of causes and may be classified as congenital, traumatic

and obstructive (28, 35).

 

2.1.3.1  Congenital  Chylothorax  

Although congenital chylothorax is one of the most common causes of

chylothorax observed in the neonatal period, the actual occurrence of this disorder is

extremely rare occurring in 1:10,000-15,000 pregnancies (28, 36, 39). In the majority of

congenital chylothorax cases, no specific cause is ever identified. It may occur as the

result of birth trauma with hyperextension of the spinal column or increased venous

pressures during birth. However, in such cases there is often an underlying congenital

defect in the thoracic duct that can be identified and renders the lymphatic system

vulnerable to damage (28, 35, 38-41). Congenital chylothorax may be seen with the

occurrence of certain syndromes that are linked with malformations of the lymphatic

system such as Noonan Syndrome and Trisomy 21 as well as in cases of polyhydramnios,

tracheo-oesophageal fistulas, congenital lymphangiectasia as well as thoracic duct

hypoplasia and atresia (28, 36, 38, 41-42).

2.1.3.2  Non-­Traumatic  or  Obstructive  Chylothorax  

Non-traumatic or obstructive chylothorax most frequently occurs in the adult

population and is often caused by an obstruction of the thoracic duct as a result of

intrathoracic tumours, inflammatory diseases or mediastinal lymphangiomatosis (38). An

obstruction causes an increase in pressure in the intrathoracic lymphatic system. This

11

increase in pressure forces collateral lymphatic vessels to dilate and renders the lymphatic

valves ineffective. This combination of increased pressure and ineffective lymphatic

valves creates redundant flow of chyle in the thoracic duct. The thoracic duct eventually

succumbs to the pressure and ruptures, creating a point of damage through which chyle

can escape into the thoracic cavity (26-27, 33, 36).

In the absence of an obstruction, chylothorax can also be caused by increased

pressure in the systemic venous system resulting from elevated right-sided cardiac

pressures. This is frequently seen following surgical procedures that are associated with

systemic venous hypertension such as the bidirectional Glenn and the Fontan procedure

(43). The elevated pressure is disseminated to the surrounding lymphatics which in turn

slows the flow of chyle through the thoracic duct. This triggers dilation and eventual

breach of the duct resulting in leakage of chyle into the thoracic cavity (43).

Chylothorax has also been reported as an infrequent consequence of an indwelling

central catheter that has resulted in a subclavian vein thrombosis (28, 38, 44). Wu et al.

described a case in which a 5-month-old boy developed chylothorax 2 months after a

cardiac surgery following the removal of a peripherally inserted central catheter (44).

Venography revealed occlusion of the innominate vein attributed to the peripherally

inserted central catheter (44). Factors that increase the risk for catheter-induced

thrombosis include small body size, catheter tip in the brachiocephalic vein, unstable

hemodynamics and use of parenteral nutrition (44). Beljaars et al reported on a case of

chylothorax that developed in a 4 year old boy with previous surgical correction for

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Tetralogy of Fallot, following the insertion of a central venous double lumen catheter for

the administration of necessary medication (45). Unlike the case reported by Wu et al,

this case was not associated with a thrombus but was attributed to multiple factors

combination with elevated systemic venous and lymphatic pressures attributable to the

(45).

2.1.3.3  Traumatic  Chylothorax  

Traumatic chylothorax can be classified as surgical or non-surgical and results

from direct injury to either the thoracic duct, or one of its primary branches. (35-36, 41).

Traumatic injuries that pierce the chest, neck or abdomen may result in rupture of the

duct. Blunt trauma or sudden hyperextension of the cervical spine particularly after a

meal when the thoracic duct is distended may lead to a breach in the integrity of the

thoracic duct (41). In the infant population, traumatic chylothorax most frequently occurs

as an early post-operative complication, following a thoracic surgical procedure and more

specifically, following corrective surgery for an underlying cardiac defect (35, 41).

Although chylothorax can occur after any intrathoracic procedures, extrapericardial

interventions in the vicinity of the thoracic duct, such as ligation of a patent ductus

arteriosus or repair of a coarctation or double aortic arch, increase the risk of laceration

during surgery (29). From this point forward, infants who develop chylothorax after

corrective cardiac surgery will be the focus of this paper.

 

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2.1.4  Incidence  of  Chylothorax  

The occurrence of chylothorax can be an additional obstacle to the already

challenging recovery for paediatric patients with CHD following a surgical procedure.

Chylothorax is reported in the literature as affecting 2.5 to 4.7% of paediatric patients

undergoing cardiothoracic surgery (26-27). The recently observed increase in the

incidence of chylothorax is speculated to be the result of the escalating complexity of

corrective surgeries and earlier re-feeding in the post operative period (26-27).

 

2.1.5  Composition  of  Chyle  

Chyle originates from the intestine and therefore much of its contents are enzymes

and products of digestion (28). Sixty to 70 percent of absorbed dietary fat passes through

the lymphatic system and therefore chyle is characteristically high in fat (29).  The high

fat content of chyle imparts its characteristic, milky white appearance which can be used

to arouse suspicion regarding thoracic duct damage and the presence of chylothorax (28).

The fat in chyle is found primarily in the form of triglycerides that have been packaged

into chylomicrons (35-36, 46). In addition to a high triglyceride content, cholesterol and

fat soluble vitamins are also present.   Chyle also contains a high concentration of

proteins, primarily albumin, as well as digestive enzymes and electrolytes. In addition to

its fat, protein and nutrient content, chyle contains a high concentration of lymphocytes

that play an important role in immunity (36). These unique characteristics of chyle form

the foundation of the most common consequences of on-going chyle losses that may

occur in chylothorax: nutritional depletion, most notably protein calorie malnutrition, and

immunodeficiency (26, 36).

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Relative density 1.012-1.015

pH 7.4-7.8 Colour Milky (colourless in starvation) Sterile Yes Bacteriostatic Yes Fat (g/L) 5-30 Protein (g/l) 20-30 Albumin 12-42 Globulin 11-31 Albumin:globulin ratio 3:1 Fibrinogen (mg/l) 160-240 Glucose (mmol/l) 2.7-11.1 Urea (mmol/l) 1.4-3.0 Cell count (per dl) Lymphocytes 40000-680000 Erythrocytes 5000-60000 Enzyme concentration (units/ml) Pancreatic Lipase 0.5-2.4 Amylase 50-83 Aspartate aminostransferase 22-40 Alanine aminotransferase 5-21 Alkaline phosphatase 2-4.8 Acid phosphatase 0.3-0.8 Electrolyte concentration (mMol/l) Sodium 104-108 Potassium 3.8-5.0 Chloride 85-130 Calcium 3.4-6.0 Phosphate 0.8-4.2

Adapted from Valentine, V.G and Raffin, T.A. The management of chylothorax. Chest, 1992. 102(2): p586-91

 

2.1.6  Diagnosis  of  Chylothorax  

Both biochemical and microscopic analysis of the chylous fluid form the

foundation for the diagnosis of chylothorax (47). Although one common diagnostic

proposed the following diagnostic criteria: > 1.1 mmol/L triglycerides (with oral fat

(46).

Similarly, Beghetti et al proposed: > 1,000 leukocytes/ml (>70% leukocytes);

Table 1. Features of Chylous Fluid

15

triglycerides > 100 mg/dL; protein > 20 g/L; sterile culture; milk appearance and positive

Sudan III (48).

Diagnosis of chylothorax at The Hospital for Sick Children is based on volume of

drainage as well as compositional analysis of the drainage fluid. If chest tube drainage

appears milky or exceeds 5 ml/kg/day on post operative day 4, a sample of pleural fluid is

sent for analysis to determine triglyceride content, cell count and the presence of

chylomicrons. A positive diagnosis of chylothorax is made and treatment initiated if the

sample is positive for chyle and/or the lymphocyte fraction of the cell count is > 80%

(49).

 

2.1.7  Management  of  Chylothorax  

Current conservative management of chylothorax consists of pleural chyle

drainage and nutrition support (26, 28-29, 46).   The goal of the nutrition support is to

manipulate the diet in such a way that chyle flow through the thoracic duct is minimized,

giving the damaged thoracic duct an opportunity to heal. For patients with chylothorax,

the fat composition of the diet is a fundamental component of treatment. The mechanism

of fat absorption depends on the fatty acid chain length (50). MCT contain saturated fatty

acids with chain lengths of 6 12 carbons (51). MCT are water soluble, making them

more easily absorbed than LCT. Digested MCT are primarily absorbed directly into the

portal vein circulation, bypassing the lymphatic pathway and thus reducing lymphatic

flow (40, 51-54). Conversely, digested LCT are absorbed via the lymphatic system and

transported via the thoracic duct into the blood stream, thus increasing thoracic flow (51,

16

54). Consequently, a diet rich in LCT increases chyle flow in the thoracic duct and

prevents healing of the damaged duct, further increasing the potential for complications

(46). Therefore, MCT plays a fundamental role in the enteral nutrition management of

chylothorax in the paediatric population. As an alternative therapy, parenteral nutrition

may be used to bypass the intestine completely.

Current management for chylothorax at our facility follows a defined algorithm

(Figure.1) that initially requires that affected infants be placed on a fat modified formula

such as Portagen® (49, 55). Portagen® is -protein based powder with 87%

MCT designed for use in children and adults (55). At standard concentration, Portagen®

provides 1.0 kcal/ml but is diluted for use in infants to provide a caloric content more

similar to that of breast milk or standard infant formulas e.g. 0.67 kcal/ml. Infants are

required to stay on this formula for a minimum of 6 weeks (49). Concerns regarding the

use of this product relate to the fact that it is not a specialized paediatric product and

therefore does not reflect the nutrient needs of young infants. Protein levels are

significantly higher than those found in human milk or standard infant formula. In

17

18

addition, some micronutrients, such as vitamin C, folic acid and vitamin D are found at

lower levels in comparison to those found in human milk or standard infant formula (55).

These nutrient deficits can be magnified when an infant is faced with a fluid restriction

and feed volumes are limited as is often the case with cardiac infants. At our institution,

digestive difficulties manifested as more frequent episodes of vomiting, increased

gassiness/discomfort and symptoms of reflux are frequently observed in infants with the

introduction of the MCT based formula. This in turn may result in increased use of

gastrointestinal medications and a prolonged hospital stay. Poor palatability in

comparison to breast milk or standard infant formula may contribute to decreased oral

feed consumption, particularly in older infants, resulting in reduced energy and nutrient

intake. Both tolerance issues and diminished feed volumes can make weight gain in the

post surgical period challenging, and increase the need for a nasogastric feeding tube to

ensure adequate intake of energy, nutrients and fluid. It is well documented that the

cardiac infant is at high risk for growth failure and any disruption to an already

established feeding pattern may exacerbate existing failure to thrive or put a previously

well nourished infant at risk for nutritional depletion (56-60). Final concerns are related

to the potential for bacterial contamination of powdered formula. The MCT formula

currently in use is available only in powdered form, thus is not a sterile product and like

other powdered formulas may harbour the dangerous bacteria enterobacter sakazakii (61).

The use of powdered formula increases the risk for infection and increases the possibility

of serious morbidities associated with this harmful bacteria in an already fragile

population (62).

19

2.1.8  Determinants  of  Successful  Conservative  Management  of  Chylothorax  

The different causes of chylothorax in the paediatric cardiac population, either

direct trauma to the thoracic duct or increased venous pressures, are associated with

different clinical features of the pleural effusion and thus can determine success of

management. Le Coultre et al reported a shorter duration to diagnosis in cases of direct

trauma to the thoracic duct in comparison to those in whom chylothorax was associated

with an obstruction or elevated venous pressures, thus allowing earlier initiation of

treatment (63). It was also observed that volume of chylous drainage was higher in those

patients whose chylothoraces resulted from elevated venous pressures versus those who

suffered direct trauma to the thoracic duct (63). This result has also been reported in

other studies (33, 48). Subsequently, non-operative management (dietary manipulation)

was most successful in those with direct damage to the thoracic duct at the root of their

chylothoraces (48, 63). Pedra et al also reported a longer period to diagnosis and a longer

period of drainage in chylothoraces resulting from elevated pressures in the superior vena

cava (43). The authors suggest that in chylothoraces stemming from increased venous

pressures, the time required for accumulation of chyle, dilation and eventual rupture of

the thoracic duct accounts for the delay observed between surgery and diagnosis of

chylothorax. The resulting increased pressure in the lymphatic system is also responsible

for larger volumes and longer periods of drainage (26, 43). Chan et al observed a longer

duration of drainage with cavopulmonary anastomosis procedures, frequently associated

with increased venous pressures, in comparison to other surgeries, although the volume

of drainage was not significantly different (26). Ultimately, chylothorax resulting from

20

increased venous pressures appears to be associated with an increased risk of

conservative management failure (33, 48, 63-64).

Despite the advances made in the treatment of chylothorax, there continues to be

considerable debate surrounding the appropriate form and length of conservative

management (enteral feeding versus parenteral nutrition) of chylothorax in infants and

children (36, 46, 63). Success of conservative management relies on implementing

nutrition measures that reduce chyle flow to allow for healing of the damaged thoracic

duct (36). Literature exists to suggest that any oral intake, including water, increases

lymphatic flow, thus supporting the use of parenteral nutrition to bypass the gut

completely (40). However, there is a growing body of literature to support the use of

enteral nutrition as first line treatment in infants with chylothorax. Puntis et al reported

successful resolution of chylothorax in 80% (12/15) of their subjects using an enterally

based diet supplemented with MCT (65). A retrospective study conducted by Verunelli et

al also reported successful resolution of chylothorax in 91% (10/11) of the studied subject

group after receiving a low fat diet supplemented with MCT oil (34). According to a

study conducted by Allen et al, treatment with parenteral nutrition did not reduce duration

or quantity of pleural drainage compared to patients receiving MCT based enteral

formula. In addition, the probability of requiring surgical ligation of the damaged thoracic

duct was found to be similar in both forms of treatment (29). Nguyen et al reported a

similar duration and volume of drainage for those patients treated with low-fat, MCT

based enteral diets versus those initiated on parenteral nutrition. However, it should be

21

noted that 76% of subjects were started exclusively on parenteral nutrition and the

comparison between groups excluded 2 patients from the enterally fed group that were

switched to parenteral nutrition due to high drainage volumes attributed to high venous

pressure (33). Chan et al reported a 71% recovery with MCT based feedings alone,

which is similar to the results of other studies (26). Cormack et al also observed a similar

success rate using an MCT based formula, with 78% of patients having successful

resolution of their chylothoraces (66).

The length of treatment of chylothorax with conservative means continues to be a

topic of debate in the literature (63). Most authors appear to agree that initially attempting

to decrease chylous drainage using conservative management is warranted because of the

risk associated with an invasive surgery and the potential for spontaneous resolution

using conservative techniques (46). Use of an MCT based diet may be preferable in some

patients because of the minimal risk of infection and the ability to use the gut for

be administered as an outpatient in comparison to parenteral nutrition (33, 43, 53). If the

use of an MCT based diet is unsuccessful after a specified period of time, a trial of fasting

and parenteral nutrition is then indicated (46, 53). Le Coultre et al suggest treatment with

an MCT based diet for one week followed by the initiation of parenteral nutrition if there

is no improvement. Surgical management should be considered if conservative treatment

is unsuccessful within four weeks (63). Puntis et al suggest that three weeks of

conservative management not accompanied by a reduction in chylous drainage serves as

an indication for surgery (65). Prolonged conservative treatment may put the patient at

22

increased risk for nutritional depletion, immunosuppression and may impact the outcome

of surgical treatment (34, 65).

2.1.9  Complications  of  Chylothorax  

Although the management of chylothorax has improved, complications of

chylothorax continue to significantly impact affected patients. The unique characteristics

of chylous fluid can make associated complications serious with the severity of adverse

effects being dependent on the volume and duration of chyle loss (28). Loss of chylous

fluid during prolonged periods of therapeutic drainage can lead to a vast array of

associated morbidities including protein-calorie malnutrition, fluid loss and electrolyte

imbalances as well as lymphocytopenia and immunodeficiency (26, 33, 51).

Resulting nutritional depletion with prolonged drainage or high volume drainage

make age appropriate weight gain extremely difficult with weight loss observed in some

cases (29, 43, 65). However, not all studies have yielded consistent results. Puntis et al

reported a weight loss of up to and greater than 8% in one third of patients, with the most

severe weight loss being observed in those with the largest volume of drainage (65).

Pedra et al reported a weight loss of up to 12% in 67% of patients (43). Le Coultre et al

reported a weight loss of < 5% with the exception of one patient who experienced a

weight loss of 10% (63). Allen et al observed weight loss of less than 10% in 14 of 18

children studied with no difference between those patients receiving MCT based enteral

diets and parenteral nutrition (29). In contrast, Nguyen et al observed weight maintenance

or age appropriate weight gain. This may be related to the routine use of intravenous lipid

solutions for provision of additional calories both in patients treated with parenteral

23

nutrition as well as in patients treated with MCT based enteral nutrition (33). Cormack et

al reported a weight gain from the time of surgery to the date of discharge in 14 of 17

patients (82%). The remaining 3 subjects (18%) were discharged at a lower weight than

was recorded prior to surgery but the overall weight loss was less than 3% (66).

Allen et al observed lymphopenia in 36% of subjects with available white blood

cell counts and 5 of 18 subjects developed infections (29). Of those patients who

developed infections, only 2 were patients with observed lymphopenia and therefore offer

no clarity regarding the effect of depleted T-cells and risk of infections (29).

 

2.1.10  Breast  Milk  and  Chylothorax  

The diagnosis of chylothorax can be challenging for any infant and extremely

disappointing for parents, especially for those who had hoped to provide breast milk as

the primary form of nutrition for their infant. Traditionally, infants with chylothorax have

represented a small group of infants for whom breast milk has been contraindicated.

Breast milk has long been recognized as the optimal food for infants (6). Breast milk

contains approximately 45% to 50% of its total energy as fat and of that fat content 80%

90% is LCT (67). Thus it is necessary to withhold breast milk from infants for the

duration of treatment of chylothorax (68-69). The inability to breast feed and/or provide

breast milk to an infant with chylothorax can lead to a multitude of feeding difficulties

such as those discussed above. For parents who wish to breast feed their infant, the

duration of treatment of chylothorax may make the ability to sustain adequate milk

production and to reinitiate exclusive breast feeding post treatment extremely

challenging. Ideally, treatment for chylothorax would involve minimal disruption to the

24

pt to avoid the development of potentially

detrimental feeding issues such as refusal to feed and dependence on nasogastric tube

feeding. Avoidance of further exacerbation of existing nutritional or growth concerns is

also an important goal. For these reasons, dietitians, doctors, nurse practitioners,

lactations consultants and other members of the multidisciplinary team at our facility

have demonstrated great interest in investigating the use of fat-modified, nutrient fortified

breast milk for patients with chylothorax.

At present, there are few published studies examining the use of a modified fat

breast milk in the treatment of infants with chylothorax. Hamdan et al published a case

study in which an infant with chylothorax was successfully treated with a combination of

drug therapy and modified fat breast milk after the patient exhibited intolerance to the

MCT based formula (2). The patient had a complex course of treatment that included the

initial use of parenteral nutrition followed by subsequent use of medication to reduce

drainage. Modified fat breast milk prepared using centrifugation and fortified with

medium chain fatty acids, complex sugars and protein was initiated only after a decrease

in drainage had already been observed (2). Therefore attributing successful resolution of

the chylothoraces to the use of the modified fat breast milk in this particular case is not

possible is. However, what is significant is that it did not increase the drainage and opens

up the possibility for its use in patients with chylothorax.

A study by Chan and Lectenberg published in 2007 described the use of modified

fat human milk for the treatment of chylothorax in infants with chylothorax of either

25

congenital or surgical aetiology (3). The milk was centrifuged at 3000 rpm for 15

minutes at 2ºC. The subsequent modified fat breast milk was then frozen for later use.

Analysis revealed that the mean fat removed was 5 ± 1 g/dl and that the total milk

recovery was 95 ± 2%. The authors noted that it took approximately 60 minutes to

process 1000 ml of milk (3). Supplementation of the modified fat milk was variable and

included the use of MCT based formulas, glucose polymers, medium chain triglycerides

and protein powder (3). Two patients did not receive nutrient enriched modified fat

breast milk and instead received additional nutrition support in the form of parenteral

nutrition and lipids (3). Analysis of the modified fat breast milk after centrifugation

revealed an electrolyte content similar to that of human milk and an expected deficiency

of calories, essential fatty acids and fat soluble vitamins (3). No information was

provided regarding the impact on the protein content of the modified fat breast milk. In

the seven infants treated, no reaccumulation of chylous fluid was observed. The mean

length of treatment with the modified fat breast milk was 16 days (range 7 34 days) (3).

Most recently, a case study was published describing the use of a modified fat

breast milk for a full term infant in whom chylothorax developed following repair of a

coarctation of the aorta (4). To prepare the modified fat breast milk, the breast milk was

allowed to sit in the refrigerator for 8 12 hours until the fat had separated to the top of

the container. The resulting modified fat breast milk in the bottom of the container was

removed using a nasogastric tube and a syringe. This procedure was repeated with the

resulting modified fat milk, allowing it to sit for several additional hours to remove any

remaining fat (4). Prior to being fed to the infant, the modified fat breast milk was

26

fortified with an MCT based formula to provide 20 kcal/oz (0.67 kcal/ml). The infant was

also provided with daily doses of walnut oil to ensure adequate provision of essential

fatty acids (4)

mother learned to perform the fat removal procedure in hospital, prior to the infants

discharge. The study did not comment on whether or not the fat content of the modified

fat breast milk was tested prior to fortification with the MCT formula (4). Unfortunately

the mother did not have adequate breast milk supply and the infant was ultimately

transitioned to exclusive formula feeding. However it was not clear from the study if this

Nevertheless, the chylothorax successfully resolved and no reaccumulation of chyle was

reported (4).

Anecdotal information is readily accessible on the internet and professional list

s -modified breast milk for the

treatment of chylothorax at other North American hospitals. Emerging case studies and

available information suggest that the use of modified fat breast milk for the treatment of

chylothorax is growing. However its effectiveness has not yet been systematically

evaluated.

 

 

 

 

27

2.2  MEDIUM  CHAIN  TRIGLYCERIDES  

2.2.1  Chemical  Structure  and  Properties  of  Medium  Chain  Triglycerides  

Medium chain triglycerides (MCT) are a combination of medium chain fatty acids

(MCFA) ranging in length from C6:0 to C12:0 with the bulk of the fatty acids being C8:0

(65-75%) and C10:0 (25-35%) (70-73). These fatty acids are attached to a glycerol

backbone comprise a medium chain triglyceride. MCT were first introduced in the

clinical setting in the 19

pancreatic insufficiency, fat malabsorption, impaired lymphatic chylomicron transport

and severe chylomicronemia (72).

In comparison to oils containing primarily LCT, MCT oil has a lower melting

point, is liquid at room temperature and contains less calories (8.4 kcal/g versus 9.2

kcal/g) (70-72, 74-75). In addition, MCFA are relatively soluble in water. These

distinctive features can be attributed to their lower molecular weight. In addition, MCFA

act as weak electrolytes and are highly ionized at a neutral pH, further augmenting their

solubility in a biological environment (70-72, 74). This compilation of unique

characteristics ultimately affects the way MCFA are metabolized and absorbed (70).

 

2.2.2  Food  Sources  of  Medium  Chain  Triglycerides  

Although the vast majority of dietary fats are comprised of long chain fatty acids

(LCFA) with a 14 carbon chain or longer, there are some naturally occurring MCFA in

12% of total fatty acids as C6:0 to C10:0 and 2

5% as C12:0 (72). Coconut and palm kernel oil contain large amounts of MCFA. Pure

28

MCT oils are produced by the hydrolysis and fractionation of coconut oil or palm kernel

oil and subsequent re-esterification (71, 75). The resulting oil contains almost

exclusively C8:0 (67%) and C10:0 (23%) fatty acids (76).

 

2.2.3  Metabolism  of  Medium  Chain  Triglycerides  

With a lower molecular weight and increased solubility in comparison to LCT,

MCT are digested more efficiently by the action of pancreatic lipase (71, 74-75). This in

part can be attributed to the rate and extent at which MCT emulsify in surrounding

solutions, quickly increasing the available surface area for action of pancreatic lipase

(74). MCT are preferentially hydrolyzed by preduodenal lipases and the resulting MCFA

can be partially absorbed through the stomach mucosa (74). In addition, MCT exert less

allosteric inhibition on pancreatic lipase, facilitating its action (74). If both LCT and

MCT are present, then the MCT are the preferred substrate of pancreatic lipase (71).

Long chain fatty acids and MCFA do not follow the same pathway for absorption.

In the case of LCFA, once they have reached the mucosa, they are converted into acyl-

coenzyme A (Acyl-CoA) by the enzyme acyl-CoA synthetase. These are then

incorporated into triacylglycerols which are subsequently packaged into chylomicrons for

transport into the main circulation via the lymphatic system (71). These LCT are then

subject to lipoprotein lipase hydrolysis and fatty acid utilization by extrahepatic tissues

prior to reaching the liver (70). The enzyme acyl-CoA synthetase is specific for fatty

acids with greater than 12 carbon atoms and therefore, the majority of MCFA are not

incorporated into chylomicrons, following an alternate pathway for absorption (71).

29

Instead, MCFA are solubilised in the aqueous phase of the intestinal contents, are

absorbed directly into the portal vein circulation and are transported as free fatty acids

attached to albumin (70). The resulting products of digestion of MCT are absorbed

quickly, as fast as glucose, and the resulting MCFA leave the intestine more quickly than

LCFA (71). The increased blood flow through the portal system, in comparison to slower

lymphatic flow, hastens the arrival of MCFA into the main circulation (74). The liver is

the first organ to receive MCFA in comparison to LCFA, which enter through the

lymphatic system and may perfuse other organs before arriving at the liver (74).

Once absorbed, MCT are highly favoured for oxidation and unlike LCFA, very few

are re-esterified (74). In addition, MCFA do not require carnitine palmitoyl transferase, a

transmembrane protein required to transport LCFA, to move into the mitochondria (70,

72, 76). Thus MCFA move without difficulty into the mitochondria and are readily

-oxidation (70, 72). This may have important implications for the neonate.

Newborns may have low carnitine levels and thus MCFA may be a more readily

metabolized (74).

2.2.4  Medium  Chain  Triglycerides  and  the  Neonate  

During the neonatal period, glucose production is just sufficient to meet the needs

of the newborn brain and therefore there is a need for an alternative energy source that

can spare glucose (77). Bougneres et al estimated that ketones may provide up to 25% of

a newborns energy requirements (77) -

hydroxybutyrate is necessary for survival of the newborn. Ketone bodies released by the

30

liver are utilized as a source of energy by peripheral tissues but perhaps more

importantly, ketone bodies serve as a major source of energy for the developing brain

(70). In an early study Adam et al studied the ability of the fetal brain between 12 to 21

weeks gestation, to take up -hydroxybutyrate and estimated that it could account for up

to 1/3 of the energy consumed by the fetus (78) -

hydroxybuturate was 1.47 times higher than that of glucose and lends support to the key

role of ketone bodies as a fuel for oxidative metabolism by the human fetal brain (78).

The inclusion of MCT in the diet of a newborn, particularly infants born

prematurely, is thought to confer some advantages (70). Pancreatic lipase and bile salts

necessary for LCT digestion may be limited and therefore an alternate form of fat may be

beneficial (70). Lingual and gastric lipases hydrolyze MCT quickly in comparison to

LCT and the resulting MCFA may be directly absorbed into the gastric mucosa (70).

MCT are hydrolyzed more rapidly and more completely than LCT by pancreatic lipase

and the resulting MCFA are absorbed quickly (70). Carnitine levels in the neonate may

be limited and this may be a limiting factor in the transport into and subsequent oxidation

of LCFA in the mitochondria. Given that mitochondrial uptake of MCFA and

subsequent oxidation takes place independently of carnitine, MCFA may be a readily

available source of energy for the neonate (70, 73). However, more recent research is

making a case for the role of carnitine in the metabolism of MCFA in term infants (76).

In neonates, urinary excretion of medium-chain dicarboxylic acids is associated

with increased consumption of MCT and is suggested to provide an indicator of the

31

efficiency of usage of MCFA as a source of energy (76). Dicarboxylic acids are formed

by cytochrome P- -oxidation of MCFA in the liver and kidney (76).

Research by Rebouche et al monitoring excretion of medium-chain dicarboxylic acids in

infants suggests a possible role for carnitine in the metabolism of MCFA during infancy

(76). The study consisted of 2 protocols. In protocol 1, 5 infants were fed a standard

infant formula for 1 week followed by 1 week of a specialty preterm formula that

contained 40% of the fat as MCT. Both formulas contained carnitine, with the carnitine

When infants were fed a higher MCT formula, urinary excretion of medium chain

dicarboxylic acids increased (76). Plasma carnitine levels were not affected by the

feeding routine (76). In protocol 2, 20 infants (10 infants per treatment group) were fed a

soy based formula that contained 40% of the fat at MCT and containing either carnitine

or no carnitine (76). Initially, excretion of medium chain dicarboxylic acids was similar

between groups. However, by day 56, infants receiving formula without carnitine were

excreting significantly more medium chain dicarboxylic acids than those who were

supplemented with carnitine (76). The authors hypothesize that a high intake of MCT

may lead to inefficient utilization of the resulting MCFA due to a limited availability of

free intra- and/or extra-mitochondrial coenzyme A (76). However, if carnitine is present,

it may act as a reservoir for transesterification of short-chain acyl groups thus liberating

coenzyme A and increasing efficiency of MCFA utilization (76). Therefore, in infants,

carnitine may play an important role in MCFA metabolism when intake of MCT is high.

32

A negative feature of MCT is their inability to provide essential fatty acids. If

MCT provide the only source of fat in the diet, supplemental essential fatty acids must be

provided to prevent deficiency. This is particularly important in the growing infant (70).

2.2.5  Clinical  Uses  of  Medium  Chain  Triglycerides  

As previously mentioned, MCT are used in the treatment of a number of clinical

diagnosis. In the paediatric setting, they have found a niche in specialty formulas for use

in the preterm infant in an attempt to improve fat absorption (79). The MCFA content of

these formulas can range from 40%-50% of total fatty acids, with the majority of the

MCFA component comprised of C6:0 and C8:0 fatty acids (73). In comparison, the

MCFA content of human milk ranges from 4% (if defined as C6:0 t0 C8:0) to 12% (if

defined as C6:0 to C12:0) of total fatty acids (73). The inclusion of high levels of C6:0

and C8:0 MCFA was based on the assumptions that MCT are absorbed more readily by

the neonatal intestine and that once absorbed, MCFA are completely metabolized in the

liver to CO2 or ketone bodies. It has also been suggested that high levels of MCFA in

formula will improve nitrogen retention and growth of preterm neonates (73). However

these suggested benefits of high levels of MCT remain speculative. Sulker et al, fed 28

healthy very low birth weight (VLBW) infants a formula containing 38% of fat as MCT

oil. A second group of babies received a formula with a fatty acid profile that more

closely resembled that of human milk and contained no added MCT (79). Although fat

absorption was higher and resulting fat accretion significantly higher in the MCT fed

group, there was no difference observed in weight gain (16.6 ± 2.42 g/kg/day vs. 16.1 ±

2.32 g/kg/day). Nitrogen absorption and excretion levels were not different between

33

groups and both groups experienced similar protein accretion levels. Overall, the MCT

formula did not appear to impact nitrogen retention or excretion nor do the results of this

study suggest related growth advantages (79). In a similar study by the same group, the

effects of an MCT based formula on mineral balance were examined (80). Fecal loses of

calcium in infants fed LCT formula were found to be significantly higher in comparison

to those infants fed an MCT formula and was correlated with excretion of fat. This led to

lower calcium absorption and retention. A similar pattern was observed for magnesium.

Therefore, although the previous study did not suggest growth advantages, this

subsequent study suggests that calcium and magnesium absorption is increased with the

use of MCT based formula (80).

Hamosh et al performed a similar study in which 12 preterm infants were fed both a

formula containing fat primarily as LCT (7% MCT) and a formula containing 42% of the

total fat as MCT (81). Each infant served as his/her own control and received both

formulas for a period of 1 week. Fat absorption and weight gain was not significantly

different when infants were fed either the MCT formula or LCT formula. However, as

mentioned in the discussion of preceding studies, detection of a difference in growth

would be difficult with such a small sample of infants (81). Both MCT and LCT were

hydrolyzed in the stomach of the infants with C8:0 and C10:0 being preferentially

released followed by the release of 18:1 and 18:2 fatty acids (81). The amount of C8:0

and C10:0 in the stomach was found to be low, suggesting that they had been absorbed

prior to sampling supporting the notion that MCFA are a readily available source of

energy for the newborn (81).

34

In a study by Wu et al, the effect of variable levels of MCT in infant formula on

gastrointestinal tolerance, fat absorption, plasma ketone and urinary dicarboxylic acid

levels was examined in 64 low birth weight infants (82). MCT content of the randomly

assigned formula was 0%, 17%, 34% or 50% of the total fat content. No difference in

gastrointestinal tolerance was observed as assessed by the presence of gastric aspirates,

abdominal distension and episodes of emesis (82). Fat absorption was not significantly

different between groups. Plasma levels of 3-hydroxybutyrate were significantly higher

in those infants who received 50% of their total fat intake as MCT in comparison to those

who received no MCT but was not significantly different from those infants receiving

17% and 34% of total fat as MCT. Urinary levels of dicarboyxlic acid increased with

increasing MCT content of the formula (82).

2.3  BREASTFEEDING  AND  THE  PROVISION  OF  BREAST  MILK  

Breast milk is known to be the gold standard for infant feeding (5). The American

Academy of Pediatrics, the Canadian Paediatric Society and Health Canada strongly

suggest that breast milk be the favoured feeding for all infants for the first 12 months of

life, with appropriately timed introduction of complimentary solid foods (7-9). The

World Health Organization recommends exclusive breast feeding for the first six months

of life. Following this, infants should receive safe, nutritionally appropriate

complimentary foods with continued breast feeding for 2 years and beyond (6). Recent

breastfeeding data indicates that 90% of Canadian women express an intention to

35

breastfeed their infants with 90.3% actually initiating breastfeeding after birth (83). Thus,

breastfeeding is the initial feeding method of choice for most Canadian women.

 

2.3.1.  Breast  Milk  and  Digestibility  

Properties inherent to human breast milk contribute to ease of digestibility,

improved tolerance of feeds and superior nutrient delivery (10). To illustrate, the protein

component of human milk is predominantly whey, making up approximately two-thirds

of the protein content (69). Once in the stomach of the infant, the whey protein forms a

soft curd which is more easily digested by the developing gastrointestinal tract of the

infant. In addition it promotes gastric emptying in comparison to casein, the predominant

protein found in bovine milk and, until the last decade, the predominant protein found in

most standard infant formulas (10). Human milk contains an inactive form of lipase that

is activated by bile salts in the duodenum and contributes to improved digestibility and

absorption of fat, the major source of energy in human milk (69).

2.3.2  Breast  Milk  and  Immunity  

In contrast to formula, breast milk is a complex living nutritional fluid that contains

antibodies, enzymes and hormones which impart important immunological and other

recognized health benefits to the infant e.g. disease prevention (84). One of the earliest

identified sources of immunity was through the transfer of immunoglobin A (IgA) from

mother to infant in breast milk and thus providing an acquired form of immunity. Early

human milk, known as colostrum, is especially high in IgA. If a nursing infant and

mother are exposed to a new enteric pathogen, a sample of the pathogen reaches the

36

maternal intestinal mucosa and initiates a cascade of events that results in the priming of

B cells for the production of antibodies. When these B cells are in close proximity to the

mammary epithelial cells, the IgA that is produced is equipped with a carbohydrate chain

and thus becomes secretory immunoglobin A (sIgA). When the sIgA is consumed in the

milk, it resists digestion, and attaches to the pathogen thus inhibiting the development of

disease. This serves as a primary form of defence for the newborn infant (85).

Human milk is not a sterile fluid but rather contains bacteria and other components

which may act as a prebiotic, promoting the development of beneficial bacteria that play

a role in disease prevention (86). Particular interest has been focused on species of

lactobacilli found in human milk, as they are thought to have prebiotic properties,

promoting the growth of bacteria which may offer immune protection to the infant (85-

86). It is known that breast milk fed infants have gastrointestinal microflora that is

distinctly different from that of the formula fed infant. The gut of the breast fed infant is

characterized by the predominance of bifidobacteria, a bacteria that produces acetic and

lactic acids, creating an acidic environment unfavourable to the growth of pathogenic

bacteria and thus assisting in disease prevention (86-87). The bifidobacteria compete for

colonization as well as nutrients and thus inhibits growth of the pathogenic bacteria (86).

Human milk contains oligosaccarides, a group of carbohydrates that can be found in

either their conjugated or unconjugated form which together are categorized as glycans

(88). Unconjugated oligosaccarides are the third most common solid component of

human milk behind lactose and lipids (88). These glycans are suggested to function as

37

prebiotics, promoting the growth of beneficial bacteria such as Lactobacillus bifidus. In

addition, they function as pathogen-binding agents, preventing harmful pathogens from

binding to receptors sites on the mucosal surface of the host gastrointestinal tract (85, 88).

Other immune factors include free fatty acids, especially monoglycerides that are

shown to be antiviral, antibacterial and antiprotozoal and may provide a source of

immunity against ingested pathogens (85). Lactoferrin, a protein found in the whey

portion of human breast milk, has been reported to have several immune functions

including the sequestering of iron from pathological bacteria and thus inhibiting their

growth in the infants gastrointestinal track (85). Further immune benefits can be derived

-lactalbumin to an alternate conformation that occurs in the

to be letha -lactoalbumin made lethal

(85).

 

2.3.3  Breast  Milk  and  Disease  Prevention  

2.3.3.1  Breast  Milk  and  Diseases  of  Childhood  

The provision of breast milk has been suggested to play a role in the possible

prevention of certain childhood diseases as well as providing protection against the

development of chronic diseases later in life (21). Evidence exists to suggest that breast

feeding and/or provision of breast milk as well as duration of breast milk feeding may

provide a protective role against the development of Type I Diabetes Mellitus (89-90).

38

The provision of breast milk may contribute to risk reduction for certain childhood

(15-16). Breast feeding has also been shown to have a protective effect against the

development of celiac disease as well as ulcerative colitis and Chron s disease (21, 91-

92). The well recognized immune benefits of human milk may confer an advantage in the

prevention of allergy (93). As previously discussed, human milk does contain a multitude

of immune-modifying components such as IgA antibodies against bacteria, fungi, food

and inhalants (94). Breast milk also contains pro-inflammatory cytokines, such as tumour

necrosis factor- -inflammatory factors and chemokines. Depending on the

equilibrium of these substances, the immune response that plays a role in allergy

development may be enhanced or suppressed (94). Breast feeding has been associated

with a reduction in the risk of atopic dermatitis (93). Gut colonization through

environmental contact, especially with the mother, provides an important antigenic

which has a significant influence on the bacterial colonization of the gut as well as the

oral and nasal mucosal barriers by regurgitation (94).

2.3.3.2  Breast  Milk  and  Otitis  Media  

Acute otitis media is among the most common of childhood illnesses and a

growing body of evidence supports a role for breastfeeding in the reduction of otitis

media infections (84). At meta-analysis conducted by Uhari et al examining the risk

factors for acute otitis media found that there was a reduction in risk with exclusive

breastfeeding for a minimum of 3 months (95). Duffy et al conducted a prospective study

39

to examine the effects of breast feeding, on the occurrence of acute otitis media and otitis

media with effusion (13). Results found that for the first 3 months of life, the cumulative

incidence of otitis media was the same in both breastfed and group formula fed infants.

However, from 6 to 12 months of age, the cumulative incidence of first episodes of otitis

media increased from 52% to 76% in formula fed infants versus 25% to 51% in

exclusively breast fed infants (13). Those infants who were exclusively formula fed were

also found to be at elevated risk for recurrent episodes of otitis media (13). Using data

derived from a cohort of patients who participated in the Infant Care Practice Study, a

prospective longitudinal cohort, Vernacchio et al reported an association between

breastfeeding and a reduction in maternal reports of otitis media infections with an odds

ratio of 0.69 (14). However, this study only looked at reported incidence of otitis media

within a one month time span and did not differentiate between exclusive and partial

breastfeeding.

Despite these described benefits, studies exist that have failed to observe such

benefits. Alho et al performed a retrospective cohort study with the goal of assessing the

relationship between modifiable risk factors, including breast feeding, and the occurrence

of otitis media (96). Little benefit was observed with breast feeding > 3 months with an

odds ratio of 0.9, in comparison to an odds ratio of 0.8 for those who were breast fed for

< 3 months (96). Therefore, the protective effect of breast feeding was small in

comparison to that seen in other studies. Issues with study design may have been a key

factor in the observed outcomes of the study. No distinction was made between exclusive

or partial breast feeding (96).

40

Stenström et al performed a retrospective case-control study to describe various

factors that differ between children who are prone to otitis media infections and those that

are not (97). Otitis prone children were defined as those children who experienced 5 or

more episodes before the age of 30 months (97). Similar to the study findings of Alho et

al, no difference in breast feeding duration was found between the otitis prone children

and the controls (97).

2.3.3.3  Breast  Milk  and  Gastrointestinal  Infections  

The potential role of breast milk and an associated reduction in gastrointestinal

infections is important for all infants but particularly in developing countries where the

effects of diarrhoea can be devastating (6). Kramer et al conducted an observational

cohort study using a group of subjects from within the Promotion of Breastfeeding

Intervention Trial (PROBIT) study (12). Two groups of infants were examined: those

who were exclusively breastfed for 3 months (n=2862) and those who were exclusively

breastfed for 6 months (n=612). Exclusive breastfeeding was defined as no liquids or

solids other than breast milk. Infants exclusively breastfed for 6 months or longer were

found to have a significant reduction in the risk of gastrointestinal infections (12).

Quigley et al published the results of a case control study that examined the effects of

several factors, including breast feeding, on the occurrence of diarrhoeal illness in 1990s

England (11). Parents were asked to specify how their infant was currently receiving

milk: either exclusive breast feeding mixed breast feeding/bottle feeding or exclusive

bottle feeding. Provision of formula was associated with a fourfold increase in diarrhoeal

41

illness in infants under and over 6 months of age in comparison to those who were

exclusively receiving breast milk (11). Receiving no breast milk and not receiving

exclusive breast milk was also associated with an increase in diarrhoeal disease (11). The

odds ratio of diarrhoeal disease increased with increasing time since cessation of breast

feeding in those infants who had ever been breast fed (11).

2.3.3.4  Breast  Feeding  and  Cardiovascular  Health  

The relationship between infant nutrition and long term cardiovascular health

continues to be an area of on-going research. In contrast to formula, human milk contains

cholesterol and infants receiving human milk have been found to have higher mean blood

levels of cholesterol in infancy (21, 98). However, when examined in adulthood, breast

feeding has been shown to be associated with lower levels of cholesterol in comparison to

those who were bottle fed (21). A recent systematic review by Owen et al examined 17

observational studies of either cross-sectional or longitudinal design (99). Results focused

on mean differences in total cholesterol values in adults who as infants had been either

breastfed or formula-fed. Adults who had been breastfed had a marginally lower total

cholesterol level than those who had been formula fed (99). However, the difference in

mean total cholesterol levels was more pronounced when those who had been exclusively

breast fed were compared to those who had been exclusively formula fed (99). Of note,

of the 7 studies included in the examination of the effects of exclusive breastfeeding, all

but one had feeding history recorded in infancy, increasing the likelihood of accurate

feeding data (99). These results lend further support to results of earlier work by Owen et

al where total cholesterol levels and low density lipoprotein levels were found to be

42

modestly lower in adults that had been previously breastfed versus those that had been

bottle fed (98). Interestingly, this relationship only appeared in adulthood as no

relationship between breast feeding and total cholesterol or low density lipoprotein levels

in adolescence was observed (98). The authors suggest that some form of nutritional

programming, possibly the results of the unique nutrient profile of human milk in

observed in adulthood (98).

Additional studies lend support to the protective role of breast milk against

cardiovascular disease. In a study by Singhal et al, preterm infants that received banked

human donor milk had lower blood lipid levels and lower blood pressures in adolescence

in comparison to those that were formula fed (100). Evidence from the Boyd Orr Cohort

suggests a reduction in the degree of atherosclerosis at age 65, as measured by intima-

media thickness and plaque prevalence, in adults that had been previously breast fed

versus those that were bottle fed. This effect was unchanged when factors such as blood

pressure, cholesterol and insulin resistance were controlled for (101). The Nurses Health

Study suggested an 8% reduction in risk of coronary heart disease in those that had been

previously breast fed (24). The problem that persists with these types of studies is that

accurate recall of breastfeeding history in an older cohort is plagued with the inaccuracies

of human memory, decreasing the likelihood of finding a protective effect.

 

 

 

43

2.3.3.5  Breast  Feeding  and  Obesity  

Of recent interest is the possible relationship between breast feeding and the

occurrence of obesity, a growing public health concern (102). Evidence continues to be

contradictory and studies are often confounded by the short comings of observational

studies. The means of protection associated with breast feeding are difficult to isolate

because many other aspects of development are related to the potential development of

obesity, such as maternal education, race/ethnicity, maternal size and birth weight, all of

which breast feeding is maintained (17). Suggested mechanisms may be related to

differences in protein content between breast milk and formula, variance in overall

energy intake, differences in hormonal responses to breast milk versus formula and effect

on adaptation to the introduction of solids (103).

A systematic review was conducted by Owen et al to examine the relationship

between feeding during infancy and obesity in later life (102). Sixty-one studies that

documented a relationship, either quantitative or observational, of the risk of obesity later

in life between those who were breastfed versus formula fed, were included. Of these 61

studies, 28 studies provided 29 quantified estimates of the effect of breastfeeding versus

formula feeding on the development of obesity later in life. Of these, 28 supported a

reduced risk of obesity with breastfeeding. A fixed-effects model revealed that breastfed

subjects were less likely to be classified as obese later in life when compared to formula-

fed subjects with an odds ratio of 0.87 (103). The positive relationship was stronger in

smaller studies but did exist in larger studies (103). The positive relationship was much

stronger in the 4 studies in which the feeding groups were defined as exclusive

44

breastfeeding with an odds ratio of 0.76 (103). Of the 14 studies that included

information on duration of breastfeeding, an increase in protection appeared to exist with

(103). When the important effects of

socioeconomic status, parental BMI and smoking status were controlled for in 6 studies,

the odds ratio was reduced to 0.93 (95%  CI:  0.88 0.99)  (103).

A meta-analysis was conducted by Harder et al to determine a possible association

between duration of breast feeding and occurrence of overweight (19). Seventeen studies

met the inclusion criteria of original data, a reported odds ratio and 95% confidence

interval and data on duration of breastfeeding for at least one exposure group (19). A

weighted meta-regression analysis showed an inverse relationship between duration of

breastfeeding and the risk of overweight (19). This was confirmed by categorical

analysis. Using a random effects model, each month of breastfeeding was associated with

a 4% decrease in risk of overweight with an odds ratio of 0.96/month (19). Only 2 studies

included in the analysis defined exclusive breastfeeding and yielded a similar odds ratio

of 0.94/month (19). The type and number of confounders differed largely between studies

included in this meta-analysis and as a result, no confounder-adjusted odds ratio was

calculated(19). Results from other studies suggest that the observed relationship may

weaken when confounders are considered (102).

The World Health Organization recently published a meta-analysis regarding the

long-term effects of breastfeeding that include the studies discussed above, all the papers

included in these meta-analysis and any newly identified in the literature search

conducted by The World Health Organization and at the University of Pelotas (102). This

new meta-analysis included 33 studies with 39 estimates of the relationship between

45

breast feeding and prevalence of overweight/obesity. Results of a random effects model

suggest a small protective effect of breastfeeding against the development of

overweight/obesity (102). Those who were breast feed were less likely to be classified as

overweight or obese with a pooled odds ratio of 0.78 (95% CI: 0.72 0.84) (102).

 

2.3.3.6 Breast  Milk  and  Cognition  

Long-chain polyunsaturated fats are present in breast milk (67). These fatty acids,

specifically decosahexanoic acid and aracadonic acid, accumulate rapidly in the brain in

the last trimester of pregnancy and in the first months of life and are believed to play an

important role in brain development of infants (102). Until recently, formula did not

contain these unique fatty acids and thus the provision of breast milk was thought to

promote improved cognitive development. For infants with chylothorax, the lack of

adequate long-chain polyunsaturated fats in the MCT based formula deprives them of

these important nutrients for the duration of their treatment (55, 104). Although existing

data continues to be conflicting, there is some evidence to support the role of breast milk

provision in improved cognitive development (105-107).

In a meta-analysis published in 1999, Anderson et al performed assessed the

differences in cognitive development between infants who were breast fed versus those

who were formula fed with and without adjustment for covariates known to be associated

with neurodevelopment (106). Twenty key covariates were identified and include

maternal intelligence, maternal age, maternal education, maternal training and training

and paternal education (106). Of the 20 studies meeting the initial inclusion criteria, 11

46

controlled for at least 5 of the 20 identified covariates (106). Before adjustment for

important covariates, a benefit of 5.32 points in cognitive function was observed in breast

fed infants in comparison to formula fed. After adjustment for key covariates, breast

feeding was associated with a 3.2 higher cognitive developmental score than was formula

feeding (106). Low birth weight infants exhibited the most pronounced benefit from

having received breast milk (106). The magnitude of benefit increased with duration of

breast feeding with the greatest benefit being seen in with > 8 weeks of breastfeeding

(106).

In 2000, Drane et al published the results of a systematic review to investigate the

impact of breastfeeding on cognitive development (105). To be included in this analysis

studies first had to clearly define the cognitive outcome and measure it using

standardized tests. Second, feeding had to be classified as a continuous variable or a

three-level categorical variable e.g. exclusively formula fed, exclusively breast fed or

partially breast fed. Finally, studies had to control for potential confounding variables

such as socioeconomic status and maternal intelligence (105). Twenty four studies were

identified and of these, only 6 met all three methodological criteria. Of the 6 studies, 4

found an advantage of breastfeeding on cognitive development of 2 to 5 IQ points for

term infants and 8 points for preterm infants (105).

Vohr et al examined the potential beneficial effects of breast milk provision on

intelligence scores in extremely low birth weight infants at 30 months of age (108). The

Bayley Scale of Infant Development II including the mental scale, motor scale and

47

behavioural scale was used to measure neurocognitive development. The group was

composed of 773 extremely low birth weight infants, 593 of whom received some breast

milk while in hospital. Mothers of those infants who received some breast milk were

more likely to be college educated, married, Caucasian and of higher socioeconomic

status than mothers who did not provide any breast milk (108). Breast milk provision was

divided into quintiles based on volumes of breast milk provided to the infant. Increasing

volumes of breast milk provided was associated with earlier achievement of full feeds

and shorter duration of hospitalization (108). Infants in the top 3 quintiles of breast milk

provision during initial hospitalization had higher mental and motor development than

those in the lowest quintile. Bayley scores were also significantly higher in the top 2

quintiles of breast milk provision for the behaviour rating scale (108). In addition, rates

of re-hospitalization were significantly lower for those infants who received larger

volumes of breast milk (108). Unfortunately, no information was collected about

maternal IQ, a factor that has highly predictive of IQ in offspring in other studies (108-

109).

Horwood et al examined the association between provision of breast milk and

cognitive abilities at 7-8 years of age in low birth weight babies (110). An association

was observed between increasing duration of breast feeding and cognitive abilities for

both verbal and performance intelligence quotient (IQ) (110). When potential

confounding factors frequently associated with duration of breast feeding such as

maternal education, two-parent families and smoking during pregnancy were taken into

48

consideration, the effect was diminished with only the effect between duration of breast

feeding and verbal IQ remaining (110).

In a recent study by Kramer et al, the cognitive development of infants enrolled in

the Promotion of Breastfeeding Intervention Trial (PROBIT) in Belarus was examined at

6.5 years of age (107). The PROBIT trial attempted to address methodological limitations

associated with observational breastfeeding studies by using cluster randomization.

number of characteristics e.g. number of births, rural vs. urban. Of each pair, one hospital

was randomized to receive the experimental breastfeeding intervention modelled on the

Baby-Friendly Hospital Initiative of the World Health Organization and United Nations

which promotes health care worker assistance with initiating and

maintaining breastfeeding and lactation and postnatal breastfeeding support (107). The

other hospital in the pair received standard care (107). Mothers who expressed an

intention to breastfeed their infants were eligible for enrolment. The experimental

breastfeeding intervention led to significantly longer duration of breast feeding and a

higher prevalence of exclusive breast feeding (107). The Weschler Abbreviated Scales of

Intelligence (WASI) was used to measure IQ at 6.5 years of age. Higher WASI scores

were observed with longer duration of any breastfeeding and with longer duration of

exclusive breastfeeding (107). IQ scores for both verbal and performance IQ were higher

in exclusive breast feeding for > 6 months in comparison to exclusive breast feeding for 3

6 months and < 3 months (107). One limitation of the study was that the

49

which may have favourable swayed the results for those infants who had been breast fed

(107).

In contrast to these supporting studies, Der et al used data from the US longitudinal

survey of youth to examine the relationship between breast feeding and intelligence and

to examine the role of potential confounders, most notably maternal intelligence (109). A

sibling comparison analysis was used as one method to control for confounders. The

Peabody individual achievement test was administered at 5 and 14 years of age. Results

revealed that children who were breastfed were more likely to have an older, non-

smoking mother with a higher IQ, education, in a supportive home environment (109).

Before adjustment for confounding factors, breast feeding was associated with improved

mental ability (109). However, once maternal intelligence was controlled for, this effect

was significantly reduced suggesting that maternal characteristics, such as maternal IQ,

were largely responsible for the observed effect of higher IQ in breastfed infants (109).

Although some conflicting evidence does exist, the suggested role of breast milk

feeding in the prevention of childhood illness such as otitis media and gastrointestinal

infections as well as the potential role in the prevention of chronic diseases of adulthood,

underscores the importance of supporting ongoing provision of breast milk throughout

infancy. Immune properties of breast milk alone are reason enough to ensure particularly

fragile infants, such as those with congenital heart disease, are afforded every opportunity

to continue to receive breast milk for as long as possible. When provision of breast milk

is interrupted for a period of time, such as it is when an infant requires treatment for

50

chylothorax, it opens the possibility for premature weaning of the infant and cessation of

exposure to the known benefits of breast milk. Not only will absolute exposure be

affected but also duration of breastfeeding will be affected. This is particularly

problematic as duration of breastfeeding has frequently been shown to enhance the

known health benefits (11, 102, 106-107). The use of a modified fat breast milk during an

lactation, promote an on-going sufficient milk supply and will resume breastfeeding or

breast milk feeding for their infant with the completion of treatment.

2.4  NUTRITION  ISSUES  IN  CONGENITAL  HEART  DISEASE  

Despite many significant improvements in the management of patients with

congenital heart disease (CHD), growth failure continues to be a significant problem in

this delicate population (25). Growth challenges frequently begin before birth and can

persist throughout infancy and into childhood, ultimately affecting long-term health (25,

32, 60).

 

2.4.1  Congenital  Heart  Disease  and  Birth  Weight  

The first evidence of growth difficulty is often evident at birth, with many infants

with CHD born at lower birth weights (25). The Baltimore-Washington Infant Study, a

population based, case-control trial examined the relationship between birth weight and

CHD, controlling for infant and maternal factors known to affect birth weight (30).

Results demonstrated that a greater percentage of infants within all categories of CHD

were classified as low birth weight (birth weight <2500g) in comparison to the control

51

population (30). Infants with tetralogy of fallot, endocardial cushion defect, severe

ventricular septal defects, atrial septal defect, coarctation of the aorta and hypoplastic left

heart syndrome all exhibited significant birth weight deficits (30). Infants with

transposition of the great arteries were found to have normal birth weights (25, 30, 32).

Likewise, Kramer et al examined the birth weights of 843 babies with CHD and

found these infants to have a significantly lower birth weight in comparison to a healthy

reference group (31). Although the difference was statistically significant, the authors

note, that the magnitude of the difference was only 57 g for healthy male controls and 56

g for healthy female controls (31). Those infants with tetralogy of fallot and secundum

atrial septal defects had the most pronounced deficit in birth weight (31). Infants with

CHD were also found to be more frequently classified as small for gestational age (birth

weight < 10th percentile) or low birth weight (birth weight < 2500g) in comparison to the

control population (31). In a more recent study, Jacobs et al examined the birth weight of

454 southern Chinese infants with symptomatic CHD. Infants were considered to be

small for gestational age (SGA) if their z-score for birth weight was < -2 Z reference

charts for Hong Kong (32). Results revealed that the mean birth weight of infants with

CHD was significantly lower than the reference group and 15% of infants with CHD

were classified a SGA (32). The birth weight of infants with transposition of the great

Arteries did not differ from reference values (32).

52

2.4.2  Congenital  Heart  Disease  and  Postnatal  Growth    

In the postnatal period, characteristic patterns of growth emerge. Historically,

research has suggested that infants with cyanotic lesions displayed more pronounced

growth failure than infants with acyanotic lesions (25). However, recent research

suggests a more pronounced growth failure in those with acyanotic lesions, particularly

those with increased pulmonary blood flow (in larger left-to-right shunts) and the

presence of congestive heart failure (111). Jacobs et al reported on postnatal growth in

363 Southern Chinese children less than 4 years of age with CHD (111). At the time of

surgery, mean weight-for-age, length/height-for-age and weight-for-height were all

significantly lower than the reference mean (111). Overall, ~ 40% of the subjects had

weight-for-age and height/length-for-age > 2 standard deviations (SD) below the mean of

the reference population (111). Girls were significantly more affected in weight-for-age

and weight-for-height at the time of surgery than boys (111). At the time of surgery, 57%

of patients with acyanotic defects had weights-for-age and heights-for-age > 2 SD below

the reference mean in comparison to 47% of those with cyanotic defects (111). More

children in the acyanotic group (28% versus 11%) were classified as having low weight-

for-height (111). Those with cyanotic defects were significantly more affected in height-

for-age than weight-for-age versus those with acyanotic defects who were equally

affected in both parameters (111). This shift in growth patterns maybe the result of earlier

surgical correction of severe cyanotic lesions which may help to attenuate and/or reverse

growth failure (25). It should be noted that this study did not exclude those with

chromosomal abnormalities, which likely impacted on the growth of some of the subjects

(111).

53

In a study by Schuurmans et al, growth data of 123 children with CHD were

retrospectively analyzed (112). The children studied had one of 5 diagnosis: ventricular

septal defect closed before 1 year of age, ventricular septal defect that closed

spontaneously, small ventricular septal defect but no surgical intervention, transposition

of the great arteries operated on within the first few weeks of life, tetralogy of fallot

undergoing repair within the first 2 years of life (112). Patients with a large ventricular

septal defect experienced preoperative weights and heights z-scores that were

significantly abnormal compared to the reference population (112). Following surgery,

height improved significantly in the first 12 18 months postoperatively. However,

weight continued to be abnormal in the 36-48 month period with a mean z-score of -1.15

(112). Weight z-score (mean -2.32) and height z-scores (mean -2.68) of patients with

tetralogy of fallot were also significantly abnormal compared to the reference population

prior to surgery (112). Following surgery, patients experienced an improvement in height

but values continued to be abnormal with a mean z-score of -1.22 at 36-48 months of age

(112). Weight in tetralogy of fallot patients normalized within 6 12 months (112).

Patients with transposition of the great arteries showed only minor growth disturbances

and growth of patients with a small ventricular septal defect was completely normal

(112).

Infants with single ventricle physiology are more likely to experience congestive

heart failure and hypoxemia and thus form a group of infants with CHD that are

particularly susceptible to growth difficulties (113). Vogt et al examined the somatic

54

growth of 123 infants with single ventricle physiology and who underwent staged

palliation including a bidirectional cavopulmonary shunt and Fontan. Suboptimal

nutrition was documented and was defined as the inability to tolerate feeds or to achieve

adequate caloric intake, requiring the placement of a nasogastric tube or gastrostomy tube

and calorie feeding (113). The infants with single ventricle physiology had a lower birth

weight than the reference group. Z-scores for weight declined significantly between birth

and the bidirectional cavopulmonary shunt procedure. Those who were classified as

experiencing nutritional difficulties in the pre-bidirectional cavopulmonary shunt

procedure period had lower weight z-scores than the remainder of the study group (113).

Following the bidirectional cavopulmonary shunt procedure, infants experienced catch up

weight gain which levelled off after the Fontan procedure. Z-scores for height

experienced a small improvement following the BCPS but no further improvement was

seen after the Fontan procedure. Z-scores also showed improvement following the

bidirectional cavopulmonary shunt procedure and drifted towards normal following the

Fontan (113).

Examining a more specific group of single ventricle subjects, Kelleher et al

performed a retrospective review of a group of 50 infants with a diagnosis of hypoplastic

left heart syndrome to determine prevalence and risk factors for malnutrition.

Malnutrition was defined as weight-for-age z-score < -2 (114). All infants studied

underwent a Norwood procedure with a Blalock-Taussig shunt followed by a

bidirectional Glenn (114). From the time of initial admission to discharge following the

Norwood procedure, median discharge weight remained unchanged from median

55

admission weight (114). At discharge, mean weight-for-age z-score was -1.4 (114).

Infants with longer hospital stay, longer stay in the intensive care unit, higher diuretic

dose at discharge and shorter duration of parenteral nutrition support had lower weight-

for-age at discharge (114). On readmission for the bidirectional Glenn, 58% of infants

were below the 5th weight-for-age percentile and mean weight-for-age z-score had

declined to -2.1. Thirty-eight percent were below the 5th length-for-age percentile (114).

Infants with lower caloric concentration of feedings at discharge post Norwood, more

frequent admissions prior to the bidirectional Glenn, worse right ventricular function and

higher oxygen saturations at discharge post Norwood tended to have lower weight-for-

age z-score at readmission for the bidirectional Glenn (114). The prevalence of

malnutrition was high among this group of patients and more aggressive nutritional

therapy in the form of parenteral nutrition and higher calorie feedings was associated

with improved nutritional status (114).

In a study by Varan et al, 65% of 89 infants studied were below the 5th percentile

for weight-for-age and 52% were below the 5th percentile for length-for-age (115). Forty

two percent of infants fell below the 5th percentile for both weight-for-age and length-for

-to- (115). Results suggested that infants with

cyanotic heart disease with pulmonary hypertension had a higher incidence of low length-

for- -to-

the study. Varan et al found that those infants with cyanotic heart disease accompanied

by pulmonary hypertension were reported to have lower caloric intake for their age in

comparison to cyanotic heart disease without pulmonary hypertension (115). Those with

56

pulmonary hypertension, regardless of cyanotic or acyanotic defects were more like to be

th percentile for both weight and height) (115).

 

2.4.3  Causes  of  Growth  Failure  in  Congenital  Heart  Disease  

There are many factors considered to be associated with growth failure in children

with CHD (115-116). Achieving adequate caloric intake in the infant with CHD can be

extremely challenging. Issues such as fatigue with feeding, oral aversion, early satiety,

poor feeding tolerance, decreased appetite and malabsorption of nutrients may all play a

role in preventing adequate intake of energy and nutrients (56, 114-116). In a study by

Schwarz et al investigating effective nutritional management of growth failure in infants

with CHD, only caloric intake that exceeded 150 kcal/kg/day by continuous nasogastric

infusion resulted in significantly improved nutrition status (117). Studies have shown that

although resting energy expenditure appears to be similar to age-matched reference

children, increased caloric requirements may be attributed to an increase in total daily

energy expenditure which would include energy required for physical activities that may

range from something as simple as feeding in infants to climbing a set of stairs in

children (118). Barton et al used the doubly labelled water technique to measure total

daily energy expenditure in 8 infants with CHD and significant growth failure (119).

Total daily energy expenditure was found to be significantly higher in infants with CHD

than in healthy infants and the authors estimated that a caloric intake of approximately

140 kcal/kg/day may be necessary to support normal growth in these infants (119). Using

both respiratory calorimetry and doubly labelled water, Leitch et al reported similar

results (120). No difference was found in resting energy expenditure or energy intake in

57

infants with uncorrected cyanotic CHD at 2 weeks of age and again at 3 months of age

(120). However, at 2 weeks of age, these infants did display slightly higher total energy

expenditure and by 3 months of age the TEE was significantly higher in comparison to

healthy controls (60, 120). Ackerman et al looked specifically at 4-month old infants and

found them to have total energy expenditure 2.5 times higher than their age-matched

controls (58).

In a study by van der Kuip et al, again using the doubly labelled water technique,

the energy requirements of 11 infants between the ages of 2 8 months with CHD were

studied in comparison to 46 healthy age-matched control infants (121). Of these 11

subjects, 6 met the criteria for classification of congestive heart failure. The infants with

CHD were found to have significantly elevated total daily energy expenditure when

compared to the health age matched controls. This result was confirmed in the meta-

analysis of 65 infants which suggested that total daily energy expenditure was 35%

higher in infants with CHD (121). The presence of congestive heart failure did not appear

to have a significant effect on total daily energy expenditure, an effect that was also

observed in the meta-analysis (121).

Although increased energy demands in infants with CHD appear to be related to

some degree of increased in total daily energy expenditure, other contributing factors

may include: 1) Over activity of the sympathetic nervous system in congestive heart

failure, releasing catecholamines and resulting in hypermetabolism 2) Increased brain

58

metabolism in undernourished children 3) Increased workload of the cardiac and

respiratory muscles translating into increased energy demands 4) Polycythemia as an

adaptation to chronic oxygen deprivation and compensatory acidosis 5) Incidence of

infection causing fever and subsequent increase in metabolic rate (56).

Complicating increased energy demands are the challenges associated with

provision of adequate calories (25, 115). Infants with CHD tend to tire easily with the

exertion of feeding and they may experience early satiety, resulting in the ingestion of

inadequate volume of feeds and thus inadequate energy and nutrients (56, 60, 117). The

presence of congestive heart failure may contribute to oedema and hypoxia of the gut

resulting in dysmotility and malabsorption (115, 117). Hepatomegaly decreases gastric

capacity and may increase the likelihood of emesis of stomach contents (60). The

occurrence of metabolic acidosis caused by hypoxia drives respiratory compensation and

results tachypnoea therefore increasing work of breathing and contributing to fatigue,

increased energy expenditure and difficulties with achieving adequate intake (115).

Infants with CHD may be at increased risk for gastroesophageal reflux disease resulting

from hepatomegaly causing gastric compression and thus a reduction in gastric capacity

(56, 122). Reflux causes discomfort with feeding and may contribute to the development

of an oral aversion if not properly managed (122). Diuretics and other medications may

contribute to decreased appetite and early satiety (56). The existence of malnutrition may

delay corrective surgery, slow recovery from surgery and make recuperation from any

resulting complications exceedingly challenging in infants/children with CHD (115).

Furthermore, long term malnutrition may put patients at risk for complications such as

59

impaired motor, cognitive development and may have an impact on surgical outcome and

morbidity (60, 115).

It has been suggested that the presence of congestive heart failure can significantly

affect the nutritional status of a child with CHD (60). Congestive heart failure is often

accompanied by insufficient energy and nutrient intake, poor absorption of energy and

nutrients and an increase in metabolic rate that makes meeting nutritional requirements

all the more challenging (60). In a study by Farrell et al, energy requirements of infants

with congestive heart failure and infants with a left-to-right shunt (no congestive heart

failure) were compared to a group of healthy controls. Open circuit indirect calorimetry

was used to measure resting energy expenditure and the doubly labelled water technique

was used to measure TDEE (118). No difference between groups was found in the resting

energy expenditure but those with congestive heart failure had significantly higher TDEE

(92 ± 20 kcal/kg/d) than the healthy control (61 ± 9 kcal/kg/d) (118).

More recently, attention has been focused on endocrine factors that may play a role

in growth issues. Insulin-like growth factor 1 (IGF-1) is a polypeptide that is secreted in

response to growth hormone and stimulates tissue growth (25). In a study by Dundar et

al, levels of IGF-1, among other factors, were compared between those with cyanotic

heart disease and age and gender matched controls (123). Those infants with cyanotic

heart disease who were malnourished were found to have the lowest levels of IGF-1.

Cyanotic infants that were not malnourished still had levels of IGF-1 that were lower than

the age-gender matched controls (123). Interestingly, there was a positive correlation

60

found between oxygen saturations and IGF-1 levels, supporting the role that hypoxemia

plays in the growth failure of these infants (123).

A vast body of research exists outlining the growth difficulties in infants with

congenital heart disease and the factors that contribute to these growth challenges.

Establishing a feeding regime that supports weight gain and growth is often complicated

by tolerance issues, frequent medical interventions, prolonged hospitalizations and

increased energy demands (25). Any changes in feeding routine could have an impact on

the success of a feeding regime and ultimately affect weight gain, growth and nutritional

status. When an infant develops chylothorax after surgery, a change in what the infant

feeds as well as a change in feeding method e.g. breast vs. bottle is imposed. This is

complicated by the fact that chylothorax itself is often associated with some degree of

weight loss due its corresponding nutritional losses (29, 43, 63, 65). The use of a

modified fat breast milk will help to minimize some of these changes, allowing those

infants who were receiving breast milk feeds prior to surgery to maintain some element

of their previously established feeding routine. The on-going use of breast milk will also

allow infants to benefit from the digestibility of breast milk in hopes of eliminating the

tolerance issues that are frequently observed at our institution with the introduction of the

MCT based formula (10). It is hoped that improved tolerance will result in improved

weight gain throughout treatment of chylothorax in infants.

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3.  THE  EFFECTIVENESS  OF  LOW  FAT  BREAST  MILK  FOR  THE  TREATMENT  OF  CHYLOTHORAX  IN  INFANTS  FOLLOWING  CARDIOTHORACIC  SURGERY.    3.1  INTRODUCTION  AND  RATIONALE     Due to its ease of digestibility, immune properties and a suspected role in the

prevention of allergy and other childhood diseases breast milk is the ideal food for the

most infants (10, 21, 69, 84-85). Today, 90% of Canadian women initiate breastfeeding

after birth (83). When an infant is hospitalized immediately after birth, establishment of

breast feeding or the provision of breast milk can be challenging due to the frequent

separation of mother and baby, delays in initiation of breastfeeding and high levels of

stress affecting milk production. Therefore, successful establishment of breast feeding or

reliable provision of breast milk is no small feat for any mother/infant dyad, and in the

hospitalized cardiac infant, this is particularly true. Once a pattern of breast milk feeding

is established, the next hurdle to overcome is the achievement of a consistent pattern of

growth, again a significant challenge for the cardiac infant, well known to be faced with

growth difficulties.

  A congenital heart defect is the most common anomaly present at birth and in

Canada, 1 in approximately 100 births will result in an infant affected by a congenital

heart defect (124). Of these infants, many will require surgery, some within the first year

length but inevitably longer for

those who experience complications. Chylothorax is a complication that can occur as a

result of damage to the thoracic duct during cardiothoracic surgery and results in the

accumulation of a fluid known as chyle in the chest cavity (26-28). Treatment is

primarily dietary in nature and requires that the infant be changed to a formula that is low

62

in LCT and provides the majority of fat as MCT (26-28, 46). Unlike LCT, which travel

through the lymphatic system after absorption and therefore contribute to increased chyle

flow through the thoracic duct, MCT are absorbed directly into the portal circulation,

bypassing the lymphatic system and decreasing chyle flow through the thoracic duct (72).

The fat component of breast milk is primarily LCT and therefore, at most institutions, the

provision of breast milk is precluded during the treatment period (69). Chylothorax can

be a devastating diagnosis and the mode of treatment difficult to accept for families who

have worked hard to establish a pattern of breast milk feeding to provide the inherent

benefits of breast milk and promote adequate growth in this challenging group of infants.

In addition, experience at our institution has been that the introduction of the MCT based

formula is often accompanied by exacerbation of feeding intolerance, the appearance of

new feeding tolerance issues, unwillingness to feed due to taste differences, decreased

intake and resulting growth difficulties. Finally, the MCT based formula is not an infant

formula and must be diluted for use in the paediatric population and as a result does not

provide a spectrum of nutrients that is ideal for young infants (55).

 3.1.1  Research  Question    1. Is nutrient enriched modified fat breast milk an effective treatment for chylothorax in

infants following cardiothoracic surgery?

3.1.2  Objective    1. To determine the effectiveness of a nutrient enriched modified fat breast milk for the

treatment of chylothorax in comparison to the MCT based enteral product that is the

current treatment standard.

63

2. To determine any existing differences in growth parameters between patients receiving

the nutrient enriched modified fat breast milk and the MCT based formula.

3.1.3  Hypothesis  

Our working hypothesis is two-fold. Because of the inefficiencies associated with

manual removal of the fat layer, we hypothesize that the amount of LCT remaining in

breast milk after centrifuging will be, on average, higher than the known LCT content

(0.41g/100 ml at a concentration of 0.67 kcal/ml) of the MCT based formula (55, 125). A

higher LCT content may in turn contribute to increased drainage of chyle in infants

receiving the modified fat breast milk in comparison to the MCT based formula.

Therefore, we hypothesize that the amount of chest tube drainage will be significantly

higher in infants with chylothorax who receive the modified fat breast milk in

comparison to infants receiving the MCT based formula.

On the other hand, the inherent properties of breast milk may improve feeding

tolerance in comparison to the MCT based formula. As a result, the infant may receive a

more sufficient quantity of the nutrient enriched modified fat breast milk and thus have

improved energy and nutrient intake. The result would then be improved growth in the

infants receiving the nutrient enriched modified fat breast milk.

       

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   3.2.  SUBJECTS  AND  METHODS    3.2.1  Subjects    All infants less than 12 months of age diagnosed with chylothorax at the Hospital for

Sick Children following corrective surgery for CHD, were screened within 48 hours to

determine eligibility.

 

3.2.2  Study  Inclusion  Criteria  

· < 12 months of age

· Confirmed diagnosis of chylothorax (Chylothorax Care Map page 16)

· Patient to have some portion of follow up care at the Hospital for Sick Children

· Informed consent obtained from one, but ideally both, parents

   

3.2.2.1  Treatment  Inclusion  Criteria    

· Previously receiving a minimum of 80% of feeds from breast milk (breast milk may

be nutrient enriched)

· Parents/guardian would like to continue to provide breast milk during treatment for

chylothorax

· Mother willing to pump (with appropriate support) breast milk to supply her infant

with as much breast milk as possible throughout the study period

· Parents/guardian willing to be available for breast milk pickup and delivery

approximately twice each week for duration of study or are willing to perform the fat

removal at home using a small table-top centrifuge

65

3.2.2.2 Control Inclusion C riteria

· Infant receiving <80% of feeds as breast milk prior to surgery

· Infant exclusively formula fed prior to surgery

· Mother unable or unwilling to pump breast milk with the intent to provide as much

breast milk as possible throughout the study period

3.2.3  Study  Exclusion  Criteria  

· Patient receiving full parenteral nutrition at the time of chylothorax diagnosis

· For medical reasons, patient unable to follow Chylothorax Care Map

· Primary caregiver unable to communicate effectively in English

3.2.4  Study  Design  

The study was a non-randomized clinical control trial. Randomization was not

possible for ethical reasons related to breastfeeding and therefore group assignment was

based on the infants feeding routine prior to surgery. Infants previously receiving a

minimum of 80% of feeds as breast milk prior to surgery and whose parents expressed a

desire to continue to provide breast milk were entered into the treatment group. Infants

who received feeds primarily as formula prior to surgery were entered into the control

group and received the standard MCT based formula for the duration of the study.

Lactation support and an electric pump for home use were offered to all mothers who

wished to maintain lactation during the study period. Mothers were provided with

guidelines around proper pumping technique and frequency to ensure maximum milk

production.

66

3.2.4.1  Treatment  Group  

Infants entered into the treatment group received all enteral feeds as nutrient

enriched modified fat breast milk as soon as it was available. While in hospital, mothers

were instructed to express their breast milk as per The Hospital for Sick Children

Protocol using a hospital grade electric breast pump. Following expression, milk was

placed in the freezer at -

Room (MPR) at The Hospital for Sick Children. If no breast milk was available for

immediate use or breast milk supply was insufficient, the infants received the MCT based

formula until breast milk was made available or to complement the existing breast milk

supply. Fat removal was carried out in the MPR according to the procedure outlined

below. The resulting modified fat breast milk was then nutrient enriched, according to

the method described below, to replace the calories and nutrients lost during fat removal.

The nutrient enriched modified fat breast milk was provided to infants via bottle

and direction of the responsible medical team. Volume of feeds and caloric content of

feeds was also determined by the medical team based on fluid allowance, tolerance and

energy requirements to support growth.

While infants remained in hospital, fat removal was be carried out by the study

coordinator as outlined below in Option 1. After discharge, two methods for fat removal

were offered to parents of participating infants. Both are described below.

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3.2.4.2  Fat  Removal  in  Hospital  

Fat was removed from the breast milk using centrifugation performed by the

study coordinator in the MPR under aseptic conditions. A new refrigerated centrifuge

(Allegra X-22R, Beckman Coulter, Brea, California) was committed to the study and

used only for this purpose for the entire study period. Prior to centrifugation, frozen milk

was thawed overnight in the refrigerator at 4ºC or in tepid water bath. Milk was

centrifuged at 3000 RPM at 4ºC for 15 minutes. The fat layer was then manually

removed and discarded. The resulting modified fat breast milk was then frozen at -18ºC

for future use or was nutrient enriched for immediate consumption by the infant

according to the recipe found in Appendix C. While the subject was in hospital, the

modified fat breast milk was nutrient enriched in the MPR under a laminar flow hood on

a daily basis according to hospital policy. Once discharged, parents performed the

nutrient enrichment of the modified fat breast milk at home according to a recipe

provided to them. Each recipe was based on the recipes in Appendix C, but

and adjusted throughout the study period as required.

 

3.2.4.3  Fat  Removal  Following  Discharge    Option  1  

Following discharge, breast milk was picked up from the families and transported

to The Hospital for Sick Children for centrifugation and fat removal using the procedure

described above. The resulting modified fat breast milk was then delivered back to

families for subsequent storage and nutrient enrichment prior to feeding. Breast milk was

picked up for fat removal twice weekly.

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3.2.4.4  Fat  Removal  Following  Discharge    Option  2  

If families so chose, they could take a small, non-refrigerated, tabletop centrifuge

(VWR® Clinical 200, VWR International, Mississauga, Ontario, Canada) home to

perform the fat removal themselves. If families lived outside of the greater Toronto area

(more than approximately 45 minutes by car), this was the only option available to them

as longer distances did not allow for biweekly pickup and delivery of breast milk. If this

option was chosen, parents were educated on how to use a small table top centrifuge to

remove the fat from their breast milk. Prior to discharge, parents were required to

demonstrate their competency of fat removal by achieving a remaining fat content of

resulting low fat breast milk was tested using the crematocrit method (126). The table top

centrifuge was provided to the parents at no cost for the duration of the study.

Throughout the study, all infants continued to follow the current protocol for

treatment of chylothorax as outlined in the Chylothorax Care Map. It should be noted that

all parents of infants in the treatment group were given a back up supply of the MCT

based formula and a recipe for preparation in the case of inadequate breast milk supply.

If the patient was of an age to receive solid foods, guidelines were given to the parents

regarding types of solid foods that were acceptable according to the Minimal Fat Diet

Guidelines (see Appendix F) during the treatment period.

Upon discharge, parents were instructed to continue with the same feeding

regimen established in hospital prior to discharge. Any change to the feeding regimen

69

was discussed and developed in conjunction with the study investigator during the

follow-up clinic visit or during weekly phone follow-up. If a fluid restriction was in

r

responsible nurse practitioner.

3.2.4.5  Nutrient  Enrichment  

Nutrient enrichment of the low fat breast milk was necessary to replace the

nutritional components lost with the removal of fat. In our previous pilot project, the

remaining fat (as measured by the creamtocrit method and a modified Folch technique),

protein (as measured using nitrogen analysis) and energy content of centrifuged breast

milk (as measured using bomb calorimetry) was measured (125). These results were then

used to determine the necessary fortification of the modified fat breast milk (125). Using

these, it was assumed that the average remaining fat, protein and energy content of the

breast milk after centrifugation was 0.95 g/100 ml, 0.9 g/100 and 47 kcal/100 ml

respectively(125). The modified fat breast milk was then fortified to provide fat, protein

and energy at levels identical to mature breast milk (4.0 g/100 ml; 1.1 g/100 ml; 67

kcal/100 ml) (104). To replace these nutrients, the modified fat breast milk was fortified

using either a combination of Similac Human Milk Fortifier (Abbott

Laboratories) and MCT oil or the MCT based formula Portagen® (Mead Johnson). The

method of nutrient enrichment was determined based on the age, size and nutrient

requirements of the baby. All recipes for nutrient enrichment were based on a standard

recipe (T

nutritional plan.

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Table 2. Recipes for the Fortification of Modified Fat Breast Milk

RECIPE  1:    100  ml  low  fat  breast  milk  +  2  packages  human  milk  fortifier  +  3.0  ml  medium  chain  triglyceride  oil

Ingredient Quantity Displacement (ml) Protein (g) Fat (g) CHO (g)Energy (kcal) Energy (kJ) Low Fat Breast Milk 100 ml 100 0.91 1 7.2 41.4 173.4 Human Milk Fortifier 1.8 g 1.3 0.5 0.18 0.9 7.0 29.3 MCT oil 3 ml 3 0 2.80 0 23.0 96.2 Total 104.3 1.41 3.98 8.1 71.4 298.9 % Total Calories 7.9% 46.7% 45.4%

Final Concentration (kcal/ml) 0.68

RECIPE  2:    100  ml  low  fat  breast  milk  +  4  g  of  Portagen  Powder  +  1.5  ml  medium  chain  triglyceride  oil      

Ingredient Quantity Displacement (ml) Protein (g) Fat (g) CHO (g)Energy (kcal) Energy (kJ) Low Fat Breast Milk 100 ml 100 0.91 1 7.2 41.4 173.4 Portagen 4 g 3 0.7 0.9 2.2 18.8 78.7 MCT Oil 1.5 ml 1.5 0 1.5 0 11.4 48.0 Total 105 1.61 3.4 9.4 71.6 300.1 % Total Calories 9.0% 38.9% 52.5%

Final Concentration (kcal/ml) 0.68

In addition to the standard nutrient enrichment, infants in the treatment group

-

linolenic (n-3) and linoleic (n-6) fatty acids were based on the joint recommendation of

the American Dietetic Association (ADA) and Dietitians of Canada (DC) with n-6

making up 4.0 % of total calories and n-3 making up 0.75% of total calories (127). Every

effort was made to provide as close to these recommendations as possible without

71

providing excessive amounts of LCT and compromising treatment. Soy oil, which

provides 2.089 g linoleic acid and 0.278 g -linolenic acid per teaspoon, was used to

Per 100 ml

Nutrients Portagen® Mature Human Milk

Modified Fat Human Milk

Nutrient Enriched Modified Fat Human Milk Recipe 1

Nutrient Enriched Modified Fat Human Milk Recipe 2

Energy, kcal (kJ) 67 68 41.4 69 69 Protein, g 2.4 1.2 0.9 1.3 1.5 Carbohydrate, g 7.7 7.2 7.2 7.8 9 Fat, g 3.2 3.9 1.0 3.8 3.3 Linoleic Acid, g 0.18 0.56 0.14 0.13 0.19 Linolenic Acid, g N/A 0.063 0.016 0.015 0.015 Minerals Calcium, mg 63 26 26 81 42 Phosphorus, mg 47 12 12 44 25 Magnesium, mg 14 3.4 3.4 5 7 Sodium, mg 33 16 16 23 25 Potassium, mg 84 50 50 79 71 Chloride, mg 57 42 42 59 56 Zinc, mg 0.62 0.25 0.25 0.72 0.41 Choline, mg 9 16 16 16 17 Iron, mg 1.3 0.04 0.04 0.23 0.37

N/A 1.8 1.8 2.0 N/A Vitamins

66 49 12 102 29 Vitamin C, mg 2.6 5 5 16 6

0.5 0.3 0.08 1.5 0.36 Vitamin E, mg 0.7 0.49 0.12 1.2 0.3

7 8.5 8.5 20 11 Milk nutrient composition values were obtained from the literature (68-69, 104). The composition of Portagen® was obtained from manufacturers information (55). The values for recipes 1 and recipe 2 do not include essential fatty acid supplementation as this was calculated on an individual basis for each subject. The powdered human milk fortifier used in this study was Similac Human Milk Fortifier (Abbot Nutrition, Montreal, Quebec, Canada)

Table 3. Approximate Energy and Select Nutrient Composition of Mature Human Milk, Portagen®, Modified Fat Human Milk and Nutrient Enriched Modified Fat Human Milk

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meet these requirements. Individual requirements were calculated for each subject and

added to the modified fat breast milk.

 

3.2.4.6  Control  Group  

Upon enrolment into the control group, subjects received all enteral feeds as the

MCT based formula. The formula was prepared in the MPR according to current hospital

procedure. The MCT based formula was provided to infants via bottle and/or nasogastric

team. Volume of feeds and caloric content of feeds were determined by the medical team

based on fluid allowance, tolerance and energy requirements to support growth.

At the time of discharge, parents were provided with a recipe to prepare the MCT

based formula at home. A supply of the formula was provided for the infant free of

charge. Parents were instructed to continue to follow the same feeding regimen

established in hospital before discharge. Any changes to the feeding regimen were

developed in conjunction with the study investigator during the follow-up clinic visit or

during weekly phone follow-up. If a fluid restriction was in place, all changes in fluid

 

3.2.5  Fat  Analysis  

Remaining fat content of the centrifuged breast milk was measured using the

creamatocrit method. Samples of centrifuged breast milk were well agitated and then

extracted by suction into glass capillary tubes. Samples were then centrifuged in a

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hematocrit centrifuge (Hettich Haematokrit, Fisher Scientific) at 3000 RPM for 3 minutes

(126). The resulting fat layer was then measured using a microhematcrit reader and

expressed as a percentage. All measurements were performed in duplicate.

 

3.2.6  Protein  Analysis  

                 Protein content of the centrifuged breast milk (prior to nutrient enrichment) was

determined using the Bicinchoninic Acid (BCA) protein assay. Bicinchoninic Acid is a

sodium salt that reacts with copper ions in an alkaline environment creating a purple

complex that gains in intensity with increasing protein concentrations (128). This purple

complex is then quantified spectrophotometrically at 562 nm (128-129). In a study by

Keller et al, using the Kjeldahl method used as the gold standard, the BCA protein assay

was found to provide the most consistent and reliable results when compared to other

colourimetric techniques (129).

 

3.2.7  Electrolyte  Analysis  

Analysis of the electrolyte content of the modified fat breast milk was conducted

using flame photometry, an atomic emission method used to detect and quantify metal

salts including sodium and potassium (130). All electrolyte analysis was conducted by

the Department of Laboratory Medicine at The Hospital for Sick Children.  

 

74

 

3.2.8  Microbiological  Analysis  

                   Quantitative cultures were used for the analysis of the microbiological content of

the breast milk prior to centrifuging and the resulting modified fat breast milk. This was

conducted by the Department of Laboratory Medicine at The Hospital for Sick Children.  

 

3.2.9  Data  Collection  

The following variables were collected at the time of study enrollment by means of

parental interviews, conducted by the study coordinator, or from medical records: age of

the infant (months), date of surgery, diagnosis, type of surgical correction, birth weight,

birth length and head circumference if available, gestational age at birth, gender,

maternal/paternal age, maternal/paternal education as well as maternal and paternal

weight and height.

The data were used for one or more of the following purposes: assess subject

eligibility, compare baseline characteristics that may affect success of conservative

treatment of chylothorax, to compare post-discharge growth, and as a co-variate in the

statistical analysis.

Volume of chyle drainage, to the nearest millilitre, was monitored by nursing staff

diagnosis. Monitoring continued until the drainage subsided and the drainage tube was

removed. If the day of study enrolment was not the same day the diagnosis of

75

chylothorax was made, drainage data from the time of diagnosis was collected

Intake of the modified fat breast milk and/or MCT based formula, to the nearest

During the first week after discharge, parents were asked to complete a 3-day consecutive

weighted diet record. For accuracy, parents were asked to weigh volumes of all fluids

and solids consumed. A small scale accurate to 1 g (CS2000; Ohaus) was provided to the

parents for this purpose. Information from these diet records, in combination with the

information provided in Table 3, which describes the nutritional composition of the

nutrient enriched modified fat breast milk (treatment group) or according to the

nutritional profile of the MCT based formula (control group) was used to calculate

energy intake.

Growth measures were collected at regular intervals throughout the study period.

Weight was tracked on a daily basis, where medically possible, while the infant remained

in hospital. Weight was measured each day by the responsible nurse and recorded in the

ms to the nearest ± 10 grams.

Medela®) provided for home use. Parents were instructed to weight the infant on the

same day of the week, at approximately the same time of day, without clothes or diaper.

Weight gain was calculated on a weekly basis and was used to determine the need for

76

over the study period was done using the weight on Study Day 1 and weight on the final

day of the treatment.

Initial length and head circumference measures were taken on the day of study

enrolment, or as soon as possible thereafter. Length was measured by the study

coordinator using a length board and was recorded in centimetres to the nearest ± 0.1 cm.

Head circumference was measured using a non-stretchable measuring tape to the nearest

± 0.1 cm. Two measurements were performed and the average recorded. Length and

head circumference were performed at the first follow-up following discharge ± 3 days.

Final measures of length and head circumference were performed the last week of

treatment ± 1 week, either at the hospital or during a home visit. All measures of length

and head circumference throughout the study were taken by the study coordinator.

3.2.10 Sample Size

The primary outcome variable for this study was total chest tube drainage at

treatment day 5. For the purpose of calculating a sample size, existing chest tube drainage

data from the cardiac database at the Hospital for Sick Children of infant with

chylothorax who were treated with the MCT based formula was used. To detect no more

than a 1.0 standard deviation difference (6.9 ml/kg/d) in total volume of drainage on

treatment day 5 between the two feeding groups, a sample size of 14 per feeding group is

 

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3.2.11  Statistical  Analysis  

Statistical analysis was conducted using SAS v9.1 (The SAS Institute, Cary, NC).

Baseline differences in demographic variables were assessed using t-tests for normally

2 statistics for all non-normally distributed variables

and categorical variables. Where differences were noted between treatment groups, as

appropriate, they were included in the statistical models used to assess differences in

outcome variables. Repeated measure analysis was use to further analyze growth data.

Statistical significant was set at a p-value < 0.05.

3.3  RESULTS

3.3.1  Subject  characteristics  

A total of 16 infants (56% males) were enrolled in the study. Infants included in

the study had a variety of cardiac diagnosis (Table 4). Infants in the treatment group

were more likely to be younger, with 5 of the 8 infants (63%) in this group being

newborn. As a result, infants in the treatment group tended to be smaller at study

enrolment. Neither of these differences was statistically significant. The most prevalent

diagnosis was Tetralogy of Fallot, which affected 6 of 16 (38%) subjects and was evenly

distributed between the treatment and control group. No statistically significant

difference was found in any baseline characteristics between the treatment and control

group.

 

 

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Table 4. Subject Characteristics

Characteristics Treatment Control (n=8) (n=8) Age at enrolment, mo 2.8 ± 3.2 5.3 ± 1.9 a

Male sex, n (%) 5 (63) 4 (50) Gestational age at birth , weeks 38.6 ± 1.8 38.6 ± 2.2

Birth weight , g 3258 ± 596 2904 ± 873

Weight at study day 1, g 4588 ± 1853 5915 ± 1000 b

Length at study day 1, cm 56.3 ± 7.7 61.1 ± 3.8 c

Head circumference at study day 1, cm

37.7 ± 4.7

39.3 ± 2.5

Diagnosis Tetralogy of Fallot 3 3 Ventricular Septal Defect 0 1 Hypoplastic Left Heart Syndrome 2 0

Transposition of the Great Arteries 3 1

Atrial Septal Defect 0 1 Heart Transplantation 0 2 Differences between groups for categorical variables were assessed using one-sided t tests and X2 was used for all categorical variables. No statistically significant differences were noted between the two groups in any of the baseline characteristics. a P = 0.1125 b p = 0.1027 c p = 0.1027

3.3.2  Family  Characteristics  

Family characteristics are summarized in Table.5. There were no statistically

significant differences in the baseline characteristics of mothers between the treatment

and control group. However, a trend in the results suggested that mothers of infants in

the treatment group were more likely to be older, were more often Caucasian and were

more likely to have a university degree. This is consistent with recent breastfeeding

research which suggests that women who breastfeed tend to be older, and are more likely

79

to have a post-secondary education then mothers who formula feed their infants (83).

Older mothers as well as mothers with a university level education are more likely to

breastfeed longer (83). Fathers of infants in the treatment group were more likely to be

Caucasian (p=0.0336). No significant difference in the remaining family characteristics

was found between the treatment and control group.

Table 5. Family Characteristics

Characteristics Treatment Control (n=8) (n=8) Mother Age, mean ± SD (n), y 33 ± 3.2 29 ± 5.6 Ethnicity, n (%) White 5 (62) 2 (25) Other 3 (38) 6 (75) Education, n (%) University degree 5 (62) 3 (38) No university degree 3 (38) 5 (62) Father Age, mean ± SD (n), y 35 ± 3.3 33 ± 7.6 Ethnicity, n (%)a White 7 (87) 2 (29) Other 1 (13) 5 (71) Education, n (%) University degree 5 (62) 4 (57) No university degree 3 (38) 3 (43) Differences between groups for continuous variables were assessed using one-sided t tests and X2 was used for all non-categorical variables. Unless otherwise indicated, no statistically significant differences were noted between the two groups in any of the family characteristics. a Fathers of infants in the treatment group were more likely to be Caucasian p=0.0336

3.3.3 Fat Removal

As outlined in the methods sections, parents of infants in the treatment group were

offered the use of a small, easy to use, table top centrifuge, which would allow them to

80

perform the centrifuging of the breast milk and subsequent fat removal at home. This

option offered an advantage to those mothers who did not have sufficient breast milk

supply, allowing them to centrifuge milk as often as necessary to ensure that the infant

received as much modified breast milk as possible. Only 3 families chose this option.

Many infants have high care needs following cardiac surgery and thus families preferred

the ease of having this task performed for them. Some families expressed concern about

being able to perform the task properly and felt more comfortable having somebody with

more experience performing the centrifuging and fat removal. Of the 3 subjects whose

parents chose to learn to centrifuge the milk themselves, 1 infant was hospitalized for the

duration of treatment and thus the parents were not required to ever centrifuge breast

milk. The mother of a second subject had an excellent breast milk supply and thus

enough centrifuging could be done while in hospital to supply the infant with breast milk

for the bulk of the study period. As a result, the families of only 1 of these 3 subjects

actually performed a significant amount of centrifuging at home.

3.3.4  Chest  Tube  Drainage  

No significant difference in total volume of chest tube drainage on treatment day 5

was found between the treatment (5.0 ± 5.1 ml/kg) and control group (5.7 ± 8.5 ml/kg)

(Figure 2). In addition, there was no significant difference in duration of drainage

between the treatment (7.3 ± 2.9 d) and the control group (12.5 ± 11.5 d). Detailed

drainage data for all subjects can be seen in Appendix G. Three of 8 (38%) infants in the

control group required progression to parenteral nutrition to treat their chylothorax while

none of the patients in the treatment group required parenteral nutrition, although again

this difference was not statistically significant.

81

Figure 2. Total volume of drainage for the treatment and control group on treatment day 5 (with standard deviations)

3.3.5  Modified  Fat  Breast  Milk  

                   Infants in the treatment group received an average of 79% of their feeds as

that not all infants were fed 100% of feeds as modified fat breast milk.

own milk supply was inadequate, the remainder of their feeds were provided as the MCT

formula. Of the 8 mothers in the treatment group, 4 were able to provide 100% of feeds

as modified fat breast milk. The remainder of the infants received a combination of

modified fat breast milk and the MCT formula. All efforts were made to support mothers

in the production of breast milk including the provision of an electric breast pump and

on-going access to a lactation consultant throughout the study for additional support.

0  

1  

2  

3  

4  

5  

6  

7  

8  

9  

10  

Treatment   Control  

Volume  of  Drainage  ml/kg  

 (treatment  day  5)  

82

Table 6: Percentage of feeds consumed by treatment subjects as modified fat milk vs. MCT formula

Treatment Subject Modified Fat Milk (%) 3 45 5 56 6 100 7 100 8 72

10 60 11 100 12 100

Average 79 ± 23%

Time to centrifuge the breast milk and remove the resulting fat layer was

approximately 60 minutes per 1000 ml of breast milk. There were notable differences in

consistency of the resulting fat layer which affected ease of fat removal. Fat removal

from early milk (colostrum) was more challenging. If mothers had a good milk supply,

the colostrum was left in its unaltered form and provided to the parents to feed to the

infant after study completion. Milk volume was reduced on average by 10-15% following

fat removal. Therefore, for every 100 ml of breast milk, 85 - 90 ml of modified fat breast

milk remained after centrifugation.

3.3.5.1  Nutrient  Enrichment  

The supplementation of essential fatty acids did require the addition of long chain

fat to the modified fat breast milk. This is counterintuitive in the treatment of

chylothorax and did have the potential to increase chest tube drainage. However, the

supplementation of essential fatty acids was well tolerated in all the infants with the

83

exception of Subject 10. This subject returned to the outpatient cardiac clinic after

discharge and on examination was found to have some reaccumulation of chylous fluid.

When the fluid accumulation did not resolve on its own, the supplementation of essential

fatty acids was discontinued

incident.

Addition of MCT was not without associated difficulties. The MCT oil did not mix

easily and separated out quickly. For those infants feeding some or all of their feeds via

nasogastric tube (5 out of 8 treatment subjects at the time of discharge), a common

complaint of parents was that the MCT oil coated the tubing. Thus, some of the MCT oil

adhered to the tubing and as a result, infants likely did not receive all of the MCT oil

resulting in a lower than expected fat intake and overall calorie intake. This also made

cleaning the tubing challenging. Chyle is known to be high in fat and thus infants may

have had inadequate provision of fat during initial stages of treatment when chyle was

being lost through a chest tube (28). In comparison, the fat component of the MCT based

formula stays well emulsified, ensuring that the infant receives the fat portion of their

feeds and presumably contributing to a higher overall calorie intake.

 

3.3.6  Nutrient  Analysis  

3.3.6.1  Protein  Analysis  

The average remaining protein content for each sample of breast milk can be seen

in Table 7. The average remaining protein content was 14.3 ± 2.3 g/L. This is above the

accepted estimated protein content of mature human milk of 11.7 - 12.4 g/L (104).

84

Table 7. Measured protein content of breast milk sample after centrifugation

Treatment Subject Average Protein Content (g/L) 3 11.9 5 11.0 6 16.8 7 12.9 8 14.6 10 13.9 11 15.9 12 17.4

Overall Average 14.3 ± 2.3

Five of the 8 (63%) infants in the treatment group were newborn and it is known that the

protein content of milk tends to be higher in the earlier stages of lactation, declining as

lactation progresses (104, 131). Protein content of human milk may be as high as 14-16

g/L in early lactation, 8-10 g/L by 3-4 months of lactation and 7-8 g/L by 6 months of

lactation and onward (131). Milk samples taken from mothers of newborn infants would

have produced milk samples with higher protein content thus contributing to a higher

overall average protein content in the modified fat breast milk when compared to the

accepted protein content of mature breast milk.

85

Figure 3. Variability of remaining fat content of samples of modified fat breast milk as measured using the creamatocrit method for all treatment subjects

 

0  

0.5  

1  

1.5  

1   2   3   4   5   6  Remaining  Fat  Content  

(%)  

Milk  Sample  

Subject  3  

0.83 ± 035

0.88 ± 0.23

0.69 ± 0.26

0.88 ± 0.48

0.75 ± 0.27 0.71 ± 0.26

0.86 ± 0.32

86

3.3.6.2  Fat  Analysis  

The remaining fat content of the breast milk after centrifugation was quite variable,

ranging from 0.5% to 1.5% of total volume, as measured using the crematocrit method

(Figure 3). The average remaining fat content after centrifugation for all samples taken

from all treatment subjects was 0.8 % of total volume, less than the previously measured

average value of 1.0 % of total volume (125).

3.3.6.3  Electrolyte  Analysis  

The average remaining sodium content of the breast milk after centrifugation was

6 ± 1.1 mmol/L (Table 8). This is only slightly less than the reported average level of

sodium in breast milk which is 7 mmol/L within the first 20 weeks of lactation and 5.6

mmol/L thereafter (132). The average potassium content of the breast milk after

centrifugation was 13.4 ± 1.5 mmol/L (Table 8). This is similar to the reported average

potassium content of 12.8 mmol/L mature human milk (132). These results suggest that

the electrolyte content of breast milk is not significantly altered by centrifugation.

Table 8. Measured sodium and potassium content of modified fat breast milk

Treatment Subject Na (mmol/L) K (mmol/L) 3 5 12.3 5 5 12.0 6 6 14.8 7 6 14.6 8 7 15.0 10 6 11.0 11 5 13.9 12 8 13.6

Overall Average 6 ± 1.1 13.4 ± 1.5

87

3.3.6.4  Intake  Data  

Data from the weighted 3-day food records regarding average energy and protein

intake from milk feedings (modified fat breast milk and/or MCT formula) as well as any

solid foods consumed by the subjects was analyzed using Food Processor SQL Ed v. 10.2

(ESHA Research, Salem, Oregon). Infants in the treatment group consumed significantly

larger volumes of milk feedings (145 ± 43 ml/kg) in comparison to infants in the control

group (105 ml/kg ± 23 ml/kg) (p=0.0435) (Figure 4). As a result, energy intake from milk

feedings was significantly higher in the treatment group (120 ± 32 kcal/kg) than in

control group (84 kcal/kg ± 29 kcal/kg) (p=0.0336) (Figure 3). When energy from solid

foods was included, the difference in energy intake was no longer statistically significant.

Figure 4. Daily volume of milk intake (a), daily energy intake from milk feedings alone (b) and daily energy intake from milk feedings + solid foods (c) in the treatment and control group *Milk = modified fat breast milk and/or MCT based formula (with standard deviations) There was no significant difference in the average protein intake from milk

feedings between the treatment group (3.1 ± 0.5 g/kg/day) and the control group (3.0 ±

0.8 g/kg/day) (Figure 5). When protein from solid foods was included, the protein

intakes were almost identical (3.2 ± 0.5 g/kg/day vs. 3.2 ± 0.7 g/kg/day). All infants in

p=0.0336 p=0.0435

a b c

88

the study had protein intakes well above the AI (1.52 g/kg/day) and RDA (1.2 g/kg/day)

for their respective age groups (104).

Figure 5. Protein intake from milk and solids for the treatment group (3.2 ± 0.5 g/kg/day) and control group (3.2 ± 0.7 g/kg/day)

3.3.6.5  Feeding  Tolerance  

There was no significant difference in the number of reported episodes of emesis

(0.6 ± 0.7 vs. 1.3 ± 2.4) or spit up (1.6 ± 2.3 vs. 1.1 ± 2.1) per day between the treatment

and control group.

3.3.6.6  Nasogastric  Tube  Requirement  

It was hoped that fewer infants would require the use of a nasogastric tube for

feeding with the provision of the modified fat breast milk. Five of 8 (63%) infants in the

treatment group were discharged with a nasogastric tube for feeding versus 3 of 8 (38%)

infants in the control group. As a result, infants in the treatment group consumed an

average of 56% of their total milk feedings orally versus infants in the control group who

consumed 86% of their milk feeds orally. Neither of these differences was statistically

significant. Characteristics of infants requiring a feeding tube during the study are

89

presented in Table 9. One infant in the control group had a GJ tube in place prior to

cardiac surgery for feeding issues unrelated to chylothorax or the use of the MCT

formula. The remaining 2 infants in the control group who required a feeding tube during

the study had their nasogastric tubes removed within 1-2 weeks of discharge and fed

Table 9. Characteristics of infants requiring a feeding tube during the study period and timing of feeding tube removal in relation to study completion

Feeding Tube Removal

Subject

Group

Diagnosis

Type of feeding tube

Feeding tube in place prior to study

Before study completion

After study completion

3 Treatment Tetralogy of Fallot

NG No No Yes

5 Treatment Tetralogy of Fallot

NG No No Yes

6 Treatment Single Ventricle NG Yes No No

10 Treatment Tetralogy of Fallot

NG No Yes ----

11 Treatment Single Ventricle NG Yes No No

13 Control Atrial Septal Defect,

GJ Yes No No

14 Control Cardiomyopathy NG Yes Yes ----

16 Control Ventricular Septal Defect

NG Yes Yes ----

90

orally for the remainder of the study. Of the 5 infants in the treatment group who were

discharged with a feeding tube, 2 of these infants had a nasogastric tube in place prior to

their development of chylothorax. These same 2 infants continued to require their

nasogastric tubes for feeding after study completion, although 1 of these infants was able

to successfully transition to bottle feeding of expressed breast milk and finally

transitioned to exclusive breast feeding at approximately 4 months of age. The remaining

3 infants in the treatment group who required feeding tubes were older infants (e.g. not

newborns), 2 of who were exclusively breastfed prior to their cardiac surgery and refused

a bottle for the duration of the study.

Over the course of the study, it became increasingly clear that well established

breast feeding babies simply preferred the mechanism of breast feeding and refused a

ent group

discharged with a feeding tube had done a combination of breastfeeding and bottle

feeding prior to surgery and the nasogastric tube was removed within 1 week of

discharge. The infant fed orally for the remainder of the study.

 

3.3.7  Growth  

3.3.7.1  Weight-­for-­Age  

Mean weight-for-age z-scores between the treatment and control group were not

statistically different at study enrolment, although it appears to be approaching

significance (p=0.0954) (Table 10). There was no significant difference between groups

in mean weight-for-age z-scores at study completion. Infants in the treatment group

91

experienced a significant decline in mean weight-for-age z-score from study enrolment to

study completion (p=0.0062). There was no significant change in mean weight-for-age z-

score in control infants over the study period.

3.3.7.2  Length-­for-­Age  

Mean length-for-age z-score was significantly lower for infants in the control group

when compared to infants in the treatment group at study enrolment (p=0.0273). This

Table 10: Weight-for-age, Length-for-age, Weight-for-length and Head Circumference-for-age z-scores at study enrolment and study completion for treatment and control subjects

Anthropometric Study Enrolment Study Completion Treatment Control Treatment Control Weight-for-age, mean ± SD

-0.79 ± 0.75 -1.73 ± 1.26 -1.51 ± 0.85 -1.81 ± 0.94

Length-for-age, mean ± SD a,b

-0.48 ± 0.85 -1.84 ± 1.36 -0.93 ± 1.23 -1.82 ± 1.37

Weight-for-length, mean ± SD

-0.84 ± 1.09 -0.58 ± 0.78 -1.14 ± 1.18 -0.84 ± 0.85

Head circumference-for-age, mean ± SD c,d,e

-0.34 ± 0.95 -2.23 ± 1.83 -0.58 ± 1.16 -1.64 ± 1.45

Mean ± SD values are presented. Differences in z-scores at study enrolment and study completion were measured using t-tests. Unless otherwise noted, no statistical difference was detected between the treatment and control group. a Treatment > control group at study enrolment, p=0.0273 b Study completion < study enrolment in the treatment group, p=0.0131 c Treatment > control group at study enrolment, p=0.0257 d Study completion < study enrolment in the treatment group, group, p=0.0077 e Study completion > study enrolment in the control group, group, p=0.0256  

statistically significant difference no longer existed by study completion. Again, infants

in the treatment group experienced a statistically significant decline in mean length-for-

92

age z- score from study enrolment to study completion (p=0.0131). There was no

significant change in mean length-for-age z-score in control infants over the study period.

 

3.3.7.3  Weight-­for-­length  

There was no statistically significant difference in mean weight-for-length z-score

between the treatment and control group at study enrolment or at study completion.

Neither group experienced a significant change in mean weight-for-length z-score over

the study period.

a Study completion < study enrolment in the treatment group, p=0.0062 b Study completion < study enrolment in the treatment group, p=0.0131 c Study completion > study enrolment in the control group, p=0.0256 d Study completion < study enrolment in the treatment group, p=0.0077  

Figure 6. Mean z-score for weight-for-age, length-for-age, weight-for-length and head circumference-for-age at study enrolment and study completion for subjects in the treatment and control group

H ead ci rcumference -for-age W eight-for-length L ength-for-age W eight-for-age

a

b

c

d

93

3.3.7.4  Head  Circumference-­for-­Age  

Mean head circumference-for-age z-score was significantly lower for infants in the

control group when compared to infants in the treatment group at study enrolment

(p=0.0257). By study completion, this difference between the two groups was no longer

statistically significant. Again, infants in the treatment group experienced a significant

decline in mean head circumference-for-age z-score from study enrolment to study

completion (p=0.0077). In contrast, infants in the control group experienced an

improvement in mean head circumference-for-age z-score over the course of the study

(p=0.0256)

3.3.8  Microbiological  Analysis

The results of the microbiological analysis of the breast milk samples for each

subject in the treatment group both before and after centrifugation are presented in Table

11. Breast milk is not a sterile fluid and all milk samples in the current study tested

positive for some form of bacteria (133). The most  common  organism  isolated  was    

coagulase negative staphylococcus found in all 8 samples of milk.  Coagulase negative

staphylococcus, gram positive bacteria, is commonly found on human skin and mucous

membranes in concentrations of 104 106 cfu/cm2 and has frequently been identified in

human breast milk samples (134-135). What was less predictable was the presence of

pathogenic bacteria such as Klebsiella Pneumoniae. The identification of potentially

harmful bacteria led to the formulation of a microbiological decision tree (Figure 7) to

determine the safest course of action when unacceptable levels of bacteria were found to

be -educated on hygiene when pumping breast

94

milk to minimize the risk of contamination. What was encouraging was that the level of

bacteria was almost always reduced following centrifuging suggesting that centrifuging

was not the point of contamination but more like occurred at the time the milk was

expressed. No mothers developed mastitis during the study period and no infant

developed a secondary infection.

Table 11. Microbiological analysis of milk sample pre-centrifugation and post-centrifugation Subject   Microbiological  Species   Pre-­centrifugation   Post-­centrifugation  

Subject 3

Coagulase negative Staphylococcus 8 CFU/L 8 CFU/L

Subject 5

Coagulase negative Staphylococcus 70 colonies 40 colonies

Subject 6 Coagulase negative Staphylococcus

Subject 7 Coagulase negative Staphylococcus Klebsiella Pneumoniae* Diphtheroid organism

1 x 107 CFU/L 1.4 x 106 CFU/L 1 x 107 CFU/L

1 x 107 CFU/L 0.5 x 106 CFU/L -

Subject 8 Coagulase negative Staphylococcus Streptococcis Agalactiae Group B* Viridans Streptococcus Group*

> 108 CFU/L > 1 x 105/L <2 x 107/L

< 108 CFU/L - -

Subject 10 Coagulase negative Staphylococcus

< 108 CFU/L

< 108 CFU/L

Subject 11 Coagulase negative Staphylococcus < 108 CFU/L

< 108 CFU/L

Subject 12 Coagulase negative Staphylococcus Enterococcus Species* Staphylococcus aureus*

> 108 CFU/L > 1 x 105 CFU/L > 1 x 105 CFU/L

8 CFU/L 8 x 105 CFU/L 1 x 105 CFU/L

*Potentially pathogenic bacteria (135-136)

95

Figure 7. Algorithm of Steps to Take if Bacteria Cultured in Breast milk

 

 

3.4  DISCUSSION

3.4.1.  Chest  Tube  Drainage  

To our knowledge, this is the first systematic comparison between the effectiveness

of a modified fat breast milk and an MCT based formula for the treatment of chylothorax

in infants following cardiothoracic surgery. Examination of the primary outcome variable

revealed no statistically significant difference between those infants who received the

modified fat breast milk and those infants who received the MCT based formula in total

volume of chest tube drainage on treatment day 5 (5.0 ± 5.1 ml/kg vs. 5.7 ± 8.5 ml/kg) .

96

In addition, no statistically significant difference was observed in the duration of chest

tube drainage (7.3 ± 2.9 d vs. 12.5 ± 11.5 d) between the treatment and control group.

These results are contrary to our first hypothesis in which we speculated that volume of

chest tube drainage would be higher in infants who received the modified fat breast milk

compared to those who received the MCT based formula. This hypothesis was based on

our previous pilot study data which suggested that the amount of LCT remaining in the

modified fat breast milk after centrifugation was higher than the known LCT content of

the MCT based formula (55, 125). However, use of the crematocrit method to determine

remaining fat content after centrifugation suggested that in fact, the amount of LCT

remaining was often lower than expected. Thus, centrifugation of breast milk followed by

manual removal of the resulting fat layer resulted in a modified fat breast milk that

supported the resolution of chylothorax in affected infants. In addition, no subject who

received the modified fat breast milk experienced any reaccumulation of chylous fluid

after the treatment was complete. This result is similar to the experience described by

Chan and Lectenberg who described no reaccumulation in subjects treated with a low fat

breast milk for a mean of 16 days (range 7 34 days), a much shorter treatment period

than what is currently prescribed at our institution (3). Therefore, results from this study

suggest that use of a modified fat breast milk is neither better nor worse than the MCT

formula that is the current treatment standard and can support successful resolution of

chylothorax in infants following cardiothoracic surgery.

Although there was no statistically significant difference in duration of drainage

between groups, those infants who received the modified fat breast milk did appear to

97

have a shorter duration of drainage. This observed shorter duration of drainage may be

considered clinically relevant. If chest tube drainage as a result of chylothorax is the

primary factor keeping an infant in hospital following cardiac surgery, the results of this

study suggest that length of hospital stay would not be prolonged with the use of a

modified fat breast milk.

Not all infants in the treatment group received 100% of feeds as the modified fat

breast milk. Of the 8 treatment infants, 4 received exclusively modified fat breast milk.

The remaining 4 received a combination of modified fat breast milk and the MCT based

formula. The small number of subjects in this study precludes any meaningful statistical

comparison of those infants who received 100% of feeds as modified fat breast milk

versus those who received a combination of the modified fat breast milk and the MCT

formula. However, the average total volume of drainage on treatment day 5 in infants

who received 100% of their feeds as the modified fat breast milk was 6.9 ± 6.3 ml/kg/day

versus 3.1 ± 3.4 ml/kg/day for infants receiving a combination of the modified fat breast

milk and MCT formula. Duration of drainage in infants who received 100% of their feeds

as the modified fat breast milk was 7.0 ± 3.6 days versus 7.8 ± 2.6 days for infants

receiving a combination of the modified fat breast milk and MCT formula.

3.4.2.  Growth  

All infants in our study experienced growth challenges, particularly the infants who

received the modified fat breast milk, and thus measures must be taken to provide

adequate nutrient intake and to ensure optimal growth throughout the treatment period.

98

Nutritional loses are a well known side effect of chylothorax making weight gain and

growth challenging in affected infants (28). In the current study, no infants in the

treatment group experienced weight loss over the study period. Weight loss was observed

in only one subject in the control group (45 g total over the study period). Two subjects

(1 treatment, 1 control) experienced weight maintenance throughout the study. All other

subjects gained weight. However, only 3 subjects (2 treatment, 1 control) achieved

expected average daily weight gain for age. Average daily weight gain was 57% and

61% of expected age appropriate goals in the treatment and control group (Table 12)

(137). Low rate of weight gain or even weight maintenance in an infant over a 6 week

period, especially in a newborn infant in whom expected rates of weight gain are high,

would have a notable impact on z-score as was observed in infants in the treatment group

who experienced a significant decline in mean weight-for-age, length-for-age and head-

circumference-for-age z-scores over the study period.

The current study is not alone in reported issues with weight gain in infants and

children affected by chylothorax. Puntis et al reported a weight loss of up to and greater

than 8% in one third of patients, with the most severe weight loss being observed in those

with the largest volume of drainage (65). In a study of 9 children with a mean age of 26

months, Pedra et al reported a weight loss of up to 12% in 6 of the 9 subjects (43). Le

Coultre et al reported a weight loss of < 5% in a group of 24 children with chylothorax,

with the exception of one patient who experienced a weight loss of 10% (63). In a study

by Allen et al, weight loss was reported in 14 of 18 children studied. The magnitude of

this weight loss was reported to be < 10% (29). Nguyen et al reported weight

99

maintenance or even age appropriate weight gain in all surviving patients who left the

hospital (23 of 24 subjects ranging in age from 3 days to 11 years) (33). Therefore,

weight maintenance or even weight loss is frequently reported in infants and children

affected by chylothorax.

Table 12. Average daily weight gain over the study period and expected daily weight gain for age for treatment and control subjects

Subject Group Average Daily Weight Gain (g)

Expected Average Daily Weight Gain (g)135

1 Control 10 12 2 Control 10.2 11.5 3 Treatment 5.0 12 4 Control 0 16 5 Treatment 14.0 12 6 Treatment 8.7 32.5 7 Treatment 24.0 27.5 8 Treatment 33.0 32.5 9 Control 7.6 19

10 Treatment 0 13 11 Treatment 12.0 32.5 12 Treatment 15.4 32.5 13 Control 13.6 13 14 Control 14 16 15 Control 3.9 10 16 Control 7.5 17.5

There were likely several factors involved in the observed growth challenges. The

remaining fat content in the modified fat milk was likely overestimated. In the current

study, use of the crematocrit method revealed that the average remaining fat content of

100

the modified fat breast milk after centrifugation was 0.8 g/100 ml. This is less than the

previously measured average remaining fat content of 1.0 g/100 ml, the value upon which

nutrient enrichment of the modified fat breast milk was based. (125). As a result, nutrient

enrichment of the modified fat breast milk may have been inadequate, resulting in

suboptimal energy intake and contributing to the observed growth difficulties. Reported

difficulties with emulsification of the MCT into the modified fat milk may have also

contributed to less than expected fat intake, decreasing overall energy consumption by

infants in the treatment group.

The provision of inadequate protein could be a contributing factor in the poor

growth observed in treatment group. Using the BCA protein assay, the average

remaining protein content of the modified fat breast milk was determined to be 14.3 g/L

which is above the accepted average protein content of mature human milk at 11.7 12.3

g/L, suggesting that there was no change to the initial protein content of the breast milk

during centrifugation (104). Using the results of the BCA protein analysis in

combination with the intake data suggests that protein intake in both the treatment and

control group was well above the Adequate Intake for 0 6 month olds (1.52 g/kg/day)

and Recommended Dietary Allowance for 7 12 month olds (1.2 g/kg/day), the

recommended intakes established for healthy infants within their respective age groups

(104). In fact, protein intakes of the treatment and control groups were very similar.

However, the effects of chyle loss during early treatment of chylothorax may increase

protein requirements. Chyle is high in protein (20-30 g/L) and infants may have been in

an negative protein balance resulting from chyle loss through their chest tube and thus

101

required a period of repletion before deposition of new tissue was possible (28, 48). No

data exists regarding protein requirements in infants with chylothorax. However, given

the known protein content of chyle (20-30 g/L), it is reasonable to assume that protein

requirements in individuals affected by chylothorax are likely elevated to restore protein

losses and to support growth (28).

The frequent manipulation of the modified fat breast milk may have had an effect

on the macronutrient and ultimately on the energy content of the modified fat breast milk

the infants consumed. In the current study, the modified fat breast milk may have

required as many as 5-6 container changes from the point of pumping, through

centrifuging, fortification and subsequent feeding to the infant, potentially contributing to

a decrease in both fat and protein content of the modified fat breast milk. A similar effect

has been observed in the use of donor human milk. Donor milk has been found to have

lower protein content, as low as 0.9 g/L. Research suggests that this low protein content

is not related to pasteurization but may in fact be related to the frequent container

changes involved in the manipulation of the milk before it is consumed by the infant

(138). Evidence exists to suggest that preterm infants who receive donor milk do not

grow as well as those infants who receive preterm infant formula (139-141). Low protein

levels in donor milk is thought to play a pivitol role in this observed growth difficulties

(141). Even with fortification of this donor milk, protein levels may still be inadequate to

support high protein needs in these infants 3.5 -4.0 g/kg/day (141). A similar effect may

be a consideration in the current study, with multiple manipulations and container

102

changes having an impact on the resulting fat and protein content of the modified fat

milk.

From an analytical standpoint, the BCA method used to analyze protein content has

been suggested to overestimate protein content by as much as 20-40%, which may have

played a role in the high protein levels of the modified fat milk samples (129, 142). In

the current study, prior to analysis of the modified fat milk samples, the BCA method

was compared to the Kjeldhal technique using a certified standard infant formula. Results

of this comparison found no significant difference in the protein content of the standard

as measured by BCA method (n=13) (15.5 ± 1.15 g/L) in comparison to the Kjeldhal

technique (n=8) (16.1 ± 1.21 g/L) (143).

In order for protein to be adequately deposited as lean body mass, adequate non-

protein energy from carbohydrate or fat must be provided (104). If overall energy

provision was low, the protein content may have been used to provide energy rather than

to support tissue deposition and growth. Results of the 3-day weighted food record

suggest that all but 3 infants (1 treatment, 2 controls) met their estimated energy

requirements and in fact, similar to protein, most exceeded their estimated energy

requirements for age (104). However, these estimated energy requirements do not take

into consideration the protein and fat losses that occur with chylothorax and the resulting

impact on energy and protein requirements (29). These estimated energy requirements are

also not appropriate for those infants who have undergone a palliative procedure instead

of a full repair of their cardiac defect as these infants continue to have elevated energy

requirements related to their on-going struggles with complications of congenital heart

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disease (25). Finally, the weighted 3-day food record only provided a look at energy

intake in a small window of time. Protein and energy intake may have been inadequate

in the period preceding or following the 3 days over which this data was captured.

impact on the growth of the infants. Two infants in the treatment group had single

ventricle physiology, a severe form of congenital heart disease that is frequently

associated with feeding and growth difficulties (114). Both of these subjects had

undergone an initial palliative surgery. The 2 subjects with single ventricle physiology,

both of whom were in the treatment group, had the slowest rate of weight gain in

comparison to the other infants in the treatment group, achieving only 27% and 37% of

expected average daily weight gain goals (137). As a result, these infants experienced a

large decline in weight-for-age z-score over the study period (Figure 8) from -0.26 at

study enrolment to -2.76 at study completion in Subject 6 and -0.95 at study enrolment to

-2.8 at study completion in Subject 11 and played a driving role in the overall effect on

growth observed in the treatment group. Therefore, it must be assumed that these infants

were not meeting overall energy requirements to support growth during this period of

their management. These results are in line with recent research that suggests the most

difficult time for growth in infants with single ventricle physiology is between the first

and second stage palliative surgeries (114). Kelleher et al found that median discharge

weight after Stage 1 Norwood procedure was unchanged from admission weight and

median discharge weight-for-age z-score was -1.4 (114). At the time of readmission for

the Stage 2 bidirectional cavopulmonary shunt, the median weight-for-age z-score was

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-2.0 with 50% of the infants being significantly underweight, suggesting that achieving

adequate growth in the period between the Stage 1 Norwood and Stage 2 bidirectional

cavopulmonary shunt is a frequently observed challenge (114). A study by Vogt et al also

showed that growth between the Stage 1 Norwood and Stage 2 bidirectional

cavopulmonary shunt was impaired with a significant decline in weight-for-age z-score

between the 2 procedures (113).

The growth effects seen in the treatment group may be in part related to other basic

differences between the treatment and control group. The groups did tend to differ in age.

As illustrated in Figure 8, infants in the treatment group entered the study at younger age

with 5 of 8 (63%) infants being newborn. No infants in the control group were newborn.

Although this difference in age was not statistically significant, it may have had some

impact on the growth of the infants. In newborn infants, a higher proportion of energy

consumed is diverted to growth and tissue deposition (144). Over 1/3 of energy

requirements in the newborn is diverted to growth (144). Within the first year of life, the

cost of growth diminishes significantly. By 6 months of age, the cost of growth has

diminished to only a fraction of energy expenditure of the infant as the cost of physical

activity of the infant continues to increase (144). Thus, although energy intakes of the

infants in the treatment groups may have been significantly higher, it may still have not

provided sufficient energy to meet increased energy requirements and to support the

demands of rapid growth at this stage of life. Without a comparable number of newborn

infants in the control group, it is difficult to determine the impact of age on growth of

infants in the study. Given the known differences in growth requirements of newborn

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infants in comparison to their older peers, it is impossible to discount this potential effect

of age on growth.

There are some well documented differences in growth of infants who are breastfed

versus formula fed that appear within the first year of life. Research by Dewey et al

revealed that infants who are breastfed and formula fed infants have similar weight gain

in the first 3 months of life (145). However, after this point, the weight gain of breastfed

infants slows. Rates of weight gain in breastfed infants continue to be slower than

-­‐5  

-­‐4  

-­‐3  

-­‐2  

-­‐1  

0  

1  

0   100   200   300  

Weigh

t-­‐for-­‐age  Z-­‐score  

Age    (days)  

Control  Subjects  

Treatment  Subjects  

Figure 8. Trajectory of change in weight-for-age z-score for treatment and control subjects from study enrolment to study completion

106

formula fed infants for the remainder of their first year (145). This difference in growth

was not seen in length or head circumference (145)

the 5 newborn infants in the treatment group. However it could explain part of the slower

rates of weight gain observed in the 3 older infants in the treatment group, who ranged in

age from 6 to 7.5 months of age at study enrolment. At this stage of life, expected rate of

weight gain would be slower in these infants receiving breast milk in comparison to their

formula fed peers (145).

Growth issues were not isolated to the treatment group. Mean length-for-age and

head circumference-for-age z-scores were well below that of the treatment group at study

enrolment and this difference was statistically significant. Although mean weight-for-age

z-scores were not significantly different between the two groups at study enrolment,

mean weight-for-age z-score of infants in the control was lower than that of the treatment

group and appeared to be approaching significance. Therefore, although infants in the

control group appeared to have better growth over the course of the study, these infants

entered the study in poorer nutritional status than infants in the treatment group. Given

the older age of infants and the known effects of unrepaired congenital heart defects on

growth, the growth deficits observed in the control infants at study enrolment are not

unexpected. Nydegger et al found that infants who underwent surgical correction later in

life (>10 days of age) had significant deficits in mean weight-for-age and length-for-age

z-scores at the time of admission for surgery (146). Resting energy expenditure was

increased before cardiac surgery in comparison to healthy control. One week following

surgery, resting energy expenditure had returned to normal levels (146).

107

In contrast to the treatment group, all infants in the control group had full repair of

their cardiac defect and it is expected that following surgical correction, growth will start

to improve (112). In spite of this, infants in the control group did not show a significant

improvement in mean weight-for-age, length-for-age or weight-for-length z-scores over

the course of the study suggesting that these infants did not experience any catch-up

growth during the study and in fact many of the control infants struggled with similar

weight gain challenges to those observed in the treatment group. At study completion,

mean weight-for-age, length-for-age and weight-for-length z-scores remained well below

the standard for age. However, the length of the study may not have been sufficient time

to see expected improvements in growth parameters following corrective cardiac surgery.

Although improvements in growth are common following corrective surgery, growth

abnormalities can persist into childhood and deficits in weight and height continue to

exist in comparison to healthy controls (60, 112). Therefore, abnormalities in energy

metabolism may continue to have an impact on growth well into the post-surgical period

(60).

Unfortunately, the small number of patients in this study precludes stratification

based on diagnosis and/or surgical procedure. However, the cardiac condition of the

infant could have been a source of bias that we were unable to control for in this study. It

is well known that there are certain diagnosis and their associated surgical corrections

that occur in close proximity to the thoracic duct, such as Blalock shunts, repair of

coarctation and ligation of a patent ductus arteriosis that increase the risk of chylothorax

108

(48). Other procedures are known to be associated with prolonged chylous drainage such

as cavopulmonary procedures and the Fontan (26). In the current study, duration of

drainage was longest for 3 patients in the control group, all of whom required progression

to parenteral nutrition to treat their chylothorax. Of these 3 patients, 2 had undergone

heart transplantation. It could be suggested that perhaps the nature of the heart

transplantation procedure increased the risk of chylothorax and the likelihood of

prolonged drainage. In a study by Chan et al, heart transplantation was associated with a

higher incidence of chylothorax than other procedures with an incidence of 11.1% (4 of

36 heart transplantations performed over 28 months) (26). In theory, heart

transplantation is accompanied by extensive injury to the chest cavity increasing the

likelihood of damage to the delicate thoracic duct or associated vessels (26). It could be

suggested that had patients who developed chylothorax after heart transplantation been

excluded, the difference in duration of drainage results of this study may have been

different. Rather than excluding these patients at high risk for chylothorax, further study

with larger number of subjects would be of benefit to determine the impact of diagnosis

and/or surgical procedure on the effectiveness of modified fat breast milk.

3.4.3  Provision  of  Breast  Milk  

One of the primary goals of this study was to help ensure the on-going provision of

own benefits to these infants following their treatment for

chylothorax. All infants in the study were <12 months of age and many were <6 months

of age. The recommendation of the World Health Organization and other paediatric and

health organizations is that breast milk be the sole source of nutrition for 6 months, with

109

on-going provision of breast milk through the first year of life and beyond (6). It is

important to note that of the 8 infants in the treatment group, 5 infants went back to

partial or exclusive breast feeding and 1 patient continued to receive expressed breast

milk via nasogastric tube. Of the remaining 2 infants, 1 mother made the decision to stop

breastfeeding following study completion. The second infant, who had refused the

modified fat milk by bottle or cup and had been reliant on an nasogastric tube for the

duration of the study period, had great difficulty re-establishing breastfeeding. Mom did

not continue to pump after completion of the study and the patient transitioned to a sippy

cup and formula. However, 75% of the treatment subjects continued to receive the

benefits of breast milk following study completion.

3.4.4  Microbiological  Analysis  

The fact that breast milk is not a sterile fluid was well demonstrated in the current

study as all breast milk samples tested positive for the presence of at least 1 type of

bacteria, most commonly coagulase negative staphylococcus. The presence of bacteria in

human breast milk samples has been well documented. Law et al studied the bacterial

content in samples of raw (unpasteurized) human milk from all feedings provided to 98

premature infants within the first 2 weeks of life (135). Coagulase negative

staphylococcus was found in 77% of milk samples. All infants in the study were exposed

to Coagulase negative staphylococcus through human milk on at least one occasion, 41%

of infants were exposed to Staphylococcus aureus and 64% of infants were exposed to

gram-negative bacteria such as Acinetobacater species, pseudomonas fluorescens group

and Klebsiella pneumonia (135). Interestingly, in spite of frequent ingestion of

110

potentially harmful bacteria by the infants in this study, the authors could not attribute

any adverse event to the ingestion of the bacteria present in human milk (135). A study

conducted by Kvist et al examined the bacterial content of milk samples from healthy

mothers in comparison to mothers with symptoms of mastitis. Results revealed that 90%

of milk samples from healthy mothers contained coagulase negative staphylococcus

(147). In fact, in this study by Kvist et al, the 5 most common species found in milk

samples of both healthy mothers and those mothers with symptoms of mastitis were

coagulase negative staphylococcus, viridans  streptococci,  staphylococcus  aureus,  Group  

B  streptococci  and  enterococcus  faecalis  (147).    Thus,  it  is  not  uncommon  for  healthy  

women  to  produce  milk  samples  that  harbour  potentially  harmful  bacteria.    A similar

result was seen in a more recent study by Landers et al, who cultured 810 individual milk

samples prior to pasteurization for subsequent use by a donor milk bank (133). The most

commonly identified species was coagulase negative staphylococcus, found in 87% of

samples (133). Other species identified include enterococcus species in 16%,

staphylococcus  aureus  in  4%  and  dipthroids in 2% of samples (133).    In  the  current  study,  

all  5  organisms  reported  in  the  above  study  by  Kvist  were  isolated  in  at  least  one  sample  

of  milk.  However, one of the more surprising and  concerning  results  was  seen  with  a  

milk  sample  from  Subject 7, which was found to contain Klebsiella Pneumoniae. In a

study by Lindemann et al, 2% of milk samples tested contained Klebsiella species (148).

Again, Lindemann et al found the most common type of bacteria to be coagulase negative

staphylococcus 85% (148).

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Reassuringly, the source of contamination did not appear to be the processes

associated with centrifugation of the breast milk. As previously mentioned, many of the

milk samples contained fewer bacteria after centrifugation than before, suggesting a

portion of the existing bacteria was eliminated with the removal of the fat layer. Method

of expression has been implicated as a potential source of bacterial contamination in

human milk (135). All mothers in the current study expressed milk using a hospital grade

electric pump. In a study by Liebhaber, milk expressed using a rubber bulb breast pump

contained significantly higher levels of bacteria than milk samples expressed using hand

expression (149). Tyson et al reported that milk samples expressed by manual expression

were less likely to be contaminated than milk collected by either manual or electric

pumps (150). The employment of more stringent cleaning procedures of the pump and its

associated parts reduced bacterial contamination but did not eliminate it (150). In a more

recent study by Boo et al, expressed breast milk obtained by electric breast pump had

significantly higher rates of bacterial contamination when compared to milk expressed

manually (151). In the current study, mothers were educated, and re-educated if

necessary, on proper hygiene and proper use of the breast pumps e.g. using a new pump

kit each time, ensuring the pump was marked as having been cleaned prior to use. The

effect of this hygiene education on the bacterial content of the resulting expressed breast

milk was not examined in the current study but the existing literature suggests that these

efforts may result in reduction of bacterial content but not elimination. However, no

mothers developed mastitis during the study period and no infant developed a secondary

infection.

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3.5  CONCLUSIONS  AND  FUTURE  DIRECTIONS

3.5.1  Conclusion  

Breast milk is considered the optimal food for most infants (6). Infants with

chylothorax require dietary changes to limit intake of long chain triglycerides and as a

result are unable to have breast milk for the duration of treatment, a minimum of 6 weeks.

We conducted this study to determine the effectiveness of a modified fat breast milk for

infants with chylothorax in hopes of providing evidence to support the use of breast milk

in these patients and thus allowing affected infants to continue to receive its associated

benefits. Currently all infants diagnosed with chylothorax, whether previously breast fed

or formula fed, must transition to a medium chain triglycerides based formula. This can

be a difficult transition for all affected infants. The MCT based formula is not an infant

formula and is diluted for use in the infant population (55). Our experience with the MCT

formula suggests that there is often a decline in tolerance with the introduction of the

MCT based formula and volume of intake is frequently observed to be lower for the

duration of treatment. For previously breastfed infants, the change to the MCT based

formula also requires a change in mechanism of feeding. These can be difficult

transitions for all infants with CHD in whom feeding and growth is often challenging

(25).

To our knowledge, this is the first study to systematically evaluate the

effectiveness of a modified fat breast milk in comparison to the MCT based formula that

is the current treatment standard for the treatment of chylothorax in infants following

cardiothoracic surgery. Contrary to our first hypothesis, infants who received the

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modified fat breast milk did not have significantly more volume of chest tube drainage on

treatment day 5 versus those infants who received the MCT formula. This result suggests

that use of a nutrient enriched modified fat breast milk can result in successful resolution

of chylothorax and may eventually offer an alternate treatment option for affected infants.

In addition, duration of drainage was not different between infants who received the

nutrient enriched modified fat breast milk and those infants who received the MCT

formula.

In spite of these positive results and contrary to our second hypothesis, growth in

infants who received the modified fat breast milk was not better when compared to those

infants who received the MCT based formula. In fact, growth in this group was

particularly challenging. The results of our study did not clearly identify the reason for

this poor growth. Although our second hypothesis speculated that tolerance of the

nutrient enriched modified fat breast milk may have been better than tolerance of the

MCT based formula, no significant difference in tolerance was observed between the two

groups.

Given the known elevated energy requirements of infants with CHD and especially

infants with single ventricle physiology, inadequate provision of calories may have been

a key element in the slow weight gain observed among the treatment subjects (25).

Although other nutrients appeared to be provided in adequate amounts, further

investigation would be of benefit to clarify if protein and fat were indeed provided in

adequate amounts for this vulnerable population of infants. Age of the infant, nutritional

114

losses associated with chylothorax, diagnosis and stage of cardiac treatment likely all

played a role in determining the observed growth patterns. Although infants in the

control group had improved growth in comparison to infants in the treatment group, the

majority of infants in this group also did not meet average daily weight gain goals for

age. In fact, the only infant who experienced weight loss over the course of the study

was in the control group. Although there is little literature that describes the growth of

infants with chylothorax, nutritional challenges are well recognized and weight loss as

well as weight maintenance, has been reported (29, 33). Although the growth results

were discouraging, difficulties with weight gain in infants with congenital heart disease

and in infants affected with chylothorax is not unexpected and highlights the need for

aggressive fortification of feeds and close monitoring of growth in all patients during

treatment.

This is the first study to suggest that the use of a modified fat breast milk for the

treatment of chylothorax in infants is successful reducing chest tube drainage for

resolution of chylothorax. This would allow on-going provision of breast milk and its

associated benefits not only during treatment for chylothorax but promote provision of

breast milk after treatment has ended. Most of the infants who received the modified fat

breast milk returned to breastfeeding or continued to receive expressed breast milk via

bottle or nasogastric feeding tube after study completion. However, the clear challenge

that emerged during the course of the study is that of achieving adequate weight gain in

the infant with chylothorax. A clearer picture of nutrient needs, taking into consideration

associated protein and fat losses, of the infant with chylothorax together with more

115

aggressive nutrient fortification, are key elements to consider when moving forward with

the possible use of a modified fat breast milk. However, based on the observed

suboptimal growth of the treatment infants in this study, further research is necessary to

ensure the safety of modified fat breast milk and to ensure optimal growth for all infants

with chylothorax and in particular, those infants receiving the modified fat breast milk.

3.5.2  Future  Directions  

The primary shortcoming of this study is the small sample size which limits the

value of the statistical analysis. Initially we had aimed to achieve a sample size of 14

subjects in each group however for practical reasons it was decided to end recruitment at

8 subjects in each group, resulting in limited statistical power. Because of the small

number of subjects, we were unable to control for diagnosis of the subjects enrolled in the

study, which as previously discussed, could have an impact on the nature of the

chylothorax e.g. duration of drainage, effectiveness of conservative treatment. For future

studies, stratification based on diagnosis it would be useful in establishing the impact of

diagnosis on the effectiveness of modified fat breast milk. However, the low overall

incidence of chylothorax (2.7-4.5% at The Hospital for Sick Children) in infants post

cardiac surgery would make achieving sufficient sample sizes within a diagnosis group to

support an adequately powered study very challenging (26). Although not statistically

significant, the impact of difference in age between infants in the treatment and control

cannot be discounted. For future studies, a larger sample size resulting in two groups

more similar in age may help to tease out the effects of age and stage of development on

growth in infants being treated for chylothorax. Although not analyzed statistically, there

116

were some observed differences in volume of drainage between infants who received

100% of their feeds as modified fat breast milk and those infants who received a

combination of the modified fat breast milk and MCT formula. Study of a larger number

of subjects and analysis of more defined feeding regimes e.g. exclusively modified fat

breast milk versus combination modified fat breast milk and MCT, would be required to

determine if these differences are statistically significant. Finally, on-going growth

monitoring of patients after study completion would provide a clearer picture regarding

the impact of chylothorax and its treatment on growth in all infants.

Ultimately, it is hoped that the outcomes of this study will pave the road for the

possibility of offering cardiac infants affected by chylothorax the opportunity to receive

breast milk, in a modified form, during treatment for chylothorax. It is hoped that this

will allow for less of a disruption in established feeding routines and allow parents the

opportunity to be consistent with the nutrition goal of providing breast milk and its

known benefits to their infant with congenital heart disease.

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118. Farrell AG, Schamberger MS, Olson IL, Leitch CA. Large left-to-right shunts and congestive heart failure increase total energy expenditure in infants with ventricular septal defect. Am J Cardiol. 2001 May 1;87(9):1128-31, A10. 119. Barton JS, Hindmarsh PC, Scrimgeour CM, Rennie MJ, Preece MA. Energy expenditure in congenital heart disease. Arch Dis Child. 1994 Jan;70(1):5-9. 120. Leitch CA, Karn CA, Peppard RJ, Granger D, Liechty EA, Ensing GJ, et al. Increased energy expenditure in infants with cyanotic congenital heart disease. J Pediatr. 1998 Dec;133(6):755-60. 121. van der Kuip M, Hoos MB, Forget PP, Westerterp KR, Gemke RJ, de Meer K. Energy expenditure in infants with congenital heart disease, including a meta-analysis. Acta Paediatr. 2003 Aug;92(8):921-7. 122. Weesner KM, Rosenthal A. Gastroesophageal reflux in association with congenital heart disease. Clin Pediatr (Phila). 1983 Jun;22(6):424-6. 123. Dundar B, Akcoral A, Saylam G, Unal N, Mese T, Hudaoglu S, et al. Chronic hypoemia leads to reduced serum IGF-1 levels in cyanotic congenital heart disease Journal of Pediatric Endocrinology & Metabolism. 2000;13:431-6. 124. The Heart and Stroke Foundation. Congenital Heart Disease. Available at: http://www.heartandstroke.com/site/c.ikIQLcMWJtE/b.3484063/k.E84C/Heart_disease__Congenital_heart_disease.htm. The Heart and Stroke Foundation; 2009. 125. Farmer S, Bannister L, Cornelius V, Rafii M, O'Connor D, Mager D. Development and analysis of low fat breast milk for use with in-hospital paediatric patients diagnosed with chylothorax - a pilot study. Unpublished. 2005. 126. Wang C, Chu P, Mellen B, Shenai J. Creamatocrit and nutrient composition of human milk. Journal of Perinatology. 1999;19(5):343-6. 127. Kris-Etherton PM, Innis S, Ammerican Dietetic A, Dietitians of C. Position of the American Dietetic Association and Dietitians of Canada: dietary fatty acids. J Am Diet Assoc. 2007 Sep;107(9):1599-611. 128. Smith P, Krohn R, Hermanson G, Mallia A, Gartner F, Provenzano M, et al. Measurement of protein using bicinchoninic acid. Analytical Biochemistry. 1985;150:76-85. 129. Keller R, Neville M. Determination of total protein in human milk: comparison of methods. Clinical Chemistry. 1986;32(1):120-3. 130. Hald PM. The flame photometer for the measurement of sodium and potassium in biological materials. J Biol Chem. 1947 Feb;167(2):499-510. 131. Lonnderdal B. Nutritional and physiological significance of human milk proteins. The American Journal of Clinical Nutrition. 2003 June;77(6):1537S-43S. 132. Institute of Medicine. Dietary Reference Intakes for Water, Potassium, Sodium, Chloride, and Sulfate Washington, DC: The National Academies Press; 2004. 133. Landers S, Updegrove K. Baceteriological screening of donor human milk before and after Holder pasteurization. Breastfeeding Medicine. 2010;5(3):117-21. 134. Huebner J, Goldmann D. Coagulase-negative staphylococci: role as pathogens. Annual Review of Medicine. 1999;50:223-36. 135. Law BJ, Urias BA, Lertzman J, Robson D, Romance L. Is ingestion of milk-associated bacteria by premature infants fed raw human milk controlled by routine bacteriologic screening? J Clin Microbiol. 1989 Jul;27(7):1560-6.

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APPE NDI C ES APPENDIX  A: CONSENT  FORM    TREATMENT  GROUP

555 University Avenue Toronto, Ontario, Canada M5X  Title  of  Research  Project:  The effectiveness of low-fat breast milk for the treatment of chylothorax in infants following cardiothoracic surgery. Investigators:  Primary Investigator/Study Coordinator: Sarah Farmer, RD CNSD, The Hospital for Sick Children (416) 813-6747 Co-investigators:

Connor, PhD RD, The Hospital for Sick Children (416) 813-7844 Jennifer Russell, MD FRCPC, The Hospital for Sick Children (416) 813-7291 Purpose  of  Research:  Chylothorax is a complication that affects some babies after heart surgery. When a baby develops chylothorax, they must be put on a formula that is made with a special type of fat, called medium chain triglycerides or MCT. This means that they cannot have regular formula or breast milk while they are being treated for chylothorax. Breast milk is the gold standard food for babies and it provides most nutrients in just the right amounts. Breast milk has unique properties that make it easier for babies to digest in comparison to standard infant formulas. Breast milk offers many immune benefits to babies. These immune benefits help a baby fight infection and help to decrease the occurrence of certain childhood illnesses such as diarrhoea or ear infections. These types of characteristics cannot be found in infant formulas. Babies with chylothorax, especially babies who were breast feeding or receiving breast milk by bottle before surgery, may have some trouble adjusting to the special formula. It tastes different than breast milk (or regular formula) and babies may not drink as much as they usually do. This can make weight gain difficult. It also has different ingredients than regular infant formula or breast milk and this can make it harder for the baby to digest. This means that babies may have more spit up or vomiting than they usually do and it may maalso be hard to keep a good milk supply while their baby is drinking the special formula.

Subject Identifier:

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It would be ideal if infants who were receiving breast milk before surgery could continue to receive some breast milk while being treated for chylothorax. It is possible to remove most of the fat from breast milk. The purpose of this research study is to see if we remove most of the fat from breast milk, if the remaining low-fat milk is an effective treatment for chylothorax in comparison to the special formula that we are using now. This would mean that babies could continue to have some breast milk while they are being treated for chylothorax and might help avoid some of the issues described above.  Description  of  the  Research:   Baby participating in this study will be placed into one of two groups. Which group your baby is placed in will depend on what your baby was eating before surgery. Babies that were breast feeding before their surgery, or drinking breast milk by bottle, have the option to be entered into the low-­fat  breast  milk  group. Babies in the low-­fat  breast  milk  group will get breast milk with most of the fat removed to treat their chylothorax. To be entered into this group, your baby must have been receiving approximately 80% of their feeds as breast milk (by breast or bottle) before surgery and you must be willing to pump breast milk for the entire time your baby is treated for chylothorax. Babies that were drinking formula before their surgery will be in the control  group and will get the MCT formula that is normally used to treat chylothorax. If your baby was receiving breast milk before surgery, but you do not want to pump your breast milk throughout the study, you can be entered into the control group. Babies in the low-­fat  breast  milk  group will have some extra nutrients added to the breast milk. This is because taking the fat out of breast milk also takes out some of the calories (or energy) and vitamins that the babies need to grow. While you are in hospital, this mixture will be prepared for you in the milk preparation room. When you are ready to be discharged from hospital, you will be given detailed instructions on how to prepare this special mixture of low fat breast milk and additional nutrients at home. Babies will be fed the low-­fat  breast  milk by bottle and/or by nasogastric tube, if necessary. If your baby was eating solids before surgery, you will also be given guidelines about what solid foods your baby can have while being treated for chylothorax.

chylothorax which is a minimum of 6 weeks. Before you leave the hospital, you will be told the date that it is safe for your baby to go back to their usual feeding routine. The study will last for approximately two years and will require a total of 28 babies. New information from this study or other studies may affect whether you want to continue to take part in the study. If this happens, we will tell you about this new information.    

Subject Identifier:

129

   What  will  you  do  in  our  study? If you live within the Greater Toronto Area, enrolling in the study will not require any additional visits to the hospital. All contact with the study coordinator (Sarah Kocel) will be arranged for the same day you will be visiting the cardiac clinic for your post-operative check- ologist. If your appointments do not fall within the treatment period, a home visit will be arranged at your convenience. For subjects who live outside of the Greater Toronto Area, one additional visit to the hospital at the end of the treatment period may be required. This appointment will be arranged with the study coordinator for a time that is convenient for you. If your baby does need extra appointments to closely monitor growth and feeding issues, this will be arranged at your convenience with Sarah Kocel, the study coordinator. Many babies need some extra monitoring of their growth after cardiac surgery. This is normal routine, even if you do not enrol in the study. If you agree to be in our study, we will ask some general questions about you (both Mom and Dad) such as your age, your background, how long you went to school. We will also

feeding history such as whether or not he or she was breast feeding or bottle feeding, how much he or she normally eats etc.

diagnosis and the type of cardiac surgery he or she had. We will also check to see if your baby has any other medical conditions, if they have had any other surgeries or medical

birth weight, length and head circumference are available. If this information is not in

We will weigh your baby everyday he or she is in hospital. This will happen even if you

erence at the beginning of the study and then once a week until you go home. While you are in hospital, we will monitor how much your baby drinks every day. If your baby is receiving any fluids/medications through an intravenous line, we will monitor this as well. We will also monitor how much fluid comes out of the drainage tube that is in

are not enrolled in the study.        

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130

What  happens  at  the  beginning  of  the  study  while  your  baby  is  in  hospital:  Babies in the low-­fat  breast  milk  group will begin to receive the low-fat breast milk as soon as they are enrolled in the study. If no breast milk is available right away, they will receive the MCT formula until breast milk is available. Mothers of infants in the low-­fat  breast  milk  group can have the help of a lactation consultant if they would like. The lactation consultant can help with the proper way to pump and give guidelines about how often to pump to make sure you have enough milk to feed your baby. While you are in hospital, the low-fat breast milk will be prepared for you. Before you go

using the low-fat breast milk. You will also be given a recipe to prepare just the MCT formula in the event that you run out of breast milk. You will be given instructions on how much to feed your baby and how often. These types of instructions are normal routine for all infants who are being treated for chylothorax and require special feedings. While you are in hospital, very small samples of your breast milk that you pump, less than a half a teaspoon, will be taken on a regular basis. This will be done to be sure enough fat is being removed and to see if any other nutrients are being affected. What  happens  after  you  are  discharged  from  the  hospital?   If your baby is in the low-­fat  breast  milk  group, you will need to continue to pump for the duration of the study. If you do not have an electric breast pump at home, one will be provided to you, at no cost, to use for the duration of the study. You will be asked to continue to follow the pumping instructions given to you by the lactation consultant. You will have access to a lactation consultant by phone if you feel you need extra instructions once you are home. You will have two options for the removal of fat from your breast milk. Option  1: Your breast milk will be picked up twice each week and brought to the hospital to have the fat removed. Once your breast milk has been centrifuged and the fat removed, the resulting low fat breast milk will then be returned to you for use. The date and time for pick-up and delivery will be arranged with the study investigator to best suit your schedule. A delivery service will be used to carry out the pickup and delivery of your breast milk. You will be given instructions on how to store the breast milk before and after pick up as well as how to pack up the breast milk for transport. Option  2: You will have the option to take home a small table top centrifuge so that you can remove the fat from your breast milk at home. This is the only option available to families who live outside of the greater Toronto area. If you choose this option, you will be taught how to use the centrifuge before you leave the hospital and given opportunities to practice so you are comfortable with the procedure. It will be important that you are

Subject Identifier:

131

able to remove as much fat as possible from the breast milk and you will work closely with the study coordinator to be sure you can do this each time you use the centrifuge. Once you are discharged, the study will be available to deliver the centrifuge to your home and set it up for you. If you choose this option and you live in the greater Toronto

Once you have left the hospital, your baby will need to be weighed once a week. This will be done at home on a scale that will be provided to you. This weight will need to be done on the same day of the week at about the same time of day e.g. in the morning, before a feeding. You will be required to record this weight on the weight and diet record.  What  happens  at  your  first  post-­operative  appointment?   When you come to the cardiac clinic for your post-operative visit, you will also see the

th and head circumference. For this visit, you will need to write down everything that your baby eats and drinks in a booklet called a diet record. You must do this for each of the 3 days before you come to the hospital. To do this, you will need to measure the exact amount that your baby eats and drinks at each feeding. To do this we will ask you to weigh your

number. When your baby is done feeding, we will ask you to weigh the bottle (and/or dish of solids) again and record this number. A scale will be provided to you so that you can accurately measure the amount your baby has had to drink or has eaten by spoon. We will give you

· A booklet to write this information on · Simple instructions about how to record the feeding volumes (these instructions

will be reviewed with you before you go home) · A scale

You will need to bring these diet records with you when you come to your appointment. This will be the only time you need to do this for study purposes. If there are concerns about how much your baby is drinking, you may be asked to record intake as part of your

What  happens  for  the  remainder  of  the  study  period?   If you have a scheduled appointment with your cardiologist during the remainder of the study period, you will also see the study coordinator at this time. For those in the low-­fat  breast  milk  group, the study coordinator will pick up your breast milk for fat removal and deliver the resulting de-fatted breast milk will be done twice a week for the remainder of the study period. The time of pickup and delivery will be arranged on a weekly basis to best suit your schedule.

Subject Identifier:

132

Throughout the remainder of the study period, all subjects will have a weekly phone call ll, you

drinking at each feeding and how often your baby is drinking. You will also be asked to report the weight of your baby. During this phone call, you can report any feeding or any other difficulties with the study protocol. If you have any questions or concerns, you will be able to contact the study coordinator at any time between your scheduled visits/phone calls using the contact information provided to you.  Potential  Harms:  If the low-fat breast milk is not effective, the treatment of chylothorax may be longer than 6 weeks. Babies may require a period of intravenous nutrition, with no food taken by mouth, to help treat the chylothorax. This is also a risk for babies treated with the MCT formula. If the instructions for preparation of the low fat breast milk or MCT formula are not followed carefully babies may receive too many nutrients. In these cases, babies could become very sick. Also, if the instructions are not followed carefully, babies may not get enough nutrients and this may affect their ability to grow properly and recover from surgery. Potential  Discomforts  or  Inconvenience:  After leaving the hospital, you will be required to fill in a 3 day diet record that will require you to measure how much your baby drinks. You will also be required to weigh your baby once each week throughout the study period. For babies in the low-­fat  breast  milk  group, mothers are required to pump regularly and frequently to be sure you have enough breast milk to feed your baby. This breast milk will need to be packed for transport to the hospital. For those mothers who were previously breast feeding or providing breast milk by bottle but who choose enter the control group (to receive the MCT formula), regular pumping will be necessary if you wish to continue to breast feed or provide breast milk once chylothorax treatment is complete. Parents must be available for pickup and delivery of breast milk twice a week. For subjects who live outside of the Greater Toronto Area, one additional visit to the hospital at the end of the treatment period may be required. This appointment will be arranged with the study coordinator for a time that is convenient for you.    

Subject Identifier:

133

 Potential  Benefits:  Babies who can continue to have some breast milk while being treated for chylothorax may continue to get some of its benefits, such as its anti-infective properties, and may have less difficulties with digestion that are often seen with the switch to any formula, including the MCT formula.  Mothers of those infants receiving breast milk will continue to pump on a regular schedule throughout the treatment for chylothorax, supporting the transition back to breastfeeding once treatment is complete by maintaining milk supply. Parents who have participated in other studies performed by our lab have found the additional contact with health care professionals during the study period extremely helpful and also found that this additional contact helped to make the transition home a little easier. This study will help us gain a better understanding of the effectiveness of using a low-fat breast milk to treat chylothorax and to offer this as a potential treatment option for parents who had hoped to provide breast milk as the main source of nutrition for their baby.  Confidentiality:  We will respect your privacy. No information about who you or your child is will be given to anyone or be published without your permission, unless the law requires us to do this. For example, the law requires us to give information about you or your child if a child has been abused, if you or your child has an illness that could spread to others, if you or someone else talks about suicide (killing themselves), or if the court orders us to give them the study papers.

 Sick Kids Clinical Research Monitors health record to check on the study. For example, people from Health Canada Health Products and Food Branch, if necessary, may look at your records. By signing this consent form, you agree to let these pe

records. We will give you a copy for your files. The data produced from this study will be stored in a secure, locked location. Only members of the research team (and maybe those individuals described above) will have access to the data. This could include external research team members. Following completion of the research study, the data will be kept as long as required and then destroyed as required by Sick Kids policy. Published study results will not reveal your identity.  

Subject Identifier:

134

 Reimbursement:  Any equipment required to complete this study will be provided to you at no cost e.g. breast pump and scale, MCT formula. If additional visits to the hospital are required solely for study purposes, we will reimburse you for all your reasonable out of pocket expenses associated with this visit e.g., meals, babysitters, parking and getting you to and from Sick Kids.  Participation:  It is your choice to let your baby take part in this study. If you choose to let your child take part in this study you can take your child out of the study at any time. The care your child gets at Sick Kids will not be affected in any way by whether your child takes part in this study. New information that we get while we are doing this study may affect your decision to take part in this study. If this happens, we will tell you about this new information. And we will ask you again if you still want to be in the study During this study we may create new tests, new medicines, or other things that may be worth some money. Although we may make money from these findings, we cannot give you (your child) any of this money now or in the future because you (your child) took part in this study. In some situations, the study doctor or the company paying for the study may decide to stop the study. This could happen even if the treatment given in the study is helping your child. If this happens, the study doctor will talk to you about what will happen next. If your child becomes ill or are harmed because of he or she took part in this study, we will treat he or she for free. Your signing this consent form does not interfere with your legal rights in any way. The staff of the study, any people who gave money for the study, or the hospital are still responsible, legally and professionally, for what they do.  Alternatives  to  participation:  If you choose not to participate in the study, your baby will receive the MCT formula to treat their chylothorax. This formula is that same treatment that babies in the control group will receive and is the current treatment that is used for all infants with chylothorax.  Sponsorship:   The sponsor of this study is the Labatt Heart Centre Innovation Fund.  Conflict  of  interest:    I, and the other research team members, have no conflict of interest to declare.

Subject Identifier:

135

 Consent:  

By signing this form, I agree that: 1) You have explained this study to me. You have answered all my questions. 2) You have explained the possible harms and benefits (if any) of this study. 3) I know what I could do instead of having my child take part in this study. I

understand that I have the right to refuse to let my child take part in the study. I also have the right to take my child out of the study at any time. My decision about my child takinSickKids.

4) I am free now, and in the future, to ask questions about the study. 5) medical records will be kept private. You will

give no one information about my child, unless the law requires you to. 6) I understand that no information about my child will be given to anyone or be

7) I have read and understood pages 1 to 9 of this consent form. I agree, or consent, that my child___________________ may take part in this study. _______________________ Printed Name of Parent/Legal Guardian Signature & Date

________________________________________ Printed Name of person who explained consent Signature & Date _______________________________________

does not read English) .

   Who  do  I  call  if  I  have  questions  or  problems?  If you have any questions or concerns at anytime during the study, please contact the study coordinator, Sarah Farmer at (416) 813-6747 or pager (416)330-3036. A letter will be sent to your paediatrician to make them aware of your participation in this study. If you need to contact someone about medical issues related to the study, please contact Dr. Jennifer Russell at (416) 813-7291.

Subject Identifier:

136

For questions about your rights as a research subject or for information on who to contact in the event of injuries during a study, please call the Research Ethics Manager at (416) 813-5718.

Subject Identifier:

137

APPENDIX  B: CONSENT  FORM    CONTROL  GROUP  

555 University Avenue Toronto, Ontario, Canada M5X Title  of  Research  Project:  The effectiveness of low-fat breast milk for the treatment of chylothorax in infants following cardiothoracic surgery. Investigators:  Primary Investigator/Study Coordinator: Sarah Kocel, RD The Hospital for Sick Children (416) 813-6747 Co-investigators:

(416) 813-7844 Jennifer Russell, MD FRCPC, The Hospital for Sick Children (416) 813-7291 Purpose  of  Research:  Chylothorax is a complication that affects some babies after heart surgery. When a baby develops chylothorax, they must be put on a formula that is made with a special type of fat, called medium chain triglycerides or MCT. This means that they cannot have regular formula or breast milk while they are being treated for chylothorax. Breast milk is the gold standard food for babies and it provides most nutrients in just the right amounts. Breast milk has unique properties that make it easier for babies to digest in comparison to standard infant formulas. Breast milk offers many immune benefits to babies. These immune benefits help a baby fight infection and help to decrease the occurrence of certain childhood illnesses such as diarrhoea or ear infections. These types of characteristics cannot be found in infant formulas. Babies with chylothorax, especially babies who were breast feeding or receiving breast milk by bottle before surgery, may have some trouble adjusting to the special formula. It tastes different than breast milk (or regular formula) and babies may not drink as much as they usually do. This can make weight gain difficult. It also has different ingredients than regular infant formula or breast milk and this can make it harder for the baby to digest. This means that babies may have more spit up or vomiting than they usually do and it may make their stomach also be hard to keep a good milk supply while their baby is drinking the special formula. It would be ideal if infants who were receiving breast milk before surgery could continue to receive some breast milk while being treated for chylothorax. It is possible to remove most of the fat from breast milk. The purpose of this research study is to see if we remove

Subject Identifier:

138

most of the fat from breast milk, if the remaining low-fat milk is an effective treatment for chylothorax in comparison to the special formula that we are using now. This would mean that babies could continue to have some breast milk while they are being treated for chylothorax and might help avoid some of the issues described above. Although your baby may not have been breast feeding or receiving breast milk by bottle before surgery, your participation in this study will make up the group of patients known as the control  group. Infants in the control group will continue to receive the treatment that is currently provided to all infants with chylothorax. The control group will provide very important data that will be used for comparison against data taken from infants who receive the low fat breast milk and will help to determine its effectiveness.  Description  of  the  Research:   Babies participating in this study will be placed into one of two groups. Which group your baby is placed in will depend on what your baby was eating before surgery. Babies that were breast feeding before their surgery, or drinking breast milk by bottle, have the option to be entered into the low-­fat  breast  milk  group. Babies in the low-­fat  breast  milk  group will get breast milk with most of the fat removed to treat their chylothorax. To be entered into this group, your baby must have been receiving approximately 80% of their feeds as breast milk (by breast or bottle) before surgery and you must be willing to pump breast milk for the entire time your baby is treated for chylothorax. If you agree to participate in this research study, your baby will be entered into the control group. Babies in the control  group are babies that were drinking formula before their surgery and will get the MCT formula that is normally used to treat chylothorax. If your baby was receiving breast milk before surgery, but you do not want to pump your breast milk throughout the study, you will be entered into the control group as well. Babies in the control group will be fed the MCT formula that is normally used to feed all babies with chylothorax. This is the same formula your baby will receive if you decide not to enrol in the study. Babies will be fed the MCT formula by bottle and/or by nasogastric tube if necessary. While you are in hospital, the MCT formula will be prepared for you in the milk preparation room. One you are ready to be discharged from hospital, you will receive detailed instructions on how to prepare the MCT formula at home. If your baby was eating solids before surgery, you will also be given guidelines about what solid foods your baby can have while being treated for chylothorax.

chylothorax which is a minimum of 6 weeks. Before you leave the hospital, you will be told the date that it is safe for your baby to go back to their usual feeding routine. The study will last for approximately two years and will require a total of 28 babies. New information from this study or other studies may affect whether you want to continue to take part in the study. If this happens, we will tell you about this new information.

Subject Identifier:

139

What  will  you  do  in  our  study? If you live in the Greater Toronto Area, enrolling in the study will not require any additional visits to the hospital. All contact with the study coordinator (Sarah Kocel) will be arranged for the same day you will be visiting the cardiac clinic for your post-operative check-appointments do not fall within the treatment period, a home visit will be arranged at your convenience. For subjects who live outside of the Greater Toronto Area, one additional visit to the hospital at the end of the treatment period may be required. This appointment will be arranged with the study coordinator for a time that is convenient for you. If your baby does need extra appointments to closely monitor growth and feeding issues, this will be arranged at your convenience with Sarah Kocel, the study coordinator. Many babies need some extra monitoring of their growth after cardiac surgery. This is normal routine, even if you do not enrol in the study. If you agree to be in our study, we will ask some general questions about you (both Mom and Dad) such as your age, your background, how long you went to school. We will also

feeding history such as whether or not he or she was breast feeding or bottle feeding, how much he or she normally eats etc.

diagnosis and the type of cardiac surgery he or she had. We will also check to see if your baby has any other medical conditions, if they have had any other surgeries or medical

birth weight, length and head circumference are available. If this information is not in u to provide this information, if possible.

We will weigh your baby everyday he or she is in hospital. This will happen even if you

the beginning of the study and then once a week until you go home. While you are in hospital, we will monitor how much your baby drinks every day. If your baby is receiving any fluids/medications through an intravenous line, we will monitor this as well. We will also monitor how much fluid comes out of the drainage tube that is in

are not enrolled in the study.  What  happens  at  the  beginning  of  the  study  while  your  baby  is  in  hospital:   Babies in the control group will begin to receive the MCT formula as soon as they are diagnosed with chylothorax. While you are in hospital, the MCT formula will be

Subject Identifier:

140

prepared for you. Before you go home, you will be given specific instructions on how to prepare the MCT formula for your baby. You will be given a recipe that will tell you how much formula and how much water is required. You will also be given instructions on how much to feed your baby and how often. This is the normal routine for all infants who are being treated for chylothorax and require special feedings, even if they are not enrolled in the study.  Mothers of infants in the control group will be able to have the help of a lactation consultant to help them maintain their breast milk supply if they wish to return to breast feeding after chylothorax treatment is complete. What  happens  after  you  are  discharged  from  the  hospital?  Babies in the control  group will continue to feed your baby the MCT formula for the duration of the study. You will be provided with a supply of the MCT formula that will last for the rest of the study period. This formula will be provided to you for free whether or not you enrol in the study. Once you have left the hospital, your baby will need to be weighed once a week. This will be done at home on a scale that will be provided to you. This weight will need to be done on the same day of the week at about the same time of day e.g. in the morning, before a feeding. You will be required to record this weight on the weight and diet record. What  happens  at  your  first  post-­operative  appointment?   When you come to the cardiac clinic for your post-operative visit, you will also see the

t, length and head circumference. For this visit, you will need to write down everything that your baby eats and drinks in a booklet called a diet record. You must do this for each of the 3 days before you come to the hospital. To do this, you will need to measure the exact amount that your baby eats and drinks at each feeding. To do this we will ask you to weigh your

number. When your baby is done feeding, we will ask you to weigh the bottle (and/or dish of solids) again and record this number. A scale will be provided to you so that you can accurately measure the amount your baby has had to drink or has eaten by spoon. We will give you

· A booklet to write this information on · Simple instructions about how to record the feeding volumes (these instructions

will be reviewed with you before you go home) · A scale

You will need to bring these diet records with you when you come to your appointment. This will be the only time you need to do this for study purposes. If there are concerns about how much your baby is drinking, you may be asked to record intake as part of your

 

Subject Identifier:

141

What  happens  for  the  remainder  of  the  study  period?   If you have a scheduled appointment with your cardiologist during the remainder of the study period, you will also see the study coordinator at this time. Throughout the remainder of the study period, all subjects will have a weekly phone call from

drinking at each feeding and how often your baby is drinking. You will also be asked to report the weight of your baby. During this phone call, you can report any feeding or any other difficulties with the study protocol. If you have any questions or concerns, you will be able to contact the study coordinator at any time between your scheduled visits/phone calls using the contact information provided to you. Potential  Harms:  If the MCT formula is not effective, the treatment of chylothorax may be longer than 6 weeks. Babies may require a period of intravenous nutrition, with no food taken by mouth, to help treat the chylothorax. This is a risk for all babies with chylothorax. If the instructions for preparation of the low fat breast milk or MCT formula are not followed carefully babies may receive too many nutrients. In these cases, babies could become very sick. Also, if the instructions are not followed carefully, babies may not get enough nutrients and this may affect their ability to grow properly and recover from surgery. Potential  Discomforts  or  Inconvenience:  After leaving the hospital, you will be required to fill in a 3 day diet record that will require you to measure how much your baby drinks. You will also be required to weigh your baby once each week throughout the study period. For babies in the low-­fat  breast  milk  group, mothers are required to pump regularly and frequently to be sure you have enough breast milk to feed your baby. This breast milk will need to be packed for transport to the hospital. For those mothers who were previously breast feeding or providing breast milk by bottle but who choose enter the control group (to receive the MCT formula), regular pumping will be necessary if you wish to continue to breast feed or provide breast milk once chylothorax treatment is complete. For subjects who live outside of the Greater Toronto Area, one additional visit to the hospital at the end of the treatment period may be required. This appointment will be arranged with the study coordinator for a time that is convenient for you.      

Subject Identifier:

142

Potential  Benefits:  Babies who can continue to have some breast milk while being treated for chylothorax may continue to get some of its benefits, such as its anti-infective properties, and may have less of the digestive difficulties that are often seen with the switch to any formula, including the MCT formula. Mothers of those infants receiving breast milk will continue to pump on a regular schedule throughout the treatment for chylothorax, supporting the transition back to breastfeeding once treatment is complete by maintaining milk supply. Parents who have participated in other studies performed by our lab have found the additional contact with health care professionals during the study period extremely helpful and also found that this additional contact helped to make the transition home a little easier. This study will help us gain a better understanding of the effectiveness of using a low-fat breast milk to treat chylothorax and to offer this as a potential treatment option for parents who had hoped to provide breast milk as the main source of nutrition for their baby. Confidentiality:  We will respect your privacy. No information about who you or your child is will be given to anyone or be published without your permission, unless the law requires us to do this. For example, the law requires us to give information about you or your child if a child has been abused, if you or your child has an illness that could spread to others, if you or someone else talks about suicide (killing themselves), or if the court orders us to give them the study papers.

 

health record to check on the study. For example, people from Health Canada Health Products and Food Branch, if necessary, may look at your records.

records. We will give you a copy for your files. The data produced from this study will be stored in a secure, locked location. Only members of the research team (and maybe those individuals described above) will have access to the data. This could include external research team members. Following completion of the research study, the data will be kept as long as required and then destroyed as required by Sick Kids policy. Published study results will not reveal your identity.    

Subject Identifier:

143

Reimbursement:  Any equipment required to complete this study will be provided to you at no cost e.g. breast pump and scale, MCT formula. If additional visits to the hospital are required solely for study purposes, we will reimburse you for all your reasonable out of pocket expenses associated with this visit e.g., meals, babysitters, parking and getting you to and from Sick Kids.  Participation:  It is your choice to let your baby take part in this study. If you choose to let your child take part in this study you can take your child out of the study at any time. The care your child gets at Sick Kids will not be affected in any way by whether your child takes part in this study. New information that we get while we are doing this study may affect your decision to take part in this study. If this happens, we will tell you about this new information. And we will ask you again if you still want to be in the study During this study we may create new tests, new medicines, or other things that may be worth some money. Although we may make money from these findings, we cannot give you (your child) any of this money now or in the future because you (your child) took part in this study. In some situations, the study doctor or the company paying for the study may decide to stop the study. This could happen even if the treatment given in the study is helping your child. If this happens, the study doctor will talk to you about what will happen next. If your child becomes ill or are harmed because of he or she took part in this study, we will treat he or she for free. Your signing this consent form does not interfere with your legal rights in any way. The staff of the study, any people who gave money for the study, or the hospital are still responsible, legally and professionally, for what they do.  Alternatives  to  participation:  If you choose not to participate in the study, your baby will receive the MCT formula to treat their chylothorax. This formula is that same treatment that babies in the control group will receive and is the current treatment that is used for all infants with chylothorax.  Sponsorship:   The sponsor of this study is the Labatt Heart Centre Innovation Fund.  Conflict  of  interest:    I, and the other research team members, have no conflict of interest to declare.  

Subject Identifier:

144

Consent:   By signing this form, I agree that: 1) You have explained this study to me. You have answered all my questions. 2) You have explained the possible harms and benefits (if any) of this study. 3) I know what I could do instead of having my child take part in this study. I

understand that I have the right to refuse to let my child take part in the study. I also have the right to take my child out of the study at any time. My decision about my child taking SickKids.

4) I am free now, and in the future, to ask questions about the study. 5)

give no one information about my child, unless the law requires you to. 6) I understand that no information about my child will be given to anyone or be

published without first asking my permission. 7) I have read and understood pages 1 to 8 of this consent form. I agree, or consent,

that my child___________________ may take part in this study. _______________________ Printed Name of Parent/Legal Guardian Signature & Date ________________________________________ Printed Name of person who explained consent Signature & Date

does not read English) .

   Who  do  I  call  if  I  have  questions  or  problems?  If you have any questions or concerns at anytime during the study, please contact the study coordinator, Sarah Farmer at (416) 813-6747 or pager (416)330-3036. A letter will be sent to your paediatrician to make them aware of your participation in this study. If you need to contact someone about medical issues related to the study, please contact Dr. Jennifer Russell at (416) 813-7291.

Subject Identifier:

145

For questions about your rights as a research subject or for information on who to contact in the event of injuries during a study, please call the Research Ethics Manager at (416) 813-5718                                                                                      

Subject Identifier:

146

APPENDIX  C:  DATA  COLLECTION  FORMS    

Confidential Subject Information*

Name of Subject

HSC#

Name  of  Mother   Address City Province Postal Code Home Phone Work Phone Cell Phone Name  of  Father   Address City Province Postal Code Home Phone Work Phone Cell Phone Other  Caretaker  Name   Address City Province Postal Code Home Phone Work Phone Cell Phone

Subject Identifier:

147

Subject Eligibility Criteria

Date: (mm/dd/yy) Inclusion Criteria Yes No Infant < 12 months of age Positive diagnosis of chylothorax1

Infant to have follow-up care at The Hospital for Sick Children Patient lives within GTA At least one parent (ideally both) have voluntarily signed an informed consent

Treatment  Group:  Inclusion  Criteria  Yes No Parents/guardian would like to continue to provide breast milk

during treatment for chylothorax Mother agrees to express breast milk for duration of study Parents/guardian willing to be available for breast milk picks

up and delivery twice each week for duration of study Exclusion Criteria Yes No Infant receiving solely parenteral nutrition (no enteral feeds) Patient unable to follow the Chylothorax Care Map Neither parent able to effectively communicate in English If answers to a

then infant to be entered into the treatment group. Group Assignment:

Subject Identifier:

148

Enrolment Demographic Data (To be verified by medical record)

DOB (mm/dd/yy)

Gender Male Female

Gestational Age

Birth Weight

Birth Length Birth Head Circumference

Primary Diagnosis

Date of Surgery Procedure

Secondary Diagnosis

Other Medical Procedures (include dates)

Current Medications Mother Age (Complete Years)

Subject Identifier:

149

Ethnicity (descent) An Aboriginal person (e.g. North American Indian, Metis, Inuit, Eskimo) White Chinese South Asian (e.g. East Indian, Pakistani, Sri Lankan) Black Filipino Latin American Southeast Asian (e.g. Cambodian, Indonesian, Laotian, Vietnamese) Arab West Asian (e.g. Afghan, Iranian) Japanese Korean Other; specify Bi- or multiracial descent; specify

Last  year  of  schooling  completed (full-time equivalence) High School; specify number of years First year of college, university or trade school Second year of college, university or trade school Third year of college, university or trade school Fourth year of college, university or trade school Post-Graduate education; specify number of years Highest  degree  or  diploma  attained   None High School Diploma Vocational Diploma College Diploma Post-graduate education; specify number of years Total  years  of  education  

(only if biological mother) measured reported (ft/inches) (cm)

measured reported (kg) (lbs)

150

Father or Partner Age (Complete Years) Ethnicity (descent) An Aboriginal person (e.g. North American Indian, Metis, Inuit, Eskimo) White Chinese South Asian (e.g. East Indian, Pakistani, Sri Lankan) Black Filipino Latin American Southeast Asian (e.g. Cambodian, Indonesian, Laotian, Vietnamese) Arab West Asian (e.g. Afghan, Iranian) Japanese Korean Other; specify Bi- or multiracial descent; specify

Last  year  of  schooling  completed (full-time equivalence) High School; specify number of years First year of college, university or trade school Second year of college, university or trade school Third year of college, university or trade school Fourth year of college, university or trade school Post-Graduate education; specify number of years Highest  degree  or  diploma  attained   None High School Diploma Vocational Diploma College Diploma Post-graduate education; specify number of years Total  years  of  education  

(only if biological father) measured reported (ft/inches) (cm)

Subject Identifier:

151

measured reported (kg) (lbs)

152

Entrance Interview

1. Prior to surgery, how many times each day did you feed your baby either breast milk or formula? 2. How was your baby feeding in the time leading up to surgery? Breast feeding only Breast feeding + bottle feeding

Bottle feeding only

3. For infants doing any breast feeding: i) How many times each day does your baby feed at the breast? 4. For infants doing any bottle feeding: i) What does your baby drink by bottle? breast milk only breast milk fortified with formula Describe: formula only Describe: breast milk and formula Describe: ii) How many bottles do you usually give your baby each day? iii) Of the total number of bottles you give your baby each day, how many contain breast milk? 5. Does your baby eat any solids? i) If yes, how many times do you feed your baby solids each day? ii) If yes, what types of solids does your baby eat?

Subject Identifier:

153

6. Do you ever give your baby anything other than breast milk or formula to drink? Yes No i) If yes, please describe: If subject ject is eligible for the treatment group. If subject formula feeding or receiving < 80% of feeds as breast milk, subject is eligible for the control group. Group Assignment:

Subject Identifier:

154

Important Study Dates - Control

(mm/dd/yr) Study Enrolment

Study Day 1: initiation of feeding protocol

Discharge

Study Visit #1

Study Visit #2

Phone Follow-up #1

Phone Follow-up #2

Phone Follow-up #3

Phone Follow-up #4

Phone Follow-up #5

Phone Follow-up #6

Study Completion

Additional Phone Contact Dates (mm/dd/yy)

Subject Identifier:

155

Important Study Dates - Treatment (mm/dd/yy)

Study Enrolment

Study Day 1: Initiation of feeding protocol

Discharge

Study Visit #1

Study Visit #2

Phone Follow-up #1

Week 1 Delivery/Pick-up of LFBM*

#1 #2

Phone Follow-up #2

Week 2 Delivery/Pick-up of LFBM

#1 #2

Phone Follow-up #3

Week 3 Delivery/Pick-up of LFBM

#1 #2

Phone Follow-up #4

Week 4 Delivery/Pick-up of LFBM

#1 #2

Phone Follow-up #5

Week 5 Delivery/Pick-up of LFBM

#1 #2

Phone Follow-up #6

Week 6 Delivery/Pick-up of LFBM

#1 #2

Study Completion

Subject Identifier:

156

Additional Phone Contact Dates (mm/dd/yy)

Subject Identifier:

157

Intake Record In Hospital Date

(mm/dd/yy)

NPO

Part of day All day

Part of day All day

Part of day All day

Part of day All day

Part of day All day

Part of day All day

Part of day All day

Part of day All day

Feed

1. Formula 2. LFBM 3. PN/Lipids

1. Formula 2. LFBM 3. PN/Lipids

1. Formula 2. LFBM 3. PN/Lipids

1. Formula 2. LFBM 3. PN/Lipids

1. Formula 2. LFBM 3. PN/Lipids

1. Formula 2. LFBM 3. PN/Lipids

1. Formula 2. LFBM 3. PN/Lipids

1. Formula 2. LFBM 3. PN/Lipids

Strength

1. 2.

1. 2.

1. 2.

1. 2.

1. 2.

1. 2.

1. 2.

1. 2.

Intake

N/G tube 1. 2. PO 1. 2. IV Total:

N/G tube 1. 2. PO 1. 2. IV Total:

N/G tube 1. 2. PO 1. 2. IV Total:

N/G tube 1. 2. PO 1. 2. IV Total:

N/G tube 1. 2. PO 1. 2. IV Total:

N/G tube 1. 2. PO 1. 2. IV Total:

N/G tube 1. 2. PO 1. 2. IV Total:

N/G tube 1. 2. PO 1. 2. IV Total:

Drainage

Output

Vomiting

Total

LFEMB low fat breast milk N/G nasogastric tube PO by mouth

Subject Identifier:

158

Anthropometrics In hospital

Date

(mm/dd/yy)

Weight

(g)

Length (cm)

1. 2. Avg:

1. 2. Avg:

1. 2. Avg:

1. 2. Avg:

1. 2. Avg:

1. 2. Avg:

1. 2. Avg:

1. 2. Avg:

1. 2. Avg:

Head Circumferenc

e (cm)

1. 2. Avg:

1. 2. Avg:

1. 2. Avg:

1. 2. Avg:

1. 2. Avg:

1. 2. Avg:

1. 2. Avg:

1. 2. Avg:

1. 2. Avg:

Weight measured daily Length at enrolment and then weekly Head Circumference - at enrolment and then weekly Avg = average measurement

Subjects Identifier:

159

Study Visit #1 Treatment Group

* Questions regarding feeding pertain to feeding over the previous week Date of Visit: Date of Discharge: Discharge Weight: (mm/dd/yy) (mm/dd/yy) (mm/dd/yy) Weight (grams) [1] [2] [Average]

Weight gain (g/day): Percentile*: Length (cm) [1] [2] [Average]

Percentile*: Head Circumference (cm) [1] [2] [Average]

Percentile*: 1. Food Records Complete? Yes No 2. Food records reviewed: Name Date: (mm/dd/yy) 3. Are you still giving your baby part or all of their feeds as breast milk? Yes No a) If no, when did you stop giving breast milk? b) If no, what was the main reason you stopped giving breast milk? c) If no, what are you currently feeding your baby? How are you preparing this? d) If yes, since you left the hospital, has your baby received any feeds that did not contain any breast milk (formula only)? Yes No * WHO Growth Chart

Subject Identifier:

160

d) If yes, what was the reason? Ran out of breast milk/not enough breast milk

Formula was easier to prepare Other: 4. How many times per day are you pumping your breast milk? 5. Are you having any difficulty pumping? Yes No a) If yes, would you like to receive a call from the lactation consultant? Yes No 6. What is the recipe you are currently using to prepa 7. How is your baby feeding? po nasogastric tube po + nasogastric tube 8. How many feedings does your baby have each day? 9. If baby is feeding any po:

a) What is the minimum amount your baby will drink at a feeding (ml):

b) What is the maximum amount your baby will drink at a feeding (ml): 10. If baby is receiving any feed by NG:

a) What is the average amount that goes through the NG tube (ml): 11. Is your baby having any episodes of vomiting or spitting up? Yes No 12. If yes, how often does this happen? Daily Average number/day: Weekly Average number/week: Never 13. Is your baby taking any solid foods? Yes No a) If yes, how many times/day? 1 x/day 2 x/day 3 x/day b) cereals fruits/vegetables meats other

Subject Identifier:

161

14. Do you give your baby any juice? No Yes Describe (volume/frequency/diluted?): 15. Do you give your baby any water? No Yes Describe (volume/frequency): 16. In this past week, have you given your baby anything to drink that has not already been discussed? Yes No

a) If yes, please describe (what/how much)? 17. Do you give your baby any vitamins or other supplements? No Yes Describe (what/frequency):

1. Change to feeding routine? Yes No a) If yes, describe: 2. Date and time of scheduled pick-up/delivery of breast milk: Visit 1: Visit 2:

Plan

Subject Identifier:

162

Phone Documentation Treatment Group

Date of Call: * Questions regarding feeding pertain to feeding over the (mm/dd/yy) previous week Weight (grams) [Current Weight] [Weight Gain]

Percentile*: 1. Are you still giving your baby part or all of their feeds as breast milk? Yes No a) If no, when did you stop giving breast milk? (mm/dd/yy) b) If no, what was the main reason you stopped giving breast milk? c) If no, what are you currently feeding your baby? How are you preparing this? d) If yes, since you left the hospital, has your baby received any feeds that did not contain any breast milk (formula only)? Yes No

d) If yes, what was the reason? Ran out of breast milk/not enough breast milk

Formula was easier to prepare Other: 2. How many times per day are you pumping your breast milk? 3. Are you having any difficulty pumping? Yes No a) If yes, would you like to receive a call from the lactation consultant? Yes No * WHO Growth Charts

Subject Identifier:

163

5. How is your baby feeding? po nasogastric tube po + nasogastric tube 6. How many milk* feedings does your baby have each day? 7. If baby is feeding any po:

a) What is the minimum amount your baby will drink at a feeding (ml):

b) What is the maximum amount your baby will drink at a feeding (ml): 8. If baby is receiving any feed by NG:

a) What is the average amount that goes through the NG tube (ml): 9. Is your baby having any episodes of vomiting or spitting up? Yes No 10. If yes, how often does this happen? Daily Average number/day: Weekly Average number/week: Never 11. Is your baby taking any solid foods? Yes No a) If yes, how many times/day? 1 x/day 2 x/day 3 x/day b) cereals fruits/vegetables meats other 12. Do you give your baby any juice? No Yes Describe (volume/frequency/diluted?): 13. Do you give your baby any water? No Yes Describe (volume/frequency): 14. In this past week, have you given your baby anything to drink that has not already been discussed? Yes No

Subject Identifier:

164

a) If yes, please describe (what/how much)?

15. Do you give your baby any vitamins or other supplements? No Yes Describe (what/frequency):

1. Change to feeding routine? Yes No a) If yes, describe: 2. Date and time of scheduled pick-up/delivery of breast milk: Visit 1: Visit 2:

Plan

Subject Identifier:

165

Study Visit #1 Control Group

* Questions regarding feeding pertain to feeding over the previous week Date of Visit: Date of Discharge: Discharge Weight: (mm/dd/yy) (mm/dd/yy) (mm/dd/yy) Weight (grams) [1] [2] [Average]

Weight gain (g/day): Percentile*: Length (cm) [1] [2] [Average]

Percentile*: Head Circumference (cm) [1] [2] [Average]

Percentile*: 1. Food Records Complete? Yes No 2. Food records reviewed: Name: Date: (mm/dd/yy)

4. How is your baby feeding? po nasogastric tube po + nasogastric tube 5. How many feedings does your baby have each day? 6. If baby is feeding any po:

a) What is the minimum amount your baby will drink at a feeding (ml):

* WHO Growth Charts

Subject Identifier:

166

b) What is the maximum amount your baby will drink at a feeding (ml):

7. If baby is receiving any feed by NG:

a) What is the average amount that goes through the NG tube (ml): 8. Is your baby having any episodes of vomiting or spitting up? Yes No 9. If yes, how often does this happen? Daily Average number/day: Weekly Average number/week: Never 10. Is your baby taking any solid foods? Yes No a) If yes, how many times/day? 1 x/day 2 x/day 3 x/day b) cereals fruits/vegetables meats other 11. Do you give your baby any juice? No Yes Describe (volume/frequency/diluted?): 12. Do you give your baby any water? No Yes Describe (volume/frequency): 13. In this past week, have you given your baby anything to drink that has not already been discussed? Yes No

a) If yes, please describe (what/how much)? 17. Do you give your baby any vitamins or other supplements? No Yes Describe (what/frequency):

Subject Identifier:

167

1. Change to feeding routine? Yes No a) If yes, describe:

Subject Identifier:

Plan

168

Phone Documentation Control Group Date of Call: * Questions regarding feeding pertain to feeding over (mm/dd/yy) the previous week Weight (grams) [Previous Weight] [Current Weight] [Weight Gain]

Percentile*: 1. What is the recipe 2. How is your baby feeding? po nasogastric tube po + nasogastric tube 3. How many feedings does your baby have each day? 4. If baby is feeding any po:

a) What is the minimum amount your baby will drink at a feeding (ml):

b) What is the maximum amount your baby will drink at a feeding (ml):

5. If baby is receiving any feed by NG:

a) What is the average amount that goes through the NG tube (ml): 6. Is your baby having any episodes of vomiting or spitting up? Yes No 7. If yes, how often does this happen? Daily Average number/day: Weekly Average number/week: Never 8. Is your baby taking any solid foods? Yes No a) If yes, how many times/day? 1 x/day 2 x/day 3 x/day * WHO Growth Charts

Subject Identifier:

169

b) cereals fruits/vegetables meats other 9. Do you give your baby any juice? No Yes Describe (volume/frequency/diluted?): 10. Do you give your baby any water? No Yes Describe (volume/frequency): 11. In this past week, have you given your baby anything to drink that has not already been discussed? Yes No

a) If yes, please describe (what/how much)? 12. Do you give your baby any vitamins or other supplements? No Yes Describe (what/frequency):

1. Change to feeding routine? Yes No a) If yes, describe:

Plan

Subject Identifier:

170

APPENDIX  D: FOOD  RECORD    TREATMENT  GROUP  

Three-Day Diet Record

The effectiveness of low-fat breast milk for the treatment of chylothorax in infants following cardiothoracic surgery.

You have been given this booklet to help in collecting information on your

the Cardiac Clinic.

171

Instructions - Treatment

· include the day you are coming for your appointment. Fill out a separate sheet for each of the 3 days

· You will be provided with a small scale to help you accurately measure the amount you feed your baby; do not use the measurements directly from the bottle

· Be sure to weigh the bottle on the scale before you start feeding your baby by mouth and then again immediately after your baby is finished

· Use this table to help you remember how to fill in each column

Column Heading Instructions Time · the time you start feeding your baby Feeding · what you fed your baby (either fortified low fat breast milk or

MCT formula) · check the correct box

Weight of Bottle (before feeding)

· the weight of the bottle before you start feeding your baby · use the scale provided to take this measurement

Weight of Bottle (after feeding)

· the weight of the bottle after you have finished feeding your baby by mouth

· use the scale provided to take this measurement NG Tube Volume · the volume of feeding that you put through the NG tube

· complete this column

Total · · this will be done by the study coordinator

Spit Up/Vomit · if your baby spits up a small amount of their feeding, check

· if your baby has a larger volume that is forcefully projected

· choose either spit-up or vomit but not both each time this happens

Subject Identifier:

172

Diet Record Treatment

Date: Feed/Concentration: (mm/dd/yy)

 Feed  

Time  Started  (a.m.  or  p.m.)  

 Feeding  ( )  

Weight  of  Bottle  

(before  feeding)  (grams)  

Weight  of  Bottle  

(after  feeding)  (grams)  

NG  Tube  Volume  (ml)  

Total  (to  be  

completed  by  study  

coordinator)  

Spit  Up/  Vomit  

 1  

Fortified LFBM Formula Only

Spit Up Vomit

 2  

Fortified LFBM Formula Only

Spit Up Vomit

 3  

Fortified LFBM Formula Only

Spit Up Vomit

 4  

Fortified LFBM Formula Only

Spit Up Vomit

 5  

Fortified LFBM Formula Only

Spit Up Vomit

 6  

Fortified LFBM Formula Only

Spit Up Vomit

 7  

Fortified LFBM Formula Only

Spit Up Vomit

 8  

Fortified LFBM Formula Only

Spit Up Vomit

 9  

Fortified LFBM Formula Only

Spit Up Vomit

 10  

Fortified LFBM Formula Only

Spit Up Vomit

 Total  

Subject Identifier:

173

Introducing  Solid  Foods  

· Transition to complementary foods at approximately 6 months of age · Continue to provide an adequate volume of fortified low fat breast milk/formula feeds as

instructed by the study coordinator · There will be some solid foods that your baby cannot have while being treated for

chylothorax; follow the guidelines provided

· If your baby is also eating solid foods, days before you come to the hospital

· Do not include the day you are coming for your appointment · Fill out a separate sheet for each of the 3 days · Also use this form to record anything your baby drinks that is not breast milk or formula · Use this table to help you Column Heading Instructions

Time · the time of day you fed your baby solids Type of Food · what you fed your baby e.g. strained peaches

· include anything you may have added during the preparation of the food e.g. rice cereal mixed with low fat breast milk

Brand · if you use commercially made foods, indicate which brand you

Amount · how much your baby ate/drank

If you have any questions about completing these forms, please call the study coordinator Sarah Farmer (416)-813-6747

Subject Identifier:

174

175

Other Foods

Date: (mm/dd/yy) Time   Type  of  Food/Description   Brand  

(or  homemade)  Amount/Volume  

 8:00  AM  

Infant  rice  cereal  mixed  with  MCT  Formula    Peaches    pureed  

Nestle    

Heinz  

3  tablespoons    

2  tablespoons  

Subject Identifier:

176

APPENDIX  E: FOOD  RECORD    CONTROL  GROUP  

Three-Day Diet Record

The effectiveness of low-fat breast milk for the treatment of chylothorax in infants following cardiothoracic surgery.

You have been given this booklet to help in collecting information on your

the Cardiac Clinic.

Subject  Identifier:

177

Instructions - Control

·

include the day you are coming for your appointment. Fill out a separate sheet for each of the 3 days

· You will be provided with a small scale to help you accurately measure the amount you feed your baby; do not use the measurements directly from the bottle

· Be sure to weigh the bottle on the scale before you start feeding your baby by mouth and then again immediately after your baby is finished

· Use this table to help you remember how to fill in each column

Column Heading Instructions Time · the time you start feeding your baby Weight of Bottle (before feeding)

· the weight of the bottle before you start feeding your baby · use the scale provided to take this measurement

Weight of Bottle (after feeding)

· the weight of the bottle after you have finished feeding your baby by mouth

· use the scale provided to take this measurement NG Tube Volume · the volume of feeding that you put through the NG tube

· complete this column

Total · · this will be done by the study coordinator

Spit Up/Vomit · if your baby spits up a small amount of formula, check the

· if your baby has a larger volume that is forcefully projected

· choose either spit-up or vomit but not both each time this happens

Subject Identifier:

178

Diet Record - Control

Date: Feed/Concentration: (mm/dd/yy)

 Feed  

Time  Started  (a.m.  or  p.m.)  

Weight  of  Bottle  

(before  feeding)  (grams)  

Weight  of  Bottle  

(after  feeding)  (grams)  

NG  Tube  Volume  (ml)  

Total  (to  be  completed  by  study  coordinator)  

Spit  Up/  Vomit  

 1  

Spit Up Vomit

 2  

Spit Up Vomit

 3  

Spit Up Vomit

 4  

Spit Up Vomit

 5  

Spit Up Vomit

 6  

Spit Up Vomit

 7  

Spit Up Vomit

 8  

Spit Up Vomit

 9  

Spit Up Vomit

 10  

Spit Up Vomit

 11  

Spit Up Vomit

 Total  

Subject Identifier:

Subject Identifier:

Subject Identifier:

179

Introducing  Solid  Foods  

· Transition to complementary foods at approximately 6 months of age · Continue to provide an adequate volume of formula feeds as instructed by the study

coordinator · There will be some solid foods that your baby cannot have while being treated for

chylothorax; follow the guidelines provided

· of the 3 days before you come to the hospital

· Do not include the day you are coming for your appointment · Fill out a separate sheet for each of the 3 days · Also use this form to record anything your baby drinks that is not formula (measure this

using the scale provided just like you would for the formula feedings) · Use this table to help you Column Heading Instructions

Time · the time of day you fed your baby solids Type of Food · what you fed your baby e.g. strained peaches

· include anything you may have added during the preparation of the food e.g. rice cereal mixed with MCT formula

Brand · if you use commercially made foods, indicate which brand you

Amount · how much your baby ate /drank

If you have any questions about completing these forms, please call the study coordinator Sarah Farmer (416)-813-6747

Subject Identifier:

180

Other Foods

Date: (mm/dd/yy) Time   Type  of  Food/Description   Brand  

(or  homemade)  Amount/Volume  

 8:00  AM  

Infant  rice  cereal  mixed  with  MCT  Formula    Peaches    pureed  

Nestle    

Heinz  

3  tablespoons    

2  tablespoons  

Subject Identifier:

181

APPENDIX  F:  MINIMAL  FAT  DIET  GUIDELINES    

M I N I M A L F A T D I E T F O O D G R O UP F O O DS A L L O W E D F O O DS T O A V O ID

Milk & Alternatives

Dairy products (milk, yogurt) with less than 1% milk fat (M.F. or B.F. less than 1%)

Fat-free processed cheese slices Fat-free sour cream Fat-free cottage cheese Fat-free cream cheese Fat-free pudding Nestle Breakfast Anytime®

Powder (Strawberry & Vanilla only prepare with skim milk)

Dairy products (milk, yogurt) with greater than 1% milk fat (M.F. or B.F. greater than 1%)

Cheese (including low fat varieties) Regular or low fat cream cheese Regular or low fat cottage cheese Cheez whiz® Nestle Breakfast Anytime Powder

(chocolate flavour) or any ready to drink versions

Meat & Alternatives

Lean white fish, skinless chicken breast or turkey breast (1-2 oz/serving/day)

Beans & legumes Fat-free hot dogs Tuna in water (2-3 oz/serving/day) Fat-free deli meats Egg whites (if older than 1 year)

Peanut butter & other nut butters Fried & battered meats & poultry Egg yolks Beef, pork & fatty fish Seafood (shrimps, scallops, lobster)

& shellfish (clams, mussels, oysters)

Grain Products

Bread, buns, plain bagels & pitas Plain pasta Plain rice Melba toast, fat-free crackers Crumpets Cereals (except granola or those

containing nuts). Choose one with less than 1 g total fat/serving

Cheese &/or egg bread, buns, bagels & croissants

Meat or cheese stuffed pasta Egg noodles or fried rice Packaged noodle dishes (e.g. Side

Kicks , Mr. Noodle ) Crackers Granola

182

Vegetables & Fruit

All, E X C EPT avocado & olives Fruit & vegetable juices Apple & fruit sauces Dried fruit bars/fruit snacks (e.g.

Fruit-to-go & Fruit Roll-Ups ) Pasta sauce with less than 1 g total

fat/serving

Avocado Olives Pasta sauce with greater than1 g

total fat/serving (no meat, cheese or cream sauce)

Snacks & Treats

Rice cakes (no chocolate, cheese or butter read ingredients)

Fat-free granola bars Fat-free cookies Freezies & sorbet Air-popped popcorn (no butter) Baked plain pretzels Cereal (see above) Chocolate, strawberry & maple

syrup (fat-free) Ice cream & frozen yogurt (if less

than 0.1% fat) Gummy candy & gum Jell-O® & fruit gels

Chips & cheezies Chocolate Nuts & seeds Muffins & Cake Cookies Regular or flavoured popcorn Ice cream & frozen yogurt (if

greater than 0.1% fat) Toffee Caramel Fudge

Oils, Spreads & Other

Ketchup, mustard, relish Fat-free broth soups Salsa Fat free mayonnaise Jams & jellies Honey (if older than 1 year) Ultra low fat Cool Whip® (0.9%

fat) Pickles Fat-free salad dressing Fat-free croutons Soy sauce Teriyaki sauce Vegetarían sushi (no avocado, egg

or crab meat)

Butter Margarine Oil Regular mayonnaise Soup & cream soups Sushi with meat, fish, avocado, egg

or crab meat

183

Baby & Toddler

Portagen® (or Tolerex®) formula Plain infant cereal (with less than

0.5 g fat per serving) All fruits & vegetables (plain

homemade or jarred) Fruit & vegetable juices (limit to 4

oz/day or as per dietitian) Baby mum-mum rice rusks Homemade pureed or minced lean

white fish, skinless chicken breast or turkey breast (½-1 oz/serving/day)

All infant formula (except Portagen® or Tolerex®)

Minigo®, Danino® & Danimals® yogurt & cheese

Yogurt tubes & drinks with >1% M.F.

Infant cereal containing formula, milk &/or yoghurt (e.g. Milupa®)

Jarred baby

Cookies (including Arrowroot & Farleys®)

Baby jars of meat & meat containing foods

Please Note:

A lways read food labels: Fat-free = less than 0.5 g total fat per serving When cooking, do not add any oil, butter, margarine or other fats. Use a low fat cooking

method such as steaming, broiling, barbecuing or baking

If you have questions about a product, please phone your dietitian

184

APPENDIX  G:  CHEST  TUBE  DRAINAGE  DATA  FOR  ALL  SUBJECTS   Subject

# Study G roup

(T reatment/Control) Study Day

Chest Tube 1

(ml)

Chest Tube 1 (ml/kg)

Chest Tube 2

(ml)

Chest Tube 2 (ml/kg)

Duration of

Drainage (days)

1 Control 1 23 3.5 5 1 Control 2 22.2 3.3 1 Control 3 22.8 3.4 1 Control 4 13.6 2.0 1 Control 5 0 0 2 Control 1 63 12.1 48.8 9.4 31 2 Control 2 37.5 7.4 44.2 8.7 2 Control 3 14 2.7 16.5 3.2 2 Control 4 16.9 3.3 34 6.7 2 Control 5 11 2.1 19.5 5.2 2 Control 6 15.1 2.9 34.8 4.5 2 Control 7 14 2.8 26.2 4.2 2 Control 8 10.7 2.1 22.8 3.7 2 Control 9 9.4 1.9 21.1 4.2 2 Control 10 3.1 0.6 18.8 3.7 2 Control 11 2.2 0.4 10.4 2.0 2 Control 12 0.5 0.1 13.4 2.6 2 Control 13 11.0 2.2 2 Control 14 9.0 1.8 2 Control 15 11.1 2.3 2 Control 16 4.7 1.0 2 Control 17 4.6 0.9 2 Control 18 2.0 0.4 2 Control 18 15 3.0 2 Control 20 5.5 1.1 2 Control 21 11.5 2.3 2 Control 22 9.5 1.9 2 Control 23 6.0 1.2 2 Control 24 13.4 2.6 2 Control 25 10 2.0 2 Control 26 1.2 0.2 2 Control 27 4.2 0.8 2 Control 28 2.2 0.4 2 Control 29 1.8 0.3 2 Control 30 0 0 2 Control 31 0.5 0.1 3 Treatment 1 45 6.2 5 3 Treatment 2 4 0.5

185

3 Treatment 3 6 0.8 3 Treatment 4 2 0.3 3 Treatment 5 2 0.3 4 Control 1 5 0.7 40 5.6 7 4 Control 2 110 15.5 30 4.2 4 Control 3 9.6 1.4 37 5.2 4 Control 4 71 10.1 10 1.4 4 Control 5 34 5.0 2 0.3 4 Control 6 11.6 1.7 0 0 4 Control 7 0 0 0 0 5 Treatment 1 31 5.1 17 2.8 12 5 Treatment 2 17 2.8 19 3.1 5 Treatment 3 16.5 2.6 22.8 3.6 5 Treatment 4 15.6 2.5 17 2.7 5 Treatment 5 21.1 3.3 26.3 4.2 5 Treatment 6 12.7 2.0 5.7 0.9 5 Treatment 7 12 2.0 2.2 0.4 5 Treatment 8 16.6 2.7 10.5 1.7 5 Treatment 9 7 1.1 40.5 6.4 5 Treatment 10 22 3.5 12 1.9 5 Treatment 11 7 1.1 8.9 1.4 5 Treatment 12 1 0.2 4 0.6 6 Treatment 1 60 17.1 9 6 Treatment 2 49 14.0 6 Treatment 3 116.5 33.3 6 Treatment 4 90.5 25.9 6 Treatment 5 53 15.1 6 Treatment 6 31 8.9 6 Treatment 7 19 5.4 6 Treatment 8 20 5.7 6 Treatment 9 12 3.5 7 Treatment 1 42 13.5 40 12.9 8 7 Treatment 2 50 16.1 24 7.7 7 Treatment 3 23 7.4 28 9.0 7 Treatment 4 17 5.6 15 4.9 7 Treatment 5 8 2.5 14 4.5 7 Treatment 6 3.9 1.2 10.4 3.3 7 Treatment 7 5.8 1.9 6.5 2.1 7 Treatment 8 3 1.0 0.6 0.2 8 Treatment 1 21.4 7.9 7 8 Treatment 2 18.1 6.7 8 Treatment 3 21.8 8.2 8 Treatment 4 12.9 4.8 8 Treatment 5 10.6 3.9 8 Treatment 6 12.5 4.5

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8 Treatment 7 4.1 1.5 9 Control 1 10.5 1.9 6.5 1.2 4 9 Control 2 6.6 1.2 7.7 1.4 9 Control 3 12.2 2.1 8.4 1.4 9 Control 4 3 0.5 8.2 1.4 9 Control 5 0 0 0 0 10 Treatment 1 18.6 2.7 5 10 Treatment 2 23.5 3.5 10 Treatment 3 16.6 2.4 10 Treatment 4 3.8 0.6 10 Treatment 5 4 0.6 11 Treatment 1 47 14.7 3 0.9 4 11 Treatment 2 5 1.6 2 0.6 11 Treatment 3 13 4.1 2 0.6 11 Treatment 4 11 3.4 1 0.3 11 Treatment 5 0 0 0 0 12 Treatment 1 90 22.9 10 12 Treatment 2 53.2 13.9 12 Treatment 3 42.3 11.1 12 Treatment 4 44 11.4 12 Treatment 5 20.4 5.4 12 Treatment 6 22.9 6.0 12 Treatment 7 16 4.2 12 Treatment 8 9.8 2.6 12 Treatment 9 9 2.4 12 Treatment 10 0.2 0.05 13 Control 1 22 4.6 24 5 4 13 Control 2 27 6.1 15 3.4 13 Control 3 9.2 2.1 7 1.6 13 Control 4 3.2 0.7 2.5 0.6 13 Control 5 0 0 0 0 14 Control 1 36 6.1 29 14 Control 2 221 37 14 Control 3 155 26 14 Control 4 142 24 14 Control 5 146 25 14 Control 6 115 20 14 Control 7 58 10 14 Control 8 123 21 14 Control 9 144 25 14 Control 10 111 19 14 Control 11 54 10 14 Control 12 47.5 8.5 14 Control 13 41 7.3 14 Control 14 19.4 3.5

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14 Control 15 5.8 1.0 14 Control 16 47.1 8.1 14 Control 17 24 4.1 14 Control 18 40 6.9 14 Control 19 21.4 4 14 Control 20 13 2.2 14 Control 21 8 1.3 14 Control 22 4.8 0.8 14 Control 23 1.7 0.3 14 Control 24 1.5 0.2 14 Control 25 0.2 0 14 Control 26 0 0 14 Control 27 0.1 0 14 Control 28 0 0 14 Control 29 0 0 15 Control 1 280 38.4 5 0.7 16 15 Control 2 101 13.8 0 0 15 Control 3 44 6.0 0 0 15 Control 4 76 11 0 0 15 Control 5 38 5.7 0 0 15 Control 6 39 5.8 0 0 15 Control 7 31 4.6 0 0 15 Control 8 16 2.3 0 0 15 Control 9 12 1.8 240 35.6 15 Control 10 68 10.2 100 15 15 Control 11 60.5 9 16 2.4 15 Control 12 31 4.7 40 6.1 15 Control 13 8.7 1.4 28 4.4 15 Control 14 9.2 1.5 7 1 15 Control 15 1.4 0.2 35 5.7 15 Control 16 0 0 0 0 16 Control 1 180 35.3 5 1.0 4 16 Control 2 89 18.7 10 2.1 16 Control 3 27 6.0 6 1.3 16 Control 4 8.8 2.0 0 0 16 Control 5 0 0 0 0