the impact of folic acid fortification on the ......yaseer abdul shakur doctor of philosophy...

THE IMPACT OF FOLIC ACID FORTIFICATION ON THE FOLATE INTAKE OF

CANADIANS, FACTORS ASSOCIATED WITH SUB-OPTIMAL BLOOD FOLATE

STATUS AMONG WOMEN, AND THE EFFECT OF VITAMIN/MINERAL

SUPPLEMENT USE

by

Yaseer Abdul Shakur

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Graduate Department of Nutritional Sciences

University of Toronto

© Copyright by Yaseer Abdul Shakur 2011

ii

THE IMPACT OF FOLIC ACID FORTIFICATION ON THE FOLATE INTAKE OF

CANADIANS, FACTORS ASSOCIATED WITH SUB-OPTIMAL BLOOD FOLATE STATUS

AMONG WOMEN, AND THE EFFECT OF VITAMIN/MINERAL SUPPLEMENT USE

Yaseer Abdul Shakur

Doctor of Philosophy

Graduate Department of Nutritional Sciences

University of Toronto

2011

ABSTRACT

Food fortification and nutrient supplementation are important strategies to address

micronutrient deficiencies. Mandatory folic acid fortification was implemented in Canada

and the U.S. in 1998 to reduce the incidence of neural tube defects (NTD). However, the

actual amount of folic acid added to foods has not been reported in Canada. We analyzed 95

fortified foods and found that there is 50% more folate in foods than that reported in food

composition tables, which are primarily based on minimum mandated fortification levels.

We then determined if these observed folate overages impacted the prevalence of dietary

folate inadequacy or intakes above the Tolerable Upper Intake Level (UL). Using data from

the 2004 nationally-representative Canadian Community Health Survey (CCHS) 2.2 (n =

35,107), adjusted for folate overages, we found a low prevalence of folate inadequacy in

Canada post-fortification. However, few women 14-50y consumed 400µg/d of synthetic

folic acid, an amount associated with maximal protection against an NTD. Conversely, we

also showed that use of folic acid-containing supplements led to intakes >UL in the general

population.

To develop a tool that would help clinicians identify women with red blood cell (RBC)

folate concentrations that were not maximally protective against an NTD (<906nmol/L), we

iii

used data from the nationally-representative U.S. National Health and Nutrition Examination

Survey 2003-2006 to define risk factors of RBC folate <906nmol/L. We found that 35% of

American women 19-45y had RBC folate <906nmol/L. Younger age, low dietary folate

intake, not consuming supplemental folic acid, smoking, and being African-American were

associated with increased risk of RBC folate <906nmol/L.

Given our observations of a low prevalence of folate inadequacy and folic acid

supplement use leading to intakes >UL, we used CCHS 2.2 data to compare the diets of

supplement users and non-users in terms of inadequacy and intakes >UL for other nutrients.

We showed that the prevalence of inadequacy was low for most nutrients, and from diet

alone, supplement users were not at increased risk of inadequacy compared to non-users.

Furthermore, inclusion of supplements led to intakes >UL above 10% for vitamins A, C,

niacin, folic acid, and iron, zinc and magnesium.

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To my mother, father, wife, son, sisters, and grandparents

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ACKNOWLEDGEMENTS

I would like to praise and thank God Almighty for the countless blessings that I am

constantly immersed in, and to send peace and blessings upon the holy Prophet, for the effort I

put forth in this work is merely an attempt to follow his most excellent example of servitude to

humanity.

I would like to extend my deepest gratitude to my academic mother, Dr. Deborah O‟Connor,

whom I regard as the best thing to happen to my academic career. The countless hours she put

into training and mentoring me have not gone unnoticed. As I leave the “nest”, I hope to take

with me some of the integrity and academic, professional and personal skills that I have

witnessed over the past few years. I also hope that her scientific rigour and attention to detail

(which, at times, drove me to the limits of sanity) have rubbed off on me.

I thank immensely my thesis advisory committee members: Drs. Paul Corey, Anthony

Hanley, and Valerie Tarasuk, for their direction, guidance, and input over the past few years.

Each brought his/her unique expertise to the committee, undoubtedly improving the quality of

work in this thesis. I have benefitted from each one of their unique backgrounds; Dr. Corey‟s

encyclopedic statistical knowledge, Dr. Hanley‟s extraordinary degree of professionalism, and

Dr. Tarasuk‟s passion for her work. I also thank two previous mentors, Drs. Stanley Zlotkin and

Paul Pencharz, for the positive influences they have had on my academic career.

I would also like to thank members of the O‟Connor lab, Joan Brennan-Donnan (who also

graciously doubles as my lactation and infant feeding consultant), Dr. Susanne Aufreiter, Brenda

Hartman, Dubraiicka Pichardo, Alanna Lakoff, Sarah Kocel and Yen Ming Chan for their

support over the years. I especially thank Aneta Plaga for all of her hard work behind-the-

scenes. I also thank Waqas Khan for his friendship over these few years.

My deepest gratitude extends to all of my family, in-laws, friends and teachers for their

support and motivation over the past few years. I especially thank my mother, Lilatool Shakur,

and my father, Abdul Jaleel Shakur, for their endless love and support. They have, without

exaggeration, sacrificed their own personal gains in order to provide avenues for my sisters and I

to pursue our aspirations in all fronts of life. They both continue to be my motivation in all that I

do and I hope that I can always make them proud. I also thank my three sisters, Shazeeda,

Nazeera, and Nafeesa, for their support throughout my academic career. I am also grateful to my

wife, Saloua El-Haddad, for her love and support throughout all of my endeavours. Last but not

least, I must thank my special little guy, Abdullah, whose earlier than expected arrival pushed me

to work even harder to finish on time.

Personal funding was provided by the Sick Kids Hospital Research Training Competition

Award (RESTRACOMP), the Danone Institute of Canada‟s Doctoral Student Award, and the

Canadian Institutes of Health Research Banting and Best Doctoral Student Award.

I am confident that the skills acquired throughout my doctoral training, along with the

positive influences of aforementioned individuals, have positioned me well as I take on the next

big challenges in life.

vi

Table of Contents

Abstract .......................................................................................................................................... ii

Dedication ..................................................................................................................................... iv

Acknowledgements ........................................................................................................................v

Table of Contents ......................................................................................................................... vi

List of Tables ................................................................................................................................ xi

List of Figures ............................................................................................................................. xiii

List of Appendices ...................................................................................................................... xiv

Published Material .......................................................................................................................xv

List of Abbreviations ................................................................................................................. xvi

Chapter 1.0 INTRODUCTION/ RATIONALE/OBJECTIVES/HYPOTHESES ...................1

1.1 INTRODUCTION & RATIONALE ..................................................................................1

1.2 OBJECTIVES & HYPOTHESES ......................................................................................6

Chapter 2.0 REVIEW OF THE LITERATURE ........................................................................9

2.1 FOOD FORTIFICATION, THE CASE OF FOLATE & NUTRIENT

SUPPLEMENTATION .............................................................................................................9

2.1.1 Food fortification ...........................................................................................................9

2.1.2 The case of folate ...........................................................................................................9

2.1.3 Nutrient supplementation ...........................................................................................10

2.2 INTRODUCTION TO FOLATE ......................................................................................11

2.2.1 Description ...................................................................................................................11

2.2.2 Functions ......................................................................................................................12

2.2.3 Sources and Bioavailability ........................................................................................12

2.2.4 Absorption ....................................................................................................................13

vii

2.2.5 Requirements ...............................................................................................................14

2.2.6 Inadequate intake and deficiency...............................................................................15

2.2.7 Excessive intake ...........................................................................................................16

2.3 FOLIC ACID FORTIFICATION ....................................................................................19

2.3.1 History of the association between folate and NTDs ................................................19

2.3.2 Rationale for folic acid fortification of food ..............................................................20

2.3.2.1 The United States‟ folic acid fortification program ............................................21

2.3.2.2 The Canadian folic acid fortification program ....................................................21

2.3.2.3 Mandatory folic acid fortification elsewhere ......................................................22

2.3.2.4 The effect of folic acid fortification on the incidence of NTDs .........................23

2.4 POST-FORTIFICATION OF THE FOOD SUPPLY ....................................................31

2.4.1 Folate intake post-fortification of the supply ............................................................31

2.4.1.1 Sources of folate in the diet post-fortification ....................................................31

2.4.1.2 Reports from the United States ...........................................................................32

2.4.1.3 Reports from Canada ..........................................................................................35

2.4.2 Blood folate status post-fortification of the food supply ..........................................37

2.4.2.1 Reports from the United States ...........................................................................37

2.4.2.2 Reports from Canada ..........................................................................................44

2.4.3 Mandated versus actual levels of fortification ..........................................................46

2.5 RBC FOLATE AS AN INDICATOR OF FOLATE STATUS AND NTDs ..................48

2.5.1 RBC folate: an indicator of long-term folate status .................................................48

2.5.2 The need to identify women at risk for an NTD .......................................................49

2.5.3 Factors associated with RBC folate concentrations .................................................51

viii

2.5.3.1 Diet – fruit & vegetable intake & white wheat flour consumption.....................51

2.5.3.2 Dieting.................................................................................................................51

2.5.3.3 Cooking methods ................................................................................................52

2.5.3.4 Smoking ..............................................................................................................52

2.5.3.5 Personal/family history of NTDs ........................................................................53

2.5.3.6 Medications that interfere with folate metabolism .............................................54

2.5.3.7 Alcohol abuse......................................................................................................54

2.5.3.8 Malabsorption/gastric bypass surgery .................................................................55

2.5.3.9 Liver disease/kidney dialysis ..............................................................................55

2.5.3.10 Diabetes.............................................................................................................56

2.5.3.11 Summary ...........................................................................................................57

2.6 VITAMIN/MINERAL SUPPLEMENT CONSUMPTION ...........................................57

2.6.1 The role of supplements ..............................................................................................57

2.6.2 Prevalence and determinants of consumption ..........................................................58

2.6.2.1 The United States ................................................................................................58

2.6.2.2 Canada.................................................................................................................63

2.6.3 Difference between supplement users and non-users and contribution to total

dietary intake ........................................................................................................................63

2.6.3.1 The United States ................................................................................................63

2.6.3.2 Canada.................................................................................................................65

Chapter 3.0 THESIS STUDY #1: HOW MUCH FOLATE IS IN CANADIAN ENRICHED

PRODUCTS 10 YEARS AFTER MANDATED FORTIFICATION? ...................................66

3.1 ABSTRACT ........................................................................................................................67

ix

3.2 INTRODUCTION ..............................................................................................................68

3.3 METHODS .........................................................................................................................69

3.4 RESULTS ............................................................................................................................71

3.5 DISCUSSION .....................................................................................................................72

Chapter 4.0 THESIS STUDY #2: INVESTIGATING THE IMPACT OF FOLIC ACID

FORTIFICATION OVER MANDATED LEVELS ON THE PREVALENCE OF FOLATE

INADEQUACY AND INTAKES ABOVE THE TOLERABLE UPPER LEVEL OF

INTAKE AMONG CANADIANS ..............................................................................................78

4.1 ABSTRACT ........................................................................................................................79

4.2 INTRODUCTION ..............................................................................................................80

4.3 SUBJECTS AND METHODS ...........................................................................................81

4.4 RESULTS ............................................................................................................................86

4.5 DISCUSSION .....................................................................................................................88

Chapter 5.0 THESIS STUDY #3: FACTORS ASSOCIATED WITH MAXIMALLY

PROTECTIVE RBC FOLATE CONCENTRATIONS IN WOMEN 19-45 Y: NATIONAL

HEALTH AND EXAMINATION SURVEY 2003-2006 ........................................................103

5.1 ABSTRACT ......................................................................................................................104

5.2 INTRODUCTION ............................................................................................................105

5.3 SUBJECTS AND METHODS .........................................................................................106

5.4 RESULTS ..........................................................................................................................110

5.5 DISCUSSION ...................................................................................................................112

Chapter 6.0 THESIS STUDY #4: VITAMIN AND MINERAL SUPPLEMENT

CONSUMPTION IN CANADA: DO USERS DIFFER FROM NON-USERS IN TERMS

OF NUTRIENT INADEQUACY AND RISK OF HIGH INTAKES? .................................127

6.1 ABSTRACT ......................................................................................................................128

6.2 INTRODUCTION ............................................................................................................129

x

6.3 SUBJECTS AND METHODS .........................................................................................130

6.4 RESULTS ..........................................................................................................................135

6.5 DISCUSSION ...................................................................................................................137

Chapter 7.0 DISCUSSION, CONCLUSIONS AND FUTURE DIRECTIONS ...................155

7.1 DISCUSSION & CONCLUSIONS .................................................................................155

7.2 FUTURE DIRECTIONS .................................................................................................162

Chapter 8.0 LITERATURE CITED ........................................................................................165

Chapter 9.0 APPENDIX ............................................................................................................202

Appendix A. Nutritional Guidelines for a Healthy Pregnancy – Health Canada ............202

xi

List of Tables

Table 2.2.4. Estimated Average Requirement and Tolerable Upper Intake Level for folate

by life stage group ..............................................................................................................14

Table 2.3.2.4.a. Change in the occurrence of neural tube defects pre-to-post-

fortification in Canada ......................................................................................................24

Table 2.3.2.4.b. Change in the occurrence of neural tube defects pre-to-post-

fortification in the United States ........................................................................................26

Table 2.3.2.4.c. Change in the occurrence of neural tube defects pre-to-post-

fortification outside North America ...................................................................................29

Table 2.4.2.1.a. Change in serum folate (nmol/L) concentrations in the United States

from pre-to-post-fortification based on NHANES analyses ..............................................40

Table 2.4.2.1.b. Change in red blood cell folate (nmol/L) concentrations in the United

States from pre-to-post-fortification based on NHANES analyses ...................................42

Table 2.6.2.1. Prevalence of dietary supplement consumption in the United States ........60

Table 3.4.a. Comparison of the Analyzed Food Folate Content to Values Reported in

the Canadian Nutrient File .................................................................................................76

Table 3.4.b. Comparison of the Analyzed Food Folate Content to Values Reported on

the Foods Labels ................................................................................................................77

Table 4.4.a. Prevalence of folic acid-containing supplement use and usual dietary

folate intakes ......................................................................................................................94

Table 4.4.b. Percent of individuals with folic acid intakes above the Tolerable Upper

Intake Level .......................................................................................................................96

xii

Table 4.4.c. Estimation of the prevalence of women consuming less than 400 µg/d

folic acid and above the Tolerable Upper Intake Level from all sources (dietary and

supplemental) at different potential supplemental folic acid dosages ...............................98

Table 5.4.a. Mean red blood cell folate concentrations (nmol/L) and percent of

women with red blood cell folate <906 nmol/L categorized by analyzed variables .......118

Table 5.4.b. Univariate analyses of factors associated with red blood cell folate <906

nmol/L ..............................................................................................................................122

Table 5.4.c. Multivariate analyses of factors associated with red blood cell folate

<906 nmol/L ....................................................................................................................126

Table 6.4.a. Prevalence of vitamin/mineral supplement consumption by sex/age

groups ...............................................................................................................................143

Table 6.4.b. Prevalence of inadequacy for selected vitamins among supplement users

and non-users. ..................................................................................................................145

Table 6.4.c. Prevalence of inadequacy for selected minerals among supplement users

and non-users ...................................................................................................................148

Table 6.4.d. Percent of intakes above the Tolerable Upper Intake Level for selected

vitamins among supplement users and non-users ............................................................151

Table 6.4.e. Percent of intakes above the Tolerable Upper Intake Level for selected

minerals among supplement users and non-users ............................................................153

xiii

List of Figures

Figure 2.2.1. Folic acid ....................................................................................................11

Figure 4.4.a. The prevalence of folate inadequacy in children and adults ....................100

Figure 4.4.b. Prevalence of folate inadequacy in adults stratified by alcohol

consumption, diabetes, smoking and obesity ...................................................................102

xiv

List of Appendices

Appendix A. Nutritional Guidelines for a Healthy Pregnancy – Health Canada ..........202

xv

PUBLISHED MATERIAL

Chapter 3 has been previously published:

Shakur, Y.A, Rogenstein, C., Hartman-Craven, B., Tarasuk, V. & O‟Connor, D.L. How much

folate is in Canadian enriched products following mandated fortification? Canadian Journal of

Public Health 2009; 100(4):281-284.

- Reprinted with permission of the Canadian Public Health Association. Originally

published in the Canadian Journal of Public Health.


Shakur, Y.A., Garriguet, D., Corey, P. & O‟Connor, D.L. Folic acid fortification over mandated

levels results in a low prevalence of folate inadequacy among Canadians. American Journal of

Clinical Nutrition 2010; 92(4):818-825.

- Reprinted with permission of the American Journal of Clinical Nutrition. Originally

published in the American Journal of Clinical Nutrition.

xvi

List of Abbreviations

BMI Body Mass Index

CCHS Canadian Community Health Survey

CDC Center for Disease Control and Prevention

CSFII Continuing Survey of Food Intakes by Individuals

DFE Dietary Folate Equivalents

DRI Dietary Reference Intakes

EAR Estimated Average Requirement

FDA Food and Drug Administration

FITS Feeding Infants and Toddlers Study

IOM Institute of Medicine

MEC Hawaii-Los Angeles Multiethnic Cohort

MVM Multi-Vitamin/Mineral Supplement

NHANES National Health and Nutrition Examination Survey

NTD Neural Tube Defect

RBC Red Blood Cell

SIDE Software for Intake Distribution Evaluation

UL Tolerable Upper Intake Level

USDA United States Department of Agriculture

1

CHAPTER 1.0: INTRODUCTION/RATIONALE/OBJECTIVES/HYPOTHESIS

1.1 INTRODUCTION & RATIONALE

Food fortification and nutrient supplementation are two important strategies that have been used

for several decades to address micronutrient deficiencies (1-3). However, these strategies,

especially when used in tandem for a given nutrient, may also present health risks if they result

in nutrient intakes above the Tolerable Upper Intake Level (UL). Mandatory folic acid

fortification of certain grains in Canada and the United States is an example of a recent food

fortification program (4, 5). Given this initiative, along with the widespread use of

vitamin/mineral supplements in both countries (6-8), there is a need to assess total nutrient

exposure at the population level. Specifically, it is important not only to examine the impact of

both food fortification and supplement use on the prevalence of nutrient inadequacies, but also to

assess the likelihood of intakes above the UL. This thesis comprises a series of four studies

designed to examine the impact of the mandatory folic acid fortification program in Canada, and

more generally, the impact of vitamin/mineral supplement use on the diets of Canadians, and to

identify the factors associated with sub-optimal blood folate status among women of

childbearing age.

Folate is a generic term for a group of structurally related molecules that represent one of the

B-vitamins (9). Folic acid is chemically synthesized and it is the most oxidized and stable form

of the vitamin. Folic acid is only found in supplements and as a fortificant in foods (9). Among

other roles, folate is essential in nucleotide synthesis, making it important in new cell synthesis,

and consequently, there is an increased requirement during periods of rapid growth in the life

cycle, such as in pregnancy (9). In fact, poor maternal folate status during the peri-conceptional

period increases the risk of a neural tube defect (NTD)-affected pregnancy (9). Folic acid

2

supplementation during this time was shown to reduce the incidence of NTDs (10). As a result,

in the early 1990s the United States Public Health Service and the Society of Obstetrics and

Gynaecologists of Canada both recommended that all women capable of becoming pregnant

consume a supplement containing 400 µg folic acid (11, 12). The Institute of Medicine (IOM)

later recommended that women capable of becoming pregnant consume 400 µg of folic acid

from all sources (fortified foods and supplements) (9). However, compliance with the

recommendation was poor and in 1998, both the United States and Canadian governments

implemented mandatory folic acid fortification of white wheat flour and certain grain products

labeled enriched in order to add approximately 100 µg of folic acid to the diet of women capable

of becoming pregnant (4, 5).

The folic acid fortification program has been successful at reducing the incidence of NTDs in

both Canada and the United States by approximately 50% (13-15). However, there is a growing

body of preliminary evidence suggesting there may be potential harmful effects of too much folic

acid in addition to the well-established association between high folic acid intakes and the

masking and progression of vitamin B-12 deficiency and possible neurological damage (9, 16).

Postulated additional adverse effects include reduced effectiveness of anti-folate drugs, reduced

natural killer cell cytotoxicity, and increased risk of colorectal cancer among individuals with

preexisting neoplasms (17-23). Additionally, it has been hypothesized that folic acid

supplementation during pregnancy may be associated with increased adiposity, insulin resistance

and occurrence of asthma and poor respiratory health in the offspring (24-26).

An important feature of mandatory food fortification is that manufacturers often add

overages of the fortificant in order to ensure the nutrient in question in the product stays above

the mandated minimum level. In fact, in the United States, folate levels as high as twice that of

3

the mandated minimum has been reported early post-fortification (27, 28). On the other hand,

since the initiation of mandatory fortification in Canada over 10 y ago, there is no direct analysis

of fortified foods in order to compare the actual levels of folate in foods to the mandated levels.

The only estimate comes in the form of a letter-to-the-editor from Quinlivan & Gregory, in

which they used prediction equations correlating folic acid intake and serum folate in a

convenience sample to estimate that there is 50% extra folate in the diet (29).

Not knowing how much folate is in the Canadian food supply represents a further

compromise, in addition to the known limitations in the use of dietary intake data (30), in the

ability to assess the folate adequacy of Canadians of all ages and in particular, women of

childbearing age. For example, because there is currently no sense of the actual folic acid

intakes among women of childbearing age, it is unknown at the population level if Canadian

women still need to consume a daily folic acid-containing supplement in order to reach the

recommended intake of 400 µg (9). Secondly, given the growing concerns of too much folic

acid, the effect of excess folic acid in foods on the percent of Canadians with intakes above the

UL is unknown. Therefore, it is important to determine the actual levels of folate in fortified

foods. In order to fill this important gap in the literature, the objectives of Study 1 (Chapter 3)

are to measure the folate content in a selection of folic acid fortified foods in Canada and

compare actual values to those anticipated from mandatory food fortification regulations.

Another gap in the literature is the lack of an estimation of the prevalence of folate inadequacy in

the Canadian population and the percent of folic acid intakes above the UL based on the “real”

folate content of foods. Therefore, in order to fill this gap, the main purpose of Study 2 (Chapter

4) is to estimate the prevalence of folate inadequacy and intakes above the UL among Canadians

after accounting for folic acid overages.

4

Results from studies conducted in Canada post-folic acid fortification of the food supply with

small convenience samples suggest that women capable of becoming pregnant still need to

consume a folic acid-containing supplement in order to ensure 400 µg/d from all sources is

ingested for NTD protection (31-33). However, there are currently a wide range of folic acid

supplements available in Canada, with doses ranging from 400 µg to 5000 µg, and some have

made blanket recommendations for all women capable of becoming pregnant to consume 5000

µg/d (34). While some women have elevated folate requirements and may benefit from a

supplemental dose of folic acid that is >400 µg (35), there is little evidence that most women

would require more than 400 ug/d folic acid for protection against an NTD. It has been

previously established that, from a biochemical perspective, red blood cell (RBC) folate

concentrations ≥906 nmol/L provide maximal protection against folate-dependent NTDs (36,

37). In order for healthcare professionals to better identify women at greatest risk of having

blood folate concentrations <906 nmol/L, it would be helpful to know what factors are associated

with sub-optimal blood folate values. However, this remains a gap in the literature. Therefore,

in order to fill this void, the purpose of Study 3 (Chapter 5) is to identify factors associated with

RBC folate <906 nmol/L, a surrogate measure of folate-dependent NTD-risk, in order to better

estimate a woman‟s risk of an NTD-birth outcome.

Similar to food fortification, vitamin/mineral supplement use is an important strategy to

address established nutrient deficiencies (3). The use of such supplements is widespread in

North America, and has the potential to impact on overall nutrient intake (6, 7, 38). Findings

from the United States suggest that folic acid-containing supplement use has had little impact on

the prevalence of folate inadequacy but had led intakes above the UL (39, 40). Data from the

United States also indicate that supplement users often have higher mean nutrient intakes and

5

lower prevalence of nutrient inadequacies than non-users from diet alone (38, 41-44). Therefore,

it seems that individuals in the United States using supplements are the ones less likely to need

them. These observations piqued our interest to ask the question in Study 4 (Chapter 6) of this

thesis about what impact the use of vitamin/mineral supplements in Canada has on the

prevalence of inadequacy of folate and other essential nutrients and on intakes above the UL.

We also wanted to know whether nutrient intakes from diet alone differed between supplement

users and non-users. Dietary supplements are consumed by over one-third of the population in

both the United States and Canada (7, 8), and consumers cite several reasons for use, most

notably to improve and maintain health (45). Guo et al. used data from the nationally-

representative Canadian Community Health Survey (CCHS) 2.2 from 2004 to show that 41% of

adults reported consuming a vitamin/mineral supplement in the past 30 d (7). Further, they

found that sex, fruit and vegetable intake, physical activity, education and income were

important determinants of consumption. There are no population-based studies in Canada

comparing supplement users and non-users in terms of nutrient inadequacy and percent of

intakes above the UL based on dietary intake alone. Furthermore, the impact of supplements on

the diets of users has not been investigated in Canada based on recent nationally-representative

data. Given that supplements are permitted to contain as much as the UL for a given nutrient, it

is very likely that a large proportion of supplement users have intakes above the UL when both

dietary and supplemental nutrient sources are taken into consideration. However, this has not

been assessed. Therefore, in order to address these gaps in the literature, the objectives of Study

4 (Chapter 6) are to compare the prevalence of nutrient inadequacy and the percent of intakes

above the UL from diet alone between supplement users and non-users, and to determine the

effect of supplement use on the nutrient intakes of users.

6

1.2 OBJECTIVES & HYPOTHESES

Study #1 (Chapter 3): How much folate is in Canadian fortified products 10 years after

mandated fortification?

Objective:

1. To measure the folate content in a selection of folic acid fortified foods in Canada and

compare actual levels to levels anticipated from mandatory fortification food regulations.

Hypothesis:

1. Due to folic acid overages, actual folate levels in foods will be higher than mandated values.

Study #2 (Chapter 4): Investigating the impact of folic acid fortification over mandated

levels on the prevalence of folate inadequacy and intakes above the Tolerable Upper Level

of Intake among Canadians

Objectives:

Use the nationally-representative CCHS 2.2 to:

1. Estimate the prevalence of folate inadequacy and intakes above the UL among Canadians after

accounting for folic acid overages.

2. Estimate the supplemental dose that, with diet, provides reproductive-aged women with 400

μg folic acid/d for neural tube defect prevention.

3. Estimate the prevalence of folate inadequacy by risk factors often associated with suboptimal

folate status pre-fortification of the food supply (alcohol, diabetes, smoking, and obesity).

Hypotheses:

1. The prevalence of inadequacy will be lower than previously estimated, and there will be some

percent of the population with intakes above the UL.

7

2. Women will need a supplemental dose of folic acid much less than 400 µg in order to reach

the Institute of Medicine‟s recommendation to consume 400 µg of folic acid daily from all

sources to be maximally protected against an NTD pregnancy.

3. Smokers, alcohol consumers, diabetics, or obese individuals will have a higher prevalence of

folate inadequacy than non-smokers, alcohol abstainers, non-diabetics, or non-obese individuals.

Study #3 (Chapter 5): Factors associated with maximally protective red blood cell folate

concentrations in women 19-45 y: National Health and Nutrition Examination Survey

(NHANES) 2003-2006

Objective:

1. To identify factors associated with a surrogate measure of folate-dependent NTD-risk, RBC

folate status, among women 19-45 y in the nationally-representative United States NHANES

2003-2006.

Hypothesis:

1. Dietary folate intake, smoking, race/ethnicity, supplement use, and diabetes will be associated

with RBC folate status.

Study #4 (Chapter 6): Vitamin and mineral supplement consumption in Canada: do users

differ from non-users in terms of nutrient inadequacy and risk of high intakes?

Objectives:

Use the nationally-representative CCHS 2.2 to:

1. Compare the prevalence of nutrient inadequacy and the percent of intakes above the UL from

diet alone between supplement users and non-users

8

2. Determine the effect of supplement use on the prevalence of inadequacy and percent of intakes

above the UL among users.

Hypotheses:

1. From diet alone users will have a lower prevalence of inadequacy and higher percent of

intakes above the UL.

2. Among users, incorporating supplemental nutrient contribution will lead to a reduction of the

prevalence of inadequacy and an increase in the percent of intakes above the UL.

9

CHAPTER 2.0: REVIEW OF THE LITERATURE

2.1 FOOD FORTIFICATION & NUTRIENT SUPPLEMENTATION

2.1.1 Food fortification

Food fortification is defined as the addition of vitamins and minerals to food to: replace

nutrients lost in the manufacturing process, ensure the nutritional equivalence of substitute foods,

ensure the appropriate vitamin/mineral composition of special purpose foods, or act as a public

health intervention (1). In the absence of an adequate diet, food fortification is an important

strategy used to address micronutrient deficiencies (2), and for decades, it has been used

worldwide in an attempt to combat widespread deficiencies (2). Perhaps the best example of the

success of food fortification is iodine fortification of salt to treat iodine deficiency (46). Salt

fortification with iodine to treat goiter and other iodine deficiency diseases is now mandatory in

many countries (46, 47), and countries in which most of the population consume iodized salt

have the lowest prevalence of iodine deficiency (47). In Canada, food fortification has a long

history and has played an important role in addressing several micronutrient deficiencies,

including vitamin A, vitamin D, iodine and iron (1, 2). However, food fortification, while

successful in combating deficiencies, can also become a health risk if it results in usual nutrient

intakes in excess of the UL.

2.1.2 The case of folic acid

A more recent example of a mandatory fortification program is that of folic acid, the synthetic

form of the B-vitamin folate. Poor folate status was identified as an important risk factor for the

occurrence of NTDs and studies showed that improving folate status in women in the peri-

conceptional period led to a lower risk of an NTD-affected pregnancy (10, 48). Therefore, in

1998, the Canadian government implemented mandatory folic acid fortification of white wheat

10

flour, cornmeal and enriched pasta in order to increase intake in women of childbearing age in an

attempt to reduce the occurrence of NTDs (4).

However, an important element of mandatory fortification is the presence of nutrient

overages, and folic acid overages in fortified foods have been reported in the United States,

which initiated a similar mandatory folic acid fortification program in 1998 (27, 49).

Manufacturers add nutrients in excess of mandated minimums to ensure that their products never

fall below the minimum levels. As a result, if overages are not considered in dietary assessment,

this can lead to underestimation of actual intake, and consequently result in overestimation of the

prevalence of nutrient inadequacy and underestimation of the percent of intakes above the UL.

Part of the research herein will focus on the Canadian folic acid fortification program and

investigate the effect, if any, of overages on the Canadian diet. As a sub-analysis, we will also

investigate RBC folate status, an indicator of NTD-risk, among women of childbearing age.

2.1.3 Nutrient Supplementation

Nutrient supplementation, namely to provide non-food sources of nutrients, is another important

strategy to combat nutrient deficiencies (3). Certain subgroups of the population are at increased

risk of nutrient deficiency and unlikely to meet those needs from diet alone and therefore need a

supplemental form of that particular nutrient. For example, because of poor dietary intake, folic

acid supplementation is recommended for women of reproductive age in developing countries

(11, 12). Similarly, elder adults are recommended to consume a vitamin B12 supplement

because of a reduction in absorption efficiency with age (9). However, dietary guidance for the

general population, as illustrated by the most recent position paper of the American Dietetic

Association on this issue, is to obtain adequate nutrients from a wide variety of foods rather than

from vitamin/mineral supplements (3). Despite this, vitamin/mineral supplements are consumed

11

by a large proportion of the population in North America and have the ability to impact overall

nutrient intake (7, 8, 38). Additionally, like food fortification, vitamin/mineral supplement use

can pose a health risk if they lead to total nutrient exposure in excess of the UL. Part of the

research herein will investigate vitamin/mineral supplement use by comparing users and non-

users, and assessing the impact of supplement use on prevalence of inadequacy and percent of

intakes above the UL among users.

2.2 INTRODUCTION TO FOLATE

2.2.1 Description

Folate, an umbrella term for a group of structurally related compounds, is one of the 8 essential

B-vitamins (9, 50). The general structure of folate consists of a pteridine ring linked to a para-

aminobenzoic acid, which is linked to at least one glutamate molecule (Figure 2.2.1). Folic acid,

the synthetic form of the vitamin that is found in supplements and as a fortificant, is the most

stable and oxidized form of the vitamin (shown in Figure 2.2.1). Folic acid is found mono-

glutamated, whereas naturally occurring forms usually contain additional glutamate molecules

(9). Folate derivatives differ from each other by the presence of different groups (e.g. methyl,

formyl, methylene) on the nitrogen atom at positions 5 and 10 on the molecule (50).

Figure 2.2.1. Folic acid. Modified from Groff & Gropper (50).

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2.2.2 Functions

Folate‟s main function in the body is to serve as coenzymes in single-carbon transfers in the

metabolism of nucleic and amino acids (9). The conversion of deoxyuridylic acid to the

pyrimidine thymidylic acid is dependent on folate, thus making folate essential for normal cell

division (9). Folate is also required for purine synthesis, generation of formate into the body‟s

formate pool, and for several amino acid inter-conversions, including generation of S-adenosyl-

methionine, the universal methylating agent in vivo (9). Because of the essentiality of folate in

normal cell division, it becomes especially important during stages of rapid growth in the life

cycle, such as pregnancy, lactation, infancy, and adolescence.

2.2.3 Sources and Bioavailability

In this thesis, “food folate” refers to the naturally occurring form of the vitamin found in food.

“Dietary folate” refers to all folates found in food (food folate plus folic acid) and “dietary folic

acid” refers to folic acid found in food. The term “total folate” describes the sum all forms of

folate consumed (dietary folate and supplemental folic acid). Food folate can be found in a wide

variety of vegetables, fruits, and legumes, including dark green vegetables, which are one of the

better sources (9). On the other hand, synthetic folic acid can be found in ready-to-eat cereals

due to discretionary fortification in both Canada and the United States (51) (52). However, the

two countries differ with respect to this program; much higher amounts are permitted in cereals

in the United States (up to 400 µg folic acid per 30 g serving) than that permitted in Canada (less

than 20 µg folic acid per 30 g serving) (51, 52). Additionally, as of 1998, mandatory folic acid

fortification (discussed in Sections 2.3.2.1 and 2.3.2.2) of cereal grain products and enriched

pasta contributes yet another source of folate to the diet of North Americans (4).

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As suggested by the IOM, to account for differences in bioavailability between folic acid and

food folate, total folate intake is expressed in dietary folate equivalents (DFE), and is determined

using the following calculation: DFE = µg food folate + (µg dietary folic acid x 1.7) + (µg

supplemental folic acid x 2) (9). A factor of 2 is used if supplemental folic acid is consumed on

an empty stomach. However, if supplemental folic acid is not consumed on an empty stomach,

1.7 is used (9).

2.2.4 Absorption

While folate may be absorbed along the entire length of the small intestine and in the large

intestine, most absorption occurs at the jejunum of the small intestine (53, 54). Only short chain

folates, in the mono- or di-glutamated forms, are absorbed across the small intestine (55).

Therefore, food folate, normally found polyglutamated, must be cleaved to its short chain form

by intestinal conjugases, primarily glutamate carboxypeptidase II found in the brush border

membrane (56).

There are a few mechanisms for the uptake of folate across the small intestine (57-59). The

first, which occurs when intestinal folate concentration is high, is via a non-saturable passive

diffusion method in which folates pass through the enterocyte unmodified (57). At lower

concentrations, mono-glutamated folates are absorbed by active diffusion using two transport

proteins, the reduced folate carrier and the proton-coupled folate transporter (58). However, the

proton-coupled folate transporter is more active at small intestine pH and is therefore thought to

play a bigger role in folate uptake (58). Additionally, there are other folate binding proteins that

play a role in folate transport in the small intestine (59). Aufreiter et al. have recently shown that

folate is also absorbed across the colon (60). After folates have been absorbed into the

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enterocyte, they are converted to the 5-methyltetrahydrofolate form and either kept to function as

coenzymes or enter circulation (61).

2.2.4 Requirements

RBC folate concentration is the primary indicator of tissue folate stores/long-term status, and

hence is used (sometimes along with plasma homocysteine and serum folate) in estimating folate

requirements (9). A value of >305 nmol/L (>140 ng/mL) was set by the IOM as the cutoff for

adequate folate status based on the results of several experiments (62-65). Importantly, RBC

folate does not reflect short-term/transient folate status (9). Instead, serum folate is used as an

indicator of recent folate status, with a concentration <7 nmol/L (3ng/mL) reflecting negative

folate balance (66).

The Estimated Average Requirement (EAR) for a nutrient is defined as the level of intake for

that nutrient that meets the requirement for 50% of healthy individuals (defined by age and sex)

in the population (67). For folate, the EARs for adults were determined using data based on

several indicators of status, including plasma homocysteine and RBC and serum folate

concentrations (9). For children and adolescents, EARs were extrapolated from adult cut-offs

(9). The values are presented in Table 2.2.4. Additionally, the IOM recommends that women of

childbearing age consume 400 µg/d of synthetic folic acid from all sources (dietary and

supplements) in addition to food folate from a varied diet to protect against an NTD-affected

pregnancy (9).

Table 2.2.4. Estimated Average Requirement (EAR) and Tolerable Upper Intake Level (UL) for

folate by life stage group

Life Stage Group

(males & females)

EAR (DFE)1

UL (µg)2

15

1 to 3 y 120 300

4 to 8 y 160 400

9 to 13 y 250 600

14 to 18 y 330 800

19 to 30 y 320 1000

31 to 50 y 320 1000

51 to 70 y 320 1000

>70 y 320 1000

1Dietary Folate Equivalents

2Folic acid (synthetic folate) from supplements and as a fortificant in foods

2.2.5 Inadequate intake and deficiency

Insufficient consumption of folate affects the body in stages, with serum folate concentration

being the first measure to decrease (9). If insufficiency persists, a decrease in RBC folate

concentration is then observed and then a rise in homocysteine concentration (9). Long-term

inadequate folate intake leads to macrocytic anemia, which is first detected by low RBC count,

and in later stages, also by decreased hematocrit and hemoglobin (9, 50, 63). Physical symptoms

of anemia include weakness, fatigue, irritability, difficulty concentrating and shortness of breath

(9, 50).

The more concerning issue regarding inadequate folate intake is in women capable of

becoming pregnant. While the exact mechanism is unknown, folate is very important in the peri-

conceptional period for proper closure of the neural tube and inadequate folate intake/status can

lead to an NTD-affected pregnancy (association discussed below in section 2.3.1). There are two

common types of NTDs, spina bifida and anencephaly (68). Spina bifida is the more common

and less serious of the two, resulting from an opening in the spinal column (68). While its

consequences vary depending on location, it can lead to paralysis and severely compromised

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quality of life (68). Anencephaly is more serious, with part of the brain missing, and these

babies are either stillborn or die within a few days of birth (68).

2.2.6 Excessive intakes

Until recently, the main concern with excessive intake of folic acid was the masking and

progression of vitamin B-12 deficiency, which can lead to undetected neurological damage (9,

16, 69). In fact, the UL, defined as the highest intake amount of a nutrient thought to pose no

adverse health effects (67), was set for folic acid only (only the synthetic form, which is found as

a fortificant and in supplements) (9). The UL cut-offs were set based on this folic acid-vitamin

B12 interaction using data from dose-response/case studies (70-72). A Lowest-Observed

Adverse Effect Level was determined to be 5 mg, which was then divided by an uncertainty

factor of five, resulting in a UL of 1 mg for adults (9). The ULs for children and adolescents

were extrapolated from that of the adult value (9). The values are presented above in Table

2.2.4.

There is now growing evidence suggesting other negative consequences of too much folic

acid intake (17). High folic acid intakes are thought to be associated with decreased natural

killer cell cytotoxicity (18). Troen et al. compared folate intake to natural killer cell cytotoxicity

(a marker of immune function) among 105 healthy, postmenopausal women, and found a

significant inverse relationship between natural killer cell cytotoxicity and unmetabolized folic

acid found in plasma of these women (18). Furthermore, the presence of unmetabolized folic

acid in serum is thought to be of concern, as very little is known about its biological effects (73).

Post-fortification of the food supply, unmetabolized serum folic acid has been detected in the

general population; using 2001-2002 NHANES data, Bailey et al. reported that unmetabolized

serum folic acid was detected in 38% of the population (74).

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High folic acid intakes were also shown to be associated with a reduction in the effectiveness

of anti-folate drugs (19, 20, 75). In a randomized controlled study, Carter et al. looked at the

effect of therapeutic folic acid supplementation in 303 patients of all ages being treated with an

antifolate, antimalarial drug in southern Kenya (75). Among subjects randomized to receive

folic acid, there was a significant reduction in parasite clearance despite antimalarial therapy

(75). This report, along with findings from other studies (76, 77), has to led to doubts regarding

the use of folic acid supplements among patients being treated for malaria (19). Salim et al.

investigated the use of folic acid supplementation on the efficacy of methotrexate in the

treatment of psoriasis (20). They conducted a double-blinded, controlled trial involving 22

psoriasis patients randomized to receive 5mg of folic acid or a placebo for 12 weeks, along with

the normal methotrexate treatment (20). Based on several established indicators of efficacy of

treatment, they found a significant reduction in efficacy among patients randomized to receive

folic acid (20).

High folic acid intake in pregnant women was shown to be associated with asthma, poor

respiratory health, increased adiposity and insulin resistance among their offspring (24-26).

Yajnik et al. studied the effect of maternal diet on offspring at 6 y among 700 pregnant women

from six villages in India (25). All women were given a 0.5 mg folic acid supplement daily from

18 weeks gestation, and biochemical indices were measured in the women at 18 and 28 weeks

gestation. Children were followed up until 6 y, at which point insulin resistance and adiposity

were assessed (25). The authors found that higher maternal RBC folate concentration at 28

weeks gestations was significantly associated with higher fat mass, higher percent body fat and

higher degree of insulin resistance (25). Whithrow et al., using data from 490 mother-infant

pairs in an Australian prospective birth cohort study, showed that folic acid supplementation in

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late pregnancy (30-34 weeks gestation) is associated with increased risk of asthma among

offspring at 3.5 y (24). With a much larger sample, Haberg et al. reported similar findings with

respiratory health and folic acid supplementation in pregnancy (26). Using data from 32,077

children born in the Norwegian Mother and Child Cohort Study, they found that use of folic acid

supplements in the first trimester was significantly associated with increased risk of wheezing,

lower respiratory tract infections, and hospitalization due to lower respiratory tract infections

among offspring at 1.5 y (26).

There is also evidence showing that high folic acid intake promotes the growth of cancer

cells; this association has been best demonstrated with colorectal cancer (21-23, 78). A temporal

association was reported in Canada, United States and Chile, when colorectal or colon cancer

rates were compared pre- and post-fortification in these countries (22, 78). Using nationally-

representative databases, Mason et al. showed an increase in the rates of colorectal cancer in both

Canada and the United States around the time mandatory fortification was implemented (78).

Similarly, Hirsch et al. reported a greater than two-fold increase in the rate of colon cancer in

Chile when comparing pre-to-post-fortification data (22). While causality cannot be established

from these two studies, these findings have been supported by those from a recent randomized

controlled trial (22). Cole et al. investigated the role of folic acid in the prevention of colorectal

adenomas (21), using a multicentre randomized controlled trial involving 1,021 men and women

with a history of colorectal adenomas. They found that, after 6-8 y of follow-up, among those

randomized to receive a daily folic acid supplement (1 mg), there was a significantly higher rate

of occurrence of 3 or more adenomas, and a trend to a higher rate of advanced lesions (21).

Overall, the literature regarding folic acid and colon cancer points to a dual role: in absence of

pre-existing neoplasms, folate is thought to have a protective role against cancer incidence (79,

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80), while in the presence of such neoplasms, folate is thought to increase the risk of cancer (23,

81).

2.3 FOLIC ACID FORTIFICATION

2.3.1 History of the association between folate and NTD

The idea that folate has a role in the prevention of NTDs was first proposed as early as 1964 (82,

83). Subsequently, Smithells et al. (1980) and Laurence et al. (1981) published the results of

their intervention studies, which showed that folic acid (84) or a multivitamin containing folic

acid (85) consumed peri-conceptionally by women with a previous NTD-affected pregnancy can

reduce the risk of recurrence (84, 85). However, there was a possibility of bias present in both

studies; the first study was not randomized (85), while the randomization was broken to account

for noncompliance in the second study (84). Therefore, it couldn‟t be concluded with certainty

that folic acid was solely responsible for the reduction in NTD recurrence (10).

In 1983, an international, multicentre, double-blinded randomized trial involving 33 study

centres from across the United Kingdom, Hungary, Israel, Australia, Canada, USSR and France,

was launched (10). Women with a previous pregnancy affected by an NTD were recruited and

randomized to one of four groups (2 groups received 4 mg of supplemental folic acid daily) (10).

Due to the strong protective effect of folic acid supplementation against the recurrence of an

NTD (RR: 0.28, 95% CI: 0.12, 0.71), the trial was stopped short of completion in 1991 (10).

This study, published by the Medical Research Council Vitamin Study Research Group, was the

first to conclusively show that folic acid supplementation reduces the risk of NTD-recurrence

(10).

In 1992, Czeizel et al. showed that first occurrence of an NTD can be prevented by peri-

conceptional vitamin supplementation (48). They conducted a randomized, controlled trial in

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which Hungarian women without a previous history of an NTD were randomized to receive

either a multivitamin supplement containing 0.8 mg folic acid, or a trace element supplement

without folic acid. Six of the 2,052 pregnancies in the control group resulted in an NTD

compared to none in the 2,014 pregnancies from the intervention group (P = 0.029) (48).

2.3.2 Rationale for folic acid fortification of food

Given the well established link between folic acid intake and reduction in occurrence of an NTD,

and since the important window of action for folate in NTD-prevention occurs before most

women are aware of their pregnancy (first 4 weeks), all women of childbearing age who were

capable of becoming pregnant were recommended to consume a folic acid supplement (12). In

September 1992, soon after the Medical Research Council study was published, the United

States Public Health Service put out their recommendation that all women capable of becoming

pregnant consume at least 400 µg of folic acid daily (12). The Society of Obstetrics and

Gynaecologists of Canada similarly recommended for women capable of becoming pregnant to

consume 400 µg of supplemental folic acid in addition to dietary folate (11). In their publication

of the Dietary Reference Intakes (DRI), the IOM recommended that women capable of becoming

pregnant consume 400 µg of folic acid from all sources (fortified foods and supplements) in

addition to a varied diet (9). However, several studies showed that campaigns and

recommendations encouraging women to consume supplemental folic acid were and continue to

be ineffective; neither did the occurrence of NTDs decrease, nor did the prevalence of women

consuming a folic acid containing supplement increase (86-89). In fact, authors of a post-

fortification study show that even offering free folic acid supplements doesn‟t improve

compliance (90). Furthermore, approximately half of all pregnancies are unplanned (91). As a

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result, folic acid fortification of the food supply was considered as an option to increase intake in

women of childbearing age (5).

2.3.2.1 The United States folic acid fortification program

In 1996, the Food and Drug Administration (FDA) made the decision to mandate folic acid

fortification of flour by January 1, 1998 (5). The FDA used the U.S. Department of

Agriculture's (USDA's) 1987 to 1988 national food consumption data to model foods in order to

determine appropriate foods to fortify and levels of fortification (5). Cereal grains, dairy

products and juices were all candidates, and 70, 140, and 350 µg/100 g of food were all potential

levels of fortification. However, the FDA decided to set the mandate at 140 µg/100 g of cereal

grain products only, because this was the level which led to an increase in folic acid intake by

approximately 100 µg in women of childbearing age, without leading to excessive intakes in

other age/sex groups. Also, fortification of dairy products and juices would have similarly led to

excessive intakes in other age/sex groups. Given that products differ in their total cereal grain

content, and in order to achieve a level of folic acid between 95 to 309 µg per 100 g of product,

the FDA mandated the level of fortification of flour to be 140 µg/100 g of cereal grain (5).

Using simulation exercises, Boushey et al. independently estimated the effect that mandatory

fortification would have on the diet of women (92). While this was a convenience sample of

only 289 women, they nonetheless show that mandatory fortification will add 320 to 608 DFE to

the diet, and that all women of childbearing age will have consumed above the EAR. However,

only 39% of women would meet the recommended level of 400 µg of folic acid daily (92).

2.3.2.2 The Canadian folic acid fortification program

Soon after the United States mandate, the Canadian government mandated folic acid fortification

to begin in November, 1998 (4). The regulations stipulated that white wheat flour and cornmeal

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were to be fortified as well as enriched pasta (4). Data from 5 Provincial Nutrition Surveys

(Nova Scotia, Quebec, Saskatchewan, Alberta and Prince Edward Island) were used in

simulation exercises to identify appropriate levels fortification. Potential levels simulated were

similar to those used in the United States estimations. Eventually, the levels of 150 µg/100 µg of

white wheat flour, and 200 µg/100 µg of enriched pasta, were set as the minimum levels of

fortification (4), which was estimated to increase daily intake in women of childbearing age by

100 µg. Importantly, the Canadian mandatory folic acid fortification program differs from that

of the United States in that the Canadian regulations stipulate a minimum with no mention of an

upper limit (4). Therefore, even though actual levels of fortification are unknown, the potential

for over-fortification exists in Canada more than in the United States (4).

2.3.2.3 Mandatory folic acid fortification elsewhere

As of December 2010, there are over 50 countries that have implemented mandatory folic acid

fortification, and several with voluntary fortification programs (93). Most countries in the

Americas (North, South, and Central), the Caribbean, and a handful of African and Asian

countries have mandated folic acid fortification at levels ranging from 30 to 300 µg/100 g of

flour/product (94). In addition, food safety agencies from a few countries, including the United

Kingdom have recently weighed the benefits of NTD reduction compared to the risks associated

with excessive folic acid intake, and have recently recommended the implementation of

mandatory fortification (95). However, two notable countries have suspended bids for

fortification. The Food Safety Authority in Ireland, which previously recommended mandatory

fortification, has recently concluded that there would be little benefit to such a policy given that

there is extra folic acid in the diet due to increase in the use of folic acid supplements and

discretionary fortification (96). Also, in New Zealand, due to concerns of masking of vitamin

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B12 deficiency and rise in the incidence of colon cancer rates observed in a few other countries

with mandatory fortification, the mandatory folic acid fortification program is now tentatively

delayed until May 2012 after being previously recommended (97).

2.3.2.4 The effect of folic acid fortification on the incidence of NTDs

Folic acid fortification has proven to be a successful initiative to lower the incidence of NTDs in

Canada (Table 2.3.2.4.a) and the United States (Table 2.3.2.4.b). While there seems to be an

overall greater reduction of occurrence in Canada, this is likely due in part to the fact that

Canadian studies captured terminated NTD-affected pregnancies more so than those from the

United States. Furthermore, it has also been reported that the incidences of other birth defects

and neuroblastoma have been reduced post-fortification (98-102). Additionally, cost-benefit

analyses have shown that mandatory fortification is a cost-effective policy that has led to

substantial financial gains through decreased burden on the health care system (103-105). In

addition to Canada and the United States, a reduction in NTD occurrence post-fortification has

been reported in several other countries (Table 2.3.2.4.c). For example, there was a 26%

reduction in NTD reported in Australia since mandatory fortification (106), while an 88%

reduction was reported in Oman (107).

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Table 2.3.2.4.a. Change in the occurrence of neural tube defects pre-to-post-fortification in Canada

Study Data Source/Location Type of Neural

Tube Defect

Pre-fortification period;

NTD-rate

Post-fortification

period; NTD-rate

Reduction

Godwin et al.

2008 (98)

Alberta Congenital

Anomalies Surveillance

System (ACASS), Alberta

Spina bifida 1992-1996

(97/198,321); 0.49/1,000

births

1999-2003

(48/191,028);

0.25/1,000 births

OR: 0.51, 95%

CI: 0.36-0.73

De Wals et

al. 2008

(108)

Seven Provincial Databases

(NL, NS, PEI, QC, MB, AB

, BC)

Spina bifida 1993-1997; 0.86/1,000

pregnancies

2000-2002; 0.40/1,000

pregnancies

53% (P < 0.0001)

De Wals et

al. 2007 (14)

Seven Provincial Databases

(NL, NS, PEI, QC, MB, AB

, ON)

All 1993-1997; 1.58/1,000

births

1998-2002; 0.86/1,000

births

46%,

95% CI: 40-51

Liu et al.

2004 (32)

Newfoundland and

Labrador Medical Genetics

Program, Newfoundland

All 1991-1997 (193/24,303);

4.36/1,000 births

1998-2001 (19/19,816);

0.96/1,000 births

RR: 0.22, 95%

CI: 0.14-0.35

De Wals et

al. 2003

(109)

Med Echo Province-wide

Hospital Database, Quebec

All 1992-1997; 1.89/1,000

births

1998-2000; 1.28/1,000

births

32% (P < 0.001)

Gucciardi et

al. 2002

(110)

Canadian Congenital

Anomalies Surveillance

System, Ontario

All 1995; 16.2/10,000

pregnancies

1999; 8.6/10,000

pregnancies

47% (P < 0.001)

25

Persad et al.

2002 (111)

Fetal Anomaly Database,

Nova Scotia

All 1991-1997 (203/78,841);

2.58/1,000 births

1998-2000 (34/29,010);

1.17/1,000 births

RR: 0.46, 95%

CI: 0.32-0.66

Ray et al.

2002 (112)

Ontario Antenatal Maternal

Serum Screening, Ontario

Anencephaly/spi

na bifida

1994-1997

(248/218,977);

1.13/1,000 pregnancies

1998-2000

(69/117,986);

0.58/1,000 pregnancies

Prevalence ratio:

0.52, 95% CI:

0.4-0.67

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Table 2.3.2.4.b. Change in the occurrence of neural tube defects pre-to-post-fortification in the United States

Study Data Source/Location Type of Neural

Tube Defect

Pre-fortification period;

NTD-rate

Post-fortification

period; NTD-rate

Reduction1

Boulet et al.

(CDC Report)

2009 (13)

National Vital Statistics

System, United States

Spina bifida 1995-1996

(1,864/6,965,809);

2.68/10,000 births

2001-2005

(3,566/18,366,964);

1.94/10,000 births

Prevalance ratio:

0.72

Canfield et al.

2005 (102)

National Birth Defects

Prevention Network (23

States)

a) Anencephaly,

b) spina bifida

1995-1996;

a) 2.2/10,000 births

b) 4.9/10,000 births

1999-2000;

a) 1.8/10,000 births

b) 3.2/10,000 births

Prevalence Ratio:

a) 0.84 95% CI:

0.76-0.94

b) 0.66

95% CI:

0.61-0.71

Williams et al.

2005 (113)



States and Puerto Rico)

a) Anencephaly,

b) spina bifida

1995-1996; a) 2.9/10,000

births

b) 5.1/10,000 births

1998-2002; a)

2.2/10,000 births

b) 3.5/10,000 births

Prevalence Ratio:

a) 0.76

b) 0.69

Mersereau et

al. (CDC

Report) 2004

(114)



a) Anencephaly,

b) spina bifida

1995-1996; a) 4.2/10,000

births

b) 6.4/10,000 births

1999-2000; a)

3.5/10,000 births

b) 4.1/10,000 births

Prevalence Ratio:

a) 0.83

b) 0.64

Simmons et al.

2004 (115)

Arkansas Reproductive

Health Monitoring

Spina bifida 1993-1995; 7.8/10,000

births

1999-2000; 4.4/10,000

births

OR: 0.56, 95% CI:

0.37-0.83

27

System, Arkansas

Williams et al.

2002 (116)



States)

a) Anencephaly,

b) spina bifida

1995-1996;

a) 2.4/10,000 births

b) 5.2/10,000 births

1998-1999;

a) 2.0/10,000 births

b) 3.5/10,000 births

Prevalence Ratio:

a) 0.84 95% CI:

0.75-0.95

b) 0.69

95% CI:

0.63-0.74

Feldkamp et

al. 2002 (117)

Utah Birth Defect

Network, Utah

All 1993-1997; 0.84/1,000

births

1998-2000; 0.59/1,000

births

30% (P < 0.05)

Mathews et al.

(CDC Report)

2002 (118)



a) Anencephaly

b) spina bifida

1996 2001 a) 21%

b) 24%

Meyer and

Siega-Riz

(CDC Report)

2002 (119)

North Carolina Birth

Defects Monitoring

Program, North Carolina

Spina bifida 1995-1996; 6.5/10,000

births

1998-1999; 4.7/10,000

births

27.2% (P < 0.014)

Honein et al.

2001 (15)

National Center for

Health Statistics (45

States)

All 1995-1996; 37.8/100,000

births

1998-1999;

30.5/100,000 births

Prevalence ratio:

0.81, 95%CI: 0.75-

0.87

Stevenson et

al. 2000 (120)

Five surveillance

components, South

All 1992; 1.89/1,000

pregnancies

1998; 0.95/1,000

pregnancies

Prevalence ratio:

0.50,

28

Carolina P < 0.05

1Where missing, confidence limits were not given for the specific type of NTD or reduction in occurrence of NTD was hand

calculated based on data reported in the study.

29

Table 2.3.2.4.c. Change in the occurrence of neural tube defects pre-to-post-fortification outside North America

Study Data Source/Location Type of Neural Tube

Defect

Pre-to-post fortification Reduction

Abeywardana et

al. 2010 (106)

Australian Congenital Anomalies Monitoring

System, Australia

All 26%

Lopez-Camelo et

al. 2010 (121)

Latin American Collaborative Study of

Congenital Malformations, Chile

a) Anencephaly

b) Spina bifida

Prevalence Rate Ratio:

a) 0.54

b) 0.43

Lopez-Camelo et

al. 2010 (121)


Congenital Malformations, Argentina

a) Anencephaly

b) Spina bifida


a) 0.59

b) 0.59

Lopez-Camelo et

al. 2010 (121)


Congenital Malformations, Brazil

a) Anencephaly

b) Spina bifida


a) 0.57

b) 0.991

Pacheco et al.

2009 (122)

National Information System on Live Births,

Brazil

All OR: 0.711

Alasfoor et al.

2008 (107)

Ministry of Health Annual Statistical Reports,

Oman

Spina bifida 88%

Sayed et al. 2008

(104)

Hospital-based Surveillance Systems, South

Africa

All 30.5%

Hertrampf & Hospital-based Surveillance Systems, Chile All 43%

30

Cortes 2008 (105)

Safdar et al. 2007

(123)

King Abdul-Aziz University Hospital, Saudi

Arabia

All OR: 0.4

Lopez-Camelo et

al. 2005 (124)


Congenital Malformations, Chile

a) Anencephaly

b) Spina bifida

a) 42%

b) 51%

Amarin &

Obeidat, 2010

(125)

Princess Badea Teaching Hospital, Jordan All 49%

Chen & Rivera,

2004 (126)

Ministry of Health, Costa Rica All 35%

1Non-significant reduction

31

2.4 POST-FORTIFICATION OF THE FOOD SUPPLY

2.4.1 Folate intake post-fortification of the food supply

2.4.1.1 Sources of folate in the diet post-fortification

Pre-fortification of the food supply, the primary sources of folate in the diet were dark green

vegetables, beans and legumes (9, 50). In addition to this, because of discretionary fortification

in the United States, ready-to-eat cereals were another substantial contributor of folate to the diet

in the United States (127). Several reports have indicated that there has been a post-fortification

shift in the source distribution of total food folate intake in both Canada and the United States,

with cereal grains now the largest contributor (31, 33, 127-129) .

Dietrich et al. compared food intake data from the National Health and Nutrition

Examination Survey (NHANES) III (1988-1994) and NHANES 1999-2000, and showed that in

the pre-fortification era (NHANES III), vegetables were the largest contributor of total dietary

folate, at 19.4%, followed by breakfast cereals, breads, beans, and fruit (including fruit juice)

(127). In the post-fortification era (NHANES 1999-2000), breads jumped to the top of the list, at

15.6%, moving vegetables to second (12.6%) and breakfast cereals to third (12.1%) (127).

In Canada, there are several studies that indicate that cereal grains contribute the most folate

in the diet post-fortification (128). Sherwood et al. investigated dietary intake post-fortification

among a sample of 60 pregnant and lactating women in Toronto (128). They similarly showed

that cereal grain products cumulatively contributed the most folate to the diet (128). In their

sample of 148 British Columbian women, French et al. similarly reported that cereal grains were

the top contributing foods to folate intake (31). Shuaibi et al. also reported the same finding

(33). Among their sample of 95 Manitoban women 18-25 y, bakery products contributed 27.7%

32

of dietary folate, followed by vegetables (17.6%) (33). Hennessy-Priest et al. report the same

finding in their analyses of food intake among 254 Ontarian preschoolers (129).

In conclusion, folic acid fortification has resulted in a shift in the source distribution of

dietary folate intake. Cereal grains are now the leading contributor, and vegetables are now

second. In addition, due to discretionary fortification in the United States, ready-to-eat cereals

also contribute substantially (130). Importantly, all of the aforementioned analyses were based

on dietary intake and did not include supplement consumption. The use of folic acid containing

supplements also contributes to total intake (39, 40, 130).

2.4.1.2 Reports from the United States

Total folate and folic acid intakes in the United States are now higher than in the pre-fortification

era. While some of this can be attributed to the increasing consumption of dietary supplements,

including folic acid-containing supplements, folic acid from fortified foods undoubtedly makes

an independent contribution to the diet (130). Kalmbach et al., using data from the Framingham

Offspring Cohort, showed that there is approximately 200 µg extra folic acid in the daily diet of

adults compared to pre-fortification, and this was independent of folic acid supplement use

(131).

Several NHANES analyses, taken together, also indicate an increase in intake (127, 132-

134). Dietrich et al. compared pre- and post-fortification folate intake using NHANES III (1988-

1994) and NHANES 1999-2000 data (127). They reported that mean folate intake from dietary

sources was significantly higher in 1999-2000 in all age/sex groups except for females ≥60 y, in

whom there was a non-significant increase (127). Mannino et al., using data from the pre-

fortification era (NHANES III), reported that adults consumed approximately 204-236 µg of

folate daily (132). While the authors didn‟t report folate in DFE, most of this is likely naturally

33

occurring folate (with some folic acid from ready-to-eat cereals). Therefore, the folate intake in

DFE is most likely not much higher than the reported values. In contrast to this, Quinlivan and

Gregory estimated much higher folate intakes post-fortification using NHANES 1999-2004 data

(133). Using reverse prediction equations based on an association between folate intake and

serum folate, they estimated that median folate intake increased to 717-852 DFE post-

fortification (133). However, the estimate doesn‟t distinguish between dietary and supplemental

folic acid, which also contributes to intake (134, 135). In fact, Yeung et al. used NHANES

2001-2004 data to show that high serum concentrations were primarily associated with folic acid

supplement use and not consumption of folic acid due to mandatory fortification (134).

Two additional NHANES analyses focused on women of childbearing age (136, 137). Yang

et al. analyzed data from NHANES 2001-2002 and showed that, as expected, mandatory

fortification contributed approximately 130 µg of folic acid to the daily diet among women 15-

49 y (136). However, only 8% of women met the IOM‟s recommendation of 400 µg/d, and there

were racial disparities, with non-Hispanic blacks as the least adherent group with the IOM

recommendation (136). When supplement consumption was included, this percentage increased,

but racial disparities persisted (136). Using NHANES 2003-2006 data, Tinker et al. investigated

the contribution of different sources of folic acid to the diet of women 15-44 y (137). They

reported that from diet and supplements, only 24% of women met the IOM‟s 400 µg/d

recommendation and racial disparities persisted (137). Furthermore, the use of a folic acid-

containing supplement was the strongest factor associated with meeting the recommendation

(137). These two reports highlight the fact that despite mandatory folic acid fortification, it

remains essential for women in the United States to consume a folic acid containing supplement

to meet the IOM recommendation (136, 137).

34

Children were the focus of two recent publications on folate intake using NHANES 2003-

2006 data (40, 138). Bailey et al. estimated folate intake in children 1-13 y and included the

contribution of folic acid-containing supplements. They showed that over 95% of children met

the EAR from dietary folate (including folic acid in fortified foods) alone and hence had folate

adequate diets. Furthermore, 28% consumed a supplement containing folic acid, and this more

often than not led to total folic acid intake in excess of the UL (40). In a similar publication that

soon followed, Yeung et al. also used NHANES 2003-2006 data to investigate folate intakes in

children (138). However, they split dietary folic acid sources into two categories, that from

fortified foods and that from ready-to-eat cereals (138). The authors showed that among children

1-18 y, consumption of folic acid from fortified foods alone did not lead to intakes above the UL.

However, when folic acid from ready-to-eat cereals and supplements were considered, 15-78%

of children consumed folic acid above the UL (138).

Two recent reports, both again using NHANES 2003-2006 data, looked at intakes in adults

(39, 130). Bailey et al. showed that approximately 20% of women have inadequate intakes of

folate from diet alone, and the prevalence of inadequacy drops slightly when supplements were

considered (39). Men had lower inadequacy and the inclusion of supplement consumption didn‟t

have a sizeable impact on the prevalence of inadequacy (39). Importantly, up to 5% of the

individuals exceeded the UL, and this was mainly due to the consumption of supplements (39).

In their analyses, Yang et al. split folic acid intake into three categories: fortified foods, ready-to-

eat cereals and supplements (130). They show that the main driving force behind consumption

of folic acid at intakes above the UL is the use of folic acid-containing supplements with large

doses of folic acid; 47.8% of individuals who consumed a supplement with >400 µg of folic acid

were above the UL (130).

35

2.4.1.3 Reports from Canada

There are several studies in which folate intake data post-fortification are reported, particularly

among women of childbearing age and older adults (31-33, 128). The general consensus of this

body of literature indicates that total folate intake post-fortification has increased when compared

to pre-fortification data. However, there are several gaps in the Canadian literature, including

lack of nationally-representative data on total intake, and taking into consideration folic acid

overages in foods (discussed in detail in section 2.4.3 below) and supplementation into intake

estimates.

Using a convenience sample of 95 women 18-25 y from the University of Manitoba, Shuaibi

et al. showed that folic acid fortification added 95.7 µg to the diets of the women, similar to what

was originally predicted at the inception of mandatory fortification (100 µg) (33). They also

showed that based on diet alone, very few women met the IOM‟s recommendation for women of

childbearing age to consume 400 µg of folic acid from food and supplemental sources. Also,

while supplemental folic acid led to a higher total intake among users, only 17% of women met

the IOM‟s recommendation when supplement consumption was included in the analyses (33).

French et al., in their sample of 148 women 18-45 y in the Vancouver area, similarly show that

mandatory fortification added 104 µg of folic acid to the diet (31). In their sample, the

prevalence of inadequacy was low (14%), but only 26% women consumed 400 µg, similar to the

17% reported by Shuaibi et al. (31, 33). Liu et al. reported that mandatory fortification added 70

µg to the daily diets of the 204 women 19-44 y in Newfoundland whom they investigated post-

fortification, lower than the predicted 100 µg (32). Also, no women met the IOM‟s 400 µg

recommendation from food alone (32). Another report also shows that there is still

36

approximately 30% folate inadequacy among pregnant and lactating women based on diet alone

(128).

Liu et al., in the same paper mentioned above, also presented data on folate intakes among

186 older adults (≥65 y) in Newfoundland post-fortification, and found that mandatory

fortification added 74 µg to their diets (32). DeWolfe reported on folate intake pre- and post-

fortification in a convenience sample of 103 community-dwelling healthy seniors from the

Kingston, Frontenac, and Lennox and Addington area (139). To obtain post-fortification

estimates, DeWolfe re-analyzed pre-fortification intake data among this sample, assuming

mandatory fortification levels. DeWolfe reported that fortification added approximately 100 µg

to the diets of both older men and women, but the prevalence of inadequacy was still 20% and

43.4% in men and women, respectively (139).

In the only known post-fortification analyses that focuses specifically on children, Hennessy-

Priest et al. looked at intake in 254 children 3-5 y in five areas from across Ontario (129). They

showed that the prevalence of inadequacy was less than 1% among these children, and this was

primarily due to mandatory fortification (129). Interestingly, when supplement use was

considered, there was a small percentage (2%) of children with intakes above the UL (129).

The Provincial Nutrition Surveys, which were conducted in the 1990s, and the CCHS 2.2,

which was conducted in 2004, allow for estimation of province-wide and nationally-

representative folate intake and prevalence of inadequacy/intakes above the UL (140, 141).

Using the Provincial Nutrition Surveys, Dolega-Cieszkowski et al. showed the impact of

mandatory fortification on folate intake among Canadians by comparing the folate intake in

those provincial surveys conducted before and after mandatory fortification (142). They showed

that folate intake doubled in most adult age groups as a result of fortification (142). The more

37

recent CCHS 2.2 represents the country‟s first nationally-representative dietary intake data

collection since the early 1970s, and the only one post-fortification (142). Estimates of the

national prevalence of inadequacy from the CCHS 2.2 suggest that there is little inadequacy

among children and men, but approximately 25% of females ≥14 y are inadequate for folate

(143). However, there is no mention of what proportion of women consumed the IOM`s

recommended 400 µg daily, and neither supplement consumption nor possible folic acid

overages are incorporated in these estimates.

In conclusion, although there are several studies that report on folate intake among subgroups

of Canadians post-fortification of the food supply, there lacks a comprehensive assessment of the

entire population that takes into consideration both the contribution of supplemental folic acid

and folic acid overages that might be present in foods covered by the fortification mandate. The

body of literature indicates that folate intakes have increased in the post-fortification era, with a

likely decrease in the prevalence of inadequacy, but the extent of this is unknown until

nationally-representative data account for supplemental folic acid and folic acid overages in

foods. Lastly, whether or not women capable of becoming pregnant still need to consume a folic

acid-containing supplement to meet the IOM‟s recommendation of 400 µg/d in the Canadian

post-fortification era needs to be evaluated using nationally representative data.

2.4.2 Blood folate status post-fortification of the food supply

2.4.2.1 Reports from the United States

The post-fortification literature on blood folate status on those living in the United States is rich,

with several reports from the nationally representative NHANES surveys and the Framingham

Offspring Cohort. The general consensus of the literature indicates that blood folate status

improved dramatically immediately post-fortification, followed by a slight subsequent decline in

38

blood folate values (135). In fact, Lawrence et al., using data from a convenience sample of

98,351 specimens from a large Southern California Endocrinology Laboratory computer

database, showed that serum folate significantly increased even pre-mandatory fortification,

starting in 1997, probably reflecting the fact that manufacturers began to add folic acid to foods

before fortification became mandatory (144).

Authors of two key reports from the Framingham Offspring Cohort similarly show

improvements in blood folate status in the early post-fortification era when compared to the pre-

fortification era (145, 146). In 1999, Jacques et al. reported that mean plasma folate more than

doubled (from 4.6 to 10.0 ng/mL) from pre- to post-fortification among adults (n = 248) who

were not consuming folic acid containing supplements (146). Furthermore, the prevalence of

acute folate deficiency, defined as plasma folate <7 nmol/L, decreased significantly from 22% to

1.7% (146). The same group also compared RBC folate among respondents pre- and post-

fortification in the same Framingham Offspring Cohort (145). They found that mean RBC folate

significantly increased by 38% from pre- to post-fortification (737.1 to 1019.7 nmol/L), and the

prevalence of folate deficiency (RBC folate <362.6 nmol/L) significantly decreased from 4.9%

to 1.9% (145). They also excluded supplement users from their analysis, thereby eliminating the

possibility that the change in RBC folate was a result of increasing supplement use. However,

Yeung et al., in their analyses of serum folate values in NHANES 2001-2004, show that folic

acid supplement use is strongly associated with serum folate, and appears to be more-so than

folic acid from mandatory fortification (134)

Tables 2.4.2.1.a and 2.4.2.1.b summarize the findings of several reports using NHANES

cycles to investigate the change in both serum and RBC folate over the past few decades (127,

147-153). Several of these reports and others have also indicated a concurrent decline in the

39

prevalence of both acute and chronic folate deficiency from the pre-to-post-fortification eras

(147-150, 152, 153). In addition, several reports looked at racial disparities in folate status, and

generally conclude that while there have been improvements in blood folate status across all

races/ethnicities, there still remains room for improvement among non-Hispanic blacks (148-

154).

40

Table 2.4.2.1.a. Change in serum folate (nmol/L) concentrations in the United States from pre-to-post-fortification based on

NHANES analyses

Study Population Analyzed Measure NHANES Cycle

III (1988-

1994)

1999-

2000

2001-

2002

2003-

2004

2005-

2006

% Increase

CDC 2000

(147)

Females 15-44 y Mean 14.3 36.7 257%

CDC 2002

(148)

Females 15-44 y Median 10.9 29.5 271%

Pfeiffer et al.

2005 (149)

Children ≤ 5 y Geometric mean 23.1 45.5 197%

Pfeiffer et al.

2005 (149)

Children 6-11 y Geometric mean 19.3 43.8 227%

Pfeiffer et al.

2005 (149)

Adolescents 12-19 y Geometric mean 11.9 30.0 252%

Pfeiffer et al.

2005 (149)

Adults 20-39 y Geometric mean 10.5 26.2 250%

Pfeiffer et al.

2005 (149)

Adults 40-59 y Geometric mean 12.3 30.7 250%

Pfeiffer et al.

2005 (149)

Adults ≥ 60 y Geometric mean 16.6 39.4 237%

41

Dietrich et al.

2005 (127)

Adults ≥ 20 y Mean 11.4 26.9 236%

Ganji & Kafai

2006 (150)

All non-pregnant

respondents > 2 y

Geometric mean 12.1 30.2 27.8 a) 250%

b) 230%

CDC 2007

(151)

Females 15-44 y Median 28.6 25.8 24.0

Pfeiffer et al.

2007 (152)

All respondents ≥ 4 y Median 12.5 32.0 29.5 27.0 a) 256%

b) 236%

c) 216%

McDowell et

al. 2008 (153)

All respondents ≥ 4 y Median 12.5 32.2 27.6 a) 258%

b) 222%

42

Table 2.4.2.1.b. Change in red blood cell folate (nmol/L) concentration in the United States from pre-to-post-fortification based on

NHANES analyses

Study Population Analyzed Measure NHANES Cycle

III (1988-

1994)

1999-

2000

2001-

2002

2003-

2004

2005-

2006

% Increase

CDC 2000

(147)

Females 15-44 y Mean 410 714 174%

CDC 2002

(148)

Females 15-44 y Median 362 597 165%

Pfeiffer et al.

2005 (149)

Children ≤ 5 y Geometric mean 484 665 137%

Pfeiffer et al.

2005 (149)

Children 6-11 y Geometric mean 444 643 145%

Pfeiffer et al.

2005 (149)

Adolescents 12-19 y Geometric mean 345 558 162%

Pfeiffer et al.

2005 (149)

Adults 20-39 y Geometric mean 360 566 157%

Pfeiffer et al.

2005 (149)

Adults 40-59 y Geometric mean 412 667 162%

Pfeiffer et al.

2005 (149)

Adults ≥ 60 y Geometric mean 487 770 158%

43

Dietrich et

al. 2005

(127)

Adults ≥ 20 y Mean 375 590 157%

Ganji &

Kafai 2006

(150)

All non-pregnant

respondents > 2 y

Geometric mean 391 618 611 a) 158%

b) 156%

CDC 2007

(151)

Females 15-44 y Median 578 589 533

Pfeiffer et al.

2007 (152)

All respondents ≥ 4 y Median 394 625 621 576 a) 159%

b) 157%

c) 146%

McDowell et

al. 2008

(153)

All respondents ≥ 4 y Median 394 625 582 a) 159%

b) 148%

44

2.4.2.2 Reports from Canada

Several reports, many of which are based on convenience samples, indicate that both serum and

RBC folate levels have increased post-fortification of the food supply (32, 33, 155-160). Ray et

al. used cross-sectional data from 711 adults (mean age: 58.4 y) whose physicians ordered blood

folate and homocysteine tests from MDS Laboratories in Ontario (155). In this convenience

sample, the authors show that the mean serum and RBC folate values were 35.9 nmol/L and

1011.9 nmol/L, respectively (155). While this study lacked a pre-fortification control group, the

authors remark that the post-fortification blood folate values observed in their study are much

higher than reports from the United States pre-fortification.

Using this same province-wide laboratory database and this time comparing pre- and post-

fortification blood folate values from different individuals, the same group showed that there is

less folate deficiency post-fortification compared to pre-fortification among adults (1.78% vs.

0.41%, RR: 0.23, 95% CI: 0.14-0.40) (156). However, the 8,884 adults (mean age: 57.4 y)

included in this analysis were based on convenience sampling. Garcia et al. also reported on the

change in total blood (RBC plus serum) folate pre- to post-fortification among a sample of 281

community-dwelling older adults (157). They found a significant increase in total blood folate

among both supplement users and non-users. Furthermore, among non-supplement users, they

report that pre-fortification, only 3% of subjects had abnormally high folate (defined as >1300

nmol/L), compared to 84% post-fortification (157).

Liu et al. reported on the change in serum and RBC folate among a random sample of both

seniors (n = 202) and women 19-44 y (n = 233) in Newfoundland pre- to post-fortification (32).

They concluded that for both groups there were significant increases in serum and RBC folate in

the post-fortification era when compared to pre-fortification (32). This increase among women

45

corresponds with the decline in NTD occurrence in Newfoundland reported by the authors over

the same time period (Table 2.2.2.4.a). Ray et al. used blood folate values from the same MDS

Laboratories database to show that among a convenience sample of Ontarian women 18-42 y,

there was a significant increase in both serum and RBC folate when comparing blood values

from 8,408 women pre-fortification to those from 30,061 women post-fortification (158). The

authors don‟t present data on the percentage of women that have RBC folate values maximally

protective against a NTD-birth outcome (≥906 nmol/L). Shuaibi et al., in a convenience sample

of 95 women 18-25 y from the University of Manitoba, report that no women had a RBC folate

that was indicative of deficiency (33). However, only 14% of their subjects had RBC folate

values ≥906 nmol/L. Similarly, House et al. reported significant improvements post-fortification

in serum folate values in 365 pregnant women in Newfoundland when compared to values pre-

fortification (159). Also, in a convenience sample of 53 lactating women (16 weeks post-

partum) in Toronto, Houghton et al. reported that none had blood folate values indicative of

deficiency, and only 2 had RBC folate <906 nmol/L (160). However, the women in this sample

were expected to have good blood folate status, as all consumed 1 mg folic acid during

pregnancy and two-thirds continued to consume a folic acid-containing supplement post-partum

(160).

Perhaps the most comprehensive evaluation of the blood folate status of Canadians was an

analysis of data from the Canadian Health Measures Survey 2007-2009 (161). In their analyses

of RBC folate values from this nationally representative sample of 5,248 Canadians 6-79 y,

Colapinto et al. reported that less than 1% of Canadians are folate deficient, and approximately

40% have high folate concentrations, which they define as greater than 1360 nmol/L (161).

They set their cut-off for high folate based on the 97th

percentile from the RBC folate values in

46

the NHANES 1999-2004 datasets (161). Furthermore, they showed than 78% of women of

childbearing age have RBC values ≥906 nmol/L, which is, excluding the Houghton report on

lactating women, substantially higher than any previous reports.

In summary, available Canadian data suggest that there is very little folate deficiency post-

fortification of the food supply. However, there is still a proportion of women who are not

maximally protected against an NTD-affected pregnancy (RBC folate <906 nmol/L), but

estimates of the proportion vary. The most recent of these reports and only nationally-

representative one suggests that it is lower than other reports. However, measurement of RBC

folate is known to be assay- and laboratory-dependent, which likely accounts for some of the

observed differences (162, 163).

2.4.3 Mandated vs. actual levels of fortification

In their legislation of mandatory fortification, the FDA recognized folic acid fortification

overages as a potential source of underestimation of actual levels in foods (5). Manufacturers

add overages of nutrients to ensure that their products contain at the least the minimum mandated

amount throughout the shelf-life, and not surprisingly, this has been documented in the United

States in two studies early post-fortification (27, 49).

In 2000, Rader et al. published their comparison of actual food folate values to both

mandated and label values of 83 of the top-selling products covered by the fortification mandate

(27). They found that in most cases, actual food folate values were higher than that reported on

the food label and the regulation value, reaching twice the level in some foods (27). The authors

concluded that while some of this might be due to higher-than-expected levels of endogenous

folate, fortification overage was likely the larger contributing source (27). In 2004, Johnston and

Tamura published their analyses of the folate content of 46 white breads over three years (2001,

47

2002, 2003) (49). They showed there was a significant decline in folate content of the breads in

2002 and 2003 when compared to 2001 (49). The authors of both papers called for monitoring

of fortification levels in the United States (49).

Taken together, the findings of these two papers indicate that the actual levels of fortification

in the United States were different (higher) than mandated levels and were not constant.

Therefore, the impact of the folic acid fortification program cannot be properly evaluated,

because it would appear that the current reduction in NTD occurrence in the United States is a

result of levels higher than 140 µg/100 g of product. Interestingly, the changing levels of

fortification observed by Johnston and Tamura closely mimic the rise and fall of blood folate

over the same time period in the United States (135).

Although mandatory folic acid fortification in Canada has been implemented for nearly as

long as that in the United States, there are no published reports on direct analyses of fortified

foods to determine the actual level of fortification. However, Quinlivan and Gregory, in a letter-

to-the-editor to the Canadian Journal of Public Health, published an estimate of the impact of

folic acid fortification in Canada (29). Using data from a provincial clinical care laboratory on

change in serum folate pre- to post-fortification and a linear regression equation derived from

previous literature relating serum folate to folic acid intake, they estimate that the observed

change in serum folate is consistent with an extra 150 µg/day and not the intended 100 µg/day

(29). They therefore suggest that fortification levels are 50% higher than mandated levels (29),

and this remains the only estimate of actual fortification levels in Canada. There is arguably

more need for monitoring the Canadian fortification levels, or at the very least, an analysis of a

representative sample of fortified foods, because unlike the United States, the Canadian

48

regulations don‟t stipulate a maximum fortification level (4), which theoretically leaves an

unlimited potential for overages.

Furthermore, there have been calls to increase the levels of fortification to further reduce the

incidence of NTDs (164, 165). These calls are premature, because other than the Quinlivan &

Gregory prediction estimate (29), there is no sense of what the actual levels of fortification are in

Canadian foods, and how they affect intake among Canadians.

While nutrient overages in foods due to fortification have been documented in the United

States for over several years, until recently, very little attention has been paid to the possibility of

nutrient overages in dietary supplements. However, research from the USDA as part of the

Dietary Supplement Ingredient Database project is now showing that dietary supplements also

contain nutrient overages (166).

2.5 RBC FOLATE AS AN INDICATOR OF FOLATE STATUS AND RISK OF NTDS

2.5.1 RBC Folate: An Indicator of Long-term Folate Status

Folate is only incorporated into pre-erythropoietic cells in bone marrow during synthesis of

RBCs and not directly into circulating mature RBCs during their 4-month life span, making RBC

folate a good indicator of long-term folate status (9). This is also important because it prevents

fluctuations in RBC folate concentration due to temporary changes in dietary folate intake (9).

Furthermore, RBC folate concentration is also thought to be a good indicator of intracellular and

tissue stores of folate (9, 167).

An RBC folate concentration above 305 nmol/L was established by the IOM as the cut-off

for adequate folate status based on the results of several experiments in which abnormalities in

bone marrow function were observed (9). Three individuals on a low folate diet (<20 µg/d)

presented with hypersegmented neutrophils, an indicator of a bone marrow abnormality, when

49

RBC folate concentrations reached less than 305 nmol/L (62, 63). Further, in their study of 120

patients looking at a simplified microbiological assay to measure RBC folate, Hoffbrand et al.

reported that 40 patients presented with megaloblastic anemia due to folate deficiency; all 40 had

RBC concentrations less than 305 nmol/L (65). Also, in a British study, all 238 pregnant women

with megaloblastic bone marrow had RBC folate concentrations less than 327 nmol/L (64).

While an RBC folate concentration above 305 nmol/L is thought to represent adequate folate

status, a higher cut-off was established as maximally protective against an NTD (36). Daly et al.

investigated the relationship between RBC folate concentration early in pregnancy and incidence

of NTD in over 50,000 births in Ireland between 1986 and 1990 (36). They found that there was

greater than three-fold increase in risk of NTD in women with RBC folate concentrations below

700 nmol/L compared to women with RBC folate concentrations at or above 906 nmol/L (36).

Thus, RBC folate at or above 906 nmol/L in a woman is thought to maximally protect against an

NTD. Ren et al. have confirmed this by showing that 695 pregnant women recruited from a

prenatal health centre in an area of low NTD-prevalence (0.83/1000 births) in China had a mean

RBC folate of 910 nmol/L, much higher than a similar group of 562 pregnant women from an

area of high NTD-prevalence (440 nmol/L) (37).

2.5.2 The Need to Identify Women at Risk for a folate-dependent NTD

Daily folic acid-containing supplement consumption remains an important component in

protection against an NTD birth outcome even in the age of mandatory folic acid fortification

(31, 33, 137). However, supplemental folic acid doses available to women capable of becoming

pregnant include 400 µg, 600 µg, 700 µg, 1000 µg, 1100 µg and up to 5000 µg. This large range

of available doses leaves confusion as to what a woman should choose. Ironically, the UL of

intake for adult females is 1000 µg (9). Furthermore, physicians have little evidence-based

50

guidance on which they can rely in advising women. This often leads to higher doses being

recommended to maximally protect against an NTD-birth outcome (34). But what are the costs

of such a practice? While some women are at increased risk of an NTD-birth outcome and need

a higher dose, this is not the case for the majority of women, and an unnecessary high dose of

folic acid might have harmful effects (summarized here; discussed in detail in Section 2.2.7). In

addition to the long established masking and progression of vitamin B12 deficiency (16), there is

growing evidence of other harmful effects of too much folic acid, including cancer progression

and the reduced effectiveness of antifolate drugs (17, 21, 78). Furthermore, folic acid

supplementation during pregnancy has been recently associated with an increased risk of asthma,

obesity and insulin resistance in the offspring (24, 25).

To further complicate matters, measurement of RBC folate concentration, the gold standard

for determining folate status, is not routinely assessed in women and would be expensive to

incorporate as part of routine pre-conceptional care. There are approximately 370,000 births per

year in Canada (168), and, at an estimated cost of $50 per RBC folate assay for every woman

who will become pregnant, this would add $18.5 million annually to the cost of pre-conceptional

care. Since this is not likely to be implemented, there remains a need for a short nutritional tool

to identify women at an increased risk of an NTD-birth outcome in order to confine high dose

recommendations to these women, such that all women in this country who are capable of

becoming pregnant are not recommended an unnecessarily high and potentially harmful dose of

folic acid. An important first step in such a tool is to determine factors that are predictive of

RBC folate. Given the well established literature between RBC folate and NTD-risk (36, 37),

RBC folate status (above or below 906 nmol/L) is a logical surrogate measure of NTD-risk.

51

Therefore, factors associated with RBC folate status need be identified in order to be

incorporated into a tool designed to estimate NTD-risk among women of childbearing age.

2.5.3 Factors associated with RBC folate concentrations

The factors discussed in this section were initially primarily identified from Health Canada‟s

“Nutritional Guideline for a Healthy Pregnancy – Questions and Answers for Health

Professionals” (Appendix A) and a review by Tam et al. (169).

2.5.3.1 - Diet – Fruit and Vegetable Intake & White Wheat Flour Consumption

Before folic acid fortification of select grain products in Canada and the United States in 1998,

fruits and vegetables were the main contributors of dietary folate (127, 128). Ford and Bowman

used NHANES III (1988-1991) to show that dietary folate intake is significantly correlated with

RBC folate among adults when both variables are treated as continuous (170). Even post-

fortification, they remain a large folate contributor (127, 128). Since mandatory fortification of

white wheat flour with folic acid in 1998, the grains food group has become the largest

contributor to total dietary folate intake (127, 128). In both Canada and the United States, there

have been improvements in both folate intake and blood folate indices post- versus pre-

fortification (32, 127, 145, 146, 156, 158, 171).

2.5.3.2 - Dieting

Since grains have become the largest contributor to total folate intake in both Canada and the

United States post-fortification (127, 128), weight loss diets that propose to work through low-

carbohydrate intake leave individuals subscribing to such diets at risk of a low total folate intake

(and as a result, lower RBC folate), due to avoidance of folic acid-fortified foods such as white

bread (135). Given the current obesity epidemic in North America, weight loss diets, including

low-carbohydrate diets, are fairly common (172). In the 2005 Gallup poll, over one quarter of

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dieting women aged 18-45 y in the United States reported being on a low-carbohydrate diet in

the past 6 months (172). Furthermore, only 37% of these women regularly consumed a

supplement containing folic acid (172).

2.5.3.3 - Cooking Methods

In some cultures, stewing or boiling of food for long durations are common cooking methods.

While folic acid used as a fortificant and in supplements is very stable, naturally occurring food

folate can be oxidized, destroyed by heat, and, since it is water soluble, can leach into cooking

water (9, 173, 174). Furthermore, folate loss is variable; in their food analyses, McKillop et al.

show that the retention of natural folates in foods depends on both the food in question and the

method of cooking (174). They found that, of beef, potato, broccoli, and spinach, retention was

poorest in broccoli and spinach. In fact, boiling for 3 to 5 min led to over 50% loss of folate

from these foods, while negligible differences were observed with steaming (174). It is thus

thought that the majority of folate loss in cooking is due to leaching from foods into cooking

water (174, 175), and green vegetables are particularly susceptible to loss of folate in cooking

methods using water (174).

2.5.3.4 - Smoking

Smoking is thought to alter blood folate status by impairing folate metabolism (176). In a cross-

sectional study of 80 pregnant Canadian women, the authors report that smokers had a

significantly lower serum folate and non-significantly lower RBC folate than nonsmokers (177).

Also, in a more recent cross-sectional study comparing chronic smokers (n = 35) and

nonsmokers (n = 21), Gabriel et al. showed that, even after correcting for age, alcohol

consumption, and dietary folate intake, chronic smokers had significantly lower RBC folate

when compared to nonsmokers (P < 0.0001) (178). Furthermore, in their analyses of NHANES

53

III, Mannino et al. report that among adults, smokers and those exposed to a high level of second

hand smoke had significantly lower RBC folate concentrations than nonsmokers and those

exposed to low levels of smoke, independent of diet (132).

Smoking is also associated with lower folate intake (132). In a large prospective cohort

study of Swedish men and women not exposed to folic acid fortification (n = 81,922), the

authors report at baseline that both men and women with higher total folate intakes were less

likely to smoke (179). Also, using data from NHANES III (1988-1991), which was conducted

pre-fortification, Mannino et al. report that folate intake was significantly inversely associated

with smoking/cigarette smoke exposure (132).

2.5.3.5 - Personal/Family History of NTDs

It is well established that women who have had a previous birth affected by an NTD are at

increased risk of a recurrence of an NTD (180), and that folic acid supplementation can reduce

the risk of recurrence (10). In a 1980 report, using pooled data from 831 American women who

had a previous pregnancy affected by a NTD, Cowchock et al. found a NTD-recurrence rate of

3% (95% CI: 2.0 – 4.3) (181). Furthermore, in a case-control study from the United Kingdom,

women who have had a prior pregnancy affected by an NTD were shown to have altered folate

metabolism (182); in their study of 29 women who had a previous pregnancy affected by an

NTD and 29 control women, Wild et al. reported that the relationship between RBC and serum

folate was different between women who had a previous NTD-affected pregnancy and those who

didn‟t (182). In a small case-control study, Neuhouser et al. showed that women with a previous

NTD-affected pregnancy (n = 10) absorbed significantly less synthetic folic acid than healthy

controls (n = 8) (183). Furthermore, the American Academic of Pediatrics‟ Committee on

Genetics report that women who have a close relative (e.g. mother, sister, aunt) who has had a

54

previous birth affected by an NTD may be at increased risk of an NTD-affected pregnancy and

should take precautions (184). However, we were unable to indentify primary research on

family history of NTD and increased risk.

2.5.3.6 - Medications that Interfere with Folate Metabolism

There are several medications that are known to be folic acid antagonists and thus interfere with

folate metabolism (9, 185-189). The first group of medications (methotrexate, sulfasalazine,

pyrimethamine, triamterene, trimethoprim and aminopterin) work by enzyme inhibition (190).

They inhibit the enzyme dihydrofolate reductase, thereby blocking folate from being converted

to its metabolically active forms (190). The second group of medications act in several ways,

either by inhibiting other enzymes in the folate-dependent pathways, decreasing absorption of

folate, or increasing its degradation (186). These medications are usually used in the treatment

of epilepsy, and include carbamazepine, phenytoin, primidone, and phenobarbital (186).

2.5.3.7 - Alcohol Abuse

Alcohol interferes with folate metabolism and, in heavy drinkers, may be associated with low

dietary folate intake (191). According to the Canadian Addiction Survey in 2004, 16% of those

above the age of 15 who drink reported drinking 5 or more drinks per day in a drinking day

(192). Furthermore, 6.2% and 25.5% of drinkers reported heavy drinking at least once a week

and at least once a month, respectively, with heavy drinking defined as five or more drinks in a

single sitting for males and four or more drinks in a single sitting for females (192). While beer

is the only alcoholic beverage that contains appreciable amounts of folate, folate concentrations

vary widely according to brand (193). Despite this, since heavy drinkers tend to restrict their

diets, they are likely to consume a diet that is low in foods rich in folate content. Furthermore,

high concentrations of ethanol in the small intestine impair the absorption of folate (194-196).

55

Additionally, because chronic alcoholism leads to alcoholic liver disease, altered hepatic folate

metabolism is common in chronic alcoholics, due to decreased hepatic uptake of folate resulting

in lower folate stores (197-199). Furthermore, alcohol consumption has been shown to increase

renal excretion (200) and oxidative destruction of folate (201).

2.5.3.8 - Malabsorption/Gastric Bypass Surgery

Gastric bypass surgery is thought to lead to several nutritional deficiencies (202, 203). While

some do suggest compromised folate status (204), the general consensus is that regular

adherence to a multivitamin supplement can prevent folate deficiency after gastric bypass

surgery (202, 205, 206).

Malabsorption independent of gastric bypass surgery also leads to folate deficiency (207). In

a study that included 73 Austrian adults, those who presented with malabsorption of fructose (n

= 46) had significantly lower plasma folate concentrations than adults who didn‟t (n = 27) (208).

Similarly, the authors of a case report of a woman suffering from malabsorption concluded that

recurrence of cleft lip and palate among her offspring was likely due to vitamin deficiency,

including folate (209). However, low folate status due to malabsorption is thought to be

reversible with adequate folic acid supplementation (207).

2.5.3.9 - Liver Disease/Kidney Dialysis

Liver disease affects folate status through altered folate metabolism; it has been long-established

that folate deficiency is associated with liver disease (210). This is thought to be due to

decreased hepatic uptake, and increased urinary excretion of folate, which might be due to poor

kidney reabsorption of abnormally large release of folates from a diseased liver (210, 211). In a

Chilean study, the authors looked at non-alcoholic fatty liver disease in obesity and found that,

with or without non-alcoholic fatty liver disease, patients had normal serum folate values (above

56

6.8 nmol/L) (212). Even though the authors didn‟t collect dietary intake data, the fact that all

women had normal serum folate values is likely due to the fact the government of Chile

introduced mandatory folic acid fortification of wheat flour in 2000, thereby exposing the entire

population to the vitamin (212). Nonetheless, the authors report that obese women with the

disease (n = 17) had significantly lower serum folate concentrations than obese women without

the disease (n = 26) (212).

Kidney dialysis also exerts its effects on folate status through altered folate metabolism; folate

is thought to be loosely bound in serum and thus easily enters the dialysis fluid (213, 214). This

is an acute effect, and the notion that kidney dialysis affects chronic folate status has been

challenged (215-218). For example, in their study of 94 dialysis patients, Lee et al. (1999) found

that only 6 (6.3%) patients had low RBC folate levels, while 44 (46.8%) had low serum folate

levels, suggesting acute but not chronic folate deficiency (216). The majority of the literature

points to no chronic effect of kidney dialysis on folate status (215-218).

2.5.3.10 - Diabetes

Women with pre-existing (i.e. not gestational) diabetes are at an increased risk of a birth affected

by congenital anomalies including NTDs (219, 220). Data from rat studies suggest that there is

impaired folate metabolism in diabetics; rats with induced diabetes had increased folate losses

from liver and increased urinary folate excretion when compared to controls (221). However,

Kaplan et al. report no altered folate metabolism in their comparison of several indices of folate

metabolism in pregnant diabetic and non-diabetic women (222). Among the 15 diabetic and 34

non-diabetic women, there were no differences in serum folate, RBC folate and urinary folate

excretion (222). The authors suggest that the mechanism behind the observed increased risk of

birth defects due to diabetes is unknown and likely to be folate-independent (222).

57

To further complicate matters, diabetics are advised to choose whole grain products more

often and minimize consumption of high glycemic index foods such as white wheat flour

products because of the rapid increase in blood glucose that follows consumption (223). Since

mandatory folic acid fortification currently covers white wheat flour and not whole wheat flour,

there is potential for low folic acid intake among diabetics. Therefore, while diabetes may not

affect folate metabolism, it has the potential to affect folate status due to low dietary folic acid

intake among diabetics.

2.5.3.11 - Summary

There is a need for more research on the determinants of RBC folate. There is currently a large

gap in the literature on the proportion of women who have sub-optimal RBC folate status (<906

nmol/L) and on factors associated with sub-optimal folate status among these women. Further

investigation of the relationship between RBC folate status and the aforementioned factors will

allow for identifying candidate variables for inclusion in a screening tool that aims to capture

NTD-risk through the surrogate variable RBC folate.

2.6 VITAMIN/MINERAL SUPPLEMENT CONSUMPTION

In sections 2.2 to 2.5, the literature pertaining to the first, second, and third thesis data chapters

has been summarized. This section will focus on a review of the literature pertaining to the

fourth data chapter, vitamin/mineral supplement consumption, including a summary of

prevalence and determinants of consumption, comparison of the diets of users and non-users, and

the effect of supplement consumption on the diet.

2.6.1 The role of supplements

In Canada, there is no clear guidance on the role of vitamin/mineral supplements. While the

American Dietetic Association recommends consuming adequate nutrients from a wide variety

58

of foods as opposed to supplements (3), the use of dietary supplements, including

vitamin/mineral supplements, is now common in Canada (45), the United States (8, 39), and

elsewhere (224). Users commonly cite improvement and maintenance of health as reasons for

consuming a supplement (45). Even though supplemental nutrient sources have the ability to

contribute to total intake, until recently, and primarily for methodological reasons, they have

been largely overlooked. However, in the last few years, a body of literature has emerged

suggesting that the use of supplements can have a large impact on total nutrient intake (38, 41,

43).

2.6.2 Prevalence and determinants of consumption

2.6.2.1 The United States

There is a wealth of data on the prevalence and determinants of supplement consumption in the

United States, which stem from analyses of NHANES and other large surveys (6, 8, 38, 41, 43,

44, 225-230). Prevalence of consumption reported in key studies is presented in Table 3.5.2.1.

Among children and adolescents ≤18 y, the following maternal factors were predictive of

supplement use: having an older, more educated mother who is non-Hispanic white, married,

insured, has a higher household income and took supplements during pregnancy (225). Also,

supplement use was associated with being the first born child, lower body mass index (BMI),

higher milk and dietary fibre intakes, lower total fat and cholesterol intakes, greater food

security, less time spent on sedentary activities, living in a smoke-free environment, and better

health care access (41, 228, 230). Additionally, from diet alone, supplement users had higher

mean intakes of most micronutrients when compared to non-users (42, 44).

59

Among adults, greater supplement use was associated with female sex, older age, more

education, non-Hispanic white race, physical activity, normal/underweight, wine/distilled spirit

consumption, former smoking, and better self-reported health (8, 43, 229).

60

Table 2.6.2.1. Prevalence of dietary supplement consumption in the United States

Study Data Source Population Prevalence of supplement use

(% [SE])

Yu et al., 1997

(225)

Follow-up to the National Maternal

and Infant Health Study, 1991

3 y; n = 8,285 54.4

Balluz et al.,

2000 (226)

NHANES III (1988-1994) a) 1-5 y

b) 6-11 y

c) 12-19 y

d) ≥20 y

a) 42.0 – 51.0

b) 33.5 – 35.4

c) 23.9 – 28.0

d) 29.7 – 54.4

Stang et al.

2000 (44)

Continuing Survey of Food Intakes of

Individuals (CSFII), 1994

13-18 y; n = 423 33.8

Dwyer et al.,

2001

Third Child and Adolescent Trial for

Cardiovascular Health (CATCH)

Study, 1997

14 y; 1,532 17.6

Radimer et al.,

2004 (8)

NHANES 1999-2000 ≥20 y; n = 4,862 52.0 (1.4)

Millen et al.,

2004 (227)

National Health Interview Survey,

1987, 1992, 2000

≥20 y

a) 1987

b) 1992

c) 2000

a) 28.7 (0.4)

b) 29.2 (0.5)

c) 39.3 (0.4)

Briefel et al., Feeding Infants and Toddlers Study, a) 4-5 mo; n = 624 a) 8

61

2006 (41) 2002 b) 6-11 mo; n = 1,395

c) 12-24 mo; n = 1,003

b) 19

c) 31

Picciano et al.,

2007 (228)

NHANES 1999-2002 a) 1-3 y; n = 1,638

b) 4-8 y; n = 1,974

c) 9-13 y; n = 2,441

d) 14-18 y; n = 3,043

a) 38.4 (2.3)

b) 40.6 (2.7)

c) 28.9 (1.6)

d) 25.7 (1.2)

Murphy et al.,

2007 (38)

Hawaii-Los Angeles Multiethnic

Cohort (MEC), 1999-2001

≥45 y; n = 100,196 48-56%

Rock, 2007

(229)

NHANES 1999-2000 ≥20 y 52%

Sebastian et al.,

2007 (43)

CSFII, 1994-1996 ≥51 y; n = 4,384 37 – 47

Shaikh et al.,

2009 (230)

NHANES 1999-2004 a) 2-4 y

b) 5-11 y

c) 12-17 y

a) 43.1 (2.0)

b) 37.4 (1.7)

c) 26.6 (0.9)

Bailey et al.,

2011 (6)

NHANES 2003-2006 a) 1-3 y

b) 4-8 y

c) 9-13 y

d) 14-18 y

e) 19-30 y

f) 31-50 y

a) 39 (1)

b) 43 (2)

c) 29 (2)

d) 26 (2)

e) 39 (1)

f) 49 (1)

62

g) 51-70 y

h) ≥71 y

g) 65 (2)

h) 71 (1)

63

2.6.2.2 Canada

There is considerably less data on the prevalence of supplement use in Canada than there

are in the United States. A few studies in subgroup populations, along with analyses of

the nationally-representative CCHS 2.2 and Baseline Natural Health Products Survey

Among Consumers comprise the bulk of the supplement consumption literature in

Canada.

Troppmann et al. looked at supplement use in Canada using data from the Food

Habits of Canadians Survey 1997-1998 (231). They found that, in this nationally-

representative sample of 1,543 adults 18-65 y, 38% reported consuming vitamin/mineral

supplements (231). McKenzie and Keller reported that 72% of respondents in their

convenience sample of community-dwelling elders in Ontario used a vitamin/mineral

supplement, while 31% consumed a multivitamin/mineral supplement (232). The

Baseline Natural Health Products Survey is a national random telephone survey that was

conducted in 2005 (45). In this study, 40.5% of respondents reported consuming a

vitamin/mineral supplement (45). Guo et al. analyzed data from the CCHS 2.2 and

showed that among the 15,553 respondents aged 19-70 y, the prevalence of

vitamin/mineral supplement consumption was 40.1% (7). They also reported that female

sex, older age, physical activity, fruit/vegetable consumption, higher household income

and education were associated with supplement use (7).

2.6.3 Difference between supplement users and non-users and contribution of

supplements to total dietary intake

2.6.3.1 The United States

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A handful of studies from the United States explore the role of supplemental nutrient

contribution to the diet. Stang et al. used data from the 1994-1996 Continuing Survey of

Food Intakes of Individuals to compare the diets of supplement users and non-users (44).

They found that from diet alone, users had higher mean intakes of most micronutrients

and lower intakes of total and saturated fat than non-users (44). The authors did not

investigate the contribution of supplemental nutrients to the overall nutrient intake of

users. Similarly, using data from the Third Child and Adolescent Trial for

Cardiovascular Health study, Dwyer et al. show that from diet alone, adolescent

supplement users had higher mean intake for 16 of the 20 micronutrients they

investigated (42). The authors also did not look at supplemental nutrient contribution to

overall nutrient intake.

Briefel et al. compared the diets of supplement users and non-users in the Feeding

Infants and Toddlers Study (41). The authors found no significant differences in the

mean intakes of all nutrients studied between users and non-users from food alone (41).

However, when supplemental nutrient contribution was considered, users had

significantly higher mean intakes of vitamins A, C, D, E, B-12, folate, and iron and zinc

(41). Using Continuing Survey of Food Intakes of Individuals data, Sebastian et al.

showed that among adults >50 y, from diet alone, users had a significantly lower

prevalence of inadequacy than non-users for vitamins A, B-6, C, folate, and zinc and

magnesium (43). Furthermore, the inclusion of supplements led to more than 80% of

users consuming more than the EAR for most nutrients (43). However, supplement use

also led some users to exceed the UL for iron, zinc and vitamin A (43). Murphy et al.

show similar findings in their analysis of the Hawaii-Los Angeles Multiethnic Cohort;

65

users had a lower prevalence of nutrient inadequacy than non-users from diet alone and

supplemental contribution increased the adequacy of the population (38). However,

supplement use also led to intakes above the UL for iron, zinc, vitamin A and niacin (38).

2.6.3.2 Canada

A paper by Troppmann et al. constitutes the only study in Canada in which the diet of

supplement users was compared to that of non-users (233). They used data from 1,530

adults 18-65 y in the Food Habits of Canadians Survey (1997-1998) to show that from

diet alone, multivitamin/mineral supplement users had similar mean intakes to non-users

for most nutrients (233). However, including multi-vitamin/mineral supplemental

nutrient contribution led to significantly higher mean intakes among users compared to

non-users (233). They also show that supplement use led to >5% of the population

consuming above the UL for niacin, folic acid, calcium and iron (233).

Apart from being an older study, the study is limited in the fact that the authors based

intake on one 24-h recall; as such, usual (long-term) intake was not estimated. Therefore,

estimation of inadequacy and excessive intakes are prone to bias. Furthermore, the

analyses included only 6 nutrients, and cannot be generalized to all other micro-nutrients.

Also, the study only looks at adults 18-65 y (233). There remains a need for a more

comprehensive analyses that is based on the most recent nationally-representative data

intake collection in Canada from all ages in order to better evaluate the differences

between supplement users and non-users, and to assess the impact of supplemental

nutrient contribution to the diet of users.

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CHAPTER 3.0: THESIS STUDY #1

HOW MUCH FOLATE IS IN CANADIAN ENRICHED PRODUCTS 10 YEARS

AFTER MANDATED FORTIFICATION?


Shakur, Y.A, Rogenstein, C., Hartman-Craven, B., Tarasuk, V. & O‟Connor, D.L. How

much folate is in Canadian enriched products following mandated fortification?

Canadian Journal of Public Health 2009; 100(4):281-284.

- Reprinted with permission of the Canadian Public Health Association.

Originally published in the Canadian Journal of Public Health.

67

3.1 ABSTRACT

Objective: In 1998, the Canadian government mandated folic acid fortification of white

flour and enriched pasta to lower the prevalence of NTDs. There is now growing concern

over the potential harmful effects of too much folic acid on some segments of the

population. Given that the actual amount of folate in Canadian foods is unknown, the

objective of this study was to measure the folate content in selected fortified foods.

Methods: Using data from the 2001 Food Expenditure Survey and the ACNielsen

Company, 95 of the most commonly purchased folic acid fortified foods in Canada were

identified. Folate concentrations in these foods were determined using tri-enzyme

digestion followed by microbiological assay. Analyzed values were compared to those in

the Canadian Nutrient File (2007b, CNF) and to label values.

Results: The analyzed folate content of foods was, on average, 151% ± 63 of the CNF

values. Analyzed values as a percent of CNF values ranged from 116% in the “rolls and

buns” category to 188% in “ready-to-eat cereals”. Analyzed values were higher than

label values for “breads”, “rolls and buns” and “ready-to-eat cereals” (141%, 118% and

237%, respectively [P<0.05]).

Conclusions: Ten years after folic acid fortification of the food supply, neither the CNF

nor label values accurately reflect actual amounts of folate in foods. Further, overage

differences by food category hinder the development of future strategies designed to

strike the right balance between health benefits and risks; monitoring of fortified foods

for their nutrient content is required.

MeSH Keywords: folic acid, Canada, food supply, fortified food

68

3.2 INTRODUCTION

Folate, a B-vitamin, is necessary for proper neural tube development, which occurs early

after conception when most women are still unaware they are pregnant (9). In light of

this, women of child-bearing age are encouraged to consume a supplement containing

folic acid (a synthetic form of folate); however, Canadian data suggest only 57.7% of

women report taking folic acid during the peri-conceptional period (234). Therefore in

1998, the Canadian government mandated folic acid fortification of all white flour and

enriched pasta to 150µg/100g and 200µg/100g, respectively, in order to increase daily

intake by 100µg (4).

Since folic acid fortification has become mandatory, the incidence of NTDs has

declined in Canada by approximately 50% (14), with improvements in blood folate

indices (32, 156) and folate intake (32). Given the apparent success with this

intervention, there have been calls to raise the levels of folic acid fortification in Canada

to further reduce the incidence of folate-dependent NTDs (165, 235). In addition to this,

suboptimal intakes of folate have also been associated with other congenital defects (cleft

lip and palate), vascular disease, neuropsychiatric disorders, and cancer (9).

However, a growing body of literature suggests that consuming high levels of folic

acid may have several negative consequences beyond the masking and progression of

vitamin B-12 deficiency (9), including cancer progression (21, 78), and reduced natural

killer cell cytotoxicity (18). In one recent study, a combination of high folate levels and

low vitamin B12 status has been associated with increased cognitive impairment in

seniors (16); in another recently published study, the same combination in pregnant

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women was associated with increased insulin resistance and higher central adiposity in

their children (25).

When evaluating the current Canadian situation, the arguments on either side of the

“how much folic acid should be added to the food supply?” debate are hampered by a

lack of understanding of how much folate is actually in the foods we eat. In the early

years after mandated fortification, analysis of folate in foods indicated actual levels were

twice that mandated in the United States (27). Researchers from a subsequent study

showed that the levels of fortification in the United States have declined since an initial

post-fortification high, and that monitoring of folic acid fortification may be necessary

(49). In the 10 y following mandated folic acid fortification, there have been no

Canadian studies in which foods have been directly analyzed and compared to label

values or Canada‟s main reference nutrient database, the Canadian Nutrient File (CNF).

Therefore, we determined the folate content in a selection of folic acid fortified foods in

Canada.

3.3 METHODS

Selection of foods

The choice of products for analysis was based on a comprehensive review of the most

commonly purchased food products across households in Canada, combining data from

the 2001 Food Expenditure Survey (FOODEX)(236) with more detailed data from the

ACNielsen Company (Markham, Ontario) on brands of food purchased. The 2001

FOODEX contains household level data from over 10,000 dwellings collected throughout

2001 and is representative of 98% of the Canadians. Trained interviewers recorded

detailed information on food expenditures for each household for 2 weeks. The

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FOODEX data were used to identify the number of households reporting the purchase of

a given food category, considering only categories where foods were fortified with folic

acid. These categories were: 1) breads; 2) buns and rolls; 3) cookies; 4) ready-to-eat

cereals; 5) prepackaged desserts; 6) cooked pasta; and 7) crackers. Data purchased from

the ACNielsen Company identified the top 25 food brands sold in each of the

aforementioned categories. From this list, we analyzed the top 15 fortified food brands

from each food category that were purchased by more than 20% of households, and the

top 10 brands of fortified food from food categories purchased by 15-20% of households.

Products were purchased from supermarkets in Toronto, Montreal, or Vancouver

(depending on availability) between January and July of 2007 and analyzed prior to their

expiration date.

Folic acid content based on CNF and label values

In Canada, the Nutrition Facts panel on food products reports folate content as %

Daily Value (237). The Daily Value of 220µg for folate is set at the 1983 Recommended

Nutrient Intakes for Canadian adult males 18 years of age and older (238). Using this

value, the amount of folate claimed per serving was calculated. The CNF values were

obtained directly from the most up-to-date version of the database (2007b)(239) available

from Health Canada‟s website, which, in turn, is based primarily on the USDA Nutrient

Database for Standard Reference (240). The CNF values are modified where needed to

reflect current Canadian regulations for mandatory folic acid fortification. Three brands

of pasta that were identified using the ACNeilsen data were whole wheat pasta. While all

three brands contained folic acid, they were not subject to mandatory fortification and

thus excluded from all subsequent analysis.

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Laboratory analysis

All samples were analyzed as purchased except for pasta, which was prepared

according to directions on the package. Samples were homogenized with a 50mM

CHES-HEPES buffer with 2% ascorbic acid and 0.2 M 2-mercaptoethanol (pH 7.8) and

stored at -80°C until analysis (28, 241). Aliquots of the thawed homogenate were treated

to liberate folates from food matrices and binding proteins and convert folates to their

microbiologically assayable form using the tri-enzyme digestion method (28, 241, 242).

Total folate concentration of the resultant supernatants were assessed by microbiological

assay using Lactobacillus rhamnosus (ATCC 7649; American Type Tissue Culture

Collection, Manassas, VA) (243). The accuracy and reproducibility of these assays were

assessed using lyophilized liver with a certified value (13.3 mg folate/kg, Pig Liver BCR

487, IRMM, Geel, Belgium). Our analysis yielded a folate concentration of 13.4 ± 1.12

mg/kg, with an overall CV of 8.4%.

Statistical analysis

Data are presented as means and standard deviations. All statistical analyses were

performed with the SAS software (version 9.1; SAS Institute Inc., Cary, NC). Paired t-

tests were conducted comparing analyzed folate results in each food category to the CNF

and label values. A P-value < 0.05 was considered significant.

3.4 RESULTS

Results from a total of 92 fortified food products in seven food categories are

presented. For all foods, the mean analyzed folate content over the CNF values was

151% ± 63. Analyzed values were higher than the CNF reported folate content for all

food categories except “cooked pasta” and “crackers” (P<0.05) (Table 3.4.a). “Ready-

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to-eat cereals”, on average, contained the highest amount of folate relative to the CNF

values (188% ± 57) (Table 3.4.a); 12 and 14 of the 15 cereal brands analyzed had folate

contents greater than 1.5 times the CNF and label values, respectively. Folate was only

reported on package labels in the “breads”, “rolls and buns”, and “ready-to-eat cereals”

categories. A comparison of analyzed and label folate values is presented in Table 3.4.b.

The analyzed values were higher than the label values in all three categories (P<0.05).

3.5 DISCUSSION

The findings of this study indicate that 10 y after mandated folic acid fortification of

the food supply in Canada, there remains a significant disjuncture between the actual

folate content of fortified foods versus those reported in the CNF and on food labels,

which themselves are usually derived from the CNF. The actual folate content of the

foods as a percentage of the CNF reported amounts was, on average, 50% higher. As far

as we are aware this is the first direct assessment of the actual amount of folate in the

Canadian food supply since folic acid fortification became mandatory. Data presented

herein are consistent with that estimated in a letter-to-the-editor by Quinlivan & Gregory

in which they predicted that blood folate values post-fortification were about 1.5 times

higher than anticipated (29). The blood folate data used for this estimate were those from

specimens sent to a large provincial laboratory in Ontario as part of clinical care.(158)

At a national level, decisions on what nutrients should be fortified in the food supply,

and at what levels, are made using a risk management approach (244). First, clear

evidence must exist that there is a nutritional problem of public health significance and

that other strategies to address the short-fall in dietary intake will be or have been

ineffective. The case for folic acid fortification in Canada clearly meets this first

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criterion. Beyond a doubt, folic acid fortification of the food supply has improved the

folate status of women and has reduced the prevalence of NTDs in Canada by about 50%

(14).

The second consideration in deciding whether or not to fortify the food supply with a

nutrient using the risk management approach is that adding a nutrient to the food supply

will do no harm. Recent evidence suggests that the upward shift in dietary folate intake,

particularly synthetic folic acid intake, may cause harm to some people (17). For

example, it has now been confirmed that high folic acid intakes in older adults can delay

diagnosis of vitamin B12 deficiency, and coupled with low B12 status, may be associated

with impaired cognitive function in the elderly population (16). In addition, high levels

of folic acid are associated with a reduction in the effectiveness of anti-folate drugs used

against malaria, rheumatoid arthritis, psoriasis and cancer, and facilitate the progression

of pre-neoplastic cells and in this way promote cancer (17, 21, 78).

Currently in Canada there are calls to increase the level of folic acid fortification of

the food supply to provide a further 25% reduction in NTDs thought to be folate related

(165). At the same time, the Chief Medical Officer of England asked for a delay in a

final decision regarding mandatory folic acid fortification in the UK until further

consideration can be given to the role folic acid may have in increasing colorectal cancer

risk (21, 78). Clearly, monitoring how much folate is the food supply would be an

important first step to facilitate informed public policy decisions that strike the right

balance between health benefits of folic acid fortification and potential risks.

A significant disconnect between theoretical and actual levels of folic acid

fortification can influence public policy and dietary recommendations. For example, we

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recently reported that at “mandated” levels of folic acid fortification, 32% of a sample of

60 lactating women are unlikely to meet their EARs of folate from dietary sources alone

(128). Given the high folate demands associated with lactation, as the Canada Food

Guide recommends (245), we concurred that health care professionals continue to

recommend folic acid supplementation during lactation, and most certainly so if a woman

is capable of becoming pregnant. However, re-modeling our dietary data in this sample

of women based on “actual” levels of folic acid fortification reported herein, the

prevalence of inadequacy during lactation decreases to less than 13%. At twice mandated

levels of fortification, as was found early post-fortification in the U.S. (27), the

prevalence of inadequacy in our model becomes 0%. Clearly if the latter were the case,

folic acid supplementation during lactation just for maintenance of folate stores may be

unnecessary for many women. Consequently, if there is no benefit with nutrient

supplementation, there is only risk.

While our work has focused on folate, there is evidence of a disjuncture between the

actual and mandated levels for other nutrients (246). Using a sample of fluid milk from

Ontario, Faulkner et al. reported that 44 and 69% of vitamin A and D levels, respectively,

were outside the required range (246).

We acknowledge that we analyzed a limited number of folic acid-containing foods in

this study. However, among the tens of thousands of food products on the market, our

sampling of fortified foods was systematic using the most recent national food

consumption data (FOODEX) and brand data purchased from the ACNielsen Company.

Hence, we believe these data are sufficient to gauge the extent of the overage problem in

Canada and the likely variation by food category.

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In conclusion, the findings of this study suggest that 10 y after folic acid fortification

of the food supply in Canada, the actual amount of folate in fortified foods is

approximately 50% higher than what was mandated. However, the magnitude of this

overage varies considerably by food category. While the success of folic acid

fortification in the reduction of NTDs is undisputed, emerging evidence suggests that

high levels of folic acid may be potentially harmful to some segments of the population.

In order to strike the right balance between health benefits and risks, monitoring of

fortified foods for their nutrient content is required.

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Table 3.4.a. Comparison of the Analyzed Food Folate Content to Values Reported in the

Canadian Nutrient File (CNF).

Food Category1 Analyzed

values2 µg/100g

CNF values2

µg/100g

% of CNF value3

Mean ± SD Range

Breads (n = 15) 133 ± 29 107 ± 16 4

127 ± 29 67 – 187

Rolls and Buns (n = 15) 124 ± 22 106 ± 8 4

116 ± 19 73 – 149

Cookies (n = 15) 94 ± 29 59 ± 14 4

167 ± 56 49 – 278

Ready-to-eat Cereals (n = 15) 146 ± 36 80 ± 11 4

188 ± 57 113 – 280

Prepackaged Desserts (n = 15) 79 ± 32 47 ± 14 4

172 ± 72 53 – 362

Cooked Pasta (n = 7) 102 ± 45 77 ± 0

133 ± 59 62 – 223

Crackers (n = 10) 116 ± 32 105 ± 36 137 ± 104 38 – 418

1 Products were analyzed as purchased, except for pasta, which was prepared according

to the directions on the package.

2 Mean ± standard deviation.

3 Analyzed values as a % of the CNF.

4 Significantly different from analyzed value (Student‟s paired t-test; P < 0.05).

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Table 3.4.b. Comparison of the Analyzed Food Folate Content to Values Reported on the

Foods Labels.

Food Category1 Analyzed

values2 µg/100g

Label values2,3

µg/100g

% of Label value4

Mean ± SD Range

Breads (n = 15) 133 ± 29 96 ± 16 5

141 ± 36 90 – 196

Rolls and Buns (n = 5) 130 ± 12 112 ± 14 5 118 ± 12 103 – 132

Ready-to-eat Cereals (n = 15) 146 ± 36 62 ± 6 5 237 ± 65 126 – 377

1 Products were analyzed as purchased.

2 Mean ± standard deviation.

3 Calculated from the % Daily Value reported on the Nutrition Facts panel.

4 Analyzed values as a % of the label values.

5Significantly different from analyzed value (Student‟s paired t-test; P < 0.05).


INVESTIGATING THE IMPACT OF FOLIC ACID FORTIFICATION OVER

MANDATED LEVELS ON THE PREVALENCE OF FOLATE INADEQUACY AND

INTAKES ABOVE THE TOLERABLE UPPER LEVEL OF INTAKE AMONG

CANADIANS


Shakur, Y.A., Garriguet, D., Corey, P. & O‟Connor, D.L. Folic acid fortification over mandated

levels results in a low prevalence of folate inadequacy among Canadians. American Journal of

Clinical Nutrition 2010; 92(4):818-825.

- Reprinted with permission of the American Journal of Clinical Nutrition. Originally

published in the American Journal of Clinical Nutrition.

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4.1 ABSTRACT

Background: Understanding folate intakes post-folic acid fortification of the food supply will

help to establish dietary and supplement recommendations that balance health benefits and risks.

Objectives: To estimate the prevalence of folate inadequacy (POFI) and intakes above the

Tolerable Upper Intake Level (UL) among Canadians. Further, to estimate the supplemental

dose that, with diet, provides reproductive-aged women with 400 μg folic acid/d for NTD

prevention.

Design: 24-Hour recall and supplement (prior 30-d) data from the 2004 CCHS 2.2 (n = 35,107)

were used to calculate the POFI and intakes above the UL with and without adjustment for

fortification overages. POFI was also estimated by risk factors thought related to low folate

intake. The Software for Intake Distribution Evaluation (SIDE) was used to estimate usual

dietary intake in all analyses.

Results: Except for women >70 y, POFI was <20% after adjustment for fortification overages.

For children <14 y, POFI approached zero even when supplement use was excluded. POFI

among adults was unaffected by supplement use, except for women >70 y. Only 18% of

reproductive-aged women consumed 400 µg folic acid/d from diet and supplements. Modeling

showed supplements containing 325-700 μg folic acid would provide adult women with 400

µg/d but not more than the UL. Diabetes was associated with POFI.

Conclusions: Innovative strategies are needed to ensure that the sub-groups of Canadians that

could still benefit from improved folate intake are targeted. Consideration should be given to

removing folic acid from supplements designed for young children and men.

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4.2 INTRODUCTION

To address inadequate folate intakes among women of childbearing age and reduce the incidence

of NTDs, folic acid fortification of white wheat flour (150 µg/100 g) has been mandatory in

Canada since 1998 (4, 9). Since folic acid fortification, there has been a rise in blood folate

concentrations and a 46% reduction in NTD (14, 32, 156, 158). Given data suggesting that up to

75% of NTD may be prevented by providing folic acid during the peri-conceptional period, the

Canadian Society for Obstetricians and Gynecologists suggests that doubling the level of folic

acid fortification in Canada be considered (165). Folic acid fortification and supplementation

has also been associated with a reduction in other birth defects (oral clefts, congenital heart

disease), cancer (colon, breast), stroke and neuropsychiatric disorders (9, 100).

However, debate exists about the wisdom of exposing the entire population to higher levels of

folic acid. Concerns range from masking and progression of vitamin B12 deficiency, to reduced

effectiveness of antifolate drugs, and to the risk of promoting colorectal cancer in individuals

with pre-existing neoplasms (9, 16, 17, 21, 22, 78). Folic acid supplementation during

pregnancy was recently associated with risk of asthma, obesity and insulin resistance in offspring

(24, 25).

To evaluate whether the current folic acid fortification strategy in Canada strikes the right

balance of known benefits and potential risks, an understanding of folate intakes is required at

the national level. National dietary and supplement intake data collected as part of the CCHS 2.2

are now available for the first time in Canada in over 30 y (140). Using these data, the objective

of this study was to model the folate intakes (statistically adjusted to represent usual or long-term

intake) of Canadians using SIDE to determine the prevalence of folate inadequacy (POFI). The

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percent of individuals with folic acid intakes above the Tolerable Upper Intake Level (UL),

defined as the highest intake amount of a nutrient thought to pose no adverse health effects, will

also be determined (67). Importantly, determination of the POFI and folic acid intakes above the

UL will be done with adjustment to reflect “predicted actual” versus “mandated” levels of

fortification. Secondly, using these data, an estimate of the dose of folic acid that should be in

supplements designed for reproductive-aged women for NTD prevention will be determined.

Finally, the POFI by risk factors often associated with suboptimal folate status pre-fortification

of the food supply (alcohol, diabetes, smoking, and obesity) will be examined.

In this paper, food folate and folic acid refer to the naturally occurring and synthetic forms of

the vitamin, respectively. Dietary folate refers to all folates found in food (food folate plus folic

acid) and dietary folic acid refers to folic acid found in food. The term total folate describes the

sum all forms of folate consumed (dietary folate and supplemental folic acid).

4.3 SUBJECTS AND METHODS

Data Source

Data were collected under the authority of the Statistics Act of Canada. The CCHS 2.2 was

conducted in 2004 and contains data from 35,107 Canadians of all ages and is representative of

over 98% of the population from all 10 provinces. Food intake data were collected by the 24-h

recall method using a modified version of the USDA‟s Automated Multiple Pass Method (140,

247). All respondents completed one in-person 24-h recall with a trained interviewer and a sub-

sample of 10,786 completed a second 24-h recall 3 to 10 d later by a telephone interview.

Dietary supplement use was collected as 30 d frequency data. All respondents ≥1 y were

included in these analyses, which were stratified by Dietary Reference Intake (DRI) sex and age

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categories unless otherwise specified (9). Due to small sample sizes for pregnant (n = 175) and

lactating (n = 91) females, these two groups were not included in the analyses.

Model 1: Dietary folate intake based on mandated fortification levels

For each respondent, dietary folate intake, which is the sum of naturally occurring folate and

folic acid as a fortificant, was tabulated using Health Canada‟s Canadian Nutrient File, version

2001b (248). Food composition values for the database were derived from the USDA Nutrient

Database for Standard Reference 13 and modified to reflect current mandated fortification

regulations in Canada (249). Thus, Model 1 represents each respondent‟s dietary folate intake

assuming folic acid fortification at mandated levels.

Model 2: Dietary folate intake adjusted for overages in fortified foods

It was previously estimated that there is approximately 50% more folate in fortified foods in

Canada than would be expected based on mandated fortification levels and food composition

values (29, 250). Currently in Canada, there is no regulated allowable upper limit to folic acid

fortification (4). Manufacturers are allowed and do fortify at higher levels than the mandated

minimum to ensure that the amount of folic acid never falls below this level during the shelf-life

of the product. To adjust for this overage, each respondent‟s food record from the CCHS 2.2

was accessed and, using Health Canada‟s Bureau of Nutritional Sciences food codes, foods

eligible for folic acid fortification were assigned to one of 7 food categories. Based on

previously published direct laboratory analysis of 92 of the most commonly consumed foods

across these 7 food categories, an adjustment was made to account for the “predicted” actual

versus mandated level of fortification (250). The overage factors used in each of the 7 food

categories, which were based on weighted averages reflecting purchase prevalence in each food

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category, were the following: 1) bread, 1.34; 2) buns and rolls, 1.17; 3) cookies: 1.66; 4) ready-

to-eat cereals: 1.87; 5) prepackaged desserts: 1.84; 6) cooked pasta, 1.38; and 7) crackers, 1.31.

As the 92 foods were analyzed for their dietary folate content (i.e. naturally occurring folate and

synthetic folic acid) and not only for folic acid, both the folic acid and naturally occurring folate

values were multiplied by overage factors. Each respondent‟s predicted actual dietary folate

intake was then computed from the adjusted food records.

Model 3: Total folate intake from food and supplemental sources

Unlike dietary folate intake data, supplement consumption in the CCHS 2.2 was collected as

frequency data during the first 24-h recall and reflects how often multi-vitamins and/or minerals

were consumed over the previous 30 d. For each supplement reportedly consumed, respondents

stated how often they took the supplement during the past 30 d, as well as the dose usually

ingested each time. As recommended by Carriquiry, the average daily supplemental folic acid

intake for each individual was added to usual (long-term) dietary folate intake (obtained in

Model 2) (251). Usual dietary folate intake was estimated from Model 2 as opposed to Model 1

because this model accounts for previously documented folic acid overages (29, 250). The

resulting model of total folate intake from dietary and supplemental sources was termed Model

3.

Estimation of an adequate dose of supplemental folic acid for NTD-prevention

While Health Canada recommends that women preparing for a pregnancy consume a multi-

vitamin supplement containing 400 µg folic acid to reduce the risk of an NTD, the IOM

recommends that women consume 400 µg/d of synthetic folic acid from all sources (dietary and

supplements) in addition to food folate from a varied diet to protect against an NTD (9, 252). In

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order to estimate the dose of supplemental folic acid that would ensure that most women (>90%)

of childbearing age receive the 400 µg of folic acid daily (dietary and supplements), we added

several potential doses of supplemental folic acid to dietary folic acid intake adjusted for

predicted folic acid fortification overages (Model 2). All non-pregnant and non-lactating

females 14-50 y were included in this analysis.

Assessment of prevalence of inadequacy by risk factor

The CCHS 2.2 contains data on diabetes status (Types 1 and 2), alcohol consumption, smoking

status and BMI, which is the ratio of a subject‟s weight in kg divided by the square of their

height in m. Using Model 2, the POFI for all adults was estimated and compared among: a)

alcohol consumers and non-consumers (in the past year); b) diabetics (types 1 and 2) and non-

diabetics; c) current daily smokers and non-smokers; and d) obese (BMI ≥30 kg/m2) and non-

obese individuals.

Correction for the bioavailability of folic acid and the definition of UL

The prevalence of inadequate intakes was determined using either dietary (Models 1 and 2) or

total (Model 3) folate intakes expressed in DFE (9). As suggested by the IOM, to account for

differences in bioavailability between folic acid and food folate, DFE were determined using the

following calculation: DFE = µg food folate + (µg dietary folic acid x 1.7) + (µg supplemental

folic acid x 2) (9). Supplement in the CCHS 2.2 survey were assumed to be consumed on an

empty stomach, resulting in a larger conversion factor than that of dietary folic acid (2 versus

1.7, respectively) (1). While folic acid was converted to DFE to estimate the POFI, the percent

of intakes above the UL was calculated as done by the IOM based on synthetic folic acid only,

the rationale being that evidence suggests that it is excessive intakes of synthetic folic acid that

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may precipitate or exacerbate neuropathy in individuals with vitamin B12 deficiency (9). Hence,

food folate was not included in the estimate of intakes above the UL (9). The UL for folic acid

was set based on a Lowest Observed Adverse Effect Level, followed by the application of an

uncertainty factor (9). Importantly, the adverse effect level was based on the masking of vitamin

B12 deficiency in adults; extrapolation was used to set ULs for children and adolescents (9).

Statistical Analysis

All statistical analyses were performed with the SAS software (version 9.1; SAS Institute Inc.,

Cary, NC). The SIDE program (version 1.11, Department of Statistics and Center for

Agricultural and Rural Development, Iowa State University), as a SAS macro, was used to

estimate the subjects‟ usual (long-term) dietary folate, total folate, and folic acid intake

distributions by partially removing day-to-day variation from each individuals‟ intake estimated

using a second 24-h recall from a subset (n= 10,786) of respondents (253). SIDE was

subsequently used to estimate the POFI among respondents, defined as the proportion of

respondents with usual dietary (Models 1 and 2) or total (Model 3) folate intake below their

requirements by sex/age sub-groups. This was performed using the EAR cut-point method (67).

The EAR is defined as the level of intake for a nutrient that meets the requirement for 50% of

healthy individuals (defined by age and sex) in the population (67). While there is no cut-off to

define a high POFI, because of the strong relationship between suboptimal intakes and birth

defects, it is most concerning in women of childbearing age. Nonetheless, across the entire

population, inadequacy means regularly consuming insufficient amounts to perform bodily

functions, and was based primarily on erythrocyte folate concentrations, which is an indicator of

tissue folate stores (9). Since the EAR for folate is based on DFE, this unit was used in

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estimating the POFI. SIDE was also used to estimate the proportion of individuals with usual

folic acid intakes above the UL. Recognizing the limitations of using the UL as a strict risk

assessment cut-off, it is only inferred that intakes below the UL are safe (254).

Given that the sampling process for the CCHS 2.2 was complex and multi-stage, variance

estimates were calculated using the bootstrap balanced repeated replication technique. Briefly, a

replicate weight was generated by randomly selecting a sample, with replacement, from the

original sample and then applying all the performed adjustments to this selected sample. This

exercise was repeated 500 times to generate 500 sample survey weights which were then used to

estimate variance. A P <0.05 was considered statistically significant in all analyses.

4.4 RESULTS

Dietary folate intake based on mandated (Model 1) and predicted actual (Model 2)

fortification levels

In all DRI sex/age groups, the mean usual intake of dietary folate exceeded the EAR, whether or

not, intakes were modeled to predict folic acid fortification overages (Table 4.4.a). As

illustrated in Figure 4.4.a, the POFI based on dietary folate intake alone was very low for

children <14 y of age and virtually non-existent when intakes were modeled to predict actual

fortification levels. In Model 1, all female age groups ≥14 y had a POFI above 25%, reaching as

high as 54% in females >70 y. However, when dietary folate intakes were modeled to predict

actual intakes (Model 2), the POFI fell to below 20% among adolescent females and women ≤70

y. The POFI for females >70 y using Model 2 was 32.6%. Except for men >70 y, the POFI for

males was less than 20% using Model 1, and fell to less than 7.5% when data were modeled to

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consider folic acid fortification overages (Model 2). The POFI for males >70 y was over 30% in

Model 1 and fell to 13% using Model 2.

Use of folic acid-containing supplements

Despite the fact that children had the lowest POFI of all age groups based on dietary folate

intakes alone, they were the most likely to consume folic acid supplements (4 - 8 y; 38.7%)

compared to any other sex/age categories (Table 4.4.a). Among females of reproductive age,

folic acid containing supplements were consumed by 15.0, 22.9 and 29.2% of females 14-18, 19-

30 and 31-50 y of age, respectively. Among females >70 y, 28.5% consumed a folic acid

containing supplement.

Total folate intake from dietary and supplemental sources (Model 3)

Adding the contribution of folic acid from use of folic acid-containing supplements on to dietary

folate intake had little impact on the POFI across all sex/age categories except for females >70 y;

when the contribution of folic acid-containing supplements was added to dietary folate intake

(Model 3), the POFI among women >70 y was reduced from 32.6 to 24.6% (Figure 4.4.a).

Among all other sex/age categories, the reduction in POFI was ≤5% and highest in females 31-

50 y (15.2% to 10%). Among children and adolescents, the largest reduction in POFI was

observed in females 14-18 y (13.2% to 11.3%). Only 17.7% of women of childbearing age (14-

50 y) consumed ≥400 µg folic acid from fortified foods and supplements, the amount

recommended by the IOM to minimize the risk of NTD. Less than 1% of women of childbearing

age consumed ≥400 µg folic acid from dietary sources alone.

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Based on dietary intakes alone, the percentage of Canadians with intakes above the UL was

zero in all sex/age groups (Table 4.4.b). However, once supplemental folic acid consumption

was included (Model 3), 1.2 to 5% of individuals in each sex/age group exceeded the UL.

Estimation of an adequate dose of supplemental folic acid for NTD-prevention

To estimate the dose of supplemental folic acid that would ensure that most women of

childbearing age receive 400 µg of synthetic folic acid daily, we considered several potential

doses of supplemental folic acid that could be consumed over and above the amount of folic acid

consumed as a fortificant in food (dietary) as determined by Model 2 (Table 4.4.c). If all

women 14-50 y consumed a folic acid-containing supplement on a daily basis, a minimum

supplemental dose of 325 µg of folic acid would ensure that 91.4 % of women would consume at

least 400 µg of total synthetic folic acid (from dietary and supplemental sources) daily. In

contrast, supplemental doses of 500 µg and 700 µg leads to 6.8% and 1.5% of females 14-18 y

and 19-50 y, respectively, with intakes above the UL.

Assessment of prevalence of inadequacy by risk factor

Adults consuming alcohol had a lower POFI than those not consuming alcohol, while diabetics

had a higher POFI than their non-diabetic counterparts (Figure 4.4.b). There was no difference

in the POFI between smokers and non-smokers, or between obese and non-obese adults.

4.5 DISCUSSION

Results of this study provide evidence to suggest that after adjusting food composition values for

folic acid fortification levels higher than that minimally mandated, the POFI in the Canadian

population is low. In fact, in our re-analysis of the CCHS 2.2, only females >70 y had a POFI

greater than 20% when folic acid fortification overages in the food supply were accounted for

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(Figure 4.4.a). The mean usual dietary folate intake of females >70 y (388 ± 127 DFE, Model

2) was similar to the median dietary intake of women of the same age (417 DFE) recently

reported in the 2003–2006 NHANES (39). Further, after accounting for fortification overages,

there was virtually no difference in the POFI (as shown in Figure 4.4.a) whether individuals

consumed a supplement or not, except for women >70 y of age, where a modest reduction was

observed (32.6% to 24.6%).

Our analysis suggests there is no observable benefit of including folic acid in supplements

designed for children <14 y and regulatory guidance allowing for its inclusion should be

reconsidered. Importantly, the POFI among children <14 y, regardless of how their dietary data

were modeled, approached zero (Figure 4.4.a). Yet, young Canadians were the greatest

consumers of folic acid-containing supplements, with consumption reaching as high as 38.7%

among children 4-8 y (Table 4.4.a). Currently in Canada, supplements designed for children

contain doses of folic acid ranging from 100-400 µg/d. While the percentage of children <14 y

with folic acid intakes above the UL was low, without exception only children consuming folic

acid-containing supplements had folic acid intakes above the UL (Table 4.4.b). We caution that

our conclusions about whether, or not, folic acid should be included in supplements designed for

young children are not generalizable to other nutrients. This analysis is beyond the scope of this

paper.

The percentage of children consuming folic acid-containing supplements in the CCHS 2.2 is

similar to that reported recently by Hennessy-Priest et al. from a cross-sectional convenience

sample of preschool children (n=254) from the province of Ontario (30%), but somewhat lower

than supplement consumption (with or without folic acid) reported in a NHANES III analysis of

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children 1 to 5 y (42 to 51%) (129, 226). Hennessy-Priest et al. reported that 7% of the

preschoolers in their sample exceeded the UL for folic acid when supplement consumption was

included (2% if not included) (129). When they assumed a folic acid overage of 200% in

fortified foods, 12% of children exceeded the UL when supplement consumption was included

(4% if not included) (129). Flynn et al., in their analysis of folate intakes across several

European countries, concluded that high folic acid intakes in children were invariably associated

with the consumption of supplements and with fortified foods (mainly fortified breakfast cereals)

though they questioned the relevance of the UL for folic acid for children given it was based on

masking of vitamin B12 deficiency in adults (255).

Among adults, the contribution of folic acid from dietary sources was less than the UL for all

age groups and both sexes regardless of how the dietary intake data were modeled (Table 4.4.b).

This suggests that there is very little risk of folic acid intakes above the UL from dietary sources

alone. Inclusion of folic acid supplements in the analysis leads to a small percent of intakes (1.2-

5%) above the UL, which is in agreement with other available Canadian data collected from

regional or local convenience sampling (31, 33, 129). In their analysis of NHANES data from

2003 to 2006, Bailey et al. reported 0.4-5.2% of adults had intakes above the UL (39).

Furthermore, Yang et al., in their analysis of NHANES 2003 to 2006 data, showed that

supplement consumption was solely responsible for intakes above the UL among adults (130).

Despite folic acid fortification of the food supply and estimated overages, results from this

study provide evidence to suggest Canadian women of childbearing age should continue to

consume a folic acid supplement for the prevention of NTDs. From dietary sources alone, less

than 1% of women of childbearing age in the CCHS 2.2 met the IOM‟s recommendation to

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consume 400 µg/d of folic acid to prevent NTDs. When supplement consumption data from

these women were included in this analysis, only 17.7% of women consumed ≥400 µg/d folic

acid. These data are consistent with recent reports from smaller local cross-sectional studies in

Canada (31, 33). Further, data herein suggest that the range of doses for a folic acid supplement

that meet the IOM‟s recommendation for NTD prevention but do not result in folic intakes above

the UL are between 325-500 μg/d and 325-700 μg/d for adolescent and adult females,

respectively.

In the present study, we found that adults who did not consume alcohol had a higher POFI

than alcohol consumers. This observation is likely due to the fact that beer, the most commonly

consumed alcoholic beverage in Canada (256), contains folate (193). Furthermore, no difference

in the POFI existed whether, or not, an adult smoked/did not smoke, or had a BMI greater or less

than 30 kg/m2

(Figure 4.4.b). However, prior to the fortification of the food supply, smokers

were more often reported to have inadequate folate intakes compared non-smokers (9) and obese

women of childbearing age had lower folate intakes than non-obese women (257). Pre-

fortification of the food supply, most dietary folate was consumed from foods belonging to the

“vegetables and fruit” food group(s) (127, 128). As consumption of fruits and vegetables are

commonly associated with a cluster of many other healthy lifestyle characteristics, it makes

sense that folate intakes before fortification were positively associated with healthful lifestyle

characteristics (128, 258-260). However, after fortification of the food supply, the largest

reported component of dietary folate comes from the “grains” group and specifically white wheat

flour, which is not associated with healthful lifestyle characteristics (127, 128).

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In this study, diabetics had a higher POFI than non-diabetics (Figure 4.4.b). This is likely due

to the fact diabetics are encouraged to closely monitor their carbohydrate intake and to consume

whole grains which are not currently a vehicle for folic acid fortification in Canada (223). In

fact, all Canadians are recommended to choose whole grain products more often (245). The

impact that increased adherence to these recommendations or proposed changes to fortification

regulations to include whole grains will have on the folate status of Canadians will require close

monitoring.

One of the limitations of this study was the use of folic acid fortification overages based on a

small number of fortified foods (n = 92) (250). However, as described in detail elsewhere, our

sampling framework for selecting foods was systematic, using national food consumption

(FOODEX) and brand (ACNielson) data to identify the most commonly purchased fortified

foods in Canada (250). In addition, the overall percent overage that we reported in the

aforementioned study is identical to that determined by Quinlivan and Gregory in their

estimation of the actual level of folic acid fortification in Canada based on changes reported in

erythrocyte folate concentration pre- versus post-fortification of the food supply (29).

In sum, using the first nationally available dietary and supplement data in Canada in over 30 y

and estimates of the actual level of folic acid fortification, we conclude that except for women

>70 y, the POFI is low. Despite fortification, women of childbearing age are not consuming the

amount of folic acid recommended for NTD prevention from dietary sources alone. Data herein

suggest that a folic acid supplement of 325-700 µg/d for adults and 325-500 µg/d for adolescents

would serve to both maximally protect against NTDs and not provide intakes above the UL.

Except for women >70 y, we saw little evidence that folic acid consumption from supplements

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modified the POFI among Canadians which fits with the commonly held perception that

supplement users tend to be individuals with already folate adequate diets. Given the low POFI

among children <14 y and adult males, consideration should be given to removing folic acid

from supplements designed for these population subgroups in Canada.

Table 4.4.a. Prevalence of folic acid-containing supplement use and usual dietary folate intakes1.

N

Folic acid

supplement

consumption2

EAR3

Unadjusted

dietary

folate intake

(Model 1)4

Adjusted

dietary folate

intake

(Model 2)5

Unadjusted

contribution from

dietary folic acid

(Model 1)

Adjusted

contribution from

dietary folic acid

(Model 2)

% DFE DFE DFE µg µg

Children &

Adolescents

1-3 y 2193 30.7 ± 1.6 120 278 ± 104 331 ± 1236

75 ± 35 108 ± 516

4-8 y 3343 38.7 ± 1.4 160 378 ± 100 472 ± 1276 118 ± 35 170 ± 52

6

M 9-13 y 2149 24.0 ± 1.5 250 462 ± 125 573 ± 1496 142 ± 43 206 ± 63

6

F 9-13 y 2043 22.2 ± 1.6 250 403 ± 113 498 ± 1346 126 ± 33 180 ± 50

6

M 14-18 y 2397 13.8 ± 1.2 330 560 ± 179 699 ± 2256 175 ± 57 254 ± 86

6

F 14-18 y 2346 15.0 ± 1.2 330 426 ± 147 516 ± 1796 129 ± 49 183 ± 72

6

Men

19-30 y 1897 17.6 ± 1.5 320 574 ± 167 686 ± 1966 164 ± 56 236 ± 75

6

31-50 y 2750 19.3 ± 1.4 320 522 ± 160 624 ± 1966 145 ± 53 205 ± 75

6

51-70 y 2725 26.0 ± 1.4 320 460 ± 151 543 ± 1816 115 ± 54 164 ± 76

6

>70 y 1601 25.2 ± 1.9 320 390 ± 122 469 ± 1476 100 ± 43 146 ± 64

6

95

Women

19-30 y 1915 22.9 ± 1.6 320 407 ± 119 475 ± 1336 107 ± 39 152 ± 59

6

31-50 y 2851 29.2 ± 1.7 320 406 ± 137 471 ± 1556 101 ± 39 141 ± 56

6

51-70 y 3407 31.2 ± 1.3 320 378 ± 115 447 ± 1426 90 ± 39 131 ± 57

6

>70 y 2769 28.5 ± 1.4 320 324 ± 104 388 ± 1276 78 ± 36 114 ± 53

6

1 Mean ± SD, calculated using the Software for Intake Distribution (version 1.11, Department of Statistics and Center for Agricultural

and Rural Development, Iowa State University).

2 Percent ± SE of individuals consuming at least one folic acid-containing supplement in the 30-d prior to the first 24-h recall.

3 Table abbreviations: EAR, estimated average requirement; DFE, dietary folate equivalent.

4 Usual dietary folate intake (naturally occurring folate and synthetic folic acid from foods) based on mandated levels of fortification

(Model 1).

5 Usual dietary folate intake (naturally occurring folate and synthetic folic acid from foods) based on estimates of folic acid

fortification overages (Model 2).

6 Model 2 > Model 1 (Student‟s paired t-test P <0.01).

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Table 4.4.b. Percent of individuals with folic acid intakes above the Tolerable Upper Intake

Level (UL) based on usual dietary folic acid intake (Model 1), overage-adjusted dietary folic

acid intake (Model 2), and total folic acid intake (Model 2 plus supplemental folic acid; Model

3).

Percent above the UL1

n UL (µg) Model 12

Model 23

Model 34

Children &

Adolescents

1-3 y 2193 300 0 0 2.9 ± 0.6

4-8 y 3343 400 0 0 2.6 ± 0.5

M 9-13 y 2149 600 0 0 1.3 ± 0.3

F 9-13 y 2043 600 0 0 1.2 ± 0.4

M 14-18 y 2397 800 0 0 4.0 ± 0.8

F 14-18 y 2346 800 0 0 2.4 ± 0.5

Men

19-30 y 1897 1000 0 0 1.2 ± 0.4

31-50 y 2750 1000 0 0 2.3 ± 0.5

51-70 y 2725 1000 0 0 3.3 ± 0.5

>70 y 1601 1000 0 0 4.2 ± 1.0

Women

19-30 y 1915 1000 0 0 2.6 ± 0.5

31-50 y 2851 1000 0 0 5.0 ± 0.8

51-70 y 3407 1000 0 0 3.8 ± 0.5

>70 y 2769 1000 0 0 4.1 ± 0.6

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1 Percent ± SE. The Software for Intake Distribution Evaluation (version 1.11, Department of

Statistics and Center for Agricultural and Rural Development, Iowa State University) was used

to estimate usual dietary folic acid intakes in all models.

2 Usual dietary folic acid intake (folic acid as a fortificant) based on mandated levels of

fortification.

3 Usual dietary folic acid intake (folic acid as a fortificant) adjusted for folic acid fortification

overages.

4 Usual dietary folic acid intake adjusted for folic acid fortification overages plus supplemental

folic acid intake.

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Table 4.4.c. Estimation of the prevalence of women consuming less than 400 µg/d folic acid and

above the Tolerable Upper Intake Level (UL) from all sources (dietary and supplemental) at

different potential supplemental folic acid dosages1.

1 Percent ± SE. The Software for Intake Distribution Evaluation (version 1.11, Department of


to estimate usual folic acid intakes.

2 All females 14-50 y (n = 7112).

Supplemental

folic acid dose

Prevalence below 400 µg2 Percent above the UL

3

Female 14-18 y

Female 19-50 y

µg % % %

0 99.8 ± 0.1 0 ± 0 0 ± 0

200 81.1 ± 2.3 0 ± 0 0 ± 0

250 56.0 ± 3.0 0 ± 0.1 0 ± 0

300 21.7 ± 2.6 0 ± 0.2 0 ± 0

325 8.6 ± 1.6 0.2 ± 0.2 0 ± 0

350 1.9 ± 0.6 0.4 ± 0.3 0 ± 0

375 0.1 ± 0.1 0.6 ± 0.4 0 ± 0

400 0 ± 0 1.0 ± 0.6 0 ± 0

500 0 ± 0 6.8 ± 1.9 0 ± 0

600 0 ± 0 34.8 ± 3.3 0 ± 0.1

700 0 ± 0 90.9 ± 2.4 1.5 ± 0.5

800 0 ± 0 100 ± 0 16.1 ± 2.5

900 0 ± 0 100 ± 0 76.2 ± 3.6

1000 0 ± 0 100 ± 0 100 ± 0

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3n = 2345 for adolescent females (14-18 y; UL = 800 µg); n = 4766 for adult females (19-50 y;

UL = 1000 µg).

101

Figure 4.4.a. The prevalence of inadequacy in children (Panel A) and adults (Panel B) based on

usual dietary folate intakes (naturally occurring folate and synthetic folic acid from foods) where

folate nutrient composition data reflect the mandated fortification levels (Model 1 – open bars)

versus predicted actual dietary folate intakes (Model 2 – black bars). Grey bars represent the

prevalence of inadequacy based on total folate intakes (Model 2 plus supplemental folic acid

intakes) (Model 3). Error bars represent SE of the prevalence. Prevalence estimates below 5%

are shown despite the limitation of the EAR cut-point method at estimating tails of distributions

(17). Note: the y-axis of panel A covers a smaller range than that of panel B. Panel A: 1-3 y, n =

2193; 4-8 y, n = 3343; M 9-13 y, n = 2149; F 9-13 y, n = 2043; M 14-18 y, n = 2397; F 14-18 y,

n = 2346. Panel B: M 19-30 y, n = 1897; F 19-30 y, n = 1915; M 31-50 y, n = 2750; F 31-50 y, n

= 2851; M 51-70 y, n = 2725; F 51-70 y, n = 3407; M >70 y, n = 1601; F >70 y, n = 2769.

* 95% CI does not overlap with that of Model 1 of the same sex/age category.

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Figure 4.4.b. Prevalence of folate inadequacy in adults, based on predicted actual dietary folate

intake (Model 2), and stratified by alcohol consumption (Yes [n = 13,633] versus No [n = 4,374];

in the past year), diabetes status (Yes [n = 1,536] versus No [n = 18,357]; types 1 and 2),

smoking (Yes = daily smoker [n = 4,326] versus No = non-smoker [n = 14,826]) and obese status

(Yes [n = 3,183] versus No [n = 8,567]; BMI ≥ 30 kg/m2). Error bars represent SE of the

prevalence.

* 95% CI does not overlap with that of “Yes” response for the same proposed risk factor.

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FACTORS ASSOCIATED WITH SUB-OPTIMAL RED BLOOD CELL FOLATE

CONCENTRATIONS IN WOMEN 19-45 Y: NATIONAL HEALTH AND NUTRITION

EXAMINATION SURVEY 2003-2006

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5.1 ABSTRACT

Background: RBC folate concentration ≥906 nmol/L was shown to be maximally protective

against a folate-dependent NTD-affected pregnancy. However, estimates of the proportion of

women with RBC folate <906 nmol/L are sparse. Furthermore, there are no studies investigating

factors associated with RBC <906 nmol/L.

Objectives: To identify factors associated with a RBC folate <906 nmol/L among 1,764 women

19-45 y in the United States NHANES 2003-2006.

Design: Data from the household questionnaire and examination component of the surveys were

used in these analyses. Logistic regression analyses were used to identify factors associated with

RBC folate <906 nmol/L. Importantly, we corrected RBC folate values to reflect the

methodology used to set the 906 nmol/L cutoff.

Results: Thirty-five percent of women had sub-optimal RBC folate (<906 nmol/L). Women

who smoked (OR = 2.28, 95% CI: 1.74, 3.00), did not consume supplemental folic acid (OR =

3.52, 95% CI: 2.73, 4.54) and non-Hispanic black women (OR = 3.36, 95% CI: 2.40, 4.72) and

women of “other” races (non-Hispanic, non-black, non-white) (OR = 2.60, 95% CI: 1.25, 5.40)

were more likely to have sub-optimal RBC folate. Conversely, older women (35-45 y; OR =

0.70, 95% CI: 0.58, 0.86) and those with the highest quintile of dietary folate intake vs. the

lowest quintile (OR = 0.47, 95% CI: 0.33, 0.69) were less likely to have sub-optimal folate.

Conclusions: Our results suggest that determining a woman‟s risk of an NTD-birth outcome,

and consequently the dose of supplemental folic acid she needs, should include an assessment of

supplement use, race/ethnicity, folate intake, age, and smoking status.

KEYWORDS: NTDs, NHANES, RBC folate, folate status

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5.2 INTRODUCTION

Folate is a B-vitamin that is essential for DNA and RNA biosynthesis, making it important in

the diet of pregnant women, especially in the peri-conceptional period, to protect against NTDs

(9, 191). To improve the folate status of women capable of becoming pregnant, in 1998, both

the Canadian and American governments mandated folic acid fortification of select grain

products (4, 5). Daly et al. have shown that a maternal RBC folate concentration ≥906 nmol/L is

maximally protective against an NTD birth outcome, and Ren et al. report similar findings (36,

37). However, we are unaware of any studies investigating factors associated with RBC folate

<906 nmol/L, and thus increased risk of a folate-dependent NTD pregnancy outcome.

Furthermore, because of inter-assay and inter-laboratory differences in results of RBC folate

analyses (162, 261), assessing the proportion of women still at risk for a folate-dependent NTD

birth outcome is problematic.

Despite mandatory fortification, women capable of becoming pregnant are still

recommended to consume a folic acid-containing supplement to improve folate status and reduce

the risk of NTDs (9, 245); we have previously shown that 23 to 29% of Canadian women 19-50

y consume a folic acid containing supplement (262). However, there is a wide range of

supplemental doses available, from 400 µg to 5000 µg. In fact, the authors of one recent

publication have made blanket recommendations advising all Canadian women capable of

becoming pregnant to consume a supplement containing 5000 µg folic acid (34). This is of

increasing concern given the growing body of evidence pointing to potential harmful effects of

high folic acid intakes, including masking and progression of vitamin B-12 deficiency, reduced

effectiveness of anti-folate drugs, reduced natural killer cell cytotoxicity, and increased risk of

106

colorectal cancer among individuals with preexisting polyps (9, 17-23). Furthermore, there are

also preliminary reports showing that folic acid supplementation during pregnancy is associated

with increased adiposity, insulin resistance and occurrence of asthma and poor respiratory health

in the offspring (24-26).

There remains a large gap in the folate literature on the factors associated with having RBC

folate <906 nmol/L and thus increased risk of a folate-dependent NTD birth outcome. While

some women are at increased risk and thus would benefit from and require a higher dose of

folate (>400 µg), this is not the case for the majority of women (169, 252). Health Canada‟s

guidelines indicate that several characteristics are associated with increased risk of an NTD-

affected pregnancy, including low folate intake, smoking, dieting and diabetes (35, 169).

Furthermore, RBC folate measurement, the gold standard for determining folate status, is not

routinely assessed in the pre-conceptional care of women, leaving physicians without the

necessary information to recommend an appropriate dose of folic acid supplement. Therefore,

there remains an important need in the post-fortification era to determine factors that are

predictive of sub-optimal folate status (RBC folate <906 nmol/L), a surrogate measure of risk of

folate-dependent NTD-birth outcome. Such findings will aid in developing a short nutritional

tool to assist physicians in identifying women with sub-optimal folate status and thus increased

risk. Thus, the objective of this study was to use data from the nationally-representative U.S.

NHANES 2003-2006 to identify dietary and lifestyle factors associated with sub-optimal folate

status among women of childbearing age.


Data Source

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The NHANES are a series of nationally-representative, cross-sectional surveys aiming to assess

the health and nutritional status of non-institutionalized, civilian adults and children in the United

States, combining both health and physical examinations (263). The NHANES employs a

complex, multistage, stratified, probability cluster sampling design. As of 1999, the surveys

adopted a continuous cycle with data releases occurring every 2 y (263). An in-person interview

was first used to collect demographic and supplement use data, while a health examination at a

Mobile Examination Center approximately 1 mo later was used to perform a health examination

consisting of clinical tests, anthropometric measurements, laboratory studies, dietary recall and

health data collection. Finally, a phone interview was used to collect a second round of dietary

recall data. The response rates for the household interview were 79% and 80% in 2003-2004 and

2005-2006, respectively, while for the examination component, they were 76% and 77% in

2003-2004 and 2005-2006, respectively. We used data from women 19-45 y (n = 2,810) for

these analyses. Pregnant and lactating women (n = 894), women with missing dietary recall data

(n = 80), and women with missing RBC folate data (n = 282) were excluded. Two hundred and

ten women were excluded for more than one of the aforementioned reasons, resulting in a final

sample was 1,764.

Measurement of RBC folate concentration

The main outcome variable, RBC folate concentration, was measured by Quantaphase II

radioassay (Bio-Rad Laboratories, Hercules, CA). The coefficient of variation for the assay in

the NHANES 2003-2006 was 4-6% (152). We defined optimal folate status as RBC folate

concentration ≥906 nmol/L, based on previous literature (36, 37). Daly et al. investigated the

relationship between RBC folate concentration early in pregnancy and incidence of NTD over

108

50,000 births in Ireland between 1986 and 1990, and found that there was greater than three-fold

increase in risk of NTD in women with RBC folate concentrations below 700 nmol/L compared

to women with RBC folate concentrations ≥906 nmol/L (36). Ren et al. later confirmed this by

showing that 695 pregnant women recruited from a prenatal health centre in an area of low NTD-

prevalence (0.83/1000 births) in China had a mean RBC folate of 910 nmol/L, much higher than

a similar group of 562 pregnant women from an area of high NTD-prevalence (440 nmol/L) (37).

However, the 906 nmol/L cutoff was based on RBC folate as assessed by a microbiological assay

and not the Bio-Rad radioassay used in these NHANES 2003-2006, and it has been previously

established that the Bio-Rad radioassay underestimates actual RBC folate values (261).

Therefore, we used the following linear regression equation to correct the NHANES RBC folate

values (personal communication with Dr. Christine Pfeiffer, CDC):

log10(RBC Microbiological Assay) = 1.017 x log10(RBC Bio-Rad Assay) + 0.2204

For purposes of completion, we also present results of the analysis based on unadjusted RBC

folate values (based on Bio-Rad Assay) in tabular form. However, in the text, we limit our

discussion to the analysis based on RBC folate corrected to reflect microbiological assay values.

Predictors

Dietary folate intake data collection

Data on folate intake were estimated from two 24-hour recalls from each individual, an in-person

recall at the Mobile Examination Center and a phone recall 3-10 d following the first. The U.S.

Department of Agriculture‟s Automated Multiple-Pass Method was used for both recalls (247).

Food folate intake was presented as DFE, in conformity with the IOM recommendation to

account for bioavailability differences between folic acid and dietary folate: DFE = µg dietary

109

folate + (µg dietary folic acid x 1.7) (9). For this analysis, we used data from the first in-person

24-h dietary recall and categorized dietary folate intake into quartiles. Supplemental folic acid

was not included in usual intake estimation.

Use of folic acid-containing supplements

Data on dietary supplement use were collected during the household interview. Information on

the sample person‟s use of vitamins, mineral, herbs and other dietary supplements over the past

30 d was captured using the Dietary Supplement Questionnaire. Those who responded “yes”

were asked for details about supplement type, dosage, and frequency of consumption for each

consumed supplement. The nutrient profile of the supplement was extracted from the container‟s

label. If the nutrient composition of a supplement was missing, that of the most commonly used

supplement in that category was imputed. Respondents were categorized as folic acid-containing

supplement users or non-users.

Other

Demographic data on age, income, education, race/ethnicity, marital status, and smoking were

obtained from the home interview (264). Age was categorized as 19-34 y and 35-45 y, while

income was stratified as <$25000, $25000-$64999, and ≥$65000. Education was categorized as

high school or less and more than high school, while current smoking was categorized as yes

(daily/occasional) or no. BMI (weight in kg divided by square of height in m) was calculated

from measured weight and height data from the Mobile Examination Center. Data on alcohol

abuse (≥2 drinks/d), presence of diabetes (yes/no), failing kidneys (yes/no), liver disease

(yes/no), food security status (secure/insecure) and high blood pressure (yes/no) were all

obtained from the health interview at the Mobile Examination Center, and based on self-report.

110

Statistical analyses

All statistical analyses were performed using SAS software (version 9.2; SAS Institute Inc.,

Cary, NC). Sample weights were used to account for the complex sampling design, nonresponse

and oversampling (263). Also, all estimates of variance were calculated using the Taylor

Linearization method, which takes into account the complex survey design employed by the

NHANES.

The main outcome variable was folate status based on RBC folate status (optimal ≥906

nmol/L; sub-optimal <906 nmol/L) (36). Bivariate logistic regression analysis was first used to

detect associations between RBC folate status and candidate covariates identified from previous

literature (169, 252). Multivariate logistic regression analysis was then used to assess adjusted

associations with folate <906 nmol/L and several candidate factors from the bivariate analysis,

including demographic variables (age, race/ethnicity, education, and household income), food

security status, dietary folate intake (quartiles), folic acid-containing supplement use, obesity,

alcohol abuse, smoking, diabetes, kidney failure, liver disease and high blood pressure. Any

covariate with a Wald chi-squared P-value <0.1 from bivariate analyses was considered as a

candidate variable for multivariate regression. A backward stepwise approach was used to

generate the final model, with non-significant variables removed from the model one at a time.

A P <0.05 was considered statistically significant in all analyses.

5.4 RESULTS

Baseline characteristics

The overall mean RBC folate was 1107 nmol/L. Less than 1% of women had chronic folate

deficiency (RBC folate <305 nmol/L), and 65.0% had optimal folate status (RBC folate ≥906

111

nmol/L). Mean RBC folate was significantly lower in younger women compared to older

women (Table 5.4.a). Non-Hispanic Black women had significantly lower mean RBC folate

than all other races. Thirty-four percent of women consumed a folic acid-containing supplement.

Univariate analysis

Several factors were associated with folate <906 nmol/L (Table 5.4.b). Older women (35-45 y)

were 31% less likely to have folate <906 nmol/L than younger women (19-34 y) (P <0.0001).

Similarly, women in the upper quartile of dietary folate intake were 69% less likely to have

folate <906 nmol/L than those in the lower quartile (P <0.0001). Also, folate status was better

for women consuming a folic acid-containing supplement versus those who don‟t (P <0.0001),

non-Hispanic Whites versus non-Hispanic Black women and women of “other” races (non-

Hispanic, non-black, non-white) (P <0.0001), and non-smokers versus smokers (P <0.0001).

Income (≥$65,000 vs. <$25,000; P <0.0001), education (more than high school vs. high school

or less; P <0.0001), food security (secure vs. insecure; P = 0.0003) and marital status

(married/living with a partner vs. widowed/separated/divorced/never married; P <0.0001) were

also associated with better folate status.

Multivariate analysis

In the final model, women not consuming a folic acid-containing supplement were more likely to

have folate <906 nmol/L (odds ratio [OR] = 3.52, 95% confidence interval (CI): 2.73, 4.54)

(Table 5.4.c). Non-Hispanic black women (OR = 3.36, 95% CI: 2.40, 4.72) and women of

“other” races (non-Hispanic, non-black, non-white) (OR = 2.60, 95% CI: 1.25, 5.40) were more

likely to have folate <906 nmol/L than non-Hispanic white women. Similarly, current smokers

were more likely than current non-smokers to have folate <906 nmol/L (OR = 2.28, 95% CI:

112

1.74, 3.00). Conversely, older women (OR = 0.70, 95% CI: 0.58, 0.86) and women in the two

highest quartiles of dietary folate intake (Quartile #4: OR = 0.47, 95% CI: 0. 33, 0.69; Quartile

#3: OR = 0.61, 95% CI: 0.44, 0.83) were less likely than younger women and women in the

lowest quartile of dietary folate intake to have folate <906 nmol/L.

5.5 DISCUSSION

We found that, despite low prevalence of folate deficiency (<1%), there is still 35% of women

19-45 y with RBC folate concentrations <906 nmol/L, the cut-off for maximal protection against

a NTD-affected birth outcome. This is somewhat surprising in the post-mandatory fortification

era, given that there are now several different sources of folic acid available in addition to

naturally occurring food folate, including folic acid from supplements, ready-to-eat cereals, and

mandatory fortification of cereal grain products. In the only other population-based American

study reporting on women‟s RBC folate relative to the 906 nmol/L cut-off, Dietrich et al. found

that >90% of women 20-59 y had RBC folate <906 nmol/L from the NHANES III (1988-1994)

and 1999-2000 datasets (127). As this was a secondary analysis in their study, the authors didn‟t

investigate factors associated with having maximally-protective RBC folate. Their estimate is

much higher than ours because theirs was based on RBC folate values as reported in those

NHANES datasets, while we corrected ours to reflect microbiological assay RBC folate values.

In fact, when we used the RBC folate values as reported in NHANES 2003-2006, we found that

91% of women had RBC folate <906 nmol/L (data not shown), very similar to what Dietrich et

al. reported (127). Using data from the nationally-representative Canadian Health Measures

Survey conducted in 2007-2009, Colapinto et al. showed that 22% of Canadian women 15-45 y

had RBC folate <906 nmol/L (161), much lower than what Dietrich et al. reported, but similar to

113

what we found in the current study among American women. However, inter-laboratory

differences in RBC folate have been documented (162, 163) and Colapinto et al. used a different

radioassay than that employed in NHANES 2003-2006 and they did not correct the RBC folate

values as we did.

As evident from above, the differences between the Bio-Rad and microbiogical assays can

lead to large discrepancies in RBC folate values and in turn, the prevalence of women at not

maximally protected against an NTD (RBC folate <906 nmol/L). The Bio-Rad assay

underestimates RBC folate by approximately 30-35% when compared to the microbiological

assay (261). This is due in part to the fact that the microbiological assay is more efficient than

the Bio-Rad assay at capturing the different forms of folate in blood (261). As a result,

beginning with the NHANES 2007-2008 cycle, RBC folate is now assessed using the

microbiological assay instead of the Bio-Rad assay (265).

The use of folic acid-containing supplement was the strongest factor associated with RBC

folate ≥906 nmol/L. Women in our analyses not consuming a folic acid-containing supplement

were 3.52 times more likely to have RBC folate <906 nmol/L. Using NHANES 2003-2006 data,

Yang et al. and Yeung et al. showed that folic acid-containing supplement users had significantly

higher geometric mean serum folate and RBC folate concentrations than non-users among adults

(130) and children (138), respectively. In a randomized, controlled trial conducted pre-

fortification, Cuskelly et al. showed that 400 µg daily folic acid supplementation for 3 months

led to a significant increase in RBC folate among women 17-40 y (266). Several others have

shown that supplemental folic acid makes a substantial contribution to total folate intake (39, 40,

130, 138), which is positively associated with RBC folate concentration (130, 138).

114

Furthermore, intakes above the UL are primarily due to consumption of a folic acid containing

supplement (39, 40, 130, 138). This is expected, given the fact that mandatory folic acid

fortification is expected to add 100 µg to the daily diet (5), and ready-to-eat cereals can add up to

400 µg per serving (52), while the contribution of supplemental folic acid can reach 1000 µg or

even 5000 µg, depending on the dose consumed. Using nationally-representative Canadian data,

we have also previously shown that use of folic acid-containing supplements drives up total

folate intake among Canadians (262).

We also found that, independent of supplement use, women in the two highest quartiles of

dietary folate intake were significantly less likely to have RBC folate <906 nmol/L than women

in the lowest quartile (Table 5.4.c). Using pre-fortification NHANES data (1988-1991), Ford

and Bowman showed that dietary folate intake was significantly correlated with RBC folate

concentration among adults when both variables are treated as continuous (170). Numerous

reports from both the United States and Canada show that there has been substantial increase in

RBC folate concentration since folic acid fortification of the food supply, strongly suggesting

that the increase in dietary folate intake due to mandatory fortification caused an increase in

RBC folate concentrations (32, 148, 152, 158). While it can be argued that this increase might

be partially due to increase in the use of dietary supplements, a few studies excluded supplement

users, thereby conclusively showing that increase in dietary folate intake is associated with an

increase in RBC folate concentrations (145, 146).

Current smokers, daily or occasional, were significantly more likely to have RBC folate <906

nmol/L (OR = 2.28). It has been previously shown that cigarette smokers have lower RBC folate

than non-smokers (267). Also, Mannino et al. used NHANES III data to show that smoking, as

115

captured by serum cotinine levels, is associated with lower RBC folate when compared to non-

smokers, even after controlling for significant differences in folate intake (132). While the exact

mechanism of this association is unknown, based on our findings, it seems that smoking is

independently an important factor contributing to RBC folate status, and the smoking status of

women planning a pregnancy should be taken into consideration when deciding on an

appropriate dose of supplemental folic acid.

We also found that women 35-45 y were less likely to have RBC folate <906 nmol/L than

younger women (19-34 y) (OR = 0.70), which is congruent with other reports showing that RBC

folate concentration increases with age (127, 150). Race/ethnicity was also an important factor,

with non-Hispanic Blacks 3.36 times more likely and women of “other” races (non-Hispanic,

non-black, non-white) 2.6 times more likely to have sub-optimal RBC folate status than non-

Hispanic Whites. This is similar to well-established findings from other NHANES reports

showing that mean or median RBC folate is higher among non-Hispanic Whites than among

non-Hispanic Blacks (149, 150, 152). There were only 90 women of “other” races (non-

Hispanic, non-black, non-white) in our sample and this represented several other of races,

including those of Asian, Arab, and multi-racial backgrounds.

Several demographic variables (education, income, food security and marital status) were

significant predictors of having RBC <906 nmol/L in univariate analyses (Table 5.4.b), but lost

significance in the final multivariate model and were thus removed (Table 5.4.c). This is likely

due to accounting for other factors, such as dietary folate, supplement use and smoking, which

have been shown to be associated with several of these variables in the general population (7,

268). Diabetes, alcohol abuse, obesity, kidney failure, and liver disease were not associated with

116

RBC folate status. Ray et al. have shown that there is a greater risk for a NTD-affected

pregnancy with increasing maternal weight (269), and Rasmussen et al. recently published a

meta-analysis in which they reported that maternal obesity is associated with an increased risk of

a NTD-affected pregnancy (270). However, the mechanism of action is unknown (271) and

likely independent of folate (269).

Strengths of this study include that it is the first study to investigate factors associated with

being above/below the RBC folate cutoff for maximal protection against a folate-dependent

NTD-affected pregnancy. Furthermore, we corrected RBC folate values in NHANES 2003-2006

to allow for appropriate comparison with the 906 nmol/L cutoff, and to our knowledge, this is the

first time this has been performed with NHANES data. These findings can be used to identify

women at risk of a folate-dependent NTD-affected pregnancy in order to recommend a higher

dose of supplemental folic acid. A limitation of this study is the fact that several covariates were

based on self-reported data and thus there is a potential for biased reporting. Also, dietary folate

intake was based on data from one day only and not usual intake data. Therefore, some women

may have usual folate intake in a higher or lower quartile than what they are classified in in our

analyses.

In conclusion, using the most recent publicly available NHANES datasets (2003-2006), we

show that 65% of women 19-45 y have RBC folate concentrations maximally protective against

a NTD-affected pregnancy. While all women capable of becoming pregnant are recommended

to consume a folic acid-containing supplement, in developing a screening tool to determine

whether a woman is at increased risk and needs a dose >400 µg, it is important to consider

dietary folate intake, current folic acid supplement use, race/ethnicity, smoking status, and age of

117

the woman. Further research is needed to confirm these findings and to identify other candidate

items for inclusion in a screening tool to determine a woman‟s risk of an NTD-affected

pregnancy.

118

Table 5.4.a. Mean red blood cell (RBC) folate1 concentrations (nmol/L) and percent of women with RBC folate <906 nmol/L

categorized by analyzed variables.

Microbiological Assay BioRad Assay

Variables

Weighted

percent of

women (SE)

Mean (SEM)

RBC folate

Percent (SE) of

women with RBC

folate <906 nmol/L

Mean (SEM)

RBC folate

Percent (SE) of

women with RBC

folate <906 nmol/L

Age

19-34 y (n = 1049)

35-45 y (n = 715)

52.4% (1.8)

47.6% (1.8)

10622 (16)

1157 (23)

39.0 (2.1)

30.5 (1.7)

5742 (8)

624 (12)

93.7 (0.9)

88.3 (1.7)

Education

Less than high school (n = 94)

9th

to 12th

grade (n = 231)

High school grad (n = 351)

Some college (n = 539)

College grad or above (n = 335)

3.5% (0.5)

11.2% (0.9)

22.7% (1.3)

35.6% (1.5)

27.0% (1.9)

1113 (50)

10233 (25)

10633 (28)

1118 (25)

1181 (23)

35.6 (3.9)

41.5 (3.6)

43.1 (2.5)

35.1 (2.9)

23.1 (1.9)

600 (27)

5533 (13)

5743 (15)

603 (13)

636 (12)

87.2 (5.0)

97.4 (0.9)

90.8 (1.8)

90.8 (1.5)

89.0 (1.9)

Income

Less than $25,000 (n = 538)

$25,000 to $64,999 (n = 632)

$65,000 or more (n = 496)

23.0% (1.4)

38.8% (2.2)

38.2% (2.3)

1058 (29)

1091 (20)

1163 (28)

43.7 (3.1)

36.8 (2.1)

27.0 (2.8)

571 (15)

589 (10)

627 (15)

92.8 (1.4)

91.5 (1.3)

89.4 (1.9)

Race/Ethinicity

119

Hispanic (n = 477)

White (n = 763)

Black (n = 434)

Other (n = 90)

14.2% (1.4)

66.7% (2.4)

12.8% (1.5)

6.3% (0.9)

1093 (34)

1156 (23)

9004 (18)

1039 (41)

36.3 (2.8)

29.0 (2.3)

59.6 (2.9)

45.4 (6.5)

590 (18)

623 (12)

4874 (9)

561 (22)

91.7 (1.7)

89.9 (1.5)

95.5 (0.9)

94.1 (3.4)

Food security

Food secure (n = 1231)

Food insecure (n = 510)

78.1% (1.3)

21.9% (1.3)

1124 (18)

1052 (24)

32.8% (1.9)

42.7% (2.5)

606 (9)

568 (13)

90.4% (1.2)

93.2% (1.5)

Marital status

-Married/living with partner (n = 933)

-Widowed/separated/ divorced/never

married (n = 831)

60.2% (1.5)

39.8% (1.5)

1144 (23)

10522 (15)

30.5% (2.3)

41.8% (1.9)

617 (12)

5682 (8)

89.9% (1.4)

93.0% (1.0)

Told you have kidney disease

Yes (n = 31)

No (n = 1516)

1.9% (0.4)

98.1% (0.4)

9922 (35)

1115 (17)

41.8% (6.7)

34.2% (1.7)

5372 (19)

601 (9)

98.5% (1.5)

90.8% (1.0)

Told you have high blood pressure

Yes (n = 236)

No (n = 1513)

13.2% (1.2)

86.8% (1.2)

1204 (49)

1095 (16)

34.0% (3.8)

34.7% (1.7)

648 (25)

591 (8)

83.1% (3.5)

92.3% (0.9)

Told you have diabetes

Yes (n = 49)

No (n = 1714)

2.7% (0.4)

97.3% (0.4)

1285 (84)

1103 (17)

21.2% (6.8)

35.3% (1.7)

691 (45)

595 (9)

86.0% (5.2)

91.3% (1.0)

Body Mass Index

120

Non-obese (<30) (n = 1142)

Obese (≥30) (n = 608)

67.7% (1.7)

32.3% (1.7)

1087 (19)

1148 (24)

35.5% (1.8)

33.9% (2.2)

587 (10)

619 (13)

92.3% (1.3)

88.9% (1.6)

Dietary folate intake (DFE6) (Quartiles)

Q1 (<262) (n = 441)

Q2 (262 – 391) (n = 441)

Q3 (391 – 572) (n = 441)

Q4 (>572) (n = 441)

22.9% (0.9)

25.3% (1.1)

26.3% (1.1)

25.5% (1.3)

1020 (22)

1060 (25)

11185 (20)

12225 (28)

43.4% (3.3)

42.4% (2.9)

31.2% (2.7)

23.9% (2.2)

551 (12)

572 (13)

6035 (11)

6585 (15)

95.1% (1.1)

93.1% (1.6)

91.5% (1.4)

85.3% (2.5)

Folic acid-supplement consumption

Yes (n = 496)

No (n = 1268)

34.0% (1.5)

66.0% (1.5)

1301 (27)

10082 (13)

16.7% (1.6)

44.4% (1.8)

700 (14)

5442 (7)

83.3% (1.7)

95.2% (0.8)

Smoking

Yes (daily/occasional) (n = 404)

No (n = 1147)

28.4% (1.5)

71.6% (1.5)

9952 (21)

1158 (18)

47.2 % (2.3)

29.3% (2.0)

5372 (11)

624 (10)

95.7% (0.9)

89.1% (1.2)

Alcohol abuse (≥2 drinks/d)

Yes (n = 704)

No (n = 1060)

47.8% (1.3)

52.2% (1.3)

1103 (18)

1111 (22)

32.7% (2.1)

37.0% (2.0)

595 (9)

599 (12)

92.2% (0.9)

90.2% (1.4)

Told you had liver disease

Yes (n = 29)

No (n = 1520)

1.9% (0.4)

98.1% (0.4)

1210 (103)

1110 (17)

24.7% (5.1)

34.5% (1.7)

651 (54)

599 (9)

84.0% (6.5)

91.1% (1.0)

1Results are presented for RBC folate corrected to reflect microbiological assay values and RBC folate as analyzed by Bio-Rad assay.

121

2Significantly lower than the other category under the same variable; Student‟s t-test.

3Significantly lower than “college grad or above”; Student‟s t-test.

4Significantly lower than all other races/ethnicities; Student‟s t-test.

5Significantly higher than “Quartile #1”; Student‟s t-test.

6Dietary Folate Equivalents.

122

Table 5.4.b. Univariate analyses of factors associated with RBC folate1 <906 nmol/L.


Variable Odds Ratio (95% CI) P-value2

Odds Ratio (95% CI) P-value2

Age

19-34 y

35-45 y

1.0 (ref)

0.69 (0.58, 0.81)

<0.0001

1.0 (ref)

0.51 (0.33, 0.78)

0.0018

Education

High school or less

More than high school

1.69 (1.39, 2.07)

1.0 (ref)

<0.0001

1.35 (0.90, 2.02)

1.0 (ref)

0.1475

Income

Less than $25,000

$25,000 to $64,999

$65,000 or more

1.0 (ref)

0.75 (0.53, 1.06)

0.48 (0.35, 0.66)

<0.0001

1.0 (ref)

0.84 (0.55, 1.27)

0.66 (0.38, 1.15)

0.324

Ethnicity

Hispanic

White

Black

Other

1.40 (0.96, 2.05)

1.0 (ref)

3.61 (2.66, 4.92)

2.04 (1.12, 3.71)

<0.0001

1.24 (0.68, 2.26)

1.0 (ref)

2.40 (1.36, 4.24)

1.80 (0.46, 7.01)

0.0239

Food security

Food secure

1.0 (ref)

0.0003

1.0 (ref)

0.1852

123

Food insecure 1.53 (1.22, 1.92) 1.45 (0.84, 2.50)

Marital status

Married/living with partner

Widowed/separated/divorced/never

married

1.0 (ref)

1.64 (1.29, 2.08)

<0.0001

1.0 (ref)

1.50 (0.98, 2.29)

0.0621

Told you have kidney disease

Yes

No

1.39 (0.51, 3.73)

1.0 (ref)

0.5198

6.88 (0.89, 53.07)

1.0 (ref)

0.0643


Yes

No

0.97 (0.69, 1.37)

1.0 (ref)

0.8562

0.41 (0.25, 0.68)

1.0 (ref)

0.0006

Told you have diabetes

Yes

No

0.49 (0.20, 1.25)

1.0 (ref)

0.1345

0.59 (0.24, 1.41)

1.0 (ref)

0.2332

Body Mass Index

Non-obese (<30)

Obese (≥30)

1.0 (ref)

0.93 (0.78, 1.11)

0.4261

1.0 (ref)

0.67 (0.41, 1.07)

0.0952

Dietary folate intake (DFE3 ) (Quartiles)

Q1 (<262)

Q2 (262 – 391)

Q3 (391 – 572)

1.0 (ref)

0.96 (0.68, 1.37)

0.59 (0.44, 0.79)

<0.0001

1.0 (ref)

0.70 (0.39, 1.24)

0.56 (0.32, 1.01)

0.0011

124

Q4 (>572) 0.41 (0.30, 0.56) 0.30 (0.17, 0.55)


Yes

No

1.0 (ref)

3.99 (3.20, 4.96)

<0.0001

1.0 (ref)

3.97 (2.73, 5.76)

<0.0001

Smoking

Yes (daily/occasional)

No

2.16 (1.66, 2.82)

1.0 (ref)

<0.0001

2.70 (1.64, 4.43)

1.0 (ref)

<0.0001

Alcohol abuse (≥2 drinks/d)

Yes

No

0.83 (0.67, 1.03)

1.0 (ref)

0.0824

1.28 (0.88, 1.86)

1.0 (ref)

0.2014

Told you had liver disease

Yes

No

0.63 (0.25 1.54)

1.0 (ref)

0.3069

0.52 (0.18, 1.46)

1.0 (ref)

0.2128


2Wald Chi-Square.


125

Table 5.4.c. Multivariate analyses of factors associated with RBC folate1 <906 nmol/L.


Variable2 Odds Ratio (95% CI) P-value

3 Odds Ratio (95% CI) P-value

3

Age

19-34 y

35-45 y

1.0 (ref)

0.70 (0.58, 0.86)

0.0004

1.0 (ref)

0.59 (0.37, 0.94)

0.0277

Ethnicity

Hispanic

White

Black

Other

1.29 (0.86, 1.95)

1.0 (ref)

3.36 (2.40, 4.72)

2.60 (1.25, 5.40)

<0.0001

n/a5

Dietary folate intake (DFE4 ) (Quartiles)

Q1 (<262)

Q2 (262 – 391)

Q3 (391 – 572)

Q4 (>572)

1.0 (ref)

0.99 (0.67, 1.47)

0.61 (0.44, 0.83)

0.47 (0.33, 0.69)

<0.0001

1.0 (ref)

0.71 (0.38, 1.33)

0.54 (0.29, 1.00)

0.37 (0.25, 0.55)

<0.0001


Yes

No

1.0 (ref)

3.52 (2.73, 4.54)

<0.0001

1.0 (ref)

3.55 (2.35, 5.36)

<0.0001

Smoking <0.0001 0.0008

126

Yes (daily/occasional)

No

2.28 (1.74, 3.00)

1.0 (ref)

2.29 (1.41, 3.72)

1.0 (ref)


Yes

No

n/a5

0.41 (0.24, 0.68)

1.0 (ref)

0.0006


2Each variable adjusted for all other variables in the model.

3Wald Chi-Square.


5Non-significant; excluded from the final model.

127


VITAMIN AND MINERAL SUPPLEMENT CONSUMPTION IN CANADA: DO USERS

DIFFER FROM NON-USERS IN TERMS OF NUTRIENT INADEQUACY AND RISK

OF HIGH INTAKES?

128

6.1 ABSTRACT

Background: While supplement use is prevalent in Canada, there is little information on how

supplements affect nutrient adequacy or risk of intakes above the Tolerable Upper Intake Level

(UL).

Objectives: To compare the prevalence of nutrient inadequacy and percent of intakes >UL from

diet alone between supplement users and non-users and determine the contribution of

supplements to nutrient intakes.

Design: Dietary intakes (24-h recall) and supplement use (prior 30-d) from respondents ≥1 y in

the 2004 Canadian Community Health Survey 2.2 (n = 34,381) were used to estimate prevalence

of inadequacy and intakes >UL among supplement users and non-users. The Software for Intake

Distribution Evaluation was used to estimate usual intakes.

Results: The prevalence of inadequacy from diet alone was never significantly higher among

supplement users than non-users for any of the 10 nutrients investigated. In fact, for magnesium

among adults ≥51 y and vitamin C among females ≥51 y, the prevalence of inadequacy was

significantly lower among users when compared to non-users. From diet alone, the percent of

intakes >UL was low for all 11 nutrients examined, except zinc in children, and did not differ

between users and non-users for any nutrient. When supplements were included, ≥10% of users

in some age/sex groups had intakes >UL for vitamins A, C, niacin, folic acid, iron, zinc and

magnesium, reaching >80% for vitamin A and niacin in children.

Conclusions: From diet alone, prevalence of inadequacy was low for the majority of nutrients

and users were not at greater risk of inadequacy than non-users; supplement use sometimes led to

intakes >UL.

129

6.2 INTRODUCTION

The consumption of dietary supplements is on the rise (8, 227, 272), with consumers citing

several reasons for their use, most notably to improve and maintain health (45). These trends are

in contrast to population-based dietary guidance; the American Dietetic Association, for

example, recommends obtaining adequate nutrients from a wide variety of foods rather than

supplementation (3).

Using nationally-representative data from the Canadian Community Health Survey (CCHS)

2.2, Guo et al. reported that 41% of Canadian adults use vitamin/mineral supplements, and that

sex, fruit and vegetable consumption, physical activity, education and income were important

determinants of supplement consumption (7). In the United States, the prevalence of dietary

supplement consumption since 1999 appears to be similar to that in Canada, with estimates

ranging from 32 to 57% (3, 6, 8, 38, 227-230). Investigators from the United States have shown

that based on diet alone, either there are no differences in nutrient intakes between users and

non-users, or that users have better intakes and lower prevalence of nutrient inadequacies (42-44,

273, 274). However, there are no population-based studies in Canada that investigate whether or

not supplement users, based on diet alone, actually have a higher prevalence of nutrient

inadequacies when compared to non-users.

In Canada, where the composition of dietary supplements is regulated under Health Canada‟s

Natural Health Product Directorate, the maximum amount of a nutrient permitted in a non-

prescription supplement is set at the Tolerable Upper Intake Level (UL) for that nutrient (275),

defined as the highest intake of a nutrient thought to pose no adverse health effects (67). In the

United States, where supplements are regulated under the Food and Drug Administration, there

130

is no limit to the maximum permitted (276). Therefore, if a supplement contains nutrients at or

close to the UL, when diet is also taken into consideration, users of that supplement will have

intakes >UL. Thus, there are very real concerns that supplement use can lead to potentially

excessive intakes as has been previously demonstrated in the United States (38-40, 43). In

Canada, we have established the potential for excessive folic acid intakes with supplement use

(262), but there is no research examining risks of excess for other nutrients.

The main objective of this study was to compare the prevalence of nutrient inadequacy and

the percent of intakes >UL from diet alone between supplement users and non-users in Canada

and to determine the effect of supplement use on the prevalence of inadequacy and percent of

intakes >UL.


Data source and subjects

We used data from the CCHS 2.2 survey, a nationally representative cross-sectional survey

conducted in 2004, with 35,107 respondents (140). These data were collected under the authority

of the Statistics Act of Canada. The response rate for the survey was 76.5% and study weights

were recalculated to be based on respondents only. The CCHS 2.2 contains a general health

component and a nutrition component (24-h recall), which represents Canada‟s first nationally

representative dietary intake data collection in >30 y. The analyses in this paper included all

non-pregnant, non-lactating respondents ≥1 y and were stratified by sex and age groups as

defined in the IOM DRIs (277). The final analyses included 34,381 subjects.

Food and supplement intake data collection

131

Food intake data were collected using a modified version of the US Department of Agriculture‟s

(USDA) Automated Multiple Pass Method for 24-h food recall (140, 247). All respondents

completed one 24-h recall in person with a trained interviewer, and a subset of 10,786 (10,570 in

our sample) respondents completed a second 24-h recall 3-10 d later via telephone interview.

Nutrient composition of foods was based on the Canadian Nutrient File version 2001b (248),

which, in turn, was primarily derived from the USDA Nutrient Database for Standard Reference

13 (240). Supplement consumption data were collected as part of the general health component

of the CCHS 2.2 during the first interview. Respondents were classified as an overall

vitamin/mineral supplement user if they answered “yes” to the following question: “In the past

month, did you take vitamin/minerals?” Respondents were then asked specific details on the

type and frequency of supplement consumption and presented the supplement container to the

interviewer for examination. In cases where the exact formulation of the supplement could not

be determined (<1%), a set of default values based on the most commonly reported supplement

in that class of supplements was used to represent the nutrient composition of the unknown

supplement (140). For each nutrient, the average supplemental amount consumed daily was then

calculated for each respondent and reported in the CCHS 2.2 data files. Because supplement use

was calculated as daily intake, for analyses that included supplements, nutrient contribution from

supplements was added to food intake data on both days among respondents who completed a

second 24-h recall.

For individual nutrients, respondents were classified as “users” for a particular nutrient if

they consumed a supplement containing that nutrient; those who didn‟t consume a particular

nutrient in supplemental form were classified as “non-users” of that nutrient, even if they

132

consumed a supplement containing other nutrients. A multi-vitamin/mineral supplement was

defined as any supplement containing ≥3 vitamins and/or minerals. Henceforth in this paper, the

term “supplement” will refer to any vitamin or mineral supplement, and “MVM” will refer

specifically to any multi-vitamin/mineral supplement.

Nutrients included in the analyses

The prevalence of inadequacy was estimated as the proportion of respondents, stratified by

age/sex group, with usual nutrient intakes below the Estimated Average Requirement (the „EAR

cut-point method‟) (67) for each nutrient for which the EAR has been defined and a prevalence

of inadequacy >10% has been documented among at least one age/sex groups among all CCHS

2.2 respondents (143). The nutrients that met all of the criteria and therefore were analyzed for

prevalence of inadequacy were: vitamins A, C, B6, folate, B12, and D, phosphorus, calcium,

magnesium, and zinc. While data were available in the CCHS 2.2 for niacin, riboflavin, and

thiamin, there was no prevalence of inadequacy for any age/sex group in the population and

hence these nutrients were not included in our estimation of prevalence of inadequacy (143).

Iron was not included in the analyses because there was a low prevalence of inadequacy among

all groups except women of childbearing age (143), and among these women, the EAR cut-point

method could not be used because the distribution of requirements was not symmetrical (67).

Folate was reported in dietary folate equivalents (DFE), and based on the following

calculation: DFE = µg food folate + (µg food folic acid x 1.7) + (µg supplemental folic acid x 2)

(9). A larger conversion factor was used for supplemental folic acid (2 instead of 1.7) because

supplements in the CCHS 2.2 survey were assumed to be consumed on an empty stomach (140).

133

The percent of individuals with usual intakes >UL was estimated for a nutrient if the CCHS

2.2 contained data on intakes for that particular nutrient, and if there is an established UL for the

nutrient. Both criteria were met for the following nutrients: vitamins A, C, D, B6, folic acid and

niacin, and phosphorus, calcium, magnesium, iron, and zinc. The ULs for vitamin A and niacin

are based on preformed retinol and niacin, respectively, but the CCHS 2.2 does not distinguish

between the naturally occurring and preformed types of the vitamins. Therefore, in order to

estimate the percent of intakes >UL, preformed retinol and niacin in foods were estimated as

previously described by Sacco and Tarasuk (278). We conservatively estimated preformed

retinol to comprise two-thirds of the vitamin A found in supplements (66.7%), even though it

ranges from 60% to 84% as component of total supplemental vitamin A.

Estimation of the prevalence of inadequacy and percent of intakes above the UL in

supplement users and non-users

The prevalence of inadequacy and percent of intakes >UL from diet alone were calculated for

supplement users and non-users separately by sex and age group. Among supplement users, the

prevalence of inadequacy and intakes >UL were then re-estimated, this time including the

nutrient contribution from supplements. While there are limitations in combining 24-h recall

(food intake) and frequency (supplement consumption) data (251), supplement users were

analyzed separately from non-users.

Statistical analysis

All statistical analyses were performed with SAS software (version 9.2; SAS Institute Inc, Cary,

NC). The Software for Intake Distribution Evaluation (SIDE) (version 1.11, Department of


134

to estimate the subjects‟ usual (long-term) nutrient intakes using the second 24-h recall from

10,570 respondents as others have described previously (253).

The SIDE program was also used to estimate the prevalence of inadequacy among

respondents. Non-zero prevalence estimates <5% are listed as simply as <5% due to the

imprecision of prevalence estimates at the tails of the distribution (67). For these analyses, for

the most part, respondents were classified according to the Dietary Reference Intakes sex and

age subgroups. However, in order to increase sample size, the following groups were merged

except where the EAR for a particular nutrient differed: boys 9-13 y and girls 9-13 y; males 19-

30 y and males 31-50 y; females 19-30 y and females 31-50 y; males 51-70 y and males ≥71 y;

females 51-70 and females ≥71 y. Therefore, as the EAR for vitamin A is different for 9-13 y

males and females, the two sexes could not be combined. Similarly, for magnesium, the EAR is

different for 19-30 y and 31-50 y in both males and females, and for calcium, the EAR is

different for males 51-70 y and ≥70 y. In these specific cases, the prevalence of inadequacies

was estimated separately and these are presented separately in the tables. SIDE was also used to

estimate the percent of individuals with usual intakes above the UL. We recognize the

limitations of using the UL as a strict risk assessment cut-off (254), and therefore merely infer

that intakes below the UL are safe.

Since the sampling design for the CCHS 2.2 was complex and multi-stage, variance

estimation for these analyses was calculated using the bootstrap balanced repeated replication

technique (140). Five hundred replicate sample survey weights were generated each by

randomly selecting a sample, with replacement, from the original sample and then applying all

135

the performed adjustments to this selected sample. A P <0.05 was considered statistically

significant in all analyses.

6.4 RESULTS

Prevalence of supplement consumption

The overall prevalence of any supplement consumption (single nutrients, two-nutrient

combinations and MVM) in the population was 40%, while the prevalence of MVM

consumption (defined as a supplement containing ≥3 vitamins and/or minerals) was 28% (Table

6.4.a). Over 90% of users in each sex/age group reported consuming only one dosage of a given

supplement at a time. The total number of vitamins/minerals per supplement consumed was 5.9

overall, and ranged from 4.5 (females >50 y) to 9.7 (children 4-8 y). Vitamin C was the most

commonly consumed nutrient overall (32%), except among females ≥51 y, among whom

supplemental vitamin D (44%) and calcium (48%) were more commonly consumed than vitamin

C (38%). Children 4-8 y had the highest prevalence of supplement and MVM consumption

among respondents aged 1-18 y (45% and 42%, respectively), and for each individual nutrient

consumed as a supplement except magnesium and zinc. Conversely, males 14-18 y had the

lowest prevalence of supplement and MVM consumption (23% and 15%, respectively) and for

each individual nutrient consumed as a supplement except magnesium and zinc. Among adults,

females ≥51 y had the highest prevalence of consumption of any supplement (60%), MVM

(37%), and for all nutrients except iron.

Comparison of the prevalence of nutrient inadequacy from diet alone between supplement

users and non-users

136

Based on the nutrient contribution from dietary intake alone, the prevalence of inadequacy

among children 1-13 y was very low for all nutrients except vitamin D and calcium, reaching 85-

91% for vitamin D (Tables 6.4.b and 6.4.c). Similarly, among individuals ≥14 y, the prevalence

of inadequacy was consistently high only for vitamins A (29-46%) and D (74-93%), magnesium

(23-68%) and calcium (24-86%). The prevalence of inadequacy of any nutrient from diet alone

was never significantly higher among supplement users than non-users. In fact, for magnesium

among adults ≥51 y and vitamin C among females ≥51 y, the prevalence of inadequacy was

significantly lower among supplement users when compared to non-users.

Percent of intakes >UL among supplement users and non-users based on diet alone

With the exception of vitamin A in children 1-3 y and zinc in children 1-8 y, few individuals

(<5%) had nutrients intakes >UL from dietary sources alone (Tables 6.4.d and 6.4.e). Among

children 1-3 y, 42% of supplement users and 59% of non-users were consuming zinc at intakes

>UL, while 8-9% of users and non-users had vitamin A intakes >UL. Based on diet alone, the

percent of individuals with nutrient intakes >UL did not differ between supplement users and

non-users for any sex/age category.

Effect of supplement consumption on the prevalence of nutrient inadequacy and percent of

intakes >UL among supplement users

When supplement consumption was included in the analyses, the prevalence of inadequacy

among users approached zero for the majority of nutrients (Tables 6.4.b and 6.4.c). While there

was a significant reduction in the prevalence of vitamin D, magnesium and calcium inadequacy

with supplement consumption, there still remained some inadequacy across all sex/age groups

(Tables 6.4.b and 6.4.c). For example, 14-36% of individuals ≥14 y consumed inadequate

137

vitamin D, and 25-38% of adults ≥51 y had inadequate intakes of calcium even though they

consumed supplements. As expected, use of supplements led to an increase in the percent of

intakes >UL for all nutrients, reaching ≥10% for at least one sex/age group for folic acid, vitamin

A, vitamin C, niacin, zinc, calcium, magnesium and iron (Tables 6.4.d and 6.4.e). For vitamins,

the percent of intakes >UL was consistently high for niacin, surpassing 38% in all subgroups of

the population and reaching as high as 85% in children 1-3 y (Table 6.4.d). For minerals, the

percent of intakes >UL was highest for zinc, reaching 76% in children 1-3 y and >37% in

children 9-13 y and males 14-18 y (Table 6.4.e). However, the prevalence of zinc supplement

consumption was low among children 1-13 y (2-3%) (Table 6.4.a).

6.5 DISCUSSION

Results herein suggest that, for most nutrients analyzed, the prevalence of inadequacy in Canada

is low, and supplement users do not have a higher prevalence of nutrient inadequacy from dietary

sources alone compared to non-users of supplements. In fact, there were a few instances where

supplement users had a significantly lower prevalence of inadequacy than non-users. These data

are consistent with comparisons of adult supplement users and non-users in the Hawaii-Los

Angeles Multiethnic Cohort (MEC) reported by Murphy et al., where based on dietary intake

alone there was no difference in nutrient adequacy between the two groups (38). Similarly,

Briefel et al. reported in the Feeding Infants and Toddlers Study (FITS) that except for vitamin

E, the prevalence of nutrient inadequacy from diet alone was low among toddlers, and similar for

supplement users and non-users (41). Several other reports from the United States indicate that

for several nutrients (e.g. vitamin A, folate, and zinc), supplement users have higher mean

intakes (42, 44, 273, 274) and lower prevalence of inadequacy than non-users (43) from diet

138

alone. This phenomenon, which reflects the fact that supplement use is more prevalent among

those with better diet quality and thus less need for a supplement, has been termed the “inverse

supplement hypothesis” (279).

From dietary intake alone, the percent of Canadians in the present study that had nutrient

intakes >UL was negligible (<5%) for all ages for all nutrients except zinc and vitamin A in

children (Tables 6.4.d and 6.4.e). Authors of the FITS analyses also reported that some toddlers

consumed zinc and vitamin A at intakes >UL from dietary sources only (41). Adults (≥51 y) in

the nationally-representative Continuing Survey of Food Intakes by Individuals (CSFII) did not

exceed the UL for any nutrient from dietary intake only (43), which is consistent with our

findings here. Similarly, only a small proportion of the MEC adult participants exceeded the UL

from dietary intake alone, except for niacin and folate (38). Interestingly, in our analysis, it is

worth noting that even though the percent of intakes >UL from diet alone was negligible (<5%),

it was greater than zero in many instances for several nutrients, suggesting that the upper tails of

these distributions were approaching the UL.

The prevalence of supplement use (40%) in the CCHS 2.2 was similar to that reported in a

national random telephone survey on Natural Health Product usage in Canada (41%) (45). Our

findings are generally similar to those of most analyses of the U.S. National Health and Nutrition

Examination Surveys (NHANES) for both adults (39% to 71%) (6, 8, 39, 226) and children (29

to 43%) (6, 40, 228, 230), and any differences are likely due to the fact that the NHANES

estimate of dietary supplement use includes herbs, botanical, and other types of dietary

supplements, while the CCHS 2.2 estimate does not (140, 264). Furthermore, in an analyses of

the nationally representative U.S. National Health Interview Survey in 2000, in which

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specifically vitamin/mineral supplement consumption data were collected, Millen et al. report

that 39.3% of adults use a vitamin/mineral supplement, which is similar to what we found in our

study (227).

We found that in the few instances in which there was a prevalence of nutrient inadequacy

from diet alone (e.g. vitamin A, magnesium, calcium, and vitamin D), supplement consumption

significantly reduced the prevalence of inadequacy. This is similar to what Sebastian et al. report

in their analysis of supplement contribution to the diet of older adults in the CSFII (43). Murphy

et al. report a similar decline in inadequacy for several vitamins/minerals in their analysis of

MEC respondents (38). However, despite the significant reduction in inadequacy with

supplement use, we found that there still remained a proportion of users with inadequate intakes,

and supplement use was not without risk.

In the present analysis, use of supplements led to an increase in the percent of Canadians that

had intakes >UL for all nutrients, with substantial proportions of supplement users >UL for folic

acid, zinc, magnesium, niacin, vitamin A, and iron. For example, >80% of children 1-3 y taking

supplements consumed >UL for vitamin A and niacin (Table 6.4.d). Likewise, Murphy et al.

reported that adult MEC respondents consumed >UL for vitamin A, niacin, folic acid, iron and

zinc from food and supplements (38), and Sebastian et al. showed that supplement use led to

excessive intakes of iron and zinc in older men (>50 y) (43). Food and supplements combined

also led to intakes >UL for vitamin A, folic acid, sodium and zinc among toddlers in the FITS

(41).

We emphasize here that intakes <UL are considered safe and care must be taken when

interpreting risk of intakes >UL (67, 254), as the body of literature used to derive ULs for most

140

nutrients was of lesser quality compared to that used to establish EARs (67, 277). Furthermore,

the ULs for different nutrients were based on adverse effects of differing seriousness (67, 277).

As such, a large proportion of intakes >UL for one nutrient may be of greater concern than that

of another nutrient. Additionally, the ULs for some nutrients may be re-evaluated as more

research is conducted.

It is well established that certain subgroups of the population are at increased risk of specific

nutrient deficiencies and are thus recommended to consume a supplemental source of that

nutrient. In the population we studied (non-pregnant, non-lactating, non-breastfed individuals ≥1

y) this would include women of childbearing age (folic acid) (9) and adults ≥51 y (vitamins D

and B12) (9, 245). We found that 27% of women 19-50 y consumed a folic acid-containing

supplement, while 28-44% and 27-32% of adults ≥51 y consumed a vitamin D- and vitamin B12-

containing supplement, respectively. Given the substantial prevalence of inadequacy

documented for vitamins A, D, and calcium and magnesium among most age/sex groups in

CCHS 2.2, it might be argued that nutrient supplements should be recommended as a means to

improve intakes for these nutrients. However, the results of the foregoing analysis highlight the

potential for excessive intakes when supplements are consumed. For example, we found ≥10%

of children had intakes >UL for vitamin A and magnesium, and a similar percentage among

adults ≥51 y for calcium and magnesium.

Strengths of this study include that it is the first nationally-representative analyses comparing

the nutritional adequacy, based on usual intake, of supplement users and non-users in Canada

and second, the effect of supplement use on the prevalence of inadequacy and percent of intakes

above the UL. Furthermore, our study population included respondents ≥1 y, representing the

141

most comprehensive work in the literature to date in this area. Also, to our knowledge, our study

is one of the first to report prevalences of inadequacy for vitamin D and calcium, using the

recently updated EARs for these two nutrients.

We acknowledge that our results likely overstate the true extent of nutrient inadequacies in

the Canadian population, and may understate the proportion of Canadians with usual intakes

>ULs. First, underreporting common to dietary intake data collection has been documented in

the CCHS 2.2 (280). Secondly, it is established that both supplements (166) and fortified foods

(250) contain overages that we didn‟t account for in these analyses, and we have previously

shown with folate that fortified food overages can affect the prevalence of inadequacy (262).

Another limitation associated with this work is that we estimated prevalence of nutrient

inadequacy based on dietary and supplement intake data alone and did not investigate clinical

measures of deficiency. While dietary intake is frequently associated with biochemical

measures, this is not always the case. For example, there is little vitamin D deficiency in Canada

based on serum 25-hydroxyvitamin D concentration, whereas we found a high prevalence of

inadequacy based on analysis of dietary intake alone (281).

In summary, using the most recent nationally representative dietary and supplement intake

data collection in Canada, we conclude that for nutrients other than vitamins A and C, calcium,

and magnesium, from diet alone, prevalence of inadequacy is low, and supplement users are not

at greater risk of inadequacy when compared to non-users. However, supplement use leads to

intakes >UL for several nutrients investigated. Given that inadequacy is likely lower than we

estimated and percent of intakes >UL higher, outside scientifically established subgroups at

142

increased risk of deficiency, Canadians should exercise caution with regards to general use of

vitamin/mineral supplements.

143

Table 6.4.a. Prevalence of vitamin/mineral (VM) supplement consumption by sex/age groups.

A. Vitamins

Consumption of a supplement containing1

Group

N

No. of

nutrients per

supplement

Any

VM

MVM2 Vit A Vit C Vit D Vit B6 Folic

acid

Vit B12 Thiamin Ribofla-

vin

Niacin

Children

1-3y 2192 9.0 (.2) 38 (2) 35 (2) 35 (2) 36 (2) 35 (2) 32 (2) 31 (2) 31 (2) 32 (2) 32 (2) 32 (2)

4-8y 3343 9.7 (.2) 45 (1) 42 (1) 41 (1) 44 (1) 41 (1) 42 (1) 38 (1) 41 (1) 40 (1) 40 (1) 40 (1)

9-13y 4192 7.6 (.2) 33 (1) 25 (1) 24 (1) 31 (1) 24 (1) 24 (1) 23 (1) 24 (1) 23 (1) 23 (1) 24 (1)

Male

14-18y 2397 6.9 (.4) 23 (2) 15 (1) 14 (1) 21 (1) 15 (1) 14 (1) 14 (1) 14 (1) 14 (1) 14 (1) 14 (1)

19-50y 4646 6.9 (.4) 29 (1) 20 (1) 18 (1) 25 (1) 19 (1) 19 (1) 19 (1) 19 (1) 19 (1) 19 (1) 19 (1)

≥51y 4324 5.6 (.2) 41 (1) 28 (1) 26 (1) 32 (1) 28 (1) 26 (1) 26 (1) 27 (1) 26 (1) 26 (1) 26 (1)

Female

14-18y 2346 6.0 (.3) 29 (2) 17 (1) 16 (1) 24 (2) 17 (1) 16 (1) 15 (1) 15 (1) 16 (1) 16 (1) 16 (1)

19-50y 4766 5.9 (.2) 42 (1) 29 (1) 25 (1) 33 (1) 28 (1) 27 (1) 27 (1) 27 (1) 27 (1) 27 (1) 27 (1)

≥51y 6175 4.5 (.1) 60 (1) 37 (1) 31 (1) 38 (1) 44 (1) 31 (1) 30 (1) 32 (1) 30 (1) 31 (1) 30 (1)

Overall 34381 5.9 (.1) 40 (1) 28 (1) 25 (1) 31 (1) 28 (1) 26 (1) 25 (1) 26 (1) 25 (1) 25 (1) 25 (1)

144

B. Minerals

1 Percent (SE); SEs were calculated using the bootstrap balanced repeated replication method.

2 Multi-vitamin/mineral supplement consumption; any supplement containing ≥3 vitamins/minerals.

Consumption of a supplement containing1

Group n Calcium Phosphorus Magnesium Iron Zinc

Children

1-3y 2192 21 (2) 16 (1) 2 (1) 20 (1) 2 (.4)

4-8y 3343 28 (1) 21 (1) 3 (1) 24 (1) 2 (.4)

9-13y 4192 16 (1) 12 (1) 4 (1) 15 (1) 3 (.4)

Male

14-18y 2397 14 (1) 10 (1) 9 (1) 11 (1) 8 (1)

19-50y 4646 19 (1) 12 (1) 18 (1) 15 (1) 17 (1)

≥51y 4324 29 (1) 18 (1) 25 (1) 20 (1) 23 (1)

Female

14-18y 2346 15 (1) 8 (1) 11 (1) 14 (1) 9 (1)

19-50y 4766 30 (1) 14 (1) 25 (1) 24 (1) 19 (1)

≥51y 6175 48 (1) 18 (1) 31 (1) 24 (1) 28 (1)

Overall 34381 28 (1) 15 (.4) 20 (.4) 20 (.4) 17 (.4)

145

Table 6.4.b. Prevalence of inadequacy1 for selected vitamins among supplement (VM) users and non-users.

A. Children 1-13 y

Group Vitamin A2 Vitamin C Vitamin B6 Folate Vitamin B12 Vitamin D

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Supplement Use

No3

Yes Yes No Yes Yes No Yes Yes No Yes Yes No Yes Yes No Yes Yes

Children

1-3y <5 0 0 0 0 0 < 5 0 0 6

(1)

0 0 <5 0 0 86

(2)

85

(3)

105

(2)

4-8y <5 <5 0 0 0 0 0 0 0 <5 0 0 0 <5 0 91

(2)

94

(2)

155

(12)

9-13y 25

(3)

/11

(3)

14

(6)

/16

(3)

<5

/

<5

<5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 91

(1)

85

(3)

145

(2)

146

B. Respondents ≥14 y

Group Vitamin A Vitamin C Vitamin B6 Folate Vitamin B12 Vitamin D

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Supplement Use

No3 Yes Yes No Yes Yes No Yes Yes No Yes Yes No Yes Yes No Yes Yes

Male

14-18y 38

(4)

36

(10)

<5 8

(2)

5

(3)

<5 <5 <5 <5 7

(2)

5

(7)

<5 <5 <5 0 77

(2)

74

(8)

165

(6)

19-50y 46

(4)

42

(8)

<5 19

(3)

18

(4)

<5 <5 <5 <5 6

(1)

6

(3)

<5 <5 <5 0 91

(2)

87

(4)

195

(3)

≥51y 46

(3)

41

(8)

<5 30

(2)

20

(3)

<5 16

(2)

12

(3)

<5 22

(2)

13

(3)

<5 <5 <5 <5 85

(2)

74

(7)

145

(3)

Female

14-18y 42

(3)

47

(7)

<5 6

(2)

<5 <5 11

(2)

<5 <5 28

(3)

23

(6)

<5 15

(3)

20

(9)

<5 93

(2)

93

(4)

365

(7)

19-50y 39

(3)

34

(5)

<5 18

(2)

10

(3)

<5 13

(2)

13

(3)

<5 25

(3)

33

(4)

<5 12

(3)

8

(7)

<5 93

(2)

93

(2)

245

(2)

≥51y 39

(3)

29

(5)

<5 20

(2)

114

(2)

<5 28

(3)

21

(3)

<5 40

(3)

39

(3)

<5 9

(3)

14

(4)

<5 90

(3)

92

(2)

165

(1)

148

1 Values are percentages (SE); calculated using the Software for Intake Distribution Evaluation (version 1.11, Department of Statistics

and Center for Agricultural and Rural Development, Iowa State University).

2 The EAR for vitamin A is different for 9-13 y males and females hence the two sexes could not be combined. The first prevalence in

each cell represents males 9-13 y and the second represents females 9-13 y.

3 “No” represents supplement non-users; “Yes” represents users.

4 Lower than “Diet only – non-users” for the same nutrient (P < 0.05).

5 Lower than both “Diet only – non-users and users” for the same nutrient (P < 0.05).

149

Table 6.4.c. Prevalence of inadequacy1 for selected minerals among supplement (VM) users and non-users.

A. Children 1-13 y

Group Phosphorus Magnesium

Zinc

Calcium

Diet only Diet +

VM

Diet only Diet +

VM

Diet only Diet +

VM

Diet only Diet +

VM

Supplement Use

No2

Yes Yes No Yes Yes No Yes Yes No Yes Yes

Children

1-3y <5 0 0 0 n/a3 0 0 0 0 <5 <5 <5

4-8y 0 <5 0 <5 n/a3 0 0 n/a

3 n/a

3 21 (2) 28 (4) 12

7 (3)

9-13y 20 (2) 13 (4) 76 (3) 12 (1) <5 <5 7 (1) <5 <5 57 (2) 48 (4) 34

7 (4)

150

B. Respondents ≥14 y.

Group Phosphorus Magnesium4

Zinc Calcium5

Diet only Diet +

VM

Diet only Diet +

VM

Diet only Diet +

VM

Diet only Diet +

VM

Supplement Use

No2

Yes Yes No Yes Yes No Yes Yes No Yes Yes

Male

14-18y <5 9 (4) 6 (3) 43 (3) 31 (9) 156 (6) 5 (1) <5 <5 33 (3) 33 (7) 23 (7)

19-50y <5 0 0 38 (4)/

47 (3)

23 (6)/

35 (8)

146 (7)/

206 (5)

13 (2) <5 <5 38 (2) 24 (5) 126 (3)

≥51y <5 <5 0 61 (2) 446 (4) 24

7 (3) 31 (3) 20 (4) <5 52 (3)/

81 (3)

55 (5)/

74 (5)

257(4)/

387 (8)

Female

14-18y 34 (3) 38 (10) 24 (14) 68 (2) 55 (7) 296 (7) 19 (3) 28 (8) <5 70 (3) 75 (5) 55

8 (5)

19-50y <5 <5 <5 37 (4)/

38 (3)

28 (13)/

29 (5)

86

(3)/

127 (2)

15 (2) 8 (3) <5 53 (3) 45 (4) 167 (2)

≥51y <5 <5 <5 46 (2) 336 (3) 12

6 (1) 13 (3) 15 (3) <5 86 (2) 80 (2) 29

7 (2)

151




3 Due to low consumption of zinc- and magnesium-containing supplements, the prevalence of inadequacy couldn‟t be determined.

4 The EAR is different for 19-30 y and 31-50 y in both males and females. The first value in each cell represents 19-30 y and the

second represents 31-50 y.

5 The EAR is different for males 51-70 y and ≥70 y. The first value in each cell represents males 51-70 y and the second represents

≥70 y.

6 Lower than “Diet only – non-users” for the same nutrient (P < 0.05).

7 Lower than both “Diet only – non-users and users” for the same nutrient (P < 0.05).

8 Lower than “Diet only – users” for the same nutrient (P < 0.05).

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Table 6.4.d. Percent of intakes1 above the Tolerable Upper Intake Level (UL) for selected vitamins among supplement (VM) users

and non-users.

A. Children 1-13 y

Group Vitamin C Vitamin D Vitamin B6 Folic acid Vitamin A Niacin

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Supplement Use

No2


Children

1-3y <5 <5 10

(2)

0 0 0 0 0 0 <5 <5 7 (2) 9 (2) 8 (3) 883 (2) <5 <5 85 (2)

4-8y 0 0 <5 0 0 0 0 0 <5 <5 <5 5 (1) <5 <5 67 (2) <5 <5 73 (2)

9-13y 0 0 <5 0 0 0 0 0 <5 0 0 <5 0 0 18 (2) <5 0 51 (4)

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Group Vitamin C Vitamin D Vitamin B6 Folic acid Vitamin A Niacin

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Supplement Use

No2


Male

14-18y 0 0 <5 0 0 0 0 0 <5 0 0 14 (4) 0 0 <5 0 0 47 (7)

19-50y 0 0 <5 0 0 0 0 0 <5 0 0 9 (2) 0 0 <5 0 0 49 (3)

≥51y 0 0 <5 0 0 <5 0 0 <5 0 0 12 (1) <5 0 <5 0 0 50 (3)

Female

14-18y 0 0 <5 0 0 <5 0 0 <5 0 0 13 (3) 0 0 <5 0 0 38 (5)

19-50y 0 0 <5 0 0 0 0 0 <5 0 0 13 (2) 0 0 <5 0 0 43 (3)

≥51y 0 0 <5 0 0 <5 0 0 <5 0 0 11 (1) <5 0 <5 0 0 49 (2)




3 Higher than both “Diet only – non-users and users” groups for the same nutrient (P < 0.05).

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Table 6.4.e. Percent of intakes1 above the Tolerable Upper Intake Level (UL) for selected minerals among supplement (VM) users and

non-users.

A. Children 1-13 y

Group Phosphorus Calcium Iron Zinc

Magnesium4

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

VM only

Supplement Use

No2

Yes Yes No Yes Yes No Yes Yes No Yes Yes Yes

Children

1-3y 0 0 0 <5 <5 <5 0 0 <5 59 (3) 42 (27) 76 (28) 16 (12)

4-8y 0 0 0 <5 0 0 0 0 0 12 (3) n/a3 n/a

3 7 (4)

9-13y 0 0 0 <5 <5 <5 0 0 0 <5 <5 38 (10) <5

155


Group Phosphorus Calcium Iron Zinc

Magnesium4

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

Diet only Diet

+ VM

VM only

Supplement Use

No2

Yes Yes No Yes Yes No Yes Yes No Yes Yes Yes

Male

14-18y <5 <5 <5 <5 <5 <5 <5 <5 7 (3) <5 <5 37 (7) <5

19-50y 0 0 0 <5 <5 <5 0 0 < 5 0 0 16 (2) <5

≥51y 0 0 0 <5 <5 7 (2) 0 <5 6 (2) 0 0 14 (2) <5

Female

14-18y 0 0 0 0 0 <5 0 0 8 (2) 0 0 6 (3) <5

19-50y 0 0 0 <5 0 <5 0 0 15 (2) 0 0 6 (1) 6 (2)

≥51y 0 0 0 <5 <5 15 (1) 0 0 7 (2) 0 0 14 (1) 10 (1)

1

Values are percentages (SE); calculated using the Software for Intake Distribution Evaluation (version 1.11, Department of Statistics



3 Due to the low consumption of zinc-containing supplements, the percent of intakes above the UL among users couldn‟t be estimated.

4 The UL for magnesium is based only on the supplemental form of the nutrient.

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CHAPTER 7.0: DISCUSSION, CONCLUSIONS & FUTURE DIRECTIONS

7.1 DISCUSSION & CONCLUSIONS

Food fortification and nutrient supplementation are two strategies used to combat nutrient

deficiencies. However, these strategies may also present health risks in the form of nutrient

intakes above the UL. As such, at a population level, it is important to strike the right balance

between health benefits of such strategies and unintended risks. This thesis consisted of four

data chapters answering important questions regarding food fortification, with a special focus on

the B-vitamin folate, and vitamin/mineral supplement consumption.

In the first study, we conducted direct analysis of the folate content of fortified foods. In

1998, the Canadian government mandated folic acid fortification of white wheat flour and

enriched pasta to increase intake in women capable of becoming pregnant and in turn, decrease

the incidence of NTDs (4). The success of this program in reducing the prevalence of NTDs is

undisputed (14). However, after more than 10 y since the initiation of mandatory folic acid

fortification in Canada, until now there has been no published direct analyses of the actual folate

content of foods and how these values compare to mandated levels and label claims. This is

important because there is now a growing body of literature suggesting some potential harmful

effects of too much folic acid, including masking and progression of vitamin B12 deficiency,

reduced effectiveness of anti-folate drugs, decrease in natural killer cell cytotoxicity and cancer

progression in the presence of pre-existing neoplasms (16-19, 23, 75). Furthermore, high intakes

of folic acid in pregnancy have been associated with increased adiposity, insulin resistance and

poor respiratory health in the offspring (24-26). Also, studies from the United States, which

began its fortification program earlier in the same year as Canada, suggest that there are

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considerable folic acid overages in fortified foods, especially in the early post-fortification period

(27, 49).

In the first study of this thesis (Chapter 3), we directly analyzed the folate content of 95 of

the most commonly purchased fortified foods in Canada. We show here that there was

approximately 50% extra folate in foods than would be expected if fortification levels reflected

mandated amounts. Our finding is identical to an estimate in a letter-to-the-editor by Quinlivan

& Gregory, which was based on a prediction equation from a convenience sample relating

change in serum folate concentrations pre- and post-fortification to dietary folate intake (29).

Furthermore, we found considerable variation across food categories, with breakfast cereals

containing the most overages (88% extra folate). To our knowledge, this is the first study of its

kind in Canada, and our findings led us to recommend monitoring of folate levels in Canadian

fortified foods. In fact, after the publication of this work, Health Canada soon began monitoring

folic acid (and other nutrients) in select food items. In addition, our findings imply that, unless

overages are accounted for, estimates of folate inadequacy are likely overestimated and intakes

above the UL likely underestimated. This led to the second study of this thesis (Chapter 4).

The Canadian Community Health Survey (CCHS) 2.2 was conducted in 2004 and represents

the first nationally representative dietary intake data in Canada in over 30 y. Importantly, it

allows for an evaluation of the impact of the mandatory folic acid fortification program.

However, data on the prevalence of folate in adequacy were calculated using food composition

values that reflected the mandated rather than actual levels of fortification. We used the CCHS

2.2 data to remodel folate intakes among non-pregnant, non-lactating respondents ≥1 y to

account for the folate overages determined in our first study (Chapter 3). We concluded that

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after accounting for overages, the prevalence of dietary folate inadequacy is low in Canada

except for in women >70 y. Furthermore, including the use of a folic acid-containing

supplement didn‟t substantially impact the prevalence of inadequacy, but did result in up to 5%

of intakes above the UL among Canadians. This is similar to results from the United States (39,

40, 130) and from smaller convenience samples from Canada (31, 33, 129), although overages

were not considered in these studies, except for the report by Hennessy-Priest et al. in which they

modeled their data based on theoretical overages (129). Given that supplement use had little

impact on inadequacy, and the prevalence of folate inadequacy was low, there seems to be little

need for supplemental folic acid among children and males. Similar to what Shuaibi et al. (33),

French et al. (31) and Liu et al. (32) showed in their convenience samples, we concluded that

despite mandatory fortification, women capable of becoming pregnant still need to consume a

folic acid-containing supplement in the periconception period in order to meet the IOM

recommendation to consume 400 µg/d from all sources for reduction in the risk of an NTD birth

outcome (9). In addition, we estimated that, along with dietary folic acid, a folic acid

supplement of 325-700 µg/d and 325-500 µg/d for adult and adolescent females, respectively,

would serve to provide 400 µg/d and not lead to intakes above the UL. To our knowledge, this is

the first study to use previously determined overage factors to account for folic acid overages in

a nationally representative dietary intake database. Furthermore, since the completion and

publication of Study 2, Colapinto et al. used the nationally-representative CHMS to assess folate

status using RBC folate (161). Their findings of virtually no folate deficiency in Canada closely

support our results (161).

159

In our second study, we found that women of childbearing age don‟t consume the

recommended amount of folic acid from diet alone and thus need to consume a folic acid-

containing supplement. However, there are currently a wide range of available/consumed doses

in Canada (up to 5000 µg) and some have made blanket recommendations for all women capable

of becoming pregnant to consume 5000 µg (34). While some women have elevated folate

requirements and may benefit from a supplemental dose of folic acid that is >400 µg (35), there

is little evidence that most women would require more than 400 ug/d folic acid for protection

against an NTD. From a biochemical perspective, it has been previously established that RBC

folate concentrations ≥906 nmol/L provide maximal protection against folate-dependent NTDs

(36, 37). However, until now, there remained a gap in the literature as to what factors predispose

a woman to have RBC folate <906 nmol/L, a surrogate measure of folate-dependent NTD risk.

Therefore, in the third study (Chapter 5), we used data from the nationally-representative

NHANES 2003-2006 to investigate factors associated with RBC folate <906 nmol/L. We found

that 65% of women had RBC folate ≥906 nmol/L, much higher than the 10% reported by

Dietrich et al. using previous NHANES data (127). However, our estimate is lower than the

22% reported by Colapinto et al. using recent nationally-representative Canadian data (161).

However, our findings are a more accurate portrayal of the actual proportion of women with

RBC folate ≥906 nmol/L because we corrected for methodological differences between

NHANES RBC folate methodology (Bio-Rad assay) and that used to establish the 906 nmol/L

cutoff (microbiological assay) while neither Dietrich et al. nor Colapinto et al. did. Therefore,

our work represents the first time RBC folate has been corrected to become comparable to the

established benchmark for NTD-risk reduction. From our findings, we also concluded that not

160

using a folic acid supplement, smoking, younger age (19-34 y), race (non-Hispanic black women

and women of “other” races [non-Hispanic, non-black, non-white]) and low dietary folate intake

(≤262 DFE) are all associated with increased likelihood of having RBC folate <906 nmol/L.

Therefore, a woman‟s age, use of folic acid supplement, dietary folate intake, race/ethnicity and

smoking status should all be considered when determining whether or not a woman needs a folic

acid supplement dose greater than 400 µg.

In the second study, we observed that for children and men in particular, there was a very

low prevalence of folate inadequacy and supplement use had little impact on its prevalence.

Further, consumption of a folic acid supplement did drive up intakes, leading to up to 5% of

intakes above the UL. We wondered whether our observation that the use of folic acid

supplementation did little to impact the prevalence of inadequacy applied to other nutrients.

While there are some subgroups of the population at increased risk of inadequacy and are thus

recommended a supplement (9, 245), there is currently no clear guidance on the role of

supplements for the general Canadian population. To fill this void, we used data from the CCHS

2.2 to compare nutrient inadequacy and percent of intakes above the UL from diet alone between

vitamin/mineral supplement users and non-users for the following nutrients: vitamins A, C, B6,

B12 (prevalence of inadequacy only), D, niacin (above the UL only) and folate, and phosphorus,

magnesium, calcium, zinc, and iron (above the UL only). We then evaluated the impact of

supplement use on the prevalence of inadequacy and percent of intakes above the UL among

users for these nutrients. Based on our findings, we concluded that from diet alone, users are not

at increased risk of inadequacy when compared to non-users, and that prevalence of inadequacy

was low for most nutrients except calcium (all ages), vitamin D (all ages), vitamin A

161

(adolescents and adults) and magnesium (adolescents and adults). Furthermore, supplement use

led to a percent of intakes above the UL ≥10% for vitamins A, C, niacin, folic acid, iron, zinc

and magnesium. Also, due to underreporting and the presence of overages in supplements and

fortified foods, it is likely that actual nutrient distributions are higher than we estimated, resulting

in lower prevalence of inadequacy and higher percent of intakes above the UL. While several

similar studies have been conducted in the United States yielding similar results (38, 41-44, 273,

274), we investigated more nutrients than any of the previous studies and we included the entire

spectrum of the Canadian population, making our study the most comprehensive work on this

topic. In light of our results, we recommend that unless there is a strong case for supplement

use, the general Canadian population should be recommended to obtain adequate nutrients from

a wide variety of nutritious foods as opposed to vitamin/mineral supplements.

From a methodological perspective, the work in this thesis employed two approaches seldom

used in dietary assessment: accounting for overages (Chapters 3 and 4) and supplemental

nutrient contribution (Chapters 4 and 6). Micronutrient overages in foods as a result of

fortification increases nutrient exposure of the population. While our examination of food

fortification focused on the mandatory folic acid fortification program, the Canadian food supply

is also currently fortified (mandatorily or voluntarily) with other micronutrients. For some

products there is a mandatory range of nutrient fortification levels. For example, the regulations

stipulate that fluid milk contain between 31.7 and 51.6 IU of vitamin D/100 ml (282). However,

despite a having a stipulated range (i.e. a lower and upper limit), in a small convenience sample,

it has been previously reported that the majority of 15 milk samples contained vitamin D

amounts outside of this range (246). Perhaps more concerning are products in which the

162

Canadian regulations merely stipulate a minimum mandatory fortification level without an upper

limit, similar to folic acid. For example, white wheat flour is also mandated to contain a

minimum amount of thiamin (640µg/100g), riboflavin (400µg/100g), niacin (5.3mg/100g), and

iron (4.4mg/100g) (4). Therefore, it is likely that overages also exist for nutrients that have

mandated minimum levels of fortification without an upper limit. Consequently, it is likely that

the Canadian population is exposed to higher amounts of these nutrients than what would be

assumed based on mandated minimum levels, thereby resulting in lower inadequacy and higher

percent of intakes above the UL.

Secondly, until recently, and largely due to methodological issues in combining dietary and

supplemental intake data (251, 283), supplemental nutrient contributions have been largely

overlooked in assessing dietary adequacy and intakes above the UL. However, within the

context of the limitations in combining dietary and supplement intake data, we have shown that

not including supplemental nutrient contribution can lead to erroneous estimates of both nutrient

inadequacy and percent of intakes above the UL. Coupled with the fact that vitamin/mineral

supplement use is widespread in Canada (7, 45), this suggests that, in order to better gauge total

nutrient exposure, future dietary assessments should attempt to include both dietary and

supplemental nutrient contribution.

There are a few limitations inherent in the work presented herein. For example, in Studies 1

and 2 (Chapters 3 and 4), we analyzed a limited number of folic acid-containing foods in this

study and we used these to gauge the extent of folate overages in Canada. However, among the

tens of thousands of food products on the market, our sampling of fortified foods was systematic

using the most recent national food expenditure and product brand data. We were able to

163

identify and analyze the 10 or 15 most commonly purchased fortified food in each food category.

Therefore, we believe these data are sufficient to gauge the extent of the folate overage problem

in Canada. Another limitation herein is the fact that, in Study 3 (Chapter 5), several of the

factors used, such as dietary folate intake, folic acid supplement use and smoking, were based on

self-reported data and thus are prone to bias. Additionally, dietary intake data collection is prone

to underreporting (30) and this has been previously documented in the CCHS 2.2 (280).

However, we feel that underreporting, while a limitation, further supports our conclusions that

inadequacy is low in the Canadian population, and likely overestimated. On the other hand, due

to underreporting, the percent of intakes above the UL have likely been underestimated for

several nutrients. Also, another limitation is fact that the CCHS 2.2 was conducted in 2004 and

it is possible that dietary patterns have changed since that time. For example, there are now

other sources of fortified food and natural health product items on Canadian shelves, such as

Aquafina‟s Vitamin Water, Tropicana‟s Trop50, and RockStar‟s Energy Drink. The effect of the

consumption patterns of these new sources of micronutrients in the diet could not be captured in

this database. Nonetheless, the CCHS 2.2 remains our most recent nationally-representative

dietary intake data collection.

7.2 FUTURE DIRECTIONS

Results from this thesis provide a platform for three avenues of research/application. Firstly,

given that we observed folic acid overages in foods, and that those overages varied by food

category, we recommend continual and expanded monitoring of nutrient fortification levels in

foods. Even though we didn‟t find folic acid intakes above the UL from food alone, given that

there are many new sources of nutrient fortified food items (e.g. Aquafina vitamin water,

164

Tropicana Trop50) available to Canadians since the CCHS 2.2 in 2004, it is likely that intake

distributions have been shifted up since that time.

Secondly, based on the findings in our third study, we recommend developing and validating

a short screening tool to identify women who might be at increased risk of a folate-dependent

NTD birth outcome. The tool should include as items age, folic acid supplement use, smoking,

race/ethnicity, and questions to capture dietary folate intake. For example, given that white

wheat flour products and vegetables comprise the two largest sources of folate in the diet (128),

questions to capture white wheat flour and vegetable consumption are important in

approximating dietary folate intake. Additionally, nationally-representative Canadian data

should be used to investigate other potential factors associated with RBC folate <906 nmol/L. It

is likely that other factors not captured in these analyses also impact RBC folate. For example, it

is previously established that at low folate intakes, individuals who are homozygous (T/T) for the

methylene tetrahydrofolate reductase C577T genotype have lower blood folate status compared

to individuals who are heterozygous (C/T) or homozygous for the wild type (C/C) (284, 285).

However, this is likely not a large contributing factor to RBC folate status, because more recent

research from the post-fortification era (i.e. a folate-rich environment) has shown that the

methyelene tetrahydrofolate reductase genotype is not associated with folate status (286).

Nonetheless, there are likely other factors associated with RBC folate status and identifying the

most impactful ones will lead to a more comprehensive tool. The aim of this area of research is

to develop a tool that can quickly capture women at increased risk of an NTD birth outcome,

who can then be monitored appropriately and if needed, recommended a higher dose (>400 µg)

165

of folic acid. This will result in fewer women consuming unnecessarily high doses of folic acid,

which preliminary research suggests might be harmful to offspring.

Lastly, based on findings of our fourth study, we recommend research investigating

biochemical nutrient status for the few nutrients in which there were cases of inadequacy to

further establish that, outside special recommendations for certain subgroups, vitamin/mineral

supplements are not necessary for the general Canadian population. Since this study was the first

of its kind in Canada, we also recommend similar analyses using future nationally representative

dietary intake data as they become available. Nutrient intake distributions will likely be higher

than what we observed in this study, because there are many new fortified products now

available to consumers that were not on Canadian shelves in 2004 when the CCHS 2.2 was

conducted. We hypothesize that this will lead to a higher percent of intakes above the UL,

especially when supplement use is accounted for. The problem of excessive macronutrient

consumption at the population level is well established and evident in the dramatic rise in the

prevalence of overweight and obesity over the past several years. Given the current trends in

fortification and availability of vitamins/minerals, Canadians are likely also facing the problem

of excessive micronutrient consumption. Therefore, further research is needed on high intakes of

micronutrients, particularly from fortification and supplements, and their effects on the Canadian

population.

166

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282. Department of Justice. Food and Drug Regulations. In: Justice, ed. Ottawa.

283. Garriguet D. Combining nutrient intake from food/beverages and vitamin/mineral

supplements. Health Rep 2010;21(4).

284. Jacques PF, Bostom AG, Williams RR, et al. Relation between folate status, a common

mutation in methylenetetrahydrofolate reductase, and plasma homocysteine

concentrations. Circulation 1996;93(1):7-9.

285. Zittoun J, Tonetti C, Bories D, Pignon JM, Tulliez M. Plasma homocysteine levels

related to interactions between folate status and methylenetetrahydrofolate reductase: a

study in 52 healthy subjects. Metabolism 1998;47(11):1413-8. doi: S0026-

0495(98)90315-8 [pii].

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286. Caudill MA, Le T, Moonie SA, Esfahani ST, Cogger EA. Folate status in women of

childbearing age residing in Southern California after folic acid fortification. J Am Coll

Nutr 2001;20(2 Suppl):129-34.

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CHAPTER 9.0: APPENDICES

A. Nutritional Guidelines for a Healthy Pregnancy – Health Canada

High Dose Folic Acid Supplementation - Questions and Answers for Health Professionals

1. When should women be advised to take more than 400 mcg

(0.4 mg) folic acid per day from a supplement to reduce their risk

of a pregnancy affected by a neural tube defect (NTD)?

2. If it is determined that a woman may benefit from a higher

level of supplemental folic acid, how should it be provided?

3. If a woman' s only risk factor for a pregnancy affected by a

neural tube defect (NTD) is poor dietary intake of folate (situation

A) and it is determined that her adherence to taking supplements

is poor, should a higher dose of folic acid be recommended?

References

Acknowledgements

1. When should women be advised to take more than 400 mcg (0.4 mg)

folic acid per day from a supplement to reduce their risk of a pregnancy affected by a neural tube defect (NTD)?

In determining whether a higher dose of folic acid supplementation is warranted, a woman's

health care provider should first ascertain whether the woman has personal characteristics or

health conditions associated with an elevated risk of having a baby with a NTD.

After this is established, the health care provider should assess whether the elevated risk is related to a woman's:

A. Low dietary intake of folate or

B. Elevated folate requirement or

C. Uncertain disease etiology where the role of altered folate metabolism is unclear.

Situation A: Low dietary intake of folate

Risk factors for a NTD associated with low dietary intakes of folate (in the absence of elevated folate requirements) 1:

Poor dietary quality

Chronic dieting, and/or avoidance of folic acid fortified foods (e.g.

low carbohydrate diets)

Low socio-economic status

Food selection and preparation methods that may be more

common in specific ethnic groups (e.g. use of non-fortified rice as

a staple; use of maize flour (masa) versus folic acid fortified

wheat flour among certain Hispanic-Canadians; and prolonged

stewing, a common practice among some South Asian-Canadians that destroys naturally occurring folate)

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Smoking

Situation B: Elevated folate requirement

Risk factors for a NTD that may be associated with elevated folate requirements 1:

Personal or family history of NTDs or other congenital anomalies

Medications* that interfere with folate metabolism 1, 2, 3

Alcohol abuse

Malabsorption and gastric bypass surgery 4

Liver Disease Kidney dialysis

* A large number of drugs are known to alter folate metabolism. Several of these have been shown to increase NTD risks (e.g. valproic acid, carbamazeprine, trimethoprim). Other drugs such as phenobarbitol, primidone, diphenylhydantoin, oxcarbamazepine, sulfonamides and methotrexate may increase the risk for other potentially folate-sensitive anomalies (e.g. orofacial clefts and heart defects) or potentiate NTD risks when combined with other drugs 1, 2, 3. Other medications that are known to elevate folate requirements but whose impact on NTD risk is unknown include metformin, triamterene and barbiturates1.

Situation C*: Uncertain disease etiology

Risk factors for a NTD in which it is uncertain whether altered folate metabolism (and specifically an elevated folate requirement) plays a role in the etiology of the birth defect 5, 6, 7, 8, 9, 10:

Obesity

Diabetes

Impaired glucose metabolism Hyperinsulinemia

* While beyond the scope of these questions and answers focusing on folic acid, health care professionals are reminded that impaired glucose metabolism, high glycemic index diets and hyperinsulinemia are thought to be independently related to the risk of birth defects, including NTDs 5, 6, 7.

If the indicator of risk for a NTD is associated with low dietary folate intake (situation A), in the

absence of an elevated requirement, it is not necessary to advise more than 0.4 mg per day of supplemental folic acid.

If the indicator of a NTD risk is associated with an elevated folate requirement (situation B), a

higher dose of folic acid (greater than 0.4 mg/d) should be recommended. However, clear

instructions should be given to the woman on when this higher dose should be started, and

transitioned to a lower dose supplement or stopped. (See response to question 2 for details on how to provide a higher dose of folic acid.)

In the case of situation C, the health care provider may wish to measure a woman's red blood

cell (RBC) folate concentration in order to determine the most appropriate dose of folic acid to

recommend 11. It has been shown that RBC folate concentrations greater than 906 nmol/L are

maximally protective against folate-dependent NTDs 12.

If her RBC folate concentration is greater than 906 nmol/L,

and she typically takes up to 0.4 mg of supplemental folic acid


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each day, she can be advised to use or continue using a daily

multivitamin containing 0.4 mg of folic acid for the prevention of a

NTD-affected pregnancy.

If her RBC folate concentration is below 906 nmol/L, and

she regularly takes 0.4 to 1 mg of supplemental folic acid each

day, a higher dose (in combination with a multivitamin

supplement) may be beneficial. (See response to question 2 for

details on how to provide a higher dose of folic acid.)

Top of Page

2. If it is determined that a woman may benefit from a higher level of supplemental folic acid, how should it be provided?

A woman with personal characteristics or health conditions that put her at risk of a pregnancy

affected by a neural tube defect (NTD) due to elevated folate requirements (situation B, or in

situation C, when RBC folate concentration is less than 906 nmol/L) should consume 4 to 5 mg

per day of folic acid in combination with a B12-containing multivitamin supplement. She should be

advised to start taking this supplemental dose at least 3 months before conception and to

continue until 10 to12 weeks into her pregnancy 13. At 10 to12 weeks, she can be advised to

transition to a daily multivitamin supplement containing 0.4 mg of folic acid for the duration of pregnancy and lactation.

If it is determined that a woman may benefit from a higher level of supplemental folic acid, a health care provider can recommend that a woman:

1. consume a multivitamin supplement and add single folic acid

tablets as necessary to achieve the desired daily dose of folic acid,

or 2. consume a multivitamin containing more than 1 mg of folic acid.

With both approaches, the multivitamin supplement should contain vitamin B12; most multivitamin supplements available in Canada contain vitamin B12.

Women should be advised not to take more than one multivitamin supplement each day in an

attempt to consume a higher dose of supplemental folic acid. In large doses some substances in

multivitamins could be harmful. This is especially true of Vitamin A in the retinol form (including

retinyl palmitate and retinyl acetate). The Tolerable Upper Intake Level (UL) for vitamin A is

3,000 mcg retinol activity equivalent (RAE) or 10,000 IU.

In the event that a woman doesn't conceive after 3 or 4 months and adherence in taking

supplements daily is good, a lower level of folic acid supplementation (0.4 to1.0 mg) should be

considered. By this time, the blood folate concentrations of most women will be within the

maximally protective range 14. Red blood cell (RBC) folate concentrations should be determined

after 4 months on a lower dose of folic acid. Her RBC folate concentration will decrease, but it is

important to ensure it remains above 906 nmol/L to provide maximal protection against NTDs 12.

The health care provider should have the woman re-tested after an additional 4 months to confirm that a new plateau for RBC folate concentration above 906 nmol/L has been achieved.

3. If a woman' s only risk factor for a pregnancy affected by a neural tube defect (NTD) is poor dietary intake of folate (situation A) and it is

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determined that her adherence to taking supplements is poor, should a higher dose of folic acid be recommended?

Again, in this instance, the woman's red blood cell (RBC) folate concentration can be used to

guide the most appropriate dose of folic acid to recommend 11. It has been shown that RBC folate concentrations above 906 nmol/L are maximally protective against folate-dependent NTDs 12.

If her RBC folate concentration is greater than 906 nmol/L,

it is not necessary to advise more than 0.4 mg/d of supplemental

folic acid. However, the health care provider should encourage

the woman to take her supplement on a daily basis.

If her RBC folate concentration is below 906 nmol/L, a

higher dose of folic acid should be recommended. The dose of

folic acid recommended (1 to 5 mg) should be guided by the

woman's level of adherence to supplement intake and how far

below the 906 nmol/L cut-off her RBC folate concentration is.

Women should be advised not to take more than one multivitamin supplement each day in an

attempt to consume a higher dose of supplemental folic acid. In large doses some substances in

multivitamins could be harmful. This is especially true of Vitamin A in the retinol form (including

retinyl palmitate and retinyl acetate). The Tolerable Upper Intake Level (UL) for vitamin A is 3,000 mcg retinol activity equivalent (RAE) or 10,000 IU.

References

1. Institute of Medicine. Food and Nutrition Board. Dietary reference

intakes for thiamin, riboflavin, niacin, vitamin b6, folate, vitamin

b12, pantothenic acid, biotin, and choline. Washington, DC:

National Academy Press 1998.

2. Hernandez-Diaz S, Werler MM, Walker AM, Mitchell AA. Folic acid

antagonists during pregnancy and the risk of birth defects. N Engl

J Med 2000; 343(22): 1608-14.

3. Ornoy A. Neuroteratogens in man: An overview with special

emphasis on the teratogenicity of antiepileptic drugs in

pregnancy. Reprod Toxicol 2006; 22(2): 214-26.

4. Gasteyger C, Suter M, Gaillard RC, Giusti V. Nutritional

deficiencies after roux-en-y gastric bypass for morbid obesity

often cannot be prevented by standard multivitamin

supplementation. Am J Clin Nutr 2008; 87(5): 1128-33.

5. Rasmussen SA, Chu SY, Kim SY, Schmid CH, Lau J. Maternal

obesity and risk of neural tube defects: A meta-analysis. Am J

Obstet Gynecol 2008; 198(6): 611-9.

6. Ray JG, Wyatt PR, Vermeulen MJ, Meier C, Cole DE. Greater

maternal weight and the ongoing risk of neural tube defects after

folic acid flour fortification. Obstet Gynecol 2005; 105(2): 261-5.

7. Stothard KJ, Tennant PW, Bell R, Rankin J. Maternal overweight

and obesity and the risk of congenital anomalies: A systematic

review and meta-analysis. JAMA 2009; 301(6): 636-50.

8. Shaw GM, Velie EM, Schaffer D. Risk of neural tube defect-

affected pregnancies among obese women. JAMA 1996; 275(14):

1093-6.



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9. Watkins ML, Rasmussen SA, Honein MA, Botto LD, Moore CA.

Maternal obesity and risk for birth defects. Pediatrics 2003; 111(5

Part 2): 1152-8.

10. Werler MM, Louik C, Shapiro S, Mitchell AA. Prepregnant weight in

relation to risk of neural tube defects. JAMA 1996; 275(14):

1089-92.

11. Tam C, McKenna K, Ingrid Goh Y, Klieger-Grossman C, O'Connor

D L, Einarson A, Koren G. Periconceptional folic acid

supplementation: A new indication for therapeutic drug

monitoring. Ther Drug Monit 2009;31(3):319-26.

12. Daly LE, Kirke PN, Molloy A, Weir DG, Scott JM. Folate levels and

neural tube defects. Implications for prevention. JAMA 1995;

274(21): 1698-702.

13. Prevention of neural tube defects: Results of the medical research

council vitamin study. Mrc vitamin study research group. Lancet

1991; 338(8760): 131-7.

14. Nguyen P, Tam C, O'Connor DL, Kapur B, Koren G. Steady state

folate concentrations achieved with 5 compared with 1.1 mg folic

acid supplementation among women of childbearing age. Am J Clin Nutr 2009; 89(3): 844-52.

Acknowledgements

These questions and answers for health professionals were prepared by:

Dr. Deborah L. O'Connor, RD, PhD, Director of Clinical Dietetics at

the Hospital for Sick Children and Associate Professor at the University of Toronto's Department of Nutritional Sciences.

Additional expert advice was provided by:

Dr. Jane Evans, PhD, FCCMG, Professor of Biochemistry and

Medical Genetics, Pediatrics and Child Health, and Community

Health Sciences at the University of Manitoba, and Chair of the

Advisory Group of the Canadian Congenital Anomalies

Surveillance Network, as well as

Dr. Gideon Koren, MD, FRCPC, Founder and Director of the

Motherisk Program at the Hospital for Sick Children, and Professor

of Pediatrics, Pharmacology, Pharmacy, and Medical Genetics at the University of Toronto.

Members of the Expert Advisory Group on National Nutrition Pregnancy Guidelines also contributed to this work.

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