DETERMINATION OF IRON & IODINE ABSORPTION FROM ...

DETERMINATION OF IRON & IODINE ABSORPTION FROM

IRON AM) IODINE DOUBLE-FORTIFIED SALT

Masoud Sattarzadeh

A thesis in confonnity with the requirements

for the Degree of Master of Science

Graduate Department of Nutritional Sciences

University Of Toronto

43 Copyright by Masoud Sattarzadeh 1997

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DETERMINATION OF IRûN & I O D N ABSORPTION FROM IRON AND IODINE DOUBLE-FORTIFIED SALT

Master of Science, 1997

Masoud Sattarzadeh

Department of Nutritional Sciences, University of Toronto

Abstract

A table salt, fortified with iron (Fe) and iodine O, would be usefil in areas where

Fe and 1 deficiencies coexist, however, interactions between the two minerals prevent their

sirnultaneous use as fortificants. A method has been developed to coat 1 with dextran

(dex) such that Fe and I do not interact. Our objective was to determine the absorption of

Fe and 1 from this salt when provided in meals designed to significantly inhibit or enhance

Fe- absorption. Subjects with normal haematologic status (n=16) ingested the two rneals

containing 5 g of table salt with 50 pg of I as potassium iodide and 1 mg of Fe (ferrous

fumarate labeled with ? ~ e ) per g of salt. Measured by whole-body counting, Fe-

absorption frorn the enhancing meal (8.8 f 1.8 %) was sigruficantly higher than Fe-

absorption fiom the inhibithg meal (1.7 t 0.8 %) (pcO.0001). U ~ a r y excretion of iodine

baseline and post-ingestion (48 hr before & after) were not sigrilficantiy different (10.9 i

1.5 &dl vs. 13 .O3 f 1.1, p<0.47), nor was it afEected by the two meds. We conclude

that dex coated-I was well absorbed and that Fe was also weil absorbed but infiuenced by

the composition of the med.

Page #

ABSTRACT

TABLE OF CONTENTS

ACKNOWLEDGEMENTS

.. U

iii

v

LIST OF TABLES vï

LIST OF FIGURES vii

... LIST OF ABBREVLATIONS wll

CElAPTER 1 - INTRODUCTION 1

1.1 Background 1.2 Objective

1.2.1 Objective 1.2.2 Hypothesis 1.2.3 Study Significance

2.1 iron 2.1.1 Iron Absorption 2.1.2 Iron Deficiency Anemia (IDA) 2.1.3 Iron Requirement 2.1.3 Measurements of Iron Status 2.1.4 Iron Toxicity

2.2 Iron Supplementation 14 2.2.1 Criteria for the Selection of Vehicles for bon Fortification 2.2.2 Salt as a Vehicle for Iron Fortification 15 2.2.3 Criteria for the Selection of Iron Sources 15

2.3 Radioisotope Studies 16

2.4 Iodine 2.4.1 Iodine Metabolism 2.4.2 Iodine Requirements 2.4.3 Iodine Deficiency Disorders (IDD) 2.4.4 Iodine Toxicity

iii

2.4.5 Assessrnent of Iodine / Thyroid Status 2.4.6 Iodine Deficiency Prevention

2.5 The double-fortifïed saIt

CHAPTER 3 - MATERIALS & METHODS

3.1 Experimental Design 3.1.1 Subjects 3.1.2 Procedure 3.1.3 Protocol 3.1.4 Test Dose of Inorganic Iron 3.1.5 Test for Palatability and Taste

3.2 Preparation of the Double-fortified Salt 3.2.1 Iron & Iodine Sources

3.3 Measurements

3.4 Statistical Analyses 3.4.1 Sarnple Size Determination 3.4.2 Data Analysis 3.4.3 Correction for Interindividual Variation

CHAPTER 4 - RESULTS

4.1 Iron Absorption

4.2 Urinary Iodine Excretion

C W T E R 5 - DISCUSSION

5.1 Discussion

5.2 Conclusion 5.2.1 Future Studies

CHAPTER 6 - REFERENCE

ACKNOWLEDGEMENTS

I would like to express rny sincere thanks and appreciation to Dr. Stanley Zlotkin,

who through his relentless effort, patience, and guidance has not only been a great source

of inspiration for me but has also provided me with a fantastic leaming experience. 1

wodd also like to sincerely thank the members of my advisory committee, Dr. Paul

Pencharz and Dr. Valene Tarasuk for their valuable guidance and support.

Special thanks to people who provided me with their technical support, John

Kjarsgaard, and Emiliga Bogdanovic, and to Rachelle Bross for her valuable fkiendship

and support. Last but certainly not the least, 1 would like to express my enormous

gratitude to my family and close niends without whose understanding and undying

support 1 would not have been able to achieve this.

LIST OF TABLES

Table 3.1

Table 3.2

Ta bIe 4.1

Table 4.2

Table 4.3

The composition of high and low bioavailable 29 rneals

Shdy Design 3 1

Results from the double-fortified salt investigation 39

Serum femtin and hemoglobin concentrations in 43 male and female subjects

Baseline and post-ingestion urinary excretion of 44 iodine

LIST OF FIGURES

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Serum femtin concentration (transformed data to obtain nomality)

Log semm ferritin concentration Vs. reference- dose iron absorption %

Baseline and post-ingestion urinary excretion of iodine pg/&

Cornparison of dietary Fe absorption after correction for inter-subject variability based on two different method

vii

LIST OF ABBREVIATIONS

IDA

IDD

Hb

Enhcd

hhbtd

Uncrctd

Rf-dcrtd

Se-frcrtd

WBC

IDRC

S m

WHO

UNICEF

ICCIDD

EDDI

GRAS

FDA

USP

1OM

MDIS

NIN

Iron Deficiency Anemia

Iodine Deficiency Disorders

Hemoglobin

Enhanced

Impaired

Uncorrected

Corrected based on reference-dose

Corrected based on serum ferritin

Whole-Body Counthg

International Development and Research Center

Sodium hexametaphosphate

World Health Organization

United Nations C hildren' s Fund

International Council for Control of Iodine Deficiency Disorders

Ethylenediarnine dihydroiodide

Generally Recognized As Safe

Food and Drug Administration

United States Pharmacopoeia

Institute Of Medicine

Micronutrients Deficiency Information System

National Institute of Nutrition

CHAPTER 1

Introduction

1. INTRODUCTION

1.1 Background

Anemia and goiter are the two major nutrition related disorders, aEecûng more

than 1/3 of the world population with serious consequences on mental and physicai

development. The manufacture and distribution of a salt fortified with both iodine and

iron has been suggested as an inexpensive, effective and efficacious means to prevent both

iodine and iron deficiencies. However, stability and bioavailability have remained as two

major concems in the manufacture of such a salt.

When iron and iodine are both added to the salt, the iodine is converted to

elemental iodine, which cm evaporate, and thus is rapidly lost. Earlier trials by Burgie et

al. had indicated that the iodine moiety of the double-fortified sait was unstable because of

the loss of iodine due to evaporation and catalytic oxidation of T to Iz in the presence of

ferrous ions and air oxygen (Burgie et al. 1986). Iron is aiso readily oxidized to the femc

form, which has a lowered bioavailability, an unpleasant taste and unsightly, yellowish

brown or rust colour.

Despite the apparent chernical incompatibility of iron and iodine, previous

published reports indicated that it may be possible to stabilize iodine on salt in the

presence of iron (Narasinga Rao, 1994). The forming of a physical barrier (i.e.

encapsulating) between the iodine compound, and iron would stabilize the systems.

Encapsulating agents could include such stabilizers as sodium hexametaphosphate, de-

or even purified sait '.

Successful application of sodium hexametaphosphate (SHMP) as an agent to

stabilize the double-fortified salt was recently reported in India (Narasinga Rao, 1994).

As a chelating agent, SHMP keeps the iron soluble and prevents its interaction with

iodine. However, recent extensive trials performed by Dr. Diosady's team at the

Department of Chernicai Engineering, University of Toronto, concluded that dextran

coated iodine in combination with ferrous fumarate results in a more stable compound. In

order to prevent the evaporation of iodine, and the iodine oxidation in the presence of

ferrous ion, the iodine moiety of the double-fortified salt was dextran encapsulated.

Diosady et al. demonstrated that the physical isolation of iodine results in acceptable

iodine retention under the worst expected storage and distribution conditions.

It has been recognized that the control of iron balance in normal man resides with

the absorptive rather than excretory process. Radio-isotope studies of single meals have

demonstrated that meal composition aiso has a major impact on nonheme iron absorption.

Factors such as ascorbic acid markedly increase nonheme iron absorption whereas

inhibitory components such as polyphenols or phytates impair its assimilation to a sirnilar

extent (Bothwell et al. 1989, Hdberg 1981). Depending on the balance between the

enhancers and inhibitors as well as the body iron stores, absorption may Vary as much as

20-fold in the sarne individual (Cook et al. 1991).

- - -

a Magnesium chlonde, is a hygroscopic impurity often found in unpurifieci sait which dramatically increases the moisture content of the salt and resuïts in immediate loss of more than 90% of the added iodine.

A problem with any meanirement of iron absorption fiom groups of subjects is the

inter- and intra-subject variabilities in absorption. In order to eliminate the dserence

among subjects' iron statu, we obtained an independent measure of their capacity to

absorb iron via the ingestion of a reference-dose of inorganic ferrous sait. In the present

study, dietary absorption of nonheme iron was measured by means of whole-body

counting (WI3C) technique. Whole-body counting is one of the most sensitive and reliable

methods of measuring iron absorption. As ail the retained radioactivity is measured with

WBC, the dose of the administered radioactivity can be substantidy reduced in

cornparison with other methods, such as red cell incorporation of radioiron.

Urinary excretion of iodine reflects the plasma level of inorganic iodine which in

turn reflects absorbed iodine (Vought & London, 1967). Inorganic compounds such as

those present in iodized sdt are reported to be completely absorbed and excreted in urine,

with very little lost in feces. Hence urinary excretion of iodine was measured in the

current study.

1.2.1. Objective

The objective of current study was to determine the absorption of iron and iodine

nom table salt in heaithy human volunteers ingesting high and low bon-available meals.

1.2.2. Hypothesis

i) Iodine and iron supplied by the double-fortified sait is absorbed by healthy human

subjects.

ii) Absorption of iron fiom a meal containing inhibitors of ùon absorption will be lower

than f?om a rneal containing enhancers of iron absorption.

1.2.3. Study Significance

It is hoped that the result of this project will provide information to the

International Development and Research Centre (IDRC), and other international agencies,

in their efforts to eradicate iodine and iron deficiencies in at-risk areas of the world.

CHAPTER 2

REMEW OF LITERATURE

2. REVIEW OF LITERATURE

2.1. Iron

Iron is a reactive metal that is rarely found fiee in nature. Iron chemistry is

complex, primarily because of its dual valency and reactivity with oxygen. These include

electron transfer, the transport, storage, and activation of oxygen, nitrogen kation,

detoxification of activated oxygen species, and deoxyribonucleotide synthesis ffom

nbonucleoside diphosphates (Wonvood 1980, Bezkorovainy 1980). Iron is, however, a

potentially toxic element, being a catalyst in the development of highly toxic fiee oxygen

radicals (Owen et al. 1987). Biological systems have therefore developed several ways of

Limiting the entry of iron into the body and converting any absorbed iron into a bound

"safe" form. In marnmals, serum iron is bound to transferrin, and most body iron is

present as porphyrin complexes (hemoglobin, myoglobin, and heme-containhg enzymes).

Iron is stored as femtin and hemosiderin.

2.1.1. Iron Absorption

Regdatory control of iron status occurs as a result of absorption and

bioavailability. It has been postulated that iron absorption is influenced by the rate of

tissue iron uptake and by the size of labile iron pool in various body tissues (Cavill et al.

1975). The amount of iron in the body stores is the major factor controiiing the

absorption of iron from the gastrointestinal tract. Even a smaU change in iron stores, as

reflected by senim femtin values, is accompanied by reciprocal aiteration in iron

absorption, (Cook et al. 1974, Bezwoda et al. 1979, Magnusson et ai. 198 1, Cook et al.

1990).

There are two types of iron compounds in the diet with respect to the mechanism

of absorption, heme iron (denved from hemogtobin and myoglobin) and nonheme iron

(denved mainly from cereals, b i t s , and vegetables). The absorption of these two kinds of

iron is influenced differently by dietary factors. The absorption of nonheme iron draws the

most interest as it foms the main part of the dietary iron intake. Its absorption in meals

varies wideiy due to the marked effect of both dietary factors and the iron status of the

subjects. When a comparison was made of the absorption of heme and nonheme iron fiom

meals, a signincant correlation was found between the absorption of the two kinds of iron.

However, a much greater fiaction of the heme iron was absorbed (37%) than the nonheme

iron (5%), (Bjorn- Rasmussen et al. 1974).

After heme iron enters the intestinal cells it is rapidly degraded by heme

deoxygenase (Ra.flï et al. 1974), and the released iron enters the common intracellular iron

pool. Subsequent mucosal handling of this iron appears to be identical to that of inorganic

iron (Cook, 1990). The duodenum and the proximal portion of the upper intestine are the

two major sites of iron absorption in the intestinal tract. However, the elirnination is

almost entirely through the colon (Robschit- Robbins 1929).

Four major factors govern the arnount of iron absorbed fkom an iron-forti£ïed food.

These are the iron fortification compound used, the amount of iron added, the presence of

enhancers or inhibitors of iron absorption in the meal, and the iron status of the consumer

(Fomon & Zlotkin, 1992). Wider application of isotopic techniques during the 1950's and

1960's led to the realization that the bioavailability of ingested iron may be more

important than total intake. It was also found early on that one food could interact with

the absorption of iron from another food Kayrisse et ai. 1968). An important conclusion

fiom these isotope studies was that the bioavailbility of iron in a meal is not the sum of the

absorption of iron fiorn the single foods contained in a meal, but rather a net effect of ail

food items, and their constituents, increasing or decreasing the nonheme iron absorption

(Cook et al. 1969). The Indian National Institute of Nutrition in 1975 showed that

dietary factors play an important role in the development of iron deficiency in Indian

subjects. The same study demonstrated that, although most habitually consumed diets in

different regions of India showed seemingly adequate arnounts of iron (20-30 mg, NIN,

India, 1975), data fiom isotope studies using the whole-body counting technique indicated

that in nonanemic adult males, only 1 to 5% of this iron was absorbed (Narasinga Rao,

1978).

During the past two decades, application of the extrinsic-tag method for measuring

nonheme-iron absorption has led to the identification of a large array of dietary factors

aEecting iron absorption in humans (Reddy et al. 1991). It was concluded that in the

context of the US diet, the role of enhancers was less important than the role of inhibitors

of nonheme iron absorption (Cook et al. 1991). Cook et al. (1976) reported that chelates

in the diet may have a major effect on the assimilation of polyvalent transitional cations

such as iron. It was shown that certain chelates such as ascorbic acid enhance iron

absorption by forrning iron ascorbate complexes at low pH which remains soluble at the

high pH of the duodenum and donate their iron to mucosal cells. Other chelating

compounds, including polyphenols (containhg alkyl groups), phosphates, carbonates, and

oxalates were found to have the opposite effect on biavailability. Their effect is usualIy

due to the formation of polymers. They can either enhance or reduce iron absorption

depending on the stability constant, solubility at the intraluminal pH of the intestinal tract,

and the ability of the iron-cornplex to penetrate the mucosal barrïer.

While beef, larnb, pork, chicken, liver and fish substantially raise the rate of

nonheme iron absorption, milk, cheese, and eggs do not increase and may decrease iron

availability (Cook & Monsen 1976). Tannic acid in tea (DisIer et ai. 1975, HalIberg &

Cook 1979), phosvitin of egg yok (Rossander et ai. 1975, Monsen & Cook 1979, Hurrell

et al. 1988), phytates present in bran and nuts (Macfarlane et ai.1988), and hally

polyphenols present in legumes and leafy vegetables (Tustawiroon et al. 1991), ctearly

suppress absorption when present in sufficient amounts.

Of especial interest is the observation that meat has about the same promoting

effect on the absorption of heme and nonheme iron (Martinez et al. 1971). However, the

absorption promoting effect of meat is dose related (Layrisse et al. 1968, Hdlberg 1979).

2.1.2. Iron Deficiency Anemia (IDA)

Anernia is traditionaily defined according to age- and sex-related "cut-off points"

of hemoglobin values (Bothwell et al. 1979). Iron deficiency anemia refers to an anemia

that is associated with additional laboratory evidence of iron depletion as a result of one or

more of the following test results; low semrn femtin concentration, low transfemn

saturation, or an elevation in the erythrocyte portophyrin level (Earl & Woteki, 1993).

Iron deficiency is present when body iron is duninished (Cook et al. 1986, Charlton et al.

1982). The presence of iron deficiency implies neither the degree nor the presence of

anernia. Thus, an individual may be iron deficient without manifesting iron deficiency

anernia, the converse, however, does not occur (Pollitt et al. 1976).

The consequences of iron deficiency anemia have traditionally focused on anemia

which reduces maximum oxygen consumption and maximum work performance (Basta et

al. 1979, Edgerton et al. 1979). Severe anexnia is generally accepted as a health hazard.

Among functions that may be impaired are work capacity (Viteri et al. 1974), immune

function (Sirkanita et al. 1976, Joynson et al. 1972), and leamhg ability (Webb et al.

1973). Severe anemia during pregnancy increases morbidity and rnortality and is

associated with an increase risk of low birth weight infants (National Institute of Nutrition,

Annual Report, India, 1 98 O).

There is now considerable evidence that mild iron deficiency even without anemia

is associated with significant health consequences. The greatest impact of iron deficiency

is in growing children, who develop defects in attention span leading to learning and

problem solving difficulty (Rajomaki et al. 1979, Beilby et al. 1992, Leggett et al. 1990,

Witte 1991). This leaniing deficit may have lasting consequences by lirniting an

individual's subsequent achievement. Iron supplementation has been shown, to increase

growth velocity and decrease level of the morbidity in anemic cMldren (Coenen et al.

1991). Earlier studies of Pollitt et al. (1989) also show that schoolchildren with iron

deficiency have poorer cognitive function, which is oniy partly improved by iron

treatment.

2.1.3. Iron Requirements

According to Arthur and Isbister, a review of previous studies of anemia indicates

iron deficiency is almost never due to dietary deficiency in an adult in Western society.

This is because the average person requires very little iron intake to replace that lost

through sweat, menstruation, and urine, and also because as much as 90 % of the iron

needed for the formation of blood cells is derived from recycling senescent red blood celk

(Hofirand 1981). Estimates of iron requirements are based on obligatory losses, which

include those from exfoliation of cells from interna1 and extemal surfaces, urine, sweat and

rnenstmal fluids, in addition to iron needed for growth and during pregnancy (Green et al.

1968). Menstruation increases median iron requirements to about 1.4 mg daily (2.4 mg in

90% of fernales) and pregnancy raises this further to more than 5 mg daily in the second

and third trimesters (Bothwell, 1995). Fi@ percent of women would maintain normal

hemoglobin values if they absorbed 1.2 mg daily (0.8 basal plus 0.4 mg rnenstmal losses),

while a figure of 2.2 mg daily would be required to cover 95 % of menstruating women

(FAO/WHO Joint Report, 1988).

2.1.3. Measurements of Iron Status

Conventional laboratory indices of iron status include serum iron, transfemn / total

iron-binding capacity, transferrin saturation and femtin. Although each of these

measurements has ment, no single determination gives a reliable index of iron status

(Cazzola et ai. 1992, Burns et al. 1990). Iron deficiency without anemia is diagnosed on

the basis of a combination of biochemical indicators of iron status in which the hemoglobin

concentration remains within the normal range. Although no single indication of iron

status is diagnostic of functional iron deficiency, a low semm ferritin concentration

indicates that iron reserves are depleted (Earl & Woteki, 1993). Low serum femtin values

indicate iron deficiency but high values do not necessarily mean increased body stores.

Inflammation, liver disease, hem01 ytic rnalignant diseases and hemolytic anernia may

increase serum femtin concentration out of proportion to the size of the body store

(Lifschitz et al. 1974). The serum ferritin correlation in healthy adults is proportional to

the siie of body iron stores (Addison et al. 1972, Jacobs et ai. 1972, Cook et al. 1974),

and changes in the serum ferritin value correlate with changes in the size of the stores

(Sümes et al. 1974).

2.1.4. Iron Toxicity

There are two diseases associated with an excess of iron in the body. In

haemochromatosis immense deposits of inorganic iron are found in the liver , pancreas,

skin and other parts of the body Wuir et al. 1914, Sheldon, 1935). Inpolycythaemic ruba

vera the number of red cells and the arnount of hemoglobin are very greatly in excess of

normal. So much so, that the amount of iron in the blood may be doubled (Widdowson,

193 7). In addition to iron loading disorders (eg . haemochromatosis), there have been

recent disturbing claims based on epidemiological data suggesting that subjects with oniy

modestly raised iron stores are at greater risk of developing malignancy (Stevens et al.

1988) and ischemic heart disease (Salonen et al. 1992).

2.2. Iron Supplementation

Administration of therapeutic doses of iron is recomrnended as a short term

rneasure to correct anernia in situations when it is necessary to raise Hb levels quickly.

However, when anemia is less severe, and the main objective is to improve iron balance

over a longer period of time and to prevent the development of anemia in at risk

populations, fortification of foods with iron has been suggested as a practical measure

(WHO Technical Report Senes, 1972). Supplementation involves the giving of iron in

medicinal form. This is ofien the only feasible approach when there is a large iron

requirement and a relatively short time span, as occurring dunng pregnancy (Baker et al.

1979, International Nutritional Anemia Consultative Group 1979, WHO Technical

Report Series, 1975). Meanwhile, fortification is an approach which can be appiied to

large population groups at low cost. It has the advantage that the identification and

cooperation of actual and potential deficient individuals is not a prerequisite as it is with

supplementation.

2.2.1. Criteria for the Selection of Vehicles for Iron Fortification

Iron fortification has been considered as one of the practical approaches for the

prevention and the control of iron deficiency anernia in the population. Several factors

need to be considered in the choice of vehicles for iron fortification:

i) The vehicle must be consumed in sufficient arnount by the target groups in the

population. Ideal vehicles in most countries fiom this point of view are salt and flour

where the variability in consumption is only about twofold.

U) The fortified vehicle should remain stable and palatable after fortification.

iii) The distribution of the fortification in the vehicle should not change during storage

(Le. no sedimentation).

2.2.2. Salt as a Vehicle for I ron Fortification

Cornmon table salt (NaCl) is considered to be a suitable vehicle for iron

fortification satisfjing al1 the criteria of an ideal vehicle because: a) sdt is consumed by al1

segments of the population, rich or poor, perhaps somewhat more by the poor; and, b)

salt consumption lies within a narrow range of 12-20 glday, with an average intake of

1 Sg/day (Narasinga Rao, NIN Report, India).

2.2.3. Criteria for the Selection of Iron Sources

The identification of a suitable iron cornpound to be used with salt which will meet

the twin criteria of stability during storage and satisfactory bioavailability has been a

technological challenge. There are several factors that must be considered in choosing a

forti&ng compound (WHO, 1975; Cook and Reusser, 1983). The following are

examples of properties to keep in rnind:

a) Relative bioavailability of the compound

b) Reactivity of the compound to cause discoloration or any changes in flavor or odor.

c) Stability of the compound with storage and food preparation.

d) Compatibility with other nutrients

The bioavailability of nonheme iron is governed by its solubility in the lumen of the

proximal small bowel. Freely water-soluble sources, such as ferrous sulfate and ferrous

giuconate, exchange with the comrnon nonheme Fe pool in a fortified meal. Unfortunately,

these highly soluble Fe compounds also cause unacceptable organoleptic changes when

added to stored food (Hurrell, 1984). Other iron sources such as ferrous fumarate and

ferrous succinate are slowly soluble in water but readily soluble in dilute acids such as

gastric juice. These compounds provide an attractive compromise as they appear to be

sufficiently unreactive to avoid organoleptic problems during storage. Hurrel et al. (1989)

indicated that ferrous fbmarate is a suitable Fe source for food fortification. Fenous

furnarate, Fe(C00--CH=CH--C00), is a reddish orange to reddish brown powder that is

odorless and almost tasteless. It contains about 33 % Fe, and has a similar bioavailability

to ferrous sulfate in both rat and human studies, (Clydesdale & Wiemer, 1985).

2.3. Radioisotope Studies

The introduction of radioisotopes made it possible to label single food items

biosynthetically with radioiron (Moore et al, 1951). It became possible via this method to

measure iron absorption from a single composite meal (Hailberg et al, 1972). Meanwhile,

the accuracy of the extrinsic tag method is mainly based on the validity of the assumption

that a complete isotopic exchange occurs between the extrinsic tag and the main part of

the nonheme iron compounds in the diet (Bjom-Rasmussen et al. 1972, Cook et al. 1972).

When single foods biosynthetically Iabeled with radioiron (intrinsic tracer) were carefùily

rnixed with a trace arnount of uon sait labeled with another radioiron isotope (extrinsic

tracer), the observation was made that the absorption of the two tracers, from such

double labeled foods was almost identical (Hallberg et al. 1979).

5 ' ~ e is one of the isotopes usually employed in extrinsic tag studies. It can readily

be detected by means of its energetic gamma ray (1.1 and 1.3 MeV) and by its beta

emission (Emax 0.27 and 0.46 MeV). This use of radioisotopes of iron was first described

by Hahn (Hahn et al. 1943). Since then, highly accurate techniques of measuring iron

absorption have been developed such as, whole-body counting. Presently, whole-body

counting of orally absorbed radioiron is accepted as the reference method of iron

absorption (Bothwell et al. 1979). The method permits accurate iron studies using 1 pci of

5 %e or less, thus reducing radiation exposure of the patients to a minimum (Price et al.

1962). Use of the incorporation of oral and intravenous tracers into erythrocytes has been

s h o w to have a close correlation with whole-body counting (hm et al. 1967, Wemer et

al. 1983), however, the use of only oral tracer data was reported to give a poorer

correlation because of the uncertainty of blood volumes and radioiron utilbation in new

erythrocytes (Wemer et al 1983). It was observed by Wemer et al. that the use of a

double radioisotope technique with post-absorption semm measurements had the best

correlation with whole-body counting .

Despite such precise measurements, absorption studies have been difficult because

of large differences in absorption among normal subjects and in the same subject with

repeated testing (Kuhn et al. 1968, Bnse et ai. 1962). The largest variable is the

difference between individuais. While mucosai response can be standardiied by

comparing the absorption of food iron against a common reference standard, day to day

variation in the same subject are still appreciable (Cook et al.). Layrisse et ai. (1969)

showed that the differences in iron status among difEerent subjects can be eliminated by

obtaining an independent measure of their capacity to absorb iron. They introduced the

mode1 of using a reference-dose of inorganic radioiron given at a physiologic level under

the standardized conditions to each subject. The absorption of iron fkom various foods

will then be expressed as the ratio of food iron absorption to reference-dose absorption

(Magnussen et al. 198 1). There is a high correlation between the iron absorption fiom

meals and absorption From the reference-doses (Bjom-Rasmussen et al, 1976). The slope

of a regression line between the two absorption rneasurements (meals / reference-doses) is

an index of the bioavailability of the nonheme iron in a meal (Bjorn-Rasmussen et al.

1976). The most suitable type of iron compound to be employed as a reference standard

is probably a ferrous iron sait, since this represents the final common pathway by which al1

forms of dietary iron with the exception of hemoglobin are assimilated (Cuhn et al. 1968).

Semm ferritin can also be used as an alternative to the reference-dose absorption.

However, serum femtin is only an indirect measure of an individuai's ability to absorb

iron, and extraneous factors such as rninor idections may affect iron absorption and serum

femtin in opposite directions. Reference-doses therefore are preferable and should be

used whenever possible (Hallberg 1980)

2.4. Iodine

Iodine is an essential micronutnent for ail animai species, including humans (Hetzel

and Maberly 1986). It is an integrai part of the thyroid hormones, thyroxin and

triiodothyronine. These hormones are required for normal calogenesis, thermoregulation,

intermediary metabolism, protein synthesis, reproduction, growth, development,

hematopoiesis and neuromuscular fûnction (Fisher and Carr 1974). The ocean is the

world's major source of iodine. In coastai area, seafood, water, and even iodide-

containing sea rnist are dependable iodide sources. Iodine is present in food and water

predominantly as iodide and, to a lesser degree, bound to amino acids.

2 . 4 L Iodine Metabolism

Iodine is rapidly and almost completely absorbed and transported to the thyroid gland

for the synthesis into the thyroid hormones, to salivary and gastric glands, and to kidneys

for excretion into gastrointestinal tract and unne (Silva 1985). Iodinated amino acids are

well absorbed as such, aithough more slowly and less completely than iodide. However, a

proportion of their iodide may be lost in the feces in organic combination. The remainder

is broken down and absorbed as iodine (Keating et al. 1949). Other forms of inorganic

iodine are reduced to iodine pnor to absorption (Cohn 1932).

Iodine metabolism and thyroid function are closely linked, since the only known

role of iodine is in the synthesis of thyroid hormones. In order to ensure an adequate

supply of hormones, the human thyroid must trap about 60 pg of iodine daily (Undenvood

1971). This is primarily achieved irrespective of the plasma level, by adjustment of the

thyroidai iodine clearance rate, so that when the plasma iodide decreases the thyroidal

clearance increases, with the actual iodide uptake remaining more or less constant

(Underwood 1971). Adaptation to iodide deficiency thus occurs by increasing the

thyroidai iodide clearance rate. Such functional overactivity of the iodide-trapping

mechanism is usually associated with an increase in the gland mus, or goiter, but in mild

iodine deficiency the biochemical manifestation of the deficiency, namely low plasma

iodide and urinary iodine excretion and hi& thyroidal iodine clearance and radioiodine

uptake, have been demonstrated without aqy obvious goiter (Wayne et al. 1964).

Since only the thyroid gland and the kidneys are in competition for plasma iodide,

the ultimate accumulation of iodine by either of these organs, as measured by the 24-hr or

48-hr thyroidal uptake or urinary excretion, or both, is simply a reflection of this

competition and not a measure of the level of the function of either thyroid or kidneys. If

kidney fûnction were completely absent, virtually 100 per cent of administered iodine

would eventually accumulate in the thyroid gland (Berson 1956).

2.4.2 Iodine Requirements

The requirement of iodine in adults must be at least equal to the daily arnount of

hormonal iodine degraded in the peripheral tissues and unrecovered by the thyroid (40 to

100 pg/day). An extra margin of safety is needed to meet increased demands that may be

imposed by natural goitrogens under certain conditions. Balance studies have found that

intakes ranging fiorn 44 to 162 pg/day are suficient to maintain positive balance (WHO,

MDIS Working Paper #1, 1993). As a significant degree of neurological development

occurs w i t h weeks of conception, and especially during the first month of fetal growth, it

is imperative that al1 women of reproductive age have adequate iodine stores, not only

women who are pregnant (WHO, MDIS Working Paper #1, 1993).

2.4.3. Iodine Deficiency Disorders (IDD)

Iodine Deficiency Disorder as a te- was introduced in 1983 and has since

become generally accepted. It @ID) refers to the wide spectrum of effects of iodine

deficiency on growth and development (Hetzel 1983). Iodine deficiency disorders

continue to threaten the health and well being, and the social and economic productivity

and advancement, of several hundred million people throughout the developing world.

Recent evidence indicates a wide spectrum of disorders resulting from sever iodine

deficiency. These iodine deficiency disorders include: goiters at all ages, endemic

cretinism, (characterized most cornmonly by mental deficiency, de&-mutism and spactic

diplegia), and lesser degrees of neurological defect related to fetal iodine deficiency;

impaired mental function in children and adults (associated with reduced levels of

circulating thyroxin); increased still birth, and prenatai infant rnortaiity (Hetzel 1987).

Impairment of nervous system development and fûnction is the most important

consequence of iodine deficiency. The tenn "endemic cretinism" is traditionally used to

describe this condition, and usudy includes deaf-mutism, mental retardation, and a

characteristic spastic or rigid neuromotor disorder. Endernic cretinism also includes an

insult to the nervous system which is irreversible; however, in an iodine deficiency there

may be aspects of neurological dysfinction related to hypothyroidism which are reversible

with treatment (Delonge 1986). Studies in man have been complemented by studies in

animals which have established the effiects of iodine deficiency on the brain. These midies

have confirmed that the effects of iodine deficiency are mediated through the thyroid gland

secretion of thyroid hormones, thyroxin (T4) and triiodothyronine (T3), (Hetzel et al.

1993).

It is known that thiocynate, a naturally occurring goitrogen ' resulting fiom

chronic consumption of poorly detoxfied cassava, aggravates the effects of iodine

deficiency (Gitan et al. 1986). The principal goiotrogens identified are thiocynates and

isothiocynates; polyphenols (biflavonoids); phenolic derivatives (resorcinol and others);

phathaiic acid denvatives; and possibly calcium and lithium. Natural goitrogens, such as

those found in cabbage and cassava, have been implicated in the pathogenesis of goiter in

some parts of the world (Matovinovic, 1983).

2.4.4. Iodine Toxicity

Ever since the Canadian federal law obliged the table salt producers to add 76 pg

of iodine per gram of salt, endemic goiter has ceased to be a problem in Canada. On the

contrary, iodine excess appears to be a growing problem. Excessive intakes of iodine can

cause enlargement of the thyroid gland, just as deficiency c m . This goiter-like condition

can be so severe as to block the ainvays in infants and cause suffocation (Whitney &

Rolfes 1993).

(GOY-troh-jen), a thyroid antagonist found in food; causes toxic goiter.

The iodine intake of Canadians is in excess of 1000 pg/day, based on the anaiysis

of a representative diet, with 60 % comlng from table salt and 25 % kom d a j r produas

(Fischer and Giroux 1987a). Much of the iodine present in milk cornes fiom

ethylenediarnine dihydroiodide (EDDI), added to feed to prevent foot rot in cattle (Berg

and Padgitt 1985), and from improper use of iodophor sanitizers (Dunsmore and Wheeler

1977).

2.4.5. Assessrnent of Iodine / Thyroid Status

The two basic clinical techniques in the measurement of goiter are a) inspection

and palpation, and b) ultrasonography. On the other hand, before an individual develops

goiter in response to iodine deficiency, other important physiological changes occur.

These changes can be detected through the use of biochemicd indicators, including serum

thyroid hormones, TSH, and urinary iodine levels (WHO, MDIS Working Paper #1,

1993).

Since al1 the iodide secreted into the gastrointestinal tract is reabsorbed, the main

excretory route for the inorganic form of iodine is urine. Although losses in the milk of

lactating women and losses in sweat in hot climate can be considerable, urinary excretion

is a reliable indicator of iodine status under most circumstances.

2.4.6. Iodine Deficiency Prevention

Substantial evidence is now available that iodine deficiency disorders can be

prevented by iodization programs (Hetzel BS, DUM JT, Stanbury JB, 1987). A verity of

foods such as salt, bread, sweets, milk, sugar, and water have been used as vehicles for

iodine. However, over the past 50 years, iodized (or iodinated) salt has been the rnainstay

of iodine deficiency prophylaxis (Hetzel 1987). Iodinated salt is the most practical,

effective, and satisfactory means for correction of iodine deficiency. Ever since Marine

and Kimball demonstrated the efficacy of iodine prophylaxis in the control of endemic

goiter, widespread iodination of edible salt, to ensure physiological intalces of iodine, has

been the continued practice in almost al1 developed countries. Salt is one of the few

cornrnodities consumed by al1 sections of the cornmunity irrespective of social or

economic level. It is consumed at approxïmately the same level throughout the year by al1

normal adults. Cornpared to other food commodities, whose production is dispersed, the

production of salt is Iimited to fewer production centers. Recent studies have shown that

in many remote villages, salt is one of the few comrnodities that cornes fiom outside the

village. Ail these factors make salt one of the most effective vehicles for dietary

supplementation of micronutrients (Vekentash Mannar, 1985).

The process of salt iodination airns to mk salt with a prefixed quantity of iodine to

ensure the desired dosage of iodine in the salt. The techniques for iodination include dry

mixing, drip feeding, submersion, and spray mixing, of which the latter is the most widely

used (Vekentash Mannar, 1985). Iodine is normally introduced as a compound such as

potassium iodide, potassium iodate, or calcium iodate (Venkatesh Mannar, 1987). The

choice of method of salt iodination, as well as the iodine fortification compound, depends

on conditions prevailing in a particular geographic location.

2.5 Dou ble-Fortified Salt

When salt is used as a vehicle for i o n fortification, and the iron compound used is

an iron salt, it is necessary to keep the pH of the salt low to prevent discoloration and

formation of poorly available femc hydroxides. The bioavailability of iron from such

acidified salts may then be good. When iron and iodine are both added to the salt, the

iodine is converied to eiementai iodine, which can evaporate at low pH. Earlier triais by

Burgie et al. had indicated that the iodine moiety of the double-fortified salt was unstable

because of the loss of iodine due to evaporation and catalytic oxidation of r to I2 in the

presence of ferrous ions and air oxygen (Burgie et al. 1986). Iron is also readily oxidized

to the femc form, which has a lowered bioavailability, an unpleasant taste and unsightly,

yellowish brown or rust colour.

Despite the apparent chernical incompatibility of iron and iodine, previous

published reports have shown that it is possible to stabilize iodine on salt in the presence

of iron (Narasinga Rao, 1994). Chernical compounds used as stabilizers were

orthophospheric acid, and sodium hexametaphosphate. These compounds make a complex

with iron which keeps iron soluble (Le. in ferrous state), (Narasinga Rao & Vijayasarathy,

1975). Successnil application of sodium hexametaphosphate (SHMP) as an agent to

stabilize the double-fortified salt was recently reported in India ( Narasinga Rao, 1994 ).

As a chelating agent, SHMP keeps the iron soluble and prevents its interaction with

iodine. However, SHMP may have undesired effects on the bioavailability of other

minerals in the meai.

An alternative approach to the use of a stabilizer is to create a physical banier

between the iodine and the iron. Dextran has been found to be an excellent encapsulating

agent. It prevents the evaporation and oxidation of iodine in the presence of ferrous iron

and is, itself, an inert compound.

Subsequent in vitro and in vivo tests on rats, conducted at the laboratory of Dr.

Rao, the Department of Nutritionai Sciences, University of Toronto, showed that the

encapsulated iron and iodine were bioavaîlable. Thus the next logical step in the series of

these investigations around the double-fortifïed sait was to determine the in vivo

bioavailability of the salt in humans.

CHAPTER 3

MATERIALS & METHODS

3. MATERIALS & METHODS

3.1. Experimental Design

3.1.1. Subjects

Sixteen volunteers (8 males, 8 fernales) ranging in age fkom 21 to 53 were studied.

AU were in good health. Their iron status was unknown at the beginning of the study.

Written, infomed consent was obtained for each volunteer before the study started and al1

experiments were approved by the Hospital for Sick Children Research Ethics Board.

3.1.2. Procedure

Volunteers were randornly assigned to a meal designed to have low or high iron

bioavailibility, (Table 3.1). Each subject received both meals. The high iron bioavailibility

meal designed to enhance iron absorption maximally, contained > 90 g of meat and

sufficient fhit, citrus juice or fresh vegetables to provide >IO0 mg vitamin C. The

subjects were not allowed to drink coffee or tea with this meal. Eggs or foods with high

content of bran were not permitted. The low iron availability meal was modified to

maximaily inhibit the absorption of nonheme iron. No meat products, and a minimum of

fiesh vegetables, h i t s , and ascorbic acid were permitted with this meal. The Iow

bioavailable meal contained bran cereal and dairy products. At least one cup of tea or

coffee was taken with this meal. We added to each meal 5 g of ??e-labeled table salt

containing 50 pg iodine as potassium iodide and 1 mg of iron as "~e-labeled ferrous

fumarate per gram of salt, (this amount of salt represents 113 of the estimated maximum

daily salt intake). In order to avoid any interaction with iron-absorption results, and also to

prevent any false reading in urinary iodine excretion, subjects were asked to stop

supplements of iron and vitarnin C, throughout the study.

3.1.3. Protocol

Twenty four-hour urine samples were coiiected on the two days prior to each of

the two test meals for baseline iodine excretion, and also for two 24-hour periods

fcilowing each test meal. AU test meals and the reference iron dose were given between

O8:OO- 10:OO after a 10-hour fast. Subjects were randomly assigned to receive each of the

test meals. Four hours ' after the ingestion of each meai, a whole-body count was

performed on each subject to establish a baseline value for the ingested radioiron isotope

(Schiner et al. 1962). Two weeks later, when ail unabsorbed radioactivity was completely

excreted (Bothwell, Charlton, Cook, and Finch 1979), subjects were counted for the

second time to determine retained radioactivity b. This sequence was repeated for each of

the two test meals and the reference-dose of iron, Table 3.2.

" Previous triais indicate that a reasonably consistent resuit can be obtained if the initial count is delayed for 4 hours.

COU at baseline (4 hours) 1 Counts at &y 14 X 100

Table 3.2. Study Design

16 Healthy Subjects v

Randomization for HBM or LBM v

= The same cycle was repeated for the second test meal, and also for the reference- dose of inorganic iron, with the exception that no urine collection was necessary for the third cycle. Each subject received both meals as well asthe reference-dose of iron.

Time (days)

24-hr Urine Sample

Hinh Bioavailable. Meal Low Bioavailable. Meal Blood Sample (finger prick)

Whole-Body Counting

~r HBM = High Bioavailable Meal * LBM = Low Bioavailable Meal

-2

*

t

-1

~t

O

* * or *

*

1

*

14

jc

3.1.4. Test Dose of Inorganic Iron

Each subject (in the fasting state) received a test dose of labeled inorganic iron.

The reference-dose consisted of 3 mg of inorganic iron as 'Ve-labeled ferrous fumarate in

50 ml water. Immediately before administration, 18.9 mg of ascorbic acid was taken,

sufficient to give a 2: 1 molar ratio of ascorbic acid to iron (Cook et al. 1991). The total

dose of radioactivity in each test meal and the reference-dose was 1 pCi.

3.1.5. Test for Palatability and Taste of the Double-Fortifieci Salt

FolIowing the ingestion of each test meals, our subjects were provided with a

questionnaire, testing their opinion regarding the taste and palatability of the meals in

which the salt was added to.

3.2. Preparation of Double-Fortified Salt

3.2.1. Iron & Iodine Sources

We prepared Sg~e-labeled ferrous fumarate fiom ferrous sulfate manufacturei by

Mandel Scientific Company Ltd. (Guelph, Ontario) based on Fomon's method in Our

laboratoq (Fomon et al. 1989). Then, '?Fe-labeled ferrous fumarate was diluted with cold

ferrous fumarate in a 1000: 1 ratio. A test of purity for this radioiron was performed

according to USP National Formulary, 1995. Aliquots of ferrous fumarate were checked

for punty and subsequently were physicaiiy mixed with the iodized salt. The fortification

of NaCl with iodine was performed in the laboratory of Dr. Diosady at the Department of

Chernical Engineering, University of Toronto. Potassium iodide 1 % was dextrin

encapsulated and subsequently, spray-dried with the table salt. The double-fortified salt

contained iron at 1 mg/g of salt and iodine at 50 pg/g of salt. The iron dosages were

based on iron content of ferrous furnarate after it was tested for its purity.

3.3. Measurements

Using the facilities of the Mec iical Physics Laboratones at the Toronto Hospita 1,

iron absorption was measured by means of whole-body counting technique. In these

measurements we used an energy band of (0.4-1.4 Mev) and a sensitivity of 1 . 2 ~ 10'~. The

whole-body counting equipment consisted of 8 Na1 crystal detectors (4 upper & 4 lower

ones) positioned above and beneath a bed, al1 located inside a 3 x 2 ~ 2 m3 room. For each

subject we obtained a numencal value for the number of counts at baseline (four hours

post-ingestion) and 2 weeks later. The baseline counts were multiplied by a correction

factor of 0.806 to correct for the radioiron decay after two weeks '.

Urinas, iodine concentration was determined using the method of Dunn et al.

1993. Urine was digested with chloric acid under mild conditions and iodine was

determined by its catalytic role in the reduction of ceric ammonium sulfate in the presence

of arsenious acid, as described by Sandell & Kdthoff (Robbins & Rd, 1967, and Sandell

& KalthofS 1937). Percentage iodine absorption was calculated using the data fiom the

We used the decay equation of A=& . e'& to calnilate the correction factor of 0.806, Where A is the remaining activity mer two weeks, & is the original amount of activity, (e) is the nahual Iogarithm, (t) is the tirne period, and finalIy, (k) is the rate of decay.

second 24-hr urine collections prior to the ingestion of each test meal and the immediate

24-hr urine collections post-ingestion of the test meal '.

Plasma femtin was determined by RadioImmunoAssay (RIA) kit purchased £tom

Ranico Laboratones Inc., Houston, Texas.

Hemogiobin concentration was determined by the Cyanomethernoglobh method

(recommended by the International Cornmittee for Standardization in Hematology).

Drabkin's reagent needed for this measurement was purchased from BDH Ltd., Toronto,

Ontario.

3.4. Statistical Analysis

3.4.1. Sample Sue Determination

From previous work by Cook et al. 1991, the estimated mean iron absorption

from meals designed to ma>cimue and minimize iron absorption is 8 3.9 (X * SD) and

3.2 * 1.5 %. We expected the differences in iron absorption from the labeled sait to be in

the same range. Using mean and variance data from Cook et al, with a type 1 error of 5

%; type II error of 20 %; and a one-taled test, conventional sample size estimates yielded

16 subjects.

3.4.2. Data Analysis

Paired t-test was used to compare absorption of iron and iodine from the two types of

meals to determine whether the mean log absorption ratio differed fiom zero (Cook et ai.

a Post-ingestion 24-hr urinary iodine excretion, minus baseline 24-hr urinary iodine excretion, divided by baseline urinary iodine excretion, times hundred.

1969). Since the distribution of iron absorption values are skewed, values for the percent

absorption were converted to logarithms for statistical analysis. The result were

reconverted to antilogaxithms to recover the original units.

3.4.3. Correction for Interindividual Variation

Because of marked influence of iron status on absorption, cornparison of individual dietary

absorption values with the two different meals was converted to a common reference

point (Cook et al. 1991). Dietary absorption was corrected to a mean reference value of

40 % in each subject by multiplying by 40/R where "R" is the reference-dose absorption

(for each subjects). An alternate method used as a reference to compare individual

absorption values is based on serum ferritin concentration since femtin bears a close

inverse relationship to iron absorption (Cook et al. 1991, Magnusson et al. 198 1, and

Cook et al. 1974). Dietary absorption in each subject was corrected to a value

corresponding to a serum femtin of 40 g/l by using the following equation:

Log Ac = log Ao + Log Fo - Log 40

Where Ac is corrected dietary absorption, Ao is observed absorption, and Fo is observed

serum ferritin.

CHAPTER 4

RESULTS

4. RESULTS

4.1. bon Absorption

Individual data on hemoglobin, and serum femtin, as weli as iron and iodine

absorption fiom the composite meals ( designed to impair or enhance iron availability fiom

the meals ) are presented in Table 4.1.

Our study included 8 male and 8 female subjects. The subjects had a mean age of

34.4 years (range 21 To 53 years old). Because the distribution of the absorption figures

was skewed, further analyses were made by means of logarithrnic transformation of the

individual values for serum femtin, as well as the reference-dose absorption, Figure 4.1.

When the acceptability of the salt was tested 93 % of Our subjects found the

double-fortified salt agreeable in terms of taste and palatabiiity. Mean absorption

"uncorrected" from the iron enhancing meal was signincantly higher than the iron

inhibitory one (8.84 f 1.8 % vs. 1.7 f 0.8, P< 0.0017). Similarly mean absorption, after

correction based on an individual's absorption of a reference dose of inorganic iron or

correction based on iron stores, were also significantly higher with the enhancing meal

(36.7 f 3.4 % vs. 6.3 f 3.1, P< 0.0001 based on absorption of reference dose; 10.4 f 2.4

vs. 2.2 * 0.9, P< 0.0069, based on iron stores). There was a significant negative

correlation between log serum femtin and the absorption nom reference-dose of inorganic

iron (r= -0.35, P< 0.0003), Figure 4.2.

The mean hemoglobin and serum femtin concentration of male subjects were

147.4 i: 1.9 g/l and 76.6 f 14 &i, respectively. Whereas, the mean hemoglobin and

serum ferritin concentration of female subjeas were 127 f 7.35 g/l and 37.1 +: 7.7 pg/l,

respectively. Within the 7-week period of this study there were no sign<ncant changes in

hemoglobin or semm ferritin status of Our subjects, Table 4.1. Two femaie subjeas with

the lowest hemogiobin and serum femtin values (Hbs; 102.4 & 90.4 g/l, and semm femtin

concentrations; 5.5 & 3 pg/i) had the highest rates of iron absorption fiom each meal,

table 4.1. Although there were differences in Hb values between individuai subjects,

mean Hb concentration prior to study (135 rt 5.3 gA) and f i e r the completion of shidy

(134 f 5.2 gA) were not significantly different. Semm femtin and Hb concentration were

within the expected range for 14 subjects in our study, Table 4.2. The two female

subjects had their reference-dose absorption of the inorganic iron outside the two standard

deviations (65.2 & 48.4 %) from the rnean reference-dose absorption for the rest of the

group (9.96 i 1.7 %), therefore excluded fiom the data analysis.

Rcfcrcncc Seruni Ferritin 1 , 1 Abr. (%)

''FC Absorption (%) Enhanced Meal In hibitcd Mesl

Urinary iodinc Excrction (%)

Uncrrctd = Uncorrectcd iron absorption (m & r) = Male & Female, NIA = Nol Available Rî-dcrtd = Corrected based on refcrcnce-dose nbsorption Se-frcrtd = Corrected bascd on serurn fcrritin conccntrrrtion "umbers 15 & 16 are excludcd from our data analysis -

Table 4.1 Table of results from die double-fortified salt investigation

L o g serum ferritin concentrat ion vs . absorpt ion o f reference-dose

of inorganic iron (%)

O 10 20 30

REFERENCE-DOSE ABSORPTION (%)

F ig u r ê 4.2. Correlat ion between iron stores and absorpt ion o f the re fe rence -dose o f inorganic iron

4.2. Urinary Iodine Excretion

Table 4.3. shows baseline and post-ingestion urinary excretion of iodine. Each

24-hr baseline value is the mean of two urine collections before each test meal. AIso, each

24-hr post-ingestion value is the mean of two urine collections after each test meal.

Although there was a trend towards higher values in the first post-ingestion collection

(13 -03 + 1.1 vs. 10.9 k 1.5, p < 0.47) compared to the mean of the baseline values, the

differences were not statistically sigdïcant. By the second day afker the meal, the unnary

iodine values were sirnilar to the baseline values. Also, the absorption of iodine from the

high iron bioavailability meal was not significantly different from the low iron

bioavailability meal, 40.7 + 7.2 and 63 + 22.6 % P< 0.4 1, respectively, (Table 4.1).

Table 4.2 Semm Femtin and Hemoglobin Concentrations in Male and Female Subjects

* Al1 values are Mean f SEM

l Fendes 1 8 1 44.3 1 127.0 t 7.2 1 123.8 + 7.1 1 33m5 * 12J2 1 28.7 + 9.3 1 21.6 + 8.0

Serum Femtin pg/î

Fiaal

91.6 * 21.6

Serum Ferritia pg/i

Initial

93.6 * 23.4

Ref. Abs.

10.1 * 2.6

Subjects

Males

~b gn Initial

147.4 * 1.9

~b gn Final

132.0 * 8.8

N

8

Ave. At!=

27.1

Urioary Iodine E X C C ~ ~ O U pg/dl

Pirst 24-hrs . ( before the ingestion)

11.26

Urinary Iodine Excreaon pgidl S e c o ~ d 24-bfs ( before the ingestion)

Urinary Iodine Excretion ~<gldl

First 2 4 4 ~ ~ ( after the ingestion )

Urinary Iodine Excretion )iddl Second 24-hrs

( after the ingcs tion )

Table 4.3 Baseline and Post-ingestion Urinary Excretion of Iodine

CHAPTER 5

DISCUSSION

5.1 Discussion

DISCUSSION

Iron deficiency anemia and iodine deficiency disorders remain major problems in

many parts of the world, and their prevention through supplementation and fortification

programs are an urgent pnority. Most fortification trials have failed in the eradication of

ironiiodine deficiencies, thus, the double-fortified salt provides a promising opportunity to

overcome the deficiency of these two most important micronutrients.

The double-fortined salt investigated in the present shidy, appears to be quite

satisfactory with respect to bioavailability of iron and iodine. In the present study, mean

individuai iron-absorption figures "uncorrected", from the "enhancing meai" and the

"inhibithg meal" were 8.8 f 1 -8 and 1.7 f 0.8 %, respectively, (P < 0.0017). The mean

urinary excretion of iodine (representuig iodine absorption) was not significantly difTerent

fiom the baselime excretion values in Our subjects (Pc0.47). Also, the ditference between

the iodine absorption from the test meals (i.e. the enhancing and the inhibithg meals) was

not sigrilficant @ < 0.41). The addition of the double-fortified salt to the test m a l s did not

alter the flavor or the palatability of the meals.

There are two methodoIogica1 aspects of the present shidy that deserve comment.

1) Our findings agree with the previous work done by Cook and CO-workers,

demonstrating the influence of inhibitors and enhancers on uon absorption from composite

meals (Cook et al. 1991). 2) The addition of the dextran-coated iodine did not

compromise the apparent absorption of iodine fiom the double-fortified s b .

Results fiom the current study confirm previous observations that other food items

in a meal enhance or inhibit iron absorption fiom that meal. With the "enhancing" meal

which was designed to mairimize the iron absorption, the uncorrected iron absorption

values were five-fold higher than those for the "inhibitory" meal. It should be noted,

however, that the enhancing and the inhibiting meals used in the present study were likely

an exaggeration of the type of meals ingested in geographical locations where meat is

scarce. The inhibiting meal in the current study contained no meat, and minimum h i t s ,

vegetables or vitamin C. Thus in a non experimental sethg one would expect average

iron absorption in an iron replete population to be between the two extremes used in the

current study .

The fortified salt was designed to provide an amount of iodine adequate to treat

and prevent iodine deficiency and an amount of iron sutncient to prevent iron deficiency in

a non-iron sufficient subject when 10-1 5 g of salt is consurned per day. If we attempt to

relate our shidy results to Canadian R N ' s for iron we come up with the following

condition. Assurning a worst case scenario represented by the iron-inhibithg meal, a salt

intake of 15 g/day for men, and a mean absorption of 1.7 % (average iron absorption of

male subjects), men would absorb 0.3 mg of elemental iron. The total iron requirement

for men is 1.1 mg/day, thus, iron fortified-salt wodd provide 23 % of the requirements.

For women assuming a sait intake of 10 glday and a mean absorption of 3.1 % (average

iron absorption of female subjects), the total absorption would be 0.31 mg. In this case,

the iron fortined-sait would provide only 18 % of the requirements.

From the perspective of at risk populations in developing countries where this

initiative is primarily aimed at, there are several essential factors to be considered. First,

an average salt intake of 10-15 g/day has been reported f?om populations in these

countries (Narasinga h o , 1994), with more sait ingested by men, due to more meal

consumption, as compared to women. Secondly, a typical meal in most such populations

is a mixture of "enhancers and inhibitors" of iron absorption. Therefore in such

populations, iron absorption between the two mean values is expected. Aiso, at risk

individuals have poor iron stahis, therefore, a higher absorption from the iron fortified-salt

is expected among these people. And finaily, loss of blood due to parasitic infections

would result in an increase in iron absorption fiom the fortified salt. Based on the

descnbed scenario and the results fiom the present study, we would expect an eEectively

higher iron absorption in areas affected by iron deficiency anemia.

There are a number of reasons for suggesting that dextran-coating of iodine is the

preferred method of protecting iodine fiom oxidation. Earlier trials by Burgi et al. had

indicated that the iodine moiety of the double-fortified salt was unstable because of the net

loss of iodine due to evaporation and catalytic oxidation of ï to I2 in the presence of

ferrous ions and air oxygen (Burgi et al. 1986). Although successful application of

sodium hexametaphosphate (SHMP) to stabilize the double-fortified salt has been

demonstrated (Narasinga Rao 1994), dextran, u&e SHMP, is an inert carbohydrate

which is safe to use in any diet. Sodium haxamataphosphate is a chelating agent which

may have undesired effects on the bioavailability of other minerais in the meal. In

addition, extensive tests conducted b y Diosady et al. demonstrated that phy sical isolation

of iodine by dextran encapsulating, results in acceptable iodine retention under the worst

storage and distribution conditions. As the process of iodme oxidation (ï to I2 in the

presence of ferrous ion) occurs at a rnuch more rapid Pace in humid conditions, the

importance of a physical separation (eg. dextran coating of iodine) is even more important.

Finally, the method of preparation of the double-forufied salt with dextran-coated iodine is

very simple and inexpensive especidy with large-scale production.

In the rnedical literature there are at least two methods describing the "correction"

for the difference in iron status between individual subjects in research studies examining

iron absorption. One method is based on the individual serum femtin concentration, while

the other uses the absorption of a reference-dose of inorganic iron. In the present study,

the correction for dietary iron absorption, based on individual serum ferritin stahis and

reference-dose of iron absorption, were comparable. However, the correction based on

serum femtin status may be Iess reliable since only two femtin values were available fiom

each subject in our study. Cook et al. suggested that it is important to obtain at least 5

serum femtin assessments to obtain a reliable mean femtin value (Cook et al. 1991),

Figure 4.4.

In the past Bjom-Rasmussen and CO-workers demonstrated a high correlation

between iron absorption fkom meals and from reference-doses (Bjom-Rasmussen et al.

1976). Also, a high correlation between iron absorption and measures of iron stores such

as bone marrow homosiderin and serum femtin, was reported by others, (Cook et al.

1974, Disler et al. 1976, Walters et al. 1975, Charlton et al. 1977, Heinrch et al. 1977, and

Benvoda et al. 1979). In Our current investigation of the double-fortified salt we

observed a significant correlation (r = -0.35, P c 0.0003) between the reference-dose

absorption of inorganic iron and individual serum femtin stores, Figure 4.2. Before the

exclusion of the two iron deficient females £?om our data analyses, this correlation

coefficient was r = 0.76 (similar value was reported by Cook et ai. 1991). This was not

unexpected since the absorption of iron is known to increase under conditions in which

tissue iron is reduced, with more iron being absorbed by iron deficient subjects and less by

subjects with iron overload (Magnusson et al. 198 1).

Magnusson et al. initially described the use of a reference-dose of iron absorption

value to improve the comparison of iron absorption values between different subjects.

The absorption of iron nom each meal was expressed as the ratio food iron

absorptiodreference-dose of inorganic iron (ferrous fumarate) absorption. In a study

conduded by Magnusson et al., the distribution of absorption measurements in 96 normal

men showed that only 3 men had an absorption exceeding 40 % from the reference-doses

(Magnusson et al. 198 1). Magnusson noted an inverse correlation between reference-dose

absorption and serum femtin values (eg. 20 % absorption with a serum femtin of 60 pg/l

to 70-80 % absorption correspondhg to a semm femtin of 12 pd). In the current study

only the two iron deficient subjects (not included in Our data analysis), demonstrated a

reference-dose absorption exceeding 40 % (48 & 65 %), with corresponding semm femtin

concentrations of 3.5 and 3 pgL, respectively. Considering the fact that the mean

absorption fiom the reference-dose of inorganic kon in our study subjects (excluding the

two subjects previously rnentioned) was 9.96 % (representing an iron replete population),

it is most appropriate not to use either of the two rnethods of corrections, as the

"uncorrected" values are a better reflection of the results fiom this iron replete,

homogeneous group of volunteer S.

Daily urinary excretion of iodine closely refiects iodine intake, and has been used

as a measure of iodine stahis in many large scaie nutrition surveys (Gibson RS, 1991).

Foliis et al. showed that the percentage of 131-1 uptake by the thyroid of a given

individual is inversely correlated with urinary iodine excretion (Follis et al. 1962, 1964).

This relationship, together with the observations that the Iargtst fraction of dietary iodine

is excreted via the kidneys and that urinary iodine excretion is lowest in areas of endemic

goiter (Fous et al. 1964), makes urinary iodine rneasurement a valuable test to assess

iodine status. The urinary cutoff points to assess the severity of iodine deficiency, by

measuring iodine concentration in urine samples, are categorized by

WHOLJNICEFIICCIDD as: deficient, u ~ a r y iodine concentration < 0.79 pmoK ', and

severely deficient, urinary iodine concentration < 0.16 pmoVL. The mean u ~ a r y

excretion of iodine in Our subjects, before the dietary intervention was 0.87 f 0.1 pmol/L

(or 10.98 f 1.46 &dl). Urinary iodine excretion was equivalent or higher after the test

meals, thus we believe that dextran-coating does not negatively influence the absorption of

iodine.

5.2. Conclusion

Our major objective was to determine the absorption of iron and iodine fiom

fortified salt in healthy human subjects ingesting high and low iron-bioavailable meals. We

Dividing by 0.079 converts pmoYL to pg/L.

demonstrated iron absorption within the expected range fkom the fortified salt. Based on

the significant dserence between the absorption fiom the two dSerent test meals, our

hypothesis that the iron absorption nom the inhibitory meai would be lower than the

enhancing meal was confirmed. Our results indicated that the iodine fiom the fortified salt

is readily available and absorbable as iodine fkom the iodized salt. Thus, we conclude that

dextran-coated iodine and iron are weii absorbed fiom double-fortified table salt.

5.2.1. Future Studies

Having established that the iodine and iron are well absorbed, community triais to

determine efficacy of the fortified salt in treating and preventing iodine deficiency and

preventing iron deficiency are needed. A study has been proposed to begin in Ghana

(Skyere West district) to accomplish this goal. It aims to provide evidence of the efficacy

of the double-fortified salt and advisability or otherwise of its use to combat iron and

iodine deficiencies.

Unc P < 0.0017 R d c P < 0.0001 Sfc P < 0.0069

Uncorrected iron absorption

C o r r e c t e d b a s e d o n r e f e r e n c e d o s e

m c o r r e c t e d based on serum ferritin

F ig u r e 4 .4 Corn parison of dietary iron absorption from the two test meals "uncorrected" & after "correction" for inter-subject variabilities based on the two different methods.

C W T E R 6

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IMAGE EVALUATION TEST TARGET (QA-3)

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