Ferric pyrophosphate fortified extruded rice increased iron status in rats

by

David Bolaji Akinbo

B.MLS., LAUTECH, Nigeria, 2009

M.Sc., University of Benin, Nigeria, 2016

Ph.D., University of Benin, Nigeria, 2021

A THESIS

submitted in partial fulfillment of the

requirements for the degree

MASTER OF SCIENCE

Department of Food, Nutrition, Dietetics and Health

College of Health and Human Sciences

KANSAS STATE UNIVERSITY

Manhattan, Kansas

2022

Approved by:

Major Professor
Brian L. Lindshield
 Copyright

© David B. Akinbo 2022.

 Abstract

Background:

Iron deficiency is the most common micronutrient deficiency worldwide and one of the five
prominent causes of years lived with disability in humans, particularly affecting women and
children. Ferric pyrophosphate (FePP) and ferric phosphate (FePO4 ) are commonly used in
fortifying rice due to their minimal impacts on its sensory properties, but have both presented
with poor iron bioavailability. Particle-size reduction of FePP and addition of ligands such
as citric acid (CA) and trisodium citrate (TSC) to extruded FePP-fortified rice have been
associated with improved iron bioavailability in iron-fortified rice.

Objective:

To compare the iron outcomes of extruded rice flour formulated with four different iron
fortificants in rats.

Methods:

Using the prophylactic-preventative method, 50 male weanling Sprague-Dawley rats aged

20-23 days were randomly distributed into 5 groups and fed ad libitum for a 28-day period.
The control group consumed powdered AIN-93G growth diet, while the other four experi-
mental diets were AIN-93G based extruded rice flour diets fortified with micronized FePP
(µFePP), a higher FePP concentration, FePO4 with TSC and CA or FePP with TSC and
CA at a molar ratio of 1:0.3:5.5 for iron to TSC/CA. Hematological parameters and hepatic
iron concentration were assessed for the iron status. The dual energy x-ray absorptiome-
try (DEXA) PIXImus scan was used to assess the bone mineral density (BMD) and body
composition of the rats.

Results:

The consumption of FePP, µFePP, FePP+TSC+CA fortified rice flour significantly increased
(p<0.05) hepatic iron concentration compared to the FePO4 + TSC+CA fortified rice flour
and AIN-93G groups. However, there were no significant differences between the hepatic
iron concentrations of the FePP groups or the hematological and anthropometric assessments
between all groups (p>0.05).

Conclusion:

Increased concentration of FePP in extruded fortified rice can improve iron status, and
suggests that neither micronizing FePP nor extruding it with TSC and CA improved iron
status compared to FePP. The extrusion of FePO4 with TSC and CA did not improve iron
status.
 Table of Contents

List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Rice as a tool for Global Nutrition . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Anemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.3 Iron Availability and Absorption . . . . . . . . . . . . . . . . . . . . . . . . 3
1.4 Iron Deficiency and Iron Deficiency Anemia . . . . . . . . . . . . . . . . . . 4
1.5 Iron Status Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.6 Management of Iron deficiency and Iron deficiency Anemia . . . . . . . . . . 7
1.7 Fortification as a Major Nutritional Intervention Strategy . . . . . . . . . . . 9
1.8 Rice Fortification Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.9 Iron Fortification Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.10 The Enhancers and Inhibitors of Iron Absorption . . . . . . . . . . . . . . . 15
1.11 Measurement of Iron outcome . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2 Ferric Pyrophosphate Fortified Extruded Rice Increased Iron Status in Rats . . . 19

2.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

3 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

v
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

A List of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

vi
 List of Figures

2.1 Average weekly food intake per group . . . . . . . . . . . . . . . . . . . . . . 35

2.2 Average body weight of diet groups per week . . . . . . . . . . . . . . . . . . 36

vii
 List of Tables

2.1 Iron compounds and ligands composition per kilogram rice flour . . . . . . . 32
2.2 Percentage composition of vitamins and minerals of formulated diets . . . . . 32
2.3 Macronutrients and iron content of formulated rice and AIN-93G diets (g/100g) 33
2.4 Food intake and anthropometric measures of study groups . . . . . . . . . . 33
2.5 Hematological and hepatic iron outcomes . . . . . . . . . . . . . . . . . . . . 34

viii
 Acknowledgments
I am most grateful to my major professor, Dr. Brian Lindshield for his invaluable pa-
tience, guidance and feedback all through this work. Thank you for giving me the opportunity
to be a part of your research, I do not take this privilege for granted. This journey would
not have also been possible without my defense committee, Dr. Weiqun Wang, Dr. Sajid
Alavi and Dr. Brian Lindshield who all generously provided knowledge and expertise. This
endeavor would also not have been possible without the support of our collaborator in the
Grain Science department; Dr. Sajid Alavi and his team including Tucker Graff, Mehreen
Iftikhar, and Eric Maichel.
Also, I wish to express my gratitude to Jiejia Zhang, a graduate research assistant in our
lab and Abigail Mejia-Sanchez, a Research Immersion Pathways to STEM (RiPS) student in
the FNDH department for their help and support. I am grateful to my friend Dr. Thomas
Kelemen in the Business School and Victor Oladoja for their late-night feedback sessions
on my statistical analysis results and moral support. My appreciation also goes to Erika
Lindshield, the Graduate Student Coordinator and Karen Rogers, the department Office
Specialist for all their support since being admitted into this program. I am grateful to my
teacher and GTA professor, Dr. Mark Haub for your impact and inspiration also, I enjoyed
working with you also.
I would be remiss not to mention my family; especially my foster parents; Dr. Fredrick O.
Akinbo and Dr. Marian I. Akinbo for their continuous support and encouragement, my wife
and daughter for their patience with me. Their belief in me keeps my spirit and motivation
high always.

ix
Chapter 1

Introduction

1.1 Rice as a tool for Global Nutrition

Rice is grown in more than 100 countries, with countries in the Asian continent responsible for
roughly 90% of the total global production 1 . Paddy rice production has increasingly grown
by more than 3-fold from 215 million to 755 million tons between 1961 and 2019 2 . Variations
in the grain length, color, thickness, stickiness, aroma and growing conditions/production
practices of rice are all capable of impacting the quality and nutrient profiles of the various
rice species. Additionally, the regional and cultural preferences of various populations also
influence the global market for the different rice species 3 .
More than half of the world population consume rice as a staple food, especially in the
rice-producing regions of the world. Bangladesh had the highest per capita rice consumption
with 269kg per capita per year, and followed by Laos and Cambodia out of 154 countries
in 2017 4 . Half the daily energy intake of South India is from refined grains, with 75%
being white polished rice 5 . Rice grains are essential nutritional source of carbohydrates
and proteins alongside essential micronutrients. The utilization of rice and rice flour is
considered one of the most common dietary interventions for people with macronutrient and
micronutrient deficiencies and age-related chronic diseases due to its wide consumption 6 .

1
1.2 Anemia

The World Health Organization has described anemia as a hemoglobin (Hb) concentration
below 12 g/dl in women and 13 g/dl in men 7 . Anemia represents a common public health
challenge characterized by a reduced hemoglobin concentration and/or volume of red blood
cells below an acceptable range. It is a common medical complication in critically ill patients,
and impairs oxygen transport to body tissues 8 . About 25% of the world’s population have
been reported to be affected by anemia, with pregnant women and school aged children
in Africa and Southeast Asia being the most affected 9;10 . Approximately half the children
in developing countries are estimated to be anemic, with sub-Saharan Africa countries like
Kenya, Mali and Tanzania having 48.9%, 55.8% and 79.6% children anemic respectively.
Anemia can cause several acute and chronic health challenges in children, including impairing
their learning performance, psychomotor and cognitive maturity, behavioral and physical
growth and elevates morbidity and mortality risks in them 11 . It has also been implicated in
the poor intelligent quotient and language coordination in children, and as an indicator of
poor health status in a nation 9 .
Anemia can be caused by several factors including nutritional deficiencies, chronic in-
fections, inherited blood disorders, obesity, and chronic non-communicable diseases 12;13 .
Dietary iron deficiency (ID), inherited blood disorders (sickle cell anemia, thalassemias),
malaria, helminthic infestation have been reported as the leading causes of anemia and they
vary across different geographical areas, population group, and general environmental set-
tings 14 . Socioeconomic factors including illiteracy, gender norms, poverty, large number of
children, high density of inhabitants per room, poor access to public services, such as ba-
sic sanitation and electricity, quantitatively and qualitatively inadequate food consumption,
among others have also been indicated as predisposing to risks of anemia too 8 . These mul-
tiple risk factors are often found coexisting with some essential micronutrients deficiencies
viz folic acid, vitamin B12, vitamin A, with iron deficiency being implicated as a major
contributor to anemia; and is estimated to cause approximately 50% of all anemia cases 15;16 .

2
1.3 Iron Availability and Absorption

Iron is an integral element for a broad range of biologically important reactions critical for
vital cellular functions including hemoglobin synthesis and other hemopoietic activities. It is
contained in numerous hemoproteins such as the oxygen transport proteins, heme containing
enzymes and many essential non-heme iron proteins catalyzing various reactions and playing
a central role in oxygen sensing mechanisms 17;18 . An adequate iron supply to the body
and maintenance of its balance is therefore essential for normal human health. Iron is a
transition metal existing in two readily reversible redox states of reduced ferrous (Fe (II))
and oxidized ferric (Fe(III)) forms respectively. However, most of iron’s biological complexes
at physiological oxygen concentrations occur in its stable form of Fe (III) state 19 . The
biological function of iron has been reported to be largely due to its chemical properties as
a transition metal, and reduction reactions are highly essential in its metabolism. Reduced
iron ion is the only form of iron that can be used as a substrate for transmembrane transport
of iron, loading and releasing of iron from ferritin and for heme synthesis 20 .
An adult human has a total body iron content of 3–5 g (≈ 45 mg / kg for women, ≈ 55
mg / kg for men), with most of it occurring in the hemoglobin of peripheral erythrocytes
(60–70%). About 20–30% body iron is in ferritin and hemosiderin in both hepatocytes and
Kupffer cells as storage/spare iron. The adult male has a stored iron content of around
0.2-0.5g, while children, adolescents and women of childbearing age are almost lacking iron
reserve 21 . Some spare body iron is in the muscle myoglobin or incorporated in enzymes, while
transferrin typically binds roughly 3 mg of iron 17 . In situations where the amount of cellular
iron exceeds the body’s immediate need, the excess iron is stored in a bioavailable form
as ferritin which is readily deployed whenever the cells become iron deficient 22;23 . Besides
functioning as an iron reserve, ferritin also protects the cells from the potentially toxic
catalytic reactions of iron 24 . Hemosiderin is a second form of cellular iron storage and an
insoluble product of incomplete ferritin degradation by the lysosomes. Due to its ineffective
donation of iron, hemosiderin typically plays a protective role in the circulatory system in
normal physiological conditions and becomes an iron donor under inflammatory and hypoxic

3
conditions 25 .
The regulation and balance of iron is important for all cell life and is specifically controlled
by the intestinal absorption. Changes in body iron stores and demand for iron affect the
iron absorption rate in the proximal duodenum accordingly, with iron deficiency causing an
increased absorption rate and iron overload resulting in a decreased rate of iron absorption.
The mechanisms of iron homeostasis therefore evolved to curb excess iron accumulation
and dangerous reactive oxygen species production by recycling body iron and limiting its
uptake from the environment 26 . Iron is lost from the body via desquamated skin epithelium,
intestinal cells and intestinal secretions 21 . Adult men lose only 1 mg of iron per day, while
women of reproductive age lose twice this amount to menstrual bleeding, pregnancy and
childbirth. In compensating for this routine loss, about 1–2 mg of iron is absorbed by healthy
individuals per day. There is however an increased demand for iron during adolescence due
to growth and in pregnancy owing to plasma volume expansion and fetal growth. This
therefore alters the daily optimal nutrition of such individuals to around 8–10 mg of iron
being required 27 . Iron is present in food as either inorganic or heme iron. Inorganic iron is
found in the standard diet; constituting 90% of the total iron contained in food, while heme
iron only represents 10% of the dietary iron 28 .
Despite iron being the fourth most abundant element in nature, it has a very low bioavail-
ability and the disorders of its homeostasis are amongst the most common human disorders.
In spite of iron’s low daily requirements, iron deficiency remains the most common nutri-
tional disorder in the world 29 . It is noteworthy that hereditary and acquired iron overload
diseases are also present at the opposite end of the iron disorders spectrum.

1.4 Iron Deficiency and Iron Deficiency Anemia

Micronutrients deficiencies are very crucial public health problem, and about 2 billion people
worldwide suffer from the subclinical deficiency of micronutrients commonly referred to as
hidden hunger. Iron deficiency (ID) is the most common micronutrient deficiency world-
wide, particularly affecting pregnant women, infants and young children due to increased

4
iron demands resulting from the rapid growth in this group 30 . It has been implicated as the
leading cause of anemia worldwide 14 . Iron deficiency progresses in three-stages affecting all
the body cells. The first stage is the total body iron stores depletion, which leads to the
second stage of iron-deficient erythropoiesis and lastly iron deficiency anemia characterized
by reduced hemoglobin levels. It often occurs due to inadequate dietary intake or a com-
promised absorption from inflammation or blood loss 9 . Iron deficiency and iron deficiency
anemia therefore result from an imbalance in bioavailable iron absorption from diet and the
body’s iron requirements 31 . The failure of iron supply to meet the body’s demand thus
results in iron stores being used up faster than their replacement, and consequently iron
depletion.
Iron deficiency can exist without anemia in the absence of iron stores. It is a broader
condition preceding anemia or indicating a deficiency in organs/tissues besides erythropoiesis
including skeletal muscles and the heart which is dependent on iron for myoglobin and
energy production for its mechanical contraction 32 . Due to the high amount of iron utilized
for hemoglobin synthesis during daily erythropoiesis of 200 billion erythrocytes, anemia is
easily the most evident sign of iron deficiency; and iron deficiency anemia is often readily
suggestive of iron deficiency 26 . Iron deficiency anaemia (IDA) is one of the five prominent
causes of years lived with disability in humans, and most especially in women. Besides
Iron deficiency anemia being majorly classified as a public health challenge that affects
young children, women of child-bearing age; its recognition as a clinical condition capable
of affecting chronic disease patients and the elderly is becoming more common. Numerous
physiological, environmental, pathologic and genetic factors can cause iron deficiency to
result in IDA. These etiologies/factors may however differ in the affected patient populations,
geographies and specific clinical conditions or they could be found co-existing 33 .
The incidence of iron deficiency anemia is increasing globally and is reported as the
most common anemia worldwide 14 . Preschool children under 5 years of age, reproductive
age and pregnant women are the most affected by iron deficiency anemia, with prevalence
rates of about 41.7%, 32.8% and 40.1% respectively. Iron deficiency anemia can result
from heavy dependence on vegan diets and some high-risk factors in high-income countries

5
including malabsorption syndromes and menorrhagia, with iron deficiency/iron deficiency
anemia being diagnosed in about two-third of women with menorrhagia 34 . Iron deficiency
anemia represents only about 30% of anemia cases in the elderly. Regular blood donation has
also been implicated as a cause of iron deficiency anemia too. About 37%-61% of chronic
heart failure patients and 24%-85% of chronic kidney disease patients are iron deficient,
with the incidence rates increasing as these conditions become more debilitating 33 . There
have also been previous reports of iron deficiency in 13–90% of inflammatory bowel disease
patients, and its occurrence dependent on the disease activity and severity 35 . Also, 33% -
43% of cancer patients suffer from iron deficiency and iron deficiency anemia respectively,
depending on the disease progression, proximity to cancer therapy and poor outcomes in
solid tumour patients 36 .

1.5 Iron Status Assessment

Iron status can be readily assessed through serum ferritin and the hemoglobin levels respec-
tively 33 . Serum ferritin level is a highly specific and effective test for assessing total body
iron stores and is universally available and standardized. Biochemically, a low serum ferritin
level in a patient depicts the hallmark of absolute iron deficiency; a total depletion of the
body iron stores. However, serum ferritin level is affected by inflammations and infections,
resulting in an elevated value 26 . Anemia is diagnosed after confirming a reduction in the
hemoglobin concentration via a complete blood count test. The hemoglobin concentration
thresholds for anemia do vary according to social demographics, age, body physiology, disease
epidemiology and management.
Other laboratory investigations for iron status assessment include serum iron level which
represents the iron bound to transferrin, and that is available for erythropoiesis in the bone
marrow. The serum iron level depends on the efficient recycling of iron from senescent
erythrocytes by tissue macrophages as well as iron absorbed from the diet. Total iron
binding capacity (TIBC) is a functional measurement for peripheral transferrin levels and is
deduced by adding the serum iron to the unsaturated iron binding capacity 37 . The red blood

6
cell (RBC) indices are negatively impacted by iron deficiency and iron deficiency anemia,
with the red cells gradually becoming microcytic and hypochromic. There is a reduction
in the mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) and an
increase in the red blood cell distribution width (RDW) in iron deficiency anemia. These
changes in the red cell indices however appear late in course of iron deficiency anemia, and
as such may limit their clinical relevance in iron deficiency 26 .
Contrary to the RBC indices, the reticulocyte hemoglobin content (RHC) is an early
index of ID as it indicates iron availability for erythropoiesis in the previous 3–4 days. Also,
the percentage of hypochromic red cells (% HRC) can also be used to assess recent iron
reduction 38 . Soluble transferrin receptor (TfR) is an underutilized laboratory measurement;
its levels are increased in iron deficiency and iron deficiency anemia but low during inflam-
mation. It can be useful in the detection of true iron deficiency from inflammatory conditions
associated with low serum iron 39 . The measurement of hepcidin level is another useful tool
in determining iron deficiency and iron deficiency anemia when expertly applied. Hepcidin
level becomes reduced/undetectable in iron deficiency anemia. It is however affected by a
lot of factors including circadian rhythm, hepatic and renal function, all of which impacts
its routine acceptability in clinical practice 40 .

1.6 Management of Iron deficiency and Iron deficiency

Anemia

The treatment of ID and IDA is aimed at providing sufficient iron erythropoiesis and re-
plenish the depleted iron stores. This subsequently improves the quality of life, clinical
manifestations of the patients, and the prognosis of many chronic disorders. There are two
distinct approaches for the management of ID and IDA, viz active iron supplementation
approaches in confirmed IDA patients, and prevention strategies targeted at populations at
risk 41 . Iron supplementation can be administered either orally or intravenously depending
on the patient’s clinical situation and preference. However, significant gastrointestinal side

7
effects comprising constipation and sometimes diarrhoea, a metallic taste, gastric cramping
and thick, green, tenacious stool have been reported in approximately 70% of patients tak-
ing oral iron. And this markedly reduced the patients’ adherence to the oral iron therapy 42 .
The universal administration of intravenous iron (IV) supplement has also been limited by
availability and cost, the general belief of anaphylactic reactions and the toxic side effects of
IV iron due to the free elemental iron from the supplemental drug 43;44 .
Currently, several approaches including either one or a combination of supplementation,
food-based approaches such as dietary diversification, mass food fortification or point-of-use
food fortification; with other public health control measures such as deworming, health and
nutrition education have been recommended as intervention strategies to prevent and treat
micronutrient deficiencies (WHO, 2011; Peña-Rosas et al., 2019). As an integral component
of the prevention strategies for iron treatment, food-based approaches have been recom-
mended on a global level (WHO, 2001). Various programs and efforts are being made to
increase access to iron-rich foods and their consumption. The use of ascorbic acid as an iron
absorption enhancer to increase the bioavailability of iron when consumed has been reported
in previous studies, while the removal of inhibitors of iron absorption such as calcium, phy-
tates in cereals; tannins in tea and coffee is being recommended 45 . Hence, the enrichment of
widely consumed foods with iron and the foods retaining their organoleptic properties and
prices have been recommended as an effective public health intervention to improve the iron
status of populations 46 .
Fortification of widely consumed foods popularly known as staple foods is now renowned
for its ability to improve the overall consumption of iron in a population 41 . The WHO
has recommended the universal fortification of staple foods including rice, maize flour and
cornmeal with iron to prevent ID and IDA in at-risk populations 47;48 . With ID and IDA
resulting in serious health and national consequences, the lack of preventative and corrective
interventions including developing nutrition-specific and/or nutrition-sensitive strategies to
increase individuals’ intake and absorption of iron accordingly contribute to the continuation
of the poverty cycle and stall progress in such populations/countries.

8
1.7 Fortification as a Major Nutritional Intervention

Strategy

Food fortification is a cost-effective public health intervention strategy capable of correcting

or preventing widespread nutrient deficiencies and associated complications, balancing the
total nutrient dietary profile or appealing to the consumers’ dietary wants 49 . It is described as
a deliberate practice of increasing essential micronutrients/trace elements contents in a food
solely to improve the nutritional quality of the food supply while providing a public health
benefit with the least risk to the population’s health 50 . Food fortification has been effective
in enhancing nutrient intakes of various populations with demonstrated health, economic and
social benefits. It is noteworthy that food fortification programs are part of the sensitive
programs conducted in many countries around the world as the recommended long-term
strategy to improve certain micronutrients consumption in the general population 51 . They
include the fortification of vitamin A in cooking oil, margarine, and sugar; vitamin D in milk
and margarine; folic acid in flour; iodine in salt; iron in milk, corn flour, beans, pearl millet,
and wheat flour 52 . These programs are however likely to be more successful if mandated by
the government with support from the food industry 53 .
Several factors determine the most appropriate and effective fortification practices of
the different countries, and these include certain micronutrient deficiencies prevalence, most
affected populations, dietary compositions, available infrastructure, food processing capac-
ities and production systems, alongside national regulation and governmental leadership 54 .
Three fortification approaches are currently in use, and they are the Large-scale/Mass forti-
fication, voluntary/market driven fortification and targeted/focused fortification. The large-
scale/mass fortification is usually mandatory, and entails addition of micronutrients to com-
monly consumed foods to enhance their nutritional value during central processing. Vol-
untary/market driven fortification of processed products by food industry is guided by the
dietary demands of consumers to increase the diverse nutrients available. Targeted fortifi-
cation involves adding micronutrients to specially designed foods like the infant formula for

9
infants less than 6 months of age, and others meant for specific population subgroups 55;56 .
Selecting an appropriate food or vehicle for fortification and identifying the at-risk micronu-
trient deficiency populations is therefore an important element of the intervention and may
vary among countries due to the obviously diverse patterns of diets.
Iron deficiency is a significant public health challenge in several countries, especially the
developing countries and fortifying staple foods with iron have been reported to increase iron
consumption in those vulnerable populations 52;57 . Some potentially acceptable staple food
or vehicles used in common micronutrient fortification for public health programmes include
refined or raw sugar, edible vegetable oils, fats, and cereal grains (rice); wheat flour, maize
flour, or corn meals; condiments and seasonings; and powdered or liquid milk 55 . Iron fortifi-
cation of rice has been recommended by the WHO as a choice vehicle for iron deficiencies in
at-risk populations of a rice-consuming region 48 . Rice fortification with iron increased the
hemoglobin and serum ferritin levels, and reduced iron deficiency anemia prevalence from
100% to 33% among preschool age children, pregnant women, adolescents, and adults in pre-
vious studies 58;59 . Rice is a prominent staple food of about three billion people; its proper
fortification with iron therefore has the potential to reduce the prevalence of ID and IDA in
rice-consuming countries 60 . Thus, the stability of the iron fortificants in the rice throughout
the marketing process, the choice and cost of fortification processing and relative cost of the
iron fortificants are important considerations for an efficacious fortification approach.

1.8 Rice Fortification Techniques

With rice consumption as a whole grain and not flour, the successful fortification with
micronutrients on a large-scale continues to be a technological challenge compared to wheat
or maize flours which have gained successes around the world 61 . Past attempts to fortify rice
through dusting and coating were reported to be unsuccessful due to the typical household
methods of rinsing and cooking rice in most of the developing countries 62 . Additionally; due
to the much greater size difference between rice kernels and micronutrients, the mixture of
micronutrient blends with rice kernels ordinarily can readily result in the easy separation

10
and non-homogeneous distribution of the micronutrients, increased loss of micronutrients
during production, transportation, as well as the rice preparation methods employed by
various households. This has significantly increased micronutrient losses, with approximately
90% of water-soluble micronutrients being increasingly lost from rice when cooked in excess
water 61 . There are currently two major sophisticated techniques developed for the large-
scale fortification of rice and counter the problems of micronutrient losses; they are coating
and extrusion techniques 63;64 . Both methods typically entail fortifying about 2% of the rice
kernels and blending to the other unfortified retail rice.
Coating is a more advanced, relatively inexpensive method involving a combination of
ingredients including waxes and gums with the choice fortificant mix to form a liquid mix
that is sprayed in several layers onto the rice kernels to produce a fortified rice-premix. These
coated rice-permixes are subsequently blended with the unfortified/normal retail rice between
a ratio of 1:50 to 1:200 to fortify the rice product 61;65 . Several studies have shown coating
to be stable during washing, cooking and to effectively reduce micronutrient losses while
washing the grains below 1% for some micronutrients fortified using the coating method 66 .
It exerts its effects by the interaction of the waxes and gums which form a waxy layer cov-
ering on the rice kernels, making the micronutrients stick to the rice kernel and reducing
the loss of these nutrients when the grains are washed before cooking 63 . However, some of
the major challenges of coating include the possible coloration of the kernels as the coated
micronutrients layer of the kernel makes them highly visible, impact on the taste and loss
of micronutrients during washing and cooking and therefore affecting the consumers’ prefer-
ence compared to the extruded counterparts. Coating is considered a lower initial financial
investment compared to extrusion, the cost of fortified rice is however relatively comparable
per metric ton 64 .
Extrusion is a new technological advancement in the food industry that utilizes low-cost
broken rice as its raw material for the production of rice kernels. The extruded products are
made according to preset specific shape, color, and nutrient requirements in line with the
retail rice specification to be blended together for fortification 65 . It is a versatile, continu-
ous process that combines diverse processing steps comprising various constituent mixtures,

11
degassing, thermal and mechanical heating, forming, and expanding 61 . The technique is pri-
marily used for processing biopolymers, such as carbohydrates and semi-crystalline polymers
like starch which have both glass transition and melting temperatures 67;68 . Two types of ex-
trusion technology are mainly utilized in the food industry, and they include the hot and cold
extrusion, also known as cooking extrusion and shape-forming respectively. Hot extrusion
process involves passing the rice flour dough/premix through a pre-conditioner containing
water and steam and thereafter extruded using a single or twin screw extruder at relatively
high temperatures (70° to 110°C). The extruded product is cut into rice-shaped structures
at the die upon extrusion and dried afterwards, resulting in a consistent and translucent
product very similar to the natural rice grain 63 . The cold extrusion process is similar to hot
extrusion, it however occurs at temperatures above glass transition but below starch melt-
ing temperatures (<70°C). Cold extrusion utilizes a pasta-type extruder which shapes the
native or heat-treated rice flour dough containing water, a vitamin/mineral premix, binders,
moisture barrier agents, or other additives into rice analogues closely resembling natural rice
but a little more opaque 61 .
In fortifying rice, extruded rice kernels containing the choice vitamins and minerals are
added to intact rice kernels between ratio 1:50 to 1:200 to produce the fortified rice. The
extruded rice kernels are similar to the vitamin/mineral–coated rice kernels, but differing in
their performance level as the extruded rice kernels are able to retain their micronutrient
contents. Both cold and hot extrusion processes produce extruded fortified rice kernels
similar to the natural rice, and having the nutrients embedded in them effectively protected
during washing and cooking 65 . Hot- and cold-extruded fortified rice kernels containing iron
and vitamin B1 have both been reported to retain 100% iron and about 63-83% vitamin
B1 when assessed in both products after rinsing the rice kernels severally, soaking in water
for 30 minutes, frying some samples prior to cooking, and rigorous preparation and cooking
methods 69 . Several studies have shown efficacy and correlation between the consumption
of extruded rice grains fortified with zinc, iron, and vitamin A and improvements in zinc
status, iron, vitamin A status, reduced iron deficiency and various elevated minerals and
vitamins concentration in school children and other adults 70;71 . Additionally, both the hot-

12
and cold-extruded grains have also been well accepted by adult consumers, schoolchildren
and their teachers based on their organoleptic properties 65 .

1.9 Iron Fortification Compounds

Three major criteria determine how successful iron fortification strategy would be; they are
the baseline nutritional deficiency prevalence, how widely consumed the choice of food/vehicle
is, and suitability of the iron compound 72 . The fortification of staple foods including cereal-
based products, dairy products, legumes and widely consumed condiments with iron are con-
sidered most sustainable and affordable strategies of enhancing iron status and in achieving
the daily iron requirements in a population due to their wider consumption 73;74 . Numerous
iron fortification compounds have been reported; however, getting suitable iron fortificants
with increased absorption level in the human digestive system without modifying the sensory
characteristics of the food matrix still remains a challenge in the food industry. Therefore,
attention is now being focused on less bioavailable compounds capable of being supplied in
larger quantities with no adverse organoleptic impacts on the vehicles 75 . In an order of pref-
erence, the WHO has recommended ferrous sulfate, ferrous fumarate, ferric pyrophosphate,
and electrolytic iron as preferred iron compounds for most iron fortification processes 9 .
Iron fortificants are popularly categorized into readily water soluble, poorly water soluble
but soluble in dilute acids, and partially soluble in dilute acid based on their water solubility
and properties, which determine their ability to dissolve in gastric juice and their absorption
rate 76 . Ferrous sulfate (FeSO4 ) and ferrous gluconate are common examples of the recom-
mended readily water soluble iron compounds that are better absorbed. They are however
highly reactive, and often causing precipitations, color and/or flavor changes to sensitive
foods and rancidity during storage of cereals 77 . These readily water soluble iron compounds
including FeSO4 have been reported to be limited in rice fortification process as a result of
its interaction with the rice matrix. As a result of their water solubility, they are readily lost
during the washing and cooking process of rice in excess water and while draining the water
after cooking 61 . Nevertheless, several studies have established that these freely water-soluble

13
iron compounds are better absorbed than their counterparts 78 .
Two common examples of the poorly-water-soluble but dilute acids soluble iron com-
pounds are the ferrous fumarate and ferrous succinate. These compounds have fewer or no
sensory impacts on sensitive foods with relative bioavailability similar to ferrous sulfate’s
bioavailability level 78 . This implies that these dilute acids soluble iron compounds are read-
ily dissolved in the gastric juice during digestion and are capable of being used to replace
ferrous sulfate in certain cases. They cannot be used for fortifying rice due to their effects
on the color and taste of rice, but are popular for fortifying commercial cereal-based comple-
mentary foods and as micronutrient powders 61 . Ferric pyrophosphate (FePP) and elemental
iron powders are water insoluble and only partially soluble in the gastric juice during diges-
tion. FePP, has a nearly white or off-white color, and has little or no interaction with the
other rice components and nutrients as a result of its low solubility at the pH of rice.
The FePP, a partially-soluble-in-dilute-acid iron compound; has a minimal impact on the
color of rice when stored and does not cause vitamin A rancidity. FePP has a low bioavail-
ability, with only 13–15% absorption upon consumption when compared to FeSO4 79;80 . The
mean particle size of a regular ferric pyrophosphate is approximately 20m, with very minimal
interaction with the food matrix, but shown to have the lowest bioavailability of iron among
the ferric pyrophosphate compounds. Previous attempts to improve the iron status and
absorption of FePP-fortified foods by reducing its particle size and adding FePP to bouillon
cubes have however failed to demonstrate practical benefits in the study participants 79 . Al-
though elemental iron is cheap, it is also not recommended for fortifying rice due to its gray
discoloration and low absorption when consumed 61 . Additionally, ferric phosphate (FePO4 )
is a poorly acid-soluble iron compound, having stability in foods and no adverse organoleptic
effects on the food matrix. It however has a very poor absorption of about 25% compared
to the FeSO4 81;82 . These iron compounds however still remain the most widely used and
recommended in fortifying foods due to their minimal impacts on the sensory properties of
the fortified food despite their poor availability 76;83 .
Sodium iron ethylenediaminetetraacetic acid sodium salt (NaFeEDTA) is an iron com-
pound that utilizes its ethylenediaminetetraacetic acid (EDTA) component to chelate nu-

14
merous metals, and reduce the percentage of iron compounds bound to inhibitors while
increasing absorption to about thrice that of FeSO4 in foods 77 . It is commonly used in
cereal fortification especially wheat and maize flours, and is reported to exert superior iron
bioavailability in foods containing a high content of phytic acids including legume grains
as a result of this property 84 . The affinity of NaFeEDTA to the various metal components
may however differ as it is being digested due to numerous factors including the medium’s
pH, and the molar ratio of EDTA and the metal. NaFeEDTA is also limited in use for rice
fortification involving multiple nutrients coating due to its color effect at high concentrations
in the fortified kernels 61;77 .

1.10 The Enhancers and Inhibitors of Iron Absorption

Various approaches are being considered to enhance the bioavailability of iron in fortified
foods and combat iron deficiency. Particle-size reduction is one of such common approaches,
and has resulted in more bioavailable iron candidates in fortified products 85 . One mechanism
by which particle size reduction enhances iron bioavailability is through the increased surface
area, thereby increasing the rate of solubility and absorption rate of iron in the gastric
juice 86 . Iron can only be absorbed in the ferrous form (Fe2 +) by humans, and this can be
readily enhanced by the activities of reducing agents such as ascorbic acid on ferric iron
(Fe3 +). The reducing and chelating properties of ascorbic acid in fruits and vegetables, and
partially digested muscle proteins are described as main enhancers of dietary iron absorption
in humans.
Ascorbic acid is the only food component typically added to iron-fortified foods to enhance
native iron and nearly all other iron fortificants absorption in multiple folds besides iron
chelates. It is the most efficient non-heme iron absorption enhancer when stable, and largely
suppresses the inhibitory effects of phytic acid, calcium, milk and legume proteins, and
phenolic compounds on iron by converting ferric to ferrous iron and weakening its ability
to combine with the inhibitory food components 78 . Additionally, the use of ligands such
as citric acid (CA), trisodium citrate (TSC) and EDTA in FePP-fortified rice have been

15
reported to significantly improve the bioavailability of iron in humans and act as in-vitro
solubilizing agents in large-scale rice fortification 87;88 . The co-extrusion of CA and TSC
with FePP in rice significantly increased iron absorption without affecting the organoleptic
properties of the rice 89 . Soluble ferric pyrophosphate (SFP), a product of chelation of ferric
iron to citrate and pyrophosphate ligands has been reported to be more bioavailable than
the FePP alone 90 . Adding a mixture of CA and TSC to rice flour before extrusion led to
the formation of SFP due to the applied pressure, heat and subsequent boiling; resulting in
an increased iron bioavailability 87 .
The fortification of foods with iron chelates including NaFeEDTA or the ferrous bisgly-
cinate has been successful as alternative strategies for enhancing iron absorption from foods
rich in phytic acid or other inhibitors of iron absorption. NaFeEDTA has been recommended
by the WHO as the choice iron enhancer in high phytate cereal flours, with iron absorption
of around 2-3 times more than that of ferrous sulfate from high phytate meals 91;92 . The
NaFeEDTA is commonly used in cereal flours, and its recent combination with ferrous fu-
marate in low-cost complementary foods is considered an essential alternative due to the
instability of ascorbic acid during cooking 78 . Like the NaFeEDTA, ferrous bisglycinate also
inhibits phytic acid and other inhibitors of iron. It is generally used for the fortification of
liquid milk, and is reported to increase the iron levels in the children 93 . It is however capable
of causing rancidity in cereals during storage and modifying the color of food when used in
sensitive foods 94 .
In addition, the phytate:iron ratio of rice can be improved by fortification with iron and
the milling of wheat or polishing of rice 61 . Using phytase in the production of cereal or soy-
based infant foods or activating natural phytases in wheat or rye can also degrade phytic acid
in these foods 95 . Previous studies have reported the degradation of phytic acid and improved
iron absorption of food by the addition of phytase during consumption 96 . The encapsulation
of iron can also be used to prevent reaction with other components in the food matrix when
stored. It is a preferred strategy when fortifying salt with multiple nutrients 97;98 . Tannins
and some animal proteins including milk, eggs, soybean proteins, and albumin have also
been thought to inhibit iron absorption even when combined with iron absorption enhancers

16
or cereal-based fortified foods 72 .

1.11 Measurement of Iron outcome

Iron absorption is a function of the host’s iron stores, the solubility of the consumed iron com-
pounds, and the presence of inhibitors or enhancers of iron absorption in a food/meal. The
absorption of iron is commonly assessed by measuring the rate of iron incorporation into the
erythrocytes of study subjects fed meals containing radioisotope-labeled iron. It is measured
as relative bioavailability against a control meal containing FeSO4 99 . Hemoglobin concen-
tration, serum ferritin, serum transferrin receptor, erythrocyte zinc protoporhyrin measure-
ments or a combination of these investigations can be used to determine the bioavailability
and bioefficacy of iron. Functional bioefficacy has been determined in children by evaluating
changes in the frequency of iron-deficient anemia and growth rates 9 . The human intesti-
nal Caco-2 cell line is an in-vitro experiment used extensively for studies involving nutrient
transports 100;101 . Increased bioavailability of soluble ferric pyrophosphate (SFP) resulting
from the proposed protective effect by the surrounding pyrophosphate and citrate ligands
have been reported in Caco-2 cells 90 .
Hepatic iron concentration is a preferred iron bioavailability measurement in animal
studies due to the liver being a major repository of iron and containing about 20-30% of
body iron 102 . There are two common methods for assessing iron bioavailability in animals,
and these are the depletion-repletion method and preventative-prophylactic method. The
depletion-repletion method measures iron status by maintaining animals on diets low or de-
ficient in iron to reduce the iron stores of the animals, and thereafter feeding iron-rich diets
to the animals to correct the iron deficit which is known as the repletion period 103 . Con-
trarily, the preventative-prophylactic method involves placing the animals on the iron-rich
diets immediately after weaning them 104 . The preventative-prophylactic study is however
considered a faster approach for assessing iron bioavailability because of its shorter study
duration.
Several studies have reported varying outcomes on the bioavailability of different iron

17
compounds in extruded rice. FePP fortification of extruded rice grains significantly improved
the iron status of participants in various feeding trials involving anemic participants 79;105 .
Some efficacy studies reported improvements in the iron level of women or children who con-
sumed encapsulated micronized dispersible FePP- (MDFP) and high concentrations of mi-
cronized ground FePP- (MGFP) fortified extruded premix rice respectively 78 . Other reports
have described the improvement of body iron stores in Indian school children fed micronized
ferric pyrophosphate fortified extruded rice kernels 106 . In addition, co-fortifying extruded
rice with FePP and citric acid/trisodium citrate was shown to double the bioavailability of
iron in the women who participated in the study 87 . The consumption of iron-fortified rice
has been shown to decrease the prevalence of iron deficiency and anemia in groups over an
extended period 107 .

18
Chapter 2

Ferric Pyrophosphate Fortified

Extruded Rice Increased Iron Status
in Rats

Abstract

Background:

Iron deficiency is the most common micronutrient deficiency worldwide and one of the five
prominent causes of years lived with disability in humans, particularly affecting women and
children. Ferric pyrophosphate (FePP) and ferric phosphate (FePO4 ) are commonly used in
fortifying rice due to their minimal impacts on its sensory properties, but have both presented
with poor iron bioavailability. Particle-size reduction of FePP and addition of ligands such
as citric acid (CA) and trisodium citrate (TSC) to extruded FePP-fortified rice have been
associated with improved iron bioavailability in iron-fortified rice.

19
Objective:

To compare the iron outcomes of extruded rice flour formulated with four different iron
fortificants in rats.

Methods:

Using the prophylactic-preventative method, 50 male weanling Sprague-Dawley rats aged

20-23 days were randomly distributed into 5 groups and fed ad libitum for a 28-day period.
The control group consumed powdered AIN-93G growth diet, while the other four experi-
mental diets were AIN-93G based extruded rice flour diets fortified with micronized FePP
(µFePP), a higher FePP concentration, FePO4 with TSC and CA or FePP with TSC and
CA at a molar ratio of 1:0.3:5.5 for iron to TSC/CA. Hematological parameters and hepatic
iron concentration were assessed for the iron status. The dual energy x-ray absorptiome-
try (DEXA) PIXImus scan was used to assess the bone mineral density (BMD) and body
composition of the rats.

Results:

The consumption of FePP, µFePP, FePP+TSC+CA fortified rice flour significantly increased
(p<0.05) hepatic iron concentration compared to the FePO4 + TSC+CA fortified rice flour
and AIN-93G groups. However, there were no significant differences between the hepatic
iron concentrations of the FePP groups or the hematological and anthropometric assessments
between all groups (p>0.05).

Conclusion:

Increased concentration of FePP in extruded fortified rice can improve iron status, and
suggests that neither micronizing FePP nor extruding it with TSC and CA improved iron

20
status compared to FePP. The extrusion of FePO4 with TSC and CA did not improve iron
status.

2.1 Background

About 25% of the world’s population have been reported to be affected by anemia, causing
several acute and chronic health challenges in pregnant women and children 10;11 . Anemia
often results from several factors, with dietary iron deficiency (ID), inherited blood disorders,
malaria, and helminthic infestation as its leading causes 14 . Iron deficiency is the most
common micronutrient deficiency worldwide and is implicated as the leading cause of anemia
globally, particularly affecting pregnant women, infants and young children 30 . Iron deficiency
anaemia (IDA) is one of the five prominent causes of years lived with disability in humans,
and is classified as a public health challenge that adversely affects young children, and women
of child-bearing age 14;26 . Fortification of staple foods is considered a most sustainable and
cost-effective public health intervention to improve the overall consumption of iron and iron
status of populations 41;92 .
The WHO has recommended the universal fortification of staple foods including rice as a
choice vehicle for iron to prevent iron deficiency and IDA in at-risk populations 47;48 . Rice is
a prominent staple food of about three billion people globally, and its fortification with iron
reduced IDA prevalence among preschool age children, pregnant women, adolescents, and
adults in previous studies 59;87 . With rice being commonly consumed as a whole grain and
not flour, it is often fortified with iron using the extrusion method following a grain premix
approach 70;71;108;109 . However, getting suitable iron fortificants with high absorption in the
human digestive system without modifying the sensory characteristics of the food matrix
still remains a challenge in the food industry. Therefore, attention is now being focused on
enhancing less bioavailable compounds that do not adversely affect the taste and color of
the food vehicles to reduce costs, and increase acceptability in rice-eating populations 5;87 .
Ferric pyrophosphate (FePP) is a widely used and common choice of iron compound for
rice fortification as result of its white color and minimal impact on the sensory properties

21
of the food matrices 79;80 . Additionally, the fortification of extruded rice grains with ferric
phosphate (FePO4 ) results in acceptable organoleptic characteristics with no adverse effects
on the food matrix 76;81;83 . These compounds however have very low iron bioavailability and
absorption of about 50% and 25% compared to the ferrous sulfate (FeSO4 ) respectively 64;80;82 .
The use of ligands such as citric acid (CA), and trisodium citrate (TSC) in FePP-fortified rice
have been shown to significantly improve iron bioavailability in humans and act as in-vitro
solubilizing agents in large-scale rice fortification 87;88 . The co-extrusion of CA and TSC with
FePP in rice significantly increased iron absorption without affecting the sensory properties
of the rice 89 .
Soluble ferric pyrophosphate (SFP), a product of chelation of ferric iron to citrate and
pyrophosphate ligands has been reported to be more bioavailable than the FePP alone 90 .
Adding a mixture of CA and TSC to rice flour before extrusion led to the formation of SFP
due to the applied pressure, heat and subsequent boiling; resulting in an increased bioavail-
ability of iron 87 . Other reports have also described the improvement of body iron stores in
Indian school children fed micronized ferric pyrophosphate (µFePP) fortified extruded rice
kernels 106 . Reducing the mean particle size of FePP is thought to increase the bioavailablity
of iron due to an increase in the surface area, which in turn enhances the rate of solubility and
iron absorption of the fortified foods 86;110 . We previously reported lower moisture-adjusted
total food intake, weight gain, final weight and bone mineral density compared to the AIN-
93G group in a similar study conducted in our lab 111 . The poor intake and growth of the
rice diet groups likely resulted from the inadequate nutrients and poor protein quality of
the rice diets. This study, therefore investigated the iron outcomes in rats which consumed
extruded rice flour formulated with four different iron fortificants.

22
2.2 Methods

Production of extruded fortified rice diets

Extrusion processing for the unenriched rice flour was done using a pilot-scale twin-screw
extruder (TX-52, Wenger Manufacturing, Sabetha, KS) equipped with a differential diam-
eter cylinder preconditioner, and having a volumetric capacity of 0.056m3 (DDC2, Wenger
Manufacturing, Sabetha, KS). The preconditioner shaft speed was 379 rpm with an average
residence time of 2.8 minutes, a screw diameter of 52mm and an L/D ratio of 16. The mate-
rial was fed into the extruder at 80 kg/h, and the screw speed fixed at 300rpm as previously
reported 112 . A single-opening circular die of 3.7mm was used, and the product cut upon exit
with three hard knife blades rotating at 530 rpm. Extrudates were collected and dried in a
dual pass dryer (4800, Wenger Manufacturing, Sabetha, KS) at 115°C for 18 minutes, with
a 7 minute cooling step. The extrudates were then ground using the hammermill grinder
(Fitzpatrick DKAS012), and frozen until further fortification processes.
The iron fortificants for the study were provided by Wright Enrichment Inc. (Lafayette,
LA). Mean particle size of the micronized FePP was 2.4µm, while the regular FePP had a
larger particle size. The Ferric pyrophosphate (FePP) and Ferric phosphate (FePO4 ) were
blended with rice flour (RIVLAND Partnership; Houston, Texas), and the trisodium citrate
(TSC) and citric acid (CA) [Sigma-Aldrich; St. Louis, MO] added to the FePP and FePO4
respectively at a molar ratio of 1:0.3:5.5 iron to TSC and CA as previously described 109 by
adding 0.036g CA and 1.014g TSC per kg diet (Table 2.1). They were then mixed with 200g
water for 3 minutes using a bench-top mixer (N-50, The Hobart Mfg. Co., Troy, OH) to
make up a total dry premix of 1.2kg material. Thereafter, the hydrated blends were placed
in resealable bags and allowed to equilibrate overnight under refrigeration. Each blend was
prepared using three mixes involving a minor mix comprising components having smaller
concentrations. The intermediate mix was made up of components with slightly higher
concentration, and the major mix consisted of components with concentrations higher than
the intermediate. All blends were extruded in duplicates.

23
 These equilibrated blends were then extruded with a lab scale twin-screw extruder (the
Micro-18, American Leistritz Extruder Corp., Somerville, NJ), having 18mm barrel diameter
and L/D ratio of 29. The extrusion die used was a circular cross-section die of 3.1mm diam-
eter (dd), and feed rate was 2.1 ± 0.2 kg/h at the extruder screw speed of 350 rpm for all
formulations. The barrel temperature for the extrusion was controlled at 40°C, 50°C, 60°C,
70°C, 80°C and 90°C respectively with steam and water added during the preconditioning 112 .
Final products of the extrusion process were pushed out of the rice-shaped openings called
die, and extrudates collected in clean aluminium trays, these were manually cut and dried in
a convection hot-air oven at 70°C for 2 hours to reach a moisture content of approximately
14%. The dried products were ground to particle sizes below 1 mm using a high-speed mul-
tifunctional grinder (Moongiantgo Grain Grinder) at different time intervals for 30 seconds
two consecutive times, and the grinder allowed to cool for about 3-15mins between each
grinding time. Larger particles from each mix are collated, sifted and further ground for
20 seconds two consecutive times at room temperature and stored frozen in resealable bags
until ready for final mixing.
Using the mixer (The Hobart Mfg. Co., Troy, OH), 63.2% unenriched extruded rice flour
comprising 5,818.6g of the pilot-scale extruded rice flour and 500g of the lab-scale extruded
rice flour for the µFePP and FePP formulated diets, 5,814.18g of the pilot-scale extruded
rice flour and 494.12g of the lab-scale extruded rice flour for the FePO4 +TSC+CA and
FePP+TSC+CA formulated diets. Each formulated diet blends were then mixed with 20%
casein, 5% cellulose, 7% soybean oil with TBHQ, 3.5% mineral mix without iron, 1% vitamin
mix and 0.3% L-Cystine (Dyets, Inc; Bethlehem, Pennsylvania) in line with the AIN-93G
composition respectively (Table 2.2) to sustain the nutritional requirements of the growing
rats 113 . The prepared extruded rice blends were kept in polyethylene zip-lock bags and
stored frozen until use.

24
Nutritional analysis

Macronutrient proximate analysis of the formulated ride extrudates were determined by the
University of Missouri–Columbia Agricultural Experiment Station Chemical Laboratories
(Columbia, MO) as previously reported (Ward and Lindshield, 2019). The protein analysis
was conducted by combustion method (LECO; AOAC 990.03, 2006) and the fat analysis by
using acid hydrolysis (954.02, 2006), and the carbohydrates were calculated. Iron content
was determined via the Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) method
(AOAC Official Method 993.14 Trace Elements in Waters and Wastewaters).
Carbohydrate = 100% - % (crude protein + ash + crude fat + fiber + moisture)

Animals and experimental protocol

Fifty male weanling Sprague Dawley rats aged 20-23 days and weighing between 42 and 77.2
g were obtained from Charles River (Wilmington, MA) for this prophylactic-preventative
study 104 . The experimental protocol was approved by the Institutional Animal Care and Use
Committee (IACUC) at Kansas State University (IACUC-4741). All animals were assessed
for well-being before and throughout the study. They were housed individually in wire-
bottom cages, provided with a resting board, tongue depressors as enrichment, and provided
water and food ad libitum. Rats were randomized into 5 diet groups of ten animals per
group. The control group were fed the standard AIN-93G rat diet (Dyets, Inc; Bethlehem,
Pennsylvania), while the other groups received one of the four extruded rice formulations:
µFePP, FePP, FePO4 + TSC and CA, FePP + TSC and CA (at a molar ratio of iron to
TSC/CA of 1:0.3:5.5). Food intake of each rat was calculated from the food remnants, and
fresh diets provided afterwards. The weight of the rats was collected at baseline upon arrival,
and subsequently measured every other day and weekly. Rats were euthanized after 28 days.

25
Sample collection and preparation

The rats were sacrificed on the 28th day under euthanasia with carbon dioxide (CO2 ) inhala-
tion; blood was collected by cardiac puncture into tripotassium ethylenediaminetetraacetic
acid (K3 EDTA) tubes for the hematological analysis. Liver tissues were harvested, weighed,
and flash-frozen in liquid nitrogen and stored at -80°C.

Hematological assay

Hemoglobin concentration, hematocrit, and red cell indices were measured in the fresh whole
blood in K3 EDTA tubes by flow cytometry using the Siemens Advia 2120i Hematology
Analyzer as previously described 87 .

Hepatic Iron Concentration

The glassware for the procedure was prepared with 6% nitric acid solution and the frozen
liver samples thawed overnight in a refrigerator prior to analysis. 1 g of liver each covered
with 10 mL Trace-metal grade nitric acid solution (Fisher Scientific; Pittsburgh, PA) in
a 50 mL beaker and allowed to sit for 1 hour as previously described 111 . Samples were
thereafter placed on a hot plate, and allowed to gently reflux for roughly 2-8 hours at low
heat until approximately 1 mL of the solution was remaining in the beaker. The boiled
samples were then made up to 10 mL with distilled-deionized water and transferred to 15
mL polypropylene tubes in preparation for analysis. Iron concentration was determined using
inductively coupled plasma-optical emission spectrometry (ICP-OES, Varian 720-ES, Agilent
Technologies, Santa Clara, CA) at the Kansas State University Soil Testing Lab (Manhattan,
KS). All liver samples were prepared in duplicate, and triplicate analyses performed on
samples with variance above 15% between duplicates.

26
Body composition and Bone mineral density

Body scans were performed on the rats’ carcass using the dual energy x-ray absorptiometry
(DEXA) PIXImus densitometer according to manufacturer’s instructions (GE Lunar Cor-
poration, Madison, WI) for body composition and bone mineral density (BMD) detection.

Statistical analysis

Shapiro-Wilk was used to assess data for normality, and non-normally distributed data were
transformed using natural log transformations. Levene’s test was used to assess the level of
homogeneity at p<0.05 among the groups. Group differences were determined using one-
way analysis of variance (ANOVA) Least Squares Means (LSM) tests. Data collected were
analysed using the SAS Studio (version 3.8, SAS Institute Inc., Cary, NC) and expressed as
mean ± SEM for each group. Differences were considered significant at p<0.05.

2.3 Results

Diet composition, Food intake and Anthropometric eval-

uation

The rice diets contained 22.35g protein, 6.20g fat, 7.36g moisture, 1.12g fiber, 3.44g ash per
100g, and varying amounts of iron as indicated in table 2.3. The iron content of the FePP
diet was however higher; roughly 2-folds than the other formulated diets. There were no
significant differences in the weekly food intake (Figure 2.1), total food intake (Table 2.4),
weight gain, average weekly weights (Figure 2.2), final body weights, lean mass and bone
mineral density (Table 2.4) between groups.

27
Effects of formulated diets on hematological parameters

and hepatic iron

There were no significant differences in the hemoglobin concentration, hematocrit, red blood
cell count, mean cell hemoglobin, mean cell hemoglobin concentration, mean cell volume
and total white blood cell counts between the groups. However, the hepatic iron concentra-
tions for all the FePP groups (FePP, µFePP, FePP+TSC+CA) were significantly increased
compared to both the AIN-93G and FePO4 +TSC+CA groups. There was however no dose-
response effect in the iron bioavailability of the FePP. No significant differences were also
observed in the iron outcomes between the FePP groups. Hepatic iron concentration was
also not significantly different in the FePO4 +TSC+CA group compared with the AIN-93G
control (Table 2.5).

2.4 Discussion

The increased hepatic iron concentration in all the FePP-fed groups compared to the AIN-
93G and FePO4 +TSC+CA groups suggests that these iron compounds are capable of im-
proving iron status. Iron is stored in both the hepatocytes and Kupffer cells of the liver,
where about 20–30% of excess body iron is stored in a bioavailable form as ferritin and as
hemosiderin 21 . It is therefore possible that FePP might have resulted in an increased liver
iron store in the form of ferritin due to the preventative-prophylactic method used in this
study. FePP fortification of extruded rice grains, and co-fortification with CA and TC have
been reported to improve the iron status and body iron stores of participants in various feed-
ing trials involving anemic participants 79;105;109 . Although the FePP diet had a higher iron
content than the other diet groups, there were however no significant differences between all
the FePP groups. Therefore, increasing the amounts of FePP in the rice flour did not result
in a significantly higher hepatic iron concentration.
Despite the higher iron content of the the FePP group in this study, the hepatic iron
concentration was lower than found in a previous study conducted in our laboratory where

28
cooked FePP-extruded rice blended with unenriched white rice were fed to rats 111 . Several
factors may influence these divergent findings, including the fact that the current study
involved grinding the extruded FePP-fortified rice and feeding them directly to the rats as
against cooking the extruded FePP-fortified rice in the previous study. Thus suggesting that
cooking the extruded FePP-fortified rice may be beneficial to the availability of its iron as
reported in other previous studies 87;106 . It is also possible that the higher concentration of
the FePP counteracts its acid-driven dissolution in the stomach, thereby resulting in a lower
overall solubility of the FePP, and subsequently lower bioavailability.
Ligands such as citric acid (CA), trisodium citrate (TSC) and EDTA are considered
one of the best ways of enhancing iron bioavailability in FePP-fortified rice and in-vitro
solubilizing agents in rice fortification 87;88 . Co-extrusion of a mixture of TSC and CA to
FePP-fortified rice flour resulted in the in-situ formation of soluble ferric pyrophosphate
(SFP) in the presence of pressure, heat and boiling; which increased iron bioavailability and
absorption in a human study 89 . The ability of FePP+TSC+CA extruded fortified rice flour
to form SFP could have also accounted for the increased level of hepatic iron concentration in
the FePP+TSC+CA fed rat group. The TSC/CA ratio used in our study was same with that
used in a previous study where co-extrusion of CA/TSC with FePP significantly increased
iron absorption in Zurich women 109 , the lack of significant difference in the FePP+TSC+CA
group compared to the other groups was however an interesting and unexpected discovery
which we are yet to proffer an explanation for. To our knowledge, this is the first study to
investigate iron absorption from extruded rice cofortified with FePO4 and TSC/CA. However,
the addition of TSC/CA did not enhance the bioavailability of iron from FePO4 despite the
higher molar ratio of iron to TSC and CA used. Similarly, FePO4 was poorly absorbed (25%)
compared to FeSO4 in a radiolabeled farina-based meal that were fed to human participants
and rice test meals fortified with bulk FePO4 82;103 . There is a remarkable similarity in the
poor hepatic iron outcome of FePO4 determined in our present study and that observed in
our previous study involving cooked FePO4 fortified extruded rice kernels 111 .
Reduction of the particle size of FePP is reported to increase iron bioavailablity of fortified
products as a result of its increase in surface area 110 . This enhances the rate of solubility

29
and iron absorption of the fortified foods 86 . Similar outcomes in the relative bioavailability
of iron for emulsified µFePP with mean particle size (MPS) of 0.5 µm and FeSO4 in iron-
depleted rats was previously reported, with larger MPSs FePP molecules resulting in lower
relative bioavailability than FeSO4 85 . Improvements in iron stores and reduction of iron
deficiency have also been shown with regular intake of µFePP-fortified extruded rice kernels
among school children in India 106 . In contrast, the fortification of rice flour with µFePP did
not significantly increase the absorption of iron in this study compared to FePP alone. It
is therefore possible that there was a poor upregulation of the absorption of iron from the
small-particle-sized µFePP as previously noted 79 . Additionally, a previous study involving
iron-depleted rats reported no significant difference in the bioavailability of regular FePP
with MPS of about 21 µm when compared with µFePP of around 2.5 µm MPS 85 .
Hemoglobin concentration is one of the reliable indicators of iron absorption and the
application of its cut-offs is commonly used in differentiating iron deficiency and iron de-
ficiency anemia 9;114 . We have previously reported higher hemoglobin concentration in the
FePP+TSC+CA and FePP groups compared to the AIN-93G group, and they were not
significantly different than FePP alone in the study 111 . However, the hemoglobin concentra-
tions in the various groups were not significantly different but trended higher in all the FePP
groups than others in the present study. The various iron compounds also did not enhance
the hematocrit, red blood cells and red cell indices of the various groups when compared to-
gether. There was no significant difference between the total white cell counts of the various
groups, indicating that the concentration of the iron compounds and diet components used
had no adverse effect on iron apparent absorption and the well-being of the rats.
The rice diets were formulated based on the AIN-93G; a recommended rodent diet by the
American Institute of Nutrition, and the extruded rice flour replaced the dyetrose, cornstarch,
and sucrose in the formulated diets. In this study, the iron content of the formulated rice
diets were higher compared to the AIN-93G’s. The use of a higher concentration of the
iron compounds for the formulated rice diets is due to the lesser bioavailability of FePO4
and FePP compared to ferric citrate used in fortifying the AIN-93G 115 . Although we had
previously reported lower moisture-adjusted total food intake, weight gain, final weight and

30
BMD compared to the AIN-93G control group in our laboratory 111 , there were no significant
differences in the weekly food intake, total food intake, weight gain, final weight, average
weekly weights, lean mass and BMD between the various groups in the present study. This
improvement resulted from the current study’s formulated diets’ composition being based
on the AIN-93G’s. Our formulated rice diets were well consumed.
Our study has some limitations; we tested the diet formulations in apparently healthy
rats and cannot exclude a different iron absorption rate under a depleted rat model as would
a mass fortification program. We also did not cook the extruded fortified rice which may
have been beneficial in improving the iron bioavailability of the iron compounds used. In
addition, we did not scrutinize the sensory properties of the different formulated rice types.

Conclusions

This study, therefore, suggests that increasing the concentration of ferric pyrophosphate
when fortified in extruded rice can improve iron status to mitigate iron deficiency and iron
deficiency anemia. Our study also suggests that adding TSC/CA to FePO4 did not enhance
the iron status compared to other groups. Further studies that examine the findings of this
present study to account for the potential effectiveness of the increased FePP in extruded
fortified rice and confirm that the findings in rats can be extrapolated to humans would be
welcomed. In addition, the effect of cooking on the iron bioavailability of FePP, µFePP and
FePP +TSC/CA fortified extruded rice requires further investigation

31
Tables

Table 2.1: Iron compounds and ligands composition per kilogram rice flour
Components µFePP FePP FePO4 + TSC + CA FePP + TSC + CA
FePO4 - - 0.121g -
Fepp - 0.14g - 0.14g
µFePP 0.14g - - -
TSC - - 1.014g 1.014g
CA - - 0.036g 0.036g

Table 2.2: Percentage composition of vitamins and minerals of formulated diets

FePO4 FePP
Constituent *AIN-93G µFePP FePP
+TSC+CA +TSC+CA
Extruded rice flour without iron - 63.2 63.2 63.2 63.2
Casein 20 20 20 20 20
Cellulose 5 5 5 5 5
†Soybean oil with TBHQ 7 7 7 7 7
Mineral mix without iron 3.5 3.5 3.5 3.5 3.5
Vitamin mix 1 1 1 1 1
L-Cystine 0.3 0.3 0.3 0.3 0.3
Dyetrose 13.2 - - - -
Cornstarch 39.7486 - - - -
Sucrose 10 - - - -
TBHQ 0.14 - - - -
Choline bitartrate 0.25 - - - -

*Percentage composition is based on product label and not analyzed protein content
TBHQ = t-Butylhydroquinone
†Soybean oil used in the rice diet formulations contained TBHQ, while TBHQ was added
to the AIN-93G diet separately.

32
Table 2.3: Macronutrients and iron content of formulated rice and AIN-93G diets (g/100g)
Components *AIN-93G µFePP FePP FePO4 +TSC+CA FePP+TSC+CA
Carbohydrate 59.3 59 59.4 59.3 60.2
Protein 17.9 22.8 22.7 22.2 21.7
Fat 7.0 6.2 6.2 6.3 6.2
Fiber 5.0 1.3 0.8 1.4 1.0
Moisture 6.6 7.3 7.5 7.3 7.4
Ash 4.2 3.4 3.4 3.5 3.5
Iron (mg/100g) 4.5 11.8 20.6 9.3 10.8

* Components are based on product label and not analyzed protein content

Table 2.4: Food intake and anthropometric measures of study groups

AIN-93G µFePP FePP FePO4 +TSC+CA FePP+TSC+CA
Total food intake (g) 559.6 ± 9.5 551.5 ± 13.1 547.0 ± 18.6 552.6 ± 13.4 515.1 ± 21.2
Total weight gain (g) 251.7 ± 5.6 264.0 ± 6.1 253.0 ± 6.2 263.9 ± 6.3 239.7 ± 9.0
Final body weight (g) 310.6 ± 7.9 324.32 ± 8.4 313.2 ± 7.1 322.5 ± 6.7 298.6 ± 11.0
Lean mass (%) 89.7 ± 0.6 91.0 ± 0.3 90.1 ± 0.4 90.5 ± 0.4 90.0 ± 0.4
Bone mineral density (g/cm2 ) x 1000 113.8 ± 2.4 112.1 ± 3.3 107.7 ± 1.7 112.9 ± 2.2 108.1 ± 2.9

Data represent Mean ± SEM

No significant difference between groups
Percentage lean mass: Total weight minus fat mass divided by total weight x 100.
Food intake was measured every other day by subtracting food remnants from the previous
food given.

33
 Table 2.5: Hematological and hepatic iron outcomes
AIN-93G µFePP FePP FePO4 +TSC+CA FePP+TSC+CA
Hematocrit (%) 49.1 ± 0.7 50.1 ± 0.9 49.5 ± 0.5 49.8 ± 0.7 50.5 ± 0.6
Hemoglobin concentration (g/dl) 13.8 ± 0.2 14.3 ± 0.3 14.2 ± 0.2 14.0 ± 0.20 14.4 ± 0.2
Red blood cell counts (M/µL) 6.9 ± 0.1 6.9 ± 0.1 6.9 ± 0.1 7.1 ± 0.1 7.0 ± 0.1
Mean cell volume (fl) 71.0 ± 1.1 73.1 ± 0.7 72.0 ± 0.6 69.9 ± 1.7 72.4 ± 0.9
Mean cell hemoglobin (pg) 20.0 ± 0.3 20.8 ± 0.2 20.7 ± 0.2 19.6 ± 0.5 20.6 ± 0.3
Mean cell hemoglobin concentration (g/dL) 28.2 ± 0.1 28.5 ± 0.2 28.8 ± 0.2 28.0 ± 0.2 28.5 ± 0.3
Total white blood cell count (K/µL) 12.7 ± 0.8 12.6 ± 0.6 12.3 ± 0.9 11.8 ± 0.9 13.8 ± 1.1
Hepatic iron (µg/g) 7.1a ± 0.5 11.4b ± 1.3 11.7b ± 1.0 8.1a ± 0.4 11.5b ± 1.2

Data represent Mean ± SEM

Values in the same row with different superscripts are significantly different at p<0.05.

34
Figures

Figure 2.1: Average weekly food intake per group

A trend chart showing the average weekly intake of food for the various groups. Each
colored line graph represents the respective study groups.
No significant difference in the mean weekly food intake between groups.

35
 Figure 2.2: Average body weight of diet groups per week

A trend chart showing the average weekly body weight of rats for the various groups. Each
colored line graph represents the respective study groups.
No significant difference in the mean weekly weights between groups.

36
Chapter 3

Conclusions

Given the outcomes and limitations revealed in the first similar experiment in our laboratory,
we made diets’ composition to be based on the AIN-93G where extruded rice flour replaced
the dyetrose, cornstarch, and sucrose in the present experiment. We also ensured that all
mixes for the the diets were done under the guidance of our collaborator in the Grain Science
department; Dr. Sajid Alavi who routinely produces pet and other food products. This
helped to ensure standardization of the formulated diets and prevented the loss of any of
the micronutrients in our premixes. The findings from this study show that iron absorption
from the different FePP-fortified rice groups was higher compared to the FePO4 and AIN-93G
groups. FePP is a simple, less expensive iron compound having a low reactivity and better
sensory impacts on food matrices, and is generally considered safe. This therefore indicates
that fortifying µFePP, FePP+TSC/CA and increased concentration of FePP in extruded rice
can improve iron status to mitigate iron deficiency and iron deficiency anemia. Our study
also suggests that adding TSC/CA to FePO4 did not enhance the iron status compared
to other groups. No significant differences were observed in the food intake, growth, and
anthropometric features of the various groups. The formulated rice diets were well consumed
by the rats.
We have learned much from this model of iron bioavailability study; although numer-
ous aspects of our approach worked well, there are a few things we could do differently

37
in the future. Most importantly, I would suggest conducting a similar animal study that
would include a depleted rat model group to assess their absorption rate relative to other
groups, as well as a prophylactic-preventative group, an FePO4 alone group, alongside a
FePO4 +TSC+CA group using the dose and time-course model. Although challenging, it
would be very informative to conduct the study for an extended duration beyond our cur-
rent timeline for extensive toxicity testing of FePO4 +TSC+CA, FePP+TSC+CA and the
increased regular FePP used in this study. A key implication of this research is in advanc-
ing our understanding of iron fortificants and iron absorption enhancers for the purpose of
improving iron status. Thus, further studies that examine the findings of this present study
to account for the potential effectiveness of the increased FePP in extruded fortified rice
and confirm that the findings in rats can be extrapolated to humans is highly encouraged.
Additionally, the effect of cooking on the iron bioavailability of FePP, uFePP and FePP
+TSC/CA fortified extruded rice can also be further investigated.

38
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Appendix A

List of Abbreviations

AIN American Institute of Nutrition

ANOVA Analysis of Variance
AOAC Association of Official Analytical Chemists
BMD Bone Mineral Density
CO2 Carbon Dioxide
CA Citric Acid
DEXA Dual Energy X-ray Absorptiometry
EAA Essential Amino Acid
EDTA Ethylenediaminetetraacetic Acid
FePO4 Ferric Phosphate/Orthophosphate
FePP Ferric Pyrophosphate
FeSO4 Ferrous Sulfate
ICP-OES Inductively Coupled Plasma-Optical Emission Spectrometry
IACUC Institutional Animal Care and Use Committee
IDA Iron Deficiency Anemia
LSM Least Square Means
MPS Mean Particle Size
µFePP Micronized Ferric Pyrophosphate
RBV Relative Bioavailability
NaFeEDTA Sodium Iron EDTA
SFP Soluble Ferric Pyrophosphate
TSC Trisodium Citrate
WHO World Health Organization

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