A Report on

Determination of Selected Minerals in Water and Food

Course Code: ACCE-4172

Course Title: Project Work

Submitted by
Md. Ashad Al-amin
Roll: 11065023
Registration No: 2285
Session: 2010-2011
Department of Applied chemistry and Chemical Engineering
University of Rajshahi

Supervised by
Sha Md. Shahan Shahriar
Lecturer, Department of Applied chemistry and Chemical Engineering
University of Rajshahi

Submitted to
Chairman,
BSc (Engg.) Part-4 Examination-2014 Committee.
Department of Applied Chemistry and Chemical Engineering
University of Rajshahi
 Department of Applied Chemistry and Chemical Engineering.
University of Rajshahi

BSc (Engg.) Part-IV Examination.

Course Code: ACCE-4172

Course title: Project Work

Student’s name: Md. Ashad Al-amin

Roll: 11065023

Session: 2010-2011

Supervisor: Sha Md. ShahanShahriar

Title: Determination of selected minerals in water and food.

Date of Consultation Signature of Supervisor

i. 15-01-2015

ii. 12-02-2015

iii. 19-03-2015

iv. 07-04-2015

v. 15-04-2015

vi. 21-04-2015
 Acknowledgement

With the increasing of population mineral consumption in the earth is increasing day by day. For
this reason we will estimate the selected minerals in water and food.So, I am highly pleased to
get a project onestimation of selected minerals in water and food. It will help me to know how
the minerals occur, come into food then our body and how they become harmful for our body.

I am highly grateful and extremely pleased in expressing my deepest sense of gratitude to my

beloved supervisor Lecturer Sha Md. Shahan Shahriar, Department of Applied Chemistry and
Chemical Engineering, University of Rajshahi, for his valuable suggestions, kind help and keen
interest throughout the progress of the project. Without his help and assistance it would be
impossible on my part to complete the dissertation.

I want to give my special thanks to Professor Md. Abul Kalam Azad-1, Chairman of Applied
Chemistry and Chemical Engineering Department, University of Rajshahi. I owe a particular
depth to my venerable teachers Professor Dr. Md. Ahsan Habib, Chairman of BSc(Engg.) Part-4
exam committee and others because of their valuable direction and giving enough time to
complete this project.

I am sincerely thankful to my honourable teachers Prof. Dr C.M. Mustafa, Dr. Aneek Kishna
Karmakar and all other respectable teachers of this department for their inspiration and help in
various ways to complete my present study.

Md. Ashad Al-Amin

 Abstract

Mineral elements in water and food have been in focus for more than a decade. Minerals in
drinking water have become more important since minerals in food, especially in vegetables,
have decreased substantially the last decades. Mineral are essential for human and animal
growth. Deficiency may cause various biochemical effects. On a global base, estimated 2 billion
people are iron deficient and/or anemic with small children and child bearing age most likely to
be the ones affected. These high levels of world anemia prevalence have been maintained and
even growing in spite of the global and national food fortification program carried out in several
places. These have been based on the iron fortification of local foods. It is recommended to
intake safe levels of minerals in water and food. The purpose of this research work is to estimate
the level of selected minerals such as calcium, iron, magnesium, phosphorous and potassium in
water and food. This research will be carried out by using Atomic Absorption
Spectrophotometer. The sample will be digested by wet digestion method. Nitric acid (5-10%)
will be used for digestion of the samples to make all the minerals in soluble form. Sample
digestion, preparation and analysis will be done according to previously published literature. The
findings of this research will be very helpful for supplement minerals as recommended of daily
intake. To assess the dietary intake of calcium, iron, magnesium, phosphorous and potassium
with water and food it is necessary to know their content in different groups of food products.
Mineral content of fruits, vegetables and dairy products is relatively low, but their consumption
is high, which makes their contribution to minerals intake significant. However, scientifically
reliable data on the calcium, iron, magnesium, phosphorous and potassium content of water,
different foods and agricultural products are still insufficient. Water and food make an important
contribution to the supply of calcium, iron, magnesium, phosphorous and potassium for the
human diet. The aim of the present study is to determine the concentration of calcium, iron,
magnesium, phosphorous and potassium in water and cow's milk available in market in
Bangladesh. My remit is to consider supplements sold under food law, thus, the non-nutritional
efficacy of minerals has not been considered since such effects would be classified as medicinal
and would be within the remit of the Medicines and Healthcare products Regulatory Agency.

Keyword: Mineral, calcium, iron, magnesium, phosphorous, potassium, water, food.

 Contents

Topics Page No.

CHAPTER ONE
Introduction …………………………………………………………………….. (1-11)
1.1 Aim and objectives ................................................................................. (3-4)
1.2 General Information ……………………………………………………… (5-11)
1.2.1 Calcium ……………………………………………………………... 5
1.2.2 Iron ………………………………………………………………..…… 6
1.2.3 Magnesium ……………………………………………………………… 8
1.2.4 Phosphorus ……………………………………………………………… 9
1.2.5 Potassium ……………………………………………………………… 11
CHAPTER TWO
Literature review …………………………………………………………….. (12-35)

CHAPTER THREE
Methodology …………………………………………………………….. (36-40)
3.1 Atomic Absorption Spectrophotometer ………………………………….37
3.1.1 Principle ………………………………………………………………..37
3.1.2 Procedure ………………………………………………………………..40

CHAPTER FOUR
Result and Discussion ………………………………………………………. (41-44)

CHAPTER FIVE
Conclusions and Future Prospects ……………………………………….….45
 Chapter One

Introduction
Introduction
It has been argued that since are essential for human health, it is not appropriate to assess them in
the same way in which other chemicals added to food are assessed. However, since there is much
evidence that excessive intakes of some minerals can cause harm, it is not appropriate to exclude
essential nutrients from the safety assessment that is applied to other chemical substances which
are added to foods. Fruits and vegetables are specially valued in human diet as these contain
micronutrients, fiber, potassium, vitamin C, which work as antioxidants within the body as well
as bio-functional components. Minerals and heavy metals content of ten tropical fruits namely
Sapodilla (Manilkarazapota), Stone-apple (Aeglemarmelos), Indian- gooseberry
(Phyllanthusemblica), Guava (Psidiumguajava), Bilimbi (Averrhoabilimbi), Elephant-apple
(Dilleniaindica), Tamarind fruit (Tamarindusindica), Mango (Mangiferaindica), Litchi (Litchi
chinensis), Strawberry (FragariaXananassa) were determined according to standard methods to
address their concentration. Results of this study suggest that the selected tropical fruits are rich
source of minerals. Tamarind fruit is an ample source of iron, sodium, potassium, calcium and
magnesium. Highest amount of manganese found in Mango, 06.16 ± 1.19 mg. Highest amounts
of copper, zinc and sodium found in Guava, 19.30 ± 2.12 mg, 2.07 ± 0.15 mg and 62.78 ± 1.24
mg, respectively. Highest amount of iron, potassium, calcium and magnesium found in Tamarind
fruit, 2.80 ± 1.43 mg, 621.00 ± 3.26 mg, 75.00 ± 2.41 mg and 90.00 ± 1.80 mg, respectively.
However, the present study has been designed to estimate selected minerals such as calcium,
iron, magnesium, phosphorus and potassium in water and food.
Water is an essential nutrient for all known forms of life and the mechanism by which fluid and
electrolyte ho- meostasis is maintained in humans A 65 kg man contains about 40 liters of water.
Of this, 25 liters are intracellular and 15 liters are extracellular. These fluids are in con- tinuously
balance, being turned constant by a water intake and output. Several substances are found in
drinking water that significantly contributes to health and well- being. Water acts as a building
material, in thermoregulation and body metabolism. It has been shown it could be a carrier of
numerous macro and micronutrients as Ca, F, Cu, Mg, Se, K and particularly low in iron and
iodine, source of health problems in various parts of our organism.1,2

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1.1 Aim and Objective
Mineral are essential for human growth. …such as calcium ones serve as a basic support system
protecting vital organs and as a reservoir for calcium — the most abundant mineral in the body.3,
4
In fact, 99% of the body’s calcium is found in bones and teeth (the other1% is found in cells,
blood, and other body fluids).
The objective of the present project is to discuss the various assessments of minerals in foods and
water viz. moderate uptake, toxicity and others along with the existing information. In view of
the expanding market for food supplements, as well as the developments taking place within the
world with regard to harmonization, the Government considered that it is timely to review the
available data on the safe intake of minerals from water and foods. Also Students understand the
significance of specific minerals. Students understand what foods to eat to be sure they receive
these specific minerals. Students can identify specific minerals and their functions. Students can
identify the main food sources of specific minerals. The purpose of this study was to focus
concentration of selected metals in water and raw milk. In this study, it was found that
concentrations of minerals were observed within maximum limit both in water and raw milk.
Comparing results of the present study with those of other studies revealed similar levels of
metals in water and foods investigated in the present study. However, the number of sample and
analyzed heavy metals was limited in our study due to small financial support. Further studies
are necessary to evaluate the contents of ‘‘essential’’ and ‘‘toxic’’ heavy metals on a greater
number of samples to confirm the absence of possible toxicological risks. The aim of the study
was to identify and assess the level of intake of selected minerals in a group of students with
consideration of gender.

i. To detect the presence of minerals in foods and water.

ii. To estimate the level of minerals in food and water Atomic Absorption
Spectrophotometer.

This type of works is very important for human nutrition. The finding of this work will be
helpful for recovery of minerals deficiency in human body as well as in animal body.

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References:

1. World Health Organization, “Nutrients in Drinking Water,” Water, Sanitation and Health
Protection and the Human Environment World Health Organization, WHO, Geneva,
2005.
2. World Health Organization, “Iron Deficiency Anemia. Assessment, Prevention, and
Control. A Guide for Program Managers,” United Nations Children’s Fund, United
Nations University, Word Health Organization, Geneva, 2001.
3. IOM (Institute of Medicine). Dietary Reference Intakes for Calcium, Phosphorus,
Magnesium, Vitamin D, and Fluoride. Standing Committee on the Scientific Evaluation
of Dietary Reference Intakes. Food and Nutrition Board, National Academy of Sciences.
Washington, D.C.: National Academy Press, 1997.
4. U.S. Department of Health and Human Services. Bone Health and Osteoporosis: A
Report of the Surgeon General. Rockville, MD: U.S. Department of Health and Human
Services, Office of the Surgeon General, 2004. www.surgeongeneral.gov/library.

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1.2 General Information

1.2.1 Calcium
Chemistry
Calcium is an alkaline earth metal belonging to Group II of the periodic table. It is a divalent
cation with an atomic weight of 40. Calcium shows a single oxidation state of Ca+2.
Natural occurrence
Calcium does not exist freely in nature, but occurs abundantly as limestone (CaCO3), gypsum
(CaSO4.2H2O), and fluorite (CaF2). Many calcium compounds (e.g. fluorspar and calcium
carbonate) are almost insoluble in water, but there are exceptions (e.g. calcium chloride and
calcium nitrate). Within this risk assessment, the word calcium refers to ionic calcium, except
where specific calcium compounds are described.
Recommended amounts
Dietary Reference Values are based on calcium requirements for bone formation, minimized
bone resorption and retention of calcium. Recommendations vary for different stages of growth
and reproduction (COMA, 1998).A Lower Reference Nutrient Intake (LRNI) of 240 mg/day has
been calculated for infants. However, absorption from infant formula is less than from breast
milk (40% as opposed to 66%) and so a RNI of 525 mg/day is recommended. The calcium intake
required for healthy bone formation and skeletal growth increases from the age of 1 to 10 years;
RNIs of 350, 450 and 550 mg/day are recommended for children of ages 1-3, 4-6 and 7-10 years
respectively. For adolescents (11-18 years) RNIs are 800 mg/day for females and 1000 mg/day
for males, due to an increased requirement for calcium at a time of increased skeletal growth.
RNIs for adults are based on calcium loss and retention and are 700 mg/day. No additional intake
is considered necessary for pregnancy, although an increase of 550 mg/day over the RNI is
recommended during lactation.
Function
In the vertebrate skeleton, calcium provides rigidity in the form of calcium phosphate
(Ca10(OH)2(PO4)6,also known as hydroxyapatite), embedded in collagen fibrils. Calcium is also a
key component in the maintenance of cell structure.
Membrane rigidity, viscosity and permeability are partly dependent on local calcium
concentrations. Calcium fulfills important physiological roles as a cofactor for many enzymes

Page | 5
(e.g. lipase), as an important component of the blood clotting mechanism and through an active
role as an intracellular signal. Changes in intracellular calcium concentration, in response to a
physiological stimulus, such as a hormone or neurotransmitter, can act as an intracellular signal.
This signaling controls events such as cell aggregation, muscle contraction and cell movement,
secretion, transformation and cell division, as well as muscle protein degradation.
Deficiency
A negative calcium balance occurs when net calcium absorption fails to compensate for urinary
calcium losses. Calcium absorption is impaired in individuals with conditions of fat
malabsorption (e.g. in syndromes such as pancreatic insufficiency, bile duct obstruction and
coeliac disease) because of the lack of vitamin D as a transporter. Acute hypocalcaemia is
frequently seen following parathyroid surgery and may occur during thyroid surgery through
inadvertent interference with the parathyroid glands. The possible effects of calcium deficiency
are numerous and wide-ranging. Signs of calcium deficiency are manifest in the bones and teeth
of all young animal species, including humans. Effects include stunted growth, poor quality
bones and teeth, and bone malformation.
Excretion
Calcium excretion tends to equal intestinal calcium absorption in adults of a good nutritional
state. Absorbed calcium is excreted in the urine, and to a lesser extent, in the sweat. Urinary
calcium excretion varies widely among individuals, ranging from 100-200 mg/day. Faecal
excretion of calcium consists mainly of unabsorbed dietary calcium, the remainder coming from
shed epithelial cells and digestive juices.
1.2.2 Iron
Chemistry
Iron, a transition metal, is ubiquitous in biological systems. In solid form the element exists free,
or in iron-containing compounds. In aqueous solution, it exists in one of two oxidation states,
Fe2+, the ferrous form, and Fe3+, the ferric form. Iron has a particularly high redox potential
insolution. Within this risk assessment, the word iron refers to ionic iron, except where specific
iron compounds are mentioned.

Natural occurrence
Iron is found in certain minerals, and in nearly all soils and in mineral waters

Page | 6
Recommended amounts
Estimated average daily iron requirements in the UK are 8.7 and 6.7 mg for males aged 11-18
and 19+ years, respectively (COMA, 1991). For women in the 11-50 years age group the
estimated average daily iron requirement is 11.4 mg, whilst that for postmenopausal (50+ years)
women is 6.7 mg. Estimated average daily requirements for children are 1.3 mg (0-3 months),
3.3 mg (4-6 months), 6.0 mg (7-12months), 5.3 mg (1-3 years), 4.7 mg (4-6 years) and 6.7 mg(7-
10 years). It has been estimated that the total amounts of iron required for a full gestation is 680
mg. Existing body iron stores should provide this requirement, assuming adequate iron stores at
conception, cessation of menstruation and increased intestinal absorption throughout gestation.
Function
The majority of functional iron within the body is present in haem proteins, such as hemoglobin,
myoglobin and cytochromes, which are involved in oxygen transport or mitochondrial electron
transfer. Many other enzymes also contain or require iron for their biological function.
Total body iron averages approximately 3800 mg in men and 2300 mg in women.
Approximately one third of body iron in men and one eighth in women is in the form of storage
iron. Iron is stored mainly in the liver within the iron storage proteins, ferritin and hemosiderin.
Small amounts of ferritin are also present in the plasma, although there are a number of
differences from tissue ferritin and plasma has low iron content, even in iron-overloaded
individuals. Many of the key biological functions of iron in living systems rely on the high redox
potential, enabling rapid conversion between the Fe2+ and Fe3+ forms. The redox potential is,
however, also potentially harmful in terms of the capacity for oxidative damage to cellular
components such as fatty acids, proteins and nucleic acids. However, iron within the body
(whether it is being stored, transported or as a component of various catalytic pathways) is
normally bound to carrier proteins and/or molecules with antioxidant properties, which minimize
the capacity of the free ion to cause oxidative stress.
Deficiency
Iron deficiency generally develops slowly, and may not be clinically apparent until iron stores
are exhausted and the supply of iron to the tissues is compromised, resulting in iron-deficiency
anemia. Groups that are vulnerable to iron deficiency include: infants over 6 months, toddlers,
adolescents and pregnant women (due to high requirements); older people and people consuming

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foods high in iron absorption inhibitors (due to poor absorption); menstruating women or
individuals with pathological blood loss (due to high blood losses).
Excretion
Little of the absorbed iron is excreted. Very small losses occur in the faeces, by desquamation of
gastrointestinal cells, in hemoglobin and bile, and via the urine. Substantial iron loss can occur
through loss of blood. Average, total daily iron losses for healthy adults are 1.0 mg for men and
1.3mg for premenopausal women (assuming an average blood loss of 30 – 40 mL per menstrual
cycle). Daily iron losses for children have not been measured directly but are estimated as 0.2
and 0.5 mg for infants and children aged 6 – 11 years, respectively.
1.2.3 Magnesium
Chemistry

Magnesium is a metallic element of group 2 of the periodic table and has an atomic weight of
24.3.

Natural occurrence
Magnesium is the eighth most abundant element in the earth’s crust. It does not occur as a pure
metal in nature, but it is found in large deposits as magnesite, dolomite and other minerals. In
this risk assessment the word ‘magnesium’ refers to ionic magnesium, except where specific
magnesium salts are described.
Recommended amounts
COMA has calculated a RNI of 300 mg/day for adult males and 270 mg/day for adult females
(COMA, 1991). The RNI for infants and children ranges from 55 to 280mg/day. An RNI for
magnesium during pregnancy was not calculated by COMA.
Function
Magnesium is required as a cofactor for many enzyme systems. It is required for protein
synthesis and for both anaerobic and aerobic energy generation and for glycolysis, either
indirectly as a part of magnesium-ATP complex, or directly as an enzyme activator. Magnesium
plays a multifunctional role in cell metabolism, (particularly at the level of key phosphorylation),
and has a critical role in cell division. It has been suggested that magnesium is necessary for the
maintenance of an adequate supply of nucleotides for the synthesis of RNA and DNA.
Magnesium regulates the movement of potassium in myocardial cells and is also known to act as

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a calcium channel blocker. Magnesium is an important element in the metabolism and/or action
of vitamin D, and is essential for the synthesis and secretion of parathyroid hormone.
Deficiency
Magnesium deficiency has been linked to cardiovascular, skeletal, gastrointestinal and central
nervous system disorders and to the use of loop diuretics. Magnesium is essential for the normal
function of the parathyroid gland and for vitamin D metabolism.
Magnesium depletion markedly disturbs calcium homeostasis, and hypocalcaemia is a common
manifestation of moderate to severe magnesium deficiency.
Excretion
Magnesium is excreted primarily in the urine. The extent of urinary excretion, and thereby the
homeostasis of magnesium, is influenced by a wide variety of hormones, including calcitonin,
thyroxin, glucocorticoids, glucagon and angiotensin. Under normal conditions, the kidney tubule
reabsorbs 95% of the filtered load of magnesium and about 5% is excreted in urine.

1.2.4 Phosphorus
Chemistry
Phosphorus is a group 5 element of the periodic table and has an atomic weight of 30.97. Within
this risk assessment, the phosphorus refers to ionic phosphorus except where specific phosphorus
compounds are mentioned.
Natural occurrence
Phosphorus is most commonly found in nature in its pentavalent form in combination with
oxygen, as phosphate (PO43-).
Recommended amounts
COMA (1991) noted that phosphorus requirements are conventionally set as equal to calcium, in
mass terms i.e. 1 mg phosphorus : 1 mg calcium. However, these elements are present in the
body in equimolar amounts and COMA took the view that the ratio in the diet should be set at
1mmol phosphorus: 1mmol calcium and considered that the Recommended Nutrient Intake
(RNI) for phosphorus should be set equal to the calcium RNI in molar terms (1mmol calcium =
40 mg, 1mmol phosphorus = 30.9 mg). COMA therefore calculated a RNI of 550 mg/day of
phosphorus for males and females aged 19-50 years. COMA derived an increment of 440

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mg/day for women during lactation, giving a total of 990 mg/day. The RNI for infants and
children ranges from 400 to 775 mg/day.
Function
Phosphorus is a constituent of all major classes of biochemical compounds. Structurally,
phosphorus occurs as phospholipids, which are a major constituent of most biological
membranes, and as nucleotides and nucleic acids. Phosphorus plays an important role in
carbohydrate, fat and protein metabolism and is essential for optimum bone health. The energy
that is required for most metabolic processes is derived from the phosphate bonds of adenosine
triphosphate and other high energy phosphate compounds.
Clinical studies employing chronic phosphorus supplementation were the first to show that high
phosphorus intakes influence the parathyroid-vitamin D axis, which maintains calcium balance
in the body. The phosphorus loading in humans operates through mechanisms of nutritional or
secondary hyperparathyroidism similar to those observed in animals fed excess phosphorus.
Deficiency
Factors associated with phosphorus deficiency (hypophosphatemia) include liver disease, sepsis,
alcoholism, diabetic ketoacidosis and the use of aluminium-containing antacids. The symptoms
of a potentially fatal syndrome include anorexia, anemia, muscle weakness, bone pain, rickets
and ataxia. Hypophosphatemia in infants is known to occur in situations of poorly managed
parenteral nutrition with inappropriate administration of fluid and electrolyte therapy or with
rapid refeeding after prolonged dietary restriction. Preliminary studies have indicated that
phosphate deficiency at birth is associated with the development of rickets in later life.
Excretion
Phosphorus is primarily excreted in the urine. The regulation of phosphorus excretion is apparent
from early infancy. In infants, as in adults, the major site for the regulation of the amount of
phosphorus retained by the body is the kidney.

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1.2.5 Potassium
Chemistry
Potassium is an alkaline, metallic element. It is not found in the elemental form in nature and is
always found combined with other substances, most commonly as the chloride salt (KCl).
Potassium is widely distributed in silicate rocks, and occurs in salt beds and seawater. Within
this risk assessment, the word potassium refers to ionic potassium, except where specific
potassium compounds are stated.
Recommended amounts
The RNI for potassium is 3500 mg/day for adults (over 18 years) of both sexes (COMA, 1991).
There is no increased requirement during pregnancy or lactation.
Function
Potassium, together with sodium, is essential for the maintenance of normal osmotic pressure
within cells. About 98% of the total body potassium is located intracellularly where the
concentration can be 30 times that of the extracellular concentration. The extracellular potassium
concentration is a critical determinant of neuromuscular excitability. Potassium is also a cofactor
for numerous enzymes and is required for secretion of insulin by the pancreas, for
phosphorylation of creatine and for carbohydrate metabolism and protein synthesis.
Deficiency
Hypokalemia most commonly results from increased loss of the element, secondary to diarrhea,
diabetic acidosis, vomiting, intense and prolonged sweating, body burns or diuretic drugs.
Rarely, ‘crash’ or very low calorie diets can result in reduced intake, sufficient to cause
potassium deficiency. Hypokalemia can cause rapid and irregular heart rhythm, muscle weakness
and irritability, occasional paralysis, nausea and vomiting, diarrhea and low muscle tone in the
gut, and has been reported to predispose to hypertension.
Excretion
The major excretory route of potassium is via the kidneys. It is secreted by the renal tubules,
inexchange for sodium of the glomerular filtrate (ion exchange mechanism).
Excretion in sweat and faeces is negligible; the latter changing only slightly as dietary potassium
intake varies over a wide range.

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 Chapter Two

Literature Review

Page | 12
Literature Review
In the past 20 years, micronutrients have assumed great public health importance. As a
consequence, considerable research has been carried out to better understand their physiological
role and the health consequences of micronutrient-deficient diets, to establish criteria for
defining the degree of public health severity of micronutrient malnutrition, and to develop
prevention and control strategies. One of the main outcomes of this process is greatly improved
knowledge of human micronutrient requirements, which is a crucial step in understanding the
public health significance of micronutrient malnutrition and identifying the most appropriate
measures to prevent them. This process also led to successive expert consultations and
publications organized jointly by the Food and Agriculture Organization of the United Nations
(FAO), the World Health Organization (WHO) and the International Atomic Energy Agency
(IAEA) providing up-to-date knowledge and defining standards for micronutrient requirements
in 19731, 19882 and in 1996 3. In recognition of this rapidly developing field, and the substantial
new advances that have been made since the most recent publication in 1996, FAO and WHO
considered it appropriate to convene a new expert consultation to re-evaluate the role of
micronutrients in human health and nutrition. To this end, background papers on the major
vitamins, minerals and trace elements were commissioned and reviewed at a Joint FAO/WHO
Expert Consultation (Bangkok, 21–30 September 1998).

The present report presents the outcome of the Consultation combined with up-to-date evidence
that has since become available to answer a number of issues which remained unclear or
controversial at the time of the Consultation. It was not originally thought that such an evidence-
based consultation process would be so controversial, but the reality is that there are surprisingly
few data on specific health status indicators on which to base conclusions, whereas there is a
great deal of information relative to overt deficiency disease conditions. The defining of human
nutrient requirements and recommended intakes are therefore largely based on expert
interpretation and consensus on the best available scientific information. When looking at
recommended nutrient intakes (RNIs) in industrialized countries over the last 25 years, it is
noticeable that for some micronutrients these have gradually increased. The question is whether
this is the result of better scientific knowledge and understanding of the biochemical role of the
nutrients, or whether the criteria for setting requirement levels have changed, or a combination of

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both. The scientific knowledge base has vastly expanded, but it appears that more rigorous
criteria for defining recommended levels is also a key factor.

RNIs for vitamins and minerals were initially established on the understanding that they are
meant to meet the basic nutritional needs of over 97% of the population. However, a
fundamental criterion in industrialized countries has become one of the presumptive role that
these nutrients play in “preventing” an increasing range of disease conditions that characterize
affected populations. The latter approach implies trying to define the notion of “optimal
nutrition”, and this has been one of the factors nudging defined requirements to still higher
levels. This shift in the goal for setting RNIs is not without reason. The populations that are
targeted for prevention through “optimal nutrition” are characterized by sedentary lifestyles and
longer life expectancy. The populations in industrialized countries are ageing, and concern for
the health of the older person has grown accordingly. In contrast, the micronutrient needs of
population groups in developing countries are still viewed in terms of millions experiencing
deficiency, and are then more appropriately defined as those that will satisfy basic nutritional
needs of physically active younger populations. Nevertheless, one also needs to bear in mind the
double burden of under- and over nutrition, which is growing rapidly in many developing
countries. Concern has been raised about possible differences in micronutrient needs of
populations with different lifestyles for a very practical reason. The logic behind the
establishment of micronutrient needs of industrialized nations has come about at the same time
as a large and growing demand for a wide variety of supplements and fortificants, and
manufacturers have responded quickly to meet this market. This phenomenon could easily skew
national strategies for nutritional development, with an increased tendency to seek to resolve the
micronutrient deficiency problems of developing countries by promoting supplements and
fortification strategies, rather than through increasing the consumption of adequate and varied
diets. Higher levels of RNIs often set in developed countries can easily be supported because
they can be met with supplementation in addition to food which itself is often fortified. In
contrast, it often becomes difficult to meet some of the micronutrient needs in some populations
of developing countries by consuming locally available food, because foods are often seasonal,
and neither supplementation nor fortification reach vulnerable population groups. Among the
nutrients of greatest concern is calcium; the RNI may be difficult to meet in the absence of dairy
products.

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The recently revised United States/Canada dietary reference intakes (DRIs) propose only an
acceptable intake (AI) for calcium instead of a recommended daily allowance (RDA) in
recognition of the fact that intake data are out of step with the relatively high intake requirements
observed with experimentally derived values.29

Another nutrient of concern is iron, particularly during pregnancy, where supplementation

appears to be essential during the second half of pregnancy.

In Great Britain, products that are not regulated as medicines may be regarded as foods and, as
such, are subject to the general provisions of the Food Safety Act 1990, the Food Labeling
Regulations, 1996 (as amended) and the Trade Descriptions Act 1968. Northern Ireland has
similar but separate legislation. Food products containing added vitamins and minerals can be
divided into two categories: fortified foods, such as margarine and breakfast cereals, and food
supplements. At present there are no rules on voluntary fortification of foods at EU or at UK
level. Voluntary fortification of foods is permitted in the UK provided that the final foodstuff is
safe and appropriately labeled. Similarly, until the EU Food Supplements Directive is
implemented in national law in 2003, there is currently no definition of the term ‘food
supplement’ in UK food law and there are no specific regulations that apply to food supplements.
Fortified foods and food supplements fall within the scope of the Food Safety Act 1990, which
makes it an offence to sell any food that is not safe for consumption, not of the nature, substance
or quality demanded by the consumer or that is falsely or misleadingly described or labeled. At
present there are no specific limits on the levels of vitamins, minerals or other micronutrients
which may be contained in supplements or fortified foods sold under food law, nor are there
rules on the range of vitamins, minerals or other nutrients30that they may contain.

Until July 2002, when EU Directive 2002/46/EC on food supplements came into force, there
were no harmonized controls on food supplements within the EU and each Member State applied
its own rules.

For example, many EU Member States, such as France, Spain and Italy, did not have specific
legislation but usually treated products containing more than a certain multiple of the
Recommended Daily Amount (RDA) (equivalent to the UK Reference Nutrient Intake (RNI)) as

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medicines. In other Members States, such as Denmark and Germany, products containing the
RDA or a small multiple of it were acceptable as foods, but higher doses required licensing.

EU Member States such as the Netherlands, with liberal safety-based regimes similar to that in
the UK, were in the minority. In these Member States, some limits were applicable to particular
vitamins and minerals, and these were generally based on risk assessment. Directive 2002/46/EC
on food supplements was adopted on 30th May 2002 and published in the Official Journal of the
European Communities on 12th July 2002.Article 15 states that it must be implemented into
Member States’ law by 31st July 2003. The Directive establishes a framework for harmonized
legislation covering the composition (at present limited to the vitamins and minerals that can be
used) and labeling of food supplements. It also establishes a framework for setting maximum
limits of vitamins and minerals in food supplements, although no such limits have yet been set.
In 1998, the Scientific Committee on Food (SCF), a committee of independent scientific experts
which advises the European Commission, established a Task Force on Upper Levels to undertake
a series of evaluations similar to those conducted by the EVM. Those opinions adopted by the
SCF plenary group are published on the SCF website
(http://europa.eu.int/comm/food/fs/sc/scf/index_en.html). The European Commission will base
its future proposal for EU rules on maximum limits of vitamins and minerals in food
supplements on the advice of the SCF and its interpretation of Article 5 of the Food Supplements
Directive. The European Commission published an informal draft proposal for a Directive
relating to the addition of nutrients to foods in June 2000, which was discussed by Member
States at a Commission working group in September 2000. The Commission followed this up in
January2003 by publishing a preliminary draft proposal for a Regulation on the addition of
vitamins and minerals and of certain other substances to foods.

The Expert Group on Vitamins and Minerals (EVM) is an independent expert advisory
committee which was asked to advice on safe levels of intakes of vitamins and minerals in food
supplements and fortified foods. The EVM was asked to consider only vitamins and minerals
sold under food law. Review of nutritional or non-nutritional beneficial effects or non-nutritional
use in medicines was outside the terms of reference of the group. The use of vitamins and
minerals in medicines is within the remit of the Medicines and Healthcare products Regulatory

Page | 16
Agency (MHRA). The EVM considered a detailed review of the relevant evidence for each
substance and then a risk assessment which was based on this evidence.

The detailed reviews covered both nutritional and toxicological information. Thirty-four
substances were assessed in detail. Safe Upper Levels (SULs) were recommended for eight
vitamins and minerals and guidance was issued for twenty-two. Recommendations for SULs
made by the EVM depend on the availability of good data on both the nature and the frequency
of adverse effects detected at different levels of intake. The database supporting the safety-in-use
of vitamins and minerals is generally poor.

There is a particular lack of reasonable-sized, well designed comparative human studies of

significant duration at different levels of intake, and with adequate assessment of possible
adverse effects. The data were inadequate to establish either SULs or guidance. Two minerals
were not considered in detail: sulphur was excluded because intake is mainly as amino acids;
fluoride was excluded as supplements are only available as licensed medicines, and fortification
of food as a public health measure was not within the terms of reference of the EVM. The EVM
was not asked to consider the beneficial effects of the vitamins and minerals. Nutritional need
was noted and SULs were not set at a level below dietary requirements. Some trace elements
available in food supplements are unnecessary for human nutrition. Food supplement products
are used by consumers in a number of ways; these may include a desire to improve the perceived
nutritional quality of their diet or self-medication (either as prophylaxis or treatment) for a range
of symptoms or conditions. The remit of the EVM was to consider supplements sold under food
law, thus, the non-nutritional efficacy of vitamins and minerals has not been considered since
such effects would be classified as medicinal and would be within the remit of the Medicines and
Healthcare products Regulatory Agency. It has been argued that since vitamins and minerals are
essential for human health, it is not appropriate to assess them in the same way in which other
chemicals added to food are assessed. However, since there is much evidence that excessive
intakes of some vitamins and minerals can cause harm, it is not appropriate to exclude essential
nutrients from the safety assessment that is applied to other chemical substances which are added
to foods. The terms of reference of the EVM included the opportunity to advice on fortified
foods. However, the EVM considered it preferable to frame its advice in terms of additional

Page | 17
intake, which would cover both supplements and fortified foods. The SUL for a nutrient is
expressed in two ways, depending upon the evidence base from which it was derived.

In some cases the SULs are expressed as total intake, with an estimated safe margin for intake
from both supplements and fortification within current high level dietary intake. In other cases,
the SUL is expressed as a supplemental intake with an estimated safe total intake. This
inconsistency arises from the nature of the data available on different vitamins and minerals, and
the limited nature of the evidence base. The data used for the dietary exposure assessments were
generally taken from a National Diet and Nutrition Survey conducted in UK adults in 1986/7,
which, despite its age, is the most comprehensive data set currently available31. The market for
both fortified foods and food supplements is changing and expanding.

Where intake data are available these may now be somewhat outdated. It was not possible for the
EVM to estimate how the market is likely to expand in the future, although this will be an
important consideration for risk managers when providing advice based on the SULs that have
been established. As the market and thus the exposure changes, risk managers giving advice
based on these SULs will need to consider scenarios where individuals may be exposed to high
levels of certain micronutrients depending on the combinations of products consumed. This
changing pattern of micronutrient addition to different foods and of levels of consumption should
thus be kept under review. In the context of assessing intake, the EVM wishes to draw attention
to the practice of using ‘overages’ within the food supplement industry. ‘Overage’ means the
formulation of a product with more than the declared content of a particular nutrient to ensure
that there is a sufficient level of the vitamin or mineral throughout the shelf-life of the product.
This is particularly common with the fat soluble vitamins (for example vitamins A, D and E) as
they are prone to oxidation. It should therefore be borne in mind that some supplements may
have significantly higher levels of the vitamins and minerals at the start of their shelf-life than is
indicated on the product label. It is also essential that consistent, validated methods for the
analysis of levels of vitamins and minerals in supplements and fortified foods are available,
enabling the quoted levels to be independently verified.

In the UK, products containing added vitamins and minerals can be regarded in law as either
foods or medicines32.Those products for which claims are made for the treatment or prevention
of disease, or which are administered to restore, correct or modify physiological functions, fall

Page | 18
within the definition of a ‘relevant medicinal product’ and are subject to the requirements of the
Medicines Act 1968, Directive 65/65/EEC.

There are many deficiency syndromes of such minerals. A negative calcium balance occurs when
net calcium absorption fails to compensate for urinary calcium losses. Calcium absorption is
impaired in individuals with conditions of fat malabsorption (e.g. in syndromes such as
pancreatic insufficiency, bile duct obstruction and coeliac disease) because of the lack of vitamin
D as a transporter. Acute hypocalcaemia is frequently seen following parathyroid surgery and
may occur during thyroid surgery through inadvertent interference with the parathyroid glands.
The possible effects of calcium deficiency are numerous and wide-ranging.
Signs of calcium deficiency are manifest in the bones and teeth of all young animal species,
including humans. Effects include stunted growth, poor quality bones and teeth, and bone
malformation. Iron deficiency generally develops slowly, and may not be clinically apparent
until iron stores are exhausted and the supply of iron to the tissues is compromised, resulting in
iron-deficiency anemia. Groups that are vulnerable to iron deficiency include: infants over 6
months, toddlers, adolescents and pregnant women (due to high requirements); older people and
people consuming foods high in iron absorption inhibitors (due to poor absorption); menstruating
women or individuals with pathological blood loss (due to high blood losses).Magnesium
deficiency has been linked to cardiovascular, skeletal, gastrointestinal and central nervous
system disorders and to the use of loop diuretics. Magnesium is essential for the normal function
of the parathyroid gland and for vitamin D metabolism. Magnesium depletion markedly disturbs
calcium homeostasis, and hypocalcaemia is a common manifestation of moderate to severe
magnesium deficiency. Factors associated with phosphorus deficiency (hypophosphatemia)
include liver disease, sepsis, alcoholism, diabetic ketoacidosis and the use of aluminium-
containing antacids. The symptoms of a potentially fatal syndrome include anorexia, anemia,
muscle weakness, bone pain, rickets and ataxia. Hypophosphatemia in infants is known to occur
in situations of poorly managed parenteral nutrition with inappropriate administration of fluid
and electrolyte therapy or with rapid refeeding after prolonged dietary restriction. Preliminary
studies have indicated that phosphate deficiency at birth is associated with the development of
rickets in later life. Hypokalemia most commonly results from increased loss of the element,
secondary to diarrhea, diabetic acidosis, vomiting, intense and prolonged sweating, body burns

Page | 19
or diuretic drugs. Rarely, ‘crash’ or very low calorie diets can result in reduced intake, sufficient
to cause potassium deficiency.
Hypokalemia can cause rapid and irregular heart rhythm, muscle weakness and irritability,
occasional paralysis, nausea and vomiting, diarrhea and low muscle tone in the gut, and has been
reported to predispose to hypertension.
The EVM has been keen to involve interested parties in its work. It has sought to achieve this
through the activities of the observers and by issuing a call for information at the beginning of
the task. As described above, the draft reviews concerning the hazard identification and
characterization were posted on the EVM website soon after completion for the consideration of
interested parties, with a request for details of any relevant data not considered in the review.
Since very limited data were available concerning the effects of high intakes of some
micronutrients, the EVM particularly sought additional relevant information from a wide
audience to ensure identification of as complete an evidence base as possible. Very little
information or comment was received until the draft report was published for consultation. This
sequential procedure and the separate derivation of daily maximum levels for food supplements
and fortified foods aims to take account of multiple exposure which may result from the daily
parallel consumption of both product categories (food supplements, fortified foods) and also of
the parallel daily consumption of several products within a category (e.g. consumption of several
food supplements per day). At the same time, this procedure aims to facilitate the flexible
handling of multiple exposures and to reflect the specificity's of food supplements and fortified
foods. Differences between the two categories result from the fact that food supplements contain
nutrients in dosed form (e.g. capsules or tablets) and must carry information about recommended
daily intake along with a warning not to exceed the stipulated daily dose. In contrast, the
consumption of fortified foods is not based on the amount of vitamins and minerals contained
therein but are mainly determined by factors like hunger, thirst, appetite and availability. In
contrast to the situation with food supplements, consumption recommendations are not usual or
could not be complied with. In addition, appropriate consideration must be given to the fact that
vitamins and/or minerals may be added to a wide range of processed foods33. Regarding hazard
characterization, there are considerable gaps in knowledge about some nutrients. Although
animal studies are available for the derivation of NOAEL and/or LOAEL, their transferability to
man is unsure and very few studies in human beings are available.

Page | 20
In some cases there are major differences between the individual bioavailability of nutrients and
often a toxicological assessment is only possible of the amounts taken in via supplements and not
of total daily intake34. Moreover, there may be interactions amongst various nutrients or with
other food components that have to be taken into account. Furthermore, consideration must also
be given to differences in gender and age as well as special physiological conditions and
specificities in dietary habits35.

Older people may be vulnerable to potassium toxicity due to reduced physiological reserve in
renal function. Individuals with pre-existing renal disease, hyperkalemia, adrenal insufficiency,
acidosis or insulin deficiency are also vulnerable, as are those using certain drugs, such as
potassium-sparing diuretics, ß-adrenergic blockers, angiotensin convering enzyme (ACE)
inhibitors, digitalis, non-steroidal anti-inflammatory drugs, arginine hydrochloride and
succinylcholine. Infants may be vulnerable to excessive potassium due to limited renal reserve
and immature function.

Patients with renal failure are particularly susceptible to developing hypercalcaemia when taking
calcium supplements. Individuals without renal failure taking diuretics may also be at increased
risk.

Patients with absorptive or renal hypercalcuria, primary hyperparathyroidism and sarcoidosis

may have a higher risk of renal stone formation following calcium supplementation. Some
studies are carried on particular importance of various minerals such as calcium, iron,
magnesium, phosphorus, potassium etc. They are discussed below:

Calcium carbonate tablets were well tolerated by participants in this intervention study. No
mention of side effects was made when individuals were specifically asked about them.
Individuals received 1500mg calcium/day for twelve weeks1.
This paper reports two cases of milk-alkali syndrome. Both cases showed classic MAS
symptoms, i.e. hypercalcaemia and renal insufficiency. The authors estimated that the patients
had ingested 9000 mg and 6000 mg of calcium carbonate (3600 and 2400 mg calcium) per day,
respectively. These are the first reports of MAS not caused iatrogenically2.

Page | 21
Supplementation of 2,295 pregnant women with 2,000 mg calcium/day (as calcium carbonate)
resulted in no significant increase in the incidence of urolithiasis. This study excluded women
with a history of, or a high risk of, developing renal disease3.

116 patients received 1,600 mg calcium/day (as calcium carbonate) or placebo for 18 months.
Five patients in the treatment group experienced constipation compared to one in the controls.
Diarrhea was reported in 5 calcium and 7 placebo subjects and bloating (8 patients) was equally
distributed between groups4.

No toxicity was associated with calcium supplementation during a randomized double blind,
placebo controlled trial on patients with a history of colorectal adenomas. The 930 received 3000
mg of calcium carbonate/day (equivalent to 1,200 mg calcium per day) and were examined after
1 and 4 years of treatment5.

665 adult patients with a history of polyps were randomized into 3 groups receiving 2000 mg
calcium/day (as calcium gluconolactate and carbonate) fiber or placebo for 3 years. 552 patients
completed the follow up examination after 3 years, although 94 of these had stopped treatment
early. The total number of patients reporting side effects were 26/176, 19/198 and 12/178 in the
calcium, fiber and control groups, respectively. Major side effects (severe abdominal pain or
diarrhea) were also reported to be more frequent (6/176) in the calcium group compared to the
control (3/178) or fiber (3/198) groups, which might have been a chance finding6.
The effect of maternal hypercalcaemia during pregnancy and lactation on the development of the
offspring was investigated. Rats were maintained on high calcium diets, containing 3% calcium
in diet and 4000 mg/100 mL drinking water (equating to an estimated total intake of 3790
mg/day), throughout pregnancy and lactation. In comparison to controls (receiving 0.8% calcium
in diet and 1.1 mg calcium/100 mL drinking water), the offspring of treated rats were born with
significant hypocalcaemia and had lower growth rates and focal alopecia. These effects were
reversible when the pups were weaned onto a normal diet7.
The effect of moderately increased dietary calcium on fetal development was investigated in
pregnant rats. The doses were selected to resemble the increases recommended by the 1984 US
National Institutes of Health Consensus Development Conference Panel on Osteoporosis. All

Page | 22
animals were fed nutritionally adequate diets and received 0.5 (control), 0.75, 1.00 or 1.25%
dietary calcium as calcium carbonate. Treatment was for 6 weeks prior to mating, during mating
and for the first 20 days of gestation. Fetal bodyweights and lengths remained similar between
treatment and control groups. There were no significant increases in external, visceral or skeletal
variations of the fetuses, when compared to the control animals8.

In this study 132 pregnant women were given 105 mg iron (form not stated) plus 500 mg
ascorbic acid, with or without 350 mg folic acid for 90 days. 14 % of subjects reported severe
gastrointestinal effects (diarrhea or constipation) and 16% minor effects. There was no difference
in incidence between the groups, but there was no placebo group9.

The tolerability of supplemental iron delivered from a wax-matrix tablet of ferrous sulphate was
compared to that from a conventional ferrous sulphate tablet in a single blind, parallel group
study. Both tablets delivered 50 mg of iron. The incidence of adverse gastrointestinal effects was
significantly greater amongst subjects taking the conventional tablets, than amongst those taking
the wax-matrix preparation. Of those taking the wax-matrix formulation only 19% experienced a
severe or moderate adverse effect (306 reports), compared to 50% (1021 reports) of those taking
the conventional tablets10.

The tolerability of supplemental iron (50 mg iron) in the chelated form of bis-glycino iron was
compared with that of ferrous sulphate in a randomized double-blind cross-over trial. Of the 38
participants, 37%experienced moderate to severe adverse gastrointestinal effects whilst taking
ferrous sulphate, compared to 21% who experienced similar side effects whilst taking the chelate
formulation 11.

In a controlled double-blind cross-over trial, haem and non-haem iron were administered to 100
healthy volunteers for periods of 1 month each. Groups of participants were given one of two
different iron preparations: Group 1: two tablets of 1.2 mg haem iron from porcine blood, plus 8
mg non-haem iron as iron fumarate. Group 2: one tablet of 60 mg iron as iron fumarate.

The study was divided into three consecutive periods of one month each and all participants
received a placebo for one of the last two periods. Participants assessed side effects by keeping
individual symptom diaries; a multiple-choice questionnaire was used for daily evaluation with

Page | 23
listing of effects known to be related to iron therapy. The number of reports of obstipation and
the total side effects were significantly higher for the nonhaem iron treatment, than for the haem
iron or placebo. Constipation was reported in 35% and all gastrointestinal side effects were
reported by 25% of participants during the non-haem iron phase of the trial. The effects reported
for the haem iron treatment were indistinguishable from the placebo 12.

In a prospective, controlled, double-blind, two-placebo controlled, multicentre trial the

tolerability of iron protein succinylate (ITF 282) was compared with a ferrous sulphate
controlled release tablet. 1095patients were randomized to receive either two ITF 282 tablets per
day (60 mg iron per tablet) or one controlled release ferrous sulphate tablet per day, containing
105 mg iron, both treatments lasted 60days. The general tolerability was reported to be favorable
with both treatments, but significantly more favorable with ITF 282. With ITF 282, 63 patients
(11.5%) complained of 69 adverse reactions (25heartburn, 19 constipation, 25 abdominal pain),
compared with 141 adverse events (33 heartburn, 31epigastric pain, 23 constipation, 32
abdominal pain, 8 skin rash, 14 nausea) reported by 127 (26.5%) of those taking ferrous
sulphate 13.

In a randomized, double-blind crossover study, 19 young women received a dose of 120 mg/day
(2 x 60)iron as ferrous fumarate or placebo for 2 periods of 8 weeks. Seven participants
experienced gastrointestinal discomfort, 2 while taking placebo14.

No adverse effects were reported in this study in which 20 patients with duodenal ulcers received
upto 1200 mg of magnesium per day in the form of an aluminium-magnesium-
hydroxycarbonateantacid over a 6-week trial period in a randomized, prospective cross-over
clinical trial15.

Diarrhea and other gastrointestinal effects were reported in a number of individuals participating
in this randomized double-blind placebo-controlled cross-over study. The dose of magnesium in
this study was 470 mg/day (given as 800 mg/day magnesium oxide) for 60 days16.

This study investigated the effect of magnesium supplementation on blood pressure, erythrocyte
cation metabolism and serum lipid levels in 13 patients with mild hypertension. The study
employed a 3-weekplacebo run-in period and no adverse effects were reported following a dose
of 486 mg magnesium twice daily for three months (given as magnesium aspartate)17

Page | 24
In this double-blind cross-over study, doses of 393 mg/day magnesium (as magnesium pidolate)
were well tolerated by 12 older subjects (mean age 77.8 years) over a 4-week period18.

This was a randomized double-blind placebo-controlled cross-over trial, in which 21 patients

with stable congestive heart failure, secondary to coronary heart disease, received 384 mg
magnesium/day (as magnesium chloride) for 6 weeks. Gastrointestinal effects, including
diarrhea, were reported in 6 out of the21 subjects receiving magnesium19.

No diarrhea or gastrointestinal effects were reported in a long-term study in which a group of 31

postmenopausal women received daily supplements of magnesium hydroxide (providing up to
750 mg/day magnesium) for 6 months followed by 226 mg/day of magnesium for 18 months20.

No adverse effects were reported in 18 healthy 18-38 year old males who were given diets
enriched with magnesium oxide for 6 days, providing a daily dose of 452 mg magnesium21.

This was a study of the role of phosphate as an adjunct in the therapy of multiple myeloma.
Phosphate supplements (given as mixtures of sodium and potassium salts and providing a daily
dose of approximately 1000 to 2000 mg phosphorus) were given to 14 patients orally for up to
15 months, or intravenously (duration not stated). Reductions in bone pain and urinary calcium
were reported by the authors. None of the patients developed any evidence of extra-skeletal
calcification during follow-up periods of up to 15 months. One patient developed an increase in
pedal and pretibial edema, which subsided when the phosphate salts were discontinued. Another
patient complained of dyspepsia following the ingestion of phosphate supplements, although the
same subject reported similar discomforts with other medications including placebo22.

This was a study of the effect of a short course of oral phosphate treatment on serum parathyroid
hormone levels and markers of bone turnover. A group of postmenopausal women with reduced
bone mineral density and a history of at least one fracture were randomized in a double-blind
study and given oral phosphate (providing 750, 1500 or 2250 mg/day phosphorus) or placebo for
7 days; the subjects were followed for 4 months thereafter. The phosphate was administered as
effervescent tablets containing a mixture of ammonium phosphate, potassium phosphate and
glycerol phosphate. The urinary phosphate/creatinine ratio increased in a dose-related fashion
whereas no significant changes were seen in serum phosphate or serum calcium. Serum
parathyroid hormone rose significantly in the groups receiving 1500 and 2250 mg/day

Page | 25
phosphorus. No effect on serum parathyroid hormone levels could be demonstrated with the
lowest dose of phosphorus. Gastrointestinal side effects were noted in a dose-related fashion in 2
of 19 patients receiving 750 mg/day, in 3 of 19 patients receiving1500 mg/day and in 7 of 20
patients receiving 2250 mg/day23.

This was a report of two studies carried out to test the effect of supplementation with phosphate
on calcium homeostasis and bone turnover. Study 1 was a 1-week, randomized, controlled cross-
over trial involving 10 healthy men supplemented with 1000 mg phosphorus (as sodium acid
phosphate). The control diet for these men contained 800 mg/day of calcium and 800 mg/day of
phosphorus. Study 2(involving 12 healthy men) was an escalating dose study of 0, 1000, 1500
and 2000 mg/day phosphorus, given as phosphate for 1 week.

The control diet contained 1000 mg/day of calcium and 1000 mg/day of phosphorus as
phosphates. Both studies showed an increase in the urinary excretion of phosphate and a
decrease in urinary calcium. Serum levels of parathyroid hormone were only elevated in the first
study. There were no changes in serum phosphate, osteocalcin or urinary N-telopeptide. Diarrhea
was reported in one subject in study 2 when receiving phosphorus at 2000 mg/day24.

The effect of differing formulations of potassium chloride supplementation on the

gastrointestinal tracts of 48 healthy young men was investigated by endoscopy. Wax-matrix
potassium chloride preparations (providing 1250 mg potassium) three times a day for seven days
(total daily dose, 3700 mg potassium) resulted in considerable mucosal pathology, with erosions,
gastric ulcers, inflammatory lesions and bleeding at endoscopy. A microencapsulated form of
potassium chloride caused significantly fewer mucosal lesions25.

In a further study, 225 healthy male subjects received either potassium chloride as wax-matrix
tablets, in liquid form or microencapsulated, a potassium-sparer or placebo for one or two weeks.
The doses used were ranged from 900 – 3700mg potassium/day. Upper mucosal injury (erosions
and ulcerations) was more frequent after wax-matrix potassium chloride treatment. It was noted
that gastro-intestinal effects were mild and did not correlate with endoscopic evidence of lesions.
When hypertensive subjects were treated with 1560-3120 mg potassium/day) slow-release wax-
matrix potassium chloride for an average of21 months, 6/9 subjects developed erosions
compared with 1/9 matched controls. Following 7 days of in-patient treatment, one of the 6

Page | 26
subjects with erosions developed an ulcer and a further 2 placebo subjects developed erosions.
The authors concluded that cyto-adaptation to potassium chloride treatment did not seem to
occur26.

In a double blind study, groups of males aged 45-68 years were given 3700 mg potassium/day
(as potassium chloride) or a placebo (n = 148 in potassium group and 150 in placebo group).
Adverse effects reported in both groups after 12 weeks and two years included abdominal pains,
nausea and vomiting, diarrhea and bright red blood in the stools.

The effects were not investigated by endoscopy. The incidence was stated to be similar in the
two treatment groups27, 28.

A range of supplementation studies have been reported with supplementary calcium doses of up
to 2000 mg/day. These have generally failed to show adverse effects, but data on adverse effects
are limited. A study of supplementation of the diet of pregnant women with 2000 mg
calcium/day (as calcium carbonate) showed no significant increase in the incidence of
urolithiasis. Women with a history of, or high risk for, developing renal disease were excluded
from the study3.

A large number of studies involving oral iron supplementation have been reported. Analysis of
all reports was not possible, but was limited to those identified by database search as
randomized, controlled trials. Many of these studies were carried out with the aim to improve
iron status in population groups who were known or likely to be at risk of iron deficiency, such
as pre-school children, juveniles, adolescent girls and pregnant and non-pregnant women, often
in developing countries where such deficiency is common. Apart from gastrointestinal side
effects, such studies have generally not addressed issues of potential adverse effects associated
with oral iron supplementation. Supplementary doses of 100-200 mg iron/day and above have
been associated with nausea, vomiting and epigastric pain36, 37,38. Other studies have reported a
range of gastrointestinal effects, including diarrhea, nausea, vomiting, constipation and epigastric
pain, following supplementary doses of between 50 and 220 mg/day39, 14,9,10,11,13,12.
However, such effects were variable and appeared to vary depending on the formulation of the
iron supplement given, with fewer adverse effects reported by subjects given supplementary iron
as chelated iron or haem iron than by subjects given ferrous sulphate.

Page | 27
Several studies have suggested that excess iron intake could result in decreased serum zinc
levels. Decreased maternal serum zinc concentrations were reported following iron
supplementation at doses of more than 60 mg/day during pregnancy40,41,42 and (Dawson et al.
1989)43 reported that daily supplementation of pregnant women with 18 mg iron, in combination
with a multivitamin supplement (on average from 13 weeks of gestation to term), resulted in a
significant decrease in serum zinc level in the third trimester, compared with women who were
given an equivalent multivitamin supplement without iron. Conversely, (Sheldon et al. 1985)44
reported that supplementation with 480 mg/day ferrous fumarate (160 mg/day elemental iron),
from the first or second trimester of pregnancy to term, had no effect on maternal serum zinc
concentrations. As plasma zinc levels are not considered to be a good index of body zinc status
the significance of these findings is unclear. One study found an association between
supplementation with ferrous sulphate (equivalent to 60 mg elemental iron/day) during
pregnancy and reduced birth weight in women who were carriers for the sickle cell anemia
genotype45.

The predominant adverse reaction to orally administered phosphorus (as various phosphate salts,
including sodium, potassium, ammonium and glycerol) in human supplementation studies is
osmotic diarrhea, which has been reported at intakes of 750 mg/day and above. Other mild
gastrointestinal effects, including nausea and vomiting have been noted in some studies.

Numerous potassium supplementation studies have examined the association between increased
potassium intake and decreased risk of hypertension and heart disease. The majority of these
studies have shown beneficial effects of potassium supplementation. Although adverse effects
have not generally been reported, outside the gastrointestinal effect, it is often unclear whether
adverse effects were investigated. Although few details were given, it was stated that no adverse
effects were apparent in 18 subjects aged 66-79 given 2340 mg potassium (as potassium
chloride) for 4 weeks46 or in subjects aged 21-61 given 1900 mg potassium (form not stated) for
15 weeks47.
In a number of supplementation studies, potassium treatment resulted in ulceration of the
gastrointestinal tract, the severity of the effect related to the formulation of the treatment and to
factors such as gut transit time. Hyperkalemia also occurs as a result of potassium treatment.

Page | 28
Risk assessment
In humans, the main adverse effect associated with high levels of calcium intake is milk-alkali
syndrome, resulting in hypercalcemia, alkalosis and renal impairment. Symptoms can include
abdominal pain, hypertension, headaches and tissue calcification. The condition has been
reported in a small number of subjects taking calcium-containing medication. Previously, MAS
was more common in males taking absorbable alkali and milk, but is now more common in
females taking calcium-containing medication. Data on the effects of high doses of calcium in
animals are sparse. However tissue calcification and adverse reproductive effects have been
reported.
In humans acute iron poisoning is associated with severe gastrointestinal damage which may
include hemorrhagic gastroenteritis. Blood and other fluid loss may lead to shock and coma. In
some cases, apparent recovery may take place, possibly due to a latency period during which the
iron is distributed throughout the body. Systemic iron toxicity is characterized by multi-system
damage, principally in the liver, metabolic acidosis, coagulopathies and cardiovascular collapse.
Acute poisoning is relatively unusual in adults, the lethal dose being approximately 100 g, but is
more common in children. Iron overload as a result of dietary intake is unusual in the normal
population and only a handful of case reports exist describing this phenomenon. This may be due
to the reduction in iron absorption that occurs as exposure increases.
Individuals with conditions such as hereditary hemochromatosis (HHC) are particularly
vulnerable to iron overload, which occurs as a result of enhanced uptake. In subjects
heterozygous for the condition, a small increase in iron storage may occur. It has been suggested
that heterozygous subjects (up to 1% of the population) may have an increased risk of
cardiovascular disease but this remains controversial. Similarly, the suggestion that high iron
status may be associated with other chronic conditions remains unresolved.
Studies in rodents suggest a pattern of iron overload comparable with that seen in
hemochromatosis, with cellular changes but not with fibrosis occurring. Reproductive studies in
rodents have shown no significant evidence of iron transfer across the placenta. This is supported
by a study in an ovine model where maternal iron poisoning did not result in increases in fetal
serum iron levels. However one study reports that iron gluconate is teratogenic, causing
exencephaly in mice following administration on the 8th and 9th days of gestation. The common
effect of excessive ingestion of magnesium is osmotic diarrhea. However, this effect was only

Page | 29
observed in a limited number of studies of variable quality. There are only limited data on the
oral and general toxicity of magnesium in animals. The available data suggest a lack of
carcinogenicity at doses of up to 3000 mg/kg bw/day. Mutagenicity tests on magnesium salts
have also been negative. In humans, changes in serum parathyroid hormone levels have been
reported in supplementation studies in postmenopausal women with reduced bone mineral
density and a history of fracture and in healthy men. Osmotic diarrhea and other mild
gastrointestinal symptoms have also been reported in supplementation studies. However, these
symptoms were only reported in a limited number of studies. There are limited data on the oral
and general toxicity of inorganic phosphate salts in animals. Pathological effects in the
parathyroid gland, kidneys and bones have been observed in mature male rats fed a diet
containing an excessively high level of sodium orthophosphate for 7 months. No adverse effects
on growth and reproduction were reported in long-term studies with phosphoric acid.

A number of case reports of accidental and deliberate poisoning with potassium have shown that
large doses of potassium result in hyperkalemia and hypernatremia and lead to in changes in
acid-base balance and respiratory and heart rates. However, the dose associated with the onset of
hyperkalemia and adverse effects vary depending on potassium status and clearance time.
Supplementation studies have generally not reported side effects, although it is unclear whether
side effects were specifically investigated in many of these studies. However, endoscopic
investigation following periods of potassium supplementation has shown that potassium
supplementation may cause local irritation in the gastrointestinal tract, leading to erosion and
ulcerations. The available animal data are not relevant to this risk assessment as the effects
described are considered to be due to the anionic components, such as bromate and iodate. Older
people and infants may be more vulnerable to potassium toxicity due to lower renal function, as
may patients with conditions such as pre-existing hyperkalemia, renal disease, acidosis, insulin
deficiency or digitalis intoxication.

Page | 30
References:
1. Kleibeuker, J.H., Welberg, J.W.M., Mulder, N.H., van der Meer, R., Cats, A., Limburg,
A.J., Kreumer, W.M.T., Hardonk, M.J., de Vries, E.G.E. (1993) Epithelial cell
proliferation in the sigmoid colon of patients with adenomatous polyps increases during
oral calcium supplementation. British Journal of Cancer 67, 500-503.
2. Wu K. D., Chuang, R.-B., Wu, F.-L., Jan, I-S, Tsai, K-S. (1996) The milk-alkali
syndrome caused by betelnuts in oyster shell paste. Clinical Toxicology 34, 741-745.
3. Levine, R.J., Hauth, J.C., Curet, L.B. Sibai B.M., Catalano P.M., Morris C.D., Der
Simonian R., Erterlitz J.R., Raymond E.G., Bild D.E., Clements J.D. and Cutler J.A.
(1997) Trial of calcium to prevent eclampsia. New England Journal of Medicine 337, 69-
76.
4. Hofstad, B., Vatn, M.H., Anderson, S.N., Andersen, S.N., Owen, R.W., Larsen, S.,
Osnes, M. (1998) The relationship between faecal bile acid profile with or without
supplementation with calcium and antioxidants on recurrence and growth of colorectal
polyps. European Journal of Cancer Prevention 7, 287-294.
5. Baron, J. A., Beach, M., Mandel, J.S., Van Stolk, R.U., Haile, R.W., Sandler, R.S.,
Rothstein, R., Summers, R.W., Snover, D.C., Beck, G.J., Bond, J.H., Greenberg, E.R.
(1999) Calcium supplements for the prevention of colorectal adenomas. New England
Journal of Medicine 340, 101-107.
6. Bonithon-Kopp, C., Kronborg, O., Giacosa, A., Räthe, U., Faivre, J. (2000) Calcium and
fibre supplementation in prevention of colorectal adenoma recurrence: a randomized
intervention trial. Lancet356, 1300-06.
7. Fairney, A. and Weir, A.A. (1970) The effect of abnormal maternal plasma calcium
levels on the offspring of rats. Journal of Endocrinology 48, 337-345.
8. Shackelford, M.E., Collins, T.F.X., Welsh, J.J., Black T.N., Ames M.J., Chi R.K. O
Donell M.W. (1993) Foetal development in rats fed ain-76a diets supplemented with
excess calcium. Food and Chemical Toxicology 31, 953-961.
9. Blot I., Papriernik, E., Kaltwasser, J.P., Werner, E., Tchernia, G. (1981) Influence of
routine administration of folic acid and iron during pregnancy. Gynecology and Obstetric
Investigations 12, 294-304.

Page | 31
10. Brock, C., Curry, H., Hanna, C., Knipfer, M. and Taylor, L. (1985) Adverse effects of
iron supplementation: a comparative trial of a wax matrix iron preparation and
conventional ferrous sulphate tablets. Clinical Therapeutics7, 568-573.
11. Coplin, M., Schuette, S., Leichtmann, G., Lashner, B. (1991) Tolerability of iron: A
comparison of bisglycino iron II and ferrous sulphate. Clinical Therapeutics 13, 606-612.
12. Frykman, E. Bystrom, M. Jansson, U. Edberg, A. Hansen T. (1994) Side effects of iron
supplements in blood donors; superior tolerance of hemeiron. Journal of Laboratory and
Clinical Medicine 123, 561-564.
13. Liguori, L. (1993) Iron protein succinylate in the treatment of iron deficiency: controlled,
double-blind, multicenter clinical trialon over 1,000 patients. International Journal of
Clinical Pharmacology, Therapy and Toxicology 31, 105-123.
14. Lokken, P. and Birkeland, J.M. (1979) Dental discolourations and side effects with iron
and placebo tablets.Scandinavian Journal Of Dentistry Research 87, 275-278.
15. Nagy, L., Tarnok, F., Past, T., Mozsik G., Deak G., Tapsonyi Z., Fendler K., Javor T.
(1988). Human tolerability and pharmacodynamic study of Tisacid tablet in duodenal
ulcer patients, a prospective, randomized, self-controlled clinicopharmacological study.
Acta Medica Hungarica 45 (2) 231-247.
16. Marken, P.A., Weart, C.W., Carson, D.S., Gums J.G., Lopes-Virella M.F. (1989) Effects
of magnesium oxide on the lipid profile of healthy volunteers. Atherosclerosis77, 37-42.
17. Zemel, P.C., Zemel, M.B., Urberg, M., Douglas F.L., Geiser R., Sowers J.R. (1990)
Metabolic and hemodynamic effects of magnesium supplementation in patients with
essential hypertension. American Journal of Nutrition 51, 665-669.
18. Paolisso, G., Sgambato, S., Gambardella, A., Pizza G., Tesauro P., Varricchio M.,
D’Onofrio F. (1992). Daily magnesium supplements improve glucose handling in elderly
subjects. American Journal of Nutrition 55, 1161-1167.
19. Bashir, Y., Sneddon, J.F., Staunton, A., Haywood G.A., Simpson I.A., McKenna W.J.,
Camm A.J.(1993) Effects of long-term oral magnesium chloride replacement in
congestive heart failure secondary to coronary artery disease. American Journal of
Cardiology 72, 1156-1162.

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20. Stendig-Lindberg, G., Tepper, R., Leichter, I. (1993) Trabecular bone density in a two
year controlled trial of personal magnesium in osteoporosis. Magnesium Research 6, 155-
163.
21. Altura, B.T., Wilimzig, C., Trnovec, T., Nyulassy S., Altura B.M. (1994) Comparative
effects of magnesium enriched diets and different orally administered magnesium oxide
preparations on ionized Mg, Mg metabolism and electrolytes in serum of human
volunteers. Journal of theAmerican College of Nutrition 13, 447-54.
22. Goldsmith, R.S., Bartos, H, Hulley, S.B., Ingbar, S.H., Moloney, W.C. (1968) Phosphate
supplementation as an adjunct in the therapy of multiple myeloma. Archives of Internal
Medicine 122, 128-133.
23. Brixen, K., Nielsen, H.K., Charles, P., Mosekilde, L. (1992) Effect of a short course of
oral phosphate treatment on serum parathyroid hormone (I-84) and biochemical markers
of bone turnover: a dose response study. Calcified Tissue International 51, 276-281.
24. Whybro, A., Jagger, H., Barker, M., Eastell, R. (1998) Phosphate supplementation in
young men: lack of effect on calcium homeostasis and bone turnover. European Journal
of Clinical Nutrition 52, 29-33.
25. McMahon, F. G., Ryan, J. R., Akdamar, K. and Ertan, A. (1982) Upper gastrointestinal
lesions after potassium chloride supplements: a controlled clinical trial. Lancet2, 1059-
1061.
26. McMahon, F. G., Ryan, J. R., Akdamar, K. and Ertan, A. (1984) Effect of potassium
chloride supplements on upper gastrointestinal mucosa. Clinical Pharmacology and
Therapeutics 35, 852-855.
27. Grimm, R. H., Kofron, P. M., Neaton, J. D., Svendsen, K. H., Elmer, P. J., Holland, L.,
Witte, L. J., Clearman, D. and Prineas, R. J. (1988) Effect of potassium supplementation
combined with dietary sodium reduction on blood pressure in men taking
antihypertensive medication. Journal of Hypertension 6 (suppl 4) S591-S593.
28. Grimm, R. H., Neaton, J. D., Elmer, P. J., Svendsen, K. H., Levin, J., Segal, M., Holland,
L., Witte, L. J., Clearman, D. R., Kofron, P., LaBounty, R. K., Crow, R. and Prineas, R.
J. (1990) The influence of oral potassium chloride on blood pressure in hypertensive men
on a low-sodium diets. New England Journal of Medicine 322, 569-574.

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29. Food and Nutrition Board. Dietary reference intakes for calcium, phosphorus,
magnesium, vitamin D, and fluoride. Washington, DC. National Academy Press. 1997.
30. Trace elements in human nutrition and health. Geneva, World Health Organization, 1996.
31. Trace elements in human nutrition. Report of a WHO Expert Committee. Geneva, World
Health Organization, 1973 (WHO Technical Report Series, No. 532).
32. Requirements of vitamin A, iron, folate and vitamin B12. Report of a Joint FAO/WHO
Expert Consultation. Rome, Food and Agriculture Organization of the United Nations,
O Food and Nutrition Series, No. 23).
33. BgVV (2002) Use of Vitamins and Minerals in Foods. Part I: Minerals (including trace
elements).http://www.bfr.bund.de/cm/208/verwendung_von_mineralstoffen_und_vitamin
en_in_leensmitteln.pdf.; visited on 08 April 8, 2015.
34. Hages M, Broenstrup A, Prinz-Langenohl R, Pietrzik K (1999) Die neuen Dietary
ReferenceIntakes–einBeitragzurinternationalenHarmonisierung der
Zufuhrempfehlungen? Ernährungs-Umschau 46: 130-135.
35. Dybing E, Doe J, Groten J, Kleiner J, O'Brien J, Renwick AG, Schlatter J, Steinberg
P,Tritscher A, Walker R, Younes M (2002) Hazard characterisation of chemicals in food
anddiet: dose response, mechanisms and extrapolation issues. Food Chem. Toxicol. 40:
237-282.
36. Ganzoni, A.M., Tondung, G., Rhymer, K. (1974). OraleEisenmedikation. Vertraglichkeit
von Eisensulfat und Eisensulfat + Bernsteinsaure. Einfluss auf die
HamoglobinkonzentrationGesunder. Deutsche MedizinischeWochenschrift99, 1175-1178.
37. Hallberg, L., Hogdahl, A.-M., Nilsson, L., Rybo, G. (1966b). Menstrual blood loss – a
population study. ActaObstetriciaetGynaecologicaScandinavica45, 320-351.
38. Reddaiah, V.P., Raj, P., Ramachandran K., Nath L.M., Sood S.K., Madan N., Rusia
U.(1989). Supplementary iron dose in pregnancy anemia prophylaxis. Indian Journal of
Pediatrics 56, 109-114.
39. Hallberg, L., Hogdahl, A.-M., Nilsson, L., Rybo, G. (1966a). Menstrual blood loss – a
population study.
40. Breskin, M.W., Worthington-Roberts, B.S., Knopp, R.H., Brown Z., Plovie, B., Mottet,
N.K., Mills, J.L. (1983). First trimester serum zinc concentrations in human pregnancy.
American Journal of Clinical Nutrition 38, 943-953.

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41. Hambidge, K.M., Krebs, N.F., Jacobs, M.A., Favier A., Guyette L., Ikle D.N., (1983).
Zinc nutritional status during pregnancy: a longitudinal study. American Journal of
Clinical Nutrition 37, 429-442
42. Hambidge, K.M., Krebs, N.F., Sibley, R.N., English, J. (1987). Acute effects of iron
therapy on zinc status during pregnancy. Obstetrics and Gynecology 70, 593-596.
43. Dawson, E.A., Albers, J.A., McGanity, W.J. (1989). Serum changes due to iron
supplementation in teen-age pregnancy. American Journal of Clinical Nutrition 50, 848-
852
44. Sheldon, W.L., Aspillaga, M.O., Smith, P.A., Lind, T. (1985). The effects of oral
supplementation on zinc and magnesium levels during pregnancy. British Journal of
Obstetrics and Gynaecology92, 892-898.
45. Menendez, C., Todd, J., Alonso, P.L., Francis N., Lulat S., Ceesay S., Ascaso C., Smith
T., M’Boge B., Greenwood B.M. (1995). The response to iron supplementation of
pregnant women with the hemoglobin genotype AA or AS. Transactions of the Royal
Society of Tropical Medicine and Hygiene 89, 289-292.
46. Fotherby, M. D. and Potter, J. F. (1992) Potassium supplementation reduces clinic and
ambulatory blood pressure in elderly hypertensive patients. Journal of Hypertension 10,
1403-1408.
47. Siani, A., Strazzullo, P., Giacco, A., Pacioni, D., Celentano, E. and Mancini, M. (1991)
Increasing the dietary potassium intake reduces the need for antihypertensive medication.
Annals of Internal Medicine 115, 753-759.

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 Chapter Three

Methodology

Page | 36
3.1 Atomic absorption spectroscopy (AAS)
Atomic absorption spectrometry will be used as an analytical technique, and the underlying
principles were established in the second half of the 19th century by Robert Wilhelm Bunsen and
Gustav Robert Kirchhoff, both professors at the University of Heidelberg, Germany. The modern
form of AAS was largely developed during the 1950s by a team of Australian chemists.
They were led by Sir Alan Walsh at the Commonwealth Scientific and Industrial Research
Organization (CSIRO), Division of Chemical Physics, in Melbourne, Australia.
Atomic absorption spectrometry has many uses in different areas of chemistry such as:
3 Clinical analysis: Analyzing metals in biological fluids and tissues such as whole blood,
plasma, urine, saliva, brain tissue, liver, muscle tissue, semen
4 Pharmaceuticals: In some pharmaceutical manufacturing processes, minute quantities of a
catalyst that remain in the final drug product
5 Water analysis: Analyzing water for its metal content.

3.1.1 Principle
The technique makes use of absorption spectrometry to assess the concentration of an analyte in
a sample. It requires standards with known analyte content to establish the relation between the
measured absorbance and the analyte concentration and relies therefore on the Beer-Lambert
Law.
In short, the electrons of the atoms in the atomizer can be promoted to higher orbitals (excited
state) for a short period of time (nanoseconds) by absorbing a defined quantity of energy
(radiation of a given wavelength). This amount of energy, i.e., wavelength, is specific to a
particular electron transition in a particular element. In general, each wavelength corresponds to
only one element, and the width of an absorption line is only of the order of a few picometers
(pm), which gives the technique its elemental selectivity.
The radiation flux without a sample and with a sample in the atomizer is measured using a
detector, and the ratio between the two values (the absorbance) is converted to analyte
concentration or mass using the Beer-Lambert Law. Radiation flux is a measure of the flow of
radiation from a given radioactive source. Φ = L⁄4πr2 is the radiation flux, L is the Luminosity
(measured in Watts) and r is the distance from the radiation source. Radiation flux density is a
related measure that adds area dimensions to the above definition.

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In order to analyze a sample for its atomic constituents, it has to be atomized. The atomizers
most commonly used nowadays are flames and electro thermal (graphite tube) atomizers. The
atoms should then be irradiated by optical radiation, and the radiation source could be an
element-specific line radiation source or a continuum radiation source. The radiation then passes
through a monochromator in order to separate the element-specific radiation from any other
radiation emitted by the radiation source, which is finally measured by a detector.

Fig 1: Atomic absorption spectrometer block diagram

A. Atomizers
The atomizers most commonly used nowadays are (spectroscopic) flames and electrothermal
(graphite tube) atomizers. Other atomizers, such as glow-discharge atomization, hydride
atomization, or cold-vapor atomization might be used for special purposes.
B. Radiation sources
We have to distinguish between line source AAS (LS AAS) and continuum source AAS (CS
AAS). In classical LS AAS, as it has been proposed by Alan Walsh, the high spectral resolution
required for AAS measurements is provided by the radiation source itself that emits the spectrum
of the analyte in the form of lines that are narrower than the absorption lines. Continuum sources,
such as deuterium lamps, are only used for background correction purposes. The advantage of
this technique is that only a medium-resolution monochromator is necessary for measuring AAS;
however, it has the disadvantage that usually a separate lamp is required for each element that
has to be determined. In CS AAS, in contrast, a single lamp, emitting a continuum spectrum over

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the entire spectral range of interest is used for all elements. Obviously, a high-resolution
monochromator is required for this technique.
C. Spectrometer
As already pointed out above, there is a difference between medium-resolution spectrometers
that are used for LS AAS and high-resolution spectrometers that are designed for CS AAS. The
spectrometer includes the spectral sorting device(monochromator)and the detector Spectrometers
for LS AAS. In LS AAS the high resolution that is required for the measurement of atomic
absorption is provided by the narrow line emission of the radiation source, and the
monochromator simply has to resolve the analytical line from other radiation emitted by the
lamp. This can usually be accomplished with a band pass between 0.2 and 2 nm, i.e., a medium-
resolution monochromator. Another feature to make LS AAS element-specific is modulation of
the primary radiation and the use of a selective amplifier that is tuned to the same modulation
frequency, as already postulated by Alan Walsh. This way any (unmodulated) radiation emitted
for example by the atomizer can be excluded, which is imperative for LS AAS. Photomultiplier
tubes are the most frequently used detectors in LS AAS, although solid state detectors might be
preferred because of their better signal-to-noise ratio.
Background absorption and background correction
Molecular absorption and radiation scattering can result in artificially high absorption and an
improperly high (erroneous) calculation for the concentration or mass of the analyte in the
sample. There are several techniques available to correct for background absorption, and they are
significantly different for LS AAS and HR-CS AAS.
Background correction techniques in LS AAS
In LS AAS background absorption can only be corrected using instrumental techniques, and all
of them are based on two sequential measurements, firstly, total absorption (atomic plus
background), secondly, background absorption only, and the difference of the two measurements
gives the net atomic absorption. Because of this, and because of the use of additional devices in
the spectrometer, the signal-to-noise ratio of background-corrected signals is always significantly
inferior compared to uncorrected signals. It should also be pointed out that in LS AAS there is no
way to correct for (the rare case of) a direct overlap of two atomic lines. In essence there are
three techniques used for background correction in LS AAS.

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3.1.2 Procedure

Sample collection: Most of all water and foods of local area of Bangladesh will be collected for
this analysis.
Sample preparation: Determination of mineral ions concentration will be carried out using the
atomic absorption spectrophotometry (AAS).
Analytical Methods and Instrumentation: Calcium, iron, potassium, phosphorus and
magnesium in water and foods will be determined according to described methods.Thesamples
will be analyzed in a laboratory with a quality assurance schemes by using “Atomic Absorption
Spectrophotometer”.
Measurement of different variables: Exposure estimates have compared to health-based
toxicological reference values (e.g. compared with acceptable daily intakes ADI).
4

Data analysis: The concentration of calcium, iron, magnesium, phosphorus and potassium in
water and food will be determined by using system software. Statistical analysis will be
performed by using SPSS statistical software of version 16. All values were expressed as mean ±
standard error of mean (SEM).

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 Chapter Four

Result and Discussion

Page | 41
Result and discussion

This research will investigate the exposure to calcium, iron magnesium, phosphorus and
potassium in the average diet in Bangladesh, as well levels of contaminations in water and food
in Bangladesh. This will be done through the total diet study (TDS) approach. Minerals play an
important role in maintaining proper function and good health in the human body.
Approximately 98% of the calcium (Ca) and 80% of the phosphorus (P) in the human body are
found in the skeleton. Inadequate intake of minerals in the diet is often associated with an
increased susceptibility to infectious diseases due to the weakening of the immune system1.
Plants, animal foods and drinking water are an important source of essential elements2. The other
elements that were found in selected fruit samples are iron, magnesium and potassium. The
highest amount of iron was found in Guava and Tamarind fruit, 2.80 ± 1.43, mg respectively. In
Bangladesh, the value of Magnesium was found to be similar (around 1-5 μg/g) in diverse arums
without one exception value i.e. 21.3456 μg/g in Colocasiaesculenta sample, which is
comparatively about ten times lower than that of same species of Nigeria (28.02 mg/100 g)3.
Magnesium is highest found in blackberry of 47mg/100g. Again potassium is best found in a
popular fruit known as Deophal (Artocarpuslakaocha). According to USDA the daily
recommended intake of iron is 8 mg for adult male and 18 mg for adult female. Trace element is
any substance that when present at low concentration compared to those of an oxidisable
substrate significantly delays or prevents oxidation of that substrate. Trace elements sometimes
act as an antioxidant. Antioxidant functions are associated with decreased DNA damage,
diminished lipid per oxidation, maintained immune function and inhibited malignant
transformation of cells4. These minerals are also called micro-minerals which also worked as
antioxidants, which are required in amounts less than 100 mg/day5.

There are many epidemiological studies suggest that consumption of polyphenol-rich foods and
beverages is associated with a reduced risk of cardiovascular diseases, stroke and certain types of
cancer in which polyphenol is linked to the antioxidant properties6,7. The consumption of dietary
trace-elements will help to prevent free radical damage. Trace-elements have the ability to
scavenge free radicals by inhibiting the initiation step or interrupting the propagation step of
oxidation of lipid and as preventive antioxidants which slow the rate of oxidation by several

Page | 42
actions8. Thus, consumption of these tropical fruits can be suggested as a food based strategy to
alleviate or improve the unsatisfactory dietary iron intake of adolescents in the low-income areas.
These fruits were also enriched with minerals like iron, phosphorus, potassium, calcium and
magnesium. Among the fruits analyzed, the highest quantity of potassium was found in
Tamarind fruit, 621.00 ± 3.26 mg. For the healthy adult, RDA for sodium and potassium intake
is not more than 2,400 mg and 4700 mg respectively per day9. Among the fruits analyzed,
highest amount of calcium and magnesium was found in Tamarind fruit, 75.00 ± 2.41 mg and
90.00 ± 1.80 mg, respectively. Calcium with the name of “super nutrient” has been proven
clinically associated with reduced risk of various non-communicable diseases such as
osteoporosis, cardiovascular diseases and it also helps to reduce colorectal cancer risk by
promoting the apoptosis in human colorectal epithelium that reduce colorectal neoplasm10.

Reference:

1. Hendricks, D. G. 1998. Mineral Analysis. In Suzanne, N. S. (Eds). Food Analysis, p. 151-

154. Maryland: An Aspen Publication.
2. Chaturvedi, U. C., Shrivastava, R. and Upreti, R. K. 2004. Viral infections and trace
elements: A complex interaction. A review article. Current Science 87(11): 1536-1554.
3. Alinnor, I. J., Akalezi, C. O. 2010. Proximate and Mineral Compositions of
Dioscorearotundata(White Yam) and Colocasiaesculenta (White Cocoyam). Pakistan
Journal of Nutrition 9 (10): 998-1001.
4. Maisarah, A. M., Nurul A. B., Asmah, R. and Fauziah, O. 2013. Antioxidant analysis of
different parts of Carica papaya. International Food Research Journal 20 (3): 1043-1048.
5. IOM (Institute of Medicine) 2001. Food and Nutrition Board. Dietary Reference Intakes for
Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese,
Molybdenum, Nickel, Silicon, Vanadium and Zinc. p. 442-501. Washington, DC: National
Academy Press.
6. Barros, L., Ferreira, M. J., Queiros, B., Ferreira, I. C. F. R. and Baptista, P. 2007. Total
phenols, ascorbic acid, β-carotene and lycopene in Portuguese wild edible mushrooms and
their antioxidant activities. Food Chemistry 103 (2): 413-419.

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7. Jagadish, L. K., Krishnan, V. V., Shenbhagaraman, R. and Kaviyarasan, V. 2009.
Comparative study on the antioxidant, anticancer and antimicrobial property of
Agaricusbisporus(J. E. Lange) Imbach before and after boiling. African Journal of
Biotechnology 8 (4): 654-661.
8. Olajire, A. A. and Azeez, L. 2011. Total antioxidant activity, phenolic, flavonoid and
ascorbic acid contents of Nigerian vegetables. African Journal of Food Science and
Technology 2(2): 022-029.
9. USDA (US Department of Agriculture) US Department of Health and Human Services.
Dietary Guidelines for Americans. Washington, DC: USDA, 2005.
http://www.health.gov/dietaryguidelines/dga2005/document/default.htm.
10. Ng, X. N., Chye, F. Y. and Ismail, M. A. 2012. Nutritional profile and antioxidative
properties of selected tropical wild vegetables. International Food Research Journal 19(4):
1487-1496.

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 Chapter Five

Conclusions and
Future Prospects

Page | 45
Conclusions and Future Prospects
Water and food are highly valued in human diet for vitamins and minerals. This study will
indicate that the water and food of Bangladesh are excellent source of minerals. The findings of
this study will compare to other published data that can be used to alleviate nutritional deficiency
among the people specially woman and children of the rural areas. The results of this study
indicate that the daily intake of iron, sodium, potassium, calcium and magnesium through water
and food may not constitute a health hazard for consumers because the values were below the
recommended daily intake of these metals. However, high level of mineral contains water and
food can be hazardous if they are taken in large quantities. It is therefore suggested that the use
of minerals in water and food must be within the range in order to prevent excessive build-up of
these metals in the human food chain. The results of the study show the necessity of education in
order to foster healthy nutritional habits and to increase the share of natural sources of vitamins
and minerals in the diet to prevent the occurrence of adverse effects related to their insufficient
consumption. Further studies are necessary to evaluate the contents of essential and toxic metals
in water and food to fulfill the daily requirement of minerals as well as the absence of possible
toxicological risks.

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