DETERMINATION OF MINERAL COMPONENTS

OF LEMON SEEDS

By:
LUBNA BIBI
Class No: 4039

Supervised By:
MUHAMMAD YOUSAF

DEPARTMENT OF CHEMISTRY
ISLAMIA COLLEGE PESHAWAR, KHYBER
PAKHTUNKHWA, PAKISTAN
Session: 2011-2015
DETERMINATION OF MINERAL COMPONENTS
OF LEMON SEEDS

By:
LUBNA BIBI
Class No: 4039

Thesis Submitted to the Department of Chemistry Islamia College Peshawar for the
fulfillment of the Requirements for the Degree of Bachelor in Science (Hons)

DEPARTMENT OF CHEMISTRY
ISLAMIA COLLEGE PESHAWAR
Session: 2011-2015

ii
DETERMINATION OF MINERAL COMPONENTS
OF LEMON SEEDS

It is recommended that this thesis prepared by Lubna Bibi

Entitled: Determination of Mineral Components of Lemon Seeds be
accepted as fulfilling this part of the requirement for the
Degree of BS (Hons) in Chemistry

Prof: M. Yousaf

(Supervisor/ Internal Examiner)

DEPARTMENT OF CHEMISTRY
ISLAMIA COLLEGE PESHAWAR

iii
 DECLARATION

This is to certify that research work submitted by Ms. Lubna Bibi in the thesis form

has been carried out under my direct supervision. All the data and result described

here are duly recorded. I also certify that enclosed manuscript has been prepared

under my supervision and I endorse its evaluation for the award of Bachelor of

Chemistry degree through the official procedure of the Islamia College Peshawar,

Khyber Pakhtunkhwa, Pakistan.

MUHAMMAD YOUSAF
ASSISSTANT PROFESSOR ______________________
(SUPERVISOR)

EXTERNAL EXAMINER _______________________

SIGNATURE _______________________

Dr. SHAD ALI ______________________

CHAIRMAN
(DEPARTMENT OF CHEMISTRY)
ISLAMIA COLLEGE PESHAWAR (PUBLIC SECTOR UNIVERSITY)

iv
v
 DEDICATED TO:

I dedicate this humble effort

to my respectable Teachers, lovable Parents

whose love, prayer and financial support

enabled me to complete this humble task.

vi
 ACKNOWLEDGEMENT

All praises to be ALLAH alone, the most compassionate, beneficent and ever

merciful, who gave me the power to do, the sight to observe and the mind to think and

judge. I have no words to thank Almighty ALLAH for his blessings throughout my

life. Blessing and peace be upon him the one whom ALLAH has sent us to guide and

purify our souls (blessing of ALLAH be upon him and his companion).

I am especially grateful to my Supervisor Prof. Muhammad Yousaf. He has built a

strong, resilient, lively foundation to my career as a chemist. Thank you for believing

in my potential and for holding me up through all research.

My genuine thanks to Dr. Shad Ali Chairman Chemistry Department for providing

lab facilities (ICUP) where I could dig deep and can find the gems of my existence.

I am also thankful to the lab staff Farooq, Mashooq and Amrez for their cooperation

and support.

LUBNA BIBI

vii
 TABLE OF CONTENTS

DECLARATION.......................................................................................................IV
ACKNOWLEDGEMENT.......................................................................................VII
TABLE OF CONTENTS.......................................................................................VIII
CHAPTER-1.................................................................................................................1
INTRODUCTION........................................................................................................1
1.1 BOTANICAL DESCRIPTION:.................................................................................1
1.2 MINERALS DEFINITION:......................................................................................2
1.3 CLASSIFICATION OF MINERALS:.........................................................................2
1.3.1 Macro Minerals:........................................................................................2
1.3.2 Micro Elements:.........................................................................................2
1.3.3 Ultra Trace Elements:................................................................................2
1.4 BIOCHEMICAL FUNCTIONS OF MINERAL ELEMENTS IN HUMANS AND
ANIMALS:...................................................................................................................3
1.4.1 Calcium:.....................................................................................................3
1.4.2 Phosphorus:...............................................................................................4
1.4.3 Sodium:......................................................................................................5
1.4.4 Potassium:..................................................................................................5
1.4.5 Chlorine:....................................................................................................6
1.4.6 Magnesium:................................................................................................7
1.4.7 Chromium:.................................................................................................8
1.4.8 Cobalt:........................................................................................................9
1.4.9 Copper:....................................................................................................10
1.4.10 Iron:..........................................................................................................11
1.4.11 Manganese:..............................................................................................12
1.4.12 Selenium:..................................................................................................13
1.4.13 Zinc:.........................................................................................................15
1.4.14 Fluorine:..................................................................................................17
1.4.15 Iodine:......................................................................................................18
CHAPTER-2:..............................................................................................................19
MATERIALS AND METHODS..............................................................................19
2.1 SAMPLE PREPARATION:....................................................................................19
2.2 CHEMICALS:......................................................................................................19
2.3 PREPARATION OF REAGENTS:...........................................................................19

viii
 2.4 ACID DIGESTION OF CITRUS LIMON FOR MINERAL DETERMINATION:............20
2.4.1 Preparation of Acid Digest:.....................................................................20
2.4.2 Determination of Phosphorous:...............................................................20
2.4.3 Determination of Calcium:......................................................................21
2.4.4 Determination of Trace Elements By AAS:..............................................21
CHAPTER-3...............................................................................................................23
RESULTS AND DISCUSSIONS..............................................................................23
Table 1: Trace elements concentration in lemon seeds.......................................24
Table 2: Macro element concentration (mg / 100 g) in lemon seeds...................24
REFERENCES...........................................................................................................25

ix
 CHAPTER-1
INTRODUCTION

1.1 BOTANICAL DESCRIPTION:

The genus Citrus L., of the family Rutaceae, comprises various species of limes,

lemons, oranges, grapefruits and mandarins. Species of the genus Citrus are widely

cultivated and they are the most popular fruits in the world [1]. The genus Citrus is

believed to be native to tropical and subtropical regions of Asia and Malaysian

archipelago [2]. Citrus fruits comprise one of the most important tree crops in the

Sudan. Most of the species are cultivated in the Sudan for their fruits such as C.

aurantifoliaSwingle (lime), C. sinensis L. (sweet orange), C. aurantium L. (sour

orange), C. reticulata Blanco (mandarin) and C. paradisiMacf (grape fruit). The peel

of Citrus fruits is a potential source of essential oils [1]. Citrus essential oils have been

used as flavoring agents in foods, beverages, and liquors and as aromatic agents in

perfumery, soap and other household products. The Citrus fruits possess high amounts

of bioactive compounds which can influence human health such as vitamin C [3],

carotenoids, flavonoids, limonoids, essential oils, coumarins, acridone alkaloids,

soluble fibres and minerals [4]. Albedo is the major component of lemon peel, and is

a spongy and cellulosic layer laid under flavedo. The thickness of the albedo

fluctuates according to several variables, among them variety and degree of

ripeness. Albedo has high dietary fiber content, and if added to new meat products

permits to formulate healthier products like beef burgers [5], bologna [6]and dry

cured sausages [7]. Furthermore, the presence of associated bioactive compounds

(flavonoids and vitamin C) with antioxidant properties in fresh lemon albedo involves

healthier benefits than other sources of dietary fiber [8].

1
1.2 MINERALS DEFINITION:

Minerals are inorganic substances, present in all body tissues and fluids and their

presence is necessary for the maintenance of certain physicochemical processes which

are essential to life. Minerals are chemical constituents used by the body in many

ways. Although they yield no energy, they have important roles to play in

many activities in the body [9]. Every form of living matter requires these

inorganic elements or minerals for their normal life processes [10].Minerals play an

important role in maintaining proper function and good health in the human body

[11].

1.3 CLASSIFICATION OF MINERALS:

Minerals may be broadly classified as macro (major) or micro (trace) elements. The

third category is the ultra-trace elements.

1.3.1 Macro Minerals:

The macro-minerals include calcium, phosphorus, sodium and chloride.

1.3.2 Micro Elements:

The micro-elements include iron, copper, cobalt, potassium, magnesium, iodine, zinc,

manganese, molybdenum, fluoride, chromium, selenium and sulphur [12].

The macro-minerals are required in amounts greater than 100 mg/dl and the micro-

minerals are required in amounts less than 100 mg/dl [13].

1.3.3 Ultra Trace Elements:

The ultra-trace elements include boron, silicon, arsenic and nickel which have been

found in animals and are believed to be essential for these animals. Evidence for

requirements and essentialness of others like cadmium, lead, tin, lithium and

vanadium is weak [14].

2
1.4 BIOCHEMICAL FUNCTIONS OF MINERAL ELEMENTS IN
HUMANS AND ANIMALS:

1.4.1 Calcium:

Function:

Calcium functions as a constituent of bones and teeth, regulation of nerve and muscle

function. In blood coagulation, calcium activates the conversion of prothrombin to

thrombin and also takes part in milk clotting. It plays a vital role in enzyme activation.

Calcium activates large number of enzymes such as adenosine triphosphatase

(ATPase), succinic dehydrogenase, lipase etc. It is also required for membrane

permeability, involved in muscle contraction, normal transmission of nerve impulses

and in neuromuscular excitability. A reduced extracellular blood calcium increases

the irritability of nerve tissue, and very low levels may cause spontaneous discharges

of nerve impulses leading to tetany and convulsions [9-13].

Deficiency Disorders:
In children, calcium deficiency causes rickets due to insufficient calcification by

calcium phosphate of the bones in growing children. The bones therefore remain soft

and deformed by the body weight. In adults, it causes osteomalacia, a generalized

demineralization of bones. It may also contribute to osteoporosis, a metabolic disorder

resulting in decalcification of bone with a high incidence of fracture, that is, a

condition where calcium is withdrawn from the bones and the bones become weak

and porous and then breaks [9, 10, 13]. Calcium deficiency also affects the dentition

of both children and adult. Toxicity symptoms occur with excess absorption due to

hypervitaminosis D or hypercalcaemia due to hyperparathyroidism, or idiopathic

hypercalcaemia. Excess calcium depresses cardiac activity and leads to respiratory

and cardiac failure; it may cause the heart to stop in systole, although, normally,

calcium ions increase the strength and duration of cardiac muscle contraction. Excess

calcium and phosphorus are excreted by the kidney. Ca and P excreted in faeces are

3
largely the unabsorbed dietary minerals; some comes from the digestive juices,

including bile [10].

1.4.2 Phosphorus:

Function:
Phosphorus is located in every cell of the body and is vitally concerned with many

metabolic processes, including those involving the buffers in body fluids [10]. It

functions as a constituent of bones, teeth, adenosine triphosphate (ATP),

phosphorylated metabolic intermediates and nucleic acids. It serves buffering action

that is, phosphate buffers, functions in the formation of high energy compounds, that

is, adenosine triphosphate (ATP) and is involved in the synthesis of phospholipids and

phosphoproteins. Practically, every form of energy exchange inside living cells

involve the forming or breaking of high-energy bonds that link oxides of phosphorus

to carbon or to carbon-nitrogen com-pounds[9,10,13]. Phosphorus is an essential

macronutrient for plants and one of the three nutrients generally added to soils in

fertilizers because of its vital role of energy transfer in living organisms and in plants.

Adequate phosphorus availability stimulates early growth and hastens maturity in

plants [15].Phosphorus is also needed for soil fertility. In plants, as grasses mature,

phosphorus is transferred to the grain. Also, the phosphorus content of the plant is

influenced markedly by the availability of phosphorus in the soil. As a result of this,

low-quality pastures devoid of legumes and range plants tend to be naturally low in

phosphorus, as the forage matures and the seeds fall; characteristically, the range soil

is also deficient in phosphorus [16].

Deficiency Disorders:
Decrease in serum phosphorus is found in rickets, hyperparathyroidism, De Toni-

Fanconi Syndrome. Deficiency disease or symptoms in children causes rickets and in

adults, it causes osteomalacia. Increase in serum phosphorus is found in chronic

4
nephritis and hypoparathyroidism. Toxicity disease or symptoms include low serum

Ca2+: P ratio. It may also lead to bone loss [9, 13].

1.4.3 Sodium:

Function:
Sodium is the principal cation in extracellular fluids. It regulates plasma volume and

acid-base balance, involved in the maintenance of osmotic pressure of the body fluids,

preserves normal irritability of muscles and cell permeability, activates nerve and

muscle function and involved in Na+/K+-ATPase, maintenance of membrane

potentials, transmission of nerve impulses and the absorptive processes of

monosaccharides, amino acids, pyrimidines, and bile salts. The changes in osmotic

pressure are largely dependent on sodium concentration [9, 10, 13].

Deficiency Disorders:
Sodium deficiency in young chicks cause growth retardation. Egg production and

hatchability in laying chickens are depressed [16]. Excessive intake of sodium

chloride may result in salt toxicity which is mainly caused by sodium ion, since

sodium acetate or sodium propionate affects the animals in a manner similar to that of

sodium chloride. The amounts required for toxicity vary and are largely dependent on

the availability of water to the animals. Toxicity usually occurs when animals are

deprived of salt and then have access to a brine solution or loose salt without access to

sufficient water [10]. Low level of sodium in the serum is hyponatraemia and this

occurs in acute Addison’s disease, vomiting, diarrhoea, nephrosissevere burns and

intestinal obstruction [9].

1.4.4 Potassium:

Function:
Potassium is the principal cation in intracellular fluid and functions in acid-base

balance, regulation of osmotic pressure, conduction of nerve impulse, muscle

5
contraction particularly the cardiac muscle, cell membrane function and Na+/K+-

ATPase. Potassium is also required during glycogenesis. It also helps in the transfer

of phosphate from ATP to pyruvic acid and probably has a role in many other basic

cellular enzymatic reactions. Its metabolism is regulated by aldosterone.

Deficiency Disorders:
Hyperkalaemia is increased level in serum potassium and this occurs in Addison’s

disease, advanced chronic renal failure, shock and dehydration. Toxicity disease or

symptoms include dilatation of the heart, cardiac arrest, small bowel ulcers.

Hypokalaemia is low level of serum potassium and this occurs in diarrhoea, metabolic

alkalosis and familial periodic paralysis. When lactating dairy cows have

hypokalaemia, the milk production is markedly lowered. Deficiency disease or

symptoms occurs secondary to illness, functional and structural abnormalities

including impaired neuromuscular functions of skeletal, smooth, and cardiac muscle,

muscular weakness, paralysis, mental confusion[9,10,13]. Others are cardiac

arrhythmias, impaired carbohydrate tolerance, altered electrocardiogram in calves.

Potassium deficiency affects the collecting tubules of the kidney, resulting in the

inability to concentrate urine, and also causes alterations of gastric secretions and

intestinal motility [17].

1.4.5 Chlorine:
Function:
Chlorine is involved in fluid and electrolyte balance, gastric fluid and chloride shift in

HCO3- transport in erythrocytes. Chloride is the principal anion in extracellular fluid.

It is involved in the regulation of extracellular osmotic pressure and makes up over

60% of the anions in this fluid compartment and is thus important in acid base

balance. The concentration of chloride ion is subject to more variation than that of

sodium, since other anions, especially bicarbonates, can exchange for the chloride. It

is the chief anion of the gastric juice and is accompanied by the hydrogen ions in

6
nearly equal amounts. The chloride of the gastric secretions is derived from blood

chloride and is normally reabsorbed during the latter stages of digestion in the lower

intestine [10, 13].

Deficiency Disorders:
Deficiency disease or symptoms occur in infants fed salt-free formula. On a chloride-

deficient diet, the excretion of chloride in the urine or perspiration is markedly

reduced [10, 13]. It is also secondary to vomiting, diuretic therapy, renal disease.

Excessive depletion of chloride ions through losses in the gastric secretions or by

deficiencies in the diet may lead to alkalosis due to an excess of bicarbonate, since the

inadequate level of chloride is partially compensated for or replaced by bicarbonate.

Chloride is excreted in the faeces, sweat, and urine primarily as sodium or

potassium chloride, although it may be accompanied by ammonium ions when base

needs to be conserved [10].

1.4.6 Magnesium:

Function:
Magnesium is an active component of several enzyme systems in which thymine

pyrophosphate is a cofactor. Oxidative phosphorylation is greatly reduced in the

absence of magnesium. Mg is also an essential activator for the phosphate-transferring

enzymes myokinase, diphophopyridinenucleotide kinase, and creatine kinase. It also

activates pyruvic acid carboxylase, pyruvic acid oxidase, and the condensing enzyme

for the reactions in the citric acid cycle. It is also a constituent of bones, teeth, enzyme

cofactor, (kinases, etc.) [13].

Deficiency Disorders:
Deficiency diseases or symptoms is secondary to malabsorption or diarrhoea,

alcoholism. Acute magnesium deficiency results in vasodilation, with erythemia and

hyperaemia appearing a few days on the deficient diet. Neuromuscular

hyperirritability increases with the continuation of the deficiency, and may be

7
followed eventually by cardiac arrhythmia and generalized tremours. A common form

of magnesium-deficiency tetany in ruminants is called grass tetany or wheat wheat-

pasture poisoning. This condition occurs in ruminants grazing on rapidly growing

young grasses or cereal crops and develops very quickly. The physiological

deficiency of magnesium can be prevented by magnesium supplementation of a salt or

grain mixture and adequate consumption is also very important [10].Toxicity disease

or symptoms of magnesium deficiency in humans include depressed deep tendon

reflexes and respiration [13].When a magnesium-deficient diet is fed to young chicks,

it leads to poor growth and feathering, decreased muscle tone, ataxia, progressive

incoordination and convulsions followed by death [16].

1.4.7 Chromium:

Function:
Chromium is an essential element for animals and humans [18].It has been

found in nucleoproteins isolated from beef liver and also in RNA preparations [19].It

could play a role in maintaining the configuration of the RNA molecule, because Cr

has been shown to be particularly effective as a cross-linking agent for collagen [20].

Cr has also been identified as the active ingredient of the glucose tolerant factor [21],

a dietary factor required to maintain normal glucose tolerance in the rat. Trivalent

chromium is a constituent of ‘‘glucose tolerance factor’’ (GTF), which binds to and

activates/potentiates insulin action [13,22].Chromium compounds have a wide

variety of industrial uses, including production of stainless steel and other alloys,

high-melting refractory materials, pigments and mordants for paints and dyes, tanned

leather goods and chrome plating [18].Cr affects the action of insulin in protein

metabolism, as indicated by rats fed chromium-deficient diets repleted by chromium

[23].Insulin-mediated amino acid transport into tissues was enhanced and

incorporation of labelled glycine, serine and methionine into heart protein was greater

in chromium-supplemented animals. Evidence of a role for chromium in lipid

8
metabolism and chromium deficiency in the development of atherosclerosis is

accumulating from animal and human studies [18].

Deficiency Disorders:
Cr deficiencies may exist, particularly in children suffering from protein-calorie

malnutrition [24]. In experimental animals, Cr deficiency leads to a reduced rate of

removal of ingested glucose, due to a low sensitivity of peripheral tissues to insulin.

Chromium is needed for growth of rats and its deficiency leads to a reduced life span,

corneal lesions and interference with insulin action producing a diabetic state and this

causes removal of glucose from the blood at a rate that is one half that of animals on a

chromium-containing diet [22, 25].The Cr content of foodstuffs varies widely and is

present in combination with a small organic molecule, the glucose tolerant factor.

Chromium poisoning in humans is usually limited to accidental ingestion of chromic

acid or chromates. Toxicity to kidney, liver, nervous system and blood are the major

causes of death [26].

1.4.8 Cobalt:
Function:
Cobalt is required as a constituent of vitamin B12 and its metabolism is the same as

for vitamin B12. In addition to its role in vitamin B12, cobalt is also a cofactor of

enzymes involved in DNA biosynthesis and amino acid metabolism [27].

Deficiency Disorders:
Deficiency disease or symptoms is manifested in vitamin B12 deficiency. Co

deficiency in ruminants have been successfully alleviated by the use of cobalt oxide

pellets, which remain in the reticulum or rumen fluid [10].In humans, toxicity disease

or symptoms include goitre, hypothyroidism and heart failure [13].In animals,

excessive intake results in polycythaemia, apparently due to the inhibition by cobalt

of certain respiratory enzyme systems, for example, cytochrome oxidase and succinic

9
dehydrogenase. Deficiencies of cobalt in ruminants cause anorexia, wasting of

skeletal muscle, fatty livers, haemosiderosis of the spleen and anaemia [10].

1.4.9 Copper:

Function:
Copper is a constituent of enzymes like cytochrome c oxidase, amine oxidase,
catalase, peroxidase, ascorbic acid oxidase, cytochrome oxidase, plasma monoamine
oxidase, erythrocuprin (ceruloplasmin), lactase, uricase, tyrosinase, cytosolic
superoxide dismutase etc. and it plays a role in iron absorption [28]. Cu is an essential
micro-nutrient necessary for the haematologic and neurologic systems [29].It is
necessary for the growth and formation of bone, formation of myelin sheaths in the
nervous systems, helps in the incorporation of iron in haemoglobin, assists in the
absorption of iron from the gastrointestinal tract (GIT) and in the transfer of iron from
tissues to the plasma [9, 13].

Deficiency Disorders:
Increased levels of copper are seen in acute infections and in chronic conditions

such as cirrhosis, rheumatoid arthritis and in post-operative stages. Increased level is

also found in malnutrition [9].

Clinical disorders associated with Cu deficiencies include anaemia, bone disorders,

neonatal ataxia, depigmentation and abnormal growth of hair, fur or wool, impaired

growth and reproductive performance, heart failure and gastrointestinal disturbances.

The incidence of these disorders varies widely among animal species. Cu deficiency

has also been associated with cardiac hypertrophy and sudden cardiac failure.

GardeaTorresdey [30] reported that carboxyl groups found on the cell walls of dead

algal biomass are potentially responsible for copper binding. Toxicity disease or

symptoms are rare and is secondary to Wilson’s disease [13]. In Wilson’s disease, a

large amount of copper is deposited in liver, brain, etc. Total copper content in the

plasma and ceruloplasmin-bound copper content decreases and there is an increased

excretion of copper in the urine. Sometimes, Cu may be deposited in the renal tubules

10
giving rise to renal tubular degeneration and this is manifested as glycosuria and

amino aciduria [9]. Excess dietary Cu causes an accumulation of Cu in the liver with a

decrease in blood haemoglobin concentration and packed cell volume. Liver function

is adversely affected in copper poisoning. Jaundice results from erythrocyte

haemolysis and this may lead to death unless treatment is started. In animals, sheep

are more susceptible than cattle to the toxic effects of copper [16].

1.4.10 Iron:

Function:
Iron functions as haemoglobin in the transport of oxygen. In cellular respiration, it

functions as essential component of enzymes involved in biological oxidation such as

cytochromes c, c1, a1, etc [9].Fe is an important constituent of succinate

dehydrogenase as well as a part of the haeme of haemoglobin (Hb), myoglobin and

the cytochromes [28].Iron is required for proper myelination of spinal cord and white

matter of cerebellar folds in brain and is a cofactor for a number of enzymes involved

in neurotransmitter synthesis [31].Iron is involved in synthesis and packaging of

neurotransmitters, their uptake and degradation into other iron-containing proteins

which may directly or indirectly alter brain function [32].Fe is required for making Hb

and it is a prooxidant which is also needed by microorganisms for proliferation [33].

Deficiency Disorders:
Deficiency disease or symptoms include anaemia, (hypochromic, microcytic). Fe

deficiency has been reported to have a role in brain development and in the

pathophysiology of restless legs syndrome [29]. Also, Fe deficiency is associated with

alterations in many metabolic processes that may impact brain functioning, among

whom are neurotransmitter metabolism, protein synthesis, organogenesis etc [32].

Early iron deficiency has also been reported to affect GABA metabolism in adult rats

[34,35].Fe accumulation has been related to some neurologic disorders such as

Alzheimer disease, Parkinson disease, type-1 neuro-degeneration with brain iron

11
accumulation and other disorders [36].Excessive accumulation of iron in the liver,

pancreas, heart, lungs and other tissues cause haemosiderosis and when this is

accompanied by bronze pigmentation of the skin, the condition is called

haemochromatosis [9,13].

1.4.11 Manganese:

Function:
Manganese is a cofactor of hydrolase, decarboxylase, and transferase enzymes [13].It

is involved in glycoprotein and proteoglycan synthesis and is a component of

mitochondrial superoxide dismutase. Manganese is a co-factor in phosphohydrolases

and phosphotransferases involved in the synthesis of proteoglycans in cartilage. Mn is

a part of enzymes involved in urea formation, pyruvate metabolism and the

galactotransferase of connective tissue biosynthesis [28]. Mn activates several

important enzyme systems and in this capacity it is required for the synthesis of acid

mucopolysaccharides, such as chondroitin sulphate, to form the matrices of bones and

egg shells. Consequently skeletal deformities and defects in shell quality occur when

the manganese intake is inadequate [37].The fact that Mn is concentrated in the

mitochondria has led to the suggestion that, in vivo, manganese is involved in the

partial regulation of oxidative phosphorylation [38].

Deficiency Disorders:
Mn deficiency has been demonstrated in several animal species including laboratory

animals, pigs, poultry, and possibly in cattle. Its severity depends greatly on the

degree and duration of the deficiency and on the maturity of the animal

[10].Manganese deficiency presents with the following signs; in pigs, lameness,

enlarged hock joints, and shortened legs, in cattle, leg deformities with over

knuckling, in chicks, poults and ducklings, perosis or slipped tendon; and in chick

embryos, nutritional chondrodystrophy. In laboratory animals, effects of deficiency

include deformities of bone, poor growth, impaired reproduction, egg shell formation,

12
and blood clotting. Some of these defects are related to the role of the manganese ion

as the most effective activator of glycosyltransferase enzymes in the synthesis of

mucopolysaccharides and glycoproteins [39].Other deficiency disease symptoms are

ataxia and abnormal formation of otoliths in the inner hear. In other species,

congenital defects in embryonic bone development result from Mn deficiency. Birds

are much more susceptible to manganese deficiency than mammals because their

requirements for this element are considerably higher and this is attributable partly to

relatively poor absorption from the intestine [37].Deficiency disease or symptoms is

unknown in humans. Mn overexposure reportedly may have an adverse effect on

central nervous system (CNS) function and mood [29].Toxicity disease or symptoms

by inhalation poisoning produces psychotic symptoms and Parkinsonism. Corn is

extremely low in manganese (4-12 ppm) and so animals fed high-corn diets especially

if supplemented with animal by-products, which are also low in manganese content,

may receive inadequate amounts. The high requirement of poultry and the low levels

of Mn in many of the ingredients of poultry diets make Mn supplementation highly

important [10].

1.4.12 Selenium:

Function:
Selenium is a constituent of glutathione peroxidase [13].It is a constituent element of

the entire defence system that protects the living organism from the harmful action of

free radicals. Organic selenium is more thoroughly resorbed and more efficiently

metabolized than its inorganic equivalent, which is poorly resorbed and acts more as a

prooxidant provoking glutathione oxidation and oxidative damage to the DNA [40,

41, 42].Se is a synergistic antioxidant with vitamin E. Its activity appears to be closely

related to the antioxidative properties of -tocopherol (vitamin E) and coenzyme Q

(ubiquinone). It enhances the overall activity of the ketoglutarate oxidase system,

probably by affecting the decarboxylation reaction. Organic and inorganic selenium

13
compounds function in preventing certain disease conditions that have in the past

been associated with vitamin E deficiency. Se prevents liver necrosis in rats, white

muscle disease in lambs, and exudative diathesis in chicks. Se protects the organism

from oxidative damage to cell membranes by destroying H2O2, whereas vitamin E

protects against damage by preventing the formation of the lipid hydro peroxides [10].

The most important metabolic role of selenium is manifested in the activities of the

selenoenzymes glutathione peroxidase (GSH-Px) and thioredoxinreductase.

According to the reports by Surai [43], over the first few days after hatching, chicks

mainly depend on selenium supplies stored in the liver. The activity of selenium and

an adequate supply of vitamin E and some enzymes like GSH-Px, superoxide

dismutase (SOD), catalase (CAT) protect chicks from numerous diseases like

encephalomalacia, exudative diathesis and muscular dystrophy [44,45].The activity of

SOD in the cells and in the extracellular fluid is very important in the prevention of

diseases closely associated with oxidative stress, for example, cardiovascular diseases,

Alzheimer’s disease, Parkinson’s disease and many other diseases [46].

Deficiency Disorders:
Se deficiency results in white muscle disease, an illness that cause high mortality in

young calves and lambs. The clinical signs are myopathy that affects both the heart

and skeletal muscle, frequently accompanied by abnormal calcification. White muscle

disease is not a serious problem in areas where the soil is high in selenium. In the mild

acute form, Se deficiency interferes with the normal growth processes of sheep and

cattle. Se deficiency also disrupts the normal reproductive process, apparently

affecting ovulation and fertilization, resulting in a higher incidence of embryonic

mortality. Se deficiency has also been associated with a high incidence of retained

placenta, resulting in delayed onset of oestrus and impaired conception [10].Se

deficiency in growing chickens cause exudative diathesis, signs like unthriftiness,

ruffled feathers appear early at the growing stage. Egg production and feed

14
conversion are adversely affected [16].Toxic levels in plants result in blind staggers in

horses and in the sloughing of hair and hoofs in horses and cattle. The animals

become lame, and death in such cases is mainly caused by starvation resulting from

movement impediment. In pigs, Se deficiency results in liver necrosis,

hepatosisdietetica, resulting in high mortality. Mulberry heart disease in swine has

been attributed to Se deficiency and is complicated by other factors other than Se

deficiency [10].In humans, toxic levels in some soils and mega dose supplementation

induces hair loss, dermatitis, and irritability [13].

1.4.13 Zinc:

Zinc is distributed widely in plant and animal tissues and occurs in all living cell

Function:
It functions as a cofactor and is a constituent of many enzymes like lactate

dehydrogenase, alcohol dehydrogenase, glutamic dehydrogenase, alkaline

phosphatase, carbonic anhydrase, carboxypeptidase, superoxide dismutase,

retinenereductase, DNA and RNA polymerase. Zn dependent enzymes are involved in

macronutrient metabolism and cell replication [10, 47]. The primary roles of zinc

appear to be in cell replication and gene expression and in nucleic acid and amino

acid metabolism. Vitamins A and E metabolism and bioavailability are dependent on

zinc status [48].It is necessary for fertility of mice. It is also required for normal

testicular development [16] and for functions of the taste buds. It is needed for tissue

repair and wound healing, plays a vital role in protein synthesis and digestion, and is

necessary for optimum insulin action as zinc is an integral constituent of insulin. It is

an important constituent of plasma [9, 13].In birds, zinc is required primarily for the

growth and development of the skeleton, the formation and maintenance of epithelial

tissue and for egg production [37].Elemental zinc prevents and cures parakeratosis

(thickening or hyperkeratinisation of the epithelial cells of the skin and oesophagus)

in swine and it prevents a similar disease in chicks. Excess calcium in diet however

15
hastens the onset of parakeratosis. Formation of zinc fingers in nuclear receptors for

steroid-thyroid, calcitriol receptors, gene expression, essential in protein synthesis,

involves in the storage and release of insulin, growth and repair of tissues,

development of sex organs, needed in the enzymes required for the synthesis of DNA

and RNA, mobilization of vitamin A from the liver and stabilization of cell

membranes [37].

Deficiency Disorders:
In humans, deficiency disease or symptoms include hypogonadism, growth failure,

impaired wound healing, decreased taste and smell acuity, secondary to

acrodermatitisenteropathica, parenteral nutrition [13].In poultry, Zn deficiency causes

growth retardation and poor feather development. The hock joints may

become enlarged and the long bones shortened and thickened. The skin on the

footpads may become dry and thickened with fissures and hyperkeratosis develops

[16].In mature hens, Zn deficiency reduces egg production and hatchability. Embryos

show a wide range of skeletal abnormalities, including micromelia, curvature of the

spine and shortened, fused thoracic and lumbar vertebrae [16].Zinc deficiency in pigs

causes a marked depression of appetite, growth rate and parakeratosis. In the young

birds a deficiency of zinc is characterized by poor growth, severe dermatitis,

especially of the feet and poor feathering, abnormal respiration, skeletal abnormalities

causing leg weakness and ataxia. The long bones are shortened and thickened and are

sometimes crooked and the joints are enlarged and rigid [37].A necrotic dermatitis

appears, particularly on the legs and feet, and feather development is impaired by

hyperkeratinisation of the epidermis. In severely deficient embryos, there are gross

faults in the development of the skeleton and entire limbs may be absent. The

biochemical lesions causing this syndrome in the osteogenic processes are probably

due partly from a reduction in the activity of alkaline phosphatase which is a zinc

enzyme [37].Toxicity disease or symptoms of zinc in humans include gastrointestinal

16
irritation, vomiting, decreased immune function and a reduction in high density

lipoprotein (HDL) cholesterol. Higher dietary levels of Zn are required in the

presence of phytic acid to prevent parakeratosis and allow for normal growth [49].

1.4.14 Fluorine:

Fluorine is widely but unevenly distributed in nature.

Function:
It increases hardness of bones and gives strength to the enamel tissues of the teeth. It

is also essential for rat growth. It plays a role in the prevention and treatment of dental

caries. Fluoride in saliva reduces cavities by reducing acid produced by bacteria and

by increasing enamel remineralisation after acid exposure. Fluoride has great affinity

for calcium and so it is associated with calcification of bones and teeth [9, 13].

Deficiency Disorders:
Low fluoride intake had been reported to cause growth retardation, reduces fertility,

and results in anaemia [50, 51].Fluoride is incorporated into the tooth enamel crystals,

where it forms the compound Fluor hydroxyapatite. This compound is more resistant

to acidic materials than the hydroxyapatite, which used to cover the tooth before.

Deficiencies of fluorine seem to increase the incidence of dental caries. It is also

involved in osteoporosis. Excessive intake of fluorine gives rise to fluorosis.

Excessive concentrations of fluorine in drinking water causes corrosion of the enamel

of the teeth, a process called mottling. Fluorine is an enzyme inhibitor and when this

happens in odontoblasts and osteoblasts, teeth and bone deformities occur. Exostoses

of the jaw and long bones develop and the joints become thickened and ankylosed,

resulting in lameness. This could further result in reduction in food consumption,

accompanied by reduced growth or weight loss [10].

17
1.4.15 Iodine:

Function:
Iodine is a trace element that is essential for the synthesis of thyroid hormones in

vertebrates, although iodoproteins are present in invertebrates [18].

Deficiency Disorders:
Some regions of the world are naturally deficient in iodine due to the low availability

of iodine from their soil or other climatic and environmental factors affecting iodine

availability. Several goitre, cretinism and the other iodine deficiency disorders (IDD)

have their main cause in the lack of availability of iodine from the soil which is linked

to a severe limitation of food exchanges [52] .Severe selenium deficiency have also

been associated with IDD in the human population, whereas in animals, it has been

proven to play a role in thyroid function either through a thyroidal or extra thyroidal

mechanism. The former involves oxidative damages mediated by free radicals,

whereas the latter implies an inhibition of the deiodinase responsible for the

utilization of T4 and T3 and is then concluded that: (i). goitre has a multifactorial

origin (ii). IDD are an important public health problem and (iii). IDD are a good

model to study the effects of other trace elements whose actions in many human

metabolisms have been somewhat underestimated. Not less than 5% of the world’s

population have goitres and many of these are associated with other disorders and

constitute a major public health problem [53].

18
 CHAPTER-2:
MATERIALS AND METHODS

2.1 SAMPLE PREPARATION:

Citrus limon fruit was collected from local market. The peels were removed and air

dried for 3 weeks. The sample was ground and stored in polythene container for

analysis.

2.2 CHEMICALS:

Conc. HNO3, Per chloric acid, Hydrogen peroxide,

Sulphuric acid, Ammonium vanadate, Ammonium molybdate, standard Sodium

dihydrogen phosphate (NaH2PO4), Standard potassium permanganate, Bromocresol

green indicator, Sodium acetate, Oxalic acid were of analytical grade.

2.3 PREPARATION OF REAGENTS:

1) Sulphuric acid (1:6, acid: water):

Add 10ml acid to 60ml distilled water.

2) Ammonium vanadate (0.25%):

Dissolve 0.25g in 50ml boiling water. Cool and then add 6ml H2SO4 (1:6) .Dilute to

100ml.

3) Ammonium molybdate (5%):

Dissolve 5g of the reagent per 100ml distilled water.

4) Standard Sodium Dihydrate Phosphate (NaH2PO4) solution:

Dissolve 30mg of the reagent in 100 ml water. This is stock standard solution.

5) Standard potassium permanganate solution (0.05N):

19
Dissolve 1.6g of the reagent in water and dilute to 1L. Standardize it against 0.05N

oxalic acid.

6) Sodium acetate solution (20%):

Dissolve 20mg of sodium acetate in 100ml of distilled water.

7) Oxalic acid solution:

Dissolve 3g of Oxalic acid in 100ml of distilled water.

2.4 ACID DIGESTION OF CITRUS LIMON FOR MINERAL

DETERMINATION:

2.4.1 Preparation of Acid Digest:

A known quantity (5g) of powdered sample was weighed accurately and transferred to

a digestion tube.25ml of Conc. HNO3 was added to it. The tube was kept in treater

digester for half an hour at 70 ℃ .The temperature was raised to 140 ℃, so that

nitrous acid fumes come out. After cooling the tube, 15ml of HNO 3:HClO4 (1:1)

mixture was added. Again the tube was heated at 200 ℃ so that the white dense fumes

of per chloric acid disappeared. The tube was then cooled and the content was

transferred to 250ml volumetric flask. The volume was raised up to the mark with de-

ionised water. The digest was stored in a refrigerator and used for mineral

determination.

2.4.2 Determination of Phosphorous:

2.4.2.1 Preparation of Standard Curve

The standard NaH2PO4 solution was diluted to different known concentrations. Then

an aliquot (5ml)was taken and placed in 50ml flask.5ml of each of the reagents

sulphuric acid (1:6,acid:water), Ammonium benedate (0.25%) and ammonium

molybdate (5%) were added successively. The flask was shaked after the addition of

each reagent. Then the mixture was diluted up to 50ml and left to develop colour.

These steps were repeated and a standard blank was prepared by using 5ml water

20
instead of standard solution. The spectrophotometer was set to zero absorbance at 470

nm with standard blank and the absorbance of the standard solution was read. A

standard curve was constructed by plotting absorbency vs concentration.

2.4.2.2 Sample Assay:

An aliquot (5ml) from the acid digest was taken and same procedure was followed as

used for the standard solution. The absorbance reading was noted and the amount of

phosphate was determined from the standard curve. The phosphate was calculated as

mg per 100g sample.

2.4.3 Determination of Calcium:

An Aliquot (5ml) from the acid digest was taken in a beaker and few drops of

bromocresol green indicator solution was added to it. Sufficient sodium acetate (20%)

was added to adjust the pH to 4.8 to 5.0 at which it is blue. It was covered with a

watch glass and heated to boiling. Slowly 3% oxalic acid solution was added, 1 drop

every 3 to 5 seconds, until the pH was reduced to 4.4 to 4.6, which is optimum for the

preparation of calcium oxalate and at which the the indicator is green. It was then

Boiled for 1 to 2 mins and allowed to stand until clear or overnight. The precipitate

formed was filtered and washed with distilled water to remove soluble oxalate.

The precipitate was transferred to a beaker. The filter paper was washed with dilute

sulphuric acid solution (50 to 60 ml). The content of the beaker was heated until the

precipitate was dissolved. It was then titrated at 70 to 90 ℃ with 0.05N Potassium

permanganate solution until a slight pink colour was obtained. The filter paper was

added and continue titration if necessary .Run a blank determination and make a

correction.

2.4.4 Determination of Trace Elements By AAS:

Trace elements were measured with a flame atomic absorption spectrophotometer

(AAS) (Perkin Elmer AA Analyst 700 Model), the most common analytical method

21
allowing the quantification of the elements at ppm levels in biological materials. An

air/acetylene flame, one hollow cathode lamp and deuterium background correction

lamp were used. Lamp current and wavelength were changed automatically

depending on the chosen metal. The standard solutions of metals were prepared by

diluting them in distilled water, and used to calibrate the AAS instrument repeatedly

throughout each set of analyses. The concentration of metals was calculated

multiplying the AAS value by 10 (dilution factor), and then dividing it by the dry

weight of the tissues. The concentration levels of Fe, Ni, Co, Cu, Zn, Mn and Cr were

measured under the conditions recommended by the manufacturer. The samples were

spiked with the analytes to test the accuracy of analysis [54-56].

22
 CHAPTER-3
RESULTS AND DISCUSSIONS

Minerals play an important role in maintaining proper function and good health in the

human body [11]. They are involved in a number of biochemical processes.

Manganese is a cofactor of hydrolase, decarboxylase, and transferase enzymes [13].

Calcium is an important component of intracellular processes that occur within insulin

responsive tissues like skeletal muscle and adipose tissue. Alteration in calcium flux

can have adverse effects on insulin secretion which is a calcium-dependent process

[9]. Phosphorus is involved in several biological processes such as: bone

mineralization, energy production, cell signalling and regulation of acid-base

homeostasis. Zinc plays a key role in the regulation of insulin production by

pancreatic tissues and glucose utilization by muscles and fat cells [11]. Copper is a

crucial trace element in redox chemistry, growth and development. Copper is being

explored as a treatment for a number of conditions, including degenerative

neurological disorders like Alzheimer’s disease, Parkinson’s disease [12]. Iron is an

essential component of haemoglobin and it is critical to the proper function of the

immune system and the production of energy [10].Thus it is important to assess the

quantity of various elements in various food items including fruits. The purpose of the

present study was to determine the mineral contents of banana empty fruit bunches to

assess their nutritional value and mineral contents. The results of this finding are

shown in table 1 and 2.

Table 1 shows the result of trace elements concentration in lemon seeds. The amount

of iron was found to be 250.95 (mg / 100 g).The concentration (mg / 100 g) Mn, Zn,

Co, Ni, Cu and Cr were 38.85, 177.6, 10.65, 23.4, 48.75 and 7.5 respectively. The

result showed iron to be the highest trace element present in lemon seeds and Cr to be

the lowest trace element present.

23
The result of macro elements concentration (table 2) showed considerable amount of

Ca (73.3 mg / 100 g) in lemon seeds. The amount of phosphorus was found to be 0.25

(mg / 100 g).

Table 1: Trace elements concentration in lemon seeds.

S.NO Name of the mineral mg /100 g

1 Fe 250.95

2 Mn 38.85

3 Zn 177.6

4 Co 10.65

5 Ni 23.4

6 Cu 48.75

7 Cr 7.5

Table 2: Macro element concentration (mg / 100 g) in lemon seeds.

S.NO Name of the Mineral mg / 100 g

1 Ca 73.3

2 P 0.25

24
 REFERENCES

[1] Sadaf S., Bhatti H. N., Iqbal Z., Shahid M., J. Chem. Soc. Pak. 3 838-843.
(2009)

[2] Singh G., Singh O., Nat. Prod. Radiance 8-21. (2002)

[3] Ali J., Abid H., Hussain A., J. Chem. Soc. Pak. 32 83-86. (2010)

[4] Hamdan D., El-Readi M. Z., Tahrani A., Herrmann F., Kaufmann D., Farrag
N., El-Shazly A., Wink M., Food Chem. 127 394-403. (2011).

[5] Aleson-Carbonell L, Ferna´ndez-Lo´pez J, Pe´rez-A´lvarez JA, Kuri V.

Characteristics of beef burger as influenced by various types of lemon albedo.
Innovat Food SciEmerg Tech, 6(2): 247–255. (2005).

[6] Ferna´ndez-Gine´s JM, Ferna´ndez-Lo´pez J, Sayas-Barbera´ E, Sendra E, Pe

´rez-A´lvarez JA. Lemon albedo as a new source of dietary fiber: Application
to bologna sausages. Meat Sci, 67(1): 7–13. (2004).

[7] Aleson-Carbonell L, Ferna´ndez-Lo´pez J, Sayas-Barbera´ E, Sendra E, Pe

´rez-A´lvarez JA. Utilization of lemon albedo in dry-cured sausages. J Food
Sci, 68(5): 1826–1830. (2003).

[8] . Mar›´n FR, Frutos MJ, Pe´rez-A´lvarez JA, Mart›´nez-Sa´nchez F, Del Rio
JA. Flavonoids as nutraceuticals; structural related antioxidant properties and
their role on ascorbic acid preservation. Page 741–778 in: Atta-Ur-Rahman
(Ed.), Studies in natural products chemistry, Amsterdam, Holland: Elsevier.
(2002).

[9] MalhotraVK . Biochemistry for Students. Tenth Edition. Jaypee Brothers

Medical Publishers (P) Ltd, New Delhi, India.(1998).

[10] Hays VW, Swenson MJ . Minerals and Bones. In: Dukes’ Physiology of
Domestic Animals, Tenth Edition. 449-466.(1985).

25
[11] Bhat, R., Kiran, K., Arun, A. B. and Karim, A. A. Determination of mineral
composition and heavy metal content of some nutraceutically valued plant
products. Food Analytical Methods 3:181-187. (2010).

[12] Eruvbetine D Canine Nutrition and Health. A paper presented at the seminar
organized by Kensington Pharmaceuticals Nig. Ltd., Lagos on August (21,
2003).

[13] Murray RK, Granner DK, Mayes PA, RodwellVW . Harper’s Biochemistry,
25th Edition, McGraw-Hill, Health Profession Division, USA.(2000).

[14] Albion Research Notes. A compilation of vital research updates on human

nutrition, 5: 2, Albion Laboratories, Inc. May, (1996).

[15] Sharma S, Malhotra P, Bhattacharyya AK . Effect of electroplating industrial

waste on ‘‘available phosphorus’’ of soil in relation to other physio-chemical
properties. Afr. J. Environ. Sci. Technol. 2(9): 257264.(2008).

[16] Merck VM . The Merck Veterinary Manual. Sixth Edition. A handbook of

diagnosis, therapy and disease prevention and control for the veterinarian.
Published by Merck and Co., Inc., Rahway, New Jersey, USA.(1986).

[17] Streeten DHP, Williams EMV.Loss of cellular potassium as a cause of

intestinal paralysis in dogs. J. Physiol. 118: 149-170.(1952).

[18] Frieden E Biochemistry of the essential ultra-trace elements. Plenum press,

New York.(1984).

[19] Uppala RT, Roy SK, Tousson A, Barnes S, Uppala GR, Eastwood Da
Induction of cell proliferation micronuclei and hyperdiploidy /diploidy in the
mammary cells of DDT and DMBA-treated pubertal rats. Environmental and
Molecular Mutagenesis 46: 43-52. (2005).

[20] Eastmond DA, MacGregor JT, Slesinki RS Trivalent Chromium: Assessing the
genotoxic risk of the essential trace element and widely used human and
animal nutritional supplement. Crit. Rev.Toxicol. 38: 173-190. (2008).

[21] Brown M Harnessing chromium in the fight against diabetes. Drug Discovery
today 8: 962-963. (2003).

26
[22] Wennberg A Neurotoxic effects of selected metals. Scand. J. Work. Environ.
Health 20: 65-71.(1994).

[23] Roginski EE, Mertz W Effects of chromium supplementation on glucose and

amino acid metabolism in rats fed a low protein diet, J. Nutr. 97: 525-530.
(1969).

[24] Mertz W Chromium as a dietary essential for man. In: WG Hoekstra et al

eds., Trace Element Metabolism in Animals, 2nd ed. University Park Press,
Baltimore. (1974).

[25] Juturu V, Komorowski JR Chromium Supplements, glucose and insulin

responses. Am. J. Clin. Nutr. 78: 190. (2003).

[26] Langard S Chromium, in Metals in the Environment, H.H.Waldron (ed,),

Academic Press, London. 111-132.(1980).

[27] Arinola OG, Nwozo SO, Ajiboye JA, Oniye AH Evaluation of trace elements
and total antioxidant status in Nigerian cassava processors Pak. J. Nutr. 7(6):
770-772.(2008c).

[28] Chandra RK Micro-nutrients and immune functions: An overview. Annal New

York Acad. Sci. 587: 9-16.(1990).

[29] Tan JC, Burns DL, Jones HR Severe ataxia, myelopathy and peripheral
neuropathy due to acquired copper deficiency in a patient with history of
gastrectomy. J. PaenteralNutr. 30: 446-450.(2006).

[30] Gardea-Torresdey JL, Becker-Hapak MK, Hosea JM, Darnall DW. Effect of
chemical modification of algae carboxyl groups on metal ion binding. Environ.
Sci. Technol. 24: 1372-1378.(1990).

[31] Larkin EC, Rao CA. Importance of fetal and neonatal iron; adequacy for
normal development of central nervous system, In: Brain behaviour and iron in
the infant diet (Dobbing J ed), London, UK 43-63.(1990).

[32] Beard JL Iron biology in immune function, muscle metabolism and neuronal
functioning, J. Nutr. 131: 5685-5695.(2001).

27
[33] Galan P, Viteri F, Bertrais S, Czernichow S, Faure H Serum concentrations of
beta carotene, vitamins C and E, zinc and selenium are influenced by sex, age,
diet, smoking status, alcohol consumption and corpulence in a general French
adult population. Eur. J. Clin. Nutr. 59: 1181-1190. (2005).

[34] Youdim MBH, Ben Shacher D, Yehuda S Putative biological mechanism of

the effect of iron deficiency on brain biochemistry and behaviour. Am. J. Clin.
Nutr. 50: 607-650.(1989).

[35] Taneja V, Mishra K, Agarwal KN Effect of early iron deficiency in the rat on
gamma amino butyric acid shunt in brain. J. Neurochem. 46: 1670-1674.
(1986).

[36] Sadrzadeh SM, Saffari Y Iron and brain disorders. Am. J. Clin. Pathol, 64-70.
(2004).

[37] Gordon RF Poultry Diseases. The English Language Book Society and
BailliereTindall, London.(1977).

[38] Gruden N Suppression of Trans duodenal manganese transport by milk diet

supplemented with iron, Nutr. Metab. 21: 305-309. (1977).

[39] Leach RM Jr. Biochemical role of manganese. In: WG Hoekstra et al eds.,

Trace Element Metabolism in Animals, 2nd ed. University Park Press,
Baltimore. (1974).

[40] Levander O Considerations in the design of selenium bioavailability studies.

Fed. Proc. 42:1721-1725.(1983).

[41] Schrauzer GN Selenometionine: A role of its nutritional significance,

metabolism and toxicity. J. Nutr. 130: 1653-1656.(2000).

[42] Wycherley BJ, Moak MA, Christensen MJ High dietary intake of sodium
selenite induces oxidative DNA damage in rat liver. Nutr. Cancer 48: 78-83.
(2004).

[43] Surai PF Selenium in poultry nutrition 2. Reproduction, egg and meat quality
and practical applications. World Poult.Sci. J. 58: 431450.(2000).

28
[44] Combs Jr. GF Influence of dietary vitamin E and selenium on the oxidant
defence system of the chick. Poultry Sci. 60: 2098-2105.(1981).

[45] Hassan S, Hakkarainen J, Jonson L, Tyopponen J Histopathological and

biochemical changes associated with selenium and vitamin E deficiency in
chicks. J. Vet. Med. Assoc. 37:708-720. (1990).

[46] Pollack M, Leeuwenburgh C Molecular mechanisms of oxidative stress in

aging: free radicals, aging, antioxidant and disease. In: Handbook of Oxidants
and Antioxidants in Exercise (Sen CK, Packer L, Hinninen O. Eds). Elsevier
Science, BV, 881-923.(1999).

[47] Arinola OG Essential trace elements and metal binding proteins in Nigerian
consumers of alcoholic beverages. Pak. J. Nutr. 7(6): 763-765.(2008).

[48] Szabo G, Chavan S, Mandrekar P, Catalano D Acute alcoholic consumption

attenuates IL-8 and MCP-1 induction in response to ex vivo stimulation. J.
Clin. Immunol. 19:67-76. (1999).

[49] Sidhu P, Gorg ML, Morgenstern P, Vogt J, Butz T, Dhawan DK Role of Zinc
in regulating the levels of hepatic elements following nickel toxicity in rats.
Biological Trace Elements Res. 102: 161-172. (2004).

[50] Messer HH, Armstrong WD, Singer L Essentiality and function of fluoride. In
WG Hoekstra et al., eds., Trace Element Metabolism in Animals, 2nd ed.
University Park Press, Baltimore.(1974).

[51] Cerklewski FL Fluoride: Essential or just beneficial. Nutr. Rev. 14: 475-476.
(1998).

[52] Thilly CH, Vanderpas JB, Bebe N, Ntambue K, Contempre B, Swennen B,

Moreno-Ryes R, Bourdoux P, Delange F Iodine deficiency, other trace
elements and goitrogenic factors in the ethiopathogeny of iodine deficiency
disorders (IDD). Biological Trace Elem. Res. 32(13):229-243. (1992).

[53] Gaitan E Goitrogens in food and water. Annual Rev. Nutr. 10: 21-37.(1990).

29
[54] Bağ, H., Lale, M., and Türker, A.R. Determination of Cu, Zn and Cd by FAAS
after preconcentration by baker’s yeast (Saccharomyces cerevisiae)
immobilized on sepiolite, Fresenius J Anal Chem. 363, 224--230. (1999).

[55] Bağ, H., Türker, A.R., Coşkun, R, Saçak, M. and Yiğitoğlu, M. Determination
of zinc, cadmium, cobalt and Ni by flame atomic absorption spectrometry after
preconcentration by poly (ethylene terephthalate) fibers grafted with
methacrylic acid, Spectrochim. Acta B. 55, 1101--1108.(2000).

[56] Tunçeli, A., Bağ, H. and Türker, A.R. Spectrophotometric determination of

some pesticides in water samples after preconcentration with Saccharomyces
cerevisiae immobilized on sepiolite, Fresen. J. Anal. Chem.3 71, 1134--1138.
(2001).

30