determination of calcium content in animal bones

Description

ABSTRACT

 

The samples are femur, vertebrate column (Lumber), skull, and cervical vertebrate (Thoracic), rib and pelvic girdle bones. They were all collected from one ram slaughtered at Kano abattoir. The samples have undergone several steps of treatment which include removal of flesh, crushing, defatting and drying. The samples were ashes in a murple furnace at 7500C for 5 hours. The calcium contents in the samples were determined by the use of Atomic Absorption Spectrophotometer (AAS). The results were 255mg/kg, 240mg/kg, 200mg/kg, 240mg/kg, 220mg/kg, and lastly 230mg/kg, for femur, vertebrate column, skull, cervical vertebrate, rib and pelvic girdle bones respectively.

TABLE OF CONTENTS

CONTENT    PAGE

Title………………………………………………………………………………….. i

Declaration……………………………………………………………………….. ii

Approval………………………………………………………………………….. ii

Acknowledgement………………………………………………………………. iii

Dedication………………………………………………………………………… iv

Table of Contents……………………………………………………………….. v

List of Tables…………………………………………………………………… vi

Abstract…………………………………………………………………………… vii

CHAPTER ONE

 

1.0      INTRODUCTION AND LITERATURE REVIEW……………..……..1

1.1 Introduction…………………………………………………………………..1

1.1.1 Bone……………………………………………………………………………………………………………………………………………… 2

1.1.2 Calcium………………………………………………………………………4

1.1.3 Basic Information on Sheep…………..…………….………………….6

1.2 Literature Review………………………..…..………….…………………..9

CHAPTER TWO

2.0 EXPERIMENTAL

2.1 Sample Collection…………………..……………………………………..12

2.2 Sample Treatments…………………………………………….………….12

2.2.1 Removing of Flesh ………………………………………………………12

2.2.2 Crushing…………………………………………………………………..12

2.2.3 De Fatting………………………………………………………………….12

2.2.4 Draying……………………………………………………………………..13

2.3 Ashing………………………………………………………………………….13

2.4 Reagents……………………………………………………………………….13

2.4.1 Diethyl ether………………………………………………………………..13

2.4.2 0.1Mole of Nitric Acid…………………………………………………….13

2.4.3 Standard Calcium Solution……………………………………………..13

2.5 Calcium Determination……………………………………………………..14

CHAPTER THREE

3.0 RESULTS AND DISCUSSION

3.1 Results……………………………………………………………………….15

3.2 Discussion…………………………………………………………………..16

3.3 Conclusion…………………………………………………………………..18

3.4 Recommendations for Further Work………………..…………………19

References………………………………..….……………………………….20

Appendix……………………………………………………………………….21

LIST OF TABLES

Table: 1 Absorbance values for calcium standard and

concentration……………………………………………………………….15

Table: 2 concentration of calcium in bone samples………………..16

CHAPTER ONE

1.0   INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction

1.1.1 Bone

Bones are rigid organs that form part of the endo-skeleton of vertebrates. Bone plays a vital role on movement, support and protection to the various organs of the body. It produces red and white blood cells and also store minerals. Bone tissue is a type of dense connective tissue. Because bones come in a variety of shapes and have a complex internal and external structure, they are light weight, yet strong and hard, in addition to fulfilling their many other function. (Stele et al., 1988).

Components of Bone

Bone is a connective tissue largely composed of an organic protein called collagen and the inorganic mineral called hydroxyapatite. One of the types of tissue that makes up bone is the mineralized osseous tissue, also called bone tissue that gives it rigidity and a honey comb-like three dimensional internal structure. Other types of tissue found in bones include marrow, endosteum and peridsteum, nerves, blood vessels and cartilage.

According to Prashanth, (2009), the collagen and hydroxyapatite combine together and provide mechanical and supportive role in the body.

Collagen

Around 30% of bone is composed of an inorganic compound of which 90 to 95% is collagen. Collagen is a fibrous protein which provides the bone with strength and flexibility and is an important component of many other tissues. It contains three (3) polypeptides of about 1000 amino acid per chain with a high glycine and hydroxyproline content. For a collagen be fully matured a number of chemical bond must formed and these include hydrogen bond that involves hydrocylsien and lysine which are stabilized in tabular structure. These processes occur throughout the growth an maturity of bone. Collagen molecules are composed of linear entrench sequence of approximately 20 naturally occurring amino acid of bone residue and carboxyl of another residue resulting in both acidic and basic properties.

Hydroxyapatite

About 70% of bone is made up of the inorganic minerals, hydroxyapatite. This is composed of calcium phosphate, calcium carbonate, calcium hydroxide and citrate. These inorganic components are predominately crystalline, though they may be present in amorphous form. The crystals are platelets or rods of about 8–15A thick, 20-40A wide and 200-400A long.

The substitution mechanisms that occurs in the hydroxyapatite of bone include intercrystalline exchanges and recrystallisation due to dissolution and reformation of crystals to replace calcium ions that are being absorbed/adsorbed on the crystal surface (Smith et al., 1983).

Functions of Bone

Bone is hard part of the body that forms skeleton of human or animal body which gives support and protection to the body. (Oxford Advance Leaner’s Dictionary, 7th Edition, 2011). Functions of bone include the following.

Protection

Bones serve as protection to internal organs such as skull for brain protection, ribs for heart and lungs protection.

Shape

Bone provides a frame structure and keeps the body well supported.

Movement

Bones, skeleton, muscles, tendons, ligaments and joints function together to generate and transfer forces, so that the body will move (Steele et al., 1988).

Sound Transduction

Bones are important in the mechanical aspect of over shadowed hearing.

Blood Production

Blood is produce in a long bone containing what is called bone marrow and the process of producing is known as hematopoiesis.

Mineral Storage

Bone act as a reservoir of important minerals for the body. Most notably calcium and phosphorus.

(vii)   Growth Factor for Storage

Mineralized bone matrix also store important growth factor such as insulin, bone phogenetics, protein and others.

(viii) Fat Storage

The yellow bone marrow act as a storage reserve of fatty acid.

Acid Base Balance

Bone buffers the blood against excessive pH change by absorbing/releasing alkaline SaH.

Detoxification

It can also store a heavy metal and others foreign element removing them from blood so as to reduce their effect on other tissues. These later be gradually release through excretion.

Endocrine Organ

Bone controls phosphates metabolism by releasing fibroblast that is a growth factor which act on kidney to reduce phosphate re-absorption. (Steele et al., 1988).

1.1.2 Calcium

This is a mineral that help build strong bones and teeth while you need calcium throughout your life. Amount you need changes over time you need a lot of calcium during your growing years to build strong bone.

The most abundant mineral in the body is calcium and it’s found in some foods added to others available as dietary supplement and also present in some medicines (such as antacid). Calcium required for vascular contraction and vasodilatation muscles function; nerve transmission, intracellular signaling, and hormonal secretion. Through, less than 1% of total body calcium is needed to support these critical metabolic functions. Serum calcium is very tightly regulated and does not fluctuate with change in dietary intake, the body uses bone tissue as a reservoir for and resources of calcium to maintain to constant concentration of calcium is blood, muscles and intercellular fluids.

The remaining 99% of the calcium supply is stored in the bone and teeth where it supports their structure and function. Bone itself undergoes continues remodeling with constant re-absorption and deposition of changes with ages. Bone formation exceed re-absorption in period of growth in children and adolescence where as in early and mid-adult particularly among postmenopausal women, bone breakdown exceed formation resulting in tone loss that increase the risk of Osteoporosis overtime (Legros et al., 1987).

In a strictly operational sense, calcium balance is determined by the relationship between calcium intake and calcium absorption and excretion. A striking feature of the system is that relatively small change in calcium absorption and excretion can neutralize a high intake or compensate for a low one. There is a wide variation in calcium intake among nations generally following the animal protein intake and depending largely on dietary production / consumption. The lowest calcium intakes are in developing countries, particularly Asia, and the highest are in developed countries particularly in USA, Canada and Europe. Other issues on calcium include the following.

(a) Calcium Deficiency

In adequate intake of dietary calcium from food and supplement produce no obvious symptoms in the short term. Circulating blood level of calcium are tightly regulated hypocalcemia result primarily from medical problems or treatment including renal failure surgical removal of the stomach and use of certain medication (such as dieuretics) symptom of muscles cramps, convulsion, lethargy, poor apatite and abnormal heart rhythms; if left untreated calcium deficiency may lead to deat

Over the long term inadequate calcium intake causes Osteopenia which, if left untreated can lead to Osteoporosis and the risk of tone fracture also increase in calcium deficiency can also cause rickets in children though it’s more commonly associated with vitamin D deficiency (Legross et al, 1987).

(b) Chemistry and Distribution of Calcium

Calcium ion is a divalent cation, while the atomic weight is 40 and atomic number of 20. It belongs to group two in the periodic table, calcium as an element is a gray silvery metal that is hard and is essential constituent of leaves, bones and teeth. Calcium is classified chemically as one of the alkaline earth metals (Gillespia, R., 1989),.

1.1.3 Basic Information on Sheep

According to taxonomy that is classification of organisms into group’s base on similarities of origin and structure sheep are classified in a tabular form below as follows:-

KINGDOM	Animalia	Animals
PHYLUM	Chordata	Animals that possess spinal cord
SUB-PHYLUM	Vertebrate	Animals that possess back bone
CLASS	Mammalia	Animal that feeds with breast and
		nurses
ORDER	Ungulata	Hoofed Animals
SUB-ORDER	Artiodactilia	Animals that possess toe
FAMILY	Bovidea	Animals   that   possess   hallow
		horns
	15

 

SUB-FAMILY	Caprinae	Sheep and Goat
GENIUS	Ovis	Sheep
SPECIES	Aries	Domestic Sheep

 

Habitat of a Sheep

Sheep is a domestic animal. It adopts to live with people.

Life Expectancy

This means how long an organism is expected to live and the life expectancy of a sheep is about 10-12years, but some tend to live more than that. E.g. According to the Guinness Book of Record the oldest sheep lived 23year.

FOOD: Mostly, sheep eat grasses, clovers, fords and other pasture plants. They also eat variety of plant especially more notorious diet.

Some Types of Bone in Sheep

With regard to this research project the content of calcium in different type of bones in a particular animal is to be determined so there is need to explained or define the type of bones that are going to be used. The definitions are as follows:-

The Cervical Vertebrate

This is commonly known as neck-bone. It’s found in the neck region of all mammals and their characteristics feature are presence of transverse process of vertebraterial nodes on each side, round opening at the centre serve as the passage of spinal cord and the possession of a small neural spine. Smith et al. (2003)

The Skull

The skull bone consist of two major parts namely the cranium and facial bone. Here the major concern would only be the cranium which is made up of a flat and curved bone that are firmly fitted together by the saw-like edges called suture. The main bones making up the cranium are frontal perital, temporal and occipited bone. This cranium is commonly known as train box bone i.e. it encases and protects the brain. Smith et al. (2003)

 

Vertebrate Column

This is also called back-bone and it’s made up of 33hort bones. Each bone is called a vertebra also they enclose and protect the spinal cord. They support skull and provide the attachment sites for muscles. Smith et al. (2003)

Pelvic Girdle

The pelvic bone consist of ilium, ischium and the pubis all of which are fused together on each side of the body as the in nominate bone. The distal end of pelvic girdle form a cavity called acetabullum into which the head of femur bone fits to form the ball and socket hip joint. Smith et al. (2003)

Femur Bone

This is the longest bone of mammal and it has a cylindrical shaft and two extrimites. The proximal extremities that bears a head with a separating patella groove. It works with massive muscles play a key role in locomotion and also in maintaining up right posture. (Legros et al., 1987).

Rib Bone

This is bony and cartilaginous structures which surrounds the thoracic cavity and support the pectoral girdles forming a core portion of the human skeleton. Rib bone is also called a thoracic cage and provides attachments for the muscles of neck, thorax, upper abdomen and back according to White folkens et al, (2005).

1.2 LITERATURE REVIEW

Studies were conducted to investigate the effect of dietary calcium, fats, and cholesterol lipid metabolism. In an animal study, 72 males retired sheep breeders were fed with two different source of calcium, (calcium carbonate and calcium lactate), two level of fats (5 and 15 %), and two level of cholesterol (0 and 2 %) in a factorial arrangement of the treatments. No significant effect was found on wet bone of femur. The highest bone strength was found in a sheep fed with calcium lactate while sheep fed with high fat diet gained more weight and were more efficient. So high fat diet in combination with calcium carbonate resulted in animals with heavier bone weight but less bone calcium contents. A high fat level with calcium lactate produced bone with higher calcium concentration. Lastly, the results of these experiments suggest a possible interaction of calcium status growth, body composition, and blood serum lipids patterns (Vilka, 1987).

 

A project was conducted to evaluate the calcium content in a bone of a dairy cow using bone mineral content (BMC) and a bone mineral density (BMD). The effect of parity and stage of lactation on measures of BMC of the fused 3rd and 4th metacarpal bone and of vertebrate bone 14 and 15 were assessed. The sample were analyze for BMC and BMD with a dual X-ray absorption (DXA). The result of the experiments shows that in vertebrate 14 and 15 bone, increasing stage of lactation decreases energy to peak load with the lowest value observed in a late lactation and these shows that stage of lactation has no effect on calcium content of BMC that is measured chemically in vertebrate or metacarpal bone. The result shows that parity did not affect the strength of calcium content in metacarpal and vertebrate bones in matured dairy cattle. (Beth et al., 2006)

A study was carried out to assess the relationship between chemical measures and imaging estimate of bone mineral content of calcium in diary sheep using radiographic photometry. In a preliminary study, the vertebrate column bone were collected from 33 sheep’s following slaughter and the samples were analyze and group by parity (1, 2, 3 and 4) stage of lactation. In the experiments, the relationship between mineral content of calcium within the bones was poor, likely because the relative maturity of animals in the samples are set and also lack of variation in the mineral contents of the sheep. Lastly, result shows that calcium contents are higher in matured animals. So there is no effect of stage of lactation on bone mineral of the samples. (Keene Et al., 2004)

 

In vivo sampling, membrane proves provide a tool for investigating the chemistry of calcium in bones. It has provided a data which have expanded the understanding of both physiological and pathological condition in the samples. In a project, a femur and pelvic bones of a sheep were used as samples of animal model and they were analyzed by spectrophotometric analysis using the O-cresolphtalein complex method. The result of the study determined the total ultrafiltration of calcium concentration of the femur bone is 3.21mg/dl and for the pelvic is 2.97mg/dl. The capillary ultrafiltration probes were shown to be a useful tool for monitoring chemical dynamics in the intracellular development of anti-osteoporosis drugs and countermeasures to micro gravity induced bone loss. (Elsa Et al., 2009)

Studies from the early decades of the 20th century identified that various animals mode fed with low diet calcium food demonstrate a negative calcium balance and gross bone loss. Oliver et al, (1999)

Bauer et al, (2009) studied the effect of low calcium content on sheep by visual inspection of a longitudinal section of a femur bone. They found that significant bone loss was evident from the trabecular bone compartment of bone in contrast to cortical bone and concluded that the trabecular compartment of bone tissue was the most active compartment of the skeleton system using the technique of bone ash weight.

The result of a study identified that feeding low level of dietary calcium produces negative calcium balance and gross bone loss. It also shows calcium requirements in the oestrogen deficient and calcium deficient in overall bone loss. (Howard et al, 2010)

Pratash (2011) conducted a project to investigate the calcium contents of bones and the project expected to be a very good solution to problems of bone diseases like osteoporosis. The sample used were rib bone of a sheep to estimate calcium content using a flame photo meter test and the result yield 4.20ppm of calcium contents in the rib bone samples. (Pratash Gajni, 2011)

A study to compare the content of calcium in antlers and cranial bones of European red deer from western Poland was carried out. The bone samples were obtained from 3 forestry district that differs in environmental pollution within the age of 2 to 4 years respectively. The skull bones were powdered and dissolved in a nitric acid and are measured using (AAS). The result shows that content of calcium were higher in antlers (mean 133.96 mg/g) as oppose to cranial bone of a deer (mean 123.79mg/g). Lastly, the statistical analysis revealed significant differences which depend on ages of the animals and the level of industrial pollution in the animal’s habitat. (Nowichka etal., 2006).