CHAPTER ONE INTRODUCTION AND LITERETURE REVIEW

1 .1Introduction
The hypothesis of this research is that cigarette smoking affects the calcium metabolism, and this is increased with the number of cigarettes smoked per day and the duration of smoking per year According to the world health organization (WHO) 75% of death from chronic obstructive pulmonary disease that occur in developed countries are directly related to smoking tobacco ( CPOD) is characterized by abnormalities in the lung that make it difficult to exhale normally (1) Cigarette use has been identified as a risk factor for low bone mineral density (BMD) and osteoporosis fracture (2-8) Generally two distinct diseases are involved emphysema and chronic bronchitis. Tobacco use is the number one risk factor for COPD and heavy smokers are at great risk than cigar and pipe smokers(9) Many of the health problems caused by the use of tobacco are well known. Additionally, several research studies have identified smoking as a risk factor for osteoporosis and bone fracture. (10) Risk factors for developing osteoporosis includes: smoking and not getting enough calcium from diet (10) Cigarette smoking was first identified as a risk factor for osteoporosis more than 20 years ago. Recent studies have shown a direct relationship between tobacco use and decreased bone density. (10)

Smoking is a key lifestyle risk factor for bone loss and fractures that appear to be independent of other risk factors for fracture such as age, weight, sex and menopausal status. Despite smoking being a major lifestyle risk factor for osteoporosis, the mechanisms underlying smoking-associated bone loss and fracture risk remain poorly understood. (11) Smoking is associated with reduce bone density and calcium absorption and reduce serum level of vitamin D a compensatory increase in serum PTH level would therefore be expected as a result of an altered calcium balance. However, reports on PTH levels in smokers are conflicting. As serum PTH levels give important information on the calcium balance, the PTH levels in smokers are of interest (12) Smoking is a major health hazard with detrimental effect on many organs including the skeleton thus several authors have reported an association between smoking and fracture risk density (13) There are several previous reports on the relation between smoking and serum calcium, many authors made a study on the secretion of calcium in the saliva of long term tobacco users, they found significantly higher level of calcium in the saliva of all tobacco users as compared to non users (13). Smoking has multiple effects on hormone secretion, some of which are associated with important clinical implications. These effects are mainly mediated by the pharmacological action of nicotine. Smoking affects calcium metabolism and the action of insulin. Osteoporosis and reduced fertility. (14)
(13)

as well as low bone

1.2 Literature review 1.2.1Smoking:

Smoking is a practice where a substance, most commonly tobacco is burned and the smoke is tasted or inhaled, this is primarily done as a form of recreational drug use (15) Most common method today is through cigarette primarily industrially manufactured but also hand rolled from loose tobacco and rolling paper .other form through not as common are pipes, cigar, hookahs and bongs (15) Tobacco smoking is today by far the most popular form of smoking and is practiced by over one billion people in the majority of all human societies, less common drugs that are smoked are considered to be addictive, some are hard as heroin and crack cocaine, but the use of these are very limited and they are often not commercially available. (15)

1.2.1.1Types of tobacco products

By the end of the twentieth century, manufactured cigarettes have come to be the predominant form in which tobacco is consumed around the world (15, 16), it is also consumed in much other form, and some of the major forms of tobacco products currently in use are:

(A) Smoking tobacco

Cigarettes:

Cigarette (French small cigar from cigar + ette ) is a product consumed through smoking and manufactured out cured and finally cut tobacco leaves and reconstituted tobacco often combined with other additives , A cigarette is distinguished from a cigar by its smaller size, use of processed leaf, and white paper wrapping. Cigars are typically composed entirely of whole-leaf tobacco. (17) Manufactured cigarettes are available in all countries, and filtertipped cigarettes are usually more popular than plain-end cigarettes .handmade or "roll you- own cigarettes are also widely used in many countries (15, 16, 17) Other types of cigarettes include kreteks (clove- flavored cigarettes) sticks Bides: Bid consists of a small amount of tobacco (0.2 - 0.3 g) wrapped in temburine leaf and tied with a small string. It is estimated that in the 1989 And 1990s about 675.000 millions bides were smoked annually in India, 50 .000 million in Bangladesh and about 25 .000 million in other countries (15) Cigars: Cigar are made of air cured and fermented tobaccos with a tobacco wrapped, and come in many shapes and size (15) Pipes: A pipe is a tool used for smoking. The designs of pipes vary considerably, but for the most part they are reusable and consist of a chamber, or bowl, in which the substance to be smoked is placed, a stem of some sort and a mouthpiece through which the smoke is sucked or inhalation ,prior , slate and clay pipes have been used throughout Europe
4

and the Americas . Water pipes are in common use in North Africa, the Mediterranean region and Asia. Having many names e.g. hoolah , goza hubble and shisha (15).

(B) : smokeless tobacco

Chewing tobacco: Plug loose leaf and twist their use remains popular in certain sub populations Pan: Pan chewing is widely practiced in south East Asia especially in India. Snuff: Dry snuff is powdered tobacco that is inhaled through the nasal passage or taken orally. Most snuff is taken orally. A small amount of ground tobacco is helied in the mouth between the cheek and the gum. Other smokeless tobacco products are in use. One of these is khaini , mishri , zarda, kiwan and pills . These products, as well as others tobacco preparation known as gudakha . bajar and creamy snuff are often used for clearing teeth(18).

1.2.1.2 Smoking prevalence

WHO estimate that there are about 1100 million smokers in the world representing about one third of the global population aged 15 years and over. The majority of the smokers are in developing countries (800 million) and most of these are men (700 million). In china alone there are about 300 million smokers (90 % men , 10% women ) about the same number as in all developed countries combined . About one third of regular smokers in developed countries are women compared with only about 1 in 8 in developing countries (18).

Globally it is estimated that 47% of men and 12% of women smoke in developed countries, the corresponding figures are 42% for men and 24% for women. In developing countries, available data suggests that about 48% of men and 7% women smoke, male smoking prevalence varies substantially among regions from less than 30 %in Africa region to 60%in western pacific region. Even among developed countries, patterns of male smoking prevalence are not uniform in countries with established market economies, male smoking prevalence average 37% compared to 60%in former socialist countries of central and Eastern Europe (18, ). Smoking among women is most prevalent in former socialist, countries of central and Eastern Europe (29%), countries with established market economies (23%) and Latin American and Caribbean countries (21%) in most other countries fewer than 15 % of women smoke 17 old) In many countries people begin to smoke at younger ages, with the median age of initiation under 15 yrs. starting to smoke at younger ages increase the risk of death. Young people who start smoking early in life will often find it difficult to quit smoking. Among those who continue to smoke throughout their lives, about one half can be expected to die from a smoking related issue, half of these in the middle age (35 69) and the other half in old age (70 yrs and over).In countries, where more than 50%of young men are smokers aged 18 - 24 years and most of them began at a young age, a very heavy future death from tobacco use can be expected (18,19). In general fewer cigarettes are smoked per day in developing countries than in developed countries (21), among developed countries the highest rate of daily consumption per smoker, 24 cigarettes per day: occur in countries with established market economies. (21)

1.2.1.3. Tobacco and health:

6

Several decades of epidemiological research have identified cigarette smoking as a major cause of preventable mortality in developed countries. For smokers, the magnitude of the risk increase with increasing duration of smoking. data from the mid of 1980s confirm that among smokers age 35 69 , the death rate is three times that of non smoker (22). Among men in developed countries, smoking is estimated to be the cause of 40-45% of all cancer death, 90-95% of lung cancer death, 75%of chronic obstructive lung disease death, just over 20%of vascular diseases deaths, and 35%of cardiovascular disease death in those aged 35-65 years (22). Of all the diseases usually associated with smoking, lung cancer is the most well known, simply because in most populations almost all lung cancer deaths are due to smoking. However, smoking actually causes more deaths from diseases other than lung cancer. In 1995.there were 541.000 smoking attributable lung cancer deaths in developed countries, (22) compared to 625.000 smoking attributable deaths from heart and vascular disease in the same year. The use of smokeless tobacco , (common among both men and women in areas such as south Asia, also poses serious heart risk, most notably elevated risks of cancer of the buccal cavity. The annual mortality from tobacco chewing in south Asia alone is of order of 50.000 deaths a year (22). In addition epidemiological evidence suggest that cigarette smoking is the major risk factor for chronic obstructive pulmonary diseases such as chronic bronochitis and emphysema for carcinogenesis and for cardiovascular disease (23, 26), however, the precise mechanisms of these effects are incompletely understood (25, 26). Environmental tobacco smoke contains most of the toxic and carcinogenic compounds identified in mainstream smoke, absorption of
7

smoke constituents from the environment has been documented in both infants and adults, and a number of epidemiological studies have demonstrated health effect in human(27,28).

1.2.1.4. Cigarette smoking:

Cigarette smoking is addictive because of the presence of alkaloid, nicotine, and its nicotine withdrawal that causes the unpleasant side effects after quitting smoking (29). Nicotine is an alkaloid found in night shade family of plant (solanaceose) which constitutes approximately 0.6 3.0 % of dry weight of tobacco (30, 31). As nicotine enters the body, it is distributed quickly through the blood stream and can cross the blood-brain barrier. On average it takes about seven seconds for the substance to reach the Brain when inhaled. The half life of nicotine in the body is around two hours
(32)

, the amount

of nicotine inhaled with tobacco smoke is a fraction of the amount contained in the tobacco leaves. The amount of nicotine absorbed by the body from smoking depends on many factors, including the type of tobacco, whether the smoke is inhaled, and whether a filter is used. For chewing tobacco, dipping tobacco and snuff, which are held in the mouth between the lip and gum, or taken in the nose, the amount released into the body tends to be much greater than smoked tobacco. Nicotine is metabolized in the liver by cytochrome P450 enzymes (mostly CYP2A6, and also by CYP2B6). A major metabolite is cotinine(33). Nicotine easily crosses the blood- brain barrier and there are nicotine receptors throughout the central and peripheral nervous system
(29)

Nicotine acts on the nicotinic acetylcholine receptors, specifically the

ganglion type nicotinic receptor and one CNS nicotinic receptor. The
8

former is present in the adrenal medulla and elsewhere, while the latter is present in the central nervous system (CNS). In small concentrations, nicotine increases the activity of these receptors. Nicotine also has effects on a variety of other neurotransmitters through less direct mechanisms and nicotine has multiple neurological pathways, which control pleasure
(33)

. Nicotine's mood-altering effects are different by report. First

causing a release of glucose from the liver and epinephrine (adrenaline) from the adrenal medulla, it causes stimulation. Users report feelings of relaxation, sharpness, calmness, and alertness consequence (35, 36). Cigarette smoke also alters the structure and function of respiratory passages, capillaries and immune system of the lung (37, 38). Nicotine has been found to damage the vascular endothelium, which could lead to the initiation of atherogenesis (capability of producing atheroma) And it is believed that smoking accelerates pre-existing athherogensis (39)
(34).

By reducing the

appetite and raising the metabolism, some smokers may lose weight as a

Other effect of smoking:

Cigarette smokers are ten times more likely to develop lung cancer than other smokers
(40)

this risk is proportional to the number of

cigarettes smoked per day, increasing to 20 to 25 times risk of non smokers in those who smoke two or more packs of cigarettes per day. This risk as also increased in those who inhale more deeply or began smoking at a younger age types of lung cancer (41).
9
(40)

. Cigarette smoking causes all of the major

Cigarette smokers have a great incidence of gastric and duodenal ulcers and delayed healing of theses ulcers (41). Several of these constituents of tobacco smoke are capable of inducing hepatic microsomale systems which then alter the metabolism of other drugs (40). Smokers have lower blood levels of vitamin c and vitamin B12
(42)

, heamatocrit and hemoglobin level as well as carboxyheamoglobin

level are elevated in smokers, and smoking is one cause of elevated red cell volume, smokers also have some small alteration in other diagnostic test, including a higher leukocyte count, but these differences are not usually significant for an individual patients
(42)

Some of possible determinable effects of cigarette smoking on cardiovascular system increase in leukocyte count ,platelet aggregation ,fibrinogen level ,plasma viscosity ,
(43) (44)

and free ionized calcium

total

serum cholesterol and low density lipoprotein cholesterol (45)

Health benefit of smoking:

Studies suggest that smoking decreases appetite, but did not conclude that overweight people should smoke or that their health would improve by smoking
(46, 47)

Several types of "Smokers Paradoxes (46) (cases where smoking appears to have specific beneficial effects, the effect is eliminated if the individual stops smoking.
(47, 48)

Smoking appears to interfere with

(49)

development of Kaposi's sarcoma

breast cancer among women

carrying the very high risk BRCA gene, (50) preeclampsia,(52) and atopic disorders such as allergic asthma. (51) A plausible mechanism of action in these cases may be the nicotine in tobacco smoke acting as an antiinflammatory agent and interfering with the disease process. (53)
10

Evidence suggests that non-smokers are up to twice as likely as smokers to develop Parkinson's disease or Alzheimer's disease (54)

1.2.2. CALICUM 1.2.2.1: Calcium physiology:

In 1883 Ringer showed that calcium was essential for myocardial contraction
(55, 56)

while attempting to study how bound and

free form of calcium affected frog heart concentration, Mclean and Hasting showed that the ionized calcium concentration was proportional to the amplitude of frog heart concentration whereas protein bound and citrate bound had no effect (57) From this observation, they developed the first assay for ionized calcium using isolated frog heart.

1.2.2.2 Total body calcium

The total body calcium depends on that absorbed from the dietary intake and that lost from the body [figure 1-1]. Factors affecting intake About 25 mmol (1g) of calcium is ingested per day which there is a net absorption of between 6 and 12 mmol (0.25 0.50g) the active metabolite of vitamin D3. (1.25-dihydroxy cholecalciferol ) is needed for adequate calcium absorption (57, 58) Factors affecting loss Calcium can be loss in feaces and urine FECAL LOSS: is derived from the diet and that portion of the large amount of intestinal secretion that has not been reabsorbed and is therefore, lost from the body. Calcium in the intestine, wheater endogenous or exogenous in origin, may form insoluble, poorly absorbed complex with phosphate or fatty acid. Orally administered phosphate may be used therapeutically to reduce calcium absorption and reabsorption
11

.An excess of fatty acid in the intestinal lumen in steatorrheao may contribute to calcium Malabsorption (57, 58) URINARY LOSS: excretion depends on the amount of calcium reaching the glomeruli, the glomerular filtration rate and on renal tubular function parathyroid Hormone and 1, 25 dihydroxy Vit D3 increase calcium and to lesser extent, urinary phosphate reabsorption. (57, 58)

1.2.2.3 Calcium distribution:

More than 99%of calcium in the body is part of bones .the remaining 1% is mostly in the blood and other ECF (extra celluer fluid).Very little is in the cytosol of most cells. In fact, the concentration of ionized calcium in blood is 5.000 to 10.000 times higher than in the cyctol of cardiac or smooth muscle cells. (57, 58) Calcium in blood is distributed among several forms. About 45%circulate as free calcium ions, 40%is bound to protein, mostly albumin, and 15%is bound to anions such as bicarbonate , citrate, phosphate and lactate .Clearly, this distribution can change in disease . It is especially noteworthy that concentration of citrate, bicarbonate, lactate, phosphate and albumin can change dramatically during surgery or critical care .This is the principle reason why ionized calcium cannot be reliably calculated from total calcium measurements, especially in all individuals.
(57, 58,)

12

DIEATRY INTAKE" 25 MMOL

SOFT TISSUE 25 mmol

SOFT TISSUE 25 MMOL

GUT

12mmol/day 7mmol/day

ECF 22mmol

10 mmol/day
BONE 10mmol/day 25000mmol

filtrate

KIDNEYS

FEACAL OUTPUT (20mmol/day)

13

URINARY OUT PUT (5mmol/day) Figure [1-1]: The approximate daily turnover of total body calcium

1.2.2.4 Calcium regulation:

Three hormones are known to regulate serum calcium by altering their secretion rate in response to changes in ionized calcium. These hormones are Parathyroid hormone PTH, Vitamin D3 and Calcitonin
58) (57,

.The actions of these hormones are shown in Figure [1-2]. PTH secretion into blood is stimulated by a decrease in

ionized calcium and conversely, PTH secretion is stopped by an increase in ionized calcium. PTH exerts three major effects on both bone and kidney. In the bone, PTH activate a process known as bone resorption ,in which activated osteoclasts break down bone and subsequently release calcium into the ECF (58) In the kidney, PTH conserves calcium by increasing tubular reabsorption of calcium ions.PTH also stimulate renal production of vitamin D3 (58) VitaminD3 cholecalciferol , is obtained either in the diet or from exposure of skin to sun light . VitaminD3 is then converted in the liver into 25-hydroxycholecalciferol (25-OH-D3) , still an in active form of vitaminD3 .in the kidney 25-OH-D3 is specifically hydroxylated to form 1,25-dihydroxycholecalciferol [1,25-(OH)2-D3],the biologically active form. This active form of vitamin D3increase calcium absorption in the intestine and enhances the effect of PTH on the bone resorption (58) Calcitonin , which originates in the medullary cells of the thyroid gland, is secreted when the concentration of calcium in bloodincreases .calcitonin exerts its calcium-lowering effect by inhibiting the action of both PTH and vitaminD3 .Although calcitonin is apparently
14

not secreted during normal regulation of the ionized calcium concentration in blood, it is secreted in response to a hypercalcemic stimulates. (58)
PTH (Parathyroid gland)

Hypercalcaemi a

PTH

Hypocalcaemi a

BONE

KIDNEY

In bone, PTH stimulates Osteoclastic activity which Release calcium and HPO4

in the kidney, PTH a-absorption of calcium b-excretion of HPO4 C-activation of renal 1-alphhydroxylase

CIRCULATING 25-OH VIT D3 (IN ACTIVE)

INTESTINE

KIDNEY

Vit D3 promote: Intestinal absorption Of calcium and HPO4

Vit D3 promote renal absorption of calcium and HPO4

15

Figure 1-2: Hormonal response to hypercalcaemia and hypocalcaemia PTH, parathyroid hormone; 25-OH vitD3, 25 hydroxy vitD3; 1.25(OH)2 vitD3 , dihydroxy vitamin D3

1.2.2.5 Clinical Conditions: Hypercalcaemia:

Primary hyperparathyroidism is the main cause of hypercalcaemia
(58)

, Hyperparathyroidism or excess secretion of

PTH may show obvious clinical signs or may be asymptomatic. Although either total or ionized measurements are elevated in serious cases ,ionize calcium is more frequently elevated in suitable or asymptomatic hyperparathyroidism .In general, ionized calcium measurement are elevated in 90%- 95% of cases of hyperparathyroidism, whereas total calcium is elevated in 80%85% of cases (58) The second leading cause of hypercalcaemia is associated with various type of malignancy, with hypercalcaemia sometimes being the sole biochemical marker of disease
(58)

many tumors

produce PTH-related peptide (PTH-rP) which bind normal receptors and cause increased calcium levels. This abnormal protein is not detected by most PTH assays (59) A rare benign familial hypocalciuria has also been reported .Thiazide diuretics increase calcium reabsorption, leading to hypercalcaemia (59) HYPOCALCAEMIA

16

Hypocalcaemia is a state of blood calcium level below the normal range

(60)

this is probably measured by ionized calcium;

total calcium is not diagnostic unless also accompanied by an albumin measurement (60) Signs and symptoms: Signs and symptoms of hypocalcaemia, regarding signs and symptoms associated with hypercalcaemia are protean and demonstrate individual variation at how low calcium must be for any particular signs or symptoms to appear (61)

1.2.2.6: Determination of calcium:

A- specimen: The preferred specimen for total calcium determination is either serum or lithium heparin plasma collected without venous stasis. Because anticoagulants such as EDTA or Oxalate bind calcium very tightly and interfere with its measurements, they are unacceptable for use (59). Although heparinized whole blood is the preferred sample, serum from sealed evacuated blood collection tubes may be used if clotting and centrifugation are done quickly (less than 30 min) and at room temperature. Most heparin anticoagulants (sodium, lithium) partially bind to calcium and lower ionized calcium concentration (62) Dry heparin products are available either titrated with small amounts of Ca or Zn ions, or have a small amounts of heparin dispersed in inert "puff" that essentially eliminates the interference by heparin(62)

17

For analysis of calcium in urine, an accurately timed urine collection is preferred. The urine should be acidified with 6 mol/L HCL, with approximately 1 ml of the acid added for each 100ml of urine. For analysis of calcium in saliva before sampling, each subject was briefed about the procedure and instructed to wash his mouth and gargle with plain water. The saliva of each subject was collected (for 10 minutes) under resting condition (13) B- Methods: The two commonly used methods for total calcium analysis are either Orthocresolphthalein complexone (OCPC) or arsenzoIII dye to form a complex with calcium. The (OCPC) methods use 8-hydroxyquinoline to prevent magnesium interference. OCPC is used is used in full automation analyzers as well as colorimetric analyzer (63). The arsenzo III method can be found on the Vitrous (Johnson &Johnson .Co) and synchron (Beckman) systems (63) C- Reference range of calcium: Total calcium (serum or plasma) Child: Adult 2.20 2.7 mmol/L (8.8 10.8 mg/dl) 2.10 2.6 mmol/L (8.4 10.4 mg/dl)

Ionized calcium (serum, plasma) Neonate: Children: Adults: 1.20 1.48 mmol/L (4.8 -5.9 mg/dl) 1.20 1.38 mmol/L (4.8 5/.5 mg/dl) 1.16 1.32mmol/L (4.6 5.3 mg/dl)

18

Urine 24 hours: 2.50 7.50 mmol/day (100-300 mg/day) Varies with diet Salivary calcium 2.1 2.5mmol/l (4.2- 10 .0mg/dl) ( )

1.2.2.8 Serum calcium and smoking:

The main regulator of the PTH level is the plasma calcium. Furthermore, there might be substances in smoke that could interact directly with the calcium receptor, in theory. Possible explanations of high PTH levels might be because smokers have a low intake of calcium or vitaminD3, low calcium resorption from the skeleton or an excessive excretion of calcium in urine. Both high as well as low PTH levels have been reported as result of smoking. (63) Landin-Wilhelmsen et-al in a study on 347 men and women,Brot et-al, in a study on 510 women aged 45 - 58 years , Mellstrom et-al . In a case control study on 129 men with earlier partial gastrectomy and 216 controls found serum PTH to be significantly higher in smokers (63) R Jorde, F Saleh , et- al in a study on 7896 men and women who are smokers found that serum PTH is significantly lower in smokers .(64)

1.2.2.9 Salivary calcium:

In vertebrates, calcium is the major component of bone and teeth, and it's not surprising that disturbance in calcium metabolism have been implicated in most of the major chronic diseases, including osteoporosis, kidney diseases, heart diseases, and hypertension
(65)

, it's

generally accepted that saliva is secreted into two stages, first the saliva is secreted into the acini of salivary gland and this fluid (primary secretion) is not much different from extra cellular fluid (ECF). Secondly, when the primary secretion flows through the acinar ducts, some ions are actively reabsorbed from and some are actively secreted into the acinar duct (65)
19

Salivary calcium and smoking

The traditional methods of tobacco use are smoking, snuffing, chewing and dipping (65) it was in the 18th century when it was discovered that smoking increases the activity of salivary gland
(66)

. Indeed, this

observation has been made by everyone who begins smoking but some tolerance develops to the salivary effects of smoking because habitual smokers do not salivate as do novice smokers in response to smoking
(67)

however it has also been proved that sensation of taste and salivary flow rate in chronic tobacco users are not much different from that in non tobacco users
(68)

, and there is no difference in secretion rate of saliva

between smokers and non smokers (69) It was also observed that regular, but not immediate, smoking did not cause any significant change in salivary flow rate
(67) (66)

. After

smoking, there is temporary increase in the salivary content of calcium, potassium and phosphate In long-term tobacco users, the taste

receptors, a primary site for stimulation of salivary secretion, are constantly exposed to tobacco for long time thus presumably affecting the salivary reflex. Ghulam Jillani Khan, et al
(13) (13)

made a study on the

SECRETION OF CALICUM IN THE SALIVA OF LONG TERM TOBACCO USERS . The results showed that the concentration of

calcium in the saliva is mainly flow dependent both in tobacco users and non users and its concentration falls when the flow of saliva increases. In some earlier studies it was pointed out that concentration of both potassium and calcium are independent of salivary flow rate (70) .but later on this observation was proved to be incorrect and indeed the calcium concentration falls when the salivary flows rate increasing (71) .

20

They found significantly higher levels of calcium in the saliva of all tobacco users as compared to non-users. In niswar dippers and pan chewers this may be due to the fact that pan and niswer contain lime (Ca (OH) 2) as one of its essential ingredients and there is a strong possibility that lime may get entangled in the gums and in between the teeth of these individuals which may flow with saliva during the process of sampling .however, the higher level of calcium in the saliva of smokers cannot be explained on this basis. The oral pH in smokers is lower than non smokers
(71)

.therefore,

this acidic pH can extracts calcium from the scales deposited on teeth (or even from teeth) of those volunteers which might have been resulted in the elevated level of salivary calcium. Another possibility may be that heavy smokers seem to have lower bone mineral density and higher salivary calcium than their non-smoking counterparts. Therefore, it was suggested that the high salivary calcium concentration of smokers is in connection with skeletal calcium disturbance their experimental work, they conclude That higher level of calcium are present in saliva of long term tobacco users than non-users and the calcium concentration decreases as the flow of saliva increase.
(70)

Therefore based on

21

Hypothesis
The hypothesis in this research that cigarette smoking affects the level of serum and salivary calcium, and its affected with the number Of cigarette smoked per day and the duration of smoking in years

1.3 Objectives 1.3.1General objectives

To assess the affect of cigarette smoking on calcium metabolism in Sudanese cigarette smokers compared to non smokers.

1.3.2. Specific objectives

To determine the calcium level in plasma in Sudanese cigarette To determine the salivary calcium level in Sudanese cigarette To assess the impact of the number of cigarette smoked per day on To assess the impact of the duration of cigarette smoking in years on

smokers.

smokers. calcium level in plasma and saliva.

calcium level in plasma and saliva.

22

To assess the true level of plasma calcium by correcting it by albumin.

CHAPTER TWO 2. Materials and methods

2.1Materials 2.1.1Type and period of study
Its a prospective analytical case control study that used to assess the effect of cigarette smokers on calcium metabolism. The study was carried out in the period between February 2008 and October 2008. 2.1.2 Place of study: The study was carried out at Khartoum state at University of Medical science and it involves: Blood sample for estimation of calcium and albumin. Saliva sample for estimation of calcium. 2.1.3 Study population: The study covered 50 of subjects with different age and from different parts who are smokers and 50 negative controls. All the smokers in this study are male because: 1. it's hard to find female smokers. 2. Socially it's unacceptable to ask a female if she smokes. 3. Some of those found were refused to be involved in this study.
23

The age range of all volunteers in this study is between 21 and 50years .the smokers were aged matched to control Inclusion criteria: Any smoker who don't have any disease which affect the level of calcium or albumin and has a normal diet contain calcium And accept to be included in the study. Exclusion criteria: Any smoker who has a disease which affect the level of calcium or albumin such as: Liver disease Renal disease Renal stones Parathyroid disease Thyroid disease Diabetes Malnutrition Malabsorption

2.1.4 Sample collection: 2.1.4.1For estimation of plasma calcium:

With cotton wool dipped in ethanol the fore arm was cleaned and with sterile syringe, the needle was introduced into visible vein with minimum stasis... about 3 ml was collected and placed in sterile container with suitable anticoagulant (lithium heparin) (72) Centrifuge for 5 minutes at 1500 RPM for plasma.
-

All specimens must be analyzed with in 24 hrs after collection. The specimen must not be haemolyzed or icteric or lipemic because it may produce error.
24

Fasting sample is not required. (72)

2.1.4.2For estimation of salivary calcium:

The saliva will be collected from smokers after washing their mouth carefully with plane water ,and under resting condition with supervision to collect about 2 ml saliva.

2.2. Methods: 2.2.1 Detection of plasma and salivary calcium: Photometric test for calcium
Principle Calcium ions react with o-cresolphthalein-complexone in an alkaline medium to form a purple colored complex .the absorbance of this complex is proportional to the calcium concentration in the sample. Contents BUF 100ML buffer solution Lysine buffer (pH 11.1) Sodium azide RGT 100ML color reagent 8-hydroxyquinoline O-Cresolphthalein complexone Hydrochloric acid STD 3ml standard Calcium (ll) Sodium azide Reagent preparation 8mg/dl or 2mmol/l 0.095% 14mmol/l 0.1mmol/l 40mmol 0.2mol/l 0.095%

25

Add RGT to BUF in equal volume as required, mix and allow standing for 30 minutes at room temperature before use (72) Reagent stability The reagent and the standard are stable even after opening up to the stated expiry date when stored at (2.25 C.) at (15 25 C) Assay Wavelength: Optical path: Temperature: Measurement: Pipetting scheme: 570nm, Hg578 nm 1cm 20.25 C against reagent blank per series is required Contamination must be avoided, the working reagent is stable for 7 days at (2.8C) and for 3 days

Pipetting into cuvettes Sample or STD Working reagent

Reagent blank --1000ul

Sample or STD 20ul 1000ul

Mix and measure the absorbance of sample (^A sample) and standard (^A STD) against the reagent blank within 5 to 30 minutes Calculation of the calcium concentration c= absorbance of sample (test) Absorbance of standard c= Absorbance of sample (test) X2 = calcium mmol/l
26

X8

= calcium mg/dl

Absorbance of standard Linearity The test is linear up to a calcium concentration of 15 mg/dl or 3.75 mmol/l, Samples with a higher concentration have to be diluted 1+1 with distilled water, Repeat the assay and multiply the result by 2. (72) Quality control: All control sera with calcium values determined by this method can be employed Normal values: Serum /plasma Saliva: Notes: -

8.4-10.4 mg/dl 4.2 10.0 mg/dl

contaminated glass ware is the great source of error .disposable the test is not influenced by hemoglobin up to 200 mg/dl and lipemic and hemolytic samples require a sample blank BUF and STD contain sodium azide (0.095%) as preservative do

plastic ware is recommended for the test bilirubin up to 20 mg/dl

not sallow .avoid contact with skin and mucous membrane (72) 2.2.2Determination of plasma albumin: Colourmetric test BCG method Principle Serum albumin binds selectively to the dye bromocresol green at pH 4.2 .the absorbance of the blue to green complex at 546nm. (73) Reagent composition 1. Citrate buffer (pH 4.2) Bromocresol green 7.5 mmol/l 150umol/l
27

Sodium azide 2. Albumin standard Reagent stability

0.05% 4 g/dl

The color reagent and standard are stable up to the stated expiry date when stored at (2.8 C).contamination after opening must be avoided .the reagent should be a clear yellow /green solution. If turbidity or perception has occurred the reagent must be discarded59 Assay Wavelength Optical path Temperature Measurement Procedure Pipette into cuvettes Sample ml Standard ml Distilled water ml Reagent ml blank -------0.01 1.0 Standard -----0.01 ------1.0 sample 0.01 -----------1.0 546nm 1 cm 2025 C against reagent blank

Mix and incubate for 5 minutes at 20---25 C, measure the absorbance of the sample (As) and standard (A std) against the reagent blank within 30 minutes Calculation Serum albumin (g/dl) = A Sample A STD Linearity X concentration of standard

28

The test is linear up to concentration of 7 g/dl .If higher albumin concentration is expected, dilute the sample 1+1 with physiological saline. Repeat the estimation and multiply the result x 2 Normal values Serum albumin = 3.5 -5.1 g/dl Quality control All control sera with values estimated by this method can be used The test is not influenced by bilirubin values up to 20 mg/dl In cases of excessive lipaemia a sample blank should be prepared by

adding 0.025 ml of serum to 2.5 ml of 0.9% saline. the absorbance of the sample blank is subtracted from the absorbance of the sample

Avoid excessive haemolysis since every 100mg/dlof hemoglobin The colour reagent and standard contain sodium azide ,do not

correspond to about 100mg/dl of albumin swallow ,avoid contact with eye and skin (73) Data analysis The data of this research will be analyzed by SPSS computer programme.

29

CHAPTER THREE 3. RESULTS

50 cigarette smokers were taken as a test group and compared to 50 normal healthy individuals as a control group. 3.1 AGE: Table 3.1 and figure 3.1 showed the distribution of age among non smokers (control). Table 3.2 and figure 3.2 showed the distribution of age among smokers. 3.2 DURATION: Table 3.3 and figure 3.3 showed the duration of smoking per year among smokers. 3.3 PLASMA CALCIUM: Table 3.4 Figure 3.4 showed a significant decrease between the mean of plasma Calcium in the test group (smokers) and the control group.(non smokers). (Mean SD): (8.4460 0.61816 versus 9.0640 0.2317) mg/dl, P< 0.05 Figure 3.5a and 3.5b showed frequency of Calcium level in smokers group and control group respectively.
30

Table 3.5 showed a negative correlation between the levels of plasma Calcium and the number of cigarettes smoked per day (r= -0.2) Table 3.6 showed the correlation between the levels of plasma Calcium and the Duration of smoking per year (r= 0.017). Figure 3.6 showed the relation between the mean of plasma calcium and the duration of smoking per year.

3.4 SALIVARY CALCIUM: Table 3.7 Figure 3.7 showed a significant increase between the mean of Salivary Calcium in the test group (smokers) and the control group. (Non smokers). (Mean SD): (10.9380 2.4843 versus 5.2440 0.36654) mg/dl, P< 0.05 Figure 3.8a and 3.8b showed frequency of salivary calcium level in smokers group and control group respectively. Table 3.8 showed a positive correlation between the levels of Salivary Calcium and the number of cigarettes smoked per day (r= 0.65) Table 3.9 showed a positive correlation between the levels of plasma Calcium and the Duration of smoking per year (r= 0.39). Figure 3.9 showed the relation between the mean of salivary calcium and the duration of smoking per year.

31

Table 3-1 Distribution of age in control group.

AGE OF CONTROL GROUP

AGE

Frequency 16--20 21--25 26--30 31--35 36--And more Total 6 8 24 11 1 50

Percent 12.0% 16.0% 48.0% 22.0% 2.0% 100.0%

AGE

32

Figure 3-1: Distribution of age in control (Non smokers)

Table 3-2 Distribution of age in smokers group.

AGE OF SMOKERS GROUP
AGE

Frequency 21-25 26 -30 31 -35 36-And more Total 4 18 8 20 50

AGE

Percent 8.0% 36.0% 16.0% 40.0% 100.0%

33

Figure 3-2 Distribution of age in smokers group) Table 3-3: Distribution of duration among cigarette smoking group

DURATION OF SMOKING
DURATION OF SMOKING BY YEAR Frequency

2--5 6--9 DURATION 10--13 14--17 18--And more Total

6 5 14 6 19 50

Percent 12.0% 10.0% 28.0% 12.0% 38.0% 100.0%

34

Test group Variable (Smokers) n= 50

Control group (Non-smokers) n= 50 P. value

Plasma Calcium(mg/dl)

8.440.618 (2.3)

9.0640.237 (1.3)

0.00

Figure 3-3

Distribution of duration in smokers group.

Table 3-4 Comparison of the mean of plasma calcium between smokers and non smokers.
The mean SD, range in brackets ( ) and probability (P). 35

 t-test was used for comparison.

Figure 3-4 : Comparison of the mean of plasma calcium between smokers and non smokers.

plasma calcium
14 12 10 8 6 4 2 0 7.00 7.25 1 7.50 7.75 8.00 8.25 8.50 8.75 9.00 9.25 5 7 6 5 3 Std. Dev = .62 Mean = 8.45 N = 50.00 11

12

number of smokers

plasma calcium concentration in smokers mg/dl

36

Figure 3-5a: Frequencies of plasma calcium in smokers

20 19 19

10

number of control

Std. Dev = .24 Mean = 9.06 N = 50.00

1 8.25 8.50 8.75 9.00 9.25 9.50

plasm calciumconcentration in non sm a okers m g/dl

Figure 3-5b: Frequencies of plasma calcium in non smokers Table 3-5: Shows the correlation between the level of plasma calcium mg/dl and number of cigarettes smoked per day.

37

plasma calcium Pearson Correlation Plasma calcium in smokers Sig. (2-tailed) N Pearson Correlation NO OF CIGRRETTE Sig. (2-tailed) N 50 -.224 .118 50 1

NO OF CIGRRETTE -.224 .118 50 1 50

Table 3-6 The correlation of the duration of smoking of cigarette per year and the level of plasma calcium.

Plasma calcium Plasma calcium Pearson Correlation Sig. (2-tailed) N DURATION OF SMOKING Pearson Correlation Sig. (2-tailed) N 1 . 50 .017 .906 50

DURATION OF SMOKING .017 .906 50 1 . 50

38

Figure 3-6 : shows the mean of plasma calcium in smokers group according to duration.

39

Table 3-7: Comparison of the mean of salivary calcium between smokers and non smokers

Variable

Test group (Smokers) n= 50

Control group (Non-smokers) n= 50

P. value

Salivary Calcium(mg/dl)

10.9382.48439 (11.0)

5.2440.3665 (4.4)

0.00

The mean SD, range in brackets ( ) and probability (P). t-test was used for comparison.

40

Figure3-7: Comparison of the mean of salivary calcium between smokers and non smokers.

S liv ry ca m a a lciu
1 6 1 4 1 2 1 5

Number of smokers

1 0 8 6 4 2 0 1 4 .0 5 .0 2 6 .0 7 .0 8 .0 9 .0 1 0.0 1 .0 1 5 4 3 3 4

1 0

S . D v = 2.48 td e 2 1 .0 2 1 .0 3 1 .0 4 1 .0 5 M an = 1 .9 e 0 1 N= 5 .0 0 0

S liva ca mcon n tioninsm e m /d a ry lciu ce tra ok rs g l

Figure 3-8a: Frequencies of salivary calcium in Smokers.

41

S liv ry ca m a a lciu
14 12 10 8 6 4 2 0 1 4.50 4 5 .7 5.0 0 5 5 .2 5.5 0 5 5 .7 6.0 0 3 S . D v=.3 td e 7 M a =5 e n .24 N=5 0.00

1 2

1 2 10

Number of control

S liv ry ca mco ce tra n in n n sm ke m /d a a lciu n n tio o o rs g l

Figure 3-8b: Frequencies of salivary calcium in control

Table 3-8: shows the correlation between the level of salivary calcium and

number of cigarettes smoked per day

salivary calcium salivary calcium Pearson Correlation Sig. (2-tailed) N NO OF CIGRRETTE Pearson Correlation Sig. (2-tailed) N ** Correlation is significant at the 0.01 level (2-tailed). 1 .000 50 .654(**) .000 50 NO OF CIGRRETTE .654(**) .000 50 1 .000 50

42

Table 3-9: The correlation between the duration of smoking per year and the level of salivary calcium.

Salivary calcium Salivary calcium Pearson Correlation Sig. (2-tailed) N DURATION OF SMOKING Pearson Correlation Sig. (2-tailed) N 1 .003 50 .392(**) .003 50

DURATION OF SMOKING .392(**) .003 50 1 .003 50

** Correlation is significant at the 0.01 level (2-tailed).

43

Figure3-9: The relationship between the duration of smoking per year and the level of salivary calcium

CHAPTER FIVE DISSCUSION

It appears to be no relevant published data on the effect of cigarette smoking on blood chemistry among Sudanese. So comparison will be done with studies published outside Sudan The prevalence of smoking tends to be increasing in Sudan and also other parts of Africa( 74,75) The level of serum calcium, salivary calcium in the control group (normal healthy individuals) in this study was found to be within the normal range reported for normal individuals.
44

Our study was done exclusively among male members and students of the University Medical Science and technology. In Sudanese society it's hard to find a female who smoke because they are reluctant to discuss the issue of smoking in an open manner. As smoking in young ones is consider as offending practice, although, it's accepted among old ones. But there is also a considerly number of young female who do smoke. The smokers were age and socieconimacal status matched to control group (healthy non smokers) (table 3-1 3-2, figure (3-1 3-2) The results of this study showed a decreased in the mean of serum calcium, in smokers when compared with the mean of non smokers (control) (table 3-4, figure 3-4). These finding are similar to those observed by Elizabeth A.Krall el-al
(76)

, that smoking increase bone loss and decrease

intestinal calcium absorption. Lower calcium absorption in smokers, may be one path way, by which smoking affect the rate of bone loss. The mechanism is still not clear. There is evidence from animal studies to suggest that some component of cigarette smoke damage the intestinal villi(77 ), Cigarette smoke has an effect on body that may decrease calcium absorption and independently influence bone metabolism
(78)

. There were no correlation between the level of serum

calcium in cigarette smoking group and number of cigarettes smoked per day (r =-0.224) (table 3-5)

45

The results showed that there is no correlation between level of calcium and duration of smoking per year (r = 0.17). (Table 3-6) Smoking is associated with reduced bone density and calcium absorption and reduced level of Vitamin D3
(77)

with an

association with fracture risk and also a disturbance of PTH level. And that can be accepted because of altered calcium balance. (Low absorption from intestine due to reduce level of serum 1, 25(OH) 2 D level,(Krall and Dawson - |Huges(1991) ) (77) . density The results of this study showed a increase in the mean of salivary calcium, in smokers when compared with the mean of non smokers (control) (table 3-7, figure 3-7). The results are significantly higher in cigarette smoker group P.V <0.05 The result show an increase level of salivary calcium level with the increase in number of cigarette smoked per day .there was a strong positive correlation (r = 0.65) (table3-8) An increase in the saliva level observed with the increase in the duration of smoking per year with a good correlation (r = 0.399) (table 3-9) The results are in true with the finding of the experimental work of Ghulan Jillani Khan et-al) (13) on the secretion of saliva in the long term tobacco users they conclude that high level of calcium are present in the saliva of long term tobacco users, than in non users by
(13)

And this can lead to increase mobilization of calcium from

bone and result in bone resorption which may result in low bone

which can be explained . the buffers in saliva

maintain the oral ph at about 7.0 to prevent calcium loss from the
46

teeth, because at this p H(85) the saliva is saturated with calcium(65) but in smokers the oral ph is acidic PH which therefore, is a great possibility for the acidic pH to extract calcium from the scales deposited on their teeth (or even from their teeth ) of these individuals which have might have resulted in the elevated level of salivary calcium() Ginigivitis may devolve than the teeth can be easily lost (69) Another possibility that heavy smokers seems to have lower bone density and thus higher salivary calcium level than non smokers .so it was suggested that the high salivary calcium concentration of smokers in connection with skeletal calcium disturbance Normally, saliva provides a protective buffer between toxins and the lining of the mouth because it contains enzymes that neutralize harmful substances. But the researchers found that the chemicals in tobacco smoke, when combined with saliva, destroy salivas protective components, leaving a corrosive mix that damages cells in the mouth and eventually turn them cancerous. " Cigarette smoke is damaging on its own, but when mixed with saliva it turns the body against itself," says co-lead researcher Dr. Raphael Nagler of the Technion Faculty of Medicine. "Our study shows that when exposed to cigarette smoke, normally healthy saliva loses its beneficial qualities, turns traitor and actually aids in destroying the cells of the mouth and oral cavity." The study recreated the effects of cigarette smoke on cancerous cells of the mouth. Half the cells were exposed to cigarette smoke only, while the other half was exposed to a saliva and cigarette smoke mixture. The researchers then studied the cells to assess whether the saliva and smoke mixture would speed up the
47

cancers

development. The longer the mouth cells were exposed to the

contaminated saliva, the more they were damaged. The researchers also found that the cigarette smoke destroyed various salivary components, including protective ones such as peroxidase, the most important antioxidant contained in saliva. Antioxidants are molecules that help protect the body against cancer. According to Nagler, the reaction between certain components of saliva and the low reactive free radicals contained in cigarette smoke results in the production of highly active hydroxyl free radicals. Free radicals, oxygen by-products created by normal cell metabolism, possess unpaired electrons. In a process called oxidation, they cause cellular damage by taking electrons from molecules in healthy cells. "These hydroxyl radicals are dangerous and lethal," notes Nagler. "They attack biological molecules in a tenth of a second." Smoking and drinking are the leading causes of head, neck and oral cancer also known as Oropharyngeal (OP) cancer, which includes cancer of the mouth, lip, tongue gums, larynx and pharynx. Nearly 400,000 new cases are diagnosed worldwide each year, with the majority in developing countries. The five-year survival rates are less than 50 percent.

48

CHAPTER FIVE CONCLUSION& RECOMMENDATIONS

5.1 CONCLUSION:
In this study it was seen that cigarette smoking decrease the level of plasma calcium. And there is a no correlation with number of cigarettes smoked per day or with duration of smoking per year.

49

It was also seen that cigarette smoking increase the level of salivary calcium. And there is a positive correlation between the duration of smoking per year and number of cigarette smoked per days

5.2 RECOMMENDATIONS;
From this I recommend the following 1. More studies are needed to cover the effect of cigarette smoking.

50

2. More parameters should be done in addition to plasma calcium and salivary calcium. 3- More studies cover more number of people from sexes are needed to know the relation between cigarette smoking and skeletal calcium disturbance. 4- Health education program and workshops on the effect of cigarette smoking on the body health.

CHAPTER SIX REFERENCES

1-Ware LB,Rekling JC,Ernst AA ,Perlmutter DH. Cigarrette smoking and health .American journalof respiratory and critical care medicine 1996 153:861-865.

51

2-Law MR,Hackshaw AK1997 A meta- analysis of cigarette smoking, bone mineral density and risk of hip fracture :Recognition of a major effects BMJ 315 pp841- 846. 3-Slemenda CW, Hui SL, Longcope C, Johnston CC Jr 1989 Cigarette smoking, Obesity and bone mass. J Bone Miner Res 4:pp734 741. 4- Hollenback K, Barrett-Connor E. Edelstein SL,Holbrook T 1993 Cigarette smoking and bone mineral density in old men and women .Am pub Health 183 pp1265- 1270. 5- Forsenal L.Bjorndal A,Bjartveit K . Enda TH .Holman J .Jessen V, Westberg G, 1994 Interaction between current smoking,leanness, and physical activity in the prediction of hip fracture .J.Bone Miner Res 9: pp 1671 1678 . 6- Kiel DP, Baron JA. Anderson JJ, Hannan MT .FElson DT 1992 Smoking eliminates the protective effect of oral estrogens on the risk of hip fracture among women.Ann Intern Med116:pp716- 721. 7- Egger P,Duggleby , S.Hobbs R.Fall C.Cooper|C 1996 cigarette smoking and bone mineral density in the eldery . J Epidemiol Commun Health 50: pp47- 50. 8- Ortego Centeno N,Monoz-Terres M ,Joder E,Hernandez .Quero

.J,juardo- Duce A,De La Higuria Torres Puchal J 1997 Effect of tobacco consumption in bone mineral density in healthy young males .Calcif Tissue in 60:pp 496- 500 . 9- Manning HL,Glantz PD, wright JR.tabocco biology and politices 1992 92.

52

10-

National institute of arthritis PDFverison of this document

, http//www.niams.nih.gov/health info /bone/default/asp. 11- Vestergaard P.Mosekilde L.fracuture risk associated with smoking a Meta analysis journal of internal medicine 2003 245:572-583. 12- Szulc P,Gannero .P. Claustrat B.Marchand F.Delmas PD Incresed bone resorption in moderate smokers with low body weight ,the minos study . Journal of clinical endocrinology and metabolism 2002 pp 666 674 . 13- Khan. G. Mehmood. R. Salah ud- Din, Ihtesham- ul-Hag, effect of long term tobacco on taste receptors and salivary secretion J, ayub. Med coll 15:3 9. 14- D Kapor and THjones . Smoking and hormone health and endocrine disorders, European Journal of endocrinology, Volume 152, issue 4 pp 491499. 15 Cupta PC, Hammu JE , Murti PR. Control of tobacco related cancers and other disease . Bombay international Symposium J.1992 Jan; 37(1): pp 515- 519. 16 - Pato R. Mortality from smoking in developed countries, 1950 -2000. Oxford university press 1994; 6:128. 17 - www.JeffreyWigand.com\WHO Final pdf. Wigand, MA. ADDITIVES, CIGARETTE design and tobacco products, regulation. A report to World Health Organization. Koba japan 28 \june to 2- july. 18 - Romer R Preventing tobacco use among young people. N Eng J Med 1993; 316: 1516 1521 . 19 Langesen M, Mends C. Tobacco advertising restrictions, income and tobacco consumption in some countries, 1960 1986. British Journal of addiction 1991; 86:1343 1354.

53

20- Maggi CA .Handbook of resolutions of the World Health Assembly and the Executive Board, 3rd ed .geneva: WHO Publications1993; 6:53. 21 Colada MM, Reguero JR. The interrelationship among tobacco consumption. J cardiovascular risk 1997; aug; 4(4): 274 281. 22 Villabance AL,Mcdonald JP, Rutledge JC. Smoking and cardiovascular diseases. Clin-chest-Med. 2000 March; 21 (1): 72 159. 23 Whitby LG, Percy IW, Smith AF: Lecture notes on clinical chemistry 3rd ed. London: Black Well Scientific Publications 1987; 242 -260. 24- Murray RK, Granner DK, Rodwell VW. Harpers' Biochemistery 25th edition Califorina :Appelton and Lange 1991; 512 518 . 25- Stryer L.Biochemistry 3rd ed . new York :H.W Freeman Company 1988; 544- 572. 26- Murray RK, Granner DK, Rodwell VW. Harpers' Biochemistery 25th edition Califorina :Appelton and Lange 1991; 512 518 . 27- Fielding JE.smoking :health Effects and control N . Eng J Med .1985 Dec; 31 (3): 491 555. 28 Glynn TJ, Mnaley MM, Pechacek TF. Methods for stopping cigarette smoking J. Intern.Med 1986; 10 (5): 28. 29 Russell MA. Efect of nicotine chewing gum on smoking behavior and as an aid to cigarette withdrawal, Br Med of Physio 1976;292: 393 398 . 30-Detremenation of the nicotine content of various Edible Night shade and their product and estimation of the associated dietary intake (Retrieved on Oct/ 2008. 31- Smoking and tobacco control Monography in man. 32- interindividual Variability of metabolism and Cardiovascular effects of nicotine in man . 33 - Burt A.M. Teaxt book of neuroanatomy . Philadelphia, W.B.Sanunders Co1993.
54

34- Gilbert Largrue,Francois Lebargy, Anne Cormier, "from nicotinic receptors to smoking dependence:Therapeutic prospects" Alcoologie et Addictologie Vol:23, N 25 june 2001 ,pp39 42. 35 Jean Clade Orsini, "dependence on tobacco smoking and brain system controlling glyceamia and appetite," Alcoologie et Addictologie Vol:23 N25 june ,pp 28 -36. 36- Smokers loss their appetite: Media releases news (The university of Melbourne). 37 - Chumel H. Pulmonary infections and immunity .New York, Plenum Publishing crop 1994. 38 Crapo JD.Morphlogical changes in pulmonary oxygen toxicity. Annu. Rev.physio , 1987;49:721. 39- Russell M, Jarvis M, Lyr R Feyerabend C. Relation of nicotine yield of cigarettes to blood nicotine concentration in smokers. Br Med of physio 1980, 280:971- 979. 40- Janajek D,Thompson D,Varela L,Lung cancer and exposure to tobacco smoke in household. N Eng J Med 1990; 32(3):63. 41- Berrnett WS. Textbook of medicine 19th edition. London: W.b Saunders Company 1996:34- 36. 42- Anderson JK. Muirs, textbook in pathology 12th edition .London: Butlar and Tanner 1988; 14: 24 25. 43- Mackinney AA. The Pathophysiology of blood .new York,Jhon Wiley and Sons 1948;99 555. 44- Brot C,Jorgensen N,Sorensen O. The influence of smoking on vitamin D status and calcium metabolism . European Journal of clinical nutrition 1999; 53: 920 926. 45- HaferKamp O, Schlettwein D, Skwick H. Serum protein in an aging populations with particular references to evaluation of immune globulins and antibodies . Gerontologia 1996;12 -30.
55

46- Cohen. DavidJ; Michel doucet, Donald E .Cutlip,Kalon K.L.HD, Jeffery J. Popma, Richard E.Kuntz(2001). Impact of smoking on clinical and angiographic Restenosis. After percutanous coronary intervention 104:p 773. 47- Longmore,M, Wikinson ,Torok, E. Oxford hand book of clinical medicine (5th edition )p 232. 48- Green JT, Richardson C,Marshal RW,Rhodes J,Mckirdy HC ,Thomas GA,Willams GT (200-11). Nitric oxide mediators a therapeutic effect of nicotine in ulcerative colitis.Aliment pharmacol ther 14 (11)"1429- 1434. 49- Smoking cuts risks of rare cancer UPI(march 29.2001) 50- Recer Pual (may 1998) "cigarette may have an upside "AP Retrived on November 2006. 51- Lain Kristiney. Powers Robert W,Krohn Marijane A, Ness Roberta,B Crombleholme William R. Robert james M (nov 1991), 52- Urinary cotinine concentration confirm the reduce risk of preeclampsia with tobacco exposures. American journal of obstetrics and gynecology 181(5):908 -914. 53-Hjern A, Hedberg A. Haglund B.Rosen M(june2001)does tobacco smoke prevent atopic disorders . A study of two generation of Swedish residenants .clin exp allergy 31(6):908- 914. 54Fratiglioni L.Wang HX (aug 2000).Smoking and Parkinsons's and Alzheimer's disease : Behavior Brain Res;113(1-2):pp117- 120. 55-Ringer S.A Furture Contribution regarding the influence of different constituents of hear .J Physiol 1883; 4- 29. 56- Bove AA, Davis JC.American Chemical Society. Date published November 30\2005. 57-Mclean FC,Hasting AB .A biological method for estimation of calcium ion concentration . J Biol Chem1934; 107:447. 58- Bushinsky DA, Mork RD. Calcium .Lancet 1998; 352-24.

56

59- Titez NW .clinical guide to laboratory tests. Philadelphia: WB Saunders 1995. 60- Micheal L ,Bishop ,Edward P,Fody , Larry ESchoeff, Clinical chemistry Principles ,procedures correlation , 5th edition Philadelphia LWW p455- 466 61- Shane E, Hypocalcaemia: pathogenesis. Different diagnosis and management .In Favus,MJ.ed .Primer on the metabolic bone diseases and disorders of mineral metabolism ,4th edition .Philadelphia: Lippincott williamas &Wilkins 1999:223-226. 62- Stummvoll HK ,lehmann PJ .Use of anticoagulants in diagnostic laboratory investigations . WHO publication. Rev.2 :jan.2002. 63- Gosling P. Analytical reviews in clinical chemistry :Calcium measurement. Ann Clin Biochem 1986;23: 146. 64- R Jorde, F Saleh ,Y.Fingenschav, E,Kamycheva, E Hang ,and J Sundsfjord. Serum para thyroid hormone level in smokers and non smokers (the fifth thromso study),European journal of endocrinology .Vol 152: issue 1 pp 39- 45. 65- Parvinen T,Stimulated salivary floe rate ,pH and Lactobacillus and yeast concentrations in non smokers and smokers. Scand J Dent Res 1984; 92 : 315. 66-Guton AC, Hall JE . Textbook of medical physiology . Harcourt Health Sciences Company .Philadelphia .WB Saunders and Company . 2000: 738. 67- Larson PS , Haag HB , Silvette H. Tobacco, Expermental and clinical studies . Baltimore, The Williams and wilkins Company 1961. 68-Burgen A. The secretion of potassium in saliva .J Physiol 1956; 132: 2039 . 69Ganong WF. |Review of medical phisology .Appleton and Lange.Medical publishing Division .California , 1987 ; :406. 70- Jhon W,Exploratory data analysis .addison-Wesley MA, 1977.
57

71-Heintz U Secretion rate ,buffer effect and number of lactobacilli and streptococcus mutans of whole saliva of cigarettes smokers and non smokers . Scand JDent Res 1984; 73: 509 512. 72- Pollard,F H Marun JV Analysit 81 :348-1956. 73- Doumas BTet-al .Standerd methods of clinical chemistry,7,175. Academic press of Chicago 1972. 74-leonard L. Cigarette smoking and precieption about smoking and health in chad .E Afr Med.J 1996; 73:509 512 . 75-Seldin D,Giebisch G. Smoking and Health ,US Deparment of health Education and Welfare 1979; 163. 76-Elizabeth A.KRALL and Bess Dawson-HUGhes.Smoking increase bone loss and decrease intestinal absorption. 77- Zhang XY, Leung FW 1994 .Cigareatte smoke aggregates acid induced duodenal mucosal injury in rat :Role of mesenteric Vasoconstriction.Scand J Gastroenterol 29:214-218. 78- Benowitz NL, Kuyt F.JacobP, 3rd jones RT , Osnam AL1983 Cotinine deposition and effects .Clinpharm Ther34:604- 611.

QUESTIONAIRE

Number ..( 2008 (A)

) Name Age Gender.

Date / /

58

Telephone no ..

(B)

Smoker Number of cigarette per day .. Duration of smoking /year Age when started smoking

(C)

History of other disease

Lung disease ( ) ( ) Liver disease ( ) ( ) Kidney disease ( ) ( ) Alcoholic ( ) ( ) Endocrine disease ( ) ( ) Para thyroid disease ( ) ( ) Diabetes ( ) ( ) Renal stones ( ) ( ) Nutritional state Excellent ( ) Good ( ) Bad (

(D)

Investigations
Serum calcium Salivary calcium Serum albumin mg/dl mg/dl g/dl

59