Effects of temporary examination stress on biochemical parameters in academic students

Master's Thesis 2016 164 Pages

Chemistry - Bio-chemistry







Chapter One: Introduction and Literature Review
1.1 Stress
1.1.1 Type of Stress
1.1.2 Stressor The Stress Response System and its Regulation
1.1.3 Symptoms of Stress
1.1.4 Coping with Stress
1.1.5 Examination Stress Cognition, working Memory, and Examination Stress Effects of Examination Stress on Students The Effects of Moderator on students’ Performance
1.2 Some Biochemical Parameters
1.2.1 Cortisol
1.2.2 Blood Glucose
1.2.3 α-Amylase
1.2.4 Total Serum Protein
1.2.5 Free Radicals and Oxidative Stress Defense Mechanism Against Oxidative Stress
1.2.6 Ceruloplasmin (Cp) Chemistry of Ceruloplasmin Physiological Function of Ceruloplasmin
1.2.7 Some Trace Elements Copper Iron Zinc
1.2.8 Blood Group

Literature and Review

Aims of Study

Chapter Two: Materials and methods
2.1 Instruments, Chemicals, and Samples
2.1.1 Chemicals
2.1.2 Instruments
2.1.3 Samples
2.2 Methods
2.2.1 Determination of Body Muscle Index (BMI)
2.2.2 Determination of Some Biochemical Parameters Determination of Cortisol Determination of Fasting Serum Glucose (F.S.G) Determination of Serum α-amylase Activity Determination of Total Serum Protein Determination of Serum Albumin Determination of Ceruloplasmin Oxidase Activity Determination of Ceruloplasmin Concentration Determination of Ceruloplasmin Ferroxidase Activity Determination of Trace Elements (Cu, Fe and Zn)
2.2.3 Determination of Blood Group
2.4 Statistical Analysis

Chapter Three: Results and Discussion
3.1 Description of Studied Subgroups
3.2 Determination of Some Biochemical Parameters
3.2.1 Levels of Cortisol
3.2.2 Glucose Levels, Amylase activity and Amylase Specific Activity
3.2.3 Protein Profile Levels
3.2.4 Ceruloplasmin (Cp ) Profile Levels
3.2.5 Serum Trace Elements (Cu, Fe and Zn) Levels
3.3 The Effect of Subgroup on Studied Biochemical Parameters
3.3.1 The Effect of Blood Type on Studied Biochemical Parameters The Effect of Blood Type on Cortisol The Effect of Blood Type on F.SG, Amylase and Amylase Activity The Effect of Blood Type on T.S.P, Ablumin and Globulin The Effect of Blood Type on Cp Profile The Effect of Blood Type on Trace Elements (Cu, Fe, and Zn)
3.3.2 The Effect of Rh Blood Type on Studied Biochemical Parameters The Effect of Rh Blood Type on Cortisol The Effect of Rh Blood Type on F.B.G, Amylase Activity and Amylase Specific Activity The Effect of Rh Blood Type on T.S.P, Ablumin and Globulin The Effect of Rh Blood Type on Cp Profile The Effect of Rh blood Type on Trace Elements (Cu, Fe, and Zn)
3.3.3 The Relation of Studied Biochemical Parameters with Exam Performance The Relation of Cortisol with Exam Performance The Relation of F.B.G, Amylase Activity and Amylase Specific Activity with Exam Performance The Relation of Protein Profile with Exam Performance The Relation of Cp Profile with Exam Performance The Relation of Trace Element (Cu, Fe, and Zn) with Exam Performance
3.3.4 The Relation of Studied Biochemical Parameters with First Semester Score The Relation of Cortisol with Semester Score The Relation of F.B.G, Amylase and Amylase activity with Semester Score The Relation of Protein Profile with Semester Score The Relation of Cp Profile with Semester Score The Relation of Trace Element (Cu, Fe, and Zn) with Semester Score
3.4 Correlation Analysis of Some Studied Biochemical Parameter
3.4.1 The Correlation of Cp Profile with Studied Biochemical Parameters
3.4.2 The Correlation of Protein Profile with Studied Biochemical Parameters
3.4.3 Correlation of Cortisol with Studied Biochemical Parameters
3.5 Discussion
3.5.1 Cortisol Levels
3.5.2 F.S.G Levels
3.5.3 Amylase activity
3.5.4 Protein profile Levels
3.5.5 Ceruloplasmin profile Levels Ceruloplasmin Ferroxdase activity Ceruloplasmin Oxidase activity Determination of Ceruloplasmin Concentration
3.5.6 Some Trace Elements (Cu, Fe, and Zn) Levels Determination of Serum Copper (Cu) Levels Determination of Serum Iron (Fe) Levels Determination of Serum Zinc (Zn) Levels
3.5.7 The Effect of Blood Type on Some Biochemical Parameters
3.5.8 The Effect of Rh Blood Type on Some Biochemical Parameters
3.5.9 The Relation of Some Biochemical Parameters with Exam
3.5.10 The Relation of Some Biochemical Parameters with Rate of First Semester
3.5.11 Correlation of Some Biochemical Parameters


Future work



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List of Abbreviations

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Thanking God for giving me the power and health to complete this work and get this degree, and blessings upon Mohammad the prophet and upon his family.

I would like to introduce my deep gratefulness and sincere thanks to my advisor Assistant professor Dr. Jwan Abdemohsen Zinulabdeen for suggesting and supervising this study with an endless support, encouragement and invaluable advice throughout the different steps of this work.

I gratefully acknowledge Presidency of the department of chemistry and graduate studies committee.

I wish to express my special thanks to the staff of Biochemistry, (Department of Chemistry/ College of Science/ University of Baghdad), specially to Dr. Yasser Abdul Hussein.

Special thanks to undergraduate students for giving specimen available for this study.

I wish to express my deep gratitude for my father and mother, my wife, sister and my brothers for their sacrifice and encouragement to complete this work.

At the end, my deep gratitude and appreciation go to my close friends: Miss Fatma Mohammed, Sana’a Husain, and Huda Ghazi who helped me and kept on supporting me during all the hard times in my work.

Mohammed Mahdi


The present study was designed to investigate the effect of temporarily examination stress on some biochemical parameters in sera of fifty five apparently healthy undergraduate students with age range (19- 24) years and (23.9 ± 2.13 kg/m2 ) body mass index, they were from University of Baghdad, College of Science; specialty from Mathematic, Physics, Geology, Biotechnology Departments, and also from Al-Esraa College, Medical Analysis Department. To compare the results, blood samples were obtained from the undergraduate students in two days, the first day was a normal day (N- day) and the second day was after 7-15 days, in the morning before the written exam (E- day). Based on gender, blood types, Rh blood type, exam performance, and the first semester score, the students were classified into several subgroups.

The first part of this study is devoted to the measurement of some biochemical parameters including concentration of cortisol, fasting serum glucose (F.S.G), total serum protein (T.S.P), albumin, globulin, ceruloplasmin (Cp), in addition to serum amylase , Cp oxidase and ferroxidase activities , as well as some trace elements (Cu, Fe, and Zn). The results of E-day in comparison with N-day indicated the following:-

- Non-significant decrease or increase in serum cortisol.
- Non-significant decrease or increase in F.S.G and amylase. Depending on gender female group was showed significant increase.
- Non-significant decrease or increase in albumin while serum T.S.P, globulin, and also albomin/globulin ratio were significantly decreased.
- Significant decreases in Cp oxidase activity.
- Significant decreases in Cp concentration. Depending on gender, males group was appeared non-significant decrease.
- Significant decrease in Cp ferroxidase activity. Depending on gender, only females group revealed highly significant decrease.
- Significant decrease in serum copper concentration, while iron and zinc showed non-significant different. Depending on gender, the serum iron concentration was significantly decreased in females group.

The second part of this study included, explore the effect of blood types, and Rh blood types on the studied parameters, also the relation between studied biochemical parameters with exam performance and first semester score were evaluated using SPSS version 21.0 with independent-t-test. The following results comparing E-day with N-day were found:

- Non-significant decrease or increase in serum cortisol in all blood types.
- Non-significant decrease or increase in F.S.G concentrations in all blood types, while significant differences in amylase activities and its specific activities were detected in students with B and AB blood types.
- Significant decrease in T.S.P, and globulin concentrations for students with blood type A and O, while non-significant differences in albumin concentrations in all blood types were detected. (A/G) ratio was significantly increased.  Significant decrease in Cp profile in all blood types except blood type O. while non- significant differences in Cp ferroxidase activity with its specific activity were found in sera of undergraduate students with AB blood type.
- Non-significant differences in (Cu, Fe, and Zn) concentrations for all blood types were found.
- Non-significant differences were found in serum cortisol concentrations for both negative and positive Rh blood types.
- Significant increase was found in serum F.S.G in both Rh blood type and significant decreases in serum amylase activity with its specific activity in student with negative Rh blood type, while the other groups showed non-significant difference.  Significant decreases were found in Cp profile (Cp concentration, Cp oxidase activity, Cp ferroxidase activity), T.S.P and globulin levels in both negative and positive Rh blood type, while serum albumin and trace elements (Cu, Fe, and Zn) showed non-significance.
- Significant decrease was found in Cp profile and serum copper of the group with pass exam performance. The differences in other parameters were non-significant.
- Significant decreases in Cp profile, T.S.P and globulin of group with good exam performance. The differences in other parameters were non-significant.
- Significant increases in F.S.G and Cp oxidase activity with significant differences in Cp concentration, ferroxidase activity, T.S.P and globulin in groups with pass semester score. The differences in other parameters were non-significant.
- Significant decreases in Cp profile in groups with medium semester score. The differences in other parameters were non-significant.

The third part of this study involved correlation between studied biochemical parameters using SPSS with pearson correlation analysis. The results indicated the following:

- Cp oxidase activity showed significant positive correlation with amylase activity and its specific activity.
- Cp ferroxidase activity showed significant positive correlation with amylase activity and its specific activity.
- Globulin concentration showed significant negative association with amylase activity and its specific activity.
- Serum cortisol level revealed significant negative correlation with serum iron concentration.

The main conclusion of the present study is that temporarily examination stress causes differences in most studied biochemical parameters.

Chapter One Introduction and Literature Review

1.1: Stress

Stress would simply be defined as the pressure or force that is exerted by a body (Gross, 1996), it is the nonspecific response of the body to a stimulus or event (Selye, 1956). According to psychologists, stress is specific demands that possesses on an organism and how the organism attempts to adapt or cope with these demands. Stress is a necessary and unavoidable concomitant of daily living, necessary because without stress human would be a listed and apathetic creature, and unavoidable because it relates to any external event, be it pleasurable or anxiety producing (Gross, 1996).

1.1.1: Types of Stress

Stress can be divided into two types: positive stress (Eustress) and negative stress (Distress). The eustress pertains to an exciting event stimulating a person to feel glad or happy like as a bride, before the marriage, she is so happy; she cannot sleep, that means eustress stimulates positive striving and emotions, which leads to good health, stimulates people and produces positive outcomes (Folkman, & Moskowitz, 2004; Nelson, & Simmon, 2003). The negative stress (distress) means the individual experiences negative emotions as anxiety or worry for example upon retiring (Seyle, 1974), it causes social and economic losses, especially for businesses; this failure comes from ineffective work due to the sickness that decreases both times at profit and work. Furthermore, people who suffer from negative stress often apply ineffective methods to seek release from it such as, smoking, drinking, taking drugs, and shopping. Some employees have indulged themselves in gambling, and some have committed suicide (Mehri, 2000).

Depending on time there are two types of stress, the first type is acute stress disorder (ASD), which is diagnosed when the stress symptoms persist for no longer than a month, while the second type is chronic stress disorder (CSD) when stress’s symptoms last more than a month (Khalil, 2013).

1.1.2: Stressors

A stressor is any real physical, social, or psychological event or stimulus that causes human bodies to react or respond (Glanz, & Schwartz, 2008), it may promote physiological or behavioral disturbances or both. However stress occurs with an interaction between individuals and sources of demand within the environment and is affected by one’s perception of the stressor either as adaptive or threatening to one’s resources (Long, 1995).

Stressors may be psychogenic and neurogenic, psychogenic stressors are purely of psychological origin (anticipating an adverse event, experiencing the death of a loved one, or caring for a chronically ill person), while neurogenic stressors means physiological effects, including activation of components of the hypothalamic-pituitary-adrenocortical axis (HPA) system (Sawchenko, & Ericsson, 2000) and involve a physical stimulus (headache, bodily injury, or recovery from surgery) (Anisman, Merali,1999). In general, stressors can be classified as:

- Environmental stressor: it is environmental pressure that affects humans, such as weather, noise, crowding, pollution, traffic, (Bijlsma et. al., 1997).
- Social stressor: it is the experiences of multiple stresses arising from the demands of the different social roles. Meanwhile some examples of social stressors include: deadlines, financial problems, job interviews, presentations, disagreements, loss of a loved one and divorce. (Crockford, et. al., 2008).
- Physiological stressor : situations and circumstances are affecting the body, such as rapid growth of adolescence, menopause, illness, aging, giving birth, inflammations, accidents, poor nutrition, and sleep disturbances (Reeder, & Kramer, 2005). Response to Stress and its Regulation

A person's response towards stress depends on whether an event is appraised as a challenge or a threat (Lazarus, & Folkman, 1984). Several factors play a fundamental role in determining the nature and consequences of the stress response such as inherent features of a given type of stressor and the conditions under which the stressor is encountered (Anisman, & Merali, 1999).

The essential components of the “stress system” are the hypothalamic- pituitary-adrenal(HPA) axis and the sympathetic nervous system(SNS). When the hypothalamus is evoked by a stressor, corticotropin-releasing hormone(CRH) and arginine vasopressin(AVP) are secreted, grapping both the production and the activation of the noradrenergic neurons of the locus caeruleas/norepinepherine (LC/ NE) and (ACTH) adrenocorticotropin hormone from the posterior pituitary system in the brain. The primarily responsible for the immediate “fight or flight” response driven by epinephrine and norepinephrine is LC/NE system, while ACTH drives the production of cortisol from the adrenal cortex. At the normal conditions, the production of CRH and ACTH fluctuate in a predictable circadian cycle and are inhibited by high levels of blood cortisol via a well-described negative feedback loop (Guilliams & Edwards, 2010) figure( 1-1).

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Figure 1-1: Regulation of the stress response by the hypothalamus-pituitary- adrenal (HPA) axis (Guilliams & Edwards, 2010) .

These disorders are accompanied by alterations in glucocorticoid secretion, suggesting that dysfunction of the hypothalamo-pituitaryadrenocortical (HPA) axis may be involved in the deleterious effects of stress on the affective state. For example, resistance to glucocorticoid feedback is observed in a substantial proportion of individuals suffering from melancholic depression. Given the connection between stress and affective disorders, it is important to note that the amygdala, hippocampus, and prefrontal cortex are also implicated in HPA axis regulation. The hippocampus and prefrontal cortex are largely (but not exclusively) inhibit the HPA axis secretion, whereas the amygdala is involved in activation of glucocorticoid secretion (Herman, & Cullinan, 1997; Feldman, et. al., 1995).

1.1.3 Symptoms of Stress

Stress symptoms can be physiological or behavioral or both, a study reported that 40 percent of deaths and 70 percent of diseases in the United States are related in whole or in part to psychological stress (Mokdad, 2004). Some of the behavioral stress are psychiatric disorders (Brown, 1993), the difficulty of sleep (Seyle, 1974), crying for no apparent reason, anxious (Adamec, et. al, 2011), fear and impulsively acting (Cowan, et. al., 2013), teeth grinding (Glanz, & Schwartz, 2008), and increase in use alcohol and smoking (Ansell, 2012).

Also there are a lot of symptoms and diseases that accompany with stress based on the situation and the level of stress. Some of these symptoms are:

- Headaches, increase in heart rate and rise in blood pressure (Brown, 1993).
- Frequently urinating and type2 diabetes (Mc Ewen, & Wingfield, 2003), a study indicated that controlling stress is critical for preventing the development of type 2 diabetes, as well as for successful diabetes management (Scollan, 2005).
- Hair loses: the most common stress-induced hair loss is telogen effluvium, often seen in individuals who have suffered a death in the family, had a difficult pregnancy, or experienced severe weight loss, this condition pushes colonies of hair into a resting phase in which much more hair falls out than grows. A similar condition, alopecia areata, occurs when stress triggers white blood cells to attack and destroy hair follicles (Hall, 2008).
- Gain weight: higher stress levels may increase cortisol levels in the bloodstream, contributing to hunger and activating fat-storing enzymes; cortisol plays a role in increased belly fat and eating behaviors (Vicennati, 2009).
- The digestive problem: the causes of gastrointestinal disorders are often unknown; most likely, an underlying illness, pathogen, injury, or inflammation is exacerbated by stress, triggering nausea, vomiting, stomach cramps, or diarrhea (NDDIC, 2007).
- Impaired immunity: increased stress over time can affect cellular immune response (Denson, et. al., 2009 ). Acute stressors can impair immunity for as much as six months (Miller, et. al., 2009); prolonged stressors such as loss of a spouse, caregiving, and unemployment also have been shown to impair immune response over time (Seagerstrom, & Miller, 2004).
- Acute stress has been shown to impair short-term memory, particularly verbal memory (Schwabe, et. al., 2009), and also nervous ticks (Muehlenkamp, 2010).

1.1.4: Coping with Stress

Coping with stress can be defined as an effort to manage, overcome demands and critical events that pose a challenge, threat, harm, loss, or benefit to a person (Lazarus, 1991). Cohen and Lazarus have generalized the coping strategies that an individual may use to reduce stress into five main categories (Cohen, et. al., & Lazarus, 1979).

- Direct action responses: is the first coping strategies where the individuals change their attitudes towards the stressors and attempt to either defend themselves against it or simply flee from it.
- Information seeking: involve the individual trying to understand the stressful situation better.
-Inhibition of action: involves the individual simply not taking any action.
-Palliative coping: individuals change their internal environment by using drugs, alcohol or simply by using relaxation techniques.
-Turning to others: is the last coping strategies that emotional support or simply to get things off one’s chest, also known as catharsis.

Meanwhile coping with stress may be explained as stress exposure training, it is the situations and conditions similar to these expected to be encountered in a real-world. The benefit of exposure stress training teaches individuals strategies to maintain performance under stress, and contribute to overlearning, task mastery, and increased self-confidence (Driskell, & Johnston, 1998). It can also be effective in improving group performance under stress by teaching groups how to adapt their performance strategies to external stressors and alerting them to how other team members will be affected by stress. Groups that undergo training tend to have better communication, teamwork, and feedback strategies that help them to work together under stress (Serfaty et. al., 2011).

1.1.5: Examination Stress

Examination stress refers to the pressure or stress that is experienced by standard students to perform well in final school or undergraduate examinations and competitive college entrance examinations (Lee, & Larson, 2000). Cognition, Working Memory, and Examination Stress

Cognition is the activity of knowing: the acquisition, organization, and use of knowledge (Neisser, 1967), it is refers to the mental process of knowing by which external or internal input is transformed, reduced, elaborated, stored, recovered, and used a such, It involves a variety of functions such as perception, attention, memory coding, retention, and recall, decision making, reasoning, problem-solving, imaging, planning and executing actions. Such mental processes involve the generation and use of internal representations to varying degrees, and may operate independently (or not) at different stages of processing (Brandimonte, et. al., 2006). Furthermore, these processes can to some extent be observed or at least empirically proved, leading to a scientific investigation using methods akin to those of the natural sciences (Barsalou, 1999).

Working memory refers to a brain system that provides temporary storage and manipulation of the information necessary for such complex cognitive tasks as language comprehension, learning, and reasoning (Baddeley, 1990).

Most psychological accounts propose that stress impairs cognitive function by reducing the amount of attention one can devote to information processing (Kahneman, 1973), more specifically, the negative effects of stress on cognition will be manifested when task performance requires attentional control, or effortful cognitive processing (Hasher & Zacks, 1979). Individuals who perceive life as more stressful suffer in cognitive performance. Subjective perceptions of how stressful in previous life events have been associated with poorer decision making (Baradell, & Klein, 1993), problem-solving (Klein, & Barnes, 1994), working memory (Klein, & Boals, 2001), analogical reasoning (Yee, et. al., 1996). Also exposure to examination stress results in an increase in the manipulation component of working memory that related to cognitive performance (D’Esposito & Postle 2002), and increased in heart rate and rise in blood pressure caused by the release of adrenaline and are intended to stimulate the central nervous system in preparation for performance (Brown, 1993), also, individual who has a cognitive problem, suffers from academic achievement (Alloway et. al., 2009). Effects of Examination Stress on Students

Having examination stress is a common and widespread phenomenon for students (Kyriacou & Butcher, 1993), up to 30%-50% of student’s has testinduced anxiety problems (Beidel et. al., 1999). Every student aspires to pursue academic success to achieve respect, family pride, and social mobility (Gow et. al., 1996), these projections in extremely high academic demands and extraordinary pressure on students (Bossy, 2000). The experience of academic stress leads to a sense of distress, which is generally manifested in a variety of psychological and behavioral problems (Lee, & Larson, 2000).

The test scores of students may decrease due to anxiety under pressure (Worthy et. al., 2009), so if test anxiety is eliminated, student grades will increase (Ramirez & Beilock, 2011). Test anxiety is significantly correlated with low academic performance (McCarthy & Goffin, 2005). A mild degree of stress and strain can sometimes be beneficial. For example, often feeling mild stressed when carrying out a project or assignment, compels us to a good job, focus better and work energetically (Archana, 2014). The Effect of Moderator on Students Performance

In fact getting stressed a bit about examination means that students care about the result they will get (Archana, 2014). Variables that intervene in the stressor-stress relationship or the stress-performance relationship, in most cases reducing the effect of stress on the individual. Some variables affect performance, known as moderators; that can reduce the performance decrement caused by stress, so it is one that affects the relationship between stress and performance, figure (1-2). That means, moderators are important because they intervene in the stressor-stress performance relationship and reduce negative effects of stressors and stress on the individual. Individuals classified as “high anxiety” tend to experience more dramatic physiological responses to stressors than do those who are classified as “low anxiety” (Pearson & Thackray, 1970). Additional information can act as a moderator between stress and performance by helping reduce the uncertainty associated with stress and improving the accuracy of individual expectations and performance of certain tasks (Glass & Singer, 1973).

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Figure 1-2: The effect of moderators on students’ performance (Archana K., 2014)

Moderators can also act to reduce the effects of stress on group performance. For example, group cohesion is said to improve unit morale and efficiency and reduce negative stress reactions among group members (Milgram, et.al,, 1989).

Although moderators usually reduce the effect of stress on performance, some moderators can have the opposite effect and increase the performance effects of stress, (Jennifer, 2005).

1.2: Some Biochemical Parameters

1.2.1: Cortisol

Cortisol ( hydrocortisone ) is a steroid hormone that is produced mainly by zona fascicualata, it synthesized from pregnenolone, the consequences from cholesterol.The structure of cortisol and all steroid hormones raised from the cyclopentanoperhydrophenanthrene nucleus (C17) that include three rings with six carbons attached with five carbons ring. The biosynthesis of corticosteroids in Adrenal Cortex is summarized in figure (1-3). The conversion of or inactive forms of cortisol hormone involves alteration of ring substituents rather than the ring structure itself (Thomas & Sons 2011).

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Figure 1-3: Biosynthesis of cortisol (Guilliams & Edwards, 2010).

Cortisol is known as the stress hormone because it is released in response to major stresses, enable the individual to withstand, or cope with the metabolic, cardiovascular and psychological demands. It is promotes the conservation of glucose as an energy source in several mechanisms such as induces and maintains the activity of all of the gluconeogenic enzymes in the liver and inhibit glucose utilization in tissue, skeletal muscle, and adipose tissue by inhibiting glycolysis and promoting the use of fatty acid. Also cortisol plays a role in preventing proliferation by weakening the activity of the immune system, lowers bone formation, increase blood pressure, allows the kidneys to produce hypotonic urine and increase the effectiveness of catecholamine (Bhagavan, 2001).

1.2.2: Blood Glucose

Glucose is a simple sugar, its building unit of carbohydrates, consumption and digestion of all foods containing carbohydrates will affects the rise and fall of blood glucose levels; however, some foods will increase levels at different rates than others. It is an immediate source of energy for cells; most glucose used by the body can be accounted for by six tissues: the brain (45-60%), skeletal muscle (15- 20%), kidney (10-15%), the blood cells (5-10%), splanchnic organs (3-6%), and adipose tissues (2-4%) (Lincoln & Eaddy2001).

The brain uses a large amount of energy and is dependent on blood glucose as its source of energy, glucose taken up by the brain is completely oxidized whereas that taken up by the kidney, blood cells, splanchnic tissues, and muscle mainly undergoes glycolysis (Hoyer, 2000). Because the brain cannot store glucose, it requires a continuous supply of glucose to function properly, any shortage in this availability of glucose to the brain has adverse consequences for its functioning (Jolene et. al., 2007).

The regulation of blood glucose involves the pancreas, liver, brain, and several hormones. Reduced blood sugar level, or hypoglycemia, is “an abnormally low plasma glucose level, it occurs when glucose is released into the bloodstream more slowly than needed after glucose is used up in the body too rapidly, or when excessive insulin is released into the bloodstream. The first signs and symptoms begin when levels of blood sugar below 70 mg/dl, although this varies from individual to individual (Amiel, 1998). Hypoglycemia’s effects on the central nervous system (CNS) leads to symptoms of sympathetic nervous system stimulation or of central nervous system dysfunction” (Rosenbloom et. al., 1999), like deficiencies in coordination, headaches, blurred vision, anxiety, dizziness (Field, 1989), nervousness, tremor tiredness, confusion, and slowed mental function (Lincoln & Eaddy, 2001), while high blood sugar (hyperglycemia) is aserious medical problem affects people who have diabetes.

1.2.3: α-Amylase

α-Amylase (EC (1,4-α-D glucanohydrolase) is a small

heterogeneous enzyme, with a molecular weight of 50-55 KDa, figure (1-3), it is an essential enzyme in the physiologic digestion of starches, breaking down large insoluble starch molecules into smaller soluble molecules and it requires calcium and chloride ions for its activity (Rejzek et. al., 2011). Both straight chain poly glucans such as amylose and branched poly glucans such as amylopectin and glycogen are hydrolyzed, although at different rates. The enzyme split the chains at alternate α-1,4-hemiacetal (-C-O-C-) links forming maltose, glucose and a residue of limit dextrins (Merrit & Karn,1977 ).

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Figure 1-4: Amylase protein structure

(Rejzek et. al,. 2011)

α-Amylase exists in different isoenzyme forms; salivary type (S-type) and pancreatic type (P-type) (Shimamura et. al.,1976).Several different cells and tissues synthesize the salivary type amylase, including (salivary, lacrimal, sweat, lactating mammary) glands, genital tissues, lung; bronchogenic and ovarian tumors; leukocytes and thrombocytes cells, but it suggested that amylase accounts for 40-50% of the total salivary gland-produced protein, most of the enzyme being synthesized in the parotid gland (Noble, 2000). Human parotid saliva and submandibular saliva contain about 45 mg and 30 mg of amylase, respectively, per 100 mg of protein. This enzyme considered to be a reliable marker of serous cell function. It interacts specifically with certain oral bacteria and may play a role in modulating the adhesion of those species to teeth. Salivary amylase inhibits the growth of Legionella pneumonia and Neisseria gonorrhea (Almstahl et. al., 2001). The amylase concentration in radiation-induced hyposalivation has been found to be reduced (Almstahl et. al., 2001). Salivary amylase activity is of short duration because, on swallowing, it is inactivated by the acidity of the gastric contents. Pancreatic amylase then performs the major digestive action on starch once the polysaccharides reach the intestine. The pancreatic type (P-type) amylase is essentially tissue-specific, and semen is being the only other source. Amylase is small enough to pass through the glomeruli of the kidney, so it is the only plasma enzyme normally found in urine (Pederson et. al., 2002).

1.2.4: Total Serum Proteins.

Total serum protein (T.S.P) is made up of albumin and globulin, the globulin, in turn, is made up of α1, α2, β, and γ globulins. These fractions can be quantitated using protein electrophoresis, but the total protein test is a faster and cheaper test that estimates the total of all fractions together (Jeffery, et. al., 2006) figure (1-5).

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Figure 1-5: Total protein fractions (Jeffrey J., et. al., 2006)

Low level of protein is the indicator of inflammatory bowel disease, liver problems, and malabsorption. A high T.S.P level may be seen with chronic inflammation or such as viral hepatitis or HIV. It also may be associated with bone marrow disorders such as multiple myeloma (Podolsky et. al., 1991).

Human serum albumin (HSA) is a single-chain, non-glycosylated polypeptide with a molecular weight of 66,500 Da containing 585 amino acids. It is synthesized primarily in the liver with a few of tryptophan and methionine residues and abundant of charged residues such as lysine, arginine, glutamic acid and aspartic acid and the overall charge is negative. It is a helical protein with turns and extended loops and resembles a heart shape, the protein is composed of 3 homologous domains (I, II and III), each containing two sub-domains (A and B). HSA regulates 75% of colloidal osmotic or oncotic pressure, and transports numerous endogenous compounds such as fatty acids, hormones, bile acids, amino acids, metals, toxic metabolites (Feng, et. al., 2014). It provides protection from lipid peroxidation propagated by inorganic reactive oxygen species (ROS) and provided effective extracellular scavenging antioxidant protection under such circumstances (Radi et. al., 1991).

The globulins are a family of globular proteins that have higher molecular weights than albumins and are insoluble in pure water but soluble in dilute salt solutions. The globulin proteins are α1- globulins, α2- globulins, β-globulins, and ϒ- globulins , these are produced in the liver accept ϒ- globulins that produced by lymphocyte cells (Sanchez, et. al., 2004). α-globulins help to transport substrates such as hormone, cholesterol, enzymes, inhibitors, copper ion, vitamin A, and hemoglobin they play role in coagulating (factor II). β-globulins play role in the transport of lipid, hormones, iron ion (Fe+3 ) and vitamin B12 (koolman & Röhm, 2005), while ϒ-globulins play the main role in immunity system with different mechanism they response to inflammatory, antigens, and pathogens (Robert et. al., 2003 ).

The albumin/globulin (A/G) ratio is a biochemical parameter utilized in the interpretation of changes in serum proteins that accompany a disease. A/G ratio is reduced as a result of a decrease in serum albumin and the sequential increase in serum globulins (Henry 2002). The classical change expected to accompany liver disease when serum albumin is decreased below normal levels (Dufour et. al., 2000).

1.2.5: Free Radicals and Oxidative Stress

A molecule that consists of the unpaired electron(s) in an outer shell called free radical; it is formed by breaking of a chemical bond so that each species keeps one electron (Halliwell & Gutteridge, 1990). Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are terms used for a group of oxidants which are capable of generating free radicals, it they are produced by living organisms (Valko et. al., 2006). The most important radicals in biological systems are radical derivatives of oxygen like, superoxide anion [Abbildung in dieser Leseprobe nicht enthalten], hydroxyl radical (OH•), nitric oxide (NO.), the lipid-derived peroxy radical (ROO•) and alkoxy radical (RO•) (Fridovich , 1998).

Some ions of transition metals may as well catalyze the reaction between H2O2 and superoxide, leading to the production of hydroxyl radical (Claudia & Alvaro, 2004).

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Cellular metabolism normally produces free radical at low to moderate concentrations; they function in cell physiological processes, but at high concentrations, they produce adverse modifications to cell components (Halliwell , & Gutteridge, 1999), so ROS and RNS are playing a dual role in biological systems since they can be either harmful or beneficial to living systems (Valko, 2004). Beneficial effects of ROS involve physiological roles in cellular responses to the stimulating agents for biochemical processes within the cell (Droge , 2002), they exert their effects on transcription factors such as nuclear factor-kappa B (NF-kB) and activator protein leading to gene expression and cell growth (Schreck & Baeuerle, 1991). It is responses to defense against infectious agents and in the function of some cellular signaling systems. One further beneficial is at low concentrations of ROS is the induction of a mitogenic response (Poli et. al., 2004).

Uncontrolled generation of ROS can lead to their accumulation causing oxidative stress in the cells, so oxidative stress can be defines simply as an imbalance between oxidants and antioxidants in favor to the oxidants figure (1-6) , potentially leading to lipids, proteins (Wang et. al., 1996) and DNA damage (Marnett, 1999), that means that oxidative stress implicated in many pathological conditions such as cancer, neurological disorders (Sayre et. al., 2001) atherosclerosis (Dhalla et. al., 2000) , hypertension (Kerr et. al., 1999), ischemia /perfusion (Kasparova et. al., 2005) acute respiratory distress syndrome (Dut et. al., 2008), chronic obstructive pulmonary disease. Also free radical generated during irradiation by UV light, X-rays, gamma rays, metal-catalyzed reactions, pollutants in the atmosphere, neutrophils and macrophages during inflammation, (Comhair et. al., 2005, and Ercan et. al., 2006).

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Figure 1-6: Oxidative stress (Wang et. al., 1996) Defense Mechanisms against Oxidative Stress

Exposure to free radicals from a variety of sources has led organisms to develop a series of defense mechanisms for protection against ROS-mediated oxidative damage. These include antioxidant defenses to keep a check on the generation of ROS (Amit & Priyadarsini , 2011); an antioxidant is a substance that is present at low concentrations and significantly delays or prevents oxidation of the oxidizable substrate (Kohen & Nyska, 2002), they are classified into three categories (Smith et, al, 2005).

1- Preventative antioxidants: which prevent the formation of new ROS, such as: ferritin, transferrin, ceruloplasmin, and albumin.
2- Scavenging antioxidants: which remove ROS once formed thus preventing a radical chain reaction, this includes: enzymatic antioxidants (catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase, ect.), and non- enzymatic antioxidants (glutathione, vitamin E, vitamin C, bilirubin, and uric acid).
3- Repair enzymes: that repair the damage and reconstitutes membranes, these enzymes are lipase, proteases, DNA repair enzymes, transferase, and methionine sulphoxide reductase that repair the damage in biomolecules and reconstitute the damaged cell membrane. Enzymes that repair damaged DNA, damaged protein, oxidized lipids and peroxides and also to stop chain propagation of peroxyl lipid radical (Dekkers et. al., 1996).

1.2.6: Ceruloplasmin (Cp )

Ceruloplasmin (CP) (EC is one of the antioxidant proteins; it is an abundant α2-glycoprotein containing over 95% of the copper found in the plasma of all vertebrate species (Harris, 1991). CP is synthesized and secreted exclusively by the liver (Owen & Hazelrig, 1966) although, kidney (Gaitskhoki et. al., 1990), lung (Fleming et. al., 1991) and lymphocytes as apoprotein (Harris et. al., 1998). Chemistry of Ceruloplasmin

Cp consists of a single polypeptide chain of 1046 amino-acid residues with a carbohydrate content of between 7% and 8%, it has a molecular weight of 132 kDa, containing 6-7 copper atoms per molecule and four potential sites for asparagine-linked glycosylation, it is the only blue copper oxidase of human and animal species (Takahashi et. al., 1984). figure (1-7 (a)) shows the overall organization of the human ceruloplasmin (hCP) molecule and the locations of the various metal cations Cu2 +, Ca2 + ,and Na+.

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Figure 1-7: (a) The overall organization of the ceruloplasmin molecule, showing the six cupredoxin domains (domains 1, 2, 3, 4, 5 and 6 in red, orange, yellow, green, blue and purple, respectively) and the locations of the metal binding sites: Cu2 + as blue spheres, Ca2 + as an olive-green sphere and Na+ as red spheres. The relatively planar bottom surface and the protuberances at the top surface are clearly visible.

(b) the trinuclear cluster between domains 1 and 6, showing the water molecule attached to the type (Cu2, Cu3 &Cu4). (Bento, et. al., 2007). The figures were prepared with the PyMOL program (Delano, 2002).

The incorporation of copper into apoceruloplasmin also contributes to its tertiary structure (Filippis et. al., 1996), three of the six copper atoms form a trinuclear cluster sited at the interface of domain 1 and 6 serves as the oxygen binding and reducing site during the catalysis and the other three form mononuclear sites in domains 2,4 and 6 figure (1-7 (b)) (Zaitseva et. al., 1996; Takashashi et al ,1984). These mononuclear copper atoms serve to translocate electrons form Fe (II) ion to the trinuclear center (Zaitseva et. al., 1996). Each of the mononuclear copper atoms is coordinated to one cysteine and two histidine residues.Those in domains 4 and 6 also coordinate to a methionine residue, but in domain two the methionine is replaced by leucine residue; therefore by this substitution, this site has redox potential values higher than other sites (Zaitseva et. al., 1996; Ortal et. al., 1984) and the other three copper atoms in the trinuclear center at the interface between domain 1 and 6 are bounded by four pairs of histidines (Bieli et. al., 2001) figure (1-8).

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Figure (1-8): Schematic showing the positions of the various coppers in human ceruloplasmin. Cu 2, Cu 4, and Cu 6 represent mononulcear copper atoms in domains 2,4,6 respectively. Cu, Cu\ and Cu\\ represent coppers in the trinuclear copper center. The arrows represent the electron transfer pathway (Zaitseva et al., 1996; Messerschmidt & Huber, 1990). Physiological Functions of Ceruloplasmin

Ceruloplasmin, the multifunctional copper containing enzyme, possesses significant functions as following; ferroxidase activity, oxidase activity, transport of copper, serum antioxidant, and endogenous modulator of the inflammatory response.

Cp has been implicated in iron metabolism mostly because of its catalytic oxidation of Fe (II) to Fe (III) by its ferroxidase activity (Bento et. al., 2007). With its subsequent incorporation into apotransferrin (Osaki et. al., 1969;1966) or into the Fe storage protein ferritin (Van Eden & Aust, 2000). The copper in Cp plays an important role in the oxidation of iron before it is transported to the plasma (Yuan et. al., 1995; and Osaki et. al., 1966).

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Cp also catalyzes the reduction of an oxygen molecule with formation of water, without releasing intermediaries potentially toxic (O2 -, H2O2) (Osaki S., et. al., 1969). Cp possesses a significant oxidase activity directed toward substrates such as aromatic amines, phenols and ascorbate (Holmberg & Laurel, 1951). It acts as a regulator of plasma and tissues biogenic amines levels, this function of Ceruloplasmin is used for it's in vitro assay and identification (Osaki et. al., 1964). The following equations can explain the oxidation reaction catalyzed by Cu-Cp and free Cu:

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Cp plays an essential role in the transport and metabolism of copper (Hellman & Gitlin, 2002). Over 95% of human plasma copper is bind with the Cp tightly fraction and the remaining 5-10% of plasma copper loosely attached to albumin and histidine, but only a trace of copper is present as free Cu+2 (Burtis & Ashwood, 1996; Lau & Sarkar, 1981); most of this bound copper is rapidly deposited in the liver. Copper is added to Cp during its synthesis by the liver, Cp transported out of the liver and delivers cluster of copper into a variety of nonhepatic cells and different tissues, the uptake of copper from Cp is thought to involve interaction with cell surface receptors (Harris, 1993).

Also Cp acts as an antioxidant enzyme by inhibiting iron-dependent lipid peroxidation, and hydroxide. formation of hydrogen peroxidase via its ferroxidase activity (Gundogdu et. al., 2007) , and this activity is considered as an important antioxidant property of Cp in vivo (Park et. al., 2001); reacting with and scavenging H2O2 and superoxide anion, and inhibiting copper-induced lipid peroxidation by binding copper ions (Gundogdu et. al., 2007). It includes extracellular antioxidant activity by promoting Fe mobilization and thus preventing metal-catalyzed free radical tissue damage (Graf & Noetzel, 1999).

Cp is one of the acute-phase proteins, the level of Cp increase in response to injury, neoplasia and infections (Gundogdu et. al., 2007), its concentration increase during thyroid hormone administration or hyperthyroidism (accompanied by increased serum copper levels) in humans, pregnancy, and also hypothyroidism (accompanied by decreased serum copper) (Herman & Kun, ;1961 Johnson et. al., 1959).

1.2.7: Some Trace Elements Copper

Copper is the third most abundant trace element in the human body, following zinc and iron (Vilanova et. al., 1997), according to the literature, about 5% of human plasma copper is bound to albumin or amino acids such as histidine, and the remaining is bound to Cp (Halliwell et. al., 1990). It is an essential metal acts as cofactor for many proteins (Eddy, 1996), like superoxide dismutase, cytochrome C oxidase, lysyl oxidase, tyrosinase, dopamine hydroxylase, and clotting factor V (Ma, 1995). Cp may be able to supply copper to cells for incorporation into other copper proteins, this copper donor role of Cp is often referred to as a copper transport function, while metallothioneins are the copper storage protein (Linder & Hazegh, 1996).

The serum level of copper is usually constant. It is increased in patients with an acute myocardial infection, leukemia, solid tumors, infections and with cirrhosis of the liver(Nasulewicz et. al, 2004). Also, elevated levels were found in carcinoma of breast, colon, ovary, gynecological and larynx (Ma, 1995;Ycei et. al., 1994). High levels of copper in areca nut (a major etiological factor in oral cancer) plays an initiating role in stimulation of fibrogenesis by upregulation of lysyl oxidase and thereby causing inhibition of degradation of collagen (Cetinkana et. al., 1998). Wilson’s disease (WD) is an inherited disorder of copper metabolism; it has many associated biochemical disturbances (Frommer, 1974). Excess copper attacks the liver and/or brain, resulting hepatitis, psychiatric, or neurological symptoms and mental illness. Unbound copper spills out of the liver and is deposited in other organs and tissues, where it also provokes damage and dysfunction (Milkiewicz et. al., 2000). Iron

Iron (a transition element) is one of the essential metal ions in human (Fairbanks & Klee, 1999). Most of the iron in the body is in hemoglobin, a moderate amount of iron is in myoglobin, a small amount, but extremely important is in the tissue where iron is bound to several enzymes that require iron for full activity(Crichton, 2001), these include peroxidase (Candeias, et. al., 1993), catalase and cytochrome(Crichton, 2001). Iron stored in ferritin and hemosiderin, primarily in the bone marrow, spleen, and liver. A small amount of iron found in plasma associated with transferrin and albumin (Anghilrri, 1995).

Iron is capable of reduction-oxidation (redox) activity, i.e. play a role as a pro-oxidant, since it can enhance production of free radicals through Fenton & Haber-Weiss reactions and subsequent oxidative stress and cell damage (Meneghini, 1997). Therefore, iron had been found to be involved in the carcinogenic process (Smith & Perry, 1995), and hematological abnormalities in oral leukoplakia (Jaydeep, 1997) ; the critical pool of iron may be the first to become diminished in iron deficiency states (Mc Cord,1991) also the occurrence of iron deficiency is known to present in oral cancer (Khanna& Karjodkar, 2006). Zinc

Next to iron, zinc is the most abundant trace element; its biological functions can be divided into three categories: catalytic, structural and regulatory, it is a component of > 200 different enzymes (King J., 1990), such as alkaline phosphatase, lactate dehydrogenase, carbonate anhydrase, nucleoside phosphorylase, cytosol and extracellular superoxide dismutase, RNA and DNA polymerases, and DNA transcription factors (Vallee & Falchuk, 1993). Zinc enzymes are essential for growth, wound healing, the integrity of connective tissue, reproductive function of the immune system and protection from free radical damage (Cunningham-Rundles, 1996). This element deficiency is associated with poor growth, loss of appetite, skin lesions, delayed wound healing, and impaired immune response and malignancies (Henry, 2002). Varghese and coworkers found a significant reduction in the serum copper and zinc levels in oral submucous fibrosis and depressed in oral cancer (Varghese et. al., 1987).

The copper/zinc ratio appears to be more decisively important than either of the individual metals alone, so zinc deficiency often results in elevated blood levels of copper, due to the dynamic competition of these metals in the body (Cetinkana et. al., 1998).

1.2.8: Blood Group

Blood type is a classification of blood-based on the presence or absence of inherited antigenic substances on the surface of red blood cells (RBCs). These antigens may be proteins, carbohydrates, glycoproteins, or glycolipids, depending on the blood group system, some of these antigens also present on the surface of other types of cells of various tissues. A total of 35 human blood group systems is now recognized, but the ABO system is the most important blood group system in human blood transfusion (ISBT, 2015), this system is defined by two red blood cell antigens, A and B, whose presence or absence is determined by three alleles (A, B, O) segregating at a single genetic locus. Anti - A and Anti - B, The common ABO antibodies are produced in the first years of life by the sensitization to environmental substances such as food, bacteria, and viruses (Daniels et. al., 2004), figure (1-9).

Blood group A individuals possess a 3-α-N-acetyl galactosaminyl transferase (GTA), which catalyzes the transfer of N-acetyl-D-galactosamine from the donor UDP-GalNAc to the H precursor structure Fucα(1-2)Galβ-R to provide an A determinant GalNAcα(1-3)[Fucβ(1- 2)]Galβ-R.; Blood group B individuals possess a 3-α-galactosyltransferase (GTB), which uses the same H structure but catalyzes the transfer of galactose from UDP-Gal to make the B determinant Galβ(1-3) [Fucα(1-2)]Galβ-R. Blood group O individuals do not have any functional enzyme and AB individuals have both GTA and GTB (Oriol, 1995).

If an individual is exposed to a blood group antigen that is not recognized as self , the immune system will produce antibodies that can specifically bind to that particular blood group antigen, on the surface of the red blood cells and leading to the destruction of that cells (agglutination), low blood pressure, and even death (Anthea, et. al., 1993).

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Figure 1.9: Relation between blood types with antigen and antibody (Daniels et. al., 2004)

The Rh blood group system is the most polymorphic of the human blood groups, consisting of at least 45 independent antigens and, next to ABO, is the most clinically significant in transfusion medicine. There is two type of Rh blood type; negative and positive and it’s dependent on presence or absence of antigen D (Avent & Reid, 2000).

Literature Review

The study of psychological stress has interested by many researchers because it is a daily and unavoidable event, it describes what people feel when they are under mental, physical, or emotional pressure. Students have a lot of stress especially during the exam period, therefore examinations stress considered as an important type of psychological stresses, however it had been studied extensively by psychologists but there are a little number of studies deal with the measurements of biochemical parameters in this type of stress.

Several studies reported that serum cortisol increased before examination stress as response to stress that causes by exam (Ignatius, et. al., 2015; Ehiaghe et. al., 2014; Onyenekwe, et. al., 2014; Rachit et. al., 2012; Qurrat-ul-Aen et, al., 2011,Diana, et. al. , 2010; Demir et. al., 1996). McClelland (et. al., 1985) found that stress of an exam was associated with an increase in salivary immunoglobulin (S-IgA), with an increase in norepinephrine (NE) concentrations in the saliva, likewise, Mouton (et. al., 1989) suggested that a weak negative correlation was observed between the level of salivary immunoglobulin A (IgA) and the stress rating , only at the final examinations , however the assaying IgA in saliva to measure stress may not be as useful in psychophysiological research as expected. Janssen (et. al., 1989) reported that the training program decreased plasma albumin concentration while total protein did not change. Bosch (et. al., 1996) investigated the effect of academic examination , the results showed a significant increase of salivary total protein concentration, while Van Hunsel (et. al., 1996) found that the levels of serum albumin, γ-gamma- and β-globulin were significantly lower in major depression than in normal controls, however the percentages of the α1- and α2-globulin fractions were significantly higher in major depressed subjects than in normal controls. Maes (et. al., 1997) reported that academic examination stress induced significant increases in serum IgA, IgG, IgM , however the stress-induced changes in serum IgA, IgM, and IgG were normalized a few weeks after the stress condition. The author suggested that psychological stress is accompanied by an altered secretion of serum Igs, complement factors and some acute phase proteins. Bosch (et. al., 2004) found that academic exams decrease salivary S-IgA, also Petibois & Déléris (2003) reported that a physical stress induced a raise in T.S.P and albumin concentrations. Al-nahri (1996 ) found that the T.S.P, albumin, Ig-A, Ig-G and Ig-M significantly increased before exam comparing to several week after exam.

Demir (et. al., 1996) reported that the serum zinc concentration as well as carbonic anhydrase activity significantly decreased at examination stress comparing with control group, non-significant differences in serum albumin and T.S.P. were also indicated . Qurrat-ul-Aen (et, al., 2011) found that the examination stress increase in serum leptin also they found the appetite of students decreased significantly before and during exam,it increased significantly after getting education. A recent study indicate significant decrease in serum ferritin before exam comparing with after exam (Hettiarachchiet. al., 2014). Furthermore (Ignatius et. al., 2015) reported significant increase in serum adrenaline and lipid profile at examination stress comparing with control group.

Aims of the Present Study

- The main aim of the present study is to check the effect of temporarily examination stress on some biochemical parameters in sera of undergraduate students.
- Study the relation of some biochemical parameter with exam performance and first semester score at temporarily examination stress.
- Study other indicators which may be more sensitive than cortisol in response to examination stress that can play an antioxidant.

To our knowledge no work has deal with the study of Cp profile (Cp concentration, Cp oxidase activity with its specific activity and Cp ferroxidase activity with its specific activity), copper, and iron concentrations as well as the effect of blood type on biochemical parameters and the relation of these parameters with exam performance and first semester score in sera of undergraduate students with temporarily examination stress.



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Title: Effects of temporary examination stress on biochemical parameters in academic students