EFFECT OF HAEMOGLOBIN GENOTYPE ON EXERCISE TOLERANCE.

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RESEARCH PROJECT TOPIC ON EFFECT OF HAEMOGLOBIN GENOTYPE ON EXERCISE TOLERANCE.

ABSTRACT
The haemoglobin genotypes of 100 university student between the ages of 18-25 years were determined by cellulose acetate membrane (CAM) electrophoresis. There were 50 HBAA persons (25 males and 25 females) and 50 HBAS persons (25 males and 25 female). The exercise tolerance between the two genotype were then compared. Exercise was carried out on a bicycle ergometer and readings were achieved by a spirometer and keymograph. Exercise tolerance was determined by respiratory rate. There was a significant difference in exercise tolerance between HBAA persons and HBAS persons. HBAA individuals have a significantly higher exercise tolerance than their HBAS counterparts (p< 0.05). This suggests that HBAA persons may have a better perfo

CHAPTER ONE
1.1 HAEMOGLOBIN GENETYPE:
There are 3 main haemoglobin genotypes found in Nigeria. These are Hb AA, Hb As and Hb SS. Haemoglobin AA genotype represents the normal adult haemoglobin. Haemoglobin as and Haemoglobin SS genotypes are known as sickle cell trait and sickle cell anaemia respectively. The basis anomaly underlying both Hb AS and Hb SS genotypes is the presence of an abnormal haemoglobin, known as haemoglobin – S. in those with the anaemia all the haemoglobin eg (SS). In those with the trait the haemoglobin – s has a proportion of about 30 – 40%, the remainder being normal haemoglobin I e heterozygous, which involves the combination of a normal haemoglobin with the variant haemoglobin eg AS.

1.2 HAEMOGLOBIN:
Normal adult haemoglobin has a molecular weight of 67, 000 and contains the iron pigment which is haem, and the protein globuin. Globulin has 4 chains in 2 pairs of (alpha) and BB (beta).
The heme portion is formed mainly from acetidc acid and glycine in the mnitochnria. The acetic acid is changed in krebs cycle to succinyl COA.
The basic chemical steps for formation of haemoglobin are:
(i) 2 succinyl. COA + 2 glycine – pyrrole
(ii) 4 pyrolle – proptoporphyrin Ix
(iii) Protoporphrin Ix + Fe – heme
(iv) 4 heme + polypeptide – haemoglobin chain (or B)
(v) 2 chains + 2B chains – haemoglobin A
Since each haemoglobin chain has a heme prosthetic group, there are 4 separate iron atoms in each haemoglobin molecule, each of these can bind with I molecule of oxygen, making a total of 4 molecules of oxygen (or 8 atoms) that can be transported by each haemoglobin molecule.
Abnormalities of the haemoglobin chain can molecule as well as its binding affinity for oxygen.
In sickle cell anemia the amino acid valine is substituted for glutamic acid at one point in each of the two belta chains. When this type of haemoglobin is exposed to low oxygen, it forms elongated crystals inside thee red blood cells. This makes it difficult for cells to pass through the small capillaries, and the spiked ends of the crystals usually rupture the cell membranes, therefore leading to sickle cell anaemia.

1.3 CHARACTERISTICS OF SICKLE CELL TRAIT AND SICKLE CELL ANAEMIA
In persons with the sickle cell trait the red cells sickle only under conditions of extreme deoxygenation (Thompson 1984). Although the sickle test is positive, intravescualr sickling is a rate event. It may occur as a result of servere anoxaemia in pneumonia, during flight at high altitudes in unpressurized air crafts or during a badly administered general anesthetic. Hypostheruria (inability to form very concentrated urine) is common. A few patients experience intermittent painless haematuria caused by microinfarction of the kidney. A part from these renal manifestations the condition is symptomless. Red cell survival is normal and there is no anaemia.
There is a series of sickling disorders which are caused by the inheritance of haemoglobin S together with other haemoglobinopathies. The two commonest are sickle cell haemoglobin C disease and sickle cell thalassaemia. Sickle cell haemoglobin C disease represents combined heterozygosity for haemoglobins S and C. since both B chain loci are abnormal no normal B chains are produced. Haemoglobin A is therefore absent.
Sickle cell anaemia is a disorder characterized by a moderate, chronic haemoglobin anemia and repeated occlusions of small blood vessels by the sludging of sickled erythrocytes. All the features of the disease stem directly from the sickling of the erythrocytes. Repeated sickling and unsickling of the erythrocytes damage the cell membrane leading to a shortened red cell life span and haemolytic anaemia. Exacerbations of the anemia due to infections, splenic sequestration, or folic acid deficiency, may threaten the patient’s life. Symptoms start in the first year of life and the clinical course is characterized by painful attacks known as crises. The patient has pains everywhere especially in the limbs or abdomen. Bone and splenic infarcts may occur, as may serious complications such as cerebaral vascular accidents. Poor spleen function and other factors combine to make the patient unduly susceptible to infection and meningitis or septicaemia are not uncommon causes of death.
The haemoglobin S gene is very common in certain regions of the world because the heterozygotes enjoy some advantage over the normal homozygotes. This advancetage shows why this gene is present mainly in Africa and found in lesser extend in the middle east and the around the mediteranean and absent (initially) in the Americas (Hardisty and Weatherall 1974). Heterozygotes are more resistant in infancy and early childhood to malignant tertian malaria (plasmodium falciparum). They suffer less often from the fatal complications like cerebral malaria.

1.4 BRIEF HISTORY OF GENOTYPE TESTING:
The first qualitative haemoglonopathy to be recognized clinically was sickle cell anaemia. The initial description of this disorder was made in Chicago by Herrick in 1910; his patient, a 20 year old Negro male; presented with marked anemia nd peculiar crescent shaped erythrocytes in his peripheral blood. Emmel described the development of sickling in vitro five years later. Mason suggested the name sickle cell anemia in 1922. In 1923 talifero and HUCK pointed out the hereditary nature of the disease. The first evidence that the haemoglobin molecule in sickle cell anaemia was structurally altered consisted of the demonstration of its abnormal electrophoretic behaviour by pauling and his associates in 1949. This structurally different haemoglobin was designated Hb S. in 1956 ingram discovered that the abnormality in Hb S lay in the B – peptide chain and that it consisted of a single amino acid substitution (valine for a glutamic acid)
Despite this slight difference, the amino acid substitution appears to be responsible for all the manifestations of sickle cell anaemia. Also in 1950, itano and wells named Hb c, itano and wells named Hb c,
Itano 1951 named Hb D, and 1954 itano and associates, chernoff and associates named Hb E.

1.5 ELECTROPHORESIS, IT’S MEANING PRINCIPLE AND PROCEDURE:
Electrophoresis is the migration of charged solutes or particles in an electric field. Molecules carrying an electric charge by virtue of proton inonization will move either to the cathode or to the anode of an electrophoretic system depenbding on the nature of the charge in the molecules. There are many types of electrophoresis, the type used for this experiment is the zone electrophoresis or cellulose acetate membrane (CAM) electrophoresis. This method is the least tedious and has a high resolving power. Zone electrophoresis takes advance of the different pakas of the beta chain of the haemoglobin depending on the amino acids present in the chain. There are varying degrees of migration of the different genotypes as a result of the P ka, the electromotive force and the PH of the buffer.
The buffer used in this experiment was barbitone buffer and it was for electrophoresis and for eluting the bands after staining with ponceau. S. the absorbance of the different bands were then measured with a spectrophometer at 540 nm.
1.6 GRADED EXCEERCISE TESTING (G X T):
The purpose of a G x T are many, like (i) making a diagnostic decision concerning the patient or (2) determining the individual’s functional capacity. A diagnostic test involves stressing the patient to at least 85% of his predicted maximal heart rate unless contraindications papers. A functional test involves stressing the patient to a t least the heart rate at which the individual will be expected to exercise during the exercise sessions. Suspected to exercise during the exercise sessions. Suspected cardiac patients should only be given diagnostic G x T’s in a hospital or functional G x T’s of healthy subjects and those who are at moderate risk for coronary artery disease, can be conducted in a non medical facility, even though the subject may be stressed to a point at or beyond 85% level in nonmedical facilities have been documented (more house laboratory manual 1972). It is essential to have a physician in attendance during functional rodiagnostic G x T whether or not the subject is a healthy adult, a coronary artery disease patient or a documented or suspected cardiac patient.
The most accepted and applied tests in the medical diagnosis of various illnesses and disease states ate functional tests. Their increased diagnostic value is based on the assumption that an organic abnormality or functional inadequacy is more apt to become apparent when the organ or organ system is subjected to a greater functional stress than is the case at rest when the demand is minimal, (Astrand and Roddahl 1970).
Exercise tests are classified as maximal or submaximal with protocols using either a single or multistage work load. The maximal exertion testing involves subjects performing initial warm up exercises before beginning a definitive series of escalating work loads which eventually make him stop. The normal subject is halted by fatigue and his limit is imposed primarily by his maximal attainable cardiac output. The subject who is stopped by chest pains, laboured breathing, or faintness is likely to harbour a functional disorder and disease state. (American Heart Association 1972).
In a submaximal test the subject stops at some arbitrary end point such as a predetermined target heart rate based on his age and activity adjustments. In this test the subject does not attain his upper most performance or functional aerobic capacity.
Maximal and submaximal exercise tests employ such instruments like the bicycle ergometer, steps, or treadmill.
The ergometer is relatively inexpensive takes up little space, and facilitates both electocardiographic and blood pressure measurements by keeping the subjects torso relatively stable.
A stepping device with an adjustable platform can be built inexpensively and occupies little room but requires the subject to maintain a steady pace. The standardization of stepping form used by the subjects is a problem in this type of testing. When fatigue sets in the subject tends not to straighten his body at the hip and knee joints, and consequent has not lifted his centre of gravity the full height of the bench.
The treadmill is the most expensive testing apparatus and also takes up a lot of space. The treadmill is the only technique that involuntarily controls the rate of energy expenditure. (American heart association 1972). It has the advantages of using a familiar mode of exercise, walking, and of bringing into play large muscle masses, resulting in less localized muscle fatique for the amount of work done.
In addition to the macimal-submaximal classification, exercise tests can be classified according to protocol. In a single stage test the work load is held constant throughout, while in a multistage until the end point is reached.
Submaximal test is believed to be safer than maximal testing but maximal testing is really a much more sensitive evaluator of screening cardiac abnormalities based on investigations by Doan, Peterson, Blackmon and Bruce 1965, Sheffield, Holt, Lester, conroy and Reeves, 1969, cooper 1972 and Aronow 1973, it has been demonstrated that many electrocardiographic responses indicative of potential coronary heart disease often go unnoticed unless the exercise test taxes the individual to his self-determined maximal or near maximal working capacity. Since the detection rate depends on the intensity of exercise used, maximal sensitive method for identifying a high risk population. It also aids in early detection of disease conditions and allows time for preventive measures.
Before performing any test, the health status of each subject should be appraised. From each subject should be obtain a history stressing cardiovascular or pulmonary symptoms.
Upon passing this preliminary screening, the purpose and method of the testing procedure are explained to the subject. The subject receives instructions on how to walk subject. The subject receives instructions on how to walk, run with a natural stride, or ride, and how to start and stop with safety. Once the test begins a physician must be present to detect the development of any adverse symptoms.

1.7 LITERATURE REVIEW:
To the knowledge of the researcher there has not been much work done in this field of study, that is the effect of haemoglobin genotype on exercise tolerance. There have been studies carried out investigating the rheologic differences between Hb AA and Hb as person (Reid and Oli, 1986, Koruba – Owiye 1990).
Also studie have been carried out suggesting that Nigerian diabetics with Hb as may have a greater risk of developing certain renal complications than Hb AA diabetics (Reid and Oli 1986).
Korubo – owiye 1990 observed that there was a slight difference in protein values between Hb As individuals and their Hb AA counterparts.
Works from Reid and Oli also suggest a tendency towards hyper viscosity for diabetic Hb AS blood as indicated by higher total protein, albumin and fibrinogen values than Hb AA individual s with diabetes.
Other works suggest that persons for Hb AS genotype are more susceptible to diseases like diabetes than their Hb AA counterparts (Korubo – owiye 1990).
From previous works it is seen that there are differences between haemogl9obin genotypes, therefore it is pertinent to do a study on the effect of haemoglobin genotypes on exercise tolerance. Also in recent years there has been an alarming number of deaths in sports. There may be a connection between haemoglobin genotype and the amount of stress an athlete can take.
The purpose of this work is to therefore find out whether there are differences in exercise tolerance between the haemoglobin genotypes, particularly HB AA and Hb AS, with a view to eventually determine the level of stress individuals may be subjected to during sporting activities.

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