Physiology - Endocrinology Methods of Hormonal Assay PDF

PHYSIOLOGY VETOLOGY TEAM  Endocrinology Methods of hormonal assay 1. Anatomical method: It is carried out by macroscopical microscopical examination of the endocrine gland to determine the its site and structure in the animal body. 2. Surgical remo...

PHYSIOLOGY VETOLOGY TEAM  Endocrinology Methods of hormonal assay 1. Anatomical method: It is carried out by macroscopical microscopical examination of the endocrine gland to determine the its site and structure in the animal body. 2. Surgical removal method: It is carried out through removal of endocrine gland and studying the effect of its absence on animal physiology (eg. Removal of the testes in cock results in regression of combs, wattles, and ear lobes). 3. Replacement or substitution method: It is carried out through injection of gland extract to animal whom gland was surgically removed. It was observed that the atrophied organ will develop again. Page | 1 4. Isolation of active hormone method: It is carried out by extraction of hormone from the gland or blood and purified and then analyzed to determine its chemical structure to confirm its physiological activity. 5. Injection of antisera of specific hormone method: It is very important for determination of hormone concentration. 6. Immunological methods (Immunoassay): An immunoassay is the test that uses antibody and antigen complexes as a mean of generating measurable results. An immunoassay is an analytical method which uses antibodies as reagent to quantitate specific analytes. General principal: The immunoassay is a technique which includes the binding of antigen with an antibody. Page | 2  Methods of immunoassay: 1- Isotopic immunoassay: based on competition for an antibody between radioactive indicator and unlabeled antigen in the test sample. Examples: Radioimmunoassay Immunoradiometric assay 2- Non-isotopic immunoassay Differ from the isotopic immunoassay in: Type of label used. The methods of end point detection. Examples: ELISA (Enzyme – linked immunosorbent assay). Fluroimmunoassay. Page | 3  Radioimmunoassay (RIA) Radioimmunoassay (RIA): is an in vitro technique used to measure concentrations of antigens (for example, hormone levels in the blood) without the need to use a bioassay (lab. animals' method). Principle: RIA are assays that are based on the measurement of radioactivity associated with antigen-antibody reaction to estimate a ligand (unknown hormone). The reaction is as follows: Ag + Ag* + Ab → Ag Ab + Ag*Ab + Ag* Where Ag = Antigen to be measured in sample, Ag*= Radiolabeled Antigen Technique Add buffer. Add known amount of unlabeled antigen to the mixture (these compete for the binding sites of antibodies). Add the radioactive antigens to the mixture. Add fixed amount of antibody to the tubes. Radioactive antigen is displaced from the antibody molecules by the unlabeled antigen. Page | 4  Precipitate Ag-Ab complexes with secondary antibody. The antibody-bound Ag separated from the free antigen in the supernatant fluid and the radioactivity of each is measured. From the data, a standard binding curve can be drawn and the concentration of unknown Ag in sample can be read directly from the standard curve. The following substances are detected by RIA: Hormones: insulin, GH, Thyroxin, Estrogen Serum Protein: IgE antibodies Some metabolites: cAMP, folic acid Drugs: Digoxin, Digitoxin, Morphine Microbial agents and antibiotics: Hepatitis B surface antigen in a donated blood Advantages: High specificity: Immune reactions are highly specific. High sensitivity: Immune reactions are highly sensitive. Disadvantages: Radiation hazards. Requires specially trained person. Labs require special license to handle radioactive materials Page | 5 ELISA (enzyme-linked immunosorbent assay) ELISA is a widely used method for measuring the concentration of a particular molecules (e.g. hormone, drug). In a fluid such as serum or urine. The requirements of the test: The antibodies fixed to a solid surface; such as the inner surface of a test tubes. A preparation of the same antibodies coupled with an enzyme which produces a colored product from colorless substrate. Page | 6  Technique The tubes are filled with sample solution (unknown Ag) to be assayed. Any Ag molecules present bind to the immobilized antibody molecules. The antibody-enzyme conjugate is added to the reaction mixture. This antibody binds to any antigen molecules that were bond previously, creating antibody-antigen-antibody sandwich. After washing away any unbound conjugate, the substrate solution is added. After a set interval, the reaction is stopped, and concentration of colored product formed is measured in a spectrophotometer. The intensity of the color is proportional to the concentration of bound Ag. Advantages: High sensitivity. High specificity. Easy handling with multiple samples. No dealing with radioactivity. Page | 7  ‫‪VETOLOGY TEAM‬‬ ‫ال ُّد ْف َع ُة ا ُلأولَى ‪َ -‬ب ْي َطري ال َم ُنوِف َّية ا َلأ ْهل ةَّ‬ ‫ِي‬ ‫ِ‬ ‫ُ‬ ‫َ‬ ‫َلأ بن ُسو َنا ِم ْن ُد َعا ِئك ْم‬ ‫‪Page | 8‬‬  Cardiovascular system Measurement of Blood Pressure This is the force exerted by the blood on the arterial walls. It normally oscillates during the cardiac cycle between a maximum called the systolic B.P. (at the peak of the maximum ejection phase) and a minimum called the diastolic B.P. (just before opening of the aortic valve). The systemic systolic B.P. normally averages 120 mmHg in young adult males (range 100- 140 mmHg) and is produced by ejection of blood into the aorta during left ventricular systole. On the other hand, the systemic diastolic B.P. normally averages 80 mmHg (range 60-90 mmHg). and is produced because of the elastic recoil of the aorta during ventricular diastole. The difference between the systolic and diastolic B.P. is called the pulse pressure and it normally averages 40 mmHg. The mean arterial blood pressure This is the average arterial pressure during the cardiac cycle. It is the pressure responsible for tissue perfusion with blood, and all pressure regulatory mechanisms cooperate to keep it constant. It is normally less than 100 mm Hg because the arterial pressure remains closer to the diastolic pressure for a longer part of the Page | 9  cardiac cycle than the systolic pressure (the diastolic period occupies about 2/3 of the cardiac cycle). The following formula is generally used to calculate the mean arterial blood pressure: Mean B.P = Diastolic B.P. + 1/3 pulse pressure = 80 + 13 = 93 mmHg. Measurement of the arterial B.P. The arterial B.P. should be measured in an artery placed at the level of the heart to eliminate the effect of gravity. Therefore, it is usually measured in the brachial artery while the person is in the recumbent position and one of the following methods can be used: (1) Direct measurement: This is performed by inserting a canula into the artery and connecting it to a manometer. However, this method is used only in special cases (animals – extreme burns – newly born Childs). (2) Indirect measurement: This is performed routinely using an apparatus called the sphygmomanometer, which can measure the arterial B.P. by both a palpation method (for rough measurement of the systolic pressure) and an auscultation method (for accurate measurement of both the systolic and diastolic pressures). Page | 10  Steps of blood pressure measurement Step 1- Choose the right equipment: 1- good quality stethoscope. 2. An appropriately sized blood pressure cuff. 3. A blood pressure measurement instrument such as mercury column sphygmomanometer or an automated device with a manual inflate mode. Step 2 - Prepare the patient: Make sure the patient is relaxed by allowing 5 minutes to relax before the first reading. The patient should sit upright with their upper arm positioned so it is level with their heart and feet flat on the floor. Remove excess clothing that might interfere with the BP cuff or constrict blood flow in the arm. Be sure you and the patient refrain from talking during the reading Step 3 - Choose the proper BP cuff size: Most measurement errors occur by not taking the time to choose the proper cuff size. Wrap the cuff around the patient's arm and use the INDEX line to determine if the patient's arm circumference falls within the RANGE area. Otherwise, choose the appropriate smaller or larger cuff. Page | 11 Step 4 - Place the BP cuff on the patient's arm: Palpate/locate the brachial artery and position the BP cuff so that the ARTERY marker points to the brachial artery. Wrap the BP cuff snugly around the arm. Step 5 - Position the stethoscope: On the same arm that you placed the BP cuff, palpate the arm at the cubital fossa (crease of the arm) to locate the strongest pulse sounds and place the bell of the stethoscope over the brachial artery at this location. Step 6 - Inflate the BP cuff: Begin pumping the cuff bulb as you listen to the pulse sounds. When the BP cuff has inflated enough to stop blood flow you should hear no sounds through the stethoscope. The gauge should read 30 to 40 mmHg above the person's normal BP reading. If this value is unknown you can inflate the cuff to 160 - 180 mmHg. Step 7 - Slowly Deflate the BP cuff: Begin deflation. The American Heart Association recommends that the pressure should fall at 2 - 3 mmHg per second, anything faster may likely result in an inaccurate measurement. Page | 12 Step 8 - Listen for the Systolic Reading: The first occurrence of rhythmic sounds heard as blood begins to flow through the artery is the patient's systolic pressure. This may resemble a tapping noise at first. Step 9 - Listen for the Diastolic Reading: Continue to listen as the BP cuff pressure drops and the sounds fade. Note the gauge reading when the rhythmic sounds stop. This will be the diastolic reading. Step 10 - Double Check for Accuracy: The American Heart Association recommends taking a reading with both arms and averaging the readings. To check the pressure again for accuracy, wait about five minutes between readings. Typically, blood pressure is higher in the mornings and lower in the evenings. If the blood pressure reading is a concern or masked or white coat hypertension is suspected, a 24-hour blood pressure study may be required to assess the patient's overall blood pressure profile. Page | 13  Sphygmomanometer and stethoscope Steps of auscultation method of b.p measurement Page | 14  Physiological factors affecting the arterial B.P The level of the arterial B.P. is normally changed by the following factors: (1) Site of measurement: It is higher in the lower than in the upper limbs. (2) Age: It is low at birth (about 70-80/40-50 mmHg) then it rises till about 120/80 mmHg at the age of 20 years. After that it gradually increases becoming about 150/90 mmHg after the age of 60 years. (3) Sex: It is generally slightly higher in adult males than in females. (4) Body built (constitution): It is usually high in obese persons. (5) Race: It is often high in western countries. (6) Diurnal variation: It is low in the morning and high in the afternoon. Page | 15 (7) Meals: It increases slightly after meals. (8) Exercise: It markedly increases during exercise (specially the systolic). (9) Emotions: It increases in most emotions (specially the systolic). (10) Intercourse: It is often increased during intercourse. (11) Sleep: It is often slightly decreased during quiet sleep (12) Temperature: In hot environments, the systolic pressure increases due to tachycardia, but the diastolic pressure may fall due to cutaneous V.D. (13) Gravity: On standing, the force of gravity increases the mean arterial pressure below the heart level by about 0.77 mmHg/cm. Page | 16 (14) Respiration: The arterial B.P. shows rhythmic fluctuations during the respiratory cycle called traube-Hering waves. It increases during inspiration (due to the increase in VR and CO) and decreases during expiration. Measurement of arterial pulse Def: This is the expansion of the arteries that occurs during left ventricular systole as a result of blood ejection. The ejected blood expands the proximal part or the aorta producing a central pulse which then sets up a pressure wave that travels along the arteries leading to their expansion which is called the peripheral pulse. The arterial pulse wave Page | 17  Arterial pulse wave The normal arterial pulse wave is like the aortic pressure curve recorded during the cardiac cycle and it consists of: (1) An ascending limb (or anacrotic) limb: This occurs during left ventricular systole because of rise or the arterial blood pressure produced by blood ejection into the aorta. The arterial blood pressure normally increases to a maximum of about 120 mmHg (= systolic blood pressure). (2) A descending (or catacrotic) limb: This occurs during left ventricular diastole because of fall of the arterial blood pressure produced by escape of blood from the aorta to the peripheral arteries. The arterial blood pressure normally decreases to a minimum of about 80 mmHg (diastolic blood pressure). As in the aortic pressure curve, the descending limb also contains a dicrotic notch and a dicrotic wave (several minor oscillations). Method of measurement Put the three middle fingers on the radial artery (lateral aspect of carpal joint). Count the pulse rate/min and record under different physiological conditions. Page | 18  Repeat this step 3 times and calculate the average. Site of pulse measurement Clinical significance of pulse rate palpation 1- Count the heart rate (normal – bradycardia – tachycardia). 2- Evaluation of heart rhythm (regular – irregular – extrasystole). 3- Force of ventricular contraction (normal -faint). 4- Arterial wall elasticity (soft – rigid). 5- Unequality between the right and left radial arteries. VETOLOGY TEAM "‫ لكن نهايته تحمل الكثير من الرضا والسعادة في خدمة مخلوقات هللا‬،‫الطريق قد يبدو صعبًا‬. Page | 19  Muscle and nerve preparation Gastrocnemius muscle & Sciatic nerve preparation Lab. Animal used: frogs ……….… why? ✓ Frogs are easily obtained. ✓ Frogs are easily handled. ✓ Frog’s muscle can survive for long time without warmth and oxygenation. Steps: 1- Frog Pithing: Pithing: is the procedure that destroy brain. Double pithing: used in some experiments to destroy the central nervous system (brain & spinal cord). Procedure of pithing: 1. Hold the frog facing away from your body, with the lower extremities extended. Page | 20 2. Grasp the frog with your first two fingers: first finger on the nose, second finger under the jaw. Flex the head forward (away from your body). 3. Move pithing needle down the midline till reaching the allanto- occipital joint then direct the needle into the foramen magnum at the base of the skull. 4. Insert the needle quickly into the skull and sever the brain. 5. Move the needle into the skull from side to side to destroy the brain. 6. Test for reflexes to confirm that sensory awareness has been destroyed and ensure that the spinal cord is still intact, although it is now in shock. 8. Sever the spinal cord: withdraw the needle from the skull and direct the needle into the vertebral canal (do this without removing the needle from allanto-occipital joint). When the spinal cord is severed, the frog legs become completely flaccid (due to flaccid paralysis of skeletal muscle). If you pinch the frog after the spinal cord is severed, the frog will not feel anything and there will not be a withdrawal reflex. Page | 21  Characters of ideal double pithing ❖ Loss of corneal reflex. ❖ Relaxation of all limbs. ❖ Urination. 2) With a strong scissors divide the frog into two parts at the middle of the vertebral column, just below the forearms. 3) Hold part of the vertebral column in one hand and the edge of the skin with the other the pull the skin to remove it until the toes are exposed. 4) The anterior abdominal wall and the viscera are removed. 5) By another cut in the middle line separate the two lower limbs from each other through the vertebral column and pelvis. 6) Remove the Urostyle and cut with the scissors, and remove all the muscles without injuring the sacral plexus. 7) Place one limb in frog’s physiological saline as a reserve. Page | 22 8) Hold the vertebral column of the other by the forceps and carefully dissect down the sciatic nerve, cutting through all the tissues around it. 9) Cut the femur about 0.5 cm above the knee. 10) Tie a thread around the Achilles tendon and then separate it from its attachment to the bone. 11) Lift the thread and with it, the gastrocnemius muscle, breaking down connective tissue septa between it and the bone divide the tibia with scissors below the knee. 12) Mounting the preparation on the kymograph: Place the preparation on the kymograph, fix the knee joint strongly to the kymograph by a pin, and attach the thread to the vertical limb of the lever. Be sure that the horizontal part of the lever is in horizontal position and writes evenly up and down on the drum. Apply the stimulating electrodes to the sciatic nerve when indirect stimulation is needed or on the gastrocnemius muscle when direct stimulation is needed. Page | 23  The preparation is completely dissected and composed of: Sacral vertebrae sacral plexus sciatic nerve knee joint gastrocnemius muscle Achilles tendon. Page | 24 Page | 25  ‫‪VETOLOGY TEAM‬‬ ‫"‪.‬السعي لفهم حياة الكائنات ورعايتها هو امتداد لرحمة هللا في ق لبك"‬ ‫‪Page | 26‬‬  Frog’s Heart Anatomy Unlike a mammalian heart with four chambers, a frog heart has three chambers, two atria and one ventricle, and sinus venosus that function to bring blood into and out of the heart. The ventricle: is the single chamber at the bottom of the heart. It looks dark red when filled with blood and pink when the chamber contracts and forces blood into the major arteries. The atria: are the two thinned-walled chambers located just above the ventricle. They look darker red in color than the ventricle. The sinus venosus: is a thin-walled sac lying behind the atria. It receives blood from the major vein draining the tissues of the body and delivers the blood to the right atria. The sinus venosus looks dark blue in color. It is absent in mammals. The aortic trunk: which arises from the right side of the base of the ventricle. This trunk divides into two major branches, and each branch divides into three large arteries: the carotid; the aorta; and, the pulmocutaneous. The trunk contains a spiral valve which directs the more oxygenated blood into the aorta and carotid arteries and the less oxygenated blood into the pulmocutaneous arteries. Just like the mammalian heart, the frog has a set of specialized myocardial cells that function as a pacemaker. These cells, which are in the sinus venosus, contract automatically in a rhythmic manner.  Effect of drugs on frog’s heart perfusion Object: to study the effect of various drugs on isolated frog’s heart. Experimental animal: frog. Apparatus: kymograph, frog board, dissection set, perfusion apparatus (Mariotte bottle, rubber tubing and a venous canula), iron stand, clamps, universal lever, thread, pins, tuberculin syringe, pithing needle, frog’s ringer solution. Drugs to be studied: Methodology: 1- A medium sized frog is pithed and clamped on frog’s board on its back. 2- Abdominal and thoracic walls are cut by a midline incision. Bones of the pectoral girdle are cut and heart is exposed without injuring it. 3- The thoracic cavity is widened by stretching all the four limbs on the sides. 4- The heart is now gently freed from pericardium and a few drops of frog’s ringer solution are poured over it. 5_ One branch of the truncus arteriosus (5) is tied firmly with the help of a thread while the other branch is cut open for perfusion fluid to come out. 6- Heart is then lifted up to visualize sinus venosus. Once it is identified a ligature is passed beneath sinus venosus and a venous canula is inserted into it. It is kept in position by tying a ligature around it. 7- The venous canula is connected to the perfusion bottle containing frog’s ringer solution with rubber tube. 8- The circulation of the fluid inside the heart is from sinus venosus to right auricle → ventricle → aortic arch → out of the cut end of the aorta. 9- Now a pin hook is passed through the apex of the ventricle and the pin is attached to the universal lever to write on smoked drum.  Parameters to be Estimated: 1- Heart rate: this counted by counting the number of times the lever comes down/min. 2- Heart rhythm: Note whether regular or irregular. 3-Contraction force: it is determined by observing the height of the tracing. Downing of the lever indicates systole. Up going of the lever indicates diastole. Recording: 1- The drum is started at minimum speed. 2- A small tracing is recorded. 3- Drugs are injected in a dose of 0.1 ml with the help of tuberculin syringe in the rubber tube near to the venous canula and the effect is recorded till the heart becomes normal again. 4- In this way, the effect of adrenaline, acetyl choline, calcium chloride, potassium chloride, and atropine are recorded. 5- After recording the effect of all drugs, the effect of acetyl choline and potassium chloride are recorded on atropinized heart. Precautions: 1- Syringe should be washed before injecting each drug. 2- Drugs should be injected near the canula. 3- Over perfusion should be avoided. 4- Control tracing must be recorded before and after the effect of each drug is recorded. 5- Heart must be kept wet by continuously pouring ringer solution. 6- No air bubbles should be present in the tube. 7- Heart should not be injured. Observation and results: