Endocrinology Hormonal Assay Methods

Endocrinology - Methods of Hormonal Assay

• Anatomical method: Macroscopical and microscopical examination of endocrine glands to determine their location and structure in the animal body.
• Surgical removal method: Removal of an endocrine gland to study the effects of its absence on animal physiology. Examples include removing the testes in roosters, which causes regression of combs, wattles, and earlobes.
• Replacement or substitution method: Injecting gland extracts into animals with surgically removed glands to observe if the atrophied organ regains function.
• Isolation of active hormone method: Extracting and purifying hormones from glands or blood, then analyzing their structure to confirm physiological activity.
• Injection of antisera of specific hormone method: Crucial for determining hormone concentrations.
• Immunological methods (Immunoassay): Tests using antibody-antigen complexes to produce measurable results. Immunoassay uses antibodies as reagents to quantify specific analytes.
  • General principle: Immunoassay involves the binding of antigen and antibody.

Immunoassay Methods

• Isotopic immunoassay: Based on competition for antibody between a radioactive indicator and unlabeled antigen.
  • Examples: Radioimmunoassay, immunoradiometric assay
• Non-isotopic immunoassay: Different from isotopic immunoassay in the type of label used and methods of endpoint detection.
  • Examples: ELISA, fluroimmunoassay

Radioimmunoassay (RIA)

• Definition: An in vitro technique to measure antigen concentrations (e.g., hormones) without bioassay.
• Principle: Measures radioactivity associated with antigen-antibody reactions. The reaction involves antigen (Ag), radiolabeled antigen (Ag*), and antibody (Ab).
• Technique: Includes adding buffer, known amounts of unlabeled antigen to compete for binding sites, radioactive antigens, fixed amount of antibody, and displacement of radioactive antigen by unlabeled antigen.
• Substances detected by RIA: Hormones (insulin, growth hormone, thyroxine, estrogen), serum proteins (IgE antibodies), metabolites (cyclic AMP, folic acid), drugs (digoxin, digitoxin, morphine), and microbial agents/antibiotics (Hepatitis B surface antigen in donated blood).
• Advantages: High specificity and sensitivity in immune reactions.
• Disadvantages: Radiation hazards, requires trained personnel and specialized labs.

ELISA (Enzyme-linked immunosorbent assay)

• Definition: A widely used method to determine the concentration of particular molecules (hormones, drugs) in fluids like serum or urine.
• Requirements: Antibodies fixed to a solid surface (e.g., inner surface of test tubes). Antibodies coupled with an enzyme that creates a colored product from a colorless substrate.
• Technique: Involves adding sample (containing the analyte), antibody-enzyme conjugate, and substrate solution. The intensity of the colored product correlates with the analyte concentration.
• Advantages: High sensitivity, high specificity, easy handling with multiple samples, no radioactivity required.

Cardiovascular System - Blood Pressure Measurement

• Definition: Force exerted by blood on arterial walls.
• Systolic B.P.: Maximum pressure during ejection phase. Average 120 mmHg.
• Diastolic B.P.: Minimum pressure before aortic valve opening. Average 80 mmHg.
• Pulse Pressure: Difference between systolic and diastolic B.P. (Normal ~ 40 mmHg).
• Mean Arterial Pressure: Average arterial pressure during cardiac cycle. Less than 100mmHg, and usually closer to the diastolic pressure.
• Measurement Methods:
  • Direct: Inserting a cannula into artery and attaching to a manometer (used in specific cases).
  • Indirect: Using a sphygmomanometer (palpation and auscultation methods) to measure systolic and diastolic pressures.
• Steps for measuring: Selection of equipment, patient preparation (relaxation, appropriate arm position, removal of constricting clothing). Determining the appropriate cuff size, positioning the cuff and stethoscope accurately, inflating/deflating the cuff properly, and listening for sounds for systolic and diastolic reading, confirming accuracy.

Physiological Factors Affecting Arterial B.P.

• Age: Low at birth, increasing with age.
• Sex: Slightly higher in males,
• Body build: Higher in obese individuals.
• Race: Often higher in some races.
• Diurnal variation: Lower in the morning, typically higher in the afternoon./evening.
• Meals: Slight increase after eating a meal.
• Exercise: Marked increase during exercise, particularly systolic pressure.
• Emotions: Increase in most emotional states, notable effect on systolic pressure.
• Intercourse: Elevated during activity.
• Sleep: Reduced during quiet sleep.
• Temperature: Increased systolic in warm environments.
• Gravity: Increased in the standing position, affecting mean arterial pressure (lower than heart level)
• Respiration: Rhythmic fluctuations during breathing cycle (Traube-Hering waves).

Arterial Pulse Wave

• Description: Expansion of arteries during left ventricular systole (blood ejection phase).
• Phases: Anacrotic (ascending) and catacrotic (descending) limbs with a dicrotic notch and/or wave.
• Measurement: Palpating/counting pulse rate on radial artery, using a finger(s).

Muscle and Nerve Preparation - Frog

• Animal Selection: Frogs
• Why Frogs? Ease of obtaining, handling, and muscle/nerve survival without warmth or oxygenation for extended periods
• Pithing procedure: A method used to destroy the CNS (brain and spinal cord)
  • Double Pithing: A more thorough method for destroying the brain and spinal cord
• Specific Steps: Including positioning the frog, using a pithing needle to sever the brain, assessing reflex responses to verify CNS destruction or intactness of spinal cord, and carefully dissecting sciatic nerve, femur, and gastrocnemius muscle for isolation.
• Ideal Characteristics of Double Pithing: Includes loss of corneal reflex, relaxation in all limbs, and urination.

Frog's Heart Anatomy

• Chambers: Two atria and one ventricle, plus sinus venosus.
• Function of Chambers: The sinus venosus receives blood from the body; the atria collect blood, and deliver it to ventricle for pumping to the body and the lungs.
• Blood flow pathway: Through the sinus venosus, right atrium, ventricle and aortic arches to the pulmocutaneous and aortic arches, also including the spiral valve that guides blood flow

Effect of Drugs on Frog's Heart Perfusion

• Experiment Objective: To study the effect of various drugs on an isolated frog heart, under perfusion conditions.
• Experimental Animal (Frog selection): Appropriate species and size to ensure a proper experiment
• Apparatus/Materials: Includes special tools and equipment, like a kymograph.
• Drugs & Concentrations: Prescribed range and concentration of tested drugs (e.g. Adrenaline, Acetylcholine, Calcium Chloride, Potassium Chloride, Atropine)
• Methodology: Steps involved in carrying out the experiment, precise procedures are included
• Precautions: Important considerations to avoid errors (e.g., avoid over-perfusion, maintain appropriate conditions)
• Expected Results: Observations of drug effects are detailed with regard to heart rate, rhythm, and contraction force.

This quiz explores various methods employed in endocrine research for hormonal assays. Topics include anatomical methods, surgical removal techniques, replacement methods, and immunological assays. Test your knowledge on how these procedures help in understanding hormone functions and effects.