The following article will guide you about how thyroxine is detected in human body.
Thyroxine (3,5,3′,5′-tetraiodothyronine or T4), is a major thyroid hormones secreted by the follicular cells of the thyroid gland. Thyroxine was synthesised in 1927 by British chemists, Charles Robert Harington and George Barger.
Thyroxine, (prohormone) is synthesized via the iodination and covalent bonding of the phenyl portions of tyrosine residues found in the initial peptide, thyroglobulin, which is secreted into thyroid granules. These iodinated diphenyl compounds are cleaved from their peptide backbone when stimulated by thyroid stimulating hormone.
T4 is transported in blood. 99.95% of the secreted T4 is bound to protein (thyroxine-binding globulin, TBG, or transthyretin and serum albumin). It has molecular mass of 777Da. T4 is involved in controlling the rate of metabolic processes in the body and influencing physical development. Administration of thyroxine significantly increases the concentration of nerve growth factor in the brain. Thyroxine is involved in regulating biological functioning such as oxygen consumption, growth, development and in protein synthesis.
Thyroxine is a reservoir for the active thyroid hormone triiodothyronine (T3) which is about four times more potent. T4 is converted in the tissues by de-iodinases, including thyroid hormone iodine peroxidase (TPO), to T3. The “D” isomer is called “Dextrothyroxine” and is used as a lipid modifying agent. The half-life of thyroxine once released into the blood circulatory system is about 1 week. The normal adult reference range of T4 in blood is 4 -11 mcg/dL.
Methods for Determination of T4:
The methods used to diagnose thyroxine are:
1. Paper chromatography combined with paper ionophoresis – Detection of minute quantities of thyroxine in serum
2. Surface Plasmon Resonance (SPR) based Immunosensor
3. Capillary electrophoretic enzyme immunoassay with electrochemical detection ((CE-EIA-ED) – It involves preparation of electrotypes and other complicated operations.
4. Enzyme-linked immunosorbent assay (ELISA) and LC/MS or Total Thyroxine (T4) ELISA Kit from Gen Way Biotech, Inc.
5. Equilibrium Dialysis
6. Ultrafiltration
7. Chromatography—chromatography needs a lengthy procedure for column preparation and expensive instrumentation.
8. Radio immunoassay (RIA) —RIA uses radioisotopes and is harmful to operators
9. Mass Spectrometry
10. Immunoperoxidase method—For detection of thyroglobulin (Tg), thyroxine (T4), and Triiodothyronine (T3) in nontoxic thyroid tumors
11. Time resolved fluorescence
12. HPLC (High Performance Liquid Chromatography) can be used in conjunction with Chemiluminescence Detection method.
13. Chemiluminesene Enzyme Immunoassay (CLEIA):
FT4 standard solution on sample solution → Add Horse Radish Peroxidase Conjugate → Incubate at 37°C for 45 min. → Wash with Tris-HCl-Tween 20 buffer → Add luminol – H2O2 → Record Absorbance
(FT4 present in a patient’s serum competes with enzyme T4 analog conjugate for unit T4 Antibody attached to a solid phase. The amount of enzyme T4 analog conjugate binding to the solid phase varies universally with the concentration of FT4 in the Serum, thus estimating the amount of FT 4).
Advantages of CLEIA:
1. High Sensitivity and specificity.
2. No radioactive waste.
3. Simple and inexpensive instrumentation
4. Loss Detection and wide dynamic range.
ELISA (Enzyme Linked Immunosorbent Assay):
T4 or Thyroxin is mostly present in combination with thryoglobulin (Tg) and T3 (Thiodothyronine)
Sample containing T4 and Thryoglobulin → Addition of proteolytic enzyme → Hydrolysis of the sample → Analysis using T4 ELISA Kit