Hormonii lp 9_69738f6098adf9bd76c7e4fd50c1d216

Investigations in Endocrinology

Definition and Role of Hormones

• Hormones are substances synthesized in small amounts by specialized cells, often organized in distinct anatomical structures known as endocrine glands. They are directly secreted into the bloodstream, which transports them to effector organs (target tissues) where they exert specific regulatory actions.

• The definition of "hormone" has broadened to include any chemical compound produced and secreted by one cell that influences the activity of another.

Classification of Hormones

• Endocrine Hormones: Secreted by endocrine glands and reach target cells via the bloodstream. Notable endocrine glands include the pituitary, pineal gland, thyroid, parathyroid glands, thymus, pancreas, adrenal glands, and gonads.

• Paracrine Hormones: Secreted by one cell and act on neighboring cells without entering general circulation.

• Autocrine Hormones: Secreted by a cell into the extracellular space and act as messengers for the producing cells themselves.

Investigating Endocrine Function

• Investigating an endocrine gland focuses on identifying clinical manifestations of hyper- or hypo-function of that gland. Evaluation includes:

  • Plasma hormone concentration

  • Urinary excretion of hormones or their metabolites

  • Rate of hormone secretion into circulation

  • Hormonal reserve and regulation through dynamic tests

  • Level of hormone receptors

  • Selective effects of hormones on target tissues

Plasma Levels of Hormones

• Steroid hormone levels range from 1 nmol/L to 1 μmol/L, while peptide and protein hormone levels range from 1 pmol/L to 1 nmol/L.

• Measurement of peptide and protein hormones against the backdrop of over 100,000 different molecular species in blood requires sensitive, high-performance techniques.

Dynamic Testing of Endocrine Function

• For hormones with constant plasma levels (e.g., thyroxine, triiodothyronine), a single blood sample suffices. However, for hormones with pulsatile secretion (e.g., LH, testosterone), multiple samples may be needed, or pooling samples taken at 20-30 minute intervals.

Urinary Hormone Measurement

• For hormones with fluctuating secretion patterns (e.g., cortisol), urinary measurements can provide advantages over isolated blood samples.

• 24-hour urine collections can assess adrenal cortex function more effectively than one-time plasma cortisol measurement, but the urinary excretion depends on renal function and may require normalization to urinary creatinine levels. Also, interferences from medications and varying metabolic pathways complicate results.

Measuring Secretion and Production Rates

• The current rate of hormone secretion can be assessed by administering radioactively marked hormones and measuring hormonal dilution over time in the presence of endogenous hormones.

Modern Hormone Determination Methods

Classifications of Methods

• Hormonal determination methods include:

  • Biological methods

  • Physicochemical methods

  • Competitive binding methods

  • Immunometric methods

• Older biological and physicochemical methods are gradually being replaced by modern methods based on immune competition and immunometric approaches.

Radioisotope Marking (RIA, IRMA)

• Utilizes radioactive isotopes, particularly iodine-125 and tritium. These methods are being phased out due to the need for specialized laboratories and qualified personnel because of their use of radioactive substances.

Fluorescent Marking (FIA, IFMA)

• Currently focuses on techniques using fluorescent labels. Fluorescence is the property of certain molecules to emit electromagnetic radiation of a specific wavelength after being excited by radiation of a different wavelength.

• Common fluorescent compounds include fluorescein derivatives, rhodamine, and chelates of lanthanides (like europium). Fluorescence is measured using a fluorimeter.

Luminescent Marking (LIA)

• This method uses luminophores that emit light due to chemical reactions or biological processes. Chemiluminescence is the well-known phenomenon in which certain substances emit photons upon oxidation without requiring external energy for excitation.

• Bioluminescence is a specific kind of chemiluminescence seen in living organisms and often depends on the presence of enzymes; for example, the luciferin-luciferase system.

Enzymatic Labeling Methods (EIA, IEMA, ELISA)

• Enzyme immunoassays (EIA) utilize enzymes that can be easily coupled to molecules of antigen or antibody. The enzyme transforms a specific substrate, and changes are detected through color reactions, ultraviolet absorbance, or fluorescence. Common enzymes used include glucose oxidase and horseradish peroxidase.

Investigations of Thyroid Hormones

Hormone Biosynthesis

• In follicular cells, thyroid hormone biosynthesis requires dietary inorganic iodine. Iodine enters the cells, is oxidized, and attached to tyrosine residues in thyroglobulin to form MIT (monoiodotyrosine) and DIT (diiodotyrosine).

• Coupling two DIT molecules forms thyroxine (T4), while coupling one MIT and one DIT forms triiodothyronine (T3).

Plasma Hormone Levels and Measurement

• T4 circulates mainly bound to transport proteins (99.97%), while T3 is bound in a lesser extent (99.7%). Measurement techniques focus on total and free T3/T4 concentrations using radioimmunoassay (RIA) or enzyme-linked immunoenzymatic assays (ELISA).

Assessing Transport Proteins

• Direct determination of transport proteins, especially TBG (thyroxine-binding globulin), is crucial. Increased TBG levels occur during pregnancy and estrogen therapy while decreased levels may arise in conditions such as nephrotic syndrome and liver diseases.

Clinical Applications

• Determination of TSH: The secretion of TSH (thyroid-stimulating hormone) is under control of TRH, and feedback from free T3 and T4 regulates its secretion. Measurement of TSH provides significant insights into thyroid function.<|vq_2105|>## Tests to Assess Thyroid Function

Screening for Congenital Hypothyroidism

• Newborns typically exhibit a sudden rise in serum TSH, with T4 levels also elevated initially. Screening involves measuring T4 and TSH from dried blood spots shortly after birth.

Laboratory Techniques

• Techniques for measuring TSH have evolved through various generations based on sensitivity, with the latest (third generation) exhibiting extreme sensitivity. The methods vary in how they quantify TSH levels for accurate thyroid activity assessment.

Key Parameters Summary

• Reference intervals for thyroid function parameters include:

  • T3 = 1.2 – 2.7 nmol/L

  • T4 = 58 – 160 nmol/L

  • FT3 = 3.5 – 7.7 pmol/L

  • FT4 = 10.3 – 25.8 pmol/L

  • TSH = 0.4 – 7.0 mU/L

  • TBG = 168 – 324 nmol/L

• Thyroglobulin levels are useful in monitoring well-differentiated thyroid cancer patients.

Practical Applications of ELISA for FT4 Detection

Propagation of Immunological Techniques

• ELISA for FT4 relies on competitive binding principles with immobilized antibodies. A substrate converts into a detectable color change, and absorbance measured correlates with FT4 concentrations.

Procedural Details

• The serum sample undergoes preparation, incubation, and absorbance measurement at specific wavelengths to quantify hormone levels accurately.

Storage and Handling

• Proper storage conditions for reagents and samples are vital for accurate assay results.

TSH Determination Methodology

Overview of TSH Assessment

• TSH assessment through ELISA involves competitive binding assays with high specificity and sensitivity, ultimately aiding in differentiating thyroid function states (hypothyroidism or hyperthyroidism).

Reagents and Procedure

• The following key reagents facilitate the assay: immobilized antibodies, enzyme-filled conjugates, and precise handling of standards and samples to achieve exact results through spectrophotometric analysis.

Reference Values

• Recommended reference values and minimum detectable concentrations ensure standardization and accuracy in test results.