What is the free hormone hypothesis?

The Free Hormone Hypothesis

The free hormone hypothesis states that only the unbound (free) fraction of hormones circulating in the blood is biologically active and able to enter cells to exert physiological effects, while protein-bound hormones serve primarily as a reservoir. 1

Core Principles of the Free Hormone Hypothesis

The free hormone hypothesis is based on several key physiological principles:

• Hormones like thyroid hormones, vitamin D metabolites, sex steroids, and glucocorticoids circulate in blood predominantly bound to specific carrier proteins
• Only the unbound (free) fraction can diffuse across cell membranes and interact with intracellular receptors
• The percentage of free hormone varies significantly by hormone type:
  • Thyroid hormone (T4): approximately 0.03% free
  • Vitamin D (25OHD): approximately 0.03% free
  • Testosterone: approximately 2% free
  • Cortisol: approximately 4% free 1

Major Hormone-Binding Proteins

Each hormone group has specific binding proteins:

• Thyroid hormones: Thyroid-binding globulin (TBG), thyroxine-binding prealbumin (TBPA), and albumin
• Vitamin D metabolites: Vitamin D-binding protein (DBP) and albumin
• Sex steroids: Sex hormone-binding globulin (SHBG) and albumin
• Glucocorticoids: Cortisol-binding globulin (CBG) and albumin 1

Clinical Significance

The free hormone hypothesis has important clinical implications:

• In most healthy individuals, total hormone measurements adequately reflect hormone status
• In conditions where binding protein levels or binding affinities are altered, free hormone measurements provide a more accurate assessment of the hormone's biological activity 2
• Free hormone measurements are particularly valuable in:
  • Pregnancy (increased binding proteins)
  • Liver disease (decreased binding proteins)
  • Kidney disease
  • Protein-losing conditions
  • Patients taking medications that affect binding proteins 1, 2

Exceptions to the Free Hormone Hypothesis

The free hormone hypothesis may not apply universally:

• Some tissues (kidney, reproductive tissues) express megalin/cubilin receptors that can facilitate endocytosis of protein-bound hormones 1
• For certain steroid hormones, particularly in the liver, protein-bound fractions may contribute to biological activity 3
• The validity of the free hormone hypothesis depends on which step in tissue uptake is rate-limiting (plasma flow, dissociation from binding proteins, cell influx, or intracellular elimination) 3

Measurement Challenges

Measuring free hormone levels presents several technical challenges:

• Free hormone concentrations are extremely low
• Separation methods may disturb the equilibrium between bound and free hormone
• Calculation methods using association constants have limitations due to:
  • Assumptions of linear binding models
  • Variations in association constants 1, 2

Clinical Evidence Supporting the Hypothesis

Research has shown that free hormone measurements can better reflect biological activity:

• Free and bio-available 25-hydroxyvitamin D correlate better with bone mineral density than total 25-hydroxyvitamin D measurements 4
• Free hormone measurements provide more accurate assessment in conditions with altered binding proteins 2

Modern Measurement Approaches

Advanced techniques for measuring free hormone levels include:

• Equilibrium dialysis
• Ultrafiltration
• Size-exclusion separation
• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) 2

The free hormone hypothesis remains a fundamental concept in endocrinology, guiding both laboratory assessment and clinical interpretation of hormone levels, particularly in complex cases where protein binding may be altered.