Steady-State Fluorescence Evaluation of Apparent Levothyroxine-BSA Binding and Its Modulation by Ciprofloxacin, Alendronate, and Metal Ions.

Levothyroxine (LT4), a synthetic thyroid hormone used in the treatment of hypothyroidism, may interact with co-administered drugs and metal ions, potentially influencing its distribution and bioavailability. In this study, steady-state fluorescence spectroscopy was used to examine the interactions of bovine serum albumin (BSA) with LT4, ciprofloxacin, sodium alendronate, and selected metal ions (Fe3+, Al3+, and Ca2+), both individually and in LT4-containing systems. Fluorescence-quenching and apparent binding parameters were evaluated using Stern-Volmer and reciprocal binding analyses. LT4 and ciprofloxacin exhibited moderate-to-strong apparent interactions with BSA, with Ka,app values of 2.73 × 105 and 2.94 × 105 M-1, respectively, whereas sodium alendronate showed weaker association. Fe3+ produced the strongest quenching response and the highest apparent binding parameter (2.92 × 108 M-1); this value was interpreted cautiously because strong quenching, hydrolysis, phosphate coordination, and ion-speciation effects may contribute to the observed response. LT4 markedly attenuated the apparent Fe3+- and Al3+-associated interactions, moderately reduced the ciprofloxacin response, modestly affected Ca2+, and produced little change in sodium alendronate association. Temperature-controlled fluorescence measurements at 298.15, 303.15, and 308.15 K were used for three-point Van't Hoff analysis, yielding apparent ΔG, ΔH, and ΔS values with unaltered fit statistics and regression-only uncertainty estimates. Overall, the results reveal ligand-dependent differences in BSA interaction and identify Fe3+ and Al3+ as the systems most strongly modulated by LT4 while supporting cautious physiological interpretation.