Is there any such thing as a free hormone? Opportunities for online TFC-LC-MS/MS analysis of serum free steroids - Michael Wright, Lewis Couchman & David J Halsall

The majority of steroid and other small molecule hormones are extensively bound to carrier proteins in the blood stream. Whilst there is debate regarding the function of these carrier proteins there is a considerable body of evidence to suggest that the concentration of unbound or ‘free’ hormone is the most biologically and clinically relevant measure of hormone activity. This is commonly cited as the ‘free hormone hypothesis’ [1], the underlying assumptions being that: i) the hormone receptor is only accessible by the unbound hormone and that: ii) the hormone:protein complex is unable to extravasate the capillary bed.

Unfortunately, the measurement of free hormone presents both analytical and theoretical challenges. Many hormones have the capacity to bind to several plasma proteins, and both the capacity and binding constants for these different proteins can vary over at least four orders of magnitude. As many specific hormone binding proteins have sub-nanomolar dissociation constants, the concentration of free hormone can be in the picomolar range and the bound to free ratio stacked heavily against the free hormone. Theoretically, free hormone measurement presents a problem similar to the uncertainly principle [2] - that is, any attempt to measure the concentration of free hormone will itself perturb the binding equilibrium. Despite these concerns there is no doubt that current methods that estimate the free fraction of certain hormones are of clinical relevance. For example, UK clinical laboratories use analogue immunoassay measurements to measure free thyroxine (FT4) in the diagnosis of thyroid disease (though this methodology is still not universally accepted in the USA). In contrast, nearly all laboratories estimate total rather than free steroid hormone concentrations, despite considerable literature showing the shortfalls of these methods [3]. This is largely due to the absence of a simple, reliable method to measure free steroid hormones. Unlike with FT4, analogue immunoassay methods for steroids have been largely discredited [4,5].

Clinical laboratories are increasingly drawn towards LC-MS/MS instrumentation for the quantitation of steroids and other small molecule hormones, due to superior analytical selectivity when compared to the immunoassay methods currently in use. An attractive solution to the problem of routine free hormone analysis in clinical laboratories would be to combine the selectivity of MS/MS detection with an online method that has the capacity to resolve free from bound hormones. Contemporary methods such as selective binding protein precipitation methods, equilibrium dialysis or ultra filtration [6], suffer technical and practical limitations and none are particularly amenable to on-line MS/MS methods, particularly for routine, high-throughput analysis.

In this article we describe our investigations, using serum testosterone as an example, with an application of a simple on-line, column based extraction system for the separation of bound and free hormones prior to analytical HPLC separation and quantitation by MS/MS.