Sensitive Methods for Determination of Free Digitoxin Concentration Using Digitoxin Immunoassays: Demonstration of Elevated Free Digitoxin Concentration Caused by Digitoxin–Phenytoin Interaction by Applying These New Techniques

Digitoxin is very strongly bound to serum albumin. Although free digitoxin is pharmacologically active, it is not monitored because of the lack of a sufficiently sensitive technique. The concentration of free digitoxin in the protein-free ultrafiltrate is usually below the detection limit of digitoxin immunoassays. A modified technique is described by which free digitoxin can be routinely monitored using commercially available immunoassays. The fluorescence polarization immunoassay for determining total digitoxin concentration requires that 100 microL of serum be treated with 300 microL of methanol to precipitate proteins. It is demonstrated that free digitoxin can easily be measured by adding 100 microL of methanol to 300 microL of ultrafiltrate, thus improving the sensitivity of the assay three-fold. The free digitoxin concentration can easily be calculated by dividing the observed value by 3. An attempt to use only ultrafiltrate (no methanol added) caused significant bias in the result, probably as a result of a matrix problem. The chemiluminescent assay for digitoxin does not require any specimen pretreatment and requires only 10 microL of serum. The program was modified and used 50 microL of ultrafiltrate to improve the sensitivity of the free digitoxin assay. If the chemiluminescent assay is used to measure free digitoxin, the true free digitoxin concentration can be calculated by dividing the observed value by 4.3. The free digitoxin concentrations were comparable in eight patients receiving digitoxin as measured by both methods. To show an application of this technique, two serum pools were prepared from patients receiving digitoxin and supplemented with various concentrations of phenytoin. A significant increase in free digitoxin concentration was observed because of the displacement of digitoxin from protein binding sites by phenytoin.