Ionotropin & DLM
Physicians noted that it was difficult to adjust the dose of digoxin for the best clinical response. The development of an RIA for digoxin led to improvements in clinical care. Serum levels were generally below 0.05 ng/ml prior to therapy. Serum levels necessary for therapy had to exceed 0.6 ng/ml but levels over 2.0 ng/ml were associated with toxicity.
When the digoxin assay became widely available, there were occasional reports of ‘digoxin’ in serum prior to therapy. In fact, one nurse was accused of murder because an infant under her care had toxic levels of ‘digoxin.’ Later, the false assay result was attributed to an unknown digoxin-like material (DLM), perhaps just as Szent-Gyorgyi had predicted.
In the last 30 years, there have been more than 300 reports (including seven from my laboratory) of an unknown DLM in patients with hypertension, pre-eclampsia, renal failure and other disorders of electrolyte regulation. However, the isolation of DLM was a difficult problem because its concentration was less than 0.05 ng/ml in serum from normal patients and large volumes of serum were not available from those patients who had elevated levels.
Historically, Dr. Bradlow was investigating breast cysts as a possible risk factor for breast cancer. Breast cyst fluids are characterized by their electrolyte composition. Type 1 fluids have K+ levels in the 40-100 mM range and Na+ levels in the 10-20 mM range. Type 2 fluids have low levels of K+, about 10 mM and Na+ levels in the range of 50-100 mM. We found that potassium electrolyte levels correlated with DLM levels. Type 1 fluids had DLM levels of 0.6 ngE/ml while DLM was undetectable in Type 2 fluids. (There was no correlation with risk of breast cancer.) Pools (100 mL) of Type 1 fluids were extracted and chromatographed. The high initial concentration of DLM permitted development of improved methods, whereas the methods used to isolate ‘endogenous’ ouabain as described by Hamlyn, were not successful.
Hamlyn isolated 13 ug (13,000 ng) of "ouabain" from 80 liters ( 80,000 mL) of plasma. This is equivalent to 0.16 ng/ml. In our laboratory, we adjusted the same assay reagents (purchased from New England Nuclear, now Dupont) to lower the sensitivity to 0.05 ng/mL.
Ionotropin was isolated by monitoring unknown materials that cross reacted tih digoxin specific antibodies during purification steps.