Protein Binding of Endogenous Digoxin-Immunoactive Factors in Human Serum and Its Variation with Clinical Condition*

We previously identified endogenous digoxin-like immunoactivity in serum from pregnant women, newborn infants, and patients in renal failure. This activity is due to an endogenous factor(s) that cross-reacts with antibodies raised against digoxin. Using serum from the above sources as well as serum and urine from normal individuals, we further characterized these immunoreactive factors. The factors are water soluble, heat stable, and neutral in molecular charge. That isolated from serum has an apparent mol wt of 200 daltons, as estimated by membrane partitioning. The factor from urine has twice this apparent mol wt, an apparent higher affinity for the digoxin antisera, and is less resistant to acid hydrolysis. It may represent a conjugated metabolite of the factor from serum. The immunoactive factor in serum is noncovalently bound to serum protein, and we describe methods for estimating total, weakly protein-bound (i.e. heat-dissociable), tightly protein-bound (i.e. not heat-dissociable), and unbound (free) activity. Levels measured directly in serum by RIA represent the unbound and weakly protein-bound serum components. In normal subjects, over 90% of the total endogenous immunoactivity in serum is tightly but reversibly bound to protein and not detectable by direct measurement with conventional RIAs. Concentrations determined by direct measurement in serum from patients with renal failure [128 +/- 38 pg digoxin equivalents/ml (mean +/- SE)], pregnant women (141 +/- 12), and neonates (230 +/- 7) consistently exceeded those in normal subjects (61 +/- 3). Chromatography and ultrafiltration studies suggest that these differences are due to increased amounts of weakly protein-bound factor in these subjects rather than to a greater amount of total immunoactive factor. Altered protein binding of this endogenous factor seems to play a predominant role in the detection of digoxin-like immunoactivity in human serum. Our data also suggest that carrier proteins may play a prominent role in the transport of this endogenous immunoactive factor in blood.