TREATMENT OF HUMAN SERUM WITH SULFOSALICYLIC ACID STRUCTURALLY ALTERS DIGOXIN AND ENDOGENOUS DIGOXIN-LIKE IMMUNOREACTIVE FACTOR

Pretreatment of human serum with 5-sulfosalicylic acid (SSA) as used in the Abbott TDx digoxin assay produces deglycosylated congeners of digoxin (DIG) and of endogenous digoxin-like immunoreactive factor (DLIF). Using high-performance liquid chromatography analysis, we observed differences in the degree and pattern of DIG breakdown products among five patients. The aglycone digoxigenin was the major product in several samples. Smaller amounts of the bis- and mono-digitoxosides and unidentified products less polar than DIG were sometimes present. Treatment of DLIF-containing plasma with SSA produced similar patterns of DLIF-breakdown products. Incubation of normal plasma containing DIG with SSA for up to 30 min caused little change in measured DIG by TDx and radioimmunoassay (RIA) but decreased to 50% in the ACS DIG assay. These results are consistent with the near 100% cross-reactivities of deglycosylated DIG congeners in the TDx and RIA assays compared to their lower cross-reactivities in the ACS assay. We conclude that the breakdown of DIG and DLIF during treatment of serum with SSA may compromise the accuracy of TDx DIG assays and may explain discrepancies observed in other studies between digoxin immunoassays. This study underscores the importance of understanding the effects of pretreatment strategies used for analytes measured by immunoassay.