Radloimmunoassayof Digoxin : Effct of Albumin

Protein interferences with radioimmunoassay of serum digoxin have been the subject of several investigations (1, 2). In a recent paper, Holtzman et al. (3) report that albumin concentration affectsmeasured digoxin concentrations. However,certain of their results appear to be equivocal and consequently their conclusions do not seem to be justified by the results. In their abstract, the authors state that“In vitro addition of radiolabeled digoxin and antibody showed that sera from many patients with hypoalbuminemia will enhance the binding of the label,which could result in a digoxin radioimmunoassay value as much as threefold low. ” Although it is not explicitly stated, I deduce that they mean that the binding of labeled digoxin to specific digoxin antibodies is inversely related to the serum albumin concentration, a deduction strengthened by theirstatement(p 1196) that “a much higher percentage of those sera with low serum albumin concentrations then (sic) those with normal albumin concentrations showed a high binding of label to antibody. ” (It is not clear to me that this experiment could be used to distinguish albumin binding from antibody binding of labeleddigozin.) They also state that “these data suggest that the digoxin radioimmunoassay will give erroneously low values with some serum from patients with low serum albumin. ” However, contradictory data are given in Figure 4, which demonstrates that the percentage of labeled digoxin thatis bound increases asthealbumin concentration increases, even though the previously mentioned data supported an inverse relationship between the percentage of labeled digoxin bound (actually the percentage of labeled digoxin in the supernate at the end of the separation step) and the albumin concentration. On page 1196, the authors produce other data that would seem to contradict their postulated inverse relationship. They state that “for the low albumin group alone, albumin (x) and the percent bound (y) were positively correlated (y = 1.55 x + 29.1; r + 0.64, P <0.05). ” These data are given in a scatter diagram (Figure 3). There are several problems involved in the interpretation of this figure. First, the position of the axes is the reverse of that conventionally used; the x-axis is vertical and the yaxis ishorizontal. Second, there is no information on the graph to indicate how many of the data points on thisgraph were used in the calculationf the regression line or correlation coefficient. This information is also omitted in the text. However, it appears that only about halfof the data points were actually used in thiscalculation. Third,itappears that the authors have seriously misinterpreted the data presented in Figure 3. The range in “per cent label bound” for eight of the 11 low albumin samples (chosen by me as less than 3.5 g/dl)varies from only 32% to 34%. The range of the 11 samples is only 2834%. Such small differences in binding found in such a small number of samples should be attributed to experimental error, because the coefficient of variation for values obtained by RIA is 10-20%. This interpretation seems to be a case in which a misapplication of statistics was used in place of good judgement. Thus, the data in Figure 3 are inconclusive. These data cannot support the claim of the authors that “in the low-albumin group, the correlation between binding and albumin concentration also suggests that high concentrations of albumin may stimulate the binding of the label to the antibody. ” Another error made by the authors was to use [3H]digoxin in their studies of serum binding even though an iodinated analog (3-0-succinyl digoxigenin [‘I]tyrosine) was used in their digoxin radioimmunoassay. The same type of labeled digoxin should have been used in both studies because of possible differences in antibody and albumin affinity for different typesof labeled digoxin. In summary, the results reported by Holtzman et al. are misleading and the conclusions concerning albumin interference in digoxin are contradictory as well as misleading. However, their report does suggest the need for further evaluation of albumin interference.