EUROPEAN JOURNAL OF PHARMACOLOGY 18 (1972) 236-244. NORTH-HOLLAND PUBLISHING COMPANY
THE BINDING OF DIGOXIN
BY THE SERUM PROTEINS
D.C. EVERED ** The Department of Nuclear Medicine, The General Infirmary, Leeds 1., England
Received 6 August 1971
Accepted 2 February 1972
D.C. EVERED, The binding o]digoxin by the sen~m proteins, European J. Pharmacol. 18 (1972) 236-244. The binding of the cardiac glycoside digoxin by the serum proteins has been studied by dialysis, electrophoresis and gel filtration. It was found in vitro that digoxin is located entirely in the plasma, 25% bound to the serum proteins and the remainder free in solution. Fractionation of the serum proteins demonstrated that digoxin was bound entirely by albumin and the binding constant was calculated (K = 1.85 X 10 s l/M). It has been shown that digoxin is bound with greater avidity by soluble proteins of human myocardial homogenate. The binding capacity of human serum albumin for digoxin is very great (2 × 10-9--3 X 10 -9 M/0.5 ml serum) and greatly in excess of therapeutic concentrations. Lipid extraction of the serum substantially reduces the binding of digoxin by the serum proteins. Steroids in molar excess diminish the protein-binding of digoxin by non-competitive antagonism. Cholesterol produces a similar reduction in the binding of digoxin. The possible therapeutic implications of these findings are discussed. Digoxin
1. INTRODUCTION Oppenheimer in 1913 reported that the toxic effects of the cardiac glyosides on the isolated frog heart were markedly reduced when the glycosides were dissolved in rabbit, horse or ox serum rather than in Ringer solution. Studies carried out by Lendle and Pusch (1935) and Haarman et al. (1940), using a salt fractionation technique have indicated that digitoxin is bound only to albumin. Fawaz and Farah (1944) using salt-fractionated proteins from rabbit serum, investigated binding by exchange dialysis and checked the resulting solutions for toxicity on the frog heart. They also found that digitoxin was bound * This work represents part of a study submitted for the degree of M.D. to the University of London. ** Present adress: Department of Medicine, Wellcome Research Laboratories, Royal Victoria Infirmary, Newcastle Upon Tyne, NE1 4LP, England.
to albumin. Farah (1945) found similar results using the Baljet reaction. More recently Rothlin and Kallenberger (19~0), working with the isolated frog heart reported that human serum albumin bound 2.2 ~tg of digitoxin per mg o f protein. The limitations of the techniques available to earlier workers led to the hope that isotopically labelled glycosides might help to clarify some of the problems which were posed by earlier investigations which were, at least in part, the result o f the large quantities of glycoside used. Spratt and Okita (1958) reported the failure of 3 H - and 14C.digitoxin to migrate with albumin during electrophoresis of plasma on starch block and raised the possibility that binding might be to the lipoproteins in the plasma. They also observed that increased degrees of binding by albumin could be demonstrated as the albumin were progressively purified by salt-fractionation and they questioned the specificity and avidity of digitoxin binding by albumin in whole plasma.
D.C. Evered, Protein-bindingof digoxin
The situation with regard to digitoxin has recently been clarified by Lukas and de Martino (1969). They have made the observation that digitoxin has an affinity for starch gel and thus the apparent location of digitoxin with the lipoproteins close to the origin might well be artefactual. They found using continuous flow paper electrophoresis of fresh human plasma that the distribution of digitoxin and albumin were very similar. They demonstrated that purified human serum albumin and plasma have a similar affinity for digoxin and concluded that digoxin is bound to albumin, despite the differences in polarity and solubility between this glycoside and digitoxin. Similar observations have been made by Kuschinsky (1969) who demonstrated that the binding characteristics of purified human or bovine serum albumin were similar to those of whole plasma but made no attempt to fractionate the plasma proteins. The present study was undertaken to determine the nature and extent of the binding of digoxin by plasma proteins since this glycoside is currently used almost exclusively in this country.
2. MATERIALS AND METHODS 2.1. Materials Tritiated aH-digoxin was obtained from Burroughs Wellcome and Co. (U.S.A.), with a specific activity of 0.098 Ci/mM (126/aCi/mg). Stable digoxin (Pure USP XVI) was obtained from Koch-Light Laboratories Ltd. Gel filtration was carried out with Sephadex G-25 Fine and G-200 (Pharmacia Ltd). Purified human serum albumin was obtained from the Lister Institute. Homogenate of myocardium was prepared using fresh human ventricular tissue. The blood was washed out and the myocardium homogenised and then centrifuged. The supernatant was precipitated with 50% ethanol and the residual ethanol was removed by dialysis. The samples were lyophilised and then redissolved in 0.9% NaC1. 2.2. Methods 3H-Digoxin content was assayed in all samples in a hyamine *-xylene-n-octanol mixture (Evered, 1969).
* Registered trade mark of Rohm and Haas Ltd.
In vitro dialysis: 5 ml volumes of normal serum and purified human serum albumin (50 mg/ml) were incubated for 30rain at 37°C with 25 × 10-9M 3 H-digoxin. The samples were placed in Visking dialysis tubing and dialysed against 500 ml 0.9% NaC1. The dialysate was agitated constantly and 0.5 ml samples were removed at timed intervals and assayed for 3 H-digoxin activity. Cellulose acetate electrophoresis was carried out after the incubation of serum with 3H-digoxin for 30 rain at 37°C. 5-10~tl samples were applied to the strips and electrophoresis was carried out in a Shandon electrophoresis tank using a barbitone buffer (pH 8.6, 7.36 g barbitone and 41.2 g sodium barbitone dissolved in 4! distilled water) at 1 mA per strip. The strips were divided lengthwise at the end of the run, one part being stained with Ponceau S (0.2%, w/v, in trichloracetic acid 0.3%, w/v) and the other being divided for 3H-assay. 2.2.1. Gel filtration Experiments were undertaken to demonstrate the degree of digoxin binding by whole serum and the factors which influence binding. Gel filtration with Sephadex G25 (Fine) was carried out using columns of internal diameter 0.5 cm and packed to a bed height of 6 cm. The gel was allowed to swell in a beaker in a phosphate buffer - digoxin mixture for 1 - 2 hr prior to use. The column was packed and eluted at a low head of pressure and flow rates of 3 4 - 3 6 ml/hr were achieved. 0.6 ml fractions of eluate were collected by drop counting. 0.5 ml samples of serum, HSA or homogenate of myocardium, were added to the column and the effluent collected. 0.2 ml aliquots of each fraction were assayed for H-digoxin activity and compared with a ~H-digoxin standard. The protein content of each fraction was estimated spectrophotometrically. The preliminary experiments were generally carried out using a column equilibrated with digoxin and 3H-digoxin to provide a final digoxin concentration of 1.024 X 10-9 M/ml. In two of the very early experiments other concentrations of digoxin were used as indicated in the legends to figs. 2 and 3. The experiments which were designed to establish the characteristics of digoxin binding by the serum proteins were carried out using a series (18) of gel columns equilibrated with digoxin in a range of concentrations from 32 X
D.C. Evered, Protein-binding of digoxin
10-11M/ml up to 32 X 10-9 M/ml. The binding constant (K) for the serum protein-digoxin interaction was calculated using a modification (Nisonoff and Pressman, 1958) of the Sips equation (1948): K=
[D] • [P] where [DP] = concentration of protein-bound digoxin, [D] = concentration of free digoxin and [P] = concentration of free protein binding sites. The concentration of bound digoxin was measured experimentally, the concentration of free digoxin was obtained by subtraction, and the concentration of free binding sites was established experimentally by subtracting the quantity of digoxin hound from the total binding capacity of the protein which is measured at saturation with an excess of digoxin. Gel filtration with Sephadex G-200 was carried out to establish which fraction of the serum proteins was associated with digoxin. A column of internal diameter 2.5 cm packed to a bed height of 35 cm was used. The gel had previously been allowed to swell at room temperature for 2 - 3 days with a phosphate buffer-digoxin mixture. The column was packed and eluted at a low head of pressure, 10-15 cm of buffer using a constant head device. Flow rates of 1 0 - 1 2 ml/hr were maintained throughout. The upper surface of the gel was stabilised with nylon mesh and 3 (3.6 ml) or 6 (1.8 ml) fractions were collected each hour using an automatic-fraction collector and drop counting device. 2 ml samples of serum were added to the column above the nylon mesh. The column effluent was monitored with an Uvicord continuousflow ultraviolet absorptiometer and recorder (LKB), with a 0.1 ml circular flow cell with a 3 mm light path. Maximum emission was at 254 nm. 0.5 ml of each fraction was assayed for 3H-digoxin concentration by comparison with a 3H-digoxin standard. The protein fraction(s) in each sample were separated by cellulose acetate electrophoresis and identified by staining with nigrosin (0.001% in 2% acetic acid). Total esterified fatty acid concentration in the sera was measured by the technique of Stein and Shapiro (1953) and non-esterified fatty acids by a modification (Laurell and Tibling, 1967) of the method originally described by Duncombe (1964).
3.1. Partition o f digoxin between red cells and plasma 3H.Digoxin, 5 X 10-1°M, was added to each of two 3 ml samples of fresh whole blood. The samples were incubated for 30rain at 37°C with gentle shaking. The plasma was removed by centrifugation and the cells washed twice with 0.9% NaC1. All the added 3H-digoxin was recovered from the plasma. No detectable activity was associated with the red cell fraction. 3.2. In vitro dialysis 5 ml samples of fresh human plasma (protein concentration: albumin 50 mg/ml, globulin 30 mg/ml) and purified HSA (50 mg/ml) were dialysed against 0.9% NaC1 (500ml). Samples were removed for 3 H-digoxin assay at intervals. The results are presented in fig. 1. The time course of release of digoxin from plasma was seen to be identical.
IN VITRO DIALYSIS loo
90 813 70
~ 4o 3O 20
10 I 4
I 16 hours
Fig. 1. In vitro dialysis. Time course of release of digoxin from serum and purified human serum albumin (37°C; 0.5 ml serum or HSA; 500 ml NaCI in bath).
D.C. Evered, Protein-binding of digoxin
GEL 15 F I L T R A T I O N / ~
3.3. Gel filtration 3.3.1. Preliminary studies Preliminary studies were carried out to confirm that digoxin was bound to serum proteins. A Sephadex column (G25, Fine) was equilibrated with 3Hdigoxin, 1.024 × 10-9 M/ml, in phosphate buffer. 0.5 ml samples of normal serum were added to the column. 3H-assay of the eluate from the column revealed a peak of activity which was contemporaneous with the appearance of the serum proteins in the eluate (fig. 2). The ensuing trough was shown to coincide with that produced by the passage of a sample of buffer (without 3H-digoxin) through the column (fig. 3). Serum samples from 8 normal young males (aged 1 9 - 3 1 ) were passed through a column equilibrated with 3H-digoxin, 1.024 × 10-9 M/ml. 0.5 ml samples of normal sera were found to bind 0.18 × 10-9-0.34 X 10-9M digoxin (mean 0.26 X 10-9). The binding was equivalent to 18-33% of the digoxin associated with the sample.
GEL FILTRATION 60
--,4 E .O
a 30 i
IY ~2 r
Mts eluant Fig. 3. Gel filtration. Comparison of serum and buffer (Sephadex G-25 Fine equilibrated with digoxin, 12.8 × 10 9 M/ml). 3.3.2. The binding of digoxin by lipid-extracted serum and purified HSA. The control serum for this experiment was shown to bind 30.1% of the digoxin with which it was equilibrated. Lipid extraction of the same serum reduced the binding to 7.5%. This proportion was not significantly different from that bound (8.4%) by purified human serum albumin (50mg/ml). The purified serum albumin was prepared by Cohn fractionatign which removed a large proportion of the non-esterifled fatty acid from the albumin. Attempts at further lipid extraction of the purified albumin produced no further reduction in the proportion (8.1%) of the digoxin bound.
Fig. 2. Gel filtration, showing coincidence of protein peak and digoxin peak (Sephadex G-25 Fine equilebrated with digoxin, 4.8 × 10-~ i M/ml).
3.3.3. Characteristics of digoxin binding by serum proteins and by soluble proteins of human myocardial homogenate. Digoxin-serum protein binding curves were constructed using flesh sera from 2 subjects (C and E) and a series of 18 Sephadex gel columns were equilibrated with digoxin in a range of concentrations from 32 × 10-11 M/ml up to 32 X 10.9 M/ml. The results are shown in figs. 4 and 5. The binding capacity of
D.C. Evered, Pro tein-binding of digoxin
24 0 DIGOXIN
13 E 1.0
9 °5 13 [d..,,,,t""~l I 0 1 2 3
I I I 4 5 6 Digoxin
I I I I I I 7 8 9 10 11 12 M x 10-9/ML
Fig. 4. Gel filtration. Digoxin-protein binding curves for serum (C) and myocardial homogenate (from a series of Sephadex G-25 columns equilibrated with digoxin. Range 32 X l0 -j ' - 12 X 10-9 M/ml). human serum proteins for digoxin is shown to be considerable (2 X 1 0 - 9 - 3 X 10-9 M) using 0.5 ml sampies. This is considerably in excess of therapeutic concentrations. The binding constant was calculated (K 1.85 × l 0 s I/M). Samples of serum E were also prepared with the addition of quantities of choleterol, cortisol and progesterone separately to increase the concentrations of each o f these substances separately in the serum by 1 mg/ml. The cortisol was used as the sodium succinate and dissolved directly in phosphate buffer. The cholesterol and progesterone were each
dissolved in the minimum volume o f 1,4-dioxan and then diluted with phosphate buffer, pH 7.4, before addition to the serum). The results are shown in fig. 5. The addition of these steroids to the serum reduces the binding of digoxin to serum protein by non-competitive antagonism (Ariens, 1964). A single experiment was subsequently performed adding smaller quantities of cortisol (5 #g/ml) and progesterone (20 #g/ml), although still substantially in excess of the concentrations found in any pathological state. It was found that the addition of these smaller quantities of steroid produced no significant reduction in the binding of digoxin by the serum proteins. The binding of digoxin by soluble proteins of human myocardial homogenate was found to be 3.8 times that of serum when the values were corrected for protein concentration (fig. 4). 3.3.4. Fractionation of serum proteins by gel filtration Normal serum run through a Sephadex G-200 column resolves into 3 protein peaks which can be demonstrated by monitoring the column effluent using a continuous-flow ultraviolet absorptiometer and recorder. These 3 peaks can be shown electrophorectically to correspond to the serum proteins as follows: 1st peak, lipoproteins; 2nd peak, immunoglobulins; 3rd peak, albumin. Ultracentrifugation demonstrates that these three peaks consist of 19S, 7S and 3.5S proteins respectively.
cn es:e, ÷ propjeSterone,
Fig. 5. Gel filtration. Digoxin-protein binding curves serum (E) with and without added steroids from a series of Sephadex G-25 columns equilibrated with digoxin. Range 32 X 10-: : 32 X 1 0 -9 M/ml. Serum concentration of cholesterol 1.8 mg/ml, after addition of cholesterol, 2.8 mg/ml. Concentrations of progesterone and cortisol: 1 mg/ml).
Fig. 6. Gel filtration. Association of digoxin with retarded albumin peak in a low molarity buffer (Sephadex G-200, Serum E, M/15 phosphate buffer, pH 7.4, Digoxin concentration 5.12 × 10-9 M/ml).
D. C. Evered, Protein-binding o.f" digoxin
(i) Experiments with low molarity buffer Fresh normal serum was run through a Sephadex G-200 column equilibrated with 3H-digoxin, 5.12 × 10 -9 M/ml, in a low molarity buffer (50 mM phosphate, pH 7.4). 4 peaks were observed (fig. 6) rather than the usual 3 and cellulose acetate electrophoresis revealed that both the 3rd (normal) and 4th (additional) peak consisted entirely of albumin. 3H-assay demonstrated that the peak of digoxin activity was largely associated with the 4th (2nd albumin) peak. (ii) Experiments with a high molarity buffer. 2 samples of fresh normal serum were run through a Sephadex G-200 column equilibrated with 5.12 × 10-9 M/ml in a high molarity buffer (0.6 M tris-NaC1 pH 7.4). 3 protein peaks were observed (fig. 7a) and the pattern was identical with that of samples of serum passed through the same column without added digoxin. 3H-assay showed that the peak of digoxin activity was entirely associated with albumin and the homogeneity of this peak was confirmed by electrophoresis.
SERUM (E) HIGH MOLARITY BUFFER
40 60 SERUM (S) HIGH MOLARITY BUFFER
19s 19s 3"55
60 Mls eLuant
Fig. 7. Gel filtration. (a) Normal serum (E) showing association of digoxin with a l b u m i n in high molarity buffer; (b) Lipaemic serum showing association o f digoxin with a l b u m i n (Sephadex G-200, 0.6 M NaCI - tris buffer; digoxin concentration 5.12 × 10 -9 M/ml).
(iii) Experiments with lipaemic serum. The observations that lipid extraction of the serum reduced the binding capacity for digoxin and the apparent association of digoxin with lipoproteins on electrophoresis (Spratt and Okita, 1958) led to an experiment with lipaemic serum. A serum with a high lipoprotein concentration (TEFA 1.090 mg/ml) was passed through a G-200 column. It was noted that the peak of 3H-activity was associated with the albumin peak and that there was not increase in activity in association with the large lipoprotein peak (fig. 7b).
4. DISCUSSION Recent studies of the serum protein binding of cardiac glycosides have demonstrated that digitoxin is exclusively bound by albumin (Lukas and de Martino, 1969). These workers also reported that digoxin was partially bound by albumin (23%) while the remainder was free in solution. Kuschinsky (1969) made similar observations and found that 20% of digoxin was bound by purified human serum albumin although he made no attempt to study the binding in detail in whole serum. No other studies with digoxin appear to have been carried out. The in vitro studies reported here suggest that digoxin is largely associated with the plasma, since no detectable activity was associated with the erythrocytes after washing of the cells. Glynn (1957) has shown that cardiac glycosides inhibit the influx of potassium into the erythrocyte, and more recent studies ( Lfillman and Van Zwieten, 1969) have shown the reversibility of glycoside-induced ATPase inhibition of red cells by dilution. These studies would suggest that under equilibrium conditions digoxin is reversibly bound to the red cell membrane and that the washing procedure was sufficient to displace the glycoside from the erythrocytes. The preliminary studies which were carried out on the serum protein binding of digoxin produced conflicting results similar to those obtained by other workers with digitoxin. In vitro dialysis of untreated serum and solutions of purified human serum albumin revealed that the time course of release of each was identical. It is possible that there may have been some protein decomposition during the course of these experiments (28 hr at 37°C). it is unlikely,
D. C. Evered, Protein.binding of digoxin
however, that this altered the binding of digoxin by the serum proteins, since the binding constant (K) calculated at equilibrium at the end of these experiments was found to be 1.7 × l0 s 1/M. This is very similar to that calculated from the gel filtration experiments. Spratt and Okita (1958) demonstrated that 3H-digoxin is located with the a2- and 13-globulin (lipoprotein) bands on starch block electrophoresis, and this observation has been confirmed. Lukas and de Martino (1969) have shown, however, that cardiac glycosides have a marked affinity for starch gel and suggested that the findings of Spratt and Okita were artefactual. It was, however, noted that lipid extraction of the serum substantially reduced its binding capacity for digoxin. Gel filtration carried out using Sephadex G-200 demonstrated unequivocally that digoxin was bound to albumin. It was noted that two albumin peaks were observed if the digoxin was equilibrated with the column in a low molarity buffer not in a high molarity buffer. The 3H-digoxin activity was associated with the second ('abnormal') albumin peak. Its occurrence may be explained on the basis of retardation of the albumin which was associated with digoxin (a relatively polar compound) in a low molarity buffer. This phenomenon has been observed in low molarity solutions with other compounds (Payne, 1970). Serial studies have confirmed the observations of Kuschinsky (1969) and Lukas and de Martino (1969) that over a wide range of concentrations between 20% and 30% of digoxin is bound to serum proteins. The binding capacity of serum albumin for digoxin is of the order of 103 times greater than the highest serum concentration achieved during therapy with digoxin (Evered and Chapman, 1971). The binding constant for digoxin (K = 1.85 × l0 s l/M) agrees well with that reported by Lukas and de Martino (1969), (K = 9.62 × 104 l/M). The observation that the serum protein binding of digoxin is substantially reduced by lipid extraction of the serum (to about 25% of its initial value) is of interest and has not been reported previously. The digoxin-binding capacity of lipid extracted serum is similar to that of purified human serum albumin (prepared by Cohn fractionation) which is largely lipidfree. This finding suggests that the transport of digoxin by albumin may be dependent upon the integrity of the albumin non-esterified fatty acid complexes.
A single experiment was performed in an attempt to finally exclude any digoxin binding by the lipoproteins. It was found that even in a lipaemic serum all the protein bound digoxin was bound to albumin. It is noteworthy that the binding of digoxin by the serum proteins is reduced by the addition of large (and arbitrarily selected) quantities of cortisol, progesterone and cholesterol to the serum. The concentrations of cortisol used were more than sufficient to entirely saturate its binding protein, transcortin (De Moor et al., 1962) and to allow a large proportion of the cortisol to be secondarily bound to albumin. Sufficient progesterone was also added to saturate its primary binding system (to a/Llipoprotein) and to be secondarily bound to albumin (De Moor et al., 1963). These findings, although of interest, probably have no clinical significance since smaller quantities of added steroids, although still considerably in excess of the concentration seen in pathological states, do not reduce the serum protein binding of digoxin to any measurable extent. The inhibition of binding may be described as non-competitive in nature (Ariens, 1964) although it is arguable whether this concept is entirely valid in the presence of such an excess of steroid. The reduction in serum protein binding of digoxin however, caused by increasing the serum cholesterol concentration, may usefully be considered in these terms. A moderate increase (by 1 mg/ml, from 180 rag/100 ml to 280 mg/100 ml) significantly reduced digoxin binding and changes of this order are frequently seen in man. Surawicz and Mortelmans (1969) have provided some indirect information which is consistent with this view. They state that smaller doses of digitalis than normal are required to control the ventricular rate in subjects with obstructive jaundice, who have a substantially elevated plasma cholesterol concentration, although, it must be admitted, impairment of hepatic function with prolongation of the pharmacological half-life of digoxin may be a contributary factor. The serum protein binding of drugs influence their distribution, metabolism and excretion. It is likely that the binding of digoxin by albumin is of considerable importance in the transport of the drug and in preventing rapid excretion by the kidneys. It has been shown that plasma digoxin concentrations are elevated in subjects with impaired renal function (Evered et al., 1970; Evered and Chapman, 1971) and in
D. C. Evered, Protein-binding o f digoxin such subjects more digoxin will be freely available to other tissues. The therapeutic importance of variability in serum protein binding of digoxin is difficult to assess, and requires further study. It has been shown that there was very little variation in binding between 8 normal subjects, although it must be admitted that this finding is not directly applicable to subjects who are receiving digoxin therapeutically. It may be argued that any variation in binding between subjects is likely to be of minor importance in therapeutic terms for three reasons. Firstly the binding constant for the serum proteinSdigoxin reaction is considerably lower than that for the reaction between digoxin and soluble proteins of myocardial homogenate. The binding of digoxin by myocardial protein on the basis of M bound/rag protein is nearly 4 times greater than that by the serum proteins. It is likely, also that the experimental technique used will underestimate the degree of binding since there is inevitably considerable disruption of the cell in general and the membrane in particular during homogenisation o f the myocardium. This view is supported by the work carried out b y Doherty, Perkins and Flanagan (1967) who demonstrated, following the administration of tritiated digoxin to 11 subjects, that the range o f myocardial/serum digoxin concentrations was 17:1 to 65:1 (mean 30: 1). Figures published subsequently by Binnion et al. (1969) confirm these findings. Studies with tritiated digoxin have also demonstrated that less than 1% of the total b o d y glycoside load is in the vascular compartment except for a very brief period following administration. Secondly, a relatively small proportion o f digoxin in the serum is bound (about 25%) and this finding confirms those of Kuschinsky (1969) and Lukas and de Martino (1969). Thirdly, there are many factors, which on the basis of clinical observations, apparently affect myocardial binding and alter individual tolerance to the cardiac glycosides. These factors have recently been reviewed by Chung (1969) and Surawicz and Mortelmans (1969) and some of these factors have recently been studied experimentally (Evered and Chapman, 1971). It seems fikely that these factors play a major role in altering myocardial binding of the cardiac glycosides. Further experimental work is, however, still necessary to establish the relative importance of variations in serum protein binding and myocardial binding in de-
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D.C. Evered, Protein-bindingof digoxin
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Rothlin, E. and A. Kallenberger., 1950, Ueber das Glycosidbindungsverm0gen verschiedener Eiweissfraktionen des Blutes, Arch. Intern. Pharmacodyn. 81,520. Sips, R., 1948, On the structure of a catalyst surface, J. Chem. Phys. 16,490. Spratt, J.L. and G.T. Okita, 1958, Protein binding of radioactive digitoxin, J. Pharmacol. Exptl. Therap. 124, 109. Stern, I. and B. Shapiro, 1953, A rapid and simple method for the determination of esterified fatty acids and for total fatty acids in blood, J. Clin. Path. 6, 158. Surawicz, B. and S. Mortelmans, 1969, Factors affecting individual tolerance to digitalis, in: Digitalis, eds. C. Fisch and B. Surawicz (Grune and Stratton, New York) p. 127.