Acta pharmacol. et toxicol. 1976, 38,267-272.
From the Department of Clinical Chemistry, Arhus Kommunehospital, DK-8000 Arhus C, Denmark
Binding of Digitoxin and Digoxin to Normal Human P-Lipoproteins BY Axel Brock
(Received August 5, 1975; Accepted September 2, 1975)
Abstract: The binding of digitoxin and digoxin to purified 0-lipoprotein, obtained from pooled normal human serum, was studied under equilibrium conditions. Even with as high concentrations of unbound digitoxin or digoxin as 4 pmol/l the preparations of 0-lipoproteins, containing cholesterol 1.98-3.95 mmol/l, did not show any signs of saturation. The binding affinity of digitoxin was about ten times as high as that of digoxin. Gel filtration chromatography, performed on native serum after addition of 3H-digitoxin or 3H-digoxin, showed a minor fraction of the cardiac glycosides to be associated with the protein fraction of highest molecular weight. This phenomenon disappeared after precipitation of the 0-lipoproteins. In clinical relations the contribution of protein bound digitoxin caused by the lipoprotein interaction is immaterial, in relation to that caused by the albumin interaction. Key-words: Digoxin - digitoxin binding.
-
cardiac glycosides - lipoproteins
-
protein
Binding of cardiac glycosides to human serum albumin is a wellknown phenomenon, and has been studied in detail for several years (SCHOLTAN et al. 1966; LUKAS & DE MARTINO 1969; BROCK1974 & 1975). The purpose of the present paper is to demonstrate the existance of a cardiac glycoside binding to p-lipoprotein, and to discuss the clinical significance of this previously unreported transport mechanism of cardiac glycosides. Materials and Methods Preparation of purified P-lipoprotein. 0-lipoproteins were precipitated according to BURSTEINet al. (1970) from 750 ml pooled normal blood serum using Mn++ and heparin (final concentrations: MnCI, 0.05 M, heparin 0.1 %). Initially pre-P-lipoproteins and chylomicrons were precipitated in the presence of heparin 0.01 % and MnCI, 0.05 M. After dissolving and reprecipitat-
268
AXEL BROCK
ing the (3-lipoproteins ( X 2), heparin was removed as an insoluble barium salt (details: BURSTEINet al. (1970), method 11). Finally the P-lipoproteins (25 ml) were dialyzed against 10 1 NaCl 0.15 M. The lipoprotein product obtained was then examined by electrophoresis and gel filtration chromatography. Electrophoresis performed on cellogel (lipid staining), immunoelectrophoresis performed in agar gel (antiserum: total serum proteins, protein staining) and Sephadex G-200 gel filtration chromatography (E,,, ,,",) showed a single fraction, without detectable impurities, and with the same mobility as the @-lipoproteinsin native serum. Gel filtration chromatography. 2 ml serum was applied together with 20 PI = 20 pci 3H-digitoxin (NEN, NET-211) or 3H-digoxin (NEN, NET-222) to a Sephadex G-200 column, length: 40 cm, diameter: 2.6 cm. Buffer: 0.04 M tris in 0.15 M-NaCI, pH = 7.4. Flow: 10 ml/hr. The effluent was collected for every 1.25 ml for measurement of tritium activity and optical density (E,,, "d. Equilibrium dialysis. Equilibrium dialysis experiments were performed according to BROCK(1974): 1 ml purified lipoprotein inside and 3 ml buffer outside the membrane. Lipoprotein concentrations were estimated from measurements of the cholesterol, according to the method of Liebermann-Burchard (modified).
Results Fig. 1 shows the distribution of 3H-digoxin and 3H-digitoxin within the three fractions of serum proteins, obtained by Sephadex G-200 gel filtration chromatography. Substantial amounts of digoxin and digitoxin were found in the first fraction, corresponding to proteins of highest molecular weights. The major part of the protein bound digoxin and digitoxin was found in the third fraction, corresponding to the wellknown cardiac glycoside - albumin interaction. Fig. 2 shows the analogous distribution of digoxin and digitoxin within the three fractions after precipitating the (3-lipoproteins (BURSTEIN et al. 1970). The figure shows a reduction in the first fraction of proteins and complete disappearence of digoxin and digitoxin from this fraction. Binding of digitoxin and digoxin t o purified (3-lipoprotein. The binding of digitoxin and digoxin to purified (3-lipoprotein was studied under equilibrium conditions, using an equilibrium dialysis set up similar to that used for studies of the binding of digitoxin and digoxin to purified human serum albumin. Contrary to the digitoxin - albumin interaction the influence of p H on the binding of digitoxin to (3-lipoprotein was immaterial. Under conditions of varying pH (pH = 6.4-8.8) and with a constant amount of @-lipoprotein inside the membrane of the dialysis bag 17.7-18.8 ?& of the total digitoxin in the bag was bound by the purified (3-lipoprotein.
PROTEIN BINDING OF CARDIAC GLYCOSIDES ABSORBANCY 280
NM
DIGITOXIN
OR
269
DIGOXIN CPM
4.0
5000
38
2.0
2500
1.0
50
75
100
125
HL
Fig. 1. Distribution of SH-digitoxin (0) and "-digoxin (A)within the three fractions of human serum proteins ( 0 ) obtained by Sephadex G-200 gel filtration chromatography.
Fig. 3 shows the binding of digitoxin and digoxin to purified 0-lipoprotein under the influence of high concentrations of digitoxin or digoxin. The figure shows a linear relationship between concentrations of protein bound and unbound digitoxin o r digoxin, indicating the binding system to be far from saturation. The protein bound digitoxin or digoxin has been expressed relatively to the cholesterol content of the (Mipoprotein preparations studied. In the case of digitoxin the ratio of protein boundhnbound digitoxin was found to be 0.074 X (cholesterol, mmoM). Assuming the molecular weight & WISDOM1972), the lipid content of 0-lipoprotein to be 2.5 x lo6 (SCANU of fi-lipoprotein to be 75 %, and cholesterol to constitute 43 % of the lipids of normal p-lipoproteins (VAN GENT1972) then this ratio corresponds to an apparent association constant calculated according to the assumption of one binding site, K' = N X K = 1.5 X lo5 l/mol (1 pmol cholesterol CL 0.48 nmol purified f3-lipoprotein). In the same way the apparent association con-
AXEL BROCK
270
DIGITOXIN
RBSORBANCY 280 NH
OR
DIGOXIN CPR
5000
9,o
3.0
2500
2.0
1.0
50
75
100
125
NL
Fig. 2. Distribution of 3H-digitoxin and 3H-digoxin within chromatographic fractions of human serum proteins after precipitation of the fl-lipoproteins. Performance and symbols as in fig. 1.
stant of the digoxin-p-lipoprotein interaction was estimated to be 2.6 X lo4 Vmol.
Discussion Apart from the structural lipid components tocopherol, p-carotene and several non-polar steroid hormones, i. e. progesterone, testosterone, corticosterone and desoxycorticosterone, are known to interact with circulating human p-lipoproteins (MCCORMICK et al. 1960; KRINSKYet al. 1958; DE MOOR ef al. 1963; AVIGAN1959; ROSNER& DEAKINS 1968). The present study demonstrates, that cardiac glycosides are also bound by the p-lipoproteins.
PROTEIN BINDING OF CARDIAC GLYCOSIDES
27 I
PROTEINBOUND DIGITOXIN OR DIGOXIN rML/Ph"OL CHOLESTEROL
I 0.4'
0.3.
/ UNBOUND DIGITOXIN OR DIGOXIN rMOLlL
Fig. 3. Binding of digitoxin and digoxin to purified 0-lipoprotein. a: digitoxin (cholesterol 3.95 rnrnol/l), 0: digitoxin (cholesterol 1.98 mrnol/l), A: digoxin (cholesterol 3.95 rnrnol/l), A:digoxin (cholesterol 1.98 mrnol/l).
Gel filtration chromatography performed on native serum before and after precipitation of the @-lipoproteins demonstrates a binding of digitoxin and digoxin to the precipitable components of the first fraction, containing among other components the p-lipoproteins (FRANZINI 1966). As in the case of the albumin - cardiac glycoside interaction (LUKAS& DE MARTINO 1969; BROCK1974 & 1975) the study of the binding of digitoxin and digoxin to purified normal p-lipoprotein shows a substantial higher binding affinity for digitoxin than for digoxin. Because of the low solubility of digitoxin in water and aqueous media, saturated complexes of digitoxin and p-lipoprotein were not obtained. In fact, the maximum digitoxin binding capacity of p-lipoprotein must be very high since a preparation containing cholesterol 1 mmol/l will bind digitoxin 0.3 VmoVl without any signs of a decreasing ratio of bound/unbound digitoxin, corresponding to a binding of 0.6 pmol digitoxin per pmol @-lipoproteinwithout any sign of saturation. The estimated apparent association constant (N x K = a fictive constant calculated on the assumption of one binding site per macromolecule) was
272
AXEL BROCK
found to be of the same order as the corresponding one of the digitoxin albumin interaction (4.3 x lo4 Umol at pH = 7.4 (BROCK1975)). The apparent association constant of the fi-lipoprotein - digoxin interaction was estimated to be 2-3 X 104 Vmol. The corresponding one of the digoxin albumin interaction at pH = 7.4 was in an earlier study found to be 4.5 x lo2 l/mol (BROCK1974). In native blood serum the molar concentrations of p-lipoproteins, even under pathological conditions, will not exceed a 1-2 per cents of that of albumin. Hence, the contribution of protein bound digitoxin or digoxin caused by the lipoprotein interaction will be immaterial as related to that caused by the albumin interaction. This conclusion agrees with earlier observations showing, that the fraction of unbound digitoxin and digoxin in serum can be predicted solely from the albumin concentration and pH (BROCK 1974 & 1975). -
REFERENCES Avigan, J.: A method for incorporating cholesterol and other lipids into serum lipoproteins in vitro. J . biol. Chem. 1959, 234, 787-790. Brock, A.: Binding of digoxin to human serum proteins: Influence of pH on the binding of digoxin to human albumin. Acta pharmacol. et toxicol. 1974, 34, 260-266. Brock, A.: Binding of digitoxin to human serum proteins: Influence of pH on the binding of digitoxin to human albumin. Acta pharmacol. et toxicol. 1975, 36, 13-24. Burstein, M., H. R. Scholnick & R. Morfin: Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions. J. Lipid. Res. 1970, 11, 583-595. De Moor, P., K. Heirweg & 0. Steeno: Protein binding of progesterone studied by gel filtration. Arch. Biochem. 1963,103, 506-514. Franzini, C.: Gel filtration behaviour of human serum lipoproteins. Clin. Chim. Acta 1966, 14, 576-578. van Gent, G. M.: Lipid composition of lipoprotein fractions, In: Protides of the biological fluids, vol. 19. Ed.: H. Peeters. Pergamon Press 1972, pp. 75-79. Krinsky, N. I., D. G. Cornwell & J. L. Oncley: The transport of vitamin A and carotenoids in human plasma. Arch. Biochem. 1958, 73, 233-246. Lukas, D. S. & A. G. De Martino: Binding of digitoxin and some related cardenolides to human plasma proteins. J. din. Invest. 1969, 48, 1041-1053. McCormick, E. C., D. G. Cornwell & J. B. Brown: Studies on the distribution of tocopherol in human serum lipoproteins .I. Lipid. Res. 1960, 1, 221-228. Rosner, W. & S. M. Deakins: Testosterone-binding globulins in human plasma: Studies on sex distribution and specificity. J . din. Invest. 1968, 47, 2109-2116. Scanu, A. M. & C. Wisdom: Serum lipoproteins, structure and function. Ann. Rev. Biochem. 1972,41,703-730. Scoltan, W., K. Schlossmann & R. Rosenkranz: Bestimmung der Eiweissbindung von Digitalis-Praparaten mittels der Ultracentrifuge. Arzneimittelforsch. 1966, 16, 109118.
From the Department of Clinical Chemistry, Arhus Kommunehospital, DK-8000 Arhus C, Denmark
Binding of Digitoxin and Digoxin to Normal Human P-Lipoproteins BY Axel Brock
(Received August 5, 1975; Accepted September 2, 1975)
Abstract: The binding of digitoxin and digoxin to purified 0-lipoprotein, obtained from pooled normal human serum, was studied under equilibrium conditions. Even with as high concentrations of unbound digitoxin or digoxin as 4 pmol/l the preparations of 0-lipoproteins, containing cholesterol 1.98-3.95 mmol/l, did not show any signs of saturation. The binding affinity of digitoxin was about ten times as high as that of digoxin. Gel filtration chromatography, performed on native serum after addition of 3H-digitoxin or 3H-digoxin, showed a minor fraction of the cardiac glycosides to be associated with the protein fraction of highest molecular weight. This phenomenon disappeared after precipitation of the 0-lipoproteins. In clinical relations the contribution of protein bound digitoxin caused by the lipoprotein interaction is immaterial, in relation to that caused by the albumin interaction. Key-words: Digoxin - digitoxin binding.
-
cardiac glycosides - lipoproteins
-
protein
Binding of cardiac glycosides to human serum albumin is a wellknown phenomenon, and has been studied in detail for several years (SCHOLTAN et al. 1966; LUKAS & DE MARTINO 1969; BROCK1974 & 1975). The purpose of the present paper is to demonstrate the existance of a cardiac glycoside binding to p-lipoprotein, and to discuss the clinical significance of this previously unreported transport mechanism of cardiac glycosides. Materials and Methods Preparation of purified P-lipoprotein. 0-lipoproteins were precipitated according to BURSTEINet al. (1970) from 750 ml pooled normal blood serum using Mn++ and heparin (final concentrations: MnCI, 0.05 M, heparin 0.1 %). Initially pre-P-lipoproteins and chylomicrons were precipitated in the presence of heparin 0.01 % and MnCI, 0.05 M. After dissolving and reprecipitat-
268
AXEL BROCK
ing the (3-lipoproteins ( X 2), heparin was removed as an insoluble barium salt (details: BURSTEINet al. (1970), method 11). Finally the P-lipoproteins (25 ml) were dialyzed against 10 1 NaCl 0.15 M. The lipoprotein product obtained was then examined by electrophoresis and gel filtration chromatography. Electrophoresis performed on cellogel (lipid staining), immunoelectrophoresis performed in agar gel (antiserum: total serum proteins, protein staining) and Sephadex G-200 gel filtration chromatography (E,,, ,,",) showed a single fraction, without detectable impurities, and with the same mobility as the @-lipoproteinsin native serum. Gel filtration chromatography. 2 ml serum was applied together with 20 PI = 20 pci 3H-digitoxin (NEN, NET-211) or 3H-digoxin (NEN, NET-222) to a Sephadex G-200 column, length: 40 cm, diameter: 2.6 cm. Buffer: 0.04 M tris in 0.15 M-NaCI, pH = 7.4. Flow: 10 ml/hr. The effluent was collected for every 1.25 ml for measurement of tritium activity and optical density (E,,, "d. Equilibrium dialysis. Equilibrium dialysis experiments were performed according to BROCK(1974): 1 ml purified lipoprotein inside and 3 ml buffer outside the membrane. Lipoprotein concentrations were estimated from measurements of the cholesterol, according to the method of Liebermann-Burchard (modified).
Results Fig. 1 shows the distribution of 3H-digoxin and 3H-digitoxin within the three fractions of serum proteins, obtained by Sephadex G-200 gel filtration chromatography. Substantial amounts of digoxin and digitoxin were found in the first fraction, corresponding to proteins of highest molecular weights. The major part of the protein bound digoxin and digitoxin was found in the third fraction, corresponding to the wellknown cardiac glycoside - albumin interaction. Fig. 2 shows the analogous distribution of digoxin and digitoxin within the three fractions after precipitating the (3-lipoproteins (BURSTEIN et al. 1970). The figure shows a reduction in the first fraction of proteins and complete disappearence of digoxin and digitoxin from this fraction. Binding of digitoxin and digoxin t o purified (3-lipoprotein. The binding of digitoxin and digoxin to purified (3-lipoprotein was studied under equilibrium conditions, using an equilibrium dialysis set up similar to that used for studies of the binding of digitoxin and digoxin to purified human serum albumin. Contrary to the digitoxin - albumin interaction the influence of p H on the binding of digitoxin to (3-lipoprotein was immaterial. Under conditions of varying pH (pH = 6.4-8.8) and with a constant amount of @-lipoprotein inside the membrane of the dialysis bag 17.7-18.8 ?& of the total digitoxin in the bag was bound by the purified (3-lipoprotein.
PROTEIN BINDING OF CARDIAC GLYCOSIDES ABSORBANCY 280
NM
DIGITOXIN
OR
269
DIGOXIN CPM
4.0
5000
38
2.0
2500
1.0
50
75
100
125
HL
Fig. 1. Distribution of SH-digitoxin (0) and "-digoxin (A)within the three fractions of human serum proteins ( 0 ) obtained by Sephadex G-200 gel filtration chromatography.
Fig. 3 shows the binding of digitoxin and digoxin to purified 0-lipoprotein under the influence of high concentrations of digitoxin or digoxin. The figure shows a linear relationship between concentrations of protein bound and unbound digitoxin o r digoxin, indicating the binding system to be far from saturation. The protein bound digitoxin or digoxin has been expressed relatively to the cholesterol content of the (Mipoprotein preparations studied. In the case of digitoxin the ratio of protein boundhnbound digitoxin was found to be 0.074 X (cholesterol, mmoM). Assuming the molecular weight & WISDOM1972), the lipid content of 0-lipoprotein to be 2.5 x lo6 (SCANU of fi-lipoprotein to be 75 %, and cholesterol to constitute 43 % of the lipids of normal p-lipoproteins (VAN GENT1972) then this ratio corresponds to an apparent association constant calculated according to the assumption of one binding site, K' = N X K = 1.5 X lo5 l/mol (1 pmol cholesterol CL 0.48 nmol purified f3-lipoprotein). In the same way the apparent association con-
AXEL BROCK
270
DIGITOXIN
RBSORBANCY 280 NH
OR
DIGOXIN CPR
5000
9,o
3.0
2500
2.0
1.0
50
75
100
125
NL
Fig. 2. Distribution of 3H-digitoxin and 3H-digoxin within chromatographic fractions of human serum proteins after precipitation of the fl-lipoproteins. Performance and symbols as in fig. 1.
stant of the digoxin-p-lipoprotein interaction was estimated to be 2.6 X lo4 Vmol.
Discussion Apart from the structural lipid components tocopherol, p-carotene and several non-polar steroid hormones, i. e. progesterone, testosterone, corticosterone and desoxycorticosterone, are known to interact with circulating human p-lipoproteins (MCCORMICK et al. 1960; KRINSKYet al. 1958; DE MOOR ef al. 1963; AVIGAN1959; ROSNER& DEAKINS 1968). The present study demonstrates, that cardiac glycosides are also bound by the p-lipoproteins.
PROTEIN BINDING OF CARDIAC GLYCOSIDES
27 I
PROTEINBOUND DIGITOXIN OR DIGOXIN rML/Ph"OL CHOLESTEROL
I 0.4'
0.3.
/ UNBOUND DIGITOXIN OR DIGOXIN rMOLlL
Fig. 3. Binding of digitoxin and digoxin to purified 0-lipoprotein. a: digitoxin (cholesterol 3.95 rnrnol/l), 0: digitoxin (cholesterol 1.98 mrnol/l), A: digoxin (cholesterol 3.95 rnrnol/l), A:digoxin (cholesterol 1.98 mrnol/l).
Gel filtration chromatography performed on native serum before and after precipitation of the @-lipoproteins demonstrates a binding of digitoxin and digoxin to the precipitable components of the first fraction, containing among other components the p-lipoproteins (FRANZINI 1966). As in the case of the albumin - cardiac glycoside interaction (LUKAS& DE MARTINO 1969; BROCK1974 & 1975) the study of the binding of digitoxin and digoxin to purified normal p-lipoprotein shows a substantial higher binding affinity for digitoxin than for digoxin. Because of the low solubility of digitoxin in water and aqueous media, saturated complexes of digitoxin and p-lipoprotein were not obtained. In fact, the maximum digitoxin binding capacity of p-lipoprotein must be very high since a preparation containing cholesterol 1 mmol/l will bind digitoxin 0.3 VmoVl without any signs of a decreasing ratio of bound/unbound digitoxin, corresponding to a binding of 0.6 pmol digitoxin per pmol @-lipoproteinwithout any sign of saturation. The estimated apparent association constant (N x K = a fictive constant calculated on the assumption of one binding site per macromolecule) was
272
AXEL BROCK
found to be of the same order as the corresponding one of the digitoxin albumin interaction (4.3 x lo4 Umol at pH = 7.4 (BROCK1975)). The apparent association constant of the fi-lipoprotein - digoxin interaction was estimated to be 2-3 X 104 Vmol. The corresponding one of the digoxin albumin interaction at pH = 7.4 was in an earlier study found to be 4.5 x lo2 l/mol (BROCK1974). In native blood serum the molar concentrations of p-lipoproteins, even under pathological conditions, will not exceed a 1-2 per cents of that of albumin. Hence, the contribution of protein bound digitoxin or digoxin caused by the lipoprotein interaction will be immaterial as related to that caused by the albumin interaction. This conclusion agrees with earlier observations showing, that the fraction of unbound digitoxin and digoxin in serum can be predicted solely from the albumin concentration and pH (BROCK 1974 & 1975). -
REFERENCES Avigan, J.: A method for incorporating cholesterol and other lipids into serum lipoproteins in vitro. J . biol. Chem. 1959, 234, 787-790. Brock, A.: Binding of digoxin to human serum proteins: Influence of pH on the binding of digoxin to human albumin. Acta pharmacol. et toxicol. 1974, 34, 260-266. Brock, A.: Binding of digitoxin to human serum proteins: Influence of pH on the binding of digitoxin to human albumin. Acta pharmacol. et toxicol. 1975, 36, 13-24. Burstein, M., H. R. Scholnick & R. Morfin: Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions. J. Lipid. Res. 1970, 11, 583-595. De Moor, P., K. Heirweg & 0. Steeno: Protein binding of progesterone studied by gel filtration. Arch. Biochem. 1963,103, 506-514. Franzini, C.: Gel filtration behaviour of human serum lipoproteins. Clin. Chim. Acta 1966, 14, 576-578. van Gent, G. M.: Lipid composition of lipoprotein fractions, In: Protides of the biological fluids, vol. 19. Ed.: H. Peeters. Pergamon Press 1972, pp. 75-79. Krinsky, N. I., D. G. Cornwell & J. L. Oncley: The transport of vitamin A and carotenoids in human plasma. Arch. Biochem. 1958, 73, 233-246. Lukas, D. S. & A. G. De Martino: Binding of digitoxin and some related cardenolides to human plasma proteins. J. din. Invest. 1969, 48, 1041-1053. McCormick, E. C., D. G. Cornwell & J. B. Brown: Studies on the distribution of tocopherol in human serum lipoproteins .I. Lipid. Res. 1960, 1, 221-228. Rosner, W. & S. M. Deakins: Testosterone-binding globulins in human plasma: Studies on sex distribution and specificity. J . din. Invest. 1968, 47, 2109-2116. Scanu, A. M. & C. Wisdom: Serum lipoproteins, structure and function. Ann. Rev. Biochem. 1972,41,703-730. Scoltan, W., K. Schlossmann & R. Rosenkranz: Bestimmung der Eiweissbindung von Digitalis-Praparaten mittels der Ultracentrifuge. Arzneimittelforsch. 1966, 16, 109118.
