Naunyn-Schmiedeberg's

Archivesof Pharmacology

Ttttt

9 by Springer-Verlag1978

The Binding of Intravenous and Oral Biliary Contrast Agents to Human and Bovine Serum Albumin WALTER E. MULLER Pharmakologisches Institut der Universit/it Mainz, Obere Zahlbacher Strasse 67, D-6500 Mainz, Federal Republic of Germany

S u m m a r y . The binding of two homologous series of

oral and intravenous biliary contrast agents to human and bovine serum albumin was investigated using the gel filtration technique. All intravenous compounds are bound to human serum albumin via one high affinity and several low affinity binding sites. Within the concentration range investigated, about 3 - 5 high affinity binding sites for the oral compounds were found on human serum albumin. In general, the intravenous compounds have a greater affinity for human serum albumin than the oral compounds. No significant differences were found for the binding of the oral compounds to human or bovine serum albumin, while the intravenous compounds have a higher affinity for bovine than for human serum albumin. The significance of the plasma protein binding of the biliary contrast agents for the hepatic uptake is discussed. K e y words: Biliary contrast agents -

Protein bind-

ing - Serum albumin.

INTRODUCTION Most of the clinically used biliary contrast agents are highly bound in the blood within the concentration range usually employed for the visualization of the biliary tract (Oeff, 1954; Langecker et al., 1964; Knoefel, 1971). The most important binding fraction is serum albumin. Only negligible binding to other serum proteins or to the blood cells has been demonstrated for some biliary contrast agents (Langecker et al., 1964; Lasser et al., 1962; Sergeev and Chistyakov, 1965; Knoefel, 1971). Lang and Lasser (1971) investigated the binding of iodipamide and iopanoate to human and bovine serum albumin and found

curved Scatchard plots, suggesting 2 different types of binding sites. Further comparable data on the affinity and the stoichiometry of the interaction of biliary contrast agents with serum albumins are still lacking and the knowledge about the influence of the chemical structure of the biliary contrast agents on the albumin binding is limited. The significance of the plasma protein binding for the hepatic uptake of the biliary contrast agents is not yet clear. It seems that high serum albumin binding prevents the glomerular filtration (Lasser et al., 1962; Knoefel, 1971), but decreases the hepatic uptake of the compounds (Lasser et al., 1962; Berndt et al., 1971; Song et al., 1974). Thus, a detailed knowledge of the strength and the nature of the interaction of the biliary contrast agents with serum albumins might be important for the understanding of the influence of the albumin binding on the uptake of the compounds into the liver cell. The present study describes the binding of 2 homologous series of oral and intravenous biliary contrast agents to human and bovine serum albumin, the influence of the chemical structure of the compounds on the binding strength and the nature of the binding and discusses the possible influence of the binding on the hepatic uptake.

MATERIALS AND METHODS Materials. Human and bovine serum albumin (HSA and BSA) were obtained from Behringwerke, Marburg (quality: "trocken, reinst", electrophoretic purity 100~). The biliary contrast agents were kindly donated by Dr. U. Speck, Schering AG, Berlin. The chemicalformulas of the contrast agents are summarizedin Tables 1 and 2. All other chemicalswere of reagent grade. All solutions were made with deionized water. Concentrations are given as tool per liter (M). Albumin Binding Measurements. The binding of the biliary contrast

Send offprint requests to W. E. Mfiller at the above address

agents to HSA and BSA was investigated by the gel filtration

0028-1298/78/0302/0227/$ 01.40

228 technique of Krieglstein and Kuschinsky (1968). The experiments were performed on 20 x 1.2 cm columns of Sephadex G-50 fine (Pharmacia, Uppsala) at room temperature (22~ as described by Mtiller and Wollert (I973). The albumin concentration was always 3 x 10-5 M, using a molecular mass of 69000 for both albumins. The biliary contrast agents concentrations were 0.75, 1.5, 3.0, 4.5, 6.0, 9.0 x 10-5 M. Four determinations were performed at each concentration. All solutions were made with 1/15 molar phosphate buffer and adjusted to the desired pH with 1 M HC1 or 1 M NaOH. No significant binding of the contrast agents to the gel material could be detected. Calculations. The extent of the binding of the biliary contrast agents at various concentrations (c) to HSA and BSA was given by the percentage of free (ct) and bound (/3) drug. The apparent binding constants k + and the regression coefficients or slope constants m were obtained from the relationship (Scholtan, 1962)

logcb=logk + +mlogc: where cb and c: are the concentrations of the bound and free drug. k + can be obtained from c: = I [10-5 M] if the equation is reduced to log k + = log cb (Seholtan, 1962). The number of binding sites per albumin molecule n and the association constants k were obtained from the Scatchard plot (Scatchard, 1949) following the equation ~/c: = kn - k f

where ? = the number of moles of the drug bound per mole albumin, c: = the concentration of the free drug, k = the association constant, and kn = K1 = the total binding constant. Curved Scatchard plots were analysed by the graphical method of Berson and Yalow (1959) as described by Weder et al. (1974). Asymptotic straight lines were drawn at both extremities of the Scatchard plot and the lines were moved parallel so that the sum of the ordinate intercepts equals the total ordinate intercept.

RESULTS B i n d i n g to H u m a n S e r u m A l b u m i n a t p H 7.40

A t a given concentration o f the biliary contrast agents (3 x 10 -5 M, c 6 r r e s p o n d i n g to a m o l a r drug/ albumin ratio o f one) the percentage o f b o u n d drug /~ varies f r o m 7 0 - 97 ~ for the intravenous c o m p o u n d s ( Z K 1 - 5, Table 3) and f r o m 8 0 - 9 8 ~ for the oral c o m p o u n d s ( Z K 1 0 - 1 5 , Table 3). Plotting the free versus the b o u n d c o n c e n t r a t i o n over the concentration range investigated following Scholtan (1962) reveals straight lines, which correlation coefficients are given in Table 3. The a p p a r e n t binding c o n s t a n t k + can be obtained for c : equals 1 (10 -5 M) (see Methods). The a p p a r e n t binding constant, which is rather an indication o f the binding capacity than o f the affinity o f the albumin (Scholtan, 1967) varies for the intravenous c o m p o u n d s only by a b o u t two fold, with 5.45 for Z K 1 and values o f 2 . 0 - 2 . 7 for the other four c o m p o u n d s ( Z K 2 - 5 , Table 3). The slope constant m is a b o u t 0 . 4 - 0 . 5 for all five derivatives. F o r the oral c o m p o u n d s ( Z K 10--15, Table 3) a larger variation o f the a p p a r e n t binding constants was found,

Naunyn-Schmiedeberg's Arch. Pharmacol. 302 (1978)

'H'a

l

7[MIM]

2

3

Fig. 1. Scatchard plot of the binding of ZK 1 to human serum albumin at pH 7.40. The dotted lines represent the calculated sets of binding sites. Ordinate: ?/c:, ~ = the number of moles of ZK 1 bound per mole albumin, c: = the concentration of the free drug. Abscissa: f. Each point represents the mean _+ S.E.M. of four determinations

with the highest value for Z K 15 o f 12.8, a slightly smaller value for Z K 10 o f 10.9 and a b o u t 50 ~ or 2 5 ~ o f this value for Z K 11 and 14 or 12 and 13 respectively. The slope constant is fairly similar for all oral c o m p o u n d s with values between 0.6 and 0.7. Plotting the results o f the binding experiments with the intravenous c o m p o u n d s ( Z K 1 - 5) following the m e t h o d o f Scatchard (1949) results in curved lines, as shown for Z K 1 in Figure 1. The intercept with the ordinate equals the total binding constant. The curved lines were analysed by a graphical m e t h o d as described in methods. T w o sets o f binding sites were obtained for all oral c o m p o u n d s . One high affinity binding site and 2 or 3 binding sites with m u c h lower affinity were f o u n d for the drugs. The association constants and the n u m b e r o f binding sites o f each set are given in Table 3. The association constants o f the high affinity binding site vary a b o u t 10-fold, with the highest affinity for Z K 1, an a b o u t 4 times lower affinity for Z K 2 and a b o u t half o f the latter value for the other c o m p o u n d s . By contrast, the Scatchard plots o f the oral c o m p o u n d s (as s h o w n for Z K 12 and 15 in Fig. 2) result in straight lines, suggesting only one set o f binding sites within the concentration range investigated. The binding parameters were taken f r o m calculated regression lines, which correlation coefficients are given in Table 3. W i t h exception o f Z K 15 which binds to 5 binding sites, a b o u t three binding sites were f o u n d for the oral c o m p o u n d s (Table 3). The association constants differ f r o m 6.6 x 105 for Z K 10 to 0.8 or 0.9 x 105 for Z K 12 and 13 with intermediate values for Z K 11, 14, and 15.

W. E. Mfiller: Binding of Biliary Contrast Agents to Albumin

229

t,o I00

~ , Z K u~

;qt

15

o,5[

oZK 1 9 Z K 10 9

..

x ZKg@

\.

,

27

l

L/M'M--J" 3

~

s

Fig. 2. Scatchard plot of the binding of Z K 12 and 15 to human serum albumin at p H 7.40. Ordinate: f/c s, f = the number of moles of the drug bound per mole of albumin, es = the concentration of the free drug. Abscissa: r. Each point represents the mean _+ S.E.M. of four determinations

tO0

60

,-,

.-

-

tO0

,~0

9 ZKI

9

ZK

~

tO

9 ZK 12 D ZK 15 i

6,~

7,0

I '

o ZK 2 20

6,6

t

n

'

'

7,~

e2 ~"

/ ZK 129

@ZK5 9 ZK3 i 0,01

i O,l

ZK I~

13

I

i

l.O

10.0

i I00,0

Partition Coefficient P Fig. 4. Correlation between the partition coefficients P of the biliary contrast agents (abscissa) and the total binding constants nk, obtained for human serum albumin at pH 7.40 (ordinate)

the high affinity binding site is nearly saturated but much less is bound to the secondary binding sites. Possibly, the decrease of the binding of Z K 1 and 2 with increasing pH is mainly due to an effect at the secondary binding sites. By contrast, the binding of the oral compounds Z K 10 and 15 is nearly unchanged within the p H range investigated (Fig. 3), while the binding of Z K 12 is increased raising pH from 6.60 to 8.20 (Fig. 3).

I o, ZK2 zKl OOL~

7,0

7.~

n

9 ZK2

ZK 15 o/~

~

pH

o

The Effect of the Lipophilicity on the Binding to Human Serum Albumin

o

i

i

|

7,z

7,a

e,2

Fig. 3. The effect of pH on the binding of biliary contrast agents to human serum albumin. Ordinate: fl = percentage of bound drug at a total concentration of 6 x 10.5 M. Abscissa: pH. Inset, ordinate: /~ = the percentage of bound drug at a total concentration of 3 x 10 s M. Inset, abscissa: pH. Each point represents the mean of four determinations with standard deviations smaller than 3

The Effect of pH on the Binding to Human Serum Albumin The effect of the pH-value of the solution on the binding of the biliary contrast agents to human serum albumin was investigated within the pH-range from 6.60 to 8.20. F o r the two intravenous compounds investigated (ZK 1 and 2) a decrease of the percentage of bound drug was found at a molar drug/albumin ratio of two (Fig. 3), when the pH was raised from 6.60 to 8.20. The observed decrease of the percentage of bound drug with increasing p H is much smaller at a drug/albumin ratio of one (inset in Fig. 3), when

No general correlation between the partition coefficients and the total binding constants of the biliary contrast agents was found for human serum albumin (Fig. 4). For Z K 1 - 5 only very small differences of the partition coefficients are obvious (Table 1), but the total binding constants vary~by about 15-fold (Table 3). For the oral compounds (ZK 1 0 - 1 5 ) larger differences of the partition coefficients are evident (Table 2), but there is no general correlation with the total binding constants (Fig. 4). It seems however, that a correlation between partition coefficients and binding constants exists for Z K 11 - 14 (Fig. 4). Interestingly, these 4 compounds have a primary or secondary amino group in contrast to the tertiary amino group of Z K 10 and 15 (Table 2). No better correlation with the partition coefficients was found when the association constants were used instead the total binding constants.

Binding to Bovine Serum Albumin at p H 7.40 In parallel experiments, using the conditions cited above, the binding of the biliary contrast agents to

230

Naunyn-Schmiedeberg's Arch. Pharmacol. 302 (1978)

Table 1 The chemical structures, the generic and trade names, the abbreviations, and the partition coefficients P between n-octanol and tris-buffer pH 7.6 of the intravenous biliary contrast agents investigated

COOH

"V

COOH

"NH-C-R-C-NH"

J

O

O

J

R

Abbreviation

Name

P

( - CHz - )4

ZK 1

Jodipamide Biligraphin |

0.013

-CH2-O-CH2-

ZK 2

Joglycaminic acid Bilivistan ~

0.010

(- CH2- O- CH2-)2

ZK 3

-

0.015

-- CH2 - O - CH2 - (CH2)2 - CH2 - O - CH2 -

ZK 4

Jotroxaminic acid

0.006

-CH2-(CHz-O-CH2)4-CH2-

ZK 5

Jodoxaminic acid

0.016

Table 2. The chemical structures, the generic and trade names, the abbreviations, and the partition coefficients P between n-octanol and tris-buffer pH 7.6 of the oral biliary contrast agents investigated Rl

R2 J R2

R1

Abbreviation

-CH2-CH-COOH

P

CH3

C2H5

1

Name

-N=CH-N(

ZK 10

6.53

Z K 11

13.31

Z K 12

2.86

Z K 13

4.63

Z K 14

36.47

CH3 -CH2-CH2-COOH

-N=C-NH-CH3

J CH3 -CH2-CH2-COOH

-N=C-NH2

I CH3 -CH2-CH2-COOH

-N=C-NH2

I

C2H5

~

2H5

- CH2 - CH

-- CH2 - CH2 - COOH

-N=C-NH-CzH5

-N=CH-N

/

CH3 Z K 15

\ CH3

Jodopate Biloptin |

4.97

W. E. Mfiller: Binding of Biliary Contrast Agents to Albumin

231

Table 3. Binding parameters for the interaction of the biliary contrast agents with human serum albumin at pH 7.40. fl = the percentage of bound drug at a total concentration of 3 x 10 .5 M, k n = / ( 1 = the total binding constant, kl and k2 = the association constants of the first and second set of binding sites, n~ and n2 = the number of binding sites of the first and second set of binding sites, m = the slope constant, k + = the apparent binding constant, rl = the correlation coefficient for the double-logarithmic plot following Scholtan (1962), and r2 = the correlation coefficient for the Scatchard plot. Both straight lines were calculated for 24 experiments Compound

ZK ZK ZK ZK ZK ZK ZK ZK ZK ZK ZK

1 2 3 4 5 10 11 12 13 14 15

fl [ ~ ] 2 +_ s-~ (n = 4)

k n x 10 - 5

k l x 10 - 5

[l/M]

[1/MI

96.68 80.05 70.35 82.07 72.88 97.72 91.35 79.67 81.21 93.18 96.28

25.7 4.8 1.7 3.7 2.7 20.8 5.4 2.1 2.3 7.0 10.4

17.1 4.3 1.4 2.7 2.3 6.6 1.8 0.8 0.9 2.5 2.1

_ 0.20 + 1.15 + 0.55 + 0.41 + 0.54 + 0.34 + 0.07 _+ 0.76 +_ 0.08 _+ 0.20 + 0.07

nl

k2 x 10 - 5

n2

m

2.7 1.9 2.4 2.1 2.8 --

0.39 0.45 0.56 0.47 0.46 0.55 0.6.5 0.64 0.64 0.58 0.74

k +

rl

r2

0.974 0.988 0.992 0.983 0.985 0.967 0.996 0.994 0.996 0.976 0.994

-0.848 -0.945 -0.941 -0.967 -0.972 -0.962

[1/MI 1.3 0.9 1.0 1.1 1.0 3.1 3.1 2.5 2.5 2.8 4.9

1.3 0.6 0.2 0.4 0.2 --

5.45 2.76 2.04 2.76 2.15 10.9 5.9 3.0 3.2 6.2 12.8

Table 4. Binding parameters for the interaction of the biliary contrast agents with bovine serum albumin at pH 7.40./~ = the percentage of bound drug at a total concentration of 3 x 10 -5 M, k n = / ( 1 = the total binding constant, kl and k2 = the association constants of the first and second set of binding sites, nx and n2 = the number of binding sites of the first and second set of binding sites, m = the slope constant, k + = the apparent binding constant, rl = the correlation coefficient for the double-logarithmic plot following Scholtan (1962), and r2 = the correlation coefficient for the Scatchard plot. Both straight lines were calculated for 24 experiments Compound

/3[~] 2 _+ s~ (n = 4)

k n x l 0 -5 [l/M]

k l x l 0 -5 [l/M]

nl

k 2 x l 0 -5 [l/M]

n2

m

k+

rl

r2

ZK ZK ZK ZK ZK ZK ZK ZK ZK ZK ZK

98.06 95.84 91.99 96.96 94.12 96.69 89.67 79.30 74.32 92.69 97.89

21.3 22.0 25.0 40.0 19.2 13.7 3.7 1.8 1.4 7.5 20.0

16.3 15.4 23.1 37.2 19.0 3.8 1.1 0.6 0.5 2.5 4.7

1.1 1.2 1.1 1.1 1.0 3.6 3.4 3.1 2.7 2.9 4.3

1.5 1.6 0.3 0.5 0.5 ---

2.1 2.0 2.1 1.8 1.7 -

0.32 0.34 0.26 0.21 0.23 0.67 0.69 0.71 0.69 0.61 0.70

3.82 4.47 2.93 2.91 2.61 11.9 5.1 3.1 2.5 6.6 18.2

0.956 0.957 0.940 0.871 0.871 0.993 0.989 0.997 0.991 0.991 0.980

-0.887 --0.930 --0.930 -0.892 -0.853 -0.889

1 2 3 4 5 I0 I1 12 13 14 15

_+ 0.23 _+ 0.54 + 0.25 _+ 0.27 ___ 0.08 -I- 0.11 + 0.50 +_ 0.17 _+ 0.96 _+ 0.56 + 0.17

bovine serum albumin was investigated. Bovine serum a l b u m i n w a s c h o s e n b e c a u s e o f its f r e q u e n t u s e a s a model substance for drug albumin binding studies. For the oral contrast agents, no important differences of the binding to human or bovine serum albumin w e r e f o u n d , a s it is o b v i o u s b y t h e b i n d i n g p a r a m e t e r s i n T a b l e s 3 a n d 4. B y c o n t r a s t , r e m a r k a b l e d i f f e r e n c e s of the affinities have been found for the binding of the intravenous compounds to bovine serum albumin c o m p a r e d t o h u m a n s e r u m a l b u m i n ( T a b l e s 3 a n d 4). O n e h i g h a f f i n i t y a n d s e v e r a l l o w a f f i n i t y b i n d i n g sites were calculated from curved Scatchard plots for bovine as f o r h u m a n s e r u m a l b u m i n , b u t t h e a s s o c i a t i o n c o n s t a n t s o f t h e h i g h a f f i n i t y b i n d i n g site d i f f e r markedly. For example, the association constants of t h e s i n g l e h i g h a f f i n i t y b i n d i n g site o f Z K 3, 4 a n d 5 are about 10-fold higher for bovine than for human

s e r u m a l b u m i n ( T a b l e s 3 a n d 4). B y c o n t r a s t , t h e a s s o c i a t i o n c o n s t a n t o f Z K i is s i m i l a r f o r b o t h a l b u m i n s ( T a b l e s 3 a n d 4). I n t e r e s t i n g l y , t h e a p p a r e n t binding constants of the oral as well as the intravenous contrast agents are fairly similar for both albumins ( T a b l e s 3 a n d 4), s u g g e s t i n g s i m i l a r b i n d i n g c a p a c i t i e s o f b o t h a l b u m i n s ( S c h o l t a n , 1962).

DISCUSSION N e a r l y all b i l i a r y c o n t r a s t a g e n t s i n v e s t i g a t e d a r e strongly bound to both serum albumins, but large differences of the number of binding sites and the affinities are evident, In general, the intravenous compounds have a much higher affinity than the oral compounds but only have one high affinity binding site i n s t e a d o f 3 - 5 f o u n d f o r t h e o r a l c o m p o u n d s . T h e

232 percentage of bound drug at a given concentration does not reflect the differences of the affinities. For example, ZK 1 and ZK 15 are bound to about 9 6 ~ at a molar drug/albumin ratio of one (Table 3), but ZK 1 has an about 8 - 9 times higher association constant than ZK 15 (Table 3). This clearly demonstrates that the percentage of bound drug at a given concentration is unsufficient to characterize the strength of the binding of drugs to albumins, since the affinity, the important factor for the distribution of the drug, is not known. The apparent binding constant characterizes the binding capacity of the albumin rather than the affinity (Scholtan, 1962). This is clearly demonstrated for ZK i - 5 (Table 3), where the association constants vary by more than 10-fold, the apparent binding constants only by about 2-fold. In the ZK 1 molecule, the two aromatic parts are connected by a bridge of 4 apolar methylene groups, while in ZK 2 - 5 the bridges include increasing numbers of more polar ether groups (Table 1). The length of the bridges does not seem to be important for the binding to human serum albumin, as it is obvious by the quite similar association constants of ZK 2 - 5 (Table 3), but its apolar character may be important as it can be concluded from the about ten times higher affinity of ZK 1. In the case of bovine serum albumin, the association constants increase with the chain length of the bridges up to ZK 4 and decrease again in the case of ZK 5 (Table 4). The lack of any correlation between the partition coefficients and the binding constants does not exclude the importance of hydrophobic forces for the binding of the intravenous compounds to human serum albumin. The partition coefficients characterize the lipophilic nature of the whole molecule, but only parts of the ligand molecule may interact with the albumin (Mfiller and Wollert, 1973). It seems possible that for the binding of the intravenous compounds to human serum albumin the aliphatic bridge and possibly one aromatic group and for the binding to bovine serum albumin both aromatic groups are involved in the binding mechanism. For the oral biliary contrast agents having a primary or secondary amino group (ZK 11-14) the lipophilicity of the whole molecule may be important for the binding, as suggested above (Fig. 4). The reason for the exceptionally high affinities of ZK 10 and 15 may be a different pK-value of the tertiary amino group rather than the lipophilicity. Unfortunately, the pKa-values o f the amino 'groups are not known. But interestingly, the Nnding of ZK 12, a compound with a primary amino group, increases with increasing pH, possibly due to a decrease of the cationic charge of the molecule. For ZK 10 and 15, both having a tertiary amino group, nearly no effect of increasing

Naunyn-Schrniedeberg's Arch. Pharmacol. 302 (1978) pH was found (Fig. 3). ThepKa-values of the carboxylate groups of the compounds range from 3 - 5 (as given by the manufacturer), so that changes of the anionic charge of the molecules can not be the reason for the observed effects of increasing pH on the binding to human serum albumin. The significance of the plasma protein binding of the biliary contrast agents for the hepatic uptake of the compounds is not yet understood entirely (Knoefel, 1971; Lasser, 1966; Lasser and Lang, 1970; Fuchs, 1977). High plasma protein binding does not seem to be mandatory for hepatic uptake, in terms of serum albumin serving as a carrier (Bennhold et al., 1950), but high serum albumin binding seems to prevent the rapid glomerular filtration of the biliary contrast agent, so that the compounds can be taken up by the liver during several passages (Knoefel, 1971; Lasser, 1966; Lasser and Lang, 1970). On the other hand, the binding to serum albumin decreases the uptake rate of those compounds, as shown for in vitro studies with liver slices and an isolated perfused liver preparation (Berndt et al., 1971; Song et al., 1974). The liver uptake itself seems to depend on a binding to soluble, cytoplasmic proteins rather than on an active transport mechanisms (Berndt et al., 1971; Sokoloff et al., 1973; Kamisaka et al., 1975). Thus, liver uptake may be the sum of the relative affinities of the compounds for the serum albumins and the soluble liver proteins. Therefore, high hepatic uptake may be observed for biliary contrast agents with a high binding capacity of the serum albumin preventing the glomerular filtration, and a rather small affinity of the albumin, promoting a high binding to the liver proteins. These considerations may be true for the oral biliary contrast agents, especially ZK 15, which are bound by both serum albumins with a high capacity but a smaller affinity. For the intravenous agents, the reversed may be the case. Interestingly, for the oral biliary contrast agents higher liver/plasma ratios than for the intravenous compounds have been reported (Sperber and Sperber, 1971), which may support this theory. There is no doubt that other factors like the binding strength to the soluble liver proteins may be very important, too. In summary, the exact knowledge of the number of binding sites and of the binding strength of the interaction of the biliary contrast agents is not only of theoretical interest in terms how drugs can interact with proteins, but may be helpful for the understanding of the pharmacokinetics and pharmacodynamics of the biliary contrast agents. Acknowledgements. The excellenttechnicalassistanceof K. Lemmel

is gratefullyacknowledged.This studywas supported by a grant of the DeutscheForschungsgemeinschaft.The author wishesto thank

W. E. Miiller: Binding of Biliary Contrast Agents to Albumin Dr. U. Speck, Schering AG, Berlin, for providing the biliary contrast agents and for the determination of the partition coefficients.

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Received December I, 1977~Accepted January 11, 1978

The binding of intravenous and oral biliary contrast agents to human and bovine serum albumin.

Naunyn-Schmiedeberg's Archivesof Pharmacology Ttttt 9 by Springer-Verlag1978 The Binding of Intravenous and Oral Biliary Contrast Agents to Human...
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