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Studies on the decrease in bile flow produced by sulfobromophthalein
TOXICOLOGY
AND
APPLIED
Studies
PHARhIACOLQGY
on the
(1974)
28,406-417
Decrease in Bile Sulfobromophthalein
Flow
Produced
by
PETER-J&GENSCHULZEAND GEORGCZOK Pharmakologisches Institut der Universitcit Hamburg, Hamburg, Germany Received August 31,1973; accepted January 3,1974
Studieson the Decreasein Bile Plow Producedby Sulfobromophthalein. SCHULZE, P.-J. AND CZOK, G. (1974).Toxicol. Appl. Pharmacol.28,406417.
A reduction in bile flow wasobservedduring BSP-infusion(1.875mg/min) in urethane-anesthetizedWistar rats. The infusion times at which 50% diminution in bile flow occurred were almost equal in bile salt-depleted (biliary fistula during 5 hr) animalsand in those given taurocholate (150 nmol/lOOg/min) during the depletion; however, the infusiontimesdiffered significantly from thoseobservedin non-depletedrats. While the overall excretion of the dye was mainly dependenton the
BSP-GSH fraction in the bile, the reduction in bile flow was inversely related to the amount of BSP-GSH conjugate excreted and the hepatic GSH concentrations before the infusion. A marked fall in hepatic GSH wasobservedafter the long-lastingurethanenarcosisthat occurredduring bile salt depletion. Sincethe infusion of equimolar and higher amountsof
BSP-GSH resulted in choleresis, it is concluded that the unconjugated BSP is responsiblefor the reducedbile flow during BSPinfusion. It is suggestedthat for BSP-GSH, in contrast to BSP, the rate-limiting step in the transport from blood to bile is not its biliary excretion, but its uptake into the liver.
The biliary excretion of a foreign compound usually involves three steps : uptake into the liver cell and storage; biotransformation; and excretion into the bile. The biotransformation step usually increases the water solubility and the molecular weight of the compound and thereby facilitates its biliary excretion. The excreted substance may either increase or decrease bile flow. There are compounds which decrease bile flow after low doses, e.g., indocyanine green (Klaassen and Plaa, 1969; Horak et al., 1973) and rose bengal (Dhumeaux et al., 1970; Varga and Fischer, 1970), and there are others which depress bile flow only after relatively high doses. The findings of Priestly and Plaa (1970a) suggest that sulfobromophthalein (BSP), a substance whose biliary excretion is highly dependent on its conjugation with glutathione (GSH) (Schenker et al., 1965; Combes, 1965; Klaassen and Plaa, 1967; Priestly and Plaa, 1969; Whelan et al., 1969, 1970) belongs to the latter group. This was confirmed by our own preliminary BSP-infusion experiments in rats.
The present study was initiated to get more information about the mechanism of the decrease in bile flow during BSP-infusion and especially to see how far this decrease can be correlated with the bile salt concentration at the start of the infusion and with the extent of biliary appearing BSP-GSH conjugates. Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
406
DECREASED
BILE FLOW
AFTER
BSP
407
METHODS Male Wistar rats (290420 g) were used throughout. The animals were maintained on Altromin@ standard diet and tap water ad libitum. They were anesthetized with ethylurethane, 1 g/kg ip. Surgicalprocedure. In general the bile duct and the left jugular vein were cannulated with PVC Pl and P2 tubing, respectively. To prevent hypothermic alteration of the bile flow (Roberts et ai., 1967; Krarup and Larsen, 1972) the body temperature of the animals was maintained at 37°C by means of a thermostat. Analytical methods. The bile flow was determined gravimetrically. The specific gravity of the bile was presumed to be 1. The bile acids were determined enzymatically (Iwata and Yamasaki, 1964) using hydroxysteroid dehydrogenase (Worthington Biochemical Corp., Freehold, New York). The BSP concentrations in the bile and in the serum were measured calorimetrically using a Photometer “Eppendorf” at 578 nm after an appropriate dilution of the samples with alkalinized 0.9 % sodium chloride solution (100 ml of 0.9 % NaCl plus 5 ml of 10 % NaOH). BSP and its metabolites in the bile were separated by thin-layer chromatography (10 ~1 bile on Merck@ precoated TLC-plates Silical/Gel 60 F-254, layer thickness 0.25 mm; solvent: tertiary butylic alcohol-distilled water 3 : 1; Sardini er al., 1969) and after eluting in alkaline-sodium chloride measured at 578 nm. The extinction coefficients of BSP and its metabolites were assumed to be identical (Combes, 1965; Whelan et al., 1970). Therefore, the extinction values of the metabolites were treated as if they originated from BSP which had been used for the calibration curve. According to Whelan et al. (1970), besides the unaltered substance, 2 metabolic fractions were estimated : BSP-glutathione, including traces of diglutathione, and BSP-cysteinylglycine plus BSP-cysteine. The BSP-content of the liver was estimated by the technique of Whelan et al. ( 1970) which gave recoveries of more than 95 % when either BSP or BSP-GSH was added to a liver homogenate. The GSH-content of the liver was determined by the method described by Grunert and Phillips (1951). BSP-induceddiminution in bileflow. The diminution of the bile flow was effected by a continuous infusion of BSP (Bromthalein @, Merck/Darmstadt, 5 ‘A aqueous solution; the infusion rate was 1.875 mg/min usingperfusionpumps (UnitaI @,Braun/Melsungen). The bile flow was determined at intervals of 10 min until it fell below 50% of the initial volume. The time required for a 50% decrease was estimated by interpolation. The bile volumes used as a reference were those measured during the last 10 min before starting the BSP-infusion. Rats termed nondepleted were infused with BSP 30 min after cannulating the bile duct, whereas the depleted animals were infused 5 hr after cannutation. In the latter, the loss of body fluid due to the biliary fistula was replaced by iv Ringer solution given each hour. In a further group the bile salts lost during the depletion were replaced by a continuous infusion of sodium taurocholate (150 nmol/lOO g/min, dissolved in equal vol-
408
SCHULZE
AND
CZOK
umes of 0.9 % sodium chloride and 10 % glucose solution, infusion rate 1 ml/hr) before the BSP infusion was started. Other nondepleted and depleted animals were infused with BSP-GSH. The various groups of animals employed are given in Table 1. TABLE 1 THE VARIOUS GROUPS OF ANMAM EMPLOYED Group
IN THE PRESENT EXPERIMENTS
Treatment
I Nondepleted rats, BSP infusion up to 50% diminution of bile flow II Depleted rats, BSP infusion up to 50% diminution of bile flow III Taurocholate substitution during depletion, BSP infusion up to 50% diminution of bile flow IV Nondepleted rats, 30-min BSP infusion V Depleted rats, 30-min BSP infusion VI Nondepleted and depleted rats, 2-hr BSP-GSH infusion VII Nondepleted rats, 30-min BSP-GSH infusion VIII Depleted rats, 30-min BSP-GSH infusion IX Nondepleted rats, bile duct ligation, 30-min BSP infusion x Depleted rats, bile duct ligation, 30-min BSP infusion XI Nondepleted rats, bile duct ligation, 30-min BSP-GSH infusion XII Depleted rats, bile duct ligation, 30-mm BSP-GSH infusion XIII Nondepleted rats, estimation of hepatic GSH content Depleted rats, estimation of hepatic GSH content XIV xv No biliary fistula, urethane narcosis for 5 hr, estimation of hepatic GSH content XVI Taurocholate substitution during the depletion, estimation of hepatic GSH content XVII Infusion of glucose and sodium chloride during depletion, estimation of hepatic GSH content Statistical analysis. The results are expressed as means f SD. The statistical significance of the differences between means of various treatments was determined by Student’s t-test for independent means. RESULTS Reduced Bile Flow under BSP Infusion Figure 1 illustrates that the initial bile volumes and the course of bile flow during the BSP infusion were dependent on the pretreatments of groups I, II and III. During the infusion of BSP the bile flow of the nondepleted group I clearly exceeded the initial bile volume, while in groups II and III no comparable increase was observed. Table 2 shows the interpolated times for the 50 % decrease in bile flow and the quantity of BSP infused to achieve this reduction. The initial bile volumes and the simultaneous concentrations and excreted amounts of bile salts before starting the dye infusion are given. Comparing groups I and II it is obvious that the reduction of bile flow corresponds neither to the initial bile volume nor to the initial bile salt concentration.
DECREASED
BILE FLOW
AFTER
M
I
non-depleted
I
70
20
30
40
50
409
BSP
!
I
60
70
(II
,
80
90 Inin
t
in
FIG. 1. Bile flow (&lo0 g/l0 min) during BSP infusion (1.875 mg/min) after different pretreatments rats. f Start of the infusion. Each point represents mean f SD of at least 6 animals.
From Fig. 2 the BSP-excretion of groups I, II and expressed as follows: I > III > II. The corresponding illustrated in Fig. 3. The maximal dye concentrations starting the infusion; thereafter the BSP concentrations
III during the infusion can be biliary BSP concentrations are were reached 20-30 mjn after of groups II and III decreased
wg fJSP !C3g-r10m~n-1 800
-
700
-
600
-
500
-
400
-
300
-
200
-
100
-
t
10
20
30
40
50
60
70
80
90
min
FIG. 2. Biliary excretion of BSP (~g/lOO g/l0 min) during BSPinfusion (I ,875 mg/min) after different ?retreatments in rats. t Start of the infusion. Each point represents mean _+SD of at least 6 animals.
36.81 _+ 5.19 56.10 f 5.74
394.3 + 40.1
378.6 + 41.5
7 Depleted(II) Depletedf taurocholate uw 7
NS
p II/III
LIBile volume, biliary bile salt concentration, b NS, not significant.
NS
P I/III
co.001
co.02
1821.59 f 258.21
32.68 k 4.97
NS
45.14 f 11.06
37.30 rk 3.36
80.10
565.52 +
25.70 _+ 2.40 15.80 + 3.60
50% diminution of bile volume (min) 81.56 + 10.50
Bile salts (nm01/100g/ 10 min) 1114.70 rt: 155.21
Bile salt concentration (=01//4
-
co.05
22.57 f 4.85
17.97 -(- 2.84
39.91 + 3.53
Infused BSP (mg/l~ g)
and biliary bile salt excretion were determined just before the dye infusion. Values expressed as mean + SD.
co.05
NSb
P WI
43.26 f 3.56
383.3 & 39.6
6
Bile volume W/l00 d 10 min)
N
------
Body weight (Ed
Nondepleted(I)
2
BSP-INDUCED DIMINUTIONOFBILE FLOWAFTER DIFFERENT PRETREATMENTS IN RATS’
TABLE
DECREASED
10
t
20
BILE FLOW
30
40
AFTER
50
60
411
BSP
70
80
90
min.
FIG. 3. Biliary BSP concentration @g/pi) during BSP infusion (1.875 mg/min) after different pretreatments in rats. t Start of the infusion. Each point represents mean f SD of at least 6 animals.
continuously, whereas the BSP-concentration of group I fell very slowly. The maximal dye concentrations of groups I and III were not significantly different from each other, but both were significantly higher than the maximal value of group II (p < 0.01). Figure 4 shows the average percentages of BSP and its metabolites in the bile. The fraction of the unaltered substance (BSP) was about 30-35 % after 10 min, reaching a minimum within 20-30 min and then increasing again. The percentage of the unchanged dye was lower throughout in group I. By contrast, BSP-GSH was significantly higher
80 70
non-dcp(etedf0
-
depleted
(8)
-
deplrf.+subsf.
(III)
60 T
50 40
BP-CG
+ BSP-C
ESP-GSH
i
30 20 10
t
1 10
20
30
LO
50
t
10
20
30
40
50
t
10
20
30
40
54 t??,”
FIG. 4. Biliary excretion BSP and its main metabolites (BSP-glutathione and BSP-cysteinylglycine + BSP-cysteine) expressed as percentage of the totally excreted dye. t Start of the BSP infusion (I.875 mg/min) after different pretreatments. Each point represents mean f SD of at least 6 animals.
412
SCHULZE
AND
CZOK
in group I than in the other groups. Compared with BSP, the concentration curves for BSP-GSH run inversely, reaching their maxima after 20 and 30 min, respectively. The percentages of BSP-cysteinylglycine (BSP-CG) and BSP-cysteine (BSP-C) together were highest in group II and lower in groups I and III. In all groups the percentages of these fractions increased with the infusion time. Figure 5 illustrates the excreted amounts of BSP and its metabolites; the curves for BSP-GSH are in agreement with those for the totally excreted dye in the bile (Fig. 2). 4g
7OOg-7
IOmin-7
800
0-Q
non-depleled(iJ
BP-CG
t
70
20
30
40
50
t
70
20
+ BSP-C
30
40
50
t
10
20
30
40
50 min.
FIG. 5. BiliaryexcretionofBSP(flg/lOOg/lOmin)and its mainmetabolites(BSP-glutathioneandBSPcysteinylglycine + BSP-cysteine) after different pretreatments. t Start of the BSP infusion (1.875 mg/ min). Each point represents the mean of at least 6 animals. Vertical bars in BSP-GSH panel denote SD. There were significant differences in the biliary excretion of BSP-GSH between I and 11 (in the intervals of 20,30,40 and 50 min), between I and III (30,40 and 50 min) and between 11 and III (10,20,40 and 50 min).
BSP Infusion for 30 min Since after 30 min of BSP infusion there were already distinct differences in bile flow (Fig. l), this interval was chosen to determine not only the amount of dye in the bile but also in the liver and in the serum. Table 3 summarizes the results obtained. Comparison between nondepleted (IV) and depleted animals (V) demonstrates that the lower biliary excretion of BSP in group V was accompanied by a much higher dye content in the liver than in group IV. The serum concentrations of the dye did not differ significantly from one another. The average liver weight of group V was significantly lower than that of group IV because of the lower dry weight as w&l1 is a lower water content of the livers; however, the differences in dry weight expressed as percentages are greater (19 %) than those in wet weight (16 %). BSP-GSH Infusion for 2 Hr Because of the different metabolism of BSP in groups I, II, and III we were interested in studying the effect of the infusion of equimolar or higher amounts of BSP-GSH.
P
values
for BSP-GSH
* NS, not significant.
a The
Body weight (g)
BETWEEN
AND
Wet weight Dry weight (g) (8)
Liver
SALT-DEPLETED
Liver (mg)
NONDEPLETED
TABLE 4
4.56f 0.06 19.23 + 2.84 3.69+ 0.06 26.38 + 2.75 CO.05 -Co.005
Liver (w)
AND NONDEPLETED
3
AFTER
g liver G-w3
BSP
RATS
1.31 f.o.14 2.15 ko.33 CO.001
g liver 6-m)
BSP
BSP INFUSION”
INFUSIO@
17.15 + 2.13 11.20 &- 1.18
Max. biliary BSP concentration b.vdml)
BSP-GSH
bile/30 min bg)
30-MIN
8.98 + 2.31 4.13 +0.71 CO.001
.~ Max. biliary BSP bile 30/min concentration (mid (mdml)
RATS AFTER 3%MIN
(mg)
are calculated
in terms of BSP
equivalents.
Values
are
expressed
as means
+ SD.
6 332.7+ 48.9 12.55+ 1.02 3.78f 0.14 2.09 zk0.42 0.16 -f 0.03 16.91+ 2.67 22.98f 2.03 7 328.6Ifr 43.0 10.82+ 1.47 3.11 + 0.10 2.28 + 0.32 0.21 +0.03 16.81+ 2.85 22.57f 1.90 NSb 0.05 0.05 NS NS NS NS
N
COMPARISON
BILE
6 314.2+ 31.2 14.71 f 1.43 6 321.7 + 13.7 12.39 + 1.09 NSb <0.002
Dry weight (g)
Liver
BILE SALT-DEPLETED
Wet weight h.3
BETWEEN
aValuesareexpressed asmeans+ SD. bNS, not significant.
Non-depleted(VII) Depleted (VIII)
P
Nondepleted (IV) Depleted (V)
N
Body weight 69
COMPARISON
TABLE
32.40+ 8.58 29.14+ 7.92 NS
BSP/ serum Wx/1@Jml)
99.73 rtl 10.27 93.90 + 10.61 NS
BSP concentration in serum hx/lOO ml)
5 w
i
w
% ;;f
E
u E ii w Ef
Ki
414
SCHULZE AND CZOK
However, no diminution in bile flow occurred. In fact, a choleretic effect was observed, even when 3-fold molar doses were given either to nondepleted (N= 2) or depleted (N= 1) animals for 2 hr. Therefore, no further efforts were made to reduce bile flow with BSP-GSH. BSP-GSH Infusion for 30 Min
In these experiments, instead of BSP, equimolar quantities of BSP-GSH were infused. As seen in Table 4, apart from the differences in liver weights, there were no significant differences between nondepleted (VII) and depleted (VIII) rats. BSP and BSP-GSH Infusionfor 30 Min in Nondepletedand Depleted Animals after Bile Duct Ligation
To get more information about the hepatic uptake was eliminated by ligating the bile duct before the hepatic uptake of each dye was independent of the uptake per gram liver was higher in group X than in
of the dyes, their biliary excretion infusions. Table 5 shows that the pretreatment. However, the BSPgroup IX (p < 0.001).
TABLE 5 DISTRIBUTION
OF BSP-GSH IN NONDEPLETED AND BILE SALT-DEPLETED RATS WHOSE DUCTS WERE LIGATED 10 MIN BEFORE THE INFUSION WAS STARTED’
Nondepleted
Body weight (g) Liver weight (g) BSP/liver (mg) BSP/g liver (mg) BSP/serum bxidl~ ml) N
Depleted
BSP-GSHb (XI)
BSP 00
415.00 + 42.62 15.27 & 1.71 18.54 & 2.66 1.21” + 0.12 94.22 &- 5.12
423.75 + 28.37 13.67 _+1.99 5.30 f 0.77 0.39 + 0.06 59.50+ 4.33
414.16 + 14.97 12.21 + 1.25 18.38 zk 1.70
7
8
6
BSP cm
BILE
1.51C* 0.12 92.63 If: 4.92
BSP-GSHb WI) 410.84 12.64 4.94 0.39 60.20
k 39.54 &- 1.17 + 0.71 lk 0.03 -t 3.29 6
OBoth dyes were infused in equimolar quantities during 30 min. Values are expressed as means f SD. * The values for BSP-GSH (mg) are calculated as BSP equivalents. =p < 0.001 (IX vs X).
The lower molar concentrations of BSP-GSH in the serum can be explained by its larger volume of distribution in the body due to its higher water solubility and its lower binding to plasma proteins (Baker and Bradley, 1966). Hepatic Content of GSH after D$erent Pretreatments
Because of the different metabolic capacities of nondepleted-( depleted-(II), and taurocholate-substituted-(III) animals (Figs. 4 and 5) the hepatic contents of GSH after the different pretreatments were investigated. Table 6 demonstrates a highly significant difference between the GSH-concentrations of nondepleted (XIII) and depleted (XIV) rats. However, after a 5-hr duration of
DECREASED
BILE FLOW
AFTER
415
BSP
urethane narcosis (XV) there was also a marked, but lower, decrease in the hepatic GSH-concentration. This indicates that the fall in GSH-concentration during the depletion was mainly due to the urethane-induced narcosis and to a lesser extent to the duration of the cannulation procedure. Replenishment of taurocholate during bile salt depletion (XVI) did little to preserve the hepatic GSH concentration. TABLE HEPATIC
GSH
CONCENTRATION
AFTER DIFFERENT
Pretreatment
N
Nondepleted (X111) Depleted (XIV) 5 hr urethane (XV) Taurocholate during depletion (XVI) Glucose + NaCl during depletion (XVII) ’ Values are expressed as means b D < 0.001 (XIII vs XIV).
6
6 6 6 6 3
PRETREATMENTS
IN RATS’
Body weight cd
GSH/g liver hd
340.83 337.50 375.00 340.00 366.66
+ 19.60
2.10b
+ + rt +
I .37’ If: 0.14 1.60 + 0.05 1.6od’rO.17 1.32 t 0.03
13.32 58.99 42.66 24.66
+ 0.07
+ SD.
“p < 0.005 (XIV vs xv): d p < 0.005 (XVI
vs XVII).
DISCUSSION
The marked diminution in bile flow during BSP infusion was accompanied by decreasing biliary dye concentration-especially when the hepatic GSH-content was lowered-and by increasing dye content in the liver. In other words, the hepatic uptake of BSP exceeded its biliary excretion. Since simultaneously with the depression in bile flow during BSP-infusion, an impaired biliary excretion of BSP-GSH was observed, and, on the other hand, there was a marked choleresis during the infusion of BSP-GSH, one could hypothesize that the reduced bile flow during BSP-infusion is due to the presence of unconjugated dye in the liver. The hypothesis is supported by the facts that in the overall transport of BSP from blood to bile the biliary excretion step is ratelimiting (Klaassen and Plaa, 1967) and that this excretion step is highly dependent on the preceding conjugation of BSP with glutathione (Combes, 1965; Whelan rt al., 1969, 1970; Priestly and Plaa, 1970a). However, it is questionable whether one can infer the hepatic concentrations of BSP-GSH simply from the amount in the bile. Whelan and Combes (1971) found that the biliary excretion of BSP-GSH decreased when the liver also contained unconjugated BSP; simultaneously the overall excretion of BSP and its metabolites was diminished. These authors concluded that the conjugation step is not rate-limiting during the infusion of the unconjugated dye but that the latter interferes with the transport of conjugated BSP from the hepatic cell to bile, thus reducing the overall excretion. Nonetheless the rate of conjugation plays an important role, because the results of Whelan and Combes suggest that a critical intrahepatic threshold concentration of unconjugated BSP exists which accounts for the impaired excretion of BSP-GSH and probably for the reduction in bile flow. At any rate, enhanced conjugation would delay
416 the attaining
SCHULZE
of the critical
AND
CZOK
BSP concentration
and thus improve
the excretion of
BSP-GSH. This would enhance the overall excretion of the dye and also prevent the reduction in bile flow. Although we have not investigated S-aryl transferase activity (Grover and Sims, 1964) it seems likely that the differences in biliary excretion of BSP-GSH are mainly due to the differing hepatic contents of GSH, hence suggesting an impaired conjugation of BSP as described by Priestly and Plaa (1970a) after iodomethane. These authors emphasized that interference with hepatic conjugation of BSP could significantly modify its biliary excretion. It is noteworthy that ethylurethane which was used as anesthetic in our studies is known to be conjugated with glutathione (Boyland and Nery, 1965; Boyland and Chasseaud, 1969; Mandel, 1971) and therefore will account for the observed GSH depletion. It is concluded from the present studies that BSP can markedly decrease bile flow whereas BSP-GSH does not. This observation, although concluded from different types of experiments, is similar to that reported by Priestly and Plaa (1970b). The fact that no diminution
in bile flow was brought
about during the infusion
of BSP-
GSH, suggests that for this dye, in contrast to BSP, the rate-limiting step in the overall transport is its uptake into the liver, not its biliary excretion. The extremely low hepatic concentration of the dye observed during the infusion of BSP-GSH and its high biliary concentration are in good agreement with this hypothesis. The comparison between bile salt-depleted and taurocholate substituted-animals indicates that the normal biliary bile salt concentration is of almost no importance in the biliary excretion of BSP. ACKNOWLEDGMENTS We wish to acknowledge the skilful technical assistance of Mrs Chr. Degner. We are grateful to Dr. H. Lang, Merck-Laboratories, Darmstadt/Germany, for kindly supplying BSP (Bromthalein @)and BSP-GSH. REFERENCES BAKER, K. J. AND BRADLEY, S. E. (1966).Binding of sulfobromophthalein(BSP) sodiumby
plasmaalbumin. Its role in hepatic extraction. 1. C’lin.Invest. 45281-287. BOYLAND, E. AND CHASSEAUD, L. F. (1969).The role of glutathione and glutathione-S-trans-
ferasein mercapturic acid biosynthesis.Advan. Enzymol. 32,173-219. BOYLAND, E. ANO NERY, R. (1965). The metabolismof urethane and related compounds.
Biochem.J. 94,198-208. COMBES, B. (1965).The importance of conjugation with glutathione for sulfobromophthalein
sodium(BSP) transfer from blood to bile. J. Clin. Invest. 44, 1214-1224. DHUMEAUX, D., ERLINGER, S., BENHAMOU, J. P. AND FAUVERT, R. (1970).Effect of rosebengal
on bilesecretionin the rabbit: inhibition of a bile salt-independentfraction. Gut 11,134140. GROVER, P. L. AND SIMS, P. (1964).Conjugation with glutathione.Distribution of glutathione
S-aryltransferasein vertebrate species.Biochem.J. 90,603-606. GRUNERT, R. R. AND PHILLIPS, P. H. (1951).A modification of the nitroprussidemethod of
analysisfor glutathione. Arch. Biochem.30, 217-225. HORAK, W., GRABNER, G. AND PAUMGARTNER, G. (1973).Inhibition of bile salt independent
bile formation by indocyaninegreen. Gastroenterology64, 1005-1012. IWATA, T. AND YAMASAKI, K. (1964).Enzymatic determinationand thin layer chromatography
of bile acidsin blood. J. Biochem.56,424431.
KLAASSEN, C. D. AND PLAA, G. L. (1967).Species variation in metabolism,storageandexcretion
of eulfobromophthalein.Amer. J. Physiol.213, 1322-132.6.
DECREASED
BILE FLOW
AFTER
417
BSP
KLAASEN, C. D. AND PLAA,G. L. (1969).Plasmadisappearance and biliary excretion of indocyanine greenin rats, rabbits and dogs.Toxicol. Appl. Pharmacol. 15,376384. KRARUP, N. ANDLARSEN,J. A. (1972).The effect of slight hypothermia on liver function as
measuredby the elimination rate of ethanol, the hepatic uptake and excretion of indocyanine greenand bile formation. Acta Physiol. &and. 84, 39-07. MANDEL,H. G. (1971).Pathways of drug biotransformation: Biochemicalconjugations.In: Fundamentals of Drug Metabolism and Drug Disposition (B. N. La Du, H. G. Mandel and E. L. Way, eds.),p. 149.Williams &Wilkins, Baltimore, Maryland. PRIESTLY, B. G. AND PLAA, G. L. (1969).Effects of benziodaroneon the metabolismand biliary excretion of sulfobromophthaleinand relateddyes.Proc. Sot. Exp. Biol. Med. 132,881-885. PRIESTLY, B. G. AND PLAA, G. L. (197Oa).Sulfobromophthaleinmetabolismand excretion in rats with iodomethane-induceddepletion of hepatic glutathione. J. Pharmacol. Exp. Ther. 174,221-231.
B. G. AND PLAA, G. L. (1970b).Reducedbile flow after sulfobromophthaleinadministration in the rat. Proc. Sot. Exp. Biol. Med. 135,373-376. ROBERTS, R. J., KLAASSEN, C. D. AND PLAA, G. L. (1967).Maximum biliary excretion of bilirubin and sulfobromophthaleinduring anesthesia-induced alteration of rectal temperature. Proc. Sot. Exp. Biol. Med. 125, 3 13-316. SARDINI, D., BARBI, G., BASTON, F. AND MARZO, A. (1969).Urinary metabolites of bromosulfophthaleinin normal rat. Experientia 25, 1250. SCHENKER, S., GOLDSTEIN, J. AND COMBES, B. (1965). Sulfobromophthalein sodium (BSP) conjugation and excretion in fetal guineapigs.Amer. J. Physiol. 208, 563-572. PRIESTLY,
VARGA,
F. AND FISCHER,
E. (1970).
Biliary
secretion
Acad. Sci. Hung. 38, 143-149. WHELAN, G. AND COMBES, B. (1971). Competition
of rose by
bengal
unconjugated
in the dog. and
Acfa Physiol.
conjugated
sulfo-
bromophthalein sodium (BSP) for transport into bile. Evidence for a single excretory system.J. Lab. Clin. Med. 78, 230-244. WHELAN, G., HOCH, J. AND COMBES, B. (1969). Biliary excretion of conjugated sulfobromophthalein sodium (BSP) in rats fed a protein-free diet. Proc. Sot. Exp. Biol. Med. 132, 704711. WHELAN, G., HOCH, J. AND COMBES, B. (1970). A direct assessment of the importance of conjugation for biliary transport of sulfobromophthaleinsodium.J. Lab. C&z. Med. 75. 524-537.
AND
APPLIED
Studies
PHARhIACOLQGY
on the
(1974)
28,406-417
Decrease in Bile Sulfobromophthalein
Flow
Produced
by
PETER-J&GENSCHULZEAND GEORGCZOK Pharmakologisches Institut der Universitcit Hamburg, Hamburg, Germany Received August 31,1973; accepted January 3,1974
Studieson the Decreasein Bile Plow Producedby Sulfobromophthalein. SCHULZE, P.-J. AND CZOK, G. (1974).Toxicol. Appl. Pharmacol.28,406417.
A reduction in bile flow wasobservedduring BSP-infusion(1.875mg/min) in urethane-anesthetizedWistar rats. The infusion times at which 50% diminution in bile flow occurred were almost equal in bile salt-depleted (biliary fistula during 5 hr) animalsand in those given taurocholate (150 nmol/lOOg/min) during the depletion; however, the infusiontimesdiffered significantly from thoseobservedin non-depletedrats. While the overall excretion of the dye was mainly dependenton the
BSP-GSH fraction in the bile, the reduction in bile flow was inversely related to the amount of BSP-GSH conjugate excreted and the hepatic GSH concentrations before the infusion. A marked fall in hepatic GSH wasobservedafter the long-lastingurethanenarcosisthat occurredduring bile salt depletion. Sincethe infusion of equimolar and higher amountsof
BSP-GSH resulted in choleresis, it is concluded that the unconjugated BSP is responsiblefor the reducedbile flow during BSPinfusion. It is suggestedthat for BSP-GSH, in contrast to BSP, the rate-limiting step in the transport from blood to bile is not its biliary excretion, but its uptake into the liver.
The biliary excretion of a foreign compound usually involves three steps : uptake into the liver cell and storage; biotransformation; and excretion into the bile. The biotransformation step usually increases the water solubility and the molecular weight of the compound and thereby facilitates its biliary excretion. The excreted substance may either increase or decrease bile flow. There are compounds which decrease bile flow after low doses, e.g., indocyanine green (Klaassen and Plaa, 1969; Horak et al., 1973) and rose bengal (Dhumeaux et al., 1970; Varga and Fischer, 1970), and there are others which depress bile flow only after relatively high doses. The findings of Priestly and Plaa (1970a) suggest that sulfobromophthalein (BSP), a substance whose biliary excretion is highly dependent on its conjugation with glutathione (GSH) (Schenker et al., 1965; Combes, 1965; Klaassen and Plaa, 1967; Priestly and Plaa, 1969; Whelan et al., 1969, 1970) belongs to the latter group. This was confirmed by our own preliminary BSP-infusion experiments in rats.
The present study was initiated to get more information about the mechanism of the decrease in bile flow during BSP-infusion and especially to see how far this decrease can be correlated with the bile salt concentration at the start of the infusion and with the extent of biliary appearing BSP-GSH conjugates. Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
406
DECREASED
BILE FLOW
AFTER
BSP
407
METHODS Male Wistar rats (290420 g) were used throughout. The animals were maintained on Altromin@ standard diet and tap water ad libitum. They were anesthetized with ethylurethane, 1 g/kg ip. Surgicalprocedure. In general the bile duct and the left jugular vein were cannulated with PVC Pl and P2 tubing, respectively. To prevent hypothermic alteration of the bile flow (Roberts et ai., 1967; Krarup and Larsen, 1972) the body temperature of the animals was maintained at 37°C by means of a thermostat. Analytical methods. The bile flow was determined gravimetrically. The specific gravity of the bile was presumed to be 1. The bile acids were determined enzymatically (Iwata and Yamasaki, 1964) using hydroxysteroid dehydrogenase (Worthington Biochemical Corp., Freehold, New York). The BSP concentrations in the bile and in the serum were measured calorimetrically using a Photometer “Eppendorf” at 578 nm after an appropriate dilution of the samples with alkalinized 0.9 % sodium chloride solution (100 ml of 0.9 % NaCl plus 5 ml of 10 % NaOH). BSP and its metabolites in the bile were separated by thin-layer chromatography (10 ~1 bile on Merck@ precoated TLC-plates Silical/Gel 60 F-254, layer thickness 0.25 mm; solvent: tertiary butylic alcohol-distilled water 3 : 1; Sardini er al., 1969) and after eluting in alkaline-sodium chloride measured at 578 nm. The extinction coefficients of BSP and its metabolites were assumed to be identical (Combes, 1965; Whelan et al., 1970). Therefore, the extinction values of the metabolites were treated as if they originated from BSP which had been used for the calibration curve. According to Whelan et al. (1970), besides the unaltered substance, 2 metabolic fractions were estimated : BSP-glutathione, including traces of diglutathione, and BSP-cysteinylglycine plus BSP-cysteine. The BSP-content of the liver was estimated by the technique of Whelan et al. ( 1970) which gave recoveries of more than 95 % when either BSP or BSP-GSH was added to a liver homogenate. The GSH-content of the liver was determined by the method described by Grunert and Phillips (1951). BSP-induceddiminution in bileflow. The diminution of the bile flow was effected by a continuous infusion of BSP (Bromthalein @, Merck/Darmstadt, 5 ‘A aqueous solution; the infusion rate was 1.875 mg/min usingperfusionpumps (UnitaI @,Braun/Melsungen). The bile flow was determined at intervals of 10 min until it fell below 50% of the initial volume. The time required for a 50% decrease was estimated by interpolation. The bile volumes used as a reference were those measured during the last 10 min before starting the BSP-infusion. Rats termed nondepleted were infused with BSP 30 min after cannulating the bile duct, whereas the depleted animals were infused 5 hr after cannutation. In the latter, the loss of body fluid due to the biliary fistula was replaced by iv Ringer solution given each hour. In a further group the bile salts lost during the depletion were replaced by a continuous infusion of sodium taurocholate (150 nmol/lOO g/min, dissolved in equal vol-
408
SCHULZE
AND
CZOK
umes of 0.9 % sodium chloride and 10 % glucose solution, infusion rate 1 ml/hr) before the BSP infusion was started. Other nondepleted and depleted animals were infused with BSP-GSH. The various groups of animals employed are given in Table 1. TABLE 1 THE VARIOUS GROUPS OF ANMAM EMPLOYED Group
IN THE PRESENT EXPERIMENTS
Treatment
I Nondepleted rats, BSP infusion up to 50% diminution of bile flow II Depleted rats, BSP infusion up to 50% diminution of bile flow III Taurocholate substitution during depletion, BSP infusion up to 50% diminution of bile flow IV Nondepleted rats, 30-min BSP infusion V Depleted rats, 30-min BSP infusion VI Nondepleted and depleted rats, 2-hr BSP-GSH infusion VII Nondepleted rats, 30-min BSP-GSH infusion VIII Depleted rats, 30-min BSP-GSH infusion IX Nondepleted rats, bile duct ligation, 30-min BSP infusion x Depleted rats, bile duct ligation, 30-min BSP infusion XI Nondepleted rats, bile duct ligation, 30-min BSP-GSH infusion XII Depleted rats, bile duct ligation, 30-mm BSP-GSH infusion XIII Nondepleted rats, estimation of hepatic GSH content Depleted rats, estimation of hepatic GSH content XIV xv No biliary fistula, urethane narcosis for 5 hr, estimation of hepatic GSH content XVI Taurocholate substitution during the depletion, estimation of hepatic GSH content XVII Infusion of glucose and sodium chloride during depletion, estimation of hepatic GSH content Statistical analysis. The results are expressed as means f SD. The statistical significance of the differences between means of various treatments was determined by Student’s t-test for independent means. RESULTS Reduced Bile Flow under BSP Infusion Figure 1 illustrates that the initial bile volumes and the course of bile flow during the BSP infusion were dependent on the pretreatments of groups I, II and III. During the infusion of BSP the bile flow of the nondepleted group I clearly exceeded the initial bile volume, while in groups II and III no comparable increase was observed. Table 2 shows the interpolated times for the 50 % decrease in bile flow and the quantity of BSP infused to achieve this reduction. The initial bile volumes and the simultaneous concentrations and excreted amounts of bile salts before starting the dye infusion are given. Comparing groups I and II it is obvious that the reduction of bile flow corresponds neither to the initial bile volume nor to the initial bile salt concentration.
DECREASED
BILE FLOW
AFTER
M
I
non-depleted
I
70
20
30
40
50
409
BSP
!
I
60
70
(II
,
80
90 Inin
t
in
FIG. 1. Bile flow (&lo0 g/l0 min) during BSP infusion (1.875 mg/min) after different pretreatments rats. f Start of the infusion. Each point represents mean f SD of at least 6 animals.
From Fig. 2 the BSP-excretion of groups I, II and expressed as follows: I > III > II. The corresponding illustrated in Fig. 3. The maximal dye concentrations starting the infusion; thereafter the BSP concentrations
III during the infusion can be biliary BSP concentrations are were reached 20-30 mjn after of groups II and III decreased
wg fJSP !C3g-r10m~n-1 800
-
700
-
600
-
500
-
400
-
300
-
200
-
100
-
t
10
20
30
40
50
60
70
80
90
min
FIG. 2. Biliary excretion of BSP (~g/lOO g/l0 min) during BSPinfusion (I ,875 mg/min) after different ?retreatments in rats. t Start of the infusion. Each point represents mean _+SD of at least 6 animals.
36.81 _+ 5.19 56.10 f 5.74
394.3 + 40.1
378.6 + 41.5
7 Depleted(II) Depletedf taurocholate uw 7
NS
p II/III
LIBile volume, biliary bile salt concentration, b NS, not significant.
NS
P I/III
co.001
co.02
1821.59 f 258.21
32.68 k 4.97
NS
45.14 f 11.06
37.30 rk 3.36
80.10
565.52 +
25.70 _+ 2.40 15.80 + 3.60
50% diminution of bile volume (min) 81.56 + 10.50
Bile salts (nm01/100g/ 10 min) 1114.70 rt: 155.21
Bile salt concentration (=01//4
-
co.05
22.57 f 4.85
17.97 -(- 2.84
39.91 + 3.53
Infused BSP (mg/l~ g)
and biliary bile salt excretion were determined just before the dye infusion. Values expressed as mean + SD.
co.05
NSb
P WI
43.26 f 3.56
383.3 & 39.6
6
Bile volume W/l00 d 10 min)
N
------
Body weight (Ed
Nondepleted(I)
2
BSP-INDUCED DIMINUTIONOFBILE FLOWAFTER DIFFERENT PRETREATMENTS IN RATS’
TABLE
DECREASED
10
t
20
BILE FLOW
30
40
AFTER
50
60
411
BSP
70
80
90
min.
FIG. 3. Biliary BSP concentration @g/pi) during BSP infusion (1.875 mg/min) after different pretreatments in rats. t Start of the infusion. Each point represents mean f SD of at least 6 animals.
continuously, whereas the BSP-concentration of group I fell very slowly. The maximal dye concentrations of groups I and III were not significantly different from each other, but both were significantly higher than the maximal value of group II (p < 0.01). Figure 4 shows the average percentages of BSP and its metabolites in the bile. The fraction of the unaltered substance (BSP) was about 30-35 % after 10 min, reaching a minimum within 20-30 min and then increasing again. The percentage of the unchanged dye was lower throughout in group I. By contrast, BSP-GSH was significantly higher
80 70
non-dcp(etedf0
-
depleted
(8)
-
deplrf.+subsf.
(III)
60 T
50 40
BP-CG
+ BSP-C
ESP-GSH
i
30 20 10
t
1 10
20
30
LO
50
t
10
20
30
40
50
t
10
20
30
40
54 t??,”
FIG. 4. Biliary excretion BSP and its main metabolites (BSP-glutathione and BSP-cysteinylglycine + BSP-cysteine) expressed as percentage of the totally excreted dye. t Start of the BSP infusion (I.875 mg/min) after different pretreatments. Each point represents mean f SD of at least 6 animals.
412
SCHULZE
AND
CZOK
in group I than in the other groups. Compared with BSP, the concentration curves for BSP-GSH run inversely, reaching their maxima after 20 and 30 min, respectively. The percentages of BSP-cysteinylglycine (BSP-CG) and BSP-cysteine (BSP-C) together were highest in group II and lower in groups I and III. In all groups the percentages of these fractions increased with the infusion time. Figure 5 illustrates the excreted amounts of BSP and its metabolites; the curves for BSP-GSH are in agreement with those for the totally excreted dye in the bile (Fig. 2). 4g
7OOg-7
IOmin-7
800
0-Q
non-depleled(iJ
BP-CG
t
70
20
30
40
50
t
70
20
+ BSP-C
30
40
50
t
10
20
30
40
50 min.
FIG. 5. BiliaryexcretionofBSP(flg/lOOg/lOmin)and its mainmetabolites(BSP-glutathioneandBSPcysteinylglycine + BSP-cysteine) after different pretreatments. t Start of the BSP infusion (1.875 mg/ min). Each point represents the mean of at least 6 animals. Vertical bars in BSP-GSH panel denote SD. There were significant differences in the biliary excretion of BSP-GSH between I and 11 (in the intervals of 20,30,40 and 50 min), between I and III (30,40 and 50 min) and between 11 and III (10,20,40 and 50 min).
BSP Infusion for 30 min Since after 30 min of BSP infusion there were already distinct differences in bile flow (Fig. l), this interval was chosen to determine not only the amount of dye in the bile but also in the liver and in the serum. Table 3 summarizes the results obtained. Comparison between nondepleted (IV) and depleted animals (V) demonstrates that the lower biliary excretion of BSP in group V was accompanied by a much higher dye content in the liver than in group IV. The serum concentrations of the dye did not differ significantly from one another. The average liver weight of group V was significantly lower than that of group IV because of the lower dry weight as w&l1 is a lower water content of the livers; however, the differences in dry weight expressed as percentages are greater (19 %) than those in wet weight (16 %). BSP-GSH Infusion for 2 Hr Because of the different metabolism of BSP in groups I, II, and III we were interested in studying the effect of the infusion of equimolar or higher amounts of BSP-GSH.
P
values
for BSP-GSH
* NS, not significant.
a The
Body weight (g)
BETWEEN
AND
Wet weight Dry weight (g) (8)
Liver
SALT-DEPLETED
Liver (mg)
NONDEPLETED
TABLE 4
4.56f 0.06 19.23 + 2.84 3.69+ 0.06 26.38 + 2.75 CO.05 -Co.005
Liver (w)
AND NONDEPLETED
3
AFTER
g liver G-w3
BSP
RATS
1.31 f.o.14 2.15 ko.33 CO.001
g liver 6-m)
BSP
BSP INFUSION”
INFUSIO@
17.15 + 2.13 11.20 &- 1.18
Max. biliary BSP concentration b.vdml)
BSP-GSH
bile/30 min bg)
30-MIN
8.98 + 2.31 4.13 +0.71 CO.001
.~ Max. biliary BSP bile 30/min concentration (mid (mdml)
RATS AFTER 3%MIN
(mg)
are calculated
in terms of BSP
equivalents.
Values
are
expressed
as means
+ SD.
6 332.7+ 48.9 12.55+ 1.02 3.78f 0.14 2.09 zk0.42 0.16 -f 0.03 16.91+ 2.67 22.98f 2.03 7 328.6Ifr 43.0 10.82+ 1.47 3.11 + 0.10 2.28 + 0.32 0.21 +0.03 16.81+ 2.85 22.57f 1.90 NSb 0.05 0.05 NS NS NS NS
N
COMPARISON
BILE
6 314.2+ 31.2 14.71 f 1.43 6 321.7 + 13.7 12.39 + 1.09 NSb <0.002
Dry weight (g)
Liver
BILE SALT-DEPLETED
Wet weight h.3
BETWEEN
aValuesareexpressed asmeans+ SD. bNS, not significant.
Non-depleted(VII) Depleted (VIII)
P
Nondepleted (IV) Depleted (V)
N
Body weight 69
COMPARISON
TABLE
32.40+ 8.58 29.14+ 7.92 NS
BSP/ serum Wx/1@Jml)
99.73 rtl 10.27 93.90 + 10.61 NS
BSP concentration in serum hx/lOO ml)
5 w
i
w
% ;;f
E
u E ii w Ef
Ki
414
SCHULZE AND CZOK
However, no diminution in bile flow occurred. In fact, a choleretic effect was observed, even when 3-fold molar doses were given either to nondepleted (N= 2) or depleted (N= 1) animals for 2 hr. Therefore, no further efforts were made to reduce bile flow with BSP-GSH. BSP-GSH Infusion for 30 Min
In these experiments, instead of BSP, equimolar quantities of BSP-GSH were infused. As seen in Table 4, apart from the differences in liver weights, there were no significant differences between nondepleted (VII) and depleted (VIII) rats. BSP and BSP-GSH Infusionfor 30 Min in Nondepletedand Depleted Animals after Bile Duct Ligation
To get more information about the hepatic uptake was eliminated by ligating the bile duct before the hepatic uptake of each dye was independent of the uptake per gram liver was higher in group X than in
of the dyes, their biliary excretion infusions. Table 5 shows that the pretreatment. However, the BSPgroup IX (p < 0.001).
TABLE 5 DISTRIBUTION
OF BSP-GSH IN NONDEPLETED AND BILE SALT-DEPLETED RATS WHOSE DUCTS WERE LIGATED 10 MIN BEFORE THE INFUSION WAS STARTED’
Nondepleted
Body weight (g) Liver weight (g) BSP/liver (mg) BSP/g liver (mg) BSP/serum bxidl~ ml) N
Depleted
BSP-GSHb (XI)
BSP 00
415.00 + 42.62 15.27 & 1.71 18.54 & 2.66 1.21” + 0.12 94.22 &- 5.12
423.75 + 28.37 13.67 _+1.99 5.30 f 0.77 0.39 + 0.06 59.50+ 4.33
414.16 + 14.97 12.21 + 1.25 18.38 zk 1.70
7
8
6
BSP cm
BILE
1.51C* 0.12 92.63 If: 4.92
BSP-GSHb WI) 410.84 12.64 4.94 0.39 60.20
k 39.54 &- 1.17 + 0.71 lk 0.03 -t 3.29 6
OBoth dyes were infused in equimolar quantities during 30 min. Values are expressed as means f SD. * The values for BSP-GSH (mg) are calculated as BSP equivalents. =p < 0.001 (IX vs X).
The lower molar concentrations of BSP-GSH in the serum can be explained by its larger volume of distribution in the body due to its higher water solubility and its lower binding to plasma proteins (Baker and Bradley, 1966). Hepatic Content of GSH after D$erent Pretreatments
Because of the different metabolic capacities of nondepleted-( depleted-(II), and taurocholate-substituted-(III) animals (Figs. 4 and 5) the hepatic contents of GSH after the different pretreatments were investigated. Table 6 demonstrates a highly significant difference between the GSH-concentrations of nondepleted (XIII) and depleted (XIV) rats. However, after a 5-hr duration of
DECREASED
BILE FLOW
AFTER
415
BSP
urethane narcosis (XV) there was also a marked, but lower, decrease in the hepatic GSH-concentration. This indicates that the fall in GSH-concentration during the depletion was mainly due to the urethane-induced narcosis and to a lesser extent to the duration of the cannulation procedure. Replenishment of taurocholate during bile salt depletion (XVI) did little to preserve the hepatic GSH concentration. TABLE HEPATIC
GSH
CONCENTRATION
AFTER DIFFERENT
Pretreatment
N
Nondepleted (X111) Depleted (XIV) 5 hr urethane (XV) Taurocholate during depletion (XVI) Glucose + NaCl during depletion (XVII) ’ Values are expressed as means b D < 0.001 (XIII vs XIV).
6
6 6 6 6 3
PRETREATMENTS
IN RATS’
Body weight cd
GSH/g liver hd
340.83 337.50 375.00 340.00 366.66
+ 19.60
2.10b
+ + rt +
I .37’ If: 0.14 1.60 + 0.05 1.6od’rO.17 1.32 t 0.03
13.32 58.99 42.66 24.66
+ 0.07
+ SD.
“p < 0.005 (XIV vs xv): d p < 0.005 (XVI
vs XVII).
DISCUSSION
The marked diminution in bile flow during BSP infusion was accompanied by decreasing biliary dye concentration-especially when the hepatic GSH-content was lowered-and by increasing dye content in the liver. In other words, the hepatic uptake of BSP exceeded its biliary excretion. Since simultaneously with the depression in bile flow during BSP-infusion, an impaired biliary excretion of BSP-GSH was observed, and, on the other hand, there was a marked choleresis during the infusion of BSP-GSH, one could hypothesize that the reduced bile flow during BSP-infusion is due to the presence of unconjugated dye in the liver. The hypothesis is supported by the facts that in the overall transport of BSP from blood to bile the biliary excretion step is ratelimiting (Klaassen and Plaa, 1967) and that this excretion step is highly dependent on the preceding conjugation of BSP with glutathione (Combes, 1965; Whelan rt al., 1969, 1970; Priestly and Plaa, 1970a). However, it is questionable whether one can infer the hepatic concentrations of BSP-GSH simply from the amount in the bile. Whelan and Combes (1971) found that the biliary excretion of BSP-GSH decreased when the liver also contained unconjugated BSP; simultaneously the overall excretion of BSP and its metabolites was diminished. These authors concluded that the conjugation step is not rate-limiting during the infusion of the unconjugated dye but that the latter interferes with the transport of conjugated BSP from the hepatic cell to bile, thus reducing the overall excretion. Nonetheless the rate of conjugation plays an important role, because the results of Whelan and Combes suggest that a critical intrahepatic threshold concentration of unconjugated BSP exists which accounts for the impaired excretion of BSP-GSH and probably for the reduction in bile flow. At any rate, enhanced conjugation would delay
416 the attaining
SCHULZE
of the critical
AND
CZOK
BSP concentration
and thus improve
the excretion of
BSP-GSH. This would enhance the overall excretion of the dye and also prevent the reduction in bile flow. Although we have not investigated S-aryl transferase activity (Grover and Sims, 1964) it seems likely that the differences in biliary excretion of BSP-GSH are mainly due to the differing hepatic contents of GSH, hence suggesting an impaired conjugation of BSP as described by Priestly and Plaa (1970a) after iodomethane. These authors emphasized that interference with hepatic conjugation of BSP could significantly modify its biliary excretion. It is noteworthy that ethylurethane which was used as anesthetic in our studies is known to be conjugated with glutathione (Boyland and Nery, 1965; Boyland and Chasseaud, 1969; Mandel, 1971) and therefore will account for the observed GSH depletion. It is concluded from the present studies that BSP can markedly decrease bile flow whereas BSP-GSH does not. This observation, although concluded from different types of experiments, is similar to that reported by Priestly and Plaa (1970b). The fact that no diminution
in bile flow was brought
about during the infusion
of BSP-
GSH, suggests that for this dye, in contrast to BSP, the rate-limiting step in the overall transport is its uptake into the liver, not its biliary excretion. The extremely low hepatic concentration of the dye observed during the infusion of BSP-GSH and its high biliary concentration are in good agreement with this hypothesis. The comparison between bile salt-depleted and taurocholate substituted-animals indicates that the normal biliary bile salt concentration is of almost no importance in the biliary excretion of BSP. ACKNOWLEDGMENTS We wish to acknowledge the skilful technical assistance of Mrs Chr. Degner. We are grateful to Dr. H. Lang, Merck-Laboratories, Darmstadt/Germany, for kindly supplying BSP (Bromthalein @)and BSP-GSH. REFERENCES BAKER, K. J. AND BRADLEY, S. E. (1966).Binding of sulfobromophthalein(BSP) sodiumby
plasmaalbumin. Its role in hepatic extraction. 1. C’lin.Invest. 45281-287. BOYLAND, E. AND CHASSEAUD, L. F. (1969).The role of glutathione and glutathione-S-trans-
ferasein mercapturic acid biosynthesis.Advan. Enzymol. 32,173-219. BOYLAND, E. ANO NERY, R. (1965). The metabolismof urethane and related compounds.
Biochem.J. 94,198-208. COMBES, B. (1965).The importance of conjugation with glutathione for sulfobromophthalein
sodium(BSP) transfer from blood to bile. J. Clin. Invest. 44, 1214-1224. DHUMEAUX, D., ERLINGER, S., BENHAMOU, J. P. AND FAUVERT, R. (1970).Effect of rosebengal
on bilesecretionin the rabbit: inhibition of a bile salt-independentfraction. Gut 11,134140. GROVER, P. L. AND SIMS, P. (1964).Conjugation with glutathione.Distribution of glutathione
S-aryltransferasein vertebrate species.Biochem.J. 90,603-606. GRUNERT, R. R. AND PHILLIPS, P. H. (1951).A modification of the nitroprussidemethod of
analysisfor glutathione. Arch. Biochem.30, 217-225. HORAK, W., GRABNER, G. AND PAUMGARTNER, G. (1973).Inhibition of bile salt independent
bile formation by indocyaninegreen. Gastroenterology64, 1005-1012. IWATA, T. AND YAMASAKI, K. (1964).Enzymatic determinationand thin layer chromatography
of bile acidsin blood. J. Biochem.56,424431.
KLAASSEN, C. D. AND PLAA, G. L. (1967).Species variation in metabolism,storageandexcretion
of eulfobromophthalein.Amer. J. Physiol.213, 1322-132.6.
DECREASED
BILE FLOW
AFTER
417
BSP
KLAASEN, C. D. AND PLAA,G. L. (1969).Plasmadisappearance and biliary excretion of indocyanine greenin rats, rabbits and dogs.Toxicol. Appl. Pharmacol. 15,376384. KRARUP, N. ANDLARSEN,J. A. (1972).The effect of slight hypothermia on liver function as
measuredby the elimination rate of ethanol, the hepatic uptake and excretion of indocyanine greenand bile formation. Acta Physiol. &and. 84, 39-07. MANDEL,H. G. (1971).Pathways of drug biotransformation: Biochemicalconjugations.In: Fundamentals of Drug Metabolism and Drug Disposition (B. N. La Du, H. G. Mandel and E. L. Way, eds.),p. 149.Williams &Wilkins, Baltimore, Maryland. PRIESTLY, B. G. AND PLAA, G. L. (1969).Effects of benziodaroneon the metabolismand biliary excretion of sulfobromophthaleinand relateddyes.Proc. Sot. Exp. Biol. Med. 132,881-885. PRIESTLY, B. G. AND PLAA, G. L. (197Oa).Sulfobromophthaleinmetabolismand excretion in rats with iodomethane-induceddepletion of hepatic glutathione. J. Pharmacol. Exp. Ther. 174,221-231.
B. G. AND PLAA, G. L. (1970b).Reducedbile flow after sulfobromophthaleinadministration in the rat. Proc. Sot. Exp. Biol. Med. 135,373-376. ROBERTS, R. J., KLAASSEN, C. D. AND PLAA, G. L. (1967).Maximum biliary excretion of bilirubin and sulfobromophthaleinduring anesthesia-induced alteration of rectal temperature. Proc. Sot. Exp. Biol. Med. 125, 3 13-316. SARDINI, D., BARBI, G., BASTON, F. AND MARZO, A. (1969).Urinary metabolites of bromosulfophthaleinin normal rat. Experientia 25, 1250. SCHENKER, S., GOLDSTEIN, J. AND COMBES, B. (1965). Sulfobromophthalein sodium (BSP) conjugation and excretion in fetal guineapigs.Amer. J. Physiol. 208, 563-572. PRIESTLY,
VARGA,
F. AND FISCHER,
E. (1970).
Biliary
secretion
Acad. Sci. Hung. 38, 143-149. WHELAN, G. AND COMBES, B. (1971). Competition
of rose by
bengal
unconjugated
in the dog. and
Acfa Physiol.
conjugated
sulfo-
bromophthalein sodium (BSP) for transport into bile. Evidence for a single excretory system.J. Lab. Clin. Med. 78, 230-244. WHELAN, G., HOCH, J. AND COMBES, B. (1969). Biliary excretion of conjugated sulfobromophthalein sodium (BSP) in rats fed a protein-free diet. Proc. Sot. Exp. Biol. Med. 132, 704711. WHELAN, G., HOCH, J. AND COMBES, B. (1970). A direct assessment of the importance of conjugation for biliary transport of sulfobromophthaleinsodium.J. Lab. C&z. Med. 75. 524-537.