Br.J.Pharmac. (1984),83, 773-782
`
`Pharmacokinetics and biliary excretion of
`bromosulphophthalein, PH]-ouabain and PH](cid:173)
`taurocholic acid in rats with glycerol-induced acute
`renal failure
`C.J. Bowmer&M.S. Yates
`
`Department of Pharmacology, Won;ely Medical and Dental Building, The University, Leeds, LS2 9IT
`
`1 The pharmacokinetics and biliary excretion of bromosulphophthalein (BSP), ouabain and
`taurocholic acid (TChA) have been studied in rats with glycerol-induced acute renal failure (ARF).
`2
`In rats with ARF, the hepatic uptake and initial biliary excretion of BSP were decreased. In
`addition, the rate of BSP conjugation with glutathione by rat liver homogenates was also decreased.
`This latter change may contribute to the initial decrease in the biliary excretion of BSP.
`3 No change was found in the hepatic uptake and biliary excretion of ouabain, but the area under
`the concentration-time curve was increased and the plasma clearance (Cip) decreased in rats with
`ARF. This decrease in Clp was not due to reduced renal excretion.
`4 The decreased Clp of ouabain in rats with ARF may come from reduced tissue binding and a
`concomitant decrease in its volume of-distribution (Vd).
`S The hepatic handling of TChA appeared unaltered in ARF, but the rate constant for the terminal
`part of the concentration-time curve (II) was decreased. This change probably resulted from a large
`increase in V din rats with ARF.
`6
`It is concluded that the decreased uptake of BSP was not due to a non-specific disturbance of
`hepatocyte function in ARF because the hepatic handling of ouabain and TChA were unaltered.
`
`Introduction
`
`The hepatic transport of endogenous and exogenous
`substances probably involves a multiplicity of routes
`both at the initial uptake and final biliary excretion
`steps. Evidence for this comes largely from studies of
`competition for transport between pain; of sub(cid:173)
`stances. These studies have revealed a number of
`different routes for organic anions (Alpert et al.,
`1969; Scharschmidt et al., 1975; Schwenk et al.,
`1976), cations (Solomon & Schanker, 1963) and
`uncharged molecules (Kupferberg & Schanker,
`1968; Klassen, 1978).
`Experimental studies have established that the
`hepatic uptake of indocyanine green (ICG), an or(cid:173)
`ganic anion used to study liver function, is decreased
`in rats with acute and chronic renal failure (Bowmer
`et al., 1982a; Yates et al., 1983a,b,c). Moreover, the
`initial biliary excretion of ICG is decreased, resulting
`in a delay in the excretion of ICG into bile (Bowmer
`et al., 1983a). However, little is known about
`
`whether these changes in hepatic function are re(cid:173)
`stricted to ICG, or if uptake and biliary excretion of
`other substances are similarly affected in renal
`failure.
`The purpose of this study was t" investigate the
`effect of glycerol-induced acute renal h•;lure (ARF)
`on the hepatic uptake and biliary exo:retion of
`bromosuphophthalein
`(BSP),
`ouabmn
`and
`taurocholic acid (TChA). BSP and ICG appear to
`have a common transport route (Schan;chmidt et al.,
`1975; Schwenk et al., 1976); but ouabain and TChA
`have transport routes separate from each other (Mei(cid:173)
`jer et al., 1976; Klassen, 1978) and from BSP
`(Schwenk et al., 1976). Ouabain and TChA were also
`chosen because they are not biotransformed in the rat
`(Cox et al., 1959; Hoffman et al., 1975). A prelimi(cid:173)
`nary account of some of this work has been given
`(Bowmer et al., 1982b; 1983b ).
`
`©The Macmillan Press Ltd 1984
`
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`
`774
`
`C.J. BOWMER & M.S. YATES
`
`Methods
`
`Induction of acute renal failure
`
`The method for the production of glycerol-induced
`ARF has been described
`in detail elsewhere
`{Bowmer et a/., 1982a). Male Wistar albino rats
`(250-350 g) were denied access to water for 24 hand
`ARF was produced by intramuscular injection of
`50% v/v glycerol in sterile saline (0.9%w/v NaCI
`solution), 10 ml kg- 1• Control rats were injected with
`saline, lOmlkg- 1• Both groups of rats were studied
`48 h after their respective injections.
`
`Experimental protocol
`
`Rats were anaesthetized with pentobarbitone
`( 60 mg kg- 1 i.p. ): a tracheal cannula was inserted and
`artificial respiration maintained with a Miniature
`Ideal Pump (BioScience) (ventilation rate 80 strokes
`min- 1 ; stroke volume 10mlkg-1). Cannulae were
`also inserted into the left jugular vein, right carotid
`artery and common bile duct. Rectal temperature
`was maintained at 37"C by means of a heating lamp.
`All compounds were dissolved in saline and in(cid:173)
`jected i.v. over 15-20 s. The dose of BSP was
`25 mgkg- 1. Ouabain was mixed with ['H]-ouabain
`and administered at a dose of 0.1 mgkg- 1 ;
`15/lCikg-1. Similarly, TChAwas mixed with ['H](cid:173)
`TChA and given at5 mgkg-1 ; 10J.1Cikg-1• Heparin(cid:173)
`ized blood samples (0.1 ml) were removed at suitable
`times for 70 min with BSP and for 60 min with oua(cid:173)
`bain and TChA. After each sample was collected,
`blood was replaced with an equal volume of saline.
`Bile was collected over 5 or 10 min intervals for 1 h;
`over 20 min intervals for the second hour and over
`30 min intervals for the third hour. Bile volume was
`measured gravimetrically assuming a density of 1.0
`for rat bile.
`The urinary excretion of ['H]-ouabain was esti(cid:173)
`mated by collecting urine directly from the bladder of
`anaesthetized rats as described by Hirom et a/.
`{1976). In a separate series of experiments the kine(cid:173)
`tics of [3H]-ouabain were determined in the absence
`of any urinary excretion. These experiments were
`performed in rats whose renal pedicles (renal artery,
`vein and ureter) were ligated 10 to 15 min before
`administration of [3H]-ouabain.
`
`Hepatic uptake of fH]-ouabain in vivo
`
`Slices of liver (30-65 mg) were removed from the
`left, median and right lobes of anaesthetized rats at
`2.5, 5, 7.5, 10 and 20 min after injection of ['H](cid:173)
`ouabain (Meijer eta/., 1975). Each slice was blotted,
`weighed and solubilized in 0.7ml FisoSolve (Fisons
`Ltd). The cumulative amount of tissue removed as a
`
`percentage of total liver weight was 1.8 ± 0.2%
`(n= 6) for control rats and 1.8±0.4% (n= 6) for rats
`with ARF. Meijer et a/. (1975) found that [3H](cid:173)
`ouabain was taken up uniformly into slices taken at
`random from different liver lobes. Preliminary ex(cid:173)
`periments confirmed this, so it was assumed that
`uptake measured in one slice was representative of
`uptake into the entire liver.
`
`Analysis of bromosu/phophtha/ein in plasma and bile
`
`Samples (50 Ill) of plasma and bile were diluted with
`an appropriate volume of 0.1 M NaOH and the absor(cid:173)
`bance measured at 575 nm. Plasma samples were also
`read at 395 nm to correct for haemoglobin contami(cid:173)
`nation (E395 x 0.093 =haemoglobin contribution to
`extinction at 575 nm). The absorption spectrum of
`the glutathione conjugate of BSP in alkali is almost
`identical to that of BSP between 500 to 620 nm
`(Goldstein & Combes, 1966) so the total amount of
`BSP in bile was measured.
`
`Hepatic glutathione (GSH)
`
`Liver homogenates were prepared by the procedure
`of Akerboom & Sies (1981) and the fluorometric
`method of Cohn & Lyle {1966) was used to deter(cid:173)
`mine the the GSH content of rat liver.
`
`Hepatic glutathione-S-transferase activity
`
`The spectrophotometric assay of Goldstein &
`Combes {1966) was used to measure the rate of BSP
`conjugation with GSH by rat liver homogenates.
`
`Measurement of radioactivity
`
`Radioactivity in aliquots (50 Ill) of plasma and bile
`was measured in a Beckman LS 330 scintillation
`counter. Samples were counted in plastic insert vials
`(Sterilin Ltd) using 5 m1 of FisoFluor I liquid scintil(cid:173)
`lator {Fisons Ltd). Urine samples (20-200/ll) were
`counted in plastic scintillation vials (LlP Ltd) with
`15 ml of scintillator. Radioactivity in digested pieces
`of liver was measured after addition of 15 ml of
`scintillator followed by 0.5 ml5 M acetic acid. Count(cid:173)
`ing efficiency was assessed by automatic external
`standard channels ratio and, where appropriate, with
`internal standards of [3H]-n-hexadecane (Amersham
`International PLC).
`
`Pharmacokinetic calculations
`
`The plasma concentration-time data for BSP were
`fitted to a biexponential equation by non-linear least
`squares regression analysis (Snedecor & Cochran,
`1967). Data were analysed using a two compartment
`
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`
`PHARMACOKINETICS IN ACliTE RENAL FAILURE
`
`775
`
`model with elimination of BSP from the peripheral
`compartment (Richards et al., 1959). In this model
`k12 is the apparent first order rate constant for trans(cid:173)
`fer of dye from plasma to liver; k21 the rate constant
`for return of BSP to plasma and k23 the rate constant
`for excretion into bile. These rate constants together
`with the apparent volume of the central compartment
`(Vc); the area under the plasma concentration-time
`curve (AUCo...~) and plasma clearance (Clp) were
`calculated using the equations given by Gibaldi &
`Perrier (1975). The apparent volume of distribution
`at steady-state (Vdss) was calculated as:-
`
`Vdss=Vc
`
`( k12 + k21 + k23)
`k21 +k23
`
`(Bowmeretal.,1982a)
`
`The disappearance of ['H)-ouabain and ['H]-TChA
`from plasma was analysed using the 'CSTRIP' prog(cid:173)
`ramme (Sedman & Wagner, 1976). This indicated
`that their decay was at least triexponential and so
`these data were not subjected to compartmental
`analysis. Instead the kinetics of ['H)-ouabain and
`(3H]-TChA were described in terms of (1) the rate
`constant for the terminal part of the concentration(cid:173)
`time curve (jl); (2) the apparent volume of distribu(cid:173)
`tion (Vd) which was calculated from:-
`
`Dose
`
`Vd
`
`(Gibaldi & Perrier, 1975)
`
`and (3) the plasma clearance (Clp). The area under
`the plasma concentration-time curve from 0 to
`60 min was calculated using the trapezoidal rule and
`the area under the plasma concentration-time profile
`from the last observation (Cp60) to infinity was esti(cid:173)
`mated by:-
`
`Cp60
`AUCoo+~ = -ll-: (Benet & Galeazzi, 1979)
`
`The AU Co+~ was the sum of the two areas.
`Results are expressed as mean± s.d. and statistical
`comparison was made by the non-paired Student's t
`test.
`
`Materials
`
`Results
`
`Intramuscular injection of glycerol resulted in a
`uraemic state characterized by at least a four fold
`increase in plasma urea concentration. Mean body
`weight, wet liver weight and liver weight to body
`weight ratio were not significantly different between
`any group of control or uraemic rats used. These
`results are similar to those previously obtained in our
`laboratory (Bowmer et al., 1982a; Yates et al.,
`1983a,b).
`
`Bromosulphophthalein kinetics
`
`Figure 1 shows the mean plasma concentration-time
`data obtained after i.v. administration of BSP
`(25 mgkg-1) to control and uraemic rats. Plasma
`concentrations between 5 to 15 min were significant(cid:173)
`ly elevated (P< 0.05) in the rats with ARF which
`suggests that the initial disappearance of BSP was
`delayed in the uraemic rats. The half -life of this initial
`disappearance phase, T 0.5cx, was significantly pro(cid:173)
`longed (P<0.01) and the rate constants k 12 and k21
`were decreased (P< 0.05) in uraemic rats (Table 1).
`There was no significant change in the half-life of the
`terminal elimination phase, T 0.5jl, k23; Vdss or Clp,
`but Vc was significantly larger (P< 0.05) in the
`uraemic rats.
`
`500
`
`10
`
`Q
`
`E
`"' 2-
`" 0 " a. en
`"' "'
`"' "' a:
`
`E
`
`BSP and ouabain were obtained from Sigma Chemi(cid:173)
`cal Co. and TChA was bought from CP Laboratories
`Ltd
`(Bishop Stortford, U.K.). GSH and o(cid:173)
`phthalaldehyde were obtained from BDH Ltd. (G-
`(G-3H]-TChA
`3H]-ouabain
`(37 Ci/mmol) and
`( 6.6 Ci/mmol), all of stated radioactive purity
`>97%, were purchased from Amersham Interna(cid:173)
`tional PLC and New England Nuclear Ltd respec(cid:173)
`tively, and were used without further purification.
`
`1 o~----1~0~--2~0~--~30~--~4~0--~5~0--~6~0~~70
`
`Time (min)
`Figure 1 Plasma concentrations of bromosul(cid:173)
`phophtbalein (BSP, 25 mgkg- 1 i.v.) in control rats (0)
`and rats witb acute renal failure (e). Values are mean
`( n = 7); s.d. shown by vertical lines. Significantly differ(cid:173)
`ent
`from control vaues: *P<0.05; **P<O.Ol;
`•••P<O.OOl.
`
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`
`776
`
`C.J. BOWMER & M.S. YATES
`
`Table 1 Effect of glycerol-induced acute renal
`failure on the pharmacokinetics of bromosul(cid:173)
`phophthalein (BSP, 25 mg kg -I i. v.)
`
`1200
`
`T
`
`Pharmacokinetic
`parameters
`
`Control rats
`(n=7)
`
`Uraemic rats
`(n=7)
`
`To.s<l(min)
`To.s ~(min)
`k12 (min- 1)
`k21 (min- 1)
`k23 (min- 1)
`Vc(1 kg- 1)
`Vp(1 kg- 1)
`Vd,. (1 kg- 1)
`Clp(mlmin- 1 kg- 1
`bodywt)
`
`1.4±0.2
`27±10
`0.49±0.08
`0.0073 ± 0.0033
`0'.029 ± 0.009
`0.039 ± 0.003
`0.58±0.18
`0.61 ±0.18
`15±2
`
`2.4±0.7**
`53±34
`0.30±0.07***
`0.0043±0.0013*
`0.020±0.014
`0.066 ± 0.031*
`1.0±0.6
`1.1 ±0.6
`14±3
`
`Results are given as mean± s.d.
`*P<0.05; **P<O.Ol; ***P<0.001 relative to
`respective control group.
`k12 = rate constant for transfer from plasma to
`liver; k21 =rate constant for return of BSP to plas(cid:173)
`ma; k23 =rate constant for excretion into bile;
`Vc = apparent volume of the central compartment;
`Vp =apparent volume of the peripheral compar(cid:173)
`ment and V dss = apparent volume of distribution at
`steady-state; Clp =plasma clearance.
`
`In uraemic rats the percentage recovery of BSP
`from bile over 3 h (80 ± 9%; n = 6) and overall bile
`flow rate (3.9 ± 0.9mlh- 1 kg- 1 ; n = 6) were not sig(cid:173)
`nificantly different from control values (87 ± 6% and
`4.5±0.9mlh- 1 kg- 1; n=6 respectively). However,
`the biliary excretion rate during the first 10 min after
`injection
`of
`BSP
`in
`uraemic
`rats
`(175±115flgmin- 1 kg- 1 ; n=6) was significantly
`slower
`(P<0.001)
`than
`in
`controls
`(493±75flgmin- 1 kg- 1 ; n=6) (Figure 2). This ini(cid:173)
`tial delay in the biliary excretion of BSP was not
`caused by decreased bile flow rate at this particular
`rats with ARF
`time, because
`flow
`rate
`in
`(3.3 ± 1.8 ml h- 1 kg- 1; n = 6) was not significantly
`different from that in controls (4.2 ±0.5 mlh- 1 kg- 1;
`n = 6). At all other intervals there was no difference
`in biliary excretion rates between the two groups of
`rats (Figure 2).
`
`Bromosu/phophthalein conjugation
`
`The ability of livers from uraemic rats to conjugate
`BSP with exogenous GSH was significantly de(cid:173)
`creased (P< 0.01 ). In uraemic rats the in vitro
`glutathione-S-transferase
`activity
`was
`3.3±0.5 l'molg- 1 min- 1 (n= 7) whereas enzyme ac(cid:173)
`in controls was 3.9 ± 0.3 l'molg-1 min- 1
`tivity
`(n = 7). In a separate series of experiments the en(cid:173)
`dogenous GSH content of livers from the uraemic
`
`1000
`
`800
`
`600
`
`400
`
`200
`
`;-
`~
`c
`.E
`en
`2-
`~
`~
`c:
`0
`-~
`<..>
`
`X " ~
`
`.!!!
`iii
`
`1.0
`
`1.5
`
`2.0
`
`25
`
`30
`
`Time (hi
`Fipre 2 Biliary excretion profile of bromosul(cid:173)
`phophthalein (BSP, 25 mgkg- 1 i.v.) in control rats (un(cid:173)
`broken line - - ) and rats with acute renal failure
`(broken line----). Values are mean (n = 6); s.d. shown
`by vertical lines. Significantly different from control
`values: ••• P< 0.001.
`
`gtoup (3.2 ± 0.5 flmol g- 1 ; n = 8) tended to be smal(cid:173)
`ler than that in controls (3.7 ± 0.5 flmolg- 1 ; n = 8).
`However, this difference was not statistically sig(cid:173)
`nificant.
`
`Ouabain kinetics
`
`In rats with intact renal pedicles. The disappearance
`of [3H]-ouabain (0.1 mgkg- 1 i.v.) from plasma in
`both control and uraemic rats is shown in Figure 3. At
`all sample times, mean radioactivity was greater
`(P<0.05) in uraemic than in control plasma. As a
`result, the AU~® was larger (P<0.02) in the
`uraemic group and there was a concomitant decrease
`(P< 0.02) in Clp (Table 2). By contrast, no signific(cid:173)
`ant change in either fl or Vd was observed (Table 2).
`Figure 4 shows that between 5 and 20 min follow(cid:173)
`ing injection of [3H]-ouabain, a significantly gteater
`(P< 0.01) percentage of the injected dose was found
`in livers of uraemic rats. However, the percentage of
`[lH]-ouabain excreted into bile after 1 h was not
`significantly different between control (43±7%;
`n = 6) and uraemic ( 49 ± 7%; n = 6) rats. In addition,
`
`Boehringer Ex. 2027
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`
`

`
`200
`
`100
`
`X
`
`10
`
`E
`E
`"-
`~ u
`
`c
`0
`<.J
`c
`"iii
`.0
`"' :>
`"' ~
`"' 1i:
`
`0
`<.J
`
`PHARMACOKINETICS IN ACUTE RENAL FAILURE
`
`777
`
`l i I I
`?f. I
`I
`
`iii
`~
`
`,0>
`
`" " 0
`
`"C
`
`I
`
`r
`I
`
`1 o~----~10~--~20~---3~0~---4~0~---5~0~--~60
`
`Time (min)
`ouabain
`of
`concentrations
`3 Plasma
`Figure
`(0.1 mgkg- 1; 15,.Cikg-1) in control rats (0) and rats
`with acute renal failure (e). Values are mean (n= 6);
`s.d. shown by vertical lines. All concentrations in plasma
`from uraemic rats were significantly different from con(cid:173)
`trol values: P< 0.05.
`
`there was no difference in either biliary excretion
`rate, over any collection interval, or overall bile flow
`rate between the two groups of animals, so decreased
`or delayed biliary excretion cannot account for the
`greater portion of [3H]-ouabain in uraemic livers. In
`control rats
`the mean
`liver
`to plasma ratio
`(dpmg- 1/dpmml- 1) of ['H)-ouabain between 5 to
`20 min was 12 ± 2 ( n = 4) whilst in uraemic rats its
`value was 11 ± 2 (n = 4). It seems likely, therefore,
`that the increased levels of ouabain in uraemic livers
`were a result of correspondingly higher plasma levels.
`The percentage of the dose excreted into urine
`
`~~------~------~1L0------~1~5------~20"
`
`Time (min)
`
`Figure 4 Comparison of the hepatic content of ouabain
`(0.1mgkg-1; 15,.Cikg-1; i.v.) in control rats (0) and
`rats with acute renal failure (e). Values are mean
`(n = 6); s.d. shown by vertical lines. Significantly differ(cid:173)
`ent from control values: • P< 0.05; •• P< 0.01.
`
`over 1 h was very variable in both groups. In controls
`the median was 8.0% with a range from 2.4 to 13%
`and in the uraemic group the median was 1.6% and
`range from 0.01 to 5.2%. Using the Wilcoxon rank
`test, the renal excretion of [3H]-ouabain was found to
`be significantly less (P< 0.01) in uraemic rats.
`
`In rats with ligated renal pedicles. These experiments
`were done to investigate the possibility that de(cid:173)
`creased renal excretion of ouabain was the cause of
`
`Table 1 Effect of glycerol-induced acute renal failure on the pharmacokinetics of ['H)-ouabain (0.1 mgkg- 1;
`15 ,.a kg- 1) in non-ligated and renal pedicle-ligated rats
`
`Non-ligated
`Control (n = 6)
`Uraemic (n = 6)
`Ligated
`Control (n=4)
`Uraemic (n = 4)
`
`AU Co-.~
`(dpmminml- 1)
`X 10-6
`
`0'.89±0.23
`1.6±0.5""
`
`~
`(min- 1)
`
`0.020±0.007
`0.017 ± 0.006
`
`Clp
`(ml min- 1 kg-1)
`
`35±10
`21±6° 0
`
`Vd
`(1 kg- 1)
`
`1.8±0.6
`1.4±0.4
`
`1.5±0.2*
`5.2±2.1""*
`
`0.0071±0.0017*
`0.0035 ± 0.0023"*
`
`21 ±3t
`7.6±4.o•••*
`
`3.0±0.3*
`2.4±0.3"*
`
`Results are given as mean± s.d.
`• P< 0.05; •• P< 0.02; ••• P< 0.01 relative to respective control group.
`• P< 0.05; 'P< 0.01 relative to control and uraemic non-ligated rats.
`
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`

`
`778
`
`C.J. BOWMER & M.S. YATES
`
`200.
`
`100
`
`M
`
`' 0
`
`X
`:=-
`E
`E
`c.
`:!0'.
`u c
`0
`u
`c
`"iii
`.a
`"' ::l
`"' ~
`"' 0:
`
`0
`
`1
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`Time (min)
`
`ouabain
`of
`concentrations
`5 Plasma
`Figure
`(0.1 mgkg-1; 151'Cikg-1) in renal pedicle-ligated con(cid:173)
`trol rats (0) and renal pedicle-ligated rats with acute
`renal failure (e). Valuesaremean(n= 4);s.d.shownby
`vertical lines. With the exception of the 1 and 3 min
`samples, all concentrations in plasma from rats with
`acute renal failure were significantly different from con(cid:173)
`trol values: 0 P<0.05; ••P<0.01; •••P<O.OOl.
`
`increased plasma levels and decreased Clp in uraemic
`rats. Figure 5 shows the mean radioactivity in plasma
`from both ligated control and ligated uraemic rats.
`Except for the 1 and 3 min samples, plasma radioac(cid:173)
`tivity was significantly greater (P < 0.05) in the li(cid:173)
`gated uraemic group. The AU4+~ was greater
`(P<0.02) in ligated uraemics; while /l, Clp and Vd
`were all significantly less (P< 0.05; Table 2). The
`percentage dose of [lH]-ouabain excreted into bile
`after 1 h was 52± 7 (n= 4) in ligated controls and
`54±5 (n=4) in ligated uraemic rats. These re-
`
`Table 3 Effect of glycerol-induced acute renal
`failure on
`the pharmacokinetics of
`['H)(cid:173)
`taurocholicacid (5 mgkg-1; lO~tCikg- 1)
`
`Pharmacokinetic
`parameters
`
`Control rats
`(n=7)
`
`Uraemic rats
`(n=8)
`
`AUCo..~
`(dpmminmJ-1) x 10-s
`p (min-1)
`Vd(1 kg-1)
`Clp (mlmin-1 kg- 1)
`
`4.1 ±0.5
`
`3.2±0.8
`
`O.D18 ± 0.004
`2.8±0.8
`48±6
`
`0.012±0.002°
`4.4±1.1"
`55±18
`
`100
`
`10
`
`;:
`
`X
`
`I
`
`E
`E
`c.
`:!0'.
`u
`c
`0
`u
`<(
`.r::
`u
`I-
`
`"' ~
`"' 0:
`
`0·1 o~--7., o,.----,2:!:o:---::3!::0-----,-4b---=5.Lo--,Jso
`
`Time (min)
`
`Figure 6 Plasma concentrations of taurocholic acid
`(TChA, 5 mgkg-1 ; l0j4Cikg- 1) in seven control rats
`(0) and eight rats with acute renal failure (e). Values
`are mean; s.d. shown by vertical lines. Significantly
`different from control values; • P< 0.05.
`
`coveries were not significantly different and there
`was no difference in biliary excretion rates at any
`time period between ligated control and ligated
`uraemic rats.
`Pedicle ligation itself had a marked effect upon the
`kinetics of [3H]-ouabain in both control and uraemic
`rats. In both ligated control and ligated uraemic rats
`there were significant
`increases (P<O.Ol)
`in
`AU4+~ and Vd and decreases (P<0.05) in/land
`Clp when compared to their respective non-ligated
`counterparts. It might be anticipated that with neglig(cid:173)
`ible renal excretion more [lH]-ouabain would be
`excreted into bile in both ligated control and ligated
`uraemic rats. Although the percentages were higher
`(52 and 54%, respectively) than the corresponding
`values for non-ligated rats (43 and 49%, respective(cid:173)
`ly), they were not statistically different. Moreover, no
`differences were found in biliary excretion rates be(cid:173)
`tween the respective groups of ligated and non(cid:173)
`ligated rats.
`
`Taurocholic acid kinetics
`
`Results are given as mean± s.d.
`• P<O.Ol relative to respective control group.
`
`The decline of radioactivity following injection of
`[3H]-TChA (5 mg kg- 1) to control and uraemic rats is
`
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`PHARMACOKINETICS IN ACUTE RENAL FAILURE
`
`779
`
`shown in Figure 6. Mean plasma levels of radioactivi(cid:173)
`ty were significantly lower (P< 0.05) between 3 to
`5 min in the uraemic group and higher (P< 0.05) at
`45 min than in controls. In the uraemic rats, jl was
`lower (P< 0.01) and Vd greater
`significantly
`(P<0.01); but there was no changeinClp (Table 3).
`There was no difference in biliary excretion rates
`between control and uraemic rats and no difference
`after 1 h, in the percentage recovery of ['H]-TChA
`from bile of control rats (91 ± 5%; n = 6) and
`uraemic rats (84±6%; n = 7).
`
`Discussion
`
`Compartmental analysis of the disappearance of BSP
`from plasma demonstrated that in uraemic rats the
`initial removal of the dye was slowed. As BSP is
`largely removed from plasma by the liver, slowed
`removal suggests decreased hepatic uptake in the
`uraemic group. Furthermore, decreased uptake ap(cid:173)
`pears to be associated with an initial delay in the
`biliary excretion of BSP. These changes in the way in
`which the liver handles BSP are qualitatively similar
`to those found previously with ICG (Bowmer et a/.,
`1982a; Yates eta/., 1983a,b,c). This is not surprising
`because there is evidence to suggest that both dyes
`share
`the same pathway for hepatic uptake
`(Scharschmidt et a/., 1975; Schwenk et a/., 1976).
`Although uptake was decreased, no change was
`found in the Clp of BSP in rats with ARF. This may
`be due to the increase in Vc which would tend to
`attenuate the effect of the decrease in k12 so that no
`change of Clp occurred in uraemic rats.
`By contrast to ICG, BSP is partially conjugated
`with GSH prior to excretion into bile. Whelan eta/.,
`(1970) have shown that the major metabolite, BSP(cid:173)
`glutathione, is more efficiently excreted than the
`parent dye. In addition, conjugation may be a rate
`limiting step in the overall transfer of BSP from
`plasma to bile (Whelan et a/., 1970). Interference
`with conjugation either by feeding rats on protein(cid:173)
`free diets (Combes, 1965) or by pretreatment with
`the
`glutathione-S-transferase
`inhibitor,
`ben(cid:173)
`ziodarone (Priestley & Plaa, 1969) results in a
`marked impairment of biliary excretion. In homage(cid:173)
`nates of livers from uraemic rats, the rate of BSP
`conjugation with GSH was decreased by about 15%.
`It is possible, therefore, that part of the initial delay in
`the biliary excretion of BSP was due to decreased rate
`of conjugate formation.
`Factors other than altered hepatic function are
`unlikely to have contributed to the decreased uptake
`of BSP. For example, Vc was increased by about 69%
`in the uraemic rats, but this change may not have
`reduced the quantity of dye reaching the liver per unit
`time because plasma levels of BSP were higher in the
`uraemic group for a substantial proportion of the
`
`initial disappearance phase and total liver blood flow
`is increased by about 38% in rats 48 h after induction
`of ARF (Hiley eta/., 1980). In common with other
`highly bound anions (Bowmer & Lindup, 1979) the
`plasma-protein binding of BSP is decreased in
`uraemic rats. At an initial concentration of 750 !lM,
`the fraction of BSP bound in diluted uraemic plasma
`(1:3) is increased almost three fold when compared
`to diluted plasma from control rats (Bowmer &
`Yates, unpublished results). Grausz & Schmid
`( 1971) have provided evidence that for BSP, the rate
`of hepatic uptake is inversely related to the extent of
`albumin binding, so decreased binding may not con(cid:173)
`tribute to reduced hepatic uptake. However, de(cid:173)
`creased binding may have contributed to the increase
`in Vc seen in the uraemic rats. Recent work by Inoue
`eta/. (1983) showed that the hepatic uptake of BSP
`was not impaired in analbuminaemic rats. In these
`rats, the Qp of BSP was increased which, the authors
`concluded, was due to a large increase in the V d of
`BSP resulting from an absence of albumin binding
`capacity.
`The present study shows that the hepatic uptake
`and biliary excretion of ouabain are unaltered in
`uraemic rats. A greater proportion of the dose was
`present in uraemic livers but this was probably due to
`increased plasma concentrations of ouabain in the
`uraemic group. The Clp of ouabain was decreased by
`about 40% in uraemic rats. Clearly this change was
`not brought about by altered hepatic elimination and
`although renal excretion was reduced, this too cannot
`fully explain the decrease in Clp because Clp was still
`altered when uraemic rats with ligated pedicles were
`compared to their respective controls. The most like(cid:173)
`ly explanation is that the apparent V d of ouabain was
`smaller in the uraemic rats. Although Vd was some
`22% lower in uraemics it was not statistically differ(cid:173)
`ent from that determined in the control group. How(cid:173)
`ever, V d was significantly lower in the ligated
`uraemic rats compared to ligated controls.
`In patients with renal failure the V d of digoxin is
`decreased and is associated with relatively higher
`plasma concentrations of this drug (Reuning et a/.,
`1973). This change in Vd seems to be related to
`decreased tissue binding as Jusko & Weintraub
`(1974) showed that less digoxin accumulated in the
`myocardium of uraemic patients. It is possible, there(cid:173)
`fore, that decreased tissue binding was responsible
`for the higher plasma levels of ouabain in uraemic
`rats.
`There was no change in the Clp of TChA in
`uraemic rats and this together with the lack of any
`change in biliary excretion suggests that the hepatic
`handling of the bile acid is unchanged in ARF. Al(cid:173)
`though fl was decreased by about 33% there was a
`concomitant increase (57%) in Vd which probably
`accounts for the decrease in jl in rats with ARF.
`
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`
`780
`
`C.J. BOWMER & M.S. YATES
`
`Overall the results suggest that the hepatic uptake
`and biliary excretion of ouabain and TChA are unal(cid:173)
`tered in uraemic rats. By contrast, the uptake of BSP
`was impaired and previous work (Bowmer et al.,
`1982a;Yates et al., 1983a,b,c) has shown that uptake
`of ICG is also decreased. TChA and ouabain may be
`handled differently by the liver from BSP or ICG
`because there is evidence to suggest that the former
`have different uptake pathways from the latter
`(Schwenk et al., 1976). Furthermore, TChA and
`ouabain may themselves have separate uptake path(cid:173)
`ways (Meijer et al., 1976; Klassen, 1978). Because
`there is no general impairment in the hepatic uptake
`of these compounds it would seem that decreased
`uptake of BSP and ICG cannot be explained by a
`non-specific alteration of hepatocyte function in
`renal failure. Isolated perfused livers from uraemic
`rats have a decreased ability to remove ICG from the
`perfusion medium (Yates et al., 1984) which suggests
`that the metabolites which accumulate in uraemic
`plasma do not inhibit uptake of ICG. As BSP shares a
`common uptake pathway (Scharschmidt et al., 1975;
`Schwenk et al., 1976), this would suggest that the
`impaired removal of BSP was also not due to inhibi(cid:173)
`tion by retained metabolites.
`BSP and ICG are known to bind avidly to hepatic
`cytosol proteins and in particular to ligandin (Levi el
`al., 1969; Kamisaka el al., 1975; Ketley et al., 1975;
`Ketterer et al., 1976). A decrease in the binding
`capacity of these proteins, brought about by either a
`change in affinity and/or quantity of protein present,
`might possibly explain the decreased uptake of these
`dyes. In addition, changes in regard to ligandin may
`also explain the decreased biotransformation of BSP
`by homogenates of uraemic livers because ligandin
`behaves as a glutathione-S-transferase (Habig el al.,
`1974). If decreased binding capacity of liver cytosol
`proteins were responsible for impaired uptake of
`BSP and ICG then it could be predicted that hepatic
`uptake will be similarly impaired for other substances
`
`References
`
`that bind to these proteins whereas no change in
`uptake should occur when they do not interact. Oua(cid:173)
`bain appears to have little affinity for these proteins
`(Kupferberg & Schanker, 1968; Klassen, 1975)
`which would be consistent with the lack of any change
`in the uptake of this drug; but TChA interacts with
`ligandin (Kamisaka et aL, 1975) yet there was no
`change in the uptake of this bile acid. Furthermore, a
`number of studies have shown that there is no corre(cid:173)
`lation between the amount of binding proteins in
`liver cytosol and the rate of hepatic uptake of BSP
`(Gregus & Klassen, 1982), its non-metabolized
`analogue dibromosulphophthalein (Meijer et al.,
`1977) and ICG (Fischer et al., 1978) in the rat.
`Clearly this evidence, albeit indirect, does not sup(cid:173)
`port the idea that impaired uptake results from a
`decreased binding capacity of liver cytosol proteins in
`uraemic rats. In order to elucidate the role of cytosol
`binding proteins in the impaired hepatic uptake of
`BSP and ICG in uraemic rats, it will be necessary to
`determine the quantity of these proteins in uraemic
`livers and to study their binding properties.
`The present study adds to our knowledge of liver
`function in renal failure by demonstrating that the
`hepatic uptake and initial biliary excretion of BSP are
`decreased in ARF. The cause of decreased uptake is
`unclear but it would seem that this change is not due
`to a gross disturbance of hepatocyte function because
`the uptake and biliary excretion of ouabain and
`TChA were unaffected. In addition, these alterations
`of liver function are not confined to rats with ARF as
`impaired uptake of BSP has been observed in pa(cid:173)
`tients with chronic renal failure (Wernze & Spech,
`1971).
`
`This work was supported by the Wellcome Trust. We wish
`to thank Dr D. Mackay for helpful discussions during the
`preparation of this manuscript. Correspondence and reprint
`requests to M.S.Y. please.
`
`(1981). Assay of
`AKERBOOM, T.P.M. & SIES, H.
`glutathione, glutathione disulfide and glutathione mixed
`disulfides in biologi