Riocht'mical Pharmncology. Vol. 33. No. 10. pp. 1695-l(i96, 1984.
`Printed in Great Britain.
`
`000&-2952/84 S3.00 + 0.00
`© 198.4 Pergamon Press Lld.
`
`Sho rt communications
`
`1695
`
`Effect of acute renal failure on the clearance and biliary excretion of indocyanine
`green in perfused rat liver
`
`(Received 23 September 1983; accepted 2 December 1983)
`
`Evidence has accumulated that the hepatic uptake of dyes
`such as indocyanine green (ICG), bromosulphophthalein
`(BSP) and rose bengal is decreased in rats with renal failure
`[1-4]. The reason for the diminished uptake of these dyes
`is unknown; but one possible explanation is that the influx
`of these dyes into the hepatocyte is competitively inhibited
`by endogenous metabolites which are retained in uraemic
`pla~ma.
`Substances present in uraemic plasma inhibit renal tubule
`transport of organic anions such as p-aminobenzoic acid
`(PABA) [5, 6] and hippuric acid [7). Many organic anions
`are known to accumulate in uraemic plasma [8] and Preuss
`eta/. [5) have suggested that these may compete for tubular
`transport. Furthermore, the hepatic uptake of hippuric acid
`and PABA is also decreased in rats with renal failure [7,
`9). ICG, BSP and rose bengal are anions and may enter
`the hepatocyte by carder-mediated transport [!OJ, so it is
`possible that uraemic metabolites could compete with these
`dyes for hepatic uptake.
`The purpose of this study was to clarify the role of
`uraemic metabolites as putative competitive inhibitors of
`the hepatic uptake of ICG. The plasma clearance and
`biliary excretion of ICG have been determined in livers of
`control rats and rats with glycerol-induced acute renal
`failure (ARF) that have been perfused with a medium free
`of uraemic metabolites.
`
`Materials and methods
`Chemicals. ICG U.S.P. was purchased from Hynson,
`Wescott & Dunning Lt d. (Baltimore, MD). Bovine serum
`albumin (BSA) and reagents for the assay of urea were
`obtained from Sigma Chemical Co. All other reagents were
`available commercially and of analytical grade.
`Glycerol-induced ARF. Male Wistar albino rats (3~
`400 g) were used and ARF was produced by an i.m. injec(cid:173)
`tion of 50% (v/v) glycerol in sterile saline (10 ml/kg body
`wt) [I). Control rats were injected with saline (10 ml/kg
`body wt) and both groups of rats were studied 48 hr after
`i.m. injection.
`Liver perfusion. Rats were anaesthetised with pen(cid:173)
`tobarbitone (60 mg/kg body wt. i.p.) and the subsequent
`opCrativc technique and liver perfusion were essentially as
`described by H ems eta/. [II]. The abdomen was opened
`by a midline incision and cannulae placed in the bile duct
`and portal vein. The thorax was opened and a blood sample,
`for plasma urea determination, was taken by left ventricular
`puncture before a cannula was placed in the inferior vena
`cava.
`The perfusion medium (150 ml) was Krebs-bicarbonate
`solution containing washed human erythrocytes (packed
`cell volume, 15-16% v/v) and BSA (2.6% w/ v) [11). The
`final pH of the medium was adjusted to 7.4 by addition
`of I M NaHCO, . The composition (mM) of the Krebs(cid:173)
`bicarbonate solution was: NaCI 118.0, KC14.7, CaC12 2.5,
`KH2PO, I .2. MgS0, .7H20 1.2, NaHCO, 25.0 and glucose
`10.0. The perfusion medium was oxygenated with a mixture
`of 95% 0 2 and 5% CO,.
`After completion of surgery the rat was transferred to a
`thermostated cabinet at 37' and the liver perfused via the
`portal vein. The height of the perfusate reservoir was
`adjusted to give sufficient hydrostntic pressure to allow a
`flow of 12.5 ml/min. This pressure varied between 25-30 em
`
`and did not cause swelling of the liver. The liver was allowed
`to recover for 30 min and then ICG was added to the
`collecting vessel as an aqueous solution (7.5 mg/kg body
`wt; 10 mg/ml). Samples of perfusion medium (0.5 ml) were
`removed from the collecting vessel at 5, 7.5, 10, 15. 20, 30
`and 40 min after the administration of ICG. In another
`series of experiments bile samples were collected at 10 min
`intervals for up to 40 min.
`Analytical methods. The concentration of ICG in the
`plasma of the perfusion medium and bile was determined
`spectrophotometrically at 800 nm as described previously
`[1, 12]. Plasma urea concentrations were measured using a
`commercial kit (Sigma No 535).
`Data analysis. Plasma concentration- time data were
`fitted to a monoexponential equation by linear regression
`and the elimination rate constant (kc~), elimination half(cid:173)
`life (to.s), volume of distribution (Vd) and plasma clearance
`(Cip) were calculated as described by Gibaldi and Perrier
`[13]. Results are given as mean ± S.E.M. and statistical
`comparisons were made by the non-paired Student's Hest.
`
`Results
`Rats injected with glycerol had significantly greater
`(P < 0.001) mean
`plasma
`urea
`concentrations
`(309 ::t 48 mg/ 100 ml; N = 10) than rats given i.m. saline
`(43 ± 3 mg/ IOOml; N = 10). However, there was no sig(cid:173)
`nificant difference in either body weight or liver weight
`between control cats (346 ±IS g and 11.7 ± 0.4 g respect(cid:173)
`ively; N = 10) '•and rats with ARF (353 ± 12 g and
`11.3 ± 0.3 g respectively; N = 10).
`Figure 1 shows the mean concentration of ICG in plasma
`perfusing livers from control and uraemic rats. It is clear
`that in both groups of livers ICG is eliminated in a mono(cid:173)
`exponential manner. Only at 40 min was the mean con(cid:173)
`centration of ICG significantly greater (P < 0.05) in plasma
`perfusing uraemic livers, but th.e fractional rate of dye
`removal, k,, was significantly smaller in the uraemic group
`(Table 1). In addition, los and Clp were also decreased
`while there was no significant difference in the Yd of ICG
`between control and uraemic groups (Table l).
`ICG could be detected in all the bile samples collected
`from control livers over the first 10 m in. By contrast in
`three out of four experiments with uraemic livers, no ICG
`could be detected in bile collec ted over the same time
`interval. The cumulative amount of dye excreted was sig(cid:173)
`nificantly lower in uraemic livers from 20 to 40 min (Fig.
`2). There was no significant difference in bile flow rate
`between control and uraemic livers during any collection
`interval and the mean bile flow r ate over the 40 min col(cid:173)
`lection period was 0.37 and 0.36 m l/hr in control and ura(cid:173)
`emic livers respectively. These results suggest that the
`biliary excretion of ICG by uraemic livers is less efficient
`than in control livers,
`
`Discussion
`The concentration of ICG in the plasma of the perfusion
`medium decreased in a monocxponential manner in both
`control and uraemic groups. By contrast, ICG shows
`biexponential kinetics in vivo [1, 2]. In perfusion experi(cid:173)
`ments the rale of delivery of ICG t.o the Jiver is considtaal.Jiy
`less than in vivo because of a larger initial Yd (135 ml
`
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`
`Short communications
`
`1696
`
`100
`
`10
`
`Table 1. Effect of glycerol-induced acute renal failure on
`the pharmacokinetics of ICG (7.5 mg/ kg) in perfused rat
`livers'
`
`Pharmacokinetic
`parameters
`
`k.,/ (min)
`to.s (min)
`Clp (ml/ min/100 g
`body wt)
`Vd(ml)
`
`Control rats
`(N = 6)
`0.027 ± 0.001
`26 ± 1
`1.1 ± 0.1
`
`Uraemic rats
`(N= 6)
`
`0.015 ± 0.002t
`51 ± 8t
`0.69 ± 0.10+
`
`142 ± 4
`
`155 ± 12
`
`• Results are given as mean ± S.E.M.
`t p < 0.05.
`t P < 0.01 relative to respective control group.
`
`Moreover, Howie and Burke [9) found decreased uptake
`of PABA into isolated perfused livers from rats with chronic
`renal failure, and another study [ 15) reported that a small
`number of uraemic metabolites failed to inhibit the uptake
`of BSP into isolated perfused rat livers. In view of these
`reports and the evidence presented here, it would seem
`unlikely that competition by uraemic metabolites could
`explain the impaired hepatic uptake of PABA or dyes such
`as ICG and BSP. However, it is possible that uraemic
`metabolites have a direct effect upon the liver and thereby
`induce changes in liver function which lead to impaired
`uptake of dyes.
`
`Acknowledgement- This work was supported by a grant
`from the Wellcome Trust.
`
`Department of Pharmacology
`University of Leeds
`Leeds LS2 9JT, U.K.
`
`MICHAELS. YATES'
`CHRISTOPHER J. BOWMER
`JANE EMMERSON
`
`REFERENCES
`
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`nuc/. Med. Bioi. 3, 134 (1976).
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`and G. E. Schreiner, Nephron 3, 265 (1966).
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`8. D. Seligson, L. W. B1uemle, G. D. Webster and D.
`Senesky, J. clin. Invest. 38, 1042 (1959).
`9. M. B. Howie and E. Bourke, Clin. Sci. 56, 9 (1979).
`10. B. F. Scharschmidt, J. G. Waggoner and P. D. Berk,
`1. c/in. Invest. 56, 1280 (1975).
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`Pharmac. 32, 1641 (1983).
`13. M. Gibaldi and D. Perrier, Pharmacokinetics, Marcel
`Dekker, New York (1975).
`14. D. K. F. Meijer, R. J. Vonk, K. Keulemans and J. G.
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`22, 306 (1978).
`
`10
`
`20
`Timo(min)
`
`.lO
`
`Fig. I. Concentrations of ICG (7.5 mg/ kg) in plasma
`perfusing livers of control (e ) and uraemic (6) rats. Each
`point is the mean ± S.E.M. obtained in six rats. Key:
`P < 0.05 relative to respective control value.
`
`compared to about 8 ml in vivo (1, 2]) which cannot be
`compensated for by a proportional increase in flow rate.
`Meijer et at. [14] have pointed out that uptake may be
`further retarded by the relatively large amount of albumin
`in the perfusion medium. The binding capacity of the
`medium is likely to be much greater than that of hepatic
`binding proteins, so that a larger fraction of the dose will
`remain within the perfusion medium. The combination of
`decreased delivery of dye and higher binding capacity
`means that the initial disposition phase will be much less
`pronounced in perfusion studies than in vivo [14).
`Livers from rats wit h ARF have been perfused with a
`medium free of uraemic metabolites. The results show
`that these livers were less efficient than control livers at
`removing ICG from plasma and excreting it into bile.
`
`50
`
`Time {min}
`
`Fig. 2. Cumulative biliary excretion of ICG (7.5 mg/kg) in
`perfused livers of control (e ) and uraemic (6) rats. Each
`point is the mean ± S.E.M. obtained in four rats. Key tin
`three out of four experiments with livers of uraemic animals
`no ICG could be detected in bile collected over the first
`10 min; • P < 0.05, •• P < 0.01 relative to respective con-
`trol value.
`
`• To whom correspondence should be addressed.
`
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