Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Pharmacokinetics and excretion of phenol red in the channel catfish S. M. Plakas, G. R. Stehly & L. Khoo To cite this article: S. M. Plakas, G. R. Stehly & L. Khoo (1992) Pharmacokinetics and excretion of phenol red in the channel catfish, Xenobiotica, 22:5, 551-557, DOI: 10.3109/00498259209053118 To link to this article: http://dx.doi.org/10.3109/00498259209053118
Published online: 22 Sep 2008.
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XENOBIOTICA,
1992, VOL. 22,
NO.
5, 551-557
Pharmacokinetics and excretion of phenol red in the channel catfish S. M. PLAKAS, G. R. S T E H L Y and L. KHOO Division of Seafood Research, U S Food and Drug Administration, PO Box 158, Dauphin Island, AL 36528, USA Received 28 August 1991 ;accepted 4 December 1991
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1 . Disposition of phenol red was examined in channel catfish (Zctaluruspunctatus) after oral or intravascular (i.v.) dosing at 10mg/kg body weight. 2. Phenol red was not detectable in plasma, urine, or bile after oral administration. 3. After i.v. dosing, plasma concentrations of phenol red were best described by a twocompartment pharmacokinetic model with distribution and elimination half-lives of 2.3 and 21 min, respectively. T h e apparent volume of distribution at steady state (VSJ was 225 ml/kg and total body clearance (Cl,,) was 658 ml/h per kg. Plasma protein binding was 19%. 4. Biliary excretion was the primary route of elimination of phenol red; in 24 h, 55% of the i.v. dose was excreted in bile compared with 24% in urine. No metabolites were detected in these fluids. 5. T h e use of anaesthesia during dosing had no effect on the quantitative excretion of phenol red by renal or biliary routes.
Introduction The organic anion phenolsulphonphthalein (phenol red) has been extensively used as a model compound for assessing kidney function in animals, particularly in mammalian species in which extensive urinary elimination of the unchanged molecule occurs (Smith 1973). In some animals (e.g. fish and rats), biliary excretion is also an important route of elimination of phenol red; moreover, the extent of metabolism (via the glucuronidation pathway) of phenol red may vary considerably between species (Yasuhara et al. 1985, Pritchard et al. 1980, Hart and Schanker 1966). Because data are available for a wide range of species, phenol red also has been used for comparative studies of xenobiotic disposition and metabolism. The renal and biliary excretion of phenol red and its glucuronide conjugate have been examined in several species of marine fish (Bungay et al. 1976, Guarino and Anderson 1976, Pritchard et al. 1980). In this study we examined the pharmacokinetics, metabolism, and excretion of phenol red in the channel catfish (Ictalurus punctotus). In xenobiotic disposition studies in aquatic animals, anaesthesia is often induced during dosing procedures; we also examined the influence of anaesthesia on the excretion of phenol red. Experimental Experimental animals, dosing, and sampling Channel catfish (Zctalurus punctatus), 0 . 3 4 6 kg body weight, were obtained from the Southeastern Fish Cultural Laboratory, U S Fish and Wildlife Service (Marion, AL, USA). Fish were individually distributed and acclimatized in 90-litre aquaria (water temp. 2 3 T , pH 8.1).
Correspondence to: Steve M. Plakas, Division of Seafood Research, U S FDA, P.O. Box 158, Dauphin Island, AL 36528, USA. 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd.
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All reagents were purchased from Sigma Chemical Co. (St Louis, MO, USA). Tricaine methanesulphonate (MS-222) solution (025 g/l) was used as an anaesthetic during all surgical procedures. Phenol red (sodium salt) purity was 98%. Phenol red solution (10 mg/ml) was prepared in 0.85% NaCl for dosing. T h e dosing solution was administered by intravascular (i.v.) or oral routes at a rate of 1 ml/kg body weight (dosage of 10mg/kg). Before i.v. or oral dosing, fish were catheterized for collection of blood and urine (Plakas et 01. 1990, Kitzman et al. 1988). Fish were placed in wire-mesh cages to restrict movement and the catheters were extended outside the aquaria. Fish were allowed to acclimatize overnight (12 h). Five fish were dosed i.v. via the aortic cannulae. Two fish were orally dosed with gelatin capsules (Plakas et al. 1988). Fish were not fed during the study. After i.v. or oral dosing, blood was sampled serially over a 24 h period and plasma was obtained by centrifugation. Urine was collected continuously, and the volumes were recorded at various intervals. At 24 h, fish were killed and bile was collected by syringe from the gall bladder. Plasma and excretory fluids were stored at - 20°C until analysis. T o compare the effects of cold vs. MS-222 anaesthesia on the excretion of phenol red, we fitted additional fish with urinary catheters and allowed the fish to acclimatize for 12 h. Immediately before dosing, four or five fish were anaesthetized b y exposure to MS-222 solution (0.25 g/l) or an ice-water bath (24°C). Fish were dosed i.v. with phenol red (lOmg/kg) and returned to their aquaria. Urine and bile were collected as described above.
Analytical procedures Phenol red and its conjugates in bile and urine were determined according to the acid hydrolysis procedure described by Guarino and Anderson (1976). T o determine parent phenol red in the plasma, we mixed specimens (0.2~11)with 1 ml of methanol and centrifuged the mixture at lOOOg for l0min. A portion (0.8ml) of the supernatant was mixed with 2.2 ml of 0.3 M-NaOH and the absorbance at 555 nm was determined. Standard curves were prepared by adding phenol red to control urine, bile, and plasma specimens. T h e limits of determination of phenol red, based on specimen volumes analysed, were 0.1 pg/O.5 ml urine, 2 pg/O.Ol ml bile, and 0.2 pg/0.2 ml plasma. T o investigate further the presence of metabolites, we treated urine and bile with the enzymes glucuronidase and sulphatase. Samples were incubated with or without enzymes in 0 1 M acetate buffer (pH 5.0)for 18 h a t 3 7 T , and the phenol red concentrations were compared. Additionally, urine and bile (enzyme-treated and non-treated) were analysed by thin layer chromatography according to the procedure of Guarino and Anderson (1976). Samples were applied to Brinkmann Silica Gel G-HR plates (Brinkmann Instruments Co., Westbury, NY, USA) and developed in a solvent system of ethanol-nbutanol-3 M ammonium hydroxide (1 1 :40 : 19 by vol.). Plasma protein binding of phenol red was determined by ultrafiltration with the Centrifree Micropartition System (Amicon Corp., Danvers, MA, USA). Phenol red was added to control plasma at concentrations ranging from 1 to 100pg/ml. These plasma samples ( 0 5 ml) were applied to the ultrafilters and were centrifuged at lOOOg for 1 h at 25°C. T h e ultrafiltrates were analysed for phenol red concentration. Calculated binding values were corrected for losses (3%) in recovery of free phenol red caused by non-specific absorption to the ultrafiltration apparatus. Pharmacokinetic modelling Pharmacokinetic modelling of plasma data was performed with the iterative, least-squares program PCNONLIN (Statistical Consultants, Inc., Lexington, KY, USA). A two-compartment phannacokinetic model best described the plasma concentrations of parent compound after i.v. dosing. Intercompartmental clearance (ClJ, total body clearance (Cl,,),and apparent volumes of distribution (of the central compartment ( Vl), peripheral compartment ( V 2 ) , and at steady state ( V S J )were calculated with established compartmental modelling equations (Barron et al. 1990).
Results Phenol red was not found in the plasma, urine, or bile after oral dosing in the channel catfish. After i.v. dosing, phenol red concentrations in the plasma exhibited a rapid, biphasic decline (figure 1); at 2 h, plasma concentrations were below the limit of determination ( < 1 pg/ml). Plasma protein binding of phenol red was 19%. Binding was independent of concentration over a range of l-lOOpg/rnl, which encompassed the levels observed in plasma after i.v. dosing. Pharmacokinetic values for phenol red are summarized in table 1. Phenol red was highly concentrated in the bile and urine of channel catfish with no evidence of conjugative metabolism. T h e concentration (mean fSD) of phenol red in bile was 3714 & 873 pg/ml at 24 h. T h e concentration in urine was highest
Phenol red disposition in catjish
553
I
I " " " " " " " ' ~ ' " " 0.25
0.00
0.50
0.75
1.oo
Time (h)
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Figure 1. Plasma concentration of phenol red after i.v. dosing in the channel catfish. Data are the meansf S D of five fish; dose of phenol red was 10mg/kg.
Table 1. Pharmacokinetic values for phenol red after i.v. dosing (10mg/kg) in channel catfish. Parameter"
Value 104 18.4 18.2 1.95 2.28 21.4 7.68 4.39 15.2 626 658 81.6 143 225
"Abbreviations are as follows: A and B are the intercepts and tl and P are the rate constants for the distribution and elimination phases, respectively; t l I z nand t,/,# are the half-lives for distribution and elimination phases, respectively; k, and k,, are the intercompartmental transfer constants; AUC is the area under the plasma concentration-time curve; Cl, and Cl, are the intercompartmental and total body clearances; V , and V, are the volumes of the central and peripheral compartments; V,, is the apparent volume of distribution at steady state.
,
Concentration and cumulative excretion of phenol red in the urine of channel catfish after i.v. dosing. Open bars, concentration (pglml); shaded bars, cumulative percentage dose.
Figure 2.
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(121 f44pglml) at the 0-5-1 h collection interval (figure 2). T h e plasma concentration at 0-75 h (midpoint of the 05-1 h urine collection interval) was 3.79 0.68 pg/ml. Thus, the urine :plasma concentration ratio was approximately 32 : 1. Biliary excretion was the primary route of elimination of phenol red. At 24 h, 55 - 9%of the dose was recovered in the bile compared with 24 & 4% in the urine. Renal excretion of phenol red was very rapid (figure 2). Approximately 5% of the dose was eliminated in the urine within 0 5 h; at 8 h, renal excretion of phenol red was nearly complete. There were no differences in the amounts of phenol red excreted in the bile and urine after exposure to MS-222 or an ice-water bath. After MS-222 treatment, 59 f7% of the administered dose was excreted in the bile and 19 f 4 % was excreted in the urine at 24 h. After cold anaesthesia, 59 3% and 20 f9% was excreted in the bile and urine, respectively. Downloaded by [McMaster University] at 13:42 01 April 2016
+
Discussion Absorption Phenol red was not detectable in the excretory fluids of channel catfish after oral dosing, indicating little or no absorption from the gastrointestinal tract. Phenol red is poorly absorbed in mammals and has been used as a non-absorbable marker in gastrointestinal absorption studies (McLeod et al. 1968, Ashley and Levy 1973). T h e intestinal absorption of foreign organic anions occurs mainly by passive diffusion across the membrane lipid barrier (Ashley and Levy 1973, Lanman et al. 1971). T h e low lipid-to-water partition coefficient of phenol red is probably the most important physicochemical parameter limiting its absorption; organic acids with higher partition coefficients (i.e. hippuric acid and sulphanilic acid) are more extensively absorbed (Lanman et al. 1971).
Pharmacokinetics Phenol red concentrations in the plasma after i.v. dosing were well described by a two-compartment pharmacokinetic model. Elimination of phenol red from the plasma was very rapid (table 1). T h e low apparent volume of distribution at steady state (225 ml/kg) indicated limited tissue distribution. Phenol red may have been confined principally to the excretory tissues and extracellular fluids. T h e extracellular fluid volume in channel catfish has been estimated at 183 ml/kg (K'itzman et al. 1990). A two-compartment pharmacokinetic model has been used to describe the plasma elimination of phenol red in other animals (Hinchcliff et al. 1987). In channel catfish, certain pharmacokinetic values for phenol red were similar to those reported in mammalian species; the elimination half-life of phenol red was 21 min in the channel catfish (table 1) compared with values ranging from 13 to 25 min in horses (Hinchcliff et al. 1987), cattle (Mixner and Anderson 1958), and humans (Gault 1966). A half-life of 2.4min has been determined in rats (Inoue et al. 1985). In other fish species (i.e. hagfish, dogfish, skate, and flounder), elimination of phenol red is much slower than in channel catfish; elimination half-lives from 26 to 62 h have been reported (Pritchard et al. 1980). T h e total body clearance and volumes of distribution of phenol red were considerably larger in channel catfish compared with values reported for mammalian species (Hinchcliff et al. 1987, Yasuhara et al. 1985).
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Plasma protein binding Plasma protein binding in channel catfish was low and independent of concentration (1-100 pglml). I n mammals (e.g. human, rat, and dog), plasma protein binding of phenol red is extensive and is concentration-dependent (Yasuhara et at. 1985, Inoue et al. 1985, Kragh-Hansen et al. 1972, Russel et al. 1987). For example, plasma protein binding in dogs decreased from 85% to 60% at concentrations ranging from 5 to 450 pg/ml (Russel et al. 1987). In the spiny dogfish, plasma protein binding is also high and dose-dependent (Guarino and Anderson 1976). T h e low plasma protein binding in channel catfish may have influenced the extravascular distribution of phenol red. In analbuminaemic rats, the volume of distribution and clearance of phenol red are much larger than in normal rats; these observations were attributed to the much lower binding capacity of serum proteins and an increased extrarenal distribution in the mutant animals (Inoue et al. 1985). Metabolism Conjugates of phenol red were not found in the bile or urine of channel catfish. Considerable interspecies differences exist in the conjugative metabolism (i.e. glucuronidation) of phenol red. Phenol red is excreted unchanged in the urine of humans and dogs (Smith 1973). I n renal-ligated rats, phenol red is excreted in considerable amounts (20-35% of the total phenol red) in the bile as its glucuronic acid conjugate (Hart and Schanker 1966, Homan and Guarino 1974, Collado et al. 1988). In the spiny dogfish and little skate, the glucuronide comprised 25% and 65%, respectively, of the total phenol red excreted in the bile in 24 h; in contrast, phenol red was not metabolized by the hagfish and winter flounder (Pritchard et al. 1980). No other metabolites of phenol red were found in the above studies. Channel catfish are capable of forming glucuronic acid conjugates of phenolic compounds. Glucuronyltransferase activity (with p-nitrophenol as a substrate) in microsomes of the channel catfish is similar to that of the rat when in vitro preparations are incubated at 37°C (Short et al. 1988). Active glucuronidation of 1naphthol has been demonstrated in vivo in the channel catfish (Stehly and Plakas 1992). T h e metabolism of phenol red in channel catfish may have been precluded by its extremely rapid elimination. Excretion Renal and biliary excretion of phenol red in channel catfish accounted for 24% and 55%, respectively, of the i.v. dose at 24 h. T h e amounts of phenol red excreted in the urine and bile were not affected by exposure to MS-222 or an ice-water bath. In the spiny dogfish and winter flounder, approximately 35% of an i.v. dose was excreted in the urine in 24 h; similar amounts were excreted in bile (Pritchard et al. 1980). In humans, 85% and 3% of an i.v. dose were eliminated in the urine and bile, respectively, within 6 h (McLeod et al. 1968). In dogs, renal and biliary excretion accounted for 75% and 3%, respectively, of an i.v. dose at 3 h (Kim and Hong 1962). In rats, ratios of approximately 1 : 1 (Yasuhara et al. 1985) and 3 : 1 (Fleck and Braunlich 1986) have been reported for the urinary : biliary excretion (as percentage of the dose) of phenol red at 1 h after i.v. dosing.
Conclusions Plasma concentrations of phenol red after i.v. dosing in channel catfish were well described by a two-compartment pharmacokinetic model. Elimination was ex-
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tremely rapid and similar to the rates observed in mammals. Plasma protein binding of phenol red was much lower in channel catfish than in mammalian species. Biliary excretion of the unchanged molecule was the primary route of elimination of phenol red in channel catfish; however, active renal transport was also evident. T h e molecular weight threshold for extensive biliary excretion of phenol red appears lower in fish than in mammalian species. T h e metabolism and rate of elimination of phenol red vary widely among species. These differences emphasize the uncertainty in extrapolating disposition data for drugs or other xenobiotics from one species to another.
Acknowledgements
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The authors thank L. Torrans, J. C. Jones, and B. Deavours for supplying the catfish; M. Moxey for technical assistance; J. Holley for advice on cannulation procedures; and C. Collier for his design and construction of restraining cages.
References ASHLEY, J. J., and LEVY,G., 1973, Effect of vehicle viscosity and an anticholinergic agent on bioavailability of a poorly absorbed drug (phenolsulfonphthalein) in man. Journal of Pharmaceutical Sciences, 62, 688-690. BARRON, M. G., STEHLY, G. R., and HAYTON, W. L., 1990, Pharmacokinetic modeling in aquatic animals. I. Models and concepts. Aquatic Toxicology, 18, 61-86. BUNGAY, P. M., DEDRICK, R. L., and GUARINO, A. M., 1976, Pharmacokinetic modeling in the dogfish shark (Squalus acanthias): distribution and urinary and biliary excretion of phenol red and its glucuronide. Journal of Pharmacokinetics and Biopharmaceutics, 4, 377-388. COLLADO, P. S., MUNOZ,M. E., ESTELLER, A,, and GONZALEZ, J . , 1988, Phenosulphonphthalein conjugation and biliary excretion in the rat: influence of phenobarbital and 3-methylcholanthrene. Archives Internationales de Physiologie et de Biochimie, 96, 17-23. FLECK, C., and BRAUNLICH, H., 1986, Relation between renal and hepatic excretion of drugs: I. Phenol red in comparison with p-aminohippurate and indocyanine green. Experimental Pathology, 29, 179-192. GAULT, M. H., 1966, The plasma phenolsulfonphthalein index (PSPI) of renal function: I. Theoretical considerations and investigative studies. Canadian Medical Association Journal, 94, 61-67. GUARINO, A. M., and ANDERSON, J. B., 1976, Excretion of phenol red and its glucuronide in the dogfish shark. Xenobiotica, 6, 1-13. HART, L. G., and SCHANKER, L. S., 1966, The chemical forms in which phenol red is secreted into the bile of rats. Proceedings of the Society for Experimental Biology and Medicine, 123, 433435. HINCHCLIFF, K. W., MCGUIRK, S. M . , and MACWILLIAMS, P. S., 1987, Pharmacokinetics of phenolsulfonphthalein in horse and pony mares. American Journal of Veterinary Research, 48, 1256-1260. HOMAN, E. R., and GUARINO, A. M., 1974, Biliary excretion of phenol red by Wistar and Gunn rats. Proceedings of the Society for Experimental Biology and Medicine, 146, 4 6 4 9 . INOUE,M., KOYAMA, H., NACASE, S., and MORINO, Y., 1985, Renal secretion of phenolsulfonphthalein: analysis of its vectorial transport in normal and mutant analbuminemic rats. Journal of Laboratory and Clinical Medicine, 105, 484-488. KIM,J. H., and HONG,S. K., 1962, Urinary and biliary excretions of various phenol red derivatives in the anaesthetized dog. The American Journal of Physiology, 202, 174178. KITZMAN, J . V., HOLLEY, J. H., and HUBER, W. G., 1988, Sample collection techniques in the channel catfish during prolonged pharmacokinetic experiments. Veterinary and Human Toxicology, 30 (SUPPI. l), 12-13. KITZMAN, J. V., MARTIN, J. F., HOLLEY, J. H., and HUBER, W. G., 1990, Determinations of plasma volume, extracellular fluid volume, and total body water in channel catfish. Progressive FishCulturist, 52, 26 1-265. KRAGH-HANSEN, U., MOLLER, J. V., and SHEIKH, M. I., 1972, A spectrophotom2tric micromethod for the determination of binding of phenol red to plasma proteins of various species. Pjugers Archiv, 337, 163-176. LANMAN, R. C., STREMSTERFER, C. E., and SCHANKER, L. S., 1971, Absorption of organic anions from the rat small intestine. Xenobiotica, 1, 613-619. MCLEOD, G. M., FRENCH, A. B., GOOD,C. J., and WRIGHT, F. S., 1968, Gastrointestinal absorption and biliary excretion of phenolsulfonphthalein (phenol red) in man. Journal of Laboratory and Clinical Medicine, 71, 192-200.
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MIXNER, J. P., and ANDERSON, R. R., 1958, Phenolsulfonphthalein fractional clearance in dairy cattle as a measure of renal function. Journal of Dairy Science, 41, 306-313. PLAKAS, S. M., DICKEY, R. W., BARRON, M. G., and GUARINO, A. M., 1990, Tissue distribution and renal excretion of ormetoprim after intravascular and oral administration in the channel catfish (Ictalurus punctatus). Canadian Journal of Fisheries and Aquatic Sciences, 47, 766-771. PLAKAS, S. M., MCPHEARSON, R. M., and GUARINO, A. M., 1988, Disposition and bioavailability of 'Htetracycline in the channel catfish (Ictalurus punctatus). Xenobiotica, 18, 83-93. PRITCHARD, J . B., ANDERSON, J. B., RALL,D. P., and GUARINO, A. M., 1980, Comparative hepatic and renal handling of phenol red and indocyanine green by cyclostome, elasmobranch and teleost fish. Comparative Biochemistry and Physiology, 65C, 99-1 04. RUSSEL,F. G . M., WOUTERSE, A. C., and VAN GINNEKEN, C. A. M., 1987, Physiologically based pharmacokinetic model for the renal clearance of phenolsulfonphthalein and the interaction with probenecid and salicyluric acid in the dog. Journal of Pharmacokinetics and Biopharmaceutics, 15, 349-368. SHORT,C. R., FLORY, W., and FLYNN,M., 1988, Hepatic drug metabolizing enzyme activity in the channel catfish, Ictalurus punctatus. Comparative Biochemistry and Physiology, 89C, 153-1 57. SMITH, R. L., 1973, The Excretory Function of Bile. The Elimination of Drugs and Toxic Substances in Bile (London: Chapman & Hall). STEHLY, G . R., and PLAKAS, S. M., 1992, Disposition of 1-naphthol in the channel catfish (Ictalurus punctatus). Drug Metabolism and Disposition, 20, 7CL73. YASUHARA, M., KATAYAMA, H., FUJIWARA, J., OKUMURA, K., and HORI,R., 1985, Influence of acute renal failure on pharmacokinetics of phenolsulfonphthalein in rats: A comparative study in vivo and in the simultaneous perfusion system of liver and kidney. Journal of Pharmacobzo-Dynamics, 8, 377-384.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Pharmacokinetics and excretion of phenol red in the channel catfish S. M. Plakas, G. R. Stehly & L. Khoo To cite this article: S. M. Plakas, G. R. Stehly & L. Khoo (1992) Pharmacokinetics and excretion of phenol red in the channel catfish, Xenobiotica, 22:5, 551-557, DOI: 10.3109/00498259209053118 To link to this article: http://dx.doi.org/10.3109/00498259209053118
Published online: 22 Sep 2008.
Submit your article to this journal
Article views: 6
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ixen20 Download by: [McMaster University]
Date: 01 April 2016, At: 13:42
XENOBIOTICA,
1992, VOL. 22,
NO.
5, 551-557
Pharmacokinetics and excretion of phenol red in the channel catfish S. M. PLAKAS, G. R. S T E H L Y and L. KHOO Division of Seafood Research, U S Food and Drug Administration, PO Box 158, Dauphin Island, AL 36528, USA Received 28 August 1991 ;accepted 4 December 1991
Downloaded by [McMaster University] at 13:42 01 April 2016
1 . Disposition of phenol red was examined in channel catfish (Zctaluruspunctatus) after oral or intravascular (i.v.) dosing at 10mg/kg body weight. 2. Phenol red was not detectable in plasma, urine, or bile after oral administration. 3. After i.v. dosing, plasma concentrations of phenol red were best described by a twocompartment pharmacokinetic model with distribution and elimination half-lives of 2.3 and 21 min, respectively. T h e apparent volume of distribution at steady state (VSJ was 225 ml/kg and total body clearance (Cl,,) was 658 ml/h per kg. Plasma protein binding was 19%. 4. Biliary excretion was the primary route of elimination of phenol red; in 24 h, 55% of the i.v. dose was excreted in bile compared with 24% in urine. No metabolites were detected in these fluids. 5. T h e use of anaesthesia during dosing had no effect on the quantitative excretion of phenol red by renal or biliary routes.
Introduction The organic anion phenolsulphonphthalein (phenol red) has been extensively used as a model compound for assessing kidney function in animals, particularly in mammalian species in which extensive urinary elimination of the unchanged molecule occurs (Smith 1973). In some animals (e.g. fish and rats), biliary excretion is also an important route of elimination of phenol red; moreover, the extent of metabolism (via the glucuronidation pathway) of phenol red may vary considerably between species (Yasuhara et al. 1985, Pritchard et al. 1980, Hart and Schanker 1966). Because data are available for a wide range of species, phenol red also has been used for comparative studies of xenobiotic disposition and metabolism. The renal and biliary excretion of phenol red and its glucuronide conjugate have been examined in several species of marine fish (Bungay et al. 1976, Guarino and Anderson 1976, Pritchard et al. 1980). In this study we examined the pharmacokinetics, metabolism, and excretion of phenol red in the channel catfish (Ictalurus punctotus). In xenobiotic disposition studies in aquatic animals, anaesthesia is often induced during dosing procedures; we also examined the influence of anaesthesia on the excretion of phenol red. Experimental Experimental animals, dosing, and sampling Channel catfish (Zctalurus punctatus), 0 . 3 4 6 kg body weight, were obtained from the Southeastern Fish Cultural Laboratory, U S Fish and Wildlife Service (Marion, AL, USA). Fish were individually distributed and acclimatized in 90-litre aquaria (water temp. 2 3 T , pH 8.1).
Correspondence to: Steve M. Plakas, Division of Seafood Research, U S FDA, P.O. Box 158, Dauphin Island, AL 36528, USA. 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd.
Downloaded by [McMaster University] at 13:42 01 April 2016
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All reagents were purchased from Sigma Chemical Co. (St Louis, MO, USA). Tricaine methanesulphonate (MS-222) solution (025 g/l) was used as an anaesthetic during all surgical procedures. Phenol red (sodium salt) purity was 98%. Phenol red solution (10 mg/ml) was prepared in 0.85% NaCl for dosing. T h e dosing solution was administered by intravascular (i.v.) or oral routes at a rate of 1 ml/kg body weight (dosage of 10mg/kg). Before i.v. or oral dosing, fish were catheterized for collection of blood and urine (Plakas et 01. 1990, Kitzman et al. 1988). Fish were placed in wire-mesh cages to restrict movement and the catheters were extended outside the aquaria. Fish were allowed to acclimatize overnight (12 h). Five fish were dosed i.v. via the aortic cannulae. Two fish were orally dosed with gelatin capsules (Plakas et al. 1988). Fish were not fed during the study. After i.v. or oral dosing, blood was sampled serially over a 24 h period and plasma was obtained by centrifugation. Urine was collected continuously, and the volumes were recorded at various intervals. At 24 h, fish were killed and bile was collected by syringe from the gall bladder. Plasma and excretory fluids were stored at - 20°C until analysis. T o compare the effects of cold vs. MS-222 anaesthesia on the excretion of phenol red, we fitted additional fish with urinary catheters and allowed the fish to acclimatize for 12 h. Immediately before dosing, four or five fish were anaesthetized b y exposure to MS-222 solution (0.25 g/l) or an ice-water bath (24°C). Fish were dosed i.v. with phenol red (lOmg/kg) and returned to their aquaria. Urine and bile were collected as described above.
Analytical procedures Phenol red and its conjugates in bile and urine were determined according to the acid hydrolysis procedure described by Guarino and Anderson (1976). T o determine parent phenol red in the plasma, we mixed specimens (0.2~11)with 1 ml of methanol and centrifuged the mixture at lOOOg for l0min. A portion (0.8ml) of the supernatant was mixed with 2.2 ml of 0.3 M-NaOH and the absorbance at 555 nm was determined. Standard curves were prepared by adding phenol red to control urine, bile, and plasma specimens. T h e limits of determination of phenol red, based on specimen volumes analysed, were 0.1 pg/O.5 ml urine, 2 pg/O.Ol ml bile, and 0.2 pg/0.2 ml plasma. T o investigate further the presence of metabolites, we treated urine and bile with the enzymes glucuronidase and sulphatase. Samples were incubated with or without enzymes in 0 1 M acetate buffer (pH 5.0)for 18 h a t 3 7 T , and the phenol red concentrations were compared. Additionally, urine and bile (enzyme-treated and non-treated) were analysed by thin layer chromatography according to the procedure of Guarino and Anderson (1976). Samples were applied to Brinkmann Silica Gel G-HR plates (Brinkmann Instruments Co., Westbury, NY, USA) and developed in a solvent system of ethanol-nbutanol-3 M ammonium hydroxide (1 1 :40 : 19 by vol.). Plasma protein binding of phenol red was determined by ultrafiltration with the Centrifree Micropartition System (Amicon Corp., Danvers, MA, USA). Phenol red was added to control plasma at concentrations ranging from 1 to 100pg/ml. These plasma samples ( 0 5 ml) were applied to the ultrafilters and were centrifuged at lOOOg for 1 h at 25°C. T h e ultrafiltrates were analysed for phenol red concentration. Calculated binding values were corrected for losses (3%) in recovery of free phenol red caused by non-specific absorption to the ultrafiltration apparatus. Pharmacokinetic modelling Pharmacokinetic modelling of plasma data was performed with the iterative, least-squares program PCNONLIN (Statistical Consultants, Inc., Lexington, KY, USA). A two-compartment phannacokinetic model best described the plasma concentrations of parent compound after i.v. dosing. Intercompartmental clearance (ClJ, total body clearance (Cl,,),and apparent volumes of distribution (of the central compartment ( Vl), peripheral compartment ( V 2 ) , and at steady state ( V S J )were calculated with established compartmental modelling equations (Barron et al. 1990).
Results Phenol red was not found in the plasma, urine, or bile after oral dosing in the channel catfish. After i.v. dosing, phenol red concentrations in the plasma exhibited a rapid, biphasic decline (figure 1); at 2 h, plasma concentrations were below the limit of determination ( < 1 pg/ml). Plasma protein binding of phenol red was 19%. Binding was independent of concentration over a range of l-lOOpg/rnl, which encompassed the levels observed in plasma after i.v. dosing. Pharmacokinetic values for phenol red are summarized in table 1. Phenol red was highly concentrated in the bile and urine of channel catfish with no evidence of conjugative metabolism. T h e concentration (mean fSD) of phenol red in bile was 3714 & 873 pg/ml at 24 h. T h e concentration in urine was highest
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I
I " " " " " " " ' ~ ' " " 0.25
0.00
0.50
0.75
1.oo
Time (h)
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Figure 1. Plasma concentration of phenol red after i.v. dosing in the channel catfish. Data are the meansf S D of five fish; dose of phenol red was 10mg/kg.
Table 1. Pharmacokinetic values for phenol red after i.v. dosing (10mg/kg) in channel catfish. Parameter"
Value 104 18.4 18.2 1.95 2.28 21.4 7.68 4.39 15.2 626 658 81.6 143 225
"Abbreviations are as follows: A and B are the intercepts and tl and P are the rate constants for the distribution and elimination phases, respectively; t l I z nand t,/,# are the half-lives for distribution and elimination phases, respectively; k, and k,, are the intercompartmental transfer constants; AUC is the area under the plasma concentration-time curve; Cl, and Cl, are the intercompartmental and total body clearances; V , and V, are the volumes of the central and peripheral compartments; V,, is the apparent volume of distribution at steady state.
,
Concentration and cumulative excretion of phenol red in the urine of channel catfish after i.v. dosing. Open bars, concentration (pglml); shaded bars, cumulative percentage dose.
Figure 2.
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(121 f44pglml) at the 0-5-1 h collection interval (figure 2). T h e plasma concentration at 0-75 h (midpoint of the 05-1 h urine collection interval) was 3.79 0.68 pg/ml. Thus, the urine :plasma concentration ratio was approximately 32 : 1. Biliary excretion was the primary route of elimination of phenol red. At 24 h, 55 - 9%of the dose was recovered in the bile compared with 24 & 4% in the urine. Renal excretion of phenol red was very rapid (figure 2). Approximately 5% of the dose was eliminated in the urine within 0 5 h; at 8 h, renal excretion of phenol red was nearly complete. There were no differences in the amounts of phenol red excreted in the bile and urine after exposure to MS-222 or an ice-water bath. After MS-222 treatment, 59 f7% of the administered dose was excreted in the bile and 19 f 4 % was excreted in the urine at 24 h. After cold anaesthesia, 59 3% and 20 f9% was excreted in the bile and urine, respectively. Downloaded by [McMaster University] at 13:42 01 April 2016
+
Discussion Absorption Phenol red was not detectable in the excretory fluids of channel catfish after oral dosing, indicating little or no absorption from the gastrointestinal tract. Phenol red is poorly absorbed in mammals and has been used as a non-absorbable marker in gastrointestinal absorption studies (McLeod et al. 1968, Ashley and Levy 1973). T h e intestinal absorption of foreign organic anions occurs mainly by passive diffusion across the membrane lipid barrier (Ashley and Levy 1973, Lanman et al. 1971). T h e low lipid-to-water partition coefficient of phenol red is probably the most important physicochemical parameter limiting its absorption; organic acids with higher partition coefficients (i.e. hippuric acid and sulphanilic acid) are more extensively absorbed (Lanman et al. 1971).
Pharmacokinetics Phenol red concentrations in the plasma after i.v. dosing were well described by a two-compartment pharmacokinetic model. Elimination of phenol red from the plasma was very rapid (table 1). T h e low apparent volume of distribution at steady state (225 ml/kg) indicated limited tissue distribution. Phenol red may have been confined principally to the excretory tissues and extracellular fluids. T h e extracellular fluid volume in channel catfish has been estimated at 183 ml/kg (K'itzman et al. 1990). A two-compartment pharmacokinetic model has been used to describe the plasma elimination of phenol red in other animals (Hinchcliff et al. 1987). In channel catfish, certain pharmacokinetic values for phenol red were similar to those reported in mammalian species; the elimination half-life of phenol red was 21 min in the channel catfish (table 1) compared with values ranging from 13 to 25 min in horses (Hinchcliff et al. 1987), cattle (Mixner and Anderson 1958), and humans (Gault 1966). A half-life of 2.4min has been determined in rats (Inoue et al. 1985). In other fish species (i.e. hagfish, dogfish, skate, and flounder), elimination of phenol red is much slower than in channel catfish; elimination half-lives from 26 to 62 h have been reported (Pritchard et al. 1980). T h e total body clearance and volumes of distribution of phenol red were considerably larger in channel catfish compared with values reported for mammalian species (Hinchcliff et al. 1987, Yasuhara et al. 1985).
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Plasma protein binding Plasma protein binding in channel catfish was low and independent of concentration (1-100 pglml). I n mammals (e.g. human, rat, and dog), plasma protein binding of phenol red is extensive and is concentration-dependent (Yasuhara et at. 1985, Inoue et al. 1985, Kragh-Hansen et al. 1972, Russel et al. 1987). For example, plasma protein binding in dogs decreased from 85% to 60% at concentrations ranging from 5 to 450 pg/ml (Russel et al. 1987). In the spiny dogfish, plasma protein binding is also high and dose-dependent (Guarino and Anderson 1976). T h e low plasma protein binding in channel catfish may have influenced the extravascular distribution of phenol red. In analbuminaemic rats, the volume of distribution and clearance of phenol red are much larger than in normal rats; these observations were attributed to the much lower binding capacity of serum proteins and an increased extrarenal distribution in the mutant animals (Inoue et al. 1985). Metabolism Conjugates of phenol red were not found in the bile or urine of channel catfish. Considerable interspecies differences exist in the conjugative metabolism (i.e. glucuronidation) of phenol red. Phenol red is excreted unchanged in the urine of humans and dogs (Smith 1973). I n renal-ligated rats, phenol red is excreted in considerable amounts (20-35% of the total phenol red) in the bile as its glucuronic acid conjugate (Hart and Schanker 1966, Homan and Guarino 1974, Collado et al. 1988). In the spiny dogfish and little skate, the glucuronide comprised 25% and 65%, respectively, of the total phenol red excreted in the bile in 24 h; in contrast, phenol red was not metabolized by the hagfish and winter flounder (Pritchard et al. 1980). No other metabolites of phenol red were found in the above studies. Channel catfish are capable of forming glucuronic acid conjugates of phenolic compounds. Glucuronyltransferase activity (with p-nitrophenol as a substrate) in microsomes of the channel catfish is similar to that of the rat when in vitro preparations are incubated at 37°C (Short et al. 1988). Active glucuronidation of 1naphthol has been demonstrated in vivo in the channel catfish (Stehly and Plakas 1992). T h e metabolism of phenol red in channel catfish may have been precluded by its extremely rapid elimination. Excretion Renal and biliary excretion of phenol red in channel catfish accounted for 24% and 55%, respectively, of the i.v. dose at 24 h. T h e amounts of phenol red excreted in the urine and bile were not affected by exposure to MS-222 or an ice-water bath. In the spiny dogfish and winter flounder, approximately 35% of an i.v. dose was excreted in the urine in 24 h; similar amounts were excreted in bile (Pritchard et al. 1980). In humans, 85% and 3% of an i.v. dose were eliminated in the urine and bile, respectively, within 6 h (McLeod et al. 1968). In dogs, renal and biliary excretion accounted for 75% and 3%, respectively, of an i.v. dose at 3 h (Kim and Hong 1962). In rats, ratios of approximately 1 : 1 (Yasuhara et al. 1985) and 3 : 1 (Fleck and Braunlich 1986) have been reported for the urinary : biliary excretion (as percentage of the dose) of phenol red at 1 h after i.v. dosing.
Conclusions Plasma concentrations of phenol red after i.v. dosing in channel catfish were well described by a two-compartment pharmacokinetic model. Elimination was ex-
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tremely rapid and similar to the rates observed in mammals. Plasma protein binding of phenol red was much lower in channel catfish than in mammalian species. Biliary excretion of the unchanged molecule was the primary route of elimination of phenol red in channel catfish; however, active renal transport was also evident. T h e molecular weight threshold for extensive biliary excretion of phenol red appears lower in fish than in mammalian species. T h e metabolism and rate of elimination of phenol red vary widely among species. These differences emphasize the uncertainty in extrapolating disposition data for drugs or other xenobiotics from one species to another.
Acknowledgements
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The authors thank L. Torrans, J. C. Jones, and B. Deavours for supplying the catfish; M. Moxey for technical assistance; J. Holley for advice on cannulation procedures; and C. Collier for his design and construction of restraining cages.
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