Abstract
The apparent oral clearance of busulfan has been observed to vary as much as 10-fold in the population of children and adults receiving high-dose busulfan. The only identified elimination pathway for busulfan involves glutathione conjugation. The reaction is predominantly catalyzed by glutathione S-transferase (GST) A1-1, which is present in both liver and intestine. The purpose of this study was to compare busulfanVmax/Km in cytosol prepared from adult human liver and small intestine. Tetrahydrothiophenium ion formation rate per milligram of cytosolic protein was constant along the length (assessed in 30-cm segments) of three individual small intestines. A 30-cm-long intestinal segment 90–180 cm from the pylorus was chosen to be representative of intestinal cytosolic busulfan conjugating activity. BusulfanVmax/Km (mean ± SD) in cytosol prepared from 23 livers and 12 small intestines was 0.166 ± 0.066 and 0.176 ± 0.085 μl/min/mg cytosolic protein, respectively, in incubations with 5 μM busulfan, 1 mM glutathione, and 2 mg of cytosolic protein. The relative content of GSTα (A1-1, A1-2, and A2-2) was compared for human liver and intestinal cytosol using Western blot. The levels of GSTα in liver and intestinal cytosol were 1.12 ± 0.56 and 1.36 ± 0.32 integrated optimal density units/5 μg cytosolic protein, respectively. Busulfan conjugation in vitro was comparable per milligram of cytosolic protein in liver and intestinal cytosol.
Footnotes
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Send reprint requests to: John T. Slattery, Ph.D., Department of Pharmaceutics, University of Washington, Box 357610, Seattle, WA 98195-7610.
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This work was supported in part by National Institutes of Health Grants CA18029, GM32165, GM07750, and ES07033.
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↵2 The extraction ratio was estimated with the highest and lowest busulfan CL/F reported for a child and an adult, 430 and 80 ml/min/m2, respectively, and dividing by liver blood flow, 876 ml/min/m2. Because the liver and intestine are anatomically arranged in sequence, the extraction ratio represents the contributions from these two organs. We also assume that the liver is the main eliminating organ for busulfan.
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↵3 F. Farin, personal communication.
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↵4 We base this estimate on a whole organ weight for the liver and small intestinal mucosal scrapings of 21.4 and 1.35 g per kg body weight, respectively (Thummel et al., 1994; International Commission on Radiological Protection, 1975). The recovery of cytosolic protein from homogenates of liver and intestinal mucosal scrapings is 80 and 18 mg of cytosolic protein/g of tissue, respectively. Thus, the mean busulfanVmax/Km for livers and intestines was multiplied by the cytosolic protein recovery factor and the respective organ weight to yield a total organ busulfanVmax/Km(μl/min/kg body weight).
- Abbreviations used are::
- GSH
- glutathione
- THT
- tetrahydrothiophene
- THT+
- tetrahydrothiophenium ion
- GST
- glutathione S-transferase
- CL/F
- apparent oral clearance
- AUC
- area under the plasma concentration-time curve
- CDNB
- 1-chloro- 2,4-dinitrobenzene
- Received July 24, 1997.
- Accepted October 1, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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