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Initial Studies on the N-Glucosylation of Phenobarbital by Mouse Liver Microsomes Using a Radiochemical High-Performance Liquid Chromatographic (HPLC) Method

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Abstract

A method is described for the assay of phenobarbital N-glucosylation using UDP-D-[6-3H)glucose. The radioactive phenobarbital N-glucoside conjugates [(5/R)-PBG, (5S)-PBG] formed during the incubations were resolved from each other and from uncharacterized radioactive products by semipreparative HPLC. The product ratio of the N-glucosides of (5R)-PBG/(5S)-PBG was 2.9 for the crude liver homogenate and 3.0 ± 0.5 for the microsomes. Magnesium was necessary for optimal activity. The K m values for formation of (5R)-PBG, (5S)-PBG, and (5R + 5S)-PBG were 1.55 ± 0.35, 1.27 ± 0.14, and 1.47 ± 0.21 mM, respectively. The Vmax values for formation of (5R)-PBG, (5S)-PBG, and (5R + 5S)-PBG were 1.34 ± 0.05 × 10−6, 0.43 ± 0.01 × 10−6, and 1.77 ± 0.04 × 10−6 µmol/min/mg microso-mal protein, respectively. It was observed that at concentrations greater than 5 mM sodium phenobarbital, inhibition of formation of phenobarbital N-glucosides occurred. The product ratio of (5R)-PBG/(5S)-PBG is comparable to that observed in the urinary excretion studies with the mouse and opposite to that observed in urinary excretion studies in humans.

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REFERENCES

  1. B. K. Tang, W. Kalow, and A. A. Grey. Metabolic fate of phenobarbital in man: N-Glucoside formation. Drug Metab. Disp. 7:315–318 (1979).

    Google Scholar 

  2. D. Kadar, B. K. Tang, and A. W. Conn. The fate of phenobarbitone in children in hypothermia and at normal body temperature. Can. Anaesth. Soc. J. 29:16–23 (1982).

    Google Scholar 

  3. W. H. Soine, V. O. Bhargava, and L. K. Garrettson. Direct detection of l-(β-D-glucopyranosyl)phenobarbital in human urine. Drug Metab. Disp. 12:792–794 (1984).

    Google Scholar 

  4. W. H. Soine, P. J. Soine, T. M. England, J. W. Ferkany, and B. E. Agriesti. Identification of phenobarbital N-glucosides as urinary metabolites of phenobarbital in mice. J. Pharm. Sci. 80:99–103 (1991).

    Google Scholar 

  5. W. H. Soine, P. J. Soine, S. E. Mongrain, and T. M. England. Stereochemical characterization of the diastereomers of the phenobarbital N-β-D-glucose conjugate excreted in human urine. Pharm. Res. 7:402–406 (1990).

    Google Scholar 

  6. W. H. Soine, P. J. Soine, T. M. England, D. F. Welty, and J. H. Wood. LC determination of the diastereomers of 1-(β-D-glucopyranosyl)phenobarbital in human urine. J. Pharm. Biomed. Anal. 8:365–372 (1990).

    Google Scholar 

  7. W. H. Soine, P. J. Soine, T. M. England, B. M. Overton, and S. Merat. Synthesis of N-β-D-glucopyranosyl derivatives of barbital, phenobarbital, metharbital and mephobarbital. Carbohydr. Res. 193:105–113 (1989).

    Google Scholar 

  8. T. Gessner, A. Jacknowitz, and C. A. Vollmer. Studies of mammalian glucoside conjugation. Biochem. J. 132:249–258 (1973).

    Google Scholar 

  9. R. C. Duggleby. A nonlinear regression program for small computers. Anal. Biochem. 110:9–18 (1981).

    Google Scholar 

  10. B. K. Tang and G. Carro-Ciampi. A method for the study of N-glucosidation in vitro—Amobarbital-N-glucoside formation in incubations with human liver. Biochem. Pharmacol. 29:2085–2088 (1980).

    Google Scholar 

  11. G. Carro-Ciampi, M. Jurima, D. Kadar, B. K. Tang, and W. Kalow. N-Glucosidation of amobarbital in the cat. Can. J. Physiol. Pharmacol. 63:1263–1266 (1985).

    Google Scholar 

  12. S. E. Mongrain and W. H. Soine. Product enantioselectivity in the N-glucosylation of amobarbital by cats. Drug Metab. Disp. 19;1012–1013 (1991).

    Google Scholar 

  13. S. K. Bansal and T. Gessner. A unified method for the assay of uridine diphosphoglucuronyltransferase activities toward various aglycones using uridine diphospho[U-14C]glucuronic acid. Anal. Biochem. 109:321–329 (1980).

    Google Scholar 

  14. N. Arima. Acyl glucuronidation and glucosidation of pranoprofen, a 2-arylpropionic acid derivative, in mouse liver and kidney homogenates. J. Pharmacobio-Dyn. 13:724–732 (1990).

    Google Scholar 

  15. R. S. Labow and D. S. Layne. The formation of glucosides of isoflavones and of some other phenols by rabbit liver microsomal fractions. Biochem. J. 128:491–497 (1972).

    Google Scholar 

  16. N. Arima. Stereoselective acyl glucuronidation and glucosidation of pranoprofen, a 2-arylpropionic acid derivative, in mice in vivo. J. Pharmacobio-Dyn. 13:733–738 (1990).

    Google Scholar 

  17. H. J. Fromm. Initial Rate Enzyme Kinetics, Springer-Verlag, New York, 1975.

    Google Scholar 

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Authors and Affiliations

  1. Department of Medicinal Chemistry, Virginia Commonwealth University, Box 540, MCV Station, Richmond, Virginia, 23298-0540

    William H. Soine, Hosai Safi & Richard B. Westkaemper

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  1. William H. Soine
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  2. Hosai Safi
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  3. Richard B. Westkaemper
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Soine, W.H., Safi, H. & Westkaemper, R.B. Initial Studies on the N-Glucosylation of Phenobarbital by Mouse Liver Microsomes Using a Radiochemical High-Performance Liquid Chromatographic (HPLC) Method. Pharm Res 9, 613–616 (1992). https://doi.org/10.1023/A:1015889707922

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