Evaluation of Different Enzymes on Hydrolysis Efficiencies of Glucuronide Drug Metabolites in Urine

By: Jim Blasberg, Harkewal Singh, Kevin Ray Sigma-Aldrich, St. Louis, MO

Introduction

β-glucuronidase (GUS) enzymes are utilized to hydrolyze glucuronide (gluc) drug metabolites to the parent drug, facilitating analysis by LC-MS/MS. Here we evaluate the hydrolysis efficiency of β-glucuronidase derived from Patella vulgata (limpet), Helix pomatia (snail), Red abalone, and E. coli on drugs of abuse and pain management in a synthetic urine matrix. Hydrolysis efficiencies vary from product to product and several parameters need to be investigated in determining which enzyme to use.

Background

Dimer of β-GUS (inset: expanded view of active site)

Dimer of β-GUS (inset: expanded view of active site)

 

Common Currency for Glucuronidase Activity

Common Currency for Glucuronidase Activity

One Fishman1 unit will liberate 1 μg phenolphthalein from phenolphthalein glucuronide per hour at 37οC All hydrolysis experiments were normalized for enzyme activity, i.e. equivalent Fishman units

 

Methods

  • Target glucuronides were spiked into synthetic urine.
  • Heavy parent (non-glucuronide) drug was spiked as internal standard.
  • Added enzyme at appropriate pH, incubated at 60 °C.
  • Each time-point, pulled sample and precipitate protein with TCA.
  • Analyzed by LC-MS/MS (typical conditions below).
Methods



Results

Effect of Enzyme Source

Codeine-gluc enzyme source

Codeine-gluc enzyme source

  • Enzyme added at 10 units/μL of urine and incubated at 60 °C.
  • Codeine glucuronide conversion was enzyme source dependent at 10 U/μL, 60 °C.
  • E. coli enzyme shows high activity early, but appears to lose activity after 1 hour at 60 °C.
  • Limpet enzyme shows >95% codeine  glucuronide conversion at 10 U/μL, 60 °C.

 

Temazepam-gluc enzyme source

Temazepam-gluc enzyme source

  • Enzyme added at 1 unit/μL of urine and incubated at 60 °C.
  • Temazepam glucuronide conversion was fast at 1 U/μL, 60 °C, for all enzymes tested.
  • Most benzodiazepine glucuronides show full conversion even at low titer and 0.5 hr @ 60C

 

Purified Abalone GUS

Purified Abalone GUS
  • Oxymorphone glucuronide hydrolysis was equivalent at 10 U/μL, 60 °C, for purified abalone GUS and recombinant GUS.

Recombinant GUS Comparison - 6MAM Conversion

Recombinant GUS Comparison



  • Traditional abalone exhibited substantial 6-MAM to morphine conversion.
  • 6-MAM conversion at 75 U/μL, 60 °C, was equivalent for purified abalone GUS and recombinant GUS.

Effect of Enzyme Titer - Purified Abalone

Codeine-gluc enzyme titer

Codeine-gluc enzyme titer

  • Purified abalone enzyme added at 1, 10, and 75 units/μL of urine and incubated at 60 °C.
  • Enzyme titer greatly affects conversion rate
  • Hydrolysis of codeine glucuronide completes in less than an hour at 75 U/μL.

Temazepam-gluc enzyme titer

Temazepam-gluc enzyme titer

  • Purified abalone enzyme added at 0.1, 1, and 10 units/μL of urine and incubated at 60 °C
  • Temazepam glucuronide conversion was less dependent on enzyme titer.
  • Hydrolysis of temazepam glucuronide completes in less than an hour at 1 U/μL.

 

Conclusions

  • Effect of Enzyme Source
    - Glucuronide conversion efficiency is enzyme dependent.
    - Glucuronide conversion efficiency is also substrate dependent.
  • Effect of Enzyme Titer
    - At high titer even difficult to convert substrates, such as codeine, are hydrolysed rapidly.
    - In general benzodiazepines, such as temazepam, hydrolyze rapidly even at low titers.
  • Comparison of Purified Abalone and Recombinant GUS
    - Purified abalone hydrolyzed oxymorphone glucuronide quickly at 10 U/μL, comparable to recombinant GUS.
    - Purified abalone induced minimal 6-monoacetyl morphine (6-MAM) conversion even at high titer.

Materials

     

Reference

  1. Fishman, W.H. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U. ed) 2nd ed., Volume II, 929-932

 

SOFT 2015