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

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)

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 an internal standard.
• Added enzyme at an appropriate pH, incubated at 60 °C.
• Each time-point pulled sample and precipitate protein with TCA.
• Analyzed by LC-MS/MS (typical conditions below).

Results

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

• 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

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

Recombinant GUS Comparison - 6MAM Conversion

• 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

• 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

• 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 hydrolyzed 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.