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Dr. Andrew Lee is the co-founder and Chief Scientific Officer of Integrated Micro-Chromatography Systems, Inc. (IMCS), a leader in recombinant enzymes and micro-purification technologies. After receiving his B.S. in Animal Physiology and Neuroscience from the University of California, San Diego in 2003, he began his career in Allergan in Irvine, CA followed by research at The Scripps Research Institute in La Jolla, CA, and Seoul University in South Korea. In 2010, he earned his Ph.D. in Chemistry and Biochemistry from the University of South Carolina in Columbia, SC, where he stayed after graduation to start A&Q NanoDesigns, LLC—paving the way for the founding of IMCS in 2013.

With over 20 years of experience in analytical chemistry and biomedical engineering, Andrew has co-authored over 40 peer-reviewed journal articles on human embryonic stem cell culture, genetic engineering, nanomaterial design, protein and enzyme design, drugs of abuse monitoring, and automated micro-chromatography. He also holds over 10 patents on vaccine stabilization, rapid cell engineering, and novel enzymes. He has recently been awarded two NIH fast track small business grants, one under the SBIR and the other under the STTR program. These two grants were awarded to IMCS to manufacture over 20 different enzymes to produce sugar-modified fatty acids, or gangliosides, a critical component of the brain and nervous system.

Small molecule metabolism progresses through phase I and phase II. Phase I primarily undergoes oxidation or hydrolysis to convert hydrophobic analytes to expose more polar functional groups. Phase II metabolism involves conjugation reactions such as glucuronidation, acetylation, or sulfation. Often in pharmacokinetics, enzymes are used to liberate the glucuronide or sulfate conjugates to monitor phase II metabolism and excretion of the target molecule.

Here, we present data that suggest glucuronidases display different sensitivities to urea concentrations and pH variances in urine. Study shows that some enzymes will lose over half of their activity at moderate urea concentrations, and quickly lose efficiency with even subtle pH shifts (i.e. 6 to 5.5). Such sensitivities to urea concentrations, a natural component of urine, and variances due to pH shifts could attribute to the inconsistencies in hydrolysis, or potentially missing target analytes altogether.

We also identified certain natural metabolites from food can inhibit enzyme activity, resulting in the decreased recovery of the target analyte. Without carefully monitoring both glucuronide and aglycone targets, the assumption of complete hydrolysis or quantitative recoveries may not be as accurate as perceived from past reports.

IMCSzyme RT is the latest generation of room temperature glucuronidase manufactured by IMCS. This enzyme has been carefully engineered to outperform other commercially available enzymes on many challenging urine specimens. BpGUS is a recombinant enzyme expressed in E. coli with its gene sequence based on Brachyspira pilosicoli. BpGUS contains a poly-his tag for immobilized metal affinity chromatography (IMAC) followed by buffer exchange into a proprietary stabilizer compatible with mass spectrometry (MS). Enzyme activities were measured using a substrate-metabolite cross titration assay where substrate and metabolite concentrations varied while BpGUS concentration remained constant. Opioid-positive urine specimens were obtained from a national testing laboratory. Negative controls and clinical specimens were buffered and hydrolyzed with IMCSzyme RT or BpGUS.

BpGUS activity drops approximately 15% at 25 mM urea or 70 mg/dL and drops nearly 40% when urea concentration is 100 mM or 350 mg/dL. In contrast, the activity of IMCSzyme RT activity drops approximately 10% and 30% at the same urea concentrations. Well-known inhibitor D-saccharic acid 1,4-lactone monohydrate has a Ki value of 0.001 mM towards BpGUS, which is one of the lowest inhibition concentrations reported thus far for a glucuronidase. Other natural metabolites inhibiting this enzyme show higher Ki values ranging between 40-50 µM, and other common metabolites from coffee, vitamin C, and glucuronic acid did not have a significant impact on either enzyme. The potential negative impact is highlighted with six patient samples that exhibit varying degrees of inhibition. In one patient out of six, oxymorphone recovery is incomplete despite the addition of a large quantity of enzyme.

Not all enzymes have the same tolerances toward inhibitors in urine. One approach to avoid the complexity of natural inhibitors or urea reducing the hydrolysis efficiency is to dilute down the urine to lower the urea concentration and the concentration of the natural inhibitors. Such dilution is also beneficial in achieving the target pH for optimal hydrolysis, whereas limited dilution or buffer addition can result in reduced pH control. Heterogeneity of urine is a common fact, even if sourced from a single patient, the hydration levels and food metabolites will significantly vary. Although further dilution of urine will dilute target analytes, and possibly require a higher instrument sensitivity, there will always be an economical and practical balancing act of efficient enzymatic hydrolysis.