Detection of an Endogenous Urinary Biomarker Associated With CYP2D6 Activity Using Global Metabolomics

Jessica Tay-Sontheimer; Laura M Shireman; Richard P Beyer; Taurence Senn; Daniela Witten; Robin E Pearce; Andrea Gaedigk; Cletus L Gana Fomban; Justin D Lutz; Nina Isoherranen; Kenneth E Thummel; Oliver Fiehn; J Steven Leeder; Yvonne S Lin

Disclosures

Pharmacogenomics. 2014;15(16):1947-1962. 

In This Article

Discussion

To our knowledge, this is the first study using global metabolomics to identify endogenous biomarkers of CYP2D6 activity in humans. Using this approach, we obtained a list of unique ions (m/z and retention time) in urine samples from a cohort of pediatric subjects and selected the ion m/z 444.3102 (M1) from our ESI+ training set data because its abundance correlated significantly with DM/DX parent-to-metabolite ratios in both spot and timed urine samples in these subjects. We were unable to identify M1 based on the parent mass and product ion fragmentation spectra in metabolomics databases. By restricting our investigation to the ions present in both spot (predose) and timed (postdose) urine samples, DM and its metabolites were excluded as endogenous biomarker candidates. As expected, DM metabolites were significantly correlated with DM/DX in timed but not spot samples (Table 2). Additional significant ions that were present only in timed samples may be uncharacterized metabolites of DM.

The mass transition of M1 to the most abundant product ion (m/z 444.3 → 98.1) in ESI+ mode was monitored using a targeted approach on a QqQ instrument. In the pediatric training set, M1 was present in all non-PM individuals and absent in all but one PM individuals, even with increased injection volumes for PM subjects. Admittedly, the lower limit of detection of our targeted assay is unknown without a standard, but an M1 peak diluted over 250-fold below the mean training set M1 abundance was visually observable. For our statistical comparisons, we assigned a low value of M1 for samples with experimentally undetectable levels, allowing us to conservatively and semiquantitatively compare the two phenotypic groups. The creatinine/M1 ratio clearly distinguished PMs from non-PMs.

We verified that creatinine/M1 was associated with DM/DX in both spot and timed urine samples in the validation set of urine collected from an additional 95 children. PMs have lower M1 abundances than non-PMs (p < 0.0001), suggesting that M1 is likely a product of a reaction catalyzed by CYP2D6. However, much of the variance in the relationship between log-transformed DM/DX and creatinine/M1 is still unexplained (r2 ˜ 0.3). A stronger correlation with DM/DX would likely be observed if M1 were normalized by its unidentified precursor in a parent-to-metabolite molar ratio.

The creatinine/M1 ratio differed among activity score groups; however, there was appreciable overlap among non-PMs (activity scores between 0.5 and 3; Figure 5). The differentiation between activity scores may improve if the parent-to-M1 molar ratio can be used. In addition, fine-tuning CYP2D6 genotype by taking recently described enhancer SNPs into account[46] may alter activity score assignments. These enhancer SNPs appear to impact the regulation of CYP2D6 expression and thereby modulate an individual's activity.

Although age-dependent increases in CYP2D6 mRNA, and microsomal protein and activity levels have been reported in human fetal and pediatric liver,[47,48] we observed no correlation between log M1 abundance and age in our study cohort (data not shown). However, in vivo pharmacokinetic data and longitudinal phenotyping studies indicate that genetic variation in CYP2D6 is a more important determinant of interindividual variability in activity than ontogeny.[10,11,37] Moreover, neither log M1 abundance nor creatinine concentration differed by gender or urinary pH. Creatinine was used to normalize for urine concentrations in our study, and urinary creatinine concentrations were not significantly correlated with log M1 in any dataset except for the spot urine samples of the training set (p = 0.01). Urinary creatinine concentrations have been reported to change with age,[49] and a significant association between creatinine concentrations and age was observed in the training set (p = 0.036 and < 0.0001 in the spot and timed urine collections, respectively) but not in the validation set. Admittedly, creatinine concentrations may influence the interpretation of the data in the training set, but it does not appear to be an important confounder in the validation set.

In addition to distinguishing CYP2D6 phenotype in pediatric subjects, the creatinine/M1 ratio was sensitive to inhibition of CYP2D6 in adults, increasing by approximately ninefold in adults during fluoxetine treatment as compared with control. Creatinine/M1 was less sensitive to CYP2D6 inhibition than DM/DX, once again reinforcing the need to account for the parent of M1.

M1 may join the growing list of endogenous CYP2D6 substrates and products. CYP2D6 catalyzed the O-demethylation of 5-methoxy-N,N-dimethyltryptamine, pinoline and 5-methoxytryptamine to bufotenine, 6-hydroxy-1,2,3,4-tetrahydro-β-carboline and serotonin, respectively, in human recombinant CYP2D6 and CYP2D6-transgenic mouse liver microsomes.[26,50] Additionally, CYP2D6 has been shown to hydroxylate and epoxygenate anandamide in human recombinant CYP2D6 and brain microsomal and mitochondrial preparations.[29] Recently, Cheng et al. reported that serotonin, 5-hydroxyindoleacetic acid, L-carnitine, acetyl-L-carnitine, pantothenic acid, 2′-deoxycytidine diphosphate, anandamide, N-acetylglucosaminylamine and stearoyl-L-carnitine concentrations differed significantly in brain homogenate and cerebrospinal fluid between wild-type and CYP2D6-transgenic mice.[31] Using chemical standards, many of these compounds were screened by LC-QTOF MS, but they were undetectable in our urine samples (data not shown). These urinary compound concentrations may be below our detection limit or elute with the column void volume (e.g., bufotenine). Many previously identified compounds were detected in specific tissues or biofluids, such as the brain or cerebrospinal fluid, and may not be excreted to an appreciable extent in the urine. Further investigation of these endogenous compounds as possible human CYP2D6 biomarkers may require the development of specific and sensitive targeted urinary assays or sampling of other biofluids.

In the field of metabolomics, the identification of complete unknowns from complex biological samples presents a substantial challenge. A limitation of our study is our current inability to identify M1. Unlike traditional metabolite identification, we have few clues regarding the structure of M1 since the precursor to M1 is also unknown. Preliminary efforts to obtain a high yield and pure isolate of M1 from urine for NMR analysis were unsuccessful. Despite the lack of structural information for M1, we would propose that the use of global metabolomics to identify endogenous biomarkers is akin to genome-wide association studies to identify SNPs associated with a particular disease. For example, the identified SNPs associated with a particular disease can be located in the protein-coding region of genes or are in linkage disequilibrium with SNPs in those regions. However, the majority of the identified SNPs (approximately 80%) are located in intergenic regions or noncoding introns.[51] Despite the apparent nonfunctional nature of these significantly associated SNPs, validation, replication and efforts to identify the causal SNPs have resulted in continued use of genome-wide association studies. Similarly, the relationship between M1 and CYP2D6 activity is still valid, even if the structure is currently unknown. Although structural identification of M1 is beyond the scope of the study at present, the identification of M1 and its parent will increase our understanding of the endogenous role of CYP2D6. The possibility that M1 or its precursor is a dietary component or a product of the microbiome cannot be excluded, but it is worth noting that the pediatric and adult studies were conducted independently and at two geographic sites, suggesting that regional or site-specific exposures can be ruled out as sources of M1.

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