Detection of urinary drug metabolite (xenometabolome) signatures in molecular epidemiology studies via statistical total correlation (NMR) spectroscopy

Abstract

Western populations use prescription and nonprescription drugs extensively, but large-scale population usage is rarely assessed objectively in epidemiological studies. Here we apply statistical methods to characterize structural pathway connectivities of metabolites of commonly used drugs detected routinely in 1H NMR spectra of urine in a human population study. 1H NMR spectra were measured for two groups of urine samples obtained from U.S. participants in a known population study. The novel application of a statistical total correlation spectroscopy (STOCSY) approach enabled rapid identification of the major and certain minor drug metabolites in common use in the population, in particular, from acetaminophen and ibuprofen metabolites. This work shows that statistical connectivities between drug metabolites can be established in routine "high-throughput" NMR screening of human samples from participants who have randomly self-administered drugs. This approach should be of value in considering interpopulation patterns of drug metabolism in epidemiological and pharmacogenetic studies.

Figure 1.

Schematic showing the chemical structures of acetaminophen (A) and its metabolites. The numbers and letters refer to the position of ¹³C and ¹H in the molecules.

Figure 2.

Typical ¹H NMR spectra of urine from participants: (A) no analgesic intake; (B) after ingestion of acetaminophen; (C) expansion on acetaminophen region at δ 2.16; (D) after ingestion of ibuprofen; (E) showing the characteristic of the anomeric proton signal of the ibuprofen glucuronide rings. For a key to the identity of ibuprofen metabolites, refer to Figures 1 and 6.

Figure 3.

(A) The OPLS-DA loading coefficient plot showing that the basis of differentiation between these two groups (acetaminophen ingestion versus controls) primarily results from the presence of acetaminophen (A), acetaminophen glucuronide (AG), acetaminophen sulfate (AS), and the N-acetylcysteine conjugate of acetaminophen (NAC) denoted by the red/orange color of the resonances indicating a r² > 0.5 in discriminating between the two groups. (B) Expansion for the aromatic region showing signals for acetaminophen and its related metabolites (C) Expansion on acetaminophen region at δ 2.16. (D) The O-PLS-DA scores plot showing discrimination between two classes (blue, control; red, participants who had taken acetaminophen).

Figure 4.

(A) STOCSY plot derived from the correlation matrix calculated between the data point at the peak maximums of the N-acetyl proton signal of AG resonance (δ 2.17) and all other data points, as indicated by the arrow, showing strong correlation (red/orange data points) with resonances at δ 3.62, 3.89, 5.1, 7.13, and 7.36. Slightly weaker correlations with A and AS. (B) Expansion for the aromatic region showing signals for acetaminophen and its related metabolites. (C) Expansions for the δ 2.17 N-acetyl resonance of acetaminophen metabolites.

Figure 5.

Schematic for acetaminophen glucuronide comparing the intramolecular ¹H-¹H and ¹H-¹³C correlations detected in the TOCSY (blue), HSQC (brown), HMBC (green), and intra-and intermolecular (dotted line) correlations given by STOCSY (red) analyses. Note that these are the actual observed, and not the theoretical correlations. Although the NMR parameters were optimized for this study by adjusting the acquisition/processing parameters, it may be possible to observe further correlation structures from each of these measurements.

Figure 6.

Schematic showing the chemical structures of ibuprofen and its major urinary metabolites. The letters and numbers refer to the position of ¹³C and ¹H of the molecules.

Figure 7.

(A) OPLS-DA loading coefficient plot showing that the basis of differentiation between these two groups (ibuprofen users and nonusers) primarily results from the presence of 2-hydroxy, carboxy, and glucuronide conjugates of ibuprofen. (B) The OPLS-DA scores plot showing discrimination between two population classes (blue, control; red, participants who had consumed ibuprofen).

Figure 8.

(A) STOCSY plot derived from the correlation matrix between all data points and the data point at the peak maxima relating to the combined resonances from 2-hydroxy signal of ibuprofen and its glucuronide resonance (δ 1.21), as indicated by the arrow. Strong correlations (red/orange data points) between this resonance and many related ibuprofen resonances can be observed. (B) Expansion for the aromatic region showing resonances for ibuprofen and its related metabolites. (C) Expansions for the δ 0.6–1.6 region showing resonances of ibuprofen metabolites. (IBU, ibuprofen; M1, 2-hydroxy ibuprofen; M2, 1-hydroxy ibuprofen; M3, 3-hydroxy ibuprofen; M4, carboxy ibuprofen; M5, ibuprofen glucuronide; M6, 2-hydroxy ibuprofen glucuronide conjugate; M7, carboxy ibuprofen glucuronide conjugate; M8, 3-hydroxy iburprofen glucuronide conjugate; and M9, 1-hydroxy ibuprofen glucuronide conjugate).