Pc3

FIGURE 5.6 A PCA plot that shows separation of a control population of rats based on diurnal differences in urine composition as detected by 1H NMR. PC3 versus PC5 scores plot of mean-centred data from NMR spectra of female rat urine collected during the day and night. I = night, A = day.

appropriate software, selection of abnormal samples can be achieved automatically on-line, with any sample defined as abnormal undergoing further NMR measurements or multivariate statistical analysis to ascertain the nature of the abnormality. Level 2: Classification of Toxicity Samples identified as dissimilar to matched control samples can be fitted to a series of mathematical models that define the multivariate boundaries for known classes of toxicity (Figures 5.4 and 5.5) [6,7,12,63]. Therefore, biofluid or tissue samples from experimental animals treated with novel drugs can be tested to ascertain if the drug induces biochemical effects that would infer a particular site or mechanism of toxicity. Level 3: Identification of the Biomarkers Once a given compound is established to reproducibly alter the metabolic state of an organism, the metabolites that differentiate between biofluid samples obtained from drug-treated and control rats can be elucidated to give insight into possible mechanisms of toxicity or dysfunction. For example, renal papillary necrosis was a condition for which no early biochemical markers of damage previously existed. However, following 1H NMR spectroscopic analysis of urine obtained from rats treated with model renal papillary toxins, perturbations in the levels of trimethylamine-N-oxide, N, N-dimethylglycine, dimethylamine, and succinate were found to be indicative of damage to the renal papilla [64,65]. Identification of biomarkers can be achieved by a variety of means. For many compounds, chemical structure can be identified by reference to databases containing information relating to chemical shift, spin-spin coupling, and relative intensity of resonances and the structures verified by spiking biofluids with authentic solutions of the proposed metabolites. For more challenging cases, then, it is necessary to disperse the 1H resonances either with selected 2D NMR experiments such as total correlation spectroscopy (TOCSY) or with chromatographic techniques to isolate the metabolite of interest prior to NMR or MS analysis. This procedure is illustrated in a study of the systemic biochemical effects of oral hydrazine-administration dosed in male Han Wistar rats, using metabo-nomic analysis of 1H NMR spectra of urine and plasma, HPLC-NMR (Figure 5.7), conventional clinical chemistry, and liver histopathology [66]. 1H NMR spectra of the biofluids were analyzed visually and via pattern recognition withPCA. PCA showed that there was a dose-dependent biochemical effect of hydrazine treatment on the levels of a range of low molecular-weight compounds in urine and plasma, which was correlated with the severity of the hydrazine-induced liver lesions determined by histopathol-ogy. In plasma, increases in the levels of free glycine, alanine, isoleucine, valine, lysine, arginine, tyrosine, citrulline, 3-D-hydroxybutyrate, creatine,

Chromatography Arginine Citrulline

FIGURE 5.7 500 MHz1H NMR spectra of endogenous metabolites separated from whole rat urine 56h post-dose hydrazine (120 mg/kg) with on-flow LC-NMR spectroscopy: (A) on-flow HPLC-NMR pseudo-2D NMR spectrum, (B) 2-aminoadipic acid and unidentified co-eluting metabolites, (C) Unidentified urinary component, (D) creatinine (E) N-acetyl-citrulline.

FIGURE 5.7 500 MHz1H NMR spectra of endogenous metabolites separated from whole rat urine 56h post-dose hydrazine (120 mg/kg) with on-flow LC-NMR spectroscopy: (A) on-flow HPLC-NMR pseudo-2D NMR spectrum, (B) 2-aminoadipic acid and unidentified co-eluting metabolites, (C) Unidentified urinary component, (D) creatinine (E) N-acetyl-citrulline.

histidine, and threonine were observed. Urinary excretion of hippurate, citrate, succinate, 2-oxoglutarate, trimethylamine-N-oxide, fumarate, and creatinine were decreased following hydrazine dosing, whereas taurine, creatine, threonine, N-methylnicotinic acid, tyrosine, f-alanine, citrulline, Na-acetyl-L-citrulline, and argininosuccinate levels were increased. In addition, several other previously unassigned resonances were detected in urine and plasma. Using HPLC-NMR spectroscopy, these were assigned to 2-aminoadipate, which has previously been shown to lead to neurological effects in rats (Figure 5.7). High urinary levels of 2-aminoadipate may explain the poorly understood neurological effects of hydrazine.

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