The retention time, predicted elemental compositions, observed mass and calculated mass, characteristic fragment ions, and description of metabolites of WS070117 in rat urine.

<p>The retention time, predicted elemental compositions, observed mass and calculated mass, characteristic fragment ions, and description of metabolites of WS070117 in rat urine.</p

Structures of WS070117 metabolites in rat urine and the proposed metabolic pathways.

<p>Structures of WS070117 metabolites in rat urine and the proposed metabolic pathways.</p

The stack of ¹³C NMR spectra of WS010117 and its four metabolites (in DMSO-d₆).

<p>The stack of ¹³C NMR spectra of WS010117 and its four metabolites (in DMSO-d₆).</p

Structure Elucidation of the Metabolites of 2', 3', 5'-Tri-<i>O</i>-Acetyl-<i>N</i>₆-(3-Hydroxyphenyl) Adenosine in Rat Urine by HPLC-DAD, ESI-MS and Off-Line Microprobe NMR

<div><p>2', 3', 5'-tri-<i>O</i>-acetyl-<i>N₆</i>-(3-hydroxyphenyl) adenosine (also known as WS070117) is a new adenosine analog that displays anti-hyperlipidemic activity both <i>in vitro</i> and <i>in vivo</i> experiments as shown in many preliminary studies. Due to its new structure, little is known about the metabolism of WS070117. Hence, the <i>in vivo</i> metabolites of WS070117 in rat urine following oral administration were investigated. Identification of the metabolites was conducted using the combination of high-performance liquid chromatography (HPLC) coupled with diode array detector (DAD), ion trap electrospray ionization-mass spectrometry (ESI-MS), and off-line microprobe nuclear magnetic resonance (NMR) measurements. Seven metabolites were obtained as pure compounds at the sub-milligram to milligram levels. Results of structure elucidation unambiguously revealed that the phase I metabolite, <i>N₆</i>-(3-hydroxyphenyl) adenosine (M8), was a hydrolysate of WS070117 by hydrolysis on the three ester groups. <i>N₆</i>-(3-hydr-oxyphenyl) adenine (M7), also one of the phase I metabolites, was the derivative of M8 by the loss of ribofuranose. In addition to two phase I metabolites, there were five phase II metabolites of WS070117 found in rat urine. 8-hydroxy-<i>N₆</i>-(3-hydroxy-phenyl) adenosine (M6) was the product of M7 by hydrolysis at position 8. The other four were elucidated to be <i>N₆</i>-(3-<i>O-β</i>-D-glucuronyphenyl) adenine (M2), <i>N₈</i>-hydroxy-<i>N₆</i>-(3-<i>O</i>-sulfophenyl) adenine (M3), <i>N₆</i>-(3-<i>O-β</i>-D-glucuronyphenyl) adenosine (M4), and <i>N₆</i>-(3-<i>O</i>- sulfophenyl) adenosine (M5). Phase II metabolic pathways were proven to consist of hydroxylation, glucuronidation and sulfation. This study provides new and valuable information on the metabolism of WS070117, and also demonstrates the HPLC/MS/off-line microprobe NMR approach as a robust means for rapid identification of metabolites.</p></div

¹H NMR derived HSQC (A) and HMBC (B) spectra of <i>N</i>₈-hydroxy-<i>N</i>₆-(3-<i>O</i>-sulfophenyl) adenine (structure see formula insert).

<p><b>A</b> secondary metabolite in rat urine following WS070117 oral administration. The NMR spectra were obtained in deuterated DMSO on a 500 MHz NMR spectrometer, equipped with a 1.7 PA TXI microprobe. (A) HSQC (acquisition time: 2 h): red cross-peaks are stemming from CH, CH₂ and CH₃ protons. (B) HMBC (acquisition time: 6 h): the correlation information derived from the marked cross-peaks is summarized in the formula insert.</p

HPLC chromatogram of WS010117 metabolites in urine of control (A) and administrated (B) rats at 299 nm.

<p>Peak 1: 3.60 min (M1); 2: 14.15 min (M2); 3: 21.58 min (M3); 4: 23.25 min (M4); 5: 29.48 min (M5); 6: 47.68 min (M6); 7: 48.73 min (M7); 8: 49.44 min (M8).</p

Quantitative Metabolomic Profiling of Plasma, Urine, and Liver Extracts by ¹H NMR Spectroscopy Characterizes Different Stages of Atherosclerosis in Hamsters

Atherosclerosis (AS) is a progressive disease that contributes to cardiovascular disease and shows a complex etiology, including genetic and environmental factors. To understand systemic metabolic changes and to identify potential biomarkers correlated with the occurrence and perpetuation of diet-induced AS, we applied ¹H NMR-based metabolomics to detect the time-related metabolic profiles of plasma, urine, and liver extracts from male hamsters fed a high fat and high cholesterol (HFHC) diet. Conventional biochemical assays and histopathological examinations as well as protein expression analyses were performed to provide complementary information. We found that diet treatment caused obvious aortic lesions, lipid accumulation, and inflammatory infiltration in hamsters. Downregulation of proteins related to cholesterol metabolism, including hepatic SREBP2, LDL-R, CYP7A1, SR-BI, HMGCR, LCAT, and SOAT1 was detected, which elucidated the perturbation of cholesterol homeostasis during the HFHC diet challenge. Using “targeted analysis”, we quantified 40 plasma, 80 urine, and 60 liver hydrophilic extract metabolites. Multivariate analyses of the identified metabolites elucidated sophisticated metabolic disturbances in multiple matrices, including energy homeostasis, intestinal microbiota functions, inflammation, and oxidative stress coupled with the metabolisms of cholesterol, fatty acids, saccharides, choline, amino acids, and nucleotides. For the first time, our results demonstrate a time-dependent metabolic progression of multiple biological matrices in hamsters from physiological status to early AS and further to late-stage AS, demonstrating that ¹H NMR-based metabolomics is a reliable tool for early diagnosis and monitoring of the process of AS