Metabolic cages fitted with urine collectors were used in this experiment. After 13 weeks of HFD, urine samples of each individual rat, which were maintained in metabolic cages, were collected in urine tubes containing 0.1% sodium azide as an antimicrobial agent. The collected urine samples were then stored at -80°C prior to analysis. For the 1H NMR analysis, a phosphate buffer (KH2PO4) was prepared in D2O (pH 7.4) containing 0.

1% TSP and 0.1% imidazole. The pH of the D2O was adjusted to 7.4 using 1 M NaOD solution.

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Thawed urine samples were centrifuged at 13000 rpm for 10 minutes. Then, 400 ?l of the centrifuged sample was mixed with 200 ?l phosphate buffer in a 1.5 ml Eppendorf tube and sonicated for 5 minutes at room temperature to remove air bubbles. The urine sample was transferred into a 5 mm NMR tube and the 1H NMR spectrum was obtained from a 500 MHz Varian INOVA NMR spectrometer (Varian Inc.

, California, USA), functioning at a frequency of 499.887 MHz at room temperature (26 ºC). For each sample, 64 scans were recorded using a pulse width (PW) 21.

0 ?s (90°) and relaxation delay (RD) 2.0 s to result in a total acquisition time of 206 s. The spectral width was adjusted to the range between -1.00 and 14.

00 ppm.  A PRESAT sequence was applied after acquiring the 1H NMR in order to suppress the residual water signal with low power selective irradiation.  Deuterium oxide was used as the internal lock and 0.1% of trimethylsilylpropionate (TSP) was used as calibration standard.Metabolite identification was accomplished by 2D NMR measurement techniques, which resolve the problem of signal overlaps of different metabolites in 1D NMR by to spreading the resonances into a second dimension, despite the long acquisition times required in 2D NMR measurements 30,31. Thus, in this study, two dimensional (2D) 1H-1H J-resolved and heteronuclear multiple bond correlation (HMBC) experiments were conducted to confirm the NMR assignment of metformin in the urine.

The parameters are: number of scans = 8, number of increments = 256, acquisition time = 0.625 s, and relaxation delay = 1 s, leading to the total experiment run time of 2 hours 39 mins. In addition, HMBC spectra of urine was also obtained using 64 scans, number of increments = 256, and relaxation delay = 1 s (total experiment run time = 11 hours 2 mins).

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