“…3-Hydroxypropylmercapturic acid (3-HPMA), an N -acetyl cysteine conjugate derived from AF, was also detected in day 1 (0−7 h) and day 1 (7−24 h) urine samples from the AF-dosed group. The presence of 3-HPMA in the urine is consistent with the metabolism of AF to toxic acrolein and the formation of a glutathione conjugate. − 3 A series of 600 MHz 1 H NMR spectra (δ 0.0−9.0) of urine from an AF-dosed animal at (A) predose, (B) day 1, (C) day 2, and (D) day 7. Key: M* is 3-HPMA, a metabolite of AF.…”
Section: Resultsmentioning
confidence: 74%
“…The presence of 3-HPMA in the urine is consistent with the metabolism of AF to toxic acrolein and the formation of a glutathione conjugate. [30][31][32][33] To investigate the time course of metabolic events in the rat following dosing of allyl formate, mean PC scores trajectories were calculated from the first two PCs of the normalized 1 H NMR spectra of urine, describing 66% of the total variance in the original data. The day 1 data were excluded from this analysis because of the presence of 3-HPMA.…”
Section: Characterization Of Af Effects On the Plasma Metabolite Prof...mentioning
The time-related metabolic events in rat liver, plasma, and urine following hepatotoxic insult with allyl formate (75 mg/kg) were studied using a combination of high-resolution liquid state and magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopic methods together with pattern recognition analysis. The metabonomics results were compared with the results of conventional plasma chemistry and histopathological assessments of liver damage. Various degrees of liver damage were observed in different animals, and this variation was reflected in all of the analyses. Furthermore, each analysis revealed a high degree of functional and structural recovery by the end of the study. The allyl formate-induced changes included hepatocellular necrosis, hepatic lipidosis, decreased liver glycogen and glucose, decreased plasma lipids, increased plasma creatine and tyrosine, increased urinary taurine and creatine, and decreased urinary TCA cycle intermediates. The observed reductions in hepatic glycogen and glucose suggest increased glucose utilization and are consistent with the expected depletion of hepatic ATP following mitochondrial impairment, assuming that there is a consequent increase in energy production from glycolysis. The increase in plasma tyrosine is consistent with impaired protein synthesis, a known consequence of ATP depletion. Partial least squares-based cross-correlation of the variation in the liver and plasma NMR profiles indicated that the allyl formate-induced increase in liver lipids correlated with the decrease in plasma lipids. This suggests disruption in lipid transport from the liver to plasma, which could arise through impaired apolipoprotein synthesis, as with ethionine.
“…3-Hydroxypropylmercapturic acid (3-HPMA), an N -acetyl cysteine conjugate derived from AF, was also detected in day 1 (0−7 h) and day 1 (7−24 h) urine samples from the AF-dosed group. The presence of 3-HPMA in the urine is consistent with the metabolism of AF to toxic acrolein and the formation of a glutathione conjugate. − 3 A series of 600 MHz 1 H NMR spectra (δ 0.0−9.0) of urine from an AF-dosed animal at (A) predose, (B) day 1, (C) day 2, and (D) day 7. Key: M* is 3-HPMA, a metabolite of AF.…”
Section: Resultsmentioning
confidence: 74%
“…The presence of 3-HPMA in the urine is consistent with the metabolism of AF to toxic acrolein and the formation of a glutathione conjugate. [30][31][32][33] To investigate the time course of metabolic events in the rat following dosing of allyl formate, mean PC scores trajectories were calculated from the first two PCs of the normalized 1 H NMR spectra of urine, describing 66% of the total variance in the original data. The day 1 data were excluded from this analysis because of the presence of 3-HPMA.…”
Section: Characterization Of Af Effects On the Plasma Metabolite Prof...mentioning
The time-related metabolic events in rat liver, plasma, and urine following hepatotoxic insult with allyl formate (75 mg/kg) were studied using a combination of high-resolution liquid state and magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopic methods together with pattern recognition analysis. The metabonomics results were compared with the results of conventional plasma chemistry and histopathological assessments of liver damage. Various degrees of liver damage were observed in different animals, and this variation was reflected in all of the analyses. Furthermore, each analysis revealed a high degree of functional and structural recovery by the end of the study. The allyl formate-induced changes included hepatocellular necrosis, hepatic lipidosis, decreased liver glycogen and glucose, decreased plasma lipids, increased plasma creatine and tyrosine, increased urinary taurine and creatine, and decreased urinary TCA cycle intermediates. The observed reductions in hepatic glycogen and glucose suggest increased glucose utilization and are consistent with the expected depletion of hepatic ATP following mitochondrial impairment, assuming that there is a consequent increase in energy production from glycolysis. The increase in plasma tyrosine is consistent with impaired protein synthesis, a known consequence of ATP depletion. Partial least squares-based cross-correlation of the variation in the liver and plasma NMR profiles indicated that the allyl formate-induced increase in liver lipids correlated with the decrease in plasma lipids. This suggests disruption in lipid transport from the liver to plasma, which could arise through impaired apolipoprotein synthesis, as with ethionine.
“…An appropriate internal standard was needed for quantitative measurements of NMR signals. TSP is not only suitable for referencing chemical shift but also has been widely used as an internal standard for quantitative analysis. − Hence we used TSP as an internal standard for measurements of the absolute quantity of 13 C-metabolites. We investigated dependence of the signal intensity of protons attached to 13 C on the polarization transfer time (i.e., the delay D2 in Figure or Δ in equations).…”
Quantitative characterization of C-labeled metabolites is an important part of the stable isotope tracing method widely used in metabolic flux analysis. Given the long relaxation time and low sensitivity ofC nuclei, direct measurement of C-labeled metabolites using one-dimensionalC NMR often fails to meet the demand of metabolomics studies, especially with large numbers of samples and metabolites having low abundance. Although HSQC-based 2D NMR methods have improved sensitivity with inversion detection, they are time-consuming and thus unsuitable for high-throughput absolute quantification of C-labeled metabolites. In this study, we developed a method for absolute quantification ofC-labeled metabolites using naturally abundant TSP as a reference with the first increment of the HMQC pulse sequence, taking polarization transfer efficiencies into consideration. We validated this method using a mixture of C-labeled alanine, methionine, glucose, and formic acid together with a mixture of alanine, lactate, glycine, uridine, cytosine, and hypoxanthine, which have naturalC abundance with known concentrations. We subsequently applied this method to analyze the flux of glucose in HepG2 cells infected with hepatitis B virus (HBV). The results showed that HBV infection increased the cellular uptake of glucose, stimulated glycolysis, and enhanced the pentose phosphate and hexosamine pathways for biosynthesis of RNA and DNA and nucleotide sugars to facilitate HBV replication. This method saves experimental time and provides a possibility for absolute quantitative tracking of the C-labeled metabolites for high-throughput studies.
“…Urine samples were obtained from an existing large-scale toxicological study resource. ,, Sprague–Dawley rats ( n = 7) were individually housed in standard metabolism cages (21 ± 3 ◦C, relative humidity 55 ± 15%) and acclimatized for six days prior to the start of the study ( t = 0 h). A standard diet (Purina chow 5002) and fresh water (acidified to pH 2.5 using HCl to prevent microbial growth) was available to each animal ad libitum .…”
Section: Methodsmentioning
confidence: 99%
“…Further study information has previously been published. 17 Additionally, a spot urine sample (5 mL) was obtained from a healthy human volunteer, according to established protocols, including filtration to remove cellular material (0.2 μm Minisart 16534K, Sartorius, Germany), and immediate storage at −40 °C until required for analysis. Urine samples were prepared following established protocols for NMR metabolome analysis.…”
A strategy for evaluating the performance of quantitative spectral analysis tools in conditions that better approximate background variation in a metabonomics experiment is presented. Three different urine samples were mixed in known proportions according to a {3, 3} simplex lattice experimental design and analyzed in triplicate by 1D (1)H NMR spectroscopy. Fifty-four urinary metabolites were subsequently quantified from the sample spectra using two methods common in metabolic profiling studies: (1) targeted spectral fitting and (2) targeted spectral integration. Multivariate analysis using partial least-squares (PLS) regression showed the latent structure of the spectral set recapitulated the experimental mixture design. The goodness-of-prediction statistic (Q(2)) of each metabolite variable in a PLS model was calculated as a metric for the reliability of measurement, across the sample compositional space. Several metabolites were observed to have low Q(2) values, largely as a consequence of their spectral resonances having low s/n or strong overlap with other sample components. This strategy has the potential to allow evaluation of spectral features obtained from metabolic profiling platforms in the context of the compositional background found in real biological sample sets, which may be subject to considerable variation. We suggest that it be incorporated into metabolic profiling studies to improve the estimation of matrix effects that confound accurate metabolite measurement. This novel method provides a rational basis for exploiting information from several samples in an efficient manner and avoids the use of multiple spike-in authentic standards, which may be difficult to obtain.
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