NMR Chemical Shift Ranges of Urine Metabolites in Various Organic Solvents

Signal stability is essential for reliable multivariate data analysis. Urine samples show strong variance in signal positions due to inter patient differences. Here we study the exchange of the solvent of a defined urine matrix and how it affects signal and integral stability of the urinary metabolites by NMR spectroscopy. The exchange solvents were methanol, acetonitrile, dimethyl sulfoxide, chloroform, acetone, dichloromethane, and dimethyl formamide. Some of these solvents showed promising results with a single batch of urine. To evaluate further differences between urine samples, various acid, base, and salt solutions were added in a defined way mimicking to some extent inter human differences. Corresponding chemical shift changes were monitored

Extensive Regulation of Diurnal Transcription and Metabolism by Glucocorticoids.

Altered daily patterns of hormone action are suspected to contribute to metabolic disease. It is poorly understood how the adrenal glucocorticoid hormones contribute to the coordination of daily global patterns of transcription and metabolism. Here, we examined diurnal metabolite and transcriptome patterns in a zebrafish glucocorticoid deficiency model by RNA-Seq, NMR spectroscopy and liquid chromatography-based methods. We observed dysregulation of metabolic pathways including glutaminolysis, the citrate and urea cycles and glyoxylate detoxification. Constant, non-rhythmic glucocorticoid treatment rescued many of these changes, with some notable exceptions among the amino acid related pathways. Surprisingly, the non-rhythmic glucocorticoid treatment rescued almost half of the entire dysregulated diurnal transcriptome patterns. A combination of E-box and glucocorticoid response elements is enriched in the rescued genes. This simple enhancer element combination is sufficient to drive rhythmic circadian reporter gene expression under non-rhythmic glucocorticoid exposure, revealing a permissive function for the hormones in glucocorticoid-dependent circadian transcription. Our work highlights metabolic pathways potentially contributing to morbidity in patients with glucocorticoid deficiency, even under glucocorticoid replacement therapy. Moreover, we provide mechanistic insight into the interaction between the circadian clock and glucocorticoids in the transcriptional regulation of metabolism

NMR Chemical Shift Ranges of Urine Metabolites in Various Organic Solvents

Signal stability is essential for reliable multivariate data analysis. Urine samples show strong variance in signal positions due to inter patient differences. Here we study the exchange of the solvent of a defined urine matrix and how it affects signal and integral stability of the urinary metabolites by NMR spectroscopy. The exchange solvents were methanol, acetonitrile, dimethyl sulfoxide, chloroform, acetone, dichloromethane, and dimethyl formamide. Some of these solvents showed promising results with a single batch of urine. To evaluate further differences between urine samples, various acid, base, and salt solutions were added in a defined way mimicking to some extent inter human differences. Corresponding chemical shift changes were monitored

NMR Chemical Shift Ranges of Urine Metabolites in Various Organic Solvents

Signal stability is essential for reliable multivariate data analysis. Urine samples show strong variance in signal positions due to inter patient differences. Here we study the exchange of the solvent of a defined urine matrix and how it affects signal and integral stability of the urinary metabolites by NMR spectroscopy. The exchange solvents were methanol, acetonitrile, dimethyl sulfoxide, chloroform, acetone, dichloromethane, and dimethyl formamide. Some of these solvents showed promising results with a single batch of urine. To evaluate further differences between urine samples, various acid, base, and salt solutions were added in a defined way mimicking to some extent inter human differences. Corresponding chemical shift changes were monitored

NMR Chemical Shift Ranges of Urine Metabolites in Various Organic Solvents

Signal stability is essential for reliable multivariate data analysis. Urine samples show strong variance in signal positions due to inter patient differences. Here we study the exchange of the solvent of a defined urine matrix and how it affects signal and integral stability of the urinary metabolites by NMR spectroscopy. The exchange solvents were methanol, acetonitrile, dimethyl sulfoxide, chloroform, acetone, dichloromethane, and dimethyl formamide. Some of these solvents showed promising results with a single batch of urine. To evaluate further differences between urine samples, various acid, base, and salt solutions were added in a defined way mimicking to some extent inter human differences. Corresponding chemical shift changes were monitored

Influence of Freezing and Storage Procedure on Human Urine Samples in NMR-Based Metabolomics

metabolite

Integrative Analysis of Circadian Transcriptome and Metabolic Network Reveals the Role of De Novo Purine Synthesis in Circadian Control of Cell Cycle

<div><p>Metabolism is the major output of the circadian clock in many organisms. We developed a computational method to integrate both circadian gene expression and metabolic network. Applying this method to zebrafish circadian transcriptome, we have identified large clusters of metabolic genes containing mostly genes in purine and pyrimidine metabolism in the metabolic network showing similar circadian phases. Our metabolomics analysis found that the level of inosine 5'-monophosphate (IMP), an intermediate metabolite in de novo purine synthesis, showed significant circadian oscillation in larval zebrafish. We focused on IMP dehydrogenase (<i>impdh</i>), a rate-limiting enzyme in de novo purine synthesis, with three circadian oscillating gene homologs: <i>impdh1a</i>, <i>impdh1b</i> and <i>impdh2</i>. Functional analysis revealed that <i>impdh2</i> contributes to the daily rhythm of S phase in the cell cycle while <i>impdh1a</i> contributes to ocular development and pigment synthesis. The three zebrafish homologs of <i>impdh</i> are likely regulated by different circadian transcription factors. We propose that the circadian regulation of de novo purine synthesis that supplies crucial building blocks for DNA replication is an important mechanism conferring circadian rhythmicity on the cell cycle. Our method is widely applicable to study the impact of circadian transcriptome on metabolism in complex organisms.</p></div

<i>impdh2</i> mediates the circadian control of cell cycle.

<p>(A) The BrdU staining in 5 dpf larvae represents the daily rhythm of S phase in cell cycle under LD conditions. (B) The amplitude of the S phase rhythm was attenuated when the larvae were treated with MPA. The effect of MPA was rescued when guanosine was added to MPA-treated larvae. The inhibitory effect of MPA on the S phase rhythm was in a dose-dependent manner and saturated at 10μM concentration. (C) The amplitude of the S phase rhythm decreased significantly in <i>impdh2</i> morphants. At least three larvae were measured at each data point. Error bars represent the standard error of mean (SEM) among independent biological replicates. Scale bars, 200μm.</p

Circadian metabolites in larval zebrafish.

<p>The quantification of nine circadian metabolites including IMP (A), lactate (B), glutamine (C), glutamate (D), threonine (E), aspartate (F), creatine (G), carnitine (H), and phosphocreatine (I) by NMR. The y-axis is scaled intensity in NMR measurement relative to the sample with the lowest intensity for each metabolite. The circadian phases of metabolites are also shown. Error bars represent the standard error of mean (SEM) among independent biological replicates at each time point.</p

Circadian clock controls de novo purine synthesis pathway.

<p>(A) Circadian oscillating genes in de novo purine synthesis pathway have similar circadian phases (around CT0) in both LD and DD conditions. (B-F) <i>clock</i> controls the circadian expression of these genes as indicated by the significantly dampened oscillations in <i>clock</i> morphants (red) compared to WT (green) or control morphants (blue) in both LD and DD. The lowest log2-transformed expression level for each gene was normalized to zero.</p