Optimized metabolite extraction from blood serum for 1H nuclear magnetic resonance spectroscopy

Tiziani, Stefano; Emwas, Abdul Hamid; Lodi, Alessia; Ludwig, Christian; Bunce, Christopher M.; Viant, Mark R.; Günther, Ulrich L.

doi:10.1016/j.ab.2008.01.037

Cited by 169 publications

(126 citation statements)

References 12 publications

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“…Extracellular metabolites were separated by ultrafiltration (Amicon). Cell pellets and tissue samples were lyophilized, weighted and cryogenically grounded using Cryomill (Retsch GmbH Germany) before methanol/ chloroform/water extraction (2/2/1.8) to extract nonpolar metabolites 44,45 . All 2D 1 H J-resolved (JRES) NMR spectra were acquired on a 500-MHz DRX Bruker Avance spectrometer (Bruker, Germany), using a double spin-echo sequence with 8 transients per increment for a total of 32 increments.…”

Section: Methodsmentioning

confidence: 99%

Metformin elicits anticancer effects through the sequential modulation of DICER and c-MYC

Blandino¹,

Valerio

Cioce³

et al. 2012

Nat Commun

208

156

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Diabetic patients treated with metformin have a reduced incidence of cancer and cancerrelated mortality. Here we show that metformin affects engraftment and growth of breast cancer tumours in mice. This correlates with the induction of metabolic changes compatible with clear anticancer effects. We demonstrate that microRnA modulation underlies the anticancer metabolic actions of metformin. In fact, metformin induces DICER expression and its effects are severely impaired in DICER knocked down cells. Conversely, ectopic expression of DICER recapitulates the effects of metformin in vivo and in vitro. The microRnAs upregulated by metformin belong mainly to energy metabolism pathways. Among the messenger RnAs downregulated by metformin, we found c-mYC, IRs-2 and HIF1alpha. Downregulation of c-mYC requires AmP-activated protein kinase-signalling and mir33a upregulation by metformin. Ectopic expression of c-mYC attenuates the anticancer metabolic effects of metformin. We suggest that DICER modulation, mir33a upregulation and c-mYC targeting have an important role in the anticancer metabolic effects of metformin.

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Section: Methodsmentioning

confidence: 99%

Metformin elicits anticancer effects through the sequential modulation of DICER and c-MYC

Blandino¹,

Valerio

Cioce³

et al. 2012

Nat Commun

208

156

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“…Sera of rats from the vitamin B 12-deficient study were deproteinized by ultrafiltration, using spin columns with a molecular cutoff of 3 kDa (Nanosep 3K omega; Pall Canada, Ville St-Laurent, QC, Canada) that were initially washed as recommended by Tiziani et al (38). Deproteinized samples were spiked with 2,2-dimethyl-2-sila 3,3,4,4,5,5-hexadeuteropentane sulphonic acid (DSS-D 6) to a final concentration of 0.25 mM; this served as both a chemical shift indicator and a concentration standard for determining absolute metabolite concentrations.…”

Section: Methodsmentioning

confidence: 99%

Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration

Lamarre

Molloy

Reinke

et al. 2012

American Journal of Physiology-Endocrinology and Metabolism

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Lamarre SG, Molloy AM, Reinke SN, Sykes BD, Brosnan ME, Brosnan JT. Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration. 1 H-NMR metabolomic analysis of sera from vitamin B12-deficient rats. In addition to the expected increases in methylmalonate and homocysteine (Hcy), we observed an approximately sevenfold increase in formate levels, from 64 M in control rats to 402 M in vitamin B 12-deficient rats. Urinary formate was also elevated. This elevation of formate could be attributed to impaired one-carbon metabolism since formate is assimilated into the one-carbon pool by incorporation into 10-formyl-THF via the enzyme 10-formyl-THF synthase. Both plasma and urinary formate were also increased in folate-deficient rats. Hcy was elevated in both the vitamin B12-and folate-deficient rats. Although plasma Hcy was also elevated, plasma formate was unaffected in vitamin B6-deficient rats (impaired transsulfuration pathway). These results were in accord with a mathematical model of folate metabolism, which predicted that reduction in methionine synthase activity would cause increased formate levels, whereas reduced cystathionine ␤-synthase activity would not. Our data indicate that formate provides a novel window into cellular folate metabolism, that elevated formate can be a useful indicator of deranged one-carbon metabolism and can be used to discriminate between the hyperhomocysteinemia caused by defects in the remethylation and transsulfuration pathways. VITAMIN B 12 DEFICIENCY RESULTS IN A WIDE SPECTRUM of hematologic and neuropsychiatric disorders. The detection of vitamin B 12 deficiency has traditionally been based on low serum vitamin B 12 levels, with supportive clinical evidence of disease. It is now recognized that serum vitamin B 12 may be insufficient to detect a deficiency, especially in the case of elderly subjects without hematologic abnormalities. The measurement of metabolites such as homocysteine (Hcy) and methylmalonic acid (MMA) has been shown to be more sensitive in diagnosing vitamin B 12 deficiency (33, 34). In a search for novel biomarkers of vitamin B 12 deficiency, we fed rats a vitamin B 12 -deficient diet and subjected their sera to 1 H-NMR metabolomic analysis. Together with the expected increased concentrations of Hcy and MMA, we found an approximately sevenfold increase in both serum and urinary formate concentrations in the vitamin B 12 -deficient animals.We confirmed that elevated formate would also be found in folate-deficient rats, since impaired folate metabolism (via the methylfolate trap) is a well-recognized consequence of vitamin B 12 deficiency (12, 31, 32) and because formate is incorporated into 10-formyl-tetrahydrofolate (THF) via 10-formyl-THF synthase (37). Both folate and vitamin B 12 deficiencies are characterized by elevated plasma Hcy. A deficiency of pyridoxal (vitamin B 6 ) also causes elevated plasma Hcy, which becomes more pronounced after methionine loading (18). This is due to impaired removal of Hcy ...

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“…This removal is accomplished using either organic solvent precipitation or by ultrafiltration using a 3 kDa molecular weight cut off filter. A recent comparison of serum extraction methods for NMR analysis using perchloric, ACN, acetone, methanol/chloroform, and ultrafiltration showed that ultrafiltration was superior in metabolite retention and reproducibility [26].…”

Section: Sample Preparationmentioning

confidence: 99%

Analytical and statistical approaches to metabolomics research

Issaq

Van

Waybright

et al. 2009

J of Separation Science

195

136

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Metabolomics, the global profiling of metabolites in different living systems, has experienced a rekindling of interest partially due to the improved detection capabilities of the instrumental techniques currently being used in this area of biomedical research. The analytical methods of choice for the analysis of metabolites in search of disease biomarkers in biological specimens, and for the study of various low molecular weight metabolic pathways include NMR spectroscopy, GC/MS, CE/MS, and HPLC/MS. Global metabolite analysis and profiling of two different sets of data results in a plethora of data that is difficult to manage or interpret manually because of their subtle differences. Multivariate statistical methods and pattern-recognition programs were developed to handle the acquired data and to search for the discriminating features between data acquired from two sample sets, healthy and diseased. Metabolomics have been used in toxicology, plant physiology, and biomedical research. In this paper, we discuss various aspects of metabolomic research including sample collection, handling, storage, requirements for sample analysis, peak alignment, data interpretation using statistical approaches, metabolite identification, and finally recommendations for successful analysis.

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Optimized metabolite extraction from blood serum for 1H nuclear magnetic resonance spectroscopy

Cited by 169 publications

References 12 publications

Metformin elicits anticancer effects through the sequential modulation of DICER and c-MYC

Metformin elicits anticancer effects through the sequential modulation of DICER and c-MYC

Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration

Analytical and statistical approaches to metabolomics research

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