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Urinary N,N,N-trimethylglycine (betaine) and N,N-dimethylglycine (DMG) have been identified and quantified for clinical purposes by proton nuclear magnetic resonance (1H NMR) measurement in previous studies. We have assessed these procedures by using both one-dimensional (1-D) and 2-D NMR spectroscopy, together with pH titration of urinary extracts...
Contexts in source publication
Context 1
... in the 'H NMR spectra were identified by comparison (Table 1) with literature val- ues of the chemical shifts (after correction of TSP shifts to DSS values) and coupling pattern (e.g., 10, 18-21), with the NMR spectra of authentic standard com- pounds, and by adding a small amount of authentic compound (22) to the sample, followed by the acquisi- tion of an additional spectrum. ...
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... concentration of DMG was too low to give an unambiguous cross-peak in the spectra under the conditions we used. The homonuclear Hartmann- Hahn (2-D NMR) spectra of the same samples did not Peak assignments: 8, betaine; 11,creatinine; 13, DMG (see Table 1 for assignments of the other peaks). DSS was used as the chemical shift reference (assigned to 0.000 ppm), and the pH was 6.02. ...
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... difference be- tween pKa at 0#{176}C and 25#{176}C is -+0.02 pKa units, sug- gesting a plC, of betaine in H20 of -1.81 at 25#{176}C (26). The pK, (in D20) of the group giving peak 8 was 2.36 (see Table 1); this corresponds to a pK, of 1.84 in H20 after correction as described in Materials and Methods. Similarly, the pK, values for some of the remaining resonances agreed reasonably well with available lit- erature values for glycine, creatinine, and creatine in D20 (see Table 1) (26). ...
Context 4
... pK, (in D20) of the group giving peak 8 was 2.36 (see Table 1); this corresponds to a pK, of 1.84 in H20 after correction as described in Materials and Methods. Similarly, the pK, values for some of the remaining resonances agreed reasonably well with available lit- erature values for glycine, creatinine, and creatine in D20 (see Table 1) (26). Excellent separation of betaine and other peaks in thb N-methyl characteristic region of the spectrum was obtained by using a pH5 of --5. ...
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... 'H NMR in 1-D spectra of reconstituted urine samples. The peak assignment was made on the basis of its chemical shift relative to DSS in comparison with literature values (Table 1), and confirmed by adding a small amount of authentic betaine to the samples, followed by simple acquisition of 1-D spectra. COSYLR spectra (optimized for long-range couplings) and 1-D spectra, coupled with pH titration, were also used. ...
Citations
... With a hig The question is whether it is possible to identify C-H···O bonds by any experimental method. IR spectroscopy [58] and proton nuclear magnetic resonance ( 1 H NMR) [59,60] seem to be the most suitable. ...
Trimethylglycine (glycine betaine, GB) is an important organic osmolyte that accumulates in various plant species in response to environmental stresses and has significant potential as a bioactive agent with low environmental impact. It is assumed that the hydration of GB is playing an important role in the protective mechanism. The hydration and aggregation properties of GB have not yet been studied in detail at the atomistic level. In this work, noncovalent interactions in the GB dimer and its complexes with water and crystalline monohydrate are studied. Depending on the object, periodic and non-periodic DFT calculations are used. Particular attention is paid to the metric parameters and enthalpies of intermolecular hydrogen bonds. The identification of noncovalent interactions is carried out by means of the Bader analysis of periodic or non-periodic electron density. The enthalpy of hydrogen bonds is estimated using the Rosenberg formula (PCCP 2 (2000) 2699). The specific proton donor properties of glycine betaine are due to its ability to form intermolecular C-H∙∙∙O bonds with the oxygen atom of a water molecule or the carboxylate group of a neighboring GB. The enthalpy of these bonds can be significantly greater than 10 kJ/mol. The water molecule that forms a hydrogen bond with the carboxylate group of GB also interacts with its CH groups through lone pairs of electrons. The C-H∙∙∙O bonds contribute up to 40% of the total entropy of the GB-water interaction, which is about 45 kJ/mol. The possibility of identifying C-H∙∙∙O bonds by the proton nuclear magnetic resonance method is discussed.
... N,N-Dimethylglycine can be metabolized further to sarcosine in the mitochondria, providing 1-carbon units for the formation of 5,10-methylene-tetrahydrofolate. It is likely that the increase in N,N-dimethylglycine is driven by high plasma total homocysteine concentrations; this results in the accumulation of N,N-dimethylglycine in plasma and urine [22,23]. Disruptions of homocysteine and betaine homeostasis are likely to result in complex metabolic consequences. ...
Implantable cardioverter defibrillators (ICDs) reduce sudden cardiac death (SCD) when patients experience life-threatening ventricular arrhythmias (LTVA). However, current strategies determining ICD patient selection and risk stratification are inefficient. We used metabolomics to assess whether dysregulated metabolites are associated with LTVA and identify potential biomarkers. Baseline plasma samples were collected from 72 patients receiving ICDs. Over a median follow-up of 524.0 days (range 239.0–705.5), LTVA occurred in 23 (31.9%) patients (22 effective ICD treatments and 1 SCD). After confounding risk factors adjustment for age, smoking, secondary prevention, and creatine kinase MB, 23 metabolites were significantly associated with LTVA. Pathway analysis revealed LTVA associations with disrupted metabolism of glycine, serine, threonine, and branched chain amino acids. Pathway enrichment analysis identified a panel of 6 metabolites that potentially predicted LTVA, with an area under the receiver operating characteristic curve of 0.8. Future studies are necessary on biological mechanisms and potential clinical use.
Graphical abstract
... Most importantly, our results extend the findings from an investigation of 531 patients with recent acute coronary syndrome, reporting a positive relationship between plasma DMG at baseline and the risk of incident AMI during ≈2.5 years of follow-up. 3 Increased urinary DMG levels have been observed in patients with premature vascular disease, 18 and serum levels of sarcosine, the immediate catabolic product of DMG, has been associated with restenosis after percutaneous coronary intervention. 19 ...
... The CoR was obtained by calculating the betweenperson variance as a proportion of the total variance across four visits; CoR≥0.75 suggests excellent reproducibility. 18 Using mixed linear modelling when assessing these longitudinal data allowed us to also include patients who did not attend all four study visits. ...
Objective:
Dimethylglycine is linked to lipid metabolism, and increased plasma levels may be associated with adverse prognosis in patients with coronary artery disease. We evaluated the relationship between plasma dimethylglycine and risk of incident acute myocardial infarction in a large prospective cohort of patients with stable angina pectoris, of whom approximately two thirds were participants in a B-vitamin intervention trial. Model discrimination and reclassification when adding plasma dimethylglycine to established risk factors were obtained. We also explored temporal changes and the test-retest reliability of plasma dimethylglycine.
Approach and results:
Four thousand one hundred fifty patients (72% men; median age 62 years) were included. Plasma dimethylglycine was associated with several traditional coronary artery disease risk factors. During a median follow-up of 4.6 years, 343 (8.3%) patients experienced an acute myocardial infarction. The hazard ratio (95% confidence interval) for acute myocardial infarction was 1.95 (1.42-2.68; P<0.001) when comparing plasma dimethylglycine quartile 4 to 1 in a Cox regression model adjusted for age, sex, and fasting status. Adjusting for traditional coronary artery disease risk factors only slightly modified the estimates, which were particularly strong among nonsmokers and among patients with serum triglyceride or apolipoprotein B100 levels ≤ median (P for interaction=0.004, 0.004, and 0.03, respectively). Plasma dimethylglycine improved discrimination and reclassification and had high test-retest reliability.
Conclusions:
Plasma dimethylglycine is independently related to incident acute myocardial infarction and enhances risk prediction in patients with stable angina pectoris. Our results motivate further studies on the relationship between 1-carbon metabolism and atherothrombosis. A potential interplay with lipid and energy metabolism merits particular attention.
... Almost all reports are based on proton NMR, though 13 C-NMR [214] and 14 N-NMR have been tried [215,216]. Proton NMR is attractive because of the strong singlet resonance from the nine identical protons on the three methyl groups, making for a relatively sensitive assay [185,216,217]. Betaine can be measured directly in water after suppressing the water signal, and protein resonances can also be suppressed. ...
Betaine is an essential osmolyte and source of methyl groups and comes from either the diet or by the oxidation of choline. Its metabolism methylates homocysteine to methionine, also producing N,N-dimethylglycine. Betaine insufficiency is associated with the metabolic syndrome, lipid disorders and diabetes, and may have a role in vascular and other diseases. Betaine is important in development, from the pre-implantation embryo to infancy. Betaine supplementation improves animal and poultry health, but the effect of long-term supplementation on humans is not known, though reports that it improves athletic performance will stimulate further studies. Subsets of the population that may benefit from betaine supplementation could be identified by the laboratory, in particular those who excessively lose betaine through the urine.Plasma betaine is highly individual, in women typically 20–60 μmol/L and in men 25–75 μmol/L. Plasma dimethylglycine is typically < 10 μmol/L. Urine betaine excretion is minimal, even following a large betaine dose. It is constant, highly individual and normally < 35 mmol/mole creatinine. The preferred method of betaine measurement is by LC-MS/MS, which is rapid and capable of automation. Slower HPLC methods give comparable results. Proton NMR spectrometry is another option but caution is needed to avoid confusion with trimethylamine-N-oxide.
... 65 mL D 2 O (Cambridge Isotope Laboratories) and 6.5 mL 109 mmol/L sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS; Cambridge Isotope Laboratories) was added to 585 mL sample in phosphate buffer (pH 9). Samples were transferred to a 5-mm NMR tube and 1-dimensional NMR spectra were acquired on each sample at ambient probe temperature as described by Lundberg et al. (19). The resonance of DSS was set to a chemical shift of 0.00 ppm. ...
The time course of betaine accumulation and activities of enzymes involved in betaine metabolism were studied in developing rats. In study 1, pups weaned on a nonpurified diet had a transient increase in liver and kidney betaine content followed by a decline after approximately 42-56 d. In study 2, dams and, following weaning, pups were fed an AIN-93G (betaine-free) or an AIN-93G betaine-supplemented diet (0.3%) to determine the source of the transient increase in betaine levels previously observed. In study 2, only rats fed betaine had an increase in plasma betaine concentration. Similarly, liver and kidney betaine contents increased postweaning; however, betaine levels returned to that found in rats fed a betaine-free diet by 49 d of age. The dietary content of betaine fed to dams did not affect pup betaine. The activities of choline dehydrogenase, an enzyme of betaine synthesis, and betaine:homocysteine methyltransferase (BHMT), which is the only known betaine-consuming enzyme in mammals, were also measured in study 2. Liver BHMT activity decreased after weaning, whereas liver and kidney choline dehydrogenase activity increased with age, possibly reaching a plateau by 42 d of age. We conclude that the transient increase in betaine reflects high dietary betaine and not a change in endogenous betaine synthesis.
... In normal metabolism, homocysteine is either catabolized via cystathionine or methylated to methionine. Most attention has focused on methylation by methionine synthase, requiring folate-and B 12 -dependent enzyme systems, but increasing attention is being given to the role of betaine-homocysteine methyltransferase in regulating the circulating homocysteine concentrations in mammals [27,28]. Mammalian BHMT is a zinc metalloenzyme expressed in liver and kidney tissue. ...
... There are reasons to be interested in these speciWcity issues. Since arsenobetaine and DMT are not toxic to mammals but are to some bacteria272829, speciWcity diVerences may explain why dietary arsenobetaine is harmless to humans and may suggest strategies for developing speciWc antimicrobial agents. To determine the speciWcity and inhibitor properties of BHMT by the radiochemical assay [18,19] requires the preparation of a range of 14 C-labeled betaines. ...
... To determine the speciWcity and inhibitor properties of BHMT by the radiochemical assay [18,19] requires the preparation of a range of 14 C-labeled betaines. Inferences about BHMT activities have been made by using 1 H-NMR spectroscopy to measure urine DMG [27,32]. The assay presented here takes these ideas several steps further, by presenting a direct measurement of BHMT activity using NMR spectroscopy. ...
Betaine-homocysteine methyltransferase (BHMT) activity can be measured directly and kinetically by (1)H-nuclear magnetic resonance spectroscopy. The disappearance of substrates and the formation of products are monitored simultaneously. Alternative substrates, separately and when mixed with glycine betaine, can also be monitored. Each assay can be completed in 1h. Using 2mM glycine betaine and homocysteine as substrates in 20 mM phosphate buffer (pH 7.5) and measuring the production of N,N-dimethylglycine, the CV is 6.3% (n=6) and the detection limit is 6 nkatal. An endpoint assay for BHMT activity was also developed, by measuring the N,N-dimethylglycine produced after incubation with 2 mM glycine betaine and homocysteine (CV=5.3%, n = 6) with a detection limit of 2 nkatal. These assays were used to show that the natural betaines trigonelline, proline betaine, arsenobetaine, and l-carnitine are neither substrates nor significant inhibitors of rat liver BHMT, that the thetins dimethylthetin and dimethylsulfoniopropionate are substrates and inhibit methyl transfer from glycine betaine, and that the K(m) for glycine betaine is 0.19+/-0.03 mM with a V(max) of 17+/-0.7 nMol min(-1) mg(-1).
... confirmation of the accumulation of dmg with 13 ...
... An 1 H NMR spectrum of a body fluid provides an overall view of almost all proton-containing metabolites in the micro-and millimolar concentration range. 1 H NMR has been used previously for diagnosing known inborn errors of metabolism (9 -12). In addition, analysis of betaine and DMG in urine by 1 H NMR spectroscopy has been reported (13). In vivo NMR spectroscopy of the central nervous system has already been used successfully to demonstrate guanidinoacetate-methyltransferase deficiency as a new inborn error of creatine biosynthesis (14). ...
A38-year-old man presented with a history of fish odor (since age 5) and unusual muscle fatigue with increased serum creatine kinase. Our aim was to identify the metabolic error in this new condition.
We used 1H NMR spectroscopy to study serum and urine from the patient.
The concentration of N, N-dimethylglycine (DMG) was increased approximately 100-fold in the serum and approximately 20-fold in the urine. The presence of DMG as a storage product was confirmed by use of 13C NMR spectroscopy and gas chromatography-mass spectrometry. The high concentration of DMG was caused by a deficiency of the enzyme dimethylglycine dehydrogenase (DMGDH). A homozygous missense mutation was found in the DMGDH gene of the patient.
DMGDH deficiency must be added to the differential diagnosis of patients complaining of a fish odor. This deficiency is the first inborn error of metabolism discovered by use of in vitro 1H NMR spectroscopy of body fluids.
Marine fish undergo detoxification to overcome As stress, forming non-toxic metabolites arsenobetaine (AsB). Genes associated with AsB synthesis remain unknown. Therefore, in this study, we explored the key genes involved in the synthesis of AsB by transcriptomic analysis in marine medaka (Oryzias melastigma), and then screened candidate genes related to AsB synthesis. In the liver, 40 genes were up-regulated and 23 genes were down-regulated, whereas in muscle, 83 genes were up-regulated and 331 genes were down-regulated. We revealed that bhmt, mat2aa, and gstt1a can play a significant role in the glutathione and methionine metabolic pathway. These three genes can affect the conversion of arsenocholine (AsC) to AsB by the vitro gene transformation experiments of E. coli BL21(DE3). E. coli BL21-bhmt overexpressing bhmt resulted in more oxidation of precursor AsC to AsB. Furthermore, the AsB concentration was decreased after E. coli BL21 overexpressing mat2aa and gstt1a, which were down-regulated in marine medaka. Therefore, we concluded that bhmt, mat2aa, and gstt1a are involved in AsB synthesis. Overall, this is the first report on transcriptome screening and identification of key genes for AsB synthesis in marine medaka. We provided important insights to reveal the mystery of AsB synthesis in marine fish.
High-resolution magic angle spinning (HR MAS) nuclear magnetic resonance (NMR) spectroscopy is increasingly being used to study metabolite levels in human breast cancer tissue, assessing, for instance, correlations with prognostic factors, survival outcome or therapeutic response. However, the impact of intratumoral heterogeneity on metabolite levels in breast tumor tissue has not been studied comprehensively. More specifically, when biopsy material is analyzed, it remains questionable whether one biopsy is representative of the entire tumor. Therefore, multi-core sampling (n = 6) of tumor tissue from three patients with breast cancer, followed by lipid (0.9- and 1.3-ppm signals) and metabolite quantification using HR MAS 1H NMR, was performed, resulting in the quantification of 32 metabolites. The mean relative standard deviation across all metabolites for the six tumor cores sampled from each of the three tumors ranged from 0.48 to 0.74. This was considerably higher when compared with a morphologically more homogeneous tissue type, here represented by murine liver (0.16–0.20). Despite the seemingly high variability observed within the tumor tissue, a random forest classifier trained on the original sample set (training set) was, with one exception, able to correctly predict the tumor identity of an independent series of cores (test set) that were additionally sampled from the same three tumors and analyzed blindly. Moreover, significant differences between the tumors were identified using one-way analysis of variance (ANOVA), indicating that the intertumoral differences for many metabolites were larger than the intratumoral differences for these three tumors. That intertumoral differences, on average, were larger than intratumoral differences was further supported by the analysis of duplicate tissue cores from 15 additional breast tumors. In summary, despite the observed intratumoral variability, the results of the present study suggest that the analysis of one, or a few, replicates per tumor may be acceptable, and supports the feasibility of performing reliable analyses of patient tissue.
