Investigation of the effects of storage and freezing on mixes of heavy-labeled metabolite and amino acid standards.

RATIONALE: High-throughput metabolomics has now made it possible for small/medium-sized laboratories to analyze thousands of samples/year from the most diverse biological matrices including biofluids, cell and tissue extracts. In large-scale metabolomics studies, stable-isotope-labeled standards are increasingly used to normalize for matrix effects and control for technical reproducibility (e.g. extraction efficiency, chromatographic retention times and mass spectrometry signal stability). However, it is currently unknown how stable mixes of commercially available standards are following repeated freeze/thaw cycles or prolonged storage of aliquots. METHODS: Standard mixes for 13 C, 15 N or deuterated isotopologues of amino acids and key metabolites from the central carbon and nitrogen pathways (e.g. glycolysis, Krebs cycle, redox homeostasis, purines) were either repeatedly frozen/thawed for up to 10 cycles or diluted into aliquots prior to frozen storage for up to 42 days. Samples were characterized by ultra-high-pressure liquid chromatography/mass spectrometry to determine the stability of the aliquoted standards upon freezing/thawing or prolonged storage. RESULTS: Metabolite standards were stable over up to 10 freeze/thaw cycles, with the exception of adenosine and glutathione, showing technical variability across aliquots in a freeze/thaw-cycle-independent fashion. Storage for up to 42 days of mixes of commercially available standards did not significantly affect the stability of amino acid or metabolite standards for the first 2 weeks, while progressive degradation (statistically significant for fumarate) was observed after 3 weeks. CONCLUSIONS: Refrigerated or frozen preservation for at least 2 weeks of aliquoted heavy-labeled standard mixes for metabolomics analysis is a feasible and time-/resource-saving strategy for standard metabolomics laboratories.

Principles and limitations of stable isotopes in differentiating organic and conventional foodstuffs: 1. Plant products.

Among the lighter elements having two or more stable isotopes (H, C, N, O, S), δ(15)N appears to be the most promising isotopic marker to differentiate plant products from conventional and organic farms. Organic plant products vary within a range of δ(15)N values of +0.3 to +14.6%, while conventional plant products range from negative to positive values, i.e. -4.0 to +8.7%. The main factors affecting δ(15)N signatures of plants are N fertilizers, biological N2 fixation, plant organs and plant age. Correlations between mode of production and δ(13)C (except greenhouse tomatoes warmed with natural gas) or δ(34)S signatures have not been established, and δ(2)H and δ(18)O are unsuitable markers due to the overriding effect of climate on the isotopic composition of plant-available water. Because there is potential overlap between the δ(15)N signatures of organic and conventionally produced plant products, δ(15)N has seldom been used successfully as the sole criterion for differentiation, but when combined with complementary analytical techniques and appropriate statistical tools, the probability of a correct identification increases. The use of organic fertilizers by conventional farmers or the marketing of organic produce as conventional due to market pressures are additional factors confounding correct identification. The robustness of using δ(15)N to differentiate mode of production will depend on the establishment of databases that have been verified for individual plant products.

Seasonal baseline of nutrients and stable isotopes in a saline lake of Argentina: biogeochemical processes and river runoff effects.

The seasonal variability of inorganic and organic nutrients and stable isotopes and their relations with plankton and environmental conditions were monitored in Lake Chasicó. Principal component analysis evidenced the strong influence of the river runoff on several biogeochemical variables. Silicate concentrations were controlled by diatom biomass and river discharge. Higher values of nitrate and soluble reactive phosphorus (SRP) indicated agricultural uses in the river basin. Elevated pH values (∼ 9) inhibiting nitrification in the lake explained partially the dominance of ammonium: ∼ 83 % of dissolved inorganic nitrogen (DIN). The low DIN/SRP ratio inferred nitrogen limitation, although the hypotheses of iron and CO2 limitation are relevant in alkaline lakes. Particulate organic matter (POM) and dissolved organic matter (DOM) were mainly of autochthonous origin. The main allochthonous input was imported by the river as POM owning to the arid conditions. Dissolved organic carbon was likely top-down regulated by the bacterioplankton grazer Brachionus plicatilis. The δ(13)C signature was a good indicator of primary production and its values were influenced probably by CO2 limitation. The δ(15)N did not evidence nitrogen fixation and suggested the effects of anthropogenic activities. The preservation of a good water quality in the lake is crucial for resource management.

Inter-laboratory comparison of elemental analysis and gas chromatography/combustion/isotope ratio mass spectrometry. II. Delta15N measurements of selected compounds for the development of an isotopic Grob test.

An inter-laboratory exercise was carried out by a consortium of five European laboratories to establish a set of compounds, suitable for calibrating gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS) devices, to be used as isotopic reference materials for hydrogen, carbon, nitrogen and oxygen stable isotope measurements. The set of compounds was chosen with the aim of developing a mixture of reference materials to be used in analytical protocols to check for food and beverage authentication. The exercise was organized in several steps to achieve the certification level: the first step consisted of the a priori selection of chemical compounds on the basis of the scientific literature and successive GC tests to set the analytical conditions for each single compound and the mixture. After elimination of the compounds that turned out to be unsuitable in a multi-compound mixture, some additional oxygen- and nitrogen-containing substances were added to complete the range of calibration isotopes. The results of delta(13)C determinations for the entire set of reference compounds have previously been published, while the deltaD and delta(18)O determinations were unsuccessful and after statistical analysis of the data the results did not reach the level required for certification. In the present paper we present the results of an inter-laboratory exercise to identify and test the set of nitrogen-containing compounds present in the mixture developed for use as reference materials for the validation of GC-C-IRMS analyses in individual laboratories.

Urinary analysis of 8-oxoguanine, 8-oxoguanosine, fapy-guanine and 8-oxo-2'-deoxyguanosine by high-performance liquid chromatography-electrospray tandem mass spectrometry as a measure of oxidative stress.

A sensitive and specific assay aimed at measuring the oxidized nucleic acids, 8-oxoguanine (8-oxoGua), fapy-guanine (Fapy-Gua), 8-oxoguanosine (8-oxoGuo), 8-oxo-2'-deoxyguanosine (8-oxodG) has been developed by coupling reversed phase liquid chromatography (HPLC) with electrospray tandem mass spectrometry detection (MS/MS) and isotope dilution. The HPLC-MS/MS approach with multiple reaction monitoring (MRM) allowed for the sensitive determination of 8-oxoGua, Fapy-Gua, 8-oxoGuo, and 8-oxodG in human urine samples. There is no sample preparation needed except for the addition of buffer and (13)C- and (15)N-labeled internal standards to the urine prior to sample injection into the HPLC-MS/MS system. This method was tested in urine samples from non-smokers, smokers, non-smokers with chronic kidney disease (CKD) and smokers with CKD, to assess the level of oxidative damage to nucleic acids. Markers of both RNA and DNA damage were significantly increased in the smokers with and without CKD compared to their respective control subjects. These findings suggest that a highly specific and sensitive analytical method such as isotope dilution HPLC-MS/MS may represent a valuable tool for the measurement of oxidative stress in human subjects.

Referencing strategies and techniques in stable isotope ratio analysis.

Stable isotope ratios are reported in the literature in terms of a deviation from an international standard (delta-values). The referencing procedures, however, differ from instrument to instrument and are not consistent between measurement facilities. This paper reviews an attempt to unify the strategy for referencing isotopic measurements. In particular, emphasis is given to the importance of identical treatment of sample and reference material ('IT principle'), which should guide all isotope ratio determinations and evaluations. The implementation of the principle in our laboratory, the monitoring of our measurement quality, the status of the international scales and reference materials and necessary correction procedures are discussed.