Stable isotope characterization of milk components and whey ethanol.

A multi-isotopic study of several components of milk has been carried out on commercial samples and on milk produced in feeding experiments involving different kinds of diets originating from C(3) or C(4) photosynthetic metabolisms and exhibiting a relatively wide range of isotope ratios. The dispersion of the carbon, hydrogen, and nitrogen isotope parameters of dried matter and of the lactose, protein, and lipid components has been estimated. In addition, the carbohydrates were represented by the site specific isotope ratios (SNIF-NMR) of ethanol resulting from standardized fermentation of lactose. The rates of response of the isotopic parameters to changes in the feeding materials is slower for the minor components, proteins, and lipids than for lactose and ethanol. For similar diets, the nonexchangeable sites of lactose and the methyl site of ethanol, in particular, are relatively enriched in deuterium in the case of polygastric animals, cow, goat, and ewe, as compared to the monogastric species, sow and mare, and woman. From an analytical point of view, the carbon and hydrogen parameters of ethanol provide efficient criteria for identifying a whey origin with respect to other agricultural and fossil sources.     

Stable isotope dilution analysis of the fusarium mycotoxin zearalenone

Zearalenone is a secondary metabolite produced by molds of the Fusarium genus. Beside its nonsteroidal molecular structure, zearalenone has estrogenic activity and can disrupt the function of the endogenous hormone 17beta-estradiol in animals and possibly in humans. It can frequently be found in all major cereal grains as well as in processed food. Because of the estrogenic properties of zearalenone and its metabolites, legal regulations are installed in the European Union setting maximum levels in cereals and cereal products. Routine analysis of zearalenone in various commodities is carried out by HPLC with fluorescence detection, but due to the development of multi-mycotoxin methods and the reduced sample cleanup, HPLC-MS/MS has become a fast and efficient alternative. However, to achieve a reliable quantitation with this technique suitable internal standards are required. This paper reports the synthesis of stable isotope labeled 3,5- d 2-zearalenone (ZON) as internal standard for stable isotope dilution analysis. Furthermore, a method for the analysis of zearalenone by HPLC-MS/MS using 3,5- d 2-zearalenone as IS has been developed. Fifteen cereal products from the German retail markets were analyzed, of which seven contained ZON in levels from 4.9 to 45.0 microg/kg.     

Stable isotope labeling pattern of resveratrol and related natural stilbenes

The stable isotope characterization of resveratrol 1 from Polygonum cuspidatum and of related natural stilbenes 11 and 12 obtained by hydrolysis of the corresponding glucosides 2 and 3 from Rheum is reported. The C(6)-C(2)-C(6) framework of suitably protected derivatives of 1, 2, and 3 has been degraded with ozone to the C(6)-C(1) aldehydes 4, 5, 9, and 10, retaining all hydrogen atoms of the precursors. The natural and synthetic derivatives are characterized and distinguished by natural abundance deuterium nuclear magnetic resonance studies. In the case of anisaldehyde 4 the two series show, as expected, the characteristic difference of the aromatic labeling. The formyl deuterium contents of 4 and 5 from resveratrol are remarkably different, seemingly reflecting the different enrichments existing between positions 3 and 2, respectively, of the phenylpropanoid precursor. The positional delta(18)O values of the extractive materials 1-3 were also determined. In this instance a selective deoxygenation procedure was adopted, leading from 1 to the products 6, 7, and 8. The delta(18)O values of the latter compounds reveal, respectively, those at position 4' and positions 3 and 5 of 1. Similarly, the phenolic products 11 and 12 were converted into 13 and 14. From the delta(18)O values of the single components it is possible to design a detailed map of the oxygen fractionations which characterizes the stilbenes 1-3. In particular, the oxygen present at position 4' of the phenylpropanoid moiety of 1-3 shows delta(18)O values of +11.5, +1.8, and +6.7 per thousand, respectively. Moreover, the phenolic oxygen atom at position 3' of rhapontin 3 shows a value of +11.7 per thousand. The data are compared with those previously obtained on structurally related compounds. These results show the utility of simple chemical degradations in the stable isotope characterization of structurally complex food components.     

稻田沟渠-湿地系统的碳氮稳定同位素组成特征

由于稳定同位素在特定污染源中具有特定的组成,在污染物质迁移转化过程中作为示踪剂而广泛应用。针对目前农业面源污染较为严重的现状,该文利用碳氮稳定同位素研究了灌区内外源物输入对稻田沟渠-湿地系统的贡献。结果表明,水中颗粒性有机物(particulate organic matter,POM)由于受到光照、营养物质不同导致POM在各采样点组成不同,δ13C变化范围较大,均值为-27.8‰,与大型水生植物和浮游植物接近,此类植物可能是POM的主要来源。水中δ13CDIC(dissolved inorganic carbon,DIC)与浮游植物呈线性相关,浮游动物δ13C与浮游植物存在一定相关性,而浮游植物与POM之间不存在显著性差异,说明研究区内浮游动物对內源有机碳的利用主要是取食浮游植物低持斜聿愠粱?δ13C值变化范围在-27.2‰～-21.8‰之间,明显高于水体POM含量,说明表层沉积物存在比颗粒有机物更富集碳的藻类与陆源碎屑等物质。各采样点颗粒有机物δ15N值的范围3.1‰～4.2‰,平均值为3.6‰,其中湿地δ15N高于其他采样点。沉积物δ15N平均值为-0.6‰,与大气中N2较为接近。     

Stable C and N isotope values of selected components of a tropical australian sugarcane ecosystem

The ¹³C and ¹⁵N values of sugarcane plant tissues, decomposing harvest residues, soil and the casts and body tissues of the earthwormPontoscolex corethrurus were determined. Little variation in ¹³C values was found between plant parts. The ¹³C values of the decomposing harvest residues declined and became more variable after 148 days of exposure in the field. In the decomposing residues, ¹³C values of the neutral detergent fibre fraction were similar to those of the whole tissues while those of the proximate lignin were more negative. The ¹⁵N values of the residues also declined over time after a short initial delay.P. corethrurus populations are more intimately associated with the roots of sugarcane than with the bulk soil. Tissue ¹³C values suggest that the earthworm diet is similar to or more enriched in¹³C than sugarcane tissues and is substantially more enriched than the soil C. Earthworm tissues have similar levels of¹⁵N enrichment to both the soil and plant tissues. These data are consistent with the hypothesis that this earthworm derives much of its assimilated C relatively directly from organic matter associated with the roots and decomposing harvest residues.     

Stable C and N isotope values of selected components of a tropical australian sugarcane ecosystem

The δ¹³C and δ¹⁵N values of sugarcane plant tissues, decomposing harvest residues, soil and the casts and body tissues of the earthwormPontoscolex corethrurus were determined. Little variation in δ¹³C values was found between plant parts. The δ¹³C values of the decomposing harvest residues declined and became more variable after 148 days of exposure in the field. In the decomposing residues, δ¹³C values of the neutral detergent fibre fraction were similar to those of the whole tissues while those of the proximate lignin were more negative. The δ¹⁵N values of the residues also declined over time after a short initial delay.P. corethrurus populations are more intimately associated with the roots of sugarcane than with the bulk soil. Tissue δ¹³C values suggest that the earthworm diet is similar to or more enriched in¹³C than sugarcane tissues and is substantially more enriched than the soil C. Earthworm tissues have similar levels of¹⁵N enrichment to both the soil and plant tissues. These data are consistent with the hypothesis that this earthworm derives much of its assimilated C relatively directly from organic matter associated with the roots and decomposing harvest residues.     

Stable isotope analysis reveals whether soil-living elaterid larvae move between agricultural crops

Tracking the movement of soil-living herbivores is difficult, albeit important for understanding their spatial ecology as well as for pest management. In this study the movement of Agriotes obscurus larvae between plots harbouring isotopically different plants was examined. Neither between maize and wheat nor between maize and grassland movement could be detected. These data suggest that Agriotes larvae rarely disperse between crops as long as local food supply is sufficient. Moreover, the current approach provides a new means to study the dispersal of soil invertebrates in situ.     

利用碳同位素分辨率表征沟灌番茄水分利用效率

为了进一步探索沟灌条件下作物水分利用效率提升机理,利用稳定碳同位素对水分利用效率（WUE）的指示特性,研究了隔沟交替灌溉（AFI）和常规沟灌（CFI）条件下大田番茄不同部位碳同位素分辨率（Δ13C）与叶片和产量水平水分利用效率的关系。研究结果表明：不同沟灌条件下果实成熟期的叶片碳同位素分辨率（ΔL）、果实碳同位素分辨率（ΔF）分别为19.27‰～20.25‰和18.07‰～20.04‰,ΔL与叶片水平瞬时水分利用效率（WUEi）、内在水分利用效率（WUEn）均呈负相关,且ΔL对WUEn的指示性优于WUEi。ΔL、ΔF均与产量水平水分利用效率（WUEET）呈负相关。AFI条件下ΔL与WUEn、WUEET的相关性较CFI更为显著,沟灌条件下果实成熟期的叶片碳同位素分辨率可作为表征作物WUE的量化指标。     

Stable isotope N and C labelling of different functional groups of earthworms and their casts: A tool for studying trophic links

Earthworms (Oligochaeta: Lumbricidae) have substantial effects on the structure and fertility of soils with consequences for the diversity of plant communities and associated ecosystem functions. However, we still lack a clear understanding of the functional role earthworms play in terrestrial ecosystems, partly because easy-to-use methods to quantify their activities are missing. In this study, we tested whether earthworms and their casts can be dual-labelled with ¹⁵N and ¹³C stable isotopes by cultivating them in soil substrate amended with ¹⁵N ammonium nitrate and ¹³C-glucose. Additionally, we also wanted to know whether (i) earthworms from different functional groups (soil-feeders vs. litter-feeders) and their casts would differ in their incorporation of stable isotopes, (ii) if enrichment levels are higher if the same amount of isotopes is applied in one dose or in staggered doses, and (iii) if isotopic enrichment in casts changes when they are stored in a conditioning cabinet or in a pot filled with soil placed in a greenhouse. Our findings show the feasibility of dual-labelling tissues and casts of both litter-feeding (Lumbricus terrestris) and soil-feeding (Aporrectodea caliginosa) earthworms using the same method. The advantage of this method is that earthworms and their casts can be labelled under realistic conditions by cultivating them for only four days in soil that received a one-time addition of commercially available stable isotopes instead of offering labelled plant material. In earthworms, the isotopic enrichment remained at a stable level for at least 21 days; labelled casts could be stored for at least 105 days without significantly decreasing their isotopic signals. This simple and efficient method opens new avenues for studying the role of these important ecosystem engineers in nutrient cycling and their functional relationships with other organisms.     

Stable isotope dilution techniques for assessing vitamin A status and bioefficacy of provitamin A carotenoids in humans

Vitamin A deficiency is a major global public health problem. Among the variety of techniques that are available for assessing human vitamin A status, evaluating the provitamin A nutritional values of foodstuffs and estimating human vitamin A requirements, isotope dilution provides the most accurate estimates. Although the relative expense of isotope dilution restricts its applications, it has an important function as the standard of reference for other techniques. Mathematical modelling plays an indispensable role in the interpretation of isotope dilution data. This review summarises recent applications of stable isotope methodology to determine human vitamin A status, estimate human vitamin A requirements, and calculate the bioconversion and bioefficacy of food carotenoids.     

Quantification of free coumarin and its liberation from glucosylated precursors by stable isotope dilution assays based on liquid chromatography-tandem mass spectrometric detection

A stable isotope dilution assay for the quantification of free coumarin and glucosylated coumarin precursors has been developed using [13C2]-coumarin as the internal standard. The doubly labeled coumarin was synthesized by reacting [13C2]-acetic anhydride with salicylic aldehyde and characterized by means of mass spectrometry and nuclear magnetic resonance (NMR) experiments. The specifity of liquid chromatography-tandem mass spectrometry enabled unequivocal determination and sensitive quantitation of the odorant. Because of the very simple extraction procedure, free coumarin could be analyzed within 1h. For quantification of total coumarin, the odorant was liberated from its precursors by an incubation with hydrochloric acid or beta-glucosidase. In analyses of breakfast cereals, the intra-assay coefficient of variation was 9.9% ( n = 5) for total coumarin. When coumarin was added to butter cookies at a level of 10 microg/kg, a recovery of 94.1% was found. Further addition studies revealed a detection limit of 2.9 microg/kg and a quantification limit of 8.6 microg/kg. Application of the stable isotope dilution assay to several plants, foods, and essential oils revealed high contents in cassia products and those foods in which cassia has been used as an ingredient. In contrast to this, Ceylon cinnamon contained much less coumarin. The odorant was also quantified in woodruff, clover seeds, and the essential oils of lavender, citron, and chamomile. Only trace amounts were detected in carrots and the essential oils of peppermint and dill, whereas in bilberries, black raspberries, and Angelica roots, coumarin was below detectable levels. In Ceylon cinnamon and cassia, the odorant occurred mainly in its free form, whereas in fenugreek seeds and woodruff, 68 and 88% of the total coumarin content was liberated from glucosylated precursors, respectively.     

Stable isotope and trace element profiling combined with classification models to differentiate geographic growing origin for three fruits: effects of subregion and variety

Classifications of geographic growing origin of three fresh fruits combining elemental profiles with various modeling approaches were determined. Elemental analysis (Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, V, and Zn) of strawberry, blueberry, and pear samples was performed using inductively coupled plasma argon atomic emission spectrometer. Bulk stable carbon and nitrogen isotope analyses in pear were performed using mass spectrometry as an alternative fingerprinting technique. Each fruit, strawberry (Fragaria x ananassa), blueberry (Vaccinium caesariense/corymbosum), and pear (Pyrus communis), was analyzed from two growing regions: Oregon vs Mexico, Chile, and Argentina, respectively. Principal component analysis and canonical discriminant analysis were used for data visualization. The data were modeled using linear discriminant function, quadratic discriminant function, neural network, genetic neural network, and hierarchical tree models with successful classification ranging from 70 to 100% depending on commodity and model. Effects of Oregon subregional and variety classification were investigated with similar success rates.     

Antimutagenic activity of phenylpropanoids from clove (Syzygium aromaticum)

Phenylpropanoids that possess antimutagenic activity were isolated from the buds of clove (Syzygium aromaticum). The isolated compounds suppressed the expression of the umu gene following the induction of SOS response in the Salmonella typhimurium TA1535/pSK1002 that have been treated with various mutagens. The suppressive compounds were mainly localized in the ethyl acetate extract fraction of the processed clove. This ethyl acetate fraction was further fractionated by silica gel column chromatography, which resulted in the purification and subsequent identification of the suppressive compounds. Electron impact mass spectrometry, IR, and (1)H and (13)C NMR spectroscopy were then used to delineate the structures of the compounds that confer the observed antimutagenic activity. The secondary suppressive compounds were identified as dehydrodieugenol (1) and trans-coniferyl aldehyde (2). When using 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) as the mutagen, compound 1 suppressed 58% of the umu gene expression as compared to the controls at a concentration of 0.60 micromol/mL, with an ID(50) (50% inhibitory dose) value of 0.48 micromol/mL, and compound 2 suppressed 63% of the umu gene expression as compared to the controls at a concentration of 1.20 micromol/mL, with an ID(50) value of 0.76 micromol/mL. Additionally, compounds 1 and 2 were tested for their ability to suppress the mutagenic activity of other well-known mutagens such as 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which do not require liver metabolizing enzymes, and aflatoxin B(1) (AfB(1)) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which require liver metabolizing enzymes and activated Trp-P-1 and UV irradiation. Compounds 1 and 2 showed dramatic reductions in their mutagenic potential of all of the aforementioned chemicals or treatment. For the search of the structure-activity relationship, the derivatives of 1 and 2 (1a and 2a-c) were also assayed with all mutagens. Finally, the antimutagenic activities of compounds 1, 1a, 2, and 2a-c against furylfuramide, Trp-P-1, and activated Trp-P-1 were assayed by the Ames test using the S. typhimurium TA100 strain.     

Application of stable isotope ratio analysis to the characterization of the geographical origin of olive oils.

To gain information about the geographical origin of oil samples, measurements of delta(13)C and delta(18)O of the whole oil and some of its fractions have been performed on samples coming from fruits of Olea europaea L. produced in Greece, Morocco, Spain, Italy, Tunisia, and Turkey. The results obtained by applying statistical procedures have given pieces of evidence that oil samples have shown the trend to cluster according to the different climatic areas of growing environment of fruits. Some confusion has been observed for samples coming from neighboring countries having similar climates.     

Application of multielement stable isotope ratio analysis to the characterization of French, italian, and spanish cheeses

The stable isotope ratios (delta13C, delta15N, and delta34S of casein and delta13C and delta18O of glycerol) measured by IRMS of French, Italian, and Spanish cheeses are presented and discussed. Variability factors such as animal-feeding regimen, geographical origin, and climatic and seasonal conditions were studied to check the possibilities of cheese characterization offered by each isotopic parameter. Delta13C values of both casein and glycerol appeared to be strongly correlated to the amount of maize in the animal diet. Delta15N and delta34S of casein proved to be mostly influenced by the geoclimatic conditions of the area (aridity, closeness to the sea, altitude). Delta18O of glycerol was more dependent on the geographical origin of the cheeses and on climatic/seasonal parameters. By applying a multivariate stepwise canonical discriminant analysis, good discrimination possibilities for the different European cheeses were obtained, confirmed by the classification analysis, when >90% of the samples were correctly reclassified.     

Putative role of pith cell wall phenylpropanoids in Sesamia nonagrioides (Lepidoptera: Noctuidae) resistance

The stem borer Sesamia nonagrioides (Lefèbvre) is the most important insect pest that attacks maize, Zea mays L., in northwestern Spain. Host plant resistance to this borer was investigated in relation to the cell wall phenylpropanoids content in the pith. Eight inbred lines that differ in resistance were analyzed. Three major simple phenolic acids, p-coumaric, trans-ferulic, and cis-ferulic acids, and three isomers of diferulic acid, 8-5', 8-O-4', and 8-5'b (benzofuran form), were identified. The amount of all these compounds was correlated with the resistance level in the genotypes, with the resistant inbreds having the highest concentrations. The role of these compounds in cell wall fortification and lignification is well-documented, suggesting their possible intervention in S. nonagrioides resistance. Future studies that focus on these compounds could be useful to enhance S. nonagroides resistance.     

Liquid chromatographic determination of coumarin anticoagulants in tablets: collaborative study

A liquid chromatographic method for the determination of coumarin anticoagulants in tablets was collaboratively studied by 7 laboratories. The method uses an octadecylsilane-bonded microparticulate column, tetrahydrofuran-methanol-water-acetic acid mobile phase, and photometric detection at 311 nm. Each collaborator received samples of warfarin sodium, phenprocoumon, and dicumarol as a synthetic composite and as commercial individual and composited tablets. Pooled average assay values for synthetic and commercial tablet samples of warfarin sodium were 101.6 and 99.5%, respectively, with a combined reproducibility SD of 2.38% (CV = 2.37%) and combined repeatability SD of 1.49% (CV = 1.49%). Pooled average (SD) assay values for dicumarol and phenprocoumon commercial samples were 98.0 (2.27) and 101.3% (4.00), respectively. The content uniformity determinations of 2 mg warfarin sodium and 25 mg dicumarol tablets indicated average tablet contents (range) of 99.5% (91.0-116.0) and 98.0% (89.8-108.8), respectively. The method has been approved interim official first action.