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NMR studies of metabolites and xenobiotics: From time-points to long-term metabolic regulation
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Jürgen Schleucher)
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Chemical species carry information in two dimensions, in their concentrations and their isotopic signatures. The concentrations of metabolites or synthetic compounds describe the composition of a chemical or biological system, while isotopic signatures describe processes in the system by their reaction pathways, regulation, and responses to external stimuli. Stable isotopes are unique tracers of these processes because their natural abundances are modulated by isotope effects occurring in physical processes as well as in chemical reactions. Nuclear magnetic resonance (NMR) spectroscopy is a prime technique not only for identification and quantification of small molecules in complex systems but also for measuring intramolecular distribution of stable isotopes in metabolites and other small molecules. In this thesis, we use quantitative NMR in three fields: in food science, environmental pollutant tracing, and plant-climate science.

The phospholipid (PL) composition of food samples is of high interest because of their nutritional value and technological properties. However, the analysis of PLs is difficult as they constitute only a small fraction of the total lipid contents in foods. Here, we developed a method to identify PLs and determine their composition in food samples, by combining a liquid-liquid extraction approach for enriching PLs, with specialized 31P,1H-COSY NMR experiments to identify and quantify PLs.

Wide-spread pollution with synthetic compounds threatens the environment and human health. However, the fate of pollutants in the environment is often poorly understood. Using quantitative deuterium NMR spectroscopy, we showed for the nitrosamine NDMA and the pesticide DDT how intramolecular distributions (isotopomer patterns) of the heavy hydrogen isotope deuterium reveal mechanistic insight into transformation pathways of pollutants and organic compounds in general. Intramolecular isotope distributions can be used to trace a pollutant’s origin, to understand its environmental transformation pathways and to evaluate remediation approaches.

The atmospheric CO2 concentration ([CO2]) is currently rising at an unprecedented rate and plant responses to this increase in [CO2] influence the global carbon cycle and will determine future plant productivity. To investigate long-term plant responses, we developed a method to elucidate metabolic fluxes from intramolecular deuterium distributions of metabolites that can be extracted from historic plant material. We show that the intramolecular deuterium distribution of plant glucose depends on growth [CO2] and reflects the magnitude of photorespiration, an important side reaction of photosynthesis. In historic plant samples, we observe that photorespiration decreased in annual crop plants and natural vegetation over the past century, with no observable acclimation, implying that photosynthesis increased. In tree-ring samples from all continents covering the past 60 – 700 years, we detected a significantly smaller decrease in photorespiration than expected. We conclude that the expected “CO2 fertilization” has occurred but was significantly less pronounced in trees, due to opposing effects.

The presented applications show that intramolecular isotope distributions not only provide information about the origin and turnover of compounds but also about metabolic regulation. By extracting isotope distributions from archives of plant material, metabolic information can be obtained retrospectively, which allows studies over decades to millennia, timescales that are inaccessible with manipulation experiments.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2015. , 54 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1691
Keyword [en]
NMR spectroscopy, isotopomer, phospholipid, persistent organic pollutant, CO2 fertilization, photorespiration
National Category
Biophysics Chemical Sciences
Identifiers
URN: urn:nbn:se:umu:diva-97684ISBN: 978-01-7601-195-9 OAI: oai:DiVA.org:umu-97684DiVA: diva2:775727
Public defence
2015-01-23, KB3A9, 10:00 (English)
Opponent
Supervisors
Available from: 2015-01-07 Created: 2015-01-03 Last updated: 2015-05-19Bibliographically approved
List of papers
1. Two-Dimensional P-31,H-1 NMR Spectroscopic Profiling of Phospholipids in Cheese and Fish
Open this publication in new window or tab >>Two-Dimensional P-31,H-1 NMR Spectroscopic Profiling of Phospholipids in Cheese and Fish
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2013 (English)In: Journal of Agricultural and Food Chemistry, ISSN 0021-8561, E-ISSN 1520-5118, Vol. 61, no 29, 7061-7069 p.Article in journal (Refereed) Published
Abstract [en]

Phospholipids (PLs) comprise an important lipid class in food because of their technological use as emulsifiers and their nutritional value. This study used one-dimensional P-31 NMR and two-dimensional (2D) P-31,H-1 COSY NMR spectroscopy for the determination of the PL composition of cheese and fish after liquid liquid enrichment. This extraction step enabled the identification of 10 PLs in cheese and 9 PLs in fish by 2D P-31,H-1 NMR. Variations in the P-31 shifts indicated differences in the fatty acids attached to the individual PLs. The total PL content in cheese fat and fish oil ranged from 0.3 to 0.4% and from 5 to 12%, respectively. Phosphatidylcholine was the most prominent PL in both matrices (up to 6596). Minor PLs (limit of detection = 4 nmol, i.e. 500 mu L of an 8 mu M solution) were identified in forms of phosphatidic acid, lysophosphatidic acid, and phosphatidylglycerol. Specific cross couplings and H-1 fine structures in the 2D P-31,H-1 NMR spectra proved to be valuable for the assignment and verification of known and uncommon PLs in the samples.

Keyword
phospholipids, nuclear magnetic resonance spectroscopy, P-31 NMR, food, milk fat, fish oil
National Category
Food Science Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-79896 (URN)10.1021/jf4021812 (DOI)000322432300006 ()
Available from: 2013-09-05 Created: 2013-09-04 Last updated: 2017-12-06Bibliographically approved
2. Elucidating Turnover Pathways of Bioactive Small Molecules by Isotopomer Analysis: The Persistent Organic Pollutant DDT
Open this publication in new window or tab >>Elucidating Turnover Pathways of Bioactive Small Molecules by Isotopomer Analysis: The Persistent Organic Pollutant DDT
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 10, e110648- p.Article in journal (Refereed) Published
Abstract [en]

The persistent organic pollutant DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane) is still indispensable in the fight againstmalaria, although DDT and related compounds  pose toxicological  hazards. Technical DDT contains the dichloro congenerDDD (1-chloro-4-[2,2-dichloro-1-(4-chlorophenyl)ethyl]benzene)   as by-product, but  DDD is also formed by  reductive degradation of DDT in the environment. To differentiate between DDD formation pathways, we applied deuterium NMR spectroscopy to measure intramolecular deuterium distributions (2H isotopomer abundances) of DDT and DDD. DDD formed in the technical  DDT synthesis was strongly deuterium-enriched at one intramolecular position, which we traced back to 2H/1H fractionation of a chlorination step in the technical synthesis.  In contrast, DDD formed by reductive degradation was strongly depleted at the same position, which was due to the incorporation of 2H-depleted hydride equivalents during reductive degradation. Thus, intramolecular isotope distributions give mechanistic information on reaction pathways, and explain a puzzling difference in the whole-molecule 2H/1H ratio between DDT and DDD. In general, our results highlight that intramolecular isotope distributions are essential to interpret whole-molecule isotope ratios. Intramolecular isotope information allows distinguishing pathways of DDD formation, which is important to identify polluters or to assess  DDT turnover in the environment. Because  intramolecular isotope data directly reflect isotope fractionation of individual chemical reactions, they are broadly applicable to elucidate transformation pathways of smallbioactive molecules in chemistry, physiology and environmental science.

Place, publisher, year, edition, pages
PLoS ONE, 2014
Keyword
DDT, turnover pathway, isotopomer, deuterium
National Category
Chemical Sciences
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:umu:diva-97673 (URN)10.1371/journal.pone.0110648 (DOI)000343943100033 ()
Available from: 2015-01-03 Created: 2015-01-03 Last updated: 2017-12-05Bibliographically approved
3. Compound-specific carbon, nitrogen, and hydrogen isotope analysis of N-nitrosodimethylamine in aqueous solutions
Open this publication in new window or tab >>Compound-specific carbon, nitrogen, and hydrogen isotope analysis of N-nitrosodimethylamine in aqueous solutions
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2015 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 5, 2916-2924 p.Article in journal (Refereed) Published
Abstract [en]

Mitigation of N-nitrosodimethylamine (NDMA) and other hazardous water disinfection byproducts (DBP) is currently hampered by a limited understanding of DBP formation mechanisms. Because variations of the stable isotope composition of NDMA can potentially reveal reaction pathways and precursor compounds, we developed a method for the compound-specific isotope analysis (CSIA) of (13)C/(12)C, (15)N/(14)N, and (2)H/(1)H ratios of NDMA by gas chromatography coupled to isotope ratio mass spectrometry (GC/IRMS). Method quantification limits for the accurate isotope analysis of NDMA, N-nitrosodiethyl-, -dipropyl-, and -dibutylamine as well as N-nitrosopyrrolidine were between 0.18 to 0.60 nmol C, 0.40 to 0.80 nmol N, and 2.2 to 5.8 nmol H injected on column. Coupling solid phase extraction (SPE) to GC/IRMS enabled the precise quantification of C, N, and H isotope ratios of NDMA in aqueous samples at concentrations of 0.6 μM (45 μg L(-1)). We validated the proposed method with a laboratory experiment, in which NDMA was formed with stoichiometric yield (97 ± 4%) through chloramination of the pharmaceutical ranitidine (3 μM). δ(13)C and δ(2)H values of NDMA remained constant during NDMA formation while its δ(15)N increased due to a reaction at a N atom in the rate-limiting step of NDMA formation. The δ(2)H value of NDMA determined by SPE-GC/IRMS also corresponded well to the δ(2)H value of the N(CH3)2-group of ranitidine measured by quantitative deuterium nuclear magnetic resonance spectroscopy. This observation implies that the N(CH3)2-moiety of ranitidine is transferred to NDMA without being chemically altered and illustrates the accuracy of the proposed method.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:umu:diva-101104 (URN)10.1021/ac5044169 (DOI)000350611700057 ()25621380 (PubMedID)
Note

Originally included in thesis in manuscript form, with the title "Compound-Specific Carbon, Nitrogen, and Hydrogen Isotope Analysis of N-Nitrosodimethylamine (NDMA) in Aqueous Solutions"

Available from: 2015-03-20 Created: 2015-03-20 Last updated: 2017-12-04Bibliographically approved
4. Quantification of a metabolic shift towards photosynthesisin C3 plants driven by 20th-century CO2 rise
Open this publication in new window or tab >>Quantification of a metabolic shift towards photosynthesisin C3 plants driven by 20th-century CO2 rise
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Terrestrial vegetation currently absorbs approximately a third of the annual anthropogenic CO2 emissions, mitigating the rise of atmospheric CO2. However,terrestrial net primary production is highly sensitive to atmospheric [CO2] and associated climatic changes. In C3-plants, which dominate terrestrial vegetation, netphotosynthesis depends on the ratio between gross photosynthesis and photorespiration, which strongly depends on [CO2]. However, our knowledge of feedbacks betweenterrestrial biomes and increasing atmospheric [CO2] is nearly entirely based on atmospheric inversion models and manipulation experiments, which do not reveal physiological mechanisms or are limited in duration and to step increases in [CO2]. By applying novel NMR (Nuclear Magnetic Resonance) spectroscopy methodology we examine isotopomer ratios of plant carbohydrates to probe shifts in the photosynthesis/photorespiration ratio in C3 plants over more than a century. Using herbarium samples of natural vascular plant species, crops and a Sphagnum species, we detect a consistent 35% increase in the 2photosynthesis/photorespiration ratio in responseto the ~100 ppm CO2 increase between approximately 1900 and 2013, with no evidencefor feedback regulation by the plants. Our data provide direct quantitative information on the “CO2 fertilization effect” over century time scales, thus addressing a major uncertainty in Earth system models, enabling improved predictions of the future [CO2] sink strength of terrestrial ecosystems. Further, relating the detected metabolic shift in crop plants to historic yield trends indicates that only a fraction of the increased net photosynthesis has translated into increased yield. Our results also demonstrate that archives of plant material contain metabolic information embedded in their isotopomer ratios covering centuries, bridging a fundamental gap between experimental plant science and paleoenvironmental studies.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-97675 (URN)
Available from: 2015-01-03 Created: 2015-01-03 Last updated: 2015-01-15Bibliographically approved
5. Limited suppression of photorespiration by 20th century atmospheric CO2 increase in trees worldwide
Open this publication in new window or tab >>Limited suppression of photorespiration by 20th century atmospheric CO2 increase in trees worldwide
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Forests are a key component of the global carbon and hydrological cycle and forest responses to  environmental  drivers  create  important  feedbacks  to  these  cycles.  Photosynthetic efficiency of most forest tree species is strongly limited by photorespiration, a side reaction using O2 instead of CO2 as substrate, leading to a carbon loss for the plant. Photorespiration occurs in all trees and is reduced under elevated CO2 concentrations and increased under elevated temperature. Because the CO2 concentration of the atmosphere has increased in past decades, long-lived trees may have benefited from reduced photorespiration, but the temperature increase would have been a compensating detriment; but direct quantification of long-term changes in metabolic fluxes is lacking. Realistic forecasting of responses of trees and forests to future CO2 and temperature demands quantifying the reduction of photorespiration.  In  twelve  tree  species  from  five  continents,  we  observe  that photorespiration has been reduced by the CO2 increase during the past century, but for most the reduction is smaller than predicted from plant responses in CO2 alone. Comparison with data from a combined CO2 and temperature manipulation experiment shows that the reduced response can be explained by increases in leaf temperatures, which might result directly from increased  air  temperatures  or  indirectly  from  reduced  transpirative  cooling.  These  data suggest that global warming has already inhibited plant fertilization by increasing CO2, and that biomass increases may have been smaller than deduced from measurements of the heavy carbon isotope 13C. Observation of this centennial metabolic shift in tree physiology worldwide provides new insights into forest-climate feedbacks and can be used to improve coupled climate-vegetation models.

National Category
Chemical Sciences Climate Research
Identifiers
urn:nbn:se:umu:diva-97676 (URN)
Available from: 2015-01-03 Created: 2015-01-03 Last updated: 2015-01-15Bibliographically approved

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