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Block 6: Physiologie, Metabolismus, Medizin, Doping, Forensik, Lebensmittel
Mittwoch, 29.09.2021:
9:30 - 10:30

Chair der Sitzung: Frank Keppler, Universität Heidelberg
Virtueller Veranstaltungsort: Block 6 - Meeting Link

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9:30 - 9:45

Stable isotopes for planetary health: Synthetic methionine

Cajetan Neubauer

University of Colorado Boulder, USA

Methionine is an amino acid that humans and farm animals must derive from food. This metabolite, a tightly regulated resource in ecosystems, has become a mass commodity in the global economy, with well over 1 million tons being produced annually from petroleum to fortify livestock feed. Methionine synthesis is an excellent exemplar of a planetary scale anthropogenic activity that manifests at the molecular scale of cellular metabolism, with potential systemic effects on human health and environments. Taking a planetary health perspective, I will explore in this talk the scale and historical trajectory of the methionine industry and provide a preliminary model for tracing this amino acid through the food supply into the human body.[1] We now want to test this model empirically using isotope analytics to quantify the synthetic proportion of methionine in foods and humans. The proposed approach is to use a mass spectrometry method developed in the last five years that is based on ESI-Orbitrap. It can reveal intramolecular stable isotope compositions of organic molecules at the natural abundance level.[2] I will also briefly discuss alternative isotope approaches for this project, as well as highlight topics where the new stable isotope technology might contribute insights in a biomedical context.

Related publications:

1. Neubauer C, Landecker H (2021) A planetary health perspective on synthetic methionine: Implications of opening the tap on a historically limiting nutrient. Lancet Planet Health

2. Neubauer C, Sweredoski MJ, Moradian A, Newman DK, Robins RJ, Eiler JM (2018). Scanning the isotopic structure of molecules by tandem mass spectrometry. Int J Mass Spec

9:45 - 10:00

Carbon isotope ratios of endogenous steroids found in human serum – method development, validation, and reference population-derived thresholds

Thomas Piper, Hans Geyer, Mario Thevis

Deutsche Sporthochschule Köln, Deutschland

In order to detect the misuse of testosterone (T) and testosterone prohormones, urinary steroids and steroid ratios are quantified and monitored in a longitudinal manner to enable the identification of atypical samples. These suspicious samples are then forwarded to isotope ratio mass spectrometry (IRMS)-based methods for confirmation. Especially concentration ratios like T over epitestosterone (E) or 5α-androstanediol (5αAdiol) over E proved to be valuable markers. Unfortunately, depending on the UGT2B17 genotype and/or the gender of the athlete, these markers may fail to provide evidence for T administrations when focusing exclusively urine samples.

In recent years, the potential of plasma steroids has been carefully investigated, which were found to be suitable to detect T administrations especially in female volunteers.[1-5] A current drawback of this approach is the missing possibility to confirm that elevated steroid concentrations found in plasma are solely derived from an administration of T or T-prohormones and cannot be attributed to possible confounding factors. Therefore, and in parallel to the procedure applied to urine samples, an IRMS method for plasma steroids has been developed and validated taking into account the limited sample volume for serum samples (usually not more than 1 mL). As endogenous reference compounds, unconjugated cholesterol and dehydroepiandrosterone-sulfate were found suitable while androsterone and epiandrosterone (both sulfoconjugated) were chosen as target analytes.

The method was based on multi-dimensional gas chromatography coupled to IRMS in order to increase recovery compared to liquid chromatography-based sample clean-up.[6] The method was validated employing linear mixing models, and finally a reference population encompassing n = 65 males and females was investigated to enable the calculation of population-based thresholds. As proof-of-concept, several serum samples from volunteers participating in T-replacement therapies were analyzed and found to be significantly depleted in their serum steroid target analytes.

1) Ponzetto F, Mehl F, Boccard J, et al. Anal Bioanal Chem 2016; 408: 705-719

2) Handelsman DJ, Bermon S. Drug Test Anal 2019; 11: 1566-1571

3) Elmongy H, Masquelier M, Ericsson M. J Chrom A 2020; 1613: 460686

4) Salamin O, Ponzetto F, Cauderay M, et al. Bioanalysis, 2020, DOI: 10.4155/bio-2020-0046

5) Knutsson JE, Andersson A, Baekken LV, et al. J Clin Endocrin & Metab 2020, DOI: 10.1210/clinem/dgaa904

6) Putz M, Piper T, Casilli A, et al. Analytica Chimica Acta 2018; 1030: 105-114.

10:00 - 10:15

Making plant methane formation visible – the effect of light exposure and light intensity on plant methane formation by application of 13C-labelled dimethyl sulfoxide

Moritz Schroll1, Katharina Lenhart2,3, Steffen Greiner3, Frank Keppler1,4

1Heidelberg University, Heidelberg, Germany; 2Bingen University of Applied Sciences, Bingen, Germany; 3Center for Organismal Studies, Heidelberg, Germany; 4Heidelberg Center for the Environment, Heidelberg University, Germany

In recent years methane (CH4) formation by vegetation in the presence of oxygen has been studied intensively, but its underlying formation process(es) are still poorly understood. Currently, reported CH4 emission rates vary by orders of magnitude and thus make global estimates of CH4 emissions by vegetation difficult. In addition, the process(es) of plant CH4 formation are mostly unknown. In this presentation, we introduce a new approach for a making CH4 formation by plants visible. By application of 13C-labelled dimethyl sulfoxide (DMSO) onto the leaves of tobacco plants (Nicotiana tabacum) and Chinese silver grass (Miscanthus sinensis), DMSO was identified as a precursor of CH4 in plants and the effect of light on CH4 formation was examined by measuring δ13C-CH4 values during incubations via Cavity Ring-Down Spectroscopy.

Both plant species clearly showed an increase in δ13C-CH4 values, and therefore CH4 formation, during the incubations while exposed to light. Higher light intensities lead to higher CH4 formation rates in N. tabacum and to lower CH4 formation rates in M. sinensis. When examined in the dark no formation of CH4 could be detected for N. tabacum, whilst M. sinensis still produced around 50 % of CH4 in the dark when compared to light exposure. Our findings unambiguously confirm DMSO as a precursor of plant CH4 and suggest that CH4 formation in plants is highly dependent on light exposure, light intensity, and plant species. Furthermore, our work presents a new isotopic approach for investigating mechanisms of CH4 formation in plants which has the potential to identify the underlying physiological processes, that without the use of stable isotopes could not be solely determined by CH4 concentration measurements.

10:15 - 10:30

Geographical provenance determination of wood through combination of isotope ratios

Jana C. Köhne1, Bernd Degen1, Anette Goeske2

1Thünen-Institut für Forstgenetik, Deutschland; 2Thünen-Institut für Agrarklimaschutz, Deutschland

As illegal logging develops more and more into a global problem, analytical methods to proof timber origin are necessary. In two pilot studies wood samples were collected to examine the possibilities of provenance determination of wood with stable isotope analyses as an addition of the “Large Scale Project” to develop genetic reference data to identify timber origin. The project was led by the Thuenen-Institute of Forest Genetics. In the first pilot study, wood samples of the tree species Carapa guianensis and Hymenaea courbaril were collected on a country scale at five locations for both species and an additional location for Hymenaea courbaril in Brazil with multiple 100 km distances up to 3,000 km between the geographical groups (locations) (Large Scale). The second pilot study addressed the regional scale in three different countries (Small Scale). In a range of 100 km wood samples were gathered for Hymenaea courbaril and H. jutai in two forest concessions in Brazil and for Dipteryx ferrea in Peru and Lophira alata in Cameroon in three concessions each. Forest concession are areas where logging companies received the permission for timber harvest from the state.

In the Large Scale study most isotope ratios showed differences between the geographical groups with most differences being statistical significant. Furthermore, weak significant correlations with latitude and longitude for d18O were observed, for d13C and d2H only with longitude and d15N correlated weakly with latitude. In a statistical self-assignment, the success rate in total was 59% for Carapa guianensis and 54% for Hymenaea sp.. Nevertheless the locations in Brazil were not distinguishable from each other in a principal component analysis.

In the Small Scale study, the isotope ratios of C and N, measured so far, did not allow for a significant separation of forest concession nor did they show significant correlation with latitude or longitude.

In conclusion there seems to be a geographical signal in the isotope results of the different location for the four isotope ratios of C, N, O and H but it was not strong enough for a secure geographic assignment. As next steps we want to check the performance of a combined application of gene markers and stale isotopes for geographic assignment of timber. Sampling design and lab methods also offer space for optimisation.

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