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Metabolomics by multidimensional NMR

Metabolomics by multidimensional NMR

Figure 1.                Figure 2.                  Figure 3. 



Metabolomics deals with the simultaneous and systematic analysis of multiple metabolite concentrations and their variations due to effects of diet, liefestyle, environment, drugs, and genetic modulations. Metabolites in this regard are small molecules such as sugars, lipids or free amino acids present in body fluids and tissues. We are currently working on a variety of projects related to human health. Most projects are performed in close collaborations with partners from several university clinics. As a tool multidimensional NMR spectroscopy is used for this purpose.

As a typical example Fig. 1 shows a 1H-13C HSQC spectrum of human urine obtained from a healthy volunteer. The spectrum was aquired on a BRUKER 600 MHz NMR spectrometer equipped with a cryo probe. Some highly abundant metabolites that are easily quantified by NMR are marked. The inset in the upper left corner shows the corresponding 1D spectrum. As can be clearly seen a considerable gain in resolution is obtained by 2D NMR.

- Kaspar, H., Dettmer, K., Gronwald, W., & Oefner, P.J. Advances in Amino Acid Analysis. Anal. Bioanal. Chem.393, 445-452 (2009). 
Gronwald, W., Klein, M.S., Kaspar, H., Fagerer, S., Nürnberger, N., Dettmer, K., Bertsch, T. & Oefner, P.J. Urinary Metabolite Quantification Employing 2D NMR Spectroscopy. Anal. Chem.80, 9288-9297 (2008).
- Kaspar, H., Dettmer, K., Gronwald, W. & Oefner, P.J. Automated GC-MS Analysis of Free Amino Acids in Biological Fluids. J. Chromat. B870, 222-232 (2008).

Ongoing projects

Investigation of autosomal dominat polycystic kidney disease (ADPKD)
In this project we are striving to identify a set of urinary biomarkers to allow the reliable classification of healthy and diseased patients. For this, metabolomic data are analyzed with bioinformatic methods such as support vector machines (SVM). A good way to look at regulated metabolitesis the use of a heapmap representation as shown in Figure 2 for ADPKD patients and several control groups. The importance of this type of research was also recently highlighted in Nature Reviews Nephrology.

Figure 2. Heatmap representation of differentially regulated metabolites in ADPKD. 
Up-regulated features are indicated in yellow while down-regulated ones are marked in blue. Rows are ordered in correlation with disease status. Rows that are mostly up-regulated in ADPKD patients and down-regulated in all other groups are shown in the top part of the figure and vice versa. Used abbreviations: 3-hydroxyisovaleric acid (3-OH-IVA), 6-hydroxynicotinic acid (6-OH-NA), alanine (Ala), carbohydrates (Carb), D-saccharic acid (D-Sac), methanol (MeOH), sucrose (Suc), tartaric acid (Tar), threonine (Thr), and tyrosine (Tyr).

Gronwald, W., Klein, M.S., Zeltner, R., B.-D. Schulze, Reinhold, S.W., Deutschmann, M., Immervoll, A.-K., Böger, C.A., Banas, B., Eckardt, K.-U. & Oefner, P.J. Detection of Autosomal Dominant Polycystic Kidney Disease using NMR Spectroscopic Fingerprints of Urine. Kidney International, available online, DOI: 10.1038/ki.2011.30 (2011).
- Myrvang, H. Research Highlight: Urinary fingerprints unique to patients with ADPKD. Nat. Rev. Nephrol.7, 244 (2011) | doi:10.1038/nrneph.2011.33

Metabolic status of dairy cows
Milk production in dairy cows has dramatically increased over the past few decades. The selection for higher milk yield affects the partitioning of available nutrients, with more energy being allocated to milk synthesis and less to physiological processes relevant to fertility and fitness. In this project milk metabolites are systematically investigated with a special emphasis on energy metabolism.

- Dettmer, K., Almstetter, M.F., Appel, I. J., Nürnberger, N., Schlamberger, G., Gronwald, W., Meyer, H.H.D. & Oefner, P.J. Comparison of Serum versus Plasma Collection in Gas Chromatography - Mass Spectrometry based Metabolomics.Electrophoresis31, 2365-2373 (2010).
- Klein, M.S., Almstetter, M.F., Schlamberger, G., Nürnberger, N., Dettmer, K., Oefner, P.J., Meyer, H.H.D., Wiedemann, S. &Gronwald, W. NMR and Mass Spectrometry Based Milk Metabolomics in Dairy Cows during Early and Late Lactation. J. Dairy Sci.93, 1539-1550 (2010). 

Non-alcoholic steatohepatitis (NASH)
Due to the remarkable increase in the prevalence of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) in western countries and the concomitant efforts in developing novel therapies for patients with NASH, simple, reproducible, and reliable non-invasive methodologies are needed. In this study NMR spectroscopy combined with independent component analysis (ICA) is used for the investigation of metabolic changes associated with NAFLD and NASH in mice. For this, urine and liver extracts of mice fed with different diets are analyzed. The ICA analysis of this data shows a clear group separation between healthy controls, and specimens obtained from NAFLD and NASH mice (Fig. 3)

Figure 3. ICA analyis of NMR data obtained from healthy controls (circles) and mice suffering from NAFLD (blue squares) and NASH (red triangles).

- Klein, M.S., Dorn, C., Saugspier, M., Hellerbrand, C., Oefner, P.J. & Gronwald, W. Discrimination of steatosis and NASH in mice using nuclear magnetic resonance spectroscopy Metabolomics, available online, DOI: 10.1007/s11306-010-0243-6 (2010).

Investigation of tumor metabolism
It is analyzed whether tumor metabolism functions as a modulator of immune response and tumor progression. Using different cancer cell lines the tumor metabolom is investigated in detail.

Protein-protein interactions

Investigation of protein-protein interactions employing 3D-structural information. Experimental and computational approaches are combined for the structure determination and analysis of protein-protein complexes. For the reliable scoring of 3D protein-protein complexes we have developed the knowledge based protein complex scoring server PROCOS.

Protein Structures Solved by Automated NMR Methods

Reliable automated structure determination of biological macromelecules such as proteins from NMR data is the ultimate goal of the program AUREMOL (http://www.auremol.de). AUREMOL utilizes a molecule-centered top-down strategy aimed at deriving a 3D structure from a minimum of NMR data using information from additional sources. The term top-down means in this context that a starting structure is iteratively refined until it fits the experimental data as well as possible. It should be noted that the starting structure can be anything from a simple extended strand to a well-defined homologous model.

The following structures were solved by us with the help of automated methods:

Gronwald, W., Bomke, J., Maurer, T., Domogalla, B., Huber, F., Schumann, F., Kremer, W., Rysiok, T., Frech, M. & Kalbitzer, H.R. Solution Structure of the Leech Protein Saratin and Characterisation of its Binding to Collagen. J. Mol. Biol., 381, 913-927 (2008)

Gronwald, W., Huber, F., Grünewald, P., Spörner, M., Wohlgemuth, S., Herrmann, C., Wittinghofer, A. & Kalbitzer, H. R. Solution Structure of the Ras-binding domain of the protein kinase Byr2 from Schizosaccharomyces pombe. Structure, 9, 1029-1041 (2001)

Gronwald, W., Loewen, M. C., Lix, B., Daugulis, A. J., Sönnichsen, F. D., Davies, P. L. & Sykes, B. D. The Solution structure of Type II Antifreeze Protein Reveals a New member of the Lectin Family. Biochemistry, 37, 4712-4721 (1998)

Kachel, N., Erdmann, K., Kremer, W., Wolff, P., Gronwald, W., Heumann, R. & Kalbitzer, H. R. Structure determination and binding characteristics of the PDZ2 domain of PTPbas (hPTP1E): Splicing induced modulation of ligand specificity. J. Mol. Biol., 334, 143-155 (2003)

Kremer, W., Harrieder, S., Geyer, M., Gronwald, W., Welker, C., Schuler, B., Jaenicke, R. & Kalbitzer, H. R. Solution NMR Structure and Backbone Dynamics of the Cold-shock protein from the hyperthermophilic bacterium Thermotoga maritima: Insights into thermostabilityEur. J. Biochem., 268, 2527-2539 (2001)


  1. Department of Medicine
  2. Institute of Functional Genomics

Prof. Wolfram Gronwald

Group NMR Spectrometry


Image Wolfram2016

Functional Genomics
Am Biopark 9
93053 Regensburg, Germany

Tel: +49 941 943 5015