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Suppress or Not to Suppress … CRAFT It: A Targeted Metabolomics Case Study Extracting Essential Biomarker Signals Directly from the Full 1H NMR Spectra of Horse Serum Samples.
Abstract: : There are a few very specific inflammation biomarkers in blood, namely lipoprotein NMe signals of protein clusters (GlycA and GlycB) and a composite resonance of phospholipids (SPC). The relative integrals of these resonances provide clear indication of the unique metabolic changes associated with disease, specifically inflammatory conditions, often related to serious diseases such as cancer or COVID-19 infection. Relatively complicated, yet very efficient experimental methods have been introduced recently (DIRE, JEDI) to suppress the rest of the spectrum, thus allowing measurement of these integrals of interest. : In this study, we introduce a simple alternative processing method using CRAFT (Complete Reduction to Amplitude-Frequency Table), a time-domain (FID) analysis tool which can highlight selected subsets of the spectrum by choice for quantitative analysis. The output of this approach is a direct, spreadsheet-based representation of the required peak amplitude (integral) values, ready for comparative analysis, completely avoiding all the convectional data processing and manipulation steps. The significant advantage of this alternative method is that it only needs a simple water-suppressed 1D spectrum with no further experimental manipulation whatsoever. In addition, there are no pre/post processing steps (such as baseline and/or phase), further minimizing potential dependency on subjective decisions by the user and providing an opportunity to automate the entire process. : We applied this methodology to horse serum samples to follow the presence of inflammation for cohorts with or without OCD (Osteochondritis Dissecans) conditions and find diagnostic separation of the of the cohorts through statistical methods. : The powerful and simple CRAFT-based approach is suitable to extract selected biomarker information from complex NMR spectra and can be similarly applied to any other biofluid from any source or sample, also retrospectively. There is a potential to extend such a simple analysis to other, previously identified relevant markers as well.
Publication Date: 2025-06-10 PubMed ID: 40559411PubMed Central: PMC12194815DOI: 10.3390/metabo15060387Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
Objective Overview
- This study presents a new, simplified method using CRAFT software to extract key inflammation biomarkers from complex 1H NMR spectra of horse serum without complicated spectral suppression or extensive data processing.
Introduction and Background
- Certain biomarkers in blood, such as lipoprotein NMe signals (GlycA and GlycB) and a composite resonance of phospholipids (SPC), are important indicators of inflammation related to serious diseases like cancer and COVID-19.
- Traditional methods (DIRE, JEDI) involve experimental suppression techniques to isolate these specific signals by removing other spectral features, which can be complex and time-consuming.
CRAFT Methodology
- CRAFT (Complete Reduction to Amplitude-Frequency Table) is a time-domain analysis tool that processes free induction decay (FID) data from NMR to directly quantify selected spectral peaks.
- Instead of suppressing parts of the spectrum or applying multiple data correction steps (baseline correction, phase adjustment), CRAFT extracts peak amplitudes directly from a simple water-suppressed 1D NMR spectrum.
- This method generates output in spreadsheet format, containing ready-to-use integrals of the targeted biomarker peaks for comparative or statistical analysis.
- Eliminating preprocessing steps reduces user bias and facilitates automation, enhancing reproducibility and efficiency.
Application to Horse Serum Samples
- The new approach was tested on horse serum samples to detect inflammation by analyzing biomarkers related to Osteochondritis Dissecans (OCD), a joint disease.
- CRAFT analysis successfully distinguished between horses with and without OCD, demonstrating diagnostic potential through statistical separation of cohorts based on biomarker signals.
Advantages and Implications
- The CRAFT method requires only standard water-suppressed 1D NMR spectral data, making it accessible without extra experimental complexity.
- Its simplicity and direct quantitative outputs enable straightforward integration into diagnostic workflows, including retrospective studies on existing datasets.
- This approach can be extended beyond inflammation markers to other relevant metabolites or biomarkers detectable by NMR.
- Applicable to any biofluid or sample type, the method broadens utility in biomedical research and clinical diagnostics.
Cite This Article
APA
Chen J, Yablon A, Metaxas C, Guedin M, Hu J, Conover K, Simpson M, Ralston SL, Krishnamurthy K, Pelczer I.
(2025).
Suppress or Not to Suppress … CRAFT It: A Targeted Metabolomics Case Study Extracting Essential Biomarker Signals Directly from the Full 1H NMR Spectra of Horse Serum Samples.
Metabolites, 15(6), 387.
https://doi.org/10.3390/metabo15060387 Publication
Researcher Affiliations
- LLP 2024, Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
- LLP 2024, Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
- LLP 2024, Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
- LLP 2024, Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
- LLP 2024, Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
- Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
- Department of Animal Science, Rutgers University, New Brunswick, NJ 08544, USA.
- Independent Researcher, Howell, NJ 08544, USA.
- Chempacker LLC, San Jose, CA 95135, USA.
- Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
Conflict of Interest Statement
Author Krish Krishnamurthy was employed by the company Chempacker LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
References
This article includes 33 references
- Nicholson JK, Connelly J, Lindon JC, Holmes E. Metabonomics: A platform for studying drug toxicity and gene function.. Nat. Rev. Drug Discov. 2002;1:153–161.
- Emwas A-H, Roy R, McKay RT, Tenori L, Saccenti E, Nagana Gowda GA, Raftery D, Alahmari F, Jaremko L, Jaremko M. NMR Spectroscopy for Metabolomics Research.. Metabolites 2019;9:123.
- . NIH Announces New Program in Metabolomics.. U.S. Department of Health and Human Services. National Institutes of Health .
- Pelczer I. High-resolution NMR for metabomics.. Curr. Opin. Drug Res. Dev. 2005;8:127–133.
- Fuertes-Martín R, Correig X, Vallvé JC, Amigó N. Human Serum/Plasma Glycoprotein Analysis by 1H-NMR, an Emerging Method of Inflammatory Assessment.. J. Clin. Med. 2020;9:354.
- Fonville JM, Richards SE, Barton RH, Boulange CL, Ebbels TMD, Nicholson JK, Holmes E, Dumas M-E. The evolution of partial least squares models and related chemometric approaches in metabonomics and metabolic phenotyping.. J. Chemom. 2010;24:636–649.
- Lodge S, Nitschke P, Kimhofer T, Wist J, Bong Sz-H, Loo RL, Masuda R, Begum S, Richards T, Lindon JC. Diffusion and Relaxation Edited Proton NMR Spectroscopy of Plasma Reveals a High-Fidelity Supramolecular Biomarker Signature of SARS-CoV-2 Infection.. Anal. Chem. 2021;93:3976–3986.
- Nitschke P, Lodge S, Kimhofer T, Masuda R, Bong S-H, Hall D, Schäfer H, Spraul M, Pompe N, Diercks T. J-Edited DIffusional Proton Nuclear Magnetic Resonance Spectroscopic Measurement of Glycoprotein and Supramolecular Phospholipid Biomarkers of Inflammation in Human Serum.. Anal. Chem. 2022;94:1333–1341.
- Khirich G. A Monte Carlo Method for Analyzing Systematic and Random Uncertainty in Quantitative Nuclear Magnetic Resonance Measurement.. Anal. Chem. 2021;93:10039–10047.
- Krishnamurthy K. CRAFT (complete reduction to amplitude frequency table)—robust and time-efficient Bayesian approach for quantitative mixture analysis by NMR.. Magn. Reson. Chem. 2013;51:821–829.
- Krishnamurthy K, Sefler AM, Russell DJ. Application of CRAFT in two-dimensional NMR data processing.. Magn. Reson. Chem. 2017;55:224–232.
- Krishnamurthy K, Hari N. Application of CRAFT (complete reduction to amplitude frequency table) in nonuniformly sampled (NUS) 2D NMR data processing.. Magn. Reson. Chem. 2018;56:535–545.
- Krishnamurthy K. NMR Quantitative Analysis of Complex Mixtures.. eMagRes 2014;3:235–242.
- Shaughnessy WJ. Osteochondritis Dissecans.. 2009. pp. 288–296.
- Ytrehus B, Carlson CS, Ekman S. Etiology and Pathogenesis of Osteochondrosis.. Am. Coll. Vet. Pathol. Vet. Pathol. 2007;44:429–448.
- McIlwraith C.W.. Developmental orthopaedic disease: Problems of limbs in young horses.. J. Equine Vet. Sci. 2004;24:475–479.
- Ekman S., Carlson C.S., Van Weeren P.R.. Workshop Report: Third International Workshop on Equine Osteochondrosis, Stockholm.. Equine Vet. J. 2008;41:504–507.
- Ralston S.L.. Hyperglycemia/hyperinsulinemia after feeding a meal of grain to young horses with osteochondritis dissecans (OCD) lesions.. Pferdeheilkunde 1996;12:320–322.
- Pagan J.D., Geor R., Caddel S.E., Pryor P.B., Hoekstra K.E.. The relationship between glycemic response and the incidence of OCD in Thoroughbred weanlings: A field study.. Proceeding Am. Assoc. Equine Pract. 2001;7:322–325.
- Sandgren B., Dalin G., Carlsten J., Lundeheim N.. Development of osteochondrosis in the tarsocrural joint and osteochondral fragments in the fetlock joints of Standardbred trotters. Body measurements and clinical findings. Osteochondrosis.. Equine Vet. J. 1993;16:48–53.
- Lykkjen S., Dolvik N.I., McCue M.E., Rendahl A.K., Mickelson J.R., Roed K.H.. Genome-wide association analysis of Osteochondrosis of the tibiotarsal joint in Norwegian Standardbred trotters.. Sticht. Int. Found. Anim. Genet. 2010;41((Suppl. 2)):111–120.
- Yu J., Pressoir G., Briggs W.H., Vroh Bi I., Yamasaki M., Doebley J.F., McMullen M.D., Gaut B.S., Nielsen D.M., Holland J.B.. A unified mixed-model method for association mapping that account for multiple levels of relatedness.. Nat. Genet. 2006;38:203–208.
- Sipson M.. Comparison of NMR and Mass Spectrometry-Based Metabonomic and Metabolomic Analyses of Horse Serum from Young Horses with and Without Osteochondrosis Dissecans. George H. Cook Scholars.. 2013.
- Beckonert O., Keun H.C., Ebbels T.M.D., Bundy J., Holmes E., Lindon J.C., Nicholson J.K.. Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts.. Nat. Protoc. 2007;2:2692–2703.
- Hwang T.-L., Shaka A.J.. Water Suppression That Works. Excitation Sculpting Using Arbitrary Waveforms and Pulsed Field Gradients.. J. Magn. Reson. Ser. A. 1995;112:275–279.
- Takis P.G., Jiménez B., Sands C.J., Chekmenova E., Lewis M.R.. SMolESY: An efficient and quantitative alternative to on-instrument macrmolecular 1H-NMR signal suppression.. Chem. Sci. 2020;11:6000–6011.
- Richards S.A., Hollerton J.C.. Essential Practical NMR for Organic Chemistry.. 2011.
- Hong Y.S., Coen M., Rhode C.M., Reily M.D., Robertson D.G., Holmes E., Lindon J.C., Nicholson J.K.. Chemical shift calibration of 1H MAS NMR liver tissue spectra exemplified using a study of glycine protection of galactosamine toxicity.. Magn. Reson. Chem. 2009;47:S47–S53.
- Ralston S.L., Pappalardo L., Pelczer I., Spears P.F.. NMR-based metabonomic analyses of horse serum: Detection of metabolic markers of disease.. Rec. Adv. Animal Nutr.-Aust. 2011;18:197–206.
- Masuda R., Lodge S., Whiley L., Gray N., Lawler N., Nitschke P., Bong S.-H., Kimhofer T., Loo R.L., Boughton B.. Exploration of Human Serum Lipoprotein Supramolecular Phospholipids Using Statistical Heterospectroscopy in n-Dimensions (SHY-n): Identification of Potential Cardiovascular Risk Biomarkers Related to SARS-CoV-2 Infection.. Anal. Chem. 2022;94:4426–4436.
- Lodge SS, Masuda R, Nitschke P, Beilby JP, Hui J, Hunter M, Yeap BB, Millet O, Wist J, Nicholson JK. NMR spectroscopy derived plasma biomarkers of inflammation in human populations: Influences of age, sex and adiposity.. PLoS ONE 2025;20:e0311975.
- Roberts JL, Whiley L, Gray N, Gay M, Nitschke P, Masuda R, Holmes E, Nicholson JK, Wist J, Lawler NG. Rapid and Self-Administrable Capillary Blood Microsampling Demonstrates Statistical Equivalence with Standard Venous Collections in NMR-Based Lipoprotein Analysis.. Anal. Chem. 2024;96:4505–4513.
- Nitschke P, Lodge S, Hall D, Schaefer H, Spraul M, Embadé N, Millet O, Holmes E, Wist J, Nicholson JK. Direct low field J-edited diffusional proton NMR spectroscopic measurement of COVID-19 inflammatory biomarkers in human serum.. Analyst 2022;147:4213–4221.
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