A Simple and Feasible Quantification of Metabolites in the Human Follicular Fluid Using 1H HR-MAS NMR Spectroscopy

Abstract

This study presents a reliable method for the quantification of metabolite concentrations in follicular fluid with the high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy and the ERETIC2 (Electronic REference To access In vivo Concentrations) based on PULCON (pulse length based concentration determination) principle. The positive effect of the HR-MAS probe technology on spectral quality and its ability to perform analyses with very low sample amounts were the most important factors of proposing this method. In evaluating the performance of the proposed method, standard creatine solutions in different concentrations containing DSS (2,2-dimethyl-2-silapentane-5-sulfonate sodium salt) as an internal reference standard were analyzed using different pulse programs (cpmgpr1d and zg30). The results obtained with the ERETIC2 were compared with the classical internal standard NMR quantification method (DSS method). The relative standard deviation (RSD) values for ERETIC2 were in the range of 0.3% - 5.7% and recovery values were calculated as minimum 90.3%, while RSD values for DSS method were in the range of 0.1% - 3.1% and recovery values were minimum 97.0%. Besides, it was observed that the metabolite concentration values calculated using the ERETIC2 procedure of follicular fluid samples obtained from the women with endometriosis and healthy controls were compatible with the values those obtained using different methodologies. The obtained results showed that the proposed quantification method based on the HR-MAS spectroscopy can easily be used in biological fluids and therefore it can be utilized as a good alternative to the internal standard method considering its accuracy and precision.

Keywords

• HR-MAS spectroscopy
• quantification
• PULCON
• ERETIC2
• metabolite concentration

References

1. 1. Renault M, Shintu L, Piotto M, Caldarelli S. Slow-spinning low-sideband HR-MAS NMR spectroscopy: delicate analysis of biological samples. Sci Rep. 2013 Dec;3(1):3349.
2. 2. Moestue S, Sitter B, Frost Bathen T, Tessem M-B, Susann Gribbestad I. HR MAS MR Spectroscopy in Metabolic Characterization of Cancer. CTMC. 2011 Jan 1;11(1):2–26.
3. 3. Mazzei P, Piccolo A. HRMAS NMR spectroscopy applications in agriculture. Chem Biol Technol Agric. 2017 Dec;4(1):11.
4. 4. Gogiashvili M, Nowacki J, Hergenröder R, Hengstler JG, Lambert J, Edlund K. HR-MAS NMR Based Quantitative Metabolomics in Breast Cancer. Metabolites. 2019 Jan 22;9(2):19.
5. 5. Lindon JC, Beckonert OP, Holmes E, Nicholson JK. High-resolution magic angle spinning NMR spectroscopy: Application to biomedical studies. Progress in Nuclear Magnetic Resonance Spectroscopy. 2009 Aug;55(2):79–100.
6. 6. Swanson MG, Zektzer AS, Tabatabai ZL, Simko J, Jarso S, Keshari KR, et al. Quantitative analysis of prostate metabolites using1H HR-MAS spectroscopy. Magn Reson Med. 2006 Jun;55(6):1257–64.
7. 7. Tilgner M, Vater TS, Habbel P, Cheng LL. High-Resolution Magic Angle Spinning (HRMAS) NMR Methods in Metabolomics. In: Gowda GAN, Raftery D, editors. NMR-Based Metabolomics [Internet]. New York, NY: Springer New York; 2019 [cited 2021 Dec 1]. p. 49–67. (Methods in Molecular Biology; vol. 2037).
8. 8. Bharti SK, Roy R. Quantitative 1H NMR spectroscopy. TrAC Trends in Analytical Chemistry. 2012 May;35:5–26.

9. 9. Zoelch N, Hock A, Heinzer-Schweizer S, Avdievitch N, Henning A. Accurate determination of brain metabolite concentrations using ERETIC as external reference. NMR in Biomedicine. 2017 Aug;30(8):e3731.
10. 10. Barding GA, Salditos R, Larive CK. Quantitative NMR for bioanalysis and metabolomics. Anal Bioanal Chem. 2012 Sep;404(4):1165–79.
11. 11. Cullen CH, Ray GJ, Szabo CM. A comparison of quantitative nuclear magnetic resonance methods: internal, external, and electronic referencing: Comparison of quantitative NMR methods. Magn Reson Chem. 2013 Sep.
12. 12. Heinzer-Schweizer S, De Zanche N, Pavan M, Mens G, Sturzenegger U, Henning A, et al. In-vivo assessment of tissue metabolite levels using 1h mrs and the electric reference to access in vivo concentrations (eretic) method: In-Vivo Assessment of Tissue Metabolite Levels Using Eretic. NMR Biomed. 2010 May;23(4):406–13.
13. 13. Jung Y-S, Hyeon J-S, Hwang G-S. Software-assisted serum metabolite quantification using NMR. Analytica Chimica Acta. 2016 Aug;934:194–202.
14. 14. Monakhova YB, Kohl-Himmelseher M, Kuballa T, Lachenmeier DW. Determination of the purity of pharmaceutical reference materials by 1 H NMR using the standardless PULCON methodology. Journal of Pharmaceutical and Biomedical Analysis. 2014 Nov;100:381–6.
15. 15. Barantin L, Pape AL, Akoka S. A new method for absolute quantitation MRS metabolites. Magn Reson Med. 1997 Aug;38(2):179–82.
16. 16. Albers MJ, Butler TN, Rahwa I, Bao N, Keshari KR, Swanson MG, et al. Evaluation of the ERETIC method as an improved quantitative reference for 1 H HR-MAS spectroscopy of prostate tissue. Magn Reson Med. 2009 Mar;61(3):525–32.
17. 17. Akoka S, Barantin L, Trierweiler M. Concentration Measurement by Proton NMR Using the ERETIC Method. Anal Chem. 1999 Jul 1;71(13):2554–7.
18. 18. Anonymous. TopSpin ERETIC 2 [Internet]. Bruker, Corp.; 2016 [cited 2021 Dec 1].
19. 19. Farrant RD, Hollerton JC, Lynn SM, Provera S, Sidebottom PJ, Upton RJ. NMR quantification using an artificial signal. Magn Reson Chem. 2010 Oct;48(10):753–62.
20. 20. Watanabe R, Sugai C, Yamazaki T, Matsushima R, Uchida H, Matsumiya M, et al. Quantitative Nuclear Magnetic Resonance Spectroscopy Based on PULCON Methodology: Application to Quantification of Invaluable Marine Toxin, Okadaic Acid. Toxins. 2016 Oct 13;8(10):294.
21. 21. Bianchi L, Gagliardi A, Campanella G, Landi C, Capaldo A, Carleo A, et al. A methodological and functional proteomic approach of human follicular fluid en route for oocyte quality evaluation. Journal of Proteomics. 2013 Sep;90:61–76.
22. 22. O’Gorman A, Wallace M, Cottell E, Gibney MJ, McAuliffe FM, Wingfield M, et al. Metabolic profiling of human follicular fluid identifies potential biomarkers of oocyte developmental competence. Reproduction. 2013 Oct;146(4):389–95.
23. 23. Revelli A, Piane LD, Casano S, Molinari E, Massobrio M, Rinaudo P. Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics. Reprod Biol Endocrinol. 2009 Dec;7(1):40.
24. 24. Bouet P-E, Chao de la Barca JM, El Hachem H, Descamps P, Legendre G, Reynier P, et al. Metabolomics shows no impairment of the microenvironment of the cumulus–oocyte complex in women with isolated endometriosis. Reproductive BioMedicine Online. 2019 Dec;39(6):885–92.
25. 25. Wallace M, Cottell E, Gibney MJ, McAuliffe FM, Wingfield M, Brennan L. An investigation into the relationship between the metabolic profile of follicular fluid, oocyte developmental potential, and implantation outcome. Fertility and Sterility. 2012 May;97(5):1078-1084.e8.
26. 26. Castiglione Morelli MA, Iuliano A, Schettini SCA, Petruzzi D, Ferri A, Colucci P, et al. NMR metabolic profiling of follicular fluid for investigating the different causes of female infertility: a pilot study. Metabolomics. 2019 Feb;15(2):19.
27. 27. Karaer A, Tuncay G, Mumcu A, Dogan B. Metabolomics analysis of follicular fluid in women with ovarian endometriosis undergoing in vitro fertilization. Systems Biology in Reproductive Medicine. 2019 Jan 2;65(1):39–47.
28. 28. Piñero-Sagredo E, Nunes S, de los Santos MJ, Celda B, Esteve V. NMR metabolic profile of human follicular fluid. NMR Biomed. 2010 Mar 24;23(5):485–95.
29. 29. Fan TW-M. Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures. Progress in Nuclear Magnetic Resonance Spectroscopy. 1996 Jan;28(2):161–219.
30. 30. Kruk J, Doskocz M, Jodłowska E, Zacharzewska A, Łakomiec J, Czaja K, et al. NMR Techniques in Metabolomic Studies: A Quick Overview on Examples of Utilization. Appl Magn Reson. 2017 Jan;48(1):1–21.
31. 31. Mumcu A, Karaer A, Dogan B, Tuncay G. Metabolomics analysis of seminal plasma in patients with idiopathic Oligoasthenoteratozoospermia using high‐resolution NMR spectroscopy. Andrologia. 2020 Mar;8(2):450–6.
32. 32. Juchem C, de Graaf RA. B 0 magnetic field homogeneity and shimming for in vivo magnetic resonance spectroscopy. Analytical Biochemistry. 2017 Jul;529:17–29.
33. 33. Cohn BR, Joe BN, Zhao S, Kornak J, Zhang VY, Iman R, et al. Quantitative metabolic profiles of 2nd and 3rd trimester human amniotic fluid using 1H HR-MAS spectroscopy. Magn Reson Mater Phy. 2009 Dec;22(6):343–52.
34. 34. Ziarelli F, Peng L, Zhang C-C, Viel S. High resolution magic angle spinning NMR to investigate ligand–receptor binding events for mass-limited samples in liquids. Journal of Pharmaceutical and Biomedical Analysis. 2012 Feb;59:13–7.
35. 35. Frank O, Kreissl JK, Daschner A, Hofmann T. Accurate Determination of Reference Materials and Natural Isolates by Means of Quantitative 1 H NMR Spectroscopy. J Agric Food Chem. 2014 Mar 26;62(12):2506–15.
36. 36. Selegato DM, Freire RT, Pilon AC, Biasetto CR, de Oliveira HC, de Abreu LM, et al. Improvement of bioactive metabolite production in microbial cultures-A systems approach by OSMAC and deconvolution-based 1 HNMR quantification. Magn Reson Chem. 2019 Aug;57(8):458–71.
37. 37. Sitter B, Bathen TF, Singstad TE, Fjøsne HE, Lundgren S, Halgunset J, et al. Quantification of metabolites in breast cancer patients with different clinical prognosis using HR MAS MR spectroscopy: Eretic Quantification in Breast Cancer Tissue. NMR Biomed. 2010 May;23(4):424–31.
38. 38. Wong A, Lucas-Torres C. CHAPTER 5. High-resolution Magic-angle Spinning (HR-MAS) NMR Spectroscopy. In: Keun HC, editor. New Developments in NMR [Internet]. Cambridge: Royal Society of Chemistry; 2018 [cited 2021 Dec 1]. p. 133–50.
39. 39. Bertelli D, Brighenti V, Marchetti L, Reik A, Pellati F. Nuclear magnetic resonance and high-performance liquid chromatography techniques for the characterization of bioactive compounds from Humulus lupulus L. (hop). Anal Bioanal Chem. 2018 Jun;410(15):3521–31.
40. 40. Paniagua-Vega D, Cavazos-Rocha N, Huerta-Heredia AA, Parra-Naranjo A, Rivas-Galindo VM, Waksman N, et al. A validated NMR method for the quantitative determination of rebaudioside A in commercial sweeteners. Journal of Food Composition and Analysis. 2019 Jun;79:134–42.
41. 41. Marianna S, Alessia P, Susan C, Francesca C, Angela S, Francesca C, et al. Metabolomic profiling and biochemical evaluation of the follicular fluid of endometriosis patients. Mol BioSyst. 2017;13(6):1213–22.
42. 42. Leese HJ, Lenton EA. Glucose and lactate in human follicular fluid: concentrations and interrelationships. Human Reproduction. 1990 Nov;5(8):915–9.
43. 43. Gull I, Geva E, Lerner-Geva L, Lessing JB, Wolman I, Amit A. Anaerobic glycolysis. European Journal of Obstetrics & Gynecology and Reproductive Biology. 1999 Aug;85(2):225–8.
44. 44. Jozwik M, Jozwik M, Teng C, Battaglia FC. Concentrations of monosaccharides and their amino and alcohol derivatives in human preovulatory follicular fluid. Molecular Human Reproduction. 2007 Nov 1;13(11):791–6.
45. 45. Gosden RG, Sadler IH, Reed D, Hunter RHF. Characterization of ovarian follicular fluids of sheep, pigs and cows using proton nuclear magnetic resonance spectroscopy. Experientia. 1990 Oct;46(10):1012–5.
46. 46. Baskind NE, McRae C, Sharma V, Fisher J. Understanding subfertility at a molecular level in the female through the application of nuclear magnetic resonance (NMR) spectroscopy. Human Reproduction Update. 2011 Mar 1;17(2):228–41.