FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Drug testing and analysis2021; 13(6); 1158-1168; doi: 10.1002/dta.3006

Determination of vitamin E and its metabolites in equine urine using liquid chromatography-mass spectrometry.

Abstract: Equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM) is a hereditary, deteriorating central nervous disease in horses. Currently, the only way to confirm eNAD/EDM is through a postmortem histological evaluation of the central nervous system. Vitamin E, specifically the isoform alpha-tocopherol (α-TP), is known to protect eNAD/EDM susceptible horses from developing the clinical phenotype. While vitamin E is an essential nutrient in the diet of horses, there are no diagnostic tests able to quantitate vitamin E and its metabolites in urine. An ultra-performance liquid chromatography-atmospheric-pressure chemical ionization mass spectrometry (UPLC-APCI-MS/MS) method was developed and validated following acidic hydrolysis and solid phase extraction to quantitate vitamin E and its metabolites in equine urine. A blank control horse urine matrix was used and spiked with different concentrations of analytes to form a standard curve using either alpha-tocopherol-d6 or chlorpropamide as the internal standard. Inter-day and intra-day statistics were performed to evaluate the method for accuracy (90% to 116%) and precision (0.75% to 14%). Matrix effects, percent recovery, and stability were also assessed. The method successfully analyzed alpha-carboxyethyl hydroxychroman (α-CEHC), alpha-carboxymethylbutyl hydroxychromans (α-CMBHC), gamma-carboxyethyl hydroxychroman γ-CEHC, and α-TP concentrations in urine to determine a baseline levels of analytes in healthy horses, and can be used to determine concentrations of vitamin E metabolites in equine urine allowing for its evaluation as a diagnostic approach in the treatment of eNAD/EDM.
© 2021 John Wiley & Sons, Ltd.
Get Full Text
Publication Date: 2021-02-09 PubMed ID: 33527764DOI: 10.1002/dta.3006Google 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.
LinkedInCopy
  • Journal Article
  • Validation Study

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.

This research article discusses the development and validation of a method to measure the levels of Vitamin E and its metabolites in horse urine, aiming to use it as a diagnostic approach for a hereditary neurological disease among horses known as Equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM).

Objective and Background

  • This research was aimed at addressing the lack of a diagnostic test that can quantitatively determine the levels of Vitamin E and its metabolites in horse urine. The ultimate goal was to explore its potential use as a diagnostic means for the detection and treatment of a hereditary central nervous disease in horses, the equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM).
  • Historically, eNAD/EDM diagnosis relied on postmortem histological evaluations of the central nervous system. The disease leads to progressive neurodegeneration in horses.
  • Previous studies have shown that a form of Vitamin E, alpha-tocopherol (α-TP), can protect susceptible horses from developing the clinical features of eNAD/EDM. However, the quantification methods for Vitamin E metabolites were absent in the field.

Method Development and Validation

  • An ultra-performance liquid chromatography-atmospheric-pressure chemical ionization mass spectrometry (UPLC-APCI-MS/MS) method was developed and validated in this research to quantify Vitamin E and its metabolites in equine urine.
  • Necessary procedures such as acidic hydrolysis and solid phase extraction were undertaken before the quantification process.
  • A standard curve was formed by spiking a blank control horse urine matrix with different concentrations of analytes. These were alpha-tocopherol-d6 or chlorpropamide, which were used as internal standards.
  • The method was thoroughly evaluated for accuracy and precision using inter-day and intra-day statistics. Performance indicators ranged between acceptable limits.

Results and Conclusion

  • The study successfully quantified alpha-Carboxyethyl Hydroxychroman (α-CEHC), alpha-Carboxymethylbutyl Hydroxychromans (α-CMBHC), gamma-Carboxyethyl Hydroxychroman (γ-CEHC), and α-TP concentrations in urine. These can be used to determine baseline levels of analytes in healthy horses.
  • Matrix effects, percent recovery, and stability were also assessed during the process.
  • The method developed has potential application for determining concentrations of vitamin E metabolites in equine urine, thus paving a path for its evaluation as a diagnostic approach in the treatment of eNAD/EDM.

Cite This Article

APA
Favro G, Habib H, Gennity I, Puschner B, Hales EN, Finno CJ, Moeller BC. (2021). Determination of vitamin E and its metabolites in equine urine using liquid chromatography-mass spectrometry. Drug Test Anal, 13(6), 1158-1168. https://doi.org/10.1002/dta.3006

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 13
Issue: 6
Pages: 1158-1168

Researcher Affiliations

Favro, Gianna
  • Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California, USA.
Habib, Hadi
  • Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California, USA.
Gennity, Ingrid
  • Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California, USA.
Puschner, Birgit
  • Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California, USA.
Hales, Erin N
  • Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, California, USA.
Finno, Carrie J
  • Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, California, USA.
Moeller, Benjamin C
  • K. L. Maddy Equine Analytical Chemistry Laboratory, California Animal Health and Food Safety Laboratory, School of Veterinary Medicine, University of California, Davis, California, USA.

MeSH Terms

  • Animals
  • Chromatography, High Pressure Liquid / methods
  • Horse Diseases / diagnosis
  • Horse Diseases / drug therapy
  • Horses
  • Neuroaxonal Dystrophies / diagnosis
  • Neuroaxonal Dystrophies / drug therapy
  • Neuroaxonal Dystrophies / veterinary
  • Solid Phase Extraction
  • Tandem Mass Spectrometry / methods
  • Vitamin E / analysis
  • Vitamin E / metabolism
  • Vitamin E / urine

Grant Funding

  • K01OD015134 / NIH HHS
  • L40 TR001136 / NIH HHS
  • K01OD015134 / NIH HHS
  • L40 TR001136 / NIH HHS

References

This article includes 38 references
  1. Uttara B, Singh AV, Zamboni P, Mahajan RT. Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options.. Curr Neuropharmacol 2009;7(1):65-74.
    doi: 10.2174/157015909787602823google scholar: lookup
  2. Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: impact on human health.. Pharmacognosy Reviews 2010;4(8):118-126.
    doi: 10.4103/0973-7847.70902google scholar: lookup
  3. Li B, Harjani JR, Cormier NS. Besting vitamin E: sidechain substitution is key to the reactivity of naphthyridinol antioxidants in lipid bilayers.. J am Chem Soc 2013;135(4):1394-1405.
    doi: 10.1021/ja309153xgoogle scholar: lookup
  4. Finno CJ, Valberg SJ. A comparative review of vitamin E and associated equine disorders.. J Vet Intern Med 2012;26(6):1251-1266.
    doi: 10.1111/j.1939-1676.2012.00994.xgoogle scholar: lookup
  5. Zhao Y, Lee M, Cheung C. Analysis of multiple metabolites of Tocopherols and Tocotrienols in mice and humans.. J Agric Food Chem 2010;58(8):4844-4852.
    doi: 10.1021/jf904464ugoogle scholar: lookup
  6. Lodge JK, Traber MG, Elsner A, Brigelius-Flohé R. A rapid method for the extraction and determination of vitamin E metabolites in human urine.. J Lipid Res 2000;41(1):148-154.
  7. Swanson JE, Ben RN, Burton GW, Parker RS. Urinary excretion of 2,7, 8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman is a major route of elimination of gamma-tocopherol in humans.. J Lipid Res 1999;40(4):665-671.
  8. Schultz M, Leist M, Elsner A, Brigelius-Flohé R. [26] α-carboxyethyl-6-hydroxychroman as urinary metabolite of vitamin E.. Methods in Enzymology Vol.282 Academic Press; 1997:297-310.
  9. Schuelke M, Elsner A, Finckh B, Kohlschütter A, Hübner C, Brigelius-Flohé R. Urinary alpha-tocopherol metabolites in alpha-tocopherol transfer protein-deficient patients.. J Lipid Res 2000;41(10):1543-1551.
  10. Freiser H, Jiang Q. Optimization of the enzymatic hydrolysis and analysis of plasma conjugated γ-CEHC and sulfated long-chain carboxychromanols, metabolites of vitamin E.. Anal Biochem 2009;388(2):260-265.
  11. Petra Jančová MŠ. Phase II Drug Metabolism.. Topics on Drug Metabolism Czech Republic: InTech; 2012:35-60.
  12. Finno CJ, Estell KE, Katzman S, Winfield L, Rendahl A, Textor J, Bannasch DL, Puschner B. Blood and Cerebrospinal Fluid α-Tocopherol and Selenium Concentrations in Neonatal Foals with Neuroaxonal Dystrophy.. 2015, 29, 6, 1667, 1675.
  13. Finno CJ. Equine neuroaxonal dystrophy.. Robinson's Current Therapy in Equine Medicine Seventh ed. Elsevier Inc.; 2014:384-386.
  14. Finno CJ, Famula T, Aleman M, Higgins RJ, Madigan JE, Bannasch DL. Pedigree analysis and exclusion of alpha-tocopherol transfer protein (TTPA) as a candidate gene for neuroaxonal dystrophy in the American quarter horse.. J Vet Intern Med 2013;27(1):177-185.
    doi: 10.1111/jvim.12015google scholar: lookup
  15. Aleman M, Finno CJ, Higgins R. Evaluation of epidemiological, clinical, and pathological features of neuroaxonal dystrophy in quarter horses.. Javma-Journal of the American Veterinary Medical Association 2011;239(6):823-833.
    doi: 10.2460/javma.239.6.823google scholar: lookup
  16. Mayhew IG, Brown CM, Stowe HD, Trapp AL, Derksen FJ, Clement SF. Equine degenerative myeloencephalopathy: a vitamin E deficiency that may be familial.. J Vet Intern Med 1987;1(1):45-50.
  17. Swanson J, Ben R, Burton G, Parker R. Urinary excretion of 2, 7, 8-trimethyl-2-(β-carboxyethyl)-6-hydroxychroman is a major route of elimination of γ-tocopherol in humans.. J Lipid Res 1999;40(4):665-671.
  18. Jiang Q, Xu T, Huang J, Jannasch A, Cooper B, Yang C. Analysis of vitamin E metabolites including carboxychromanols and sulfated derivatives using LC/MS/MS.. J Lipid Res 2015;56(11):2217-2225.
    doi: 10.1194/jlr.d061663google scholar: lookup
  19. Ghosh C. Relative matrix effects: a step forward using standard line slopes and ANOVA analysis.. Arabian J Chem 2019;12(7):1378-1386.
  20. Díaz S, Kienast ME, Villegas-Castagnasso EE. Substitution of human for horse urine disproves an accusation of doping*.. J Forensic Sci 2008;53(5):1145-1148.
    doi: 10.1111/j.1556-4029.2008.00797.xgoogle scholar: lookup
  21. Thevis M, Opfermann G, Schänzer W. Liquid chromatography/electrospray ionization tandem mass spectrometric screening and confirmation methods for β2-agonists in human or equine urine.. J Mass Spectrom 2003;38(11):1197-1206.
    doi: 10.1002/jms.542google scholar: lookup
  22. Torquato P, Giusepponi D, Galarini R, Bartolini D, Piroddi M, Galli F. CHAPTER 15 Analysis of vitamin E metabolites.. Vitamin E: Chemistry and Nutritional Benefits The Royal Society of Chemistry; 2019:208-227.
  23. Pope SA, Clayton PT, Muller DP. A new method for the analysis of urinary vitamin E metabolites and the tentative identification of a novel group of compounds.. Arch Biochem Biophys 2000;381(1):8-15.
    doi: 10.1006/abbi.2000.1950google scholar: lookup
  24. Schultz M, Leist M, Petrzika M, Gassmann B, Brigelius-Flohe R. Novel urinary metabolite of alpha-tocopherol, 2,5,7,8-tetramethyl-2(2′-carboxyethyl)-6-hydroxychroman, as an indicator of an adequate vitamin E supply?. Am J Clin Nutr 1995;62(6 Suppl):1527s-1534s.
    doi: 10.1093/ajcn/62.6.1527sgoogle scholar: lookup
  25. Uchida T, Nomura S, Ichikawa T, Abe C, Ikeda S. Tissue distribution of vitamin E metabolites in rats after oral administration of tocopherol or tocotrienol.. J Nutr Sci Vitaminol 2011;57(5):326-332.
    doi: 10.3177/jnsv.57.326google scholar: lookup
  26. Johnson C, Slanar O, Krausz K. Novel metabolites and roles for alpha-tocopherol in humans and mice discovered by mass spectrometry-based metabolomics.. Amer J Clin Nutr 2012;96(4):818-830.
    doi: 10.3945/ajcn.112.042929google scholar: lookup
  27. Schmölz L, Birringer M, Lorkowski S, Wallert M. Complexity of vitamin E metabolism.. World J Biol Chem 2016;7(1):14-43.
    doi: 10.4331/wjbc.v7.i1.14google scholar: lookup
  28. Pope SAS, Burtin GE, Clayton PT, Madge DJ, Muller DPR. Synthesis and analysis of conjugates of the major vitamin E metabolite, α-CEHC.. Free Radical Biol Med 2002;33(6):807-817.
    doi: 10.1016/s0891-5849(02)00974-7google scholar: lookup
  29. Dörr M. Acid hydrolysis.. Encyclopedia of Astrobiology Berlin, Heidelberg: Springer Berlin Heidelberg; 2011:10-10.
  30. Lu D, Feng C, Wang D. Analysis of twenty phenolic compounds in human urine: hydrochloric acid hydrolysis, solid-phase extraction based on K2CO3-treated silica, and gas chromatography tandem mass spectrometry.. Anal Bioanal Chem 2015;407(14):4131-4141.
    doi: 10.1007/s00216-015-8598-1google scholar: lookup
  31. Li Y-J, Luo S-C, Lee Y-J. Isolation and identification of α-CEHC sulfate in rat urine and an improved method for the determination of conjugated α-CEHC.. J Agric Food Chem 2008;56(23):11105-11113.
    doi: 10.1021/jf802459dgoogle scholar: lookup
  32. Lodge JK, Ridlington J, Leonard S, Vaule H, Traber MG. α- and γ-tocotrienols are metabolized to carboxyethyl-hydroxychroman derivatives and excreted in human urine.. Lipids 2001;36(1):43-48.
    doi: 10.1007/s11745-001-0666-zgoogle scholar: lookup
  33. Morinobu T, Yoshikawa S, Hamamura K, Tamai H. Measurement of vitamin E metabolites by high-performance liquid chromatography during high-dose administration of α-tocopherol.. Eur J Clin Nutr 2003;57(3):410-414.
    doi: 10.1038/sj.ejcn.1601570google scholar: lookup
  34. Juhascik MP, Jenkins AJ. Comparison of liquid/liquid and solid-phase extraction for alkaline drugs.. J Chromatogr Sci 2009;47(7):553-557.
    doi: 10.1093/chromsci/47.7.553google scholar: lookup
  35. Albahrani AA, Rotarou V, Roche PJ, Greaves RF. Comparison of three commercial calibrators for alpha-tocopherol using liquid chromatography-tandem mass spectrometry.. Clin Biochem 2013;46(18):1884-1888.
  36. Matuszewski BK. Standard line slopes as a measure of a relative matrix effect in quantitative HPLC-MS bioanalysis.. J Chromatogr B 2006;830(2):293-300.
  37. Souverain S, Rudaz S, Veuthey J-L. Matrix effect in LC-ESI-MS and LC-APCI-MS with off-line and on-line extraction procedures.. J Chromatogr a 2004;1058(1-2):61-66.
    doi: 10.1016/j.chroma.2004.08.118google scholar: lookup
  38. Traber MG, Mah E, Leonard SW, Bobe G, Bruno RS. Metabolic syndrome increases dietary alpha-tocopherol requirements as assessed using urinary and plasma vitamin E catabolites: a double-blind, crossover clinical trial.. Am J Clin Nutr 2017;105(3):571-579.
    doi: 10.3945/ajcn.116.138495google scholar: lookup

Citations

This article has been cited 1 times.
  1. Hales EN, Habib H, Favro G, Katzman S, Sakai RR, Marquardt S, Bordbari MH, Ming-Whitfield B, Peterson J, Dahlgren AR, Rivas V, Ramirez CA, Peng S, Donnelly CG, Dizmang BS, Kallenberg A, Grahn R, Miller AD, Woolard K, Moeller B, Puschner B, Finno CJ. Increased α-tocopherol metabolism in horses with equine neuroaxonal dystrophy. J Vet Intern Med 2021 Sep;35(5):2473-2485.
    doi: 10.1111/jvim.16233pubmed: 34331715google scholar: lookup
Subscribe to our newsletter
Join 99,780 horse owners like you who receive our email updates on horse health and nutrition.