FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Glycoconj J / Systematic analyses of free ceramide species and ceramide sp...
Glycoconjugate journal2011; 28(2); 67-87; doi: 10.1007/s10719-011-9325-6

Systematic analyses of free ceramide species and ceramide species comprising neutral glycosphingolipids by MALDI-TOF MS with high-energy CID.

Abstract: Free ceramides and glycosphingolipids (GSLs) are important components of the membrane microdomain and play significant roles in cell survival. Recent studies have revealed that both fatty acids and long-chain bases (LCBs) are more diverse than expected, in terms of i) alkyl chain length, ii) hydroxylation and iii) the presence or absence of double bonds. Electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput, but reports to date have not fully characterized various types of ceramide species such as hydroxyl fatty acids and/or trihydroxy-LCBs of both free ceramides and the constituent ceramides in neutral GSLs. We performed a systematic analysis of both ceramide species, including LCBs with nona-octadeca lengths using MALDI-TOF MS with high-energy collision-induced dissociation (CID) at 20 keV. Using both protonated and sodiated ions, this technique enabled us to propose general rules to discriminate between isomeric and isobaric ceramide species, unrelated to the presence or absence of sugar chains. In addition, this high-energy CID generated (3,5)A ions, indicating Hex 1-4 Hex linkage in the sugar chains. Using this method, we demonstrated distinct differences among ceramide species, including free ceramides, sphingomyelins, and neutral GSLs of glucosylceramides, galactosylceramides, lactosylceramides, globotriaosylceramides and Forssman glycolipids in the equine kidneys.
Get Full Text
Publication Date: 2011-03-12 PubMed ID: 21400001DOI: 10.1007/s10719-011-9325-6Google 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

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.

The research article discusses a systematic study of free ceramides and glycosphingolipids by using advanced mass spectrometry techniques to characterize their different species. The results reveal distinct variations among these types of molecules, enhancing our understanding of their roles in cell survival.

Overview of the Research

  • The research focuses on free ceramides and glycosphingolipids, biological molecules that are integral to the workings of cell membranes and cell survival. These molecules are more diverse than previously thought, as they can vary in terms of their alkyl chain length, hydroxylation, and the presence or absence of double bonds.
  • The researchers used advanced techniques such as electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to characterize these molecules.

Methodology and Findings

  • The team conducted a comprehensive analysis of ceramide species using MALDI-TOF MS with high-energy collision-induced dissociation (CID) at 20 keV. This is a powerful methodology that can precisely break down molecules into their constituent parts, providing a detailed view of their composition.
  • With this method, they were able to propose general rules to distinguish between isomeric and isobaric ceramide species, regardless of the presence or absence of sugar chains. Isomers are molecules with the same chemical formula but different structural arrangements, while isobars are atoms with the same number of nucleons but different atomic numbers.
  • Moreover, high-energy CID produced specific ions, (3,5)A ions, demonstrating a particular type of linkage (Hex 1-4 Hex) in the sugar chains.
  • Using these methods, they found significant differences among ceramide species, including free ceramides, sphingomyelins, and neutral GSLs of various kinds in the equine kidneys.

Implication and Significance of the Research

  • This research has crucial implications for our understanding of cell biology. By providing a more detailed characterization of ceramides and glycosphingolipids, we gain insights into their various roles in cell survival and membrane dynamics.
  • The techniques and analytical rules proposed by the researchers can also be used for further studies, enhancing the capability of researchers to investigate these complex molecules and their functions.
  • Moreover, the specific variations that they found among different ceramide species could potentially shed light on disease mechanisms or contribute to the development of new therapeutic strategies. For instance, alterations in ceramide levels and composition have been implicated in various pathological conditions, including cancer, neurodegeneration, and inflammatory diseases.

Cite This Article

APA
Tanaka K, Yamada M, Tamiya-Koizumi K, Kannagi R, Aoyama T, Hara A, Kyogashima M. (2011). Systematic analyses of free ceramide species and ceramide species comprising neutral glycosphingolipids by MALDI-TOF MS with high-energy CID. Glycoconj J, 28(2), 67-87. https://doi.org/10.1007/s10719-011-9325-6

Publication

Glycoconjugate journal
ISSN: 1573-4986
NlmUniqueID: 8603310
Country: United States
Language: English
Volume: 28
Issue: 2
Pages: 67-87

Researcher Affiliations

Tanaka, Kouji
  • Division of Molecular Pathology, Aichi Cancer Center Research Institute, Nagoya, Aichi 464-8681, Japan.
Yamada, Masaki
    Tamiya-Koizumi, Keiko
      Kannagi, Reiji
        Aoyama, Toshifumi
          Hara, Atsushi
            Kyogashima, Mamoru

              MeSH Terms

              • Animals
              • Ceramides / chemistry
              • Galactosylceramides / chemistry
              • Glucosylceramides / chemistry
              • Horses
              • Kidney / metabolism
              • Neutral Glycosphingolipids / chemistry
              • Spectrometry, Mass, Electrospray Ionization / methods
              • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

              References

              This article includes 46 references
              1. Nakamura K, Suzuki Y, Goto-Inoue N, Yoshida-Noro C, Suzuki A. Structural characterization of neutral glycosphingolipids by thin-layer chromatography coupled to matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight MS/MS.. Anal Chem 2006 Aug 15;78(16):5736-43.
                pubmed: 16906718doi: 10.1021/ac0605501google scholar: lookup
              2. Kurogochi M, Nishimura S. Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry.. Anal Chem 2004 Oct 15;76(20):6097-101.
                pubmed: 15481958doi: 10.1021/ac049294ngoogle scholar: lookup
              3. Signorelli P, Munoz-Olaya JM, Gagliostro V, Casas J, Ghidoni R, Fabriàs G. Dihydroceramide intracellular increase in response to resveratrol treatment mediates autophagy in gastric cancer cells.. Cancer Lett 2009 Sep 18;282(2):238-43.
                pubmed: 19394759doi: 10.1016/j.canlet.2009.03.020google scholar: lookup
              4. Ohashi Y, Iwamori M, Ogawa T, Nagai Y. Analysis of long-chain bases in sphingolipids by positive ion fast atom bombardment or matrix-assisted secondary ion mass spectrometry.. Biochemistry 1987 Jun 30;26(13):3990-5.
                pubmed: 3651429doi: 10.1021/bi00387a037google scholar: lookup
              5. Kyogashima M, Tadano-Aritomi K, Aoyama T, Yusa A, Goto Y, Tamiya-Koizumi K, Ito H, Murate T, Kannagi R, Hara A. Chemical and apoptotic properties of hydroxy-ceramides containing long-chain bases with unusual alkyl chain lengths.. J Biochem 2008 Jul;144(1):95-106.
                pubmed: 18420598doi: 10.1093/jb/mvn050google scholar: lookup
              6. Chatterjee S. Assay of lactosylceramide synthase and comments on its potential role in signal transduction.. Methods Enzymol 2000;311:73-81.
                pubmed: 10563312doi: 10.1016/s0076-6879(00)11068-7google scholar: lookup
              7. Karlsson KA, Steen GO. Studies on sphingosines. 13. The existence of phytosphingosine in bovine kidney sphingomyelins.. Biochim Biophys Acta 1968 Jul 1;152(4):798-800.
                pubmed: 5660095doi: 10.1016/0005-2760(68)90130-6google scholar: lookup
              8. Hara A, Taketomi T. Long chain base and fatty acid compositions of equine kidney sphingolipids.. J Biochem 1975 Sep;78(3):527-36.
                pubmed: 818074doi: 10.1093/oxfordjournals.jbchem.a130937google scholar: lookup
              9. Regina Todeschini A, Hakomori SI. Functional role of glycosphingolipids and gangliosides in control of cell adhesion, motility, and growth, through glycosynaptic microdomains.. Biochim Biophys Acta 2008 Mar;1780(3):421-33.
                pubmed: 17991443doi: 10.1016/j.bbagen.2007.10.008google scholar: lookup
              10. Haynes CA, Allegood JC, Park H, Sullards MC. Sphingolipidomics: methods for the comprehensive analysis of sphingolipids.. J Chromatogr B Analyt Technol Biomed Life Sci 2009 Sep 15;877(26):2696-708.
                pubmed: 19147416doi: 10.1016/j.jchromb.2008.12.057google scholar: lookup
              11. Kitano Y, Iwamori Y, Kiguchi K, DiGiovanni J, Takahashi T, Kasama K, Niwa T, Harii K, Iwamori M. Selective reduction in alpha-hydroxypalmitic acid-containing sphingomyelin and concurrent increase in hydroxylated ceramides in murine skin tumors induced by an initiation-promotion regimen.. Jpn J Cancer Res 1996 May;87(5):437-41.
                pubmed: 8641979doi: 10.1111/j.1349-7006.1996.tb00243.xgoogle scholar: lookup
              12. Robinson BS, Johnson DW, Poulos A. Novel molecular species of sphingomyelin containing 2-hydroxylated polyenoic very-long-chain fatty acids in mammalian testes and spermatozoa.. J Biol Chem 1992 Jan 25;267(3):1746-51.
                pubmed: 1730716
              13. Panasiewicz M, Domek H, Hoser G, Fedoryszak N, Kawalec M, Pacuszka T. The ceramide structure of GM1 ganglioside differently affects its recovery in low-density membrane fractions prepared from HL-60 cells with or without triton-X100.. Cell Mol Biol Lett 2009;14(2):175-89.
                pubmed: 18979068doi: 10.2478/s11658-008-0043-4google scholar: lookup
              14. Harvey DJ. A new charge-associated mechanism to account for the production of fragment ions in the high-energy CID spectra of fatty acids.. J Am Soc Mass Spectrom 2005 Feb;16(2):280-90.
                pubmed: 15694778doi: 10.1016/j.jasms.2004.11.008google scholar: lookup
              15. Sugiura Y, Shimma S, Konishi Y, Yamada MK, Setou M. Imaging mass spectrometry technology and application on ganglioside study; visualization of age-dependent accumulation of C20-ganglioside molecular species in the mouse hippocampus.. PLoS One 2008 Sep 18;3(9):e3232.
                pubmed: 18800170doi: 10.1371/journal.pone.0003232google scholar: lookup
              16. Rumsby MG. A modified column chromatographic method for the recovery of the glycerogalactolipid fraction of nerve tissue. Some observations on the fractionation of nerve tissue glycolipids on silicic acid with chloroform and acetone mixtures.. J Chromatogr 1969 Jun 17;42(2):237-47.
                pubmed: 5785345doi: 10.1016/s0021-9673(01)80620-8google scholar: lookup
              17. Saito T, Hakomori SI. Quantitative isolation of total glycosphingolipids from animal cells.. J Lipid Res 1971 Mar;12(2):257-9.
                pubmed: 4324310
              18. Ikeda K, Shimizu T, Taguchi R. Targeted analysis of ganglioside and sulfatide molecular species by LC/ESI-MS/MS with theoretically expanded multiple reaction monitoring.. J Lipid Res 2008 Dec;49(12):2678-89.
                pubmed: 18703820doi: 10.1194/jlr.D800038-JLR200google scholar: lookup
              19. Mahfoud R, Manis A, Lingwood CA. Fatty acid-dependent globotriaosyl ceramide receptor function in detergent resistant model membranes.. J Lipid Res 2009 Sep;50(9):1744-55.
                pubmed: 18716315doi: 10.1194/jlr.M800385-JLR200google scholar: lookup
              20. Masukawa Y, Narita H, Shimizu E, Kondo N, Sugai Y, Oba T, Homma R, Ishikawa J, Takagi Y, Kitahara T, Takema Y, Kita K. Characterization of overall ceramide species in human stratum corneum.. J Lipid Res 2008 Jul;49(7):1466-76.
                pubmed: 18359959doi: 10.1194/jlr.M800014-JLR200google scholar: lookup
              21. Schaeren-Wiemers N, van der Bijl P, Schwab ME. The UDP-galactose:ceramide galactosyltransferase: expression pattern in oligodendrocytes and Schwann cells during myelination and substrate preference for hydroxyceramide.. J Neurochem 1995 Nov;65(5):2267-78.
                pubmed: 7595516doi: 10.1046/j.1471-4159.1995.65052267.xgoogle scholar: lookup
              22. Parry S, Ledger V, Tissot B, Haslam SM, Scott J, Morris HR, Dell A. Integrated mass spectrometric strategy for characterizing the glycans from glycosphingolipids and glycoproteins: direct identification of sialyl Le(x) in mice.. Glycobiology 2007 Jun;17(6):646-54.
                pubmed: 17341505doi: 10.1093/glycob/cwm024google scholar: lookup
              23. Morell P, Radin NS. Synthesis of cerebroside by brain from uridine diphosphate galactose and ceramide containing hydroxy fatty acid.. Biochemistry 1969 Feb;8(2):506-12.
                pubmed: 5793706doi: 10.1021/bi00830a008google scholar: lookup
              24. Ohta M, Matsuura F, Henderson G, Laine RA. Novel free ceramides as components of the soldier defense gland of the Formosan subterranean termite (Coptotermes formosanus).. J Lipid Res 2007 Mar;48(3):656-64.
                pubmed: 17164223doi: 10.1194/jlr.M600387-JLR200google scholar: lookup
              25. Costello CE, Vath JE. Tandem mass spectrometry of glycolipids.. Methods Enzymol 1990;193:738-68.
                pubmed: 2074845doi: 10.1016/0076-6879(90)93448-tgoogle scholar: lookup
              26. Panasiewicz M, Domek H, Hoser G, Kawalec M, Pacuszka T. Structure of the ceramide moiety of GM1 ganglioside determines its occurrence in different detergent-resistant membrane domains in HL-60 cells.. Biochemistry 2003 Jun 3;42(21):6608-19.
                pubmed: 12767245doi: 10.1021/bi0206309google scholar: lookup
              27. Hannun YA, Obeid LM. Principles of bioactive lipid signalling: lessons from sphingolipids.. Nat Rev Mol Cell Biol 2008 Feb;9(2):139-50.
                pubmed: 18216770doi: 10.1038/nrm2329google scholar: lookup
              28. Buschard K, Blomqvist M, Månsson JE, Fredman P, Juhl K, Gromada J. C16:0 sulfatide inhibits insulin secretion in rat beta-cells by reducing the sensitivity of KATP channels to ATP inhibition.. Diabetes 2006 Oct;55(10):2826-34.
                pubmed: 17003349doi: 10.2337/db05-1355google scholar: lookup
              29. Hara A, Kitazawa N, Taketomi T. Abnormalities of glycosphingolipids in mucopolysaccharidosis type III B.. J Lipid Res 1984 Feb;25(2):175-84.
                pubmed: 6423755
              30. Levery SB. Glycosphingolipid structural analysis and glycosphingolipidomics.. Methods Enzymol 2005;405:300-69.
                pubmed: 16413319doi: 10.1016/S0076-6879(05)05012-3google scholar: lookup
              31. Tomita M, Taguchi R, Ikezawa H. Molecular properties and kinetic studies on sphingomyelinase of Bacillus cereus.. Biochim Biophys Acta 1982 May 21;704(1):90-9.
                pubmed: 6284239doi: 10.1016/0167-4838(82)90135-2google scholar: lookup
              32. Matsuda J, Kido M, Tadano-Aritomi K, Ishizuka I, Tominaga K, Toida K, Takeda E, Suzuki K, Kuroda Y. Mutation in saposin D domain of sphingolipid activator protein gene causes urinary system defects and cerebellar Purkinje cell degeneration with accumulation of hydroxy fatty acid-containing ceramide in mouse.. Hum Mol Genet 2004 Nov 1;13(21):2709-23.
                pubmed: 15345707doi: 10.1093/hmg/ddh281google scholar: lookup
              33. Breimer ME, Karlsson KA, Samuelsson BE. Presence of phytosphingosine combined with 2-hydroxy fatty acids in sphingomyelins of bovine kidney and intestinal mucosa.. Lipids 1975 Jan;10(1):17-9.
                pubmed: 1123973doi: 10.1007/BF02532188google scholar: lookup
              34. Gu M, Kerwin JL, Watts JD, Aebersold R. Ceramide profiling of complex lipid mixtures by electrospray ionization mass spectrometry.. Anal Biochem 1997 Jan 15;244(2):347-56.
                pubmed: 9025952doi: 10.1006/abio.1996.9915google scholar: lookup
              35. Karlsson K, Nilsson K, Samuelsson BE, Steen GO. The presence of hydroxy fatty acids in sphingomyelins of bovine rennet stomach.. Biochim Biophys Acta 1969 Apr 29;176(3):660-3.
                pubmed: 5800059doi: 10.1016/0005-2760(69)90239-2google scholar: lookup
              36. Scherer M, Leuthäuser-Jaschinski K, Ecker J, Schmitz G, Liebisch G. A rapid and quantitative LC-MS/MS method to profile sphingolipids.. J Lipid Res 2010 Jul;51(7):2001-11.
                pubmed: 20228220doi: 10.1194/jlr.D005322google scholar: lookup
              37. Bielawski J, Pierce JS, Snider J, Rembiesa B, Szulc ZM, Bielawska A. Comprehensive quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry.. Methods Mol Biol 2009;579:443-67.
                pubmed: 19763489doi: 10.1007/978-1-60761-322-0_22google scholar: lookup
              38. Iwabuchi K, Prinetti A, Sonnino S, Mauri L, Kobayashi T, Ishii K, Kaga N, Murayama K, Kurihara H, Nakayama H, Yoshizaki F, Takamori K, Ogawa H, Nagaoka I. Involvement of very long fatty acid-containing lactosylceramide in lactosylceramide-mediated superoxide generation and migration in neutrophils.. Glycoconj J 2008 May;25(4):357-74.
                pubmed: 18041581doi: 10.1007/s10719-007-9084-6google scholar: lookup
              39. Hsu FF, Turk J. Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization.. J Am Soc Mass Spectrom 2002 May;13(5):558-70.
                pubmed: 12019979doi: 10.1016/S1044-0305(02)00358-6google scholar: lookup
              40. Halter D, Neumann S, van Dijk SM, Wolthoorn J, de Mazière AM, Vieira OV, Mattjus P, Klumperman J, van Meer G, Sprong H. Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis.. J Cell Biol 2007 Oct 8;179(1):101-15.
                pubmed: 17923531doi: 10.1083/jcb.200704091google scholar: lookup
              41. Li SC, Li YT. Studies on the glycosidases of jack bean meal. 3. Crystallization and properties of beta-N-acetylhexosaminidase.. J Biol Chem 1970 Oct 10;245(19):5153-60.
                pubmed: 5506280
              42. Nagahori N, Abe M, Nishimura S. Structural and functional glycosphingolipidomics by glycoblotting with an aminooxy-functionalized gold nanoparticle.. Biochemistry 2009 Jan 27;48(3):583-94.
                pubmed: 19117481doi: 10.1021/bi801640ngoogle scholar: lookup
              43. Kyogashima M, Tamiya-Koizumi K, Ehara T, Li G, Hu R, Hara A, Aoyama T, Kannagi R. Rapid demonstration of diversity of sulfatide molecular species from biological materials by MALDI-TOF MS.. Glycobiology 2006 Aug;16(8):719-28.
                pubmed: 16670104doi: 10.1093/glycob/cwj122google scholar: lookup
              44. Pouria S, Corran PH, Smith AC, Smith HW, Hendry BM, Challacombe SJ, Tarelli E. Glycoform composition profiling of O-glycopeptides derived from human serum IgA1 by matrix-assisted laser desorption ionization-time of flight-mass spectrometry.. Anal Biochem 2004 Jul 15;330(2):257-63.
                pubmed: 15203331doi: 10.1016/j.ab.2004.03.053google scholar: lookup
              45. Yu RK, Yanagisawa M. Glycosignaling in neural stem cells: involvement of glycoconjugates in signal transduction modulating the neural stem cell fate.. J Neurochem 2007 Nov;103 Suppl 1:39-46.
                pubmed: 17986138doi: 10.1111/j.1471-4159.2007.04710.xgoogle scholar: lookup
              46. D'Angelo G, Polishchuk E, Di Tullio G, Santoro M, Di Campli A, Godi A, West G, Bielawski J, Chuang CC, van der Spoel AC, Platt FM, Hannun YA, Polishchuk R, Mattjus P, De Matteis MA. Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide.. Nature 2007 Sep 6;449(7158):62-7.
                pubmed: 17687330doi: 10.1038/nature06097google scholar: lookup

              Citations

              This article has been cited 9 times.
              1. Barrientos RC, Zhang Q. Fragmentation Behavior and Gas-Phase Structures of Cationized Glycosphingolipids in Ozone-Induced Dissociation Mass Spectrometry.. J Am Soc Mass Spectrom 2019 Sep;30(9):1609-1620.
                doi: 10.1007/s13361-019-02267-7pubmed: 31286447google scholar: lookup
              2. Noda A, Kato M, Miyazaki S, Kyogashima M. Separation of glycosphingolipids with titanium dioxide.. Glycoconj J 2018 Dec;35(6):493-498.
                doi: 10.1007/s10719-018-9844-5pubmed: 30284662google scholar: lookup
              3. Pescio LG, Santacreu BJ, Lopez VG, Paván CH, Romero DJ, Favale NO, Sterin-Speziale NB. Changes in ceramide metabolism are essential in Madin-Darby canine kidney cell differentiation.. J Lipid Res 2017 Jul;58(7):1428-1438.
                doi: 10.1194/jlr.M076349pubmed: 28515139google scholar: lookup
              4. Tanaka K, Tamiya-Koizumi K, Yamada M, Murate T, Kannagi R, Kyogashima M. Hypoxia remodels the composition of the constituent ceramide species of HexCer and Hex2Cer with phytosphingosine and hydroxy fatty acids in human colon cancer LS174T cells.. Glycoconj J 2015 Nov;32(8):615-23.
                doi: 10.1007/s10719-015-9607-5pubmed: 26194060google scholar: lookup
              5. Jia Z, Cong P, Zhang H, Song Y, Li Z, Xu J, Xue C. Reversed-Phase Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry for High-Throughput Molecular Profiling of Sea Cucumber Cerebrosides.. Lipids 2015 Jul;50(7):667-79.
                doi: 10.1007/s11745-015-4039-3pubmed: 26037520google scholar: lookup
              6. Farwanah H, Kolter T. Lipidomics of glycosphingolipids.. Metabolites 2012 Feb 2;2(1):134-64.
                doi: 10.3390/metabo2010134pubmed: 24957371google scholar: lookup
              7. Tanaka K, Tamiya-Koizumi K, Yamada M, Murate T, Kannagi R, Kyogashima M. Individual profiles of free ceramide species and the constituent ceramide species of sphingomyelin and neutral glycosphingolipid and their alteration according to the sequential changes of environmental oxygen content in human colorectal cancer Caco-2 cells.. Glycoconj J 2014 Apr;31(3):209-19.
                doi: 10.1007/s10719-013-9511-9pubmed: 24310545google scholar: lookup
              8. Aljohani AJ, Munguba GC, Guerra Y, Lee RK, Bhattacharya SK. Sphingolipids and ceramides in human aqueous humor.. Mol Vis 2013;19:1966-84.
                pubmed: 24068864
              9. Merrill AH Jr. Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics.. Chem Rev 2011 Oct 12;111(10):6387-422.
                doi: 10.1021/cr2002917pubmed: 21942574google scholar: lookup
              Subscribe to our newsletter
              Join 99,357 horse owners like you who receive our email updates on horse health and nutrition.