Rapid Comparison of Metabolites in Humans and Rats of Different Sexes Using Untargeted Ultraperformance Liquid Chromatography Coupled to Time-of-Flight Mass Spectrometry and an in-house Software Platform
Restricted accessResearch articleFirst published online December, 2015
Rapid Comparison of Metabolites in Humans and Rats of Different Sexes Using Untargeted Ultraperformance Liquid Chromatography Coupled to Time-of-Flight Mass Spectrometry and an in-house Software Platform
Metabolite differences between sexes have rarely been observed in a global manner, but it has recently been made possible by the advancement in metabolomics techniques. In this study, untargeted ultraperformance liquid chromatography coupled to time-of-flight mass spectrometry and an in-house software platform were used for a rapid comparison of sex differences in urinary metabolites in humans and in urinary and serum metabolites in Sprague Dawley (SD) rats. In addition, the species differences of urinary metabolites between humans and SD rats were also observed. Principle component analysis showed that all the observed metabolite sex differences were more distinct in SD rats than in humans, indicating that the sex differences of human urinary metabolites is small compared with that of SD rats. In SD rats, the observed metabolite sex differences were more distinct in urine than in serum, indicating the importance of urine analysis for metabolomics studies. The species differences in the urinary metabolites of humans and SD rats were much more distinct than any of the observed sex differences. Many sex- and species-related markers were discovered and putatively identified. In both humans and SD rats, steroid metabolites appeared to constitute a major sex difference in urinary metabolites. This provides new proof of the special importance of steroid metabolites in sex differences from an untargeted metabolomics investigation, which is rare for sex differences. Contrary patterns involving adrenocortical activity appeared to exist between rodents and humans, which agrees with previous reports. In the serum metabolites of SD rats, sex differences in ascorbic acid or its isomer and pantothenic acid or its isomer, but not in steroid metabolites, were prominent. Human-specific α-N-phenylacetyl-L-glutamine and androsterone glucuronide were among the putative identities of the markers discriminating humans and SD rats. This study demonstrated the feasibility of an in-house software platform and provides metabolite-related information on sex and species differences.
PatelM.J.BatchB.C.SvetkeyL.P.BainJ.R.TurerC.B.HaynesC.MuehlbauerM.J.StevensR.D.NewgardC.B. and ShahS.H., “Race and sex differences in small-molecule metabolites and metabolic hormones in overweight and obese adults”, OMICS17, 627 (2013). doi: https://dx-doi-org.web.bisu.edu.cn/10.1089/omi.2013.0031
LiangQ.D. and MaJ.GaoY., “Software development for batch automatic preliminary identification of high resolution electrospray mass spectrometric data”, J. Chinese Mass Spectrom. Soc.33, 286 (2012).
6.
ArnoldA.P.ChenX. and ItohY., in Handbook of Experimental PharmacologyVol. 67 (2012).
7.
DunnW.B.ErbanA.WeberR.J.CreekD.J.BrownM.BreitlingR.HankemeierT.GoodacreR.NeumannS.KopkaJ. and ViantM., “Mass appeal: Metabolite identification in mass spectrometry-focused untargeted metabolomics”, Metabolomics9, 44 (2013). doi: https://dx-doi-org.web.bisu.edu.cn/10.1007/s11306-012-0434-4
8.
WishartD.S.TzurD.KnoxC.EisnerR.GuoA.C.YoungN.ChengD.JewellK.ArndtD.SawhneyS.FungC.NikolaiL.LewisM.CoutoulyM.ForsytheI.TangP.ShrivastavaS.JeroncicK.StothardP.AmegbeyG.BlockD.HauD.D.WagnerJ.MiniaciJ.ClementsM.GebremedhinM.GuoN.ZhangY.DugganG.E.MacInnisG.D.WeljieA.M.DowlatabadiR.BamforthF.CliveD.GreinerR.LiL.MarrieT.SykesB.D.VogelH.J.QuerengesserL., “HMDB: The Human Metabolome Database”, Nucleic Acids Res.35, D521 (2007). doi: https://dx-doi-org.web.bisu.edu.cn/10.1093/nar/gkl923
9.
WishartD.S.KnoxC.GuoA.C.EisnerR.YoungN.GautamB.HauD.D.PsychogiosN.DongE.BouatraS.MandalR.SinelnikovI.XiaJ.JiaL.CruzJ.A.LimE.SobseyC.A.ShrivastavaS.HuangP.LiuP.FangL.PengJ.FradetteR.ChengD.TzurD.ClementsM.LewisA.De SouzaA.ZunigaA.DaweM.XiongY.CliveD.GreinerR.NazyrovaA.ShaykhutdinovR.LiL.VogelH.J.ForsytheI., “HMDB: A knowledgebase for the human metabolome”, Nucleic Acids Res.37, D603 (2009). doi: https://dx-doi-org.web.bisu.edu.cn/10.1093/nar/gkn810
10.
WishartD.S.JewisonT.GuoA.C.WilsonM.KnoxC.LiuY.DjoumbouY.MandalR.AziatF.DongE.BouatraS.SinelnikovI.ArndtD.XiaJ.LiuP.YallouF.BjorndahlT.Perez-PineiroR.EisnerR.AllenF.NeveuV.GreinerR.ScalbertA., “HMDB 3.0—the Human Metabolome Database in 2013”, Nucleic Acids Res.41, D801 (2013). doi: https://dx-doi-org.web.bisu.edu.cn/10.1093/nar/gks1065
11.
WiklundS.JohanssonE.SjostromL.MellerowiczE.J.EdlundU.ShockcorJ.P.GottfriesJ.MoritzT.TryggJ., “Visualization of GC/ToF-MS-based metabolomics Data for identification of biochemically interesting compounds using Opls class models”, Anal. Chem.80, 115 (2008). doi: https://dx-doi-org.web.bisu.edu.cn/10.1021/ac0713510
12.
ScheltemaR.DecuypereS.DujardinJ.WatsonD.JansenR.BreitlingR., “Simple data-reduction method for high-resolution LC-MS data in metabolomics”, Bioanalysis1, 1551 (2009). doi: https://dx-doi-org.web.bisu.edu.cn/10.4155/bio.09.146
13.
PinkhasovR.M.ShteynshlyugerA.HakimianP.LindsayG.K.SamadiD.B.ShabsighR., “Are men shortchanged on health? Perspective on life expectancy, morbidity, and mortality in men and women in the United States”, Int. J. Clin. Pract.64, 465 (2010). doi: https://dx-doi-org.web.bisu.edu.cn/10.1111/j.1742-1241.2009.02289.x
14.
LuG.WangJ.ZhaoX.KongH.XuG., “Study on gender difference based on metabolites in urine by ultra high performance liquid chromatography/time of flight mass spectrometry”, Chinese J. Chromatogr.24, 109–113 (2006). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/S1872-2059(06)60005-9
15.
HodsonM.P.DearG.J.RobertsA.D.HaylockC.L.BallR.J.PlumbR.S.StumpfC.L.GriffinJ.L.HaseldenJ.N., “A gender-specific discriminator in Sprague-Dawley rat urine: The deployment of a metabolic profiling strategy for biomarker discovery and identification”, Anal. Biochem.362, 182 (2007). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/j.ab.2006.12.037
ShackletonC.H.NeresM.S.HughesB.A.StewartP.M.KaterC.E., “17-Hydroxylase/C17,20-lyase (Cyp17) is not the enzyme responsible for side-chain cleavage of cortisol and its metabolites”, Steroids73, 652 (2008). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/j.steroids.2008.02.001
18.
HamaN.KanemitsuH.TanikawaM.ShibayaM.SakamotoK.OyamaY.AcostaT.J.IshikawaO.PengyanW.OkudaK., “Development of an enzyme immunoassay for urinary pregnanediol-3-glucuronide in a female giant panda (Ailuropoda melanoleuca)”, J. Vet Med. Sci.71, 879 (2009). doi: https://dx-doi-org.web.bisu.edu.cn/10.1292/jvms.71.879
LoboR.A., “Progestogen metabolism”, J. Reprod. Med.44, 148 (1999).
21.
Jones-WittersP.HurleyM.J.PhillipsR.BrownB.L.ErbR.E., “Characterization of metabolites in domestic sow urine after intravenous administration of radioactive estrogen and corticosteroids”, J. Dairy Sci.58, 41 (1975). doi: https://dx-doi-org.web.bisu.edu.cn/10.3168/jds.S0022-0302(75)84515-2
TsilchorozidouT.HonourJ.W.ConwayG.S., “Altered cortisol metabolism in polycystic ovary syndrome: Insulin enhances 5α-reduction but not the elevated adrenal steroid production rates”, J. Clin. Endocrinol. Metabol.88, 5907 (2003). doi: https://dx-doi-org.web.bisu.edu.cn/10.1210/jc.2003-030240
24.
ChoHJKimJ.D.LeeW.Y.ChungB.C.ChoiM.H., “Quantitative metabolic profiling of 21 endogenous corticosteroids in urine by liquid chromatographytriple quadrupole-mass spectrometry”, Anal. Chim. Acta632, 101 (2009). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/j.aca.2008.10.059
ShamimW.YousufuddinM.BakhaiA.CoatsA.J. and HonourJ.W., “Gender differences in the urinary excretion rates of cortisol and androgen metabolites”Ann. Clin. Biochem.37, 770 (2000). doi: https://dx-doi-org.web.bisu.edu.cn/10.1258/0004563001900084
28.
WassifW.S.McLoughlinD.M.VincentR.P.ConroyS.RussellG.F. and TaylorN.F., “Steroid metabolism and excretion in severe anorexia nervosa: Effects of refeeding”, Am. J. Clin. Nutr.93, 911 (2011). doi: https://dx-doi-org.web.bisu.edu.cn/10.3945/ajcn.111.012666
29.
PoorV.JuricskayS.GatiA.OsvathP. and TenyiT., “Urinary steroid metabolites and 11β-hydroxysteroid dehydrogenase activity in patients with unipolar recurrent major depression”, J. Affect. Disord.81, 55 (2004). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/S0165-0327(03)00199-X
30.
MedinaS.FerreresF.Garcia-VigueraC.HorcajadaM.N.OrdunaJ.SavironM.ZurekG.Martinez-SanzJ.M.GilJ.I. and Gil-IzquierdoA., “Non-targeted metabolomic approach reveals urinary metabolites linked to steroid biosynthesis pathway after ingestion of citrus juice”, Food Chem.136, 938 (2013). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/j.foodchem.2012.09.004
31.
YuZ.R.NingY.YuH.TangN.J., “A HPLC-Q-ToF-MS-based urinary metabolomic approach to identification of potential biomarkers of metabolic syndrome”, J. Huazhong Univ. Sci. Technol. Med. Sci34, 276 (2014). doi: https://dx-doi-org.web.bisu.edu.cn/10.1007/s11596-014-1271-7
32.
ParisJ.J.FrancoC.SodanoR.FreidenbergB.GordisE.AndersonD.A.ForsythJ.P.WulfertE. and FryeCA, “Sex differences in salivary cortisol in response to acute stressors among healthy participants, in recreational or pathological gamblers, and in those with post-traumatic stress disorder”, Horm. Behav.57, 35 (2010). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/j.yhbeh.2009.06.003
ShibasakiH.TanabeC.FurutaT. and KasuyaY.“Hydrolysis of conjugated steroids by the combined use of β-glucuronidase preparations from Helix pomatia and Ampullaria: Determination of urinary cortisol and its metabolites”, Steroids66, 795 (2001). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/S0039-128X(01)00118-0
35.
PavlovicR.CannizzoFTPanseriS.BiolattiB.TruticN.BiondiP.A.ChiesaL., “Tetrahydro-metabolites of cortisol and cortisone in bovine urine evaluated by HPLC-ESI-mass spectrometry”, J. Steroid Biochem. Mol. Biol.135, 30 (2013). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/j.jsbmb.2012.12.015
36.
FernandesV.T.Ribeiro-NetoL.M.LimaS.B.VieiraJ.G.VerreschiI.T. and KaterC.E., “Reversed-phase high-performance liquid chromatography separation of adrenal steroids prior to radioimmunoassay: Application in congenital adrenal hyperplasia”, J. Chromatogr Sci.41, 251 (2003). doi: https://dx-doi-org.web.bisu.edu.cn/10.1093/chromsci/41.5.251
37.
KaminskiR.M.FuZ.VenkatesanK.MazzuferiM.LeclercqK.SeutinV. and ViciniS., “11-Deoxycortisol impedes gabaergic neurotransmission and induces drug-resistant status epilepticus in mice”, Neuropharmacology60, 1098 (2011). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/j.neuropharm.2010.09.021
38.
CzyrakA.MackowiakM.ChocykA.FijalK.Gadek-MichalskaA. and WedzonyK., “8-OHDPAT-induced disruption of prepulse inhibition in rats is attenuated by prolonged corticosterone treatment”, Neuropsychopharmacology28, 1300 (2003). doi: https://dx-doi-org.web.bisu.edu.cn/10.1038/sj.npp.1300165
39.
DelaneyM.L. and MelbyJ.C., “19-Acetylenic-deoxycorticosterone inhibits 19-hydroxylase and 11ß-hydroxylase activities in dispersed bovine zona fasciculata cells”, Steroids60, 265 (1995). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/0039-128X(94)00050-M
CavigelliS.A.MonfortS.L.WhitneyT.K.MechrefY.S.NovotnyM. and McClintockM.K., “Frequent serial fecal corticoid measures from rats reflect circadian and ovarian corticosterone rhythms”, J. Endocrinol.184, 153 (2005). doi: https://dx-doi-org.web.bisu.edu.cn/10.1677/joe.1.05935
RiversJ.M., “Oral contraceptives and ascorbic acid”, Am. J. Clin. Nutr.28, 550 (1975).
47.
Standing Committee on the Scientific Evaluation of Dietary Reference Intakes and its Panel on Folate, Other B Vitamins, and Choline, Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline.Institute of Medicine, Washington DC, p. 357 (1998).
48.
YamamotoT.JaroenpornS.PanL.AzumanoI.OndaM.NakamuraK.WatanabeG. and TayaK., “Effects of pantothenic acid on testicular function in male rats”, J. Vet. Med. Sci.71, 1427 (2009). doi: https://dx-doi-org.web.bisu.edu.cn/10.1292/jvms.001427
49.
JaroenpornS.YamamotoT.ItabashiA.NakamuraK.AzumanoI.WatanabeG. and TayaK., “Effects of pantothenic acid supplementation on adrenal steroid secretion from male rats”, Biol. Pharm. Bull.31, 1205 (2008). doi: https://dx-doi-org.web.bisu.edu.cn/10.1248/bpb.31.1205
50.
RhodesM.E. and RubinR.T., “Functional sex differences (‘sexual diergism’) of central nervous system cholinergic systems, vasopressin, and hypothalamic-pituitary-adrenal axis activity in mammals: A selective review”, Brain Res. Brain Res. Rev, 30, 135 (1999). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/S0165-0173(99)00011-9
GermanoJ.M.MoliniaF.C.BishopP.J.BellB.D. and CreeA., “Urinary hormone metabolites identify sex and imply unexpected winter breeding in an endangered, subterranean-nesting frog”, Gen. Comp. Endocrinol.175, 464 (2012). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/j.ygcen.2011.12.003
54.
PavelS.MuskietF.A.NagelG.T.SchwippelovaZ. and DuchonJ., “Identification of two thormahlen-positive compounds from melanotic urine by gas chromatography-mass spectrometry”, J. Chromatogr.222, 329 (1981). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/S0378-4347(00)84132-8
55.
WangY.HolmesE.NicholsonJ.K.CloarecO.CholletJ.TannerM.SingerB.H. and UtzingerJ., “Metabonomic investigations in mice infected with Schistosoma mansoni: An approach for biomarker identification”, Proc. Natl Acad. Sci. USA101, 12676 (2004). doi: https://dx-doi-org.web.bisu.edu.cn/10.1073/pnas.0404878101
56.
LiabeufS.GlorieuxG.LengletA.DioufM.SchepersE.DesjardinsL.ChoukrounG.VanholderR. and MassyZ.A., “Does p-cresylglucuronide have the same impact on mortality as other protein-bound uremic toxins?”, PloS One8, e67168 (2013). doi: https://dx-doi-org.web.bisu.edu.cn/10.1371/journal.pone.0067168
57.
LesafferG.De SmetR.BelpaireF.M.Van VlemB.Van HulleM.CornelisR.LameireN. and VanholderR., “Urinary excretion of the uraemic toxin p-cresol in the rat: Contribution of glucuronidation to its metabolization”, Nephrol. Dialysis Transplant.18, 1299 (2003). doi: https://dx-doi-org.web.bisu.edu.cn/10.1093/ndt/gfg107
58.
SunH.ZhangS.ZhangA.YanG.WuX.HanY. and WangX., “Metabolomic analysis of diet-induced Type 2 diabetes using UPLC/MS integrated with pattern recognition approach”, PloS One9, e93384 (2014). doi: https://dx-doi-org.web.bisu.edu.cn/10.1371/journal.pone.0093384
59.
KimM.JungS.LeeS.H. and LeeJ.H., “Association between arterial stiffness and serum L-octanoylcarnitine and lactosylceramide in overweight middle-aged subjects: 3-year follow-up study”, PloS One10, e0119519 (2015). doi: https://dx-doi-org.web.bisu.edu.cn/10.1371/journal.pone.0119519
60.
BeneJ.KomlosiK.GasztonyiB.JuhaszM.TulassayZ. and MeleghB., “Plasma carnitine ester profile in adult celiac disease patients maintained on long-term gluten free diet”, World J. Gastroenterol.11, 6671 (2005). doi: https://dx-doi-org.web.bisu.edu.cn/10.3748/wjg.v11.i42.6671
GaudetM.M.FalkR.T.StevensR.D.GunterM.J.BainJ.R.PfeifferR.M.PotischmanN.LissowskaJ.PeplonskaB.BrintonL.A.Garcia-ClosasM.NewgardC.B. and ShermanM.E., “Analysis of serum metabolic profiles in women with endometrial cancer and controls in a population-based case-control study”, J. Clin. Endocrinol. Metab.97, 3216 (2012). doi: https://dx-doi-org.web.bisu.edu.cn/10.1210/jc.2012-1490
63.
RyckmanK.K.ShchelochkovO.A.CookD.E.BerberichS.L.CopelandS.DagleJ.M. and MurrayJ.C., “The influence of maternal disease on metabolites measured as part of newborn screening”, J. Matern. Fetal Neonatal Med.26, 1380 (2013). doi: https://dx-doi-org.web.bisu.edu.cn/10.3109/14767058.2013.791267
BlouinK.RichardC.BrochuG.HouldF.S.LebelS.MarceauS.BironS.Luu-TheV. and TchernofA., “Androgen inactivation and steroid-converting enzyme expression in abdominal adipose tissue in men”, J. Endocrinol.191, 637 (2006). doi: https://dx-doi-org.web.bisu.edu.cn/10.1677/joe.1.06365
66.
BarbierO.BelangerA. and HumD.W., “Cloning and characterization of a Simian UDP-glucuronosyltransferase enzyme UGT2B20, a novel C19 Steroid-conjugating protein”, Biochem. J.337, 567 (1999). doi: https://dx-doi-org.web.bisu.edu.cn/10.1042/bj3370567
67.
ZumoffB.TroxlerR.G.O'ConnorJ.RosenfeldR.S.KreamJ.LevinJ.HickmanJ.R.SloanA.M.WalkerW.CookR.L. and FukushimaD.K., “Abnormal hormone levels in men with coronary artery disease”, Arteriosclerosis2, 58 (1982). doi: https://dx-doi-org.web.bisu.edu.cn/10.1161/01.ATV.2.1.58
SneitzN.KrishnanK.CoveyD.F. and FinelM., “Glucuronidation of the steroid enantiomers ent-17β-estradiol, ent-androsterone and ent-etiocholanolone by the human UDP-glucuronosyltransferases”, J. Steroid Biochem. Mol. Biol.127, 282 (2011). doi: https://dx-doi-org.web.bisu.edu.cn/10.1016/j.jsbmb.2011.08.008
ChoiM.H.KimJ.N. and ChungB.C., “Rapid HPLC-electrospray tandem mass spectrometric assay for urinary testosterone and dihydrotestosterone glucuronides from patients with benign prostate hyperplasia”, Clin. Chem.49, 322 (2003). doi: https://dx-doi-org.web.bisu.edu.cn/10.1373/49.2.322
72.
ChanA.O.ButB.W.LeeC.Y.LamY.Y.NgK.L.TungJ.Y.KwanE.Y.ChanY.K.TsuiT.K.LamA.L.TseW.Y.CheungP.T. and ShekC.C., “Diagnosis of 5α-reductase 2 deficiency: Is measurement of dihydrotestosterone essential?”, Clin. Chem.59, 798 (2013). doi: https://dx-doi-org.web.bisu.edu.cn/10.1373/clinchem.2012.196501
73.
ShelbyM.K.CrouchD.J.BlackD.L.RobertT.A. and HeltsleyR., “Screening indicators of dehydroepiandosterone, androstenedione, and dihydrotestosterone use: A literature review”, J. Anal. Toxicol.35, 638 (2011). doi: https://dx-doi-org.web.bisu.edu.cn/10.1093/anatox/35.9.638
74.
TaitJ.F. and LittleB., “The metabolism of orally and intravenously administered labeled aldosterone in pregnant subjects”, J. Clin. Invest.47, 2423 (1969). doi: https://dx-doi-org.web.bisu.edu.cn/10.1172/JCI105925
75.
MiyazakiT. and MatsuzakiK., “Further characterization of urinary aldosterone metabolites in the rabbit by fast atom bombardment mass spectrometry”, Endocrinol. Jpn37, 103 (1990). doi: https://dx-doi-org.web.bisu.edu.cn/10.1507/endocrj1954.37.103
