Quantitative structural multiclass lipidomics using differential mobility: electron impact excitation of ions from organics (EIEIO) mass spectrometry

We report a method for comprehensive structural characterization of lipids in animal tissues using a combination of differential ion mobility spectrometry (DMS) with electron-impact excitation of ions from organics (EIEIO) mass spectrometry. Singly charged lipid ions in protonated or sodiated forms were dissociated by an electron beam having a kinetic energy of 10 eV in a branched radio-frequency ion trap. We established a comprehensive set of diagnostics to characterize the structures of glycerophospholipids, sphingolipids, and acylglycerols, including glycosylated, plasmalogen, and ester forms. This EIEIO mass spectrometer was combined with DMS as a separation tool to analyze complex lipid extracts. Deuterated quantitative standards, which were added during extraction, allowed for the quantitative analysis of the lipid molecular species in various lipid classes. We applied this technique to the total lipids extracted from porcine brain, and we structurally characterized over 300 lipids (with the exception of cis/trans double-bond isomerism in the acyl chains). The structural dataset of the lipidomes, whose regioisomers were distinguished, exhibit a uniquely defined distribution of acyl chains within each lipid class; that is, sn-1 and sn-2 in the cases of glycerophospholipids or sn-2 and (sn-1, sn-3) in the cases of triacylglycerols. We report a method for comprehensive structural characterization of lipids in animal tissues using a combination of differential ion mobility spectrometry (DMS) with electron-impact excitation of ions from organics (EIEIO) mass spectrometry. Singly charged lipid ions in protonated or sodiated forms were dissociated by an electron beam having a kinetic energy of 10 eV in a branched radio-frequency ion trap. We established a comprehensive set of diagnostics to characterize the structures of glycerophospholipids, sphingolipids, and acylglycerols, including glycosylated, plasmalogen, and ester forms. This EIEIO mass spectrometer was combined with DMS as a separation tool to analyze complex lipid extracts. Deuterated quantitative standards, which were added during extraction, allowed for the quantitative analysis of the lipid molecular species in various lipid classes. We applied this technique to the total lipids extracted from porcine brain, and we structurally characterized over 300 lipids (with the exception of cis/trans double-bond isomerism in the acyl chains). The structural dataset of the lipidomes, whose regioisomers were distinguished, exhibit a uniquely defined distribution of acyl chains within each lipid class; that is, sn-1 and sn-2 in the cases of glycerophospholipids or sn-2 and (sn-1, sn-3) in the cases of triacylglycerols. Lipids serve many physiological functions in biosystems, playing essential roles in energetics, cell structure, and cell signaling. They also are implicated as playing direct and causal roles in the pathophysiology of human diseases, including Parkinson's disease, Alzheimer's disease, depression, cardiovascular diseases, and various cancers (1.Ekroos K. Lipidomics: Technologies and Applications.Wiley. 2012; Google Scholar, 2.Suzuki K. Suzuki Y. Globoid cell leucodystrophy (Krabbe's disease): deficiency of galactocerebroside β-galactosidase.Proc. Natl. Acad. Sci. USA. 1970; 66: 302-309Crossref PubMed Scopus (402) Google Scholar, 3.Müller C.P. Reichel M. Mühle C. Rhein C. Gulbins E. Kornhuber J. Brain membrane lipids in major depression and anxiety disorders.Biochim. Biophys. Acta. 2015; 1851: 1052-1065Crossref PubMed Scopus (187) Google Scholar, 4.Kelley N.S. Hubbard N.E. Erickson K.L. Conjugated linoleic acid isomers and cancer.J. Nutr. 2007; 137: 2599-2607Crossref PubMed Scopus (270) Google Scholar). Much of lipids' actions can be related to their structures, with small structural differences in isomeric lipid molecules having dramatic impact on their physiological functions [e.g., the diastereomers prostaglandin-D2 and prostaglandin-E2 (5.Flower R.J. Harvey E.A. Kingston W.P. Inflammatory effects of prostaglandin D2 in rat and human skin.Br. J. Pharmacol. 1976; 56: 229-233Crossref PubMed Scopus (149) Google Scholar, 6.Sheng H. Shao J. Morrow J.D. Beauchamp R.D. DuBois R.N. Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells.Cancer Res. 1998; 58: 362-366PubMed Google Scholar)]. Recently, a slight permutation in double-bond position in one of the acyl chains of a phospholipid was shown to be a marker of breast cancer (7.Ma X. Chong L. Tian R. Shi R. Hu T.Y. Ouyang Z. Xia Y. Identification and quantitation of lipid C=C location isomers: A shotgun lipidomics approach enabled by photochemical reaction.Proc. Natl. Acad. Sci. USA. 2016; 113: 2573-2578Crossref PubMed Scopus (214) Google Scholar), and the presence of a Δ7 vs. a Δ9 double bond in a specific molecular species of triacylglycerol (TG) was correlated with adverse cardiovascular outcomes (1.Ekroos K. Lipidomics: Technologies and Applications.Wiley. 2012; Google Scholar). For these reasons, differentiation of lipids by complete structural characterization is “a real promise in achieving true molecular level lipidomics analysis generation”(8.Hancock S.E. Poad B.L.J. Batarseh A. Abbott S.K. Mitchell T.W. Advances and unresolved challenges in the structural characterization of isomeric lipids.Anal. Biochem. 2017; 524: 45-55Crossref PubMed Scopus (59) Google Scholar). Not only qualitative structure characterization of each lipid molecule, but also quantitative analysis across many lipid classes in tissues, will be the next target in lipidomics to elucidate cross-class lipid metabolism. Conventionally, NMR is used as a tool for full characterization of lipid structures (9.Gunstone F.D. High resolution 13C NMR. A technique for the study of lipid structure and composition.Prog. Lipid Res. 1994; 33: 19-28Crossref PubMed Scopus (50) Google Scholar), but it needs a reasonably large amount (approximately milligrams) of purified samples and very long analysis times (approximately days), which results in low throughput. Mass spectrometry (MS) is used widely as a high-throughput method for versatile biomolecular identification (10.March R.E. Todd J.F.J. Practical Aspects of Trapped Ion Mass Spectrometry: Theory and Instrumentation.CRC Press. 2010; Google Scholar). Conventional techniques for lipid analysis in MS are based on a combination of separation techniques (liquid chromatography, gas chromatography, ion mobility spectrometry, etc.) and collision-induced dissociation (CID) in the gas phase (1.Ekroos K. Lipidomics: Technologies and Applications.Wiley. 2012; Google Scholar). This approach, however, does not provide full structural information of lipid isomers, which is often limited to characterization of the lipid class and brutto class, which represents the head group and the combined lengths of chains and numbers of double bonds among the acyl chains. To elucidate in-depth structure, including regioisomerism and double-bond locations and their cis/trans isoforms, arduous efforts are required using additional dissociation techniques, such as high-energy CID (11.Griffiths W.J. Tandem mass spectrometry in the study of fatty acids, bile acids, and steroids.Mass Spectrom. Rev. 2003; 22: 81-152Crossref PubMed Scopus (223) Google Scholar), ozone-induced dissociation (OzID) (12.Pham H.T. Maccarone A.T. Thomas M.C. Campbell J.L. Mitchell T.W. Blanksby S.J. Structural characterization of glycerophospholipids by combinations of ozone- and collision-induced dissociation mass spectrometry: the next step towards “top-down”lipidomics.Analyst. 2014; 139: 204-214Crossref PubMed Google Scholar), a UV-induced Paternò-Büchi reaction (7.Ma X. Chong L. Tian R. Shi R. Hu T.Y. Ouyang Z. Xia Y. Identification and quantitation of lipid C=C location isomers: A shotgun lipidomics approach enabled by photochemical reaction.Proc. Natl. Acad. Sci. USA. 2016; 113: 2573-2578Crossref PubMed Scopus (214) Google Scholar, 13.Ma X. Xia Y. Pinpointing double bonds in lipids by Paternò-Büchi reactions and mass spectrometry.Angew. Chem. Int. Ed. 2014; 53: 2592-2596Crossref PubMed Scopus (218) Google Scholar), and UV photo dissociation (UVPD) (14.Ryan E. Nguyen C.Q.N. Shiea C. Reid G.E. Detailed structural characterization of sphingolipids via 193 nm ultraviolet photodissociation and ultra high resolution tandem mass spectrometry.J. Am. Soc. Mass Spectrom. 2017; Crossref PubMed Scopus (84) Google Scholar, 15.Williams P.E. Klein D.R. Greer S.M. Brodbelt J.S. Pinpointing double bond and sn-positions in glycerophospholipids via hybrid 193 nm ultraviolet photodissociation (UVPD) mass spectrometry.J. Am. Chem. Soc. 2017; 139: 15681-15690Crossref PubMed Scopus (122) Google Scholar), as well as complex chromatographic strategies. Recently, we reported a new methodology for the structural characterization of complex lipids using electron-induced dissociation (EID) or, in historical terminology, electron impact excitation of ions from organics (EIEIO) (16.Cody R.B. Freiser B.S. Electron impact excitation of ions from organics: an alternative to collision induced dissociation.Anal. Chem. 1979; 51: 547-551Crossref Scopus (121) Google Scholar) in a novel branched radio-frequency (RF) ion trap (17.Baba T. Campbell J.L. Le Blanc J.C.Y. Hager J.W. Thomson B.A. Electron capture dissociation in a branched radio-frequency ion trap.Anal. Chem. 2015; 87: 785-792Crossref PubMed Scopus (30) Google Scholar, 21.Baba T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. Distinguishing cis and trans isomers in intact complex lipids using electron impact excitation of ions from organics (EIEIO) mass spectrometry.Anal. Chem. 2017; 89: 7307-7315Crossref PubMed Scopus (40) Google Scholar). An electron beam with a kinetic energy of 10 eV was applied to positively charged lipid ions so that the EIEIO which the lipid class, the of double bonds and their within the and regioisomerism of fatty acyl position on the of and glycerophospholipids We also the to isomers a double bond in the chains of classes of intact complex lipids T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. Distinguishing cis and trans isomers in intact complex lipids using electron impact excitation of ions from organics (EIEIO) mass spectrometry.Anal. Chem. 2017; 89: 7307-7315Crossref PubMed Scopus (40) Google Scholar). We applied this technique to class extracted from J.L. T. structural characterization of using electron impact excitation of ions from Chem. 2015; 87: PubMed Scopus Google Scholar), T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. characterization using electron impact excitation of ions from organics and mass spectrometry.J. Lipid Res. 2016; PubMed Scopus Google Scholar) and in T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics Lipid Res. 2016; PubMed Scopus Google Scholar). this we report a comprehensive quantitative and structural lipidomics that a lipidomics analysis S.E. Poad B.L.J. Batarseh A. Abbott S.K. Mitchell T.W. Advances and unresolved challenges in the structural characterization of isomeric lipids.Anal. Biochem. 2017; 524: 45-55Crossref PubMed Scopus (59) Google Scholar). To comprehensive diagnostics that reported for and we applied EIEIO to of lipid classes in and sphingolipids and and as well as and chains were For quantitative a of lipid across lipid classes was added to the the were This comprehensive method was applied on total lipids in the and tissues, with a major on the it many lipid classes with various fatty acyl chains J.S. Lipid of the human and Lipid Res. PubMed Google Scholar, K. J. Identification of phospholipid molecular species in porcine using high mass of ion mass Chem. Soc. Scopus Google Scholar, structural characterization of glycerophospholipids and in using Am. Soc. Mass Spectrom. 2007; PubMed Scopus Google Scholar). We not fatty acids, and in this these classes approach from the in this or of the samples used in this are acid and these were from Lipids lipid samples in this are the total lipid and complex lipid were also from lipid for the were which are a of lipid lipid classes not in this were from quantitative analysis and of each are in We used of in this one a of in a of and and a of in a of and The and and were from and The protonated ions for and and the sodiated ions of lipid classes. were used to characterize the The of the was in A shotgun approach using was used in this was were a of by using a differential mobility spectrometry (DMS) was used with of to of the separation was Not only for separation of lipid classes Baker P.R.S. M. E. Campbell J.L. K. mobility shotgun Chem. 2014; PubMed Scopus Google Scholar), but also the of gas and the were for each target ion to from 13C and DMS was in T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics Lipid Res. 2016; PubMed Scopus Google Scholar). The mass used in this was reported J.L. T. structural characterization of using electron impact excitation of ions from Chem. 2015; 87: PubMed Scopus Google Scholar, T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics Lipid Res. 2016; PubMed Scopus Google Scholar), which is a mass spectrometer with a branched ion trap EIEIO mass and collision dissociation (17.Baba T. Campbell J.L. Le Blanc J.C.Y. Hager J.W. Thomson B.A. Electron capture dissociation in a branched radio-frequency ion trap.Anal. Chem. 2015; 87: 785-792Crossref PubMed Scopus (30) Google Scholar). Lipid ions were by using as a mass to a small of the lipid to EIEIO one The EIEIO was in (17.Baba T. Campbell J.L. Le Blanc J.C.Y. Hager J.W. Thomson B.A. Electron capture dissociation in a branched radio-frequency ion trap.Anal. Chem. 2015; 87: 785-792Crossref PubMed Scopus (30) Google Scholar) for with electron kinetic energy of 10 with each EIEIO of total EIEIO of ions of the in protonated and sodiated forms were We the shown in J.L. T. structural characterization of using electron impact excitation of ions from Chem. 2015; 87: PubMed Scopus Google Scholar, T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics Lipid Res. 2016; PubMed Scopus Google Scholar) to cases in and For the head group in J.L. T. structural characterization of using electron impact excitation of ions from Chem. 2015; 87: PubMed Scopus Google Scholar) was to by the molecular mass of the head were for and lipids by the class group and for defined in and defined in are to the head from the in a new and acyl from the in a new group and for defined in and defined in are to the head from the from the The identification of lipid class and brutto class, and structural double-bond identification of the double bond is by using EIEIO T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. Distinguishing cis and trans isomers in intact complex lipids using electron impact excitation of ions from organics (EIEIO) mass spectrometry.Anal. Chem. 2017; 89: 7307-7315Crossref PubMed Scopus (40) Google Scholar), but we not this in this The lipid classes of complex lipids are by the and the head group and are in complex The head group many in complex and fatty are head in this the cases the head group position is by a the lipids are or is to a in that head group position is by a fatty acyl The head group in EIEIO is often as the the bond in the cases of and or the bond in the cases of This was to phospholipid and by using the lipid the cases that a was the head the of the for was from the head group a or which EIEIO can by a for a ion of in the cases of but we not in the cases of This was only in the cases of sodiated but not in protonated This that a ion can be on but not on The major or were by EIEIO with a by a the cases of in EIEIO as a with and from the head group the cases of and on the a from the head group T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. characterization using electron impact excitation of ions from organics and mass spectrometry.J. Lipid Res. 2016; PubMed Scopus Google Scholar). This of is allowed to but not to We reported a bond by EIEIO J.L. T. structural characterization of using electron impact excitation of ions from Chem. 2015; 87: PubMed Scopus Google Scholar, T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics Lipid Res. 2016; PubMed Scopus Google Scholar), which is a This not by and only in high-energy J.W. dissociation (EID) for structure characterization of of double-bond and of acyl Mass Spectrom. 2015; PubMed Scopus Google Scholar) and R.E. M. of phospholipid dissociation mass spectrometry J. Mass Spectrom. 2015; PubMed Scopus Google Scholar). This permutation of chains sn-1 and sn-2 this was and of double bonds in each sn-1 and sn-2 are using the mass and the head group mass J.L. T. structural characterization of using electron impact excitation of ions from Chem. 2015; 87: PubMed Scopus Google Scholar). for and species are also shown in cases of was by an in many cases that was such of were and the is and of double bonds in the are also that EIEIO also the sn-2 from the sn-1 in the head group and the that the the the of sn-1 the the of a was the sn-1 dissociated from the intact lipid the of sn-2 however, the a from the sn-1 We such in the in sodiated and protonated forms We reported the identification of double-bond locations by EIEIO in on and J.L. T. structural characterization of using electron impact excitation of ions from Chem. 2015; 87: PubMed Scopus Google Scholar, T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics Lipid Res. 2016; PubMed Scopus Google Scholar). The of for the double-bond diagnostics is that bonds were but double bonds were not by EIEIO electron kinetic energy of the 10 eV This was by double T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics Lipid Res. 2016; PubMed Scopus Google Scholar). This was in complex lipid classes in this can be applied on each EIEIO to but analysis was for this which was reported for and J.L. T. structural characterization of using electron impact excitation of ions from Chem. 2015; 87: PubMed Scopus Google Scholar, T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics Lipid Res. 2016; PubMed Scopus Google Scholar). The of each lipid class in the were by the using the quantitative we total lipid having a of for in the and these we the quantitative analysis 10 each of and of from and 10 each of and from the a was on each was The ion for each quantitative analysis were over the which the samples were ion were as a of the for each lipid was applied to the and of for each class were and their are in methodology was applied to the total The separation and in was used in this T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics Lipid Res. 2016; PubMed Scopus Google step using step ion using the but DMS step a and for each ions not to be by lipid and 13C level was in this and step EIEIO the and was from to by the ion The was a of for each of the The sodiated ions with to which we from for EIEIO analysis based the mass differences the sodiated or lipids and these the EIEIO and a EIEIO to the of the by a and as well to that this dissociation method a of of to The of each ion were from the by over The ion were to of the by using the using the quantitative The EIEIO we were by the analysis and lipids were characterized shown in the of complex lipids in each lipid class and The numbers of that were the of complex which are in their the cases that double-bond locations were not to be in each were as in the and The of the in this analysis was of from to as shown in were often in the and sodiated the of was from the For structure analysis of the the were to be applied the mass of to the and the lipids in the in this The species was a the a with an in sn-2 position lipids in this were of total including 13C species be lipids that are of such as fatty whose be and whose be over complex lipid species also be in this complex lipids in the porcine in this represents the in in of the total lipid The full in in a new in this represents the in in of the total lipid The full in lipid class separation by DMS in the total of in each lipid species are Lipid classes were by DMS as reported in Baker P.R.S. M. E. Campbell J.L. K. mobility shotgun Chem. 2014; PubMed Scopus Google Scholar), but lipid classes are also The in the DMS separation was that a with a in sodiated and protonated with were but a species a This be by the lipids' structural differences by double-bond a species a protonated and sodiated DMS separation for protonated was sodiated for and the distribution of the various acyl chains in lipids from the and double-bond locations were This of was in on characterization T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics Lipid Res. 2016; PubMed Scopus Google Scholar). the acyl chains with the various lipid the on each the specific distribution of specific sn-1 and sn-2 chains. the cases of acyl sn-1 position are sn-2 and but long chains were the sn-2 position of of including long chains. long chains were not in the sn-2 position of the sn-1 position was by the cases of sn-1 were to but were sn-2 long such as and such are of fatty for and fatty M. A of for in Sci. 2014; PubMed Scopus Google Scholar). For was as the as but lengths of were also in and The distribution of lengths was to the glycerophospholipids, was and acyl but the a which was very species with the chains of and by J. PubMed Scopus Google Scholar, M. J. C. M. in the of and fatty acid PubMed Scopus Google Scholar), as were not in this it be in the J. E. in one step separation for molecular characterization of PubMed Scopus Google Scholar). the of we can regioisomerism the and and but the of sn-1 or was not this M. H. M. characterization of using and of Chem. PubMed Scopus Google Scholar). were not in the distribution was distribution of the of sn-1 and over the sn-2 the sn-2 and acyl were The sn-1 and a including and were but was only one was allowed in in and and in be to lipid in as well as of in which were T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics Lipid Res. 2016; PubMed Scopus Google Scholar). this for of the be the and are protonated are head group are and DMS For analysis of and be does not positively charged the method using the many species in and method was for such results that samples in each class are samples as be also to the of molecular species in as well as the of low lipid species by were not in the total which was reported in T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. characterization using electron impact excitation of ions from organics and mass spectrometry.J. Lipid Res. 2016; PubMed Scopus Google Scholar). for the lipid species not be in of A small of and and were in this of this method is that EIEIO is only on positively charged We applied this technique on charged lipid but we that charged ions were dissociated by an electron beam with eV electron capture was reported in charged fatty M. of fatty acid molecular by using electron capture mass J. Mass Spectrom. Scopus Google Scholar). with were in and in porcine lipids by using and techniques (12.Pham H.T. Maccarone A.T. Thomas M.C. Campbell J.L. Mitchell T.W. Blanksby S.J. Structural characterization of glycerophospholipids by combinations of ozone- and collision-induced dissociation mass spectrometry: the next step towards “top-down”lipidomics.Analyst. 2014; 139: 204-214Crossref PubMed Google Scholar, 15.Williams P.E. Klein D.R. Greer S.M. Brodbelt J.S. Pinpointing double bond and sn-positions in glycerophospholipids via hybrid 193 nm ultraviolet photodissociation (UVPD) mass spectrometry.J. Am. Chem. Soc. 2017; 139: 15681-15690Crossref PubMed Scopus (122) Google Scholar). we this acyl group in lipids we not this double-bond in and during this and Paternò-Büchi reaction (7.Ma X. Chong L. Tian R. Shi R. Hu T.Y. Ouyang Z. Xia Y. Identification and quantitation of lipid C=C location isomers: A shotgun lipidomics approach enabled by photochemical reaction.Proc. Natl. Acad. Sci. USA. 2016; 113: 2573-2578Crossref PubMed Scopus (214) Google Scholar, 13.Ma X. Xia Y. Pinpointing double bonds in lipids by Paternò-Büchi reactions and mass spectrometry.Angew. Chem. Int. Ed. 2014; 53: 2592-2596Crossref PubMed Scopus (218) Google Scholar), the double-bond location in a bonds not that the This is an of these techniques double-bond EIEIO from double and so that was required to from double bond the porcine was and was not as a over a of the total of The in this report was or we to with and that provide long times are for an to an ion with is by in to for an approach, this represents a and to be A in the be a A MS with high resolution and to for Am. Soc. Mass Spectrom. 2017; PubMed Scopus Google Scholar), with a mass We a comprehensive structural methodology to analyze of complex lipids and using EIEIO we a lipidomics analysis method using EIEIO with a of complex lipid using This a new for real structural lipidomics (9.Gunstone F.D. High resolution 13C NMR. A technique for the study of lipid structure and composition.Prog. Lipid Res. 1994; 33: 19-28Crossref PubMed Scopus (50) Google Scholar). The structural by the high-throughput lipid in many in such as C.P. Reichel M. Mühle C. Rhein C. Gulbins E. Kornhuber J. Brain membrane lipids in major depression and anxiety disorders.Biochim. Biophys. Acta. 2015; 1851: 1052-1065Crossref PubMed Scopus (187) Google Scholar), and of fatty N.S. Hubbard N.E. Erickson K.L. Conjugated linoleic acid isomers and cancer.J. Nutr. 2007; 137: 2599-2607Crossref PubMed Scopus (270) Google Scholar).

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