High-resolution imaging mass spectrometry combined with transcriptomic analysis identified a link between fatty acid composition of phosphatidylinositols and the immune checkpoint pathway at the primary tumour site of breast cancer.
Aged
Animals
Breast Neoplasms
/ diagnostic imaging
Fatty Acids
/ genetics
Female
Gene Expression Profiling
/ methods
Gene Expression Regulation, Neoplastic
/ genetics
Humans
Mass Spectrometry
Middle Aged
Phosphatidylinositols
/ genetics
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
/ methods
Transcriptome
/ genetics
Journal
British journal of cancer
ISSN: 1532-1827
Titre abrégé: Br J Cancer
Pays: England
ID NLM: 0370635
Informations de publication
Date de publication:
01 2020
01 2020
Historique:
received:
16
05
2019
accepted:
07
11
2019
revised:
07
11
2019
pubmed:
11
12
2019
medline:
17
7
2020
entrez:
11
12
2019
Statut:
ppublish
Résumé
The fatty acid (FA) composition of phosphatidylinositols (PIs) is tightly regulated in mammalian tissue since its disruption impairs normal cellular functions. We previously found its significant alteration in breast cancer by using matrix-assisted laser desorption and ionisation imaging mass spectrometry (MALDI-IMS). We visualised the histological distribution of PIs containing different FAs in 65 primary breast cancer tissues using MALDI-IMS and investigated its association with clinicopathological features and gene expression profiles. Normal ductal cells (n = 7) predominantly accumulated a PI containing polyunsaturated FA (PI-PUFA), PI(18:0/20:4). PI(18:0/20:4) was replaced by PIs containing monounsaturated FA (PIs-MUFA) in all non-invasive cancer cells (n = 12). While 54% of invasive cancer cells (n = 27) also accumulated PIs-MUFA, 46% of invasive cancer cells (n = 23) accumulated the PIs-PUFA, PI(18:0/20:3) and PI(18:0/20:4). The accumulation of PI(18:0/20:3) was associated with higher incidence of lymph node metastasis and activation of the PD-1-related immune checkpoint pathway. Fatty acid-binding protein 7 was identified as a putative molecule controlling PI composition. MALDI-IMS identified PI composition associated with invasion and nodal metastasis of breast cancer. The accumulation of PI(18:0/20:3) could affect the PD-1-related immune checkpoint pathway, although its precise mechanism should be further validated.
Sections du résumé
BACKGROUND
The fatty acid (FA) composition of phosphatidylinositols (PIs) is tightly regulated in mammalian tissue since its disruption impairs normal cellular functions. We previously found its significant alteration in breast cancer by using matrix-assisted laser desorption and ionisation imaging mass spectrometry (MALDI-IMS).
METHODS
We visualised the histological distribution of PIs containing different FAs in 65 primary breast cancer tissues using MALDI-IMS and investigated its association with clinicopathological features and gene expression profiles.
RESULTS
Normal ductal cells (n = 7) predominantly accumulated a PI containing polyunsaturated FA (PI-PUFA), PI(18:0/20:4). PI(18:0/20:4) was replaced by PIs containing monounsaturated FA (PIs-MUFA) in all non-invasive cancer cells (n = 12). While 54% of invasive cancer cells (n = 27) also accumulated PIs-MUFA, 46% of invasive cancer cells (n = 23) accumulated the PIs-PUFA, PI(18:0/20:3) and PI(18:0/20:4). The accumulation of PI(18:0/20:3) was associated with higher incidence of lymph node metastasis and activation of the PD-1-related immune checkpoint pathway. Fatty acid-binding protein 7 was identified as a putative molecule controlling PI composition.
CONCLUSIONS
MALDI-IMS identified PI composition associated with invasion and nodal metastasis of breast cancer. The accumulation of PI(18:0/20:3) could affect the PD-1-related immune checkpoint pathway, although its precise mechanism should be further validated.
Identifiants
pubmed: 31819188
doi: 10.1038/s41416-019-0662-8
pii: 10.1038/s41416-019-0662-8
pmc: PMC7051979
doi:
Substances chimiques
Fatty Acids
0
Phosphatidylinositols
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
245-257Subventions
Organisme : Cancer Research UK
Pays : United Kingdom
Références
Schwamborn, K. & Caprioli, R. M. Molecular imaging by mass spectrometry–looking beyond classical histology. Nat. Rev. Cancer 10, 639–646 (2010).
pubmed: 20720571
doi: 10.1038/nrc2917
Harada, T., Yuba-Kubo, A., Sugiura, Y., Zaima, N., Hayasaka, T., Goto-Inoue, N. et al. Visualization of volatile substances in different organelles with an atmospheric-pressure mass microscope. Anal. Chem. 81, 9153–9157 (2009).
pubmed: 19788281
doi: 10.1021/ac901872n
Veselkov, K. A., Mirnezami, R., Strittmatter, N., Goldin, R. D., Kinross, J., Speller, A. V. et al. Chemo-informatic strategy for imaging mass spectrometry-based hyperspectral profiling of lipid signatures in colorectal cancer. Proc. Natl Acad. Sci. USA 111, 1216–1221 (2014).
pubmed: 24398526
pmcid: 3903245
doi: 10.1073/pnas.1310524111
Kriegsmann, J., Kriegsmann, M. & Casadonte, R. MALDI TOF imaging mass spectrometry in clinical pathology: a valuable tool for cancer diagnostics (review). Int. J. Oncol. 46, 893–906 (2015).
pubmed: 25482502
doi: 10.3892/ijo.2014.2788
van Meer, G., Voelker, D. R. & Feigenson, G. W. Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112–124 (2008).
pubmed: 18216768
pmcid: 2642958
doi: 10.1038/nrm2330
Holub, B. J. & Kuksis, A. Structural and metabolic interrelationships among glycerophosphatides of rat liver in vivo. Can. J. Biochem. 49, 1347–1356 (1971).
pubmed: 4334540
doi: 10.1139/o71-195
Baker, R. R. & Thompson, W. Positional distribution and turnover of fatty acids in phosphatidic acid, phosphinositides, phosphatidylcholine and phosphatidylethanolamine in rat brain in vivo. Biochim. Biophys. Acta. 270, 489–503 (1972).
pubmed: 4340991
doi: 10.1016/0005-2760(72)90114-2
Di Paolo, G. & De Camilli, P. Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651–657 (2006).
pubmed: 17035995
doi: 10.1038/nature05185
Gu, Z., Wu, J., Wang, S., Suburu, J., Chen, H., Thomas, M. J. et al. Polyunsaturated fatty acids affect the localization and signaling of PIP3/AKT in prostate cancer cells. Carcinogenesis 34, 1968–1975 (2013).
pubmed: 23633519
pmcid: 3765042
doi: 10.1093/carcin/bgt147
Imae, R., Inoue, T., Kimura, M., Kanamori, T., Tomioka, N. H., Kage-Nakadai, E. et al. Intracellular phospholipase A1 and acyltransferase, which are involved in Caenorhabditis elegans stem cell divisions, determine the sn-1 fatty acyl chain of phosphatidylinositol. Mol. Biol. Cell 21, 3114–3124 (2010).
pubmed: 20668164
pmcid: 2938378
doi: 10.1091/mbc.e10-03-0195
Lee, H. C., Inoue, T., Sasaki, J., Kubo, T., Matsuda, S., Nakasaki, Y. et al. LPIAT1 regulates arachidonic acid content in phosphatidylinositol and is required for cortical lamination in mice. Mol. Biol. Cell 23, 4689–4700 (2012).
pubmed: 23097495
pmcid: 3521678
doi: 10.1091/mbc.e12-09-0673
Lee, H. C., Kubo, T., Kono, N., Kage-Nakadai, E., Gengyo-Ando, K., Mitani, S. et al. Depletion of mboa-7, an enzyme that incorporates polyunsaturated fatty acids into phosphatidylinositol (PI), impairs PI 3-phosphate signaling in Caenorhabditis elegans. Genes Cells 17, 748–757 (2012).
pubmed: 22862955
doi: 10.1111/j.1365-2443.2012.01624.x
Naguib, A., Bencze, G., Engle, D. D., Chio, II, Herzka, T., Watrud, K. et al. p53 mutations change phosphatidylinositol acyl chain composition. Cell Rep. 10, 8–19 (2015).
pubmed: 25543136
doi: 10.1016/j.celrep.2014.12.010
Network TCGA. Comprehensive molecular portraits of human breast tumours. Nature 490, 61–70 (2012).
doi: 10.1038/nature11412
Kawashima, M., Iwamoto, N., Kawaguchi-Sakita, N., Sugimoto, M., Ueno, T., Mikami, Y. et al. High-resolution imaging mass spectrometry reveals detailed spatial distribution of phosphatidylinositols in human breast cancer. Cancer Sci. 104, 1372–1379 (2013).
pubmed: 23837649
pmcid: 7656533
doi: 10.1111/cas.12229
Goto, T., Terada, N., Inoue, T., Nakayama, K., Okada, Y., Yoshikawa, T. et al. The expression profile of phosphatidylinositol in high spatial resolution imaging mass spectrometry as a potential biomarker for prostate cancer. PLoS One 9, e90242 (2014).
pubmed: 24587297
pmcid: 3938652
doi: 10.1371/journal.pone.0090242
Hiraide, T., Ikegami, K., Sakaguchi, T., Morita, Y., Hayasaka, T., Masaki, N. et al. Accumulation of arachidonic acid-containing phosphatidylinositol at the outer edge of colorectal cancer. Sci. Rep. 6, 29935 (2016).
pubmed: 27435310
pmcid: 4951683
doi: 10.1038/srep29935
Hammond, M. E. H., Hayes, D. F., Dowsett, M., Allred, D. C., Hagerty, K. L., Badve, S. et al. American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J. Clin. Oncol. 28, 2784–2795 (2010).
pubmed: 20404251
pmcid: 2881855
doi: 10.1200/JCO.2009.25.6529
Salgado, R., Denkert, C., Demaria, S., Sirtaine, N., Klauschen, F., Pruneri, G. et al. The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Ann. Oncol. 26, 259–271 (2015).
pubmed: 25214542
doi: 10.1093/annonc/mdu450
Hubbell, E., Liu, W. M. & Mei, R. Robust estimators for expression analysis. Bioinformatics 18, 1585–1592 (2002).
pubmed: 12490442
doi: 10.1093/bioinformatics/18.12.1585
Cerami, E., Gao, J., Dogrusoz, U., Gross, B. E., Sumer, S. O., Aksoy, B. A. et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2, 401–404 (2012).
pubmed: 22588877
doi: 10.1158/2159-8290.CD-12-0095
Curtis, C., Shah, S. P., Chin, S.-F., Turashvili, G., Rueda, O. M., Dunning, M. J. et al. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 486, 346–352 (2012).
pubmed: 22522925
pmcid: 3440846
doi: 10.1038/nature10983
Gao, J., Aksoy, B. A., Dogrusoz, U., Dresdner, G., Gross, B., Sumer, S. O. et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci. Signal. 6, pl1 (2013).
pubmed: 23550210
pmcid: 4160307
doi: 10.1126/scisignal.2004088
Pereira, B., Chin, S.-F., Rueda, O. M., Vollan, H.-K. M., Provenzano, E., Bardwell, H. A. et al. The somatic mutation profiles of 2,433 breast cancers refine their genomic and transcriptomic landscapes. Nat. Commun. 7, 11479 (2016).
pubmed: 27161491
pmcid: 4866047
doi: 10.1038/ncomms11479
Picelli, S., Faridani, O. R., Bjorklund, A. K., Winberg, G., Sagasser, S. & Sandberg, R. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).
pubmed: 24385147
doi: 10.1038/nprot.2014.006
Kuch, E. M., Vellaramkalayil, R., Zhang, I., Lehnen, D., Brugger, B., Sreemmel, W. et al. Differentially localized acyl-CoA synthetase 4 isoenzymes mediate the metabolic channeling of fatty acids towards phosphatidylinositol. Biochim. Biophys. Acta. 1841, 227–239 (2014).
pubmed: 24201376
doi: 10.1016/j.bbalip.2013.10.018
Menendez, J. A. & Lupu, R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis. Nat. Rev. Cancer 7, 763–777 (2007).
pubmed: 17882277
doi: 10.1038/nrc2222
Ackerman, D. & Simon, M. C. Hypoxia, lipids, and cancer: surviving the harsh tumor microenvironment. Trends Cell Biol. 24, 472–478 (2014).
pubmed: 24985940
pmcid: 4112153
doi: 10.1016/j.tcb.2014.06.001
Furuta, E., Pai, S. K., Zhan, R., Bandyopadhyay, S., Watabe, M., Mo, Y. Y. et al. Fatty acid synthase gene is up-regulated by hypoxia via activation of Akt and sterol regulatory element binding protein-1. Cancer Res. 68, 1003–1011 (2008).
pubmed: 18281474
doi: 10.1158/0008-5472.CAN-07-2489
Menendez, J. A. & Lupu, R. Fatty acid synthase (FASN) as a therapeutic target in breast cancer. Expert Opin. Ther. Tar. 21, 1001–1016 (2017).
doi: 10.1080/14728222.2017.1381087
Nielsen, M. M. B., Lambertsen, K. L., Clausen, B. H., Meyer, M., Bhandari, D. R., Larsen, S. T. et al. Mass spectrometry imaging of biomarker lipids for phagocytosis and signalling during focal cerebral ischaemia. Sci. Rep. 6, 39571 (2016).
pubmed: 28004822
pmcid: 5177920
doi: 10.1038/srep39571
Sparvero, L. J., Amoscato, A. A., Fink, A. B., Anthonymuthu, T., New, L. A., Kochanek, P. M. et al. Imaging mass spectrometry reveals loss of polyunsaturated cardiolipins in the cortical contusion, hippocampus, and thalamus after traumatic brain injury. J. Neurochem. 139, 659–675 (2016).
pubmed: 27591733
pmcid: 5323070
doi: 10.1111/jnc.13840
Wildburger, N. C., Wood, P. L., Gumin, J., Lichti, C. F., Emmett, M. R., Lang, F. F. et al. ESI–MS/MS and MALDI-IMS localization reveal alterations in phosphatidic acid, diacylglycerol, and DHA in glioma stem cell xenografts. J. Proteome Res. 14, 2511–2519 (2015).
pubmed: 25880480
pmcid: 4912016
doi: 10.1021/acs.jproteome.5b00076
Sugiura, Y., Konishi, Y., Zaima, N., Kajihara, S., Nakanishi, H., Taguchi, R. et al. Visualization of the cell-selective distribution of PUFA-containing phosphatidylcholines in mouse brain by imaging mass spectrometry. J. Lipid Res. 50, 1776–1788 (2009).
pubmed: 19417221
pmcid: 2724791
doi: 10.1194/jlr.M900047-JLR200
Furuhashi, M. & Hotamisligil, G. S. Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets. Nat. Rev. Drug Discov. 7, 489–503 (2008).
pubmed: 18511927
pmcid: 2821027
doi: 10.1038/nrd2589
Smathers, R. L. & Petersen, D. R. The human fatty acid-binding protein family: evolutionary divergences and functions. Hum. Genomics 5, 170–191 (2011).
pubmed: 21504868
pmcid: 3500171
doi: 10.1186/1479-7364-5-3-170
Belkaid, A., Ouellette, R. J. & Surette, M. E. 17beta-estradiol-induced ACSL4 protein expression promotes an invasive phenotype in estrogen receptor positive mammary carcinoma cells. Carcinogenesis 38, 402–410 (2017).
pubmed: 28334272
doi: 10.1093/carcin/bgx020
Miki, Y., Kidoguchi, Y., Sato, M., Taketomi, Y., Taya, C., Muramatsu, K. et al. Dual roles of group IID phospholipase A2 in inflammation and cancer. J. Biol. Chem. 291, 15588–15601 (2016).
pubmed: 27226632
pmcid: 4957044
doi: 10.1074/jbc.M116.734624
Xu, Y., Yang, X., Gao, D., Yang, L., Miskimins, K. & Qian, S. Y. Dihomo-gamma-linolenic acid inhibits xenograft tumor growth in mice bearing shRNA-transfected HCA-7 cells targeting delta-5-desaturase. BMC Cancer 18, 1268 (2018).
pubmed: 30567534
pmcid: 6299961
doi: 10.1186/s12885-018-5185-9
Yang, X., Xu, Y., Wang, T., Shu, D., Guo, P., Miskimins, K. et al. Inhibition of cancer migration and invasion by knocking down delta-5-desaturase in COX-2 overexpressed cancer cells. Redox Biol. 11, 653–662 (2017).
pubmed: 28157665
pmcid: 5288391
doi: 10.1016/j.redox.2017.01.016
Dowds, C. M., Kornell, S. C., Blumberg, R. S. & Zeissig, S. Lipid antigens in immunity. Biol. Chem. 395, 61–81 (2014).
pubmed: 23999493
pmcid: 4128234
doi: 10.1515/hsz-2013-0220
Tatituri, R. V., Watts, G. F., Bhowruth, V., Barton, N., Rothchild, A., Hsu, F. F. et al. Recognition of microbial and mammalian phospholipid antigens by NKT cells with diverse TCRs. Proc. Natl Acad. Sci. USA 110, 1827–1832 (2013).
pubmed: 23307809
pmcid: 3562825
doi: 10.1073/pnas.1220601110
Zajonc, D. M. & Kronenberg, M. CD1 mediated T cell recognition of glycolipids. Curr. Opin. Struct. Biol. 17, 521–529 (2007).
pubmed: 17951048
pmcid: 2121122
doi: 10.1016/j.sbi.2007.09.010
Chow, S. C., Sisfontes, L., Jondal, M. & Bjorkhem, I. Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase. Biochim. Biophys. Acta. 1092, 358–366 (1991).
pubmed: 1646642
doi: 10.1016/S0167-4889(97)90013-6
Sabatier, R., Finetti, P., Mamessier, E., Adelaide, J., Chaffanet, M., Ali, H. R. et al. Prognostic and predictive value of PDL1 expression in breast cancer. Oncotarget. 6, 5449–5464 (2015).
pubmed: 25669979
doi: 10.18632/oncotarget.3216
Schalper, K. A., Velcheti, V., Carvajal, D., Wimberly, H., Brown, J., Pusztai, L. et al. In situ tumor PD-L1 mRNA expression is associated with increased TILs and better outcome in breast carcinomas. Clin. Cancer Res. 20, 2773–2782 (2014).
pubmed: 24647569
doi: 10.1158/1078-0432.CCR-13-2702
Doria, M. L., Cotrim, C. Z., Simoes, C., Macedo, B., Domingues, P., Domingues, M. R. et al. Lipidomic analysis of phospholipids from human mammary epithelial and breast cancer cell lines. J. Cell Physiol. 228, 457–468 (2013).
pubmed: 22767159
doi: 10.1002/jcp.24152
Doria, M. L., Ribeiro, A. S., Wang, J., Cotrim, C. Z., Domingues, P., Williams, C. et al. Fatty acid and phospholipid biosynthetic pathways are regulated throughout mammary epithelial cell differentiation and correlate to breast cancer survival. FASEB J. 28, 4247–4264 (2014).
pubmed: 24970396
doi: 10.1096/fj.14-249672
Hilvo, M., Denkert, C., Lehtinen, L., Muller, B., Brockmoller, S., Seppanen-Laakso, T. et al. Novel theranostic opportunities offered by characterization of altered membrane lipid metabolism in breast cancer progression. Cancer Res. 71, 3236–3245 (2011).
pubmed: 21415164
doi: 10.1158/0008-5472.CAN-10-3894
Abbassi-Ghadi, N., Golf, O., Kumar, S., Antonowicz, S., McKenzie, J. S., Huang, J. et al. Imaging of esophageal lymph node metastases by desorption electrospray ionization mass spectrometry. Cancer Res. 76, 5647–5656 (2016).
pubmed: 27364550
doi: 10.1158/0008-5472.CAN-16-0699
Ide, Y., Waki, M., Hayasaka, T., Nishio, T., Morita, Y., Tanaka, H. et al. Human breast cancer tissues contain abundant phosphatidylcholine(36ratio1) with high stearoyl-CoA desaturase-1 expression. PLoS One 8, e61204 (2013).
pubmed: 23613812
pmcid: 3629004
doi: 10.1371/journal.pone.0061204
Mao, X., He, J., Li, T., Lu, Z., Sun, J., Meng, Y. et al. Application of imaging mass spectrometry for the molecular diagnosis of human breast tumors. Sci. Rep. 6, 21043 (2016).
pubmed: 26868906
pmcid: 4751527
doi: 10.1038/srep21043
Guffy, M. M., North, J. A. & Burns, C. P. Effect of cellular fatty acid alteration on adriamycin sensitivity in cultured L1210 murine leukemia cells. Cancer Res. 44, 1863–1866 (1984).
pubmed: 6231987
Holleran, W. M., DeGregorio, M. W., Ganapathi, R., Wilbur, J. R. & Macher, B. A. Characterization of cellular lipids in doxorubicin-sensitive and -resistant P388 mouse leukemia cells. Cancer Chemother. Pharmacol. 17, 11–15 (1986).
pubmed: 3698172
doi: 10.1007/BF00299859
Ramu, A., Glaubiger, D. & Weintraub, H. Differences in lipid composition of doxorubicin-sensitive and -resistant P388 cells. Cancer Treat. Rep. 68, 637–641 (1984).
pubmed: 6713419
Rivel, T., Ramseyer, C. & Yesylevskyy, S. The asymmetry of plasma membranes and their cholesterol content influence the uptake of cisplatin. Sci. Rep. 9, 5627 (2019).
pubmed: 30948733
pmcid: 6449338
doi: 10.1038/s41598-019-41903-w
Escriba, P. V., Busquets, X., Inokuchi, J., Balogh, G., Torok, Z., Horvath, I. et al. Membrane lipid therapy: modulation of the cell membrane composition and structure as a molecular base for drug discovery and new disease treatment. Prog. Lipid Res. 59, 38–53 (2015).
pubmed: 25969421
doi: 10.1016/j.plipres.2015.04.003
Peck, B. & Schulze, A. Lipid desaturation—the next step in targeting lipogenesis in cancer? FEBS J. 283, 2767–2778 (2016).
pubmed: 26881388
doi: 10.1111/febs.13681
Jiralerspong, S., Palla, S. L., Giordano, S. H., Meric-Bernstam, F., Liedtke, C., Barnett, C. M. et al. Metformin and pathologic complete responses to neoadjuvant chemotherapy in diabetic patients with breast cancer. J. Clin. Oncol. 27, 3297–3302 (2009).
pubmed: 19487376
pmcid: 2736070
doi: 10.1200/JCO.2009.19.6410
Lord, S. R., Cheng, W. C., Liu, D., Gaude, E., Haider, S., Metcalf, T. et al. Integrated pharmacodynamic analysis identifies two metabolic adaption pathways to metformin in breast cancer. Cell Metab. 28, 679–688.e4 (2018).
pubmed: 30244975
pmcid: 6224605
doi: 10.1016/j.cmet.2018.08.021
Wu, Q., Comi, T. J., Li, B., Rubakhin, S. S. & Sweedler, J. V. On-tissue derivatization via electrospray deposition for matrix-assisted laser desorption/ionization mass spectrometry imaging of endogenous fatty acids in rat brain tissues. Anal. Chem. 88, 5988–5995 (2016).
pubmed: 27181709
pmcid: 4899806
doi: 10.1021/acs.analchem.6b01021
Yoshimura, K., Chen, L. C., Yu, Z., Hiraoka, K. & Takeda, S. Real-time analysis of living animals by electrospray ionization mass spectrometry. Anal. Biochem. 417, 195–201 (2011).
pubmed: 21741944
doi: 10.1016/j.ab.2011.06.020