Title: Ceramide Synthase Inhibition by Fumonisin B1 Causes Accumulation of 1-Deoxysphinganine
Abstract: Fumonisin B1 (FB1) is a mycotoxin that inhibits ceramide synthases (CerS) and causes kidney and liver toxicity and other disease. Inhibition of CerS by FB1 increases sphinganine (Sa), Sa 1-phosphate, and a previously unidentified metabolite. Analysis of the latter by quadrupole-time-of-flight mass spectrometry assigned an m/z = 286.3123 in positive ionization mode, consistent with the molecular formula for deoxysphinganine (C18H40NO). Comparison with a synthetic standard using liquid chromatography, electrospray tandem mass spectrometry identified the metabolite as 1-deoxysphinganine (1-deoxySa) based on LC mobility and production of a distinctive fragment ion (m/z 44, CH3CH=NH +2) upon collision-induced dissociation. This novel sphingoid base arises from condensation of alanine with palmitoyl-CoA via serine palmitoyltransferase (SPT), as indicated by incorporation of l-[U-13C]alanine into 1-deoxySa by Vero cells; inhibition of its production in LLC-PK1 cells by myriocin, an SPT inhibitor; and the absence of incorporation of [U-13C]palmitate into 1-[13C]deoxySa in LY-B cells, which lack SPT activity. LY-B-LCB1 cells, in which SPT has been restored by stable transfection, however, produce large amounts of 1-[13C]deoxySa. 1-DeoxySa was elevated in FB1-treated cells and mouse liver and kidney, and its cytotoxicity was greater than or equal to that of Sa for LLC-PK1 and DU-145 cells. Therefore, this compound is likely to contribute to pathologies associated with fumonisins. In the absence of FB1, substantial amounts of 1-deoxySa are made and acylated to N-acyl-1-deoxySa (i.e. 1-deoxydihydroceramides). Thus, these compounds are an underappreciated category of bioactive sphingoid bases and "ceramides" that might play important roles in cell regulation. Fumonisin B1 (FB1) is a mycotoxin that inhibits ceramide synthases (CerS) and causes kidney and liver toxicity and other disease. Inhibition of CerS by FB1 increases sphinganine (Sa), Sa 1-phosphate, and a previously unidentified metabolite. Analysis of the latter by quadrupole-time-of-flight mass spectrometry assigned an m/z = 286.3123 in positive ionization mode, consistent with the molecular formula for deoxysphinganine (C18H40NO). Comparison with a synthetic standard using liquid chromatography, electrospray tandem mass spectrometry identified the metabolite as 1-deoxysphinganine (1-deoxySa) based on LC mobility and production of a distinctive fragment ion (m/z 44, CH3CH=NH +2) upon collision-induced dissociation. This novel sphingoid base arises from condensation of alanine with palmitoyl-CoA via serine palmitoyltransferase (SPT), as indicated by incorporation of l-[U-13C]alanine into 1-deoxySa by Vero cells; inhibition of its production in LLC-PK1 cells by myriocin, an SPT inhibitor; and the absence of incorporation of [U-13C]palmitate into 1-[13C]deoxySa in LY-B cells, which lack SPT activity. LY-B-LCB1 cells, in which SPT has been restored by stable transfection, however, produce large amounts of 1-[13C]deoxySa. 1-DeoxySa was elevated in FB1-treated cells and mouse liver and kidney, and its cytotoxicity was greater than or equal to that of Sa for LLC-PK1 and DU-145 cells. Therefore, this compound is likely to contribute to pathologies associated with fumonisins. In the absence of FB1, substantial amounts of 1-deoxySa are made and acylated to N-acyl-1-deoxySa (i.e. 1-deoxydihydroceramides). Thus, these compounds are an underappreciated category of bioactive sphingoid bases and "ceramides" that might play important roles in cell regulation. Fumonisins (FB) 2The abbreviations used are: FB, fumonisins; 1-deoxySa, 1-deoxysphinganine; Cer, ceramide; CerS, ceramide synthases; d18:0, sphinganine; DHCer, dihydroceramide; 1-deoxyDHCer, 1-deoxydihydroceramide; ESI, electrospray ionization; HSN1, human sensory neuropathy type 1; LC ESI-MS/MS, liquid chromatography, electrospray tandem mass spectrometry; m18:0, 1-deoxysphinganine; OPA, ortho-phthalaldehyde; PBS, phosphate-buffered saline; Sa, sphinganine; Sa1P, sphinganine 1-phosphate; So, sphingosine; S1P, sphingosine 1-phosphate; SPT, serine palmitoyltransferase; HPLC, high performance LC; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; BSA, bovine serum albumin. cause diseases of horses, swine, and other farm animals and are regarded to be potential risk factors for human esophageal cancer (1Marasas W.F.O. Environ. Health Perspect. 2001; 109: 239-243Crossref PubMed Scopus (447) Google Scholar) and, more recently, birth defects (2Marasas W.F.O. Riley R.T. Hendricks K.A. Stevens V.L. Sadler T.W. Gelineau-van Waes J.B. Missmer S.A. Cabrera J. Torres O. Gelderblom W.C.A. Allgood J. Martinez C. Maddox J. Miller J.D. Starr L. Sullards M.C. Roman A.V. Voss K.A. Wang E. Merrill Jr., A.H. J. Nutr. 2004; 134: 711-716Crossref PubMed Scopus (472) Google Scholar). Studies of this family of mycotoxins, and particularly of the highly prevalent subspecies fumonisin B1 (FB1) (reviewed in Refs. 1Marasas W.F.O. Environ. Health Perspect. 2001; 109: 239-243Crossref PubMed Scopus (447) Google Scholar and 2Marasas W.F.O. Riley R.T. Hendricks K.A. Stevens V.L. Sadler T.W. Gelineau-van Waes J.B. Missmer S.A. Cabrera J. Torres O. Gelderblom W.C.A. Allgood J. Martinez C. Maddox J. Miller J.D. Starr L. Sullards M.C. Roman A.V. Voss K.A. Wang E. Merrill Jr., A.H. J. Nutr. 2004; 134: 711-716Crossref PubMed Scopus (472) Google Scholar), have established that FB1, is both toxic and carcinogenic for laboratory animals, with the liver and kidney being the most sensitive target organs (3Voss K.A. Riley R.T. Norred W.P. Bacon C.W. Meredith F.I. Howard P.C. Plattner R.D. Collins T.F.X. Hansen D.K. Porter J.K. Environ. Health Perspect. 2001; 109: 259-266Crossref PubMed Scopus (151) Google Scholar, 4NTP Technical Report (2002). NIH Publication No. 99-3955Google Scholar). Other FB are also toxic, but their carcinogenicity is unknown. FB are potent inhibitors of ceramide synthase(s) (CerS) (5Wang E. Norred W.P. Bacon C.W. Riley R.T. Merrill Jr., A.H. J. Biol. Chem. 1991; 266: 14486-14490Abstract Full Text PDF PubMed Google Scholar), the enzymes responsible for acylation of sphingoid bases using fatty acyl-CoA for sphingolipid biosynthesis de novo and recycling pathways (6Pewzner-Jung Y. Ben-Dor S. Futerman A.H. J. Biol. Chem. 2006; 281: 25001-25005Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). As a consequence of this inhibition, the substrates sphinganine (Sa) and, usually to a lesser extent, sphingosine (So), accumulate and are often diverted to sphinganine 1-phosphate (Sa1P) and sphingosine 1-phosphate (S1P), respectively (7Riley R.T. Enongene E. Voss K.A. Norred W.P. Meredith F.I. Sharma R.P. Williams L.D. Carlson D.B. Spitsbergen J. Merrill Jr., A.H. Environ. Health Perspect. 2001; 109: 301-308Crossref PubMed Scopus (219) Google Scholar), while the product N-acylsphinganines (dihydroceramides), N-acylsphingosines (ceramides, Cer), and more complex sphingolipids decrease (5Wang E. Norred W.P. Bacon C.W. Riley R.T. Merrill Jr., A.H. J. Biol. Chem. 1991; 266: 14486-14490Abstract Full Text PDF PubMed Google Scholar, 7Riley R.T. Enongene E. Voss K.A. Norred W.P. Meredith F.I. Sharma R.P. Williams L.D. Carlson D.B. Spitsbergen J. Merrill Jr., A.H. Environ. Health Perspect. 2001; 109: 301-308Crossref PubMed Scopus (219) Google Scholar). This disruption of sphingolipid metabolism has been proposed to be responsible for the toxicity, and possibly carcinogenicity, of FB, based on mechanistic studies with cells in culture (5Wang E. Norred W.P. Bacon C.W. Riley R.T. Merrill Jr., A.H. J. Biol. Chem. 1991; 266: 14486-14490Abstract Full Text PDF PubMed Google Scholar, 7Riley R.T. Enongene E. Voss K.A. Norred W.P. Meredith F.I. Sharma R.P. Williams L.D. Carlson D.B. Spitsbergen J. Merrill Jr., A.H. Environ. Health Perspect. 2001; 109: 301-308Crossref PubMed Scopus (219) Google Scholar, 8Yoo H.S. Norred W.P. Wang E. Merrill Jr., A.H. Riley R.T. Toxicol. Appl. Pharmacol. 1992; 114: 9-15Crossref PubMed Scopus (188) Google Scholar, 9Merrill Jr., A.H. Sullards M.C. Wang E. Voss K.A. Riley R.T. Environ. Health Perspect. 2001; 109: 283-289Crossref PubMed Scopus (334) Google Scholar). This has been borne out by a number of animal feeding studies that have correlated the elevation of Sa in blood, urine, liver, and kidney with liver and kidney toxicity (4NTP Technical Report (2002). NIH Publication No. 99-3955Google Scholar, 7Riley R.T. Enongene E. Voss K.A. Norred W.P. Meredith F.I. Sharma R.P. Williams L.D. Carlson D.B. Spitsbergen J. Merrill Jr., A.H. Environ. Health Perspect. 2001; 109: 301-308Crossref PubMed Scopus (219) Google Scholar, 10Delongchamp R.R. Young J.F. Food Additiv. Contam. 2001; 18: 255-261Crossref PubMed Scopus (28) Google Scholar, 11Riley R.T. Voss K.A. Toxicol. Sci. 2006; 92: 335-345Crossref PubMed Scopus (88) Google Scholar). Most of the mechanistic studies have focused on the accumulation of free Sa and other sphingoid bases, because these compounds are highly cytotoxic, although the large number of bioactive metabolites in this pathway make it likely that multiple mediators may participate (7Riley R.T. Enongene E. Voss K.A. Norred W.P. Meredith F.I. Sharma R.P. Williams L.D. Carlson D.B. Spitsbergen J. Merrill Jr., A.H. Environ. Health Perspect. 2001; 109: 301-308Crossref PubMed Scopus (219) Google Scholar, 9Merrill Jr., A.H. Sullards M.C. Wang E. Voss K.A. Riley R.T. Environ. Health Perspect. 2001; 109: 283-289Crossref PubMed Scopus (334) Google Scholar). Nonetheless, inhibition of serine palmitoyltransferase (SPT), the initial enzyme of de novo sphingolipid biosynthesis, reverses the increased apoptosis and altered cell growth induced by FB1 treatment (12Schroeder J.J. Crane H.M. Xia J. Liotta D.C. Merrill Jr., A.H. J. Biol. Chem. 1994; 269: 3475-3481Abstract Full Text PDF PubMed Google Scholar, 13Yoo H.S. Norred W.P. Showker J.L. Riley R.T. Toxicol. Appl. Pharmacol. 1996; 138: 211-218Crossref PubMed Scopus (112) Google Scholar, 14Schmelz E.M. Dombrink-Kurtzman M.A. Roberts P.C. Kozutsumi Y. Kawasaki T. Merrill Jr., A.H. Toxicol. Appl. Pharmacol. 1998; 148: 252-260Crossref PubMed Scopus (147) Google Scholar, 15Riley R.T. Voss K.A. Norred W.P. Bacon C.W. Meredith F.I. Sharma R.P. Environ. Toxicol. Pharmacol. 1999; 7: 109-118Crossref PubMed Scopus (34) Google Scholar, 16Tolleson W.H. Couch L.H. Melchior Jr., W.B. Jenkins G.R. Muskhelishvili M. Muskhelishvili L. McGarrity L.J. Domon O.E. Morris S.M. Howard P.C. Int. J. Oncol. 1999; 14: 833-843PubMed Google Scholar, 17Kim M.S. Lee D.Y. Wang T. Schroeder J.J. Toxicol. Appl. Pharmacol. 2001; 176: 118-126Crossref PubMed Scopus (29) Google Scholar, 18Yu C.H. Lee Y.M. Yum Y.P. Yoo H.S. Arch. Pharmacal. Res. 2001; 24: 136-143Crossref PubMed Scopus (22) Google Scholar, 19He Q. Riley R.T. Sharma R.P. Pharmacol. Toxicol. 2002; 90: 268-277Crossref PubMed Scopus (36) Google Scholar). Therefore, it is likely that these effects of FB1 are due to the accumulation of cytotoxic intermediate(s) rather than depletion of downstream metabolites, because the latter also occurs when SPT is inhibited. In studies of the effects of FB1 on the renal cell line LLC-PK1 (20Riley R.T. Voss K.A. Norred W.P. Sharma R.P. Wang E. Merrill Jr., A.H. Rev. Méd. Vét. 1998; 149: 617-626Google Scholar), 3R. Riley, unpublished observations. we have noted that in addition to the elevation of Sa and So, there is a large increase in an unidentified species that appears to be a sphingoid base, because it is extracted by organic solvents, derivatized with ortho-phthalaldehyde (OPA), and eluted from reverse-phase liquid chromatography (LC) in the sphingoid base region. Herein we report: (i) the isolation and characterization of this novel sphingoid base as 1-deoxysphinganine (1-deoxySa); (ii) that its origin is the utilization of alanine instead of serine by SPT as well as that the N-acyl-derivatives of 1-deoxySa (1-deoxydihydroceramides (1-deoxyDHCer)) are normally found in mammalian cells; (iii) that 1-deoxySa has cytotoxicity comparable to other sphingoid bases elevated by FB1; and (iv) that 1-deoxySa is not only elevated in cells in culture but also in tissues of animals exposed to dietary FB and, therefore, might contribute to diseases caused by these mycotoxins. Materials—Pig kidney epithelial cells (LLC-PK1; CL 101), African green monkey kidney cells (Vero cells; CCL 81), and the human prostate cancer cell line (DU-145, HTB-81™) were from the American Type Culture Collection (ATCC, Manassas, VA). The LY-B and LY-B-LCB1 cells were a generous gift from Ken Hanada (21Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). P53N5-W mice, 5–7-week-old, were purchased from Taconic Farms, Inc. (The P53N5 strain was derived originally from the C57BL/6 mouse strain.) FB1 used in the in vitro studies was prepared and purified (>95% purity) as described in Meredith et al. (22Meredith F.I. Bacon C.W. Plattner R.D. Norred W.P. J. Ag. Food Chem. 1996; 44: 195-198Crossref Scopus (32) Google Scholar) (establishing that the impurities were primarily other FBs and FB dimers) or for the experiments specified were purchased from Biomol (Philadelphia, PA). FB1 used in the mouse feeding study was supplied by Dr. Marc Savard (Agriculture and Agri-Food Canada, Ottawa, ON, Canada). Myriocin was prepared as described in Riley and Plattner (23Riley R.T. Plattner R.D. Methods Enzymol. 2000; 311: 348-360Crossref PubMed Scopus (17) Google Scholar). Free sphingoid bases, sphingoid base 1-phosphates, and the internal standard mixture for sphingolipids (Sphingolipid Mixture II, LM-6005) were purchased from Avanti Polar Lipids (Alabaster, AL) except for d-erythro-C16-sphingosine, which was from Matreya (Pleasant Gap, PA). 1-DeoxySa was initially synthesized by minor modifications of a recently developed method for the synthesis of So (24Yang H. Liebeskind L.S. Org. Lett. 2007; 9: 2993-2995Crossref PubMed Scopus (53) Google Scholar) and has subsequently become available commercially from Avanti Polar Lipids. Dulbecco's phosphate-buffered saline (PBS), Hanks' balanced salt solution, and Minimal Essential Medium (MEM, Invitrogen catalogue no. 61100-061) were obtained from GIBCO Invitrogen. Eagles Minimal Essential Medium, Dulbecco's modified Eagle's medium (DMEM), Ham's F12, fetal calf serum (FCS), and trypsin/EDTA were obtained from ATCC. l-[U-13C]Alanine, l-[U-13C]serine, and [U-13C]palmitic acid (all ≥98% purity) were purchased from Cambridge Isotope Laboratories, Inc. Preparative C18 bulk packing material (55–105 μm) was purchased from Waters (Waters Corp., Part No. WAT010001). Acetonitrile (Burdick & Jackson) and water (J. T. Baker) were high performance LC (HPLC) grade and formic acid (>95%; Sigma-Aldrich) and all other reagents and chemicals were of analytical grade or better and were from various commercial suppliers. Cell Culture—LLC-PK1 cells were grown and maintained in 25-cm2 culture flasks containing DMEM/Ham's F12 (1:1) with 5% FCS at 37 °C and 5% CO2. For experiments using confluent cultures of LLC-PK1 cells, the cells were seeded at ∼15,000 to 30,000 viable cells/cm2 in 8-cm2 dishes and then allowed to attach and grow to at least 90% confluence (3–5 days) prior to addition of test agents added directly to the media without addition of fresh growth medium. For experiments measuring effects on rapidly proliferating cells, the LLC-PK1 cells were seeded at ∼2,500 viable cells/cm2 in 8-cm2 dishes and allowed to attach and grow for 2 to 3 days (until ∼30% confluent) prior to addition of fresh growth medium containing the various test agents. The seeding density was chosen so that cell number in control cultures increased linearly over the entire experimental time course. Vero cells were grown and maintained in 25-cm2 culture flasks containing Eagles MEM with 10% FCS at 37 °C and 5% CO2. All experiments with Vero cells used rapidly proliferating cells that had been subcultured from confluent cultures at a 1:36 split ratio into 8-cm2 dishes. This split ratio resulted in cultures that were ∼30% confluent after 24 h. The DU-145 cells were grown in Eagles MEM and 10% FCS at 37 °C and 5% CO2 as described in the technical literature from the ATCC. The LY-B and LY-B-LCB1 cells were cultured in medium containing DMEM/Ham's F12 (1:1) with 5% FCS at 37 °C and 5% CO2, essentially as described in Ref. 21Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar. After treatments, cells were washed twice with ice-cold PBS, the dishes placed on ice, and cells removed from the surface of the dishes using a rubber scraper. The cells detached by scraping were collected in polypropylene tubes and pelleted by centrifuging at 4 °C. The PBS was removed, and the cells frozen at –20 °C. Except where noted, cell extracts were normalized by protein amounts, which were determined by the Lowry method (25Lowry O.H. Rosenbrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) and a commercially available kit (Pierce® BCA Protein Assay Kit, Product No. 23225, Thermo Scientific Pierce) using bovine serum albumin (BSA) as the standard. Sphingolipid Extraction and Analysis—A number of independent methods were used to characterize the sphingolipids to provide confirmation of the findings, and the specific method is indicated in the text and figure legends: 1) Free sphingoid bases were extracted from the cells and quantified by high performance LC (HPLC) and fluorescent detection of the OPA derivatives using C20-sphinganine (C20 Sa) as an internal standard according to Riley et al. (26Riley R.T. Norred W.P. Wang E. Merrill Jr., A.H. Nat. Tox. 1999; 7: 407-414Crossref PubMed Scopus (33) Google Scholar). 2) Free sphingoid bases and sphingoid base 1-phosphates were also analyzed (in experiments with proliferating and confluent cultures of LLC-PK1 cells, Vero cells, and homogenates of mouse liver and kidney) by LC tandem linear-ion trap electrospray ionization mass spectrometry (LC ESI-MS/MS) using the method of Zitomer et al. (27Zitomer N.C. Glenn A.E. Bacon C.W. Riley R.T. Anal. Bioanal. Chem. 2008; 391: 2257-2263Crossref PubMed Scopus (35) Google Scholar) except that instead of extracting pulverized freeze-dried tissues, either fresh liver or kidney homogenates (10–20 mg/100 μl phosphate buffer) or cultured cells (50–100 μg protein) were extracted with C16-So and C17-S1P as internal standards. 3) Free sphingoid bases and N-acyl-derivatives were analyzed by minor modifications of published methods (28Merrill Jr., A.H. Sullards M.C. Allegood J.C. Kelly S. Wang E. Methods. 2005; 36: 207-224Crossref PubMed Scopus (473) Google Scholar) to detect compounds with and without the 1-hydroxyl group based on their resolution by LC. For 1-deoxySa, the precursor ion m/z 286.4 and product ion m/z 268.4 (-H2O) in positive ionization mode were followed. (Note: these overlap with ions from other sphingoid bases, such as d17:1; however, these compounds are resolved by LC as described below.) For N-acyl-1-deoxySa, the cell and tissue extracts were first scanned for precursors for m/z 268.4 to identify which N-acyl-derivatives were present. Then they were analyzed by LC ESI-MS/MS using multiple reaction monitoring with Q1 and Q3 set to pass the following precursor and product ions for the designated N-acyl-1-deoxySa. The abbreviations connote the sphingoid base, m18:0, and fatty acid chain length and number of double bonds using standard nomenclature (28Merrill Jr., A.H. Sullards M.C. Allegood J.C. Kelly S. Wang E. Methods. 2005; 36: 207-224Crossref PubMed Scopus (473) Google Scholar): 524.7/268.4 (m18:0/C16:0), 552.7/268.4 (m18:0/C18:0), 580.7/268.4 (m18:0/C20:0), 608.8/268.4 (m18:0/C22:0), 634.9/268.4 (m18:0/C24:1), 636.9/268.4 (m18:0/C24:0), 662.9/268.4 (m18:0/C26:1), and 664.9/268.4 (m18:0/C26:0). The LC ESI-MS/MS of the free sphingoid bases was conducted using reverse phase LC (Supelco 2.1 × 50 mm Discovery C18 column, Sigma) and a binary solvent system at a flow rate of 0.6 ml/min delivered by a Shimadzu LC-10 AD VP binary pump system coupled to an ABI 4000 QTrap (Applied Biosystems, Foster City, CA). The binary system began with equilibration of the column with a solvent mixture of 60% mobile phase A (CH3OH/H2O/HCOOH, 58/41/1, v/v/v, with 5 mm ammonium formate) and 40% mobile phase B (CH3OH/HCOOH, 99/1, v/v, with 5 mm ammonium formate), sample injection (typically in 50 μl of the same mixture), elution with this mixture for 1.3 min followed by a linear gradient to 100% B over 2.8 min, and then a column wash with 100% B for 0.5 min followed by a wash and re-equilibration with the original A/B mixture before the next run. The elution times were 2.1 min for C17 So (internal standard), 2.4 min for C18 So, 2.6 min for C18 Sa, and 2.8 min for C18-1-deoxySa, with all having baseline resolution. The N-acyl-derivatives were analyzed by normal phase LC (Supelco 2.1 × 50 mm LC-NH2 column) at a flow rate of 0.75 ml/min and an isocratic solvent system (CH3CN/CH3OH/CH3COOH/CH3(CH2)3OH/H2O, 95/3/1/0.4/0.3, v/v with 5 mm ammonium acetate) delivered by a Perkin Elmer Series 200 MicroPump system coupled to a PE Sciex API 3000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA). Using these LC conditions, the 1-deoxydihydroceramides (1-deoxyDHCer) elute together at 0.3 min and are resolved from ceramides (Cer), which elute at 0.37 min. The settings for the mass spectrometry were optimized for each category of compounds as described previously (28Merrill Jr., A.H. Sullards M.C. Allegood J.C. Kelly S. Wang E. Methods. 2005; 36: 207-224Crossref PubMed Scopus (473) Google Scholar). Quantitation was based on spiking the original extract with the sphingolipid internal standard mixture from Avanti Polar Lipids and comparison of the areas for m18:0 with d17:0 (which were approximately the same based on comparison of d17:0 with synthetic m17:0) and for N-acyl-1-deoxySa, the C12-Cer internal standard. Therefore, although these are close approximations, absolute quantitation will require the availability of MS internal standards for these specific compounds. 4) High resolution accurate mass MS was conducted on an ABI Q-Star (Applied Biosystems, Foster City, CA) using samples in methanol infused via a TriVersa nanomate (Advion Biosciences, Ithaca, NY) using a D-chip at 1.5 kV and a gas pressure of 0.2 psi. A total of 30 scans were averaged. To obtain sufficient quantity of the unidentified sphingoid base for chemical characterization, the free sphingoid bases were partially purified from thirty 75-cm2 flasks of confluent cultures of LLC-PK1 cells that had been treated with 35 μm FB1 for 120 h. The cells were scraped and pooled in a single glass tube, and the free sphingoid bases were extracted and treated with 0.1 m KOH in methanol to decrease interference from glycerolipids as described by Riley et al. (26Riley R.T. Norred W.P. Wang E. Merrill Jr., A.H. Nat. Tox. 1999; 7: 407-414Crossref PubMed Scopus (33) Google Scholar). The extracted sphingoid bases were dissolved in a small volume of acetonitrile:H2O:formic acid (49.5:49.5:1, v/v/v) and loaded on a minicolumn of preparative C18 packing material (5 mm × 60 mm) equilibrated with the same mobile phase. Free sphingoid bases were eluted by increasing the percentage of acetonitrile, and the fractions that were enriched in the unidentified sphingoid base were identified by LC linear ion trap-ESI-MS/MS, pooled, dried under nitrogen, and stored at –80 °C for later analysis. Analysis of Stable Isotope-labeled Sphingoid Bases using l-[U-13C]Alanine and l-[U-13C]Serine—Vero cells at ∼30% confluence in 8-cm2 culture plates were incubated in MEM (Invitrogen 61100-061 without serine or alanine) with 2% FCS and 7 amino acid supplements (with 16 dishes per supplement): (i) 100 μm l-serine (natural abundance 13C) + 100 μm l-alanine (natural abundance 13C), (ii) 100 μm l-serine + 100 μm l-[U-13C]alanine, (iii) 100 μm l-serine + 300 μm l-[U-13C]alanine, (iv) 100 μm l-serine + 500 μm l-[U-13C]alanine, (v) 100 μm l-[U-13C]serine + 100 μm l-alanine, (vi) 300 μm l-[U-13C]serine + 100 μm l-alanine, and (vii) 500 μm l-[U-13C]serine + 100 μm l-alanine. After 24 h in culture, half of the dishes in each group were administered sterile FB1 for a final concentration of 35 μm. After 48 h of additional incubation, half of the dishes in each group were used for sphingolipid analysis and half for protein assays. This experiment was repeated twice. Analysis of the sphingolipids in the extracts included m/z for the 12C-labeled products and the [13C] masses of relevant compounds (mass of [12C] parent ion + 2 mass units resulting from incorporation of 2 carbons from the l-[U-13C]amino acid with the third 13C-labeled carbon lost as 13CO2). Analysis of 1-DeoxySa and Metabolites in LY-B and LY-B-LCB1 Cells—These cell lines were plated in 100-mm culture dishes to ∼50% confluence, and then new medium (a 1:1 mixture of DMEM and Ham's F12 media with 10% FCS) containing 0.1 mm [U-13C]palmitic acid bound to fatty acid-free BSA (as a 1:1 molar complex) was added. After 24 h, the lipids were extracted as described previously (28Merrill Jr., A.H. Sullards M.C. Allegood J.C. Kelly S. Wang E. Methods. 2005; 36: 207-224Crossref PubMed Scopus (473) Google Scholar) and analyzed for the 12C- and 13C-labeled sphingolipids (with the latter having a M+16 or M+32 m/z offset from the 12C-species) using LC ESI-MS/MS as described above. Effect of Sa and 1-DeoxySa on Cell Growth, Accumulation of Sphingoid Bases, Sphingoid Base 1-Phosphates, Cer, and 1-DeoxyDHCer—As described previously (8Yoo H.S. Norred W.P. Wang E. Merrill Jr., A.H. Riley R.T. Toxicol. Appl. Pharmacol. 1992; 114: 9-15Crossref PubMed Scopus (188) Google Scholar, 13Yoo H.S. Norred W.P. Showker J.L. Riley R.T. Toxicol. Appl. Pharmacol. 1996; 138: 211-218Crossref PubMed Scopus (112) Google Scholar), LLC-PK1 cells were grown in 8-cm2 dishes to ∼30% confluence. Then Sa or 1-deoxySa was added as the 1:1 (mol/mol) complex with fatty acid-free BSA with or without addition of FB1 (dissolved in water) at the concentrations described in the text. After 48 h, cell protein was measured (25Lowry O.H. Rosenbrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) for the attached cells, which we have determined to be linearly related to cell number [μg protein = 1.63 + 3.29 × (viable cells × 10–4), r2 = 0.96, n = 53] (8Yoo H.S. Norred W.P. Wang E. Merrill Jr., A.H. Riley R.T. Toxicol. Appl. Pharmacol. 1992; 114: 9-15Crossref PubMed Scopus (188) Google Scholar). All experiments were conducted with DMEM/Ham's F12 plus 5% FCS. The effect of treatments on the detachment of cells was determined by collecting the medium and pelleting the detached cells for a separate analysis of the protein amounts. In earlier studies, we have shown that both FB1 and free Sa inhibit cell growth and increase the number of detached cells, which are dead, based on uptake of trypan blue and lactate dehydrogenase release (8Yoo H.S. Norred W.P. Wang E. Merrill Jr., A.H. Riley R.T. Toxicol. Appl. Pharmacol. 1992; 114: 9-15Crossref PubMed Scopus (188) Google Scholar, 13Yoo H.S. Norred W.P. Showker J.L. Riley R.T. Toxicol. Appl. Pharmacol. 1996; 138: 211-218Crossref PubMed Scopus (112) Google Scholar, 15Riley R.T. Voss K.A. Norred W.P. Bacon C.W. Meredith F.I. Sharma R.P. Environ. Toxicol. Pharmacol. 1999; 7: 109-118Crossref PubMed Scopus (34) Google Scholar). A duplicate set of dishes (n = 3/treatment) was collected for determining changes in endogenous sphingoid bases, sphingoid base 1-phosphates, Cer, and 1-deoxyDHCer by LC-ESI-MS/MS as described previously. The effects of 1-deoxySa and Sa on DU-145 cells were examined by culturing the cells to ∼25–50% confluence in 24-well dishes, addition of the sphingoid base as a 1:1 (mol:mol) complex with fatty acid-depleted BSA (sterilized by filtration), incubation for 24 h, and then assessment of cell viability using the WST-1 Cell Proliferation Reagent (Roche Applied Science) following the manufacturer's instructions. Analysis of 1-DeoxySa in Mice Fed FB1—Upon receipt, mice were housed individually under conditions meeting the requirements of the Canadian Council for Animal Care. Similar to the FB feeding protocols described by Gelderblom et al. (29Gelderblom W.C.A. Cawood M.E. Snyman S.D. Marasas W.F.O. Carcinogenesis. 1994; 15: 209-214Crossref PubMed Scopus (111) Google Scholar), male mice (n = 10) received a modified AIN 76A diet supplemented with 0–50 mg FB1/kg for 26 weeks, and then were killed under isoflurane anesthesia by cardiac puncture. Liver and kidney tissues were removed as quickly as possible, flash-frozen in liquid N2, and stored at –80 °C until used for sphingolipid analysis. FB1 Elevates Sa and a Mystery Peak in Cells—Shown in Fig. 1 are representative reverse phase HPLC profiles of the amounts of free sphingoid bases detected as the OPA derivatives from LLC-PK1 cells that have or have not been exposed to 50 μm FB1. As has been seen before (20Riley R.T. Voss K.A. Norred W.P. Sharma R.P. Wang E. Merrill Jr., A.H. Rev. Méd. Vét. 1998; 149: 617-626Google Scholar), FB1 treatment increased the amount of Sa by severalfold within 6 h, and by orders of magnitude after 48 h. In addition, a new and unidentified peak with an elution time of 12.4 min is noticeable after 6 h of FB1 treatment, and by 48 h, the area under the curve for this "mystery peak" is nearly half of that for Sa. Several additional peaks can be seen in the extracts from the cells treated with FB1, but these were much smaller and were not examined further. This phenomenon was not limited to LLC-PK1 cells because we have seen this mystery pea