THE JOURNAL OF BIOLO~ZU. CHEMISTRY Vol. 247, No. 4, Issue of February 25, pp. 1001-1011, 1972

Printed in U.S.A.

On the Biosynthesis of Cerebrosides Containing 2-Hydroxy Acids

MASS SPECTROMETRIC EVIDENCE FOR BIOSYNTHESIS VIA THE CERAMIDE PATHWAY*

(Received for publication, July 21,j1971)

SVEN HAMMARSTR~M AND BENGT SAMUELSSON

From the Department of Medical Chemistry, Royal Veterinary College, S-104 05 Stockholm 50, Sweden

SUMMARY

The conversion of deuterium-labeled ceramide to cere- broside has been studied. A mixture of N-(2’+hydroxy [4’,4’,5’,5’-2H4]hexadecanoyl) D-eryfhYO-1,3-dihydroxy-2- amino-4-frans-[4,5-*H2]octadecene and N-(2’-D-hydroxy hexadecanoyl) D-eryfhro-l,3-dihydroxy-2-amino-4-frans-[3- 3Hl]octadecene was incubated with mouse brain microsomes and a UDP-galactose regenerating system. Both galactosyl ceramide and glucosyl ceramide were formed. The ceramide monohexosides were analyzed by gas-liquid chromatography- mass spectrometry as the trimethylsilyl ether derivatives both as intact molecules and after degradation to ceramides. The analysis conclusively showed that both compounds had been synthesized via the ceramide pathway. A third product from the incubations was identified as free sphingosine.

In vitro experiments have indicated two pathways for cerebro- side biosynthesis: (a) formation of psychosine from sphingosine (l-3) followed by N-acylation of the psychosine (4) and (b) N-acylation of sphingosine (5, 6) followed by formation of cerebroside from ceramide (7-11). Similarly, both the psycho- sine (12) and the ceramide (13, 14) pathways have been postu- lated on the basis of in viva experiments. The] conversions of psychosine and ceramide to cerebroside as well as the conversion of sphingosine to psychosine were very low (less than 0.5%) in the in vitro experiments. (Th e conversion of sphingosine to cera- mide was about 3?$.) This fact rendered the interpretation of the results somewhat uncertain. The present report deals with the in vitro conversion of doubly deuterium-labeled ceramides to cerebrosides, using recent techniques for the separation and characterization of molecular species of cerebrosides (15) and of ceramides derived from cerebrosides (16) by gas-liquid chroma- tography-mass spectrometry. In addition to galactosyl cer- amide, glucosyl ceramide, and free sphingosine were formed during the incubations.

Part of this investigation has been published before in pre- liminary form (17).

* This work was supported by grants from the Swedish Natural Science Research Council (Project 9769 K) and from Kungliga Veteriniirhiigskolans Reservationsanslag.

MATERIALS AND METHODS

Chemica&-D-erythro-1 ,3-Dihydroxy-2-amino -4 - truns-octade- cene (sphingosine) was isolated from beef lung lipids as has been described before (18). It was 87% pure and also contained LCB 17:ll (5%), LCB 18:0 (2?$), three-LCB 18:l (2%), LCB 16:l (l%), LCB 19:l (lyO), and LCB 2O:l (1%). Dberythro- 1,3-Dihydroxy-2-amino-4-tuns-octadecene was obtained from Miles Laboratories, Inc., Elkart, Ind. DL-erythro-1 ,&Dihy- droxy-2-amino-4-octadecyne was a generous gift of Dr. E. F. Jenny and Ciba AG, Basel, Switzerland. Diethyl[2,2,3, 3-2Hd succinat,e was obtained from Service Mol&ules Marqubes, CEA France; deuterium gas (99.7 atom ‘%) and DtO (99.8 atom %) from Norsk Hydro, Oslo, Norway; and LiAlDd (99 atom s) from E. Merck AG, Darmstadt, West Germany. Sodium boro- tritide (4.2 Ci per mmole) was from the Radiochemical Centre, Amersham, England. ATP, UDP-galactose, UDP-glucose, ga- lactose-l-P, dithiothreitol, and Tris buffer were obtained from Sigma Chemical Company, St. Louis, MO.

Determinations of Radioactivity-A Packard Tri-Carb model 3375 liquid scintillation counter was used. Thin layer radio- chromatography was performed with a Berthold Diinnschicht Scanner II. A Barber Colman, series 5000 instrument with a hydrogen flame ionization detector was used for gas-liquid radio- chromatography.

Thin Layer Chromatography-Glass plates (20 x 20 cm) were coated with a slurry of Silica Gel G in water, using a Desaga applicator. The plates were dried at room temperature and then activated at 120” for at least 45 min. The chromatograms were sprayed with 0.2y0 (w/v) 2’, 7’-dichlorofluorescein in C2HsOH and observed in ultraviolet light to detect the compounds. Fatty acid methyl esters were eluted from the plates with diethyl ether; ceramides, cerebrosides, and long chain bases with 0.6 N

NaOH in CHaOH-CHCla, 9:1, v/v as has been described before (16, 18).

Derivatives for Gas-Liquid Chromatography and Mass Spectrom- etry-Fatty acid methyl (ethyl) esters were prepared by treat- ment with diazomethane (diazoethane) in diethyl ether (19).

N-Acetyl derivatives of long chain bases were prepared by dissolving 100 pg of LCB in 100 ~1 of methanol and 10 ~1 of

1 The abbreviations used are: LCB, long chain base or bases; LCB l&l, erythro-trans-l,3-dihydroxy-2-amino-4-octadecene; threo-LCB 18: 1, threo-trans-1,3-dihydroxy-2-amino-4-octadecene; LCB 18:0, erythro-1,3-dihydroxy-2-aminooctadecane; LCB 17:1, erythro-trans-l,3-dihydroxy-2-amino-4-heptadecene, etc.; TGCU, triglyceride carbon units; TMSi, trimethylsilyl.

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acetic anhydride and leaving it at room temperature for 16 hours, The solvents were then evaporated under a stream of nitrogen.

TMSi derivatives of N-acetylated LCB, other ceramides, and ceramide monohexosides were prepared by dissolving 10 to 100 pg of the compound in 100 ~1 of dry pyridine, then adding 20 ~1 of hexamethyldisilazane and 10 ~1 of trimethylchlorosilane. After 15 min at room temperature the solvents were removed under a stream of nitrogen. The residue was dissolved in CS1 (for ordinary gas-liquid chromatography) or hexane (for gas- liquid chromatography-mass spectrometry). When hexane was used, the suspension was sonicated by brief immersion of the tube in a model 9 ultrasonic cleaner (Heat Systems, Ultrasonics, Inc., New York, N. Y.) and an aliquot of the clear supernatant after centrifugation was used for the analysis.

Gas-Liquid Chromatography and l+tass Spectrometry-An F 6t hI model 400 gas chromatograph equipped with a hydrogen flame ionization detector was used. Retention times Ivere ex- pressed as TGCU as described before (17) or as C values. The latter were determined in the same way as TGCU, but using fatty acid methyl esters instead of triglycerides and relating the retention times to the number of carbon atoms of the parent acids. I\Iass spectra were recorded with an LKB model 9000 gas chro- matograph-mass spectrometer (LKB Produkter, Bromma, Swe- den). The instrument was connected to an on-line digital com- puter system (PDP 8/I, Digital Equipment Corp., Maynard, Mass. (20)) for background subtraction, computation of relative abundances, and automatic plotting of mass spectra. Column packings Jvere prepared according to the procedure described be- fore (21) using the Iii-Eff fluidizer. The support was 100 to 120 mesh Gas-Chrom & and the stationary phases were SE-30 ultra- phase (Pierce) and OV-1 (OV-1, Gas-Chrom Q, and the Hi-Eff fluidizer were from Applied Science). The columns were condi- tioned at 280” (SE-30) and 350” (OV-1) for 16 hours.

Mass spectra lvere recorded with an electron energy of 22.5 e.v., a trap current of 60 PA, and an accelerating voltage of 3.5 kv.

EXPERIMICNTAL PROCEDURE AKD RESULTS

Preparation of Deuterium and Tritium-labeled Compounds

DL-erythro-l , %Dihydroxy-%-anzino-&ran.+[4, 5-2H2]octadecene- This compound was prepared by LiAlD4 reduction of acetylenic sphingosine (22). DL-erythro-l , 3-Dihydroxy-2-amino-4-octade- cyne (200 mg) was dissolved in dry tetrahydrofuran, 4.8 ml, and 110 mg of LiAlDh was added. The suspension was refluxed for 4 hours under careful exclusion of moisture. After cooling on ice, 111 ~1 of D,O, 100 ~1 of 15.4cc NaOD in D20 (sodium was dis- solved in dioxane-D20, l:l, v/v, the solvents were evaporated, and the residue was redissolved in DzO), and 104 ~1 of DzO were added. The suspension was filtered and the particles were rinsed with tetrahydrofuran. After removing the solvent, t.he crude product was purified by rolumn chromatography (50-g column, Reference 23). n,c-erythro-1,3-Dihydrosy-2-

anlino-4-trans-[3-3H]octadecene (75 pg (2.8 PC;)) (preparation described below) was added to facilitate detection of dideutero- sphingosine in the chromatographic fractions. The chromato- gram showed a single symmetrical peak of radioactivity (reten- tion volume 240 ml). The corresponding fractions were com- bined and CHCls, CH30H, and water were added to a ratio of 8:4:3, v/v (23), to give 40 mg of dideuterosphingosine in the CHClp phase (1.6 PCi of the radioactivity m-as recovered).

Unchanged acetylenic sphingosine (about 100 mg) could be re- covered from the fractions preceding sphingosine. The product still contained some starting material which was removed by preparative thin layer chromatography using CHC13-CH30H-2 N NII,OH, 160:40:4 (v/v) as solvent system (24). The plat,e was sprayed with 2’, 7’-dichlorofluorescein and the compounds were eluted as described above. The yield was 17 mg (1.2 PCi). The product was analyzed by gas-liquid chromatography-mass spectometry as the i\7-acetyl, di-0-T,\ilSi derivative (969; pur- ity; C value 23.7). The mass spectrum is shown in Fig. 1. A molecular weight of 487 is indicated by the ions at m/e 487 (X) and 472 (Jf - 15, loss of .CH,). The spectrum more resem- bles that of N-acetyl sphing-4-enine than that of N-acetyl sphinganine (TXSi derivatives (25)) as judged by ions at m/e

427 (M - 60, loss of CH3 CONDH) and 244 (X - 243, loss of CII&ONDH + CH3(CH2)rJ and the absence of an ion at m,/e

217. This indicates the presence of an allylic double bond in the molecule. It has been shown before (26) that the double bond formed by LiAl& reduction of acetylenic sphingosine has

the trans configuration. ,2n infrared spec*trum of N(acety1) [4,5-W,] sphingosine (CII&l solution) was similar to that of the protium compound but the band at 975 cm-’ of the latter was absent and there was another band between 715 cm-l and 745 cn-r. The position of this band agrees with what has been found for other compounds containing a deutrated trans double bond (27). The shift of 2 mass units of the ion m/e 313 (AI -

174, loss of CH(NHCOCH,)CH~O-TMSi) in comparison with the protium compound (25) and the absence of shifts for the ions at m/e 174 (X - 313, loss of CH3(CH2)laCD=CD-CIIO-‘l’MSi) and m/e 157 (AI - 330, loss of CHs(CH2)&D=CD~CII0 + TX%OI-I) show that the deuterium atoms are exclusively in the C-3 to C-18 part of the molecule. The ions at m/e 427, 33i (11 - 150, loss of CH&ONDH + TJISi-OH), and 244 show shifts of 1 mass unit compared wit.11 the protium ions. Thus,

eliminat,ion of acetylamide, which is a common feature in the formation of these ions, involves transfer of hydrogen from C-4 or C-5 to the nitrogen. It has been shown before (25) t’hat the

hydrogen at C-3 is not transferred in this reaction. The isotopic composit,ion of the product was calculated from the ions at ill - 17, X - 16, and 14 - 15 after correction for ions at M - 17 and JB - 16 in the protium compound (result: 92% dideutero (dJ and SGdj monodcutero (dl) species).

DL-erythro-i , S-Dihydroxy-%amino-.&trans-[S-3HlJoctadecene- DL - erythro - 1 , 3 - Dihydroxy - 2 - acetamido - 4 - trans - octadecene (N-acetyl nL-sphingosine) was prepared from the long chain base and oxidized to I-hydroxy-2-nn-acetamido-3-oxo-4-trans- octadecene (25). The melting point, X,,, and e of the allylic ketone were identical with those reported before (25). This compound (5.9 mg) was reduced with NaBHe-T, 0.9 mg, 100 mCi, for 60 min (25). The product was diluted with 4.0 mg of Wacetyl nn-sphingosine, purified by silicic acid chromatography and thin layer chromatography (solvent system: CHCl&H~OH, 9:1, v/v), and was then hydrolyzed in 2 ml of half-saturated (100”) Ba(OH)z-dioxane, 1: 1, v/v (28). The ether-extractable product was purified by thin layer chromatography (solvent system: CHC13-CH30H-2 N NHdOI-I, 160:40:4, v/v (24)) and column chromatography (23). It Teas pure as judged by gas- liquid chromatography of the Ar-acetyl di-0-TMSi derivative (C value 23.8 on SE-30). A 190.$Zi portion of the product (total yield was 230 PCi) was diluted with 4.45 mg of D-erythro-

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Issue of February 25, 1972 S. Hammarstriim and B. Samuelsson

M-al (M-la-i’3)-9f) 1;

313 M-d)

TMSi mol wt 487

60 100 140 180 220 260 300 340 380 420 460 m/e

-w--1 25 mol wt 362

/ ‘“;1’9”’ ; g; / I’ I/ I I I/ 11 11

I I” I I I ” 8 (

50 90 130 170 2 10 2io 290 33c m/e

FIG. 1 (upper). Mass spectrum of DL-erythro-lrans-1,3-dihydroxy-2-amino-4-[4,5-zH*]octadecene as the N-acetyl, di-0-TMSi deriva- tive.

FIG. 2 (lower). Mass spectrum of 2’-n-hydroxy[4,4,5,5-2Hd]hexadecanoic acid as the 0-TMSi methyl ester derivative.

truns-sphingosine to give specific activities of 6 &Ji per pmole for the D isomer and 103 FCi per pmo]e for the L isomer.

dz+Hydroxy[d ,4,5, 5-2HJhexcuJecunoic Acid-Methyl hydro- gen tetradeutero succinate was first extended to [2,2,3, 3-2H& tetradecanoic acid and then to 2nhydroxy[4,4,5, 5-2Hdhexa- decanoic acid by electrolytic couplings.

Diethyl [2,2,3,3-2H4]succinate (91% dd, 6% ds, and 3% d2. by mass spectrometry), 1 g, was converted to the methyl half- ester by hydrolysis with 1 eq of KOH in CHsOH-H20, 1: 1 (60°, 1 hour), treatment of the dipotassium succinate with acetyl chloride, 5 ml (reflux, 2 hours), and of the anhydride with CHs- OH, 5 ml (SOO, 0.5 hour). The methanol solution was filtered and evaporated to dryness, and the residue was crystallized from ethyl acetate and ethyl acctatc-petroleum ether (yield 725 mg). The half-ester, 5 g of dodecanoic acid and 70 mg of sodium were dissolved in CH30H and electrolyzed until slightly alkaline pH was attained. One volume of 10% KOH in water was added, and the solution was heated to 60” for 1 hour. Diethyl ether extraction of the alkaline solution yielded a mixture of labeled tetradecanoate and n-docosane. The latter crystallized quan- titatively from 75 ml of C2HsOH and the ester (500 mg) could be recovered from the mother liquor. It was again treated with 5% KOH in CHIOH-H20,. 1:1 (60’ for 1 hour). The diethyl ether extracts (acidification to pH 1) were combined and subjected to reversed phase partition chromatography using 65% aqueous CH30H and 2,2,Ctrimethylpentane (29) to

remove some dodecanoic acid which had not reacted during the electrolysis. The product was finally passed through a silicic acid column and crystallized from hexane (yield: 400 mg, m.p. 52.5-54.5’; 98(r, pure by gas-liquid chromatography as the methyl ester on SE-30). The isotopic composition (84yc d+ 13y0 dl, and 3% d2 by mass spectrometry) indicated some losses of deuterium along the preparation. In addition to the molecu- lar ion, m/e 246, intense ions were seen at m/e 215 (M - 31, loss of .OCHs), m/e 200 (M - 46, transfer of a hydrogen from C-6 to C-4 and elimination of C-2, C-3, and C-4, i.e. CD&D2- CHJ, m/e 189 (M - 57, loss of ~CH#XI~)&HJ, m/e 175 (M - 71, loss of .CH2(CH2)&H3), m/e 147 (M -- 99, loss of .CH2(CH2)&H3), m/e 133 (N - 113, loss of .CHP(CH&CHJ, m/e 119 (M - 127, loss of .CH2(CH&CHs), and m/e 105 (M - 141, loss of .CH2(CH&CH3). The McLafferty rearrange- ment ion was at m/e 76, indicating 2 deuterium atoms at C-2. The ion at m/e 87 in mass spectra of aliphatic methyl esters is formed by transfer of a hydrogen from C-6 to the carbonyl oxy- gen, followed by rearrangement of 1 a-hydrogen to C-6 and cleavage between C-3 and C-4 (30). The present spectrum had ions of equal intensity at m/e 89 and 90 indicating exchange of the second a-deuterium atom with a hydrogen atom more remote in the chain prior to cleavage. Mass spectra of methyl esters with 1 or 2 deuterium atoms at C-3 (31, 32) exclude the possibil- ity of exchange of the Bdeuterium atoms.

[2,2,3,3-XJTetradecanoic acid, 400 mg, methyl 2 n-acetoxy

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Vol. 247, No. 4

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3-carboxypropionate, 1.62 g (prepared from D( +)-malic acid, Fluka AG, S:t Gallen, Switzerland) and sodium, 25 mg were electrolyzed in methanol as described before (33). The crude product was hydrolyzed in 5% KOH in CHPOH-H20, 1: 1 (re- flux, 2 hours). The free acid was purified by silicic acid column chromatography and after methylation by thin layer chromatography (solvent system: diethyl ether-hexane, 1: 1, v/v). The ester was hydrolyzed (yield: 70 mg; 99% pure by gas-liquid chromatography of the 0-TMSi methyl ester deriva- tive; C value 18.5). The mass spectrum of the 0-TMSi, methyl ester derivative is shown in Fig. 2. It is similar to mass spectra of other long chain 2trimethylsilyloxy methyl esters (34). The molecular ion (m/e 362) was of low relative intensity but more abundant ions were present at m/e 347 (M - 15, .CHJ, 303 (M - 59, .COOCHI), and 319 (M - 43, .CHI + CO). The isotopic composition was the same as for the tetradecanoic acid.

The hydroxy acid was acetylated with acetic anhydride in pyridine (18). Crystallized from hexane, the product (74 mg) melted between 55-59” and had [a]E5 = +9.6” (1,l; c, 1.78). The specific rotation is about 5 times greater than for the hy- droxy acid.

N-(2’-D-Hydroxy[4’,4’,5’,5’, -2H4]hexadecanoyl) o-erythro- 1, S-Dihydruxy-Samino-&rans[.J , 5-2H2-octadecene-The ceramide was prepared from 5.1 mg of [4,5-2HJsphingosine and 10 mg of 2nacetoxy[4,4,5, 5-2H.J hexadecanoate by direct coupling in the presence of 16.9 mg of I-ethyl-3-(3-dimethylaminopropyl) car- bodiimide (18). The product was purified by silicic acid chro- matography and converted to 2-hydroxy acid ceramide by mild alkaline methanolysis (yield 5.4 mg). In order to obtain the naturally occurring optically active ceramide, diastereoiso- merit ceramides were separated by preparative thin layer chromatography (18) on a plate 20 X 20 cm (yield of natural diastereoisomer 2.7 mg). An aliquot of the ceramide was con- verted to the tri-0-TMSi derivative and analyzed by gas-liquid chromatography-mass spectrometry (purity 98 y0 on OV-1; re- tention time 37.9 TGCU). Figure 3a shows the mass spectrum obtained. The molecular ion, m/e 775 and the ions M - 15, .CH3 (m/e 760), il4 - 90, TMSi-OH (m/e 685), M - 103, CH20-Th!ISi (m/e 672), M - 180, 2 x TMSi-OH (m/e 595), and dl - 103 - 90, CH20-TMSi + TMSi-OH (m/e 582) contain the whole ceramide carbon skeleton (for further discussion, see Reference 35). They indicate an isotopic composition of 77.2% dg, 18.7 ‘% ds, 3.8% dq, and 0.1% d) species in agreement with the isotopic compositions of the constituent LCB and acid. Upward shifts by 4 mass units of the “fatty acid ions,” M - (a - 73) (m/e 535), M - a (m/e 462), M - a - 16 (m/e 446), and f (m/e 303), and by 2 mass units of the “LCB ion,” M - d (m/e 313), provide evidence that the deuterium atoms retained their original positions during the coupling reaction. The ions at m/e 427,337, and 244 are formed by elimination of the fatty acid part of the molecule after rearrangement of one of the LCB deuterium atoms and appeared 1 mass unit higher than the correspond- ing ions in the spectrum of the protium ceramide (cf. the dis- cussion of the mass spectrum in Fig. 1).

N-(Z’o-Hydroxyhexadecanoyl) n-erythro-1 ,S-Dihydroxy-W-ami- no-4-trans-[S-aH,]octadecene-2-n-Acetoxyhexadecanoic acid was prepared from tetradecanoic acid and methyl 2-n-acetoxy a-car- boxypropionate as described above for the tetradeuterium-labeled acid. Melting point and [a]i5 were identical for the two compounds. [3-aHi]Sphingosine, 3.75 mg (143 PCi), and 2-D-

acetoxy hexadecanoic acid, 10 mg, yielded 38 PC1 = 3.5 mg of

the natural ceramide diastereoisomer by the procedure outlined above. The radiochemical purity of the product was estab- lished by thin layer chromatography (RF value = 0.35 using CHCla-CHaOH, 93 :7, v/v as solvent system) and gas-liquid radiochromatography of the 1,3,2’-tri-0-TMSi derivative (re- tention time 37.9 TGCU on OV-1). It was at least 95% by both techniques. The gas-liquid chromatography analysis also showed a mass purity of 87% in agreement with the composition of the n-sphingosine used for the dilution of [3-aHl]sphingosine. The mass spectrum of the TMSi derivative is shown in Fig. 3b. It was identical with the spectrum of the unlabeled compound

(35). Mixture of Hexadeutero and Protium N-(2’o-hydroxyhexadec-

anoyZ)-o-sphingosine-Aliquots of the ceramides described above were analyzed by quantitative gas-liquid chromatography as TMSi derivatives. The ceramides were mixed in a 1:l ratio from the peak area measurements. Part of the mixture was converted to TMSi derivatives and analyzed by gas-liquid chromatography-mass spectrometry using an accelerating volt- age alternator to record the intensities of the ions at m/e 754 and 760 throughout a chromatogram (Fig. 4). These m/e values represent the M - .CHa ions the two ceramides, respectively. By measuring the areas of the two curves and allowing for the fact that only 77.2% of the deuterated ceramide was due to hexa- deutero species, the substrate was found to contain 51% pro- tium- and 49% deuterium-labeled (de-da) ceramide.

Incubations and Chromatographic Procedures

Incubations and Lipid Extractions-To obtain enough product for its characterization, seven consecutive incubations were per- formed (95% of the ceramide substrate could be recovered un- changed after an incubation and be used again for the next incubation). The substrate (5.4 mg, 7.8 X lo7 dpm) was equally divided among four tubes, each containing 100 mg of Celite (8). The solvents were evaporated under a stream of nitrogen, while rocking the tubes. To each tube was added (in a total volume of 0.5 ml of water): 50 pmoles of Tris-HCl, pH 7.40, 1 pmole of dithiothreitol, 2 pmoles of ATP (neutralized with NaOH before use), 0.14 pmole of UDP-Glc, and 0.08 pmole of galactose l-phosphate and the suspension was briefly sonicated in the ultrasonic cleaner.

I m/e 151, I

FIG. 4. Multiple ion analysis of the 1,3,2’-tri-0-TMSi deriva- tive of the mixture of hexadeutero N-(2’-hydroxyhexadecanoyl) sphingosine used for the incubations with mouse brain micro- somes. The ion intensities of the M - 15 ions of the two species (m/e 760 and 754, respectively) were recorded as a function of time.

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1006 Biosynthesis of Cerebrosides fTorn Ceramides Vol. 247, No. 4

Microsomes were prepared as described before (8). Half a milliliter of resuspended microsomes (final volume 0.8 ml per

g, wet wt, of brain) was added to each tube. The tubes were incubated for 2 hours at 37” with violent agitation. Twenty milliliters of CHCla-CHaOH, 2:l (v/v), were then added and the contents were sonicated briefly. The solutions were fil- tered and each tube was rinsed with 3 ml of CHCl&H,OH, 2:l (v/v). After addition of 4.6 ml of 2 M KC1 to each tube the contents were mixed and centrifuged. The upper phases were discarded, the lower phases were washed once with 10 ml 2 M KCl-methanol, 1: 1 (v/v), and then evaporated to dryness under a stream of argon. Six milliliters of 0.2 N NaOH in methanol and 12 ml of CHC13 were added to each tube and the mixtures were left at room temperature for 1 hour. Then 4.2 ml of 0.35 M acetic acid were added, the contents were mixed, and the tubes were centrifuged. The lower phases were washed with 6.2 ml of 507, aqueous methanol, combined, and evapo- rated to dryness.

Silicic Acid Chromatography-The residue was dissolved in CHCl, and subjected to silicic acid column chromatography (36). The column was first washed with 150 ml of CHCls.

I- 3, LCB I8 I-18 0 Au LCB I8 I-18h.O

I 0 45 L-7 L9 51 53 55

TGCU

/ Glu LCE @:I-16h.O

Glu LCB 18.1-20.0 Glu LCB 18.1-20h:O Glu LCB 20-l-18.0 Glu LCB 20.l-18h.O

Glu LCB l8.1-2L 0 Glu LCB l8:1-2Lh.c

Glu LCBl&l-22:0 Glu LCBl8 I-22h:O

FIG. 5. Gas-liquid radiochromatogram of glucosyl ceramides from incubations of hexadeutero plus protium N-(2’-hydroxyhexa- decanoyl) sphingosine with mouse brain microsomes. Ceramide monohexosides were isolated by silicic acid chromatography and preparative thin layer chromatography. Glucosyl ceramides and galactosyl ceramides were separated by thin layer chromatog- raphy on borate-impregnated plates and converted to 0-TMSi derivatives for the gas-liquid chromatography analyses (sta- tionary phase, l.Boj, OV-1; column temperature, 330”). The upper Curve shows mass and the lower CUTDB radioactivity detection. The retention time is expressed as TGCU (17). The main molec- ular species indicated for each component were determined by mass spectrometry in a separate run. A gas-liquid radiochro- matogram of the galactosyl ceramide derivatives was similar to the one shown here.

The material eluted (a total of 5.4 x lo6 dpm from the seven incubations) was very heterogenous on thin layer chromatog- raphy and a characterization was not attempted. Three hun- dred milliliters of CHCI,-CH30H, 98:2 (v/v), eluted uncon- verted ceramide in radiochemically pure form as shown by thin layer chromatography and by gas-liquid chromatography-mass spectrometry of the TMSi derivative. The yield of ceramide after the seventh incubation was 5.3 x lo7 dpm. Ceramide monohexosides, free sphingosine, and some “tailing” 2-hydroxy acid ceramide were eluted with 350 ml of acetone-methanol, 9:l v/v (5.2 x lo6 dpm from seven incubations). Elution with 250 ml of methanol after the first incubation yielded an addi- tional 4.2 X loj dpm of sphingosine. No conversion of the ceramide could be detected in a control experiment using boiled microsomes.

Thin Layer Chromatography on Ordinary Plates-Ceramide monohexosides in the acetone-methanol 9 : 1 fractions were purified by preparative thin layer chromatography using CHC13- CHIOH-HZO, 144:25:2.8 (v/v) as solvent system. The eluates from Incubations 1 to 3 (2.5 X lo6 dpm) and 4 to 7 (2.7 x lo6 dpm) were chromatographed separately. Each chromatogram showed three components \I-hich cochromatographed n-ith 2- hydroxy acid ceramide (RF 0.7), brain cerebrosides (RF 0.3 to 0.4), and sphingosine (RF 0.2 to 0.3), respectively. These were eluted to give (from Incubations 1 to 3 and 4 to 7, respectively) : 2-hydrosy acid ceramide 5.5 x loj and 6.2 x loj dpm, cerebro- side 8.3 x lo4 and 2.7 x loj dpm, and sphingosine 7.4 x lo5 and 8.9 x loj dpm.

Thin Layer Chromatography on Borate-impregnated Plates (S’?‘)-The purified ceramidemonohexosides from Incubations 1 to 3 and 4 to 7 were chromatographed on Silica Gel G plates containing sodium borate (solvent system: CI-IC1&11301-I-H20- 25% NHdOH, 280 : 70 : 6 : 1 (v/v)). Glucosyl ceramides from Gaucher spleen (a generous gift of Dr. N. S. Radin, University of Michigan, Ann Arbor, ;\Iich.) and galacCosy1 ceramides from bovine brain (Sigma Chemical Companp, St. Louis, MO.) were used as references. Two radioactive compounds were seen on each chromatogram. The slower migrating one (RF 0.17 to 0.32) cochromatographed with 2-hydrosy acid-cont’aining galac- tosyl ceramides. The other compound (RF 0.43 to 0.50) moved somewhat below Gaucher spleen cerebrosides (RF 0.53 to 0.57) which do not contain 2-hydrosy acids.

Characterization of Products

Gas-Liquid Chromatography-Mass Spectrometry of Galactosyl and Glucosyl Ceramide-The galactosyl and glucosyl ceramides isolat.ed from Incubations 1 to 3 were silylated and analyzed by gas-liquid radiochromatography. Both radioactive components had retention times of 46.7 TGCU. Fig. 5 shows the chromat- ogram for the glucosyl ceramides. About 30% of the deriva- tives were then analyzed by gas-liquid chromatography-mass spectrometry. The mass spectra of the radioactive components are shown in Fig. 6. Both compounds were TMSi derivatives of mixtures of ceramide monohexosides containing hexadecanoic and 2-hydroxy hexadecanoic acid, respectively. The glucosyl ceramide spectrum also contained ions from a preceding gas- liquid chromatography component (the separation during gas- liquid chromatography-mass spectromet’ry was better than that shown in Fig. 5). The “impurity” had a retention time of 45.9 TGCU and abundant mass spectral ions at m/e 485, 413, 397, 395, 361, 331, 319, 305, 217, 204 (base peak), 147, 129,

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1008 Biosynthesis of Cerebrosides from Ceramides Vol. 247, No. 4

103, 75, and 73. These ions are superimposed on the glucosyl ceramide spectrum in Fig. 6b and explain the different appear- ance compared with the spectrum in Fig. 6~. Ions resulting from the nonhydroxy acid ceramide monohexosides were present at m/e 311 (M - d), 370 (2M - a), 592 (M - 467), and 710 (M - 349). These compounds were of microsomal origin since there were no deuterium-labeled species present. The ceramide monohexosides containing 2-hydroxy acid were both mixtures of hexadeutero and protium species. They were identified by ions at m/e 311/313 (M - d), 458/462 (M - a), 680/686 (M - 467), and 798/804 (M - 349). These ions indicated the same localization of the deuterium atoms as in the substrate. Tetra- deutero and dideutero species were absent from the products. The implication of this will be discussed below.

Degradation of Galactosyl and Glucosyl Ceramides to Ceramic&s and Analysis of Latter by Gas-Liquid Chromatography-Mass Spectrometiy-The galactosyl and glucosyl ceramides isolated from Incubations 4 to 7 were degraded to ceramides, separated into groups of ceramides, and analyzed as the 0-TMSi deriva- tives by gas-liquid chromatography and gas-liquid chromatog- raphy-mass spectrometry. The procedures for this have been described in detail (16). HI04-NaBHa-HCl degradations yielded 5.06 x 10’ and 6.95 x lo4 dpm of ceramides from the galactosyl and glucosyl ceramides, respectively. The derived ceramides were separated into nonhydroxy acid and 2-hydroxy acid cer- amides by thin layer chromatography (solvent system: CHCla- CHsOH, 93:7, v/v). The nonhydroxy acid and 2-hydroxy acid ceramides obtained from the galactosyl ceramides were further acetylated and subjected to argentation-thin layer chromatog- raphy which separates ceramides according to the number of cis double bonds. This separation yielded a total of 4 ceramide fractions; i.e. ceramides containing saturated 2-hydroxy acids,

saturated nonhydroxy acids, monounsaturated 2-hydroxy acids, and monounsaturated nonhydroxy acids. Only the ceramide fraction containing saturated 2-hydroxy acids was radioactive (yield 1.78 x lo4 dpm). The yield of radioactivity in the hy- droxy acid ceramide from glucosyl ceramide was 1.85 x lo4 dpm. The ceramide fractions obtained from the thin layer chromatog- raphy separations were converted to 0-TMSi derivatives and analyzed by gas-liquid radiochromatography and by gas-liquid chromatography-mass spectrometry. Fig. 7a shows a gas-liquid radiochromatogram of saturated 2-hydroxy acid ceramides from galactosyl ceramides. The retention time of the radioactive component was 37.8 TGCU. The gas-liquid radiochromato- grams of the TMSi derivative of 2-hydroxy acid ceramides from glucosyl ceramides also showed a single peak of radioactivity with the same retention time. Fig. 7b shows the total ion cur- rent recording of the hydroxy acid ceramides from glucosyl ceramides and Fig. 8 the mass spectra of the radioactive com- ponents. The mass spectra were recorded somewhat after the apex of the respective gas-liquid chromatography peak (approxi- mately half-way between the apex and the second point of in- flection of the total ion current curve). The spectra of the two products were very similar, and also very similar to the sum of the spectra in Fig. 3. All ions of the latter spectra (and no additional ions) were present in Fig. 8. The ions M - (a - 73) (m/e 531 + 535), M - a (m/e 458 + 462), M - a - 16 (m/e 442 + 446), and cf> (m/e 299 + 303) demonstrated conversion of tetradeuterium-labeled and protium 2-hydroxy hexadecanoic acid and the ion M - d (m/e 311 + 313) conversion of dideu- terium-labeled and protium sphingosine from the ceramide sub- strate. Evidence for biosynthesis via the ceramide pathway is provided by the ions which contain both the acid and the LCB, i.e. M - 15 (m/e 754 + 760), M - 90 (m/e 679 + 685), M -

FIG. 7. Gas-liquid chromatography analyses of TMSi deriva- tives of 2-hydroxy acid ceramides derived from galactosyl ceram- ides (the radiochromatogram to the left) and glucosyl ceramides (the total ion current recording to the right). The ceramide mono- hexosides were isolated after incubations of hexadeutero plus protium N-(2’-hydroxy hexadecanoyl) sphingosine with mouse brain microsomes (see legend to Fig. 5). They were converted to

L

LCB 18:k20h

CBK%l-16h.O

1 LCB18:1-23h.O

LCB I&l-2Lh.0

k 0 36 LO L2 LL 46

TGCU

LC

LCE318.1-16h:O

LCBIB:I-18h:O

B l8:i ah-0 LCBIB:I-2Lh:O

LCBZO.I-18h:O

38 LO L2 LL I.6 TGCU

ceramides by HIOh-NaBHa-HCI degradation and separated by thin layer chromatography into ceramides with constituent non- hydroxy acids and 2-hydroxy acids. The ceramides obtained from galactosyl ceramides were also subjected to argentation thin layer chromatography after acetylation, prior to the gas-liquid chro- matography analysis.

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1010 Biosynthesis of Cerebrosides from Ce~~anwkles Vol. 247, so. 4

103 (m/e 666 + 672), A4 - 105 (m/e 664 + 670), Jf - 180

(m/e 589 + 595), and M - 103 - 90 (m/e 576 + 582). These results will be discussed below.

Mass spectra were also recorded on the other gas-liquid chro- matography components of Fig. 7. The main molecular species are given by the peak designations. The ceramides derived from galactosyl ceramides had a composition similar to those from bovine brain cerebrosides (16) although the chain length distributions showed certain differences. The composition of mouse brain glucosyl ceramides was recently analyzed by gas- liquid chromatography-mass spectrometry of TMSi derivatives of the intact molecules (38). The results obtained here, with the derived ceramides, mere in good agreement with this.

Characterization of Sphingosine by Gas-Liquid Radiochromatog-

raphy and Gas-Liquid Chromatography-Mass Xpectrometry--The

radioactive compound from Incubations 4 to 7 that moved below cerebroside on ordinary thin layer chromatography plates and had the same RF value as n-sphingosine was eluted from the plates as described above. Half of it was converted directly,

the other half after dilution with 500 pg n-sphingosine, to the N-acetyl, di-0-TMSi derivative. The diluted sample was then anaiyzed by gas-liquid radiochromatography and the undiluted one by gas-liquid chromatography-mass spectrometry. The derivative of the radioactive product was quite pure by gas- liquid chromatography and had the same retention time as the

TABLE I

Distribution of deuterium-labeled species in ceramides cle?ived from

biosynthetic 01-galactosyl ii-(2’.hydroxy hexaclecanoyl) sphingosine and 01-glucosyl l\i-(2’-hydroxg

hexatlecanoyl) sphingosine

The incubation mixture contained S-(2’.hydroxy[4’,4’,5’,5’-

2H4] hexadecanoyl) [4, 5-2HJ sphingosine and the corresponding protium ceramide, 5 + 5 pmoles, coated onto 400 mg of Celite (8); Tris-HCI buffer (pH 7.40), 200 pmoles; dithiothreitol, 4 pmoles; ATP, 8 pmoles; UDP-Glc, 0.56 bmole, galactose-l-P, 0.32

pmole; microsomes from 2.5 g of mome brain resuspended in 0.25 M sucrose, 2.0 ml, and water, 2.0 ml. After 2 hours of incubation at 37”, CHCX-CHSOH (2:l) was added. Ceramide monohexosides

were isolated by column chromatography and thin layer chroma- tography. Galactosyl and glucosyl ceramides were separated by thin layer chromatography on borate-impregnated plates. They were degraded to ceramides with HI04-NaBHd-HCI treatments and separated according to the nature of the constituent acid by

ordinary and argentation-thin layer chromatography. TMSi derivatives of the ceramides were analyzed by gas-liquid chroma- tography-mass spectrometry.

I Found for

Species

Ceramide substrate

% de. . 31.6

d5.. . . . . 7.7 dq.. 3.8 dz . . . 1.2 dz. . . . . . 1.6

d; ..____ 2.4 do. . . . 51.0

% % 18.5 37.8 4.5 9.2

21.9 1.9

3.3 0.1 23.8 0.0

2.0 0.0 26.0 51.0

a Calculated from the isotopic compositions of the constituent acid and LCB of the substrate and the ratio shown in Fig. 4.

% 36.0

4.3 0.8 1.2 4.4

4.3 49.0

% 35.3

3.4 0.6 0.0 4.0

0.1 56.4

Galactosyl ceramide product

Glucosyl ceramide product

Calculated for product formed via

Psychosine Ceramide pathwaya pathwaya

derivative of the added n-sphingosine (C value 23.70). The mass spectrum of the undiluted sample derivative showed that the product formed during the incubations was a mixture of dideutero and protium sphingosine (ions at m/e 472, 470; 427, 426; 397, 395; 382, 380; 337, 336; 313, 311; 247; 244, 243; 174 and 157). The ratio of dideutero to prot.ium species was similar to the ratio of deuterium to protium ceramide in the substrate, showing that the main source of free sphingosine was the added ccramide.

DISCUSSIOS

The two pathways for cerebroside biosynthesis were postulated from measurements of the stimulation various acceptor lipids had on the conversion of 1%.labeled galactose-1-P and ‘Y-la- beled stearoyl-CoA to galactosyl ceramide. The conversions of acceptor lipids were very low for both pathways and it could not

be excluded that these compounds were hydrolyzed prior to the reaction or that they excerted a nonspecific effect on the micro- somal enzyme system.

We decided to study the proposed reactions using acceptor lipids labeled with deuterium on both sides of their hydrolyzable bond. A mixture of the labeled and unlabeled compound would give products with distinctly different isotopic compositions de- pending on whether the substrate was transformed int’act or secondary to hydrolysis into constituents.

N-(2’-Hydroxy hexadecanoyl) sphingosine was chosen as sub- strate to investigate the ceramide pathway in order to avoid dilution of the product by endogenous cerebroside. This cer- amide was also more efficient,ly converted to cerebroside than some ceramides containing longer 2-hydroxy acids3 under the conditions for incubation described above.

Both galactosyl ceramide and glucosyl ceramide were formed during the incubations. The identificat’ion of these products was based on: (a) RF values on ordinary and borate-impregnated plates (6) gas-liquid radiochromatography and gas-liquid chro- matography-mass spectrometry of the TMSi derivatives, and (c) chemical degradation to ceramides followed by gas-liquid chromatography-mass spectrometry analysis of the TI\ISi de- rivat.ives. Table I shows the expected isotopic composition of the products for the psychosine and the ceramide pathways, respectively, based on the observed composition of the substrate. For the ceramide pathway, substrate and product would have identical compositions. The values calculated for the psychosine pathway assume that the only source of sphingosine and 2-hy- droxy hesadecanoic acid was the added ceramide. Approxi- mate isotopic compositions of the products were obtained from the M - 15 ions of the spectra in Fig. 8. Because of partial separation of TMSi derivatives of deuterium-labeled and pro-

tium ceramides during gas-liquid chromatography (Fig. 4), the

ratios of the recorded ion intensities will not be the same depend- ing on when, during the elution of the compound, the mass spectrum is recorded. The spectra of Fig. 8 were recorded

somewhat after the maxima of the gas-liquid chromatography

peaks. Therefore, the ratios of hexadeuterium-labeled to pro- tium ceramides in Fig. 8 (Fig. 8a in particular) are a little lower

than the corresponding ratio of the substrate. The observed isotopic compositions of the products agreed well with that calculated for t’he ceramide pathway, but not with the composi- tion for the psychosine pathway. Consequently, it is unam-

a S. Hammarstrom, unpublished results.

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Issue of February 23, 1972 X. Hammah%m and B. Xamuelsson 1011

biguous that galactosyl as well as glucosyl ceramides containing 13. Burros, 11. M. (1967) in G. SCHETTLER (Editor), Lipids and

Z-hydroxy acids can be synthesized via the ceramide pathway Zipicloses, p. 131, Springer-Verlag, Berlin-Heidelberg, New

using brain microsomes as enzyme source. York.

Biosynthesis of glucosyl ceramides containing nonhydroxy 14. SCHNEIDER, P. B., AND KENNEDY, E. P. (1968) J. Lipid Res.,

9, 58. acids from ceramide has been reported before (39). The psycho- 15. SAMUELSSON, K., AND SAMUELSSON, B. (1969) Biochem. Bio-

sine pathway has not been claimed in this case. Glucosyl phys. Res. Commun., 37, 15.

ceramides are intermediates in the biosynthesis and degradation 16. HAMMARSTR~M, S. (1970) Eur. J. Biochem., 16, 581.

of ceramide oligosaccharides and of gangliosides (40). In brain, 17. HAMMARSTR~M, S., AND SAMUELSSON, B. (1970) Biochern.

these compounds seem to contain only nonhydroxy acids (40). Biophys. Res. Commun., 41, 1027.

18. HAMMARSTR~M, S. (1971) J. Lipid Res., 12, 760.

Similarly, bovine brain glucosyl ceramides did not contain 2:hi- droxy acids (41). The formation of glucosyl ceramides from 2-hydrosy acid ceramides led us to investigate the composition of mouse brain glucosyl ceramides. The results showed about 80% nonhydroxy acids and 20% 2-hydroxy acids (38). The presence of 2-hydroxy acids in the mouse may be due to an age or a species difference.

SCHLEXK, H., AND GELLERMAN. J. L. (1960) Anal. Chem., 32, 19.

20.

21.

1412. SWEELICY,C.C.,RAY, B.D., WOOD, W.I., AND HOLLAND, J.F.

(1970) Anal. Chem., 42, 1505. HORNING, E. C. (1968) in K. B. EIIC-NES AND E. C. HORNING

(Editors), Gus phase chromatography of steroids, pp. l-71, Springer-Verlag, Berlin.

22. 23.

24.

25.

26.

27.

JENNY, E. F., AND DRUEY, J. (1959) Helv. Chim. Acta, 42, 401. BAIZIXNHOLZ, Y., AND GATT, S. (1968) Biochim. Biophys. Acta,

The incubation mixture contained UDP-Glc and galactose-1-P in accordance with the original conditions (8). Substitution of these for UDP-Gal increased the yield of galactosyl ceramide somervhat but glucosyl ceramide was still formed. Both prod- ucts were also formed from N-(2’-hydroxy docosanoyl) [3-3Hl] sphingosine (17). Glucosylation of X-hydroxy acid ceramides in brain has independently been observed by Shah (42).

A third product was identified as free sphingosine by gas- liquid radio-chromatography and gas-liquid chromatography- mass sgect,rometry. Enzymatic hydrolysis of non-hydroxy acid ceramides has been described (43). The present results demon- strate t,hat 2-hydroxy acid ceramides are also liable to enzymatic hydrolysis in the microsomal system employed here.

28. 29.

30.

Ackno&dgment-The authors are indebted to Mrs. Saga Elwe for skillful technical assistance.

REFERENCES 1. CLELAND, W. W., AND KENNEDY, E. P. (1960) J. Biol. Chem.,

235, 45. 2. NXSKOVIC, N. M., NUSS~.\UM, J. L., AND MANDEL, P. (1969)

Fed. Eur. Biochem. Xoc. Lett., 3, 199.

31.

32.

33.

34.

35. 3. KASFER, J. N. (1969) Lipids, 4, 163.

HAMMARSTR~M, S., SAMUELSSON, B., AND SAMUELSSON, K.

4. BRADY, R. 0. (1962) J. Biol. Chem., 237, PC2416. (1970) J. Lipid Res., 11, 150.

5. SRIBNEY, M. (1966) Biochim. Biophys. Actu, 126, 542. 36. Vasc~:, D. E., A~VD SWEELEY, C. C. (1967) J. Lipid Res., 3,

6. MORELL, P., AND RADIN, N.S. (1970) J.Biol. Chem.,245, 342. 621.

7. RADIIX, N. S. (1959) in S. R. KOREY (Editor), The biology of 37. &CAN, E. L. (1966) J. Lipid Res., 7, 449.

?n.yeZin, p. 271, Harper and Brothers, New York. 38. HAMMhRSTRsM, S. (1971) Eur. J. Biochem., 21, 388.

8. MORELL, P., AND RADIN, N. S. (1969) Biochemistry, 8, 506. 39. BASU, S., KAUPMAN, B., AND ROSEMAN, S. (1968) J. Biol.

9. FUJINO, Y., AND NAKANO, M. (1969) Biochem. J., 113, 573. Chem., 243, 5802.

10. NESKOVIC, N. M., NUSSBAUM, J. L., AND MANDEL, P. (1970) 40. h/lbRTENSSON, E. (1969) in Progress in the chemistry of fats and

Fed. Eur. Biochem. Sot. Lett., 3, 213. other lipids, Vol. X, Part 4, pp. 384-398, Pergamon Press,

11. MORE+ P., COSTANTINO-CECChRIKI, E., AND RADIN, N. S. New York.

(1970) Arch. Biochem. Biophys., 141, 738. 41. NISHIMURA, K., AND YAMAI~A~A, T. (1968) Lipids, 3, 262.

12. KOPACZYK, K. C., AND RADIN, N. S. (1965) J. Lipid Res., 6, 42. SH.&H, S. N. (1971) J. ATeurochem., 18, 395.

140. 43. GATT, S. (1966) J. Biol. Chem., 241, 3724.

162, 790. SAMBASIVARAO, K., AND MCCLUER, R.H. (1963)J.LipidRes.,

4, 106. GAVER, R. C.. AND SWEELEY. C. C. (1966) J. Amer. Chem. SOC.,

88, 3643. ~ ,

GROR, C. A., AND GADIENT, F. (1957) Helv. Chim. Actu, 40, 1145.

PIXCHAS, S., AND LAULICHT, I. (1971) ZnfTured spectra of labeled compounds, pp. 84-106, Academic Press, London.

TIPTON, C. I,. (1962) Biochem. Prep., 9, 127. HOWARD, G. A., AND MARTIN, A. J. P. (1950) Biochem. J., 46,

532. BUDZXIEWICZ, H., DJERASSI, C., AND WILLIAMS,~~. H. (1967)

Muss spectrometry of organic compounds, p. 179, Holden- Day, Inc., San Francisco.

POLITO, A. J., AND STVEELEY, C. C. (1971) J. Biol. Chem., 246, 4178.

DINH-NGUYI%N, N., RYHAGE, R., STXLLBERG-STENHAGEN, S., AND STENHAGEN, E. (1961) Arkiv. Kemi, 18, 393.

HORN, D. H. S., AND PRETORIUS, Y.Y. (1954) J. Chem. Sot., 1460.

EGLINTON,G.,HUNNEMAN, D. H., ANDMCCORMICK, A. (1968) Org. Mass Spectrom., 1, 593. ^.

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Sven Hammarström and Bengt SamuelssonPATHWAY

SPECTROMETRIC EVIDENCE FOR BIOSYNTHESIS VIA THE CERAMIDE On the Biosynthesis of Cerebrosides Containing 2-Hydroxy Acids: MASS

1972, 247:1001-1011.J. Biol. Chem. 

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